Peptidomimetics Protocols

1 Synthesis and Use of Pseudopeptides Derived from 1,2,4=Oxadiazole-, 1,3,4=0xadiazole-, and 1,2,4=Triazole=based Dipept...

Author: Wieslaw M. Kazmierski

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1 Synthesis and Use of Pseudopeptides Derived from 1,2,4=Oxadiazole-, 1,3,4=0xadiazole-, and 1,2,4=Triazole=based Dipeptidomimetics Kristina Luthman, Susanna Borg, and Uli Hacksell 1. Introduction This chapter focuses on the tsostertc replacement of peptide bonds with three different types of heterocycltc rmg systems (I); 1,2,4-oxadtazole (2), 1,3,4oxadtazole (3), and 1,2,4-trtazole rmgs (4-6). The rmg systemsare stmtlar m size and shape but show variations m aromattc, electrostatic, and hydrogen bondmg properties These varrattons provide opportunittes to study properties of importance for amtde bond mimicry. The derivatives are synthestzed from protected natural ammo acids, and the reactton condttrons have been chosen so that the enanttopurtty IS retained during the reaction sequences.Two series of mtmettcs will be described, one in which the carboxyhc acid functronality 1s directly attached to the heterocychc rmg (2) and one series with a methylene group inserted between the rmg and the carboxylic acid group (7). Since we have focused on the design and synthesis of Phe-Gly mtmetrcs, the synthetic examples described here start from t-phenylalanme. However, we have used the same synthetic scheme also for other ammo acids and notes will be given when other dertvattves require differences m reaction condtttons (I) The use of the dipepttdomtmetrcs as burldmg blocks in pseudopepttde synthesis will also be described (7). These synthesesare performed on solid phase using Bocchemtstry. Also, the deprotectton and purtficatton of the pseudopeptides by reversed phase HPLC wtll be dtscussed.

From

Methods m Molecular Medune. Vol 23 Pepfdomfmebcs Protocols Edlted by W M Kazmrerskl OHumana Press Inc , Totowa, NJ

1

Luthman, Borg, and Hacksell

2 2. Materials 2.1. Overview

‘H and 13C NMR sp ectra were recorded at 270 and 67.5 MHz with tetramethylsilane as the internal standard. Thin layer chromatography (TLC) was carried out on aluminum sheets precoated with silica gel 60 F,,, (0.2 mm, E. Merck); spots were visualized with UV detection and by spraying with a 2% ethanol solution of ninhydrin, followed by heating. Column chromatography was performed on silica using Kieselgel 60 (230400 mesh, E. Merck). Solvents were in some cases dried prior to use. THF and diethyl ether were distilled from sodium/benzophenone, and dichloromethane was dried over MgSO+

2. I. 1, HPLC Columns, Conditions, and Necessary Reagents 1. Analyses of the optical purity of 1,2,4-oxadiazole and 1,3,4-oxadiazole derivatives: J. T. Baker Chiralcel OD-H column, Detection wavelength: 254 or 2 11 nm. Ambient temperature. Mobile phase: Hexane/2-propanol/diethylamine (90: 10:O. 1 (2 and 5/ or 95:5:0.1 /9/). Flow rate: 0.5 mL/min. Reagents 1. Ethyl 2-chloro-2-(hydroximino)acetate. 2. Diethyl ether. 3. Liquid or gaseous ammonia (NH,). 4. Hexane. 5. N,hr-Dicyclohexylcarbodiimide (DCC). 6. tert-Butyloxycarbonyl(Boc)-L-phenylalanine. 7. Dichloromethane. 8. Pyridine. 9. Pentane. 10. Ethyl acetate. Il. NaHCO,. 12. Aqueous HCl (pH 2). 13. Aqueous NaCl (Brine). 14. MgSO,. 15. Methanol (MeOH). 16. tert-Butyldiphenylchlorosilane. 17. 3-Hydroxypropionitrile. 18. Triethylamine. 19. 4-Dimethylaminopyridine (DMAP). 20. Tetrahydrofuran (THF). 2 1. Citric acid. 22. Hydroxylamine hydrochloride. 23. K2C03, 24. Ethanol.

Synthesis and Use of Peptides

3

25. Na$O,, 26. Chloroform (CHCI,). 27. 1 M Tetrabutylammonium fluoride in THF. 28. Acetic acid. 29. Acetone. 30. Na$r,O,.2 H,O. 3 1. H,SO, (97%). 2. Analyses of the optical purity of 1,2,4-triazole derivatives: J. T. Baker Chiralpak AD column. Detection wavelength: 225 nm. Temperature: 30°C. Mobile phase: Hexane/2-propanol/diethylamine (90: 1O:O.1 /14/ or 80:20:0.1 /Z 71). Flow rate: 0.5 or 1 mlimin. Reagents 1. Boc-L-phenylalanine methyl ester. 2. Methanol (MeOH). 3. Hydrazine hydrate. 4. Chloroform. 5. Triethylamine. 6. Tetrahydrofuran (THF). 7. Ethyl oxalyl chloride. 8. Diethyl ether. 9. NaHCOs. 10. NaHSO,. I I. MgSO,. 12. Hexane. 13. Pyridine. 14. Thionyl chloride (SOCI,). 15. Toluene. 16. Dichloromethane. 17. Ethyl acetate. IS. N-Methylmorpholine (NMM). 19. Methyl malonyl chloride. 20. Citric acid. 2 I. Aqueous NaCl (Brine). 3. Purification of pseudopeptides with reversed-phase semi-preparative HPLC: Waters pBondapak C-18 column, 25 x 100 mm. Detection wavelength: 254 nm. Ambient temperature. Mobile phase: A gradient of 0 to 60% acetonitrile in water/ I%TFA in 60 min. Flow rate: 5 mL/min. Reagents 1. Ethyl thiooxamate. 2. Ethanol. 3. Hydrazine hydrate. 4. Activated charcoal. 5. Dichloromethane. 6. Celite.

4

Luthman, Borg, and Ha&se/l 7 Pentane 8 Ethyl chloroformate 9 Boc-t,-phenylalanme 10 Triethylamine. 11 Tetrahydrofuran (THF) 12 NJ-Dtmethylformamtde (DMF) 13 Xylenes (mixture of isomers) 14 Chloroform 15 Methanol (MeOH) 16 Ethyl acetate 17 Ethyl cyanoacetate 18. Gaseous HCl 19 Dtethyl ether 4 Analyses of pseudopepttdes wtth reversed-phase HPLC Waters pBondapak C18 column, 8 x 100 mm Detectton wavelength 254 nm Ambtent temperature Mobile phase A gradient of 0 to 60% acetomtrtle m water/l%TFA in 30 mm Flow rate 1 mL/mm Reagents 1 4-Methylbenzhydrylamme (MBHA) resin 2 KOH 3 Ethanol. 4 Methanol 5 Hf-ton exchange resm (Dowex’ 50 W x 8) 6 Drethyl ether 7. MgS04. 8 Dtchloromethane (DMF) 9 NJ-Dtmethylformamtde 10 Benzene 11 NaHCOs 12 Aqueous HCl (I M) 13 Trtfluoroacetlc actd (TFA) 14 2-( lH-Benzotrtazol1-yl)- 1,1,3,3-tetramethyluromum hexafluorophosphate (HBTU) hydrate (HOBt). 15 1-Hydroxybenzotrtazole 16 N-Ethyl-N,N-dnsopropylamme (DIPEA) 17 2-( lH-Benzotrtazol-1 -yl)-1 ,1,3,3-tetramethyluromum tetrafluoroborate (TBTU) 18 Ethyl chloroformate 19 N-methylmorpholme (NMM) 20 Iodotrtmethylstlane (TMSI) 2 1 Thtoamsole

5

Synthesis and Use of Peptldes

N/OH (Boc-L-Phe)aO

+

pyrldine

Et0 2

0

-

BocHN

A

COOEt

1

2

Figure 1

TBDPSO (Boc-L-Phe)20

+ 3

N’OH

BocHN

TBAF

Jones’

BocHN

BocHN

oxidation 6

Figure 2 22 1,2-Ethanedtol 23 Trlfluoromethanesulfomc acid (TFMSA)

3. Methods 3.1. Preparation

of 1,2,4-Oxadiazoles

(Figs. 1 and 2)

1,2,4-Oxadlazole dlpeptldomlmetlcs (see Note 1) can be obtained from amldoxlmes and Boc-protected ammo acid anhydrides (see Note 2) (8-10). Symmetric anhydrldes are synthesized from Boc-protected amino acids by treatment with N,N-dlcyclohexylcarbodnmlde (DCC) (see Note 3) (II). The amidoxlme (12) and the anhydnd? are reacted in pyndme, initially forming an O-acylamldoxlme (see Note 4). This intermediate is not isolated but directly heated to reflux to achieve the cychzatlon (23) (see Note 5) The use of a symmetric anhydride as activating group requires two equivalents of the protected ammo acid, however, it IS possible to recover one equivalent from the reaction mixture.

Luthman, Borg, and Hacksell

6 3 I. 7 Ethyl Z-Amino-2-(hydrox/mlno)Acetate

I (14)

1 Dissolve ethyl 2-chloro-2-(hydroxlmmo)acetate (1 0 g, 6 6 mmol) m anhydrous diethyl ether Cool the solution to 0 “C 2 Pass gaseous NH, through the solution for approx 3 mm (see Note 6) Filter off the precipitated ammomum chloride and concentrate the filtrate to an 011 Solldlfy by addition of hexane Filter off the precipitate to obtain approx 660 mg (76%) of 1 Mp 75-76°C (8)

3 1 2. Ethyl 5-[(S)-[(tert-Buty/oxycarbonyl)Amino]-2-Phenylethyl]7,2,4-Oxadlazole-3-Carboxylate 2 (Fig. 7) DCC (239 mg, 1 16 mmol) to a solution of Boc-L-phenylalanme (6 14 mg, 2 32 mmol) m dry dlchloromethane (30 mL) at room temperature Stir the reaction mixture at 0°C for 1 h Filter off the precipitated N,N’-dlcyclohexylurea (DCU) and concentrate the filtrate In vucuo Redissolve the crude symmetric anhydride m pyrldme Add dropwise a solution of 1 (102 mg, 0 77 mmol) m pyrldme Heat the mixture to reflux for 2 h (TLC, pentaneiethyl acetate, 3.2) Add water when the reaction IS complete, and evaporate the solvent (see Note 7) Partition the residue between dlchloromethane and water and wash the organic layer with saturated aqueous NaHCO,, aqueous HCl (pH = 2), brine, and water Pun@ the crude product (see Note 8) by column chromatography using pentane/ethyl acetate (8 5 1 5) as eluent Recrystalhzatlon from dlethyl etherkexane affords 200 mg (72 %) of pure 2 Mp 75-76°C; [a]o = -29 4” (c 1 0, MeOH), tR 17 2 mm (see Subbeading 2.), ‘H NMR (CDC&) 6 7 3G7 00 (m, 5H), 5 39 (m, 1H), 5 17 (br d, IH), 4 5 1 (q, J = 7 2,2H), 3.27 (AB system, 2H), 1 44 (t, 3H), 1 40 (s, 9H), ‘)C NMR (CDCl,) S 1809,1618,1575,1547,1346,1292,1289,1276,808,632,495,399,282,141

3. I .3. Preparation of Amidoxlmes from Nitrrles (3-tert-Butyld~phenyls~lyloxypropanamide

Ox/me 3

can be synthesized from mtrlles by treatment with hydroxylamme In this specific case we use 3-hydroxyproplomtnle as starting material, and the reaction proceeds m two steps. In the first step, the alcohol functlonallty IS protected as a sllyl ether (151, and m the second step the mtrlle IS converted mto the amldoxlme (see Note 9) Amldoxlmes

1 Add tert-butyldlphenylchlorosdane (18 5 mL, 73 mmol) to a mixture of 3-hydroxyproplomtrile (2 46 mL, 36 mmol), trlethylamme (12 7 mL, 91 mmol), and dlmethylammopyridme (DMAP) (178 mg, 1 5 mmol) m dry THF (75 mL) under a nitrogen atmosphere 2 Stir the mixture at room temperature for 3 5 h (TLC, dlethyl ether/pentane, 1 3) Filter off the salts and evaporate the solvent uz V~CUO Redissolve the residue m dlchloromethane. Extract with saturated aqueous NaHC03, saturated aqueous cltrlc acid, and brine

Synthesrs and Use of Peptides

7

3. Purify the crude product (see Note 8) by column chromatography using dlchloromethane/pentane (2.3) as eluent Concentrate the appropriate fractions to obtain a solid residue Recrystalllzatlon from dlchloromethane/pentane affords 8 6 g (76%) of pure 3-tert-butyldlphenylsllyloxyproplomtrlle Mp 50-51°C (15), 1HNMR (CDCI,) 6 7.70-7 35 (m, lOH), 3 84 (t,J= 6 3,2H), 2 53 (t, 2H), 1 08 (s, 9H), 13CNMR (CDC13) 6 135 5,132 7,129 9,127 8, 117 9, 59 0, 26.7, 21 4, 19.1 4 Add a solution of hydroxylamme hydrochloride (3.8 g, 55 mmol) and K&O, (3.8 g, 27 mmol) in water to a solution of 3-tert-butyldlphenylsllyloxyproplonitrlle (8 5 g, 27 mmol) m ethanol (see Note 10) 5 Stir the mixture for 23 h at room temperature Evaporate the volatlles In vacw and add dlethyl ether to the residue Filter off the solids and dry the filtrate with Na,SO, Filter off the drying agent and concentrate the filtrate zn vucuo The residue can be crystallized by addition of dlethyl ether/pentane However, recrystalllzatlon can be difficult (see Note 11) You will obtain 5 4 g (57%) of pure 3 Mp 83-84”C, ‘H NMR (CDCl,) 6 7 70-7 25 (m, lOH), 5.04 (br s, 2H), 3 88 (t, .I=5 6,2H), 2 37 (t, 2H), 1 06 (s, 9H), 13CNMR (CDCl,) 6 153 9, 135 5, 1329, 1300, 1279,624,336,268, 19 1

3.1 4. 2-[5-[(S)- 7-(tert-Butyloxycarbonylammo-2-Pheny/ethyl]1,2,4-Oxadlazol-3-yl]Acetic Acid 6 (Fig. 2) Prepare the symmetric anhydride as described m (Subheading 3.1.2.) using 1 43 g (5 4 mmol) of Boc-L-phenylalanme and 558 mg (2 7 mmol) of DCC Dissolve the crude anhydride m pyndme. Add dropwlse a solution of 3 (926 mg; 2.7 mmol) m pyrldme at room temperature. Heat the mixture to reflux for 3 h (TLC, dichloromethane/pentane, 4 1) Add water and evaporate the solvent zn V~CUO(see Note 7). Redissolve the resldue in dlethyl ether Extract with water, 10% aqueous citric acid, and saturated aqueous NaHC03 Purify the crude product (see Note 8) by column chromatography using dlchloromethane/pentane (4 1) as eluent to obtain 940 mg (63%) ofpure 5-([(S)-ltert-butyloxycarbonylam~no-2-phenylethyl]-3-(2-~e~~-butyld~phenyls~lyloxyethyl)1,2,4-oxadlazole 4 as an 011. [a],,=-9 4” (c 1.2, CHCI,), ‘H NMR (CDCl,) 6 7.65-7.00 (m, 15H), 5 29 (m, IH), 5.06 (m, IH), 4.00 (t, J= 5.4, 2H), 3 29-3 08 (m, 2H), 2 96 (t, 2H), 1 40 (s, 9H), 1 01 (s, 9H); 13C NMR (CDClJ F 178 3, 168 2, 154.7, 135 5, 135.0, 133.4, 129.7, 129 2, 128.6, 127 7, 127.2, 80.4, 60.8, 49 3,40.0, 29 5,28 2, 26 7, 19.1 Dissolve 4 (285 mg, 0 49 mmol) m dry THF (50 mL) Keep the solution under nitrogen atmosphere Add a mixture of tetrabutylammomum fluoride (810 &; 0 80 mmol, 1 M solution in THF) and 30 pL of acetic acid m THF (see Note 12). Stir the reactlon mixture for 2 h at room temperature (TLC, dlethyl ether), Evaporate the solvent and redissolve the residue in dlethyl ether. Extract with 10% aqueous citric acid, saturated aqueous NaHC03, and brine

8

Luthman,

Borg, and Hacksell

7 Put@ the crude product (see Note 8) by column chromatography usmg first dlethyl ether/pentane ( 1.2) and then dlethyl ether as eluents Concentration of appropriate fractlons and recrystalhzatlon m dlethyl etheripentane should afford 125 mg (75%) of pure 5-([(S)-l-tert-butyl-oxycarbonylam~no-2-phenylethyl]-3-(2-hydroxyethyl)-1,2,4oxadiazole (5). Mp 84-85”C; [a],=-20.8” (c 0 9, CHC13), tR 30.9 mm (2 11 nm) (see Subheading 2.), ‘H NMR (CDCl,) 6 7 30-7 00 (m, 5H), 5 3 (br, lH), 5 1 (br, lH), 3 95 (t, J= 5 9,2H), 3 31-3 14 (m, 2H), 2 96 (t, 2H), 2 30 (t, IH), 141 (s, 9H), 13C NMR (CDC13) 6 178 8, 168.5, 154 9, 135 0, 129 2, 128 7, 127 4, 80 6, 59.4, 49 4, 39 8,29 2,28 2 8 Dissolve purltied 5 (50 mg, 0 15 mmol) m acetone and chill the solution to 0°C Add Jones’ reagent (26) (457 mL, 0 3 1 mmol, 0 67 M) (see Note 13) 9 Stir the solution for 12 h (TLC, dlethyl ether) Filter off the sohds and evaporate the solvent Redissolve the residue m dlethyl ether and extract wtth saturated aqueous NaHC03. Acid@ the aqueous phase with 1 Maqueous HCl and extract with dtethyl ether Recrystallize the crude product (see Note 8) from chloroform to obtain 34 mg (64%) ofpure 6 Mp 163 5-164.5”C; [aID=5’ (c 1 1, CHCI,), ‘H NMR (acetone-d,) 6 7 25-7 15 (m, 5H), 6 70 (br d, lH), 5 22-5 10 (m, IH), 3 78 (s, 2H), 3 29 (dd, J= 13 7, 6 0, lH), 3 16 (dd, J= 9 2, IH), 1 28 (s, 9H), 13CNMR (acetone-d,) 6 181 0, 169 4, 166 3,156 2,137 8, 130 5, 129 4, 127 9,80.0,50 8,39 8,32 6,28 7

3.2. Preparation

of 1,3,#-Oxadiazoles

(Figs. 3 and 4)

1,3,4-Oxadlazole dipeptldomlmetics (see Note 14) can be obtained by dehydration of the correspondmg dlacylhydrazmes (I 7) The dlacylhydrazmes are obtained from ammo acid hydrazldes, which are conveniently synthesized from amino acid esters and hydrazme (Z&19) (see Note 15) Boc-protected ammo acid esters are either commercially available or can be synthesized from Boc-protected ammo acids by treatment with DCC/DMAP and the appropriate alcohol (20) (see Note 16). The esters are reacted with hydrazme hydrate to produce the hydrazldes that are treated with an acid chloride (or another activated carboxyhc acid denvatlve) to obtain the diacylhydrazmes (22). The dehydration condltlons should be mild and compatible with the properties of the ammo acid protecting group (see Note 17) Therefore, we use a mixture of thlonyl chloride (SOCI,) and pyrldme at 0°C This leads to the formation of a 1,2,3,4-oxathladmzole-S-oxide intermedlate, which can be isolated or dn-ectly heated m a high-bollmg solvent to form the 1,3,4oxadlazole derivative by thermal ehmmatlon of sulfur dioxide (22) 3.2.1. Preparation of Amino Aad Hydrazides ([(S)-2-[(tert-Butyloxycarbonyl)Amlno]-3-Phenylpropanoyl] Hydrazine, 7) (Fig. 3) 1 Dissolve

Boc-L-phenylalanme

methyl ester (14 g, 0 05 mol) m methanol (see

Note 18) For preparation of ammo acid esters, see Note 16 and ref. 20

Synthesis and Use of Peptldes

9

1 DCClEtOH c 2 Hydrame

Boc-L-Phe

BocHN 0

ClCOCOOEt Et3N

1 SOCl2, pyrldine 2. Toluene, reflux

BocHN

BocHN

N-N 9

8

Ftgure 3

7

+ “TCOOMe 0

1. SOCI,, pyndme w 2. Toluene, reflux

Et3N

*

BocHN

BocHN N-N 11

Figure 4

2 Add hydrazme hydrate (7 3 mL; 0.15 mol). 3 Stir at room temperature for I8 h (TLC, chloroform/MeOH, 9.1) 4 Put the reaction flask m the refrigerator for 1 h to allow the hydraztde to precipitate (see Note 19) The precipitate is filtered off to afford 11.5 g (82%) of pure 7 Mp 120-122°C (see Note 20) [a]n = +9 7” (c 1 01, MeOH); ‘H NMR (CDCls) 6 7 70 (br s, lH), 7 3c7.15 (m, 5H), 5 32 (br d, lH), 4 36 (dd, IH), 3.8 1 (br s, 2H), 3 02 (AB system, 2H), 1.39 (s, 9H), 13C NMR (CDC13) 6 17 1 9, 155 2, 136 4, 129 2, 128 8, 127 1, 80.5,54 8,38 4,28 3

10

Luthman, Borg, and Hacksell

3.2.2 Preparation of Diacylhydrazines (1 -(Ethoxyoxalyl)-2-[(S)-2[(tert-Butyloxycarbonyl)-Amino]-3-Phenylpropanoyl] Hydrazme, 8) (Fig. 3) Dtssolve 7 (1 3 g, 4 65 mol) and triethylamme (777 $; 5 58 mmol) m dry THF under nitrogen atmosphere Chtll the solutton to -30°C Add dropwtse ethyl oxalyl chloride (624 mL, 5 58 mmol) dissolved m 5 mL of THF, and let the reaction stu for 20 h The temperature should be gradually mcreased to room temperature (TLC, chloroform/MeOH, 9 1) Filter off the precipitated trtethylammomum hydrochlortde and concentrate the filtrate zn vucuo Partmon the residue between dtethyl ether and water and extract the organic layer with saturated aqueous NaHCO, and 1 M NaHSO, Crystallize the crude product (see Note 8) m dtethyl etherihexane to obtain 1 0 g (60%) of pure 8 (see Note 21). Mp 88-89°C [cY.]~= -21.7” (c 1 01, MeOH), ‘H NMR (CDC13) 6 9 20 (br s, 2H), 7 35-7 20 (m, 5H), 5 07 (br d, 1H), 4 5 1 (m, lH), 4 37 (q, .I= 7 0, 2H), 3 18 (dd, lH), 3 05 (dd, J= -14 0, 6.0, lH), 1.39 (s, 9H), 1 38 (t, 3H), 13CNMR (CDC13) 6 169.0, 158 7, 155 7, 152.9, 136 0, 129 3, 128 7, 127 1, 80 8,65 6,54 3,37 9,28 3, 13 9

3.2.3 Ethyl 5-[(S)-[(tert-Butyloxycarbonyl)Amlno]-2-Phenylethyl]1,3,4-Oxadiazole-2-Carboxylate 9 (Fig. 3) Dissolve 8 (1 .O g, 2 64 mmol) m dry dtethyl ether (75 mL) under nitrogen atmosphere Chill the solutron to O’C (see Note 22) Add pyrtdme (550 mL, 6 85 mmol) and thereafter SOCl* (250 mL, 3 43 mmol) dissolved in drethyl ether (see Note 23) Let the reactton stir for 2 h at 0°C Filter off the precipitated pyndunum salt as quickly and carefully as possible (seeNote 24) Concentrate the filtrate WIvacua without heating Redtssolve the residue m dry toluene and heat the solutton to reflux for 2 h (TLC, chloroform/MeOH/hexane, 9.1 1) Evaporate the solvent zn vucuo and purify the product by column chromatography with dtchloromethane/ethyl acetate (19 1) as eluent Recrystalhzatron from drchloromethane/ pentane affords 590 mg (62%) of pure 9 Mp 122-123”C, [a],, = -3 1 9” (c 10, MeOH), tR 45 7 mm (see Subheading 2.); ‘H NMR (CDCl,) 6 73>705(m,5H),536(m, lH),509(brd, lH),451 (q,J=72,2H),332(dd, lH), 3 22 (dd, lH), 1 45 (t, 3H), 1 40 (s, 9H), 13CNMR (CDCl,) 6 168 5, 156 9, 154 6, 1541,134.7,1292,1288,1274,808,636,486,396,282,140

3.2 4 Methyl 2-~5-[(S)-l-(tert-5utyloxycarbonylamino-2-Phenylethyl]I, 3,4-Oxadlazol-2-yl]Ace ta te 11 (Fig. 4) 1 Dissolve 7 (433 mg; 1.6 mmol) (Subheading 3.2.1.) m dry THF under nitrogen atmosphere. Add N-methylmorpholme (NMM) (255 mL; 2 3 mmol) and chill the solution to -30°C (see Note 25) 2 Add methyl malonyl chloride (248 pL, 2.3 mmol) and let the reaction stir for 4 h whtle the temperature 1sallowed to increase to room temperature (TLC, ethyl acetate)

11

Synthesis and Use of Peptides

Filter off the salt and concentrate the filtrate zn wcuo The residue is partitioned between ethyl acetate and water and the organic layer is then extracted with saturated aqueous NaHCO,, 10% aqueous citric acid, and brine Purify the crude product (see Note 8) by column chromatography using ethyl acetate/pentane (2 1) as eluent You should obtam about 340 mg (58%) of pure 10 Mp 158-159°C [a]o = -10 0” (c 1.0, CHCl,), ‘H NMR (CDCl,) 6 10 21 (bs, lH), 10 05 (bs, lH), 7 27-7 15 (m, 5H), 5 78 (d, lH), 4 79 (m, ‘H), 3 74 (s, 3H), 3 44 (s, 2H), 3 18-2 90 (AB system, 2H), 1.34 (s, 9H), r3C NMR (CDCl,) 6 168 2, 168.0, 161 5, 155.6, 136 5, 129 4, 128 4, 126.8,80 0,53.8,52 6,39 9,38 6,28.3 Dissolve pure 10 (253 mg, 0 67 mol) in dry THF under a mtrogen atmosphere Chill the solution to 0°C Add first pyridine (140 mL, 1.7 mmol), then SOCl, (62 mL, 0 87 mmol) dissolved m THF. Let the reactron stir at 0°C for 2 h. Filter off the precipitated salt as quickly and carefully as possible (see Note 24). Concentrate the filtrate zn vucuo without heating Redissolve the residue m dry toluene and heat the solution to reffux for I 5 h (TLC, diethyl etheripentane, 9 1) Evaporate the solvent and purify the residue by column chromatography with drethyl ether/pentane (2 1) as eluent Recrystalhzation from drethyl ether/pentane affords 75 mg (3 1%) of pure 11 Mp 59 5-61”C, [a]n = -31 5” (c 1 0, CHCl,), ‘H NMR (CDCl,) 6 7 27-7 08 (m, 5H), 5 27 (m, lH), 5 18 (m, lH), 3 93 (s, 2H), 3 75 (s, 3H), 3 32-3 15 (AB system, 2H), 140 (s, 9H), r3C NMR (CDCl,) 6 167 1, 166,5, 160 6, 154 7, 135 1, 1293,1286,1271,804,528,483,397,315,281

3.3. Preparation

of 1,2,4-Triazoles

(Figs. 5 and 6)

1,2,4-Trtazole dipeptrdomimetics (see Note 26) can be obtained via an intramolecular condensation of acylamrdrazones, which dehydrate spontaneously upon heating (23,24). Acylamidrazones are obtained by reacting mixed anhydrides of protected ammo actds with amidrazones (24) or by reaction of a hydrazide with an imidate ester. The cyclization reaction IS performed m xylenes (mixture of isomers) at a temperature above the melting point (> 1SO’C) of the acylamrdrazone. Xylene does not usually dissolve the acylamidrazones but functions as a “carrier” solvent and dissolves the products (25).

3.3.7. Ethyl Oxamdrazonate

12 (26,27)

1 Dissolve 2 0 g of ethyl thiooxamate (15.5 mmol) m 50 mL of ethanol 2 Add a solution of hydrazme hydrate (0 76 mL, 15 5 mmol) m 10 mL of ethanol 3. Stir for 6 h at room temperature under H$-elimmatron. Put the flask m the refrigerator overnight Filter off the precipitate and wash the solid with ethanol. Concentrate the filtrate zn V~CUO If a dark yellow solid is obtained, it could be treated with activated charcoal m dichloromethane for purification Filter off the charcoal through a Celite-pad and evaporate the solvent 4 Crystallize from dichloromethane/pentane to afford 12

Luthman, Borg, and Hacksell

12

*

Xylenes

A

COOEt

BocHN HN-N

Figure 5

7

+

Et3N

EtO’fhOOEt NH

0

15

Xylenes

A 17

Figure 6

3.3.2. Preparation of Acylamidrazones (1 -(Ethoxyoxal/midyl)-2[(S)-Z-[(tert-Butyloxycarbonyl)Amino]-3-Phenylpropanoyl] Hydrazlde, 13) (Fig. 5) 1. Add ethyl chloroformate (48 mL, 0 50 mmol) to a chilled solution (-5°C) of BocL-phenylalanme (111 mg, 0.42 mmol) and trlethylamme (70 &, 0 50 mmol) m dry THF 2 Stir the mixture for 30 mm at -5°C Filter off the precipitated tnethylammomum chloride. The mixed anhydrlde thus formed IS used immediately m the next step (28). 3 Add a solution of 12 (46 mg, 0 35 mmol) m dry THF to the filtrate at room temperature Let the reactlon mixture stir for 5 h at room temperature.

Synthess and Use of Peptides

13

4 The precipitated product 13 1s filtered off and recrystallized from ethanol/pentane (see Note 27) You should afford about 100 mg (80%) of pure 13. Mp 176178’C, [a],= +105 7” (c 1 05, DMF), ‘H NMR (DMSO-d,) 6 9 96 (br s, 0 5 H), 9.84 (br s, 0 5 H), 7 42-7 13 (m, 5H), 7.08 (br d, 0.5 H), 6 94 (br d, 0.5 H), 6.50 (br s, 2H), 4 86 (m, 0.5 H), 4.37-4 17 (m, 2 5H), 3 07-2.54 (m, 2H), 1.29 (s, 9H), 1 38-1 24 (m, 3H), t3C NMR (DMSO-d,) 6 173 3, 168 1, 162.2, 161.9, 155 6, 155 4, 139 6, 139 3, 137 9, 136 1, 129.2, 128 1, 127 9, 126 3, 126.2,78 1, 77 8,61 8,61 6,54 9, 54 2,37 5,35 9,28.2, 14.1

3.3.3 Ethyl 5-[(S)-[(tert-Butyloxycarbonyl)Amino]-2-Phenylethyl~-1,2,#Triazole-3-Carboxylate 14 (Fig. 5) Heat the acylamidrazone 13 (3 18 mg; 0.84 mmol) m xylenes (mixture of ISOmers) at 190°C for 6 h (see Note 28) (TLC, chloroform/MeOH/pentane, 9.1. I). 2 Evaporate the solvent and purify the residue by column chromatography using pentane/ethyl acetate (7 3) as eluent Concentration of appropriate fracttons and recrystalhzatton from dichloromethane/pentane affords 196 mg (65%) of pure 14 Mp 154-154 5”C, [aID=-8” (c 1 0, CHCI,); tR 11 6 mm (see Subheading 2), ‘H NMR (CDCl,) 6 13 0 (br s, lH), 7 6&7 00 (m, 5H), 5 76 (br d, lH), 5 25 (app dd, lH), 4 48 (q, J= 7 0,2H), 3 26 (AB system, 2H), 1.41 (t, 3H), 1 34 (s, 9H), 13C NMR (CDCI,) d 159 6, 159 5 (w), 155 8, 153 9, 136 2, 129.2, 128.6, 127 0, 80 6,62 2,49 2, 40.3,28.2,

14.2

3.3 4. Ethyl P-Ammo-p-Ethoxyacrylate

Hydrochloride

15 (29)

1 MIX 5 g (44 2 mmol) of ethyl cyanoacetate and 2.8 mL (48 6 mmol) of ethanol at 0°C 2 Add 7 5 mL (48 6 mmol) of a solutton of HCl m dtethyl ether (HCl-concentratton 236 mg/g solutron) (see Note 29). 3 Stir at 0°C for 24 h. Filter off the prectpttate and wash with dtethyl ether You should obtain 3.7 g (55%) of pure 15 Mp lOl-102°C.

3.3.5. Ethyl 2$5-[(S)- 1-(tert-Butyloxycarbonylammo-2-Phenylethylj1,2,4-triazol-3-yl]Acetate 17 (Scheme 6) 1 Add a solutton of 7 (143 mg, 0 5 1 mmol) (Subheading 3.2.1.) m ethanol to a solution of 15 (100 mg, 0 5 1 mmol) and triethylamine (7 1 pL, 0.5 1 mmol) m ethanol 2 Star the mtxture for 12 h at room temperature. Add water and cool the flask m the refrigerator. Filter off the precipitated 16. 3 Heat a mixture of crude 16 m xylenes at 155°C for 30 mm 4 Evaporate the solvent and purify the residue by column chromatography usmg first ethyl acetateipentane (2 3), then ethyl acetate/pentane (1: 1) as eluents. Concentratton of approprtate fractions and recrystallization of the restdue from dtethyl ether/pentane affords 110 mg (57%) of pure 17. Mp = 106 5-107°C [alo= -24.2” (c 1 0, CHCls), tR 22 1 mm (see Subheading 2.); ‘H NMR (CDC13) 6 12 6 (br, lH), 7 95-7 00 (m, 5H), 5 78 (br s, IH), 5 13 (m, IH), 4.20 (q, J=7 2,2H), 3 85 (s, 2H), 3 28-3 07 (m, 2H), 1.34 (s, 9H), 1 27 (t, 3H), t3C NMR (CDCI,) 6

Luthman, Borg, and Hacksell

14 169 2, C-3 and C-5 not vwble, 49 5,40 4, 33 5,28 2, 14.0

155 5, 136.7, 129 4, 128 8, 126.6, 79.9, 61 6,

3.4. Use of Heterocyclic

Dipeptidomimetics as Building Blocks in Pseudopeptide Synthesis

The heterocycltc drpeptldomtmetrcs synthestzed above may be used as building blocks m pseudopepttde synthesis using the sohd phase strategy and Boc-chemistry (30,31). Srde cham functtonal groups were protected. The coupling reactions were run manually m disposable syringes (5 mL), with a porous polyethylene disc as a filter (approx 50 pm pore size) at room temperature. A stainless steel needle was attached to the syringe (see Note 30) The syringe was rotated m a carousel during the deprotectton and couplmg steps. We used approx 50 pm01 of the MBHA resin (32) m each synthesis (see Notes 31 and 32). The couplmg of naturally occurring ammo acids to the resin-bound pepttde chain and the deprotectton of the N-terminal ammo acid can be done as described m section 3 4 2

3.4.1 Hydrolysis of the Azole Carboxylic Esters 2, 9, 11, 14, and 17 The free carboxyhc acids of the dlpepttdomtmettcs are used m the pseudopeptide synthesis. Therefore, the dipeptidomimetic esters have to be hydrolyzed before couplmg to the growing pepttde chain. Hydrolysis of 2: The 1,2,4-oxadtazole derivative 2 (Subheading 3.1.2.) can be hydrolyzed m a solution of 1 M aqueous KOH (approx 1.2 mmol) m ethanol by stirring for 20 mm at room temperature. Concentrate the mixture and redtssolve the residue m methanol Add a H+-ion exchange resin (Dowex@ 50 W (8) and stir for 5 mm. Filter and concentrate the filtrate. Redissolve the residue m diethyl ether. Dry with MgS04, filter, and concentrate the filtrate zn vacua (see Note 33) A solution of the crude acid m a mixture of dtchloromethane/DMF (4: 1) is used m the pepttde synthesis. Hydrolysis of 9 and 11. The 1,3,4-oxadtazole dertvattves 9 and 11 (Subheading 3.2.3. and 3.2.4.) can be hydrolyzed m a solution of 1 A4 aqueous KOH (approx 1.2 mmol) n-rethanol by stn-rmg for 30 mm at room temperature Work-up of hydrolyzed 9 Evaporate the solvent and dry the restdue carefully by repeated addition and evaporation of benzene, thereafter drying on a vacuum pump without heating (see Note 34). A solutton of the crude carboxylate m a mixture of dtchloromethane/DMF (4.1) 1s used m the pepttde syntheses. Work-up of hydrolyzed 11. Evaporate the solvent and partmon the residue between dtethyl ether and water. Extract the organic layer with saturated NaHC03. Acidify the basic water phase with 1 A4 aqueous HCl and extract

15

Synthesis and Use of Peptides

with diethyl ether. Dry the orgamc phase with MgS04, filter, and concentrate. A solution of the crude acid m DMF is used in the peptide synthesis. Hydrolysis of 14 and 17. The 1,2,4-triazole derivatives 14 and 17 (Subheadings 3.3.3. and 3.3.5.) can be hydrolyzed m a solution of 1 M aqueous KOH (approx 2.5 mmol) m ethanol by stnrmg for 14 h at 65°C and 5 h at room temperature, respectively The solvent is evaporated and the residue is carefully dried as described above for the hydrolysis of 9 (see Note 35). A solution of the crude carboxylate in DMF is used m the peptide synthesis. In the hydrolysis of 17, the restdue obtained after evaporation should be redissolved m ethanol. The treatment with ion exchange resin and the work up procedure is the same as that described above for the hydrolysis of 2 A solution of the crude acid m DMF is used m the peptide synthesis.

3.42. Activation and Couplrng of Heterocyck In Pseudopeptide Synthesis The general procedure Note 36).

Dipeptidomimetics

for solid phase peptrde synthesis is as follows

(see

1 Load the resin (-50 pm01 = 1 eq) into the syringe and wash with DMF (2 x 5 mm). 2 If the N-terminal ammo acid is protected, remove the Boc-group by treatment of the resin with cmc TFA for 8 mm (Caution: see Note 37) Remove the liquid from the syringe and wash the resin with DMF (4 x 1 mm + 2 x 3 mm) (see Note 38) 3 Add a solution of 3 eq of the Boc-ammo acid, 3 eq of HOBt, 3 eq of HBTU or TBTU and 6 eq of DIPEA in DMF to the resin. Run the coupling reaction for 30 mm Remove the liquid from the syringe and wash the resin with DMF (4 x 1 mm + 2 x 3 mm) Steps 2 and 3 are repeated until all ammo acids are coupled to the resin (see Note 39). 4 Wash the resin with dichloromethane (Caution: see Note 40) Remove the plunger and air-dry the resin. The dipeptidomimetics 9,14, and 17 were coupled to the pepttde resin using HBTU/HOBt/DIPEA or TBTU/HOBt/DIPEA as coupling reagents, whereas 2, 6, and 9 were activated as mixed anhydrides by treatment with ethyl chloroformate/triethylamme or NMM, as described m Note 15. Modified ammo acid derivatives (as the heterocyclic dipeptidomimetics described here) are known to react slower than the naturally occurrmg amino acids in coupling reactions. Therefore, long reaction times (12-24 h) are recommended (33). The couplmgs were generally performed with an excess of the fragment and were repeated twice when possible Boc-protectmg groups are generally removed from ammo acids by treatment with TFA. However, we have observed complete or partial epimerization at the a-carbon of the 1,2,4oxadiazole derivatives using these conditions (see Note 41). Therefore, the

16

Luthman, Borg, and Hacksell

removal of the Boc-group of all the azole derivatives described above should be performed using lodotrlmethylsllane (TMSI) (34,35) (procedure described below). Boc-deprotectlon steps subsequent to the couplmg of one ammo acid to the ammo function of the azole building block, can be performed using TFA without eplmerlzatlon Deprotectlon

using TMSI

1 Add a solution of 2 eq of TMSI m dlchloromethane to the resin and rotate the syringe for 20 mm. 2 Remove the llqulds from the syrmge. Quench the reaction by adding a solution of 6 eq methanol m dlchloromethane (Caution: see Note 42) and rotate for an addltional 10 mm 3 Remove the hqulds from the syringe and wash the resin with dlchloromethane (2 x 1 mm + 3 mm) and DMF (2 x 1 mm + 3 mm)

Before coupling of the next amino acid, the resin should be treated with a solution of NMM (3 eq) m DMF. 3.4.3 Cleavage of the Pseudopeptlde

from the Resin

1, 2 3 4 5

Transfer the resin from the syringe to a roundbottom flask. Add thloamsole (100 mL) and 1,2-ethanedlol (50 &) to the resin Stir the mixture at room temperature for 10 mm and add TFA (1 mL) Stir for 10 mm and add TFMSA (100 pL) Stir for an additional 2 h Add cold dlethyl ether (O’C) and filter off the precipitated peptlde together with the resin. 6 Wash the solids carefully with TFA to dissolve the peptlde, the resin will remam on the fret 7 Add cold dlethyl ether to the filtrate to precipitate the peptlde Filter off the precipitate (see Note 43)

3.4.4. Pseudopeptide

Purification and Analysis

Purification of the pseudopeptldes 1s performed by semi-preparative reversed phase HPLC using gradients of acetonitrrle (O-60 %) m water/O, 1% TFA. Pool the fractions containing the pseudopeptlde and evaporate the acetonitrile znvacua Lyophlllze the residue to obtain a white powder. We have analyzed the pseudopeptldes by reversed phase HPLC, plasma desorptlon mass spectrometry, and ammo acid analysis. Interestingly, the ammo acid analysis of the pseudopeptldes containing the 1,2,4-oxadlazole or the 1,3,4-oxadlazole based mlmetlcs resulted m the formation of fragments analyzed as phenylalanine. However, the trlazole rmg system was not affected by the condltlons used m the hydrolysis and was not detected m the amino acid analysis.

Synthesis and Use of Peptrdes

17

3.5. Discussion The heterocychc dipeptidomtmetrcs described here can be conveniently obtained m optrcally pure form m moderate to good yields from common ammo acids. Then phystcochemical properties make them interesting as amide bond replacements, and their use in pseudopeptrde synthesis can be relevant m studies of structural mtmrcry or when an increased chemical stability of a peptide IS desirable 4. Notes 1 1,2,4-Oxadtazole mtmetics have been synthesized starting from Boc-protected glycme, L-phenylalanme, L-alamne, L-cysteine(Bn), L-serme(Bn), L-prolme, L-aspartate(Bn), and L-argmme(Boc)z. The different reaction times, chromatographic condtttons, yields, meltmg points, NMR spectral data, and optical rotations not given here are presented m ref. 1. 2. Also, the mtxed anhydrtde formed by reacting Boc-L-phenylalanme wtth ethyl chloroformate has been tried for activation. However, only Boc-L-Phe-OEt was produced In the synthesis using Boc-aspartate(Bn), we attempted both the symmetric anhydrtde and the 4-mtrophenyl ester activation procedures The two methods gave similar yields of 1,2,4-oxadtazole products, but the enantiopurtty was constderably lower after 4-mtrophenyl ester activation than that resultmg from symmetrtc anhydride acttvatton. For acttvatton of ammo acids, see ref. II The symmetric anhydrides are quite stable and could be stored for short pertods of time without extenstve degradation, However, tt is recommended that they are used immediately after preparation We have observed that the product obtained from Boc-L-phenylalamne ~111 be partially or completely racemtzed if the cychzation reaction IS performed in the presence of strong bases such as NaH or BuLi (36). Neither tsolatton of the intermediate nor addttton of a dehydratmg agent were required Gaseous NH, can be generated from hqutd NH3. Liqutd NH3 IS added to a threenecked flask, cooled to -78°C under a mtrogen atmosphere. A cannula IS connected to another three-necked flask wtth ethyl 2-chloro-2-(hydroximmo)acetate dtssolved m dtethyl ether at 0°C. When the coolmg bath is removed from the ltqutd NHs, and the temperature increases, gaseous NH3 IS slowly starting to bubble through the reaction flask The water IS added to facthtate the evaporation of pyridme by azeotropic disttllation After extraction, the organic layer should be dried wtth MgS04. The drymg agent is filtered off, and the filtrate IS concentrated zn vacua We have used both tert-butyldtmetylstlyl (TBDMS) and tert-butyldtphenylsilyl (TBDPS) ethers and found that both work well The TBDMS ether is formed m higher yield than the TBDPS ether. However, the latter 1seasier to detect on TLC plates, and tt 1s a solid and thereby caster to obtam m pure form 10. The ratio of ethanol/water should be such that a clear solutton IS obtained

18

Luthman, Borg, and Hacksell

11 The amtdoxtme formatton seems to be reversible at elevated temperatures We have observed that during crystalltzatron from a heated solutton of 3, we obtamed a considerable amount of the startmg mtrtle 12 The acetic acid 1sadded to prevent racemrzatton caused by the basic fluortde ton. 13 A 0 67 M solutton of Jones’ reagent was prepared accordmg to reference 16, dtssolve 10 g (33 mmol) of Na2Cr,0,*2H20 m 30 mL of water. Add 13 6 g concentrated sulfurtc acid (134 mmol) Drlute the solutton with water to a 50-mL total volume 14 1,3,4-Oxadtazole mtmettcs have been synthesized from Boc-protected glycme, L-phenylalanme, L-alanme, L-cysteme(Bn), L-serme(Bn), and L-prolme The dtfferent reaction times, chromatographtc condtttons, yields, melting points, NMR spectral data, and optical rotations not given here are presented m ref. 2 15 The mtxed anhydrtdes of ammo acrds can be used Instead of the esters to prepare the hydraztdes Mtxed anhydrtdes (28) can be prepared by addition of ethyl chloroformate (1.1 eq) to a chilled (-5°C) solutton of the Boc-protected ammo acid (1 eq) and NMM or trtethylamme (1 s1 eq) m dtchloromethane/DMF (4 1) The mixture 1s stirred for 30 mm at -5°C. Filter off the ammonmm salt 16 Esters of ammo acids can be prepared from a Boc-protected ammo acrd (1 eq) dissolved m dry dtethyl ether with DCC (1 1 eq), DMAP (0 1 eq), and the appropriate alcohol (2 5 eq) (20) Star the reactton mixture at room temperature (TLC, dtethyl ether/ pentane, 1.1) Filter off the DCU and extract the filtrate with 1 A4 NaHSO,, saturated aqueous NaHCOs, and water Dry the orgamc layer with MgSO,, filter and concentrate the filtrate zn vacua to obtain the crude ester Thts reaction sequence has been used to prepare Boc-protected glycme, L-alanme, L-cysteme(Bn), L-serme(Bn), and L-prolme ethyl esters m high yields 17 Generally, the dehydratton of the dtacylhydraztdes IS performed using an efficient dehydrating agent such as methanesulfomc acid, concentrated sulfuric acid, acetic anhydride, phosphorus oxychlortde, thtonyl chlortde, or phosphorus pentoxide (37,38) However, these reaction condmons are not compatible with the use of the acid labile Boc-protecting group 18 Ethanol 1s the appropriate solvent, if an ethyl ester IS used 19. If the hydrazrde IS not prectpttatmg in the cold, the solvent 1sevaporated, and the residue IS recrystallized from ethyl acetate/pentane The hydrazides derived from Boc-L-prolme and Boc-L-serme(Bn) were not isolated as solids but were used m the next step as crude 011s 20 The melting point observed (120-122‘C) IS different from that given m ref. 39 (93-95°C) 21 The dracylhydrazldes derived from Boc-protected L-alanme, L-serme(Bn), and L-prolme were used m the next step without crystalhzatton 22 It 1s Important to have dry condtttons to opttmtze the yield 23 Hugh qualtty pyrtdme and SOCl* should be used to Improve the yield. The presence of pyrtdme m the cycltzatton IS Important, since no product was obtained when the reaction was performed m neat SOCl*, and only 26% yield of cychzed product was obtained when a solutton of SOCl;! m dtchloroethane was used.

Synthesis and Use of Peptldes

19

24 The pyrtdmmm salt from the formation of the oxathtadtazole-S-oxide has to be completely removed from the chilled solubon to Improve the yield m the reaction The salt is very hygroscoptc and, therefore, the filtration has to be performed quickly. Do not wash the solid wtth THF since that might redtssolve the salt 25 Trrethylamme can be used instead of NMM m this reaction 26 1,2,4-Trtazole mtmetrcs have been synthesized starting from Boc-protected glytine, L-phenylalanine, t,-alanme, L-cysteme(Bn), L-serine(Bn), and L-proltne The different reaction times, chromatographtc condrttons, yields, melting pomts, NMR spectral data, and optrcal rotations not given here are presented m ref. 1. 27 For those acylamtdrazones that do not precipitate (e g , the Boc-glycme and Boc-L-prolme derived acylamidrazones), the crude product obtained after evaporation of the solvent can be used in the next step without further purtficatton However, most amtdrazones did precipitate and were obtamed m good yields followmg filtratton 28. The 011bath temperature should be kept above the meltmg point of the acylamtdrazonate, 1e , between 18&2Oo”C Use a Dean-Stark apparatus and a silicon 011bath 29 To make the etheral HCl, wergh an Ehrlenmeyer flask with a known amount of drethyl ether Bubble gaseous HCl through the ether and weigh the flask again to get the concentratron 30 A disposable syringe of an all-plastic type without a rubber seal should be used (e g., Fortuna@), 5 mL with a Luer connectton) The disc used as a filter should be made of polyethylene/polypropylene (e.g , Vyon@). The needle attached to the Luer connectton can be e g ONCE 18G x 2, I,2 x 50,50-12 31 The 4-methylbenzhydrylamme (MBHA) resin has been used for C-terminally amtdated pepttdes (32) The “Merrtfield resin” (40,41) and the PAM resm (30) can be used for nonamtdated peptrdes 32 Dtpeptidomtmetlc 6 has been used successfully also m automated peptrde syntheses 33 The free carboxylic acid dertvatrves of the 1,2,4-oxadrazoles slowly decarboxylate spontaneously and can not be stored Hence, the ester hydrolysis should be performed mnnedtately before the peptrde synthesis (42) 34 The free carboxylic acid dertvatives of the 1,3,4-oxadrazoles can not be isolated owing to immediate decarboxylatton (43,44) 35 The free carboxyhc acid dertvattves of the 1,2,4-trtazoles cannot be isolated owmg to immediate decarboxylatlon (45,46) 36 The solvent volume should be -750 pL The syrmge IS rotated during the dlfferent reaction and washing steps Hugh quahty reagents and solvents should be used m the pepttde synthesis 37. Heat IS generated when TFA gets m contact with DMF To avoid overpressure m the syringe the resin can be qutckly rinsed (2 x 10 s) with TFA before starting the deprotectton 38 A sequence of rinses IS needed to efficiently remove the TFA. Deprotectton condtttons involving cone TFA are rather harsh Preferably 30% TFA m dlchloromethane should be used.

20

Luthman, Borg, and Hacksell

39

For a good result, the synthesis should not be interrupted but contmued without delay until all ammo acids are coupled. 40 Heat 1s generated when dichloromethane gets m contact with DMF To avoid overpressure m the syringe the resin can first be quickly rinsed (2 x 10 s) with dichloromethane 41 In an attempt to determine the enantiopurity of 2, we wanted to make the diastereomeric amide dertvattves using (-)-Mosher acid chloride We performed the Boc-deprotection usmg TFA (30% m dtchloromethane) The free amme did not show any optical rotation and HPLC-analysts of the amide indicated that racemizatton had occurred durmg the Boc-deprotection. 42 Elimmatton of CO, occurs 43 More lipophihc peptides can be soluble m dtethyl ether Be careful to not dlscard the ether phase unttl the result of the cleavage reaction has been analyzed. Preferably a different work-up procedure should be used Perform the procedure described m l-4 Thereafter, filter off the resin. Partition the filtrate between 20% HOAc and dtethyl ether. Lyophihze the water phase to obtain a white solid.

References Borg, S , Estenne-Bouhtou, G , Luthman, K , Csoregh, I , Hesselmk, W., and Hacksell, U (1995) Synthesis of 1,2,4-oxadtazole-, 1,3,4-oxadiazole, and 1,2,4triazole-derived dipeptidomlmetics J Ovg Chem 60, 3 112-3 120 Clapp, L B (1984) 1,2,3- and 1,2,4-Oxadiazoles, m Comprehenswe Heterocyclrc Chemzstry, vol 6 (Potts, K T , ed ), Pergamon, Oxford, pp 365-392 Hill, J (1984) 1,3,4-Oxadiazoles, m Comprehenszve Heterocyclzc Chemzstry, vol 6 (Potts, K T , ed ), Pergamon, Oxford, pp 427-466 Polya, J B (1984) 1,2,4-Trtazoles, zn Comprehenswe Heterocyck Chemzstry, vol 5, part 4A, (Potts, K T , ed ), Pergamon, Oxford, pp. 733-790 Burrell, G , Evans, J M , Hadley, M S , Hicks, F , and Stemp, G (1994) Benzopyran potassmm channel activators related to cromakahm-Heterocychc amide replacement at position 4 Broorg Med Chem 4, 1285-1290 Thompson, S K , Eppley, A M , Frazee, J S , Darcy, M G , Lum, R T , Tomaszek, T A, Jr, Ivanoff, L A, Morris, J F , Sternberg, E J , Lambert, D M , Fernandez, A V , Petteway, S R , Jr, Meek, T D , Metcalf, B W , and Gleason, J G (1994) Synthesis and antiviral activity of a novel class of HIV-l protease mhibttors contammg a heterocychc Pl ‘-P2’ amide bond isostere Bloorg Med Chem Lett 4,2441-2446 Borg, S , Vollmga, R. C , Teremus, L , and Luthman, K (1997) Heterocychc PheGly mimetics as building blocks m pseudopeptides Design, synthesis, and evaluanon J Med Chem , submitted Eloy, F and Lenaers, R (1966) Synthese d’ammo-oxadtazoles-1,2,4 Helv Chum Acta 49, 1430-1432 Paulder, W W. and Kuder, J E (1967) The conversion of imtdazo[ 1,5-alpyridmes mto 3-(2-pyrtdyl)-1,2,4-oxadiazoles J Org Chem 32, 243CL2433

Synthesis and Use of Peptides

21

10 Yurngi S , Miyake, A , Fushtmi, T , Imamtya, E , Matsumura, H , and Imai, Y (1973) Studies on the syntheses of N-heterocyclic compounds III. Hypercholeserolemic 1,2,4-oxadiazole dertvattves. Chem Pharm Bull. 21, I64 l-l 650. 11 Jones, J. (199 1) Peptide bond formation, m The Chemzcal Syntheszs of Peptzdes (Halpern, J , Green, M L K., Mukatzama, T., eds.), Oxford University Press, New York, pp 42-75 12 Eloy, F and Lenaers, R. (1962) The chemistry of amidoxtmes and related compounds Chem Rev 62, 155-183 13 Chiou, S and Shine, H J (1989) A stmphfied procedure for preparing 3,5-disubstttuted- 1,2,4-oxadtazoles by reaction of amidoxtmes with acyl chlorides m pyrtdine solutton J Heterocyclzc Chem 26, 125-128 14 Ungnade, H E and Kissmger, L W (1958) The structure of amidoximes. J Org Chem 23, 1794-1796 15. Loubmoux, B., O’Sulhvan, A C , Smnes, J -L , and Wmkler, T (1994) A new method for the synthesis of 2-carboxymethyl-2-hydroxy-tetrahydropyrans, Tetrahedron 50,2047-2054 16. Brown, H. C., Garg, C P , and Liu, K -T (1971) The oxidation of secondary alcohols m diethyl ether with aqueous chromic acid A convenient procedure for the preparation of ketones m high enantiomeric purity J Org Chem 36, 387-390 17 Muller, E (1971) Umwandlung von DI-, Tri- und Tetraacylhydrazmen, m Methoden der Organzschen Chemze, vol 1012 (Houben-Weyl) (Muller, E , ed), Georg Thleme Verlag, Stuttgart, pp 163-l 68 18 Stelzel, P (1974) N-Acyl-azoimme (N-Acyl-ammosaure-aztde, Azid-Methode), m Methoden der Organzschen Chemze (Houben- Weyl), vol XVI2 (Muller, E , ed), Georg Thieme Verlag, Stuttgart, pp. 296-322 19 Rzeszotarska, B , Nadolska, B , and Tarnawski, J. (198 1) Synthese von Peptiden mu 3’-Iod-I-tyrosm ohne Blockierung der Phenolfunktion J Lzebzgs Ann Chem 1294-1302 20 Hassner, A and Alexaman, V. (1978) Direct room temperature esterificatlon of carboxyhc acids Tetrahedron Lett 4475-4478. 21 Boesch, R. (1978) 1,3,4-Oxadiazolverbmdungen, deren Herstellung und ihre Verwendung Ger Pat 28 08 842 22 Golfier, M and Guillerez, M.-G. (1976) Cychsations dipolau-es I. Mecamsme de la cychsatton des dihydrazides par la reactif SOCl,/pyridine. Tetrahedron Lett 267-270 23 Neilson, D G , Roger, R , Heathe, J. W M , and Newlands, L. R (1970) The chemistry of amidrazones. Chem Rev 70, 15 l-l 70 24. Watson, K. M. and Neilson, D. G. (1975) The chemistry of amidrazones, m The Chemzstry of Functzonal Groups, part “The Chemistry of Amzdines and Imzdates ” (Patat, S , ed ), John Wiley, New York, pp. 491-544. 25 Francis, J. E , Gorczyca, L. A., Mazzenga, G. C , and Meckler, H (1987) A convenient synthesis of 3,5-dtsubstttuted1,2,4-triazoles. Tetrahedron Lett 28, 5133-5136

22

Luthman, Borg, and Hacksell

26 Schmidt, P and Druey, J (1955) Heilmittelchemtsche studten m der heterocycllschen rethe. 1,2,4-trtazme. Helv Chzm Acta 38, 156&l 564 27 Ratz, R and Schroeder, H. (1958) Products from reaction of hydrazme and thtonooxamlc acid and then conversion mto heterocychc compounds J Org Chem 23, 193 1-1934 28 Chen, F M. F and Benotton, N L (1987) The preparation and reactions of mixed anhydrtdes of N-alkoxycarbonylammo acids Can J Chem 65,6 1962.5 29 Ghckman, S A and Cope, A C (1945) Structure of p-ammo derivatives of a$unsaturated lactones and esters J Am Chem Sot 67, 10 17-l 020 30 Merrtfield, R B (1963) Solid phase peptide synthesis. I The synthesis of a tetrapeptide J Am Chem Sot 85,2149-2154 3 1 Barany, G , Knelb-Cordomer, N , and Mullen, D G. (1987) Solid phase peptide synthesis. a silver anniversary report /nt J Pep&de Protean Res 30, 705-739 32 Matsueda, G R and Stewart, J M (198 1) A p-methylbenzhydrylamme resin for improved solid-phase synthesis of peptide amides Peptzdes 2,45-50 33 Benz, H (1994) The role of solid-phase fragment condensation (SPFC) m pepttde synthesis Syntheses 337-358 34 Jung, M E and Lyster, M A (1978) Conversion of alkyl carbamates mto ammes via treatment with trimethylsilyl iodide J Chem Sot , Chem Commun 3 15-3 16 35 Lott, R S , Chauhan, V S , and Stammer, C, H (1979) Trtmethylsilyl iodide as a peptide deblockmg agent J Chem Sot , Chem Commun 495496 36 Borg, S , Luthman, K , Nyberg, F , Terenius, L , and Hacksell, U. (1993) 1,2,4Oxadtazole derivatives of phenylalamne potential mhibttors of substance P endopeptidase Eur J Med Chem 28,801-8 10 37 Rlgo, B and Couturier, D (1986) Studies on pyrrohdmones A convenient synthesis of 2-methyl-5-(5-0x-l -benzyl-2-pyrrohdmyl)1,3,4-oxadrazoles J Heterocyclzc Chem 23,253-256 38 Cauhez, P , Couturier, D , Rigo, B , Fasseur, D , and Halama, P (1993) Studies on pyrrohdmones Synthesis of 2-(5-oxo-2-pyrroltdmyl)1,3,4-oxadiazoles and 2-(5-oxo-2-pyrroltdmyl)benzlmldazoles J Heterocyclzc Chem 30,921-927 39 Wleland, T , Lewalter, J , and Birr, C (1970) Uber peptidsynthesen, XLIV Nachtraghche aktivierung von carboxyl-derivaten durch oxydation oder ehmmierung und ihre anwendung zur peptid-synthese an fester phase sowie zur cychsierung von peptrden Lrebzgs Ann Chem 740, 3 147 40 Bodanszky, M., Klausner, Y S , and Ondetti, M A (1976) Peptlde Synthesu, 2nd ed., Wiley, New York, Chapter 7, pp 158-176 41 Barany, G and Merrifield, R B (1980) Sohd-phase peptide synthesis, m The Peptldes Analysu, Synthesu, Bzology, ~012 (Gross, E. and Metenhofer, J., eds ), Academic, New York, l-284. 42. Brachwltz, H (1972) Hydroxtmsaurederivate; versuch zur darstellung der 5-phenyl- 1,2,4-oxadlazol-3-carbonsaure. 2 Chem. 12, 130-l 32 43 Spmellt, D , Noto, R., Consigho, G , Werber, G., and Bucchert, F (1977) Kmettc study of the decarboxylation of 5-ammo- 1,3,4-oxadiazole-2-carboxylic acid to 2-

Synthesis and Use of Peptides ammo-1,3,4-oxadlazole

23

m water as a function of proton actlvtty J Chem Sot ,

Perkln Trans 2,639%642

44 Noto, R , Bucchert, F , Constgho, G , and Spmelb, D (1980) Studies on decarboxylatton reactions. Part 4. Kmetic study of the decarboxylatton of some N-alkylor N-phenyl-substituted S-ammo- 1,3,4-oxadtazole-2-carboxyhc acids J Chem Sot , Perkm Trans 2, 1627-1630 45 Dost, J , Stem, J , and Heschel, M (1986) Zur herstellung von 5-substitmerten 1,2,4-trtazol-3-carbonsaurederivaten aus oxalsaureethylester-N 1-acylamtdrazonen Z Chem 26,203-204. 46 Heschel, H., Stem, J , and Dost, J. (1987) Uber cychsterungsversuche von oxalsaureethylester-Nl-acyl-amtdrazonen zu 5-R- 1,4-trtazol-3-carbonsauredertvaten Wzss Z Paedagog Hochsch

“Karl Llebknecht”

Potsdam 31,45-52

2 Syntheses of FMOC=2,3=Methanoleucine Stereoisomers and Their Incorporation into Peptidomimetics Kevin Burgess and Wen Li 1. Introduction 2,3-Methanoamino acids resemble protein ammo acids. The only difference between these groups of compounds IS that the former have one addrtronal carbon whrch spans the a- and p atoms (Fig. 1). Thus cyclopropane ring constraint of 2,3-methanoammo acids allows four side chain orientations; drfferent conformatrons are therefore favored when stereotsomeric cyclopropane ammo acrds are mcorporated mto pepttde sequences Pepttdomlmetics contaming 2,3-methanoammo acids are also more hindered, and this imparts proteolytrc stabrbty (I-4). Overall, both these factors (I.e., the conformatronal constraints and enhanced proteolytic stabrhtres, make 2,3-methanoammo acids excellent probes for medrcmal chemrstry and brophysrcal experiments.) Described here are multrstep syntheses of all four 2,3-methano-analogs of leucme. This procedure illustrates some steps that have also proved to be very effective for syntheses of several other 2,3-methanoammo acids Specrfically, thus protocol shows how cychc sulfates may be transformed into cyclopropanes and how the two esters of a dialkyl cyclopropane- 1,l -drcarboxylate can be drfferentrated and elaborated mto amino acid functionalitres. 2. Materials 2.1. Reagents 1 2 3 4

L-Valme D-Valme Sodmm mtrlte (NaNO,) Sodmm borohydrlde (NaBH,) From

Methods m Molecular Medune, Vol 23 Pepbdommet/cs Protocols EdIted by W M Kazmferskl OHumana Press Inc , Totowa, NJ

25

Burgess and LI

26

H~N..,c$H

SLIU

FMocNyy~

2R,3S-cyclo-LeU

FMocNy

(1)

2S,3R-cyclo-Leu

FMomHpo$i

(2)

2S,3S-cycle-LeU

Fig 1 Leu and the four stereolsomers

FMOCNI-I~O~H

(3)

2&W-cycle-Leu

(4)

of cycle-Leu

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19.

Iodme Thlonyl chloride (SOCl*) Sodium perlodate (NaI04) Ruthenium trlchlorlde (RuC13 3H,O) Sodium hydride (NaH) Dlmethyl malonate Sodmm carbonate (Na,C03) Trlethylamme (NEt,) tert-Butanol (‘BuOH) Dlphenyl phosphorazldate (DPPA, (PhO),P(O)N,) Sodium hydroxide (NaOH) Sodmm bicarbonate (NaHCO,) Trlfluoroacetlc acid (CF,CO,H) 9-Fluorenylmethyloxycarbonyl-N-hydroxysucc~nim~de (FMOC-OSu). Benzotnazole1-yl-oxy-trls(dlmethylamlno)phosphonlumhexafluorophosphate (BOP) 20. 1-Hydroxybenzotnazole (HOBt) 21 4-Methylmorpholme (NMM) 22 Plperldme 23 Dlethyl glutaconate

3. Methods 3.1. Preparation of FMOC-(2R,3S)-2,3-Methanoleucine 3.1.1 (2S)-3Methylbutan- 1,2-Cyclic Sulfate 5 (Fig. 2)

1

1 A 1 L three-necked, round-bottomed flask was equipped with a reflux condenser/ CaCl, drying tube connected to a HCl trap, charged with 14.1 g (136 0 mmol, 1 0 eq) of (2S)-3-methylbutan-1,2-diol (see Note 1) m CC& (140 mL) 2 Thlonyl chloride (11 9 mL, 163 0 mmol, 1.2 eq) was added via syrmge to the flask, and the resulting solution was refluxed for 1 h 3. The solution was cooled to 0°C usmg an Ice-water bath CH,CN (140 mL), 22 0 mg of RuCl, 3H,O (0 1 mmol, 0 0006 eq) and 43 62 g of NaIO, (204 0 mmol, 1 5 eq) were added to this solution, followed by water (200 mL) The resulting orange mixture was stirred at 25°C for 2 h. TLC at this stage indicated that no starting material remamed

27

Syntheses of FMOC-2,3-Methanoleucine 02

(1) soc12, cc14

S

*

H?OH

Oa

(II) NaIO,, cat R&I3 MeCN(,,,, 25 “C

A.

3 5

MeWk,J%Me

Me02C

NaH, DME.

C02Me

85 “C

:::::

-

Y,

(I) NafZO3. MeOH 25 “C. 60 h * (II) DPPA, NEt3 ‘BuOH, 85 ‘C. 12 h

6

BOCH

G02Me f’ ‘G,

NaOH. MeOH

BOCHN

25 ‘C, 7 d

+ c,,

C02H

v

t

8

7

(I) TFA, CC14, 0 ‘C, 50 min (II) FMOC-OSu,

Ir DMF, 25 ‘C, 6 h

FMOCNH +*+ “Q,

CO,H

yr 2R,3S-cycle-Leu

(1)

Ftg 2 Syntheses of 2R,3S-cycle-Leu.

The mixture was extracted with diethyl ether (1 L). The orgamc layer was washed wtth water (50 mL), saturated aqueous NaHCOs (2 x 50 mL), and brme (50 mL) It was then dried over MgSO,, filtered through a small pad of stltca gel (removed the dark green color), and concentrated to afford the product (17 7 g, 78%) as an 011 ‘H NMR (200 MHz, CDCls) 6 0 95-O 99 (3H, d, 6.9 Hz), 1 07-l 10 (3H, d, 6 7 Hz), 2 00-2 17 (lH, m), 4 35-4 46 (lH, m), 4 6@-4 73 (2H, m), 13C NMR (50 MHz, CDCl,) 6 16 7 (CH,), 17 9 (CH,), 3 1 0 (CH), 71 5 (CH& 87 1 (CH) Thts maternal was used m the next step wtthout further purtficatton

3.1.2. (S)-D/methyl Z-(1 -Methylethyl) Cyclopropane- 1, I -Dlcarboxylate 6 1 A 1 L, two-necked, round-bottomed flask was equipped with a reflux condenser, flushed with N,, and charged with dtmethyl malonate (10 2 mL, 89 0 mmol, 1 0 eq) and freshly dtsttlled DME (150 mL)

28

Burgess and Li

2 Sodmm hydrtde (4 5 g, 188 0 mmol, 2 1 eq) was added to the above solution m one portton (2S)-3-Methylbutan-1,2-cychc sulfate 5 (14 8 g, 89 4 mmol, 1.O eq) tn DME (50 mL) was added via a cannular. Restdues m the cannula were then washed through using addmonal DME (2 x 50 mL) The resultmg solutton was stirred at 25°C for 1 h, then refluxed for 30 h 3 The VISCOUS solutton formed was cooled to amblent temperature, then water (150 mL) and ether (150 mL) were added The water layer was extracted wtth ether (2 x 100 mL) and ethyl acetate (2 x 100 mL) The combmed orgamc layers were dried over Na$O,, and concentrated m vacua to afford the crude product (17 1 g ) as an 011.Thts material was used m the next step wtthout further purtficatron, but a small sample was purified via flash chromatography (4.1 hexane EtOAc) to obtain the charactertzatron data that follows 4 ‘H NMR (200 MHz, CDCl,) 6 0.97 (3H, d, 5.5 Hz), 1.01 (3H, d, 5.6 Hz), 1 05 (lH, m), 135 (2H, m), 1 68 (lH, dd), 3.69 (3H, s), 3.73 (3H, s), 13C NMR (50 MHz, CDCl,) 6 20 6 (CH,), 21 6 (CH,), 22 4 (CH), 28 6 (CH,), 34 3 (C), 36 6 (CH), 52 3 (OCH,), 52 5 (OCH,), 168 8 (CO), 170 8 (CO)

3.1.3. (1 R,2S)-Methy/2-(7-Methylethy/)-I-(A!-(tert-Butoxycarbony/) Ammo)Cyclopropane- 1-Carboxyla te 7 The crude dunethyl dtcarboxylate derlvattve 6 prepared as descrtbed above (16.4 g, 82 0 mmol, 1 0 eq) and Na2C03 (19.1 g, 180 3 mmol, 2 2 eq) were mixed m MeOH-H,O (300 mL/75 mL) and stirred at 25°C for 60 h The reaction mixture was concentrated and extracted with dtethyl ether (200 mL) then EtOAc (500 mL). The aqueous layer was actdtfied with 2 M HCl, extracted wrth EtOAc (2 x 500 mL), then dried over Na,SO, After filtration, and evaporatton of the solvent, 13.2 g of the crude (lS,2S)-2(methylethyl)1-(methoxycarbonyl) cyclopropane1-carboxyltc acid was obtamed (87%) as a colorless 011 The crude carboxybc acid derrvattve prepared in step 3 (12 3 g, 66 0 mmol, 1 0 eq) and NEt, (11 0 mL, 79 1 mmol, 1 2 eq) were mixed wtth dry t-BuOH (200 mL) at 25°C under N, Dtphenyl phosphoraztdate (15 6 mL, 72 6 mmol, 1 1 eq) was added via syringe over 1 mm and the reaction mixture was stirred at reflux for 12 h The solutton was concentrated to dryness, and the residue purified vta flash chromatography (4 1 hexane EtOAc) to give 13 4 g of the product as a colorless 011 NMR studies of thus sample showed that tt contained trace tmpurmes derived from the dlphenyl phosphorazidate Nevertheless, this sample was used m the next reaction wrthout further purrficatron. A small amount of this product was purtfied by recrystalhzatton from dtchloromethane/hexanes to obtain the characterization data which follows ‘H NMR (200 MHz, CDCI,) 6 0 89 (3H,d, 6 6 Hz), 0 99 (3H, d, 6 6 Hz), 1 20 (3H, m), 1 42 (9H, s), 1 53 (lH, m), 3 70 (3H, s), 5 12 (lH, b); r3C NMR (50 MHz, CDCl,) 6 22 0 (CH,), 22 2 (CH,), 22 8 (CH), 22 9 (CH), 26 8 (CH,), 28 3 (CH,), 39 5 (C), 52 2 (OCH,), 79 95 (C), 172 45 (CO), [c1]*~,=-17 10”

Syntheses of FMOC-2,3-Methanoleucme (c = 0.83, CH,CI,), 258 33, found* 258

MS (FABIDP)

29

m/z calculated

for C,,H,,O,N

(M + H),

3.1 4. (1 R,2S)-2-(Methylethyl)- 1-(N-(tert-6utoxycarbonyl)Amino) Cyclopropane- 1-Carboxylic Acid 8 1 A 1 NNaOH solutton (61 mL, 61 mmol, 1 2 eq) was added to a solutton of the ester 7 (13 0 g, 5 1 0 mmol, 1 0 eq) in MeOH (50 mL) and the reaction mtxture was stirred for 6 d at 25°C (see Note 2). 2 The hydrolyses as described m step 1 was mcomplete Consequently, the reaction mrxture was concentrated, then extracted with ether. This ether layer contained 2.0 g of the startmg materral 7 , and the destred product remamed m the aqueous layer 3 The aqueous layer was extracted wtth EtOAc (3 x 20 mL) to remove any restdual organic soluble tmpurrttes The aqueous layer was then actditied wtth 2 M HCl, extracted wtth EtOAc (3 x 250 mL), then drted over Na$O,+ After filtratton, evaporatton of the solvent, and drying m vacua , the crude actd (7 2 g) was obtamed as a white sohd 4 The followmg data was obtamed for the compound Isolated m step 3 ‘H NMR (200 MHz, CDCl,) 6 0 93 (3H, d, 6 6 Hz), 0.98 (3H, d, 6 7 Hz), 1 25 (2H, m), 1 42 (9H, s), 1.65 (2H, m), 5.25 (IH, br s), 13C NMR (50 MHz,CDCls) 6 22 1 (CH,), 22 3 (CH,), 23 2 (CH), 26 8 (CH,), 28 3 (CH,), 39 00 (CH), 40 5 (C), 80 3 (C), 158.8 (CO), 177 0 (CO); MS (FAB/DP) m/z calculated for C,2H2,04N (M + H) ,244 13, found 244, [a1200 =-63 85” (c = 1 3, CH,CI,), m p 14&14l”C

3.7.5. FMOC-(2R,3S)-Methanoleucine

I

A sample of the material (s) prepared as descrtbed above (0 5 g, 2 1 mmol) was dissolved m Ccl, (10 mL) at 0°C TFA (4 5 mL) was added dropwtse over approximately 1 min, and the reaction was stirred for 50 mm at 25°C. The solutron formed above was evaporated to dryness, neutralized with 2 M NaOH, and lyphohzed, resultmg m a crude amme Thus amme was dissolved m 1 M Na2C0s (3 7 mL). DMF (5 0 mL) and H,O (1 3 mL) were added, and the solution was cooled to 0°C FMOC-OSu (0 6 g, 1 8 mmol, 1 0 es) was added m one portion, and the mixture was stirred for 8 h at 25°C Water (75 mL) was added, and the aqueous layer was washed with Et20 (5 x 30 mL) to remove the lypophthc rmpurmes The aqueous layer was then acrdrfied wtth crtrtc acid (5 g), and the product was extracted into EtOAc (2 x 60 mL) The EtOAc solutton from step 4 was washed with H20 (50 mL), brute (50 mL), and drred (Na,SO,) Thus solutron was filtered, and the solvent was removed m vacua The white sohd remammg was drred under vacuum to give 0.6 g of the product 1 This material was pure on the basis of TLC and NMR analyses A small sample was recrystallized from dtchloromethane/hexanes to obtam the data gtven below. ‘H NMR (200 MHz,CDCl,) 6 1 05 (6H, b), 1.35 (2H, m), 1.65 (2H, m), 4 2 1 (lH, t), 4,45 (2H, d), 5 48 (IH, b), 6.26 (IH, b), 7.34 (4H, m), 7 56 (2H, d), 7 74 (2H,

Burgess and LI

30

d), 13C NMR (50 MHz, CDCls) 6 22.0 (CH,), 22 1 (CH,), 23 6 (CH), 26 7 (CH,), 38 90 (CH), 41 0 (C), 47 I (CH), 67 0 (CH,), 119 9 (CH), 125 O(CH), 127 0 (CH), 127 7 (CH), 141 3 (C), 143 6 (C), 156 7 (CO), 177 6 (CO), MS (FAB/DP) m/z calculated for CZ2HZ304N (M + H), 366 43, found 366, [a120D -26 32”(c = 1 00, CH,Cl,)

3.2. Preparation of FMOC-(2S,3R)-2,3-Methanoleucine 2 FMOC-(2S,3R)-Methanoleucme was prepared using the same procedures that were applied m the synthesis of FMOC-(2R,3S)-methanoleucme 1 described above, except that the starting material was derived from o-valme instead of L-valme Characterization data for the final product 2 follows ‘H NMR (300 MHz, CDCl,) F 1.04 (6H, b), 1 35 (2H, m), 1 65 (2H, m), 4 25 (lH, t), 4 45 (2H, d, J=6 0 Hz), 5 49 (IH, b), 6 18 (lH, b), 7 35 (4H, m), 7 62 (2H, d, J = 7.2 Hz), 7 78 (2H, d, J = 7.2 Hz); 13CNMR (75 MHz, CDCl,) 6 22.0 (CH,), 22.2 (CH,), 23.6(CH,), 26.7 (CH), 38 9(C), 41.0 (CH), 47 1 (CH), 66.9 (CH,), 119.9 (CH), 125 0 (CH), 127.0 (CH), 127 6 (CH), 141 2 (C), 143.8 (C), 156.5(CO), 177.7 (CO), MS (FAB/DP) m/z calculated for Cz2HZ304N (M + H), 366.43; found: 366, [a]200 = 28 61” (c = 1.05, CH,Cl,). 3.3. Preparation of FMOC-(2S,3S)-2,3-Methanoleucine 3.3 1. (lR,2S)-Ethyl2-(I-Methylethyl)-l-(3(E-ethyl Propenylate))Cyclopropane1-Carboxylate 9 (Fig. 3)

3

1 Dtethyl glutaconate (4.0 g, 21 0 mmol) was added to a well-strrred solutton of NaH (1 1 g, 45 0 mmol, 2 1 eq) m DME (70 mL) at 25°C under N, 2 After 10 mm of stnrmg at 25°C (2S)-3-methylbutan-1,2-cyclic sulfate 5 (3 6 g, 21 5 mmol) was added over 10 mm 3 After sturmg at 25°C for 1 h, the solution was heated to reflux for another 4 h 4 The vtscous solution after reflux was cooled to ambtent temperature, then water (150 mL) and ether (150 mL) were added The water layer was extracted wtth ether (2 x 100 mL) and ethyl acetate (2 x 100 mL) The combined organic layers were dried over Na$SO,, then concentrated 5 The residue was purified by flash chromatography (6 1 hexane EtOAc), and 3 3 g of the product were obtamed as an 011 Thts sample was a mrxture of two isomers m a ratio of 6 1 The followmg spectral data was obtained for this material 6 ‘H NMR (200 MHz, CDCl,), 6 for the predominant Isomer 0 86 (3H, d, 6 1 Hz), 1 01 (3H, d, 6 0 Hz), 1.19 (2H, m), 1.27 (6H, m), 1 65 (2H, m), 4 17 (4H, m), 5 76 (lH, d, 15 84 Hz), 7 40 (lH, d, 15 84 Hz) The followmg were conspicuous peaks for the minor isomer 6 5 60 (d, 15 80 Hz), 7 55 (d, 15 80 Hz)

3.3.2. (lS,2S)-Ethyl Z-(I-Methylethyl)-1-[N-(tert-Butoxycarbonyl AmmojCyclopropane- I -Carboxylate 10 1 A catalytic amount of RuC13 3H,O (0 05 g, 0 2 mmol, 0 02 eq) was added to a vigorously stirred mixture of the cyclopropane dertvattve prepared as above

31

Syntheses of FMOC-2,3Metbanoleucine

so2

0’ ‘0 J-J

EtO,CACO,Et NaH. DME,

Et02C 85 ‘C

-3

(I) NaIO,, cat RuCI,, Ccl,, MeCN, 25 ‘C, 12 h

NHSOC

EtO& .Y

*

(II) DPPA, NBt3, $uOH,

NaOH,

reflux

EtOH, 25 “C, 4 d w

(I) TFA. Ccl,+ 0 ‘C, 50 mm (II) FMOC-OSu,

* DMF, 25 ‘C, 8 h

FMOCNH

,.C02H

P 2 $3~cycle-Leu

(3)

Fig 3 Syntheses of 2S, 3S-cycle-Leu

(3 2 g, 12 8 mmol, 1 0 eq) and NaIO, (22.0 g, 102.0 mmol, 8.0 eq) In Ccl, (28 mL), CHJN (28 mL) and HZ0 (42 mL) at 25°C. After stnrmg for 6 h, the mtxture was filtered, the precipttate was washed with HZ0 and CH2C12, and the aqueous layer was extracted wtth CHQ, (2 x 50 mL) The combined orgamc extracts were drted (MgSO& and the solvent was removed m vacua to yteld a mixture of actds A sample of the crude mixture generated above (2 3 g, 14 2 mmol, 1 0 eq) was mixed wtth dtphenylphosphorylaztdate (4 3 g, 15.6 mmol, 1 1 eq) and trtethylamme (1 8 g, 17 7 mmol, 1 2 eq) m ‘BuOH (24 mL), and heated to reflux for 12 h. The mtxture was cooled to 25°C and concentrated under vacuum The residue was purtfied vra flash chromatography (6.1 hexane EtOAc) to give 2.8 g of a white sohd which proved to be the desired product contaminated with traces of matertals derrved from dtphenylphosphorylazidate A small amount of thts compound was recrystalhzed from MeOH to obtain the data that follows.

32

Burgess and LI

6 ‘H NMR (300 MHz, CDCI,), F 0 95 (lH, m), 1 04 (3H, d, 6 0 Hz), 1 07 (3H, d, 6 0 Hz), 1.27 (3H, t), 1 43 (lH, m), 1.49 (9H, s), 1 70 (2H, m), 4 14 (2H, q), 4 99 (lH, b), 13CNMR (75 MHz, CDC13), 6 14 1 (CH& 22 2 (CH,), 22 6 (CH,), 23 5 (CH,) 27 8 (CH), 28 2 (CH,), 35.9 (CH), 38 7 (C), 61 1 (CH,), 79 9 (C), 156 4 (CO), 173 2 (CO)

3.3 3. (lS,2S)-2-(1 -Methylethyl)- I-[N-(tert-Butoxycarbonyl) Amino]Cyclopropane- 1-Carboxyk Acd 1 I 1 A 1M NaOH solutton (13 3 mL, 13.3 mmol, 1 2 eq) was added in one portion to a solution of the product prepared above (2.4 g, 8 9 mmol, 1 0 eq) n-r EtOH (20 mL) 2 This reaction mtxture was starred for 4 d at 25°C then concentrated and extracted wrth EtOAc to remove lypophtltc rmpurmes 3 The aqueous layer was actdlfied wtth 2 M HCl, extracted with EtOAc (3 x 20 mL), then dried over Na,SO, The acid was obtamed as a whrte solid (1 1 g) after evaporatton of the solvent under vacuum A small amount of this product was purified by recrystallization from MeOH to obtam the following charactertsttc data 4 ‘H NMR (200 MHz, methanol-d4), 6 0.90 (lH, b), 1 12 (6H, t), 1 45 (3H, m), 1 51 (9H, s), 7 03 (lH, b), 13C NMR (50 MHz, methanol-d& 6 22 5 (CH,), 22 8 (CH,), 22 9 (CH,), 28 6 (CH,), 29 1 (CH), 37 0 (CH), 39 6 (C), 80 4 (C), 159 0 (CO), 176 9 (CO); (FAB/DP) m/z calculated for C12H2,04N (M + H) 244 13, found 244, [IY.]~~~ = -21 40” (c = 0 86, MeOH), m p 182-183°C

3.3 4 FMOC-(2S,3S)-2,3-Methanoleucine

3

1 FMOC-(2&3X)-2,3-Methanoleucme was prepared usmg same procedure as outlmed for the syntheses of FMOC-(2R,3S)-2,3-methanoleucme 1 from its BOC derrvatrve 8 A small sample was recrystallrzed from methanol to obtam the following data 2 ‘H NMR (300 MHz, methanol-d,), 6 0 90 (lH, b), 1 07 (6H, t), 1 29 (lH, m), 1 55 (2H, b), 4 24 (lH, t), 4 39 (2H, d, 6 6 Hz), 7 34 (4H, m), 7 69 (2H, d, 6 9 Hz), 7 79 (2H, d, 7 5 Hz), 13CNMR (75 MHz, methanol-d,), F 22 1 (CH2), 22 3 (CH,), 22 6 (CH,), 28 6 (CH), 36 8 (CH), 39 3 (C), 47 9 (CH), 67 3 (CH,), 120 4 (CH), 125.7 (CH), 127 6 (CH), 128 2 (CH), 142 1 (C), 144 8 (C), 159 2 (CO), 176 3 (CO), MS (FAB/DP) m/z calculated for CZ2H2s04N (M + H), 366 43, found 366, [u]~~,, =6.55” (c = 1 08, MeOH)

3.4. Preparation

of FMOC-(2R,3R)-2,SMethanoleucine

4

FMOC-(2R,3R)-Methanoleucme was prepared usmg the same procedures used for FMOC-(2&3S)-methanoleucme 3, except that the starttng materral was derived from D-valme instead of L-valme. Data for charactcrizatlon follows.

33

Syntheses of FMOC-2,344ethanoleucine

‘H NMR (300 MHz, methanol-d& 6 0.89 (IH, b), 1.08 (6H, t), 1.28 (1 H, m), 1.54 (lH, m), 4.26 (lH, t), 4.40 (2H, d, 6.3 Hz), 7.38 (4H, m), 7.70 (2H, d, 7.2 Hz), 7.81 (2H, d, 5.7 Hz); t3C NMR (75 MHz, methanol-d& 6 22.0 (CH,), 22.3 (CH3), 22 6 (CH,), 28.6 (CH), 36.8 (CH), 39 3 (C), 47.9 (CH), 67.3 (CHJ, 120.4 (CH), 125.8 (CH), 127.6 (CH), 128.2 (CH), 142.1 (C), 144.8 (C), 159.1 (CO), 176.2 (CO); MS (FABIDP) m/z calculated for CZ2HZ304N (M + H), 366.43; found: 366; [a] 2oD= 6.41’ (c = 0.96, MeOH). 3.5. Solid Phase Syntheses of Peptidomimetics Containing 2,,SMethanoamino Acids Peptidomimetics containing 2,3-methanoamino acids can be prepared by stepwise couplings of FMOC-amino acid derivatives (5,6) on Rink amide resin (7). Manual peptide syntheses (8) using 30 mL reaction vessels fitted with coarse glass frits are convenient. These vessels are agitated using a wrist shaker (Burred, model 75). Combinations of BOPiHOBtiNMM (premixed with FMOC-protected amino acids in DMF) can be used for the coupling steps (9). A DMF washing cycle (10 x 1 min, ca. 10 mL) is executed after each

coupling and deprotection. A negative ninhydrin test (IO) indicates completion of each coupling, Cleavage

of 2,3-methanoamino

acid containing

peptidomimetics

from

Rink’s amide resin is conveniently effected by using a mixture of trifluoroacetic acid (81.5%), phenol (5%), water (5%), 1,2-ethanedithiol (2.5%), and thioanisol (1%). The resin is filtered, then washed with trifluoroacetic acid then CH,Cl,. The filtrate is then concentrated. Water is added, and the aqueous layer is washed with Et,O. Crude peptidomimeitcs generated in this way can be purified by preparative RP-HPLC. 3.6. Discussion The syntheses described herein were designed to minimize the number of chromatographic purifications required to obtain pure final products. A trained chemist can produce one of these products in approximately three weeks. Both chiral centers are derived from valine; therefore, the starting materials are cheap, and there are no steps in which asymmetric induction may vary. Cycle-Leu derivatives are relatively simple members of the 2,3-methanoamino acid family. Asymmetric synthesesof other members of this series have been summarized in a review (II). 4. Notes 1. The starting material diol was made from valine through two literature steps: (1) deamination of the amino group by using NaNO* and 1NH,SO,, the concentration of H2S04 is important; crystals of the resulting a-hydroxy acid could not

34

Burgess and 11’

be obtained if 5 M H,SO, was used. (12) (ii) reduction of the a-hydroxy acid to the diol by using NaBH, and I, in THF. (13) The latter procedure was convenient, since the reducing agent NaBH,/I, is easier to handle than LiAlH,. Other syntheses of 2,3-methanoamino acids require dials from other sources. One convenient and flexible method is to use mannitol as a starting material to give cyclopropanes with functionalized side-chains (24,15). Elaboration of this functionality facilitates syntheses of the cycle-Met, (15) cycle-Arg, (26) truncated cycle-Arg, (2 7) and cycle-Glu/Gln amino acids (28). 2. Subsequently, we have found that this hydrolysis is complete in l-2 d when the reaction temperature was 50°C.

References 1. Kimura, H., Stammer, C. H., Ren-Lin, C., and Stewart, J. (1983) The synthesis, bioactivity, and enzyme stability of D-Ala2, E-cycle-Phe4, Leu5 enkephalins. Biochem. Biophys. Res. Commun. 115, 112-l 15. 2. Ogawa, T., Shimohigashi, Y., Yoshitomi, H., Sakamoto, H., Kodama, H., Waki, M., and Stammer, C. H. (1988) The molecular design of stereospecific chymotrypsin inhibitors with structurally constrained amino acid. Pept. Chem. 25-30. 3. Ogawa, T., Shimohigashi, Y., Shiota, M., Waki, M., Stammer, C. H., and Ohno, M. (1989) 2,3-Methanophenylalanine and a,P-dehydrophenylalanine derivatives as chymotrypsin inhibitor. Pept. Chem. 43-4 6. 4. Ogawa, T., Yoshitomi, H., Kodama, H., Waki, M., Stammer, C. H., and Shimohigashi, Y. (1989) Enzyme inhibition by dipeptides containing 2,3-methanophenylalanine, a sterically constrained amino acid. Febs Lett. 250, 227-230. 5. Atherton, E. and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis, A Practical Approach, Oxford University Press, Oxford, UK. 6. Fields, G. B. and Noble, R. L. (1990) Solid Phase Peptide Synthesis Utilizing 9-Flourenylmethoxycarbonyl Amino Acids. Int. J. Peptide Protein Res. 35, 161-214. 7. Rink, H. (1987) Solid-Phase Synthesis of Protected Peptide Fragments Using a Trialkoxydiphenyl-Methylester Resin. Tetrahedron Lett. 28, 3787-3790. 8. Stewart, J. M. and Young, J. D. (1984) Solid Phase Peptide Synthesis 9. Felix, A. M., Wang, C.-T., Heimer, E. P., and Fournier, A. (1988) Applications of BOP Reagent in Solid Phase Synthesis II. Solid Phase Side-chain to Side-chain Cyclizations Using BOP Reagent. Int. J. Pept. Prot. Rex 31,23 l-238. 10. Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. BioChem. 34,595-598. 11. Burgess, K., Ho, K.-K., and Moye-Sherman, D. (1994) Asymmetric Syntheses of 2,3-Methanoamino Acids. SYNLETT. 8, 575-583. 12. Koch, P., Nakatani, Y., Luu, B., and Ourisson, G. (1983) A Stereoselective Synthesis and a Conveniant Synthesis of Optically Pure (24 R)- and (24 St24hydroxycholesterols. Bull. Sot. Chim. France. II, 189-l 94.

Syntheses of FMOC-2,3- Methanoleucine

35

13. McKennon, M. J., Meyers, A. I., Drauz, K., and Schwarm, M. (1993) A Convenient Reduction of Amino Acids and Their Derivatives. J. Org. Chem. 58, 3568-3571. 14. Burgess, K., Ho, K.-K., and Ke, C.-Y. (1993) Synthesis of a Valuable Cyclopropyl Chiron for Preparations of 2,3-Methanoamino Acids. J. Org. Chem. 58,3767-3768. 15. Burgess, K., and Ke, C.-Y. (1996) Large Scale Syntheses of N-Protected 2,3Methanomethionine Stereoisomers. Synthesis. 1463-1467. 16. Burgess, K. and Ho, K.-K. (1992) Asymmetric Syntheses of Protected Derivatives of Ornithine- and Arginine-2,3-Methanologs. Tetrahedron Lett. 33, 5677-5680. 17. Burgess, K., Lim, D., Ho, K.-K., and Ke, C.-Y. (1994) Asymmetric Syntheses of Protected Derivatives of Carnosadine and its Stereoisomers as Conformationally Constrained Surrogates for Arginine. J. Org. Chem. 59, 2 179-2 185. 18. Burgess, K., and Lim, D. Y. (1995) Asymmetric Syntheses of the Stereoisomers of Protected 2,3-Methanoglutamine. Tetrahedron Lett. 36, 78 15-78 18.

3 Synthesis of an Esterase-Sensitive Cyclic Prodrug of a Model Hexapeptide Having Enhanced Membrane Permeability and Enzymatic Stability Using an Acyloxyalkoxy Promoiety Sanjeev Gangwar, Giovanni M. Pauletti, Teruna J. Siahaan, Valentino J. Stella, and Ronald T. Borchardt 1. Introduction The clmtcal development of orally active peptide drugs has been hmtted by their unfavorable phystcochemlcal characteristtcs (e g., charge, hydrogen bondmg potential, size), whrch prevent them from permeating btologtcal barrters such as the mtestmal mucosa, and also therr lack of stabthty against enzymatic degradation (I-12). Unfortunately, many of the structural features of pepttdes (t.e , the N-terminal ammo group and C-terminal carboxyl group, and side chain carboxyl, ammo, and hydroxyl groups) that bestow upon the molecule affinity and spectficrty for its pharmacologtcal receptor severely restrict tts ability to permeate btological barriers and make the molecules substrates for pepttdases Therefore, successful oral delivery of peptides depends on strategies designed to alter the physicochemtcal charactertsttcs of these potential drugs wtthout changing their btologtcal acttvtty m order to circumvent the intestinal epttheha1 cells. This chapter describes a novel prodrug strategy for preparing cycltc peptides that have increased metaboltc stabihty and cell membrane permeabtllty compared to linear peptrdes We will demonstrate this methodology using a model hexapepttde (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) derrved from deltasleep mducmg pepttde (13) to prepare a cycltc prodrug by lmkmg the N-termtnal ammo group to the C-termmal carboxyl group vta an acyloxyalkoxy promoiety. This cycltc pepttde prodrug 1s designed to be susceptible to esterase From

Methods in Molecular Medicme, Vol 23 Pepbdom~met~cs Protocols Edited by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

37

Gangwar et al.

38

HCHO

+ COP

1

Chemical Fast

1

H,‘N-[=)-COO

Figure 1

metabohsm (slow step) leading to a cascade of chemical reactions and resultmg m the generation of the linear peptlde (Fig. 1) (14-18) 2. Materials ‘H NMR spectra were recorded on a Bruker AM-500 (500 MHz) or a Varlan XL 300 (300 MHz) instrument. Chemical shifts are expressed m parts per mlllion (6) relative to tetramethylsllane (TMS), with either TMS or residual solvent as an internal reference. Abbreviations are as follows* s, singlet, d, doublet, t, triplet; q, quartet; b, broad. High performance hquld chromatography (HPLC) was conducted usmg a Raimn gradient system with a Dynamax UV detector The desired products were purified by preparative reversed-phase HPLC using a C- 18 column (flow rate of 5 mL/min) and elutmg with a gradient of solvent A (0.1%TFA/H20:5% ACN) and solvent B (ACN) from 20% to 100% solvent B over 65 mm The desired peptlde was analyzed by analytical reversed-phase HPLC, using a C- 18 column (flow rate of 1 mL/mm) and elutmg with a linear gradient of solvent A (0 1%TFA/H20*5% ACN) and solvent B (ACN) from 10% to 50% solvent B over 14 mm. Starting matertals were purchased from Aldrich Chemical Co , Sigma, Fluka Chemicals or Bachem Bioscience Inc. and used as received 2.1. Reagents for Method 3.1 1 2 3 4 5 6 7 8

I-Chloromethyl chloroformate p-N\rltrophenol N-Methyl morpholme (NMM) Sodium Iodide (NaI) 1-Hydroxybenzotnazole (HOBt) Hexamethylphosphoramlde (HMPA) 10% Palladmm on Carbon (10% Pd/C). Ethyl acetate (EtOAc)

39

Acy/oxyalkoxy Promoiety 9 10 11 12. 13 14 15 16 17 18 19 20 21 22 23 24

Dtchloromethane (CH,CI,) NJ-Dtmethylformamtde (DMF) Acetomtrlle (ACN) Sodmm carbonate (Na,COs) Sodmm bicarbonate (NaHCO,) Acetone Sodmm bisulfite (NaHSO,) Cttrrc acid Sodmm chloride (NaCl) Sodmm hydroxtde (NaOH) Cesmm carbonate (C&CO,) Sodium sulfate (Na,SO,) Chloroform (CHCl,) Methanol (CHsOH) Dtethylether (Et20) Ethanol (EtOH).

2.2. Reagenfs

8 9 10 11 12 13

for Method 3.2

2,2,2-Trrchloroethanol 4-(N,N-dtmethyl)ammopyrldlne (DMAP). I-(3-dtmethylammopropyl)-3-ethylcarbodtimtde Ethyl acetate (EtOAc) Sodium bicarbonate (NaHCO,). Sodmm chlortde (NaCl) Sodmm sulfate (Na,SO,) Trrfluoroacetic actd (TFA) Dtchloromethane (CH,Cl,) Drethylether (Et20) 1-Hydroxybenzotrtazole (HOBt). N-Methyl morpholme (NMM) Cttrtc acid.

hydrochlortde

(EDC)

hydrochlortde

(EDC)

2.3. Reagents for Method 3.3 2 3 4. 5 6 7 8 9 10 11

l-(3-dtmethylammopropyl)-3-ethylcarbodiimide l-Hydroxybenzotrtazole (HOBt) N-Methyl morpholme (NMM) Dtchloromethane (CHQ,) Citric acid Sodium bicarbonate (NaHCOs) Sodium chloride (NaCI) Sodium sulfate (Na,SO,) Zmc dust (Zn) Acetic acid (AcOH) Trtfluoroacettc actd (TFA)

40 12 13 14 15 16 17 18

Gangwar Phenol Ethanedtthtol Dlethylether (Et,O) N,N-Bts(2-oxo-3-oxazohdmyl)-phosphmlc 4-(N,N-dtmethyl)ammopyrtdme (DMAP) 10% Palladmm on Carbon (10% Pd/C) Absolute ethanol (EtOH)

et al

chloride (BOP-Cl)

2.4. Reagents for Method 3.4 1 2 3 4 5 6 7 8 9 10 11 12 13.

Potassmm bttartrate Tartartc actd Sodium acetate Acetic actd (AcOH) Sodmm phosphate Phosphortc acid Sodmm borate Sodmm hydroxtde Sodium chlortde (NaCl) Human plasma Hanks’ balanced salt solution (HBSS) Guamdmmm hydrochlortde Paraoxon (dtethyl p-mtrophenyl phosphate).

2.5. Reagents for Method 3.5 1 2 3 4

Caco-2 cells Hanks’ balanced salt solutton (HBSS) Acetomtrtle (ACN) Phosphortc acrd

2.6. HPLC Columns 1 Reversed-phase semr-preparatrve column: Ramm C-l 8 column (12 ~.tm, 300 A, 25 cm x 21 4 mm ID) 2 Reversed-phase analyttcal column Dynamax C- 18 column (5 pm, 300 A, 25 cm x 4 6 mm ID)

3. Methods 3.1. Preparation of Boc-AlanineoxymethylCarbonyl-N-Tryptophan 6 The approach which 1s pursued for the synthesis of the cycltc prodrug 1 mvolves msertton of the acyloxyalkoxy promotety between two ammo acids m this sequence before the final cycllzatton step. In the model hexapepttde used tn these studies, we Insert the promotety between the Ala and the Trp residues,

Acyloxyalkoxy Promoiety

0

p-mtrophenol

L

CI~OXCI

C,AOB0.Q.N02

JfL

,~(fo4=J~02

2 Boc-Ala-COOCs+

I H

0

0

BcE’~‘+O~O~N CH,

H B~~~~-./+~-‘o~o’0

H

tH,

0 -

0

\

/

-No2

4

5 R = Bzl Pd.C/

H2

6 R=H

Fig 2 Synthesisof the key intermediate6 using a convergent approach to the synthesis of the cychc prodrug 1 mvolvmg fragments 6 and 10. Figure 2 shows the syntheses of the key intermediate 6 m which the promorety has been inserted between Boc-Ala and Trp. The synthesis of 6, was begun by reacting 1-chloromethyl chloroformate withp-mtrophenol m the presence of N-methyl morpholine (NMM) to afford 1-chloromethyl-p-mtrophenol carbonate 2. Substrtutron of the chloride m 2 with iodide is achieved by reaction with NaI to give the iodo compound 3. Compound 3 is then reacted with the cesmm salt of Boc-Ala in DMF to give Boc-alanineoxymethyl-p-mtrophenol carbonate 4. This reaction was shown to afford better yields of 4 using the cesmm salt of Boc-Ala than the sodium or potassium salts of this ammo acid. Compound 4, was coupled with Trp-OBzl m the presence of NMM and HOBt m HMPA to afford Boc-alanmeoxymethyl-carbonyl-N-tryptophanbenzyl ester 5. Removal of the benzyl group n-rcompound 5 was achieved by hydrogenolysis using 10% Pd/C as a catalyst under an Hz atmosphere m EtOH to give 6 m 96% yield. Compound 6 was purified by preparative reversedphase HPLC 3.1.1. Preparation of l-Chloromethyl-p-Nitrophenol

Carbonate 2

1 Add a solutron of 1-chloromethyl chloroformate (3.2 mL, 0 036 mol) dropwrse to an ice-cold reaction mrxture of p-mtrophenol (5 g, 0 036 mol) and N-methylmorpholme (3.6 mL, 0 036 mol) m CHCls (50 mL) 2. Stir the reaction mixture at 0°C for 1 h and at ambient temperature for 24 h 3 After stu-rmg overmght, wash the reaction mrxture successively with 10% aqueous citric acrd (2 x 20 mL), H,O (2 x 50 mL), saturated aqueous NaHCOs (2 x 40 mL), Hz0 (2 x 50 mL) and saturated aqueous NaCl(20 mL)

42

Gangwar et al.

4

Dry the CHCl, layer over anhydrous Na$O,, and then decant and evaporate to gave pure 1-chloromethyl-p-mtrophenol carbonate 2 (6 9 g, 84%) as a light yellow 011

3.1 2. Preparation of 1-lodomethyl-p -Nitrophenol Carbonate 3 1 Add m one portron NaI(5 g, 0 03 mol) to a solutton of 1-chloromethyl carbonate 2 (6 9 g, 0 03 mol) m acetone (50 mL), and stir the reaction mixture at 50°C for 24 h 2 After stnrmg ovemrght, evaporate the solvent and dissolve the residue m Et,0 (100 mL) Wash the Et,0 layer successively with 10% aqueous Na$O, (2 x 20 mL), HZ0 (2 x 50 mL), saturated aqueous NaCl(20 mL) and dry over anhydrous Na*SO, 3 Remove the solvent under reduced pressure to afford 1-todomethyl-p-mtrophenol carbonate 3 (9 1 g, 94%) as a pure yellow 011 ‘H-NMR (CDCl,, S) 6 03 (2H, s), 739(2H,d,J=9Hz),824(2H,d,J=9Hz)

3 7 3 Preparation of Boc-Alanmeoxymethyl-CarbonylN-Tryptophan Benzyl Ester 5 1 Prepare the cesmm salt of Boc-Ala by reacting Boc-Ala (2 g, 0 01 mol) with C&O? (1 7 g, 5 2 mmol) m CH30H (30 mL). After sttrrmg the-reaction mixture for 1 h, remove the solvent under reduced pressure to afford the cesmm salt of Boc-Ala as a white powder Add a solution of the cesmm salt of Boc-Ala (0 96 g, 3 mmol) m DMF (20 mL) slowly to an me-cold and stirred solution of iodomethyl-p-mtrophenol carbonate 3 (1 g, 3 mmol) m DMF (50 mL), followed by addmon of Boc-Ala (2 g, 0 01 mol) over a pertod of 2 h After stirring the reaction mixture for 24 h at room temperature, remove the solvent under reduced pressure to afford an oily residue Dissolve the oily residue m EtOAc (100 mL), and wash successively with 10% NaHCO, (2 x 20 mL), HZ0 (2 x 50 mL) and saturated aqueous NaCl(20 mL) Dry the organic layer over anhydrous Na2S04, and decant out the solid Evaporate the EtOAc under reduced pressure to give Boc-alanmeoxymethyl-pmtrophenol carbonate 4 (2 7 g, 70%) as a yellow or1 Add NMM (0 12 mL, 1 05 mmol) to a stirred solutton of Boc-alanmeoxymethylp-mtrophenol carbonate 4 (0 4 g, 1 04 mmol), TrpOBzl HCl(0 35 g, 1 05 mmol) and HOBt (0 142 g, 1 05 mmol) m HMPA (50 mL) After sttrrmg for 24 h at room temperature, drlute the reaction mixture wrth CH,Cl, (200 mL), and wash with cold 10% NaOH (2 x 50 mL) and H20 (2 x 100 mL) Separate the CH@, layer, dry over anhydrous Na2S04 and concentrate under reduced pressure to yield Boc-alamneoxymethyl-carbonyl-N-tryptophan benzyl ester 5 (0.42 g, 75%) as a pale yellow 011

3.1 4. Preparation of Boc-AlanineoxymethylCarbonyl-N-Tryptophan 6 1 Dissolve Boc-alanmeoxymethyl-carbonyl-N-tryptophan benzyl ester 5 (0 2 g, 0 37 mmol) m absolute EtOH (25 mL), and add 10% Pd-C (0 03 g) Stir the

Acyloxyalkoxy Promo/&y

43

L

OH

Boc-N H

COOBzl

COOBzl

COOBzl Tnchloroethanol

0

-

Boc-N L OCHPCC13 H 0

-

TFA

TFK

H3+N L

7

OCH2CC13 0 8

I

Boc-Ala-Gly-Gly-OH

CH3 HO

TFA‘H3+N

4

H~

TFA

NAN?fNhCH2CC13 0 H o 10

-

Boc-

‘COOBzl

9

‘COOBzl

Ftg 3 Synthesis of the tetrapeptide 10 reaction mtxture under a H2 atmosphere using a hydrogen balloon on the top of the round bottom flask 2 After 10 h, filter the reactron mixture, and concentrate the filtrate under reduced pressure to furnish Boc-alamneoxymethyl-carbonyl-N-tryptophan 6 (0 16 g, 96%) 3 Purify the product by semi-preparatrve reversed-phase HPLC and monitor the eluent at h = 280 nm. Analyze pepttde 6 by analyttcal reversed-phase HPLC and monitor the eluent at h = 280 nm The retention time of peptrde 6 1s 15 26 mmutes ‘H NMR ([CD&CO, S) 1 30 (3H, d, J = 6 9 Hz), 1.39 (9H, s), 3 19-3 43 (2H, m), 4 16 (IH, d, J = 6 2 Hz), 4 53-4 54 (IH, br), 4 78 (lH, br), 5 62-5.74 (2H, br), 7 03 (IH, t, J = 6 8 Hz), 7 10 (lH, t, J = 6 8 Hz), 7 38 (lH, d, J = 7.5 Hz), 7 63 (lH, d, J = 7 8 Hz)

3.2. Preparation

of Boc-Ala-G/y-G/y-Asp(OBzl)-OTce

9

Figure 2 tllustrates the solutron phase synthesis of tetrapeptide 10 using standard Boc-ammo acid chemtstry (19). The key to this solution-phase approach 1s the selecttve protection of the a-and P-carboxyl groups of the Asp restdue. We successfully used the trtchloroethyl (Tee) ester protecting group for the a-carboxyl group of the Asp residue, which 1squite stable to actdrc condttrons and can be removed by zmc m AcOH (20). Treatment of Boc-Asp(OBzl)-OH

with trichloroethanol in the presence of EDC and HOBt gave Boc-Asp(OBzl)OTce 7. Compound 7 IS then treated with 50% TFA m DCM to give 8 m quantrtattve yield. The tripeptide Boc-Ala-Gly-Gly-OH (synthesized by standard Boc-ammo acid chemistry with EDC and HOBt as couplmg reagents) IS then coupled to the Tee ester 8 m the presence of EDC and HOBt to yield tetrapeptide 9. Treatment of 9 with 50% TFA m DCM, provides 10

44

Gangwar et al

3 2 1. Preparation of Boc-Asp(OBzl)-OTce

7

1 Dtssolve Boc-Asp(OBzl)-OH (1 g, 3 mmol), 2,2,2-trrchloroethanol (0 4 mL, 3 mmol) and DMAP (0 18 g, 1 5 mmol) m CH2C12 (30 mL) and cool to O’C 2. Add EDC (0 57 g, 3 mmol) to this cooled solutton, and stir the reactton mixture at 0°C for 3 h and at ambtent temperature for 21 h 3 Falter out the prectprtate and drlute the filtrate wrth EtOAc (100 mL). 4 Wash the EtOAc layer successrvely with saturated NaHC03 (2 X 20 mL), Hz0 (2 x 50 mL) and saturated aqueous NaCl (20 mL) Dry the EtOAc layer over anhydrous Na2S04 and concentrate under reduced pressure to yteld BocAsp(OBzl)-OTce 7 (1 13 g, 83%) as a yellow 011 ‘H-NMR (CDCls, 6) 1 45 (9H, s), 2.93 and 3 15 (2H, dd, J = 17 Hz and 4 5 Hz), 4 67 and 4 75 (2H, dd, J = 12.3 Hz), 4 74 75 (lH, m), 5 13 (2H, s), 5 56 (lH, d, J = 9 Hz), 7 34-7 38 (5H, m)

3.2.2 Preparation of H-Asp(OBzl)-OTce

8

1 Add TFA (5 mL) to a stirred solution of Boc-Asp(OBzl)-OTce 7 (1 g, 2 2 mmol) m CHzClz (5 mL) Stir the reaction mrxture at room temperature for 45 mm 2 Remove volattle compounds m the reaction mixture using a rotary evaporator under vacuum 3 Triturate the residue and wash with anhydrous Et,0 and isolate the solid by decantatton Dry the solid Asp(OBzl)-OTce 8 (0 78g, 100%) under vacuum to remove residual Et,O, and use 8 m the next step wrthout further purrfrcatron

3.2.3. Preparation of Boc-Ala-G/y-G/y-Asp(OBzl)-OTce

9

1. Add EDC (0.99 g, 5 16 mmol) m one portion to a cooled (O’C) and stirred solution of Boc-Ala-Gly-Gly-OH (1 6 g, 5.13 mmol), Asp(OBzl)-OTce 8 (2 4 g, 5.13 mmol), HOBt (0 69 g, 5.13 mmol) and NMM (0 5 mL, 5 13 mmol) m CH,Cl, (100 mL) 2 Star the reaction mixture at 0°C for 4 h and at ambient temperature for 24 h Dilute the reaction mixture wtth CH,Cl, (250 mL) and wash with 10% aqueous crtrtc acid (2 x 50 mL), HI0 (100 mL), saturated NaHCO, (2 x 50 mL), HZ0 (100 mL) and saturated aqueous NaCl(50 mL) 3 Dry the organic layer over anhydrous Na2S04 and concentrate under reduced pressure to furmsh Boc-Ala-Gly-Gly-Asp(OBzl)-OTce 9 (2.8 g, 86%) as an pale yellow oil

3.2.4 Preparation of H-Ala-G/y-G/y-Asp(OBzl)-OTce

10

1 Dissolve Boc-Ala-Gly-Gly-Asp(OBzl)-OTce 9 (0.29 g, 0 44 mmol) m CH& (5 mL) and cool the solutron to 0°C 2 Add TFA (5 mL) mto thus clear solutton and stir the reactton mtxture at room temperature for 1 h Remove volattle compounds m the reactton mtxture by rotary evaporator under vacuum

45

Acy/oxyalkoxy Promoiety

s

Boc-

11

BOP-Cl

H

13 R=Bzl

Pd-Cl Hp

C

1 R=H

Fig 4 Synthesis of the cychc prodrug 1

3 Trlturate the residue, and wash with anhydrous Et,O, and isolate the solid by decantatlon Dry H-Ala-Gly-Gly-Asp(OBzl)-OTce 10 (0 27 g, 95%) under vacuum to remove the residual Et,O, and use in the next step without further purlfkatlon

3.3. Preparation

of Cyclic Prodrug

7

Frgure 4 shows the assembly of fragments 6 and 10 for the synthesis of the cyclic prodrug 1. The tetrapeptide 10 is reacted with Boc-alanineoxymethylcarbonyl-N-tryptophan 6 m the presence of EDC, HOBt, and NMM to give the fully protected linear hexapeptide 11 in 70% yield. Both of the protecting groups on 11 are removed by first treating with zinc m AcOH to remove the Tee protecting group, then with 50% TFA m DCM to remove the Boc protectmg group. This afforded 12 m 60% overall yield. The linear hexapeptlde 12 1s purified by semi-preparative reversed-phase HPLC. Cychzatlon 1s accomplished by the standard high-dllutlon

technique using BOP-Cl

as an activating

46

Gangwar et al.

reagent (21) in the presence of NMM and DMAP to give the cyclic pepttde 13 m 20% yield. Hydrogenolysts of cyclic pepttde 13 to remove the Bzl protectmg group from the Asp residue 1sthen achteved with 10% Pd/C as a catalyst under a HI atmosphere m EtOH to yield the desired cyclic prodrug 1. The crude cychc prodrug 1 IS purified by preparative reverse-phase HPLC and was analyzed by analyttcal reverse-phase HPLC 3.3.1. Preparation of Boc-Ala-(OCH,OCO)Trp-Ala-G/y-G/y-Asp(OBzl)-OTce 11 1 Add EDC (0 09 g, 0 44 mmol) to a cooled (O’C ) and strrred so!utton of Bocalamneoxymethyl carbonyl-N-tryptophan 6 (0 2 g, 0 44 mmol), Ala-Gly-GlyAsp(OBzl)-OTce 10 (0 29 g, 0.44 mmol), HOBt (0 6 g, 0 44 mmol) and NMM (0 09 mL, 0 89 mmol) m CH2C12 (50 mL) 2. Stir the mixture for 2 h at 0°C and 30 h at ambient temperature 3 Dilute the reactton mixture with CH2C12 (250 mL) and wash successtvely wrth 10% aqueous cttrrc acid (2 x 50 mL), H20 (100 mL), saturated NaHCO, (2 x 50 mL), H,O (100 mL) and saturated aqueous NaCl(50 mL) 4. Dry the organic layer over anhydrous Na2S04, and concentrate under reduced pressure to furmsh an or1 of Boc-Ala-(OCH20CO)-Trp-Ala-Gly-Gly-Asp(OBzl)OTce 11 (0.3 g, 70%).

3.3.2. Preparation of Ala-(OCH,OCO)-Trp-Ala-G/y-G/y-Asp (OBzl)-OH 72 Add Zn dust (1 g) to a stnred solutron of Boc-Ala-(OCH,OCO)-Trp-Ala-Gly-GlyAsp(OBzl)-OTce 11 (0 2 g, 0 2 mmol) m AcOH (50 mL) over a period of 1 h Stn the reactron mrxture for 24 h at room temperature, then filter out the msoiuble maternal, and concentrate the filtrate under reduced pressure to grve an 011yresidue Dissolve the resultmg residue m CH,Clz (10 mL), and cool the solutton to 0°C Add TFA (5 mL), phenol (2 g) and ethanedtthrol (0.2 mL) to thus clear and stirred solutton. After stnrmg at room temperature for 2 h, evaporate CH$l,, and trtturate the restdue with anhydrous Et,O. Wash the sohd with anhydrous Et20, and dry under reduced pressure to grve Ala(OCH20CO)-Trp-Ala-Gly-Gly-Asp(OBzl)-OH 12 (0 10 g, 60%) Purify the product by semr-preparatrve reversed-phase HPLC and momtor the eluent at h = 280 nm Analyze peptrde 12 by analytical reversed-phase HPLC and momtor the eluent at h = 280 nm The retention trme of linear peptrde 12 is 13 03 mm

3.3.3. Preparation of Cyck Hexapeptlde Prodrug 7 1 Add a solution of linear peptrde 12 (0 138 g, 0 186 mmol) and NMM (0 2 mL, 1 98 mmol) m CH,Cl, (100 mL) dropwlse to a cooled (O’C ), sot-red solutron of BOP-Cl (0 236 g, 0.93 mmol) and DMAP (0 023 g, 1 86 mmol) m 50 mL of CH,Cl, over 2 h

47

Acyloxyalkoxy Promoiety

After stmmg the reaction mtxture for 3 days at room temperature, evaporate the solvent under reduced pressure, and dtssolve the residue m CH,Cl, (100 mL). Wash successtvely the organic layer with 10% aqueous citric acid (2 x 20 mL), H,O (50 mL), saturated NaHCO, (2 x 20 mL), H,O (50 mL) and saturated aqueous NaCl(20 mL) Dry the organic layer over anhydrous Na,S04, and concentrate under reduced pressure to grve cyclic peptide prodrug 13 (0.027 g, 20%). Analyze pepttde 13 by analyttcal reversed-phase HPLC and momtor the eluent at h = 280 nm. The retention ttme of cyclic peptide 13 IS 14 3 minutes MS (FAB) m/z 722 (Mf + 1) Use cychc prodrug 13 dtrectly in the next step wrthout further purificatton. Add m one portion 10% Pd-C (10 mg) to a solution of cyclic hexapepttde 13 (0 027 g, 0 037 mmol) m absolute EtOH (15 mL) Stir the reaction mixture under a H, atmosphere for 24 h Filter out the reaction mtxture to remove the Pd-C, and remove the solvent under reduced pressure to gave cychc prodrug 1 (0 023 g, 100%) Purify the product by semi-preparatrve reversed-phase HPLC, and monitor the eluent at h = 280 nm Analyze by analytical reversed-phase HPLC and momtor the eluent at h = 280 nm The retention of cychc prodrug 1 IS 11 2 minutes ‘H NMR (D,O, 8) 0 98 (3H, d,J = 7 2 Hz), 1.37 (3H, d, J = 7.2 Hz), 2 65-2 85 (2H, m), 3 10-3 45 (2H, m), 3 654.40 (8H, m), 5.34 (lH, d, J = 6.3 Hz), 5 95 (lH, d, J = 6 6 Hz), 7 11 (lH, t, J = 7.2 Hz) 7 20 (lH, t, J = 7.2 Hz) 7.27 (lH, s), 7 46 (IH, d, J = 6 3 Hz), 7 53 (lH, d, J = 7 8 Hz)

3.4. Chemical and Enzymatic Stability of Cyclic Prodrug

1

The chemical stability of the acyloxyalkoxycarbamate prodrug was determined at 37°C over the pH range 3.0-9.6. In aqueous buffered soluttons (37’(Z), pH 3.0-9.6, cyclic prodrug 1 degrades quantitatively to the linear hexapeptide (mass balance 297.2%). Maxtmum stab&y was found at pH values I4 With mcreasmg pH, cychc prodrug 1 degrades progresstvely faster to the linear hexapeptide (22). The stability of the acyloxyalkoxycarbamate prodrug was also assessed m 90% human plasma at 37°C in the presence and absence of paraoxon, a potent esterase inhibitor. In 90% human plasma (t,,* = 100 + 4 min), the disappearance of cyclic prodrug 1 is significantly faster than m buffered solution, pH 7.4 (t,,, = 206 + 11 mm).

3.4. I. Stability of Cyclic Prodrug 1 m Aqueous Buffered Solutions, pH 3.0-9 6 1 Prepare soluttons (500 )JL) of cyclic prodrug 1 (-15 pg/mL) m buffers at an tome strength of 0 5 A4 adjusted with NaCl The buffers used for each pH are as follows: potassmm bttartrate, pH 3.0, sodmm acetate, pH 4 0, sodmm phosphate, pH 5 0, 6 0, 7 0, 8.0, HBSS, pH 7 4, sodmm borate, pH 9.0, 9 6 2 Mamtam samples at 37.0 rt 0.5”C m a temperature-controlled shaking water bath (60 rpm).

48

Gangwar et a/

3 Remove 20 pL ahquots periodically SIS conditrons see Note 1)

and analyze mnnediately by HPLC

(analy-

3.4.2. Enzymatic Stability of Cyclic Prodrug 1 in 90% Human Plasma Incubate cychc prodrug 1 at a final concentration of -24 @4 m 90% human plasma (see Note 2) in a temperature-controlled (37.0 * 0 5’C) shaking water bath (60 rpm) Remove ahquots (20 pL) at various time points up to 360 mm Quench immediately the esterase activity of the sample by adding 150 p.L of a freshly prepared 6 N guamdmmm hydrochloride solution m acidrfied HBSS (HBSS contammg 0 01% (v/v) phosphoric acid) Transfer ahquots (150 pL) of that acidic mixture (pH -3) to an Ultrafree@-MC 5000 NMWL filter urnt (Mrlhpore, Bedford, MA) and centrifuge at 5000g for 60 mm (4°C) Dilute aliquots (150 p.L) of the filtrates with mobile phase and Inject on a Dynamax C- 18 HPLC column (for analysis conditions see Note 1) Stability studies m the presence of paraoxon are performed identically, except that the 90% human plasma is premcubated with paraoxon (final concentratron 1 mM) for 15 mm at 37°C

3.5. Transport Properties of Cyclic Prodrug Across Biological Membranes

1

To evaluate the cellular permeation characterrsttcs of the cyclic prodrug 1 and the linear hexapeptide, transport studies can be conducted using monolayers of Caco-2 cells, an zn vitro cell culture model of the mtestmal mucosa (23) Caco-2 cells, which spontaneously undergo differentiation mto confluent monolayer-s, have been shown to exhibit both the physical and metabohc barrier properties of mtestmal mucosal cells to peptides (24-26). When applied to the apical (i.e., lummal) side of Caco-2 cell monolayers, the linear hexapeptide rapidly disappears (t,,* = 14 mm), suggesting high susceptibihty to enzymatic degradation by peptidases. Smce the actual transport of this hexapeptide to the basolateral (i.e., serosal) compartment was not detected, the apparent permeability coefficient (P,,,) of the linear model hexapeptide was estimated to be less than 1.7 x 10m9cm/s based on the sensitivity of our analytical methods (22). In contrast, cyclic prodrug 1 is sigmficantly more stable (no degradation up to 3 hours of mcubation with Caco-2 cells) agamst metabolic degradation and exhibits a Pap,,value of 1.30 + 0.15 x10” cm/s

3.5 1. Transport of Cychc Prodrug 1 Across Caco-2 Cell Monolayers 1 Grow Caco-2 cells on collagen-coated polycarbonate membranes (Transwells@) to confluent monolayers of differentiated enterocytes (see Note 3) 2 Wash cell monolayers 3 times with prewarmed HBSS, pH 7 4, and 1 5 ml of the peptide solution (-100 @4 m HBSS) is applied to the donor (i.e., apical) compartment Add 2 5 mL of HBSS to the receiver (I e , basolateral) compartment

Acyloxyalkoxy Promoiety

49

3. Maintain cell monolayers in a temperature-controlled (37.0 f O.S°C) shaking water bath (60 rpm). 4. Remove at various times up to 180 min, samples (120 pL, receiver side; 20 a, donor side) from both sides. Replace the volume removed from the receiver side with fresh, prewarmed HBSS. 5. Add an aliquot of ACN and diluted phosphoric acid (final concentrations 10% (v/v) and 0.01% (v/v), respectively) to stabilize the samples. 6. Freeze the acidic mixture (pH -3) immediately in a dry-ice/acetone bath and keep at -80°C until HPLC analysis (for analysis conditions see Note 1).

3.6. Discussion To be effective, a prodrug should degrade chemically and/or enzymatically to the parent drug in vivo. Cyclic prodrug 1 is expected to undergo esterase-

mediated hydrolysis of the ester bond of the carbamate moiety, followed by two fast chemical steps to release the linear hexapeptide (WAGGDA) (Fig. 1). Since ester bonds are not only enzymatically but also chemically labile, we determined the stability of the cyclic prodrug 1 both in aqueous buffered solutions pH 3.0-9.6, and in human plasma known to contain esterase activity. With respect to chemical instability, this cyclic prodrug was more stable under moderate acidic conditions than in basic solution. Maximum stability was found at pH values 54. With increasing pH, cyclic prodrug 1 degrades progressively faster to the linear hexapeptide. At physiological pH (7.4), apparent half-lives (t& calculated from the pseudo-first order rate, constants are t1,2= 206 f 11 min for the disappearance of cyclic prodrug 1 and t 1,2 = 213 + 13 min for the formation

of the linear

hexapeptide.

In 90%

human plasma (t1,2 = 100 f 4 min), the disappearance of cyclic prodrug 1 is significantly faster than in buffered solution, pH 7.4 (t,,, = 206 + 11 min). This suggests that the disappearance of the cyclic prodrug may be catalyzed by esterases.Additional experimental evidence for the proposed mechanism was obtained from studies using paraoxon, a potent esterase inhibitor. After preincubation of human plasma with paraoxon, the disappearance of cyclic prodrug 1 is substantially slower (tri2 = 171 + 13 min). This strongly suggests that the parent peptide is released from cyclic prodrug 1 by an apparent esterase-catalyzed reaction. In human blood, cyclic prodrug 1 was 25-fold more stable than the linear hexapeptide. Transport studies indicate that cyclic prodrug 1 is at least 76-fold more able to permeate this model of the intestinal mucosa than is the linear hexapeptide (22). In conclusion, we have described a methodology for preparation of an acyloxyalkoxy-linked cyclic prodrug of a model hexapeptide via the N- and C-terminal ends. However, it should be feasible to use this methodology to cyclize other biologically active peptides by linking the C-terminal

Gangwar et al.

50

carboxyl group to a side-chain amino (e.g., Lys, Arg) or hydroxyl (e.g., Ser, Thr, Tyr) group, or by linking a side-chain carboxyl group (e.g., Asp, Glu) to a side chain amino (e.g., Lys, Arg), or hydroxyl (e.g., Ser, Thr, Tyr) group. 4. Notes 1. Elute the peptides at the flow rate of 1 mL/min using 74.0% (v/v) ACN in water with TFA (O.l%, v/v) as the tor the eluent with a fluorescence detector at emission h = 285 nm). 2. Dilute human plasma with HBSS, pH 7.4 to 90% (v/v). 3. For a detailed description of the cell culture conditions,

a gradient from 10.8 to ion-pairing agent. Monih = 345 nm (excitation

see refs. 22 and 23).

References 1. Amidon, G. L. and Lee, H. J. (1994) Absorption of peptide and peptidomimetic drugs, Annu. Rev. Pharmacol. Toxicol. 34,32 1-34 1. 2. Burton, P. S., Conradi, R. A., Hilgers, A. R., Ho, N. F. H., and Maggiora, L. L. (1992) The relationship between peptide structure and transport across epithelial cell monolayers. J. Controlled Release 19, 87-98. 3. Burton, P. S., Conradi, R. A., and Ho, N. F. H. (1993) Evidence for a polarized efflux system for peptides in the apical region of Caco-2 cells. Biochem. Biophys. Res. Commun. 190,760-766. 4. Bocci, V. (1990) Catabolism of therapeutic proteins and peptides with implications for drug delivery. Adv. Drug Delivery Rev. 4, 149-169. 5. Burton, P. S., Hill, R. B., and Conradi, R. A. (1987) Metabolism and transport of peptides across the intestinal mucosa. Proc. Int. Symp. Control. Rel. Bioact. Muter. 14,&7. 6. Conradi, R. A., Hilgers, A. R., Ho, N. F. H., and Burton, P. S. (1992) The influence of peptide structure on transport across Caco-2 cells. II. Peptide bond modification which results in improved permeability. Pharm. Res. 9, 435-439. 7. Conradi, R. A., Wilkinson, K. F., Rush, B. D., Hilgers, A. R., Ruwart, M. J., and Burton, P. S. (1993) In vitro/in vivo models for peptide oral absorption: comparison of Caco-2 cell permeability with rat intestinal absorption of renin inhibitory peptides. Pharm. Res. 10, 1790-1792. 8. Lee, V. H. L. (1988) Enzymatic barriers to peptide and protein absorption. Crit.

Rev. Ther. Drug Carrier syst. 5,69-97. 9. Lee, V. H. L. and Yamamoto, A. (1990) Penetration and enzymatic barriers to peptide and protein absorption. Adv. Drug Delivery Rev. 4, 17 l-207. 10. Lee, V. H. L., Dodda-Kashi, S., Grass, G. M., and Rubas, W. (1991) Oral route of peptide and protein delivery, in Peptide and Protein Drug Delivery (Lee, V. H. L., ed.), Marcel Dekker, New York, pp. 691-738. 11. Lee, V. H. L., Traver, R. D., and Taub, M. E. (1991) Enzymatic barriers to peptide and protein drug delivery, in Peptide and Protein Drug Delivery (Lee, V. H. L., ed.), Marcel Dekker, New York, pp. 303-357.

Acyloxyalkoxy Promoiety

51

12. Zhou, X. H. (1994) Overcoming enzymatic and absorption barriers to non-parenterally administered protein and peptide drugs. J. Controlled Release 29, 239-252. 13. Gray, R. A., Vander Velde, D. G., Burke, C. J., Manning, M. C., Middaugh, C. R., and Borchardt, R. T. (1994) Delta-sleep-inducing peptide: solution conformation studies of a membrane-permeable peptide. Biochemistry 33, 1323-l 33 1. 14. Alexander, J., Fromtling, R. A., Bland, J. A., Pelak, B. A., and Gilfillan, E. C. (199 1) (Acyloxy)alkyl carbamate prodrugs of norfloxacin. J. Med. Chem. 34,7%8 1. 15. Folkmann, M. and Lund, F. J. (1990) Acyloxymethyl carbonochloridates. New intermediates in prodrug synthesis. Synthesis 1159-l 166. 16. Gangwar, S., Pauletti, G. M., Siahaan, T. J., Stella, V. J., and Borchardt, R. T. (1996) Synthesis of a novel prodrug of a hexapeptide using acyloxyalkoxy promoiety which has increased stability to peptidase metabolism and increased cellular permeability. J. Org. Chem. 62, 1356-1362. 17. Gogate, U. S., Repta, A. J., and Alexander, J. (1987) N-(Acyloxyalkoxycarbonyl) derivatives as potential prodrugs of amines. I. Kinetics and mechanism of degradation in aqueous solutions. Int. J. Pharm. 40, 235-248. 18. Gogate, U. S. and Repta, A. J. (1987) N-(Acyloxyalkoxycarbonyl) derivatives as potential prodrugs of amines. II. Esterase-catalyzed release of parent amines from model prodrugs. J. Pharm. Sci. 40,249-255. 19. Atherton, E. and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis: A Practical Approach, 2nd ed., IRL, Oxford. 20. Greene, T. W. and Wuts, P. G. M. (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley, New York, pp. 240-241. 21. Diago-Meseguer, J., Palomo-Coll, A. L., Fernandez-Lizarbe, J. R., and ZugazaBilbabo, A. (1980) A new reagent for activating carboxyl groups: Preparation and reaction of N,N-Bis(2-oxo-3-oxazolidinyl)-phosphinic chloride. Synthesis 547-55 1. 22. Pauletti, G. M., Gangwar, S., Okumu, F. W., Siahaan, T. J., Stella, V. J., and Borchardt, R. T. (1996) Esterase-sensitive cyclic prodrugs of peptides: Evaluation of the acyloxyalkoxy promoiety in a model hexapeptide. Pharm. Res. 13, 1615-1623. 23. Hidalgo, I. J., Raub, T. J., and Borchardt, R. T. (1989) Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96, 736-749. 24. Wilson, G., Hassan, I. F., Dix, C. J., Williamson, I., Shah, R., and Mackay, M. (1990) Transport and permeability properties of human Caco-2 cells: An in vitro model of the intestinal epithelial cell barrier. J. Controlled Release 11,254O. 25. Pinto, M., Robine-Leon, S., Appay, M.D., Kedinger, M., Tradou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J., and Zweibaum, A. (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47, 323-330. 26. Artursson, P. (1990) Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells. J. Pharm. Sci. 79,476-482.

Synthesis of an Esterase-Sensitive Cyclic Prodrug of a Model Hexapeptide Having Enhanced Membrane Permeability and Enzymatic Stability Using a 3-(2’-Hydroxy-4’,6’-Dimethylphenyl)-3,3=Dimethyl Propionic Acid Promoiety Binghe Wang, Sanjeev Gangwar, Giovanni Teruna Siahaan, and Ronald T. Borchardt

Pauletti,

1. Introduction One of the major obstacles to the development of btologically active peptides as clinically useful therapeuttc agents has been then low permeation through btological barriers (e.g., Intestinal mucosa, blood-brain barrier) and their metabolic lability (1,2). Overcommg these problems 1sa very contemporary issue for the development of peptide pharmaceuticals. In the preceding chapter, we have indicated that masking the C- and N-terminal polar functional groups of a peptide through cyclizatton with an acyloxyalkoxy linker can greatly enhance the membrane permeation and metabolic stability of the linear pepttde (3). In this chapter, we wish to report a method for the preparation of esterase-sensitive cyclic prodrugs of peptides by taking advantage of a unique “trtmethyl lock”-facthtated lactomzatton system (Fig. 1). Substituted phenol propiomc acid derivatives such as 2, upon unmasking of the hydroxyl group, undergo a facile spontaneous mtramolecular cychzatton to release the moieties attached to the carboxyl functtonal group (Fig. 1) (46). The facile cychzation reaction 1sthe result of the “trimethyl lock”, which was shown earlier to mcrease the rate of the cychzatton reaction in the order of 105-7 (4-7). The result of such facthtatton is that compound 2 has a half-life of only approximately 100 s at room temperature in aqueous solution (8,9). Such systemshave

From

Methods Edlted

by

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Medwne,

W M Kazmterskl

Vol 23 Peptrdom/met/cs

OHumana

53

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Inc , Totowa,

Protocols NJ

Wang et al.

54

Esterase Slow

0 Chetmcal Fast H3C7

4

Ftg 1 The destgn of an esterase sensitive prodrug system for the cychc dertvattzation of pepttdes been used to develop prodrugs of ammes and alcohols (8-20) and redox-sensitrve protectmg groups of ammes (21). This “trimethyl lock” system can also be used as a linker for the preparation of cychc prodrugs of peptides and peptide mtmetics In this approach, the C- and N-terminal ends of a linear pepttde can be masked by forming an ester and an amtde bond with the phenol hydroxyl and stde cham carboxyl groups, respectively, of the lmker (Fig. 1) Here we use a model hexapeptide (HTrp-Ala-Gly-Gly-Asp-Ala-OH) derived from delta-sleep-mducmg pepttde (12,13) as an example to show how the cychc prodrug of a peptide can be prepared by linking the N-terminal ammo group to the C-terminal carboxyl group vta this “trtmethyl lock” linker. The overall concept of the design 1sshown m Fig. 1.

2. Materials ‘H-NMR spectra were recorded on either a 500 MHz or a 300 MHz mstrument. High performance hqutd chromatography (HPLC) was conducted using a dual pump system with a UV detector All starting materials were purchased from Aldrich Chemical Co., Sigma, Fluka Chemicals or Bachem Bioscience Inc and used as received Wang’s resin for solid phase peptide synthesis was obtained from BACHEM Btosctence Inc. with 0.95 mmol/g substttution.

2.7. Reagents

for Method 3.7

I 4-Dtmethylammopyridme 2 3,5-Dtmethylphenol 3 Acetic acid.

(DMAP)

Propionic Acid Promoiety 4 Acetone 5 Celite 52 1, 6 Drchloromethane (CH,Cl,, DCM) Dtmethylacryhc acid 8 Dtmethylformamrde (DMF) 9 Ethyl acetate (AcOEt) 10 Hexanes. 11 Hydrochlortc acid (36 5-38 0%) 12 Lithium alummum hydride (LtAlH,) 13 Magnesium sulfate (anhydrous, MgSO,) 14 Methanol 15 N-Boc-L-Alanme-OpNP 16 Potassmm permanganate(KMn0,) 17 Pyrtdmmm chlorochromate (PCC) 18. Silica gel 200-400 mesh 19 Sodium chloride (NaCl) 20 Sodium hydrogen carbonate (NaHCO,) 21 Sulfuric actd (H,SO,) 22 teut-Butyldtmethylsilylchlor~de (TBDMS-Cl) 23 Tetrahydrofuran (THF) 24 TLC (silica gel). 25 Trlethylamme (TEA)

2.2. Reagents for Method 3.2 2 3 4 5 6 7 8 9 10 11 12 13. 14 15 16 17 18 19 20

1-Hydroxybenzotrrazole hydrate (HOBt) 4-Drmethylammopyridme (DMAP) 1,3-Dusopropylcarbodllmlde (DIC) Acetomtrtle (ACN) P-Benzyl-aspartrc acid Brs(2-oxo-3-oxazohdmyl)-phosphnnc chloride (Bop-Cl). Dlmethylformamtde (DMF) Ethanol Ethyl acetate (AcOEt) Ethyl ether (Et,O) Hydrogen gas L-Phenylalanme-benzyl ester. HCl Magnesium sulfate (anhydrous, MgS04). Methanol N-Boc-L-Alanme-OpNP N-Fmoc-Asp( P-OBzl) N-Fmoc-Glycme N-Fmoc-L-Alanme N-Fmoc-L-Tryptophan N-Methylmorpholme (NMM).

55

Wang et al.

56 2 1. 22 23 24. 25 26 27 28 29 30 3 I, 32.

Nmhydrm Palladmm on activated carbon (10% PdK) Plpertdme Potassmm cyanide (KCN). Silica gel 200-400 mesh. Sodmm chloride (NaCl) Sodmm hydrogen carbonate (NaHCOs) Sodmm sulfate (anhydrous, Na,SO,) TLC (silica gel) Trrethylamme (TEA) Trtfluoroacettc acid (TFA) Wang’s alkoxy resin

2.3. Reagents 1 2 3. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19. 20 21 22 23

for Method 3.3

1-(3-Dtmethylaminopropyl)-3-ethylcarbodnmtde 1-Hydroxybenzotrtazole hydrate (HOBt). 4-Dtmethylammopyrtdme (DMAP) 2,2,2-Trrchloroethanol Acettc actd Acetomtrtle (ACN) Boc-Alanme Boc-P-Benzyl-asparttc acrd Boc-Glycme Boc-Ttyptophan Drchloromethane (CH,CI,, DCM) Ethanedtthtol Ethyl ether (Et,O) Glycme-benzylester HCl N-Methylmorpholme (NMM) Phenol Sodmm chlortde (NaCl) Sodmm hydrogen carbonate (NaHCO,) Sodium sulfate (anhydrous, Na,SO,). Tetrahydrofuran (THF) TLC (srltca gel) Trtfluoroacettc actd (TFA) Zmc

Note: Please see Subheadings additional reagents.

HCl (EDC)

2.4. and 2.5. of the preceding chapter for

2.4. HPLC Columns Reversed-phase preparative column. Ramin C- 18 column (12 J-WI,300 A, 25 cm x 214 mm ID).

Propionic Acid PromoIety

5

6

57

8

7

10

9

12

11

Fig. 2 Synthetic approach to the intermediate with Boc-Ala attached to the Linker (a) Dlmethacryhc acid/ toluene,con H,SO,, (b) LAH/THF, (c) TBDMS-Cl/TEA/ DCM; (d) BocAla-OpNP, DMAP/DCM, reflux, (e) HOAc/THF/H,O, (f) PCC, (g) KMnO,

Reversed-phase analytical 25 cm x 4 6 mm ID).

column:

Dynamax

C- 18 column

(5 pm, 300 A,

3. Methods The synthetic approach to the esterase-sensitive cychc prodrug of the model lmear hexapeptlde can be divided mto three parts: the synthesis of the linker with the first ammo acid attached (Fig. 2); solid phase approach to the synthesis of the cyclic prodrug (Fig. 3); and solution phase approach to the synthesis of the cyclrc prodrug (Fig. 4)

3.7. Preparation of the Critical lnfermediate with i3oc Alanine Attached to the Promoiety Due to the facile cychzatlon of the “trimethyl lock” system (Fig. l), the mcorporatlon of this promoiety mto the cyclic system first requires the protection of the phenolic hydroxyl group. Therefore, a key intermediate for the successful synthesis of the cychc prodrug 1 (Fig. 1) 1s the promotety (Prom) with the appropriate ammo acid attached (Boc-Ala-Prom, 12) (Fig. 2). This key intermediate can then be incorporated mto the peptide by condensing with the prepared linear peptlde. The synthesis of 12 starts by reacting commercially available 3,5-dlmethylpheno15 with dlmethacrylate in the presence of concentrated sulfuriq acid to give the lactone 6, that can be reduced with LiAlH, to give the dlol7 (8,9). Selective protection of the primary hydroxyl group with t-butyldimethyl sllyl (TBDMS) chloride m the presence of trlethylamme (TEA) gives the protected dlol 8 (Fig. 2) (9). Reaction of the activatedp-mtrophenol (pNP) ester of N-

Wang et al.

58

Boc-alanine with 8 m the presence of dtmethylammopyrtdme (DMAP) m refluxmg drchloromethane (DCM) gives 9 m 80% yield. Treatment of the TBDMS ether 9 wtth acetic acid m the presence of water, and THF yields the primary alcohol 10, which can be converted m a two-step oxtdatton with pyrtdmmm chlorochromate (PCC) and KMn04 to the key intermediate 12 3 1 1 4,4,5,7- Tetramethyl-3,4-dlhydrocoumarin

6

1. Add 770 mL of toluene and 20 mL of concentrated sulfuric acid to the mixture of 45 g (368 mmol) of 3,5-dtmethylphenol5 and 50 g (438 mmol) of dlmethacryhc acid methyl ester 2 Reflux the reaction mixture under nitrogen for 7 h wtth a Dean-Stark trap 3 Cool the reaction mixture to room temperature 4 Wash the reactron mtxture with water (100 mL x 3), 5% sodrum bicarbonate (100 mL x 2), and water (50 mL x 2), and dry the solution over anhydrous MgS04 overmght 5 Evaporate the solvent on a rotoevaporator, and add 75 mL of hexanes Allow the product to crystallize m a freezer at least overnight 6 Collect the crystals (see Note l), and wash with cold hexanes to give the desired product m about 65% yield (50 g)

3.1 2 4,4,5,7- Tetramethyl-3,4-dihydrocoumarin-diol

7

1 Add a solutton of 20 g (98 mmol) of 4,4,5,7-tetramethyl-3,4-dthydrocoumarm 6 m 100 mL of anhydrous THF m an addition funnel dropwtse to a suspension of 5 6 g (147 mmol) of LrAlH4 (see Note 2) m 50 mL of anhydrous THF m a roundbottom flask equtpped with a magnetic stir bar and immersed m an me bath Rinse the addrtron funnel with more anhydrous THF (50 mL x 2), and add the washings to the reaction mixture 2 Remove the ice bath, and stir the reaction mixture overnight at RT 3 Cool the reactton mixture with an ice bath, and quench the reaction by slowly adding me-cold 10% HCI solution with sttrrmg. 4 Separate the organic layer, and wash It with water (50 mL x 2) 5 Evaporate the solvent, and recrystallize the residue m a mixture of DCM and hexanes to give the pure product m 75% yield ( 15 4 g)

3.1.3. 4,4,5,7-Tetramethyl-3,4-dihydrocoumarin

TBDMS-drol8

1 Add dropwlse a solution of 30 mL of DCM and 31 mL (222 mmol) of TEA during a period of 1 h with sturmg to a mixture of 11 63 g (55 9 mmol) of dtol 7 and 9 3 g (61 7 mmol) of TBDMS chlortde in 70 mL of methylene chloride cooled with me bath. 2 Remove the ice bath, and stir the reactron at RT for 1 day 3 Evaporate the solvent, and dissolve the residue m 250 mL of methylene chloride 4 Wash the solution wtth water (20 mL x 4), and dry over anhydrous MgS04 for 1 h 5. Solvent evaporation should give about 18.1 g of a whrte solid product (100%)

Prop/on/c Acid Promoiety

59

3 1.4. Boc-Ala-TBDMS-d/o19 MIX 5.18 g (16 1 mmol) of TBDMS-dlol8,5 g (16.1 mmol) of Boc-Ala-OpNP, 2 g (16 4 mmol) of DMAP, and 20 mL of anhydrous methylene chloride Reflux the reaction mixture with stirring for 7 h, and then add an additional 0 4 g of DMAP, and reflux for an additIonal h Evaporate the solvent, and dissolve the residual m 200 mL of methylene chloride Wash the solution with saturated NaHC03 (20 mL x 4), 1% HCI (20 mL x 2), and brine (20 mL x 2) Then dry the solution over Na2S04 overmght Purify the residue after solvent evaporation by column chromatography (silica gel) using a gradient of 5 to 10% of ethyl acetate m hexanes to give a white solid product (6 87 g, 84%) ‘H-NMR (CDC,3) 680(1H,s),6.50(1H,s),5.18(1H,m),451(1H,m),347 (2H,t,J=76Hz),251(3H,s),221(3H,s),201(2H,m),l54(3H,d,J=7.3 Hz), 1 45 (15 H, s), 0 83 (6 H, s), -0.04 (6 H, s)

3 1.5 Boc-Ala-alcohol

10

1 Add 9 mL of THF, 19 mL of HI0 and 58 mL of HOAc to 5 0 g (9.9 mmol) of the Boc-Ala-TBDMS-dlol9 2 Stir the reaction at RT for 1 h 3 Evaporate the solvent to give a colorless oily residue (4 8 g), that IS used directly for oxldatlon without purification (see Note 3) 4 ‘H-NMR(CDCl,)679(lH,s),65l(lH,s),535(1H,m),4.47(lH,m),349 (2 H, m), 2 49 (3 H, s), 2 20 (3 H, s), 2.04 (2 H, m), 1 54 (3 H, d,J= 7 4 Hz), 1 46 (6 H, s), 1 42 (9 H, s)

3 1.5 Boc-Ala-amd 12 Add a solution of alcohol 10 (3 90 g, 9 9 mmol) m 200 mL of DCM dropwlse to a solution of 4 38 g (20 3 mmol) of PCC m 200 mL of DCM The reaction color changes from orange to black during addition. Stir the reaction mixture at RT for 1 h, then filter the reaction solution through Cellte and evaporate the solvent Purify with a short silica gel column to give 3 08 g of an oily product (79%) Dissolve the oily product m 40 mL of acetone and add this solution dropwise to a solution of 1 42 g (9 0 mmol) of KMn04 m 40 mL of acetone-H,0 (1.1) Strr at RT for 17 h Evaporate the acetone from the reaction mixture, and filter the residue through Cehte After filtration, adjust the pH of the solution to pH 3 with dilute (3 6%) HCl Then, extract the mixture with ethyl acetate (100 mL x 3), and dry the combined organic extracts over anhydrous MgSO, overnight Evaporate the solvent to give a white solid product (2 9 g, 88%) ‘H-NMR (CDC13) 6 72 (1 H, s), 6.44 (lH, s), 5.09 (1 H, m), 4 30 (1 H, m), 2 74 (2 H, b), 2 44 (3 H, s), 2 13 (3 H, s), 1 46 (9 H, m), 1 35 (9 H, s)

Wang et al

60

Trp-Ala-Gly-Gly-Asp@-BzL)OR (H)-Trp-Ala-Gly-Asp(fSBzL)-Resin

b

+

13 14a R = Rem

Fig 3 Solid phase approach to the synthesis of a cyhc prodrug of the model hexapeptide (a) 12, DCC/HOBt, (b) 50% TFA, (c) Bop-CL/DCM, (d) H,/Pd-C

3.2. Solid Phase Approach to the Synthesis of the Cyclic Prodrug 77 (Fig. 3) 3.2.7. Synthesis of the Linear Peptide 15 A typical synthesis uses Wang’s alkoxy resin (Bachem Btoscience) (14) Fmoc protected ammo acids were used for the solid phase peptide synthesis unless otherwise indicated (15). The couplmg of the first ammo acid (p-benzyl-asparttc actd) requires direct acttvation of the carboxyl groups with DIC for optimal yields The couplmg of subsequent ammo acids used the DIC/HOBt method, and the reaction was monitored by the nmhydrm test (161, as well as by the weight of the resin The final cleavage of the peptide from the resin was accomplished by treatment with 50% TFA/DCM. A TYPICAL COUPLING PROCEDURE 1 Mrx 3.6 mL of a 0 63 M solution of DIC m DCM and 1 1 g (3.4 mmol) of BocAsp@-OBzl)-OH, and stir for 20 mm 2 Add this solution to 0 50 g of Wang’s resin (0 95 mmol/g), and react for 2 5 h 3 Dram the reaction solution, and wash the resin with DMF (20 mL x 2) 4 Mix 0 55 g of Boc-Asp@-OBzl)-COOH and 1 8 mL of 0 63 M DIC m DCM for 10 mm 5 Add this solution to the resin and react for 2 5 h 6. Dram the reaction solution and wash the resin with DMF (20 mL x 4) and DCM (30 mL x 3)

A TYPICAL DEPROTECTION PROCEDURE 1. Add 20 mL of 20% pipendme m DCM to the resin (0.5 g scale), and react for 20 mm 2 Dram the solution, add 20 mL of 20% pipendine m DCM to the resin, and react for 5 nun

Propionic Acid PromoIety

61

3 Dram the solution, and wash the resm with DMF (20 mL x 3) and DCM (20 mL x 3). 4 The nmhydrm test shows dark blue color (see Note 4) CLEAVAGE OF THE PEPTIDE FROM THE RESIN AND Boc DEPROTECTION

1 Add 10 mL of 50% TFA m DCM to the resin 14a (100 mg scale), and react for 30 mm 2 Dram the TFA solution, and add 10 mL of 50% TFA in DCM, and react for 30 mm 3 Combine the TFA solution, and evaporate the volatile components under reduced pressure with a rotoevaporator to give linear peptlde 15 4 ‘HNMR(CD,OD) 754(1 H,d),7.31 (6H,m),7 10(1 H,m),702(1 H,m), 6 98 (1 H, b), 6.81 (1 H, b), 6.58 (1 H, b), 5.10 (2 H, s), 4 83 (1 H, t), 4 54 (1 H, t), 4 37 (1 H, m), 4.15 (1 H, m), 3 88 (2 H, b), 3.61 (2 H, b), 3 17-2 89 (4 H, m), 263(2H,s),243(3H,s),219(3H,s),171(3H,d),1.46(6H,s),121(3H,d)

3 2.2. Cyck Peptide with Aspartk Acid Protection 16 1 Add 10 mL of anhydrous DMF, 240 mL of anhydrous DCM, 100 mL (0 9 1 mmol) of NMM, and 158 mg (0.62 mmol) of Bop-Cl to 94 mg (0.11 mmol) of linear peptlde 15. Stir the reactlon solution at RT for 34 h. 2 Wash the reaction mixture with water (20 mL x 2) and dry over anhydrous MgS04 for 2 h. 3 Purify the cyclic peptlde with HPLC (reversed-phase, 70% methanol m water) to give 7 mg of a pure cychc peptlde 4 ‘H-NMR (CDCl,). 6 5-7 6 (19 H, NH), 5 14 (2 H, m), 4 82 (1 H, m), 4.52 (1 H, m),438(1H,m),3.66-403(5H,m),2.88-3.11(4H),271(1H,d),251(3H, s), 2 40 (1 H, d), 2 17 (3 H, s), 1 5-l 7 (9 H, m), 1.13 (3 H, d)

3.2.3. Cyclic Peptide 77 1 Add 20 mL of absolute ethanol and 3 mg of Pd-C 10% (see Note 5) to 7 mg (8 2 pool) of the cychc peptlde 16 with benzyl protection of the P-carboxyl group of aspartlc acid Then stir the reaction at RT under 1 atm of hydrogen for 22 h 2 Filter the reactlon mixture, and wash the Pd-C with methanol (10 mL x 4) Wash the colorless residue left after solvent evaporation with chloroform. Dry the product under vacuum to give 3 5 mg of product. 3 ‘H-NMR (CD,OD): 6.5s7.51 (7 H), 4 84 (1 H, m), 4 40-4 62 (2 H, m), 3 6O4 23 (5 H, m), 2.78-3 20 (4 H, m), 2 44 ( 1 H, d), 2 36 (3 H, s), 2 18 (3 H, s), 2 04 (1 H,d), 1 l&l 70(12H,m)

3.3. Solution Phase Approach of the Cyclic Prodrug 17

to the Synthesis

In the solutlon phase approach (Fig. 4), linear pentapeptide 18 was prepared usmg standard Boc-ammo acid chemistry (16,17). The key to this solutlonphase approach was the selective protection of the a-and P-carboxyl groups of the Asp residue. We successfully used the trlchloroethyl (Tee) ester (18) pro-

tecting group for the a-carboxyl group of Asp residue. Compound 18 was

62

Wang et al. a_

Boc-Trp-Ala-Gly-Gly-Asp(OBzl)-OTce 18

b

14b, R = Tee

cd

*

H-Trp-Ala-Gly-Gly-Asp(OBzl)-OTce 19 15

e_

16 -

f

17

Fig 4 Solutron-phase synthetic approach to cychc prodrug 17. (a) TFA, CH&, (b) EDC, HOBT, NMM, (c) ZnIAcOH, (d) TFA, CH,Cl,, (e) Bop-Cl, NMM, DMAP, (f) H,/Pd-C. treated with 50% TFA tn DCM to gave 19 in quantttattve yield. Pentapepttde 19 was reacted with 12 tn the presence of EDC, HOBt and NMM to gave the fully protected linear hexapeptrde 14b m 68% yield. The protectmg groups were removed using zmc/AcOH and 50% TFA/DCM to provide 15 m 50% overall yield. Compound 15 was purified by preparative reversed-phase HPLC. Cychzatton was then accomplished by a standard high-dtlutton technique using BOP-Cl as an acttvatmg reagent (19) n-t the presence of NMM and DMAP to afford cychc pepttde 16 with Asp-j3-benzyl protectton m 15% yield Hydrogenolysis of the protected cychc pepttde provided the desired cychc prodrug 17 m quantttattve yield.

3.3. I. Boc-Asp(OBzl)-OTce 1 Dissolve Boc-Asp(OBzl)-OH (1 g, 3 mmol), 2,2,2-trrchloroethanol (0 4 mL, 3 mmol), and DMAP (0 18 g, 1 5 mmol) m DCM (30 mL). 2 Cool the solutton to 0°C To this cooled solution, add EDC (0 57 g, 3 mmol) 3 Stir the reaction mixture at 0°C for 3 h, and then at ambient temperature for 2 1 h 4 Filter out the precipitate, and dilute the filtrate with EtOAc (100 mL) 5 Wash the EtOAc layer successively wrth saturated NaHCOs (2 x 20 mL), H,O

(2 x 50 mL) and brine (20 mL) 6 Dry the EtOAc layer over anhydrous Na,SO,, and evaporate the organic solvent under reduced pressure to yield Boc-Asp(OBzl)-OTce (1 13 g, 83%) as a yellow or1 7 ‘H-NMR (CDCls) 1.45 (9H, s), 2.93 and 3 15 (2H, dd, J= 17 Hz and 4.5 Hz), 4 67 and4 75 (2H, dd, J= 12.3 Hz), 4 7-4 75 (lH, m), 5 13 (2H, s), 5 56 (lH, d, J = 9 Hz), 7 34-7 38 (5H, m)

3.3.2 H-Asp(OBzl)-OTce 1 Add TFA (5 mL) to a stn-red solutton of Boc-Asp(OBzl)-OTce

(1 g, 2 2 mmol) m

DCM (5 mL) Stir the reaction mixture at RT for 45 minutes. 2 Remove volatile components m the reaction mixture using a rotary evaporator

under vacuum 3 Trlturate and wash the residue with anhydrous

decantanon 4 Dry the solid H-Asp(OBzl)-OTce used m the next step without

Et,O, and isolate the solid by

(0 78 g, 100%) under vacuum The product 1s

further purttication

MS (FAB)

354 (M+ + 1)

Propionic Acid Promoiety

63

3.3.3. Boc-Ala-G/y-G/y-Asp(OBzl)-OTce 1. Add EDC (0 99 g, 5 16 mmol) m one portion to a cooled (O’C) and stirred solutron of Boc-Ala-Gly-Gly-OH (1 6 g, 5 13 mmol), Asp(OBzl)-OTce (2.4 g, 5.13 mmol), HOBT (0.69 g, 5.13 mmol), and NMM (0.5 mL, 5.13 mmol) m DCM (100 mL) Stir the reaction mtxture at 0°C for 4 h and at ambient temperature for 24 h. Dilute the reaction mrxture wtth DCM (250 mL). Wash the solutton successrvely with 10% aqueous citric acrd (2 x 50 mL), Hz0 (100 mL), saturated NaHCO, (2 x 50 mL), Hz0 (100 mL) and brine (50 mL) Dry the organic layer over anhydrous Na2S04, and evaporate the solvent under reduced pressure to furnish Boc-Ala-Gly-Gly-Asp(OBzl)-OTce (2 8 g, 86%) as a pale yellow oil 6 ‘H-NMR (DMSO-d,): 1 17 (3H, d, J = 7.2 Hz), 1.37 (9H, s), 2 85 and 2 97 (2H, dd, J= 17 Hz and 6 2 Hz), 3 71-3 79 (3H, br), 3.98 (2H, br), 4 86 (2H, d, J = 4 3 Hz), 5.12 (2H, s), 7 01 (lH, d, J= 6.5 Hz), 7 33-7 37 (5H, br), 8.07 (2H, d, J= 6 Hz), 8.54 (lH, d, J= 7.8 Hz).

3.3.4. H-Ala-G/y-G/y-Asp(OBzl)-OTce 1 Dissolve Boc-Ala-Gly-Gly-Asp(OBzl)-OTce (0 29 g, 0 44 mmol) in DCM (5 mL), and cool the solutton to 0°C 2. Add TFA (5 mL) to the above clear solutton and stir the reaction mtxture at RT for 1 h 3 Remove volatde components m the reactton mixture using a rotary evaporator under vacuum 4 Triturate and wash the residue with anhydrous Et,O, and isolate the solid by decantanon 5 Dry H-Ala-Gly-Gly-Asp(OBzl)-OTce (0.27 g, 95%) under vacuum. The product IS used as is m the next step 6 ‘H-NMR (DMSO-d6)* 1 36 (3H, d, J = 7 2 Hz), 2 85 and 2 98 (2H, dd, J = 17OHzand6OHz),37&401 (6H,m),486(2H,d,J=4 1 Hz),5 12(2H,s), 7 36-7 37 (5H, br), 8 09 (2H, br), 8 24 (lH, br), 8.64 (IH, d, J= 7 2 Hz)

3.3.5. Boc-Trp-Ala-G/y-G/y-Asp(OBzl)-OTce

18

1 Add EDC (0.99 g, 5.16 mmol) in one portron to a cooled (O’C) stirred solutton of H-Ala-Gly-Gly-Asp(OBzl)-OTce (2.2 g, 5.13 mmol), HOBT (0.69 g, 5 13 mmol), Boc-Trp (1 56 g, 5 13 mmol), and NMM (0.5 mL, 5 13 mmol) m DCM (100 mL) 2 Stn the reaction mrxture at 0°C for 4 h and at ambrent temperature for 24 h 3 Dilute the reaction mixture wtth DCM (250 mL), and wash the solutton successively with 10% aqueous crtrtc acrd (2 x 50 mL), HZ0 (100 mL), saturated NaHCO, (2 x 50 mL), H,O (100 mL) and brine (50 mL) 4. Dry the organic layer over anhydrous Na,SO,, and concentrate the solutton under reduced pressure to furnish Boc-Trp-Ala-Gly-Gly-Asp(OBzl)-OTce (3 6 g, 86%) as a pale yellow oil 5 ‘H-NMR (CDCI,): 1.21 (3H, d, J= 7.2 Hz), 1 43 (9H, s), 2.99 and 3.17 (2H, m), 3.23 (2H, d, J= 6 8 Hz), 3 52-3 59 (lH, m), 3.73-3 89 (lH, m), 3 97 (2H, d, J=

64

Wang et al 5 9 Hz), 4 234 27 (lH, m), 4 41-4.48 (IH, m) 4 75 (lH, d, J= 11 8 Hz), 4 41 (lH,d,J= 11 8Hz),5.02-5 09(lH,m),5 14(2H,s),6,25(lH,m),649(lH,m), 7 04-7 40 (9H, m), 7 64 (lH, d, J= 7 4).

3.3 6. H-Trp-Ala-G/y-G/y-Asp(OBzl)-OTce

79

1 Dissolve Boc-Trp-Ala-Gly-Gly-Asp(OBzl)-OTce (0 29 g, 0 44 mmol) m DCM (5 mL), and cool the solutton to 0°C 2 Add TFA (5 mL) to this clear solution, and star the reactton mixture at RT for 1 h 3 Remove volatile components m the reaction mixture using a rotary evaporator under vacuum 4. Trtturate and wash the residue with anhydrous Et,O, and Isolate the solid by decantatton 5 Dry H-Trp-Ala-Gly-Gly-Asp(OBzl)-OTce (0 3 g, 95%) under vacuum to remove residual Et,O, and use the crude material as 1s m the next step

3.3.7. Boc-Ala-(Prom)-Trp-Ala-G/y-G/y-Asp(OBzl)-OTce

74b

1. Add EDC (0.09 g, 0 44 mmol) to a cooled (O’C ) stnred solutton of Boc-Ala(Prom)-OH 12 (0 17 g, 0 44 mmol), H-Trp-Ala-Gly-Gly-Asp(OBzl)-OTce (0 32 g, 0 44 mmol), HOBT (0 6 g, 0 44 mmol), and NMM (0 09 mL, 0 89 mmol) m DCM (50 mL) 2 Stir the mixture for 2 h at 0°C and for 30 h at ambient temperature 3 Dilute the reactton mtxture with DCM (250 mL), and wash the solutton successively wtth 10% aqueous cttrtc acid (2 x 50 mL), H,O (100 mL), saturated NaHCO, (2 x 50 mL), H,O (100 mL), and brine (50 mL) 4 Dry the organic layer over anhydrous Na$SO,, and concentrate the solutton under reduced pressure to hnnish an 011of Boc-Ala-(Prom)-Trp-Ala-Gly-Gly-Asp(OBzl)OTce (0 33 g, 68%) 5 Analyze the pepttde by an analyttcal reversed-phase HPLC usmg a C-l 8 column (flow rate 1 mL/mm), eluting with a gradtent of solvent A. 0 1% TFA/H,O*5% ACN and solvent B ACN The retentton time of the linear pepttde 1s 16 85 mm ‘H-NMR (CDCl,) 0 99 (3H, d, J= 6.7 Hz), I 38 (9H, s), 1 49-l 72 (9H, m), 2.11 (3H, s), 2 43-2 71 (2H, m), 2 98-3 17 (4H, m), 3.71-382(1H,m),392-402(4H,m),411-4.18(1H,m),433(1H,t,J= 6 8 Hz), 4 534 71 (3H, m), 5 13 (2H, s), 6 39 (2H, s), 6 71 (2H, s), 6 95 7 53 (9H, m), 8 32 (lH, s)

3.3.8. H-Ala-(Prom)-Trp-Ala-G/y-G/y-Asp(OBzl)-OH

15

1 Add Zn dust (1 g) over 1 h to a stnred solutton of Boc-Ala-(Prop)-Trp-Ala-GlyGly-Asp(OBzl)-OTce (0 22 g, 0 2 mmol) m AcOH (50 mL) 2 After stnrmg the reaction mtxture for 24 h at RT, filter out the msoluble material, and concentrate the filtrate under reduced pressure to give an oily restdue 3 Dissolve the residue m DCM (10 mL), and cool the solutton to 0°C 4 Add TFA (5 mL), phenol (2 g), and ethanedtthtol(0 2 mL) to this clear solutton

PropionIc Aad Promorefy

65

5 After stirrmg at room temperature for 2 h, evaporate DCM and trrturate the resldue with anhydrous Et,0 6. Wash the solid with anhydrous Et,O, remove the ether by decantanon, and dry the remnant under reduced pressure to afford H-Ala-(Prom)-Trp-Ala-Gly-GlyAsp(OBzl)-OH (0 09 g, 50%) 7. Purrfy the product further by preparattve reversed-phase HPLC using a C- 18 column (flow rate 5 mL/mm), and elutmg with a gradient of solvent A. 0 1% TFA/ Hz0 5% ACN and solvent B. ACN. 8 Analyze the peptide by analytical reversed-phase HPLC using a C- 18 column (flow rate 1 mL/mm) and elutmg with a gradient of solvent A. 0 1% TFA/ H,O:5% ACN and solvent B. ACN. The retention time of linear peptrde IS 14 97 mm 9 Cychze linear pepttde 15 following procedure 3.2.2 to grve 16. 10 Cleave the benzyl protectmg group of aspartrc acrd followmg procedure 3.2.3 to give 17

3.4. Chemical and Enzymatic Stability of Cyclic Prodrug

17

A fundamental requirement for the prodrug approach to be successful IS reliable converston to the parent drug by either enzyme-catalyzed or nonenzymattc reactrons The cyclic phenylpropiomc acid prodrug 1s designed to release the lmear hexapeptide by an esterase-mediated hydrolysis of the ester bond followed by a fast chemical step, resulting m the release of the parent peptide and formatton of a lactone (Fig. 1). In Hanks’ balanced salt solution (HBSS), a physrologrcal buffer system, pH 7.4, cycbc prodrug 17 degrades quantitatively to the linear hexapepttde and the lactone Mass balance was achieved 294.8% for the lmear hexapeptrde and 285.5% for the lactone, respectively (20). Apparent half-lives (t,,.J calculated from pseudo first-order rate constants were tr, 2 = 1795 + 289 min for the dtsappearance of cyclrc prodrug 17 and t1,2 = 1834 +_ 296 mm for the appearance of the parent pepttde. Formation of the lactone was kinetrcally equivalent to the disappearance of the cyclic prodrug (t,,, = 2180 + 370 mm) suggesting that the rate-limrtmg step m the cascade of reactions leading to the release of the lmear hexapeptide IS indeed the hydrolyses of the ester moiety. In 90% human plasma (tl,Z = 508 + 24 min), the disappearance of cyclic prodrug 17 was significantly faster than that determined in HBSS, pH 7.4 (tI,2 = 1795 +_289 mm). Preincubation of this blologrcal medium with paraoxon, a potent esterase mhtbltor, resulted In a substantially slower disappearance of cyclic prodrug 17 (tt,* = 1729 I!I 245 min). This Implies that the linear hexapeptrde IS released from cyclic prodrug 17 by an apparent esterasemediated reaction.

66

Wang et al

3.4.1. Stabihty of Cyclic Prodrug 77 In HBSS, pH 7.4 1 Preparesoluttons(500 uL) of cyclic prodrug 17 (-18 pg/mL) m HBSS,pH 7.4. 2 Maintarn samplesat 37 0 + 0 5°C m a temperature-controlledshakingwater bath (60 rpm> 3 Remove 20 uL samplespertodically, and analyzethem unmedtately by HPLC (analystscondtttonsseeNote 6). 3.4 2. Enzymabc Stabihty of Cychc Prodrug 17 rn 90% Human Plasma Follow experimental procedures descrtbed in Chapter 3, Subheading 3.4.2. 3.5. Transport Properties of Cyclic Prodrug 77 Across Biological Membranes The cell permeatton charactertsttcs of cycltc prodrug 17 and the lmear hexapepttde were assessed across Caco-2 cell monolayers, an m vttro cell culture model of the intestinal mucosa (20). Caco-2 cells, that spontaneously undergo dtfferentratlon into confluent monolayers have been shown to exhibit both the physical and metaboltc barrier properties of mtestmal mucosal cells to peptides (21-23). When applted to the aptcal side (1 e , lummal side) of the cell monolayer, the linear hexapepttde rapidly dtsappears (tt12 = 14 mm), suggesting high suscepttbiltty to enzymatic degradation by pepttdases (24) Since the actual transport of thts hexapeptlde to the basolateral (1.e , serosal) compartment was not detected, the apparent permeability coeffrctent (P,,,) of the linear model hexapepttde was estimated to be less than 1.7 x 1Op9cm/s based on the senstttvrty of our analytical methods (2#J. In contrast, cycbc prodrug 17 was stgmftcantly more stable agamst metabolic degradation. After a 3-h incubation pertod, 103 f 6.3% of the prodrug mlttally applied to the apical side of the cell monolayers was still present (20). The Pappvalue calculated for cycltc prodrug 17 was 1.2 1 -I 0.12 x 1O-’ cm/s, indicating that cycltc prodrug 1s at least 71 times more able to permeate this model of the mtestmal cellular barrier than IS the linear hexapeptrde. 3.5 1. Transport of Cyclic Prodrug 77 Across Caco-2 Cell Monolayers Follow experimental procedures described m Chapter 3, Subheading 3.5.1. 3.6. Discussion We have described a general strategy for preparing a cychc prodrug of a model hexapepttde via the N- and C- terminal ends by uttltzmg a unique “trimethyl lock”-facilitated lactonizatlon system However, it should be feasible to use thts methodology to cyclize other brologically active pepttdes by lmkmg the C-termmal carboxyl group to a side chain ammo (e g., Lys, Arg) or

Propionic Acid Promolefy

67

hydroxyl (e.g., Ser, Thr, Tyr) group, or linking a side chain carboxyl group (e.g., Asp, Glu) to a side chain amino (e.g., Lys, Arg) or hydroxyl (e.g., Ser, Thr, Tyr) group. Apphcatton of this methodology to biologically active peptides (e.g., opiotd pepttdes) and pepttde mtmettcs (e.g., RGD analogs) IS currently under mvestigatton. 4. Notes 1 Grind large crystals before washing 2 Handle LiAlH4 with care because contact with moisture may cause fire. 3 The BOC-Ala group attached to the phenol hydroxyl group can migrate to the primary hydroxyl group durmg column chromatography. This is the reason that 10 was used for oxidation without further purification 4 For a detailed description of the nmhydrm test see. Soled Phase Peptlde Syntheszs, Stewart, J M and Young, J D (1984) Pierce Chemical, Rockford, IL, pp 105,106 5. Pd-C has a tendency to catch tire when dry Wet Pd-C with the reaction solvent as soon as it is taken out of the origmal container 6 Elute the peptides at the flow-rate of 1 mL/mm using a gradient from 11 6 to 90.0% (v/v) ACN m water with TFA (O.l%, v/v) as the ion-panmg agent. Momtor the eluent with a fluorescence detector at emission h = 345 nm (excitation h = 285 nm)

References 1. Ollyai, R and Stella, V J ( 1993) Prodrugs of peptides and proteins for improved formulation and delivery Annu Rev Pharmacol Tox~ol 32, 521-544 2 Taylor, M D. and Amidon, G. L (ed. ) (1994) Peptlde-Based Drug Design Controllzng Transport and Metubolzsm American Chemical Society, Washmgton, D C 3. Gangwar, S., Paulette, G. M , Siahaan, T. J., Stella, V. J., and Borchardt, R. T. (1999) Synthesis of an esterase-sensitive cyclic prodrug of a model hexapeptide having enhanced membrane permeability and enzymatic stability using an acyloxyalkoxy promoiety. Meth Mol Blol Vol 23, Peptldomzmetzcs Protcols (Kazmierski, W. M , ed ), Humana Press, Totowa, NJ, pp 37-51. 4 Borchardt, R T. and Cohen, L A. (1972) Stereopopulation control III. Facihtation of mtramolecular coqugate addition of the carboxyl group. J Am Chem Sot 94,9175-9182 5 King, M M and Cohen, L A (1983) Stereopopulation control. 7 Rate enhancement in the lactomzation of 3-(o-hydroxyphenyl)propionlc acids dependence on the size of aromatic ring substituents J Am Chem Sot 105, 2752-2760 6 Milstem, S. and Cohen, L. A. (1972) Stereopopulation control. I. Rate enhancement m the lactomzation of o-hydroxyhydrocinnamic acids J Am Chem Sot 94,9158-9165

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7 Wang, B , Nicolaou, M G , Liu, S , and Borchardt, R T (1996) Structural analySISof a facile lactomzation system facthtated by a “trtmethyl lock ” Broorg Chem 24,3949 8. Amsberry, K. L. and Borchardt, R. T. (1990) The lactomzatton of 2’-hydroxyhydrocmnamtc acid amides potential prodrugs for amines. J Org Chem 55, 5867-5877 9 Amsberry, K. L , Gerstenberger, A L , and Borchardt, R T (1991) Amme prodrugs which utthze hydroxy amide lactomzatton II A potential esterase-sensmve amide prodrug Pharm Res 8,45546 1 10 Ueda, Y , Mtkktlmem, A B., Kmp, J 0 , Rose, W C , Casazza, A M , and Vyas, D M. (1993) Novel water soluble phosphate prodrugs of taxol possessing zn vlvo antttumor actlvny Bzoorg Med Chem Lett 3, 1761-1766 11 Wang, B , Lm, S , and Borchardt, R T (1995) Development of a novel redoxsensitive protectmg group for ammes which utthzes a facthtated lactomzatton reaction J Org Chem 60,539-543 12 Raetsst, S and Audus, K L (1989) Permeabthty of delta sleep-mducmg pepttde through monolayers of bovine brain mtcrovessel endothehal cells J Pharm Pharmacol 41,848-852 13 Gray, R , Vander Velde, D., Burke, C. J , Mannmg, M , Mlddaugh, C R , and Borchardt, R T (1994) Delta sleep-mducmg pepttde: solutton conformatlonal studtes of a membrane-permeable peptide Blochemzstry 37,293-304 14 Wang, S. S (1973) p-Alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydraztde resin for solid phase synthesis of protected pepttde fragments J Am Chem Sot 95, 1328-1333. 15 Atherton, E and Sheppard, R C (1989) Soled Phase Peptlde Syntheses A Practlcal Approach IRL, New York 16 Stewart, J M and Young, J D (1984) Solrd Phase Peptzde Syntheses Pierce Chemical, Rockford, IL. 17 Bodanszky, M and Bodanszky, A (1984) The Practice of Peptlde Syntheses Sprmger Verlag, New York 18 Greene, T W and Wuts, P G M (199 1) Protective Groups VI Organic Syntheses John-Wiley, New York, pp 240-24 1. 19 Tung, R. D and Rich, D H (1985) Bis(2-oxo-3-oxazohdmyl) phosphmtc chloride (1) as a couplmg reagent for N-alkyl ammo acids J Am Chem Sot 107, 4342-4343 20. Paulette, G M , Gangwar, S , Wang, B., Stahaan, T J , and Borchardt, R T (1996) Esterase-sensitive cychc prodrugs of pepttdes. evaluation of a phenylproptomc acid promoiety m a model hexapepttde. Pharm Res 14, 1 l-l 7 21 Wtlson, G , Hassan, I F., Dtx, C J , Wtlhamson, I , Shah, R , and Mackay, M (1990) Transport and permeabthty properties of human Caco-2 cells an zn vztro model of the mtestmal eptthehal cell barrier J Control Release 11,25-40. 22 Pinto, M., Robme-Leon, S., Appay, M.-D , Kedmger, M., Tradou, N , Dussaulx, E , Lacrotx, B , Stmon-Assmann, P , Haffen, K , Fogh, J , and Zweibaum, A

Propionic Acid PromoIety

69

(1983) Enterocyte-hke differentiation and polarization of the human colon carcinoma cell lme Caco-2 m culture Bzol Cell 47,323-330. 23 Arthursson, P (1990) Epithehal transport of drugs m cell culture I* A model for studying the passive diffusion of drugs over intestmal absorptive (Caco-2) cells J Pharm Scl 79,47&482

24 Paulette, G M , Gangwar, S , Okumu, F. W., Siahaan, T. J., Stella, V. J , and Borchardt, R T (1996) Esterase-sensitive cyclic prodrugs of peptides evaluation of an acyloxyalkoxy promoiety m a model hexapeptide. Pharm Res 13,16 13-162 1

5 Synthesis of Coumarin-Based, Esterase-Sensitive Cyclic Prodrugs of Opioid Peptides with Enhanced Membrane Permeability and Enzymatic Stability Binghe Wang, Daxian Shan, Wei Wang, Huijuan Zhang, Olafur Gudmundsson, and Ronald T. Borchardt 1. Introduction With the discovery of an increasing number of biologically active pepttdes and peptide mimetics (1-3), there is a pressmgneed for the development of strategiesto deliver thesebiologically active compoundsto the desired siteof action.The preceding two chapters have described two methods of making esterase-sensitivecychc prodrugs of peptides. In this chapter,we wish to describe a third method of making esterase-sensitivecychc prodrugs of peptides using DADLE, an opioid peptide (4 7), as an example. The design takesadvantage of the facile cyclization reaction of coumarmlc acid and its denvatlves (8,9). Becauseof the presenceof a 2-phenohc hydroxyl group and the as-geometry of the double bond, coumarmic acid and derivatives can undergo a facile lactonlzatlonwith an effective molar@ of 3.7 x 10” (8-11). Sucha systemhas been used for the development of esterase-sensitiveprodrugs of amlnes (IO) This coumarmic acid moiety can also be used as a lurker for the preparation of esterase-sensitive cychc prodrugs of peptides and peptide mimetics. The basic design principle is shown m Fig. 1, where the C- and N-termmal ends of a linear peptide can be masked by forming an ester and amide bonds with the phenol hydroxyl and side chain carboxyl groups of the lurker, respectively. The coumarm-based prodrug systemhas several unique features. First, the final product of the promoiety after the drug is released is coumarm, of which the toxicity has been extensively studied, and it was found to be relatively nontoxic (12-21). The availabihty of the toxicity data of coumarm eliminates one major uncertainty with the clinical development of any prodrug moiety SecFrom

Methods !n Molecular Medmne, Vol 23 Peptrdommehcs Edlted by W M Kazmlerskl @Humana Press Inc , Totowa,

71

Protocols NJ

Wang et al.

72

‘OOC-Peptide-NH,+

Fig 1 The destgn of an esterase-senstttve cychc prodrug system for pepttdes and peptide mimetics

ond, the synthesis of coumarm-based prodrugs starts from a coumarm, readily available mexpensive material. Third, the coumarm-based prodrugs, because of their structural differences compared to the “trtmethyl lock”-based prodrug systems (see Chapter 4), are expected to have somewhat different release ktnettcs and enzyme (esterase) spectfictty, which would complement the “trtmethyl lock”-based prodrug system m terms of potential apphcattons

2. Materials ‘H-NMR spectra were obtained at 300 MHz. All ‘H chemical shifts are reported m ppm relative to the internal standard of tetramethylstlane (TMS, 6 0 00). All protected ammo acids were obtained from BACHEM Bioscience Inc. All solvents were purchased from Fisher Scientific Co. All other chemical reagents were purchased from Aldrich Chemical Co.

2.1. Reagents for Method 3. I 1-Hydroxybenzotriazole (HOBt) 4-Dimethylammopyrrdme (DMAP) 1,3-Dicyclohexylcarbodllmlde (DCC) Acetic acid Coumarin Ethyl acetate Ethyl ether 8 H202 (30%). 9 Hexanes 10 Hydrochloric acid (HCI, 36%) 2. 3 4. 5 6.

Synthesis of Coumarin-Based 11 12 13. 14 15 16 17 18 19 20 21 22

LlAlH, Manganese (IV) oxide Methanol (MeOH). Methylene chloride (CH,Cl,). MgSO, N-Boc-L-Leu H,O NaHCO, Silica gel Sodium chlorite Sodium sulfide tert-Butyld~methyls~lylchlorlde Tetrahydrofuran (THF)

2.2. Reagents 1 2 3 4. 5 6 7 8 9 10 11 12 13. 14 15

Cyclic Prodrugs

(TBDMS-Cl)

for Method 3.2

10% Palladium on activated carbon (10% Pd-C) 4-Dlmethylammopyndme (DMAP) 1,3-Dlcyclohexylcarbodllmlde (DCC) CH,Cl, Ethyl acetate. Hexanes Hydrochloric acid (HCl, 36%) Hydrogen gas L-Phenylalamne-t-butyl ester HCl (H-Phe-0-t-Bu MeOH MgSO, N-Cbz-Glycme (N-Cbz-Gly) NaHCO,. THF Trlethylamme (TEA)

HCl)

2.3. Reagents for Method 3.3 1 2 3 4 5 6 7 8 9 10. 11 12 13

1-Hydroxybenzotnazole (HOBt) 4-Dimethylammopyndme (DMAP) 1,3-Dicyclohexylcarbodilmide (DCC). Bls(2-oxo-3-oxazolidmyl)-phosphmlc chloride (Bop-Cl) Cltrlc acid Ethyl acetate MeOH MgSO, N,N-Dlmethylformamlde (DMF) NaHCO, Tetrahydrofuran (THF) Trlethylamme (TEA) Trlfluoroacetlc acid (TFA)

73

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Wang et al

2.4. Reagents

for Method 3.4

1 (NH&-Q 2 3 4 5 6 7

De-ionized water DMSO Dry ice MeOH Phosphate buffer Porcine liver esterase

2.5. Reagents for Method 3.5 See Subheading

2.5. of the preceding

chapter

2.6. HPLC Columns Reversed-phase 25cmx46mmID.)

analytical

column: Vydac C- 18 column (particle size* 5 urn,

3. Methods The synthetic approach to the coumarm-based, cychc prodrug of DADLE can be divided mto three parts. 1) the synthesis of the lmker with the first ammo acid attached 9, 2) the synthesis of the linear peptide 16, and 3) the formation of the final cychc prodrug 18. Owing to the facile cyclization of coumaruuc acid derivatives 2 (Fig. l), the mcorporation of this promoiety mto the cychc system first requires the protection of the phenollc group. Therefore, a key intermediate for the successful synthesis of the cychc prodrugs is the promoiety with the appropriate ammo acid attached 9 (Fig. 2)

3.7. Synthesis

of the Key Intermediate

with Boc-D-Leu

Attached

Starting from commercially available coumarm, 2-(Z-3’-tert-butyldimethylsdyloxyl- 1‘-propenyl)-phenol5 can be prepared in two steps through reduction and selective protection of the primary hydroxyl functional group (Fig. 2) (I&12) The couplmg of the free phenol hydroxyl group with the carboxyl functional group of Boc-D-Leu can be accomplished by using DCC as the couplmg reagent m the presence of DMAP (22). The free allyhc hydroxyl group after cleavage of the stlyl group using acetic acid can be converted to the carboxyl group m a two-step oxidatron to give 9 (23) The promoiety with the appropriate Boc-D-Leu attached 9 can then react with the linear peptide prepared

3.1.1. Z-3-(2’-Hydroxyl)phenyl-2-propen-

l-01 4

1 In a round-bottom flask, suspend LIAIH, (see Note 1) (0 76 g, 80 mmol of hydride) in 20 mL of anhydrous ethyl ether.

Synthesis of CoumarvM3ased

Cyck Prodrugs

75

Fig 2 Preparation of the key Intermediate. LIAIH,; b TBDMS-Cl, DMAP, c BocD-Leu, DCC, DMAP; d HOAc, THF, H,O, e MnO,; f NaClo,, H,O, Dilute 1.76 g (55 mmol) of methanol with 10 mL of dry ethyl ether Dropwtse add the solutton mto the suspension of LtA1H4 with stu-ring at 0°C Transfer the mixture (see Note 2) mto the flask charged with coumarm (1 46 g, 10 mmol) m 10 mL of ethyl ether with strrring at 0°C Continue stn-rmg for 30 min, and then remove the ice bath Stir the mixture for another 30 mm at RT Pour the reaction mixture mto 50 g of ice containing 5 mL of concentrated hydrochlortc acid (see Note 3), and extract the product with ether (30 mL x 3) Wash the combined ether layers with cold water (30 mL x 3), and dry the organic extracts over MgSO, Solvent removal gives a residue. Separate the residue on a slhca gel column with hexanes/EtOAc (2: 1, v/v) as the eluent to provide 0 86 g (59%) of white crystals. ‘H-NMR (CDCl,) 6 7.186 77 (4H, m), 6.63 (lH, d, J= 11 7 Hz), 5 80 (lH, dt, J=11.7 Hz, 6 6 Hz), 4.26 (2H, d, J=6.6 Hz) 3.1.2.

2-(Z-3’-tert

-Butyld~methyLsilyloxyl- 1‘-propenyl)-phenol5

1. To the sttrred solution of Z-3-(2’-hydroxyl)phenyl-2-propen-l-01 4 (3.87 g, 25 8 mmol) m 40 mL of dry THF, add a solution of TBDMS-Cl (4 28 g, 28.4 mmol) m 30 mL of dry THF at 0°C followed by a dropwrse addmon of DMAP (4.73 g, 38 7 mmol) m 50 mL of dry THF 2. After stirring for 14 h at 0°C filter the mixture and evaporate the filtrate zn vacm Then, add ethyl acetate (50 mL) mto the residue 3. Wash the resultmg mixture with IN HCl solution (30 mL x 2), 5% NaHCOt solution (25 mL) and water (25 mL) Then dry over anhydrous MgSO,. 4 Filter and remove the solvent on a rotary-evaporator at reduced pressure to gave 7 63 g of a yellow oily product 5 Purify the crude product on a s&a gel column with ethyl acetate/hexane (1.15, v/v) as eluent to afford 5 63 g (83%) of a white solid. 6 ‘H-NMR(CDCl,).67.21-6.87(4H,m),6.52(1H,d,J= 11.7Hz),6.04( lH,m), 4.18 (2H, dd, J= 7 2 Hz, 0.9 Hz), 0 90 (9H, s), 0 07 (6H, s)

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Wang et al

3.1.3. N-Boc-D-Leu-2-(Z-3’-tert-Butyldimethyk~lyloxylphenyl Ester 6

1‘-propenyl)-

1. To a solution of?&Boc-D-Leu HZ0 (1.43 1 g, 6 95 mmol) m 65 mL of dry methylene chloride at 0°C add DCC (1 730 g, 6 95 mmol) (Note 4) 2 After stirrmg for 5 mm, add compound 5 (1 530 g, 5 80 mmol) and DMAP (0 707 g, 5 80 mmol) Continue stn-rmg for 2 h at 0°C and 3 h at RT 3 Evaporate the mixture to 25 mL, and store m a freezer (-18’C) for 30 mm Remove the white precipitate dicyclohexylurea (DCU) by filtration 4 Dilute the filtrate with methylene chloride to 80 mL and wash the solution with saturated NaHCO, (30 mL x 2), 1 NHCl(30 mL x 2 ) and brme (30 mL), and dry over MgSO, 5 Evaporation zn vucuo gives an oil. Purify the oil on a sihca gel column with EtOAc/Hexane (1:5, v/v) as eluent to afford 2.7 10 g of a viscous product (98%) 6 ‘H-NMR (CDCl,) &726707(4H,m),639(1 H,d,J= 11 7),589(1 H,m), 4 53 (1 H, m), 4 30 (2 H, d,J= 6 0 Hz), 1.79 (2 H, m), 1 64 (1 H, m), 1 46 (9 H, s), 101 (6H,d,J=6OHz),O89(9H,s),O03(6H,s)

3.1.4. N-Boc-D-Leu

2-(Z-3’-hydroxyl-

1‘-propenyl)-phenyl

Ester 7

1 To a solution of 6 (3 33 g, 6 52 mmol) m 9 mL of THF, add a mixture of acetic acid (27 mL) and water (9 mL). Stir the resultmg mixture at RT for 4 h 2 Evaporate the solvent zn vucuo Dtssolve the residue m 100 mL of EtOAc and wash the solution with saturated NaHCO, (40 mL x 2), water (40 mL) and brme (40 mL) (see Note 5) 3 Dry the organic layer over anhydrous MgS04 Removal of the solvent affords an oil (2 332 g, 99%) 4 ‘H-NMR (CDCl,) 6 7 25-7 06(4 H, m), 6 44 (1 H, d, J= 11 1 Hz), 5 95 (1 H, m), 450(1 H,m),420(2H,d,J=63Hz), 179(2H,m), 163(1 H,m), 146(9H, s), 1 01 (6 H, d,J= 5 4 Hz)

3.1.5. 3-(2’-N-Boc-D-Leuanyloxy)pheny/-Z-propenal 1. Add activated manganese (IV) oxide (9.191 g) mto a solution of 7 (2 33 g, 6 42 mmol) m 20 mL of dry CH,Cl, m one hour intervals (during a period of 7 h) TLC (hexanes ethyl acetate = 2.1) shows all startmg material 7 has disappeared (see Note 6) 2 Filter the reaction mixture through silica gel, and then wash the silica gel with CH,Cl, (150 mL) Evaporate the solvent zn wcuo to afford 1 932 g of a slightly yellow residue (83%) that ISused m next stepwithout further purification 3 ‘H-NMR(CDC1,)*6982(1H,d,J=8 lHz),756-730(4H,m),7 17(1H,d,J =84Hz),621 (1 H,dd,J=g.lHz, 11 4Hz),449(1 H,m), 174(2H,m), 162 (1 H, m), 146 (9 H, s), 101 (6H, d, J= 5 4 Hz)

3.7.6. 3-(2’-N-Boc-D-Leuciny/oxy)phenyl-Z-propenoic

AC/d 9

1 Dropwise add a solution of sodmmchlorite (80%, 0 848 g, 7.50 mmol) m 7 5 mL of water withm 4 h into a stirred solution of aldehyde 8 (1 932 g) m acetomtrile

Synthesis of Coumann-Based N-CBZ-Gly

+ PheOBt-t

A

77

Cyclrc Prodrugs

N-CBZ-Gly-PheOBt-t

k

H2NGly-L-PheOBu-t 11 D-H2NAla-Gly-L-PheOBu-t

10

L

CBZ-N-D-Ala-Gly-L-Phe-OBut

11

12

13

-% CBZ-N-L-Tyr-D-Ala-Gly-L-PheOBu-t

A

L-H2NTyr-D-Ala-Gly-L-PheOBu-t

14

15

Fig 3. Preparation of L-HzN-Tyr-n-Ala-Gly-L-PheOBu-t a. DCC, DMAP;b H,, 10% Pd-C, c N-CBZ-D-Ala, DCC, DMAP; d N-CBZ-L-Tyr, DCC, DMAP (5 4 mL), water (2 2 mL), and 30% hydrogen peroxide aqueous solutron (0 63 mL) Keep the temperature below 10°C with an me-water bath during addition. When the reaction starts, oxygen evolves Continue stirring the reaction mixture for 2 5 h while keeping the temperature unchanged. Add sodium sulfide (2.8 g) into the reaction mixture Acrdificatron with IN HCI (to pH = 1) gives a slightly yellow solution Extract the solution with ethyl acetate (75 mL x 2). Wash the combined organic phases with water (40 mL) and brme (40 mL x 2), and dry the organic layer over anhydrous MgSO, Ftltratton and solvent evaporation afford a crude product (1 886 g) that 1srecrystallized m ethyl acetate and hexane (1.3 v/v) to give 1 4 1 g (62%) of a pale yellow solid product ‘H-NMR (CDCl,) 6 7.51-7 10 (4 H, m), 6.99 (1 H, d,J= 12.3 Hz), 6 05 (1 H, d, J= 12.3 Hz), 4 51 (1 H, m), 1.70 (3 H), 1.46 (9 H, s), 1.00 (6 H, d, J= 5 7 Hz)

3.2. Synthesis

of the Linear Peptide 15

The preparation of L-Tyr-D-Ala-Gly-L-Phe t-butyl ester 15 is shown m Fig. 3. L-Phe t-butyl ester IS coupled with N-CBZ-Gly m the presence of DCC

and HOBt to give N-CBZ-Gly-L-Phe t-butyl ester 10. Hydrogenolysrs of 10 produces Gly-L-Phe t-butyl ester 11, which IS coupled with N-CBZ-D-Ala m the presence of DCC and HOBt to produce N-CBZ-o-Ala-Gly-L-Phe t-butyl ester 12. N-CBZ group can be removed from 12 by hydrogenolysis using a palladium catalyst. Tripeptide 13 reacts with N-CBZ-L-Tyr to give N-CBZ-LTyr-o-Ala-Gly-L-Phe t-butyl ester 14. This protected tetrapeptide 14 IS hydrogenolyzed to provide 15. 3 2 1. N-CBZ-Gly-L-Phe

t-butyl ester 10

1 To a mixture of IV-CBZ-Gly (837 mg, 4.0 mmol) and HOBt (536 mg, 4 0 mmol) (see Note 7) m 30 mL of ethyl acetate chilled m an me-water bath, add DCC (906 mg, 4.4 mmol) m one portion Stir the mixture for 30 mm, and remove the ice bath Formatton of white precrprtate (DCU) is observed. 2 Add L-Phe-0-t-Bu (103 1 mg, 4.0 mmol) and TEA (556 pL, 4 0 mmol). Continue strrrmg the mrxture at RT for 2 h Pour 15 rnL of hexanes into the rmxture with shakmg.

78

Wang et al

3 Store the mixture m a freezer overnight. Remove DCU by fItratIon Wash the preclpltates on a fretted funnel with 10 mL of ethyl acetate 4 Wash the combined filtrates with 4% HCl(30 mL x 2), saturated NaHCO, solution (25 mL x 3) and brine (30 mL x 3), and dry the mixture over anhydrous MgSO, Filtration and solvent evaporation give 1 55 g (94%) of dlpeptlde 12

3.2.2. Preparation of G/y-L-fhe

t-Butyl Ester 71

1. Prepare a solution of N-CBZ-Gly-L-Phe t-butyl ester 10 (1540 mg, 3.74 mmol) m methanol (10 mL) (see Note 8) m a round-bottom flask equipped with a magnetic stirrer and a gas inlet-outlet tube (see Note 9) 2 Displace the air by a slow stream of nitrogen, and add 10% palladium-on-charcoal (100 mg) (see Note 10). Once again, pass a slow stream of nitrogen through the flask for a few mm, and then start the mtroductlon of a slow stream of hydrogen 3 Keep the catalyst m suspension by vigorous stirring. After being stirred for 2 h, the starting material disappears on TLC with ethyl acetate as eluent, and then stop the mtroduction of hydrogen. 4. Displace the remaining hydrogen by nitrogen, and remove the catalyst by filtration Wash the catalyst with methanol (5 mL), and store it under water until it 1s discarded or regenerated 5 Evaporate the filtrate 112V~CUOto give 1.028 g (99%) of a solid product.

3.2.3. N-CBZ-D-Ala-G/y-L-Phe-0-t-Bu

12

1 Cool a solution of N-CBZ-D-Ala (692 mg, 3 1 mmol) m 15 mL of dry CH,Cl, m an ice-water bath with stirring under N2 Add DCC (639 mg, 3 1 mmol) to the solution After 5 mm add HOBt (541 mg, 3 1 mmol) 2 Stir the mixture at 0°C for 10 mm and then add Gly-L-Phe-0-t-Bu 11 (860 mg, 3 1 mmol) and DMAP (378 mg, 3 1 mmol). After stirring at 0°C for 2 5 h and at RT for 4 h, store the mixture m a freezer (-20°C) for 0 5 h 3 Remove the white precipitate by filtration Dilute the combined organic filtrates to 150 mL with CH2C12, then wash with 10% citric acid (30 mL x 2), 5% NaHCO, (30 mL x 2) and brine (40 mL x 2), followed by drying over MgS04. 4. Filtration and evaporation furnish an oil Dissolve the residue m a mixture of 10 mL of ethyl acetate and 5 mL of hexane, and filter to get rid of DCU Evaporate the solvent to afford 1.481g of a clear oily product (99%)

3.2.4. D-Ala-G/y-L-Phe-0-t-Bu

13

1 Bubble N2 gas mto a solution of N-CBZ-n-Ala-Gly-L-Phe-0-t-Bu 12 (1 47 g) m 25 mL of methanol for 15 mm, and then add 250 mg of 10% Pd-C. 2 Once again, pass a slow stream of mtrogen gas and then introduce H2 into the reaction mixture. Stir the mixture vigorously at RT for 1.5 h. TLC (ethyl acetate) shows the starting material has disappeared 3 Remove the catalyst by filtration Collect the filtrate Evaporate the solvent to afford the desired product as a foam (0.969 g, 9 1%)

Synthesis of Coumann-Based

Cyclic Prodrugs

3 2.5. N-CBZ-L-Tyr-D-Ala-G/y-L-Phe-0-t-Bu

79

14

1 Chtll a solution of N-CBZ-L-Tyr (848 mg, 2 69 mmol) and HOBt (363 mg, 2 69 mmol) m 20 mL of dry THF in an ice-water bath with stnrmg under N, Add DCC (554 mg, 2 69 mmol) to the solution. 2. Stir the mixture at 0°C for 10 mm and then add o-Ala-Gly-L-Phe-0-t-Bu 13 (940 mg, 2 69 mmol) and DMAP (328 mg, 2.69 mmol). After sttrrmg at 0°C for 2 h and RT for 3 h, concentrate the mixture to 10 mL. Store the mixture m a freezer (-2O’C) for 0.5 h 3 Remove the white precipitate by filtration Dtlute the filtrates to 150 mL wtth CH2C12, and then wash the resulting mixture with 10% cttrtc actd (30 mL x 2), 5% NaHCOa (30 mL x 2), 10% citric acrd (30 mL) and brine (50 mL) Dry the organic layer over anhydrous MgS04. 4 Filtration and evaporatton furnish an 011.Redtssolve the residue m the mtxture of 15 mL of ethyl acetate and 5 mL of hexane, and filter to remove DCU Evaporate the solvent to afford a clear od(1 74 g, 100%).

3.2.6. L-Tyr-D-Ala-G/y-L-Phe-0-t-Bu

15

1 Bubble N2 gas mto a solutton of N-CBZ-L-Tyr-o-Ala-Gly-r.-Phe-0-t-Bu 14 (1.74 g) m 20 mL of methanol, and add 300 mg of 10% Pd-C. Flush the reaction flask with H, gas, and then connect a H2 balloon with the flask 2 Vigorously star the mixture at RT under H, for 1 5 h TLC (ethyl acetate) analysts shows the starting maternal has disappeared. Filter off the catalyst and then wash the catalyst with methanol (5 mL x 2) 3 Collect the filtrates, and evaporate the solvent to afford the desired product as a white solid (1 296 g, 94%)

3.3. Preparation

of the Final Cyclic Prodrug

18

The key rnterrnedtate 9 can be attached to the tetrapeptide 15 by usmg DCC as the activating reagent. Boc and t-butyl groups can be removed by TFA (24-

26). After deprotectlon, cychzation can be carried out using Bop-Cl as the activating

agent (27) to produce the final compound

18 (Fig. 4).

3 3.1. Linear Peptide 16 1 Cool a solution of 9 (226 mg 0.6 mmol) and HOBt (8 1 mg, 0.6 mmol) m 3 0 mL of dry THF m an ice-water bath under NZ. Add DCC (124 mg, 0.6 mmol). 2 After stnrmg for 10 mm, add a solution of tetrapepttde 15 (257 mg, 0 5 mmol) and DMAP (73 mg, 0 6 mmol) m dry THF (5.0 mL) Stir the mtxture at 0°C for 2 h and at RT for 4 h 3. Store the mixture m a freezer (-18’C) for 0.5 h Remove the white precipitate (DCU) by filtration 4 Evaporate the filtrate, and dissolve the residue in 60 mL of ethyl acetate. Wash the solution with 10% cttric acid (15 mL x 2), 5% NaHC03 (15 mL x 2) 10% cltrlc acid (15 mL) and brme (15 mL x 2) and dry the organic layer over MgSO,.

Wang et al.

80 COOH

+ H2N-Tyr-D-Ala-Gly-PheOBu-t 9 Boc-D-Leu-0

15 CO-L-Tyr-D-Ala-Gly-PheOBu-t \

a , D-Ala 16 CO-L-Tyr-D-Ala-Gly-PheOH

Phe

C

17

ii

Fig 4 Synthesis of the cychc prodrug of the pentapeptlde a DCC, HOBt, DMAP, b TFA, c. Bop-Cl, TEA, DCM, DMF

5 Solvent removal zn vucuo affords a crude product that IS purified on a silica gel column with CH,Cl, and CH30H (20.1, v/v) as eluent to give a white solid 16 (240 mg, 48%)

3.3.2. Cychc Prodrug 18 Stir a solution of 16 (240 mg, 0 275 mmol) m 15 mL of 25% TFA m CH,Cl, at RT for 2 5 h under N, TLC (ethyl acetate) analysis shows that the starting matereal has disappeared (see Note 11) Evaporate the solvent to afford a residue 17. Dissolve the residue m 5 mL of DMF and 240 mL of CH,Cl, Add Bop-Cl(470 mg, 1 85 mmol) and TEA (0.37 mL, 2 66 mmol) into the solution Stir the solution at RT for 20 h under NZ. Evaporate the solvent Dissolve the residue m 80 mL of ethyl acetate Wash the resultmg solution with water (15 mL), 10% cltrlc acid (15 mL), 5% NaHC03 (15 mL), and water (15 mL). Dry the organic layer over MgS04 Remove the solvent zn vac~o to afford a white residue Purify the residue on a silica gel column with CH,Cl, and CH30H (20 1, v/v) as eluent to give the final product 18 (69 mg, 38%) ‘H-NMR (CD,OD)* 6 7 35-7 19 (8 H, m), 7.05 (2 H, d, J= 7 8 Hz), 6 93 (2 H, d, J=87Hz),6.78(1 H,d,J= 12.0Hz),669(2H,d,J=87Hz),620(1 H,d,J =12OHz),463(2H,m),435(lH,t,J=7,2Hz),395(1H,q,J=72Hz),371 (1 H, d,J= 17 1 Hz), 3 33 (1 H, d,J= 17 1 Hz), 3.29 (1 H, dd, J= 13 8 Hz, 6 6 Hz),3 OS(1 H,dd,J=13.8Hz,8 7Hz),2.80(2H,m), 173(3H,m), 1 17(3H,d, J=72Hz),O93(6H,dd,J=l38Hz,6,3Hz)

Synthesis of Coumarin-Based

Cyclic Prodrugs

81

3.4. Chemical and Enzymatic Stability Test of the Cyclic Prodrug 18 Bioreversibility is the essenceof any prodrug strategy. The coumarin-based cyclic prodrug 18 is designed to release the original peptide, DADLE, by an esterase-mediated hydrolysis of the ester bond followed by a fast chemical step (Fig. 1). In phosphate buffer pH 7.4, cyclic prodrug 18 degrades to DADLE and coumarin 3 with an apparent half-lives (t1,2) of 1206 min. The presumed intermediate 2 was not observed, indicating that the rate-limiting step in the cascade of reactions leading to the release of DADLE is indeed the hydrolysis of the ester moiety. As designed, the disappearance of cyclic prodrug 18 was significantly faster in the presence of porcine liver esterase (t,,, = 761 min) than that determined in phosphate buffer, pH 7.4. 3.4.1. Chemical Kinetics 1. Preparea solution of cyclic prodrug 18 ( 1W4M) by diluting a 0.01 M stocksolution of the prodrug in DMSO with 0.05 Mphosphate buffer (pH 7.4). 2. Keep the solution at 37 + 0.5”C in a water bath. 3. Periodically remove aliquots, and immediately freeze them in a dry ice/acetone bath to stop the reaction. 4. Analyze the samples by reversed-phase HPLC by monitoring the disappearance of the cyclic prodrug 18 and the appearance of coumarin 3. Detection wavelength is 285 nm. Mobile phase consists of HPLC grade methanol and deionized water.

3.4.2. Purified Esterase Kinetics 1. Dilute 1.5 microliters of purified porcine liver esterase (carboxylic-ester hydrolase, EC 3.1.1.1; E2884, Sigma) suspension (in 3.2 M [NH4]$04 solution, pH 8,) to 9.9 mL with phosphate buffer (0.05 A4, pH 7.4). The resulting solution contains 6800 units of enzyme per mL. 2. Combine 100 microliters of the 0.01 M stock solution of cyclic prodrug 18 in DMSO with the above solution. 3. Shake the mixture for 30 s and then keep it at 37 + 0.5”C in a water bath. 4. Periodically remove aliquots from the reaction mixture, and immediately freeze them in a dry ice/acetone bath to stop the reaction. 5. Analyze the samples using reversed-phase HPLC.

3.5. Transport properties of Cyclic Prodrug 18 Across Biological Membranes The permeation characteristics of the cyclic prodrug 18 and its parent peptide, DADLE, are conducted across Caco-2 cell monolayers, an in vitro cell culture model of the intestinal mucosa (28). Caco-2 cells, which spontaneously undergo differentiation into confluent monolayers, have been shown to exhibit both the physical and metabolic

barrier properties of intestinal

mucosal cells to

82

Wang et al.

peptides (2%32). Neither the parent peptide, DADLE, nor the cyclic prodrug 18 shows any degradation when they are applied to the apical side (i.e. luminal side) of the cell monolayer and incubated for 180 min. The cyclic prodrug 18 itself cannot be detected by fluorescence because of significant quenching. Therefore aliquots from the transport studies are treated briefly with high pH (Subheading 3.5.1., step 7) to convert the cyclic prodrug to DADLE, which can be detected by fluorescence, and coumarin 3, which can be detected by UV. The Pap,, values for the DADLE and the cyclic prodrug 18 are 9.9 1 f 0.5 1 x IO-*cm/s and 1.34 + 0.26 x lO-‘j cm/s, respectively. This indicates that cyclic prodrug 18 is 13.5 times more permeable through this model of intestinal cellular barrier than is DADLE.

3.5.1. Transport of Cyclic Prodrug 18 Across Caco-2 Monolayers 1. Grow Caco-2 cells on a collagen-coated polycarbonate membrane (Transwells@) to confluent monolayers of differentiated enterocytes (see Note 12). 2. Wash cell monolayers 3 times with prewarmed HBSS, pH 7.4, and 1.5 mL of the peptide solution (140 pA4 in HBSS and 1% MeOH) is applied to the donor (i.e., apical) compartment. Add 2.5 mL of HBSS to the receiver (i.e., basolateral) compartment. 3. Maintain cell monolayers in a temperature-controlled (37.0 + 0.5”C) shaking water bath (60 rpm). 4. Remove at various times up to 180 min, taking samples (120 mL, receiver side; 20 pL, donor side) from both sides. Replace the volume removed from the receiver side with fresh prewarmed HBSS. 5. Add an aliquot of ACN and diluted phosphoric acid (final concentration 10% (v/v) and 0.01% (v/v) respectively) to stabilize the samples. 6. Freeze the acidic mixture (pH 3) immediately in a dry-ice/acetone bath and keep at -80°C until analysis. 7. Thaw samples rapidly in a temperature-controlled (37.0 f 0.5”C) water bath. Add an aliquot of 1 N NaOH (10 mL) and put in a temperature-controlled (37.0 * 0.5”C) shaking water bath (60 rpm) for 15 min. Then add an aliquot of 1 N HCl (20 pL), and analyze immediately by HPLC (see Note 13).

3.6. Discussion We have described a general strategy for preparing a cyclic prodrug of DADLE, an opioid peptide, via the N- and C-terminal ends by utilizing a unique coumarinic acid linker. The final product of the promoiety is coumarin 3, which is known not to be toxic. This eliminates one major uncertainty in the development of this prodrug strategy for practical applications. Furthermore, it should be feasible to use this methodology to cyclize other biologically active peptides by linking the C-terminal carboxylic group to a side chain amino (e.g., Lys, Arg) or hydroxyl (e.g., Ser, Thr, Tyr) group, or linking a side chain car-

Synthesis of Coumarin-Based

Cydic Prodrugs

83

boxy1 group (e.g., Asp, Glu) to a side chain amino (e.g., Lys, Arg) or hydroxyl (e.g., Ser, Thr, Tyr) group. Application of this methodology to other biologically active peptides (e.g., opioid peptides) and peptide mimetics (e.g., RGD analogs) is currently under investigation. 4. Notes 1. Lithium aluminum hydride must be handled with care, because contact with moisture may cause fire. 2. There may be some solid particles left in the LiAlH, mixture. You may take a spatula and transfer it to the coumarin solution. 3. Add reaction mixture dropwise into the cold acidic water. If addition is reversed, gas and heatproduction may causea spill. 4. Carbodiimides, such as dicyclohexylcarbodiimide and diisopropylcarbodiimide, are known for causing severe allergic reactions. They should be handled with care. Contact with the skin and the eyes should be avoided. 5. Before being purified on a column, the solution must be washed to remove acetic acid. 6. The total reaction time is about 7 h. If starting material has not disappeared, it is necessary to add more MnO, to complete the reaction. 7. HOBt is a monohydrate. Accordingly, the amount of HOBt must be adjusted. In this preparation, the monohydrate sample is used. 8. In the hydrogenolysis, it is generally advisable to use ethanol rather than methanol because the palladium catalyst ignites methanol more readily than other solvents. 9 The inlet tube needs not reach below the surface of the solution. It is better to pass the gases just above the surface. The outlet tube is connected through a rubber tubing with a pipet that is immersed in water. This allows the monitoring of H2 as it leaves the flask. A steady bubbling should be maintained. 10. Before adding palladium catalyst, be sure that the air is displaced completely by nitrogen. Otherwise remaining oxygen may cause ignition once hydrogen is passed. 11. Even though, the starting material has already disappeared on TLC, it is necessary to check the t-butyl group by ‘H-NMR at about 1.5 ppm. Be sure that this peak completely disappears before going on to the cyclization step. If necessary, repeat the treatment with TFA. 12. For a detailed description of the cell culture conditions see (29). 13. The peptidks are eluted at a flow rate of 1 mL/min using a gradient from 26-90% (v/v) ACN in water with TFA (0.1% v/v) as the ion pairing agent. Monitor the eluents with both a fluorescence detector (emission = 305 nm and excitation + 280 nm) and a UV (= 254 nm) detector.

Acknowledgments This work was supported by a grant from the Presbyterian Health Foundation (PHF-987). Mass spectra were obtained at the Mass Spectrometer Labora-

tory for Biotechnology. Partial funding for the Facility was obtained from the North Carolina Biotechnology Center and the National Science Foundation Grant9111391.

Reference V. J. and Gehrig, C. A. (1989) Recent Development in the design of receptor specific opioid peptides. Med. Res. Rev. 9,343AO 1.

1. Hruby,

2. Schiller, P. W. (1991) Development of receptor-specific opioid peptide analogs. Prog. Med. Chem. 28,301-340. 3. Schiller, P. W. (1993) Development ofreceptor-selective opioid peptide analogs as pharmaceutical tools and as potential drugs. Handbook Exp. Pharmacol. 104,68 l-7 10. 4. Bedded, C. R., Clark, R. B., Hardy, Cl. W., Lowe, L. A., Ubatuba, F. B., Vane, J. R., Wilkinson, S., Chang, K. J., Cuatrecasas, P., and Miller, R. J. (1977) Structural requirements for opioid activity of analogs of the enkephalins. Proc. R. Sot. London 198, 149-265.

5. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A., and

6. 7.

8.

9.

Morris, H. R. (1975) identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 577-579. Ehrenpreis, S. and Sicuteri, F. (1983) Degradation of Endogenous Opioids. Raven, New York. Schiller, P. W., Nguyen, T. M.-D., Maziak, L., and Lemieux, C. (1985) A novel cyclic opioid peptide analog showing high preference for receptors. Biochem. Biophys. Rex Comm. 121,558-564. Hershfield, R. and Schmir, G. L. (1973) The lactonization of ring-substituted coumarinic acids. Structural effects on the partitioning of the tetrahedral intermediates in esterification. J. Am. Chem. Sot. 95, 7359-7368. Hershfield, R. and Schmir, G. L. (1973) Lactonization of coumarinic acids. Kinetic evidence for three species of the tetrahedral intermediate. J. Am. Chem. Sot. 95,8032-8040.

10. Wang, B., Zhang, H., and Wang, W. (1996) Chemical feasibility 11.

12. 13. 14. 15.

studies of a

potential coumarin-based prodrug system. Bioorg. Med. Chem. Lett. 6, 945-950. Wattenberg, L. W., Lam, L. K. T., and Fladmoe, A. V. (1979) Inhibition of chemical carcinogen-induced neoplasia by coumarins and a-angelicalactone. Cancer Res. 39, 165 l-1660. Wang, B., Wang, W., Zhang, H., Shari, D., and Smith, T. D. (1996) Coumarinbased prodrugs 2. Synthesis and bioreversibility studies of an esterase-sensitive cyclic prodrug of DADLE, an opioid peptide. Bioorg. Med. Chem. Lett. 6,2823-2826. Berkarda, B., Bouffard-Eyuboglu, H., and Dermand, U. (1983) The effect of coumarin derivatives on the immunological system of man. Agents Actions 13,5&55. Thornes, R. D. (1983) Coumarins, Melanoma and Cellular Immunity. Protective Agents in Cancer, 43-56. Marshall, M. E., Mendelsohn, L., Butler, K., Cantrell, J., Harvey, J., and Macdonald, J. S. (1987) Treatment of non-small cell lung cancer with coumarin and cimetidine. Cancer Treat. Rep. 71,91-92.

Synthesis of Coumarin-Based

Cyclic Prodrugs

85

16 Marshall, M E , Mendelsohn, L , Butler, K., Riley, L., Cantrell, J , Wiseman, C , Taylor, R , and Macdonald, J. S. (1987) Treatment of metastatlc renal cell carcinoma with coumarm and cimetldme a ptlot study J Clan Oncol. 5, 862-866 17 Marshall, M. E , Butler, K , Cantrell, J , Wiseman, C , and Macdonald, J S. (1989) Treatment of advanced mahgnant melanoma with coumarm and clmetidine a pilot study Cancer Chemother Pharmacol 24,65%i6 18 Marshall, M E , Conley, D , Hollmgsworth, P , Brown, S., and Thompson, J S (1989) Effects of coumarm on lymphocyte, natural killer cell, and monocyte function m vitro. J Blol Response Mod 8,7&85 19 Nan, R V , Fisher, E P , Safe, S. H., Cortez, C , Harvey, R G , and DiGlovanrn, J (1991) Novel coumarnrs as potential anticarcmogemc agents Carcznogeneszs 12,6S69 20 National Toxicology Program (1993) Toxzcology and Carcznogenesls Studzes of Coumann, U S. Department of Health and Human Servtces Pubhc Health Service and National Institutes of Health, Bethesda, MD 2 1. Tseng, A (1991) Chemopreventlon of tumors in MTV-H-ras transgemc mice with coumarin Am Assoc Cancer Res Proc 32, Abstract No 2257. 22 Stewart, J. M and Young, J D (1984) Solid Phase Peptzde Syntheses Pierce, Rockford, IL 23. Dalcannale, E and Montanart, F. (1986) Selecttve oxrdation of aldehydes to carboxyllc acid with sodium chlorate-hydrogen peroxrde J Org Chem 52, 567-569 24 Bryan, D B (1977) Nuclear analogues of p-lactam antibiotics 2 The total synthesis of 8-oxo-4-thia-1 -azablcyclio[4 2 0 ]oct-2-ene-2-carboxyhc acid. J Am Chem Sot 99,2353-2355 25 Lundt, B F (1978) Removal of tert-butoxylcarbonyl protectmg groups with trtfluoroacetlc acid Int J Pepttde Protean Res 12,258-268. 26 Bodanszky, M and Bodanszky, A. (1984) The Practice of Pep&de Syntheses Springer-Velag, New York. 27 Diago-Meseguer, J , Palomo-Coll, A L , Femandez-Lizarbe, J. R , and Zugaza-Bilbao, A (1980) A new reagent for activatmg carboxyl groups: preparation and reactrons of N,N-bls[2-oxo-3-oxazohdmyl]phosphoro dlamldlc chloride. Syntheszs ,547-55 1, 28 Hrdalgo, I J , Raub, T. J., and Borchardt, R. T. (1989) Characterization of the human colon carcinoma cell lme (Caco-2) as a model system for mtestmal epnheha1 permeablhty Gastroenterology 96, 736749 29 Wilson, G , Hassan, I F., Dlx, C. J , Williamson, I., Shah, R , and Mackay, M (1990) Transport and permeabihty properties of human Caco-2 cells an m vitro model of the intestinal eplthehal cell barrier. J Control. Release 11,25-40. 30 Pmto, M , Robme-Leon, S , Appay, M.-D , Kedmger, M , Tradou, N , Dussaulx, E , Lacroix, B , Simon-Assmann, P , Haffen, K., Fogh, J , and Zweibaum, A. (1983) Entrocyte-hke differentration and polarization of the human colon carcinoma cell lme Caco-2 m culture Blol Cell 47, 323-330 3 1 Artursson, P (1990) Epnhehal transport of the drugs m cell culture I. a model for studying the passrve diffusion of intestinal absorpttve (Caco-2) cell J Pharm Sci 79,47&482

Azatides as Peptidomimetics Solution and Liquid Phase Syntheses Hyunsoo

Han, Juyoung

Yoon, and Kim D. Janda

1. Introduction Peptidomimetics have become immensely important for both orgamc and medicmal chemists (1). The alteration of peptides to peptidomimetics has included peptide side cham manipulations, amino acid extensions (Z), deletions (3), substitutions (la,b), and most recently backbone modifications (4). It is this latter development that has been exploited for the synthesis of biomimetic polymeric structures. Such progress has been fueled by the suggestion that peptidomimetics may provide novel scaffolds for the generation of macromolecules with new properties of both biological and chemical mterest (4). We reported recently (5) an efficient method for the solution and liquid phase syntheses of a biopolymer mimetic consisting of “a-aza-amino acids” lurked m a repetitive manner to form what we term an azatide ohgomer. To construct this biopolymer mimetic, three stagesof research were pursued: (1) the development of general synthetic procedures that allowed the synthesis of a wide variety of Boc-protected aza-ammo acid monomers; (2) optimization of solution phase procedures for the coupling of aza-amino acids m a repetitive manner; and (3) design and synthesis of a linker that would support azatide synthesis using a soluble polymer that we term a liquid phase synthetic format. The successful completion of these three phases of research demonstrates that ohgoazatides can now be rapidly assembled on a homogeneous polymeric support. The long-term prospectus of this new biopolymer is the exploration of peptide structure, as well as a potential source of new peptidomimetic libraries. From

Methods Edlted

by

m Molecular

Medxine,

W M Kazmlerskl

Vol 23 Peptldomimebcs

OHumana

87

Press

Inc , Totowa,

Protocols NJ

88

Han et al.

2. Materials Methylene chloride and methanol were dried over CaH, and powered magnesium, respectively. NMR spectra were obtained on a Bruker AM-300 spectrometer. 2.7. Reagents for Method 3.7 Methylene chloride, pentafluorophenyl carbonate, 4-dimethylammopyrtdme (DMAP), Boc-protected methylhydrazine, Boc-protected tsopropylhydrazme, Boc-protected isobutylhydrazme, Boc-protected benzylhydrazme and Boc-protected p-(O-benzyl)hydroxybenzylhydrazine. 2.2. Reagents for Method 3.2 Isobutylene, sulfuric acid (H2S04), methyl p-hydroxymethylbenzoate, diethyl ether, 0 8 A4 lithium hydroxide (LiOH), 1 N hydrochloric acid (HCl), methanol, methylene chloride, magnesium sulfate (MgSOJ, polyethylene glyco1 monomethyl ether (MeO-PEG), 4-drmethylammopyrrdme (DMAP), dlcyclohexylcarbodnmide (DCC), phosphorus pentaoxide (P205), trifluoro acetic acid (TFA), ethanol, dnsopropylethylamme (DIPEA) and 10% Pd/C 3. Methods 3.1. Preparation of Boc-Protected Alkylhydrazine and Solution Phase Diazatide Synthesis

Monomers

For the synthesis of ohgoazatides, an alphabet of suitably protected azaammo acid constituents needed to be prepared Our approach was to synthesize de ~OVOBoc-protected alkylhydrazme monomers substituted with a variety of functronal groups Two principal routes are used m their syntheses(Fig. 1). (1) Reduction of Boc-protected hydrazones (61, derived from the reaction of Boccarbazate with either an aldehyde or ketone, and (2) Alkylization of hydrazme with an alkylhahde, followed by Boc-protection of the resulting alkylhydrazme (7). The outgrowth of thesemethods is the transient protection of either the “ammo or carboxy-terminal” functionality of the aza-ammo acid and an ability to create a unique alphabet of a-aza-ammo acid R-groups. To convert these Boc-protected aza-amino acids mto acylating agents that would allow stepwise chain lengthening, the hydrazme portion of the molecule had to be activated (Fig. 2). Activation of this moiety is a challenge, since the Boc-alkylhydrazmes are poorer nucleophiles than simple ammes or ammo acids Consequently, we required a highly activated carbonyl synthon that would allow facile couplmg of two Boc-protected aza-amino acids to form the azattde-linkage Furthermore, this couplmg reaction had to be controllable, so that symmetrical drmer formation could be mmimized. Our mmal attempts to

Pep tidomime tic Aza tides

89

HAN1

o-t-Butyl

H +

-

0-t-Butyl ,lj N--If 4 0 R’ R2

H2

HN ,I: .f-t-W

--GE--R’

A

R2

(,)

0

H2N-NH2 + R-Br

-

or R-Cl

R’

1

‘NH2

-

(W20

t-Butyl-0

ty R

.W

(2)

Fig 1 Preparation of Boc-protected alkylhydrazme monomers (Reprinted from ref. 5 with permission )

acids together usmg p-nitrophenyl chloroformate, carbonyldnmldazole, bzs(2,4-dmitrophenyl) carbonate, or trlchloromethyl chlorofomate were unsuccessful, as they suffered from comphcated side reactlons, poor reaction yields, and/or prolonged reaction time. We reasoned that these results were caused by either the msufflclent leaving ability of p-mtrophenol and lmidazole, or sterlc hindrance of the o-mtro group m the case of &x(2,4-dinitrophenyl) carbonate. To overcome these problems, we opted to use bu-pentafluorophenyl carbonate 1 (Fig. 2) as the carbonyl actlvatlon element (8). Our decision to use this reagent was based on three factors. First, the pentafluorophenol functlonallty 1sa powerful electron-withdrawing group, while the fluoro substltuents mmlmize sterlc problems. Second, the bw pentafluorophenyl carbonate can be readily prepared from phosgene and a sodmm pentafluorophenolate solution Finally, the compound IS a highly crystallme solid that IS easily handled. Shown in Fig. 2 are two solution phase routes to dlazatldes. In the first case, carbamate 2 (Fig. 2) 1s utilized for the coupling reaction. Thus, a Boc-protected aza-ammo acid 1s added dropwise to 1 (Fig. 2), actlvatmg the l-R4 hydrazinecarboxyllc acid, 1,l -dlmethylethyl ester. The activated complex formed, 2 (Fig. 2), is not isolated but instead immediately reacted via the addltlon of a second Boc-protected alkylhydrazme to complete the dlazatlde coupling. This coupling procedure provides diazatldes in good yield with few side reactions m an acceptable reaction time. Results using this couplmg method are summarized m Table 1 From this table, it is evident that the coupling process seems to be quite general, as both simple (Glya-Glya [superscript a refers to an aza-ammo acid linkage]) and sterlcally demanding (Vala-Vala) azatldes can be synthesized m less than an hour. The latter result is extremely couple

two aza-ammo

Han et al

90 1 Starbng

from I-Fl’-Hydrazme

Carboxyllc

Acid, l,l-Dtmethylethyl

Ester

1, DMAP *

t-Butyl-0

t-Butyl-0 c

t-Butyl-0

DMAP 5; R2=H, R4=H 7; R%nsthyl, R4=methyl 8; F?=methyl, R4=benzyl 9; R’=methyl, R4=lsobutyl 10; R2dsobutyl, R4=lsobutyl 11; R2=lsopropyl, R4=lsopropyl ( seeTable 1)

2 Starting

from 2-R’-Hydrazme

Carboxyk

Acid, 1.6Dimethylethyl

Ester

1, DMAP t-Butyl-0

D

t-Butyl-0

Fig 2 Routes for solution phase dlazatlde synthesis (Reprinted with ref. 5 )

important, as it dictates whether the stepwlse couplmg of aza-ammo acids IS feasible. Whereas coupling through activated l-R4 hydrazmecarboxyhc acid, 1,l-dlmethylethyl ester was successful, the couplmg of activated 2-R2 hydrazinecarboxyhc acid, I,1 -dlmethylethyl ester was not (Fig. 2). From these

Pep tidomimetic Aza tides

91

Table 1 Preparation of diazatides starting from l-R’-Hydrazinecarboxylic Acid, l,l-Dimethylethyl

Compound

R’

R2

R3

5 6 7 8 9 10 11

H Methyl H H H H H

H H Methyl Methyl Methyl Isobutyl Isopropyl

H H H H H H H

Ester

R4 H Methyl Methyl Benzyl

Isobutyl Isobutyl Isopropyl

Yield (%) 92 91 90 85 84 82 84

(Reprinted from ref. 5 with permlsslon )

findmgs, we surmise that the activated complex IS not carbamate 2 (Fig. 2) but rather the rsocyanate 3. For an acttvated 2-R’ hydrazmecarboxylic actd, l,ldtmethylethyl ester, the Intermediate tsocyanate 9 IS untenable because of the carbamate’s substitution pattern.

3.1.1. General Sol&Ion Phase Dlazatide-Coupling

Procedure (Fig. 2)

1 To a stirred

solution of pentafluorophenyl carbonate (50 0 mg, 13 0 mmol) see Notes 1 and 2) tn methylene chloride (5 mL), add a solution of 1-(N-Boc)-alkylhydrazme (1 eq) (see Note 3) and DMAP (1 eq) m methylene chloride (2 mL) dropwise over a period of 20 mm. 2 Upon completton of the addttron, add a solution of 2-(N-Boc)-alkylhydrazme (1 eq) and DMAP (1 eq) m methylene chloride (2 mL) to the reaction mixture

(Caution:

Stir the reaction mixture for 30 mm at room temperature 3 Removal of solvent and flash chromatography metrical diazatide

Boc-Glya-Glya-Boc

(10) provides the desired unsym-

5

‘H-NMR (300 MHz, CDCl,) 6 1.35 (s, 18H), 7.33 (broad s, 2H), 7.77 (s, 2H), ‘,C-NMR (75 MHz, CDCl,) 6 27.9, 81 3, 156.2, 157 1; HRMS (FAB) calculated for [C, ,HZ2N405 + Cs’] 423 0645, found 423.0655 (Table 1). Boc-Alaa-Alaa-Boc

6

‘H-NMR (300 MHz, CDCl,) 6 1.42 (s, 18H), 3.09 (s, 6H), 7 43 (s, 2H); ‘,C-NMR (75 MHz, CDCl,) 6 28.1, 38.3, 81 5, 156.1, 157 0; HRMS (FAB)

92

Han et al

calculated for [C13HZ6N405 + Cs’] 45 1.0958, found 45 1.0976 (symmetrical: see Note 4). Boc-Alaa-Alaa-Boc

7

‘H-NMR (300 MHz, CDCls) 6 1.43 (s, 9H), 1.45 (s, 9H), 3.08 (s, 3H), 3.09 (s, 3H), 6.45 (broad s, lH), 7.05 and 7.62 (broad s, 1H); 13C-NMR (75 MHz, CDCl3) 6 28.2, 28 3, 37.9, 81.2, 81.4, 155.4, 156 6, 156.8; HRMS (FAB) calculated for [C, ,HZ2N405 + Cs’] 451 0958, found 451.0965 (Table 1). Boc-Alaa-Phea-Boc

8

‘H-NMR (300 MHz, CDCl,) 6 1.37 (s, 9H), 1.41 (s, 9H), 3.1 (s, 3H), 4.50 (broad s, 2H), 6.10 and 6.59 (broad s, lH), 7 22 (m, 5H), 7 37 and 7 55 (broad s, 1H); 13C-NMR (75 MHz, CDCl,) 6 27.8,28.2, 38.5, 54.3, 81 5, 81.6, 127.3, 128.3, 128.6, 137.3, 155.5, 156 4, 156.5, HRMS (FAB) calculated for [C19H30N405 + Cs’] 527 1271, found 527.1289 (Table 1). Boc-Alaa-Leua-Boc

9

‘H-NMR (300 MHz, CDCl,) 6 0.90 (d, J= 7 Hz, 6H), 1.40 (s, 9H), 1 45 (s, 9H), 1.84 (m, lH), 3.07 (s, 3H), 3.35 (broad s, 2H), 6.30 and 6.56 (broad s, IH), 7.20 and 7.36 (broad s, 1H); 13C-NMR (75 MHz, CDCl,) 6 19.9, 26 3, 28.1, 28.3, 38.0, 55.7, 81.1, 82.0, 154.5, 156.1, 157,6; HRMS (FAB) calculated for [C16H32N405 + Cs’] 493.1427, found 493.1447 (Table 1) Boc-Leua-Leua-Boc

10

‘H-NMR (300 MHz, CDC13) 6 0.88 (d, J= 7 Hz, 6H), 0.90 (d, J= 7 Hz, 6H), 1 42 (s, 9H), 1 46 (s, 9H), 1.86 (m, 2H), 3 27 (broad s, 4H), 6.33 and 6.57 (broad s, lH), 7.11 and 7.23 (broad s, 1H); 13C-NMR (75 MHz, CDCl,) 6 20.4, 20.4,27.6,27.8,28.5,28.6,56.7,59.5,

(FAB) calculated for [C,,H,sN,O, Boc-Vala-Vata-Boc

81.8, 82.3, 156.3, 156.5, 158.1,HRMS

+ Cs’] 535.1897, found 535.188 1 (Table

1).

11

m p lOl-102°C; ‘H-NMR (300 MHz, CDCl,) 6 1.09 (broad s, 12H), 1 40 (s, 9H), 1 45 (s, 9H), 4.32 (broad s, IH), 4.61 (m, 2H), 6.27 (broad s, IH), 6.79 (broads, 1H); 13C-NMR (75 MHz, CDC13) 6 19.3, 19.8,28.0, 28 3,48.4,48 6, 8 1.O, 8 1.6, 157.1, 157.4, 157.9; HRMS (FAB) calculated for [C,7H34N405 + Cs’] 507.1584, found 507.1599 (Table 1).

3.2. MeO-PEG-Supported

Leu-Enkephalin

Azatide Synthesis

The techniques described above allow a-azatlde cham building to be performed in an iterative manner. To prepare a small well-defined a-azatlde, we chose to use polymer-supported hqmd phase syntheses (11) Llqurd phase syn-

Peptldomimetic Azatides

93

lhcsrs uses a soluble lmear homopolymer (polyethylene glycol monomethyl cthcr [MeO-PEG]) which serves as a terminal protecting group for the compound to be synthestzed. The essence of this technology is that tt avoids a number of difliculttes found m solid-phase synthesis and preserves the positive aspects of solutton phase synthesis. We have demonstrated the advantages of using liquid phase synthesis through the constructton of both peptide and small molecule combmatorral libraries (12). A leucine-enkephalm peptide sequence YGGFL was chosen as the first azatide mimetic to be synthesized. This pentamer was selected, as the N-terminal sequence within this unit YGGF is common to most natural opioid peptides (13). The successful diazatlde couplmg procedure described m Fig. 3 implies N-to-C-terminal construction of the azatide Ap-substituted benzyl ester spacer unit that would accommodate directional synthesis on MeO-PEG and withstand the rigors of Boc-chemistry was designed (14, Fig. 3). It was reasoned that 14 (Fig. 3) attached to MeO-PEG would be stable against acidolysis owing to the presence of the para-benzoate substituent, and the ohgoazatide could be liberated by catalytic hydrogenation generating a free ammo group. Thus, methyl p-(hydroxymethyl) benzoate was O-protected as the t-butyl ether by treatment with isobutylene and acid. Subsequent hydrolysis of the methyl ester with lithium hydroxide provided 14 (Fig. 3). Lurker 14 (Fig. 3) was coupled to MeO-PEG with the aid of DCUDMAP, and upon deprotection with trifluoroacetic acid (TFA) gave the MeO-PEG-benzyl-OH (15, Fig. 3) support ready for azatide synthesis. Synthesis of the azatide pentamer YaGaGaFaLa was accomplished m a repetitive stepwise fashion as shown m Fig. 3. Because of the unique physical properties of the MeO-PEG homopolymer, each coupling/ deprotection reaction could be purified by precipitation of the modified homopolymer. Furthermore, MeO-PEG allows the reaction progress to be conveniently monitored by either proton-NMR spectroscopy or the Kaiser nmhydrm test (14). Based on our linker strategy, the pentamer and the benzyl protectmg group of aza-tyrosme could be liberated m a single step using catalytic hydrogenation to give the Boc-protected pentamer (overall yield 56.7% from 15, Fig. 3). This compound was converted to the desired Leu-enkephalm azatide by treatment with trrfluoroacetic acid.

3.2.1 Linker Preparatron Methyl p-(0-t-butyl) Hydroxymethylbenzoate 73 1 Liquefy lsobutylene (8 mL, exess) in a sealed bottle at -78°C 2 Add a solution of sulfuric acid (0 5 mL) and methyl p-hydroxymethylbenzoate (12, Fig. 3, see Note 5) (2 00 g, 12.0 mmol) in dry ethyl ether (20 mL) to the lsobutylene solution (8 mL) at -8°C and stir overnight at room temperature 3 Cool the resulting mixture to 4°C Then, add ice-cooled water to the reaction mixture

MeO-PEG-Om

DMAP

16

I) TFAKH2C12,

DIPEA 0 0x

I:/4 ‘I;’

0-t-Butyl , DMAP

I

III) Repeat a cycle v&h Gv. Phe”,

of above I) and and Leua

II)

95

96

Han et al.

4

Dry the ether layer over magnesmm sulfate Concentrate the ether layer to a white solid A yteld of 2 59 g (96 8%) of desired product should be obtained m p 34-36”C, ‘H-NMR (300 MHz, CDCl,) 6 1 29 (s, 9H), 3 89 (s, 3H), 4 49 (s, 2H), 7 40 (d, J = 6 7 Hz, 2H), 7 98 (d, J= 6 7 Hz, 2H), 13C-NMR (75 MHz, CDCI,) 6 27 5, 51 8, 63 4, 73 6, 126.8, 128 7, 129.5, HRMS (FAB) calculated for [C,,H,,05 + Cs’] 355 0310, found 355 0323

3.2 2. p-(0-Butyl)Hydroxymethylbenzolc

Acid 14

1 Dissolve methylp-(O-t-butyl)hydroxymethylbenzoate (13, Fig. 3) (2 02 g, 9 10 mmol) m a 0 8 M LlOH solution m methanol and water (34 ml, methanol H20, 3 1) 2 Star the reaction mixture until the startmg maternal disappears as Judged by TLC (methylene chloride: ethyl ether = 9 1) Acidify the reactton mixture by the addttton of 1 N-HCl and extract with methylene chloride 3 Dry the methylene chlorrde layer over magnesmm sulfate Concentrate the methylene chloride layer to a whtte soltd A yield of 1 72 g (90 9%) of 14 (Fig. 3) should be obtained m p 147-149”C, ‘H-NMR (300 MHz, CDCl,) 6 1 39 (s, 9H), 4.50 (s, 2H), 7.42 (d, J= 6 8 Hz, 2H), 8 06 (d, J= 6.8 Hz, 2H); 13C-NMR (75 MHz, CDCls) 6 27 6,63 6,73.9, 127 0, 128 0, 130 3, 146 4, 171 8, HRMS (FAB) calculated for [Ct2Ht603 + Na+] 231 0997, Found 23 1 0986

3 2 3 Synthesis of MeO-PEG-Linker-YaGaGaPLa Attachment of p-(0-Buty/)Hydroxymethy/benzoic to MeO-PEG; [MeO-PEG-Benzyl-OH] 15

Acid 74

1 Dissolve 14 (Fig. 3) (125 mg, 601 mnol), MeO-PEG (1 00 g, 200 unrol) (see Note 6), DMAP (6 11 pg, 50 0 pool) and DCC (124 mg, 601 pool) in methylene chloride (10 mL) 2 Stir the resultmg mtxture for 12 h and filter the prectpnated urea through cellte Add dlethyl ether slowly to the filtrate m order to precipitate the polymer Filter and wash the polymer precipitate wtth cold absolute ethanol and ether, and dry the precipitate over P205 under vacuum 3 Drssolve this soltd m trtfluoroacettc acid and stir the resulting solution for 9 mm at room temperature Pour the reactton mtxture onto an me-cold drethyl ether solutton with vrgorous strrrmg Collect and wash the precrpttate with cold absolute ethanol and drethyl ether A yield of 935 mg (9 1 1%) should be obtained upon drymg over P,Os under vacuum ‘H NMR (300 MHz, CD,OD) 6 4 45 (t, J= 7 Hz, 2H), 4 71 (s, 2H), 7 41 (d, J= 7Hz, 2H), 7 98 (d, J = 7 Hz, 2H)

3.2 4. Synthesis of the Leu-Enkephalin Azatide 17. Construct/on of (0-Benzy/)TyFG/y-G/p-Phea-Lena-Boc on MeO-PEG-Benzyl-OH 75 1 Stir a mixture of 15 (Fig. 3) (195 mg, 38.0 mmol), pentafluorophenyl carbamate of Boc-lp-(0benzyl)hydroxybenzyl]hydrazme (102 mg, 5 eq), and DMAP (23 2 mg, 5 eq) m methylene chloride (5 mL) for 24 h at room temperature

Peptidomimetic Azatides

Y/

2 Add diethyl ether slowly to this mixture to preclpltate the polymer product 16. Wash the polymer product with absolute ethanol (see Note 7) and diethyl ether and dry the product over P,O, under vacuum. ]H NMR (300 MHz, CD,OD) 6 1.40 (s, 9H), 4 45 (2H), 4 55 (2H), 5.00 (2H), 5.15 (2H), 6 55 (IH), 6 88 (2H), 7 15 (2H),7.38 (7H), 8.00(2H) 3. Dissolve the polymer 16 m TFA/methylene chloride and stir the resultmg mixture for 30 mm to cleave the Boc-group. 4. Add diethyl ether slowly to this mixture to precipitate the polymer product, trlfluoro acetate salt of Lp-(O-benzyl)hydroxybenzyl]Tyla-O-benzyl-PEG-OMe. Wash this salt with absolute ethanol and dlethyl ether and dry the product over P,O, under vacuum. 5. Dissolve this salt m methylene chloride, and neutralize with dllsopropylethylamme (DIPEA, 1 eq) Add the pentafluorophenyl carbamate of Boccarbazate (5 eq) and DMAP (5 eq) to the resulting mixture. Stir the reactlon mixture for 4 h 6 Add diethyl ether slowly to this mixture to precipitate the polymer product, Boc-Glya-(U-benzyl)-Tyra-O-benzyl-PEG-OMe Wash the polymer product with absolute ethanol and dlethyl ether, and dry the product over P,O, under vacuum. After repetltlon of this cycle of deprotection, neutralization, and coupling wtth Glya, Phea, and Leua, the Leu-enkephalm azatide 17 should be obtained (137 mg, 62 4%) from 15: ‘H NMR (300 MHz, CD30D) 6 0 87 (6H), 1.42 (9H), 1 91 (lH), 4.43 (2H), 4 96 (2H), 5 12 (2H), 6.83 (2H), 7 13 (2H), 7.37 (7H), 7 95 (2H) The multlpllclty of peaks is not described due to the peak broadenmg

3.2.5. Ty+Glp-G/p-PheVe@-Boc

18

1 Hydrogenate compound 17 (Fig. 3) (137 mg, 23 7 pool) with 10% Pd/C (100 mg) m methanol (5 mL) under a balloon contammg one atmosphere of hydrogen for 4 h. 2 Remove all volatlles zn vacua, and extract the residue with absolute ethanol 3 Concentrate this ethanol solution. After the purification of this crude product by preparative thm layer chromatography, the desired material 1sobtained as a smgle band Rf = 0 4 (13.25 mg, 90.7%, TLC solvent , methylene chlorrde, methanol = 9. 1) IH-NMR (300 MHz, CD,OD) 6 0.93 (d,J= 7 Hz, 6H), 1 43 (s, 9H), 1 47 (s, 9H), 1 95 (m, 1H), 3.27 (broad s, 2H), 4.17 and 5.19 (broad s, 2H), 4.50 (broad s, 2J3), 6 76 (d, J= 6 7 HZ, 2H), 7.12 (d, J= 6.7 Hz, 2H), 7 33 (m, 5H); m/z (ESI, poslttve) 639 (M + Na)+, 617 (M + I)‘,

3.2.6. Tyla-G/y-G/p-Phea-Lena

* 2CF3COOH 19

1. Dtssolve Tyld-Glya-Gly”-Phea-Leua-Boc (18, Fig. 3) (13.25 mg, 21.5 mmol) m TFA/methyIene chloride (5 mL), and stir for 30 min 2, Remove all volatrles IPI vucuo to give the desired product as a white hygroscoplc solid (16 0 mg, 100%). ‘H NMR (300 MHz, CD,OD) 6 1.05 (d, J= 6 7 HZ, 6H),

Han et al.

98

2.09 (m, lH), 3.07 (broad s, 2H), 4.22 and 5.26 (broad s, 2H), 4.65 (broad s, 2H), 6.77 (d, J= 6.8 Hz, 2H), 7.17 (d, J= 6.8 Hz, 2H), 7.35 (m, 5H); m/z (ESI, positive) 539 (M + Na)+, 5 17 (M + l)+.

3.3. Discussion We have shown solution phase and liquid phase methodologies for the stepwise synthesis of azatides. Tandem mass spectrometry technique (IS) was used for the sequence determination of our azatide (see Note 8). While a competition ELISA method was used to investigate if the Ty?-Glya-Glya-PheaLeua sequence could bind to IgG 3-E7 (12) (see Note 9). Unfortunately, at 1 mM the azatide pentamer showed no propensity to compete with the natural peptide for 3-E7. We assume from the available data (2649) (see Note 9) that 19 should adopt a more extended conformation within the critical glycine region, so that this azatide oligomer would have difficulty in achieving the orientation displayed by the antigenic determinant (Tyr-Gly-Gly-Phe-Leu) that elicited IgG 3-E7. In spite of our inability to show biological activity for azatide 19, we believe that azatides will be of considerable interest, as they are new materials with the potential for novel biological properties. Furthermore, the structural and pharmacological properties of these azatides may provide important leads for the drug industry, and biophysical studies of these polymers could enhance our understanding of receptor-ligand interactions. Combinatorial library construction of this new biomimetic polymer may provide a means to fabricate global peptidomimetic libraries. 4. Notes 1. Pentafluorophenyl carbonate is prepared according to literature procedure (9). 2. Phosgene, which is used for the synthesis of pentafluorophenyl carbonate, is extremely toxic, so it should be handled under the hood with care. 3. Boc-protected methylhydrazines, isopropylhydrazines, isobutylhydrazines, benzylhydrazines, andp-(0-benzyl)hydroxybenzylhydrazines (7,s) are prepared according to literature procedures. 4. Symmetrical Boc-Alaa-Alaa-Boc (6, Table 1) representsBoc-N(CH,)-NH-CONH-N(CH,)-Boc. 5. p-Hydroxymethylbenzoate(12, Fig. 3) is available from Aldrich. 6. MeO-PEG should be dried over P,O, in a vacuum before use. 7. Using absoluteethanol is very important, becauseMeO-PEG hasgood solubility in water. 8. The Leu-enkephalin azatide was subjected to ESI-tandem massspectrum analysis. As shown in Fig. 4, proton transfer would preferentially occur on the more basic tertiary amide nitrogen for an azatide over the secondary amide nitrogen. The protonation of a tertiary amide nitrogen causesbond-cleavage between the

99

Peptidomimetic Aza tides Preferential

I-

cleavage 7

Azatides

A-type:

Peptides

0 +$~H

B-type: R’

$&drd~ 1

Fig. 4. Fragmentation patterns of (M + l)+ ion of peptides and azatides. (Reprinted from ref. 5 with permission). a-nitrogen and carbonyl carbon to generate X- and A-type fragments (Fig. 4). This prediction was manifested in the collision-induced dissociation (CAD) pattern of Leu-enkephalin azatide 19 shown in Fig. 5. The MS-MS of the (M + H+) ion at 5 17 produced daughter peaks at 403,255,197 (A-type), 32 1,263 (X-type) and MS-MS-MS on 403 (M-Leu” + H+) gave granddaughter peaks at 255, 197, 139 (A-type), 207, 149 (X-type), 239, 197, 123 (Y-type). Peaks at 297, 149, 107, 91 represent A-type fragments involving cleavage of side-chain of Tyf. Mass difference between homologous A-type ions corresponds to elements -CONHNR-. Predicted m/z values for AI-As fragments were obtained by sequentially adding the incremental masses of Glya, Glya, Phea, and Leua to that for A’ at 139. A similar argument can be made for X-type and Y-type fragments, confirming the Tyra-Glya-Glya-Phea-Leua sequence of Leu-enkephalin azatide. 9. Further details can be found in ref. 5.

References la. Spatola, A. F. (1983) Peptide backbone modifications: a structure-activity analysis of peptides containing amide bond surrogates, in Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins (Weinstein, B., ed.), Marcel Dekker, New York, pp. 267-357. lb. Sherman, D. B. and Spatola, A. F. (1990) Compatibility of thioamides with reverse turn features: synthesis and conformatioinal analysis of two model cyclic pseudopeptides containing thioamides as backbone modifications. J. Am.Chem. Sot. 112,433-441. lc. Hirschmann, R. (1990) Medicinal chemistry in the golden age of biology: lessons from steroid and peptide research. Angew. Chem. Znt. Ed. Engl. 29, 1278-I 30 1. Id. Gante, J. (1994) Peptidomimetics-tailored enzyme-inhibitors. Angew. Chem. Int. Ed. Engl. 33, 1699-1720.

Han et a/.

(139)197 255 403 517 Tyr%y'Glf-Phea-Leua 517(379)321 263 (115)

139 197 255 403 TyBGv-Glya-Phe* 403 (265) 207 149

a’I

Tyt%iiy"-Glya-Phe' 403 239 197 123

Fig. 5. CAD spectra of m/z 517 (M + 1)’ and 403 peaks for compound (Reprinted from ref. 5 with permission).

19.

Pep tidomlmetic Aza tides

101

2a Fretdmger, R M., Veber, D F , Perlow, D S., Brooks, J R , and Saperstem, R (1980) Btoactive conformation of lutemtzmg hormone-releasing hormone evidence from a conformationally constrained analog Sczence 210, 656-658 2b Stachowiak, K., Khosla, M. C., Plucmska, K , Khairallah, P A., and Bumpus, F. M. (1979) Synthesis of angiotensm II analogues by mcorporatmg P-homoisoleucme residues J Med Chem 22, 1128-l 132. 3 Sarantakis, D , McKmley, W , and Jaunakais, I (1976) Structure activity studies on somatostatm. Chn Endocrmol 5,275S-2768 4a Hagihara, M , Anthony, N J , Stout, T. J , Clardy, J , and Schretber, S. J (1992) Vmylogous polypeptides an alternative pepttde backbone J Am Chem Sot 114,6568A570

4b Simon, R J , Kama, R S , Zuckerman, R N , Huebner, V. D , Jewell, D A, Banvtlle, S , Ng, S , Wang, L , Rosenberg, S., Marlowe, C K., Spellmeyer, D C., Tan, R., Frankel, A D , Santa, D V , Cohen, F E , and Bartlett, P A (1992) Peptoids a modular approach to drug discovery. Proc Nat1 Acad Scz USA 89, 9367-937 1 4c

4d

4e

4f

5 6.

7

8

9

10

Smith, A B , III, Keenan, T P , Holcomb, R. C., Sprengeler, P A., Guzman, M. C , Wood, J L , Carroll, P J , and Hirschmann, R (1992) Design, synthesis, and crystal structure of a pyrrolinone-based peptidomimetic possesmg the conformation of a P-strand potential application to the design of novel mhibitors of proteolytic enzymes J Am Chem Sot 114, 10,672-10,674 Cho, C Y , Moran, E J , Cherry, S R , Stephans, J C , Fodor, S P A, Adams, C L., Sundaaram, A, Jacobs, J W., and Schultz, P G (1993) An unnatural btopolymer Sczence 261, 1303-l 305 Liskamp, R M J (1994) Opportunities for new chemical libraries unnatural biopolymers and diversomers Angew Chem Int. Ed Engl 33, 633-636 Burgess, K , Lmthtcum, D S , and Shin, H (1995) Solid-phase syntheses of unnatural biopolymers containing repeating urea units Angew Chem Znt Ed Engl 34,907-909 Han, H and Janda, K. D (1996) Azatides. solution and liquid phase syntheses of a new peptidomimetic J Am Chem Sot 119,2539-2544. Dutta, A S , and Morley, J. S (1975) Polypeptides Part XIII Preparation of a-azaammo-acid (cabazic acid) derivatives and mtermediates for the preparation of a-aza-peptides. J Chem Sot Perkln Trans 1, 1712. Biel, J H., Drukker, A E , Mitchell, T. F , Sprengeler, E P., Nuhfer, P A , Conway, A C , and Honta, A. (1959) Central sttmulants Chemistry and structureactivity relationships of aralkyl hydrazmes J Am Chem Sot 81,2805-28 13 Efimov, V A , Kalmkma, A. L , and Chakhmakhcheva, 0 G. (1993) Dipentafluorophenyl carbonate-a reagent for the synthesis of ohgonucleotides and their conJugates Nucleic Acids Res 21, 5337-5344. Magrath, J. and Abeles, R H (1992) Cysteme protease mhtbition by azapepttde esters. J A4ed Chem 35,4279+283 Still, W C , Kahn, M , and Mitra, A. (1978) Rapid chromatographic techmque for preparative separation with modern resolution J Org Chem 43,2923-2925

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11 Geckeler, K E (1995) Soluble polymer supports for hqutd-phase synthesis, in Advances zn Polymer Sczence, vol 121 (Abe, A , et al , ed ), Sprmger-Verlag, Berlm, p 3 1. 12 Han, H , Wolfe, M M , Brenner, S , and Janda, K. D (1995) Liquid-phase combinatorial synthesis Proc Nat1 Acad Scl USA 92,64196423. 13 Meo, T., Gansch, C , Inan, R , Hollt, V , Weber, E , Herz, A , and Riethmuller, G (1983) Monoclonal anttbody to the message sequence Tyr-Gly-Gly-Phe of opioid pepttdes exhibits the specificity requirements of mammalian optold receptors. Proc Natl Acad Sci USA 80,4084-4089 14. Kaiser, E , Colescott, R L., Bossmger, C D , and Cook, P I (1970) Color test for detection of free terminal ammo groups m the solid-phase synthesis of peptides Anal Blochem 34,595-598 15 Hunt D F , Yates III, J R., Shabanowttz, J , Winston, S , and Hauser, C R (I 986) Protein sequencing by tandem mass spectrmetry Proc Nat1 Acad Scz

USA 83,62334237 16 Garner, B , Nakamsht, H , and Kahn, M (1993) Conformationally constrained nonpepttde S-turn mtmetics of enkephalin Tetrahedron 49,3433-3448 17a Lowe, G H and Bart, S K (1978) Energy conformatton study of met-enkephalm and its D-Ala analogue and then resemblance to rigid opiates Proc Nat1 Acad Scz USA 75,7-l 1 17b Manavalan, P and Momany, F A (1981) Conformational energy calculations on enkephalms and enkephalm analogs. Classificatton of conformations to dtfferent configurational types Int J Pept Protem Res 18,256-275 18a Smith, G D , and Griffin, J F (1978) Conformatton of [Leua] Enkephalm from X-ray diffraction. features important for recognition at opiate receptor Sczence 199, 1214-1216 18b Ishida, T., Kenmotsu, M., Mmo, Y , Inoue, M , FuJiwara, T , Tomtta, K., Ktmura, T , and Sakaktbara, S (1984) X-ray diffraction studies of enkephalms Blochem J 218,677-689 19a Ohvato, P R and Guerrero, S A (1983) Conformational studies of a-substituted carbonyl compounds Part I Conformatton and electronic mteractton m hetero-substttuted acetones by infrared and ultravtolet spectroscopy, J Chem Sot , Perkzn Trans II, 1053-1058. 19b Graybill, T. L , Ross, J. T , Gauvm, B. R , Gregory, J S , Harris, A L , Ator, M A., Rmker, J. M , and Dolle, R E (1992) Structure-activity relationships of the pyrtdazmone series of 5-hpoxygenase mhtbttors Bzoorganzc A4ed Chem Lett 2, 1357-l 360

7 Synthesis of Cbz-Protected Ketomethylene Dipeptide lsosteres Robert V. Hoffman

and Junhua Tao

1. Introduction Ketomethylene pepttde tsosteres have been the focus of a variety of synthetic studies because of then potential as therapeutic agents m the treatment of medical condittons mediated by proteases (Z-3) The dtpeptide core umts 1 are chemtcally characterized by a 1,4-disposition of the carbonyl groups (ketone and carboxylate) and have an alkyl group at C-2 that is a chiral center. Protecting groups at ammo nitrogen (typtcally carbamate such as Cbz or Boc) and the carboxyl group (typically ester) are usually present to make processing easier but must be readily and independently removable to permit extension m either the N-terminal or C-terminal directions (Fig. 1). Most syntheses of these compounds have utilized constructton of the 3,4 carbon-carbon bond to assemble the carbon backbone of the isostere core unit (3-11). Usually, a protected a-ammo aldehyde 1s reacted wtth an ester homoenolate equivalent. The resulting 4-hydroxy ester 1slactomzed and alkylated at C-2 wtth choral mduction to give hydroxyethylene pepttde tsosteres that give ketomethylene peptide isosteres upon oxidation A less common approach is to construct the 2,3-bond of the tsostere core unit. Development of this strategy requires an ammoketone enolate equivalent and an a-ketone electrophile (12-16), and alkyl groups at C-2 must still be installed by alkylation of a lactone enolate (17). It was subsequently found that reaction of scalemic 2-triflyloxy esters with pketoester enolates provided an excellent new method for the synthesis a variety of 2-alkylated y-ketoactds 3, the core unit of 2-substituted ketomethylene peptide tsosteres, m generally high enantiomeric excess (18,19). This strategy has been brought to fruition by its extension to the From

Methods

m Molecular

Medune,

E&ted by W M Kazmlerskl

Vol

@Humana

103

23

Peptldom\mehcs

Press Inc , Totowa,

Protocols

NJ

104

Hoffman and Tao

z-L,,,p+ 4 1

0

Frgure 1 preparation of N-protected ketomethylene drpepttde rsostere esterswtth high enanttoselecttvtty and dtastereoselectlvtty. 2. Materials Solvents used m this work were HPLC grade and used as received Tetrahydrofuran was dried by dtstillatton from benzophenone ketyl. Thm layer chromatography was performed on Stltca Gel 60 F,,, plates from EM reagents Vtsualizatton was by UV madration of todme Preparative layer chromatography was performed on Sthca Gel 60 F2=+,preparative plates from EM reagents. Vtsuahzatton was by UV nradtatton. Flash chromatography (20) was performed on Sthca Gel 60 (230-400 mesh) from EM reagents Normal phase HPLC was performed on a Rainen Dynamax 60-A sthca column (4.6mm x 25cm). ‘H and r3Cnmr spectra were taken m CDCl, and chemical shifts 6 are reported m ppm downfield from TMS. IR spectra were recorded as thm films (011s)or as KBr disks (solids) Elemental analysesfor all new compounds were within a0 4% of the theoretical value 2.1. Reagents for Method 3.1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5

N-Carbobenzyloxy-L-leucme (N-Cbz-LeuOH) N-Carbobenzyloxy-L-prolme (N-Cbz-ProOH). N-Carbobenzyloxy-L-valme (N-Cbz-ValOH) N-Carbobenzyloxy-O-benzyl-L-tyrosme (N-Cbz-Tyr N-Carbobenzyloxy-L-tryptophan (N-Cbz-TrpOH) N-Carbobenzyloxy-L-phenylalamne (N-Cbz-PheOH) N-Carbobenzyloxy-L-alamne (N-Cbz-AlaOH) Carbonyl dumldazole (CDI) n-Butyl hthmm (2 5 M m hexane) Dusopropylamme tert-Butyl acetate Tetrahydrofuran 1NHCl Ethyl acetate Magnesium sulfate (MgSOJ

(0Bn)OH)

Cbz-Protected Ketomethylene 2.2. Reagents

105

Dipeptide lsosferes

for Method 3.2

1 Methyl D-lactate 4a 2 D-Norvahne

3 4 5 6 7 8 9 10 11 12. 13 14 15

D-Phenylalanme D-Cyclohexylalanme D-Leucine D-Vahne Trtfhc anhydride 2,6-Lutidme Sodium nitrite H2S04 Acetone Anhydrous potassium carbonate Iodomethane Dichloromethane Pentane

2.3. Reagents for Method 3.3 1 2 3 4 5. 6 7 8 9

Sodium hydride (60% in mineral 011)(NaH). Tetrahydrofuran Dichloromethane. 1N HCI solution Ethyl acetate MgS04 (anh). Trifluoroacetic acid Saturated sodium chloride solution (brine). Saturated sodmm bicarbonate solution (NaHC03) 10. Hexane 11 Europnun trls[3-(heptafluoropropylhydroxymethylene)-(-)-camphorate]

(Eu(hfc)J

3. Methods The preparation of Cbz-protected ketomethylene drpeptrde rsosteres 1 mvolves three steps (Fig. 2). Step a 1s the synthesis of 4-Cbz-ammo-3-ketoesters 3 from Cbz-protected ammo acrds 2. Step b IS the synthesis of scalemic 2-trrflyloxy esters 5, from a-hyclroxy esters 4 (themselves prepared from ammo acids) Step c is the chiral alkylation of the enolate of 3 with triflyloxy esters 5 followed by decarboxylatlon to give the dipeptlde isostere 1

3.7. Preparation of Cbz-Protected from Cbz-Amino Acids 2

p-Ketoesters

3

The first operatton (step a, Fig. 1) converts Cbz-protected ammo acrds 2 to the correspondmg t-butyl P-ketoesters 3 by reaction wtth CDI, followed by treatment with the lrthmm enolate of t-butyl acetate (Fig. 3) (21,22) . A vart-

Hoffman and Tao

106

A “+H

++

“+,,bBu

CbzNH

CbzNH

Ot-Bu

3

Tf20

OH iHB

c

4

.BU+

‘Ot Bu CbrNH

3

OCH3

OCH3

5

R2J

R+,/( CbzNH

R2iji,

OCH3 2,6-lut;d,ne iTf

1. OTf

OCH3

TFA t

Fig 2 Three step sequence for the synthesis of ketomethylene

dlpeptlde lsosteres

0 Rl -2 CbzNH

‘OH ‘Ot-Bu

2a, RI= I-Bu (Leuclne) b, R,=(CH,), (Proline)

3a-g

(79-85%)

c, R,=I-Pr (Vahne) R,= BnOBn (Tyrosme, OBn)

d,

H&

d

(Tryptophan)

i-l

f, R,=Bn (Phenylalanme) g, RI= Me (Alanme)

Figure 3

ety of branched and unbranched stdechains, as well as heteroatom-contammg amino acids 2a-g were utthzed to demonstrate the generality of this conversion, whxh was found to be very good The ee’s of the p-keto esters were not determined, as the lrterature suggests that this conversron takes place without any racemrzatton of the a-posttton (21,22). Moreover, the optical purities determined for the ulttmate y-ketoester products also show that racenuzatron 1smstgmticant.

Cbz-Protected Ketomethylene Dipeptide lsosteres

107

3.1 1. (S)-tert-Buty/4-[(BenzyIoxycarbonyI) Amino]-3-Oxo-6-Methylheptanoate 3a Add CD1 (1 71 g, 10 5 mmol) to a stn-red solutton of N-Cbz-L-Leucme 2a (2 65 g, 10 0 mmol) in THF (20 mL) at room temperature under a N2 atmosphere You should see vigorous bubbling which disappears within 10 mm Stir the resultmg solutton for 40 mm at room temperature, and use tt for the next reaction wtthout purificatton. Durmg thts time, prepare a solution of hthtum tert-butoxycarbonylmethamde Add BULI (2 50 M, 12.6 mL, 3 1 5 mmol) (see Note 1) by syringe to a sol&ton of dusopropylamme (4 55 mL, 3 1 5 mmol) m dry THF (30 mL) under mtrogen that has been cooled to 0°C. Stir for 10 mm at 0°C and then cool to -78°C Add tertbutyl acetate (4.27 mL, 3 1 5 mmol) dissolved m dry THF (10 mL ) dropwtse by syrmge, and stir the resultmg mixture for 15 min Add the acyl tmtdazole solutton prepared m step 2 dropwrse to the pale yellow solutton of the hthmm enolate at -78°C under a N2 atmosphere. The reactton IS very exothermic, and you should see the boiling of the acetone-dry tee bath durmg the additton Stir the resultmg pale yellow mixture at -78°C for 40 mm Quench the reactton at -78°C wtth 1 N HCl(lO0 mL), extract wtth ethyl acetate (3 x 50 mL), combme the orgamc extracts, wash with brme (100 mL), dry (MgSOJ, filter the solution through a short pad of sthca gel (about 2 cm) on a fretted funnel, and concentrate by rotary evaporation to provide 3a as a pale yellow 011 Purify by flash chromatography (about 6 Inches of stltca gel) usmg hexane ethyl acetate 4 1 v/v) as the eluent You should get about 2.83 g, 76% (based on 2a) of 3a as pale yellow 011 [a]*50 = 4.38” (c 1 05, CHCl& ‘H NMR (CDC13) 6 0 93 (d, 3H,J= 6 2 Hz), 0 96 (d, 3H,J= 6 3 Hz), 1 36 (m, lH), 1 45 (s, 9H), 1 62 (m, 2H), 3 38 (d, lH,J= 13 6 Hz), 3.50 (d, lH,J= 13 6 Hz), 4 46 (m, lH), 5 10 (s, 2H), 5.41 (d, lH, J= 8.7 Hz), 7.33 (s, 5H); 13C NMR (CDCl$ 6 21 8,236,25 1,282,404, 479, 590, 674, 827, 1285, 366, 1566, 1665, 203 3; FTIR (neat) 3356,2977, 1715 (br), 1630 (m) cm-’

3.1.2. (S)-tert-Buty/3-[(N-Benzyloxycarbonyl) PyrrolWne-2]-3-Oxo-Propanoate 3b Use the same general procedure described above for 3a with the followmg amounts of reagents 1 React N-Cbz-L-Prolme (2.49 g, 10.0 mmol) wtth CD1 (1.71 g, 10.5 mmol) in THF (20 mL) 2 Prepare a solutton of hthtum tert-butoxycarbonylmethamde usmg dttsopropylamme (4.55 mL, 3 1 5 mmol), BuLt (2 50 M, 12 6 mL, 3 1 5 mmol), and tert-butyl acetate (4 27 mL, 31.5 mmol) m dry THF (30 mL +10 mL) 3. React these two soluttons at -78°C and work up as m step 5 of Subheading 3.1.1. 4. Purify by flash chromatography (hexane ethyl acetate 4.1 v/v). You should get about 2.95 g, 85% (based on 2b) of 3b as a colorless 011. [r~]~~,, =-64.4”

Hoffman and Tao

108

(c 3 85, CHCl,), ‘H NMR (CDCl,) ( ma J or rotamer) 6 3 56 (s, 2H), 3 60 (m, 2H), 4 48 (dd, lH, J= 7 4,9 0 Hz), 5.09 (s, 2H), 7 3 1 (m, 5H), 13CNMR (CDCI,) (mixture of rotamers) 6 23.9,24 6,28 3,28 8, 29 9,47 2, 48 2, 59.6, 65 4, 65 7, 676, 757, 81 3, 822, 128.1, 1289, 1366, 1369, 1547, 1555, 1665, 1668, 203 1, FTIR (neat) 2976, 1720 (br), 1642 (m) cm-’

3.7.3. (S)-tert-Butyl4-[(Benzyloxycarbonyl) Am/no]-3-Oxo-5Methylhexanoate 3c Use the same general procedure described above for 3a with the followmg amounts of reagents. 1 React N-Cbz-L-Vahne (2.51 g, 10 0 mmol) with CD1 (1 71 g, 10 5 mmol) m THF (20 mL) 2 Prepare a solution of llthmm tert-butoxycarbonylmethamde usmg dllsopropylamme (4 55 mL, 3 1 5 mmol), BuLl (2.50 A4, 12 6 mL, 3 1 5 mmol), and tertbutyl acetate (4 27 mL, 3 1 5 mmol) m dry THF (30 mL +10 mL). 3. React these two solutions at -78°C and work up as m step 5 of Subheading 3.1.1. 4 Purify by flash chromatography (hexane.ethyl acetate 4.1 v/v) You should get about 2 86 g, 82% (based on 2c) of 3c as a colorless 011. [u]~‘~ = +30 9” (c 2 80, CHCI,), ‘H NMR (CDC13) 6 0 80 (d, 2H, J = 6.9 HZ), 1.02 (d, 2H, J = 6.7 Hz), 1 45 (s, 9H), 1 48 (enol) (s, 9H) 2 26 (m, lH), 3 44 (s, 2H), 4 46 (dd, lH,J= 3 8, 8 9 Hz), 5 10, (s, 2H), 5 48 (d, IH, J= 8 9 Hz), 7 34 (s, 5H), 13C NMR (CDCl,) (major)6 16 9,20 4,28 4,30 1,48 7,65 2,67 5,82.8, 128 6, 136 7, 157 0, 166 3, 202 5, FTIR (neat) 3335,2987, 1731 (br), 1648 (m) cm-’

3.7 4. (S)-tert -Buty/ 4-[(Benzyloxycarbonyl) Amino]-3-Oxo-5-(4-Benzyloxy)Phenylpentanoate

3d

Use the same general procedure described above for 3a with the followmg amounts of reagents. 1 React N-Cbz-L-Tyrosine (3 00 g, 7 40 mmol) with CD1 (1 26 g, 7 77 mmol) m THF (20 mL) 2 Prepare a solution of llthmm tert-butoxycarbonylmethamde usmg dnsopropylamine (3 37 mL, 23 3 mmol), BULI (2 50 M, 9 32 mL, 23 3 mmol), and tert-butyl acetate (3 16 mL, 23 3 mmol) m dry THF (30 mL + 10 mL) 3 React these two solutions at -78”C, and work up as in step 5 of Subheading 3.1.1. 4 Purify by flash chromatography (hexane ethyl acetate 4.1 v/v) You should get about 2 94 g, 79% (based on 2d) of 3d asa white sohd. mp 63.5-64 5“C, [a]25D = +15 7” (c 6 25, CHCl,), ‘H NMR (CDCI,) 6 1 42 (s, 9H), 3 05 (m, 2H), 3 38 (s, 2H), 4 62 (dd, lH, J= 6 7,8 8 Hz), 4 95 (s, 2H), 5 03 (s, 2H), 5 56 (br, lH), 6 827.36 (m, 10H); 13CNMR (CDCl,) 6 28 4, 36 6, 48 6, 61 5, 70 3, 82 7, 1155, 128.5, 129.0, 130 8, 136 8, 137.5, 156 4, 158 3, 166 6, 202.4, FTIR (neat) 3356, 2987,172l (br), 1661 (m) cm-’

Cbz-Protected Ketomethylene

Dipeptide lsosteres

109

3 1.5. (S)- tert- Butyl4-[(BenzyIoxycarbonyI) Ammo]-3-Oxo-5-(3-indolyl) Pentanoate 3e Use the same general procedure described above for 3a with the following amounts of reagents 1 React N-Cbz-L-Tryptophan (3 38 g, 10 0 mmol) with CD1 (1 7 1 g, 10 5 mmol) m THF (20 mL) 2 Prepare a solution of lithium tert-butoxycarbonylmethamde usmg dnsopropylamme (4.55 mL, 3 1.5 mmol), BuLt (2.50 M, 12.6 mL, 3 1.5 mmol), and tertbutyl acetate (4.27 mL, 3 1.5 mmol) m dry THF (30 mL +lO mL) 3 React these two solutions at -78°C and work up as m step 5 of Subheading 3.1.1. 4 Purify by flash chromatography (hexane:ethyl acetate gradient 4:l to 2:l v/v). You should get about 3.40 g, 78% (based on 2e) of 3e as a glassy colorless oil [a]25D = +12 0” (c 0 55, CHCl$; ‘H NMR (CDC13) 6 1.38 (s, 9H), 3.18 (m, 2H), 3 32 (s, 2H), 4 72 (m, lH), 5 00 (s, 2H), 5.67 (d, lH, J= 6 6 Hz), 6 81-7 56 (m, lOH),8,59(br, lH), ‘3CNMR(CDC13)627.4,28.4,48.6,60.9,67.5, 82 8, 109.8, 112 0, 119.0, 120.2, 122 6,123 8, 127 9, 128 6, 129 2, 136 8, 156 6, 166.9,203.3, FTIR (neat) 3376,2947, 1725 (br), 1636 (m) cm-l

3.1.6. (S)-tert -Buty/ 4-[(BenzyloxycarbonyI) Amino]-3-Oxo-5-Phenylpentanoate 3f Use the same general procedure described above for 3a with the followmg amounts of reagents. I

React N-Cbz-L-Phenylalanme (2.99 g, 10.0 mmol) wrth CD1 (1 7 1 g, 10 5 mmol) m THF (20 mL). 2. Prepare a solutton of hthmm tert-butoxycarbonylmethatude usmg dnsopropylamine (4.55 mL, 3 1.5 mmol), BuLi (2.50 M, 12 6 mL, 3 1 5 mmol), and tert-butyl acetate (4 27 mL, 3 1 5 mmol) m dry THF (30 mL +lO mL) 3. React these two solutions at -78°C and work up as m step 5 of Subheading 3.1.1. 4 Purify by flash chromatography (hexane:ethyl acetate 4 1 v/v) You should get about 3 21 g, 81% (based on 2f) of 3f as a pale yellow 011 [IX]~~~ = +ll.l” (c 5.00, CHCI-J, ‘H NMR (CDCl,) 6 1.44 (s, 9H), 3 09 (m, 2H), 3 38 (s, 2H), 4 66 (m, lH), 5.03 (s, 2H), 5 59 (d, 2H, J= 7.5 Hz), 7.24 (m, 5H); 13CNMR (CDCl,) 6 284, 382, 48 5, 61 3, 674, 82 7, 1274, 128.5, 1292, 129 8, 1365, 1564, 166 4,202.3, FTIR (neat) 3317,2976, 1720 (br), 1661 (m) cm-’

3.7 7. (S)-tert -B&y/ 4@enzy/oxycarbony/) Ammo]-3-Oxo-Pentanoate 3g Use the same general procedure described above for 3a with the followmg amounts of reagents. 1 React N-Cbz-L-Alanine THF (20 mL)

(2 23 g, 10.0 mmol) with CD1 (1.71 g, 10.5 mmol) in

110

Hoffman and Tao

2 Prepare a solution of lithium tert-butoxycarbonylmethamde using dnsopropylamine (4.55 mL, 3 1 5 mmol), BuLi (2 50 M, 12 6 mL, 3 1.5 mmol), and tertbutyl acetate (4 27 mL, 3 1.5 mmol) in dry THF (30 mL +lO mL) 3. React these two solutions at -78°C and work up as m step 5 of Subheading 3.1.1. 4. Purify by flash chromatography (hexaneaethyl acetate 4.1 v/v) You should get about 2 57 g, 80% (based on 2g) of 3g as a pale yellow oil* [a]250 = +lO 2” (c 3 60, CDCl,), ‘H NMR (CDCl,) 6 1 35 (d, 3H, .I= 7 2 Hz), 1.44 (s, 9H), 3 46 (s, 2H), 4.45 (m, lH), 5 09 (s, 2H), 5.73 (d, lH,J= 6.7 Hz), 7.32 (s, 5H), 13CNMR (CDCl,) 6 17 5, 28 3, 47 5, 56 2, 67.3, 82 7, 128 5, 128 9, 136 6, 156 3, 166 5, 202 8, FTIR (neat) 3326,2977, 1710 (br), 1648 (m) cm-’

3.2. Preparation of (2R) 2-Triflyloxy from R-Amino Acids

Esters 5

The second step (step b, Fig. 2) is to prepare a-triflyloxy esters 5 from a-hydroxy esters 4. Optically pure a-hydroxy esters 4 are available by a variety of methods (24). Methyl lactate 4a 1savailable commercially in either configuration. For the other hydroxy esters 4b-f needed in this work, conversion of ammo acids to optl-

tally pure a-hydroxy esters1ssimple and convenient and provides a wide range of a-hydroxy esters m a choice of configurations (Fig. 4). The configuration of the ahydroxy ester; hence, the a-triflyloxy ester will be the same as that of the startmg

ammo acid.Reaction of the a-hydroxy ester with trlflic anhybde and lutidme as an acid scavenger gives the a-tnflyloxy ester in excellent yields. In some previous studies, the solution of the a-tr~flyloxy ester and the lutidmmm salt was used directly m the next step (I&19) This protocol does not work for the synthesis of Cbz-protected ketoesters 3 because of the presence of the lutldmmm salt by-prod-

uct. Consequently a very simple method was developed to isolate the a-triflyloxy ester m very pure form. Excellent results could then be obtained m the subsequent chiral alkylatlon. ~Triflyloxy esters 5&were prepared m order to have a selec-

tion of R2 groups to include m the final dipeptlde lsosteres. 3.2.1. (RI-Methyl 2-Hydroxypentanoate

46

Add a solution of NaN02 (4 30 g, 62 3 mmol) m H20 (20 mL) dropwlse to an icecooled solution of D-norvalme (5.00 g, 42.7 mmol) m 1 N H2S04 (68 3 mL, 68 3 mmol) with vigorous stirring Stir the resultmg solutlon at the same temperature for 1 h, and then at room temperature for 12 h. Extract the aqueous reactlon mixture with ethyl ether (5 x 100 mL), combme the ethereal extracts, and concentrate by rotary evaporator Dry the residue by addmg benzene (200 mL) and refluxmg with a Dean-Stark trap (see Note 2) Concentrate by rotary evaporation. Dissolve the pale yellow, crude 2-hydroxy acid m acetone (100 mL), and add K2C03 (6 49 g, 47 0 mmol). Stir the white suspension at room temperature

111

Cbz-Protected Ketomethylene Dipeptlde lsosteres (TflzO 3 lutldme 4a, 4b, 4c, 4d, 4e, 4f,

%=Me

RJOCH dTf

3

5a-f (&S-95%)

Rpn-Pr q=Bn

%=C,H,,CH, R.p I-Bu R.p I-Pr

(70-75%) Figure 4

for 1 h, and then add todomethane (3 42 mL, 54.6 mmol) by a syringe. Reflux the resultmg mrlky solutton under mtrogen overnight, filter through a short pad of sllrca gel (about 2 cm) on a fretted funnel, and remove the solvent by rotary evaporation 5 Purify the product by Kugelrohr dtsttllatton (bath temperature 60-65°C 0 07 mmHg) (You might need to take several fractions). You should get about 4 06 g, 72% (based on the ammo acid) of 4b as a colorless 011, [u]~~,, = -9 68” (c 0 95, CHCls); ‘H NMR (CDCl,) 6 0 94 (t, 3H, J= 7 3 Hz), 1.45-1 71 (set of m, 4H), 2 76 (d, lH, J= 5 7 Hz), 3 79 (s, 3H), 4.20 (m, 1H)

3.2.2. (I?)-Methyl 2-Hydroxy-4-Phenylpropanoate

4c

Use the same general procedure described above for 4b with the followmg amounts of reagents. D-3-phenyllactic acid was purchased commercially, so it was not prepared from the ammo acid by drazottzatton. 1 Dissolve D-3-phenyllacttc acid (5 00 g, 30.1 mmol) in acetone (100 mL), and add K2C03 (4 58 g, 33 1 mmol) Stir the white suspension at room temperature for 1 h, and then add todomethane (2.41 m, 38.5 mmol) by syringe Reflux the resulting milky solutton under nitrogen overnight, filter through a short pad of srhca gel (about 2 cm) m a fretted funnel, and remove the solvent by rotary evaporation 2 Purify the crude 2-hydroxy ester by Kugelrohr dtstillatton (bath temperature 12OWO 07 mmHg) You should get about 5.15 g, 95% of 4c as a white solid, mp 45-46”C, [al250 = +11 2” (c 2 8, CHCls); ‘H NMR (CDCl,) 6 2.74 (d, lH, J= 9 0 Hz), 3 05 (m, 2H), 3.77 (s, 3H), 4.45 (m, lH), 7 27 (m, 5H)

3.2.3. @)-Methyl 2-Hydroxy-3-Cyclohexylpropanoate

4d

Use the same general procedure descrtbed above for 4b with the followmg amounts of reagents.

112

Hoffman

and Tao

1 Add a solution of NaNO, (2.92 g, 42 3 mmol) m H,O (20 mL) to an Ice cooled solution of D-3-cyclohexylalanine (5 00 g, 29 0 mmol) dissolved m 1 N H,SO, (46.4 mL, 46 4 mmol) Stir and workup as described m step 3 of Subheading 3.2.1.

2 Dissolve the crude hydroxy acid m acetone (100 mL), and add K&O, (4 4 1 g, 3 1 9 mmol) followed by lodomethane (2.32 mL, 37 1 mmol) 3 After the normal reaction time and work up as m step 4 of Subheading 3.2.1., purify the crude hydroxy ester by Kugelrohr dlstlllatlon (bath temperature 150-l 8OWO 07 mmHg) You should get about 3 78 g, 70% (based on ammo acid) of 4d as a colorless 011 [alz5,, = +0 24” (c 2 1, CHCl,), ‘H NMR (CDCI,) 6 0 78- 1.92 (set of m, 13H), 2 68 (br, lH), 3 78 (s, 3H), 4 24 (dd, lH, J= 4 5, 9 2 Hz) 3.2 4. (R)-Methyl

2-Hydroxy-4-Methylpentanoate

4e

Use the same general procedure described above for 4b with the following amounts of reagents 1 Add a solution of NaN02 (5 04 g, 73 0 mmol) m H20 (20 mL) to an Ice-cooled solution of D-leucme (6 56 g, 50 0 mmol) dissolved m 1 N H$O, (80 0 mL, 80 0 mmol) Stir and work up as described m step 3 of Subheading 3.2.1. 2 Dissolve the crude hydroxy acid m acetone (100 mL), and add K&O3 (7 60 g, 55.0 mmol) followed by lodomethane (4.00 mL, 64 0 mmol) 3 After the normal reactlon time and work-up as m step 4 of Subheading 3.2.1,, purify the crude hydroxy ester by Kugelrohr dlstlllatlon (bath temperature 6OWj5”C/O 07 mmHg) You should get about 5 12 g, 70% (based on ammo acid) of 4e as a colorless 011 [a]250 = -2 2” (c 2 2, CHCI,), IH NMR (CDClj) 6 0 92 (d, 3H, J= 2 2 Hz), 0.96 (d, 3H, J= 6 5 Hz), 1 54 (t, 2H, J = 6 5 Hz), 1 89 (m, lH), 2.91 (d, lH,J= 6 1 Hz), 3 78 (s, 3H), 4 22 (m, 1H) 3.2 5. (R)-Methyl Z-Hydroxy-3-Methylbutanoate

4f

Use the same general procedure described above for 4b with the followmg amounts of reagents. 1 Add a solution of NaN02 (5 12 g, 74 2 mmol) m H,O (20 mL) to an icecooled solution of D-valme (6.00 g, 50.8 mmol) dissolved In 1 N H2S04 (81 3 m, 81 3 mmol) Stir and work up as described m step 3 of Subheading 3.2.1.

2 Dissolve the crude hydroxy acid m acetone (100 mL), and add K2C03 (7 72 g, 55 9 mmol), followed by lodomethane (4 06 mL, 65 0 mmol) 3 After the normal reactlon time and work up as m step 4 of Subheading 3.2.1., purl@ the crude hydroxy ester by Kugelrohr dlstlllatlon (bath temperature 5&7O”C/ 0 07 mmHg). You should get about 4.06 g, 71% (based on ammo acid) of 4f as a colorless oil: [cI]~~, =-23 1” (c 2.5, CHCl,); ‘H NMR (CDC13) 6 0 87 (d, 3H, J = 6 8 Hz), 1 02 (d, 3H, J= 7 0 Hz), 2 07 (m, lH), 2 78 (d, lH, J= 6.1 Hz), 3.80 (s, 3H),405(dd,lH,J=37,61Hz)

Cbz-Protected Ketomethylene

113

Dipeptide lsosteres

3 2 6. @)-Methyl 2-Triflyloxypropanoate

5a

1 Add trlfllc anhydride (0 90 mL, 5.25 mmol) (see Note 3) to a stirred solution of (R)-methyl 2-hydroxypropanoate 4a (520 mg, 5 00 mmol) m dichloromethane (20 mL) at 0°C under a mtrogen atmosphere 2 Slowly add 2,6-lutldme (0.61 mL, 5 25 mmol) by syringe You should see white vapor form over the solution 3 Stir the resulting pmk solution for 20 min, and then concentrate by rotary evaporation. Dissolve the residue m pentane (150 mL), and cool the solution m dry ice for several minutes to help precipitate the lutldmlum salt Filter on a Buchner funnel, and concentrate the filtrate to provide trlflate Sa as a pale pmk 011. Isolated samples of the triflate can be stored m a freezer for months without decomposition, as monitored by ‘H NMR You should get about I .05 g, 89% (based on 2-hydroxy ester). ‘H NMR (CDCl& 6 1 72 (d, 3H, J= 7 0 Hz), 3 86 (s, 3H), 5 25 (q, lH, J= 7 0 Hz)

3 2.7 (R)-Methyl2-Tnflyloxypentanoate

56

Use the same general procedure described above for 5a with the followmg amounts of reagents. 1 Add trlfllc anhydrlde (1 80 mL, 10 5 mmol) to (R)-methyl2-hydroxypentanoate 4b (1.32 g, 10 0 mmol) m dlchloromethane (40 mL) 2 Slowly add 2,6-lutldme (1 22 mL, 10 5 mmol) by syringe 3 Isolate trlflate 5b by dlssolutlon in pentane, filtration, and concentration as m step 3 of Subheading 3.2.6. You should get about 2 48 g, 94% (based on 4b), ‘H NMR (CDC13) 6 0 99 (t, 3H, J = 7 4 Hz), 1 50 (m, 2H), 1 97 (m, 2H), 3 85 (s, 3H),5 14(t, lH,J=67Hz)

3.2 8. (R)-Methyl2-Triflyloxy-3-Phenylpropanoate

5c

Use the same general procedure described above for 5a with the following amounts of reagents. 1 Add trlfllc anhydride (1 80 mL, 10.5 mmol) to (@-methyl 2-hydroxy-3phenylpropanoate) 4c (1 80 g, 10 0 mmol) in drchloromethane (40 mL) 2 Slowly add 2,6-lutldme (1 22 mL, 10.5 mmol) by syringe. 3 Isolate trlflate 5c by dlssolutlon m pentane, filtration, and concentration as m step 3 of Subheading 3.2.6. You should get about 2 95 g, 95% (based on 4~); ‘H NMR (CDCl,) 6 3 29 (m, 2H), 3 83 (s, 3H), 5 25 (dd, lH, J= 4 2,4 5 Hz), 7 32 (m, 5H)

3.2.9. (R)-Methyl2-Triflyloxy-3-Cyclohexylpropanoate

5d

Use the same general procedure described above for 5a with the followmg amounts of reagents. 1 Add trlfllc anhydride (1 OS mL, 6 30 mmol) to (Q-methyl 2-hydroxy-3cyclohexylpropanoate 4d (1 12 g, 6 00 mm01 ) in dlchloromethane (40 mL) 2 Slowly add 2,6-lutldme (0 73 mL, 6 30 mmol) by syringe.

Hoffman and Tao

114

3. Isolate triflate 5d by dissolution in pentane, filtration, and concentration as in step 3 of Subheading 3.2.6. You should get about 1.64 g, 86% (based on 4d); ‘H NMR(CDCl,)G0.97-1.71 (setofm, llH), 1.90(m,2H),3.84(~,3H),5.20(dd, lH, J= 4.5, 5.4 Hz).

3.2.10. (~)-Methy~2-Triflyfoxy-4-Methylpentanoate

5e

Use the same general procedure described above for 5a with the following amounts of reagents. 1. Add triflic anhydride (1.80 mL, 10.5 mmol) to (R)-methyl 2-hydroxy-4methylpentanoate 4e (1.46 g, 10.0 mmol) in dichloromethane (40 mL). 2. Slowly add 2,6-lutidine (1.22 mL, 10.5 mmol) by syringe. 3. Isolate triflate 5e by dissolution in pentane, filtration, and concentration as in step 3 of Subheading 3.2.6. You should get about 2.50 g, 90% (based on 4e); ‘H NMR (CDCl,) 6 0.95 (d, 6H, J = 8.1 Hz), 1.80 (m, 2H), 1.97 (m, lH), 3.85 (s, 3H), 5.16 (dd, lH, J= 3.6, 5.4 Hz).

3.2. Il. (R)-Methyl2-Triflyloxy-3-Methylbutanoate

5f

Use the same general procedure described above for 5a with the following amounts of reagents. 1. Add triflic anhydride (1.80 mL, 10.5 mmol) to (@-methyl 2-hydroxy-4methylpentanoate 4f (1.32 g, 10.0 mmol) in dichloromethane (40 mL). 2. Slowly add 2,6-lutidine (1.22 mL, 10.5 mmol) by syringe. 3. Isolate triflate 5f by dissolution in pentane, filtration, and concentration as in step 3 of 3.2.6. You should get about 2.5Og, 90% (based on 40; ‘H NMR (CDCl,) 6 1.00 (d, 3H,J= 2.2 Hz), 1.03 (d, 3H, J= 2.3 Hz), 2.40 (m, lH), 3.85 (s, 3H), 4.98 (d, lH, J= 4.0 Hz).

3.3. Preparation of C&-Protected Ketomethylene Dipeptide lsosteres

7

The third step (step c, Fig. 2) is the chiral alkylation of the enolate of pketoester 3 with a scalemic a-triflyloxy ester 5 followed by decarboxylation to give 1 (Fig. 5). This reaction is successful for a variety of substitution patterns (R, and RJ. Not all combinations of R, and R2 groups have been employed; rather, a representative group was chosen to demonstrate the range of structures that can be accessed. The overall yields are acceptable and not greatly different for different structural combinations. The diastereoselectivity of the process is very good with de’s generally ranging from 84-96% (Table 1). The diastereomers of 1 are readily separable by flash chromatography, and the major diastereomer was examined with the chiral lanthanide shift reagent Eu(hfc), in order to determine the ee’s. In most cases, only a single enantiomer was detected (ee > 95%) (Table 1).

115

Cbz-Protected Ketomethylene Dipeptide lsosteres

R, CbzNH

Ot-Bu 3a-g 5a-f OMe

laa, lbb, ICC, ldc, led, lfe, lgf Figure 5

3.3.1. Cbz-LeuY[COCH~(R,S)Ala-OMe

laa

1. Add a solution of 3-oxo ester 3a (726 mg, 2.00 mmol) in THF (10 mL) dropwise to a stirred suspension of NaH (84.0 mg of 60% in oil, 2.10 mmol) in dry THF (30 mL) at O°C under a nitrogen atmosphere. (You should see the bubbling from the evolution of Hz). 2. Stir the resulting mixture for 10 min to complete the formation of the enolate of 3a. 3. Add a solution of triflate 5a (949 mg, 4.02 mmol) in dichloromethane (10 mL) dropwise to the gray suspension of the 3-0~0 ester enolate. 4. Stir the resulting mixture at room temperature for 24 h. Quench the reaction with 1 NHCl(50 mL) and extract with ethyl acetate (3 x 50 mL). Combine the organic extracts, wash with brine (100 mL), dry (MgSOJ, filter through a short pad of silica gel (about 2 cm) in a fritted funnel, and concentrate by rotary evaporation to provide a pale yellow oil. 5. Without further purification, dissolve the oil in dichloromethane (10 mL) and treat with TFA (1.5 mL) (see Note 4) at room temperature for 24 h. 6. Dilute the resulting pale yellow solution with dichloromethane (50 mL), wash with saturated NaHCOs (2 x 50 mL) and brine (50 mL), dry (MgSOJ, and concentrate to provide laa as a colorless oil. 7. Purify the crude product by flash chromatography (6 inch column; hexane:ethyl acetate gradient from 4: 1 to 2: 1 v/v). You should get about 3 14 mg, 48% (based on 3a) of a mixture of diastereomers of 96% de, based on ‘H NMR. HPLC analysis (normal phase, 1.5 mL/min, hexane:ethyl acetate 1: 1 v/v, UV = 254 nm) should corroborate the diastereoselectivity found by ‘H NMR. tR 3.99 (major), 4.16 (minor); ratio 98:2. 8. Separate the major diastereomer by flash chromatography (15 inch column; hexane:ethyl acetate gradient from 4: 1 to 2: 1 v/v). The major diastereomer should have 95% e.e. by a chiral LIS study using Eu(hfc), in comparison with a racemic sample (see Note 5); [a]250 = -6.00” (c 0.40, CHCl,); ‘H NMR (CDCls) 6 0.92 (d,

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Table 1 Stereochemical Results of the Preparation of Peptide lsosteres by Chiral Alkylation Entry 1 2 3 4 5 6 7

Product

Yield (%)

de(%)b

ee(%)c

96 92 84 88 90 90 74

95 >95 >95 >95 >95 >95 92

48 45 52 45 43 62 40

laa lbb ICC ldc led lfe lgf

Qolated yields of chromatographed mixtures of dlastereomers *DetermIned by ‘H nmr and HPLC LDetermmed by choral LIS study on separated major dlastereomer

3H, J= 6 3 Hz), 0 97 (d, 3H,J=

6 0 Hz), 1 18 (d, 3H, J= 6 8 Hz), 1 40 (m, IH), 1 62 (m, 2H), 2 57 (m, lH), 2 96 (m, 2H), 3 65 (s, 3H), 4 39 (m, lH), 5.10 (s, 2H), 5 46 (d, lH, J= 8.1 Hz), 7 34 (s, 5H), 13C NMR (CDC&) 6 17 4,22 0,23 6,

25 2,35 0,40 6,42 9,52 3,58 8,67 3, 128 5, 128 9, 136 8, 156 6, 176 4,208 7,

FTIR (neat) 3336,2947,

1736, 1721 (br) cm-‘.

3.3 2 Cbz-ProY[COCHJ(R,S)Nva-OMe

Ibb

Use the same general procedure described above for laa with the followmg amounts of reagents. Add a solution of 3b (520 mg, 1 50 stu-red suspension of NaH (63 0 mg, After stu-rmg 10 mm, add a solution (7 5 mL) Stu- 24 hours and work up as m step

mmol) m dry THF (7 5 mL) dropwlse to a 1 58 mmol) m THF (25 mL) of triflate 5b (4 16 mg, 3 15 mmol) m THF 4 of Subheading

3.3.1.

Dissolve the residue m dlchloromethane (7 5 mL), treat with TFA (1 12 mL) for 24 hours, and work up the reaction mixture as m step 6 of Subheading 3.3.1. Purify the crude product by flash chromatography

(7 mch column, hexane-ethyl

acetate gradlent from 9 1 to 3 2 v/v) You should get about 244 mg, 45% of lbb, a colorless 011, as a mixture of dlastereomers with 92% de, based on ‘H NMR analysis of the mixture HPLC analysis (normal phase, 1.5 mL/mm, hexane ethyl acetate 1 1 v/v, UV = 254 nm) should corroborate the dlastereoselectlvlty by ‘H NMR t, 2.89 (minor), 5 87 (major), ratlo 96 4

found

Separate the major dlastereomer by flash chromatography (15 mch column, hexaneeethyl acetate gradlent from 9 1 to 3 2 v/v) The major dlastereomer has e e > 95% determined by a choral LIS study. [a]250 = -46 8” (c 0 50, CHC13), ‘H NMR (CDC13) 6 0 86 (major rotamer) (t, 3H, J = 7 3 Hz), 0 90 (mmor)

C&-Protected

Ketomethylene Dipeptlde isosteres

117

(t, 3H, J= 7 2 Hz), 1 27 (m, 2H), 1.50 (m, 2H), 2 14 (m, 2H), 2.25 (m, 2H), 2.61 (m, lH), 2 97 (m, 2H), 3 56 (m, 2H), 3 64 (minor) (s, 3H), 3.66 (major) (s, 3H), 4.37(mmor)(dd, lH,J=45,5.OHz),4.47(ma~or)(dd, lH,J=4.4,5 1 Hz),5.02 (d, lH, J= 11 5 Hz), 5 12 (d, lH, J= 11.5 Hz), 5 07 (d, lH, J= 14 0 Hz), 5 16 (d, lH, J= 14.0 Hz), 7 33 (m, 5H), t3C NMR (CDCl,) (mtxture of rotamers) 6 14 3, 20 5, 23 9, 24 6, 28 8, 30 0, 34 4, 39 6, 41 7, 42 1, 47 0, 47 6, 52 1, 64 8, 65 1, 67.6, 128.5, 128 9, 136 6, 136 8, 154.7, 155.4, 176 2, 176 5,208 0,208 3; FTIR (neat) 2957, 1727, 1711 cm-‘.

3.3.3. Cbz- VaiY[COCH2](R, S) Phe-OMe 1cc Use the same general procedure described above for laa with the followmg amounts of reagents. 1 Add a solution of 3c (524 mg, 1 50 mm01 ) in dry THF (7.5 mL) dropwrse to a stu-red suspension of NaH (63 0 mg, 1.58 mmol) in THF (25 mL) 2 After stmmg 10 mm, add a solutton of trtflate SC(570 mg, 3.16 mmol) m THF (7 5 mL) 3 Stir 24 hours, and work up as m step 4 of Subheading 3.3.1. 4. Dissolve the residue m drchloromethane (7 5 mL), treat with TFA (1 12 mL) for 24 hours, and work up the reaction mixture as m step 6 of Subheading 3.3.1. 5 Purify the crude product by flash chromatography (7 mch column; hexane ethyl acetate 4.1 v/v). You should get about 320 mg, 52% of ICC, a colorless 011,as a mixture of drastereomers with 84% de, based on ]H NMR analysts of the mixture HPLC analysts (normal phase, 1.5 mL/mm, hexaneeethyl acetate, 1 1 v/v, UV = 254 nm) should corroborate the diastereoselectivrty found by ‘H NMR tR 2.96 (major), 4.07 (minor); ratio 92 8 6 Separate the major drastereomer by flash chromatography (15 mch column, hexane ethyl acetate, 4: 1 v/v) The major dtastereomer has e e. > 95% determmed by a choral LIS study. [a]25n = +32 3” (c 0.65, CHC13); ‘H NMR (CDC13) 6 0.74 (d, 3H, J= 6 9 Hz), 0 99 (d, 3H, J= 6 6 Hz), 2 22 (m, lH), 2 60 (m, IH), 3 02 (m, IH), 3 17 (m, 2H), 3 62 (s, 3H), 4 30 (dd, lH, J= 4 5,6 7 Hz), 5 06 (s, 2H), 5 28 (d, lH, J= 4 5 Hz), 7 32 (m, lOH), 13C NMR (CDC13) 6 16 6, 19 2,30 6,38 2, 41 9,59 1,64 5,67.4,74.0, 128 6, 129 0, 127 3, 136 7, 1.56 9, 175 4,207 5, FTIR (neat) 3366,2977, 1728, 1711 cm-’

3.3.4. Cbz-Tyr(Bzi)Y(COCi-i,)(R,S)Phe-OMe Idc Use the same general procedure described above for laa with the followmg amounts of reagents. 1 Add a solution of 3d (951 mg, 1 89 mm01 ) m dry THF (7 5 mL) dropwtse to a stirred suspension of NaH (79.4 mg, 1 98 mmol) m THF (25 mL) 2 After stirring 10 mm, add a solution of t&late 5c (715 mg, 3 97 mmol) m THF (7 5 mL) 3 Stir 24 hours, and work up as m step 4 of Subheading 3.3.1. 4 Dissolve the residue in dtchloromethane (7.5 mL), treat wtth TFA (1.42 mL) for 24 hours, and work up the reaction mrxture as m step 6 of Subheading 3.3.1.

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5. Purify the crude product by flash chromatography (7 mch column; hexane:ethyl acetate gradient from 4.1 to 2.1 v/v) You should get about 480 mg, 45% of ldc, a colorless oil, as a mixture of diastereomers with 88% de, based on ‘H NMR analysis of the mixture HPLC analysis (normal phase, 1 5 mL/mm, hexane ethyl acetate 1 1 v/v, UV = 254 nm) should corroborate the dtastereoselectivtty found by ‘H NMR, t, 2 10 (minor), 2.86 (major), ratto 94.6 6 Separate the major dtastereomer by flash chromatography (15 inch column, hexane’ethyl acetate gradient from 4.1 to 2: 1 v/v) The major diastereomer has e e > 95% determmed by a choral LIS study [cx]~~~ = +24 4” (c 0 45, CHCl& ‘H NMR (CDCls) (major) 6 2 50 (m, 2H), 2 66-3 12 (set of m, 5H), 3 60 (s, 3H), 4 53 (dd, lH, J= 6 7,7 2 Hz), 5 00 (s, 2H), 5 06 (s, 2H), 6 52-7 40 (set of m, 19 H), i3C NMR (CDC13) (major) F 37 2, 37 7, 38 0, 41 4, 42 4, 52 4, 61.2, 67 3, 1154,1272,1279,1285,1290,1306,1368,1373,1387,1562,1583,1752, 207 4; FTIR (neat) 3346,2967, 1726 (br), 1611 (m) cm-’

3.3 5. Cbz-TrpY[COCH&?,S)Cha-OMe

led

Use the same general procedure described above for laa with the followmg amounts of reagents 1 Add a solution of 3e (654 mg, 1 50 mmol) m dry THF (7 5 mL) dropwise to a stirred suspension of NaH (63 0 mg, 1 58 mmol) m THF (25 mL) 2. After stirring 10 mm, add a solution of tnflate 5d (586 mg, 3 15 mmol) in THF (7 5 mL) 3 Stir 24 hours, and work up as m step 4 of Subheading 3.3.1. 4 Dissolve the restdue m dichloromethane (7 5 mL), treat with TFA (1 12 mL) for 24 hours, and work up the reaction mixture as m step 6 of Subheading 3.3.1. 5. Purify the crude product by flash chromatography (7 mch column, hexane ethyl acetate gradient from 4: 1 to 2 1 v/v). You should get about 325 mg, 43% of led, a colorless oil, as a mixture of diastereomers with 90% de, based on ‘H NMR analysis of the mixture HPLC analysis (normal phase, 1 5 mL/mm, hexane ethyl acetate 1 1 v/v, UV = 254 nm) should corroborate the diastereoselectivity found by ‘H NMR. tR 2 80 (malor), 3 24 (mmor); ratto 95 5 6. Separate the malor diastereomer by flash chromatography (15 mch column, hexane ethyl acetate gradient from 4 1 to 2 1 v/v) The major diastereomer has e e >95% determined by a choral LIS study [c~]~~n= +I 1 0” (c 0 01, CHCl,), ‘H NMR (CDCl,) (major) 6 0.741 64 (set of m, 13H), 2 37 (m, lH), 2 82 (m, 2H), 3 22 (d, 2H, J= 6 5 Hz), 3 62 (s, 3H), 4.68 (m, lH), 5 08 (s, 2H), 5 54 (d, lH, J= 7 2 Hz), 6 927 10 (set of m, 9H), 8 22 (br, IH), 13CNMR (CDCls) (major) F 26 5,28.0,33 4,35 4, 37 7, 39.9, 43 0, 52.2, 60 4, 67 3, 110.4, 111.7, 119 0, 120 3, 122 8, 123 2, 128 6, 136.7, 156 3, 176 7,208 6, FTIR (neat) 3416,2927, 1711 (br), 1648 (m) cm-’

3.3 6 Cbz-PheY[COCHJ(R,S)Leu-OMe

lfe

Use the same general procedure described above for laa with the followmg amounts of reagents.

Cbz-Protected Ketomethylene Dipeptide lsosteres

119

1 Add a solution of 3f (794 mg, 2 00 mmol ) m dry THF (7 5 mL) dropwise to a stirred suspension of NaH (84 0 mg, 2 10 mmol) m THF (25 mL) 2 After stirrmg 10 min, add a solution of triflate 5e (614 mg, 4.20 mmol) m THF (7 5 mL) 3 Stir 24 hours and work up as in step 4 of Subheading 3.3.1. 4. Dissolve the residue m dichloromethane (7 5 mL), treat with TFA (1 50 mL) for 24 hours, and work up the reaction mixture as m step 6 of Subheading 3.3.1. 5 Purify the crude product by flash chromatography (7 mch column; hexane ethyl acetate gradient from 9 1 to 4 1 v/v) You should get about 527 mg, 62% of lfe, a colorless 011, as a mixture of dlastereomers with 90% de, based on ‘H NMR analysis of the mixture. HPLC analysts (normal phase, 1 5 mL/min, hexanesethyl acetate 1.1 v/v, UV = 254 nm) should corroborate the diastereoselectivity found by ‘H NMR. t, 2 12 (mmor), 2.64 (major); ratio 95.5 6 Separate the major diastereomer by flash chromatography (15 inch column, hexane ethyl acetate gradient from 9:l to 4 1 v/v) The major diastereomer has e.e >95% determmed by a chit-al LIS study. [a]25t, = +15 5” (c 1.95, CHCl,), ‘H NMR (CDCl,) 6 0 85 (d, 3H, J= 6 0 Hz), 0 87 (d, 3H, J= 6 1 Hz), 1 20 (m, lH), 1 55 (m, 2H), 2 34 (m, lH), 2 83 (m, 2H), 3 07 (m, 2H), 3.64 (s, 3H), 4 60 (m, IH), 5 08 (s, 2H), 5 32 (d, lH, J= 6 7 Hz), 7 33 (m, lOH), t3C NMR (CDCl,) 6 21 8, 22 8, 23 5, 26 1, 38 4, 41 5, 52 2, 61 0, 67.3, 72 2, 127 4, 128 5, 128 9, 129.6, 136.6, 156 3, 176.4,208.0; FTIR (neat) 3346, 2967, 1716 (br) cm-’

3.3.7. Cbz-Ala Y[COCHJ(R, S) Val-OMe Igf Use the same general procedure described above for laa with the followmg amounts of reagents. Add a solution of 3g (250 mg, 0 78 mmol ) m dry THF (7 5 mL) dropwtse to a stirred suspension of NaH (32.8 mg, 0.82 mmol) m THF (25 mL). After stirring 10 mm, add a solution of triflate 5f (217 mg, 1 64 mmol) m THF (7 5 mL) Stir 48 hours, and work up as in step 4 of Subheading 3.3.1. Dissolve the residue m drchloromethane (7.5 mL), treat with TFA (0 59 mL) for 24 hours, and work up the reactron mtxture as m step 6 of Subheading 3.3.1. Purify the crude product by flash chromatography (7 mch column, hexane*ethyl acetate gradient from 9 1 to 4 1 v/v) You should get about 104 mg, 40% of lgf, a colorless 011, as a mrxture of drastereomers with 74% de, based on ‘H NMR analysis of the mixture HPLC analysis (normal phase, 1 5 mL/mm, hexane:ethyl acetate 1.1 v/v, UV = 254 nm) should corroborate the diastereoselectivity found by ‘H NMR. tR 4.22 (major), 5.44 (minor); ratio 87: 13 Separate the major dtastereomer by flash chromatography (15 mch column, hexane.ethyl acetate gradient from 9 1 to 4.1 v/v) The major diastereomer has e.e. 92%, determined by a choral LIS study. [a]25i, = +60 0” (c 0 05, CHCl,), ‘H NMR (CDCl,) 6 0.85 (d, 3H, J= 4 4 Hz), 0 88 (d, 3H, J = 4.5 Hz). 1.34 (d, 3H, J = 7 0 Hz), 2.05 (m, lH), 2 50 (m, lH), 2.88 (m, 2H), 3 66 (s, 3H), 4 42 (m, lH),

120

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5 11 (s, 2H), 5 57 (d, lH, J = 5 4 Hz), 7 40 (s, 5H), 13CNMR (CDCl,) 6 17 0, 18.6, 19 5, 30 0, 36 8, 46.3, 52 1, 55 8, 66 8, 128 1, 1363, 155.6, 174 9, 208 3, FTIR (neat) 3346,2967, 1726(br) cm-] 3.4. Discussion The above procedures constitute an exceedingly simple, general, and stereoselective method for the preparation of ketomethylene dipeptide isosteres from Cbz-protected ammo acids and scalemic 2-triflyloxy esters The method is short (three steps), efficient, and highly diastereoselective and enantioselective While not every combination of P-ketoester 3 and 2-triflyloxy ester 5 was used, the very similar yields for all casesstudied (Table 1) indicate that this method is very structurally tolerant. Similar results would be expected for other combmattons of interest. Two equivalents of the 2-triflyloxy ester 5 were used m the choral alkylanon, although no real attempts were made to opttmize the yield of this step. The excess 2-triflyloxy ester cannot be isolated after the normal reaction time of 24 h, thus, it is suspected that the 2-triflyloxy ester 5 first alkylates the P-ketoester enolate and the second equivalent alkylates the Cbz group to produce an imidate salt. The imtdate IS hydrolyzed back to the Cbz group and an a-hydroxyester on workup. The diastereoselecttvity of the process is very good with de’s generally rangmg from 74-96% (Table 1). The lowest de (74%) was found for alkylatton using triflate 5f that contains a branched chain at the a-position The diastereomers of 1 are readily separable by flash chromatography. In most cases,only a single enantiomer of the major diastereomer was detected (ee > 95%) (Table 1). (See also Note 6.) Thus, there IS usually no epimerization of the ammo acid unit during the entire sequence. Longer reaction times lead to degradation of the optical purity of the product (Entry 7, Table 1) This study utilized all (S)-ammo acids and (R)-trtflyloxy esters Since it IS well estabhshed that the choral alkylation proceeds with inversion of configuration (19), the major product diastereomer has the 2R, 5s configuration. Thus, the ketomethylene peptide isostere products have the same stereochemical sense as normal dipeptides (see Note 7). Based on the apparent structural generality of the method, it should be possible to prepare dipeptide rsosteresof any needed configurations by the appropriate choice of starting Cbz-ammo acids 2 and trrflyloxy esters 5. The products are easily unmasked for couplmg into longer peptide sequencesby standard means. Heterocychc ketomethylene peptide isosteres 6 have been prepared by semilar protocol (19, Fig. 6) The mcorporation of a heterocychc rmg could have several advantages including enhanced water solubrhty, hence, bioavailabihty (25,26), or specific bindmg interactions (27,28) The heterocycles chosen to

127

Cbz-Protected Ketomethylene Dlpeptlde lsosteres

w 6

Het

ROH

7a, Het= 2-pyndyl

1 CDI* 2

4

1 NaH Het uOt-Bu

OLl Ot-Bu

2 5a

3 TFA 4. LiOH

8a-c

7b, Het = 2-tluenyl 7c, Het = a-fury1

*

Het’

” 6a, (51%, 52% ee) 6b, (65%, 68% ee) 6c, (46%, 56% ee)

Figure 6 illustrate thts approach were Z-pyrldyl (X = N, n = I), 2-fury1 (X = 0, n = 0), and 2-thienyl (X = S, n = 0) (see Note 8)

4. Notes A good BULI solutton should be pale yellow It might take 4-5 h to remove all of the water When dry, the solutton should be transparent The trtflic anhydrtde should be colorless rather than pmk Use of pmk material IS usually unsuccessful, although the origm of the pmk color 1s not known Opening a bottle of TFA that 1sof satisfactory reactivity should give white vapor You should see a downfield shaft of all of the methyl protons with mcreasmg amounts of added Eu(hfc),. The best spectral resolution of the mdtvtdual enatiomers can be obtained at a chnal shift reagent:substrate molar ratio of 1.1 Authentic racemic samples of laa and lgf were prepared for comparison purposes The prtortty rules change m the ketomethylene tsostere, thus, the 2R configuration m the rsostere cham corresponds to the 2s configuration m an analogous pepttde chain. The preparatton of heterocycltc ketomethylene pepttde isosteres 6 IS summarized m Fig. 6 Heterocyclic carboxyhc acids 7a-c were converted to t-butyl pketo esters 8a-c by the CD1 couplmg procedure Chit-al alkylation with 5a followed by decarboxylatton and saponificatton gave the heterocychc y-ketoacld tsosteres 6a-c as mdtcated in Fig. 6. Although the chemical yields are acceptable, the optical purity of the products IS only modest In this procedure, the trtflyloxy ester 5a was prepared and used zn srtu from the reactton of (S)-methyl lactate, trifltc anhydrtde, and 2,6-luttdme in methylene chloride (19) The use of tsolated 2-trtflyloxy esters m the choral alkylation IS an tmprovement over thts earlier procedure for the chit-al alkylatton (28) Thus, tt IS likely

122

Hoffman and Tao that the reactton of Isolated 5a with the enolates of heterocychc substrates 8a-c by the procedure described above would lead to improved results If these heterocychc pepttde tsosteres are of interest, tt 1s recommended that the chit-al alkylanon procedure described for la be employed

References

5

6

10

11

12 13.

Sandler, M and Smith, H. J (1989) Design of Enzyme Znhzbztors as Drugs, Oxford, Oxford, UK, pp. 573-649. Rich, D. H. (1990) Pepttdase Inhibttors, m Comprehenszve kfedzcmal Chemzstry (Sammes, P G., ed.), Pergamon, Oxford, UK, pp. 391-441. Gante, J (1994) Pepttdomimettcs-tailored enzyme mhtbttors Angew Chem Int Ed Engl 33,1699-1720 DCzlel, R , Plante, R , Caron, V , Gremer, L , Llmas-Brunet, M , Duceppe, J -S , Malenfant, E , and Moss, N (1996) A practical and dtastereoselecttve synthesis of ketomethylene dtpepttde tsosteres of the type AAY[COCH,]Asp J Org Chem 61,2901-2903 Castmir, J R , Turetta, C , Ettouatt, L , and Paris, J (1995) First application of the Dakm-West reaction to Fmoc chemistry. syntyhests of the ketomethylene trtpepttde F-mot-Na-Asp(tBu)-(R,S)Tyr(tBu)YCO-CH*)Gly-OH Tetrahedron Lett 36, 47974800 Baker, W R and Pratt, J K (1993) Dtpepttde tsosteres 2 synthesis of hydroxyethylene dtpeptide tsostere dtastereomers from a common y-lactone intermediate. Preparation of remn and HIV-l protease mhtbttor transttton state mtmtcs Tetrahedron 49,8739-8756. Diederich, A M and Ryckman, D M (1993) Stereoselecttve synthesis of a hydroxyethylene tipepttde tsostere Tetrahedron Lett 34,6 169-6 172 Jones, D M , Ntlsson, B , and Szelke, M. (1993) A short, stereocontrolled synthesis of hydroxyethylene dtpepttde tsosteres J Org Chem 58,228&2290 D’Amello, F and Taddet, M. (1992) A Stereoselecttve method of the preparation of HIV-l protease mhibttors based on the Lewis acid mediated reaction of allylstlanes and N-Boc-a- ammo aldehydes J Org Chem 57,5247-5250 Vara Prasad, J V N , and Rich, D. H (199 1) Synthesis of hydroxyethylene dtpeptide tsosteres that mtmtc a cychc ammo acid at the Pl’ substte Tetrahedron Lett 32,5857-5860 DeCamp, A E , Kawagucht, A T , Volante, R P , and Shmkai, I (1991) Stereocontrolled addition of proptonate homoenolate equivalents to choral ammo aldehydes. Tetrahedron Lett 32, 1867-1870. Hoffman, R V. and Kim, H.-O (1992) A simple synthetic approach to Cbz-Phe-Y(CH2)Gly-Pro-OMe and related pepttde tsosteres Tetrahedron Lett 33,3579-3582 Gonzalez-Muniz, R , Garcia-Lopez, M T , Gomez-Monterrey, I., Herranz, R , Jtmeno, M. L., Suarez-Gea, M. L., Johansen, N. L., Madsen, K., Thagersen, H , and Suzdak, P. (1995) Ketomethylene and (Cyanomethylene)ammo pseudopepttde analogues of the C-terminal hexapeptrde of neurotensm J bled Chem 38, 1015-1021

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14. Lygo, B and Rudd, C N. (1995) Synthesis of Xaa-Gly-Xaa’ keto-methylene trrpeptide rsosteres mcorporatmg phenylalanme, tyrosme, and valme units Tetrahedron Lett 36, 3577-3580 15 Lagu, B R and Lrotta, D C (1994) Diastereoselective syntheses of the key lactone intermediate for the preparation of hydroxyethylene dipeptrde isosteres Tetrahedron Lett 35,547-550 16 Lygo, B. (1992) Use of an alanme derived P-ketosulfone m the synthesis of peptide rsosteres Synlett 793-795 17 Askm, D , Wallace, M A, Vacca, J P., Reamer, R A , Volante, R P , and Shmkar, I (1992) Highly diastereoselectrve alkylations of choral amide enolates new routes to hydroxyethylene dipeptide isostere mhibrtors of HIV- 1 protease J Org Chem 57,2771-2773 18 Hoffman, R V and Kim, H -0 (1993) A new chnal alkylation methodology for the synthesis of 2-alkyl4-ketoacrds m high optical purity using 2-trrflyloxy esters Tetrahedron Lett 34,205 l-2054 19 Hoffman, R V and Kim, H -0 (1995) The stereoselective synthesis of 2-alkyl-yketoacid and heterocychc ketomethylene peptrde rsostere core umts using choral alkylatron by 2-triflyloxy esters J Org Chem 60, 5 107-5 113 20 Still, W. C , Kahn, M , and Mrtra, A (1978) A rapid chromatographrc method J Org Chem 43,2923-2925 21 Harris, B D , Bhat, K L., and Jourlle, M. M (1987) Synthetic studies of drdemmns II Approaches to statme drastereomers Tetrahedron Lett 25, 2837-2840 22 Hamada, Y , Kando, Y , Shrbata, M , and Shioirr, T ( 1989) Efficient total synthesis of didemnms A and B J Am Chem Sot , 111,669473 23 Hoffman, R. V and Kim, H -0 (1992) Preparation of (2Rk2-azidoesters from 2@-Nrtrobenzenesulfonyl)oxy esters and then use as protected ammo acid equrvalents for the synthesis of di- and trrpeptides contammg d-ammo acid constituents Tetrahedron 48,3007-3013 24 Xrang, Y B , Snow, K., and Belley, M (1993) a-(Arylsulfonamrdo)borneols as auxrlarres m asymmetric synthesis an efficient and highly stereoselective method for the reduction of a-ketoesters J Org Chem 58, 993-994 25. Kempf, D J., Codacovr, L , Wang, X C , Kohlbrenner, W E , Wideburg, N E , Saldiver, A , Vasavannonda, S., Marsh, K C., Bryant, P., Sham, H L , Green, B E , Betebenner, D A , Erickson, J , and Norbeck, D W (1993) Symmetry-based mhrbrtors of HIV protease structure-activity studies ofacylated 2,4-drammo-l,Sdrphenyl-3-hydroxypentane and 2,5-drammo-1,6-drphenylhexane-3,4-dial J Med Chem 36,320-330 26 Jendralla, H , Henning, R , Seurmg, B., Herchen, J., Kulrtzscher, B , and Wunner, J (1993) Short and efficient large scale synthesis of (Rb2-benzylsuccnuc acid 4[4-(BOC-ammo)1-piperidrde] monoamide. N-terminal component of remn mhrbrtors by asymmetric hydrogenation. Synlett 155-l 57 27 Thompson, W J , Ghosh, A K , Holloway, M K , Lee, H Y , Munson, P M , Schwermg, J E , War, J , Darke, P L , Zugay, J , Emmi, E A, Schlref, W A.,

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Huff, J R , and Anderson, P. A (1993) 3’-Tetrahydrofuranylglycme as a novel, unnatural amino acid surrogate for asparagine in the design of inhibitors of the HIV protease. J Am Chem Sot , 115,801-803 28 Beckett, R P., Brown, P D., Crnnmm, M J., and Galloway, W A. (1993) Paper No 147, Medlcmal Chemistry, 204th National Meeting of the American Chemlcal Society, Denver, CO

8

(&AI kene Peptide Bond lsosteres by Cuprate Opening of Vinyl Aziridines Todd C. Henninger

and Peter Wipf

1. Introduction Alkene tsosteres are important nonhydrolyzable and rtgidtfied analogs of pepttde bonds. The (E)-alkene effectively mtmtcs the three-dimensional structure of the amide bond, especially the C(a), -C(a),+, distance (Fig. 1). The mcorporatton of an alkene tsostere mto a brologrcally active peptide provrdes a pepttdomtmetic that should have improved resistance to proteolysts and simtlar conformattonal preferences. The low polarity of the alkene is useful m mcreasmg lipophihctty, but hydrogen bonding or dtpolar interactions are generally not possible Highly stereoselective methods for the preparation of alkene isosteres are needed, if they are to be mcorporated mto biologtcally active pepttde sequences. Ideally, the synthetic route should be amenable to the mtroduction of a variety of side chain residues late m the sequence to facilitate SAR studies. This combmatton of stereoselectivity and versatility may be achieved via the SN2’ reaction of organocuprates with alkenyl aziridines The reaction of organocuprates with alkenyl aztrtdmes can, m prmctple, result in a variety of different products (Fig. 2). Fortunately, the desired SN2’ product can be favored almost exclusively by approprtate choice of cuprate and solvent (I-3). Consequently, this strategy constitutes an extremely versatile and stereocontrolled approach to (E)-alkene isosteres. Dtpepttde tsosteres prepared by this route have a high degree of diastereomeric purity. The preparation of alkene isosteres from alkenyl aztrrdmes was mdependently developed by two research groups (1-3). The results of these studies indicate several important criteria for successful tsostere formation. The most important is the nature of the organocopper reagent used to effect the key reacFrom

Methods ,n Molecular Medtone, Vol Edited by W M Kazmlerskt QHumana

125

23

Pepfhmfmebcs Press Inc , Totowa,

Protoco/s NJ

Henninger and Wipf

126

Fig. 1. Geometry of peptide bond and alkene isostere.

reduction

/

conjugate

addition

OR

y-alkylation 8-alkylation

0

\

OR

R2 R3

R534’& HN k’ k R4 ’

6

Fig. 2. Possible pathways for the addition of organocuprates to alkenyl aziridines.

tion. It was found that Gilman-type (R&uLi) and cyanocuprate [R,CuLi * LiCN] reagents provide predominantly S,2 and reduction products. However, several types of cuprates were found to be very effective for the desired E&2’ opening of the alkenyl aziridine. These include RCu * BF,, RCu(CN)Li . BF,, RCu(CN)Li, and RCu(CN)MgBr. Organozinc reagents were also found to be effective in the combined presence of lithium salts and catalytic amounts of

(E)-Alkene Peptide Bond lsosteres

127

copper. The effectiveness of several different types of cuprates allows for the easy mtroduction of many different side chains at the C-terminus of the isostere. Another requirement for successful reactions is the presence of an electronwithdrawing group on the azn-idme nitrogen. The best groups identified for this purpose are the tosyl(1,2) and Boc (1) groups. Presumably, other sulfonyl and carbamate groups would also be effective. Acyl moieties, mcludmg protected ammo acid residues, can also be used, but yields and selectivmes are lower than for the Boc- and tosyl-protected substrates (r) The nonracemic alkenyl azmdines required for this isostere synthesis can be prepared by a variety of routes. P-Hydroxy ammo acids such as set-me,threonine, and allo-threomne can serve as convenient precursors (1-I). Aziridmes with other substitutron patterns can be accessed from optically active epoxy alcohols that are available vta the Sharpless protocol (2,5). The procedures outlmed below illustrate both of these strategies. 2. Materials IR spectra were recorded on a IBM IW32 spectrophotometer. NMR spectra were recorded on Bruker AM-500 or AM-300 spectrometers m CDC13 unless otherwise noted. Optical rotations were measured on a Perkm-Elmer 24 1 polarimeter. Mass spectra were obtained on a VG-70-70 HF. Anhydrous solvents were freshly distilled from either sodium benzophenone ketyl, P205, or CaH,. Oxalyl chloride and BF3 etherate were distilled before use. Commercially available reagents were used as is. All reactions were performed m oven-dried glassware under an argon or nitrogen atmosphere Analytical TLC used Merck silica gel 60 F-254 plates, and flash chromatography (6) was used to separate and purify the crude reaction mixtures 2.1. Reagents for Method 3.7 1 Methanesulfonyl chloride 2 N,N-Drisopropylethylamme 3 Boc-threonmemethyl ester 4 Lithium borohydride (LIBH~) 5 Potassiumcarbonate(K&OX) 6 Pyridme 9SO3complex 7 Dimethyl sulfoxlde (DMSO) 8 Triethylamine (EtjN) 9. (Carbethoxymethylene)triphenylphosphorane. 10 Copper sulfate (CuSOJ. 2.2. Reagents for Method 3.2 1 L-(+)-Dusopropyl tartrate 2 Titanium(IV) isopropoxide

128 3 4 5 6 7 8 9 10 11 12 13 14 15

Henninger and Wipf Powdered 4A molecular sieves trans-2-Buten- l-01. tert-Butyl hydroperoxtde (3 0 A4 m 2,2,4-trimethylpentane) Trtbutylphosphme. Anhydrous cttrlc actd Cehte 545 Dtmethyl sulfoxtde (DMSO) Oxalyl chlortde (Carbethoxymethylene)trtphenylphosphorane Ammonmm chloride (NH&l) Sodmm azrde (NaN,) Trtphenylphosphme Dt-tert-butyl dtcarbonate

2.3. Reagents for Method 3.3 1 2 3 4 5

Methylhthmm (1 24 M m Et,O) n-Butylhthmm (2 3 Mm hexanes) Copper cyanide (CuCN) Copper iodide (CuI) Boron trtfluortde etherate (BF, OEt,)

3. Methods 3.1. Synthesis of (2R,3S)-2-([l Methyl-Aziridine-1-Carboxylic 3.1.1. (1 R,2R)-Methanesulfonlc Hydroxy- 1-Me thy/- Propyl Ester

E]-Z-EthoxycarbonylVinyl)-3Acid tert-Butyl Ester 12 Acid 2-tert-Butoxycarbonylamlno-39

1 Add a solution of 4 50 mL (58 15 mmol) of methanesulfonyl chloride m 20 mL of CH,C12 dropwtse to a stirred solutton of 7 53 g (32 30 mmol) of Boc-threonme methyl ester (8) and 10 13 mL (58 15 mmol) of N,N-dnsopropylethylamme m 35 mL of CHzClz at 0°C 2 Stir an addmonal40 mm at 0°C and then pour the reaction mixture mto me water (70 g) Separate the layers, and extract the aqueous layer with CH&l, (40 mL) Wash the combined organic layers with brme, and dry over Na,SO, Evaporate the solvent to obtain the mesylate as an amber 011that 1s used lmmedtately wtthout further purtficatron 3 Add portionwise a total of 985 mg (45 2 mmol) of LtBH4 to a solutton of the crude mesylate m 100 mL of Et,0 at 0°C. Remove the cooling bath for 15 mm, replace the coolmg bath, and carefully quench the reaction with saturated aqueous NH4Cl (90 mL) followed by 10% HCl (15 mL) Extract the aqueous layer with Et,0 (2 x 75 mL), dry the combined organic layers over MgS04, and concentrate zn vacua (Fig. 3) 4 Purify the crude material by chromatography on StO, (60% EtOAc/hexanes) to obtain 7 3 1 g (73%) of 9 as an 011 [a] “o= +7 4” (c 0 6, CH,Cl,), IR (neat) 3383,

129

(E)-Alkene Peptide Bond lsos teres HO v

0

1. MsCI, (i-Pr),NEt. OMe

2. LiEHI,

NHBoc a

79%

Et20

Mso -+

73%

S03’Pyridine DMSO,

CH,CI,

K,C03, OH NHBoc 9

CHO

70 “C 67%

-

OH

HA-BOC’ NH 10

OEt

Et3N 11

CH3CN

71%

Fig. 3. Synthesis of an alkenyl aziridine from L-threonine.

2980, 1695, 1522, 1346, 1250, 1172, 1061,978,910 cm-‘; ‘H NMR 6 5.11 (d, 1 H, J= 6.2 Hz), 4.83 (d, 1 H, J= 8.5 Hz), 3.88-3.70 (m, 2 H), 3.60 (dd, 1 H, J= 10.9, 7.7 Hz), 3.00 (s, 3 H), 2.7-2.4 (b, 1 H), 1.49-1.45 (m, 12 H); t3C NMR 6 155.5,79.5,76.1,60.7,54.7,37.6,27.7, 17.7; MS (EI) m/e (rel intensity) 252 (3), 210 (4), 196 (4) 160 (lo), 152 (40), 114 (15), 100 (30), 57 (100); HRMS m/e calculated for &H,,NO,S (M-CH,O): 252.0906, found: 252.0926.

3.1.2. (2S, 3s) -2-Hydroxymethyl-3-MethylAziridine- 7-Carboxy/ic Acid tert -Buty/ ester 10 1. Heat a slurry of 283.3 mg (1.0 mmol) of 9 and 276.5 mg (2.0 mmol) of finely pulverized K&O, in 4.5 mL of CH,CN for 7.5 h at 75”C, and then cool to room temperature. Remove the insoluble material by filtration, wash the solids with CH&l, (2 x 5 mL), and concentrate the filtrate and washings in vucuo. 2. Purify the residue by chromatography on SiOz (40% EtOAc/hexanes) to afford 98.0 mg (52%) of 10 and 62.1 mg (22%) of recovered starting material 9 (see Note 1). 10: [u]*‘~ = -1.9” (c 0.7, CH,Cl,); IR (neat) 3427, 2980, 2936, 1720, 1456, 1394, 1369, 1304, 1238, 1163, 1099, 1043 cm-‘; ‘H NMR 6 3.68-3.64 (m, 2 H), 2.86 (b, 1 H), 2.61-2.50 (m, 2 H), 1.41 (s, 9 H), 1.23 (d, 3 H, J= 5.5 Hz); i3C NMR 6 162.5, 81.3, 60.1,42.3, 37.2,27.8, 12.9; MS (EI) m/e (rel intensity) 114 (6), 87 (4), 84 (3), 69 (lo), 59 (15), 57 (loo), 43 (lo), 41 (28); HRMS m/e calculated for C,HsN02 (M-C4H90): 114.0555, found: 114.0558.

3.1.3. (2R,3S)-2-([l El-2-Ethoxycarbonyl- Vinyl)-3-MethylAziridine- 1-Carboxylic Acid tert-B&y/ Ester 12 1. Add a solution of 2 16 mg ( 1.33 mmol) of pyridine . SO3 complex in 1.33 mL of DMSO dropwise to a solution of 82.8 mg (0.442 mmol) of alcohol 10 and 246 pL (1.768 mmol) of Et,N in 3.3 mL of CH,Cl, at 0°C. Stir the mixture for 1 h at 0°C (see Note 2). 2. Partition the mixture between hexanes/Et*O (2: 1, 55 mL) and saturated aqueous NaHC03 (17 mL). Extract the aqueous layer with hexanes/Et*O (2: 1,7 mL), and

Henninger and Wipf

130

3. 4.

5.

6.

wash the combined organic extracts with 1 MNaH,PO, solution (35 mL) and brine (35 mL). Dry the organic layer over Na,SO,, and concentrate in vucuo. Purify by chromatography on SiO, (15% EtOAc/hexanes) to afford 64.4 mg (79%) of aldehyde 11 that was used without further purification. Add a solution of 180 mg (0.490 mmol) of (carbethoxymethylene)triphenylphosphorane in 1.0 mL of CH,C12 to a solution of 36.3 mg (0.196 mmol) of 11 in 1.O mL of CH,Cl, and stir overnight at room temperature. Dilute the solution with 40 mL of CH,Cl, and wash with H,O (7 mL), brine (7 mL), and saturated aqueous CuSO, (7 mL). Dry the organic layer over Na2S0, and concentrate in vucuo. Purify by chromatography on SiO, (lO%EtOAc/hexanes) to afford 35.7 mg (71%) of 12 as a solid: Mp. 34°C; [a] 2’D = -177.3” (c 3.0, CH,Cl,); IR (neat) 2982,1724,1392,1369,1300,1228,1161,1043 cm-‘; ‘H NMRG 6.74 (dd, 1 H, J= 6.8, 15.6 Hz), 6.13 (d, 1 H, J= 15.5 Hz), 4.20 (q, 2 H, J= 7.1 Hz), 3.03 (dd, 1 H, J= 6.6, 6.7 Hz), 2.73 (m, 1 H), 1.45 (s, 9 H), 1.30 (t, 3 H, J= 7.1 Hz), 1.22 (d, 3 H, J = 5.6 Hz); 13C NMR 6 165.5, 161.6, 141.7, 125.0, 81.2, 60.3, 41.1, 39.8, 27.7, 14.0, 13.2; MS (EI) m/e (rel intensity) 182 (3), 155 (6), 112 (15), 82 (20), 57 (100); HRMS m/e calculated for C,H,,NO, (M-C,H,O): 182.0818, found: 182.08 10.

3.2. Synthesis of (2R,3R)-2-([lE]-2-Ethoxycarbonyl-Vinyl)-3Methyl-Aziridine-l-Carboxyiic Acid tert-Butyl Ester 79 3.2.1. (2S,3S)-(3-Methyl-Oxiranyl)-Methanol

14

1. Cool a solution of 1.96 mL (9.3 mmol) of L-(+)-diisopropyl tartrate in 300 mL of CH2C12 to -22°C and add 2.3 1 mL (7.75 mmol) of titanium(W) isopropoxide, followed by 4.6 g of powdered 4a molecular sieves. Stir at -22°C for 15 min. 2. Add 13.2 mL (155 mmol) of Iruns-2-buten-l-01, and stir the mixture for 15 min at -22°C. 3. Add 103 mL (310 mmol) of tert-butyl hydroperoxide (3.0 M in 2,2,4-trimethylpentane) dropwise over 10 min. Maintain the reaction mixture at-20°C overnight (see Note 3). 4. Quench the reaction by the slow addition of 38.6 mL (155 mmol) of tributylphosphine over 30 min at -22°C. Add a solution of 10% acetone/Et,0 containing 1.49 g (7.75 mmol) of anhydrous citric acid, allow to warm to room temperature, filter through a pad of Celite 545, and concentrate the filtrate under reduced pressure (Fig. 4). 5. Purify the residue first by distillation (aspirator vacuum, 6O”C), and then by chromatography on Si02 (4&60% EtOAc/hexanes) to afford 9.466 g (69%) of epoxy alcohol 14 (see Note 4) as an oil: [cx,]2’D = 46.0” (c 0.9, CH,Cl,), Lit. (7) -55 (c 0.22, C,H,); IR (neat) 3412, 2972, 2930, 2872, 1452, 1383, 1103, 1039, 989, 866,819,723 cm-‘; ‘HNMR63.95-3.85 (m, 1 H), 3.663.55 (m, 1 H), 3.04 (dq, 1 H, J= 2.1,5.1 Hz), 2.90-2.85 (m, 1 H), 2.18 (b, 1 H), 1.33 (d, 3 H, J= 5.2 Hz); 13C NMR 6 61.6, 59.5, 51.9, 16.9.

(E)-Alkene Peptide Bond lsosteres

131

H r-BuOOH, -OH

L-(+)-DIPT, 13

1. Swern

Ti(O-bPr)d 4

69% (64%

A MS

OEI

2. Ph,P=CHCO,Et 14

ee)

Ox.

OH

+ O“H

56%

;z”o”H”: ZZ’-,*qA,,, +,2qA,,, Ph-yNw,,, N3

65%

N3

16 (19:

17

1)

62%

HN H

10

Boc*O t&N. 100%

THF

-

$+OEt '3Oc'

19

Fig. 4. Synthesis of an alkenyl aziridine from an optically active epoxy alcohol.

3.2.2. (2E)-3-([2S,3S]-3-Methyl-Oxirany/)-Acrylic

Acid Ethyl Ester 15

1. Add dropwise 1.41 mL (19.92 mmol) of DMSO to a solution of 0.87 mL (9.96 mmol) of oxalyl chloride in 110mL of CH2C12at 60°C. Stir for 10min at -60°C. 2. Add dropwise a solution of 73 1.4 mg (8.30 mmol) of epoxy alcohol 14 in 5.0 mL of CH,C&. Stir for 20 min at -60°C. 3. Add dropwise 3.47 mL (24.9 mmol) of Et,N. Stir for 5 min at 60°C and then allow the reaction mixture to warm to room temperature over 30 min. 4. Add a solution of 7.63 g (20.8 mmol) of (carbethoxymethylene)triphenylphosphoranein 10 mL of CH2C12,and allow to stir for 24 h. 5. Quench with aqueousNH,Cl (50 mL), separatethe layers, and extract the aqueous layer with CH,Cl, (2 x 50 mL). Dry the combined organic layers over Na$O,, and concentrate in vacua. 6. Purify by chromatography on Si02 (10% EtOAc/hexanes) to afford 723.1 mg (56%) of 15 and 92.3 mg (7.1%) of (2Z)-3-([2S,3S]-3-methyl-oxiranyl)-acrylic acid ethyl esteras oils. 15: [a]230 =-13.8” (c 1.3, CH,Cl,); IR (neat) 2984, 1720, 1304, 1261, 1240, 1188,1142, 1034, 837 cm-‘; ‘H NMR 6 6.67 (dd, 1 H, J= 15.5,7.1 Hz), 6.13 (d, 1H,J=15.6Hz),4.20(q,3H,J=7.1Hz),3.18(dd,1H,J=7.1,1.8Hz),2.97 (dq, 1 H, J = 5.1, 1.9 Hz), 1.39 (d, 3 H, J= 5.1 Hz), 1.29 (t, 3 H,J= 7.1 Hz); 13C NMRG 165.6, 144.6, 123.7, 60.5, 57.4, 57.2, 17.5, 14.2.

3.2.3. (ZE,4R,5S)-4-Azido-5-Hydroxy-Hex-Z-Enoic Ethyl Ester 16

Acid

1. Add 4.029 g (75.33 mmol) of NH4Cl and 4.897 g (75.33 mmol) of NaN3 to a solution of 3.923 g (25.11 mmol) of epoxy ester 15 in 55 mL of EtOH. Slowly heat the mixture to reflux over 1.5 h, continue heating at reflux for 50 min, and then allow to cool to room temperature.

132

Henninger and Wipf

2. Filter the reaction mixture, wash the solid residue with EtOH, and evaporate the solvents. Dissolve the residue in Et,0 (220 mL), wash with H,O (60 mL), and extract the aqueous layer with Et,0 (2 x 60 mL). Dry the combined organic layers over Na,SO,, and evaporate. 3. Purify by chromatography on SiO, (30% EtOAc/hexanes) to afford 4.27 1 g (85%) of a 19: 1 mixture of 16 and its (4S,5s) epimer 17 as an oil (see Note 5). 16: [cz]*~~= -35.4” (c 1.2, CH,Cl,); 1R (neat) 3447, 2982, 2106, 1718, 1371, 1273,1182,1095,1039,983cm-‘;1HNMR66.87(dd, lH,J=l5.8,7.lHz),6.11 (dd, 1 H,J= 15.8,0.9Hz),4.23 (q,2 H, J=7.1 Hz),4.1&4.05 (m, 1 H), 3.95-3.88 (m, 1 H), 2.05 (d, 1 H,J= 4.5), 1.31 (t, 3 H,J= 7.1 Hz), 1.20 (d, 3 H,J= 6.3 Hz); 13C NMR 6 165.6, 140.9, 125.0, 69.0, 67.9, 60.8, 18.4, 14.0; MS (EI) m/e (rel intensity) 154 (5), 127 (8), 109 (lo), 98 (40), 82 (30), 72 (20) 54 (25), 45 (100); HRMS m/e calculated for C,H,N302 (M-C,H,O): 154.06 17, found: 154.062 1.

3.2.4. (2E)-3-([2R,3R]-3-Methyl-Aziridin-2-y/)Acrylic Acid Ethyl Ester 18 1. Add 1.047 g (3.99 mmol) of triphenylphosphine in portions over 30 min to a solution of 724.0 mg (3.63 mmol) of a 19: 1 mixture of azido alcohol 16 and its (4S,SS)-epimer 17 in 16 mL of CH3CN. Heat the reaction at reflux for 3 h. 2. Evaporate the solvents, and then dissolve the residue in Et,O. Add hexane, filter the precipitates, and then evaporate the filtrate. 3. Purify by kugelrohr distillation (90°C 0.1 Torr) to afford 462.1 mg (82%) of a 19: 1 mixture of 18 and its (2S,3R) epimer as an oil. 18: [IX]*%= +96.6” (c 1.3, CH2Cl2); IR (neat) 3287,3230, 2980, 2930,1707, 1647, 1367, 1340, 1232, 1157, 1095, 978, 821 cm-‘; IH NMR 6 6.43 (b, 1 H), 6.05 (d, 1 H,J= 15.5 Hz), 4.19 (q, 2 H, J= 7.2), 2.27 (d, 1 H,J= 8.7) 2.12 (b, 1 H), 1.3s1.26 (m, 6 H); i3C NMR 6 165.8, 148.5, 121.3, 60.1, 38.7, 35.6, 18.3, 14.0; MS (EI) m/e (rel intensity) 155 (M+,l), 126 (1 l), 112 (40), 94 (35), 82 (loo), 73 (8), 67 (12), 61 (75), 54 (30), 43 (100); HRMS m/e calculated for C,H,NO, (M-C,H,): 126.0555, found: 126.0563.

3.2.5. (2R,3R)-2-(11 El-2-Ethoxycarbonyl-Vinyl)-3Methyl-Aziridine-1 -Carboxylic Acid tert-Butyl Ester 19 1. Add a solution of 140.6 mg (0.644 mmol) of di-tert-butyl dicarbonate in 1.O mL of THF to a solution of 54 pL (0.386 mmol) of Et,N and 52.2 mg (0.336 mmol) of a 19: 1 mixture of aziridine 18 and its (2S,3R) diastereomer in 1.O mL of THF. 2. After 2 h, dilute with 7 mL of Et20, and wash with H20 (2 mL). Extract the aqueous layer with Et20 (2 x 3 mL), dry the combined organic layers over Na2S04, and concentrate in vucuo. 3. Purify by chromatography on Si02 (15 % EtOAc/hexanes) to afford 85.8 mg (100%) of a 19: 1 mixture of 19 and its (2S,3R) diastereomer as an oil. 19: [a]**o = +5.8” (c 1.0, CH,Cl,); IR (neat) 2970, 1718, 1655, 1369, 1304, 1257, 1157 cm-‘; ‘H NMR 6 6.48 (dd, 1 H, J= 15.6, 8.7 Hz), 6.13 (d, 1 H, J=

(E)-Akene Peptrde Bond lsosteres

OEI

MeCu(CN)LI*BF3 THF, -78 “C

133

BocNLCozEt H

+

BocNLC02Et H :

: 20

77%

21 (8

1)

BuCU(CN)LI*BF, OEt

THF, -78 ‘C 52%

Bac~-co2Et

l

Bocpeco2Et

22

23 \

(55

1)

\

Fig 5 Preparation of dlpeptlde alkene lsosteres by reactlon of alkenyl azuldmes with organocuprates

15 6Hz),427-4.11 (m,2H),2.78(dd, 1 H,J=8.5,2.8Hz),2 .55(dq, 1 H,J=5 7, 3 1 Hz), 1 46 (s, 9 H), 1 33 (d, 3 H, J= 5 6 Hz), 1 29 (t, 3 H,J = 5 0), 13C NMR 6 165 5, 1599, 144 0, 123 9,81.6,60.4,43 9,41.4,27 8, 16.1, 14 l,MS(EI)m/e(rel mtenslty) 182 (3), 154 (6), 126 (4), 112 (16), 94 (4), 82 (20), 67 (3), 57 (loo), 41 (20); HRMS mjecalculated for C,H,2N03 (M-C,H,O). 182 08 17, found 182 0827

3.3. Synthesis

of lsosteres by Organocuprate

Reactions

3.3.7. (2R,3E,5S)-5-tert-Butoxycarbonylamino-ZMethyl-Hex-3-Enolc Acid Ethyl Ester 20 1 Add a solution of 0 96 mL (1 185 mmol) of methylhthmm (1 24 Mm Et,O) to a slurry of 106 1 mg (1 185 mmol) of CuCN m 3 3 mL of THF at -30°C (see Note 6). Warm the solution to 0°C over 10 mm and then cool to -70°C Add 146 pL (1 185 mmol) of BF, OEt,, stir for 10 mm at -7O”C, and then warm to -35°C 2 Add a solution of 100 8 mg (0 395 mmol) of 12 In 1 2 mL of THF, stir for 10 mm at -35”C, and quench with saturated aqueous NH,CI (4 0 mL) Extract with Et,0 (3 x 20 mL), dry the combined organic layers over MgS04, and concentrate zy1V~CUO (Fig. 5) 3 Purify by chromatography on SiOZ (10% EtOAc/hexanes) to afford 82.0 mg (77%) of an 8.1 mixture of 20 and y-alkylation 21 product as an 011(see Notes 7-9) 20 IR (neat) 3364,2978,2934, 1716, 1518, 1454, 1390, 1367, 1250, 1174, 1049,970,858,781 cm-‘, ‘H NMR 6 5.67 (dd, 1 H, J= 7.1, 16 0 Hz), 5 52 (dd, 1 H,J=48,156Hz),443(b,1H),425-4.1(m,1H),412(q,2H,J=71Hz), 3 15-3 05 (m, 1 H), 1 44 (s, 9 H), 1 38-l 19 (m, 9 H), 13C NMR 6 174 3, 154 9, 133 2, 128 4. 78 9, 60 3, 47 1, 42 3, 28.2, 20 7, 17 1, 14.0, MS (EI) m/e (rel intensity) 215 (7), 200 (4), 155 (18), 142 (25), 128 (lo), 114 (15), 98 (14), 88 (12), 82 (20), 70 (loo), 57 (86), 44 (25); HRMS m/e calculated for C,,H,,NO, (M-C4Hs) 215 1158, found 215 1171 Character&c peaks for 21 ‘H NMR 6 6 88 (dd, 1 H, J = 8 1, 15.6 Hz), 5 83 (d, 1 H, J = 15 6 Hz), 2 60-2 45 (m, 1 H)

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3.3.2. (2R,3E,!%)-5-tert-Butoxycarbonylamino-2Bufyl-Hex-3-Enoic Acid Ethyl Ester 22 1 Add 0 26 mL (0 600 mmol) of a solution of n-butylhthmm (2 3 Mm hexanes) to a slurry of 114.3 mg (0 600 mmol) of CuI m 3.0 mL of THF at -35°C Stir the reaction mixture for 5 mm at -35’C, and then cool to -70°C Add 73 8 pL (0 600 mmol) of BF, OEt, and stir for 5 mm 2 Add a solution of 76 6 mg of a 19 1 mixture of azlrldme 19 and Its C(3) eplmer m 2.0 mL of THF, and stir for 10 mm at -78°C Quench by addmon of 5 0 mL of saturated aqueous NH&l, and extract with Et,0 (3 x 40 mL) Dry the combined organic layers over Na2S04, and concentrate zn vucuo 3 Purify by chromatography on S102 (ZO%EtOAc/hexanes) to afford 48.9 mg of a 5 5 1 mixture of 22 and y-alkylatlon product 23 (see Notes 8,9). 22. IR (neat) 3366,2972,2932, 1714,1516, 1365, 1248, 1174 cm-‘, ‘H NMR 6 5 61-5.43 (m, 2 H), 4 43 (b, 1 H), 4 25-4 1 (m, 1 H), 4 13 (q, 2 H, J = 7 l), 2 99-2.91 (m, 1 H), 1.78-1 62 (m, 1 H), 1 55-l 45 (m, 1 H), 1 44 (s, 9 H), 1 33128(m,10H),O88(t,3H,J=71Hz),‘3CNMR61742,1550,1344,1274, 79 1, 60 3, 48 8, 32.2,29 4,28 3,22 3,20 9, 14 1, 13 8, MS (EI) m/e (rel mtenslty)257(15),242(4),212(4), 195(15), 184(13), 170(10), 140(11), 114(12), 96 (16), 88 (17), 81 (15), 70 (loo), 67 (15), 59 (lo), 57 (80), 55 (20), 53 (8), 44 (50), 41 (36), HRMS m/e calculated for C,3H23N04 (M-C,H,) 257 1627, found 257 1621 Characteristic peaks for 23. ‘H NMR 6 6 76 (dd, 1 H, J= 9 7, 15 6 Hz), 5 84 (d, 1 H,J= 15 6 Hz), 2 25-2 15 (m, 1 H) 4. Notes 1. Attempts to drive this reactlon to completion by extendmg the reflux period resulted m lower yields owmg to product decomposltlon Based on recovered starting material, the yield of this reaction amounts to 67%. Swern oxldatlon of 10 followed by Wlttlg condensation resulted m rmg opening of the azmdme Oxldatlon with catalytic tetrapropylammonmm perruthenate and 4-methylmorpholme-Noxlde gave a low yield of the azmdme aldehyde, DessMartin oxidation of 10 resulted m Improved yields, but Pankh-Doermg oxldatlon gave the best results This can be conveniently achieved by securely stoppermg the flask and allowmg it to stand, unstirred, m a freezer The enantlomerlc excess was determmed to be 84% by comparison of the optical rotation with the literature value (7). If desired, material of higher optical purity (>98% ee) can be obtained by recrystalhzatlon of the p-mtrobenzoate derlvatlve followed by hydrolysis (‘5) Alternatively, the epoxldatlon can be performed with stolchlometrlc amounts of catalyst to obtam material with >95% ee (7) Chromatographlc separation of 16 and 17 was not possible at this stage. The ratlo of 16:17 was determined by integration of the crude 1H NMR of the correspondmg azmdmes 18 and (28-18 (resonances at 6 56 and 6 92 ppm) The stmcture of

(E)-Alkene Peptide Bond lsosteres 17 was asslgned based upon Its fully characterized azlrldme derivative (29-18. The nonstereoselectlve [3,3] slgmatroplc rearrangement of allyhc azldes 1spossibly responsible for the formation of this minor dlastereomer The ratio of 16: 17 depends on the reaction temperature and can be increased by shortening the reaction time Reaction with 3 eq of NaN, and 3 eq of NH,Cl m refluxmg ethanol for 5 h resulted m a 4.1 mixture of dlastereomers Further modification of reaction condltlons by changing the solvent to MeCN and usmg LICIO, or NaClO, as Lewis acids led to decomposition of starting material In the absence of NH&l, no desired product could be isolated in ethanol The reduction product IS a frequent side product m S,2 and S,2’ reaction of organocopper reagents. It 1spossibly owing to electron transfer or enolization of the transient Cu(III)-species Catalytic use of copper salts can significantly reduce the amount of reduction product formed Another side product m addition to vmyl azlrldmes that we observed was the oxazolme rearrangement product Formation of oxazolme from acylazlrldme was especially pronounced with PhCu BF, In dlethyl ether The exclusive use of THF suppressed this undesired rearrangement product Dlethyl ether m general seems to be an inadequate choice for S,2’ additions There 1s a correlation between the electron-withdrawing effect of the acyl substltuent on the azn-ldme and the yield of a-alkylatlon product Benzoylatlon or simple acylatlon, especially if the acyl component contains additional functional groups capable of coordmatmg to metal ions, provide the poorest results and the most side-products Carbamates, especially, sulfonyl functions, lead to the highest yields of SN2’-addltlon product Antilsyn ratios m the SN2’-addition of organocuprates are > 15 1 with N-carbamate protected trans-azlndmes such as 19 With cu-azlndmes such as 12, antl/syn addition ratios generally exceed 98.2 Similar to Note 7, an increase m the electron-withdraw m effect of the N-protective group leads to an improvement m the antl/syn ratio The CZ-and y-alkylatlon products 20 and 21, and 22 and 23, have to be separated by HPLC No y-alkylatlon products have been observed with y-bo-alkylated analogs of 12 and 19, or when N-sulfonyl groups are used m place of carbamates (lb,2).

References la Wlpf, P and Fntch, P C (1994) S,2’ reactions of peptlde azn-ldmes A cupratebased approach to (E)-alkene lsosteres. J Org Chem 59,4875-4886 1b Wlpf, P and Hennmger, T (1997) Solid-phase synthesis of peptide mlmetlcs with (E)-alkene amide bond replacements derived from alkenyl azn-ldmes J Org Chem 62, 1586-1587 2 FUJII, N , Nakal, K , Tamamura, H , Otaka, A, Mimura, N , Miwa, Y , Taga, T , Yamamoto, Y , and Ibuka, T (1995) S,2’ ring openmg of azlridmes bearing an a&unsaturated ester group with organocopper reagents A new stereoselective synthetic route to (E)-alkene dlpeptlde lsosteres J Chem Sot Perk Trans I, 1359-1371

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3 Ibuka, T , Nakai, K , Habashita, H , Hotta, Y , FUJII, N , Mimura, N , Miwa, Y., Taga, T , and Yamamoto, Y. (1994) A novel route to drastereomerlcally pure (E)alkene dlpeptide lsosteres from P-aznidinyl-a$-enoates by treatment with organocopper reagents Angew Chem Int. Ed Engl 33,652-654 4. FUJU,N., Nakai, K , Habashrta, H , Hotta, Y , Tamamura, H., Otaka, A , and Ibuka, T. (1994) Synthesis of optically pure 2-aznidmemethanols. Versatde synthetic building blocks Chem Pharm Bull 42,224 l-2250 5. Gao, Y., Hanson, R M , Klunder, J M , Ko, S. Y , Masamune, H., and Sharpless, K B (1987) Catalytic asymmetric epoxldatlon and kinetic resolutron* modified procedures mcludmg m situ derivatizatton. J Am Chem Sot 109, 5765-5780 6 Still, W C , Kahn, M , and Mitra, A (1978) Raptd chromatographtc technique for preparative separations with moderate resolution J Org Chem 43,2923-2925 7 Rossiter, B A , Katsuki, T , and Sharpless, K B. (1981) Asymmetric epoxidation provides shortest routes to four chnal epoxy alcohols which are key mtermediates m syntheses of methymycm, erythromycm, leukotriene C-l, and drsparlure J Am Chem Sot 103,464-465

9 Syntheses of Norstatine, Its Analogs, and Dipeptide lsosteres by Means of P-Lactam Synthon Method lwao Ojima and Francette

Delaloge

1. Introduction Nonprotein amino acids are the amino acids that are not found 1n protelnmain chains and mostly originate 1n plants, microorganisms, and marine products. Certain nonprotein amino acids exhibit blologlcal activities by themselves, and many of them are important constituents of biologically active compounds of medicinal interest For this reason, in addition to the naturally occurring nonprotein amino acids, synthetic nonprotein amino acids have been studled extensively, especially 1n connection with the design and synthesis of various enzyme 1nhrbttors. Indeed, statlne, norstatlne, and their analogs, as well as a variety of dlpeptlde isosteres, have been developed and incorporated 1n various inhibitors of enzymes such as renin and HIV-I protease with great success (I), These amino acid residues and their isosteres provide effective transition-state mimics of the substrates for peptidases that bind to these enzymes tightly and inhibit their actions. Although a number of methods has been reported for the synthesis of statlne and 1ts analogs (2-71, to date, only a few synthetic methods are available for norstatlne and its analogs (8-12). The p-lactam synthon method developed by these laboratories (13-17) can be effectively applied to the asymmetric synthesis of norstatlne and its analogs, as well as various dlpeptlde 1sosteres. In this chapter, the preparation of 3-slloxyp-lactams will be described first, followed by those of norstatlne and 1tsanalogs, as well as dlhydroxyethylene, hydroxyethylamine and other dlpeptide isosteres.

2. Materials 1 Melting points Thomas Hoover Capillary melting point apparatus (uncorrected). 2 NMR spectra Bruker AC-250 NMR spectrometer using CDC13 as the internal standard 3 Optical rotations Perkin-Elmer 241 polanmeter. From

Methods m Molecular Mechcme, Vol 23 Pepbdommebcs Protocols Edited by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

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4 5 6 7

IR spectra* Perkm-Elmer FTIR 1600 series spectrophotometer Thm layer chromatography Merck DC-alufohen with Kieselgel 60F254 Column chromatography* Silica gel 60 (23@400 mesh ASTM, Merck) Chnal HPLC analysis Waters HPLC assembly, consistmg of a Waters M45 solvent dehvery system, a Waters Model 680 gradient controller, and a Waters M45 solvent delivery system, a Water Model 680 gradient controller, and a Waters M440 detector (at 254 nm), equipped with a Spectra Physics model SP4270 mtegrator using a chval column J T Baker DACEL-CHIRACEL OD. 8 Solvents (ethyl ether, methanol, drchloromethane, ethyl acetate, hexane, chloroform, dimethylformamide, dioxane, toluene) are dried and distilled according to known procedures

2.7. Reagents 1 2 3 4. 5 6. 7 8. 9 10 11 12 13 14 15 16 17

for Method 3.1

Sodium Benzyl alcohol. (-)-trans-2-Phenylcyclohexanol (18) Benzyloxyacetrc acid p-Toluenesulfomc acid (PTSA) Palladium on carbon (Pd/C, 10%) Imidazole (Im) Trnsopropylsilyl chloride (TIPSCl) Pyridmmm chlorochromate (PCC) p-Amsidme (p-methoxyamlme) Sodium sulfate Dnsopropylamme. n-Butylhthmm (n-BuLt, 2 5 A4 solution m hexanes) Cermm (IV) ammonium nitrate (CAN) Triethylamme (TEA). 4-Dtmethylammopyridme (DMAP) Dt-t-butyldlcarbonate (BoQO)

2.2. Reagents for Method 3.2 2 3 4 5 6

7 8 9 10. 11 12 13

Hydrochloric acid (6N) Tetra-n-butylammonmm fluoride (TBAF) (5’)-Phenylalanme methyl ester (S)-Tryptophan methyl ester (5’)-Prolme methyl ester. Citric acid Fmoc-Phe-Wang resin Piperidme Trifluoroacettc acid (TFA) Dnsopropylamme n-Butyllithmm (n-BuLi, 2 M solution m hexanes) Acetophenone. Ethyl acetate

Synthesis of Norstatine, Its Analogs, and Dipeptide lsosteres

139

2.3. Reagents for Method 3.3 1. 2 3. 4. 5. 6 7 8 9 10. 11 12. 13 14. 15. 16 17

4-Dimethylammopyrrdme (DMAP). Triethylamme (TEA) Hydrogen fluoride-pyrtdme (HF/pyr). Pyrtdme. Copper sulfate (saturated aqueous solution) Pyridimum p-toluenesulfonate (PPTS) Dimethoxypropane Dusobutylalummum hydride (DIBAH) Rochelle’s salt (sodmm potassium tartrate) Oxalyl chloride. Dimethyl sulfoxlde (DMSO) Tm tetrachlorlde (SnC&) Allyltrtbutyltm Palladium chloride (PdC12) Cuprous chloride (CuCl) Dioxygen Sodium hypochlorite (NaOCl)

2.4. Reagents for Method 3.4 1 2 3 4 5 6 7 8 9 10 11 12 13

Lithium alummum hydride (LiAlH,). Triphenylphosphme (PPh,) Diethyl azodicarboxylate (DEAD) (S)-Phenylalanme methyl ester hydrochloride (S)-Prolme methyl ester hydrochloride Triethylamine (TEA) Allylmagnesmm bromide Cuprous iodide (Cm) Pyridmmm p-toluenesulfonate (PPTS) Dimethoxypropane Ozone Hydrogen peroxide (30% wt solution m water) (H202) Sodium hydroxrde (pellets)

3. Methods

3.1. Stereoselective

Preparation of 3-Siloxy-P-Lactams

3-Siloxy-P-lactams with excellent enantiomerrc purity can be easily prepared through cyclocondensation of chiral enolates with imines (13-18). (-)-trans-2-Phenylcyclohexanol is used as the choral auxiliary (19) m thus process to achieve excellent enanttoselectrvity. The syntheses of (3R,4S)- lBoc-4-(2-methylprop-l-enyl)-3-triisopropylsiloxyazetidin-2-one 9a and (3R,4S)-l-Boc-4-cyclohexylmethyl-3-triisopropylsiloxyazet~dm-2-one 9b are described here as representative examples.

O]ima and Delaloge

140 Na.

HO..

BrCHg20&l

2

150%

PhCH20H

PD

BnOCH$OOH 75%

6”o~“~ PD

tol, PTSA, reflux

1

3

96%

Pd/C (lo%), THF, 45”C,

H2

TIPSCI. Im, DMF

HO?‘,

12 h

96%

TIPSO>” Ph 40

RT, 21 h

P to

67%

4

5

Fig. 1. Preparation of ester 5

5, LDA. TIPSO, ,R -BO”C, THF ofiPMP

H2N

NPMP

R-CHO

NazS.04, CH$& RT, 1 h

6a-c

a

R=

b

9a-c

CAN, CH&N, TIPSO, H20. -10 “C, 3h

Ba-c c

R=

9a 65%

ee = 90%

9b

75%

ee=

9c

85%

ee = 92%

96%

/--I

,R

10a 62% lOb76% 1Oc 65%

Ftg. 2 Preparation of 3-Stloxy-P-Lactams

lla

96%

llb60%

lit

65%

lla-c

(-)-trans-2-Phenylcyclohexanol(2) 1s prepared m a large scale accordmg to the literature procedure (19). Esterlficatlon of benzyloxyacetlc acid 1 (20) with chn-al alcohol 2 gives compound 3. The benzyloxy group 3 IS then deprotected by hydrogenolysls and reprotected as s11yl ether 5 (Fig. 1). The cyclocondensatron with freshly prepared imtne 7 or 8 (see Note 1) affords the czs-j3lactams 9 with 90 - 96% of enantlomerlc excess (ee) (determined by chn-al HPLC analysis using hexane/2-propanol(97/3, v/v) as the eluant) (Fig. 2). Other cls-p-lactams can be prepared followmg the same procedure, and vartous C-4 substituted cis-p-lactams 9 (4-phenyl, phenylethylene, 2-furyl, 2-methylpropyl) are obtained m good yields with excellent enantlomerlc excess 3.7.1.

Benzyloxyacetic Acid 7 (19,20)

1. To 6.3 g (0.30 mol) of sodmm metal at room temperature under nitrogen atmosphere, add 113 mL (1 09 mol) of benzyl alcohol with stnrmg Heat the reaction

Synthesis of Norstatme, Its Analogs, and Dipeptide lsosteres

141

mtxture to 150°C until the sodmm 1scompletely consumed. Then, add dropwtse 17 8 g (0 13 mol) of bromoacettc acid m THF (25 mL) 2 Star the reaction mixture at 150°C for 3 h, cool down to room temperature, add cold water (400 mL), and separate the two resulting layers. Extract the aqueous phase with two 100 mL porttons of dtchloromethane to remove any remaining benzyl alcohol Acidify the water layer with 10% HCl unttl pH -2-3 Extract the aqueous layer with three 100 mL portions of ether Combme the extracts, dry over MgSO,, and concentrate zn vacua. 3 Distill the oily residue under reduced pressure to obtain 15 98 g (75%) of 1 as a colorless 011.bp 130-135”C/O 3 mmHg; ‘H NMR (CDCl,) 6 4 17 (s, 2 H), 4.67 (s, 2 H), 7.38 (m, 5 H)

3 1.2. (7R,2S)-(-)-2-fhenylcyclohexyl

Benzyloxyacetate 3 (18)

In a 250 mL flask equipped with a Dean-Stark apparatus, reflux overmght a solution of 15 98 g (0 091 mol) of (-)-trans-2-phenylcyclohexanol 2 (19), 15 98 g (0 096 mol) of benzyloxyacetic acid 1, and a catalytic amount (approx 850 mg) of p-toluenesulfomc acid m toluene (150 mL) 2 Remove toluene by a rotary evaporator, add ether (200 mL) to the reaction mixture, and wash it with saturated aqueous NaHCO, Combme the extracts, dry over MgS04, and concentrate zn vacua to obtam 49 0 g (98%) of 3 as a white sohd. mp 52-53”C, ]H NMR (CDCl,) 6 1.26-l 63 (m, 4 H), 1 761 99 (m, 3 H), 2 l&2 20 (m, 1 H), 2.70 (td, J= 4.1, 11.0 Hz, 1 H), 3.73 (d, J= 16.5 Hz, 1 H), 384(d,J=l65Hz,lH),425(s,lH),5.13(td,J=4.l,ll.0Hz,lH),7l~ 7 39 (m, 5 H).

3.7 3. (lR,2S)-(-)-2-Pheny/cyc/ohexy/

Hydroxyacetate

4 (19)

To 2 1 32 g (0 066 mol) of (-)-benzyloxyacetate 3 m 200 mL of THF, add 4 98 g of palladmm on carbon, and stir the mixture overnight at 45°C under hydrogen atmosphere 2 Filter the reaction mixture through a Cehte pad, wash the pad with THF, and concentrate zn vacua to obtam 15 1 g (96%) of 4 as a white solid* mp 59-60°C, ‘H NMR (CDCl,) 6 1 30-1.66 (m, 4 H), 1.78-2 00 (m, 3 H), 2.10-2 20 (m, 2 H), 2.67 (td, J= 4 2, 11 0 Hz, 1 H), 3 72 (d, J= 17 0 Hz, 1 H), 3 93 (d, J= 17.0 Hz, 1 H), 5 07 (td, J= 4 2, 11 0 Hz, 1 H), 7.16-7 32 (m, 5 H)

3 1 4. (1 R,2S)-(-)-2-Phenylcyclohexyl Triisopropylsiloxyace ta te 5 (18) 1 To a solution of 5.72 g (0 083 mol) of tmidazole and 8 05 g (0.035 mol) of hydroxyacetate 4 m 18 mL of DMF, add 10.4 mL (0 048 mol) of TIPSCl and stir under nitrogen for 2 1 h 2 Wash the reaction mixture wtth 1% HCl, and remove the solvent by a rotary evaporator to obtain 5 as a pale yellow oil Dtstill the crude product under vacuum to obtam 11.7 g (87%) of a clear, vtscous 011: bp 15O”C/l nnnHg, ‘H NMR (CDCls) 6 0 94-1.25 (m, 21 H), 1.35-l 70 (m, 4 H), 1 80-2 05 (m, 3 H), 2 lO-

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3.15. 2-Cyclohexylethanal6a

(21)

1 To a suspension of pyrldmmm chlorochromate (PCC) (16 8 g, 77.99 mmol) and Cellte (16 8 g) in CH,C12 (150 mL) at room temperature, add 2-cyclohexylethanol (4 00 g, 3 1 2 mmol), and stir the mixture for 2 h 2 Filter the reaction mixture through a Flonsil@ column and concentrate in vacua to obtain aldehyde 6a as a colorless oil (3 37 g, 83%) ‘H NMR (CDC13) 6 0 9G 1.90 (m, 2 1 H), 2.28 (m, 2 H), 9 83 (s, 1 H).

3.1.6. (3R, 4S)- 1-p-Methoxyphenyl-3-Triisopropylsiloxy-4Cyclohexylmethylazetldin-Z-One 9a, (3R, 4S)- 1-p-Methoxyphenyl-3Triisopropylsiloxy-4-(Z-Methylprop1-Enyl)Azetidin-P-One 9b and (317, 4S)- 1-p-Methoxyphenyl-3-Tr~~sopropylsiloxy-4-(2-Methylpropyl) azetidin-Z-One 9c (18) 1. Prepare lmme Sa by mixing a solution ofp-amsldme 7 (7 68 mmol) (see Note 2) m 5 mL of dichloromethane with aldehyde 6a (9 98 mmol) m the presence of sodium sulfate (2 8 g, 20 mmol) (see Note 1) Stir the mixture for 1 h, and then filter the sodium sulfate, and concentrate the reaction mixture to obtam 8a as an orange 011 2 To a solution of dusopropylamme (7 68 mmol, 1 074 mL) m THF (40 mL), add 3 1 mL of n-butyllithmm (2 5 A4 solution, 7 68 mmol) at -2O”C, and cool the solution to -85’C (the use of a Cryocool@ IS convenient). To this LDA solution, add dropwlse vza cannula a solution of (-)-2-phenylcyclohexyl tnlsopropylslloxyacetate (5) (2 5 g, 6 4 mmol) m 25 mL of THF over the period of 2 5 h Stir the mixture at -85’C for 30 mm 3 Add very slowly (over the penodof 3 h) vza cannula a solutionof lmme8a (7 68 mmol) m 25 mL of THF Stir the mixture for additional 2 5 h at -85”C, then allow the mixture to warm up slowly to room temperature,and stir overnight (seeNote 3) 4. Quench the reaction with saturated aqueousammonium chloride (50 mL), and extract the mixture with three 25 mL portions of ether Wash the organic phase with two 30 mL portions of brme, dry over MgSO,, and concentrate zn vacua 5 Purify the crude product by column chromatography on slllca gel using ethyl acetate/hexane (l/l 0, v/v) asthe eluant Concentrate the appropriate fractions to obtain 2.43 g of 9a (85% yield, 90% ee) as a yellow solid 6. P-Lactams 9b and 9c can be prepared m the samemanner. Recrystalllzatlon of 9b from cyclohexane improves its enantlomerlc purity to 96% ee 9a. Low meltingpoint solid, ‘H NMR (CDCl,) 6 0.80-l 80 (m, 22 H), 3 65 (s, 3 H), 405(m,lH),490(d,J=5.0Hz,lH),6.75(d,J=8.8Hz,2H),7 17(d,J=88Hz,2H) 9b. Pale yellow solid; mp 94-95”C; ‘H NMR (300 MHz, CDCl,) 1.0&l 18 (m, 21H), 1 79 (s, 3H), 1 84 (s, 3H), 3.80 (s, 3H), 4.74 (dd, J= 4 9,9.9 Hz, lH), 4 99 (d, J= 4 9 Hz, lH), 5 27 (d, J= 9.9 Hz, lH), 6 78 (d, J= 8.9 Hz, 2H), 7 26 (d, J= 8 9 Hz, 2H) 9c Pale yellow solid; mp 59-6O”C, ‘H NMR (CDCl,) 6 0.96 (d, J = 6 4 Hz, 3 H), 1 03 (d, J= 6.4 Hz, 3 H), 1 10-l 30(m, 21H), 1 60-1.68 (m, 1 H), 1 70-l 921

Synthesis of Norstatine, Its Analogs, and Dipeptide lsosteres (m, 2 H), 3.75 (s, 3 H), 4 la.22 ~.OHZ,~H),~~~(~,J=~OHZ,~H).

143

(m, 1 H), 5.06 (d,J= 5.1 Hz, 1 H), 6.86 (d,J=

3.1.7. (3R, 4S)-4-Cyclohexylmethyl-3-Triisopropylsiloxyazetidin2-One IOa, (3R,4S)-4-(2-Methylprop-I-Enyl)-3Tnisopropylsiloxyazetidin-2-One lob and (3R,4S)-4(2-Methy/propy/)-3-Triisopropy/si/oxyazetidin-2-0ne 10~ (18) 1 To a solution of 9a (3.116 mmol) m acetomtrlle (100 mL) at -1 O”C, add slowly a solution of cermm ammonium nitrate (10.596 mmol, 5.809 g) m 115 mL of water over the period of 1 h with stlrrmg (see Note 4) 2 Stir the mixture at -10°C for 2 h, then add 50 mL of water. Extract the reaction mixture with three 30 mL portions of ethyl acetate. Combme the extracts, wash the combined extracts sequentially with water (20 mL), 5% aqueous sodium sulfite (30 mL), saturated aqueous sodium carbonate (30 mL), and brine (20 mL) Then, dry over MgSO,, and concentrate in vacua 3 Purify the crude product by column chromatography on silica gel using ethyl acetateihexane (l/4, v/v) as the eluant to obtain 868 mg (82%) of 10a as a yellow 011 4 P-Lactams lob and 1Oc can be prepared in the same manner (18) 10a Yellow 011,[cz],,*~ =-12 4” (c 1.46, CHCl,); IR (neat) 1184, 1465, 1759, 3238 cm-‘, ‘H NMR (CDCI,) 6 0.97-l .25 (m, 32 H)1.4&1.70 (m, 2 H), 3 80 (dt, J= 4.8, 8.4 Hz, 1 H), 4 95 (dd, J= 2.4, 4.8 Hz, 1 H), 6.05 (bs, 1 H), 13C NMR (CDC13) 6 12 0, 17 7, 26 1, 26.2, 26.4, 33.2, 33 7, 34 7, 37 7, 53 9, 77 8, 170 4. Analysis calculated for C,9H3702NS1* C, 67 20; H, 10 98; N, 4 12. Found* C, 6731;H, 1089;N,407 lob 76% yield, mp 84-85”C, [a],,*O = +50 0” (c 1 00, CHCl,), ‘H NMR (CDC13) 0 95-l 10 (m, 21H), 1.59 (s, 3H), 1.67 (s, 3H), 4.36 (dd, J= 4.5, 9 5 Hz, lH), 4 96 (d, J= 4 5 Hz, iH), 5 22 (d, J= 9 5 Hz, lH), 6 52 (bs, lH, NH); 13CNMR (CDCl,) 6 11 9, 17.6, 18.2,25.9,53.5,79.4, 121.5, 137.8, 169 9 Analysis calculated for C,,jH31N02S1. C, 64 59, H, 10 50; N, 4.71. Found. C, 64.45, H, 10 25, N, 4 58 lOc* Colorless oil; 85% yield; [cx]~*’ = -35.4” (c 1.33, CHCl,), ‘H NMR (CDCl,) 0.93 (d, J= 6 6 Hz, 3H), 0.96 (d, J= 6.6 Hz, 3 H), 1 05-l 25 (m, 22H), 152(m, lH),l 67(m, lH),3.78(m,lH),4.96(dd,J=2.4,4.8Hz,lH),602(bs, 1H); 13C NMR (CDCl,) 6 12 1, 17.7, 17.8, 22.3, 23 1, 25.3, 39.1, 544, 78 0, 170 0 Analysis calculated for C,6H33N02S1 C, 64.16; H, 11.10; N, 4.68. Found C, 64.17, H, 10.96, N, 4 47

3.1.8. (3R,4S)- 7-tert -Butoxycarbonyl-3- Triisopropylsiloxy-4Cyclohexylmethylazetidin-2-One Ila, (3R,4S)-7-tertButoxycarbonyl-3- Triisopropykiloxy-4-(2-Methylprop1-enyl) Azetidin-2-One I lb and (3R,4S)- 7-tert-Butoxycarbonyl-3Triisopropylsiloxy-4-(2-Methylpropyl)azetidin-2-One 1Ic (18) 1. To a solution of 10a (2.38 mmol), trlethylamine (1.65 mL, 11.88 mmol) and 4dlmethylarnmopyrldme (6.0 mg, 0,047 mmol) in dry dlchloromethane (15 mL) at room temperature, add portlonwlse dl-tert-butyl dicarbonate (1.3 1 g, 6.0 mmol)

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2 Stir the mtxture for 6 h, then add saturated aqueous ammonmm chloride (20 mL) Separate the organic phase and wash it wtth two 20 mL porttons of brme, dry over MgSO,, and concentrate zn vacua to obtain a yellow oil 3 Purify the crude product by column chromatography on silica gel using a gradient of ethyl acetate/hexane (O/10 to l/10, v/v) as the eluant to obtain 1 024 g (98% yield) of lla as a transparent oil 4 P-Lactams llb and 1lc can be prepared m the same manner lla. [al,,20 = +41.4” (c 0.70, CHCl,); IR (neat) 2926, 1810, 1726, 1342, 1156 cm-‘, ‘H NMR (CDCI,) 6 0 8&l 30 (m, 24 H), 143 (s, 9 H), 1 5&l 90 (m, 10 H), 4 06 (m, 1 H), 4 90 (m, 1 H), i3C NMR (CDCl,) 6 11 9, 17 6, 17 7,26 1,26 2, 27 9, 33 0, 33 8, 34 2, 35 5, 56 4, 76.2, 82.8, 148 3, 166.2. Analysis calculated for C,,H,,O,NSt. C, 65.56, H, 10 3 1, N, 3 19 Found C, 65 55, H, 9 97, N, 3 17 lib* 80% yield, colorless 011, [cz]~~~ = -7 61” (c 0.92, CHCl,), ‘H NMR (CDCl,) 0.94-l 00 (m, 21H), 140 (s, 9H), 1 68 (s, 3H), 1 71 (s, 3H), 4 68 (dd, 5 7,998 Hz, lH), 4 88 (d, 5 7 Hz, lH), 5 19 (d, 9 8 Hz, 1H); t3C NMR (CDCl,) 6 118,175,182,260,280,568,773,828,1184,1395,1481,1663 llc 85% yield, colorless oil, ‘H NMR (CDC13) 6 0 96 (d, J = 6 37 Hz, 6 H), 107(m,18H),1.19(m,5H),150(s,9H),160(m,1H),411(m,1H),497(d, J= 5 86 Hz, 1 H), 13C NMR (CDCl,) F 12.0, 17.6, 17.7, 22 5, 22 9, 25 0, 28 0, 37 2, 57 2, 76 2, 83 1, 148 5, 166 5 Analysis calculated for C,,H,,NO,Si C, 63 11, H, 10 34, N, 3 50 Found. C, 63 12, H, 10.08, N, 3 47

3 2 Preparation of Norstatine and Its Analogs Cyclohexylnorstatme 12a and norstatme 12c, key components of rerun mhrbrtors, are prepared through ring-openmg hydrolysis of the corresponding plactams 10a and lOc, as shown in Fig. 3 (IS). Other norstatme analogs can be obtained m the same manner (18).

3.2. I. (2R,3S)-3-Am/no-4-Cyc/ohexy/-2-Hydroxybutanoic Acid 72a and (2RJS)-3-Amino-2-Hydroxy-5-Methylhexano/c Acid 12b (18) 1. To p-lactam 10a or 1Oc (2.0 mmol) add 6 N HCl (8 0 mL) at room temperature, and stir for 3 h 2. Concentrate the reactton mtxture zn vacua to obtain the ammo acid 12a or 12c as their hydrogen chloride salts. 12a White solid; 82% yield, mp 192’C; [cx],,~~ = -11 01” (c 0 32, 6N HCl), IR (KBr dtsk) 1727 cm-‘; ‘H NMR (D,O) 6 0.89-l 68 (m, 13 H), 3 72 (m, 1 H), 4 42 (d, J= 3.2 Hz, 1 H), ‘H NMR (CDsOD) 6 0.43-l 26 (m, 13 H), 2 95 (m, 1 H), 3 72 (d, J= 3 5 Hz, 1 H), 13CNMR (CD,OD) 6 26 7,26 8,27.1,33 9,34 3,38 0, 51 9, 70 2, 174.1 Analysis calculated for C,0H2003NC1. C, 50 50; H, 8 48, N, 5 89 Found. C, 50.30, H, 8.59, N, 5 66 12c* White solid, 88% yteld, mp 2OO”C, [CZ]~~~= +7 21” (c 2 08, 1N HCl), IR (KBr disk) 1757 cm-i, ‘H NMR (CD,OD) 6 0.93 (d, J= 6.9 Hz, 3 H), 0 95 (d, J = 6.9 Hz, 3 H), 1.33 (dt, J= 6 9, 13 7 Hz, 1 H), 1.47 (dt, J= 6 9, 13 7 Hz, 1 H),

Synthesis of Norstatine, its Analogs, and Dipeptide lsosteres

TIPSO,,,

,..s -0

6N HCI. FIT

mz,,

o&H

OH 1Oa

TIPSQ,

145

,... --c

82%

6N HCI, RT

128 flz,,

o&

dH 1OC

66%

Fig. 3. Preparation of cyclohexylnorstatine

12c

12a

and norstatine12~.

1.75(m,lH),3.19(m,1H),3.83(d,J=2.3Hz,1H);’3CNMR(CD~0D)G22.8, 23.3,25.7,42.7,52.9,74.7, 179.2. Analysis calculated for C,H,,O,NCl: H, 8.16; N, 7.09. Found: C, 42.39; H, 7.99; N, 6.88.

C, 42.54;

Dipeptides containing norstatine residue and its analogs can be obtained directly through ring-opening coupling of the p-lactams 13 with a-amino acid esters (22). Various a-amino acid esters are used for the ring-opening coupling

with different /3-lactamsin excellent yields without epimerization. The preparations of norstatine dipeptides 14-16 are described as representative examples (Fig. 4). Solid state synthesis of norstatine dipeptides such as 16 can be performed using a-amino acids linked to the Wang resin (Fig. 4). 3.2.2. (3R,4S)-l-tert-8ufoxycarbonyl-4-Cyclohexylmethyl3-HydroxyAzetidin-Z-One 73a (22) 1. To a solution of lla (0.53 mmol) in THF (3 mL) at room temperature under nitrogen atmosphere, add TBAF (1 .OmL, 1 Msolution in THF), and stir the mixture for 25 min. 2. Pour the reaction mixture into water (10 mL), and extract with three 10 mL portions of ethyl acetate. Combine the extracts, dry over MgS04, and concentrate in vacua. 3. Purify the crude product by flash chromatography on silica gel using ethyl acetate asthe eluant to obtain 13a as a white solid in 92% yield: mp 13 l-132°C; [cx]~*~= +173.5” (c 0.98, CHCI,); IR (neat) 3616,3019,2976, 1807, 1726, 1601, 1522, 1422, 1333, 1212, 1152 cm-‘; ‘H NMR (CDC13) 6 1.40 (s, 9 H), 2.70 (bs, 1 H), 5.08 (d, J= 5.9 Hz, 1 H), 5.14 (d, J= 5.9 Hz, 1 H), 7.27 (d, J= 6.1 Hz, 2 H), 7.38 (m, 3 H); 13CNMR (CDC13) 6 27.9,61.6,77.0,83.8, 127.2, 128.8, 128.8, 133.1, 147.7, 169.5. Analysis calculated for C14H1704N: C, 63.87; H, 6.51; N, 5.32. Found: C, 63.71; H, 6.38; N, 5.12.

3.2.3. (2R,3S)-3-tert-Butoxycarbony/amino-2-Hydroxy4-Cyclohexylbutanoyl-(S)-Phenylalanine Methyl Ester 14a (22) 1. To a solution of p-lactam 13a (0.047 mmol) in dichloromethane (2.0 mL) at room temperature under nitrogen atmosphere, add (S)-phenylalanine methyl ester (17 mg, 0.094 mmol) (see Note S), and stir the mixture for 5 h.

Ojhna and Delaioge

146

13c

Fig. 4. Preparation acid esters.

2 h, 72 %

16

of 14-16 through ring opening of p-lactams

with a-amino

2. Treat the reaction mixture with 10% citric acid (10 mL) to remove excess amino ester, and extract with three 20 mL portions of dichloromethane. Combine the extracts, dry over MgSO,, and concentrate in VUCUO. 3. Purify the crude product by flash chromatography on silica gel using ethyl acetate/ hexane (l/4, v/v) as the eluant to obtain 14a in 94% yield as a colorless oil (see Note 6): [c.x]~~~= +25.81” (c 0.341, CH,CI,); IR (neat) 3374, 2923, 2849, 1744, 1708, 1678, 1499, 1447, 1365, 1171 cm-i; ‘H NMR (CDCI,) S 0.85-1.75 (m, 13 H), 1.37 (s, 9 H), 3.11 (m, 2 H) 3.67 (s, 3 H), 3.81 (m, 1 H), 4.06 (d,J= 2.9 Hz, 1 H), 4.82 (m, 1 H), 5.13 (bd, J= 7.7 Hz, 1 H), 7.14-7.31 (m, 6 H); t3C NMR (CDC13) S 26.1, 26.3, 26.6, 28.2, 32.8, 33.9, 34.4, 37.2, 38.1, 51.7, 52.1, 53.1, 74.6, 80.2, 127.2, 128.6, 129.2, 135.8, 157.2, 171.5, 172.5. Analysis calculated for C25H3806N2: C, 64.91; H, 8.28; N, 6.06. Found: C, 65.13; H, 8.07; N, 5.82. In the same manner, (2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-4cyclohexylbutanoyl-(S)-tryptophan methyl ester 14b and (2R,3S)-3-tert-butoxycarbonylamino-2-hydroxy-5-methylhexanoyl-(~-proline methyl ester (25) are obtained (22). 14b: Colorless solid; 92% yield; mp 91-92°C; [a]n20 = +30.46” (c 0.174, CH,Cl,); IR (neat) 3365,1738,1693, 1661, 1525, 1503, 1440, 1362, 1249, 1167 cm-‘; ‘H NMR (CDC13) S 0.85-1.79 (m, 13 H), 1.51 (s, 9 H), 3.32 (m, 2 H), 3.63 (s, 3 H), 3.86 (m, 1 H), 4.03 (d, J= 2.3 Hz, 1 H), 4.89 (m, 1 H), 5.15 (bd, J= 8.2 Hz, 1 H), 7.07-7.20 (m, 4 H), 7.32 (d, J= 7.8 Hz, 1 H), 7.54 (d,J= 7.8 Hz, 1 H), 8.39 (bs, 1 H); 13C NMR (CDCls) S 26.1,26.3,26.5,27.8,28.2, 32.8, 33.7, 34.2, 37.5,51.6,52.4,52.58,74.2,80.1, 110.0, 111.4, 118.5, 119.7, 122.3, 123.1, 127.5,

Synthesis of Norstatine, Its Analogs, and Dipeptide isosteres

147

136.1, 170.0, 170.9, 171.7. Analysis calculated for C,,H,sO,N,: C, 64.78; H, 7.65; N, 8.39. Found: C, 64.74; H, 7.74; N, 8.19. 15: White solid; 92% yield; mp 34-35°C; [alo*’ = +58.8” (c 0.25, CH,Cl,); IR (neat) 3369, 1747, 1702, 1641, 1518, 1451, 1368, 1172 cm-‘; ‘H NMR (CDCI,) 6 0.93 (d, J= 6.4 Hz, 6 H), 1.37 (s, 9 H), 1.56 (m, 2 H), 1.68 (m, 1 H), 2.04 (m, 4 H), 2.17 (m, 2 H), 3.69 (m, 1 H), 3.72 (s, 3 H), 4.10 (m, 1 H), 4.22 (s, 1 H), 4.34 (bd, J= 7.9 Hz, 1 H), 4.62 (bd, J= 9.9 Hz, 1 H); 13C NMR (CDCl,) 6 22.4,22.9,24.8,25.1,28.4,28.8,41,9,46.3,51.9, 52.2,60.0,71.9,79.3, 155.8, 170.9, 171.4. Exact mass calculated for C,sH,,06N2: m/z 373.2339. Found: 373.2324.

3.2.4. (2R,3S)-3-Amino-2-Hydroxy-5-Methy/hexanoy/(S)-Phenylalanine. TFA 76 (22) 1. Set one cartridge of Fmoc-Phe-Wang resin (80 mg, 0.042 mmol) on the RaMPSTM system. After deprotection of the Fmoc group by the standard procedure (50% piperidine in DMF for 9 min), dry the resin in vacua for 2 h. Then transfer it to a 5 mL round-bottomed flask containing a solution of 13~ (20 mg, 0.082 mmol) in dioxane (2 mL). 2. Heat the mixture at 60°C for 2 h , transfer to the cartridge fitted with a filter, wash with DMF and methanol to remove excess 13c and possible side-products, and carry out the ninhydrin test for confirming the absence of free amino group. 3. Dry the resin, transfer it to a 5 mL round-bottomed flask, and add TFA (5 mL). Stir the suspension for 2 h at room temperature. Filter the suspension, and dry the filtrate in vacua to obtain the TFA salt of dipeptide 16 (14 mg, 72%): mp 238’C; ‘HNMR(CD30D)G0.85(d,J=7.4Hz,6H), 1.31 (m, 1 H), 1.53(m,2H),2.98 (m,2H),3.41 (m, 1 H),4.00(d,J=2.7Hz, 1 H),4.68(dd, J=5.8,9.8Hz, 1 H), 7.12-7.24 (m, 5 H); MS m/z 309 (99.4%) [M-TFA+l]+. IV-Boc-P-lactams react also with ketone and ester enolates to yield hydroxy(keto)ethylene dipeptide isosteres (23). 4-Cyclohexylmethyl-P-lactam lla and 4-(2-methylpropyl)-P-lactam llc give ring-opening coupling products in good to excellent yields by reactions with lithium enolates of ketones and esters such as acetone, acetophenone, phenyl ethyl ketone, ethyl acetate at low temperature (24). The reaction is faster with ester enolates than ketone enolates. Two examples are described for this efficient ring-opening coupling reaction (Fig. 5).

3.2.5. (4R,5S)-5-(N-tert-Butoxycarbony/)Aminol-Phenyl4- Triisopropylsiloxyhexan- 1,3- Dione Enol 17 (23) 1. In a 25 mL round-bottomed flask under nitrogen, cool a solution of diisopropylamine (0.03 mL, 0.22 mmol) in THF (1 mL) to -10°C for 10 min. Then add n-butyllithium (0.09 mL, 0.22 mmol). Stir the mixture for 30 min. 2. Cool the resulting LDA solution to -78°C and add acetophenone (0.03 mL, 0.22 mmol). Stir the reaction mixture for an additional 30 min, then add a solution of p-la&am lla (75 mg, 0.17 mmol) in THF (1 mL) at -10°C for 20 min.

148

Ojima and Delaloge

THF, -10 “C, 20 min then -> FIT, 1 h &TIPS

66% TIPSO,,,

.a i

THF, -78 “C. 15 min

o!&c

OLi 1lC

E,OA

&TIPS 10

96%

Fig. 5. Preparation of 17 and 18 through reaction of p-lactams 11 with lithium enolates.

Monitor the reaction by TLC, and allow the reaction mixture to warm to room temperature over the period of 1 h. 3. Quench the reaction with saturated aqueousammonium chloride (10 mL), and extract the aqueouslayer with two 10mL portions of ether. Combine the extracts, and wash the combined extracts with two 25 mL portions of brine. Dry over MgS04, and concentrate in vac~o to obtain the crude product asan orange liquid. 4. Purify the crude product by flash chromatography on silica gel using ethyl acetate/ hexane (l/12, v/v) as the eluant to obtain 17 asan orange oil (82 mg, 86% yield): ‘H NMR (300 MHz, CDCl,) 6 0.87-1.71 (m, 13 H), 1.12 (m, 21 H), 1.38 (s, 9H),4.09 (m, 1 H), [4.32 (d,J= 1.82Hz),4.38 (d, Jz3.41 Hz), 1 H], [4.83 (d, J= 10.5 Hz), 4.93 (d, J= 11.5 Hz), 1 H], 6.63 (s, 1 H), 7.49 (m, 3 H), 7.89 (d, J= 7.44 Hz, 2 H); 13CNMR (300 MHz, CDCl,) 6 12.4, 18.1, 26.2, 26.5, 26.6, 28.3, 32.4, 34.2, 34.4, 38.06, 52.0, 94.5, 127.1, 128.7, 132.5, 135.0, 155.5, 183.4, 198.4. Analysis calculated for Cs2H5sN05Si: C, 68.65; H, 9.54; N, 2.50. Found: C, 68.64; H, 9.31; N, 2.48.

3.2.6. Ethyl (4S,5R)-5-(N-tert-Butoxycarbonyl) Amino-7-Methyl-3-0xo-4-Triisopropylsiloxyoctanoate

18 (23)

1. Cool a solution of diisopropylamine (0.11 mL, 0.75 mmol) in THF (2 mL) to -10°C in a round bottomed flask, then, add n-butyllithium (0.3 1 mL, 2.5 A4 in hexane, 0.75 mmol) to the solution, and stir the mixture for 30 min. 2. Cool the resulting LDA solution to -78°C in a dry-ice acetone bath. Add ethyl acetate (0.06 mL, 0.63 mmol) dropwise via cannulu to the solution, and stir the mixture for 15min. Add dropwise a solution of p-lactam 1lc (90 mg, 0.23 mmol) in THF (1.5 mL) to the mixture via cannulu, and stir the mixture for 15 min. 3. Check the completion of the reaction by TLC, and quench with saturated aqueous NH&l. Extract the product with Et,O, wash the organic layers with brine, dry over MgSO,, and concentrate in vucuo. 4. Purify the crude product by flash chromatography on a silica gel using ethyl acetate/hexane (l/20, v/v) as the eluant to obtain 18 as a colorless oil (106 mg,

Synthesis of Norstatine, Its Analogs, and Dipeptide lsosteres TIPSQ, ofiBoc

P

MeOH, DMAP, TEA, reflux. 2 d g4%

BocHN sT;r

BocHN \-

19

11 MP”;;~$‘z, 1 90%

“Eoyr

I

C02Me

20

DISAH,

18 h

RT, 3 h

B,Na-,, %”

92%

1) SnCI4,

Swem oxidation

149

CH&

2) +-us 3) sat NaHC03 57%

PdCl2.

(9

24 1 mixture)

CuCI, 02

H20/DMF, 86%

RT, 3 h 25

Fig 6 Preparation of dlhydroxyethylene

71%

26

isostere 26 (R = 2-methylprop-

1-enyl)

96% yield)* [a]020 = +53 9” (c 0 104, CHC13), ‘H NMR (CDCl,) 6 0 92 (m, 6 H), 108(~,18H),l 10(m,5H),126(t,J=715Hz,3H),140(s,9H),166(bm,1H), 3.59(d,J=164Hz,1H),3.70(d,J=16.6Hz,1H),404(m,1H),4.17(q,J=144, 7.17Hz,2H),[433(d),442(d),J=312Hz,1H],474(bd,J=101Hz,1H),’3C NMR(CDC13) 6 12 4, 12 6, 14 1, 14 2, 18 1,21 8,23.4,24-g, 28 3,40 0,46 4, 51 8, 61 2, 79 3, 80 1, 155 4, 167 3. Analysis calculated for C,,H,,NO$i C, 61 56, H, 10 13, N, 2.87 Found C, 61 47, H, 9 97, N, 2.72.

3.3. Preparation

of Dihydroxyethylene

lsosteres

Dlhydroxyethylene isosteres with high enantiomerlc purity are synthesized m several steps from 3-hydroxy+-lactams 11. The aldehyde 23 (R = z-Bu, cyclohexylmethyl) 1san important intermediate that has been used for the syntheses of potent renin inhibitors (25) Figure 6 illustrates the synthesis of 26, in which R 1s 2-methylprop-1-enyl, but this process has been successfully applied to other j3-lactams 11 bearmg a phenyl, benzyl, cyclohexylmethyl or 2-methylpropyl as the substltuent R (26). The reaction of p-lactam 11 with methanol under reflux for two days m the presence of triethylamme, and DMAP gives 19 m excellent yield. The TIPS group 1s removed, and the resulting ammo alcohol 20 is protected as oxazohdme 21. Subsequent reduction of the ester moiety followed by Swern oxldatlon affords aldehyde 23. Addition of an allyltm reagent, followed by

150

Ojima and Delaloge

Wacker oxldatlon (27) gives the N,O-protected dlhydroxyethylene lsostere 26. should be noted that when the substltuent R is a saturated alkyl, cycloalkyl or an aromatic group, direct ozonolysis followed by oxldatlve work-up can be used to obtain 26 (see Subheading 3.4.7.). It

3.3 7. (2R,3S)-3-(tert-Butoxycarbony/amino)-2-Triisopropy/siloxy5-Methylhex-4-Enoic Acid Methyl Ester 796 (26) 1 To a solution of Boc-P-lactam llb (0 6 g, 1.5 mmol) m freshly distilled methanol (17 mL), add DMAP (60 mg) and trlethylamme (0 426 mL, 3 0 mmol) Stir the mixture under reflux for 48 h 2 Remove the solvent under vacuum, and redissolve the residue m ethyl ether Wash the orgamc layer with water, dry over MgS04, and concentrate zn vucuo 3 Purify the crude product by flash chromatography on slllca gel using ethyl acetate/hexane (l/5, v/v) as the eluant to obtain 19b (0 607 g, 94% yield) as a colorless oil* [a]nzO = -20” (c 1 10, CHCI,), IR (neat) 2945, 2896, 1761, 1493, 1389,1366,1270,1249,1164,998,882,848,680,655 cm-‘, ‘H NMR (CDCl,) 6 109(bs,12H),1.41(s,9H),171(d,J=10Hz,6H),3.70(s,3H),434(s,1H), 4 70 (bs, 1 H), 4.95 (bs, lH), 5 16 (bd, J= 8.5 Hz, 1 H), 13C NMR (CDC13) 12 2, 17 6, 18 0, 25 3, 27.4, 28 0, 51 5, 52 1, 75 0, 78 8, 122 9, 134 9, 154 8, 171 8 Analysis calculated for C22H4205NS1 C 61 64, H 9 88, N 3.27 Found C 6 1 81, H 10.00, N 3 33

3.3.2. (2R,3S)-3-tert-Butoxycarbony/amino-2-Hydroxy/5-Methylhex-4-Enoic Acid Methyl ester 20 (26) 1 To a magnetically stirred solution of 19b (330 mg, 0 77 mmol) m pyrldme (13 mL), add dropwlse HF/pyndme (2.25 mL, 1 0 Min pyndme) at 0°C Stir the mixture at room temperature for 14 h. 2 Quench the reactlon with saturated aqueous CuS04 To remove excess pyridme, wash with saturated &SO4 until no color change 1s observed Extract the reaction mixture with three 20 mL portions of ethyl acetate. Combme the extracts, wash the combmed extracts wrth water, brme, dry over MgS04, and concentrate zn vucuo 3 Purify the crude product by flash chromatography on slhca gel using ethyl acetate/ hexane (l/5, v/v) as the eluant to obtam the product 20 (197 mg, 93%) as a white sohd mp 53-54”C, [a]n*O = +0 83” (c 1.20, CDCl,), IR (neat) 3396,2980,293 1, 1693, 1682, 1505, 1455, 1392, 1317, 1250, 1168, 880, 754 cm-‘, ‘H NMR (CDCl,) 6 1 42 ( s, 9 H), 1 75 (s, 6 H), 3.09 (bs, 1 H), 3 81 (s, 3 H), 4 14 (d, J=5 7 Hz, 1 H), 4 77 (bs, 1 H), 5 20 (bs, 1 H), 13C NMR (CDCl,) 6 18 4, 25.6, 28 2, 51.3, 52.8, 73 4, 76.5, 79.6, 121.3, 136.9, 155.1, 173.6, 193.2. Analysis calculated for C13H2305N: C 57 13, H 8 48, N 5 12, Found C 57 09, H 8 17, N 5 06

3.3.3. (4R,SS)-3-tert-Bu~oxycoxycarbony/-5-Carbomethoxy2,2-Dimethyl-4-(2-Methylprop-l-Enyl)-1,3-Oxazo/~dine 21 (26) 1 To a solution of 20 (100 mg, 0 36 mmol) m toluene (5 mL), add pyrldmmm p-toluenesulfonate (10 1 mg) as a single solid portion with stirring, followed by

Synthesis of Norstatme, Its Analogs, and Dipeptide lsosteres

151

dropwlse addltlon of 2,2-dlmethoxylpropane (35 mL, 8.93 mmol), and stir the reaction mixture at 90-95°C for 18 h 2 Add ether (25 mL) to the reaction mixture, wash with saturated aqueous NaHCO,, and extract with three 20 mL portions of ether Combme the extracts, and wash the combined extracts successively with saturated aqueous NaHCO,, brme, dry over MgSO,, and concentrate in vacua 3 Purify the crude product by flash chromatography on silica gel usmg ethyl acetate/hexane (l/5, v/v) as the eluant to obtain the product 21 (101 mg, 89% yield) as a yellow oil* [c1]*O= -48” (c 0.5, CDCI,), IR (neat) 2982, 2270, 1758, 1737, 1699, 1478, 1452, 1440, 1376, 1359, 1251, 1208, 1171, 1122, 1074, 858 cm-‘, ‘H NMR (CDCI,) 6 1 35 (s, 9 H), 1 54 (d, J= 10 5 Hz, 6 H), 1.66 (d, J= 7 5 Hz, 6 H), 3 72 (s, 3 H), 4 20 (d, J= 5 0 Hz, 1 H), 4 80 (s, 1 H), 5.15 (d, J= 7 5 Hz, 1 H), 13CNMR (CDCI,) 17 9,25.6, 27.0,28 3, 52.4, 58 4, 77 6,79.0,79.8,95.9, 124.3, 151 3, 171 0 Analysis calculated for C,6H2705N C 61 32, H 8.68, N 4 47, Found C 61.36, H 8 47, N 4 55

3.3 4. (4R,5S)-3-tert-Butoxycarbonyl-2,2-Dimethy/5-Hydroxymethyl-4-(2-Methylprop1-by/)- l,3-Oxazolidine

22 (26)

1 To a solution of 21 (100 mg, 0.32 mmol) m THF (2 mL), add dropwlse dilsobutylalummum hydride (1.10 mL, 0.96 mmol, I 0 A4 solution m toluene) with stirring (see Note 7) at 0°C under nitrogen atmosphere Stir the mixture at room temperature for 3 h 2 Quench the reaction with a saturated solution of Rochelle’s salt (sodium potassium tartrate), and extract with three 20 mL portions of ethyl acetate Combme the extracts, wash the combmed extracts with brme, dry over MgSO,, and remove the solvent under reduced pressure 3 Purify the crude product by flash chromatography on slhca gel using ethyl acetate/hexane (l/5, v/v) as the eluant to obtam the product 22 (83 6 mg, 92%) as a colorless 011. [a120 = -48” (c 0 5, CDCI,); IR (neat) 3478, 2977, 2933, 2871, 1701,1460,1394,1372,1263,1184,1074,916,864,784cm-‘; ‘HNMR(CDC1,) 6 1 38 (s, 9 H), 1.52 (s, 3 H), 1.60 (s, 3 H), 1.69 (d, J= 15 Hz, 6 H), 2.07 (m, 1 ), 3 55 (m, 1 H), 3.75 (m, 1 H), 4 35 (t, J= 10 Hz, 1 H), 4 98 (d, J= 9 Hz, 1 H), 13C NMR (CDCl,) 17 9, 25 7, 26 6, 28.3, 29.6, 56 2, 61.2, 79 3, 80 1, 94 4, 124.5, 134 5, 152 1 Analysis calculated for C,,H,,O,N. C 63 13, H 9 54, N 4 9 1, Found C 62.96, H 9 41, N 4.86

3.3 5. (4R,5S)-3-tert-Butoxycarbonyl-2,2-Dimethy/-5-Formyl4-(2-Methylprop- 7-Enyl)- 7,3-Oxazolidine 23 (26) 1 To a solution of oxalyl chloride (0.063 mL, 0.72 mmol) m 1.7 mL of CH2C12 at -70°C, add a solution of DMSO (0 102 mL, 1 44 mmol) in dlchloro-methane ( 0 3 mL). Stir the reaction mixture at -70°C for 10 mm To the mixture, add a solution of oxazolldme 22 (66 mg, 0.24 mmol) m 1 mL of dlchloromethane, stir the mixture at -70°C for 25 mm, and add tnethylamine (0.5 mL, 3 6 mmol).

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2 After 10 mm allow the resulting mixture to warm to 0°C over the period of 90 mm Quench the reaction with 5 mL of water Extract with three 10 mL portions of dlchloromethane Combme the extracts, wash with water (10 mL), 0 IN HCl sol&Ion (10 mL), water (10 mL) , 5% Na,CO, (10 mL), water (10 mL), brme, dry over MgSO,, and concentrate zn vacua 3 Purify the oily crude product by flash chromatography on slllca gel using ethyl acetate/hexane (l/4, v/v) asthe eluant to obtain aldehyde 23 This compound 1s used for the next step without further purification

3 3.6 (4R,5S)-3-tert-Butoxycarbonyl-2,2-Dimethyl-5-(2-HydroxyBut-3-Eny/)-4-(2-MethylpropI-Eny/)- 1,3-Oxazolidine 24 (26) 1 To a solution of tm tetrachlorlde (0 387 mL, 0 387 mmol, 1 M solution m dlchloromethane) at -78’C under mtrogen atmosphere, add allyltrlbutyltm (0 143 mL, 0.369 mmol). Stir the mixture at -78°C for 30 mm, and add aldehyde 23 (40 mg, 0 147 mmol) dropwlse (see Note 8) 2 Stir the solution at-78’C for an additional 45 mm, and quenchwith saturatedaqueous NaHC03 Extract the reaction mixture with three 20 mL portions of CH,C&, combmethe extracts, washwith brine, dry over MgSO,, and concentrate zn vaczzo 3 Purify the crude product by flash chromatography using hexane/ether (10/l, v/v) as the eluant to obtain compound 24 (40 mg , 87% yield, antl/syn = 9/l) as a white solid mp 4546”C, [a]o*O = 47 8” (c 1 15, CDCl,), IR (neat) 3012,2974, 2929,2359,2338,1698,1675,1541,1457,1388,1365,1250,916 cm-l; ‘HNMR (250 MHz, CDCI,) 6 1 40 (s, 9 H), 1 53 (d,J= 1 75 Hz, 3 H), 1 60 (s, 3 H), 1 69 (~,6H),206(m,lH),223(m,2H),3.6l(m,1H),372(m,lH),445(m,lH), 5 00 (m, 1 H), 5 13 (m, 1 H), 5 80 (m, 1 H) Calculated for ClsH3,04N C 66 43, H 9 60, N 4 30 Found C 66 28, H 9 83, N 4 23

3.3.7. (4R,5S)-3-tert-Butoxycarbony/-2,2-D/methy/-5-[(S)I-Hydroxy3-Oxobutylf4-(2-MethylpropI-Enyl)- 1,3-Oxazolldine 25 (26) 1 At the top of a flask contammg PdCl, (2 mg, 0 01 mmol) and CuCl (11 mg, 0 11 mmol) m 1 4 mL DMF and 0.2 mL H20, attach an oxygen balloon Stir the reactlon mixture at room temperature for 1 h so that the oxygen up-take occurs slowly. Add compound 24 (28 mg, 0.091 mmol) dropwlse via syrmge Stir the solution vigorously at room temperature for 2 5 h (monitored by TLC), and then add dlchloromethane (15 mL) 2 Filter the green pastethrough a Cehte pad, and evaporate the filtrate zn vacua 3 Purify the crude product by flash column chromatography on slhca gel usmg ethyl acetateihexane (l/4, v/v) asthe eluant to obtain 25 (34 mg, 86% yield) as a colorless 011.[a]o = -65 2” (c 1 35, CDC13), IR (neat) 3085, 2982, 1718, 1698, 1458, 1388, 1365, 1256, 1175, 1115, 1065, 887, 774 cm-‘, ‘H NMR (CDCI,) 6 1 37 (s, 9 H), 1 48-l 75 (m, 12 H), 2.17 (s, 3 H), 2.49-2.80 (m, 3 H), 3.53 (dd,J =78,5.8Hz,1H),404(bm,lH),4,47(t,J=83Hz,lH),500(dd,J=9l,93 Hz, 1 H) Calculated for ClsH3,05N C63 32, H 9.15, N 4 10 Found C 63 60, H 9 26, N 4 25

153

Synthesis of Norstatine, Its Analogs, and Oipeptide lsosteres

SccHN

a

LAH. ether CO&b

DEAD,

reflux, 4 h

OH

oTlPS

198 19b 1Bc

CHCl3,

PPh3

reflW

36 h

OH

R =cyclohexylmethyl R = kbutenyl R = kbutyl

27a 27b 27~

76% 66% 82%

;g 26c

;yf

00 70%

Fig.7 Preparation of the key intermediates 28a-c

3.3.8. (#R,5S)-3-tert-Butoxycarbony/-2,2-Oimethyl5-[(S) -2- Carbohydroxy- 1-Hydroxye thy/]4-(2-Methyl-Prop-l-Enyl)-l,3-Oxazolidine 26 (26) 1 To a stirred sol&Ion of compound 25 (33 mg, 0.1 mmol) m dloxane (3.4 mL) and water (1 mL), add sodmm hypochlorlte solution (0.4 mL) Stir the reactlon mixture at room temperature for 30 mm, and add solid Na2S03 (63 mg, 0 5 mmol). 2 Acidify the mixture qmckly to pH -3-4 by addmg 0 1 NHCl Extract the aqueous phase with three 10 mL portions of dlchloromethane, combme the extracts, and wash with brine. Dry over MgSO, and concentrate zn vucuo 3. Purify the resulting crude product by flash column chromatography on s&a gel using ethyl acetatejhexane (l/5-1/1, v/v) followed by CHCl,/MeOH(9/1, v/v) as the eluant to obtain 26 (23 mg, 7 1% yield) as colorless oil* [a]020 = -37.9” (c 0 9, CDCI,), IR (neat) 3513,3408,3380,3300,3267,3088,3018,2936,2358,2341, 1732, 1698,1652, 1435, 1379,1301,1258, 1210, 1117, 1087,906,885 cm-‘; ‘H NMR(CDCI,)G 140(s,9H), 144-1.80(m, 14H), 3.35 (d, J=4.3 Hz, 1 H),3 48 (s, 1 H), 3.70 (m, 1 H), 4 43 (m, 1 H), 5.02 (d, J= 9.1 Hz, 1 H), 6.44 (bs, 1 H), 13C NMR (CDCl,) 6 18 1, 25 7, 26.1, 28 3, 29.7, 57.1, 57 6, 57.9, 58 4, 79 9, 95 2, 103 0, 118 3, 123.6,151 9, 170 2, 185.1 Calculated for C,6H2906N C 59.46, H 8 57, N 4 08 Found C 59 30, H 8.35, N 3.89

3.4. Preparation of Hydroxyethylamine and Hydroxyethylene lsosteres The N-Boc-epoxldes 28 are important intermediates for the syntheses of various nonprotein ammo acids (28). The epoxides 28a-c can be readtly prepared from the correspondmg esters 19a-c m two steps (Fig. 7).

3.4.1. (2R,3S)-3-tert-Butoxycarbony/amino-4-Cyc/ohexy/butane1,2-Oiol27a, (2R,3S)-3-tert-Butoxycarbonylamino5-Methylhex-4-En-1,2-Oiol27b and (2R,3S)3-tert-Butoxycarbonylamino-5-Methylhexan1,2-Oiol27c (26) 1 To a suspension of lithium aluminum hydride (1.52 mmol) (see Note 9) m dry ether (4 mL), add slowly a solution of compound 19a-c (1 2 mmol) m ether (2 mL) at room temperature Warm the reaction mixture to 3&35”C for 4 h (see Note 10)

154

OIlma and Delaloge

2 Quench the react’on carefully, and filter the resultmg white precipitate Wash the filtrate with 0 1 N hydrochloric acid, and extract the aqueous layer with three 20 mL portions of ether Combme the organic extracts, wash the combined extracts with brine, dry over MgS04, and concentrate zn vucuo 3 Purify the crude product by column chromatography on silica gel using ethyl acetate/hexane (l/l, v/v) as the eluant to obtam 27a-c (76-86%) 27a* White solid, mp 94-9S’C, [a]n20 = -30 8” (c 0 325, CHCl,); IR (neat) 3348,2916, 1682, 1171 cm-‘, ‘H NMR (CDCl,) 6 0.8&l 80 (m, 13 H), 1 43 (s, 9H),280(bs,1H),360-370(m,4H),380(m,1H),472(d,J=9OHz,1H), 13C NMR (CDCl,) 6 26 1, 26 3, 26.5, 28 3, 32.7, 33.8, 34 2, 39 5, 48.4, 63 6, 73.6, 79 9, 157 2 Analysis calculated for C,5H2904N+ C, 62 69; H, 10 17; N, 4 87 Found C, 63 00, H, 9 72, N, 4 79 27b. 0’1, [aID20 = +6 0” (c 0.5, CHC13); ‘H NMR (CDCl,) 6 1 45 (bs, 9 H), 1.65 (d,J= 8 5 Hz, 6 H), 3.49 (bs, 1 H), 3 55 (bs, 1 H), 3 79 (bs, 1 H), 4 35 (bs, 1 H), 5 13 (d,J= 8.5 Hz, 1 H); 13CNMR (CDC13) 6 15 1, 18 3,25.6,28 2, 50 5, 63 3, 65 7, 74 5, 79 8, 122 0, 136 3, 156 7. Analysis calculated for C12H4205NS1 C 58 75, H 9 45, N 5 71 Found C 58 73, H 9.46, N 5 80 27c White solid, mp 87-88”C, [alD20 = -55 3” (c 0 85, CHC13), IR (neat) 3387, 3360, 1682, 1171 cm-‘, ‘H NMR (CDCl,) 6 0.92 (s, 3 H), 0 94 (s, 3 H), 12~1.60(m,11H),1.68(m,1H),292(bs,2H),352(m,2H),3.60(m,1H), 3.76 (bs, 1 H), 4 70 (bs, 1 H), 13C NMR (CDC13) 6 22 0, 23.1, 24 8, 28 3,40 9, 49 5, 63 7, 73 7, 79 2, 157 0 Analysis calculated for C,,H,,O,N. C, 58 27, H, 10 19, N, 5 66 Found C, 58 26, H, 10 02; N, 5 71

3.4.2 (2R,3S)-3-tert-Butoxycarbonylamino-4-Cyclohexylbut-7-fne Oxrde 28a, (2R,3S)-3-tert-Butoxycarbonylamino5-Methylhex-1,4-Dlene I-Oxide 28b and (2R,3S)3-tert-Butoxycarbonylamino-5-Methylhex-I-Erie Ox/de 28~ (26) 1 To a solution of 27a-c (0.49 mmol) and tr’phenylphosphme (141 mg, 0 539 mmol) m CHCl, (5 mL), add dropw’se diethyl azodlcarboxylate (0.135 mL, 0 539 mmol) with stlrrmg Reflux the reactlon mixture for 36 h 2 After removal of the solvent under reduced pressure, purify the crude product by column chromatography on silica gel pretreated with triethylamme (2.5% v/v) using ethyl acetate/hexane (l/3, v/v) as the eluant to give 28a-c (56-700/o) (see Note 11) 28a [(rlD20 =-14.0” (c 0 5, CHCl,); IR (neat) 2926, 1713, 1520, 1172 cm‘, ‘H NMR (CDC13) 6 0 80-l 80 (m, 22 H), 2 57 (s, 1 H), 2.70 ( t, J= 4.5 Hz, 1 H), 2 95 (s, 1 H), 3.97 (m, 1 H), 4.29 (m, 1 H), 13C NMR (CDC13) 6 26 1, 26 3,26 5, 28.3, 32.8, 33.8,34 1,40.9,44.4,46.5, 54 0, 79 3, 155 7. Analysis calculated for C’,H2,03N. C, 66.88; H, 10 10; N, 5.20. Found, C, 66 64, H, 9.95; N, 5 16. 28b: Colorless 0’1, [alD20 = +3 1.5’ (c 8.6, CHCl,); IR (neat) 3351,2966,2933, 2864,1708,1505,1371,1251,1064,1016,878 cm-‘, ‘HNMR(CDC1,) dl 40 (s, 9 H), 1.73 (s, 6 H), 2 60 (m, 1 H), 2 71 (t, J= 4 5 Hz, 1 H), 2.98 (m, 1 H), 4 5&4.60 (m, 2 H), 5 15 (bd, J = 8 7 Hz, 1 H), 13C NMR (CDCl,) 6 18 4, 25 6, 28 3,44 2,

Synthesis of Norstatme, Its Analogs, and Dipeptide lsosteres

28b

76%

29

74%

30

155

HCI HzN

TEA, M~OH, reflux,lOh

26b

Fig 8 Preparation of hydroxyethylamme

lsosteres 29 and 30.

47 7, 54 0, 121 8, 136 6, 155 3, Analysis calculated for C,,H,,O,NSi C 63.4 1, H 931,N6 16.FoundC635,H9 19,N626. 28c Colorless 011; [alD2’ = -17 2” (c 2.85, CHCI,); IR (neat) 3342, 1714, 1171 cm-‘; ‘H NMR (CDCl,) 6 0 92 (s, 3 H), 0 94 (s, 3 H), 1 40 (s, 11 H), 1 631 77 (m, 1 H), 2.56 (s, 1 H), 2.70 (t,J= 4.4 Hz, 1 H), 2 96 (t, 1 H), 3 95 (bs, 1 H), 4 30 (m, 1 H); 13C NMR (CDC13) 6 22 1,23.1,24 7,28.3,42 3,44.4,47 2, 54 0, 79 2, 156 6 Analysis calculated for C,,H,,O,N C, 62.85, H, 10 11, N, 6 11 Found C, 62.60; H, 9 86, N, 6.07 Hydroxyethylamme lsosteres are readrly prepared from epoxtdes 28. For example, epoxlde 28b undergoes facile ring-opemng reactions with the methyl esters of (S)-phenylalanme and (S)-prolme to give the correspondmg dlpeptlde lsosteres 29 and 30, respectively m good isolated yields (Fig. 8).

3 4 3. N-[(2R,3S)-3-tert-Butoxycar6onylammo-2-Hydroxy5-Methylhex-4-Enyl]-(S)-Phenylalanme Methyl Ester 29 (26) 1 To a solution of (S)-phenylalanme methyl ester hydrochloride (112 mg, 0.5 19 mmol) in 2 mL of dry MeOH, add dropwlse trlethylamme (0 122 mL, 0 875 mmol) at room temperature Stir the reactlon mixture for 20 mm, and add epoxlde 28b (20 mg, 0 088 mmol) m MeOH (see Note 12) 2 Heat the solution at 65°C for 10 h (momtored by TLC), Then evaporate the methanol under reduced pressure, add 25 mL of ether to the reactlon mixture, wash with 10% citric acid, brine, dry over MgSO,, and concentrate zn vacua 3 Purify the 011~crude product by flash column chromatography on slllca gel usmg ethyl acetateihexane (l/3, v/v) as the eluant to obtain 29 (27 mg, 76% yield) as a colorless 011. [a]o = -0 96” (c 3 2, CHC13); ‘H NMR (CDCl,) 6 1.42 (s, 9 H), 1 67 (d, J= 7 5 Hz, 6 H), 2 55 (d, J= 7.5 Hz, 2 H), 2.94 (m, 3 H), 3.48 (m, 3 H), 367(s,3H),416(bm,1H),476(bs,1H),513(bd,J=91Hz,1H),7.16(d,J = 6.6 Hz, 2 H), 7.29 (m, 3 H), 13C NMR (CDCl,) 6 18.4, 25.7, 28.4, 39.8, 50 1,

Ojjma and Delaloge

156

&MS-

E120, -30 ‘C

L BocHN

\

2.2~Dlmethoxypropane PPTS, 90-95 ‘C, 4 h

dH

65%

31 -

91%

-78’c

1) OS, MeOH

CH!sk,

70% 32

Fig 9 Preparation of hydroxyethylene

lsostere 33.

51 8, 62.2, 71 8, 87.3, 94 9, 123 1, 126 8, 128.5, 129.1, 135.5, 137 5, 157.5 HRMS Calculated for C,,H,,O,N, m/z 407 2546 Found. 407 2554 (A = 0 0008 mu>

3.44. N-[(2R,3S)-3-(tert-Butoxycarbonylamino)-2-Hydroxy5-Methylhex- 7-Eny/)]-(S)-Pro/he Methyl Ester 30 (26) 1 To a solution of (S)-prohne methyl ester hydrochloride (91 mg, 0 55 mmol) m 2 mL of dry MeOH, add dropwlse trlethylamme (0 150 mL, 1 1 mmol) at room temperature Stir the reaction mixture for 20 mm, and add epoxlde 28b (25 mg, 0 11 mmol) in MeOH (see Note 12). Heat the solution at 65°C for 10 h (monitored by TLC). 2 After removal of methanol under reduced pressure, purify the oily crude product by flash column chromatography on silica gel using ethyl acetate/hexane (l/10, v/v) as the eluant to obtain 30 (19 mg, 74% yield) as a colorless 011 ‘H NMR (CDCl,) 6 140 (s, 9 H), 1.68 (d, J= 4 7 Hz, 6 H), 1 80 (m, 2 H), 2 10 (m, 1 H), 2 16 (m, 1 H), 2 40 (m, 2 H), 3 21 (m, 1 H), 3 24 (m, 1 H), 3 58 (s, 3 H), 4 16 (bs, 1 H), 4 92 (bs, 1 H), 5 26 (d, J= 9 0 Hz, 1 H), 13CNMR (CDCl,) 6 18.0,23 9, 25 7,28 4,29 5,52 0,53 3,58 1,65 7,71 2,76 5,77 0,78 6, 123 9, 156 7, 178 0 Hydroxyethylene lsosteres can be prepared from epoxtdes 28. An example 1s shown m Fig. 9. The reaction of an ally1 Grtgnard reagent with epoxtde 28~ gives alcohol 31 m excellent yield. The hydroxyl and the ammo groups are protected as an oxazolidine, and the subsequent ozonolysls and oxldattve workup afford the correspondmg N, U-protected hydroxyethylene tsostere 33 m good isolated yield (Fig. 9).

3.45

(5S,6S)-6-tert-Butoxycarbonylamino-

5-Hydroxy-B-Methylnon-

1 -Ene 31 (26)

1 To a solution of epoxlde 28c (45 mg, 0 196 mmol) m THF (2 mL), add dropwlse a catalytic amount of CuI (5 mg) and allylmagneslum bromide (1 0 Mm ethyl ether, 0 59 mL, 0 588 mmol) at -30°C Stir the reaction mixture for 1 h, and quench with saturated aqueous NH4Cl Extract the reactlon mixture with three 10 mL portions of ether, dry over MgSO,, and concentrate zn vacm

Synthesis of Norstatine, Its Analogs, and Dipeptide lsosteres

157

2 Purify the crude by column chromatography on silica gel using ethyl acetate/ hexane (l/20, v/v) as the eluant to obtain 31(48 mg, 9 1% yield) as a colorless 011 [a]020 = 43.6” (c 0 55, CHCl,), IR (neat) 3074,2956, 2868, 2344, 1713, 1685, 1508, 1458, 1391, 1366, 1251, 1169, 1049, 910 cm-‘; ‘H NMR (CDCI,) 6 0 92 (d, J= 6.0 Hz, 6 H), 1.24 (m, 2 H), 1 43 (s, 9 H), 1.56 (m, 2 H),l.69 (bs, 1 H), 2 17 (m, 2 H), 3 54 (bs, 2 H), 3 75 (bs, 1 H), 4.57 (bs, 1 H), 5.00 (m, 2 H), 5 81 (m, 1 H), 13C NMR (CDCl,) 6 22 2,23 0,23.2,24 6,28.3,30.1,33.4,41 7,53.5,73.7, 116 0, 138.4, 172 0 HRMS Calculated for C,5H,o0,N: m/z 272.2226 Found. 272 2230 (A = 0 0004 m u )

3.4.6. (4S,5S)-3-tert-Butoxycarbonyl-5-(But-3-En-l-Y/)2,2-Dimethyl-4-(Z-Methylpropylj1,3-Oxazolldine (32) (26) 1 To a solution of ammo alcohol 31 (70 mg, 0.26 mmol) m 4 mL of toluene, add a catalytic amount of PPTS (5 mg) and 2,2-dlmethoxypropane (0 337 mL, 2.75 mmol) Stir the reactlon mixture at 90-95’C for 4 h (momtored by TLC), and then allow the reactlon mixture to cool to room temperature 2. Add 25 mL of ether to the reactlon mixture and wash with saturated aqueous NaHC03 Extract the aqueous layer with three 15 mL portions of ether, wash the combined ether extracts with saturated aqueous NaHCOJ and brme, dry over MgS04, and concentrate zn vucuo 3 Purify the crude product by column chromatography on slllca gel usmg ethyl acetatejhexane (l/4, v/v) as the eluant to obtain 32 as a colorless oll(5 1 mg, 65% yield) [a]020 = +33 3” (c 1 5, CHCl,); IR (neat) 3106, 3067, 3037, 2947, 1699, 1653, 1558, 1540, 1456, 1386, 1366, 1255, 1175, 1083, 914 cm’, ‘H NMR (CDCI,) 6 0 93 (d, J= 6 3 Hz, 6 H), 1 46 (s, 9 H), 1 49-l 57 (m, 11 H), 2 11 (m, 2 H), 3 83 (dd,J= 6.0, 8 0 Hz, 1 H), 4 07 dd, J= 6 7,7.1 Hz, 1 H), 4.94-5 07 (m, 3 H), 5 73-5.89 (m, 1 H), 13C NMR (CDCl,) 6 25.6,27 8, 28.5,29 9, 34 0,35 1, 43 2, 59.5, 60 8, 80 2, 114 9, 137.8, 172 1 HRMS Calculated for C,,H,,O,N m/z 312 2539. Found 312.2542 (A = 0.0003 cl)

3.4.7.

(4S,5S)-3-tert-Butoxycarbonyl-2,2-Dimethyl-5-

(Z-Hydroxycarbony/ethy/)-4-(Z-Methy/propy/)-

7,3-Oxazo/id/ne 33 (26)

1 To a solution of oxazohdme 32 (20 mg, 0 064 mmol) m 4 mL of dichloromethane, bubble a flow of N2 for 5 mm at -78°C. Introduce ozone (generated using a Welxbach Ozomzer) until the colorless reaction mixture turns mto a light blue solution (due to excess ozone). Bubble N2 again through a ptpette to get nd of excess ozone until the blue color disappears 2 Immerse the reactlon flask m an Ice bath and add H202 (0 1 mL, 30% solution m water), NaOH (20 mg, 0 5 mmol) pellets and Adogen 464 (0 1 mL, MW - 410) at 0°C (see Note 13) Stir the reaction mixture for 26 h at room temperature, and acidify the reaction mixture with 0 1 N HCl to pH 3-4 3 Extract the aqueous phase with three 10 mL portions of dlchloromethane, combine the organic extracts, wash with brine, dry over MgS04, and concentrate

zn vacua

158

Ojima and Delaloge

4 Purify the crude product by flash column chromatography on siltca gel usmg ethyl acetate/hexane (l/15-1/5, v/v) as the eluant to obtain 33 as a colorless oil (15 mg, 70% yield). [c~]o*~ =-114” (c 0 14, CHCl,), ‘H NMR (CDCl,) 6 0 92 (d, J= 6.2 Hz, 6 H), 1 25 (m, 2 H), 1 47-l 90 (m, 20 H), 3.70 (bs, 1 H), 3 81 (t,J= 5 7 Hz, 1 H), 4 05 (m, 2 H), r3C NMR (CDCl,) 6 21 4, 24 0, 25 5, 28 5, 28 6, 327,606,609,776,947,171 0,1992,IR3478,3228,3163,3130,2940, 1696, 1388,1174 Calculated forC,,H3,0sN C 61 98, H 9 48, N 4 25 Found C 61 79, H933,N4 13

4. Notes 1 The imines are very air-sensitive m general and must be prepared Just before use except for the rmine derived from benzaldehyde, which can be recrystallized from ethyl acetatelhexane and stored at room temperature for a few weeks as long as It IS protected from hght 2 Thep-amsrdme must be recrystalhzed from methanol, which gives a light brown flaky sohd. Sodmm sulfate must be drted m an oven at 150°C prior to use In general, the chu-al ester enolate-tmme cyclocondensatlon 1s highly motsture- and au-sensmve, thus, all precautions to prevent motsture and au must be taken 3 The rate of addmon is very important Both addmon of ester 5 as well as that of imme must be very slow, smce these processes are exothermic Fast addition will result m the reduced enanttoselectrvtty 4 The temperature control is important for the deprotection. The yield may decrease dramatically without proper temperature control N-termmus free a-ammo acid esters should be generated Just prior to use For the purtficatton of the crude product by column chromatography, srhca gel must be pretreated with sodmm carbonate DIBAH 1s a hazardous chemtcal and should be handled wtth care Alkyltm derrvatives are mostly toxic Use bleach to clean the glassware after use A sufftctent amount of stlrca gel must be used for column chromatography m order to get rid of tm residues that appear as blue spots at the baseline of TLC plate 9 LrAlH, should be handled with care, particularly during hydrolyses 10 More than 4 eq of LAH are necessary m order to reduce the ester moiety and deprotect the hydroxyl group 11 Silica gel is stirred with the eluant (ethyl acetateihexane) and trlethylamme (4.5%) and then poured into the column This protocol prevents the decomposition of acid-sensitive compounds 12 Methanol and trtethylamme must be freshly distilled m the presence of methylmagnesium iodide and calcmm hydride, respectrvely 13 Adogen IS a phase transfer catalyst. Generally, ozonolysts 1s performed m a mixture of dtchloromethane and methanol However, the presence of methanol leads to the formation of side-products m this reaction Accordmgly, the reaction is carried out m dtchloromethane in the presence of the phase transfer catalyst

Synthesis of Norstatine, Its Analogs, and Dipeptide lsosteres

159

Acknowledgment This research was supported by grants from the National Institutes of Health (NIGMS). The authors would hke to thank Hong Wang, Tao Wang, Edward Ng, Chung Ming Sun, Young Hoon Park, and Thierry Brigaud for then- excellent technical contributions.

References 1 Huff, J R (1991) HIV Protease. A novel chemotherapeutic target for AIDS J Med Chem 34,2305-23 14 2 Koot, W -J , Van Gmkel, R , Kranenburg, M., Hiemstra, H , Louwrier, S , Moolenaar, M J , and Speckamps, W N. (1991) Synthesis of statine from (s)mahc acid; stereocontrol via radtcal cychzatton. Tetrahedron Lett 32,40 l-404 Nebois, P and Greene, A E. (1996) Novel enanttoselective approach to y-lactams from chnal enol ethers: Synthesis of (-)-statme. J Org Chem 61,52 10-52 11 Takemoto,Y., Matsumoto,T , Ito, Y , andTerashima,S (1990)An expedmoussynthesisof (3S,4Qstatme and(3S,4S)-cyclohexylstatine Tetrahedron Lett 31,2 17-2 18 Kumeda, T., Ishizuka, T , Higuchi, T , and Hirobe, M (1988) Versatile chnal synthons for vie-ammo alcohols-Facile synthesisof (2S,3R)-3-hydroxyglutamic acid and (+) statme. J Org Chem 53,3381-3383 Joum, P , and Castro, B (1987) Stereospecific synthesis of N-protected statme and its analoguesvia choraltetramic acid J Chem Sot Perkzn Trans I, 1177 Yanagisawa, H., Kanazakt, T , and Nishi, T (1989) Synthesis of Statme and Its Analogues Chem Lett 687490 Pasto, M , CasteJon,P , Moyano, A , Pericas, M A , and Riera, A (1996) A catalytic asymmetric synthesisof cyclohexylnorstatme. J Org Synth 61,6033-6037 Matsumoto, T , Kobayashi, Y , Takemoto, Y , Ito, Y., KamiJo, T., Harada, H., and Terashima, S ( 1990) A stereoselectivesynthesisof cyclohexylnorstatme, the key component of a Renm Inhibitor Tetrahedron Lett 31,4 175-4 176 10 Kobayashi, Y , Takemoto, Y , Ito, Y., and Terashtma, S. (1990) A Novel Synthesisof (2R,3S)- and (2S,3R)-3-dmmo-2-hydrocarboxylic acid derivatives, the key componentsof a renin mhibitor and bestatin, from methyl (R)- and (S)-mandelate

Tetrahedron Lett 31,303 l-3034 11 Ito, Y., Kamqo, T., Harada, H , and Terashima, S. (1990) A novel synthesis of xyclohexylnorstatine isoprpylester, the C-terminal component of a renm inhibitor Heterocycles 30, 299-302 12 Iizuka, K , KamiJo, T , Harada, H , Akahane, K , Kubota, T , Uneyama, H , Ishida, T., and Kiso, Y (1990) Orally potent human renm mhibitor derived from angiotensmogentransition state*design, synthesisandmode of interaction J Med Chem 33,2707-27 14 13 OJima, I. (1995) Recent advances m p-lactam synthon method. Act Chem Res 28,383-389. 14

Olima, I (1995) Advances zn Asymmetrzc Syntheses by Means of the P-Lactam Synthon method, vol 1 JAI, New York, pp. 95-146

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15 OJtma, I ( 1992) fi-Lactam synthon method-enantiomerically pure l3-lactams as Synthetic mtermedtates, m The Organic Chemzstry of PLactam Antlobzotzcs (Georg, G I , ed ), VCH, New York, pp 197-255 16 OJtma, I, Habus, I , Zhao, M., Zucco, M , Park, Y H , Sun, C -M , and Bngaud, T (1992) New and efficient approaches to the semisynthesis of taxol and its C-13 side chain analogs by means of p-lactam synthon method Tetrahedron 48,6985-70 12 17 OJtma, I , Habus, I, Zhao, M , Georg, G. I., and Jayasmghe, R (1991) Efftctent and practical asymmetric synthesis of the taxol C- 13 side chain, N-Benzoyl-(2R,3,!+3-phenyltsoserme, and its analogs via choral 3-hydroxyl4-aryl-P-lactams through chiral ester enolate-imme cyclocondensatton J Org Chem 56, 1681-1684 18. OJtma, I , Park, Y H., Sun, C M , Brtgaud, T , and Zhao, M (1992) New and efficient routes to norstatme and its analogs with high enantiomertc purity by plactam synthon method Tetrahedron Lett 33,5737-5740 19 Whttesell, J K and Lawrence, R M (1986) Practical enzymatic resolution of choral auxtllartes-+nanttomertcally pure trans-2-phenylcyclohexanol and trans2-(a-Cumyl)cyclohexanol. Chzmza 40,3 18 20 Kukla, M .I , and Fortunato, 3 M (1984) Oxygenated analogues of 4-[(lHtmtdazol4-yl)methyl]-2,5-dtmethyloxazole. J Org Chem 49, 5003-5006 2 1 Corey, E J and Schmidt, G. (1979) Useful procedures for the oxidation of alcohols mvolvmg pyridmmm dtchromate m aprottc media Tetrahedron Lett 5,399 22 OJrma, I , Sun, C M , and Park, Y H (1994) New and efficient couplmg method for the synthesis of peptides bearmg norstatme residue and their analogs J Org Chem 59,1249-1250 23 OJtma, I , Ng, E W , and Sun, C M (1995) Novel route to hydroxy(keto)ethylene dipeptide tsosteres through the reaction ofN-‘Boc-P-lactams with enolates Tetrahedron Lett 36,4547-4550 24 Ng, E W (1996) Asymmetric Synthesis of Isoserme-Contammg Dlpeptides Isosteres via the P-Lactam Synthon Method PhD, Dissertation, State University of New York at Stony Brook 25 Thatsrivongs, S , Pals, D T , Kroll, L T , Turner, S R , and Han, F -S (1987) Remn mhibitors Design of angiotensmogen transition-state analogues containing novel (2R,3R,4R,5~5-amlno-3,4-dlhydroxy-2-lsopropyl-7-methyloctanolc acid J Med Chem 30,97&982 26 Wang, H , Wang, T , Delaloge, F , and OJtma, I (1997) Unpublished results 27 TSUJI, J (1984) Synthetic apphcattons of the palladium-catalyzed oxtdatton of olefins to ketones Syntheses 369-384 28 Luly, J R , Dellaria, J F , Plattner, J J , Soderquist, J L , and Yi, N (1987) A synthesis of protected ammoalkyl epoxtdes from a-ammo acids J Org Chem 52, 1487-1492

10 Synthesis

of a Versatile Peptidomimetic

Stephen Hanessian

Scaffold

and Grant McNaughton-Smith

1. Introduction The need to replace natural ammoacids in peptides with nonprotemogemc counterparts in order to obtain drug-like target molecules has stimulated a great deal of mnovatlon on several fronts (1-3) One of the more exciting areas of research m drug design hasbeen the synthesisof so-called secondarystructure peptldomlmetlc molecules that are expectedto have the sametherapeutic effects as natural peptlde counterparts, with the added advantage of metabolic stab&y ($5). Of particular interest to us has been the replacement of a dipeptlde motif m a given natural substratewith a constramed or rtgldified counterpart that stimulates p-turn formation (67). In particular, we have been mvestlgatmg the generation of a general synthetic pathway towards a variety of substitutedpeptidomlmetic core structures. Various appendages could then be attached to these central core units either by standard iterative conditions or, more preferably, by using combmatorlal techniques. This chapter will describethe preparation of one suchnovel peptldomimetlc nucleus and Its elaboration to a fully extended p-turn peptldomlmetlc. 2. Materials 1 Flash chromatography Column sizes/amount; 42 mm x 30 cm for >l g, 32 mm x 30 cm for ~1 g and 22 mm x 30 cm for
All the reactions described below except for the conversion of 7 mto 8 and the oxldatlon of 14 were performed under an inert atmosphere of either argon or From

Methods m Molecular Medme, Vol 23 Peptidomunebcs Protocols EdIted by W M Kazmlerskt @Humana Press Inc , Totowa, NJ

161

Uanesslan and McNaughton-Smith

162

mtrogen. Solvents typlcally used for extraction and/or chromatography include ethyl acetate, hexane, diethyl ether, methanol, and chloroform 2.7. Reagents for Method 3.1.1 1 Dnsobutylalummmm 2. Toluene 3 Methanol (MeOH).

hydnde (DIBAL-H)

2.2. Reagents for Method 3.1.2 1 (+)-Camphorsulphomc 2 Methanol (MeOH) 3 Triethylamme (TEA)

2.3. Reagents 1 2 3 4

acid (CSA)

for Method 3.1.3

2-(Trtmethylstlyloxy)furan (TMSOF). Boron trtfluortde etherate (BFs Et,O) Dtchloromethane (CH&l,) 1 N Hydrochlortc actd (HCl)

2.4. Reagents for Method 3.1.4 1 10% Palladium-on-carbon 2 Hydrogen. 3 Ethyl acetate (EtOAc)

(PdK).

2.5. Reagents for Method 3.1.5 1 B-Bromocatecholborane (BCB) 2 Dtchloromethane (CH,Cl,) 3 Sodmm hydrogencarbonate (NaHCO,)

2.6. Reagents for Method 3.1.6 1 Methanol (MeOH) 2 Sodium methoxtde (MeONa) 3 Ammonmm chlortde (NH&X)

2.7. Reagents for Method 3.7.7 1 2 3 4

Dichloromethane (CH,Cl,) Dess-Martm pertodmane Dtethyl ether (Et20) 1.3 A4 Sodmm hydroxide (NaOH)

2.8. Reagents

for Method 3.1.8

1 1,8-Dtazobtcyclo[S 4 Olundec-7-ene (DBU) 2 Benzene 3 2 N Hydrochlorrc actd (HCl)

Versatile Peptidomimetic Scaffold

163

2.9. Reagents for Method 3.1.9 1. 2. 3. 4.

Tetrahydrofuran (THF). Lithium triethylborohydride (LiBEt,H). Sodium hydrogencarbonate (NaHCOJ. Hydrogen peroxide (H202).

2. IO. Reagents for Method 3.1.10 1. 2. 3. 4.

Tetrahydrofuran (THF). Sodium hydride (NaH). Benzyl bromide (BnBr). Tetrabutylammonium iodide (TBAI).

2. II. Reagents for Method 3.1.11 1. 2. 3. 4.

Tetrahydrofuran (THF). tertiary-Butyllithium (tBuLi). Trimethylphosphite (Me0)3P. Oxygen.

2.12. Reagents for Method 3. I. 12 1. 2. 3. 4.

Tetrahydrofuran (THF). Triphenylphosphine (Ph3P). Diethyl azodicarboxylate (DEAD). Diphenylphosphoryl azide (PhO),PON3.

2.13. Reagents for Method 3. I. 13 1. Hydrogen fluoride-pyridine (HF-py). 2. Acetonitrile (MeCN). 3 Sodium hydrogencarbonate (NaHCO,).

2.14. Reagents for Method 3.1.14 1. 2. 3, 4. 5. 6. 7.

Sulphuric acid (H,SO,). Chromium trioxide (CrO,). Acetone. Dichloromethane (CH,Cl,). l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide 4-NJ-Dimethylaminopyridine (DMAP). 2-(Trimethylsilyl)ethanol (TMSE).

2.15. Reagents 1. 2. 3. 4.

for Method 3.2. I.

Methanol (MeOH). Ammonium acetate (NH,OAc). 10% Palladium-on-carbon (Pd/C). Hydrogen.

hydrochloride (EDCI).

164

Hanessian and McNaughton-Smith I) DiSAL-H

TMSOF,

SF3.Et20

2) CSA , MeOH OTSDPS 1

OTBDPS Z:R-H 3: R =Me

o ““1 0

HH

BOC OTSDPS 4 /_N Ii?, Pd /C

6b

6s

5

Figure 1. Synthesis of 5S/R-(5S-tert-butyldiphenylsilyloxymethyl-pyrrolidin-2Syl)dihydro-furan-2-one 6alb. 5. Acetonitrile (MeCN). 6. Indole-3-acetic acid. 7. Benzotriazol-l-yloxy-tris(dimethylamino)phosphonium (BOP). 8. 10% Hydrochloric acid (HCl).

hexafluorophosphate

2.15. Reagents for Method 3.2.2. 1. 2. 3. 4. 5. 6. 7.

Tetrahydrofuran (THF). Tetrabutylammonium fluoride (TBAF). 10% Hydrochloric acid (Hcl). Acetonitrile (MeCN). Cyclohexylamine hydrochloride. Diisopropylethylamine (DIPEA). 10% Hydrochloric acid (HCl).

3. Methods 3.1. Preparation of 6R, 3S-Azido-SR-BenzyloxyAza-2-Oxobicyclo[4.3.O]Nonane-SS-Carboxylic Acid 2-Trimethylsilylethyl Ester 16 The transformation of 2S-tert-butyldiphenylsilyloxymethyl-5-oxopyrrolidine-1 -carboxylic acid tert-butyl ester 1 (8) to SS/R-(SS-tertbutyldiphenylsilyloxymethyl-pyrrolidin-2S-yl)-dihydro-furan-2-one 6a/b is depicted in Fig. 1 (7). Reduction of the original pyroglutamic acid derivative, 1 followed by acid-catalyzed methoxylation, gives the activated hemiaminal 3. Treatment of 3 with a catalytic amount of boron trifluoride etherate generates a transitional cyclic iminium species that is trapped in situ by 2-(trimethylsilyloxy)furan to afford 4 in a highly regioselective fashion (9). Reduction then affords the lactone 5. Selective removal of the Boc amine protecting group in 5

Versatile Peptidomimetic Scaffold

165

OTBDPS Wb

Ph3P, DEAD PhOkJ”W

13 Fig 2 Synthesis nonane-9S-carboxyllc

14

15 R-H 16 R = TMSE

of 6R, 3S-azldo-SR-benzyloxy-aza-2-oxoblcyclo[4 acid 2-trimethylsllyethyl ester 16

3 0]

(IO) leads to the two dtastereotsomers 6a and 6b, which can be separated by column chromatography, If so desired. Fig. 2 highlights the conversron of 6a/b mto the trtsubstttuted azabtcyclo [4.3.0]nonane system 16 (see Note 1) A mild cycltzatton procedure (see Note 2) generates the new btcychc system 7. An oxtdatton-eptmertsatton-reduction sequence of reactions leads to the construction of the desned new stereocenters at the ring Junctton and at the nerghbormg hydroxy sues. 0-Benzylatton (II), followed by a stereoselectlve hydroxylatton (12) at C-3 vta the corresponding enolate and introductton, of an azrdo group by a Mttsunobu-type mversron (13) provides 12, which possessesall the requned stereocentersin the target molecule Removal of the TBDPS group and oxidatton to the correspondmg acid, usmg the Jones reagent (I#), furnishes the intended p-turn pepttdomtmettc nucleus 15. For purposes of functtonal group dtverstficatlon and combmatortal methods, 15 IS transformed mto 16, which encompassesthree orthogonally protected functional groups. 3 1.1. 5S-tert-RutyldiphenylsilyloxymethylZ-Hydroxy- Pyrrolidine- 1-Carboxylic Acid tert-Butyl Ester 2 1 To 10 76 g (23 7 mmol) of 1 dissolved m 60 mL of anhydrous toluene and cooled to-78°C (dry ice/acetone), add dropwlse 30.8 mL (30 8 mmol) of DIBAL-H. Stir the reaction for 1 h at -78°C

166

Hanessian and McNaughton-Smith

2. Quench the reaction by adding 30 mL of methanol Replace the cold bath with an ice bath, and contmue stnrmg for 20 mm Add 50 mL of anhydrous drethyl ether and 10 drops of water Star the reaction untrl a thick slurry appears (see Note 3) Filter the slurry, and wash with 80 mL of hot EtOAc. 3 Collect the filtrate, and concentrate to dryness (see Note 4) Purrfy the residue by passage through a short silica plug, using a l/l (v/v) mrxture of hexane and drethyl ether. Concentrate the appropriate fractrons to a free following colorless 011 One obtams about 10 0 g (22 0 mmol, 98%) of 2 (see Note 5)

3.1 2. 5S-tert-ButyldiphenylsIlyloxymethyl-2-MethoxyPyrrolrdine- 1-Carboxyk Acrd tert-Butyl Ester 3 1 To 10.0 g (22 0 mmol) of 2 dissolved m 55 mL of methanol and cooled to 0°C (me bath), add 255 mg (1 1 mmol) of CSA Stir the reaction at 0°C for 30 mm (see Note 6), then add 10 drops of TEA 2 Concentrate to dryness, and purify the resrdue by passage through a short srltca plug using a 4/l (v/v) mixture of hexane and drethyl ether Concentrate the appropriate fracttons One should get about 10 3 g (22 0 mmol, 100%) of 3

3.1.3. 2S-tert-Butyld~phenylsilyloxymethyl-5S-(5-0xo-2,5-DihydroFuran-2-yl)-Pyrrolidine- 1-Carboxylic Acid tert-Butyl Ester 4 1. To 10 3 g (22.0 mmol) of 3 and 5 55 mL (33 0 mmol) of 2-(trrmethylsrlyloxy)furan (see Note 7) dissolved m 120 mL of dry CH2C12, and cooled to -78°C (dry ice/acetone), add dropwise 1 62 mL (13 2 mmol) of boron trtfluorrde etherate Continue stirring the bright yellow reaction solutron for an addrtronal 40 mm 2 Quench the reaction by adding 24 mL of 1 N HCl Remove the cold bath, and allow the reaction mrxture to warm to room temperature Separate the orgamcs, and wash again with 20 mL of 1 N HCl followed by 20 mL of brine Dry the organic phase with MgS04, filter, and concentrate to dryness 3 Purify the residue by passage through a short sthca plug usmg a l/l (v/v) mrxture of hexane and drethyl ether as the eluant Concentrate the appropriate fractions to a VISCOUScolorless 011 (see Note 8) One should get about 10 4 g (20.1 mmol, 91%) of 4

3.1.4. 2S-tert-Butyldiphenylsilyloxymethyl-5S -(5-Oxo-TetrahydroFuran-2-yl)-Pyrrolrdine- 1-Carboxylrc Acid tert-Butyl Ester 5 1 To 6 4 g (12 3 mmol) of 4 dissolved m 10 mL of EtOAc add 0.5 g of 5% palladrum-on-carbon (see Note 9) Stir the reaction mrxture at room temperature under 1 atmosphere pressure (balloon) of hydrogen gas for 6 h 2 Carefully filter the reaction mrxture through a pad of Cehte and wash wrth a further 20 mL of EtOAc Concentrate the filtrate to dryness One obtams about 6 4 g (12 3 mmol, 100%) of 5 as a colorless or1

Versa tile Peptidomime tic Scaffold

167

3.1.5, 5S-(5S-tert -ButyldiphenyMyloxymethylPyrrolidin-2S-yl)-Dihydro-Furan-2-One 6a/b 1. To 6.4 g (12.3 mmol) of 5 dissolved m 62 mL of dry CH2C12 and shielded from light, add dropwrse a solutton of 4 90 g (24 6 mmol) B-bromocatecholborane in 62 mL of dry CH,CI, at room temperature Stir the reaction for 2 h 2 Cool the reactton m an ice bath, and carefully neutralize with 40 mL of saturated aqueous NaHCOs Star the reaction mixture vigorously for 30 mm Separate the organics, and wash repeatedly with 5 x 20 mL of saturated aqueous NaHC03 (see Note 10) Finally, wash the orgamcs with 20 mL of brine and dry, using Na$04 Filter and concentrate to dryness 3 Purify the residue by column chromatography on sthca gel using a 99/l (v/v) mixture of EtOAc and TEA as the eluant with a flow rate of 20 mL/min, if you desire to separate the two drastereomers, or a flow rate of 40 ml/mitt, rf not One should obtain 3 98 g (9 4 mmol, 77%) of 6a and 0 41 g (0 96 mmol, 7%) of 6b, both as colorless 011s

3.1.6. 6S, 9S-tert-Butyldiphenylsilyloxymethyl5FE -Hydroxy-Aza-2-Oxobicyclo[4.3.O]Nonane

7

1 To 0 47 g (1 1 mmol) of 6a/b dissolved m 8 mL of dry MeOH cooled to 0°C (me bath) add 80 mL of a 25% solution of sodium methoxlde m methanol Stir the reaction at 0°C for 1 h (see Note 11) 2 Quench the reaction by adding 8 mL of a saturated aqueous solution of NH&l, followed by the addition of 8 mL of drethyl ether Extract the organrcs twice with a 20 mL of drethyl ether and combine. Dry the organics with Na,SO,, filter and concentrate to dryness 3 Purify the residue by passage through a short stlica plug using EtOAc as the eluant. Concentrate the appropriate fractions You should get 0 44 g (1 .O mmol, 93%) of 7 as a colorless solid.

3.1.7. 6S, 9S-tert-ButyldiphenylsilyloxymethylAza-2,5-Dioxobicyc/o[4,3.]Nonane 8 1 Add 1.24 g (2.9 mmol) of 7 drssolved m 8 mL of CH& to a solutron of 2 5 g (5 85 mmol) of Dess-Martin pertodmane (16) in CH2C12 (see Note 12). Stir the reaction mixture for 2 h at room temperature. 2. Cool the reaction mixture using an me bath, and add 50 mL of drethyl ether followed by 20 mL of 1 3 M aqueous sodium hydroxide Contmue sttrrmg until the organic phase becomes clear, typically 20-30 mm 3. Separate the orgamcs, and wash with 10 mL of brine, dry with MgS04, and filter Evaporate to dryness. Purrfy the residue by column chromatography on srhca gel using a 3/7 (v/v) mixture of EtOAc and hexane as the eluant and a flow rate of 40 mL/mm. Concentrate the appropriate fractions. One obtains about 1.13 g (2 68 mmol, 92%) of 8 as a colorless 011.

768

Hanesslan and McNaughton-Smith

3.1.8. 6R, 9S-tert-ButyldiphenylsilyloxymethylAza-2,5-D/oxobicyclo[4.3.O]Nonane 9 1. To 1 60 g (3.79 mmol) of 8 dissolved m 10 mL of dry benzene, add 20 rnL of 1,8dlazoblcyclo[5.4 Olundec-7-ene (DBU), and heat the reaction at reflux for 1 h (see Note 13). Allow the reaction to cool to room temperature and concentrate to dryness 2 Add 20 mL of EtOAc and wash the orgamcs twice with 20 mL of aqueous 2 N HCl Dry with MgSO,, filter and concentrate to dryness 3 Separate the two dlastereoisomers by column chromatography on silica gel using a 3/7 (v/v) mixture of EtOAc and hexane as the eluant and a flow rate of 10 mL/mm You should get about 0.75 g (1.8 mmol, 47%) of 9 as a colorless 011 and about 0.85 g (2 0 mmol, 53%) of recovered 8

3.1.9. 6R, 9S-tert-ButyldiphenylsilyloxymethylSR-Hydroxy-Aza-2-Oxobicyc/o[4.3.O]Nonane

10

To 0 68 g (1 6 1 mmol) of 9 dissolved m 12 mL of dry THF and cooled to -78°C (dry ice/acetone), add dropwise 1 94 mL (1 94 mmol) of lithium trlethylborohydrlde Stir the reaction for 1 5 h at -78”C, then add 2 4 mL of saturated aqueous NaHCO,. Allow the reaction mixture to warm to O’C (ice bath), and add 12 drops of a 30% aqueous solution of hydrogen peroxide Stir the resultant mixture for 30 mm at 0°C. Extract the orgamcs with repeated washings with 5 x 10 mL of EtOAc Wash the combined orgamcs with 10 mL of brine, and dry with Na2S04 Filter and concentrate to dryness Purify the residue by column chromatography on silica gel using EtOAc as the eluant and a flow rate of 40 mL/mm One should obtain 0 636 g (1 50 mmol, 93%) of 10 as a 14: 1 mixture of lsomerlc p a hydroxy compounds as a colorless 011

3. I. 70 6R, 5R-Benzy/oxy-9S-tert-8uty/d~phenyls~/yloxymethylAza-2-Oxobicycio[4.3.O]Nonane 17 1 To 5 25 g (12 4 mmol) of 10 dissolved m 20 mL of dry THF and cooled to 0°C (ice bath), add portlonwlse 0 45 g (18 9 mmol) of sodium hydride Stir the suspension for 20 mm at O”C, and then allow to warm to room temperature After 20 mm at room temperature, add 10 mg (0 03 mmol) of tetrabutylammomum iodide (TBAI) and 2 91 mL (24 8 mmol) of benzyl bromide 2 After 4 h, dilute the reaction mixture with 50 mL of dlethyl ether, and quench the reactlon by adding 20 mL of saturated aqueous NH&l. Extract the orgamcs with 20 mL of dlethyl ether, and wash them with 20 mL of brine Dry the organic phase with Na2S04, filter, and concentrate to dryness 3 Separate the two dlastereolsomers by column chromatography on slhca gel using a l/l (v/v) mixture of hexane and EtOAc and a flow rate of 20 mL/mm One should get 0 42 g (0 8 mmol, 7%) of the a-benzyloxy product and 5 8 1 g (11 3 mmol, 9 1%) of 11, both as colorless 011s

769

Versa tile Peptidomime tic Scaffold 3.7.1 I. 6R, 5R-Benzyloxy-9S -tert -5uty/d/pheny/s//y/oxymethy/3R-Hydroxy-Aza-Z-Oxobicyc/o[4.3.O]Nonane 12

1 To 0 77 g (1.43 mmol) of 11 drssolved m 10 mL of dry THF and cooled to -78°C add 1 27 mL (2.15 mmol) of tert-butyllithium (tBuLi) After 5 mm, add 0 34 mL (2 87 mmol) of trimethylphosphite After an additional 2 min an oxygen-filled balloon, fitted with a wide-bore needle (see Note 14) was inserted mto the reactton solutton After 10 mm, carefully remove the needle from out of the reaction (see Note 15) 2 Quench the reactton by adding 10 mL of saturated aqueous NH&l Add 20 mL of EtOAc, and extract the orgamcs twice with 20 mL of EtOAc. Dry the combined orgamcs with MgS04, filter, and concentrate to dryness 3 Separate the product from the starting maternal by column chromatography on stllca gel using a l/l (v/v) mixture of hexane and dtethyl ether and a flow rate of 30 mL/mm. One should get 0.62 g (1.16 mmol, 81%) of 12 and 0.12 g (0.24 mmol, 17%) of 11, both as colorless oils

3.7.72. 6R, 3S-Azido-5R-Benzyloxy-9S-tertButy/d~pheny/si/y/oxymethy/-Aza-2-Oxob~cyc/o[4.3.O]Nonane

73

1 To 0 44 g (0.83 mmol) of 12 and 1 08 g (4.16 mmol) of triphenylphosphme (see Note 16) dissolved m 6 mL of dry THF and cooled to 0°C (ice bath), add 0.67 mL (4 16 mmol) of dtethylazadicarboxylate (DEAD) Star for 5 mm, after which add dropwlse 0 90 mL (4 16 mmol) of dtphenylphosphoryl azide Contmue sttrrmg the reaction mixture at 0°C for an addtttonal 1 h and at room temperature for 20 mm 2. Concentrate the reaction mixture to dryness. Purify the residue by column chromatography on silica gel using a 7/3 (v/v) mrxture of hexane and EtOAc and a flow rate of 10 mL/mm Concentrate the appropriate fractions. You should obtain about 0 36 g (0 65 mmol, 79%) of 13 as a colorless 011

3.7.73. 6R, 3S-Azide-5R -Benzyloxy-9S -Hydroxymethy/Aza-2-Oxobicyclo[4.3.O]Nonane 14 To 0.36 g (0 66 mmol) of 13 dtssolved m 12 mL of dry acetomtrtle and cooled to 0°C (ice bath), add 2 mL of 70% solutton of HF-pyrtdme m acetomtrlle (see Note 17). Star at O’C for 30 min, then at room temperature for an additional 5 h Quench the reaction by carefully pouring the reaction solutton into a mtxture of 10 mL EtOAc and 10 mL of saturated aqueous NaHCOs Repeatedly extract the organrcs with 5 x 10 mL of EtOAc Wash the combined organics with 20 mL of brine and dry with Na2S04 Filter, and concentrate to dryness Purify the residue by column chromatography on silica gel usmg EtOAc and a flow rate of 30 mL/mm Concentrate the appropriate fractions. One should obtain about 0 19 g (0.60 mmol, 92%) of 14 as a colorless solid.

170

Hanessian and McNaughfon-Smith

3.1.14. 6R, 3S-Azido-SR-benzy/oxy-Aza-2-Oxobicyc/o[4.3.O]NonaneSS-Carboxyk Acid 2-Trimethylsily~ethyl Ester 16 1 To a solution of 1 mL (2 63 mmol) of Jones reagent (see Note 18) m 3 mL of acetone cooled to 0°C (ice bath), add dropwise a solution of 100 mg (0 32 mmol) of 14 dissolved m 2 mL of acetone Stir the reaction solution for 1 h 2 Quench the reaction by adding 3 mL of isopropanol Stir the reaction mixture for 30 mm Dilute the reaction mixture with 10 mL of diethyl ether, and pass through a pad of Cehte, then wash with 10 mL of diethyl ether Add 5 mL of brme to the filtrate, process the organic layer, then concentrate to dryness 3 Drssolve the residue 15 m 2 mL of CH$l,, cool to 0°C (ice bath), and add 90 mg (0.48 mmol) of I-(3-drmethylaminopropyl)-3-ethylcarbodumrde hydrochloride (EDCI) and 39 mg (0 32 mmol) of DMAP Stir the reaction solution for 10 mm, and then add dropwise 68 mL (0 48 mmol) of 2-(trimethylsilyl)ethanol Stir the reaction for 3 h 4 Add 10 mL of EtOAc, and wash the orgamcs with 10 mL brine, dry over Na,SO, and filter. Concentrate the filtrate to dryness 5 Purify the residue by chromatography on silica gel usmg a 713 mixture of hexane and EtOAc as the eluant and a flow rate of 30 mL/mm One should get about 91 mg (0 2 1 mmol, 66%) of 16 as a colorless oil

3.2. Preparation of 6R, !%I -Benzy/oxy4S -[(7 H-indo/3-y/)Acetoamino]-Aza-2-Oxobicyclo[4.3.O]Nonane-SS-Carboxylic Cyclohexylamide 18 Figure

3 shows a representative

sequence for the elaboration

Acid of (16) mto a

fully functionalized p-turn peptidomlmetlc system 18 (see Note 19), via a serves of selective deprotectlons and amide bond formations using the BOP reagt:r1t 15 3.2. 6R, 5R -Benzy/oxy-3S -[( 1H-lndol-3-y/)Acetoaminol-Aza-2-Oxobicyclo[4.3. OjNonane9s -Carboxyk A c/d 2- Tnme thy/s//y/e thy/ Ester 17 1 To 100 mg (0.233 mmol) of 16 dissolved m 1 mL of dry methanol, add 0 mg (0 02 mmol) of ammomum acetate 17 and 10 mg of 10% palladium-on-carbon (see Note 19) Stir the reaction mixture under 1 atmosphere pressure of hydrogen for 10 mm 2 Dilute the reaction mixture with 10 mL of EtOAc, and pass through a pad of Celite Wash the filtrate with 5 mL of brine, and dry the orgamcs over Na2S04. Evaporate to dryness, and drssolve the residue m 0.5 mL of dry acetonitrile 3 Dissolve a mixture of 134 mg (0 30 mmol) of BOP (see Note 20) and 53 mg (0 303 mmol) of mdole-3-acetic acid m 0.5 mL dry acetomtrile Cool this reaction mixture to 0°C and stir for 10 min Add the solution of the amme to this mixture. Finally, add 122 mL (0.7 mmol) of dirsopropylethylamine (DIPEA), and stir the reaction for 1 h at 0°C

Versa tile Pep tidotnitne tic Scaffold

771

4 Quench the reactlon by adding 1 mL of 10% aqueous HCl. Extract the orgamcs with 3 x 10 mL allquots of EtOAc The combined organics are washed with 10 mL of saturated aqueous NaHC03 and 10 mL of brine Dry the organic phase with Na,SO,, filter, and evaporate to dryness. 5. Purify the residue by chromatography on silica gel using a 95/5 (v/v) mixture of EtOAc and methanol and a flow rate of 20 mL/mm One should obtain about 95 mg (0.17 mmol, 73%) of 17 as a colorless oil

3 2.1. 6R, 5R-Benzyloxy-3S -[( 1H-lndol-3-yl)-Acetoamino]-Aza2-Oxobicyclo[#.3.O]Nonane-9S -Carboxyl/c Acid Cyclohexylamlde

18

1 To 90 mg (0 16 mmol) of 17 dissolved m 8 mL of dry THF and cooled to 0°C (ice bath), add 0 24 mL (0.24 mmol) of a solution of TBAF m THF Stir the reaction mixture for 30 mm 2 Quench by adding 2 mL of 10% aqueous HCl Extract the orgamcs with 3 x 10 mL of EtOAc, and dry the combined organics over Na$O+ Filter, and concentrate to dryness to give a white crystallme solid 3 Dissolve the white solid m 4 mL of dry acetonitrile and cool to 0°C (ice bath) Add 100 mg (0.224 mmol) of (BOP) and 22 mg (0 224 mmol) of cyclohexylamme hydrochloride Stir for 10 min, and then add 84 mL (0.48 mmol) of dnsopropylethylamme (DIPEA) to the new reaction mixture. Stir the reaction for I h at 0°C 4 Quench the reaction mixture by adding 4 mL of 10% aqueous HCl Extract the orgamcs with 3 x 10 mL allquots of EtOAc Wash the combmed orgamcs with 10 mL of saturated aqueous NaHCO, and 10 mL of brine Dry over Na2S04, filter, and evaporate to dryness 5. Purify the residue by chromatography on slhca gel using a 97/3 (v/v) mixture of chloroform and methanol and a flow rate of 20 mL/mm You should obtam about 78 mg (0 14 mmol, 86%) of 18 as a colorless 011

3.3. Discussion This procedure hlghhghts the utlhty of trapping an lmmlum Ion with the four carbon synthon 2-(trimethylsllyoxy)furan to gain entry mto aza-2oxoblcyclo[4,3,0]nonane systems. Several of the mtermedlates generated throughout this procedure can also be elaborated mto stereochemlcally dlfferent aza-2-oxobicyclo[4.3.O]nonanes, compounds 7 and 10, bemg obvious choices (the latter could be azidated directly to furnish the 3R-azido compound) Although several chromatographlc separations are necessary, the reaction sequences encompass highly stereoselective transformations leading to products that reqwre only minimal purificatlon. The presence of the C-5 hydroxy functionality m 16 offers the possibility of utlhzing solid phase combmatorlal techniques m order to incorporate the peptldomimetlc nucleus mto a variety of p-turn bearing scaffolds (see Note 21).

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4. Notes 1 We will use the azabrcyclo[x y zlalkane nomenclature for our brcychc lactam systems 2 If thermal acttvatlon is used to cyclize the ammolactones 5, only the threo isomer undergoes lactamizatton. 3 If the slurry does not appear after 20 mm, remove the ice bath and allow to warm to room temperature If slurry is still not present add another 10-20 drops of water and 20 mL of diethyl ether 4 At this stage, the concentrated filtrate should appear as a clear solution. Do not be tempted to methylate this material before passage through the silica plug, as some aluminates remam that will disrupt the next step 5 The pure hemtammal product 2 can be stored for several days at temperatures below +5”C, however, before starting the conversion of 2 into 3, you should allocate enough time for the TMSOF condensation step as well Compound 3 IS less stable m storage 6 The reaction is typically very quack; do not let the reaction temperature increase above 0°C or use a longer reactton time than 1 h, as decomposmon products ~111 start to appear 7 The excess 2-(trtmethylstlyloxy)furan will decompose upon work-up to produce 2,5-dihydrofuran-2-one, which will be observed on tic analysts as a spot below the products. In order to reduce the number of chromatographtc separations, one can carry this by-product through the next hydrogenation step, at which point the new by-product (butyrolactone) is relatively volatile and will be removed with the rest of the solvent 8 Be careful when removmg the last traces of the solvent from compounds 4 to 11, as they will often form fme foams that ~111raptdly fill the evaporatmg apparatus 9 Palladium-on-carbon should be handled and disposed of carefully, as it is pyrophortc 10 Keep washing the organic phase until the basic aqueous phase is not colored pink 11 In order to momtor the reactton, remove a small amount of the reaction material and perform a micro work-up, then check by tic analysts 12 Depending on the quality of the Dess-Martin periodmane, some may remam undissolved The addmon of a few drops of water often removes this suspenston and speedsup the reaction 13 Prolonged heating should be avoided, as it leadsto decomposition 14 Use a wide-bore needleto avoid blockage of the oxygen flow by morgamc material that tends to build up around the needlettp 15 Do not tap the needle m order to remove the morgamc residuesattached to the needle, as this may mtttate the releaseof a sharp and unsettling violet-colored flash. The flask and contentsnormally remamintact, however, an exploston guard should always be used 16. The use of fewer equivalents of trtphenylphosphme and DEAD results in a reduction of the yield

Versa tile Pep tidomime tic Scaffold

~)BOP,C$II,NH~

173

HCI

16

Fig 3 Syntheses of GR,SR-benzyloxy-3S-[ lH-mdol-3-yl)-acetoammol-aza-2oxobicyclo[4.3 Olnonane-9S-carboxylrc acrd cyclohexylamrde 18 17 HF-pyndme is highly toxic and corrodesglass Use a plastic (PTFE) vesselfor thts reactron 18 Jones reagent should be handled and disposed of carefully, as it IS highly toxic A 2 63 M stock solution of Jones reagent can be easily prepared by adding 26 3 g of chrommm trioxide (CrO,) to a solutron of 20 mL of concentrated sulphurtc acrd m 78 mL of water 19 Only one of the two possible routes is described m Fig. 3 Inverting the order of the reaction sequence will generate 18 via 19 20. The by-product from amide couplmg reactions using BOP is hexamethylphosphoramrde (HMPA), which 1shighly toxic It should be removed m the workup, but care should be taken during the course of the reaction to avoid contact with skin. 21 If a PMB group is used Instead of the benzyl group for the protection of the hydroxyl functionalrty, it can be selectively removed wtth DDQ to reveal the hydroxy group, which can be attached to a solid support vta an approprtate spacer

References 1 Liskamp, R M J (1994) Conformationally restricted ammo acids and dtpeptrdes, (non) peptrdomrmetrcs and secondary structure mrmetrcs Reel Trav Chzm PaysBas 113,1-19

2 Adang, A E P , Hermkens, P. H H., Lmder, J. T M., QttenhelJm, H. C. J , and van Staveren, C J (1994) Case histories of peptidomtmetrcs progression from peptrdes to drugs Reel Trav Chzm Pays-Bas 113,63-78 3 Gianms, A and Kolter, T (1993) Peptidomimetrcs for receptor ltgands--drscovery, development, and medical perspectives Angew Chem Znt Ed Engl 32, 1244-l 267

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4 Kahn, M , ed. (1993) Peptide secondary structure mnnetics. Tetrahedron 50,49, 3433-3689. ’ 5. Hanessian, S , McNaughton-Smith, G. A , Lombart, H G , and Lubell, W D. (1996) Design and synthesis of conformatronally constramed ammo acids as versatile scaffolds and peptidomimetics Tetrahedron 53, 12789-12854 6 Hanessian, S and Ronan, B (1994) Design and synthesis of a prototype model antagomst of tachykmm NK-2 Receptor Btoorg Med Chem Lett 4,1397-1400 7 Hanesstan, S. and McNaughton-Smith, G A (1996) A versatile synthesis of a pturn pepttdomimettc scaffold. an approach towards a designed model antagonist of the tachykmm NK-2 receptor. Bloorg A4ed Chem Lett 6, 1567-1572 8 Woo, K -C and Jones, K. (199 1) Asymmetric synthesis from a-amino acids, some reactions of (S)-pyroglutamate Tetrahedron Lett 32,6949-6952 9 Casiraght, G and Spanu, P. (1995) Furan-, pyrrole-, and throphene-based silyloxydtenes for synthesis of densely functionahsed homochnal compounds Syntheses 607-626 10 Boeckman, R K and Potenza, J. C (1985) Catechol boron halides mild and selective reagents for cleavage of common protecting groups. Tetrahedron Lett 26, 1411-1414. 11 Czernecki, S , Georgouhs, C , Provelenghiou, C., and Fusey, G (1976) Nouvelle methode de benzylation d’hydroxyles glucidiques encombres Tetrahedron Lett 39,3535-3536 12 Hat-twig, W and Born, L J (1987) Diastereoselective and enantioselective total synthesis of the heptaprotective agent clausenamide J Org Chem 52,4352-4358 13 Hughes, D L. (1992) The Mitsunobu reaction. Org React 42,335-657 14 Bowers, A , Halsall, T G , Jones, E. R H., and Lemm, A J (1953) The chemistry of the triterpenes and related compounds. Part XVIII Elucidation of the structure of polyporemc acid C. J. Chem Sot 2548-2556. 15 Coste, J and Campagne, J.-M (1995) Apropos de l’esterification des acldes carboxyllc par le BOP ou le PyBOP. Tetrahedron Lett 36,4253-4256 16 Ireland, R E. and Longbm, L (1993) An improved procedure for the preparation of the Dess-Martin periodinane J Org Chem 58,2%99 17 SaJiki, H. (1995) Selective inhibition of benzyl ether hydrogenolysts with Pd/C due to the presence of ammmoma, pyridme or ammonmm acetate Tetrahedron Lett 36,3465-3468

11 An Asymmetric Synthesis Protocol to Prepare Topographically Constrained P-Substituted Aromatic Amino Acids Subo Liao and Victor J. Hruby 1. Introduction The side cham groups of ammo acid residues m polypeptlde hormones, neurotransmltters, growth factors, substrates,antigens, and other bloactlve peptldes have been demonstrated to be extremely important pharmacophores for receptor/ acceptor binding and for signal transduction (I). Therefore, complete understandmg of the stereochemlcal requirements of side chain groups important m peptlde hgand-receptor/acceptor interactions plays a crucial role m the rational design of bloactlve peptides and nonpeptlde mlmetlcs. Topographlcal conslderatlons provide a major approach to explore the structure-activity relationships of the side chains of the ammo acid residues m the bloactlve peptlde analogues (2). This approach can be realized by mcorporation of conformatlonally constrainedunusual ammo acids mto a peptide or nonpeptlde template. Thus, design and synthesis of unusual ammo acids with specific biased side cham conformations have tremendous impact on the exploration of the mechanism of peptide ligand-receptor recognition and of activating signal transductton. Our laboratory has beenengaged in the design and synthesis of conformationally constrained amino acids for almost a decade.One representative family among the specialammo acidswe have designed ISthe P-substitutedunusual ammo acids,in which substitution of the dlastereotopic P-hydrogens of natural a-ammo acids provides an approach to topographic control of side chain conformation (3). Incorporation of these P-substitutedunusual ammo acids into bioactive peptide hormones has produced highly selective and potent peptide hormone analogues and provided new insights mto the stereochemlcal requirements m peptIde-receptor interactions (4). We beheve that the summaFrom

Methods m Molecular Medtcme, Vol 23 fepbdom~metrcs Edited by W M Kazmlerskl @Humana Press Inc , Totowa,

175

Protocols NJ

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Liao and Hruby

WCOO” ZH,

OSJR)-P-MePhe

Qpg;ooH

H”~cooH CH,

H,C-CH,

(ZSJR)+-IPrPhe

$H,

(ZS,JR)-T,MT

Fig 1 Chemical structures of the representative P-substituted novel ammo acid (2S,3R)-P-methylphenylalanme (P-MePhe) (34 (2S,3R)-/3-lsopropylphenylalanme (P-1PrPhe) (7), and (2S,3R)-P-methyl-2’,6’-dlmethyltyrosme (TMT) (3e)

rized detailed synthetic procedures included herem might be very helpful to extend the applicatton of these types of P-subsmutedunusual ammo acids m the design of topographically constrainedbroactive peptrde analogues For this purpose, we have chosen three typical P-substttutedunusual ammo acids to exemphf) the detatled synthetic protocol to these special ammo acids (Fig. 1) Detatled conformatronal studiesbasedon temperature-dependent ‘H NMR studies and computer-assisted molecular modeling revealed that P-substttuted alkyl groups could provrde srgmficant constraint to bias the preference of the low energy conformation for the different dtastereoisomersof theseunusual ammo acids ($6) For example, the xl torstonal angle m (2S,3R)-P-MePhe prefers trans, m (2S,3R)P-tPrPhe tt prefers gauche (+), and m (2S,3R)-TMT tt prefers truns, but with a greater energy barrrer One of the most interestmg features m P-tPrPheis the presence of two bulky side chain groups, isopropyl and phenyl Therefore, it can be considered as either P-isopropylphenylalamne or P-phenyleucme. The combmanon of two ammo acid side chain groups in this chlmenc unusual ammo acid not only provides a strong constramtto the side chain conformation, but tt also provides addrttonal hydrophobic@ of the pepttde ligand to interact with receptor. A complete analysis of all isomers of [TMT’IDPDPE has provided new insights into the topographical requirement of the 6 optotd receptorand a conformation templatefor design of nonpepttde 6 opiord receptor hgands(6). More recently, mcorporatton of P-rPrPhe mto the btoacttve optotd peptide Deltorphin I has already produced some very interesting topographically constrained peptrde analogues that currently are under extenstve conformational studiesand pharmacologtcal evaluation (7). 2. Materials All reagents may be obtained from Aldrich Chemical (Milwaukee, WI) and can be used without further pmfication

unless otherwlse noted The followmg

sol-

vents should be freshly dtsttlled prior to use: THF from Na/benzophenone ketyl, and CH& from CaH2. Water should be distilled and delomzed before use. The recommended sthca gel for column chromatography IS the sthca gel 60 with 230-

fl-Substituted

177

Aromatic Ammo Acids Ph 1)

-78T RdCooH

2)

‘n

W&C DT *

Ph, r-i

R-NKo

CuBrSMq

0

- IOT

-78T Za. 2b

la. R=CH,

lb.

0

ArMSBr

R=CH(CH,)*

R=CH, R=CH(CH&

Ph

Ph I)

‘n

(n-Bu),BOTf/DIEA

-78°C 2)

ki^aNYo

00~

NBS, -78°C

ArnNyo 0

0 %l

3b 3C

da

R=CH,. ArPh R=CH,. Ad’-MeO-2’6’.MqPh R=CH(CH&. Ar=Ph

4b 4~

R=CH,. Ar=Ph R=CH,, Ar=.4’-McO-2’.6’-MqPh R=CH(CH,)*, AsPh

Ph

‘n

kfiNyo

TMGA/CH,CN

H202L~OH c h&/OH H20/THF

0 5a Sb SC

NH2 HCI IO% PdC

Ay+OH

7b 7~

0

R=CH,, Ar=Ph R=CH3. Ad’-MeO-2’.6’-MqPh R=CH(CH3)2. Ar=Ph

CF,SO,H

IrEI) R

7a

6a 6b 6c

R=CH,, Ar;Ph R=CH3. And’-MeG2’.6’-Me?Ph R=CH(CH,)2, Ar=Ph

H”~~ooH CH,

R=CH,. Ar=Ph R=CH, Ar=4’-MeO-2’.6’-Me2Ph R=CH(CH,): Ar=Ph

Fig. 2. The general synthetic scheme to prepare topographlcally stituted aromatic ammo acids

AH3

8

constramed P-sub-

400 mesh ASTM from EM Science (Gibbstown, NJ) for best results. Ethyl acetate (AR grade) and hexane (GR grade) for column chromatography may be obtained from EM Science and Malhnckrodt Chemical (ParIs, KY), respectively. The TLC plates we used for analytical TLC have been the Merck precoated Klesegel 60 F254 plates from EM Science. DetectIon of organic compounds can be made usmg

178

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I*, ninhydrm, or UV light. TheJacketed column for ion exchange chromatography may be obtained from ACE Glass Inc (Vmeland, NJ) All deuterated solvents for NMR may be obtained from Aldrich Chemical Corporation. All reactions should be carried out under the protection of argon or mtrogen, unless otherwise noted. The reaction temperatureslisted are the bath temperatures. All reaction flasks should be flame-dried under vacuum or dried m oven over 110°C for two hours before use. Melting points listed here are determmed in open capillary tubes with a Thomas-Hoover melting point apparatusand are uncorrected All elemental analysis data are wtthm 0.4% from the calculated values. All MS data are as predicted All reagents specifically required for each synthesis m Method 3 are listed below. 2.7. Reagents for Method 3.7 1 2 3. 4. 5 6 7

(E)-Crotomc acid (99+%) (see Note 1) or (E)-2-isohexenoic acid (8) Trtethylamine (99%) (see Note 2) Trimethylacetyl chloride (99%). n-Butylhthium (1.6 M m hexane) 4-Phenyl-2-oxazohdmone (3b). Ammomum chloride (AR grade) from Mallmckrodt Chemical Anhydrous magnesium sulfate (powder, AR grade) from Mallmckrodt Chemical

2.2. Reagents for Method 3.2 1 2 3 4 5 6 7

Copper bromide dimethyl sulfide complex (CuBr * S[CH&) (99%) (see Note 3) Phenylmagnesmm bromide (3 .OMm ethyl ether), or 4-bromo-3,5-dtmethylamsole (3e) I2 (crystal, resubhmed, 99 8%) Dimethyl sulfide (anhydrous, 99+%) and magnesnan turmngs (98%) (see Note 6) Ammonium chloride. Sodium chloride (powder, AR grade) from Mallmckrodt Chemical Anhydrous magnesmm sulfate

2.3. Reagents for Method 3.3. 1 2 3 4 5. 6.

Dnsopropyethyllamme (99%) Di-n-butylborontriflate (1 0 M m dichloromethane) N-bromosuccmtmtde (99%) (see Note 5) Sodium btsulfate (94%) Sodium thtosulfate (crystal, AR grade) from Mallmckrodt Anhydrous magnesium sulfate.

Chemical

2.4. Reagents for Method 3.4 1 Acetomtrtle (HPLC grade) from Baxter Healthcare (McGawn 2 Tetramethylguamdinium azide (9) 3. Anhydrous magnesium sulfate

Park, IL)

@Substituted Aromatic Amino Acids

179

2.5. Reagents for Method 3.5 1 Hydrogen peroxide (30%, ACS reagent) from VWR Sctentitic (Phoemx, AZ) 2 Ltthium hydroxide monohydrate (98+%). 3. Sodmm sulfite (98+%)

2.6. Reagents 1 2. 3 4 5

Glacial acetic acid (AR grade) from Mallinckrodt Chemical 10% palladmmcarbon (Pd/C) Ammonmm hydroxide (aq. 28 3% NH,) from EM Science Methanol (AR grade) from Mallmckrodt Chemtcal. Ethyl ether (AR grade) from EM Science

2.7. Reagents 1 2 3 4.

for Method 3.6

for Method 3.7

Trtfluoroacettc acid (99%) Thtoamsole (99%) Trtfluoromethanesulfomc acid (98%) Ammonmm hydroxide.

3. Methods 3.1. Synthesis of (4R,Z’E)-3-(2’-Butenyl)-4-Phenyl2-Oxazolidinone 2a and (4R,Z’E)-3-(2’~lsohexenyl)4-Phenyl-2-Oxazolidinone 2b 1 Place (E)-crotomc actd (1 89 g, 2 1 9 mmol, one eq) or (I?)-2-tsohexenotc acid (2 49 g, 21 9 mmol, 1.0 eq) and THF (124 mL) into a three-necked round-bottomed flask (250 mL) with magnetrc stnrmg bar. 2 Cool the solution at -78°C in dry Ice/acetone bath for 15 mm 3 Add trtethylamme (3 7 mL, 24 3 mmol, 1 1 eq) via syringe into the solution above, followed by addition of trtmethylacetyl chloride (3 0 mL, 24 1 mmol, 1.1 eq) via syringe. 4 Star the resultmg whrte suspensron at-78°C for 15 mm, 0°C m an me bath for 1 h, then at -78°C for 15 mm 5 Add n-butyhthmm (15 mL, 24 0 mmol, 1 1 eq) mto a solutton of (4R)d-phenyl2-oxazolidinone (3 82 g, 23 4 mmol, 1 07 eq) in THF (74 mL) m a three-necked round-bottomed flask (300 mL) at -78°C m a dry me/acetone bath. 6. Transfer the white suspension from step 4 vta cannula mto the hght yellow slurry of ltthtated 4(R)-4-phenyl-2-oxazoltdmone at -78°C that was prepared m step 5 about 10 mm m advance. 7 Stir the resultmg slurry at -78°C for 15 mm, and then 4 h at room temperature 8 Quench the reaction by addmon of 80 mL saturated aqueous ammomum chloride. 9 Remove the volattle by rotary evaporatton, dilute the restdue by addmon of 50 mL water, and extract mtxture with dtchloromethane (3 x 80 mL) Then, dry the combined organic phases over anhydrous magnesium sulfate, and remove the solvent of the drred organic solutron by rotary evaporator

Liao and Hruby

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10 Purify the crude product by using slhca gel chromatography with 1 9 EtOAc hexane mixtures as eluent to give the titled product 2a as white sohd (3e), yield = 94%, Mp 77 O-79 O”C, [a]220 = -111 8” (c = 1 08, CHCl,), ‘H NMR (CDC13) (ppm 7.4&7 25(m, 6H), 7 15-6.90(m, IH), 5 47(dd,J= 8 8,3 9H2, lH), 4.67(t, J= 8 8Hz, lH), 4 24(dd, J= 8.8,3 9H2, lH), 1 92(d, J= 6 7Hz, 3H) Elemental analysis for C,,H13N0, (Found) C, 67 25, H, 5 67, N, 6 13 2b as white solid (7), yield = 90%, Mp 103.5-104 O”C, [cI]~~~ = +103 1” (c = 1 0, CHCI,), ‘H NMR (CDCl,) 6 ppm 7 39-7 28(m, 5H), 7 20(dd, J= 16 4,0 8Hz, lH), 7 03(dd, J= 164,65Hz, lH),546(dd,J=87,38Hz, lH),467(t,J=87Hz, lH),425 (dd, J= 8 7,3 8Hz, lH), 2 49(m, lH), 1 04(d, J= 6 7,6H), Elemental analysis for C,,H1703N (Found) C, 69 46; H, 6 57; N, 5 36

3 2.1. Synthesis of (3’R,4R)-3-(3’-Pheny/butany/)-4-Pheny/2- Oxazolldinone 3a and (3’R,4R)-3-(3’-Phenyl/sohexanyl)4-Phenyl-2-Oxazolidinone 3c Place CuBr (CH,),S complex (4 84 g, 23 5 mmol, 1 5 eq), anhydrous dimethyl-sulfide (39 mL) via syringe and THF (75 mL) mto a three-necked roundbottomed flask (500 mL) fitted wtth a magnetic stlrrmg bar and eqmhbrmm addition funnel. Cool the mixture to -15°C (see Note 6), and then add a solution of PhMgBr (7 3 mL, 22 0 mmol, 1.4 eq) via syrmge mto the cooled mixture Stir the resulting gray mixture for 10 mm at -15”C, and then add dropwlse a solution of 2a or 2b (15 7 mmol, 10 eq) m THF (37 mL) through the equlhbrmm addition funnel (see Note 7) to the gray mixture Stir the resultmg mixture for an addItIonal 90 mm at-IO”C, and then warm It to room temperature, and stir overnight Quench the reactlon by slow addition of saturated NH4C1(aq) (70 mL), and stir the resultmg heterogeneousmixture for 30 mm Set the organic phase aslde, and extract the aqueous phase with ethyl ether (50 mL x 3) Then wash the combmedorganic phaseswith saturatedNH4Cl(aq ) (50 mL x 3), brine (50 mL), water (50 mL), and dry It over anhydrous MgSO, Evaporate the volatde of the dried organic solution on a rotary evaporator, and then purify the residue with slhca gel column chromatography usmg 2 8 (v/v) EtOAc/Hexanes aseluent to give the titled compound 3a asa white solid, yield = 76%, Mp 145 2-147 5”C, [u]~~,, =-93.3” (c = 1 0, CHCl,), ‘H NMR (CDC13) (6 ppm) 7 37-7 lO(m, lOH), 5 24(dd, J = 8 8, 3 5Hz, IH), 4 47(t, J = 8 8Hz, lH), 4 15(dd, J= 8 8, 3 5Hz, lH), 3 39-3 24(m, 3H), 1 24(d, J= 6 8Hz, 3H), MS for C19HIgN0, (Found) 327 1696 for (M+ + NH,+) 3c as a white solid, yield = 83%, Mp 102.0-103.5”C, [a] *OD= -54.6” (c = 1.0, CHCl& ‘H NMR. 6 ppm7 2M 77(m, lOH), 5.29(dd,J= 8 7,4 lHz, lH), 4 56(t, lH,J= 8 7 Hz), 4 07 (dd, J= 8 7,4 lHz, IH), 3.74(dd, J= 15 7, lOHz, lH), 3 13(dd, J= 15 7,5 2Hz, IH), 2 95(m, lH), 2 88(m, lH), 0.96(d, J= 6.7Hz, 3H), 0 74(d,J= 6 7Hz, 3H), Elemental analysisfor C21H2303N(Found) C,74 34; H, 6 97, N, 4 34

181

P-Substituted Aromattc Amino Acids 3.2.2. Synthesis of (3R:4R)-3-[3’-(4-Methoxy2,6-Dimethylphenyl)-Butanyl]-4-Phenyl-2-Oxazol~dinone

3b

1. Placed magnesium turnmgs (2.6 g, 108 mmol, 2.5 eq), THF (50 mL), 4-bromo3,5-drmethylamsole (20 5 g, 93 mmol, 2.1 eq) and I, (about 0 2 g) as mmator mto a three-neck round bottom flask (200 mL), fitted with magnetic sttrrmg bar, and a refluxmg condenser. 2 Heat the mrxture with stirring to reflux under Argon for 4 h, and then cool rt to -4°C (see Note 6) 3 Transfer the cooled top transparent solution (some magnesmm residue 1sstill left in the bottom of the flask) via cannula to a -40°C (see Note 6) stnrmg slurry of CuBr SMe2 complex (13.3 g, 65 mmol, 1.5 eq) in freshly drsttlled THF (150 mL) and anhydrous dimethylsulfide (70 mL) in a three-necked round-bottomed flask (500 mL) fitted with a equrhbrium addmon funnel 4 Warm the resulted gray mixture to -10°C (see Note 6) 5 Add dropwrse a solution of 2a (10 g, 43.0 mmol, 1.0 eq) m THF( 110 mL) from the equthbrmm addition funnel to the gray mixture at -10°C over 60 mm 6 Stir the resulting mixture for an additional 90 mm at -10°C and then warm the reaction mixture to room temperature, and stir it overnight 7. Quench the reaction by slow addttton of saturated NH&l (aq) (100 mL) and stir the heterogeneous mixture for 30 mm 8 Set the organic phase aside, and extract the aqueous phase with ethyl ether (100 mL x 3) 9 Wash the combined orgamc phases with saturated NH&l (aq ) (100 mL x 3), brine (150 mL), water (150 mL), and dry over anhydrous MgS04. 10 Remove the volatile of the dried organic solutron by rotary evaporation, then purify the residue by sihca gel column chromatography using 2 8 (v/v) EtOAc/ Hexanes as eluent to give the titled compound as a white solid, yield = 65%, Mp 99 5-101 O’C, ‘H NMR (CDCl,) (6 ppm) 7 33-7 24(m, 5H), 6 52(s, 2H), 5 36(dd,J= 8 7,3 7H2, IH), 4 60(t, J= 8 6Hz, lH), 4,23(dd, J= 8 7,3 9Hz, lH), 3 86(m, lH), 3 74(s, 3H), 3 41(s, lH), 3.39(d, J = 1 lHz, 1H ), 2,43(s, broad, 3H), 2 23(s, broad, 3H), 1 25(d, J = 7 3Hz, 3H) [a]**,, = -88.5” (c = 1 07, CHCl,). Elemental analysis for C22H25N04 (Found) C, 71 95; H, 6 80, N, 3 86

3.3. Synthesis of (2’R,3’R,4R)-3-(2’-Bromo=3’-Phenylbutanyl)4-Phenyl-2-Oxazolidinone 4a, (2’R,3’R,4R)-3-[2’-Bromo-3’(4-Methoxy-2,6-Dimethylphenyl)Butanyl]-4-Phenyl2-Oxazolidinone 46, and (2’R,3’R,4R,)-3-(2’-Bromo3’-Phenylisohexanyl)-4-Phenyl-2-Oxazolidinone 4c 1 Place 3 (10 53 mmol, 1 0 eq) and dichloromethane (50 mL) into a three-necked round-bottomed flask (300 mL) fitted with a magnetic stirring bar 2 Cool the resulting solution to -78°C in a dry ice/acetone bath for 10 mm. 3. Add a solutron of diisopropylethylamme (2 2 mL, 12 6 mmol, 1 2 eq) vta syringe to the solutron, followed by addition of dt-n-butylborontrtflate (11 mL, 11 mmol, 1.1 eq) via syringe.

182

Lrao and k/ruby Stir the resultmg suspension at -78°C in dry ice/acetone bath for 15 mm, and then warm It to O’C in an Ice both for 1 h, and then cool it to -78°C for 15 mm Transfer the cooled suspension via cannula to a suspension of NBS (2 81 g, 15 8 mmol, 1 5 eq) m dlchloromethane (40 mL) m a three-necked round-bottomed flask (500 mL) at -78°C m a dry ice/acetone bath Stir the resulting purple mixture at -78°C for 2 h, and at 0°C m an Ice bath for 3 h or longer (see Note 8) Quench the reaction with the slow addltlon of 1 3 N aq sodium blsulfate solution (100 mL) Separate the organic phase, and wash it with 1 N sodium thlosulfate (3 x 50 mL), water (3 x 50 mL), and then dry it over anhydrous magnesium sulfate Remove the volatile of the dried organic solution with evaporator, and then purify the remammg residue by silica gel chromatography with 2.8 (v/v) ethyl acetate/ hexanes as eluent to give the titled compound. 4a as white solid, Yield = 88%, [a]220 = -30 0” (c = 1 6, CHCl,), ‘H NMR (CDCI,). (6 ppm) 7 45-7 20(m, IOH), 5.95(d,J= 10.7Hz, lH), 5.51(dd,J= 8 8,4 8Hz, lH), 4 74(t,J= 9.OHz, lH), 4.29(dd,J= 8.9,4 9Hz, lH), 3 43-3 52(m, lH), 1 35(d,J= 6 9Hz, 3H) 4b as white solid, yield = 83%, Mp* 186.0-187 5”C, [cx]~~~ =-I 5” (c = I 03, CHCl,), ‘H NMR (CDCl,), (6 ppm) 7 45-7.29(m, 5H), 6.56(s, 2H), 6 44(d, J = 11 9HZ, lH), 5 54(dd, J = 8 9,5 OHz, IH), 4 77(t, J = 8 6Hz, IH), 4 29(dd, J= 8 9, 5 OHz, lH), 4 12-3 98(m, lH), 3 76(s, 3H), 2.47(s, 3H), 2.31(s, 3H), 1.31(d, J = 7 3Hz, 3H) Elemental analysis for C22H24N04Br (Found). C, 59 23, H, 5 52, N, 3 01 4c as white solid, yield = 85%, Mp 158-159”C, [a1200 = +7 2” (c = 1 0, CHCl,) ‘H NMR (6 ppm) 7 36-7 16(m, 1OH), 6 33(d, J = 11 2Hz, lH), 5 47(dd,J= 8 9, 4 7 Hz, lH), 4 75 (t, J= 8 8Hz, lH), 4.28(dd, J= 8 9,4 7H2, lH), 3.37(dd,J= 10 8,4 5Hz, lH), 2 02(m, lH), 0 84(d, J= 6 7 Hz, 3H), 0.78(d, J = 6 7 Hz, 3H). Elemental analysis for C21H2203NBr (Found) C, 60 56; H, 5 36; N, 3 33

3.4. General Synthetic Procedure To Prepare (2’S,3’R,4R)3-(2’-AzidoS’-Phenylbutyl)-4-Phenyl-2-Oxazolidione Sa, (2’S,3’R,4R)-3-[2’-Azido-3’-(4-Methoxy2,6-Dimethylphenyl)Butanyl]-4-Phenyl-2-Oxazolidione 5b and (2’S,3’R,4R)-3-[2’-Azido-3’-Phenylisohexanyl]4-Phenyl-2-Oxazolidinone 5c 1 Place 4 (4.9 mmol, 1 0 eq), and acetomtrlle (40 mL) (20) mto a round-bottomed flask ( 100 mL) 2

Cool the solution to 0°C m an ice bath, and add tetramethylguarudmmm

azlde

(3 1 g 19.7 tnmol, 4 0 eq) mto the solution m one portlon 3 Warm the slight yellow solution to room temperature, and stir it until the reaction 1s complete. The reaction 1s monitored with ‘H NMR by the disappearance of signal (doublet) for the proton adjacent to Br at 6 2-6 4 ppm and appearance of signals for the proton adjacent to the azldo group at 5 365 5 ppm.

/Mubstituted

Aromatic Amino Acids

183

4. Quench the reaction by the addition of 30 mL of saturated aqueous bicarbonate 5 Extract the resulting mixture with ethyl ether (3 x 30 mL), and dry the combined organic extractants with anhydrous magnesium sulfate. 6 Remove the volattle of the dried combined organic solutton by rotary evaporatron, and then purify the remaining residue by silica gel chromatography with 2.8 (v/v) ethyl acetate/hexane as eluent to give the titled compounds. 5a as a white solid, yield = 93%, [c~]~~o= +64.4” (c = 0.9, CH,CI), ‘H NMR (CDCI,). (6 ppm) 7 26-6 80(m, IOH), 5 42(d, J= 8.3Hz, lH), 5.40(dd, J= 8 5,4 OHz, IH), 4 68(t, J= 9 1 Hz, lH), 4 17(dd, J= 5.5, 9.0 Hz, lH), 3.40-3.27(m, lH), 1.28(d, J= 7 lHz, 3H), IR(CHC1,). 2103, 1771, 1698 cm-‘; MS for C,,H,sN,O, (Found). 368.1736 (Mf + NH,+) 5b as a white solid, yield = 90% Mp. 102 5-103 5°C [a]22,, =A1 9’ (c = 1 01, CHCl,), ‘H NMR (CDCl,)* (6 ppm) 7 22-6 58(m, 5H), 6 52(s, broad, lH), 6 25(s, broad, lH), 5.73(d, J= 11.6HZ, IH), 5.34(dd, J= 8 9, 4 9Hz, IH), 4 72(t, J = 8 9Hz, lH), 4 61(dd, J = 8 9, 4 3Hz, lH), 3 78(s, 3H), 3 77(m, lH), 2 49(s, 3H), 1 93(s, 3H), 1.48(d, J= 7.3Hz, 3H), IR(KBr)* 2100, 1790, 1690 cm-’ Elemental analysts for C22H2,N,0, (Found). C, 64.93, H, 5.83; N, 13 50 SCas a white solid, yield = 80%, Mp 159-l 5 1‘C, [o-J200 = -43 2” (c = 1 0, CHCl,) ‘H NMR(CDCl,)* (6 ppm) 7 37-6 44(m, lOH, aromatrc protons), 5 76(d, J = 11 5, lH), 5 30(dd, J = 8.9, 4 7Hz, lH), 4 68(t, J = 8 8Hz, lH), 4 03(dd, J= 8 9,4 7Hz, IH), 3 13(dd, J= 4.1,4 0 Hz, IH), 2 lO(m, lH), 0 92(d, J = 6 8 Hz, 3H), 0 82(d, J = 6 8 Hz, 3H) IR(KBr). 2103, 1763, 1699 cm-’ Elemental analysis for C21H2203N4 (Found) C, 66 61; H, 5 68, N, 14 66

3.5. Synthesis of (2S,3R)-2-Azido-3-Phenylbutanoic Acid 6a, (2S,3R)-2-Azido-3-(4-Methoxy-2,6-Dimethylphenyl)butanoic Acid 66 and (2S,3R)-2-Azido-3-Phenylisohexanoic Acid 6c 1 Place 5 (4.9 mmol, 1.0 eq), THF (60 mL), and water (20 mL) mto a three-necked round-bottomed flask (250 mL) fitted with a stnrmg bar 2 Cool the resulting solution to 0°C m an ice bath for 15 mm 3 Add a solution of hydrogen peroxide (30%, 3 4 mL, 30 mmol) dropwise mto the cooled solutton, followed by the dropwtse addition of a solution of lithmm hydroxide monohydrate (0 44 g, 10 5 mmol, 2.1 eq) m water (3 mL) 4 Stir the resulting light yellow solutron for 2 h or longer at 0°C until TLC mdicates the complete dtsappearance of the startmg material 5 Quench the reaction with a solutton of 1 3 A4 Na2S03 (30 mL), and warm the mixture to room temperature, and stir it for 30 mm 6. Remove the volattle by rotary evaporatton, and extract the remaining aqueous phase with dtchloromethane (40 mL x 3) to remove the choral auxiliary 7 Cool the remaining aqueous phase to 0°C in an ice bath, and acidify tt to pH = 1 5 with 6 N HCl, and extract it with dichloromethane (40 mL x 3), and dry the combmed organic extracts over anhydrous magnesium sulfate 8 Remove the solvent of the dried organic solution using a rotary evaporator Purify the remaining residue using silica gel chromatography with a mixture of hexane/ ethyl acetate/acetic acid (7.2.9:O 1, v/v) as eluent to give titled compounds 6a as

184

Liao and Hruby a slight yellow 011, yield = 90%, [a] 22,, = -11” (c = 1 01, CHCl,), ‘H NMR (CDCI,). (6 ppm) 9.10(s, lH), 7 26-7.33(m, 5H), 4.06(d, J= 7 Hz, lH), 3 34 3 46(m, lH), 1 37(d, J = 7 2Hz, 3H), IR(CHC1,) 2113, 1712 cm-‘, Elemental analysis for C,,H,,N,O, (found). C, 58 05, H, 5 61 6b as a slight yellow oil, yield >90%, [a]22n = +84 8” (c = 1 66, CHCl,), ‘H NMR (CDCl,) (6 ppm) 9.83(s, IH), 6.53(s, 2H), 4.14(d, J = 10.4Hz, lH), 3 75(s, 3H), 3 65(m, lH), 2 38(s, broad, 3H), 2.29(s, broad, 3H), 1 44(d, J= 7 0,3H), IR(KBr) 2 100, 1720, 1600 cm-‘, MS for C,,H,,N,O, (Found). 264 1330(M+ + H). 6c as slight yellow oil, yield = 85%, [cx]~~,, = +17 5’ (c = 1 2, CHCl,), ‘H NMR(CDC1,) (6 ppm) 9 13(s, broad, IH), 7 31-7 15(m, 5H), 4 18(d, J= 7 0 Hz, lH), 2 94(t, J= 7 3, lH), 2 30(m, lH), 0 95(d, J= 6 7Hz, 3H), 0 78(d, J= 6 7Hz, 3H) IR(film) 2116, 1713, 1600 cm-’

3.6. Synthesis of (2S,3R)-P-Methylphenylalanine of (2S,BR)-(-Methyl-2’,6’-Dimethyl-Methoxytyrosine and of (2S,3R)-2-Amino-3-Phenylisohexanoic

7a, 7b, Acid 7c

1 Place 6 (13 2 mmol), glacial acetic acid (110 mL) and 30 mL of water m a hydrogenation vessel 2 Bubble argon through the solutton for 10 mm to remove au before 10% Pd/C ( 1 0 g) was added (see Note 9) 3 Evacuate and refill the hydrogenatton vessel with H2 three times, and shake it under 36 PSI of H2 for 24 h Add 100 mL of water to the mixture m the hydrogenation vessel Filter out the Pd/C catalyst through cehte Remove the volatile of the filtrate by rotary evaporation at 50°C m a water bath Add 20 mL of 6 N hydrochloric acid to the residues Freeze the suspension or solution from step 7 m acetone/dry Ice bath Put the frozen suspension or solution from step 8 on lyophilizer to get rid of all water 10 Further purification of 7a and 7c is carried out by a cooledlacketed ion exchange column chromatography (2 5 cm x 46 cm) (Amberhte, IR-120, H+) with 20% NH,OH as eluent (see Note lo), combme the fractions containing the product, and evaporate It to remove NH3 wtth rotary evaporatton, then freeze it and lyophthze it to give the tttled compounds. 7a as off-whtte solid (3rr), Mp* 190-192°C [a]22r, = -5 3” (c = 0 75, H20). ‘H NMR (D,O, dioxane as standard at 3 55) (6 ppm) 7 15-7 25(m, 5H), 3 73(d, J= 4 9Hz, lH), 3 33(m, lH), 1 18(d, J= 7 3Hz, 3H) 7b as an off-white solid (3e), yield ~90% mp. 110 &113 0°C [a]220 = +62 53” (c = 0 84, MeOH) ‘H NMR (D,O)* (6 ppm) 6 44(s, 2H), 3 85(d, J = 11 OHz, lH), 3 54(s, 3H), 3.36(m, lH), 2.23(s, 3H), 2 16(s, 3H), 1 22(d, J = 7 3Hz, 3H) MS for C13H1903N (found) 238 1439 7c as an off-white solid (7), yield = 85%, Mp* decomposed at 186” [a1200 = +3.8’; ‘H NMR (D,O) (6 ppm) 7 25-7 08(m, 5H), 4 05(d, J = 4.6Hz, lH), 2 52(dd, J = 10 0, 5 4Hz, IH), 2 28(m, lH), 1 02(d, J = 6.4Hz, 3H), 0 54(d, J = 6 4Hz, 3H) MS for C,2H,702N (Found) 208 1335 (M + H+).

185

/?-Substituted Aromatic Amino Acids

3.7. Synthesis of (2S,3R)-(-Methyl-2’,6’-Dimethyltyrosine

6

1 Dissolve 7b (1.15 g, 4 85 mmol) m trlfluoroacetlc acid (50 mL) m a three-necked round-bottomed flask (150 mL) 2 Cool the solution to 0°C in an ice bath, and add thioamsole (3 98 mL, 33.92 mmol, 7 eq) to the cooled solution, and stir it for 10 mm. 3 Add tnfluoromethanesulfomc acid (6 43 mL, 72 69 mmol, 15 eq) via syringe to the solution 4 Stir the light yellow cloudy solution at 0°C for 30 mm, and at room temperature for 2 h 5 Remove the volatlles by rotary evaporation. 6. Dissolve the dark brownish tar residue m 100 mL of water, and extract the aqueous solution with ethyl acetate (3 x 30 mL) to remove the thioamsole 7 Load the remaining aqueous solution on a cooled Jacketed ion exchange column with Amberhte IR-120 (H+) resin (see Note lo), wash the ammo acid out with 20% NH,OH solution (this process was monitored by TLC). Fractions containing the product were combined, evaporated to remove excessWOH, frozen m dry ice/acetone bath, and lyophilized to give 1 02 g of the title compound as an off-white solid (3e) Yield = 96%, Mp: 175.0-178 O”C, decomposed. [a]**o = +46 6°C (c = 0.79, MeOH) ‘HNMR (D,O) (6 ppm) 6.36(s, 2H), 4 1l(d, J= 1 l.OHz, lH), 3SO(m, lH), 2.15(s, 3H), 2 02(s, 3H), 123(d, J= 7 2Hz, 3H) MS for C12H17N03 (Found). 224,1266(M+ + H)

4. Notes 1 The trans-crotomc acid which 1s from Aldrich Chemical Co. was dried over pentaphosphorous oxide m a vacuum desiccator overnight before use 2. Trlethylacetyl chloride (99%) and trlethylamme (99%) could be used directly without further purification but needed to be kept m a desiccator over a drying reagent such as blue slllca gel and sealed with parafilm. 3 The copper complex Cu(I)Br Me$ IS air and moisture-sensitive and IS best handled m a glove box. The asymmetrical Michael addition is very moisturesensltlve, all reagents and glassware need to be dried, especially the THF, which should be used immediately after dlstllled from blue Na/benzophenone ketyl The magnesium tunings were activated by washing with 0 5 N HCl, ethanol and dried 112vacua, and stored m argon before use. N-Bromosuccmimlde (NBS) was recrystallized from water and dried over pentaphosphorous oxide m a vacuum desiccator overnight before use The reaction was carried out m an acetone bath in which the temperature was monitored with a low temperature thermometer and controlled by addition of small amounts of dry Ice from time to time 7 The addition rate is important to get the highest stereoselectlvlty Slow dropwlse addition 1srecommended for best stereoselectlvlty In addition, temperature control 1s crucial to obtain a reasonable reaction rate and good stereoselectlvlty 8 The reaction must be kept stlrrmg for 3 h or longer at 0°C to complete the reaction, less reactlon time often causes mcomplete brommatlon, especially m large scale reactions.

186

Llao and Hruby

9 Bubbling argon or nitrogen through the organic solutron to remove air eliminates the potential danger of tire when Pd/C IS added into the hydrogenation vessel 10 The ion exchange resin packed m a column needs to be washed until the washing turns neutral before loading the crude ammo acrd sample Then, the crude sample IS washed with detomzed water until the eluent 1s neutral again, 20% NH,OH solutron IS then used to wash out the pure ammo acid

References 1 Hruby, V. J (1982) Conformattonal restrtctrons of brologrcally active peptrdes via ammo acid side chain groups Lzfe Scz 31, 189-l 99 2a Hruby, V J (1993) Conformatronal and topographrcal consrderatrons m the design of btologrcally active peptides. Blopolymers 33, 1073-1082. 2b Tomolo, C (1990) Conformatronally restrrcted peptrdes through short-range cychzatron Int J Pep Protein Res 35, 287-300 3a Dharampragada, R , Van Hulle, K , Bannister, A , Bear, S , Kennedy, L , and Hruby, V. J (1992) Asymmetric synthesis of unusual ammo acids an efficient synthesis of opttcally pure isomersof /3-methylphenylalanme Tetrahedron 48, 4733-4748.

3b Nrcolas, E , Russell, K C., and Hruby, V J (1993) Asymmetric I,4-Addmon of Organocuprates to Choral a&unsaturated N-Acyl-4-phenyl-2-oxazohdmones a new approachto the synthesisof choralP-branchedcarboxyhc acids J Org Chem 58,76&770

Nrcolas, E , Russell, K C , Knollenberg, J , and Hruby, V J (1993) Efficrent method for the total asymmetric synthesisof the isomersof P-methyltyrosme J Org Chem 58,7565-7571 3d BoteJu, L W , Wegner, K , Qran, X , and Hruby, V J (1994) Asymmetric synthesis of unusual ammo acids synthesis of optrcally pure isomers of N-mdole(2-mesrtylenesulfonyl)-P-methyltryptophan Tetrahedron 50,2391-2404 3e Qran, X., Russell, K C , Boteju, L. W., and Hruby, V. J. (1995) Stereoselectrve total synthesis of topographrcally constrained designer ammo acids 2’,6’-Drmethyl-p-methyltyrosine Tetrahedron 51, 1033-1054. 4a Qran, X., Kover K E , Shenderovrch, M D., Lou, B-S , Mrsrcka, A , Zalewska, T , Horvath, R , Davis, P., Brlsky, E. J , Porreca, F , Yammura, H I , and Hruby, V J (1994) Newly discovered stereochemrcalrequirementsm the side-chain conformation of b-optotd agonistsfor recognizing oproid &receptors J A4ed Chem 3c

37,174&1757

4b Nikrforovrch, G V , Prakash, 0 M , Gehrrg, C. A , and Hruby, V J. (1993) Solution conformation of the peptrde backbone for DPDPE and us P-MePhe4-substttuted analogs Int J Peptlde Protem Res 41, 347-361 4c Huang, Z., He, Y.-B., Raynor, K , Tallent, M., Rersme, T , and Goodman, M (1992) Mam chain and side chain choralmethylated somatostatmanalogs synthesesand conformatronal analyses J Am. Chem Sot 114,939&9401 4d. Toth, G , Russell, K C , Landis, G., Kramer, T H., Fang, L., Knapp, R , Davis, P , Burk, T F , Yamamura, H., and Hruby, V J (1992) Rmg substituted and other

/?-Substituted Aromatic Ammo Acids

5 6.

7 8 9

10

187

conformattonally constramed tyrosine analogues of [D-Pen*,D-Pen5]enkephalm with 6-optoid receptor selecttvtty J A4ed Chem 35,2383-2391 Jtao, D , Russell, K C , and Hruby, V. J (1993) Locally constrained tyrosme analogues with restricted side chain dynamics. Tetrahedron 49,35 1 l-3520 Qran, X , Shenderovtch, M D., Kover, K E , Davis, P , Horvath, R , Zalewska, T , Yamamura, H I , Porreca, F., and Hruby, V J (1996) Probing the stereochemical requirements for receptor recognmon of 6 optotd agonists through topographic modifications m posttton 1 J Am Chem Sot 118, 7280-7290 Ltao, S , Shenderovtch, M D , and Hruby, V J , unpublished results Ptrrung, M C , Han, H , and Ludwig, R T (1994) Inhrbttors of thermus thermophtlus rsopropylmalate dehydrogenase J Org Chem 59,243&2436 Papa, A J (1966) Synthesis and azrdolysis of 2-chlorotetramethylguamdme. Synthetic utility of hexa- and tetramethylguanidmmm aztde J Org Chem 31, 14261430 Hruby, V J , BoteJu, L W , and Lt, G. (1993) Chemical safety* explosion with sodrum azlde Chem Eng News 71,2

12 Synthesis of Oligopeptides Containing an Oxirane Ring in the Place of a Peptidic Bond Maurizio Taddei 1. introduction Ohgopepttdes contammg an oxirane rmg have recently been identified as mhtbttors of a variety of proteases (Z-3). These pepttdomtmettcs have the potential to coordinate with metal present m the active site and, after nucleophthc rmg opening, nreverstbly blocking the enzyme For thts reason, oxnane containing pepttdomrmetrcs are good candidates to became transition states analogs or sutctde mhibttors with long term efficacy m VIVO (3). Synthetic routes to a variety of terminal (4-8) and internal epoxide pepttdomrmettcs (!JII) have been reported but there are no examples of incorporatron of such epoxtdes mto ohgopepttdes The focus of this chapter will be on the preparation of ohgopeptrdes (up to a three-peptrde) contammg an epoxide m the place of the pepttde bond The structures prepared here can be identified, using the notatton suggested by Spatola (22) as AAxY[trans-epoxy]-AAy. The general synthetic approach described in this chapter is based on the aldol type reaction of a stlylketene thtoacetal and a p-ammo a-selenyl aldehyde derived from an olrgopepttde. This reaction stereoselecttvely generates a vtcmal hydroxy selenide which can be further oxidized to epoxtde (Fig. 1).

2. Materials 1 TLC plates. Merk silica 60 S F254developed with a KMn04/Na2C03 aqueous soluttons Eluents employed are the same used for column chromatography 2. Slhca gel for flash chromatography. 220-440 mesh ASTM. The choice between flash and gravrty column chromatography IS reported In the text. 3 Meltmg pomts are measured m a sealed capillary and are uncorrected 4 Methyl alcohol (MeOH) 5. Dtethyl ether From

Methods

in Molecular

Med/one,

Edlted by W M Kazmlerskl

l/o/

OHumana

189

23

Pept/dom/mehcs

Press Inc , Totowa,

Protocols

NJ

190

Taddei

Ollgopeptlde

4

‘-NNH OH &&COSt-Bu Ph.&

i R2

Fig 1 Retrosynthettc

R;$COOH H

i,

analysts of oxtrane pepttdomtmetrtcs

6 Toluene, prevtously distilled over Na 7 Benzene, previously distilled over Na 8 Tetrahydrofuran, d&Red over Na and subsequently before the use (THF) 9 Hexane 10 Ethyl acetate 11. Dtchloromethane, dried over CaCl, (CHQ) 12 Dimethylformamtde (DMF) 13 Dtoxane 14. Pyrtdme dried over KOH pellets 15 Sodmm carbonate, 10% solutton (Na$Os) 16 Aqueous HCl 17 Ammonmm chloride, saturated solutton (NH&l) 18 Sodmm hydroxide, 0 1 A4 solubon (NaOH) 19 Calcmm chloride (CaCl,). 20 Anhydrous sodmm sulfate (Na,SO,) 21. Celite 22 Phosphorous pentoxrde 23 Sodmm hydrogen sulfate, 15% solution (NaHSOJ

distilled over LIAIH,

2.1. Reagents for Method 3. I 1 2. 3 4 5 6. 7. 8 9 10 11 12

Acetyl Chloride N-Cbz-L-Alamne Dnsobutyl alummmm hydrtde, 1 M solution m toluene (DIBALH) (Methoxymethyl)trtphenylphosphonmm chloride Sodmm bts(trtmethylsllyl)amtde, 1 A4 solutton m THF (NaHMDS) Anhydrous sodium carbonate. Phenylselenyl chloride L-Serme HCl gas DI tert-butyl dtcarbonate 2,2-Dtmethoxypropane p-Toluensulfomc acid

Just

191

Synthesis of Oligopeptides 2.2. Reagents for Method 3.2 1 2 3. 4 5 6. 7 8 9 10

N-Boc-L-Phenylalanme N-Methylmorpholme Isobutyl chloroformate N,O-Dimethylhydroxylamine hydrochloride Trtfluoroacetic actd (TFA) Tnethylsilane. N-Boc-L-Isoleucme. Dnsopropylethyl amme (rPr,EtN) Dtethyl cyanophosphonate (DEPC) Lrthrum alummrum hydrrde (LIAIHJ.

2.3. Reagents 1 2 3 4. 5 6.

for Method 3.3

4-Methyl Pentanoic Acid Thtonyl chloride (SOCl,) tevt-Butyl thiol (t-BUSH) Buthylhthmm, 1 M solution in hexane (BuLt). Dnsopropyl amme Chlorotrtmethylstlane

2.4. Reagents for Method 3.4 1 2 3 4 5 6 7 8 9

Boron trlfuorlde etherate Potassium carbonate (l&CO,) meta-Chloroperbenzoic acid (MCPBA) Sodmm thtosulfate, 10% solutton (Na2S203) L-Phenylalanme tert-butylester hydrochlortde Dnsopropylethyl amme (iPr*EtN) Dtethyl cyanophosphonate (DEPC) Trifluoroacettc acid (TFA). Trtethylstlane

3. Methods 3.1. Preparation of (2-rac,3S)-3-(Benzyloxycarbonyl)amino2-PhenykButanal5 and (4S, 1‘R)-3-(tert-Butoxycarbonyl)amino2,2-Dimethyl-4-(1 ‘-Phenylselenyl-2’-Formyl)1,3-Oxazolidine

8

of (S)-N-Cbz-alanine 1 into the p-ammo-a-seleno aldehyde 5 is shown in Fig. 2 The protected ammo acid 1 IS transformed into aldehyde 3 after methylatton of the carboxylrc acid and subsequent reduction wrth DIBALH (13). In this reaction, the choice of the solvent and of the reaction temperature IS crtttcal to prevent overreduction of the methyl ester to alcohol and/or deprotectron at the nitrogen. The resultmg aldehyde 3 1simmediately transformed mto the vmyl ether 4 through a Wittig reaction. The yltde of The transformatron

Taddel

192 MeCOCI, MeOH

YHCbz

tjHCbz

-

/;-COO,

DISALH

/\COOMe 2 PhSeCI,

ijHCbz

NHCbz -

*

tjHCbz

Na2C03

-0Me 4

NaHMDS MeOCH2PPh,CI

*

-cCHO 5 SePh

Fig. 2 Syntheses of seleno aldehyde derived from L-alamne. NaHMDS MeOCH,PPh&I

DIBALH

t

PhSeCl Na2C03

a

9

&?Ph

Fig 3 Synthesis of seleno aldehyde derived from L-serme

methoxymethyl trtphenylphosphomum chloride 1s generated using NaHMSD in THF, and the reactton with aldehyde 3 gives product 4 as a mixture of E/Z isomers. If desired, the tsomers can be separated by column chromatography, although the stereoselectton of the next reaction 1sunaffected by the configuration of the double bond of 4. The addition of phenylselenyl chloride to the methoxy-alkene IS carried out at -78°C m the presence of Na2C03 and gives product 5 as a mixture of rsomers not separable by column chromatography. Analogous preparatron of the heterocyclrc intermediate 8 IS described m Fig. 3. The Garner aldehyde 6 1s prepared as descrrbed m the literature (I#) and transformed mto compound 9 followmg the same procedure as used for 5. The selenyl aldehyde 9 1s obtained as a single diastereorsomer

3 1.1. Cbz-Alanme Methyl Ester 2 1 Charge a two-necked flask, equipped with a condenser carrying a CaCl, tube and a dropping funnel with 2.5 g of acetyl chloride (32 mmol) and 17 mL of MeOH and cooled to 0°C After sttrrmg for 5 mm at O”C, add 2 5 g of Cbz-L-alanme (11 mmol) as a solid m small portions. 2 Reflux the mtxture for 3 h, and then cool to room temperature and pour m a 300 mL beaker contammg 150 mL of diethyl ether and 20 mL of Na$O, (saturated solution) The mixture IS transferred into a separatory funnel, the lower aqueous phase 1s separated and washed with 30 mL of dtethyl ether The organic phases are collected and dried over anhydrous Na,SO, The solvent IS evaporated to dryness to give 2 3 g of 2 as a waxy material

193

Synthesis of Oligopeptides 3.1.2. (S)-N-(Benzyloxycarbonyl)Amino

Propanol3

1 In a three-necked flask, equipped with a mechanical stirrer under N2, 2.3 g of the ester 2 (9.7 mmol) was dissolved m 30 mL of toluene and cooled to -78°C (see Note 1) From a droppmg funnel, 10 mL of DIBALH (a 1 2 A4 solution m toluene) was slowly added and the mixture stirred at -78°C for 1 h St111at -78’C, add 6 mL of MeOH m order to destroy the excess of the reducing agent. (Note 2) Remove the freezmg bath, and stir the mixture at room temperature for 3 h 2 Transfer the milky mixture mto a separatory funnel contaming 100 mL of HCl 1.5 N. The orgamc phase is separated, and the aqueous phase is extracted with three portions of 50 mL of dtethyl ether. Combme the orgamc extracts, dry over anhydrous Na2S04, and evaporate to dryness to give 1 9 g of product 3 as a pale yellow vtscous oil that needs to be used unmediately m order to avotd racemizatton on standing for longer periods, even m the refrigerator

3 1.3. (3S, 1E/Z)- 1-Methoxy-3-(BenzyloxycarbonyJAmino-

1-Butene 4

1 Place m a three-necked flask under nitrogen and mechanical stn-rmg, 3 96 g of (metoxymethyl)triphenylphosphomum chloride (11 55 mmol) and 25 mL of dry benzene and cool to 0°C From a dropping funnel, add 12 mL of a 1 Msolution of NaHMDS in THF Stir the mixture at 0” C for 2 h, and then add a solution of 1 6 g of aldehyde 3 (7.7 mmol) dissolved mto 10 mL of benzene Stir the mixture for 3 h at room temperature (see Note 3) 2 Filter the content of the flask through a small path of cehte, and wash the residue several times with diethyl ether The filtered liquor is washed two times with water (10 mL), dried over anhydrous Na2S04 and the solvent evaporated to dryness 3 The residue is fractionated through a column tilled with 200 g of silica gel and eluted with hexane / ethyl acetate l/l (see Note 4). The appropriate fractions are collected and the solvent evaporated to dryness to give 0 65 g of the E isomer of 4 (Rf = 0.6) and 0.39 g of the Z isomer of 4 (Rr = 0 5) (Eluent. hexane ethyl acetate l/l)

3. I. 4. (ZRS, 3S)-3-(Benzyloxycarbonyl) Amino-2-Phenylselenyl-Butanal In a two-necked flask under nitrogen and magnetic stnrmg, dissolve 0 86 g of 4 (3.6 mmol) m 20 mL of CH,Cl, and cool to -78°C Add 0 8 g of anhydrous Na&O, . Dissolve 1 03 g of phenylselenyl chloride (5.4 mmol) (see Note 5) mto 10 mL of CH,Cl,, and add this solutron to the flask cooled to -78°C through a rubber septum, using a syringe Stir the mixture for 3 h at -78°C then add, with a syringe through the rubber septum, 10 mL of NaHCO, (10% water solution), followed by 50 mL of CH,Cl, Let the mtxture warm up to room temperature under vigorous sturmg (see Note 6) Separate the CH,Cl, phase and wash it with water (2 x 10 mL) Dry the CH,Cl, over anhydrous Na$O, and evaporate the solvent to dryness

194

Taddel

5 The residue (1 6 g) 1s fractionated over a column of 130 g of silica gel (Eluent hexane ethyl acetate 2/l) (see Note 4) and the appropriate fractions (R, = 0.6) collected to give after evaporation of the solvent 0 88 g of compound 5 as a yellow 011 ‘H NMR (300 MHz, CDCl,, 25’C) 6 9.53 (d, J = 2 6 Hz, 1H) and 9 49 (d, J=3Hz, lH),76and7,4(m, lOH),S 3(bd, lH),5 13(s-like,2H),433(m, lH), 3.8 (m, lH), 1 36 and 1.35 (d, J= 7 Hz, 3H)

3 1.5 (S)-3-(tert-Butoxycarbonyl) Am/no-2,2-Dimethyl-4-Carboxymethyl-

1,3-Oxazolldlne 6

1 Fill a two-necked flask that IS equipped with a reflux condenser carrying a CaCl* tube and a gas inlet apparatus with 100 mL methanol and, after coolmg to O”C, saturate with gaseous HCl (see Note 7) To this solution add 25 g of L-Serme (0 23 mol) and reflux for 6 h 2 Evaporate the solvent to dryness, add 200 mL of methanol to the residue, and evaporate again to dryness Repeat this last operation a second time (see Note 8) to obtain 35 5 g of serme methyl ester hydrochloride as a white powder 3 Dissolve the solid mto 100 mL of water cooled to O”C, and add a solution of NaOH 1 N to reach pH = 12 4. To this solution, add 250 mL of THF containing 55 g of dl-tert-butyl dlcarbonate (0 25 mol) under mechanical stlrrmg, and stir the mixture for 12 h. 5 Transfer the mixture mto a 2 L separatory funnel and add 500 mL of dlethyl ether The organic layer is separated, washed with brme, and dried over anhydrous Na,SO, The solvent is evaporated to dryness to give 48 4 g of N-Bocserme methyl ester as a white solld 6 The solid is dissolved mto 100 mL of dimethoxypropane, 150 mg of p-toluensulfomc acid is added, and the mixture 1s refluxed for 12 h 7 The cooled solution 1sdiluted with 200 mL of dlethyl ether and washed two times with 50 mL of NaHCO, (saturated solution). The organic layer 1s separated, dried over anhydrous Na,SO,, and the solvent evaporated to dryness. 8 Distill the residue using a rotatory vacuum pump (1 mmHg), and collect the fraction that boils at 9&94”C to obtam 9 1 g of product 6 (86% yield)

3.7 6. (S)3-(tert-Butoxycarbonyl) Ammo-2,2-D~methyl-4-Formyl-1,3-Oxazohd~ne

7

1 Dissolve 10 g of ester 6 (39 mmol) mto a 1 L three-necked flask contammg 180 mL of dry toluene, and then cool to -78°C under nitrogen and mechanical stmmg 2 Into a separate single-necked flask stopped with a rubber septum and cooled to -78”C, place 77 mL of DIBALH (solution 1 Mm toluene) under N, 3. By means of a cannula and a balloon filled with nitrogen placed on the rubber septum of the flask transfer the contents mto the flask containing the solution of 6 (see Note 9) 4 Stir the mixture at -78” for 1 h After a check of the reaction via TLC (eluent.hexane*dlethyl ether 7/3, R, = 0.3), add 60 mL of methanol previously cooled to -78°C through the cannula, and stir the mixture at -78°C for 30 mm

Synthesis of Oligopeptides

195

5 Warm to -2O”C, and add 150 mL of HCl(1 A4 solution). Warm to 0°C and transfer the contents of the flask into a 1 L separatory funnel contammg 200 mL of ethyl acetate 6 Quickly separate the orgamc layer, wash with 200 mL of brme, and dry the organic layer over anhydrous Na,SO, 7. Evaporate the solvent to dryness and distill the residue by means of a rotatory vacuum pump (1 mmHg), collectmg the fraction that distills at 80”-88”C, to obtain 6 3 g of aldehyde 7

3.1.7. (4S, 1‘E,Z)-3-(tert-Butoxycarbonyl)Amino-2,2-Dimethyl-4-(2’Methoxy- 1‘-Ethenyl)- 1,3-Oxazolidine 8 1 In a three-necked flask, under nitrogen and mechamcal stirring, place 3 96 g of metoxymethyl trlphenyl phosphomum chloride (11.55 mmol) and 25 mL of dry benzene and cool to 0°C. From a dropping funnel, add 12 mL of a 1 Msolutlon of NaHMDS m THF Stir the mixture at 0” C for 2 h, then add a solution of 1.76 g of aldehyde 7 (7 7 mmol) dissolved mto 10 mL of benzene The mixture 1s stirred for 12 h at room temperature (see Note 3) 2 The contents of the flask 1sfiltered through a small path of cehte, and the residue washed several times with dlethyl ether. The filtered liquor IS washed two times with 10 mL of water, dried over anhydrous Na$O,, and the solvent evaporated to dryness 3 The residue IS fractionated through a column filled with 200 g of slhca gel and eluted with hexane / diethyl ether l/2 (Note 4). The appropriate fractions are collected and the solvent evaporated to dryness to give 0 25 g of the E isomer of 8 (Rf = 0.6) and 0.43 g of the Z isomer of 8 (R, = 0 5)

3.1.8. (4S, 1‘R)-3-(tert-Butoxycarbonyl)Amlno-2,2-Dlmethyl4-[(Formyl), (Phenylselenyl)-Methyl]- 1,3-Oxazolidine 9 1 In a two-necked flask under nitrogen and magnetic stnrmg, dissolve 0 92 g of product 8 (3 6 mmol) m 20 mL of CH,Cl*, and cool to -78°C. Add 1 g of anhydrous Na,CO,. 2 Dissolve 1.03 g of phenylselenyl chloride (5.4 mmol) (see Note 5) mto 10 mL of CH,Cl,, and add this solution to the flask, cooled to -7X”C, through a rubber septum using a syringe 3 Stir the mixture for 3 h at -78°C and then add from the rubber septum using the syringe 10 mL of NaHCO, (10% solution) followed by 50 mL of CH,Cl, 4 Let the mixture warm up to room temperature under vigorous stirring (see Note 6) Separate the CH,Cl, phase, and wash it with water (2 x 10 mL). Dry the CH,Cl,, over anhydrous Na,SO, and evaporate the solvent to dryness 5. The residue (1 6 g) IS fractionated over a column of 150 g of silica gel (eluent hexane/diethyl ether l/2) (see Note 4), and the appropriate fractions (R, = 0 6) are collected to give 0.88 g of compound 9 as a waxy material after evaporation of the solvent ‘H NMR (300 MHz, DMSO, 5O’C) 6 9.1 (d, J= 2 Hz, lH), 7 4 and 7.2 (m, 5H), 4 3-3 8 (bm, 4H), 1 6 and 1.7 (s, 6H), 1 5 (s, 9H).

196

Taddel IBuOCOCI. N-Me.Morpholme

TFA, Et&H CH2C12

MeOMeNHHCl

t ”

10

MedN-OMe

Boc-b-OH LIAIH.,, THF * I2

Me’N’OMe ‘3

MeJ’J.

OMe

x

.NHEoc

16

SePh

Fig 4 Synthesisof selenoaldehyde derived from drpeptide 3.2. Preparation of (2-rac,3S,2’S,3’S)-4-Phenyi2-Phenylselenyl-3-[2’-(tert-Butoxycarbony)amino3’-Methyl-PentanoylamidoJ-Butanal 16 The same procedure applied for the syntheses of 5 and 8 can be applied m the case of products derived from a polypetide. Figure 4 features efficient preparation of an aldehyde derived from its dipeptide (15) The aldehyde functton IS obtained by LlA1H4 reduction of an N-methoxy N-methylamide (Wemreb amide) (16). Thts group can be also used as the protection of the carboxyhc function durmg the peptide couplmg. Commercially avatlable N-Boc-phenylalamne 10 is transformed mto the Wemreb amide 11 using the mixed anhydride procedure with isobutyl chloroformate. Deprotectron at the mtrogen with TFA and Et$tH, as tertbutyl cation scavenger, gives product 12 as ammomum trifluoroacetate, which can be directly coupled with Boc-IleOH using DEPC as the couplmg agent Direct reduction of the amide with LiAIH4 at 0°C m THF gave aldehyde 13, which can be employed m the next step without any purification. The Wrttig reaction and the addition of PhSeCl are carried out as previously described m Subheading 3.1. for the preparation of compound 9.

Synthesis of Oligopeptides 3.2.1. (S)-2-(tert-Butoxycarbonyl) Amino-3-Phenyl-N-Methyl-N-Methoxy-Propanamide

197 11

Add to a solution of 5 g of (S)-N-Boc-phenylalamne 10 (18 9 mmol) dissolved m 35 mL of ethyl acetate and cooled to O”C, 1 92 g of N-methyl-morpholme (18 9 mmol) and 2 5 g of lsobutyl chloroformate Stir the mixture for 30 mm at O”C, and add 1 92 g of N,O-dimethylhydroxylamme hydrochloride (18 9 mmol) followed by additional 1.9 g of N-methyl-morpholme (18 9 mmol) Warm the mixture to room temperature and stir for 24 h Transfer the contents of the flask mto a separatory funnel containing 60 mL of dlethy ether, wash with 10 mL of HCl (3 M solution), followed by 10 mL of Na,CO, (10% solution) Separate the organic layer and dry over anhydrous Na,SO, Evaporation of the solvent to dryness gave an oil that was purified over a column of 250 g of silica gel (eluent hexane/ethyl acetate l/l) (see Note 4) and the appropriate fractions (R,= 0 6) collected to give 4 9 g of compound 11 as a dense 011after evaporation of the solvent

3.2.2. (S)-2-Ammonium-3-Phenyl-N-Methyl-N-MethoxyPropanamide Trifluoroacete 12 1, To a flask containing 4 9 g of amide 11 (15 9 mmol) dissolved mto 25 mL of CH2C12 under nitrogen atmosphere, add 18.1 g of TFA (160 mmol) (see Note lo), followed by 4.6 g of trlethylsllane (40 mmol) Stir the mixture for 24 h 2 Evaporate the solvent, and add 150 mL of dlethyl ether. Evaporate the ether under vacuum, add to the residue additional 150 mL of ether, evaporate under vacuum, add 150 mL of ethyl acetate and evaporate again under vacuum. 3 Collect the resulting solid mto a cup, and dry it mto a desslcator over phosphorous pentoxide (see Note 11) At the end of this procedure, 4 8 g of product 12 are collected

3.2 3. (2S,2’S,3’S)-3-PhenyI-2-(2’-(tert-Butoxycarbonyl) Am/no-3’-Methyl-Pentanoylamido)-Propanal 14 1 Dissolve 4.8 g of compound 12 (14 9 mmol) into 50 mL of CH,Cl, under mtrogen atmosphere, cool to O”C, and add 3 8 g of (S)-N-Boc-Isoleucme (16 4 mmol) followed by 5 8 g of (I-Pr)*EtN (45 mmol) and 2.7 g of DEPC (16 6 mmol). 2 Stir the mixture at room temperature for 15 h, and then add 25 mL of NH,Cl (saturated solution) (see Note 12). Transfer the mixture mto a separatory funnel, add additional 50 mL of CH,Cl,, separate the organic layer, and wash with 50 mL of water. 3 Dry the separated CH,CI, solution over anhydrous Na2S04, and evaporate the solvent under vacuum Add to the residue yellow 011 150 mL of dlethyl ether to separate 5 1 g of product 13 as a solid (Note 13). 4 Dissolve 5 1 g of product 13 (12.1 mmol) into 50 mL of THF under nitrogen and magnetic stirring, and cool to 0°C ,

Taddei

198

5 Add 0 176 g of LlAlH, (6.3 mmol) m small portions, and stir the resulting grey mixture for 3 h at room temperature 6 Cool again to 0°C and add 15 mL of NaHSO, (15% solution) followed by 50 mL of dlethyl ether (see Note 14) Separate the organic layer, wash the aqueous phase wrth additional 15 mL of dlethyl ether, and combme the ether fraction. Wash them with 40 mL of HCl(3 M solution), separate the orgamc layer, and dry over anhydrous Na,SO, 7 Evaporate the solvent under vacuum to give 3 6 g of product 14 sufficiently pure to be used m the next step (see Note 15)

3.2.4. (2-rac,3S,2’S,3’S)-#-Pheny/-2-Pheny/se/eny/-3-(2’-(tertButoxycarbonyl)-Amino-3’-Methy/-Pentanoy/am/do)-Butana/

76

In a three-necked flask under nitrogen and mechanical stlrrmg, place 5 14 g of (metoxymethyl)tnphenylphosphomum chloride (15 mmol) and 35 mL of dry benzene, and cool to 0°C. From a dropping funnel, add 16 mL of NaHMDS (1 M solution m THF). The mixture 1s stirred at 0” C for 2 h, and then add a solution of 3 6 g of aldehyde 14 (10 mmol) dissolved mto 15 mL of benzene The mixture IS stirred for 12 h at room temperature (see Note 3) The content of the flask 1s filtered through a small path of cellte, and the residue washed several times with dlethyl ether The filtered liquor 1s washed two times with water (25 mL), dried over anhydrous Na,SO,, and the solvent 1s evaporated to dryness The residue (4 5 g) 1s fractionated through a column filled with 250 g of slhca gel and eluted with hexane/ethyl acetate 512 (see Note 4) The appropriate fractions (R, = 0 7) are collected and the solvent evaporated to dryness to give 2.25 g of product 15 as a mixture of E and Z isomers In a two-necked flask under nitrogen and magnetic stlrrmg, dissolve 1 g of product 15 (2 6 mmol) in 20 mL of CH,Cl, and cool to -78°C Add 1 g of anhydrous Na,CO, Dissolve 0.5 g of phenylselenyl chloride (2 6 mmol) (see Note 5) into 10 mL of CH,Cl, and add this solution to the flask cooled to -78”C, through a rubber septum using a syringe Stir the mixture for 3 h at -78°C and then add, from the rubber septum usmg the syringe, 10 mL NaHCO, (10% solution) followed by 50 mL of CH,Cl, Let the mixture warm up to room temperature under vigorous stu-rmg (see Note 6) Separate the CH,Cl, phase, and wash it with water (2 x 10 mL) Dry the CH,Cl, over anhydrous Na,SO,, and evaporate the solvent to dryness The residue (1.6 g) 1s fractionated over a column of 150 g of slllca gel (eluent hexane/ethyl acetate 3/l) (see Note 4), and the appropriate fractions (R,= 0.6) are collected to give 0.83 g of compound 16 as a pale yellow solld after evaporation of the solvent. ‘H NMR (300 MHz, CDCl,, 25°C) 6 9 4 (m, lH), 7 5 and 7 3 (m, IOH), 6 6 and 6 1 (bd, lH), 5 0 (bd, lH), 4 8 and 4 6 (m, lH), 3 9 (m, lH), 3 7 (m, lH), 3.0 (m, 2H), 1 8 (m, lH), 1 5 (s, 9H), 1 3-O 8 (m, 8H)

Synthesis of Oiigopeptides

199 t-BUSH, Pyridme

soap

L

COOH

-

COCI

17 LDA. Me&Cl

S J-f 19

-

18

0

7(

, J-f 20

OStMea

St-au

Ftg 5 Synthesis of stlyl thtoketene acetal

3.3. Preparation

of the Silyi Thioketene

Acetal20

The choice of the nucleophlle for the aldol type reactlon that builds up the skeleton of the oxirane peptrdomrmetics is crucial for the control of the stereochemtstry of the final products, as in this reaction, two new stereogentc centers are formed. Literature data suggest that Lewis acid mediated reaction of silyl thtoketene acetals with choral aldehydes 1s stereoconvergent providing the anti isomer mdependently from the enol ether double bond geometry (17). Product 20 1s prepared starting from tsovaleryl chloride 18, which IS transformed mto the thtoester by reaction with t-BUSH tn pyridine (Fig. 5). After purtficatton by distlllatton, 19 reacts with LDA and Me,SiCl to give product 20 that can be used directly m the next step without any purtficatron.

3 3.1 tert-Butylthio-4-Methyl-Pentanoate

19

Place 5 g of 4-methyl-pentanolc acid 17 (43 mmol) m a flask equipped with a condenser and a CaCI, tube, and add one drop of DMF and 25 mL of SOCI, (see Note 16)

Reflux the mixture for 12 h, and then evaporate the SOCl, at atmospheric pressure, transfer the residue mto a dtsttllatton apparatus and dtsttl at 760 mmHg collectmg 18 as the fraction (4 3 g) that boils at 140-142°C Dissolve 4 3 g of the dlsttlled chloride 18 (32 mmol) mto 40 mL of CH,Cl, , cool to 0°C and add 2.9 g of t-BUSH (32 2 mmol) (see Note 17) and 2.6 g of pyrtdme (32 9 mmol) Stir the mixture at room temperature for 15 h, filter away the solid using a small pad of Cehte, and wash the CH,Cl, solution with 20 mL of NH&I (saturated solution) and with 20 mL of brine. Dry the solvent over anhydrous Na$O, Evaporate the solvent (see Note 18) and dtsttll the residue by means of a water pump (18 mmHg), collectmg the fractron (4.0 g) that bolls at 82%85°C

3 3 2. I-tert-Butylthio-4-Methyl1- Trime thylsilyloxy- 1-Pen tene 20 1 Dissolve 2 g of dusopropyl amme (20 mmol) into 15 mL of THF under nitrogen and magnetic stnrmg, and cool to -78°C. Add 20 mL of BuLt (1 M solutton m hexane), and stir for 15 mm at -78°C and 15 mm at room temperature.

200

Taddei BF, OEt, Chromaiographyo

CbzHNJ&OSt-s” e

2, PhSt

’

Y

aq NaOH MeOH

Fig 6 Synthesis of Z-Ala-v [trans-epoxy]LeuOH 2 Cool down again the flask at -78°C and add 3 9 g of the thioester 19 (20 mmol) dtssolved in 10 mL of THF Star the mrxture for 1 h at -78°C 3 Add slowly 3 3 g of chlorotrlmethylsllane (30 mmol), and allow the mixture to reach the room temperature under stirring The solid formed 1sfiltered away usmg a small Cehte pad, and the ltquor is evaporated to dryness to gave 4 1 g of the product 20 as a 95/5 mixture of the Z/E isomers.

3.4. Preparatioh Peptidomimetcs

of the Oxirane 23,26, and 31

The key step of our synthesis was performed reactmg 20 and compounds 5 or 16 at -50°C m the presence of 2 eq of BF, * OEt, A mixture of only 2 isomers is obtained after the work-up, and the major component 1s separable by column chromatography on stltca gel. Oxidattve elimmatton of the phenylselenyl group 1s cart-ted out using MCPBA m MeOH and a buffer envtronment. The reaction is stereospecific (18,19), giving the epoxy compound as a single isomer. The last step of the synthesis is the deprotectton of the carboxyhc function that 1s performed in an aqueous basic medmm (Fig. 6 and 7). The same protocol of reaction can be applied to the dertvattve of the serme 9 with the obvtous advantage to start from a single diasterotsomer (Fig. 8). The BF, mediates aldol type reaction with 20 and results m the formatton of a single isomer that 1s transformed mto the epoxy derivative with the same procedure as employed for 24. In this case, after alkaline hydrolysis of the ester functton, tt 1spossible to couple the acid 29 with a different ammo actd (carrymg a actdsensitive protectmg group on the carboxyhc function). This procedure allows to carry out the final deprotectton m acidic medium to give the free ammo acid that can be employed m the enzyme mhtbitton tests. 3 4.1. (2R,3S,4R,5S)-2-(2’-Methy/-l’-Propyl)5-(Benzyloxycarbonyl)amino-3,4-Epoxy-3-Hexenoic

Acid

Cbz-Ala-v[trans-epoxy]-LeuOH 23 1. Dissolve 1.5 g of compound 5 (4 mtnol) tn 40 mL of CH2C12 under nitrogen and magnetic stirring. Cool the flask to -78°C and add 1 28 g of boron trifluortde

201

Synthesis of Ol/gopeptides .NHBoc o

BF, OEt, Chromatography0 separation

CHO SePh

16

mCPBA, Na&Os

0

MeOH

BocHNAN .

*

H

----A

Fig 7 Synthesis of BocIle-Phe-v

BF, OSIMe3 20

St-Bu

mCPBA,

MeOH

+

[trans-epoxy]-LeuOH

OEt, _

-

. =

Y

PhSt 9

&Ph

27

aq NaOH Dloxane, MeOH

K&03

COOH 29

H-Phe-Ot-Bu,

Ph

DEPC, N-Me-Motphollne

COOt-BuMe

31

Ph

TFA, Et&H,

H

Y

CH$& *

HY Fig 8 Synthesrs of Ser-v [trans-epoxy]-Leu-PheOH.

etherate (9 mmol). After 15 mm, add 1.17 g of compound 20 (4.5 mmol) dlssolved m 5 mL of CH,CI,. Stn the mixture at -40°C for 3 h, and then add 10 mL of phosphate buffer, and allow the mixture to reach the room temperature. The content of the flask 1s transferred in to a 250 mL separatory funnel, and 50 mL of CH,Cl, 1sadded The orgamc layer 1sseparated, washed with 50 mL of water and 50 mL of brine, and then separated and dried over anhydrous Na2S04 Evaporate the solvent to dryness and fractionate the residue (1.8 g) over a column of 180 g of silica gel (eluent: hexane/ethyl acetate 3/l) (see Note 4), collectmg the appropriate fractions to give 0.75 g of compound 21 (Rf = 0 5) as a pale yellow solid and 0 30 g of the other isomer (Rr= 0.4), after evaporation of the solvent.

202

Taddel

5 Dissolve 0 75 g of product 21 (1 3 mmol) m 5 mL of methanol 6

7.

8 9

10

Cool to -15”C, then add 1 1 g of K&O3 (8 mmol) followed by 0 415 g of MCPBA (2 4 mmol) Stir the mixture for 2 h at -15”C, then add 30 mL of dlethyl ether and 15 mL of water. Warm the mixture to room temperature and separate the orgamc layer Extract the aqueous phase with addltlonal 10 mL of dlethyl ether, collect the ethereal fractions, and wash with 10 mL of Na,S,O, (10 % solution) and 20 mL of brme After drymg over anhydrous Na,SO,, the crude (0 60 g) 1s fractionated over a column of 60 g of slhca gel (eluent hexanelethyl acetate 512) (see Note 19), and the appropriate fractions collected to give 0 48 g of product 22 as a white waxy material, after evaporation of the solvent. Dissolve this product m 2mL of MeOH and add 5 mL of NaOH (0 1 A4solution) Stir for 3 h at room temperature Evaporate the methanol under vacuum, add 10 mL of ethyl acetate and 5 mL of water, and acldlfy with HCl 3N to pH 4 Separate the organic layer, dry over anhydrous Na,SO,, and evaporate the solvent to dryness The crude 23 (0 38 g) IS sufficiently pure but can be addltlonally punfied by crystalhzatlon from acetoneihexane m p 8688°C ‘H NMR (300 MHz, CDCl,, 45°C) 6 10 0 (s, lH), 7 4 (m, 5H), 5 1 (s-hke, 2H), 4 8 (bs, lH), 4 l-3 9 (m, lH), 3 0 (A part of an ABX system, J’= 7 Hz, J” = 1 8 Hz, lH), 2 7 (B part of an ABX system, J’ = 8 Hz, J” = 1 8 Hz), 2 8-2 5 (m, 3H), 1,2-l 0 (m, 9 H)

3 4.3 (2R,3S,4R,5S,2’S,3’S)-5-[2’-tert-(Butoxycarbonyl)amino3’-Methyl-Pentanamrdoj-2-(2’Methyk I ‘-Propy/)-6-Phenyl3,4-Epoxy-3-Hexenoic Acid, Boc-lie-Phe-+v[trans-Epoxy]-LeuOH

25

1 Dissolve 0 6 g of compound 16 (1 13 mmol) m 10 mL of CH,Cl, under mtrogen and magnetic stlrrmg. Cool the flask to -78°C and add 0 64 g of boron trlfluorlde etherate (4 5 mmol) After 15 mm, add 1 17 g of compound 20 (4 5 mmol) dlssolved m 5 mL of CH,Cl, 2 Stir the mixture at 40°C for 10 h, and then add 10 mL of phosphate buffer and allow the mixture to reach the room temperature. 3 Repeating the points 3-10 of Subheading 3.4.2., you should obtain approxlmately 0 2 g of product 26 that can be further purified usmg a column of 10 g of silica gel (eluent CH2C12/MeOH 20/l) (see Note 19), and collect the appropriate fractions (Rf= 0 3) ‘H NMR (300 MHz, CDC13, 50°C) 6 9 0 (s, lH), 7 2 (m, SH), 5 9 (bs, IH), 4 8 (bs, lH), 4 54 3 (m, lH), 3 9-3 7 (m, lH), 3 O-2 7 (m, 4H), 2 4-2.3 (m, IH), 1 8 (m, lH), 1.7-1 0 (m, 12H), 0 9-O 7 (m, 14H)

3 4.4 (4S, 1‘R,2’S,3’R)-3-[tert-(Butoxycarbonyl)Amino]-2,2-Dimethyl4-(3’Carboxy-S’Methyl- I ‘,2’-Epoxy- 1‘-Hexeny/)- 7,3-Oxazolidine 28 1. Dissolve 0 88 g of compound 9 (2.2 mmol) m 20 mL of CH2C12 under nitrogen and magnetic stirring Cool the flask to -78”C, and add 0 64 g of boron trlfluorlde etherate (4.5 mmol). After 15 mm, add 1 17 g of compound 20 (4 5 mmol) dlssolved m 5 mL of CH,CI,

Synthesis of Oligopeptldes

203

Stir the mixture at -60°C for 8 h, than add 10 mL of phosphate buffer and allow the mixture to reach the room temperature The content of the flask IS transferred m to a 250 mL separatory funnel, and 50 mL of CH,Cl, IS added The organic layer IS separated, washed with 50 mL of water and 50 mL of brme, and then separated and dried over anhydrous Na$O, Evaporate the solvent to dryness, and the residue (1.35 g) IS fractionated over a column of 130 g of slhca gel (eluent hexane/ethyl acetate 3/l) (see Note 4), and the appropriate fractions are collected to give, after evaporation of the solvent, 0 55 g of compound 27( R, = 0 5) Dissolve 0.55 g of product 27 (0 94 mmol) m 5 mL of methanol. Cool to -15°C than add 0 5 g of K&O, (4 mmol) followed by 0 2 g of MCPBA (1 2 mmol) Stir the mixture for 2 h at -15”C, than add 10 mL of dlethyl ether and 15 mL of water Warm the mixture to room temperature and separate the orgamc layer Extract the aqueous phase with addltlonal 10 mL of dlethyl ether, collect the ethereal fractions and wash with 10 mL of Na,S,O, ( 10 % solution) and 20 mL of brine After drymg over anhydrous Na,SO,, the crude (0 40 g) 1s fractionated over a column of 40 g of silica gel (eluent hexane/ethyl acetate 5/3) and the appropriate fractions collected to give, after evaporation of the solvent, 0 28 g of product 28 as a pale yellow material

3 4.5 (2R,3S,4R,5S, 1‘S)5-Am/no-N-(1 ‘-Carboxy-2’-Phenyl1‘-Ethyl)-6-Hydroxy-2-(2’-Methyl1‘-Propyl)-3,4-Epoxy3-Hexanamlde, Ser-v[Epoxy]-Leu-PheOH, 31 Dissolve 0.28 g of product 28 (0.65 mmol) m 2 mL of dloxane, add 5 mL of NaOH (0 1 A4 solution) Stir for 8 h at room temperature Evaporate the dloxane under vacuum, add to the residue 10 mL of ethyl acetate, cool to O”C, and add 5 mL of water Acldlfy with HCl 3 N to pH 4 under vigorous stirrmg Separate the organic layer, dry over anhydrous Na,SO,, and evaporate the solvent to dryness to obtain 0 196 g of 29 Dissolve 0.196 g of crude 29 (0 55 mmol) mto 5 mL of CH2C12 under mtrogen atmosphere, cool to O’C, and add 0 14 g of (S)-phenylalanme tert-butyl ester hydrochlorrde (0 55 mmol), followed by 0 167 g of (I-Pr)*EtN (1 3 mmol) and 0.097 g of DEPC (0 6 mmol) Stir the mixture at room temperature for 15 h, then add 5 mL of NH&l (saturated solution) (see Note 12) Transfer the mixture mto a separatory funnel, add addltlonal 10 mL of CH,Cl,, separate the organic layer, and wash with 5 mL of Na2C03 (10% solution) followed by 5 mL of brine Dry the separated CH,Cl, solution over anhydrous NagSO,+ and evaporate the solvent under vacuum The crude (0.3 g) is fractionated over a column of 30 g of silica gel (eluent’ CH,Cl,/methanol IO/l) (see Note 19), and the appropriate fractions (Rf = 0.4) are collected to give, after evaporation of the solvent, 0 18 g of product 30 as a white sohd

204

Taddei

7 To a 5 mL vial stopped with a rubber septum, which contams 0 18 g of protected peptlde 30 (0.35 mmol) dissolved mto 0 55 mL of CH,CI, under mtrogen atmosphere, add with a syrmge 0.18 g of TFA (1 60 mmol) followed by 0.46 g of trlethylsllane (4 mmol). Stir the mixture for 24 h 8 Place the vial mto a larger flask, and evaporate the solvent at the rotatory film evaporator Add m the vial 3 mL of dlethyl ether. Evaporate the ether, add to the residue additional 3 mL of dlethyl ether, evaporate, add to the residue 3 mL of ethyl acetate, and evaporate again under vacuum (see Note 20) 9 Place the vial mto a desslcator over phosphorous pentoxide, and store under vacuum for at least 5 h At the end of this procedure, the vial should contam about 60 mg of compound 31 as ammonium trlfluoroacetate (Note 21) HMRS 365 2079 (M+ + 1)

3.5. Discussion The synthesis of the oxlranes descrtbed above was designed as a stereoselective route to compounds structurally very slmllar to the model peptlde. Unfortunately,

apart from the case of the heterocycllc

serine derlvatlve

9, the

addition of PhSeCl to the alkoxy aldehyde was not stereoselective. That means that at least one chromatographlc separation was needed to obtain a single lsomer, thus limiting the amount of product that can be prepared with this method On the other hand, the protocol descrrbed here allows the lsolatlon of an oxirane

contammg ohgopeptlde free from protecting group on terminal ammlc and acidic functions, a form very suitable for enzyme mhlbltlon tests In prmclple, this method could be applied to the synthesis of several oxn-ane polypeptldes and might be apphed to solid phase, as the reaction conditions are very mild

and always employ homogeneous reagents. 4. Notes 1. The temperature of -78°C 1s generated using the acetone dry Ice bath 2 The addltlon of methanol to destroy the excess of DIBALH must be done at -78”C, followed by additional stirring for 3 h. Caution: addltlon of protlc solvent to solutions contammg DIBAL may produce exothermlc reactions with rapid evolution of gas 3 A deep red color mdlcates the correct formation of the yhde 4 The separation 1sperformed followmg the protocol of flash chromatography (20) 5 Caution phenyl selenly chloride 1sa highly toxic and odorous and must be handled through gloves under a fume cupboard. All the glassware must be treated with sodmm hypochlorlte to remove organic selemdes and the liquors collected for disposal 6 The addition of an aqueous solution at -78°C generates a dense slurry that must be stirred vigorously to efficiently quench the reagent This step has a strong influence on the final yield of this reaction 7. The amount of HCl dissolved m methanol is monitored by weighting the flask at interval during HCl bubbling to reach a constant weight

Synthesis of Oligopeptides

205

8 Addition

9 10 11. 12

13

14 15 16 17

18

19 20

21.

and evaporation (with the aid of a rotatory film evaporator) must be repeated until a white sohd is obtained. Precooling the solutions that have to be mixed and use of cannula for the addltlon are indispensable for getting high yields of 7. This large excess of TFA IS needed. Caution TFA 1s a highly corrosive hquld Dry until a constant weight IS reached. Caution: durmg this step, DEPC liberates HCN. The reaction need to be carried out under a fume cupboard. A KOH/KMnO, absorption station 1s recommended to destroy the HCN If there 1sno formatlon of solid, dissolve again m CH,Cl, and wash with a basic aqueous solution, separate, wash with an acidic aqueous solution Separate, dry, evaporate under vacuum, and add diethyl ether Repeat these operations until a solid separates Caution* hydrolyze LlAlH, slowly to prevent rapid evolution of gas and foaming out of the flask To prevent racemlzatlon, It IS better to prepare the ohgopeptidlc aldehydes Just before the use The reaction must be carried out under a fume cupboard, as gaseous HCl and SO, are formed t-BUSH IS extremely odorous. Any operation involving its use must be carried out under fume cupboard Everythmg that has been m contact with it must be thoroughly washed with sodium hypochlorlte solution (bleach) before being carried out from the fume cupboard The exhaust of the pump connected either with the rotatory film evaporator or with the dlstlllation apparatus must be bubbled through a beaker containing bleach that 1s located under the fume cupboard. It is the only way to prevent the security service of the bulldmg from evacuating the laboratory because it beheves there IS a leak m the gas pipes (Thiols are used as smell detector in the gas lines) The separation 1s performed using the flash silica at atmospheric pressure and collecting fractions of approximately 1 mL The procedure of addition of dlethyl ether and further evaporation at the rotatory film evaporator must be repeated until a solid is obtamed. Sometimes it may be useful to connect the flask containing the vials to a high vacuum pump to remove traces of water This compound 1ssufficiently stable at room temperature as a solid and in water solution, If maintained at pH <7. At higher pH decomposition occurs, probably through rmg opening of the epoxlde

References 1. Meara, J P and Rich, D. H (1996) Mechamstlc studies on the mactlvatlon of papain by epoxysuccmyl mhybltors. J Med. Chem 39, 3357-3366 2 Park, C , Koh, J S , Son, Y C , Choi, H., Lee, C S , Choy, N , Moon, K Y , Jung, W H , Krm, S C , and Yoon, H (1995) Ratlonale design of lrreverslble pseudoCz-symmetric HIV- 1 protease inhibitors Bioorg. Med. Chem Lett 5,1843-l 848

206

Taddel

3 Albeck, A., Persky, R , and Khper, S (1995) Design of a new selective cysteme protease mactlvator and its mechamstlc lmpltcatlons Bzoorg Med Chem. Lett 5, 1767-l 772 4 Gulllerm, D , Lavrador, K , and Gudlern, G. (1995) Enantloselectlve synthesis1 of epoxy ammo acids Synth Comm 25,877-882 5 Berkowitz, D B and Pedersen, M. L. (1995) Free a-oxlranyl ammo acids J Org Chem. 60,5368-5369 6. Parkes, K E , Bushnell, D J , Crackett, P H , Dunsdon, S J , Freeman, A C , Gunn, M P Hopkins, R. A., Lambert, R. W , Martin, J A , Merret, J. H., Reshaw, S , Spurden, W C , and Thomas, G J (1994) Studies toward the large scale synthesis of the HIV protemase inhibitor Ro 3 l-8959. J Org Chem 59,3656-3664 7 Askin, D , Eng, K K , Rossen, K , Punk, R M , Wells, K M., Volante, R P , and Relder, P J (1994) Highly dlastereoselectlve reaction of a choral, non racemlc amide enolate with (S)-glycldyl tosylate. Synthesis of the orally active HIV-1 protease mhlbltor ~-735,524 Tetrahedron Lett 35,673%676. 8 Luly, J R , Dellaria, J. F., Plattner, J J , and Soderqulst, J L. (1987) A synthesis of protected ammoalkyl epoxldes from a-amino acids J Org Chem 52 1478-1492 9 Jenmalm, A, Berts, W , Ll, Y L , Luthman, K., Csoregh, I, and Hacksell, U (1994) Stereoselective epoxldatlon of Phe-Gly and Phe-Phe vmyl lsosteres J Org Chem 59, 1139-l 148 10 Scholz, D , Bllhch, A , Charplot, B., Ettmayer, P , Lehr, P , Rosenwlrth, B , Schremer, E , and Gstach, H. (1994) Inhibitors of HIV- 1 protemase containing 2heterosubstituted 4-ammo-3-hydroxy-5-phenylpentanolc acid; synthesis, enzyme inhibition and antiviral activity J Med Chem 37, 3079-3089 11 Mann, A , Quaranta, L., Regmato, G , and Taddel, M. (1996) A general synthesis of ohgopeptldes containing an oxlrane rmg m the place of a peptldlc bond Tetrahedron Lett 37,265 I-2654 12 Spatola, A F (1983) Peptlde backbone modlficatlon* a structure-activity analysis of peptldes containing amide bond surrogates, conformatlonal constraints and related bakbone replacements Chem. Blochem Ammo Aczds, Pept Protems 7,267-357 13 Jurczak, J and Goleblowskl, A (1989) Optically active N-protected a-ammo aldehydes m orgamc synthesis Chem Rev 89, 149-164 14 Garner, P and Park, J M. (1987) The synthesis and configurational stablllty of differentially protected P-hydroxy-a-ammo aldehydes J Org Chem 52, 2361-2364 15. Clapettl, P , Taddel, M., and Uhvi P (1994) CrCl, mediated allylatlon of N-protected a-ammo aldehydes A versatile synthesis of polypeptldes contammg an hyfroxyethylene lsostere. Tetrahedron Lett 35,3 183-3 186 16. Nahm, S. and Wemreb, S. M. (1981) N-Methoxy-N-methylamldes as effective acllatmg agents Tetrahedron Lett. 22,38 15-38 18 17. Annunziata, R., Cmquml, M., COZZI, F , Cozzl, P. G , and Consolandl, E. (1992) Stereocontrol m the Mukalama aldol addition of choral a- and P-thlo-substituted aldehydes J Org Chem 57,45&461

Synthesrs of Ol/gopeptides

207

18 Uemura, S , Ohe, K , and Sugrta, N. (1988) Oxidative conversion of P-hydroxyselemdes to epoxrdes and ketones with m-chloroperbenzotc acid J Chem Sot Chem Commun 111-112 19. Ceccherelh, P , Curmr, M , Eprfamo, F., Marcotulho, M. C., and Rosa& 0. (1995) Selenoxrdes as leaving groups synthsis of epoxrdes. Tetrahedron Lett 36, 5079-5080 20. Stall, W. C., Kahn, M., and Mrtra, A (1978) Rapid chromatographtc technique for preparative separatrons wtth moderate resolutton J Org Chem 43,2923-2925

Synthesis

of Protected Lactam-Bridged

Dipeptides

Debra S. Perlow and Roger M. Freidinger 1. Introduction Lactam-bndged drpeptrdesare useful tools for the mtroduction of conformattonal constraint m higher peptides.Generalmethodshave beendevised for the synthesisof drpeptideshaving five-, six-, and seven-memberedring constramts(1,2). This chapter will focus on four syntheticpathsfrom protected chnal a-ammo acidsto lactams that involve intramolecular alkylatron, mtermolecular alkylatron, intramolecular acylation, and condensattonwith formaldehyde for a one carbon unit msertion. 2. Materials 2.1. Reagen Is for Method 3.1 1 2 3 4 5 6 7. 8 9 10 11 12 13 14 15 16 17

(tert-Butoxycarbonyl)-methronme (Boc-Met). Glycme methyl ester hydrochloride. Drmethylformamtde (DMF) Trrethylamme Hydroxybenzotrlazole hydrate (HBT, HOBT) Drcyclohexylcarbodnmde (DCC). Methylene Chloride (CH&) Crtrrc acid (0 5 M aqueous solution) Sodium bicarbonate (NaHCOs) (2 N aqueous solutton). Sodium sulfate (Na2S0,) Methyl Iodide Sodium hydride (50% mineral 011dispersion). Methyl acetate Leucme methyl ester hydrochlorrde Phenylalamne methyl ester hydrochlorrde. Hydrochlorrc acid (HCl) gaseous. (tert-Butoxycarbonyl)-D-Alamne (Boc-D-Ala) From

Methods m Molecular Medune, Vol 23 Pepbdommebcs Profocols Edlted by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

209

Perlow and Freidmger

210 2.2. Reagents 2 3. 4 5 6 7 8 9. 10 11 12 13 14

Dtmethylformamtde (DMF). Triethylamme Methylene Chloride (CH,CI,). Citric acid (0 5 M aqueous solution) Sodium bicarbonate (NaHCOs) (1 N aqueous solution) Sodmm bicarbonate (NaHCO,) (solid) Sodium sulfate (Na,SO,) Hydrochloric acid aqueous (1 N) NE-(Benzyloxycarbonyl)-L-Lysme methyl ester hydrochloride Isopropyhdene cyclopropane- I,1 -dicarboxylate (Fluka) 1,8-Diazabicyclo[5 4 Olundec-7-ene (DBU) Gramme methiodide tert-butyl alcohol Drphenylphosphorylaztde (DPPA)

2.3 Reagents 1. 2 3 4. 5 6. 7 8.

for Method 3.2

for Method 3.3

Drmethylformamide (DMF) (tert-Butoxycarbonyl)-N*-(benzyloxycarbonyl)-ornlthlne Glyoxyhc acid hydrate 10% Pd on carbon catalyst. Acetic acid (glacial) Hydrogen (H,) gaseous Supercel@ Acetomtrile

2.4. Reagents for Method 3.4 1. 2 3 4 5 6 7 8 9 10 11 12 13 14. 15 16

Glycme methyl ester hydrochlortde Dimethylformamide (DMF) Triethylamme Methylene Chloride (CH,ClJ Sodium bicarbonate (NaHCOs) (1 N aqueous solution) Hydrochloric acid aqueous (I N) Hydrochloric acid concentrated aqueous (12 N) S-(Acetamidomethyl)~L-Cysteme Sodium carbonate (Na&Os) anhydrous N-ethoxycarbonylphthallmide Diphenylphosphorylazide (DPPA) p-Toluenesulfomc acid Paraformaldehyde. 1,1,2-Trichloroethane Chloroform (CHC13) Tetrahydrofuran (THF)

Protected Lactam-Bridged

Dlpeptides

R=H R=CH2CH(CH3)2

+

H&,CHJ s

/‘3

1 2

211

3 4

R=H R=CH&H(CH&

5 6

Fig. 1 Synthesis of y-lactam dtpeptldes via mtramolecular

H&,CH~

R-H R=CH#HXH&

alkylatton.

2.5 Reagents for Chromatography 1 Thin-layer chromatography (TLC) plates- Quantum Industrres, Q-l plates 2 Thin-layer chromatography (TLC) development systems---Components were vtsuallzed either by tert-butyl hypochlonte-KI reagents, or nmhydrm 3 Sihca Gel Column Chromatography-E Merck silica gel 60 (23@400 mesh) 4 Chromatography solvent systems. a. 5.5.1-3 ethyl acetate-pyridine-acetlc acid-water (EPAW) b. 10 5: 1 3 ethyl acetate-pyrtdme-acetic acid-water (EPAW) c 30 5: 1*l ethyl acetate-pyndine-acetxz acid-water (EPAW). d 20.5: 1: 1 ethyl acetate-pyridme-acettc acrd-water (EPAW). e 95 5 OS.0 1 chloroform-methanol-water-acetic acid f 90 2 0 1 chloroform-methanol-water

3. Methods 3.1. Preparation and Tripeptides

of y-Lactam Dipeptides via Intramolecular Alkylation

Protected methtonine dtpeptrde esters are useful startmg materials for mtramolecular alkylation since they can be prepared m optically pure form by conventronal methods, and the methylthlo group can be readily converted into a leaving group (Fig. 1). Their sulfonium salts are formed via methyl iodide, and subsequent cyclizatlon to the lactams is induced by sodrum hydride in 1: 1 DMF/methylene chloride m 40-60% yreld (3). The methyl ester is almost completely hydrolyzed under the condmons of the cyclizatron. The major isolated

product IS the acid with only small amounts of the correspondmg ester bemg found (see Note 1). This synthetic route provrdes ready access to a variety of lactams of defined stereochemistry (see Note 2) simply by changing the starting ammo acid ester. This route is also applicable to the methionme sulfomum salts of larger peptides (see Fig. 2). The expected lactam tripeptide was obtamed from the sulfomum salt of Boc-D-Ala-Met-Phe-OMe (Subheading 3.1.3.).

212

Perlo w and Freidinger

10 0 H3C,S,CH3 s ,‘=

1 1 DMF/CH2CI~

Fig 2 Synthesis of y-lactam trlpeptldes via mtramolecular

alkylatlon.

3.7 7 Preparation of (S)-3-[(tert-Butoxycarbonyl) Amino]-2-Oxo-I-PyrrolidineAcetic Acid 5 3 1 1 1. BOC-MET-GLY METHYL ESTER 1 1 Combme (tert-butoxycarbonyl)-methlonme (24 9 g, 0 1 mol) and glycme methyl ester hydrochloride (12 6 g,O 1 mol) m 150 mL of degassed DMF (see Note 3) Add trlethylamme (13 9 mL, 0.1 mol) and HBT (15 3 g, 0 1 mol) to the mixture followed by DCC (20 6 g, 0 1 mol) Stir the mixture under mtrogen at room temperature overnight 2 Filter the mixture, and concentrate the filtrate zn V~CUO Redissolve the residue m methylene chloride (150 mL) Extract this solution with 0 5 M citric acid (3 x 50 mL) and 2 N aqueous NaHC03 (3 x 50 mL), and dry the orgamcs over Na*SO, 3 Filtration and concentration zn wcuo will result m a crude product that crystallizes This can be recrystallized from ethyl acetate-hexane to obtain about 24 1 g of crystalline 1 with a melting pomt of 87-89°C (0 075 mol, 75%)

3.1 1 2 BOC-MET-GLY METHYL ESTER METHYLSULFONIUM IODIDE 3 1, Dissolve [(tert-Butoxycarbonyl)methlonyl]glycme methyl ester l(5 g, 15.6 mmol), while stirring m 30 mL of methyl iodide at room temperature under nitrogen A gummy solid will separate over a period of 6.5 h 2 The supernatant can be drawn off and the residue dried zn vacua to give a hygroscoplc foam of 3 m 100% yield (7.34 g, 15.6 mmol)

Protected Lactam-Bridged 3.1.1.3.

Dipeptides

(S)-3-[( WV-B~T~XYCARB~NYL)AMINO]-2-0x0-1

213 -PYRROLIDINEACETIC

ACID 5

Dissolve [(tert-Butoxycarbonyl)methlonyl]glycme methyl ester Methylsulfomum Iodide 3 (7 34 g, 15 6 mmol) m 3 12 mL of 1 1 DMF: CH,CI, under nitrogen, and cool to 0°C (see Note 4) Add, m one portion sodium hydride (1 5 g of a 50% mineral 011 suspension, 3 1 5 mmol), and allow the mixture to stir at 0°C for 2 5 h. Add methyl acetate (104 mL), followed by water (24 mL), and allow this mixture to stand overnight at room temperature Concentrate the solution to a small volume zn wcuo and partition the residue between water (50 mL) and methylene chloride (50 mL) Separate the phases. Acid@ the (pH 8) aqueous phase to pH 4 with 0.5 Mcltric acid. Repeated extraction with methylene chloride followed by concentration in vacua will yield a crystallme product 5 (2.06 g, 5 1% yield) that melts with decomposltlon at 171-172°C IR (NuJo~) 3350, 1745, 1705, 1670, 1530, 1170 cm-‘, NMR (Me$O-&) 6 1 37 (s, 9), 1 7-2 5 (m, 2), 3.30 (m, 2), 3.77 (d, 1 J= 17 Hz), 4.04 (d, 1, J= 17 Hz), 4.0 (m, l), 7 05 (br d, 1, J= 7 5 Hz) 12.87 (br m, 1); [cx]~~~ = -3 1 2” (c 1 0, MeOH)

3.1.2. Preparation of (S)-3-[(tert-Butoxycarbonyl) Am/no]-2-0x0- 1-Pyrrol/dine-(S)-4-Methyl-2-Pentanoic 3 1 2 1. BOC-MET-LEU

METHYL

Acid 6

2

ESTER

1 Combme (tert-butoxycarbonyl)-methlomne (12 45 g, 0 05 mol) and leucme methyl ester hydrochloride (9 08 g, 0 05 mol) m 75 mL of methylene chloride under nitrogen Add trlethylamme (13 9 mL, 0 1 mol) to the mixture, followed by DCC (20 6 g, 0.1 mol) Stir the mixture at room temperature overnight 2 Filter the mixture and extract the filtrate with 0.5 Mcitrlc acid (3 x 50 mL), and 2 N aqueous NaHC03 (3 x 50 mL) and dry the orgamcs over Na,SO, 3 Filtration and concentration zn wcuo will result m a crude product that crystallizes This can be recrystallized from ethyl acetate-hexane to obtain about 15 1 g of crystalline 2 with a melting point of 108-109°C (0 04 mol, 80%) 3 1 2 2 BOC-MET-LEU METHYL

ESTER

METHYLSULFONIUM

IODIDE

4

1 Dissolve [(tert-Butoxycarbonyl)methlonyl]leucine methyl ester l(960 mg, 3 mmol) while stirring m 6 mL of methyl iodide at room temperature under nitrogen Stir for 16 h 2 Remove the methyl iodide by concentration In vacua, followed by concentration from 10 mL of methylene chloride, three times to obtain the product 4 as a syrup in 100% yield 3.1.2.3.

(S)-3-[(

TERT-BUTOXYCARBONYL)AMlNO]-2-0X0-1

METHYL-2-PENTANOIC

ACID

-PYRROLIDINE-(S)-4

6

1 Dissolve the sulfomum salt 4 (9 13 g, 17 7 mmol) m 300 mL of 1: 1 DMF: CH,Cl,, and cool the solution to 0°C under nitrogen (see Note 4)

Per/o w and Freidinger

214

2 Add m one portlon a 50% mineral oil suspension of sodium hydride (1 69 g, 35.4 mmol), and star at 0°C for 2 5 h 3 Add methyl acetate (100 mL), followed by water (25 mL), and allow the resultmg solution to stir overnight 4 Concentrate the solution to a small volume WI WCUO, and partition the residue between water (50 mL) and methylene chloride (50 mL) Separate the phases 5 Acidify the aqueous phase (pH 8) with 0 05 Mcltnc acid to pH 4 Repeated extractlons with methylene chloride, followed by concentration zn WCUO, ~111 result m the desired product 6 m about 40% yield (2.2 gm), mp 154-l 55°C (see Note 5) IR (NuJo~) 3400,1755,1725, 1675,1525,1170 cm-‘, NMR (MezSO-d,) 6 0 84 (d, 3, J= 7 Hz), 0 91 (d, 3, J= 7 Hz), 1 39 (s, 9), 1 5-l 9 (m, 4), 2 28 (m, l), 3 26 (dd, 2, Jl = 8 Hz, J2 = 4 Hz), 4.12 (dd, 1, J, = 18 Hz, J2 = 8 Hz), 4 56 (dd, 1, J, = 12 Hz, J2 = 5 Hz), 7 20 (d, 1, J= 8 Hz), 13 (br s, l), [IX]*~~ = +22 1” (c 1.0, MeOH)

3.1 3 Preparation of (S)-3-[(tert-Butoxycarbonyl)-(R)A/any/amino]-2-0x0- 1-Pyrrolidine-(S)-3-Phenyl-2-PropIonic

Acid 9

3 1 3 1 BOC-D-ALA-MET-PHE METHYLESTER7 3 1 3.1.1. Boc-Met-Phe Methyl Ester 1 Combme (tert-butoxycarbonyl)-methlomne

(14 g, 0 056mol) and phenylalamne methyl ester hydrochloride (12 g, 0 056 mol) m 75 mL of methylene chloride under nitrogen Add trlethylamme (15 3 mL, 0 11 mol) to the mixture, followed by DCC (22 6 g, 0 11 mol) Stir the mixture at room temperature overnight 2 Filter and extract the filtrate with 0.5 A4 citric acid (3 x 50 mL) and 2 N aqueous NaHCO, (3 x 50 mL), and dry the organics over Na,SO, 3 Filtration and concentration m vucuo will result in a crude product that crystallizes. This can be recrystallized from ethyl acetate-hexane to obtain about 11 9 g of crystalline Boc-Met-Phe Methyl Ester with a melting point of 8345°C (0.029 mol, 52%) 3 1 3 1.2. Methlonylphenylalanine

Methyl Ester Hydrochloride

1 Suspend the Boc-Met-Phe Methyl Ester (11.9 g, 0 029 mol) m 300 mL of ethyl acetate under nitrogen and cool to 0°C (see Note 4). 2 Bubble m HCl gas for 15 mm (see Note 6) 3. Remove from the ice bath and pass nitrogen m through the bubbler for about 20 mm, while allowing the solution to warm to room temperature. 4 Remove the bubbler and add 100 mL of hexane The desired methlonylphenylalanme methyl ester hydrochloride will precipitate out as a solid m high yield Filter and dry the product zn vucuo (see Note 7) 3 1 3 1 3 Boc-D-Ala-Met-Phe

Methyl Ester 7

1, Combme (tert-butoxycarbonyl)-o-Alanine (1.14 g, 6 mmol) and methlonylphenylalamne methyl ester hydrochloride (2 18 g, 6 mmol) m 10 mL of methylene chloride under nitrogen. Add tnethylamme (0.9 mL, 6.6 mmol) to the mixture followed by DCC (1.36 g. 6 6 mmol). Stir the mixture at room temoerature overnight.

Protected Lactam-Bridged

Dlpeptides

215

2. Filter and extract the filtrate with 0 5 A4 cttrrc acid (3 x 15 mL), and 2 N aqueous NaHCO, (3 x 15 mL) and dry the orgamcs over Na,SO, 3 Frltratron and concentratron 112vucuo will result m a crude product that crystallizes This can be recrystallized from ethyl acetate-hexane to obtain about 1 54 g of crystallme Boc-D-Ala-Met-Phe Methyl Ester with a melting point of 6566°C (3 1 mmol, 52%)

3 1.3 2. BOC-D-ALA-MET-PHE

METHYL

ESTER

METHYLSULFONIUM

IODIDE

8

1. Drssolve [(tert-Butoxycarbonyl)-D-alanyl] methronyl-phenylalamne methyl ester 7 (1 53 g, 3.1 mmol), with stnrmg, m 6 mL of methyl iodide at room temperature under nitrogen Stir for 48 h. 2. Remove the methyl iodide by concentratron zn vucuo, followed by concentratron from 10 mL of methylene chloride three trmes The product 8 will be obtamed as a syrup (1 93 g, 3.1 mmol) m 100% yield

3 1 3 3. (S)-3-[( TERT-6UTOXYCAREONYL)-(R)-ALANYLAMINOI-2-0X01 -PYRFiOLIDINE-(S)-3-PHENYL-2-PROPIONIC

ACID

9

1 Dissolve [(tert-Butoxycarbonyl)-n-alanyl] methtonyl-phenylalanme methyl ester Methylsulfonmm Iodide 8 (1 93 g, 3 1 mmol) m 3 1 mL of 1.1 DMF CH$l,, and cool the solution to 0°C under nitrogen (see Note 4). 2 Add in one portron a 50% mineral oil suspension of sodmm hydride (0 178 g, 3.72 mmol), and star at 0°C for 7 h 3. Add methyl acetate (10 mL), followed by water (2.5 mL), and allow the resulting solutton to stir overmght 4 Concentrate the solution to a small volume zn vucuo, and partition the resrdue between water (20 mL) and methylene chloride (20 mL) Separate the phases 5 Acidify the aqueous phase (pH 8) wrth 0 05 M citric actd to pH 4. Repeated extractions with methylene chloride followed by concentratton zn vacua ~111 result m the desired product 9 m about 46% yield (500 mg, 1 44 mmol) (see Note 5). IR (CH,Cl,) 3000, 1720, 1690, 1500,117O cm-r, NMR (CDCl,) 6 1 32 (d, 3, J= 6 Hz), 141 (s, 9), 1.89 (m, 1), 2 45 (m, 1), 3.03 (t, 1, J= 14 Hz), 3.25 (m, I), 338(t,l,J=9Hz),347(dd,l,J,=8Hz,J~=5Hz),428(m,l),503(dd,l,J, = 6 Hz, J2 = 4 Hz), 5 51 (m, l), 7 18-7.30 (m, 5), 7 43 (m, 1); [~]*~n = -25.7” (c 1, MeOH); TLC Rf 0 33 (system F)

3.2. Preparation of y-Lactam Dipeptides and Tripeptides via Intermolecular Alkylation The method described above for the synthesis of y-lactam drpepttdes ~111, by desrgn, result m a constrained peptrde of the type Gly-X. It IS of general interest to similarly constrain dlpeptide units not contammg glycme. A specific example is the tryptophyllysme umt of somatostatin. A method was derived for the synthesis of the y-lactam of the type Trp-Lys as outlined m Fig. 3. This approach has potential generality for a variety of y-lactam

216

Per/o w and Freidinger

0

+ G

HCI R2N

00

C02CH3

-

H

DMF Et3N

HOOC G

65’ C

10

0

11 1

NHCbr

7

DPPA,

*

DMF,

H 0

12

H

DBU,

COzCHl

N

NHCbr

Et,N w

80’ C

t-BuOH,

13

80’ C

NHCbz

1Sa Rl = CH2-3 mdoly,, R2 = COOH 1Sb Rl = COOH.RZ = CHz-s ,ndoly,

NHCbz

14

NHCbz

15

Fig. 3. Synthesis of y-lactam dlpeptides via mtermolecular

alkylatlon

drpepttdes. The synthesis IS outlmed m Fig. 3 NE-Cbz-L-lysme methyl ester hydrochlortde IS warmed with the electrophtltc cyclopropane derivative 10 (4) to produce the a-carboxylactam 12 as a mixture of dtastereomers (see Note 8). The mdolylmethyl side chain IS next introduced by alkylatton with gramme methrodtde m the presence of DBU. The result IS a separable mixture of dtastereomers 13a,13b (see Note 9). The separated acids are converted stereospecifically to the target compounds 14 and 15 by a modified Curtms rearrangement with DPPA m tert-butanol (5). The lactams are obtained m a protected form that 1s suttable for use m pepttde synthesis.

Protected Lactam-Bridged

Dlpeptides

217

3.2.7. Preparation of 6-([Benzyloxycarbonyl]Amino)-Z(S)[3(t? or S)-(~tert-butyloxycarbony~Amino)-3-(lndol-3-y~methyl)2-0x0- 1-Pyrrolidinyl)Hexanoic Acid Methyl Ester 14,15 32

1 1

6-([BENZYLOXYCARBONYL]AMlNO)-2(S)-(3-CARBOXY-~-~XO-

1 -PYRROLIDINYL)HEXANOIC

ACID

METHYL

ESTER

12

1, Dissolve NE -(Benzyloxycarbonyl)-L-lysme methyl ester hydrochloride 11 (43 3 g, 0 13 mol), trlethylamme (18.2 mL, 0 13 mol), and lsopropylldene cyclopropane- 1,l -dlcarboxylate 10 (22 2 g, 0.13 mol) m 330 mL of freshly degassed DMF and heat at 65°C under nitrogen 2 After 1 h of heating, add solid sodium bicarbonate (11 g, 0 13 mol) and continue heating 3 Continue to heat for a total of 98 h, with additional cyclopropane reagent 10 added (4 3 g, 25 mmol, and 4.8 g, 28 mmol) after 49 and 55 5 h, respectively 4 Remove the DMF 112vacua, and redissolve the brown residuem 500 mL of methylene chloride 5 Extract this solution successivelywith 1 NHCl(3 x 200 mL), water (2 x 200 mL), and saturated aqueoussodium chloride (200 mL). Isolate the orgamc layer, and dry over Na2S04. 6. Filter and concentrateto obtam a brown 011to be usedasthe crude product 12 m the next step

3 2.1 2.

6-[(BENZYLOXYCARBONyL)AMlNo]-2(S)@-CARBOXY-3-(

INDOL-3-YLMETHYL)-2-0X0-

1 -PYRROLIDINYL)HEXANOIC

ACID

METHYL

ESTER

13

1 Redissolve the crude acid 12 (63 g, 0.13 mol basedon 11) m 630 mL of degassed DMF under mtrogen, addDBU (23 mL, 0 15 mol), and grammemethlodlde(58 8 g, 0 19 mol), and heat at 80°C 2 After 45 mm, add addltlonal gramme methlodlde (58.8 g, 0 19 mol), and continue heating 3 After 2 h, add addltlonal gramme methlodlde (12 g, 0 04 mol) and continue heating. 4. Stop heating at 135 mm, and allow to cool to room temperature 5 Pour the mixture into 2 L of methylene chloride and extract the orgamcs successively with 1 N HCl (3 x 750 mL), water (2 x 750 mL), and saturated aqueous sodium chloride (750 mL) Isolate the organic layer and dry over Na2S04 6 Filter and concentrate to obtain a brown 011 7. Slllca gel column chromatography on a 40 gm portion of the crude using 2 5 kg of E. Merck Silica gel 60 (230-400 mesh), and elutmg with solvent system E will give 5 0 g of the more mobile dlastereomer 13a and 4 8 g of the less mobile dlastereomer 13b, along with 0.3 g of the mlxed fractions

Perlow and Freidmger

218 32

i

3

6-[(BENZYLOXYCARBoNyL)AMlNo]-2(S)-[3(s)([ JERJ-&JTYLOXYCARBONYL]AhnlNO)-3-(

2-0x0-1

-PYRROLIDINYL)HEXANOIC

INDOL-%YLMETHYL)ACID

METHYL

ESTER

14

1 Dissolve the acid 13a (4.5 g, 8 4 mmol) m 127 mL of dry tert-butyl alcohol, and add DPPA (2 0 mL, 9 3 mmol), followed by triethylamme (1 3 mL, 9 3 mmol) Heat at reflux under nitrogen for 48 h and then cool to room temperature 2 Concentrate the solution zn V~CUO and redissolve the residue m 150 mL of methylene chloride Wash the orgamcs successively with 0 5 Mcitnc acid (2 x 150 mL), 1 N NaHCOs (3 x 150 mL), and water (150 mL) and dry the organic layer over Na2S04 3 Filter and concentrate to obtain 4.6 g of a yellow-brown foam This foam can be chromatographed on E Merck Sihca gel 60 (23&400 mesh) (500 mL), elutmg with solvent system F to obtain 3 36 g (66%) of 14 as a white foam (see Note 10) IR (CHCls) 3400,2900,1695 cm-‘, ]H NMR (CD,OD) 6 0 69 (m, 1 H), 0.85 (m, 1 H), 12-l 6(m,4H), 143(s,9H),244(m,2H),296(m,2H),3 11 (d, 1 H,J = 14 Hz), 3 18 (d, 1 H, J= 14 Hz), 3 l-3.2 (m, 2 H), 3 64 (s, 3 H), 4.30 (t, 1 H, J =7Hz),507(s,2H),660(m,<1H),689(m,<1H),700(t,1H,J=6Hz),7 10 (t, 1 H, J= 6 Hz), 7 16 (s, 1 H), 7 36 (s, buried d, 6 H), 7.59 (d, 1 H, J= 8 Hz), [al240 = -33.20” (c 0 4, MeOH)

32

1

4

6-[(BENZYLOXYCARBoNyL)AMlNo]-2(S)-[3(R)([ JfRJ-&JTYLOXYCARBONYL]AhnlNO)-3-(

2-0x0-1

-PYRROLIDINYL)HEXANOIC

INDOL-$YLMETHYL)ACID

METHYL

ESTER

15

1 Dissolve the acid 13b (4 5 g, 8 4 mmol) m 127 mL of dry tert-butyl alcohol, and add DPPA (2 0 mL, 9.3 mmol), followed by trtethylamme (1 3 mL, 9 3 mmol) Heat at reflux under nitrogen for 48 h and then cool to room temperature 2 Concentrate the solution zn vucuo, and redissolve the residue m 150 mL of methylene chloride Wash the orgamcs successively with 0 5 Mcitrtc acid (2 x 150 mL), 1 N NaHCOs (3 x 150 mL) and water (150 mL) and dry the organic layer over Na2S04 3 Filter and concentrate to obtam 4 6 g of a yellow-brown foam This foam can be chromatographed on E Merck Srhca gel 60 (230-400 mesh) (500 mL), elutmg with solvent system F to obtain 3 36 g (66%) of 15 as a white foam (see Note 10) IR (CHCl,) 3400, 2900, 1700 cm-‘, ‘H NMR (CD30D) 6 1.30, 1 54, and 1 75 (m, 6 H), 1 39 (s, 9H), 2 39 (br t, 2 H, J= 8 Hz), 2 79 (m, 1 H), 3 05 (d, 1 H, J=14Hz),3 15(d,lH,J=14Hz),305-33(m,3H),350(s,3H),464(d ofd,1H,J,=6Hz,J~=11Hz),5.07(s,2H),703(t,1H,J=8Hz),7 13(t,lH, J= 8 Hz), 7.14 (s, 1 H), 7 36 (m, 5 H), 7 61 (d, 1 H, J= 8 Hz), [~]~~o =-1 69” (c 0 4, MeOH)

3.3. Preparation of 6- and 9Lactam Intramolecular Acyla tion

Dipeptides

via

The 6- and a-lactams are prepared via intramolecular acylatton according to the scheme shown in Figs. 4 and 5, smce the two homologs of methionine

Protected Lactam-Bridged

H N.Cbz

Dipeptides

219

H X,,COOH Hz, PdK MeOH H10 OHC-COOH

HOAc

17

16 WC

DMF

/

18

Fig 4 Synthesis of S-lactam dlpeptldes via mtramolecular

,Cbz

H,

acylatlon

PtiC N

MeOH. Hz0 OHC-COOH

BLK.

HOAe

20

19 A, /

CH3CN

EbN

21

Fig 5 Synthesis of E-lactam dlpeptldes via mtramolecular

necessary for mtramolecular

alkylatlon

acylation

are not readily avallable.

The commer-

cially available protected ormthmes and lysines are hydrogenolyzed over palladmm-carbon catalyst to remove the side chain protection followed by reductive alkylatton znsztu with glyoxylic acid. The reaction is stopped when the monoalkylatron product IS dominant. The Slactam 18 (Fig. 4) IS formed by spontaneous cychzatron of 17 m DMF at 55°C without carboxyl actlvatron Cychzation to the s-lactam 1s considerably more difficult (see Fig. 5) No cyclrzatton IS observed with a dtacld at 55°C m DMF or other solvents. The half-acid ester 20 (Fig. 5) cychzes slowly to 21 m refluxmg acetomtrtle m the presence of triethylamine The overall yield is only 25%, but no racemization IS observed

Perlow and Freidinger

220

3.3.1. Preparation of (S)-3-([tert-Butoxycarbonyl] Amino)-2-0x0- 1-Plpend/neacetlc Acid 18 3 3 1 1 Na-( JERT-BUTOXYCARBONYL)-~~~~~~~~~~~~~~~~~~~~~~~~~~~

17

1 Mix Na-(tert-butoxycarbonyl)-N”-(carbobenzyloxy)-omlthlne (11 g, 0.03 mol), glyoxyhc acid hydrate (6 7 g, 0 072 mol), and 10% Pd on carbon catalyst m methanol (50 mL), water (35 mL), and acetic acid (5 mL) (seeNote 11) 2 Place the mixture m a Parr apparatus(seeNote 12) and hydrogenate under 55 psi H, pressurefor 16 h (seeNote 13). 3 Filter the mixture through Supercel (seeNotes 15 and 16) to remove the catalyst, and wash the bed with 1: 1 methanoLwater. Concentrate the filtrate zn wcuo 4 Crystallization of the restdue from methanol-ether should yield the desiredproduct (4 66 g, 54% yield) that melts wtth decomposmonat 150°C

3.3 1.2 (S)-3-([ JERJ-BUTOXYCARBONYL] AMINO)-2-0X0-1

-PIPERDINE-ACETIC

ACID 18

1 Dissolve the diacid Na-(tert-Butoxycarbonyl)-N”-(carboxymethyl)o~lthlne 17 (4 66 g, 0 016 mol) m DMF (200 mL) and heat under nitrogen to 55°C for 2 h 2 Concentrate the solutton zn wcuo to an 011that can be crystalltzed from ethyl acetate-hexane to give the desired product 18 m 94% yield, mp 113-l 16’C IR (smear) 3300,2950, 1720, 1555, 1495, 1360, 1070 cm-‘; NMR (CDCls) 6 1 44 (s, 9), 1.75 (m, l), 1 99 (m, 2), 2 42 (m, 1) 3.363 56 (m, 2), 3 89 (d, 1,J= 15 Hz), 4 15 (m, l), 4 34 (d, 1, J= 15 Hz), 5.62 (m, l), 7.8 (br m, l), [c~]~~o-22 14” (c 0 998, MeOH)

3.3.2. Preparation of (S)-3-(jtert-Butoxycarbonyl] Ammo)-2-0x0- 1-Azepmeacetic Acid 21 3 3 2.1 Na-( JERJ-BUTOXYCARBONYL)-NE-(CARBOXYMETHYL) LYSINE METHYL ESTER 20

1 Mix Na-(tert-butoxycarbonyl)-NE-(carbobenzyloxy)-lyslne methyl ester 19 (5 9 g, 15 mmol), glyoxyhc acid hydrate (1 7 g, 18 5 mmol) and 10% Pd on carbon catalyst m methanol (40 mL) (seeNote 11) 2 Place the mixture m a Parr apparatus(seeNote 12) and hydrogenate under 55 psi H2 pressurefor 3 h (seeNote 14) 3 Filter the mixture through Supercel (seeNotes 15 and 16) to remove the catalyst, and wash the bed with 1: 1 methanol.water 4 Concentrate the filtrate zn vucuo This will give the desiredproduct 20 asa crude oil in 97% yield that can be used without purtficatton m the cychzaton below

3.3 2 2. (S)-3-([ JERJ-BUTOXYCARBONYL] AMINO)-2-0X0-1

-AZEPINEACETIC

ACID 21

1 Redissolve crude Na-(tert-Butoxycarbonyl)-NE-(carboxymethyl)lyslne methyl ester 20 (4 6 g, 14 5 mmol) m acetonitrile (1 65 L), add trtethylamme (2.7 mL, 17 9 mmol), and stir under mtrogen Heat this mixture at reflux for 3 d

Protected Lactam-Bridged

Dipeptides

221

* DMF DCC/HBT or DPPA (CHzO), TaOH CHClzCHzCl

HCI

3 2 enantlomenc

THF&O

25

mm

24

Fig 6 Synthesis of &lactam dlpeptldes via carbon insertion 2 Allow

the solution to cool, and concentrate zn vacua Partition the residue chloride (100 mL) and 0.5 M cltrlc acid (3 x 30 mL) Extract the organic layer with 1 iV aqueous NaHC03 (2 x 30 mL) and combme the basic layers 3 Acldlfy the basic extracts with 0 5 Mcltrlc acid Extract with methylene chloride (6 x 50 mL), combme the orgamcs and dry over Na,SO, 4 Filter and concentrate to obtain the desired product 21 as a white foam m 25% yield, (1 05 g, 3 7 mmol) IR (CH,Cl,) 3400,2900, 1710, 1650, 1490, 1360, 1170, 1060, 1020, 975 cm-‘, NMR (CDCl,) 6 1 44 (s, 9), 1.5-2.1 (m, 6), 3.21 (dd, l,J, -4Hz,J~=16Hz),35-3.9(m,1),421(s,2),448(td,1,J,=15Hz,J~=5H~), 6 00 (d, 1, .J = 6 Hz), 9 87 (s, l), [alz40 -3 6” (c 0 98, MeOH)

between methylene

3.4. Preparation of &Lactam Dipeptides by Condensation with Formaldehyde

via Carbon Insertion

An alternative method for formation of 6-lactam dlpeptldes using the 4-0x05-ammo- I ,3-thiazme system IS shown in Fig. 6 (6). The template for the lactam synthesis IS a protected cysteme 22 (Fig. 6) which IS prepared from commercially available Acm-Cys, according to the phthalylatlon procedure of Nefkens (see Note 17) (7). The desired dlpeptlde can be formed by standard peptlde couplmg procedures. The carbon insertion 1s accomplished by heating the dlpeptlde with paraformaldehyde and p-toluenesulfonic acid in 1,1,2-tnchloroethane with azeotroplc water removal (see Note 18). The lactam ester can then be selectively deprotected to the acid by hydrolysis using 4% HCl m refluxmg aqueous THF (8). A hmltatlon of this method IS that some racemlza-

Pet-low and Freidinger

222

tion occurs at the a-carbon of cysteme under the cyclization condltlons (see Note 22). The mtramolecular acylatlon route previously described m Subheading 3.3. tends to be limited to the synthesis of &lactams that are X-Gly denvatlves, since mtroductlon of side chains on the glycme umt would require the use of pyruvlc acid derivatives that have limited avallablllty. The use of the 4-oxo-5-amino-1,3-thiazme system as described can potentially provide a broader range of &lactam dlpeptldes from available ammo acids.

3.4.1. Preparation of 4-Oxo-5-Phthalyl-1,3-Thiazmeacetlc 3 4 1 .l

Acid 25

S-(ACETAMIDOMETHYL)-~~~~~~~~~~~~~~~~~~~~~~~~

1 Slurry S-(acetamldomethyl)-L-cysteme free base (3 84 g, 0 02 mol), anhydrous sodium carbonate (2 12 g, 0 02 mol) and N-ethoxycarbonylphthallmlde (4 5 g, 0 021 mol) m water (30 mL) Stir at room temperature for 90 mm, and then filter the remaining msolubles 2 Acidify the filtrate to pH 4 with aqueous HCI, and extract with methylene chloride containing 10% ethanol (2 x 30 mL) Readjust pH with more HCl, and extract again (2 x 30 mL) 3 Combme the orgamcs, and dry over NaTSO+ Filter and concentrate to obtain an amorphous solid 4 Purify by column chromatography usmg 500 g of silica gel beginning with solvent system C, and gradually Increasing polarity to solvent system D Concentratmg the product fractions will result in the desired product 22 (5 71 g, 90% yield), which can be crystallized from 1: 1 methanol water (mp 186188°C) 3.4 1.2. S-(ACETAMIDOMETHYL)-N~-PHTHALYL-L-CYSTEINYLGLYCINE METHYL ESTER 23 Dissolve S-(acetamldomethyl)-Na-phthalyl-1-cysteme 22 (18 g, 55 9 mmol) m degassed DMF (560 mL) under nitrogen, and cool m an Ice water bath Add DPPA (16 9 g, 61 4 mmol), followed by trlethylamme (6 2 g, 61 4 mmol) Dissolve glycme methyl ester hydrochloride (7 1 g, 56 6 mmol) m degassed DMF (240 mL) under nitrogen m a separate flask, and cool m an ice water bath Add this solution rapldly to the first solution (see Note 19) AddItIonal ahquots of trlethylamme totallmg 12 mL will be necessary to mamtam a neutral “pH ” (of DMF solution) m the first 2 h (see Note 20) The resulting mixture should be stirred, overnight while warmmg slowly to room temperature Concentrate the reaction mixture ZII vac~o and redissolve the residue m methylene chloride (250 mL) and extract with 1 N HCl(3 x 100 mL) and 1 N NaHCO, (3 x 100 mL). Dry the organic layer over Na2S04, filter, and concentrate zn vacua The residue should spontaneously crystallize. The crystals can be washed with 1.1 methylene chlonde:hexanes and dried zn vacua to give the desired product (16.9 g, 77% yield), which melts at 152-160°C Recrystalhzatlon from methylene chloride-methanol-hexane ~111 give crystallme 23, which melts at 160-161.5°C

Protected Lactam-Bridged 3 4.1.3.

METHYL

4-0X0-5-PHTHALYL-1

Dipeptides

223 24

,%THIAZINEACETATE

I

Suspend S-(acetam~domethyl)-Nu-phthalyl-~-cysteinylglyc~ne Methyl Ester 23 (16 g, 40 7 mmol), p-toluenesulfomc acid (1 5 g) and paraformaldehyde (5 g, 55.6 mmol) in 150 mL of 1,1,2-trichloroethane under nitrogen. 2 Heat to reflux using a Dean-Stark trap to separate water (see Note 21) At 2 and 4 5 h of reflux, it will be necessary to add additional paraformaldehyde (12 and 1.7 g, respectively) m order to complete the reaction Reflux for a total of 5 h 3. Cool the reaction, and concentrate w vucuo to a small volume The material can be chromatographed on 500 g silica gel and eluting with CHCl, 4 The product 24 can be crystallized from ethyl acetate to obtain a total of approximately 7 6 g (56% yield) with a melting point of 18&l 82’C The product will be a mixture of enantiomers of approximately 3.2 (see Note 22). 3 4.1.4.

4-0X0-5-PHTHALYL-1

,$THIAZINEACETIC

ACID

25

1 Dissolve under nitrogen, Methyl 4-Oxo-5-phthalyl- 1,3-thtazmeacetate 24 (4 5 g, 13.5 mmol) m 210 mL of THF, 70 mL of water, and 35 mL of concentrated hydrochloric acid. Reflux for 4.25 h. 2 Allow the reaction to cool then dilute wrth 350 mL of water and extract the product out with methylene chloride (4 x 200 mL) Dry the combined extracts over Na2S04, filter and concentrate rn vucuo 3 Crystallization from methylene chloride/THF will give multrple crops of crystalline 25 totallmg 3 65 g (85% yield) with a melting point of 225-227°C IR (NuJo~) 1780, 1750, 1725, 1625, 1200, 1175, 1110 cm-‘, NMR (Me,SO-d,) S 3.16 (ddd, 1 J, = 14 Hz, J2 = 6 Hz, J3 = 2.5 Hz), 3 57 (t, 1, J = 12 Hz), 3 99 and 4.2 1 (AB system, 2, J= 18 Hz), 4 46 and 4 76 (AB system, 2, J= 13 Hz, S at 4 46 split mtodd, J1=2.5Hz),4.92(dd, 1 J, = 12Hz,J2=6Hz),7.90(s,4), [a]24,=-020 (c 1 0, DMF) 4. Notes 1. The source of this ester cleavage was briefly investigated It appears likely that the saponification m the lactam forming cycbzatron IS due to NazO contammatmg the NaH. This IS supported by the fact that cychzation with LDA produces ester rather than acid 2 Strongly basic protic conditions during work-up cause extensive epimerizatton and should be avoided 3 Dimethylformamide is best stored over 4 angstrom sieves and then degassed by applying a 25 mm vacuum with stnrmg m a round bottom flask until bubbling ceases This will remove oxygen and the dimethylamme that forms on standing. 4. A salt water ice bath IS used to obtain 0°C. 5. Repeated extractions refers to as many extractions as are necessary to completely remove the product from the aqueous phase. This is monitored by spotting the organic extracts on a TLC plate and exposing the plate to a developmg agent 6. During this time, the starting material will go into solutton; subsequently, the product will begin precipitatmg out as the HCl salt

Perlo w and Freidinger 7 Filter quickly to recover the product, since the HCl salt will be very hygroscoptc 8 This reactton presumably occurs by initial attack of the lysine ammo group at a cyclopropane methylene to open the three-membered rmg This mtermediate then cychzes on one of the lactone carbonyls to expel acetone. 9 The more mobtle diastereomer was assigned the configuratton correspondmg to L-tryptophan, based on the analogy of the chemical shifts of the y-methylene protons of the lysme side chains of 13a and 13b m methanol with the correspondmg shifts of L-Trp-Lys and D-Trp-Lys m water. Previous NMR studies have shown conststently more pronounced upfield shifts of side chain ahphatic protons for D,L than L,L compounds (9). 10 A byproduct of the reaction (12%) IS the symmetrical urea that presumably arises from the reaction of trace water with the mtermediate isocyanate 11 It 1s a good safety practice to first wet the 10% Pd on carbon catalyst with some water before adding to the reaction This should be done only when water 1s already a component m the desned reactton. Palladmm catalysts can easily catch tire when opened to the atmosphere and should be handled under nitrogen or argon where possible. 12 A Parr apparatus is necessary to safely hydrogenate under pressure 13, At the indicated time, the monoalkylation product should be predominant by thm layer chromatography using solvent system A 14 At the indicated time, the monoalkylatton product should be predominant by thm layer chromatography, using solvent system B 15 A 3 cm deep bed of dry Supercel IS placed m a fritted funnel and wetted with water prior to the start of the filtration The Supercel is for the removal of the fines associated with the catalyst Never let the bed go dry, since pullmg oxygen through the catalyst will ignite tt An Inverted funnel with nitrogen blowing slowly through it will blanket the outside of the funnel for the safest filtration method 16 Palladium catalysts can easily catch fire when opened to the atmosphere and should be handled under mtrogen or argon where possible. It is imperattve to flush the reactton solution with mtrogen before adding dry catalyst to any flammable solution In additton, an Inverted funnel with a nitrogen lme blowmg slowly through it will blanket the outside of the flask, so that the dry catalyst can be safely added to a flammable solutton 17 The phthaltmtde protecting group is necessary to preclude cyclizatton to a thtoprolme under the conditions for lactam formation 18 The formaldehyde functions as an electrophile, asststmg m the removal of the Acm group as well as furnishing the methylene group to complete the cychzatton 19. This addition should be a simple dump, not a cannula transfer. 20 The term pH only refers to aqueous solutions The determmatton of neutrality m a DMF solution can be performed by spotting the DMF solutton on water-wetted E Merck pH sticks A reading of “pH 8” on these sticks approximates neutrality 21 Initially, the collectmg water can be tapped off of the Dean-Stark trap When no further water appears to be separating, the addition of molecular sieves (4 angstrom) to the base of the trap ~111 help remove the last of the water.

Protected Lactam-Bridged 22

Dipeptides

225

The extent of racemtzation can be examined with an NMR study of the relative heights of the methyl ester peaks using Kiralshift E7 reagent.

References 1, Freidmger, R. M., Perlow, D. S., and Veber, D F. (1982) Protected lactam-bridged drpeptldes for use as conformational constraints m pepttdes J. Org Chem 47, 104-109 2 Freidmger, R M. (1985) Synthesis of y-lactam-constrained tryptophyllysme derlvattves J Org Chem 50, 3631-3633. 3 Simtlar conditions have been used to prepare p-lactams from P-chloro amides Cf.: Baldwin, J E., Au, A, Christre, M , Haber, S B , and Hesson, D (1975) Stereospecific conversion of peptides mto p-lactams J Am Chem Sot 97, 5957-5958 4 Damshefsky, S and Smgh, R K. (1975) A highly activated cyclopropane for homoconJugate reactions J Am Chem Sot. 97,3239-3241, 5. Nmomtya, K , Shtori, T , and Yamada, S (1974) Phosphorus m organic synthesis IX On the mechanism of estertfication of malomc acid half esters by diphenyl phosphorazidate Chem Pharm Bull 22, 1795-1799 6 Kametam, T , Kigasawa, K , Htiragt, M , Wagatsuma, N , Kohagizawa, T , and Inoue, H (1978) Studies on the syntheses of heterocychc compounds 749 A simple synthesis of 4-thiazolidmones, tetrahydro- 1,3-thiazin-4-one and hexahydro- 1,3-thiazepm-4-ones from amide thiols Heterocycles 9, 83 l-840 7 Nefkms, G. H. L , Tesser, G I , and Nivard, R. J. F (1960) Simple preparation of phthaloylammo acids via a mild phthaloylation Red Trav Chum Pays-Bas 79, 688698. 8 Sheehan, J C., Goodman, M , and Hess, G P (1956) Peptide derivatives contammg hydroxyammo acids J Am Chem Sot 78, 1367 9. Arison, B. H , Hirschmann, R , and Veber, D F (1978) Inferences about the conformation of somatostatm at a biologic receptor based on NMR studies. Broorg Chem 7,447.

Synthesis of Peptidomimics Through Sugar-Based Scaffolds Yves Le Merrer, Lydie Poitout, and Jean-Claude

Depezay

1. Introduction Poor oral btoavarlabthty, low metabolic stability towards proteolysts and rapid excretion via both liver and kidneys displayed by innumerable peptides of potential therapeutic value has generated an Intensive search for pepttdomtmtcs (I-2). A possible approach of such nonpeptrdal pepttdomtmtcs 1sto replace the peptrde by a scaffold that drstrlbutes m the space the pepttdal side chains of ammo acids essential for biological activity and mlmlcs the broactlve conformation of the peptlde. To our knowledge, bicycloE2.2 2]octane (3) was the first scaffold successfully employed as peptldomtmlcs of endocrine receptors More recently, a steroid nucleus, with a cyclopentanoperhydrophenanthrene skeleton, has been used as a peptidomlmrc that binds to the fibrmogen receptor (4). An Ideal scaffold with multiple possibilities to mimic various peptldes must satisfy at less three crrterra. First, from a practical point of view, these skeletons should be easily available to make them versatile chiral building blocks. Second, these skeletons should be highly functronahzed to introduce various side chains of ammo acids. Thud, the conformatton, configuratton, flexibility, or rigidity could be modified to put the side chains m a particular spatial order to make contact with the receptor protein. Thus, the use of sugars, such as o-glucose (57), o-allose (7) and o-xylose (8), or 1,4-benzodiazepin-2-one as scaffolds (9) 1san area that has previously been exammed. Polyhydroxylated ammoheterocycles, azasugars with a pyrrohdine, prperrdme, or azepane framework, offer an additional posstbthty to mtroduce a side chain to the nitrogen. The focus of this chapter will be on the preparation of polyhydroxy-pyrrohdmes, pipertdmes, and azepanesfrom o-manmtol (20) and From

Methods m Molecular Med/nne, Vol 23 Pepf/dom/met/cs Protocols Edlted by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

227

228

Le Merrer,

Somatostatin-14:

Poitout,

and Depezay

Ala’-GlyZ-Cys3-Lys4-Asns-Phe6-Phe7-T~*-Lys9-~’o-Phe11-Thf2-Ser’3-Cys14 I

I

H-D-Phe’-CysZ-Phe3-D-Trp4-Lyss-Thr6-Cys7-Trp*-OH I

Sandostatu?

Fig 1 Somatostatm, Sandostatm@, and nonpeptlde mimics

the functtonalization to obtain nonpeptide mtmrcs of somatostatm and Sandostatm@(11,12). Somatostatm-14 (SRIF-14) 1sa cyclic tetradecapepttde (Fig. 1) that serves not only to regulate the release of growth hormone or other pttuttary hormones, but also plays a role m neuronal transmtsston (13) During the last few years, five SRIF receptor subtypes have been identified (14) Owing to the rapid proteolyttc degradation of somatostatm, several cyclic-pepttdes (25) and nonpeptide mtmtcs (5,6,8,9) of SRIF- 14 have been prepared. One of them, Sandostatm, which has a longer half-life, a greater potency, and a higher selectivity of action than native SRIF-14, is used m the treatment of acromegaly, carclnomas, and gastrenteropancreattc tumors (16). Extensive mvesttgations of the structure-acttvtty relattonships of the SRIF-14 pepttde showed that the tetrapepttde sequence Phe7-n-Trp’-Lys9-Thrio 1sthe most relevant for btologtcal activity (17). NMR studies of the synthetic somatostatm analog c[Pro-Phe-nTrp-Lys-Thr-Phe]

showed a type II’+

turn at Trp*-Lys9

and a type IV-P turn at

Phe”-Pro6 (15). Based on these stuctural models, on one hand, and on extensive structurebased molecular modeling mvestigations, on the other hand, compounds I-VI (Fig. 2), were designed as nonpeptide mimics of somatostatm. Compounds IIV are prepared by aminoheterocychzation with tryptamine of D-mannitol basepoxtde and subsequent functtonahzatton to mtroduce the side chain of the lysme, whereas compounds V and VI are prepared by ammo-heterocychzatton with benzylamlne

of L-iditol

bzs-epoxlde and subsequent functlonahzatlon

introduce the side chains of the lysine and tryptamme

to

Sugar- Based Scaffolds

229

Fig 2 Nonpeptide mimics of somatostatm

F”

r

L?&

1

Bn0~

Fig 3 Synthesis of L-gulo or o-gluco-pipendine

r,

g Bn

AOH

HO,,,,

and D-manno or L-ldo-azepane

I. 1, Azasugars: Polyhydroxy- Pyrrolidines, Piperidines, and Azepanes 1 1.1. Synthesis of Enantiomencally Pure Azasugars from o-Mannitol

obtained by the opening of homochlral C+ymmetric bls-epoxidesissued from D-mannitol by a primary amme (Fig. 3). This approach, which involves a reglospeciflc openmg of Enantlomerlcally

pure azasugars

can be

230

Le Merrer, Poitout, and Depezay

one epoxy function followed by a spontaneous ammocyclrzatlon, leads to the polyhydroxyplperidme with mverslon of conflguratlon at CZ, and the polyhydroxyazepane. Thus, ammocychzation of 1,2:5,6-dianhydro-3,4-0-benzyl+manmtol leads to a mixture of L-gulo-piperidme and o-manno-azepane that could be easily separated by column chromatography. The two isomers ratlo depends on the experimental condltlons. Under aprotlc solvent (chloroform reflux), the pipendme IS the mam product, whereas m aqueous solution m presence of perchlorlc acid, the azepane IS the major product. Under the same experimental condltlons, the 1,2:5,6-dlanhydro-3,4-O-benzyl-L-lditol gives analogous results. This ammocychzatlon can be performed with various primary amines, such as benzylamme or tryptamme (see Note 1). For example, m the parttcular case of benzylamme, the yield of pure L-gulopipendme, o-manno-azepane, or D-gluco-piperidine, L-zdo-azepane IS about 50%, 45%, 45%, and 33% m aprotlc solvent, respectively and 28%, 67%, 9%, and 66% m aqueous solution m presence of perchlorlc acid, respectively from o-manmtol-bzs-epoxide or L-ldltol-bzs-epoxlde. 1.1.2. lsomerizatron and Functionalization

of Azasugars

Having m hand the L-gulo- and D-gluco-piperidmes, and the ~mannoand L-ido-azepanes, we then functionalized heterocycles with side chains of ammoacids This can be performed by activation of the hydroxyl groups and nucleophlllc substitution (Fig. 4). This S, can proceed either by a direct nucleophlhc substitution with inversion of configuration, or by neighboring nitrogen particlpatlon to lead, via an azlrldmmm Intermediate followed by nucleophihc opening, either to the SN product with overall retention of configuration, or to the product of rmg size modlficatlon (IO) From the Cz-symmetric azepane, the nucleophlllc substitution can be controlled to choose the desired evolution From the o-manno-azepane (Fig. 5), reaction with 1 4 eq of mesyl chloride in tnethylamme at 0°C affords directly the chloromethyl-L-gulo-plpendme m about a 60% yield This result shows that m these experimental conditions, the 3-O-mesylazepane IS unstable and proceeds via an azlndmmm chloride to give a more stable rearranged product, after nucleophlhc opening by the chloride counter-ion In this

case,two products(chloroazepaneand chloromethyl-plpendme)could be nntlally formed, and the 3-chloroazepane was ikther converted, via the azmdmmm ion, to the more stable rearranged product (thermodynamic control) On the other hand, reaction with 1 2 eq of tnphenyl-phosphme-dlethyl azodlcarboxylate-hydrazolc acid (18) (see also Note 2) under Mltsunobu condltlons (19) yields, after chromatographlc punficatlon, a mixture of monoazzdo-azepane and azldomethyl-plpendme (55%, or 73% correctedyield after recovery of the starting azepane).As above, this result

Sugar- Based Scaffolds

237

S$il wth ~nvers,on of conflguratlon l

l

Rmg contraction

ZO = leavmg group

Ftg 4 Prmclple of functionahzatton

En0 HO J-7

of azasugars

p ..,\OH l-

(60%)

A D-munno-uepane

c ( ,“:z, Fig. 5. Functronalization

c( ;::, of the n-manno-azepane

shows that the reaction undergoes vta an azuidmmm intermediate the nucleophthc opening that gives rtse to the formation of the two tsomers (kmetic control). Hydrogenolysts of this mixture m presence of palladium on carbon affords the corresponding amines that can be separated by chromatography The 3-ammo-3-deoxy-3o-manno-azepane and the ammomethyl-L-gulo-ptperidine are isolated m 39 and 32% overall yields, respectively, from the starting o-manno-azepane Consequently, from the u-manno-azepane, chain extension can be performed either by nucleophthc substmmon of the halogen on the chloromethyl+guZo-ptpertdme, or from the ammo group of the 3-ammo-3-deoxy-D-manno-azepane. From the L-zdo-azepane (Fig. 6), similar treatment with mesyl chloride yields the chloromethyl-o-gluco-ptpertdme m about a 60% yield. On the other hand, Mttsunobu reaction [Ph,P-DEAD] affords only the chiral bridged morpholine (94%) which results of an mtramolecular displacement of the alkoxy-phosphomum mtermedtate by the other free hydroxyl group of the azepane. If the mtrogen atom of the azepane IS

232

Le Merrer, Poitout, and Depezay

a

(56%) En0

OBn -k 0

-x/

Bn

c

P=Boc

P=Boc

f

P=H

Fig 6 Functtonalization

f

c

P=H

of the L-zdo-azepane

deactivated by a electron-withdrawmg protecting group (I e., a Boc group) by selective hydrogenolysis of the N-benzyl bond m presence of di-tert-butyldicarbonate, the di-Omesyl azepane can be isolated (9.5%) Substitution ofthe dimesylate by a primary amme affords the diaz-abicyclo[2 2 21 and [3 2 l] structures m 60% and 15% yield, respectively Formation of these bicychc compounds results of the non regioselective opening of the azuidmmm intermediate and subsequent mtramolecular displacement of the second mesylate As a result, from the L-zdo-azepane, chain extension can be performed either by nucleophihc substitution of the halogen on the chloromethyl-o-gluco-pipendme or from the ammo group of the diazabicyclo structures

From the plperidmne,side chain extension can be controlled and performed either at C6 on the primary alcohol function or at C2on the secondaryalcohol fimctlon: Functionahzation at C, (Fig. 7) From the L-gulo-pipendme, this requires the transformation, a primary alcohol mto a mesylate, followed by nucleophihc displacement with a pnmary amme For this transformation, better yields are obtained through silylation of the two hydroxyl groups, followed by selective hydrolysis of the primary silyl ether and subsequent mesylation. In this manner, the ammomethyl-L-g&-piper+ dme can be obtained m 66% overall yield from the startmg L-gulo-pipendme

Sugar-Based Scaffolds

233

E~O~OTBDMS

R’NH b

6

P’=P’=TBDMS C

P’=H,

PZ=TBDMS

Cc

P’=Ms.

P*=‘IBDMS

Fig 7 Functlonahzatlon

\

at C, of the L-gulo-plpendme

* y(&-; HO

Fig 8 Functlonahzatlon

A

2

at C, of the L-gulo-plpendme

Functlonahzatlon at C2 (Fig. 8). Selective sllylatlon of the pnmary hydroxyl group, and mesylatlon of the secondary hydroxyl group with subsequent substitution a pnmary amme affords the L-zdo-pyrrolidme skeleton (66% overall yield), via an azmdmmm salt and regloselectlve openingatthelesssubstituted side Furthermore,the secondary alcohol function can be transformed into an azldo group wtth mverslon of configuration under Mltsunobu condltlons [Ph,P-DEAD] in the presence of hydrazolc acid (61% yield) Catalytic reduction of the azido group yields the amine from which alkylatlon can be carned out

2. Materials Prior to use, THF and Et,0 were distilled from sodium-benzophenone and CH,C& from P,O,. CH,Cl, and ethyl acetatewere filtered through K2C03 prior to use. Flash column and thm layer chromatography (TLC) require Kleselgel 60 (2OcrSOOhm) or 60H (5-40 hm) and 60FZS4(Merck), respectively For momtormg reactions and checking purity of products, the TLC plates should be viewed under shortwave UV irradlatlon. ‘H and 13CNMR spectr a were recorded with a Bruker 250AM m CDCl, (unless otherwlse indicated). Chemical shifts are reported m 6 (ppm), and coupling constants are given m Hertz, (see Note 3).

Le Merrer, Poitout, and Depezay

234

Specific rotations were measured on a Perktn Elmer 241 C polartmeter sodium (589 nm) lamp at 20°C.

2.1. Reagents for Method 3. I 2 3 4 5 6 7 8 9 10 11 12 13

1,2-5,6-dt-0-isopropylidene-D-manmtol Imldazole Sodmm hydride (NaH) Benzyl bromtde Tetrabutylammomum todtde Acetrc acid Trtphenylphosphme Duspropyl-azodtcarboxylate (DIAD). tert-Butyldrmethylsllyl chlorrde (TBDMSCI) Trtethylamme Methanesulphonyl chloride (MsCl) HCl KOH

2.2. Reagents for Method 3.2 1 Perchlortc acid (HC104) 2 Benzylamine 3 Tryptamme

2.3. Reagents for Method 3.3 1 2 3. 4 5 6. 7 8 9 10

NaOH Tetra-n-butylammon~um hydrogen sulphate. Dt-tert-butyl dtcarbonate (Boc,O) Imtdazole Trtetylamme tert-Butyldrmethylstlyl chloride (TBDMSCI) Methanesulphonyl chloride (MsCl) 1,6-Hexanedtamme 2-(tert-butoxycarbonyloxylmlno)-2-phenylacetonltrlle HCl

2.4. Reagents for Method 3.4 1 2 3 4 5 6 7

Trlphenylphosphme Dtethylazodtcarboxylate Sodmm aztde HISO

(DEAD).

H2

10% Palladmm on carbon N-ethyldusopropylamme

(BOC-ON)

wrth

235

Sugar-Based Scaffolds 8 9 10 11

6-Ammo- 1-hexanol Carbon tetrabromrde Dt-tert-butyl dicarbonate (Boc,O) HCl

2.5. Reagents for Method 3.5 1 2 3 4 5 6 7 8

NaOH Tetra-n-butylammonmm hydrogen sulphate Di-tert-butyl dtcarbonate (Boc,O) Trtetylamme Methanesulphonyl chlorrde (MsCl) 1,6-Hexanedtamme 2-(tert-butoxycarbonyloxylmlno)-2-phenylacetonitr~le HCl

(BOC-ON)

2.6. Reagents for Method 3.6 1 2 3 4 5 6 7 8

Trrphenylphosphme Dtethylazodrcarboxylate Sodrum azrde. H2S04

(DEAD)

H2

10% Pd/C. N-ethyldnsopropylamme HCl

2.7. Reagents for Method 3.7 H2

20% Palladium hydroxide on carbon. Dr-tert-butyl drcarbonate (Boc20). Trrethylamine Methanesulphonyl chlortde (MsCl). Tryptamme. HCl 8 N-ethyldnsopropylamme 9 6-Ammo- 1-hexanol. IO Carbon tetrabromide

3. Methods 3.1. Preparation of 1,2:5,BDianhydro3,4-Di-0-BenzybMannitol4 and L-iditol 7 The preparation

of the flexible

optically

pure C2-symmetric

bzs-epoxides 4

and 7, on multigram scales, from the commercially available 1,2:5,6-di-Oisopropylidene-o-manmtol 1, via the previously described 3,4-di-O-benzyl-o-

Le Merrer, Poitout, and Depezay

236

Ftg. 9 Preparatton of o-mannitol bzs-expoxlde 4 and L-ldltol

bzs-expoxrde7

manmtol3 (20) 1sshown m Fig. 9. Using Mrtsunobu condmons (19), by reaction with trtphenylphosphme and dnsopropyl azodrcarboxylate, the tetro13 was directly transformed m high yield (86%) to the 1,2.5,6-dranhydro-3,4-dt-0benzyl-n-manmtol4, with retention of configuratton at C2 and C5 On the other hand, the tetro13 afforded the bls-epoxrde 7 with mversron of contiguratron at C2 and C5 m 59% overall yield by the followmg reactlons. selective sllylatlon at the primary 1,6-hydroxyl functions, mesylatlon at the secondary 2,5hydroxyl functions, and acidic removal of sllyl groups, followed by a base promoted mtramolecular SN2.

3.1. I. 3,4-Di-O-Benzyl- 1,2:5,6-Di-0-Methylethylidene-o-Mannitol2 To NaH (5 83 g, 243 mmol) suspended m anhydrous tetrahydrofuran (90 mL), add the commercially available 1,2*5,6-di-0-tsopropylidene-o-manmtol (25 g, 96 1 mmol) at 0°C and two crystals of tmtdazole Stir at 20°C for 2 h, add tetrabutylammomum todtde (1 75 g, 4 75 mmol), then add dropwlse benzyl bromide (27 5 mL, 230 mmol)

Stir for an addltlonal

15 h at 2O”C, and then cool the mixture

Add methanol

(250 mL) and concentrate to dryness

Add dlchloromethane to the residue, filter to dzscard the salts, and wash the filtrate with brme. Dry the orgamc layer over anhydrous MgSO,, filter, and concentrate to dryness You should get about 42 5 g (quantztatzve yield) of 2 ‘H NMR 7 25(m, lOH, Ph), 4.70(s, 4H, CH,Ph); 4 21(m, 2H, HZ), 3 98(dd, 2H, H,), 3 84(dd, 2H, H,,); 3.77(d, 2H, H,), 1.40, 1 33(2s, 12H, CMe2), J2, 3 = 5.5, J,,* = 6, & = 7, J,,,n= 8 Hz.

3.7.2. 3,#-D/-O-BenzybMannitol3 1 Stn 2 (42 47 g, 96 mmol) for 2 5 h at 40°C m a 30/70 (v/v) aqueous solution of acettc actd (600 mL). 2 Concentrate to dryness, and purtfy the restdue by chromatography on srltca gel usmg a 75125 mtxture (v/v) of cyclohexanelethyl acetate to pure ethyl acetate

Sugar-Based Scaffolds

237

Concentrate the appropriate fracttons, you should get about 30.2 g (87%) of3. ‘H NMR 7.32(m, lOH, Ph), 4 6O(s, 4H, CH,Ph); 3 94(m, 2H, HZ), 3 8O(s, 2H, H,); 3 77(d, 2H, H,), 3 72(d, 2H, H,); .I,,1 = 3, J,,,, = 3.5, .I,*,$ = 11 5 Hz

3.1.3. 1,2:5,6-D~anhydro-3,4-D~-O-5enzyl-o-Mannitol4 1 To 3 (12 g, 33 mmol) m toluene (150 mL) dried twrce by evaporatron of toluene 112vucuo and dtssolved m 150 mL of toluene, add trlphenylphosphme (20 2 g, 77 mmol) and concentrate agam zn V~CUOto avoid any trace of water (see Note 4) until it remains a volume of ca 60 mL 2 Cool to 0°C and add dropwlse dirsopropylazodtcarboxylate (DIAD, 15 4 mL, 78 mmol) After 30 mm, concentrate to a paste zn vucuo (P = 0 01 mm Hg) and heat to 130°C for 2 5 h 3 Purify the residue by chromatography on sthca gel usmg a 4/l mixture (v/v) of cyclohexane/ethyl acetate. Concentrate the appropriate fractions (Rr 0.35). You should get about 9 2 g (86%) of 4. [a] 2oD= +5” (c 1 0, CH,Cl,), ‘H NMR. 7.26(s, lOH, Ph), 4.66, 4 55(AB, 4H, CH,Ph); 3.38(m, 2H, H,); 3 ll(m, 2H, H,); 2 73(dd, 2H, H,), 2 61(dd, 2H,H,s); J,,2 = 4, J,‘,2 = 2.5, J1,,s = 5.5, JA,B = 12Hz, 13C NMR. 138 0, 130.8, 128.7, 127 9, 127 8(Ph), 78.5(C3), 73 S(CH,Ph), 50.6(C2), 46 O(Cl).

3.1 4. 3,4-Di-O-Benzyl-l,6-Di-O-Tertbutyldimethylsilyl-o-Mannitol5 To 3 (12 g, 33 mmol) m DMF (32 mL) cooled to 0°C add lmtdazole (9 g, 132 mmol) and tert-butyldtmethylstlyl chloride (10 2 g, 69 3 mmol). Stn for 2 h Add a saturated aqueous solution of NH,Cl (60 mL), and extract using three 60 mL portlons of dlchloromethane Wash the combined organic layers, and dry over anhydrous MgSO,, filter, and then concentrate to dryness. Purify the residue by chromatography on silica gel using a 85/15 mixture (v/v) of cyclohexane/ethyl acetate. Concentrate the appropriate fractions (Rf 0 30) You should get about 15 6 g (80%) of 5. [cx]~~~ = +30” (c 1 0, CH,CI,), ‘H NMR* 7 35(s, IOH, Ph), 4 72,4 60(AB, 4H, CH,Ph); 3 9-3 8(m,4H, H,,,), 3.76(dd, 2H, H,); 3,62(dd, 2H, H,,), 0.90(s, 18H, tBuSi); 0.07(s, 12H, Me,St); J,,2 = 3, J,t,2 = 5, Jl,,* = 10, JA,B = 12Hz

3.1.5. 3,4-Di-0-benzyl-2,5-Di-O-Mesyl0-Tertbutyld~methylsilyl-o-Mannitol6

1,6-Di-

1. To 5 (14 45 g, 24 4 mmol) in dtchloromethane (30 mL) cooled to 0°C add trrethylamme (13 8 mL, 100 mmol) and methanesulphonyl chloride (5 7 mL, 73 5 mmol) Stir for 15 mm 2 Add water (30 mL) and dmhloromethane (40 mL), decant, wash the organic layer with brme, dry over anhydrous MgS04. Filter and concentrate the filtrate. You should get about 18.3 g (99O/) of 6 This material should be pure by TLC and can be used below without further purification [a1200 = +20” (c 1 3, CH,CI,), ‘H NMR 7.30(m, lOH, Ph), 4.80(m, 2H, H,); 4.76,4 66(AB, 4H, CH,Ph); 4,12(dd,

Le Merrer, Poitout, and Depezay 0 BnO OBn 1

4

0

0 BnO OBn 0 I

f

Ftg 10 Synthesis of L-gulo or D-gluco-piperidme

and D-manno or L-zdo-azepane

2H, H,), 3 99(s, 2H, Hs); 3.24(dd, 2H, H,,); 0 9O(s, 18H, tBuSt), 0 07(s, 12H, Me,Si), J,,* = 3 5, J,,,, = 5.5, J,,,, = JA,B = 12Hz

3.1.6. 1,2:5,6-Dlanhydro-3,4-Di-0-Benzyl-L-lditol

7

1 To a cooled solutton at 0°C of 6 (18.2 g, 24 2 mmol) m methanol (41 mL), add dropwtse concentrated HCl(5.2 mL, 58 8 mmol), stir the mixture at 20°C for 2 h, then cool to 0°C and add dropwtse 42 mL of a 20% aqueous solutton of KOH (147 mmol) Make sure that the pH of the reaction mixture remams basic after 1 h. Otherwtse, add more KOH 2 After 3 h at 20°C concentrate the mixture to dryness and add water (25 mL) and dlchloromethane (50 mL) Decant and extract the aqueous layer using three 60 mL porttons of dtchloromethane 3 Wash the combined organic layers with a saturated aqueous solutron of NH&l, and dry over anhydrous MgSO, Filter and concentrate to dryness 4 Purify the residue by chromatography on slhca gel using a 713 mixture (v/v) of cyclohexane/ethyl acetate Concentrate the appropriate fractions (R, 0 30) You should get about 6 19 g (78%) of 7 Mp 28”C, [a]*Oo ~43” (c 1 1, CH2C12), ‘H NMR: 7 3O(s, lOH, Ph); 4 85,4 61(AB, 4H, CH2Ph), 3 29(m, 2H, H3), 3 2 l(m, 2H, H,); 2 74(dd, 2H, H,); 2 53(dd, 2H, H,!), J,,2 = 4.5, J,‘,* = 2 5, J,,,, = 5, JA,B =

12Hz, 13C NMR 52 4(C&

137 9, 128.3, 127.9, 127.7(Ph),

80 7(C3), 72 3(CH,Ph);

43.1(C,)

3.2. Aminocyclization

of bis-Epoxides

The aminocyclization of his-epoxides 4 or 7 (Fig. 10) can be carried out with primary amtne in aprotic or m protic solvent. Under refluxtng chloroform, the azepane and the piperidine are isolated tn about 45% and 50% yield, respectively, after chromatographic separation. In contrast, m an aqueous solution tn presence of perchlorlc acid, fast reactton occurs at 25°C and affords

Sugar-Based Scaffolds

239

the azepane (about 65% yield); the piperidme can be also isolated but m a lower yield (about 25%). Under the same experimental condtttons, the dtastereomertc his-epoxides 4 or 7 give analogous results. In the field of the synthesis of somatostatin pepttdomtmtcs, the ammocychzatton as been performed with benzylamme and tryptamine, and the mam physical data are reported below (Subheading 3.2.2.). mainly

3.2 1 General Procedure for Aminocyclization in Aprotic Solvent 1 Reflux a solutton of 4 or 7 and prtmary amme (4 eq) m chloroform (5 mL for 1 mmol of bu-epoxlde) during 24 h (or 48 h, determined by TLC analysts of the reaction mtxture) 2. Cool the mixture and concentrate. 3. Purify the residue by chromatography on stltca gel, and concentrate the appropriate fracttons You should get the correspondmg pipertdme (50%), and azepane (45%)

3.2.2 Genera/ Procedure for Aminocyclization in Protic So/vent 1. To 4 or 7 m water (6.5 mL for 1 mm01 of Bu-epoxtde) add prtmary amme (IO eq) and perchlortc actd m a 70% aqueous solution (5 eq). 2. After stmmg for 24 h at 20°C, add dtchloromethane and decant Extract the aqueous layer using four portions of dtchloromethane 3. Wash the combined orgamc layers wtth brine, dry the combined orgamc layers over anhydrous Na2S04, and concentrate 4 Purify the residue by chromatography on sihca gel, and concentrate the approprtate fractions. You should get the correspondmg azepane (about 65%), and the ptperrdme Isomer N-Benzyl-3,4-d~-O-benzyl-l,5-d~deoxy-l,5-~m~no-~-gul~tol8* RfO 35, CH,Cl,/ acetone 9/l(v/v), mp 75°C [a] 2oD= -9” (c 1 0, CHCl& ‘H NMR 7 30(m, 15H, Ph), 4 7&4 57(m, 4H, OCH,Ph), 4.06(dd, lH, H4), 4 02(m, lH, H2), 3.92(d, 2H, NCH,Ph), 3.84(dd, lH, H6), 3.72(dd, lH, Hs); 3 55(dd, lH, He’), 3 ll(m, lH, H,), 2,76(m,2H, HI,,*), J4,5 = 5, & = 8, J5,6 = 6, J5,6’ = 7, J6,& = 11, J2,3 = 3, 138 9, 138.1, 137 9, 128.9, 128.5, 127.9, 127.7, Jiwwh = 2, 13C NMR 127 2(Ph), 78 1, 75 4(C,,,), 73 3, 72.5(OCH,Ph), 67 9(C,), 59.8(C5), 59.3(C,), 57 9(NCH,Ph), 49.3(C) N-Benzyl-3,4-d~-O-benzyl-l,6-d~deoxy-l,b~m~no-~-mann~tot 9’ R, 0.30, CH2C12/acetone 9/l(v/v), [c~]~~u = -6” (c 1.0, CHC13); ‘H NMR 7.30(m, 15H, Ph), 4 75,4 60(AB, 4H, OCH,Ph), 4 08(m, 2H, H2), 3.84(s, 2H, H3), 3 68(s, 2H, NCH,Ph), 2 83(dd, 2H, H,), 2.7l(dd, 2H, H,,), JA,B = 12, J,,, = 4,J,,,, = 6.5, J,,,, = 13, 13C NMR 138.3, 128 7, 128.3, 127.8, 127 6, 127 2(Ph), 809(C3), 73 1 (OCHzPh), 68.6(&), 63 4(NCHzPh), 57.O(Ct). 3,4-D1-O-benzyl-l,5-d~deoxy-I,5-[2’-(IH-~ndol-3-yi-ethyl]-rm~no-~-gul~tol

10 Rf 0 35, CH$l,/acetone 7/3(v/v); [cx]~~~ = -9” (c 1.0, CHCl,); ‘H NMR: 7.94(s, lH, NH); 7 57(d, IH, H ,ndole); 7.2-6.96(m, 14H, Ar); 4.71-4.52(m, 4H, OCH,Ph); 4.02(m, lH, H2), 3 96(dd, lH, H4); 3.80(dd, 2H, H6); 3 64(dd, lH,

Le Merrer, Poitout, and Depezay

240

H3), 3.60(dd, lH, H,,); 3 23-2 80(m, 7H, H, IO, ,’ 5a ,,), J2,3 = 3 5, J3,4 = 8, J4,5 = 5, J5,6= 6, J5,6’ = 7,J6,@ = 11Hz; 13CNMR. 138 0, 137 9, 136 2, 128 4, 128 0, 127 9, 127 7, 127 3, 121 7, 119 1, 118.7, 113 7, 111 l(Ar); 77 8, 75 7(C,,,), 73 2, 72 4(0CH,Ph), 67.6(C,), 59 9(C,), 58,O(C,), 55 2(C,); 50.2(C,), 23 6(C,) 3,4-D~-O-benzyl-I,6-d~deoxy-1,6-[2’-(IH-~ndol-3-yl-ethyl]-~m~no-o-mann~tol 11 R, 0 30, CH,Cl,/acetone 7/3(v/v), [a120D = 4” (c 0 9, CHCl,), ‘H NMR 8 06(s, IH, NH), 7 54(d, lH, Hlndole); 7 3-6.96(m, 14H, Ar), 4 72-4 60(m, 4H, OCH,Ph); 4 08(m, lH, HZ), 3 77(s, lH, H,); 2 93(dd, 2H, H,), 2.92-2 85(m, 4H, 1383, Ha,,,), 2 Wdd, 1JL 6); JA,B = ~~,J,,,~=~~,J,,,=~,J,~,.=~Hz,‘~CNMR 1362, 1284, 1283, 1278, 1276, 1272, 1219, 1216, 1189, 1184, 1132, 111 3(Ar); 80 8(C3), 73 l(OCH,Ph); 68 5(C2); 59 4(C,), 56 6(C,), 23.O(C& N-Benzyl-3,4-d~-O-benzyl-l,5-d~deoxy-l,5-~m~no-D-gluc~tol 12 Rf 0 30, CH,Cl,/acetone 9/l(v/v), mp 109 ‘C; [a120D = +4” (c 1.1, CHCl,), ‘H NMR 7.30(m, 15H, Ph), 4 90(dd, 2H, OCH,Ph), 4 71(m, 2H, OCH,Ph), 4 50, 3 86(AB, 2H, NCH,Ph), 4 Ol(dd, lH, H6), 3.69(t, lH, H4), 3 6l(ddd, lH, H2), 3.39(t, lH, H,), 3 36(d, lH, H6’), 3 05(dd, lH, H,,), 2 44(m, lH, H,), 2 15(dd, =95,J6,5=3,J,,,‘=11,J6,6’=12, 1H,H,,),J,,,=J,,,=J2,3=85,J2,,=4,J,,,, J A,B = 13, 13CNMR 138 6, 138 3,‘138.0, 128 8, 128 5, 128 4, 127 8, 127 4(Ph), 85.8, 78 3(C,/,), 74 8, 74 6(0CH,Ph), 69,3(C,), 65 6(C,), 58 2(C,), 57 3 (NCH,Ph), 55 2(C,) N-Benzyl-3,4-d~-O-benzyl-l,6-d~deoxy-l,6-~m~no-~-id~tol13 R,O 35, CH,Cl,/ acetone 9/l(v/v), mp 77 “C, [a120D = +15” (c 1 6, CHC13), ‘H NMR 7 30(m, 15H, Ph), 4 76, 4.62(AB, 4H, OCH,Ph), 3 8 l(m, 2H, H2), 3 7 l(d, 2H, NCH,Ph), 3 63(dd, 2H, H,), 2 92(d, 2H, H,), 2.63(dd, 2H, H,,), JA,* = 11 5, J,,,, = 12 5, J,,,, = 8, J2,3 = 4, J3,4 = 1.5, 13CNMR 138 1, 137 8, 129 1, 128 5, 127 8, 127 6(Ph), 86 7(C,), 73 7(0CH,Ph), 68 3(C,), 63 6(NCH,Ph), 57,6(C,)

3.3. Preparation of 1-Amino- 1-N-(6’-Hexylamine)3,GDi-0-Benzyl-7,2,5-Trideoxy-2,5-[2’(1 H-lndol-3-yl)-Ethyl]-Imino-L-lditol I N-Boc protectlon

of the mdole moiety of the plperldme 10 (Fig. 11) gives precursor of targets molecules I, II, and III. Selective sllylatlon of the primary hydroxyl group of 14 and mesylatron of the secondary hydroxyl group afford 16, which IS then subjected to nucleophlllc displacement by N-Boc- 1,6-hexanediamine to give the pyrrolldme 17, via an azlrldmlum salt, After cleavage of both sllyl ether and carbamates of 17, the pyrrolidine-type scaffold I IS isolated in 28% overall yield from bls-epoxlde 4 14 as a common

3.3.1. 3,4-Di-0-Benzyl-1,5-Dideoxy-1,5-[2’(1 N-Tertbutoxycarbonyl-Indol-3-yl)-Ethyl]-Imino-~-Gulitol

14

1 To a mixture of 10 (300 mg, 0.6 17 mmol), NaOH (123 mg, 3.07 mmol) and tetran-butylammonmm hydrogen sulphate (39 mg, 0.115 mmol) m dlchloromethane

Sugar- Based Scaffolds

241

Fig. 11 Synthesis of the nonpeptide mimic I

(45 mL), add dropwtse dt-tert-butyl dicarbonate (147 mg, 0 674 mmol) m drchloromethane (15 mL) 2 Stir the reaction mixture at 20°C for 5 h, and then add water (20 mL) Decant and extract with four 20 mL porttons of drchloromethane. 3 Wash the combined orgamc layers with brine, dry over anhydrous Na,SO,, filter, and concentrate to dryness You should get about 361 mg (quantitative yield) of 14 that can be used m the next step without purificatton. ‘H NMR 8 10,7 50(2d, 2H, H ,ndole); 7 35-7 15(m, 13H, Ar); 4 72-4.55(m, 4H, OCH,Ph), 4 04(m, lH, H2), 3 96(dd, lH, H4), 3 82(dd, 2H, H6), 3 67-3 57(m, 2H, H3,6p); 3 2&2 96(m, 3H, H,,,,%,), 2 88-2 77(m, 4H, Ha& 1 63(s, 9H, tBu), J3,4 = 8,J,,, = 4 5, J6,6t = 11, J6,5 = 6Hz.

3.3 2. 3,4-Di-O-Benzyi- l$Dideoxy- 1,5-[2’-(1 N-TertbutoxycarbonylIndol-3-yl)-Ethyl]-im~no-6-0-Tertbutyidimethyisiiyi-r.-Guiitoi 15 1, To a cooled solution at 0°C of 14 (360 mg, 0 614 mmol) m DMF (2 2 mL), add successively imtdazole (84 mg, 1.23 mmol) and tert-butyldtmethylstlyl chlortde (111 mg, 0 737 mmol) 2 After 2 h at O”C, add 10 mL of an aqueous solution of NH4Cl and 20 mL of dmhloromethane Decant, and extract with four 20 mL portions of dtchloromethane Wash the combmed orgamc layers with a saturated aqueous solution of NH,Cl, dry over anhydrous Na2S04, filter, and concentrate to dryness. 3 Purify the residue by chromatography on silica gel using a 3/2 mixture (v/v) of cycIohexane/ethyl acetate, Concentrate the approprtate fractrons (Rr 0.35) You should get about 305 mg (71%) of 15 [a]20,, = -9” (c 1 1, CHCl,), ‘H NMR: 8 10, 7.50(2d, 2H,H ,ndote),7 38-7 15(m, 13H, Ar), 4 664 44(m, 4H, OCH2Ph), 4.0l(m, lH, H2);4 0-3.9(m,2H,H4,& 3.78(dd, lH,H,); 3,6l(dd, lH, Hs); 3 06 2 92(m, 4H, Ha,& 2 9-2 8(m, 2H, H,,,); 2.75(dd, lH, H,,); 1.63(s, 9H, tBu), 0 86(s, 9H, tBuSi); 0 03(s, 6H, Me,Si); J6,s, = 1 l’, J6a,5= 6’, J3,4 = 7’, J2q3= 4’, J,,* = 7’, J,,lt = 1lHz

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3.3.3. 3,4-Di-0-Benzyl-l,5-Dideoxy-l,5-[2’(1 N-Tertbutoxycarbonyl-Indol-3-yl)-Ethyl]-lmino2-0-Methanesulphonyl-6-O Tertbutyldimethylsilyl-L-Gulitol

16

1 To a cooled solution at 0°C of 15 (50 mg, 0.071 mmol) m dlchloromethane (700 pL), add successively trlethylamme (20 pL, 0 14 mmol), and methanesulphonyl chorlde (8 pL, 0.106 mmol) 2 After 10 mm at O”C, add water (10 mL) and dlchloromethane (10 mL) Decant, and extract with four 10 mL portions of dlchloromethane Wash the combmed organic layers with brine, dry over anhydrous Na$O,, filter, and concentrate to dryness You should get a quantltatlve yield of 16 (56 mg) This product IS used wlthout further purification It’s strongly recommended to use 16 without delay m the next step; otherwise some shght decomposition can be observed IH NMR 8 30, 7 55(2d, 2H, H ,,,d&, 7 42-7 22(m, 13H, Ar), 5 lO(m, lH, HZ), 4 714 60(m, 4H, OCH,Ph), 4.0-3 9(m, 3H, H3,4,6)r 3 80(dd, IH, He’), 3 22 82(m,7H,H, >In(3% 5 a & 2 96(s, 3H, MeSO& 1 68(s, 9H, tBu), 0.90(s, 9H, tBuSl), 0.06(s, 6H, Me2SI), J6,6p= 11, J6’,5 = 5Hz

3 3 4 l-Am~no-7-N-[N’-Tertbutoxycarbonyl-6’-Hexylamine]3,4-Di-0-Benzyl-1,2,5-Tndeoxy-2,5-[2’-(1 N-TertbutoxycarbonylIndol-3-yl)-Ethyl]-lmino-6-0-Tertbutyldimethyls~lyl-~-Iditol 17 1 To 16 (52 mg, 0 067 mmol) m absolute ethanol (300 pL) add N-tert-butoxycarbonyl1,6-hexanedlamme (72 mg, 0.335 mmol, see Note 5) dissolved m hot ethanol (600 pL) 2. After 18 h at 2O”C, concentrate the mixture to dryness, and add water (10 mL) and dlchloromethane (10 mL) Decant and extract the aqueous layer with four 10 mL portions of dlchloromethane 3 Dry the combined organic layers over anhydrous Na,SO,, filter, and concentrate to dryness 4. Purify the residue by chromatography on slllca gel usmg a 92/8 mixture (v/v) of dlchloromethane/methanol Concentrate the appropriate fractions (Rf 0 25) You should get about 57 mg (93%) of 17 (011) This compound can be observed as two spots on TLC which is owmg to the protonatlon of the amme on silica gel [a120,, = +3” (c 1.0, CHCl,), ‘H NMR: 8 11,7 55(2d, 2H, Hlndole), 7 4-7 2(m, 13H, Ar), 4 77-4 45(m, 4H, OCH,Ph); 4 5O(s, lH, NHBoc), 4 50(m, lH, H4), 4 25(t, lH, H3), 3.93(dd, lH, H6), 3.68(dd, lH, H,,); 3.55(m, 2H, H,,,), 3.2-242(m, lOH), 1 65, 1 42(2s, 18H, tBuO), 1.33-1 O(m, SH, (CH,),), 0 83(s, 9H, Me,%), 0.015, 0 001(2s, 6H, Me2Si), J3,4 = J2,3 = 7, J6,6’ = 11, J6,5 = 2, J6n,5= 1Hz

3.3.5. 1-Amino- 1-N-[6’-Hexylaminef3,4-Di-O-Benzyl1,2,5-Trideoxy-2,5-[2’-(7 H-lndol-3-yl)-Ethyl]-Imino-L-lditol

I

1 Stir 17 (40 mg, 0 044 mmol) for 4 hat 20°C m presence of concentrated HCI (3 M m AcOEt, 300 pL) 2 Cool the reaction mixture at O’C, and add a saturated aqueous solution of NaHCO, to neutralize

Sugar-Based Scaffolds

243

3 Add dtchloromethane (10 mL), decant and extract the aqueous layer using four 10 mL portrons of dichloromethane. Wash the combined organic layers with brine, dry over anhydrous Na2S04, and concentrate to dryness. 4. Purify the residue by chromatography on silica gel usmg a 3/1/l mrxture (v/v/ v) of dtchloromethane/methanol/ammonium hydroxide (30%) Concentrate the appropriate fracttons (Rf 0.25) You should get about 22 mg (85%) of I (011). [a]*Oo = +3” (c 0 7, CHCI,); ‘H NMR* 8.72(s, lH, Hrndole), 7 55(d, IH, Hlndole), 7 4-7 2(m, 14H, Ar), 4 724 52(m, 4H, OCH,Ph), 4.32(t, lH, H4); 4 28(t, lH, Hs), 3 74(dd, lH, H,J, 3 68(dd, lH, H6’), 3 48(ddd, lH,H,), 3 36(ddd, lH,H,), 3 17-2.60(m, 8H); 2 5-2.3(m, 2H), 1 48-l.l5(m, 8H,(CH,),); &, = J2,s = J4,5 = 7, J6,6’ = 11, J6,5 = 4 5, J6t,5 = 3, J,,* = 4, J]‘,* = 3 5Hz; 13C NMR. 137 9, 1363, 1285, 1279, 122 3, 121 8, 119.1, 118 5, 113 7, 111 5(Ar); 842, 83 8(C,,,), 73 1, 72.7(OCH2Ph), 61.4, 59.6, 58.7(C,,,,,); 49.4, 48.6, 46 2, 41 3(C,,+J, 31 5, 28 2, 26.3, 26 1, 24.2(Ct,,(CH,),)

3.4. Preparation of 2-Amino-2-N-(6’-Hexylamine)-3,4-Di-O-Benzyl1,2,5-Trideoxy-1,5-[2’-(1H-lndol-3-yl)-Ethyl]-Imino-~-Iditol II The secondary hydroxyl group of the prpertdme 15 can be transformed mto an azrdo group (Fig. 12), with mversron of configuratton, under Mttsunobu condrtrons in the presence of hydrazotc acid to give 18. Catalytic reduction of the azldo group into the amme followed by alkylation with 6-bromo-N-Bochexylamme, and removal of srlylether and carbamate groups yields the piper-rdine-type

scaffold II (12% overall yield from the bu-epoxide

4).

3.4.1 2-Azido-3,4-Di-O-Benzyl- 1,2,5-Trideoxy1,5-[2’-(1 N-Tertbutoxycarbonyl-Indol-3-y/)Ethyl]-Imino-6-O-Tertbutyldimethylsilyl-L-Iditol 18 1 To a cold solutton at 0°C of trtphenylphosphine (900 mg, 3 42 mmol) m THF (1 mL), add dropwrse diethylazodtcarboxylate (540 pL, 4 17 mmol), (see Note 4) 2. After 5 mm at 0°C add dropwise hydrazoic actd (see Note 2) m benzene (0 95 M, 3 mL, 2.85 mmol) and 15 (396 mg, 0.571 mmol) m THF (1 mL). 3 Stn the reaction mrxture for 2 h at 0°C then concentrate to dryness 4 Dtssolve the resrdue m ethyl acetate, and then filter through a srhce pad 5 Purtfy the restdue by chromatography on silica gel using a 96/4 mtxture (v/v) of toluene/acetone, and concentrate the appropriate fracttons (R, 0.38) You should get about 250 mg (61%) of 18, (see Note 6). [a]*OD = 40” (c 1 0, CHCls), ‘H NMR 8.12, 7.50(2d, 2H, H ,ndole), 7.39-7 17(m, 13H, Ar), 4 88, 4 74(AB, 2H, OCH,Ph); 4.62(s, 2H, OCH,Ph); 3 97(dd, lH, H6), 3 88(dd, lH, He’), 3 743 64(m, 2H, H3,4), 3 54(dt, lH, H2), 3.17(m, lH, H,), 3.13-2.74(m, 6H), 1.65(s, 9H, tBu); 0.89(s, 9H, tBuSr), 0.06(s, 6H, Me2Sr), JA,B = 12, J6,6’ = 11, J6,5 = 5.5, J6’,5 = 2.5, J2,3 = J2,,a = 10, J2,,e = 5.5Hz.

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Le Merrer, Poitout, and Depezay

Ftg 12 Synthesis of the nonpeptide mimic II

3.4 2. 2-Ammo-3,4-DI-O-Benzyl- 1,2,5Tndeoxy1,5-[2’-( 1N-Tertbutoxycarbonyl-lndol-3-y/)Ethyl]-lmino-6-O-Tertbutyld~methylsilyl-L-lditol 19 1 Hydrogenate a mixture of 10% Pd/C (50 mg) m absolute ethanol (1 mL) prtor to the addition of 18 (104 mg, 0.143 mmol) m ethanol (1 mL) under atmospheric pressure of H, 2 After 4 h under H,, filter the mixture (celite), and concentrate the filtrate You should get about 100 mg (quantttattve yield) of 19 This product can be used m the next step without further purification (see Note 7). [c~]*~n = -7” (c 1 0, CHCI,); ‘H NMR. 8 10, 7 50(2d, 2H, H ,ndole); 7 36-7 17(m, 13H, Ar), 4 89(d, lH, OCH,Ph), 4 60(m, 3H, OCH,Ph); 3 99(dd, lH, H6); 3 83(dd, lH, He), 3.70(dd, lH, H4), 3.39(t, IH, HJ; 3 23(m, lH, H,), 3.10-2 80(m, 6H), 2.68(t, lH, H,); 1 64(s, 9H, tBu); 0 87(s, 9H, tBuSt), 0 04(s, 6H, Me2Si), J3,4 = J3,2 = 8, J4,5 = 5, J6,5 = 6, J6’,5 = 2, J6,@= 11, Jla,2 = J,a,,e = 1OHz

3.4.3. 2-Amlno-2-N-[N’-Tertbutoxycarbonyl-6’-Hexylamine]-3,4DI-O-Benzyl- 1,2,5-Tndeoxy- 1,5-[2’-(1 N-TertbutoxycarbonylIndol-3-yl)-Ethyl]-lmino-6-O-Tertbutyldimethyls~~yl-~-ld~tol20 1 Reflux a mixture of 19 (100 mg, 0 143 mmol), absolute ethanol (1 mL), N-ethyldnsopropylamme (75 uL, 0.429 mmol), and 6-bromo-N-tert-butoxycarbonylhexylamme (80 mg, 0 286 mmol, see Note 8) for 36 h, then cool to 20°C and concentrate to dryness. 2 Purify the residue by chromatography on stltca gel usmg a 95/5 mtxture (v/v) of dtchloromethane/methanol Concentrate the appropriate fractions (Rr 0 30, see Note 9) You should get about 79 mg (62%) of 20 This product can be observed as two spots on TLC whtch is owmg to the protonation of the amme on stlica gel [ a]*oD = +l 5” (c 1,O, CHCls), ‘H NMR 8.10,7 50(2d, 2H, Hlndole); 7 37-7 17(m, 13H, Ar), 4.85(d, lH, OCH,Ph), 4.58(m, 3H, OCH,Ph), 4 5O(s, IH, NHBoc), 4 O(dd, lH, H6), 3,78(dd, lH, H,); 3 74(dd, lH, H,); 3 60-3,40(m, lH, H3), 3 25(m, lH, H,); 3 lO--2.40(m, 11H); 1.64(s, 9H, tBu), 143(s, 9H, tBu), 1.4

Sugar-Based

Scaffolds

1 15(m, 8H), 0 87(s, 9H, tBuSt), 0 04(s, 6H, Me+); J4,5 = 4 5Hz

245

J6,5 = 6 5, J6,@= 10, J3,4 = 7,

3 4 4. Z-Am~no-Z-N-[6’-Hexyiamine]-3,4-Di-O-Benzyi-l,2,5-Trideoxy1,5-[2’-(1 H-indoi-3-yi)-Ethyl]-imino-L-iditoi II 1 Stir 20 (43 mg, 0 0479 mmol) for 4 h at 2OT m presence of concentrated HCl(3 Mm AcOEt, 600 pL) 2 Cool the reaction mixture to 0°C and add a saturated aqueous solutton of NaHCO, to neutralize 3 Add dtchloromethane (10 mL), decant, and extract the aqueous layer using four 10 mL porttons of dtchloromethane Wash the combmed organic layers with brine, dry over anhydrous Na2S04, and concentrate to dryness 4 Purify the restdue by chromatography on stltca gel using a 60/10/2 mixture (v/ v/v) of dichloromethane/methanol/ammonium hydroxide (30%). Concentrate the appropriate fractions You should get about 22 mg (79%) of II [c~]~~o = +18” (c 0 9, CHCl& ‘HNMR 8 37(s, lH, NH,ndo,e); 7.55(d, lH, Hlndole), 7 356 96(m, 14H, Ar), 4 92(d, lH, OCH,Ph); 4 59(m, 3H, OCH,Ph), 3 83(m, lH, H4), 3 76(dd, lH, Hs), 3 64(t, lH, H,); 3 47(t, lH, H3), 3 20(m, lH, H,), 3 l& 2 30(m, 1 lH), 1 38-l 20(m, 8H), J2,3 = J3,4 = 8 5, J6n,5= J6,6a= 9 5, J6,5 = 5Hz, 13C NMR 138 5, 137 9, 136 3, 128.5, 127.9, 127 8, 127 3, 121 9, 121 7, 119 2, 118 6, 113 7, 111,2(Ar), 81 0, 793(C,,,), 75.1, 72 7(0CH,Ph), 61 5(C,), 57 l(C& 56 4(C2); 55 2, 48 6, 47 4, 41.9(C&&, 33 2, 30.1, 26 9, 26.6, 25 2(%(cH2h>.

3.5. Preparation of 6-Amino-6-N-(6’-Hexylamine)-3,4-Di-O-Benzyl1,5,6-Trideoxy-1,5-[2’-(1 H-lndol-3-yl)-Ethyl]-Imino-r-Gulitol Ill The plperldine scaffold III can be obtamed from the azepane 11 (Fig. 13) by treatment with mesyl chloride to give the rearranged chloromethyl plperldme 22, and subsequent nucleophlllc substitution with IV-Boc- 1,&hexanediamine (44% overall yield of III from 11, see Note 10).

246

Le Merrer, Poitout, and Depezay

3.5.1. 3,4-DI-O-Benzyl- 1,6-Dideoxy- 1,6-[2’-( 1N-TertbutoxycarbonylIndol-3-yl)-Ethyl]-lmino-c-Mannitol21 1 To a mixture of 11 (226 mg, 0 465 mmol), NaOH (102 mg, 2 3 mmol) and tetran-butylammomum hydrogen sulphate (30 mg, 0 084 mmol) m dlchloromethane (34 mL), add dropwlse dl-tert-butyl dlcarbonate (I 11 mg, 0 509 mmol) m dlchloromethane (11 mL) 2 Stir the reaction mixture at 20°C for 15 h, and then add water (10 mL), decant, and extract with three 15 mL portlons of dlchloromethane 3 Wash the combined organic layers with brine, dry over anhydrous Na,SO,, filter, and concentrate to dryness. You should get about 272 mg (quantltatlve yield) of 21 This product can be used m the next step without further purlflcatlon ‘H NMR 8.07, 7 48(2d, 2H, Hlndole), 7 4-7 2(m, 13H, Ar), 4 76, 4 62(AB, 4H, OCH,Ph); 4 lO(m, 2H, H,), 3 81(s, 2H, H,); 2 96(dd, 2H, H,), 2 90(m, 4H, Ha,J; 2 83(dd, 2H, H,,); 1 65(s, 9H, tBu), JA,B = J,,,. = 12, J,,, = 4, J,,,, = 6Hz

3.5 2. 3,4-Di-0-Benzyl-6-Chloro1,5,6-Trideoxy1,5-[2’-(1 N-Tertbutoxycarbonyl-Indol-3-yl)-Ethyl]-Imino-~-Gul~tol22 1 To the dlol 21 (120 mg, 0 204 mmol) m dlchloromethane at O”C, add successively trlethylamme (56 pL, 0.408 mmol), and methanesulphonyl chloride (23 pL, 0.28 mmol) 2 After 1 5 h at O”C, add water (5 mL) and dlchloromethane (15 mL) Decant, and extract the aqueous layer using four 15 mL portions of dlchloromethane 3 Wash the combined organic layers with brine, dry over anhydrous Na2S04, then filter, and concentrate to dryness 4 Purify the residue by chromatography on silica gel using a 65135 mixture (v/v) of cyclohexane/ethyl acetate. Concentrate the appropriate fractions (Rf 0 30) You should get about 74 mg (60%) of 22. [a]*Oo = -10” (c 1.0, CHCI,), IH NMR 8 09, 7 50(2d, 2H, H ,ndole);7.4&7 15(m,13H, Ar); 4 67-4 52(m, 4H, OCH,Ph), 4 06(m, lH, H2); 3 97(dd, lH, H4); 3 76(d, 2H, H,,,), 3.54(dd, lH, H3), 3 27(dt, lH, H,), 3 07-2 77(m, 6H), 1 65(s, 9H, tBu); J3,4= 7 J4,5 = 4, J5,6= J5,@= 6, J2,3 = 3 5Hz

3.5 3. 6-Amino-6-N-[N’-Tertbutoxycarbonyl-6’-Hexylam~ne]3,4-DI-O-Benzyl- 1,5,6-Tndeoxy- 1,5-[2’-( 1N-TertbutoxycarbonylIndol-3-yl)-Ethyl]-Imino-L-Gulitol23 1 Warm a mixture of 22 (60 mg, 0 0993 mmol), and N-tert-butoxycarbonyl1,6hexanedlamine (214 mg, 9.93 mmol, see Note 5) m absolute ethanol (500 I-IL) at 60°C for 15 h, then cool to 2O”C, and concentrate to dryness 2 Add water (5 mL), and dlchloromethane (15 mL), decant, and extract the aqueous layer usmg four 15 mL portions of dichloromethane Then, dry over anhydrous Na2S04 the combmed organic layers, filter, and concentrate to dryness 3. Purify the residue by chromatography on slhca gel using a 92 5/7 5 mixture (v/v) of dlchloromethane/methanol Concentrate the appropriate fractions (Rf 0 23)

Sugar-Based

Scaffolds

247

Fig 14 Synthesis of the nonpepttde mimic IV You should get about 67 mg (86%) of 23 [a]20n =-lo” (c 1 5, CHCls), ‘H NMR 8 08,7 52(2d, 2H, H ,ndole)r7 4@-7 18(m, 13H, Ar); 4.74-4 58(m, 4H, OCH,Ph); 4 5O(s, lH, NHBoc); 4 02(m, lH, Hz); 4 O(dd, lH, H,); 3.58(dd, lH, Hs), 3 36(m, lH, H,); 3.24(dd, lH,H& 3 09-2.71(m, 9H);2.48(t,2H,Hc); 1 64(s,9H, tBu), 142(s, 9H, tBu); 1.40-l.O5(m, 8H), J4,s = 5.5, J3,4 = 8.5, J2,3 = 3.5, J6,5 = 6, J6,@= 14Hz.

3.5.4. 6-Amino-6-N-(6’-Hexylamine)-3,4-Di-O-Benzyl-7,5,6-Tr/deoxy1,5-[2’-(1 H-lndol-3-yl)-Ethyl]-lmino-L-Gulitol lll 1 Stir 23 (50 mg, 0.064 mmol) for 4 h at 20°C in presence of concentrated HCl(3 A4 m AcOEt, 500 uL) 2 Cool the reaction mixture to 0°C and add a saturated aqueous solutron of NaHCOs to neutralize 3 Add dichloromethane (10 mL), decant, and extract the aqueous layer using four 10 mL porttons of dtchloromethane Wash the combmed orgamc layers wtth brine, dry over anhydrous Na2S0,, and concentrate to dryness 4 Purify the residue by chromatography on sdlca gel using a 6012012 mixture (v/v/ v) of dtchloromethane/methanol/ammonmm hydroxide (30%). Concentrate the appropriate fractions (RrO 3), you should get about 27 mg (85%) of III [a120D = -17” (c 0.6, CHCls); ‘H NMR: 8 75(s, lH, NHlndole), 7 55(d, IH, Hlndole); 7 367 03(m, 14H, Ar); 4 69-4.54(m, 4H, OCH,Ph); 4.08(m, lH, H2); 3.94(dd, lH, H4); 3 57(dd, lH, H3), 3 26(m, lH, H6), 3 22(m, lH, Hs); 3 O(dd, lH, H6’), 3 052 62(m, 8H); 2 37(m, 2H), 1 43(m, 2H); 1.30-l lO(m, 6H), J3,4 = 8, J4,5 = 4 5, J2,3 = 3 5, J6,5 = 7, J6’.5 = 6, J6,@= 13.5Hz, 13C NMR. 138.3, 138 2, 136 3, 128 5, 128 0, 127 7, 127 5, 122.1, 121 7, 119 1, 118 7, 113 8, 111 3(Ar); 78 1, 75 O(C,,,), 73 1, 72 4(0CH,Ph), 67 6(C2); 58 5(Cs); 55 0, 49.7, 49.4, 44.7, 41.5(C1,6,a,c,d); 3 1 9, 28 7, 26 6,26 3,23 7(C,,(CH,),).

3.6. Preparation of 2-Amino-2-N-(6’-Hexylamine)-3,4-Di-O-Benzyl7,2,6-Trideoxy-7,6-[2’-(1H-lndo/-3-y/)-Ethy/]-/mino-o-Mannito/

/V

The azepane-type scaffold IV IS obtamed from the &-symmetric azepane 11 (Fig. 14). Treatment of 11 under Mitsunobu condltlons m the presence of

248

Le Merrer, Poitout, and Depezay

hydrazotc acid affords a mixture of the aztdo azepane 24, and of the correspondmg azidomethyl plpertdme via an azmdmmm mtermediate (see Subheading 1.1.2.) Catalytic reduction of the aztdo group into the amine and separation of the undesired ammomethyl-prpertdme, followed by alkylatton with 6-bromo-NBoc-hexylamme and acidic cleavage of the Boc-protectmg group, provide the azepane-type scaffold IV m 15% overall yield from the bu-epoxtde 4.

3 6.1. 2-AZ/do-3,4-DI-O-benzylEthyl]-imino-c-Mann/to/ 24

1,2,6-Trideoxy- 1,6-[2’-(1 H-lndol-3-y/)-

1 To a cooled solution at 0°C of trlphenylphosphme (168 mg, 0 640 mmol) in THF (400 yL), add dropwtse dtethylazodicarboxylate (103 pL, 0 640 mmol), (see Note 4) 2 After 5 mm at 0°C add dropwise hydrazoic acid (seeNote 2) m benzene (0 9 IV, 776 pL, 0 698 mmol) and 11 (283 mg, 0 582 mmol) in THF (300 uL) 3. Star the reaction mixture for 2 h at -10°C. If the reaction is not complete (momtormg by TLC), one eq or more of each reagent is added 4 Concentrate to dryness, and purify the residue by chromatography on slhca gel usmg a 91/9 mtxture (v/v) of dichloromethane/acetone Concentrate the appropriate fractions (RrO 30) You should get about 163 mg (55%) of a mixture of 24, and Its 6-aztdomethyl+-gulo-ptperidme isomer (seeNote 11)

3.6 2. 2-Ammo-3,4-Di-0-Benzyl-1,2,6-Trideoxy1,6-[2’-(1 H-Indoi-3-yi)-Ethyl]-imino-o-Mann/to/

25

1 Hydrogenate a mixture of 10% Pd/C (55 mg) in absoluteethanol (1 mL) prior to the addmon of the previous mixture of 24 and its 6-aztdomethyl-L-gulo-ptpendme isomer(104 mg, 0.143 mrnol) m ethanol(1 mL) under atmosphericpressureof H2 2. After 3 h under H,, filter the mixture (cehte), and concentrate the filtrate 3 Purify the residue by chromatography on silica gel using a 87/l 3 mixture (v/v) of dichloromethane/methanol. Concentrate the appropriate fracttons (Rr 0 30) The azepane and piperidine isomers have the sameRr on TLC, however, they are easily separatedby chromatographtc column You should get about 54 mg (52%) of 25, and 44 mg (42%) of its 6-ammomethyl-L-gulo-ptpertdme isomer 25, [a1200 = +1 5’ (c 14, CHCl,), ‘H NMR 8 6O(s, lH, NH); 7 53(d, lH, Hlndole),7 387 02(m, 14H, Ar); 4 64-4 50(m, 4H, OCH,Ph), 4 12(m, lH, Hs), 3 77(dd, lH, Hs), 3 53(brd, lH, H4), 3 36(m, lH, H2), 3 06(dd, lH, H6), 3.&2 83(m, 6H), 2 78(dd, lH, H6), &, = 6, J2,s= 2,J,,, = 3, J5,6t= 5, J6,@= 13Hz

3.6.3. 2-Amino-2-N-[N’- Tertbutoxycarbonyi6’-Hexyiaminel-3,4-Di-0-Benzyi-7,2,6-Trideoxy7,6-[2’-(1 H-indoi-3-yi)-Ethyl]-imino-o-Mannitoi

26

1 Reflux a mixture of 25 (40 mg, 0 082 mmol), absolute ethanol (1 mL), N-ethyldnsopropylamme (57 pL, 0 33 mmol), and 6-bromo-N-tert-butoxycarbonylhexylamme (70 mg, 0 218 mmol, see Note 8) for 24 h, then cool to 20°C and concentrate to dryness

Sugar-Based Scaffolds

249

2 Purtfy the restdue by chromatography on stlrca gel using a 93/7 mtxture (v/v) of dtchloromethane/methanol. Concentrate the approprtate fracttons (R,O 25) You should get about 45 mg (81%) of 26 [cx]~~~ = +13O (c 1 3, CHCl,), ‘H NMR 8.40(s, lH, NH), 7 52(d, lH, H ,ndole)r 7.35-7 06(m, 14H, Ar), 4 69(m, 3H, OCH,Ph); 4 50(m, lH, NHBoc); 4.47(d, lH, OCH,Ph); 4 18(m, lH, H,), 3 88(s, 2H, H,,,), 3 13-2 72(m, 11H); 2 24(m, 2H,H,), 1 42(s, 9H,tBu), 1.4-I O(m, 8H)

3.6.4. 2-Amino-2-N-(6’-Hexylamine)-3,4-Di-O-Benzyl-l,2,6-Tr~deoxy1,6-[2’-( 1H-lndol-3-yl)-Ethyl]-lmino-c-Mannitol IV Stir 26 (40 mg, 0 0584 mmol) for 30 mm at 20°C m presence of concentrated HCl(3 Mm AcOEt, 300 pL) Cool the reaction mixture to 0°C and add a saturated aqueous solutton of NaHCOs to neutralize Add dtchloromethane (15 mL), decant, and extract the aqueous layer usmg four 15 mL portions of dtchloromethane Wash the combined orgamc layers with brute, dry over anhydrous Na2S04, and concentrate to dryness Purify the residue by chromatography on stltca gel using a 60/10/l 75 mixture (v/v/v) of drchloromethane/methanol/ammonmm hydroxide (30%). Concentrate the appropriate fractions. You should get about 34 mg (quantrtattve yield) of IV [cz]~‘~ = +2 5” (c 0.9, CHCl,), ‘H NMR 8 18(s, lH, NH), 7 55(d, lH, H ,ndole)r 7.35-7 O(m, 14H, Ar); 4.68-4 62(m, 3H, OCH,Ph), 4 48(d, lH, OCH,Ph), 4 ll(m, lH, H,); 3 82(m, IH, Hs), 3 74(t, lH, H4), 3 lO(br t, lH, H,), 2.95-2 80(m, 6H), 2 75(d, 2H, H,), 2 64(t, 2H, H,), 2 43(m, 2H, Hd), 1 431 22(m, 8H), J3,4 = J4,5 = 4, J,,, = J,,,, = 6Hz, 13C NMR 138 6, 138 4, 136 2, 128.4, 127 9, 127 8, 127 5, 121.9, 121 7, 119.0, 118 6, 114.0, lll.l(Ar); Sl.O(C,,,), 72 6(0CH,Ph), 67 9(C,), 56 9(C,), 59 5, 56 0,47.7, 42.0(C,,c,,,,,,), 33 3, 30 2, 27 1, 26 7, 23.2.

3.7. Preparation of (1 R,4R, 7R,8R)-6’-{7,8-Bis-Benzyloxy-5-[Z-(1 HIndol-3-yl)-Ethyl]-2,5-Diazabicyclo[2.2.2]0ct-2-yl}-Hexylamine V and of (1 R,5S,6R, 7R)-6’-f6,7-bis-Benzyloxy-8-[Z-(1 H-lndol-3-y/)Ethyl]-3,8-Diazabicyclo[3.2.7]Ocf-3-y/}-Hexylamine VI The btcycltc-type scaffolds V and VI are prepared starting from the Czsymmetric azepane 13 issued of the heterocyclrzatton of the L-tdttol bzsepoxtde 7 wtth benzylamtne (Fig. 15). Selective hydrogenolysts of the

benzyl-mtrogen bond m presence of dl-tert-butyl dlcarbonate, followed by mesylation yteld 28. Treatment of 28 with an excess of tryptamme m ethanol gives a mixture of 29 and 30 that are isolated after separation by chromatography in 60% and 15% yield, respectively. Finally, acldrc cleavage of the Boc-protectmg group, followed by alkylatron with 6-bromo-N-Boc-hexylamine and subsequent acidic cleavage of the carbamate afford the bicycllc derivatives V or VI (33% and 8% overall yteld respectively from the bisepoxide 7).

Le Merrer, Poitout, and Depezay

Ftg 15 Syntheses of the nonpeptlde mtmtcs V and VI

3.7.1. N- Tertbutoxycarbonyl-3,4- Dt-O-Benzyl7,6-Dideoxy-l,6-lmino-L-lditoi 27 Hydrogenate a mixture of 20% palladium hydroxide on carbon (Pearlman’s catalyst, 100 mg) m ethyl acetate (5 mL) prior to the addition of 13 (200 mg, 0 462 mmol) and dl-tert-butyl dlcarbonate (111 mg, 0 509 mmol) m ethyl acetate (1 mL) under atmosphertc pressure of HZ. 2 After 2 h under H,, filter the mixture (cehte), and concentrate the filtrate You should get about 205 mg (quantnatlve yield) of 27. This product can be used m the next step without further purtticatron Mp 91°C [a120n = -1” (c 0 7, CHCl,), ‘H NMR 7 30(m, lOH, Ph); 4 8&4 48(m, 4H, OCH,Ph); 4 &3 O(m, 8H), 1 36(s, 9H)

3 7.2 N-Tertbutoxycarbonyl-3,4-Di-O-BenzylMethanesulphonyl- 1,6-lmino-L-lditol28

1,6-Dideoxy-2,3-D/-0-

1 To a cooled solutton at 0°C of 27 (170 mg, 0.383 mmol) m dlchloromethane (2 5 mL), add trlethylamme (208 ,uL, 1 53 mmol) and methane&phony1 chloride (74 pL, 0 965 mmol) 2 After 10 mm at O”C, add water (6 mL), and dtchloromethane (15 mL) Decant, and extract the aqueous layer with four 15 mL portions of drchloromethane Then, wash the combined organic layers with brine, dry over anhydrous Na2S04, and concentrate to dryness

Sugar-Based Scaffolds

251

3 Purtfy the residue by chromatography on siltca gel using a 3/2 mixture (v/v) of cyclohexane/ethyl acetate Concentrate the appropriate fracttons (R, 0 35) You should get about 218 mg (95%) of 28. [cx]~~,, = -5” (c 0.7, CHCl,), ‘H NMR 7 30(m, lOH, Ph), 4 82,(m, 2H, H,,,), 4.64-4 38(m, 4H, OCH,Ph), 3.93(2s, 2H, H,,,), 3.83-3 64(m, 4H, HI ,6), 3 IO,2 80(2s, 6H, MeSO,), 1 45(s, 9H, tBu)

3 7.3. Substitution with Tryptamine 1 Reflux a solution of 28 (180 mg, 0 3 mmol) and tryptamme (721 mg, 4 5 mmol) m absolute ethanol (3 mL) during 18 h 2 Cool the mixture, concentrate, and add water (8 mL) and dtchloromethane (20 mL) 3 Decant, extract the aqueous layer with four 20 mL portions of drchloromethane, and dry the combmed orgamc layers over anhydrous Na,SO,. Concentrate to dryness 4 Purify the residue by chromatography on stlrca gel using a 55/45 mixture (v/v) of ethyl acetate/cyclohexane. Concentrate the appropriate fractions (RfO 35 and 0 30), you should get about 103 mg (60%) of 29, and 26 mg (15%) of 30, respectively. 29 ‘H NMR 8 09(s, lH, NH), 7 59(dd, lH, Hlndole), 7 4-7 O(m, 14H, Ar), 4 814 42(m, 4H, OCH,Ph), 4 54-4 32(2s, lH, H,); 3 83(s, lH, H4); 3 73-3.67(m, lH, HI), 3 65(m, lH, HI), 3 32-3 22(2dd, lH, HI,), 3 2-3 l(m, 3H, H2,6,6’), 3 04 2 89(m,4H), 1.49, 1 46(2s, 9H) 30. ‘HNMR 7 94(s,lH,NH), 7.56(d, lH, Hlndole), 7 3-7 O(m, 14H, Ar), 4 60-4 45(m, 4H, OCH,Ph); 4 18(m, lH, H4), 3 92,3 85(2m, lH, Hs), 3 80(d, lH, H,), 3 68(d, lH, Hs), 3 45-3 40(2m, lH, H5), 3 38-3 32(2s, 6H, H2), 3 22-2 86(m, 6H), 1 4O-1.37(2s, 9H), J,,,, = J6,6s= 13Hz

3.7.4. (1 R,4R, 7R,8R)-8-his-Benzyloxy-5-[2-(1 Ethyl]-2,5-Dlazablcyclo[2.2 2]0ctane 31

H-lndol-3-y/)-

1 Stir 29 (64 mg, 0 113 mmol) for 30 min at 20°C m presence of concentrated HCl (3 A4 m AcOEt, 500 uL) 2 Cool the reactton mixture to 0°C and add a saturated aqueous solutron of NaHCOs to neutrabze 3. Add dtchloromethane (15 mL), decant, and extract the aqueous layer usmg four 15 mL portrons of dlchloromethane Wash the combined organic layers with brine, dry over anhydrous Na2S04, and concentrate to dryness You should get about 52 mg (quantttatrve yield) of 31 This product can be used m the next step wtthout further purification [cx]~~,,= -13” (c 1 I, CHCI,), ‘H NMR* 8,25(s, IH, NH), 7 60(d, lH, H ,ndole);7 37-7 09(m, 14H, Ar); 4 62,4 61,4 54,4 52(2AB, 4H, OCH,Ph), 3 82(t, lH, H4), 3.64(t, lH, Hs), 3 39(dd, lH, HI); 3 18(m, lH, H5); 3 12(dd, lH, H6), 3 04(dd, lH, H6’); 2 95(m,lH, H2), 3.02-2,88(m,4H), 2 82(dd, lH, H,,), JA,B = 12, J3,4 = J4,5 = J2,3 = J,z = J,‘,, = 2, J,,,, = 12, J6,5 = 2, J6’,5= 2 5, Jh,@= 1 1Hz

3.7 5. (1 R, SS, 6R, 7R)-6,7-his-Benzyloxy-8-[2-( Ethyl]-3,8-Diazabicyc/o[3.2.l]Octane 32

1H-lnciol-3-y/)-

1, Stir 30 (24 mg, 0 042 mmol) for 30 mm at 20°C m presence of concentrated HCl (3 A4 m AcOEt, 300 pL), and follow the same work-up as above (see 29 + 31)

252

Le Merrer, Poitout, and Depezay

2 Purify the residue by chromatography on slhca gel using a 90/10 mixture (v/v) of dlchloromethane/methanol. Concentrate the appropriate fractions (R, 0 3), you should get about 20 mg (quantitative yield) of 32 [a120D = -16” (c 0 6, CHCl,), ‘H NMR* 8 13(s, lH, NH); 7 59(d, IH, Hlndole); 7 40-7 Ol(m, 14H, Ar); 4 634 51(m, 4H, OCH,Ph); 4.39(dd, lH, H4), 4 21(d, lH, H3), 3 38(m, lH, H,), 3 22(m, 2H, H1,2); 3 13(dd, lH, H6), 3 07-2 92(m, 4H, Ha& 2 89(dd, lH, H6’), 2 74(dd, lH, H,,); & = J6,5 = 2, J4,5 = 6, J,s,, = 2 5, J,,,m = 13, J6m,5= 1 5, J6,6’ = 13Hz

3.7.6 (lR,4R,7R,8R)-6’-N-Tertbutoxycarbonyl(7,8-bis-Benzyloxy-5-[Z-(1 H-lndol-3-y/)Ethyl]-2,5-Diazabicyclo[2.2.2]0ct-2-y~-Hexylamine

33

Reflux a mixture of 31 (52 mg, 0 111 mmol), absolute ethanol (200 pL), Nethyldllsopropylamme (78 vL, 0.444 mmol), and 6-bromo-N-tert-butoxycarbonylhexylamme (93 mg, 0.333 mmol, see Note 8) for 18 h, then cool to 20°C and concentrate to dryness 2 Purify the residue by chromatography on slhca gel usmg a 94/6 mixture (v/v) of dlchloromethane/methanol Concentrate the appropriate fractions (Rf 0 3) You should get about 65 mg (88%) of 33. [a120D = 4” (c 1 4, CHCQ, ‘H NMR. 7.85(s, lH, NH); 7 57(d, lH, H,ndole), 7 40-7 02(m, 14H, Ar), 4.60(m, 4H, OCH,Ph), 4 46(s, 1H, NHBoc); 3 82(s, 2H, H3,4), 3 05(m, 2H), 3 &2 8(m, 1OH), 2 50(m, 2H), 1 42(s, 9H, tBu), 1 4-l 2(m, 8H)

3.7.7 (1 R,5S,6R, 7R)-6’-N-Tetibutoxycarbonyl-{6,7-bis-Benzyloxy-8-[2(1 H-lndol-3-yl)-Ethyl]-3,8-Diazabicyclo[3.2 l]Oct-3-yo-Hexylamine 34 Reflux a mixture of 32 (18 mg, 0 038 mmol), absolute ethanol (300 pL), Nethyldllsopropylamme (27 pL, 0 154 mmol), and 6-bromo-N-tert-butoxycarbonylhexylamme (32 mg, 0 114 mmol, see Note 8) for 18 h, and then cool to 2O”C, and concentrate to dryness 2 Purify the residue by chromatography on slhca gel using a 95/5 mixture (v/v) of dlchloromethane/methanol Concentrate the appropriate fractions (R, 0.25) You should get about 22 mg (88%) of 34. [a120D = -12” (c 0 9, CHCl,), ]H NMR 8 02(s, lH, NH), 7 58(d, lH, Hlndole); 7 38-7 O(m, 14H, Ar), 4 55(m, 4H, OCH,Ph), 4 44(s, lH, NHBoc); 4.22(m, 2H, H3,J, 3.37(m, lH, H,), 3 27(s, lH, HZ); 3 2-2.85(m, 6H), 2 72(dd, lH, H6); 2.53(s, 2H, HI,,,), 2 34(dd, IH, H6,), 2 30(m, 2H), 1 45(s, 9H, tBu); 1 4-1 2(m, 8H), J6n,5= J6,5 = 2, J6,6r = 11Hz

3.7.8. (1 R,4R, 7R,8R)-6’-{7,8-bis-Benzyloxy-5-[Z-(1 2,5- Diazabicyclo[Z.Z.Z]Oct-2-yl}-Hexylamine V

H-lndol-3-y/)-Ethyl]-

1 Stir 33 (43 mg, 0 065 mmol) for 30 mm at 20°C m presence of concentrated HCl (3 A4 m AcOEt, 400 pL) 2 Cool the reactlon mixture to OT, and add a saturated aqueous solution of NaHC03 to neutrahze

Sugar- Based Scaffolds

253

3 Add dichloromethane (15 mL), decant, and extract the aqueous layer using four 15 mL portions of dichloromethane Wash the combmed orgamc layers with brine, dry over anhydrous Na2S04, and concentrate to dryness. 4 Purify the residue by chromatography on silica gel using a 70/15/l mixture (v/v/ v) of dichloromethane/methanol/ammonmm hydroxide (30%) Concentrate the appropriate fractions (RF0 3), you should get about 35 mg (quantitative yield) of V [~]*~o =-7” (c 1 3, CHCl& ‘H NMR* 7.90(s, lH,NH), 7.56(d, lH, Hlndole), 7 40-7 04(m, 14H, Ar); 4.62(m, 4H, OCH2Ph); 3.82(s, 2H, H3,& 2.98-2.80(s, lOH), 2 63(m, 2H), 2 52(t, 2H); 1.4-1 2(m, 8H); 13C NMR 138.5, 136.1, 128 3, 127 9,127 5,122.1,121.7,119 0,118 7,114 1,ll l.l(Ar), 83 9(C,,&71.0(0CH2Ph); 55.8(C,), 54 9,54 4(C&, 50 0,49 6,41 2,31.4,28 0,26 8,26.5,24.2

3 7.9. (1 R,5S,6/?, 7R)-6’16,7-his-Benzyloxy-8-[2-(1 3,8-Diazabicyclo[3.2.l]Oct-3-y/j-Hexylamine VI

H-lndol-3-y/)-Ethyl]-

1 Stir 34 (20 mg, 0 03 mmol) for 30 mm at 20°C m presence of concentrated HCI (3 M in AcOEt, 300 PL), and follow the same work-up as above (see 33 -+ V) 2 Purify the residue by chromatography on silica gel using a 70/15/l mixture (v/v/ v) of dichloromethane/methanol/ammonium hydroxide (30%) Concentrate the appropriate fractions (Rf 0 3). You should get about 17 mg (quantitative yield) of VI [a]*‘o =-15” (CO 7,CHCl,), ‘HNMR. 8 07(s, lH,NH); 7.56(d, lH, Hlndole), 7 386 99(m, 14H, Ar), 4 52(m, 4H, OCH,Ph), 4 2O(s, 2H, H3,4), 3 36(m, lH, H,); 3 25(s, lH, H2), 3 2-2 6(m, 7H), 2 52(s, 2H, H,,,,); 2 29(m, 3H), 1 5-l 2(m, 8H), 13C NMR 138 4, 136 0, 128 1, 127.6, 127 3, 121 6, 119 0, 118 7, 114 2, 110 9(Ar), 87 4, 86 2(C3,,& 72 0, 71 2(0CH,Ph), 61.7, 60 2(C2,s), 57 4, 51 9, 50 0,47 3,41-O, 30.9, 26.6,26.2,26.0,24.2.

3.8. Discussion The binding affinity of the azasugars derivatives I to VI was measured by means of radiohgand binding studies on rat cerebral cortex usmg (3-[i251]Trp’l-SRIF). These compounds have a comparable affinity for the somatostatin receptor with IC,a values of 14, 12, 11, 10, 15, and 12 fl for I to VI, respectively. They displayed moderate affinities to the SRIF- 14 receptors (IC50 E 12 I.1M), regardless of the srze (five to seven member rings), the flexibtlity, or the rigidity of the mtrogen heterocycle-based scaffolds used These results show that the orientations of the varrous side chains are quite similar whatever the scaffold used. For comparison, the IC,, values obtamed for compounds I to VI are analogs to that reported for the p-glucose, b-xylose or benzodrazepmone based mrmics, respectrvely 16, 23, and 7 PM (9). Taken together, our results, and those concerning other sugar-based derivatives or more recently a benzodrazepmone derrvatrve exhibiting affinities for the SRIF-14 receptors strongly support the validity of the concept of the utllizatlon of nonpeptrde scaffold synthesis of nonpeptrde molecules as peptrde analogs.

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The flexible strategyreported here allows the preparation of azasugar-scaffolds with various skeletons (pyrrohdine, plperidine, azepane,or bicychc structures) This straightforward method provides a highly practical route to the synthesisof other peptldormmics by modlficatlon of the side chains of the ammo acids. 4. Notes For heterocychzatton with other primary ammes, see ref. IO Cauttonf This reaction should be carried out m a fume hood to avoid accidental exposure to toxic hydrazotc acid, see ref. 18 Care should also be taken m handling sodmm azide. To the sodmm aztde (3.25 g, 0 05 mmol) m water (3.25 mL), add benzene (20 mL) and cool to 0°C. To this paste, add dropwtse concentrated H,S04, and decant The benzemc solutton of HNs IS stored at 4°C m a glass bottle This solutron (about 0 9 M) can be tnered by an aqueous solutton of NaOH (0 1 M) m presence of phenol phtaleme To facilitate the notatton of ‘H and r3C NMR data, the numbering system used corresponds to the nomenclature rules of sugars, and a, b, c and 6 to the methylene groups of the side chams

HO OBn

b :I\

123 4 **lOEn tfN5 a

‘6

NH-C&-(CH&-Cd.(2-NH2 =

d HN

As the Mttsunobu 1sa very moisture sensittve reactton, all the reagents have to be water free Ph,P was crushed and drted under vacuum. Preparation of N-tert-butoxycarbonyl1,6-hexanedtamme: to a solutton of 1,6hexanedtamme (8 36 g, 72 mmol) and trtethylamme (2 5 mL, 18 mmol) in chloroform, add dropwtse a solutton of BOC-ON (3g, 12 mmol) m chloroform (6 mL) Stir for 5 h at 20°C filter, and concentrate the filtrate Purify the residue by chromatography on sthca gel using a 80/12/2 mixture (v/v/v) of dtchloromethane/ methanol/ammomum hydroxide (30%) You should get about 2 28 g (88%) of N-tert-butoxycarbonyl-1,6-hexanedtamme. Mp 95’C, ‘H NMR 4 75(s, lH), 3 06(q, 2H), 2 66(t, 2H), 1 4O(s, 9H), 1 50-1.20(m, 8H) The fraction of Rr 0.30 contam a mixture of the 1-azzdo-L-zdo-pyrrohdme isomer and the 2-azzdo-L-gulo-pipertdme isomer They can be separated by a second chromatography on sthca gel using a 85/l 5 mixture (v/v) of pentane/dtethylether You should get about 86 mg (25%) of the pyrrobdne Isomer (RrO 30), and 41 mg (10%) of the ptpertdme isomer (Rr 0 25) A sample of 19 can be purified by chromatography on stltca gel using a 92 517 5 mtxture (v/v) of dtchloromethane/methanol (R, 0 29)

Sugar-Based Scaffolds

255

8 Preparatron of 6-bromo- 1-N-tert-butoxycarbonylhexylamme to a cooled solution at 0°C of 6-ammo-1-hexanol (lg, 8.5 mmol) m dichloromethane (5 mL), add dropwtse a solution of di-tert-butyldicarbonate (2.4 g, 11 1 mmol) m dichloromethane (5 mL) Stir for 15 h at 20°C and add water (10 mL), and dichloromethane (10 mL). Decant, and extract the aqueous layer using three 30 mL portions of dtchloromethane. Wash the combined organic layers with brine, dry over anhydrous Na2S04, and concentrate to dryness. To the residue m diethylether (9 mL) at 0°C add carbon tetrabromide (3 05 g, 9 2 mmol), and triphenylphosphine (2.4 g, 9 2 mmol) After 3 h at 20°C filter, and concentrate the filtrate. Purify the residue by chromatography on sihca gel using a 4/l mixture (v/v) of cyclohexane/ethyl acetate You should get about 2 35 g (97%) of 6-bromo-lN-tert-butoxycarbonylhexylamine Mp 20°C ‘H NMR 4 56(s, lH), 3 32(t, 2H); 3 03(q, 2H); 1 77(m, 2H), 1 5&l 25(m, 15H) 9 The fractions (R, 0 35) contam the correspondmg N-dialkylation product, about 17 mg (11%). 10 The pipertdme-type scaffold III can also be obtained, but in a lower yield, from the piperidme 14 (43% overall yield of III from 14) by selective activatton of the primary alcohol mto a mesylate followed by nucleophilic displacement with N-Boc-1,6-hexanediamme For the selective acttvation of the primary alcohol, better ytelds were obtamed through stlylation of the two hydroxyl groups of 14 (TBDMSCI, DMF, imtdazole, 1 5 h, 2O”C), followed by selective hydrolysis of the prrmary srlylether [ 13/7/3 (v/v/v) AcOH/H,O/THF, 3 d, 3O”C] and subsequent mesylation (MsCl, NEt,, CH+Zl,, 10 mm, O’C) 11 After chromatography, wash the column with a l/l mixture (v/v) of dichloromethane/acetone to recover the starting maternal (about 70 mg, 25%)

Acknowledgments This work has been done as part of the Bra Avemr program supported by Rh6ne-Poulenc Rorer with the participation of the French Ministry of Research and the French Ministry of Industry We are grateful to Doctors D Damour and S. Mignani of Rhone-Poulenc Rorer for discussions and assistance

References 1 Holzemann, G (1991) Peptide conformation mimetics Kontakte (Darmstadt) 3-12and55-63 2. Gianms, A. and Kolter, T (1993) Pepttdomimetics for receptor hgands-discovery, development, and medical perspectives Angew Chem Int Ed Engl 32, 1244-1267 3. Belanger, P C. and Dufresne, C. (1986) Preparation of exo-6-benzyl-exo-2(m-hydroxyphenyl)-1-dimethylaminobicyclo[2 2.2loctane A non-peptide mimic ofenkephalms. Can J Chem 64, 1514-1520. 4 Hirschmann, R., Sprengeler, P A , Kawasaki, T., Leahy, J W , Shakespeare, W. C , and Smith, A B., III (1993) The versatile steroid nucleus. design and

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5.

6

7

8

9 10

11

12

13. 14

15

16

Le Merrer,

PO/tout,

and Depezay

synthesis of a peptidomimetic employmg this novel scaffold. Tetrahedron 49, 3665-3676 Hrrschmann, R., Ntcolaou, K C., Ptetramco, S , Salvmo, J , Leahy, E. M , Sprengeler, P A., Furst, G., Smith, A.B , III, Strader, C D., Cascteri, M A , Candelore, M R , Donaldson, C., Vale, W., and Maechler, L (1992) Nonpeptidal peptidomimetics with a P-D-glucose scaffoldmg. A partial Somatostatm agomst bearmg a close structural relationship to a potent, selective substance P antagonist J Am Chem Sot 114,9217-9218 Hirschmann, R , Ntcolaou, K C., Ptetranico, S , Leahy, E M., Salvmo, J , Arison, B., Cichy, M A , Spoors, P. G., Shakespeare, W.C , Sprengeler, P.A , Hamley, P , Smith, A B , III, Reisme, T , Raynor, K , Maechler, L , Donaldson, C , Vale, W , Fretdmger, R M , Cascteri, M. R., and Strader C D (1993) De novo design and synthesis of Somatostatm non-peptide pepttdomimetics uttltzmg P-n-glucose as a novel scaffoldmg J Am Chem Sot 115, 12,550-12,568 Le Dtguarher, T , Boudon, A , Elwell, C , Paterson, D E , and Billmgton, D C (1996) Synthesis of potenttal peptidomimettcs based on highly substituted glucose and allose scaffolds Bzorg Med Chem Lett 6, 1983-1988 Papageorgiou, C , Haltmer, R , Bruns, C , and Petcher, T J (1992) Design, synthesis, and bmdmg affinity of a nonpepttde mimic of somatostatm. Bzoorg Med Chem Lett 2, 135-140 Papageorgiou, C and Borer, X. (1996) A non-peptide hgand for the somatostatm receptor having a benzodiazepme structure. Bioorg Med Chem Lett 6,267-272 Le Merrer, Y , Poitout, L,, Depezay, J -C , Dosbaa, I , Geoffroy, S., and Foglietti, M -J (1997) Synthesis of azasugars as potent mhibitors of glycosidases Bzoorg Med Chem 5,519-533 Damour, D , Barreau, M , Blanchard, J -C , Burgevm, M -C , Doble, A , Herman, F , Pantel, G , James-Surcouf, E , Vuilhorgne, M , Mignam, S , Poitout, L., Le Merrer, Y , and Depezay, J -C (1996) Design, synthesis and bmdmg affinities of novel non-peptide mimics of Somatostatm/Sandostatm 8 Bzoorg Med Chem Lett 6, 1667-1672 Damour, D , Depezay, J -C , Le Merrer, Y , Mignam, S , Pantel, G , and Poitout, L (1995) Derives de la tryptamme, leur preparation et les mtdxaments les contenant French Patent apphcation # 95 055 10 Lamberts, S W. J , Krenmng, E P , and Reubt, J -C (199 1) The role of somatostatm and its analogs m the diagnoses and treatment of tumors Endocrme Rev 12,45&482 Hoyer, D , Bell, G I , Berelowttz, M , Epelbaum, J., Femuk, W , Humphrey, P P A , O’Caroll, A -M., Patel, Y. C , Schonbrunn, A , Taylor, J E , and Reisme, T (1995) Classification and nomenclature of somatostatm receptors Trends Pharmacol Scz 16, 8&88 Huang, Z., He Ya-Bo, Raynor, K., Tallent, M , Reisme, T , and Goodman, M (1992) Mam chain and side chain choral methylated somatostatm analogs synthesis and conformational analyses J Am Chem Sot 114,939&9400 Moreau, J P and DeFeudts, F V (1987) Pharmacological studies of somatostatm and somatostatm-analogs therapeuttc advances and perspectives Life Scz 40,4 19437

Sugar-Based

Scaffolds

257

17 Hocart, S J , Reddy, V , Murphy, W. A and Coy, D. H (1995) Three-dtmenstonal quantitattve structure-acttvty relattonships of somatostatm analogs 1 Comparative molecular field analysts of growth hormone release-mhtbitmg potencies J Med Chem 38, 1974-1989 18 Wolf, H. (1947) Reactions and generation of hydrazotc acid. Org React 3,845 19 Hughes, D. L (1992) The Mnsunobu reactton Org React 42,335S656 20 Jurczak, J , Bauer, T., and Chmtelewskt, M. (1987) A general approach to the syntheses of2,3-dt-O-protected dertvatrves of o-glyceraldehyde Carbohydr Res 164,493498

The Synthesis of Bicyclic Piperazinone and Related Derivatives Yvette M. Fobian and Kevin D. Moeller 1. Introduction We have been exammmg synthetic approaches to conformationally restrtcted peptrde building blocks that would allow for the constructton of a variety of possible structures in a strarght-forward, general way (I). To date, this effort has focused on developmg the chemrstry needed to rapidly lmbed sections of a pepttde backbone mto a bicychc or polycycl~c ring skeleton (24). Such a transformation 1saccomplrshed by replacing spactally close hydrogens m a desired conformatton with an appropriately sized carbon brtdge. The potenttal advantages of this approach include the preservation of both the peptide backbone and the side chains m the analog, the ability to control the ortentatton of the side chains relative to each other, the increased hydrolytic stability of the analog, and the ease with which new analogs can be designed. The potential disadvantages of this approach mclude the difficulty associated with synthestzmg the analogs and the steric size of the bridges added. In this chapter, a convenient preparation of analogs having the general structure of I (Fig. 1) IS described All of the synthesesdescribed involve two key steps; the transformation of t-Boc protected prolme methyl ester mto a vinyl or ally1 substituted bmldmg block like 3a-c (Fig. 2), and the converston of the building blocks mto the desired brcycltc derivatives 7a-d (Fig. 3) or lla,b (Fig. 4). 2. Materials All proton magnetic resonance spectra were recorded using a Varlan Gemini 300, Vartan VXR-500, Vartan Unity-Plus 500, or Vartan Unity 600 spectrometer. Chemical shifts for proton spectra have been reported as parts per mtlhon From

Methods m Molecular Medrone, Vol 23 Pepbdomunebcs Protocols Edlted by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

259

Fobian and Moeller

260

RI

I

Figure 1

3a

d,c 3b Hg=a 3c H5 = p

Fig 2 Reagents, (a) Carbon rod anode, Pt wire cathode, undivided cell, 0 03 M Et,NOTs m MeOH electrolyte solution, constant current of 26 8 mA (3 0 F/mole), 95%, (b) (CH&C = CHLt, CuBr Me+, BF, * Et,O, Et,O, -78°C to O”C, 89%, (c) i TBDMSOTf, 2,6-luttdme, CH,Cl,, 11 n-Bu,NF, H,O, THF, O”C, 68% for 3a and 49% over the two steps (d and c) for 3b and 3a, (d) CH, = CHCH,TMS, BF, Et,O, Et,O, 40°C to 0°C.

(ppm) downfield from tetramethylsilane m 6 units, and couplmg constants have

been reported m cycles per second (hertz). The sphttmg patterns have been designated

as s for singlet, 6 for doublet,

t for triplet,

p for pentet, and m for

multlplet. Carbon spectra were obtained using either the Gemmt 300 at 75 MHz or the Vat-tan VXR-500 spectrometer at 125 MHz and have been reported m ppm with the center lme of chloroform-d

trtplet referenced at 77.00 ppm or

methanol& septet at 49.0 ppm. Routme t3C NMR spectra were fully decoupled by broad and Waltz decoupling. Two dimensional NMR experiments (HMQC, HMBC, COSY, and NOESY) were carrted out at 25’C on the Vartan Unity-Plus 500 equtpped with an mdlrect detection probe (Nalorac Corp , Martinez, CA). Data was processed using Vartan Software (VNMR) employmg a Spark 10 computer (Sun Mtcrosystems, CA) Typtcal 90” flip angle pulses were 7.5 msec (‘H) and 12 msec (13C) (Sj,

261

Bicyclic Plperazinone and Related Derivatives

0 Cbz \

3a N-ethylmorpholine F

* b MqS.

ws’2

room

temp

4a R,=H, R,=CH,, n=O 4b R,=H. R2=CH Ph. n=O 4c R,=CH,. R,= d , n=O 4d. R,=H, Rz=H. n=l

6a 6b 6c 6d

‘2p3”’

*

;;;;BaSO),

99% 98% 97% (99%)

1

,N H 7a R,=H, R2=CH3, n=O 79% 7b. R1=H, R2=CH2Ph, n=O 62% 7c R,=CH,, Rz=H, n=O 82% 7d R,=H, R,=H. n=l 79%

Figure 3 EDC. Cbzx’JOH

3c HOBT,

CH2C12 w

a 03, MeOH, b Me2S,

eH2R

6a R=H 6b. R = Ph

-78 ‘C t room temp

9a. R=H, 96% 9b R=Ph, 95%

Hz/ Pd(BaSO)., N MeOH H Ila Ilb

R=H, R=Ph,

79% over 76%

two steps

Figure 4 Infrared spectra (IR) were obtained using either a Mattson Polaris FT-IR or an Analect FX-6 160 FT-IR spectrophotometer. Low resolution mass spectral data were obtained using either a HP 9500 GUMS spectrometer or a ZAB-SE spectrometer with an 8 keV acceleration

262

Fobian and Moeller

voltage and resolution of 1000. High resolution mass spectral data were obtained using a MS-50 spectrometer with an 8 keV acceleration voltage and resolution of 1000. Reactions were monitored as a function of time by either TLC with E. Merck silica gel 60 F254 plates or capillary GC. Capillary GC data were obtained using an HP Model 5890A instrument equipped with a HP 3396A integrator and an HP 20-m ULTRA II (5% phenyl methyl silicone) column. Gravity flow and flash chromatography were carried out using E. Merck silica gel 60(70-230 mesh) or ICN slhca gel. The solvents used for chromatography were mixed by volume and have been reported for each experiment Unless otherwise noted, common chemical reagents, starting materials, and solvents were purchased from Aldrich Chemical Co., Sigma Chemical Co., or Bacham Inc. and used wlthout further purification. Dusopropylamme, NJ-dlmethylformamlde, acetomtnle, toluene, and dlchloromethane were distilled over calcium hydride. Tetrahydrofuran was distdled from sodium benzophenone ketyl. Anhydrous ether was purchased from Fisher and used without further purification. Chloroform-d and methanol-& were purchased from Cambridge Isotope Laboratories (CIL) and used with no further purification All reactions were run under an inert atmosphere of nitrogen or argon m flame-dried glassware unless otherwise specified. The purity of all compounds was determined by proton and carbon NMR (>90-95%) data.

2.7. Reagents for Method 3.7 1 Preparative electrolyses were accomplished using a Mode1 410 potentlostatlc controller, a Model 630 coulometer, and a Model 420A power supply purchased from The Electrosynthesls Company, Inc 2 Platinum and carbon rod electrodes were purchased from The Electrosynthesls Company, Inc 3 Tetraethylammomum tosylate was purchased from Aldrich and stored in a vacuum desiccator (approx 0 5 mm Hg) 4. Anhydrous methanol was purchased m Sure/Seal bottles from Aldrich and used without further purification 5 Lithium metal 6 1-Bromo-2-methylpropene. 7. Copper (I) bromide-dlmethyl sulfide complex 8 BF3 Et,O. 9 2,6-Lutldine 10 Allyltrlmethylsilane and tert-butyldlmethylsllyl trlfluoromethanesulfonate were purchased from United Chemical Technologies, Inc 11 Titanium (IV) chloride was purchased as a 1 0 M solution m dlchloromethane 12 1 0 M Tetrabutylammonmm fluoride in tetrahydrofuran

263

Sicyck Piperazinone and Related Derivatives 2.2. Reagents for Method 3.2 1 2 3 4 5 7 8 9 10

N-Carbobenzyloxy-L-alanme Cyanuric fluoride was purchased from Fluka N-ethylmorpholine Ozonolysis was accomplished using the Welsbach ozonator T-S 16 from The Welsbach Corporation Methyl sulfide Palladmm on barium sulfate N-Carbobenzyloxy-L-phenylalanme N-Carbobenzyloxy-n-alanme N-Carbobenzyloxy-P-alamne

2.3. Reagents for Method 3.3 1 1-Hydroxybenzotriazole 2 Ethyl-3-(3-dimethylammopropyl)carbodumide

hydrochloride

3. Methods 3.1. Synthesis of Vinyl and Ally/ Substituted Proline Derivatives 3a-c Building blocks 3a-c are synthesized by first oxrdrzmg t-Boc protected proline methyl ester to form the N-a-methoxy amide 2 (6,7) The methoxylated amide is then treated with 2-methylpropenyllithium, copper bromtdedtmethylsulftde complex, and BF, + Et,0 at -78°C and the t-Boc group removed to form 3a (s). Similarly, the methoxylated amrde is treated with allyltrrmethylsrlane and BF, * Et,0 at -40°C to 0°C and then the t-Boc group removed to afford 3b and 3c. In this case, the rsomers were separated after the deprotectton step.

3 1.1 N-tert-Butyloxycarbonyl-5Methoxy-L-Proline

Methyl Ester 2

1 Add 7 00 g (30.5 mmol) of the t-Boc protected prolme methyl ester m 61 mL of a 0 03 M solution of tetraethylammonmm tosylate in methanol to a 100 mL three-necked round-bottomed flask equipped with carbon rod anode and platinum wire cathode 2. Degas this reaction mixture by somcation under a slow stream of nitrogen for 10 min, and then electrolyze the solution at a constant current of 26 8 mA until 3 0 F/mol of charge has been passed (see Note 1) 3 Concentrate the solution m VLICUO,and then chromatograph the residue through 225 g of silica gel that was slurry packed with 20% hexane m ether (see Note 2). Elution with the same solvent should afford 7 5 1 g (95%) of the desired product and 270 mg (4%) of the recovered starting material 4. The spectral data should be consistent with that reported previously (10) and should have the followmg NMR data for the mixture of rotomers and methoxy

264

Fobian and Moeller isomers obtamed ‘H NMR (300 MHz, CDCl,) 6 5 30 and 5 29 (2 d, J = 4.6 Hz andJ=45Hz,O7H),5.20(d,J=45Hz,0.15H),515(d,J=46Hz,O15H), 4 32 (m, lH), 3 75 and 3 72 (2 s, 3H), 3 44,3.41,3 39, and 3.36 (4 s, 3H), 2 531 70 (m, 4H), 1 49, 1 43, and 1.41 (3 s, 9H), 13CNMR (75 MHz, CDC13) 6 173.2, 172.9, 172 3, 154 1, 153 9, 89 2, 89 1, 88 4, 88 3, 80 7, 80 5, 59 5, 59 1, 58 8, 58 6, 56 1, 55 8, 55 3, 54 9, 52 0, 51 9, 48 1, 32 8, 32 1, 31 0, 30.0, 28 2, 28 0, 27 9,27 0,26 9

3.1.2. (5s)N-tert-Butyloxycarbonyl-5-(2’-MethylI- Propeny/)L-Proline Methyl Ester (Reaction 6 in Fig. 2 Leading to 3a) 1 To a flame-dried 250 mL flask under argon add 3 1 g ( 154 5 mmol) of hthmm as a 30% dlsperslon m mineral 011, and wash three times with hexane. Then add anhydrous ether (155 mL), and cool the mixture to -20°C 2 To this solution add 10 4 g (77 0 mmol) of 1-bromo-2-methylpropene. 3 After 2 h at -2O”C, cannulate the solution into a 1 L flask contammg a heterogeneous mixture of 15.9 g (77 3 mmol) of copper (I) bromide-dlmethyl sulfide complex m 190 mL of anhydrous ether at 4O“C. 4 Stir the resulting dark brown solution at -40°C for 1 h, cool to -78”C, and then treat the solution with 11 0 g (77.2 mmol) of boron trlfluorlde etherate 5 After 5 mm, add 10 0 g (38 6 mmol) of 2 to the solution and then remove the -7PC bath 6 After I5 mm of additional time, quench the reaction mixture with a 1 1 solution of ammonium hydroxide and saturated ammomum chloride, and dilute with dlchlormethane (see Note 3) 7. Separate the layers, and extract the aqueous layer three times with dlchloromethane Combme the organic layers, dry them over magnesium sulfate, and concentrate them zn vacua 8 Chromatograph the crude product through 500 g of slhca gel that was slurry packed with a 2 1 petroleum ether m ethyl acetate solution Elutlon with the same solvent should afford 9 7 g (89%) of the desired product 9 The spectral data for the mixture of rotomers ‘H NMR (300 MHz, CDCl,) 6513and5.09(2d,J=10OHzandJ=113Hz,1H),474(t,J=83Hz, 05H),463(t,J=77Hz,O5H),439(d,J=83Hz,O5H),431(d,J=77 Hz, 0 5H), 3 72 (s, 3H), 2 35-2 00 (m, 2H), 1 92 (m, IH), 1 74 (s, 1 5H) 1 69 (s, 4.5H), 1 59 (m, IH), 1 41 (s, 1 5H), 1 40 (s, 1 5H), 13C NMR (75 MHz, CDCl,) 6 173.7, 173 4, 154 4, 153 3, 132.8, 131 4, 126.2, 126 1, 79 5, 59.4, 59 0, 55.7, 55.6,51 9, 51.8, 31 5, 30 9,28 8,28 3, 28.2,27 9,25.7,25 5, 17.9, 17.8; IR (neat/NaCl) 2975, 2933, 1748, 1709, 1694, 1448, 1436, 1392, 1366, 1256, 1171, 1151, 1125, 1100; FAB MS m/z (rel intensity) 284 (M + 1,34), 228 (M +I
265

Sicyclic Piperazinone and Related Derivatives 3.1.3. (5S)-5-(2’-Methyl-1 (Reaction C m Fig. 2)

‘-Propenyl)-L-Proline

Methyl Ester 3a

1 To a 100 mL flask, add 3 50 g (12 40 mmol) of the protected methoxylated amide made above m 25 mL of dlchloromethane along with 2.02 g (18.89 mmol) of 2,6-lutldine To this mixture, add 3 57 g (13.5 mmol) of tert-butyldimethylsllyl tnfluoromethanesulfonate After 15 min, quench the reaction by adding saturated ammonium chloride, and dllutmg with ether Separate the layers, and then wash the organic layers with brine, dry over magnesium sulfate, and concentrate zn vacua. Take the resultmg residue up m 25 mL of tetrahydrofuran and cool to 0°C To this mixture, add 450 mg (25.0 mmol) of water, followed by 12 4 mL of a 1 0 M tetrabutylammonmm fluoride m tetrahydrofuran. After 1 h at O”C, quench the reaction with water and dilute with ether Extract the aqueous layer twice with ether, dry the combined organic layers over magnesium sulfate, and concentrate zn vacua. Chromatograph the crude product through 45 g of silica gel that was slurry packed with 10% methanol m ether. Elutlon with the same solvent should afford 1 53 g (68% over 2 steps) of the desired product 3a (see Note 4) 10 The spectral data should be as follows. ‘H NMR (300 MHz, CDCl,) 6 5 11 (d with fine couplmg, J= 8.6 Hz, lH), 3.93 (m, 2H), 3 73 (s, 3H), 2 27 (m, lH), 2.16 (bs, lH), 1 90 (m, 2H) 1 70 (s, 3H), 1 68(s, 3H), 1.43 (m, lH), 13CNMR (75 MHz, CDCI,) 6 175 7, 134.4, 126 6, 58 8, 56 1, 52 0,32.2,29 4, 25 7, 18 I The product was carried on without further characterization

3.7.4. (5R,S)-N-tert-Buty/oxycarbony/-5-(2’-Propenyl)-~-Pro/ine Methyl Ester (React/on D in Fig. 2 Leading to 36 and 3c) 1 In a 500 mL flask, dissolve 15 0 g (57 8 mmol) of 2 m 116 mL of anhydrous ether and cool to -40°C 2. To this solution, add 29.8 g (261.1 mmol) of allyltrimethylsllane, followed by 8.7 g (6 1.O mmol) boron trifluorlde etherate 3. Remove the coolmg bath, and after 30 mm, quench the reaction by adding saturated sodium bicarbonate 4. Extract the aqueous layer twice with ether. 5. Dry the combined organic layers over magnesium sulfate, and then concentrate the crude reaction mixture zn vacua 6 Chromatograph the crude product through 500 g of silica gel that 1sslurry packed with a 3:l petroleum ether m ethyl acetate solution. Elutlon with the same solvent should afford 11 3 g (72%) of the desired product (19) as an -2.1 mixture of isomers (see Note 5) 7 The spectral data for the 2.1 mixture of isomers with rotomers will be as follows. ‘H NMR (300 MHz, CDClJ 6 5 82-5 60 (m, lH), 4 99 (m, 2H), 4 28-4 11 (m, lH), 4.02 (m, 0.2H), 3 90 (bm, 0.6H), 3 78 (bm, 0 2H), 3.67 and 3.65 (2 s, 3H),

Fobian and Moeller

266

2 72-2.34 (bm, lH), 2 20 (m, 2H), 194 (m, 2H), 1.76 (m, lH), 1.40 (s, 3 5H), 1 35 (s, 5 5H); 13CNMR (75 MHz, CDCl,) 6 173 5, 173 2, 153.0, 135 2, 134 9, 134.8, 117 2, 117 1, 116 8, 79 8, 79 7, 79 6, 59.9, 59 7, 59 6, 59.3, 57 9, 57 8, 57 4, 57 3, 51 9, 51 7, 39 0, 38.8, 38 0, 29 4, 28 6, 28 2, 28 1, 27.9, 27 7, 27 3, 26 7 ; IR (neat/NaCl) 3109,2982, 1745, 1640, 1478, 1451, 1437, 1391, 1366, 1332, 1319, 1276, 1258, 1201, 1170, 1125, 1107, 1081, 1036, 1017, 994, 964, 947, 9 13, 860, 77 1,664, FAB MS m/z (rel mtensity) 270 (M + 1, 17), 228 (M CH,CHCH,, 22), 214 (66), 170 (M + 2 +ZH,),COCO, 66), 168 (39), 154 (20) 128 (loo), 110 (22), HRMS FAB calculated for C,,H,,NO, (M + 1) 270 1705, found 270 1696

3.1.5. (5S)-5-(2’-Propenyl)-L-Proline and (5R)-5-(2’-Propenyl)-L-Proline

Methyl Ester 36 Methyl Ester 3c

Deprotect 3 59 g (13.33 mtnol) of the protected ammes generated m the previous step using a procedure identical to that described for the synthesis of compound 3a above. Chromatgraph the crude product through 100 g of silica gel that was slurry packed with 5% methanol m ether Elution with the same solvent should afford a total of 1 27 g (56% over 2 steps) of the desired product as a 1 2 mixture of isomers (trans cu) The isomers can be separated wrth careful chromatography (see Note 6)

The spectral data for 3b were as follows ‘H NMR (300 MHz, CDCl,) 6 5 81 (ddt,J=70,101,171Hz,1H),515and5.05(2d,J=17OHzandJ=106Hz, 2H), 3 86 (dd,J=5 5,8.3 Hz, IH), 3 72 (s, 3H), 3.30 (app p,.J-6 4 Hz, lH), 2 22 (m, 3H), 1 88 (m, 2H), 1 44 (m, 1H); t3C NMR (75 MHz, CDC13) 6 176 1, 135 8, 116.5, 59 0, 57 7, 52 0, 40.7, 30.8, 29 4, FAB MS m/z (rel mtenstty) 171 (M + 2, ll), 170 (M + 1, loo), 168 (ll), 128 (M -CH2CHCH2, 27), 110 (M -CO&H,, 29); HRMS FAB calculated for CgH,6N02 (M + 1) 170 118 1, found 170 118 1 The product was carried on without further characterization (see Subheading

3.3.)

The spectral data for the cis isomer 3c were as follows ‘H NMR (300 MHz, CDCl3)G5.9~575(ddt,J=70,103,171Hz,1H),512and506(2d,J=171 Hz and J= 13 1 Hz, 2H), 3 76 (m, lH), 3 73 (s, 3H), 3 13 (m, lH), 2.38-2 00 (m, 4H), 2 00-l 84 (m, 2H), 1 36 (m, lH), t3C NMR (75 MHz, CDCl,) 6 175 3,135 5, 117 0, 59 8, 59 0, 52 0, 40 0, 3 1.0, 30.0, FAB MS m/z (rel. Intensity) 275 (19), 205 (19), 183 (17), 182 (60), 171 (25), 170(M+ 1, Sl), 156 (loo), 129 (19), 128 (M -CH,CHCH,, 47); HRMS FAB calculated for CgH16N02 (M + 1) 170 118 1, found 170.1186. The product was carried on wlthout further charactertzation (see Subheading

3.2. Synthesis

3.3.)

of the Bicyclic Piperazinone

Derivatives

Once obtained, building block 3a can be converted into a series of btcyclic peptlde mtmetics 7a-d using a simple reductive ammation based procedure (Fig. 3) In the first step of this process, bullding block 3a 1s coupled to the

Bicyclic Piperazinone and Related Derivatives

267

acid fluoride derivative of a second Cbz protected ammo acid derivative. The olefin IS then cleaved by ozonolysis, and the secondary ozonide treated with dlmethylsulfide. The resulting cychzed amino lactol derivative IS reduced to afford the desired building block. 3.2.1. (5S)-N-Benzyloxycarbonyk-Alanme-5-(Z-Methyl1-Propenyl)-L-Proline Methyl Ester 5a 1 In a 50 mL flask, cool a solution of 1.04 g (4.66 mmol) of N-carbobenzyloxyL-alanme and 0 36 g (4.5 1 mmol) of pyrldme m 11 mL of dlchloromethane to -20°C 2 To this mixture, add 2 25 g (16 66 mmol) of cyanuric fluoride 3. After 1 5 h, quench the reactlon by the addition of ice, and then extract the mixture twice with dlchloromethane 4. Dry the organic layer over magnesium sulfate and the concentrate the crude reaction mixture in vucuo Do not concentrate to dryness 5 Take the resulting residue up in 10 mL of dlchloromethane, and then add the mixture to a solution of 652 mg (3.56 mmol) of 3a and 471 mg (4.09 mmol) of N-ethylmorpholine in 6 mL of dlchloromethane at room temperature. 6 After 18 h, wash the reaction mixture with 0 5 M citric acid and 1 N sodium bicarbonate Dry the mixture over magnesmm sulfate, and then concentrate the reactlon zn vucuo Chromatograph the crude product through 30 g of silica gel that was slurry packed with 10% hexane m ether Elutlon with the same solvent should afford 993 mg (79%) of the desired product 5a (see Note 7) The spectral data for the two major rotomers (the product IS a mixture of rotomers) will be as follows. ‘H NMR (300 MHz, CDC13) 6 7.33 (app. bs, 5H), 5.67 (bd, J -7 6 Hz, IH), 5 21 (bd, J-8.8 Hz, lH), 5 07 (s, 2H), 4 72-4.50 (m, 3H), 3 72 (s, 3H), 2.40-2 10 (m, 2.5H), 2.10-1.90 (m, 1.5H), 1.80 and 1.75 (2 s, 6H), 1.40 (d, J= 6.7 Hz, 3H); 13CNMR (75 MHz, CDC&) 6 172.2, 172.0, 155 2, 136 5, 134.9, 128.4, 127.9, 127 8, 124 7, 66.4, 59.1, 56.1, 52 2, 47 8, 32 4, 26.9, 25.6, 18 8, 17 9; IR (neat/NaCl) 2976, 1745, 1722, 1645, 1522, 1507, 1501, 1454, 1435, 1375,1337, 1303, 1284, 1244, 1213,1175,1066,1047,1028; FAB MS m/z (rel mtenslty) 777 (2M + 1, 7), 425 (7), 390 (M + 2, 26), 389 (M + 1, IOO), 274 (3), 182 (6); HRMS FAB calculated for CZ1HZ9N205 (M + 1) 389.2076, found 389 2077

3.2 2. (3S, 6S, 9S)-N-Benzyloxycarbonyl-1,4-Diaza9-Carbomethoxy-5-Hydroxy-3-Methyl-Z-Oxobicyclo[4.3.O]Nonane

6a

1 In a 50 mL flask, dissolve 993 mg (2 56 mmol) of 5a m 14 mL of anhydrous methanol, and cool the mixture to -78’C 2 Bubble ozone through the solution until a blue color 1s observed. After an additional 10 mm, bubble oxygen through the reaction mixture until the blue color disappears

268

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3 Fit the reaction flask with a nitrogen bubbler, and add 609 mg (9 80 mmol) of methyl sulfide to the reaction 4 Star the reaction at room temperature for 18 h, and then concentrate the reaction zn vacua

5 Chromatograph the crude product through 30 g of sthca gel that was slurry packed wtth 5% methanol m ether Elutton with the samesolvent should afford 925 mg (99%) of the desiredproduct (6a) asthe cycbzed hydroxy compound with a mixture of isomers(seeNote 8) 6 The spectral data for the mixture of isomers will be as follows* ‘H NMR (300 MHz, CDCl,) 6 7 35 (bs, 5H), 6.00 and 5 91 (2 bs, lH), 5 16 (s, 2H), 4 53 (2 d, J= 8.2 Hz and J= 7 8 Hz, 2H), 4.06 (bs, 0 5H), 3.92 (m, 1.5H), 3 73 (s, lH), 2 35 (bm, lH), 2.06 (m, 2H), 1 84 (m, lH), 1.54 (d, J= 6 8 Hz, 3H), t3C NMR (75 MHz, CDCls) 6 172 3, 167 2, 154 6, 135 5, 128 8, 128 6, 128 4, 128 3, 128 1, 1279, 736, 732, 679, 677, 61 6, 589, 524, 51 3, 276, 274, 202, 19 3, IR (neat/NaCl) 3378 2991,2952, 2918, 2361, 2340, 1749, 1734, 1700, 1684,1675,1668,1662,1653,1646,1636,1617,1558,1473,1465,1456, 1447, 1437, 1405, 1365, 1313, 1282, 1203, 1178, 1112, 1086, 1072, 1049,997,979; FAB MS m/z (rel mtenstty) 364 (M + 2, 15) 363 (M + 1, loo), 345 (M *H, 17), 149 (19), 128(15), HRMS FAB calculated for C,8H23N206 (M + 1) 363 1556, found 363 1558

3.2.3. (3S, 6S, 9S)- 1,4-Diaza-9-Carbomethoxy3-Methyl-2-Oxoblcyclo[4.3.O]Nonane 7a 1 In a 25 mL flask, mix 846 mg (2 33 mmol) of 6a and -250 mg of palladium on barium sulfate m 6.0 mL of anhydrous methanol (seeNotes 9 and 10) 2. Ftt the reaction flask with a hydrogen balloon, and stir the reaction under a hydrogen atmospherefor 60 h 3 Filter the reaction mixture through a plug of cehte 4 Concentrate the filtrate 112vacua and chromatograph the resulttng oil through 15 g of silica gel that was slurry packed with a 2 1 ether m methanol solution Elution with the same solvent should afford 391 mg (79%) of the desired product 7a with -10% racemizatton of the bridgehead stereocenter (see Note 11) 5 The spectral data for major isomer (6s) will be as follows (see Note 12) ‘H NMR (300 MHz, CDCl,) 6 4 53 (t, J= 8 6 Hz, IH), 3 80 (m, lH), 3 75 (s, 3H), 3.63(q,J=71Hz,1H),323(dd,J=13.1,3.8Hz,1H),270(dd,J=113,94Hz, lH), 2 38 (m, lH), 2 28 (bs, lH), 2.10 (m, lH), 1 82 (m, lH), 1 51 (m, lH), 1 37 (d,J=7.2Hz,3H), ‘3CNMR(75MHz,CDC13)6 1723, 1705,59.8,57.9,51 8, 51 7,43 8,30.1,27 3, 18 5, IR (neat/NaCl) 2939,2908,2876,2853,2803,2794, 2774,2721,2703,2663,2640,2593,2541,2516,2469,2452, 1753, 1668, 1661, 1646, 1576, 1559, 1462, 1438, 1394, 1198, 1177, FAB MS m/z (rel intensity) 425 (2M + I), 213 (M + 1, loo), 185 (34); HRMS FAB calculated for C,,,HN203 (M + 1) 213 1239, found 213 1239

Bicyclic Piperazinone and Related Derivatives 3.2.4. (5S)-N -Benzy/oxycarbonyk 5-(2-Methyl- 1-Propenyl)-L-Proline

269

-PhenylalanineMethyl Ester Sb

1 In a 50 mL flask, couple 972 mg (3 25 mmol) of N-carbobenzyloxy-L-phenylalanine and 468 mg (2.55 mmol) of 3a, usmg a procedure identical to that descrtbed for the synthesis of Sa 2 Chromatograph the crude product obtamed through 30 g of silica gel that was slurry packed with 10% hexane m ether. Elutton with the same solvent should afford 997 mg (84%) of the desired product 5b as a mixture of rotomers 3 The spectral data for the two major rotomers will be as follows. ‘H NMR (300 MHz, CDC13)6 7 29 (m, lOH), 5.66 and 5.52 (2 app. bd, J-9 Hz, HI), 5 16-4 90 (m, 3H), 4.83 and 4 69 (2 m, 1I-Q 4 55 (app bd, J-9 Hz, lH), 4 36 and 4.17 (2 m, IH), 3 76 (bs, 3H), 3 16-284(m,2H),226-200(m,2H), 1.95-l 70(m, H-I), 178,1.75,1.69,andl 64(4s, 6H), 1.65 (munderthe s, 1I-Q t3CNMR(75 MHz,CDC13)G 172.2, 170 9, 155.1, 136 5, 1363, 134 7, 129 8, 128.4, 128 1, 128 0, 127.9, 127 8, 127 0, 126.8, 1243, 66.5, 66.4, 59 4,59 0,56 3,56.1,54 2,53.4,53 1,52.8,52.2,41.1,39 8,32.5,29.6,29 3,27 0,25 5, 180,IR(neat/NaC1)3029,2969,2951,1744,1737,1725,1713,1692,1657, 1649,1642, 1632,1529,1501,1494,1461,1444,1433,1427,1416,1210,1173, ; FAB MS m/z (rel intensity) 466 (M + 2, 33), 465 (M + 1, loo), 421 (15), 210 (32), 184 (29), 182 (31), HRMS FAB calculated for C27H33N205(M + 1) 465.2389, found 465.2383.

3.2.5. (3S, 6S, 9S)-N -Benzyloxycarbonyl- 1,4-Diaza-3-Benzyl9-Carbomethoxy-5-Hydroxy-2-Oxobicyclo[4.3.O]Nonane 6b 1 In a 50 mL flask, cleave the double bond m 896 mg (1 93 mmol) of Sb using a procedure tdenttcal to the one described for the ozonolysts of 5a above. 2 Chromatograph the crude product 6b through 25 g of silica gel that was slurry packed with 10% methanol m ether Elutton with the same solvent should afford 834 mg (98%) of the desired cychzed hydroxy compound 6b as a mixture of rsomers. 3 The spectral data for the mixture of isomers will be as follows ‘H NMR (300 MHz, CDCl,) 6 7 34 (m, 3 5H), 7 19 (m with a s at 6 7.16,6.5H), 5 99 (bs, 0 7H), 5.72 (bs, 0 3H), 5 26 (A of an AB pattern, J= 18 Hz, 0.5H), 5 11 (B of an AB pattern, J=l8Hz,O5H),503(d,J=ll8Hz,lH),480(mwtthanapp tatd478,lH), 4.55 (m, 2H), 3.85 (m, lH), 3 73 (s, 3H), 3.32 (m, 2H), 2.31 (m, lH), 2 01-1.60 (m, 3H), t3C NMR (75 MHz, CDCI,) 6 172.3, 166.1, 154 6, 138.5, 135 2, 130 1, 129 8, 128 3, 128 2, 126 7, 126 4, 73 1, 67.8, 61.7, 61 4, 59.0, 56 8, 52.3, 39.7, 36.3,27 5,27.3; FAB MS m/z (rel. Intensity) 440 (M + 2,27), 439 (M + 1, loo), 438 (M, lo), 421 (MaH, 8), 349 (8), 303 (8), 154 (14), 149 (9), 137 (lo), 136 (12), HRMS FAB calculated for C,,H,,N,O, (M + 1) 439.1869; found 439.1877 The product was carried on without further characterization.

3.2.6. (3S, 6S, 9S)-1,4-Diaza-3-Benzyl9-Carbomethoxy-2-Oxobicyc/o[4.3.O]Nonane

7b

1. In a 25 mL flask, reduce 2 10 mg (0.48 mmol) of 6b in a fashion identical to the reduction of 6a outlined above

270

Fobian and Moeller

2 Chromatograph the crude product through 15 g of sihca gel that was slurry packed with a 2.1 ether in methanol solution. Elutton with the same solvent afforded 78 mg (57%) of the desired product 7b with -10% racemtzation of the bridgehead stereocenter (see Note 11) 3 The spectral data for the maJor isomer (6s) ~111be as follows* ‘H NMR (300 MHz, CDCl,) 6 7 27 (m, 5H), 4.56 (t, J= 8 5 Hz, IH), 3 76 (m, 2H), 3 76 (s, 3H), 3 24 (dd,J= 13 7,3.3Hz, lH),3 ll(dd,J= 12.6,3 8Hz, lH),2 89(dd,J= 134,104Hz, lH), 2.56 (dd,J= 12.4, 10.2 Hz, lH), 2.38 (m, lH), 2.06 (m, lH), 1 84 (m, lH), 1 44 (m, lH), i3C NMR (75 MHz, CDCl,) 6 172 5, 169 3, 138.7, 129.2, 128 5, 126 5, 59.9, 58 0, 57 9, 52.2,44.1, 37 7, 30.2, 25 6, IR (neat/NaCl) 295 1, 2925, 2869,2853,2361,2340,1745,1700,1644,1604,1497,1452,1436,1368, 1322, 1278, 1201, 1177, 1139, 1094, 1082, 1030; FAB MS m/z (rel intensity) 289 (M + 1, loo), 261 (54) 197 (35), 142 (39); HRMS FAB calculated for ClgH2,N203 (M + 1) 289 1552, found 289 1553

3.2.7. (5S)-N -Benzyioxycarbonyi-o-Aian~ne5(2-Methyl- 1-Propenyi)-L-Proline Methyl Ester 5c 1. In a 50 mL flask, couple 724 mg (3 24 mmol) of N-carbobenzyloxy-n-alanme and 494 mg (2 70 mmol) of 3a m a fashion tdenttcle to that described for the syntheses of Sa. 2 Chromatograph the crude product through 30 g of sthca gel that was slurry packed with 10% hexane m ether. Elutron with the same solvent should afford 792 mg (75%) of the desired product 5c 3 The spectral data for the major rotomer will be as follows: ]H NMR (300 MHz, CDCl,) 6 7.34 (app. bs, 5H), 5.50 (d, J= 8 8 Hz, lH), 5 22-4 95 (m with a s at 6 5 10,4H), 4 56-4.45 (m, 2H), 3 70 (s, 3H), 2 44-2 14 (m, 2H), 2 05 (bs, lH), 2 02-l .92 (m, IH), 1 83 and 1 74 (2 s, 6H), 1 74 (m under the s, lH), 1 23 (d, J= 6 7 Hz, 3H); 13C NMR (75 MHz, CDCl,) 6 172 4, 172 1, 155 4, 136.4, 134 1, 128 4, 127 9, 127 8, 125.3, 66 5, 59.3, 56 5, 52.1, 47 9, 32 2, 27 2, 25 6, 18 9, 18.0; IR (neat/NaCl) 3291, 2927, 2855, 1646, 1636, 1617, 1576, 1559, 1540, 1533,1522,1506, 1497, 1490, 1473, 1457, 1448, 1436, 1430, 1424, 1420, 1374, 1242, 1211, 1173, 1067, FAB MS m/z (rel Intensity) 777 (2M + 1, 7), 425 (7), 390 (M + 2,23), 389 (M + 1, loo), 274 (4), 182 (12); HRMS FAB calculated for C,,H,,N,O, (M + 1) 389 2076; found 389.2073

3.2.8. (3R, 6S, 9S)-N-Benzyloxycarbonyl- 7,4-Diaza9-Carbomethoxy-5-Hydroxy-3-Methyl-2-Oxobicycio[4.3.O]Nonane

6c

1. In a 50 mL flask, cleave the double bond in 699 mg (1.80 mmol) of 5c by ozonolysts using the procedure outlined above for the synthesis of 6a 2. Chromatograph the crude product through 20 g of silica gel that was slurry packed with 10% methanol m ether Elution with the same solvent should afford 635 mg (97%) of the desired product 6c as the cychzed hydroxy compound The product will be a single isomer with no apparent racemizatton of the bridgehead stereocenter

B~cyclrc Piperazlnone and Related Deriva bves

271

3 The spectral data should be as follows: ‘H NMR (300 MHz, CDCI,) 6 7.36 (app bs, 5H), 5 18 (app s, 1 5H), 5 11 (app d, J-8.4 Hz, 1.5H), 4 83 (bm, lH, OH), 4 64 (bm, IH), 4 53 (dd, J= 5 2,8.1 Hz, lH), 3.98 (dd, J= 7 1, 15.1 Hz, lH), 3 75 (s, 3H), 2 47 (m, lH), 2.27 (m, lH), 1.97 (m, 2H), 1.52 (d, J= 7 4 Hz, 3H), t3C NMR(75 MHz, CDCl,) 6 171 6, 1684, 135.5, 1286, 1284, 1280, 809, 67 8, 58 1, 54 0, 52 4, 30 2, 27 4, 18 1; FAB MS m/z (rel mtenstty) 364 (M + 2, 20), 363 (M + 1, loo), 345 (MaH, 17), 273 (17), 154 (21), HRMS FAB calculated for C,,H,,N,O, (M + 1) 363 1556; found 363.1568 The product was carried on without further charactertzatlon.

3.2.9. (3R, 6S, 9S)-1,4-Dlaza-3-Benzyl9-Carbomethoxy-2-Oxobicyclo[4.3.O]Nonane

7c

1 In a 25 mL flask, reduce 89 mg (0 25 mmol) of 6c m a fashion identmal to the reduction of 6c descrtbed above 2 Chromatograph the crude product through 2.5 g of sthca gel that was slurry packed with a 2.1 ether m methanol solution. Elution with the same solvent should afford 43 mg (82%) of the desired product 7c with retention of the brtdgehead stereocenter (see Note 13) 3 The spectral data will be as follows. ‘H NMR (300 MHz, CDC13) 6 4 57 (t, J= 8 8 Hz, IH), 3 82 (m, lH), 3 76 (s, 3H), 3.46 (q, J= 7.6 Hz, lH), 3 37 (dd, J = 12 8, 4 0 Hz, lH), 2 59 (dd, J= 12 8, 10.4 Hz, lH), 2 39 (dt, J= 13, 8 Hz, lH), 2 09 (m, lH), 1 83 (m ,2H), 1.48 (m, lH), 1.42 (d, J= 7 0 Hz, 3H); i3C NMR (75 MHz, CDCI,) 6 172 5, 170 3,60 2,58.0,54 4,52 3,48.4,30 4,27 8, 18 3, IR (neat/NaCl) 2953,2944,2917, 1734,1653,1641, 1616, 1576, 1560,1540,1457, 1437, 1387, 1374, 1363, 1202, 1177, 1100, 1060 ; FAB MS m/z (rel intensity) 425 (2M + 1, 6), 392 (22), 344 (25), 213 (M + 1, loo), 185 (29), HRMS FAB calculated for C,,H,,N,O, (M + 1) 213 1239, found 213 1240

3.2 70. (5S)-N -Benzy/oxycarbony/-P-A/anine5-(Z-Methyl- 1-Propenyl)-L-Proline Methyl Ester 5d 1 In a 25 mL flask, couple 723 mg (3.24 mmol) of N-carbobenzyloxy-P-alanme to 479 mg (2 61 mmol) of 3a usmg the procedure described above for the syntheses of 5a. 2. Chromatograph the crude product through 30 g of sihca gel that was slurry packed wtth 10% hexane in ether Elutton with the same solvent should afford 92 1 mg (9 1%) of the desired product 5d 3 The spectral data for the maJor rotomer will be as follows ‘H NMR (300 MHz, CDCl,) 6 7 34 (m, 5H), 5 70 (app bt, J-5.8 Hz, lH), 5 11 (d under the s, lH), 5 09 (2 s, 2H), 4 63 (t, J= 8 2 Hz, lH), 4.54 (d, J= 8 7 Hz, lH), 3.72 (s, 3H), 3.46 (m, 2H), 2 47 (m, 2H), 2 23 (m, 2H), 1.95 (m, IH), 1.71 and 1.68 (2 s, 6H), 1.69 (m under the 2 s, IH), i3C NMR (75 MHz, CDC13) 6 172.6, 171 2, 156 3, 136 7, 133 9, 128.3, 127.8, 124 9, 66.3, 58 9, 56.3, 52.5, 36.7, 33 8, 32.3, 27.1, 25.5, 17.8; IR (neat/NaCl) 3291, 2945, 2855, 1632, 1613, 1547, 1529, 1513, 1502, 1460,1440,1433,1416, 1376, 1366,1327,1304, 1257, 1212, 1191, 1175,1140,

272

Fobian and Moeller 1076, 1066, 1009; FAB MS m/z (rel intensity) 390 (M + 2, 32), 389 (M + 1, loo), 345 (8), 255 (8); HRMS FAB calculated for CZIHZ9N205 (M + 1) 389 2076, found 389 2079.

3.2.17. (7S, lOS)-N-Benzyloxycarbonyl-l,XIiazalo-Carbomethoxy-6-Hydroxy-2-Oxobicyclo[S 3.O]Decane 6d 1 In a 50 mL flask, ozonolyze 806 mg (2 08 mmol) of 5d using the procedure described above for the synthesis of 6a 2 Chromatograph the crude product through 20 g of silica gel that was slurry packed with 5% methanol m ether. Elution with the same solvent should afford 665 mg (88%) of the product 6d. 3 The spectral data will be as follows ‘H NMR (300 MHz, CDCI,) 6 7 35 (bs, 5H), 5.72 (bs, lH), 5 14 (s, 2H), 4 57 (d,J= 8 2 Hz, lH), 4 23 (bs, lH), 4 14 (d, J= 8 7 Hz, lH), 4 02 (bm, lH), 3 71 (s, 3H), 3 48 (m, lH), 2 62 (m, 2H), 2 35 (m, 2H), 2 01 (bm, lH), 1 84 (bm, 1H); 13CNMR (75 MHz, CDC13)6 172 7, 172.3,155 0,135.9, 128 6, 128 3,127 7,78 5,67.8,61 6,60 9,52 2,39 8,38 5,35 8,29 9,27 2; FAB MS m/

z (rel intensity) 363 (M + 1, loo), 279 (45), 2465 (46), 185 (52), 154 (65), 137 (56), 136 (51), HRMS FAB calculated for C,8H23N206 (M + 1) 363 1556, found 363.1557 The product was carried on without further characterlzatlon

3.2.12. (7s + 7R, lOS)-N-1,5-Dlazalo-Carbomethoxy-2-Oxoblcyc/o[5 3.O]Decane 7d 1, In a 25 mL flask, reduce 344 mg (0 95 mmol) of 6d usmg the procedure used to synthesize 7a above 2 Chromatograph the crude product through 6 g of slhca gel that was slurry packed with a 1 1 methanol in dlchloromethane solution. Elutlon with the samesolvent should afford 159 mg (79%) of the desired product 7d with -33% racemlzatlon of the bridgehead stereocenter(see Notes 12 and 14). 3 The spectral data for the 2 1 mixture of isomers(H7a H7b) will be asfollows. ‘H NMR (300 MHz, CD,OD) 6 4 58 (d, J= 8 3 Hz, 0 33H), 4 48 (dd, J= 7 4,4 2 Hz, 0 67H), 4.18-3 9 1 (m, 0 67H), 3 90 (m 0 33H), 3 72 and 3 70 (2 s, 3H), 3 172 96 (m, 2H), 2 95-2 56 (m, 2H), 2 47 (m, lH), 2 40-l 84 (m, 4H), 1 79-l 58 (m, lH), 13CNMR (75 MHz, CD30D) 6 175 9, 174 1, 67 6, 62 4, 62 3, 62 1, 61 7, 550,545,528,527,444,44.1,416,413,31.1,310,286,28.5,IR,FABMS m/z (rel mtenslty) 425 (2M + 1, 5), 391 (18), 371 (24), 213 (M + 1, loo), 199 (17), 150 (45), 130 (83), HRMS FAB calculated for C,,H,,N,03 (M + l), 213 1239; found 213 1238

3.3. Synthesis of 1,4-Diazabicyclo[5.3.O]Decane Building Blocks The chemistry reported above was also very effective for making 1,4dlazablcyclo[5.3.0]decane building blocks (Fig. 4). This sequence took advantage of the 5-allyl-substituted prolme methyl ester derlvatlves 3b and 3c

Sicyclic Plperazinone and Related Deriva twes

273

described above The reductive ammation sequence used to convert 3b and 3c mto the blcyclic analogs lla,b was virtually Identical to that described for the synthesis of 7a-d (see Note 15). 3.3.1. (5/?)-N-Benzy/oxycarbonyl-~-A/anine5-(2’-Propenyl)-L-Proline Methyl Ester 9a In a 50 mL flask, dissolve 502 mg (2 97 mmol) of 3c, 726 mg (3.25 mmol) of N-carbobenzyloxy-L-alanme, and 48 1 mg (3.56 mmol) of 1-hydroxybenzotriazole in 12 mL of dichloromethane and cool the mixture to 0°C To this solution, add 681 mg (3 55 mmol) of ethyl-3-(3-dimethylammopropyl) carbodnmtde hydrochloride Stir the reaction mixture at room temperature for 66 h Wash the crude reaction with 1 N sodium bicarbonate, 0 5 M citric acid, 1 N sodmm bicarbonate, and water. Dry the organic layer over magnesium sulfate and then concentrate it zn vacua. Chromatograph the crude product through 35 g of sihca gel that was slurry packed with ether. Elution with ether should afford 1.07 g (96%) of the desired product 9a The product will be obtained as a mixture of rotomers The spectral data for the two maJor rotomers will be as follows ‘H NMR (300 MHz, CDCI,) 6 7 30 (app s, 5H), 6 30 (d, J-8 4 Hz, 0.75H), 6 05 (d, J-8 3 Hz, 0 25H), 5.90-5.66 (m, IH), 5 21-4.95 (m, 4H), 4.58 (app t, J -8 Hz, 0 75H), 4.3&4 26 (m, 2H), 4 15 (m, 0 25H), 3.68 (s, 3H), 2 88 (m, 0.25H), 2 48-2.05 (m, 3H), 2 00-l 64 (m, 2 75H), 1 36 (d, J = 6.8 Hz, 2 25H), 1.27 (d, J = 6 8 Hz, 0 75H), 13C NMR (75 MHz, CDC13) 6 172.1, 171 8, 170.8, 155 5, 154 8, 136 0, 135 9, 134.3, 133 6, 127 9, 127 4, 127.3, 117.8, 116.6, 66 0, 59 1, 58.8, 58 2, 57.6,52 1,51 6,48.1,470,38.8,373,29.1,287,277,26.3,182,18 l,IR(neat/ NaCl) 3284,3033,2978,2952,1748,1713,1640,1529,1501,1453, 1434,1370, 1327, 1288, 1245, 1214, 1202, 1175, 1117, 1068, 1045, 1021, 918, 743, 698, 664; FAB MS m/z (rel. intensity) 377 (M + 3, 14), 376 (M+2, 51), 375 (M + 1, loo), 331 (lo), 267 (9), 260 (31), 218 (30), 197 (lo), HRMS FAB calculated for C2sH2,N205 (M + 1) 375 1920; found 375 1919.

3.3.2. (5R)-N -Benzyloxycarbonyl-L-Alanine5-(2’-Oxoethyl)-L-Proline Methyl ester 1Oa 1. In a 100 mL flask, cleave the double bond m 1.10 g (2 94 mmol) of 9a using the ozonolysis procedure outlmed above for the synthesis of 6a. 2 Chromatograph the crude product through 35 g of silica gel that was slurry packed with 10% methanol m ether Elutton with the same solvent should afford the desired product 10a as a mixture of the open chain aldehyde and products that appeared to have picked up methanol. 3. Carry the product forward without further purification. 4. As a check, the mass spectral data for the mixture of products should be as follows: FAB MS m/z (rel. intensity) 391 (50), 377 (M + 1,23), 315 (17), 218 (22), 188 (29), 154 (43), 149 (24), 142 (38), 137 (28), 136 (40), 134 (24), 128 (loo),

Fobian and Moeller

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HRMS FAB calculated for C,,H,,N,O, (M + 1) 377.1712, found 377.1704 Obtain full characterization data following the next step

3.3.3. (3S, 7R, 1OS)- 1,4-Diaza- lo-Carbomethoxy3-Methyl2-Oxo-Bicyclo[S. 3.O]Decane 1la 1 In a 25 mL flask, reduce 606 mg (1.6 1 mmol) of 10a using the hydrogenolysls procedure described above for the synthesis of 7a 2. Chromatograph the crude product through 15 g of silica gel that was slurry packed with a 1.1 methanol m ethyl acetate solution Elution with the same solvent should afford 302 mg (79% over the 2 steps) of the desired product lla A minor product (-10%) will be observable. This product has been tentatively assigned as isomerlc at the methyl side cham (see Note 16). 3 The spectral data for the major isomer will be follows ‘H NMR (300 MHz, CDCls) 6 4.52 (d, JH = 7.5 Hz, lH), 4 09 (app bq, lH), 3 73 (s, 3H), 3.64 (app q, lH), 3 04 (dd,J= 8 2,4.9 Hz, 2H), 2.71 (bs, lH), 2 18-1 78 (m, 6H), 1.43 (d, J= 7 2 Hz, 3H), 13C NMR (75 MHz, CDC13) 6 174 5, 173 0, 61 1, 57.6, 57.1, 52 0, 43 4,35 1,32 3,27 1, 19 6, IR (neatNaC1) 3460,3337,2852,2879,1743, 1657, 1650,1640,1632, 1621,1614, 1440, 1433, 1422, 1416, 1367, 1330,1303, 1288, 1199, 1177, 1131, 1095, 1175, 1019, 1001,789,760, FAB MS m/z (rel mtensity) 453 (2M + 1, 16), 241 (14), 228 (lo), 227 (M + 1, 100) 226 (M+, 124), 199 (36), 197 (19), 183 (23), 142 (67), 139 (32 ), 128 (23), HRMS FAB calculated for C, ,H,,N,O, (M + 1) 227 1396, found 227 1396

3.3.4. (5R)-N -Benzyloxycarbonyl-L -Phenylalanine5-(2’-Propenyl)-L-Proline Methyl Ester 9b 1 In a 50 mL flask, couple 708 mg (4 18 mmol) of 3c to 1 40 g (4 74 mmol) of N-carbobenzyloxy-L-alanme using the procedure descrtbed for the synthesis of 9a 2 Chromatograph the crude product through 75 g of silica gel that was slurry packed with ether Elution with ether should afford 1 79 g (95%) of the desired product 9b The product was a mtxture of rotomers 3 The spectral data for the two maJor rotomers will be as follows ‘H NMR (300 MHz, CDC13) 6 7 24 (m, 10H) ,6 14 (d, J= 9 0 Hz, 0 6H), 5 86 (d, J= 9 0 Hz, 0.4H), 5 69 (m, J = 8 2 Hz, lH), 5 12-4.88 (m, 4H), 4.70 (app. q, J -7.7 Hz, 0 6H), 4 57-4.34 (m, 1.6H), 4 04-3 90 (m, 0 4H), 3 70 and 3.62 (2 s, 3H), 3.54 (m, 0 4H), 3 19 (A of an ABX pattern, J= 6.6, 13 7 Hz, 0.6H), 3 10-2 86 (m, 1 6H), 2.77 (m, 04H), 2.20-l 75 (m, 4,4H), 1.60-1.40 (m, 1H); i3C NMR (75 MHz, CDC13) 6 172.3, 171 8, 171.0, 170.2, 155 8, 155 0, 136 5, 136 3, 136 1, 136 0, 134 5, 133.9, 129.3, 129 2, 128 4, 128.2, 127 7, 127 5, 126.8, 126.6, 117.9, 117 0, 66 4, 59.3, 59.1, 58 0, 54.0, 53 3, 52 4, 51.9,40.6, 38 9, 38.6, 37.2, 29 2, 28 0, 27 6,26 6,1R(neat/NaC1)3278,3063,3030,2977,2951,1747,1720,1714,1638, 1632,1585,1562, 1530,1498, 1445, 1435, 1366, 1324, 1280, 1248, 1202, 1174, 1114, 1082, 1040, 1028, 1002,918, 786,749, 699,665, FAB MS m/z (rel mtensny) 452 (M + 2,23), 451 (M + 1, loo), 343 (M -C6H5CH20, S), 260 (lo), 2 18

Sicyck Piperazinone and Related Derivatives

275

(12), 210 (19); HRMS FAB calculated for C&H3,N205 (M + 1) 451 2233; found 451 2231

3.3.5. (5R)-N-Benzy/oxycarbony/-~-Pheny/a/anine5-(2’-Oxoethy/)-L-Proline Methyl Ester 106 1. In a 50 mL flask, cleave the double bond m 1.53 g (3 40 mmol) of 9b using the ozonolysis procedure described above for the synthesis of 6a 2. Chromatograph the crude product through 50 g of silica gel that was slurry packed with 5% methanol m ether Elution with the same solvent should afford 1.33 g (86%) of the desired product lob which was carried on without characterization

3.3.6. (3S, 7R, 1OS)- 1,4-Diaza-3-Benzyllo-Carbomethoxy-2-Oxo-Bicyclo[5.3.O]Decane

7 Ib

1 In a 25 mL flask, reduce 1 33 g (2 94 mmol) of lob usmg the procedure described above for the synthesis of 7a 2 Chromatograph the crude product through 30 g of sihca gel that was slurry packed with a 1 1 methanol

m ethyl acetate solutlon

Elution with the same solvent should afford

673 mg (76%) of the desired product llb as a 1: 1 mixture of stereoisomers that have tentatively been assigned as isomeric at the benzyl side chain (see Notes 17 and 18) 3 The spectral data for the isomers ~111be as follows. ‘H NMR (300 MHz, CDCls) 6 7.3 1 (m, 5H), 5 99 (bs, 0.5H), 4.49 (d, J= 8 1 Hz, IH), 4.23 (dd,J= 3 0, 10.3 Hz, 0.5H), 4 10 (m, 0 5H), 4.01 (m, 0 5H), 3 84 (bs, 1.5H), 3 76 (s, 3H), 3 73 (m, 0 5H), 3.60 (dd, J= 3 5, 14 6 Hz, 0 5H), 3 46 (dd,J= 3 8, 14 2 Hz, 0 SH), 3 lo2 76 (m, 2 5H), 2 41-I 82 (m, 5.5H); i3C NMR (75 MHz, CDC13) 6 173 2,170.1, 166 2, 138 0, 135 9, 129 2, 129 1, 128 7, 127 6, 126 8, 63.4, 61.4, 60 0, 56.5, 54 1, 52.2, 44.1, 38 1, 37 2, 36 1, 34 3, 32.1, 29 0, 27 2, 25 3; IR (neat/iVaCl) 2951,2879,2850,1734,1699,1684,1675,1669,1663,1653,1646,1636, 1456, 1436, 1419, 1399, 1280, 1201, 1178, 1030; FAB MS m/z (rel. intensity) 304 (M +2,22),303(M~1,100),275(19)215(18),211(M+-C~H,CH~,34),183(18), 154 (23), 142 (55), 134 (25), 128 (27); HRMS FAB calculated for C17HZ3Nt03 (M + 1) 303 1710, found 303.1708 4. Notes 1 For convenience, this reaction can be readily accomplished with the use of a 6volt lantern battery (9) 2. The methoxylated amides are not stable for long periods, if left crude. They have a much better shelf life if purified. 3 If this reaction is left for longer time pertods, a rearrangement occurs that converts the product from the 5-methoxylated derivative to the t-Boc protected 2-methoxyL-proline methyl ester It appears that the addition of the 2-methylpropenyl amon to the N-acyhminmm is reversible 4. This two step procedure led to much higher yields than the more common one step deprotection conditions utilizing trifluoroacetic acid For the 5-ally1 substi-

276

5.

10.

11

12.

13 14

15

16.

17.

Fobian and Moeller tuted cases (3b and 3c), the deprotection could also be done usmg BF, * Et,0 m Et,0 at room temperature overnight In this case, a small amount of the 2-methoxy-L-prolme methyl ester was agam formed However, m this case, the formation of the 5-allylsubstituted derivative was found to not be reversible Extended reaction times led to no Increase m the extent of 2-methoxy product generated The ham product eluted off the column first The stereochemistry of the two isomers were assigned using NOESY experiments on the final bicyclic products. The yields using the acid fluoride derivative were much higher than either procedures that utilized HOBT esters or mixed anhydrides The products from the ozonolysis were carefully chromatographed m order to remove all traces of Me+ This was done to avoid poisonmg the Pd catalyst m the subsequent hydrogenolysis reaction. The choice of catalyst was crucial to the success of these reactions Hydrogenolysis of the bicychc precursor with either palladmm on carbon or palladium hydroxide on carbon (Pearlman’s catalyst) resulted m only recovery of the startmg material m high yield The use of the Cbz protecting group was important for completion of the reductive ammation When a t-Boc group was used, the mtrogen was not deprotected and the N-a-hydroxy group was not removed during the hydrogenolysts reaction The vmyl-substnuted prolme used as the starting material for this sequence was a single isomer. Apparently, some of the material epimenzed at the bridgehead durmg the ozonolysis reaction. This epimenzation must have occured after the aldehyde was formed, but before the cychzation reaction to form the six-membered ring The complete carbon connectivities of all new bicychc products were assigned by the use of HMQC and HMBC experiments (5). The stereochemistry of the bicychc products was assigned with the use of NOESY experiments (4) In this case, no epimerization of the bridgehead stereocenter was observed. Evidently, the cychzation was faster m this example In thus case, the extent of bridgehead racemization was attributed to the longer time needed for the mtermediate aldehyde to cychze to a seven-membered rmg product This would afford greater time for the eptmerlzation reaction The route was virtually identical, except that m these cases, the ozonlysis reaction to form lOa,b did not automatically cychze to form the bicychc rmg skeleton. Instead, the open chain aldehyde was obtained. The second rmg formed successfully durmg the reductive ammation step This assignment was based on the overall appearance of the ‘H NMR spectrum of the mmor product vs the major product The chief differences m the spectra were centered around the methyl side cham Eptmerization of the prolme 2-position would have led to a trans-substituted prolme, hence, dramatic differences m the prolme region (as observed m the earher trans products) The NMR of the proime region remained consistent with cu-substitution This assignment was agam made by comparison of the proton NMR spectra for the two isomers. In this case, the major changes involved the benzyhc substituent

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18 It 1s important to note that none of the truns-substituted prolme derivatives synthesized to date have been plagued by the epimerrzation reaction that Interferes with the constructton of 1 la and llb.

References la LI, W and Moeller, K. D (1996) Conformationally restricted TRH analogs. the compatiblhty of a 6,5-btcychc lactam-based mimetic wtth bmdmg to TRH-R. J Am. Chem Sot 118, 10106-10112. lb Rutledge, L D., Perlman, J. H , Gershengorn, M C., Marshall, G R , and Moeller, K. D (1996) Conformationally restricted TRH analogs. a probe for the pyroglutamate regton J Med Chem 39, 157 l-l 574. 2a Wolf, J P and Rapoport, H (1989) Conformationally constramed peptrdes Chrrospecrfic synthesis of 4-alkyl-substituted-lactam-brtdged drpeptides from Lasparttc acid J Org Chem 54,3164-3 173 2b Frerdmger, R M , Perlow, D S , and Veber, D F (1982) Protected la&am-bndged dtpepttdes for use asconformatronal constramts m pepttdes J Org Chem 47,104-l 09 2c Freidmger, R. M (1985) Synthesis of y-lactam-constrained tryptophyllysine derivatives J Org Chem 50, 3631-3633 2d Zydowsky, T M , Dellarta, J F , Jr , and Nellans, H N. (1988) Efliclent and versatile synthesis of dipeptide isosteres contaming y- or b-lactams. J. Org Chem 53,5607-5616 2e Kemp, D. J and Condon, S L. (1990) Synthesis of rtgid, teterocychc drpepttde analogues J Org Chem 55, 1390-1394 2f Kemp, D S and McNamara, P E (1985) Conformatronally restrtcted cyclic nonapeptides denved from L-cysteme and LL-3-ammo-2-prperidone-6-carboxyhc acid (LL-Acp), a potent @turn-mducmg dtpeptrde analogue. J Org Chem 50,5834-5838 2iT Nagar, U and Sato, K. (1985) Synthesis of a brcyclrc dipeptide with the shape of p-turn central part Tetrahedron Lett 26, 647-650 2h Hinds, M G , Richards, N G. J , and Robmson, J A. (1988) Design and synthesis of a novel Peptrde p-turn mimetic. J Chem Sot , Chem. Commun 1988, 1447-1449 21 Paul, P. K C., Burney, P A., Campbell, M. M , and Osguthorpe, D J. (1992) Design of a natural cu-peptrde bond motif to form type VI s-turn mimetic. Bzoorg Med Chem Lett 2,141-144. 25 Ward, P , Ewan, G B , Jordon, C C , Ireland, S. J., Hagan, R M , and Brown, J R (1990) Potent and highly selective neurokmm antagomsts. J Med Chem 33, 1848-1851 2k Flynn, G A , Glroux, E L , and Dage, R C (1987) An acyliminmm ion cyclrzatron route to a novel conformationally restricted dipeptrde mimtc. applications to angtotensrn-converting enzyme mhrbrtlon J Am Chem Sot 109,7914-7915

21 Flynn, G. A , Burkholder, T P., Huber, E W , and Bey, P (1991) An acyhmmtum ion route to czs and trans “antl” phe-gly drpeptrde mtmettcs. Bzoorg Med. Chem. Lett 1,309-3 12.

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2m Burkholder, T. P , Huber, E W., and Flynn, G A. (1993) The synthesis of 6ammo-5-oxo-7-phenyl1,4-oxazepines as conformationally constrained gauche (-) dipeptide mimetics Bzoorg Med Chem Lett 3,23 l-234 2n Genm, M. J , OJala, W H , Gleason, W. B , and Johnson, R L (1993) Synthesis and crystal structure of a peptidomimetic containing the R-4,4-Spiro lactam typeII p-turn mimic. J Org Chem 58,2334-2337 20 Robl, J A (1994) Peptidomimetic synthesis Utilization of N-acyhmmmm ion cyclization chemistry m the generation of 7,6- and 7,5-fused bicycllc lactams Tetrahedron Lett 35,393-396

2p. Lombart, H. G. and Lubell, W D. (1994) Synthesis of enantiopure a,o-diammo dicarboxylates and azabicycloalkane ammo acids by claisen condensation of a[N-(phenylfluorenyl)ammo] dicarboxylates J. Org Chem 59,6 147-6 149.. 3a DiMaio, J and Belleau, B. (1989) Synthesis of choral piperazm-2-ones as model peptidomimetics J Chem Sot Perkzn Trans I, 1687-1689. 3b Bravo, A , Gomez-Monterrey, I., Gonzalez-Mumz, R., and Garcia-Lopez, M. T (199 1) Studies on N-Deprotection of v(CH2NH) psuedodipeptide methyl esters Cyclization to 2-ketopiperazmes, J Chem Sot Perkzn Trans I, 3 117-3 120 3c Jam, S , SuJatha, K , Rama Krishna, K V , Roy, R , Smgh, J., and Anand, N (1992) Lactam amide acetals XXI Use of pyroglutamic acid and prolme m chn-al synthesis of conformationally constramed piperazones Tetrahedron 48, 4985-4998 3d. Schanen, V , Riche, C., Chiarom, A , Qmroion, J -C , and Husson, H -P. (1994) Asymmetric synthesis. XxX1. Synthesis of 2-substituted piperazmes from choral non-racemic lactams Tetrahedron Lett 35,2533-2536 3e. Kozlowski, M D and Barlett, P A (1996) Formation of the 7-oxa- 1,4, lotrrazatricyclo[8.2 2 5,‘2]tetradecane-2,14-dione rmg system: misrouted synthesis of a peptidomimetic. J Org Chem 61,768 l-7696 4 Fobian, Y M , d’Avignon, D A., and Moeller, K. D (1996) New routes to conformationally restricted peptide buildmg blocks. a convenient preparation of bicycllc piperazmone derivatives Bzoorg Med Chem Lett 6, 3 15-3 18 5a d’Avignon, D A., Hanau, C E , Fobian, Y M., and Moeller, K D (1994) Apphcation of HMBC and HMQC-TOCSY NMR methods to assign the structure of bicyclic-peptide mimetics Coordznatzon Chem 32, 135-144 5b Kao, J , Li, W , and Moeller, K D (1997) Conformational studies and stereochemical assignment of a bicychc lactam contammg peptide fragment by twodimensional NMR spectroscopy. A4agn Reson Chem 35,207-272 6a Shono, T. (1984) Electroorgamc chemistry in orgamc synthesis Tetrahedron 40, 81 l-850. 6b Shono, T., Matsumura, Y , and Tsubata, K (1984) Anodic oxidation of Ncarbomethoxypyrrolidme: 2-methoxy-N-carbomethoxy-pyrrohdine, m Organzc Syntheszs, vol 63 (Saucy, G , ed.), Organic Synthesis, pp 206-213 6c Shono, T (1988) Synthesis of alkaloidal compounds usmg an electrochemical reaction as the key step, m Topzcs zn Current Chemzstry, vol 148 (Steckhan, E , ed ), Springer-Verlag, Berlm, Heidelberg, New York, pp 13 1-15 1

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7a Shono, T Matsumura, Y , and Inoue, K. (1983) Electroorganic chemtstry 7 1 Anodtc a-methoxylatton of N-carbomethoxylated or N-acylated a-ammo acid esters and p-ammo-lactams J Org Chem 48, 1388-1389 7b Thanmg, M. and Wtstrand, L -G. (1986) 181 Synthetic applications of electrochemtcally produced a-methoxyamtdes oxtdation of hydroxyproline dertvatives Helv Chzm Acta 69, 1711-1717 7c Papadopoulos, A , Heyer, J Gmzel K.-D., and Steckhan, E (1989) Regioselecttve anodic oxidatton of N-acyl, N-alkoxycarbonyl, and N-(2-mtrophenylsulfenyl) dtpeptide esters Chem Ber 122,2 159-2 164 7d Ginzel, K.-D , Brungs, P , and Steckhan, E (1989) Indirect electrochemical amethoxylatton of N-acyl and N-carboalkoxy a-ammo actd esters and appltcatton as cattomc glycme eqmvalents Tetrahedron 45, 169 l-l 70 1 7e Papadopoulos, A , Lewall, B , Steckhan, E , Gmzel, K.-D., Knoch, F , and Nteger, M. (199 1) Anodtc oxidation of N-acyl and N-alkoxycarbonyl dtpeptide esters as a key step for the formatton of chiral heterocychc synthetic bmldmg blocks. Tetrahedron 47,563-572. 7f Barrett, A. G. M and Ptllpauskas, D (199 1) Electrochemtcal oxidatton of prolme derivatives, total syntheses of bulgecinme and bulgecm C J Org Chem 56, 2787-2800

8 Thamng, M and Wistrand, L -G. (1992) Intermolecular stereoselective alkenylatton of chnal N-acylpyrrohdmtum tons. Acta Chemzca Scandunavzca 46, 194199 9 Frey, D. A , Wu, N , and Moeller, K D. (1996) Anodic electrochemtstry and the use of a 6-volt lantern battery. a simple method for attempting electrochemtcally based synthetic transformations Tetrahedron Lett 37, 83 17-8320 10 Shono, T , Matsumura, Y , Tsubata, K., Sugihara, Y., Yamane, S.-I , Kanazawa, T , and Aokt, T. (1982) Electroorgamc chemistry. 60. Electroorgamc synthesis of enamides and enecarbamates and their utilization m orgarnc synthesis J Am Chem Sot 104,66974703

Synthesis of Dipeptides with v[CH,O] Amide Bond Mimetics Wieslaw M. Kazmierski and Paul C. Fritch 1. Introduction This chapter describes the synthetic procedures leadmg to ether dipeptide isosteres of the Phe-v[CH20]-Spiro-C, (9a-9e, Fig. 1) and Phe-ilr[CHzO]Allylglycme (13, Fig. 2). Development of methods which lead to mtmetics of amide bonds is central to the conversion of pepttde leads mto pharmaceutically viable molecules. Our strategy utilizes template 1, which provides derivatives 6b-6e upon alkylation with I, Cl-alkanes (C+C,) Subsequent Fmklestem conversion to iodides 7b-7e is then followed by cychzation to spirocychc derivatives 5b-5e (Fig. 3) Compound 5a (cyclopropane derivative, Fig. 4) could not be synthesizedby the above route, owmg to a slow rate of alkylation with 1,2-&Cl-ethane. Instead, 1 was alkylated to 2, converted to an aldehyde by osmium tetroxide-catalyzed oxidation, and then to alcohol 3 by NaBH4-mediated reduction. Finally, compound 3-derived mesylate was converted to 5a in presenceof LDA. Two-stage acidolysis of 5a-5e and 12, resulted m the desired isosteres9a-9e and 13, respectively. 2. Materials Analytical HPLC conditions: Vydac C 18 rphplc, gradient O-l 00% B over 30 mm A = 0.1% TFA m water, B = 0.1% TFA m acetomtrile Flow rate 1 5 mL/mm, detection at 220 and 254 nm. 2 2 2 2 2

1 Reagents 2Reagents 3Reagents 4Reagents 5 Reagents

for for for for for From

Method Method Method Method Method

31 32 33 3 4: 35

23,24,25, 26,27,28, 9, 17, 34,35 1, 2, 26, 6, 7, 8, 9, 10, 12, 14, 23, 3 1,26, 15, 16, 18, 19,20, 3,4, 17,21 22 2,6, 17, 22,26,29, 30, 3 I, 32, 33

Methods m Molecular Medicine, Vol 23 Peptrdomlmetlcs Protocols Edited by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

281

Kazmierskl and Frltch

282 CF,C OzH

5a Sb 50 5d Se

6 N HCI

“=I n=2 n=3 11x4 n=5

0a 0b 8C 6d Be

9a 9b 9c 9d 9e

“=I n=z 63 1114 nS

Figure 1 Synthesis of Phev[CH20]Spiro

lsosteres 9a-9e

Figure 2. Synthesis of Pheu/[CH,O]Allylglycme O---)

13

“‘Ph NC4

LDA “ypr

“Xl n=2 “63 “c4 n=5

N’PMB -

c, SK )”

acetone

0

, 66 1112 6c n=3 6d n=4 6e n=5

THF

7b n=2 7c n=3 7d n=4 7.3 n=5

LDA THF

(0 011

5b 5c 5d se

n=2 n=3 n=4 n=5

Figure 3. Synthesis of morpholmones 1 Lithium dnsopropylethylamine (LDA), furanlethylbenzene 2 Ally1 iodide 3 NH&l 4. Na&O, 5. MgSO, 6 S102 7 OsO,, 2 5 % wt in 2-methyl-2-propanol 8 NaIO, 9 Brine

5b-5e

2 0 M solution m heptaneltetrahydro-

283

Synthesis of Dipeptides

0 THF 1

MsCI,

I

NalO,. OsO,(cat) THF-H,O

2

NaBH, THF-EtOH

HO 3

LDA

lPrzNEt

THF C H,CI,

0

kJo11 Msd

5a

4

Ftgure 4 Synthesis of morpholmone

5a.

10 Na$O, 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34. 35

Ethanol (EtOH). NaBH, Acetone NJ-dusopropylethylamme methanesulfonyl 1-Chloro-3-todopropane NaI. Ethyl acetate (EtOAc) 1-Chloro-4-iodobutane I-Chloro-5-todopentane 1-Chloro-6-todohexane Trtfluoroacetic acid (CF3C02H) HCl, 6.0 N solution. Dlethyhsopropylamme (DIEA) Chloroacetyl chlortde (S)-2-ammo-3-phenyl1-propanol Tetrahydrofurane (THF) p-Methoxy-benzyl chloride Sodium hydride 95% Acetomtrtle. dr-t-butyl-carbonate 4-Drmethylammopyrtdme (DMAP). 1 M hthmm hexamethyldrstlaztde (LHMDS) 4 N HWdioxane Potassmm t-butoxrde Methanol

chloride (MsCl)

in THF

3. Methods

3.1. Procedure for the Preparation of PMB-Morpholinone 1. Dissolve 22 3 g (0.147 M) of (S)-2-amino-3-phenyl-I-propanol, chloroacetyl chloride m 30 mL THF.

7

16.60 g (0 147 M)

Kazmierski and Fritch

284

Cool the solution down to O”C, and add 19 06 g (0 147 M) DIEA Remove the Ice bath, and stir overnight at room temperature (Note 1) Cool again to O”C, and add 18 0 g (0 16 kQ of potassium t-butoxlde Stir for 30 mm at room temperature Remove solvents, and partition residue between ethyl acetate and brine Dry the organic phase with magnesium sulfate. Purify the crude product on slhca gel usmg 1 2 0 1 v/v/v ethyl acetate/hexane/ methanol You should obtain 23 8 g (85%) of the product. Dissolve 5.73 g of the above morpholmone m 5 ml of anhydrous DMF, cool down to O”C, and add portlonwlse 0.72 g of NaH. Stir for 15 mm at room temperature, cool to O”C, and then add 4 70 g ofp-methoxybenzyl chloride Continue stlrrmg at room temperature for 2 h. Purify on slhca gel (1.1 ethyl acetatekexane). Yield (steps 6-8) of compound 1 4 72 g (5 1%). M(AP+) = 3 12.1 (M + 1) ‘H NMR (CDCl,) = 7 2&6 87 (9H, m), 5 42 (IH, d), 3 85 (lH, d), 4.34 (IH, d), 4.20 (d, lH), 3 79 (s, 3H), 3.68 (lH, d), 3 42 (1 H, d), 3 26 (lH, m), 2.95 (2H, m)

3.2. Genera/ Procedure A for the Preparation of Spiromorpholinone 5a 3.2 1 Ally/ Morpholinone 2 Add a solution of 375 5 mg (1 20 mmol) of morpholmone 1 m 1 0 mL of THF dropwlse to a solution of 0.66 mL (1 33 mmol) of 2 0 MLDA m heptaneketrahydrofuranlethylbenzene at -78°C Stir for 30 mm, and add 122 pL (1 33 mmol) of ally1 Iodide dropwlse Allow the reaction mixture to warm to -10°C over 2 h, quench with aqueous NH&l, and evaporate organic solvents zn vucuo Partltlon the aqueous solution between ether (20 mL) and aqueous NH&l (10 mL). Wash the organic phase with aqueous Na&O, (10 mL) and brme (10 mL), dry (MgSO& and concentrate Chromatograph on SiO, (22% EtOAc/hexanes) to afford 340 3 mg (8 1%) of ally1 morpholmone 2 as a mixture of dlastereomers

3.2.2. Morpholinone Alcohol 3 1 To a solution of 340 3 mg ( 970 mmol) of ally1 morpholme 2 m 40 mL of THF/ HZ0 (4/l) at room temperature, add 0.61 mL (0 049 mmol) of a 2 5% wt solution of Os04 m 2-methyl-2-propanol and 436 mg (2.04 mmol) of NaI04 2 Stir the reaction for 1 h, filter, and add 15 mL of HZ0 Remove THF zn V(ICUO, and extract the aqueous solution with CHCl, (40 mL) Wash the organic phase with brine (15 mL), dry (Na$O& and concentrate 3 Purkatlon is achieved by passmg the residue through a short plug of SlO, (1 1 EtOAc/hexanes) to afford crude aldehyde which was used lmmedlately 4 Dissolve the aldehyde m 6 mL ofTHF/EtOH (1 2), treat with 36.7 mg (0 970 mmol) of NaBH4, stir for 15 mm, and then treat with 1 mL of acetone

Synthesis of Dlpeptldes

285

5 Concentrate the reaction mixture, and chromatograph on SO2 (4.1 EtOAc/hexanes) to afford 262.0 mg (76%) of alcohol 3 as a mixture of diastereomers

3.2.3. Morpholinone

Mesylate 4

1. Add a solution of 103 PL (1.33 mmol) of methanesulfonyl chloride m 0 5 mL of CH&l, to a stirred solution of 262.0 mg (0.738 mmol) of alcohol 3 and 232 pL (1 33 mmol) of N,N-diisopropylethylamine m 1.O mL of CH,Cl, at 0°C. 2 Stir the reaction for 30 mm, pour into 5 mL of ice water, and extract with CHC& (15 mL) 3 Wash the organic phase with brine (5 mL), dry (Na,SO,) and concentrate. 4 Chromatograph on SIO~ (36-66% EtOAc/hexanes) to afford 230 mg (72%) of mesylate 4 as a mixture of diastereomers

3.2 4 Spiromorpholinone

5a

1 To a solution of 230 mg (0.529 mmol) of mesylate 4 m 53 mL of THF at -70°C add 304 pL (0.608 mmol) of a 2 0 h4 solution of LDA in heptane/tetrahydrofuran/ethylbenzene (Note 2) 2 Warm the reaction to 0°C over 3 h, and quench with aqueous NH&l 3 Remove orgamc solvents m vucuo, and extract the solution with CHCl, Wash the organic phase with HZ0 and brine, dry (Na*SO,), and concentrate 4 Chromatograph on Si02 (1.3 EtOAc/hexanes) to afford 124 8 mg (74%) of spnomorpholmone 5a as a white solid* Mp 79-80°C [cL]~ = -50.9” (c 1 1, CHCl,, 20°C); IR (neat) 1649,1512,1247 1035 700 cm-‘, ‘H NMR (CDCl,) 6 7 36-7 14 (m, 7 H), 6.90 (d, 2 H, J= 8 7 Hz), 5 43 (d, 1 H, J= 14 7), 3.91 (d, 1 H,J= 14 7), 3 80 (s, 3 H), 3 67-3 60 (m, 2 H), 3 42-3 37 (m, I H), 3 18-3.02 (m, 2 H), 1 621.55 (m, 1 H), 1 3s1.28 (m, 1 H), 1 23-1.14 (m, 1 H), 0 960 87(m, 1 H);13C NMR (CDCls) 6 169 4, 159 0, 137 5, 129.3, 129.2, 128 7, 128 6, 126.6, 114 0, 65.0,60 9,56.8,55 1,46 7,36 7, 17.2, 14 6, MS (ES+) m/e (relative intensity) 376 (M + K, lo), 360 (M + Na, loo), 338 (M + H, 32), 230 (36), 121 (25), Elemental Analysts, calculated C 74 75, H* 6 87, N: 4 15, found C. 74.69, H. 8.84, N* 4 13

3.3. Genera/ Procedure of Spiromorpholinones 3.3.1. Spiromorpholinone

B for the Preparation 5b-5e Figure 3 5b

3 3.1 .I CHLOROMORPHOLINONE 6b 1 Add a solution of 332 7 mg (1 07 mmol) of morpholmone 1 m 1.O mL of THF dropwise to a solution of 0.615 mL (1.23 mmol) of 2 0 M LDA m heptaneitetrahydrofuran/ethylbenzene at -78°C 2 Stir the solution for 30 mm, and add 0.575 mL (5.35 mmol) of 1-chloro-3iodopropane dropwise 3. Allow the reaction mixture to warm to -10°C over 1 h, quench with aqueous NH&l, and evaporate orgamc solvents zn vacua. Partition the aqueous solution between CHCl, (40 mL) and aqueous NH4CI (15 mL). Wash the organic phase with aqueous NazSzOs (15 mL) and brine (15 mL), dry (Na$O,), and concentrate.

286 4

Kazmierski and Fitch Chromatograph on SiO, (30% EtOAc/hexanes) to afford 376 7 mg (91%) of chloromorphohnone 6b as a mixture of diastereomers

33 12

IODOMORPHOLINONE

7b

1 Treat a solution of 376 7 mg (0.972 mmol) of morpholmone 6b m 10 mL of acetone with 1 46 g (9 72 mmol) of NaI 2 Heat the reaction mrxture at reflux for 5 h, cool to room temperature, filter solids, and concentrate 3 Partition the residue between EtOAc (30 mL) and HZ0 (20 mL) Wash the organic phase with brine (15 mL), dry (Na,SOJ, and concentrate 4 Chromatograph through a short plug of St02 (30% EtOAc/hexanes) to afford 413 5 mg (89%) of lodomorpholmone 7b as a mixture of diastereomers

3.3 1 3 SPIROMORPHOLINONE~~ 1 Treat a solution of 407 5 mg (0 850 mmol) of iodomorpholmone 7b m 85 mL of THF at -70°C wtth 0 49 mL (0.98 mmol) of a 2.0 h4 solution of LDA m heptane/ tetrahydrofuratiethylbenzene. 2 Allow the reaction to warm to -1 O’C over 4 h, and quench with aqueous NH&l 3 Evaporate orgamc solvents zn VI~CUO,and extract the solution with CHCl, Wash the orgamc phase with HZ0 and brine, dry (Na,SO,), and concentrate 4 Chromatograph on SIO* (1 4 EtOAc/hexanes) to afford 127 8 mg (45%) of spiromorpholmone 5b as a white sohd Mp 101 5-102 5°C [a]o =-44 6” (c 1 0, CHCls, 2O”C), IR (neat) 2938, 1647, 1513, 1248, 1034, 701 cm-‘, ‘H NMR (CDCls) 6 7.3 l-7 10 (m, 7 H), 6 87 (d, 2 H, J = 8 7 Hz), 5 42 (d, 1 H, J = 14 7 Hz), 3.83 (d, 1 H,J= 14 7,3 78 (s, 3 H), 3 54-3 40 (m, 2 H), 3 24-3 20 (m, 1 H), 3.07-2 88 (m, 2 H), 2 84-2 74 (m, 1 H), 2 66-2 59 (m, 1 H), 2 23-l 92 (m, 4 H), 13C NMR (CDC13) 6 171 2,158 9,137 5,129 2,128 9,128 5,126 5,114 0, 79 7,60 9,56 1, 55 I,46 4,36 6,34.0,32 2, 14 0, MS (ES+) m/e (relative mtensity) 390 (M + K, 12), 374 (M + Na, loo), 352 (M + H, 45), 244(45), 121 (36), Elemental Analysis calculated C 75 19, H 7 17, N* 3 99, found C 74 95, H

7 10,N 403 3 3.2 Spiromorpholinone

5c

1 According to general procedure B, 387 6 mg (1 25 mmol) of morpholmone 1, 0 7 15 mL (1 43 mmol) of a 2 0 M solution of LDA m heptane/tetrahydrofuran/ ethylbenzene,and0.765 mL (6.25 mmol) of I-chloro-4-iodobutaneafford 405 2 mg (8 1Oh)of chloromorpholmone 6c. 2 4052mg(lOl mmol)of6cand 151 g(101 mmol)ofNaIafford4372mg (88%) of todomorpholmone 7c 3. 399.2 mg (0.8 10 mmol) of 7c and 0 465 mL (0 93 1 mmol) of a 2 0 A4 solution of LDA m heptane/tetrahydrofuran/ethylbenzene afford 139 1 mg (50%) of spiromorpholmone 5c as a white sohd. Mp 114-l 14 5°C [a]o = -38.5” (c 1 0, CHC13,2O’C); IR (neat) 2950, 1646, 1513, 1457, 1247, 1175, 1117, 1034, 701 cm-‘, ‘H NMR (CDCl,) 6 7 32-7 13 (m, 7 H), 6 88 (d, 2 H, J= 8 4 Hz), 5 41 (d, 1 H,J= 14 6 Hz, 3 82 (d, 1 H, J= 14 6 Hz), 3 79 (s, 3 H), 3 52 (s, 2 H), 3 28-3 23

287

Synthesis of Dipeptldes

(m, 1 H), 3 10-2 94 (m, 2 H), 2 38-2 33 (m, 1 H), 2 16-2 08 (m, 1 H), 2 O&l 75 (m, 6 H), i3C NMR (CDCl,) 6 172.5, 158 9, 137 6, 129.2, 129.0, 128.5, 126 5, 114.0, 87.3,61.2, 56 3, 55.1,47 0, 39.5, 37 6,36 9,25.3, 25.1, MS (ES+) m/e (relative intensity) 388 (M + Na, loo), 366 (M + H, 87), 258 (50), 121 (23); Elemental Analysts calculated. C 75 59, H: 7.45, N. 3.83, found C* 75 45, H. 7.44, N 3 77

3 3.3. Spiromorpholinone

5d

1 According to general procedure B, 373.2 mg (1 20 mmol) of morpholmone 1, 0 69 mL (1.38 mmol) of a 2 0 M solution of LDA in heptane/tetrahydrofuran/ ethylbenzene, and 0 82 mL (6 00 mmol) of 1-chloro-5-iodopentane afford 464 0 mg (93%) of chloromorpholmone 6d 2 464Omg(l 11 mmol)of6dand 166g(ll 1 mmol)ofNaIafford510.0mg (9 1%) of rodomorpholmone 7d 3 504 0 mg (0 994 mmol) of 7d and 0 57 mL (1.14 mmol) of a 2 0 M solution of LDA in heptane/tetrahydrofuran/ethylbenzene afford 249 mg (70%) of spnomorpholmone 5d as a white sohd: Mp 9&94.5”C, [alo = -32.3” (c 1.0, CHCl,, 2OT); IR (neat) 2929,1646,1513,1455,1249,1108,701 cm-‘; ‘H NMR (CDCl,) S 7.31-7 14 (m, 7 H), 6 86 (d, 2 H,J= 8 4 Hz), 5 36 (d, 1 H,J= 14 7 Hz), 3 78-3 72 (m, 4 H), 3 54 (s, 2 H), 3 24-3 19 (m, 1 H), 3 05-2 93 (m, 2 H), 2 1s 1 95 (m, 2 H), 1.95-1 78 (m, 2 H), 1.70-I 50 (m, 5 H), 1 35-l 28 (m, 1 H), i3C NMR (CHC13) S 172 4, 158 8, 137.6, 129.1, 129.1, 129.0, 128.4, 126.4, 113 8, 78 0, 59 8, 56 3, 54 9, 46 8, 36 8, 34.7, 29.7, 24.9, 20.6, 20 5, MS (ES+) m/e (relative mtensrty) 418 (M + K, 20), 402 (M + Na, 75) 380 (M + H, 100) 272 (42), 121 (27) Elemental Analysis calculated: C. 75.96, H. 7.70, N. 3.69, found C* 75 86, H. 7.69, N* 3 67

3.3 4 Spiromorphollnone

5e

1 According to general procedure B, 343.0 mg (1.10 mmol) of morpholmone 1, 0.635 mL (1.27 mmol) of a 2.0 A4 solution of LDA m heptane/tetrahydrofuran/ ethylbenzene, and 0 86 mL (5 50 mmol) of I-chloro-6-rodohexane afford 359 7 mg (76%) of chloromorpholmone 6e 2 359 7 mg (0 837 mmol) of 6e and 1 25 g (8.37 mmol) of NaI afford 386 7 mg (89%) of iodomorpholmone 7e. 3 307 7 mg (0 590 mmol) of 7e and 0 34 mL (0 68 mmol) of a 2 0 M solution of LDA in heptane/tetrahydrofuran/ethylbenzene afford 97.2 mg (44%) of sprromorpholmone 5e as an oil* [a]o = -36 9” (c 1 1, CHC13, 2OT), IR (neat) 2925, 1644, 1513, 1455, 1247, 1175, 1118,1034,701 cm-‘, ‘H NMR (CDC13) S 7.32-7 14 (m, 7 H), 6.87 (d, 2 H, J= 8.4 Hz), 5.36 (d, 1 H, J= 14.7 Hz), 3 79 (s, 3 H), 3 72 (d, 1 H, J= 14 7 Hz), 3 58-3.47 (m, 2 H), 3 25-3 19 (m, 1 H), 3 072.94 (m, 2 H), 2.28-2.18 (m, 1 H), 2 12-2.02 (m, 2 H), 1 89-1.43 (m, 9 H), i3C NMR(CDC13)S 173 8, 158 9, 137 7, 129 3, 128.5, 126 5, 114.0,81 1,60 0,56 5, 55.1, 46 9, 39 5, 37 0, 34 7, 29 7, 29.6, 23 0, 22.3; MS (ES+) m/e (relative

Kazmierski and Fritch

288

mtenslty) 432 (M + K, 20), 416 (M + K, 44), 394 (M + H, loo), 286 (35), Elemental Analysis calculated: C* 76.30, H 7.94, N 3 56, found C 74 68, H 7 80, N 3 37

3.4. General Procedure C for the Preparation of Phey[CH20]lsosteres (9a-9e) 3.4.1. Phey[CH,O]lsostere 9a 3.4 1.1. MORPHOLINONE 8a 1 Heat the solution of 96 3 mg (0.286 mmol) of splromorpholmone5a m 0.5 mL of CF3C02H at reflux for 2.5 h, cool to room temperature, and concentrate (Note 3) 2 Chromatograph on SIO* (45-55% EtOAc/hexanes) to afford 61 7 mg (99%) of splromorpholmone 8a

3 4 1 2. PHEW[CH~O]ISOSTERE

9a

1 Heat a slurry of 76 3 mg (0 35 1 mmol) of 8a m 2 mL of 6 N HCl m a sealed tube at 95°C for 20 h, cool to room temperature, and evaporate to dryness Dissolve the residue m 10 mL of warm H20, wash with CHCl, (5 mL), and lyophlllze to afford 85 9 mg (90%) of splro lsostere 9a as a white hygroscoplc solld Mp. 215-216”C, [a]o = +3.2” (c 0 5, 1 0 A4 HCl, 20”(Z), IR (KBr) 3244,3163,2846, 1708, 1582, 1510, 1181, 1157, 1073,747,702 cm‘, ‘H NMR (DMSO-de) 6 12.80-12.40 (b, 1 H), 8 25-8 06 (b, 3 H), 7 367 22 (m, 5 H), 3 68-3 63 (m, 1 H), 3 60-3.5 1 (m, 1 H), 3 43-3 14 (b, 1 H + H20), 2.98 (dd, 1 H, J = 13 6, 5 0 Hz), 2 79 (dd, 1 H, J = 13.7, 9.7 Hz), 1 29-l 22 (m, 1 H), 1.19-l. 13 (m, 3 H), 13CNMR (DMSO-d,J 6 174 0, 136 2, 129.2, 128.5, 126 8, 67 4, 59 8, 51.6, 34.9, 15 7, 15.5, MS (ES+) m/ e (relative intensity) 236 (M-Cl, loo), MS (ES-) m/e (relative mtenslty) 272 (M-H, 27), 270 (M-H, 88), 234 (M-H,Cl, 45), 163 (45), 149(47), 133(50), 13 1 (100); HRMS (FAB) m/e calculated for C,,H,,NO, ([M-Cl]+) 236 1287, found. 236.1294

3.4.2. Phery[CH,O]lsostere

96

According to general procedure C, 113 2 mg (0 332 mmol) of splromorpholmone 5b and 0.5 mL of CF3C02H afford 69 3 mg (93%) of spn-omorpholmone8b 2 69.3 mg (0.300 mmol) of 8b and 2 mL of 6 N HCl afford 80 8 mg (94%) of splro lsostere9b asa hygroscoplc white sohd. Mp 155-l 57°C; [a]o = +4 1” (c 0 4, 1.O MHCl, 20”(Z), IR (KBr) 3427 (H,O), 2958,1718, 1493,1202,1159,734,696 cm‘, ‘H NMR (DMSO-de) F 12 95-12.85 (b, 1 H), 8 38-8,08 (b, 3 H), 7 367 24 (m, 5 H), 3 563 22 (m, 3 H, + H20), 3 00 (dd, 1 H, J= 13 4,4.8 Hz), 2 83 (dd, 1 H, J = 13 3,9 4 Hz), 2 36-2 10 (m, 4 H), 1 80-l 72 (m, 2 H), 13CNMR (DMS&d& 6 174.1,136.3, 129.2,128 5,126 8,78.9,62.4,51.8,34 9,30 4,30 3, 12 7, MS (ES+) m/e (relative mtenslty) 250 (M-Cl, loo), 134 (43), MS (ES) m/e (relative mtenslty) 286 (M-H, 16), 284 (M-H, 49), 163(48), 149(45), 131(loo), 117(28); HRMS (FAB) m/e calculated for C14H20N03([M-Cl]+) 250 1443, found 250 1438

289

Synthesis of Dipeptides 3.4.3. Phe&H,O]lsostere

9c

Accordmg to general procedure C, 107 0 mg (0 293 mmol) of spnomorpholinone 5c and 0.5 mL of CF3C02H afford 66.0 mg (92%) of sptromorpholmone 8c. 2 66 0 mg (0 269 mmol) of 8c and 2 mL of 6 NHCl afford 77 1 mg (96%) of Spiro isostere 9c as a hydrophihc white solid: Mp: 178-l 8 1“C, [a],, = +3 7’ (c 0 5, 1.O A4 HCl, 2OT), IR (KBr) 3423 (H,O), 2955,1718,1496,1178,1077,745,701 cm-‘, ‘H NMR (DMSO-d6) 6 12.80-12.60 (b, 1 H), 8 16-7 96 (b, 3 H), 7 35-7 22 (m, 5 H), 3 52-3 24 (m, 3 H + H,O), 2.94 (dd, 1 H,J= 13.7,5.3 Hz), 2 82 (dd, 1 H,J= 13 6,8 9 Hz), 1 92-1.81 (m, 4 H), 1.78-1.60 (m, 4 H); t3C NMR (DMSO-d6) 6 175.2, 136.4, 129.2, 128 5, 126 8, 87 5, 62 9, 52.0, 35 6, 35.1, 34.9, 23 8, 23 8; MS (ES+) m/e (relative intensity) 264 (M-Cl, loo), 134 (49), MS (ES-) m/e (relative intensity) 300 (M-H, 24), 298 (M-H, 70), 262 (55), 181 (32), 163 (39), 149 (52), 131 (loo), 117(30), HRMS (FAB) m/e calculated for C15H22N03([MCl]+). 264.1600, found 264 1611

3.4.4. Phey[CH,O]

lsostere 9d

1, According to general procedure C, 159 3 mg (0 420 mmol) of sptromorpholmone 5d and 0 5 mL of CF3C02H afford 1034 mg (95%) of sptromorpholmone 8d 2 1154 mg (0 446 mmol) of 8d and 2 mL of 6 NHCl afford 1274 mg (91%) of spno isostere9d asa hygroscopicwhite sohd Mp 104-106°C [a]o = +3 0” (c 0.5, 1.OA4 HCl, 2OT), IR (KBr) 3423(H,O), 2955,1718,1496,1178,1077,745,701 cm-‘, ‘H NMR (DMSO-d,) 6 12 90-12 70 (b, 1 H), 8 16-7 84 (b, 3 H), 7.36-7 22 (m, 5 H), 3 58-3 22 (m, 3 H + H20), 3 Ol-2,82 (m, 2 H), 1 80-l 59 (m, 4 H), 1 58-l 37 (m, 5 H), 1 3&l 07 (m, 1 H); t3C NMR (DMSO-d6) 6 175.3, 136.5, 129.2, 128.5, 1268, 78 7,61 8,52 1,35 0,3 1 7,3 1 2,24.7,21 0,20 9, MS (ES+) m/e (relative intensity) 278 (M-Cl, loo), 134(24), MS (ES-) m/e(relative intensity) 314 (M-H, lo), 3 12(MH, 23), 276 (M-H&l, 69), 235 (17), 217 (18), 181(32), 163 (58), 113(100); HRMS (FAB) m/e calculatedfor Ct6Hz4N03([M-Cl]+). 278.1756,found: 278 1758

3.4.5. Phey[CH,O]

lsostere 9e

1 Accordmg to general procedure C, 7 1.2 mg (0.18 1 mmol) of spiromorpholmone 5e and 0 5 mL of CF3C02H afford 42 5 mg (86%) of spiromorpholmone 8e 2 42 5 mg (0 156 mmol) of 8e and 2 mL of 6 N HCl afford 50 1 mg (98%) of spire isostere 9e as a hygroscopic white solid* Mp* 147-149°C; [alo = +3.4” (c 0 3, 1 0 MHCl, 20°C); IR (KBr) 3414 (H,O), 3028,2926,2859, 1718, 1496, 1457, 1170 1094,743,700 cm-‘, ‘H NMR (DMSO-de) 6 12 9&12 30 (b, 1 H), 8.4s 795(b,3H),729-7 16(m,5H),342-322(m,3H+HzO),299(dd, 1 H,J= 13 2,4 2 Hz), 2 79 (dd, I H, J= 13 4,9.5 Hz), 1 84-l 64 (m,4H), 1 58-l 28 (m, 8 H), 13CNMR (DMSO-d,) 6 175.8, 136 5, 129 2, 128 5, 126 8,82 2,62 0, 52 2, 35 4, 34.9, 34 9, 29 1, 29 1, 21.8, 21.7, MS (ES+) m/e (relative Intensity) 292 (M-Cl, loo), 134 (17), MS (ES-) m/e (relative intensity) 326 (M-H, 4), 290 (MH&l, 35), 181 (12), 163 (22), 149 (22), 131 (42), 113 (100); HRMS (FAB) m/e calculated for C,,Hz6N03 ([M-Cl]+): 292.1913, found: 292.1918.

290 3.5. Synthesis

Kazmlerski and Fitch of Pheyl[CH20]Allylglycine

13, Fig. 1

1. Dissolve 4 1 g (0.0215 IV) of the morpholmone (see Subheading 3.1. for the synthesis) m 30 mL acetomtnle. 2 Cool the solution to 0°C and add di-t-butyl-carbonate (6 95 g, 0.032 M), followed by the catalytic (0.262 g, 2 15 mM) amount of DMAP and 4 16 g (0 032 M) of DIEA 3 Stir overmght at room temperature (Note 4) 4 Remove solvent and purify on silica gel using 1 3 (v/v) ethyl acetate/hexane Yield of 10 1s 5 5 g (90 6%) Rf = 0.46 (l/3 ethyl acetate/hexane), tHpLc = 20 42 mm Dissolve 4 53 g (0 0156) of 2 in 40 mL THF, and cool down to -78°C Add 17 1 mL ( 17 1 mM, 1A4 solution m THF) LHMDS, and keep at -78°C for 45 mm Syringe-add 2 87 g (17.1 mM) of a freshly distilled ally1 iodide over 10 mm Quench at -78°C after 90 min with 1 M aqueous citric acid Purify on slllca usmg 1.4 (v/v) ethyl acetate/hexane Single dlastereomer 11 (2 4 g, 46 7% yield) 1s isolated (Note 5) 11: 75 42 MHz, CDCl,.l69 6 (CO), 151 1 (CO), 137 1 (Cq-ar), 133 81, 129 65, 128 69, 126.9, 118 0 (CH,=), 83.5 (Cq), 77 5 (CH-0), 614 (CH,), 57 2 (CH-alpha), 38 1 (CH2), 36 0 (CH,), 28.0 (CMe,), 300 MHz, CDCl, 7 23 (5H, m), 5 83 (m, lH, CH=), 5.15 (2H, CH,=), 4 36 (lH, dd, J= 4 5,8 5), 4 22 (lH, m), 377(1H,dd,J=24,124),360(1H,dd,J=26,12.3),300(2H,m),267(m, 2H), 1 57 (9H, s). Rf= 0 7 (l/3 ethyl acetate/hexane), tHPLC= 23 7 1 mm Smgle isomer by choral chromatography (Dalcel OF, 10% IPA/Hexane) 10 Dissolve 11 m 4 N HCl/dloxane 11 Evaporate after stirring for 30 mm at room temperature Quantitative yield of 12 1s obtained 12: 13C NMR, 75 42 MHz, CDC13 170 73 (CO), 135 39 (Cq-ar), 133 64, 129.10, 127 31, 117 87 (CH2=), 76 90 (CH-0), 67 84 (CH,), 53 3 (CHalpha), 38 5 (CH,), 35 9 (CH,), 300 MHz, CDCl, 7 40-7 15 (5H, m), 6 23 (lH, NH, br. s), 5 83 (m, lH, CH = ), 5 15 (2H, m, CH,=), 4 20 (lH, dd, J = 3 7,7 6), 404(lH,m),389(lH,m),3.42(lH,dd,J=94, 11 8),283 (lH,dd,J=55, 13 7), 2 76 (m, lH), 2 58 (2H, m) MS (ES+) 254 (M + Na), tHPLC= 16 14 mm) 12 Prepare a slurry of 160 mg 12 in 6 N aqueous HCl m a sealed tube 13 Heat at 120°C for 20 h 14. Cool the solution, and evacuate solvents 0 12 g of compound 13 IS obtamed

3.6. Discussion This chapter details the syntheses of a,a-splrocycllc morpholmones. Syntheses of mono-substituted morpholmones and mlmetlcs Xxx-v[CH20](choral)-Yyy derived from them have been reported (I-4). Such mlmetlcs were successfully used in replacing amrde bonds in renin inhibitors (I). The key step of that chemistry involved formation of (already functlonahzed) morpholmone by mtramolecular formation of w[CH,O] from preformed alcohol (Xxx) and alkyl bromide (Yyy) components (2). However, such a methodology appears to have a limited scope, since neither Phe-v[CH,O]-Phe nor Phe-v[CH,O]-oPhe could be prepared by that route (2). Alternative

procedures that utlllze

a

Synthesis of Dipeptides

291

direct alkylatron (or aldol condensation) of preassembled morpholmone scaffold have also been developed, mostly to introduce the benzylic and allylictype substituents m Yyy (34. 4. Notes 1 Better yields are obtained rf the intermediate chloroacetyl-phenylalaninol IS purrtied on a sihca gel using 1:2*0.1 v/v/v ethyl acetate/hexane/methanol). 2 Storchrometrtc amount of sodium hydride can alternatrvely be used in thus reaction. 3. Cermm ammomum mtrate m acetomtrrle/water can also be used The procedure described herem is more convenient, and requires only a mmtmal workup 4 Thus reactron 1s usually done wrthm 30 min 5 Under these condmons exclusrvely, the trans-drastereomer IS formed The orrgm of hrgh diastereoselectrvrty for Boc-protected morpholmone can be attributed to pseudoallylic A, ,s strams Allowing the mixture to equdlbrate at room temperature prior to quenching ~111 yield a mixture of drastereomers

Acknowledgments We are indebted to the members of Glaxo Wellcome Analytical Sciences Department (D. Mmrck, L. St. Johns-Wrlliams, A. Sefler, B. Hall) for their technical expertise. We would like to thank Drs. F. Sahturo and M. Hale of Vertex for strmulatmg drscussions (Bzoorg. Med. Chem , m press). We also thank Tom Wheeler and Gordon Hodgson for their support PCF IS the recrpient of Glaxo Wellcome postdoctoral fellowship. References 1. TenBrink, R. E , Pals, D. T , Hams, D. W., and Johnson, G. A. (1988) Remn mhrbitors containing v[CH20] pseudopeptrde mserts J Med Chem 31,671-677 2 TenBrink, R E (1987) A method for the preparatton of stereochemrcally defined v[CH20] pseudodipeptrdes J Org Chem 52,418422. 3 Anthony, N J , Gomez, R. P , Holtz, W. J., Murphy, J. S., Ball, R G., and Lee, T J. (1995) Pseudo-allylrc A,,, strain as a conformatronal control element: stereoselectrve syntheses of w[CH~O] pseudopeptrdes Tetrahedron Lett 22, 3821-3824. 4 Norman, B H and Krom, J S. (1995) Stereoselective synthesis of novel methylene ether dtpeptide rsosteres Tetrahedron Lett 36,415 l-4 154

17 SNAr-Based Cycloetherification

Methodology

Application in the Synthesis of Heterodectic Macrocyclic Peptides with Endo Aryl-Aryl and Aryl-Alkyl Ether Bonds Jieping

Zhu

1. Introduction The endo aryl-aryl and aryl-alkyl ether bonds exist m a number of biologically important macrocyclic natural products, such as vancomycm family glycopepttde antibtotics (l-31, antttumor series RA I-XIV (4), K- 13 (5), OF4949 (6), piperazmomycrn (71, cyclopeptide alkaloids (8,9), nonpeptidic diarylheptanoids (IO), and so on. Peptidomimetics incorporatmg, such structural features, have also been designed and synthesized as potential mhibitors of ACE (II), anti-HIV agents (12) and so on. From the view point of synthesis design (15), macrocyclization via formation of aryl-aryl or aryl-alkyl ether bond is unique, since it tackles two difficult synthetic problems, i.e., formation of ether bond and ring closure by a single operation. Intramolecular Ullmann ether synthesis (16), oxidative couplmg reaction (171, and so forth, have been employed for the synthesis of type A (Fig. 1) compounds. Rmg closure via the formation of aryl-aryl (type A) (18) and aryl-alkyl ether bonds (type B, Fig. 1) (19) by way of mtramolecualr S,,,Ar reaction (20) will be the focus of this chapter. 2. Materials Melting points were determined with a Kofler apparatus and were uncorrected Infrared (IR) spectra were recorded on a Nicolet-205 (WI, USA) spectrometer. ‘H NMR spectra were measured on Brucker (Wissenbourg, France) AC-200 (200 MHz), Bruker AC-250 (250 MHz), Bruker Aspect 3000 (300 MHz), and Bruker WM-400 (400 MHz) spectrometers with From

Methods m Molecular Me&one, Vol 23 Pepfldomunef/cs Edlied by W M Kazmterski @Humana Press Inc , Totowa,

293

Protoco/s NJ

Zhu

294

FGAQFGoFG

FG

A

B Figure 1

tetramethylsilane as mternal standard (6 ppm). Flash chromatography was performed using Kieselgel 60 (230-400 mesh, E. Merk) and usually employed a stepwise solvent polarity gradient, correlated with TLC moblllty. Solvents and reagents were purified according to standard laboratory techniques. Optical rotation was determmed on a Perkm-Elmer (Germany) automatic polarlmeter at room temperature. Mass spectra were run on AEI MS-50 (El), AEI MS-9 (CI), and Kratos MS-80 (FAB). All reactlons requiring anhydrous condltlons or mert atmosphere were conducted under an atmosphere of argon

2.1. A List of Reagents Specific for Method 3.1 1 2. 3 4 5 6 7 8 9 10 11 12 13 14

Sodium hydride (NaH) Dlethyl acetamldomalonate [AcNHCH(COOEt)2]. N,N-dlmethylformamlde (DMF) Hydrochloric acid (HCl). Thlonyl chloride (SOCl2) Methanol (MeOH) Trlfluoroacetlc anhydrlde [(CF,CO),O]. Trlethylamme (Et,N) Dlchloromethane (CH,Cl,) Subtlllsm carsberg (Baczllus Subtzlu) Protease type VIII-A from BacdZus lzchenzformls Dl-tert-butyl dlcarbonate [(Boc)~~] Dloxane- 1,4 (C4H80) Sodium bicarbonate (NaHC03)

2.2. A List of Reagents Specific for Method 3.2 1 2 3 4 5 6

Sodium hydride (NaH). Ally1 bromide (C3H,Br) Tetrahydrofuran (THF) Plvaloyl chloride (C,H&lO). Triethylamme (Et,N) Potassium hexamethyldlsllazide

(KHMDS).

S/b-Based 7 8. 9 10 11 12 13 14 15 16 17

Cycloetherificafion

2,4,6-trusopropylphenylsulfonyl azide (TrtsylNs). Iodomethane (MeI) Acetone (Me&O) Sodium iodide (NaI) Sodmm acetate (NaOAc) Ltthmm borohydrtde (LIBH~) Dtethyl ether (Et,O) Tm chlortde (SnCI,). Methanol (MeOH) tert-Butyldlmethylstlyl chlortde (TBDMSCI). DMF

2.3. A List of Reagents Specific for Method 3.3 1 2 3 4. 5 6. 7 8 9 10 11. 12 13. 14 15 16

Dicyclohexylcarbodumide (DCC) 1-Hydroxybenzotriazole (HOBt). THF Dichloromethane (CH$l,) Potassmm carbonate (K,COs). DMF Tnfluoroacettc acid (TFA) Sodmm bicarbonate (NaHC03) Acettc anhydrtde (Ac*O) Hydrogen (HZ) 10% Palladium on acttvated carbon (10% Pd/C) HCl Hydrogen tetrafluoroborate (Fluobortc acid, HBF,) tert-Butylmtrtte (‘BuONO) Copper (II) nitrate, hydrate [Cu(NO& 3H20]. Copper (I) oxide (CuzO).

2.4. A List of Reagents Specific for Method 3.4 1 2 3 4 5 6 7 8. 9 10 11 12

1-(3-Dtmethylammopropyl)-3-ethyl-carbodumtde hydrochlortde (EDC) HOBt. [Pd(PPh,),] Tetrakis(trtphenylphosphme)palladmm Ltthmm borohydrrde (LiBHJ. Dtphenylphosphoryl aztde (DPPA). DMF Cesmm fluortde (CsF) HCl Acetonitrile (MeCN). Dtchloromethane (CH,Cl,) THF Trtethylamme (EtsN).

295

296

Zhu

2.5. A List of Reagents Specific for Method 3.5 1 THF NaH MeI. 4 Hydrogen (HZ) 5 10% Palladrum on activated carbon ( 10% Pd/C) 6 MeOH 7 Hydrogen tetrafluoroborate (Fluoborrc acid, HBF,) 8 tert-Butylnitnte ([BuONO) 9 Copper (II) nitrate, hydrate [Cu(NO,), 3Hz0] 10 Copper (I) oxide (CuZO) 11 TFA. 2 3

2.6. A List of Reagents Specific for Method 3.6 I 1-(3-Drmethylammopropyl)-3-ethyl-carbodnmtde hydrochlortde (EDC) 2 1-Hydroxybenzotrtazole (HOBt) 3 Dtchloromethane (CH,CI,) 4 DMF 5. HCI 6 Acetomtrtle (MeCN) 7 THF 8 Et,N. 9 Benzyl bromide (BnBr) 10 Tetra-N-butylammontum fluoride, 1 A4 tn THF (TBAF) 11 Sodrum borohydrtde (NaBH,) 12 Elemental sulfur (S,) 13 tert-Butylnrtrtte (‘BuONO). 14 Boron trtfluortde etherate (BF3 OEt,) 15 Iron (II) sulfate (FeSO,).

3. Methods 3.1. Synthesis of D- and L-4-Nuoro-3-Nitrophenyl Alanine Methyl Ester Chemoenzymatic synthesis of enantiomertcally pure D- and L-4fluoro-3-mtrophenyl alanme methyl ester ts shown u-r Fig. 2 (21). Alkylation of acetamidomalonate wtth 4-fluoro-3-mtro benzyl bromide (1) gave compound 2 (see Note 1) which was decarboxylated to give racemrc ammo acid 3 m quantitative yield. Esterification of 3 followed by trifluoro-acetamtde formation afforded substrate 4 (85% overall yield from 1). Subtiltsm(see Note 2) mediated enzymatic hydrolysis gave L-N-trifluoroacetyl-4fluoro-3-mtro phenylalamne 6 and unreacted o-N-trtfluoroacetyl-4-fluoro3-mtro phenylalanme methyl ester 5 wtth htgh enanttomertc excess (>99%

Sdr-Based

Cycloethenfication

297

1 No2

F -A I ’

e

2

Fi%NH:OCFF$

4

“NHCOCF3

3

5

6

f

f

1 m

No2

02N$iN:,j: ‘NH2

F

/

1;

2

I

9

Fi

10 I

NH:!

‘c, MeOOC

7

‘NH2

8

Fig. 2. Reagents and condtttons (a) NaH, AcNHCH(COOEt),, DMF, room temperature, (b) cont. HCl, reflux, (c) SOCl,, MeOH, (d) (CF,CO),O, Et,N, CH,Cl,, 85% overall yreld, (e) subtthsm, PH = 7 5, (5) SOClz, MeOH, 90%. by chiral HPLC). the correspondmg

Treatment of 5 or 6 with SOCl* m MeOH amino esters 7 or 8, respectively.

(see Note 3) gave

3.1.1. Ethyl 2-(Acetylamino)-2-(Ethoxycarbonyl)3-(4’-Fluoro-3’-Nitrophenyl) Propanoate 2 1 To a suspension of sodium hydride (60% in 011,0.88 g, 22 mmol, washed with pentane) m DMF (NJV-dimethylformamide) (6 mL) add a solutron of dtethyl acetamidomalonate (5 1 g, 23.5 mmol) m DMF ( 15 mL) and a solution of 4-fluoro-3-mtrobenzyl bromrde (1,5 0 g, 2 1 36 mmol) m DMF (5 mL), successively (see Note 1) 2 Stir the reaction mixture at room temperature for 4 h, quench by careful addition of water (5 mL), and concentrate to dryness. Redissolve the restdue m CH$12, wash with brine, dry over Na2S04, and evaporate the organic phases to dryness 3 Purify the residue by flash chromatography (S102, heptane/ether = l/l) Concentrate the appropriate fracttons to give quantttattve yield of compound 2 as white needles (7 9 g, 100%) mp 125°C (CH,Cl,/heptane)

3.7.2. D, L-N - Trifluoroacetyl-4-Fluoro-3-Nitrophenylalanme Methyl Ester 4 1. Reflux a solution of 2 (7 9 g, 21 35 mmol) m concentrated HCl(50 mL) for 20 h Remove Hz0 in vucuo and dry the residue over P205 under reduced pressure (4 h) to obtain the crude racemtc ammo acid (f)-3.

298

Zhu

2 To a solution of SOCl, (7 mL) in methanol (40 mL) add (+) 3 and heat at 40°C for 5 h. Remove the solvent to obtain the crude ammo ester. 3 To the solution of the crude ammo ester m CH,Cl, (40 mL) add trrethylamme (3 mL, 21.38 mmol) and trrfluoroacetrc anhydride (3.3 mL, 23 4 mmol) 4. Stir the reaction mixture at room temperature for 3 h, evaporate the solvent, and purify the residue by flash chromatography (SrO,, CH,Cl,). Concentrate the appropriate fractrons to obtain the (+) 4 (6 14 g, 18 16 mmol, 85% overall yield from the alkylatron step). mp . 113-l 15°C.

3.1.3 ~-N-Trifluoroacetyi-4-Fiuoro-3-Nitrophenyiaianine 5 and ~-(S)-N-Trifiuoroacetyi-4-Fluoro-3-N~trophenyiaian~ne

Methyl Ester 6

1 To a brphasrc solution of(*) 4 (3 0 g, 8 87 mmol) m phosphate buffer (pH 7 5,500 mL) and CH,Cl, (75 mL) add a solution of subtrlism carlsberg (B subtzh, 40 mL, see Note 2) 2 Stir the reaction mixture vigorously at 37’C, and follow the reaction course by HPLC (column. hypersrl ODS, 5 pm (250 x 4 6 mm), eluent. 0 1% of TFA m MeCN, detection UV 250 nm) Stop the reaction at the 50%-of-hydrolysis point (6 h) by addrtron of methanol 3 Filter the precipitated enzyme, evaporate the organic solvents under reduced pressure, and extract the aqueous solution with EtOAc Wash the organic phases with brine, dry, and evaporate to afford the unhydrolyzed ester n-N-trrfluoroacetyl-4fluoro-3-mtrophenylalanme methyl ester 5 (1 43 g, 47 7%) [a], = -86 (c 0 5, CHCI,). 4. Acidify the aqueous phase with citric acid to pH = 4 0, and extract with EtOAc Wash the organic extracts with brine, dry with Na$SO,, and evaporate to obtam L-(S)-N-trlfluoroacetyl-4-fluoro-3-nltrophenylalanine 6 whose optical purity was determined by conversion mto L-N-trrfluoroacetyl-4-fluoro-3-mtrophenyl-alanine methyl ester (1 48 g, 49 3%). [o]n = +86” (c 0 96, CHC13)

3.1.4. c-4-Fiuoro-3-Nitrophenyiaianine and L-4-Fiuoro-3-Nitrophenyiaianine

Methyl Ester 7 Methyl Ester 8

1 Reflux a solution of L-(S)-N-tnfluoroacetyl-4-fluoro-3-nnrophenylalanme 6 (1 00 g, 3 08 mmol) m MeOH for 7 h. 2. Evaporate to dryness, redissolve the residue m H20, basrfy with aqueous Na$Os, and extract the aqueous phase with EtOAc. Wash the organic extracts with brine, dry over Na2S04 and evaporate to obtain pure compound 8 (67 1 mg, 90%). [o]n = +15” (c 1, CHCl$; IR (CHC13) 1735, 1537, 1356 cm-‘, ‘H NMR (200 MHz, CDC13) 6 1.60 (br s, 2H, NH,), 2 90 (dd,J= 13 8,7 8 Hz, lH), 3 13 (dd, J= 13 8, 5 1 Hz, lH), 3 70 (m, lH), 3.73 (s, 3H), 7.23 (dd, J= 10 6,8 5 Hz, lH), 7 50 (m, lH), 7 93 (dd, J = 7 1, 2.2 Hz, 1H); 13C NMR (CDCL,) 6 39 7, 52 3, 55 4, 1185(d,J=21Hz),1260,1340,1360,1545(d,J=262Hz),174;MSm/z (CI) 243 (M+ + l), anal calcd for CroH,,FN,O, C, 49 59, H, 4.58, Found C, 49 36, H, 4 64

S/b-Based

Cycloetherification

+Js OH 16

(-N’% cli

299

(-N’-‘P OTBDMS

17

18

Fig. 3 Reagents and conditions. (a) NaH, ally1 bromide, THF; (b) Me,CCOCl, Et,N, THF, then compound 13; (c) KHDMS, TrisylN,, then Me&O, NaI, NaOAc, (d) LIBH,, Et,O, (e) SnCl,, MeOH; (f) TBDMSCI, DMF. D-4-fluoro-3-nitrophenylalanine methyl ester 7 was prepared followmg the same procedure (see Note 3) Followmg the same synthetic scheme, D-3-fluoro-4-nitrophenylalanme methyl ester 9 and its L enantiomer 10 were prepared chemoenzymatlcally startmg from 3-fluoro-4-nltrobenzyl bromide 3.2. Synthesis

of o-(R)-3-A flyloxyphenyJ

Glycinol

(R)-3-allyloxy phenyl glycmol 17 was prepared using asymmetric electrophlhc azldation method as depicted m Fig. 3 (22,23). Ally1 protection of 3hydroxyphenyl acetic acid 11 followed by mcorporation of choral auxdlary R-13 via the mixed anhydride method afforded imide 14. Subsequent treatment of its potassium enolate with trisyl azlde (TrisylN3) (24) followed by a modified workup procedure (see Note 4) afforded 15. The de of this reaction was higher than 85%, and two dlastereolsomers were readily separated by careful flash chromatography. Reductive removal of choral oxazohdmone with LlBH4 (25) gave the corresponding alcohol 16 together with regeneration of the chn-al auxiliary. Reduction of azido alcohol (16) with tin chloride (26) gave the desired ammo alcohol 17 (see Note 5) which was transformed into TBDMS ether 18 ready for peptlde couphng.

3 2.1. 3-Allyloxyphenyl Acetic Acid 12 1. To a solution of 3-hydroxyphenylacetlc acid ll(5.0 g, 32.9 mmol) m THF (500 mL) add NaH (60% in o&3.95 g, 98.7 mmol) 2 Stir the reaction mixture at room temperature for 1 h, add ally1 bromide (11 4 mL, 13 1.6 mmol), and then reflux the resulting reaction mixture for 15 h.

300

Zhu

3 Cool the reaction mixture to 0°C quench by addition of water, and stir at room temperature for another 2 h. Evaporate the volatiles, and extract the residue with ether to remove the neutral species. 4. Acidify the aqueous phase, extract it with dichloromethane Wash the combmed organic phases with brine, dry over Na,SO, and evaporate to obtain a white solid 5 Recrystallize from ether-heptane to obtain 12 as white needles (6 2 g, 98%) mp 78°C

3.2.2. (4R)-3-[2-(3-Allyloxyphenyl)I-Oxoethylj-4-(Phenylmethyl)-2-Oxazolidinone

14

To a solution of 3-allyloxyphenylacettc acid 12 (1 92 g, 10 mmol) in dry THF (40 mL), cooled to -78°C add Et,N (1 68 mL, 12 mmol) and redistilled pivaloyl chloride (1 29 mL, 10 5 mmol), successively vza syringe (flask A) Stir the resultmg slurry at -78°C for 15 mm and at 0°C for 45 mm and then cool again to -78°C In a separate flask (B), dissolve the (4R)-4-phenylmethyl-2-oxazohdmone (2 12 g, 12 mmol) in dry THF (40 mL), cool to -78°C and add a solution of butylhthmm m hexane (7 5 mL, 1.6 M> slowly Stir at -78°C for another 5 mm after completion of the addition Transfer the thus formed yellowish metallated oxazohdmone 13 (flask B) via a cannula to the flask A containing the mixed anhydride Stir the resulting slurry at -78°C for 15 mm, and then warm up to room temperature over 90 mm Quench the reaction by addition of aqueous NH&l, remove the volatiles 112vacua, and extract the aqueous solution with EtOAc Wash the combined orgamc phases with brme, dry (Na,SO,), and evaporate in vucuo Purify the residue by flash chromatography (SiO,, EtOAc/heptane = l/6 then l/3). Concentrate the appropriate fractions to obtain compound 14 as a colorless oil (2 81 g, 80%). [CL],, = -104’ (c 2, CHCl,).

3.2.3. (2R, 4R)-3-[2-Azido-2-(3-AIlyloxyphenyl)1-Oxoethylj-4-(Phenylmethyl)-2-Oxazolidmone

15

1. To the solution of KHMDS (37 mL, 0.5 M, 18 5 mmol) m dry THF (40 mL), stirred at-78°C under argon, and add via syringe a precooled (-78’C) solution of imrde 14 (5.4 g, 15.4 mmol) m dry THF (90 mL) 2 Stir the resulting yellow solution at -78’C for 30 mm, and add via syringe a precooled (-78°C) solution of trisyl azide (5.7 1 g, 18 5 mmol) m dry THF (50 mL) 3 Stir the reaction at -78°C for 4 min (the solution became shghtly red), and quench by addition of HOAc (4 4 mL, 76.9 mmol) Remove the coolmg bath, and stir the reaction mixture at room temperature for 90 mm Add saturated aqueous NH&I (50 mL), evaporate the volatiles and extract the aqueous layer with EtOAc Wash the organic phase with brine, dry (Na,SO,) and evaporate to dryness 4. To the above-obtamed residue m acetone (250 mL) add NaI (11 5 g, 77 0 mmol) and NaOAc 3Hz0 (6.3 g, 46 1 mmol). Stir the reaction mixture at room temperature for 3 h (see Note 4).

301

Sflr- Based Cycloe therification

5 Filter off the morgamc salt, evaporate the filtrate, and then partition It between CH,Cl, and H,O. Extract the aqueous phase with CH,CI,, wash the combined organic phases with brine, dry (Na,SO,) and evaporate zn vacua 6 Purify the crude product by flash chromatography (SiO,, EtOAc/heptane = l/6). Concentrate the appropnate fractions to obtain the major dlastereolsomer 15 (4 95 g, 82%) as a colorless 011 [a]n = -202’ (c 4.1, CHCI,)

3.2 4. (1 R) 2-Hydroxy-l-(3-Allyloxyphenyl)-Ethyl

Azide 16

1 To a solution of compound 15 (1 58 g, 4 04 mmol) m Et,0 (50 mL) add distilled water (2 18 ml, 12 10 mmol) and LiBH4 (267 mg, 12 10 mmol) at 0°C. 2 Stir the reaction mixture at 0°C for 1 h, and quench by addltlon of HCl (1 N). Extract the aqueous phases (pH = 4) with EtOAc Wash the combined organic phases with brine, dry (Na$O,), and evaporate in vacua. 3 Purify the crude product by flash chromatography (S102, EtOAc/heptane = l/l) Concentrate the appropriate fractions to obtain compound 16 (725 mg, 82%) as a colorless 011. [a]o = -159” (c 0.76, CHCl,).

3.2.5. (FI) -3-Allyloxyphenyl

Glycinol 17

1. To a solution of compound 16 (190 mg, 0 86 mmol) m methanol (15 mL) add SnCl, 2H20 (582 mg, 2 58 mmol) 2 Stir the reaction mixture at room temperature for 20 h Evaporate the volatlles zn vacua, and partltlon the residue between Et,0 and H20 Extract the aqueous phase with Et,0 to remove the neutral species 3 Baslfy the aqueous solution with aq. NaHC03 and extract with CH,Cl* (see Note 5) Wash the combined organic phases with brine, dry (Na,SO,), and evaporate zn vacua to obtain pure compound 3-allyloxyphenylglycmol (Z 7) as a colorless oll(l24 mg, 74%), which can be used without further purification: [a]o =-15” (c 0.5, CHC13)

3.2.6. o-2-b Buty/dimethylsilanyloxy1-(3-Allyloxypheny/)-Ethyl Amine 18 1 To a solution of 3-allyloxyphenylglycmoll7 (220 mg, 1 14 mmol) m dry CH2C12 add Et3N (639 pL, 4 56 mmol), DMAP (139 3 mg, 1 14 mrnol) and TBDMSCl (5 15 5 mg, 3 42 mmol) at room temperature 2. Stir the reaction mixture at room temperature for 5 h, then dilute with aqueous NH&l, and extract the aqueous phase with CHzClz Wash the combined organic phases with brine, dry (Na,SO,) and evaporate zn vacua. 3 Purify the crude product by flash chromatography (SiO*, EtOAc/heptane = l/3 then l/2) Concentrate the appropriate fractions to obtain compound 18 (3 11 mg, 89%) as a colorless 011 [a]o =-1 1 (c 0.7, CHCl& IR (CHCl,) 3300-3500,2990, 1610, 1500 cm-l; ‘H NMR (200 MHz, CDC13) 6 -0 1 (s, 6H), 0.85 (s, 9H), 3 58 (dd,J= 8 0, 10 1 Hz, lH), 3 78 (dd, J=4 0, 10.1 Hz, lH), 4 03 (dd, J= 4 0,8 0 Hz, lH), 4 50 (td, J= 1 5, 5 3 Hz, 2H), 5 24 (qd, J= 1.5, 10.5 Hz, lH), 5 39 (qd, J= 1.5, 17.3 Hz, lH), 6.01 (tdd, J= 5.3, 10.5 and 17.3 Hz, lH), 6.8-6 9 (m, 3H), 7 18

302

Zhu OH OH

19

20

22

21

NO?

NO:

d

M&J

Me0 23

24 OH

Me0 25

26

Fig 4 Reagents and condltlons: (a) DCC, HOBt, CH&l,, room temperature, 85%, (b) K,C03, MeOH-H,O, room temperature 100%, (c) 10, DCC, HOBt, CH,CI,-THF, 82%; (d) K&O,, 0 02 Mm DMF, room temperature 87%; (4) TFA, room temperature, (f) NaHC03, AczO, CH2C12, room temperature, 83%; (g) H,, Pd/C, MeOH, HCl, 85%, (h) HBF4, ‘BuONO, MEOH, 0°C then Cu(NO& 3H20, Cu20, H20, room temperature, 74% (t, J= 7 5 Hz, IH), 13CNMR (CDCl,) 6 -5 4, 18 4,26 0,57 6,68 I,69 0, 113 5, 114 5, 117 7, 119.6, 129 7, 133 4, 141.7, 159 0, MS m/z 307,250, 162, anal calcd for C17H29N02S~ C, 66 40, H, 9 51, N, 4 55, Found C, 66.25, H, 9.37, N, 4 56

3.3. Synthesis

of K-13

K-13 27, a potent, non competltlve inhibitor of angiotensm I converting enzyme and weak inhibitor of ammopeptidase B, has been isolated from Mwromonospora halophytzca ssp exlllsla K-13 (5). Our synthesis usmg SNAr reaction as key ring closure step 1s shown in Fig. 4 (27). Coupling of the dipeptide 21, obtamed by condensation of tyrostne derivatives 19 and 20, with ammo acid 10, using DCC and HOBt, furnished the linear trlpeptide 22 in 80% LSOlated yield. Cycloetherlficatlon of 22 (I&CO,, DMF, 0 02 M, room temperatuare, 4 h) afforded the 17-membered cyclic peptlde 23 m 87% isolated yield

S&-Based

Cycloetherification

303

(see Note 6). Mild acid hydrolysis of the tert-butyloxycarbonyl carbamate (Boc) followed by conventional acetylation (see Note 7) and reduction of the mtro group provided the ammo compound 25 in 60% overall yield. Drazotrzanon (HBF,, lBuON0, MeOH) and subsequent oxrdative hydrolysis of the diazomum salt using Cu(NO& 3H,O and Cu20 (28) (see Note 8) afforded the protected K-13 26 in 74% yield Demethylation of 26 to natural K- 13 27 has already been reported (29). 3.3.1. IV-Boc-L-Tyr-L-Tyr (OMe) 21 To a solutton of L-Tyr (OMe) methyl ester 19 (4 18 mg, 2 mmol) and L-N-Boc Tyr 20 (562 mg, 2 mmol) m CH2C12 (5 mL) add HOBt (270 mg, 2 0 mmol) and DCC (412 mg, 2 0 mmol), successively Stir the reaction mixture at room temperature for 3 h, dilute with 0 1 N HCl, and extract wtth CH2C12 Wash the combmed organic phases wtth brine, dry (Na,SO,), and evaporate zn vacua Purify the residue by flash chromatography (St02, EtOAc/heptane = l/l) Concentrate the appropriate fracttons to obtain compound N-Boc-L-Tyr-t.-Tyr (OMe) methyl ester (802 mg, 85%) as a colorless oil [aJD = +32” (c 0.5, CHC13) To a solution of N-Boc-L-Tyr-L-Tyr (OMe) methyl ester (802 mg, 1 7 mmol) m MeOH (16 mL) and HZ0 (4 mL) add KZC03 (704 mg, 5 1 mmol) Stir the reaction mtxture at room temperature for 12 h, and then evaporate the volattles. Dissolve the residue in water, acidify with 1 N HCl, and extract with CH,Cl, Wash the combined organic phases with brine, dry (Na,SO,), and evaporate zn vucuo to obtam pure compound 21 (778 mg, 100%) as a white soled mp 89-90"C,[a]D=+300(c0 15, CHC13)

3.3.2. N-Boc-L-Tyr-L-Tyr (OMe)-L-(3’-Fluoro-4-Nitro) Phenylalanme Methyl Ester 22 1 To a solution of dipepttde 21(115 mg, 0.25 mmol) and 3-fluoro-4-nitro phenylalanme methyl ester 10 (61 mg, 0.25 mmol) m CH+& (3 mL) and THF (3 mL) add HOBt (34 mg, 0 25 mmol) and DCC (52 mg, 0 25 mmol), successively 2. Stir the reaction mixture at room temperature for 3 h, dilute with 0.1 N HCI, and extract with CH$l, Wash the combmed orgamc phases with brme, dry (Na$O,) and evaporate In vacua. 3 Purtfy the residue by flash chromatography (Si02, EtOAc/heptane = 3/2) Concentrate the appropriate fractions to obtam compound 22 (140 mg, 82%) as a yellow solid. mp 181-182°C; [a]n = -19’ (c 0.3, acetone), IR (CHCls) 3584, 3419,3331,2944,2856, 1744, 1700, 1681, 1602, 1215 cm-‘; ‘H NMR (CDCl,, 250 MHz) 6 1.30 (s, 9H), 2.96 (m, 4H), 3 16 (dd, J= 7 9, 14 2 Hz, lH), 3.30 (dd, J= 5 6, 14.2 Hz, lH), 3 69 (s, 3H), 3.74 (s, 3H), 4.22 (m, lH), 4 59 (dd, J= 7.5, 13.7Hz,lH),477(dd,J=77,134Hz,lH),597(d,J=77Hz,lH,NH),673 (d, J= 8 5 Hz, 2H), 6 80 (d, J= 8.6 Hz, 2H), 7.04 (d, J= 8.5 Hz, 2H), 7 11 (d, J =86Hz,2H),729(m,2H),739(dd,J=752Hz,lH,NH),804(t,J=82Hz,

304

Zhu lH), 13C NMR (CDCl,) 6 25.7, 28 7, 29 1, 34 5, 37 5, 37.8, 49 2, 57.0, 79 7, 114.5,1160,1199(d,J=21OHz),1267(d,J=37Hz),129.0,131 1,1312, 147 8, 150.5 (d,J= 273 Hz), 156 9, 159.4, 171 5, 171 7, 172 3

3.3.3. Methyl (9S, 12S, lSS)-15-{((I, l-Dimethylethoxy)-Carbonyl] Amino}-4-Nitro- 12-[(4-Methoxyphenyl)-Methyl]1 1,14-Dloxo-2-Oxa-10,13-Diazatricyclo(l5.2.2. 13,‘) Docosa-3,5,7(22), 17,19,20-Hexaene-9-Carboxylate 23 1 To a solution of22 (102 mg, 0 15 mmol) m DMF (7 mL, 0.02 M> add K2C03 (84 mg, 0 6 mmol) (see Note 6) 2. Stir the reaction mtxture at room temperature for 5 h, dilute wtth 0.1 N HCl(30 mL), and extract with EtOAc. Wash the combined orgarnc phases with brine, dry (Na,SO,) and evaporate m vacua. 3 Purify the residue by flash chromatography (St02, EtOAc/heptane = l/l) Concentrate the appropriate fractions to obtain compound 23 (86 mg, 87%) as a yellow sohd* [cx]o = -8” (c 6, CHCls); IR (CHC13) 3406, 2875, 2369, 1744, 1706, 1669, 1613, 1594, 1512, 1500 cm-‘, ‘H NMR (CDCl,, 250 MHz) 6 1.45 (s, 9H), 2 6-3 5 (m, 6H), 3 70 (s, 3H), 3.74 (s, 3H), 3 93 (m, IH), 4 20 (m, lH), 4 36 (dd,J= 4 2,4 7 Hz,lH),532(d,J=79Hz,lH),540(d,J=47Hz,lH),620(d,J=55Hz,lH), 6 28 (s, lH), 6 63 (d,J= 8.5 Hz, lH), 6 75 (d,J= 8 5 Hz, 2H), 6 81 (dd,J= 2 4,8 3 Hz, lH), 7.02 (d,J= 8 5 Hz, 2H), 7 15 (d,J= 8 1 Hz, 2H), 7 33 (d,J= 8 3 Hz, lH), 7 77 (d, J= 8 3 Hz, lH), t3C NMR (CDCl,) 6 28 5, 34 1, 35 9, 38 9, 39.6, 49 3, 528,533,553,563,802,1142,1199,1210,1223,1228,1256,1302,1320, 134 0, 142 9, 154.2, 158.9, 170 0, 170.6, MS (EI) m/z 662, 546

3 3.4. Methyl (9S, 12S, 15S)-15-(Acetylammo)4-Nltro-12-[(4-Methoxyphenyl)-Methyl]11,14-Dioxo-2-Oxa-10,13-Diazatricyclo(l5.2.2. 13~7) Docosa-3,5,7(22), 17,19,20-Hexaene-9-Carboxylate 24 1 Dissolve compound 23 (57 7 mg, 0 087 mmol) m TFA (1 5 mL) and CH,Cl, (1 5 mL), star at room temperature for 1 h and evaporate to dryness 2 Dtssolve the residue m THF (2 mL) add NaHC03 (22 mg, 0 26 mmol, see Note 7) and AczO (12 5 p.L, 0 14 mmol) 3. Stir the reaction mixture at room temperature for 1 h, dilute with H20, acidify with 1N HCl, and extract with EtOAc. Wash the combmed orgamc phases with brine, dry (Na,S04) and evaporate zn vacua. 4 Recrystalhze the restdue f?om CH,Cl, to give 24 (45 mg, 85%) as a white solid mp 325°C

3 3.5. Methyl (9S, 12S, 15S)-15-(Acetylamino)4-Amino- 12-[(4-Methoxyphenyl)-Methyl]11,14-Dioxo-2-Oxa-10,13-Diazatricyclo(l5.2.2. 13p7) Docosa-3,5,7(22), 17,19,20-Hexaene-9-Carboxylate 25 1 To a solution of 24 (33 mg, 0.055 mmol) in MeOH (5 mL) containing a few drops of 6 N HCI add a catalytic amount of 10% Pd/C

SJW3ased

Cycloetherificatlon

305

2. Hydrogenate the mixture at 1 atm H, for 1.5 h 3. Filter the mixture through a short pad of Cehte, concentrate the filtrate, redlssolve the residue m H,O, baslfy with aqueous NaHCO,, and extract with CH,Cl,. Wash the combmed organic phases with brine, dry (Na,SO,), and evaporate zn vucuo to obtain analytically pure 25 (3 1 5 mg, 100%) as a white sohd

3.3.6. Methyl (9S, 12S, 15S)-15-(Acetylamino)4-Hydroxy- lZ-[(#-Methoxyphenyl)-Methyl]11,14-Dioxo-2-Oxa-10,13-Diazatricyclo(l5.2.2. 13v7) docosa-3,5,7(22), 17,19,20-Hexaene-9-Carboxylate 26 1 To a solution of 25 (6 6 mg, 11 5 pool) m MeOH (3 mL) add, at O”C, HBF4 (5 & 54% solution m ether, 34.5 pool) and ‘BuONO (90% purity, 3 pL, 23 0 ~01) 2 Stir the reaction mixture at 0°C for 15 mm, and then at room temperature for another 15 mm 3. To the above reaction mixture add a solution of Cu(NO& * 3H20 (1 67 g, 6.9 mmol) and Cu,O (2 mg, 11 5 mmol) m distilled Hz0 (10 mL) Stir the reactlon mixture at room temperature for 20 mm (see Note 8) 4 Extract with EtOAc Wash the combmed orgamc phases with brme, dry (Na,SO,), and evaporate In vacua. 5 Purify the residue by preparative TLC (S102, eluent: CH,Cl,/MeOH = 20/l) Concentrate the appropriate fractions to obtain product 26 (5 mg, 7 1%) as a yellow solid. mp 26+27O”C

3.4. Synthesis

of a Modified

Vancomycin

Binding

Pocket

In the search for synthetic analogs having enhanced affinity toward the D-Ala-D-Ala (3/9), compound 34, a 16-membered cyclopeptide with an endocyclic biaryl ether bond, was designed and synthesized as a modified vancomycm bmdmg pocket. Synthesis of 34 featuring a key cycloetherificatlon reactton based on mtramolecular SNAr reaction is shown in Fig. 5 (22,23). Couplmg of 18 with L-(S)-N-allot-alamne 28 produced dlpeptlde 29 m 84% yield Simultaneous reductive removal of O-ally1 and N-allot groups using the reagent combmatlon LlBH4-Pd(PPh& (31,321 developed m this laboratory (see Note 9) furnished the correspondmg ammo alcohol 30. The crude ammo alcohol 30 thus obtamed was directly coupled with the dlpeptlde

31 (DPPA,

DMF,

Et,N) to give the tetrapeptide 32 in 55% overall yield (see Note 10). Macrocychzatlon of 32 proceeded smoothly using anhydrous CsF in dry DMF (0.01 A4) at room temperature (see Note 11). Under these conditions, deprotectlon of the primary alcohol and cychzation occurred m one pot to afford the 16-membered macrocycle 33 as a single isolable atroplsomer m 63% yield. Characteristic of the cychc structure 33 is the appearance of strongly shielded proton H-2 1 m 1H NMR spectrum (compound 33, dHe2,= 5.8 1; compound 32, dHm2,= 6.86) owing to the anisotroprc effect of aromatic E ring. The

306

Zhu

OTSDMS

18

28

32

29

30

I

Fig 5 Reagents and condltlons (a) EDC, CH,Cl,, 88%, (b) Pd(PPh,),, LlBH,, THF; (c) DPPA, 31, Et,N, DMF, 55%, (d) CsF, DMF, 63%, (f) HCI-MeCN, 82%

newly created chirahty resulting from the restricted rotation of the blaryl ether bond was determined by NOE techniques to have a P configuratlon (33). Removal of the Boc protective group of 33 was carried out with 0.1 N HCl m CH3CN (see Note 12) and after the usual acid-base extraction, an analytically pure compound 34 was obtained. 3.4 I. { 1-[I -(34//y/oxypheny/)-2-tert -Buty/dimethy/sr/any/oxyEthylcarbamoylfEthyl}-Carbamic Acid Ally/ Ester 29 1 To a solution of 18 (75 mg, 0 25 mmol), N-allot-(L)-alanme 28 (43 mg, 0 25 mmol) m CH&l, (4 mL) add EDC (53 mg, 0 27 mmol) 2 Stir the reaction mixture at room temperature for 2 h Dilute with water and extract with CH,Cl, Wash the combined organic phases with brine, dry (Na,SO,) and evaporate In vacua. 3 Purify the crude product by flash chromatography (S102, CH,Cl,) Concentrate the appropriate fractions to obtain compound 29 (10 1mg, 88%) asa colorless 011, [aID = -19 5’ (c 0 35, CHC13)

3.4.2. N-[7-(3-A//y/o~ypheny/)-2-tert-Buty/dimethylsilany/oxyEthyl]-2-Amino Proplonamide 30 1 To a solution of 29 (101 mg, 0.22 mmol) m dry THF (3 mL) add Pd (PPh& (11 mg, 0 01 mmol) and LlBH, (28 mg, 1 32 mmol) (see Note 9)

S/k-Based

Cycloethenfication

307

2 Stir the reaction mixture at room temperature for 1 h Quench by addition of 2 N HCl, neutralize to pH = 7.0, and extract the aqueous solution with EtOAc Wash the combmed orgamc phases with brine, dry (Na,SO,) and evaporate zn vacua 3 Purify the crude product by passing through a short pad of Sephadex LH 20 (CH,Cl,, then EtOAc) Concentrate the appropriate fractions to obtain compound 30.

3.4.3. o-N-Boc-N-methyl-Leu-o-(4-F/uoro-3-Nitro)fheny/alanine

37

1. To a solution of n-4-fluoro-kntrophenyl alanme methyl ester 7 (300 mg, 1 24 mmol) m CH,C12 (10 mL) add EDC (260 mg, 1.36 mmol) and N-Boc-N-methyln-Leucme (340 mg, 1.40 mmol) 2 Stir the reaction mixture at room temperature for 5 h, dilute with H20, and extract the aqueous phase with CH2C12 Wash the combined orgamc phases with brine, dry (Na,SO,), and evaporate zn vacua. 3. Purify the crude product by flash chromatography (SiOZ, CH,CI,/MeOH = lOO/ 1) Concentrate the appropriate fracttons to obtain the dipepttde D-N-Boc-Nmethyl-Leu-n-(4fluoro-3nitro)phenylalanine methyl ester (550 mg, 95%). mp 6&68’c; [a& = +50° (C 0 15, CHCls) 4 Stir a solution of o-N-Boc-N-methyl-Leu-D-(4-fluoro-3-nltro)phenylalanine methyl ester (469 mg, 1 mmol) m methanol (6 mL) and water (2 mL) at room temperature for 5 h m the presence of K,CO, (207 mg, 1 5 mmol) Evaporate the solvent, acidify the residue, and extract the aqueous solution with EtOAc Wash the combined organic phases with brine, dry (Na,SO,) and evaporate zn vacua to obtain 31 as a yellowish solid m quantitative yield mp 14&145”C, [o]o = +28’ (c 0 1, MeOH)

3.4.4. (l-f1 -[1-(3-Hydroxyphenyl)-2-tert-Butyldimethy/si/any/oxy]Ethylcarbamoyo-Ethylcarbamoyl)-2-(4-Fluoro3-Nitrophenyl)-Ethylcarbamoyl-3-Methyl-ButylMethyl-Carbamic AC/C/tert-Butyl ester 32 1 To a solution of 30 (obtained m step 3 4 2), dipeptide 31 (100 mg, 0.22 mmol) m dry DMF add DPPA (56 ml, 0.26 mmol) and Et,N (34 ml, 0.24 mmol), successively (see Note 10) 2 Stir the reaction mixture at room temperature for 3 h, then dilute with water and extract the aqueous solutron with EtOAc Wash the combined orgamc phases with brme, dry (NazSO,) and evaporate In vacua. 3 Purify the crude product by preparattve TLC (EtOAc/ether = l/2) to obtam the tetrapeptide 32 (94 mg, 55% m 2 steps), mp 85-9O”C, [a], = +I2 0” (c 0.2, CHCl,), IR (CHCI,) 320&3400, 1700, 1680, 1550 cm-‘; ‘H NMR (300 MHz, CDCls) 6 -0 05 (s, 3H, SiMe), -0 03 (s, 3H, StMe), 0 87 (s, 9H, SiBu’), 0.89 (d, J = 5 7 Hz, 3H, CH&&), 0 92 (d, J= 6 5 Hz, 3H, CH&&), 1 2 1 (d, J= 6.9 Hz, 3H, CH&), 1.43 (m, lH, CJ&CHMe& 147 (s, 9H, But), 1461 48 (m, lH, CH&HMe,), 2 70 (s, 3H, N-Me), 2 92 (dd,J= 7.0, 13.9 Hz, lH, H-15), 3.05 (dd, J=6.3, 13 9Hz, lH,H-15’),3.80(dd,J=5.4, 10.2Hz, lH,C&OTBDMS),3.88

308

Zhu (dd,J=4.5, 10 2 Hz, lH, C&OTBDMS), 4 50 (m, lH, H-l l), 4 63 (dd,J= 5 6, 9 6 Hz, lH, CHCH,CHMe,), 4 76 (m, lH, H-14), 4 86 (m, lH, H-8), 6 77 (dd, J = 2.3, 8 0 Hz, lH, H-4), 6.80 (d, J= 8 0 Hz, lH, H-6), 6 86 (s, lH, H-21), 7 09 (dd, J= 8 5, 10.5 Hz, 2H, H-19 and NH), 7 18 (t, J= 8 0 Hz, 2H, H-5 and H-20) 7.69 (dd, J=2.0,6 9 Hz, lH, H-17), 7.83 (brs, lH, NH), 7.93 (brs, lH, NH), t3C NMR (CDCl,) 6 -5 5, -5 4, 18.3, 18 6, 21.7, 23 9, 24 9, 25 9, 28 4, 29 4, 36 7, 38.0,494,53 9,55 4,57.0,660,81 1, 1147, 1183 (d,J=21 Hz), 1267, 128.8, 129.8, 132 2, 136 7, 141.2, 151.6, 154 0 (d, J = 262 Hz), 157.0, 168 3, 171 6, 175.8; FABMS (Thto/NaCl) m/z 798 (M + Na+), 776 (M + H+), anal calcd for C,,H5sFN,0,St. C, 58 82, H, 7 53; Found* C, 59 01; H, 7.81

3.4.5 {1-[8-Hydroxymethyij-1 I-Methyl-18-Nitro-10,13-Dioxo-2-Oxa9,12-Diazatricyclo-[14,2,2, 13~7]-Heneicosa-l(19),3,5,7(21), 16(20), 17-Hexaen-l4-Ylcarbamoyl]-3-MethyI-ButylJ-Methyl-Carbamic Acid tert-Butyl ester 33 1 To a solutton of compound 32 (77 mg, 0 1 mmol) m DMF (10 mL) add dry CsF (304 mg, 2.0 mmol) (see Note 11). 2 Star the reaction mixture at room temperature for 15 h, then dilute wtth water, and extract the aqueous solutton wtth EtOAc Wash the combmed organic phases wtth brine, dry (Na,SO,), and evaporate zn vacua 3 Purify the crude product by preparative TLC (ether/EtOAc = 2/l) to obtam the macrocycle 31 (40 mg, 63%), mp 120-121; [o]o =-71” (c 0.1, CHCl,); IR (CHCl,) 3300-3400,1720,1600,1530 cm-‘; ‘H NMR (400 MHz, CDCls, 264K) 6 0.94 ( d, J= 6.4 Hz, 3H, CH&), 0 97 (d, J= 6 6 Hz, 3H, CH&&), 1 35 (d, J= 7 1 Hz, 3H, CHU), 1.49 (s, 9H, But), 1 48-l 52 (m, lH, C_HMe& 1 73 (t, J= 7 4 Hz, 2H, C&CHMe& 2 75 (dd,J=5.4,131Hz,1H,H-15),291(s,3H,N~),365(dd,J=51,131Hz,1H,HIS’), 3.80 (m, lH, C&OH), 3.88 (m, lH, C&OH), 4 51 (m, lH, H-11), 4 66 (t, J= 7 4 Hz, lH, CHCH,CHMe& 5.01 (m, 2H, H-8 andH-14), 5.81 (s, lH, H-21), 6.72 (br s, lH, NH), 6.79 (br d, J= 6 9 Hz, lH, NH), 6.91 (d, J= 7.8 Hz, lH, H-6), 7 01 (d, J= 8 6Hz, lH,H-19),7 19(dd,J=2.0,7 SHz, lH,H-4),7 35(t,J=7.8Hz, lH,H-5),7 39 (br s, lH, NH-9) 7 54 (dd, J= 1 8,8 6 Hz, lH, H-20), 7 83 (br s, lH, H-17), t3C NMR (CDCl,) 6 21 3, 21 7, 23 6, 24 3, 28 2, 36.5, 37.9, 39 3, 49 6, 53 6, 55 4, 56.8, 65 2, 81 4, 112.8,116 8, 120 9,125 3,126 3, 1304, 134 5, 137.2, 139 6, 143 1,148.7, 157 1, 159 9, 168.8, 171 6, 172.3; FABMS (ThtoNaCl) m/z. 664 (M + Na+)

3.4.6 4-Methyl-2-Methylamino-Pentanoic Acid (8-Hydroxymethyl-1 I-Methyl-18-Nitro-10, 13-DIOXO2-Oxa-9,12-Diazatricyclo(l4,2,2, 13’7)Heneicosa-l (19), 3,5,7(2 I), 16(20), 17-Hexaene- 14-yl]Amide 34 1 To a solution of 33 (38.5 mg, 0.06 mmol) in acetomtrtle (6 mL) add cone HCl (0.6 mL) dropwtse (see Note 12). 2 Stir the reaction mixture at room temperature for 1 h, bastfy carefully the solutton (pH = 8 0) by addmon of aqueous NaHCOs, and extract the aqueous solution with EtOAc. Wash the combined organic phases wtth brine, and dry over Na2S04

S,,,Ar-Based Cycloetherification

41

42

Fig 6 Reagents and conditions (a) THF, 70%; (b) NaH, THF, 54%; (c) NaH, THF, MeI, 88%, (d) H,, Pd/C, MeOH; (e) (I) HBF,, ‘BuONO; (11) Cu(NO&, Cu,O, 48%; (f) TFA. 92%

3 Evaporate to dryness give the the pure product 34 (26 mg, 82%) mp 138-14O”C, [a], = -70” (c 0 1, CHC13)

3.5. Synthesis of Cycloisodityrosine Cycloisodrtyrosme 42 IS a key subunit found m a number of btoacttve natural products, such as piperazmomycm (7), btcyclic hexapepttde RA I-XIV (4), and so on. Synthesis of this molecule has been investigated by a wide group of chemists and proved to be difficult (15). Our synthesis based on mtramolecular S,Ar reaction is shown in Fig. 6 (34,35) Coupling of L-3-fluoro-4-mtro phenylalanme methyl ester 10 with L-N-Boc-N-methyl tyrosine pentafluorophenyl ester 35 gave the dipeptide 36 in 78% yield. Cycloetherification of 36 was carried out m degassed THF (see Note 13) using NaH as base at room temperature to give cycltc compound 37 (54%) together with its (9R, 12s) eptmer (19%). Once again, a strongly shielded proton H-l 9 (dH-i9 = 5.58 ppm) ts mdicattve of a cychc structure. N-methylatton of 37 m degassed THF (NaH, MeI) gave the dimethylated compound 38 m 88% yield (see Note 14). Hydrogenation of 38 (Pd/C, degassed MeOH) gave a quantitative yield of the ammo compound 39 (see Note 15), which was submitted directly to the hydroxylation condittons to provide 40 m 48% yield. Methylatton of the phe-

370

Zhu

no1 function (NaH, MeI, THF) gave 41, which was transformed into NJ-dimethyl cyclolsodityrosme 42 (92% yield) by removal of the N-Boc group. 3.5.1. L-3-Fluoro-4-Nitro-N-[N-Boc-N-Methyl-L-Tyrosyl] Phenylalanine Methyl Ester 36 1 Stir a solution of L-N-methyl-N-Boc tyrosme pentafluorophenyl ester 35 (52 4 mg, 0 11 mmol) and L-3-fluoro-4-mtro phenylalanme methyl ester 10 (24 mg, 0 1 mmol) m anhydrous THF (5 mL) at room temperature for 6 h Evaporate the solvent under reduced pressure 2 Purify the crude product by flash chromatography (S102, EtOAc/heptane = l/3 then l/I) Concentrate the appropriate fractions to obtain the dlpeptlde 36 as a white foam (37 mg, 70 %) [aID = -45” (c 1 2, CHCl,), IR (CHCI,) 3599,3409, 2981, 1743, 1687, 1616, 1609, 1532, 1518 cm-‘, ‘H NMR (300 MHz, CDCl,, mixture oftwo rotamers) 6 1 33 and 1 41 (2 br s, 9H), 2 67 and 2 75 (2 br s, 3H), 28-29(m,1H),30-32(m,2H),327(dd,J=56,139Hz,1H),371and372 (2 br. s, 3H), 4.74 (dd,J= 6 9,8.8 Hz, lH), 4 74 8 (m, lH), 5.43 (br s, lH), 6 76 8 (m, 3H), 7 s-7 1 (m, 4H), 7 97 (br t, J= 8 5 Hz, IH), 13C NMR (62 5 MHz, CDC13, mixture of two rotamers) 6 28 2, 30 8 and 3 1, 33 3 and 33 3, 37.6, 52 8, 59 8, 81.2, 115 5, 119 1 (d, J= 21 Hz), 125 5 (d, J= 3 6 Hz), 126.2, 128.3, 130, 135 9 (d, J= 20 Hz), 145.5 and 145.9, 155 1, 155 3 (d, J= 264 Hz), 156 7, 170 7 and 170 8; MS m/z 520 (M + H) Anal calcd for Cz5Hj0FN308 C, 57 79; H, 5.82, N, 8.09 Found* C, 57 61, H, 6 16, N, 7 47

3 5.2. Methyl 12-(S)-/N-(tert-Buty/oxycarbony/)-N-Methylamino]4-Nitro-I I-0x0-IO-Aza-2-Oxatricyclo(12.2.2. 13r7) Nonadeca-3,5,7( l9), 14,76,17-Hexaene-(SS)-Carboxylate 37 1 To a solution of 36 (100 mg, 0.19 mmole) m freshly distilled and degassedTHF (20 mL, 0 01 M> addNaH (17 mg, 0.42 mmol, 60 % dlsperslonm paraffin) (seeNote 13) 2 Stir the resulting slurry at 0” C for 10 mm and then at room temperature for 3 h, quench by addition of aqueousNH&l solution, and extract the aqueoussolution with EtOAc Wash the combined orgamc phaseswith brine, dry (Na,SO,) and evaporate zn vacua 3 Purify the restdue by flash chromatography (S102, EtOAc/heptane = l/2) Concentrate the appropriate fractions to obtam the cychc dlpeptlde 37 and its eplmer (9R, 12s) m the yields of 54% and 19%, respectively Compound 37 mp 10% 11lo C (dec ), [a]n = -90’ (c 0 6, CHC13), IR (CHCl,) 3031, 3013,2925, 2856, 1738, 1675, 1600, 1531cm-‘; ‘H NMR (300 MHz, CDC13)6 1 47 (s, 9H), 2.96 (t, J= 16.8 Hz ,lH), 3 09 (s, 3H), 3.3-3 1 (m, 3H), 3.57 (s, 3H), 4.29 (m, lH), 4 56 (dd, J= 5.1, 11 8 Hz, lH), 5 36 (br s, IH), 5 58 (br s, lH), 6 69 (dd, J= 1.6, 8 4 Hz,1H),715(dd,J=23,83Hz,1H),716(dd,J=23,82Hz,1H),75(m, 2H), 7 87 (d, J = 8 4 Hz, lH), 13CNMR (75 MHz, CDC13) 6 28 6, 31.1, 35.1, 36 7, 52.3, 53 1, 62, 80 7, 119 8, 122, 124 2, 125 8, 126, 130 9, 133 9, 135 9, 143, 150 9, 156, 156 6, 159 5, 168 5, 171.3; MS m/z 500 (M + H), HRMS m/z 500 2036 (C25H30N308(M + 1) requires 500 2033)

S&-Based

Cycloetherification

317

3.53. Methyl 72-(S)-~-(tert-Butyloxycarbonyll-N-Methylamino)lo-Methyl-4-Nitro- 11-0x0- lo-Aza-2-Oxatricyclo(12.2.2. 1317) Nonadeca-3,5,7( 19), 74,16,17-Hexaene-(9S)-Carboxylate 38 1 To a solution of cyclic dipeptide (9S, 129-37 (53.4 mg, 0.11 mmol) in degassed THF (10 mL) and DMF (500 pL) add freshly redistilled Me1 (670 pL, 10 7 mmol) and NaH (5 mg, 0.13 mmol, 60% dispersion in parafin ) at O’C (see Note 14) 2. Stir the resulting reaction mixture at 0°C for 10 mm and then at room temperature for 1 5 h, quench by addition of water (1 mL) and saturated aqueous NH&I solution (10 mL), and extract the aqueous solution with EtOAc. Wash the combined organic phases with brute, dry (Na,S04), and evaporate zn vucuo 3 Purify the residue by preparative TLC (toluene/EtOAc = 4/l) to obtain 38 (47 mg, 85 %) as a colorless oil* [a],, = -143” (c 0.7, CHCl,)

3.5.4. Methyl 4-Amino- lZ-(S)-[N-(tert-Butyloxycarbonyl)N-Methylamino]-IO-Methyl-7 I-0x0-lo-Aza-2-Oxatricyclo(72.2.2. Nonadeca-3,5,7( 19), 14,7 6,17-Hexaene-(9S)-Carboxylate 39

1a7)

1. Stir vigorously a suspension of 38 (18 mg, 0.035 mmol) and 10% Pd/C m 3 mL of degassed MeOH under 1 atm hydrogen atmosphere. 2 After 45 mm, filter the mixture through a short pad of cehte, and wash thoroughly with MeOH. Concentrate the filtrate under reduced pressure to obtain 39 (17 mg, quantitative) as a pmk oil. [a]o =-54.3” (c 0 4, CHCls) (see Note 15).

3.5.5. Methyl - 72-(S)-p-(tert-Buty/oxycarbony/)-N-Methy/amino]4-Hydroxy-lO-Methy/-7l-Oxo-lO-Aza-2-Oxatricyclo(12.2.2. 1317) Nonadeca-3,5,7( 19), 14,76,17-Hexaene-(SS)-Carboxylate 40 1 To a solution of 39 (17 mg, 0 035 mmol) in MeOH (3 mL) add at 0°C HBF4 (5 pL, 0.11 mmol, 54% in diethylether ) and ‘BuONO (10 pL, 0 071 mmol), successively 2. Stir the reaction mixture at 0°C for 30 min, and then at room temperature for 1 h, cool again to 0°C add a solution of Cu(NO& * 3Hz0 (5.12 g, 21 2 mmole) and CuzO (15 mg, 0.035 mmol) m distilled water (10 mL) 3 Stir for another 30 mm, filter the reaction mixture through a short pad of Cehte, and wash thoroughly with CH&l, Extract the aqueous solution with CH$l, Wash the combined organic phases with brine, dry (Na$O& and evaporate zn vacua. 4 Purify the crude product by preparative TLC (SiOz, toluene/EtOAc = 4/l) to obtain product 40 (8 mg, 47%) as colorless oil [a], = -134” (c 0.4, CHCl,)

3.5.6. Methyl - lZ-(S)-/N-(tert-Buty/oxycarbony/)-N-Methy/amino]4-Methoxy-lo-methyl-l I-oxo-lo-aza-2-oxatricyclo(12.2.2. 13,7) Nonadeca-3,5,7( 19), 14,16,7 7-hexaene-(9S)-carboxylate 41 1 To a solution of 20 (4 mg, 8.3 pmol) m dry THF (500 pL) add, at 0°C freshly redistilled CH31 (30 mL, 0 50 pool, 60 Eq) and NaH (1 mg, 0.018 mmol, 60% dispersion in paraffin)

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2 Stir the reactron mrxture at room temperature for 1 h, quench by addmon of saturated aqueous NH&l, and extract with EtOAc Wash the combmed orgamc phases with brrne, dry (Na,SO,) and evaporate zn vacua 3 Purify the crude product by preparative TLC (toluene/AcOEt 5/l) to obtam 41

(3 8 mg, 93%) as a colorless 011. [a]u = -160” (c 0 4, CHCI,) 3.5 7. Methyl -4Methoxy-

72-(S)-(N -rnethy/ammo)IO-Methyl-ll-0x0-lo-Aza-2-Oxatricyclo[12.2.2 13,‘] Nonadeca-3,5,7(19), 14,16,17-Hexaene-&IS)-Carboxylate

42

1 Stir a solutron of compound 41 (3.8 mg, 7 7 pmole) m CH2C12 (1.5 mL) and

CF,COOH (150 &) at room temperature for 1 h 2 Dilute the reaction mixture with H20, and extract with Et,0 to remove the neu-

tral species. 3 Bastfy carefully the aqueous solutron, and extract with EtOAc. Wash the combined organic phases with brine, dry (Na,SO,) and evaporate zn wcuo to obtain pure compound 42 (2 9 mg, 96 %) as a colorless or1 Compound 42 was found to extst as a mtxture of two dtstmct conformers and was readrly detected by TLC (R, = 0 41 and 0 48 m CH,Cl,/MeOH = 10/l), IR (CHCl,) 3682, 2985, 1748, 1642, 1522, 150 1 cm-‘, ‘H NMR (300 MHz, CDCI,, mixture of two conformers A and B [l/l] not assignable) 6 2 59 (s, 3H), 2 62 (s, 3H), 2 66 (s, 3H), 2 75 (s, 3H), 2 79-3 25 (m, 6H), 3.56 (dd,J= 4 2, 10.1 Hz, lH), 3.69 (s, 3H), 3.75 (s, 3H), 3.86 (m, lH), 3 94 (s, 3H), 3 95 (s, 3H), 4 27 (d,J= 2 Hz, lH), 4 41 (dd, J= 3 7, 12 lHz, lH),467(d,J=2Hz, lH),662(br d,J=8 1 Hz, lH),676(d,J=82 Hz, lH), 6 82 (d, J= 8.3 Hz, lH), 6.93 (dd, J= 2.5,8 4 Hz, lH), 7 07 (dd, J= 2 3, 8 3 Hz, lH), 7 22-7 29 (m, lH), 7.3 (m, lH), 7 4 (dd, J= 2 1, 8.3 Hz, lH), 7 48 (dd, J= 2.1, 8.3 Hz, lH), MS m/z 399 (M + H)

3.6. Synthesis Cyclopepttde

of 1CMembered

Cyclopeptide

Alkaloid

(8,9) constitute a large family of natural products (14-membered) and meta (13-membered) cyclophane unit with a characteristic aryl-alkyl ether linkage. Such structure unit has also been found m other natural products, such as anttmttottc agent ustlloxm (36), and so forth. Classically, the key rmg closure was accomplished by way of macrolactamtzatron (37). Our synthesis employmg mtramolecular SNAr reaction for the rmg closure via the formatton of aryl-alkyl ether bond was shown m Fig. 7 (19). Couplmg of 2-(3’~mtro-4’-fluoro) phenylethylamme that typically

alkaloids

contain

a para

43 with L-N-Boc-Phe 44 afforded dipeptide 45. Mild acidic deprotectron (HClMeCN) followed by coupling with N-Boc Ser (TBDMS) OH 46 gave the trtp-

epttde 47 in 68% overall yteld. Simultaneous deprotectron of N-carbamate and U-srlyl ether with HCl-MeCN gave an amino alcohol that was selectively Nbisbenzylated to afford 48 m 78% yield (see Note 16). Treatment of a DMF solution of 48 (0.01 M) with TBAF (see Note 17) m the presence of molecular sieves at room temperature gave a mixture of two separable cychc monomers

S,,,Ar-Based Cycloetherification

313

\-/ cl 53

Fig. 7 Reagents and condltlons. (a) Et,N, N-Boc-Phe, EDC, HOBt, CH,C12; (b) (I) MeCN-HCl, (11) Et,N, N-Boc-Ser(TBDMS), EDC, HOBt, CIj$lp-DMF, (c) (1) MeCN-HCl, (11) NaHC03, BnBr, DMSO-THF, (d) TBAF, 4-A molecular sieves, DMF, (e) NaBH,, elemental sulfur, THF, (f) (i) BF3 * OEt,, ‘BuONO, CH2Cl,, (il) FeSO,, DMF

49 and 50 whose structure was assigned as two atroplsomers from spectroscopic studies and was confirmed by subsequent chemtcal transformatron The lack of atropdlastereoselectivlty is of no consequence, since chiral planarity will be destroyed after removal of nitro group at the final stage. Removal of mtro group was carried out by a two step sequence. Reduction of 49 and 50 with sulfurated NaBHa (38) in THF gave the correspondmg ammo compounds 51 and 52 Dlazotizatlon of 51 and 52 (‘BuONO, BF, * Et,O) (39) followed by FeS04 (40) mediated reduction of crude dlazomum salt gave the desired cyclophane 53 m 53% nonoptlmized yield (see Note 18). 3.6.1. { l-[2-(4-Fluoro-3-Nitropheny/)-Ethyl Carbamoyi]2+heny/ethy/j-Carbamic Acid tert B&y/ Ester 45 1. To a solution of 43 (2 2 1 g, 10 mmol) in CH,Cl, (40 mL) add Et,N (2.1 mL, 15 n-m-101)

After 20 min, add L-N-Boc-Phe44 (2.9 1g, 11 mrnol), HOBt (1.49 g, 11mmol), and EDC (2.11 g, 11 mmol) at O”C, successively

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314

2 Stir the reaction mixture at 0°C for 2 h, dilute with saturated aqueous NH&I and extract with CH,Cl, Wash the combined organic phases with brine, dry (Na,SO,), and evaporate zn vacua. 3 Purtfy the crude product by flash chromatography (EtOAc/heptane = 2/3) Concentrate the appropriate fractions to obtain 45 (3 67 g, 85%) asa yellow solid. mp 139T, [aID = +5” (c 0 3, CHCl,)

3 6.2 (2-(tert-Buty~imethylsilanyloxy)-l-{l[2-(4-Fluoro-3-NitrophenyI)-Ethyl Carbamoyl)-2-Phenyl Carbamoy/}-Ethyl)-Carbamic Acid tert Butyl Ester 47

Ethyl

Stir a solution of compound 45 (2 g, 4 64 mmol) m MeOH (20 mL) and cone HCl(3 mL) at room temperature for 1 h. Evaporate to dryness Dissolve the residue m CH2C12(20 mL) and DMF (5 mL) Add Et,N (1 0 mL, 7 19 mmol) Stir the mixture at room temperature for 20 mm, add L-N-Boc-Ser (TBDMS) (1 62 g, 5 1 mmol), HOBt (688.5 mg, 5 10 mmol), and EDC (979 2 mg, 5 1 mmol), successively. Stir the reaction mixture at room temperature for another 5 h, dilute wtth saturated aqueous NH&l, and extract with CH.$12 Wash the combined organic phaseswith brine, dry (Na,SO,), and evaporate zn vacm Purify the crude product by flash chromatography (EtOAc/heptane = 213) Concentrate the appropriate fractions to obtam 47 (1.99 g, 68%) [a]u = -33” (c 0 2, CHCl,).

3.6.3 2-Dibenzylammo-N-{1-[2-(4-Fluorod-NltrophenyljEthylcarbamoyl]-2- Phenylethyl}-3- Hydroxy Proplonamide 48 1 Stir a solution of 47 (828 mg, 1 3 1 mmol) m MeOH (5 mL) and cone HCl(1 mL) at room temperature for 1 h. Evaporate to dryness 2. Dissolve the residue m THF (20 mL) and DMSO (5 mL) Add NaHCO, (330 mg, 3 93 mmol) and benzyl bromide (0 39 mL, 3.28 mmol) Heat the reaction mixture to 90°C for 12 h (see Note 16) 3 Cool the reaction mixture to room temperature, dilute with saturated aqueous NH&I, and extract with EtOAc. Wash the combined organic phaseswith brine, dry (Na$O,), and evaporate zn vacua 4. Purify the crude product by flash chromatography (EtOAc/heptane = l/l then 21 1) Concentrate the approprtate fracttons to obtain 48 (642 3 mg, 82 %): mp 49°C; [a], = -41” (c 0 1, CHCl,), IR (CHCl,) 3432, 3358, 1684, 1657, 1528, 1487, 1350 cm-‘; ‘H NMR (CDC13,300 MHz, ) 6 2 66 (dd, J= 6 9, 13 8 Hz, lH), 2 72 (dd,J=6.8, 13 8Hz, lH),3.05(m,2H),3.12(dd,J=50,8.1 Hz, lH,OH),3.28 (dd,J=4.4,7.1Hz,lH),335(dd,J=72,137Hz,lH),345(dd,J=66,137Hz, lH),352,375(ABq,J=13.7Hz,4H),394(ddd,J=44,81,12.3Hz,lH),443 (ddd, J= 5 0, 7 1, 12 3, IH), 4 55 (q, J= 7.5 Hz, IH), 5 68 (br t, J= 6 0 Hz, lH, NH), 7.1-7 4 (m, 17H), 7.73 (dd, J= 2 2,7 0 Hz, lH), 7 87 (d, J= 8 1 Hz, lH, NH), 13CNMR (CDCl,) 6 35 1,39 2,40.9,54.9,55 6,59 0,63 2, 119 2 (d, J= 21.2 Hz), 126 6, 127.8, 128 3, 129 3, 129.4, 129.9, 136 5 (d, J= 8 1Hz), 136 9, 137 9, 139 1,

S&-Based

Cycloetherification

315

154 9 (d,J= 262 5 Hz), 171 3,174 2, MS (CI) m/z 599 (M + l)‘, 581, anal calcd for C,,H,,N,O,F. C, 68.21, H, 5 89, N, 9 36, Found: C, 68.27, H, 5.91; N, 9 36.

3.6.4. (4S, 7s) 7-Benzyl-4-Dibenzylamino-14-N&o2-Oxa-6,9-Dlaza-Bicycle (10.2.2) Hexadeca-1(15), 12(16), 13-Triene-5,8-Dione 49 and 50 1 To a solution of 48 (132 mg, 0 22 mmol) in DMF (22 mL, 0 0 1 M) add TBAF (1 A4 solution in THF, 1 1 mL) and molecular sieves (3 A) (see Note 17) 2 Stir the reaction rmxture at room temperature for4 h, dilute with 100 mL ofEt,O, wash the combined organic phases with brine, dry (Na*SO,) and evaporate zizvucuo. 3. Purify the crude product by preparative TLC (EtOAc/heptane = 2/l, 2 migrations) Concentrate the appropriate fractions to obtain 49 (41 mg, 32 %) and Its atroplsomer 50 (47 mg, 37%) Compound 49. mp 119”C, [a]o = -3 13’ (CHC13, c 0.1); IR(CHC1,) 3430,3325,2938,2847,1680,1616,1560,1447,1335 cm-‘, ‘H NMR (CDCl,, 300 MHz, ) 6 2.68 (ddd, J = 3 4, 7.1, 13 7 Hz, IH), 2.82 (dd, J = 9 0, 14 2 Hz, lH), 3 O-3 2 (m, 4H), 3.05, 3.41 (AB q, J= 14 1 Hz, 4H), 4.20 (m, 1H),429(dd,J=1.5,118Hz,1H),441(brq,J=7.0Hz,1H),480(dd,J=89, 11.8Hz,1H),517(d,J=93Hz,1H,NH),562(brs,1H,NH),670(d,J=83Hz), 7.10-7 35 (m, 16H), 7.71 (d, J= 2 1 Hz, lH), 13C NMR (CDCl,) 6 33 5, 37 8, 38 1,53.6,54 8,62 2,70 6, 125 8, 127 2, 127.5, 127 6, 128.5, 128.7, 128 9, 129.3, 134.8, 135.0, 136 2, 138 8, 142 2, 150.2, 168.0, 170 4, MS (CI) m/z 579 (M + l)‘, HRMS m/z 579 2614 (M + l), (&Hj5N405 reqmres 579 2607) Compound 50. mp lOl”C, [aJo =-128” (c 0.1, CHClJ, IR (CHCI,) 3438,3395,2938,2840, 1666, 1623, 1532, 1490, 1447, 1356 cm-‘; ‘H NMR (CDC13, 300 MHz, ) 6 2 50 (m, lH), 2.82 (dd,J= 8 0, 13 8 Hz, lH), 2 9-3 1 (m, 2H), 3.21,3 62 (AB q,J= 14 1 Hz, 4H), 3.38 (dd, J= 1 5, 8 7 Hz, IH), 4 15 (ddt, J= 5.3, 11 1, 14 1 Hz, 1H),435(m,2H),5.01(dd,J=87,11.5Hz,1H),512(brd,J=111Hz,1H), 5 62 (d,J= 9.4 Hz, IH, NH), 7.02 (dd, J= 2 0, 8.5 Hz, lH), 7 10 (d,J= 8.5 Hz, lH), 7 20-7.35 (m, 15H), 7 39 (d,J= 2.0 Hz, 1H); 13CNMR (CDCl,) 6 34.3,39 1, 39.5, 54 0,55.2,62.4,72.0, 119.7, 124 9, 127 1, 127.5, 128.5, 128 7, 128 8, 128 9, 129 3, 129 4, 133.1, 135 0, 136 3, 139.0, 142 7, 148 4, 169 3, 170 2, MS (CI) m/z 579 (M + l)+, HRMS m/z 579.2605 (M + l), (Cj4Hj5N405 requires 579 2607)

3.6.5. (4S, 7s) 14-Amino-7-Benzyl-4-Dibenzylammo2-Oxa-6,9-Dlaza-Bicycle [10.2.2] Hexadeca-1(15), 12(16), 13-Triene-5,8-Dione 51 and 52 1 To a solution of 49 (14 mg, 0 024 mmol) m THF add elemental sulfur (11 5 mg) and NaBH, (4 6 mg, 0 12 mmol) at room temperature 2 Reflux the reaction mixture for 1 h, cool to room temperature, dilute with saturated aqueous NaHC03, and extract with EtOAc Wash the combined organic phases with brine, dry (Na,SO,), and evaporate zn vacua 3 Purify the crude product by preparative TLC (EtOAc/heptane = 2/l, 2 mlgratlons) Concentrate the appropriate fractions to obtain 51 (10 7 mg, 8 1%) mp 91”C, [a], =-101” (c 0 4, CHCl,)

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Following the same procedure, compound 50 was transformed into ammo derivative 52: mp 59”C, [a],, = -56” (c 0.2, CHC13). 3.6.6. (4S, 7s) 7-Benzyl-4-Dibenzylamino-2-0xa-6,9-Diaza-Bicyclo (10.2.2) Hexadeca-1(15), 12(16), 13-Triene-5,8-Dione 53 1 To the flask contammg BF3 Et,0 (17 pL, 0 13 mmol) cooled at -15°C add a solution of 51 (23 mg, 0.042 mmol) m CH& (100 pL), and then precooled ‘BuONO (90% purity, 17.2 pL, 0 13 mmol) dropwise. Stir the reaction mixture at -15°C for 10 mm then at 5°C for 1 h. Evaporate (see Note 18) to dryness 2 Dissolve the residue (dlazonium salt) in degassed DMF, and add FeS04 (0 13 mmol)

3. Stir the reaction mixture at room temperature for 1 h, dilute with aqueous NaHC03, and extractwith EtOAc.Washthecombined organic phaseswith brine, dry (Na,SO,) and evaporate in VUCUO. 4 Purify the crude product by preparative TLC (EtOAc/heptane = l/l) Concentrate the appropriate fractions to obtain 53 (20.4 mg, 91%). [a]o =-93” (c 0 2, CHC13)

The same procedure applied to compound 52 gave 53 m slmllar yield. 3.7. Discussion From the vlewpomt of rational retrosynthetic analysis, mamly two strategies are evident for the synthesis of heterodectlc peptldes with a blaryl ether or arylalkyl ether linkage. The first one consists of formation of the tinctlonallzed blaryl ether or aryl-alkyl ether followed by macrolactamlzatlon, whereas the second one involves the preparation of a linear peptlde followed by cycloethenficatlon as the key cyclizatlon step.The main attribute of the second strategy IS its convergency. However, the existing methods for thts crucial bond-formmg process are scarce, especially when mild condltlons are mandatory because of the sensltlve tinctlonahtles present m natural products. The efficiency of the newly developed mtramolecular SNAr based cycloethenficatlon methodology (see Note 19) m the construction of such structural unit IS remarkable and should find apphcatlons m the synthesisof natural products and/or deslgned bloactlve peptldomlmetlcs 4. Notes 1 Displacement of fluoride via SNAr mechanism was not observed m this alkylatlon step and this may be explained on the basis of HSAB (Hard Soft Acids Bases) principle (41). 2 Protease type VIII-A from B. lzchenzformzs (from Sigma) worked with equal efficiency 3 In our hands, SOCl,/MeOH proved to be the best condltlons to convert compound 5 mto 7 Treatment of 5 under basic condltlons, such as K$O,-MeOHH,O (SO’C), did remove the trlfluoroacetyl function However, it hydrolyze the methyl ester function as well. Moreover, displacement of fluoride by methoxlde (S,Ar) occurred also to a certain extent under these basic condltlons

Sfir-Based

Cycloefherificatron

317

4 Thts step serves to transform the triazene intermediate 5. 6. 7 8

9

10

11 12 13 14 15 16

17. 18 19

to the desired azide and was better than the origmal procedure using KOAc in THF Repeated extractions were required to ensure a good isolated yield of ammo alcohol 17. The high-dilution technique was not required, and possible side products derived from dimerization or from O- and N-transacylation were not observed. Make sure that the reaction mixture IS basic. Otherwise add more NaHCO,. At least 300 Eq of Cu(NO,)z 3H,O were required to ensure a good yield of the desired transformation We used 600 equivalents of Cu(NO& 3H,O m this experiment The use of LiBH, was essential, since other nucleophihc reagents such as NaBH, and ammes led to a sigmficant amount of N-allylated product Ltttle, if any, epimerization occurred m this reaction. However, if EDC (1-[3Dimethylammo propyll-3-ethyl-carbodiimide hydrochloride) or DCC was employed as coupling agent instead of DPPA, racemization at the chit-al center of ammo ester 3 1 was observed, leading to two separable dtastereoisomers in a 2/l ratto CsF was dried on heating to 140°C under vacuum for 5 h CsF was a better promotor than K,CO, for this macrocychzation These deprotection conditions (HCl-MeCN) are superior to the alternative ones using TFA as acid A variable amount of an oxidized product (A) wasisolated if nondegassedTHF wasused A large excess of freshly distilled Me1 should be used m order to Increase the reaction rate and thus avoid the degradation of compound 37 Compound 39 decomposes readily and thus should be used without further purification It is essential to protect the terminal ammo function as dialkylated amme to avoid the possible J3-elimmation as indicated m followmg scheme There are two driving forces, namely releasing of rmg strain and the fact that ortho mtro substituted phenol is a good leaving group, for this undesired reaction. However, when the ammo group was bisbenzylated, both stereoelectromc and steric effects will disfavor this process Other bases such as CsF, NaH, KHMDS, and so forth, were meffective for this cychzation. THF can be used as solvent, but longer reaction time was required In this case, this two-step sequence gave a better yield than the corresponding one-pot (‘BuONO, DMF) (42) reduction procedure The choice of the base that effects the macrocycllzation is substrate-dependent In general, we found that K,CO, and CsF are among the most efficient and mild bases for promoting the formation of aryl-aryl ether bond and TBAF for the arylalkyl ether bond

References 1 Williams, D H (1984) Structural studies on some antibiotics of the vancomycm group, and on the antibiotic-receptor complexes, by ‘H NMR Account Chem Res 17.364-369

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2 NagaraJan, R (1993) Structure-acttvity relattonshtps of vancomycm-type glycopeptlde antibiottcs. J Antzobzot 46, 118 l-l 195 3 NagaraJan, R (1994) Glycopeptide Anttbtottcs Marcel Dekker, Inc , New York 4 Itokwa, H. and Takeya, K. (1993) Antitumor substances from higher plants Heterocycles 35, 1467-1501. 5 Yasuzawa, T , Shirahata, K , and Sano, H (1987) K-13, a novel mhtbttor of angiotensm I converting enzyme produced by micromonospora halophytlca subsp exrhsta II. structure determination J Antzbzot 40, 455-458 6 Sano, S , Ikat, K., Katayama, K., Takesako, K., Nakamura, T , Obayasht, A., et al (1986) OF4949, new inhibitor of ammopepttdase B II Elucrdatton of structure J An&blot 39, 1685-1696. 7 Kaneda, M , Tamar, S , Nakamura, S , Hirata, T , Kusht, Y., and Suga, T (1982) Piperazinomycm, a novel antifungal anttbtottc Il. Structure determmatlon J Antzbzot 35, 1137-l 140. 8 Joullie, M J. and Nutt, R E (1985) Cyclopeptrde alkaloids, m Alkulozds Chemzcal and Bzologzcal Perspectives, vol 3 (Pelletter, S W., ed.), Wiley, New York, pp. 113-168. 9 Schmtdt, U , Lieberknecht, A , and Haslmger, E (1985) Pepttde alkaloids, m The Alkalozds, vol 9 (Brossi, A , ed ), Academic, New York, pp 299-326. 10 Keseru, G M and Nogradt, M (1995) The chemistry of natural diarylheptanoids, m Studies in Natural Products Chemistry, vol 17 (Atta-ur-Rahman, ed.), Elsevter Sctence B V , Amsterdam, pp 357-394. 11 Hobbs, D W and Still, W C (1989) Synthesis of a thioether analog of the macrocychc trtpeptide K-l 3 Tetrahedron Lett 30,5405-5408 12 Podlogar, B. L , Farr, R A., Frredrtch, D., Tarnus, C , Huber, E W., Cregge, R J, et al. (1994) Design, synthesis, and conformational analysts of a novel macrocycltc HIV-protease mhlbrtor. J Med Chem 37, 3684-3692 13 Chen, J J , Coles, P. J , Arnold, L 0 D., Smith, R. A , MacDonald, I D , Cam&e, J , and Krantz, A (1996) Synthesis and activity of conformationally-constrained macrocyclic norstatme-based mhtbitor of HIV protease Blorg Med Chem Lett 6,435-438 14. Reid, R C , March, D. R , Dooley, M J , Bergman, D A , Abbenante, G , Fairhe, D. P (1996) A novel bicychc enzyme mhtbttor as a consensus pepttdomtmetics for the receptor-bound conformations of 12 peptic mhibitors of HIV- 1 protease J Am Chem Sot 118,85 1 l-85 17 and references cued therem 15 Rama Rao, A V , GurJar, M K , Reddy, L., and Rao, A S. (1995) Studies directed toward the synthesis of vancomycm and related cyclic peptides Chem Rev 95, 2135-2167. 16 Boger, D L , Patane, M. A., and Zhou, J. (1994) Total syntheses of bouvardm, Omethylbouvardm, and 0-methyl-N9-desmethylbouvardm. J Am Chem Sot 116, 8544-8556 and references cited therem 17. Nakamura, K., Ntshiyama, S , and Yamamura, S. (1995) Synthetic studies on vancomycm. synthesis of seco-aglucovancomycins Tetrahedron Lett 36,862 l-8624 and references cued therem

S,.&-Based Cycloetherification

319

18. For a highlight, see. Burgess, K , Lim, D., and Martinez, C. I. (1996) Nucleophihc aromatic subsmutton-a possible key step m total synthesis of vancomycm Angew Chem Znt Ed Engl 35,1077-1078. For a short account, see: Zhu, J (1997) SNAr based macrocychzation via biaryl ether formation. Application in natural product synthesis SynZett 133-144. For more recent examples, see’ Beugelmans, R , BoisChoussy, M , Vergne, C , Bouillon, J. P , and Zhu, J (1996) Synthesis of a model bicyclm C-O-D-O-E ring of vancomycm by a one-pot double SNAr based macrocyclizatton. J Chem Sot Chem Commun. 1029-1030; Vergne, C , Bois-Choussy, M , Beugelmans, R , and Zhu, J. (1997) Synthesis of four atropdiastereoismers of C-O-D-O-E rmg of vancomycm by sequential cycloetherifications. Tetrahedron Lett 38, 1403-1406, Bois-Choussy, M , Vergne, C , Neuville, L , Beugelmans, R , and Zhu, J (1997) Synthesis of model tricyclic CO-D-O-E-F-O-G rmg of teicoplamn Tetrahedron Lett 38,5795-5798 19 Zhu, J , Laib, T , Chastanet, J , and Beugelmans, R (1996) A novel strategy towards the total synthesis of cyclopepttde alkaloids Angew Chem Int Ed Engl 35,2517-2519 20 For a recent book, see Terrier, F (1991) Nucleophlllc Aromatic Dzsplacement The Role of the Nltro Group VCH, New York 21 Vergne, C , Bois-Choussy, M , Ouazzatn, J , Beugelmans, R , and Zhu, J (1997) Chemoenzymatm synthesis of enantiomermally pure 4-fluoro-3-mtro and 3fluoro-4-mtro phenylalanme Tetrahedron Asymmetry 8,391-398 22 Bois-Choussy, M , Beugelmans, R , Bouillon, J P., and Zhu, J. (1995) Synthesis of a modified carboxylate-bmdmg pocket of vancomycm Tetrahedron Lett 36, 478 14784 23 Bois-Choussy, M , Neuville, L , Beugelmans, R , and Zhu, J (1996) Synthesis of modified carboxylate-bmdmg pocket of vancomycm and teicoplanm J Org Chem 61,9309-9322 24. Evans, D. A , Britton, T C , Ellman, J A , and Dorow, R L (1990) The asymmetric synthesis of a-ammo acids Electrophihc aztdation of chnal amide enolates, a practical approach to the synthesis of(R)- and (S)-a-azido carboxylic acids J Am Chem Sot 112,40114030 25 Penning, T D., DJUriC, S W , Haack, R A , Kaltsh, V J , Miyashno, J M , Rowell, B W , et al (1990) Improved procedure for the reduction of Nacyloxazohdmones Synthetic Commun 20,307-3 12 26 Main, S N , Smgh, M P , and Micetich, R. G. (1986) Facile conversion of azides to ammes. Tetrahedron Lett 27, 1423-1424 27 Beugelmans, R , Bigot, A , and Zhu, J (1994) A novel synthesis of K-13 Tetrahedron Lett 35,739 l-7394 28 Cohen, T , Dietz, A G , and Miser, J. R. (1977) A simple preparation of phenols from diazomum ions via the generation and oxidation of aryl radicals by copper salts J Org Chem 42,2053-2058 29. Evans, D. A., Ellman, J. A. (1989) The total synthesis of the isodityrosme-derived cyclic tripeptides OF4949-III and K-l 3 Determination of the absolute configuration of K-13. J Am Chem Sot 111, 1063-1072.

320

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30 Walsh, C T (1993) Vancomycm resistance. decodmg the molecular logic Sczence 261,308-309. 31 Beugelmans, R , Bourdet, S , Begot, A , and Zhu, J (1994) Reductive deprotection of aryl ally1 ether with Pd[(PPh),],/NaBH, Tetrahedron Lett. 35,4349-4350 32. Beugelmans, R., Luc, N , Bois-Choussy, M , Chastanet, J , and Zhu, J (1995) Palladium catalyzed reductive deprotection of allot transprotection and peptide bond formation Tetrahedron Lett 36,3 129-3 132 33 Eliel, E. L. and Willen, S. H. (1994) Chnahty in molecules devoid of chual centres, m Stereochemistry of Orgamc Compounds. John Wiley, New York, pp 1119-1190. 34 Beugelmans, R , Bigot, A , Bois-Choussy, M., and Zhu, J. (1996) A new approach to the synthesis of piperazinomycm and bouvardm. facile access to cycloisodityrosme via an mtramolecular S,Ar reaction J Org Chem 61, 77 l-774 35 Bigot, A , Beugelmans, R., and Zhu, J (1997) A formal total synthesis of deoxybouvardm Tetrahedron 53, 10,7.53-10,764 36 Koiso, Y , LI, Y., Iwasaki, S , Hanaoka, K , Kobayashi, T , Sonoda, R , et al (1994) Ustiloxms, antimitotic cychc peptides from false smut balls on rice panicles caused by ustilagmoidea vnens J Antlbzot 47, 765-773 37. Schmidt, U , Zah, M., and Lieberknecht, A (1991) The total synthesis of frangulanme J Chem Sot , Chem Commun 1002-1004 38 Lalancette, J M , F&he, A , Brindle, J R., and Lahbertt, M (1972) Reductions of functional groups with sulfurated borohydrides Application to steroidal ketones. Syntheses 526-532 39. Doyle, M P. and Bryker, W J (1979) Alkyl nitrite-metal hahde deammation reactions 6. direct synthesis of arenediazomum tetrafluoroborate salts from aromatic ammes, tert-butyl nitrite, and boron trtfluoride etherate m anhydrous media

J Org Chem 44, 1572-1574 40. Wassmundt, F. W and Kiesman, W F (1995) Efficient catalyst of hydrodediazomattons m dtmethylformamide J Org Chem 60, 17 13-l 7 19 41 Pearson, R G (1987) Recent advances m the concept of hard and soft acids and bases J Chem Edu 64,561-567 42 Doyle, M. P , Dellaria, J. F., Siegfried, B., and Bishop, S W (1977) Reductive deammation of arylamme by alkyl nitrites m N,N-dimethylformamtde. A direct conversion of arylammes to aromatic hydrocarbons J Org Chem 42,3494-3498

Cyclic Aromatic Amino Acids with Constrained x1 and x2 Dihedral Angles Dirk Tourwd, Koen Iterbeke, Wieslaw M. Kazmierski, and Gdza T&h 1. Introduction The concept of topographic design of peptide neurotransmitters and hormones was pioneered by Hruby (1,2). When the design involved primarily constramt of the side chains of a peptide that has a well-defined backbone conformation, the term “topographic design on a stable template” was proposed (3) The side cham x’ of aromatic ammo acids, such as Phe, Trp, Tyr, and His, can be constrained m either the gauche (-) or gauche (+) conformation by linking the nitrogen atom to the aromatic rmg through a methylene bridge (Fig. 1). For Tic, a clear preference was found for a gauche (-) conformation when it is at the N-terminus of a peptide, and for a gauche (+) conformation when it is m the peptide sequence (3). As a result of the rmg constraint, also x2 and $I are restricted (45). The concept mitially resulted m the potent p opioid antagonist D-Tic-CTAP (61, based on a somatostatm sequence Since then, TIC has been successfully applied in bradykmm antagonists (7), ACE inhibitors (8), renin inhibitors (9), and m the &opioid antagonist TIPP (10,11). Tic is a constituent of a potent farnesyl-transferase mhibttor (12), and its perhydroderivative is a constituent of a potent HIV protease mhibitor (13) Similarly, the tryptophane analog Tee is a constituent of bombesm antagonists (24-16). The histidme analog is a naturally occurring compound, spmacme. It was used m bombesm (26) and glucagon (17) analogs. Various ring-substituted analogs of Tic and of the histidine analog are From

Methods Edlted

m Molecular

Me&one,

by W M Kazmlerskl

Vol 23 fepbdom/met/cs

OHumana

321

Press

Inc , Totowa,

Protocols NJ

322

Tourwh et al

M

COOH

HN+;o ?zooH M&,C COOH

TIC

Tee

TIP (spmacme)

Fig 1 Prmciple of side-chain constraint for Phe, Trp, and His

studred as nonpeptide angiotensin II antagonists (18,29). The main synthetic method for this type of amino acids is a Pxtet-Spengler condensation reaction with formaldehyde (Fig. 2). This chapter describes the synthesis of various cyclic analogs of Phe, Tyr, Trp, and His and the analysis of their optical purittes. 2. Materials 2.7. Reagents for Method 3.1 1 2 3. 4 5. 6 7. 8 9 10 11 12 13

Acetone. Acetomtrile (CHsCN) Ammomum hydroxide (NH,OH). Dimethyl sulfoxide (DMSO) Ethanol (EtOH). Formaldehyde (CH,O) 37% solution m Hz0 Hydrochloric acid (HCl) Methanol (MeOH). L-Phenylalanme (t.-Phe). Sodium hydroxide (NaOH) 2,3,4,6-Tetra-O-acetyl-p-n-glucopyranosyl isothiocyanate (GITC). Triethylamme (NEt3) Trifluoroacetic acid (TFA)

323

Cychc Aromatrc Amino Acids

X

-

Y

RI

Rz

RT

compound

H

H

H

H

H

TIC (1)

H

H

CH?

H

H

a-Me-TIC

H

H

H

CH3

H

eryrluo-/3-Me-TIC

H

H

H

H

CH3

thrco-P-Me-Ts

H

H

CH3

CH3

H

(3s 1S)-a$-dlMe-Tic

(&I)

H

H

CH1

H

CHT

(3s JR)-a.P-dlMe-Trc

(4h)

OH

H

H

H

H

G-HE (5)

H

OH

H

H

H

7-HIC (6)

(2) (-?;I) (31,)

Fig 2 Plctet-Spengler condensation leadmg to TIC analogs

2.2. Reagents for Method 3.2 1 2 3 4 5 6 7 8

CH,CN NH40H EtOH HCI MeOH L-a-Methylphenylalanme Paraformaldehyde (CH,O), TFA

2.3. Reagents for Method 3.3 1 2 3 4 5 6 7

Acetic acid (AcOH) CH,CN Erytho-P-methylphenylalamne HCl CH20 37% solution m H,O HCI. Marfey’s reagent (N-a-[2,4-dmltro-5fluorophenyll-L-alaninamlde; MeOH

FDAA)

Tourwk et a/

324 8 9 10 11

n-Butanol (nBuOH) Sodrum bicarbonate (NaHC03) Threo-P-methylphenylalanme TFA

HCl

2.4. Reagents for Method 3.4 1 2. 3 4. 5 6 7 8. 9 10 11 12 13 14 15 16 17 18 19 20

Acetyl chloride L-Alamne HCl NH&l Benzoyl chloride (1-Bromoethyl)benzene Chloroform (CHQ) Dlchloromethane (DCM) Dnsopropylethylamme (DIEA) EtOH Ethyl acetate (EtOAc) Ethyl ether Formaldehyde (CH*O) 37% solution m HZ0 HCI Lithium dnsopropylamlde (LDA) Magnesium sulfate (MgSOJ. Nitrogen (N2) Paraformaldehyde Petroleum ether Pwalaldehyde Tetrahydrofuran (THF)

2.5. Reagents 1 2 3 4 5 6 7

Acetone CH$N CH20 37% solution m H20 HCl MeOH TFA D,L-m-Tyrosme

2.6. Reagents 1 2 3 4 5

for Method 3.5

for Method 3.6

AcOH CH,CN. 3’,5’-Dnodo-L-tyrosme 1,2-Dlmethoxyethane EtOH

(DME).

Cyclic Aromatic Amino Acids 6. 7. 8. 9 10 Il. 12 13 14.

325

CH20 37% solution m HZ0 HCI. Hydrogen (H2) MeOH nBuOH Pd-C (10%) Sodmm thtosulfate (Na$,O,). Triethylamine (NEt,). TFA

2.7. Reagents for Method 3.7 1 2 3 4 5 6 7

CHsCN NH,OH. CH,O 37% solution m HZ0 MeOH. Sulfurrc acid (H,SO,) TFA L-Tryptophane.

2.8. Reagents for Method 3.8 1 2 3 4

Ammonmm hydroxide (NH,OH) CH,O 37% solution m H,O. L-Histidine HCl

2.9. Analysis

Techniques

1. Melting point (mp) determined m open capillary tubes using a Thiele tube 2 Thin-layer chromatography (TLC): Merck (Darmstadt, Germany) 60 stlrca plates wrth fluorescent mdicator F254, solvent systems indicated Detectron with a UV lamp or m Iz-atmosphere 3 Optrcal rotation (or,)* Optical Activity AA5 (Cambridge, UK) automatic polarrmeter (h = 289 nm) 4 Reversed-phase-high performance hqurd chromatography (RP-HPLC). column* Vydac (Hesperra, CA) 218TP54 column (C-18; id = 4 6 mm, I= 25 cm); flow = 1 mL/min , detection h = 2 15 nm, unless otherwise noted 5. Mass spectrometry (MS)* VG-Quattro II (Manchester, UK) instrument. The romzatron technique IS electrospray All compounds described show the calculated Mt + 1 molecular peak. 6 ‘H-Nuclear magnetic resonance (‘H NMR)* Bruker (Karlsruhe, Germany) AM250-P (250 M Hz)

326

Tourw6 et al.

3. Methods

3.1. Preparation of 1,2,3,4-Tetrahydroisoquinoline3-Carboxylic Acid (Tic) 1 The Plctet-Spengler reaction leading to Tic (20), as shown 1n Fig. 2 (all X, Y, R = H), has been descrtbed either 1nrefluxing concentrated HCl and sulfuric acid for 2 d (21) or 1n concentrated HCl for a total time of 3.5 h (22). An evaluation of both methods with respect to yield and optical purity of the final compound led to the procedure described below This method gives rise to partial racemlzatlon. Optically pure (3s)- and (3R)Tic has been obtained by fractional crystallization of 1tsbenzyl esterp-toluenesulfonate (23). Alternatively, an asymmetric transformation of the racemate via salt formation with (IS)-lo-camphorsulfomc acid, leading to (3S)-T1c 1n 80% yield was reported (24). A synthetic method based on the base-catalyzed reaction of dlethyl-2(acetylammo)malonate with 1,24u(bromomethyl)benzene was used for largescale synthesis (25) The racemate was either transformed into the (-)-menthyl ester, followed by chromatographlc separation, or transformed into 1tsbenzyl ester and resolved by fractional crystallization of the mandellc acid salts. The method described below gives a rapid accessto (3s)- or (3R)-T1c analogs and describes a convenient method for an accurate determination of the enantiomeric composition, 3.1.1. (S)- 1,2,3,4- Tetrahydroisoqumoline-3-Carboxylic

Acid 1

To 25 g L-Phe (0 15 1 mol) add 215 mL 10 N HCl and 54 mL of a 37% aqueous formaldehyde solutton Star the reaction mixture for 30 mm m a botlmg water bath (see Note 1) Add another 53 mL 10 N HCl and 26 mL 37% aqueous formaldehyde Continue stnrmg the reaction mtxture for another 2 h m the bollmg water bath Remove the reaction flask from the boiling water bath, and allow tt to cool slowly to room temperature, then store tt overnight m a refrigerator. Filter the white precipitate, and wash with 150 mL water, then with 50 mL acetone (see Note 2) Recrystallize from hot ethanol/water (l/l) An ee of 70% IS determined (see Subheading 3.1.2.) After two recrystalltzattons opttcally pure L-Tic HCl 1s

obtained* 18 7 g, yield 72% When the pH of the recrystalhzat1onsolvent 1sadjusted to 7 0, using cont. NH,OH, the zwlttertomc form IS obtained Characterizatton of L-Tic * HCl mp 273-275°C TLC (CHsCN/H,O/MeOH)* R,= 0 53, [a]o =-177 4’ (c = 1, 1 NNaOH) *H NMR (DMSO) 7.3 (m, 4H, 4H,,,,), 4 3 (m, lH, Hs), 4 2 (s, 2H, H,), 3 3-3 0 (m, 2H, H4) HPLC: H,O + 0 1% TFA to 20% CH,CN m Hz0 with 0 1% TFA m 30 mm, k’ = 2 42

327

Cyclic Aromatic Amino Acids 3.7.2. Determ/nat/on of Enantlomenc Composit/on of TIC 7 (26,27)

1 Dissolve 1 mg Tic -1 m 2 mL of a water/acetonitrtle (l/l) solution contammg 0.4% triethylamme. 2 A 100 pL aliquot of this solutton is added to 100 pL of a 0 2% (w/v) solution of GITC m acetomtrile and allowed to react for 0.5 h 3 A 20-pL ahquot is injected onto the RP-HPLC column, with UV-detectton at 250 nm (see Note 3) Gradient 20% acetomtrile in HZ0 to 55% acetonitrile m HZ0 in 20 min k’ (GITC-L-Tic) = 4 2 1 k’ (GITC-D-Tic) = 4.42

3.2. Preparation of (3S)-Methyl1,2,3,4-Tetrahydroisoquinole-3-Carboxylic

Acid (a-MeTic) 2

1 To 250 mg (S)-a-methylphenylalanme (1.40 mmol) add 6 mL 6 N HCl and 0.5 g paraformaldehyde 2. Stir the reaction mixture overnight (18 h) at 100°C. 3 Allow the mixture to cool to room temperature, and evaporate to dryness 4 Dissolve the residue in hot ethanol/water (l/l), and adJust the pH of the solution to 7 by addmg a 1 5 N NH40H solution 5. Store m the refrigerator for one hour, and filter the white precipitate Wash with ice-cold water (see Note 4) Yield+ 145 mg (58%) Characterization: mp* 282-285’C, TLC (CHsCN/MeOH/H,O, 4/1/l))* R,= 0 62, ‘H NMR (D,O)* 7 O-6 8 (M, 4H, H ,,,,),4 18(d, lH,H,n,J= 16Hz),4O(d, lH, H,, J= 16 Hz), 2 95 (d, lH, H,, J= 16 Hz); 2.73 (d, lH, H,,, J= 16 Hz), 12 (s, CH,). HPLC (1% CH,CN m H,O, 0 1% TFA isocratic) k’ = 5 06

3.3. Preparation

of P-Methyl-Tic

Isomers 3a,3b

The preparation of the @MeTic isomers by the Prctet-Spengler reaction was performed on racemic erythro- or threo+MePhe (29). These were obtained by reaction of diethyl-2-(acetylammo)malonate with 1-bromoethylbenzene (30). The dtastereomers of P-Me-Phe were separated by fractional crystalltzation. The resulting racemtc P-MeTtc isomers can be incorporated mto the pepttde sequence, the resultmg stereoisomeric pepttdes separated, and the absolute configuration of the P-MeTtc residue can be determined after hydrolysis of the pepttde (26), as described in Subheading 3.3.3.

3.3.7. Preparation of Rae-Erythro-p-Methyl-T/c

3a

1 As described m Subheading 3.1.1., add 20 mL 12 N HCl to 3 15 g erythro (2S, 3R, and 2R, 35”)~P-methyl-Phe HCl(O.0146 mol), followed by 5 mL of a 37% aqueous formaldehyde solution 2 Stir the reaction mixture m an 011bath at 110°C for 30 mm (reflux). 3 Add another 5 mL 12 N HCI and 2 5 mL formaldehyde solution 4 Continue to reflux for an additional hour.

Tourw.6 et a/.

328

5 Remove the oil bath, cool the reactton mixture and evaporate to dryness 6 The residue IS crystalhzed from water. Yield 2 4 g, (72%) of erythro+Me-Tic HCl Charactertzatton mp 278-281°C, TLC (n-BuOHIAcOHIH,O 4.1’1) R,= 0 47, ‘H NMR (D,O)* 7 57-7.34 (m, 4H, H ,,,,),453(d,2H,H,,J= 18Hz),3 89 (m, IH, Hs), 3 54 (m, lH, H,); 1.59 (d, CH,, J= 7 5 Hz) HPLC. H,O + 0 1% TFA to 80% CH,CN in H,O + 0 1% TFA In 30 min. k’ = 2 18

3.3.2. Preparation of Rat-Threo-P-Methyl-Tic

3b

Use the procedure described above, starting from 2 g threo-P-methyl-Phe HCl(O.093 mol) Yield. 1 g (66 %) of threo-P-Me-TIC HCI Charactertzatton. m p 253-257°C TLC (n-BLJOHIA~OHIH,O 4 1 1) Rr = 0 45, ‘H NMR(D,O) 7 52-7 35 (m, 4H, H,,,,); 4 58 (d, 2H, H,,J= 18 Hz), 4.47 (m, lH, H,), 3 74 (m, lH, H4), 1 39 (d, CH,, J = 7 5 Hz) HPLC 100% H,O + 0.1 TFA to 80% CH,CN m H,O + 0 1% TFA m 30 mm k’ = 2 18

3.3 3. Determination of the Absolute Configuration of the P-Me-Tic Isomers (31) After mcorporation of the racemtc erythro- or threo-P-methyl-TIC mto the pepttde sequence, the resultmg diastereomertc pepttdes are separated by RPHPLC. The pepttdes are hydrolyzed to the amino acids m the usual way. The (2S, 3$)-P-M e-T ic was dtstmgutshed from the (2R, 3R) enanttomer by dtgestton of the racemate with L-ammo acid oxtdase, followed by choral dertvattzatton and HPLC analysts. The enzymatic dtgestion dtd not proceed for the (2R, 3s) and (2S, 3R) racemate. Therefore, a standard of (2S, 3R)-P-Me-Tic was prepared from a sample of (2S, 3R)-P-Me-Phe. Marfey’s reagent (FDAA) (33) proved to be more effictent for the separation of the enanttomers than GITC. Hydrolyze a I-mg sample of the [P-Me-Tic] contammg pepttde m 300 uL 6 N HCl (33) Dissolve the restdue m 500 @ water Take 100 uL of this solutton and add 50 pL 1 M NaHCOs Check the pH, and add addmonal NaHCO, solution until the solution 1sbasic Prepare a 1% (w/w) solutton of Marfey reagent m acetone Add a 1 5 M excess of thts solutton to the ammo actd solutton Thermostat the sample for 90 mm at 40°C, then allow the sample to cool to room temperature, and add 25 l.tL 2 M HCl Inject a 2O+L sample onto a Vydac 2 18TP54 HPLC column, and elute m tsocratlc mode with 0.1% aqueous TFA/CH$N/MeOH (51/12/37) as eluent, and UV detectton at 340 nm.

Cyclic Aromatic Amino Acids

IS)-

\h-\H\le

329

-

h

IL3

13S.4Si-4a

Fig 3 Asymmetric synthesis of (3S,4S)-a$-dimethyl-Tic Elutlon sequence, (23, 35’) (k’ = 9.36), (2R, 35')(k’ = 10 55), (2R, 3R) (k' =I 1 60), (2S, 3R) (k’ = 13 28)

3.4. Preparation of a$-Dimethyl1,2,3,4-Tetrahydroisoquinoline-3-Carboxylic

Acid 4

This dimethyl-substituted compound was deslgned to provide a Tic analog with complementary conformatlonal properties (34). In contrast to L-TX, the (3s) analogs of 4 have an energetxally favored gauche (+) conformatlon m the N-terminal free amino acid and a gauche (-) conformation in the N-acylated form. The Plctet-Spengler cycllzatlon was performed on each stereolsomer of

a$-dimethylphenylalamne 11. These were prepared by asymmetric synthesis using imidazolidinone chemistry developed by Seebach (Fig. 3). The synthesis of the (2S,35’)- and of the (2&3R)isomers is described; by symmetry rules, however, the same procedure can be applied for the preparation

of the correspond-

mg (2R,3R)- and (2R,3S)-enantiomers, 3.4.1.(2S,5S)-I-Benzoyl-2-tert-Butyl-

3,5-Dimethyl-lmidazolidin-4-One,

9a

1 Suspend 10 g (0 0722 mol) of L-alamne * HCl salt and 20 g of magnesmm sulfate in 180 mL of DCM 2 Add 10 5 mL (0 0964 M) of plvalaldehyde, followed by 18 2 mL (0 1044 mol) of DIEA, while vigorously stxrmg the reaction at 0°C 3. Continue the reaction overnight at room temperature. Filter off the solids, and remove the solvents 4 Dissolve the mune m 100 mL of absolute ethanol, and add dropwlse 8 82 g (0 112 mol) of acetyl chloride at -10°C. 5 Allow the reaction to warm up while the optically pure (2S,SS)-lmldazohdmone precipitates out Filter it out, and wash with small amounts of cold ethanol 6 To the above precipitate add 100 mL DCM and 0 16 Mof DIEA (20 48 g, 27 8 mL), followed by 0 08 M of benzoyl chloride (11.24 g)

330

Tourwk et al

7 Stir overnight at room temperature, extract 3x with 1M aqueous sodium carbonate, followed by 3x water 8 Dry the DCM layer with magnesium sulfate and purify the product on slhca gel Yield 78 4% ‘H NMR (CDCl,) 7 9-7 3 (m, 5H), 5 64 (s, lH), 4 30 (q, lH, J= 7 3), 3 03 (s, 3H), 1 06 (s, 9H), 1 00 (d, 3H, J= 7.3), 1 06 (s, 9H)

3.4 2.(2S,5S)-7-Benzoy/-2-tert-Buty/-3-Methy/5-(a-(S)-Methyl-Benzyl)lmrdazoki~n-4-One,

1Oa

Dissolve 58 6 g (0 213 mol) of 9a m 1300 mL THF, and cool to -78°C under N, blanket To the above solution slowly add 0 256 A4 of precooled LDA (see Note S), and equilibrate for 45 mm (the solution turns red). Add 38.75 g (0 213 mol) of (I-bromoethyl)benzene at -78°C Allow the reaction to warm up to a room temperature, and continue it for 60 h (see

Note 6) Pour the reaction into 300 mL of saturated aqueous ammonium chloride, and extract with 2 x 500 mL of ethyl acetate. Combine and dry the organic phases A silica gel purlficatlon (2.1 pet. ether/ethyl acetate) should result m 50 4 g of 10a and lob, m the ratio of 1.2.5, respectively Separate both chastereomers by a crystallization from 2.1 pet ether/ethyl acetate, (2$,5S,5&)-lob crystallizes first as large cubes, and (2S,5S,5-aR)-lob forms fine needles 10a ‘H NMR (CDCI,) 6 7 7-6 7 (m, IOH), 5 76 (s, lH), 3 09 (s, 3H), 3 00 (q, lH, J= 7 2), 1 26 (s, 3H), 1 09 (d, 3H, J= 7 2), 1 06 (s, 9H). [c~]*~o = 106 9” (c = 0 569, CHC13)

3.4.3. (2S,5S)-i-Benzoy/-2-tert-Buty/-3-Methy/-5-(~~-(R)Methyl-Benzyl)Imidazolidin-4-One (~S,~S,~-CXR)-~O~ This compound ISsynthesized m a manner similar to that of lOa, except that a threefold excessof (1-bromoethyl)benzene is used over 12 h. Yield (recrystallized from ethyl acetate/petroleum ether 1 2 [v/v]) 81 7% ‘H NMR (CDC13 two rotamers) 6 7 80-6 60 (m, lOH), 4 86 (s, 0 38H), 4.80 (s. 0 62H), 4.25 (q, J= 7 4,0 62H), 2 97 (q, J= 7 1,0 38H), 2 90 (s, 1 77H), 2 53 (s, 1.23H), 2 00 (s, 1 77H), 1 65 (s, 1 23H), 1 55 (d, J= 7 4, 1 77H), 1 23 (d, J = 7 14, 1 23H), 0 91 (s, 5 58H), 0.65 (s, 3 42H) [c~]~~,, = 41 4” (c = 0 5 10, CHCl,)

3 4.4. (2S,3R)-2,3-Dimethylphenylalanine,

1Ib

1 Suspend 0 40 g (1 06 mmol) of lob m 10 mL of 6 N HCl m a glass hydrolysis tube, cap, and heat at 220°C for 5 h (see Notes 7 and 8) 2 Cool down the tube to-30”C, vent, and extract the contents with ethyl ether (3x) 3 Convert the hydrochloride salt mto a zwltterlon (see Note 9) Yield 75% ‘H NMR (D*O) 6 7 08 (m, 5H), 2 94 (q, lH, J= 7 3), 1 03 (d, 3H, J = 7 3), 0 96 (s, 3H) [cx]*~~ = 20 7” (c = 0 458, MeOH)

331

Cyc/ic Aromatic Amino Acids 3.4.5. (2S,3S)-2,3-Dimethylphenylalanine,

1la

This compound 1s synthesized in a manner similar to that of 2S,3R-llb. Yield 82%. ‘H NMR (D,O): 6 7.09 (m, 5H), 3.07 (q, lH,J= 7.4), 1.14 (s, 3H), 25D = -3 1.7” (c = 0.492, MeOH). 1.11 (d, 3H, J= 7.4). [a]

3.4 6. (3S,4S)-3,4-Dlmethyl3-Carboxylic Acid, 4a

1,2,3,4-Tetrahydroisoqunoline

1 Combme 0 log of (2S,35’)-1 la, 0.50 g of paraformaldehyde (see Note 10) and 6 mL of 6 N aqueous HCI m a Pyrex tube 2. Degas, cap, and heat at 170°C for 10 h (see Note 7). 3 Cool down the tube to -30” C, vent (see Subheading 3.4.4.), and convert the hydrochloride salt to a zwltterionic form (see Note 9) to obtain 0 076 g (72%) of 4a ‘H NMR (D,O). 6 7.18-7.00 (m, 4H), 4 18 (d, lH, J= 16 0), 4 00 (d, lH, J= 16.0), 3.17 (q, lH, J= 7.4), 121 (s, 3H), 1 06 (d, 3H, J= 7 4) [c1]~~o=-16 7” (c = 0.45, MeOH)

3.4.7. (3S,4R)-3,4-Dimethy/-7,2,3,4-Tetrahydroisoquino/ine 3-Carboxylic Acid, 4b 1 Combme 5.2 g, (26.94 mmol) of llb, 20 mL of 37% aqueous formaldehyde and 60 mL of cone HCl m a round-bottom flask 2 Reflux for 1 h, evaporate, and convert the hydrochloride salt mto a zwltterlomc form (Note 9) Yield 3 9 (70.6%) of 4b ‘HNMR(D,O) 6704(m,4H),4.19(d,1H,J=160),4.07(d,1H,J=167), 2 93 (q, IH, J= 7 3), 1.18 (s, 3H), 0.95 (d, 3H, J= 7 3). [a12$,=-91.4 (c = 0 466, MeOH)

3.5. Preparation of (R,S)-1,2,3,4-Tetrahydroisoquinoline6-Hydroxy-3-Carboxylic Acid 5 The Plctet-Spengler reaction was performed on racemic meta-tyrosine (35). The reaction In the standard condltlon 1sreported to result m several byproducts. In weakly acidic conditions, the cyclic amino acid 1s obtained in excellent yield. 1 Add 18.45 g (102 mmol) D,L-m-Tyr to 75 mL 0 05 N aqueous HCl and add 14 4 mL of a 37% aqueous formaldehyde solution 2. Heat at 90°C for 45 mm, and then cool down to room temperature 3 Filter the precipitate, and wash twice with 60 mL water and twice with 60 mL acetone. 4 Dry zn vucuo. Yield 12.21 g (62%). Charactertzatlon: mp 270-274”C, TLC (CH$N/MeOH/water 4: 1-l) Rf = 0 55, ‘H NMR (DMSO + few drops D,O) 7.W.6 (m, 3H, H,,,, ), 4 06 (s, 2H, H,), 3 59 (m, lH, H3); 3 10 (dd, lH, Hq, J= 16 Hz, 7 Hz); 2.85 (dd, lH, H,,, J= 16 Hz, 7 Hz) HPLC (O-50% CH,CN m 0 1% TFA/H,O m 30 mm) k’ = 0 79

332 3.6. Preparation of 1,2,3,4-Tetrahydro7-Hydroxy-lsoquinoline-3-Carboxylic

Tourwb et al Acid 6

The preparation of this analog cannot be performed by the Plctet-Spengler reaction on tyrosine. Under these condltlons, a phenol-formaldehyde condensation gives rise to a polymerization reaction (36). The reactive 3’,5’-positions of tyrosme are therefore blocked by bromine or iodine atoms. The best overall chemical and stereochemlcal yield IS obtained for 3’,5’dnodo-tyrosine (36). The iodine can either be removed prior to Boc-nitrogen protection or after Boc-protection

3.6.7. Preparation of (S)-1,2,3,4-Tetrahydro7-Hydroxy-6,8-Diiodoisoquinoline-3-Carboxylic

Acid

1 Suspend 18 1 g 3’,5’-diiodo-L-tyrosme (40 mmol) m 180 mL cone HCl and 12 mL 1,2-dlmethoxyethane (see Note 11). 2 Add 13 2 mL of a 37% aqueous formaldehyde solution, and heat the reactlon mixture to 72°C over 0 5 h with vigorous stlrring (see Note 12). 3 Add again 80 mL cone HCl, 6 mL 1,2-dlmethoxyethane, and 6 6 mL of the 37% formaldehyde solution Continue the reaction for 18 h at 72-75°C 4 Cool the suspension m an ice bath and filter 5. Wash the filter cake thoroughly several times with 1,2-dlmethoxyethane, and dry m a vacuum desiccator. 6 The shght tan powder can be used as such, or can be crystallized from MeOH/water Yield. 8.6 g (4X%), ee = 97% (see Note 13) Characterlzatlon mp 208-209”C, TLC (nBuOH/AcOH/HzO 4/1/l) Rf = 0 63, [a],, = -88 83” (c = 0.2 AcOH), ‘H NMR (D,O/TFA)* 7 01 (s, lH, H ,,,,); 3.79 (d,J= 16 5, lH, H,,); 3 66 (dd,J = 10 8, 5.3, lH, H,), 3 54 (d, J= 16 5, lH, H,), 2.69 (dd, J= 17.0, 5.3, lH, H4!), 2 53 (dd, J= 17 0, 10.81 IH, H4) HPLC (10% CH,CN m 0 1% TFA/ H,O isocratic) k’ = 4 33

3.6.2. Preparation of (S)-7,2,3,4-Tetrahydro7-Hydroxy-Isoquinoline-3-Carboxylic Acid 6 1. Dissolve 4.45 g (9 93 mmol) of the duodo-compound described above m 150 mL EtOH and 50 mL HZ0 containing 4% triethylamme (8 mL) 2 Add 0 62 g 10% Pd-C catalyst and hydrogenate over 40 PSI hydrogen pressure for 3 h 3 Filter the catalyst, decolorize the filtrate by addlng some crystals of Na2S203, and concentrate the solution under vacuum until crystals start to appear 4 Adjust the pH of the solution to 6.0, and store overnight m a refrigerator 5 Filter the crystals, wash with Ice-cold water, and dry m a vacuum desiccator Yield 1 42 g (73%), ee = 97% (Note 13) Charactenzatlon. mp 294”C, TLC (nBuOH/AcOH/H20 4/1/l) Rr= 0 5 1, [a],, = -169 lo (c = 1 0 m AcOH), ‘H NMR (D,O/TFA): 6 86 (d, lH, H,, J= 8 3Hz); 6 54 (dd, lH, H,, J= 8 5,2 5 Hz), 6 40 (d, lH, Hs, J= 2.5 Hz); 4.09 (m, 2H, H,); 4.05 (dd, lH, H3, J= 10 5,

333

Cyclic Aromatic Am/no Acids

5 5 Hz); 3.06 (dd, lH, H‘,, J= 17.2, 5.5 Hz), 2.85 (dd, lH, H4’, J= 17 2, 10 2 Hz) HPLC (O-20% CH$N m 0.1% TFA/H*O in 20 mm) k’ = 2.08

3.7. Preparation SH-Pyrido[3,4$]

of 1,2,3,4-TetrahydroIndole-SCarboxylic Acid (Tee, 7)

The Pictet-Spengler reaction of tryptophane with formaldehyde IS reported m basic and m acldlc condltlons. A better yield (97%) is reported for the base-catalyzed reaction (38). Under acidic conditions, a yteld of 78% IS reported (39). The formation of the 9hydroxymethyl-derivative was minrmized mainly by controllmg the stolchiometry of the reaction (40) The authors checked both procedures and analyzed the crystals obtained by HPLC. Two side products were identified by MS: the reported 9-hydroxymethyl-derivative and a Tee-dlmer linked by a methylene group (see Note 14) (41). The formation of the hydroxymethyl compound can be mmlmlzed by using a slight excessof formaldehyde (1.1 Eq) and a short reaction time (1 h). The obtained crystals, however, still contain the dlmer (about 10-l 5%, by HPLC), which cannot be removed by crystalhzatlon. However, after Boc protectlon, this Impurity can be removed. Both the final yield and the occurrence of the side reactions are comparable for the acldlc and basic catalyzed reaction. 1 Add IO g L-Trp (49 mmol) to a mixture of 60 mL 1 M H,S04 and 180 mL water m an Ice bath. 2. Add 4 mL of a 37% aqueous formaldehyde solution (49 4 mmol), and stir for 1 h m the ice bath 3 Adjust the pH to 6 0 with cone NH40H 4 Filter the precipitate, wash with cold water, and crystallize from water. Yield 9 1 g (86%) of Tee, containing 10% of dlmer (HPLC) mp 304-306”C, TLC (CH3CN/H20/MeOH; 4: 1 1) Rf = 0.13, ‘H NMR (D,O/TFA). 7.3-6 7 (m, 4H, H,,,,); 4 35 (d, 2H, H,,J= 8 4 Hz), 4 1 (m, IH, H,); 3.0 (m, 2H, H4) HPLC (O-70% CH,CN m 0 1% TFA/H,O m 30 mm), 1 = 280nm. k’ (7) = 2 0, k’ (dlmer) = 3 28

3.8. Preparation of 4,5,6,7-Tetrahydro-1H-lmidazo [4,5-c]Pyridine-6-Carboxylic Acid (Tip, Spinacine,

Fig. 1)

The reaction of L-histldme and formaldehyde to give spmacine was first reported by Welhsch in 1913 (42). This amino acid was isolated from natural sources such as spinach. Klutchko et al. prepared several derivatives (43,44) with a Plctet-Spengler reaction. 1 A mixture of 15 g (100 mmol) L-hlstldme, 200 mL 12 NHCl and 30 mL formaldehyde (37% m water) was stlrred overnight at room temperature

334

Tout-w6 et al.

2 Additional formaldehyde (30 mL) was added, and the mixture was heated on an oil bath at 100°C for 3 h 3 Cool the reaction mixture, and evaporate the reaction 4. The crude reaction residue was dissolved m water, evaporated, and again dlssolved m 25 mL water. 5 Adjust the pH to 3 5 by addition of concentrated NH,OH (Note 15) Yield* 63%, mp 286287, [a] *j,,= -131 3“ (c = 2 11 m water), ‘H NMR (D,O)* 8 74 (s, lH, H2), 4 63 (d,lH, H4), 4 46 (d, lH, H4’), 4 25 (q, lH, H6), 3.47 (dd, lH, H7), 3 14 (dd, lH, H,‘) 4. Notes It IS important not to exceed the reaction temperature of 95°C in order to avoid excessive racemization The washing with acetone removes the side product lH-2-benzopyran-3-carboxyhc acid (23). A direct analysis without choral derlvatlzatlon has been performed on a Dalcell Chlralpak WH column (28). The obtained white powder is pure aMe-Tic. The formation of side products IS mmlmlzed The following side products were identified by HPLC/MS* N-methyla-methyl-Phe (tR = 15 0 mm), N-methyl-a-methyl-Tic (tR = 30.7 mm). 5 One can generate this amount of LDA zn sztu by coolmg 35 84 g (0 256 M) of dllsopropylamme in 350 mL THF to -78’C, followed by an addition of 160 mL of 1 6 Mn-BuLl m hexane Stolchlometrlc amount of the electrophlle and longer reaction time will increase the yield of kmetlcally disfavored (2S,5$5-aS)-10a High vapor pressure’ Use extreme precautions during this operation Tuck the reaction m the hood corner, and use the chemical shield This IS a heterogeneous reaction Presence of solid lmldazohdmone indicates that the reaction IS still m progress This can be done by Ion-exchange chromatography (Dowex 50x8- 100, strongly acidic) with 1 3 N aqueous ammonia. The eluent contams the ammomum salt of the ammo acid, and it needs to be hydrolyzed off to a pure ammo acid by a cycle of three to four rotary-evaporations at 40°C. 10 For the 3S,4S (and its mirror image 3R,4R) isomer, use of aqueous formaldehyde solutions gives raise to N-Methyl-a$-dlmethyl Tic This can be traced to formic acl&medlated reduction ofN-methylene lmme of a$-dlmethyl Phe, which forms first. Another molecule of formaldehyde IS then used to for the Plctet-Spengler N-methyl-a$-dlmethyl TIC product Degassmg paraformaldehyde solution ehmlnates formation of forrnlc acid. This 1s not a problem for 3S,4R and 3R,4S somers, and aqueous formaldehyde can be used instead 11 The 1,2-dimethoxyethane increases the solublhty of the starting material At the end of the reaction, thorough washing of the precipitate with this solvent IS essential to remove any remaining starting compound

Cyclic Aromatic Amino Acids

335

12 It 1s important that the reaction temperature does not exceed 75°C. Above this temperature, iodine elimmation becomes very important, and the yield drops dramatically 13. The % ee 1s determined as described for Tic 1 by GITC derivatlzatlon followed by RP-HPLC* 0.1% aqueous TFA/MeOH (l/l), flow 0 8 mL/min, k’[(S)dilodocompound] = 7 09, k’[(R)-dllodocompound] = 13.01, k’[(S)-61 = 1.45, k’[(R)-61

= 2.35

14. HPLC conditions O-50% CH$N m H,O + 0.1 TFA m 30 mm, h = 280 nm. k’(9-CH,OH) = 1 41, k’(7) = 2.0; k’(dlmer) = 3 28 MS M+ + 1 (9-CH,OH) = 247, Mt + 1 (7) = 217, M+ + 1 (dimer) = 445 15. The free amino acid of spmacme was obtained by adjusting rhe pH of a solutron of spmacme HCl crystals to 8 0 by ammomum hydroxide Recrystalhzatlon from hot water (yield 48%) m,, 235-250”, [~]~~o = 182 2” (c = 1 02 water), TLC (n-BuOHl H,O/AcOH 2 1 2) R,= 0.28

References 1 Kazmlerski, W and Hruby, V. J. (1988) A new approach to receptor ligand design synthesis and conformation of a new class of potent and highly selective p opiold antagomsts utlllzmg tetrahydrolsoquiniline carboxylic acid Tetrahedron 44(3), 697-7 10 2. Hruby, V. J , Al-Obeldl, F , and Kazmlerskl, W. (1990) Emergmg approaches m the molecular design of receptor-selective peptide ligands: conformational, topographical and dynamic conslderatlons. Biochem J 268,249-262 3 Kazmlerskl, W M , Yamamura, H I , and Hruby, V. J (199 1) Topographic design of peptlde neurotransmltters and hormones on stable backbone templates: Relation of conformation and dynamics to bioactlvity J Am Chem. Sot. 113,2275-2283. 4 Lovas, S. and Murphy, R. F (1994) Solvated structure analysis of a conformatlonally restrlcted analogue of phenylalanme m a dlpeptlde model by the AMlSM2 method. J Mel Struct (Theochem) 311,297-304 5. Valle, G , Kazmlerskl, W M , Crisma, M , Bonora, G. M., Tomolo, C , and Hruby, V. J (1992) Constrained phenylalanme analogues Preferred conformation of the 1,2,3,4-tetrahydrolsoqumoline-3-carboxyhc acid (Tic) residue. Int. J, Peptzde Protein Res 40,222-232. 6. Kazmlerskl, W , Wire, W S , Lul, G. K., Knapp, R. J , Shook, J E , Burks, T. F., et al. (1988) Design and synthesis of somatostatm analogues with topographical propertles that lead to highly potent and specific ~1opioid receptor antagonists with greatly reduced bmdmg at somatostatm receptors. J Med. Chem 31(11), 2 170-2 177 7. Kyle, D J , Martm, J. A , Farmer, S. G , and Burch, R M. (1991) Design and conformatlonal analysis of several highly potent bradykmm receptor antagomsts J A4ed Chem 34(3), 1230-1233 8 Klutchko, S., Blankley, C. J., Fleming, R. W., Kikley, J. M , Werner, A. E., Nordin, I., et al (1986) Synthesis of novel anglotensin converting enzyme mhlbltor qumaprll and related compounds A divergence of structure-actlvlty relatlonships for non-sulfhydryl and sulfhydryl types. J Med. Chem. 29(10), 1953-1961.

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29. 30

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33

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Tourwd et al.

et de la N-tosyl L-tyrosme influence sur l’activtte optique Eur Polymer Journal 8,5 13-524 Verschueren, K , Toth, G., Tourwe, D , Lebl, M., Van Bmst, G , and Hruby, V (1992) A facile synthesis of 1,2,3,4-tetrahydro-7-hydroxyqulnollne-3-carboxyllc acid, a conformationally constramed tyrosine analogue Syntheses $4588460 Ltppke, K P , Schunack, W. G., Wennmg, W , and Mullet-, W E (1983) j3carbolmes as benzodtazepme receptor hgands. 1 Synthesis and benzodtazepme receptor mteractton of esters of P-carboline-3-carboxyhc acid J Med Chem 26(4),499-503. Brossi, A., Focella, A , and Teitel, S. (1973) Alkaloids m mammalian tissues 3 Condensation of L-tryptophan and L-5-hydroxytryptophan with formaldehyde and acetaldehyde J Med Chem 16(4), 4181120 Coutts, R T , Micetich, R G., Baker, G B., Benderly, A , Dewhurst, T , Hall, T W., et al. (1984) Some 3-carboxamides of P-carbolme and tetrahydro+-carbolme Heterocycles 22(l), 131-143 Iterbeke, K , Laus, G , Verheyden, P , and Tourwe, D , Side-reactions m the preparation of 1,2,3,4-tetrahydro-P-carbolme-3-carboxyltc acid Lett Pept Scl. (1998) m press Welhsch, J (1913) Bzochem Z 49, 173-194 Klutchko, S. R., Hodges, J. C., Blankley, C J , and Colbry, N. L (1991) 4,5,6,7Tetrahydro-lH-lmldazo[4,5-c]pyrldlne-6-carboxyllc acids (spmacmes) J Heterocyclzc Chem 28,97-108 Blankley, C. J , Hodges, J C , Klutcho, S. R , Himmelsbach, R. J , Chucholowski, A., Conolly, C J., et al. (1991) Synthesis and structure-activity relationships of a novel series of non-peptide Angtotensm II receptor bmdmg mhrbitors specttic for the AT, subtype J Med Chem 34,3248-3260

19 Asymmetric Syntheses of Unnatural Amino Acids and Hydroxyethylene Peptide lsosteres Robert M. Williams 1. Introduction Unnatural and naturally occurring nonproteinogenic a-amino acids have become important building blocks for the synthesis of btologically active peptides and peptidomimetlc drug molecules. The asymmetric synthesis of aammo acids has therefore become quite important as an mdlspensable research tool m academic, government, and mdustrlal laboratories, and methodologies have been reviewed extensively The established methods for the asymmetrlc synthesis of ammo acids can be divided mto roughly SIXcategories (I). (1) The highly stereoselectlve hydrogenation of choral, nonracemic dehydro ammo acid derlvatlves or the asymmetric hydrogenation of prochlral dehydro amino acid derivatives. Chn-al glycme equivalents serve as useful a-ammo acid templates undergoing homologation vza carbon-carbon bond formation at the a-posltlon through nucleophllic carbamon alkylatlon (2) or electrophllic carbocation substitution (3). In addition both nucleophillc amination (4) and electrophlllc aminatlon (5) of optlcally active carbonyl derivatives has very recently been developed (6) Enzymatic and whole-cell-based syntheses have recently become more attractive in terms of substrate versatlllty, cost, and scale. All of these methods have their relative strengths and weaknesses; the optimum method for each individual appllcatlon must still be considered on a case-bycase basis with respect to functionality, quantity desired, cost, and time. The focus of this chapter ~111illustrate the utility of choral, nonracemlc glycinates which are commercially available and can be manipulated m a variety of ways to accessstructurally diverse classesof a-amino acids.

From

Methods Edlted

m Molecular

Medune,

by W M Kazmlerskl

Vol 23 Pept/dom/met/cs

@Humana

339

Press

Inc , Totowa,

Protocols NJ

340

Williams

2. Materials 2.1. Reagents for Method 3.7 1 tert-Butyl (2R,3S)-(-)-6-oxo-2,3-dlphenyl-4-morphollnecarboxylate 1 Aldrtch Chemical Co catalog #33-l 84-8 (CAS Registry # 112741-49-8) The enantiomer of 1 IS also commerctally available tert-Butyl (2&3R)-(+)-6-oxo-2,3-dtphenyl4-morpholmecarboxylate Aldrich Chemical Co catalog #33- 18 l-3 (CAS Regw try #112741-.50-l) 2 Ally1 iodide. 3 Tetrahydrofuran (THF) 4 Lithium bzs(trimethylsilyl)amide (LiN[SrMe,],) 5 Ethyl acetate (EtOAc) 6 Magnesium sulfate (MgSOJ 7 de-Dimethyl sulfoxide (DMSO-d6) 8 Lithium metal (LiO) 9 Liquid ammonia (NHst$ 10 Hydrochloric acid (1 N HCl, 2 N HCl) 11 Ethanol (EtOH) 12 Ammomum chloride (NH&l) 13 Methanol (MeOH) 14 Dichloromethane (methylene chlorrde, [CH,Cl,]). 13. Ether (Et,O) 14 Tetramethyl silane (TMS). 15 Sodium metal (Na”)

2.2. Reagents for Method 3.2 1 tert-Butyl (2R,3S)-(-)-6-oxo-2,3-dlphenyl-4-morphollnecarboxylate 1 Aldrich Chemical Co catalog #33- 184-8 (CAS Registry # 11274 l-49-8) The enanttomer of 1 is also commercially available tert-Butyl (2&3R)-(+)-6-oxo-2,3-diphenyl4-morpholmecarboxylate Aldrich Chemical Co catalog #33- 18 l-3 (CAS Regrstry #112741-50-l) 2 Sodium bis(trtmethylsrlyl)amide (NaN[StMe,]J 3 THF 4 Methyl iodide (CHJ) 5 EtOAc 6 MgSO‘, 7. Hexanes 8 1-Bromo-3-methyl-2-butene 9 Potassium brs(trrmethylsilyl)amide (KN[SiMe3]2). 10 CH,Cl, 11 Na’. 12 NH,(I) 13 EtOH 15 NH&l 16. CH,Cl,

Asymmetm Syntheses

341

2.3. Reagents for Method 3.3 1 Aldrich 1 tert-Butyl (2R,3S)-(-)-6- oxo-2,3-dtphenyl-4-morpholmecarboxylate Chemical Co. catalog #33- 184-8 (CAS Regtstry # 11274 l-49-8). The enantromer of 1 IS also commercrally available. tertButy1 (2S,3R)-(+)-6-oxo-2,3-drphenyl4-morpholmecarboxylate Aldrtch Chemtcal Co. catalog #33-181-3 (CAS Regtstry #112741-50-l) 2 (k)-(Drethylammo)methylphenyloxosulfonmm fluoroborate 3. Carbon tetrachloride (Ccl,) 4 N-bromosuccmtmlde (NBS). 5 tetrahydrofuran (THF) 6 Trrmethylphosphtte 7 Hexanes 8 EtOH 9 Lrthmm dnsopropyl amrde (LDA, LiN[t-Pr],) 10 Proptonaldehyde (CHsCH$ZHO). 11 Saturated aqueous NaCl (brine) 12 EtOAc 13 MgS04 14 Deutero chloroform (CDCl,) 15 Sodium hydride (NaH, as an 011dtsperston). 16 Dtmethyl sulfoxrde (DMSO). 17. Lie 18 NH,(,) 19. NH&l 20 Hydrochlorrc acid (2 JVN HCl) 21 CH2C12

2.4. Reagents for Method 3.4 1 tert-Butyl (2R,3S)-(-)-6-oxo-2,3-dlphenyl-4-morphollnecarboxylate 1 Aldrich Chemical Co. catalog #33-184-8 (CAS Regrstry #112741-49-g) The enantromer of 1 1s also commerctally available: tert-Butyl (2&3R)-(+)-6-oxo-2,3-dtphenyl4-morpholmecarboxylate Aldrich Chemical Co catalog #33-181-3 (CAS Regrstry #112741-50-l) 2 (+)-(Drethylammo)methylphenyloxosulfonmm fluoroborate. 3 ccl, 4 NBS 5 THF. 6 Trtmethylphosphtte 7. Hexanes. 8 EtOH 9 LDA, LtN(t-Pr),. 10 Benzaldehyde (C6H&HO). 11. Hexanes. 12 TFA

342 13 14 15 16 17 18. 19. 20 21 22 23 24

Wdiams Sodmm bicarbonate (NaHCOs) CH,C12 Sodmm sulfate (Na,SOJ Ltthmm hydroxtde (LiOH) Hydrochlortc actd (1 N, 2 N HCl) Dtazomethane (CH2N2) prepared from 1-methyl-3-nitro(MNNG) (Aldrich). Sodium hydroxide (NaOH, 5 N) MeOH EtOAc Lead tetraacetate (Pb[OAc14) Trtethylamme (EtsN) Di-tert-butyldicarbonate ([BOC],O)

2.5. Reagents

1-mtrosoguamdme

for Method 3.5

1 Benzyl-(2R,3S)-(-)-6-oxo-2,3-dlphenyl-4-morphollnecarboxylate 16 Aldrich Chemical Co, catalog #33,187-2 (CAS Registry #lo05 16-54-9), the enatiomer of 16 is also commercailly available* Benzyl (2S,3R)-(+)-6-oxo-2,3-diphenyl-4morpholmecarboxylate Aldrich Chemical Co catalog #33-185-6 (CAS Registry # 105228-46-4) 2 Hexamethylphosphoric trtamtde (HMPA). 3 NaN(SiMe& 4 THF 5 Isobutyl triflate 6 Sodmm bicarbonate (NaHCOs) 7 Ammomum chloride (NH&l) 8 Sodium sulfate (Na$OJ 9 EtOAc 10 CH,C12 11 Dnsobutylalummum hydride (DIBAH, 1 Mm hexane) 12 Et,N 13 Acetic anhydride (Ac*O) 14 N, N-dtmethylammo pyrtdme (DMAP) 15. Methyl t-butyldtmethylsilyl ketene acetal 16 Zmc bromide (ZnBrJ. 17. Hydrochloric acid (1 N, 2 N HCl). 18 Dioxane 19. Potassium hydroxide (KOH, 1 M) 20 Dtethyl ether (ether, EtzO) 21 tert-Butanol (r-BuOH) 22 Ammomum hydroxide (NH40H, 0 1 M)

2.6. Analytical

Considerations

and Purification

1 Elemental analyses were determined for all new crystallme compounds and are accurate to within the calculated values by &O 4%

Asymmetric Syntheses LIN(StMe&, 0

343 THF

‘-\=

IJO, NHq0

tBOCHN

C&H HY

o

THF. EtOH 3

1

Fig 1. Synthesis of N-t-BOC allylglycme 2 Column chromatography was performed usmg Merck srlica gel-grade 60, 230400 mesh, 60 A 3. Radial chromatography (PTLC) was done on l-, 2-, and 4-mm silica gel plates using E. Merck silica gel 60 PF-2.54 containing gypsum on a Harrison Research Chromatotron Model 7924. Alternatively, PTLC can be performed using standard 20 x 20 cm plates of 0 5, 1.0, or 2.0 mm thickness, and eluted m the mdtcated solvent systems 4 Low-temperature reactions reported as -78°C to -82°C are the bath temperatures of the dry me-acetone coolmg bath and reflect reaction temperatures to within t5’C 5 The optical purity of the final ammo acids were determined according to the procedure described m ref. (2) 6. C,s reverse-phase Sep-pak cartridges are used to purify zwitterlonic ammo acids. 7 Ion-exchange chromatography Dowex 50x8-200

3. Methods. 3.1 Preparation

of N-t-BOGAllylglycine

3

A typical method for alkylatmg the oxazmones 1 and converting these substances mto N-t-BOGprotected ammo acids of either the (R)- or (s) absolute configuration is illustrated below in Fig. 1 (2). After dlastereoselective enolate alkylation leading to 2, dlssolvmg metal reduction directly affords the corresponding N-t-BOGprotected ammo acid 3.

3.7.1. (3S,5S, 6R)-4-(tert-Butyloxycarbony/)-5,6-Diphenyl3-(2’-Propenyl)-2,3,5,6-Tetrahydro-4H1,4-Oxazm-2-One 2 1 To a stirred solution of 1 (300 mg, 0.849 mmol, 1 Eq) and ally1 iodide (388 ML, 4.243 mmol, 5 Eq) m THF (5 mL), add hthmm bzs(trtmethylsllyl)amlde (1019 pL, 1 0 19 mmol, 1 2 Eq, 1 M solution m THF) dropwtse via syringe at -78°C. 2. After 40 mm, pour the reaction mixture into ethyl acetate. Wash the organic layer with water and brine, and dry the organic extract over anhydrous magnesium sulfate 3. Falter, concentrate, and purify the product by column chromatography on srlrca gel (elute with EtOAchexanes, 2.5) You should get about 286 mg (85%) of 2 as white solid and 15 7 mg (5%) of unreacted 1: ]H NMR (200 MHz, DMSO-de, 393 K [see Note 11, vs TMS) 6 1 29 (9H, s), 2.82-2 92 (2H, m), 4,88 (IH, t, J =

344

Williams 700Hz),5 16-530(3H,m),5.84-605(1H,m),6 18(1H,d,J=307Hz),6556 59 (2H, m), 7 03-7.27 (8H, m); IR (NaCl, CH,Cl,) 1755, 1690 cm-‘, mp 169171”C, [cx]~~,, =-48” (c 0.1, CH,CI,).

3 1.2 (S)-N-(tert-Butyloxycarbonyl)Allylglyc~ne

3

To a solution of LI” (14 mg, 2 017 mmol, 12 Eq) m hquld ammoma (20 mL, dlstllled from Na’), add a solution of compound 2 (66 mg, 0 168 mmol, 1 Eq) and ethanol (120 mL) m THF (3 mL) at -33°C. After 10 mm, the blue color will dissipate and quench the reaction mixture with excess ammonmm chloride Allow the reaction mixture to warm to room temperature during which time the ammonia will evaporate Dilute the residue with water, extract the aqueous layer twice with ether, and acidify to pH 2 0 with 1 N HCl Extract the aqueous layer thrice with ethyl acetate. Combme the organic extracts, and dry over anhydrous magnesium sulfate Filter, concentrate, and purify the product by PTLC on slhca gel (elute with 5% MeOH m CH,Cl,) You should get about 18 mg (50%) of 3 as a colorless 011 (98% ee): ‘H NMR (200 MHz, DMSO-d6, vs TMS) 6 1.37 (9H, s), 2 27-2.46 (2H, m), 3.85-3.97 (IH, m), 5 01-5 14 (2H,m), 5.67-5 87 (lH, m), 7 01 (lH, d, D,O exch , J= 8.04 Hz); IR (NaCl, CDCl,) 3430,3050,1715 cm-‘, [IX]~~,, =-3.9” (c 1, CH2C12)

3.2. Preparation of F)-N-(tert-Butyloxycarbony/)2-(3’-Methyl-2’-Butenyl)Alanine 6 The a,a-dlsubstttuted a-ammo acids are particularly slgntficant m that they are generally more dlffcult to prepare by any of the known methods (1) with structurally diverse side-chain functlonallty. These ammo acids have attracted Increasing interest as conformatlonally restrlctlve ammo acid surrogates, which also impart desirable protease resistance in synthetic peptldes of bloioglcal interest. a,a-Dlsubstltuted ammo acids have become of significant medicinal and blochemlcal interest, being powerful enzyme inhibitors, e.g , for DOPA, ormthme, glutamate, S-adenosylmethlonme (SAM) decarboxylases, and aspartate ammo transferase. These substances have also found utlhty as conformatlonal modifiers for physiologically active peptides. a,a-Dtsubstltuted a-ammo acids can be conveniently prepared m high enantiomerlc ratios (>99% er) by the sequential enolate alkylatlon of the oxazmones as illustrated m Fig. 2 (2).

3.2.7 (3S,5S,6R)-4-(tert-Butyloxycarbonyl)-5,6-Drphenyl-3-Methyl2,3,5,6-Tetrahyciro-4H-1,4-0xazin-2-0ne 4 1 To a stirred solution of 1 (500 mg, 1.416 mmol, 1 Eq) m THF (10 mL), add sodium bis(tr~methylstlyl)am~de (1500 FL, 1S mmol, 1.06 Eq, as a 1 M solution m THF) dropwlse via syringe at -82°C

Asymmetric Syntheses NaN(SIMe3)2,

345

THF

CH3 I, -78%

1

4

6 ‘H3CACH

3

Fig 2 Synthesis of (S)-N-(tert-Butyloxycarbony1)-2-(3’-methyl-2’-butenyl)alanlne 2. After 35 mm, add methyl lodlde (900 pL, 14.46 mmol, 10 2 Eq) to the reactlon mixture Stir the resulting solutron for an additional 1 5 h at -82”C, and pour the mixture Into water 3 Extract the aqueous layer three times with ethyl acetate. Combine the organic extracts, and dry over anhydrous magnesium sulfate 4 Filter, concentrate, and purify the product by radial chromatography on silica gel (elute with EtOAc:hexanes, 1*2), you should obtain about 475 mg (91%) of 4 as white solid: ‘H NMR (200 MHz, DMSO-d6, 393 K, vs TMS) 6 1.19 (9H, s), 1.7 1 (3H, d, J= 6 99 Hz), 4 88 (lH, q, J= 7.12 Hz), 5 15 (IH, d, J= 2 81 Hz), 6 18 (lH, d, J =2 87 Hz), 6.54 (2H, m), 7 03-7.29 (8H, m); IR (NaCl, CH,Cl,) 1752, 1702 cm-‘, mp 204-206°C; [1~]*~,=-61“ (c 0.2, CH,Cl,).

3 2.2. (3S,5S,GR)-4-(tert-Buty/oxycarbonyl)-5,6-Diphenyl-3-Methy/3-(3’-Methyl-2’-Butenyl)-2,3,5,6-Tetrahydro-4H-l,4-Oxazin-2-0ne

5

1 To a stirred solution of 4 (500 mg, 1.36 mmol, 1 Eq) and I-bromo-3-methyl-2butene (791 mL, 6.804 mmol, 5 Eq) in THF (6 mL), add potassium bu(tnmethylsilyl)amlde (1944 mL, 2.72 mmol, 2 Eq, as a 1.4 Msolutlon m THF) dropwlse via syringe at -78’C 2 After 30 mm, pour the reactlon mixture Into ethyl acetate. Wash the organic layer with water and brine, and dry the organic layer over anhydrous magnesium sulfate 3 Filter, concentrate, and purify the product by column chromatography on slllca gel (elute with CH2Clz:hexanes, 2: l), you should obtain 475 mg (80%) of 5 as a white solid: ‘H NMR (200 MHz, DMSO-d6, 393 K, vs TMS) 6 1 39 (9H, s), 1 65 (3H, s), 1 74 (3H, s), 1 75 (3H, s), 2.81 (lH, dd, J= 14 36 Hz, J = 8 32 Hz), 3 27 (lH, dd, J= 14 29 Hz, J= 7 50 Hz), 5 24 (lH, t, J= 7.99 Hz), 5.44 (lH, d,J=3.17Hz),608(1H,d,J=3 24Hz),6.87-6.92(2H,m),7.10-7 25(8H, m), IR (NaCl, CH,Cl,) 1746, 1702 cm -I; mp 139-140°C; [a]250 = +55 1” (c 0 9, CH,Cl,)

3.2.3. (S)-N-(tert-Buty/oxycarbonyl)2-(3’-Methyl-2’-Butenyl)Alanine 6 1 To a solution of Na” (69 mg, 3 001 mmol, 13 Eq) in liquid ammonia (25 mL, distilled from Na’), add a solution of compound 5 (100 mg, 0.230 mmol, 1 Eq) and ethanol (200 pL) m THF (3 mL) via syringe at -33°C

Williams

346 Ph

Ph 1 NBS/CC14 retlux 2 (Me0)3P

/ THF

1

H3CO-,P,-0

3

H3a

7

8

Ph

Fig 3. Syntheses of coronamtc acid After 10 mm, quench the reaction mixture with excess ammomum chloride, and

allow the reaction mtxture to warm to room temperature Allow

the reaction mixture to warm to room temperature durmg which time the

ammonia ~111 evaporate Dtlute the mtxture wtth water, and extract the aqueous layer twice wtth ether, and actdtfy to pH 2.0 with 1 N HCI Next, extract the aqueous layer three times wtth ethyl acetate Combtne the organic extracts, and dry over anhydrous magnesium sulfate. Filter, concentrate, and purify the product by PTLC on slhca gel (elute with 5%

MeOH m CH,Cl,);

you should obtain about 38 mg (65%) of 6 as colorless or1

(>99% er) ‘H NMR (200 MHz, DMSO-d6, vs TMS) 6 1 24 (3H, s), 1 35 (9H, s), 1 56 ( 3H, s), 1 67 (3H, s), 2 3&2 57 ( 2H, m), 5 03 (lH, t, J= 7 06 Hz), 6 76 (lH, br s, D20 exchangeable), IR (NaCl, CDCl,) 1715,1653,1498 cm-‘, [a]250 = -13 4” (c 0 87, CH2C12)

3.3. Preparation

of N-t-BOGCoronamic

Acid 70

Several members of the ammocyclopropane class of ammo acids are naturally occurrmg, the first example bemg the isolatton of the parent compound 1-ammocyclopropane carboxyltc acid (ACC) from cider apples and perry pears. ACC has been found to be the biosynthetic precursor to the plant hormone ethylene and is a substrate to the PLP-linked enzyme ACPC deaminase, whtch converts ACC to ammonia and 2-ketobutyrate. This family of ammo acids 1s of tremendous interest because of tts biologrcal acttvtty and potential use m conformationally restricted peptrdes and as btosynthettc and mechamstic probes. A stereoselectrve approach to E- 1-ammocyclopropane carboxyhc acids has been developed (3) based on the phosphonate-based olefinatron of the oxazmones 1 to 8 as shown m Fig. 3. Johnson sulfoxtmme-based cyclopropanation (4) occurs wrth a high level of stereocontrol from the more hindered face of 8; m most cases, the dtastereoselectrvtty IS >99% (see Note 2). The example illustrated yields the AWBOC derivative of coronamic acid. The

347

Asymmetric Syntheses

N-t-BOC group can be convemently removed by treatment with methanohc HCI followed by propylene oxide scavenging of residual acid (follow the detailed procedure m ref. 3). 3.3.1. (3S,5S,GR)-4-(tert-Butoxycarbony/)-3-(Drmethoxyphosphory/)5,6-Diphenyl-2,3,5,6-Tetrahydro-4H1,4-Oxazin-2-One 7 1 To a flask contammg 1 (3.0 g, 8.49 mmol, 1 0 Eq) and NBS (1 7 g, 9 34 mmol, 1 1 Eq), add CC& (500 mL) Heat the mixture to reflux temperature for 1 h and cool the solution to 0°C 2 Filter through Celite to remove succinimlde, and concentrate UI wcuo to yield the bromide as a white solid This material 1s not purified further, but rather, IS carried on directly crude 3. To the crude bromide add THF (36 mL) and trimethylphosphlte (1 1 mL, 9 34 mmol, 1 1 Eq), and gently reflux the mixture for 12 h 4 Cool the mixture to room temperature and concentrate, which will provide a yellow VlSCOUS011 5. Purify the product via flash slllca chromatography (160 g silica, elute with 1 lo1 1 EtOH/hexanes) which should provide 3 4 g (86 3%, two steps) of 7 as a white crystallme sohd ‘H NMR (300 MHz, DMSO-d,, 393 K) 6 TMS 1 03(s) and 1 39(s) (9H), 3 81-3 93 (6H, m), 5.25 (d, J= 3 1 Hz) and 5 33 (d, J= 2 9 Hz) (lH),554-568(1H,m),618(d,J=29Hz)and633(d,J=3.lHz)(lH),6.556.59 (2H, m), 7 04-7 31 (8H, m); IR (KBr): 3030, 3019, 2976, 2964 7, 2921, 2856,1959,1889,1747,1703,1295,1273,1049, 1028 cm-‘, [c~]~‘o=-38 55” (c = 1.O, CH,Cl,), mp = 143-144°C.

3 3.2. (E)-(5S, 6R)-4-(tert-Butoxycarbonyl)-5,6-Diphenyl-

3-Propylidene-2,3,5,6-Tetrahydro-4H-l,4-0xazln-2-0ne

8

1 To a -15°C solution (lce/MeOH) of 7 (1 0 g, 2.17 mmol, 1.O Eq) m THF (2 mL), add 3 7 mL of a 0 6 M LDA solution m THF (2 17 mmol, 1 0 Eq) via syringe 2 Stir the resulting reaction for 1 5 h, followed by addltlon of proplonaldehyde (1 6mL,21 67mmo1, IOOEq)at-15°C. 3 Slowly warm the reaction to room temperature and contmue stirring for 20 h 4. Quench with 5 mL saturated NaCl, and extract with 3 x 5 mL EtOAc Combme the orgamc fractions, dry over MgS04, filter, and remove the solvent In vacua

5 Purify the product by flash slhca gel chromatography (68 g slllca, elute with 1 1 EtOAc/hexanes) which should provide 780 mg (91 5%) of 8 as a white crystalline solid ‘H NMR (300 MHz, CDC13)6 TMS 1.14-l 19 (12H, m), 2 51-2.74 (2H, m), 5 16 (lH, d, J= 1 95 Hz), 5 72 (lH, d, J= 2 89 Hz), 6 634 70 (3H, m), 6.96-6.99 (2H, m), 7 07-7.28 (6H, m); IR (NaCl, neat) 3089,3067,3033, 2978, 2933,2878, 1739, 1711, 1628, 1283 3, 1233, 1161 cm-‘; [c~]~~o=-135 3” (c = 1.0, CH,Cl,); mp = 158 5-160°C

Williams

348 3.3.3. (lS,3S,5S,GR)-4-(tert-Butoxycarbonyl)-5,6-Diphenyl1-Ethyl-7-0xa-4-Azaspiro[2.5]0cta-8-0ne 9

To a mtxture of (+)-(dtethylammo)methylphenyloxosulfonium fluoroborate4 (152 mg, 0 5 1 mmol, 2 0 Eq) and 50% NaH dtsperston (24 4 mg, 0.5 1 mmol, 2 0 Eq), add DMSO (2 mL) at room temperature After sturmg 1 h, transfer the yhde solution vta cannula to a partially frozen slurry of 8 (100 mg, 0.25 mmol, 1.0 Eq) in DMSO (2 mL) at 18°C Allow the reaction to thaw slowly over several hours, and contmue sturmg for 4 d at room temperature To the mixture, add brine (2 mL), followed by extraction wtth 3 x 5 mL EtOAc Combme the organic fractions, wash repeatedly wtth H20, dry over MgSO,, filter, and concentrate Purify the product via flash silica gel chromatography (9 9 g silica gel, elute with 1’ 10 EtOAc/Hexanes) which should provide 104 mg ( 100%) of 9 as a white crystallme solid ‘H NMR (300 MHz, CDCls) 6 TMS 1 03-l 3 1 (13H, m), 1 59-l 80 (3H, m), 2 74 (lH, broad s), 5 20 (lH, apparent s), 5 96 (IH, d, J= 3.2 Hz), 6 74 6 77 (2H, m), 6 99-7.28 (8H, m), IR(KBr). 3091,3033,3009,2961,2935,1751, 1706, 1457, 1386, 1365, 1243, 1155, 1100, 1063 cm-‘, [t~]~~,, = +20 5’ (c = 1 0, CH,Cl,), mp = 178-l 80°C.

3 3 4. N-t -BOC-Coronamic Acid (/El-[5S,6R]4-(tert-Butoxycarbonyl)-5,6-Diphenyl3-Propylidene-2,3,5,6-Tetrahydro-4H1,4-Oxazin-Z-One)

10

1 To a -78°C solutton of 9 (559.4 mg, 1.37 mmol, 1 0 Eq) and anhydrous EtOH (805 6 pL, 13 73 mmol, 10.0 Eq) in THF (15 mL) and NHs(,, (98 mL), add Li” (- 124 mg) until the persistent blue discharge is observed. 2 Quench with NH,Cl, and allow the reaction to warm to room temperature during which time the ammonia should completely evaporate 3 Dissolve the white residue in a mmtmum volume of H,O, wash with ether (3 x 5 mL), and carefully acidify to pH 2 5 with 2 N HCl while pertodtcally extracting the product with EtOAc 4 Combme the organic fractions, dry over MgS04, filter, and concentrate. This should provide about 203 mg (64.4%) of 10 as a white crystallme solid* ‘H NMR (CDsOD) 6 TMS: 0.98 (3H, t, J= 7.4 Hz), 1 12-1 17 (IH, m), 1.39-1 48 (1 lH, m), 1 54-l 63 (2H, m), 4 88 (2H, broad s); IR(KBr): 3303, 3253, 3100, 2975, 2936,2878,2697,2583,2492, 1700, 1649, 1478,1458,1410, 1397, 1368, 1306, 1200, 1162 cm-‘, [a]25n = +33.3” (c = 1 0, CH30H), mp = 126-127°C

3.4. Preparation of (lS,2f?)-(+)-2-Phenyl1-Amino- 1Gyclopropane1-Carboxylic

Acid 15

In some cases, removal of the oxazmone btbenzyl chiral auxrltaty cannot be accomplished under the typical reductive conditions (dissolvtng metal or catalytic hydrogenatton). An alternate procedure, mvolvtng lead tetraacetate cleav-

Asymmetric Syntheses

1) Pb(OAc)d MeOH. CH& 2) 1 M HCl(aq). 41%(3

349

H

NH~BOC

1 LIoWaq),

THF

steps)

Fig. 4 Synthesis of (1S,2R)-(+)-2-phenyl-l-ammo-l-cyclopropane-l-carboxyl~c

actd

age, can be used m some instances; this is illustrated m Fig. 4 with the preparation of the conformattonally restricted analog of phenylalanme, (l&2@-(+)-2Phenyl- 1-ammo- 1-cyclopropane- I -carboxylic acid 15 (5). The procedure involves initial hydrolytic ring-opening of the lactone and esterificatton to the methyl ester. Lead tetraacetate degrades the chiral auxiliary m the form of two equivalents of benzaldehyde. Various protecting groups can be arranged on the amino acid that mtght be useful m peptide synthesis protocols (see Note 3). The procedure for making the starting material 11 IS given m ref. 3 and follows the protocol described above in Fig. 3 using benzaldehyde. 3.4.1. (lS,3S,5S,6R)-1,5,6-Triphenyl7-0xa-4-Azaspiro[2.5]0cta-8-0ne 1 To a 0°C solution containing ll(398 mg, 0.87 mmol, 1 0 Eq) m CH$I, (15 mL), add TFA (1 35 mL, 17 47 mmol, 20 0 Eq) 2 Slowly warm the reaction to room temperature and stir the mixture an addltronal 17 h 3 Remove the solvent and excess TFA EII vacua and redissolve the crude residue m CH2C12 (15 mL) and wash with dilute NaHCOs (aq). 4 Separate the layers, and extract the aqueous layer twrce wtth 5 mL of CH2C12 5 Combme the orgamc layers, dry over Na,SO,, filter, and concentrate to about 307 mg (99%) of crude product that is used directly in the next step without further purification See ref. 5 for spectroscoprc data for this Intermediate.

3.4.2. (1S,2R, 7‘S,2R)-I-(N-(l’,2’-Diphenyk2’-Hydroxyethyl) Amino)-2-Phenylcyclopropane1-Carboxylic Acid 12 1 Heat to reflux temperature, a mixture containing the crude product obtamed in 3 4.1 (340 mg, 0 96 mmol, 1 0 Eq), LrOH H,O (52 mg, 1 24 mmol, 1.3 Eq), EtOH (5 mL), and Hz0 (5 mL) for 0.5 h 2 Cool the reaction to room temperature and add 2 A4 HCl (aq) until a thick white precipitate forms

350

Will/am.

3 Collect the crude product via Buchner tiltratton and wash thoroughly with H,O, which should provtde about 302 mg (85%) 12 as a white amorphous solid ‘H NMR(300 MHz, DMSO-d,) 6 TMS 1 33 (lH, dd, Jge,,,= 9 4 Hz, J,,, = 4 8Hz), 1.70 (lH, dd, Jgem= 8.0 Hz, J,,, = 4.7 Hz), 2.32 (lH, apparent t, J= 8 7 Hz), 4 12 (lH, d, J= 4.8 Hz), 4 90 (lH, d, J= 4 8 Hz), 6 96 (2H, d, J= 6 9 Hz), 7.07-7 25 (13H, m); IR(KBr) 3424,3268,3089,3062,3029,2873,2780,1952,1886,1630, 1604, 1569, 1498, 1454, 1390, 1353, 1327, 1190, 1099, 1066 cm-‘, [a]25,, = +82 4” (c = 0 5, 1 MNaOH); mp = 201-202°C

3.4.3. (IS,2R, 7‘S,2’R)-Methyl- 1-(N-(7 ‘,2’-Diphenyl2’-Hydroxyethyl)Amino)-2-Phenylcyclopropane1-Carboxy/ate 73 1 To a 0°C slurry containing MNNG (440 mg, 3 0 mmol, 6 0 Eq) and Et,0 (6 mL), add approx 1 mL 5 N NaOH 2 Stn the mixture for 20 mm and add the ethereal CH2N2 solution to a room temperature suspension of 12 (186 mg, 0.50 mmol, 1 0 Eq) in MeOH (2 mL) 3 Stir the reaction overmght m an open flask, and concentrate the crude ester to dryness 4 Purify the product via flash column silrca gel chromatography (10 g silica gel, elute with 1 5 EtOAc/hexanes), which should yield about 171 mg (89%) 13 as a white solid ‘H NMR(300 MHz) (CDCl,) 6 TMS. 1 41 (lH, dd, Jgem= 9 6 Hz, J,,, =45Hz),l 98(1H,dd,J,,,=79Hz,J,,, =27Hz),257(lH,apparentt,J=88 Hz),269(1H,s),3 14(3H,s),360(1H,d,J=39Hz),4.28(1H,d,J=5OHz), 4 97 (1 H, t, J = 4 5 Hz), 6 99-7.01 (2H, m), 7 07-7 26 (13H, m); IR(KBr) 3426, 3339,3236,3086,3063,3029,2956,2918,1734,1452, 1319, 1215, 1199, 1154, 1061 cm-‘; [cx]25n= +I28 4” (c = 1 0, CH,Cl,), mp = 142-143°C

3.4.4. (lS,2R)-Methyl 7-(N-/Iert-Butoxycarbonyl] Amino)-2-Phenylcyclopropane1-Carboxylate 14 To a -15°C solution containing 13 (62 mg, 0 16 mmol, 1 0 Eq) and a 1 2 mixture of MeOH/CH,Cl, (3 mL), add Pb(OAc)4 (78 mg, 0.18 mmol, 1.1 Eq). The reaction should be complete m 2 mm Quench the reaction with saturated NaHCOs (5 mL) at -15°C A heavy white precipitate should form, remove via Buchner filtration Wash the filter cake thoroughly with CH2C12 (10 mL) and separate the filtrate layers Extract the aqueous layer with CH2C12 (2 x 5 mL) and combine the orgamc fractions Dry over Na2S0,, filter, and concentrate This should provide about 5 1 mg of the actd-sensttive tmine (>lOO%, contammated by benzaldehyde and minor impurities) as a clear oil Use this material directly for the subsequent transformatton without addmonal purification or handling To a room temperature solution of the crude imme (45 mg based on theoretical yreld, 0.16 mmol, 1 0 Eq) m THF (5 mL), add 1 N HCl(,,) (5 mL) After stnrmg for 15 mm, concentrate the reaction to dryness, and thoroughly dry the residue under high vacuum overnight

Asymmetric Syntheses

351

7 To the crude hydrochloride salt m THF (5 mL) at room temperature, add Et,N (33 mL, 0.24 mmol, 1 5 Eq). Immediately, a fine white suspension should form 8 Cool the slurry to 0°C and add di-tert-butyldicarbonate (38 mg, 0 18 mmol, 1 1 Eq) Warm the reaction to room temperature and stir for 24 h. 9 Add EtOAc (5 mL) to the reaction, and then wash the solution with H,O Dry the orgamc layer over Na,SO,, filter, and concentrate 10 Purify the product via PTLC on silica gel (96 4 CH,Cl,/MeOH) which should afford 19 mg (41% from 13) 14 as a clear viscous oil. ‘H NMR(300 MHz) (CDCl,) 6 TMS 1 49 (9H, s), 1 61 (lH, dd, Jgem= 9 6 Hz, J,,, = 4 2 Hz), 2 18 (1H, dd, Jgem= 8 4 Hz, J,!,, = 2 9 Hz), 3.35 (3H, s), 5.33 (lH, broad s), 7 18-7 33 (5H, m), IR(NaCl/neat), 3358,3062,3029,3004,2977,2952,2933,1724, 1497, 1367, 1333, 1249, 1214, 1160, 1059 cm-‘; [cx]*~~ = +74 8” (c = 1 10, CH,Cl,)

3 4.5. (IS,2R)-(+)-2-Phenyl-I-Ammo1-Cyclopropane- I-Carboxylic Acid 15 1 Heat to reflux temperature a mixture of 14 (44 mg, 0 15 mmol, 1 0 Eq), LiOH H20 (63 mg, 1 50 mmol, 10 0 Eq), MeOH (4.0 mL), and H20 (2 0 mL) for 2 5 h 2 Cool the reaction to room temperature, and acidify to pH 2 5. 3. Extract the product with CH2C12 (3 x 5 mL) Combme the organic extracts, dry over Na2S04, filter, and concentrate Evaporation of the solvent should provide 40 mg (97%) of the correspondmg carboxyhc acid as a clear oil; use this material directly for the final step [a] 25D= +86 5” (c = 1 32, CH,Cl,); 4 Add a-15°C solution of anhydrous 1 NHCl m MeOH (6 0 mL, 6 00 mmol, 40 Eq) to the crude acid obtained above (40 mg, 0 15 mmol, 1 0 eq) as a neat oil Allow the resulting mixture to stir for 4 h at -15°C and 1 h at room temperature 5 Concentrate the reaction to dryness, and purify the crude ammo acid hydrochloride salt by aqueous elutron on a C,s reverse-phase Sep-pak cartridge, which should afford 28 mg (90%) of 15HCl as a whrte crystalline solid. ‘H NMR(300 MHz) (D,O) 6HODat464. 175(1H,dd,J,,= lO.OHz, J,,,=2 7Hz),2 01 (lH,apparentt, J= 7.8 Hz), 2 98 (lH, apparent t, J= 9.5 Hz), 7.20 (5H, s); IR (KBr). 3665-2000 (broad), 3435,3004,1712, 1594, 1498, 1267, 1183 cm-’ [cx]*~~ = +72.7” (c = 1.0, H20)

3.5. Preparation

of Statine 20

Since the discovery of pepstatm by Umezawa m 1970, there has been a tremendous level of Interest m the design and syntheses of nonscrssrle peptrde mrmrcs. Pepstatm 1s a naturally occurrmg pepttde produced by varrous Streptomyces sp. that was demonstrated to be a potent inhibrtor of aspartrc proteases, such as pepsin, rerun, and cathepsm D. Pepstatm contains the unusual ammo acrd statme 20 which has become the prototypical hydroxymethylene isostere of the putative tetrahedral transition state for pepttde bond hydrolysis. Most syntheses of statme and related hydroxymethylene peptrde rsosteres utilrze the natural ammo acid (m the case of statme, leucme) as a startmg material, which IS homologated by two carbons.

Willrams

352 NaN(SIMe3)2 o

-78%

/ THF

1 DIBAH.

Tfo

CH2C12,

2 (Ac)20, DMAP. CH2C12

16 17

1 KOH, dmane.

-78°C Et3N

OAc H kH3 H3C

16 MeOH

aZCH3 ZnBr2. CH2C12 -45%-tOOC

2 L?, NH3. THF t &OH

Ho

H

a2H

Fig 5 Synthesis of statme The method employed (6) relies on the ready availability of the alkylated oxazmones, such as 17 that can be prepared from the commercially available glycine templates 16. To illustrate this method, the synthesis of statine 20 1s shown m Fig. 5. The sodium enolate of 16 was alkylated with 1-lsobutyl trlflate to afford the alkylation product 17 in 78% yield. The an&-isomer (17, shown) was the only diastereomer that could be detected by ‘H NMR analysis. Reduction of the lactone carbonyl with DIBAH m methylene chloride at low temperature gave the correspondmg lactol which was nnmedlately acetylated with acetic anhydride m the presence of DMAP and trlethylamme to give the hemlacetal 18 m 79% overall yield from 17. The acetates were formed (m all cases) as a roughly equlmolar mixture of diastereomers and were used m the subsequent couplings as a mixture. The t-butyldlmethylsllyl ketene acetal of methylacetate (7) was condensed with 18 m the presence of zmc bromide m methylene chloride at -4YC + 0°C to furnish the separable couplmg products 19 and a diastereomer in a 4: 1 ratio m 65% combined yield. The major dlastereolsomer 19 was shown to have the desired 2S,3Sstereochemistry by conversion mto statme Thus, hydrolysis of the methyl ester mto the correspondmg acid was accomplished with aqueous, methanohc KOH m dloxane, the crude acid was subsequently reduced with hthmm m liquid ammonia to provide essentially optically pure (-)-statme 20 m 69% yield.

3.5.1. (3S,5S,6R)-#-(Benzyloxycarbonyl)-5,6-Diphenyl3-lsobutyl-2,3,5,6-Tetrahydro-#H1,4-Oxazm-Z-One

17

1 Add a solution of sodium bls(tr~methylsllyl)amlde (6 00 mmol, 6 mL, 1 A4 m hexane) to a solution of (+)-lactone 16 (1 94 g, 5 00 mmol) m THF (60 mL, anhydrous) and HMPA (5 mL) at -78’C 2 Stir the mixture at -78°C for 15 mm, and then add a solution of lsobutyl trlflate (1.90 g, 9 22 mmol) in THF (5 mL)

Asymmetric Syntheses

353

3 After 30 mm of stirring at -78°C add a saturated solution of NaHC03 (10 mL). Keep the temperature of the mixture at 25°C for 30 mm, and then extract with EtOAc (2 x 100 mL). Washed with a saturated solution of NH&l (200 mL) and brine (200 mL), and finally, dry the organic solution with Na,SO,; filter and concentrate under reduced pressure on a rotary evaporator 4 Purify the product by silica gel chromatography (120 g), using CH,Cl,, with 5% EtOAc This should give 1 74 g (78%) of 17 as a white solid: mp = 182-183°C [~]~~n = +36.6 (c = 1 35, CH,Cl,), ‘H NMR (DMSO-D, 393 K, 300 MHz) 6 1.OO (d, 3H,J= 6.4 Hz), 1 05 (d, 3H,J=6.4 Hz), 1.87-2.12 (m, 3H), 4.93 (dd, lH,J= 51,10OHz),498(d,lH,J=12.3Hz),505(d,lH,J=3 lHz),624(d,lH,J= 3 1 Hz), 6.62 (dd, 2H, J= 7 2, 1.5 Hz), 7.06-7.28 (m, 13H), i3C NMR (C,D,, 340 K, 75 MHz) 6 21 54, 23 46, 25 45, 44 55, 56 45, 61 51, 67 74, 79 02, 126 80, 127 76, 128.08, 128 23, 128.43, 135 30, 136 50, 154.30, 168 10

3 5.2. Compound 18 Add a solution of dnsobutylalummmm hydride (6.00 mmol, 6 mL, 1 Mm hexane) to a solution of (+)-lactone 17 (1 65 g, 3 70 mmol) m CH,Cl, (70 mL) at -78°C Stir the mixture at -78’C for 60 mm, and add Hz0 (10 mL) Stir the mixture at 25°C for 30 mm, filter through Celite, wash with a saturated solution of NH&l ( 100 mL) and brine (100 mL), dry over Na2S04, filter and concentrate. This should afford the lactol as an 011(1 60 g) which should be used without further purification. Add trtethylamine (0 75 g, 7.40 mmol) followed by acetic acid anhydride (0 76 g, 7 40 mmol) to a solution of the crude hemiacetal(l.60 g) m CH,Cl, (25 mL) at 0°C. Add a catalytic amount of DMAP (- 1 mg), and stir the mixture for 10 mm at 0°C and 20 mm at 25°C Dilute the solution with CH2C12(25 mL) wash with H,O (50 mL) and brme (50 mL), and dry over Na,SO, Filter, concentrate, and purify the product by chromatography (silica gel, 120 g), elute with CH,Cl,, EtOAc 5%), which should give compound 18, (1.43 g, 79%) as a colorless oil as a -3/2 mixture of diastereoisomers: ‘H NMR (C6D6, 340 K, 300 MHz) major isomer 6 103 (d, 3H, J= 6 0 Hz), 1.71 (s, 3H), 4.58 (m, lH), 4 90 (d (AB), IH, J= 12 3 Hz), 5.03 (d [AB], IH, J= 12.3 Hz), 5 20 (d, IH, J= 3.8 Hz), 5 40 (d, lH, J= 3 9 Hz), 6.54 (d, lH, J= 1 8 Hz), mmor isomer 6 0 68 (d, 3H, J= 6.5 Hz), 1 66 (s, 3H), 4 28 (m, lH), 5.05 (d [AB], IH, J= 12 2 Hz), 5 13 (d [AB], lH, J= 12 2 Hz), 5.70 (s (broad), lH), 6 59 (d, lH, J= 3.5 Hz), mixed signals 6 0 89 (d, 3H + 3H, J= 6.0 Hz), 1 50-2.20 (m, 3H + 3H), 6.85-7.20 (m, 15H + 15H); IR (NaCl film)* 1750, 1703 cm-’

3.5.3. Compound 79 1. Add a solution of compound 18 ( 0.500 g, 1 02 mmol) and methyl t-butyldimethylsilyl ketene acetal (7) (0 752 g, 4.00 mmol) m CH,Cl, (4 mL) to a suspension of ZnBr, (0 563 g, 2 50 mmol, previously dried under high vacuum at 150°C for an hour and at 25°C overnight) m CH2C12 (6 mL) at -45°C.

Williams

354

2 Slowly warm the mixture to -10°C Stir at -10°C for 30 mm and at 0°C for 1 5 h 3 Add a 2 N HCI solution (10 mL), and stir the mixture at 25°C for 1 h 4 Wash the orgamc layer with a 2 IV HCl solution (10 mL), a saturated solution of NaHC03 (10 mL) and brine (10 mL), and dry over Na2S0,, filter, and concentrate 5 Purify the product by chromatography (slhca gel [60 g], elute with hexane, EtOAc lo%), which should furnish compound 19 (0 122 g, 24%) as a colorless 011and a mixture of 19 and a dlastereomer (7/3, 0.218 g, 42%) ‘H NMR (C,D,, 340 K, 300 MHz) 6 0 68 (d, 3H, J= 6 5 Hz), 0.91 (d, 3H, J=6 6 Hz), 1 38 (ddd, lH, J =7.1, 13 4Hz), 1 71 (m, lH), 2 09 (ddd, lH, J= 6.2, 13 5 Hz), 2.60 (dd (AB), lH, J= 5 2, 15.0 Hz), 2 68 (dd (AB), lH, J= 7 2, 14 9 Hz), 3 41 (s, 3H), 4.00 (q, lH, J= 8.8 Hz), 4 38 (4 38 (q, lH, J= 6 8 Hz), 4 88 (d (AB), lH, J= 12 2 Hz), 5.025 07 (m, 2H), 5 44 (d, IH, J= 3 6 Hz), 6.87-7 22 (m, 15H); IR (NaCl film) 1742, 1704 cm-’ [cz]250 = +22 1” (c = 2.4, CHQ,).

3.5.4. Statirte 20 Stir a solution of ester 19 ( 0 140 g, 0.28 mmol) m dloxane (1 mL), MeOH (1 mL), and KOH 1 M (1 5 mL) at 25°C for 5 h. Remove the organic solvents under reduced pressure, dilute the aqueous layer with KOH 1 M (2 mL), and wash with Et,0 (5 mL) Acidify with 1 N HCl and extract with EtOAc (2 x 10 mL) Dry over Na*SO,, filter, and concentrate to afford the acid (0 139 g) Use the crude acid without further purlficatlon A pure sample for analysis can be obtamed by chromatography, see ref. 6 Add a solution of the acid obtamed above m l-BuOH (0 14 mL) and THF (5 mL) to a blue-black solution of llthmm (0.030 g, 4 29 mmol) m liquid NH, (10 mL) at -78°C Stir the mixture for 30 mm at this temperature, and add solld NH&l until decoloratlon Warm the mixture to room temperature, concentrate under reduced pressure, dilute with Hz0 (5 mL), keeping the pH acldlc, and wash with Et20 (10 mL) Purify the product by Ion-exchange chromatography (Dowex 50x8-200, 15 g, elute with H20, NH,OH 0 1 A4, 0 5 M, and 1 M) which affords statme 20 (0 035 g, 69%). [a] 250 = + 18 1o (c = 0 9, H20) The ‘H NMR of the synthetic substance proved to be identical to the spectra of a sample from obtained from Sigma Chemical Co

3.6. Discussion Aldrich Chemical Co. has sold the D- and L- forms of both the N-t-BOC 1 and N-CBz lactones 16 smce 1988. The IV-t-BOC lactones have been partlcularly important since these provide direct accessto the corresponding N-t-BOCa-ammo acids suitable for most peptlde-couplmg strategies. Four different protocols

for the removal

of the chiral auxiliary

have been devised; two are

Asymmetric Syntheses

355

reductive and two are oxidative. Only two of these have been illustrated m thts chapter (dissolving metal reduction and lead tetraacetate oxidation) In addition, acidic removal of the N-t-BOC group from derivatized oxazmones followed by hydrolytic rmg opening of the lactone group and sodium periodate oxidation provides an alternative oxidative removal strategy. For the N-CBz derrvatized oxazinones, the most popular deprotection involves simple catalytic hydrogenation (for example, compound 17, Fig. 5 can be directly converted mto (S)-leucme by catalytic hydrogenation); refer to ref. Ic for a more complete discussion of the appropriate substrates and methods to use. The versatility provided by the choice of these different protocols gives the researcher an extremely broad range of flexibility m selectmg functionality m the newly introduced a-“R” group. The deprotectron methods for each homologated substrate (Note 4) derived from lactones 1 and 16 are given below: Method

Condttions

Tvne of a-‘%” Grou p Ammo actd Ref

Oxazmones derived from NCBz substrates (16) 1 Catalytic hydrogenation 2 Birch reduction

(H,/Pd-C, EtOH) (Lr” or Na“, NH&

Saturated, ahphattc Unsaturated Saturated, ahphatrc Cyclopropyl

Zwtttenon Zwittenon

Zc Ic

Unsaturated Saturated, ahphattc Aromatic Saturated, ahphatic Aromatic P-Arylcyclopropyl Saturated, ahphatrc

N-t-BOC

Zc

Zwittenon

lc

Methyl ester

Zc

Oxazmones derived from N-t-BOC substrates (I) 1 Birch reduction

(Li“ or Na”, NH31n)

2 Oxidation

(1) H+, 2) NaIO,

3 Oxidation

(1) H+, 2) Pb(OAc),

It must be added that although the chiral auxiliary is sacrificed m the final deprotections, thrs system offers an important advantage over numerous other ammo acid synthesesthat require expensive, time-consummg chromatographic separations, recovery, and “recycling” of choral auxiliaries (rarely done m practice m a basic research setting), hydrolysis of esters, and so on, to obtain the ammo acids themselves In the present case, the choral auxiliaries are polar, water-soluble substancesthat even if it were possible to recover, would require a drfficult separation from the hydrophilic amino acid products. Thus, the destruction of the chnal auxiliary m this case turns out to be a significant advantage, since the final processmg converts the choral auxiliary into an innocuous substance (bibenzyl or benzaldehyde, depending on whether reduction or oxidation methods are used, respectively) of greatly different solubility properties than the ammo acids or t-BOC ammo acids, and is easily removed

Willlams

356

by trrturation or extractron The raw cost of the ammo alcohols used to prepare the lactones of course preclude the apphcatton of this chemistry to large, multrktlo mdustrral-scale syntheses. Thus, thts system IS most appropriate for the basic research chemist who needs rapid and predictable access to a large number of structurally diverse ammo acids in optically active form. 4. Notes 1. Owmg to slow conformational

exchange of the urethane groups (N-t-BOC or N-

CBz) on the NMR time scale, rt IS necessary to run the NMR spectra at high

temperature to obtain sharp, interpretable spectra NMR spectra can be conveniently recorded m DMSO-d6 at 393 K and in some instances m C6D6, at 340 K 2. The cyclopropanatron step proceeds exclusrvely from the more hindered face of the oxazmone This facial bias was observed m every case studied (-100% dr) A mechanistic explanation for thts diastereoselectivity has been offered (see ref. 3) 3. The protection of the nitrogen culminating m the preparation of compound 14 with a t-BOC group aids m the purtfication of the product followmg the lead tetraacetate cleavage step The subsequent LIOH step affords the N-t-BOC ammo acid, which can, if desired, be used m pepttde couplmg The final actdic treatment gives the free ammo acid as the HCl salt. 4 For a discusston of the most appropriate cleavage method to use on a particular substrate, please refer to ref. Zc.

References 1 For reviews on methods to prepare a-ammo actds, see (a) Williams, R M. (1994) Synthesis of Optlcalfy Actwe a-Ammo Acids Organic Chemutry Series (Baldwm, J E and Magnus, P D., eds ), Pergamon, Oxford, (b) Duthaler, R 0 (1994) Recent developments m the stereoselective synthesis of a-ammo acids Tetrahedron 50, 15391650, (c) Williams, R. M (1992) Asymmetrtc synthesis of a-ammo acids Aldrlchzmlca Acta 25, 1 l-25 2 Willtams, R. M and Im, M.-N (1991) Asymmetric synthesis of mono-substituted and o,a-disubstituted a-ammo acids via diastereoselectlve glycme enolate alkylations J Am Chem Sot 113,927&9286 3 Wtllrams, R M., Fegley, G. J , Asymmetrw Syntheses of I-Amlnocyclopropane Carboxylzc Acid Derwatwes. J Am Chem Sot (1991) 113,87968806 4 Johnson, C R and Jamga, E R. (1973) Nucleophlltc alkyhdene transfer reagents Ethylides, isopropylides and cyclopropyltdes derived from salts of sulfoximmes

J Am Chem Sot. 95,7692-7700 5. Williams, R M and Fegley, G J. (1993) Asymmetric synthesis of lS,2R-(+)-2phenyl- 1-ammocyclopropane-1-carboxylic actd J Org Chem 58,6933%6935 6 Wtllrams, R M., Colson, P -J. and Zhai, W. (1994) A new method for hydroxymethylene peptide isostere synthesis asymmetric synthesis of statme Tetrahedron Lett (1994) 35,9371-9374 7 Kita, Y., Haruta, J , FUJII, T , Segawa, J., and Tamura, Y. (1981) 0-silylated ketene acetal chemistry, a mild and effictent t-butyldtmthylsilylating agent Syntheszs 45 1.

20 Fluoroolefin

lsosteres

J. T. Welch and T. Allmendinger 1. Introduction Replacement of the amide bond m the peptide backbone can improve the activity, stability, or bioavailability of the resultant unnatural peptide (I). Amide bond surrogates cannot only impart peptidase resistance, but can also facilitate conformational control of the target peptide Although the alkene v[C=C] isostere IS an accurate mimic of the steric demand, bond lengths, and bond angles of the amide bond (2-6), it also permits construction of both the (&‘) and (2) (cu- and trans-) isomers independently. Unlike the amide bond, which has some degree of flexibility, the v[C=C] isostere is conformationally fixed (7-s). The w[CF=C] isostere retains these attributes, but accurately mimics the electronic features of the amide bond (9-11) to include dipole moment, charge distribution, and electrostatic potential. The v[CF=C] isostere has recently been employed m the preparation of a number of peptidomimetics typical preparations, which will be discussed m this chapter (see Table 1). 2. Materials Infrared (IR) spectra were obtained on a Perkm-Elmer 1600 Series FTIR spectrometer. All ‘H NMR spectra were recorded at 300 MHz on a Gemmi300 NMR spectrometer with CDCls as solvent and tetramethylsilane (TMS) or residual chloroform as the internal standard. All i3C NMR spectra were recorded at 75.429 MHz on a Gemmi XL-300 NMR spectrometer with CDC13 as solvent and TMS or residual chloroform as the internal standard. igF NMR spectra were recorded at 282.203 MHz on a Gemini XL-300 NMR spectrometer with CDCl, as solvent and chlorotrifluoromethane (CFC13) as the mternal standard Thin-layer chromatography was performed with silica gel FIs4 From

Methods m Molecular Medune, Vol Edlted by W M Kazmlerskl OHumana

357

23

fept/dom/metcs Press Inc , Totowa,

Protocols NJ

Welch and Allmendmger

358 Table 1 y[CF=C] lsostere

Containing

Peptides Ref

Pepttde Phe-v[(Z)-CF-CH]-Gly Phe-v[(E)-CF-CH]-Gly Arg-Pro-Lys-Pro-Gln-GlnPhe-Phe-v[(Z)-CF-CH]-Gly Leu-Met Phe-v[(Z)-CF-Cl-Pro Gly-v[(Z)-CF-CH]-Gly Phe-W[(Z)-CF-Cl-Phe Ala-v[(Z)-CF-CH]-Gly Gly-v[(Z)-CF-CH]-Leu Gly-v[(Z)-CF-CH]-Leu-Gly Gly-v[(Z)-CF-CH]-Leu-Ala Gly-v[(Z)-CF-CH]-Leu-Leu Gly-t+r[(Z)-CF-CH]-Leu-Phe

10 10 I2

12

10 12 13 14 14 14 14 14

(Merck) as the adsorbent on 0.2-mm thick, plastic-backed plates. The chromatograms were visualized under UV (254 nm) by stammg with a 5% solutton of phosphomolybdtc acid m tsopropanol followed by drying m an oven at 90°C or by spraying with a 95.5 mixture of 0.2% ninhydrin in n-butanol and 10% aqueous acetic acid followed by heating. Column chromatography was per-

formed using srhca gel 60 (70-230 mesh, Merck) and flash stllca gel 60 (0.04& 0.063 pm, 230-400 mesh, EM Science) Melting points were determined m open capillaries using a Buchl 5 10 melting pomt apparatus and are reported uncorrected. Botlmg points are reported m degrees centigrade at the indicated pressure m mm mercury (Hg) and are uncorrected. Optxal rotations were taken on a Perkm-Elmer 241 polartmeter with a sodium lamp (589 nm, lo-cm path length, cell, concentratton m g/100 mL) of the indicated solvent. 2.7. Reagents 1 2 3 4 5 6 7 8 9.

for Method

3.7

2,4,6-Trtmethylphenol Tetrahydrofuran n-Butyl lnhtum (2 5 A4 solutton m hexanes) Fluoroacetyl chloride. Ammomum hydroxide Dlethyl ether Sodturn sulfate. Sodtum bicarbonate Dusopropylamtne

Fluoroolefin 10 11. 12 13 14 15 16 17 18 19 20 21 22. 23 24. 25 26. 27 28 29 30 31 32. 33 34 35

lsosteres

Pentane Chlorotrtmethylsilane. Magnesmm sulfate Tartartc actd Methylhthmm (1 5 M solution m dtethyl ether) Cyclopentanone Trtmethylstlylethoxymethyl chloride. Ammonmm chlortde Hexanes Dnsobutylalummum hydride Potassmm sodium tartrate Ethyl acetate Chrommm trioxtde. Pyrtdme Neutral alumma. Dtchloromethane Trtphenylphosphme. Phthalimide Dtethylazadicarboxylate Boron trtfluorlde etherate Jones reagent Acetone. Hydrazme Ethanol Hydrochlortc acid Dowex 50-X-8 ion exchange resin.

2.2. Reagents for Method 3.2 1 2 3 4 5 6 7 8 9. 10 11. 12 13 14 15 16

(R)-(+ )-4-benzyl-2-oxazohdmone Tetrahydrofuran. 1 6 N n-butyl ltthmm m hexane 4-Methylpentanoyl chloride Ammomum chloride Dtchloromethane Magnesium sulfate Silica gel Hexane Chloroform Tttanmm tetrachlortde Dnsopropylethylamme Trtoxane. Sodmm chloride Dtmethylformamtde Imtdazole

359

Welch and Allmendinger

360 17 18 19 20. 21 22 23 24 25 26 27 28. 29 30 31 32 33

tert-Butyldtmethylstlyl chloride Ltthtum borohydrtde Pyrtdmmm chlorochromate. Dtethyl oxalate Ethanol Ethyl fluoroacetate Ammonia Methanol. Lithium alummum hydrtde Cehte Benzyl chloroformate Trtethylamme Acetomtrtle Tetra-n-butylammonmm fluoride Jones reagent Acetone. Isopropyl alcohol

2.3. Reagents for Method 3.3 1 2 3 4 5 6 7 8. 9 10 11 12 13 14 15 16. 17 18 19 20 21 22 23 24. 25

Acetamide. Methanol Ethanol tert-Butyl a-chloroacetate Potassmm fluoride Dnsopropylamme. Pentane n-Butylhthmm (2 5 M solution in hexanes). Chlorotrimethylstlane Sodmm bicarbonate Magnesium sulfate Tartartc acid Tetrahydrofuran 2-Hydroxymethyl cyclopentanone Hexanes Diethyl ether Dnsobutylalummum hydrtde Methylhthmm (1 5 M solutton m dtethyl ether) Dlchloromethane Triphenylphosphine Dtethylazodtcarboxylate Phthahmlde Methylhydrazme 1,l ,1,3,3,3-Hexamethyldtstlazane. Ammonmm chloride.

361

Fluoroolefm lsosteres 26. Dtoxane 27 Trtethylamme. 28 2-(t-Butoxycarbonyloxyimmo)-2-phenyl 29 Acettc actd 30 Jones reagent.

acetomtrtle

2.4. Reagents for Method 3.4 1 2 3 4 5.

6 7 8 9 10 11 12 13, 14 15. 16 17 18 19

20. 21 22 23 24

Drchlorofluoromethane Potassium hydroxide. Ammonium chloride Sodium laurylsulfate Hexane. Zmc Copper (I) chloride Dtethyl ether Hydrochloric actd Ethyl acetate Sodmm bicarbonate Sodmm sulfate tert- Butyl bromoacetate Lithium aluminum hydride Sodium hydroxtde Celtte Sodium hydrtde Trichloroacetomtrtle Pentane Acettc actd Methanol Drchloromethane Jones reagent Chloroform

3. Methods 3.1. Preparation 3.1 1. 2,4,6-The

of @)- and (E)-G/y-y[CF=C]-(o&Pro thylphenyl Fhoroaceta te 7

Dipeptides

1 To a solutton of 2,4,6-trtmethylphenol(36.64 g, 0.27 mol) m THF (300 mL), add dropwrse n-BuLr (129 mL, 0.32 mol, 2.5 M solutron m hexanes) at O’over a period of 30 mm. Add fluoroacetyl chloride (39 g, 0.40 mol) dropwtse at 0°C (Fig. 1). (Caution: See Notes 1 and 2.) 2 Remove the Ice bath, and allow the solutron to stir for 16 h at room temperature. Cool the reaction mixture m an me bath, and add concentrated ammonmm hydroxrde (10 mL) dropwtse

Welch and Allmendmger

362 0

1 4 equw OTMP

F31 1

0

1

LDA

0

2 6equv TMSCI 3 Saturated aq tartarlc acid

LDA

2 SEM-Cl

F’r~~~~ .QMe3

0 O-,SIW c)rc 3 (67%)

\ SIMe3

$SiMe3

4

5 (65%)

1 4 (B

SIMe3

&Me3 7

6 (97%)

(0 Ph3P,

DEAD

phthalimtde

NHphth

NHPhth

BFyEt20

HO StMe3 6

Frgure 1 3

Pour the mixture mto a saturated NaHCO, solutton (100 mL), and separate the organic Extract the aqueous layer wtth drethyl ether (3 x 100 mL) Dry the combined orgamc layers over Na$O,, filter, and evaporate 4 Recrystallrze the solid three times from hexanes After recrystalhzatron, 42.8 g (8 1%) of 2,4,6-trrmethylphenyl fluoroacetate, 1, can be obtained mp 70-7 1°C

Fluoroolefin

363

lsosteres

‘H NMR (CDCl,) S 6 93 (s, 2H, H), 5 17 (d, 2H, JH,F = 47.2 Hz), 2 3 1 (s, 3H), 2 16 (s, 6H) 19FNMR (CDCl,) S-230.15 (t, .& = 47.2 Hz).

3.1.2. 2,4,6-Trimethylphenyl

a-Fluoro-(a-Trimethylsilylacetate)

2

To a solution of dnsopropylamme (8 41 mL, 60 mmol) m THF (30 mL), add dropwlse n-butyllithlum (24 mL, 60 mmol, 2.5 Msolutlon in hexanes) at -20°C Allow the solution to stir for 10 mm at -2O”C, and then transfer with a canula Into a solution of THF (120 mL) and pentane (150 mL), contammg 2,4,6trimethylphenyl fluoroacetate 1 (2 94 g, 15 mmol) and chlorotrimethylsllane (15.23 mL, 120 mmol) at -95°C. Keep the temperature between -90°C and -95°C during the transfer (Note 3) Allow the reaction mixture to stir for 15 mm at -95”C, and then warm to -25°C over a period of 3 h At -25”C, quench the reactlon mixture with a saturated NaHC03 solution (10 mL), and separate the organic layer Extract the aqueous layer with hexanes (3 x 50 mL) Combme the organic layers, dry over MgSO,, filter, and evaporate Hydrolyze this mixture with a saturated tartaric acid solution (100 mL) at room temperature for 15 h to form 3 48 g (86 5%) of 2,4,6-tnmethylphenyl a-fluorocL-trlmethylsllylacetate, 2, after dlstlllatlon: bp 1OO- 106°C (0 1 mm Hg) ’ H NMR (CDCIJ) S 6.90 (s, 2H, H), 5 25 (d, lH, & = 45 2 Hz), 2.28 (s, 3H), 2 15 (s, 6H), 0.34 (s, 9H, ) 13CNMR (CDCl,) S 169.00 (d, .& = 18 4 Hz), 145 36, 135 45, 129.49,129 29,87 84(d,JC,F= 181 2Hz),20.57,16 54,-3 69. 19FNMR(CDC13) S -226 50 (d, & = 45 2 Hz)

3. I 3 2-[2-(Trimethykilyl)

Ethoxymethyl] Cyclopentanone

3

Add dropwlse methylhthmm (17 5 mL, 26 25 mmol, 1 5 A4 solution m dlethyl ether) to a solution of dlisopropylamme (3.68 mL, 26 25 mmol) m THF (100 mL) at -20°C After the addition was completed, allow the solution to stir for 10 min at -20°C. Cool the solution to -95’C and add cyclopentanone (2 19 mL, 25.0 mmol) dropwlse Stir the solution for 15 min at -95“C, and add dropwlse 2-tnmethylsllylethoxymethylchlorlde (5 09 mL, 28.75 mmol), so that the temperature never exceeds -90°C Allow the solution to stir for 45 min at -9O”C, and then allow to warm to room temperature Quench the reaction mixture with a saturated NaHC03 solution (20 mL), and extract with dlethyl ether (3 x 30 mL). Follow drying over anhydrous sodium sulfate by evaporation of the solvent zn vucuo. Dlstlllatlon yields 10 70 g (66 5%) of 2-[2(tr~methylsllyl)ethoxymethyl]cyclopentanone, 3, bp 70-74’C (0 20-0.25 mm Hg) ‘H NMR (CDCl,) S 3 60-3 45 (m, 4H,), 2 40-1.70 (m, 7H,), 0.89 (t, J= 6 98 Hz, 2H), -0-01 (s, 9H,) 13C NMR (CDC13) S 219.44, 68.93 and 68.29, 49.32, 38.55, 27.12, 20 78, 17.88, -1 48.

364

Welch and Allmendmger

3.1.4. (z)- 1-({I -Fluoro-2-Carboxy-[2,4,6-Trimethylphenylf 2-[2-(Trimethylsilyl) EthoxymethyljCyclopentane 4 and (E)- 1-11-Fluoro-2-Carboxy-(2,4,6-Trimethylphenyl)2-2-(Trimethylsilyl)Ethoxymethyl]lCyclopentane 5 Add dropwtse n-butylhthium (2.64 mL, 6 6 mmol, 2 5 M solution m hexanes) to a solution of dnsopropylamme (0 93 mL, 6.6 mmol) m THF (50 mL) at -20°C, and allow the solutton to stir for 10 mm at -2O’C Cool the solution to -85°C and add dropwtse 2,4,6-trrmethylphenyl a-fluoro-atrtmethylstlylacetate, 2 (1.6 1 g, 6 0 mmol), dtssolved m THF (3 mL). Allow the reaction mixture to stir for 45 mm between -85”and -90°C Add dropwtse 2-[2(tr~methyls~lyl)ethoxymethyl]lcyclopentanone, 3 (1 67 g, 7 8 mmol) as a solution m THF (3 mL) Allow the reactton mixture to stir for 5 mm at -9O”C, and then remove the cooling bath Quench the reaction mixture with a saturated ammomum chloride solutton (10 mL) Separate the organic layer, and extract the aqueous layer with hexanes (3 x 50 mL) Combme the organic layers dry over MgS04, filter, and evaporate. tgF NMR analysts of the reaction mixture showed 70% converston (4 1 ratio of Z E isomers 4 and 5) and 30% 2,4,6-trimethylphenyl a-fluoroacetate, 1 The crude product was bulb-to-bulb dtsttlled (Kugelrohr) at reduced pressure (0 25 mm Hg), and the fractron between 170 and 230°C was collected. Yteld 64 5% (3 04 g; 92 2% based on conversion) @)-isomer 4 ‘H NMR (CDCl,) 6 6 90 (s, 2H, H), 3 6c3.30 (m, 5H), 2 82 (m, 2H), 2 28 (s, 3H), 2 14 (s, 6H), 0 86 (t, J= 8 2 Hz, 2H), 0 03 (s, 9H) 13CNMR (CDCl,) F 158 55 (d, Jc,r = 35 5 Hz), 145.24, 144 16 (d,Jc,, = 14 5 Hz), 141 64 (4 JC,F = 248 4 HZ), 135 50, 129 52, 129 27, 70 39 (d, JC,F = 4 0 HZ), 67 72, 42 48,30 36 (d, JC,F = 5 4 Hz), 29 82,22 57,20 63, 18 08, 16 24, -1 52 lgF NMR (CDCls) 6 -122.92 (E)-isomer 5. ‘H NMR (CDCls) 6 6.9 1 (s, 2H, H), 3 55-3 30 (m, 5H), 2 75 (m, 2H), 2 26 (s, 3H), 2.16 (s, 6H), 0.89 (t, J= 8 2 Hz, 2H), 0.01 (s, 9H,) 13CNMR (CDC13) 6 159.10 (d, JC,F = 36.6 Hz), 145.38, 143 38 (d, Jc,r = 12 0 Hz), 141 77 (d, J,-,F = 248 0 Hz), 135.52, 129.61, 129 25, 70 12 (d, JC,F = 4.1 Hz), 68.22 (s, 2H), 44.08,31 30,29 27,24 72,20.73, 18 10, 16 30, -1 34 lgF NMR (CDCl,) 6 -127 11

3.1.5. (Z)-l-(1-Fluoro-2-Hydroxyethylidene)2-[2-(Trimethyls~lyl) EthoxymethyljCyclopentane and (E)-1-(1-Fluoro-2-Hydroxyethylidene)2-[2-(Trimethylsily~)Ethoxymethyl]Cyclopentane

6 7

1 To a solution of a mixture of esters 4 and 5 (3 00 g, 7 64 mmol) m THF (75 mL) at -70°C add dropwtse dnsobutylalummum hydride (5.45 mL, 30 57 mmol) Allow the mixture to stir for 30 min at -70°C then remove the coolmg, and allow the solution to stir for 30 mm at room temperature 2 Cool the solution to 0°C and add a saturated K&O3 solutron (1 65 mL) dropwtse Then add potassmm sodmm tartrate (17 25 g, 6 1 13 mmol) m porttons, followed

Fluoroolefin

365

lsosteres

by water (30 mL) and dtethyl ether (50 mL). Vigorously star the mtxture for 5 mm, and allow the organic layer to separate Decant the orgamc layer, and extract the aqueous layer with dlethyl ether (3 x 50 mL Combine the organic layers dry over MgSO,, filter, and evaporate. 3. Column chromatography (hexanes/EtOAc, 90: 10) yielded 1 55 g of (Z)-alcohol 6 and 0 37 g of (E)-alcohol 7 (combined yteld* 96.5%); TLC (hexanes/EtOAc; 85.15; (Z)-alcohol, Rf 0 28, @‘)-alcohol, Rf 0 1 3) (Z)-alcohol 6: ‘H NMR (CDCl,) 6 4.17 (dd, lH, Jn,r 30 4 Hz and Jn,n 15 0 Hz),), 3 98 (t, lH, Jn,r = Jn,n = 15 0 Hz), 3 47 (m, 2H),), 3 28 (m, lH), 3 13 (t, lH, J=lO 6 Hz), 2 95-2 85 (m, lH), 2 50-2.25 (m, 2H), 1 85-l 35 (m, 4H), 0 94 (dd, 2H, J = 10 6 Hz and 6 8 Hz), -0.03 (s, 9H, ) 13C NMR (CDCl,) 6 154.03 (d, Jc,r = 249 6 Hz), 123 24 (d, JC,F = 16 4 Hz), 72 83 (d, J C,F = 3 2 Hz), 68.64, 59 65 (d, JC,F = 34.1 Hz), 40 55 (d, JC,F = 5 6 Hz), 29 98, 26 69 (d, JC,F 5.4 Hz), 23.65, 17.93, -1.55). 19F NMR (CDCI,) 6 110 28 (dd, J,l,F = 30.4 Hz and 15.0 Hz). (E)-alcohol7: ‘H NMR (CDCl,) 6 4.15 (d, 2H, JH,F = 20.7 Hz), 3 65-3.35 (m, 3H), 3 28 (m, lH), 3 13 (t, lH, J= 10.6 Hz), 2.95-2.85 (m, lH), 2 5&2 25 (m, 2H), 1 85-l 35 (m, 4H), 0 94(dd, 2H, J= 10 6 Hz and 6 8 Hz),-0 03 (s, 9H, ) 13C NMR (CDCl,) 6 15 1 49 (d, JC,F = 245 8 Hz), 123 04 (d, JC,F = 15 7 Hz), 7 1 12 (d, JC,F = 5.0 Hz), 67.94,59 42 (d, JC,F = 30.9Hz),40.89,29.92,28.36(d,Jc,,=4.1 Hz), 24 74, 18 04, -1 50 19FNMR (CDCl,) 6 -119 73 (t, JH,F = 20.7 Hz),

3.1.6. (E)-N -Phtha/imide- 7-(Z-Amino- 7-F/uoroethy/idene)2-[2-(Trimethylsilyl)Ethoxymethyl]Cyclopentane 9 To a solution of alcohol 7 (2 02 g, 7.75 mmol) m THF (25 mL), add trtphenylphosphine (2 24 g, 8 52 mmol), phthaltmtde (1.37 g, 9.29 mmol), and dtethylazadrcarboxylate (DEAD) (1.58 mL, 10.07 mmol), and star the mtxture for 12 h at room temperature 2 Silica gel (8 g, 70-230 mesh) was added, and the solvent was evaporated Chromatography (hexanes/EtOAc, 4 1) yielded 2 25 g (75%) of (E)-N-phthahmtde- l[2-am~no-l-fluoro(ethyl~dene)-2-[2-(tr~methyls~lyl)ethoxymethyl]]cyclopentane (TLC: hexanes/EtOAc; 4: 1, R,0.38). ‘H NMR (CDCl,) 6 7.85-7.65 (m, 4H, H), 4.39 (d, JH,F = 19 0 Hz, lH), 4.38 (d, JH,F = 19.0 Hz, IH), 3 55-3 30 (m, 3H), 3 13 (t, J= 8 7 Hz, lH), 3 03 (br, lH), 2 60-2.40 (m, 2H), 1.85-1.60(m, 4H, 2), 0 87 (dt, J = 8 3 Hz 52 7 Hz, 2H), A) 04 (s, 9H). 13CNMR (CDCI,) 6 167.65, 146 71 (d, J,-F = 247 1 Hz), 134 04, 132 06, 124 46 (d, JC,F = 14.4 Hz), 123 37,70.90 (d, Jc,,=2.2Hz),67.89,41.25,36.85(d,Jc,,=28.6Hz),3002,28.60(d,Jc,,=4.1 Hz), 24.73, 18.08, -1.40 (s, 9H). 19FNMR (CDCl,) 6 -117.42 (t, JH,F = 19 0 Hz). 3 (Z)-fluoroolefin 8 1s hkewlse prepared m 99% yield.

3. I. 7. (E)-N -Phthalimide- l-(2-AminoI-Fluoroethylrdene)-2-(Hydroxymethyl)Cyclopentane 1 To a solution of 9 (2 20 g, 5 65 mmol) m dichloromethane BF, Et,0 (2.82 mL, 22 9 mmol) at 0°C

11 (40 mL), add dropwise

366

Welch and Allmendinger

2 Allow the solutton to warm up slowly to room temperature, and star for two more hours Quench the reaction mtxture slowly at O’C with a saturated NaHCO, solution (30 mL). 3. Separate the organic layer The aqueous layer was extracted wtth dtchloromethane (3 x 30 mL) Combine the orgamc layers dry over MgSO,, filter, and evaporate Column chromatography (hexanes/EtOAc, 1 1), yielded 1.46 g (89%) 11 as a white soled (TLC. hexanes/EtOAc; 1.1, R,O 30) mp. 9698°C. ‘H NMR (CDCl,) 6 7 9c7.70 (m, 4H, H), 4 43 (d, Ju,r = 19 6 Hz, 2H), 3.7s3.50 (m, 2H), 3 20 (br, lH), 2.98 (br, lH), 2 65-2 40 (m, 2H), 1.90-l 60 (m, 5H) t3C NMR (CDCI,) 6 167 71, 146.88 (d,Jc,, = 246 8 Hz), 134 09, 131.94, 124 18 (d,Jc,, = 13 0 Hz), 123.40, 64 06 (d, Jc,r = 2 3 Hz), 43 84, 36 92 (d, JC,F 3 1 4 Hz), 29 54, 28.82 (d, JC,F = 4 6 Hz), 24.89 . 19FNMR (CDCl,) 6 -117.27 (t, JH,r = 19 6 Hz) 4. (Z)-Fluorooletin 10 1s hkewtse prepared m 92% yield (TLC: hexanes/EtOAc, 1 1, R,-0 27) Mp 110-l 12°C. ‘H NMR (CDCl,) 6 7 80-7.70 (m, 4H, H), 4.67 (t, JH,F=JH,H=l5.3Hz,lH),449(dd,J,,,=l84Hz,JH,H=l53H~,lH),3703 40 (m, 2H), 3 20 (br, lH), 3.04 (q, J = 7 5 Hz, IH), 2 50-2 20 (m, 2H), 1 9& 1 50 (m, 4H) “CNMR (CDCl,) 6 168 0,146.7 (d, JC,F = 246 7 Hz), 134 0,13 1 8, 124 4 (d, JC,F = 15.4 Hz), 123 3, 65 3 (d, JC,F = 3 8 Hz), 43 1 (d, JC,F = 5.8 Hz), 36.9 (d, JC,F = 28 4 Hz), 29 7,27 2 (d, JC,F = 3 3 Hz), 23 4 19FNMR (CDCl,) 61139(t,J,,r= 184Hz)

3 1.8. (Z)-N-Phthalimide-I-(2-Amino1-Fluoroethylidene)-Cyclopentane

Carboxylic Acid 12

1 To a solution of Jones reagent (6 1 mL) m acetone (60 mL) at 0°C add (z)fluoroolefin 10 (1 96 g, 6 76 mmol) Allow the mtxture to star for 45 mm at 0°C and then add H,O (20 mL). 2 Extract the solutton wtth ethyl acetate (5 x 30 mL). Combme the organic layers dry over MgSO,, filter, and evaporate. Yield of (Z)-fluoroolefin 12: 1 2 1 g (59%) ‘H NMR (CDCI,) 6 7 85-7.60 (m, 4H, H), 4 52 (t, JH,F = 11 9 Hz, IH), 4 50 (d, J H,F = 24 5 Hz, lH), 389 (br, lH), 2 50-2 30 (m, 2H), 2 3&l 60 (m, 4H) 13C NMR (CDCl,) 6 179 3 (d, JC,F = 4 1 Hz), 167.5, 144.7 (d, JC,F = 251 6 Hz), 133 9, 131.7, 123 2, 122 3 (d,Jc,,=20 1 Hz),45.2(d,JC,r=4 9Hz),36.3 (d,Jc,,=27 5 Hz), 31 0, 27 5 (d, JC,F = 2.8 Hz), 24.5. 19FNMR (CDCl,) 6 -111 49 (d, JH,F = 24.5 Hz, 11 9 Hz) 3 The yield of (E)-N-phthalimlde-2-(2-amlno-l-fluoroethylldene)cyclopentane carboxyhc acid 13. IS 47%

3.1.9. (Z)-Gly- y[CF=C]-(@-Pro

Dipeptide lsostere 14

1 To a solution of Q-fluoroolefin 12 (0 37 g, 1 22 mmol) m ethanol (4 mL), add a 1 Msolutton of hydrazme m ethanol (1 22 mL) Allow the mtxture to star for 18 h at room temperature 2 Evaporate the solvent, and then add a 2 N HCl solutton (3 mL) Stir the mixture for 2 h at room temperature, and then evaporate the solvent Purtfy the crude reaction mixture on a cation-exchange column with a gradient of increasmg HCl

367

Fluoroolefin lsosteres

concentratron. Yield of (Z)-Gly-v[CF=CH](D,L)-Pro dtpeptide rsostere, 14 0 15 g (66%) ‘H NMR (D,O) 6 3 69 (d, Ju,r = 20 0 Hz, 2H), 3 34 (t, Jn,n = 6.9 Hz), 2 55-2 30 (m, 2H), 2 lo-l.95 (m, lH), 1 95-1 80 (m, lH), 1 80-l 55 (m, 2H) r9F NMR (D,O) 6 -113 53 (t, Ju,r = 20 0 Hz) 3 Slmrlarly the (E)-Gly-v[CF=CH]-(DL)-Pro drpeptide rsostere, 15. 1sprepared m 45% yield ‘H NMR (D,O) 6 3.88 (d, Ju,r = 19.1 Hz, 2H), 3.70-3.50 (m, IH), 2 5&2 30 (m, 2H), 2 2c2.00 (m, IH), 1.95-1 75 (m, 2H), 1.80-1.55 (m, 1H) ‘9FNMR(D20)6-112 18(t,J,,,= 19.1 Hz)

3.2 . N-[Benzyloxycarbonyl]-Gly-y/[CF=CH](L)-Leu Dipeptides (ref. 14) 3.2.7. (4R)-4-Benzy/-3-(4-Methyl-l-Oxopentyl)-2-Oxazolidinone

76

1 To a solution of 7 00 g (39 5 n-m-101)of (R)-(+)-4-benzyl-2-oxazohdmone m 120 mL of THF, add 24 7 mL (39 5 mmol) of 1 6 N n-BuLr m hexane over 10 min at -78°C 2 After 30 mm, add 6 38 g (47 4 mmol) of 4-methylpentanoyl chloride at the same temperature Stir for 30 mm at -78°C then allow the reaction mtxture to warm to room temperature, and add 25 mL of aqueous NH&l and 10 mL of water 3. Remove the THF WI VLICUO,then extract the residue with 100 mL of CH2C12 and wash the organic extract with 1 NNaOH, water, and brine, and dry over MgS04 4 Remove the solvent m vucuo, then chromatograph the residue on srhca gel (1.10 EtOAc/hexane) to afford 10 5 g (96%) of oxazohdmone 16 (Fig. 2) as a colorless 011. Rf 0.5 1 (1.4 EtOAc/hexane); [a] 23D= -54 7” (c 1.27, CHCl,), ‘H (CDCl,, 400 MHz 6 0 94 (d, 6, J= 6 4), 1 53-1.70 (m, 3), 2.76 (dd, l,J= 9 6, 13.4) 2 863.03 (m, 2), 3.30 (dd, 1, J= 3.2, 13 4), 4.15-4.27 (m, 2), 4 64-4 72 (m, l), 7 157 45 (m, 5), r3C NMR(CDCl,, 100 MHz) 6 22 3, 27 6, 33 1, 33.5, 37 9, 55 1, 66.1, 127 2, 128 9, 129.4, 135 3, 153.4, 173.6.

3.2.2. (4R)-4-Benzyl-3-[(2S)-2-(Hydroxymethyl]4-Methyl- 7-Oxopenty/)-2-Oxazolidinone 77 1 To a solution of 3 26 g (11 9 mmol) of oxazolidmone 16 m 50 mL of dry CH2C12 add 1 37 mL (12 5 mmol) of TICI, followed by 2 18 mL (12 5 mmol) of dusopropylethylamme at 0°C After 1 h, add 1.07 g (13 1 mmol) of trtoxane and 1 44 mL (13 1 mmol) of TICI, to this solution, and star the mrxture for an additronal2 5 h at O’C 2. Add 50 mL of aqueous NaCl and 50 mL of water, then extract the resultant mixture wtth 300 mL of EtOAc, and wash the organic layer wrth aqueous NaHCOs, water, and brine, and dry over MgSO+ 3 Remove the solvent zn VUCUO,then chromatograph the residue on srhca gel (1.5 EtOAc/hexane) to give 3.09 g (85%) of the aldol adduct as a white solid. RfO 32 (1:3 EtOAc/ hexane), [a]230 = -54 9” (c 1 02, CHC13); mp 71-72°C (recrystallized from EtOAc and hexane); ‘H (CDC13, 400 MHz) 6 0.92 (d, 3, J= 6 2), 0 93 (d, 3,5= 6 0), 1.361.46 (m, 1), 1 53-l 70 (m, 2), 2.23 (br s, 1, OH), 2.82 (dd, 1, J= 9 5, 13 4), 3.32 (dd, 1, J= 3 4, 13 4,) l), 3.87-3.93 (m, 1), 4.06-4.14 (m, 1)

Welch and Allmendmger

368

TiC14, trloxan.9

lmldazole TBDSMCI,

I-PrgNEt Ph’

Ph’ 16

o$+y DMF

:

OTBDMS

Ph’

HO 17

16 LIBH~

(65%)

EtLF

OTBDMS

+

Eto2c~

OTBDMS

21

PCC,

adza

HO

2 NaH, (COzEt)z, FCHPC02Et

t-

‘r

i

(99%)

OTBDMS

20 123

19

EZ (53%)

(76%) /

N-13, MeOH /

/ 0 l+N+

OTBDMS F

=L

1 LIAIH~ 2 CbzCI.

Et3N

CbzNH

v

OTEDMS F

t 23

22

(67%)

(69%) TBAF

JOfl&X

CbzNH

=

CbzNH

‘OzH F

-‘f-f

OH

‘k F

t 24

25 (91%)

(99%)

Figure 2. 4 184.26 (m, 2), 4 67-4 73 (m, l), 7.25-7 38 (m, 5), 13CNMR (CDCl,, 100 MHz) 6 22 3, 22 8,25 9,37 3,37 8,43 6,55.6,64 3,66 1,127 3,128 9,1294,135 2,153 5,176 1 3.2.3.

(4R)-4-Benzyl-3-{2S)-2-[(tert-Butyldimethylsilyl)

OxyJMethyl-4-Methyl-I-OxopentylJ-2-Oxazolldinone

18

1 To a solutton of 825 mg (2 70 mmol) of the aldol adduct 17 m 8 mL of DMF, add 276 mg (4.05 mmol) of imidazole and 529 mg (3 51 mmol) of tertbutyldimethylstlyl chlortde, and then star the mixture for 1 h at room temperature

Fluoroolefin

lsosteres

369

2. Add 8 mL of 1 NHCl, and then extract the mixture with 30 mL of EtOAc Wash the organic layer with aqueous NaHCO,, water, and brine, and dry over MgSO, 3 Remove of the solvent in vucuo, and then chromatograph the residue on srlica gel (15 EtOAc/hexane) to afford 1.12 g (99%) of the sdyl ether as a colorless 011:R, 0.47 (15 EtOAc/hexane), [a]23,, = 23 3” (c 1 24, CHCl,); ‘H NMR (CDCl,, 400 MHz) 6 0 06 (s, 3), 0.07 (s, 3), 0.89 (s, 9), 0 92(d, 3, J = 6 6), 0 93 (d, 3, J = 6-3), 1 30-l 37 (m, 1), 1 53-l 70 (m, 2), 2 68 (dd, 1, J= 9 9, 13 4), 3 35 (dd, 1, J= 3 2, 134),38l(dd,I,J=51,95),387(dd,l,J=77,95),4.13-425(m,3) 4684.75 (m, 1) 7 25-7 40 (m, 5); t3C NMR (CDCl,, 100 MHz) 6 -5.6, -5 5, 18 2, 22 8, 25 8, 26 1, 37.7, 38 0, 43 7, 55.4, 65 0, 65.8, 127 2, 128.9, 129 4, 135.6, 153.1, 175 5

3.24. (ZR)-2-i[(2ert-Butyldimethylsilyl) Oxy]Methyl}-4-Methylpentanol19 1 To a solution of 1 01 g (2 41 mmol) of oxazolidinone s11yl ether 18, add 52 5 mg (2 41 mmol) of LIBH, at 0°C Star the mtxture for 30 mm at room temperature, and then add 12 mL of 1 N HCI 2 Extract the resultant mixture with 30 mL of EtOAc, and wash the organic layer with aqueous NaHCO,, water, and brme 3 Remove of the solvent rn VCICUO,and then chromatograph the resrdue on sthca gel (1.10 EtOAc/hexane) to give 453 mg (76% yteld) of alcohol 19 as a colorless oil* Rr 0.46 (1.5 EtOAc/hexane), [a]23n = +10 3” (c 1 02, CHCl,), ‘H NMR (CDCl,, 400 MHz) 6 0 08 (s, 6H), 0.89 (d, 6H, J = 6.4), 0 90 (s, 9), 0 95-l 13 (m, 2), 1 55-1.67 (m, l), 1 78-l 88 (m, I), 3 56 (dd, 1, J= 7 97,9 8), 3.60 (dd, 1, J= 7 5, 10 7), 3 72 (dd, 1. J= 3 3, 10 7), 3.80 (dd, I, J= 4 0,9-g), 13C NMR (CDCI,, 100 MHz) 6 -5 7, -5 6, 18 1, 22 77, 22.8 1, 25.4,25.8, 36 9, 39.6, 67 1, 67 7

3.2.5. Ethyl (Z)-(4R)-4-~[~ert-Butyldimethylsilyl)Oxy~methyl}2-Fluoro6-Methyl-2-Heptenoate and the E-Isomer (121-20 and [El-21) 1 To a solution of 13 1 mg (0 53 1 mmol) of alcohol 19 m 2 mL ofCH2C12,add 286 mg (1.33 mmol) of PCC, and then stir the mixture for 3 h at room temperature 2. Remove the chrommm reagent by silica gel chromatography (CH2C12 as an eluant) to give 106 mg of the aldehyde as a colorless 011: Rf 0 62 (1.8 EtOAc/hexane), ‘H NMR (CDCl,, 400 MHz) F 0.05 (s, 6 H), 0 87 (s, 9 H), 0 90 (d, 3, J= 6.2) 0 91 (d, 3, J= 5 S), 1.24-1.35 (m, 1) 152-l 70 (m, 2), 2.46-2.54 (m, 1), 3 77-3.86 (m, 2), 9 69 (d 1, J = 2 8) Thts maternal was used m the subsequent reaction wtthout further charactertzation. 3. To a suspension of 5 1 0 mg (1.27 mmol) of NaH In 2.5 mL of THF, add 0.180 mL (1 33 mmol) of dtethyl oxalate, 5 mL of EtOH, and 5 mL (0 05 mol) of ethyl fluoroacetate Star the mrxture for 15 mln at 45”C, and then add 0 123 mL (1.27 mmol) of ethyl fluoroacetate. Stir the mixture for an addtttonal 1 5 h at 55’C 4 After addition of 106 mg of the aldehyde (0.434 mmol) m 1 mL of THF, heat the reaction mixture under reflux for 16 h

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5 Dilute the reaction mtxture wtth 5 mL of aqueous NH&l and 5 mL of water and extract with 40 mL of EtOAc Wash the orgamc layer with water and brme, and dry over MgSO, 6 Remove the solvent, and then chromatograph the restdue on sthca gel (1 20 ether/ hexane) to give 93.8 mg, of a mixture of the Z- and E-esters (Z)-20 and (Q-21 7 Separate thts mtxture by preparattve TLC (2 mm) to afford 64 6 mg (37% yteld from alcohol 19) of Z olefin (Q-20 and 27.6 mg (16% yteld) of E olefin (Q-21 as colorless oils (Z)-20: R, 0 41 (1 20 ether/hexane)* [c~]*~o = -29 8O (c 1 07, CHCl,), tH NMR (CDCl,, 400 MHz) 6 0 03 (s, 6), 0.86 (d, 3,J= 6 2) 0 880 (s, 9), 0 90 (d, 3, J= 6 6), 1 22-l 40 (m, 2), 1 33 (t, 3, J= 7 2), 1 46-l 60 (m, I), 2 85-2 95 (m, I), 3 52 (dd, 1, J= 5 9, 9 8), 3 57 (dd, 1, J= 5 5, 9.8), 4.22-4 34 (m, 2), 5.97 (dd, 1, J= 10 5, 33 8), 13C NMR (CDCI,, 100 MHz) 6 -5 5, 14.1, 18 2, 21 8, 23 5, 25 77 25 80, 36.4,40.1,61 5,65 8, 122 7 (d, J= 1l), 148 3 (d, J= 255), 160 9 (d, J= 37) (E)-21 Rf 0 5 1 (1:20 ether/hexane)* tH NMR (CDCL,, 400 MHz) 6 0 02 (s, 3), 0 02 (s, 3), 0 87 (d, 3, J= 6 5), 0 87 (s, 9), 0 91 (d, 3, J= 6.6), 1 19 (ddd, 1 J = 5 5,9 0,13 5), 1 30-l 40 (m, l), 1 34 (t, 3, J= 7 2), 1 47-l 60 (m, l), 3 37-3 48 (m, l), 3 51 (ddd, 1, J= l-1,5.6,9 7), 3.57 (dd, 1, J= 5.0,9.7),4.23-4.36 (m, 2), 575(dd, 1,J=109,225),‘3CNMR(CDCL3, lOOMHz)6-5 5,14 1,182,222, 23 3,257,25.8,36.5(d,J=5),408,61 3,66.1 (d,J=3), 125,8(d,J= 17), 147 3 (d, J= 251), 161 0 (d, J= 37)

3.2 6 (Z)-(4R)-4-{[(tert-Butyldimethylsilyl)Oxy]Methy/}-2Nuoro-6-Methyl-2-Heptenamlde 22 Stir a mixture of 32 6 mg (0 0980 mmol) of ester (Z)-20 and 2 mL of 2 N NH, m MeOH for 40 h at room temperature 2 Concentrate the reaction mixture zn vucuo, and then chromatograph the restdue on sthca gel (1 2 EtOAc/hexane) to give 26 5 mg (89% yield) of the amide as a white solid. R, 0 49 (1.2 EtOAc/hexane); [o]230 = -27 4” (c 0 76, CHCI,), IH NMR (CDCL,, 400 MHz) 6 0 027 (s, 3), 0.033 (s, 3), 0 87 (d, 3, J= 6 4), 0 88 (s, 9), 0 89 (d, 3, J = 6 6), 1 18-1.28 (m, l), 1.361 38 (m, 1), 1.461 60 (m, l), 280-292(m,1),350(dd,l,J=64,98),356(dd,l,J=57,98),558(brs,l), 5 96 (dd, 1, J= 10.6, 36.9), 6.10 (br s, l), t3C NMR (CDCL,, 100 MHz) 6-5.4, 182,21.7,235,257,258,364,40.1,659,1194(d,J=12),151 O(d,J=264), 162.1 (d, J= 32).

3.2.7. N -(Benzy/oxycarbonyl)-N -(@]-#RI4-{[(tert-Buty/dimethy/s//y/)Oxy]Methy~2-Fluoro-6-Methyl-2-Hepteny/)-Amine 23 1 To a suspension of 93.4 mg (2 34 mmol) of LtAlH4 (95%) m 5 mL of ether, add a solutton of 284 mg (0 934 mmol) of the amide 22 m 3 mL of ether at 0°C Stir the reaction mixture for 1 h at room temperature, and heat under reflux for 20 mm.

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2. Add 5 mL of water at 0°C to the reaction mixture Filter the mixture through Cehte, and then wash the sohd with ether (3 x 10 mL) Wash the combmed filtrate wtth brine, and dry over MgSO,. 3 Remove the solvent to gave 256 mg of crude amme, which will be used m the next step without further purtficatton 4 To a solutton of this amme m 4 mL of THF, add 0 391 mL (2 80 n-n-1101) of trtethylamme and 0 266 mL (1 87 mmol) of benzyl chloroformate at O’C Stn the mixture for 1 h at 0°C and then pour mto 5 mL of 1 NHCl Extract the mrxture with 30 mL of EtOAc, wash with aqueous NaHCO,, water, and brine, and dry over MgSO, 5 Remove the solvent In vacua, and then chromatograph the residue on silica gel (1.100 CH,CN/CH,Cl,) to afford 265 mg (67% from the amide) of the desired carbobenzyloxyamme 23 and 12 4 mg (3 3% from the amide) of defluormated maternal as colorless oils 23: R,O 58 (l.lOOCH,CNICH,Cl,), [a]230=-21.1”(c 102,CHCl,), ‘HNMR (CDCl,, 400 MHz) 6 0 02 (s, 6), 0 85 (d, 3,5= 6.5), 0 8762 (d, 3, .I= 6 l), 0 8763 (s, 9), 1.06-l 16(m, l), 123-1.32(m, 1). 1.45-1 58 (m, 1),2.67-2 78(m, l), 3 45 (d, 2, .I= 5 9). 3.89 (dd, 2, J= 5 9, 14 9), 4 59 (dd, l,J= 10 2,37 2), 4.92 (br s, l), 5 12 (s, 2), 7 3Ck7 40 (m, 5). r3C NMR (CDC13, 100 MHz) 6 -5 4, 18 3, 21 8, 23 6,25 6,25 9,35 4,40 7,41 9 (d, J= 33), 66 4,67.0, 109 9 (d, J= 14), 128 1, 128 2, 128.5,136 4, 155 7 (d, J= 254), 156 0. Desfluoro-23 R, 0 53 (1 100 CH,CN/CH,Cl,); ‘H NMR (CDCl,, 400 MHz) 6 002(s,6),083(d,3,J=6.5),085-090(m,12),1.06-1 16(m,1),1 18-128(m,l), 1.46-l 58 (m, l), 2.182 29 (m, l), 3.44 (d, 2, J= 6 2), 3 72-3.82 (m, 2), 4 66-4 74 (m, l), 5 11 (s, 2), 5 39 (dd, l,J8 4, 15.5), 5 49 (dt, 1, J= 5 8, 15 5) 7 287 38 (m, 5) 3.28.

N -(/3enzy/oxycatwty/)-N-[(i!)-(4R)-P-Fhoro-

4-(Hydroxymethyl)-6-Methyl-2-Heptenyl]Amme

24

1 To a solution of 2 01 g (4 74 mmol) of stlyl ether 23 m 25 mL of THF, add 2 48 g (9 48 mmol) of n-Bu,NF at 0°C Stir for 2 h at room temperature, then pour the reaction mixture into 30 mL of water, and extract with 120 mL of EtOAc 2. Wash the organic layer wtth brine, and dry over MgSO, Remove the solvent in vucuo, and then purify the residue by chromatography on stltca gel (1 2-2 3 EtOAc/hexane) to give 1 45 g (99%) of the deprotected alcohol as a colorless 011 Rf 0 38 (1 1 EtOAc/hexane), [a]23r, = +14.5’ (c 1 08, CHCl,), ‘H NMR (CDCl,, 400 MHz) 6 0 86 (d, 3, J=6 6), 0 89 (d, 3, J= 6 6), 1.1&l 24 (m, 2), 1 47-l 59 (m, l), 2 75-2 87 (m, I), 3 38 (dd, 1, J = 7 8, 10 5), 3.55 (dd, 1, J = 5 1, 10 5), 3 89 (dd, 2, J= 5 2, 14 2), 4.57 (dd, 1, J= 10 1, 36 9), 5.02-5 16 (m, 3), 7 307 40(m, 5), t3CNMR(CDC13, lOOMHz)G21.7,23 4,25.6,35 6,40 4,41 9(d, J=33), 66 3,67 0, 109 3 (d, J= 13), 128 1, 128 2,128 5,136 3,156.2, 156.7 (d, J= 255)

3.2.9. (Z) -(2R) -5-/N -(Benzyloxycarbonyl)Amino]4-Fluoro-2(2-Methylpropyl)3-Pentenoic Acid 25 1. To a solution of 224 mg (0 724 mmol) of the alcohol 24 m 9 mL of acetone, add 0.9 mL of Jones reagent at 0°C. Stir the reaction mtxture for 1 h at room temperature,

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2 Add isopropyl alcohol (0.4 mL) and 10 mL of water, and then extract the mixture with 40 mL of EtOAc 3. Wash the organic layer wrth water and brine, and dry over MgSO,. 4. Remove the solvent, and then chromatograph the residue on sthca gel (1 2 EtOAc/ hexane-water saturated EtOAc) to give 214 mg (91% yield) of carboxyltc acid 25 as a colorless 011 [t~]*~n = +39 8” (c 1.02, CHCl,), ‘H NMR (CDCL3, 400 MHz) 6 0 90 (d, 3, J= 6.3), 0 93 (d, 3, J= 6 3), 1 34-l 46 (m, I), 1 5&l 68 (m, 2), 3 57 (dt, 1, J= 6 7, 9 5), 3 93 (dd, 2, J= 6 1, 13 4), 4 88 (dd, 1, J= 9.5, 35 2), 4.9& 5 05 (m, I), 5 13 (s, 2), 7 32-7.42 (m, 5), 13C NMR (CDCL3, 100 MHz) F 21 8, 22 7,25 7,38.8 (d, J= 3), 41 47,41 51 (d, J= 32), 67 1, 105 4, (d, J= 12), 128 1, 128 2, 128 5, 136 2, 156.1, 156 6 (d, J= 259), 179 3

3.3. N-tert-Butyloxycarbonyl-]-(o,L)-A/a-~[CF=C]-(D,L)Pro Dipeptides 3.3.7. tert-Butyl a-Fluoroacetate 26 1 To a mixture of acetamtde (44.26 g, 750 mmol, recrystallized from MeOH/EtOH and dried) and potassium fluoride (43 58, 750 mmol, dried under vacuum using an Abderhalden apparatus at 110°C for 24 h), add tert-butyl a-chloroacetate (46.58 g, 300 mmol) 2. Heat the mixture to 9O”C, and stir well for at least 12 h to ensure complete reaction, then allow to cool to room temperature 3 Distill the reaction mixture under reduced pressure (71-72”C/145 mmHg) to give pure 3 as colorless llqutd (32 25 g, 80%) 4 The further purtfkatton by the second dtsttllatton (13 l-l 33”C/760 mmHg) removes trace amounts of starting material 2, bp 13 l-133°C ( Ltt 133-135”C, ‘H NMR (CDCl,) 6 4 68 (d, J= 48 4 Hz, 2H), 1 48 (s, 9H) 19FNMR (CDCl,) 6 -228 28 (t, J = 48.8 Hz)

3.3.2. tert-Butyl a-Fluoro-a-Trimethyls~lylacetate

27

1 To a solutton of dusopropylamme (33 4 mL, 240 mmol) m THF (120 mL), add slowly n-butylhthmm (95 4 mL, 240 mmol, 2 5 Msolutton in hexanes) at -30°C 2 Stir the solutton for 15 mm at -30°C and then transfer vta canula mto the prevtously prepared solutron of tert-butyl fluoroacetate 26 (8 00 g, 60 mmol) and chlorotrtmethylstlane (45 7 mL, 360 mmol) m 320 mL of THF and 400 mL of pentane at -78’C 3 Maintain the temperature between-78 and-80°C during the transfer Then warm to 0°C over a period of 3 h 4. Quench the mixture immedtately with NaHC03 (120 mL) at 0°C 5 Extract the separated aqueous layer with ether (3 x 120 mL) Dry the combined organic layers with MgS04, filter, and concentrate to a volume of 200 mL Analysts of the t9F NMR mdtcated a mtxture of C- and O-bzs-stlylated enol ether and monosilylated a-fluoro ester 27 6 Hydrolyze this mixture with saturated tartartc acid aqueous solutton (200 mL) at room temperature overnight to yield 7 33 g (71%) of tert-butyl a-fluoro-a-

fluoroolefin

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(trtmethylstlyl)acetate 27 after dtsttllatton. bp 62-64”C (30 mm Hg); ‘H NMR (CDCls) 6 4.5 (d, J = 47 9 Hz, 1H), 1 4 (s, 9H), 0.1 (m, 9H,) ‘)C NMR (CDC13) 61700(d,J=192Hz),87.8(d,J=181.2Hz),817,283,29,11,-37 19F NMR (CDC13) 8 -225 7 (d, J = 47 5 Hz)

3.3.3 Peterson Olefination of TBDMS-Protected (Z)-(I-fluoro-2carboxy-tert -b&y/)-2-[2 -tert -butyld/methy/sj/y/joxymethy/ cyclopentone 28 and (E)-l-(1 -fhoro-2-carboxy-tert-butyl)-2-[2-tert-butyl dime thylsi/yl]oxyme thy/ cyclopen tone 29 1 To a solutton of dusopropylamme (2 4 mL, 17 mmol) III THF (105 mL), add dropw’se n-butylhthmm (6 9 mL, 17 mmol, 2.5 A4 solutton m hexane) at -25°C 2 Stir the solutton for 15 mm at -30°C then cooled to -95’C Add tert-butyl ofluoro-a-trtmethylsdyl acetate 27 (3 10 g, 15 0 mmol ) dissolved m THF (10 mL) to LDA solutton, and star for 40 mm at -95°C Follow by addition of 2hydroxymethylcyclopentanone derivative (3 77 g, 16 5 mmol) m THF (10 mL) 3. Stir the reaction mixture quenched with saturated NH&l (25 mL) at 0°C Extract the separated aqueous layer wtth hexanes (3 x 100 mL). Dry the combmed orgamc layers with MgS04, filter and evaporate. 4 Purify the crude product by column chromatography (hexane/CH$&, 6:4) to give 2 18 g of the (Z)-fluorooletin, 28, and 1 80 g of the (E)-fluoroolefin, 29 (overall yield* 78%) TLC (50% CH,Cl, in hexanes Rr 0.46, 28, Rr 0 52, 29) (Z)-isomer, 28 ‘H NMR (CDCl,) 6 3.73 (dd, .I= 9 6,4 2 Hz, lH, ), 3.46 (t, J = 8.7 Hz, lH), 3 09 (m, lH), 2 70-2 54 (m, 2H), 1 86-l 65 (m, 4H), 1 50 (s, 9H), 0.86 (s, 9H), 0 02 (s, 3H), 0 01 (s, 3H,) 13CNMR (CDCI,) 6 160 4 (d, J= 35 0 Hz), 142 9 (d, J= 246 3 Hz), 139.5 (d, J= 13 6 Hz), 81 9, 62 9 (d, J= 4 0 Hz), 45 9, 3 1 1 (d, J= 1 7 Hz), 28 5,28 I, 25 8,24 6, 18.2, -5 4, -5 5 19FNMR (CDCl,) 6 -125.43 (s) (Q-isomer, 29, ‘H NMR (CDCl,) 6 3 62 (dd, J= 9 3,3 9 Hz, lH), 3.45 (t, J= 8.7 Hz, lH), 3 35 (m, IH), 2 51-2 42 (m, 2H, ring), 1.74-1.64 (m, 4H), 1 52 (s, 9H), 0 87 (s, 9H), 0.02 (s, 3H), 0 01 (s, 3H). ‘)C NMR (CDC13) 6 159.88 (d, J= 35 6 Hz), 142 9 (d, J= 245 9 Hz), 139 9 (d, J= 14 8 Hz), 82 0,64 0 (d, J= 4 5 Hz), 44 6 (d, J = 1 7 Hz,) 30 1, 29 5, 28 0, 25 8, 22 6, 18.2, -5.4, -5 5 19F NMR (CDC13)G -120 88 (s)

3.3.4. I-(I’-Fluoro-2’-Oxoethylidene)2-(tert-Butyldimethyls~lyloxymethyl)Cyclopentanes

30 and 31

1 Dtssolve tert-butyl ester 28 (2 00 g, 5.80 mmol) m dry drethyl ether (21 mL) Cool the resultmg solutron to -78°C and add ditsobutylalummum hydnde (1.3 mL, 7.3 mmol) dropwtse as 1 A4 solutton in pentane (7 3 mL) 2 Stir the mtxture at -78°C for 1 h, and then quench with Hz0 (2 mL) 3. Warm the reactton mtxture to room temperature, and star for an additronal 1 h, Filter the whtte solids, and wash thoroughly with ether Dry the filtrate over MgS04, and concentrate UI vacua. 4 Chromatograph the hght yellow restdue accordmg to the condmons of St111usmg hexane/EtOAc (9 1) as eluent to provide the aldehyde 30 (1 47 g, 93%) ‘H NMR

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(CDCl,) 6 9 57 (d, .I= 16.4 Hz, lH), 3 74 (dd,J= 9.7,4 0 Hz, lH), 9 7,6 8 Hz, lH), 3 20-3 10 (m, lH), 2 84-2.60 (m, 2H), 2 30-l 65 (s, 9H), 0.01 (s, 6H). 13C NMR (CDCl,) 6 182.26, (d, J= 29 1 Hz), 248 8 Hz), 147 58 (d, J = 11 2 Hz), 63.16 (d, J = 3 9 Hz), 45 82, 25.81,25 16, 1821,-5.49,-5 56 ‘9FNMR(CDC13)6-133.69(d,J=

3.64 (dd, J= (m, 4H), 0 84 149 86 (d, J= 29 05, 28 29, 162Hz)

Except when using 1.5 Eq of dirsobutylalummum hydrrde, the (IZ) aldehyde 31 can be prepared m the same manner in 78% yield ‘H NMR (CDCl,) 6 9.53 (d, J= 19 0 Hz, lH,), 3 59 (br. dd, J= 9.7,6 0 Hz, lH), 3.38 (t, J= 9.3 Hz, lH), 3.28 (br q, J= 7.4 Hz, lH), 2.64-2.58 (m, 2H), 1.901 60 (m, 4H), 0.83 (s, 9H), 0 02 (s, 6H) 13C NMR (CDCl,) 6 182.76 (d, J = 24.7 Hz), 15 1.21 (d, J = 250.2 Hz), 146.88 (d, J= 13.2 Hz), 64.80 (d,J= 3.5 Hz,), 42.77 (d, J= 5.4 Hz), 29.54,29.10,25.78,25.72, 18.23,-5.47,-5.56. 19F NMR (CDCl,) s-129.40 (d, J=

19.0 Hz).

3.3.5. I -[I ‘-Fluoro-(Z’-Hydroxy-Z-Methyl)Ethylidene]2-(tert-Butyldimethyls~lyloxymethyl) Cyclopentanes 32 and 33 1 Add methylhthmm (3 1 mL, 4.6 mmol, 1.5 Msolutton in dtethyl ether) dropwtse to a solution of (I$)-aldehyde, 31 (0 70 g, 2.6 mmol) In THF (66 mL) at -78°C under N2 2 Star the reactton mtxture at -78°C for 90 mm Remove the coohng bath, then strr the mtxture at room temperature for another hour, then retool to O”C, and quench with 30 mL of HZ0 3 To the quenched mrxture, add 150 mL of CH,C& Extract the separated aqueous layer wtth CH$& (2 x 60 mL) Dry the combined organic layers over MgS04, filter and concentrate t9F NMR analysts of the crude product show a 3 2.1 ratio of dtastereomers, which may be separated by column chromatography with hexane/EtOAc (9.1) to give 0.47 g of the major dtastereomer, 33a, and 0 15 g of the minor isomer, 33b (total yteld 83%)

MaJor dlastereomer 33a* ‘H NMR (CDCl,) S 4 44 (dq, J = 24 2 Hz, lH), 3.51 (ddd,J=10.0,5.8, 1.8Hz, lH),3.39(t,J=9.7Hz, lH),2.83(brq,J=7.2Hz, lH), 2.47-2.27 (m, 2H), 1.81-1.45 (m, 4H), 1.31 (d, J= 6.0 Hz, 3H), 0 88 (s, 9H), 0.07 (s, 6H). 13C NMR (CDCl,) 6 155.36 (d, J= 253.0 Hz), 121.18 (d, J= 17.0 Hz), 66 24 (d, J= 3.0 Hz), 64.62 (d, J= 30.4 Hz), 42.61 (d, J= 5 3 Hz), 29.83,26.76 (d, J= 3.6 Hz), 26.08,23 31, 18.68, 18.58 (d, J= 4.2 Hz),-5 53, -5 56 19F NMR (CDCl,) 6 -123.40 (d, J = 24 4 Hz). Mmor dtastereomer 33b: ‘H NMR (CDCl,) 6 4.48 (br sext., J= 6.8 Hz, 1 H), 371(dd,J=99,5.4Hz,lH),3.55(t,J=99Hz,lH),311(brq,J=74Hz, IH), 2.82 (br s, lH), 2.50-2.20 (m, 2H,), 2.16-1.50 (m, 4H), 1.34 (d, J= 6.5 Hz, 3H), 0 88 (s, 9H), 0.04 (s, 6H). 13C NMR (CDC13) 6 154.97 (d, J = 247.2 Hz), 119.41 (d, J= 16.8 Hz), 65.70 (d, J= 3.7 Hz), 65.45 (d, J= 35.0 Hz), 41.53 (d, J= 5.2 Hz), 29.61,27.01 (d,J=3.6Hz),25.93,20.70, 18.40,-5.51,-5.57.‘9F NMR (CDCl,) 6 -119.90 (d, J= 14.7 Hz).

Nuoroolefrn

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In the same method, 32 can be prepared m 80% yield as colorless 011. The major Isomer, 32a: ‘H NMR (CDCI,) 6 4.47 (dq, J = 26.0, 6.6 Hz, IH), 3.74 (dd, J= 9.8, 4.4 Hz, lH), 3.37 (t, J= 9.6 Hz, lH, ), 3.00-2.85 (m, lH), 2 402.10 (m, 2H), 1.90-1.50 (m, 4H), 1.34 (d, J= 6.6 Hz, 3H), 0.87 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H,). 13C NMR (CDCl,) 6 153.30 (d, J= 249.2 Hz), 120.70 (d, J = 15.4Hz),65.33(d,J=29.5Hz),63.85(d,J=3.1 Hz),43.50,29.18,2820(d, J= 4.9 Hz), 25.95,24 55, 19.87 (d, J= 2.2 Hz),-5 45, -5 5 1 19F NMR (CDC13) 6 -132.05 (d, J = 25.8 Hz).

3.3.6. N -Phthalimide- I-[( 1‘-Fluoro-2’-Amino) Propylidene2-(tert-f3uty/dimethy/si/y/oxy)Methy/] Cyclopentanes 34 and 35 1 Treat the major isomer, 33a (0 61 g, 2.1 mmol), triphenylphosphme (0 72 g, 2 7 mmol) and phthahmlde (0 41 g, 2 7 mmol) m THF (20 mL ) dropwlse with dlethylazodicarboxylate (0 50 g, 2 73 mmol) as a 2-mL solution m THF at room temperature 2 After stirring at ambient temperature for 4 d, evaporate the solvent zn wcuo 3 Dissolve the syrupy residue m a mmlmum amount of CH& and transfer to a column Elutlon with hexane/EtOAc (9 I) yields 0 427 g (49%) of 35a as yellow oil ‘H NMR (CDCl,) 6 7 81 (dd, J= 5 4,2.9 Hz, 2H), 7 68 (dd, J= 5 4, 3.1 Hz, 2H),537(dq,J=299,74Hz,lH),349(dd,J=74,24Hz,2H),3 lO--300(m, lH), 2 35-2 30 (m, 2H), 1.74 (d, J= 7 4 Hz, 3H), 1 7&l 52 (m, 4H), 0 91 (s, 9H), 0 09 (s, 3H), 0 08 (s, 3H,). 19F NMR (CDCl,) 6-l 18.40 (d, J = 30 2 Hz)

(.Z)-isomer, 34 may be prepared m the same manner m 29%. ‘H NMR (CDQ) d 7.84-7.65 (m, 8H), 5 27-5.09 (m, 2H), 3 70 (dd, J = 9.9, 4.2 Hz, IH), 3 66 (dd, J= 9.6,3.9 Hz, IH), 3.43 (t, J= 9.2 Hz, lH), 3.34 (dd, J= 9.0 Hz, lH), 3.00-2.85 (m, 2H), 2.38-2.27 (m, 4H), 1.67 (d, J= 6.3 Hz, 3H), 1.65 (d, J = 5.7 Hz, 3H), 1.79-1.53 (m, 8H), 0.86 (s, 9H), 0.76 (s, 9H), 0.01 (s, 6H,). 19F NMR (CDCl,) d -119.14 (d, J = 24.4 Hz), -120.09 (d, J = 24.4 Hz).

3.3.7. 1-[( 7‘- Fluoro-2’-Amino) Propylidene2-(t -Butyldimethylsilyloxy)Methyl]Cyclopentanes

36 and 37

1 Method A (Fig. 3). Add methylhydrazine (92 mg, 2 0 mmol) dropwlse to a solution of 35 (84 0 mg, 0 20 mmol) m 4 mL of freshly distilled CH& 2 Stir the reactlon mixture at room temperature, and momtor by TLC. After 48 h or when TLC analysis mdlcates that the reactlon 1scomplete, evaporate the solvent, and then treat the residue with 5 mL of EtOAc 3 Filter the white sohds, and wash with EtOAc Concentration of the filtrate will give a crude amme product, which can be purified by a column chromatography (4% MeOH in CH&l,), affordmg free amine 37 (49 mg, 86%). ‘H NMR (CDC13) 6 3 39-3.28 (m, 3H), 2.77-2.69 (m, lH), 2.362.23 (m, 2H), 1.82-1 58 (m, 6H), 1 28 (d, J= 6.5 Hz, 3H), 0.86 (s, 9H), 0.01 (s, 6H). 13C NMR (CDC&) 6 156 29 (d, J=248 5Hz), 118 81 (d,J= 14 7Hz),65 29(d, J=4 1 Hz),46 07 (d,J=25.8

376

Welch and Allmendinger

SIMe3 26 (80 %)

~TBDMS 30 ( 93 % )

30+31

Z’

c-y+

27 (71%)

OTBDMS

OTBDMS

;J;;;

OTBDMS

&BDMs 28 (43 % )

31 (78%)

t-j=&6

OTBDMS

F

OTBDMS 34 (2) (29%) 35 (E) (49 %)

32 (2) (80%) 33 (E) (63%)

29 (35 % )

‘;“;“2 OTBDMS 36 (2) (72 %) 37 (E) (66%)

0 LHMDS ether,

-30 “C

CH 3LI ether,

36

-76 ‘X

OTBDMS 30

Figure 3 Hz),43 lO(d,J=53Hz),2941,27 13,2708,2596,23 19FNMR (CDC13) 6 -129 09 (d, J = 24 4 Hz)

28, 18.41,-5 32,-5 38

(z)-isomer 36 may be prepared by followmg the same procedure m 72% yield 1 Method B (Fig. 3) Slowly add a hexane solutton of n-butylhthmm (1 5 mL, 3 8 mmol, 2 5 Msolutron m hexane) to a solutton of l,l, 1,3,3,3-hexamethyldtsdazane (0 67 g, 4 2 mmol) m dtethyl ether (32 mL) cooled m an me-water bath Remove the coolmg bath, and stir the solutton at room temperature for 30 mm 2. Cool the mixture to -30°C and to this hthmm bu(trtmethylstlyl)amtde solutton add a solutton of 0.87 g ( 3 2 rnmol) of the aldehyde 30 m 8 mL of ether Stir the mixture at -30°C for 1 h, and then cool to -78’C 3 Treat the resultmg solution contammg N-trrmethylsilyl tmine with methylhthmm (4 3 mL, 6.40 mmol, 1.5 M solutron m ether) at -78°C Stir the mixture at -78°C for 1 h and then at room temperature for an addmonal 2 h

Fluoroolefin

377

lsosteres

e -

w

F

Eoc-ON,

TEA

-

dioxane

NHBoc F

-e

OTBDMS

OTBDMS

36

36 l 39 77% from 26a

: 1 AcOHiH 38

20/THF

2 Separation by column chromatography Total yield

-

NHBoc

+

($

NHBoc

F -7 HO

Hd I -4Oa

94 %

u -4Ob

: NHBoc

u-40b

Jones

&

NHBoc % i-OF

HO

HO

u -42b

I -42a

Figure 4 4 Cool the solution to 0°C agam, quench with 32 mL of saturated aqueous NH,Cl, and extract with CH,C12 (4 x 50 mL) Dry the combined extracts (MgS04), and concentrate in vacua. 5 To remove some nonpolar impunties, subject the resrdue to a purification on a very short silica column using hexane/CH,Cl, (1’ 1) as the eluent to give a 1.3 1 ratio of dlastereomers, 36 (0 86 g, 93%). The mixture of dlastereomers can be carried to the next step without lsolatlon ‘H NMR (CDC13) F 3 73-3 56 (m, 2H), 3.37 and 3 33 (t, J= 9 4 and 9 6 Hz, lH, ), 2 95-2 82 (m, lH), 2 78-2.10 (m, 2H), 1 80-l 47 (m, 6H), 1.21 and 1 20 (d, J= 6 7,6 7 Hz, 3H), 0 86 and 0.85 (s, 9H), 0 010 (s, 6H) 13C NMR (CDC13) 6 155 51 (d, J= 249 5 Hz), 155 33 (d, J= 248 7 Hz), 117.85 (d, J= 15 9 Hz), 11774(d,J=159Hz),6400(d,J=3.4Hz),6396(d,J=34Hz),4658(d,J= 28.5 Hz), 46 47 (d, J= 28 5 Hz), 43.27,29.27,28 26 (dd, J= 5 5,4 4 Hz), 28 19, 28 13, 25 90, 24 82, 24 58, 20.56 20.12, 18 31, 18 28, -5 31, -5 36, -5 53 19F NMR (CDC13) 6 -132 08 (d, J = 25.8 Hz), -132 08 (d, J = 27 1 Hz) (E)-isomer 37 was prepared by method B m 67% yield.

3 3.8. N -t -Butyloxycarbonyl- 1-[( I’- Fluoro-2’-Amino) Propylidene2-(t-Buty/dimethyh/y/oxy) Methyl] Cyclopentanes 38 and 39 1. To a solution of amme 36 (0 86 g, 3 0 mmol) in dloxane (60 mL), add tnethylamme (0 62 mL, 4 5 mmol) and 2-(t-butoxycarbonyloxylmmo)-2-phenyl acetonitrile (Boc-ON) (0.88 g, 3.6 mmol) (see Fig. 4) 2 Stir the mixture for 20 h at room temperature, and then evaporate the solvent Elute the crude product by a careful column chromatography with hexane/EtOAc (20 1) yield 0.88 g of 38 (77% from aldehyde 30)

378

Welch and Allmend/nger

‘H NMR (CDCI,) 6 4 76 (br s, IH), 4.47 (br d, J = 24 7 Hz, lH), 3 68 and 3.63 (dd, J= 8.5,4 5 Hz and 9.5,4.5 Hz, lH), 3 38 and 3.32 (t, J= 8.3, 9.6 Hz, lH), 2.89 (m, IH), 2.38 (m, lH), 2.16 (m, lH), 1.x5-1.53 (m, 4H), 1.40 (s, 9H), 1.23and 1.22(d,J=69,6.9Hz,3H),0.86andO 85(s,9H),O,OOl (s,6H). 13C NMR (CDCl,) 6 154.80, 154.77, 153.13 (d, J= 248.2 Hz), 119.68 (br s), 79.37,63.90 (d, J= 3.6 Hz), 63.78 (d, J= 3 3 Hz), 45.76 (d, J= 28 2 Hz), 45.57 (d,J=26.0Hz),43 39,43.26,29.31,28.36(]25.91,25 88,24.79,24.44, 18.75, 18.38, 18.29, 1826,-5.37,-5.41,-5.45. ‘9FNMR(CDCl,)6-130.09(d,J= 24 4 Hz), -130.78 (d, J= 24.4 Hz). (E)-isomer 39 can be prepared by followmg the same procedure in 72% yield. The data for the more polar dlastereomer 39b. ‘H NMR (CD(&) 6 4.76 (br s, IH), 4.53 (dq, J= 30.1, 5 9 Hz, lH), 3.42-3.32 (m, 2H), 2.56 (br s, lH), 2.40-2.17 (m, lH), 1 77-1.56 (m, 4H), 1.40 (s, 9H), 1.26 (d, J= 6 9 Hz, 3H), 0.85 (s, 9H), 0.010 (s, 6H). 13C NMR (CDC13) 6 154.70, 152.57 (d, J= 247.0 Hz), 120.33 (d, J= 15.5 Hz), 79.27, 63 31 (d, J= 3.0 Hz), 45 48 (d, J= 26 2 Hz), 42.84 (d, J= 3 7 Hz), 29 39,28.37,27.08 (d, J= 3.0 Hz), 25.91,23.45, 19.25, 18 40, -5.37, -5.45. 19F NMR (CDC13) 6 -127.51 (d, J= 32.6 Hz). The data for the dlastereomer 39a: ‘H NMR (CDCl,) 6 4 95 (br d, J = 30 1, 5.9Hz, lH),45O(dq,J=27.1,77Hz, lH),3.62(brs, lH),3.38-3.26(m,2H), 2 78-2 67 (m, lH), 2.34-2.21 (m, 2H), 1.55-1 47 (m, 3H), 1.40 (s, 9H), 1.24 (d, J = 6 9 Hz, 3H), 0.85 (s, 9H), 0.03-0.01 (m, 6H). 13C NMR (CDCI,) 6 154.57, 152.57 (d, J= 246.9 Hz), 119.93 (d, J= 16.1 Hz), 79.26, 64.49 (d, J= 4.0 Hz), 45.73 (d, J= 28.0 Hz), 42.74 (d, J= 4.5 Hz), 29.32,28 36, 27.36 (d, J = 3.0 Hz), 25.95, 23 21, 19.25, 18.32, -5 37, -5.52, 19F NMR (CDC13) 6 123 74 (d, J = 29.9 Hz).

3.3.9. (Z) -N -t -Bu tyloxycarbonyl- I-[( 1‘-Fluoro-2’-Am/no) Propylldenef2-(Hydroxy)Methyl Cyclopentanes 40 1 Stir a solution of 38 (0 77 g, 2 0 mmol) m AcOH/H&I/THF (100 mL, 13.7 3) for 16 h at room temperature 2 Remove the solvent under vacuum 3 Treat the yellow hqu’d res’due with solld NaHCO, until the mixture 1s slightly basic, and then add HZ0 (8 mL) 4 Extract the mixture with EtOAc (4 x 40 mL) Dry the combmed organ’c layers (MgS04) and concentrate The residue can be purified by chromatography (hexane/EtOAc, 4.1) to provide 0 28 g of one d’astereomer 40a (5 1%) and 0 23 g of the other isomer, 40b (43%) (2) isomer 40a: ‘H NMR (CDCl,) 6 4.74 (br s, lH, ), 4.38 (br d, J = 28 5 Hz, lH), 3.57 (d, J= 4.5 Hz, 2H), 2.97-2.88 (m, IH), 2.4S2.35 (m, IH), 2.282.13 (m, lH), 1.92-1.51 (m, 5H), 1.40 (s, 9H), 1.24 (cl, J= 7.0 Hz, 3H). 13C NMR (CDCl,) 6 154.97, 151.99 (d, J= 249.3 Hz,), 119.17 (d, J= 14.7 Hz),

Fluoroolefin lsosteres

379

79.64, 64.13 (d, J= 3.8 Hz, ), 46.05 (d, J= 26.8 Hz, ), 43.45, 29.64, 28.41, 28.31,24.85, 17.90. 19F NMR (CDC13) 6 -130 50 (d, J= 28 5 Hz) Data for 40b. ‘H NMR (CDCl,) 6 4.80 (br s, lH), 4.45 (br d, J = 27.0 Hz, lH), 3.62 (dd, J= 10.5,5.1 Hz, IH), 3.44 (t, J= 9.2 Hz, lH), 2.93 (m, lH), 2.43 (m, lH), 2.19 (m, lH), 1.87 (m, lH), 1.77-1.52 (m, 4H), 1.39 (s, 9H), 1.23 (d, J= 7 0 Hz, 3H). 13C NMR (CDC13) 6 154.82, 152.67 (d, J= 248.1 Hz), I 19.32 (d, J= 14.1 Hz), 79.50, 64.18 (d, J= 3.7 Hz), 46.62 (d, J= 29.5 Hz), 43 46, 29.39,28.32,28.13 (d, J= 5.3 Hz), 24.77, 18.63. 19F NMR (CDCI,) 6-129 28 (d, J= 29 8 Hz). Elemental microanalysis calculated for C’4H24FN03. C, 61 52, H, 8.85. Found* C, 6141; H, 8 99 (Q-Isomer 41a can be prepared by follow’ng the same procedure m 74% yield (mp: 105-108°C). The data for the more polar d’astereomer 41a: ‘H NMR (CDC13) 6 4.79 (d, J= 7.1 Hz, lH), 4.59 (dq, J= 28.0, 7.4 Hz, lH), 4 22 (br t, J= 7.0 Hz, lH), 3 50 (t, J= 6 5 Hz, 2H), 2.41-2.26 (m, 2H), 1.76-1.52 (m, 3H), 1 38 (s, 9H), 1.23 (d, J= 7.0 Hz, 3H). 13C NMR (CDCl,) 6 155 44, 15 1.97 (d, J = 248 2 Hz), 120.71 (d, J = 16.2 Hz), 80.07, 65.50, 45 54 (d, J = 27.4 Hz), 43.12 (d, J= 5.1 Hz), 3047, 28.28, 27.05 (d, J= 4.2 Hz), 23.54, 18.67 19F NMR (CDCl,) 6 -125.94 (d, J = 29.9 Hz).

3 3 10. (Z)-N-t-Butyloxycarbonyl- I-](1 ‘-Fluoro-2’-Ammo) Propylldene]-2-Cyclopentane Carboxylic Acid 42a and 42b 1 Add Jones reagent (0 30 mL, 2 8 mmol) dropwlse

to a solution of alcohol 40a

(0 15 g, 0 56 mmol) m dry acetone (9 mL) at 0°C

2 Stir the react’on m’xtnre for 1 h at O”C, then quench with H20 (14 mL), and extract with EtOAc (3 x 30 mL) Dry the extracts over MgS04, filter, and concentrate ln vacua Column chromatography (hexane/EtOAc, 3.2) yielded the carboxyhc ac’d 42a (0 12 g, 73%) as a white solid mp 122-126°C ‘H NMR (CDC&) 6 4 83 (br s, 1H), 4 47 (br d, J= 28 9 Hz, 1H), 3 48 (br s, 1H), 2 62-2.40 (m, 1H), 2 32-2 17 (m, 1H), 2 0 l-l 84 (m, 4H), 141 (s, 9H), 1 25 (d, J= 6 9 Hz, 3H) 13CNMR(CDC1,) 6 179 34,154 99,153 16 (d,J=2545Hz), 11779(d,J= 139Hz),7969,4559(d,J=30OHz),4545,3165, 28 28,28 09 (d, J= 3.8 Hz), 25 74,18 00 19FNMR (CDCl,) 6-123 26 (d, J= 25 8 Hz)

The other d’astereomer 42b can be prepared m the same manner m 74% yield Mp 9GlO3”C ‘H NMR (CDC13) 64 76 (br s, IH), 4.49 (br d,J= 27.1 Hz, lH), 3 52 (m, lH), 2 60-2 47 (m, lH), 2 41-2 26 (m, IH), 2 O&l 81 (m, 4H), 141 (s, 9H), 1 27 (d, J = 7 0 Hz, 3H) 13C NMR (CDC13) 6 179 68, 154 91, 153 20 (d, J = 251 4 Hz), 117.53(d,J= 15.3 Hz),7964,45 59(d,J=30OHz),4522,31 61,28 35, 28 OO(d, J= 3 9 Hz), 25 76, 18 12 19FNMR (CDCI,) 6 -124 90 (d, J= 25 8 Hz)

3.4. Preparation of N-Trichloroacetyl-(Z)-(o,L)Phe-v[CF=CH]-G/y Dipeptides 3 4.1. 2-Fluoro-4-Phenyl-2-(Z)-Butenal45 1 The enol ether 43 was prepared m 8&96% (1975) Tetrahedron Lett 3833

yield accordmg to

J

F

Normant

Welch and Allmendinger

380 OEt

o^“““’

dOEI

0~

‘2-IpClF KOH. H 20, 18-C-6

43

U-13(CH2),,0S03Nz

44

(84-96%)

(66%)

1 NaH 2 ccl&N

H,$04/

o+

/

o,, CCI M-i 3

493

CH ,OH

CCI

45

3

(64%)

&OH

C*3/H2S04

&C02H

cc13

50 (100%)

cc13

51 (63%)

Figure 5

2. To a stirred solution of 40 g of the diethyl ketal of acrolem, 55 g KOH, 45 g water and 1 5 g 18-C-6 at -15 to -5°C add 63.2 g dichlorofluoromethane for 45 mm 3 One hour later, add 250 mL water, and extract the product with ether/pentane Wash the extracts with ammomum chloride (aq ), evaporate, and disttll the product, bp 5MO”C (15 mm) to give 37 3 g (66%) of 44 (see Fig. 5) 4. Heat 37 g of 44 and 150 mL of an 0.03 A4 sodium laurylsulfate solution under reflux for 5 5 h, cool, and extract with hexane 5 Evaporate the extracts, and then distill the residue bp 7&76”C (230 mm) to give 16 2 g (6 1%) of aldehyde 45

3.4.2. tert-Butyl-3-Fluoro-2-Hydroxy-6-Phenyl-4-(Z)-Hexenoate

46

1 To a suspension of 2 g zinc, 0 3 g copper (I) chloride m 15 mL ether (reflux), add a solution of 3 28 g (20 mmol) of 2-fluoro-4-phenyl-2(Z)-butenal and 4.88 g (25 mmol) of tert- butyl bromoacetate m 7 mL of dtethyl ether within 30 mm

Fluoroolefin lsosteres

381

2. After stnrmg for an additional 45 mm, cool the mixture, and treat with hydrochloric acid (40 mL, 0 3 N) and hexane/ethyl acetate (4.1, 30 mL) Filter the biphasic mixture through Cehte, and then separate the phases. Wash the organic phase with 0 1 N HCl, aqueous sodmm bicarbonate, and brine, dried over sodium sulfate and evaporated to dryness. 3 Chromatograph the residue on sthca (100 g), and elute with hexaneiethyl acetate (9.1) to afford 5.3 g (94%) of the desired product.

3.4.3. 4-Fluoro-6-Phenyl-4-(Z)-Hexen-1,3-Diol47 1 Add hydroxyester 46 (2.54 g, 9 1 mmol, solution m 10 mL ether) to hthmm aluminum hydride (0 76 g, 20 mmol) m 10 mL ether at 0°C within 10 mm. 2 Continue to stir for 3 h under reflux. After coolmg, add 3 mL 1 N sodium hydroxide and then stir the mixture for 1 h. 3 After filtering over Celne, evaporate the filtrate, and chromatograph the residue on silica using hexane/ethyl acetate 3.2 to afford 1.16 g (5.5 mmol, 61%) of the dial.

3.4.4. 4- Fluoro-6-Phenyl-53-(Z)-Hexen- I-01 50

Trichloroacetylammo-

1 Treat a solutton of 47 (1 13 g, 5.37 mmol) m THF (7 mL) with 30 mg of sodium hydride (suspenston m 3 mL hexane) at -lO”C, and allow to warm to room temperature 2. Add the mixture obtained to a solutton of 1 56 g (10 8 mmol) trichloroacetomtrtle m 15 mL of ether at -5 to O”C, and stir for 1 h Evaporate the solvent at room temperature, and dilute the residue with pentane (30 mL), treat with acetic acid (40 mg, 1.25 mmol), and filter Evaporate the filtrate to give 1.2 g of 4-fluoro-6-phenyl-4(Z)-hexen1,3-diol-butrichloromethyhmrdate 48 IR (CH,Cl,) 1665 cm-i. Dissolve 48 m xylene (30 mL), and heat under reflux for 3 h. Evaporate the solvent, and chromatograph the residue on silica using hexane/ethyl acetate (15 14 1) affording 1 715 g (3 44 mmol, 64%) of 4-fluoro-6-phenyl-5tnchloroacetylammo-3Z-hexen- l-01 tnchloroacetnnidate 49 IR 1665 cm-‘, 1720 cm-‘, Dissolve 49 (1.7 g, 3 4 mmol) m methanol (10 mL) and treat with cone sulfuric acid (1 g) After 10 mm add slowly 15 mL of aq sodium bicarbonate Remove the methanol under reduced pressure, and extract the aqueous phase with dichloromethane Evaporation of the solvent affords 1.11 g (100%) of 4-fluoro-6-phenyl-5trichloroacetylammo-3Z-hexen-l-01 50 ‘H-NMR (CDCls, 300 MHz) 6 7 4-7.2 (m,), 6 8 (d, 8 Hz, 1H); 4 7 (dt,Jn, =37 5 Hz, 7.5 Hz, lH), 4 75-4 6 (m, 1H); 3 54 (t, 6 Hz, 2H), 3.05 (ABX, 2H), 2.4-2.2 (m, 2H).

3.4.5. 4-Fluoro-6-Phenyl-5-Tnchloroacetylammo3-(Z)-Hexenoic Acid 51 1 Treat a solution of 1 02 g of above alcohol 50 m 12 mL of acetone with 3 mL of Jones reagent with cooling

382

Welch and Allmendinger

2 After sturmg for 1 5 h, add 2-propanol and water until the reaction mixture 1s green, mdtcatmg complete reduction of the chrommm After extraction with chloroform, evaporate the extracts, and then dissolve the residue m ether/hexane 3 Extract the ether/hexane solution with sodium hydroxide (0 1 N, 3 times) Acidify the basic extracts with sulfuric acid (2 N) and extract with dichloromethane Dry the extracts (sodium sulfate) and evaporate to afford a crystalline product (0 67 g, 1 82 mmol, 63%) mp 108109”C, ‘H NMR (CDCl,, 300 MHz) 6 7 38-7 16 (m, 5H), 6.75 (d, 8 Hz, lH), 4 97 (dt, 37 Hz, 7 5 Hz), 4 75 (sextet, 7.5 Hz, lH), 3 21 (d, 7 Hz, IH), 3 09 (ABX, 7 Hz, 4 Hz, 1H)

4. Notes 1. Caution: Sodium fluoroacetate and fluoroacetyl chloride are fatal poisons affecting the central nervous system and causmg epileptic convulstons These materials were only handled m an efficient fume hood Respiratory protection 1s advised 2 Fluoroacetyl chloride may be prepared by the addition of 1 5 Eq of phthaloyl dtchloride to sodium fluoroacetate m an apparatus prepared for dtstillatton Fluoroacetyl chloride my be drsttlled at bp 65-95°C from the reaction mixture m up to 83% yield as a colorless 011. 3 The synthesis of a-fluoro-a-sllyl-acetates 1s dependent on the bulk of the ester functron Both 2,4,6-trtmethylphenol and tert-butanol have been used conveniently Interestmgly, when the ester 1s treated with one equivalent of base and trialkylstlylhalide, stlylatlon occurs both on carbon and oxygen to form a difficultly separable mixture However, the use of excess base and stlylatmg reagent has been found to result consistently m high-yteldmg preparations of the (C, O)btssdylketene acetal. Cautious hydrolysis with tartartc acid as described then reproducibly affords very clean ct-fluoro-a-stlyl-acetates m good yield These materials are reasonably stable, but must be stored m the freezer The best results are obtained with freshly prepared materials

References 1 Fok, Y F and Yankeelow, J. A , Jr (1977) Blochem Bzophys Res Commun 74, 273-278 2 Hann, M M and Sammes, P G (1980) J Chem Sot , Chem Commun 234-235 3 Cox, M T , Heaton, D W , and Horbury, J (1980) J Chem Sot , Chem Commun

799-800 4 Cox, M. T., Gormley, J J , Hayward,

C. F , and Pettern, N. N. (1980) J Chem

Sot , Chem Commun 800-802 5 Hann, M M., Sammes, P. G., Kennewell, P D , and Taylor, J. B (1982) J Chem Sot , Perkm Trans I 307-314. 6 Miles, N. J., Sammes, P F , Kennewell, P. D., and Westwood, R (1985) J Chem Sot , Perkzn Trans I 2299-2304 7 Kaltenbronn, J. S., Hudspeth, J P , Lunney, E A , Michmewtcz, B M , Nlcolatdes, E D , et al (1990) J h4ed Chem 33,838-845

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383

8 Andres, C J , Macdonald T L , Ocam, T D , and Longhi, D (1993) J Org Chem 58,66096613 9 Abraham, R J , Elhson, S L R., Schonholzer, P , and Thomas, W A (1986) Tetrahedron 42,2101-2110 10 Allmendmger, T , Furet, P , and Hungerbtihler, E (1990) Tetrahedron Lett 31, 7297-7300. 11 Creplak, P. and Kollman, P. A (1993) J Comp. Azded Mole Deszgn 7,291-304 12. Allmendinger, T , Felder, E., and Hungerbuhler, E (1991) Fluorodefin dtpeptrde rsosteres, m Selectwe Fluortnation tn Organtc and Btoorgantc Chemtstry (Welch, J T , ed.), ACS Books, Washmgton, D.C., pp. 186-195. 13 Allmendinger, T , Angst, C., and Karfunkel, H (1994) Proc Intl Conf Fluor Chem , 155th Committee on Fluorme Chemistry (1994) Jap Sot. Prom. Scrence 149-162. 14 Bartlett, P A and Otake, A (1995) J Org Chem 60,3 107-3 111 15 Stem, R L , Schmid, F. X., et al (1993) Advances tn Protetn Chemistry, vol 44 (Anfinsen, C. B., Edsall, J T , Richards, F M and Etsenberg, D S , eds.), Academic Press, New York, pp l-66 16 Deber, C M , Madrson, V , and Blout, E. R (1976) Chem Res 9, 106 17. Crerghton, T E (1984) Protezns Structures and Molecular Prtnctples, W H Freeman, New York. 18. Schmtd, F X., Mayr, L M , Mucke, M., and Schonbrunner, E R (1993) Accessory folding protems, m Adv Protean Chem (Lorrmer, G, Ed ), Academic, New York, pp 25566 19 Fischer, G , Wrttmann-Lrebold, B , Lang, K , Krefhaber, T , and Schmid, F X (1989) Nature 337,476 20 Takahashr, N., Hayano, T , and Suzuki, M. (1989) Nature 337,476. 21 Stein, R L , (1993) Accessory folding proteins, m Adv Protein Chem, vol 44 (Lorrmer, G , ed ), Academrc, New York, pp l-24. 22 Creplak, P and Kollman, P A (1993) J Comp Atded Mole Destgn 7,291-304 23 DeCorte, B (1992) Ph D Drssertatron, Umversrty at Albany, Albany, NY 24. Boros-Gregor, L , DeCorte, B , Grmr, R , Welch, J T., Wu, Y., and HandSchumacher, R (1994) Tetrahedron Lett 33,6033-6036 25 Eschenmoser, A and Frey, A (1952) Helv Chum Acta 35, 1660. 26. Camellr, G., Gracomim, D., Panunzro, M., Martelli, G , and Spunta, G (1987) G Tetrahedon Lett 28, 5639 27 Kofron, J. L., Kuzmrc, P , Krshore, V., Gemmecker, G , Fesrk, S W , and Rrch, D. H. (1992) J Am Chem Sot 114,267O. 28. Bergmann, K , Boros, L G., and Welch, J T. (1996) Unpublished Results 29 Barth, A and Schowen, R L (1990) Dtpepttdyl IV-General and Applted Aspects, vol. 38, Instrtut fur Pharmakologrsche Forschung, Berlin 30 Yaron, A and Narder, R (1993) Crtt Rev Btochem. Mol Btol 28, 3 l-81. 3 1. McDonald, J K. and Barrett, A. J. (1986) Mammalzan Proteases A Glossary and Bibliography vol 2, Academic, London, pp 132-144. 32 Hopsu-Havu, V K. and Sarrmo, S R. (1967) Hoppe-Seyler’s Z Physiol Chem 348,1540-l 550.

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33 Mentlem, R , Heymann, E , Scholz, W , Feller, A C , and Flad, H -D (1984) Cell Zmmunol 89, 1540-1550 34 Feller, A C , Heijnen, C J , Balheux, R E , and Parwaresc, M. R (1982) 51, 227-234 35 Schon, E., Demuth, H U , Barth, A , and Ansorge, S (1984) Bzochem J 223,255-258 36 Mattern, T., Flad, H. D., Feller, A C., Heymann, E , and Ulmer, A J (1989) Leukocyte Z’yplng ZV(Knapp, E., ed ), Oxford University Press, New York, pp 4 17,418 37 Barton, R. W , Prendergast, J., and Kennedy, C. A (1990) J Leukoc Bzol48,29 l-296 38 Flentke, G R , Munoz, E , Huber, B T , Plaut, A G , Kettner, C A , and Bachovchm, W W (1991) Proc Nat1 Acad Scz USA 88, 15X-1559 39 Snow, R J, Bachovchm, W W , Barton, R. W., Campbell, S J , Coutts, S J , Freeman, D M , Gutheil, W G , Kelly, T A , Kennedy, C A , et al (1994) J Am Chem Sot 116,1086&10869 40. Demuth, H. U., Baumgrass, R., Schaper, C., Fischer, G., and Barth, A (1988) J 129-142. Enzyme Inhlb 41 Demuth, H U , Neumann, U , and Barth, A (1989) J Enzyme Znhzb 2,239-248 42 Neumann, U , Stemmetzer, T , Barth, A., and Demuth, H.-U (1991) J Enzyme Inhzb 4,2 13-226 43 Kelly, T A, Adams, J , Bachovtchm, W W., Barton, R. W., Campbell, S J , Coutts, S J , Kennedy, C A , and Snow, R J (1993) J Am Chem Sot 115, 12637, 12638 44 Powers, J. C (1977) Meth Enzymol 46, 197-208 45 Boduszek, B , Oleksyszyn, J , Kam, C -M , Selzler, J , Smith, R E , and Powers, J C (1994) J Mea Chem 37,3969-3976 46 Demuth, H -U , (1990) J Enzyme Inhtb 3,249-278 47 Demuth, H -U , Schlenzrg, D., Schterhorn, A , Groasche, G , Chapot-Chartier, M -P , and Grtpon, J -C (1993) FEBS Lett 320,23-27 48. Welch, J. T. and Lm, J (1996) Tetrahedron 52,291-304 49 Lm, J and Welch, J T (1994) Abstracts of Papers, 208th National Meeting of the American ChemzcalSoczety, Amer Chem Sot , Washmgton D C , ORGN 277 50 Frscher, G , Demuth, H U , and Barth, A (1983) Pharmazze 38,249,250 51 Hart, D J , Kanai, K , Thomas, D G , and Yang, T -K (1983) J Org Chem 48, 289-298 52 Hnao, A , Hattort, I , Yamaguchi, K , andNakahama,S (1982) Synthesu, 46 1,462 53 Itoh, M , Hagtwara, D , and Kamtya, T (1977) Bull Chem Sot Jpn 50,71%721 54 Kawar, A , Hara, 0 , Hamada, Y , and Shtom, T (1988) Tetrahedron Lett 29, 633 l-6334 55. Staab, H. A., Luking, M , and Durr, F H. Chem Ber 95, 1275-1283. 56 Brown, D A, Geraty, R. A., Glennon, J D , and Choileam, N (1985) Synth Commun 15, 1159

21 Synthesis of 3-Amino-l GarboxymethylBenzodiazepine (BZA) Peptidomimetics James C. Marsters, Jr. and Thomas E. Rawson 1. Introduction Replacement of key structural or bmdmg elements of a peptide lead with nonpeptide components can improve affimty and metabolic stability (1-S). Such a strategy was successfully applied to the generation of potent, cell-permeable inhlbltors of Ras farnesyltransferase (FTase) (6,7), The central pan of ammo acids in the CAAX tetrapeptide was replaced with the nonpeptrde scaffold 3-methylammo- 1-carboxymethyl-2,3-dihydro-5-phenyl1H- 1,4benzodiazepin-2-one, (N-Me)BZA, shown below Modeling studies have suggested that the (N-Me)BZA scaffold functions as a conformationally constramed dipeptide turn mimic (7). This chapter describes the synthesis of BZA and (N-Me)BZA scaffolds suitable for solid-phase and solution chemistries, and methods of preparation of peptidomimetic inhibitors of farnesyltransferase. 2. Materials Thin-layer chromatography was performed usmg 60 A silica gel plates (Whatman, 1 x 3 tn., #4500-lOI), and column chromatography was conducted using silica gel (EM 230-400 mesh, VWR, Westchester, PA). Analytical and preparative HPLC was performed on Vydac Cis or Dynamax 300A Cis supports eluted with acetonltrtle/water/O. 1% TFA momtormg at 2 14 nm. ‘H NMR spectra were obtained using a Varian VXR-300s MHz spectrometer, and samples are dissolved m deuterated chloroform (CDCl,, 0 001% TMS, Cambridge Isotope Labs, Andover, MA) or deuterated dimethylsulfoxide (DMSOde, Cambridge Isotope Labs). Measurement

of apparent molecular mass was performed with a PE/Sclex API-

1 mass spectrometer. Solvents (dichloromethane [DCM], ethyl acetate [EtOAc], From

Methods in Molecular Medmne, Vol 23 Pepbdommehcs Edlted by W M Kazmlerskl @Humana Press Inc , Totowa,

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hexane, methanol [MeOH], ethanol [EtOH], dlmethylformamlde [DMF], and tetrahydrofuran [THF]) were purchased from Aldrich, J. T. Baker, or Mallmckrodt and used without further purlficatlon. Unless stated otherwise, all reagents were purchased from Aldrich. 2.1. Reagents for Method 3.1 1 2 3 4 5 6 7

Bromoacetyl bromide 2-Ammobenzophenone Ammoma (Matheson Gas Products, Newark, CA) 1-Methyl-2-pyrrolidinone (NMP) tert-Butyl bromoacetate Cesmm carbonate Trifluoroacetic acid (TFA, Halocarbon Products, River Edge, NJ)

2.2. Reagents for Method 3.2 1 2 3 4 5 6 7 8

Potassium tert-butoxide Isobutyl nitrite 1,2-Dtmethoxyethane (glyme) Hydrogen gas (Matheson Gas Products) Raney Nt (Aldrich, Milwaukee, WI) dt-tert-Butyl dtcarbonate (Fluka, Ronkonkoma, NY) Methyl iodide. Sodmm hydride (60% dispersion m mineral oil, Aldrich)

2.3. Reagents for Method 3.3 1 Benzotriazol- 1-yloxy-tris-(dimethylammo)-phosphonmm hexafluorophosphate (BOP, Rmheheu Biotech , Montreal, Canada) 2 N-methylmorpholme (NMM) 3 1-Hydroxybenzotriazole hydrate (HOBt, Advanced ChemTech, Louisville, KY) 4. L-Methtonme Merrifield resin (BaChem, Torrance, CA) 5. Amsole (Aldrich). 6 Ethylmethylsulfide (EtSMe, Aldrich). 7. Triethylamme (Et,N, Aldrtch). 8 Fmoc-(S-trityl)-L-cysteme (BaChem). 9 bzs(2-Oxo-3-oxazohdmyl)phosphmtc chloride (BOP-Cl, Aldrich). 10 Dnsopropylethylamme 11 Pipertdme (Fluka). 12 Hydrogen fluoride (Matheson Gas Products)

2.4. Reagents for Method 3.4 1 L-Methtomne methyl ester (Sigma, St Louis, MO) 2 Dusopropycarbodumide (DIPC) 3. 1-(3-Dimethylammopropyl)-3-ethylcarbodumtde hydrochlortde

(EDC, Aldrich)

Synthesis of BZA

387

A

R=H,Me

co2teu 2

l

3 C 02H /

Fig 1 Synthesis of l-carboxymethyl-2,3-d~hydro-5-phenyl-1H-l,4-benzod~azeptn2-one 5 (a) BrCH&OBr, DCM, H20; (b) NH3, MeOH; (c) CsC03, tBuO-COCH,Br, NMP; (d) TFA, DCM

3. Methods 3.7. Preparation of l-Carboxymethyl-2,3-Dihydro5- Phenyl- 1H- 1,4-Benzodiazepin-2-one 5 The synthesis of the benzodlazepme core has been described (7,s) and can be convemently performed on a relatively large scale (-1 mol) using commonly avallable laboratory equipment (see Note 1). As shown m Fig. 1, acylatlon of 2-ammobenzophenone I with bromoacetylbromide is followed by

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treatment with ammonia and cychzatlon under reflux Alkyatlon with tert-butyl bromoacetate and acidic hydrolysis of the subsequent ester provides the 1-carboxymethyl-benzodlazepme product 5 as a pale yellow foam. 3 1 f.2-Bromoacetamido-benzophenone

2

A solution of bromoacetyl bromide (100 mL, 1.15 mol) dissolved m dlchloromethane (DCM, 300 mL) was added over 30 mm to a solution of 2-ammobenzophenone 1(197 g, 1.Omol) dissolved in DCM (1 3 L) and water (100 mL) at -10°C under vigorous mechanical stmmg. The resulting mixture was stirred for an additional 1 h at -YC, and then warmed to ambient temperature. The layers were separated,and the orgamcswere washedwith dilute sodium bicarbonate, and then dried over anhydrous sodium sulfate. Evaporation afforded 309.8 g (0.97 mol, 97%) of 2-bromoacetamldobenzophenone 2 as off-white crystals Selected ‘H NMR (300 MHz, CDCl,): 11.5 ppm (1 H, br s), 4.0 ppm (2 H, s). 3 1.2 2,3-Dihydro-5-phenyl-

1H- 1,4-benzodiazepm-2-one

3

A suspension of 2-bromoacetamldobenzophenone 2 (275 g, 0.86 mol) in MeOH (1 L) was treated with a solution of saturated ammonia m MeOH (3 L), and the resulting solution stirred at ambient temperature for 6 h, and then heated at reflux for an additional 4 h After cooling, water (500 mL) was added, and the solution was concentrated to about 1 L m volume, and the product filtered, yleldmg 200.7 g (0.85 mol, 98%) crystallme 2,3-dlhydro-5-phenyl- 1H- 1,4benzodlazepm-2-one 3. Selected ‘H NMR (300 MHz, CDCl,): 9.5 ppm (1 H, br s), 4 35 (2 H, br s) 3 7 3 1-tert -Buty/carboxymethy/-2,3-dihydro5-phenyl- 1H- 1,4-benzodiazepm-2-one 4 A 1-L round-bottomed flask was equipped with a magnetic stirring bar and nitrogen Inlet, and was sequentially charged with 100 g (0 423 mol) of 2,3dlhydro-5-phenyl- 1H- 1,4-benzodlazepm-2-one 3, 600 mL of 1-methyl-2pyrrohdmone, 97 mL ( 117 g, 0.60 1 mol) of tert-butyl bromoacetate, and 194 g (0 595 mol) of ceslum carbonate. After stirring overnight at ambient temperature, the reaction mixture was diluted with 2 L water and extracted with ethyl acetate (3 x 600 mL) The combmed organic extracts were washed with water (4 x 300 mL) and brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to provide 202 g crude 4. This material was recrystallized from hexanes/ethyl acetate to provide 123 g (0.35 1 mol, 83%) of tert-butylcarboxymethyl-2,3-dlhydro-5-phenyl- 1H-l ,4benzo-diazepm-2-one 4 as a white crystalline solid Selected ‘HNMR (300 MHz, CDC13): 4.8 + 3.8 ppm (2 H, d, J= 11 Hz), 4 5 + 4.2 ppm (2 H, d, J= 17 Hz), 1.5 ppm (9 H, s).

Synthesis of BZA

389

/” 02H

6

Fig 2 Synthesis of (N-Boc)BZA lsobutylmtnte, 1,2-dlmethoxyethane, THF, H20, (d) CH31, NaH, THF.

9

8 and (N-Boc)(N-Me)BZA 9 (a) KOC(CH&, (b) H,, Raney Nl, MeOH, (c) Bo+O, NaOH,

3 7.4 1-Carboxymethyl-2,3-dihydro-5-phenyl7H- I, 4-benzodlazepin-2-one 5 A solution of tert-butylcarboxymethyl-2,3-dlhydro-5-phenyl1H- 1,4-benzodlazepm-2-one 4 (58 g, 0.166 mol) m trtfluoroacetrc acid (TFA, 100 mL) was stirred overnight, followed by evaporation and retreatment with TFA (100 mL) The mixture was concentrated under reduced pressure, and the residue was dtssolved m DCM, and then washed sequentially with water and brine. The organICS were dried over anhydrous sodturn sulfate and evaporated to yield 48.4 g of 1 -carboxymethyl-2,3-dthydro-5-phenyl1 H- 1,4-benzodiazepm-2-one 5 (0 164 mol, 99%) as a yellow foam, which was used without further purificatton. Selected ‘H NMR (300 MHz, CDCls + 1 drop DMSO-d6): 10.8 ppm (1 H, br s), 4.8 + 3.85 ppm (2 H, d, J= 13 2 Hz), 4 6 + 4.4 ppm (2 H, d, J= 17.4 Hz).

3.2. Preparation of (N-Boc)-3-amino-2,3-dihydro-5-phenyl1H- 1,4-benzodiazepin-2-one1-acetic acid (/N -Boc]BZA, 8) and (N-Boc)-3-methylamino-2,3-dihydrophenyl1H- 1,4-benzodiazepin-2-one1-acetic acid (/N -Boc][N-Me]BZA,

9)

The amme functionality is introduced into the scaffold as outlined in Fig. 2. Treatment of 5 with potassium t-butoxtde and trappmg of the carbamon gener-

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ated at position 3 with lsobutylmtrlte yields the oxlme, 6, which 1sreadily converted to amme 7 via catalytic hydrogenation with Raney nickel. N-Boc protection with dl-tert-butyl dlcarbonate provides the unsubstituted BZA scaffold 8, which can be used directly in peptidomimetic synthesis. Alternatively, the amme can be protected as the N-Fmoc derlvatlve (9) (see Note 2). Methylatlon of (N-Boc)BZA 1s carried out by treatment wtth sodium hydride and methyl iodide. Incorporation of (N-Boc)(N-Me)BZA 9 mto peptlomlmetic inhlbltors of farnesyltransferase 1sdescribed below (see Note 3). It should be noted that (N-Boc)BZA and (N-Boc)(N-Me)BZA are prepared as mixtures of two enantlomers at C-3. Although it 1sbeyond the scope of this chapter, we have prevlously described the enantiospeclflc choral resolution of each isomer of (N-Me)BZA as the camphorsulfomc acid salt (10) (see Note 4). In general practice, however, mcorporatlon of the racemic BZA or (N-Me)BZA scaffolds mto peptidomlmetlcs ylelded compounds as a mixture of two diastenomers, which are easily separated by HPLC. 3.2.1 3-Oximino-2,3-dihydro-5-phenyl1H- 1,4-benzodiazepin-Z-one- 1-acetic acid 6 A solution of 2,3-dihydro-5-phenyl- 1H- 1,4-benzodlazepm-2-one- 1-acetic acid 5 (30 g, 0.102 mol) m 1,2 dlmethoxyethane (1 L) was cooled to -5°C and deoxygened with mtrogen. Solid potassium tert-butoxlde (47.7 g, 0.43 mol) was added portlonwlse and the resulting red solution was stlrred for 30 mm at 0-5°C A solution of lsobutyl nitrite (13.8 mL, 0.116 mol) in 1,2 dlmethoxyethane (20 mL) was added, producmg an orange-yellow suspension The mixture was neutralized after l/2 h with acetic acid (200 mL) and concentrated to afford crude 6 as a yellow solid (-33 g), which was used directly m the following reaction. Selected ‘H NMR (300 MHz, DMSO-d,): 11.0 ppm (1 H, s), 4.3 ppm (2 H, s). 3.2.2. 3-Amino-2,3-Dlhydro-5-Phenyl1H- 1,4-Benzodiazepin-2-One- 1-Acetic Acid 7 A solution of 3-oximlno-2,3-dlhydro-5-phenyl-lH-l,4-benzodlazepln-2one- l-acetic acid 6 (33 g, 102 mmol) m MeOH (200 mL) contammg 2 mL acetic acid was hydrogenated m a Parr shaker apparatus over Raney Nickel (1: 1 by weight to oxime, prewashed twice with water and then once with ethanol) at 65 psi and ambient temperature for 1.5 d The catalyst was removed by suction filtration through cellte, and the solution concentrated to yield crude 3-ammo-2,3-dlhydro-5-phenyl- 1H- 1,4-benzodlazepm-2-one- I -acetIc acid 7, which was used without further purification Selected ‘H NMR (300 MHz, DMSO-d&: 4.35 + 4.05 ppm (2 H, d, J - 15 Hz), 4.35 ppm (1 H, s), 3.4 ppm (br).

Synthesis of BZA 3.2.3. (N -Boc)-3-Amino-2,3-dihydro-5-phenylone- 1-acetic aad (/N -Boc]BZA), 8

391 1H- 1,4benzodiazepin-2-

A solutton of 3-ammo-2,3-dthydro-5-phenyl- 1H- 1,4-benzodiazepm-2-one1-acetic acid 7 (-I 02 mmol) m THF (100 mL) and water (100 mL) was cooled to 0°C and dt-tert-butyl dtcarbonate (28.8 g, 132 mmol) was added under a nitrogen atmosphere followed by dropwise addition of sodium hydroxide (1 N solutton) until the pH of the solutton was -10 as measured by wetted pH paper. The solution was allowed to come to ambient temperature, stirred overnight, cooled again to 0°C and actdtfied (pH -3 0) with dropwise addttton of concentrated sulfuric acid. The solutton was diluted with ethyl acetate, partitioned, and the organic extract dried over anhydrous sodium sulfate and concentrated. The residue was recrystallized from 120 mL methanol yteldmg 18 g (44 mmol, 43%) of (IV-Boc)-3-ammo-2,3-dihydro-5-phenyl- 1H- 1,4-benzodtazepin-2-one1-acetic acid 8 as cream needles. Selected tH NMR (300 MHz, CDCI,): 11.7 ppm (1 H, br), 6.55 ppm (1 H, d, J- 8 Hz), 5.4 ppm (1 H, d, J- 8 Hz), 4.6 ppm (2 H, s), 1.4 ppm (9 H, s) MS [(M + H)+]. 410.1 3.2.4. (N-Boc)-3-Methylamino-2,3-dihydro-5-phenyl1H- 1,4-benzodiazepln-2-one- 1-acetic acid (/N -Boc]/N -Me]BZA, 9) An oven-dried, 100-mL round-bottomed flask was equipped with a magnetic stn-rmg bar and nitrogen Inlet, and was sequentially charged with 4.63 g (11.3 mmol) of (N-Boc)3-amtno-2,3-dthydro-5-phenyl1H- 1,4-benzodtazepm-2-one-l-acetic acid 8, 50 mL of anhydrous THF, and 2.80 mL (6 38 g, 45.0 mmol) of methyl iodide. The reaction flask was cooled to -5°C m an me/acetone bath, and 1.18 g (29.5 mmol) of NaH (60% drsperston m mineral 011)was added m portions over a 5-min period (Caution: vigorous gas evolutton). After 50 mm., the reaction mixture was quenched wtth a 5% (w/v) aqueous solution of citric acid, dtluted with water, and extracted wtth ethyl acetate (3 x 40 mL) The combined orgamcs were washed wtth water (30 mL), brme (30 mL), dried with anhydrous sodmm sulfate, filtered, and concentrated under reduced pressure to provtde 6.27 g of a VISCOUSyellow 011.Flash chromatography of the crude material on 150 g of silica using 43:55:2 ethyl acetate/hexanes/acettc acid as eluent yielded 4.54 g (10 7 mmol, 95%) of (N-Boc) 3-methylamino-2,3-dthydro-5-phenyl- 1H- 1,4-benzodtazepm-2-one- 1-acetic acid 9 ([IV-Boc][N-Me]BZA) as a clear glass Selected ‘H NMR (300 MHz, CDCls): 8.4 ppm (1 H, br), 5 8 + 5.5 ppm (1 H, 2 s),4.7+4.4ppm(2 H,d, J= 17.7Hz), 3.35 ppm(3 H, s), 1.45 + 1.25 ppm (9 H, 2 s). MS [(M + H)+]: 424.1909; found CZ3HZ6N305.

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Marsters and Rawson

SMe

d

e-g

Fig 3 Solid-phasesynthesisof Cys-(N-Me)BZA-Met 12 (a) BOP, HOBt, NMM, DMA, (b) TFA, anisole,EtSMe, DCM, (c) Et,N, DCM, (d) Fmoc-(S-Trt)Cys, Cl, DIPEA, DCM, (e) piperidine, DMA, (f) TFA, anisole,EtSMe, DCM, (g)

BOP-

HF, am-

sole, EtSMe, 0°C 3.3. Methods of Solid-Phase

Peptide Synthesis

(N-Boc)BZA and (N-Boc)(N-Me)BZA can be conveniently mcorporated mto peptidomimetic compounds via standard solid-phase methodologtes (11) The synthetic scheme outlined m Fig. 3 uses standard Merrifield resin and N-Boc protection Equally good results can be obtained using acid-labile Wang resin and N-Fmoc protectton. BZA and (N-Me)BZA couplmgs can be performed using standard activatmg agents (DIPC, BOP, or HBTU). Use of BOP-Cl as the activating agent when couplmg to (N-Me)BZA gave somewhat higher yields 3.3.7. Preparation of Cys-(N-Me)BZA-Met

72

In the example shown m Fig. 3, (N-Boc) 3-methylammo-2,3-dihydro-5phenyl- 1H-l ,4-benzodiazepin-2-one-l -acetic acid (9, 860 mg, 2.0 mmol),

Synthesis of BZA

393

benzotnazol- 1-yloxy-trzs (dimethylamino)-phosphomum hexafluorophosphate (BOP, 900 mg, 2 0 mmol), NMM (225 p.L, 2.0 mmol), and HOBt (280 mg, 2.0 mmol) m dlmethylacetamtde (DMA, 30 mL) were added to deprotected L-methlonine-lmked Merrlfield resin (Bachem, 1.25 g, 0.71 mEq/g, 12 h). After washing (DMA, and then DCM) and deprotectlon (45% TFA/S% amsole/5% EtSMe/DCM), the resin was neutralized (20%Et,N/DCM) and washed (DCM). Next, Fmoc-(S-tntyl)-L-cysteme (2.1 g, 3 6 mmol), bls(2-oxo-3-oxazo1ldmyl)phosphinic chloride (BOP-Cl, 0.99 g, 3.9 mmol), and diisopropylethylamme (1.4 mL, 7 8 mmol) were combined and added to the resin (DCM, 30 mL, 10 h) (see Note 5). After removal of the Fmoc (20% ptpendine/DMA) and trltyl(45% TFA/S% EtSMe/S% amsole/DCM) protecting groups, the resin was washed (MeOH) and dried under vacuum. Product was cleaved from the resm (32 mL, HF/lO% amsole/5% EtSMe, O”C, 1 h) (see Note 6) and purified via HPLC. Purlficatlon of 173 mg of crude material (Vydac Cl 8, CH3CN/H,0/ 0 1% TFA) afforded the product, N- { [3-(2-ammo-3-mercapto- 1-oxopropyl) methylammo]-2,3,4,5-tetrahydro-2-oxo1H- 1-benzazepm- 1-yl acetyl} -Lmethlonlne (Cys-[N-Me]BZA-Met, 12), as two separable dlastereomers (opposite configuratlon at C-3 of the benzodiazepme) designated 12A (39 mg) and 12B (22 mg) correspondmg to the early and late elutmg peaks respectively. Mass (electrospray, M + H+) calcd: 558.35 found: 558.3 (12A), 558.3 (12B). 3.4. Methods of Synthesis in Solution Compounds containing BZA or (N-Me)BZA can also be conveniently prepared m solution Solution chemlstrles are easily scaled to afford gram quantlties of material, and can be used to generate carboxy-modified prodrugs (12). The synthetic scheme outlined m Fig. 4 shows the preparation of the cell-permeable prodrug, 15 (BZA-5B of ref. 6). Water-soluble couplmg reagents, such as EDC, yield excellent results when used m a mmlmum volume of DMF contaming HOBt (see Note 7). 3.4.1. Preparation of Cys-(N-Me)BZA-Met(OCH,)

15

As shown m Fig. 4, (N-Boc)3-methylamino-2,3-dlhydro-5-phenyl1H- 1,4benzodlazepm-2-one-l-acetic acid 9 (2.3 g, 5.4 mmol), L-methiomne methyl ester (2.25 g, 13.5 mmol), DIPC (1.04 mL, 6.6 mmol), and HOBt (0 9 g, 6.7 mmol) were combined m DCM (20 mL). After 10 h, the reaction was diluted (DCM, 90 mL), washed twice with 0.1 N sulfuric acid, twice with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to yield 3.5 g crude N- { [3-(tert-butoxycarbonyl)methylamino-2,3-dlhydro-2-oxo-5-phenyl1H- 1,4-benzodlazepin- 1-yl] acetyl}-L-methionine methyl ester. The coupled material (0.75 g, -1.3 mmol) was deprotected (30% TFA, 30 mL, 3 h), concentrated, and neutrahzed via part&Ion between ethyl acetate (100 mL)

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Fig 4. Synthesis of Cys-(N-Me)BZA-Met(OCH3) 15 (a) DIPC, HOBt, DCM, (b) TFA, DCM, (c) N-Boc-(S-SEt)-Cys, EDC, HOBt, DMF, (d) TFA, DCM, (e) DTT, ACN, H20, pH -8.&8 5

and saturated sodium bicarbonate. The orgamcs were washed with brine, dried over anhydrous sodium sulfate, and purified (silica, DCM/MeOH 99.1 with 0.2% Et,N) to yield 0.55 g (1.17 mmol, 90%) N-[(3-ammo-2,3-dlhydro-2-oxo5-phenyl- 1H- 1,4-benzodlazepm- 1-yl) acetyll-L-methlomne methyl ester 13 as a clear oil. Coupling of 13 with N-Boc-(S-ethylthlo)-cysteme (0.99 g, 3.6 mmol) (see Note S), EDC (0.68 g, 3 6 mmol), and HOBt (0.16 g, 1.2 mmol) m DMF (10 mL, 12 h), followed by aqueous workup (0.1 N H2S04, brine, sat. NaHC03, brine) m ethyl acetate and silica chromatography (DCM/l-10% MeOH, as above), yielded the product, N-( {3-[(2-(tert-butoxycarbonyl)ammo-3mercapto- 1-oxopropyl)methylamlno]-2,3-dlhydro-2-oxo-5-phenyl1H- 1,4benzodlazepm-

1-yl} acetyl)-L-methlomne

methyl ester 14 (0.82 g, 93%)

Removal of the Boc- (30% TFA as above) and ethylthlo- (60 mg dlthlothreltol, 20 ml 50% CH,CN/H20, pH 7 5) protecting groups afforded N([3-[(2-ammo-3-mercapto-1-oxopropyl) methylammo]-2,3-dlhydro-2-0x0-5phenyl- 1 H- 1, 4-benzodlazepm-

1-yl] acetyl) -L-methlomne

methyl

ester 15

(Cys-[N-Me]BZA-Met[OCHJ).

Purlficatton by HPLC (Vydac C18, CH$ZN/

Synthesis of BZA

395

H,O/O. 1% TFA) resolved the diastereomers possessing opposite configuratton at C-3. Purification of 120 mg of the crude material yielded the two drastereomers 15A (26 mg) and 15B (30 mg). Mass (electrospray, M + H+) calcd: 572.2 found: 572.3 (15A), 572.3 (15B) 4. Notes 1 Synthesis of the benzodrazepme core follows much of the early work described by Bock et al. (8) Similar scaffolds have been used in the preparation of combinatorial libraries (13) 2 The N-Fmoc-protecting group can be easily mcorporated using fluorenylmethyloxycarbonyl-N-hydroxysuccinimide (Fmoc-OSuc) (9) 3. N-methylation of the amide at C-3 has been postulated to stabilize the czs amide conformation of the mhibitor (6 ,7) 4. Enantospecific resolution can easily be performed on a large scale if a single enantiomer is preferred (10) 5. S-Trityl cysteme was found to provide better overall yields than more robust S-benzyl protection. 6 Caution: spectalized equipment is requtred for the handling and mtroductron of hydrogen fluoride Commercial labs can be contracted to provide this cleavage service 7 In general, acid-labile N-Boc protection schemes are preferable to Fmoc when used m solution. 8. S-S-ethyl side-chain protection was found to be convemently removed m aqueous DTT and simplified sample mtroduction on HPLC

Acknowledgments The authors would like to thank Daniel Burdick, Dave Oare, Ken Paris, Todd Somers, Mark Reynolds, and Martin Struble for invaluable scientific insight and technical expertme.

References 1 Freidmger, R M (1989) Non-peptide ligands for peptrde receptors. Trends Pharm Scl 10,270 2 Farmer, P. S (1980) Bridging the gap between bioactive peptides and nonpeptrdes Some perspectives m design, m Drug Deszgn, vol. X (Arrens, E J , ed ), Academic, New York, pp 119-143. 3. Ball, J B. and Alewood, P F. (1990) Conformational constraints. nonpeptide betaturn mimics J Mel Recognztlon 3, 55 4. Morgan, B. A. and Gamor, J. A. (1989) Approaches to the discovery of nonpeptide ligands for peptide receptors and peptidases. Ann Rep Med. Chem 24,243 5. Gante, J. (1994) Peptidomimetics: Tailored enzyme inhibitors. Arzgew Chem Znt Ed 22, 1699

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6 James, G L., Goldstein, J L , Brown, M S , Rawson, T E , Somers, T C , McDowell, R. S., et al (1993) Benzodiazepine peptrdomrmetrcs. Potent mhrbrtors of Ras farnesylatton m animal cells Sczence 260, 1937-42 7 Marsters, J. C , Jr , McDowell, R. S , Reynolds, M E , Oare, D. A., Somers, T C , Stanley, M. S., et al (1994) Benzodiazepine pepttdomtmetrc mhtbttors of farnesyltransferase Bloorgamc Med Chem 2,949-957 8 Bock, M G , DiPardo, R M , Evans, B. E., Rittle, K E , Veber, D F , Freidinger, R M., et al (1987) Synthesis and resolution of 3-ammo-1,3-drhydro-5-phenyl2H- 1,4-benzodrazepin-2-ones J Org Chem 52,3232 9 Paquet, A (1982) Introductton of 9-fluorenylmethyloxycarbonyl, trtchloroethoxycarbonyl, and benzyloxycarbonyl amme protecting groups mto O-unprotected hydroxy amino acids using succmtmidyl carbonates. Can J Chem 60,976 10 Rawson, T. E., Somers, T. C., Marsters, J C , Jr, Wan, D. T., Reynolds, M E., and Burdock, D J. (1995) Stereochemtstry of the benzodtazepme based Ras farnesyltransferase inhibitors Bzoorganzc Med Chem Lett 5, 1335-1338 11 Barany, G and Memfield, R B (1980) Solid-phase pepttde synthesis, u-rThe Peptzdes, vol 2 (Gross, E. and Meienhofer, J , ed ), Academic, New York, pp l-284 12 Bundgaard, H. (1985) m Deszgn ofprodrugs (Bundgaard H , ed ), Elsevter, New York, pp. l-92. 13 Thompson, L A and Ellman, J A (1996) Synthesis and applications of small molecule bbrartes. Chem Rev 96,555AOO 14 Butcher, J. W , Ltverton, N J , Selnick, H. G , Elliot, J M., Smrth, G R , Tebben, A J , et al (1996) Preparation of 3-amino- 1,4-benzodiazepme-2-ones vta drrect aztdatton wtth trtsyl aztde. Tetrahedron Lett 37, 6685-6688

22 A Conformationally Restricted P-Strand HIV Protease Inhibitor Michael C. Hillier and Stephen F. Martin 1. Introduction The use of cyclopropanes as a conformationally restricted subunits in biological systems has been the subject of intense study by our group and others (I-10). Our recent efforts have focused on the use of 1,2,3-trisubstituted cyclopropanes as novel [-NH-Co-] or [-CO-Co-] bond replacements m pseudopepttdes to restrict both side-chain orientanon and enforce backbone secondary structures. To test these assumptions, the cyclopropane contammg analog 1 (Fig. 1) was modeled after the potent HIV protease mhibitor 2, which together with a series of related derivatives was developed at Abbott Laboratories (12). This pseudopeptide contains a symmetrical diammo diol motif 8 (Fig. 2) flanked by Cbz-protected valme residues and is known to bmd m a P-strand fashion at the enzyme-active site (12). Our analog 1 was designed to restrict the orientation of the valine residues and to mimic this “extended” backbone conformation. Comparison of enzyme mhtbmon constants for both compound 1 and the parent mhibitor 2 will then elucidate the efficacy of the cyclopropane as a conformationally restrictive subunit. 2. Materials Unless otherwise noted, solvents and reagents are reagent-grade and can be used without purification Dichloromethane (CH,Cl,) is distilled from calcium hydride prior to use. The p-toluenesulfonylhydrazone of glyoxylic acid chloride is synthesized according to the procedure of Blankley et al. (13). The rhodium carboxylate catalyst Rh,[5(S)-MEPY], is synthesized according to the procedure of Doyle et al. (14). N-Benzyl-N-(4-methoxybenzyl)amme is synthesized accordmg to the procedure of Henke et al. (15). 2,5-Diammo- 1,6From

Methods in Molecular Medrcme, Vol 23 Peptldom!met/cs Protocols Edlted by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

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Ph

-“N~;&;~yOvPh Ph-0 H

0

\

OH

H

0

Ph n ‘

Fig. 1. HIV protease inhibitors diphenyl-3(R),4(R)-dial (diammo dial) 8 ts synthesized accordmg to the procedure of Kempf et al. (16). All other materials can be purchased from Aldrich Chemical Co. (Milwaukee, WI). Reactions involvmg an- or moisture-sensitive reagents or intermediates should performed under an mert atmosphere of argon m glassware that is oven- or flame-dried. Melting points are uncorrected. Infrared (IR) spectra are recorded either neat on sodium chloride plates or as solutions m CHCl, as indicated and are reported m wave numbers (cm-‘) referenced to the 1601.8 cm-’ absorption of a polystyrene film. ‘H (300 MHz) and r3C (75.5 MHz) NMR spectra are obtained as soluttons m CDC13 unless otherwise indicated, and chemical shifts are reported m parts per mtlllon (ppm, S) downfield from internal standard Me& (TMS). Coupling constants are reported in hertz (Hz). Spectral splitting patterns are designated as s, singlet; br, broad; d, doublet; t, triplet; q, quartet; m, multiplet; and camp, complex multiplet. Flash chromatography should be performed using Merck sthca gel 60 (23CL-400 mesh ASTM). Percent yields are given for compounds that were 295% pure as judged by NMR.

2.7. Reagents 1, 2. 3, 4. 5 6

7. 8 9 10. 11.

for Method 3.7

3-Methyl-2-butenol p-Toluenesulfonylhydrazone of glyoxyhc acid chloride (store in desiccator) N, N-Dimethylamlme (distilled from CaH2 and stored over KOH) Triethylamme (Et,N, distilled from CaH,). 1 Naq Hydrochloric acid (HCI). Na2S04 Rh2[5(S)-MEPY], 2 0 M Trimethylalummum (AlMe,) in hexanes N-Benzyl-N-(4-methoxybenzyl)-amine Pyridmium chlorochromate (PCC) Celite

399

P-Strand HIV Protease InhIbitor 0 Rh2[5(S)-MEPYl4 CH2C12,

HI,,

0

A

3

4

N(MPM)Bn

MPMNHBn AIMe3,

DCE

A

1) PCC,

CH2C12

2) K&03,

MeOH

HO 5

N(MPM)Bn

1) Jones 2) CF3C02H

NHBn

HO 7

6

6. EDC. HOBt

1

DMF

Ph OH

/

Rb

NH2

6

Fig 2 Synthesis of the cyclopropane contaimng mhlbltor 1 12. 13. 14. 15 16. 17 18. 19.

K&O3 MgS04 Acetone. 8 N Jones reagent. Trlfluoroacetlc acid (TFA). 2,5-Dlammo-l,6-diphenyl-3(R),4(R)-dlol (dlamino diol) 1-Hydroxybenzotnazole (HOBt) N-Ethyl-N-(dlmethylammopropyl)-carbodnmlde hydrochloride (EDC).

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and Martin

20 NaCl 2 1 Citric actd 22. NaHCO,

3. Methods 3.1. Preparation of (N -Benzyl)Amide Cyclopropane Diamine Dial HIV Protease Inhibitor 1 The constructton of the cyclopropane contammg mhtbrtor 1 mvolves couplmg of the known diammo dial 8 to the N-benzylamide cyclopropyl acid subunit 7 via a carbodnmide couplmg (Fig. 2). This carboxylic acid motety is derived from a chiral cyclopropyl lactone 4 that is prepared via an enantioselective cyclopropanation of the dtazoester 3 using a catalytic amount of Rh,[S(S)-MEPY],. Subsequent Lewis acid- (A1Me3) promoted amidation of this lactone accordmg to the Wemreb protocol affords the amide alcohol 5, which is oxidized to the correspondmg aldehyde and epimerized to give the trans compound 6 (2 7,18). Treatment of this aldehyde with Jones reagent followed by removal of thep-methoxybenzyl group with TFA affords the desired N-benzyl cyclopropyl acid subunit 7 (19,20). 3.1.1. 2,2-D/methyl

Butenyldiazoacetate

3

1 Add 1 29 g (15 0 mmol) of 3-methyl-2-butenol to 45 mL of dry CH2C12, and cool this solutton to 0°C m an me bath 2. Add 4 00 g (15 4 mmol) of the p-toluenesulfonylhydrazone of glyoxyhc acid chloride m one portion followed by 1 92 mL (2 00 mmol) ofN,N-drmethylamlme, whereupon the reaction mixture should darken 3 After sttrrmg for 15 mm at O”C, add 5 8 mL (46.2 mmol) of EtsN, and stir the mixture for 10 mm at 0°C and for 15 min at room temperature 4 Add 32 mL of water to quench the reaction, and remove the volatrles vta rotary evaporator (see Note 1). 5 Add 35 mL saturated aqueous cttrtc acid, 35 mL EtOAcihexanes (1 9) and separate the two phases Wash the organic layer with addmonal saturated aqueous citric acid (1 x 35 mL) and combme the aqueous layers Extract this aqueous solutton with a mixture of EtOAc/hexanes (1 9) (1 x 35 mL) to recover addrttonal product 6 Dry the combined organic layers over (MgSO,), and concentrate the solutton vta rotary evaporatton The resulting crude oily residue 1s then purified via flash chromatography elutmg with EtOAc/hexanes (5 95) to obtain 2 13 g (14 1 mmol,

94%) of 3 as a fluorescent yellow oil (seeNote 2) ‘H NMR (250 MHz) 6 5.385 32 (m, 1 H), 4.75 (s, 1 H), 4 67 (d, J= 7 2 Hz, 2 H), 1 76 (s, 3 H), 1 72 (s, 3 H),

13CNMR (65.2 MHz) 6 167.5, 139 3, 118 5, 61 6, 46.1, 25.7, 17.9; IR (neat) v 3080,2910,1690,1618 cm-i, massspectrum(CI) m/z 154 0728 (C7H9N202 + H requires 154 0742), 154, 153 (base), 152

P-Strand HIV Protease Inhibitor

401

3.1.2. IR-(la, 5a, 6p)-6,6-Dimethyl3-Oxablcyclo[3.l.O]Hexane-Z-One 4 1 Dissolve 0 1 g (0.13 mmol) of Rh,[S(S)-MEPY], m 610 mL dry CH,Cl, and heat the mixture to reflux m a flask fitted with a water-cooled, reflux condenser 2 Dissolve 2.0 g (13 mmol) of the yellow dlazoester 3 in dry CH2C12 (50 mL), and add this solution to the refluxmg solution of catalyst via syringe pump over 16 h (see Note 3) 3 Dlscontmue heating, and allow the reaction to cool to room temperature, whereupon the solvent 1s removed by rotary evaporation The resulting dark oily residue 1s then purified using flash chromatography elutmg with EtOAc/hexanes (1:3) to give 1 4 g (1.1 mmol, 85%, 98% ee) of 4 as a clear 011(21, see Note 4) ‘H NMR (250 MHz) 6 4 37 (dd, J= 5.4, 10.0 Hz, 1 H), 4.15 (d,J= 10 0 Hz, 1 H), 2 07-2.03 (m, 1 H), 1.61 (d,J=46Hz, 1 H), 1 18(s,3H), 1 17(s,3 H), 13CNMR(62.5MHz) 6 174.9,66.5,30 5,30 0,25 1,23 0, 14 3; IR (neat) v 2890, 1745, 1450, 1350 cm-‘, mass spectrum (CI) m/z 127 0760 (C,H,,O, + H requires 127 0759)

3 1 3 [IS-(la,Za)]-(N-Benzy/-N-p-Methoxybenzyl)2-(Hydroxymethyl)-3,3-Dlmethyl1-Carboxamide

5

1 Dissolve 1.4 g (6 0 mmol) of N-benzyl-N-(4-methoxybenzyl)amme in 25 mL 1,2-dichloroethane (DCE) m a flask fitted with a water-cooled reflux condenser. 2 Cool this mixture to 0°C m an Ice bath, and slowly add 3.0 ml (6.0 mmol) of 2 0 M trlmethylalummum m hexanes 3 Stir the reaction mixture for 30 mm at O”C, remove the coolmg bath and slowly add a solution contammg0 25 g (2 0 mmol) of the cyclopropyl lactone 4 m 17 mL dlchloroethane 4 Heat this reaction mixture under reflux for 14 h, cool to O’C m an Ice bath, and quench the mixture by slow addltlon of 5 mL 1 N HCl (see Note 5). 5 Extract this mixture with CH2C12(3 x 15 mL), and dry the combmed organic layers over MgS04. Concentrate the organic solution via rotary evaporator, and purify the crude product by flash chromatography elutmg with hexanes/EtOAc (I* 1) to afford 0 5 g (1 4 mmol, 78%) of 5 as a colorless oil ‘H NMR (rotamers) F74&705(comp,7H),692(d,J=8,6Hz, 1 H),684(d,J=8.6Hz, 1 H),5.21 and 5 16 (rotamenc d, J= 14 6 Hz, 1 H), 4 75 and 4.70 (rotameric d, J= 16 6 Hz, 1 H), 4 29 and 4 24 (rotamenc d, J= 16.6 Hz, 1 H), 4 16-3 87 (camp, 3 H), 3 83 and 3 80 (rotamenc s, 3 H), 1 64 and 1.60 (rotamenc d, J = 8 5 Hz, 1 H), 1 4 l1 3 1 (m, 1 H), 1 15 and 1 14 (rotamenc s, 3 H), 1 12 and 1.11 (rotameric s, 3 H), 13CNMR (rotamers) 6 172.1, 159.0, 158 5, 133.5, 133.0, 129.5, 128 9, 128 5, 128.1, 127 8, 127.6, 127.3, 126.4, 114.3, 113.9,59.4,55.2,55 1,49 8,49.4,47 5, 47 1, 31 9, 30 4, 27 6, 23 7, 15 9, IR v (CHCI,) 3457, 2948, 1628 cm-‘; mass spectrum (CI) m/z 354 2060 (CZ2HZ9N103+ H requires 354 2069), 336

3.7 4. [IR-(la,2/3)+ -Benzy/-N-~-methoxybenzy/aminy/carbony/)2-Formyl-3,3-Dimethyl- 1-Carboxamides 6 1 Dissolve 0 17 g (0 47 mmol) of 5 in 10 mL of dry CH,Cl,, then add 0 20 g (0.90 mmol) PCC and 0.10 g of Cehte Then stir the mixture for 24 h (seeNote 6)

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Hlll/er and Mart/n

2 Add 10 mL Et,0 to the reaction, and filter the resultant suspension through a small plug of silica gel Wash the silica with additional Et,0 (3 x 10 mL) to elute all remaming product 3 Concentrate the filtrate under reduced pressure, and purify the crude product by flash chromatography elutmg with hexanes/EtOAc (1.1) to furnish 0.15 g (0 44 mmol, 60%) of the cu-aldehyde as a clear 011(see Note 7) ‘H NMR (rotamers) 6 9 70 (d, J= 8.6 Hz, 1 H), 7 4&7 05 (camp, 7 H), 6 92 (dd, J= 8.6 Hz, 1 H), 6.85 (d, J = 8 6 Hz, 1 H), 5.02 and 4 95 (rotamenc d, J = 14 4 Hz, 1 H), 4 63 and 4 60 (rotamenc d, J = 16 6 Hz, 1 H), 4 44 and 4 35 (rotamenc d, J = 14 4 Hz, 1 H), 4 20 and 4 15 (rotamenc d, J = 16 6 Hz, 1 H) 3 80 and 3 78 (rotamenc s, 3 H), 2 32 and 2.27 (rotameric d, J= 8.7 Hz, 1 H), 2 79 and 2 75 (rotamenc t, J= 8 7 Hz, 1 H), 1 49 and 1 47 (rotamenc s, 3 H), 1 18 and 1.11 (rotamenc s, 3 H) , 13C NMR (rotamers) 6 200.7, 169.6, 159 5, 159.4, 136.7, 136 5, 129 6, 129 0, 128.6, 128 2, 127 6, 126.3, 114 4, 114 0, 55 1, 55.0, 50.0, 49.0,48 1, 48 0, 39 9, 39 5, 29 2,28.6,28.5, 15 6, 15 5, IR v (CHCl,) 2955, 1701, 1647 cm-‘, mass spectrum (CI) m/z 352 1922 (Cz2H,,N,03 + H requires 352 1912), 260, 121. 4 Dissolve 0.10 g (0 30 n-m-101)of the cis-aldehyde from the preceding experiment m 10 mL of degassed MeOH, add 0 15 g (1.08 mmol) K&O,, and then stir the suspension for 14 h at room temperature (see Note 8) 5. Add 5 mL of water to the reaction, and extract the mixture with CH,Cl, (3 x 5 mL) 6 Dry the combined organic extracts over MgSO,, filter, and concentrate under reduced pressure to afford 0.08 g (0.23 mmol, 80%) of 6 as a clear viscous 011 No further purlficatlon is required ‘H NMR (rotamers) 6 9 70 and 9 69 (rotamenc d, J= 7 4 Hz, 1 H), 7.41-7 05 (camp, 7 H), 6.90 (d, J= 8 6 Hz, 1 H), 6 84 (d, J= 8 6 Hz, 1 H), 4 85 and 4 83 (rotamenc d, J= 15 2 Hz, 1 H), 4 62 and 4.58 (rotamenc d, J = 15 2 Hz, 1 H), 4.46-4.28 (camp, 2 H), 3 81 and 3 79 (rotamenc s, 3 H), 2 772.75 (m, 1 H), 2 61 and 2 55 (rotameric d, J = 5 0 Hz, 1 H), 1.26 and 1 23 (rotamenc s, 3 H), 1 14 and 1 07 (rotamenc s, 3 H), 13CNMR (rotamers) 6 199 4, 169 1, 158 8, 158.7, 137 8, 137 2, 129.6, 129 4, 129 0, 128 6, 128 2, 127 7, 127 5, 1271,1264,1144,1139,552,551,496,495,482,481,403,353,330, 21 0,20.9,20.0, 19 9, IR v (CHCl,) 2970, 1710, 1646 cm-‘, mass spectrum (CI) m/z 352 1922 (C22H25N103 + H requires 352.1912), 260

3.1 5. [lR-(la,2~)]-(N-Benzyl)-2-Carboxyl3,3-Dimethylcyclopropane1-Carbo.xamide

7

1 Dissolve 80 mg (0.22 mmol) of 6 in 5.0 mL of acetone and cool the solution to 0°C m an Ice bath To this stirred solution, add 0 11 mL (0 88 mmol) of a freshly prepared solution of 8 J/Jones reagent, and stir the mixture for 3 h (see Note 9) 2 Quench the reaction by adding 1 mL 1 N HCl, and extract the solution with CH2C12 (3 x 5 mL) Dry the combined organic layers with MgSO,, filter, and concentrate via rotary evaporator 3 Purify the crude acid by flash chromatography elutmg with hexanes/EtOAc (l-1) containing 2% AcOH to get 72 mg (0.20 mmol, 67%) of the acid as a white solid, mp 165--167°C (see Note 10). ‘H NMR (rotamers) 6 7 40-7.01 (camp, 7 H), 6 90

p-Strand HIV Protease Inhibitor

403

(d, J= 8 6 Hz H), 6.81 (d, J= 8 6 Hz, 1 H), 4.88 and4 80 (rotameric d, J= 16 3 Hz, 1 H), 4 62 and 4 55 (rotamertc d, J= 16 3 Hz, 1 H), 4.21-4 15 (camp, 2 H), 3 80 and 3 78 (rotamenc s, 3 H), 2 44 and 2.41 (rotamenc d, J= 6 0 Hz, 1 H), 2 38 and 2 32 (rotamerrc d, J = 6 0 Hz, 1 H), 1.15 (camp, 6 H); 13C NMR (rotamers) 6 176.5, 169.1, 159 2, 159.0, 137 2, 136.5, 129 6,129.2, 128.9, 128.6, 128.5, 128.1, 1279, 127.7, 129.4, 1265, 1144, 113.9,55.3,49.8,495,48 1,480,34.9,31 6, 30.5,20.8, 19.8, 19.7, IR v (CHCl,) 2957,2622, 1731 cm-‘, mass spectrum (CI) m/z 368 1858 (C,,H,,N,O, + H requires 368 1861), 349,322,136, 121 4. To remove thep-methoxyphenyl-methyl protecting group dissolve 40 mg (0.11 mmol) of the carboxybc actd m 5 mL of neat trifluoroacettc actd (TFA) and stir for 24 h at room temperature 5 Remove the solvents under reduced pressure, and dissolve the crude restdue m 5 mL of CH,Cl, 6 Wash thts organic mixture wtth water (2 x 1 mL), and dry the organic phase over MgSO, Filter and concentrate the dried organic solvents to grve a crude yellow solid This material can be purified by flash chromatography elutmg wtth hexanes/EtOAc (1 1) containing 2% AcOH to yield 24 mg (0.10 mmol, 90%) of 7 as a white sohd, mp 178-I 80°C ‘H NMR 6 7.40-7 29 (camp, 5 H), 6 40 (t, J= 2 8 Hz, lH),44l(d,J=57Hz,2H),232(d,J=56Hz,lH),2.06(d,J=56Hz,lH), 1 32 (s, 3 H), 1 25 (s, 3 H), 13CNMR 6 176.0, 168.7, 138 0, 128 7, 127 5, 127 3, 126.7, 126.5,43.8, 35 9, 32 1, 30 5, 20 5,20 3, IR v (CHCI,) 2954, 2750, 1697, 1649 cm-‘, mass spectrum (CI) m/z 268.2528 (Ct4Ht7N103 + H requtres 268 2532), 248 (M + 1), 230,202, 184, 141,91.

3.1.6. N -Benzyl Amide Cyclopropyl HIV Protease Inhibitor 1 1 To a flame-drred, argon-flushed round-bottomed flask charged with 20 mg (0 08 mmol) of the carboxylic acid 7 in 1 1 mL of DMF, add 13 mg (0 04 mmol) of the dtammo dtol 8 and 24 mg (0 18 mmol) HOBt 2 Place the reactton flask into an rce/NaCl bath, and stir for 0.5 h before adding 17 mg (0 09 mmol) EDC 3 Remove the ice bath, and stir the mixture at room temperature for 24 h 4. Add 1 mL of EtOAc, 0.5 mL of brine, and 0 5 mL of 10% aqueous citrtc acrd to the reaction Separate the organic phase, and wash It with 0 5 mL 10% aq NaHC03 and 0 5 mL brine 5 After drymg the organic layer with MgS04, filter and remove solvents via rotary evaporator to afford 30 mg (0.04 mmol, 90%) of 1 as a white sohd, mp 264 267°C No further punficatton is required. ‘H NMR (DMSO-d6) 6 8 56 (t, J= 6 4 Hz, 2 H), 7 81 (d, J= 9 5 Hz, 2 H), 7 31-7 08 (camp, 20 H), 4.68 (s, 2 H), 4 60 (t, J= 95Hz,2H),422(d,J=6.4Hz,4H),3.17(~,2H),2.80(dd,J=6.4,9.5Hz,2 H), 2 09 (d, J= 5 5 Hz, 2 H), 2 00 (d, J= 5 5 Hz, 2 H), 1 15 (s, 6 H), 1 12 (s, 6 H), 13C NMR (CD30D) 6 172 5, 172 0, 140 2, 131 2, 131 0, 130.5, 130 2, 129 8, 129 4, 128.9, 128 3, 127 6, 74.3, 73 2, 52 5, 44.1, 39.2, 34.6, 34 2, 28.1, 21 2, 20 3, IR v (CHC13) 3050, 2952, 1685, 1678 cm-‘; mass spectrum (FAB) m/z 759.4133 (Ct6Hs4N406 + H requtres 759.4121), 741

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3.2. Discussion The preceding protocol details the synthesis of an enantiopure trisubstituted cyclopropane 4 and its mcorporation mto the HIV protease mhibitor 1. The essential features of this sequence are the highly enantioselective mtramolecular cyclopropanation, the Lewis acid-mediated lactone amtdation, and the aldehyde epimerization. When taken together, these transformations allow for the rapid assembly of “extended” cyclopropane contammg pseudopeptides. The asymmetric cyclopropanation provides for the mcorporation of any number of ammo acid residues (m this case gem dimethyl or valme) m predictable orientations depending on the vinyl substttuentand its configuration (i.e., CZ’S or truns). The Lewis acid- (AlMe,) promoted lactone amidatton allows for the incorporation of simple protected ammes and has been extended to include ammo acids m other examples (22). Finally, the facile epimerization of the aldehydic center affords the trans configuration about the cyclopropane, which may mimic “extended” or P-strand pseudopeptide secondary structure. Biologtcal mhibition data and structural information for 1 shows that this analog closely resembles the parent mhibitor 2 m potency and enzyme-bound conformation. A full exammation of these results will be reported (23). 4. Notes Failure to remove CH,CI, at this stage may result m purrficahon drfficulttes owing to the highly soluble NJ-drmethylamlme The yellow drazoester 1s extremely volatile and may evaporate under reduced pressure, so care should be taken durmg concentratron vta rotary evaporator. This matenal should also be stored m a remgerator (25°C) and under argon when not m use If any residual base or water from the previous reaction remains, lower yields of product will result This may be indicated by a color change to violet or light yellow of the refluxmg solutron during the course of the reaction Enanttomertc excesses were determined via the procedure of Martin et al (21) Alternatively, Doyle and coworkers have utrhzed a 30 m x 0 32 mm rd Chtraldex B-PH (P-cyclodextrm) column for these determmattons (14) Caution! Addition of acid to the reaction mixture 1sextremely exothermtc Lower product yields were observed when Cehte was not employed m this step The reaction suspensionshould darken to black after a few minutes of stnrmg The aldehyde should be usedimmediately. If storage1srequired, place m a freezer at -20°C under an inert atmosphere. The methanol may be degassedby bubbling argon or nitrogen through distilled methanol for approx 15mm before use This increasesthe solubtltty of the K&O, m solutton and shortensreaction times It was observed that for reaction times longer than 24 h, a srgmficant amount of product decomposttton began to occur

Cs$O,, a more soluble metal carbonate,may be substitutedfor K&O, in this reaction tf desired

P-Strand HIV Protease Inhibitor

405

9 A typical procedure for the preparation of 8 N Jones reagent IS as follows. To 0.67 g (6.7 mmol) of Cr(VI)O, m 0 66 mL H,O was added 0 34 mL (6 1 mmol) cone H,SO, For a more comprehenstve dtscussion, see ref. 18 10. Alternattvely, the actd product may be recrystalhzed from EtOAc/hexanes

References 1 Martin, S F , Austin, R E., and Oalmann, C. J (1990) Stereoselecttve synthesis of cyclopropanes as novel dipepttde isosteres Tetrahedron Lett 31,473 l-4734 2 Martm, S F , Austin, R E , Oalmann, C. J., Baker, W. R., Condon, S. L , delara, E , et al (1992) 1,2,3-Trisubstituted cyclopropanes as conformationally restrtcted pepttde tsosteres. application to the desrgn and synthesis of novel remn mhtbttors J Med Chem 35, 1710-1721 3 Baker, W R , Jae, H-S , Martin, S. F., Condon, S. L., Stem, H. H , Cohen, J , et al (1992) Conformationally restrtcted pepttde isosteres 2 Synthesis and m vitro potency of dipepttde renm mhtbitors employing a 2-alkylsulfonyl-3-phenylcyclopropane carboxamide as a P, ammo acid replacment Blo. Med Chem Lett 2, 1405-1410. 4 Martin, S F , Oalmann, C J , and Liras, S (1993) Cyclopropanes as conformattonally restricted peptide isosteres Design and syntheses of novel collagenase mhibitors. Tetrahedron 49, 3521-3532. 5 Stammer, C. H (1990) Cyclopropane amino acids (2,3- and 3,4-methanommo acids) Tetrahedron 45, 223 l-2254 6 Melmck, M J , Bisaha, S N , and Gammill, R B (1990) Conformationally restrtcted P, -P,’ transition state analogues Synthesis of 1(R), 3(R) [l(s), 2(S)] and l(s), 3(s) [l(s), 2(S)]-3-[3-cyclohexyl-2[(Boc)amlno]-l-hydroxylpropyl]-2,2Dimethylcyclopropane carboxyhc acrd Tetrahedron Lett 31, 961-964 7 Shtmamoto, K., Ishtda, M., Shmozaki, H., and Ohfune, Y J (1991) Synthesis of four diastereomeric L-2-(carboxycyclopropyl) glycines. Conformationally constramed L-glutamate analogues J Org Chem 56,4167-4176 8. de Frutos, P , Fernandez, D , Fernandez-Alvarez, E., and Bernabe, M. (1992) Synthests of asymmetric (E)-a-[2-phenyl(ethyl)cyclopropyl] glycmes from set-me by diastereoselective dibromocyclopropanatton Tetrahedron 48, 1123-I 130 9. Zhu, Y -F , Yamazaki, T , Tsang, J W , Lok, S., and Goodman, M. (1992) Synthesis and taste properttes of L-aspartyl-methylated l-ammocyclopropanecarboxylic acid methyl esters. J Org. Chem 48, 1074-1081. 10. Burgess, K and Ho, K -K (1992) Asymmetric synthesis of protected derivattves of ormthme- and argmme-2,3-methanologs Tetrahedron Lett 33, 5677-5680. 11 Kempf, D J , Codacovt, L , Wang, X C , Kohlbrenner, W E , Wideburg, N E , Saldtvar, A., et al (1993) Symmetry-based mhibitors of HIV protease. structureactivity studies of acylated 2,4-dtammo-1,5-diphenyl-3-hydroxypentane and 2,5diamino- 1,6-diphenylhexan+3,4-dial. J. Med. Chem 36, 320-330 12 Erickson, J , Neidhart, D J , VanDrie, J , Kempf, D. J , Wang, X C., Norbeck, D W., et al (1990) Design, activity and 2 8 A crystal structure of a C, symmetrtc mhtbttor complexed to HIV-l protease. Sczence 249,527-533.

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13 Blankley, C J , Sauter, F. J., and House, H 0 (1973) Crotyl dtazoacetate, m Orgunrc Synthesrs Co11 vol 5, (Baumgauter, H E , ed ), John Wiley, New York, pp 258-263 14. Doyle, M P., Winchester, W. R , Protopopava, M. N., Kazala, A P , and Westrum, L J (1996) (lR, 55’)-(-)-6,6-dimethyl-3-oxabtcyco[3 l.O]hexan-2-one. Htghly enantroselective mtramolecular cyclopropanatton catalyzed by dirhodmm(I1) tetrakrs[methyl 2-pyrrolidone-S(R)-carboxylate], m Organic Synthesu, vol 73, (Boeckman, R. K , Jr, ed ) John Wiley, New York, pp 13-24. 15. Henke, B R , Koukhs, A. J , and Heathcock, C. H (1992) Intramolecular 1,3dtpolar cycloaddmon of stabihzed azomethme yhdes to unactrvated dipolarophtles J Org Chem 57,705&7066 16 Kempf, D J , Sowm, T J , Doherty, E M., Hanmck, S M , Codavoct, L , Henry, R F , et al (1992) Stereocontrolled synthesis of C,-symmetric and Pseudo-C,symmetric diammo alcohols and dials for use m HIV protease mhibltors J Org Chem. 57,5692-5700. 17 Basha, A., Lipton, M , and Weinreb, S M (1977) General method for conversion of esters to amides, Tetrahedron Lett 4 17 1 18 Corey, E. J and Suggs, J W (1975) Pyrtduuum chlorochromate An effectent reagent for oxtdation of primary and secondary alcohols to carbonyl compounds Tetrahedron Lett 31,2647-2650 19 For a general discussion of the Jones Reagent (chromic acid), see Paquette, L A (ed ) (1995) Encyclopedia of Reagents for Organic Synthesis, vol 2, John Wiley, New York, pp 1621-1623. 20 For a comprehensive dtscussion of protecting groups, see Greene, T. W and Wuts, P G M (eds ) (1991) Protectwe Groups zn Organic Synthesu, 2nd ed , WrleyInterscience, New York. 2 1 Martin, S F., Oalmann, C J , and Ltras, S. (1992) Enantioselective rhodium catalyzed intramolecular cyclopropanatton of homoallyhc dtazoacetates Tetrahedron Lett 33,6727-6730 22 Dwyer, M P and Martin, S F. (1998) Synthesis of cyclopropane-containing LeuEnkephalm analogs, n-r Peptldomrmetlcs Protocols (Kazmterskt, W , ed ), Humana, Totowa, NJ, Chap 23 23 Martin, S F , Dorsey, G. O., Gaue, T , Htlher, M C , Kesslar, H., Baur, M. et al. (1998) Cyclopropane-derived peptidomrmetics: Design, synthesis, evaluation, and structure of novel HIV-I protease mhtbitors. J Med Chem (m press).

23 Synthesis of Cyclopropane-Containing Leu-Enkephalin Analogs Michael P. Dwyer and Stephen F. Martin 1. Introduction The use of substituted cyclopropanes as conformationally constramed peptrdomtmetics has recerved considerable attention recently (Id). The efforts from our laboratory m this area have focused on the use 1,2,3-trrsubstituted cyclopropanes as novel rsosterrc replacements m several brologtcal systems (7-20). A common theme of this program has been the use of trans-substituted cyclopropanes to enforce extended or “j3-strand” secondary structure while orientmg the ammo acid side chain m a predictable conformation (II). In an effort to explore further the utility of this novel isostere, modeling and calculatrons suggested that a &-substituted cyclopropane dipeptide subunit could stabilize a turn structure The focus of this chapter IS to describe the preparation of a novel cyclopropane-containing cis-substituted (-Glyw[CHOH-cp-CONHI-) subunit, which replaces Gly*-G1y3 subunit of the Leu-enkephalm (Tyr-GlyGly-Phe-Leu) framework shown m Fig. 1. 2. Materials Dtchloromethane (CH,Cl,) and dichloroethane (C1CH2CH2C1) were distilled from calcmm hydride prior to use. Dimethylformamide (DMF) was distilled from magnesium sulfate under reduced pressure and stored over molecular sieves. The p-toluenesulfonylhydrazone of glyoxylic acid chloride was prepared according to the method of Blankley et al. (12) and stored m a desiccator. Rh,[55’-MEPY], was prepared according to the method of Doyle (13) and stored m a desrccator. Unless otherwise noted, solvents and reagents were reagent grade and used without purrtication. Reactions involving au- or moisture-sensitive reagents or intermediates were performed under an met-t From

Methods m Molecular Medrone, Vol 23 Pepbdomlmetrcs Protocols Edlted by W M Kazmlerskl OHumana Press Inc , Totowa, NJ

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408

bH Leu-Enkephalm

Fig 1 Leu-Enkephalm

analog 1

atmosphere of argon m glassware that had been oven- or flame-dried Melting points are uncorrected. Infrared (IR) spectra were recorded either neat on sodium chloride plates or as solutions m CHC13 as indicated and are reported m wave numbers (cm-‘) referenced to the 1601 8 cm-’ absorption of a polystyrene film ‘H (300 MHz) and 13C (75.5 MHz) NMR spectra were obtained as solutions m CDCl, unless otherwise indicated, and chemical shifts are reported m parts per million (ppm, 6) downfield from mternal standard Me& (TMS). Couplmg constants are reported m hertz (Hz). Spectral splittmg patterns are designated as s, singlet; br, broad; d, doublet; t, triplet; q, quartet, m, multiplet; and camp, complex multtplet Flash chromatography was performed using Merck silica gel 60 (23WOO mesh ASTM). Percent yields are given for compounds that were 295% pure as Judged by NMR.

2.7. Reagents 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

for Method 3.7

1,4-Pentadien-3-01 Dichloromethane (CHQ,) Trlethylamme (distilled from CaHz and stored over KOH, Et,N) Dimethylanilme (distilled from CaH, and stored over KOH) p-Toluenesulfonylhydrazone of glyoxyhc acid chloride Rh2(5S-MEPY)4 Sodium borohydride (NaBH,) Methanesulfonyl chloride (distilled from CaH& Sodium azide (NaN& 2 0 M solution of trimethylalummum (AlMe,) in hexanes. Phenylalanyl-leucyl carboxyhc acid (Phe-Leu) Dichloroethane (DCE) Diazomethane (CH2N2) Ethanol (EtOH) Methanol (MeOH) Celite

Cyclopropane-Containing

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17 18 19 20. 2 1. 22. 23. 24 25 26 27. 28 29

Palladmm on carbon (10%) Hydrogen (H,) 1-Hydroxybenzotriazole (HOBt). N-Ethyl-N-(dtmethylammopropyl)-carbodnmtde hydrochlortde (EDC). N-tert-Butoxycarbonyl tyrosyl carboxyhc actd (Boc-Tyr-OH) Dimethylformamtde (DMF) Trtfluoroacetic acid (TFA) Magnesium sulfate (MgSO& Sodmm sulfate (Na,SO,) Ethyl acetate (EtOAc) Pentane. Dlethyl ether (Et,O) Hexanes 30. Acetic acid (AcOH)

3. Methods 3.1. Preparation of [lS, 4S, 5S]-4-Azidomethyl3-Oxabicyclo[3.1.0]Hexan-2-One 5 and [lS, 2R 4S]-2-(5-r-Tyrosyl-Aminoethyl-4-Hydroxy)Cyclopropyl-1-r-Phenylalanyl-L-Leucyl methyl Ester 1 The transformatton to the final product 1 via azidolactone 4 IS shown m Fig. 2. 1,4-Pentadien-3-01 is converted to the correspondmg dtazoacetate 2 according to the method of Corey and Meyers (14). Drazoacetate 2 undergoes a highly stereoselectrve cyclopropanatton reaction in the presence of Rh2[5SMEPY14 to afford the cyclopropane derivative 3 as the major product (>94% enantiometric excess; 95% dtastereometric excess) (15). The alkene of 3 IS oxtdatively cleaved with ozone followed by reduction to afford the mtermediate alcohol, which 1sconverted to azide 4 vra a two-step protocol. With azlde 4 m hand, treatment with Phe-Leu dipeptrde under modified Wemreb amrdatton conditions (16) affords the intermediate acid, which IS converted to the methyl ester 5 on treatment with diazomethane. Catalyttc hydrogenation affords 6, which is then subjected to a carbodiimide mediated peptide couplmg (EDC, HOBt) to mcorporate the tyrosme functtonahty. Removal of the Boc group with trifluoroacetrc acid affords final product 1 3.1.1. 1,4-Pentadienyl-3-Dlazoacetate

2

1 Add 1.72 mL (13 5 mmol) of N, N-dimethylamlme dropwtse to a solution of 1 02 g (12.1 mmol) of 1,4-pentadren-3-01 and 3.46 g (13 3 mmol) of p-toluenesulfonylhydrazone of glyoxyhc acid chloride m 60 mL of dry CH,Cl, at O’C 2 Stir the reaction mixture for 15 mm at 0°C add 4 73 mL (33.9 mmol) of Et,N, and stir the reaction for 12 h at room temperature. 3 Add 30 mL of water and 40 mL of CH$&, and allow the layers to separate Extract the aqueous layer with two 50-mL portrons of CH2C12, and combme the

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Rh2[(5SJ-MEPY]d nI

I

1) O,, NaBH,

1) H-Phe-Leu-OH

2) MsCI. EbN 3) NaNa, DMF

2) CYN,

Me3AI

w

4

Phe-Leu-OMe

H, Pd-C

w

HO 5

Phe-Leu-OMe

1) Boc-NH-Tyr-OH EDC. HOBt 2) CF,CO,H

HzN

-1

HO 5

Fig 2 Synthesis of azldo-lactone 4 and final product 1

organic layers Dry the organic layer over MgSO,, filter, and concentrate under reduced pressure to yield an orange/brown 011 4 Purify the crude oil by flash chromatography using pentane/Et20 (20: 1) as eluant to provide 1.19 g (7 86 mmol, 65%) of 2 as a bright yellow hquld (see Note 1) ‘H NMR 6 5 90-5 79 (camp, 3 H), 5 33-5 20 (camp, 4 H), 4.83 (s, 1 H); 13C NMR 6 165 0, 134.6, 116 7,74 6,45.6, IR (CHC13) v 2116, 1695, 1380, 1225 cm-‘, mass spectrum (CI) m/z 153 0671 (C,Hs02N2 + H requires 153 0664), 125, 113

3 7.2. [IS, 4R, 5R]-4-Vinyl-3-Oxabicyc/o[3

7 O]Hexan-2-One 3

1 Add 1 0 g (6 5 mmol) of 2 m 50 mL of dry CH2C12 by syringe pump over 12-l 8 h to 50 mg (0 06 mmol) of Rh,(SS-MEPY), m 500 mL of CH,CI, at reffux 2 After the addition, cool the mixture to room temperature, and concentrate the solvent under reduced pressure to yield a maroon 011 3 Purify the resultmg 011by flash chromatography using pentane/Et*O (1 1) as eluant to afford 0.73 g (5 9 mmol, 90%) of 3 as a colorless 011(see Note 2) ‘H NMR 6 5 7s5.68 (m, 1 H), 5 29-5.16 (camp, 2 H), 4.95 (t, J= 5.1 Hz, 1 H), 2 25-2 18 (m, 1 H), 2 03-l 97 (m, 1 H), 1 OS-l.01 (m, 1 H), 0 82-O 78 (m, 1 H); 13C NMR 6 175 2, 132.6, 117.6,78.7,20.6, 17.9,8.7; IR(neat) v 1770, 13 15, 1190, 1070 cm-l, mass spectrum (CI) m/z 125.0605 (C7H802 + H requires 125 0602), 107,97,79

Cyclopropane-Containing

Leu-Enkephalin

Analogs

411

3 7.3. [lS, 4S, 5S]-4-Azdomethyi-3-Oxabicycio [3.1.O]Hexan-2-One 4 1 Pass a stream of O3 through 0 57 g (4.6 mmol) of 3 m 30 mL of MeOH and 10 mL of CH,C12 at -78°C until the solution reaches a blue end pomt (see Note 3) 2 Purge the excess ozone by bubbling argon through the solution to afford a colorless solution, Add 0 34 g (9 2 mmol) of NaBH4 carefully to the reactlon and warm to OT, and stir for 30 mm (see Note 4) 3 CarefUlly add 10 mL of saturated aqueous NH&l, and concentrate the reaction mixture under reduced pressure (see Note 5). Extract the resultmg aqueous layer with three 15 mL portions of CH2C12, and combine the organic layers Dry the organic layer over MgSO,, filter, and concentrate under reduced pressure to yield a light yellow 011 4 Purify the crude 011by flash chromatography usmg CH$l,/MeOH (25: 1) as eluant to afford 0 36 g (2 8 mmol, 60%) of the alcohol as a pale yellow oil ‘H NMR ~476-469(m,1H),381-3.69(m,2H),358(s,1H),23~222(m,1H),2142.08 (m, 1 H), 1.19-l 12 (m, 1 H), 1.08-l 04 (m, 1 H), 13C NMR 6 176.0, 79 5, 62.2, 18 6, 17 3, 8 6, IR (CHC13) v 3470, 2885, 1770, 1192, 1042 cm-‘, mass spectrum (CI) m/z 129 0549 (C6Hs03 + H requires 129 0552), 111 (base) 5 Add 0 16 mL (1 17 mmol) of Et,N and then 0 07 mL (0 94 mmol) of methanesulfonyl chloride to 0.10 g (0 78 mmol) of the alcohol m 4 mL of CH,Cl, at OT. 6 Stir the reaction mixture for 1 h at 0°C Add 5 mL of saturated aqueous NaHCO,, and extract the aqueous layer w:th three lo-mL portions of CH2C12 7 Combme the organic layers, and wash them with 10 mL of 10% aqueous HCl and 10 mL of saturated NaCl Dry the organic layer over MgSO,, filter, and remove the solvent under reduced pressure to yield a light yellow oil (see Note 6) 8 Purify the crude residue by flash chromatography usmg hexanes/EtOAc (1 3) as eluant to afford 0 14 g (0.70 mmol, 900/) of the intermediate mesylate as a pale yellow 011 ‘H NMR 6 4 89-4 84 (m, 1 H), 4 38 (dd, J= 4.3, 11 2 Hz, 1 H), 4 27 (dd, J= 7 0,ll 2 Hz, 1 H), 3 07 (s, 3 H), 2 34-2 28 (m, 1 H), 2 22-2.16 (m, 1 H), 1 28-l 21 (m, 1 H), 1.05 (dd, J= 4 7,8 3 Hz, 1 H); 13C NMR 6 174 5,75 5,68 4, 37.6, 18 2, 17.4, 8.8 IR (CHCl,) n 2930, 1784, 1550, 1463, 1025 cm-‘; mass spectrum (CI) m/z 207 0332 (C7H1,,05S + H requires 207 0327) 9 Add 0 44 g (7 0 mmol) of NaN, to 0.14 g (0 70 mmol) of the mesylate m 3 mL of DMF, and heat the mixture at 65T for 12 h 10 Cool the mixture to room temperature, add 20 mL of Et,0 and filter the reaction mixture Wash the filtrate with two 7-mL portions of water and two 7-mL portlons of saturated NaCl Dry the orgamc layer over Na$O,, filter, and concentrate the solvent under reduced pressure to yield a light yellow 011 11 Purify the crude 011by flash chromatography usmg hexanes/EtOAc ( 1.1) as eluant to yield 96 mg (0 63 mmol, 90%) of 4 as a yellow 011. ‘H NMR 6 4 66 (dd, J = 5 3,6 5 Hz, 1 H), 3 50-3 40 (m, 2 H), 2 28-2.21 (m, 1 H), 2 15-2 09 (m, 1 H), 1 18-1 13 (m, 1 H), 102-0.98(m, 1 H), 13CNMR6 174 6,76 8,51.8,19 1,17 6, 8 7, IR (CHCl,) v 2928,2109,1778, 1227 cm-‘; mass spectrum (CI) m/z 154 06 12 (C,H,N,O, + H requires 154.06 17) (base), 126, 111, 108.

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3.1.4. [lS, 2R, 4S]-2-(5-Azidoethyl-4-Hydroxy)Cyclopropyl- 1-L-Phenylalanyl-L-Leucyl Methyl Ester 5 Add 3 0 mL (6.0 mmol) of a 2 M solution of AlMe m hexanes over 40 mm to 0 56 g (2 0 mmol) of Phe-Leu-OH m 12 mL of CH,ClCH&l (DCE) at room temperature to afford a homogenous reaction mixture (see Note 7) Stir the resulting solution for 30 mm, and then add 0 10 g (0 65 mmol) of 4 m 6 mL of DCE dropwlse Fit the round bottom flask with a reflux condenser, and heat the mixture at reflux for 24 h. Cool the mixture to OT, and carefully add 10 mL of 10% aqueous HCl (see Note 8) Extract the resulting mixture with three 15-mL portions of CH,C12, and combme the organic layers. Dry the organic layer over Na2S04, filter, and remove the solvents under reduced pressure to yield an off-white solid (see Note 9) Purify the crude pseudotetrapeptide by flash chromatography using CH,Cl,/ MeOH (25 1 with 1% AcOH) as eluant to yield 0 17 g (0 39 mmol, 60%) of the acid as a light yellow solid. mp 146-148’C dec.; ‘H NMR (CD,OD) 6 7 3&7 20 (camp, 5 H), 4 75-4 69 (m, 1 H), 4 43-4.39 (m, 1 H), 3.52 (dt, J= 4 7, 9 1 Hz, 1 H),320(dd,J=43,141Hz,1H),280(dd,J=11 1,144Hz,lH),254(d,J = 4 9 Hz, 2 H), 1 62-l-70 (camp, 5 H), 1.18-1 15 (m, 1 H), 1 07-l 01 (m, 1 H), 0 98&O 91 (camp, 7 H); 13C NMR (CD,OD) F 175.9, 174.0, 173 4, 138 4, 130 3, 129 5, 127 7, 70 1, 57 1, 55.8, 52 2, 41 7, 39.0, 25 9, 24 3, 21 9, 20 1, 11 6 IR (nujol) v 3440, 3282, 2100, 1713, 1638, 1560 cm-‘, mass spectrum (CI) m/z 432 2256 (C2,Hz9N505 + H requires 432.2247), 242 Add an ethereal solution of CH2N2 to 0 17 g (0 39 mmol) of the acid m 3 mL of EtOH at room temperature until a bright yellow color persists (see Note 10) Disperse the excess CH2N, by bubbling argon through the reaction mixture to afford a clear solution Remove the solvent under reduced pressure, and purify the crude residue by flash chromatography using CH$lJMeOH (25: 1) as eluant to yield 0 16 g (0 37 mmol, 95%) of 5 as a white sohd mp 115-l 17T, ‘H NMR (CD,OD) 6 7 3&7 21 (camp, 5 H), 4.75 (dd, J= 4 4, 10 7 Hz, 1 H), 4 5@-4 45 (m, 1 H), 3 69 (s, 3 H), 3.53(dt,J=45,99Hz,1H),317(dd,J=44,14OHz,lH),281(dd,J=105, 14 0 Hz, 1 H), 2 57 (d,J=4 9 Hz, 1 H), 1.73-1 59 (camp, 5 H), 1 18-1 12 (m, 1 H), 1 08-l 01 (m, 1 H), 0 99-0.96 (m, 1 H), 0 94 (d,J= 6 2 Hz, 3 H), 0.90 (d, J = 6 2 Hz, 3 H), 13C NMR (CD30D) F 174 4, 174 0, 173 3, 138 6, 130 3, 129 5, 127 7, 70 0, 57 2, 55.7, 52 7, 52 2,41.5, 39.0, 25 9, 24 4, 23.2, 21 9, 20 1, 11 5, IR (nuJo1) v 3271, 2104, 1746, 1639, 1555, 1076 cm-‘, mass spectrum (CI) m/z 446 2401 (Cz2H3,N505+ H requires 446 2403), 361,329, 175

3.7 5. [lS, 2R, 4S]-2-(5-Aminoethyl-4-Hydroxy)Cyclopropyl- 1-L-Phenylalanyl-L-Leucyl Methyl Ester 6 1 Add 10 mg of 10% Pd/C (cat.) to a solution of 0 16 g (0.37 mmol) of 5 m 4 mL of MeOH at room temperature 2 Degasthe solution under aspirator pressure,and fill the reaction flask with argon three times Repeat this procedure filling with hydrogen gas (see Note 11). Stir

Cyclopropane-Containing

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Analogs

413

the mtxture under H, (1 atm) for 6-8 h (until TLC shows complete consumption of startmg material) 3 Dilute the reaction mixture with 3 mL of MeOH, and filter through a Celite pad (see Note 12) Wash the pad with two 3-mL portions of MeOH 4. Concentrate the combmed filtrates under reduced pressure to yield 0 13 g (0 3 1 mmol, 85%) of 8 as a clear glass Thus compound IS >95% pure by ‘H NMR and can be used m the next transformation without further purification, mp 90-93°C ‘H NMR (CD,OD) 6 7 3G7 20 (camp, 5H), 4 74-4 69 (m, 1 H), 4.48-4 43 (m, 1 H), 3 68 (s, 3 H), 3 37-3 33 (m, 1 H), 3 14 (dd,J= 5 0,14.0 Hz, 1 H), 2 84 (dd,J= 10 1,14 0 Hz, 1 H), 2.25 (dd, J= 8 0, 13 2 Hz, 1 H), 2.15 (dd, J= 3 7, 13 2 Hz, 1 H), 1 74-l 58 (camp, 4 H), 1 10-l 06 (camp, 2 H), 1.00-O 97 (m, 1 H), 0 94 (d, J= 6 4 Hz, 3 H), 090 (d, J= 64 Hz, 3 H), “C NMR (CD,OD) 6 174.4, 174.0, 173 9, 138 6, 130.3, 129.5, 127 8, 71 5, 55 8, 52 7, 52 2, 41 5, 39 0, 25 9, 25 2, 23 3, 21 9, 20.1, 11 1; IR (nulol) v 3298, 1744, 1636, 1560, 1052 cm-‘; mass spectrum (CI) m/ z 420.2495 (C,,H,,N,O, + H requires 420 2498), 26 1,2 12 (base)

3 7.6. [7S, 2R 4S]-2-(5-L-Tyrosyl-Aminoethyl-4-HydroxyjCyclopropyl- 1-L-Phenylalanyl-L-Leucyl Methyl Ester 1 To 50 mg (0.12 mmol) of 6 m 1 2 mL of DMF at -10°C add 5 1 mg (0 38 mmol) of HOBt, 44 mg (0.16 mmol) of Boc-Tyr-OH, and 33 mg (0 16 mmol) of 1-(3dimethylammopropyl)-3-ethylcarbodnmrde hydrochloride (EDC) Stir the mixture at this temperature for 2 h, and then remove the bath and stir for 14 h at room temperature Add 3 mL of EtOAc and 1 mL of saturated aqueous NaCl, and separate the layers Extract the aqueous layer with two 3-mL portions of EtOAc, and combme the organic layers Wash the organic layer with two 4-mL portions of 10% aqueous citric acid, two 4-mL portions of saturated aqueous NaHCO,, and two 4 mL portions of saturated aqueous NaCl (see Note 13) Dry the organic layer over Na,SO,, filter, and concentrate under reduced pressure to yield a light yellow solid Purify the crude solid by flash chromatography using CH2C12/MeOH (20.1) as eluant to yield 53 mg (0 08 mmol, 65%) of the protected pentapeptide as a whrte sohd mp 130-132°C; ‘H NMR (CD,OD) 6 7 28-7 15 (camp, 5 H), 7 04 (d, J = 8 4 Hz, 2 H), 6 70 (d, J = 8 4 Hz, 2 H), 4.74-4.71 (m, 1 H), 4 474 45 (m, 1 H), 4.23-4 18 (m, 1 H), 3 67 (s, 3 H) 3 48-3 32 (m, 1 H), 3 16 (dd, J= 5 6, 14 0 Hz, 1 H), 3.00 (dd, J= 5.8, 13 9 Hz, 2 H), 2 89 (dd, J= 9 4, 13 9 Hz, 1 H), 2 7&2 56 (m, 2 H), 1 70-l 56 (camp, 4 H), 1.36 (s, 9 H), 1 30-l 21 (m, 1 H), 1 19-O 97 (camp, 2 H), 0 93 (d, J = 6 3 Hz, 3 H), 0 90 (d, J = 6 3 Hz, 3 H, i3C NMR (CD30D)G 175.4, 1745,174.3, 173 8, 1576, 157 2, 1384, 131 4, 1303, 1295, 129 3, 127 4, 116 2, 108 9, 80.7, 69 5, 68.4, 57 8, 55 8, 52 7, 45 9, 41 5, 38 9, 38 5, 28 7, 26 5, 25 9, 24 7, 23 3, 21.9, 20.3, 11 0; IR (CHC13) v 2995, 1735, 168.5, 1045 cm-‘, mass spectrum (CI) m/z 683.3640 (CjGHSON409 + H requires 683 3656), 293 (base)

Dwyer and Mart/n

414

Phe-Leu-OMe

H-Tyr-HN

8

Fig 3 Related structures 7 and 8 6. Dissolve 20 mg (0.03 mmol) of the fully protected pentapepttde m 2 mL of trtfluoroacettc acid, and stir the solutton for 30 mm at room temperature. 7 Remove the solvent under reduced pressure, and trtturate the crude residue wtth two 2-mL porttons of Et,0 to yield a light yellow solid (see Note 14). 8 Dissolve the crude solid rn 2 mL of EtOAc, and wash the organic layer with two 1-mL portions of saturated aqueous NaHCOs and one 1-mL portion saturated NaCl (see Note 15) 9 Dry the organic layer over Na2S04, filter, and concentrate the solvent under reduced pressure to yield a light yellow solid 10 Recrystallize the crude product from 0.3 mL of MeOH and 0.3 mL of Et,0 to yield 16 mg (0.028 mmol, 95%) of 1 as a white solid. mp 12&122”C, ‘H NMR (CDsOD) 6 7 32-7 18 (camp, 5 H), 7 09 (d, J= 8 5 Hz, 2 H), 6 78 (d, J= 8 5 Hz, 2 H), 4 64-4 59 (m, 1 H), 4 4994 46 (m, 1 H), 4 11 (t, J= 7 1 Hz, 1 H), 3.66 (s, 3 H) 3 55-3 53 (m, 1 H), 3 38-3 34 (m, 1 H), 3 20-3 12 (camp, 3 H), 2 97-2 85 (camp, 2 H), 1 74-l 55 (camp, 5 H), 1.05-l 00 (m, 1 H), 0.93 (d, J= 6 5 Hz, 4 H), 0 89 (d, J = 6 5 Hz, 3 H), 13CNMR (CD,OD) 6 176 6, 174 3, 173 9, 173 8, 157.4, 131 4, 130.4, 129.5, 129 4, 127 8, 116.3,69 5,57 8,55 9,52 7,52.2,45 9, 41 6,41 2,38 9,25 9,249,23 2,21 9,20.3, 11 O,IR(nulol)v2998, 1730, 1690, 1140 cm-‘, mass spectrum (CI) m/z 583 3119 (C31H42N407 + H requtres 583 3132), 294 (base), 163

3.2. Discussion The method descrrbed above detatls the efficient synthesis of a cls-substrtuted (-Glyv[CHOH-cp-CONHI-) dipeptide incorporated mto a Leuenkephalin analog. This method relies on a highly dtastereoselecttve and enanttoselecttve

cyclopropanatton

step coupled

with a novel vartant of the

Wemreb protocol to afford 1 m only 10 steps from commerctally available starting materials. This method has been used to prepare a number of related analogs of Leu-enkephalin mcludmg 7 and 8 shown m Fig. 3. Conformatronal and btologtcal studies of these novel peptrde rsosteres are under way and will be reported m due course (I 7).

Cyclopropane-Containmg

Leu-Enkephalin

Analogs

415

4. Notes 1 The yellow dtazoester should be used promptly after purtfication. For prolonged storage, keep this compound m a refrigerator 2. The diastereomertc excess was determined by ‘H NMR analysts of the crude reaction mixture, whereas the enanttomertc excess was determined by choral shift experiment with Eu(hfc)s on the correspondmg diol 3. All operations mvolvmg ozone should be conducted m a well-ventilated fume hood 4 The quality of the NaBH4 used in the reduction of the ozomde has a profound effect on the overall yield This reagent should be snow-white and free-flowing 5 The addition of the saturated solution should be done slowly, since this process IS quite exothermtc It 1salso important to concentrate the solutton completely, since the alcohol 1s somewhat soluble m aqueous solutton. 6 The crude material can be used in the next transformatton without further purttication Some mstabthty has been observed on prolonged exposure of this mtermediate to sthca gel 7 It 1s Important to use a fresh bottle of trtmethylalummum to obtain consistent yields on this reaction In addttton, use of excess trtmethylaluminum over the quantities indicated will lead to destructton of the starting material and reduced yields of product. 8 It 1s necessary to add the aqueous acid solutton to hydrolyze all the alummum salts. If this is not done, extraction of the product 1s troublesome 9 For ease of preparation, this compound can be used m the next step crude and purified after the estertficatton wtth dtazomethane. 10. The ethereal solutton of dtazomethane can be added dropwtse using either a flame-polished pipet or a plasm dropper. Sharp glass edges and ground glass Joints should be avoided 11 It is important to degas the solutton under aspirator pressure m order to get reproducible results for this reaction 12 It 1stmportant to keep the Cehte pad shghtly moist during the filtration Drying the Cehte pad completely can possibly lead to a metal fire 13 The series of aqueous washes IS necessary to remove byproducts of the reaction as well as excess reagents (such as HOBt) Failure to follow this protocol makes tsolatton of the pure coupled product virtually tmposstble. 14 The trifluoroacetate salt can be recrystallized at this stage with MeOH/EtzO to yield a white solid that can be stored for months m the freezer. 15 Great care should be taken to mmlmtze the amount of aqueous solutton used during neutrahzatton If low yields are encountered, salt the aqueous layers with sodium chloride and reextract with ethyl acetate.

References 1 Stammer, C H (1990) Cyclopropane ammo acids (2,3- and 3,4-methanommo acids). Tetrahedron 45, 223 l-2254 2 Melmck, M J , Bisaha, S N , and Gammtll, R B (1990) Conformattonally restricted PI-P,’ transitton state analogues. synthesis of l(R), 3(R) [l(S), 2(s)] and

416

3

4

5

6 7

8

9

10 11

Dwyer and Mart/n l(S), 3(S) [l(s), 2(S)]-3-[3-Cyclohexyl-2[(Boc)Amlno]-l-Hydroxylpropyl]-2,2dtmethylcyclopropane carboxyhc acid. Tetrahedron Lett 31,96 l-964 Shimamoto, K., Ishida, M., Shmozaki, H., and Ohfune, Y. J (1991) Synthesis of four dtastereomeric L-2-(carboxycyclopropyl) glycines Conformationally constrained L-glutamate analogues. J Org Chem 56, 4167-4176 de Frutos, P , Fernandez, D , Fernandez-Alvarez, E , and Bernabe, M (1992) Synthesis of asymmetric (E)-a-[2-Phenyl(Ethyl)Cyclopropyl] glycmes from serme by diastereoselective dtbromocyclopropanatton. Tetrahedron 48, 1123-l 130 Zhu, Y -F , Yamazakt, T , Tsang, J W., Lok, S , and Goodman, M (1992) Synthesis and taste properties of L-aspartyl-methylated 1-ammocyclopropanecarboxyhc acid methyl esters J Org Chem 48, 1074-108 1 Burgess, K. and Ho, K.-K. (1992) Asymmetric synthesis of protected derivatives of ornithine- and argmine-2,3-methanologs Tetrahedron Lett 33,5677-5680 Martin, S. F , Austin, R. E , Oalmann, C J., Baker, W. R , Condon, S. L , DeLara, E , et al (1992) 1,2,3-Trtsubstttuted cyclopropanes as conformationally restricted peptide isosteres* application to the design and synthesis of novel renm mhibitors J &fed Chem 35,171&1721 Baker, W R , Jae, H.-S, Martm, S F , Condon, S L , Stem, H H , Cohen, J , et al (1992) Conformationally restricted peptide isosteres 2 Synthesis and m vitro potency of dipeptide renm mhtbitors employmg a 2-alkylsulfonyl-3-phenylcyclopropane carboxamtde as a P3 amino acid replacement. BzoMed Chem Lett 2, 1045-1410 Martin, S F , Oalmann, C J., and Liras, S (1993) Cyclopropanes as conformattonally restricted pepttde isosteres. design and synthesis of novel collagenase mhibitors Tetrahedron 49, 3521-3532 Hillier, M C and Martm, S. F (1997) Synthesis of conformationally-constrained HIV-l protease mhibttors, Chapter 22, this vol Martin, S F , Austin, R E , and Oalmann, C J (1990) Stereoselecttve synthesis of 1,2,3-trisubstttued cyclopropanes as novel dipepttde tsosteres Tetrahedron Lett 31,473 14734

12 Blankley,

C J , Sauter, F. J , and House, H 0 (1973) Crotyl dtazoacetate, in collective vol V (Baumgarter, H E , ed ), John Wiley, New York, pp 258-263 Doyle, M P , Winchester, W R., Protopopava, M N., Kazla, A P , and Wenstrum, L J (1996) (lR, SS)-(-)-6,6-Dimethyl-3-oxabicyclo[3 1 Olhexanone Highly enanttoselective mtramolecular cyclopropanation catalyzed by dn-rhodmm(II) tetrakts[methyl 2-pyrrohdone-S(R)-carboxylate], m Organic Syntheses, ~0173 (Boeckman, R. K , Jr, ed.) John Wiley, New York, pp 13-24 Corey, E J and Myers, A G (1984) Efficient synthesis and mtramolecular cyclopropanatton of unsaturated dtazoacettc esters Tetrahedron Lett 23,355%3562 Martin, S F , Spaller, M R , Lnas, S , and Hartmann, B , (1994) Enantio- and dtastereoselectivtty m the mtramolecular cyclopropanatton of secondary allyhc diazoacetates, J Am Chem Sot 116,4493-4494. Basha, A , Lipton, M , and Wemreb, S M. (1977) A mild, general method for the conversion of esters to amides Tetrahedron Lett 18,4 17 l-4 174 Martin, S F. and Dwyer, M. P , unpublished results Organic 5jmthe.w

13

14 15 16 17

24 The I,5Disubstituted Tetrazole Ring as a c&Amide Bond Surrogate Janusz Zabrocki

and Garland R. Marshall

1. Introduction Prolme occuptes a special role among those amino acids mcorporated mto peptides and proteins by the normal ribosomal pathways, smce it is the only residue that leads to an N-alkyl amide bond. In peptide natural products that often have special biosynthetic pathways or unusual posttranslational modtfications, N-methyl ammo acids are common and may play a specral role because of then conformational properties, mcludmg then prochvity for cu-trans isomerism of the amide bond. Numerous peptides with important biological activittes, such as cyclosporm and dtdemnm, contain N-methyl ammo acids Cis-truns isomerism of the N-alkyl amide bond involving the ammo group can readtly be observed (1) m the NMR of prolme and N-methyl ammo acid-contaming peptides. In the caseof angiotensin and thyrohberm (TRH) analogs, the quantity of czs-isomer m aqueous solutton was correlated (2) with the biological activity. This suggested that the cu-isomer might be the one bound to the receptor and responsible for the observed biological acttvtty. Bairaktart et al. (3) have reported that the normal amide bond between an Ile and Lys reszdues m the lmear peptide, bombolitm, has the cis-conformation when bound to phosphohpid micelles. In protem crystal structures, cu-amide bond conformations are occastonally observed for the normal, nonalkylated amide bond. A czsamide bond predisposes the peptide for a reverse turn, a so-called Type VI pturn. Brand1 and Deber (4) have proposed that cis-tram isomerism of proline reszdue might play a role m transduction of transmembrane protems. Others have suggested that this mterconversion may be responsible for many of the slow kinetic events seen m enzyme reactions and protein foldmg. As one can see from all the examples cited above, the czs-conformer of the amide bond From

Methods m Molecular Me&one, Vol 23 Peptfdomimehcs Protocols E&ted by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

417

Zabrocki and Marshall

418

cannot be ignored as a possible candidate for the receptor-bound conformanon of brologically active pepttdes ThusIS certamly more likely for the amide bond of prolme and other N-alkyl ammo acids, but this possibrllty cannot be dtsmissed a prior2 for normal amide bonds as well. Synthetic replacement of the amide bond with a surrogate that would lock the conformatron etther czs or tram would address its role tn molecular recognition.

Although

the cis-olefinic

group mtght appear to be an ideal cu-amide

bond mrmtc, isomertzatton of the cu-a,y-unsaturated carbonyl system to the more stable a$-unsaturated

carbonyl system has precluded Its use m the design

of peptide analogs (5). To get around this problem, Marshall et al. (6) proposed the 1,5-dtsubstrtuted tetrazole ring system, Y[CNJ 1, as an altematrve way of mimicking the czs-conformatton of a pepttde bond. Conformattonal analysts has shown, based on the excellent geometric similarity of the 1,5-disubstituted tetrazole ring when compared to the czs-amide bond, that pepttdes contau-nng this surrogate can assume most (88%) of the conformattons available to the

parent compound (7,8). 2. Materials 2.7 - Reagents 1 Carbobenzoxy-ammo acid with side chain appropriately protected* In examples, carbobenzoxy-L-alanme (Z-Ala-OH), Z-Pro-OH and Z-Phe-OH, were used to generate dipeptides 2 tert-Butoxycarbonyl-ammo acid with side chain appropriately protected In examples, tert-butoxycarbonyl-phenylalanme (Boc-Phe-OH), Boc-Arg(Tos)-OH, Boc-Gly-OH, Boc-Pro-OH, and Boc-Ser(Bzl)-OH were used 3 Ammo acid benzyl ester with side chain appropriately protected In examples, L-alanme benzyl ester (Ala-OBzl) was used 4 Ammo acid methyl ester with side chain appropriately protected In examples, L-tyrosme methyl ester (Tyr-OMe) was used 5 Dipeptides protected with carbobenzoxy group and benzyl ester group: In examples, Z-Ala-Ala-OBzl, Z-Phe-Ala-OBzl, and Z-Pro-Ala-OBzl were used and prepared by standard solution peptide chemistry 6 Boc-Arg(Tos)-benzyl ester Merrifield resin (chloromethyl-1% divmylbenzenepolystyrene substituted with Boc-(Arg[Tos]-OH, 0 4mmol/g) 7 Phosphorus pentachloride (PC&) 8 Qumolme 9 Hydrazorc acid (II&) 10 Chloroform 11 Ethyl acetate 12. Hydrochloric acid (HCl). 13 Sodium bicarbonate (NaHC03) 14 Dichloromethane. 15 Acetone

The 1,5-D/substituted 16 17 18 19 20. 21 22 23 24 25. 26. 27. 28 29 30 3 1. 32 33 34 35 36 37 38

Tetrazole Ring

419

Methanol Ethanol Potassmm btsulfate (KHSO,). Hydrogen bromide (HBr) in acetic acid (30% solution) N-Methylmorpholme Isobutyl chloroformate. N,N-Dimethylformamide (DMF). Sodium chloride (saturated solution) Sodium sulfate (anhydrous) Ether. Petroleum ether (boiling range 3&6O”C) Acetic acid. 10% Palladium on charcoal (Pd/C) N-Hydroxybenzotriazole (HOBT). Dicyclohexylcarbodumide (DCC) Trifluoroacetic actd (TFA) Triethylamme (TEA) Amsole Hexane a-Chymotrypsm Butanol. Diphenylphosphoryl azide (DPPA) Hydrogen fluoride (HF)

2.2. Chromatography 1 TLC, 250-nm silica gel GF precoated umplates (Analtech) 2 Columns for flash chromatography packed with silica gel 60 (2 x 15 cm, 4 x 15 cm, 55x 15cm) 3 Normal-phase cartridge 8SI 10 pm Radial-PAK and (a) ethyl acetate/hexane (1: 1) or (b) ethyl acetate/hexane (1 2). 4. Reverse-phase analytical column: Vydac Cis (0.46 x 25 cm, particle size 5 urn) 5 Reverse-phase preparative column: Vydac Cis (10 mm x 25 cm, particle size 5 pm). 6. Solvents: (a) 0.05% trifluoroacettc acid m Hz0 and (b) 0.033% trifluoroacetic acid m acetomtrile/H20 (90. IO).

3. Methods The conversion of the amide bond of the protected dtpeptide 2 into a tetrazole ring 4 joining the two side chains of the dipeptide with retention of the chiral integrity at the two a-carbons requires special experimental conditions (Fig. 1). In our initial experiments (6) and in those reported by Yu and Johnson (9), epimerization of one or both chiral centers was observed. The use of PCl,/HNs alone to convert the amide bond into the tetrazole via the imidoyl chlortde 3 leads to eptmerizatlon of the amino acid N-terminal to the

420

Zabrocki and Marshall L

X=ZPht Y = OMe. OBrl

‘WW,

CwAmlde h

4

R2

X-NH-CH

CH-CO-Y

X-NH-CH

5 % CH-CO-Y

/ “\ 0

H

Figure 1 tetrazole ring. The resulting diastereomeric mixture can then be separated mto compounds 5 and 6. The simple modification of the reaction conditions by the addition of qumoline during the formation of the imidoyl chloride intermediate, as first suggested by Hirai et al. (IO), was efficient in preventmg epimerization of the N-terminal amino acid residue of the protected tetrazole dipeptide (II). Only small amounts (3-5%) of the undestrable epimer were obtained when this procedure was used. The resulting tetrazole dipeptide is sensittve to base, and, therefore, special protecting schemes had to be invoked in order to mamtam its chiral integrity during peptide synthesis. In fact, the traditional procedure of solid-phase synthesis for neutrahzatton (10% triethylamme m methylene chloride-Boc strategy) or removal of protecting group (20% piperidme m DMF-FMOC strategy) was sufficient to cause epimerization of the a-carbon on the C-terminal side of the tetrazole ring. This sensitivity to basic conditions required the use of actdolytic removable protecting groups. The use of Z for ammo protection and benzyl ester for carboxyl protection with differential removal of the Z group by HBr/ AcOH has proven to be a practical route to a wide variety of tetrazole dipeptides (12). Immediate acylation of the tetrazole dipeptide with a Boc ammo

The 1,5-Disubstituted i!-L-Ala-L-Ala-OBzl 2

Tetrazole Ring Z-L-Ala-y[CN,]-L-Ala-OBzl

TH: s’~~““a 3’

1’

421

HWHOAc

96%

HBr*L-Ala- y[CN,]-L-Ala-OBzl

I Boc-Phe-L-AlaY[CF$]

’

2

Boc-Phe-L-Alay[CN,]-L-Ala-OBzl H2/Pd

1

s

95%

-L-Ala-OH

11

1) H Phe-Arg(Tos)-Polymer 2) 4 steps SPPS

DCC,HOBT

I Boc-Arg(Tos)-Pro-Pro-Gly-Phe-L-Ala-~[CN~JL,D-Ala-Phe-Arg(Tos)-Polymer

12

1)HF 2) HPLC

I Arg-Pro-Pro-Gly-L-Alay[CN,]-L-Ala-Pro-Phe-Arg Ala- y[CN,]-Ala]” -BK 13 Arg-Pro-Pro-Gly-L-Ala,+,

and

[CN+D-Ala-Pro-Phe-Arg

[Ala- \y [CN,] -D-Alap ’ -BK

14

Figure 2

acid was necessary to prevent formation of dlketoplperazme, which 1s favored because of the cu-conformation of the amide bond surrogate of the dipeptlde.

3.1. Preparation of Protected Tetrazole Dipeptides Z-AA-Y[CN&AA-OBzl, Fig 2 1 Add qumolme(2 mmol) at room temperatureto a stirred solution of PCIS(1 mmol) m chloroform (5 mL) (a white precipitate ISformed) 2. Stir the mixture for 20 mm before the crystalline dlpeptlde (1 mmol) IS added m portions with stirring at such a rate that the temperature stays below 20°C 3 After 30 mm at 20°C (a clear solution forms), add a benzene solution of hydrazolc acid (3 mL) (Note 1) Stir the reaction mixture at room temperature for 1 h before evaporation

422

Zabrocki and Marshall

4. Par&Ion the crude residue between ethyl acetate and water (30 mL of each) Wash the organic layer with 1 N HCl (2 x 15 mL), 1 N NaHCOJ (2 x 15 mL), Hz0 (2 x 15 mL), and saturated NaCl solution (30 mL) 5 Evaporate the dried (Na$O& ethyl acetate solution, and purify the residue by flash chromatography to give the tetrazole derlvatlve as only one stereolsomer

3.1.1. Preparation of Z-Ala-Y[CN&Ala-OBzl8 Use the procedure above with Z-Ala-Ala-OBzl 7 and separate Z-AlaY[CN4]-Ala-OBzl 8 from unreacted starting material by flash chromatography (solvent system dichloromethane:acetone, 30: 1, v/v) to give isolated product (24.9%) as white crystals; mp = 142-143°C; [a]25D = -51.2” (c 1, MeOH), TLC Rf = 0.1 (hexane:ethyl acetate 4: 1, v/v) ; HPLC (a) tR = 7.9 min; FABMS m/e 4 10 (MH+), calcd for C21H2304N5409 3 1.2 Preparation of Z-Pro-Y[CN,JAla-OBzl

16, Fig. 3

Use the procedure above with Z-Pro-Ala-OBzl 15 and separate Z-ProY[CNJ-Ala-OBzl 16 from unreacted starting material by flash chromatography (solvent system dlchloromethane.acetone, 30: 1, v/v) to give the isolated product as white crystals (68.3%); mp = 97.5-98°C; [a]25D = -15.9” (c 0.5, MeOH), TLC Rf = 0.55 (hexane:ethyl acetate 1: 1, v/v; HPLC (a) t, = 12.9 mm; FABMS m/e 436 (MH+); calcd for C23H2S04N5435. 3 1.3. Preparation of Z-Phe-Y[CN,]-Ala-OBzl24,

Fig. 4

Use the procedure above with Z-Phe-Ala-OBzl 23 and separate Z-PheY[CNJ-Ala-OBz124 from unreacted starting dlpeptide ester by flash chromatography (solvent system dlchloromethane.acetone, 60:1, v/v) to give the isolated product (62%) as white crystals; mp = 145-146°C; [a]25D = -61.2” (c 1, MeOH),

TLC R, = 0.38 (hexane.ethyl

acetate 3.1, v/v); HPLC

(b) tR = 8 2

mm; FABMS m/e 486 (MH+), calcd for (&H2,04N5 485. 3.2. Preparation of Protected (Boc-AA-AA-Y[CNJAA-OBzl

Tetrazole Tripeptides and Boc-AA-AA-Y[CN&AA-Oh’)

For the synthesis of protected trlpeptldes

BOGAA-AA-Y[CN,]-AA-OBzl,

the Z group from Z-AA-Y[CN,]-AA-OBzl IS removed with HBr/AcOH (Note 3). The resulting hydrobromlde is coupled with Boc-AA-OH using the mixed anhydride procedure with isobutyl chloroformate (the method of choice for preventing cychc [AA-Y {CN4}-AA] diketopiperazme formation) to give BocAA-AA-Y[CN&AA-OBzl. The subsequent removal of the benzyl ester group by hydrogenolysls leads to the tnpeptlde acid.

The 1,5Disubstituted

Tetrazoie Ring

Z-L-Pro-L-Ala-OBzl 15

i;Td S’6~~nc”“e 3

;

423 Z-L-Pro-y[CNJ

HWHOAc

99%

1

HBr*L-Pro-yl[CN,]-L-Ala-OBzl Boc-Arg(Tos)-OH

-L-Ala-OBzl Is

17

MA, 75% I

Boc-Arg(Tos)-L-Pro-

Y [CNJ -L-Ala-OBzl

18

Boc-ArgfTos)-L-Pro-

Y [CNJ-L-Ala-OH

19

H-Gly O&l

DCCJHOBT,

Boc-Arg(Tos)-L-Pro-

51%

Y [CN,]-L-Ala-Gly-OBzI

20

HZ/Pd

I Boc-Arg(Tos)-L-Pro-

Y[CNJL-Ala-Gly-OH H Phe-Ser(BzIbPro 2) HF 31 HPLC

Phe Arg(Tos)

21 Polymer

DCWIOBT

1 Arg-L-Pro-y,[CNJL-Ala-Gly-Phe-Ser-Pro-Phe-Arg [Pro- q~ [CN,] -Aiaf bK

22

Figure 3

3.2.1. Preparation of Boc-Arg(T.os)-Pro-Y[CN,]-Ala-OH

19

3.2.1 .l BOC-ARG(Tos)-PRO-Y[CN,I-ALA-OBZL 3.2 1 1 1 Removal of Z Group from Dipeptrde Z-Pro-y[CN,l-Ala-OBzI 16 1 Treat (stirrmg) a solution of 970 mg (2 mmol) of the dlpeptlde Z-Pro-v[CNQ]-AlaOBzl16 m 1 mL of acetlc acid with 5 mL of 30% solution of HBr in acetic acid 2 After 20 mm at room temperature (Note 2), pour the solution into 50 mL of ether (precooled to -lO”C), with vigorous stirring To the resulting preapltate, add 20 mL of petroleum ether (3&6O”C), allow the mixture to stand for 15 mm at 0°C and then filter 3. Wash the sohd two times with 1: 1 ether/petroleum ether and dry EIZVQCUOto give 748 mg (98%) of dlpeptlde HBr salt 17, a nonhygroscoplc solid, mp = 152-l 53°C;

Zabrocki and Marshall

424 C rrp

I

S

v

Phe

I

Ala

‘i’(CN4)

‘OBzl

w(CN4)

---0Bzl

‘+VN,)

--OH

y(CN4)

1

U HMA

-0Me

-0Me

‘W$)

-0Me

‘W$)

-0Me

Boc

y(CN4)

TFA -

“(CN4)

DH 5 5-6 0

-OH

-OH

Figure 4 [a]250 = 44 301.

3.2.1.1.2.

8” (c 1, MeOH);

Couplmg

FABMS

m/e 302 (MH+), calcd for C,,H,,O,N,

with Boc-Arg(Tos)-OH

1 Cool a solution of 813 mg (1 9 mmol) of Boc-Arg(Tos)-OH m 5 mL dlchloromethane/DMF (1.1) to -15’C and treat with 0 21 mL (1 9 mmol) of N-methylmorpholme, followed by 0.26 mL (1.9 mmol) of lsobutylchloroformate. 2 Stir the mixture for 10 mm, then add 732 mg (1 9 mmol) of sohd HBr HN-Prov[CN4]-Ala-OBzl 17, followed by addition of 0 21 mL (1 9 mmol) of Nmethylmorpholme at such a rate that the temperature stays below -10°C Stir

The 1,5-D/substituted Tetrazole Ring

425

for 1 h at -10°C allow the mtxture to warm up slowly to room temperature, and stir overnight 3 Remove the solvents zn vucuo. Take the residue up m ethyl acetate (50 mL), and wash wtth 1 NNaHSO, (3 x 20 mL), 1 NNaHCO, (3 x 20 mL), water (2 x 20 mL) and saturated NaCl solution (20 mL). Evaporate the dried (Na,SO,) ethyl acetate solutton, and purify the residue by flash chromatography (solvent system dichloromethane/acetone, 3.1, v/v) Isolate the tripepttde derivative 18 (1 01 g, 75%) as an amorphous powder; [a] 25o = +2 0’ (c 1, MeOH); TLC R, = 0 35 (drchloromethane*acetone 3 1, v/v), R, = 0.48 (dlchloromethane methanol 10: 1, v/v), FABMS m/e 712 (MH+), calcd for C,,H,,O,N,S 7 11, tH NMR (CDCI,). 6 1.40, 1 2-l 7 (s over m, 12H, BocCHs and Argj3,yCH2), 1 95 (d, J = 7 4, 3H, AlaPCH,); 2 36 (s, 3H, TosCH,); 3.02-3 20 (m, 2H, ArgGCH2), 3 60-3 78 (m, 2H, ProGCH2), 4.28-4.40 (m, lH, ArgaCH), 5.15-5 28 (m, 3H, CH2Ph and ProaCH), 5.70 (q, J = 7 4, lH, Ala&H), 6 40 (broad s, 2H, NH); 7.20 (d, 2H, TosPh), 7 25-7.38 (m, 5H, OBzlPh), 7 74 (d, 2H, TosPh) r3C NMR (CDCl,) 17.43 (AlapC), 21 45 (tosCH3), 23.81 (ArgyC); 25.01 (ProyC), 28 37 (BocCH3), 29 28 (ArgpC), 30 94 (PropC), 40.48 (ArgGC); 47 03 (ProGC); 50 64,5 1.15 (Arg and Pro&); 56 69 (Ala&), 68.24 (CH,Ph), 79 96 (BocC), 125 91, 128 19, 128 55, 128 60, 128 98, 134 49, 140.93 (Tos and Bzl Ph); 155 3, 156 21, 156 67 (CN4, Boc GO, ArgGC), 168 07, 170.93 (Arg and Ala C=O). Anal calcd for C,,H,,O,N,S* C, 55.68, H, 6 37; N, 17.71; S, 4 50 Found: C, 55 26, H, 6.74, N, 17 30, s, 4 59 3.2 1 2 BOC-ARG(To.s)-PRO-v[CN,I-ALA-OH 1 Hydrogenate a solutton of 1 0 1 g (1.4 mmol) of the trtpepttde benzyl ester 18 m 20 mL ethanol and a few drops of acetic acid overnight m the presence of 250 mg 10% Pd/C (Note 4) 2 Evaporate the filtered solutton and take the restdue up m a small amount of ethyl acetate and sufficient 1 N NaHCO, solution (1.9) 3. Acidify the aqueous phase with solid sodium bisulfate to pH = 2.5, and extract the chromatographrcally pure (TLC) tripepttde actd with ethyl acetate (3 x 30 mL) 4 Evaporate the dried (Na,SO,) ethyl acetate solutton, and Isolate the product 19 (766 mg, 88%) as a glassy powder; [a] 250 = -7 9” (c 1, MeOH), TLC R, =0.54 (Chloroform*methanol acetic acid 9+1:0 5, v/v), Rf = 0 88 (Dtchloromethane: methanol.water 14 6 1, v/v), FABMS m/e 622 (MH+), calcd for C26H3907N$ 621 ‘H NMR (CDC13). 6 1.41, 140-1.60 (s over m, llH, Boc CH, and ArgyCH,), 1 95, 1 8-2 0 (d over m, J = 7.3(d), 5H, AlaPCH, and ArggCH2), 2 14-2.32 (m, 4H, Prop,yCH& 2 38 (s, 3H, TosCH3), 2.9-3.1 (m, 2H, ArgGCH2); 3 63.8 (m, 4H*, ProGCH2), 4 22-4.32 (m, lH, ArgaCH); 5 165 24 (m, lH, Pro&H); 5.72-5 84 (m, 2H*, Ala&H); 7.14-7 40 (broad m, 6H*, TosPh and COOH), 7 67 (d, J = 7 83,2H, TosPh). t3C NMR (CDCl,) 17 82 (AlapC); 2 1 58 (Tos CH3), 24 53 (ArgyC), 25 11 (ProyC); 28 48 (BocCH,); 28 92 (ArgpC), 30.61 (PropC), 40 72 (ArgGC); 47 26 (ProGC); 49 94, 51 89 (Arg and Pro aC), 56 16 (Ala&), 80 23 (BocCq), 126 03,129 28 (Tos Ph); 155 64,156 21,156 60 (CN,, Boc GO, and ArgGC); 170 28, 171 13 (Arg and Ala C=O). Anal calcd for

426

Zabrocki and Marshall &,Hs907N9S+ C, SO 23, H, 6 32, N, 20 28; S, 5 16 Found C, 49 60, H, 6 77, N, 20 13, s, 4 05

3.2.2. Preparation of Boc-Phe-Ala-Y[CN,JAla-OH

11

3 2.2.1. BOC-PHE-ALA-Y[CN&ALA-OBZL 3 2 2 1 1. Removal

of Z group from dipeptlde

Z-L-Ala-Y[CN,]-L-Ala-OBzl

Treat a solution of 586 mg (1 43 mmol) of Z-L-Ala-Y[CN,]-L-Ala-OBzl 8 m 1 mL of acetic acid (stnrmg) with 3 5 mL of 30% solutron of HBr m acetic acid After 20 mm at room temperature, pour the solution mto 30 mL of ether (precooled to -lO’C), with vtgorous stnrmg. Discard the oily hydrobromtde prectpttate and the upper phase Wash the or1 with ether (3 x 20 mL), and dry zn vucuo over KOH to give 490 mg (96 3%) of drpeptlde HBr salt as a very hygroscoptc glass (Note 3), FABMS m/e 276, calcd for C,3H,702N5 275

3 2.2.1.2. Coupling with Boc-Phe-OH Couple Boc-Phe-OH (358 mg, 1 35 mmol) with the HBr salt of H,N-Ala-y[CN,]Ala-OBzl (480 mg, 1 35 mmol, 9) using rsobutyl chloroformate as described above m Subheading 3.2.1. Crystallize the crude material from ethyl acetate/petroleum ether to yield 560 mg (66 6%) of white crystals 10. mp = 159-160°C [a]25n = -26 6” (c 0 5, MeOH), TLC R,= 0.67 (dtchloromethane,acetone 7.1, v/v), Rf= 0 45 (hexane.ethyl acetate 1 1, v/v), FABMS m/e 523 (MH+), calcd for C,,H,,O,N,

522 ‘H NMR (CDCl,)

6 1 37 (s, 9H, BocCHs), 1.59 (d, J = 6 9, 3H, Ala2PCH3), 1 91 (d, J = 7 3, 3H, AlasPCHs), 2 95 (d, J= 6.7,2H, PhePCH2), 4 164 28 (m, lH, PheaCH), 5.20 (m, 2H, CH,Ph), 5 35-5 48 (m, lH, Ala,aCH); 5 56 (q, J= 7 3, lH, AlagCH), 6.78 (d, lH, NH); 7 &7 4 (m, 1 lH, Phe and OBzl Ph and NH) r3C NMR (CDCls). 17 47,19.69 (AlapC); 28 23 (BocCH,), 37 66 (PhepC), 38 77 (Ala&), 55 49,56 10 (Phe and Ala UC), 68 21 (CH,Ph), 80 60 (BocCq), 126 94, 128 17, 128 57, 129 07, 134 48, 136 08 (Z and Phe Ph), 155 00, 155.52 (CN4 and Boc GO), 167 68, 170 83 (Phe and Ala C=O) Anal calcd for C&H,,OsN,. C, 62 05, H, 6 56, N, 16 08 Found C, 62 27, H, 6 63, N, 16 Il.

3 2 2 2 BOC-PHE-ALA-Y[CN,]-ALA-OH

11

Hydrogenate the trtpepttde Boc-Phe-Ala-Y [CNJ-Ala-OBzl (243 mg, 0.46 mmol, 10) with 100 mg 10% PdK to yield 208 mg (84 9%) of crystallme material 11; mp 186--187”C, [alz5,, = -24.2” (c 0.5, MeOH); TLC R, = 0.33 (drchloromethane:methanol 10: 1, v/v); FABMS m/e 433, calcd for C2uH2s05N6 432. ‘H NMR (DMSO). 6 1.28 (s, 9H, BocCH,); 1.55 (d, J = 6.8, 3H, Ala#CH& 1 76 (d, J = 7.3, 3H, AlasPCH,); 2.59-2.83 (m, 2H, PhebCH,), 4.01-4.19 (m, lH, PheaCH); 5.45-5.55 (m, lH, Ala,CH), 5.62 (q, J= 7 3, lH, Ala&H); 6.96 (d, J= 8.7, lH, NH); 7.1-7.5 (m, 5H, PhePh); 8 75 (d, J= 8.6, lH, NH). t3C NMR (DMSO): 6 17.29,19.12 (AlapC); 28 14 (BocCH,); 37 10

The i,5-Disubstituted

Tetrazole Ring

427

(PhepC); 38.05 (Ala,aC); 55.09, 55.69 (PheaCH and Ala,aCH), 77.87 (BocCq); 125.97, 127.78, 129.08, 138.12 (PhePh); 155.16, 156.20 (CN4 and Boc C=O); 169.70, 171.5 1 (Phe and Ala C=O). Anal. calcd for C20H2805N6:C, 55.54; H, 6.53, N, 19.43. Found: C, 55.71; H, 6.62; N, 19.36. 3 2.3. Preparation of Boc- Val- Phe- Y[CN,]-Ala-OH (Fig, 4) 3 2.3.1 REMOVAL OF Z GROUP FROM DIPEPTIDE Z-PHE-Y[CN4]-A~~-OBz~

24

1. Treat a solutton of 1.75 g (3 6 mmol) of Z-Phe-Y[CNJ-Ala-OBzl 24 m 1 mL acetic acid wrth 9 mL of 30% HBr m acetic acid while sturmg. 2. After 20 mm at room temperature, pour the solutton mto 70 mL of ether (precooled to -1O’C) with vtgorous stnrmg After the oily hydrobromtde precrpttates, discard the upper phase Wash the 011wrth ether (3 x 35 mL) and dry In vucuo over KOH to give 1 39 g (89%) of the dipeptrde HBr salt as a very hygroscopm glass (Note 3) 3.2.3.2

COUPLING WITH BOG-VAL-OH

1 Couple Boc-Val-OH (580 mg, 2.67 mmol) with the HBr salt of H#-Phe-Y [CN,]Ala-OBzl (1.16 g, 2 67 mmol) using tsobutylchloroformate as descrtbed above m Subheading 3.2.1. 2 Purify the crude material by flash chromatography (hexane*ethyl acetate, 3.1, v/ v) to yteld 821 mg (55 8%) of whrte crystals 25. mp 154155°C [cx]~~~=-59 0’ (c = 1, MeOH), R,= 0 64 (ethyl acetate:hexane, 1 1, v/v), R,= 0 41 (CH,Cl, acetone, 15.1, v/v), HPLC purrty 95%, tR = 16 03 mm (gradtent 45-90% B m 25 mm ), FAB-MS m/z 551 (MH+), 573 (MNa+), calcd for C2sH3s05N6 550 13C NMR (125 7 MHz, CDCl,) 6 16 46 (Ala C/3), 17.35, 19.05 (Val Cy), 28 24 (Boc CH,), 30 63 (Val CD), 41 11 (Phe Cp), 45 28 (Phe Co), 55 62 (Ala Co), 59 68 (Val Co), 68 10 (Bzl CH2), 78 0 (Boc quat C), 127 49, 128 11, 128 52, 128 58, 128 95, 128.18, 134 63, 135 33 (Phe and Bzl arom), 155.21, 155 69 (Boc C=O, CN& 167.4 1, 17 1.47 (Ala and Val C=O). 3.2.3 2 1. Boc- Val- Phe- Y[CN,]-Ala-OH Hydrogenate Boc-Val-Phe-Y[CN,]-Ala-OBz125 (7 15 mg, 1.3 mmol) m methanol (15 mL) in the presence of 10% Pd/C (150 mg) for 5 h (monitor by TLC) 2 Evaporate the filtered solutton, and wash the restdual solid several times wtth hexane to yreld 671 mg(94%); mp 161-163°C [~I~~,,=-54 6” (c = 0.5, MeOH), Rf = 0 81 (Butanol acetic acid ethyl acetate-water, 1 1’ 1’ 1, v/v); HPLC purity 97%, ta 7 15 mm (gradient 45-90% B m 25 mm); FAB-MS m/z 46 1 (MH+), 483 (MNa+), 459 (MH-); calcd for C22H32N605 460 t3C NMR (75 4 MHz, DMSO) 6 17 26 (Val Cy), 17 99 (Ala Cp), 19 08 (Val Cy), 28 19 (Boc CH,), 30 64 (Val Cp), 38 82 (Phe Cp), 43 85 (Phe Co), 55.13 (Ala Co), 59.37 (Val Co), 77 99 (Boc quat. C), 126.53,128.09, 129 19,136 36 (Phe arom.), 154 98, 155 42 (Boc GO, CN& 169 32, 171 03 (Ala and Val C=O).

428

Zabrocki and Marshall

3.3. Preparation of Protected (Boc-AA-AA-Y[CN&AA-AA-OR,

Tetrazole Tetrapeptides R q Bzl, Me)

The synthesis of protected tetrapeptldes IS achieved by couplmg trlpeptldes Boc-AA-AA-Y[CN&AA-OH with the appropriate ammo acid esters using the mixed anhydride or DCCYHOBT couplmg strategies. 3.3.1. Preparation of Boc-Arg(Tos)-Pro-Y[CN,]-Ala-Gly-OBzl ZO Activate the tnpeptlde acid, Boc-Arg(Tos)-Pro-Y[CN,I-Ala-OH 19 (62 1 mg, 1 mmol) with HOBT (135 mg, 1 mmol) and DCC (206 mg, 1 mmol) m DMF (5 mL) at 0°C Add after 30 mm, a solution of glycme benzyl ester p-toluenosulfonate (337 mg, 1 mmol) and N-methylmorpholme (0 11 ml, 1 mmol) at 0°C. Stir overmght at room temperature, and then filter off the dlcyclohexylurea and remove the solvent removed. Take up the residue in ethyl acetate (50 mL) and wash with 1 NNaHS04 (3 x 20 mL), 1 NNaHC03 (3 x 20 mL), water (2 x 20 mL), and saturated NaCl solution (20 mL) Evaporate the dried (Na$O,) ethyl acetate solution, and purify the residue by flash chromatography (solvent system dichloromethane.acetone, 3 1, v/v) to give the tetrapeptlde derivative 20 (395 mg, 5 1%) as a glassy powder, [cx]~~~ = -8 9” (c 1, MeOH); TLC Rf = 0 20 (dlchloromethane acetone, 3 1, v/v), Rf = 0 54 (dlchloromethane methanol, 10.1, v/v); FABMS m/e 769 (MH+), calcd for C35H,sOsN10S 768 ‘H NMR (CDCl,). 6 1 43 (s, 9H, BocCH& 1 3-l 7 (m, 4H, Arg&yCH& 1.8 (d, J = 7.0, 3H, AlaPCH3), 2 37, 2.08-2 42 (s over m, 7H, TosCH, and Proj3,yCH2), 3 05-3 2 (m, 2H, ArgGCH,), 3 65-3 84 (m, 2H, ProGCH,), 4 09-4 15 (m, 2H, GlyaCH,), 4 3W 48 (m, lH, ArgcKH); 5 12 (s, 2H, OBzlCH& 5 15-5 20 (m, lH, Pro&H), 5 50 (q,J= 7 2, lH, AlaaCH); 6 44 (bs, 2H, NH), 7.21 (d, 2H, TosPh), 7 28-7.36 (m, 5H, OBzlPh), 7 76 (d, J= 8 1, 2H, TosPh), 7.87 (t, J= 4 88, lH, NH) Anal calcd for Cj5H4s0sN,,S C, 54 67, H, 6.29, N, 18.22, S, 4.17. Found. C, 54 17, H, 6 61, N, 17.71, S, 4 58

3.3.2. Preparation of Boc-Val-Phe-Y[CN,]-Ala-Tyr-OMe

(Fig. 4)

1 Couple Boc-Val-Phe-Y[CN4]-Ala-OH (230 mg, 0.5 mmol) with the HCl salt of Tyr-OMe (139 mg, 0 8 mmol) using the mlxed anhydrlde procedure with lsobutylchloroformate as described above in Subheading 3.2.1. 2. Purify the crude material by flash chromatography (dlchloromethane:acetone, 7.1, v/v) to yield 287 mg (90%) of the tetrapeptlde derivative as a glassy powder. [al*% = -9 54” (c =0 5 MeOH); Rf = 0 29 (ethyl acetate.hexane, 1: 1, v/v), R, = 0.15 (dlchloromethane acetone, 7 I, v/v), Rf= 0 90 (ethyl acetate methanol, 7 1, v/ v); HPLC purity 97% (gradient 45-90% B in 25 mm); FAB-MS m/z 638 (MH’), 660 (MNa+), calcd for C,,H,,O,N, 637. 13CNMR (125.7 MHz, CDCl,) 6 16 47 (AlaCP), 16 99, 19.03 (Val Cy), 28.30 (Boc CH3), 30 86 (Val Cp), 36 26 (Tyr CD), 39 27 (Phe Cp), 47.21 (Phe Ca), 52.19 (OCH3), 54 19 (Tyr Co.), 55.69 (Ala Ca), 58 94 (Val Ca), 80.65 (Boc quat C), 115 59, 127 07, 127.77, 129 14, 130 08,

The 1,5-Disubstltuted

Tetrazole Ring

429

134.74 (Phe, Tyr arom.), 155.08, 155.24, 156.33 (Tyr XC, Boc C=O, CNJ, 165 64 (Tyr C=O), 17 1.60, 173 01 (Val, Ala C=O).

3.4. Incorporation into Biologically

of Tetrazole Tripeptides Active Peptides

Because of the high percentage of prolme residues in the nonapepttde bradykmm (BK) and the presence of N-methylalanine in a potent cyclic hexapepttde analog of somatostatm (13), synthesis of analogs of these two compounds 1s chosen to illustrate the strategy of incorporation of the 1,5-disubstttuted tetrazole dipepttde mto longer peptrde sequences. Preliminary results of the rncorporatton of tetrazole dipeptide analogs into biologically actrve peptides, such as thyroliberin (TRH) (6), somatostatin (14), BK (12,25,,, enkephalin (16, I 7), and CCK (18,19), have been reported. A tetrazole analog of deammooxytocm was prepared by Lebl et al (20) m which Leu-Y[CN&Gly-NH, was obtained by a different synthetic route in which the preformed Z-Leu-tetrazole was alkylated with methyl bromoacetate (21).

3.4.1. Preparation of Tetrazole Analogs of Bradykinin The tetrazole-containmg pepttdes, Boc-Phe-Ala-Y[CN&Ala-OH 11 and Boc-Arg(Tos)-Pro-Y[CN4]-Ala-Gly-OBzl 20, prepared m solutron are mcorporated into BK sequences, as analogs of the natural segments Phe-Ser-Pro and Arg-Pro-Pro-Gly using conventtonal solid-phase peptrde synthesis. One gram of Boc-Arg(Tos)-benzyl ester Merrttield resin (0.4 mmol/g) IS extended by the followmg CH& 50% TFA/CH& CH2C12 10% TEA/CH,C12 CH&l, Coupling 2nd Couplmg

synthetic cycle: 3 x 2 mm 5 min and 25 min 3 x2mm 5 mm and 10 mm 3 x 2 mm 3 Eq Boc-AA and 3 Eq DCC m CH,Cl, 3 Eq Boc-AA and 3 Eq DCC m DMF

After the addition of Boc-Phe, the polymer is divided into two halves. 3.4 1.1. [ALAY’[CN~]-ALA]~~~-BK 13 AND [ALAY[CNJ-D-ALA]~~~-BK

14 (FIG. 2)

1 Add Boc-Phe-Ala-y[CN&Ala-OH 11 to Phe-Arg(Tos)-polymer (Merrrfield) using DCC/HOBT m DMF. Complete the BK sequence by successrve addrtrons of Boc-Gly, Boc-Pro, Boc-Pro, and Boc-Arg(Tos) using the solid-phase protocol above This results m compound 12. 2. Cleave the protected peptrde resin 12 (720 mg) with 10 mL of HF/anisole (9.1) for 1 h at 0” (Note 5) to yield 150 mg of crude product

Zabrockt and Marshall

430

3 Punfy part of the crude peptide on a Vydac C,a preparative column (10 x 250 mm) usmg the followmg solvents: A = HZ0 (0 1% TFA), B = 90% acetomtnle/H20 (0 1% TFA) with a gradient of 1530% B in 40 min. Isolate two compounds m the ratio of 2.1 Peak 1 is assigned to (AlaY[CN,]-Ala)6,7-BK 13 and peak 2 to (AlaY[CN&oAla)6y7-BK 14 based, m part, on then relative abundance and previous epimerizatton experiments (11) Characterize the isolated peptides on analytical HPLC (Vydac C ,a) 1540% B in 25 min, ta (1) = 15.1 min and ta (2) = 15 9 min. The two peptides give the same molecular ion, MH+ = 1043, calcd for C4sH7a09N1a 1042. Ammo acid analysis Peak I ’ Arg, 2 30, Gly, 1.00; Phe, 2.03; Ala, 0.81; and Pro, 2 03 Peak 2. Arg, 2.09, Gly, 1.00, Phe, 1 98; Ala, 0 83; and Pro, 1 88. Peak 1: 13C NMR (D,O): 15.85, 16.51 (Ala PC); 21.79, 23.01 (Arg yC); 23.36,23.43 (Pro yC); 25.67 (Arg PC); 26.80,26.99 (Pro PC); 28.05 (Arg PC), 35.53, 35.90 (Phe PC); 38.19 (Ala6 aC); 39.12, 39.27 (Arg SC), 41.02 (Gly UC); 46.47, 46.75 (Pro SC); 50.09, 50.86 (Arg aC); 53.35, 54.12 (Phe aC); 55.46 (Ala7 aC); 57.83, 59.31 (Pro GE); 125.93, 125.99, 127.46, 127.79, 127.84, 134.59, 134.64 (Phe Ph); 155.18, 155.22 (Arg SC); 155.55 (CN4); 166.34, 167.57, 169.55, 170.34, 170.75, 170.91, 173.28, 174.41 (C=O). The presence of the dtastereotsomer containmg D-Ala is the result of epimerizatlon of the a-carbon on the C-terrmnal side of tetrazole rtng, whtch presumably occurred (11) on prolonged exposure to the 10% solution of triethylamme m methylene chloride (used for neutralization of the ammo group during solid-phase peptide synthesis). 3.4.1.2.

(PRoY[CN&ALA)~~~-BK

22

1, Establish the peptide sequence Phe-Ser-Pro-Phe-Arg(Tos) on the Merritield polymer by the above protocol. 2. Hydrogenate Boc-Arg(Tos)-Pro-v[CNJ-Ala-Gly-OBzl20 to give the free acid, Boc-Arg(Tos)-Pro-v[CNJ-Ala-Gly-OH 21, and then couple it to the peptide polymer (I 50 mg) using DCC/HOBT in DMF. 3 Cleave the peptide from the polymer (200 mg) with 10 mL of HF/anisole (9.1) for 1 h at 0” The crude yield is 58 mg. 4 Purify part on a Vydac C,, preparative column using the same conditions as above for the other BK analogs. Only one compound 22 is isolated with a ta = 15 3 mm and an MH+ = 1059, calcd for C4SH70010N181058 Ammo acid analysis. Arg, 2.37, Gly, 1.OO,Phe, 2 37, Ser, 0 90, Ala, 0.36; and Pro, 1.41. 13CNMR (90): 15.79 (Ala PC), 2 I 78,22.98 (ArgyC); 23 03,23.42 (Pro yC); 25 63 (Arg PC); 26.91,28 00 (Pro PC), 29 46 (Arg PC), 35 53, 36.03 (Phe SC); 39.15, 39 33 (Arg 6C); 41.44 (Gly aC), 46.5 1,46 8 1 (Pro SC); 50.09,50 19 (Arg UC, Pro2 CXC);5 1 58 (Ser PC), 5 1 57 (Arg CXC),53.56,53 62 (Phe aC), 55.91 (Ala3 UC); 59 33,59 83 (Pro7 aC, Ser aC); 125 99, 126 02, 127.51, 127.58, 127 94, 128 08, 134 91, 135.02 (Phe Ph), 155 54, 155.57 (Arg 6C, CN4); 166.88, 168.43, 168.66, 169.21, 171 32, 171.43, 172.34, 17. The three bradykmm analogs were previously assayed for agomstic/antagonistm activity on isolated rat uterus by the protocol described by Marshall et al.

The 1,5- Disubs tituted Te trazole Ring

431

(22) and m binding assaysto bovine brain membranes. The lack of activity for the three analogs implies that the c&conformer of either Pro2 or Pro’ is not likely to be the biologically active conformer recognized at the BK B2 receptor. This assumes that the additional steric bulk of the tetrazole rmg does not preclude mteraction of the correct conformer with the receptor and that specific recognition of the adjacent hydrogen bond donor and acceptor functionality of the czs-amidebond IS not a requisite for molecular recogmtlon 3.42. Preparation of a Cyc/ic Somatostatin Hexapeptide Containing a Tetrazole cis-Amide Bond Surrogate (Fig. 4) In a cychc hexapeptide, cyclo[-o-Trp-Lys-Val-Phe-WMeAla-Tyr], an analog of the somatostatm the dtpeptide unit Phe-N-MeAla is replaced by a dipeptide surrogate, Phe-Y[CN&Ala, with the amide bond locked m the CESgeometry by the 1,5-disubstituted tetrazole ring. The synthesis of Boc-Val-Phe-Y [CN,]Ala-Tyr-OMe is achieved by hydrogenolysis of the C-terminal benzyl ester group of Boc-Val-Phe-Y[CN,]-Ala-OBzl 25 and coupling of the resultmg

trlpeptlde with Tyl-OMe using the mixed anhydride procedure, as mentioned above After removal of the Boc-protecting group (TFA/CH,Cl,), the tetrapeptlde benzyl ester 1s coupled to BOC-D-Trp-Lys(.Z’)-OH giving the fully protected hexapeptide Boc-n-Trp-Lys(Z)-Val-Phe-Y[CN&Ala-Tyr-OMe. Because of the known proclivity of the C-terminal a-carbon of tetrazole dipeptides to epimerize on prolonged base exposure, removal of the methyl ester group is achieved by an enzymatic hydrolysis with chymotrypsm instead of saponification. After Boc deprotection (TFA), the unprotected hexapeptide is cyclized using DPPA m the presence of solid sodium bicarbonate, followed by removal of the 2 group of Lys (HF) HPLC purification yields both the desired product as well as a side product (7%), presumed to be the t-butyl adduct to the Trp and/or Tyr side chain. 3.4 2.1 BOC-D-TRP-LYS(CBZ)-VAL-PHE-Y[CN,I-ALA-TWOME 3.4.2.1.1. Removal of Boc Group from Tetrapeptlde Boc- Val-Phe-Y[CNJ-Ala-Tyr-OMe 1 Cool a solution of protected tetrapeptide (484 mg, 0 75 mmol) in 3.5 mL CH2C12 to 5”C, and treat with 3 5 mL TFA 2 After stmmg at 0-5°C for 35 min (TLC monitoring), pour the reaction mixture into 50 mL of ether (precooled to -20°C)

with vigorous stirring, whereupon

the

product precipitates as a white solid 3 After 5 mm, add petroleum ether (50 mL), and allow the precipitate to settle for 30 min at 0°C.

4 Filtrate and wash the precipitate with 1: 1 ether:petroleum ether (3 x 20 mL) Dry WI vucuo (over NaOH)

to yield the tetrapeptide trlfluoroacetate

salt (419 mg,

432

Zabrocki and Marshall 87 3%) as a very hygroscoptc amorphous powder, Rf= 0 47 (chloroform/MeOH/ water 90.10 1, v/v) 3.4 2 1.2 Coupling

wrfh Boc-o-Trp-Lys(Z)-OH

1 Couple Boc-o-Trp-Lys(Cbz)-OH (368 mg, 0 65 mmol) with the TFA salt of ValPhe-Y [CNJ-Ala-Tyr-OMe (4 19 mg, 0.65 mmol) using the mixed anhydrrde procedure with tsobutylchloroformate as described earher 2 Purify the crude materral by flash chromatography (CH,Cl,.acetone, 3 I, v/v) to yield the hexapeptrde derivative as an amorphous solid (590 mg, 83 6%) [c.x]~~~= -19 80” (c = 0 5, MeOH); Rf = 0 25 (CH,Cl, acetone, 3 1, v/v), HPLC purity 96%, tR 14.61 mm (gradient 45-90% B in 25 mm), tR = 9.20 (isocratrc 60% B), FAB-MS m/z 1086 (MH+), calcd for C,,H,,N, ,O,, 1085. 3.4.2 2 BOG-D-TAP-LYS(CBZ)-VAL-PHE-Y[CNd]-ALA-TYR-OH

Suspend the hexapeptide Boc-n-Trp-Lys(Cbz)-Val-Phe-‘P[CNJ]-Ala-OMe (0 543 mg, 0 5 mmol) m DMF:water (4 6,40 mL), and adjust the pH to 5 5 using 1 N acetic acid Add a-Chymotrypsin (20 mg), and stir the reaction mixture at pH 5 5-6 0 until the consumption of 0 I NNaOH ceases (16 h) and no starting material IS detectable by HPLC Termmate the reaction by the addition of methanol (50 mL), and after 1 h evaporate the solution to dryness Take the residue up m ethyl acetate (50 mL) and wash wtth water (3 x 20 mL) Dry over anhydrous MgS04, and evaporate the organic phase to grve the pure hexapepttde acid as a glassy powder. Yield 471 mg (88%), HPLC purity 97%, tR = 6.47 mm (Vydac C,s, tsocratlc 60% B), [a]25t, = -12 6” (c = 0.5, MeOH), Rf = 0 88 (butanol .acetrc acid.ethyl acetate:water 1 1 1.1, v/v); Rf = 0.70 (CH,Cl,.MeOH 5.1, v/v). 13CNMR (125.7 MHz, CDCl,) 6 16 28 (Val Cy), 18 01 (Ala Cp), 19 13 (Val Cy), 21 79 (Lys Cy), 28 39 (Boc CH,), 29 23,29 26, 29 52 (Trp, Lys, Val Cp), 3 1.02 (Lys C6), 36 34 (Tyr C/3), 39 01 (Phe Cp), 40 28 (Lys Cs), 47 28 (Phe Co), 52 16 (Trp Co), 53 96 (Tyr Co), 55 18 (Ala CCL),55 61 (Lys Co), 58 56 (Val Co), 66 81 (Cbz CH,), 80 63 (Boc quat. C), 109 41, 111 65, 11581, 118.41, 11966, 122.31, 12326, 12689, 12697, 12751, 12800, 128 18, 128 56, 128 95, 129.10, 130 16, 135.05, 136 38, 136 44 (Trp, Phe, Tyr and Cbz arom.), 154 93, 155.08, 155.87, 157 04 (Tyr Cx, Boc and Z C=O, CN& 165 70, 172.07, 172 65, 173.50 (Tyr, Ala, Val, Lys and Trp C=O).

3 4.2.3. CYCLO(D-TRP-LYS(Z)-VAL-PHE-Y![CN4]-A~~-T~@* 3 4 2.3 1 Removal of Boc from Hexapeptlde

Boc-D-T~~-L~~(Z)-V~I-P~~-Y[CN~]-

Ala-Tyr-OH.

Deprotect 500 mg (0.46 mmol) of hexapeptide usmg 4 5 tnL+ TFA: U-&Cl2 (1 1) and 0.152 mL amsole as described above to yreld 300 mg of deprotected hexapepttde (60%); Rf = 0.81 (butanol:acetic actd:ethyl acetate: water, 1 1: I : 1, v/v).

The 1,5-D/substituted

Tetrazole

433

Ring

3 4.2.3 2 Cyclization of the Hexapeptlde

D- Trp-Lys(z)-

Val- Phe- Y[CN,l-Ala-

Tyr

1. Cool a solutton of hexapepttde (250 mg, 0.23 mmol) in 28 mL DMF to 0°C and slowly add drphenylphosphoryl aztde (DPPA, 54 ml, 0.25 mmol), followed by NaHCO, (97 mg, 1.15 mmol) 2 Star the reaction mixture for 3 d (no substrate was detectable by TLC), then filter, and evaporate to dryness under hrgh vacuum. 3 Add 5 mL water to the VISCOUSoily residue, and precipitate the crude product as an amorphous solid (223 mg). 4 Purify the crude material by flash chromatography (CH,Cl,.methanol5 1, v/v) to yield 187 mg (85%) of the cyclic hexapepttde Rf = 0 17 (CHpC1,:methanol 5 1, v/v), R, = 0 70 (butanol acetic acrd:ethyl acetate’water, 1.1.1 1, v/v) This material 1s used m the deprotectron step wrthout further purrticatton. 3.4.2.4.

CYCLO(D-TRP-LYS-VAL-PHE-Y[CN&ALA-TYR)

1 Treat the cyclrc peptide cyclo(D-Trp-Lys@)Val-Phe- Y[CN,]-Ala-Tyr) (143 mg, 0 15 mmol) with HF m the presence of 10 mL amsole for 1 h at 0°C 2 Evaporate HF, and precrpnate the crude product wrth ether, wash several times with ether, and filter 3 After dissolvmg m glacial acetic acid and lyophtlwng, recover 100 mg of crude peptlde as a powdery solid, and purify 45 mg using preparative HPLC (Vydac C,s, gradient 3040% B m 40 mm ta = 14 mm) to yield 11 mg (19 9%), HPLC purity 99.5%, t, = 9.58 mm (gradient 30-50% B in 25 mm); FAB-MS m/z 820 (MH+) calcd forC43H53N1106819 ‘3CNMR(150 87MHz,DMSO)G 15 97 (ValCr), 16 79 (Ala Cp), 18.92 (Val Cy), 22 22 (Lys Cy), 26.04 (Trp Cp), 29 98 (Lys CB), 30.49 (Lys CS), 3 1 47 (Tyr Cp), 37 25 (Phe Cp), 37.74 (Lys CE), 45.97 (Phe Co), 52.98 (Trp Co), 54.45 (Tyr Co), 54.50 (Ala Co), 54 94 (Lys Co), 55 03 (Val Co), 108 92, 111.33, 114.90, 117 98, 118.10, 120 70, 123.35, 126 67, 126 94, 127.01, 128.42, 129 31, 129.76, 135 68, 136.12 (Trp, Phe and Tyr arom.), 155 53, 155 99 (Tyr Cx, CN,), 165.43, 169 56, 171.46, 171 67, 171.96 (Trp, Lys, Val, Ala, and Tyr C=O)

This synthesized analog was prevtously tested in vitro for its ability to inhibit GRF-stimulated GH release from dispersed pituitary cells and showed nearly equivalent potency (83%) as compared with natural somatostatm (14) 3.5. Discussion Because of the geometrical similarity, the I,5 disubstituted tetrazole ring IS a good conformational mimic of the c&amide bond. If the role of proline, or other N-alkyl ammo acids found in a wide variety of biologically important peptides IS to make the cu-amide conformer more energetically accessible for bmdmg and/or recognmon by the receptor, then the tetrazole analog should provide a useful probe of this role. On the other hand, the czs-amide offers a unique arrangement of an adjacent hydrogen-bond donor and acceptor,

Zabrocki and Marshall which the tetrazole does not possess and whose greater steric bulk could prevent the analog from assuming close proximity to the receptor. Retention of activity by analogs with tetrazole replacement of an amide bond provtdes strong evidence for the role of the crs-amide m receptor recognition. Lack of activtty does not exclude the czs-amrde from constderation, because of the differences pointed out above. The availability of this synthetic route to chirally pure tetrazoles and methods for mcorporation mto blologrcally actrve peptrdes facilitate the use of this conformational mimic of the cu-amide bond in studies of molecular recogmtron. 4. Notes 1 Hydrazoic actd was prepared according to von Braun (23), is extremely toxic, and should be handled with extreme caution, mcluding the use of a safety shield owing to the known explosive hazards associated with azides. A summary of the protocol follows In a 500-mL, three-necked flask containing a dropping funnel, thermometer, efficient mechanical stirrer, and glass outlet tube, prepare a paste from 13 g of sodium azide and 13 mL of warm water To this paste, add 80 mL of benzene, and cool the mixture to 0°C. While the mixture IS stirred vigorously and cooled, add concentrated sulfuric acid dropwise (5 3 mL) The temperature should not exceed 10°C. After the addition of sulfuric acid, cool the mixture to 0°C and decant the organic layer, and dry it over anhydrous sodium sulfate The concentration of hydrazoic acid in benzene is usually about 4%, and the solutton should be kept over anhydrous sodium sulfate m the refrigerator This procedure is adapted from the description of Hans Wolff in Organic Reactions, vol. 3, p 327 2 Removal of the Z group from tetrazole-contammg dipeptides using HBr/acetic acid treatment should be limited to 20 mm to munmize removal of the benzyl ester group 3. All hydrobromide salts of tetrazole peptide esters should be stored in a desiccator over solid KOH owing to then very htgh hygroscopicity 4. Removal of the benzyl esters groups by hydrogenolysis should be done at a pressure of 4-5 kg/cm2 using a Parr hydrogenation apparatus 5 HF cleavage requires special apparatus and precautions Description of experimental procedures and safety measures can be found m Stewart and Young (24).

References 1. Thomas, W A. and Williams, M. K. (1972) 13CNuclear magnetic resonance spectroscopy and cdtrans isomerism in dipeptides containing proline J Chem Sot , Chem Commun. 994

2. Liakopoulou-Kyriaktdes, M. and Galardy, R E. (1979) s-Czsand s-Trans isomerism of the his-pro peptide bond m angiotensin and thyrohberm analogues Bzochemistry

l&1952-1957.

3 Bairaktari, E., Mierke, D F., Mamma, S., and Peggion, E J. (1990) Observation of a czsamide isomer within a linear peptide. J, Am Chem Sot 112,5383

The 1,5-Disubstltuted

Tetrazole Ring

4 Brandl, C J. and Deber, C M (1986) Hypothesis about brane-buried prolme residues m transport proteins Proc 83,917-921 5. Hann, M. M., Sammes, P G , Kennewell, P. D., and Taylor, bond isosteres of the peptide bond, an enkephalin snalog.

435 the function of memNat1 Acad Scz USA

J B (1982) On double J Chem Sot , Perktn

Trans 1,307-3 14

6 Marshall, G R., Humblet, C., Van Opdenbosch, N., and Zabrocki, J (198 1) Peptide bond modification and its effect on conformational mimicry, m Peptzdes Syntheses-Structure-Functton, Proceedtngs of the Seventh American Peptide Sympostum (Rich D. H and Gross E , eds.), Pierce Chemical, Rockford, IL, pp. 6699672. 7 Zabrocki, J , Smith, D G , Dunbar, J B., Jr., IIjima, H., and Marshall G R (1988) Conformational mimicry. 1. 1,5-Disubstituted tetrazole rmg as a surrogate for the CISamide bond J Am Chem Sot 110,5875-5880. 8. Smith, G. D., Zabrocki, J , Flak, T. A., and Marshall G R. (1991) Conformational mimicry. II. An obligatory cis amide bond in a small linear peptide Znt J Pept Protein Res. 37, 19 l-l 97. 9 Yu, K-L and Johnson, R L (1987) Synthesis and chemical properties of tetrazole peptide analogs. J Org Chem 52,205 l-2059 10 Hirai, K , Iwano, Y., Saito, T., Hiraoka, T., and Kishida, Y. (1976) Functionahzation of C6(7) of penacillamms and cephalosporms via 1,3-dipolar intermediate. Tetrahedron Lett. 16, 1303-1306 11 Zabrocki, J , Dunbar, J B , Jr, Marshall, K W , Toth, M V , and Marshall, G R (1992) Conformational mimicry Part III. Synthesis and mcorporation of 1,5-disubstituted tetrazole dipeptide analogs mto peptides with preservation of choral mtegrity bradykmm. J Org Chem 57, 202-209 12 Zabrocki, J and Marshall, G R , work in progress 13. Veber, D. F., Saperstein, R., Nutt, R. F., Freidmger, R M., Brady, S F , Curley, P., et al. (1984) A Super active cychc hexapeptide analog of somatostatm. Ltfe Scl 34, 1371-1378 14 Beusen, D D , Zabrocki, J , Slomczynska, U , Head, R D , Kao, J., and Marshall, G R. (1995) Conformational mimicry: synthesis and solution conformation of a cyclic somatostatm hexapeptide containing a tetrazole cls-amide bond surrogate Btopolymers 36, 18 l-200 15 Zabrocki, J , Smith, G D , Dunbar, J. B , Jr, Marshall, K W , Toth, M , and Marshall, G R. Tetrazole peptide analogs, m Pepttdes 1988, Proceedtngs of the 20th European Pepttde Sympostum (Jung G and Bayer E., eds ), Walter de Gruyter, Berlin, pp 295-297 16. Zabrocki J and OleJniczak B. (1993) Tetrazole analogs of Leu-enkephalm 12th Poltsh Pepttde Sympostum, Karpacz, Poland, Abstract, p 98 17. Zabrocki J., Olczak J , Kaczmarek K., Maszczynska I, and Lipkowski A W (1995) The synthesis of tetrazole analogs of dipeptides containing glycme m Nterminal position Leu-enkephalm analogs 13th Poltsh Pepttde Symposium, Gdansk, Poland, Abstract, p 40.

436

Zabrocki and Marshall

18 BoteJu, L. W and Hruby, V J (1993) Tryptophan-contammg 1,5-tetrazole drpeptide analogs synthesis of TrpY [CN,]Nle as a CIS amtde bond surrogate Z’etrahedron Lett 34, 1757-1760 19 Boteju, L W , Zalewska, T., Yamamura, H I , and Hruby, V J (1993) Tryptophan-norleucme 1,5-disubstnuted tetrazoles as CIS peptlde bond mimics mvesttgatton of the broactrve conformation of a potent and selective peptide for the cholecystokmm-B receptor. Bzoorg A4ed Chem Lett. 3,201 l-2016 20. Lebl, M , Slamnova, J , and Johnson, R. L. (1990) Analogs of oxytocm contammg a pseudopeptrde Leu-Gly bond of czsand trans configuratton Int J Pept Protean Res ,33,1621 21 Valle, G , Cnsma, M , Yu, K -L , Toniolo, C , Mlshra, R K , and Johnson, R L. (1988) Syntheses and X-ray diffraction analysis of the tetrazole peptide analogue Pro-Leu-v[CN4]-Gly-NH2 Co11 Czech Chem Commun 53, 2863-2876 22 Marshall, G R , Vme, W. H , and Needleman, P (1970) A specific competitive mhibitor of angiotensm II. Proc Nat1 Acad Scz USA 67, 16241630 23 von Braun, J. (193 1) Untersuchungen uber die Bestandteile des Erdols Ann ,490, 100-179 24 Stewart, J M. and Young, J D (1984) Soled Phase Peptlde Syntheses Pierce Chemical Co., Rockford, IL

25 Synthesis of (2R, 3R, 4R, 55)~iHer& Butyloxycarbonylamino-3,4-Dihydroxy=2=lsopropyl3,4-O,O-lsopropylidene-6-Cyclohexyl-Hexanoic

Acid

Allen Scott, Brian G. Conway, and Mark A. Krook 1. Introduction The dlpepttdtc dthydroxyethylene tsostere (2R, 3R, 4R, 5S)-5-tertbutyloxy-carbonylamrno-3,4-dlhydroxy-2-isopropyl-3,4-~, O-isopropyltdene-6-cyclohexyl-hexanoic actd, 15, 1s a representative butldmg block of an important class of unnatural dtpepttde mtmtcs. This pseudo-dtpepttde of Cha-Val is considered to be a transition-state analog mimic that produces potent inhibitors of the enzyme renin when incorporated mto angiotensmogen substrate peptrde sequence (I). The same strategy has also been proven successful m the development of potent mhibttors of other aspartyl proteases, mcludmg HIV protease (2). This synthetic method has been proven successful in the preparation of 1OO-g-scalequantity of this important butldmg block, which was used in the preparation of a potent HIV protease mhrbitory peptidomimettc, PNU-75875, which was proven effective in the treatment of SIV-infected monkeys (3). This convergent synthesis (Fig. 1) is based on the key connectton m which the drastereoselective aldol addition between the aldehyde 8 and the acyloxazolidinone 11 gave the adduct 12 with complete stereochemical control of the resulting two stereogemc centers. The synthesis of compound 8 started with Boc-L-phenylalanine 1, which was reduced with borane-tetrahydrofuran (THF) to give the correspondmg alcohol 2 , the phenyl rmg of which was hydrogenated to the cyclohexyl rmg in compound 3. Oxidatton to the corresponding aldehyde 4 was followed by the addition of vinyl Grtgnard reagent to give the allyhc alcohol 5. The vinyl group served as the latent aldehyde equtvalent. From

Methods

m Molecular

Me&one,

Edlted by W M Kazmlerskl

Vol

@Humana

437

23

fepbdomrmet~cs

Press Inc , Totowa,

Protocols

NJ

438

Scott, Conway, and Krook

Synthesis of Butyloxycarbonylamino

439

Acid

Treatment with acidic 2-methoxypropene, followed by ozone, gave the aldehyde 7. The acetal group not only served as the protecting group, but also provided a rigid framework m which the aldehyde 7 could now be equilibrated to largely favor the required eptmeric aldehyde 8. The acyloxazohdinone 11 came from an acylation of the oxazohdmone 10, which in turn was prepared from (+)-norephedrme 9 The boron enolate aldol addition between 8 and 11 gave the adduct 12, the choral auxiliary of which was removed by a boron trtflate-catalyzed reduction to give the diol13. The acetal-protecting group was again strategically designed to protect the secondary hydroxyl group differentially via a mtgratton wtth acid treatment to give 14 in which only the primary hydroxyl group was unprotected. Ruthemum-catalyzed periodic acid oxrdatton of the alcohol 14 gave the desired acid 15, whtch was appropriately protected as a choral butldmg block for the mcorporatton of this dthydroxyethylene drpepttdrc tsostere for the preparation of enzyme inhibitors 2. Materials 2.1. Reagents for Method 3.1 2 3 4 5 6 7 8. 9 10

N-(tevt-Butyloxycarbonyl)-L-Phenylalanme, Borane, 1 0 Mm THF Acetrc acid Methanol THF Hydrochloric actd (HCl) Sodium bicarbonate Sodmm chloride Ethyl acetate Sodium sulfate, anhydrous

1

2.2. Reagents for Method 3.2 1. 2 3 4

N-(tert-Butyloxycarbonyl)~L-phenylalanmol, Rhodium, 5% on carbon. Hydrogen gas Ethanol, absolute.

2

2.3. Reagents for Method 3.3 1. 2. 3 4. 5 6 7

2R - (tert-Butyloxycarbonyl)ammo-3-cyclohexylPyrrdme - sulfur trloxrde complex (pyrtdme-SOs). Trtethylamme Dtmethylsulfoxtde (DMSO) Ethyl acetate Cttrrc acid Sodium bicarbonate

1-propanol, 3

Scott, Conway, and Krook

440 8 Sodium chloride 9 Magnesium sulfate, anhydrous.

2.4. Reagents 1 2. 3 4. 5 6 7 8

2R-(tert-Butyloxycarbonyl)amlno-3-cyclohexyl-l-propanal, Vmylmagnesmm bromide, 1 Mm THF THF Ethyl acetate So&urn bicarbonate Magnesium sulfate, anhydrous Slhca gel 60 (Merck 70-230 mesh) Methylene chloride

2.5, Reagents 1 2 3 4 5 6 7. 8

for Method 3.4 4.

for Method 3.5

4S -(tert-Butyloxycarbonyl)amlno-5-cyclohexyl-l-penten-3R,S-o 2-Methoxypropene. Pyrldimum p-toluene sulfonate Methylene chloride. Sodium bicarbonate. Slhca gel 60 (Merck 70-230 mesh) Ethyl acetate Hexane

2.6. Reagents

5

for Method 3.6

1 4S-Cyclohexylmethyl-5R,S-ethenyl-2,2-d~methyl-3-(tert-butyloxycarbonyl) oxazolidine, 6 2 Methylene chloride. 3. Methanol 4 Ozone 5 Acetic acid, glacial 6 Zinc dust 7 Ethyl acetate 8 Heptane 9 Slhca gel 60 (Merck 70-230 mesh)

2.7. Reagents for Method 3.7 1 2,2-D~methyl-3-(tert-butyloxycarbonyl)-4~-(cyclohexylmethyl)-5R,~-oxazol~d~ne aldehyde, 7 2 Methanol 3 Potassmm carbonate, anhydrous 4 Phosphate buffer (pH 7). 5. Acetlc acid, glacial 6 Methyl t-butylether (MTBE) 7 Magnesium sulfate, anhydrous.

Synthesis of Butyloxycarbonylamino

441

Acid

8. Sthca gel 60 (Merck 70-230 mesh). 9. Ethyl acetate 10 Heptane

2.8. Reagents 1 2 3 4 5 6. 7. 8 9

(+)-Norephedrme hydrochlorrde, 9 Sodium hydroxide. Dtethyl carbonate Potassium carbonate (finely powdered). t-Butyl methyl ether (B&J) Sodium chloride. Methylene chloride 5% NaHCOs Magnesium sulfate

2.9. Reagents 1 2 3 4 5 6 7 8 9

for Method 3.8

for Method 3.9

(4R,5,!+(+)-4-Methyl-5-phenyl-2-oxazolidmone, n-Butylhthmm (1 6 A4) Isovaleryl chloride (THF) (B&J) t-Butyl methyl ether (B&J) 5% NaHC03 1 NNaOH Brine Magnesium sulfate

2. IO. Reagents

10

for Method 3.70

1. 2,2-Dimethyl-3-(tert-butyloxycarbonyl)-4S-(cyclohexylmethy1)-5R-oxazolid~ne aldehyde, 8. 2 3-(Oxo-3-methylbutyl)-4R-methyl-5~-phenyl-2-oxazol~d~none, 11. 3 1 0 M Dtbutylboron trrflate m methylene chloride 4 Diisopropylethylamine. 5. Methylene chloride 6 pH 7 1 0 MPhosphate buffer 7 Methanol (B&J) 8 30% Aqueous hydrogen peroxide. 9 Anhydrous magnesium sulfate 10 Silica gel 60 (Merck 70-230 mesh). 11 (THF) (B&J) 12 Methyl t-butyl ether

2.11. Reagents for Method 3.7 1 1 3-(tert-butyloxycarbonyl)-4S-cyclohexylmethyl-2,2-d~methyl-5R[3-(4R-methyl-2-oxo-5S-phenyloxazol~din-3-yl)-3-oxo-2R-~sopropyl-lRhydroxylpropyloxazohdine, 12

442 2 3 4 5 6 7 8 9 10 11 12 13. 2.12.

Scott, Conway, and Krook Dusopropylethylamme 1 0 M Dlbutylboron trlflate m methylene chloride 2 0 A4 Lithium Borohydrlde m THF 30% Aqueous hydrogen peroxide pH 7 1.O M Phosphate buffer Methanol (B&J) Methylene chloride Anhydrous magnesium sulfate (THF) (B&J) Ethyl acetate Heptane Silica gel 60 (Merck 70-230 mesh) Reagents

for Method

3.12

1 2R-~sopropyl-3-(4S-cyclohexylmethyl-2,2-d~methyl-3-~e~~-butyloxycarbonyloxazohdm-5R-yl)-1,3R-propanedlol, 13 2 Anhydrous camphorsulfomc acid 3 Acetone (B&J) 4 Methylene chloride (B&J) 5 Sodium bicarbonate 6 Cehte 7 Slhca gel 60 (Merck 70-230 mesh). 8 Ethyl acetate 9 Heptane 2.73. Reagents for Method 3.73 2R-isopropyl-SS-( 1-tert-butyloxycarbonylam~no-2-cyclohexylethyl)-2,2-d~methyl-4R-dloxolaneethanol, 14 2 Periodic Acid. 3 Ruthenium trlchlorlde hydrate. 4 Acetonitrile (B&J) 5 Carbon tetrachlorlde 6 Methylene chloride (B&J) 7 Anhydrous magnesium sulfate 8 Silica gel 60 (Merck 70-230 mesh) 9 Ethyl acetate 10 Heptane (B&J)

3. Methods 3.7. N-eert-Bufyloxycarbony/)-L-Phenylalaninol,

2

1 To a cooled (O’C) solution of 2638 mL of 1 M Borane m THF, add a solution of 336 g of 1 dissolved m 633 mL of THF over the course of 1 h, while mamtammg the reaction temperature below 5°C. 2 Stir the reactlon mixture at &5”C for 1.5 h (see Note 1).

Synthesis of Butyloxycarbonylamino

Acid

443

3. Carefully add 490 mL of a solution of 10% acetic acid m methanol to the stirring reaction mixture while maintaining the reaction mixture below 10°C. 4. Concentrate the reaction mixture to dryness zn vucuo on a rotary evaporator (4O’C) 5 Dissolve the concentrate m 6.3 L of ethyl acetate, and wash the organic solution with (see Note 2): 144L a Sodium bicarbonate, saturated aqueous 115L b Hydrochloric acid, 1.O N c Sodium chloride, saturated aqueous 11.5 L 115L. d Sodmm chloride, saturated aqueous 6 Dry the organic layer over anhydrous sodium sulfate, filter, and concentrate to dryness zn vacm (40°C) 7 Dry the solids m a vacuum oven (25’C) overnight to give 284 7 g (90%) of 2 as a colorless solid, mp 94--95”C, which can be used m the next step (Note 3)

3.2. ZR -(tert -Bufyloxycar&onyl) Amino-3-Cyclohexyl1-Propanol,

3

1 Charge a nitrogen inerted 20-L stainless-steel autoclave with 58 1 2 g of N-(tertbutyloxycarbonyl)~L-phenylalamnol, 2,85 1 g of 5% rhodium on carbon, and 7 4 L of absolute ethanol 2 Purge the autoclave first with nitrogen and then with hydrogen 3 Hydrogenate the reaction mixture under 150 pslg hydrogen at 50°C until hydrogen uptake ceases (see Notes 4 and 5). 4 Filter the completed reaction mixture through filter aid, and concentrate the filtrate to dryness zizvaczdo(4O’C) to give 598 g (100%) of 3 as an 011,which can be used m the next step (see Note 6)

3.3.2&(tert-Butyloxycarbonyl) Amino-&Cyclohexyl-1-Propanal,

4

Treat 298 0 g of 3 with 3 34 L of dry DMSO and 479 mL of tnethylamine. Stir to dissolve In a separate nitrogen-inerted vessel, treat 607 g of pyrldine -SO, complex with 3 34 L of DMSO. Stir to dissolve While maintaining the reaction temperature below 25”C, add the pyndme-SO,/ DMSO solution to the solution of 3 over a 30-mm period When the addition IS complete, remove the cooling and stir the mixture at amblent temperature for an additional 2 h (see Note 7) Transfer the completed reaction to a wash tank containing 16 5 L of crushed ice/ water slurry Partition the reactlon mixture with 2 x 8.6 L of ethyl acetate Wash the combined organic layers with 2 x 8.6 L of 10% aqueous citric acid, 3 4 L of saturated aqueous sodium bicarbonate, and 3 4 L of saturated sodium chloride Dry the organic layer over anhydrous magnesium sulfate, filter, and concentrate zn vucuo (35°C) to give 234 6 g of 4 as a tan oil, which can be used m the next step (see Note 8)

444

Scott, Conway, and Krook

3.4. S-(tert-Butyloxycarbonyl) Amino-5-Cyclohexyl1-Penten-S(t?, S)-ol, 5 To a cold (-30°C) solution of vmylmagnesmm bromide (1 M m THF), add a solutlon of 234 6 g of 4 dissolved in 6.4 L of dry THF over the course of 1 h while mamtammg the reaction mixture between -25 and -3O’C throughout (see Note 9) On completion of the addition, stir the mixture at -30°C for an additional 30 mm (see Note 10) Quench the completed reaction mixture mto 1.6 L of saturated aqueous ammomum chloride by pouring the reactlon mixture slowly mto the vigorously stirring aqueous solution Partition the mixture with 3 x 4 0 L of ethyl acetate Wash the combined organic extracts with 4.0 L of saturated aqueous sodium bicarbonate and then with 2 x 4 L of saturated aqueous sodium chloride Dry the organic layer over anhydrous magnesium sulfate, filter, and concentrate the filtrate to dryness zn vacua Chromatograph the crude 5 on 12 5 kg of slhca gel elutmg with 150 L of 5% ethyl acetatejmethylene chloride, collectmg a 2 O-L forecut followed by 8-L fractions Combine and concentrate the clean cuts to give 131 g (40%) of 5, which can be used m the next step (see Note 11)

3.5. S-Cyc/ohexy/methy/-S(R,S)-Etheny/Z,Z-Dimethy/-3-~ert-Buty/oxycarbony/)Oxazo/idine,

6

1 Charge a 5-L, three-necked, round-bottomed flask with 262 7 g of 5, 186 5 mL of methylene chloride, 886 mL of 2-methoxypropene, and 12.14 g of pyndmlumptoluenesulfonate 2 Stir the reaction mixture at room temperature mamtammg the temperature under 30°C (see Note 12) 3 Treat the completed reaction nuxture with 4 32 g of sodnun bicarbonate, stir for 1 h, filter through Cehte, and concentrate m vacua (35°C) to give 410 g of crude 6 as an 011 4 Chromatograph crude 6 on 25 0 kg of silica gel, elutmg with 150 L of 3% ethyl acetate/heptane Collect 20-L fractions, poolmg the clean cuts (2628) and concentrating zn vacua (35’C) to give 249 g (83%) of 6, (see Note 13), which can be used m the next step

3.6.2,2-Dimethyl-3-(tert-Buty/oxycarbony/)4S-(Cyclohexylmethyl)-5(R,S)-Oxazo/idineakfehyde,

7

1. Charge a 2 L, three-necked, round-bottomed flask with 47 7 g of 6, 7 15 mL of methylene chloride, and 182 mL of methanol. 2 Cool the system to -70°C and then sparge oxygen through the system for 20 mm 3 From an ozone generator, sparge ozone through the reaction mixture until a pale blue color persists (see Note 14) 4 Stop ozone delivery, and sparge the cold reaction mixture with nitrogen for several minutes until the blue color of the excess ozone clears (see Note 15)

Synthesis of Butyloxycarbonylamino

445

Acid

5 Quench the reaction by pouring the reaction mixture mto a 5-L flask contammg a cold (-45°C) mixture of 895 mL of water, 895 mL of methanol, 35 8 mL of glacial acetic acid, and 35.8 g of zinc dust 6. Stir the quenched reactlon mixture for 5 min at +5”C, remove the coolmg and allow the mixture to warm to room temperature 7. Filter the mixture through Cehte, allow the phases to settle, and remove the lower (organic) layer (see Note 16). 8. Partition the aqueous layer with an additional 3 x 900 mL of methylene chloride, dry the pooled organic layer over anhydrous sodium sulfate, filter, and concentrate the filtrate to dryness zn vac~o (35“C) to give 53 5 g of crude 7 as a tan 011 9 Purify the crude 7 by column chromatography on silica gel (1 33 kg), elutmg with 15% ethyl acetate/hexane 10 Collect 350-mL fractions from the column, pool the clean cuts (18-20), and concentrate to dryness zn vucuo (40°C) to give 97 0 g of 7 as a colorless 011 (see Notes 17 and 18), which IS suitable for use m the next step

3.7.2,2-Dimethyl-3-(tert -Bufyioxycarbonyl) 4S-(Cyclohexylmethyl)~5R-Oxarolidine

Aldehyde,

8

Charge a nitrogen merted, 5-L, round-bottomed flask with 146.0 g of 7, 1.73 L of methanol, and 57 7 g of potassmm carbonate Stir the mixture at about 23T for 3 h, and cool to 0°C Add 49 14 mL of glacial acetic acid to the reaction mixture, stir for 5 mm (OT), remove the coolmg, and allow to warm to about 23°C. Add 500 mL of pH 7 0 phosphate buffer (see Note 19) and remove the methanol zn vacua (40°C)

Partltlon the aqueous concentrate with 2 37 L ofmethyl t-butylether, dry the organic layer over anhydrous magnesium sulfate, filter, and concentrate the filtrate to dryness zn vucuo (40°C) to give 134 7 g of crude 8 as a colorless solid (see Note 20) Chromatograph crude 8 over 1 9 kg of slhca gel, elutmg with 15% ethyl acetate/ heptane and collectmg 2-L fractions Combme all product containing fractions and concentrate to dryness zn vacua (40°C) to give 114 g (78%) of 8 as a white sohd (Notes 21 and 22)

3.8. (4R-cis)-4-Methyl-5-Phenyl-2-Oxazolidinone,

10

1. Dissolve (+)-norephedrme hydrochloride, 9 (155 1 g) m 75 mL water and neutralize with a solution of sodium hydroxide (49.68 g) in 750 mL water 2 Extract the mixture with t-butyl methyl ether (5 x 750 mL), dry the combined extracts over magnesium sulfate, filter, and concentrate to a pale yellow 011 m a 1-L, three-necked, round-bottomed flask 3. Outfit the flask for atmospheric dlstlllatlon, add diethyl carbonate (110 mL), potassium carbonate (5.7 g), stir, and heat at 115°C for 18 h (see Note 23). 4. Cool the mixture to room temperature, and dissolve the sohds m methylene chloride (1500 mL) Wash the organic solution with 5% sodium bicarbonate solution (2 x 375 mL), dry over magnesmm sulfate, filter, and evaporate the solvent.

446

Scott, Conway, and Krook

5 Recrystallize the sohds from ethyl acetate/hexanes to provtde 10 (112 2 g, 84% yield) as off-white crystals (see Note 24)

3.9. (4R-cis)-4-Methyl-3-~3-~ef~y/-I-Oxobutyl)5-Phenyl-2-Oxazolone, 11 Outfit a 5-L, three-necked, round-bottomed flask with a nitrogen inlet, thermometer, mechanical stirrer, and pressure-equahzmg addmon funnel Charge the flask with 111.92 g of the oxazohdmone 10, 1100 mL of THF, degassed with nitrogen and cooled to -78°C (acetone/dry Ice bath), mamtam a nitrogen atmosphere Charge the addition funnel with 456 mL of n-butyllithmm, and add dropwtse to the stirred solutron over a 90-min period (see Note 25) Rinse the addmon funnel with 50 mL of THF, and continue stn-rmg for 10 mm Charge the addmon funnel with 102 mL of tsovaleryl chlortde, and add dropwlse over a 30-mm period (see Note 26) Remove the coolmg bath, and warm the reaction to room temperature Stir for 30 mm at room temperature, then quench the reaction mixture wtth 500 mL of 5% NaHCO,, and stir for 5 mm Pour the reactton mixture into a separatory funnel contammg 500 mL of 1 N NaOH and extract with two 1-L porttons of t-butyl methyl ether. Combme the organic extracts, wash with brine (500 mL), dry over MgS04, filter, and concentrate to a yellow 011n-ra 500-mL round-bottomed flask Dtsttll the remaining yellow 011under vacuum at 164°C (0 60 mmHg) to provrde 155 28 g (86%) of 11 (see Notes 27 and 28)

3.70. 3-pert -Buty/oxycarbonyl)-4-Cyclohexylmefhyl2,2-Dimefhy/-SR-[3-(4R-Mefhyl-2-Oxo-SS-Phenyloxazolidin3-yl)-3-Oxo-2R-Isopropyl-1 R-tiydroxy]Propyloxazolidine,

12

An oven-dried 5-L, three-necked flask IS fitted with an oven-drred 1 L addition funnel, drted magnetic star bar, argon inlet, dried glass stopper, septum, and lowtemperature thermometer Purge with argon Charge 11 (84 9 g) to flask and continue purge Using an oven-dried stainless-steel needle transfer, dry methylene chloride from the bottle to the flask via the addmon funnel using argon pressure on the bottle (333 mL) Stir Cool to 0°C under argon using an ice/salt bath Transfer dtbutylboron trtflate from the septum-capped bottle to the additron funnel using an oven-dried stainless-steel needle and argon pressure on the bottle (353 mL) (see Note 29) Add triflate dropwtse to the stirred solutton m the flask over 30 mm allowing the temperature to rise from -2” to +4”C At 0” add dusopropylethylamme (66.5 mL) slowly from an oven-dried syringe over 13 mm allowing the temperature to rise from + lo to +6” Stir for 30 mm at -5” to +5”C

Synthesis of Butyloxycarbonylamino

Acid

447

10. Cool to -78°C under argon 11 Into a septum-stoppered, oven-dried Erlenmeyer flask, charge 8 (95 0 g) Purge with argon. Charge dry methylene chloride (275 mL) via dried stamless-steel needle from bottle via argon pressure on the bottle. Shake under argon to dissolve. 12 Transfer the solution of 8 to the addition funnel using argon pressure on the Erlenmeyer flask, and a dried stainless-steel needle. Rinse with methylene chloride as needed via syrmge 13 To the cloudy yellow solution m the flask, add the solution of 8 dropwise over about 90 min mamtammg the pot temperature at less than -70°C 14. Stir for 30 min at -78°C and then remove the coolmg 15. Warm to room temperature using a water bath and sample via syringe for TLC assay. Quench 0 5 mL of the yellow solution mto pH 7 1 .OM buffer (1 mL) Add methanol (l/2 mL) followed by 2.1 methanol/30% hydrogen peroxide (1 mL) Add methyl t-butyl ether (2 mL) Shake and spot 2-5 h of the upper layer on a 20cm silica gel plate. Elute with 1% tetrahydrofuran m methylene chloride, and visualize using phosphomolybdic acid in ethanol spray 16 Sample periodically, and continue until there IS no change by TLC It can be allowed to stir overnight at room temperature if necessary 17 Cool to 0” using an ice/salt bath 18. Add pH 7 0 1.0 A4 buffer (241 mL) rapidly via a graduated cylinder Warms to +5”C Add methanol (48 1 mL) rapidly via a graduated cylinder as solids precipitate Warms to +6”C. 19 Slowly add 30% hydrogen peroxide (241 mL) m methanol (481 mL) dropwise mamtammg the pot at
25. Removal of the solvent from fractions 9-20 provides 140.7 g of pure 12 (about 75% yield) (see Note 30)

3.11. 2Fl -isopropyl-3-(4S-Cyclohexylmethyl-2,2-Dimethyl3-tert-Butyloxycarbonyl-Oxazolidin-5kyl)1,3R-Propanediol,

13

1 An oven-dried, 5-L, one-necked flask is fitted with an oven-dried Claisen head, magnettc star bar, 500 mL addition funnel, septum, argon bubbler, thermometer, and coolmg bath.

448

Scott, Conway, and Krook

2 The flask IS charged with 12 (138 5 g) and purged with argon. 3 Charge via a dried stamless-steel needle dry THF (485 mL) from the bottle to the addmon funnel usmg argon pressure on the bottle 4. Add to the flask, and dtssolve the foam with stnrmg under argon 5. Cool to 0°C and add via oven-dried syringe dusopropylethylamine (51 6 mL) over 20 mm (4°C maximum) 6. Charge via an oven-dried needle dibutylboron trrflate (259 mL) from the bottle to the addition funnel using argon pressure. 7. Add trtflate dropwtse mamtammg the pot below +8”C over 60 mm usmg an me/ salt bath Rinse funnel with THF via syrmge. 8 Stir for 30 mm at 0°C. 9. Charge via an oven-dried needle 2.0 Mhthmm borohydrtde (294 mL) to the addttton funnel from the bottle using argon pressure. 10. Add dropwtse to the reaction solutton over 65 mm. Hydrogen gas IS evolved slowly during the addition The temperature IS maintained below +6”C 11. Stir 90 min at 0” and then warm to room temperature May be allowed to stir overmght The reaction IS momtored by TLC (see Note 31) 12. When complete cool to 0°C and quench by careful addmon of a solutron contammg methanol (477 mL), pH 7 0 1 0 Mbuffer (477 mL), and 30% hydrogen peroxide (239 mL). Mamtam the pot at <+8”C Foams durmg the early part ofthe addmon 13 Star for 10 mm at O’C 14. Warm to room temperature, and partttton between methylene chloride (1100 mL) and pH 7 0 buffer (1100 mL) 15 Extract the aqueous layer with methylene chloride (2 x 1 L). 16 Dry the combined organic layers over magnesmm sulfate 17 Remove the solvent zn vucuo (35’C bath) to gave 201 7 g of mixture containing 13 and the chit-al auxthary 18 Chromatograph the crude product (201.7 g + 6 9 g from a stmdar run) on srllca gel (9 kg) packed wtth 10% ethyl acetate m heptane Apply the mrxture m methylene chlorrde (200 mL) 19 Elute with 10% (20 L), 20% (40 L), and 75% (16 L) ethyl acetate m heptane taking 4-L fractions after a 20-L forecut. 20 Combme pure fractrons (8-23) as determined by TLC, and remove the solvent zn vacua to afford 93 1 g of 13 as a colorless foam (about 92% yield, but contains a UV vlstble impurity separated at the next step) (see Note 32)

3.12. 2FMsopropyMS-(l-tert-Butyloxycarbonylamino2-Cyc/ohexy/ethy/)-2,2 Dimethyl-4WDioxolaneethano1,

14

1 Purge a 5-L, round-bottomed flask containing 13 (93 1 g) with argon The flask IS fitted wtth a magnetic sturer, septum, argon inlet, and thermometer 2 Via a funnel, charge a solutton of camphorsulfomc acid (2 1 0 g) m dry methylene chloride (900 mL) 3 Stir at room temperature unttl there 1sno change by TLC assay (srbca gel GF 20-cm plate, 30% ethyl acetate m heptane, phosphomolybdlc acid char)

Synthesis of Butyloxycarbonylamino

Acid

449

4 Add dry acetone (1250 mL) via tinnel after Installing a water bath Warms to +29”C 5 Allow to stir overnight at room temperature m order to convert any trlol back to the acetonide. Follow by usmg the above TLC assay 6 Add sodium bicarbonate (15.0 g) to the solution, and continue stirring for 2 h as colorless sohd precipitates 7 Filter off the solid on a pad of Cehte rmsmg with dry acetone as needed 8. Remove the solvent zn vucuo (35°C bath) to give 14 as a tan foam (94 3 g) 9. Chromatograph the crude product (94.3 g) (m 100 mL methylene chloride) on slhca gel (2500 g) packed with 5% ethyl acetate in heptane. Elute with same takmg 1-L fractions after a 7 L forecut. 10 Combme pure fractions (14-43) as determmed by TLC (slhca gel GF lo-cm plate, 30% ethyl acetate m heptane, Rf= 0 6; vlsuahze with UV and phospho-molybdlc acid char) 11 Remove the solvent zn vacua to provide 48 8 g of 14 as a colorless sohd (52% yield) (see Note 33)

3.13. (2Ft, 3R, 4R, 5S)-54ert -Butyloxycarbonylamio3,4-Dihydroxy-2-lsopropyl-3,4-O,O-lsopropylidene6-Cyclohexyl-tfexanoic Acid, 15 1 A 3-L, round-bottomed flask IS fitted with a magnetic stirrer, septum, mtrogen inlet, thermometer probe, and coolmg bath 2 Charge 14 (48 8 g), and purge with nitrogen 3 Charge acetomtrde (220 mL) and carbon tetrachlorlde (220 mL) with stlrrmg Remove a TLC sample. 4 Add (r5”C deiomzed water (327 mL) and ruthenium trlchlorlde (0.62 g) 5 Immerse the flask m a large water bath at room temperature 6 With vigorous stmmg, add perlodlc acid (107.4 g) portlonwlse for 90 mm mamtammg the pot at <+26”C Follow the reaction to completion by TLC assay (seeNote 34) 7 Dilute the mixture with methylene chloride (1 5 L) Extract and separate 8 Extract the aqueous layer with methylene chloride (2 x 500 mL) 9 Dry the combined organic layers over magnesium sulfate 10 Remove the solvent m vucuo (35°C bath) to produce 53 8 g of crude 15 as a dark brown 011 11 Chromatograph the crude product (53 8 g) dissolved in 50 mL CH.&& on sdlca gel (5 100 g) packed m 15% ethyl acetate in heptane 12 Elute with 15% (30 L), 20% (20 L), and 30% (96 L) ethyl acetate m heptane taking 2-L fractions (l-22) and 4-L fractions (23-36) after a 10-L forecut 13 Combme pure fractions (&36) as determined by TLC assay (see Note 35) 14 Remove the solvent in vucuo (35°C bath) to give 35.95 g of 15 as gray crystals (71% yield) 15 Wash the crystals with cold heptane (200 mL), and dry m the vacuum oven at room temperature to afford 33 3 g of 15 as pale gray crystals (see Note 36) 4. Notes 1 During this addition, the time 1s not as important as mamtalmng the reaction temperature 2 After extractlon, the second so&urn chloride wash was found to be pH 6 0

450

Scott, Conway, and Krook

3 The material was found to be “one spot” by TLC (50% ethyl acetate/ 50% hexane, ninhydrm char, R, 1 = 0 to 0 56 streak, R,2 = 0 67) ‘H-NMR (CDCl,, TMS) ppm 1 30 (s, 9H), 2 75 (d, 2H), 3.20 (w, br, lH), 3 50, (d, 2H), 3.60-3.90 (m, lH), 4 85 (d, lH), 7.15 (s, 5H) 4 This reduction was allowed to contmue overnight for 18 h, but was probably complete m 8-10 h. 5. The reaction was confirmed complete by TLC assay (50% ethyl acetate/ 50% hexane, nmhydrm char, Rf3 = 0 75) 6 ‘H-NMR (CDCls , TMS). ppm 0 40-l 90 (m, 22H), 3 404.00 (m, 3H), 5 10 (d, 1H) 7 The reaction was confirmed complete by TLC (5% ethyl acetate/ 95% methylene chloride, nmhydrm char, R, 3 = 0 5, R, 4 = 0 62) The TLC sample was prepared by quenching a small sample of the reaction mtxture with 10% aqueous citric actd and ethyl acetate. After shakmg and allowing the mixture to settle, the top layer was spotted on the TLC plate. Since 4 1s known to eptmertze at the 2 posttton wtth time, this unstable mterme8 dtate should be carried on m the next step wrthm a few hours. Overmght storage at -20°C has been shown to be acceptable 9 Coolmg the solution below -30°C will cause some of the vmylmagnesmm bromide to prectpitate out of solutton and wtll cause a decrease m the chemical yteld of the reactton 10 The reactton IS followed by TLC to a pomt. However, there 1s always a famt residual spot, which coelutes with starting material, that does not dtsappear on further extending the reaction time beyond 30 mm (TLC 5% ethyl acetate/ 95% dtchloromethane, phosphomolybdic acid char, R+-4 = 0 75, Rf5 = 0 33) The TLC sample was prepared by quenching a small portion of the reaction mtxture mto saturated aqueous ammomum chloride Spot the top (organic) layer on the plate 11 Mixed fracttons were pooled and rechromatographed, which raised the recovery of clean material by 5%, up to the reported 40% ‘H-NMR (CDCl, , TMS). ppm 0 461 90 (m), 2 15 (s), 2.6 (m, br), 3.2 (m, br), 3 8 (m, br), 4 2 (q), 4 7 (m, br), 5 3 (d), 5 7@6 10 (m, br). 12 The reaction IS assayed for completion by TLC, 5% ethyl acetate / 95% hexane, phosphomolybdtc actd char, Rf 5 = 0 05, Rf unknown = 0.9. 13 The first column gave 188 g of clean 6. The mtxed cuts were pooled and rechromatographed to gave an addmonal61 g of clean 6 ‘H-NMR (CDCI, , TMS) ppm 0 4&l 90 (m), 2 1 (s), 2 2 (s), 3.70-4.60 (m, br), 5.1c5.5 (m, br), 5 [email protected] (m, br) 14 This reaction requrred 4 h to go to completion, The reactton rate was dependent on the rate of ozone generation 15 The completton of reactron was confirmed by observing the disappearance of 6 via TLC (5% ethyl acetate/95% hexane, phosphomolybdrc acrd char, Rf 6 = 0 62 16 Before concentrating the orgamc layers, they were tested with starch/iodide paper and found to be peroxtde free 17. The course of the column purtficatton was followed by TLC (5% ethyl acetate/ 95% hexane, Phosphomolybdtc acid char, Rf 6 = 0 62, Rf 7 = 0 15 ‘H-NMR (CDC13 , TMS) ppm 0 4&2 00 (m, 27H), 4 l-4.4 (d, 2H), 9 66 (s, 0 2H), 9 76 (s, 0 8H)

Synthesis of Butyloxycarbonylamino

Acid

451

18 The 011 solidified during storage at -2O’C 19. Prepare 0.1 M pH 7 0, phosphate buffer: Dissolve 6.9 g of sodium dthydrogen phosphate monohydrate m 500 mL of water. Treat with 291 mL of 0 1 Maqueous sodium hydroxide solution 20 Compound 8 has several percent residual methanol at thus pomt. The subsequent column chromatography 1semployed to reduce this methanol to a negltgible level. Methanol interferes with the next chemical step 21 Intermediate 8 IS stored at -20°C to mamtam stereochemical Integrity. 22. mp of 8 1s59-63’C ‘H-NMR (CDCla , TMS): ppm 0 462 00 (m, 27H), 4 l-4.4 (d,2H), 9 66 (s, O.O8H), 9 76 (s, 0 92H). The ratio of isomers is determmed to be 11 5(R)/l O(S) by comparing the integration of the aldehyde peaks m the ‘HNMR at ppm 9 76 and 9 66, respectively 23. Ethanol is distilled over durmg the course of the reaction 24 Physical properties for 10. mp 116118”C, ‘H-NMR (CDC13, TMS). ppm 7 30 (s, 5H), 6.65 (br s, NH), 5 67 (d, lH), 4 19 (m, lH), 0 81 (d, 3H) [a],, = +156.3” (CHCl& TLC (silica gel GF): Rf = 0 50 in ethyl acetate/hexanes (75/25). 25. A 3°C exotherm IS noted during the addttion 26 A 10°C exotherm is observed during the addition 27 The product crystallized within 24 h on standing at room temperature 28 Physical properties for 11, ‘H-NMR (CDCl,,TMS) ppm 7 3 (s, 5H), 5 61 (d, lH), 4 75 (m, lH), 2 72 (m, 2H), 2 2 (m, lH), 0 99 (d, 6H), 0 91 (d, 3H) [a]n + 44 7 (CHCI,). TLC (silica gel GF). R,= 0 78 m ethyl acetateihexanes (75.25) 29 The mole ratio of di-n-butyl boron triflate to 11 must be 1 1 to 1 0 for satisfactory results, since excess triflate reagent leads to an increase m the amount of undesired “anti”-aldol product 30. TLC analysis* 5 uL is spotted on a IO-cm silrca gel GF plate, and the plate IS eluted with 1% THF/methylene chloride The Rf of 12 is 0.6 1 The plate is vtsuahzed with phosphomolybdic acid spray 31 TLC analysis Via syringe five drops of solution are quenched in 1 mL methanol/ 1 mL, pH 7.0 buffer/l mL 30% hydrogen peroxide The quenched solution is spotted on a 20-cm silica gel GF plate and the plate is eluted with 1% THF/ methylene chloride The plate is vtsuahzed with phosphomolybdic acid spray TheRfof12is061andtheRfof13is035 32 TLC analysis Use a 20 cm silica gel GF plate eluting with 30% ethyl acetate in heptane Visualize with phosphomolybdic acid spray. The Rf of 13 IS 0.35. 33 [a]n =-30” (c = 0 97, CHCl,). ‘H-NMR (CDCl,, TMS) ppm 0 75-l 75 (m, 34H), 1 75-l 95 (m, lH), 2 1 (m, lH), 2 90 (m, lH), 3.6-3.95 (m, 5H), 4 72 (m, 1H). 34 TLC analysis Use a lo-cm silica gel GF plate elutmg with 30% ethyl acetate m heptane Visualize with phosphomolybdic acid spray Rf of 15 is 0 4 (tails) 35 Use the conditions m Note 34 after concentrating the sample to 20% of the original volume on the steam bath. 36 [a], =-lo (c = 0.01, CHCl,). ‘H-NMR (CDQ TMS): ppm 0 75-l .50 (m, 29H), 1 5s 1 75 (m, 4H), 1.82-1 92 (m, IH), 2.05-2.22 (m, lH), 2.55 (m, IH), 3.75412 (m, 3H), 4.80 (m, HI), 9 9 (s, br, HI). C, 64 58, H, 9.77; N, 3 26, calcd C, 64.60, H, 9 67; N, 3 28

452

Scott, Conway, and Krook

References Thatsrrvongs, S , Pals, D T., Kroll, L T , Turner, S R , and Han, F -S. (1987) Renm mhibitors Design of angtotensmogen transition-state analogues contammg novel (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-lsopropyl-7-methyloctanolc actd J Med Chem 30,976-982 Thaisrtvongs, S., Tomassellr, A G , Moon, J B , Hut, J 0 , McQuade, T J , Turner, S R , et al (1991) Inhrbitors of the protease from human nnmunodefictency vnus* design and modellmg of a compound contammg a drhydroxyethylene isostere Insert with high bmdmg affmty and effective anti-viral actrvny. J A4ed Chem 3423462356 Martm, L N , Sotke, K. F , Murphey-Corb, M , Bohm, R P., Roberts, E D , Kakuk, T. J., et al (1994) Effects of a peptrdomimetic mhibitor of retrovlral proteases (U-75875) on stmian tmmunodeficiency virus mfection m rhesus monkeys Antzmzcrob Agents Chemother 38, 1277-1283

Synthesis of (2S,4S$S)-5(t-Butoxycarbonylamino)4-( t-Butyldimethylsilyloxy)-2-lsopropyl7-Methyl Octanoic Acid Mark A. Lyster and Brian G. Conway 1. Introduction In an effort to produce renm mhibttory peptides, the preparation of (2S,4S,5S)-5-(t-Butoxycarbonylam~no)-4-(t-butyldimethyls~lyloxy)-2-~sopropyl-7-methyl octanotc acid, 15, was carried out (1). This pseudo-dipeptide was used to replace combinattons of leucme coupled with valine in pepttde sequences The preparation described is the final method developed, after many early less successful attempts The synthesis mvolves (Fig. 1) the coupling of I-bromo-5-methyl-2-hexene 3 and N-isovaleryl-L-prolmol 4 using the opttcal activity of the L-prolmol to induce optical activrty at the C-2 posmon of 5, a key intermediate (2). After purifying this intermediate through the formatron of its benzoate, the functionalizatlon of the C-4 and C-5 positions is carried out through an epoxtdation to give two stereoisomers. After mesylation and separation of the undesired isomer, 12, rt IS converted to the desired isomer, 11, through a hydrolysis/lactomzation process, which inverts the C-4 and C-5 centers (3). After formatron of the mesylate, from the recycled material the amme function IS introduced through azrde displacement of the mesylate, followed by reduction (4) and protectron with the t-BOC group. Finally, the lactone IS opened and the hydroxyl group 1sprotected as the t-butyldtmethylsilyl ether to give the trtle compound, 15 As can be seen in the procedure, this synthesis was carried out on a large-scale producing 4 8 kg of 15 through a 15-step sequence in an overall yield of 18.5%.

From

Methods m Molecular Medune, Vol 23 Peptldomrmebcs Protocols Edlted by W M Kazmlerskl OHumana Press Inc , Totowa, NJ

453

Lyster and Conway

Figure I

2. Materials 2.7. Reagents for Method 3.7 1. 4-Methyl- 1-pentyne 1. 2. Ethyl chlorofomate.

455

Synthesis of Octanoic Acid 3 4 5 6 7 8 9

n-Butylhthmm (1.7 N solution in hexane). Vttrtde (70% solution m toluene) Tetrahydrofuran (THF) Sulfurtc acid (98%) Ethyl acetate Sodmm btcarbonate solutton (7%) Sodium chloride solution (25%)

2.2. Reagents for Method 3.2 1 2 3 4. 5 6

5-Methyl-2-Hexenol 2 Phosphorous trtbromide Hexane Sodmm carbonate solution (7%). Sodium chloride solution (25%). Sodmm sulfate

2.3. Reagents for Method 3.3 1 2 3 4 5 6 7 8

N-Isovaleryl-L-prolmol 4 Lithmm dtisopropylamide solutton (LDA, 2&27% m tetrahydrofuran [THF]) THF. (E)- 1-Bromo-5-methyl-2-hexene 3 Sodium chlortde solution (25%) Ethyl acetate Hexane Sthca gel (Woelm, 63-200 mesh)

2.4. Reagents for Method 3.4 1. 2 3 4. 5 6 7 8. 9

(2S)-2-Isopropyl-7-methyl-4-octenoic (2R)-2-Isopropyl-7-methyl-4-octenotc Triethylamme (TEA) Benzoyl chloride Methylene chloride Sodium bicarbonate solution (5%) Ethyl acetate Hexane Sthca gel (Woelm, 63-200 mesh)

2.5. Reagents 1 2 3 4 5

acid, L-prolmol amide 5 acid, L-prolmol amide 6.

for Method 3.5

(2S)-2-Isopropyl-7-methyl-4-octenotc Potassium carbonate. Acetomtrtle. Ethyl acetate Sodtum chlortde solutton (25%)

acid, L-prolmol (0-benzoyl)amtde

7

Lyster and Conway

456 2.6. Reagents 1 2 3 4 5

(2S)-2-Isopropyl-7-methyl-4-octenoic Sulfuric acid (98%) Diglyme Hexane Methylene chloride

2.7. Reagents 1 2. 3. 4 5, 6 7 8

acid, L-prolmol amide 5

for Method 3.7

(2S)-2-Isopropyl-7-methy1-4-octenotc acid 8. Magnesium monoperoxyphthalate hexahydrate (MMPP). Isopropyl alcohol (IPA). Methylene chloride. Sodmm sulfite. Sodium chloride solution (5%) Sodium bicarbonate solutton (1%) Sodium sulfate.

2.8. Reagents 1 2. 3 4 5 6. 7 8 9 10 11

for Method 3.6

for Method 3.8

(2S,4S,5R)-4,5-Dlhydroxy-2-lsopropyl-7-methyloctanoic (2S,4R,5S)-4,5-Dlhydroxy-2-1sopropyl-7-methyloctanoic TEA Methanesulfonyl chloride Hydrochlortc acid (HCl, 37%) Sodium bicarbonate solution (5%) Sodium chloride solution (2%) Methylene chloride Methyl t-butyl ether (MTBE) Hexane. Silica gel (Merck, 7CL230 mesh)

2.9. Reagents

acid, g-lactone 9. acid, g-lactone 10

for Method 3.9

1, (2S,4R,SS)-4-Hydroxy-2-lsopropyl-5-mesyloxy-7-methyloctanoic lactone 12 2 Acetomtrtle 3. Potassmm hydroxide solution (10.2%) 4 Acetic acid (Glacial) 5 HC1(37%) 6 Sodmm chloride solution (25%) 7 MTBE 8 Sodmm bicarbonate solution (8%). 9 Methanesulfonyl chloride 10 Triethylamme (TEA) 11 Methylene chloride.

acid, y-

457

Synthesis of Octanoic Acid 2.10. Reagents for Method 3.10 1 (2S,4S,SR)-4-Hydroxy-2-tsopropyl-5-mesyloxy-7-methylocato~c lactone 11 2 Sodmm azrde 3 N,N-Drmethylacetamide (DMAC) 4 Ethyl acetate 5 Sodmm chloride solution (15%) 6. MTBE

2.7 7. Reagents

actd, y-

for Method 3.7 1

1, (ZS,4S,5S)-5-Azido-4-hydroxy-2-lsopropyl-7-methyloctanolc 10% PdK. 3. THF 4. Sodium bicarbonate 5 Sodmm hypophosphite 6 Dt-t-butyl dtcarbonate 7 Sodmm chloride solutton (15%) 8 Hexane 9 Ethyl acetate. 10 Sthca gel (Merck, 70-230 mesh)

acid, y-lactone 13

2

2.12. Reagents

for Method 3.12

1 (2S,4S,5S)-5-(t-Butoxycarbonylamlno)-4-hydroxy-2-lsopropyl-7-methyloctanolc acid, y-lactone 14 2. Acetomtrtle . 3 Potassmm hydroxtde solutron (1 03 N) 4 THF 5 Imtdazole 6 DMF 7 t-Butyldtmethylstlyl chloride (tBDMSC) 8 Hexane 9 Sodmm chloride solutton (20%) 10 Sodmm sulfate. 11 Potassium carbonate. 12. Methanol 13, Ethyl acetate. 14 Potassmm btsulfate solutton (6 5%) 15 Sodmm sulfate 13 Acetomtrtle

2.13. Analytical

Materials 5

TLC-Plate* Slhca gei, Analtech GF; Solvent systems---#l, 15% ethyl acetate/85% hexane; #2, 50% ethyl acetate/50% hexane; #3, 5% acetone/35%

458

Lyster and Conway

methylene chloride/60% hexane; #4,3% acetone/97% methylene chloride; #5, 25% ethyl acetate/75% hexane; #6, 20% ethyl acetate/80% hexane; #7, 10% ethyl acetate/90% hexane. GC column #l-10% FFAP, 6’; conditions A-inj. 250°C det. 250°C oven temp. 150°C conditions &mj. 250°C det. 250°C Oven temp. 10&2OO”C (5 mm at lOO”C, lS”C/mm, 20 min at 200°C). GC column #2-Durawax-DXI, 1.O p film, 60 m/O.317 mm ID; condttions A-mj. 250°C det. 250°C oven temp 2 lO-240°C (4 min at 21O”C, 4”C/mm, 32 mm at 240°C). GC column #3-Chirasil-Val III, 25 m, id 0 25 cm; conditions A-inj. 200°C det. 200°C oven temp. 160-220°C (16 min at 160°C 32”C/mm to 220°C); conditions B- mj. 220°C, det. 220°C oven temp 172°C GC column #4-DB-1, 15 m, mj. 250°C det. 250°C oven temp 150°C. GC column #5-DB-I,15 m, inj. 250°C det. 250°C oven temp. 200-220°C (3”CYmin). HPLC column #1-Alltech Nucleosil 100, silica, 5 b 4.6 mmx 25 cm; Eluent-1% isopropanol/99% hexane, flow rate 1 mL/mm, detector at 254 nm. Optical rotations-determmed as a 1% solution m ethanol. 3. Methods 3.1.5-Methyl-2-HexeneI To a cooled (-26°C)

1-o/2 solution of 35 5 kg of 1 (1 Eq) m 3 12 kg of THF, add

169 8 kg of n-butyllrthrum solutron (1 Eq) over a 3-h period while mamtarrung the reaction temperature at C-10°C (see Note 1). Stir the reaction mixture for 30 mm after the addttron IS complete, and cool the mixture down to -28°C 2 Add the reactton mrxture to a cooled (-25°C) solutron of 70 3 kg of ethyl chloroformate (1.5 Eq) m 3 12 kg of THF over 50 mm whrle keeping the temperature below 0°C 3 Stir the mixture for 30 min, then warm the mixture to 25°C and star overnight

(see Note 2) 4. Cool the reaction mrxture to -24°C and add 500 kg of Vrtrrde solutron (4 Eq) over a perrod of 10 h whrle mamtammg a reaction temperature below -1O’C (see Note 3) 5 Star the reaction mixture at -10°C for 30 mm, then warm the mixture to 25°C and star overnight (see Note 4) 6 Quench the reaction mixture by adding it to a solutron of 690 kg of sulfurrc acrd m 1500 L of water while keeping the temperature below 30°C (see Note 5). 7 Extract the product with 2 x 400 L of ethyl acetate and backwash the combined ethyl acetate solution with 700 L of sodium bicarbonate solution and 2 x 500 L of

sodium chloride solution. 8 Removed the solvent through a vacuum drstlllatron to give 36 5 kg (80%) of 2 as a crude or1 that can be used m the next step (see Note 6)

459

Synthesis of Octanoic Acid 3.2. 1-Bromo-5-Methyl-2-Hexene

3

1. A cooled (-19OC) solution of 90 2 kg of crude 2 (1 Eq) m 800 L of hexane, IS treated with 131 25 kg of phosphorous tribromide (0.6 Eq) over 30 min while keeping the temperature <--10°C (see Note 7). 2 Stir the reactlon mixture for 30 mm at -lO”C, warm to O”C, and stir for 3 h 3 Quench the reaction mixture by adding It to 800 L of sodium carbonate solution, mix, and separate the organic layer 4. Wash the hexane layer with 2 x 600 L of sodium chloride solution, and dry It over sodium sulfate 5 Remove the solvent through a vacuum dlstillatlon to give 100 5 kg of crude 3 (70 5%), which can be used m the next step (see Note 8)

3.3. (2S)-2-lsopropyl-7-Methyl-4-Octenoic Acid, L-Prolinol Amide 5 and (2R)-2-Isopropyl-7-Methyl4-Octenoic Acid, r-Prolinol Amide 6 1. Add a cooled (-25°C) solution of 7 1.5 kg of 4 (1 Eq) (see Note 9) in 400 L of THF to a cooled (-50°C) solution of 95 1 kg of LDA (2 3 Eq) m 700 L of THF (see Note 10) at a rate sufficient to keep the reaction temperature below -20°C 2 Stir the reaction mixture for 3 h, and cool to -50°C (see Note 11). 3 Add a cooled (-25’C) solution of 101 5 kg of 3 (1.47 Eq) m 200 L of THF at a rate sufficient to keep the reaction mixture temperature below 40°C 4 Stir the reactlon mixture for 3 h at 40 to -45°C (see Note 12) 5. Quench the reaction mixture by adding it to 2000 L of a sodmm chloride solution 6 Separate the upper organic layer, and wash the aqueous with 600 L of ethyl acetate 7. Combme the mam organic layer and the ethyl acetate wash, and remove the solvent through a vacuum dlstlllatlon. 8 Purify the crude product through a slhca gel chromatography (500 kg of slhca gel) eluted with ethyl acetate/hexane mixtures (see Note 13) Combme the appropriate cuts and concentrate to give 96.75 kg of the desired product mixture of 5 and 6 (89%).

3.4. (2S)-2-lsopropyl-7-Methyl-4-Octenoic L-Prolinol (0-Benzoyl)Amide 7

Acid,

1 Stir a mixture of 102 5 kg of 5 and 6 (1 Eq), 56.1 kg of TEA (1 5 Eq) and 67 5 kg of benzoyl chloride (1 3 Eq) in 1000 L of methylene chloride overnight (see Note 14) 2 Wash the crude reaction mixture with 2 x 430 L of sodium bicarbonate solution and concentrate under vacuum 3 Purify the crude product through silica gel chromatography (500 kg of whca gel) eluted with ethyl acetate/hexane mixtures (see Note 15). Combine the appropnate cuts and concentrate to give 101 3 kg of 7 (68% from 4)

3.5. (2S)-2-lsopropyl-7-Methyl-4-Octenoic L-Prolinol amide 5

Acid,

1 Stir a mixture of 100 kg of 7 (1 Eq), 100 kg of acetonitnle, 108 kg of potassium carbonate (3 Eq), and 800 L of water at 78°C for 39 h (see Note 16).

Lyster and Conway

460

2 Cool the reaction mixture to 25”C, and add 1200 L of ethyl acetate 3 Remove the lower aqueous phase, and wash it with 800 L of ethyl acetate 4. Combme the ethyl acetate wash with the main organic phase, and wash with 2 x 500 L of sodmm chloride solution. 5 Remove the solvent under vacuum to give 62 2 kg of crude 5 (85%), which can be used m the next step (see Note 17).

3.6. (2S)-2-lsopropyl-7-Methyl-4-Octenoic

Acid 8

1 Heat a mixture of 62 2 kg of crude 5 (1 Eq), 1300 L of dlglyme, I84 kg of sulfuric acid (8.4 Eq) and 1140 L of water to reflux for 48 h (see Note 18) 2. Cool the reactlon mixture and wash the reactlon mixture, with 2 x 550 L of hexane Combme the hexane washes 3 Wash the combined hexane washes with 4 x 400 L of water (see Note 19) 4 Concentrate the hexane solution under vacuum to give 38 5 kg (88 %) of crude 8 which can be used m the next step (see Note 20)

3.7. (2S,4S,5R)-4,5-Dihydroxy-Z-lsopropyl-7-Methyloctanoic Acid, y-lactone 9 and (2S,4R,5S)-4,5-DihydroxyP-Isopropyl-7-Methyloctanoic Acid, y-Lactone IO Add a solution of 38.2 kg of crude 8 (1 Eq) m 1000 L of IPA to a mixture of 191 kg of MMPP (2 5 Eq) m 13 10 L of water Stir the reaction mixture at 33-35°C for 18 h (see Note 21) Quench excess peroxide with the addltton of a solution of 62 kg of sodium sulfite (2 5 Eq) m 500 L of water Remove the IPA through a vacuum dlstillatlon, and add 1000 L of water Wash the aqueous mixture with three 400-L portions of methylene chloride Combined the methylene chloride washes, and wash with 1000 L of sodium bicarbonate solution and 800 L of sodium chloride solution Dry the methylene chloride solution over sodium sulfate. Remove the solvent through vacuum dlstlllatlon to give 37.7 kg of a crude product mixture of 9 and 10 (91%), which can be used m the next step (see Note 22)

3.8. (2S,4S,5R)-4-Hydroxy-Z-lsopropyl-5-Mesyloxy7-Methyloctanoic Acid, y-Lactone 17 and (2S,4R,sS)-4-Hydroxy-2Isopropyl-5-Mesyloxy-7-Methyloctanoic Acid, y-Lactone 72 1 Treat a cooled (0-2°C) solution of 37.8 kg of a crude mixture of 9 and 10 (1 Eq) and 27.6 kg of TEA ( 1.5 Eq) m 200 L of methylene chloride with 23.95 of methanesulfonyl chloride (1.15 Eq) at a rate sufticlent to mamtam a reaction temperature below 10°C (see Note 23) 2 Stir the reaction mixture at 20-25°C for 1 h (see Note 24) Quench the reaction with a solution of 10 9 kg of HCl(0 6 Eq) m 100 L of water 3 Separate the organic layer, wash with 110 L of sodium bicarbonate solution, and 110 L of sodium chloride solution and dry it over sodium sulfate

Synthesis of Octanoic Acid

461

4 Remove the solvent through a vacuum distillation to give the crude product mixture of 11 and 12. 5 The crude product was purified through silica gel chromatography (1000 kg of silica gel) eluted with MTBE/hexane mixtures (see Note 25). This gave both isomers in pure form Yield was 19.1 kg of 11 and 25.0 kg of 12 (overall 83%).

3.9. (2S,4S,5R)-4-Hydroxy-2-lsopropyl-5-Mesyloxy7-Mefhylocefoic Acid, y-Lactone 11 1 Treat a solution of 25.0 kg of 12 (1 Eq) in 200 L of acetomtrile with 95.4 kg of potassmm hydroxide solution (2 Eq) 2 Stir the reaction mixture at 22-25°C for 18-24 h (see Note 26) 3 Treat the reaction mixture with 29 5 kg of acetic acid (5.7 Eq) and 14.5 kg of hydrochloric acid (1 7 Eq) to lactomze the intermediate carboxylate (see Note 27). 4 Add 500 L of sodium chloride solution, and wash the product out with 400 L of MTBE. 5 Wash the MTBE solution with 400 L of sodium bicarbonate solution and 500 L of sodium chloride solution, and dry it over sodium sulfate. 6. Remove the solvent through a vacuum dlstlllatlon to give the crude hydroxy lactone as an oil (see Notes 28 and 29) 7 Dissolve the crude intermediate m 200 L of methylene chloride, and add 13 0 kg ofTEA(1.5 Eq). 8. Treat the mixture with 11 8 kg of methanesulfonyl chloride (1.2 Eq) and stir the reaction at 20-25°C for one h (see Note 30). 9 Add a solution of 4 2 kg of hydrochloric acid (0.5 Eq) m 500 L of water, and separate the phases Wash the organic layer with 100 L of sodium bicarbonate solution and 100 L of sodium chloride solution, and dry it over sodium sulfate 10. Concentrated under vacuum to give 20.85 kg of crude 11 (82%), which can be used m the next step.

3.10. (2S,4S,sS)-5-Azido-4-Hydroxy-2-lsopropyl7-Methyloctanoic Acid, y-lactone 73 1. Heat a mixture of 32.55 kg of 11 (a mixture of purified from 3.8 and crude from 3.9, 1 Eq), 25.2 kg of sodium azide (3.5 Eq), 100 L of MTBE, and 536 kg of DMAC to 84-86°C for 5 h (see Notes 31 and 32). 2. Cool the reaction mixture to 25’C, and add 570 L of ethyl acetate. 3 Wash the diluted reaction mixture with 400 L of sodium chloride solution, and backwash the aqueous with 380 L of ethyl acetate. Repeat this three times using the same portion of ethyl acetate for the backwash each time. 4 Combme the ethyl acetate backwash with the main organic layer, and dry the solution over magnesium sulfate 5. Concentrated the combined organic solutions through vacuum distlllatlon to give 24.57 kg of crude 13 (92.5%), which can be used m the next step.

462 3.11. (2S,4S,5S)-5-(t-Butoxycarbonylamino)4-Hydroxy-2-Isopropyl-7-Methyloctanoic

Lyster and Conway Acid, y-Lactone

14

1 Treat a mixture of 24.4 kg of crude 13 (1 Eq), 542 kg of THF, 12.8 kg of sodium bicarbonate (1 5 Eq), 10 L of water and 1.9 kg of 10% Pd/C (0.018 Eq) with a solution of 17 9 kg of sodium hypophosphlte (1.5 Eq) m 9 1 L of water 2 Heat the reaction mixture to reflux (64°C) for 12 h (see Note 33) 3 Cool the reaction mixture to 25°C and add a solution of 33 4 kg of dl-t-butyl dlcarbonate (1.5 Eq) m 33 kg of THF 4 Stir the reaction overmght at 25’C (see Note 34). 5. Add 130 L of sodium chloride solution, and filter the mixture to remove the palladium catalyst. 6 Separate the organic layer from the filtrate, and wash it with 150 L of sodium chloride solution 7 Remove the solvent under vacuum, and add hexane to slurry the crude product 8. Collect the product by filtratlon to grve 29 kg of crude 14 (see Note 35) 9. Purify the product through silica gel chromatography (600 kg of silica gel) eluted with a 10% ethyl acetate/90% hexane mixture (see Note 36) This gave 24.75 kg of purified 14 (77 5%, mp 148-149 5’C) (see Notes 37 and 38)

3.12. (2S,4S,5S)-5-(I-Butoxycarbonylamino)4-fl-6utyldimethylsilyloxy)-2-lsopropyl7-Methyl Octanoic Acid 15 1 Treat a solution of 5 kg of 14 (1 Eq) m 2 12 kg of acetomtrlle and 200 L of water with 18.8 kg of potassmm hydroxide solution (1 Eq), and stir at 25°C for l-2 d (see Note 39) Remove the solvents through vacuum dlstlllatlon, and any residual water azeotroplcally using THF Dissolve the crude potassium carboxylate intermediate m 180 kg of DMF, and treat with 6 2 kg of lmldazole (5 Eq) and 8 43 kg of tBDMSC (3.5 Eq) Stir the reaction mixture at 25’C for 70 h (see Note 40) Wash the product from the reaction mixture with SIX 50 L portlons of hexane, wash the combined hexane washes with 70 L of sodium chloride solution, and dry it over sodium sulfate 6 Remove the solvent under vacuum to give the crude bu-sllylated intermediate asan 011 7 Dissolve the crude sllylated intermediate in 140 kg of methanol, and treat the solution with a solution of 2 43 kg of potassium carbonate (1.1 Eq) m 12 L of water 8 Stir the reaction mixture at 25°C for 30 min to hydrolyze the sllyl ester (see Note 41). 9 Concentrate the reaction mixture through vacuum dlstlllatlon, and remove residual methanol azeotroplcally with ethyl acetate 10 Dissolve the residue m 70 L of sodium chloride solution, and add 140 L of ethyl acetate 11 Adjust the pH of the reaction mixture to 3 5-4.0 with the addition of sodium blsulfate solution (see Note 42)

Synthesis of Octanolc Acid

463

12. Remove the aqueous layer from the product contammg ethyl acetate layer Wash the aqueous layer with 25 L of ethyl acetate, and combine this wash with the mam ethyl acetate solution. Dry the combmed ethyl acetate solution over sodium sulfate 13. Remove the solvent through vacuum dlstlllation, and add 22 kg of acetomtrlle to crystallize the product (see Note 43) 14. Isolate the product by filtration, and dry to give 4 8 kg of 15 (67 5 %, mp 105 5107 5°C) (see Note 44).

4. Notes 1. Durmg this addition, the add time is not as crucial as mamtammg the reaction temperature below -1O’C 2 Reaction checked by GC (column #l, condltlons A), Rt (acetylemc ester mtermediate) = 3 mm 3. During this addition, the reactlon temperature 1smore crucial than the time of the addition. 4 Reaction checked by GC (column #l, conditions A), Rt (2) = 2 mm 5 This quench may take 2-3 h m order to mamtam the temperature below 30°C 6 Product, 2, was checked by CC (column #l, condltlons A). There was some 2 found m the distillate which was recovered with a second dlstlllatlon Two lots of product were obtained 36 5 kg at 94% = 34.3 kg and 7.5 kg at 60% = 4.5 kg, total = 38 8 kg (overall yield-78 5%) ‘H NMR (CDC13, TMS) 6H(D), 0 83; lH(M), 1 2-1.75; 2H(M), 1 75-2.0, lH(SS, broad), 2 45; 2H(D), 4.0;2H(M), 5 48-5 70. 13C NMR (CDC13, TMS) CH,, 22 49, CH,, 42.23 and 63.10; CH, 28 96, 130 11, 132 24 7. Care must be taken with the HBr fumes given off during this add 8 Product analyzed by GC (column #I, conditions B) Rt (3) = 3 3 mm ‘H NMR (CDC13, TMS). 6H(D), 0 88, lH(M), 1 64; 2H(T), 1.94, 2H(D), 3 94, 2H(M), 5.71 13CNMR (CDC13, TMS). 22.15, 28 06, 33.29,41 24, 127.36, 135 23 9. N-lsovaleryl-L-prolmol, 4, can be prepared by reactmg L-prolinol with 2 Eq of lsovaleryl chloride to give the bzs-acylated ester amide product. This product 1s then hydrolyzed with aqueous base to give 4 10 The solution of LDA in THF IS prepared ahead of time and chllled to remove the heat of solvatlon associated with dissolvmg the LDA in THF. 11. This transfer was done m 18 min with the initial temperature at -52°C and the final temperature at -26Y 12. The reaction was checked by TLC (solvent system #2) Rf 5 and 6 = 0 53, Rf4 = 0 27 13 A stamless steel column (2 ft diameter x 16 ft long) was dry-packed with 500 kg of slllca gel and equlllbrated with 1000 L of 1% ethyl acetate/99% hexane mixture. The crude mixture of 5 and 6 was dissolved m 200 L of 5% ethyl acetate/ 95% hexane and charged to the column The column was eluted with 2080 L of hexane, 1000 L of 10% ethyl acetate/90% hexane, 6000 L of 30% ethyl acetate/ 70% hexane, and 3000 L of 40% ethyl acetate/60% hexane, and 200-L cuts were collected Cuts 7-14 and 1654 were combined and concentrated to a neat 011to

464

14 15

16 17

18 19 20 21 22

23

24 25

Lyster and Conway give 89.5 kg of the desired product mrxture, 5 and 6, and cuts 36, 15, and 55-60 were combmed and concentrated to a neat oil to give 14 5 kg of 011, which was assayed by TLC to be 50% the desired product mixture, 5 and 6 and 50% rmpurrties Yreld: 96 75 kg (89%) The reaction was followed by TLC (solvent system #3) R, (7 and C2 R isomer) = 033,Rf(5and6)=008 A stamless-steel column (2 ft diameter x 16 ft long) was dry-packed with 500 kg of sthca gel and equrhbrated with 1000 L of 5% ethyl acetate/95% hexane The crude product mixture was drssolve in 150 L of 50% ethyl acetate/50% hexane, and the solution was charged to the column, eluted with 3000 L of 5% ethyl acetate/95% hexane, 3000 L of 20% ethyl acetate/80% hexane, and 1000 L of 50% ethyl acetate/50% hexane, and 200-L cuts were collected and checked by TLC (solvent system #3) and HPLC (column #1) Cuts 5-10 were concentrated to give 63.3 kg of 7 ( HPLC assay-99 9%), cuts 1 l-16 were concentrated to grve 62 kg of a mixture of 7 and the 2R Isomer, Cuts 17-20 were concentrated to give 1427 g of the 2R Isomer The mixed cuts were further purtfied through chromatography to give an addttronal38 kg. Total yield 101 3 kg (68% yield) The reaction was followed by TLC (solvent system #5). R,-5 = 0 35, Rf7 = 0 7 An ahquot was removed prior to concentratron, and the yield estimated from this operation was 62 2 kg of 5 Product analyzed by HPLC (column #l)* 99 2% 5 (retention time 10 5 mm), 0 5% the 2R isomer (retention time 15.0 mm) and 0 3% of material with a cis double bond. The reaction was followed by TLC (solvent system #3) Rf 5 = 0 35, Rf 8 = 0 1 These washes are needed to remove residual DMAC m the product solution ‘H NMR (CDCl,, TMS) 6H(D), 0.84,6H(D), 0 96; 2H(M), 1 39-l 72,2H(M), 1 72-1 98, 3H(M), 1 98-2.33; 2H(M), 5.3-5.5; lH(S, broad), 11 9 The reaction was followed by TLC (solvent system #3)* Rf 8 = 0 1, R, (9 and 10) = 0 4 The product mixture was analyzed by GC (column #3, conditrons A)* 9 (2S,4S,5R rsomer)-39 27% (RT = 8 56 mm), 10 (2S,4R,5S Isomer)-58 09% (RT = 7 25 mm); with mmor isomers as follows* 2S,4S,5S Isomer-0 3 1% (RT = 6.68 mm), 2S,4R,5R isomer4 45% (RT = 5 99 min); 2R,4R,5R isomer and 2R,4R,5S ISOmer (not resolved)---0 74% (RT = 6.82 mm), 2R,4R,5S isomer-l 14% (RT = 9 08 mm). The 2R,4S,5S isomer was not seen (RT = 5 77 mm) This addition was done over a period of 1.5 h to keep the reaction temperature below 10°C Thus 1s done to mmrmrze a side reaction that displaces the mesylate with chloride ion present m the reaction mixture This side reaction gives the inverted chloride which will lead to lower yields later m the sequence The reaction was followed by TLC (solvent system #4) Rf (9 and 10) = 0 45, Rf (lland12)=064 A stainless-steel column (2ft dtameter x 16 ft long) was dry-packed wrth 500 kg of silica gel and equtltbrated with 1000 L of 10% MTBE/90% hexane solvent mixture. The column was charged wrth half of the crude product and eluted with a 15% MTBE/85% hexane solvent mixture, and 200-L cuts were collected The cuts were analyzed by TLC (solvent system #I), Rf 11 = 0.20, Rf 12 = 0 16 and

Synthesis of Octanoic Acid GC (Column #4, Rt 11 = 4.24 min, Rt 12 = 4.54 min) with cuts 15-40 contammg 11, cuts 41-99 contammg 11 and 12, and cuts lO(rll7 containing 12. The sec-

26 27.

28

29 30 31

32. 33.

34

35

36.

ond half of the crude product and all mixed cuts were similarly purified with a total of four separate columns carried out Yield 19.1 kg of 11 and 25.0 kg of 12 (overall 83%) The reaction was followed by TLC (solvent system #4). R, 12 = 0 7; Rr(mtermediate carboxylate) = 0 0. This sequence inverts the C-4 and C-5 posittons, and gives the hydroxy lactone isomer 9 from the mesylate 12. The reaction was followed by TLC (solvent system #4) Rf (intermediate carboxylate) = 0 0; Rf 9 = 0.5. The crude product was analyzed by GC (column #3, conditions A)* 0.5% 2S,4S,5S isomer; 91 3% 2S,4S,5R isomer, 9, 2 0% 2S,4R,5S isomer, 2 6% 2R,4R,5S ISOmer, 3 6% 2R,4S,5R isomer. The yield (as determined by analysts of an ahquot) was 19kgof9. It IS important to make sure that there IS no residual water m the crude lactomzed intermediate 9 before attempting the mesylation reaction to produce 11 The reaction was followed by TLC (solvent system #5)* Rf (9) = 0.35; Rf 11 = 0 68 Every material used m this reaction was filtered to make sure that no metallic particles found their way into the reaction mixture. This was owing to the possible formation of heavy metal salts of the aztde ion, which can be explosive The reaction was followed by TLC (solvent system #6): Rf 11 = 0.4, R, 13 = 0.7 The reaction was followed by TLC (solvent system #6)* Rf 13 = 0 7, Rf (amme intermediate) = 0 15 Owing to incomplete reaction at 9 5 h of reaction time, an additional 500 g of 10% Pd/C catalyst were added to the reaction The reaction was complete m 10 5 h The reaction was followed by TLC (solvent system #6). Rf (amine intermediate) = 0 15; Rf 14 = 0 67 At 1 h of reaction time, 2 kg of sodium bicarbonate were added to make sure that all the amino function was free. The reaction was complete m 2 h The product was analyzed and found to contain a substanttal amount of a urea byproduct, which is the result of the t-BOC function of 14 reacting with unprotected amme mtermediate This reaction is promoted by the presence of sodium hypophosphite. The need for chromatographic purification at this time was necessary to remove this urea byproduct A stainless-steel column (2 ft diameter x 16 ft long) was dry-packed with 300 kg of silica gel and equilibrated with 750 L of 10% ethyl acetate/90% hexane. Onehalf of the crude 14 was dissolved m 60 L of methylene chloride and charged to the column. The column was eluted with 10% ethyl acetate/90% hexane and cuts (200 L each) were monitored by TLC (solvent system #6). Rf 14 = 0 67, R, (urea byproduct) = 0 28 The second half of the crude 14 was purified m the same manner through a second column, Appropriate cuts were combmed and concentrated to dryness The product was slurried in hexane, and isolated by filtration, and dried. Yield 24 75 kg (88%).

466

Lyster and Conway

37 Analytical data on 14. mp 148149 5°C ‘H NMR (CDCl,, TMS) 0 95 Hz, 6H, d, 108 Hz, 6H, d, 1 s1.9 Hz, 4H, m, 1 5 hz, 9H, s, 2 &2 7 Hz, 3H, m, 3 65-4 03 hz, lH, m, 4 Wl.6 Hz, lH, m; GC-100% U-75,103, opt. rot (1, EtOH). -38 9” (ref-41”C) 38 Samples of the other seven possible isomers of 14 were prepared and a choral GC analysts was developed (GC column #3, condtttons B) Rt (14-2S,4S,5S isomer) = 2 1 28 mm, Rt (2R,4S,5S isomer) = 2 1.74 mm, Rt (2R,4R,5R isomer) = 22 18 mm, Rt (2S,4R,5R isomer) = 23.20 mm, Rt (2R,4R,5S isomer) = 28 93 mm, Rt (2S,4S,5R Isomer & 2S,4R,5S isomer which coelute) = 29 91 mm, Rt (2R,4S,5R isomer) = 30.49 mm 39 This reaction was monitored by TLC (solvent system #7) R, 14 = 0 1, R, (carboxylate mtermedtate) = 0.0. If the reaction is mcomplete after 1 d a small portion of additional potassium hydroxide solution can be added to drove the reaction to completton 40. This reaction was monitored by TLC (solvent system #6)* R, (carboxylate mtermediate) = 0.0, R, (bzs-stlylated mtermedtate) = 0 3 Reaction time = 67 5 h 41. Thts reaction was monitored by TLC (solvent system #6): Rf (bzs-stlylated mtermediate) = 0.3, Rr(15) = 0.2. The reaction was complete m 30 mm 42 If the pH is allowed to go below 3.5, some degradation of the product is observed owing to silyl ether hydrolysis 43. After the acetomtrtle IS added, the mixture can be heated to dissolve the crude product and then cooled to 0°C to crystallize the product. 44 Analytical data. GC (column #5)-99.76% 15 (Rt = 1 37 mm), 0.24% C-2 eptmer (Rt = 4 91” mm), opt rot (1, EtOH) -33 8’; ‘H NMR (CDCls, TMS). 6H(S), 0 1, 21H(M), 0 75-1 0, 2H(M), 1 2-l 6, 9H(S), 1 48; 4H(M), 1 62 1, IH(M), 2 2-2 5; lH(M), 3 5-3 8; IH(M), 4 35-4 55, 1 lH(M), 5 4-5 65

Acknowledgments This proJect received extensive analytical support from several labs at The UpJohn Co. The authors would like to acknowledge specifically the efforts of F. M. Eckenrode, G. A. Allison, W. M. Knoll, J. W. Manskl, and C I Johnson of Firm Chemical Analytical Services. References 1 This work is based on initial efforts by many other labs at The UpJohn Co The authors would like to acknowledge spectftcally Jackson B Hester and his coworkers. 2 Thts couplmg was based on work by Evans, D. A. and Takacs, J. M , (1980) Enantioselective alkylation of chiral enolates Tetrahedron Lett 21,42334236 3. This conversion 1sbased on researchcarried out by Peter G M Wuts at The Upjohn Co 4 This reductton is based on work by Boyer, S K , Bach, J , and McKenna, J (1985) Mild hydrogen-transfer reductions using sodium hypophosphite J Org Chem 50,3048-3411.

27 Synthesis

of a-Vinyl Amino Acids

David B. Berkowitz, Jill M. McFadden, Michelle L. Pedersen

Marianne

K. Smith, and

1. Introduction This chapter presents procedures for the synthesis of a-vinyl ammo acids, m which the usual a-proton is replaced by an unsubstltuted vinyl group (Fig. 1). The parent member of this family, a-vinylglycine (R f H), is a natural product (1,2) and acts as a suicide substrate for a number of PLP-dependent enzymes (4-9) Higher members of this family (R z H) have also been synthesized (1O12). Several including a-vinyl-m-tyrosine (13-15), a-vinyl-DOPA (13-151, a-vinylglutamate (16), a-vinylormthine (171, a-vinyllysine (18), and a-vinylargmine (18) are Trojan horse inhibitors of their cognate ammo acid decarboxylases (AADCs). Such (appropriately labeled) AADC inhibitors may also have potential as reagents for positron emission tomography (29). a-Viny1 ammo acids have potential as novel building blocks for unnatural peptlde synthesis. It 1sknown that peptldes containing a-branched ammo acids often tend toward helical secondary structure (20,21) and may be resistant to proteases (22). a-Vmylglycine has been incorporated mto peptides (23), m one case to produce a suicide substrate for peptldylglycme a-hydroxylatmg monooxygenase (23). This chapter 1spresented m three parts: 1. Imtially, a convenient and readily reproducible procedure for the synthesis of racemlc higher, a-vinyl ammo acids is presented (Fig. 2) This approach mvolves the formal vmylatlon of an ammo acid-derived dlanion with ethylene oxide serving as the vinyl cation equivalent (IO). Two examples are given, syntheses of (k)-a-vmylphenylalanme and of (f)-a-vmylhrstldme The followmg two sections deal with more recent, enantloselective syntheses

From

Methods m Molecular Me&one, Vol 23 Pepbdomrmebcs Protocols Edlted by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

467

468

Berkowitz et al

Fig 1 Generic structure for a-vmyl ammo acids

(PhSe)z,

NaHB(OM+

WNz

4) 2a (85%)

la/b

(*) 2b (80%) PhSa

(*)3a (i)

(97%) 3b

(69%)

(*) 4a (99%)

(*)Sa

(i)

(i)

4b

(80%)

Fig 2 Formal a-vmylatton

Bac20.

(80%) Sb (73%)

of ammo acids

NEt,

(PhS&

NaBH4, w

CH$&

0

DMF,

(97%)

6

Ph&N,

(83%)

7

TFA

NH&x

8

9

-

CH2C12

(90%. 2 steps)

10

Frg 3 Synthesis of L-vmylglycme 2 A convenient synthesis of L-vmylglycme from L-homoserme IS detailed (Fig. 3) (36) Key features mclude racemrzatron-free, phenylselenolate-medtated lactone ring-opening (24) and the use of acid-labile protecting groups to provide smooth access to this P,y-unsaturated ammo acid wtthout double-bond rsomertzatron Operationally, this method appears to be at least as reproducrble as any of the enantroselecttve vmylglycme syntheses so far reported (3). For mtermedrate-scale

a- Vinyl Amino Aads

469

(PhSe)i,

NaBH, Ph2CN2

(*) 1 3

B.&i YY

‘SePh

(94%)

TPA CCHPh2

-

-

(96%)

4) 1 4

16

Fig. 4 Synthesis of the chu-al vmylglycme

dienolate precursor

Me

b

299 1

WA-

TL

Frg. 5. Alkylatrons of choral vmylglycme-derrved

dramon drenolates.

work (up to several grams), this procedure IS economically viable For largescale work, however, the prrce of the starting material, L-homoserme, becomes prohibitive. For such applications, procedures emanatmg from L-glutamate (3i,3j) or L-methronme (3e) may be more destrable. 3 A choral auxrlrary-mediated synthesis of enantromerrcally enrrched, hrgher a-vinyl ammo actds 1sdescribed (Fig. 4-6). Operattonally, a choral, vmylglycmederrved dranion IS generated from dehydrobutyrme derivative 16 and then alkylated with a suitable electrophrle All choral mformatton resides m the (-)-8(P-naphthyl)menthyl ester auxiliary (25)

Our working model for the reactive conformation of the chnal vmylglycmederived dranion 17 is illustrated m Fig. 5. The key structural features that have been designed mto 17 are: (1) a-Amidate chelation to lithium to enforce the E-enolate geometry and (2) potential z-stackmg of the naphthyl moiety of the auxiliary with the extended dtenolate z-system to enforce si face alkylanon. In fact, alkylatron of 17 proceeds with near-absolute drastereoselectivity,

470

Berkowitz et al.

===&$+& 0

Kn-~~~yj

)

====

18alb

=---

6Ny_ 0

(-)-a-#-naphthyl)menthol

0

19a

(69%)

2Oa

(96%)

19b

(92%)

2Ob

(96%)

Fig 6 Deprotection and recovery of the choral auxiliary particularly with “nonconjugated” electrophiles. Examples are provided here for the synthesis of L-a-vmylleucme and L-a-vmylomtthine. In these cases, exclusive deconjugative a-alkylation is seen, as opposed to y-alkylation. The chn-al auxiliary may be recovered m high yield using a modification of Gassman’s “anhydrous hydroxide” conditions for ester hydrolysis (26) (Fig. 6). Acid-catalyzed benzamide hydrolysis then yields the free, enantiomerically enriched, a-vmyl amino acid.

2. Materials 2.1. General All reactions are conducted under argon atmosphere using oven-dried glassware unless otherwise noted. Methylene chloride, pyridme, and dnsopropylamine are distilled from CaH2. THF, Et,0 PhH, and toluene are distilled from sodium benzophenone ketyl. Methanol is distilled from Mg. Other reagents are obtained from commercial sources and used without further purification. Flash chromatography is performed using Merck sthca gel 60 (230-400 mesh). Meltmg points were determined using a Meltemp II (Laboratory Devices, USA) apparatus and are uncorrected. For the spectral data reported herem, ‘H NMR spectra were recorded on a GE Omega-500, GE Omega-300, NT-360, or VXR200 instrument. Proton-(de)coupled 13C NMR spectra were acquired on a GE Omega-500, GE Omega-300, or VXR-200 instrument. Infrared spectra were obtained using an Analect RFX-65 FTIR spectrometer. Mass spectra were acquired at the Nebraska Center for Mass Spectrometry (University of Nebraska-Lincoln) and are reported as m/z (relative mtenstty). Elemental analyses were carried out by M-H-W Labs (Phoenix, AZ). All compounds gave a satisfactory elemental analysis (k 0 4% for C, H and N) or were judged to be of adequate purity by HRMS and ‘H and 13C NMR spectra.

2.2. Reagenfs for the Synthesis of (,)-a-Vinylphenylalanine

5a

1 Methyl N-benzoylphenylalanmate (19) 2 Ethylene oxide (pressure tin from Fluka most convenient, otherwise a lecture bottle from any source) 3 Dlphenyl diselenide (Ph,Se*)

471

a- l/my/ Amino Amds 4 5 6 7 8 9 10 11. 12 13. 14 15 16 17 18 19 20 21 22 23 24 25 26. 27. 28. 29 30 31 32

Sodium trtmethoxyborohydrtde [NaHB(OMe)s]. N-Methyl-N-mtroso-p-toluenesulfonamide (Dtazald@) N,N,N’,N’-Tetramethylethylenediamme (TMEDA). 1-Hexene. Dnsopropylamme n-Butyllnhmm (Aldrtch; nominally 1.6 M in hexanes) tttrated as per ref. (28), requu-es. 2,!GDimethoxybenzyl alcohol KOH NaHCO, Na,CO, NH&l MgSO‘, NH40H NaOAc NH&l Acetic acid (CHsCOOH) Trtethylamme (NEt,, Acetone Acetic Acid Benzene Chloroform Ethyl acetate (EtOAc) Dlethyl ether (Et,O) Hexanes Methyl alcohol (MeOH) Hydrochloric acid (HCl). Methylene chloride (CH,Cl,). Tetrahydrofuran (THF) NJ-Dlmethylformamtde (DMF). HCl

2.3. Reagents

for the Synthesis

of (k)-a-Vinylhistidine

5b

As for Subheading 2.2., except substitute methyl Na-benzoylhtstidmate for methyl N-benzoylphenylalanmate (ref. 21) and add: triphenylmethyl chloride and tetrabutylammomum pertodate

2.4. Reagents for the Synthesis of L-a-Vinylglycine 1 2 3 4. 5 6 7

L-Homoserme Trlfluoroacetic acid (TFA) Dt-tert-butyl-dtcarbonate (Boc)~O. Trtethylamme. Diphenyldrselenide (Ph,Se,) Sodium borohydrtde NaOAc

10

Berkowitz et al.

472 8 1-Hexene 9 Drphenyl diazomethane prepared as per ref. 30, requires. Benzophenone hydrazone, Mercurtc oxide (HgO), Na2S04, KOH, Ethyl alcohol (EtOH). 10 CH& 11 DMF 12. NaHCO, 13 NH&l 14 MgS04 15 EtOAc 16 Benzene 17 Methyl alcohol. 18 Diethyl ether (EtzO) 19 Hexene 20 HCl 2 1 Acetic acid (CHsCOOH)

2.5. Reagents for the Preparation of (7 T?,2S, 571)~8’(P-Naphfhyl)menthy/ N-Benzoyi-@)-dehydrobutyrinafe 1, 2 3 4. 5. 6 7

8

9 10 11

(f)-a-Ammo-y-butyrolactone hydrobromtde Benzoyl chlortde (BzCl). Trtethylamme Ph2Se2 Sodium borohydrtde. 1-Hexene Diphenyl dtazomethane prepared as per ref. 30, requnes Benzophenone hydrazone, NO, WSO4, KOH, EtOH. 8-(P-Naphthyl)menthol prepared as per ref. 25, requrres 2-Bromonaphthalene, (R)-(+)-Pulegone, Magnestum turmngs (Mg), Copper chlortde (I) (CuCl), Ltthmm alummum hydrtde (LAH), Et,0 TFA p-TsOH CH2C12

16

473

a- Vinyl Amino Acids 12 13 14 15. 16 17. 18 19 20. 21.

DMF. NaHCO, NH&l cuso, Methyl alcohol (MeOH) EtOAc Benzene Et,0 Hexane HCl

2.6. Reagents for the Preparafion

of L-a- Vinylornifhine

20a

1 Dusopropylamme 2. Hexamethylphosphoramtde (HMPA) 3 n-Butylhthmm (Aldrich; nommally 1.6 A4 in hexanes) trtrated as per ref. 20, requires. 2,5-dtmethoxybenzyl alcohol and 1-benzaldtmmo-3-todopropane Prepared as per ref. (17); requires 1-Ammo-3-bromopropane, Benzaldehyde, NaI 4 Potassmm tert-butoxrde (KOtBu) 5 CH,CI, 6 NaHCO, 7 Na,CO, 8 NH&l 9. MgSO, 10. NH,OH 11. EtOAc 12. 1,4-Droxane. 13 Et*0 14 Hexenes 15. MeOH 16 HCl

2.7. Reagents for the Preparation

of r-a-Vinylleucine

As for Subheading 2.6., above, except substitute for 1 -benzaldlmmo-3-rodopropane.

20b

1-rodo-2-methylpropane

3. Methods 3.1. Preparation of (+a- Vinylphenylalanine 3 7.1. (A)-N-Benzoyl-2-Benzylhomoserine Lactone 2a 1 (See Note 1 ) To a solutton of dnsopropylamme (22 4 mL, 160 mmol) and TMEDA (36 2 mL, 240 mmol) tn THF (800 mL) at -78°C is added IZ-

Berkowitz et al.

474

2

3 4

5

butylhthmm (100 mL, 1 6 A4 m n-hexane), and the resultmg solution stlrred for 30 mm at 0°C. The LDA solution thereby generated 1s cooled to -7PC and a solution of (+)-la(14 6 g, 52 0 mmol, prepared accordmg to ref. 27) m THF (350 mL) at -78°C 1sadded via cannula The resultmg bright orange solution 1s stirred for 1 h at -78°C The cooling bath 1sremoved, and excess ethylene oxide (ca. 25 g) 1sbubbled mto the solution with stirring over a period of 45 mm The reaction mixture 1spoured mto ether (500 mL) and NH,Cl (aq ,500 mL) and vigorously stirred for 15 mm After further extraction with ether (3 x 300 mL), the combined orgamcs are dried (MgSO,), filtered and evaporated Flash chromatography (50% EtOAc/hexane) gives 2a (13 0 g, 85%) as a white solid. mp 17G172’C; ‘H NMR (200 MHz, CDCl,) 6 2 66-2 77 (m, 2 H), 3 123 19(d,J=13Hz,lH),322-3.29(d,J=13Hz,lH),34LC357(appdt,J=8,9Hz, 1 H), 4 22-4.32 (app dt, J= 3,9 Hz, 1 H), 6.61 (s, 1 H), 7 15-7 68 (m, 10 H), 13C NMR (50 MHz, CDCI,): 6 33.5, 41 6, 66 0, 127 0, 127 9, 128 7, 128 9, 130 0, 132 1,1767,178 l,IR(film). 1760,1650cm-‘,MS(EI) 295(8 6,M+),204(100)

3 1 2. (*)-Methyl N-Benzoyl-2-(2’-Phenylseleno) Ethylphenylalaninate 3a (See Note 1.) To an argon-purged flask containing so&urn tnmethoxyborohydride (563 mg, 4.41 mmol) 1sadded a solution of diphenyl diselemde (687 mg, 2 20 mmol) m DMF (30 mL) The resulting orange suspension is heated to 65°C for 1 h, whereby a clear, colorless solution results To this solution at room temperature 1s added a solution of lactone 2a (1 00 g, 3 39 mmol) m DMF (30 mL), and the reaction mixture heated at 110°C (preheated oil bath) for 2 h. After cooling to room temperature, the solvent 1s removed m vacua, and the residue diluted with Et,0 (200 mL) and brought to pH 4.0 with 1 N HCl (aq ) The aqueous layer 1s extracted twice more with Et,0 (200 mL) and combined orgamcs dried (MgSOJ and filtered Esterificatlon of the crude acid 1s then carned out with dlazomethane To a solution of N-methyl-N-mtroso-p-toluenesulfonanude (Diazald@) (2 15 g, 10 1 mmol) m Et,0 (31 mL), m a dark bottle equipped with a magnetic stirrer, 1s added a cold (5OC) solution of KOH (2 3 g, 41 mmol) m H,O (4 5 mL)/EtOH (7 5 mL) The bottle 1s topped off with a one-hole rubber stopper, fitted with a fire-polished bent glass outlet tube (see Note 3) The resultmg two-phase nuxture 1s heated to 45°C with vigorous stmmg. As soon as heating commences, the gas outlet tube is directed mto an Erlenmeyer flask containing the combined ethereal extracts of the crude acid (at O°C) from step 3 The CH,N,/Et,O codlstlllate 1sbubbled directly mto the solution of the acid until approximately two-thirds of the ether has been distilled over (ca 20 mm) Then the heating bath 1sremoved, and the distillation is terminated After tiher stu-nng of the estenfication reaction mixture (typically l&30 nun), an acetic acid quench (1 mL) 1s added, and sturmg 1scontinued for another l&30 mm.

CX-Vmyl Amino Acids

475

5 Evaporation of the solvent and chromatography (O--10% EtOAc/hexane) yields 3a (1.52 g, 97%) as a white solid* mp 124-126’C, ‘H NMR (360 MHz, CDCl,). 6 2.24-2 32 (ddd, J= 6, 11, 14 Hz, 1 H), 2 43-2.51 (ddd,J= 6, 11, 12 Hz, 1 H), 2.73-2 79 (ddd, J= 5, 11, 12 Hz, 1 H), 2 97-3 01 (d, J= 13 Hz, 1 H), 3 14-3.21 (ddd, J= 5, 11, 14 Hz, 1 H), 3.64 (s, 3 H), 3.76-3.80 (d, J= 13 Hz, 1 H), 6 846 86 (m, 3 H), 7.01-7.14 (m, 6 H), 7 24-7.39 (m, 5 H), 7.51-7 55 (m, 2 H), 13C NMR(50MHz,CDCl& 621.9,36 1,40 6,52 8,66 8, 126.9, 126.9, 127.0, 128 3, 128 6, 129.1, 129 3, 129.6, 131 6, 132 4, 134.9, 135 9, 166 7, 173 4; IR (film) 1735, 1663 cm-‘, MS (EI). 467 (0 4, M+), 105 (100)

3.1.3 (+Mefhyl

N Benzoyl-2-Vinylphenylalaninate

4a

1 Ozone IS bubbled into a solution of selemde 3a (6 84 g, 14.7 mmol) in CH,Cl, (150 mL) at -78°C until a light blue color persists 2 After addition of 1-hexene (3 1 7 mL, 255 mmol; see Note 4), this cold solution IS added dropwise to refluxmg PhH (500 mL), and refluxing 1scontinued for 30 mm (see Note 5) 3 All volatlles are evaporated zn vucuo and the crude product IS subjected to flash chromatography (&lo% EtOAc/hexane) to yield 4a (4 48 g, 99%) as a white solid mp 1l&l 17OC;IR (film)* 1740,165O cm-‘; ‘H NMR (300 MHz, CDCl& 6 3 40-3 45 (d, J= 14 Hz, 1 H), 3 82 (s, 3 H), 3 89-3 93 (d, J= 14 Hz, 1 H), 5 28-5.34 (d, J= 17 Hz, 1H),532-535(d,J=llHz,1H),614-624(dd,J=11,17Hz,1H),699(s,1H), 7 06-7 09 (m, 2 H), 7 19-7.22 (m, 3 H), 7.39-7 50 (m, 3 H), 7 6+7 72 (m, 2 H), 13C NMR (50 MHz, CDCl,): 6 40 0, 53 0,65.9, 116 3, 126.9, 127 1, 128.3, 128 6, 130.0, 131.6, 134 8, 135.7, 136.3, 166 4, 172.3; MS (EI). 309 (3, M+), 105 (100)

3.1.4. (k)-a-Vmylphenylalan~ne

5a

1 A suspension of 4a (500 mg, 1 62 mmol) m 6 NHCl(l3 mL) is refluxed for 4 h. 2 After extraction with CH2C12 (25 mL), the aqueous layer 1s evaporated zn vucuo with mild heating (T < 50°C) 3 The residue is applied to a Dowex 50 X 8 Ion-exchange column After washmg with several column volumes of H20, product is eluted with 1.3 M NH,OH. Fractions are analyzed by TLC using CHZC12/MeOH/NH40H(conc. aq ) (94 5: 1) as eluent and ninhydrin staining Clean fractions are pooled and evaporated using a rotary evaporator to which a vacuum pump has been attached, Further drymg 1scamed out at 0 1 torr with heating (T = 5O’C) and a P205 sidearm One obtains 5a (247 mg, 80%). ‘H NMR (500 MHz, D,O) 6 2 99-3.04 (d, J= 14 Hz, 1 H), 3.31-3 36 (d, J= 14 Hz, 1 H), 5 19-525(d,J=17Hz,1H),5.32-536(d,J=11Hz,1H),60~.15(dd,J=11,17Hz,1 H), 7 17-7 20 (m, 2 H), 7 27-7 31 (m, 3 H); 13CNMR (50 MHz, D20) 6 41.1,65 9, 116 3, 127.6, 128 6, 129 9, 133 3, 134 9, 173 3; MS (EI) 191 (1, M+), 100 (100)

3.2. Preparation of (k)-a-Vinylhistidine 3.2.7. (&)-Methyl N93enzoykNT-Trityhistidinate

lb

1. To a solution of methyl N-benzoylhlstidinate (7.77 g, 28.4 mmol, prepared according to ref. 29) and trlphenylmethyl chloride (15 7 g, 56 9 mmol) m CH2C12 (120 mL) at room temperature is added NEt, ( 17 0 mL, 122 mmol).

476

Berkowitz et al

2 After 4 5 h, the volatrles are removed in vacua and the resrduepurified by column chromatography (30% acetoneKHC1,) to yield lb (14 3 g, 98%) as a light yellow solid mp 74-76”C, ‘H NMR (300 MHz, DMSO-d,) 6 3.07-3 14 (dd, J= 4,14Hz,1H),315-321(dd,J=5,14Hz,1H),363(s,3H),498-504(appdt, J= 5,8 Hz, 1 H), 6.60-6.60 (m, 1 H), 7.08-7 14 (m, 6 H), 7 27-7 51 (m, 13 H), 7 89-7.93 (m, 2 H), 8 38-8 41 (d, J= 8 Hz, 1 H), t3C NMR (125 MHz, CDCl,) 6 26 6, 52 0, 52.9, 75.2, 119 6, 127 2, 128 0 (2 C), 128 3, 129.6, 131 3, 134 0, 136 6, 138 7, 142 2, 166 8, 171 7, IR (film)* 1745, 1660 cm-‘, MS (FAB, 3NOBA): 516 (50, MH+), 105 (100).

3.2.2. (k)-N -6enzoyL2-[(l ‘-T&y/)-4’-lmidazolyl] Methylhomosenne Lactone 2b 1 For formation of this dramon and its reaction with ethylene oxide, Subheading 3.1.1. is followed, except that NaHC03 (aq.) IS substituted for NH4CI (aq ) m the workup 2. From lb (1 80 g, 5.50 mmol) is obtained 2b (1 48 g, SO%), as a white solid, after flash chromatography (45 :45.10 CHCls/hexane/acetone) mp 104-l 06”C, ‘H NMR (500 MHz, CDCl& 6 2 18-2 23 (ddd, J= 4,8, 13 Hz, 1 H), 2.67-2 73 (appdt, J= 9, 13 Hz, 1 H), 2.78-2 81 (d, J= 14 Hz, 1 H), 3.15-3.18 (d, J= 14Hz, 1 H),4.19-424(appq,J=9Hz, 1 H),4.56-461 (appdt,J=4,9Hz, 1 H),675 (s, 1 H), 7 08-7.18 (m, 6 H), 7 22-7.7.49 (m, 13 H), 7 90-7 92 (m, 2 H), 9 18 (s, 1 H), 13CNMR (125 MHz, CDCl,) 6 31.5, 33 6, 58 9, 64.9, 75 5, 120 7, 127 3, 128.1 (2 C), 128.2, 128 4, 129.7, 131 6, 133 2, 135 4, 139.1, 142 1, 166 1, 176 0, IR (film) 1770, 1660 cm-‘, MS (FAB, 3-NOBA). 528 (100, MH+), 243 (100)

3 2 3. ($-Methyl Na-Benzoyl-2-(2’~phenylseleno) Ethyl-N *-Tritylhlstidinate 3b 1 For phenylselenolate-mediated lactone opening and subsequentesterrficatton, Subheading 3.1.2. is followed, except that 100 mMNaOAc/HOAc buffer (aq , pH 5.0) IS substttuted for 1 N HCl m the actdtficatton step (Note 6) 2 From lactone2b (1 44 g, 2.73 mmol)is obtained3b (1 32 g, 69%) asa white solid,after flash chromatography(O-50% EtOAc/hexane). mp 6668”C, ‘H NMR (500 MHz, CDCl,) 6 2 462 51 (m, 1 H), 2 68-2.74 (m, 1H), 2 8%2 96 (m, 2 H), 3 l&3 13(d, J= 14 Hz, 1 H), 3 62-3 65 (d, J= 14Hz, 1 H), 3.73 (s, 3 H), 6 50 (s, 1 H), 6 97-6 98 (m, 6 H), 7 14-7 29 (m, 12 H), 7 34-7.49 (m, 6 H), 7 63-7.67 (m, 3 H), 13CNMR (125 MHz, CDC13)6 21.9,34 1,36 1,52.8,64 8,76 3, 1205, 126 7, 127.0, 12800 (2 C), 128.03,1285, 129.0,1296,1304, 131.4,132 1, 134 5, 1384, 142 1, 166 2, 173 6, IR (film). 1735, 1665cm-‘, MS (FAB, 3-NOBA) 700 (4, MH+), 243 (100).

3.2.4. (*)-Methyl Na-Benzoyl-Nr-Trityl-2-Vmylhistidinate

4b

1 A solution contammg 3b (995 mg, 1.43 mmol) and tetrabutylammonmm periodate (803 mg, 1 85 mmol) m CHCl, (25 mL) is refluxed for 5 h (seeNote 7) 2 After coolmg to room temperature, the volatlles are evaporated

477

CI-Vinyl Amino Acids

3 The crude reaction mixture 1schromatographed (O-SO% EtOAc/hexane) to give 4b (618 mg, 80%) as a white solid: mp 73-75°C ‘H NMR (500 MHz, CDCI,): 6 3 09-3.12 (d, J= 14 Hz, 1 H), 3.25-3 27 (d, J= 14 Hz, 1 H), 3.75 (s, 3 H), 4 985.01 (d,J= 17H2, 1 H),5 12-5.15(d,J= 11 Hz, 1 H),629-&34(dd,J= 11, 17 Hz, 1 H), 6.55 (s, 1 H), 7 05-7.09 (m, 6 H), 7.24-7.34 (m, 9 H), 7.39-7.49 (m, 4 H), 7 89-7 91 (m, 2 H), 8.79 (s, 1 H), 13CNMR (125 MHz, CDCl,) 6 35.4,52 7, 64 4, 75.3, 114 5, 120 8, 127 3, 128.0, 128.1, 128 4, 129.7, 131.4, 134 2, 135.7, 135 9, 138.5, 142.2, 165 8, 172 6; IR (film)* 1740, 1670 cm-‘; MS (FAB, 3NOBA). 542 (2, MH+), 243 (100)

3.2.5. (k)-a-Vinylhistidine

5b

1 A suspension of 4b (200 mg, 0.369 mmol) m 6 N HCl(4 mL) is heated at 150°C m a sealed glass vessel for 5 h. 2. Purification is carried out as for 5a to give 5b (48.7 mg, 73%) as a white solid mp 179-181°C; ‘H NMR (300 MHz, D,O): 6 3 063 12 (d, J= 15 Hz, 1 H), 3 32-3.37 (d, J= 15 Hz, 1 H), 5 31-5.37 (d,J= 18 Hz, 1 H), 5.40-5 43 (d,J= 11 Hz, 1 H), 6.1@-6 19 (dd, J= 11, 18 Hz, 1 H), 7 01 (s, 1 H), 7 70 (s, 1 H); 13C NMR(75MHz,D,O).6339,66.4,1176, 118.2,131.9, 1358,136.8, 1747,MS (FAB, 3-NOBA): 182 (100, MH+).

3.3. Preparation of r-a-Vinylglycine 3.3.7. N-(tert-Butoxycarbony/)-L-Homoserine

La&one 7

1. To a solution of L-homoserme lactone, trlfluoroacetate salt (2 00 g, 9.29 mmol, readily obtained from L-homoserme by treatment with neat TFA [32, Note S]) and NEt, (1 4 mL, 9 29 mmol) m CH2C12 (38 mL) at 0°C IS added dl-tert-butyl dlcarbonate (2.03 g, 9 29 mmol) 2 The reaction mixture 1s stlrred for 12 h at room temperature and then washed wlthH,O(l x25mL)andlNHCl(l x25mL) 3 The orgamc layer 1s dried (MgSO& filtered and evaporated to give 7 (1 82 g, 97%) as a white solId* mp 111-I 13°C; [a]oz4 = +8.16” (c = 2 5, CHC13), ‘H NMR (300 MHz, CDCl,) 6 1 44 (s, 9 H), 2 16-2.25 (app qumtet, J = 12 Hz, 1 H), 2 69-2.79 (m, 1 H), 4.18-4 28 (ddd, J = 5, 9, 11 HZ, 1 H), 4 294 38 (m, 1 H), 4 40-4 46 (app t, J = 9 Hz, 1 H), 5 03-5 10 (m, 1 H), 13C NMR (75 MHz, CDCl,). 6 28 1, 30 1, 50 0, 65.6, 80 3, 155.4, 175 4, IR (film) 1776, 1684 cm-‘.

3 3.2 (S)-Diphenylmethyl N-(tert-Butoxycarbonyl)2-[2’-(Phenylseleno)Ethyl]Glycinate 8 1. To an argon-purged flask contammg sodmm borohydrlde (3 10 mg, 8 19 mmol, see Note 9) IS added a solution of diphenyl dlselemde (1 58 g, 7 45 mmol) m DMF (60 mL), via cannula. 2 TO this solution 1sadded, via cannula, a solution of lactone 7 (1.50 g, 7 45 mmol) m DMF (60 mL)

478

Berkowitz et al.

3 The reaction 1s heated at 100°C for 1 h 4 After coohng to O”C, methanol (15 mL) ‘s added, and the crude mixture IS starred 5 h. 5 The solvent 1s removed zn vacua, and the residue is partitioned between Et,0 (200 mL) and 100 mA4NaOAc buffer (pH 5 0) The aqueouslayer 1sextracted tw’ce more with Et,0 (200 mL) 6 The combined organ’cs were dried (MgS04), filtered, and ester’f’ed with d’phenyl-dlazomethane (1 88 g, 9 69 mmol, prepared according to ref. 31) m EtOAc 7 Evaporat’on of the solvent and chromatography (0- 15% EtOAc/hexane) yields 8 (3.24 g, 83%) asa white solid mp 108-l lO”C, [a]D24=-12 3” (c = 2.9, CHCl,), ‘H NMR (300 MHz, CDCl,) 8 142 (s, 9 H), 1 962 06 (m, 1 H), 2 17-2 25 (m, 1 H), 2.76-2 85 (m, 2 H), 4 534 56 (app dd, J = 8,12 Hz, 1 H), 5 05-5 08 (d, J = 8 Hz, 1 H), 6 86 (s, 1 H), 7 20-7 45 (m, 15 H); 13C NMR (75 MHz, CDCl,): 6 22 93, 28 24, 33.31, 53 61, 78.05, 80 01, 126.95, 127 07 (2C), 128.0, 128 1, 128 5, 128 6 (2C), 129 1, 133 0, 1394, 139 5, 155 2, 171 2, IR (film) 1743, 1712 cm-’

3.3.3. Diphenylmethyl N-(tert-Butoxycarbonylj-L-a-Vmylglycinate 3.3 4 L-a-Vmylglycine, Trlfluoroacetate Salt 10

9

1 Ozone 1sbubbled mto a solution of selemde8 (474 mg, 0 913 mmol) m CH,Cl, (10 mL) at -78°C until a hght blue color persists 2 After addition of 1-hexene(2 mL, 16mmol, seeNote 4), this cold solution 1sadded dropwise to refluxmg PhH (36 mL, seeNote 5), andrefluxmg 1scontinued for 1 h 3 Evaporation of the volat’les gives crude 9 as an 0’1 4 If desired, an analytical sample of 9 may be obtained by recrystall’zat’on (hexanes) mp 89-91”C, [cL]o24= -10 1” (c = 3 5, CHCl,); ‘H NMR (300 MHz, CDC13) 6 1 45 (s, 9 H), 4.97-5 04 (m, 1 H), 5 19-5 23 (d, J= 7 Hz, 1 H), 5 26529(d,J=10Hz,1H),533-539(d,J=18Hz,1H),589~00(m,1H),690 (s, 1 H), 7 25-7.42 (m, 10 H), 13CNMR (75 MHz, CDC13) 6 28 19,55 88,78 09, 79 98, 117.7, 126 9, 127.1, 127 97, 128.04, 128 2, 128 5, 132 4, 139 39, 139 45, 154 9, 169 71, IR (film) 1743, 1711 cm-’ 5 It was found to be most convenient, however, to deprotect crude 9 without punfkat’on. To a solution of crude 3 m CH2C12(18 mL) at 0°C are addedCF,COOH (10 mL) and AcOH (57 a, 1 01 mmol) 6 The reaction mixture 1sallowed to warm to room temperature After 8 h at room temperature, H,O (30 mL) IS added, followed by extraction with CH2C12(3 x 20 mL) and Et,0 (3 x 20 mL) 7 Evaporation of the aqueouslayer, followed by thorough drymg (50°C, 0 5 torr, P,O, sidearm) yields 10 (176 mg, 90% for two steps, see Note 10). [c~]o*~ = +42.8” (c = 1 1, H,O); ‘H NMR (300 MHz, D20). 6 4 52-5 55 (d, J= 7 Hz, 1 H), 5 48-5 50 (d, J= 5 Hz, 1 H), 5.53-5 54 (d, J= 2 Hz, 1 H), 5 874 00 (ddd, J= 7, 10, 17 Hz, 1 H); 13CNMR (75 MHz, D20) 6 57 69,119 0 (q, J= 291 Hz), 125.7, 130 6, 165.5 (q, J= 35 Hz), 173 1

a- Vinyl Amino Acids

479

3.4. Preparation of (I ‘R,2S,STi)-8’-(p-Naphthyl) Men thy/ N-Benzoyl-(Z)-Dehydrobutyrina te 3.4.1 (k)-N-Benzoyl Homosenne Lactone 12 1 To a solution of (f)-a-ammo-y-butyrolactone hydrobromide (11, 10 g, 54 9 mmol) in CH,Cl, (222 mL) are added NEt, (30.6 mL, 2 19 mmol), and DMAP (67 1 mg, 5 49 mmol) and this solution IS cooled to 0°C 2. After dropwise addition of benzoyl chloride (6.37 mL, 54 9 mmol), allow the solution to come to room temperature and stir for 7 h. 3 The mixture is then extracted with NaHCO, (sat’d aq ,3 x 200 mL), and &SO, (aqueous, 3 x 200 mL) The orgamc layer is dried (MgSO,), filtered, and evaporated to yield 12 (10.5 g, 93%): ‘H NMR (200 MHz, CDCl,) 6 2 21-2 45 (m, 1 H), 2.82-3 00 (m, 1 H), 4 24-4.43 (m, lH), 4 48-4.59 (m, 1 H), 4 74 8 (m, 1 H), 7 15 (s, 1 H), 7 24 (t, J= 1.3 Hz, 2 H), 7.52 (t, J= 1 3 Hz, lH), 7 81 (app d, J= 1,3Hz,2H)

3.4.2. (?)-Diphenylmethyl N-Benzoyl2-[2’-(Pheny/se/eno)Ethy/]G/ycinate 13 1 To an Ar-purged flask contammg NaBH4 (9 10 mg, 24.1 mmol) is added, dropwise and via cannula, a deoxygenated solution (see Note 1) of PhzSez (2 75 g, 13.0 mmol) m DMF (75 mL) 2 To this solution is added, via cannula, a deoxygenated solution of 12 (3 80 g, 18.5 mmol) m DMF (75 mL), and the mixture is heated at 100°C for 1 h 3 After coolmg to 0°C MeOH (35 mL) is added, and the crude mixture is stirred for 3 h 4 The solvent is removed zn VCICUO,and the residue is partitioned between Et,0 (100 mL) and 1 N HCl(lO0 mL) The aqueous layer is extracted twice more with EtOAc (250 mL), and the combmed orgamcs are drted (MgSO,) and filtered 5 The crude product is triturated with hexane, and esterified with diphenyldiazomethane (3 90 g, 19 9 mmol, prepared according to ref. 31) m EtOAc 6 Evaporation of the solvent and recrystallization from hexane/EtOAc gives 13 (8 58 g, 88%) as a white solid mp 105-106°C; ‘H NMR (300 MHz, CDCl,) 6 2.18 (m, 1 H), 2 38 (m, 1 H), 2.84 (m, 2 H), 5.08 (app dt, J= 5,7 5 Hz, 1 H), 6 78 (d, J= 7 5 Hz, 1 H), 6 89 (s, 1 H), 7.19 (m, 3 H), 7 33 (m, 10 H), 7.42 (m, 4 H), 7 51 (m, 1 H), 7.76 (app d, J = 8 Hz, 2 H), i3C NMR (75 MHz, CDC13) S 22 9, 33 1, 52.8, 78 5, 126 9, 127 0, 127 1, 127 2, 128.1, 128.2, 128 5, 128 6, 129 1, 131 8, 132.9, 133.6, 167 1, 171 1; IR (film) 1734, 1652, 1639 cm-’

3.4.3. (5) -Dipheny/me thy/ N -Benzoy/-2- Vinylglycina te 74 1 Ozone IS bubbled mto a solution of selemde 13 (9 00 g, 17 0 mmol) m CH2C12 (150 mL) at -78°C until a light blue color persists 2. After addition of I-hexene (45 mL; see Note 4), this cold solution is added to refluxing PhH (600 mL; see Note 5), and refluxing is continued for 1 h 3 Evaporation of the solvent and recrystallization from hexane/BtOAc gives 14 (5 67 g, 90%) as a white sohd: mp 103-104°C; ‘H NMR (300 MHz, CDCls) S 5 32 (dd, J

480

Berkowitz et al. = 1 510 Hz, 1 H), 5 40 (dd,J= 1.5,17 Hz, 1 H), 5 49 (m, 1 H), 6 03 (ddd,J= 5 5,10, 17 Hz, 1 H), 6.85 (d,J= 7 5 Hz, 1 H), 6.93 (s, 1 H), 7 32 (m, 10 H), 7.43 (t, 7.5 Hz, 2 H), 7 51 (t,J=7.5 Hz, 1 HI),7 81 (appd,J=7.5 Hz,2H), 13CNMR(75 MHz,CDCl,)F 55 0, 78 7, 118.3, 126 9, 127 0, 127 1, 128 1, 128.2, 128 5, 128 6, 131.8, 131 9, 166.7, 169 8

3.4 4. (2)-N -Benzoy/ Vinylglycine 15 1 To a solution of 14 (5 50 g, 14 8 mmol) m CH*Cl;! (55 mL) are added amsole (1 93 mL, 17 8 mmol) and TFA (6 mL) This mixture IS allowed to stir at room temperature for 3 h 2 Workup proceeds by partmonmg between NaHCO, (aqueous, 100 mL) and CH2C12 (50 mL). The aqueous layer 1s washed with Et,0 (4 x 100 mL) and acrdttied to pH 2 0 with 3 N HCl. 3 Followmg extraction with Et,0 (6 x 75 mL), the organic layer IS drted (MgSO,), filtered, and evaporated to give 15 (2.92 g, 96%) as a white sohd mp 16%170°C ‘H NMR(500MHz,CDCl3)G530(d,J=10Hz,1H),532(m,1H),541(d,J=16Hz, 1 H), 6.01 (ddd,J= 5, 10, 16 Hz, 1 H), 7 07 (d, J= 7 Hz, 1 H), 7 42 (t, J= 7 5 Hz, 2 H),751(t,J=75Hz,lH),781(appd,J=75Hz,2H),9,94(br s,lH),13CNMR (125 MHz, CDCl,)G 55 0, 118 3, 127 2, 128.7, 131 6, 132 1, 133 2, 167 7, 173 6

3 4 5. (1 R,2’S,SR)-8’-(p-Naphthyl) Menthyl N -Benzoy/-(Z)-Dehydrobutyrmate

76

To a solution of 15 (2 67 g, 13 0 mmol) m benzene (119 mL) are added (lR,2S,SR)-8-(P-naphthyl)menthol (3 20 g, 11 3 mmol; prepared accordmg to ref. 25) and p-TsOH (862 mg, 4 53 mmol) This solution is refluxed using a Dean-Stark trap for 3 d After cooling, NaHCO, (aqueous, 100 mL) 1s added, and the solutton 1s extracted wtth Et,0 (3 x 50 mL), dried (MgS04) and evaporated, Flash chromatography (10% EtOAc/hexane) affords 16 (3.40 g, 64%): [cx]~~~ = -34.9” (c 1 09, CHCl,), ‘H NMR (300 MHz, CDC13) 6 0 87 (d, J= 6 Hz, 3 H), 0 88-l 08 (m, 2 H), 1 12 (d, J= 7 Hz, 3 H), 1 18-1.32 (m, 1 H), 1 25 (s, 3 H), 1 37-1.53 (m, 1 H), 1 42 (s, 3 H), 165-l 83 (m, 2 H), 194-l 99 (m, 1 H), 2 1% 2 27 (dt, J= 3, 12 Hz, 1 H), 4 98 (dt, J= 4, 11 Hz, 1 H), 5.90 (q, J= 7 Hz, 1 H), 6 34 (s, 1 H), 7 35-7 47 (m, 4 H), 7 49-7 54 (m, 2 H), 7 58-7 60 (m, 3 H), 7 70-7 76 (m, 3 H); 13C NMR (75 MHz, CDCl,) 6 14 1,21 7, 22 2, 26 3, 30 0, 31 3, 34 5, 39 5, 41 6, 50 1, 749, 122.7, 125 1, 125 3, 125.5, 127 2, 127 3, 127 8, 128 4, 131 2, 13 1 6,133 4,134 1,134 2,149.7,163 4,165 0 The (Z) alkene geometry IS established by exannnmg the relevant long-range C-H couphng constant usmg (proton-coupled) 13Cm J cc-u = 3 + 1 Hz between the vmyhc proton and the ester carbonyl carbon

3.5. Preparation of r-a-Vinylornithine 3.5.1. (1 R,2S,SR)-8’-(p-Naphthyl) Menthyl N-BenzoyA-a- Vinylornithinate

18a

1 To a solution of dnsopropylamine (6 1 & 0.47 mmol) and HMPA (1.2 mL; see Note 11) m THF (6 6 mL) at -78°C 1s added n-butylhthmm (Aldrich, 1 3 N by

a- Vinyl Amino Aads

2.

3 4 5 6 7 8

481

titration /28/; 0.36 mL, 0 47 mmol). The resultmg solutton 1sstirred for 20 mm at 0°C and then cooled to -78°C Then, 16 (200 mg, 0 43 mmol) m THF (6 6 mL) at -78°C is added vta cannula, followed by n-butyllithium (1 3 N; 0.73 mL, 0.94 mmol), and the resulting deep red solution IS stirred for 5 mm at -78°C. To this solutton is added, dropwtse, vta cannula, I-benzaldtmino-3-todopropane (255 mg, 0 94 mmol; prepared according to ref. 17) m THF (0.4 mL) After being stirred at -78’C for 45 mm, the reaction mtxture is poured mto ether (30 mL) and NaHCO, (aqueous, 30 mL) After further extraction with ether (3 x 15 mL), the combined orgamcs are dried (MgSO& filtered, and evaporated To the crude mixture are added THF (4 mL) and 3 N HCl(4 mL), and the resulting solution 1s allowed to stir at room temperature for 15 h 1 N Na,CO, (25 mL) is then added to the reactton mtxture, and the solutton 1s extracted with Et,0 (3 x 25 mL), dried (MgSO,), and evaporated. Chromatography 1scart-ted out initially with 20% EtOAc/hexane to remove benzaldehyde, and then with 5% MeOWCH,Cl, [ 1% NH,OH(sat’d aq)] to elute 18a (164 mg, 74%, 94% de) ‘H NMR (500 MHz, CDCl,) 6 0 8&O 90 (m, 2 H), 0 88 (d, J= 6 4 Hz, 3 H), 1 22-1 40 (m, 2 H), 1.32 (s, 3 H), 142 (s, 3 H), 1 32-l 55 (m, 2 H), 1 77-l 82 (m, 1 H), 2 05-2.08 (m, 1 H), 2 14-2.28 (m, 6 H), 2 592 66 (m, 2 H), 4.92-4 97 (dt, J= 4, 10 4 Hz, 1 H), 5 09 (d, J= 10.8 Hz, 1 H), 5.17 (d, J= 17 3 Hz, 1 H), 5 73-5 79 (dd, J= 10 8,17.3 Hz, 1 H), 7.39-7 45 (m, 5 H), 7 47-7.51 (m, 2 H), 7 56 (s, 1 H), 7 64 (s, 1 H), 7 70 (d, J= 8 8 Hz, 1 H), 7 77 (d, J= 6 Hz, 1 H), 7 79 (app d, J= 8 Hz, 2 H); 13CNMR (125 MHz, CDCl,) 6 21 7,25.6,26 8,27 3,28.4, 31.3, 31 9,344,20.2,41 3,41 3,495,644,77.8,1153,123.4,1248,1252,1258,127.0, 127 2, 127.5, 127 9, 128 4, 131 4, 131.5, 133.3, 134.7, 135 8, 148 3, 165 9, 171 8

3.5.2. N -Benzoyl-L-a- Vinylornithine 19a 1. To a solutton of 18a (65.6 mg, 0 12 mmol) m (1.6 mL) of 1,4-droxane (see Note 12) are added KOtBu (120 mg, 1 07 mmol) and H,O (22 mL, 1.2 mmol), and the solution IS heated for 8 h at 105 “C 2. After cooling, 10% HCl(15 mL) IS added and the solution 1sextracted with Et,0 (3 x 15 mL). The organic layer IS dried (MgSO,) and evaporated, to yield 3 1.6 mg (0 11 mmol, 90%) of recovered (lR,2S,SR)-8-(P-naphthyl)menthol 3 The aqueous layer 1s evaporated wtth mild heating (t 5 5O”C), and the residue obtained 1s applied to a Dowex 50 x 8 ton-exchange column After washing the column with several column volumes of H,O, elutron with 1 3 MNH,OH affords 19a (22 6 mg, 86 mmol, 69%). [a]24n =(94% ee) +l 1 6” (c 1 5 D,O), ‘H NMR (500 MHz, CDCl,) 6 1 56-1.61 (m, 1 H), 1.66-l 71 (m, 1 H), 2 16-2 21 (dt, J= 4 4, 12 7 Hz, 1 H), 2.42-2 49 (dt, J= 4 4, 12 7 Hz, 1 H), 3 03 (app t, J= 7 6 Hz, 2 H),5.25(dJ=104Hz,1H),526(d,J=17.3Hz,lH),612-6l8(dd,J=l04, 17 3 Hz, 1 H), 7.57 (t, J= 7 7 Hz, 2 H), 7 66 (t, J= 7 7 Hz, 1 H), 7 84 (app d, J= 7.7 Hz, 2 H), 13C NMR (125 MHz, CDCl,)

129.7, 133.1, 134.7, 138.9, 169.7, 177.8.

6 23.0, 31 8, 41.1, 67 0, 114 9, 127 6,

482 3.5.3.

Berkowitz et al L-CC

Vinylornithine 20a

1. A suspension of 19a (30 1 mg, 0 11 mmol) m 6 N HCl(0 9 mL) is refluxed for 4 h 2 After extraction with CH,Cl, (3 x 10 mL), the aqueous layer is evaporated zn vacua, with mild heating (T 550°C) 3 The residue is applied to a Dowex 50 x 8 ion-exchange column After washing with several column volumes of H,O, elution follows with 1 3 MNH,OH Evaporation and drying are carried out as for 5a to give 20a (17 7 mg, 97%) [IX]~~~ = (94% ee) +18 8” (c 0.8, H,O), ‘H NMR (500 MHz, D,O) 6 1 56-1.62 (m, 1 H), 1 66-l 74 (m, 2H), 1 88-l 95 (m, lH), 2.97 (app t, J = 7 Hz, 2 H), 5 22 (d, J= 108Hz,1H),525(d,J=177Hz,1H),603-608(dd,J=108,177Hz,1H), 13C NMR (125 MHz, D20) 6 22 9,35.4,40 2,64 4, 115 0, 140 4, 179 6

3.6. Preparation of L-a-Vinylleucine 3 6.1. (1 R,2’S,SR)-8’-(/3-Naphthyl) Menthyl N-Benzoyl-L-a- Vinylleucmate 18b The same general procedure as for Subheading 3.5.1. above is followed. 2 From 16 (250 mg, 0 53 mmol) and isobutyl Iodide (120 pL, 1.06 mmol), is obtained 18b (228 mg, 0 43 mmol, 81%, 298% de) after flash chromatography ( 10% EtOAc/hexane) [~]~~o = +8 lo (c 1 1, CHC13), ‘H NMR (300 MHz, CDCl,) 6 0 83 (d, J= 6 5 Hz, 3 H), 0 85 (d, J= 6.5 Hz, 3 H), 0 89 (d, J= 6 Hz, 3 H), 0 97112(m,3H),131(s,3H),142(s,3H),14~162(m,4H),167-174(ddJ= 6 4 Hz, 1 H), 2 1 l-2.22 (m, 2 H), 2.25-2.32 (dd, J= 6 4, 14 Hz, 1 H), 4.88-4 96 (dt,J=4,11Hz,1H),513(d,J=105Hz,1H),518(d,J=17Hz,1H),5655 75 (dd,J= 10.5, 17 Hz, 1 H), 7.15 (s, 1 H), 7 39-7 57 (m, 6 H), 7.64 (br s, 1 H), 7 70-7 23 (m, 1 H), 7 34-7 80 (m, 4 H), i3C NMR (75 MHz, CDCl,) 6 21 7,23 4, 23 7, 24 8, 25.2, 27 4, 28 8, 31 4, 34 4, 40 2, 41.3, 42 2, 49 6, 64 5, 78 4, 115 1, 123 4,124 8,125 3,125 9,126 9,127 3,127 6, 127 9,128 6,131.4,131 5,133 3, 134.9, 136 9, 148.2, 165 7, 172 5, IR (film) 1733, 1675, 1653 cm-i

3 6.2. N-Benzoyl-L-a-Vinylleucine

19b

1 To a solution of 18b (200 mg, 0.38 mmol) m of 1,4-dioxane (7 6 mL, see Note 12) are added KOtBu (367 mg, 3 27 mmol) and H,O (15 pL, 0 86 mmol), and the resulting solution is heated at 105°C for 8 h. 2. After coolmg, 10% HCI (20 mL) is added, and the solution IS extracted with Et,0 (3 x 10 mL) 3. The organic layer 1sextracted with NaHCO, (satd aqueous, 10 mL). The aqueous layer IS further extracted with Et,0 (2 x 10 mL) The combined orgamcs are dried (MgSO,), filtered, and evaporated to yield (98 6 mg, 92%) of recovered (lR,2S,SR)-8-(j3-naphthyl)menthol 4 The H,O layer is acidified to pH 2 0 with 6 N HCl and extracted with Et20 (3 x 10 mL) This organic layer is dried (MgSO,), filtered, and evaporated to yield 19b (73 5 mg, 74%) [cx]~~,, = -9 5” (c = 0 55 CHCl,), ‘H NMR (500 MHz, CDC1,)G090(d,J=7Hz,3H),093(d,J=7Hz,3H),171(m,1H),20~207

a- Vinyl Amino Acids

483

(dd,J=7.5, 14Hz, 1 H),2.54-261 (dd,J=5.5, 14Hz, 1 H),5,27(d,J= 105Hz, 1 H), 5.29 (d, J= 17 Hz, 1 H), 6.06-6.16 (dd, J= 10 5, 17 Hz, 1 H), 7 60 (s, 1 H), 7 42-7.47 (m, 2 H), 7 5&7 53 (m, 1 H), 7 80 (app d, J= 8 Hz, 2 H); 13CNMR (125 MHz, CDCl,) 6 22 8, 23 8, 24 7, 43.2, 64.5, 115 6, 127.0, 128 7, 131 9, 134 2, 136 9, 166.6, 176 6

3.6.3. L-a- Vinylleucme 206 1 The same general procedure as for Subheading 3.5.3. is followed, 2 From 19b (70 0 mg, 0.27 mmol) IS obtained 20b (40 4 mg, 96%): [u]~~,, = +26.3” (c = 1 1, H,O); ‘H NMR (500 MHz, D,O) 6 0 98 (d, J= 6 Hz, 3 H), 1 02 (d, J = 6 Hz, 3 H), 1 78 (m, 1 H), 1 83-l 87 (dd, J= 4 8,14.9 Hz, 1 H), 2 01-2 05 (dd, J=7,14Hz,1H),5.36(d,J=177Hz,1H),542(d,J=108Hz,1H),6126 17 (dd, J= 10 8, 17 7 Hz, 1 H); 13C NMR (125 MHz, D20) 6 22.8, 24.3, 24 3, 449,658, 1162, 1373, 1756

4. Notes In this step, all solutions were deoxygenated by freezing (hquid N2), and SubJectmg to SIX cycles of evacuation and purging with Ar Yields were found to be generally higher when the dianion was prepared and alkylated m the presence of TMEDA. Careful! Diazomethane is toxic and explosive Care should be taken to use only polished glassware and rubber stoppers m assembling the distillation apparatus Ground-glass lomts or other rough surfaces or Jagged glass edges should be avoided This reaction should be carried out m a fume hood, behind a safety shield Diazomethane-mediated esterifications performed by the general procedure described m Subheading 3.1.2. have never led to a dangerous incident m our laboratory. 1-Hexene is added as a sacrificial olefin to consume oxidants formed as byproducts. In the absence of 1-hexene, yields of protected vinyl ammo acid are generally lower and the product is contammated with byproducts arising from a Pummerer-like rearrangement Carbon tetrachloride also works quite well m the pyrolysis step However, owing to its high cost, benzene is generally substituted The N-trityl-protectmg group on the imidazole nucleus is very acid-labile To prevent loss of this protectmg group, the workup m this particular case should be carried out under carefully controlled conditions of pH (i.e , pH 5.0 buffer IS used) If one attempts to form this selenoxide with ozone, one sees oxidation of the imidazole side chain of the histidme This problem is solved by using a modified oxidation/pyrolysis procedure wherein the organic-soluble and chemoselective oxidant, Bu4NI04, is employed One may also begin with L-homoserme lactone, which IS commercially available as its hydrochloride salt However, L-homoserme is a more economical starting material.

484

Berkowitz et al.

9 Although the phenylselenolate equtvalent derived from NaHB(OMe),/PhSeSePh was found to be of advantage for the opening of a-substituted homoserme lactones (24), it led to partial racemization here This racemtzatton could be prevented by using NaBH,/PhSeSePh, whereby the product 8 was found to be 296% ee Opttcal purity was Judged by the ‘H NMR spectra of both (a) a Mosher ester (33) [derived from 8 by the sequence. [i] LtBH,, THF, &6O”C, [ii] [q-(+)-amethoxy-a-[trifluoromethyl]phenylacetyl chloride, DMAP, NEt,, CH,Cl,) and (b) a Mosher amide [obtained by replacing Ph,CN, with H&N, m step 2 and then treatment with* [i] TFA, CH,Cl,, [ii] [fl-[+]-a-methoxy-a-[trifluoromethyllphenylacetyl chloride, DMAP, NEt,, CH,Cl,) In each case, the ‘H NMR spectrum thereby obtamed was compared to that of the Mosher ester or amide derived from (f)-o-methoxy-a-(trifluoromethyl)phenylacetyl chloride 10. The final product IS Judged to be 295% ee, based on integration of a ‘H NMR spectrum of its Mosher amtde, methyl ester To obtain this Mosher amide, the final product was subJected to the following sequence (a) HCl, MeOH, A, (b) (S)-(+)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride, DMAP, NEt,, CH,CI, (33). The ‘H NMR spectrum (CDCl,, 500 MHz) of this Mosher amide was compared wtth that of the diastereomeric Mosher amides obtained by the same sequence, starting from (+)-vmylglycme The vmyhc methme protons, the methyl ether singlets and the methyl ester singlets are resolved m the spectrum of the two dtastereomers. 11 It was found that alkylatton yields increase perceptibly m the presence of HMPA here On the other hand, diastereoselectlvlty is not decreased One possible explanation that IS consistent with these results is that HMPA breaks up enolate aggregates, leading to the more reactive monomer. However, the unchanged diastereoselectivrty suggests that HMPA does not break up the internal chelation that is proposed to control enolate geometry For a related effect of HMPA with chiral alanme-derived diamons used to synthesize enantlomerically enriched a-methyl ammo acids, see ref. 25 12 Use of the actual Gassman “anhydrous hydroxide” condmons (26) gave no reaction Substitution of refluxing dloxane for refluxing Et,0 solves this problem, presumably simply owing to the increased temperature

References 1 Dardenne, G , Casimtr, J , Marher, M., Larsen, P 0 (1974) Aclde (2R)-ammo-3butenolque (binylglycme) dans les carpophores de Rhodophyllus nzdorosus Phytochemzstry 13, 1897-l 900 2. For syntheses of (+)-a-vmylglycme and derivatives see (a) a-(2-bromo)vmylglycme. Petasts, N A and Zavlalov, I. A. (1997) A new and practical synthesis of a-ammo acids from alkenyl boromc acids J Am Chem Sot 119, 445-446, (b) Fltzner, J. F , Pratt, D V , and Hopkins, P B (1985) Synthesis of protected racemrc P,y-unsaturated-cc-ammo acids via y-phenylseleno-a,P-unsaturated esters Tetrahedron Lett 26, 1959-1962, (c) Vyas, D M , Chiang, Y , and Doyle, T. W. (1984) A practical synthesis of vmylglycme from (Z)-2-

a- Viny/ Amino Acids

3

4

5

6 7.

485

butene- 1,4-dial. J Org Chem 49,2037, (d) Greenlee, W J (1984) Synthesis of P,y-unsaturated ammo acids by the Strecker reactron. J Org Chem. 49, 26322634; (e) Hudrhk, P F and Kulkarni, A. S (1981) J Am Chem Sot a-Silyl aldehydes preparation and use as stereoselective vinyl cation equivalents. 103, 62516253, (f) Baldwin, J E., Haber, S. B., Hoskms,C , and Kruse, L I (1977) Synthesis of P,y-unsaturated amino acids. J. Org Chem 42, 1239-1241, (g) Frns, P , Helboe, P , and Larsen, P 0 (1974) Synthesis and resolution of vmylglycme, a P,y-unsaturated ammo acid. Acta Chem Stand , Ser B 28, 3 17-32 1 For enantioselective syntheses of a-vinylglycine or derivatives, see. (a) Larksarp, C. and Alper, H. (1997) Palladium(O)-catalyzed asymmetric cycloaddttton of vmyloxiranes with heterocumulenes usmg choral phosphme ltgands. an effective route to vmyloxazolrdme derivatives J Am Chem Sot 119,3709-37 15, (b) Berkowitz, D. B. and Smith, M. K. (1996) A convenient synthesis of Lvmylglycine from L-homoserine lactone S’ntheszs 39-41; (c) Trost, B. M. and Bunt, R. C. (1996) On hgand design for catalytic outer sphere reactions. a simple asymmetric synthesis of vinylglycinol. Angew Chem Znt Ed Engl 35,9!%-102; (d) Gnesback, A. G. and Hut, J (1995) A photochemical route to vinylglycme and a vmylglycme drpeptide Lz’ebzgsAnn 1957-1961, (e) Carrasco, M , Jones, R J , Kamel, S , Rapoport, H., and Truong, T. (1991) N-(benzyloxycarbonyl)~L-vmylglycme methyl ester Org S’n 70,29-34, (f) Krol, W. J., Mao, S.-S., Steele, D. L., and Townsend, C A (199 1) Stereochemical correlation of proclavamic acid and syntheses of erythro and three-L-P-hydroxyormthme from an improved vmylglycme synthon. J Org Chem 56, 728-73 1, (g) Pellicciari, R., Natalmi, B , and Marmozzr, M (1988) LVmylglycme from L-homosenne S’nthetrc Commun. 18, 1715-1721, (h) Monwake, T , Hamano, S -I., Saito, S., and Torn, S. (1987) A straightforward synthesis of ally1 ammes from a-amino acids without racemization Chem. Lett 2085-2088; (i) Barton, D. H. R., Cnch, D , Herve, Y , Potier, P., and Threry, J (1985) The free radical chemistry of carboxylic esters of 2-selenopyridme-N-oxide: a convenient syntheses of Lvinylglycme Tetrahedron 41,43474357, (j) Hanessian, S and Sahoo, S P. (1984) A novel and efficient synthesis of L-vinylglycme. Tetrahedron Lett 25, 1425-1428, (k) Schdllkopf, U., Nozulak, J., and Groth, U (1984) Asymmetric syntheses via heterocychc intermediates-XXII Enantioselecttve synthesis of a-alkenylglycme methyl esters and a-alkenylglycmes Tetrahedron 40, 1409-1417 Cho, C , Ishri, R , Hyeon, S., and Suzuki, A (1987) Inhibition of serme glyoxylate ammotransferase and mttochondrial glycme oxidation m the photorespiratory glycolate pathway by vmylglycme. Agrlc Biol Chem 51, 2597-2598. Cornell, N W , Zuurendonk, P F , Kerich, M. J , and Straight, C. B (1984) Selective mhbmon of alanme ammotransferase and aspartate ammotransferase in rat hepatocytes Bzochem J 220,707-716 Griffith, 0 W (1983) Cystemesulfmate metabolism J Bzol Chem 258, 1591-1598 Soper, T S , Manning, J. M., Marcotte, P. A , and Walsh, C. T. (1977) Inactivanon of bacterial n-ammo acid transammases by the olefinic ammo acid Dvinylglycme. J, Blol. Chem 252, 1571-1575.

486

Berkowdz et al

8 Rando, R R , Relyea, N , and Cheng, L (1976) Mechanism of the irreversible mhibition of aspartate ammotransferase by the bactertal toxm L-2-ammo-4methoxy-trans-3-butenotc acid J Blol Chem 251, 3306-33 12 9 Rando, R. R. (1974) Irreversible mhtbttion of aspartate ammotransferase by 2ammo-3-butenoic acid Bzochemzstry 13,3859-3863 10 For synthetic approaches to higher a-vinyl amino acids, m racemic form, see (a) Pedersen, M L and Berkowitz, D. B (1993) Formal a-vmylation of ammo acids use of a new benzeneselenolate equivalent J Org Chem 58, 69666975, (b) Castelhano, A L , Horne, S., Taylor, G. J., Billedeau, R , and Krantz, A (1988) Synthesis of a-ammo acids with P,y-unsaturated side chains Tetruhedron 44, 545 l-5466, (b) Munster, P. and Stegltch, W (1987) Synthesis of aamino acids by reaction of t-butyl N-(t-butoxycarbonyl)tmmoacetate with C-nucleophiles Syntheses 223-225; (c) Castelhano, A L , Home, S , Btlledeau, R , and Krantz, A. (1986) Reactions of an electrophihc glycme cation equivalent with Grignard reagents. A simple synthesis of /3,y-unsaturated ammo acids. Tetrahedron Lett 27, 2435-2438; (d) Sawada, S , Nakayama, T , Esaki, N , Tanaka, H., Soda, K., and Hill, R K (1986) Synthesis of labeled (k)-2-ammo-3-butenoic acids J Org Chem. 51, 3384-3386; (e) Steghch, W and Wegmann, H (1980) Uberfuhrung von a-ammosauren m a-vmylammosauren Syntheses 48 l-483, (f) Metcalf, B. W. and Bomlavri, E (1978) Phenyl truns-2-chlorovmyl sulphone, a vinyl cation equivalent. J Chem Sot. Chem Commun 914-915; (g) Greenlee, W J , Taub, D , and Patchett, A A. (1978) A general synthesis of a-vinyl-aamino acids. Tetrahedron Lett 3999-4002, (h) Metcalf, B W and Jund, K. (1977) Synthesis of P,y-unsaturated ammo acids as potential catalytic irreversible enzyme inhibitors Tetrahedron Lett 3689-3692, (1) Taub, D. and Patchett, A A. Syntheses of a-ethynyl-3,4-dthydroxyphenylalanme and a-vinyl-3,4dihydroxyphenylalanme. (1977) Tetrahendron Lett 2745-2748. Il. For syntheses of higher a-vinyl ammo acids (a-vmylalanme, a-vmylbutyrme and a-vmylphenylalamne) m opttcally enriched form, see (a) Colson, P -J and Hegedus, L S (1993) Asymmetric synthesis of a-alkyl-a-ammo acids from a chromium-carbene-complex-derived p-lactams J Org Chem 58,59 18-5924, (b) Seebach, D., Burger, H M., and Schickli, C P. (1991) Stereoselektive umsetzungen von rat-, oder (R)- oder (S)-5-alkyltden-2-t-butyl-3-methyl-4-oxo1-tmidazolidmcarbonsaure-t-butylestern (chorale 2,3-dehydroammosaure derivate) und herstellung eimger mchtprotemogener aminosauren Lzeblgs Ann Chzm 669684, (c) Weber, T , Aeschimann, R , Maetzke, T , and Seebach, D (1986) Methionm als vorlaufer zur enantioselectiven synthese a-verzweigter vmylglycme und anderer ammosauren. Helv Chum Acta 69, 1365-1377. 12 For formal enzymatic resolution of a-vinyl ammo acids, see Berkowitz, D B , Pumphrey, J A , and Shen, Q (1994) Enanttomerically enriched a-vinyl ammo acids vta ltpase-mediated “reverse transesterification.” Tetrahedron Lett 35, 8743-8746 13 Maycock, A. L , Aster, S. D., and Patchett, A. A. (1979) Suicide macttvation of decarboxylases. Dev Blochem. 6, 115-129

a- Vinyl Amino Acids

487

14 Rtbereau-Gayon, G , Danzm, C., Palfreyman, M G., Aubry, M , Wagner, J., Metcalf, B W., et al (1979) In v&o and zn vzvo effects of a-acetylemc DOPA and a-wnyl DOPA on aromattc L-ammo acid decarboxylase Blochem Pharm 28, 1331-1335. 15. Maycock, A. L., Aster, S. D., and Patchett, A. A (1978) Studies with mhtbttors of aromatic ammo acid decarboxylase, m Enzyme-Actwated Irreversible Inhzbztars (Seder, N., Jung, M J., and Koch-Weser, J., eds ), Elsevler, North Holland, 2 1 l-220 16. Metcalf, B and Jung, M (1979) a-Vinyl dertvattves of a-ammo acids US Patent 4,147,873, Aprtl 3. 17. Danzm, C., Casara, P , Claverie, N , and Metcalf, B W (198 1) a-Ethynyl and a-Vinyl Analogues of Ormthine as Enzyme-Activated Inhibitors of Mammalian Ormthine Decarboxylase. J A4ed Chem. 24, 16-20. 18 Berkowitz, D. B., Jahng, W -J., and Pedersen, M. L. (1996) a-Vmyllysme and avmylargmme are time-dependent mhtbttors of their cognate decarboxylases Bzoorg Med Chem Lett 6,215 1-2156 19. Damhaut, P , Lemarre, C , Plenevaux, A., Brihaye, C , Chrtstiaens, L , and Comar, D (1997) No-carrier-added asymmetric synthesis of a-methyl-a-ammo acids labelled with fluorine- 18 Tetrahedron 53,5785-5796 20 Yokum, T S , Gauthier, T. J , Hammer, R P , and McLaughlin, M L (1997) Solvent effects on the 3 &a-helix equthbrmm m short amphtpathtc peptrdes rich m a,a-dtsubstttuted ammo acids. J Am Chem Sot 119, 1167-1168 2 1 Altmann, E , Nebel, K , and Mutter, M, (199 1) Conformattonal studtes on peptides containing enanttomeric a-methyl a-ammo acids Helv Chum Acta 74, 800-806 22 Khosla, A , Stachowiak, K , Smeby, R. R , Bumpus, F M , Ptrtou, F , Lmtner, K ,

et al (198 1) Synthesis of [a-methyltyrosine4]angrotensm II Proc Nat1 Acad Sa USA 78,757-760 23 Zabrtskle, T M., Cheng, H , and Vederas, J. C (1992) Mechanism-based mactivatton of peptidylglycme a-hydroxylatmg monoxygenase (PHM) by a substrate analogue, D-phenylalanyl-L-phenylalanyl-D-vmylglycme J Am Chem Sot 114, 227&2272 24 Pedersen, M L and Berkowitz, D B (1992) A reagent for the efficient cleavage

of N-benzoylhomoserme lactones. access to a-(2-phenylseleno)ethyl ammo acids. Tetrahedron Lett 33,73 15-73 18 25 d’Angelo, J and Maddaluno, J (1986) Enanttoselecttve synthesis of p-ammo esters through high-pressure-induced addition of ammes to a$-ethylemc esters J Am Chem Sot 108,8112-8114 26 Gassman, P G and Schenk, W N (1977) A general procedure for the base-promoted hydrolysis of hindered esters at ambient temperatures. J Urg Chem 42, 918-920. 27 For the preparation of N-Bz-Phe-OMe, see: Schnyder, J and Rothenberg, M (1975) Hydrazide as a carboxyl protectmg group. Deprotectton by acidolysis Helv Chum Acta 58, 521-523

488

Berkowitz et al

28 Winkle, M R., Lansmger, J M , and Ronald, R C (1980) 2,5-Dlmethoxybenzyl alcohol. a convenient self-indicating standard for the determmatron of organohthmm reagents, J C S Chem Commun 87-88 29 For the preparation of N-Bz-Hts-OMe Campbell, J. B (1983) The synthesis of N-t-(2-hydroxypropyl)histidme, N-t-(2-hydroxyethyl)hlstldlne and then deuterated analogues. J Chem Sot Perkm I, 12 13-l 2 17 30 Miller, J B (1959) Preparation of crystalline drphenyldtazomethane J Org Chem 24,560-561 3 1. To avoid the use of HgO, see. Holton, T L and Shechter, H (1995) Advantageous syntheses of drazo compounds by oxidation of hydrazones with lead tetraacetate in basic environments J Org Chem 60, 47254729 32 Lagarras, J C , Glazer, A. N , and Rapoport, H (1979) Chromopepttdes for Cphycocyanm. J Am Chem. Sot 101,503&5037. 33. Dale, J A., Dull, D. L., and Mosher, H. S. (1969) a-Methoxy-a-trrfluoromethylphenylacetrc acid, a versatrle reagent for the determmatlon of enantlomerlc composmon of alcohols and ammes J Org Chem 34,2543-2549 34 Berkowitz, D B and Smith, M K (1995) Enantiomerlcally enriched a-methyl ammo acids Use of an acyclic, chiral alanme-derived dtamon with a high diastereofacial bras J Org Chem 60, 1233-1238

28 A Novel Synthetic Protocol for the Preparation of Enantiopure 31,4-, and Mubstituted Prolines N. Andrd Sasaki 1. Introduction Stereochemlcally well-defined 3-, 4-, and 5-monosubstltuted prolines can play an important role as probes m search of potent neuroexitatory agents or m the construction of conformatlonally constrained peptldes. Mimicking distinct conformations of excitatory ammo actds may lead to a more detailed understanding of the structural requirements for binding of these ammo acids to the proteins involved m the process of neurotransmlsslon (Z-3). Meanwhile, syntheses of conformatlonally constrained peptldes are emerging as useful means for developing peptlde-derived pharmaceutical agents (4,.5). This chapter describes methodology for syntheslzmg enantlopure 3-, 4-, and 5monosubstltuted prolmes starting from the single L-serme-derived choral derivative (R)-1 (6-9).

2. Materials General: ‘H (TMS as internal reference) 300 MHz and 13C 75.5 MHz (see Note 1) TLC (precoated with 0.25 mm of silica gel contammg 60F2+, mdlcator) developed with a solvent mixture of EtOAc/heptane). Flash chromatography on 230-400 mesh slllca gel. HPLC conditions: 1. For the determmatlon of the ratio of 4a and 4b* column, column size, 3.9 x 150 mm; eluent, heptane/EtOAc: 80/20, detector, refractometer R 410 (Waters, Mllford, MA). 2 For the determination of the ratio of 6a and 6b column, column size, 3 9 x 150 mm; eluent, heptane/EtOAc/AcOH* 1 mL/mm, detector, refractometer R 410 (Waters). From

Novapack Sl 4 p, flow rate 1 mL/mm, Novapack Sl 4 pm; 80/20/O. 1, flow rate

Methods m Molecular Me&me, Vol 23 fept/dommetm Protocols Edited by W M Kazmierski @Humana Press Inc , Totowa, NJ

489

Sasakl

490

3 For the determmatton of opttcal purity of 19: column, Novapack St 4 pm, column stze, 3.9 x 150 mm; eluent, heptane/2-propanol: 95/5, flow rate, 2 mL/mm, detector, refractometer R 4 10 (Waters) THF drsttlled from sodmm benzophenone ketyl

2.7. Reagents

for Method 3.7

1, (2R)-2-(ter~-Butoxycarbonyl)am~no-3-phenylsulfonyl-l -(2-tetrahydropyranyloxy)propane (R)-1 (Acres Orgamcs, Belgmm, 27612-0010) (see Note 2,10) 2 1 6 M n-Butylllthmm (n-BuLt) in hexanes. 3 Ally1 bromide 4. 2-Bromoethanol 5 Trtfluoromethanesulfomc anhydrlde 6 2-Bromoethyl trtflate 7 Pyridmmmp-toluenesulfonate (PPTS) 8 6% Sodium-mercury amalgam (Na-Hg) 9 Jones reagent 10 10% Pd/C. 11 Acetone. 12 Dtchloromethane 13 Ethanol 14 Ethy acetate (EtOAc) 15 Heptane 16 Methanol 17 Tetrahydrofuran (THF) 18 MgS04 19 NazHP04 20 lNHC1 21 1NNaOH 22 P,O,

2.2. Reagents

for Method 3.2

1. (2R)-2-(tert-Butoxycarbonyl)amlno-3-phenylsulfonyl-l-(2-tetrahydropyranyloxy)propane (R)-1 (IO). 2 1 6 M n-Butylbthmm (n-BuLt) m hexanes 3 2-Bromoethanol 4 Trtfluoromethanesulfonic anhydrtde. 5 2-Bromoethyl trrflate 6 Ally1 bromtde 7 PPTS 8 6%Na-Hg 9 Jones reagent. 10 Dtazomethane u-rether 11 (R)-(+)-a-Methylbenzylamme 12. Acetone. 13 Dtchloromethane

491

Enantiopure Prolines 14 15 16. 17. 18 19. 20 21.

Ethanol. EtOAc. Heptane Methanol THF MgSO,. Na2HP04 1NNaOH

2.3. Reagents for Method 3.3 1 (2R)-2-(tert-Butoxycarbonyl)amino-3-phenylsulfonyl-l-(2-tetrahydropyranyI0xy)propane (R)-1. 2. 1.6 M n-Butyllithmm (n-BuLi) in hexanes. 3 Benzyl bromide 4. (R)-(-)2,2-Dimethyl1,3-dioxolane-4-methanol [(R)-2,3-O-isopropylideneglycerol (99% ee, CHEMI S p A , Cmlsello Balsamo, Italy)] 5 Trifluoromethanesulfomc anhydride 6 (q-2,2-Dimethyl-1,3-dioxolane-4-methanol triflate [(S)-2,3Gisopropyltdeneglycerol trtflate] 13. 7. Sodium hydride (NaH) (60% dispersion m mmeral oil) 8 Methanesulfonyl chloride 9. Tetrabutylammomum iodtde (n-Bu,NI). 10 Boron trlfluoride diethyl etharate 11 6% Na-Hg. 12 Jones reagent 20 Acetone 19 Dlchloromethane. 18 Ethanol 15 EtOAc. 16 Heptane 17 Methanol 2 1 Pyridme 14. THF. 13 Trlethylamme (TEA) 23 MgSO.+ 22. Na,SO+ 25. Na2HP04 24 NaHCOs 26 NH&l 27. 1 NNaOH 28. Cont. HCI.

2.4. Reagents for Method 3.4 1 2(R)-2-(tert-Butoxycarbonyl)amlno-3-phenylsulfonyl2. (R)-2,3-lsopropyhdeneglyceraldehyde.

1-propanol 11

Sasakl

492 3 4 5. 6. 7 8 9 10 11. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29. 30 31 32

Benzyl bromrde Di-tert-butyl dicarbonate. Trifluoroacetic acid (TFA) l,%-Dichloroethane Sodium triacetoxyborohydride p-Toluenesulfonyl chloride Titanmm(IV) isopropoxide Potassium bis(trtmethylsilyl)amide (0 5 A4 solution m toluene) NaH (60% dispersion m mineral oil) n-Bu,NI 10% Pd/C 6% Na-Hg Jones reagent Diazomethane m ether Acetone Dichloromethane Diethyl ether Dimethylformamide (DMF) Ethyl acetate (EtOAc) Heptane Methanol Pyridme THF CuSOc, K&O, Na+SO, NaHCO, Na,HPO, NH&l 1 NHCl m methanol

3. Methods 3.1. Synthesis of tram-3-Substituted Proline 6a The syntheses of (2S,3S)-N-(tert-butyloxycarbonyl)-3-allylprolme 6a IS shown m Fig. 1. The choice of ally1 functtonahty at C-3 posrtron 1sbased on the fact that rt is a masked group to be transformed mto another functtonahty. The dththtated amon of (R)-1 formed by 2 Eq of n-BuLt at -78°C IS treated with 1.2 Eq of ally1 bromide to give 2 m 95% yield after flash chromatography. Then the dilithiated anion of 2 in THF formed by 2 Eq of n-BuLt at -78°C 1s treated with 1.2 Eq of 2-bromoethyl trtflate to provide the mixture of pyrrolidine 3a and its C-3 eptmer 3b in 78% yield after purtficatton by flash chromatography. Treatment of 3a/3b with PPTS (0.1 Eq) gives in 94% yield the mixture of alcohol 4a and tts C-3 epimer 4b which 1seasily ehmmated by

Enantiopure Prolines S02Ph

2 equw n-BuLI

2 equw n-BSLI

AllyBr. -78°C

FJ~(CH~)~OTI. -78°C

W-1

2

S02Ph ‘“V _ Joner oxldn

xoH

_ 6%Na-Hp.

c,, N Boc

CO&l

6a I

hoc

hoc

Sa

4a

Hz, WC

Fig 1 Synthesis of (2S,3S)-N-Boc-3-allylprolme

6a

recrystalhzatlon. HPLC analysis of 4a/4b mixture showed that the ratio of the diastereomers to be 89: 11. This indicates that the pyrrolidme rmg formation takes place m a highly stereoselectlve manner (II). Desulfonylatlon of the mixture of 4a/4b by 6% Na-Hg m methanol affords a single diastereomerlc alcohol 5a m 83% yield, which is then oxidized by Jones reagent m acetone to provide 6a m 86% yield. Catalytic hydrogenation of 6a on Pd/C affords optltally pure N-Boc-truns-3-n-propyl+proline 7 in a quantitative yield. 3. I. 1. Preparation of (2R)- l-1(2- Tetrahydropyranyl)Oxy]2-[(tert-Butyloxycarbonyl)Amino]-3-(Phenylsulfony/)-5-Hexene

2

1 Add dropwlse with vigorous stn-rmg 12 6 mL (2 Eq) of 1 6 M n-BuLi (see Note 3) to 4 0 g (10 mmol) of (R)-1 m 40 mL of THF at -78’C under a nitrogen atmosphere

2 Stir the reaction mixture at this temperature for 15 mm, and then add 1 45 g (12 mmol) of ally1 bromide m 2 mL of THF 3. Allow the mixture to warm gradually to 0°C while stirring for 2.5 h. Then concentrate under reduced pressure 4 Dilute the residue with ethyl acetate, and wash with two 20-mL portions of water, dry the orgamc phase over MgS04, filter, and evaporate the solvent under reduced pressure 5 Purify the crude product by flash chromatography on silica gel elutmg with a l/2 mixture (v/v) of ethyl acetatejheptane. You should get about 4.20 g (95% yield) of 2 as a viscous oil Rf 0 28 ([EtOAc/heptane* l/2) [R, of (R)-1 0 19 (EtOAc/ heptane. l/2)]). Characterlstlc peaks ofNMR ‘H NMR (CDC&) 6 5 83-5 55 (m,

Sasakl

494 lH), 5 18-5 04 (m, 2H), 2.65-2 50 (m, 2H), 13C NMR (CDCl,) 118.4, 118.5, 30 7, 30 5

6 133 4, 133 6,

3.1.2. Preparation of (2R,3R)- 1-(tert-Butyloxycarbonyl)2-{[(2- Te trahydropyranyl) Oxy]Me thy/} 3-Ally/-3-(Phenylsulfonyl) Pyrrolidine 3a 1 Add dropwise with vigorous stirring 12 0 mL (19 2 mmol) of 1 6 M n-BULI at -78’C to 4 16 g (9.47 mmol) of 2 m 40 mL of THF under a nitrogen atmosphere. 2 Stir the mixture for 30 mm at -5O”C, allow to cool back to -78°C and then add 2 92 g (11 3 mmol) of freshly prepared 2-bromoethyltnflate in 4 mL of THF (see Note 4 and 12). 3 Allow the reaction mixture to warm to 0°C while stmmg for 2 5 h Then concentrate under reduced pressure 4. Dilute the residue with 30 mL of ethyl acetate, wash with 10 mL of water, dry over MgS04, and remove the solvent under reduced pressure 5. Purify the crude product by flash chromatography on silica gel elutmg with a l/2 mixture (v/v) of ethyl acetatejheptane Concentrate the appropriate fractions You should get about 3 43 g (78% yield) of a mixture of 3a and Its C-3 eplmer 3b as a VISCOUS011 Rf 0 20.(EtOAc/heptane l/2) (see Note 5). Characteristic peaks of NMR* ]H(CDCl,) 6 5.9&5 60 (m, lH), 5.12 (d,J= 9 5 Hz, lH), 5.00 (d, J= 17 2 Hz, IH), 2 41-2 02 (m, 2H), 13C NMR (CDC13) 6 133.9 (CH=CH& 119 4 (CH=CH,), 3 1.O (CH,CH=CH2)

3.1.3. Preparation of (2R,3R)-1 -(tert-Buty/oxycarbony/)2-(Hydroxymethyl)-3-atlyl-3-(Phenylsulfonyl)Pyrrolidine

2

4a

Dissolve 3.43 g (7.37 mmol) of the above mixture of 3a/3b m 30 mL of ethanol, and warm to 50°C for 4 h with stirring m the presence of 185 mg (0 1 Eq) of PPTS Remove the solvent under reduced pressure, dilute the residue with 30 mL of dlchloromethane, wash with 10 mL of brine, and concentrate You should get about 2 64 g (94% yield) of a mixture of 4a/4b The ratio of this mixture IS determined to be 89.11 by HPLC (see Note 6) You can obtain 2 11 g of pure 4a by l/l) mp recrystalhzatlon (twice from EtOAc/heptane) Rf 0 20 (EtOAc/heptane 133-134”C, [~]~~o = +74 5” (c 1 0, CH,OH) Characteristic peaks of NMR ‘H NMR(CDCl3)G585-559(m,1H),5.12(d,J=98Hz,1H),502(d,J=176Hz, lH), 248 (dd,J= 15.0 Hz,4 5 Hz, 0 5H), 2.33 (dd,J= 15 0 Hz, 4 5 Hz, 0 5H), 2 24-2 04 (m, lH), 13C NMR (CDCl,) 6 131 1 (CH=CH,), 120 1 (CH=CH,), 3 1 7 (CH2CH=CH2)

3.1.4. Preparation of (Z!S,3S)- 1-(tert-Buty/oxycarony/)2- (Hydroxyme thy/) -3-Allylpyrrolidine 5a 1 Add 8 6 g (22 4 mmol) of 6% Na-Hg to a solution of 2 87 g (7 52 mmol) of a mixture of 4a/4b obtained above and 3 20 g (22.6 mmol) of Na,HPO, m 30 mL of HPLC-grade methanol (see Note 7)

Enantiopure Prolines

495

2. Stir the mixture vigorously for 1 h at room temperature, remove mercury by decanting the solution, and concentrate the solution. 3 Dissolve the residue m 50 mL of dlchloromethane, wash with four 15-mL portions of brine, dry over MgS04, and concentrate (see Note 8) 4 Purify the crude product by flash chromatography on slhca gel elutmg with a l/l mixture (v/v) of ethyl acetate/heptane. Concentrate the appropriate fractions. You should get about 1 50 g (83% yield) of pure 5a as a viscous 011 R, 0 18 (EtOAc/ heptane. l/2) (see Note 9). [cI]*~~ = -20.5” (c 1 5, MeOH) ‘H NMR (CDCl,) 6 5 75 (m, lH), 5 07 (d, J= 13 7 Hz, lH), 5 05 (d, J= 7.0 Hz, IH), 4 92 (br s, lH), 3.88-3.42 (m, 3H), 3.35-3 15 (m, lH), 2 40-l 74 (m, 5H) 147 (s, 9H), 13C(CDC13) 6 135 9, 116.8, 80.3,66.9, 65.3,64 5,46.4,41 0, 37 5,29 6, 28.5.

3.1.5. Preparation of (2S,3S)-N-(tert-Butyloxycaronyl)3-Allylproline 6a 1 Add 5.5 mL (2 Eq) of Jones reagent to a solution of 1 28 g (5 3 mmol) of 5a m 120 mL of acetone, and stir the mixture very vigorously for 1 h at room temperature. 2 Evaporate acetone under reduced pressure, and dilute the residue with 10 mL of ethyl acetate. 3 Wash the orgamc phase with three lo-mL portions of water and extract with 8 mL of 1 NNaOH. 4. Extract the resultant alkaline solution with 20 mL of ethyl acetate, and then adJust the pH to about 2 0 using 1 N HCl 5 Extract the precipitate with three 10 mL portions of dlchloromethane, wash the combmed organic extracts with brine, dry over MgS04, and concentrate to dryness 6. Dry the residue under reduced pressure over P205 You should get about 1.17 g (86% yield) of 6a as a VISCOUS011.R,O 43 (CHCl,/MeOH: 9/l) [cx]*~~ = -2 3O (c 1.0, MeOH) ‘H NMR (CDCl,) (two conformers) 6 7.20 (br s, lH), 5.86 -5 70 (m, lH), 5.09 (d, J = 16 5 Hz, lH), 5.08 (d, J = 10.5 Hz, lH), 3 95 (d, J = 4.1 Hz, 0.4H, 2-H), 3.89 (d, J= 4 5 Hz, 0.6H, 2-H), 3 63-3 45 (m, lH, 5-H), 3.55-3.37 (m, lH, 4-H), 2 50-2 35 (m, lH, 3-H), 2.45-2 30 (m, lH, allyl), 2 20-2.09 (m, lH, ally]), 2 09-2 00 (m, lH, 4-H), 1.68-1 56 (m, lH, 4-H), 1.45 (s, 3H), 1 41 (s, 6H); 13C NMR (CDCl,) 6 178 1, 176 9, 155 5, 154 1, 135 2 (CH=CH,), 117 5 (CH=CH,), 80 8, 80 5, 63 9,45.8, 45 5,44 0,42 2, 37 3 (CH,CH=CH,), 29 5, 29 2,28 3

3 1.6. Preparation of (2S,3S)-N-(tert-Buty/oxycarony/)3-n -Propylproiine 7 1. Hydrogenate 60 mg (0.23 mmol) of 6a in 5 mL of MeOH m the presence of 5 mg of 10% Pd/C for 30 mm 2 Remove the catalyst by filtration and the solvent by evaporation You should get about 55 mg (89% yield) of 7 Rf 0.47 (CHC13/MeOH 9/l) mp 88-90°C (13) [a]25D = -17 8” (c 1 0, MeOH); [a] 25D = -42 5” (c 1 0, CHCl,); (13). ‘H NMR

496

Sasaki S02Ph

SOePh S02Ph BOCN L : WH

OTHP

I eqw n-BULI

2 cqvl” n-BULI lJr(CH2J20Tr -78’C

-c?,

OTHP - $Y’C”’ 0

F!

W-1

e

OTHP

N

Boc

i?oc

8

3b

1 KCH+2 3 a:H BOC

IfJoc

10

6a

2

{E” 4)IN NaOH

KOTHP +GOTHP $0,

hoc

9a

9b

Fig. 2. Synthesis of (2S,3R)-N-Boc-3-allyprohne (CDCl,) (two conformers) 6 3.98 (d, J= 4.4 Hz, 0 6H), 3.70-3 34 (m, 2H), 2 48-2 20 (m, lH), 2 182.00 (m, 0 93 (t, J = 7 lHz, 3H), 13C NMR (CDCl,) 6 179 0, 64 7,46 1,45 8,44 7,42 4,35 6,30 2,30 1,28 4,20

3.2. Synthesis

of cis-3-Substituted

6%Na Hg

methyl ester 10 3 84 (d, J= 6.2 Hz, 0 4H), lH), 1.699128 (m, 14H), 176 5, 155 9, 153 9, 80 5, 9, 14 1

Proline Derivative

10

The synthesis of (2S,3R)-N-(tert-butyloxycarbonyl)-3-allylprolme methyl ester 10 1s depicted m Fig. 2. One-pot formation of 2,3-dtsubstttuted pyrrolrdme 8 is accomplished m excellent yield by treating dththtated amon of (R)-1 with 2bromoethyl trtflate at -78OC (7). Allylation of the sulfonyl amon of 8 takes place with high dtastereofactal selectivity providmg 3b as a maJor product m 80% yield (see Note 10). Sequential desulfonylatton of the mixture 3b/3a by 6% NaHg, and removal of the THP-protecting group followed by Jones oxtdation provides the mixture of 6a/6b m 52% overall yield. The dtastereomertc ratio of 6a/ 6b is determined to be 37:63 on the basis of HPLC analysts of the derived (R)(+)-a-methylbenzylamtde. Treatment of the thus-obtained mixture of 6a/6b with dtazomethane followed by selective sapomficatton results m an easy separation of 6a and 10. Overall yield of 10 from a mixture of 6a/6b is about 55% It is to be noted that desulfonylatton without the protectmg group on the neighboring hydroxymethyl functtonahty leads to a considerable eptmerization at C-3 position gtvmg 6a as a major product (6a/6b = 84: 16) (22). 3.21. Preparation of (ZR)- 1-(tert-Buty/oxycarbonyl)2-{[(2-Tetrahydropyranyl)Oxy]MethylJ-3-(Phenylsulfonyl)Pyrrolidine

8

1, Add dropwtse with vigorous stmtng 12.6 mL (2 Eq) of 1 6 M n-BuLt (m hexanes) to 4 0 g (10 mmol) of (R)-1 tn 40 mL of THF at -78°C under a nitrogen atmosphere 2 Stir the reaction mixture at this temperature for 15 mm, and then add 3 1 g (12 mtnol) of 2-bromoethyl trtflate in 5 mL of THF

Enantiopure Prolines

497

3 Allow the reaction mixture to warm to 0°C while stlrrmg for 2 5 h, and concentrate the reaction mixture under reduced pressure. 4 Dilute the residue with 50 mL of ethyl acetate and wash with three lo-mL portlons of brme Dry the orgamc phase over MgS04, filter, and evaporate the solvent under reduced pressure 5 Purify the crude product by flash chromatography on silica gel elutmg with a l/2 mixture (v/v) of ethyl acetate/hexane. Concentrate the appropriate fractions. You should get about 3 90 g (92% yield) of 8 as a colorless 011 R, 0 20 (EtOAc/heptane l/2) Character&c peaks of NMR ‘H NMR (CDCl,) 6 2.61-2 18 (m, 2H, formation of pyrrohdme rmg), no NH peak at 5 15 ppm; 13C NMR (CDCl,) 6 44 6,44 1 (cH,N), no peak at 46.5 ppm, which 1sattributed to C-2 of (R)-1 (14)

3 2.2 Preparation of (2R,3S)- I-(tert-Buty/oxycarbonyl)2-{[(2-TeWahydropyranyl)Oxy]Methyl]-3-A//y/3-(Phenylsulfonyl) Pyrrolidine 3b Add dropwlse with vigorous stlrrmg 5.2 mL (1 Eq) of 1 6 M n-BuLi to 3 50 g (8 2 mmol) of 8 m 35 mL of THF at -78°C under a mtrogen atmosphere. Stir the reaction mixture for 30 mm at -5O”C, and allow to cool back to -78°C to add 1.2 g (9.8 mmol) of ally1 bromide m 2 mL of THF Allow the reactlon mixture to warm to 0°C while stlrrmg for 3 h, and then concentrate under reduced pressure Dilute the residue with 30 mL of ethyl acetate, wash with three lo-mL portlons of water, dry over MgS04, and concentrate under reduced pressure. Purify the crude product by flash chromatography on slhca gel eluting with a l/2 mixture (v/v) of ethyl acetatelheptane. Concentrate the appropriate fractions You should get about 3 25 g (85% yield) of a mixture of 3b/3a. Rf 0.20 (EtOAc/heptane. l/2) (see Note 5) The ratlo of 3b and 3a was estimated to be 94.6 by HPLC analysis after removal of the tetrahydropyranyl (THP) protecting group by PPTS as described m Subheading 3.1.3. Characterlstlc peaks of NMR: ‘H NMR (CDC13) 6 6.30-42 5”6.05 (m, CII=CHJ, 13C NMR (CDC13) 6 133.3 (CH=CH*, 3b), 131 5 (CH=CH,, 3a), 119 4 (CH=CH2, 3a), 118 0 (CH=CH,, 3b), 40.2 (CH,CH=CH,, 3a), 35 7 (CH,CH=CH,, 3b). Thus obtained alcohol 4b, the C-3-eplmer of 4a, can be easily purified by recrystalllzatlon (twice from EtOAc/hexane) (see Note 11)

3.2.3. Preparation of (2S)- 1-(tert-Butyloxycarbony/)2-([(2-Tetrahydropyranyl)Oxy]methyl]-3-Allylpyrrolidine

9b

1 Add 5 2 g (13.5 mmol) of 6% Na-Hg to a solution of 2.12 g (4.56 mmol) of a mixture of 3b/3a obtamed above and 1 94 g (13.68 mmol) of Na2HP0, m 30 mL of HPLC-grade methanol 2 Stir the mixture vigorously for 1 h at room temperature, remove mercury by decanting the solution, and concentrate 3 Dissolve the residue in 50 mL of dichloromethane, and wash with four 10 mL portions of brine, dry over MgS04 and concentrate

498

Sasaki

4 Purify the crude product by flash chromatography on silica gel elutmg with a l/4 mixture (v/v) of ethyl acetate/heptane Concentrate the appropriate fractions You should get about 1 14 g (77% yield) of a mixture of 9b and its C-3 eplmer 9a as a VISCOUS011 Rf 0.34 (EtOAc/heptane. l/2) Characteristic peaks of NMR ‘H NMR (CDC13) 6 5 94-5.70 (m, lH), 5 14-4 92 (m, 2H), 13CNMR (CDCl,) 6 116.1 (CH=CH2, 9a), 115.4 (CH=&H,, 9b), 38 0 (CH2CH=CH2, 9a) 33 9 (CH,CH=CH2, 9b).

3.24. Preparation of (2S,3R)-N-(tert-BAyloxycarbonyl)3-Allylproline Methyl Ester 10 1 Dissolve 1 05 g (3 2 mmol) of the above mixture of 9b/9a in 15 mL of ethanol, and warm the solution to 50°C for 4 h with stmmg m the presence of PPTS 2 Remove the solvent under reduced pressure, dilute the residue with 15 mL of dlchloromethane, wash with 10 mL of brine, and concentrate You should get

about 730 mg of a crude mixture of 5b/5a (see Note 12) 3. Dissolve the residue m 70 mL of acetone, add 3.0 mL of Jones reagent, and stir the mixture for 1 h at room temperature.

4 Concentrate under reduced pressure, dissolve the residue m 5 mL of water and extract with two 5 mL portions of ethyl acetate Dry the organic phase over MgS04, and concentrate under reduced pressure The ratlo of a mixture of 6b/6a 1s determined to be 63.37 by HPLC analysis of their (R)-(+)-amethylbenzylamlde derivatives (see Note 13) 5 Treat the residue thus obtained with dlazomethane m ether until no gas formatlon

IS observed, and then evaporate ether to obtain 430 mg of a mixture of methyl ester of 6a and 10

6 Dissolve the residue in 1 5 mL of MeOH, and add 1 2 mL of 1 NNaOH,

stir the

mixture at room temperature for 24 h

7 Concentrate the solution to remove methanol, extract with three 2-mL portions of ethyl acetate, dry the extracts over MgS04, and concentrate

You should get about

150 mg of 10 (18% overall yield from the mixture 9b/9a) as a viscous oil R,O 27 (EtOAciheptane. l/4) [IX]*~~ = +25 5” (c 1 0, CHC13) IH NMR (CDC13) (two conformers) 6 5 88-5.70 (m, lH), 5 1 l-5.00 (m, 2H), 4 35 (d, J= 8 3 Hz, 0 4H), 4 25 (d, J = 8 6 Hz, 0 6 H), 3.72 (s, 3H), 3.75-3 58 (m, lH), 1 82-l 65 (m, lH), 146 (s,4H), 1.41 (s, 5H, 13C NMR (CDCl,) 6 172.3, 135 8 (CH-CH,), 116 5 (CH-CH,),

79 8,62 5,61 9, 51.5,45.6,42.5,34.4

(cH2CH-CH2), 29 7,28 9,28.4,28 3

3.3. Synthesis of trans-5-Substituted

Proline 20

The synthesis of (2S,SS)-N-(tert-butyloxycarbonyl)-3-(benzyloxy)methylprolme 20 is shown in Fig. 3 Thus, (R)-1 IS converted to 12 VEU11 m 80% yield. Change of an OH-protecting group of (R)-1 from THP to benzyl group IS owing to the use of the O-lsopropylldene protecting group for the electrophlle 13, which IS to be removed selectively. Dllithlated amon of 12 IS treated at -78“C wtth 1.2 Eq of (S)-isopropylidmeglycerol triflate 13 to afford 14, which

Enantiopure Prolines

499

S02Ph OTHP

NallfTllF.

-k??!%.:

BnRr

-

(JO-1

16 1

15

I cqu1v n-RULI TW, 78°C

RF? (OEOz n

14

HO \W

17

BOC

20

Frg. 3 Synthesis of (2S, 5S)-N-Boc-5-(benzyloxy)methylprolme

20

is directly hydrolyzed m the presenceof a catalytic amount of HCl m methanol to provrde dial 15 m 90% yield (in two steps).Monomesylate 16 IS prepared in 77% yield. Transformation of 16 to pyrrolidine 18 can be accomplishedvia epoxide 17 by treatment with 1 Eq of n-BuLt, and then by in sztu treatment of 17 with n-BuLi

at

-78°C m the presenceof BF, etheratein about 65% yield. Desulfonylation of 18 by treatment with 6% Na-Hg followed by Jones oxtdatton affords 20 m 75% yield (m two steps).It should benoted that transformation of the hydroxymethyl group of 19 to another functionality, such as alkyl or an N-protected aminomethyl, prior to the two-step conversion of benzyloxymethyl to carboxyhc group can lead to a dtfferent (25’,5R)-or (2S,SS)-tram- proline. According to this methodology, the remaming isomersof 20 can be prepared in enantiopure form by choice of the educt 1 and isopropylideneglycerol mflate 13 with adequatestereochemistry(14) 3 3.7. Preparation of (2R)-2-flert-Butoxycarbonyl)Amino3-Phenylsulfonyl- I -P ropanol 17 (10) 1 Dtssolve 4.0 g (10 mmol) of (IQ-1 and 25 1 mg (1 mmol) of PPTS m 20 mL of ethanol. 2 Stir the reaction mixture for 6 h at 50°C and concentrate under reduced pressure 3 Dtssolve the residue m 50 mL of ethyl acetate, wash with 5 mL of water, dry over MgS04 and concentrate You should get about 3.0 g (95% yield) of 11. Rf 0.10 (EtOAc/heptane* l/l). mp 124125°C (recrystallized from EtOAc/heptane)

Sasakl

500

[cx]~~,,= -7 3” (c 1 0, MeOH) ‘H NMR (CDCl,) 6 7 95 (d, J = 7 1 Hz), 2H), 7.727 52 (m, 3H), 5 25 (br s, lH), 4 04 (m, lH), 3 90 (m, lH), 3 80 (m, lH), 3 50 (dd,J = 14.4 Hz, 6.7 Hz, lH), 3 40 (dd,J= 14.3 Hz, 5 6 Hz, lH), 2 80 (br s, IH), 1 42 (s, 9H), 13C NMR (CDCl,) 6 155 4, 133 9, 129 4, 127.9, 79 9, 63 7, 56 5,48.7,28 3

3.3.2. Preparation of (2R)- 7-O-Benzyl-2-[(tert -5utoxycarbonyl) Amino]-3-Phenylsulfonyl-7 -Propane/ 72 1. Add 2.80 g (8 9 mmol) of 11 to a suspension ofNaH (60% 1n m1neralo11) (370 mg, 9.3 mmol) 1n 50 mL of THF at O”C, and stir the mtxture for 30 m1n at 0°C 2 Add 1 80 g (10.5 mmol) of benzyl bromide and 25 mg of n-ByNI to the above reaction mixture, and stir at 0°C for 12 h 3 Dilute the mixture with 300 mL of ethyl acetate, wash the organic phase with three 50-mL portions of water and with two 50-mL port1ons of brine, dry over Na2S04, and evaporate the solvent to dryness. 4 Purify the residue by chromatography on s111cagel elutlng with a l/3 mixture (v/v) of ethyl acetate/heptane. You should get about 3 24 g (90% yield) of 12. R, 0 37 (EtOAc/heptane: l/l) Recrystallize from EtOAc/hexane mp 105°C [a1200 = -10 3” (c 1.0, MeOH) ‘H NMR (CDC13) 6 7 90 (m, 2H), 7 73-7.50 (m, 3H), 7 43-7 20 (m, 5H), 5.11 (br s, lH), 2 44 (s, 2H), 4.16 (m, lH), 3 70 (dd,J= 9 5 Hz, 3 5 Hz, lH), 3 55 (dd, J= 9 5 Hz, 5 Hz, lH), 3 45 (d, J= 6 Hz, 2H), 1 43 (s, 9H), 13CNMR (CDCI,) 6 154.7, 139 7, 133 8, 129 3, 128.5, 128 0, 127 9, 127.7,79 9, 73 3,70 5,56 7,46 7,28 3

3.3.3. Preparation of (2R,5R)- 1-O-Benzyl2-[(tert-Butoxycarbonyl)Amino]-5,6-O-lsopropy/ldene3- Phenylsulfonyl- I, 5,6- Hexanetriol 14 1. Add dropwise with vigorous stirring 7.70 mL (12.32 mmol, 2 Eq) of 1.6Mn-BuL1 to 2 50 g (6 16 mmol) of 12 in 80 mL of THF at -78°C under a nitrogen atmosphere 2 Stir the reaction mixture at -78°C for 15 mln, and then add 1.10 g (4.50 mmol) of (&1sopropylldeneglycerol triflate 13 1n 8 mL of THF (see Note 14,15) 3. Allow the reaction mixture to warm gradually to 0°C while st1rnng for 3 h, and then concentrate under reduced pressure 4 Dilute the residue with 200 mL of ethyl acetate and wash with two 50-mL portions of water and 50 mL of brine. Dry over Na,SO,, and evaporate the solvent You should get about 3 50 g of a mixture of the starting matenal12 and product 14 This mixture 1sseparable by chromatography only when the lsopropylldene group 1sremoved from 14 Charactenstlc peaks ofNMR: ‘H NMR (CDC13) F 1 30, 1 22 (2s, 6H, ketal), 13C NMR (CDCl$6 109.5 (ketal C), 26.9,26.7 (ketal CH,), 25.6,25.5 (ketal CH,).

3.3.4. Preparation of (2R,SR)-I-0-Benzyl2-[(tert-Butoxycarbonyl)Amino]-3-Phenylsulfonyl1,5,6-Hexanetriol 15 1 Dissolve 3.50 g of the above mixture of 12 and 14 1n 160 mL of methanol, add 100 pL of cont. HCI, and stir the solution for 4 h at room temperature.

Enantiopure Prolines

501

2 Add 2 mL of tnethylamme, and evaporate the solvent. 3 Purify the residue by column chromatography on stlica gel eluting with a l/l mixture (v/v) of ethyl acetate/heptane Concentrate the appropriate fractions. You should get about 1 90 g (90% yield m two steps) of 15 as a viscous oil, with recovery of 660 mg of 12 (1 6 mmol). R, 0.10 (EtOAc/heptane* l/l). ‘H NMR (CDCl,) 6 7 97-7.03 (m, lOH), 5 78-5.52 (m, lH), 4.58-4.23 (m, 2H), 4.00-3.30 (m, 8H), 2.13-1 64 (m, 2H), 1.43 (s, 9H); 13C NMR (CDCl,) 6 155.7, 137 9, 137 6, 133 8, 129.3, 129.2, 128 6, 128.3, 128.2, 127.7, 80 1, 72 9, 70 2, 694, 68 5, 66.2,66 1,62.9,61 6, 51.0,49 2,29.7,28 7,28.3

3.3.5. Preparation of (2R,SR)-I-O-Benzyl2-[(tert -Butoxycarbonyl)Amino]-3-Phenylsulfonyi6-O-(Methylsulfonylj-7,5,6-Hexanetriol 76 1 Add dropwlse 0.26 g (2 25 mmol) of methanesulfonyl chloride to a 0 05 M solution of 1 03 g of 15 (2 15 mmol) m 43 mL of pyrldine at -40°C 2. Stir the reaction mixture for 7 h at -4O”C, and then for about 12 h at -15’C Evaporate pyrldine zn VLECUO. 3 Dissolve the residue m 300 mL of ethyl acetate Wash with four 50-mL portions of 1% (v/v) aqueous HCl, 50 mL of saturated NaHC03 solution, and 50 mL of brine Dry over MgS04 and concentrate. 4 Purify the residue by column chromatography on silica gel elutmg with a 2/l mixture (v/v) of ethyl acetate/heptane. You should get about 0.93 g (77% yield) of monomesylate 16 as a colorless 011 RfO 27 (EtOAc/heptane l/l) Characteristic peaks of NMR ‘H NMR (CDC13) 6 3.00 (s, 3H, MS), 2 22-l 80 (m, 2H), ‘3CNMR(CDC13)6 155.7, 139 0, 137.7, 1374, 134.0, 129 4, 129.4, 128.9, 128 7, 128 4, 128 2, 127 8, 127 7, 80.4, 73 1, 72 8, 69.3, 67 7, 66.2, 62 7, 61 5, 50 7, 49.2, 37 4,29 5,28.6,28.3.

3.3.6. Preparation of (2F$5S)-N-(tert-Butoxycarbony/)2-[(Benzyloxy)Methly/-3-(Phenylsulfonyl)5-Hydroxymethylpyrrolidine 18 1 Add dropwlse with vigorous stirring 1 0 mL of 1 6 Mn-BuLl to 0 84 g (1.5 mmol) of 16 m 15 mL of THF at -78°C under a nitrogen atmosphere 2 Allow the reaction mixture to warm gradually to room temperature, and stir for 30 mm. When quenched at this stage by addition of saturated aqueous ammomum chloride, you can obtain 17 (see Note 15). 3. Allow the reaction mixture to cool back to -78”C, add 0.2 mL (1.6 mmol) of boron trifluorlde dlethyl etherate, and stir for 15 mm (see Note 16) 4. Add dropwise 1.O mL of 1 6 M n-BuLi to the above reaction mixture, and allow to warm gradually to 0°C. Quench the reaction by addition of 20 mL of saturated aqueous ammonium chloride, extract with three 15-mL portions of ethyl acetate, dry the combined organic phase over Na,S04, and concentrate 5 Purify the residue by column chromatography elutmg with a l/l mixture (v/v) of ethyl acetate/heptane. Concentrate the appropriate fractions You should get about

502

Sasakl 0 44 g (63% yield) of 18 as a viscous 011 R,0.22 (EtOAc/heptane: l/l) (see Note 17) ‘H NMR (CDC13) (a mixture of two C-3 dlastereomers) 6 7 85 (m, 2H), 7 68-7 50 (m, 3H), 7 4&7 15 (m, 5H), 4 51 (s, lH), 4.42 (s, 2H), 3 90 (m, lH), 3 76 (m, 2H), 3 62 (m, 2H), 3 40 (m, lH), 2 20 (m, lH), I 94 (m, lH), 140 (s, 9H), 13C NMR(CDC1,) 6 154 5, 153 7,139.4, 133 9, 129 3, 128 2, 128 1, 127 4,

80 9,80.4,73

0,67 6,66 3,65.3,63

9,63 4,59.2,58

6,58 4,294,28

2

3.3.7. Preparation of @,5S)-N -(tert-Butoxycarbonyl)2-[(Benzyloxy) Me thy&5- Hydroxyme thylpyrrol/dme 79 1 Add 1.O g (2 6 mmol) of 6% Na-Hg to a solution of 380 mg (0 82 mmol) of 18 and 0 37 g (2.6 mmol) of Na*HPO, m 10 mL of HPLC-grade methanol (see Note 18). 2 Stir the mixture vigorously for 1 h at room temperature, remove mercury by decantmg the solution, and concentrate. 3 Dissolve the residue m 20 mL of dlchloromethane and wash with four 5-mL portions of brme Dry over MgS04, and concentrate (see Note 8) 4 Purify the crude product by flash chromatography on silica gel elutmg with a l/l mixture (v/v) of ethyl acetate/heptane Concentrate the appropriate fractions You should get about 230 mg (87% yield) of 19 as a viscous 011(see Note 19) Rf0.25 (EtOAc/heptane* l/l) [~]~~o = 41” (c 1.0, chloroform). ‘H NMR (CDCl,) 6 7 3 1 (m, 5H), 4 5 1 (s, 2H), 4 05-3 90 (m, 2H), 3 743 48 (m, 3H), 3.4&3 30 (m, lH), 2.18-l 80 (m, 4H), 1 45,l 50 (2s, 9H); 13C NMR (CDCl,) 6 156 2, 138 3, 128 5, 127 7, 127 6, 80.5, 73 3, 70 2, 69 8,67 6,60 4,58 0, 28 5, 27 2, 26 7

3.3.8. Preparation of (2S,SS)-N-(tert-Butoxycarbonyl)5-(Benzyloxy)Methlyproline 20 1 Add 0.72 mL (2 Eq) of Jones reagent to a solution of 225 mg (0 7 mmol) of 19 m 15 mL of acetone, and stir the mixture very vigorously for 1 h at room temperature 2 Evaporate acetone under reduced pressure, and dilute the residue with 10 mL of ethyl acetate 3. Wash the organic phase with three IO-mL portions of water, and extract with 1 mL of 1 NNaOH 4 Extract the resultant alkalme solution with 20 mL of ethyl acetate, and then adJust the pH to about 2 0 usmg 1 NHCl 5 Extract the preclpltate with three 5 mL portions of dlchloromethane, wash the combmed organic extracts with brine, dry over MgS04 and concentrate to dryness You should get about 200 mg (85% yield) of 20 as a viscous 011.R, 0 380 40 (CHC13/MeOH* 9/l) [a] 250 = 49" (c 1 2, MeOH) ‘H NMR (CDC13) (two conformers) 6 7 30 (br s, 5H), 4.53 (s, 2H), 4 38-4 00 (m, 2H), 3 65-3 30 (m, 2H), 2 50-2 20 (m, lH), 2 20-l 88 (m, 3H), 1 43 (s, 9H), 13C NMR (CDC13) 6 178 6, 177.5, 154.4, 153 8, 1382, 138.0, 128 2, 127 5, 127 3, 805, 80 3, 73 0, 70.5, 59 7, 59.4, 57 3, 57 2, 28.8,28 2, 28 1,27 5,27 1,26 2.

3.4. Synthesis of Vans-4-Substituted Proline 30 Enantlospeclfic synthesisof 4-substitutedprohne startmgfrom an analogof choral synthon (R)-1 andeasily avallable (I?)-2,3-isopropyhdeneglycer-aldehydeISdemon-

Enantiopure Prolines

BOON

L

503

SO2Ph

SQPh Nati. THF. W’C, 3Omm m-----

BOCN L,=, En

6

SO2Ph TFA. rt. Ih

11

BnN L i HH

21

OBn

OBn

22

CHO

TSO

HO 1N HCI. MeH IRh. rt -

NaBH(OAc), CI(CHJ2CI

HO,, OBn

TsCI. py 0°C. 17h ---+

Hot* K2C0,. wet DMF overnlghl.

Bn

En

Bn

23

24

25

Ta(OiPr& THF. -78°C. 2Cmm KHMDS. -78OC. Zh

6% Na48

rt

OH Bn

Boc

27

28

OH

30

Figure 4

stratedm Fig. 4. Use of the Boc group for N-protection m this seriestumed out to be unsuccessful in the pyrrolidme ring formation step. As a consequence,the amine group is protectedby the benzyl group from the beginning. Preparation of the startmg material 22 is carried out by N-and 0- double benzylation of 11 followed by removal of Boc group with TFA in 70% overall yteld. The reductive alkylatron of 22 with (R)-2,3-isopropylideneglyceraldehyde using sodium macetoxy-borohydnde tn 1,Zdichloroethane (17) provides 23, which is in turn converted to the key mtermedtate dial 24 in 90% overall yield. In order to obtain a trans-bsubstituted proline, mono-tosylation is affected at 3’ posttion to give 25 m 70% yield. It may suffice to saythat apnori mono-tosylation at 2’ position (three-stepprocedure from 24) eventually leads to a cis-4-substituted proline. It is possible to use (Q-2,3tsopropylideneglyceraldehyde(18,191m place of its (R)-enanttomer in the reductive alkylation step to obtain the C-2’ epimer of 24, which eventually leads to the (45’)diastereomerof the final compound 30 (seeNote 20). Epoxide 26 is formed in 90% yield by treatment of 25 with potassiumcarbonate in DMF. Intermolecular epoxide opening of 26 via the sulfonyl amon takes place exclusively m exo-5 manner furnishing 27 in 70% yield as a single dtastereomer (see Note 21). Catalytic

504

Sasaki

hydrogenation under pressure(60 psi) m the presence of dl-tert-butyl dicarbonate affords Boc-protected pyrrohdme dlol28 in 93% yield. Desulfonylatlon of 28 with 6% Na-Hg proceeds smoothly to give 29, which is then subJectedto Jones oxidation followed by treatment with dlazomethane to provide dlmethyl ester of trans-4-carboxyllc-L-prolme 30 in 63% overall yield (see Note 22). 3 4.1. Preparation of (a)- I-O-Benzyl2-N-[(tert-Butoxycarbony/)Benzyl]Amu7o3-Phenylsulfonyl-l-Propanol21 Add dropwlse with vigorous stirrmg 6 72 g (2 1 33 mmol) of 11 m 35 mL of THP to a suspension of NaH (60% in mineral oil) (2 0 g, 50 n-m-101)m 70 mL of THF at O”C, and stir the mixture for 30 mm at 0°C. Add 7 67 g (44 80 mmol) of benzyl bromide and 80 mg of n-Bu4NI to the above reaction mixture and stir for 8 h at 0°C and overnight at room temperature Quench the reaction by addition of 10 mL of water, and extract with three 50-mL portions of dlethyl ether Wash the combined ether extracts with 20 mL of brine, dry over Na2S04, and evaporate the solvent Pun@ the residue by flash chromatography on slhca gel elutmg with a l/5 mixture (v/ v) of ethyl acetate/heptane You should get about 9 12 g (86% yield) of 21 Rf 0.25 (EtOAc/heptane* l/5). [a]25D=-25.7” (c 1 0, CHCI,) Characterlstlc peaks ofNMR* ‘H NMR (CDC4) 6 7.9-7 8 (m, 2H), 7 7-7 5 (m, 3H), 7.4-7 1 (m, IOH), 4 64-4 58 (m, lH), 1 37 (s, 9H), 13C NMR (CDCl,) 6 72 2,69 9,55 0,53 2,52 1,27 8.

3 4.2. Preparation of (2R)- 1-O-Benzyl2-N-(Benzyl)Am~no-3-Phenylsulfonyl1-Propanol22 1 Dissolve 4.96 g (10.0 mmol) of 21 m 10 mL of a mixture (v/v) of dlchloromethane/tnfluoroacetic acid, and stir the mixture for 1 h at room temperature 2 Remove the solvent m vacua, dissolve the residue m 15 mL of 30% aqueous potassmm carbonate and extract with three 30-mL portions of ethyl acetate. 3 Wash the combined organic extracts with 10 mL of brine, dry over Na2S04, and evaporate the solvent 4. Purify the residue by flash chromatography on sihca gel eluting with a l/3 mixture (v/v) of ethyl acetatejheptane You should get about 3 35 g (85% yield) of 22. Rf 0 27 (EtOAc/heptane l/3). [a1200 = -21 3” (c 1 .O, CHCl,). Characterlstlc peaks of NMR: ‘H NMR (CDCI,) 6 4 43 (s, 2H), 3 68 (s, 2H) 3.593 49 (m, 2H), 3 44-3 23 (m, 2H), 2 02 (br s, 1H); 13C NMR (CDC13) 6 72 7, 70 1,57 5,52 0,50 7

3.4.3 Preparation of (2F$25)-I-O-Benzyl2-N -[(2’, 3’-lsopropylidenedioxypropyl) Benzyl]Ammo-3-Phenylsulfonyl1-Propanol23 1 Add 670 mg (3.16 mmol) of sodiumtriacetoxyborohydrldeto a solution of 300 mg (2 30 mmol) of (R)-2,3-O-lsopropyhdeneglyceraldehyde

and 900 mg (2 27 mmol)

Enantiopure Prolines

505

of 22 in 10 mL of 1,2-dichloroethane Stir the reaction mixture overnight at room temperature under a nitrogen atmosphere 2 Add 5 mL of saturated aqueous sodium bicarbonate solution, and extract with three 15 mL portlons of diethyl ether 3. Wash the ether extracts with 5 mL of brine, dry over Na$O,, and evaporate the solvent 4 Purify the residue by column chromatography on slhca gel elutmg with a l/3 mixture (v/v) of ethyl acetate/heptane. You should get about 1.10 g (96% yield) of 23 RfO 33 (EtOAc/heptane* l/3) [a] 25,, = +1 .I” (c 1 3, CHCl,). Characterlstlc peaks of NMR. ‘H NMR (CDCl,) 6 7 88-7.85 (m, 2H), 7 67-7 49 (m, 3H), 7 36 7 15 (m, lOH), 4 40 (s, 2H), 4.02 (q, J = 6.20 Hz, IH), 3 92 (dd, J= 8 01,6.14 Hz, IH), 3 79 (d, .I= 14 19 Hz, lH), 3.66 (m, 2H), 3.59-3 27 (m, 5H), 2 74 (dd, J= 13 29,6.46 Hz, IH), 2 68 (dd, J= 13.29, 5 70 Hz, lH), 1.31 (s, 3H), 1 28 (s, 3H), 13CNMR (CDC13) 6 139.4, 138 9, 137 6, 133.1, 128 8, 128 1, 127 9, 127 8, 127 3, 127 1, 126.6, 108.4,74 5, 72.6, 71 0,67.7, 55 7, 54.7, 53 7, 53 2,26.4,25 1

3 4.4. Preparation of (2R,25)- 1-O-Benzy/2-N -[(2’, 3’Dihydroxypropyl)Benzylj Am/no-3-Phenylsulfonyl- 1-Propanol24 1 Dissolve 950 mg (1.86 mmol) of 23 m 3 mL of 1 N HCl/methanol, and stir the solution at room temperature for 18 h. 2 Evaporate the solvent, dissolve the residue m 10 mL of 30% aqueous potassmm carbonate and 20 mL of ethyl acetate, and extract the aqueous phase with three 10 mL portions of ethyl acetate 3. Wash the ethyl acetate extracts with 10 mL of brine, dry over Na2S04, and evaporate the solvent 4 Purify the residue by flash chromatography on silica gel elutmg first with a l/l, and then with a 3/l mixture of ethyl acetatelheptane You should get about 820 mg (94% yield) of dlol 24. R, 0 3 1 (EtOAc/heptane* 3/l) [IX]~~~ = -52 6” (c 1 0, CHCl,) Characteristic peaks of NMR* ‘H NMR (CDC13) 6 4 41 (s, 2H), 3 82 (d, J= 13 63 Hz, IH), 3 68 (d,J= 13 63 Hz, lH), 3 17 (dd, J= 14.32,3 76 Hz, IH), 2 75 (dd, J= 13.42, 9.60 Hz, lH), 2.57 (dd, J= 13 42, 3.37 Hz,lH), 13C NMR (CDCl,) 6 72 9, 68 3, 68 0, 64.1, 56 0, 54.7, 54 4, 52 2.

3 4.5. Preparation of (2R,25)-7-O-Benzyl2-N -{[(2’- Hydroxy, 3’-p - Toluenesulfonyloxy) Propyl]Benzyl}Amlno-3-Phenylsulfonyl1-Propanol25 1. Dissolve 828 mg of dlol24 (1 76 mmol) in 18 mL of pyridme, stir the solution at OT, and add 336 mg ofp-toluenesulfonyl chloride (1 76 mmol) m one portlon 2. Stir the reaction mixture for 17 h at OT, then dilute the mixture with 20 mL of water, and extract wrth three 30-mL portions of ethyl ether 3 Wash the combined ether extracts with three lo-mL portions of saturated aqueous copper sulfate, 5 mL of water, and 5 mL of brine Dry over Na2S04, and evaporate the solvent

506

Sasaki

4. Purify the residue by flash chromatography on silica gel eluting with a l/l mixture (v/v) of ethyl acetateiheptane You should get about 753 mg (68% yield) of mono-tosylate 25. R, 0.30 (EtOAc/heptane. l/l) [cx]*~~ = -35” (c 1 2, CHCl,) Characteristic peaks of NMR: ‘H NMR (CDCl,) 6 4 42 (s, 2H), 3 92 (d, J = 5 08 Hz, 2H), 3.76 (d, J= 13 68 Hz, IH), 3 64 (d, J= 13 68 Hz, lH), 3.33 (dd, J= 14 30,7 83 Hz, lH), 3.12 (dd, J= 14.30,3.89 Hz, lH), 2 71 (dd, J= 13.56,4 10 Hz, lH), 2.63 (dd, J= 13.56, 8 33 Hz, lH), 2 42 (s, 3H), 13C NMR (CDCI,) 6 72 7, 71.3, 68 2,66.0, 55 9, 54 6, 54 5,52.6,21.1

3.4.6 Preparation of (2R,25)-I-O-Benzyl2-N -[(2’, 3’- Epoxypropyl) Benzyi] Amino-3- Phenyisuifonyi- 1-Propanoi 26 1 Dissolve 627 mg (1 0 mmol) of 25 in 5 mL of dimethylsulfonamide in the presence of 90 pL of water 2 Add 417 mg (3 0 mmol) of K2C03 to the above solutton, and stir the mixture for 24 h at room temperature. 3 Dilute the reaction mixture with 10 mL of water, and extract with three 20-mL portions of diethyl ether 4. Wash the combined organic extracts with 10 mL of brme, dry over Na2S04, and evaporate the solvent 5 Purify the residue by column chromatography on slica gel elutmg wtth a l/3 mixture (v/v) of ethyl acetatelheptane You should get about 403 mg (89% yteld) of 26 Rf 0 35 (EtOAc/heptane l/3) [a] 250 = -2 lo (c 1 0, CHCI,) Characteristic peaks ofNMR ‘H NMR (CDCl,) 6 4 44 (s, 2H), 3 89 (d, J= 14 13 Hz, 2H), 3 65 (d, J= 14 13 Hz, IH), 3.46 (dd,J= 14.24,6 17 Hz, lH), 3 36 (dd, J= 14 24,4 80 Hz, lH), 2 85 (m, 3H), 2.84 (dd, J= 15 17,3.86 Hz, lH), 2.61 (dd, J=4 92,3.93 Hz, IH), 2.52 (dd, J= 15 17, 7.10 Hz, lH), 2.34 (dd, J= 4.92, 2 50 Hz, lH), 13C NMR (CDC13) 6 73.2, 70 6, 56.3, 55 5, 54.9, 53 4,51 4,45 6

3.4.7. Preparation of (2R,4R)-N-(Benzyi-)-2-[(Benzyioxy)Methyi]3-(Phenylsuifonyi)-4-iiydroxymethyipyrroirdine 27 1 Add 2 10 mg (0 7 mmol) of tuaruurn (IV) isopropoxtde to a stirred solution of 168 mg 0 36 mmol) of 26 m 8 mL of THF at -78°C and stir the mixture for 10 mm at the same temperature 2 Add dropwtse with vigorous stirring 1 5 mL (0 7 mmol) of potassmm bis(trimethylsilyl)amrde (0.5 M solution m toluene), and stir the reaction mrxture for 2 h at -78’C 3 Quench the reaction by addttton of 2 mL of saturated aqueous NH&l solution and extract the reactton with three 20-mL portions of dtethyl ether 4. Wash the combined orgamc extracts wtth 10 mL of brme, dry over Na2S0, and evaporate the solvent. 5. Purify the residue by preparative thin-layer chromatography on silica gel elutmg with a l/l mixture (v/v) of ethyl acetate/heptane You should get about 120 mg (71% yield) of 27 Rf 0.32 (EtOAc/heptane. l/l) [a]25t, = -92” (c 0 7 CHC13)

Enantiopure Prolines

507

Characteristic peaks of NMR: IH NMR (CDCl,) 6 4.35 (dd, J= 12.45,9.86 Hz, 1H),417(d,J=1200Hz,1H),404(d,J=1200Hz,1H),3.95(d,J=1281Hz, lH), 3 85 (dd, J= 12 45,4.69 Hz, lH), 3.80 (dd, J= 8 04,3 10 Hz, lH), 3 66 (d, J=1281Hz,1H),279(dd,J=9.86,433Hz,1H),273(dd,J=1164,795Hz, lH), 13C NMR (CDCl,) 6 72.8,70.8,66.6, 65 4,60 1, 59.5, 55.45,45 7

3.48. Preparation of (2R,4R)-N-(tert-ButoxycarbonyI)2-(Hydroxymethyl)-3-(Phenylsulfonyl)-4-Hydroxymethylpyrrolidine

28

1. Dissolve 100 mg (0.22 mmol) of 27 and 100 mg (0.45 mmol) of dl-tert-butyl dlcarbonate m 5 mL of methanol, and add 10 mg of palladium on activated carbon (Pd 10%) 2. Treat with stirring the above mixture with hydrogen at 60 PSI for 5 h 3. Remove the catalyst by filtration and the solvent by evaporation. 4. Purify the crude product by preparative thin-layer chromatography on slhca gel eluting with a 95/5 mixture (v/v) of dlchloromethane/methanol You should get about 76 mg (93% yield) of 28. Rf0.50 (CH&l,/MeOH: 95/5) [IX]*~,, =-86.4” (c 1 0, CHC13). Characteristic peaks of NMR: *H NMR (CDCl,) 6 4.34-4.26 (m, lH), 4 12-4 09 (m, lH), 3 94-3.70 (m, 2H), 3 163 10 (m, lH), 146 (s, 9H); 13C NMR (CDCI,) 6 80.6, 65.2, 62 9, 61 2, 60 1,48 7,43 7, 28 2

3.4.9. Preparation of (2S,4R)N -(tert -Butoxycarbonyl)-2,4-Dihydroxymethylpyrrolidine

29

1 Add 230 mg (0 6 mmol) of 6% Na-Hg to a solution of 75 mg (0.2 mmol) of 28 and 86 mg (0 6 mmol) of Na,HPO, in 3 mL of HPLC-grade methanol 2 Stir the mixture vigorously for 2 h at O”C, remove mercury by decanting the solution and concentrate 3 Dissolve the residue in 6 mL of dichloromethane, and wash with three I-mL portions of brme, dry over Na2S04, and concentrate. 4. Purify the crude product by column chromatogaphy on silica gel elutmg with a 9515 mixture (v/v) of dlchloromethane/methanol. You should get about 38 mg (83% yield) of 29. Rf 0 35 (CH2C12/MeOH. 95/5). [cx]*~~ = -32” (c 0 7, CHC13) Characteristic peaks of NMR* ‘H NMR (CDCI,) 6 4 65 (br s, 1H), 4 00 (m, 1H), 3 20 (dd, J= 10.88,7.47 Hz, lH), 2.50-2.35 (m, lH), 1 85-l 70 (m, 2H), 1 46 (s, 9H); 13CNMR (CDCl,) 6 67 3, 64 1, 59.3, 49.9, 39.3, 30.8, 28 4

3.4.70. Preparation of @,4R)-N-(tert-Butoxycarbonyl)-4-Carboxyhc Proline Dimethyl Ester 30 1 Dissolve 23 mg (0 1 mmol) of 29 m 2 mL of acetone, add 200 pL of Jones reagent, and stir the mixture for 2 h at room temperature. 2 Concentrate the reaction mixture at about IO’C under reduced pressure, dissolve the residue in 1 mL of water, and extract with three 2-mL portions of ethyl acetate, dry over Na2S04. 3 Concentrate the solution under the reduced pressure, treat the residue with dlazomethane in ether, and evaporate the solvent

508

Sasakl

4. Purify the crude product by preparative thin-layer chromatogaphy on sthca gel elutmg with a l/l mixture (v/v) of ethyl acetateheptane You should get about 18 mg (63% yteld) of 30 Rf 0 62 (EtOAc/heptane l/l) [a12*o = -42” (c 0.5, CHCl,) NMR (two conformers). *H NMR (CDC&) 6 4 45 (dd, J= 8 79,2.62 Hz, 0.45H),

4 35(dd,J=S 71,3 84Hz,O 55H),3.87-3.54(m,2H),3.74(~,3H),3

72(s,3H),

3 343 14 (m, IH), 2 57-2 37 (m, lH), 2 262 13 (m, lH), 146, 1.41 (ds, 9H), 13CNMR (CDCl,) 6 58 6,58.4,52.2,48.5,41 8,41.0,33 2,32.5,28 3,28 2. 13C NMR (CDCl,) after removal of the Boc group by TFA. 6 1712, 168 7, 59 1, 53 8, 52.9,47 5,41.3, 31 4.

3.5. Discussion The chit-al educts (R)-1 and o-1 can be easily prepared from N-Boc-L-serine methyl ester on a large scale, and both (R)- and (S)-2,3-O-isopropylidene glycerols and glyceraldehydes are available at reasonable prices. Thus, the synthetic methodology described above serves as a convenient means to provide any one of the four stereoisomers of 3-, 4-, and 5-substituted prolmes m enantiomerically pure form. Synthesis of truns-3-substrtuted prolme is particularly facile. Though the key intermediate 4a is contaminated with its epimer 4b, reductive desulfonylation provides the single desired product 5a, which IS transformed to the final prolme derivative in a stratghtforward manner. On the other hand, cis-3-substituted proline is prepared in ester form only. Whether or not this type of ammo acid ester can be incorporated into peptides by means of the corresponding aztde remains to be seen. In this chapter, only one example has been demonstrated for the synthesis of a 5-substituted prolme. However, the present methodology provides easy accessto both c&and truns-5-substituted prolmes m enantiopure form c&9). Although the widely available, truns-4-hydroxy-L-prolme has been employed m the synthesesof many 4-substituted prolme derivatives (20), yet only a few efficient synthetic protocols have been reported for the asymmetric synthesis of 4-substituted prolines (21-24). According to the present synthetic protocol, transformation of the hydroxymethyl group of 27 to another functionality will provide a new route to a variety of 4-substituted enantiopure prolmes.

4. Notes 1 In this series of asymmetric syntheses, i3C NMR is of greater utility than ‘H NMR for the identification of the reaction products, except for the characteristic proton peaks of sulfonyl, ally& and Boc groups 2 Although (R)-1 and its antipode, Q-l, are commercially available, it is very easy to prepare on 100-g scale from Boc-L-serme methylester (10) 3 During the addition of the first equivalent of n-BuLt, the solution stays colorless (lithiation of the carbamate). On addttion of the second equivalent of the base, the solution assumes a yellow-orange color (formation of lithiated sulfonyl

Enantiopure Prolines

4

5 6. 7

8 9

10.

11

509

amon) In this respect, (R)-1 can be used as a standard against which n-BuLl solution can be titrated Though this reagent IS very moisture-sensitive, washing the crude product m dichloromethane with water prior to chromatographic purification 1spermissible It can be stored at -20°C for a few days. Rp for 3a and 3b are the same m this solvent system Retention time, 4a; 9.45 mm, 4b; 10.75 mm Commercially available (In 4-5% Na content from Strem Chemicals [Newburyport, MA] 80-l 105). 6% Na-Hg can be prepared m the followmg manner Add 7 8 g of mercury to 0 5 g of thin film (about l-2 mm thick and 1 cm2) of freshly cut sodium placed in a 500-mL Pyrex round-bottomed flask under a mtrogen atmosphere. Swirl the flask gently while applymg heat to its bottom by the aid of a heat gun The amalgam is formed mstantaneously wlthm a few minutes. Since the reaction is extremely exothermic, use thermal protectlon gloves for handlmg the flask The amalgam prepared by this method can then be stored under a mtrogen atmosphere for about a month. Use of ethyl acetate as an extraction solvent results m the formatlon of small amount of acylated product In a 13C NMR of 5a, there is only one peak at 37 4 ppm, which 1s assigned as methylene carbon of ally1 group of 5a Dlastereomer 5b shows this peak at about 33 8 ppm. We postulate that this stereochemical outcome IS owmg to a preferred pyramldal sulfonyl carbamon configuration rather than mtramolecular chelation of the lithium atom in a hthlosulfone to oxygens m the nelghbormg functionality. The tetrahydropyranyloxymethyl and sulfonyl groups may be oriented m trans-configuration to each other so that the incoming alkyl group approaches from the same face with tetrahydropyranyloxymethyl group (II) 4b mp 122-122 5°C. [c~]~~o= +7 9” (c 1.O, MeOH) ‘H NMR (CDCl,) 6 7 94 (d, J= 17 5 Hz, 2H), 7 68 (t, J= 7.5 Hz, lH), 7.58 (t, J= 7.5 Hz, 2H), 6 18-5 94 (m, IH), 5.17 (d, J= 10.0 Hz, lH), 5.16 (d, J= 17.5 Hz, IH), 4.35 (m, lH), 3 95-3 55 (m, 3H), 3 41-3 21 (m, lH), 2.92-2.44 (m, 3H), 2 24-2.10 (m, lH), 1.45 (s, 9H); 13C NMR (CDC13) 6 136.3, 134 2, 132.6, 130.2, 129.2, 119.2, 80 6, 73 4, 63.3, 62 6,45 0,34.8, 30.2,28 4

12. Eplmerlzatlon at C-3 posltion during desulfonylatlon can be observed in the 13C NMR spectrum of Sb/Sa mixture. CH2CH=CH2 6 37.5 ppm for 5a, 33.8 ppm for Sb. 13. Retention time, (R)-(+)-a-methylbenzylamide of 6a; 9.66 mm, (R)-(+)-amethylbenzylamlde of 6b; 10 80 min. 14 Obtained m 85-92% yield according to the method for the preparation of glycldyl trlflate (25). [cx]~~~ = +0.8” (c = 2.4, CHCI,). ‘H NMR (CDC13) 6 4 48-4 36 (m, 5 H), 4.13 (ddd, J= 9.0 Hz, 8 3 Hz, 1.5 Hz, 1 H), 3.87 (ddd, J= 9.0 Hz, 4 8 Hz, 1.9 Hz, 1 H), 1 45, 1 37 (two s, 6 H, ketal-CH,), 13CNMR (CDCl,) 6 10 8 (ketal-Q, 75.3 (cH20Tf), 72 7 (ketal-CH), 65 5 (ketal-CH,,, 26 5 (ketalGH3), 25 0 (ketal-CH3).

510

Sasaki

15 Formation of epoxide 17 can be confirmed by TLC (R, 0.35 [EtOAc/heptane l/ l] for 16 and about 0 27 for 17) i3C NMR (CDCl,) of 17 (a mixture of two C-3 diastereomers) 6 134 0, 129.3, 128 8, 128 4, 128 2, 127.7, 79.9, 73 0, 69 7,69 2, 62 9, 61 5,50 5,50 1,49 5,49 0,48 1,47 6,29.6, 28 7, 28 3. 16 Later, it was found that titanmm(IV) isopropoxide instead of boron trifluoride diethyl etherate gave better yield for the final product. 17 There are two byproducts that are more polar than 18. One of them is oxazohdmone, and the other one is presumed to be a cychc or acychc amme without Boc-protecting group 18. Desulfonylation with SmI, m presence of HMPA also gives 19 in 65% yield (16) 19 Enanttomertc purity of 19 was determined to be more than 99% by convertmg this compound to (2S,SS)-N-(tert-butoxycarbonyl)-2,5-dlhydroxymethylpyrrohdme by catalytic hydrogenation and comparing with the authentic trans dial by HPLC Retention time 8.54 mm (5 06 mm for czs isomer) (8). 20 In this context, the C-2’ epimer of 26 can be prepared from 24 m the followmg four step sequence a Protectron of the primary alcohol group. b Tosylation or mesylation of the secondary alcohol group c Removal of the alcohol protectmg group d Epoxide formation 21 The C-3 stereochemistry of compound 27 is tentatively assigned as (S) on the basis of ‘H-‘H nuclear Overhauser effect (NOE) spectroscopy (NOESY) experiments. The spectral details of these experiments will be disclosed elsewhere 22 Chemical yields for compounds 25 and 30 were not optimized

References 1 Bridges, R J , Stanley, M. S., Anderson, M W , Cotman, C W , and Chamberlm, A R. (1991) Conformationally defined neurotransmnter analogues Selective mhibmon of glutamate uptake by one pyrrohdme-2,4-dicarboxylate diastereomer J A4ed Chem 34717-725 2 Humphrey, J. M., Bridges, R J., Hart, J A., and Chamberlm, A R. (1994) 2,3Pyrrolidmedrcarboxyles as neurotransmrtter conformer mimics* Enantioselective synthesis via chelation-controlled enolate alkylation J Org Chem 59,2467-2472 3. Hashimoto, K., Yamamoto, O., Horikawa, M., Ohfune, Y , and Shirahama, H (1994) Synthesis and neurobiological actions of pyrrohdme-2,3-dicarboxyhc acids (PRDA) Conformationally restricted analogues of L-aspartate Bzoorg. A4ed Chem Lett 4, 1851-1854 4 Yu, K -L , RaJakumar, G., Srivastava, L. K , Mishra, R. K , and Johnson, R L (1988) Dopamme receptor modulationby conformationally constrained analogues of Pro-Leu-Gly-NH2 J Med. Chem 31, 1430-1436 5. Holladay, M. W , Lm, C. W., May, C. S., Garvey, D S , Witte, D G., Miller, T R , et al. (199 1) trans-3-Propyl-L-prolme is a highly favarable, conformatronally restricted replacement for methiomne m the C-terminal tetrapeptide of cholecys-

Enantiopure Prolines

6

7

8. 9.

10.

11

12 13

14.

15 16

17

18

19. 20.

511

tokmm Stereoselective synthesis of 3-allyl- and 3-n-propyl-L-proline derivatives from 4-hydroxy-L-prolme J Med Chem. 34,455-457. Sasakl, N. A., Pauly, R., Fontaine, C , Chlarom, A , Rlche, C., and Potter, P. (1994) Enantloselectlve syntheses of (2S,3$)- and (2R,3R)-pyrrolldme-2,3-dlcarboxylic acids: Conformatlonally constrained (s)- and (R)-aspartlc acid analogues Tetrahedron Lett 35,24 l-244 Pauly, R., Sasakl, N A., and Potter, P (1994) A versatile method for the synthesis of(s)- or (R)-cycloalkylglycine, (S)- or (R)-N-Heterocychc and a&unsaturated N-heterocyclic a-ammo acids. Tetrahedron Lett 35,237-240. Sasakl, N A., and Sagnard, I (1994) A novel method for chlrospeclfic synthesis of 2,5-disubstltuted pyrrolidines. Tetrahedron 50,7093-7108. Dockner, M., Sasakl, N. A , and Potter, P. (1996) Versatile synthesis of enantlomerically pure trans-2,5-dlsubstltuted pyrrolldines Heterocycles 42, 529-532 Sasakl, N. A., Hashimoto, C., and Potier, P. (1987) A novel approach to the synthesis of optically pure non-protein a-ammo acids m both L- and o-configurations from L-serme Tetrahedron Lett. 28,606%072. Sasakl, N A , Dockner, M , Chlarom, A , Rlche, C , and Potter, P (1997) A novel stereodlvergent synthesis of optically pue cis- and trans-3-substituted proline derivatives J Org. Chem 62,765-770. Subramaman, P K., and Woodard, R. G (1987) Synthesis of (R)- and (S)-lammo[2,2-*H2]cyclopropane-1 -carboxylic acids J Org Chem. 52, 15-l 8 Chung, J Y. L., Wasicak, J. T , Arnold, W. A , May, C. S , Nadzan, A. M , and Holladay, M W. (1990) Conformationally constrained ammo acids. Synthesis and optical resolution of 3-substituted prolme denvatlves J Org Chem. 55,270-275. Dockner, M , Sasakl, N A, Rich, C., and Potter, P. (1997) Enantiospeclfic synthesis of 2,5-disubstltuted plperldme derivatives. Synthesis of (+)-pseudoconhydrme Llebigs Ann./RecuelE 1267-1272. VedeJs, E., Engler, D A., and Mlllms. M. J (1977) Reactive trlflate alkylatmg agents. J Org Chem. 42,3 lo%3 113 Kunzer, H., Strahnke, M., Sauer, G., and Wlechert, R (1991) Reductive desulfonylatlon of phenyl sulfones by samarium (II) lodide-hexamethylphosphoric trlamlde Tetrahedron Lett 32, 194%1952 Abdel-Magld, A F., Maryanoff, C.A., and Carson, K. G. (1990) Reductive ammatin of aldehydes and ketones by using sodium tnacetoxyborohydrlde Tetrahedron Lett 39,5595-5598 Jung, M. E and Shaw, T J (1980) Total synthesis of (R)-glycerol acetomde and the antiepileptic and hypotenslve drug (-)-y-ammo-P-hydroxybutyrlc acid (GABOB). Use of vltamme C as a choral starting material J Am Chem Sot 102, 6304-6311 Takano, S., Numata, A., and Ogasawara, K (1982) A simple synthesis of (R)glycerol acetomde from ascorbic acid. Heterocycles 19,207-208 Remuzon, P (1996) Trans+hydroxy-L-proline, a usetil and versatile choral starting block Tetrahedron 52, 13,803-13,835

512

Sasakl

21 Koskmen, A M P. and Rapoport, H. (1989) Synthesis of 4-substttuted prolines as conformationally constrained ammo actd analogues J Org Chem 54, 1859-l 866 22 Saucy, F Wermc, D , and Beaulieu, P (1991) Preparation of 4-alkylprolmes by mtramolecular radtcal cychzation of chiral serine derivatives. J Chem Sot Perkm Trans 1,2885-2887. 23. RoJas, A. (1994) In Utlllsatzon de l’acrde pyroglutamlque comme source de chzralztg en synthbse (D S.Thesis ) Unwerstte de Paris-Sud, France pp 77-90

24 Ezquerra, J , Pedregal, C , Yruretagoyena, B., Rubto, A , Carreiio, M. C , Escrtbano, A , and Ruano, J L G. (1995) Synthesis of enanttomerically pure 4substituted glutamrc acids and prolmes: General Aldol reaction of pyroglutamate lactam hthmm enolate mediated by Et,0 BF, J Org Chem 60, 2925-2930

29 Synthesis of Aminobenzoic Acid-Based Nonpeptide Templates Applications in Peptidomimetic Kimberly

Drug Discovery

S. Para and Tomi K. Sawyer

1. Introduction Ammobenzoic acid-based congeners are emerging as a useful class of nonpepttde templates for appltcations m peptidomtmetic drug discovery. Exemphfytng such compounds are recently described antagonists or mhtbttors of several receptor and signal transductton protein targets, mcludmg, integrin gpIIbIIIa receptor antagonists (I), Ras famesyl protein transferase mhibttors (2), Src SH2 domain antagonists (3), and growth hormone-releasing peptide (GHRP) receptor agonists (4). Representative examples of these compounds are shown in Fig. 1 This chapter will focus on synthetic intermediates of ammobenzoic acid analogs as specifically used in the design of novel peptidomtmetic antagonists of Src SH2 (3,5,6). Structural variations of ammobenzotc acid-based templates include those exemplified below (Fig. 2) as ring-modified (I), and further substituted N-homologated (II) and/or C-homologated (III) derivatives. Such ammobenzotc acid-based nonpepttde templates are important to structurebased design as well as combmatorial chemistry strategies that are focused on pepttdomtmetic drug discovery (7).

2. Materials Chromatography 1 Thin-layer chromatography (TLC) plates-EM Science Silica Gel 60 FZs4Plates. 2 TLC development systems--components were vtsuahzed either by potassium permanganate stain, UV light (250-400 nm), 12, phosphomolybdic acid stain or ammonium molybdate stain From

Methods m Molecular Medune, Vol 23 Peptrdomrmehcs Protocols Edlted by W M Kazmlerskl OHumana Press Inc , Totowa, NJ

573

Para and Sawyer Target bgand

Targer Ltgand

lntegnn Receptor Ac-Cys-MeArg-Gly-Asp-&NH, -

SIC SH2 Domam Ac-pTyr-Glu-Glu-Ile-NH2

Target Ligand

Target

Ras Famesyl Transferase Cys-Val-Ile-Met

GHRP Receptor

Fig 1 Chemical structures of mtegrm receptor antagomst, Ras farnesyl protem transferase mhlbltor, Src SH2 domam antagonist, and GHRP receptor agontst leads, which incorporate ammobenzolc acid-based nonpeptlde templates

I

II

In

Fig 2. Several aminobenzolc acid-based nonpeptlde templates lllustratmg the generic scope of fimctlonal group elaboration of the benzene ring, ammo, and/or carboxyl groups. 3. 4. a b

Silica gel chromatography-ICN Adsorbentlen Slhca 32-63,60 A Chromatography solvent systems 1 4 ethyl acetate-hexanes 1 4 ethyl acetate-hexanes

2.7. Reagents 1 2 3 4

for Method 3.7

5-Ammomethylsahcyhc acid Sodium hydroxide (NaOH) (1 N aqueous solution) Dloxane Dl-tert-butyl dlcarbonate

Aminobenzoic Acid-Based Nonpeptide Templates 5. 6 7 8. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Ethyl acetate. Potassium hydrogen sulfate (KHS04) (saturated aqueous solution) Sodmm chloride (NaCl) (saturated aqueous solutton). Sodium bicarbonate (NaHCO,) (saturated aqueous solution) Magnesium sulfate (MgSOJ Dimethyl sulfoxide (DMSO-d,) Potassium bromide (KBr) Ether Tetrahydrofuran (THF) 4-Methyl morpholme (NMM) 1-Hydroxybenzotriazole (HOBt) l-(3-Dimethylammopropyl)-3-ethylcarbodnmide hydrochlorrde (EDCI) Ammomum hydroxide (NH,OH) (concentrated aqueous) Cttric acid (5% aqueous solutton). Methanol (CHsOH). Cesmm carbonate (Cs&Os). WV-dimethylformamide (DMF) Cyclohexylmethyl bromide Hydrochlortc acid (HCI) (4 N gaseous m dioxane).

2.2. Reagents for Method 3.2 1 2 3 4. 5. 6 7 8 9 10 11, 12 13 14. 15 16 17 18 19. 20 21 22 23 24.

CH,OH 2-Hydroxyphenylacetic acid HCl (anhydrous gas). DMSO-d6. KBr Ethyl acetate Ether DMF NaCl (saturated aqueous solution) Potassium carbonate (K2C03) Iodomethane (CHJ) MgSO, Sodium bicarbonate (NaHCOs) (saturated aqueous solution) Hexanes Dtchloromethane (CH&l,). Aluminum chloride (AlCl,) Acetyl chloride (CHsCOCl) HCl(1 N aqueous) Cyclohexylmethyl bromide Pyrtdme Hydroxylamine hydrochloride. Cttric acid (5% aqueous solution) Triethylamme Raney-nickel.

515

Para and Sawyer

516 2.3. Reagents 1 2 3 4 5 6 7 8 9. 10 11 12 13 14 15 16 17 18 19

for Method 3.3

CH30H THF NaOH (1 N aqueous solution) Citric acid (5% aqueous solution). Ethyl acetate NaCl (saturated aqueous solution) MgSO, DMSO-d6 KBr DMF NMM HOBt EDCI NH40H (concentrated aqueous) NaHCOs (saturated aqueous solution) Hydroxylamme hydrochloride Pyridme Triethylamme Raney-nickel.

2.4. Reagents for Method 3.4 1 2 3 4 5 6 7 8 9. 10 11 12 13 14 15 16.

CH$l, N- Phenyltrifluoromethanesulfonamide Phenyltrimethyltm Lithium chloride Tetrakis(triphenylphosphme)palladmm(O) Triethylamme Ethyl acetate. NaOH (1 N aqueous solution) Citric acrd (5% aqueous solution) M@04 DMSO-d6 KBr Dioxane NH40H (1 N aqueous) NaHCOs (saturated aqueous solution) NaCl (saturated aqueous solutton)

2.5. Reagents 1 2 3 4

for Method 3.5

5-Acetylsalicyltc NMM HOBt EDCI

acid

Aminobenzoic Acid-Based Nonpeptide Templates 0

0

OH EDCI, HOBT

HzN (B0c)20 N%20H20 4

1:

- BacHti

1

~&03

H

BccH

NMM. NH.,OH

2

BOCH

3

4N HCI Dloxane

CsHlrCH2Br CH30H. DMF

5

4

Figure 3 5 6 7. 8 9 10. II 12. 13 14 15 16 17 18 19 20 21 22 23

NH40H (concentrated aqueous) NaHCO, (saturated aqueous solution) THF Crtrrc acid (5% aqueous solution) Ethyl acetate. DMF Ether HCl(1 N aqueous) NaCl (saturated aqueous solution) MD& DMSO-d6. KBr. K2C03

Cyclohexylmethyl bromtde Hydroxylamme hydrochlortde Pyrtdine Triethylamme. Raney-mckel Methanol (CH,OH)

3. Methods 3.1. Preparation of Monofunctionalized Analogs of Generic Temp/ate II from 5-Aminomethylsalicylic Acid 3 1.1. Preparation of 5-Aminomethyl2-Cyclohexylme thoxy-Benzamide 5 (Fig. 3) 3 1.1.1

5-(TERT-BlJTOXYCARBANOYLAMlNC-METHYL)2-~~YDROXY-BENZOIC ACID 2

1. To a solutron of 5-aminomethylsahcyclic acid 1 (8) (8.3 g, 49.70 mmol) m 100 mL of water and 100 mL of dtoxane at 0°C was added 1 NNaOH (54.7 mL, 54 7 mmol) The reaction was allowed to warm slowly to room temperature and stnred overnight

518

Para and Sawyer

2 The reaction was then concentrated under reduced pressure to 15 mL. Two hundred mllhhters of ethyl acetate were then added, and the reactlon was acldrfied to pH 2 with saturated aqueous KHS04 The aqueous layer was then extracted with ethyl acetate (3 x 150 mL) The ethyl acetate layers were combined and washed with saturated aqueous NaCl(1 x 150 mL) and dried over MgSO,. 3 The solution was filtered and concentrated under reduced pressure to yield a white powder The powder was collected and washed with ether, followed by drymg under reduced pressure to yield (11 5 g, 87%) ‘H NMR (400 MHz, DMSO-de) S 7 67 (s, IH), 7.38 (d, 2H), 6.91 (d, lH), 4.04 (d, 2H), 1.41 (s, 9H) IR (KBr) 3364, 3223,3061,2982,2935,2876,1670,1620,1593,1527,1491,1448,1365, 1300, 1222, 1163, 1051,906,842,794,675,599,511,468 cm-’

3

i .i

.2

(3-CARBAMOYL-+HYDROXY-6ENZYL)-CARBAMIC ESTER

ACID,

TERT-BUTYL

3

1 To a solution of 5-(tert-butoxycarbonylammo-methyl)-2-hydroxy benzolc acid (20 0 g, 74.9 mmol) in 700 mL THF at 0°C was added NMM (12 3 mL, 112 3 mmol), HOBt (15 1 g, 112 3 mmol) followed by EDCI (2 1.5 g, 112 3 mmol) and stirred for 1 h at 0°C Concentrated aqueous NH,OH (15 1 mL, 112 3 mmol), and this was then added The reaction was allowed to warm slowly to room temperature and stirred overnight. 2 The reactlon was then diluted with ethyl acetate and washed with 5% aqueous citric acid (3 x 250 mL), saturated aqueous NaHCO, (3 x 250 mL), aqueous saturated NaCl(l x 250 mL) and dried over MgSO, 3 The reaction was filtered and concentrated under reduced pressure to yield a pale yellow powder The powder was collected and washed with ether, followed by drying under reduced pressure to yield (14.0 g, 71%). ‘H NMR (400 MHz, DMSO-d6) 6 12.77 (bs, IH), 8 33 (bs, lH), 7 72 (d, IH), 7 28 (d, 2H), 6 84 (d, lH), 4 05 (d, 2H), 1.38 (s, 9H), IR (KBr) 1589, 1562, 1502, 1475, 1332, 1303, 1278,1249, 1174, 1103,1043,918,852,781,758,734,704,734,688,434 cm-’

3

1 i

3

(3-CARBAMOYL-4-CYCLOHEXYLMETHOXY-BENZYL)-cARBAMlC BUTYL

ESTER

ACID,

TEF1T

4

1 To a solution of (3-carbamoyl-4-hydroxy-benzyl)-carbamlcacld, tert-butyl ester (2 0 g, 7.51 mmol) m 15 mL CH,OH at room temperature was added Cs,C03 (2 6 g, 8.27 mmol) The reaction was stirred overnight The reactlon was then concentrated under reduced pressure. DMF (50 mL) was then added, and the reactlon was then reconcentrated under reduced pressure to chase off any remammg CH,OH 2 The residue was then suspended in DMF (15 mL), cyclohexylmethyl bromide (1.2 mL, 8 27 mmol) was then added, and the reaction was heated to 65°C for 4 h 3 The reaction was then concentrated under reduced pressure. The residue was diluted with ethyl acetate and 5% aqueous cltrlc acid The organic layer was washed with 5% aqueous cltnc acid (2 x 50 mL), 1 NNaOH (2 x 50 mL), 5% cltrlc acid (2 x 50 mL), and saturated aqueous NaCl(1 x 50 mL), and dried over MgSO,.

579

Aminobenzoic Acid-Based Nonpeptide Templates

6

7

6

HCI

l

NHzOH

9

HO.

Pyndine

Raney-Ni HCI .

CHaOH,

TEA

Figure 4 4

3

The reaction was filtered and concentrated under reduced pressure to yield a pale yellow powder. The powder was collected and washed with ether, followed by drying under reduced pressure to yreld (1 52 g, 57%) ‘H NMR (400 MHz, DMSO-d,) 6 7 68 (bs, lH), 7.52 (d, 2H), 7 36 (t, lH), 7 27 (d, 2H), 7 05 (d, lH), 4 04 (d, 2H), 3.88 (d, 2H), 1.76-1.61 (m, 6H), 136 (s, 9H), 128-l 02 (m,5H), IR(KBr) 3436, 3287, 3136, 2978, 2930, 2855, 1704, 1667, 1596, 1544, 1467, 1439, 1390, 1367, 1275, 1236, 1167, 1093, 1013,818,562 cm-’

1.1

4.

5

5-AMINOMETHYL-2-CYCLOHEXYLMETHOXY-BENZAMIDE

1. A solution of 4 N HCl m dtoxane was added to (3-carbamoyl4-cyclohexylmethoxybenzyl)-carban-nc actd, tert-butyl ester (1 .Og, 2.76 mmol) The reaction is stnred for 1 h. 2 The reaction was concentrated under reduced pressure to yteld a sohd quantitatively as the amine hydrochloride salt. ‘H NMR (300 MHz, DMSO-d,) 8 57 (bs, 3H), 8 11 (s, lH), 7 85 (bs, lH), 7 76 (t, 2H), 7 37 (d, lH), 4 15 (m, 4H), 1 832 00 (m, 6H), 1.18 (m, 5H)

3.2. Preparation of Difunctionalized Analogs of Generic Template Ill from 2-Hydroxyphenylacetic Acid 3 2.1. Preparation of [5-(I-Amino-Ethyl)-Z-Cyclohexylmethoxy-Phenyl]-Acetic Acid Methyl Ester 12 (Fig. 4) 3.2.1-l.

(2-!-~YDROXY-PHENYL)-ACETIC

ACID

METHYL

ESTER

7

1 To a solution of 2-hydroxyphenylacetrc acid 6 (20 0 g, 129 7 mmol) m 250 mL of CHsOH was added anhydrous HCl bubbled at room temperature for 5 mm The reaction was capped and stirred overnight.

520

Para and Sawyer

2. The reaction was then concentrated under reduced pressure to yield a pale 011 (20 5 g, 95%) ‘H NMR (300 MHz, DMSO-d,) 6 9 45 (s, lH), 7 15 (m, 2H), 6 75 (m, 2H), 3 58 (s, 2H), 3.54 (s, 3H)

3.2.1

2

(2-METHOXY-PHENYL)-ACETIC

ACID

METHYL

ESTER

8

1 To a solution of (2-hydroxy-phenyl)-acetic acid methyl ester (16 0 g, 96 4 mmol) m 200 mL of DMF was added freshly ground K,CO, (19 9 g, 144 5 mmol) followed by CH,I (6 6 mL, 106 1 mmol). The reactlon mixture was heated to 50°C ovemlght 2. The reaction was then concentrated under reduced pressure The residue was diluted with ethyl acetate and water. The orgamc layer was washed with 1 NHCl (2 x 100 mL) and saturated aqueous NaCl(1 x 100 mL), and dried over MgSO, 3. The reaction was filtered and concentrated under reduced pressure to yield a crude 011 4 Chromatography of the residue (1.4, ethyl acetate/hexanes) gave the product as a clear 011 (9.1 g, 53%). ‘H NMR (400 MHz, DMSO-d,) 6 7.25 (t, lH), 7 17 (d, lH), 6 97 (d, lH), 6 89 (t, lH), 3 74 (s, 3H), 3 58 (s, 5H) IR(KBr) 2951, 2838, 1803, 1738,1602, 1590,1496, 1463, 1437, 1342, 1290, 1247,1211,1158, 1113, 1049, 1028,85 1,754,690 cm-’ 3 2.1.3.

(5-ACETYL-%HYDROXY-PHENYL)-ACETIC

ACID

METHYL

ESTER

9

1 To a solution of AICY (20 7 g, 155 4 mmol) m CH2C12 (50 mL) was added a solution of (2-methoxy-phenyl)-acetic acid methyl ester (8 0 g, 44.4 mmol) and CH,COCl (3 4 mL, 48.8 mmol) m CH,Cl, (50 mL) dropwlse. 2 After the addition was complete, the reactlon was heated to reflux for 5 h 3 The reaction mixture was then cooled, and carefully poured onto a mixture of Ice and water. 4 The aqueous layer was then extracted with ethyl acetate (2 x 200 mL) The combmed extracts were then washed with saturated aqueous NaCl(1 x 50 mL), dned over MgSO, 5 The reaction was filtered and concentrated under reduced pressure to yield an 011 quantitatively ‘H NMR (400 MHz, DMSO-d,) 6 10 51 (s, lH), 7 79 (m, 2H), 6 88 (d, lH), 3 63 (s, 2H), 3 59 (s, 3H), 2 47 (s, 3H)

3.2.1.4.

(5-ACETYL-2-CYCLOHEXYLMETHOXY-PHENYLbACETIC ‘ESTER

ACID

METHYL

10

To a solution of (5-acetyl-2-hydroxy-phenyl)-acek acid methyl ester (4.0 g, 19 2 mmol) in 40 mL of DMF was added freshly ground K&O, (3 98 g, 28 8 mmol) followed by cyclohexylmethyl bromide (2.95 mL, 2 1 1 mmol) The reaction mixture was heated to 65°C overnight The reaction was then concentrated under reduced pressure The residue was diluted with ethyl acetate and water The orgamc layer was washed with 1 N HCl (2 x 50 mL) and saturated aqueous NaCl(1 x 50 mL), and dried over MgS04 The reaction was filtered and concentrated under reduced pressure to yield a crude 011 Chromatography of the residue (1:4, ethyl acetate/hexanes) gave the product as a clear oll(3 6 g, 61%) ‘H NMR (400 MHz, DMSO-de) 6 7.89 (d, lH), 7 84 (s, IH), 7 05 (d,

Amlnobenzoic Acid-Based Nonpepbde Templates

521

lH), 3.86 (d, 2H), 3 66 (s, 2H), 3 58 (s, 3H), 2.49 (s, 3H), 1.81 (m, 6H), 1 12 (m, 5H) IR(KBr) 3461,3004,2932,2853, 1738, 1667, 1602, 1501, 1465, 1435, 1408, 1356, 1323,1270,1200,1165,1138,1078,1012,974,933,814,758,610,576 cm-‘. 3.2

1.5

[2-~Y~L~HE~YLMETH~~Y-5-( ACID

METHYL

ESTER

i -HYDROXYIMINO-ETHYL)-PHENYL]-ACETIC

11

1 To a solution of (5-acetyl-2-cyclohexylmethoxy-phenyl)-acetic actd methyl ester (3.0 g, 9 86 mmol) m pyrrdine (66 mL) at room temperature was added hydroxylamme hydrochloride (1.02 g, 14.8 mmol). The reaction was stirred 48 h 2. The reaction was then concentrated under reduced pressure, diluted with ethyl acetate and washed with 5% citric acid (3 x 100 mL), saturated aqueous NaCl (1 x 100 mL), and dried over MgS04 3 The solution was filtered and concentrated under reduced pressure to yield a foam. ‘HNMR (400 MHz, DMSO-d6) 6 10.94 (s, lH), 7 51 (s, IH), 7 48 (d, IH), 6 93 (d, lH), 3 77 (d, 2H), 3.61 (s, 2H), 3.58 (s, 3H), 2.10 (s, 3H), 1 63-l 76 (m, 6H), 1 02-l 29 (m, 5H). IR(KBr) 3289, 3220, 3096, 2992, 2930, 2854, 2839, 1743,1610,1506,1467,1449, 1434, 1369, 1333, 1312, 1285, 1250,1210, 1167, 1138, 1086, 1021,949,877,813,743,638,613 cm-’ 3.2 1 6.

[5-(

1 -AMINO-ETHYL)-2-CYCLOHEXYLMETHOXY-PHENYL]-ACETIC

METHYL

ACID

ESTER~~

1 To a solution of [2-cyclohexylmethoxy-5-( l-hydroxyimmo-ethyl)-phenyl]-acetic acid methyl ester (1 5 g, 4.70 mmol) m CH30H (60 mL) and triethylamme (5 mL) was added wet Raney nickel (0 30 g) 2 The reaction was then pressured under an atmosphere of hydrogen (45 PSI) for 16.5 h 3 The reaction was then concentrated under reduced pressure to yield an oil Ether was added, and anhydrous HCl was bubbled through he solution to precipitate the amme hydrochloride The solid was collected and dried under reduced pressure to yield a white solid quantitatively. iH NMR (400 MHz, DMSO-d,) 6 8 32 (bs, lH), 7.38 (d, lH), 7.36 (s, IH), 7.30 (d, lH), 4.30 (m, lH), 3 77 (d, 2H), 3 58 (s, 5H), 1.64-l 76 (m, 6H), 147 (d, 3H), I .01-I 25 (m, 5H) IR (KBr) 2993,2926,2852, 1740, 1603, 1509, 1450,1386,1310, 1260,1236, 1169, 1129,1082, 1021 cm-’

3.3. Preparation of Difunctionalized Analogs of Generic Template Ill from 5-Acetyl-2-Uydroxyphenyl Acetic Acid 3.3.7. Preparation of [5-(7-Amino-Ethyl)-2-CyclohexylmethoxyPhenylj-Acetamlde 76 (Fig. 5) 3 3.1.1.

(5-ACETYL-2-CYCLOHEXYLMETHOXY-PHENYL)-ACETIC

ACID

13

1 To a solution of (5-acetyl-2-cyclohexylmethoxy-phenyl)-acetic acid methyl ester 10 (3 0 g, 9.86 mmol) m 5 mL of CH30H and 5 mL of THF is added 1 NNaOH (7.89 mL, 7.89 mmol). The reaction is stirred overnight at room temperature.

Para and Sawyer

522

1 N NaQH

EDCI,

HOBT

CHsOH

NMM.

NH,,OH

13

HCI

l

NH20H

14

Raney-NI

HO.

Pyridine

CHsOH,

TEA

W

15

16

Frgure 5 2. The reaction is concentrated under reduced pressure The oily residue was then actdttied wtth 5% citric acid The aqueous layer was extracted with ethyl acetate (3 x 150 mL) The combined organic extracts were washed with aqueous saturated NaCl(1 x 150 mL) and dried over MgS04 3 The solution was filtered and concentrated under reduced pressure to yield a solid ‘H NMR (400 MHz, DMSO-d,) 6 12 23 (bs, lH), 7 88 (d, lH), 7.81 (s, lH), 7 04 (d, lH), 3.86 (d, 2H), 3.56 (s, 2H), 2 50 (s, 3H), 1 63-1.79 (m, 6H), 1 03-l 29 (m, 5H); IR (KBr) 3074,3038,2924,2899,2857, 1711, 1670,1600,1577,1505, 1447, 1414, 1363, 1319, 1267, 1233, 1195, 1131, 1012,910,836,590 cm-’ 3 3 1.2. 2-(5-ACETYL-2-CYCLOHEXYLMETHOXY-PHENYL)-ACETAMiDE 14 1 To a solution of (5acetyl-2-cyclohexylmethoxy-phenyl)-acetic acid (0.90 g, 3 1 mmol) m 30 mL of DMF at O°C was added NMM ( 0 5 1 mL, 4 65 mmol), HOBt (0 66 g, 4.65 mmol) followed by EDCI (0.89 g, 4 65 mmol), and stirred for 1 h at 0°C. Concentrated aqueous NH40H (0 62 mL, 4 65 mmol) was then added The reaction was allowed to warm slowly to room temperature and stirred overnight 2 The reaction was concentrated under reduced pressure, then diluted with ethyl acetate and washed with 5% aqueous citric acid (3 x 50 mL), saturated aqueous NaHC03 (3 x 50 mL) and aqueous saturated NaCl(1 x 50 mL), and dried over MgS04 3 The reaction was filtered and concentrated under reduced pressure to yield a pale yellow powder. The powder was collected, washed with ether, and drred under reduced pressure ‘H NMR (400 MHz, DMSO-d,) 6 7 86 (d, lH), 7.77 (s, lH), 7 32 (bs, lH), 7.01 (d, lH), 6 88 (bs, lH), 3 84 (d, 2H), 3 42 (s, 2H), 2.50 (s, 3H), 1.63-1.82 (m, 6H), 103-l 21 (m, 5H); IR(KBr) 3398,3330,3202,2923,2852, 1666,1603, 1581, 1504, 1423,1399,1362,1322,1306,1268,1197,1125,1082,1015,963,822,600 cm-’ 3.3

1

3

[‘ii-~Y~L~HE~YLMETH~~Y-5-(1

-HYDROXYIMINO-ETHYL)-

15 1. To a solution of 2-(5-acetyl-2-cyclohexylmethoxy-phenyl)-acetamide (0 80 g, 2 76 mmol) m pyrtdme (20 mL) at room temperature was added hydroxylamme hydrochloride (0 28 g, 4.15 mmol). The reaction was stirred 24 h PHENYL]-ACETAMIDE

Aminobenzoic Acid-Based Nonpeptide Templates 0

523

0

&xHN

a PhSn(CH&

3

17

BccHN

LICI

IS

Figure 6 2 The reaction was then concentrated under reduced pressure, diluted with ethyl acetate, and washed with 5% citric acid (3 x 50 mL), saturated aqueous NaCl (1 x 50 mL), and dried over MgSO,. 3 The solutton was filtered and concentrated under reduced pressure to yield a foam. ‘H NMR (400 MHz, DMSO-d,) 6 10.93 (s, lH), 7.48 (s, lH), 7 45 (d, lH), 7 26 (bs, lH), 6 91 (d, lH), 6.87 (bs, lH), 3.77 (d, 2H), 3.37 (s, 2H), 2 10 (s, 3H), 1 63-l 79 (m, 6H), 1 03-1.29 (m, 5H); IR (KBr) 3337,3233,3209,2925,2853, 1716, 1662, 1606, 1506, 1448, 1395, 1370, 1307, 1283, 1251, 1187, 1134, 1086, 1017,959,891,814,638,592 cm-‘. 3 3 1 4 [5-(i -AMlNO-ETHYL)-2-CYCLOHEXYLMETHOXY-PHENYL]-ACETAMlDE 16 1 To a solution [2-cyclohexylmethoxy-5-( l-hydroxyimino-ethyl)-phenyl]-acetamtde (0.84 g, 2 76 mmol) m CH,OH (55 mL) and triethylamme (5 mL) was added wet Raney nickel (0 50 g). 2. The reaction was then pressured under an atmosphere of hydrogen (50 PSI) for 19 h 3 The reaction was then concentrated under reduced pressure to yield an 011 IR (KBr) 3358,3188,2924,2852,1670,1603,1577,1505,1387,1306,1252,1187, 1129, 1084, 1020,962,893,813,583 cm-t

3.4. Preparation of Monofunctionalized Analogs of Generic Template II from 5-Aminomethylsalicylic 3.4.1. Preparation of (2-Carbamoyl-Biphenyl-4-Ylmethyl)Carbamic Acid tert-Butyl Ester 18 (Fig. 6) 3.4.1 .l

Acid

ACID 4-

. TRIFLUORO-METHANESULFONIC (T&W-6UTOXYCARBONYLAMlNo-METHYL)-

17 To a solution of (3-carbamoyl-4-hydroxy-benzyl)-carbamic actd, tert-butyl ester 3 (2 0 g, 7 51 mmol) m CH2C12 at -78°C is added triethyl amine (2.09 mL, 15 02 mmol) followed by N-phenyltrtfluoromethanesulfonamide (2.95 g ,8 27 mmol) The reaction is allowed to warm slowly to room temperature and then stirred overnight The reaction is then dtluted with ethyl acetate and the orgamcs are washed with 1 N NaOH (1 x 50 mL), 5% citric acid (2 x 50 mL) and saturated aqueous NaCl (1 x 50 mL) and dried over MgSO+ The solutton was filtered and concentrated under reduced pressure to yield an oil (2.5 g, 84%). ‘H NMR (400 MHz, DMSO-d6) 6 8.06 (bs, IH), 7.70 (bs, lH), 7 56 ii!-CARBAMOYL-PHENYL

1

2 3

4

ESTER

Para and Sawyer

524 0

0

0

0

0

19

HCI . NHpOH

C,H,,

CH2Br

20

HO.

21

- Raney-NI

Pyndlne

0

kc03

EDCI, HOBT NMM, NH,,OH

H2N

CH30H

22

23

Figure 7 (bs, lH), 7 50 (m, lH), 7.45 (m, lH), 4 18 (d, 2H), 1 39 (s, 9H), IR (KBr) 3412, 3356,3186,2999,1687,1656,1620,1592, 1524,1495,1432,1395,1369,1277, 1252, 1302, 1152, 1075, 1056,918,885,858,689,657,626,610 cm-’

3 4.1.2

(2-CARBAMOYL-BIPHENYL-4-YLMETHYL)‘CARBAMIC

ACID

TERT-BUTYL

ESTER

18

To a solution of trifluoro-methanesulfomc acid 4-(tert-butoxycarbonylammomethyl)-2-carbamoyl-phenyl ester (1 0 g, 2 5 1 mmol) in dioxane was added phenyltrimethyltm (0 62 g, 2.58 mmol), lithium chloride (0 3 1 g, 7 53 mmol) and tetrakis(triphenylphosphine)palladmm(O) (0.058 g, 0.05 mmol) The solution was heated to reflux overnight The reaction is cooled and diluted with ethyl acetate The orgamcs are washed with 1 N aqueous NH40H (1 x 50 mL), 5% citric acid (1 x 50 mL), saturated aqueous NaHCO, (1 x 50 mL), and saturated aqueous NaCl (1 x 50 mL) and dried over MgSO, The solution was filtered and concentrated under reduced pressure to yield a powder (0.80 g, 97%). ‘H NMR (400 MHz, DMSO-d6) 6 7 57 (bs, lH), 7 49 (t, lH), 7 297.47 (m, 9H), 4 18 (d, 2H), 1 40 (s, 9H), IR (KBr) 3364,3184,3067,2972, 1691, 1649, 1627,1526,1483, 1447,1415, 1386, 1272, 1252, 1165, 1113, 1055, 760,702,649 cm-’

3.5. Preparation of Difunctionalized Analogs of Generic Template II from SAcetylsalicylic 3.5.1. Preparation of 5-(l-Amino-Ethy/)2-Cyclohexylmethoxy-Benzamide 23 (Fig. 7) 3.5

1 .i

i&i-tYDROXY-!?-METHYL-BENZAMIDE

Acid

20

1 To a solution of 5-acetyl sahcyhc acid 19 (5 0 g, 27 7 mmol) m 250 mL THF at O’C was added NMM (4 5 mL, 41 6 mmol), HOBt (5.62 g, 41 6 mmol), followed by EDCI (7 9 g, 41 6 mmol), and then stirred for 1 h at 0°C Concentrated aque-

Aminobenzoic Acid-Based Nonpeptide

Templates

525

ous NH,OH (5 6 mL, 41 6 mmol) was then added The reaction was allowed to warm slowly to room temperature and stirred overmght 2 The reaction was then diluted with ethyl acetate and washed with 5% aqueous citric acid (3 x 50 mL), saturated aqueous NaHCO, (3 x 50 mL), and aqueous saturated NaCl(1 x 50 mL), and dried over MgS04 3 The reaction was filtered and concentrated under reduced pressure to yield a pale yellow powder. The powder was collected, washed wtth ether, and dried under reduced pressure. ‘H NMR (400 MHz, DMSO-d,) 6 13 84 (s, IH), 8 73 (bs, lH), 8 53 (s, lH), 8 12 (bs, lH), 8 00 (d, lH), 6 99 (d, IH), 2.59 (s, 3H); IR (KBr) 3335,3194,3069,2988,2965,1669,1624,1593,1464,1442,1411,1378, 1362,1301, 1275,1239,1145,1113,1099,978,933,866,831,770,710,658,602,573,544 cm-‘.

3.5

1.2.

5-ACETYL-2-CYCLOHEXYLMETHOXY-BENZAMIDE

21

1 To a solution of 2-hydroxy-5-methyl-benzamide m 25 mL of DMF was added freshly ground K&O, (2.3 1 g, 16.7 mmol) followed by cyclohexylmethyl bromide (1.7 mL, 12.3 mmol) The reaction mixture was heated to 65°C overmght 2 The reaction was then concentrated under reduced pressure The residue was diluted with ethyl acetate and water The organic layer was washed with 1 N HCl(2 x 50 mL) and saturated aqueous NaCl (I x 50 mL), and dried over MgSO,. 3 The reaction was filtered and concentrated under reduced pressure to provide a yellow powder The powder was collected, washed with ether, and dried under reduced pressure (2 1 g, 68%). ‘H NMR (400 MHz, DMSO-d,) 6 8.3 1 (s, 1H), 8 05 (d, lH), 7 22 (d, IH), 4 04 (d, 2H), 2.53 (s, 3H), 1 83 (m, 6H), 1 14 (m, 5H), IR(KBr) 3455,3327,3284,3240,3202,2934,2853, 1672, 1538,1495, 1455, 1431,1368,1291,1253,1178,1105,1072,1004,973829,628,567,534,516 cm-’ 3 5 t -3.

2-~Y~L~HE~YLMETH~~Y-5-(

1 -HYDROXYIMINO-ETHYL)-BENZAMIDE

22

1. To a solution of 5-acetyl-2-cyclohexylmethoxy-benzamide (1 0 g, 3 63 mmol) m pyridme (25 mL) at room temperature was added hydroxylamine hydrochloride (0 38 g, 5.45 mmol) 2 The reaction was stirred 48 h at room temperature. 3 The reaction was then concentrated under reduced pressure, diluted with ethyl acetate, and washed with 5% citric acid (3 x 100 mL), saturated aqueous NaCl (1 x 100 mL) and dried over MgS04 4 The solution was filtered and concentrated under reduced pressure to yield a foam. ‘H NMR (400 MHz, DMSO-d6) 6 11.07 (s, lH), 8 08 (d, lH), 7 72 (d, lH), 7 61 (bs, IH), 7 53 (bs, lH), 7 14 (d, lH), 4 03 (d, 2H), 2 13 (s, 3H), 1 74 (m, 6H), 1.11 (m, 5H), IR(KBr) 3455, 3285, 3235, 3 189, 3082, 3049, 2928, 2853,2802, 1661, 1578, 1560, 1502, 1450, 1426, 1374, 1297, 1269, 1253, 1162, 1090, 1007,987,948,809,735,486 cm-’

3 5.1 4. 5-( 1-AMINO-ETHYL)-2-CYCLOHEXYLMETHOXY-BENZAMIDE

23

1 To a solution of 2-cyclohexylmethoxy-5-( 1-hydroxytmmo-ethyl)-benzamide (0 50 g, 1 72 mmol) m CHsOH (20 mL) and triethylamme (5 mL) was added wet Raney nickel (0.30 g)

526

Para and Sawyer

2. The reaction was then pressured under an atmosphere of hydrogen (45 PSI) for165h 3 The reaction was then concentrated under reduced pressure to yield a white solid ‘H NMR (400 MHz, DMSO-d,) 6 7 86 (s, lH), 7.59 (d, 2H), 7 47 (d, lH), 7 10 (d, lH), 4.03 (q, lH), 3 97 (d, 2H), 1.83 (m, 6H), 1 23 (m, 8H); IR(KBr) 3433, 3175,2925,2851,1663,1591, 1494,1465, 1433, 1373, 1242, 1161,1090, 1013, 813,566 cm-’

References 1. Qian, Y , Blaskovich, M. A , Saleem, M , Seong, C M , Wathen, S P., Hamilton, A D , et al. (1994) Design and structural requirements of potent peptidomimetic inhibitors of p2lras farnesyltransferase. J. Bzol Chem 269, 12,410-12,413 2 Jackson, S., DeGrado, W , Dwivedi, A , Parthasarathy, A , Higley, A , Krywko, J , et al (1994) Template-constramed cychc peptides design of high-affinity hgands for GPIIb/IIIa J Amer Chem Sot 116,3220-3230. 3 Lunney, E. A , Para, K S , Plummer, M. S , Sawyer, T. K , Shahripour, A , Smgh, J , et al (1996) Intern PCT Patent Application, Ser No W097 12903 4. Peschke, B , Madsen, K., Hansen, B. S , and Johansen, N L. (1997) Ammomethylthiophene-2-carboxylic acids as dipeptide mimetic m new growth hormone secretagogues Bloorg h4ed Chem Lett. 7, 1969-1972 Lunney, E. A , Para, K S., Rubm, J. R , Humblet, C., Fergus, J., Marks, J , and Sawyer, T. K (1997) Structure-based design of novel series of nonpeptide hgands that bind the pp60src SH2 domain J Amer Chem Sot 119, 1247 1-12476 Para, K S , Lunney, E. A , Plummer, M. S , Stankovic, C J., Shahripour, A , Holland, D , et al. (1998) Structure-based de novo design and discovery of nonpeptide antagonists of the pp60src SH2 domam, in Proceedwgs of the Fifteenth Amerzcan Peptzde Symposzum (Tam, J and Kaumaya, P , eds ), Escom Science Publishers, Leaden, the Netherlands, m press. Sawyer, T. K (1997) Peptidomimetric and nonpeptide drug discovery Structurebased drug design, m Structure-Based Drug Design (Veerapandian. P , ed ), Marcel Dekker, New York, NY, pp. 559-634 Sekiya, M , Yanaihara, N., and Masui, T (1963) Reaction of amide homologs Ammomethylation of salicychc acid with N,N’-methylenebisacetamide Chem Pharm Bull $55 1-553

30 Aminimides

as Peptidomimetics

Brian S. Fulton, Alan P. Kaplan, Joseph C. Hogan, Jr., and Steven L. Gallion

1. Introduction Highly selective and specific protein-protein and peptlde-protem mteractions are ehclted by a large number of natural specific biological responses. The mlmlcry of these natural interactions has been an important and useful strategy for the development of metabohcally stable drugs capable of bemg administered orally (I-4). We report here on the use of a novel type of peptidomimetlc based on the amirnmide linkage. Amlmmides are chemically described as hydrazmlum hydroxide inner saltsor as ammonium-lmines. (Fig. 1) This unusual class of compounds possessesa zwltterlomc nature that provides solubllity m polar media (water, methanol, ethanol, acetomtrile, DMF, DMSO) as well as m nonpolar solvents such as benzene, when substltuents are chosen to reduce the total molecular dipole moment. The amimmide bond has two resonance forms as shown m Fig. 1. ThepK, of the protonated form is generally less than 4. The predominant resonance form will place the formal negative charge on the more electronegative oxygen atom, although we typically use diagrams in which the formal charges reside on the two nitrogens to stressthe absence of the hydrogen-bond-donating ability of the aminimide as compared the amide bond in peptides. The amimmlde bond is extremely stable under both acldlc and basic conditions and IS highly resistant to proteolytic cleavage. The synthesis of aminimldes (and the related hydroxyethyl amimmide) may be accomplished by three general methods. 1 Ternary reaction of a hydrazme, epoxlde, and ester to form a hydroxyethyl amuumlde (Fig. 2a) (5). 2 Reaction of a hydrazmlumsalt with an ester(Fig. 2b) (6). From

Methods /n Molecular Medrcme, Vol 23 Pepbdomrmefrcs Protocols Edlted by W M Kazmlerskl @Humana Press Inc , Totowa, NJ

527

528

Fig 1, Resonance forms for trimethylammontacetamidate

A Rq@,R2 x’

+

~CooMs

+

NaOR

-

R;/N’m2

B I R,X R,R,NNH,

+

R&OX

-

R,R,NNHCO& 2 NaOH

C Fig 2 General solutton-phase synthesis schemes for amnumlde formatton (a) Ternary reaction of a hydrazlne, epoxide and ester, (b) Reaction of a hydrazmium salt wtth an ester, and (c) Quanternization of a hydraztde followed by treatment with alkylatmg agent and base 3. Quantemtzatton of a hydraztde and subsequent treatment with aqueous base (Fig. Zc) Formatton of hydroxyethyl ammrmtdes from dtmethyl hydrazme, 5 epoxtdes, and 11 esters was found to give crude yields of >80%, with highest yields

achieved with protlc solvents such as lsopropanol or t-butanol, or aprotlc solvents such as DMSO

(5). The rate of the reaction increased with the electron-

withdrawing capability of the carboxyhc portion of the ester. The formation of ammtmtdes from the hydrazmmm salt requires a strong base, with sodium

Aminimides as Peptidomimetics

529

(b)

Ftg 3 Representative known pharmacologically active amnnmldes (a) a simple hydroxyammrmrdeused as a vasodtlatlve agent (b) Besulfamtde,used as a dmretrc and annhypertensive. methoxtde provtdmg the best results. The best solvent for this reactron IS t-butanol. Typical crude yields for unhindered esters are 90% or greater. Quatermzatton of hydrazrdes usmg alkyl halides or tosylates give yrelds of 60-70% Final formatton of the ammlmlde by treatment with aqueous base such as sodium hydroxide has a yield near 80%. The hydraztdes are generally formed by reaction of the hydrazme with an acid chloride, anhydnde, or carboxylic acid m the presence of DCC There are a few examples of ammimrdes as drugs Figure 3a shows a hydroxyethyl ammimtde having stgmficant vasodrlative properties (7) This compound exhibited an ED50 of 2 mg/kg administered interperatonially, with a 6-h duration of actton, and the LD50 was two orders of magnitude greater than the ED50. Besulpamtde (Fig. 3b) is synergistic with Captopril and has been used for hypertension (8). The effective dose orally administered 1sl-10 mg/kg per day, which IS several orders of magnitude less than that of the LD50. Ammimtdes have also been demonstrated to possess antrmicrobral (9) and antifungal (IO) properties. We have carried out a number of synthetic, computattonal, btochemrcal, and brophystcal studies to explore the utlhty of amimmides as pepttdomtmettcs. A detailed descrrption of the conformational comparison of aminimide and peptide scaffolds has been developed and utilized in the design of peptldomlmetrc

530

Fulton et al.

compounds. Ammlmldes have demonstrated potential as mhlbltors of both serine and acid proteases m the test systemsused thus far (11,12). 2. Materials 2.7. General Unless stated otherwise, all materials were obtained from commercial suppliers and used wlthout further purifkatlon. NMR samples were obtained m CDC& unless otherwise stated. ‘H NMR spectra were obtained at 300 75 MHz on a Varlan Gemini spectrophotometer. 13CNMR were obtained at 75.5 MHz on a Varlan Gemml spectrophotometer. Mass spectroscopy was performed by either APICI (AP) or electrospray (ES) m the analytical faclllties at ArQule. HPLC was performed usmg a Shlmadzu chromatography system and reverse phase Cl8 columns unless otherwlse mentioned. Detection was at 254 nm with gradient elutlon of lO-100% or 25-100% acetonitrlle/water (0.1% trlfluoroacetic acid) for 10 mm. While absolute retentton times are given, to is typically 0.5 mm. 2.2.1 Reagents for Method 3 7 2 3 4 5 6 7 8 9 10. 11 12 13 14

Dlmethyl hydrazme. 1-Ammoplperidme Methyl benzoate (2,3-Epoxypropyl)benzene Isopropanol Propylene oxide Methyl lsomcotmate Dlethyl ether Benzoyl chloride. Glycldol. Methanol Ethyl acetate. Toluene Trlethylamme

2.2.2. Reagents for Method 3.2 1 2 3 4 5. 6

terl-Boc-L-phenylalamne-epoxlde 1-Benzyl- I-methylhydrazme Methyl benzoate Silica gel. Ethyl acetate Pentane

Aminimides as Peptidomimetics

1

2

3

531

4

Fig 4. Preparation of 1-(3-phenyl-2-hydroxypropyl)plpendlnylbenzoyl~mide

5

6

3

4

7

Fig. 5 Preparatron of 1,l -drmethyl- 1-(2-hydroxypropyl)aminebenzoyllmlde

7.

3. Methods 3. I. General Preparation of Aminimides 3.1.1. Preparation of I-(3-Phenyl-2hydroxypropyl)piperidinylbenzoylimide 4 (Fig. 4) 1 Reflux a solutton of 1 04 g (10.0 mmol) of 1-aminoptperidme, 1 35 g (10 0 mmol) of (2,3-epoxypropyl) benzene, and 1.3 7 g (10 mmol) of methyl benzoate m 6 3 mL of tsopropanol 19 h 2. Concentrate the solutton in vacua to give an oil which sohdtfied on standing Wash the solid with ether to give 2 11 g (62%) of pure 4 as a white solid ‘H-NMR. 8.0 (d, 2H), 7.1-7.4 (m, 8H), 4.95 (m, lH), 4.5 (m, 2H), 3.85 (d, lH), 3.6 (m, IH), 2.9-3.1 (m, 3H), 2.75 (m, lH), 2.1 (m, 2H), 1.6-1.8 (m, 2H), 1.3-l .5 (m, 2H) HPLC: 8.9 min.

3.7.2. Preparation of 7, I-Dimethyl-l-(2hydroxypropyl)aminebenzoylimide 7 (Fig. 5) 1 Star at room temperature a solution of 0.60 g (10 mmol) of dtmethylhydrazine, 0.58 g (10 mmol) of propylene oxide, and 1 36 g (10 mmol) of methyl benzoate m 5 mL isopropanol for 18 h. 2. Concentrate the solution m vacua to give an oil which was concentrated in vacua at 80°C for 48 h to produce 1.97 g (89%) of pure 7 as a white solid. ‘H-NMR: 7.83 (d, 2H), 7.32 (m, 3H), 4.41 (m, lH), 3.6 (s, 3H), 3.58 (s, 3H), 3.36-3.5 (m, 2H), 3.15 (d, lH), 1.22 (d, 3H)

Fulton et al.

532

Fig 6 Preparation of l,l -dtmethyl- l-(3-phenyl-2hydroxypropyl)ammeisonicotmoylimtde 9 0 OH -

bN#

10

12

41

Fig 7 Preparation of glycidyl benzoate 12 13C-NMR: 170.556, 138.510, 63.36, 55.38, 51.62,21.54 HPLC: 10.6 min

130.3, 129.8, 128.4, 128.2, 127.8, 76 17,

3.1.3. Results of a Typical Array Run: Preparatron of 1,l -Dimethyl- l(3-phenyl-2-hydroxypropyl)amineisonicotinoylimide 9 (Fig. 6) 1 Heat a solutton of 0.06 g (1 mmol) dlmethyl hydrazme, 0 134 g (1 mmol) of (2,3-epoxypropyl) benzene, and 0.137 g (1 mmol) of methyl isonmotmate m 1 mL isopropanol at 60°C for 16 h 2 Concentrate a pulled well to give an oil which was washed with 2 mL of dlethyl ether to yield 0.255 g (85%) of pure 9 as a yellow solid. MS (AP+) 299.6 HPLC 7.3 mm

3.1.4. Preparation of Glycidyl Benzoate 12 (Fig. 7) 1

Add 7 8 g ( 102 mmol) of glycidol and 10 g (100 mmol) of tnethylamme m 20 mL of toluene to an ice cold solutton of 14 5 g (120 mmol) of benzoyl chloride m 200 mL toluene Control the rate of addition to keep the temperature of the reaction below 15°C 2. Warm the mixture to room temperature and stir for 3 h Filter off the triethylammonium chloride salt and concentrare the filtrate to grve 17 8 g (100%) of pure 12 as a pale oil

‘H-NMR: 8.05 (d, 2H), 7.4-7.6 (m, 3H), 4.65 (d, lH), 4.15 (m, lH), 3 35 (s, lH), 2.9 (s, lH), 2.7 (s, 1H) 13C-NMR. 166.8, 133.8, 131.1, 130.3, 129.4, 128.9, 65.98, 50.02,45.24

Fig. 8 Preparation of l-(2,3-dihydroxypropyl)pipertdmylbenzoylimtde

+ Y*

12

(23)

-

5

14

Fig 9 Preparation of 1,l -dimethyl- 1-(2,3-dihydroxypropyl)ammobenzoyllmtde

14.

IR (neat) 1720.2 cm-l TLC: (silica gel, 10% methanol/methylene chloride) Rf= 0.2 3 1.5. Preparation of 1-(2,3-dihydroxypropyl) piperidinylbenzoylimide 13 (Fig. 8) 1

Stir a solutton of 1 8 g (10 mmol) glycrdyl benzoate and 1 g (10 mmol) of lammoptperidme m 8 mL tsopropanol for 24 h at room temperature A white soled precipitates whtle sttrrmg. 2 Concentrate the mtxture to give a white soled which 1swashed with dtethyl ether to give 2 g (76%) of pure 13 ‘H-NMR (CD, OD). 7.86 (d, 2H), 7.36 (m, lH), 4 65 (m, lH), 4.5 (d, lH), 4.25 (m, 2H), 3.2-3.6 (m, 5 H), 2.3 (m, IH), 2.05 (m, IH), 1.7 (m, 4H) 13C-NMR(CD,OD) 172.03, 140.7, 131.2, 129.3, 129.2,68.75,68.43,66.77, 65.98,62.97,23.55, 22 ?5,22.51 IR (KBr): 1560 cm-’ HPLC 7.1 mm

TLC: (sAca gel, 10% methanol, methylene chloride) Rf = 0.23 3.1.6. Preparation of l,l-dimethyl-l-(2,3dihydroxypropyl)aminobenzoylimide 14 (Fig. 9) 1 Stir a solution of IO g (56 mmol) glycrdyl benzoate and 3.7 g (62 mmol) of dtmethyl hydrazme m 50 mL isopropanol at room temperature for 18 h. 2 Concentrate the solutton m vacua to give an oil. 3. Add 100 mL of diethyl ether to the 011with sttrrmg whtch slowly yields a white solid Filter to grve 9.1 g (68%) of pure 14 as a white sohd

534

Fulton et al.

‘H-NMR: 7.85 (d, 2H), 7.35 (m, 3H), 4.3 (m, IH), 3.53.8 (m, I IH), 3.4 (d, IH) 13C-NMR: 170.785, 138.3, 130.5, 128.5, 127.8, 72.6, 68 1, 65.0, 55.8,52.1 IR(KBr) 3309,3064, 1593, and 1551 cm“ HPLC: 5.8 mm 3.2. HIV-1 Protease Inhibitor Library Design The hydroxyammimide synthetic protocol usedto construct the library is shown m Fig. 10. Synthesisof the ammo epoxide reagents mvolve considerable time and effort (13) and were therefore varied the least. Likewise, there are few commercially available 1,I-disubstituted hydrazmes,most of which did not fit the model well. However, these may be derived from the correspondmg secondary amme using the method developed by Ohme and Preuschof (14), By contrast, a large number of methyl estersare available commercially and many others may be easily synthesizedfrom the correspondmg acid or acid chloride. The largest number of variations therefore mvolved systematicreplacement of the methyl ester component. The components arestirred m isopropanol at 60°C overnight. After workup and purification, the N-terminal t-Boc can easily be cleaved and a variety of acid chlorides and carbonates can be coupled to the resulting free amme. 3.2.1. Preparation of (S,S, R)- 1-(Z-(butoxycarbonyl)amino-Z-benzylhydroxypropyl)-methyl-benzylaminobenzoylimide 15

l-

1. Add 7.00 mm01 of t-Boc-r.-phenylalanme-epoxrde and 7 69 mm01 of 1-benzyl- lmethylhydrazme to 7 69 mm01 of methyl benzoate m 20 mL tsopropanol. 2 Star overnight at 60°C HPLC analysts showed the presence of two dtastereomers, corresponding to the R and S quanternary nitrogen eptmers 3 Remove the solvent m vacua and chromatograph the crude 011by sthca gel column chromatography to yteld 500 mg of the (S,S,R) dtastereomer 4 Purify the solid by double recrystalhzatton from 1’ 1 ethyl acetate/pentane to yield 150 mg of a pure white solid. Proof of a smgle dtastereomer was shown by ‘H and 13CNMR, mass spectrometry, and reverse-phase HPLC (C-8, l&100* lmear MeOH gradtent with 0 l%TFA) ‘H-NMR (CD,OD, 300MHz) 1 43 (s, 9H), 2 87-2 99 (m,3H), 3 21-3.29 (m,lH), 3 55 (s,3H), 3 55-3 60 (m,lH), 4 23 (d,2H,J=7 2 Hz), 7 20-7 42 (m,12H), 7 86-7 89 (m,2H) t3C-NMR (CD,OD, 75 5MHz) 169.8, 155.8, 138 2, 138.0, 132.0, 130.1, 129 9, 129.7, 129 3, 129 2, 128.8, 129.0 127.2, 126.9, 79.8, 68.1, 64 9, 63.3, 55 1, 51 8, 38 8, 28 4 FAB/ MS. m/z 504 (MH’) 526 (M+Na).

The results of the array are summarized in Table 1. The synthesis yields a stereospecific hydroxyl substituent from epoxide ring-opening, but the product is racemic with respect to the amimmtde quanternary nitrogen. In most cases, the different diastereomers were separated through normal phase chromatogra-

535

Aminimides as Peptidomimetics R +

Boc-NH L

“V, 0

Me9H2

RTOOMe

+ A

60’ C, 2-propanol

R”

1. TFA 2. R”‘X

Fig. 10 Synthetic protocol for generation of hydroxyaminimldes HIV- 1 protease activity

for inhibition

of

phy for independent blologlcal testing. The R-isomer is consistently more potent than the S-isomer. In summary, ammimides appear to demonstrate significant potential as peptldomlmetlcs and, in general, may be a source of therapeutic agents.

Table 1 StructureActivity

Relationships

from the Aminimides

15 Tested

against

HIV-1 Protease

Adapted

from Ref. 11

AQl66 R

AA

AQl68

u.

“U.

*

5.oko.s

AQl69

API70

AQISS

R

AQl59

I

>bK,

AQMO

S

AQ161

R

AQkU

II

Qf

9,

fz7-f

1.0-2 wtJ.0

AQl76

s

39t4.0

Aminimides as Peptidomimetics

537

Ammlmides have demonstrated oral bloavailability in some instances and have reached late phase development m two cases. The crystallographic evidence supports the fact that amimmldes can be used as surrogates for peptldes and hydroxyethylene scaffolds. Evidence from previous studies, as well as with AQ148, have shown no inherent toxlclty problems at effective dose level. Although we are currently uncertain about the ability of aminimldes to be passively transported across cell membranes due to the high dipole moment of the ammlmlde bond, this has not been tested extensively enough to form a general conclusion. We are optimistic that the synthetic protocols described here will produce other focused combmatorial libraries that contam compounds with increased activity and pharmacokmetlc properties

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