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&OUNDED BY %RNST *UCKER
3ERIES %DITORS 0ROF $R 0AUL , (ERRLING .OVARTIS )NTERNATIONAL !' #( "ASEL 3WITZERLAND
!LEX -ATTER -$ $IRECTOR .OVARTIS )NSTITUTE FOR 4ROPICAL $ISEASES "IOPOLIS 2OAD #HROMOS 3INGAPORE 3INGAPORE
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.ATURAL #OMPOUNDS AS $RUGS 6OLUME ))
6OL %DITED BY &RANK 0ETERSEN AND 2ENÏ !MSTUTZ
"IRKHÊUSER "ASEL "OSTON "ERLIN s
s
%DITORS &RANK 0ETERSEN 2ENÏ !MSTUTZ .OVARTIS 0HARMA !' ,ICHTSTRASSE "ASEL 3WITZERLAND
,IBRARY OF #ONGRESS #ONTROL .UMBER "IBLIOGRAPHIC INFORMATION PUBLISHED BY $IE $EUTSCHE "IBLIOTHEK $IE $EUTSCHE "IBLIOTHEK LISTS THIS PUBLICATION IN THE $EUTSCHE .ATIONALBIBLIOGRAFIE DETAILED BIBLIOGRAPHIC DATA IS AVAILABLE IN THE INTERNET AT HTTPDNBDDBDE
)3". "IRKHÊUSER 6ERLAG !' "ASEL n "OSTON n "ERLIN 4HE PUBLISHER AND EDITOR CAN GIVE NO GUARANTEE FOR THE INFORMATION ON DRUG DOSAGE AND ADMINISTRATION CONTAINED IN THIS PUBLICATION 4HE RESPECTIVE USER MUST CHECK ITS ACCURACY BY CONSULTING OTHER SOURCES OF REFERENCE IN EACH INDIVIDUAL CASE 4HE USE OF REGISTERED NAMES TRADEMARKS ETC IN THIS PUBLICATION EVEN IF NOT IDENTIFIED AS SUCH DOES NOT IMPLY THAT THEY ARE EXEMPT FROM THE RELEVANT PROTECTIVE LAWS AND REGULATIONS OR FREE FOR GENERAL USE 4HIS WORK IS SUBJECT TO COPYRIGHT !LL RIGHTS ARE RESERVED WHETHER THE WHOLE OR PART OF THE MATERIAL IS CONCERNED SPECIFICALLY THE RIGHTS OF TRANSLATION REPRINTING RE USE OF ILLUSTRATIONS RECITATION BROAD CASTING REPRODUCTION ON MICROFILMS OR IN OTHER WAYS AND STORAGE IN DATA BANKS &OR ANY KIND OF USE PERMISSION OF THE COPYRIGHT OWNER MUST BE OBTAINED © "IRKHÊUSER 6ERLAG !' "ASEL q "OSTON q "ERLIN 0/ "OX #( "ASEL 3WITZERLAND 0ART OF 3PRINGER 3CIENCE "USINESS -EDIA 0RINTED ON ACID FREE PAPER PRODUCED FROM CHLORINE FREE PULP 4#& ¤ #OVER DESIGN AND LAYOUT -ICHA ,OTROVSKY #( 4HERWIL 3WITZERLAND 0RINTED IN 'ERMANY )3". E )3".
WWWBIRKHAUSERCH
#ONTENTS &OREWORD VII 'LOSSARY IX !CTIVATION OF FUNGAL SILENT GENE CLUSTERS !N NEW AVENUE TO DRUG DISCOVERY !XEL ! "RAKHAGE *ULIA 3CHUEMANN 3EBASTIAN "ERGMANN +IRSTIN 3CHERLACH 6OLKER 3CHROECKH AND #HRISTIAN (ERTWECK
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS &+ THE ANTASCOMICINS AND RAPAMYCIN -ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY !PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY ,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES #HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS ,ESSONS FROM NATURE INSPIRING THE DESIGN OF NEW DRUGS 0ETER %RTL AND !NSGAR 3CHUFFENHAUER #HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS !NDRES ,OPEZ !INSLIE " 0ARSONS #OREY .ISLOW 'URI 'IAEVER AND #HARLES "OONE %POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS n PHARMACOLOGY FERMENTATION AND STRUCTURE ACTIVITY RELATIONSHIPS +ARL (EINZ !LTMANN AND +LAUS -EMMERT (ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS 4HE !STELLAS EXPERIENCE 9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
V
4ABLE OF CONTENTS
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49 0ETER # (IESTAND -ARTIN 2AUSCH $ANIELA 0IANI -EIER AND #AROLYN ! &OSTER !RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY 4HOMAS +UHN AND 9ING 7ANG )NDEX
VI
&OREWORD 7ITH THE BOOKS .ATURAL #OMPOUNDS AS $RUGS 6OL ) AND )) WE AIM TO SHOW NOT ONLY THE COMPLEX TECHNOLOGY CATALOGUE APPLICABLE TO NATURAL PRODUCTS IN DRUG DISCOVERY AND DEVELOPMENT RELATED SCIENCE )MPORTANT ADDITIONAL MOTIVATIONS FOR THE CURRENT EDITION HAVE BEEN TO DEMONSTRATE HOW NATURAL PRODUCTS RESEARCH CAN SUCCESSFULLY BE INTEGRATED IN MOST MODERN TECHNOLO GIES AND CONCEPTS OF DRUG DISCOVERY AND TO ILLUSTRATE THE POTENTIAL WHICH NATURAL PRODUCTS HAVE EVEN TODAY FOR INNOVATIVE TREATMENTS OF HUMAN DISEASES )N THE PRESENT VOLUME WE PUT AN EMPHASIS ON NATURAL PRODUCTS RELATED CHEMICAL DIVERSITY TARGET IDENTIFICATION AND MOST RECENT EXAMPLES OF NATURAL PRODUCTS BASED THERAPIES IN CLINICAL RESEARCH AND BEYOND 4HE INCREASINGLY SYSTEMATIC SEARCH FOR NEW BIOSYNTHESIS GENE CLUSTERS OF MICROBIAL ORIGIN OR WHOLE GENOME SEQUENCING ARE PROMISING CONCEPTS TO GET ACCESS TO NOVEL NATURAL PRODUCTS RELATED CHEMODIVERSITY INDEPEN DENT OF INITIAL PHYSIOLOGICAL LIMITATIONS 3OPHISTICATED TOTAL SYNTHESES OF COMPLEX SECONDARY METABOLITES A HOME DOMAIN OF ONLY A FEW REMAINING DISTINGUISHED ACADEMIC CHEMISTRY GROUPS CREATE INNOVATIVE AND ELEGANT METHODOLOGIES IN SYNTHETIC CHEMISTRY AND THEREBY EXPAND CONTINUOUSLY THE ARMAMENTARIUM OF THIS KEY DISCIPLINE FOR THE BENEFIT OF DRUG DISCOVERY AND ORGANIC CHEMISTRY IN GENERAL &URTHERMORE THE EXPLOITATION OF THOSE SYNTHETIC ROUTES PAIRED WITH THE UNDERSTANDING OF STRUCTURAL CHARACTERISTICS OF SECONDARY METABOLITES AND THEIR BIOLOGICAL ACTIVITY IS LEVERAGED IN THE DESIGN OF MORE AND NEW NATURAL PRODUCTS LIKE MOLECULES POTENTIALLY USEFUL AS NOVEL AND BETTER DRUGS 4HE RELEVANCE OF RECENT NATURAL PRODUCTS FOR NEW MEDICAL TREATMENTS AND ONCE INTRODUCED TO THE PHARMACEUTICAL MARKET THE CONSEQUENCES FOR THE UNDERLYING SUPPLY CHAIN MANAGEMENT OF PLANT METABOLITES CONCLUDE THIS SECOND VOLUME 7E DO HOPE THAT BOTH BOOK VOLUMES WILL CONTRIBUTE TO A DIFFERENTIATED VIEW OF CURRENT ACTIVITIES AND MAY VITALIZE CONSTRUCTIVE DISCUSSIONS ON FUTURE DIRECTIONS OF NATURAL PRODUCTS RESEARCH IN ACADEMIA AND THE PHARMACEUTICAL INDUSTRY 7E APPRECIATE THE KIND SUPPORT OF +LAUS -EMMERT 2OWAN -ORRIS AND 9ING 7ANG
VII
&OREWORD
7E WARMLY THANK ALL AUTHORS FOR THEIR COMPREHENSIVE AND EXCELLENT REVIEWS GIVING AN IN DEPTH INSIGHT INTO THE FASCINATING WORLD OF NATURAL COMPOUNDS &RANK 0ETERSEN 2ENÎ !MSTUTZ
VIII
'LOSSARY !#0 !#4 !3#/ !4 !5# """ ")/3 "- #2!$! #S! #4#, #9'$ #Y0 $## $## $-$/ $-05 $-3/ $.0 $/3 %!% %'&0 %-%! &$! '!&4- '2)$ (!4 ($!# ($,0 ()& (-0! +(-$3 +3 -!0 -#2 -$2 -%, -(# -/' -0! -2)
ACYL CARRIER PROTEIN ARTEMISININ BASED COMBINATION THERAPIES !MERICAN 3OCIETY OF #LINICAL /NCOLOGY ACYLTRANSFERASE AREA UNDER CURVE BLOOD BRAIN BARRIER BIOLOGY ORIENTED SYNTHESIS BIOACTIVE MOLECULE #OOPERATIVE 2ESEARCH AND $EVELOPMENT !GREEMENT CYCLOSPORINE ! CUTANEOUS 4 CELL LYMPHOMA #OMPREHENSIVE 9EAST 'ENOME $ATABASE CYCLOPHILIN DICYCLOHEXYLCARBODIIMIDE DYNAMIC COMBINATORIAL CHEMISTRY DIMETHYL DIOXIRANE . . DIMETHYLPROPYLENEUREA DIMETHYLSULPHOXIDE $ICTIONARY OF .ATURAL 0RODUCTS DIVERSITY ORIENTED SYNTHESIS EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS ENHANCED GREEN FLUORESCENT PROTEIN %UROPEAN !GENCY FOR THE %VALUATION OF -EDICINAL 0RODUCTS &OOD AND $RUG !DMINISTRATION 'LOBAL &UNDS TO &IGHT !)$3 4UBERCULOSIS AND -ALARIA 'ENERAL 2EPOSITORY FOR )NTERACTION $ATASETS HISTONE ACETYLTRANSFERASE HISTONE DEACETYLASE HISTONE DEACETYLASE LIKE PROTEIN HYPOXIA INDUCIBLE FACTOR HEXAMETHYLPHOSPHORAMIDE POTASSIUM HEXAMETHYLDISILAZIDE KETOACYL SYNTHASE MICROTUBULE ASSOCIATED PROTEIN MULTICOMPONENT REACTION MULTIDRUG RESISTANCE MURINE ERYTHROLEUKEMIA MAJOR HISTOCOMPATIBILITY COMPLEX MYELIN OLIGODENDROCYTE PROTEIN MYCOPHENOLIC ACID MAGNETIC RESONANCE IMAGING
IX
'LOSSARY
-4 M4/2 .#% .#) .0 .203 /- /3-!# 0#! 0#0 P$#, 0+3 03! 03! 03-& 033# 2!## 2!3 2&)$ 22-3 3#/.0 3'! 3'$ 4#- 4#2 4%3 4%3#) 4%3/4F 4)03 530)/ 6(,
X
METHYLTRANSFERASE MAMMALIAN TARGET OF RAPAMYCIN NEW CHEMICAL ENTITY .ATIONAL #ANCER )NSTITUTE NATURAL PRODUCT NONRIBOSOMAL PEPTIDE SYNTHETASE ORGANIC MOLECULE ONE STRAIN MANY COMPOUNDS APPROACH PRINCIPAL COMPONENTS ANALYSIS PEPTIDYL CARRIER PROTEIN PSEUDO DYNAMIC LIBRARY POLYKETIDE SYNTHASE PROSTATE SPECIFIC ANTIGEN POLAR SURFACE AREA PROBABILISTIC SPARSE MATRIX FACTORIZATION PROTEIN STRUCTURE SIMILARITY CLUSTERING RECEPTOR ASSISTED COMBINATORIAL CHEMISTRY RECEPTOR ACCELERATED SYNTHESIS RADIO FREQUENCY IDENTIFICATION TAG RELAPSING REMITTING MULTIPLE SCLEROSIS STRUCTURAL CLASSIFICATION OF NATURAL PRODUCTS SYNTHETIC GENETIC ARRAY 3ACCHAROMYCES 'ENOME $ATABASE 4RADITIONAL #HINESE -EDICINE 4 CELL RECEPTOR TRIETHYLSILYL TRIETHYLCHLOROSILANE TRIFLUOROMETHANESULPHONATE TRIISOPROPYLSILYL ULTRA SMALL SUPERPARAMAGNETIC IRON OXIDE VON (IPPEL ,INDAU PROTEIN
0ROGRESS IN $RUG 2ESEARCH 6OL &RANK 0ETERSEN AND 2ENÏ !MSTUTZ %DS © "IRKHÊUSER 6ERLAG "ASEL 3WITZERLAND
!CTIVATION OF FUNGAL SILENT GENE CLUSTERS ! NEW AVENUE TO DRUG DISCOVERY
"Y !XEL ! "RAKHAGE *ULIA 3CHUEMANN 3EBASTIAN "ERGMANN +IRSTIN 3CHERLACH 6OLKER 3CHROECKH AND #HRISTIAN (ERTWECK -OLECULAR AND !PPLIED -ICROBIOLOGY ,EIBNIZ )NSTITUTE FOR .ATURAL 0RODUCT 2ESEARCH AND )NFECTION "IOLOGY (+) AND &RIEDRICH 3CHILLER 5NIVERSITY "EUTENBERGSTR A *ENA 'ERMANY AXELBRAKHAGE HKI JENADE "IOMOLECULAR #HEMISTRY ,EIBNIZ )NSTITUTE FOR .ATURAL 0RODUCT 2ESEARCH AND )NFECTION "IOLOGY (+) AND &RIEDRICH 3CHILLER 5NIVERSITY "EUTENBERGSTR A *ENA 'ERMANY
!CTIVATION OF FUNGAL SILENT GENE CLUSTERS ! NEW AVENUE TO DRUG DISCOVERY
!BSTRACT 4HE ONGOING EXPONENTIAL GROWTH OF $.! SEQUENCE DATA WILL LEAD TO THE DISCOVERY OF MANY NATURAL PRODUCT BIOSYNTHESIS PATHWAYS BY GENOME MINING FOR WHICH NO ACTUAL PRODUCT HAS BEEN CHARACTERISED )N MANY CASES THESE CLUSTERS REMAIN SILENT UNDER LABORATORY CONDITIONS .EW TECHNOLOGIES BASED ON GENETIC ENGINEERING ARE AVAILABLE TO INDUCE SILENT GENES (ETER OLOGOUS EXPRESSION OF A SILENT GENE CLUSTER UNDER THE CONTROL OF DEFINED PROMOTERS CAN BE APPLIED !LTERNATIVELY PROMOTERS OF BIOSYNTHESIS GENES WITHIN THE GENOME CAN BE EXCHANGED BY DEFINED PROMOTERS -OST PROMISING HOWEVER IS THE ACTIVATION OF PATHWAY SPECIFIC REGULA TORY GENES WHICH WAS RECENTLY DEMONSTRATED 3UCH REGULATORY GENES ARE PRESENT IN MANY SEC ONDARY METABOLITE GENE CLUSTERS 4HIS APPROACH IS RENDERED FEASIBLE BY THE FACT THAT ALL OF THE GENES ENCODING THE LARGE NUMBER OF ENZYMES REQUIRED FOR THE SYNTHESIS OF A TYPICAL SECONDARY METABOLITE ARE CLUSTERED AND THAT IN SOME CASES A SINGLE REGULATOR CONTROLS THE EXPRESSION OF ALL MEMBERS OF A GENE CLUSTER TO A CERTAIN EXTENT 4HE ADVANTAGE OF THIS TECHNIQUE IS THAT ONLY A SMALL GENE NEEDS TO BE HANDLED AND THAT AN ECTOPIC INTEGRATION IS SUFFICIENT BYPASSING ALL LIMITATIONS OF HOMOLOGOUS RECOMBINATION -OST CONVENIENTLY THIS STRATEGY CAN TRIGGER THE CONCERTED EXPRESSION OF ALL PATHWAY GENES 4HE VAST AMOUNT OF $.! SEQUENCES IN THE PUBLIC DATABASE REPRESENTS ONLY THE BEGINNING OF THIS NEW GENOMICS ERA 4HE ACTIVATION OF THESE GENE CLUSTERS BY GENETIC ENGINEERING WILL LEAD TO THE DISCOVERY OF MANY SO FAR UNKNOWN PRODUCTS AND THEREFORE REPRESENTS A NOVEL AVENUE TO DRUG DISCOVERY
)NTRODUCTION
.ATURAL PRODUCTS REMAIN A CONSISTENT SOURCE OF DRUG LEADS WITH MORE THAN OF NEW CHEMICAL ENTITIES REPORTED SINCE BEING DERIVED FROM MICROBIAL NATURAL PRODUCTS REVIEWED IN ;n= %VEN MORE REMARKABLE IS THAT MORE THAN OF THE ANTICANCER AND OF THE ANTI INFECTIVE ANTIBI OTICS CURRENTLY IN CLINICAL USE ARE NATURAL PRODUCTS OR NATURAL PRODUCT BASED COMPOUNDS REVIEWED IN ;= 3ECONDARY METABOLITES FROM MICROORGANISMS HAVE BEEN AND CONTINUE TO BE A LEADING SOURCE OF MOLECULES FOR DRUG DISCOVERY BUT NEW TECHNOLOGIES ARE REQUIRED TO INCREASE THE PROBABILITY OF IDENTIFYING NEW STRUCTURES REVIEWED IN ;= 7HILE BACTERIA AND FUNGI HAVE LONG BEEN KNOWN AS PROLIFIC PRODUCERS OF DRUG CANDIDATES ANALYSES OF THEIR GENOME SEQUENCES REVEALED THAT THERE ARE FAR MORE BIOSYNTHESIS GENE CLUS TERS THAN THERE ARE CURRENTLY KNOWN METABOLITES FOR A GIVEN ORGANISM 4HIS OBSERVATION STRONGLY SUGGESTS THAT THE BIOSYNTHESIS POTENTIAL FOR NATURAL PRODUCTS IN MICROORGANISMS HAS BEEN GREATLY UNDEREXPLORED BY TRADITIONAL METHODS OF NATURAL PRODUCT DISCOVERY AND THAT A MULTITUDE OF POTENTIALLY USEFUL METABOLITES STILL AWAITS DISCOVERY ;= 4HIS IS IN FULL AGREEMENT WITH THE ONE STRAIN MANY COMPOUNDS /3-!# APPROACH OF :EECK AND CO
!XEL ! "RAKHAGE ET AL
WORKERS ACCORDING TO WHICH VARYING GROWTH CONDITIONS CAN DRAMATICALLY INFLUENCE THE METABOLITE PROFILE OF BACTERIA AND FUNGI ;= -OREOVER ONLY n OF THE MICROBIAL COMMUNITY IS ESTIMATED TO HAVE BEEN CULTIVATED IN THE LAB IMPLYING THAT THERE IS A VAST BIODIVERSITY OF NATURAL PRODUCTS AND THEIR ENCODING BIOSYNTHESIS GENES IN MICROORGANISMS THAT REMAIN TO BE EXPLOITED REVIEWED IN ;=
)MPORTANCE OF GENOMIC DATA
-IXING BOTH GENOMIC DATA AND ANALYTICAL TECHNIQUES CAN BE A POWER FUL APPROACH TO THE DISCOVERY OF NOVEL AND POTENTIALLY BIOACTIVE NATURAL PRODUCTS 4ODAY WHEN THE GENOME SEQUENCE IS AT HAND IT IS POSSIBLE TO ESTIMATE THE BIOSYNTHESIS POTENTIAL FOR A GIVEN ORGANISM BY MINING THE WHOLE GENOME FOR TYPICAL SECONDARY METABOLITE BIOSYNTHESIS GENES 4HIS APPROACH IS PARTICULARLY PROMISING IN MICROORGANISMS BECAUSE MOST OF THE NATURAL PRODUCT BIOSYNTHESIS GENES ARE ORGANISED IN CLUSTERS 4HE FORMATION OF MANY IMPORTANT FUNGAL SECONDARY METABOLITES POLYKETIDES AND NONRI BOSOMAL PEPTIDES INVOLVES MULTIFUNCTIONAL ENZYMES POLYKETIDE SYNTHASES 0+3S AND NONRIBOSOMAL PEPTIDE SYNTHETASES .203S "OTH .203S AND 0+3S UTILISE SIMPLE MALONYL 0+3 OR AMINO ACID .203 BUILDING BLOCKS AND USE VERY SIMILAR STRATEGIES FOR THEIR ASSEMBLY !LTHOUGH THEY UTILISE DIFFERENT CLASSES OF SUBSTRATES 0+3S AND .203S SHOW STRIKING SIMILARITIES IN THE MODULAR ARCHITECTURE OF THEIR CATALYTIC DOMAINS ASSEMBLY LINE LIKE MECHANISMS 4HE EXTREMELY LARGE POLYFUNCTIONAL .203 ENZYME SYSTEMS ARE ORGANISED IN MODULES %ACH MODULE IS RESPONSIBLE FOR ONE DISCRETE CHAIN ELONGATION STEP AND CAN BE SUBDIVIDED INTO DOMAINS CONTROLLING THE CHOICE OF THE EXTENDER UNIT 3EVERAL TYPES OF MODIFICATIONS CAN TAKE PLACE ON EACH INTERMEDIATE !T A MINIMUM A TYPICAL .203 MODULE CONSISTS OF AN ADENYLATION ! DOMAIN RESPONSIBLE FOR AMINO ACID ACTIVATION A THIOLA TION 4 DOMAIN n ALSO KNOWN AS PEPTIDYL CARRIER PROTEIN 0#0 THAT BINDS THE COFACTOR PHOSPHOPANTETHEINE 00 TO WHICH THE ACTIVATED AMINO ACID IS COVALENTLY ATTACHED AND A CONDENSATION # DOMAIN THAT CATALYSES PEPTIDE BOND FORMATION !DDITIONALLY A VARIETY OF OPTIONAL EG METHYL TRANSFERASE -4 OR EPIMERISATION % DOMAINS HAVE ALSO BEEN DESCRIBED ;= 3IMILARLY THREE DOMAINS ARE NECESSARY AS THE BASIC EQUIPMENT OF A 0+3 ELONGATION MODULE AN ACYLTRANSFERASE !4 DOMAIN FOR EXTENDER UNIT SELEC
!CTIVATION OF FUNGAL SILENT GENE CLUSTERS ! NEW AVENUE TO DRUG DISCOVERY
TION AND TRANSFER AN ACYL CARRIER PROTEIN !#0 FOR EXTENDER UNIT LOADING AND A KETOACYL SYNTHASE +3 DOMAIN FOR DECARBOXYLATIVE CONDENSATION OF THE EXTENDER UNIT USUALLY MALONYL #O! OR METHYLMALONYL #O! WITH AN ACYL THIOESTER 4HE RESULTING β KETO THIOESTER MAY SUBSEQUENTLY BE PROCESSED BY β KETOACYL REDUCTASE +2 DOMAINS DEHYDRATASE $( DOMAINS ENOYL REDUCTASE %2 DOMAINS AND -4 DOMAINS ;= )N BACTERIA VARIOUS EXAMPLES WERE REPORTED SHOWING THAT THESE MODULAR 0+3 AND .203 SYSTEMS CAN ALSO CLOSELY COOPERATE TO FORM SO CALLED HYBRID PRODUCTS ;n= 4YPICALLY THE NASCENT POLYKETIDE OR PEPTIDE BACKBONE IS FURTHER MODIFIED BY TAILORING ENZYMES IN POST 0+3 OR POST .203 STEPS OF THE PATHWAY SUCH AS OXYGENASES AND GLYCOSYLTRANSFERASES OR OTHER TRANSFERASES IN ORDER TO IMBUE ADDITIONAL STRUCTURAL FUNCTIONALITIES TO THE FINAL NATURAL PRODUCT 4HE CONSERVED FEA TURES WITHIN THIS MACHINERY HAVE BEEN CORNERSTONES OF THE GENOMIC GUIDED DISCOVERY OF NATURAL PRODUCTS ;n= REVIEWED IN ;=
2ECENT GENOME DISCOVERY
! SYSTEMATIC GENOMIC MINING UNCOVERED THE OCCURRENCE OF CRYPTIC 0+3 ANDOR .203 BIOSYNTHESIS GENES IN ACTINOMYCETES ; = MYXOBACTERIA ; = AND 0SEUDOMONAS ;n= 2ECENTLY THE GENOMES OF THE FILAMEN TOUS FUNGI !SPERGILLUS NIDULANS ! FUMIGATUS AND ! ORYZAE WERE PUBLISHED ;n= 3EQUENCE ALIGNMENTS SUGGEST THAT ! NIDULANS HAS THE POTENTIAL TO GENERATE UP TO POLYKETIDES NONRIBOSOMAL PEPTIDES AND TWO INDOLE ALKALOIDS SIMILAR PREDICTIONS CAN BE MADE FROM THE ! FUMIGATUS AND ! ORYZAE GENOMES 4AB ;= )NTERESTINGLY THERE APPEARS TO BE ALMOST NO ORTHOLOGUES AMONG THESE GENES ACROSS THE THREE SPECIES THUS REPRESENT ING A LOSS OF SYNTENY TO A DEGREE NOT SEEN IN OTHER REGIONS OF THE GENOMES ;= 4HIS HIGH NUMBER OF PUTATIVE METABOLITES IS GREATER THAN THE KNOWN METABOLITES ASCRIBED TO THESE SPECIES AND IT MAY BE A REFLECTION OF INCOM PLETE NATURAL PRODUCT ANALYSIS IN THESE SPECIES OR FAILURE OF MANY CLUSTERS TO BE EXPRESSED AT LEAST UNDER THE CULTURE CONDITIONS COMMONLY USED IN LABORATORIES ;= &OR EXAMPLE THE AFLATOXIN GENE CLUSTER IS NOT TRANSCRIBED IN ! ORYZAE ; = REVIEWED IN ;= !LSO FOR THESE FUNGI IT IS APPARENT THAT THIS VAST NUMBER OF PREDICTED BIOSYNTHESIS GENES OF MICROORGANISMS IS NOT REFLECTED BY THE METABOLIC PROFILE OBSERVED UNDER STANDARD FERMENTATION CONDITIONS )N THE ABSENCE
!XEL ! "RAKHAGE ET AL
4ABLE 0UTATIVE SECONDARY METABOLISM GENE TYPES 0UTATIVE GENES
! NIDULANS
! FUMIGATUS
! ORYZAE
.203
0+3
$-!4
.203 NONRIBOSOMAL PEPTIDE SYNTHETASE 0+3 POLYKETIDE SYNTHASE $-!4 DIMETHYLALLYL TRYPTOPHAN SYNTHASE 'ALAGAN ET AL .ATURE n #RAMER ET AL 'ENE n GENOME PROJECT AT WWWBROADMITEDU .IERMAN ET AL .ATURE n #RAMER ET AL 'ENE n GENOME PROJECT AT WWWBROADMITEDU -ACHIDA ET AL .ATURE n #RAMER ET AL 'ENE n GENOME PROJECT AT WWWBROADMITEDU
INCLUDES ONE HYBRID 0+3.203
OF A PARTICULAR TRIGGER THESE GENE LOCI REMAIN SILENT 3INCE THESE CRYPTIC GENE CLUSTERS COULD CODE FOR THE BIOSYNTHESIS OF IMPORTANT VIRULENCE FAC TORS TOXINS OR EVEN DRUG CANDIDATES NEW STRATEGIES FOR THEIR ACTIVATION ARE URGENTLY NEEDED TO MAKE USE OF THIS LARGELY UNTAPPED RESERVOIR OF POTEN TIALLY BIOACTIVE COMPOUNDS 2ECENTLY WE REPORTED A NEW STRATEGY FOR THE SUCCESSFUL INDUCTION OF A SILENT METABOLIC PATHWAY IN THE IMPORTANT MODEL ORGANISM ! NIDULANS WHICH LED TO THE DISCOVERY OF NOVEL 0+3 .203 HYBRID METABOLITES NAMED ASPYRIDONES ! AND " ;= -INING OF THE ! NIDULANS GENOME FOR CRYPTIC OR ORPHAN GENE CLUSTERS THAT COULD CODE FOR THE BIOSYNTHESIS OF POLYKETIDES OR NONRIBOSOMAL POLYPEPTIDES WE NOTED THE PRESENCE OF A PUTATIVE HYBRID 0+3 .203 GENE APD! 4HE DEDUCED GENE PRODUCT SHOWS TYPICAL MOTIFS OF 0+3 AND .203 DOMAINS AS WELL AS AN ADDITIONAL # TERMINAL REDUCTASE DOMAIN AND OBVIOUSLY REPRESENTS A RARE FUNGAL 0+3 .203 HYBRID 4HE 0+3 .203 ENCODING GENE IS FLANKED BY UPSTREAM GENES CODING FOR SEVERAL PUTATIVE OXIDOREDUCTASES TWO CYTOCHROME 0 MONOOXYGENASES !PD" !PD% AN &!$ DEPENDENT MONOOXYGENASE !PD$ AND AN ENOYL REDUCTASE !PD# $OWNSTREAM OF APD! THE PUTATIVE EXPORTER APD& AND ACTIVATOR APD2 GENES ARE LOCATED $ESPITE THE VAST METABOLIC DATA AVAILABLE FOR ! NIDULANS THE ROLE OF THIS GENE CLUSTER COULD NOT BE CLARIFIED 4O DATE ONLY TWO RELATED FUNGAL
!CTIVATION OF FUNGAL SILENT GENE CLUSTERS ! NEW AVENUE TO DRUG DISCOVERY
0+3 .203 HYBRID SYNTHASES HAVE BEEN ATTRIBUTED TO METABOLIC FUNCTIONS 'ENE INACTIVATION EXPERIMENTS REVEALED THAT THEY ARE INVOLVED IN THE BIO SYNTHESIS OF THE TETRAMIC ACID DERIVATIVES EQUISETIN AND FUSARIN IN &USARIUM HETEROSPORUM AND &USARIUM FUJIKUROI RESPECTIVELY ; =
'ENE ANALYSIS
&OR THE FUNCTIONAL ANALYSIS OF CRYPTIC GENES GENE INACTIVATION WOULD ONLY PROVIDE AN OPTION IF THE METABOLITE WAS CONSTITUTIVELY PRODUCED BY THE STRAIN 4HIS METHOD IS CLEARLY NOT APPLICABLE FOR GENE CLUSTERS THAT ARE SILENT UNDER STANDARD FERMENTATION CONDITIONS AND THUS TOOLS ARE NEEDED TO ASSIGN THEIR BIOSYNTHETIC ROLE )N GENERAL SEVERAL STRATEGIES WOULD BE CONCEIVABLE )N THE LATTER CASE GENE EXPRESSION NEEDS TO BE INDUCED FOR EXAMPLE BY HETEROLOGOUS EXPRESSION UNDER THE CONTROL OF DEFINED PROMOT ERS &IG ! OR BY PROMOTER EXCHANGE WITHIN THE GENOME HOMOLOGOUS EXPRESSION &IG " 4HE LATTER WOULD BE FEASIBLE IN PARTICULAR FOR CLUSTERS WITH POLYCISTRONIC GENE ORGANISATION )N CASE OF FUNGAL SECONDARY METABO LISM GENE CLUSTERS PROMOTER EXCHANGE CAN BE CUMBERSOME AND IS USUALLY EMPLOYED FOR INDIVIDUAL GENES ONLY ;= /BVIOUSLY THE LATTER APPROACH HAS NUMEROUS DISADVANTAGES 4HE TECHNIQUE IS RESTRICTED TO ORGANISMS THAT ARE AMENABLE TO GENETIC TECHNIQUES &URTHERMORE MANY FUNGI ONLY SHOW A VERY LOW FREQUENCY OF HOMOLOGOUS RECOMBINATION WHICH HAMPERS PROMOTER EXCHANGE )N ADDITION TO THE CUMBERSOME HANDLING OF LARGE GENE CONSTRUCTS ANOTHER DRAWBACK IS THAT OVEREXPRESSION OF A SINGLE GENE OF A CLUSTER OFTEN LEADS TO LIMITATION OF ANOTHER GENE PRODUCT OF THE SAME CLUSTER &INALLY GENE EXPRESSION AND AS IN THE LOVASTATIN PATHWAY THE FUNC TIONING OF ENZYMES MAY BE CONTEXT DEPENDENT REVIEWED IN ;= ! SOLUTION OF THIS PROBLEM LIES IN THE EXPRESSION OF PATHWAY SPECIFIC REGULATORY GENES WHICH ARE PRESENT IN MANY SECONDARY METABOLITE GENE CLUSTERS &IG # 4HIS APPROACH IS RENDERED FEASIBLE BY THE FACT THAT ALL OF THE GENES ENCODING THE LARGE NUMBER OF ENZYMES REQUIRED FOR THE SYNTHESIS OF A TYPICAL SECONDARY METABOLITE ARE CLUSTERED AND THAT IN SOME CASES A SINGLE REGULATOR CONTROLS THE EXPRESSION OF ALL MEMBERS OF A GENE CLUSTER TO A CERTAIN EXTENT ;= 4HE ADVANTAGE OF THIS TECHNIQUE WOULD BE THAT ONLY A SMALL GENE NEEDS TO BE HANDLED AND THAT AN ECTOPIC INTEGRATION WOULD BE SUFFICIENT BYPASSING ALL LIMITATIONS OF HOMOLOGOUS RECOMBINATION
!XEL ! "RAKHAGE ET AL
&IGURE -OLECULAR METHODS TO INDUCE SILENT GENE CLUSTERS ! %XPRESSION OF GENE CLUSTER IN A HETEROLOGOUS HOST " 0ROMOTER EXCHANGE OF INDIVIDUAL GENES OF A CLUSTER USING STRONG REGULATABLE PROMOTERS # /VEREXPRESSION OF TRANSCRIPTION FACTORS
-OST CONVENIENTLY THIS STRATEGY WOULD ALLOW FOR THE CONCERTED EXPRESSION OF ALL PATHWAY GENES 2ECENTLY WE COULD SUCCESSFULLY APPLY THIS STRATEGY ;= 4HE CRYPTIC ! NIDULANS 0+3 .203 GENE CLUSTER CONTAINS A PUTATIVE ACTIVATOR GENE DESIGNATED APD2 4HE DEDUCED GENE PRODUCT IS RELATED TO A PUTATIVE # TRANSCRIPTION FACTOR OF ! FUMIGATUS AND TO A PUTATIVE REGULATOR WITH A '!, TYPE :N#YS BINUCLEAR CLUSTER $.! BINDING DOMAIN FROM ! FLAVUS 4O PROVE THE CONCEPT THAT THE HOMOLOGOUS OVEREXPRESSION OF A REGULATORY GENE CAN LEAD TO ACTIVATION OF A SILENT GENE CLUSTER THE PUTATIVE ACTIVATOR GENE APD2 WAS AMPLIFIED FROM GENOMIC $.! AND CLONED INTO AN EXPRESSION VECTOR CARRYING THE PROMOTER OF THE ALCOHOL DEHYDROGENASE OF ! NIDULANS &IG 4HIS PROMOTER CAN BE INDUCED BY THE ADDITION OF CYCLOPENTANONE TO THE MEDIUM AND REPRESSED BY THE USE OF GLUCOSE AS THE CARBON SOURCE ;=
!CTIVATION OF FUNGAL SILENT GENE CLUSTERS ! NEW AVENUE TO DRUG DISCOVERY
&IGURE !CTIVATION OF A SILENT GENE CLUSTER IN !SPERGILLUS NIDULANS BY OVEREXPRESSION OF THE TRANSCRIPTION FACTOR GENE APD2 LED TO THE IDENTIFICATION OF THE NOVEL NATURAL PRODUCTS ASPYRIDONE ! AND " ;=
4RANSFORMANTS OF ! NIDULANS CARRYING THE ALC!P APD2 GENE FUSION ECTOP ICALLY INTEGRATED INTO THE GENOME WERE CHECKED FOR TRANSCRIPTION OF GENES OF THE CLUSTER BY .ORTHERN BLOT ANALYSIS 7HEREAS THE TRANSCRIPTS WERE COMPLETELY ABSENT IN THE WILD TYPE UNDER BOTH NON INDUCING AND INDUCING CONDITIONS IN THE TRANSFORMANT STRAIN UNDER INDUCING CONDITIONS STRONG M2.! SIGNALS WERE DETECTED FOR THE GENES OF THE CLUSTER INCLUDING THE 0+3 .203 HYBRID 4HIS OBSERVATION FURTHER CONFIRMED THAT UNDER THE CONDI TIONS APPLIED THIS GENE CLUSTER WAS SILENT WITHOUT INDUCTION &URTHERMORE IT ALSO HELPED TO CONFINE THE BORDERS OF THE GENE CLUSTER ;=
#HEMICAL ANALYSIS
(0,# COUPLED TO $!$ AND -3 DETECTORS REVEALED THAT UNDER INDUCING CONDITIONS THE TRANSFORMANT STRAINS PRODUCED TWO MAIN PRODUCTS AS WELL AS TWO MINOR COMPOUNDS 4HE MAIN METABOLITES WERE ISOLATED AND THEIR STRUCTURES WERE ELUCIDATED BY $ AND $ .-2 MEASUREMENTS AS WELL AS BY )2 AND -3 ;= 4HE NOVEL METABOLITES PRODUCED BY THE TRANSFORMANT STRAIN WERE NAMED ASPYRIDONES ! AND " DUE TO THEIR PYRIDONE MOIETIES &IG 4HEY ARE SIMILAR BUT NOT IDENTICAL TO A VARIETY OF OTHER PYRIDONES ISOLATED FROM FUNGI SUCH AS MILITARINONE $ AND TENELLIN ; = )N A BROAD BIOAC TIVITY SCREENING THEY EXHIBITED MODERATE CYTOTOXIC ACTIVITIES 4HE FUNCTION
!XEL ! "RAKHAGE ET AL
OF THE ASPYRIDONES IN THE NATURAL CONTEXT AND POSSIBLE TRIGGERS FOR THE ONSET OF THEIR BIOSYNTHESIS REMAIN THE SUBJECT OF ONGOING INVESTIGATIONS (OWEVER THE RESULTS CITED HERE PROVIDE THE PROOF OF PRINCIPLE FOR A STRAT EGY THAT MAY BE GENERALLY APPLICABLE TO THE ACTIVATION OF SILENT BIOSYNTHESIS GENE CLUSTERS IN PARTICULAR IN EUKARYOYTES
#ONCLUSION
4HE ONGOING EXPONENTIAL GROWTH OF $.! SEQUENCE DATA WILL LEAD TO THE DIS COVERY OF MANY NATURAL PRODUCT BIOSYNTHESIS PATHWAYS BY GENOME MINING FOR WHICH NO ACTUAL PRODUCT HAS BEEN CHARACTERISED 4HE VAST AMOUNT OF $.! SEQUENCES IN THE PUBLIC DATABASE REPRESENTS ONLY THE BEGINNING OF THIS NEW GENOMICS ERA 4HE ACTIVATION OF THESE GENE CLUSTERS BY GENETIC ENGINEERING WILL LEAD TO THE DISCOVERY OF MANY SO FAR UNKNOWN PRODUCTS AND THEREFORE REPRESENTS A NOVEL AVENUE TO DRUG DISCOVERY
2EFERENCES
#LARDY * 7ALSH # ,ESSONS FROM NATURAL MOLECULES .ATURE n +HOSLA # (ARNESSING THE BIOSYNTHETIC POTENTIAL OF MODULAR POLYKETIDE SYN THASES #HEM 2EV n 3IEBER 3! -ARAHIEL -! -OLECULAR MECHANISMS UNDERLYING NONRIBOSOMAL PEPTIDE SYNTHESIS APPROACHES TO NEW ANTIBIOTICS #HEM 2EV n 9IN * 3TRAIGHT 0$ (RVATIN 3 $ORRESTEIN 0# "UMPUS 3" *AO # +ELLEHER ., +OLTER 2 7ALSH #4 'ENOME WIDE HIGH THROUGHPUT MINING OF NATURAL PRODUCT BIOSYN THETIC GENE CLUSTERS BY PHAGE DISPLAY #HEM "IOL n -C!LPINE *" "ACHMANN "/ 0IRAEE - 4REMBLAY 3 !LARCO !- :AZOPOULOS % &ARNET #- -ICROBIAL GENOMICS AS A GUIDE TO DRUG DISCOVERY STRUCTURAL ELUCIDATION %#/ A NOVEL ANTIFUNGAL AGENT AS AN EXAMPLE * .AT 0ROD n 0ERIC #ONCHA . ,ONG 0& -INING THE MICROBIAL METABOLOME A NEW FRONTIER FOR NATURAL PRODUCT LEAD DISCOVERY $RUG $ISCOV 4ODAY n "ODE (" "ETHE " (OFS 2 :EECK ! "IG EFFECTS FROM SMALL CHANGES POSSIBLE WAYS TO EXPLORE NATURES CHEMICAL DIVERSITY #HEMBIOCHEM n 7ALSH #4 #HEN ( +EATING 4! (UBBARD "+ ,OSEY (# ,UO , -ARSHALL #' -ILLER $! 0ATEL (- 4AILORING ENZYMES THAT MODIFY NONRIBOSOMAL PEPTIDES DURING AND AFTER CHAIN ELONGATION ON .203 ASSEMBLY LINES #URR /PIN #HEM "IOL n #ANE $% )NTRODUCTION 0OLYKETIDE AND NONRIBOSOMAL POLYPEPTIDE BIOSYNTHE SIS &ROM COLLIE TO COLI #HEM 2EV n #ANE $% 7ALSH #4 4HE PARALLEL AND CONVERGENT UNIVERSES OF POLYKETIDE SYN THASES AND NONRIBOSOMAL PEPTIDE SYNTHETASES #HEM "IOL 2n
!CTIVATION OF FUNGAL SILENT GENE CLUSTERS ! NEW AVENUE TO DRUG DISCOVERY
0AITAN 9 !LON ' /RR % 2ON %: 2OSENBERG % 4HE FIRST GENE IN THE BIOSYNTHESIS OF THE POLYKETIDE ANTIBIOTIC 4! OF -YXOCOCCUS XANTHUS CODES FOR A UNIQUE 0+3 MODULE COUPLED TO A PEPTIDE SYNTHETASE * -OL "IOL n 3ILAKOWSKI " 3CHAIRER (5 %HRET ( +UNZE " 7EINIG 3 .ORDSIEK ' "RANDT 0 "LOCKER ( (OFLE ' "EYER 3 ET AL .EW LESSONS FOR COMBINATORIAL BIOSYNTHESIS FROM MYXOBACTERIA 4HE MYXOTHIAZOL BIOSYNTHETIC GENE CLUSTER OF 3TIGMATELLA AURANTIACA $7 * "IOL #HEM n 7ENZEL 3# +UNZE " (OFLE ' 3ILAKOWSKI " 3CHARFE - "LOCKER ( -ULLER 2 3TRUCTURE AND BIOSYNTHESIS OF MYXOCHROMIDES 3 IN 3TIGMATELLA AURANTIACA EVIDENCE FOR AN ITERATIVE BACTERIAL TYPE ) POLYKETIDE SYNTHASE AND FOR MODULE SKIPPING IN NON RIBOSOMAL PEPTIDE BIOSYNTHESIS #HEMBIOCHEM n )KEDA ( )SHIKAWA * (ANAMOTO ! 3HINOSE - +IKUCHI ( 3HIBA 4 3AKAKI 9 (ATTORI - /MURA 3 #OMPLETE GENOME SEQUENCE AND COMPARATIVE ANALYSIS OF THE INDUS TRIAL MICROORGANISM 3TREPTOMYCES AVERMITILIS .AT "IOTECHNOL n ,IU 7 #HRISTENSON 3$ 3TANDAGE 3 3HEN " "IOSYNTHESIS OF THE ENEDIYNE ANTI TUMOR ANTIBIOTIC # 3CIENCE n -ENZELLA (' 2EID 2 #ARNEY *2 #HANDRAN 33 2EISINGER 3* 0ATEL +' (OPWOOD $! 3ANTI $6 #OMBINATORIAL POLYKETIDE BIOSYNTHESIS BY DE NOVO DESIGN AND REAR RANGEMENT OF MODULAR POLYKETIDE SYNTHASE GENES .AT "IOTECHNOL n ,AUTRU 3 $EETH 2* "AILEY ,- #HALLIS ', $ISCOVERY OF A NEW PEPTIDE NATURAL PRODUCT BY 3TREPTOMYCES COELICOLOR GENOME MINING .AT #HEM "IOL n :AZOPOULOS % (UANG + 3TAFFA ! ,IU 7 "ACHMANN "/ .ONAKA + !HLERT * 4HORSON *3 3HEN " &ARNET #- ! GENOMICS GUIDED APPROACH FOR DISCOVERING AND EXPRESSING CRYPTIC METABOLIC PATHWAYS .AT "IOTECHNOL n "ODE (" -ULLER 2 4HE IMPACT OF BACTERIAL GENOMICS ON NATURAL PRODUCT RESEARCH !NGEW #HEM )NT %D %NGL n "RENDEL . 0ARTIDA -ARTINEZ ,0 3CHERLACH + (ERTWECK # ! CRYPTIC 0+3 .203 GENE LOCUS IN THE PLANT COMMENSAL 0SEUDOMONAS FLUORESCENS 0F CODES FOR THE BIOSYN THESIS OF AN ANTIMITOTIC RHIZOXIN COMPLEX /RG "IOMOL #HEM n #HALLIS ', ! WIDELY DISTRIBUTED BACTERIAL PATHWAY FOR SIDEROPHORE BIOSYNTHESIS INDEPENDENT OF NONRIBOSOMAL PEPTIDE SYNTHETASES #HEMBIOCHEM 'ROSS ( 3TOCKWELL 6/ (ENKELS -$ .OWAK 4HOMPSON " ,OPER *% 'ERWICK 7( 4HE GENOMISOTOPIC APPROACH A SYSTEMATIC METHOD TO ISOLATE PRODUCTS OF ORPHAN BIOSYNTHETIC GENE CLUSTERS #HEM "IOL n 'ALAGAN *% #ALVO 3% #UOMO # -A ,* 7ORTMAN *2 "ATZOGLOU 3 ,EE 3) "ASTURK MEN - 3PEVAK ## #LUTTERBUCK * ET AL 3EQUENCING OF !SPERGILLUS NIDULANS AND COMPARATIVE ANALYSIS WITH ! FUMIGATUS AND ! ORYZAE .ATURE n -ACHIDA - !SAI + 3ANO - 4ANAKA 4 +UMAGAI 4 4ERAI ' +USUMOTO + !RIMA 4 !KITA / +ASHIWAGI 9 ET AL 'ENOME SEQUENCING AND ANALYSIS OF !SPERGILLUS ORYZAE .ATURE n .IERMAN 7# 0AIN ! !NDERSON -* 7ORTMAN *2 +IM (3 !RROYO * "ERRIMAN - !BE + !RCHER $" "ERMEJO # ET AL 'ENOMIC SEQUENCE OF THE PATHOGENIC AND ALLERGENIC FILAMENTOUS FUNGUS !SPERGILLUS FUMIGATUS .ATURE n #RAMER 2! *R 3TAJICH *% 9AMANAKA 9 $IETRICH &3 3TEINBACH 7* 0ERFECT *2 0HYLOGENOMIC ANALYSIS OF NON RIBOSOMAL PEPTIDE SYNTHETASES IN THE GENUS !SPERGILLUS 'ENE n
!XEL ! "RAKHAGE ET AL
"OK *7 (OFFMEISTER $ -AGGIO (ALL ,! -URILLO 2 'LASNER *$ +ELLER .0 'ENOMIC MINING FOR !SPERGILLUS NATURAL PRODUCTS #HEM "IOL n 3CHERLACH + (ERTWECK # $ISCOVERY OF ASPOQUINOLONES ! $ PRENYLATED QUI NOLINE ONE ALKALOIDS FROM !SPERGILLUS NIDULANS MOTIVATED BY GENOME MINING /RG "IOMOL #HEM n VAN DEN "ROEK 0 0ITTET ! (AJJAJ ( !FLATOXIN GENES AND THE AFLATOXIGENIC POTEN TIAL OF +OJI MOULDS !PPL -ICROBIOL "IOTECHNOL n :HANG 9 1 7ILKINSON ( +ELLER .0 4SITSIGIANNIS $) 3ECONDARY METABOLITE GENE CLUSTERS )N : !N ED (ANDBOOK OF )NDUSTRIAL -ICROBIOLOGY -ARCEL $EKKER .EW 9ORK n "ERGMANN 3 3CHUEMANN * 3CHERLACH + ,ANGE # "RAKHAGE !! (ERTWECK # 'ENOMICS DRIVEN DISCOVERY OF 0+3 .203 HYBRID METABOLITES FROM !SPERGILLUS NIDULANS .AT #HEM "IOL n 3IMS *7 &ILLMORE *0 7ARNER $$ 3CHMIDT %7 %QUISETIN BIOSYNTHESIS IN &USARIUM HETEROSPORUM #HEM #OMMUN #AMB n 3ONG : #OX 2* ,AZARUS #- 3IMPSON 44 &USARIN # BIOSYNTHESIS IN &USARIUM MONILIFORME AND &USARIUM VENENATUM #HEMBIOCHEM n +ENNEDY * 4URNER ' DELTA , ALPHA AMINOADIPYL , CYSTEINYL $ VALINE SYN THETASE IS A RATE LIMITING ENZYME FOR PENICILLIN PRODUCTION IN !SPERGILLUS NIDULANS -OL 'EN 'ENET n 7ARING 2" -AY '3 -ORRIS .2 #HARACTERIZATION OF AN INDUCIBLE EXPRESSION SYSTEM IN !SPERGILLUS NIDULANS USING ALC! AND TUBULIN CODING GENES 'ENE n %LEY +, (ALO ,- 3ONG : 0OWLES ( #OX 2* "AILEY !- ,AZARUS #- 3IMPSON 4* "IOSYNTHESIS OF THE PYRIDONE TENELLIN IN THE INSECT PATHOGENIC FUNGUS "EAU VERIA BASSIANA #HEMBIOCHEM n 3CHMIDT + 2IESE 5 ,I : (AMBURGER - .OVEL TETRAMIC ACIDS AND PYRIDONE ALKALOIDS MILITARINONES " # AND $ FROM THE INSECT PATHOGENIC FUNGUS 0AECILOMYCES MILITARIS * .AT 0ROD n
0ROGRESS IN $RUG 2ESEARCH 6OL &RANK 0ETERSEN AND 2ENÏ !MSTUTZ %DS © "IRKHÊUSER 6ERLAG "ASEL 3WITZERLAND
4OTAL SYNTHESIS STUDIES ON MACRO CYCLIC PIPECOLIC ACID NATURAL PRODUCTS &+ THE ANTASCOMICINS AND RAPAMYCIN
"Y -ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY $EPARTMENT OF #HEMISTRY 5NIVERSITY OF #AMBRIDGE ,ENSFIELD 2OAD #AMBRIDGE #" %7 5+ SVL CAMACUK
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
!BSTRACT 4HIS CHAPTER DERIVES ITS INSPIRATION FROM THE CHALLENGES PRESENTED TO TOTAL SYNTHESIS CHEM ISTS BY A PARTICULAR GROUP OF MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS !LTHOUGH THERE IS CONSIDERABLE EMPHASIS ON THE COMPLETED SYNTHESES OF THE MAIN CHARACTERS &+ THE ANTASCOMYCINS AND AND RAPAMYCIN THE OVERALL COMPLEXITY OF THE MOLECULAR PROB LEM HAS STIMULATED A WEALTH OF NEW KNOWLEDGE INCLUDING THE DEVELOPMENT OF NOVEL STRATEGIES AND THE INVENTION OF NEW SYNTHETIC METHODS 4HE INGENIOUS AND INNOVATIVE APPROACHES TO THESE TARGETS HAVE ENABLED NEW GENERATIONS OF ANALOGUES AND PROVIDED MATERIAL TO FURTHER PROBE THE BIOLOGY OF THESE FASCINATING MOLECULES 7ITH PHARMACEUTICAL APPLICATION AS AN IMMUNOSUPPRESSANT AS WELL AS POTENTIAL USE FOR THE TREATMENT OF CANCER AND NEURODEGENERA TIVE DISEASES THIS FAMILY OF NATURAL PRODUCTS CONTINUES TO INSPIRE NEW AND INTERESTING SCIENCE WHILE PROVIDING SOLUTIONS TO HEALTHCARE PROBLEMS OF THE WORLD
)NTRODUCTION
7HETHER IT IS THEIR FASCINATING BIOLOGICAL FUNCTION OR REMARKABLE STRUCTURAL ARCHITECTURE COMPOUNDS ISOLATED FROM NATURAL SOURCES HAVE PLAYED AN INTEGRAL ROLE IN THE INSPIRATION AND ADVANCEMENT OF SCIENTIFIC KNOWLEDGE ACROSS A WIDE RANGE OF DISCIPLINES ;n= 4HIS IS ESPECIALLY TRUE IN THE DOMIN ION OF ORGANIC CHEMISTRY WHERE THE CHALLENGE INHERENT IN MIMICKING AND RECREATING NATURES CONSTRUCTIVE MACHINERY DRIVES BOTH INTELLECTUAL AND EXPERIMENTAL DEVELOPMENT &ROM A PRACTICAL STANDPOINT THE SEEMINGLY ENDLESS STRUCTURAL VARIETY ASSOCIATED WITH THE NATURAL PRODUCT ESTATE HAS PROVED INVALUABLE FOR THE DEVELOPMENT OF FRONT LINE THERAPEUTIC AGENTS ;n= $ESPITE A RECENT FIXATION ON THE CREATION OF SYNTHETIC SMALL MOL ECULE LIBRARIES AS A DISCOVERY PLATFORM FOR DE NOVO DRUG DEVELOPMENT ; = NATURAL PRODUCTS OR NATURAL PRODUCT DERIVEDINSPIRED ENTITIES STILL OCCUPY A DISPROPORTIONATE PERCENTAGE OF NEW CHEMICAL COMPOUNDS APPROVED AS PHARMACEUTICAL AGENTS BY THE &OOD AND $RUG !DMINISTRATION &$! ;= )N AN ERA OF SYNERGISTIC ADVANCEMENT OF ALL SCIENCES COUPLED WITH VAST IMPROVEMENTS IN COMMUNICATION AND INFORMATION MANAGEMENT THE INEVITABLE LESSONS AND BENEFITS THAT NATURE HAS YET TO ENDOW THROUGH FUR THER STUDY HOLD TREMENDOUS PROMISE ! WONDERFUL SUCCESS STORY THAT BEAUTIFULLY ILLUSTRATES THESE CONCEPTS IS EXEMPLIFIED BY A CLASS OF TRICARBONYL PIPECOLIC ACID MACROLIDES &IG WHICH TO DATE ENCOMPASSES &+ TACROLIMUS ;n= &+ ASCOMY CIN ;n= , HYDROXYASCOMYCIN ; = THE ANTASCO MICINS INCLUDING AND ;= MERIDAMYCIN ;n= AND THE RAPAMY
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
&IGURE 4HE TRICARBONYL PIPECOLIC ACID MACROLIDES FAMILY OF NATURAL PRODUCTS
CINS AND ;n= !S WELL AS THE OBVIOUS STRUCTURAL SIMILARITIES WITHIN THIS FAMILY OF NATURAL PRODUCTS MOST NOTABLY INCLUDING THE MACROCYCLIC RING CONTAINING BOTH MASKED TRICARBONYL AND PIPECOLIC MOIETIES ALL OF THE ABOVE COMPOUNDS SHARE THE SAME INITIAL BIOLOGICAL TARGET WITHIN LIVING SYSTEMS )DENTIFIED BY 3CHREIBER IN THE LATE S BY AFFINITY CHROMATOG RAPHY USING A MATRIX DERIVED FROM &+ AND TERMED &+"0 FOR +$A &+ BINDING PROTEIN " &IG THIS TARGET WAS FOUND TO BE PEPTI DYLPROLYL CIS TRANS ISOMERASE 00)ASE ;= 0REVIOUSLY DESCRIBED BY &ISCHER IN SUCH ENZYMES ARE RESPONSIBLE FOR CATALYSING THE INTERCONVERSION OF CIS AND TRANS ROTAMERS OF AMIDE BONDS ADJACENT TO PROLINE RESIDUES IN PEPTIDIC SUBSTRATES AND CONSEQUENTLY PLAY AN IMPORTANT ROLE IN PROTEIN FOLDING &IG ; = )T IS BELIEVED THAT THE PIPECOLYL KETOAMIDE PORTION OF &+ FUNC TIONS AS A TRANSITION STATE MIMIC FOR THE NATURAL PEPTIDYLPROLYL SUBSTRATE /RIGINALLY IT WAS FURTHER SPECULATED THAT THE FORMATION OF A TETRAHEDRAL ADDUCT WITHIN THE ACTIVE SITE OF &+"0 WITH EITHER THE # OR THE # ELEC TROPHILIC CARBONYL OF WAS RESPONSIBLE FOR THE INHIBITION OF ITS ROTAMASE ACTIVITY # &IG ; = 4HIS WAS SUBSEQUENTLY DISPROVED BY 3CHREIBER
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
&IGURE ! 2OTAMASE ACTIVITY " 2IBBON DIAGRAM OF &+"0 RED SHORT α HELIX BLUE FIVE STRANDED ANTIPARALLEL β SHEET YELLOW FLEXIBLE CONNECTING LOOPS ;n= 2EPRINTED WITH PERMISSION FROM ;= #OPYRIGHT !MERICAN #HEMICAL 3OCIETY 0OSSIBLE MECHANISMS OF INHIBITION OF &+"0 BY &+ # A COVA LENTLY BOUND ENZYME TETRAHEDRAL INTERMEDIATE $ A TWISTED AMIDE SURROGATE
THROUGH AN ELEGANT EXPERIMENT WHEREIN A SYNTHETIC ;# #= DOUBLY RADIOLABELLED &+ ANALOGUE VIDE INFRA WAS USED TO DEMONSTRATE THAT BINDING TO &+"0 WAS REVERSIBLE AND DID NOT INVOLVE COVALENT BONDING 2ATHER IT IS NOW POSTULATED THAT THE BIOLOGICAL MODE OF ACTION INVOLVES STABILISATION OF A TWISTED PROLYL AMIDE BOND INTERMEDIATE $ &IG ;= 3UPPORT FOR THIS SUPPOSITION HAS BEEN NOTED IN THE SOLVED CRYSTAL STRUC TURE OF &+ WHICH EXHIBITS A DIHEDRAL ANGLE BETWEEN THE # AND # CARBONYL GROUPS ;= -OREOVER A VARIETY OF COMPUTATIONAL STUDIES INVOLVING BOTH MOLECULAR DYNAMIC SIMULATIONS AND MOLECULAR MECHANIC CALCULATIONS HAVE ALSO SUGGESTED THAT 00)ASE INHIBITION INVOLVES STABILISA TION OF THIS TWISTED AMIDE TRANSITION STATE ;n= 4HE &+"0 BINDING DOMAIN FOR &+ IS FORMED BY A CONVEX PORTION OF ITS β SHEET AND THREE ASSOCIATED FLEXIBLE LOOPS NORTHERN HEMISPHERE OF " &IG THAT FORMS A HYDROPHOBIC WELL APPROXIMATELY ¯ BY ¯ IN AREA AND ¯ DEEP &+ IS BOUND IN THIS POCKET WITH HIGH AFFINITY +D N- USING A COMBINATION OF ITS PIPECOLYL RING ASSOCIATED KETOAMIDE LINKAGE TO THE PYRANOSE RING AND A FRAGMENT OF THE CYCLOHEXYLPROPENYL ESTER SIDE CHAIN 4HE REMAINING PORTION OF THE MACROCYCLIC COMPOUND EXTENDS INTO THE SOLVENT ACCESSIBLE REGION OF &+"0 )N A SIMILAR FASHION ALL OF THE STRUCTURALLY RELATED TRICARBONYL MACROLIDES n ARE CLOSELY SUPER
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IMPOSABLE UPON BOUND &+ AND ASSOCIATE WITH SIMILAR LEVELS OF POTENCY TO THE &+"0 BINDING DOMAIN ;= (OWEVER NOT ALL OF THE TRICARBONYL MACROLIDES SHARE THE SAME BIOLOGICAL PROFILE 7HILST &+ &+ AND RAPAMYCIN ARE ALL KNOWN TO BE EFFICACIOUS IMMUNOSUPPRESSANTS , THE ANTASCOMICINS AND AND MERIDAMYCIN ARE NOT ; n= &URTHERMORE IT BECAME APPARENT THAT &+ AND RAPAMY CIN ARE MUTUALLY ANTAGONISTIC DESPITE HAVING THE SAME INITIAL PROTEIN TARGET SUGGESTING A DIFFERENT MODE OF ACTION %VEN MORE PUZZLINGLY THE POWERFUL IMMUNOSUPPRESSANT CYCLOSPORINE ! #S! AN UNDECAPEPTIDE FIRST ISOLATED IN ; = AND THE MAJOR PHARMACEUTICAL PRODUCT AT THE TIME FOR THE PREVENTION OF ALLOGRAFT REJECTION 3ANDIMMUNE4- .EURAL4- ; = SHARES NO STRUCTURAL FEATURES WITH &+ BUT ELICITS THE SAME BIOLOGICAL RESPONSE )N THIS CASE THE INITIAL BIOLOGICAL TARGET OF #S! WAS KNOWN TO BE CYCLOPHILIN #Y0 INCREDIBLY ALSO A 00)ASE BUT UNAMBIGUOUSLY DIFFERENT FROM &+"0 ;= &ROM THESE INTRIGUING AND APPARENTLY CONTRA DICTORY OBSERVATIONS EMERGED A HYPOTHESIS THAT THE ACTIVE SPECIES WERE IN FACT THE &+RAPAMYCIN &+"0 AND #S! #Y0 OR DRUG IMMUNOPHILIN COMPLEXES THEMSELVES AND FOLLOWING A FASCINATING CONCERTED EFFORT THIS CONJECTURE WAS SUBSEQUENTLY VALIDATED ;n= 4HE IMMUNE RESPONSE ; = RELIES ON THE RECOGNITION OF VARIOUS ANTIGENS PROTEINS CARBOHYDRATES AND NUCLEIC ACIDS DISPLAYED BY A MAC ROPHAGE ON ITS CELL SURFACE VIA A CELL SURFACE PROTEIN TERMED THE MAJOR HISTOCOMPATIBILITY COMPLEX -(# 2ECOGNITION BY 4 CELL RECEPTORS 4#2 ON OTHER 4 CELLS TRIGGERS A CASCADE OF EVENTS SIGNAL TRANSDUCTION PATHWAY ;= IN THE 4 CELL ULTIMATELY RESULTING IN PROLIFERATION AND AN AMPLIFICA TION OF THE CELLULAR IMMUNE RESPONSE &IG /NE OF THE CRUCIAL ENZYMES INVOLVED IN THIS CASCADE IS CALCINEURIN A CALCIUM DEPENDENT SERINE THREO NINE PHOSPHATASE RESPONSIBLE FOR ACTIVATION OF THE CYTOSOLIC COMPONENT OF THE NUCLEAR FACTOR OF ACTIVATED 4 CELLS .&!4C 5PON DEPHOSPHORYLA TION .&!4C IS TRANSLOCATED FROM THE CYTOSOL ACROSS THE NUCLEAR MEMBRANE INTO THE NUCLEUS EVENTUALLY RESULTING IN LYMPHOKINE GENE EXPRESSION AND PRODUCTION OF THE CYTOKINE INTERLEUKIN ), 4HE PRESENCE OF THIS SMALL PROTEIN SECRETED FROM THE 4 CELL IS RECOGNISED BY RECEPTORS ), 2 ON OTHER 4 CELLS AND ULTIMATELY RESULTS IN ACTIVATION OF THE MAMMALIAN TARGET OF RAPAMYCIN M4/2 A SERINETHREONINE KINASE WHOSE DOWNSTREAM TAR GETS INCLUDES 0 3 KINASE ;= A CENTRAL REGULATOR OF TRANSLATION AND CELL GROWTH
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
&IGURE +% 7ESSON 4HE COMPLEX OF &+ WITH &+"0 BINDS TO AND INACTIVATES CALCINEURIN THE PHOSPHA TASE RESPONSIBLE FOR ACTIVATING NUCLEAR FACTOR OF ACTIVATED 4 CELLS .&!4 WHICH IS A TRANSCRIPTION FACTOR FOR ), SYNTHESIS 4HE RAPAMYCIN &+"0 COMPLEX BLOCKS A #A INDEPENDENT PATHWAY BY INHIBITING THE ), ACTIVATION OF 0 3 KINASE THROUGH DEACTIVATION OF M4/2 THEREBY INHIBITING CELL GROWTH
!FFINITY CHROMATOGRAPHY EXPERIMENTS WITH IMMUNOPHILIN COMPLEXES WERE USED TO EXTRACT INTERACTING PROTEINS FROM CELL LYSATES 5SING THIS METHOD THE SECONDARY TARGETS OF BOTH THE &+ &+"0 AND #S! #Y0 AGGREGATES WERE DETERMINED TO BE CALCINEURIN ; = 0OTENT INHIBITION OF THIS ENZYME BY EITHER OF THESE COMPLEXES BLOCKS .&!4C TRANSLOCA TION AND THUS SUBSEQUENT PRODUCTION OF ), AND EVENTUAL PROPAGATION OF THE IMMUNE RESPONSE )N A SIMILAR WAY THE SECONDARY TARGET OF THE RAPAMYCIN &+"0 COMPLEX WAS DETERMINED TO BE M4/2 ; = ALSO KNOWN AS &2!0 2!&4 2!04 OR 3%0 WHOSE INHIBITION HALTS THE IMMUNE RESPONSE AT THE TRANSLATIONAL LEVEL BLOCKING THE ), STIMULATED ' TO 3 PHASE TRANSITION IN 4 CELLS &IG 4HE IMPORTANCE OF THIS BIOLOGICAL ACTIVITY IS EXEMPLIFIED BY THE FACT THAT BOTH &+ 0ROGRAF4- &UJISAWA AND RAPAMYCIN 2APAMUNE4- 7YETH ARE CURRENTLY USED CLINICALLY FOR THE PREVENTION OF ALLOGRAFT REJECTION AND HAVE REVOLUTIONISED THE FIELD OF ORGAN TRANSPLANTATION
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
#ONSIDERABLE STRUCTURAL INSIGHT INTO THE NATURE OF INHIBITION OF BOTH CALCINEURIN AND M4/2 VIA THE &+ OR RAPAMYCIN &+"0 COMPLEXES HAS BEEN PROVIDED BY 8 RAY ANALYSIS OF THE TERNARY COMPLEX &IG ;n= !S DESCRIBED PREVIOUSLY BOTH &+ AND RAPAMYCIN BIND TIGHTLY TO &+"0 THROUGH HYDROPHOBIC INTERACTIONS USING THE TRICARBONYL PIPECOLIC PORTION OF THEIR ARCHITECTURE WHILE THE REMAINDER OF THE MOLECULE IS SITU ATED IN A SOLVENT ACCESSIBLE REGION )T IS NOW RECOGNISED THAT IT IS THIS LATTER FEATURE WHICH IS PRIMARILY RESPONSIBLE FOR THE DIFFERING BIOLOGICAL MODES OF ACTION OF THESE TWO NATURAL PRODUCTS AND CONSEQUENTLY BOTH STRUCTURES OF AND CAN BE DESCRIBED AS HAVING A BINDING DOMAIN ;FOR ASSOCIATION WITH &+"0 ! BLUE &IG = AND AN EFFECTOR DOMAIN ;FOR DETERMINING SUBSEQUENT ACTIVITY ! RED &IG = )N THE CASE OF &+ THE DOMINANT SITE OF BINDING OF THE BINARY COMPLEX TO CALCINEURIN INVOLVES HYDROPHOBIC INTERACTIONS WITH THE EFFECTOR REGION OF # # AND # ALLYL GROUP )N ADDITION A SECONDARY RECOGNITION SITE INVOLVING SUBSTANTIAL PROTEINnPRO TEIN &+"0 CALCINEURIN INTERACTION ALSO PLAYS A ROLE IN THE FORMATION OF TERNARY COMPLEX )N CONTRAST THE AGGREGATE COMPOSITE OF RAPAMYCIN &+"0 AND M4/2 EXHIBITS ESSENTIALLY NO PROTEINnPROTEIN CONTACTS AND IT APPEARS THAT RAPAMYCIN ACTS AS A LINCHPIN INTERACTING SEPARATELY WITH THE TWO ENZYMES THROUGH HYDROPHOBIC INTERACTIONS )N THESE THE LARGER MACROCYCLE RELATIVE TO &+ AND THE TRIENE PORTION OF FULFILL AN ESPECIALLY IMPORTANT ROLE FOR BINDING TO M4/2 AS ROTATION SLIGHTLY OUT OF CONJUGATION IN THE FULL TERNARY COMPLEX ALLOWS FURTHER BURYING OF THE # METHYL GROUP INTO A SMALL CREASE IN THE SECONDARY TARGET M4/2 7HILE THE IMMUNOMODULATING EFFECTS OF BOTH &+ AND RAPAMYCIN ARE OF TREMENDOUS IMPORTANCE THE TRICARBONYL MACROLIDES ALSO HOLD GREAT POTENTIAL IN APPLICATION TO A VARIETY OF OTHER HUMAN MALIGNANCIES &OR EXAMPLE THE NON IMMUNOSUPPRESSIVE MEMBERS OF THIS FAMILY OF NATU RAL PRODUCTS PARTICULARLY MERIDAMYCIN AND THE ANTASCOMICINS AND ;= ARE OF CONSIDERABLE INTEREST FOR THE TREATMENT OF NEURODEGENERATIVE DISEASE ;n= 4O DATE THE MODE OF ACTION OF THESE MOLECULES HAS NOT BEEN FULLY ELUCIDATED HOWEVER IT APPEARS THAT INHIBITION OF &""0 ANDOR DIFFERENT ISOZYMES EG &+"0 ; = OF THE SAME FAMILY IS SOMEHOW INVOLVED ;n= !LTHOUGH BOTH &+ AND RAPAMYCIN ALSO SHARE THIS ACTIVITY THEIR IMMUNOSUPPRESSIVE PROPERTIES LIMITS CHRONIC USE IN PATIENTS (OWEVER SYNTHETIC OR ENGINEERED ANALOGUES OF &+ ;n= HAVE BEEN DEMONSTRATED TO PROMOTE NOT ONLY REGROWTH OF DAMAGED NERVES
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
&IGURE $OMAINAL ANALYSIS OF &+"0 LIGANDS ! &+ AND RAPAMYCIN BINDING DOMAINS TO &+"0 SHOWN IN BLUE EFFECTOR DOMAINS SHOWN IN RED ;= " 2IBBON DIAGRAM OF &+;WHITE BALL AND STICK= &+"0;PURPLECALCINEURIN BLUE AND ORANGE SUBUNITS OF HETERODIMER= COMPLEX ;= 2EPRIN TED BY PERMISSION FROM -ACMILLAN 0UBLISHERS ,TD .ATURE COPYRIGHT # 2IBBON DIAGRAM OF RAPAMYCIN;GREEN BALL AND STICK= &+"0;BLUE=M4/2;RAPAMYCIN BINDING DOMAIN OF M4/2 IN RED= COMPLEX &ROM ;= 2EPRINTED WITH PERMISSION FROM !!!3
IN THE PERIPHERAL NERVOUS SYSTEM BUT ALSO THE REGENERATION OF NEURONS IN THE CENTRAL NERVOUS SYSTEM 4HESE NOVEL DERIVATIVES ARE CURRENTLY BEING EVALUATED FOR THE TREATMENT OF SUCH DEBILITATING AILMENTS AS 0ARKINSONS ;= AND !LZHEIMERS ;= DISEASES AMONG OTHERS ;= 9ET ANOTHER AREA OF PROFOUND AND RECENT IMPORTANCE RELATES TO THE USE OF THE TRICARBONYL PIPECOLIC ACID MACROLIDES SPECIFICALLY RAPAMYCIN AND DERIVATIVES AS ANTICANCER AGENTS ;n= )NTERESTINGLY RAPAMYCIN WAS IDENTIFIED BY THE .ATIONAL #ANCER )NSTITUTE .#) SHORTLY AFTER ITS DIS COVERY AS A NON CYTOTOXIC AGENT THAT HAD CYTOSTATIC ACTIVITY AGAINST SEVERAL DIFFERENT HUMAN CANCERS BOTH IN VITRO AND IN VIVO (OWEVER THE DEVELOPMENT PROGRAM OF AS A POTENTIAL ANTICANCER AGENT RECEIVED LITTLE ATTENTION UNTIL THE END OF THE S FOLLOWING THE DISCOVERY OF ANALOGUES
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
WITH IMPROVED PHARMACOLOGICAL PROPERTIES ;= AND THE RECOGNITION OF THE IMPORTANCE OF M4/2 AS A CENTRAL REGULATOR OF BOTH CELLULAR CATABOLISM AND ANABOLISM /F THE THREE MAJOR SIGNALLING PATHWAYS THAT HAVE BEEN IDENTIFIED AS IMPORTANT FOR THE DEVELOPMENT OF CANCER NAMELY THE 0)+ !+4 KINASE ;= PROTEIN KINASE # FAMILY ; = AND -!0+2AS ;= SIGNALLING CAS CADES M4/2 ; = HAS BEEN IDENTIFIED AS A KEY DOWNSTREAM ENZYME IN THE PHOSPHATIDYLINOSITOL KINASE 0)+ PATHWAY 4O DATE THE UNIQUE ABILITY OF THE RAPAMYCINRAPAMYCIN DERIVATIVE &+"0 AGGREGATE COMPLEX TO EFFECTIVELY INHIBIT M4/2 HAS HAD A DEMONSTRATED VALUE IN A VARIETY OF HUMAN TUMOUR TYPES %SPECIALLY USEFUL IN CELL LINES WITH MALFUNCTION ING PHOSPHATASES AND TENSIN HOMOLOGUES DELETED ON CHROMOSOME 04%. NEGATIVE REGULATOR OF 0)+ ; = THREE CANDIDATES ##) TEMSIROLIMUS 7YETH 0HASE))) 2!$ EVEROLIMUS .OVARTIS 0HASE)) AND !0 !RIAD 0HASE)) ARE CURRENTLY IN ADVANCED CLINICAL TRIALS !LL THREE OF THESE CANDIDATES ARE # /( RAPAMYCIN DERIVATIVES THAT OFFER IMPROVED PHARMACOKINETICS AND WATER SOLUBILITY FOR FORMULATION PURPOSES /F NOTE NO IMMUNOSUPPRESSIVE EFFECTS WERE MANIFESTED WHEN ANY OF THE ABOVE DRUG CANDIDATES WERE ADMINISTERED INTERMITTENTLY IN CLINICAL SETTINGS ;= 4HE ROLE OF CHEMICAL SYNTHESIS IN BOTH TARGET AND DIVERSITY ORIENTATED CONTEXTS HAS PLAYED AN INTEGRAL ROLE IN THE ELUCIDATION OF THE FASCINATING BIOLOGICAL PROFILE OF THE MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS n AS OUTLINED BRIEFLY ABOVE 4HE REMAINDER OF THIS CHAPTER WILL FOCUS ON THE PRINCIPLE SYNTHETIC EFFORTS DIRECTED TOWARDS THE CONSTRUCTION OF &+ ;n= ANTASCOMICINS AND ;n= AND RAPAMYCINS AND ;n=
4HE -ERCK TOTAL SYNTHESIS OF &+
!S DISCLOSED IN BY RESEARCHERS AT -ERCK 3HARP AND $OHME &+ WAS THE FIRST OF THE TRICARBONYL MACROLIDE FAMILY OF IMMUNOSUPPRESSANTS OR RELATED NATURAL PRODUCTS TO SUCCUMB TO A TOTAL SYNTHESIS EFFORT ; = 4HIS LANDMARK ACHIEVEMENT FOLLOWED YEARS OF INTENSE INTEREST AND EFFORT AND PROVIDED A FOUNDATION WHICH FUTURE SYNTHESES WOULD BUILD UPON -OREOVER IT ALSO AFFORDED SYNTHETIC MATERIAL FOR FURTHER INVESTIGATION OF
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 2ETROSYNTHETIC ANALYSIS
THE INTRIGUING BIOLOGICAL ACTIVITY OF &+ AS WELL AS ADVANCED INTER MEDIATES FOR DIVERTED TOTAL SYNTHESIS ;= IN THE SEARCH FOR COMPOUNDS WITH IMPROVED PHARMACOLOGICAL PROPERTIES %XTENSIVE STUDIES ON THE STABILITY AND POTENTIAL FORMATION OF THE MASKED TRICARBONYL UNIT OF &+ INDICATED THAT THIS FUNCTIONALITY WAS EXTREMELY SENSITIVE ESPECIALLY TO BASE MEDIATED BENZILIC ACID REARRANGE MENT ; = #ONSEQUENTLY A KEY RETROSYNTHETIC STRATEGY THAT ENTAILED THE LATE INTRODUCTION OF THIS SYSTEM WAS ADOPTED -OREOVER CONCERN FOR EPIMERISATION OF THE CHIRAL CENTRE IN 3 PIPECOLIC ACID DICTATED THE CHOICE OF A MACROLACTAMISATION APPROACH FOR RING CLOSURE )N THIS FASHION THE ESTER BOND AT # COULD BE GENERATED UNDER HIGH CONCENTRATION CONDITIONS EAR LIER IN THE SYNTHESIS IN A MILD FASHION AND HOPEFULLY AVOID ANY POTENTIAL COMPLICATIONS DUE TO RACEMISATION AT THIS CENTRE 3CHEME &URTHER DISSECTION ACROSS # # WAS ENVISIONED TO INSTALL THE TRICAR BONYL PRECURSOR IN A REDUCED AND SUITABLY PROTECTED FORM TO LEAVE THE # # ALDEHYDE AS THE PRINCIPLE SYNTHETIC TARGET $ISCONNECTION AT THE CHALLENGING TRISUBSTITUTED OLEFINIC LINKAGE # # THROUGH A 7ITTIG TYPE OLEFINATION STRATEGY FURTHER SIMPLIFIED THE PROBLEM TO THE PREPARATION OF TWO MAJOR FRAGMENTS AND OF SIMILAR COMPLEXITY WHICH WOULD RELY HEAVILY ON ASYMMETRIC ALDOL METHODOLOGY ; = FOR THEIR CON STRUCTION 3CHEME )NITIAL PREPARATION OF THE # # FRAGMENT INCORPORATING THE CYCLOHEXANE MOIETY UTILISED THE CHIRAL POOL AS A SOURCE OF THE STEREOCHEMI CAL ARRAY OF FUNCTIONALITY ABOUT THIS SIX MEMBERED RING ;= 4HUS LACTONE READILY AVAILABLE FROM n QUINIC ACID IN ONE STEP WAS CONVERTED TO BIS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
THIOCARBONYL LACTONE IN PREPARATION FOR REDUCTIVE CLEAVAGE OF THE EXTRA NEOUS HYDROXYL FUNCTIONALITY UNDER RADICAL CONDITIONS ! TWO STEP PROTO COL WAS FOUND TO BE NECESSARY BUT PROCEEDED STEREOSPECIFICALLY AND WITH GOOD REGIOSELECTIVITY TO FURNISH SIMPLIFIED LACTONE 3TANDARD FUNCTIONAL GROUP MANIPULATIONS THEN AFFORDED THE KEY CYCLOHEXANE CARBOXALDEHYDE BUILDING BLOCK AS A SINGLE DIASTEREOMER &OR SUBSEQUENT INTRODUCTION OF THE # # TRISUBSTITUTED OLEFIN THE HOMOLOGATION PROTOCOL INITIALLY INTRODUCED BY #OREY AND LATER MODIFIED BY 3CHLESSINGER WAS INVESTIGATED ; = %ARLY ATTEMPTS UNDER THE REPORTED CONDITIONS USING α LITHI ATED TRIETHYLSILYL T BUTYLIMINE OFTEN RETURNED UNREACTED STARTING MATERIAL AND MORE WORRYINGLY GAVE CAPRICIOUS AND LOW %: SELECTIVITY FOR !T THIS JUNCTURE THE -ERCK TEAM MADE A CRITICAL OBSERVATION THAT TREAT MENT OF THE CRUDE IMINE MIXTURE WITH ANHYDROUS TRIFLUOROACETIC ACID PRIOR TO THE ADDITION OF WATER AFFORDS AN OPPORTUNITY FOR THE BULKY PROTONATED IMINE INTERMEDIATE TO ISOMERISE AND GREATLY IMPROVED BOTH REACTION CONSISTENCY AND SELECTIVITY ;= !N %VANS ALDOL CONDENSATION ;= OF THE RESULTING ALDEHYDE AND BORON ENOLATE OF IMIDE FURNISHED THE DESIRED ADDUCT WITH EXCEPTIONAL STEREOCONTROL 3UBSEQUENT MANIPULA TIONS GENERATED A SECOND ELECTROPHILE WHICH WAS CONDENSED WITH THE LITHIUM ENOLATE OF 3 n ACETOXY TRIPHENYLETHANOL ;n= TO AFFORD ALCOHOL WITH MODEST SELECTIVITY FOR THE DESIRED 3 CONFIGURATION AT # &URTHER STANDARD FUNCTIONAL GROUP MODIFICATIONS AND YET ANOTHER %VANS ALDOL CONDENSATION TO SELECTIVELY INSTALL THE ALLYL SIDE CHAIN AT # COMPLETED THE ASYMMETRIC SYNTHESIS OF THE # # FRAGMENT OF &+ IN A TOTAL OF STEPS 3CHEME 5PON SCALING UP THE ABOVE ROUTE A NUMBER OF SUBSTANTIAL PROBLEMS WERE ENCOUNTERED ; = 0RINCIPLE AMONG THESE WAS THE EARLY TRIBU TYLTIN HYDRIDE REDUCTION OF QUINIC ACID DERIVATIVE WHICH PROVED VERY CUMBERSOME ON LARGE SCALE 4REATMENT OF COMMERCIALLY AVAILABLE CYCLO HEXENECARBOXYLIC ACID WITH M#0"! FOLLOWED BY HIGH DILUTION THER MOLYSIS AFFORDED HYDROXYL LACTONE AND ALTHOUGH BOTH LOW YIELDING AND RACEMIC THIS PROVED TO BE MORE CONVENIENT FOR MATERIAL THROUGHPUT &URTHER ELABORATION IN A SIMILAR FASHION AS PREVIOUSLY DISCUSSED UNEVENT FULLY FURNISHED ENAL WHICH WAS THEN SUBJECTED TO THE BORON ENOLATE OF IMIDE TO GIVE THE ALDOL ADDUCT IN COMPARABLE YIELD AND SELECTIVITY 4RANSAMIDATION OF THE %VANS ALDOL ADDUCT DERIVED FROM AND FOL LOWED BY 4%3 PROTECTION AFFORDED AN INTERMEDIATE 7EINREB AMIDE WHEREIN
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
S
OH HO
O 34
O
a
30
O
+
TIPSO
MeO
MeO
17
19
E-20
CHO
OH Ph Ph Ph
O
H N
Z-20
E-21
O
MeO
OH Ph Ph Ph 25
O
O
N
m
18
H2O
TIPSO
OH O TESO 26
30
R TFA
34
O
30
17
+ N
23
MeO
O
R
R
N Li
26
H
26
O
30
34
h
16
SiEt3
H
TIPSO
34
d,e,f,g
O
O S 15
14
R
Im
O
TIPSO
34 30
O
O
O
HO b,c
34
OH
30
R
O2CCF3
H
i
O 18
22
TIPSO
34
j,k,l 30
MeO
34
30
H
26
N
26
OH O
O
TESO 24
O
N
O
O
23
n,o,p,q TIPSO MeO
TIPSO
34
r
MeO
30 26
TESO
23
CHO OTIPS
O
O N
TIPSO
34
s,t,u 30 23
26
O
OTIPS OH O
TESO 29
27
20
O
N
O
MeO
34
30 26
TESO
23
20
CHO OTIPS OTBS
11
28
3CHEME A THIOCARBONYLDIIMIDAZOLE $#% REFLUX B N"U3N( XYLENES # C N"U3N( !)". XYLENES # OVER TWO STEPS D 4)03/4F LUT #(#L # E -E-E/ .!L#L-E 0H#( OVER TWO STEPS F -E/4F DI T BUTYL METHYLPYRIDINE #(#L 24 G $)"!, ( 4(& n # OVER TWO STEPS H LITHIO TRIETHYLSILYLPROPANAL T BUTYLIMINE 4(& n # → n # THEN 4(& # THEN (/ # %: I N"U"/4F $)0%! 4(& THEN n # → # J -E-E/ .!L#L-E #(#L n # → 24 K 4%3/4F LUT #(#L # L $)"!, ( 4(& n # OVER FOUR STEPS M 3 n ACETOXY TRIPHENYLETHANOL ,$! 4(& n # THEN 3 2 N .A/-E -E/( # O 4)03/4F LUT #(#L # P $)"!, ( 4(& # Q 3/q0Y %T. $-3/ #(#L 24 OVER FOUR STEPS R N"U"/4F $)0%! 4(& THEN n # → # S -E-E/ .!L#L-E #(#L n # → 24 T 4"3/4F LUT #(#L # U $)"!, ( 4(& n # OVER THREE STEPS
THE DIASTEREOMERS ARISING ORIGINALLY FROM RACEMIC COULD BE CONVENIENTLY SEPARATED /THER SUBSTANTIAL CHANGES INVOLVED THE USE OF CHLOROIMIDE ;= AS A CHIRAL ACETATE SYNTHON IN PLACE OF $ESPITE REQUIRING AN ADDI TIONAL STEP TO REDUCTIVELY REMOVE THE CHLORIDE BOTH THE YIELD AND SELECTIV ITY WERE MUCH IMPROVED !LTHOUGH NOT EXPLICITLY CLEAR A DIFFERENT SERIES OF REACTIONS WERE EMPLOYED TO CONVERT TO PRESUMABLY AS THIS WAS HIGHER YIELDING OR MORE CONVENIENT WITH REGARDS TO PRODUCT ISOLATION OR PURIFICATION 3CHEME /VERALL THIS MODIFIED APPROACH TO IS SLIGHTLY LONGER ENCOMPASSING STEPS IN TOTAL AND DELIVERING THE DESIRED PRODUCT
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
HO
34
MeO
H
g
MeO
h 30
34
Bn 30
O
N
26
O
CHO
O
N
26
17
16
MeO
O
30
O
30
TIPSO
34
34
c,d,e,f
30
O
O
TIPSO
TIPSO
34
a,b
OH
30
O
OH O
18
O
32
Bn 31 i,j,k
TIPSO MeO
TIPSO
34
30 26
TESO 11
23
20
q,r,s,t,u,v O O
CHO OTIPS OTBS
N Bn 34
MeO
TIPSO
34
30 26
O
l,m,n,o,p O O
TESO 27
23
CHO OTIPS
Cl
N Bn 33
MeO
34
30
H
26
O
TESO
O
24
3CHEME A M#0"! #(#L # B CHLOROBENZENE # OVER TWO STEPS C 4)03/4F LUT #(#L # D !L#( (./#( #(q(#L 0H#( # E -E/4F DI T BUTYL METHYLPYRIDINE #(#L # F $)"!, ( 4(& n # G LITHIO TRIETHYLSILYL PROPANAL . CYCLOHEXYLIMINE 4(& n # THEN 4(& # THEN (/ # OVER FIVE STEPS H %T. N"U"/4F n # THEN n # I !L#( (./#( #(q(#L #(#L # J 4%3/4F LUT #(#L # SEPARATE DIASTEREOMERS OVER TWO STEPS K $)"!, ( 4(& n # L %T. N"U"/4F n # THEN n # M ZINC !C/( 4(& ULTRASOUND IRRADIATION # N !L#( (./#( #(q(#L #(#L # O 4)03/4F LUT #(#L # P $)"!, ( 4(& n # Q %T. N"U"/4F n # THEN n # R ,I/( (/ (/ 4(& # S CARBONYL DIIMIDAZOLE #(#L # THEN (./#( #( #(#L # T ,I/( (/ (/ 4(& # OVER THREE STEPS U 4"3/4F LUT #(#L # V $)"!, ( 4(& n #
IN OVERALL YIELD (OWEVER IT IS A VERY PRACTICAL AND ROBUST SYNTHETIC SEQUENCE AND CLEARLY DEMONSTRATES THE UTILITY OF %VANS ALDOL TECHNOLOGY FOR THE CONSTRUCTION OF POLYKETIDE SUBUNITS 3CHEME 4HE APPROACH TO THE # # FRAGMENT WAS ORIGINALLY DESIGNED TO HIGHLIGHT THE UTILITY OF # SYMMETRY IN BIDIRECTIONAL SYNTHESIS ; = (OWEVER TO AID IN PRODUCT STRUCTURE DETERMINATION AND TO AVOID ADDITIONAL TERMINUS DIFFERENTIATION METHODOLOGY NEAR THE END OF THE SEQUENCE THE STEREOCENTRES WERE CREATED SEQUENTIALLY ;= -ONOEPOXY BENZYL ALCOHOL READILY AVAILABLE THROUGH USE OF 3HARPLESS EPOXIDATION OF PENTADIEN OL ;= WAS ELABORATED TO FURNISH AN INTERMEDIATE BUTYROLACTONE IN GOOD OVERALL YIELD %NOLISATION FOLLOWED BY ALKYLATION WITH -E) AFFORDED WITH REASONABLE SELECTIVITY FOR THE DESIRED STEREOCHEMISTRY AT # )N CONTRAST TO ALLYLIC ALCOHOLS SUBSTRATE OR REAGENT CONTROLLED EPOXIDATION OF HOMOALLYLIC ALCOHOLS NOTORIOUSLY OCCURS WITH SUBSTANTIALLY DIMINISHED SELECTIVITY ; = 4O ADDRESS THIS THE -ERCK GROUP EMPLOYED A THREE STEP PROCEDURE INVOLVING THE INTRAMOLECULAR FOR
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME A ,I#(#. 4(& n # → 24 B . (#L -E/( REFLUX OVER TWO STEPS C ,$! 4(& n # → n # THEN -E) n # DR D ,I!L( 4(& 24 E T BUTOXYCARBONYLOXYIMINO PHENYLACETONITRILE .A( 4(& # F "R +#/ #(#L n # OVER THREE STEPS DR G .A/-E -E/( 24 H -E) .A( 4(& 24 OVER TWO STEPS I ,I#(#. 4(& n # → 24 J . (#L -E/( REFLUX K 4"3#L )M $-& 24 OVER THREE STEPS L ,$! 4(& n # → n # THEN -E) n # DR M ,I!L( 4(& 24 N -/-"R $)0%! #(#L # O -E) .A( 4(& 24 OVER THREE STEPS P 4"!& 4(& 24 Q 0$# $-& 24 R ( 0D/( %T/( 24 S !C/ #(#L REFLUX OVER FOUR STEPS
MATION OF BROMOCARBONATE VIA TREATMENT OF / "OC PROTECTED WITH BROMINE AT n # TO AFFORD THE ANTI PRODUCT IN AN RATIO )NTEREST INGLY COMPARED TO PROTOCOLS FIRST DESCRIBED BY #ARDILLO ;= AND LATER BY BOTH "ARTLETT ;= AND 3MITH ;= THE OBSERVED STEREOCHEMISTRY FOR THE PREDOMINANT PRODUCT IS OPPOSITE TO THAT TYPICALLY SEEN !PPARENTLY THE BENZYLOXY SUBSISTENT AT # DICTATES THE STEREOCHEMICAL OUTCOME OF THIS REACTION AS PREVIOUSLY NOTED BY "ABINE IN A RELATED SYSTEM ;= /F THE TWO POSSIBLE TRANSITION STATES LEADING TO THE MAJOR AND MINOR PRODUCTS RESPEC TIVELY THE ONE IN WHICH THE VINYL AND BENZYLOXY SUBSTITUENTS ARE ECLIPSED APPEARS TO BE THE MORE REACTIVE CONFORMER DUE TO THE DELOCALISATION OF THE OXYGEN LONE PAIR INTO THE DOUBLE BOND ;= &URTHER ELABORATION TO THE FULLY FUNCTIONALISED LACTONE WAS UNEVENTFUL ASIDE FROM A NECESSARY THIRD SEPARATION OF DIASTEREOMERS AFTER INTRODUCTION OF THE METHYL STEREOCENTRE AT # 3CHEME !S WAS THE CASE WITH THE # # SUBUNIT THE ORIGINAL APPROACH 3CHEME TO THE # # FRAGMENT UNDERWENT SUBSTANTIAL CHANGES TO ADDRESS THE THREE TEDIOUS SEPARATIONS OF DIASTEREOMERIC MIXTURES AND LIM ITED FLEXIBILITY FOR ANALOGUE PREPARATION ; = 4HE REVISED SYNTHESIS EMPLOYED A SERIES OF RELIABLE TRANSFORMATIONS UTILISED HYDROXY METH YLPROPIONATE AS AN INITIAL SOURCE OF CHIRALITY AND WAS ACCOMPLISHED IN
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O 18
O
N
O
18
a,b,c
OH
15
d,e
H
OPMB
18
15
44
N OPMB
N
O
45
18 13
O
Ph
O
Bn 31
f,g
OBn
O
13
TBSO OBn + 10
h,i,j,k
12
HO
O
10
12
OCH3
OH 47
49
O
O
Br
48
l
46
S
OTBS 15
S
10
OMe OMe 13
19
O PPh2
S
v,w,x
OTBS 15
S
10
OMe OMe 52
OPiv 18
r,s,t,u
OTBS 10
OTBS 15
OMe OMe 51
OPiv 18
OPMB
OTBS m,n,o,p,q 10
15
18
OBn O
OH 50
3CHEME A .A(-$3 ALLYL IODIDE 4(& n # B ,I/( (/ (/ 4(& # C ,I!L( %T/ # → 24 D .A( "N"R 4(&$-& 24 OVER THREE STEPS E /S/ .A)/ (/ ACETONE 24 F N"U"/4F %T. #(#L n # THEN n # G !L#( (./#( #(q(#L 4(&0H#( # H 4"3#L )M $-& # I $)"!, ( 4(&0H#( n # → 24 OVER TWO STEPS J -S#L %T. #(#L n # K 4"!" ACETONE REFLUX OVER TWO STEPS L ,I %T/ # THEN 4(& n # M #( ."(/!C #(#. !C/( (/ n # DR N .A( -E) 4(& # → 24 O ( 0D/( %T/!C 24 P 0IV#L 0Y # Q 4"3/4F LUT #(#L # OVER THREE STEPS R 4&! 4(&(/ # S #/#L $-3/ #(#L n # THEN %T. n # → n # T #(#(3( "&q/%T #(#L # U 4"3/4F LUT #(#L # V ,I!L( %T/ # → 24 W 0H3/#L 0Y # X 0H0/ %T N"U,I 4(& n # → #
STEPS AND OVERALL YIELD 3CHEME -ORE IMPORTANTLY ESSENTIALLY ALL STEREOISOMERS OF ARE POTENTIALLY AVAILABLE THROUGH SIMPLE MODIFICA TION OF REAGENTS A GOAL OF SIGNIFICANT IMPORTANCE FOR THE -ERCK GROUP WHO WERE UNDOUBTEDLY INTERESTED IN SUBSEQUENT STRUCTURE ACTIVITY EVALUATION #ONVERSION OF THE ADVANCED INTERMEDIATE TO THE DITHIANE PHOSPHINE OXIDE WAS INTENDED TO INSTALL TWO CONVENIENT SITES FOR FRAGMENT COUPLING THROUGH ANIONIC ADDITION AND OLEFIN FORMATION RESPECTIVELY VIDE INFRA #OUPLING OF THE # # AND # # FRAGMENTS WAS ACCOMPLISHED BY DEPROTONATION OF PHOSPHINE OXIDE FOLLOWED BY ADDITION OF THE ALDE HYDE ELECTROPHILE ;n= 4HE RESULTING DIASTEREOMERIC MIXTURE OF HYDROXYL PHOSPHINE OXIDES COULD BE CHROMATOGRAPHICALLY SEPARATED INTO TWO MAJOR ADDUCTS AND IN TURN CONVERTED TO THE CORRESPONDING OLEFINS % AND : RESPECTIVELY THROUGH EXPOSURE TO POTASSIUM HEXAMETHYL DISILAZANE +(-$3 4HIS APPROACH TO THE # # TRISUBSTITUTED ALKENE ALTHOUGH OVERALL LOW YIELDING ALLOWED ISOLATION OF GEOMETRICALLY PURE % WHICH NOTABLY IS INSEPARABLE FORM : 3CHEME 4HE DITHIANE MOIETY PRESENT IN % WAS INITIALLY TO SERVE AS AN ACYL ANION EQUIVALENT FOR REACTION WITH DIETHYL OXALATE AND EVENTUAL INTRODUC
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO MeO
TIPSO
OTBS
S
15
S
19
10
O PPh2
34
30
H
34
OTBS
26 22
+
MeO
O TESO TIPS
a,b,c
30 23
26
TESO
13
20
CHO OTIPS OTBS
OMe OMe
19
Z-53
11
OMe S S
15 10
OMe OTBS
E-53
1
N CO2H Boc 12 TIPSO MeO
TIPSO
34
30
MeO
H
TIPSO
34
30
MeO
H
OTBS
26
N H H O PMBO O O
1
i,j 19
OMe 10
H 22
15
OMe OTBS
O
O O TIPS
N O Boc PMB Bn O 10 N O
O
OH 54
OTBS
26
22
O O TIPS
OTES 55
34
30
OTBS
26
22
1
d,e,f,g
1
h
O OMe
O
O 19
N
OMe
OPMB
15
OMe OTBS
N Boc
O
19
O O TIPS
Bn 10
15
OHC 10
OMe OTBS
9
3CHEME A N"U,I 4-%$! 4(& n # THEN B SEPARATE ADDUCTS C +(-$3 4(& # OVER THREE STEPS SIMILARLY : WAS PREPARED IN YIELD OVER THE THREE STEPS D 4&! (/ 4(& # E $## $-!0 #(#L n # F !G./ .#3 LUT #(/(4(& # G GLYOXYLIC ACID HYDRATE !C/( #(#L OVER FOUR STEPS H N"U"/4F %T. 0H#( n # THEN n # I ,I/(q(/ (/ 4(&(/ # J 4%3/4F LUT #(#L # OVER TWO STEPS
TION OF THE TRICARBONYL REGION OF &+ (OWEVER MODEL STUDIES REVEALED FUNCTIONALITY PRESENT ELSEWHERE IN THE MOLECULE DID NOT TOLERATE STRONGLY BASIC NOR ACIDIC CONDITIONS CONSEQUENTLY AN ALTERNATIVE WAS SOUGHT !LTHOUGH THE STEREOCHEMICAL CONTROL AT # AND # IS ULTIMATELY IMMATE RIAL A BORON ALDOLCHIRAL IMIDE ;= STRATEGY WAS VIEWED AS ADVAN TAGEOUS FOR ISOLATION AND CHARACTERISATION PURPOSES AS WELL AS THE MILD REACTION CONDITIONS INVOLVED IN THIS METHODOLOGY 4HUS PRIOR INTRODUC TION OF THE PIPECOLIC ACID MOIETY UNDER CAREFULLY CONTROLLED CONDITIONS TO AVOID EPIMERISATION AT # WAS FOLLOWED BY TWO STEP DEPROTECTION ;= OF THE DITHIANE TO ALDEHYDE 4HIS VERSATILE SYNTHETIC INTERMEDIATE IS ALSO AVAILABLE THROUGH EFFICIENT DEGRADATION STEPS OVERALL YIELD OF THE NATURAL PRODUCT AND SUPPLEMENTED MATERIAL SUPPLIES FOR END GAME ANALYSIS AND ANALOGUE PREPARATION ;= !SYMMETRIC ALDOL CONDENSATION
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TIPSO
TIPSO MeO
30
MeO
H
HO
34
34
30
MeO
H OTBS
26
OTBS
26
N H H O
O O TIPS
O TIPS O
O
O
H O
26 22
a,b,c,d,e
1
f,g,h,i 19
OH
O
O
1
19
19
N
N
PMBO O
30
22
22
1
34
OMe 10
OTES 55
15
OMe OTBS
8 8
O
O TBSO
15
OMe
O
O OH H O 10
15
OMe
10
OCH3
OCH3
O
56
FK506 1
3CHEME A CHLORO METHYLPYRIDINIUM IODIDE %T. #(#L # B $$1 (/ #(#L # C 4&! (/ 4(& # OVER TWO STEPS D #/#L $-3/ #(#L n # THEN %T. n # → 24 E #/#L $-3/ #(#L n # THEN %T. n # → 24 OVER TWO STEPS F (& AQ #(#. # G 4%3#L 0Y # H $-0 0Y #(#L # I (& AQ #(#. #
OF WITH THE BORON ENOLATE OF FURNISHED IN EXCELLENT YIELD ; = !LTHOUGH NOT OBVIOUS THE CHOICE OF 0-" PROTECTING GROUP IN IMIDE EMBODIES A SUBSTANTIAL AMOUNT OF PRELIMINARY STUDY AND WAS CRITICAL FOR THE FUTURE INTRODUCTION OF THE TRICARBONYL REGION SPANNING # # #LEAVAGE OF THE IMIDE UNDER STANDARD CONDITIONS TO THE FREE CARBOXYLIC ACID FOLLOWED BY GENERATION OF THE FREE AMINE VIA THE METHOD OF 3HIOIRI AND /HFUNE 4%3/4F LUT ; = AND SIMULTANEOUS 4%3 PROTECTION AT # SUCCESSFULLY GENERATED THE UNSTABLE MACROCYCLIC PRECURSOR IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD 3CHEME )MMEDIATE APPLICATION OF -UKAIYAMA CONDITIONS ;= UNDER HIGH DILUTION CONDITIONS RESULTED IN SMOOTH FORMATION OF THE DESIRED LACTAM MACROCYCLE IN EXCELLENT YIELD 3EQUENTIAL DEPROTECTION AT # AND # VIA EXPOSURE TO $$1 FOLLOWED BY AQUEOUS ACID AND TWO SUCCESSIVE APPLICA TIONS OF 3WERN OXIDATION CONDITIONS THEN FURNISHED THE TRICARBONYL COM POUND IN A VERY PLEASING YIELD 3ELECTIVE DESILYLATION OF PROVED IMPOSSIBLE THUS ALL SILICON PROTECTING GROUPS WERE REMOVED WITH (& IN ACETONITRILE RESULTING IN SPONTANEOUS HEMI KETAL FORMATION /F THE REMAIN ING FREE HYDROXYL GROUPS THOSE AT # AND # EXHIBITED HIGHER REACTIVITY AND COULD BE SELECTIVELY 4%3 PROTECTED TO ALLOW OXIDATION AT # TO THE CORRESPONDING KETONE WITH THE $ESS -ARTIN PERIODINANE $-0 ;= &INALLY DESILYLATION ONCE AGAIN WITH (& FURNISHED &+ IDENTICAL IN ALL RESPECTS WITH THE NATURAL PRODUCT 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 3UMMARY OF THE -ERCK TOTAL SYNTHESIS OF &+
)N SUMMARY THE TOTAL SYNTHESIS OF &+ BY THE -ERCK GROUP WAS ACCOMPLISHED IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD 4HE FIRST TOTAL SYNTHESIS OF ANY OF THE TRICARBONYL MACROLIDE FAMILY OF NATURAL PRODUCTS THIS ACHIEVEMENT STANDS AS AN IMPORTANT MILESTONE IN THE FIELD SETTING THE STAGE FOR ALL FUTURE SYNTHESES (IGHLIGHTS INCLUDED AN EFFICIENT USE OF THE %VANS ALDOL REACTION FOR THE PREPARATION OF VARIOUS SUB UNITS CONSTRUCTION OF THE # # TRISUBSTITUTED OLEFIN THROUGH AN INTER MOLECULAR 7ITTIG TYPE REACTION AND EFFICIENT FORMATION OF THE MACROCYCLIC RING THROUGH MACROLACTAMISATION !LTHOUGH NOT THE SHORTEST NOR HIGHEST YIELDING APPROACH VIDE INFRA OVERCOMING THE UNEXPECTED CHALLENGES ASSO CIATED WITH THE FIRST SUCCESSFUL SYNTHESIS SHOULD NOT BE OVERLOOKED
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
HO MeO
34
30
TIPSO
H
MeO
O
26
34
30
O
1
N 8
O
O OH H O 10
TIPSO
MeO
H
C20-C34 aldehyde 59
34
30
H
OPMB
26
22
20
26
CHO O OPMB TIPS
22
OH
O
C10-C34 aldehyde 57
19
O
OMe 15
O
FK506 1
MeO
8
+
1
19
N Boc
O
O
+
OMe
9
OMe
OCH3
N 1 O Boc OMe
O O TIPS
15
OHC 10
OMe OTBS
58
S S
(Me2N)2P O MeO
19
15
OMe OTBS C10-C19 dithiane 60 10
3CHEME 2ETROSYNTHETIC ANALYSIS
4HE 3CHREIBER TOTAL SYNTHESIS OF &+
3TUART 3CHREIBER AND HIS RESEARCH GROUP AT (ARVARD 5NIVERSITY HAVE BEEN PROMINENT PLAYERS IN THE FIELD OF IMMUNOSUPPRESSANTS AND THEIR ASSOCI ATED IMMUNOPHILINS BOTH WITH REGARD TO TOTAL SYNTHESIS EFFORTS AS WELL AS INVESTIGATION OF THE BIOLOGICAL MODES OF ACTION 4HIS IS APTLY ILLUSTRATED BY THEIR DISCLOSURE IN ;= SHORTLY AFTER THE -ERCK REPORT ON THE TOTAL SYNTHESIS OF &+ AS WELL AS # # # RADIOLABELLED &+ WHICH PROVED USEFUL TO DELINEATE THE MECHANISM BY WHICH THE ROTAMASE ACTIVITY OF &+"0 IS INHIBITED 2ETROSYNTHETICALLY THE 3CHREIBER APPROACH TO &+ IS VERY SIMILAR TO THAT REPORTED BY -ERCK AND ENCOMPASSES THE MAJORITY OF ALL KEY STRATEGIC DECISIONS NAMELY A LATE STAGE INTRODUCTION OF THE TRICARBONYL REGION MAC ROLACTAMISATION AND A MODIFIED (ORNER REACTION FOR FRAGMENT COUPLING AND CONSTRUCTION OF THE # # TRISUBSTITUTED OLEFIN 3CHEME (OWEVER THERE ARE CONSIDERABLE DIFFERENCES IN THE WAY THAT THE INDIVIDUAL FRAGMENTS ARE PREPARED #OMPARED WITH THE -ERCK SYNTHESIS THERE IS A NOTICEABLE LACK OF ASYMMETRIC ALDOL REACTIONS AND INDEED A MORE CONVERGENT APPROACH TO FRAGMENT SYNTHESES RESULTS IN A SIGNIFICANTLY SHORTER APPROACH 3CHREIBERS SYNTHESIS OF THE # # ALDEHYDE BEGINS SIMILARLY TO THE INITIAL -ERCK SYNTHESIS OF THE # # FRAGMENT 3CHEME ;= )N THIS CASE 3HARPLESS EPOXIDATION OF FOLLOWED BY PROTECTION OF THE MONO EPOXY ALCOHOL WITH 0-""R UNDER CAREFULLY CONTROLLED CONDITIONS TO AVOID THE FORMATION OF 0AYNE REARRANGEMENT ;= PRODUCTS FURNISHED IN GOOD YIELD 2EGIOSELECTIVE RING OPENING WITH THE ANION OF ETHOXYACETYLENE AS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
OEt OH 34
c,d
32
31
O
OH
28
g
31
34
O
31
34
34
OMe
OMe 64
OMe 63
62
61
EtO2C
e,f
OPMB
34
32
O
28
28
OPMB
a,b
65
h,i TBSO
HO
34
m
31 28
MeO
MeO
70
k,l 28
SO2Ph
OMe
HO
34 31
34
28
MeO
SO2Ph 69
OH 68
34
j
31
31
O
31
34
CO2H
MeO
28
28
TBSO
67
66
3CHEME A , $)04 4I/I0R T"U//( ¯ -3 #(#L B 0-""R .A( 4(& # C ,I#≡#/%T "&q/%T n # D .A( -E) 4(& # → 24 OVER TWO STEPS E (G#L %T/( 24 F $$1 (/ #(#L 24 OVER TWO STEPS G P4S/( ¯ -3 0H( 24 H 4"3/4F %T. #(#L n # → # I 0H#( # OVER TWO STEPS J "(q4(& 4(& n # → 24 THEN (/ .A/( 24 K 0H0 0Y ) 0H( # L .A3/0H $-& # M 4"3#L %T. #(#L # → 24 OVER TWO STEPS
AN ALTERNATIVE TO AN ACETIC ACID DERIVED ANION OCCURRED SMOOTHLY IN THE PRESENCE OF "&q/%T TO AFFORD AN UNSTABLE ALCOHOL WHICH WAS IMMEDIATELY METHYLATED TO ALKYNYL ETHER #ONSTRUCTION OF THE CYCLOHEXYL RING WAS ACCOMPLISHED VIA AN INTRIGUING )RELAND #LAISEN ;n= APPROACH FOR TRANSFER OF STEREOCHEMICAL INFORMATION FROM THE READILY PREPARED LACTONE 3UBJECTING THE RESULTING CARBOXYLIC ACID TO AN EXCESS OF "(q4(& RESULTED IN BOTH REDUCTION AND THE REGIO AND STEREOSELECTIVE INTRODUCTION OF A HYDROXYL GROUP AT # ;= #OMPARISON WITH A DEGRADATION PRODUCT ;= FROM &+ CONVENIENTLY CONFIRMED THE CORRECT STRUCTURE FOR PRIMARY ALCOHOL WHICH WAS FURTHER TRANSFORMED TO SULFONE WITHOUT INCIDENT 3CHEME 5NION OF SULFONE WITH AN APPROPRIATE ELECTROPHILE WAS ORIGINALLY INTENDED TO CONSTRUCT THE # # TRISUBSTITUTED OLEFIN THROUGH AN EVEN TUAL DEHYDRATION PROTOCOL &OR THIS PURPOSE ALDEHYDE WAS PREPARED FROM THE READILY AVAILABLE β KETO ESTER AS DISPLAYED IN SCHEME !SYMMETRIC CATALYTIC REDUCTION UNDER .OYORIS CONDITIONS ;= PROVIDED β HYDROXY ESTER WITH EXCELLENT ENANTIOCONTROL EE WHICH WAS SUBJECTED TO &RATER 3EEBACH ALKYLATION ; = WITH ALLYL BROMIDE TO FURNISH AS A SINGLE DIASTEREOMER ! SERIES OF PROTECTING GROUP MANIPULATIONS WHICH INCLUDED MASKING THE ALLYL GROUP AS AN IODOETHER AND FINALLY OXIDATION FURNISHED ALDEHYDE /F THE NUMEROUS METHODS TO EFFECT ASYMMETRIC
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O MeO
O
OPMB a
O MeO
24 20
OH OPMB
O b
MeO
24
c,d,e,f,g
24 20
SnPh3
Me
PMBO
OH OPMB
O H
20
O
24
H
20
TESO
72
26 24
I
73
74
76
i,j,k,l,m,n
PMB O O
20
OH
O
I 71 3 steps ~45%
PMBO 20
75 h
Ph s,t
CHO
24
PMBO
O
O
TESO
PMBO
r
20
O
HO
27
PMBO
o,p,q
O
20
OAc
24
27
Ph
Ph
Ph
O
O
20
OAc
24
O
O
CHO
24
27
27
I
TBSO u,v,w
34 31
MeO
70
TESO 20
PMB O O
28
SO2Ph
Ph
O
H
x,y
OMe 28
24
O H34 81
77
78
79
80
31
TESO
PMB O O
Ph
O 28
20
OMe
24
H 34
OTBS 82
O
z,aa 31
PMB O O
Ph
O 28
H
20
OMe
24
H 34
OTBS
31
OTBS
83
3CHEME A 2U#L;3 BINAP= %T. ( PSI -E/( 24 EE B ,$! 4(& n # THEN ALLYL BROMIDE (-0! n # → 24 C ,I!L( 4(& # → 24 D $$1 ¯ -3 #(#L 24 E ) .A(#/ #(#. # → 24 F $)"!, ( #(#L n # OVER TWO STEPS MIXTURE OF IODOMETHYL DIASTEREOMERS G #/#L $-3/ #(#L n # THEN %T. n # → 24 H "&q/%T #(#L n # DR I 4"3/4F %T. J / #(#L THEN 0H0 K VINYL 'RIGNARD #(#L DR L N"U.& M 0H#(/ P4S/( N / $-3 +#/ O .A"( P !C/ %T. Q :N .(#L R 4%3/4F %T. S +#/ -E/( T #/#L $-3/ #(#L n # THEN %T. n # → 24 U N"U,I 4(& n # THEN n # → # V $-0 #(#L 24 W .A(G .A(0/ 4(& -E/( n # OVER TWO STEPS X -E-G"R Y "URGESS REAGENT Z N"U.& AA #/#L $-3/ #(#L n # THEN %T. n # → 24
.O YIELDS OR FURTHER EXPERIMENTAL DETAILS REPORTED
CROTYLATION OF CHIRAL OR ACHIRAL ALDEHYDES 3CHREIBER ENJOYED MOST SUCCESS WITH STANNANE IN THE PRESENCE OF "&q/%T WHICH AFFORDED THE DESIRED ALCOHOL AS A SINGLE IODOMETHYL DIASTEREOMER ;= &URTHER ELABORA TION TO AND ATTEMPTED ANIONIC COUPLING WITH SULFONE FAILED UNDER STANDARD CONDITIONS DUE TO THE PRESENCE OF THE IODOMETHYL GROUP .O SUITABLE SOLUTION COULD BE FOUND AND CONSEQUENTLY A RATHER LENGTHY SERIES OF MANIPULATIONS CONVERTED TO ELECTROPHILE WHICH WHEN COMBINED WITH LITHIATED AFFORDED AFTER OXIDATION AND DESULFONYLATION THE DESIRED KETONE !DDITION OF -E-G"R THEN GENERATED A TERTIARY ALCOHOL WHICH COULD BE SUBSEQUENTLY DEHYDRATED BY USAGE OF THE "URGESS REAGENT TO AFFORD TRISUBSTITUTED ALKENE ;= .EITHER YIELD NOR SELECTIVITY IN THIS TRANS FORMATION WAS REPORTED ALTHOUGH EARLIER MODEL STUDIES HAD REVEALED THAT
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
MeO2C
28
OMe
a,b
MeO2C
28
OMe 31
31 34
84
34
OTIPS
85
c,d
OHC
28
OMe 31
34
OTIPS
17
28
OMe
e,f
31 34
OTIPS
86
g
Br
28
OMe 31 34
OTIPS
87
3CHEME A "(q4(& 4(& n # → # THEN .A/( (/ # → 24 B 4)03/4F %T. #(#L # → 24 C ,I!L( 4(& # D #/#L $-3/ #(#L n # THEN %T. n # → 24 E T"U/+ .#(0/ /-E 4(& n # THEN n # → n # F N"U,I 4(& n # → n # THEN -E) n # G #P:R( #L 0H( # THEN . BROMOSUCCINIMIDE 24
SUCH A STRATEGY ALTHOUGH SUCCESSFUL AFFORDS ONLY MODERATE SELECTIVITY IN FAVOUR OF THE % OLEFIN ^ &URTHER ELABORATION TO THE FULLY FUNCTIONALISED # # FRAGMENT WAS READILY ACCOMPLISHED USING STANDARD METHOD OLOGY 3CHEME ALTHOUGH ONCE AGAIN NO YIELDS WERE REPORTED 4HIS ROUTE WAS ULTIMATELY ABANDONED DUE TO ITS LENGTH INABILITY TO DIFFERENTIATE THE # AND # HYDROXYL GROUPS AND PERHAPS INEFFICIENT FORMATION OF THE CRUCIAL TRISUBSTITUTED OLEFIN 4HE ALTERNATIVE APPROACH TO THE # # FRAGMENT REPORTED BY 3CHREIBER WAS BASED UPON THE STEREOSELECTIVE ADDITION OF A VINYL METAL SPE CIES TO A # ALDEHYDE FORMING THE # # BOND INSTEAD AND THUS ALLOW ING PRIOR REGIOSELECTIVE FORMATION OF THE TRISUBSTITUTED OLEFIN 4O ACHIEVE THIS 3CHREIBER FIRST SOUGHT A SYNTHESIS OF THE # # VINYL BROMIDE THROUGH AN EXTENSION TO HIS )RELAND #LAISEN APPROACH FOR THE CYCLOHEXYL MOIETY OF &+ ;= !CCORDINGLY STRAIGHTFORWARD MANIPULATION OF TO ALDEHYDE WAS FOLLOWED BY EFFICIENT HIGH YIELDING INSTALLATION OF THE TERMINAL ALKYNE USING THE 3EYFERTH DIAZOPHOSPHONATE REAGENT IN METH ODOLOGY DEVELOPED BY #OLVIN AND 'ILBERT ;= 3TANDARD METHYLATION OF THE ALKYNE THEN ALLOWED FORMATION OF THE DESIRED % BROMOOLEFIN VIA TREATMENT OF WITH 3CHWARTZ REAGENT ; = AND SUBSEQUENT TRAPPING OF THE INTERMEDIATE VINYL ZIRCONIUM SPECIES WITH AN ELECTROPHILIC SOURCE OF BROMINE ."3 TO AFFORD IN A TOTAL OF STEPS AND OVERALL YIELD 3CHEME 4HE DESIRED COUPLING PARTNER FOR VINYL BROMIDE ALDEHYDE WAS EASILY PREPARED FROM ALLYLIC ALCOHOL 3CHEME 3ILYL PROTECTION OF THE # ALCOHOL FOLLOWED BY CHEMOSELECTIVE OZONOLYSIS LIBERATED 4HE PRESENCE OF PYRIDINE IN THIS OZONOLYSIS WAS ESSENTIAL IN MODERATING THE REACTIVITY OF THE OZONE AND PREVENTING ANY OXIDATIVE REMOVAL OF THE # 0-" ETHER BY OXIDISING ITSELF PREFERENTIALLY TO PYRIDINE . OXIDE !DDITION
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O
I
a,b
24
20
Br
PMB TIPS O O O
PMB OH O
O 76
24
20
28
OMe
PMB TIPS O O OH
31
H 34
OTIPS
28
87
H
O
c
I
20
OMe
24
31
H 34
88
OTIPS
89
I
d
PMB TIPS O O O O
N Boc H O 28
H
20
OMe
24
31
H 34
59
PMB TIPS O O O
f
OTIPS
20
HO
N Boc H O
OMe 31
H 34
91
N Boc H O 28
28 24
PMB TIPS O O O
e
O
20
OMe
24
31
H 34
OTIPS
I
OTIPS
90
3CHEME A 4)03/4F %T. #(#L B / 0Y #(#L -E/( THEN -E3 24 OVER TWO STEPS C T"U,I 4(& n # THEN -G"R THEN n # → 24 αβ D . "OC , PIP $## PYRRO LIDINOPYRIDINE #(#L n # E :N .(#L %T/( AQ # F #/#L $-3/ #(#L n # THEN %T. n # → 24
TO A COLD SOLUTION OF THE VINYL 'RIGNARD DERIVED FROM VIA INITIAL LITHIUM HALOGEN EXCHANGE FOLLOWED BY TRANSMETALLATION WITH -G"R GAVE A REA SONABLE YIELD OF DIASTEREOMERIC PRODUCTS 0LEASINGLY THIS SEPARABLE MIXTURE WAS WEIGHTED IN FAVOUR OF THE DESIRED α CARBINOL ^ AS PREDICTED IN A #RAM SELECTIVE ADDITION #RUCIALLY THIS APPROACH NOW ALSO ALLOWED DIFFERENTIATION OF THE # AND # ALCOHOLS WITH THE FORMER ALREADY PROTECTED AS THE 4)03 ETHER 7ITH THIS IN PLACE THE # HYDROXYL COULD BE SELECTIVELY ACYLATED USING READILY AVAILABLE . "OC PROTECTED 3 PIPECOLIC ACID UNDER STANDARD CONDITIONS &INALLY UNMASKING THE ALLYL SIDE CHAIN ON TREATMENT WITH ZINC AND 3WERN OXIDATION OF THE RESULTANT PRIMARY ALCOHOL COMPLETED A MUCH MORE EFFICIENT SYNTHESIS OF THE # # FRAGMENT IN A TOTAL OF STEPS AND OVERALL YIELD 3CHEME 4HE COUPLING PARTNER FOR THE # # ALDEHYDE WAS THE # # PHOS PHONATE AND IT WAS ANTICIPATED THAT THE SECOND TRISUBSTITUTED OLEFIN BETWEEN # AND # WOULD BE INSTALLED IN A (ORNER TYPE OLEFINATION BETWEEN THESE TWO FRAGMENTS &OR THE SYNTHESIS OF 3CHREIBER CHOSE TO APPLY A TWO DIRECTIONAL STRATEGY ; = TAKING ADVANTAGE OF THE LATENT # SYMMETRY OF THE # METHYL AND # METHOXY GROUPS ABOUT # TO THOSE AT # AND # RESPECTIVELY 4HEREFORE BY STARTING FROM # SYM METRIC ARABITOL HE WAS ABLE TO RAPIDLY AND EFFICIENTLY BUILD UP 3CHEME ;=
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
O
O
OH OH
AcO
C(O)Cl 93
O
O 15
15
a,b,c
OH OH OH
O
d,e
O
O
OMe OMe
g,h,i
10
MeO2C
15
OTBS 95
94
O
O
f
10
15
OTBS
92
O
CO2Me
15
10
OBn
OTBS 96
97
j,k
(Me2N)2(O)P
10 19
15
n,o,p,q
10 15
O OTBS
60
S
OMe OMe
OMe OMe
OMe OMe OMe OMe
S
m
MeO2C
l
15
O
S
MeO2C
15
O
10
10
S
O
100
99
OH
98
O
3CHEME A α ACETOXYISOBUTYRIC ACID CHLORIDE #(#. # → 24 B .A/-E 4(& # → 24 C 4"3#L .A( # → 24 D (##/%T N"U,I 4(& n # THEN "&q/%T E (G#L P4S/( %T/( REFLUX OVER TWO STEPS F I0R.( N"U,I 4(& n # THEN THEN -E) n # → n # G (& #(#. H "N4#! 4F/( #(#LCHEX OVER TWO STEPS I .A/( -E/( 4(& .A( -E) $-& # → 24 J ( 0D/( %T/!C K 0043 #(#L BRSM L ,nSELECTRIDE 4(& n # M PROPANEDITHIOL "&q/%T #(#L n # → 24 OVER TWO STEPS N ,I!L( 4(& # O 0H0 ) 0Y 0H( # → 24 P 4"3/4F %T. #(#L # Q #(#(0/ .-E N"U,I 4(& n # → # THEN ALKYL IODIDE DERIVED FROM n # → #
"IS EPOXIDE READILY AVAILABLE FROM COMMERCIALLY AVAILABLE ARABITOL BY TREATMENT WITH THE -OFFATT REAGENT ;= AND THEN ACETATE HYDROLYSIS WAS OPENED REGIOSELECTIVELY AT BOTH OXIRANE RINGS WITH AN ETHOXYACETYLIDE ANION GIVING A SPECIES THAT RECYCLISED IN SITU ON WORK UP WITH WEAK ACID TO THE CORRESPONDING FIVE MEMBERED LACTONE 3CHEME -ETHYLATION OF THE LITHIUM ENOLATE OF THEN OCCURRED THROUGH SUBSTRATE CONTROL TO INSTALL THE # AND # METHYL GROUPS WITH HIGH DIASTEREOFACIAL SELEC TIVITY WITH AN UNIDENTIFIED MINOR ISOMER !FTER PROTECTING GROUP SWAP OF THE # HYDROXYL GROUP METHANOLYSIS OF THE BIS LACTONE OCCURRED WITH CONCOMITANT METHYLATION OF THE # AND # HYDROXYLS GIVING WHEREIN THE # SYMMETRY IS STILL PRESENT 4HIS WAS NOW EXPLOITED AFTER UNMASKING THE # /( USING 0EARLMANS CATALYST TREATMENT WITH 0043 PROMOTED A SELECTIVE RECYCLISATION TO GIVE THE DESIRED LACTONE 7HEN ADOPTS A CHAIR CONFORMATION ALL SUBSTITUENTS ARE EQUATORIAL GIVING RISE TO THE SELECTIVITY VERSUS THE STEREOISOMER WHICH PLACES THE BULKY # # SUBSTITUENT AXIAL ;= (AVING DIFFERENTIATED THE TERMINI SUCCESSFULLY LACTONE WAS CLEAVED VIA REDUCTION AND TRAPPING OF THE RESULTANT ALDEHYDE AS THE DITHIANE PROPANEDITHIOL "&q/%T &INALLY A SERIES OF STANDARD TRANSFORMATIONS CONVERTED LACTONE TO THE
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TIPSO MeO
34
30
OPMB
26
N
OMe OMe (Me2N)2(O)P
10 19
15
OTBS
S
S
O +
60
PMB TIPS O O O
22
Boc a
O 28
H
20
O TIPS O
O 1
OMe
24
31 34
N Boc
OTIPS
59
19
TBSO 15
S 10 S 101
OMe
OMe
8
O MeO
HO MeO
MeO
30
30
O
N 8
O
O OH H O 10
O
e,f,g,h,i
1
19
O TIPS O
22
O 19
OMe
O
8
DMBO
1
d
N Boc
N
OMe FK506 1
OPMB
26
22
OH O
1 26
30
OPMB
26
22
O
34
34
MeO
b,c
58
TIPSO
TIPSO
34
OMe
CO2Me
TBSO 15
OMe
TESO 103
HO2C 8
19
TBSO 15
DMBO
10
OMe
O TIPS O
10
OMe
OMe
OH
102
3CHEME A N"U,I n # → # THEN (-0! n # THEN n # → # 0H-E REFLUX B 0H)/#/#& -E/( #(#L (#/ #/( !C/( #(#L 24 → # C I0R.( N"U,I 4(& → n # THEN THEN ALDEHYDE DERIVED FROM THEN ,I/( n # → 24 D LUT #(#L # THEN 4%3/4F THEN 3I/ 24 . METHYL CHLOROPYRIDINIUM IODIDE %T. #(#L E 4&! 4(& (/ F $-0 #(#L G $$1 T"U/( P( BUFFER OVER SIX STEPS H $-0 #(#L I (& #(#.
PRIMARY ALKYL IODIDE WHICH WAS DISPLACED USING THE LITHIUM SALT OF ETHYL BISDIMETHYLAMINO PHOSPHONAMIDE TO GIVE THE DESIRED PHOSPHONAMIDE REAGENT 3CHEME 7ITH THE TWO KEY FRAGMENTS OF &+ NOW COMPLETED 3CHREIBERS ATTEN TION TURNED TO HIS ENDGAME STRATEGY +EY TO THIS WAS THE INITIAL COUPLING OF AND TO FORM THE # # TRISUBSTITUTED OLEFIN (OWEVER ON ADDI TION OF THE LITHIUM ANION OF TO ALDEHYDE AND SUBSEQUENT THERMAL ELIMINATION OF THE MAJOR DIASTEREOMER PAIR 0H-E # OLEFIN COULD ONLY BE OBTAINED AS THE DESIRED ISOMER IN A DISAPPOINTING YIELD 3CHEME 4HE INITIAL ADDITION ALSO GAVE RISE TO A SECOND PAIR OF DIASTEROEOMERS WHICH ON HEATING GAVE RISE TO THE : OLEFIN ALTHOUGH THEIR QUANTITIES DO NOT ACCOUNT FOR THE BULK OF THE MATERIAL 4HIS WAS UNDOUBTEDLY A
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
SIGNIFICANT PROBLEM IN THE SYNTHESIS ALTHOUGH THIS RESULT SHOULD NOT HAVE BEEN COMPLETELY UNEXPECTED THE -ERCK GROUP HAD SIMILAR TROUBLES IN THE FIRST TOTAL SYNTHESIS OF &+ YIELD OF THE DESIRED OLEFIN AND INDEED FRAGMENT COUPLINGS TO FORM TRISUBSTITUTED OLEFINS WITH ANY RESPECTABLE STEREOCONTROL OR YIELD ARE NOTORIOUSLY DIFFICULT IN NATURAL PRODUCTS SYNTHESES .EVERTHELESS THE UNION OF AND ALLOWED 3CHREIBER TO PROCEED TOWARDS &+ 5NMASKING THE # ALDEHYDE WAS ACHIEVED IN A TWO STEP PROCEDURE VIA THE DIMETHYLACETAL IN YIELD WITH STANDARD ONE STEP PROCEDURES GIVING AT BEST 4HE FINAL TWO CARBONS OF THE NATURAL PROD UCT WERE THEN INSTALLED USING AN ALDOL REACTION WITH THE LITHIUM ENOLATE OF TWO STEPS FROM COMMERCIALLY AVAILABLE α BROMOACETIC ACID AND THE ALDEHYDE DERIVED FROM GIVING AN INCONSEQUENTIAL MIXTURE OF DIASTEREOMERS !T THIS STAGE THE # # # RADIOLABELLED ANALOGUE WAS ALSO PREPARED VIA INCORPORATION THROUGH USE OF COMMERCIALLY AVAILABLE # α BROMOACETIC ACID 3CHEME (YDROLYSIS OF THE ALDOL ADDUCT FOLLOWED BY SILYL MEDIATED REMOVAL OF THE . "OC PROTECTING GROUP THEN ENABLED STANDARD -UKAIYAMA MACROLAC TAMISATION ;= TO GIVE THE PARENT MACROCYCLE OF THE IMMUNOSUPPRESSANT &+ IN REASONABLE OVERALL YIELD FROM ALDEHYDE &INALLY A DELICATE AND ORDER SENSITIVE SERIES OF PROTECTING GROUP MANIPULATIONS AND OXIDA TIONS WERE ACHIEVED RETURNING THE NATURAL PRODUCT AND COMPLETING THE SECOND TOTAL SYNTHESIS OF &+ )N COMPLETING HIS TOTAL SYNTHESIS OF &+ 3CHREIBER UNVEILED A STRAT EGY VERY SIMILAR TO THAT SUCCESSFULLY ACCOMPLISHED BY THE -ERCK GROUP )NDEED WITH THE SAME MAJOR DISCONNECTIONS OF THE NATURAL PRODUCT 3CHREIBERS APPROACH ONLY REALLY DIFFERS AT THE HIGHEST LEVEL IN WHAT STAGE THE PIPECOLATE MOIETY IS INSTALLED .EVERTHELESS THERE IS CONSIDERABLE DIFFERENCE IN THE INDIVIDUAL PREPARATION OF THE KEY FRAGMENTS THE -ERCK GROUP PLACED A STRONG EMPHASIS ON THE UBIQUITOUS AND RELIABLE ASYM METRIC ALDOL REACTION PARTICULARLY THAT OF %VANS ; = WHILE THIS IS NOTICEABLY ABSENT ABOVE !S A RESULT THE SECOND SYNTHESIS OF &+ FEATURES A GREATER VARIETY OF INTERESTING METHODOLOGY 0ARTICULARLY NOTICE ABLE ARE THE FIRST USE OF THE )RELAND #LAISEN REARRANGEMENT IN THE SYNTHESIS OF THIS CLASS OF NATURAL PRODUCTS FOR THE CONSTRUCTION OF THE CYCLOHEXYL MOIETY AND THE EXPLOITATION OF THE LATENT # SYMMETRY AROUND THE # HYDROXYL GROUP IN A TWO DIRECTIONAL SYNTHESIS OF THE # # PORTION
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3CHEME 3UMMARY OF THE 3CHREIBER TOTAL SYNTHESIS OF &+
OF THE MOLECULE (OWEVER SUCH A STRATEGY WAS INEVITABLY DISADVANTAGED FROM THE OUTSET WITH THE KEY DISCONNECTION ACROSS THE # # TRISUB STITUTED OLEFIN ALWAYS GOING TO PROVE PROBLEMATIC IN THE FORWARD SENSE 4HE LOW YIELD FOR THIS REACTION n AND CRUCIALLY FOR OTHER KEY STEPS n IS DIS APPOINTING AND MARS THE SYNTHESIS SOMEWHAT .EVERTHELESS 3CHREIBER HAS REPORTED A CONSIDERABLY SHORTER APPROACH AS WELL AS A HIGHER OVERALL YIELD 3CHEME &URTHERMORE 3CHREIBERS ACHIEVEMENTS IN HIS WORK ON &+ ARE SIGNIFI CANT 0ROBABLY MOST IMPORTANT IN HIS STRATEGY WAS THE ABILITY TO INCORPORATE # LABELS AT # AND # AND THE PREPARATION OF THIS RADIOLABELLED ANALOGUE OF &+ HAS LED TO A DRAMATIC IMPROVEMENT IN THE UNDERSTANDING OF THE MODE OF ACTION OF THIS IMMUNOSUPPRESSANT 4HE SIGNIFICANCE OF THIS ADVANCEMENT SHOULD NOT BE UNDERESTIMATED
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
4HE )RELAND TOTAL SYNTHESES OF &+
)N THE LATE S 2OBERT )RELAND DISCLOSED DETAILS OF TWO SEPARATE TOTAL SYNTHESES OF &+ WITHIN TWELVE MONTHS ;n= 4HESE PUBLICATIONS WERE THE CULMINATION OF MANY YEARS OF INVESTIGATION INTO THE CHEMISTRY OF THIS FORMIDABLE MOLECULE WITHIN HIS LABORATORIES AT THE 5NIVERSITY OF 6IR GINIA WHICH ALSO GAVE RISE TO A FLEXIBLE SYNTHESIS OF &+ !.!, AN ANALOGUE COMPRISING THE ACETONIDE OF DIHYDRO &+ ;= 7HILE )RELANDS TWO SYNTHESES OF &+ CAN BE DIFFERENTIATED SOME WHAT BY THEIR KEY DISCONNECTIONS AND THEIR ALTERNATIVE APPROACHES TO SOME OF THE FUNCTIONALITY WITHIN THE MOLECULE THERE IS UNSURPRISINGLY CONSIDERABLE OVERLAP ACROSS BOTH STRATEGIES 5BIQUITOUS IS A STRONG EMPHA SIS ON THE USE OF SPIROENONES AND THE CORRESPONDING FUNCTIONALISED SPIRO KETALS AS PRECURSORS TO THE α ALLYL ALDOL SYSTEM # # VIA FRAGMENTA TION METHODOLOGY DEVELOPED PREVIOUSLY BY )RELAND !S EXPECTED SOME OF THE KEY FRAGMENTS ARE ALSO PREPARED IN A SIMILAR FASHION AS IS THE CASE ACROSS MANY OF THE PUBLISHED SYNTHESES OF THESE RELATED NATURAL PRODUCTS .EVERTHELESS TO REALISE TWO SEPARATE AND COMPLETE ROUTES TO A MOLECULE SUCH AS &+ IS A FANTASTIC ACHIEVEMENT WITHIN THE FIELD OF NATURAL PRODUCTS SYNTHESES &OR THE PURPOSE OF THIS REVIEW THE FOCUS WILL BE PREDOMINANTLY ON THE SECOND OF )RELANDS TOTAL SYNTHESES ARGUABLY THE MORE EXCITING AND THOR OUGH OF THE TWO (OWEVER DETAILS OF HIS OTHER WORK ARE PRESENTED AFTER THIS
)RELAND TOTAL SYNTHESES OF &+ 0ART /NE 4HUS IN )RELAND DISCLOSED DETAILS OF THE FOURTH AND HIS SECOND TOTAL SYNTHESIS OF &+ ; = 4HE SYNTHETIC STRATEGY WAS AN ATTRACTIVE COMBINATION BORN OUT OF METHODOLOGIES DEVELOPED WITHIN THE )RELAND GROUP AND BENEFITING FROM PREVIOUS DISCOVERIES BY THE -ERCK GROUP AND OTHERS IN THEIR EFFORTS TO SYNTHESISE THIS POTENT IMMUNOSUPPRESSANT 3PECIFICALLY THERE WERE A NUMBER OF KEY CONCERNS IDENTIFIED BY )RELAND THAT DICTATED THE RETROSYNTHESIS ! FINAL MACROCYCLISATION WAS ENVISAGED VIA AMIDE BOND FORMATION THUS AVOIDING THE PUBLISHED DIFFICULTIES IN ATTEMPT ING MACROLACTONISATION ; = AND CRUCIALLY THE CONFIGURATIONALLY LABILE
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
HO MeO
34
TBDPSO 30
TBDPSO
34
34
H O
24
1
22
O
CO2H N Boc + 12
OH 1
MeO
O
OTES 30
H
8
O
O OH H O 10
O
OMe OMe
FK506 1
O
26
CO2Et H O
O O
15
OMe
H
105
O O PMB
CHO
MeO
21 8
N
26
21 16
CO2Et H 15 O CHO
O
OMe
C8-C34 spiroketal 104
Br +
8
O
O O
27
+
OTBS
O O PMB 107
OMe
106
3CHEME 2ETROSYNTHETIC ANALYSIS
# STEREOCENTRE WITHIN &+ NECESSITATED LATE STAGE INTRODUCTION OF THE PIPECOLATE MOIETY 3CHEME &URTHERMORE THE MASKED TRICARBONYL UNIT WAS BY THIS POINT KNOWN TO BE EXTREMELY SENSITIVE WITH A PROPENSITY FOR # # BOND CLEAVAGE ; = AND AS A RESULT )RELAND CHOSE INSTEAD TO PURSUE A LESS ELECTROPHILIC # /( ANALOGUE "Y ANALOGY WITH THE NATURAL PRODUCT PEDERIN THE GROUP SOUGHT TO APPLY AND EXTEND METHODOLOGY DEVELOPED BY -EINWALD FOR THEIR SYNTHESIS OF THE TOXIC VESICANT ;= WHEREIN HYDROLYSIS OF SPIRO KETAL ACETONIDE REVEALS AFTER MANIPULATION AN α HYDROXY β KETOAMIDE ACETAL SIMILAR TO THAT IN &+ 3CHEME 4HE # # β HYDROXY KETONE ALSO IMPARTS INSTABILITY BOTH UNDER ACIDIC AND BASIC CONDITIONS ;= !S DISCUSSED )RELAND LOOKED TO TAKE ADVANTAGE OF THE α ALLYL GROUP # BY INCORPORATING IT INTO THE PROTECT ING GROUP STRATEGY USING METHODOLOGY THAT HAD BEEN DEVELOPED PREVIOUSLY IN THE GROUP AND THAT WAS BASED ON THAT OF &ßRSTNER 4HEREIN FRAGMENTA TION OF FUNCTIONALISED SPIROKETALS OF THE TYPE IN DIRECTLY DELIVERED THE α ALLYL ALDOL SYSTEM IN THE CORRECT OXIDATION STATE ;= 4HUS DECONSTRUCTION OF REVEALED COMMERCIALLY AVAILABLE 3 PIPE COLIC ACID AND THE LATTER OF WHICH WAS DISCONNECTED FURTHER ACROSS # # AND # # VIA ANTICIPATED CARBANIONIC COUPLINGS GIVING THREE IMMEDIATE TARGETS AND 4HE SYNTHESIS OF # # TRICARBONYL PRECURSOR BEGAN WITH STANDARD CARBOHYDRATE CHEMISTRY TO CONVERT METHYL α $ GLUCOSIDE TO ALCOHOL IN STEPS ;n= TAKING ADVANTAGE OF THE INEXPENSIVE READILY AVAILABLE SOURCE OF CHIRALITY THAT SUGARS AFFORD $EOXYGENATION OF THE FREE ALCOHOL WAS ACHIEVED VIA PREPARATION OF THE UNUSUAL # PHOS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
O
O
O
O
OH
a,b
OH
O
CO2Et
CO2Et
OMe
O
OMe O
CONH2
c,d
OH
OH
ArSe
HO
O
OMe OMe
OH
109
108
OMe N H
110
pederin 111
3CHEME A O NITROPHENYLSELENOCYANATE N"U0 4(& # B !C#L -E/( C (/ 4(& # → 24 D SEPARATION .( -E/( → #
O
MeO
H
10
HO
15
OH OH 112
O
MeO
OH
H
6 steps 56%
O
15
O
10
O
10
H
O
MeO 15
a,b Ph
Ph
O O
O
O
MeO
c
15
OH
OH
P Me2N NMe2
OH 113
H
10
115
114 d,e,f,g 8
8
CO2Et O H 15 CHO O O 10
k,l
CO2Et O H O O 10
i,j
15
OBn
THPO HO
CO2Me 15 O H 10
118
h
O
O
H
15
OBn
10
OBn
OMe OMe
OMe
OMe 106
8
117
116
3CHEME A N"U,I $-% 4-%$! # THEN #L0/.-E # → 24 B (.-E C ,I %T/( %T.( 4(& # OVER THREE STEPS D .A( "N"R 4(& # E .A( -E) 4(& F !C/( (3/ (/ # G !C/ $-3/ OVER TWO STEPS H ,$! 4(& 4(0/#(#/#( n # I 0043 4(& (/ # J #(#/#( 0/ OVER TWO STEPS K ( 0D/( %T/( L $-0 #(#L
PHORODIAMIDATE USING A MODIFIED PROCEDURE ; = FOLLOWED BY DISSOLVING METAL REDUCTION ,I%T.( THAT OCCURRED WITH CONCOMITANT CLEAVAGE OF THE BENZYLIDENE ACETAL FURNISHING DIOL !FTER INSTALLATION OF THE BENZYL AND METHYL ETHERS HYDROLYSIS OF THE METHYL ACETAL AND !LBRIGHT 'OODMAN OXIDATION ;= COMPLETED A SIMPLE SCALABLE APPROACH TO LACTONE 3CHEME )NTRODUCTION OF A KETAL ESTER INTO LACTONE PROVED UNEXPECTEDLY PROBLEMATIC DUE TO STERIC HINDRANCE !FTER CONSIDERABLE EXPERIMENTATION THE ADDITION OF TETRAHYDROPYRANYL ETHYL GLYCOLATE WAS ACHIEVED TO GIVE ADDUCT THAT COULD BE PROTECTED AS THE ACETONIDE AND OXIDISED TO GIVE THE DESIRED INTERMEDIATE STEPS AND YIELD IN A REASONABLE YIELD OVER THREE STEPS 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
26
24
d
OH
BnO TBSO
OH
122 26
BnO
CHO
+
(Ipc)2B
a,b
25
BnO
c
26
26
+
25
26
119
120
e,f,g
24
BnO TBSO
121
24
BnO TBSO
OTBS OH
O
124
OH
123
3CHEME A 4(& n # THEN "&q/%T THEN %T/ THEN .A/( (/ REFLUX B 4"3#L $-!0 CAT $-& OVER TWO STEPS C /S/ .-/ ACETONE (/ 0H( D .A( 4(& # THEN 4S)M # → 24 E "Z#L $-!0 CAT 0Y F -S#L $-!0 CAT 0Y G .A/-E -E/( # OVER THREE STEPS
BnO
BnO 26
O
OMe
a
26
O
OH
O
125 6 steps ~47%
OMe 24
24
126
b,c
SEt 26
BnO TBSO
127
24
OH
SEt
d,e
26
BnO TBSO
122
24
OH
f OH
26
BnO TBSO
24
O
124
3CHEME A -E-G#L #U"Rq3-E 4(& n B %T3( (#L C 4"3#L )M $-!0 CAT $-& OVER TWO STEPS D (G#L #A#/ #(#. E .A"( 4(& (/ # → 24 OVER TWO STEPS F .A( 4(& # THEN 4S)M # → 24
&OR THE SYNTHESIS OF KEY # # SPIROKETAL FRAGMENT )RELAND IDEN TIFIED THREE KEY STRUCTURES A @LINKER DOMAIN CORRESPONDING TO # # 3CHEME THE SPIROKETAL ITSELF # # 3CHEME AND A @ DIOL # # 3CHEMES (OWEVER DESPITE THIS OBSERVA TION THE STRATEGY TO REMAINED A LARGELY LINEAR SEQUENCE 4HE FIRST PART OF THIS STRATEGY ENTAILED THE CONSTRUCTION OF THE DIOL MOTIF INITIALLY VIA AN ENANTIOSELECTIVE "ROWNS CROTYLATION ; = OF BENZYLOXY ACETALDEHYDE USING : CROTYL DIISOPINOCAMPHEYL BORANE TO INSTALL THE ABSOLUTE STEREOCHEMISTRY AT # AND # WITH DR AND EE (OWEVER ELABORATION OF HOMOALLYLIC ALCOHOL TO EPOXIDE PROVED PROBLEMATIC $IHYDROXYLATION OF THE DOUBLE BOND GAVE A MIXTURE OF DIOLS AND AT BEST AND WHILST BOTH OF THESE COULD BE EASILY TRANSFORMED INTO THE DESIRED EPOXIDE ENANTIOMER THIS REQUIRED SEPARATION OF THE DIOL ISOMERS hTEDIOUS AND ON LARGE SCALES ≥ G EVEN FORMIDABLEv $IRECTED EPOXIDATION OF THE HOMOALLYLIC ALCOHOL PRO
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
HO(CH2)n
O
R OH
128
[O]
O HO(CH2)n
O
e.g. mCPBA
OH 129
H+
HO(CH2)n HO 130
O
R
O
O
O R
+
(CH2)n-2
O 131
O
R
(CH2)n-2
O 132
O
R
3CHEME $E3HONGS SPIROKETALISATION METHODOLOGY
CEEDED WITH EVEN WORSE SELECTIVITY FURNISHING A MIXTURE OF EPOXIDES USING 6/ACAC 4"(0 ;= SEE PAGE !S AN ALTERNATIVE TO THIS LABORIOUS PROBLEM )RELAND TURNED HIS ATTEN TION TO REPORTS IN THE LITERATURE THAT READILY AVAILABLE ANHYDRO β RIBO FURANOSIDE SIX STEPS FROM $ XYLOFURANOSE ;= REACTS REGIO AND STEREOSELECTIVELY WITH -E-G#L AT THE POSITION # TO FURNISH ALCOHOL ;= WHICH BARES AN IDENTICAL STEREOCHEMICAL RELATIONSHIP TO DIOL 7HILST THE ORIGINAL CONDITIONS REPORTED %T/ REFLUX WERE LARGELY UNSUCCESSFUL FOR BENZYL ETHER IN THE PRESENCE OF #U"Rq3-E IN TET RAHYDROFURAN 4(& THE REACTION GAVE CONSISTENTLY GOOD YIELDS AT ROOM TEMPERATURE 3CHEME ;= !FTER RING CLEAVAGE OF BY THIOACETAL FORMATION SELECTIVE PROTECTION OF THE LESS HINDERED ALCOHOL AS THE SILYL ETHER GAVE #AREFUL DEPROT ECTION OF THE DITHIOACETAL USING (G#L AFFORDED AN ALDEHYDE WHICH WAS IMMEDIATELY REDUCED TO DIOL WITHOUT ISOLATION TO AVOID EPIMERISA TION &INALLY FORMATION OF THE EPOXIDE AS PREVIOUSLY COMPLETED ACCESS TO AS A SINGLE DIASTEREOMER AND IN MULTI GRAM QUANTITY 3CHEME STEPS )T WAS HOPED THAT THE SPIROKETAL MOIETY COULD BE INTRODUCED USING A VARIANT OF $E3HONGS METHODOLOGY ;= WHEREIN A SUBSTITUTED FURFUROL DERIVATIVE OF THE TYPE IS OXIDISED TO AFFORD PYRANONE WHICH SUBSE QUENTLY UNDERGOES INTRAMOLECULAR KETALISATION TO FURNISH A SPIROKETAL AND CARRYING THE ANTI RELATIONSHIP UNDER EQUILIBRATING CONDITIONS 3CHEME !CCORDINGLY THE FURFURYL GROUP WAS INTRODUCED BY TREATING EPOXIDE WITH LITHIATED FURFURYL ALCOHOL IN THE PRESENCE OF "&q/%T !CIDIC REMOVAL OF THE METHOXYISOPROPYL -)0 PROTECTING GROUP USING CATALYTIC AMOUNTS OF 0043 IN 4(& (/ GAVE ALCOHOL READY FOR THE CRUCIAL SPIRO KETALISATION !FTER OXIDATION WITH M#0"! IN SITU TRAPPING OF THE ENEDIONE DIOL ACETAL WITH METHOXYPROPENE IN THE PRESENCE OF (#L GRATIFYINGLY
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
21
R 23
BnO TBSO 124
O
133
OMIP
a,b
BnO TBSO
23
OH 134
O
OH
c
23
OH
21 27
O
OH OH
21
O OH
21
OMe
R
23
O 27
135
OTBS
BnO 137
26
O
136
O O
21
O
O O 138
3CHEME A N"U,I 4(& n # → n # THEN "&q/%T n # B 0043 4(& (/ OVER TWO STEPS C M#0"! #(#L # THEN (#L
GAVE THE SPIROKETAL PRODUCT IN YIELD AS A MIXTURE IN FAVOUR OF THE DESIRED PRODUCT )NTERMOLECULAR TRAPPING OF THE REACTIVE INTERMEDIATE IN THIS CASE WITH METHOXYPROPENE AT THE TIME REPRESENTED AN IMPORTANT EXTENSION OF $E3HONGS WORK )MPRESSIVELY THE REACTION SEQUENCE FROM EPOXIDE TO SPIROKETAL WAS ACHIEVED CONSISTENTLY IN OVERALL YIELD ON SCALES UP TO G 3CHEME 4HE ENONE FUNCTIONALITY IN PROVIDED A HANDLE FOR THE INTRODUC TION OF THE # # LINKER DOMAIN VIA CONJUGATE ADDITION AT # !LKYNE EASILY PREPARED FROM 2OCHE ESTER DERIVATIVE ;= PROVIDED THE PRECURSOR FOR THE # # OLEFIN WHICH WAS OBTAINED AS THE DESIRED % ISO MER USING .EGISHI CARBOALUMINATION ; = !FTER TRANSMETALLATION OF THE ALUMINIUM SPECIES WITH A MIXED HIGHER ORDER CUPRATE ;= ADDITION OF ENONE TO THE REACTION FURNISHED THE ELONGATED FRAGMENT AS A SINGLE GEOMETRIC AND STEREOISOMER IN AN EXCELLENT YIELD 3TEREOSELEC TIVE REDUCTION OF THE CYCLIC KETONE USING , SELECTRIDE ;= VIA EQUATORIAL ATTACK THEN ESTABLISHED THE # /( SOLELY AS THE DESIRED CONFORMER VIDE INFRA !FTER MUCH EXPERIMENTATION THIS WAS PROTECTED AS THE 0-" ETHER USING 0-" TRICHLOROACETIMIDATE AND TRITYL TETRAFLUOROBORATE AT # ;= AVOIDING THE DESTRUCTIVE PATHWAYS THAT OCCURRED IN THE PRESENCE OF THE STANDARD ACID CATALYSTS FOR THIS REAGENT 4F/( "&q/%T 3CHEME 4HE 0-" ETHER WAS USED IN )RELANDS SUCCESSFUL SYNTHESIS ALTHOUGH FOR SOME OF HIS EARLIER STUDIES THE # HYDROXYL WAS PROTECTED AS THE ETHOXY ETHYL %% ETHER VIDE INFRA 4HE ORTHOGONAL HYDROXYL PROTECTING GROUPS AT # AND # IN WERE CRUCIAL FOR ENABLING FURTHER SELECTIVE HOMOLOGATION 7ITH THIS IN MIND )RELAND INVESTIGATED TWO ALTERNATIVE PATHWAYS FOR ELABORATION AT # TO INSTALL THE OXYGENATED CYCLOHEXYL MOIETY )NITIAL ATTEMPTS WERE CENTRED ABOUT THE CONSTRUCTION OF THE # # OLEFIN BOND VIA THE ADDITION OF A # ANION TO A # KETONE MODEL FOL
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
OTBS
BnO
26
O O
21
O 16
THPO
OTs
a,b
16
TBSO
19
O
BnO
OTBS O O
138
140
OTBS
O O 21
O O 141
OTBS 26
d,e
21
c 16
139 3 steps 94%
BnO
26
16
OTBS
O O PMB 142
3CHEME A (#≡#,Iq%$! $-3/ n # B 4S/( -E/( # 4"3/4F %T. %T/ C I #P:R#L -E!L #L#(#(#L THEN 24 II (#→##( N"U,I 4(& n # THEN #U#. n → n # III %T/ n # D ,nSELECTRIDE 4(& n # → # BRSM E 0-"n4#! 0H#"& CAT 4(& #
LOWED THEN BY DEHYDRATION -ODEL STUDIES DEMONSTRATED THAT AN UNSUB STITUTED CYCLOHEXYL METHYL ANION GENERATED BY LITHIUM HALOGEN EXCHANGE OF CYCLOHEXYL METHYL BROMIDE FOLLOWED BY TRANSMETALLATION WITH CERIUM )) CHLORIDE COULD BE SUCCESSFULLY ADDED TO AN α HYDROXY METHYL KETONE ANALOGUE OF IN GOOD YIELDS (OWEVER THIS REACTION WAS UNSUCCESSFUL WITH FUNCTIONALISED CYCLOHEXANE EASILY PREPARED FROM KNOWN ESTER ;= BOTH AS THE ORGANOCERIUM REAGENT OR AS ANY OF A NUMBER OF OTHER ORGANOMETALLIC DERIVATIVES 4HESE NEGATIVE RESULTS WERE ATTRIBUTED TO THE RELATIVELY LOW REACTIVITY OF THE METHYL KETONE AND INDEED MORE POSI TIVE RESULTS WERE PROMPTLY OBTAINED FOR THE ADDITION OF THE ORGANOCERIUM SPECIES DERIVED FROM INTO THE MORE REACTIVE # ALDEHYDE MODEL 3CHEME #ONSEQUENTLY CAREFUL CLEAVAGE OF THE BENZYL ETHER IN THE REAL SYSTEM WITH 7 2ANEY NICKEL AND ( ;= AVOIDED PROBLEMS ASSOCIATED WITH THE MIGRATION OF SILYL GROUPS AND REDUCTION OF THE TRISUBSTITUTED ALKENE 3UBSEQUENT OXIDATION FURNISHED THE KEY ALDEHYDE !DDITION OF LITHIATED ALKYL IODIDE TO A SOLUTION OF THIS ALDEHYDE AT n # GRATIFY INGLY RETURNED THE DESIRED # # BACKBONE IN REASONABLE YIELD AS A MIXTURE OF DIASTEREOMERS THAT WAS DIRECTLY OXIDISED TO KETONE USING $ESS -ARTIN PERIODINANE 3UCCESSFUL ADDITIONS WERE ALSO ACHIEVED WITH THE CORRESPONDING ORGANOCERIUM AND 'RIGNARD REAGENTS ALTHOUGH FOR COM PARABLE PRODUCT YIELDS THESE REQUIRED A SIGNIFICANT EXCESS OF NUCLEOPHILE $UE TO CONCERNS ABOUT THE DIFFICULTY OF LATE STAGE CLEAVAGE OF THE # /4"3 GROUP BOTH THE # AND # /4"3 GROUPS WERE REPLACED AT THIS POINT WITH THE MORE LABILE 4%3 PROTECTING GROUP ALBEIT SOMEWHAT PROBLEMATI
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBDPSO
a,b
34 28
MeO
CO2Me
143 8 steps 26.2% c.f. Scheme 27
TBDPSO
I
MeO
28
144
TBDPSO
O
34
+
c,d
H
MeO OTBS 145
34
H 146
O 28
OTBS
3CHEME A ,I!L( %T/ # B %T. -S#L 4(& # ,I) 4(& → # C T"U,I HEXANE %T/ n # THEN #E#L THEN 4(& n # THEN -E/( n # → 24 D $-0 #(#L 24 OVER TWO STEPS
CALLY DUE TO COMPETITIVE DEPROTECTION OF THE %% ETHER !TTENTION NOW TURNED TO THE FORMATION OF THE TRISUBSTITUTED OLEFIN ALL THAT REMAINED TO COMPLETE THE RIGHT HAND SIDE OF &+ 4HE INSTALLATION OF THE MISS ING # METHYL GROUP WAS ENVISAGED VIA COUPLING OF AN ENOL TRIFLATE WITH METHYL CUPRATE AS DEVELOPED BY -C-URRY ;= 7HILST ENOLISATION OF WITH LITHIUM DIISOPROPYLAMIDE ,$! WAS UNSUCCESSFUL TREATMENT WITH EQUIVALENTS OF LITHIUM DIETHYL AMIDE FOLLOWED BY $-05 AND . PHENYLTRI FLIMIDE GAVE THE DESIRED : ENOL TRIFLATE AS A SINGLE GEOMETRIC ISOMER BY & .-2 &RUSTRATINGLY REACTION WITH -E#U,I OR HIGHER ORDER ORGANOCUPRATES DID NOT OCCUR TO ANY USEFUL LEVEL INSTEAD RETURNING THE KETONE %VEN TUALLY COUPLING WAS FINALLY PROMOTED BY SONICATING THE REACTION MIXTURE ;= BUT DESPITE THE REACTIVE NATURE OF -E#U,I THIS REQUIRED SONICATION FOR UP TO H RESULTING PRACTICALLY IN A SEVERE BOTTLENECK FOR THE PREPARATION OF ON ANY APPRECIABLE SCALE 3CHEME 7ITH THIS IN MIND THE STRATEGY TO INCORPORATE THE CYCLOHEXYL MOIETY WAS ADAPTED 4HE NEW APPROACH WAS NOW BASED ON FORMATION OF THE # # BOND VIA COUPLING OF A CYCLOHEXYL VINYL ANION DERIVED FROM VINYL BROMIDE WITH # ALDEHYDE 4HE FORMER OF THESE TWO FRAGMENTS WAS READILY AVAILABLE IN SIGNIFICANT QUANTITIES THROUGH THE SEQUENCE OF REACTIONS SHOWN IN 3CHEME ;= $IELS !LDER CYCLOADDITION BETWEEN BUTADIENE AND THE ACRYLOYL ESTER OF 3 . METHYL HYDROXYSUCCINIMIDE GAVE AS A CRYSTALLINE ADDUCT ;= 3UBSEQUENT OXYGENATION AT # AND # WAS ACHIEVED VIA INTRAMOLECULAR RING OPENING OF INTERMEDIATE OXIRANE FORMED IN HIGH STEREOSELECTIVITY BY TREATMENT OF WITH M#0"! TO GIVE LACTONE ! SERIES OF STANDARD FUNCTIONAL GROUP MANIPULATIONS GENERATED A METHYL SUBSTITUTED ALKYNE THAT AFTER HYDROZIRCONATION AND TRAPPING WITH AN ELECTROPHILIC SOURCE OF BROMINE ."3 AS A SINGLE OLEFIN ISOMER IN A TOTAL OF STEPS AND YIELD 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TBDPSO
34
TBDPSO
a,b
34
28
MeO
CO2Me
I
MeO
TBDPSO
28
34
144
143 8 steps 26.2% c.f. Scheme 27
TBDPSO
OH
OTBS
MeO
30
H
O O
e
34
O OTBS
MeO
26
28
30
H
f
26
28
O O
21
BnO
O
OTBS 26
H
O O
O EE
OTBS
147
MeO
149
150 OEE =
O
TBDPSO
OTES
34
30
H
28
34
O O
O OTES
MeO
26
21
30
i
H
26
28
O O 21
21
O
O
O 16
16
O EE
OTES
TBDPSO
OTf
OTES
MeO
j
O O
O
g,h
26
H
O
O EE 148
34
30
O EE
OTBS
21 16
TBDPSO
16
O EE
OTBS
O O
O
OTBS
O
16
OTBS 26
c,d
21 16
21
O
O EE
OTES
153
O EE
OTES
151
152
3CHEME A ,I!L( %T/ # B %T. -S#L 4(& # ,I) 4(& → # C 7 2ANEY .I ( %T/( D $-0 #(#L E T"U,I HEXANE %T/ n # THEN %T/ n # BRSM F $-0 #(#L G 4"!& ¯ -3 4(& H 4%3#L )M $-& OVER TWO STEPS I %T.( N"U,I HEXANE # THEN CONCENTRATE IN VACUO THEN 4(& n # THEN $-05 THEN 0H.4F n # → 24 J #U) -E,I 4(& # THEN ULTRASOUND
34
34
O
O
a
NMe
O
O b
NMe
O
154 2 steps 52%
TBDPSO
O
155
TBDPSO
34
30 27
O O
30
156
MeO
TBDPSO
34
30
TBDPSO
34
i,j 27
MeO
H 30
34
g,h
MeO
OMe 30
O
Br 105
30
O
O
34
c,d
OH 30
e,f
k
MeO
TBDPSO
34
O 30
158
157
O 143
3CHEME A BUTADIENE 4I#L #(#L # B ,I/( (/ 4(& # C M#0"! ##L # → 24 THEN %T. 3I/ # D 4"$03#L )M $-& E -E/( .A(#/ F -E/4F DI T BUTYLPYRIDINE #(#L OVER TWO STEPS G ,I!L( %T/ # H #/#L $-3/ #(#L n # THEN %T. n # → 24 I .A( .#(0/ /-E 4(& n # J N"U,I -E) 4(& n # K #P:R( #L 4(& # THEN ."3
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
BnO
OTBS
OH
BnO
26
O O
O O
a,b
21
O O
c
O PMB
OTBS
142
O O 21
O
16
O PMB
159
O
16
O PMB
OTBS
CHO
d
21
O
16
OTBS
26
26
21
O
16
OH
HO
26
O PMB
OTBS
160
107
TBDPSO
g
34
e
MeO 27
105 TBDPSO
MeO
TBDPSO
34
O 30
MeO
26
H
O O
OH H
O O
34
OH 30
O PMB
162
O O
+ 21
O
O OTBS
26
H
21
O
163
MeO
26
f
21
OTBS
TBDPSO
34
30
Br
O PMB
OTBS
O PMB
161
3CHEME A 4"!& ¯ -3 4(& B 4"3#L %T. $-!0 4(& C 7 2ANEY .I ( %T/( D .A)/ 4(& (/ E T"U,I 4(& n # THEN -G"Rq/%T THEN 4(& F $-0 #(#L G ,I!L( ,I) %T/ n #
&OR THE LATTER # ALDEHYDE A SERIES OF PROTECTING GROUP MANIPU LATIONS ESTABLISHED A DIOL FROM THE 0-" PROTECTED SUBSTRATE WHICH WAS THEN OXIDATIVELY CLEAVED USING SODIUM PERIODATE TO AFFORD 7HILE THIS DESTROYED THE CHIRALITY AT # IT WAS RE ESTABLISHED IN THE NEXT STEP WHEN WAS ADDED TO A COOLED SOLUTION OF THE VINYL 'RIGNARD OF FORMED VIA THE VINYL LITHIUM BY TRANSMETALLATION WITH -G"Rq/%T 4HE REACTION PROCEEDED SMOOTHLY TO GIVE AN EXCELLENT YIELD OF ADDUCTS AND ALTHOUGH AS A MIXTURE OF SEPARABLE DIASTEREOMERS AT BEST 3CHEME 5NFORTUNATELY ATTEMPTS TO IMPROVE THIS BY OXIDISING THIS MIXTURE AND THEN REDUCING THE ENONE STEREOSELECTIVELY TO THE DESIRED α ISOMER GAVE EVEN LESS SELECTIVITY )NDEED ALL ATTEMPTS TO INCREASE DIASTEREO ISOMER RATIO DR WERE MET WITH FRUSTRATION BUT NEVERTHELESS THIS MODIFIED ROUTE ENABLED THE RAPID PREPARATION OF MULTI GRAM QUANTITIES OF THE RIGHT HAND SIDE OF &+ (AVING ESTABLISHED RELIABLE SYNTHESES OF BOTH MAJOR FRAGMENTS OF &+ WORK WITHIN THE GROUP THEN TURNED TO THEIR UNION AND THE ELABORATION TO THE NATURAL PRODUCT 4HE SENSITIVE NATURE OF THE # STEREOCENTRE DICTATED THAT THE PIPECOLATE MOIETY BE INTRODUCED AS LATE AS POSSIBLE AND SO PROGRESS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
8
O
TBDPSO MeO
TBDPSO
34
OH 30
MeO
26
H
O O
34
21
OTBS
O
O O 21
8
O
O PMB
SO2Ph
161
26
H
106 e,f,g
21
O
O PMB
OTES 30
OMe
O O
a,b,c,d
34
MeO
26
H
TBDPSO
O
OTES 30
CO2Et H 15 CHO O
CO2Et H O 15
O
O O PMB
O
OMe
164
165
TBDPSO
OTES
MeO
30
h
TBDPSO
34
O O
OTES
MeO
26
H
34
30
O O
CO2Et H O 15
8
O OMe
OMe
104
O O
Me H O 13
21
21 8
26
H
i
O PMB
O O
CO2Et H O 15
O OH
O PMB
Ce
O
15
O
O 8
O 16
CO2Et
R
166
OMe
167
3CHEME A 4"!& 4(& B 4S#L 0Y $-!0 #(#L THEN # 4%3#L C ,I) 4(& # D 0H3/.A $-& # OVER THREE STEPS E N"U,I 4(& n # THEN BRSM F $-0 0Y #(#L G "U3N( !)". 0H-E # H #E#Lq(/ -E/( %T/ THEN n # .A"( I 0ROTON 3PONGE¤ -E/"& ¯ -3 #(#L
WAS FIRST FOCUSED ON COMBINING AND 4HIS WAS TO BE ACHIEVED VIA ADDITION OF A # ANION DERIVED FROM THE SULFONE TO THE # ALDEHYDE 3CHEME !CCORDINGLY BOTH TERT BUTYLDIMETHYLSILYL 4"3 ETHERS IN WERE REMOVED FOLLOWED BY FORMATION OF THE PRIMARY TOSYLATE AND REPROTEC TION OF THE # /( AS ITS TRIETHYLSILYL 4%3 ETHER IN ONE POT 3CHEME 3EQUENTIAL NUCLEOPHILIC SUBSTITUTION OF THE TOSYLATE WITH IODIDE AND THEN BENZENESULFINATE GAVE # ANION PRECURSOR IN GOOD YIELD WHICH WAS AZEOTROPED REPEATEDLY WITH TOLUENE PRIOR TO THE KEY COUPLING REACTION )N THIS ADDITION OF EQUIVALENT OF N"U,I TO AT n # GENERATED THE DESIRED YELLOW ANION THAT REACTED CLEANLY WITH FRESHLY PREPARED ALDEHYDE TO AFFORD A MIXTURE OF THE DIASTEREOMERIC SULFONE ALCOHOLS IN A PLEAS ING YIELD ESSENTIALLY QUANTITATIVE BASED ON RECOVERED STARTING MATE RIAL 4HIS MIXTURE WAS OXIDISED USING THE $ESS -ARTIN REAGENT ;= AND THE SULFONE GROUP CLEAVED RADICALLY ON TREATMENT WITH EXCESS "U3N( AND !)". IN BOILING TOLUENE TO AFFORD # KETONE IN EXCELLENT YIELD AFTER CAREFUL CHROMATOGRAPHY TO AVOID SILYL ETHER CLEAVAGE 4HIS WAS REDUCED
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
CHEMOSELECTIVELY IN THE PRESENCE OF THE # ETHYL ESTER USING ,UCHE CONDI TIONS ; = TO ESTABLISH THE LAST STEREOCENTRE IN &+ AS A SINGLE DIASTEREOMER IN QUANTITATIVE YIELD 4HE # /( WAS ASSIGNED AS THE α ISOMER BASED ON PRECEDENT IN SIMILAR SYSTEMS AND WAS BELIEVED TO OCCUR VIA FORMATION OF A SIX MEMBERED CERIUM ))) CHELATE FOLLOWED BY HYDRIDE ATTACK AT THE LESS HINDERED β FACE ;= 3UBSEQUENT METHYLATION OF THIS ALCOHOL PROVED UNEXPECTEDLY PROB LEMATIC WITH BASIC CONDITIONS RESULTING IN DECOMPOSITION AND COMMON ALTERNATIVES SUCH AS #(. OR !G/ -E) GIVING ONLY TRACES OF PRODUCT 4HE USE OF -EERWEINS SALT -E/"& AND 0ROTON 3PONGE¤ WAS MORE ENCOURAG ING ;= ALTHOUGH THE REACTION WOULD NOT GO BEYOND ^ CONVERSION AND LONGER REACTION TIMES LED TO ,EWIS ACID CATALYSED DESTRUCTION OF THE SPIROKETAL MOIETY (OWEVER SOMEWHAT SURPRISINGLY IN THE PRESENCE OF ¯ MOLECULAR SIEVES THE REACTION WENT TO COMPLETION IN ONLY MIN IN NEAR QUANTITATIVE YIELD AND FURNISHED THE DESIRED # # SPIROKETAL 3CHEME 7ITH THE ASSEMBLY OF &+ NEARING COMPLETION )RELAND TURNED HIS ATTENTION TO THE ; = SPIROKETAL SUBUNIT AND ITS USE AS A PRECURSOR FOR THE TRICARBONYL FUNCTIONALITY 4HIS WAS ANTICIPATED THROUGH CLEAVAGE OF THE # # ACETONIDE FOLLOWED BY OXIDATION AT THE RESULTING # /( (OWEVER EARLY INDICATIONS IN MODEL SYSTEMS SUGGESTED THAT THE INITIAL ACETONIDE HYDROLYSIS WOULD REQUIRE REFLUXING IN A MIXTURE OF ACETIC AND SULFURIC ACID CONDITIONS THAT WERE OBVIOUSLY PROHIBITIVE FOR A MOLECULE SUCH AS WITH A RANGE OF SENSITIVE FUNCTIONALITIES !S AN ALTERNATIVE WORK THEN FOCUSED ON A POTENTIAL ANIONIC FRAGMENTA TION OF THE SAME SPIROKETAL 4HEORETICALLY DEPROTONATION AT # WOULD BE FOLLOWED BY β ELIMINATION TO CLEAVE THE SPIROKETAL IN A SELECTIVE FASHION AS IT WAS ANTICIPATED THAT ONLY ONE OF THE TWO OXYGENS AT # WOULD BE ABLE TO ADOPT THE NECESSARY ORIENTATION FOR THIS PROCESS 'RATIFYINGLY THIS PROVED TO BE THE CASE IN MODEL SYSTEMS AND THUS ON TREATMENT WITH ,I(-$3 IN 4(& (-0! β ELIMINATION OCCURRED IN TO GIVE ENEDIOL ACETAL WHICH WAS IMMEDIATELY SILYLATED TO TO IMPROVE STABILITY IN AN IMPRES SIVE YIELD OVER TWO STEPS !FTER INSTALLATION OF THE PIPECOLATE MOIETY FURTHER ENCOURAGEMENT WAS PROVIDED WHEN TREATMENT OF WITH DIMETHYL DIOXIRANE $-$/ RESULTED IN SELECTIVE OXIDATION OF THE ENEDIOL ACETAL 4HIS SUCCESSFULLY INSTALLED THE DESIRED VICINAL TRICARBONYL THEREBY JUSTIFY ING )RELANDS NOVEL APPROACH TO THIS SENSITIVE PROBLEM 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
O
O
a 14
9
O
EtO2C H
R
O
Li
OMe
O 9
O
14
O
R
O
H
OMe
O
HO
EtO2C
H
OEt
168
9
169
b
14
R
O TBS 14 O R
O 9
OMe
H
EtO2C
OMe
H
170
171 c,d
O
OH
O
9
O
14
R
N O CO2Me 174
H
OMe
f
9
O TBS 14 O R
N O CO2Me 173
H
O OMe
e
9
O TBS 14 O R
N O CO2Me 172
OMe
H
3CHEME A ,I(-$3 4(& (-0! n # → # B 4"3#L )M $-!0 $-& OVER TWO STEPS C ,I/(q(/ -E/( D -ETHYL PIPECOLATE HYDROCHLORIDE %T. CHLORO . METHYLPYRIDINIUM IODIDE $-!0 CAT #(#L OVER TWO STEPS E $-$/ #(#L n → n # F (& -E#.
7ITH THESE RESULTS IN HAND ASSEMBLY OF THE MACROCYCLE ENSUED 3CHEME !GAIN WORK FOCUSED ON ELABORATION OF RATHER THAN INSTALLATION OF THE SENSITIVE PIPECOLATE GROUP WHICH WAS TO BE LEFT TO THE LATEST POS SIBLE STAGES !S PREVIOUSLY SPIROKETAL WAS TREATED WITH ,I(-$3 IN 4(& (-0! ALTHOUGH FRUSTRATINGLY FRAGMENTATION TO THE # ALCOHOL PRE CURSOR TO WAS NOW MUCH SLOWER AND ACCOMPANIED BY CONSIDERABLE DECOMPOSITION OF THE STARTING MATERIAL &URTHERMORE RECYCLISATION OF THE GENERATED LITHIUM ALKOXIDE TO THE # EPIMER OF THE STARTING MATERIAL WAS OBSERVED UPON PROLONGED REACTION TIME 4HIS PROCESS WAS REPORTEDLY SO FAVOURABLE THAT hWHEN A REACTION MIXTURE CONSISTING ^ STARTING MATERIAL AND THE PRODUCT WAS LET STAND AT n # OVERNIGHT THE PRODUCT DISAPPEARED COMPLETELYv )N ORDER TO INFLUENCE THE EQUILIBRIUM IN PLACE AND FAVOUR THE ELIMINA TION PRODUCT )RELAND INVESTIGATED THE USE OF ,EWIS ACIDS IN STABILISING THE RING OPENED PRODUCT VIA THE FORMATION OF STRONG OXYGEN CHELATES !FTER EXTENSIVE SYSTEMATIC STUDIES OPTIMISED CONDITIONS WERE ACHIEVED ,I( -$3 ^ EQUIV -G(-$3 ^ EQUIV (-0! $-% # MIN WHICH CONSISTENTLY GAVE THE INTERMEDIATE # ALCOHOL IN n ISOLATED YIELD 4HE PROPENSITY OF THIS INTERMEDIATE TO REVERT TO ALSO PROVED PROBLEMATIC FOR THE SUBSEQUENT SILYLATION OF THE # /( ALTHOUGH BY THE SAME RATIONALE OPTIMAL CONVERSION WAS ACHIEVED WHEN USING 4"3/4F IN THE PRESENCE OF -G3/
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBDPSO
TBDPSO
34
34
OTES
MeO
30
30
26
H
O O
TBDPSO
CO2Et H O 15
O O
H
MeO
O O
a,b
30
O O
O PMB
O
EtO2C
OMe
TBS O
H
15
OMe OMe
O
O
EtO2C
TBS O
175
15
OMe OMe H
f,g,h
TBDPSO
34
O O
26
21 1
O
8
O
176 e
34
TBDPSO
34 1
I
OMe OMe H 179
O O
26
21
k,l
1
O
NH O
O TBSO H
O PMB
MeO
MeO
O
O O O
O PMB
H
TBDPSO
O O
8
O
MeO
30
O PMB OMe OMe H
178
26
H
N Boc H O
O O
i,j
O
N HO
O TBSO H
O
N Boc H O
21
H
104
26
H
c,d
21
O OMe
1
O
26
21 8
34
OTES
MeO
21
O O HO2C
O
TBS O
H
15
OMe OMe H
O PMB
177
3CHEME A ,I(-$3 -E-G"R $-% (-0! # B 4"3/4F DI T BUTYLPYRIDINE $-!0 -G3/ #(#L C 4&! 4(& (/ D . "OC , PIP $## $-!0 CAT #(#L n # E . .A/( DIOXANE (/ # F . .A/( DIOXANE (/ # G 4&! 4(& (/ H . "OC , PIP $## $-!0 #(#L n # THEN .A/( 4(& (/ OVER THREE STEPS I 4%3/4F LUT #(#L # THEN 3I/ J METHYL CHLOROPYRIDINIUM IODIDE %T. #(#L OVER TWO STEPS K $$1 #(#L (/ L 0H0 )M 0H-E # THEN )
3ELECTIVE CLEAVAGE OF THE SECONDARY 4%3 ETHER THEN LIBERATED THE # HYDROXYL IN QUANTITATIVELY ALLOWING INSTALLATION OF THE PIPECOLATE ESTER UNDER STANDARD CONDITIONS -UKAIYAMA MACROLACTAMISATION WAS ANTICI PATED USING THE -ERCK PROTOCOL ; = HOWEVER PRELIMINARY HYDROLYSIS OF THE ETHYL ESTER TO REQUIRED HEATING AT # WITH EQUIVALENTS OF .A/( IN AQUEOUS DIOXANE FOR H 7HILE THE REACTION APPEARED TO PRO CEED CLEANLY AND WITHOUT HYDROLYSIS OF THE LABILE PIPECOLATE ESTER IT COULD NOT BE DETERMINED CONCLUSIVELY WHETHER OR NOT EPIMERISATION AT THE # STEREOCENTRE HAD OCCURRED BECAUSE OF AMBIGUITIES IN THE NUCLEAR MAGNETIC RESONANCE .-2 SPECTRA ARISING FROM THE PRESENCE OF ROTAMERS ABOUT THE . "OC BOND 4HIS UNCERTAINTY WAS ACCENTUATED AFTER INVESTIGATING THE SAME HYDROLYSIS OF WITH .A/$ $/ A REACTION THAT GAVE A PRODUCT WITHOUT THE CHARACTERISTIC SIGNAL FOR THE # PROTON IN THE ( .-2 4HIS INDICATION THAT ENOLISATION WAS OCCURRING LED )RELAND TO PURSUE A DIFFERENT ENDGAME STRATEGY F G H n 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
4HE ALTERNATIVE WAS TO HYDROLYSE ESTER FIRST FOLLOWED THEN BY # /4%3 DEPROTECTION AND ESTERIFICATION 4HIS LATTER ESTERIFICATION AFFORDED THE PRODUCT AS A MIXED ANHYDRIDE WITH THE # CARBOXYLIC ACID WHICH COULD BE SELECTIVELY HYDROLYSED TO THROUGH BRIEF EXPOSURE TO .A/( IN 4(& (/ 7HILE SPECTROSCOPIC ANALYSIS REMAINED INCONCLUSIVE THE # STEREOCENTRE HAD NOT BEEN EXPOSED TO STRONGLY BASIC CONDITIONS AND AS A RESULT IT WAS BELIEVED THAT THE STEREOCHEMICAL INTEGRITY OF THE PIPECOLATE MOIETY SHOULD HAVE REMAINED INTACT 4HUS REMOVAL OF THE "OC GROUP USING 4%3/4F FOLLOWED BY SILICA GEL ; = AND FINALLY -UKAIYAMA MACROLAC TAMISATION FURNISHED IN REASONABLE YIELD AND COMPLETED THE CARBON SKELETON OF &+ 3CHEME 4O UNMASK THE # # ALDOL MOTIF THE # 0-" ETHER WAS FIRST CLEAVED USING STANDARD CONDITIONS BEFORE CAREFUL CONVERSION OF THE # /( TO THE CORRESPONDING IODIDE 3CHEME 7HILE STANDARD CONDITIONS PROVED UNRELIABLE FOR THIS TRANSFORMATION THIS WAS EVENTUALLY ACHIEVED THROUGH A MODIFIED LITERATURE PROCEDURE ;= THAT INVOLVED METICULOUS TITRATION OF SOLUTIONS OF IN TOLUENE AT # WITH AN IODINE SOLUTION IN THE PRESENCE OF EXCESS 0H0 AND IMIDAZOLE 7ITH THE SUBSTRATE FOR THE CRUCIAL SPIROKETAL FRAGMENTATION IN HAND ADDITION TO A SUSPENSION OF FRESHLY PREPARED ZINCSILVER GRAPHITE ;= EFFECTED DEALKOXYDEHALOGENATIVE CLEAVAGE TO FURNISH THE DESIRED α ALLYL ALDOL IN A VERY IMPRESSIVE NEARLY QUANTITATIVE YIELD 4HE USE OF A HIGHLY REACTIVE METAL GRAPHITE COMBINATION TO AFFECT THIS TRANSFORMATION ALLOWED EXTREMELY MILD REACTION CONDITIONS SUPPRESSING ANY SIDE REAC TIONS MORE COMMON WHEN USING 2EIKE ZINC FOR EXAMPLE SUCH AS THE DEGRADATIVE PATHWAYS THAT ARE PREVALENT IN &+ AND ITS ANALOGUES AS DISCUSSED PREVIOUSLY 4HIS REAGENT ALSO OFFERS PRACTICAL ADVANTAGES WITH SIMPLE WORKUP SOLELY BY THE FILTRATION OF INSOLUBLES -ECHANISTICALLY THE REAGENT IS NOT COMPLETELY UNDERSTOOD #+ IS KNOWN TO BE BOTH AN EFFECTIVE ONE AND TWO ELECTRON DONOR YET IN THE PRESENCE ZINC OR OTHER METALS THIS IS SOMEWHAT AMBIGUOUS 7ITH THE FRAMEWORK OF &+ NOW UNMASKED TREATMENT OF WITH $-$/ PROCEEDED AS IN MODEL STUDIES AND WITHOUT INCIDENT TO REVEAL THE VICINAL TRICARBONYL IN QUANTITATIVE YIELD AND WITH NO TRACE OF EPOXIDATION &INALLY ALL THAT REMAINED WAS DESILYLATION WHICH OCCURRED WITH SPONTA NEOUS LACTOL FORMATION TO COMPLETE )RELANDS TOTAL SYNTHESIS OF &+ 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
HO TBDPSO
HO
34
MeO
MeO
H
MeO
O O
21
1
O
NHO
O
a
OMe OMe
OH
b,c
8
OH
TBS O
O
H
8
OMe OMe
O
O OH H O 10
OMe OMe
H
180
21
O
1
N
O
O
24
O
H O O
H 179
H
21
1
N I
O TBSO
O
O
8
H
O
24 26
O
34
34
FK506 1
3CHEME A # # THEN + # THEN :N#L !G/!C 4(& 24 → # → # THEN B $-$/ #( #/ n → n # C (& #(#. OVER THREE STEPS
3CHEME 3UMMARY OF )RELANDS TOTAL SYNTHESES OF &+ 0ART /NE
)RELANDS SECOND TOTAL SYNTHESIS OF &+ REPRESENTS AN INVENTIVE STRATEGY TO TWO OF THE SIGNIFICANT PROBLEMS POSED BY THE SYNTHESIS OF THIS NATURAL PRODUCT "Y MASKING BOTH THE TRICARBONYL AND β HYDROXY KETONE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
HO MeO
30
TBDPSO
TBDPSO
34
OH
1
O
1
8
O
O OH H O 10
8
OMe
H
H
O O
+
OH 10
OMe
O
10
O
O 182
OH H O H
20
183
H
H
15
16
181
OMe OMe FK-506 1
N Boc H O
22
I
OMe OMe
26
H 21
O
O
O
OMe
O
NH O
TESO
N
O O
26
22
O
30
30
O
24
1
O
MeO
MeO
H
34
34
H
OMe O 184 +
I
20
TMS 185
3CHEME 2ETROSYNTHETIC ANALYSIS
MOIETIES WITHIN SPIROKETALS HE HAS AVOIDED THE LATENT INSTABILITY ASSOCI ATED WITH THESE REGIONS AND BEEN ABLE TO DEVELOP A FASCINATING APPROACH TO &+ 4HIS IS COMBINED WITH GREAT EFFICIENCY IN GENERATING SOME OF THE OTHER FUNCTIONALITY FOR EXAMPLE BOTH TRISUBSTITUTED OLEFINS AS SINGLE GEOMETRIC ISOMERS AND A HIGHLY EFFECTIVE STEREOCONTROLLED COUPLING STRAT EGY TO MAKE THIS ARGUABLY THE MOST COMPLETE OF THE &+ SYNTHESES REQUIRING A LONGEST LINEAR SEQUENCE OF STEPS IN OVERALL YIELD 3CHEME
)RELAND TOTAL SYNTHESES OF &+ 0ART 4WO )RELANDS ALTERNATIVE TOTAL SYNTHESIS OF &+ ACTUALLY RELEASED ONE YEAR PREVIOUSLY ;= WAS ACHIEVED USING MUCH OF THE SAME METHODOLOGY AS THAT ALREADY DISCUSSED WITH BOTH STRATEGIES UNDOUBTEDLY BENEFITING FROM THE EXPERIENCES AND SUCCESSES OF THE OTHER )N THIS APPROACH FINAL CYCLISATION WAS AGAIN ANTICIPATED VIA MACROLACTAMISATION # . AND THE # # α ALLYL ALDOL FUNCTIONALITY REMAINED EMBEDDED WITHIN THE INITIAL FRAG MENT TARGETS AS A SPIROKETAL (OWEVER KEY DIFFERENCES INCLUDED THE MASKED VICINAL TRICARBONYL FUNCTIONALITY NOW PLANNED FROM A SILYL ENEDIOL AS WELL AS THE GENERAL RETROSYNTHESIS WHICH AFTER FURTHER # # DISCON NECTION GAVE LARGE KEY @TOP AND @BOTTOM HALVES IN AN ARGUABLY A MORE CONVERGENT APPROACH 3CHEME 2ATHER IRONICALLY THE FIRST APPROACH TO TOP HALF WAS A LENGTHY LINEAR SEQUENCE 4HIS BEGAN FROM PROTECTED LACTONE ;= A COMMON INTER
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBDPSO
TBDPSO a,b MeO
34
28
O
TBDPSO
TBDPSO
OMTM
g,h
MeO
MeO
MeO
26
26
O
186
34
34
e,f
O
156 6 steps 25.3% (c.f. Scheme 27)
26
34
c,d 28
O
TBDPSO
TBDPSO
34
CHO
187
188
189
24
TBSO
i,j
34
TBDPSO
OTBS
MeO
30
34
TBDPSO
HO
26
H
o
O O
21
O
MeO
m,n
22
TBDPSO
34
k,l
MeO
26
O
26
TBSO
OH
TBSO
192
O 193
34
BzO
MeO
24
26
O
24
TBSO
191
OH
190
TBDPSO
X
34
MeO
30 27
TBDPSO
TBDPSO N Boc H MeO O bb,cc,dd
34
O
MeO
30
26
H
TBDPSO
34
OH 30
H
O O
21
O O
21
200
O 182
OH 30
26
H
O O
+ 21
O
TBSO
O
MeO
26
199
26
O H 198
H
x
201
26
26
H
24
OBn
p,q
24 26
O 119
OH 194
OH
r,s,t
24
22
26
O
OH
u
195
O O
21
OH
O O
21
TBSO
aa
z
O
H
y
O
TBSO
H
Br 105 13 steps 13.7% (c.f. Scheme 27)
34
O O
196
v,w
O O
21
O TBSO
H 197
3CHEME A %T-G"R %T. 0H-E → # B -E/4F DI TERT BUTYL METHYLPYRIDINE #(#L C +(-$3 (-0! 4(& n → # THEN (#/%T n # → 24 D +#/ -4-#L 4(& E #E#Lq(/ .A"( -E/( # F P4S/( 4(& OVER TWO STEPS G : CROTYLDIISOPINOCAMPH EYLBORANE "&q/%T %T/ n # THEN (/ .A/( H 4"3/4F LUT #(#L OVER TWO STEPS I /S/ .-/ 4(& # → 24 J "Z#L 0Y $-!0 CAT K -S#L 0Y $-!0 CAT # L .A/-E -E/( # OVER TWO STEPS M LITHIOFURFURYL METHOXYISOPROPYL ETHER "&q/%T 4(& n # N #3! 4(& (/ OVER TWO STEPS O M#0"! #(#L # THEN METHOXY PROPENE P4S/( P : CROTYLDIISOPINOCAMPHEYLBORANE 4(& n # THEN (/ .A/( Q ,I .( n # OVER TWO STEPS R 4S#L 0Y S .A( %T/ # T LITHIOFURFURYL METHOXYISOPROPYL ETHER "&q/%T 4(& n # OVER THREE STEPS U M#0"! #(#L # THEN METHOXYPROPENE (#L → # V ,nSELECTRIDE 4(& n # W 4"3#L )M $-& X /S/ .-/ 4(& (/ THEN .A)/ (/ Y T"U,I %T/ n # THEN Z $-0 #(#L AA #E#Lq(/ .A"( -E/( n # BB . "OC , PIP $## $-!0 CAT #(#L n # CC 4"!& 4(& # DD $-0 #(#L
MEDIATE IN THE SYNTHESIS OF THE CYCLOHEXYL MOIETY OF THIS CLASS OF NATURAL PRODUCTS AND EASILY AVAILABLE ON SCALE USING ROBUST $IELS !LDER CHEMIS TRY !FTER FORMATION OF ETHYL KETONE WITH ETHYLMAGNESIUM BROMIDE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
HOMOLOGATION VIA CONDENSATION WITH ETHYL FORMATE AND METHYLTHIOMETHYL -4- PROTECTION GAVE WHICH AFTER ,UCHE REDUCTION ; = UNDER WENT ACID CATALYSED REARRANGEMENT TO GIVE THE DESIRED TRANS SUBSTITUTED ALDEHYDE PREFERENTIALLY ALTHOUGH NOT EXCLUSIVELY !FTER "ROWNS CROT YLATION ; = TO SET # AND # WITH A DR OF THE OLEFIN IN COULD BE READILY TRANSFORMED TO EPOXIDE &URTHER FUNCTIONALISATION VIA RING OPENING WITH LITHIOFURFURYL METHOXYISOPROPYL ETHER AND ELABORA TION TO THE SPIROENONE MOIETY AS DISCUSSED PREVIOUSLY GAVE WITHOUT COMPLICATION 3CHEME &RUSTRATINGLY SELECTIVE DEPROTECTION OF THE # 4"3 IN WAS ELUSIVE AT THIS POINT "IS DESILYLATION COULD BE ACHIEVED BUT ONLY IN YIELD AT BEST POLYMERIC (&q0Y AND SELECTIVE REPROTECTION OF THE # HYDROXYL WHILST POSSIBLE MADE THIS APPROACH CUMBERSOME AND UNATTRACTIVE #ON SEQUENTLY )RELAND PURSUED A MORE CONVERGENT APPROACH TO THE TOP HALF OF &+ 3CHEME !DDITION OF THE ORGANOLITHIUM SPECIES OF VINYL BROMIDE SEE 3CHEME TO ALDEHYDE EASILY PREPARED FROM ALBEIT IN NINE STEPS AS WAS USED IN )RELANDS APPROACH TO &+ !.!, ;= GAVE DIRECTLY THE FREE # /( AS A DIASTEREOMERIC MIXTURE IN FAVOUR OF THE DESIRED α /( 5NFORTUNATELY ANY ATTEMPTS TO IMPROVE THIS RATIO BY AN OXIDA TION REDUCTION SEQUENCE WERE UNSUCCESSFUL GIVING THE UNDESIRED ISOMER EXCLUSIVELY UNDER A VARIETY OF CONDITIONS .EVERTHELESS THE DESIRED DIASTE REOMER COULD BE SEPARATED AND SUBSEQUENT INSTALLATION OF THE PIPECOLATE MOIETY WAS STRAIGHTFORWARD ALLOWING RAPID CONVERGENT ACCESS TO TOP HALF OF &+ 3CHEME 4HE SYNTHESIS OF THE BOTTOM HALF BEGAN FROM DIOL ;= 3ELECTIVE METHYLATION AND OXIDATION GAVE ALDEHYDE WHICH WAS ADDED INTO A SOLUTION OF THE # # 'RIGNARD DERIVED FROM VIA LITHIUMHALOGEN EXCHANGE AND TRANSMETALLATION WITH -G"R AT n # TO GIVE AS A SINGLE DETECTABLE DIASTEREOMER VIA CHELATION CONTROL 3CHEME ; = 3TRAIGHTFORWARD ELABORATION TO LACTOL FOLLOWED BY 7ITTIG OLEFINATION INSTALLED # AND # AS AN INSEPARABLE MIXTURE IN FAVOUR OF THE DESIRED TRANS OLEFIN ^ WHICH WAS SUBSEQUENTLY REDUCED TO ALLYLIC ALCOHOL WITH $)"!, ( 5SE OF THE STOICHIOMETRIC 3HARPLESS EPOXIDATION ;= CONDITIONS THEN PROCEEDED ONLY UPON THE TRANS OLEFIN ALLOWING SEPARA TION OF THE 7ITTIG BY PRODUCT AND THE EPOXIDE THAT FORMED WAS CONVERTED SPONTANEOUSLY IN SITU TO REFORM THE TETRAHYDROPYRAN RING IN GOOD YIELD
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
OMe
OMe
O
H
O
a,b,c,d
H
H
O
16
I 115 9 steps 42% (cf. Scheme 19) OTBS OTBS H 10 O H
206
16
H 15
16
TMS
OMe OH
OMe OMe
202
203 h,i
Me Al
O m 10 20
I
OMe OMe
O
20
185 7 steps 44.6%
184
20
10
15
OMe O
OH OH
f,g H
TMS
OH
20
10
e
15
15
TMS
OMe
10
10
O H O
OH
l H
10
OH H O H
CH2OH
H
TMS 20
20
j,k
10
OH 16 15
20
AlRn
OMe OMe 205
OMe OMe 183
OMe OMe 204
3CHEME A 4R#L 0Y B .A( -E) 4(& C (#L -E/( OVER THREE STEPS D $-0 #(#L E T"U,I %T/ n # THEN -G"R n # → # THEN F .A( -E) 4(& # G !C/( (/ # H 0H0 #(#/%T %T. 0H( REFLUX I $)"!, ( 4(& n # → # J 4I/I0R $%4 4"(0 ¯ -3 #(#L n # THEN K 4"!& 4(& # L #P:R#L -E!L #(#L THEN THEN n # ) M 4"3#L )M $-!0 CAT $-&
&INALLY REMOVAL OF THE TERMINAL 4-3 GROUP COMPLETED THE SYNTHESIS OF KEY ALKYNE INTERMEDIATE )T WAS ENVISAGED THAT UNION OF THE TWO HALVES AND COULD BE ACHIEVED VIA THE # # BOND IN A CARBOALUMI NATIONTRANSMETALLATION PROCEDURE THAT HAD BEEN USED SUCCESSFULLY BY )RE LAND PREVIOUSLY 4O THIS END CARBOALUMINATION #P:R#L -E!L OCCURRED CLEANLY ALBEIT SLOWLY GIVING VINYL ALANE INTERMEDIATE 5NFORTUNATELY ALL ATTEMPTS TO ACHIEVE TRANSMETALLATION TO THE DESIRED VINYL CUPRATE GAVE NO REACTION AS JUDGED BY THE LACK OF PRODUCTS ON QUENCHING WITH SIMPLE SPIROENONES )NSTEAD ADDITION OF IODINE GAVE A VINYL IODIDE AS A SINGLE GEOMETRIC ISOMER IN PLEASING YIELD AND IT WAS ANTICIPATED THAT AFTER BIS 4"3 PROTECTION THIS IODIDE COULD BE USED AS A PRECURSOR FOR THE DESIRED VINYL CUPRATE 3CHEME !CCORDINGLY AFTER LITHIUMHALOGEN EXCHANGE TRANSMETALLATION WITH A RANGE OF CYANOCUPRATES WAS INVESTIGATED ;= $ESPITE INITIAL DISAPPOINT ING RESULTS REQUIRING EXCESS OF VINYL LITHIUM AND GIVING ONLY POOR YIELDS AN EFFECTIVE PROTOCOL WAS EVENTUALLY ACHIEVED )N THIS ADDITION OF HEXYNYL COPPER AS ITS BIS (-0! COMPLEX ;= TO THE ORGANOLITHIUM SPECIES DERIVED FROM FOLLOWED THEN BY SPIROENONE GAVE THE DESIRED KETONE IN CONSISTENT YIELDS AS A SINGLE DIASTEREOMER AND THE TRISUBSTITUTED # # OLEFIN EXCLUSIVELY IN THE TRANS CONFIGURATION 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TBDPSO
TBDPSO
N Boc H O
34
1
MeO
O 30
26
H
O O
21
MeO
OTBS OTBS H 10 O H
+
30
1
H
TBSO
O TBSO
206
O O
16
10
22
O
NHO Boc
20
OMe OMe
O 182
O O
26
a I
O
34
OMe OMe
H
H
207 g,h
b,c
TBDPSO
TBDPSO
34
MeO
MeO
30
O O
26 1
8
H
O
H
O
O
16
OMe OMe H 210
O O
26
f
1
8
H
O H
O
22
O
O O
26 1
O
16
30
d,e
O
NHO
HO
34
MeO
30
O
NH O
O
22
O
TBDPSO
34
NH O Boc
OMe H
22
O
16
O O
10
OMe OMe H 209
O
O
HO
H
OMe OMe H 208
3CHEME A T"U,I %T/ n # THEN #(###Uq(-0! n # → n # THEN 4-3#L (#L 4(& B 4"!& 4(& C .A(#/ +"R #(#L THEN 4%-0/ # THEN .A/#L .A#L/ .A(0/ METHYLBUTENE T"U/( #(. %T/ D ,I/(q(/ 4(& 4%3/4F LUT #(#L # THEN !C/( 4(& # CHLORO METHYLPYRIDINIUM IODIDE %T. #(#L REFLUX E %T. %T/( OVER TWO STEPS F $-0 #(#L G $-0 #(#L H ,I/(q(/ 4(& 4%3/4F LUT #(#L # THEN !C/( 4(& # CHLORO METHYLPYRIDINIUM IODIDE %T. #(#L REFLUX FROM
%LABORATION OF TO THE &+ MACROCYCLE STARTED WITH SELECTIVE OXIDATION OF THE DIOL TO AN α HYDROXY ALDEHYDE USING THE HINDERED CHLORO OXAMMONIUM SALT DERIVED FROM METHOXY 4%-0/ IN THE PRES ENCE OF .A/#L ;= FOLLOWED BY FURTHER OXIDATION TO THE RESULTING CARBOXYLIC ACID 4HIS WAS ESTERIFIED WITH DIAZOMETHANE TO FACILITATE PURI FICATION !FTER HYDROLYSIS OF THIS ESTER AND REMOVAL OF THE "OC GROUP WITH 4%3/4F MACROLACTAMISATION WAS ACHIEVED UNDER STANDARD CONDITIONS 5NFORTUNATELY A NUMBER OF PROBLEMS INCLUDING ISOLATION OF THE CYCLISED HYDROXYLACTAM RESULTED IN A VERY LOW YIELD FOR THIS SEQUENCE n A MERE OF DIKETONE FROM HYDROXYESTER (OWEVER BY REARRANGING THESE TRANSFORMATIONS )RELAND MANAGED TO IMPROVE THIS TO FROM 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBDPSO
TBDPSO
34
MeO
30
O O
26 1
8
H
O
a,b
1
O
O
16
O
OMe OMe
O O O
O
I
16
O
O O
NH O
TESO
8
I
O
O
OMe OMe H 181
H
H 211
H
O O
26 22 1
OMe OMe
210
30
c
22
O
NHO 8
H
H
H
30
O
NHO
O
34
MeO 26
22
O
TBDPSO
34
MeO
d
HO MeO
TBDPSO
34
30
MeO
H
TBDPSO
34
30
MeO
H
O
24
8
O
O OH H O 10
f
22
N 8
OMe
O
e
O
1
O OR H O 10
213
O
1
O
N 8
OMe
TESO
O 10
H
OMe OMe
OMe
OMe FK-506 1
O
24
OH
O
OH
O
O
N
H
22
OH
1
30
O
24
22
O
34
212
3CHEME A , SELECTRIDE 4(& n # B ) 0H0 0H-E THEN )M THEN ALCOHOL DERIVED FROM # C +(-$3 4(& n # THEN 4%3#L D # # THEN + # THEN :N#L !G/!C 4(& 24 → REFLUX → 24 THEN E $-$/ #(#L n → # F (& -E#. OVER TWO STEPS
3ELECTIVE REDUCTION OF THE KETONE IN USING , SELECTRIDE ;= AND TRANSFORMATION TO EQUATORIAL IODIDE INSTALLED THE NECESSARY FUNCTION ALITY FOR DEALKOXYDEHALOGENATIVE FRAGMENTATION OF THE SPIROKETAL 4HUS AFTER CHEMOSELECTIVE ENOLISATION OF THE # KETONE WITH POTASSIUM HEXA METHYLDISILAZIDE +(-$3 WHICH HAS BEEN SHOWN NOT TO ENOLISE ESTERS AT LOW TEMPERATURE IN THE ABSENCE OF POLAR COSOLVENTS SUCH AS HEXAMETH YLPHOSPHORAMIDE (-0! THUS RETAINING THE STEREOCHEMICAL INTEGRITY AT # ;= AND TRAPPING WITH TRIETHYLCHLOROSILANE 4%3#L GAVE 4REATMENT WITH THE SAME :N !G ALLOY ;= DISCUSSED PREVIOUSLY AFFORDED WITH THE α ALLYL ALDOL FUNCTIONALITY IN PLACE #OMPARED WITH OTHER EXAMPLES OF THIS METHODOLOGY THE YIELD WAS SOMEWHAT DISAPPOINT ING WITH DEHYDRATION OF THE DESIRED ALDOL PRODUCT PREVALENT .EVERTHE LESS THE BACKBONE OF &+ WAS NOW IN PLACE AND ALL THAT REMAINED WAS OXIDATIVE AND PROTECTING GROUP MANIPULATIONS !CCORDINGLY OXIDATION OF THE SILYL ENEDIOL WAS ACHIEVED USING $-$/ IN THE SAME WAY AS THE CYCLIC ACETONIDE ENEDIOL PREVIOUSLY AND THE RESULTING MIXTURE OF SILYLATED
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 3UMMARY OF )RELANDS TOTAL SYNTHESES OF &+ 0ART 4WO
AND DESILYLATED TRICARBONYL HEMIKETALS WAS TREATED WITH HYDROGEN FLUORIDE (& TO COMPLETE )RELANDS SECOND TOTAL SYNTHESIS OF &+ 3CHEME )RELANDS ALTERNATIVE STRATEGY TO &+ IS A MORE CONVERGENT APPROACH RELYING ON THE EFFICIENT SYNTHESIS OF TOP AND BOTTOM HALVES !LTHOUGH SUB STANTIALLY MORE CONCISE WHEN VIEWED AS A WHOLE THIS APPROACH OVERALL IS THREE FOLD LOWER YIELDING 3CHEME .ONETHELESS RELEASED AS THE FIRST OF HIS TWO TRIUMPHS OVER THIS MOL ECULE IT SUCCESSFULLY AVOIDED SOME OF THE EARLY PROBLEMS ENCOUNTERED WITH THE USE OF THE SPIROKETAL PROTECTED TRICARBONYL UNIT WHILE MANAGING TO MAINTAIN SOME OF THE INVENTIVE APPROACHES SO ATTRACTIVE IN HIS FIRST DISCUSSED WORK
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TIPSO
TBSO MeO
34
34
H
TIPSO
20
26
30
PMBO
MeO
23 CHO O OTBS TBS
34
OTBS
26
216
22
HO O TIPS
O
H
+ 19
SO2Ph 19
OMe 15
OMe
OMe OTBS
OHC 10
OMe OTBS
13 Sih (& Merck)
O
PMP
19
15 10
OTBS
26
O
30
+
S S
H 22
MeO
215
Ph2P O MeO
30
S
Merck intermediate 214
I TBDPSO MeO
15 10
S
OTBS 22
OMe OTBS
217 Danishefsky
34
+
30
PMBO
19
S
26
S
OMe 15
MeO
10
OMe 218
Smith III
OMe OTBS 219
3CHEME 2ETROSYNTHETIC ANALYSES
&ORMAL SYNTHESES OF &+
!MONG THE PLETHORA OF SYNTHETIC INVESTIGATION CARRIED OUT SURROUNDING &+ THREE FORMAL SYNTHESES HAVE BEEN REPORTED 3CHEME ;n = %ACH OF THESE WAS CENTRED AROUND AN IMPROVED SYNTHESIS OF THE KEY INTERMEDIATE OR CLOSE ANALOGUE REPORTED IN AS PART OF THE FIRST TOTAL SYNTHESIS OF &+ BY -ERCK 4HE ANALOGIES WITH THE ORIGINAL SUCCESS ARE VARIED IN EACH WORK THAT BY 3IH WAS CONCERNED PREDOMINANTLY WITH AN IMPROVED SYNTHESIS OF THE SAME INDIVIDUAL BUILDING BLOCKS AND FEATURES COUPLING REACTIONS UNDER THE SAME CONDITIONS AS PREVIOUSLY REPORTED $ANISHEFSKY TOOK THIS ONE STEP FURTHER USING THE SAME KEY DISCONNECTION OF THE -ERCK INTER MEDIATE ACROSS THE # # OLEFIN BOND BUT VARYING BOTH THE IMMEDIATE TARGETS AND THE METHOD OF CONSTRUCTION OF THE TRISUBSTITUTED ALKENE 3MITH HOWEVER PURSUED A COMPLETELY DIFFERENT DISCONNECTION CHOOSING DISCONNECTION ACROSS THE # # BOND AND ANTICIPATING ITS RECONSTRUCTION VIA COUPLING OF A # # DITHIANE WITH A PRIMARY IODIDE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
4HE 3IH SYNTHESIS OF THE -ERCK INTERMEDIATE
#HARLES 3IH FORMERLY OF THE 5NIVERSITY OF 7ISCONSINS 3CHOOL OF 0HARMACY TOOK AN UNORTHODOX APPROACH TO THE # # SEGMENT OF &+ THAT RELIED HEAVILY ON BIOSYNTHETIC METHODOLOGY ;= 7ITH THE SAME KEY OLE FINIC DISCONNECTION AS THE SUCCESSFUL GROUP AT -ERCK HIS GROUP WAS MORE INTERESTED IN THE APPLICATION OF ENZYMES FOR CHEMOSELECTIVE HYDROLYSES OR ENANTIOSELECTIVE REDUCTIONS ;= 3UCH METHODOLOGY CAN BE DEMONSTRATED IN THEIR APPROACH TO THE CYCLO HEXYL MOIETY OF &+ 3CHEME 7HILE INITIAL ATTEMPTS TO HYDROLYSE ;= ENANTIOSELECTIVELY WERE UNSUCCESSFUL AFTER A SEQUENCE OF METH ANOLYSIS ALKENE REDUCTION AND METHYLATION ATTEMPTS ON PROCEEDED QUITE DIFFERENTLY )N THIS CASE BIOCATALYTIC HYDROLYSIS OF THE # /!C WAS ACHIEVED WITH COMPLETE ENANTIOSELECTIVITY USING LIPASE !+ !MANO IN P( BUFFER TO GIVE ALCOHOL IN THE MAXIMUM YIELD ! SERIES OF STANDARD FUNCTIONAL GROUP TRANSFORMATIONS CONVERTED TO ELECTROPHILE THAT AFTER FIVE FURTHER STEPS FURNISHED ENAL 2EFORMATSKY REACTION WITH RACEMIC METHYL BROMOPROPIONATE GAVE A MIXTURE OF ALL FOUR POS SIBLE DIASTEREOMERS OF WHICH WAS MOST USEFUL FOR CONVERSION TO AS A SINGLE ANTIPODE ALTHOUGH STILL REQUIRING TEDIOUS SEPARATION AND FIVE FURTHER REACTIONS 3OME MATERIAL WAS ALSO SALVAGEABLE THROUGH AN EIGHT STEP SEQUENCE EMPLOYING THE MIXTURE OF SYN DIASTEREOMERS AND THAT FEATURES AN ENZYMATIC ACETATE HYDROLYSIS TO REMOVE THE OFFENDING DIASTE REOMER !LTHOUGH NOT SPECIFIED IT IS UNLIKELY THAT ALL COMBINED MATERIAL REPRESENTED GREATER THEN CHEMICAL YIELD FROM #ONDENSATION OF WITH THE LITHIUM ENOLATE OF ALSO PREPARED VIA ENZYMATIC MEANS ;= GAVE YET ANOTHER DIASTEREOMERIC MIXTURE IN REASONABLE YIELD BUT FAVOURING THE UNDESIRED # EPIMER 3EPARATION AND FOUR FURTHER SYNTHETIC MANIPULATIONS UPON THE DESIRED ISOMER THEN FURNISHED ALDEHYDE IN A MINIMUM OF STEPS AND APPROXIMATELY OVERALL YIELD 4HE OTHER MAJOR FRAGMENT REQUIRED FOR CONSTRUCTION OF THE -ERCK INTERMEDIATE WAS PHOSPHINE OXIDE WHOSE SYNTHESIS ONCE AGAIN FEATURED THE APPLICATION OF ENZYMES TO PREPARE OPTICALLY PURE MATERIALS 3CHEME ;= !DDITION OF THE DIANION OF TO ALDEHYDE FUR NISHED THE DESIRED COUPLED MATERIAL AS A MIXTURE OF EPIMERS AT # 3EQUENTIAL BENZOYLATION AND BENZYLATION AFFORDED AN INTER
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O
AcO
AcO
a,b,c
34
O
MeO
30
220 5 steps 56.9%
34
30
221
MeO
30
6 steps ~20% from 225
CHO OPMB
H 230
26 30
H
CO2Me
34
TBSO
e,f,g
30
MeO
26 30
H
CO2Me
OH TBSO
227
MeO
30
TBSO
H
OTBS
CO2Me
34 26
MeO
30
OH 228
i
34
h MeO
20
CHO
5 steps 60%
34
OH
34
30
224
+ TBSO
34
MeO
CO2Me
223
226
26
O 23
MeO
TBSO
34
variety 26
30
CO2Me
222
MeO 34
HO
34
+ MeO
TBSO
TBSO
AcO
d CO2Me
H
H 30
H
O
225
CO2Me
OH 229
231 7 steps 23.7% TBSO TBSO
TBSO
34 26
MeO
30
H
OTBS
23 20
PMBO 232
OH O
26
MeO
30
H
34
j,k,l,m
34
+
OTBS
23 20
PMBO 233
26
MeO
30
H
PMBO
OH O
20 23 CHO O OTBS TBS
215
3CHEME A +#/ -E/( # B 0D# ( %T/!C C #(. ("& #(#L OVER THREE STEPS D ,IPASE !+ !MANO P( # AND E 4"3/4F %T. #(#L # F ,I!L( 4(& # → 24 G $-0 #(#L OVER THREE STEPS H METHYL BROMOPROPIONATE :N 0H( REFLUX AND AND CONVERSION → IN MAXIMUM I ,$! %T/ n # THEN :N#L THEN J -E.("/!C !C/( #(#. n # K 4"3/4F %T. #(#L 24 L 4&! 4(& (/ M $-0 #(#L OVER FOUR STEPS
MEDIATE WHICH ALLOWED CONVENIENT SEPARATION OF THE DIASTEREOMERS BOTH OF WHICH COULD BE ELABORATED TO THE DESIRED PHOSPHINE OXIDE ALTHOUGH THROUGH SLIGHTLY DIFFERENT REACTION SEQUENCES 3CHEME CONVERSION OF THE UNDESIRED EPIMER NOT SHOWN ! NUMBER OF UNSPECIFIED YIELDS MAKES AN ESTIMATION OF OVERALL EFFICIENCY IMPOS SIBLE ALTHOUGH IT CAN BE SAID THE SHORTEST SEQUENCE TO REQUIRED AT A MINIMUM OF FOUR STEPS #ONDITIONS FOR THE SUCCESSFUL UNION OF MAJOR FRAGMENTS AND WERE REPORTED ALTHOUGH NO YIELD WAS GIVEN !SSUMING SIMILAR SUCCESS TO THAT OF THE -ERCK GROUP AN ESTIMATE FOR THE PROJECTED OVERALL UTILITY OF THE APPROACH TO &+ CAN BE MADE AND SUGGESTS A TOTAL YIELD OF THROUGH STEPS ! NUMBER OF TEDIOUS SEPARATIONS OF DIASTEREO MERS LONG SYNTHETIC SEQUENCES AND SOMETIMES LOW YIELDS DETRACT FROM THE OVERALL APPROACH (OWEVER 3IH HAS MANAGED TO USE THE PLATFORM OF
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
18
18
CO2Et
CO2H
a
CO2Et
CO2Et
15
15
234
S
b,c,d,e
O
18 15
S
CO2tBu
18
f,g,h,i
15
H MEMO
OH
OTBS OH
237
10
n
+
CO2Et
S
OH +
Ph2P O
15
OMe OTBS
10
13
10
OEt
m
OTBS
S S
S 10
S 240
242
OH
241
o,p,q,r,s
OBn 18
10
BzO 7 steps
O MEM
S
OTBS
19
MeO S S
S
S 239
238
HO
O
O j,k,l 10
18
15
236
235
15
t,u
OTBS
OMe OMe 244
18
10 15
BzO O
OTBS
OH 243
3CHEME A # CYLINDRACEA LIPASE /& -EITO 3ANGYO P( B .A( 4(& n # C N"U,I (3#( 3( 4(& n # → n # THEN SODIUM SALT OF D ISOBUTYLENE (3/ #(#L 24 OVER THREE STEPS E "AKERS YEAST 2ED 3TAR TAP WATER # F -%-#L $)0%! #(#L 24 G ,I!L( %T/ # → 24 H 4"3#L 0Y 24 I -E) #A#/ ACETONE (/ # OVER FOUR STEPS J / #(#L n # THEN 0H0 n # → 24 K (3#( 3( "&q/%T #(#L n # OVER TWO STEPS L 00, 3IGMA P( M ,I!L( 4(& # → 24 N N"U,I n # → # → n # THEN DR O "Z#L 4(& 0Y # P .A( .A) 4(& "N"R OVER TWO STEPS SEPARATE DIASTEREOMERS Q 4I#L #(#L HEXANE n # R 4L./ q(/ -E/( %T/ 24 S 4"3#L 0Y #(#L T -E.("/!C ACETONE !C/( n # U #(. #(#L ("& 24 OVER TWO STEPS .O YIELDS REPORTED
3CHEME %ND GAME AND SUMMARY OF 3IHS FORMAL SYNTHESIS OF &+
A FORMIDABLE MOLECULE TO DEMONSTRATE THE UTILITY OF ENZYMES IN NATURAL PRODUCTS SYNTHESIS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
PMP
OAc
OH
26
a,b,c
OAc
26
24
OAc
O
MeO
O
OH
HO
d,e
24
26
OTBS MeO
O
f,g,h
26
OTBS
O
MeO 246
247
O
24
248
24
I
O 249 i,j
TIPSO
34
TBSO MeO
30
H OTBS
26
11 steps 23.4%
MeO
34
22
O
216 PMP 27 steps 1.7%
O
O
H
O
TBSO 26
24
O
O
30
OHC
34
k,l,m
MeO
+ 30
SO2Ph 251
PMP
70 16 steps 7.1%
26
24
O
O
PMP 250 10 steps 23.8%
3CHEME A -E/( 3N#L $#% B .A/-E -E/( C 4"3#L %T. #(#L OVER THREE STEPS D M#0"! THIOBIS TERT BUTYL METHYLPHENOL #(#L REFLUX E #U#. -E,I %T/ n # → # THEN EPOXIDE FROM n # → # F P ANISALDEHYDE DIMETHYLACETAL #3! 0H( REFLUX $EAN 3TARK G 4"!& 4(& OVER THREE STEPS H 0H0 ) 0Y 0H( # I :N 0Y %T/( REFLUX J 0043 #(#L OVER TWO STEPS K N"U,I 4(& n # THEN L "". 4(& # THEN .A/( (/ M $-0 0Y #(#L 24 OVER THREE STEPS
4HE $ANISHEFSKY SYNTHESIS OF THE -ERCK INTERMEDIATE
)N THE COURSE OF EXTENSIVE INVESTIGATIONS INTO THE SYNTHESIS OF &+ $AN ISHEFSKY ARRIVED AT AN ALTERNATIVE STRATEGY TO THE # # PORTION OF THE MOLECULE ;= !S IS OFTEN THE CASE IN SYNTHESES OF SUCH ELABORATE TARGETS $ANISHEFSKY HAD ENVISAGED THE SAME KEY DISCONNECTIONS AS THE -ERCK GROUP WITH MACROCYCLISATION VIA LACTAM BOND FORMATION AND FURTHER CLEAV AGE OF ACROSS THE # # ISOLATED TRISUBSTITUTED ALKENE (OWEVER THE LATTER WAS ANTICIPATED VIA ADDITION OF THE LITHIO ANION OF SULFONE TO ALDEHYDE COMPARED TO THE 7ITTIG TYPE APPROACH ALREADY REPORTED !LDEHYDE WAS PREPARED IN COMMERCIALLY AVAILABLE GALACTOSE DERIVA TIVE IN STEPS YIELD ;= )NITIAL INSTALLATION OF THE # METHOXY GROUP USING METHANOL IN THE PRESENCE OF 3N#L GAVE WHICH AFTER PROTECTING GROUP MANIPULATIONS UNDERWENT DIRECTED EPOXIDATION TO GIVE PREDOMINANTLY β OXIRANE ;= 2EGIOSELECTIVE ORGANOCUPRATE ATTACK THEN INSTALLED THE # METHYL GROUP BEFORE A SERIES OF STANDARD TRANSFORMATIONS LED TO IODIDE 4HE SYNTHESIS OF THIS FRAGMENT WAS COMPLETED ON TREATMENT WITH ZINC 0Y %T/( REFLUX WHEREIN 6ASELLA TYPE FRAGMENTATION ;= OF THE PYRANOSE RING AFFORDED IN OVER TWO
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
HO OH O HO OMe 14 OH 252
a,b,c,d
MeO OH O OH BnO 14 OMe 253
O
e,f,g,h
OBn
O
14
18
19
MeO
S
15
10
OMe OTBS
S
217
OEt
O
OH
10
14
MeO
OMe
258
o,p,q,r
BF4 PPh2
O
OBz 18
18
OMe
255
Rh
MeO
OH
14
MeO
OMe 254
Ph2P
t,u,v,w,x,y z,aa,bb
OH
O O
SO2Ph
OMe
i,j,k,l,m,n
O
257 s
MeO
OH
OBz
14
10
MeO
18
OMe
256
3CHEME A 4"3#L %T. $-!0 #(#L 24 B N"U3N / 0H#( REFLUX THEN "N"R 4"!) # C .A( -E) 4(& 24 D - (#L 4(& REFLUX E .A"( %T/( 24 F ACETONE P4S/( ¯ -3 24 G #/#L $-3/ #(#L n # THEN %T. n # → 24 H 0H0 #-E #/-E #(#L # → 24 OVER TWO STEPS I ,I%T"( 4(& n # → n # J -S#L ,I#L S COLLI DINE $-& # K .A#. $-& 24 L $)"!, ( #(#L n # M .A"( %T/( 24 OVER TWO STEPS N .A .( LIQ n # n # O !C/( (/ 4(& REFLUX P .A)/ 4(& (/ 24 Q 0H0 #-E #/-E #(#L # → 24 "Z#L 0Y 4(& 24 OVER FOUR STEPS S ( PSI #(#L 24 T 4S/( ¯ -3 #(#L U ,IS"U "( 4(& n # V (3#( 3( "&q/%T #(#L n # → # W 4"3/4F LUT #(#L 24 X +#/ -E/( 24 Y -S#L %T. #(#L # Z .A) ACETONE REFLUX AA 0H3/.A $-& 24 BB N"U,I -E) 4(& n #
STEPS AFTER EPIMERISATION OF THE AXIAL BENZYLIDENE ACETAL TO THE DESIRED EQUATORIAL CONFORMER 4HIS WAS THEN COUPLED WITH THE SULFONE STABILISED ANION OF PREPARED USING STANDARD METHODOLOGY STEPS ;= AND THE RESULTING DIASTEREOMERIC MIXTURE ELABORATED TO KEY # # ALDE HYDE WITHOUT MAJOR INCIDENT STEPS YIELD 3CHEME 7ORTHY OF PARTICULAR NOTE IN $ANISHEFSKYS WORK IS HIS APPROACH TO THE # # FRAGMENT OF &+ ;= #ONTAINED WITHIN THE SEVEN CARBONS FROM # TO # IS DUPLICATION OF THE SYN METHYL METHOXY RELA TIONSHIP AROUND THE CENTRAL # SECONDARY HYDROXYL GROUP 2ECOGNITION THAT THIS STEREOCENTRE COULD BE EXPLOITED TO ESTABLISH BOTH RELATIONSHIPS BY CONCURRENT HYDROGENATION OF A DIOLEFIN REPRESENTS AN INTERESTING APPROACH TO SETTING THESE STEREOCENTRES 3CHEME !LTHOUGH TWO ROUTES WERE DEVELOPED THE ONE WHICH FEATURED DIFFEREN TIATED TERMINI AS IN PROVED MORE APPLICABLE TO MATERIAL THROUGHPUT 4HUS BEGINNING FROM COMMERCIALLY AVAILABLE AND INEXPENSIVE β $ GALAC TOPYRANOSIDE AS AN INITIAL SOURCE OF CHIRALITY A SERIES OF RELIABLE AND HIGH YIELDING TRANSFORMATIONS FURNISHED 4HIS WAS FURTHER ELABORATED
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3CHEME %ND GAME AND SUMMARY OF $ANISHEFSKYS FORMAL SYNTHESIS OF &+ A N"U,I 4(& n # THEN B #&#/ / 0Y $-!0 #(#L C LITHIUM NAPHTHALENIDE 4(& n # OVER TWO STEPS %: INSEPARABLE D 0043 PROPANOL #(#. # E 4)03/4F LUT
TO THE BISOLEFINIC REDUCTION SUBSTRATE THROUGH PROTECTING GROUP MANIPULATIONS AND SEQUENTIAL HOMOLOGATION AT # WITH CYANIDE FOLLOWED BY # VIA A (ORNER 7ADSWORTH %MMONS (7% REACTION ;= $IRECTED HYDROGENATION OF THE ALKENE GROUPS WITHIN WAS ACHIEVED USING ( AT PSI IN THE PRESENCE OF %VANS RHODIUM CATALYST ;= )N THIS CASE IT IS BELIEVED THAT THE ALLYLIC METHOXY GROUPS STRONGLY FAVOUR PRODUCT ;= AND INDEED THIS WAS ISOLATED IN AN IMPRESSIVE YIELD OF ! VARIETY OF STANDARD FUNCTIONAL GROUP MANIPULATIONS WERE THEN EMPLOYED TO CONVERT TO THE FULLY ELABORATED SULFONE IN A TOTAL OF STEPS AND OVERALL YIELD 3CHEME )N THE EVENT OF THE KEY # # COUPLING REACTION LITHIATION OF N"U,I 4(& n # PROCEEDED SMOOTHLY AND UPON ADDITION OF ALDEHYDE A DIASTEREOMERIC MIXTURE OF β HYDROXYSULFONES WAS PRODUCED IN GOOD YIELD 5NFORTUNATELY ATTEMPTS TO DIRECTLY INSTALL THE UNSATURATION BY REDUC TIVE ELIMINATION OF THE VICINAL PHENYLSULFONE AND HYDROXYL GROUPS PROVED PROBLEMATIC AS NO DESIRED PRODUCT WAS ISOLATED AND ANALYSIS INSTEAD SUG GESTED LOSS OF THE # ALLYL GROUP !FTER MUCH EXPERIMENTATION $ANISHEF SKY WAS ABLE TO OVERCOME THIS THROUGH MODIFICATION OF THE LEAVING GROUP AT # VIA FORMATION THE MORE LABILE TRIFLUOROACETATE !S A RESULT ELIMINATION WAS READILY ACHIEVED USING LITHIUM NAPHTHALENIDE 4(& n # GIVING A YIELD OF FOR THESE LAST TWO STEPS 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
$ESPITE THE PRESENCE OF THE COMPLETE BACKBONE OF THE -ERCK INTER MEDIATE FURTHER PROBLEMS WERE ENCOUNTERED THAT MARRED THE END OF THE STRATEGY 5NFORTUNATELY THE # # ALKENE WAS FORMED AS AN INSEPARABLE MIXTURE OF GEOMETRIC ISOMERS %VEN MORE FRUSTRATINGLY SUBSEQUENT CLEAVAGE OF THE PARA METHOXYBENZYLIDENE ACETAL PROVED ELUSIVE GIVING OF THE CORRESPONDING DIOL AT BEST 0043 PROPANOL ACETONITRILE # ALTHOUGH SEPARATION OF THE GEOMETRIC OLEFIN ISOMERS WAS AT LEAST POSSIBLE ,ATE STAGE DIFFICULTIES WITH PROTECTING GROUP STRATEGIES ARE SO OFTEN A PITFALL IN NATURAL PRODUCTS SYNTHESIS AND WHILE IN THIS CASE IT IS UNDOUBT EDLY DISAPPOINTING $ANISHEFSKY WAS NEVERTHELESS ABLE TO FURTHER ELABORATE TO A SUITABLE -ERCK INTERMEDIATE AND COMPLETE THE FORMAL SYNTHESIS OF &+ /VERALL THE PROJECTED TOTAL SYNTHESIS OF &+ ASSUMING THE SAME END GAME AS PREVIOUSLY EMPLOYED BY THE -ERCK GROUP AFFORDS IN A TOTAL OF STEPS AND OVERALL YIELD SUBSTANTIALLY LONGER AND LOWER YIELDING THEN THE ORIGINAL REPORT 3CHEME
4HE 3MITH SYNTHESIS OF THE -ERCK INTERMEDIATE
/F THE FORMAL SYNTHESES ACHIEVED !MOS 3MITH ))) HAS REPORTED THE ONLY APPROACH THAT DIFFERS IN ITS MAIN DISCONNECTION OF THE -ERCK INTERMEDIATE ;= 7HILE -ERCK 3IH AND $ANISHEFSKY ALL ATTEMPTED FRAGMENT UNION THROUGH THE # # OLEFIN 3MITH SAW THIS AS A MISJUDGEMENT "Y CHOOS ING AN ALTERNATE STRATEGY HE THOUGHT IT WAS POSSIBLE TO DEVELOP A MORE EFFECTIVE AND STEREOCONTROLLED GENERATION OF THE TRISUBSTITUTED OLEFIN AS WELL AS AN IMPROVED FRAGMENT COUPLING 4O THIS END HE ENVISAGED FORMA TION OF THE # # BOND THROUGH COUPLING OF A # # DITHIANE ;= WITH A # # PRIMARY ALKYL IODIDE ;= 3MITHS APPROACH TO THE TRISUBSTITUTED ALKENE DERIVES FROM THE READILY PREPARED ALKYNE 3UBSEQUENT TREATMENT WITH -E!L IN THE PRESENCE OF CATALYTIC #P:R#L EFFECTED CARBOALUMINATION TO GIVE THE CORRESPONDING VINYL ALANE AS A SINGLE GEOMETRIC ISOMER !FTER FORMATION OF THE ATE COMPLEX WITH N"U,I ADDITION OF SYMMETRICAL TRANS SUBSTITUTED EPOXIDE ;= GAVE ALCOHOL IN PLEASING YIELD EVEN ON LARGE SCALE 7HILE THE USE OF THIS SYMMETRICAL EPOXIDE NECESSITATED SUBSEQUENT DIFFERENTIATION OF THE TWO PRIMARY BENZYL ETHERS IT AVOIDED PROBLEMS WITH A LACK OF CHEMOSELEC TIVITY ENCOUNTERED WHEN USING ALTERNATIVE ASYMMETRIC EPOXIDES 4HUS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
22 19
+
O
BnO
TBDPSO
OBn
a
OBn OH
OBn OBn
22
22
b,c,d,e
BnO
f
19
TBDPSO
261 5 steps 65.5%
22
BnO
BnO
19
260 7 steps 68.6%
OBn OBn
MeO
O 10
PhO2S
262
263
19
CO2Me 15
OTBS
PhO2S H MeO O
264 13 steps
265
22
BnO
OMe 19
OBn
10
10 steps
O
22
H OMe 15
OMe OTBS
OMe
219
O
OTBS
OBn OBn
22
10
15
10
OTBS
I
MeO
H
MeO
19 15
HO
h,i 19
OBn OBn
MeO
267
O
H
10
266
15
O
OTBS
3CHEME A -E!L #P:R#L #L#(#(#L N"U,I HEXANES # THEN 0H( B .A( "N"R 4"!) 4(& C 4"!& 4(& D 0H3 "U0 $-& E /XONE¤ 4(& -E/( (/ F N"U,I 4(& n # THEN n # → # G "U3N( !)". 0H-E REFLUX H (&q0Y 4(& I -E."(/!C !C/( #(#. n #
WAS READILY ELABORATED TO SULFONE WHICH COULD BE DEPROTONATED UNDER STANDARD CONDITIONS AND ON SUBSEQUENT ADDITION OF ESTER RESULTED IN OF AN EPIMERIC MIXTURE OF β KETOSULFONES $ESULFONATION OF THE PRODUCT MIXTURE PROVED UNEXPECTEDLY TROUBLESOME BUT WAS EVENTUALLY ACHIEVED WITH THE NOVEL USE OF N"U3N( AND AZOBISISOBUTYLONITRILE !)". IN REFLUXING TOLUENE GIVING KETONE $ESILYLATION AND DIRECTED REDUC TION USING TETRAMETHYLAMMONIUM TRIACETOXYBOROHYDRIDE FURNISHED THE DESIRED ANTI DIOL AS THE MAJOR PRODUCT 4HIS WAS ELABORATED THROUGH TO KEY α SILOXYIODIDE USING A STANDARD SEQUENCE INVOLVING FUNCTIONAL GROUP MANIPULATIONS WHICH PROCEEDED WITHOUT INCIDENT TO FURNISH THE DESIRED FRAGMENT IN A TOTAL OF STEPS AND OVERALL YIELD 3CHEME 4HE COUPLING PARTNER FOR IODIDE WAS DITHIANE DERIVED FROM SUL FONE PREPARED IN A NEAR IDENTICAL SEQUENCE TO THAT OF $ANISHEFSKY AND ALDEHYDE ;= 4HE LATTER WAS ACHIEVED IN A SEQUENCE OF SEVEN STEPS FROM CROTYL ALCOHOL AFTER INITIAL 3HARPLESS ASYMMETRIC EPOXIDATION AND PROTECTION OF THE PRIMARY ALCOHOL ADDITION OF THE LITHIO ANION OF DITHIANE IN THE PRESENCE OF DIMETHYL TETRAHYDRO ( PYRIM
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
O
a,b
26
TBDPSO
HO
268
c
26
S
26
TBDPSO
O
d,e,f,g
OH S
269
270
MeO
TBDPSO
34
30
l
H
S
26 24
PMBO
218
S
MeO
i,j,k
H 26 TfO PMBO 273
34
SO2Ph
S 24
S
h
30
TBDPSO
34
30
24
S
271 TBSO 70 14 steps MeO
TBDPSO
S
26
H PMBO
24
MeO
34
SO2Ph 30
S
26
HO PMBO
24
S
272
3CHEME A 3HARPLESS !% $ n $%4 #(#L B 4"$03#L %T. $-!0 CAT $-& OVER TWO STEPS C LITHIO DITHIANE $-05 4(& # D 4"!& 4(& E P ANISALDEHYDE DIMETHYLACETAL P4S/( $-& # OVER TWO STEPS F $)"!, ( #(#L n # G 3/q0Y $-3/ %T. #(#L # H N"U,I 4(& n # THEN OVER TWO STEPS I 4&!! $-3/ #(#L n # THEN %T. J !L(G 4(& AQ REFLUX K ,$! . PHENYLTRIFLIMIDE $-% $-05 n # TO 24 L -E#U,I 4(& #
IDINONE $-05 RESULTED IN OPENING OF THE EPOXIDE EXCLUSIVELY AT # DUE TO THE STERIC BULK OF THE PROTECTING GROUP AND WITH COMPLETE STEREOSELECTIV ITY 3UBSEQUENT ACCESS TO WAS STRAIGHTFORWARD 3CHEME !FTER UNION OF SULFONE AND ALDEHYDE AN OXIDATION DESULFONA TION SEQUENCE RETURNED AN INTERMEDIATE KETONE AT # &ROM THIS INSTAL LATION OF THE DESIRED METHYL GROUP WAS ACHIEVED USING THE METHOD OF -C-URRY ;= WHEREIN FORMATION OF THE ENOL TRIFLATE IS FOLLOWED BY TREAT MENT WITH LITHIUM DIMETHYLCUPRATE RETURNING THE TRISUBSTITUTED OLEFIN PREDOMINANTLY IN THIS CASE AS THE DESIRED % ISOMER 4HUS THE SYN THESIS OF WAS COMPLETE READY FOR COUPLING WITH IODIDE 3CHEME 7ITH BOTH MAJOR FRAGMENTS AVAILABLE STUDY OF THEIR UNION COULD BE INITIATED !CCORDINGLY LITHIATION OF T"U,I 4(& (-0! n # AND THEN ADDITION OF IODIDE GRATIFYINGLY RETURNED A YIELD OF # # BACKBONE &INALLY REMOVAL OF THE DITHIANE MOIETY AND THEN 0-" ETHER ENABLED DIRECTED REDUCTION OF THE # KETONE USING ,I!L( IN THE PRESENCE OF ,I) n # TO AFFORD THE # CARBINOL AS A SINGLE DIASTEREOMER THAT WAS PROTECTED AS THE 4)03 ETHER TO COMPLETE 3MITHS ALTERNATIVE SYNTHESIS OF THIS VALUABLE # # INTERMEDIATE AND A PROJECTED OVERALL YIELD OF THROUGH STEPS 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3CHEME %ND GAME AND SUMMARY OF 3MITHS FORMAL SYNTHESIS OF &+ A T"U,I (-0! 4(& n # THEN B .#3 !G./ LUT ACETONE 4(& (/ n # C $$1 #(#L (/ # D ,I!L( %T/ ,I) n # E 4)03/4F LUT #(#L # OVER TWO STEPS
4HE ,EY TOTAL SYNTHESIS OF ANTASCOMICIN "
4HE ANTASCOMICINS ARE A FAMILY OF FIVE NATURAL PRODUCTS DISCOVERED AT 3AN DOZ ;= THAT BIND THE IMMUNOPHILIN &+"0 TO THE SAME DEGREE AS &+ AND RAPAMYCIN YET THEIR LIGAND &+"0 COMPLEXES DISPLAY NO IMMUNO SUPPRESSIVE PROPERTIES ;= 3UCH COMPLEXES ARE OF INTEREST IN THE TREAT MENT OF NEURODEGENERATIVE DISEASES DUE TO THE SIGNIFICANTLY HIGHER LEVELS OF &+"0 FOUND IN THE BRAIN ;= 3TUDIES ON SYNTHETIC &+"0 LIGANDS HAVE DEMONSTRATED THEIR ABILITY TO PROMOTE THE REGROWTH OF DAMAGED NEURONS BOTH PERIPHERALLY AND IN THE CENTRAL NERVOUS SYSTEM ;n= 5NDER THE DIRECTION OF 3TEVEN 6 ,EY AT THE 5NIVERSITY OF #AMBRIDGE 5+ WE HAVE REPORTED THE ONLY TOTAL SYNTHESIS OF ANY OF THE ANTASCOMICIN FAMILY THUS FAR ;= )N OUR APPROACH TO ANTASCOMICIN " WE SOUGHT A FINAL MACROCYCLISATION VIA A NOVEL CATECHOL TETHERED $IECKMANN ; = CONDENSATION ALSO SEEN LATER IN THEIR TOTAL SYNTHESIS OF RAPAMYCIN VIDE SUPRA THAT WOULD ALLOW AN EFFICIENT INTRAMOLECULAR CONSTRUCTION OF THE # # BOND 4HE TETHER ALSO CONTAINING # AND # WAS TO BE INTRODUCED THROUGH THE NITROGEN OF THE PIPECOLATE MOIETY EXPLOITING THE RELATIVE EASE OF CARBON HETEROATOM BOND FORMATION 4HE MAIN # # FRAMEWORK
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
HO
MeO
O
33 34
25
HO 1
CO2H + MeO N Fmoc
6
30
HO 26
6
1
N
O HO 11
O
O MOMO
25
O
275
O
OBn OBn
29
MeO
O
MeO
O
33
OTBS H
+ 30
Bu3Sn
O
278
276
279
+
O
TBSO
O
TBSO
OTBS
10
PhO2S 24
TBSO
TBSO
10
17
O 16
H
OPMB
+
24
16
O
277
280
281
antascomicin B 5
3CHEME 2ETROSYNTHETIC ANALYSIS
WAS DISCONNECTED FURTHER THROUGH THE OPENING OF EPOXIDE WITH A # ANION DERIVED FROM SULFONE 3CHEME !FTER DISCONNECTION VIA RING CLOSING METATHESIS ;= ACROSS # # KEY TO THE SYNTHESIS OF EPOXIDE WAS TO BE THE ADDITION OF STANNANE TO BUTANEDIACETAL "$! PROTECTED ALDEHYDE BASED ON WORK DEVELOPED WITHIN OUR GROUP ON THE USE OF THESE BUILDING BLOCKS IN THE SYNTHESIS OF ANTI DIOLS ;n= 3ULFONE DERIVED FROM THE CORRESPONDING PROTECTED # ALCOHOL WAS TO BE CONSTRUCTED VIA THE SYN HYDROZIRCONATION OF TERMINAL ALKYNE AND ITS ADDITION TO ALDEHYDE 4HIS STRATEGY WAS GREATLY SIMPLIFIED SINCE THE RESULTING # ALCOHOL IS ULTIMATELY OXIDISED IN THE NATURAL PRODUCT AND THUS THE STEREOSELECTIVITY OF THIS ADDITION WOULD NOT BE IMPORTANT 4HE SYNTHESIS OF STANNANE BEGAN WITH AN ASYMMETRIC CROTYLATION OF COMMERCIALLY AVAILABLE BENZYLOXYACETALDEHYDE TO INSTALL THE ONLY TWO STEREOGENIC CENTRES OF THE FRAGMENT WITH EE ; = !FTER PROTECTION OF THE FREE ALCOHOL OZONOLYSIS OF THE ALKENE GAVE AN UNUSUALLY STABLE OZONIDE THAT COULD NOT BE REDUCED DIRECTLY TO THE ALCOHOL WITHOUT PROCEEDING VIA THE ALDEHYDE .EVERTHELESS THIS REMAINED A HIGH YIELDING ONE POT PROCEDURE AND ALLOWED EASY ACCESS TO IODIDE WHICH AFTER DISPLACEMENT BY THE LITHIO ANION OF ALLYLPYRIDYLSULFIDE GAVE STANNANE PRECURSOR 0REPARATION OF THE STANNANE WAS COMPLETED IN ALMOST QUANTITATIVE YIELD AND AS A MIXTURE OF GEOMETRIC ISOMERS ON TREATMENT WITH ANIONIC TRIBUTYLTIN CUPRATE GENERATED IN SITU 3CHEME ! VARIETY OF "$! PROTECTED TARTRATES HAVE BEEN DEVELOPED BY OUR GROUP AS CHIRAL BUILDING BLOCKS THAT OFFER PLATFORMS FOR A VARIETY OF STEREOSELEC
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
25
O
H
a,b
26
OBn
25
c,d
26
OBn
OBn 282
25
e
29
283
OBn
25
f
26
OBn
I
25
119
OBn
26
OBn SPy 284
OBn
26 29
OBn
Bu3Sn 278
3CHEME A % BUTENE +/T"U N"U,I 4(& n # )PC "/-E %T/ "&q/%T n # B "N"R .A( $-& C / #(#L n # THEN 0H0 .A"( -E/( OVER TWO STEPS D ) 0H0 )M #(#L E ALLYLPYRIDYLSULFIDE T"U,I 4(& n # F "U3N,I #U"R 4(& n #
TIVE REACTIONS ;n= AND THESE HAVE FOUND SUBSEQUENT APPLICATION IN SEVERAL TOTAL SYNTHESES ;= 4HEIR EASE OF PREPARATION PARTICULARLY ON SCALE IS A SIGNIFICANT ADVANTAGE &OR EXAMPLE HERE IN THE CASE OF ALDEHYDE CONDENSATION OF INEXPENSIVE DIMETHYL $ TARTRATE AND BUTANE DIONE GAVE # SYMMETRIC DIESTER $ESYMMETRISATION VIA BISREDUCTION AND MONO PROTECTION OF THE RESULTANT DIOL FOLLOWED BY 3WERN OXIDATION COMPLETED A FACILE SYNTHESIS OF THAT WAS TAKEN ON WITHOUT FURTHER PURI FICATION 3UBSEQUENT ADDITION OF STANNANE TO "$! PROTECTED ALDEHYDE IN THE PRESENCE OF ZINC IODIDE GAVE WITH NEAR COMPLETE DIASTERE OSELECTIVITY AND AS AN IMPRESSIVE RATIO SYNANTI IN FAVOUR OF THE DESIRED ISOMER )T IS BELIEVED THAT CHELATION OF THE :N BETWEEN THE CARBONYL GROUP AND THE AXIAL PAIR OF ELECTRONS ON THE α OXYGEN ATOM OF THE "$! GROUP RESTRICTS THE CONFORMATION OF THE ALDEHYDE SUCH THAT ATTACK CAN ONLY OCCUR ON ONE FACE ; = 4HIS ATTACK IS IN AN ANTIPERIPLANAR ORIENTA TION WITH THE VINYLIC HYDROGEN PLACED OVER THE CHELATE RING /F NOTE THE %: RATIO OF THE STARTING STANNANE WAS SHOWN TO HAVE LITTLE EFFECT OF THE DIASTEREOSELECTIVITY OF THE ADDITION PRODUCT 3CHEME !FTER PROTECTING GROUP MANIPULATIONS AN OXIDATION METHYLENATION SEQUENCE ON THE # ALCOHOL GAVE DIENE THE RING CLOSING METATHESIS PRECURSOR WHICH ON PORTIONWISE ADDITION OF 'RUBBS SECOND GENERATION IMIDAZOLIDINE CATALYST ;= GAVE SUBSTITUTED CYCLOHEXENE IN EXCELLENT YIELD 2EDUCTION OF THE ALKENE UNDER STANDARD HYDROGENATION CONDITIONS OCCURRED WITH CONCOMITANT REMOVAL OF THE BENZYL PROTECTING GROUPS GIV ING A DIOL WHICH WAS TRANSFORMED IN ONE STEP TO EPOXIDE 4HE SYNTHESIS OF THIS KEY INTERMEDIATE WAS COMPLETED IN STEPS AND AN OVERALL YIELD OF 3CHEME !LDEHYDE WAS PREPARED IN A SEQUENCE THAT BEGAN WITH COMMER CIALLY AVAILABLE ENANTIOPURE 3 BROMO METHYLPROPAN OL 0ROTEC
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
MeO
H HO HO
a
CO2Me CO2Me
MeO
O O
b,c
CO2Me CO2Me
MeO
H
MeO
O
30
H
MeO
OBn
26
29
25
SnBu3
O
MeO
O MOMO
30
30
279
25
OBn
26
OBn
e
29
Bu3Sn 278
291
33
MeO
287
Zn2+ O R' H O H RH SnBu3
OTBS OBn
OTBS O
33
O O MeO
286 Zn2+ O O
30
MeO
285 MeO
d
OTBS OH
33
O O
j,k
29
O
MeO
O MOMO
30
25
OTBS
33
MeO
MeO
O
MeO
O MOMO
g,h,i
29
OH 25
O
MeO
33
OBn
276
MeO
O
30
29
OBn
HO
25
OH
33
f
29
30
O
25
OBn
290
OBn
289
288
3CHEME A -E#/#/-E #3! #(/-E -E/( REFLUX B ,I!L( 4(& # C .A( 4"3#L # → 24 D #/#L $-3/ #(#L n # THEN %T. n # → 24 E :N) #(#L n # TO 24 OVER TWO STEPS SYNANTI AT # # F -/-#L $)0%! #(#L 4"!& 4(& G #/#L $-3/ #(#L n # THEN %T. n # TO 24 H -E00H"R N"U,I 4(& n # → 24 OVER TWO STEPS I BIS MESITYL IMIDAZOLIDINYLIDENE DICHLOROPHENYLMETHYLENE TRICYCLO HEXYLPHOSPHANE RUTHENIUM × MOL 0H-E REFLUX J ( 0D# %T/( K .A( TRIISOPROPYLBENZENESULFONYL IMIDAZOLE 4(&
O
O N TBSO 10
I
a,b
Ph TBSO
O
TBSO 10
14
O
294 H
10
HO
O
14
16
c
O N
O
d,e
TBSO 10
TBSO
O
14
16
f SEt
TBSO
TBSO
O
14
16
10
H
Ph 292
293
295
296
280
3CHEME A !LLYL-G#L #U) 4(& B / #(#L n # THEN 0H0 C "U"/4F %T. #(#L n # THEN THEN P( PHOSPHATE BUFFERED -E/( (/ SINGLE ISOMER D 4"3#L )M $-& E %T3( N"U,I 4(& n # F %T3I( 0D# ACETONE
TION OF THE HYDROXYL GROUP FOLLOWED BY HALIDE EXCHANGE GAVE WHICH COULD BE HOMOLOGATED USING ALLYLMAGNESIUM CHLORIDE IN THE PRESENCE OF IODIDE 3TANDARD OZONOLYSIS THEN GAVE ALDEHYDE THAT WAS USED DIRECTLY IN AN %VANS ASYMMETRIC ALDOL REACTION WITH THE BORON ENOLATE OF OXAZOLIDINONE ; = TO GIVE ADDUCT AS A SINGLE DIASTEREOMER AFTER ONE RECRYSTALLISATION AND IN EXCELLENT YIELD 4HE CHIRAL AUXILIARY WAS SUBSEQUENTLY REMOVED VIA THIOESTER 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
4HE SYNTHESIS OF ALKYNE THE COUPLING PARTNER FOR BEGAN FROM 3 2OCHE ESTER 3TANDARD TRANSFORMATIONS AFFORDED ALCOHOL WHICH WAS OXIDISED AND THEN SUBJECTED TO STANDARD (7% CONDITIONS ;= TO INSTALL THE # # OLEFIN ALMOST EXCLUSIVELY AS THE TRANS ISOMER 2EDUCTION OF THE ESTER AND CONVERSION OF THE PRIMARY ALCOHOL TO THE ALLYLIC BROMIDE THROUGH AN !PPEL REACTION THEN ALLOWED INSTALLATION OF THE 4-3 PROTECTED ALKYNE MOIETY VIA ANIONIC HALIDE DISPLACEMENT TO FURNISH SKIPPED ENYNE $ESILYLATION OF THIS AFFORDED A TERMINAL ALKYNE WHICH WHEN TREATED WITH FRESHLY PREPARED 3CHWARTZ REAGENT UNDERWENT CIS SPECIFIC HYDROZIRCONATION AS EXPECTED ; = )N THE PRESENCE OF A ,EWIS ACID SILVER PERCHLORATE THIS VINYLZIRCONIUM SPECIES REACTED WITH ALDEHYDE TO GIVE AN EXCELLENT YIELD OF ALLYLIC ALCOHOLS ; = AS A SEPARABLE MIXTURE OF DIASTEREOMERS 3INCE THE # ALCOHOL ULTIMATELY FORMS THE CARBONYL OF THE ENONE IN ANTASCOMICIN " THE SELECTIVITY OF THIS ADDITION WAS NOT IMPORTANT !LTHOUGH BOTH # EPIMERS COULD BE CARRIED FURTHER IN THE SYNTHESIS FOR CONVENIENCE IN CHARACTERISA TION THE DIASTEREOMERS WERE SEPARATED AT THIS STAGE !FTER PROTECTING GROUP MANIPULATIONS ALCOHOL WAS CONVERTED INTO THE CORRESPONDING # SULFIDE WHICH WAS SELECTIVELY OXIDISED TO GIVE SULFONE ;= IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD OF 3CHEME $EPROTONATION AT # OF SULFONE USING T"U,I IN THE PRESENCE OF (-0! n # FOLLOWED BY ADDITION OF EPOXIDE AT HIGHER TEMPERATURE n # ESTABLISHED THE # # BACKBONE OF ANTASCOMICIN " IN REA SONABLE YIELD AS A DIASTEREOMERIC MIXTURE OF γ HYDROXYSULFONES EPIMERIC AT # $ESULFONYLATION USING LITHIUM IN THE PRESENCE OF DI TERT BUTYL BIPHENYL ; = REASSURINGLY THEN GAVE ALCOHOL AS A SINGLE DIASTE REOMER 3CHEME %STERIFICATION OF THE # /( WITH COMMERCIALLY AVAILABLE . &MOC PROTECTED PIPECOLIC ACID USING %$#) GAVE A MIXTURE OF ROTAMERS TREATMENT OF THIS WITH CAMPHORSULFONIC ACID FURNISHED DESIRED DESILYLATED PRIMARY ALCOHOL ALONG WITH SOME BISDEPROTECTED MATERIAL ALTHOUGH THIS WAS EASILY SEPARATED AND RECYCLED !FTER A TWO STEP ,EY ;= 0INNICK ;= OXIDATION TO THE CARBOXYLIC ACID REMOVAL OF THE &MOC PROTECTING GROUP UNDER STANDARD CONDITIONS GAVE THE SECONDARY AMINE READY FOR INSTALLATION OF THE ANTICIPATED CATECHOL TETHER 4REATMENT OF WITH BENZO; =DIOXIN ONE FORMED AMIDE WITHOUT DIFFICULTY ALSO INSTALLING THE REMAINING # AND # CENTRES AND EXPOSING THE SECOND
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TMS OPMB
HO 22
a,b
22
EtO2C
24
297
OPMB
c
HO
OPMB
22
24
Br
d
OPMB
22
24
24
298
17
Me TMS
301
24
302
TBSO
O
TBSO
10
TBSO
TBSO
10
OTBS
OH
h,i
TBSO
TBSO 10
24
16
22
300
299
OH
OPMB 24
16
17
17
304
303
OPMB
17
e
16
f H 17
280
OPMB 24
g 281
j,k MeO TBSO
TBSO
OTBS
10
24
277
O
+ MeO
OTBS TBSO
34
PhO2S
l,m O MOMO
276
OH
OTBS 24
10
OMOM O
OMe
O
OMe
25 25
O
34
305
3CHEME A #/#L $-3/ #(#L n # THEN %T. n # → 24 B 0H0#(#/%T #(#L # → 24 %: C $)"!, ( 0H-E n # D 0H0 #"R #(#L E N"U,I 4(& n # F AQUEOUS .A/( G ;:R#P ( #L = #(#L n TO # !G#L/ #(#L MIXTURES SEPARABLE H SEPARATION 4"3#L )M $-& I $$1 #(#L (/ J 0H330H 0"U 0Y OVER TWO STEPS K 0H3E3E0H (/ %T/ L N"U,I (-0! 4(& n # THEN n # M LITHIUM DI TERT BUTYLBIPHENYL 4(& n #
ALCOHOL OF THE TETHER #OMPLETION OF THE LINKING TETHER VIA HIGH DILUTION MACROLACTONISATION - WITH %$#) FURNISHED THE MACROCYCLE IN GOOD YIELD EXPLOITING THE RELATIVE EASE OF CARBON HETEROATOM BOND FORMATION &ORMATION OF THE # # BOND TO COMPLETE THE PARENT MAC ROCYCLE OF ANTASCOMICIN " WAS THEN ACHIEVED IN YIELD ON TREATMENT WITH ,I(-$3 0RESUMABLY THE $IECKMANN LIKE CONDENSATION THAT FOLLOWS DEPROTONATION IS PROMOTED BOTH BY THE SIMPLIFIED SIX MEMBERED TRANSITION STATE IN THE TETHERED SYSTEM AND ALSO THE ENTROPIC ADVANTAGES ASSOCIATED WITH THIS CATECHOL PINNING TECHNIQUE 3CHEME !FTER STRAIGHTFORWARD OXIDATIVE REMOVAL OF THE CATECHOL MOIETY THE REMAINING DEPROTECTION AND OXIDATION SEQUENCE PROVED SOMEWHAT PROB LEMATIC AND LOW YIELDING .EVERTHELESS AFTER SELECTIVE REMOVAL OF THE SILYL PROTECTING GROUPS USING (&q0Y $ESS -ARTIN OXIDATION ;= AT # AND FINALLY 4&! CLEAVAGE OF THE "$! MOIETY COMPLETED OUR TOTAL SYNTHESIS OF ANTASCOMICIN " A FIRST IN THIS PARTICULAR FAMILY OF NATURAL PRODUCTS 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
MeO
MeO
MeO
O
MeO
O
MeO
O
MeO
O
O
MeO
O
34
MeO
O
34
34
O
O
O
34
8
MOMO
MOMO 26
6
26
a,b
1
N CO2H Fmoc 275 +
MOMO
HO
6
1
N Fmoc O
9 26
c,d
O
6
1
N H
O
MOMO
O 308 e
26
6
OH
O
N 8
O
HO
TBSO
10
TBSO
HO
OH TBSO
TBSO 10
10
OTBS 305
O
O
OTBS
O
O
O
9
TBSO
1
10
OTBS
OTBS
306
307
309 f
MeO
MeO
O
O
HO
O
34
26
1
N 8
O HO
O
9
26
O
1
6
N 8
O
O
O
9
O
O
O antascomicin B 5
26
h
OH
6
1
N 8
O
O
9
26
g
O
6
O
O
TBSO
TBSO
312
8
O
311
O O
1
N 9
O
OTBS
34
MOMO
10
10 10
O
34
MOMO
i
O
MeO O
34
MOMO
HO
O
MeO
MeO HO
6
MeO
O
O
O
TBSO
10
OTBS
OTBS 310
3CHEME A %$#) $-!0 #(#L n # B #3! -E/( #(#L n # C 40!0 .-/ #(#L .A#L/ .A(0/ METHYLBUT ENE T"U/( OVER TWO STEPS D PIPERIDINE $-& E BENZO ; =DIOXIN ONE $-!0 #(#L OVER TWO STEPS F %$#) #(#L G ,I(-$3 4(& H $-0 (/ #(#L 0Y I (&q0Y 0Y 4(& $-0 #(#L 0Y 4&! (/ OVER THREE STEPS
4HIS CHALLENGING SYNTHESIS WAS ACHIEVED THROUGH A COMBINATION OF BOTH ESTABLISHED SYNTHETIC PROCEDURES AND NOVEL METHODOLOGIES BUT IS PERHAPS MOST NOTEWORTHY FOR OUR DEMONSTRATION OF THE USE OF "$! AS A PROTECTING AND STEREODIRECTING FUNCTIONALITY AND THE USE OF A NOVEL AND HIGHLY EFFEC TIVE CATECHOL PINNING STRATEGY TO ACHIEVE MACROCYCLE FORMATION THROUGH A # # BOND FORMING EVENT AND SIMULTANEOUSLY INTRODUCE FUNCTIONALITY FOR EVENTUAL FORMATION OF THE VICINAL TRICARBONYL FUNCTIONALITY 3OME DIS APPOINTING YIELDS TOWARDS THE END OF THE ROUTE DRAMATICALLY REDUCED THE OVERALL YIELD OF THE STRATEGY .EVERTHELESS IN COMPLETING THE SYN THESIS OF ONE OF THE ANTASCOMICIN FAMILY IN A LONGEST LINEAR SEQUENCE OF JUST STEPS THIS APPROACH PROVIDES A VERY CONCISE ENTRY TO AN INTERESTING MEMBER OF THE TRICARBONYL MACROLIDES 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 3UMMARY OF ,EYS TOTAL SYNTHESIS OF ANTASCOMICIN "
4HE #HAKRABORTY SYNTHESIS TOWARDS ANTASCOMICIN ! 4HE #HAKRABORTY GROUP HAVE DISCLOSED SYNTHESES OF TWO ADVANCED FRAG MENTS IN THEIR APPROACH TOWARDS ANTASCOMICIN ! ; = 4HESE REPRE SENT THE TWO KEY INTERMEDIATES IDENTIFIED FROM THEIR RETROSYNTHETIC ANALYSIS 3CHEME )N THIS THEY ANTICIPATED A FINAL MACROCYCLISATION VIA A RING CLOSING METATHESIS REACTION ;= AND FURTHER DISCONNECTION OF THE ACY CLIC TRIENE PRECURSOR THROUGH THE # / BOND TO GIVE # # AND # # FRAGMENTS AND RESPECTIVELY 4HE GROUP HAS NOT YET PUBLISHED DETAILS OF THE ENDGAME OF THEIR SYNTHESIS 4HE SYNTHESIS OF THE # # TRICARBONYL UNIT OF ANTASCOMICIN ! BEGAN WITH A TITANIUM )6 MEDIATED DIASTEREOSELECTIVE @NON %VANS ALDOL REACTION BETWEEN . PROPANOYL OXAZOLIDINETHIONE AND ALDEHYDE TWO STEPS FROM PENTANE DIOL TO FURNISH THE SYN ADDUCT 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
HO
TBSO
HO
TBSO 26
TBSO
TBSO
11
1
N
O HO
22
OH 26
26
6
30
30
30
O
O
O
O
6
O TMSO 11
1
N O
O
314 + 6
O
O
11
O
CO2H O
21
O
16
16
antascomicin A 4
1
N
O TMSO
O 313
16
315
O
3CHEME 2ETROSYNTHETIC ANALYSIS
4HE CHIRAL AUXILIARY WAS REMOVED USING SODIUM BOROHYDRIDE TO GIVE DIOL WHICH WAS SUBJECTED TO PROTECTING GROUP MANIPULATIONS AND A TWO STEP 3WERN 0INNICK OXIDATION PROTOCOL TO FURNISH ACID !FTER FORMATION OF THE MIXED ANHYDRIDE OF ADDITION OF CHIRAL AUXILIARY RESULTED IN . ACYLATION TO GIVE AMIDE WHICH COULD BE METHYLATED AT # VIA THE SODIUM ENOLATE WITH COMPLETE DIASTEREOSELECTIVITY ALBEIT IN DISAPPOINT ING YIELD &URTHER REDUCTIVE REMOVAL OF THE OXAZOLIDINONE WAS FOLLOWED BY 3WERN OXIDATION AND HOMOLOGATION WITH THE LITHIUM ENOLATE OF ETHYL ACETATE GIVING β HYDROXY ESTER AS A MIXTURE OF DIASTEREOMERS !FTER HYDROLYSIS OF USING ,I/( THE PIPECOLATE MOIETY WAS INTRO DUCED AS THE METHYL ESTER WITH %$#) AND (/"T GIVING IN MODERATE YIELD OVER TWO STEPS /XIDATION OF β HYDROXY AMIDE WITH $ESS -ARTIN PERIODINANE INSTALLED THE TRICARBONYL FUNCTIONALITY ;= AND ON DESILYLATION THIS RESULTED IN SPONTANEOUS LACTOL FORMATION )T HAS BEEN DEMONSTRATED THAT NATURAL PRODUCTS OF THIS TYPE THAT FEATURE THE TRICARBONYL FUNCTIONALITY MASKED AS A SIX MEMBERED LACTOL CAN ACTUALLY EXIST AS ISOMERIC MIXTURES WITH THE SEVEN MEMBERED LACTOL ;= AND CONSEQUENTLY TO AVOID ANY COMPLICATIONS ASSOCIATED WITH THIS THE # HYDROXYL GROUP WAS PROTECTED AS THE 4-3 ETHER THROUGH A BIS PROTECTIONMONO DEPROTECTION STRATEGY &INALLY 0ARIKH $OERING OXIDA TION OF THE PRIMARY ALCOHOL GAVE THE DESIRED ALDEHYDE ELECTROPHILE 3CHEME 4HIS SENSITIVE INTERMEDIATE WAS USED WITHOUT PURIFICATION IN A .OZAKI (IYAMA +ISHI .(+ ;n= COUPLING WITH VINYL IODIDE PREPARED IN A SERIES OF SEVEN STRAIGHTFORWARD REACTIONS FROM THE MONO TERT BUTYLDI METHYLSILYL MONO 4"3 ETHER OF PENTANE DIOL 4HIS COUPLING
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
Bn
N
O S
Bn H
16
14
10
O
OBn
+
O 316
a
N
O S
16
14
O
OH
317
b
10
c,d
14
16
318
14
16
10
10
OH
OTBS
TBSO
OBn
OH
HO
OBn
319
320
Bn
HN
Bn 16
TBSO
OEt
14
10
OTBS
OH O
j,k
16
TBSO
325
l,m
6
h,i
14
10
OTBS
OH
16
TBSO
324
N
14
10
OTBS
O
e,f
O
O 322 g
O
16
TBSO
O
10
OTBS
O
323
321
1
N H 326
CO2Me 1
6 6
6
16
TBSO
OH
14
14
10
OTBS
OH O
n,o
N 1
CO2Me
1
N O
10
O
N
1
6
CO2Me p,q
O HO
N
O TMSO
CO2Me r
O
O TMSO 10
10
O
O 14
327
14
328
16
OH
14
329
16
CO2Me
O
OH 330
16
H
O
3CHEME A 4I#L $)0%! #(#L n # THEN n # → # B .A"( %T/( # C 4"3#L )M $-!0 CAT $-& # → 24 D ( 0D# %T/!C OVER TWO STEPS E #/#L $-3/ #(#L n # THEN %T. n # → 24 F .A#L/ .A(0/ METHYL BUTENE T"U/( # → 24 OVER TWO STEPS G 0IV#L %T. 4(& THEN ,I#L n # → 24 H .A(-$3 4(& THEN -E) n # I ,I"( %T/ (/ # OVER TWO STEPS J #/#L $-3/ #(#L n # THEN %T. n # → 24 K ,$! #(#/%T 4(& n # → # OVER TWO STEPS L ,I/( 4(& -E/( (/ # → 24 M , 0IP /-E %$#) (/"T $)0%! #(#L # → 24 OVER TWO STEPS N $-0 0Y #(#L 24 O (& #(#. OVER TWO STEPS P 4-3/4F LUT #(#L # → 24 Q . (#L 24 OVER TWO STEPS R 3/q0Y $-3/ %T. #(#L #
GAVE AN ALLYLIC ALCOHOL IN YIELD AS AN UNSPECIFIED MIXTURE OF DIASTEREO MERS THAT WAS DIRECTLY OXIDISED USING $-0 TO COMPLETE THE SYNTHESIS THE FIRST OF TWO KEY INTERMEDIATES 3CHEME 4HE SYNTHESIS OF THE # # FRAGMENT BEGAN WITH CHEMISTRY DEVELOPED IN THE #HAKRABORTY GROUP FOR THEIR SYNTHESIS OF THE TOP HALF OF &+ )N THIS THEY DEVELOPED A SCALABLE ROUTE TO THE CYCLOHEXYL FRAG MENT OF THE IMMUNOSUPPRESSANT THAT PROCEEDS VIA CYCLOHEXYL METHYL ESTER 3CHEME ;= 3TARTING FROM COMMERCIALLY AVAILABLE $ n QUINIC ACID TREATMENT WITH BENZALDEHYDE IN THE PRESENCE OF ACID CATALYST FORMED BOTH THE BENZYLIDENE ACETAL AND FIVE MEMBERED LACTONE IN ONE STEP !FTER "ARTON -C#OMBIE DEOXYGENATION ; = TO A FURTHER SERIES OF RADICAL REACTIONS FIRST OPENED THE ACETAL ."3 !)". 0H( # TO GIVE AND THEN AFFECTED DEBROMINATION N"U3N( !)". 0H( #
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
1
N OTBS
OH
a,b,c,d,e
SePh
I 22
22
331
f,g
22
I 17
17
332
333
+
O TMSO
1
h,i
O 10
N
CO2Me
O
O 10
16
CO2Me 22
O TMSO O
16
H
17
O
O 330
315
3CHEME A #/#L $-3/ #(#L n # THEN %T. n # → 24 B #() #R#L 4(& # → 24 OVER TWO STEPS C #3! #(#L -E/( # D 4S#L %T. $-!0 #(#L # → 24 E 0H3E3E0H .A"( %T/( 4(& # → 24 OVER TWO STEPS F M#0"! #(#L n # G $)0%! ##L REFLUX OVER TWO STEPS H #R#L .I#L $-3/ 24 I $-0 0Y #(#L 24
&INALLY METHANOLYSIS OF THE LACTONE ACHIEVED THE DIFFERENTIATED TRI SUBSTITUTED CYCLOHEXYL MOIETY PREVALENT IN THESE NATURAL PRODUCTS ! SERIES OF OXIDATION STATE ADJUSTMENTS AND PROTECTING GROUP MANIPULATIONS LED EVENTUALLY TO ALDEHYDE WHICH UNDERWENT (7% OLEFINATION ;= WITH KETO PHOSPONATE DERIVED FROM 3 2 NOREPHEDRINE ;= (YDROGENATION OF THE RESULTING DOUBLE BOND UNDER STANDARD CONDITIONS GAVE . ACYLATED IN YIELD OVER TWO STEPS "ORON ALDOL CONDENSATION BETWEEN THE ENOLATE OF AND CHIRAL ALDE HYDE DERIVED IN SIX STRAIGHTFORWARD STEPS FROM 2 2OCHE ESTER GAVE THE EXPECTED ANTI ALDOL ADDUCT AS A SINGLE DIASTEREOMER 0ROTECTION OF THE # HYDROXYL GROUP AS THE 4%3 ETHER FOLLOWED BY REDUCTIVE CLEAVAGE OF THE CHIRAL AUXILIARY AFFORDED WHICH WAS DEOXYGENATED VIA THE TOSYLATE USING SUPERHYDRIDE )NSTALLATION OF THE TERMINAL ALKENE WAS ACHIEVED USING 7ITTIG METHYLENATION AFTER LIBERATION OF THE PRIMARY ALCOHOL AND 0ARIKH $OERING OXIDATION AND FINAL SELECTIVE DESILYLATION USING n CAMPHORSUL FONIC ACID #3! COMPLETED THE SYNTHESIS OF THE SECOND FRAGMENT AND UNMASKED THE # /( READY TO COUPLE THIS WITH 3CHEME $ESPITE SYNTHESISING BOTH OF THE KEY FRAGMENTS IN THEIR RETROSYNTHESIS THROUGH RELIABLE AND PROVEN CHEMISTRY THE #HAKRABORTY GROUP HAVE NOT REPORTED THE CONCLUSIONS OF THEIR EFFORTS IN THE TIME SINCE 4HEIR ENDGAME STRATEGY WOULD SEEM PREDICTABLE AND EFFECTIVE HOWEVER THERE IS THE POS SIBILITY THAT BY INTRODUCING THE PIPECOLATE MOIETY AT A RELATIVELY EARLY STAGE OF THEIR SYNTHESIS THEY MAY HAVE ENCOUNTERED PROBLEMS ASSOCIATED WITH THE CONFIGURATIONALLY LABILE # STEREOCENTRE AS SEEN WHEN ATTEMPTING MACROLACTONISATION IN PREVIOUS SYNTHESES OF THE RELATED &+ VIDE SUPRA )N THEIR APPROACH SCRAMBLING OF THIS CENTRE WOULD BE PARTICULARLY
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
Ph
OH
HO
Br
O
O
a
OH OH
32
Ph
O
HO
d
32
32
28 28
O
334
32
e,f
32
335
O
28
O
O
28
HO
OH
O
BzO
BzO
O
b,c
OMe
O
O
O
336
28
337
338
Ph (EtO)2(O)P
g,h
O
N
O
Ph
TBSO 32
N
TBSO
O
H
O
l
HO i,j,k
32
OH
TBSO
28
H 341
32
OH
TBSO
28
O
343 o,p,q
TBSO
32
TBSO
m,n
28
O
TBSO
342
O
340
28
339
OBn
26 22
O
TBSO
344
TBSO
HO 32
TBSO
OBn
26 22
28
OTES 345
TBSO 32
r,s
TBSO
OBn
26 22
28
32
t,u,v,w
TBSO
OTES 346
26 22
28
OH 314
3CHEME A 0H#(/ P4S/( 0H-E B +( #3 -E) 4(& C N"U3N( !)". 0H-E D ."3 !)". 0H( # E N"U3N( !)". 0H( # F +#/ -E/( # G 4"3/4F LUT #(#L # H $)"!, ( #(#L n # → # H OVER TWO STEPS I !C#L COL LIDINE #(#L n # J 4"3/4F LUT #(#L # K +#/ -E/( # → 24 OVER THREE STEPS L #/#L $-3/ #(#L n # THEN %T. n # → 24 M ,I#L $)0%! #(#. 24 N ( 0D# %T/!C 24 OVER TWO STEPS O "U"/4F %T. #(#L n # THEN n # → 24 P 4%3/4F LUT #(#L # Q ,I%T"( %T/ # OVER THREE STEPS R 4S#L %T. $-!0 #(#L # → 24 S ,I%T"( 4(& n TO # OVER TWO STEPS T ( 0D# %T/!C 24 U 3/q0Y %T. $-3/ #(#L # V 0H0#() .A.( %T/ # W #3! -E/( #(#L # OVER FOUR STEPS
PROBLEMATIC TO DETECT SPECTROSCOPICALLY DUE TO THE PRESENCE OF . CENTRED ROTAMERS IN A NUMBER OF STEPS -ORE LIKELY RING CLOSING METATHESIS MAYBE PROVING A STUMBLING BLOCK WITH EITHER SELECTIVE FORMATION OF THE # # OLEFIN GEOMETRY PROBLEMATIC OR PERHAPS DIRECT DIFFICULTY IN FORMING THE POTENTIALLY STRAINED MACROCYCLE ITSELF ;=
4HE .ICOLAOU TOTAL SYNTHESIS OF RAPAMYCIN !LTHOUGH ORIGINALLY ISOLATED IN THE MID S BY 6ÎZINA AND CO WORKERS ; = RAPAMYCIN DID NOT ATTRACT SERIOUS ATTENTION FROM THE SYNTHETIC ORGANIC COMMUNITY UNTIL APPROXIMATELY YEARS LATER FOLLOWING THE DISCLO SURE OF &+ BY THE &UJISAWA PHARMACEUTICAL COMPANY ; = 4HE OBVI
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBDPSO
HO
TBDPSO
42
MeO
MeO
38
38
H
H
O HO
9
10
O
O
Ph
Me
MeO
38
H
N
35
+
MeO
CHO
OPMB 26
O
32
32
N H1 O O
28
42
C21-C42 iodide 347
42
O
N H H
OH O
MeO
OMe
O O
OTIPS
O TES
MeO
+
16
(-)-rapamycin 7
HO HO
8
22
1
+ TBSO
N CO2H Boc 12
I
+ O Bu3Sn TIPS 10 SnBu3 O OMe + 348
O
351
OH 22
O
350
OTES 26
34
PMBO
I
352
22
10
16
29
OMe
I HO
O
C8-C18 acid 349 353
OPMB + O
+
8
OMe 16
13
I
354
SiMe3
355
3CHEME 2ETROSYNTHETIC ANALYSIS
OUS STRUCTURAL HOMOLOGY BETWEEN THESE TWO MOLECULES COMBINED WITH THE IMPORTANCE OF THEIR IMMUNOMODULATING EFFECTS INITIATED MASSIVE RESEARCH EFFORTS ACROSS A BROAD SPECTRUM OF SCIENTIFIC DISCIPLINES INCLUDING NUMEROUS TOTAL SYNTHESIS EFFORTS .ICOLAOUS APPROACH SUCCESSFULLY COMPLETED IN ; = WAS THE FIRST TO DELIVER SYNTHETIC MATERIAL AND PAVED THE WAY FOR ANALOGUE PREPARATION AS WELL AS IMPROVED UNDERSTANDING OF THE DRUGS MODE OF ACTION THROUGH DELINEATION OF FUNDAMENTAL CELL SIGNALLING PATHWAYS )N A BOLD END GAME STRATEGY .ICOLAOU INTENDED TO CONSTRUCT A FULLY DEPROTECTED ACYCLIC PRECURSOR THROUGH AMIDE BOND FORMATION BETWEEN THE # # VINYL IODIDE ;= AND THE # # TRICARBONYL PRECURSOR WHICH WOULD THEN LACK ONLY THE # # OLEFINIC UNIT 4HIS LAST TWO CARBON BRIDGE WAS ENVISIONED TO BE INTRODUCED VIA A FINAL MACROCYCLISATION USING DISTANNYLETHENE IN A DOUBLE @STITCHING 3TILLE REACTION 3CHEME ;n= !LTHOUGH THERE HAD BEEN PREVIOUS REPORTS OF 3TILLE MACROCYCLISATIONS IN THE LITERATURE ; = .ICOLAOUS INTER INTRAMOLECULAR COUPLING APPROACH WAS UNPRECEDENTED AND IF SUCCESSFUL WOULD REPRESENT A NEW METHOD FOR THE CONSTRUCTION OF POLYENE MACROLIDES &URTHERMORE THE MILD REACTION CONDITIONS AND BROAD FUNCTIONAL GROUP TOLERANCE OF THE 3TILLE REAC TION WAS ANTICIPATED TO ALLOW DIRECT PRODUCTION OF RAPAMYCIN AND AVOID PROTECTING GROUP MANIPULATIONS AND OXIDATION STATE ADJUSTMENTS NEAR THE END OF THE SYNTHESIS &INALLY THE LATE STAGE INTRODUCTION OF THE TRIENE IN THIS FASHION IS STRATEGICALLY SOUND AND WOULD MINIMISE ANY POTENTIAL PROBLEMS WITH THIS SENSITIVE PORTION OF THE MOLECULE 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
"OTH MAJOR COMPONENTS AND OF THE EVENTUAL MACROCYCLE PRECURSOR WERE SUFFICIENTLY COMPLEX TO REQUIRE FURTHER RETROSYNTHETIC ANAL YSIS 2ECOGNISING THAT THE STEREOCHEMISTRY AT # AND # OF IS ULTI MATELY INCONSEQUENTIAL AS THESE POSITIONS WILL LATER BEAR TRIGONAL CARBONYL GROUPS DISCONNECTION ACROSS THIS BOND THROUGH A RACEMIC ALDOL REACTION OF METHYL GLYCOLATE AND A SUITABLE # # ELECTROPHILE SHOULD CON VENIENTLY INTRODUCE THE OXYGEN FUNCTIONALITY OF THE TRICARBONYL PORTION OF THE MOLECULE ! 2OCHE ESTER DERIVATIVE WAS THE PLANNED SOURCE OF THE # METHYL STEREOCENTRE AS THE METALLATED PRIMARY IODIDE OUGHT TO ATTACK THE LESS HINDERED TERMINAL CARBON ATOM OF EPOXIDE FORMING THE # # CARBON CARBON BOND .OTEWORTHY IN THE DESIGN OF THIS LATTER FRAGMENT IS THE TRISUBSTITUTED VINYL SILANE WHICH EVENTU ALLY WILL SERVE TO INTRODUCE THE REQUISITE VINYL IODIDE AT # VIDE INFRA 4HIS FUNCTIONALITY MAY BE CONVERTED WITH RETENTION OF OLEFIN GEOMETRY UPON TREATMENT WITH VARIOUS SOURCES OF ELECTROPHILIC IODIDE ; = AND THUS THIS APPROACH AVOIDS CARRYING A SENSITIVE VINYL IODIDE THROUGH A LONG SYNTHETIC SEQUENCE &OR THE LARGE # # VINYL IODIDE FRAGMENT THREE DISCONNEC TIONS LEAD TO THE FOUR FRAGMENTS AND n AS POTENTIAL BUILDING BLOCKS 4HE INDICATED DISASSEMBLY AT # # THROUGH AN ASYMMETRIC %VANS ALDOL CONDENSATION ; = IS ESPECIALLY INTERESTING AS THE CHIRAL OXAZOLIDINONE OF IS TO FUNCTION AS BOTH A CONTROL ELEMENT IN SETTING THE ABSOLUTE STEREOCHEMISTRY AT # AND # AS WELL AS AN EVENTUAL PRECUR SOR TO THE METHYL SUBSTITUTION AT # 3UBSEQUENT DISCONNECTION BETWEEN # AND # THROUGH A .(+ COUPLING ;n= HOPEFULLY OCCURRING WITH HIGH DIASTEREOSELECTIVITY WAS PLANNED AS THE SECOND MAJOR FRAGMENT COU PLING REACTION LEAVING AND AS SIMPLIFIED TARGETS 4HE PREPARATION OF THE VARIOUS FRAGMENTS AND MAJOR COUPLING REACTIONS COMPRISING THE FIRST SYNTHESIS OF RAPAMYCIN ARE DISCUSSED BELOW 3YNTHESIS OF THE # # DIHYDROXY ACID COMMENCED WITH THE CONSTRUCTION OF A SUITABLY FUNCTIONALISED DERIVATIVE OF , ASCORBIC ACID THAT COMPRISES # # OF RAPAMYCIN !LTHOUGH THE SEQUENCE TO PRE PARE 7EINREB AMIDE IS SOMEWHAT LENGTHY THE STARTING MATERIAL IS INEXPENSIVE AND EMPLOYS A COMBINATION OF WELL ESTABLISHED LITERATURE PRO TOCOLS TO AFFORD IN SEVEN STEPS AND OVERALL YIELD ;n= #ON DENSATION OF WITH THE LITHIO ANION OF VINYL IODIDE AVAILABLE FROM COMMERCIAL TRIMETHYLSILYLPROPYNE VIA A REGIO AND STEREOSELECTIVE
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
OH
OH
O
O
OH H
HO
16
a,b,c
MeO
16
f,g
O
O
N
16
O
O
O
13
357
358
359 h,i
Me3Si
OH
MeO2C
O 13
O
13
356
16
O
O
OH
MeO
d,e
O
13
j
Me3Si
18
p,q,r
I
18
360
301
363 10
I
Me3Si
16
10
OMe OH
OPMB
OPMB Me3Si 354 s
m,n,o
O
I Me3Si H
bond 60o + Me rotation R
10
OMe OTIPS
OPMB
O
I
v,w
I
k,l
16
O
O
O
13
O
361
+ Me
H
R
Me3Si
O
X 16
10
OMe OTIPS
O
H
8
353 x
OH
OMe
OMe
HO HO
8
O TIPS 10 O OMe 16
366
Me3Si
362
HO 16
16
OMe
355
365
I
Me3Si
13
364
t,u
13
16
OMe
367
368
y
I
HO HO
8
10
O
O TIPS OMe 16
I
C8-C18 acid 349
3CHEME A DIMETHOXYPROPANE (#L G ACETONE 24 B #A#/ (/ (/ # → # THEN CHARCOAL 0D# STEAM BATH C .A(#/ -E) $-! 24 D O PHENYL CARBONOCHLORI DOTHIOATE 0Y #(#L # → 24 E N"U3N( !)". 0H#( # F .A/( ACETONE (/ G -E/ -E.(q(#L $## %T. #(#L 24 H N"U3N( -OALLYL "R#/ #(#. CAT 4(& 24 I ) #(#L 24 OVER TWO STEPS J T"U,I %T/ n # THEN n # K ,I!L( ,I) %T/ n # L .A( -E) $-& 24 M #3! -E/( 24 N #&3/#L %T. #(#L # O +#/ -E/( 24 OVER TWO STEPS P 0-" 4#! #3! #(#L 24 Q ,I!L( %T/ 24 R ) )M 0H0 0H( 24 S T"U,I %T/ n # THEN THIENYL#U#. ,I n # → # THEN n # → # T 4)03/4F LUT #(#L # U .)3 4(& 24 V $$1 #(#L (/ 24 W #/#L $-3/ #(#L n # THEN %T. n # → # X ,$! 4(& n # → 24 THEN (-0! n # Y ,I/( 4(& -E/( (/ #
MOLYBDENUM CATALYSED CIS HYDROSTANNYLATION FOLLOWED BY CHEMOSELECTIVE REACTION WITH IODINE AFFORDED ENONE 3CHEME /F PARAMOUNT IMPORTANCE WAS THE SUBSEQUENT STEREOSELECTIVE REDUCTION OF THE KETONE AT # OF 'ENERALLY THE DIASTEREOSELECTIVE REDUCTION OF β HYDROXY KETONES TO EITHER THE SYN OR ANTI DIOL IS A WELL ESTABLISHED AND VALUABLE SYNTHETIC TRANSFORMATION ;n= (OWEVER OF THE VARIETY OF CONDITIONS WHICH EXIST MOST ARE BY AND LARGE APPLICABLE ONLY TO UNPRO TECTED ALCOHOLS &ORTUNATELY REDUCTION OF WITH LITHIUM ALUMINUM HYDRIDE AT LOW TEMPERATURE IN THE PRESENCE OF LITHIUM IODIDE AS A COORDI NATING ,EWIS ACID WAS FOUND TO DELIVER A SINGLE ALLYLIC ALCOHOL DIASTEREOMER
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
;n= -ETHYLATION OF THE NEWLY FORMED SECONDARY ALCOHOL AT # UNDER STANDARD CONDITIONS FOLLOWED BY A CUSTOMARY THREE STEP SEQUENCE TO CON VERT ISOPROPYLIDENE KETAL TO THE CORRESPONDING OXIRANE CREATED THE ELECTROPHILIC CENTRE # NECESSARY FOR CHAIN EXTENSION &OR THIS PURPOSE A READILY AVAILABLE DERIVATIVE OF THE 2OCHE ESTER ;= WAS LITHIATED BY TREATMENT WITH T"U,I AND FOLLOWING FORMATION OF THE MIXED HIGHER ORDER CUPRATE ;= SMOOTHLY FORMED THE DESIRED # # BOND OF RAPAMYCIN UPON REACTION WITH !T THIS POINT THE VINYL IODIDE FUNCTION MASKED AS THE CORRESPONDING SILANE WAS REVEALED BY TREATMENT OF 4)03 PROTECTED WITH .)3 TO AFFORD IN EXCELLENT YIELD AND WITH RETENTION OF THE OLEFIN GEOMETRY 4HE WELL KNOWN ABILITY OF SILICON TO STABILISE A β DISPOSED POSITIVE CHARGE THE β EFFECT ;= THROUGH OVERLAP OF A CARBON SILICON σ BOND WITH A VACANT OR DEFICIENT P ORBITAL ON THE ADJACENT CENTRE HYPERCONJUGATION IS CRITICAL FOR THIS REACTION OUTCOME &OR MAXIMAL STABILISATION OF THE RESULT ING POSITIVE CHARGE A BOND ROTATION OCCURS TO PLACE THE CARBON SILICON σ BOND AND THE EMPTY P ORBITAL IN THE SAME PLANE !LTHOUGH ROTATION IN THE OTHER SENSE IE THROUGH AN ANGLE OF WOULD ALSO ACHIEVE THE OPTIMAL ORIENTATION THIS WOULD REQUIRE PASSING THROUGH A PERPENDICULAR GEOMETRY WHEREIN ALL BENEFICIAL INTERACTIONS ARE LOST AND THUS DOES NOT OCCUR #ON SEQUENTLY CLEAVAGE OF THE CARBON SILICON BOND TO REGENERATE THE π SYSTEM OCCURS FROM A DEFINED CONFIGURATION AND RETENTION OF OLEFIN GEOMETRY IS OBSERVED SEE 3CHEME 0ROTECTING GROUP MANIPULATION AND 3WERN OXIDATION THEN PROVIDED ALDEHYDE READY FOR INTRODUCTION OF THE REMAIN ING CARBON FRAMEWORK OF THE # # CARBOXYLIC ACID &OR THIS PURPOSE AN INTERMOLECULAR ALDOL REACTION BETWEEN THE DIANION OF METHYL GLYCOLATE SERVED TO INTRODUCE THE NECESSARY OXYGENATION AT # AND # WITH THE RESULTING DIASTEREOMERIC MIXTURE OF NO CONSEQUENCE DUE TO LATER OXIDATION AT THESE CENTRES &INALLY HYDROLYSIS OF THE METHYL ESTER OF UNDER BASIC CONDITIONS COMPLETED THE SYNTHESIS OF THE # # FRAGMENT WITH A LONGEST LINEAR SEQUENCE OF STEPS AND AN OVERALL YIELD 4HE FIRST KEY COMPONENT OF THE # # VINYL IODIDE IS ALDEHYDE DERIVED ULTIMATELY FROM THE CHIRAL POOL #OMMERCIALLY AVAILABLE DI / BENZYLIDENE $ MANNITOL WAS RECOGNISED AS AN INEXPENSIVE AND CONVENIENT SOURCE OF α METHOXY ALDEHYDE AND FOLLOWING PROTECT ING GROUP MANIPULATIONS LEAD TETRAACETATE CLEAVAGE OF VICINAL DIOL EFFICIENTLY GENERATED TWO MOLAR EQUIVALENTS OF THIS INITIAL BUILDING BLOCK 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
Ph OH O
28
a,b,c
28
26
O
O
OH OTBDPS
MeO
O
26
28
TBDPSO
OH
26
O d
28
H
26
OH OMe
OH OTBDPS
22
O Ph
22
OH
N O
O
O
OMe
O
22
374 25
372
25
N
376
O
i
373
Ph
375
k,l,m
CHO
Ph
OPMB q
OPMB 22
p
28
OH
26
O 22
O 28
n,o
OH OTBDPS 22
28 26
26
OMe
OMe OMe
22
351
26
Ph
O
N
O
e,f,g,h 25
26
28
j
O Li
28
22
371
369
MeO
OTBDPS OMe
370
Ph
28
26
379
378
377
3CHEME A .A( -E) $-& 24 B 0D# ( G %T/( %T/!C 24 C 4"$03#L )M $-& 24 D 0B/!C .A#/ #(#L # → 24 E M#0"! #(#L 24 F (#L/ (/ 4(& 24 G .A)/ (/ 4(& 24 H #R/ (3/ AQ ACETONE OVER FOUR STEPS I #( ##/ #L %T. %T/ n # → # THEN n # → # J N"U"/4F %T. #(#L n # → # THEN n # → n # THEN (/ AQ K ,I"( (/ %T/ # → 24 L 4S#L %T. $-!0 #(#L # M ,I%T"( 4(& # → 24 N 4"!& 4(& 24 O P ANISALDEHYDE DIMETHYLACETAL #3! #(#L 24 P $)"!, ( #(#L n # → 24 Q #/#L $-3/ #(#L n # THEN %T. n # → #
4HE REMAINING CARBON ATOMS OF THE # # ELECTROPHILE REQUIRED CON STRUCTION OF . ACYLOXYAZOLIDINONE WHOSE PREPARATION BEGINS FROM β CITRONELLENE 3ELECTIVE OXIDATION ;= OF THE MORE ELECTRON RICH TRI SUBSTITUTED OLEFIN BY TREATMENT WITH M#0"! FOLLOWED BY ACIDIC HYDROLYSIS OF THE RESULTING EPOXIDE FURNISHED A DIOL WHICH WAS SUBSEQUENTLY CON VERTED TO CARBOXYLIC ACID THROUGH SODIUM PERIODATE INDUCED OXIDATIVE CLEAVAGE AND *ONES OXIDATION OF THE INTERMEDIATE ALDEHYDE !N APPEALING ASPECT OF THIS FOUR STEP SEQUENCE IS THE ABSENCE OF ANY CHROMATOGRAPHIC PURIFICATIONS n IDEAL IN THE EARLY STAGE OF A SYNTHETIC PROCESS AS THEY MUST BE PERFORMED ON A SUBSTANTIAL SCALE )NTRODUCTION OF THE OXAZOLIDINONE ;= DERIVED FROM 3 2 NOREPHEDRINE VIA A MIXED ANHYDRIDE COMPLETED THE PREPARATION OF 3CHEME ! RECURRING STRATEGIC OPERATION WITHIN THE .ICOLAOU APPROACH TO RAPA MYCIN IS THE USE OF AN OXAZOLIDINONE AUXILIARY BOTH TO CONTROL FACIAL
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
SELECTIVITY AND TO SERVE EVENTUALLY AS A SURROGATE TO A METHYL SUBSTITUENT 4HIS IS APTLY DEMONSTRATED IN THE SYNTHESIS OF THE # # ALDEHYDE WHEREIN FRAGMENT COUPLING BETWEEN THE BORON ENOLATE OF AND SETS BOTH THE # AND # STEREOCHEMISTRY 4HE CHIRAL AUXILIARY IS SUBSEQUENTLY CONVERTED THROUGH A SHORT SEQUENCE CULMINATING WITH THE REDUCTION OF A PRIMARY TOSYLATE WITH 3UPER (YDRIDE4- TO THE REQUISITE # METHYL GROUP !LTHOUGH REQUIRING THREE SYNTHETIC OPERATIONS TO ACCOMPLISH THESE ADDI TIONAL STEPS WERE OFFSET BY THE EXCEPTIONAL SUBSTRATE CONTROL OBSERVED TYPICAL OF AN %VANS ALDOL CONDENSATION $ESILYLATION OF FOLLOWED BY SUBSEQUENT BENZYLIDENE ACETAL FORMATION VIA ADDITION OF PARA ANISALDE HYDE DIMETHYL ACETAL UNDER ACIDIC CONDITIONS GENERATED ! REDUCTIVE CLEAVAGE WAS THEN REGIOSELECTIVELY ACCOMPLISHED BY TREATMENT OF WITH $)"!, ( TO RELEASE THE # PRIMARY ALCOHOL REPRESENTING A USEFUL METHOD TO SELECTIVELY PROTECT THE MORE HINDERED ALCOHOL OF A DIOL &INALLY 3WERN OXIDATION COMPLETED THE SYNTHESIS OF IN A LONGEST LINEAR SEQUENCE OF STEPS AND AN OVERALL YIELD OF 4HE SYNTHESIS OF THE SECOND MAJOR ELEMENT OF THE # # VINYL IODIDE BEGAN WITH THE GENERATION OF ACHIRAL ALDEHYDE FROM PROPANE DIOL ;= 4HE TWO CONTIGUOUS STEREOCENTRES AT # AND # OF WERE INTRODUCED SIMULTANEOUSLY VIA THE "ROWN ASYMMETRIC CROT YLATION PROTOCOL WITH EXCELLENT SELECTIVITY ; = ! SERIES OF STANDARD FUNCTIONAL GROUP MANIPULATIONS INCLUDING METHYLATION OF AN INTERMEDI ATE LITHIUM ACETYLIDE DURING #OREY &UCHS HOMOLOGATION ;= PROVIDED AN INTERNAL ALKYNE IN PREPARATION FOR TRANSFORMATION TO THE CRITICAL TRANS VINYL IODIDE 4HIS WAS READILY ACHIEVED VIA HYDROZIRCONATION OF THE TRIPLE BOND WITH #P:R( #L FOLLOWED BY TRAPPING WITH IODINE 7HILE TERMINAL ALKYNES ARE ALWAYS HYDROZIRCONATED TO THE TERMINAL ALKENYLZIRCO NIUM PRODUCT AN INTERNAL ALKYNE CAN AT TIMES GIVE REGIOISOMERIC MIXTURES )N GENERAL ZIRCONIUM ADDS PREFERENTIALLY TO THE CARBON ATOM BEARING THE STERICALLY SMALLER SUBSTITUENT (OWEVER TO ACHIEVE GOOD SELECTIVITY A SLIGHT EXCESS OF #P:R( #L AND PROLONGED REACTIONS TIMES IS CRITICAL TO ALLOW EQUILIBRATION OF THE INITIAL KINETIC MIXTURE VIA A PROPOSED DOUBLY HYDRO ZIRCONATED INTERMEDIATE ;= 'RATIFYINGLY IN THE CASE IN QUESTION ONLY THE DESIRED ISOMER WAS OBSERVED COMPLETING THE SYNTHESIS OF THIS FRAG MENT IN EIGHT OVERALL STEPS AND YIELD 3CHEME 4HE SYNTHESIS OF THE REMAINING COMPONENT OF BEARING THE TRISUB STITUTED CYCLOHEXANE MOIETY REQUIRED EFFICIENT ACCESS TO OPTICALLY ACTIVE
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
32
O
a,b
34
OH OH
H
OH OTBS
c
34
30
H
OTBS
32
380
381
PMB OTBS O
O
d,e
34
34 30
382
383
f
RL
Zr(Cp)2Cl
RS
H
RL
H
RS
Zr(Cp)2Cl
+
TBSO
34
h
E-elimination
PMBO 352
29
I
Cp2Zr(H)Cl
Cp2Zr(H)Cl
RL RS
H
H
Zr(Cp)2Cl
TBSO
g
34 29
PMBO
385
PMB OTBS O
Br
29
34
Br 384
Zr(Cp)2Cl
3CHEME A 4"3#L %T. #(#L 24 B 0## .A/!C #(#L 24 OVER TWO STEPS C TRANS BUTENE N"U,I +/T"U )PC"/-E THEN "&q/%T THEN n # → 24 THEN . .A/( AQ (/ 24 EE D .A(-$3 0-""R 4(& $-& # E / 0Y -E/( #(#L n # THEN -E3 n # → 24 F #"R 0H0 :N DUST #(#L # G N"U,I 4(& n # → n # THEN -E) n # → # H #P:R( #L 24 THEN ) #
2 CYCLOHEXEN OL &OR THIS PURPOSE THE POWERFUL #OREY "AKSHI 3HIBATA #"3 REDUCTION OF PROCHIRAL KETONES DEVELOPED BY #OREY AND CO WORKERS WAS SELECTED ; = 4HIS METHODOLOGY HAS A NUMBER OF ADVAN TAGES WIDE SCOPE READY AVAILABILITY OF EITHER ENANTIOMERIC FORM OF THE CATALYST PREDICTABLE STEREOCHEMICAL OUTCOME HIGH YIELDS AND EXPERIMENTAL SIMPLICITY AND EASY AND EFFICIENT RECOVERY OF THE AMINO ALCOHOL CATALYST PRECURSOR (OWEVER THE #"3 REDUCTION REQUIRES SUBSTAN TIAL STERIC BIAS IN THE SUBSTRATE AND THUS THE USE OF A TEMPORARY BROMINE ATOM IN IS A CLEVER METHOD TO ACHIEVE HIGH SELECTIVITY !PPLICATION OF STANDARD CONDITIONS FOR ASYMMETRIC REDUCTION OF BROMOCYCLOHEXENONE ; = GAVE THE ENANTIOMERICALLY ENRICHED ALLYLIC ALCOHOL IN NEAR QUANTITATIVE YIELD AND WITH HIGH ENANTIOCONTROL EE 0RACTICAL ACCESS TO THE CRITICAL ENANTIOENRICHED BUILDING BLOCK 2 CYCLOHEXEN OL WAS THEN ESTABLISHED FOLLOWING REDUCTIVE CLEAVAGE OF THE # "R BOND IN WITH LITHIUM METAL 4HE FREE SECONDARY ALCOHOL PRESENT IN WAS PRODUCTIVELY USED TO SUBSEQUENTLY DIRECT OXIDATION OF THE RESULTANT ADJA CENT OLEFIN THROUGH TREATMENT WITH M#0"! IN A PROCESS TERMED THE (EN BEST EPOXIDATION ;= !FTER BENZYLATION STEREOSPECIFIC RING OPENING OF WITH METHANOL UNDER ACIDIC CATALYSIS OCCURRED REGIOSELECTIVELY AT THE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
Ph
O
N B
O Br
a
42
38
Ph
O
Ph
O
388 b
42
OH
Me
Br
42
O
B
38
386
B
N
OBn c,d,e
42
42
Ph
O
38 38
H
387
38
391
390
Br
389 f,g
NMe2 TBSO
42
O
O
MeO
42
MeO MeO
TBSO
j,k
42
TBSO
OBn 42
TBSO
h,i
42
MeO
38
MeO
38
MeO
38
38
38
396
397
l
42
NMe2
38
TBSO
O
O
OH m
TBSO
n,o
392
393
394
395
Ph
Me
(EtO)2(O)P N
TBSO MeO
42
38
OH
p,q
TBSO
MeO
42
38
CHO
O 342 r
O
TBSO
TBDPSO
42
O 38
H
N
35
O 398
224
Ph
Me
MeO
399
O
s,t,u
42
Ph
Me
MeO
38
H
N
35
O
O
O
O
350
3CHEME A 0H3E"R 0Y #(#L # B "(q$-3 4(& n # EE C ,I 4(& T"U/( REFLUX D M#0"! #(#L 24 E .A( "N"R 4"!) 4(& # OVER TWO STEPS F #3! -E/( 24 G 4"3/4F LUT #(#L # H ( 0D# %T/( 24 I #/#L $-3/ #(#L n # THEN %T. n # → n # J ,$! 4(& n # THEN 4-3#L n # → 24 K 0D/!C #(#. # OVER TWO STEPS L ,I"( #E#Lq(/ 4(& -E/( n # M . . DIMETHYLACETAMIDE DIMETHYL ACETAL XYLENES REFLUX N ,I%T"( 4(& # O ( 0D# %T/( 24 OVER TWO STEPS P O ./#(3E#. N"U0 4(& 24 THEN (/ 4(& 24 Q / -E/( #(#L n # THEN -E3 n # → 24 R ,I#L I0R.%T #(#. 24 S %T3I( NEAT 0H0 2H#L # T (& AQ #(#. 24 U 4"$03#L )M $-& 24 OVER THREE STEPS
MORE STERICALLY ACCESSIBLE POSITION OF THE OXIRANE TO FURNISH FOLLOWING SILYLATION 3CHEME 4HE RECOGNITION THAT TRANSFER OF STEREOCHEMISTRY FROM # TO # VIA A ; = SIGMATROPIC REARRANGEMENT COULD INTRODUCE THE REQUISITE CAR BON SIDE CHAIN OF THE CYCLOHEXANE SECTOR OF RAPAMYCIN DICTATED THE NEXT CHEMICAL TRANSFORMATIONS &OLLOWING THE DEBENZYLATION AND 3WERN OXIDATION OF INTRODUCTION OF THE NECESSARY UNSATURATION WAS ACCOM PLISHED VIA IMPLEMENTATION OF THE 3AEGUSA PROTOCOL ;= WITH THE KINETIC TRIMETHYLSILYL 4-3 SILYL ENOL ETHER OF 7ITH ENONE IN HAND AXIAL DELIVERY OF HYDRIDE IN A FASHION UNDER ,UCHE CONDITIONS ; = ESTABLISHED WITH EXCELLENT DIASTEREOSELECTIVITY ALLYLIC ALCOHOL IN THE
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
DESIRED STEREOCHEMICAL ARRANGEMENT FOR THE PLANNED %SCHENMOSER #LAISEN REARRANGEMENT ;= )N THE EVENT THE INTERMEDIATE KETENE . / ACETAL FORMED FROM . . DIMETHYLACETAMIDE DIMETHYL ACETAL AND IN REFLUXING XYLENES STEREOSPECIFICALLY REARRANGED TO THE CORRESPONDING γ δ UNSATURATED AMIDE IN HIGH YIELD 2EDUCTION TO THE PRIMARY ALCOHOL WITH 3UPER (YDRIDE4- FOLLOWED BY HYDROGENATION OF THE # # ALKENE AFFORDED 2EMOVAL OF THE EXTRANEOUS CARBON ATOM ATTACHED AT # WAS ACHIEVED THROUGH OZONOLYSIS OF THE ALKENE RESULTING FROM OXIDATIVE SYN ELIMINATION OF THE 'RIECO ORTHO NITROPHENYLSELENYLATION PRODUCT OF ;= 4HE RESULTING ALDEHYDE WAS THEN EMPLOYED AS THE ELECTROPHILIC PARTNER IN A (7% REACTION ;= WITH READILY AVAILABLE AND ENANTIOMERI CALLY PURE PHOSPHONATE ;= UNDER MILD -ASAMUNE 2OUSH CONDI TIONS ;= THAT OCCURRED WITHOUT RACEMISATION ANDOR OTHER DESTRUCTIVE REACTION PROCESSES &INALLY REDUCTION OF THE DOUBLE BOND IN WITH TRI ETHYLSILANE UNDER RHODIUM CATALYSIS AND PROTECTING GROUP MANIPULATIONS TO INSTALL THE MORE ROBUST TERT BUTYLDIMETHYLSILYL 4"$03 GROUP COMPLETED THE SYNTHESIS OF THE LAST MAJOR FRAGMENT OF THE # # VINYL IODIDE IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD 3CHEME 7ITH VIABLE ROUTES AVAILABLE TO ALL MAJOR FRAGMENTS OF THE RAPAMYCIN SKELETON ATTENTION WAS THEN FOCUSED ON THEIR EFFICIENT UNION REQUIR ING FIRST CONSTRUCTION OF THE FULLY ELABORATED VINYL IODIDE #ATALYSED BY TRACE AMOUNTS OF NICKEL THE BROAD FUNCTIONAL GROUP TOLERANCE IN BOTH PARTNERS AND MILD REACTION CONDITIONS ARE SOME OF THE CHARACTERISTICS WHICH MAKE THE VENERABLE .(+ ; = REACTION IDEAL FOR APPLICATION IN COMPLEX MOLECULE SYNTHESIS 4HUS TREATMENT OF A MIXTURE OF AND IN DIMETHYLSULFOXIDE $-3/ WITH #R#L AND TRACE AMOUNTS OF .I#L SMOOTHLY EFFECTED THEIR UNION FORMING THE DESIRED # # CARBON BOND IN HIGH YIELD &ORTUNATELY THE POOR OBSERVED DIASTEREOSELECTIVITY ALBEIT IN FAVOUR OF THE DESIRED # EPIMER WAS MITIGATED BY SEPARATION VIA CHROMATOGRAPHY AND RECYCLING THROUGH AN OXIDATIONREDUCTION SEQUENCE OF THE UNDESIRED STEREOISOMER )NTERESTINGLY FOR THE STEREOSELECTIVE REDUCTION OF ENONE $)"!, ( WAS EMPLOYED RATIO OF TO OBSERVED RATHER THEN :N"( WHICH IS KNOWN TO WORK WELL FOR ANTI REDUCTIONS ; = OF α METHOXY KETONES SEE FOR EXAMPLE THE 3CHREIBER AND ,EY SYNTHESES OF RAPAMYCIN VIDE INFRA )NSTALLATION OF A BULKY TRIISO PROPYLSILYL 4)03 PROTECTING GROUP ON THE # HYDROXYL GROUP OF
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
OPMB
O
OPMB
22
34
29
TBSO
26
28
OMe b
TBSO
O
OPMB
28
26
+ H
29
PMBO
I
22 29
TBSO
26
28
OMe
OMe
351
TBDPSO
+
26
28
OH OPMB
34
29
TBSO
OMe
352
MeO
22
34
a
OPMB
OH OPMB
OPMB 22
34
c
402
400
401
d,e,f
42
38
H O
TBDPSO
42
32
OH O N O Me PMB O MeO Ph
Me
g
OTIPS
MeO
38
N
35
OPMB
Ph
O
TIPS OPMB O
OPMB
22
+
H
O
H
29
OMe
O
O
350
404
26
28
34
26
403
22
h,i,j
TBDPSO
MeO
TBDPSO
42
38
1
H 32
OH O PMB
OTIPS MeO
OPMB
N CO2H Boc 12 k
MeO
TBDPSO
42
38
MeO
H
38
H
32
N H O Boc
O
32
OTIPS
O PMB
OPMB
MeO
26
405
42
l,m,n,o,p
N H H
O O
OTIPS
O TES
26
406
OTES
MeO
26
347
I 22
22
22
3CHEME A #R#L .I#L $-3/ 24 B $-0 #(#L 24 C $)"!, ( 4(& n # D 4)03/4F LUT #(#L # E (&q0Y 4(& 24 F #/#L $-3/ #(#L n # THEN %T. n # → n # G N"U"/4F %T. #(#L n # → # THEN n # → n # THEN (/ AQ H ,I"( (/ %T/ # → 24 I 4S#L %T. $-!0 #(#L 24 J ,I%T"( 4(& # → 24 K $## I0R.%T PYRROLIDINOPYRIDINE #(#L n # L /S/ .-/ ACETONE (/ 24 M 0B/!C .A#/ 0H( # → 24 OVER TWO STEPS N #() #R#L 4(& DIOXANE 24 %: O $$1 #(#L (/ 24 4%3/4F LUT #(#L # THEN 3I/ #(#L 24
FOLLOWED BY SELECTIVE CLEAVAGE OF THE MORE LABILE 4"3 GROUP AT # AND SUBSEQUENT 3WERN OXIDATION GENERATED SUITABLY ADORNED FOR COUPLING WITH 3CHEME %MPLOYING THE SAME STRATEGY AS DISCUSSED PREVIOUSLY SEE 3CHEME ADDITION OF THE BORON ENOLATE OF TO ELECTROPHILE FORMED WITH EXCELLENT DIASTEREOSELECTIVITY THE LAST CARBON LINKAGE OF THE # # VINYL IODIDE !FTER CONVERSION OF THE %VANS AUXILIARY IN TO A CORRE SPONDING # METHYL SUBSTITUENT DICYCLOHEXYLCARBODIIMIDE $## COU
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
PLING OF WITH . T "OC , PIPECOLIC ACID AT LOW TEMPERATURE TO AVOID POTENTIAL EPIMERISATION OF THE # STEREOCENTRE GAVE 4HE LESS HINDERED TERMINAL OLEFIN OF WAS THEN SELECTIVELY DIHYDROXYLATED VIA THE 5PJOHN PROCEDURE ;= AND THE INTERMEDIATE DIOL CLEAVED TO THE CORRESPONDING ALDEHYDE WITH LEAD TETRAACETATE 3UBJECTION OF THIS ELECTROPHILE TO 4AKAIS #R#L MEDIATED IODOOLEFINATION PROCESS ;n= GAVE THE DESIRED TRANS VINYL IODIDE IN BOTH EXCELLENT SELECTIVITY %: AND YIELD &INALLY DICHLORODICYANOQUINONE $$1 INDUCED PARA METHOXYBENZYL 0-" ETHER DEPROTECTION AT BOTH # AND # HYDROXYL BEARING CARBON ATOMS FOLLOWED BY FORMATION OF TRIETHYLSILYL ETHERS AT THESE SAME TWO CENTRES WITH CONCOMITANT LOSS OF THE TERT BUTOXYCARBONYL "OC GROUP ON NITROGEN WITH TREATMENT WITH TRIETHYLSILYL TRIFLUOROMETHANESULFONATE 4%3/4F FURNISHED /VERALL COMPLETION OF THE FULLY FUNCTIONALISED # # FRAGMENT OF RAPAMYCIN REQUIRED STEPS IN A LONGEST LINEAR SEQUENCE AND WAS ACCOMPLISHED IN AN EFFICIENT YIELD 3CHEME 7ITH IN HAND THE STAGE WAS NOW SET FOR THE FINAL STAGES OF THE SYN THESIS AND @STITCHING CYCLISATION LEADING TO THE COMPLETION OF .ICOLAOUS RAPAMYCIN EFFORT 5NION OF THE # # VINYL IODIDE AND # # CARBOXYLIC ACID WAS ACCOMPLISHED VIA THE ACTIVE ESTER FORMED BY TREAT MENT OF WITH $## AND (/"4 FOLLOWED BY ADDITION OF THE AMINE TO AFFORD AMIDE IN GOOD YIELD 3CHEME "ISOXIDATION AT # AND # OF THE ISOMERIC MIXTURE THROUGH THE 3WERN PROTOCOL INSTALLED THE CHARACTERISTIC TRICARBONYL REGION OF RAPAMY CIN 4HIS WAS FOLLOWED BY SELECTIVE DEPROTECTION OF THE 4%3 ETHERS AT # AND # AND YET A SECOND APPLICATION OF THE SAME OXIDATIVE CONDITIONS PRODUCED TETRAKETONE !T THIS STAGE REMOVAL OF THE MORE ROBUST REMAINING SILYL PROTECTING GROUPS AT # # AND # RESULTED IN SPONTANEOUS HEMIKETAL FORMATION AS A MIXTURE OF LACTOL EPIMERS IN GOOD OVERALL YIELD FOR FOUR SYNTHETIC OPERATIONS $ELIGHTFULLY WHEN THE RESULTING FULLY DEPROTECTED MACROCYCLIC PRECURSOR WAS COMBINED WITH ENEDISTANNANE ;= AND SUBJECTED TO 3TILLE CROSS COUPLING CON DITIONS FORMATION OF THE DESIRED MACROCYCLE WAS ACHIEVED COMPLETING THE LANDMARK FIRST TOTAL SYNTHESIS OF RAPAMYCIN 7HILST THIS NOVEL LAST SYNTHETIC OPERATION WAS NOT EXCEPTIONALLY HIGH YIELDING BOTH UNREACTED STARTING MATERIAL AND A MONO CROSS COUPLED VINYLIODIDEVINYL STANNANE INTERMEDIATE WERE RECOVERED AND BOTH COULD BE READILY RECYCLED 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TBDPSO
TBDPSO
42
TBDPSO
42
42
C21-C42 iodide 347 MeO
38
MeO
H
38
MeO
H
N H H
HO HO
8
O + TIPS 10 OMe O
O
O
O TES
a
OTIPS MeO
HO HO
OTES 26
O
N
H O O TIPS 10 OMe O 8
I
I
16
H 32
32
32
OH
38
O TES
OTIPS
N
OTES
MeO
26
I
I
16
O O TES H O O 8 MeO O O TIPS 10 OMe O
b
OH 26
I
I
16
22
22
22
408
407
C8-C18 acid 349
OTIPS
c,d
HO
HO
42
MeO
38
H 32
O HO
9
10
N H1 O O O
O
nBu3Sn
O
OH O
MeO
OMe 16
TBDPSO
42
MeO
38
MeO
H
H
32
SnnBu3
348 f
42
38
N 8
O HO
O
H
O 10
O
32
O
O
OH
MeO
O 26
OMe
22
I
16
I
e
O H O O 8 O O TIPS 10 OMe O
N
410
OTIPS
MeO
I
16
22
(-)-rapamycin 7
O
O 26
I 22
409
3CHEME A $## (/"4 #(#L # THEN # B #/#L $-3/ #(#L n # THEN %T. n # → # C (&q0Y 4(& # → 24 D #/#L $-3/ #(#L n # THEN %T. n # → # E (& AQ #(#. 24 YIELD OVER FOUR STEPS F AND 0D#(#. #L MOL I0R.%T $-&4(& - 24
)N SUMMARY THE IMPRESSIVE SYNTHETIC EFFORT BY THE .ICOLAOU GROUP IN COMPLETING THE TOTAL SYNTHESIS OF RAPAMYCIN STANDS AS A MONUMENTAL ACHIEVEMENT IN THE FIELD OF NATURAL PRODUCT SYNTHESIS )N GENERAL ALTHOUGH THE APPROACH WAS RELATIVELY CONSERVATIVE THE EFFICIENCY IN WHICH THE FRAG MENTS WERE CONSTRUCTED AND COUPLED IN A HIGHLY CONVERGENT MANOR IS MANIFEST IN THE OBSERVED OVERALL YIELD OF THROUGH A LONGEST LINEAR SEQUENCE OF STEPS 3CHEME )N FACT DESPITE BEING THE FIRST SUCCESSFUL SYNTHETIC ENTRY TO THIS NATURAL PRODUCT AND THE EMERGENCE OF FOUR SUBSE QUENT SUCCESSFUL SYNTHESES OF RAPAMYCIN THE OVERALL YIELD REMAINS THE HIGHEST OBTAINED TO DATE -OREOVER THE CORNERSTONE DOUBLE INTERINTRA MOLECULAR 3TILLE MACROCYCLISATION ON A FULLY DEPROTECTED AND HIGHLY FUNC TIONALISED PRECURSOR DEVELOPED FOR AND SUCCESSFULLY IMPLEMENTED IN STITCHING TOGETHER THE FORMIDABLE RAPAMYCIN MACROCYCLE IS INTRIGUING AND FINDING USE IN OTHER CONTEXTS ;n=
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3CHEME 3UMMARY OF .ICOLAOUS TOTAL SYNTHESIS OF RAPAMYCIN
4HE 3CHREIBER TOTAL SYNTHESES OF RAPAMYCIN )N CONTINUATION OF AN ALREADY IMPRESSIVE LIST OF CONTRIBUTIONS DIRECTED TOWARDS IMPROVED SYNTHETIC AND BIOLOGICAL UNDERSTANDING OF THE TRICAR BONYL FAMILY OF IMMUNOMODULATING NATURAL PRODUCTS 3TUART 3CHREIBER DISCLOSED HIS TOTAL SYNTHESIS OF RAPAMYCIN IN ;= WITHIN MONTHS OF THE FIRST PUBLISHED REPORT BY + # .ICOLAOU 2ETROSYNTHETICALLY IT IS CLEAR THAT EXPERIENCE GAINED IN HIS RECENTLY COMPLETED SYNTHESIS OF &+ WAS INSTRUMENTAL IN MANY OF THE STRA TEGIC DECISIONS IN THE PROPOSED SYNTHETIC APPROACH )N PARTICULAR THE FIRST MAJOR SIMPLIFICATION IDENTIFIED THE . # AMIDE BOND AS SUITABLE FOR FORMATION OF THE MEMBERED MACROCYCLE IN RAPAMYCIN VIA A MAC ROLACTAMISATION PROTOCOL )NTERESTINGLY OF ALL THE RAPAMYCIN SYNTHESES ONLY 3CHREIBER CHOSE TO PURSUE A MORE CONVENTIONAL MEANS OF MACROLIDE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO HO
42
C22-C42 aldehyde 411
42
MeO MeO
38
38
H
O HO
9
10
O
O
42
MeO
H
38
H
O
OH O
MeO
OMe
OLi
N Boc H O +
O
O Alloc
ODEIPS
N
28
35
O O 9 412 + MeO OMe TBS 10 OMe O
22
16
(-)-rapamycin 7
414
26
O
8
26
OTBS
415
PhS
+
1
CHO N Boc 416
22
22
OMe O
OAlloc
MeO
OPMB
O
O
+
32
32
N H1 O O
TIPSO
29
Br 417
H
16
21
Ph3P=CHCO2Et O 21 20 P Ph Ph
OH O 10
PMBO
16
418 C10-C21 phospine oxide 413
N
O
+
Br
OTBS
17 19
419
3CHEME 2ETROSYNTHETIC ANALYSIS
FORMATION &URTHER DISCONNECTION IN THE TRICARBONYL # # AND TRIENE # # REGIONS REVEAL MAJOR FRAGMENTS ;= AND ;= AS PRINCIPLE SYNTHETIC TARGETS 4HE PLANNED USE OF PHOSPHINE OXIDE TO EFFECT THE UNION OF THESE FRAGMENTS AND REPRESENTS THE FIRST AND ONLY 7ITTIG TYPE APPROACH TOWARDS THE MERGER OF MAJOR SUBUNITS IN THE RAPAMYCIN SERIES !N INTRIGUING ASPECT OF THE PLANNED SYNTHESIS OF THE # # ALDE HYDE INVOLVES THE USE OF A PIPECOLATE UNIT CONTAINING A # ALDE HYDE )TS ADDITION TO THE MAIN CARBON BACKBONE OF WAS ENVISIONED TO OCCUR UNDER %VANS 4ISHCHENKO ;n= REDUCTIVE CONDITIONS THAT WOULD SIMULTANEOUSLY ALSO ACCOMPLISH THE STEREOSELECTIVE ANTI REDUCTION OF THE # KETO FUNCTION &URTHER DISSECTION OF IDENTIFIED n AND AS ADVANCED TARGETS /F PARTICULAR IMPORT BOTH DISCONNECTION AT # # AND # # VIA SEQUENTIAL CARBANIONIC COUPLINGS WITH FORMAL DIANION EQUIVALENT OR VARIANTS THEREOF AND THE USE OF A STEREODEFINED EPOXIDE TO DEFINE THE # HYDROXY STEREOCENTRE PRIOR TO # # BOND FORMATION HAVE SUBSEQUENTLY BECOME POPULAR STRATEGIES ADOPTED IN A NUMBER OF OTHER SYNTHESES OF RAPAMYCIN VIDE INFRA 3CHREIBERS SYNTHESIS BEGINS WITH A COMMERCIALLY AVAILABLE 2OCHE ESTER DERIVATIVE PREPARED THROUGH THE SHORT SYNTHETIC SEQUENCE SHOWN IN 3CHEME USING STANDARD LITERATURE PROTOCOLS 7ITH ELECTROPHILE IN HAND ALKYLATION OF THE DIANION OF ACETOAC ETATE OCCURRED REGIOSELECTIVELY AND IN HIGH YIELD AT THE γ POSITION ;= 3UBSEQUENT CONVERSION OF TO ITS 7EINREB AMIDE ANALOGUE
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O O MeO
a,b,c,d,e OH
10
O 422
Br
PMBO
f
420
O
OMe
10
O 16
421
OH O
g,h
10
PMBO
OMe
10
PMBO
16
423
N
O
418
17
i,j,k,l
19
Br
OTBS
17 19
m
OH 424
MeO
O
H
OMe OTBS
10
16
OH OH
p,q
OH O
10
OTBS
HO
19
419
16
427
n,o
10
OTBS
PMBO
19
16
19
425
426
r,s,t
S S
TBSO
OMe OH
10
16
19
u,v,w MeO
OMe TBSO
OMe
10
16
x,y,z,aa CO2Et 21
MeO OMe TBS 10 O OMe 16 21
428
429
O P Ph Ph
413
3CHEME A $(0 P4S/( %T/ # → 24 B ,I!L( %T/ C .A( 0-""R 4(& 24 D P4S/( -E/( 24 OVER FOUR STEPS E ."3 0H0 #(#L # → 24 F .A( N"U,I (-0! 4(& # THEN # G 2U#L;3 BINAP=%T. MOL ( PSI -E/( # H -E.(/-Eq(#L -E!L I ,I!L( $-% J "R ##L n # K ,$! (-0! 4(& n # OVER TWO STEPS L 4"3#L )M 4(& M T"U,I 4(& n # THEN PREMIXED WITH N"U,I 4(& n # n # N $$1 P( BUFFER #(#L O %T"/-E 4(& -E/( n # THEN .A"( n # THEN !C/( P 2U#L00H 0H( AIR 24 Q .A( -E) 4(& # → 24 OVER TWO STEPS R (3#( 3( 4I#L n # → # S 4"3/4F LUT T (&q0Y 0Y 4(& 24 U 0H)/#/#& -E/( V "A-N/ CELITE #(#L W 0H0# #(#/%T #(#L 24 OVER TWO STEPS %: X $)"!, ( Y HEXACHLOROACETONE 0H0 DI T BUTYLPYRIDINE n # Z ,I00H 4(& n # AA AIR 4(& 24 OVER THREE STEPS
WAS READILY ACCOMPLISHED AS WAS CONSTRUCTION OF THE STEREODEFINED VINYL BROMIDE 'IVEN THE WELL KNOWN INCREASED STABILITY OF TETRAHEDRAL INTERMEDIATES OF 7EINREB AMIDES ;= LITHIATION OF THE LATTER SPECIES AND ADDITION TO SMOOTHLY FORMED ENONE WITHOUT ANY COMPLICA TIONS OF OVER ADDITION 4HIS σ BOND FORMING APPROACH TO THE GENERATION OF GEOMETRICALLY DEFINED TRISUBSTITUTED OLEFINS IS A VERY USEFUL ONE AS IT CIRCUMVENTS THE SOMETIMES PROBLEMATIC %: SELECTIVITY ISSUES ASSOCIATED WITH DIRECT π BOND INSTALLATION SEE 3MITHS SYNTHESIS VIDE INFRA !LSO OF INTEREST IS THE FREE HYDROXYL GROUP PRESENT IN WHICH TO AVOID UNPRO DUCTIVE CONSUMPTION OF LITHIATED MUST BE DEPROTONATED PRIOR TO REACTION !LTHOUGH ON A SMALL SCALE THIS PROTOCOL WORKED WELL WITH MATE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
RIAL IN EXCESS OF ONE GRAM SIGNIFICANT AMOUNTS OF A # BUTYL ADDUCT WERE OBSERVED .EVERTHELESS WITH AVAILABLE TREATMENT WITH $$1 FOLLOWED BY BORON MEDIATED AND SYN STEREOSELECTIVE REDUCTION UNDER %VANS 0RASAD ; = CONDITIONS FURNISHED AS A SINGLE DIASTEREOMER 4O DIF FERENTIATE THE THREE RESULTING HYDROXYL GROUPS THE # ALCOHOL WAS USED TO TRANSIENTLY PROTECT THE # ALCOHOL VIA OXIDATION ;= AND INTRAMOLECULAR HEMIACETAL FORMATION ALLOWING SELECTIVE FORMATION OF THE METHYL ETHER AT # 2ING OPENING OF THE LACTOL PROVED ESPECIALLY DIFFICULT (OWEVER EVENTUAL SUCCESS WAS ACHIEVED VIA FORMATION OF THE DITHIOLANE UNDER ,EWIS ACIDIC CONDITIONS 3TANDARD FUNCTIONAL GROUP MANIPULATIONS INCLUDING ALLYLIC OXIDATION AND HOMOLOGATION GAVE THE DIENE ESTER WITH GOOD %: SELECTIVITY &INALLY INTRODUCTION OF THE PHOSPHINE OXIDE VIA DISPLACEMENT OF AN INTERMEDIATE ALLYLIC CHLORIDE WITH ,I00H AND EXPOSURE TO AIR COMPLETED THE SYNTHESIS OF THE # # FRAGMENT IN STEPS AND OVERALL YIELD !SSEMBLY OF THE # # ALDEHYDE BEGAN BY EXPLOITING SOME PREVIOUSLY DEVELOPED CHEMISTRY FOR THE PREPARATION OF &+ WHEREIN PENTADIEN OL WAS MANIPULATED TO PRIMARY ALCOHOL #ON VERSION TO THE CORRESPONDING IODIDE AND SUBSEQUENT ALKYLATION WITH THE LITHIATED ALLYLIC SULFIDE ;= GENERATED AN α SUBSTITUTED ALLYLIC THIO ETHER WITH EXCELLENT REGIOSELECTIVITY /XIDATION ; = SIGMATROPIC REARRANGEMENT ; = OF THE RESULTING SULFOXIDE AND IN SITU CLEAVAGE OF THE SULFENATE ESTER %T.( -E/( THEN FURNISHED EXCLUSIVELY AS THE TRANS ISOMER 3UBJECTING THIS ALLYLIC ALCOHOL TO 3HARPLESS ASYMMETRIC EPOXIDATION CONDITIONS AFFORDED EPOXY ALCOHOL WHICH WHEN TREATED WITH TRIMETHYLALUMINIUM UNDERWENT REGIOSELECTIVE RING OPENING WITH INVERSION OF CONFIGURATION AT # ;= 3UBSEQUENT CONVERSION OF VICINAL DIOL TO THE CORRESPONDING OXIRANE EMPLOYED A CUSTOMARY TWO STEP SEQUENCE TO COMPLETE THE SYNTHESIS OF THE CYCLOHEXYL BEARING # # EPOXIDE IN A TOTAL OF STEPS AND YIELD 3CHEME 3TANDARD CHEMISTRY WAS USED TO PREPARE THE CENTRAL FORMAL DIANION EQUIVALENT FROM COMMERCIALLY AVAILABLE MATERIALS !LL STEPS LEADING TO WERE REASONABLY EFFICIENT AND ALLOWED PROCESSING OF SUBSTANTIAL QUANTITIES OF MATERIAL 5NFORTUNATELY THE HYDROZIRCONATION BROMINATION SEQUENCE WHICH CULMINATED THE SYNTHESIS OF OCCURRED IN POOR YIELD 3CHEME "OTH A REGIOISOMERIC PRODUCT OF AND AN OVER REDUCED BY PRODUCT WERE NOTED BUT FORTUITOUSLY THE INSEPARABLE REGIOISO
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TIPSO
13 steps 14.0%
OH
MeO
cf. Schemes 10 and 12
42
61
TIPSO
38
OH
42
MeO
H
S
42
38
H
MeO
36
33
MeO
33
38
42
O 38
OH
H
36
H
434
433
d
431 N
TIPSO
TIPSO
42
MeO
38
H
O
MeO
OH OH
437
38
TIPSO
Al
42
33
35
TIPSO
N
TIPSO
N
O
414
c
N
432
f,g 38
S
33
430
33
MeO
42
36
S
TIPSO
N
N
42
a,b
40
O
MeO
33
H
42
e
O AlMe3
AlMe2
436
38
33
OH
H 435
3CHEME A ) 0H0 )M B ,$! 4(& n # → n # THEN IODIDE DERIVED FROM 4(& n # C M#0"! #(#L # → 24 THEN %T.( -E/( D 4I/I0R , $%4 T"U//( ¯ -3 E -E!L HEXANES # F 4S#L 0Y G +#/ -E/( OVER TWO STEPS
O
OH 29
MeO
OTBS a,b,c
Br
32
29
OTBS
32
d
SPh
32
e,f
29
29
g
PhS 32
Br 363
29
Br 438
439
440
417
3CHEME A 4"3#L )M $-& # → 24 B $)"!, ( #(#L n # C #"R 0H0 :N #(#L # → 24 OVER TWO STEPS D N"U,I 4(& n # THEN -E) n # → 24 E 4&! (/ 4(& F 0H3 "U0 0H( 24 OVER TWO STEPS G #P:R( #L 0H#( 24 → # THEN "R n #
MERIC PRODUCT WAS UNREACTIVE AFTER LITHIATION IN THE SUBSEQUENT COUPLING STEP SEE 3CHEME 4HE FINAL SUB FRAGMENT REQUIRED FOR THE PREPARATION OF WAS 7EINREB AMIDE SYNTHESISED AS SHOWN IN 3CHEME /NCE AGAIN A 2OCHE ESTER WAS USED AS A CONVENIENT STARTING MATERIAL AND INITIAL SOURCE OF CHIRALITY !FTER CONVERSION TO 3 BENZYLOXY METHYLPROPANAL HOMOLOGA TION GAVE ENOATE WITH EXCELLENT %: SELECTIVITY 3TANDARD FUNCTIONAL GROUP MANIPULATIONS THEN GENERATED PRIMARY ALCOHOL WHICH WAS HYDROGENATED EMPLOYING THE CATIONIC RHODIUM) SYSTEM OF %VANS ;= TO GENERATE IN HIGH DE )N THE FORMER TRANSFORMATION SUBSTANTIAL ! ALLYLIC STRAIN HAS BEEN POSTULATED TO RESTRICT THE HYDROXYL MOIETY TO THE VICINITY OF ONE OLEFINIC FACE GIVING RISE TO THE OBSERVED FACIAL SELECTIVITY ;= 3WERN OXIDATION OF AND CONDENSATION OF THE RESULTING ALDEHYDE WITH THE BORON ENOLATE OF 3 METHOXYACETYL PHENYLMETHYL OXA
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
Rh
Ph2P O
O
OH
OBn
a,b
26
MeO
26
H
EtO
441
363
TBSO
OBn
O c
TBSO
OH
d,e,f
26
22
BF4 PPh2
22
26
444
443
442
O
O 28
22
PMBO 26
O 28
N
O
k
TBSO 22
OMe 415
TBSO
OH O 26
28
N
O
j
22
O
OH O 26
28
N
OMe Bn
OMe 448
26
g
MeO
TBSO
OH
257 22
447
N
Bn
O
h O TBSO
O
446
i
22
26
H
445
3CHEME A "N4#! 4F/( CYCLOHEXANE #(#L B $)"!, ( #(#L n # C 0H0 #-E #/%T #(#L 24 OVER TWO STEPS D $)"!, ( #(#L n # → # E 4"3#L %T. $-!0 #(#L 24 F .A .( n # OVER TWO STEPS G WT ( PSI #(#L DE H #/#L $-3/ #(#L n # THEN %T. n # → 24 I "U"/4F I0R.%T 0H#( n # THEN n # → # OVER TWO STEPS J -E.(/-Eq(#L !L-E #(#L # → 24 K 0-"4#! 4F/(
ZOLIDINONE ; = GAVE THE DESIRED SYN %VANS ALDOL ADDUCT IN A MODEST YIELD OF ! MAJOR ADVANTAGE OF THIS TRANSFORMATION IS THE DIRECT INTRODUCTION OF THE # HYDROXYL GROUP AS ITS REQUISITE METHYL ETHER AND THUS AVOIDS ANY POTENTIAL COMPLICATIONS WITH PROTECTING GROUP MANIPULATIONS AT # 5SEFUL REMOVAL OF THE CHIRAL AUXILIARY IN VIA DIRECT TRANSAMIDATION ;= TO 7EINREB AMIDE AND 0-" PROTECTION OF THE REMAINING FREE SECONDARY HYDROXYL GROUP FURNISHED IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD 7ITH ALL THREE OF THE MAJOR COMPONENTS OF IN HAND FOCUS WAS SHIFTED TO EFFECTING THE KEY CARBON CARBON BOND FRAGMENT COUPLING REAC TIONS TO CONSTRUCT THE # # ALDEHYDE 4O THIS END VINYL BROMIDE WAS LITHIATED AND INTRODUCED TO GIVE ENONE IN GOOD OVERALL YIELD ALTHOUGH MOLAR EQUIVALENTS OF THE NUCLEOPHILE WHERE REQUIRED 4O SET THE REQUIRED STEREOCHEMISTRY AT # A ANTI CHELATION CONTROLLED ZINC BOROHYDRIDE REDUCTION ; = OF WAS SELECTED AND PLEAS INGLY FURNISHED WITH SUBSTANTIALLY IMPROVED SELECTIVITY THAN OBSERVED IN THE .ICOLAOU SYNTHESIS 3CHEME &URTHER PROTECTING GROUP MANIPULATIONS AND SULFIDE OXIDATION GENER ATED THE FULLY ELABORATED # # SULFOXIDE THAT UPON α DEPROT
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBSO
PhS
+
32
PMBO
O
26
28
22
29
TBSO N
O
a
29
417
b
32
OMe
415
MeO
32
OMe
Me O
449
451 ZnX O
R 450
TIPSO
TIPSO
29 26
H
R
SPh
OH
PMBO
22
26
OMe
Br
TBSO
SPh
O
PMBO
22
c
42
42
MeO 38
H
38
H
35
35 32
OH SPh O O 454 MeO
ODEIPS
TBSO
O
415
ODEIPS
PMBO
29
22
SO2Ph
d
TBSO
32
29
26
SPh 32
26
OMe 453
OPMB
ODEIPS
PMBO
22
26
e
OMe 452
22
TBSO
f or g,h,i TIPSO MeO
TIPSO
42
38
H
35
CHO N 1 Boc
32
OH O
416 j
ODEIPS
MeO
455
R' O H
42
38
H
35
34
H
32
32
N
R
O
OPMB
O Sm
26
TBSO
MeO
H
N H Boc O
Boc
457
L 456
22
OH
O
ODEIPS
MeO
TBSO
OPMB 26
22
k
TIPSO MeO
42
38
H
TIPSO MeO
35
32
N Boc
O
H
O
O Alloc
ODEIPS
OAlloc
MeO
26
TIPSO
42
38
H
MeO 35 32
m,n N H Boc O
O O Alloc 459
411
O
ODEIPS
MeO
OAlloc 26
42
38
H
35 32
l N H Boc O
O
458
OH
ODEIPS
MeO
OH 26
22
TBSO
22
TBSO
22
H
3CHEME A T"U,I 4(& n # THEN n # B :N"( %T/ n # DR C $%)03/4F LUT #(#L # → 24 D M#0"! 0Y n # → 24 E N"U,I n # THEN THEN "&q/%T DR SEPARABLE BY (0,# MAJOR ISOMER ISOLATED IN YIELD F N"U,I n # THEN 4-3/ RECOVERED 3- G N"U,I 4(& n # THEN #() I0R-G#L n # AFTER RECYCLE OF RECOVERED SULFONE H /S/ 0Y THEN .A(3/ I .A)/ 3I/ 4RIS (#L P( BUFFER OVER TWO STEPS J 4(& # THEN 0H#(/ 3M) 3M) MOL DR K $$1 .A(#/ L !LLOC#L 0Y LUT 4(& OVER TWO STEPS M 0043 P4S/( 4(& (/ RECOVERED STARTING MATERIAL N #/#L $-3/ #(#L n # THEN %T. n # → 24
ONATION WITH N"U,I SMOOTHLY EFFECTED RING OPENING OF THE ELECTROPHILIC EPOXIDE IN THE PRESENCE OF "&q/%T ; = 3OME DIFFICULTY WAS ENCOUNTERED IN ATTEMPTING THE OXIDATIVE DESULFONYLATION ;= OF THE DIASTEREOMERIC MIXTURE OF β HYDROXY SULFONES )NITIAL SUCCESS WAS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
ACHIEVED AFTER SCREENING A MYRIAD OF CONDITIONS AND INVOLVED METALLA TION WITH N"U,I FOLLOWED BY TREATMENT WITH BISTRIMETHYLSILYL PEROXIDE 5NFORTUNATELY ONLY A ISOLATED YIELD WAS ACHIEVED EVEN AFTER ONE RECYCLE OF RECOVERED STARTING MATERIAL -OREOVER THIS TRANSFORMATION WAS ONLY SUCCESSFUL ON THE MAJOR DIASTEREOMER AND NECESSITATED SEPARATION BY (0,# 3OME IMPROVEMENT WAS REALISED BY REACTION OF THE α LITHIO CARB ANION OF AND A CARBENOID ELECTROPHILE #() I0R-G#L TO PRODUCE AN INTERMEDIATE OLEFIN THAT WAS SUBSEQUENTLY DIHYDROXYLATED AND OXIDATIVELY CLEAVED !LTHOUGH REQUIRING THREE SYNTHETIC MANIPULATIONS THE OVERALL YIELD IS AN IMPROVEMENT FROM THE EARLIER PROCEDURE AND THE SUCCESS IS FURTHER NOTABLE GIVEN THAT A SIMILAR INTERMEDIATE IN THE ,EY SYNTHESIS OF RAPAMYCIN COULD NOT BE ADVANCED VIDE INFRA 7ITH β HYDROXY KETONE IN HAND STEPS WERE TAKEN TO INTRODUCE THE PIPECOLIC SUBUNIT AND SIMULTANEOUSLY PROTECT AGAINST ITS ELIMINATION THROUGH REDUCTION OF THE # KETONE &OR THIS PURPOSE THE 3CHREIBER GROUP EMPLOYED A VERY INTER ESTING FRAGMENT UNION%VANS 4ISHCHENKO ;= REDUCTION STRATEGY WITH 3 "OC PIPECOLAL ;= )N THE EVENT MIXING AND FOLLOWED BY THE ADDITION OF PREFORMED 0H#(/ 3M) 3M) PROVIDED THE COUPLED PRODUCT IN BOTH EXCEPTIONAL YIELD AND SELECTIVITY WITH NO DETECTABLE RACEMISATION AT # 0ROTECTING GROUP MANIPULATIONS AND OXIDATION COM PLETED THE SYNTHESIS OF THE SECOND MAJOR TARGET IN THE 3CHREIBER SYNTHESIS OF RAPAMYCIN IN A TOTAL OF STEPS AND OVERALL YIELD 3CHEME 4HE STAGE WAS NOW SET FOR THE LAST MAJOR FRAGMENT COUPLING REACTION VIA AN INTERMOLECULAR 7ITTIG REACTION BETWEEN PHOSPHINE OXIDE AND ALDEHYDE 'RATIFYINGLY REACTION OF THE LITHIUM SALT OF THE FORMER WITH ELECTROPHILE GENERATED THE CRUCIAL OLEFINIC LINKAGE IN YIELD AS A MIXTURE OF %: ISOMERS 3CHEME )N ADDITION SUBSTANTIAL AMOUNTS OF β HYDROXY PHOSPHINE OXIDE ADDUCTS WERE ISOLATED WHICH COULD BE SALVAGED BY TREATMENT WITH LITHIUM HEXAMETHYLDISILAZIDE ,I(-$3 TO EFFECT CONVERSION TO THE TRIENE IN GOOD YIELD THUS FURTHER IMPROV ING MATERIAL THROUGHPUT OF THIS ADVANCED INTERMEDIATE (YDROLYSIS OF THE ACETAL AT # WITH PYRIDINIUM PARA TOLUENESULFONATE THEN FURNISHED ALBEIT IN MODERATE YIELD PERHAPS AS A CONSEQUENCE OF OTHER ACID LABILE FUNCTIONALITIES IN THE MOLECULE 3CHEME )NSTALLATION OF THE TRICARBONYL REGION AND EVENTUAL MACROCYCLE FORMA TION OF RAPAMYCIN REQUIRED PRIOR CHAIN EXTENSION AT # WHICH WAS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TIPSO
TIPSO
42
MeO
38
42
MeO
H
38
35
N H O Boc
ODEIPS
O Alloc
+
OAlloc
MeO
16
26
21
411
a,b
O P Ph Ph
O
ODEIPS
O Alloc
N H1 Boc O O TBS 10 O OMe
413
O
H 32
MeO OMe TBS 10 O OMe
32
O
OAlloc
MeO 22
22 16
H 460
O O
c
O 461
TIPSO
TIPSO
42
MeO
38
H
TIPSO
42
MeO
AllocO 10
O Alloc
N H1 O O TBS O OMe
H
38
32
32
32
O
42
MeO
H
38
ODEIPS
e
N H1 H O
Alloc OH O
OAlloc
MeO
O
22
O
O Alloc
ODEIPS
10
Alloc OEE O
OAlloc
MeO
TBS O OMe
d
O
22
N H1 Boc O
ODEIPS
10
OAllo c
MeO 22
16
463
464
O Alloc
TBS O OMe
16
16
O
OEE =
462
O
f
TIPSO
TIPSO
42
MeO
38
MeO
H
HO
42
38
H
32
N H HO 10
O
OH
16
ODEIPS
O
H 32
OH
MeO
O O 10
22
N H1 O O TBS O OMe
O
ODEIPS O
MeO
h
N H O HO
10 22
O
9
O
O
O
OH
1
O
O
MeO
OMe
22
16
16
465
38
32
g
1
O O TBS O OMe
42
MeO
466
(-)-rapamycin 7
3CHEME A N"U,I 4(& (-0! n # THEN %: B 0043 ACETONE # RECOVERED SM C ETHOXYETHYL ACETATE ,$! 4(& n # THEN THEN !LLOC#L LUT n # → 24 D 4%3/4F LUT #(#L # THEN 3I/ E CHLORO METHYLPYRIDINIUM IODIDE %T. #(#L - OVER THREE STEPS MIXTURE OF DIASTEREOMERS F 0D00H (#/.( 4(& G $-0 #(#L H (&q0Y 0Y 4(& OVER THREE STEPS
ACCOMPLISHED THROUGH CONDENSATION OF WITH THE LITHIUM ANION OF ETHOXYETHYL ACETATE !DDITION OF !LLOC#L AND WARMING AFFECTED AN IN SITU QUENCH OF THE LITHIUM ALKOXIDE AT # TO AFFORD THE PROTECTED CARBOXYLIC ACID DERIVATIVE 4REATMENT WITH 4%3/4F TO REMOVE THE "OC PROTECTING GROUP ; = FURNISHED THE REQUISITE AMINO ACID THAT WAS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 3UMMARY OF THE 3CHREIBER SYNTHESIS OF RAPAMYCIN
DIRECTLY SUBJECTED TO -UKAIYAMA MACROCYCLISATION CONDITIONS ;= TO EFFECT RING CLOSURE GIVING A MIXTURE OF TWO DIASTEREOMERS # OF THE DESIRED MACROCYCLE 0ALLADIUM CATALYSED DEPROTECTION OF ALL THREE ALLYL CARBONATES AT # # AND # FOLLOWED BY TREATMENT WITH $-0 USING THE 'OLEC PROTOCOL ;= EFFECTED OXIDATION OF ALL THREE RELEASED ALCO HOLS ALONG WITH GENERATING THE TRICARBONYL MOTIF VIA OXIDATION OF THE # METHYLENE TO YIELD &INALLY GLOBAL DEPROTECTION OF ALL REMAINING SILYL GROUPS WITH (&q0Y RESULTED IN SPONTANEOUS HEMIACETAL FORMATION AT # GIVING MATERIAL IDENTICAL IN ALL RESPECTS WITH NATURAL RAPAMYCIN )N SUMMARY THE 3CHREIBER APPROACH TO RAPAMYCIN FEATURES SOME INTRIGUING CHEMICAL TRANSFORMATIONS TO CONSTRUCT THIS NATURAL PRODUCT IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD 3CHEME .OTABLE AMONG THESE IS THE USE OF A # # SPACER AS A FORMAL DIANION
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
EQUIVALENT TO ADD SEQUENTIALLY TWO ELECTROPHILES IN BUILDING THE # # PORTION OF THE MOLECULE ! VARIETY OF STEREOSELECTIVE π BOND CONSTRUCTIONS THROUGH A σ BOND FORMING STRATEGY THE FORMATION OF THE TRIENE UNIT VIA A 7ITTIG OLEFINATION A FASCINATING %VANS 4ISHCHENKO FRAGMENT UNION AND A MACROLACTAMISATION APPROACH TO MACROCYCLE FORMATION REPRESENT OTHER ASPECTSSTRATEGIES OF CONSIDERABLE INTEREST AND USE )N ISOLATION THE CON TRIBUTION OF THE 3CHREIBER GROUP TO THE FAMILY OF TRICARBONYL NATURAL PROD UCTS THROUGH TOTAL SYNTHESIS &+ RAPAMYCIN AND ANALOGUES IS IMPRESSIVE AND ALTHOUGH THIS REVIEW IS CENTRED PRIMARILY UPON SYNTHETIC ASPECTS IT MUST BE HIGHLIGHTED THAT ARGUABLY NO RESEARCH GROUP HAS CON TRIBUTED MORE TO THE BIOLOGICAL UNDERSTANDING OF THIS FASCINATING FAMILY OF NATURAL PRODUCTS
4HE $ANISHEFSKY TOTAL SYNTHESES OF RAPAMYCIN 3AMUEL * $ANISHEFSKY THE INCUMBENT OF A %UGENE 7 +ETTERING #HAIR AT THE 3LOAN +ETTERING )NSTITUTE WHO ALSO MAINTAINS HIS POSITION AS A PROFESSOR OF CHEMISTRY AT #OLUMBIA 5NIVERSITY IS WORLD RENOWNED FOR THE SYNTHESIS OF LARGE COMPLEX NATURAL PRODUCTS ESPECIALLY THOSE WITH RELEVANT BIOLOGICAL ACTIVITY (IS CONTRIBUTION TO THE FIELD OF THE IMMUNOSUPPRESSANT MACRO LIDES IS NO EXCEPTION AND FOLLOWING YEARS OF SYNTHETIC ;n= AS WELL AS DEGRADATIVE STUDIES ; = THE THIRD INVENTIVE APPROACH TO THE TOTAL SYNTHESIS ;= OF RAPAMYCIN APPEARED SHORTLY AFTER THOSE OF .ICOLAOU AND 3CHREIBER !S WAS THE CASE IN THE .ICOLAOU APPROACH TO RAPAMYCIN $AN ISHEFSKY AND CO WORKERS ELECTED TO INVESTIGATE AN UNCONVENTIONAL STRAT EGY TOWARDS MACROCYCLE FORMATION .AMELY DISSECTION OF THE # # BOND IDENTIFIED AN ACYCLIC KETO ALDEHYDE AS THE KEY SUBSTRATE FOR A NOVEL MACROALDOLISATION REACTION 7HILE SYNTHESIS OF LARGE RINGS THROUGH CAR BON CARBON BOND FORMING EVENTS IS RELATIVELY RARE LESS SO RECENTLY DUE TO THE SPECTACULAR SUCCESS OF RING CLOSING METATHESIS ; = THOSE EMPLOYING SUCH REACTIVE AND ADVENTUROUS FUNCTIONAL GROUPS AS PRES ENT IN AN ALDOL TRANSFORMATION ARE VERY UNCOMMON ; = &URTHER DISCONNECTION THROUGH THE ESTER LINKAGE AT # REVEALS FRAGMENTS AND AS ADVANCED TARGETS .OTABLY THIS IS THE FIRST AND ONLY APPROACH IN THE RAPAMYCIN SERIES WITH THE AMBITIOUS GOAL OF EARLY INCORPORATION OF BOTH
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO
HO
C28-C42 aldehyde 468
OH
42
MeO
MeO
38
38
H
N H O HO
O
9
H
MeO
32
10
O
OH 1
OMe
N
O
MeO 22
O TMSO
9
10
O
OH
+
OH O TBS
28
N
OMe
Ph(O)S
26
OMe
14
(-)-rapamycin 7
CHO
O
16 19
C1-C27 acid 469
30
TBSO OTBS 471
1
H O O
OBn
32 36
38
470
O
1
O
42
+ HO
32
O
O
BnO
42
EtO
9
O 472
OtBu OPiv
I
O
26
17
O
+ OBn O
10
16
+ 19
H
SO2Ph O
474
H 20
23
475
473
3CHEME 2ETROSYNTHETIC ANALYSIS
THE SENSITIVE TRICARBONYL AND TRIENE PORTIONS OF THE MOLECULE VIA UNION OF AND 3CHEME &OR THE # # ALDEHYDE AN )RELAND #LAISEN REARRANGEMENT ;n= WAS PLANNED TO INSTALL THE KEY # # CARBON BOND STEREOSE LECTIVELY VIA CHIRALITY TRANSFER FROM # 4HIS FEATURE IS THE PRINCIPLE ADVAN TAGE IN THE APPROACH TO THIS FRAGMENT AS SIMPLIFIED CONSTITUENTS AND COULD BE INITIALLY JOINED THROUGH A ROBUST ESTERIFICATION REAC TION !LTHOUGH SIMILAR STRATEGIES FOR CONSTRUCTION THE CYCLOHEXYL PORTION OF RAPAMYCIN AND RELATED MOLECULES HAD BEEN PREVIOUSLY DISCLOSED SEE 3CHREIBER n &+ .ICOLAOU n RAPAMYCIN IT HAD NEVER BEEN ATTEMPTED ON SUCH A COMPLICATED SUBSTRATE &URTHER REARRANGEMENT PROCESS WOULD ALSO FEATURE IN THE DEVELOPMENT OF A VIABLE ROUTE TO ALLYLIC ALCOHOL WHILE THE OTHER REQUISITE PIECE WOULD UTILISE STANDARD POLYKETIDE BUILDING PROTOCOLS $ANISHEFSKYS ANALYSIS OF THE # # ACID FURTHER EMBODIES SOME INTRIGUING SIMPLIFICATIONS THE FIRST OF WHICH ENVISIONS INSTALLATION OF THE PIPECOLATE AND TRICARBONYL MOIETY VIA FORMATION OF THE # # LINKAGE THROUGH USE OF A SULFONE ADDITION OF TO AN APPROPRIATE # # ESTER &URTHER DISCONNECTION ACROSS # # THROUGH A .(+ COUPLING ;n= IS NOTABLE AS IT BRINGS INTO QUESTION THE STEREOCHEMICAL CONTROL OF THE EVENTUAL # METHOXY SUBSTITUENT &INALLY AN INTENDED *ULIA OLEFINATION ;n= TO CONSTRUCT THE # # OLEFIN IN FORMATION OF THE TRIENE PORTION OF THE MOLECULE # # REPRESENTS THE LAST OF THE MAJOR BOND DISCONNECTIONS 4HE SYNTHESIS OF THESE VARIOUS FRAGMENTS THROUGH
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
FEATURE SOME INTRIGUING TRANSFORMATIONS AND UNIQUE APPROACHES AS DISCUSSED BELOW 5NDOUBTEDLY THE OVERALL STRATEGY ADOPTED BY THE $ANISHEFSKY RESEARCH TEAM WAS BASED SUBSTANTIALLY UPON EXTENSIVE DEGRADATION WORK PERFORMED ON RAPAMYCIN ITSELF ; n= 3UCH INVESTIGATIONS ARE OF OBVI OUS VALUE WHEN REASONABLE SUPPLIES OF THE NATURAL PRODUCT ARE AVAILABLE AS THEY ALLOW INVESTIGATION OF THE CRITICAL END GAME OF SYNTHESES WHEN SYN THETIC MATERIAL IS EXTREMELY PRECIOUS -OREOVER IN SOME CASES LARGE INTACT ADVANCED FRAGMENTS CAN BE RECOVERED WHICH CAN SERVE AS VALIDATION OF SYN THETIC INTERMEDIATES AS WELL AS SUPPLEMENT MATERIAL 4HIS WAS CERTAINLY THE CASE FOR $ANISHEFSKY WITH RAPAMYCIN AS ILLUSTRATED IN 3CHEME 3ELECTIVE AND SEQUENTIAL PROTECTION OF THE # AND # HYDROXYL FUNC TIONS OF FOLLOWED BY TREATMENT WITH LEAD TETRAACETATE IN -E/( RESULTED IN OXIDATIVE CLEAVAGE AT # # TO GIVE IN EXCELLENT YIELD 0RESUM ABLY THIS LATTER TRANSFORMATION EXPLOITS THE PROPENSITY OF THE ELECTROPHILIC # CARBONYL TO FORM TETRAHEDRAL INTERMEDIATES AND IS BASED UPON STUDIES PERFORMED UPON &+ IN A RELATED CONTEXT ;= 4REATMENT OF KETONE WITH ,I!L(/T"U GIVES IN A HIGHLY SELECTIVE FASHION WHAT WAS LATER CONFIRMED TO BE THE 2 # HYDROXY COMPOUND THAT AFTER SELECTIVE DESI LYLATION AT # GAVE 0REVIOUS STUDIES HAD SHOWN THAT THE # # BOND IS VERY SUSCEPTIBLE TO A RETROALDOL PROCESS UNDER BASIC OR ,EWIS ACIDIC CONDITIONS ; = AND INDEED REACTION OF WITH ,$! AT LOW TEM PERATURE FURNISHED BOTH AND IN GOOD OVERALL YIELD 3CHEME !FTER 4"3 PROTECTION OF REDUCTIVE REMOVAL OF THE PIPECOLATE SUBUNIT AND ALLYLIC OXIDATION THE FIRST OF TWO MAJOR SUBUNITS INTEGRAL TO THE TOTAL SYNTHESIS EFFORT OF $ANISHEFSKY WAS OBTAINED ALBEIT OF OPPOSITE CON FIGURATION AT # RELATIVE TO SYNTHETIC MATERIAL ,ACTONE WAS CORRE SPONDINGLY CONVERTED IN SHORT ORDER VIA STANDARD FUNCTIONAL GROUP MANIP ULATIONS TO ALDEHYDE WHICH CORRESPONDS IDENTICALLY TO MATERIAL MADE IN THE FORWARD DIRECTION VIDE INFRA 4HE SAME LACTONE COULD ALSO BE PREPARED THROUGH A SHORTER DEGRADATIVE PROTOCOL BY OMITTING PROTECTING GROUP MANIPULATIONS (OWEVER IN THIS CASE OXIDATIVE CLEAVAGE WITH LEAD TETRAACETATE FOLLOWED BY EXPOSURE TO ,$! PRODUCED UNBENEFICIALLY ENONE FORMED VIA β ELIMINATION IN ADDITION TO DESIRED %FFICIENT DIRECT ROUTES TO BOTH AND AND THE OBSERVATION OF FACILE RETROALDOL PRO CESSES ABOUT THE LINKAGE BETWEEN # # WITHOUT QUESTION INFLUENCED IN THE SYNTHETIC APPROACH UNDERTAKEN BY $ANISHEFSKY AND CO WORKERS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
HO
TIPSO
42
MeO
H
38
TIPSO
42
MeO
38
H
O HO
9
10
N H1 O O
O
OH
a,b,c
N H1 O O O O
MeO
O
32
O
OTMS
10
22
O
d,e
O
MeO
OMe
(-)-rapamycin 7
10
22
O
38
477
f
HO
42
MeO
H
38
TIPSO
H
N H1 O
9
O
10
O
O
O
OMe
O
479
OH MeO
O O O
42
32
32
MeO
22
16
476
42
MeO
O
MeO
OMe
j
HO
OH
OH
9
16
16
O
N H1 O O O O
MeO
9
O
MeO
OMe
O
H
38
32
32
O
42
MeO
MeO
H
38
CHO
32
28
k
+
O 10
22
16
MeO 9
OMe
O
+
O
N H
14
OH
CHO
O
26
OMe
O 1
O
O
478
19
480
481
l,m
g,h,i
TIPSO O 10
O
O TBS O OMe
n,o
26
OMe
14
MeO
10
484
MeO
26
OMe
14 19
19
483
42
O
TBS O OMe
38
H
32
OH O TBS 482
CHO 28
3CHEME A 4)03/4F LUT n # B 4-3/4F LUT n # C 0B/!C -E/( # D ,I!L(/T"U 4(& 24 E 4"!& !C/( 4(& 24 F ,$! 4(& n # → n # n AND n G 4"3/4F 4(& n # H $)"!, ( I -N/ OVER TWO STEPS J 0B/!C -E/( # K ,$! 4(& n # → n # n AND n L -N/ -E/( 24 M 4"3#L )M N $)"!, ( O $-0 OVER TWO STEPS
.O YIELDS REPORTED
)N THE FORWARD DIRECTION THE SYNTHESIS OF THE # # ALDEHYDE COMMENCED FROM THE READILY AVAILABLE TWO STEPS BENYLIDENEAC ETAL OF DEOXY $ GLUCOSE ;= 3TANDARD MANIPULATIONS AFFORDED AN EXOCYCLIC ENOL ETHER WHICH UPON TREATMENT WITH AQUEOUS (G#L TRIGGERED A TYPE )) &ERRIER REARRANGEMENT ; = TO GIVE AS AN INCONSEQUENTIAL MIXTURE OF # HYDROXY EPIMERS 4HIS SEQUENCE APTLY DEMONSTRATES THE UTILITY OF THE &ERRIER REARRANGEMENT PROCESS AS AN EFFICIENT ENTRY TO SUBSTITUTED CHIRAL CYCLOHEXANONES ESPECIALLY GIVEN A VARIETY OF PRECURSOR ENOL ETHERS ARE EASILY PREPARED FROM INEXPENSIVE CARBOHYDRATES
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O
Ph
HO
Br
42
O
a,b
O 38
485
OMe
MeO
c,d
O 38
486
O
42
42
HO
OMe
BnO MeO
e
O 38
487
OMe
BnO MeO
O 42
38
488
HgX
BnO
O
MeO
OH 42
38
489
OH
f,g
BnO
MeO
42
38
470
3CHEME A .A( -E) $-& # → 24 B ."3 "A#/ ##L HEAT C .A/-E -E/( 24 D .A( "N"R $-& # → 24 E (G#L ACETONE (/ HEAT # EPIMERS F -S#L 0Y 24 G ,I"( #E#Lq(/ 4(& -E/( n #
&INALLY ACTIVATION VIA THE MESYLATE OF WITH CONCOMITANT ELIMINATION GAVE THE CORRESPONDING ENONE WHICH THEN UNDERWENT A HIGHLY SELECTIVE REDUCTION UNDER ,UCHE CONDITIONS ; = TO FURNISH ALLYLIC ALCOHOL IN NINE STEPS AND OVERALL YIELD &OR THE SYNTHESIS OF THE # # CARBOXYLIC ACID $ANISHEFSKY FIRST EMPLOYED -EYERS PROTOCOL TO GAIN ACCESS TO THE WELL KNOWN BUILDING BLOCK 2 BENZYLOXY METHYLPROPANAL ;= #URRENTLY A MORE MODERN APPROACH TO THIS SAME IMPORTANT STARTING MATERIAL IS AVAILABLE IN SUBSTANTIALLY FEWER SYNTHETIC OPERATIONS THAN THE FIVE REQUIRED AS DELIN EATED IN 3CHEME ; = &URTHER ADVANCEMENT OF RELIED ON OBSERVATIONS IN THE EARLY S BY BOTH 2EETZ ; = AND (EATHCOCK ;= WHEREIN HIGH DIASTEREOFA CIAL SELECTIVITY WAS NOTED IN REACTION OF A VARIETY OF ORGANOMETALLICS WITH α OR β ALKOXY ALDEHYDES UNDER CHELATION CONTROL !PPLICATION OF 2EETZS CONDITIONS UPON ALDEHYDE DID INDEED LEAD TO THE DESIRED ANTI ADDI TION PRODUCT IN BOTH GOOD YIELD AND SELECTIVITY 3ILYLATION OF THE ALLYLATED MATERIAL FOLLOWED BY OZONOLYSIS GAVE A SECOND ALDEHYDE WHICH WAS CROTYLATED UNDER SUBSTRATE CONTROL EMPLOYING THE CHIRAL AUXIL IARY BASED % CROTYLBORONATE OF 2OUSH ;n= TO YIELD EXCLUSIVELY THE ANTI ADDITION PRODUCT AS A MODEST MIXTURE OF DIASTEREOMERS 4HIS INSEPARABLE MIXTURE OF PRODUCTS NECESSITATED THE INTRODUCTION OF AN EXTRANEOUS STEP WHEREIN THE UNWANTED DIASTEREOMER COULD BE REMOVED AFTER DESILYLATION 3UBSEQUENT REPROTECTION THEN FURNISHED #ONVER SION TO THE DESIRED CARBOXYLIC ACID WAS READILY ACCOMPLISHED VIA REGIOSELECTIVE HYDROBORATION WITH "". AND STEPWISE OXIDATION IN OVERALL STEPS AND YIELD 3CHEME 5NION OF THE TWO MAJOR CONSTITUENTS AND OF ALDEHYDE UNDER STANDARD ESTERIFICATION CONDITIONS PRODUCED WITHOUT COMPLICA
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
HO
30
OH
a
O
30
O
b,c
O
d,e
32 30
H
32 30
f
34
O
OBn
491
O
492
OH OBn
OBn
493
32
OHC TBSO
30 34
O
490
g,h
32
32
32
494
30
OBn
495 CO2iPr O B
i
CO2iPr
O
496 O
HO
OBn
32 30
36
TBSO
471
OTBS
m,n
34
HO
36
OBn
32 30
TBSO 499
OTBS
l
34
TBSO
OBn
32 30
36
OTBS
498
j,k
34
OBn
32 30
36
OH OTBS 497
3CHEME A ETHYL VINYL ETHER P4S/( %T/ # → 24 B ,I!L( 4(& # → 24 C +/T"U "N"R 4(& # → 24 D . (#L 4(& # OVER TWO STEPS E #/#L $-3/ #(#L n # THEN %T. n # → n # F ALLYL TRIMETHYLSILANE 4I#L #(#L n # MIXTURE OF # EPIMERS G 4"3#L )M $-& 24 H / -E/( #(#L n # THEN -E3 n # → 24 I 0H#( n # THEN FILTER THROUGH CELITE WITH %T/ .A/( AQ 24 J 4"!& 4(& 24 MIXTURE OF DIASTEREOMERS RECRYSTALLISE OVER TWO STEPS K 4"3#L )M $-& 24 L "". 4(& 24 THEN . .A/( (/ # M #/#L $-3/ #(#L n # THEN %T. n # → 24 N +-N/ .A(0/ AQ T"U/( (/
TION 3UBSEQUENT EXPOSURE TO ,$! IN A MIXTURE OF 4(& AND (-0! AT LOW TEMPERATURE STEREOSELECTIVELY GENERATED THE CORRESPONDING KETENE ACETAL THAT WAS QUENCHED WITH TERT BUTYLDIMETHYLSILYLCHLORIDE 4"3#L TO AFFORD AFTER WARMING 4HERMOLYSIS OF IN REFLUXING XYLENES EFFECTED THE DESIRED TRANSFER OF STEREOCHEMICAL INFORMATION FROM # TO # UPON SIGMATROPIC REARRANGEMENT SEE .ICOLAOUS SIMILAR APPROACH TO THE CYCLO HEXYL MOIETY 3CHEME AND PRODUCED ACID FOLLOWING HYDROLYSIS OF THE INTERMEDIATE SILYL ESTER 3CHEME )NTERESTINGLY MUCH HARSHER CON DITIONS THEN USUALLY REQUIRED TO EFFECT THIS REARRANGEMENT WERE NECESSARY %XAMINATION OF THE EXPECTED TRANSITION STATE FOR THE SUPRAFACIAL PROCESS PROVIDES A POSSIBLE EXPLANATION AS ALL THREE RING SUBSTITUENTS MUST BE AXI ALLY DISPOSED IN THE CYCLOHEXENOL CHAIR CONFORMATION &OR THE PLANNED "ARTON LIKE FREE RADICAL DECARBOXYLATION ; = OF TO REMOVE THE SUPERFLUOUS CARBOXYLIC ACID AT # ATTEMPTS WERE MADE TO FORM THE REQUISITE THIOHYDROXAMIC ESTER OF ULTIMATELY WITH OUT SUCCESS (OWEVER PREPARATION OF THE . ACYLOXY PHTHALIMIDE ANA LOGUE UNDER THE MILD CONDITIONS OF /KADA AND /DA ;= FOLLOWED BY IRRADIATION IN THE PRESENCE OF . METHYLCARBAZOLE AND T"U3( DID SUC CESSFULLY DELIVER THE DESIRED DECARBOXYLATED PRODUCT 3EQUENTIAL
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O OH BnO
O 42
MeO
OBn
32
+ HO
30
36
TBSO
38
BnO
30
36
42
TBSO
OTBS
b
OBn
32
O
30
36
BnO
42
TBSO
OTBS
OTBS
MeO
470
TBSO
OBn
32
O
a
MeO
38
471
38
500
501 c,d
HO
BnO
42
BnO
42
g,h 36
MeO
38
H
OH
32
36
MeO
30
38
H
OTBS
TBSO 504
OBn
32
36
MeO
30
OTBS
O TBS 503
42
e,f
H HO2C
OTBS
BnO
42
42 36
MeO
30
O TBS 502 k
i,j
HO
OBn
32
38
38
H
32
28
36
OMe
MeO
38
30
TBSO
OTBS
O
30
O
OTBS
O 506
505
l,m,n
TIPSO MeO
OBn
32
H
42
38
HO s,t,u
H
32
OH O TBS 468
CHO 28
BnO
42 36
MeO
38
H
32
28
30
O H 508
OTBS O
O
OEt
o,p,q,r
42 36
MeO
38
H
OBn
32 30
I
O
OTBS O
H
507
3CHEME A %$#) $-!0 #(#L 24 B ,$! 4(& (-0! n # THEN 4"3#L n # → 24 C 0H#( REFLUX D ,I/( AQ 4(& 24 EPIMERIC MIXTURE AT # E . HYDROXYPHTHALIMIDE %$#) $-!0 #(#L 24 F Hν PYRAX . METHYLCARBAZOLE T"U3( I0R/( (/ OVER FIVE STEPS G 2ANEY NICKEL %T/( 24 THEN FILTER THEN %T/!C 0T/ ( ATM 24 H 0D# %T/!C ( 24 OVER TWO STEPS I 2U00H #L 0H( 24 J CARBOMETHOXYETHYLIDENE TRIPHENYLPHOSPHORANE 0H#( # K #/#L $-!0 #(#L L 4S.(.( $-% # THEN .A/!C (/ 24 M $)"!, ( 0H#( n # N ) 0H)/!C CYCLOHEXANE Hν OVER THREE STEPS MIXTURE OF EPIMERS AT # O 0H3N( !)". 0H#( REFLUX P ( 0D/( %T/!C Q HYDROXY 4%-0/ BENZOATE #A/#L .A(#/ AQ #(#L R CARBOETHOXYETHYLIDENE TRIPHENYLPHOSPHORA NE 0H#( # OVER TWO STEPS S 4)03/4F LUT #(#L # T $)"!, ( 0H#( n # U -N/ #(#L 24
HYDROGENOLYSIS OF THE BENZYL PROTECTING GROUPS AND HYDROGENATION GAVE OF WHICH THE PRIMARY ALCOHOL WAS SELECTIVELY OXIDISED ;= WITH TRISTRIPHENYPHOSPHINE RUTHENIUM)) CHLORIDE AND THE RESULTING ALDE HYDE HOMOLOGATED TO ENOATE 5NFORTUNATELY ALL ATTEMPTS AT FURTHER ELABORATION TO THE DESIRED # # ALDEHYDE WERE THWARTED BY AN INABILITY TO SELECTIVELY DIFFERENTIATE THE # AND # HYDROXYL GROUPS 3CHEME ! SOLUTION TO THIS DIFFICULTY WAS ACHIEVED FROM CAREFUL OBSERVATION OF THE ATTEMPTED FORMATION OF THE PREVIOUSLY MENTIONED THIOHYDROXAMIC
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
ESTERS 5NDER SOME CONDITIONS LACTONISATION THROUGH THE DESILYLATED # HYDROXYL WAS UNEXPECTEDLY OBSERVED AND ON FURTHER OPTIMISATION IT WAS FOUND TREATMENT OF WITH OXALYL CHLORIDE EFFECTED SMOOTH CONVERSION TO $IIMIDE REDUCTION ; = OF THE ALKENE TO AVOID LOSS OF THE BENZYL PROTECTING GROUPS REDUCTION TO THE LACTOL AND 3UÇREZ OXIDATIVE CLEAVAGE ;= AFFORDED A MIXTURE OF IODOFORMATES 7ITH THE # AND # HYDROXYL GROUPS NOW ORTHOGONALLY PROTECTED REDUCTIVE DEHALOGENA TION WITH TRIPHENYLTIN HYDRIDE AND STANDARD FUNCTIONAL GROUP TRANSFORMA TIONS AS BEFORE PRODUCED ENOATE &INALLY 4)03 PROTECTION OF THE # HYDROXYL GROUP SIMULTANEOUS REDUCTION OF ESTER AND REMARKABLY DURABLE FORMYL FUNCTION AND ALLYLIC OXIDATION PRODUCED ALDEHYDE IN STEPS AND OVERALL YIELD 3YNTHESIS OF THE # # ACID BEGAN WITH FRAGMENT AND EMPLOYED SOME INTRIGUING CHEMISTRY TO CONSTRUCT A FORMAL DIALDEHYDE EQUIVALENT IN SUCH A WAY THAT THE TERMINAL FUNCTIONS ARE AT ALL TIMES DIFFERENT 4HE TWO COMPONENTS AND OF THE FIRST KEY BOND FORMING EVENT NAMELY A CHELATION CONTROLLED HETERO $IELS !LDER CYCLOADDITION ; = WERE SYNTHESISED IN SHORT ORDER FROM ETHYL 3 LACTATE AND PENTA NONE RESPECTIVELY EMPLOYING LITERATURE PROTOCOLS ;n= 5PON PRECOMPLEXATION OF 3 BENZYLOXY PROPANAL WITH -G"R AND SUB SEQUENT ADDITION OF THE ACTIVATED DIENE DIHYDROPYRONE WAS PRODUCED IN GOOD YIELD WITH EXCEPTIONAL SELECTIVITY 2EDUCTION WITH DIISO BUTYALUMINIUM HYDRIDE $)"!, ( THEN GENERATED BRANCHED GLYCAL SUITABLY ADORNED FOR THE SECOND APPLICATION OF A &ERRIER TYPE REARRANGEMENT IN THIS CASE TYPE ) ; = IN THE $ANISHEFSKY SYNTHESIS !CCORDINGLY TREATMENT OF WITH ACIDIC PROPANOL INDUCED FORMATION OF ALLYLOXYCAR BENIUM ION WHICH WAS QUENCHED BY A SOLVENT MOLECULE TO GIVE THE CORRESPONDING GLYCOSIDE IN EXCELLENT YIELD 4HE FORMAL DIALDEHYDE EQUIVALENT WAS THEN DELIVERED BY A HIGHLY STEREOSELECTIVE REDUCTION OF THE # # ALKENE AND REMOVAL OF THE BENZYL PROTECTING GROUP !S IS EVIDENT BOTH # AND # OF CONTAIN FUNCTIONALITY DIOL AND ACETAL RESPECTIVELY FROM WHICH READY CONVERSION TO THE CORRESPONDING ALDEHYDE CAN BE PERFORMED SEQUENTIALLY TO AVOID FORMATION OF A MESO SPECIES 4HUS TREATMENT WITH PROPANEDITHIOL IN THE PRESENCE OF A ,EWIS ACID FORMS THE LINEAR DITHIANE AND SUBSEQUENT OXIDATIVE CLEAVAGE FOLLOWED BY HOMOLOGATION GAVE ENOATE 3IMPLE OXIDATION STATE ADJUSTMENTS AT # FURNISHED ENAL IN STEPS AND OVERALL YIELD 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
OH
OBn
a
22
CO2Et 509
O
O
OBn
b
22
22
510
511
CO2Et
c
H
512 + OMe
O
O e
23
26
f
O
g
h
22
BnO
O
H
26
O 22
BnO
OH
H
517
518
23
23 26
513
22 26
d 26
BnO
OH
26
OH
514
23
OMe
OTMS
i
516
515
HOiPr 26
H
H 22
O
26
O
O
22
BnO
H
522
519
OiPr
OiPr H
S 20
23
O
26
S
OCH3
o,p
20
S 23
O
26
S
m,n
OH
HO
S 26
S
22
l
O HO
26
22
H 525
524
523
521
j,k
O BnO
26
22
H
520
3CHEME A "N"R !G/ %T/ REFLUX B ,I!L( %T/ # OVER TWO STEPS C #/#L $-3/ #(#L n # THEN %T. n # → 24 D (#/%T .A( 0H( # → 24 THEN (#L E P4S/( -E/( 0H( REFLUX F 4-3/4F 4%! %T/ # G -G"R 4(& 24 THEN THEN !C/( (/ H $)"!, ( 0H( 24 I P4S/( I0R/( 24 OVER TWO STEPS J 0D!L/ ( PSI %T/!C 24 K 0D# ( PSI %T/!C 24 OVER TWO STEPS L (3#( 3( "&q/%T #(#L n # → n # M 0B/!C +/!C #(#. n # N METHYL TRIPHENYLPHOS PHORANYLIDENE ACETATE #(#L 24 O $)"!, ( 0H#( n # → 24 P #/#L $-3/ #(#L n # THEN %T. n # → 24
4HE ADDITION OF HYDROGEN IODIDE TO TETROLIC ACID CAN FURNISH EITHER THE % OR : IODOCROTONIC ACID PRODUCT DEPENDING UPON THE CONDITIONS EMPLOYED ; = AND FUNCTIONS AS EFFICIENT ENTRY TO THE CENTRAL SPACERDIANION EQUIVALENT OF )N THIS CASE ISOMERISATION OF THE INITIALLY FORMED : ADDITION PRODUCT GAVE AND AFTER A SERIES OF STANDARD MANIPULATIONS TO INSTALL THE SULFONE AT # FURNISHED AS A CRYSTALLINE SOLID IN OVERALL FIVE STEPS AND OVERALL YIELD 3CHEME $ANISHEFSKYS USE OF SUGAR DERIVATIVES AS A CONVENIENT SOURCE OF STARTING MATERIAL CHIRALITY CONTINUES IN HIS PREPARATION OF FRAGMENT 3CHEME $ 'LUCAL WAS READILY CONVERTED TO α METHYL DEOXYGLYCOSIDE THROUGH A SHORT REACTION SEQUENCE FEATURING A STEREOSELECTIVE IODO METHOXYLATIONREDUCTION SEQUENCE &URTHER FUNCTIONAL GROUP MANIPULA TIONS AND A STANDARD (7% ;= THEN LED TO ALLYLIC MESYLATE !LTERNATIVELY $ GALACTAL TRIACETATE WAS AN EXPEDIENT ENTRY POINT TO IN A REACTION SEQUENCE SIMILAR TO THAT EMPLOYED FOR THE % ISOMER
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
CO2H
a
17
b
c
17
I
I
I
I
I 17
17
d
17
e
17
19
19
526
CO2H
527
19
CO2Me
528
19
19
19
X
OH
529
530
SO2Ph 474
3CHEME A () AQ # SEALED TUBE THEN NEAT # B #(. %T/ # THEN !C/( C $)"!, ( 0H#( # D -S#L %T. $-!0 #(#L # 8 #L OR -S MIXTURE EMPLOYED E SODIUM BENZENE SULFINIC ACID $-& 24 OVER TWO STEPS
BUT EMPLOYING THE MODIFIED 3TILL 'ENNARI ;= PROCEDURE TO INTRODUCE THE : OLEFIN 3CHEME "ASED UPON THE WORK OF )BUKA AND 9AMAMOTO ;= EITHER OR SHOULD GIVE RISE TO THE DESIRED # METHYL STEREOCHEM ISTRY FOLLOWING METHYL CUPRATE 3. DISPLACEMENT OF THE ACTIVATED ALCOHOL )N THESE REACTIONS THE ADDITION OF "&q/%T IS CRITICAL TO EFFECTING CLEAN CHIRALITY TRANSFER !LSO OF NOTE THE % DOUBLE BOND IN THE PRODUCT IS NOT DEPENDENT ON THAT OF THE STARTING MATERIAL )NDEED UPON APPLICATION OF THE DESCRIBED OPTIMISED CONDITIONS FOR CUPRATE DISPLACEMENT THE CLOSELY RELATED ESTERS 2 %T AND 2 -E WERE ISOLATED AS SINGLE DIASTEREO MERS 3TANDARD FUNCTIONAL GROUP TRANSFORMATIONS SUCCESSFULLY CONVERTED EITHER OR TO THE SAME # # ALDEHYDE IN A SHORTEST LINEAR SEQUENCE OF STEPS AND YIELD 4HE FIRST ATTEMPT AT COUPLING THE THREE ADVANCED FRAGMENTS TOWARDS THE COMPLETION OF THE # # CARBON SKELETON OF RAPAMY CIN IS SHOWN IN 3CHEME 5NION OF ALDEHYDE AND VINYL IODIDE UNDER .(+ CONDITIONS ;n= GAVE A REASONABLE YIELD OF THE COUPLED PRODUCTS n BUT WITH NO SELECTIVITY AT THE NEWLY FORMED # HYDROXY STEREOCENTRE !LTHOUGH SOME MATERIAL COULD BE SALVAGED THROUGH AN OXIDATIONREDUC TION SEQUENCE OF THE UNWANTED EPIMER THE LATTER REACTION ONCE AGAIN OCCURRED WITH NO DIASTEREOSELECTIVITY GIVING A MIXTURE OF AND -ETHYLATION OF THE DESIRED # ISOMER FOLLOWED BY DEPROTONATION OF AND ADDITION OF THE RESULTING α LITHIOSULFONYL ANION INTO DITHIANE ENAL GAVE A MIXTURE OF STEREOISOMERS FOLLOWING ACETYLATION IN GOOD YIELD !TTEMPTED DIRECT FORMATION OF THE DESIRED # # TRIENE BY TREAT MENT OF WITH SODIUM AMALGAM IN 4(& WAS SUCCESSFUL HOW EVER THE CENTRAL # # OLEFIN WAS FORMED AS A INSEPARABLE MIXTURE OF % AND : ISOMERS AND THE PRODUCT WAS NOT SUFFICIENTLY STABLE TO ALLOW
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
HO
TBSO a,b,c
HO 12
O
HO
TBSO d,e
MsO 12
BnO
16
531
HO O
12
HO
16
16
OBn S
f
MsO
O
BnO
O
g,h
OBn S
EtO 10
S
14
533
S
OMs
OMs
OMe
532
16 12
535
534
i AcO
TBSO j,k,l,m
AcO 12
12
HO
HO
q,r 14
S
12
10
OMs
OMe
246
OBn S
OBn S 16
O 16
16
AcO
HO n,o,p
HO
O
538
MeO
O
S
OBn S
RO 10
OMs
14
S
R = Et, 536; R = Me, 537
540
539
O
s
t
OBn O w
10
TBDPSO
14
545
H
TBDPSO
OBn S
10
14
544
v
S
HO 10
OBn S 14
543
u
S
RO
O
OBn S
10
14
S
R = Et, 541; R = Me, 542
3CHEME A 4"3#L $-& 24 B "U3N/ 0H#( $EAN 3TARK THEN "N"R N"U."R 0H#( # C -S#L 0Y # → 24 D .)3 #(#. -E/( 24 E N"U3N( !)". 0H( REFLUX F (3#( 3( "&q/%T #(#L # → 24 G 0B/!C 0H( 24 H CARBOETHOXYME THYLENE TRIPHENYLPHOSPHORANE #(#L # I -E#U#. ,Iq,I"R "&q/%T 4(& n # J .)3 -E/( #(#. 24 K N"U3N( !)". 0H#( REFLUX L .A/-E -E/( 24 M 4"3#L )M $-& 24 OVER TWO STEPS MIXTURE OF αβ ANOMERS N "U3N/ 0H#( $EAN 3TARK THEN "N"R N"U."R 0H#( # O -S#L 0Y $-!0 #(#L 24 P (3#( 3( "&q/%T #(#L # → 24 Q 0B/!C 0H( 24 R #&#(/ 0/ #(#/#( # +(-$3 4(& n # S -E#U#. ,Iq,I"R "&q/%T 4(& n # T 0H0 2H#L ( 0H( 24 FOR FOR U $)"!, ( 0H#( # FOR AND V 4"$03#L )M $-& 24 W ."3 ACETONE (/ n #
REMOVAL OF THE DITHIANE MOIETY 2EVERSING THE ORDER OF EVENTS THROUGH TREATMENT WITH .#3 AND !G./ REVEALED THE ALDEHYDE FUNCTION AT # WHICH WAS SUBSEQUENTLY REDUCED WITH .A"( 3UBJECTING THE RESULTING PRI MARY ALCOHOL TO THE ACTION OF SODIUM AMALGAM FURNISHED THE DESIRED UNPROTECTED TRIENE UNFORTUNATELY ONCE AGAIN AS A MIXTURE OF OLEFIN ISOMERS 3CHEME )NTERESTINGLY A SOLUTION TO BOTH SELECTIVITY IN FORMATION AND STABILITY UPON USE OF THE TRIENE WAS ACHIEVED VIA AN INTERMEDIATE PHENYLSULFONYL ALKENE FORMED IN EXCELLENT YIELD AND SELECTIVITY BY DIAZABICYCLOUN DECANE $"5 INDUCED ELIMINATION OF ACETATE &ROM THIS SPECIES THE ALDEHYDE WAS EASILY REVEALED AND SUBSEQUENT DESULFONYLATION UNDER STANDARD CONDITIONS FURNISHED THE CENTRAL # # OLEFIN AS A SINGLE % GEOMETRY ALTHOUGH SOME EPIMERISATION AT # HAD OCCURRED
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
OBn O 10
TBDPSO
SO2Ph
14 19
548 b
I
OBn O
17
10
TBDPSO
H
14
c
OBn OH
OBn OH 10
a
+
TBDPSO
19
+
19
TBDPSO
SO2Ph
14 19
SO2Ph 547
546
474
545
10
SO2Ph
14
d OBn OMe
OAc
10 14
S
20
S
20
OBn OMe
S
H
e,f
26
19
TBDPSO
23
26
+
S
O
10
TBDPSO
SO2Ph
14 19
SO2Ph
525
550
549
g,h,i
OBn OMe
H
10
j
TBDPSO
551
OBn OMe
H
OBn OMe
S
10
TBDPSO
26
OH
14
19
14
S
O
10
k,l
26
TBDPSO
14 19
26
H
SO2Ph 552
553
3CHEME A #R#L .I#L $-3/ 24 SEPARABLE B $-0 0Y #(#L 24 C ,I"( #E#Lq(/ -E/( 4(& n # SEPARABLE D -E/4F DI T BUTYL PYRIDINE #(#L REFLUX E S"U,I 4(& n # THEN n # F !C/ %T. $-!0 #(#L 24 MIXTURE OF DIASTEREOMERS G .#3 !G./ (/ #(#. 24 MIXTURE OF DIAS TEREOMERS H .A"( #(#L %T/( # I +(0/ 4(& -E/( n # THEN .A(G OVER TWO STEPS MIXTURE OF %: OLEFIN ISOMERS J $"5 4(& 24 K .#3 !G./ (/ #(#. 24 L +(0/ 4(& -E/( n # THEN .A(G MIXTURE OF # EPIMERS
4HE COMPLETE LACK OF CONTROL OF THE # STEREOCENTER 3CHEME NECES SITATED A SUBTLE REDESIGN OF THE REACTION SEQUENCE 3CHEME HOWEVER THE LESSONS LEARNED IN THE PREVIOUS STUDY WERE CRITICAL TO THE ULTIMATE DESIGN OF A VIABLE SYNTHESIS OF )N AN APPROACH SIMILAR TO THAT TAKEN TO ADDRESS PROTECTING GROUP DIFFICULTIES IN FRAGMENT 3CHEME $ANISHEFSKY AND CO WORKERS ELECTED TO PURSUE A LACTONE FORMATION STRATEGY AS A MEANS TO OVERCOME IN THIS CASE SELECTIVITY ISSUES .(+ COUPLING OF VINYL IODIDE AND MODIFIED ALDEHYDE COUPLING PARTNER AGAIN OCCURRED WITH NO SELECTIVITY GIVING A MIXTURE OF DIASTEREOMERIC COMPOUNDS WHICH WERE CONVERTED TO THE SAME β HYDROXY KETONE BY OXIDATION AND REMOVAL OF THE BENZYL PROTECTING GROUP 3CHEME !T THIS POINT THE RESULTING FREE ALCOHOL AT # WAS EXPLOITED IN A SUBSTRATE CONTROLLED REDUCTION UNDER %VANS 0RASAD ; = CONDI
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O EtO
OBn S
EtO
S
14
10
I
OBn O
O
a
10
16
541
17
H
O
OH O
10
14
b,c,d
+ 19
EtO
474
473
SO2Ph 19
SO2Ph 554
e O TBSO TESO
OMe
i,j,k,l
SO2Ph
14
10
10
O
19
26
H
OH OH
10
14
26
23
555
CHO
26
TBSO
x,y,z
19
OPiv
14
26
23
S
20
524
OMe
10
O
558
559
S
OCH3
m,n,o,p
23
TBDPSO
TBDPSO
475
26
q,r 20
23
SO2Ph 19
19
OPiv s,t 20
20
O
TBSO
O
556 OPiv
TESO
EtO
SO2Ph
14
557
u,v,w
f,g,h
OMe
OMe
O
10
TESO
14
26
19
H
SO2Ph 560
561
aa,bb
H
O
TBSO
10
14
OMe
O
TBSO
cc,dd
26
O
OMe
10
19
OMe
TESO
14
26 19
OMe
484
562
3CHEME A B #R#L .I#L $-3/ 24 OF # EPIMERS C $-0 #(#L 24 OVER TWO STEPS D "#L #(#L n # E .A"( %T"/-E 0H#( n # F ,I/( 4(& -E/( (/ # G %$#) #(#L 24 OVER TWO STEPS H -E) !G/ 24 I +#/ -E/( 24 J 4"3/4F LUT #(#L # OVER THREE STEPS K $)"!, ( 4(& # L 4%3#L %T. #(#L OVER TWO STEPS M $)"!, ( 0H#( n # → 24 N 4"$03#L %T. #(#L O .#3 !G./ 4(& -E/( P GLYOXYLIC ACID !C/( #(#L OVER THREE STEPS Q .A"( %T/( R 0IV#L %T. #(#L S (& AQ (/ #(#. T $-0 #(#L OVER FOUR STEPS U ,$! 4(& n # THEN V !C/ %T. $-!0 #(#L 24 W $"5 4(& 24 OVER THREE STEPS X $)"!, ( 0H#( # Y +(0/ 4(& -E/( n # THEN .A(G Z $-0 0Y 24 OVER THREE STEPS AA ,I#(/#( %T/ n # BB $-0 0Y #(#L 24 OVER TWO STEPS CC !C/( (/ 4(& 24 DD $-0 0Y #(#L OVER TWO STEPS
.O YIELD OR EXPERIMENTAL DETAILS REPORTED
TIONS WHICH GAVE AS A SINGLE DIASTEREOMER 0RODUCTIVE LACTONE FORMA TION BETWEEN THE # ALCOHOL AND # ESTER ALLOWED FOR INSTALLATION OF THE METHYL ETHER AT # AND THEN DIFFERENTIATED PROTECTING GROUPS AT BOTH # AND # 0RIOR TO COUPLING WITH THE ENAL ELECTRO PHILE ALSO UNDERWENT MODIFICATION SEQUENCE M → N IN 3CHEME
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TO AVOID POTENTIAL ISSUES WITH LATE STAGE REMOVAL OF THE DITHIANE MOIETY WHICH HAD PREVIOUSLY BEEN PROBLEMATIC )N THE EVENT FORMATION OF THE LITHIO ANION OF SULFONE ADDITION TO AND ELIMINATION TO THE PHE NYLSULFONYL TRIENE GRATIFYINGLY OCCURRED IN EXCELLENT YIELD OVER THE THREE STEPS !S EXPECTED REDUCTIVE REMOVAL OF THE PIVALATE THROUGH TREAT MENT OF WITH $)"!, ( AND DESULFONYLATION YIELDED AS A SINGLE # # ALKENE &URTHER ELABORATION TO THE # # ALDEHYDE OCCURRED WITHOUT INCIDENT AFFORDING IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD OF 4HE FINAL STAGES OF $ANISHEFSKYS SYNTHESIS OF RAPAMYCIN REQUIRED INCORPORATION OF THE TRICARBONYL MOIETY UPON THE TRIENE FRAGMENT JUST DISCUSSED &OR THIS PURPOSE THE ADDITION OF THE ANION OF α PHENYLTHIOAMIDES TO A SUITABLE ALDEHYDE FOLLOWED BY OXIDATION WITH $ESS -ARTIN PERIODINANE HAD BEEN DEVELOPED PREVIOUSLY TO ACCESS SIMPLE VICINAL TRICARBONYL SYSTEMS ;= !PPLICATION TO THE REAL SYSTEM 3CHEME WITH THE READILY PREPARED , PIPECOLIC SULFOXIDE DERIVATIVE PLEAS INGLY GAVE THE DESIRED TRICARBONYL SYSTEM AFTER OXIDATION 4REATMENT OF THIS INTERMEDIATE WITH (&q0Y RESULTED IN DESILYLATION AND SPONTANEOUS HEMIACETAL FORMATION AT # -ODEL STUDIES ;= HAD INDICATED THAT A FREE HEMIKETAL HYDROXYL GROUP AT # WOULD INTERRUPT ANY ATTEMPTS TO COUPLE THE PIPECOLATE GROUP WITH # # FRAGMENT UNDER STANDARD ACTI VATING CONDITIONS SEE INSERT 3CHEME !CCORDINGLY AFTER EXCHANGING THE ACID LABILE TERT BUTYL ESTER FOR THE ALLYL ESTER THE TERTIARY HEMIKETAL HYDROXYL GROUP IN WAS PROTECTED AS ITS TRIMETHYLSILYL ETHER 4REATMENT WITH PALLADIUM NOW EFFECTED CLEAVAGE OF THE ALLYL ESTER UNDER MILD CON DITIONS AND CRUCIALLY RETAINED THE 4-3 GROUP TO FURNISH CARBOXYLIC ACID 3UBSEQUENT ESTERIFICATION OF WITH THE FREE SECONDARY ALCOHOL PRESENT AT # OF UNDER STANDARD CONDITIONS SELECTIVE DESILYLATION OF THE # AND # HYDROXYL GROUPS AND OXIDATION GAVE THE CRUCIAL PRECURSOR PRECURSOR FOR THE INTENDED MACROALDOLISATION PROCESS 3CHEME )T IS SOMEWHAT CURIOUS THAT THE LATTER DESILYLATIONOXIDATION SEQUENCE WAS PERFORMED PRIOR TO ATTEMPTING THE INTRAMOLECULAR ALDOL REACTION ESPECIALLY GIVEN THE PROPENSITY FOR ELIMINATION OF THE PIPECOLATE UNIT 7ITH COMPOUND IN HAND EXTENSIVE INVESTIGATIONS DIRECTED TOWARDS SUCCESSFUL GENERATION OF THE DESIRED ENOLATE AND STEREOSELECTIVE RING CLO SURE WERE INVESTIGATED 4HIS TRANSFORMATION PROVED EXCEPTIONALLY DIFFICULT
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O
O 1
N H
OtBu
OtBu +
1
a,b
N O
9
O
O
TBS O OMe
10
OMe
14
1 OtBu H O O 9 TBS 10 O OMe
N
PhS(O) HO
c
26
19
O 26
PhS(O)
OMe
14
563
472
19
484
564 H
O
N
1
O
O
carboxyl activation (X) absence of TMS at C10-OH
9 10
O
O HO
O
O
9
X
O d,e,f,g
O
10 14
1
N
R
R 566 14
567
TIPSO
42
38
OH
1
N MeO
H
32
+
O TMSO
9
10
OH O TBS
CHO
1
N
H O O
O
O
OMe
O HO
9
10
26
OMe
14
28
h,i
O
H O O
O
O
OMe
OMe 19
19
565
469
468
26
14
j,k,l
TIPSO
TIPSO
42
MeO
38
H
HO
42
MeO
38
H
32
N H1 O O 9
O HO 10
O
O
16
O
m
CHO O
22
isomer 570 (22%)
O HO
9
10
N H1 O O O
O
H 32
OH
O
O
MeO
OMe
n
O HO
9
10 22
N H1 O O O
O
O
OH O
MeO
OMe
22
16
16
568
38
32
MeO
OMe
42
MeO
569
(-)-rapamycin 7
3CHEME A PHENYLTHIO ACETIC ACID %$#) #(#L 24 B .A)/ (/ -E/( 24 C ,$! 4(& n # THEN n # D $-0 0Y #(#L 24 E (&q0Y 4(& 24 OVER TWO STEPS F (#/( #(#L 24 G ALLYL BROMIDE +#/ 4"!) $-& 24 OVER TWO STEPS H 4-3 )M $-!0 $-& 24 I 0D00H MOL 0H0 #(#L 24 J $## $-!0 #(#L n # K 4"!& !C/( 4(& # L $-0 0Y #(#L 24 M 4I#L/I0R #(#L %T. n # N (&q0Y 4(& 24
AND ESSENTIALLY ALL STANDARD REACTION CONDITIONS INVESTIGATED FAILED TO PRO VIDE EVEN A TRACE OF THE DESIRED PRODUCT &INALLY USE OF 4I#L IN THE PRESENCE OF %T. WAS SUCCESSFUL IN INDUCING THE DESIRED BOND FORMING EVENT ALBEIT IN LOW YIELD 3LIGHTLY IMPROVED RESULTS WERE ACHIEVED BY USE ISOPROPOXYTITA NIUM TRICHLORIDE AS THE PROMOTER BUT UNFORTUNATELY WAS NEVER ISOLATED IN MORE THEN 5NREACTED STARTING MATERIAL DEGRADATION PRODUCTS AND
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 3UMMARY OF $ANISHEFSKYS TOTAL SYNTHESIS OF RAPAMYCIN
THE CONSISTENT PRESENCE OF AN APPARENT STEREOISOMER OF ACCOUNTED FOR THE REMAINDER OF THE MASS BALANCE &INALLY DESILYLATION OF WITH (&q0Y YIELDED RAPAMYCIN IDENTICAL IN ALL REGARDS WITH SAMPLES OF THE NATURAL MATERIAL )N SUMMARY THE $ANISHEFSKY SYNTHESIS OF RAPAMYCIN REQUIRED A LONGEST LINEAR SEQUENCE OF STEPS AND AN OVERALL YIELD OF 3CHEME !LTHOUGH THE LONGEST OF THE VARIOUS APPROACHES THIS TOTAL SYNTHESIS EFFORT INCLUDED NUMEROUS INTRIGUING AND UNCONVENTIONAL STRATEGIES MOST NOTA BLY A NOVEL MACROALDOLISATION /THER HIGHLIGHTS INCLUDE IMPLEMENTATION OF A NUMBER OF GROUP DEVELOPED METHODOLOGIES EG HETERO $IELS !LDER CYCLOADDITION CONSTRUCTION OF THE TRICARBONYL REGION ELEGANT DEGRADATION STUDIES AN EFFICIENT USE OF CARBOHYDRATE STARTING MATERIALS IN COMBINATION WITH REARRANGEMENT PROCESSES TO ACCESS A VARIETY OF FRAGMENTS AND A NUM BER OF CLEVER SOLUTIONS TO UNEXPECTED DIFFICULTIES ENCOUNTERED DURING THE COURSE OF SYNTHESIS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
4HE 3MITH TOTAL SYNTHESES OF RAPAMYCIN AND DEMETHOXYRAPAMYCIN )N !MOS " 3MITH ))) DISCLOSED THE LAST OF THE SUCCESSFUL APPROACHES TO RAPAMYCIN OF THAT DECADE WITH FULL EXPERIMENTAL DETAILS FOLLOWING TWO YEARS LATER ;n= 4HE SYNTHESIS HIGHLIGHTS BOTH σ BOND FORMA TION FOR THE CONSTRUCTION OF STEREODEFINED OLEFINS AND DITHIANE CHEMISTRY FOR THE UNION OF COMPLEX FRAGMENTS ; = -OREOVER IT IS A HIGHLY MODULAR AND CONVERGENT APPROACH AND WAS SUCCESSFUL IN DELIVERING NOT ONLY RAPAMYCIN BUT ALSO ITS NATURALLY OCCURRING CONGENER DEME THOXYRAPAMYCIN ; = 2ETROSYNTHETICALLY CLEAVAGE OF THE # ESTER LINKAGE AND THE CENTRAL OLEFIN # # OF THE TRIENE AFFORDS THE TWO LARGE FRAGMENTS AND 4HIS STRATEGY WAS DESIGNED TO ALLOW MAXIMUM FLEXIBILITY IN THE FINAL MACROCYCLISATION AS EITHER A 3TILLE COUPLING OR MACROLACTAMISATION COULD BE EMPLOYED DEPENDING UPON THE ORDERING OF EVENTS &URTHER CONSIDERATION OF AND REVEALS BUILDING BLOCKS n AS INITIAL SYNTHETIC TARGETS 3CHEME 4HE SYNTHESIS OF # # STANNANE WAS ENVISIONED TO RELY HEAV ILY ON ANIONIC COUPLINGS OF SUBSTITUTED DITHIANES WITH SUITABLE ELECTRO PHILES TO BUILD THE # # AND # # SIGMA BONDS )N CONTRAST TO MUCH OF THE EARLY WORK INVOLVING TRANSFORMATIONS OF THIS TYPE ; = THE STRUCTURAL COMPLEXITY OF IS SUBSTANTIAL AND WOULD PROVE AN EXCELLENT TESTING GROUND FOR DEMONSTRATION AND ADVANCEMENT OF THIS METHODOLOGY 4HE DISCONNECTION ACROSS THE # # LINKAGE IN THIS FASHION IS ESPECIALLY INTERESTING AS IT AFFORDS AN EXCELLENT POINT OF DIVERGENCE TOWARDS THE TWO NATURAL PRODUCT TARGETS AND (OWEVER THIS IS ALSO A POTENTIALLY PROB LEMATIC STRATEGY AS CONTROL OF THE RESULTING STEREOCHEMISTRY AT # IS CRUCIAL AND FAR FROM ASSURED 4HE CHALLENGING TRISUBSTITUTED OLEFINIC LINKAGE WITH FLANKING STEREOCENTRES ON EITHER SIDE WAS TO BE CONSTRUCTED USING A σ BOND FORMATION EVENT ; = )N THIS FASHION THE UNACCEPTABLE ISOMERIC MIX TURES OFTEN OBTAINED FROM α α DISUBSTITUTED YLIDES MIGHT BE AVOIDED !S THE STEREOCHEMISTRY WITHIN THE # # REGION IS ULTIMATELY UNIM PORTANT ADDITION OF THE DIANION OF , . ACETYLPIPECOLIC ACID ; = TO ALDEHYDE FOLLOWED BY OXIDATION UNDER THE 'OLEC PROTOCOL ;= AS MENTIONED PREVIOUSLY SEE 3CHEME PAGE SHOULD RAPIDLY GENERATE THE TRICARBONYL REGION OF 4HE SILYL TERMINATED ENYNE OF WAS ENVI
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO
42
MeO
HO
38
42
C21-C42 stannane (R = OMe) 571; (R = H) 572
TIPSO
42
H
38
H
38
OH O
H
OTBS
32
R
O HO
9
N H1 O O
O
O
OH
I OPMB
O
OMe
OPMB
S
26
+
+
(MeO)2HC 22
575
32
S
OTBS 27
1
CO2H
N 10
574
O
R 22
16
O TESO
(-)-rapamycin (R = OMe) 7 (-)-27-demethoxyrapamycin (R = H) 8
Bu3Sn
576
22
O 10
S 26
33
32
MeO
S
+
MeO
CHO
O 10
O
OMe 16
H C1-C20 acid 573
1
20
N
I
CO2H
8
+
OTBS OMe 16
O 577
20
TMS 578
3CHEME 2ETROSYNTHETIC ANALYSIS
SIONED TO SERVE AS THE PRECURSOR TO A SECOND σ BOND CONSTRUCTION THROUGH HYDROMETALLATIONIODINATION )N THE FORWARD DIRECTION THE SYNTHESIS OF THE # # SULFONE BOR ROWS FROM CHEMISTRY EMPLOYED IN 3MITHS FORMAL TOTAL SYNTHESIS OF &+ 3CHEME ; = !N ASYMMETRIC $IELS !LDER REACTION OF BUTADIENE WITH HOMOCHIRAL SULTAM ;= UNDER ,EWIS ACID CATALYSIS EFFICIENTLY GENERATED CARBOXYLIC ACID IN GOOD OVERALL YIELD AND ENANTIO MERIC EXCESS FOLLOWING BASE PROMOTED HYDROLYSIS OF THE CHIRAL AUXIL IARY !LTHOUGH CERTAINLY SUITABLE FOR THE PRODUCTION OF REASONABLE AMOUNTS OF THERE ARE A NUMBER OF DRAWBACKS TO THIS APPROACH INCLUDING MOST SIGNIFICANTLY THE MULTI STEP SYNTHESIS OF A STOICHIOMETRIC ASYMMETRIC CON TROLLER 5NFORTUNATELY TO DATE DIRECT CATALYTIC ASYMMETRIC $IELS ALDER REAC TIONS OF UNSUBSTITUTED BUTADIENES AND SIMPLE DIENOPHILES REMAINS AN UNRESOLVED PROBLEM FOR THE ORGANIC CHEMISTRY COMMUNITY ; = 7ITH IN HAND IODOLACTONISATION INSTALLED THE NECESSARY OXYGENATION AT # AND SUBSEQUENT DEHYDROHALOGENATION WITH $"5 GENERATED AN OLE FINIC HANDLE FOR FURTHER MANIPULATION !FTER REDUCTION OF THE LACTONE WITH ,I!L( THE RESULTING PRIMARY ALCOHOL WAS SELECTIVELY TRANSFORMED TO THE CORRESPONDING SULFIDE BY TREATMENT WITH TRI N BUTYLPHOSPHINE AND DIPHENYLDISULFIDE 3TANDARD SYNTHETIC MANIPULATIONS THEN PRODUCED SULFONE SUITABLY ADORNED FOR THE CRUCIAL REGIO AND STEREOSELECTIVE INTRODUCTION OF THE LAST REMAINING HYDROXYL FUNCTION 0REVIOUSLY IT HAD BEEN SHOWN THAT HYDROBORATIONOXIDATION OF ALKOXYCYCLOHEXENE DERIVA TIVES IS SUITABLE FOR THE TRANS INSTALLATION OF A VICINAL HYDROXYL GROUP TO
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O2 S N
O
O2 S N
a,b
O 42
42
c,d
R
42
e,f
38
H
CO2H
38
HO
O
42
g,h
SPh
MeO
38
SO2Ph 38
O
579
30
580
581
583
582
i TIPSO
MeO
TIPSO
42
o,p 38
H
574
TIPSO
33
I OPMB
MeO
m,n 38
H
33
MeO
O
42
42
HO
TIPSO 42
585 38
H
33
OTBDPS O
414
OTBDPS
586
k,l
MeO
42
j
MeO
38
38
SO2Ph
SO2Ph
OH 584
69
3CHEME A BUTADIENE %T!L#L #(#L n # B ,I/( 4(&(/ 24 C ) +) .A(#/ (/ # D $"5 4(& REFLUX E ,I!L( %T/ # F "U0 0H3 $-& 24 G #(. "&q/%T %T/ # H /XONE¤ 4(&-E/((/ # I "(q4(& .A/( T"U/( n # → 24 J 4)03#L )M $-& 24 ^ K N"U,I 4(& n # THEN "&q/%T L .A(G .A(0/ -E/( 24 OVER TWO STEPS M -S#L %T. #(#L N .A( (-0! # → 24 O ,I) "&q/%T %T/ n # P 0-" 4#! "&q/%T #(#L n #
A NEIGHBOURING ALKOXY GROUP ; = )N THE EVENT TREATMENT OF WITH "(q4(& AT LOW TEMPERATURE GAVE THE DESIRED PRODUCT IN GOOD OVER ALL YIELD 3ILYLATION ;= FOLLOWED BY "&q/%T PROMOTED REGIOSELECTIVE RING OPENING OF THE READILY AVAILABLE EPOXIDE ;= BY THE ANION OF EFFICIENTLY COMPLETED THE CARBON FRAMEWORK AND INSTALLED THE LAST TWO STEREOCENTRES OF )NTERESTINGLY THE INCORRECT ANTIPODE AT # WAS INSTALLED PURPOSEFULLY TO ALLOW AFTER DESULFONYLATION AND MESYLATION INVERSION OF THIS CENTRE THROUGH INTRAMOLECULAR EPOXIDE FORMATION OF THE LIBERATED # HYDROXYL GROUP UPON DEPROTECTION UNDER BASIC CONDITIONS ;n= &INALLY RING OPENING OF THE OXIRANE WITH LITHIUM IODIDE AND "&q/%T GAVE AN INTERMEDIATE IODOHYDRIN WHICH WAS SUBSEQUENTLY PROTECTED AS ITS 0-" ETHER UNDER THE ACIDIC CONDITIONS OF "UNDLE ;= TO COMPLETE THE SYNTHESIS OF THE # # FRAGMENT /VERALL THIS WAS PREPARED IN TOTAL STEPS AND YIELD FROM THE CHIRAL DIENOPHILE STEPS YIELD FROM BASIC STARTING MATERIALS 3CHEME 4HE SYNTHESIS OF THE # # CENTRAL DITHIANE FRAGMENT BEGAN WITH A SERIES OF STANDARD TRANSFORMATIONS WHICH CONVERTED 2 HYDROXY METHYLPROPIONATE TO DITHIANE IN OVER THE FOUR STEPS $ESI LYLATION OF TOSYLATION AND &INKELSTEIN TREATMENT AFFORDED PRIMARY IODIDE THAT UNDERWENT CLEAN DISPLACEMENT WHEN TREATED WITH SODIUM
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
MeO
a,b
OH
32
O
S
c,d
OTBDPS
32
e,f,g
OTBDPS
32
S
S
OH
420
587
h
I
32
S
S 588
SO2Ph
32
S
589
590
OHC
O
O
i
591
o,p,q,r
32
H OTBS
S
27
32
OH
27
S 576
S
OH 595
m,n
S
27
32
O O
S 594
OH
O
OTf
O
S
l
S
27
32
O O
S 593
j,k
S
27
32
O
S O2SPh O 592
3CHEME A 4"$03#L )M B ,I"( %T/ OVER TWO STEPS C #/#L $-3/ #(#L n # THEN %T. n # → 24 D (3#( 3( "&q/%T OVER TWO STEPS E 4"!& 4(& F 4S#L %T. OVER TWO STEPS G .A) ACETONE H 0H3/.Aq(/ $-& OVER TWO STEPS I N"U,I 4(& n # → n # THEN J #/#L $-3/ #(#L n # THEN %T. n # → 24 OVER TWO STEPS K !L(G 4(&(/ REFLUX L ,I(-$3 (-0!4(& n # THEN 4F.0H M -E#U,I %T/ n # N #3! -E/( 24 O 0IV#L $-!0 P 4"3/4F LUT Q $)"!, ( R #/#L $-3/ #(#L n # THEN %T. n # → 24
BENZENESULFINATE TO GIVE THE CORRESPONDING SULFONE α ,ITHIATION AND CONDENSATION WITH ISOPROPYLIDENE , GYLCERALDEHYDE GAVE A SINGLE KETONE FOLLOWING OXIDATION AND DESULFONYLATION 4HE STEREOSELECTIVE GENERA TION OF % OR : ENOLATES IS A WELL STUDIED PROCESS ;= AS IS THE ABILITY TO TRAP THEM WITH GEOMETRICAL INTEGRITY AS EITHER SILYL ENOL ETHERS OR VINYL TRI FLATES 4HE FINDING THAT THE LATTER UNDERGO SMOOTH COUPLING ;= WITH LITH IUM DIMETHYL CUPRATES PROVIDES AN ELEGANT SOLUTION TO THE STEREOSELECTIVE GENERATION OF METHYLATED TRI SUBSTITUTED OLEFINS PRESENT IN A LARGE NUMBER OF NATURAL PRODUCTS ;n= 4HIS WAS EXPLOITED BY 3MITH IN HIS &+ SYNTHESIS AND HAS FOUND APPLICATION FOR THE CONSTRUCTION OF THE # # OLEFINIC LINKAGE PRESENT IN RAPAMYCIN #ONSIDERABLE EXPERIMENTATION WAS REQUIRED TO DETERMINE THE OPTIMUM ENOLISATION CONDITIONS FOR ;= (OWEVER WITH THESE IN HAND TRAPPING WITH . PHENYLTRIFLUOROMETHANE SULFONIMIDE ;= AND COUPLING WITH -E#U,I GAVE EXCLUSIVELY THE DESIRED : OLEFIN ISOMER IN RESPECTABLE YIELD 0ROTECTING GROUP MANIPULATIONS AND A FINAL 3WERN OXIDATION GAVE THE α SILOXY ALDEHYDE FOR WHICH A CRYSTAL STRUCTURE WAS OBTAINED CONFIRMING BOTH THE STEREOCHEMISTRY AND OLEFIN GEOMETRY 3CHEME ;= /VERALL FROM THE COMMERCIALLY AVAIL ABLE 2OCHE ESTER ALDEHYDE REQUIRED SYNTHETIC MANIPULATIONS AND WAS PREPARED IN A YIELD OF
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O
O
O
OEt
EtO
+
O
b,c
O
O
d,e
598
597
CO2Me
MeO2C
EtO2C
Br 596
CO2Et
EtO2C
a OEt
599
600
f
S S 26
m,n
HO
26
S
22
OMe
S
j,k,l
g,h,i
OH
TBDPSO
22
MeO
603
26
22
602
CO2Me
HO2C
26
22
601
575
3CHEME A .A %T/( REFLUX B (#L REFLUX C !C/ REFLUX THEN RECRYSTALLISE FROM %T/!C D -E/( REFLUX E -E/(0H( (3/ REFLUX F α CHYMOTRYPSIN - PHOSPHATE BUF FER P( 24 H G "/-E "(q$-3 4(& # H 4"$03#L )M I $)"!, ( J #/#L $-3/ #(#L n # THEN %T. n # → 24 K (3#( 3( "&q/%T L 4"!& M #/#L $-3/ #(#L n # THEN %T. n # → 24 N -E/ #( 4S/( -E/( OVER TWO STEPS
7ITH ROUTES TO BOTH THE # # AND # # FRAGMENTS IN HAND ATTENTION WAS THEN FOCUSED ON THE SYNTHESIS OF THE REMAINING MOIETY REQUIRED TO CONSTRUCT NAMELY THE # # DITHIANE &OR THIS 3MITH ELECTED TO PURSUE ENZYMATIC DESYMMETRISATION ;= OF MESO DIESTER WITH α CHYMOTRYPSIN TO AFFORD THE HALF ESTER IN GOOD YIELD AND ENANTIOSELECTIVITY !T THE TIME OF PUBLICATION THIS WAS PROBABLY THE MOST RELIABLE OF THE ENZYMATIC METHODS AVAILABLE ALTHOUGH CURRENTLY MORE EFFICIENT ALTERNATIVES EXIST /NE OTHER SALIENT POINT NOT IMMEDIATELY APPARENT IS THAT STARTING MATERIAL REQUIRES FIVE STEPS TO PREPARE ;= OF WHICH ACCESS TO THE MESO ANHYDRIDE WAS PARTICULARLY LOW YIELDING 4HIS HAS RECENTLY BEEN ADDRESSED BY -AIER THROUGH A DYNAMIC RECRYSTAL LISATION ;= AND THIS IMPROVEMENT HAS BEEN EMPLOYED IN ,EYS SYNTHESIS OF RAPAMYCIN VIDE INFRA ! SERIES OF ROUTINE FUNCTIONAL GROUP CONVERSIONS AND OXIDATION STATE ADJUSTMENTS WAS THEN EMPLOYED TO PREPARE THE FULLY ELABORATED # # DITHIANE IN AN OVERALL YIELD AND STEPS 4O AVOID OXIDATIVE METHODOLOGY LIMITATIONS DUE TO THE PRESENCE OF SULFUR AND POTENTIAL EPIMERISATION OF THE # METHYL STEREOCENTRE IN ADVANCED INTERMEDIATES THE # ALDEHYDE WAS MASKED IN THE CORRECT OXIDATION STATE AS ITS DIMETHYL ACETAL 3CHEME 4HE IMPORTANCE OF FLEXIBLY IN A LARGE SYNTHETIC ENDEAVOUR IS APTLY DEM ONSTRATED BY THE FACT THAT AND OF 3CHEME ALL PROVED TO BE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO
S
a
OTBDPS
32
TIPSO
S 588
MeO
38
H
33
b,c
OTBDPS
MeO S
H
OMs 30
605
d
TIPSO
MeO
H
33
TIPSO
42
I
38
H
PMBO
SS
PMBO 607
MeO
33
30
decomposition (MeO)2HC
33
604
I OPMB
26
?
H
Nuc
33
42
38
38
PMBO S S
OMs
S
42
PMBO S S
574
TIPSO
38
MeO
30
42
MeO
TIPSO
42
S
S
42
38
H
33
PMBO
S
S
OMs
606
608
22
575
3CHEME A T"U,I (-0!4(& n # THEN PRECOOLED n # SOLUTION IN (-0!4(& B .A( (-0! # → 24 C -S#L %T. D .A) ACETONE # DECOMPOSITION
IMPORTANT FOR THE ELUCIDATION OF A VIABLE ROUTE TO THE FULLY ELABORATED # # CARBON FRAMEWORK !LTHOUGH NOT PREVIOUSLY DISCUSSED 3MITHS INITIAL SYNTHETIC TARGET WAS FORMED EFFICIENTLY BY IMMEDIATE ADDITION OF PRI MARY IODIDE TO THE PREFORMED ALKYL DITHIANE ANION T"U,I OF IN A COLD n # (-0!4(& MIXTURE 3CHEME 4HIS PROTOCOL AND SLIGHT MODIFICATIONS THEREOF HAS PROVEN REMARKABLY GENERAL IN A WIDE VARI ETY OF COMPLEX FRAGMENT COUPLINGS AND REPRESENTS A VERY IMPORTANT ADVANCE FOR APPLICATION OF DITHIANE METHODOLOGY ;= 4HE KEY OBSERVATION WAS THE NEAR INSTANTANEOUS GENERATION MIN OF THE DITHIANE ANION UNDER THESE CONDITIONS WHICH GRADUALLY LOSES ITS REACTIVITY WITH TIME ; = NECESSITATING EITHER THE INITIAL PRESENCE OF THE ELECTROPHILE EG EPOXIDE OR ITS RAPID ADDITION TO ACHIEVE HIGH YIELDS 7ITH READY ACCESS TO SECURED THE PLAN WAS SUBSEQUENT CONVERSION TO ELECTROPHILE AND COUPLING OF A SECOND DITHIANE ANION SUCH AS 5NFORTUNATELY ALL ATTEMPTS TO PREPARE PRIMARY IODIDE FAILED UNDER THE CONDITIONS EXPLORED AND REVEALED AN INTERESTING FEATURE OF DISUBSTITUTED DITHIANES WITH PROXIMAL LEAVING GROUPS TO REARRANGEDECOMPOSE UNPRODUCTIVELY ; = 4HE DIFFERENCE BETWEEN MONOSUBSTITUTED DITHIANES WHERE SIMILAR REARRANGEMENTS ARE GENERALLY NOT PROBLEMATIC AND THEIR DISUBSTITUTED COUNTERPARTS HAS BEEN ATTRIBUTED TO THE CONFORMATIONAL CHANGES AND STERIC CONGESTION ABOUT THE DITHIANE ; = )N ANY REGARD AN INABILITY TO ACCESS REQUIRED A
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
S
27
32
O O
S
594 TIPSO a
TIPSO MeO
42
MeO
38
33
I OPMB
574
42
38
H
H
33
27
O
PMBO S S
O
609
b,c TIPSO
TIPSO
42
MeO
38
H
33
SS
PMBO
27
d,e
MeO
38
H
I OTBS
PMBO
SS
OH
610 TIPSO
S
g
27
33
MeO
42
38
H
27
33
OTs
611
S
TIPSO
42
O
SS
PMBO
613
42
S
S
26
26
f
MeO
38
h,i
H 32
(MeO)2HC
PMBO S S
22
OTBS
575
(MeO)2HC 22
575 26
S S
+ elmination products (41%) (MeO)2HC
612
22
3CHEME A T"U,I (-0!4(& n # THEN PRECOOLED n # SOLUTION IN (-0!4(& B #3! -E/( 24 C 4S#L %T. $-!0 #(#L # → 24 D .A) ACETONE E 4"3/4F LUT F T"U,I (-0!4(& n # THEN PRECOOLED n # SOLUTION IN (-0!4(& G +#/ -E/( 24 H T"U,I (-0!4(& n # THEN PRE COOLED n # SOLUTION IN (-0!4(& OVER TWO STEPS I 4"3/4F LUT n #
REDESIGN OF THE SYNTHETIC APPROACH WHICH WAS READILY ACHIEVED DUE TO THE MODULAR NATURE OF THE OVERALL STRATEGY VIDE INFRA &OR DEMETHOXYRAPAMYCIN ALKYLATION OF THE LITHIO ANION OF IN PLACE OF WITH IODIDE UNDER THE CONDITIONS DISCUSSED PREVI OUSLY AFFORDED THE DESIRED PRODUCT IN AN IMPRESSIVE YIELD OF 3UBSEQUENT FUNCTIONAL GROUP TRANSFORMATIONS CONVERTED TO THE CORRE SPONDING PRIMARY IODIDE 5NFORTUNATELY UPON ATTEMPTED COUPLING WITH THE FINAL # # FRAGMENT AN UNACCEPTABLY LOW YIELD OF WAS OBTAINED DUE TO THE FORMATION OF SUBSTANTIAL AMOUNTS OF IODO ELIMI
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO
MeO
TIPSO
42
38
H
33
a,b,c,d
27
MeO
TIPSO
42
38
[O]
H
33
O
PMBO
SS
O
PMBO
609
SS
MeO
27
OH OTBS
38
O
H
33
PMBO
614
SS
27
H OTBS
615
S
S 26
e,f
TIPSO
42
(MeO)2HC
42
22
MeO
38
H
O
575
33
PMBO O 616
27 H OTBS
g
multiple products
3CHEME A #3! -E/( 24 B 0IV#L $-!0 #(#L C 4"3/4F LUT #(#L D $)"!, ( 0H#( E -E) #A#/ -E#.4(&(/ # F #/#L $-3/ #(#L n # THEN %T. n # → 24 G T"U,I (-0!4(& n # THEN PRECOOLED n # SOLUTION IN (-0!4(& MULTIPLE PRODUCTS
NATION PRODUCTS 4O AVOID THIS ISSUE WAS CONVERTED TO EPOXIDE WHICH WAS SMOOTHLY RING OPENED AT THE OXIRANE TERMINUS BY METALLATED !FTER SILYLATION COMPLETION OF THE DEMETHOXYRAPAMYCIN # # BACKBONE REQUIRES ONLY THE INSTALLATION OF THE VINYL STANNANE MOIETY VIDE INFRA /VERALL WAS PREPARED IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD )N CONTRAST TO DEMETHOXYRAPAMYCIN FOR PREPARATION OF RAPAMY CIN # MUST BEAR OXYGENATION "OTH &ELKIN !NH ANALYSIS ;n= STERIC AND STEREOELECTRONIC ARGUMENTS CAN BE MADE TO PLACE THE /4"3 MOIETY PERPENDICULAR TO THE CARBONYL AND LITERATURE PRECEDENT FOR THE ADDITION OF METALLATED DITHIANES TO $ ISOPROPYLIDENEGLYCERALDEHYDE ; = SEEMED TO SUGGEST THE DESIRED STEREOCHEMISTRY OF THE NEW CHIRAL CENTRE WOULD PREDOMINATE )NITIALLY THE SAME COUPLING SEQUENCE AS USED SUCCESS FULLY ABOVE FOR DEMETHOXYRAPAMYCIN 3CHEME WAS ENVISIONED TO SERVE AS AN EFFICIENT ENTRY TO 4HUS AFTER HYDROLYSIS OF ACETONIDE AS PREVIOUSLY DESCRIBED THE RESULTING DIOL WAS SELECTIVELY PROTECTED AT THE PRIMARY POSITION AND FURTHER FUNCTIONAL GROUP TRANSFORMATIONS THEN AFFORDED THE OXIDATION PRECURSOR 3CHEME (OWEVER ALL OXIDA TION PROTOCOLS FAILED TO DELIVER THE DESIRED ALDEHYDE AND THROUGH A SERIES OF CONTROL EXPERIMENTS AN UNFAVOURABLE CONFORMATION IMPARTED BY THE PRESENCE OF THE DISUBSTITUTED DITHIANE SEEMED THE MOST PROB
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
S
O
S
a
26
O S
(MeO)2HC
H OTBS
S
575
S
32
H
H
Nuc
617 Observed
32
S
TBSO
(MeO)2HC
(MeO)2HC
618
619
22
576
S S
27
+
TBSO
S
OH
S
S S
27
32 27
22
OH
OTBS
R
22
b TIPSO
TIPSO
MeO
42
42
MeO 38
H
38
H
32
PMBO S S
OTBS 27
S S
MeO
574
33
I OPMB
OMe
S
32
S S
27
S
TBSO
c
(MeO)2HC 22
620 (MeO)2HC 22
621
3CHEME A T"U,I (-0!4(& n # THEN PRECOOLED n # SOLUTION IN (-0!4(& B -E) .A( # 4(& 24 C T"U,I (-0!4(& n # THEN PRECOOLED n # SOLUTION IN (-0!4(&
ABLE CAUSE .OT SURPRISINGLY THE MULTIPLE ELECTROPHILIC SITES IN KETO ALDE HYDE MADE FURTHER ELABORATION THROUGH ANIONIC CHEMISTRY UNSUITABLE 3CHEME )N AN ATTEMPT TO RESOLVE THIS ISSUE AN ALTERNATIVE FRAGMENT ASSEMBLY ORDER WAS INVESTIGATED AND PROVED TO AFFORD 3MITHS FIRST SUCCESSFUL ROUTE TO THE # # CARBON FRAMEWORK OF RAPAMYCIN !S BOTH AND CONTAIN SIMILAR FUNCTIONALITY THEIR UNION WAS COMPLICATED BY COMPETI TIVE PROTON TRANSFER 4HIS DIFFICULTY COULD BE RESOLVED BY ADDITION OF THE PRE COOLED ALDEHYDE TO AN EXCESS FIVE EQUIVALENTS OF THE PREFORMED DITHIANE ANION OF AT n # !LTHOUGH NOT IDEAL THE MAJORITY OF UNREACTED COUPLING PARTNER COULD BE RECOVERED AND AFFORDED THE ADDITION PRODUCT AS A MIXTURE OF # EPIMERS IN OVERALL YIELD !S DISCUSSED PREVIOUSLY AND ASSUMING &ELKIN !NH CONTROL IT WAS EXPECTED THAT THE MAJOR COMPONENT OF THIS MIXTURE WOULD BE THE DESIRED # 2 ALCOHOL 3URPRISINGLY 8 RAY ANALYSIS OF A DERIVATIVE OF REVEALED THAT UNDESIRED # 3 ALCOHOL WAS THE DOMINANT PRODUCT SUGGEST ING ADDITION OF THE METALLATED DITHIANE TO A CONFORMATION IN WHICH THE VINYL SIDE CHAIN WAS PERPENDICULAR TO THE CARBONYL 4HIS WAS SUPPORTED BY A PREVIOUSLY OBTAINED CRYSTAL STRUCTURE OF THE ELECTROPHILIC PARTNER
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO O S
S
576
S
a
32 27 H OTBS
CH(OMe)2
32
MeO
b
27
S
42
38
H
574 TIPSO MeO
TIPSO
42
38
H
MeO
OTBS 27
R
38
+
OTBS
33
623
I OPMB
TIPSO
H
42
32
PMBO S S
MeO
d
OTBS
38
O
H
33
27
S S
S S
HO
(MeO)2HC
22
22
PMBO
S S
e
CH(OMe)2
SS
c
(MeO)2HC R = OH, 625
33
42
32
PMBO S S
H PMBO
622 MeO
38
27
TIPSO
OTBS
42
SS
27 H OTBS
CH(OMe)2
26
22
615 575
624
R = OMe, 621
3CHEME A -E/ #( -E/( 4S/( B T"U,I (-0!4(& n # THEN PRECOOLED n # SOLUTION IN (-0!4(& C #L##/( ACETONE D T"U,I (-0!4(& n # THEN PRE COOLED n # SOLUTION IN (-0!4(& E -E) .A( # 4(&
)N THIS THE SOLID STATE CONFIGURATION CLEARLY DEMONSTRATES THAT THE STERIC INFLUENCE OF THE TRISUBSTITUTED OLEFIN OVERRIDES THAT OF THE STEREOELECTRONIC EFFECT OF THE α ALKOXY SUBSTITUENT .O MENTION OF EFFORTS TO INVERT THE # STEREOCHEMISTRY WAS MADE ALTHOUGH BY VIRTUE OF ITS α DISPOSITION TO THE BULKY DISUBSTITUTED DITHIANE SUCH A TRANSFORMATION MIGHT BE DIFFICULT )N ANY REGARD SEPARATION OF THE DIASTEREOMERS WAS POSSIBLE AND ALLOWED UNEVENTFUL TRANSFORMATION TO THE # # ADVANCED FRAGMENT AFTER A SEC OND ALKYLATION WITH PRIMARY IODIDE 3CHEME !LTHOUGH SUCCESSFUL IN DELIVERING DESIRED TARGET THE POOR SELECTIV ITY AND THUS OVERALL STEP YIELD ALONG WITH ITS TEDIOUS PURIFICA TION RADIAL CHROMATOGRAPHY NECESSITATED AN ALTERNATIVE STRATEGY 3MITH HAD ACCURATELY NOTED THE DRAMATIC EFFECT THAT A DISUBSTITUTED DITHIANE CAN HAVE ON REACTIVITY ON NUMEROUS OCCASIONS VIDE SUPRA AND CORRECTLY SPECULATED THAT THE SAME MIGHT HOLD TRUE WITH REGARD TO STEREOCHEMICAL OUTCOMES #ONSEQUENTLY THE # # FRAGMENT WAS MODIFIED TO ALLOW ACCESS TO THE PREVIOUSLY INACCESSIBLE ALDEHYDE !LKYLATION OF THE METALLATED DITHIANE DIMETHYL ACETAL WITH FOLLOWED BY HYDRO LYSIS REVEALED THE ALDEHYDE FUNCTION WITHOUT COMPLICATION 3UBSEQUENT ADDITION TO A SOLUTION OF THE PREFORMED ANION OF THEN GENERATED A
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
MIXTURE OF # α AND β EPIMERS BUT IN MUCH IMPROVED SELECTIVITY FOR THE DESIRED CONFIGURATION VERSUS ONCE AGAIN SEPARATED BY RADIAL CHRO MATOGRAPHY 4HIS IMPORTANT MODIFICATION GREATLY IMPROVED THE MATERIAL THROUGHPUT FOR THE ADVANCED # # CARBON BACKBONE AND OVERALL WAS PREPARED IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD 3CHEME !LL THAT REMAINED TO COMPLETE THE # # STANNANE FOR THE SYNTHESIS OF BOTH RAPAMYCIN AND DEMETHOXYRAPAMYCIN WAS A ONE CARBON HOMOLOGATION TO INSTALL THE EVENTUAL VINYL STANNANE AT # AND A SERIES OF PROTECTING GROUP MANIPULATIONS 4HE RECENT DEVELOP MENT OF THE 3TORK :HAO BISTRIFLUOROACETOXY IODOBENZENE PROTOCOL ;= HAS PROVED VERY VALUABLE FOR REVEALING KETONE FUNCTIONALITY IN COMPLEX MOLECULES AS EXEMPLIFIED BY THE SMOOTH CONVERSION OF TO AND TO 0ALLADIUM CATALYSED HYDROSTANNATION OF THE RESULTING BIS KETO INTERMEDIATES AND COMPLETED THE SYNTHESIS OF THE # # VINYL STANNANE FOR BOTH RAPAMYCIN AND ITS ANALOGUE IN EXCELLENT YIELD AND WITH HIGH REGIO AND STEREOSELECTIVITY /VERALL THE SYNTHESIS OF 2 ( AND 2 /-E REQUIRED A TOTAL OF AND STEPS IN AND YIELDS RESPECTIVELY 3CHEME #ONSTRUCTION OF THE REMAINING # # CARBOXYLIC ACID ONCE AGAIN UTILISED IN PART PREVIOUSLY DEVELOPED CHEMISTRY EMPLOYED IN 3MITHS SYNTHESIS OF THE LATRUNCULINS ; = "AEYER 6ILLIGER OXIDATION OF READ ILY AVAILABLE ALLYLCYCLOPENTANONE GENERATED RACEMIC LACTONE WHICH WAS THEN ENOLISED UNDER STANDARD CONDITIONS AND TRAPPED WITH -E) TO INSTALL THE # α METHYL GROUP !LTHOUGH INITIALLY A MIXTURE OF DIASTEREOMERS ORTHO ESTER FORMATION WITH 2 2 BUTANEDIOL EFFECTED BOTH RESOLUTION AND EQUILIBRATION IMPROVING THE TRANSCIS RATIO TO 4HIS UNEXPECTED YET FORTUITOUS RESULT PRESUMABLY AROSE DUE TO A MORE BIASED CONFORMATIONAL PREFERENCE FOR THE ORTHO ESTERS n VERSUS THE START ING LACTONE 4HE ISOMERS COULD THEN BE SEPARATED BY PREPARATIVE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (0,# TO AFFORD BOTH AND IN n YIELDS 7HILE WAS EMPLOYED IN THE LATRUNCULIN PROJECT ITS DIASTEREOMER WAS SUITABLE FOR FURTHER ELABORATION FOR THE RAPAMYCIN SYNTHESIS 3CHEME /ZONOLYSIS OF THE TERMINAL ALKENE FURNISHED ALDEHYDE WHICH WAS SUBSEQUENTLY CONDENSED WITH THE LITHIO ANION OF ENYNES OR PREPARED FROM THE KNOWN SILYL DIYNE ;= BY STANNYLATION ; =
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO MeO
TIPSO
42
38
MeO
H
38
32
PMBO S S
OTBS 27
R
TIPSO
42
MeO
H
38
32
PMBO S S
a
42
OTBS
H
32
PMBO S S
b,c
OTBS
27
S S
27
S S
R
S S
R
22
(MeO)2HC 22 R = H, 612 R = OMe, 621
OHC 22
R = H, 626 R = OMe, 627
R = H, 628 R = OMe, 629
d
TIPSO
MeO
TIPSO
42
38
MeO
H 32
HO
TIPSO
42
38
MeO
H 32
OTBS
O
27
R
f
HO
38
H 32
OTBS
O
27
O
42
R
e
HO S S
S S
R
22
22
Bu3Sn R = H, 572 R = OMe, 571
OTBS 27
O
22
R = H, 632 R = OMe, 633
R = H, 630 R = OMe, 631
3CHEME A 4S/( ACETONE 24 2 ( 2 /-E B #"R 0.-E 4(&(-04 n # 2 ( 2 /-E C N"U,I 4(& n # 2 ( 2 /-E D $$1 #(#L(/ # 2 ( 2 /-E E 0H)/##& 4(&(/-E/( # 2 ( 2 /-E F "U3N( 0H0 0D#L 4(& # 2 ( 2 /-E
TRANSMETALLATION )NTERESTINGLY THE : ENYNE GAVE MUCH HIGHER SELECTIV ITY VERSUS IN FAVOUR OF THE DESIRED # SECONDARY ALCOHOL STEREOCHEMISTRY HOWEVER AT THIS POINT IT WAS NOT CLEAR IF IT WOULD BE SUITABLE FOR FURTHER ELABORATION AS THE OLEFIN GEOMETRY ACROSS # # WAS OPPOSITE TO WHAT WAS REQUIRED 3CHEME 4HE POOR RATIO OBTAINED FROM ADDITION OF THE % ENYNE WAS SOMEWHAT OFFSET BY AN OXIDATION AND REAGENT CONTROLLED #"3 ; = REDUCTION PROTOCOL TO RECYCLE THE UNDESIRED EPIMER 3IMILARLY COULD BE RECYCLED IN THIS CASE WITH REASONABLE SELECTIVITY BY SIMPLE TREATMENT WITH SODIUM BOROHYDRIDE 3CHEME !TTEMPTED SYNTHESIS OF THE REQUISITE % % DIENYLSTANNANE VIA PALLADIUM CATALYSED HYDROSTANNYLATION GAVE EXCLUSIVELY THE INTERNAL STANNANE &OR TUNATELY THE OPPOSITE REGIOISOMER COULD BE GENERATED UNDER RADICAL CONDI TIONS WITH GOOD % : SELECTIVITY FOR THE TERMINAL VINYL STANNANE 4HE LATTER OLEFIN ISOMER WHEN RESUBJECTED TO THE REACTION CONDITIONS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O
O a
16
10
O b
O
10
10
O
c
O
O 10
16
O +
O
O 10
O 10
16
634
O
d
O
O
O
16
635
636
cis isomers
O
16
637
16
638
H
639
O 10
O
O 16
639
H
TMS
TMS
Me
Bu3Sn
e
20 17
20
g
f
h
TMS
TMS
Me
Bu3Sn 17
640
641
O
642
O 10
O
O
OH 16
+ 20
TMS
O 10
O
O
o,p
m,n
OH
O 10
16
20
TMS 644
640
O
TMS
O
20
OH
+
16
O 10
O
20
OH 16
TMS 643
645
i,j
646 k,l
O
O 10
O
OMe
20
SnBu3
16
647
3CHEME A M#0"! .A(#/ #(#L 24 B ,$! 4(& n # THEN -E) n # → 24 TRANSCIS C 2 2 BUTANEDIOL #3! 0H( $EAN 3TARK REFLUX TRANSCIS n ISOLATED YIELD D / #(#L n # THEN 0H0 n # → 24 E "U3N"U #U#. ,I 4(& n # THEN SAT .(#L AQ -E/( n # → 24 F "U3N"U #U#. ,I 4(& n # → n # THEN SAT .(#L AQ -E/( n # → 24 G N"U,I 4(& n # THEN n # → # H N"U,I 4(& n # THEN n # → # I 0$# #(#L J "(q4(& ) DIPHENYL METHYLTETRAHYDRO ( PYRROLO; C=; =OXAZABOROLE 0H#( K 0$# #(#L .A"( -E/( M +/( AQ $-3/ -E) N "U3N( !)". 0H#( # O +/( AQ $-3/ -E) P "U3N( !)". 0H#( #
UNDERWENT PARTIAL ISOMERISATION TO THE % % ISOMER SUGGESTING THAT A FREELY ROTATING ALLYLIC RADICAL INTERMEDIATE ;= WAS INVOLVED AND THUS PERHAPS WOULD PERMIT USE OF THE : ENYNE PREVIOUSLY DESCRIBED )NDEED SUB JECTING TO THE SAME RADICAL CONDITIONS GAVE IN REASONABLE YIELD IDENTICAL IN ALL REGARDS WITH MATERIAL PREPARED FROM 3CHEME !S THE NATURE OF THE NUCLEOPHILIC AND ELECTROPHILIC PARTNERS AS WELL AS THEIR ORDER IE # STANNANE AND # IODIDE OR THE REVERSE IS OF CONSID
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
ERABLE IMPORTANCE IN THE EVENTUAL 3TILLE COUPLING REACTION MODEL STUDIES WERE FIRST PERFORMED ! SIMPLE VINYL IODIDE READILY PREPARED FROM 3CHEME SERVED AS THE TEST FRAGMENT ALONG WITH DIENYLSTANNANE !TTEMPTED COUPLING OF THESE TWO SPECIES UNDER STANDARD CONDITIONS FURNISHED THE DESIRED TRIENE IN ONLY LOW YIELD AND OMINOUSLY AS A MIXTURE OF % AND : ISOMERS 4HE FORMATION OF SIGNIFICANT QUANTITIES OF A TETRAENE FROM HOMOCOUPLING OF WAS ALSO OBSERVED SUGGESTING THAT SLOW OXIDATIVE ADDITION OF THE VINYL IODIDE TO PALLADIUM MIGHT BE THE CAUSE -ODIFICATION OF VARIOUS REACTION PARAMETERS CATALYST LIGAND SOL VENT ETC AFFORDED NO MEANINGFUL IMPROVEMENT IN THE REACTION OUTCOME )T IS KNOWN THAT VINYL IODIDES WITH NEARBY ,EWIS BASIC FUNCTIONALITY SUCH AS ESTERS OR AMIDES CAN PERFORM POORLY IN 0D CATALYSED 3TILLE CROSS COU PLINGS ; = /NE INTERPRETATION OF THIS BEHAVIOUR IS INHIBITION THROUGH LIGATION OF THE PROXIMAL ,EWIS BASE AND THIS IS CERTAINLY A POSSIBILITY WITH SUBSTRATES SUCH AS WHICH BEAR A DITHIANE GROUP RELATIVELY CLOSE TO THE VINYL IODIDE #ONSEQUENTLY THE NUCLEOPHILIC AND ELECTROPHILIC PARTNERS WHERE INVERTED THROUGH USE OF STANDARD FUNCTIONAL GROUP MANIPULATION AND RESUBJECTED TO 3TILLE CONDITIONS 3CHEME ! DRAMATIC IMPROVEMENT IN BOTH YIELD AND ISOMERIC PURITY OF THE DESIRED TRIENE COUPLING PARTNER % WAS OBSERVED AND AS SUCH THE DECISION TO PREPARE THE # # VINYL IODIDE RATHER THEN THE CORRESPONDING STANNANE WAS MADE 4HESE OBSERVA TIONS ALSO AIDED THE ,EY GROUP VIDE INFRA IN THEIR SYNTHESIS OF RAPAMYCIN 7ITH THE SUCCESSFUL ISOMERISATION OF THE : ENYNE TO THE % % DIE NYLSTANNANE DURING RADICAL STANNATION 3CHEME BOTH AND WERE FURTHER ELABORATED TO THE FULLY FUNCTIONALISED # # CARBOXYLIC ACID &OR THE FORMER / METHYLATION OF UNDER CONDITIONS WHICH RETAINED THE 4-3 GROUP ON THE TERMINAL ALKYNE FOLLOWED BY ADDITIONAL STANDARD FUNCTIONAL GROUP TRANSFORMATIONS FURNISHED ALDEHYDE READY FOR ALDOL CONDENSATION WITH THE DIANION OF . ACETYL , PIPECOLIC ACID $OUBLE DEPROTONATION OF WITH ,I(-$3 AND ADDITION OF FURNISHED THE DESIRED ADDUCT AS AN INCONSEQUENTIAL MIXTURE OF DIASTEREOMERS WHICH AFTER METHYL ESTER FORMATION AND OXIDATION WITH $ESS -ARTIN PERI ODINANE GAVE THE INTERMEDIATE TRICARBONYL IN GOOD YIELD $ESILYLATION WITH (& IN ACETONITRILE REMOVED BOTH 4"3 AND 4%3 PROTECTING GROUPS AND ALSO INDUCED SPONTANEOUS LACTOL FORMATION AS A SINGLE ISOMER /BSERVA TIONS BY 3MITH AS WELL AS $ANISHEFSKY ;= HAD INDICATED THE NECESSITY OF PROTECTION OF THE # HEMIKETAL &URTHERMORE EVALUATION OF ENDGAME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O
O
O O
10
OMe
a
S S
SnBu3
16
O
I
+
20
647
648
b
c
O
O
OMe
+
20
O
Bu3Sn
d
S S
H
16
20
O
OMe 16
650
S S
O 10
I
16
OMe
649
O 10
O 10
S S
20
651
E-649
3CHEME A -E#. 0D#L $-& 24 MIXTURE OF OLEFIN ISOMERS B ) #(#L # C T"U,I %T/ n # THEN N"U3N) D -E#. 0D#L $-& 24
1
6
O
O 10
16
R1 = R2 =
a,b,c,d,e
OH R2
O
O
H
TBSO
10
N Ac
OMe R2 16
1
R
CO2H
N
f
R1
O
HO2C 1
OH
TBSO
OMe R2
577 10
16
R1
6
TMS , R2 = H, 643 TMS , R1 = H, 645
R1 = R2 =
TMS , R2 = H, 578 TMS , R1 = H, 652
R1 = R2 =
TMS , R2 = H, 653 TMS , R1 = H, 654
g,h,i,j
1
O
O TESO
m
O 10
N
OMe 16
H 573
20
I
N
CO2Me l
O 10
O H
658
O TESO
I
O
N
CO2Me k
O 10
OMe 16
1
1
1
CO2H
N O TESO
O TESO
16
H 657
O 10
OMe
CO2Me
O
OMe R2 16
SnBu3
H R1 = R2 =
R1
H , R2 = H, 655 H , R1 = H, 656
3CHEME A .A( # 4(& # THEN -E) # : B !C/( 4(& (/ 24 : C 4"3#L )M $-& 24 : D $)"!, ( 4(& n # → # : E #/#L $-3/ #(#L n # THEN $)0%! n # → # : F ,I(-$3 4(& n # → # THEN n # → # : G #(. %T/ # : H $-0 0Y #(#L 24 : I (& AQ #(#. 24 : J 4%3/4F %T. LUT #(#L n # : K "U3N( !)". 0H( # : L ) #(#L 24 M ,I) 0Y #
PROTECTING GROUPS THROUGH THE DEGRADATIONRECONSTRUCTION OF THE NATURAL PRODUCT LED TO THE CHOICE OF A TRIETHYLSILYL FUNCTION WHICH WAS INTRODUCED VIA 4%3/4F TO FURNISH ENYNE 3IMILARLY AN IDENTICAL SERIES OF REACTIONS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TIPSO MeO
38
1
N O
H
O
OMe
27
R
a
N H1 O O 9
O TESO
O
I
H
H 32
OTBS
O
R = H, 572 R = OMe, 571
20
16
38
32
HO
+
42
MeO
CO2H
O TESO 10
TIPSO
42
10
O
O
OTBS
O
O
R
OMe 22
573
Bu3Sn
I Bu3Sn
16
22
R = H, 659 R = OMe, 660 b
HO
TIPSO
42
MeO
38
MeO
H
TIPSO
42
38
32
O HO
9 10
N H1 O O O
O
OMe
42
MeO
H
38
H 32
32
OH
O
O
R
d
N H1 O O 9
O HO 10
22
16
(-)-rapamycin (R = OMe) 7 (-)-27-demethoxyrapamycin (R = H) 8
O
O
O
OMe 16
R = H, 663 R = OMe, 664
OTBS O
R
c
O TESO
9
10 22
N H1 O O O
O
O
OMe
OTBS O
R 22
16
R = H, 661 R = OMe, 662
3CHEME A %$#) $-!0 $-!0q(#L 24 FOR AND FOR B ; FURYL 0=0D#L $)0%! $-& 4(& 24 AND FOR C 4"!& !C/( 4(& # D (&q0Y 0Y 4(& # → 24 FOR AND FOR BOTH OVER TWO STEPS
BEGINNING FROM THE : ENYNE AFFORDED A SECOND PRECURSOR FOR APPLI CATION OF THE FREE RADICAL STANNYLATION CONDITIONS )N THE EVENT BOTH AND AFFORDED THE SAME VINYL STANNANE IN REASONABLE YIELD THAT WAS THEN FURTHER ELABORATED TO CARBOXYLIC ACID THROUGH IODODESTANNY LATION AND HYDROLYSIS OF THE METHYL ESTER 4HIS LATTER TRANSFORMATION PROVED TO BE EXTREMELY DIFFICULT AND OF A MYRIAD OF CONDITIONS INVESTIGATED ONLY THE USE OF AN EXCESS OF LITHIUM IODIDE IN ANHYDROUS PYRIDINE AFFORDED THE DESIRED PRODUCT IN ACCEPTABLE YIELD 3CHEME 7ITH ALL THE REQUISITE PIECES IN HAND FOR BOTH RAPAMYCIN AND DEMETHOXYRAPAMYCIN THE STAGE WAS SET FOR THE COMPLETION OF THEIR TOTAL SYNTHESES !LTHOUGH IN PRINCIPLE THE MACROCYCLE OF BOTH NATURAL PRODUCTS COULD BE CONSTRUCTED EITHER THROUGH LACTONISATION OR VIA A 3TILLE COUPLING REACTION MODEL STUDIES HAD SUGGESTED THAT THE LATTER WOULD PRO VIDE SUPERIOR RESULTS 4HUS UNION OF WITH WAS EFFECTED UNDER STAN DARD COUPLING CONDITIONS WITH THE ADDITION OF $-!0q(#L WHOSE PRESENCE
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3CHEME 3UMMARY OF 3MITHS TOTAL SYNTHESIS OF RAPAMYCIN
SUPPRESSED THE UNDESIRED FORMATION OF THE . ACYLUREA DERIVED FROM 3CHEME ;= )NTERESTINGLY NO β ELIMINATION ACROSS THE # # ALDOL LINKAGE WAS OBSERVED DURING ESTER FORMATION DESPITE THIS BEING A WELL DOCUMENTED TRANSFORMATION OF RAPAMYCIN UNDER BASIC CONDITIONS ; = !TTEMPTED MACROCYCLE FORMATION WITH UNDER THE PREVIOUSLY OPTI MISED 3TILLE REACTION CONDITIONS SEE 3CHEME FAILED TO PROVIDE ANY OF THE DESIRED PRODUCT &ORTUNATELY RECOURSE TO THE &ARINA 3COTT CATA LYST SYSTEM ; = RESOLVED THIS DIFFICULTY AND FURNISHED IN THE BEST YIELD AT THE TIME FOR MACROCYCLISATION IN THE RAPAMYCIN SERIES )N THIS TRANSFORMATION IT IS BELIEVED THAT THE ELECTRON POOR PHOSPHINES IN ; FURYL 0=0D#L ACCELERATE THE RATE DETERMINING STEP IN A 3TILLE SYSTEM NAMELY THAT OF TRANSMETALLATION ;n= 4REATMENT OF WITH 4"!& BUFFERED WITH !C/( EFFECTED SELECTIVE DESILYLATION TO AFFORD PRIMARILY ALTHOUGH
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 3UMMARY OF 3MITHS TOTAL SYNTHESIS OF DEMETHOXYRAPAMYCIN
A CONSIDERABLE AMOUNT OF AN INSEPARABLE SPECIES BELIEVED TO BE THE SEVEN MEMBERED # OXEPANE ;= WAS ALSO OBSERVED &INALLY FURTHER DESILYLATION OF THE # 4"3 AND # 4)03 GROUPS WITH (&q0Y FURNISHED RAPAMYCIN IDENTICAL IN ALL RESPECTS WITH THE NATURAL MATERIAL 3IMILARLY AN ANALOGOUS SET OF TRANSFORMATIONS UPON IN PLACE OF GAVE DEMOTHOXYRAPAMYCIN IN ACCORD WITH CHARACTERISTICS OF AN AUTHENTIC SAMPLE )N SUMMARY THE FLEXIBLE APPROACH BY !MOS " 3MITH ))) SUCCESSFULLY DELIVERED BOTH RAPAMYCIN AND THE NATURALLY OCCURRING DEMETHOXY CONGENER EFFICIENTLY IN AND STEPS RESPECTIVELY 3CHEMES AND )N THE RAPAMYCIN SERIES THIS LINEAR STEP COUNT IS THE SHORTEST OBTAINED TO DATE AND HIGHLIGHTS THE HIGH LEVEL OF CON VERGENCY AND EFFICIENT END GAME OF THE 3MITH SYNTHESIS /THER NOTABLE FEATURES INCLUDE THE ADVANCEMENT OF BOTH DITHIANE COUPLING REACTIONS FOR UNION OF COMPLEX FRAGMENTS AND THE USE OF σ BOND FORMING STRATEGIES FOR
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
THE STEREOSELECTIVE CONSTRUCTION OF OLEFINS 4HE IMPORTANCE OF THE FORMER METHODOLOGY WHICH IS UBIQUITOUS IN 3MITHS CHEMISTRY CANNOT BE UNDER STATED AS IT HAS PROVED INVALUABLE IN A VARIETY OF OTHER CONTEXTS &INALLY THE 3MITH APPROACH AFFORDED AT THE TIME THE HIGHEST YIELDING APPROACH TO GENERATION OF THE FORMIDABLE MACROCYCLE OF AND ALSO SERVED TO CONFIRM THE STRUCTURE OF
4HE ,EY TOTAL SYNTHESES OF RAPAMYCIN 3OME YEARS AFTER ITS ORIGINAL ISOLATION AND YEARS AFTER THE FIRST TOTAL SYNTHESIS OF RAPAMYCIN THE FIFTH AND MOST RECENT APPROACH TO THIS NATURAL PRODUCT WAS DISCLOSED BY 3TEVEN 6 ,EY IN EARLY AT THE 5NI VERSITY OF #AMBRIDGE 5+ ;= &ORTUITOUSLY TIMED WITH A RENEWED INTEREST IN THE FIELD AND REPRESENTING OVER YEARS OF EFFORT WITHIN THE GROUP THIS ACHIEVEMENT IS AN EXCEPTIONAL EXAMPLE OF THE PERSEVERANCE AND DEDICATION REQUIRED OF A PRACTITIONER IN THE ART OF TOTAL SYNTHESIS 4HIS IS THE SECOND CONTRIBUTION OF OUR GROUP TOWARDS THE CONSTRUCTION OF THE TRICARBONYL MAC ROLIDE FAMILY OF NATURAL PRODUCTS FOLLOWING SUCCESS IN THE ANTASCOMICIN SERIES VIDE SUPRA 4HE STRATEGY CONTAINS A COMBINATION OF INVENTIVE BOND FORMING EVENTS AND GROUP DEVELOPED METHODOLOGIES AS DISCUSSED BELOW 2ETROSYNTHETICALLY WE SOUGHT TO ADDRESS THE OFTEN POOR YIELDS ASSOCIATED WITH FORMATION OF RAPAMYCINS CORE VIA A TRANSANNULAR CATECHOL TEMPLATED $IECKMANN LIKE REACTION SIMILAR TO THE NOVEL APPROACH FIRST EMPLOYED IN OUR SYNTHESIS OF ANTASCOMICIN ;= &URTHER DISCONNECTION ADJACENT TO THE CENTRAL OLEFIN OF THE TRIENE THROUGH A PALLADIUM CATALYSED 3TILLE COUPLING REVEALED THE # # LACTONE AND # # VINYL STANNANE AS PRINCIPLE SYNTHETIC TARGETS 3CHEME 4HE FORMER LACTONE IS READILY SIMPLIFIED THROUGH DISCONNECTION AT # # WHEREIN THE RING OPENING OF A STEREODEFINED OXIRANE WITH A HOMOCHIRAL SULFOXIDE SUCH AS WAS ENVISIONED TO EFFECT UNION OF THESE SIMPLIFIED FRAGMENTS 4HE LARGER STANNANE IS MORE COMPLEX AND IN A STRATEGY SIMILAR TO THAT FIRST EMPLOYED BY 3CHREIBER A FORMAL DIANION SPACER UNIT SHOULD ALLOW FOR SEQUENTIAL CARBANIONIC COUPLING WITH SUITABLE # # AND # # ELECTROPHILES !S WAS THE CASE IN THE $ANISHEFSKY APPROACH TO RAPAMYCIN WE ALSO HOPED TO EXPLOIT THE RELATIVE ABUNDANCE OF NATURAL SUPPLIES OF RAPAMYCIN
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TBSO HO MeO
38
38
10
O
H 32
32
9
MeO
OH
H
N H1 O O
42
C21-C42 stannane 666 MeO
O HO
TBSO
42
42
O
1
O
OH O
MeO
OMe
22
O
OH
665
9
8
N + BocH O Br
O
O
32 29
H
33
Br
S
+
669
670
O
OTES
O
O
MeO
Br 667 + O
S 38
1
CO2H N Boc 12
Me3Sn
+
OPMB 22
R
OPMB
28
OMe 671
22 16
10
O
10
+
OMe
20
(-)-rapamycin 7
16
C10-C20 lactone 668
12
SO2Ph + O
PMBO I
13
672
OMe OBn 16
673
3CHEME 2ETROSYNTHETIC ANALYSIS
AND UNDERTAKE INSTRUCTIVE DEGRADATIVE STUDIES ; = %ND GAME PROTECTING GROUP STUDIES HAD SUGGESTED THAT DIFFERENTIATED SILYL GROUPS AT # 4"3 AND # 4%3 WERE DESIRED AND CONDITIONS WERE OPTIMISED TO INSTALL THESE SEQUENTIALLY AND IN HIGH YIELD TO AFFORD 3CHEME %XCISION OF THE PIPECOLATE AND TRICARBONYL REGIONS FROM UNDER THE CONDITIONS DEVELOPED BY ,UENGO ;= MINIMISED FORMATION OF THE ENONE BY PRODUCT FORMED ^ OBSERVED FROM β ELIMINATION ACROSS THE # # ALDOL LINKAGE AND GAVE IN GOOD YIELD 3UBSEQUENT REINSTALLATION OF A PIPECOLATE FRAGMENT IN ITS "OC PROTECTED FORM VIA STANDARD CONDITIONS FURNISHED AND EXTENSIVE STUDIES WERE INITIATED TO SELECTIVELY CLEAVE THE # # TRIENE PORTION OF THE MOLECULE )N ACCORD WITH OUR PREVIOUS SUCCESS WITH SELECTIVE OZONOLYSIS IN THE AVERMECTIN "A AND INDANOMYCIN TOTAL SYNTHESIS PROJECTS ; = TREATMENT WITH OZONE UNDER CAREFULLY CONTROLLED CONDITIONS GAVE ENAL AND ALDEHYDE AS MAJOR FRAGMENTS ALONG WITH AN INSEPARABLE MIXTURE OF LACTONE DERIVATIVES AND !LL ATTEMPTS TO CONVERT TO WERE ULTIMATELY UNSUCCESSFUL NEVERTHE LESS THE TWO PRODUCTS WERE PARTIALLY SEPARABLE AND ISOLATED ALDEHYDE WOULD PROVE VERY USEFUL IN LATER VALIDATION OF SYNTHETIC INTERMEDIATES )N ADDITION FURTHER OZONOLYSIS OF THE MIXTURE OF LACTONES GAVE PURE ENAL WHICH WAS SIMILARLY CONSTRUCTIVE IN DEVISING THE FORWARD SYNTHESIS &INALLY THE READILY AVAILABLE TRIENE DERIVATIVE ALSO INTERSECTED MATERIAL IN THE FORWARD DIRECTION AND GIVEN THE COMPLEXITY OF .-2 ANALYSIS PROVIDED ANOTHER IMPORTANT CHECKPOINT )N THE FORWARD DIRECTION TWO SYNTHESES OF THE LACTONE PORTION OF RAPA MYCIN WERE IN FACT DEVELOPED BOTH OF WHICH COMMENCE WITH CIS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBSO
HO
MeO
MeO
38
H
38
TBSO
42
42
H
O
N H1 O O
9
O
10
O
a,b
OH O
most electrophilic
MeO
OMe
N H1 O O 9
O HO 10
10
TBSO
HO NC HO
MeO 22
O
O
OTES
O
O
MeO
OMe
38
TBSO
O
MeO
O
MeO OMe
NC
H
N H1 O O O OH
O
O
9
10
22
16
38
32
OTES
d
O
22
42
O CN 677
OH O
10
O
MeO
OMe
O
N
32
22
OTES
O
675
H
N CO2H Boc 12
O
16
42
MeO
N H Boc O
N H1 O O 9
10
O
H
O
c
O
674
32
O
OTES
16
42
38
H 32
O
22
(-)-rapamycin 7
MeO
O
OMe
O
16
TBSO
38
32
32
O HO
42
MeO
O
OTES O
MeO
OMe
22
16 16
679
678
676
e various
TBSO MeO
X
42
38
TBSO
MeO
H
42
f 38
H 32
32
1
N H Boc O
O
OTES +
O
O
MeO 680
1
N H Boc O
O
O
OTES
O
O
MeO
+
10
O
O
OMe 20 16
682
681
+
CHO
10
O
OMe 18 16
CHO
683
OHC
OHC 22
22
3CHEME A 4"3#L )M $-& 24 B 4%3/4F LUT #(#L # C N"U.#. (/ 4(& n # D $## $-!0 #(#L E // 6 ,H ^ MIN MG OF #(#L n # THEN -E3 n # → 24 FOR FOR MIXTURE OF F MIXTURE OF AND RESUBJECTED TO OZONOLYSIS CONDITIONS SEE E FOR MIN OVER TWO STEPS
BENZYLOXY BUTEN OL 3CHEMES AND ;= )N THE FIRST APPROACH APPLICATION OF THE VENERABLE 3HARPLESS ASYMMETRIC EPOXIDATION FOLLOWED BY 0ARIKH $OERING OXIDATION AND HOMOLOGATION VIA THE -ASA MUNE 2OUSH PROTOCOL ;= GAVE 3UBSEQUENT TREATMENT WITH $)"!, ( EFFECTED REDUCTION OF THE ESTER AND OXIRANE FUNCTIONALITIES THE LATTER IN A REGIOSELECTIVE FASHION AFFORDING ONLY THE PRODUCT FROM HYDRIDE ADDITION AT THE MORE ELECTROPHILIC CARBON CENTRE 7ITH IN HAND STANDARD PROTECT ING GROUP MANIPULATION A SECOND 3HARPLESS EPOXIDATION OF ALLYLIC ALCOHOL
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME A 4I/I0R DIETHYL TARTRATE T"U//( n # EE B 3/q0Y %T. $-3/ #(#L # → 24 C ,I#L %T/ 0/ #(#/#( $"5 #(#. 24 D $)"!, ( #(#L n # E 0IV#L 0Y #(#L # OVER TWO STEPS F .A( -E) # G $)"!, ( #(#L n # H 4I/I0R n DIETHYL TARTRATE T"U//( ¯ -3 #(#L n # I 40!0 .-/ #(#L #(#. ¯ -3 J 0H0#("R +(-$3 4(& # → 24 K &E#/ 4(& L #/ ATM 0H( # M 0T/ ( ATM %T/!C 24 N ,$! 4(& n # O ,$! 4(& n # THEN (/
AND OXIDATIONMETHYLENATION SEQUENCE FURNISHED IN GOOD YIELD 3CHEME ! LONG STANDING INTEREST WITHIN OUR GROUP HAS BEEN THE SYNTHESIS AND SYNTHETIC APPLICATION OF η π ALLYLTRICARBONYLIRON LACTONE COMPLEXES ;= 4HESE SPECIES ARE READILY PREPARED BY THE ACTION OF DIIRONNONACARBONYL ON VINYL OXIRANES OR RELATED CYCLIC SULFITES AND CAN AFFORD EITHER β LAC TONES THROUGH OXIDATIVE DECOMPLEXATION #!. OR UNSATURATED LACTONES VIA EXHAUSTIVE CARBONYLATION !PPLICATION OF THE LATTER PROTOCOL UPON GAVE THE INTERMEDIATE ENDO COMPLEX AS THE PREDOMINANT PRODUCT AND FOLLOWING CARBONYLATION WITH #/ AT ATM AFFORDED A MIXTURE OF α β AND β γ UNSATURATED LACTONES (YDROGENATION EMPLOYING !DAMS CATALYST ;= AND METHYLATION AT # VIA THE LITHIUM ENOLATE OF GAVE THE DESIRED ADVANCED INTERMEDIATE 5NFORTUNATELY THIS PRO CEEDED WITH LITTLE SELECTIVITY AND IN FAVOUR OF THE UNDESIRED EPIMER AT THE NEWLY INTRODUCED STEREOCENTRE (OWEVER IT WAS POSSIBLE TO RECYCLE SOME OF FOLLOWING SEPARATION (0,# THROUGH A DEPROTONATIONPRO TONATION SEQUENCE TO PROVIDE FURTHER SUPPLIES OF 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3CHEME A 4I/I0R DIETHYL TARTRATE T"U//( n # EE B 3/q0Y %T. $-3/ #(#L # → 24 C 0H0#("R +(-$3 4(& # → 24 D $)"!, ( 0H#( n # OVER THREE STEPS E N"U,I %T/ 24 THEN "OC /. 4(& 24 F )"R 0H#( #(#L n # DR G +#/ -E/( 24 H -E) !G/ $-& 24 I $(0 0043 #(#L 24 J ,I!L( %T/ n # K 0-"#L .A( $-& 24 L ACID ACTIVATED AMBERLITE¤ )2 -E/( 24 M 4S#L 0Y # → 24 N 0H3( +#/ 4(& 24 THEN REFLUX O M#0"! #(#L # → 24 P N"U,I 4(& n # THEN THEN "&q/%T n # → 24 Q - LITHIUM NAPHTHALENIDE 4(& n # R $$1 (/ #(#L 24 S 40!0 .-/ #(#L ¯ -3 T $)"!, ( 0H#( n # U 4"3#L )M $-!0 $-& 24 OVER TWO STEPS V 0D/( ( ATM %T/!C 24 W 40!0 .-/ ¯ -3 #(#L #(#. X -E-G"R 4(& n # DR Y 40!0 .-/ ¯ -3 #(#L #(#. 24 Z .A(-$3 4(& # THEN n # %: AA $)"!, ( 0H#( n # BB 4"!& !C/( (/ 4(& 24 CC 40!0 .-/ ¯ -3 #(#L OVER TWO STEPS DD #R#L #() 4(& DIOXANE # %:
!S A CONSEQUENCE OF THE HIGH TOXICITY OF &E#/ HIGH PRESSURE OF CARBON MONOXIDE AND POOR SELECTIVITY EXHIBITED IN INTRODUCTION OF THE NECESSARY METHYL GROUP AT # AN ALTERNATIVE ROUTE WAS DEVELOPED 3CHEME )N MUCH THE SAME MANNER AS DISCUSSED PREVIOUSLY WAS ELABORATED TO VINYL OXIRANE WHICH WAS THEN REGIOSELECTIVELY REDUCED WITH $)"!, ( AND CONVERTED TO IODOCARBONATE FOLLOWING 3MITHS )"R ELECTROPHILIC CYCLISATION PROCEDURE ;= 3YNTHESIS OF THE FIRST SUB UNIT OF THE EVENTUAL LACTONE WAS THEN ACCOMPLISHED THROUGH EPOXIDE FORMATION AND 0URDIE METHYLATION ;= IN A TOTAL OF EIGHT STEPS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
AND YIELD 3UBSEQUENT RING OPENING OF WITH THE α LITHIO ANION OF SULFONE READILY AVAILABLE FROM THE 2OCHE ESTER IN SEVEN STEPS YIELD PROCEEDED SMOOTHLY TO PROVIDE IN QUANTITATIVE YIELD &OLLOWING REDUCTIVE DESULFONYLATION AND 0-" ETHER CLEAVAGE TREATMENT OF THE ENSUING DIOL WITH TETRAPROPYLAMMONIUM PERRUTHENATE 40!0 ;= GENERATED THE DESIRED LACTONE VIA AN INTERMEDIATE HEMIACETAL 4HIS WAS IDENTICAL IN ALL REGARDS WITH MATERIAL PREVIOUSLY PREPARED IN A SHORTER STEPS AND HIGHER YIELDING SEQUENCE THAT PROVED MORE AME NABLE TO LARGE SCALE 3CHEME &URTHER ELABORATION TO THE EVENTUAL 3TILLE CROSS COUPLING PARTNER COMMENCED WITH A NECESSARY REDUCTIONPROTECTION PROTOCOL TO FORM AND PREVENT EPIMERISATION OF THE # METHYL STEREOCENTRE IN ENSUING STEPS ! STANDARD SEQUENCE WAS THEN EMPLOYED TO CONVERT THE PRIMARY BENZYL ETHER AT # TO THE CORRESPONDING METHYL KETONE !T THIS POINT A VARIETY OF ATTEMPTS TO INSTALL THE DESIRED % % DIENYL IODIDE CONCISELY VIA AN ENYNEHYDROSTANNYLATION APPROACH WERE ULTIMATELY UNSUCCESSFUL !LTHOUGH INTRODUCTION OF THE ENYNE WAS NOT PROBLEMATIC HYDROSTAN NYLATION IN OUR SYSTEM GAVE VERY LOW YIELDS IN CONTRAST TO THE SUCCESS ENJOYED IN THE 3MITH SYNTHESIS VIDE SUPRA ;= #ONSEQUENTLY A SEQUEN TIAL HOMOLOGATION APPROACH WAS ADOPTED THAT FEATURED FIRST REACTION WITH DIETHYLCYANOMETHYL PHOSPHONATE WHICH AT LOW TEMPERATURE GAVE GOOD LEVELS OF %: OLEFIN GEOMETRY FOR THE CONJUGATED PRODUCT AS ESTABLISHED BY .UCLEAR /VERHAUSER %FFECT ./% STUDIES &URTHER FUNC TIONAL GROUP MANIPULATION CONVERTED THE CYANO GROUP TO THE CORRESPOND ING ALDEHYDE AND REGENERATED THE LACTONE WHICH UPON APPLICATION OF THE %VANS "LACK MODIFICATION ;= OF THE 4AKAI OLEFINATION ;n= GAVE AS A MIXTURE OF OLEFIN ISOMERS IN OVERALL STEPS AND YIELD )NTERESTINGLY ALTHOUGH INSEPARABLE THIS MIXTURE WAS ULTIMATELY OF NO DETRI MENT IN COUPLING OF THE ADVANCED FRAGMENTS AND 4HE CONSTRUCTION OF THE # # VINYL STANNANE COMMENCED WITH PREPARATION OF THE CYCLOHEXYL PORTION OF RAPAMYCIN ; = &OR THIS PURPOSE THE READILY PREPARED BUILDING BLOCK TRIMETHYLSILYL METHYL PROPEN OL WAS SUBJECTED TO AN ACID CATALYSED *OHNSON ORTHOESTER #LAISEN REARRANGEMENT ; = WITH TRIETHYL ORTHOACETATE TO FURNISH IN GOOD YIELD 3UBSEQUENT MONO ADDITION OF THE LITHIO ANION OF SULFONE GENERATED A RACEMIC MIXTURE OF β KETOSULFONES 4HESE WERE THEN SUBJECTED TO ASYMMETRIC REDUCTION WITH THE #"3 SYSTEM ; =
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3CHEME A N"U,I 4-%$! %T/ 4(& THEN # → 24 THEN 4-3#L n # → 24 B . (3/ 4(& 24 C -E#/%T %T#/( # D T"U,I $-% n # THEN n # → 24 E "(q$-3 #"3 4(& EE F 0$# #(#L # → 24 UNOPTIMISED G 4"3/4F 0Y $-!0 #(#L # H 3N#L #(#L n # CIS TRANS I 4"3/4F 0Y $-!0 #(#L # J 3N#L #(#L n # TRANS CIS K "". 4(& # → 24 THEN .A/( AQ (/ L #/#L $-3/ #(#L n # THEN %T. n # → 24 M n % CROTYL")PC 4(& %T/ n # THEN . .A/( (/ n # → 24 N 6/ACAC T"U//( #(#L DR ISOLATED O N"U,I 4(& n # THEN #L#3 /0H P N"U3N( !)". 0H( REFLUX
TO AFFORD SYN AND ANTI ISOMERS WITH MODEST ENANTIOSELEC TIVITY )NTERESTINGLY ALTHOUGH THE SAME INTERMEDIATE OXONIUM ION WAS EXPECTED TO FORM FROM EITHER SILYLATED SUBSTRATE UPON TREATMENT WITH TIN TETRACHLORIDE INTRAMOLECULAR COLLAPSE OF THE PENDENT ALLYL SILANE BEGIN NING FROM THE SYN ISOMER GAVE PREDOMINATELY THE CIS METHYLENE CYCLOHEXANE WHEREAS THE ANTI ISOMER RESULTED IN A MIXTURE IN FAVOUR OF THE TRANS SIX MEMBERED RING )T HAS BEEN HYPOTHESISED THAT THE UNEXPECTED STEREO OUTCOME OF THE REACTION IS A CONSEQUENCE RAPID INTRAMOLECULAR CAPTURE BY THE ALLYLSILANE AT SUFFICIENT RATE THAT THE INITIALLY FORMED OXONIUM IONS DO NOT HAVE TIME TO EQUILIBRATE ; = 4HUS THE ANTI CONFIGURED STARTING MATERIAL AFFORDS THE % OXONIUM ION WHILE THE SYN ANALOGUE FURNISHES INITIALLY THE : COUNTERPART )N THE FORMER CASE THE FAVOURABLE PSEUDOEQUATORIAL POSITION IS NATURALLY ACHIEVED GIVING RISE PREFERENTIALLY TO THE DESIRED TRANS PRODUCT /N THE OTHER
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
TBSO
MeO
42
38
H
TBSO
32 1
N H Boc O
O
OR
O
MeO
O
MeO
MeS
42
38
H
+ O
722
32
PhO2S
29
MeO
669
Me3Sn
OPMB H O 22 OR
723
22
MeO
OH
32 29
THPO
OH
32
O
363
Br
c,d
a,b
THPO
29
29
724
e
32
THPO
f
32
THPO
32
29
Br
29
I
725
726
727
3CHEME A $(0 P4S/( 4(& 24 B ,I!L( %T/ 24 OVER TWO STEPS C #/#L $-3/ #(#L n # THEN %T. n # → 24 D #"R 0H0 #(#L # OVER TWO STEPS E N"U,I 4(& n # THEN -E) n # → 24 F #P:R( #L 4(& 24 THEN )
HAND TO AVOID UNFAVOURABLE STERIC INTERACTIONS WITH THE SILYL ETHER THE : OXONIUM ION MAY PREFER TO ADOPT A PSEUDOAXIAL POSITION AND THUS FAVOUR THE FORMATION OF THE CIS PRODUCT 4HE ABOVE RATIONAL FOR THE DISPARATE STEREOCHEMICAL OUTCOME HAS BEEN SUPPORTED BY THEORETICAL CALCULATIONS AT THE -- LEVEL ALTHOUGH TO DATE THE POSSIBILITY OF COM PETITION BETWEEN 3. AND 3. REACTION MANIFOLDS HAS NOT BEEN RULED OUT 3CHEME !S ONLY THE ANTI CONFIGURED α METHOXYSULFONE FAVOURED THE DESIRED PRODUCT A TWO STEP OXIDATIONREDUCTION SEQUENCE UPON THE SYN ISOMER WAS DEVISED TO SALVAGE MATERIAL 2EGARDLESS WITH THE ROUTE TO ESTABLISHED FURTHER ELABORATION TO THE # # EPOXIDE WAS RELATIVELY STRAIGHT FORWARD 2EGIOSELECTIVE AND STEREOSELECTIVE HYDROBORA TION WITH "". FOLLOWED BY STANDARD OXIDATIVE WORK UP FURNISHED THE CORRESPONDING PRIMARY ALCOHOL AND ALLOWED FOR CONVENIENT SEPARATION OF THE MINOR CIS DIASTEREOMER DERIVED PRODUCT FROM THE CYCLISATION &URTHER OXIDATION VIA THE 3WERN PROTOCOL AND "ROWN CROTYLATION ; = OF THE RESULTING ALDEHYDE GAVE AS THE MAJOR PRODUCT READILY SEPARABLE FROM TRACES OF OTHER DIASTEREOMERS BY FLASH CHROMATOGRAPHY 4HE RESULTING FREE HYDROXYL GROUP AT # WAS THEN UTILISED TO DIRECT 6/ACAC IN A HOMOAL LYLIC EPOXIDATION ;= WHICH PROCEEDED WITH REASONABLE SELECTIVITY ;= FOR THE DESIRED ISOMER )TS FUNCTION COMPLETED A STANDARD TWO STEP "ARTON DEOXYGENATION PROTOCOL ; = REMOVED THE SUPERFLU
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
OUS # HYDROXYL GROUP PROVIDING ACCESS TO THE DESIRED EPOXIDE IN STEPS AND OVERALL YIELD /RIGINALLY THE PLANNED CONSTRUCTION OF THE # # VINYL STANNANE INVOLVED THE COUPLING OF THE SAME OXIRANE WITH AN EVENTUAL ACYL ANION EQUIVALENT 3CHEME 4HE CYCLIC ACETAL COMPRISING MUCH OF THE # # REGION WAS INTENDED TO PROTECT BOTH THE # ALDE HYDE AND THE # KETONE IN ITS REDUCED FORM !LSO IT WAS ANTICIPATED THAT THE RING SYSTEM WOULD CONFER STEREOCONTROL WHEN EXTENDING THE # # CHAIN BY INFLUENCING FACIAL SELECTIVITY ON A PENDANT # ALDEHYDE 4HE SYNTHESIS OF THE FIRST OF THE REQUISITE FRAGMENTS FOR BEGAN FROM THE 2OCHE ESTER AND EMPLOYS A STANDARD SEQUENCE OF REACTIONS TO EVENTUALLY AFFORD VINYL IODIDE 4HE KEY BUILDING BLOCK FOR THE # # ELECTROPHILES IN OUR SYNTHESIS IS MESO ANHYDRIDE WHICH ALSO FOUND USE IN 3MITHS TOTAL SYNTHESIS OF RAPAMYCIN VIDE SUPRA /RIGINALLY AVAILABLE IN ONLY MODERATE YIELD USING LITERATURE PROTOCOLS ;= RECENT IMPROVEMENTS VIA A DYNAMIC CRYS TALLISATION OF THE NORMAL MIXTURE OF MESO AND DL ISOMERS HAS MADE ACCESS TO VERY PRACTICAL ;= 2EDUCTION OF THE ANHYDRIDE TO THE CORRESPOND ING DIOL WITH ,I!L( AND DESYMMETRISATION ; = WITH COMMERCIALLY AVAILABLE AND INEXPENSIVE ,IPASE 03 GAVE THE MONOACETYLATED PRODUCT IN CONSISTENTLY HIGH ENANTIOSELECTIVITY ! SIMPLE THREE STEP PROCEDURE ACCOMPLISHED CONVERSION TO THE HYDROXYL ALKENE FROM WHICH TWO STEP OXIDATION TO THE CORRESPONDING ACID AND SELENO CYCLISATION UNDER KNOWN CONDITIONS ;= AFFORDED LACTONE IN GOOD YIELD !S EXPECTED FOR A PRESUMED CHAIR LIKE TRANSITION WHEREIN BOTH # METHYL GROUPS ADOPT AN EQUATORIAL TRANSITION STATE THIS CYCLISATION OCCURRED WITH GOOD STEREOCONTROL AND ONLY TRACE AMOUNTS OF A MINOR DIASTEREOMER WERE NOTED /XIDATIVE ELIMINATION OF THE PHENYLSELENENYL MOIETY GAVE A SINGLE DIASTE REOMER OF THE CORRESPONDING ALKENE WHICH WAS CONVERTED VIA A MIXTURE OF ANOMERIC ACETALS TO THE DESIRED ALDEHYDE !LTHOUGH EITHER OF THE # EPIMERS COULD IN PRINCIPLE BE EMPLOYED FOR FURTHER ELABORATION AT THIS STAGE THE ANOMERS WERE SEPARATED BY FLASH CHROMATOGRAPHY FOR CONVENIENCE 3CHEME ! MORE DIRECT AND HIGHER YIELDING ROUTE TO THE SAME INTERMEDIATE WAS LATER DEVELOPED WHEREBY OXIDATION OF TO THE CORRESPONDING ALDE HYDE RATHER THEN ACID AND SELENIUM MEDIATED ACETALISATION GAVE AS A MIXTURE OF ANOMERS ;= 3EPARATION AT THIS POINT FOLLOWED BY OXIDA
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
O
O
O
EtO
OEt
+
22
EtO2C
a
OEt
Br 596
22
CO2Et
O
O
b,c
597
O
598
728
H
H 28
O H
OAc
HO
599
PhSe
MeO
22
d,e
22
EtO2C
O
f,g,h
O
22
26
28
O 22
OMe
H
t,u,v
HO
O
OMe
O 22
s
H
H
i,j,k
l,m,n,o 730
OMe
O
H
r,o
PhSe 734
733
22 26
22
O
26
732
22
OMe
OH
p,q
729
731
3CHEME A .A %T/( REFLUX B (#L REFLUX C !C/ REFLUX THEN I0R.%T %T/!C 24 D ,I!L( 4(& # → 24 E ,IPASE 03 WT VINYL ACETATE $-% 24 EE n F #/#L $-3/ #(#L n # THEN %T. n # → 24 G 0H0%T"R N"U,I 4(& # H . .A/( N"U./( 4(& # OVER THREE STEPS I #/#L $-3/ #(#L n # THEN %T. n # → 24 J .A/#L +(0/ METHYL BUTENE T"U/( (/ OVER TWO STEPS K . PHENYLSELENOPH THALIMIDE 3N#L MOL #(#L L (/ 4(& # M $)"!, ( 0H#( n # N -E/( !MBERLYST MIXTURE OF ANOMERS AT # SEPARABLE O / #(#L n # THEN 0H0 n # → 24 P #/#L $-3/ #(#L n # THEN %T. n # → 24 Q . PHENYLSE LENOPHTHALIMIDE #(#L -E/( MIXTURE OF ANOMERS AT # SEPARABLE R (/ 4(& $(0 S ETHYNYLMAGNESIUM BROMIDE 0H#( 4(& n # MIXTURE OF EPIMERS AT # T :N -E/( (/ U .A( -E) 4(& V / #(#L n # THEN 0H0 n # → 24
TIVE ELIMINATION AND OZONOLYSIS AS BEFORE FURNISHED IDENTICAL TO PREVI OUSLY PREPARED MATERIAL 4HIS ALDEHYDE THEN UNDERWENT A STEREOSELECTIVE REACTION WITH ETHYNYLMAGNESIUM BROMIDE DR ACTIVATED ZINC REDUC TION OF THE ALKYNE FOLLOWED BY ALCOHOL METHYLATION AND ANOTHER OZONOLYSIS TO COMPLETE THE SYNTHESIS OF THE FIRST # # ELECTROPHILE 5NION OF WITH THE PREVIOUSLY DESCRIBED VINYL IODIDE UNDER .(+ CONDITIONS ;n= OCCURRED WITH MODERATE YIELD AND DIASTEREOSE LECTIVITY IN FAVOUR OF DESIRED # EPIMER 3CHEME !FTER SEPARATION THE ISOMER BEARING INCORRECT STEREOCHEMISTRY AT THIS CENTRE COULD BE EFFICIENTLY RECYCLED VIA OXIDATION AND :N"( ANTI REDUCTION ; = ALSO SEEN IN THE 3CHREIBER SYNTHESIS OF RAPAMYCIN !WARE OF THE DIFFICULTY ENCOUNTERED BY 3CHREIBER IN EFFECTING OXIDATIVE DESULFONYLATION AT # WE DECIDED TO CONVERT TO THE CORRESPONDING SULFENYL SULFONE THROUGH THE STANDARD SERIES OF FUNCTIONAL GROUP MANIPULATIONS "ASED UPON THE WORK OF +OTAKE ;= IT WAS HOPED THAT THIS CHANGE WOULD ALLOW EASY CONVERSION TO THE DESIRED # KETONE UPON COUPLING WITH EPOXIDE )N THE EVENT DEPROTONATION OF AT LOW TEM
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
b,c
H THPO
28 32
+ 29
O H
MeO
THPO
THPO
OMe
a 28
22
I
MeO
727
HO 32
32
O
OH H O
32
+
OH H O
28
OMe
MeO
22
d,e
OPMB H O OMe
28
OMe
MeO
22
22
734 736
735
737
f
TBSO
MeO
42
TBSO
32
38
PhO2S
42
PhS
32
32
H
OPMB H O 22 OMe
OH O
MeO
i
MeO
38
H
+ O
740
OPMB H O OMe
MeS 28
MeO
g,h 28
MeO
22
OPMB H OMe O 22
669
738
739
X j,k,l
TBSO
MeO
42
38
TBSO
TBSO
H 32 1
N H Boc O
O
MeO
OPMB
O
O
MeO
X
42
MeO
42
38
H
PhO2S
32
38
H
OH O MeO
666
OPMB H O 22 OPent
669 m
O
32
MeS
MeO
OPMB H O 22 O
742
Me3Sn 22
741
3CHEME A #R#L .I#L $-3/ SEPARABLE B 40!0 .-/ ¯ -3 #(#L C :N"( %T/ # D .A( 0-"#L .A) 4(& # → 24 E -E/( !MBERLYST F "U0 . PHENYLTHIOSUCCINIMIDE 0H( G /XONE¤ P( BUFFER 4(& -E/( H T"U,I 4(& n # THEN -E3 I T"U,I 4(& n # THEN THEN "&q/%T n # → 24 J PENTEN OL 0043 $#% # MIXTURE OF # ANOMERS SEPARABLE K /XONE¤ 4(& -E/( P( BUFFER # L T"U,I 4(& n # THEN -E3 M T"U,I 4(& n # THEN THEN "&q/%T n # → #
PERATURE FOLLOWED BY SEQUENTIAL ADDITION OF AND "&q/%T AND WARMING GAVE THE COUPLED PRODUCT IN AN UNOPTIMISED YIELD OF !LTHOUGH +OTAKE HAS OBSERVED THAT HYDROLYSIS OF DITHIOKETAL MONOSULFONES USUALLY REQUIRES MORE FORCING AND LONGER REACTIONS TIMES #U#L 3I/ OR ANODIC HYDROLYSIS WE WERE VERY PLEASED TO DIRECTLY OBTAIN THE DEPROTECTED KETONE AFTER WORK UP )N THIS INSTANCE WE SPECULATED THAT HYDROLYSIS WAS FACILITATED BY THE PRESENCE OF EXCESS ,EWIS ACID EXPLAINING THE NEED FOR MOLAR EQUIVALENTS OF "&q/%T 7ITH THE PRODUCTION OF A PRECURSOR
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
WHICH CONTAINED MOST OF THE FRAMEWORK OF VINYL STANNANE SYNTHESIS OF THIS IMPORTANT FRAGMENT SEEMED IMMINENT 4O OUR GREAT DISAPPOINT MENT ALL EFFORTS TO CONVERT THE SIX MEMBERED CYCLIC ACETAL DESIGNED TO UNMASK THE # ALCOHOL AND # ALDEHYDE TO AN ACYCLIC FORM FAILED AFFORDING ONLY STARTING MATERIAL OR DECOMPOSITION PRODUCTS IN ACIDIC MEDIA 3CHEME ! POTENTIAL SOLUTION FOLLOWED FROM REPORTS OF THE USE OF PENTENYL ACETALS BY &RASER 2EID ; = THAT SUGGESTED DEPROTECTION UNDER NEUTRAL CONDITIONS ."3 (/ MIGHT PROVIDE ACCESS TO AN INTERMEDIATE LACTOL AND THUS HOPEFULLY A RING OPENED FORM UNDER MILDER CONDITIONS THAN PREVI OUSLY ACCESSIBLE 4HUS THE METHYL ACETAL IN THIOETHER WAS EXCHANGED FOR THE PENTENYL EQUIVALENT 3UBSEQUENT FUNCTIONAL GROUP MANIPULATION AND COUPLING WITH AS DESCRIBED PREVIOUSLY GAVE A SECOND # # ADVANCED INTERMEDIATE 5NFORTUNATELY ALTHOUGH THE ANOMERIC SUB STITUENTS COULD BE INTERCHANGED AT # IN NO INSTANCE WERE CONDITIONS FOUND THAT GENERATED THE RING OPENED PRODUCT 0RESUMABLY THE RING SUB STITUTION PATTERN GREATLY STABILISES THE CLOSED STRUCTURE MAKING FURTHER ADVANCEMENT WITH THIS STRATEGY UNWORKABLE IN OUR HANDS 3CHEME )N ORDER TO CONVERT THE STUBBORN CYCLIC ACETAL INTO A USEFUL INTERMEDIATE WE RETURNED OUR ATTENTION TO THIOETHER (YDROLYSIS OF THE ACETAL UNDER ACIDIC CONDITIONS AND REDUCTION WITH $)"!, ( THEN GAVE &URTHER STANDARD FUNCTIONAL GROUP MANIPULATIONS GAVE THE LINEAR SULFENYL SULFONE WITHOUT DIFFICULTY BUT NOW PROGRESS WAS THWARTED BY EXTREMELY POOR REACTIVITY IN THE CRUCIAL COUPLING REACTIONS $ESPITE EXPLORING A VARIETY OF PROTECTING GROUPS FOR BOTH THE # AND # HYDROXYL GROUPS IN NO INSTANCE WAS UNION OF THE TWO MAJOR FRAGMENTS AND OR DERIVA TIVES EFFECTED IN GREATER THEN YIELD $EPROTONATION OF THE MORE NUCLEO PHILIC AND LESS HINDERED SULFONE FOLLOWED BY TREATMENT WITH EPOXIDE DID AFFORD THE ALKYLATED SULFONE IN GOOD YIELD AS A SINGLE ISOMER OF UNDETERMINED CONFIGURATION (OWEVER IN THIS CASE WE WERE UNABLE TO EFFECT OXIDATIVE DESULFONYLATION TO AFFORD 3CHEME 4HE LESSONS LEARNED WITH THE ADVANCED FRAGMENTS ABOVE DICTATED A NUMBER OF KEY STRATEGIC REVISIONS &IRST AND FOREMOST FOR THE CENTRAL # # FORMAL DIANION EQUIVALENT WE ELECTED TO PURSUE A DITHIANE STRATEGY IN PLACE OF THE COMPROMISED SULFONYL OR SULFENYL SULFONE APPROACH GIVEN THEIR SUCCESS IN COMPLICATED FRAGMENT COUPLING REACTIONS SEE 3MITHS SYN THESIS OF RAPAMYCIN VIDE SUPRA AND POTENTIAL DEPROTECTION UNDER A VARIETY
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
PhS
PhS 32
PhS 32
28
MeO
OPMB H OMe O
28
MeO
22
738 TBSO MeO
PhO2S 32
a
OPMB H O OH
32
OPMB H OH OH
b 28
MeO
22
743
28
MeO
22
744
22
745 d
42
38
OPMB H OH OH
c
H
TBSO
32
HO
PhO2S
42
OPMB
O
OH
MeO
X
MeO
38
+
H
PhO2S
32
28
MeO
O
748
OPMB H OH OMTM
MeS
32
e
OPMB H OH OMTM
28
MeO
22
22
669 MTMO
746 747
22
X
f
TBSO
42
TBSO
MeO
38
42
H MeO
32
HO PhSO2 749
38
H
OPMB O
OH
MeO
669
MTMO 22
3CHEME A !C/( (/ B $)"!, ( 4(& OVER TWO STEPS C /XONE¤ 4(& -E/( P( BUFFER D -E3 "Z/ #(#. E T"U,I 4(& n # THEN -E3 F T"U,I 4(& (-0! n # THEN
OF CONDITIONS )N ADDITION NEW ROUTES TO LINEAR # # ELECTROPHILES WERE DESIGNED TO ACHIEVE MORE EFFICIENT ACCESS AND GREATER FLEXIBILITY WITH REGARDS TO BOTH STEREOCHEMISTRY AND PROTECTING GROUPS SHOULD DIFFICULTIES ARISE "EGINNING WITH THE 2OCHE ESTER STANDARD FUNCTIONAL GROUP MANIPULA TION AFFORDED DITHIANE ALCOHOL AS A SINGLE ENANTIOMER !TTEMPTS TO INTRODUCE THE DESIRED HALOGENATED OLEFIN FAILED WHEN ATTEMPTING APPLICA TION OF 3CHWARTZ HYDROZIRCONATION CONDITIONS UPON 0ARTIAL SUCCESS WAS REALISED VIA PALLADIUM CATALYSED HYDROSTANNYLATION FOLLOWED BY TRAP PING WITH IODINE UNFORTUNATELY AS AN UNSUITABLE MIXTURE OF %: ISOMERS 4O RESOLVE THIS SELECTIVITY ISSUE AN INTERESTING VARIATION ;n= OF 3TILL 'ENNARI PHOSPHONATE ;= WAS EMPLOYED WHEREIN PRIOR BROMINA TION OF THE REAGENT AND REACTION WITH FRESHLY PREPARED ALDEHYDE FROM GAVE THE KINETIC TRISUBSTITUTED BROMOALKENE AS ONLY ONE DETECTABLE OLEFIN ISOMER 2EMOVAL OF THE EXTRANEOUS METHYL ESTER FUNCTIONALITY WAS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
MeO
OH
32
a,b
OTr
32
c,d
S
30
CO2Me S
S
750
420
e,f
32
29
30
OH
O
OH
32
30
S
S
751
Cp2Zr(H)Cl
OH
g
Br
32
29
753
752
e,j,k
X
h
I2 or Br2 S
32 29
S
I
l
Br
S
32 29
S
756
Br
S
S
i
32
29
S
755
Br
S
32 29
Br
S
670
754
3CHEME A 4R#L 0Y #(#L # → 24 B ,I!L( 4(& # OVER TWO STEPS C #/#L $-3/ #(#L n # THEN $)0%! n # → # D (3#( 3( "&q/%T #(#L n # → 24 OVER TWO STEPS E 3/q0Y $)0%! $-3/ #(#L 24 F #&#(/ 0/ #(#/-E +(-$3 4(& THEN "R THEN # +(-$3 THEN ALDEHYDE FROM 4(& n # G $)"!, ( #(#L n # H -S#L %T. $-!0 #(#L # THEN ,I"R $-& n I ,I%T"( 4(& # J #"R 0H0 #(#L # K N"U,I 4(& n # THEN -E) n # → 24 L "U3N( 0H0 0D#L 4(& n # THEN ) %: INSEPARABLE
BEST ACCOMPLISHED THROUGH THE ALLYLIC BROMIDE VIA REDUCTION WITH 3UPER (YDRIDE4- TO AFFORD IN NINE STEPS IN AN EFFICIENT OVERALL YIELD 3CHEME 'IVEN THAT THE STEREOCHEMISTRY OF THE # ALCOHOL IS ULTIMATELY OF NO CONSEQUENCE TWO CONCURRENT APPROACHES FOR A NEW SYNTHESIS OF A LINEAR # # ELECTROPHILE WERE PURSUED "OTH OF THESE UTILISED THE KEY ENANTIO ENRICHED DIMETHYLATED ALCOHOL PREPARED AS DISCUSSED PREVIOUSLY 3CHEME )N THE FIRST APPROACH REACTION OF READILY PREPARED ALDEHYDE WITH A γ METHOXYALLYLZINC REAGENT DERIVED FROM TRANSMETALLATION OF ZINC ONTO THE LITHIO ANION OF ALLYL METHYL ETHER WAS HIGH YIELDING BUT POORLY SELECTIVE SLIGHTLY FAVOURING THE UNDESIRED DIASTEREOMER !LTHOUGH SEPARABLE OBTAINING CLEAN BY PURIFICATION WAS TEDIOUS AND IN THE END IMPRACTICAL #ONSEQUENTLY ASYMMETRIC "ROWN ALKOXYALLYLATION ;= OF THE SAME ALDEHYDE FOLLOWED BY CLEAVAGE OF THE ACETATE PROTECTING GROUP TO FACILITATE PURIFICATION FURNISHED AS A SINGLE DIASTEREOMER !LTHOUGH THE YIELD FOR THESE TWO STEPS WAS LOWER THEN DESIRED THE REACTION UTILISED THE PRODUCT OF ENZYMATIC DESYMMETRISATION DIRECTLY AND COULD BE PERFORMED ON LARGE SCALE -OREOVER BY MASKING THE # CARBONYL AS AN OLEFIN PROTECTION OF THE # ALCOHOL AS ITS 0-" ETHER WAS FACILE AND UPON APPLICATION OF STANDARD CONDITIONS WAS ISOLATED IN EXCELLENT YIELD $IRECT OZONOLYSIS OF PROVED PROBLEMATIC THUS A TWO STEP DIHYDROXYL
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
OH
OH
OAc 22
+
22
OMe
OMe O
22
HO
OAc
b
a
OAc c,d
728 5 steps 42%
759
758
22
H
OAc
28
28
757
OH
OH
28 22
OMe 760
OPMB e
OPMB
28 22
O
f,g H
OMe
OPMB
OPMB 22
28
OMe 761
762
3CHEME A 3/q0Y $)0%! $-3/ #(#L 24 B ALLYLMETHYL ETHER S"U,I :N#L n # → n # THEN SEPARABLE C ALLYLMETHYL ETHER S"U,I 4(& n # THEN n -E/ ")PC THEN "&q/%T THEN THEN . .A/( (/ n # → 24 D +#/ -E/( 24 OVER TWO STEPS E .A( 0-"#L 4"!) $-& 24 F /S/ .-/ ACETONE (/ 24 G 0B/!C 0H( 24
ATIONCLEAVAGE PROTOCOL WAS USED TO AFFORD THE DESIRED ELECTROPHILE .OTABLY CLEAVAGE OF THE INTERMEDIATE DIOL RESULTING FROM DIHYDROXYLATION WITH 0B/!C WAS CLEAN AND HIGH YIELDING AND PURIFICATION OF THIS SENSI TIVE COUPLING PARTNER COULD BE OMITTED 3CHEME ! SECOND HIGHER YIELDING CONSTRUCTION OF THE # # CARBON BOND INVOLVED APPLICATION OF OUR RECENTLY DEVELOPED "$! VARIANT OF GLYCOLIC ACID TO EFFECT A HIGHLY SELECTIVE ALDOL CONDENSATION WITH EITHER OR ;= 4HE 0-" PROTECTED DERIVATIVE PROVED MORE PRACTICAL FOR ADVANCEMENT IN LATER TRANSFORMATIONS ALTHOUGH AS EXPECTED PROTEC TION OF THE SECONDARY ALCOHOL AT # WAS EXTREMELY DIFFICULT AS A RESULT OF THE β DISPOSITION OF ESTER CARBONYL FUNCTION AND NUMEROUS ACETAL MOIETIES PRESENT WITHIN %VENTUALLY TREATMENT WITH 0-" 4#! WITH A CATALYTIC AMOUNT OF 4R"& WERE IDENTIFIED AS SUITABLE CONDITIONS FOR PRODUCTION OF ; = AND ALTHOUGH THIS REACTION WOULD NOT GO TO COMPLETION THE STARTING MATERIAL COULD BE RECOVERED AND RECYCLED &URTHER TRANSFORMA TION TO THE DESIRED ELECTROPHILE IN THIS CASE A 7EINREB AMIDE WAS STRAIGHT FORWARD AND WAS PREPARED IN AN OVERALL YIELD OF THROUGH STEPS 3CHEME 4HE FIRST MAJOR FRAGMENT UNION INVOLVED REACTION OF TWO EQUIVALENTS OF LITHIATED WITH EITHER OR TO CONSTRUCT THE DESIRED # # BOND IN GOOD YIELDS &OR THE FORMER A PARTIALLY SEPARABLE MIXTURE OF DIASTE REOMERS WAS OBTAINED IN FAVOUR OF THE UNDESIRED CONFIGU RATION OF THE # CARBINOL !LL MATERIAL COULD BE READILY SALVAGED THROUGH AN OXIDATIONREDUCTION SEQUENCE OF THE MIXTURE WHICH OCCURRED WITH HIGH
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME A 4"3#L )M #(#L # → 24 B +#/ -E/( 24 C .A( 0-"#L 4"!) 4(& # → 24 D 4"!& 4(& # OVER FOUR STEPS E 3/q0Y $)0%! $-3/ #(#L 24 F ,I(-$3 4(& n # THEN THEN !C/( n # → 24 G 0-" 4#! 4R"& MOL 4(& 24 H #3! -E/( 24 I !G/ -E) #(#L # J ,I(-$3 -E/-E .(q(#L 4(& n # THEN n # → n # K ,I(-$3 4(& n # THEN THEN !C/( n # → 24
ANTI SELECTIVITY EMPLOYING ETHEREAL SOLUTIONS OF ZINC BOROHYDRIDE AND AFTER 4%3 PROTECTION WAS ISOLATED AS A SINGLE DIASTEREOMER READY FOR COUPLING TO THE # # EPOXIDE !LTERNATIVELY UNION OF AND GAVE A SUBSTANTIALLY IMPROVED YIELD OF DIRECTLY WITHOUT THE PROB LEMS ASSOCIATED WITH THE PREVIOUS DIASTEREOMERIC MIXTURE 2EDUCTION AND 4%3 PROTECTION AS BEFORE UNDER SLIGHTLY MODIFIED CONDITIONS PROVIDED A SECOND COMPLETED # # FRAGMENT READY FOR COUPLING TO EPOXIDE AND DIFFERING ONLY IN THE ABSOLUTE CONFIGURATION OF THE # 0-" ETHER 3CHEME 0LEASINGLY LITHIATION OF EITHER OR WITH T"U,I IN THE PRESENCE OF AND IMMEDIATE WARMING RESULTED IN SMOOTH EPOXIDE RING OPENING AND CONSTRUCTION OF THE FULL # # CARBON FRAMEWORK AFFORDING AND 4HIS PROCEDURE BASED UPON REPORTS BY 3MITH ;= WAS IMPORTANT TO ACHIEVE GOOD YIELDS AS α METHYL DITHIANES OF THIS TYPE ARE KNOWN TO FORM LITHIO ANIONS ALMOST INSTANTANEOUSLY IN A MIXTURE OF 4(&(-0! BUT THEN RAPIDLY LOSE THEIR REACTIVITY PRESUMABLY THROUGH AGGREGATION )N BOTH SERIES AND REMOVAL OF THE DITHIANE MOIETY USING THE BISTRIFLUO ROACETOXY IODOBENZENE PROTOCOL OF 3TORK AND :HAO ;= WAS NECESSARY TO
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
O O
N
32
28
S
O
OPMB
O H
S
22
OMe S
770 a
22
OPMB
OMe
OPMB
32 29
S
32
28
S
OH OPMB 775 + OMe
22
28
OMe
OPMB
OPMB
22
OMe S
OPMB
28
762
Br
i
670
S
32
28
S
OH OPMB 776
OPMB
22
772 b,c
OPMB
j,k,l OMe S
32
28
S
TESO
OMe S
22
OPMB
OPMB
32
28
S
TESO
773
777
TBSO d
MeO
TBSO
42
MeO
38
TBSO MeO
H
OTES
e,f,g,h
38
MeO
H
N H Boc O
OPMB
MeO
O
O
OTES
PMBO 22
12
38
H
32
HO S S
n,o,p,q
28
OTES 28
O
MeO
OPMB
MeO
1
N CO2H H Boc
O 42
32
1
28
774
m
H
TBSO
42
32
HO S S
38
669
O
42
38
42
MeO
H
669 TBSO
22
OPMB
OPMB
1
N CO2H H Boc
681 OHC 22
12
778 PMBO 22
3CHEME A T"U,I 4(& n # THEN n # → n # B :N"( %T/ n # H C 4%3#L )M $-& # D AND T"U,I 4(&(-0! n # → n # E 0H)/#/#& (/ -E/( 4(& 24 F $## $-!0 #(#L n # G $$1 P( BUFFER #(#L 24 Q #/#L $-3/ #(#L n # THEN %T. n # → 24 I T"U,I 4(& n # THEN n # → n # J 3/q0Y $)0%! $-3/ #(#L # K :N"( %T/ n # DAYS L 4%3/4F LUT #(#L n # M AND T"U,I 4(&(-0! n # → n # N 0H)/#/#& (/ -E/( 4(& 24 O $## $-!0 #(#L n # P $$1 P( BUFFER #(#L 24 Q #/#L $-3/ #(#L n # THEN %T. n # → 24
PERMIT ESTERIFICATION OF THE # ALCOHOL WITH 3 "OC PIPECOLIC ACID 0-" DEPROTECTION WITH BUFFERED $$1 FOLLOWED BY DOUBLE OXIDATION AT # AND # EMPLOYING 3WERN CONDITIONS CONCLUDED TWO VIABLE ROUTES TO THE SAME INTERMEDIATE IDENTICAL IN ALL RESPECTS STARTING FROM EITHER STEPS OVERALL YIELD OR STEPS OVERALL YIELD AND ALSO MATCHING MATERIAL OBTAINED THROUGH DEGRADATION SEE 3CHEME 4HE PARTICULAR USE OF THE 3WERN OXIDATION IN THIS INSTANCE WAS CRITI CAL TO AVOID SPONTANEOUS FORMATION OF THE UNDESIRED LACTOL BETWEEN # AND # /( 3CHEME ;n= 4HE FINAL STAGES OF THE SYNTHESIS
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
REQUIRED CONVERSION OF ALDEHYDE TO VINYL STANNANE WHICH WAS READILY ACCOMPLISHED VIA 4AKAI OLEFINATION AND PALLADIUM CROSS COUPLING OF THE INTERMEDIATE % VINYL IODIDE WITH HEXAMETHYLTIN AND FRESHLY PRE PARED 0D0&UR #L ;= 3UBSEQUENT UNION WITH MAJOR FRAGMENT DIENYL IODIDE EMPLOYING THE SAME CATALYST SYSTEM RESULTED IN SMOOTH FORMA TION OF THE DESIRED TRIENE WITH NO EVIDENCE OF MINOR GEOMETRICAL ISOMERS DESPITE EMPLOYING AN %: MIXTURE OF /NE EXPLANATION OF THIS FORTUITOUS RESULT IS THAT THE MINOR : COMPONENT OF MIGHT HAVE EQUILIBRATED UNDER THE REACTION CONDITIONS OR PERHAPS MAY REACT MORE SLOWLY THEN THE % ISOMER 4O GUARD AGAINST RECURRING PROBLEMS WITH β ELIMINATION ACROSS # # LATER IN THE SYNTHESIS THE # CARBONYL WAS SELECTIVELY REDUCED AND PROTECTED TO FURNISH &URTHER MANIPULATION TO α BROMOAMIDE WAS ACHIEVED VIA HYDROLYSIS OF THE LACTONE PROTECTION OF THE LIBERATED SECONDARY HYDROXYL GROUP WITH CONCOMITANT LIBERATION OF THE AMINE AND FINALLY AMIDE FORMATION WITH α BROMOACETYL BROMIDE 4HE SEQUENCE OF EVENTS IN THE ABOVE THREE REACTIONS WAS CRITICAL TO AVOID EPIMERISATION OF THE # METHYL STEREOCENTRE 7ITH IN HAND THE STAGE WAS SET FOR INTRODUCTION OF THE CATECHOL TEMPLATING MOIETY AND CRITI CAL INTRAMOLECULAR MACROCYCLE FORMATION 4O ACCOMPLISH THIS STANDARD $## COUPLING OF CATECHOL WITH THE FREE CARBOXYLIC ACID FOLLOWED BY ALKYLATIVE RING CLOSURE AFFORDED MACROCYCLIC ETHER IN GOOD YIELD )N CONTRAST TO THE PREVIOUS USE OF THE CATECHOL STRATEGY FOR MACROCYCLE FORMATION ANTASCOMICIN VIDE SUPRA WE WERE ESPECIALLY PLEASED TO DEM ONSTRATE THE FEASIBILITY OF ITS INTRODUCTION VIA A MACROETHERIFICATION ; = PROTOCOL RATHER THAN THE MORE COMMON MACROESTERIFICATION 4REAT MENT OF WITH ,I(-$3 UNDER THE PREVIOUSLY OPTIMISED CONDITIONS EFFECTED THE TEMPLATED $IECKMANN LIKE CONDENSATION TO GIVE THE NATIVE RAPAMYCIN MACROCYCLE IN AN EXCELLENT YIELD OF /F ALL THE VARI OUS APPROACHES TO CONSTRUCTION OF THIS MOTIF IN THE RAPAMYCIN SERIES THIS STRATEGY IS THE HIGHEST YIELDING MOREOVER DIRECT COMPARISON TO THE OTHER EXAMPLE OF MACROCYCLE FORMATION VIA A CARBON CARBON BOND FORMING STRATEGY $ANISHEFSKY n SERVES BEST TO ILLUSTRATE THE UTILITY OF THIS METHODOLOGY #OMPLETION OF THE TOTAL SYNTHESIS OF WAS SUBSEQUENTLY ACCOMPLISHED VIA THE FOUR STEP SEQUENCE OF !LLOC DEPROTECTION CATECHOL CLEAVAGE OXIDATION OF THE # AND # ALCOHOLS AND GLOBAL DEPROTECTION WITH (&q0Y YIELDING MATERIAL IDENTICAL IN ALL REGARDS WITH AN AUTHENTIC SAMPLE OF THE NATURAL PRODUCT 3CHEME
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
TBSO
TBSO
42
MeO
38
10
MeO
H
38
32
O
O
1
N H Boc O
a,b
OTES
H
O
N H Boc O
O
O
MeO
32
I
668 c
OTES
1
O 10
Me3Sn
OHC 22
H
38
20
16
O
42
MeO
OMe
32
1
O
MeO
TBSO
O
42
N H Boc O O
O
OTES O
MeO
OMe
22
16 22
666
681
O
679 d
TBSO
TBSO MeO
MeO
H
38
TBSO
42
42
38
H
N H O
O
O
O
O Alloc
OTES
Br
O
O
O
32
OTES
O Alloc
HO2C
1
f,g,h O
MeO
OMe
10
22
10
O
1
N H
i,j
O
MeO
OMe
O
H
38
32
32
1
42
MeO
O 10
22
N H Boc O O
OTES O
MeO 22
16
OTES 781
OTES 782
OR
OMe
16
16
O
779 R = H 780 R = Alloc
e
k,l TBSO MeO
TBSO
42
38
MeO
H
HO
42
38
H
42
MeO
32
38
32
H 32
OH N H O 10
1
O HO
O O TES O OMe O 16
OTES O
MeO
m,n
N H O 10
22
1
O
O O TES O OMe O
O
OTES O
MeO 22
o
N H O HO
O 10
O
1
O
O
OH
O
O
MeO
OMe
22
16 16
783
784
(-)-rapamycin 7
3CHEME A #R#L #() 4(& # → 24 B 0D0&UR #L -E3N .-0 DARK 24 C 0D0&UR #L .-0 DARK 24 D ,I!L(/T"U 4(& n # E !LLOC#L PYRROLIDINOPYRIDINE #(#L F - ,I/( IN (/ 4(& # G 4%3/4F LUT #(#L n # → 24 H "R#(#/ "R LUT #(#L n # I CATECHOL $## $-!0 #(#L # → 24 J +#/ $-& 24 K ,I(-$3 4(& n # → n # L 0D00H DIMEDONE 4(& 24 M 0H)/!C #(#.(/ # N $-0 0Y #(#L 24 OVER TWO STEPS O (&q0Y 4(& #
)N SUMMARY THE FIFTH AND LATEST SYNTHESIS OF RAPAMYCIN WAS COM PLETED IN A LONGEST LINEAR SEQUENCE OF STEPS AND OVERALL YIELD 3TA TISTICALLY IT IS NEITHER THE SHORTEST NOR HIGHEST YIELDING OF THE APPROACHES TO DATE AND THERE IS UNDOUBTEDLY POTENTIAL FOR IMPROVEMENT PARTICULARLY IN A SOMEWHAT LENGTHY AND OUTDATED SYNTHESIS OF THE # # FRAGMENT
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
3CHEME 3UMMARY OF ,EYS TOTAL SYNTHESIS OF RAPAMYCIN
(OWEVER IN OUR SYNTHESIS OF RAPAMYCIN WE HAVE DEMONSTRATED THE EFFICIENT APPLICATION OF A WIDE VARIETY OF GROUP DEVELOPED METHODOLO GIES INCLUDING IRON CARBONYL CHEMISTRY THE INTRAMOLECULAR ADDITION OF ALLYLSILANES TO OXONIUM IONS A "$! CONTROLLED ALDOL CONDENSATION AND MOST NOTABLY A CATECHOL TEMPLATED INTRAMOLECULAR MACROCYCLE CONSTRUC TION )N CONCERT WITH THE HIGHLY EFFECTIVE UNION OF MAJOR FRAGMENTS THIS MAKES THE MOST RECENT SYNTHESIS A VERY ENGAGING ADDITION TO A FIELD OF ENDURING INTEREST
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
#ONCLUSION 4HIS CHAPTER DERIVES ITS INSPIRATION FROM THE CHALLENGES PRESENTED TO TOTAL SYNTHESIS CHEMISTS BY A PARTICULAR GROUP OF MACROCYCLIC PIPECOLIC ACID NAT URAL PRODUCTS !LTHOUGH THERE IS CONSIDERABLE EMPHASIS ON THE COMPLETED SYNTHESES OF THE MAIN CHARACTERS &+ THE ANTASCOMICINS AND AND RAPAMYCIN THE OVERALL COMPLEXITY OF THE MOLECULAR PROBLEM HAS STIMULATED A WEALTH OF NEW KNOWLEDGE INCLUDING THE DEVELOPMENT OF NOVEL STRATEGIES AND THE INVENTION OF NEW SYNTHETIC METHODS 4HE INGE NIOUS AND INNOVATIVE APPROACHES TO THESE TARGETS HAVE ENABLED NEW GEN ERATIONS OF ANALOGUES AND PROVIDED MATERIAL TO FURTHER PROBE THE BIOLOGY OF THESE FASCINATING MOLECULES 4OTAL SYNTHESIS OF THIS TYPE IS THE ULTIMATE EXPRESSION OF OUR KNOWLEDGE OF MOLECULAR CONSTRUCTION EXPLOITING AS IT DOES THE VERY LIMITS OF OUR UNDERSTANDING OF CHEMICAL REACTIVITY 4OGETHER WITH THE CONTINUING STORY OF THEIR EXTRAORDINARY BIOLOGICAL FUNCTION THIS CLASS OF COMPOUNDS ENRICHES OUR LIVES AND SERVES AS A BEACON FOR THE DISCOVERY OF NEW NATURAL PRODUCTS IN THE PURSUIT OF CURE FOR HUMAN DISEASE
2EFERENCES
#LARDY * 7ALSH # ,ESSONS FROM NATURAL MOLECULES .ATURE n 7ILSON 2- $ANISHEFSKY 3* 3MALL MOLECULE NATURAL PRODUCTS IN THE DISCOVERY OF THERAPEUTIC AGENTS THE SYNTHESIS CONNECTION * /RG #HEM n "ALL 0 ,IFES LESSONS IN DESIGN .ATURE n ,IU $2 3CHULTZ 0' 'ENERATING NEW MOLECULAR FUNCTION A LESSON FROM NATURE !NGEW #HEM )NT %D n .EWMAN $* #RAGG '- 3NADER +- 4HE INFLUENCE OF NATURAL PRODUCTS UPON DRUG DISCOVERY .ATURAL 0RODUCT 2EPORTS n .EWMAN $* #RAGG '- (OLBECK 3 3AUSVILLE %! .ATURAL PRODUCTS AND DERIVATIVES AS LEADS TO CELL CYCLE PATHWAY TARGETS IN CANCER CHEMOTHERAPY #URR #ANCER $RUG 4ARGETS n "UTLER -3 4HE ROLE OF NATURAL PRODUCT CHEMISTRY IN DRUG DISCOVERY * .AT 0ROD n #ORDELL '! .ATURAL PRODUCTS IN DRUG DISCOVERY n CREATING A NEW VISION 0HYTOCHEM 2EV n 3TROHL 72 4HE ROLE OF NATURAL PRODUCTS IN A MODERN DRUG DISCOVERY PROGRAM $RUG $ISCOVERY 4ODAY n .EWMAN $* #RAGG '- 3NADER +- .ATURAL PRODUCTS AS SOURCES OF NEW DRUGS OVER THE PERIOD n * .AT 0ROD n
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
4ANAKA ( +URODA ! -ARUSAWA ( (ATANAKA ( +INO 4 'OTO 4 (ASHIMOTO - 4AGA 4 3TRUCTURE OF &+ A NOVEL IMMUNOSUPPRESSANT ISOLATED FROM 3TREPTOMYCES * !M #HEM 3OC n +INO 4 (ATANAKA ( (ASHIMOTO - .ISHIYAMA - 'OTO 4 /KUHARA - +OSAKA - !OKI ( )MANAKA ( &+ A NOVEL IMMUNOSUPPRESSANT ISOLATED FROM A 3TREPTOMYCES ) &ERMENTATION ISOLATION AND PHYSICOCHEMICAL AND BIOLOGICAL CHARACTERISTICS * !NTIBIOT n +INO 4 (ATANAKA ( -IYATA 3 )NAMURA . .ISHIYAMA - 9AJIMA 4 'OTO 4 /KUHARA - +OHSAKA - !OKI ( &+ A NOVEL IMMUNOSUPPRESSANT ISOLATED FROM A 3TREPTO MYCES )) )MMUNOSUPPRESSIVE EFFECT OF &+ IN VITRO * !NTIBIOT n !RAI 4 +OYAMA 9 3UENAGA 4 (ONDA ( !SCOMYCIN AN ANTIFUNGAL ANTIBIOTIC * !NTIBIOT 3ER ! n -ORISAKI - !RAI 4 )DENTITY OF IMMUNOSUPPRESSANT &2 WITH ASCOMYCIN * !NTIBIOT n (ATANAKA ( +INO 4 -IYATA 3 )NAMURA . +URODA ! 'OTO 4 4ANAKA ( /KUHARA - &2 AND &2 NOVEL IMMUNOSUPPRESSANTS ISOLATED FROM A 3TREPTO MYCES )) &ERMENTATION ISOLATION AND PHYSICOCHEMICAL AND BIOLOGICAL CHARACTERISTICS * !NTIBIOT n /R 93 #LARK 2& 8IE 1 -C!LPINE * 7HITTERN $. (ENRY 2 ,ULY *2 4HE CHEMISTRY OF ASCOMYCIN STRUCTURE DETERMINATION AND SYNTHESIS OF PYRAZOLE ANALOGS 4ETRAHEDRON n &LOROVA ' ,INDSAY 9- "ROWN -3 -C!RTHUR (!) $ENOYA #$ 2EYNOLDS +! 3TEREOCHEMICAL ANALYSES OF THE 3TREPTOMYCES HYGROSCOPICUS VAR ASCOMYCETICUS TYPE )) DEHY DROQUINATE DEHYDRATASE AND EVIDENCE FOR A ROLE OF THE ENZYME IN THE BIOSYNTHESIS OF THE SHIKIMATE DERIVED MOIETY OF ASCOMYCIN * /RG #HEM n "ECKER *7 2OTONDA * -C+EEVER "- #HAN (+ -ARCY !) 7IEDERRECHT ' (ERMES *$ 3PRINGER *0 &+ BINDING PROTEIN 4HREE DIMENSIONAL STRUCTURE OF THE COMPLEX WITH THE ANTAGONIST , * "IOL #HEM n $UMONT &* 3TARUCH -* +OPRAK 3, 3IEKIERKA ** ,IN #3 (ARRISON 2 3EWELL 4 +INDT 6- "EATTIE 42 7YVRATT 7 ET AL 4HE IMMUNOSUPPRESSIVE AND TOXIC EFFECTS OF &+ ARE MECHANISTICALLY RELATED 0HARMACOLOGY OF A NOVEL ANTAGONIST OF &+ AND RAPAMY CIN * %XP -ED n &EHR 4 3ANGLIER * * 3CHULER 7 'SCHWIND , 0ONELLE - 3CHILLING 7 7IOLAND # .OVEL &+"0 BINDING COMPOUNDS FROM A -ICROMONOSPORA STRAIN * !NTIBIOT n 3ALITURO '- :INK $, $AHL ! .IELSEN * 7U %(, +ASTNER # $UMONT &* -ERI DAMYCIN A NOVEL NONIMMUNOSUPPRESSIVE &+"0 LIGAND FROM 3TREPTOMYCES HYGROSCOPI CUS 4ETRAHEDRON ,ETT n 3UN 9 (ONG ( 3AMBOSKYY - -IRONENKO 4 ,EADLAY 0& (AYDOCK 3& /RGANIZATION OF THE BIOSYNTHETIC GENE CLUSTER IN 3TREPTOMYCES SP $3- FOR THE NOVEL NEUROPROTECT ANT POLYKETIDE MERIDAMYCIN -ICROBIOLOGY n (E - (ALTLI " 3UMMERS - &ENG 8 (UCUL * )SOLATION AND CHARACTERIZATION OF MERIDAMYCIN BIOSYNTHETIC GENE CLUSTER FROM 3TREPTOMYCES SP .22, 'ENE n 6EZINA # +UDELSKI ! 3EHGAL 3. 2APAMYCIN !9 A NEW ANTIFUNGAL ANTIBI OTIC ) 4AXONOMY OF THE PRODUCING STREPTOMYCETE AND ISOLATION OF THE ACTIVE PRINCIPLE * !NTIBIOT n
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3EHGAL 3. "AKER ( 6EZINA # 2APAMYCIN !9 A NEW ANTIFUNGAL ANTIBIOTIC )) &ERMENTATION ISOLATION AND CHARACTERIZATION * !NTIBIOT n 3WINDELLS $#. 7HITE 03 &INDLAY *! 4HE X RAY CRYSTAL STRUCTURE OF RAPAMYCIN #(./ #AN * #HEM n &INDLAY *! 2ADICS , /N THE CHEMISTRY AND HIGH FIELD NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY OF RAPAMYCIN #AN * #HEM n -C!LPINE *" 3WANSON 3* *ACKSON - 7HITTERN $. 2EVISED .-2 ASSIGNMENTS FOR RAPAMYCIN * !NTIBIOT n ,OWDEN 0!3 7ILKINSON " "OHM '! (ANDA 3 &LOSS (' ,EADLAY 0& 3TAUNTON * /RIGIN AND TRUE NATURE OF THE STARTER UNIT FOR THE RAPAMYCIN POLYKETIDE SYNTHASE !NGEW #HEM )NT %D n 3TAUNTON * 7ILKINSON " "IOSYNTHESIS OF ERYTHROMYCIN AND RAPAMYCIN #HEM 2EV n (ARDING -7 'ALAT ! 5EHLING $% 3CHREIBER 3, ! RECEPTOR FOR THE IMMUNOSUP PRESSANT &+ IS A CIS TRANS PEPTIDYL PROLYL ISOMERASE .ATURE n &ISCHER ' "ANG ( -ECH # $ETECTION OF ENZYME CATALYSIS FOR CIS TRANS ISOMERI ZATION OF PEPTIDE BONDS USING PROLINE CONTAINING PEPTIDES "IOMED "IOCHIM !CTA n 3CHMID &8 -AYR ,- -UCKE - 3CHONBRUNNER %2 0ROLYL ISOMERASES ROLE IN PROTEIN FOLDING !DV 0ROTEIN #HEM n (AMILTON '3 3TEINER *0 )MMUNOPHILINS "EYOND IMMUNOSUPPRESSION * -ED #HEM n -ICHNICK 37 2OSEN -+ 7ANDLESS 4* +ARPLUS - 3CHREIBER 3, 3OLUTION STRUC TURE OF &+"0 A ROTAMASE ENZYME AND RECEPTOR FOR &+ AND RAPAMYCIN 3CIENCE n -OORE *- 0EATTIE $! &ITZGIBBON -* 4HOMSON *! 3OLUTION STRUCTURE OF THE MAJOR BINDING PROTEIN FOR THE IMMUNOSUPPRESSANT &+ .ATURE n &ISCHER ' 7ITTMANN ,IEBOLD " ,ANG + +IEFHABER 4 3CHMID &8 #YCLOPHILIN AND PEPTIDYL PROLYL CIS TRANS ISOMERASE ARE PROBABLY IDENTICAL PROTEINS .ATURE n !LBERS -7 7ALSH #4 3CHREIBER 3, 3UBSTRATE SPECIFICITY FOR THE HUMAN ROTAMASE &+"0 A VIEW OF &+ AND RAPAMYCIN AS LEUCINE TWISTED AMIDE PROLINE MIMICS * /RG #HEM n 2OSEN -+ 3TANDAERT 2& 'ALAT ! .AKATSUKA - 3CHREIBER 3, )NHIBITION OF &+"0 ROTAMASE ACTIVITY BY IMMUNOSUPPRESSANT &+ TWISTED AMIDE SURROGATE 3CIENCE n /ROZCO - 4IRADO 2IVES *ULIAN *ORGENSEN 7, -ECHANISM FOR THE ROTAMASE ACTIV ITY OF &+ BINDING PROTEIN FROM MOLECULAR DYNAMICS SIMULATIONS "IOCHEMISTRY n &ISCHER 3 -ICHNICK 3 +ARPLUS - ! MECHANISM FOR ROTAMASE CATALYSIS BY THE &+ BINDING PROTEIN &+"0 "IOCHEMISTRY n "ACH 2$ -INTCHEVA ) +RONENBERG 7* 3CHLEGEL (" 4ORSIONAL BARRIERS IN α KETO AMIDES -ODEL STUDIES RELATED TO THE BINDING SITE OF &+ * /RG #HEM n "ELSHAW 0* -EYER 3$ *OHNSON $$ 2OMO $ )KEDA 9 !NDRUS - !LBERG $' 3CHULTZ ,7 #LARDY * 3CHREIBER 3, 3YNTHESIS STRUCTURE AND MECHANISM IN IMMUNOPHILIN RESEARCH 3YNLETT n "RAUN 7 +ALLEN * -IKOL 6 7ALKINSHAW -$ 7UTHRICH + 4HREE DIMENSIONAL
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
STRUCTURE AND ACTIONS OF IMMUNOSUPPRESSANTS AND THEIR IMMUNOPHILINS &!3%" * n )VERY -4' ! PROPOSED MOLECULAR MODEL FOR THE INTERACTION OF CALCINEURIN WITH THE CYCLOSPORIN ! CYCLOPHILIN ! COMPLEX "IOORG -ED #HEM n )VERY -4' 7EILER , -ODELING THE INTERACTION BETWEEN &+ AND &+"0 A MECHANISM FOR FORMATION OF THE CALCINEURIN INHIBITORY COMPLEX "IOORG -ED #HEM n 3CHREIBER 3, #HEMISTRY AND BIOLOGY OF THE IMMUNOPHILINS AND THEIR IMMUNO SUPPRESSIVE LIGANDS 3CIENCE n )VERY -4' )MMUNOPHILINS SWITCHED ON PROTEIN BINDING DOMAINS -ED 2ES 2EV n $REYFUSS - (AERRI % (OFMANN ( +OBEL ( 0ACHE 7 4SCHERTER ( #YCLOSPORIN ! AND # .EW METABOLITES FROM 4RICHODERMA POLYSPORUM ,INK EX 0ERS 2IFAI %UR * !PPL -ICROBIOL n 2UEGGER ! +UHN - ,ICHTI ( ,OOSLI (2 (UGUENIN 2 1UIQUEREZ # 6ON 7ARTBURG ! #YCLOSPORIN ! A PEPTIDE METABOLITE FROM 4RICHODERMA POLYSPORUM ,INK EX 0ERS 2IFAI WITH IMMUNOSUPPRESSIVE ACTIVITY (ELV #HIM !CTA n #ALNE 29 7HITE $* 4HIRU 3 %VANS $" -C-ASTER 0 $UNN $# #RADDOCK '. 0ENTLOW "$ 2OLLES + #YCLOSPORIN ! IN PATIENTS RECEIVING RENAL ALLOGRAFTS FROM CADAVER DONORS ,ANCET n "OREL *& 0HARMACOLOGY OF CYCLOSPORINE 3ANDIMMUNE )6 0HARMACOLOGICAL PROP ERTIES IN VIVO 0HARM 2EV n 3IEKIERKA ** (UNG 3( 0OE - ,IN #3 3IGAL .( ! CYTOSOLIC BINDING PROTEIN FOR THE IMMUNOSUPPRESSANT &+ HAS PEPTIDYL PROLYL ISOMERASE ACTIVITY BUT IS DISTINCT FROM CYCLOPHILIN .ATURE n +NAPP 7 2IEBER 0 $ORKEN " 3CHMIDT 2% 3TEIN ( VD "ORNE !% 4OWARDS A BETTER DEFINITION OF HUMAN LEUCOCYTE SURFACE MOLECULES )MMUNOLOGY 4ODAY n #RABTREE '2 #ONTINGENT GENETIC REGULATORY EVENTS IN 4 LYMPHOCYTE ACTIVATION 3CIENCE n (INTERDING + !LONSO $IAZ $ 7ALDMANN ( /RGANIC SYNTHESIS AND BIOLOGICAL SIGNAL TRANSDUCTION !NGEW #HEM )NT %D n 4HOMAS ' 4HE 3 KINASE SIGNALING PATHWAY IN THE CONTROL OF DEVELOPMENT AND GROWTH "IO 2ES n &RIEDMAN * 7EISSMAN ) 4WO CYTOPLASMIC CANDIDATES FOR IMMUNOPHILIN ACTION ARE REVEALED BY AFFINITY FOR A NEW CYCLOPHILIN ONE IN THE PRESENCE AND ONE IN THE ABSENCE OF #S! #ELL n ,IU * &ARMER *$ *R ,ANE 73 &RIEDMAN * 7EISSMAN ) 3CHREIBER 3, #ALCINEURIN IS A COMMON TARGET OF CYCLOPHILIN CYCLOSPORIN ! AND &+"0 &+ COMPLEXES #ELL n 3ABATINI $- %RDJUMENT "ROMAGE ( ,UI - 4EMPST 0 3NYDER 3( 2!&4 A MAM MALIAN PROTEIN THAT BINDS TO &+"0 IN A RAPAMYCIN DEPENDENT FASHION AND IS HOMOLO GOUS TO YEAST 4/2S #ELL n "ROWN %* !LBERS -7 3HIN 4" )CHIKAWA + +EITH #4 ,ANE 73 3CHREIBER 3, ! MAMMALIAN PROTEIN TARGETED BY ' ARRESTING RAPAMYCIN RECEPTOR COMPLEX .ATURE n +ISSINGER #2 0ARGE (% +NIGHTON $2 ,EWIS #4 0ELLETIER ,! 4EMPCZYK ! +ALISH 6*
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
4UCKER +$ 3HOWALTER 2% ET AL #RYSTAL STRUCTURES OF HUMAN CALCINEURIN AND THE HUMAN &+"0 &+ CALCINEURIN COMPLEX .ATURE n 'RIFFITH *0 +IM *, +IM %% 3INTCHAK -$ 4HOMSON *! &ITZGIBBON -* &LEMING -! #ARON 02 (SIAO + .AVIA -! 8 RAY STRUCTURE OF CALCINEURIN INHIBITED BY THE IMMUNOPHILIN IMMUNOSUPPRESSANT &+"0 &+ COMPLEX #ELL n #HOI * #HEN * 3CHREIBER 3, #LARDY * 3TRUCTURE OF THE &+"0 RAPAMYCIN COMPLEX INTERACTING WITH THE BINDING DOMAIN OF HUMAN &2!0 3CIENCE n (AMILTON '3 (UANG 7 #ONNOLLY -! 2OSS $4 'UO ( 6ALENTINE (, 3UZDAK 0$ 3TEINER *0 &+"0 BINDING DOMAIN ANALOGS OF &+ ARE POTENT NONIMMUNO SUPPRESSIVE NEUROTROPHIC AGENTS IN VITRO AND PROMOTE RECOVERY IN A MOUSE MODEL OF 0ARKINSONS DISEASE "IOORG -ED #HEM ,ETT n 'OLD "' .EUROIMMUNOPHILIN LIGANDS EVALUATION OF THEIR THERAPEUTIC POTENTIAL FOR THE TREATMENT OF NEUROLOGICAL DISORDERS %XP /PIN )NVEST $RUGS n 3NYDER 3( 3ABATINI $- ,AI -- 3TEINER *0 (AMILTON '3 3UZDAK 0$ .EURAL ACTIONS OF IMMUNOPHILIN LIGANDS 4RENDS 0HARMACOL 3CI n 0OULTER -/ 0AYNE +" 3TEINER *0 .EUROIMMUNOPHILINS ! NOVEL DRUG THERAPY FOR THE REVERSAL OF NEURODEGENERATIVE DISEASE .EUROSCIENCE n 'OLD "' &+ AND THE ROLE OF THE IMMUNOPHILIN &+"0 IN NERVE REGENERATION $RUG -ETAB 2EV n 'OLD "' $ENSMORE 6 3HOU 7 -ATZUK -- 'ORDON (3 )MMUNOPHILIN &+ BINDING PROTEIN NOT &+ BINDING PROTEIN MEDIATES THE NEUROTROPHIC ACTION OF &+ * 0HARMACOL %XP 4HER n 0RICE 2$ 9AMAJI 4 9AMAMOTO ( (IGASHI 9 (ANAOKA + 9AMAZAKI 3 )SHIYE - !RAMORI ) -ATSUOKA . -UTOH 3 ET AL &+ A NOVEL NON IMMUNOSUPPRESSIVE IMMU NOPHILIN NEUROTROPHIC ACTIVITY AND MECHANISM OF ACTION %UR * 0HARM n 2EVILL 70 6ODA * 2EEVES #2 #HUNG , 3CHIRMER ! !SHLEY ' #ARNEY *2 &ARDIS - #AR RERAS #7 :HOU 9 ET AL 'ENETICALLY ENGINEERED ANALOGS OF ASCOMYCIN FOR NERVE REGENERATION * 0HARMACOL %XP 4HER n 'OLD "' .UTT *' .EUROIMMUNOPHILIN LIGANDS IN THE TREATMENT OF 0ARKINSON@S DISEASE #URR /PIN 0HARMACOL n 3AUER ( &RANCIS *- *IANG ( (AMILTON '3 3TEINER *0 3YSTEMIC TREATMENT WITH '0) IMPROVES SPATIAL MEMORY AND REVERSES CHOLINERGIC NEURON ATROPHY IN THE MEDIAL SEPTAL NUCLEUS OF AGED MICE "RAIN 2ES n 3UEHIRO % 3INGLETON 2( 3TONE *2 0OVLISHOCK *4 4HE IMMUNOPHILIN LIGAND &+ ATTENUATES THE AXONAL DAMAGE ASSOCIATED WITH RAPID REWARMING FOLLOWING POSTTRAUMATIC HYPOTHERMIA %XP .EUROL n 7ULLSCHLEGER 3 ,OEWITH 2 (ALL -. 4/2 SIGNALING IN GROWTH AND METABOLISM #ELL n (UANG 3 (OUGHTON 0* 4ARGETING M4/2 SIGNALING FOR CANCER THERAPY #URR /PIN 0HARMACOL n 3MOLEWSKI 0 2ECENT DEVELOPMENTS IN TARGETING THE MAMMALIAN TARGET OF RAPAMY CIN M4/2 KINASE PATHWAY !NTI #ANCER $RUGS n &AIVRE 3 +ROEMER ' 2AYMOND % #URRENT DEVELOPMENT OF M4/2 INHIBITORS AS ANTICANCER AGENTS .AT 2EV $RUG $ISCOV n 7ALLEMACQ 0% 2ECENT DEVELOPMENTS WITH IMMUNOSUPPRESSANT MACROLIDES TAC ROLIMUS AND SIROLIMUS %XPERT /PIN )NV $RUGS n
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
6IGNOT 3 &AIVRE 3 !GUIRRE $ 2AYMOND % M4/2 TARGETED THERAPY OF CANCER WITH RAPAMYCIN DERIVATIVES !NN /NCOL n ,ARUE , "ELLACOSA ! %PITHELIAL MESENCHYMAL TRANSITION IN DEVELOPMENT AND CANCER ROLE OF PHOSPHATIDYLINOSITOL @ KINASE!KT PATHWAYS /NCOGENE n 'SCHWENDT - 0ROTEIN KINASE #δ %UR * "IOCHEM n 3TEINBERG 3& $ISTINCTIVE ACTIVATION MECHANISMS AND FUNCTIONS FOR PROTEIN KINASE #δ "IOCHEM * n &ANG *9 2ICHARDSON "# 4HE -!0+ SIGNALLING PATHWAYS AND COLORECTAL CANCER ,ANCET /NCOL n &INGAR $# "LENIS * 4ARGET OF RAPAMYCIN 4/2 AN INTEGRATOR OF NUTRIENT AND GROWTH FACTOR SIGNALS AND COORDINATOR OF CELL GROWTH AND CELL CYCLE PROGRESSION /NCOGENE n (AY . 3ONENBERG . 5PSTREAM AND DOWNSTREAM OF M4/2 'ENES $EV n +ISHIMOTO ( (AMADA + 3AUNDERS - "ACKMAN 3 3ASAKI 4 .AKANO 4 -AK 47 3UZUKI ! 0HYSIOLOGICAL FUNCTIONS OF 04%. IN MOUSE TISSUES #ELL 3TRUCT &UNCT n 2OMER , +LEIN # $EHNER ! +ESSLER ( "UCHNER * P n ! NATURAL CANCER KILLER STRUCTURAL INSIGHTS AND THERAPEUTIC CONCEPTS !NGEW #HEM )NT %D n 6OLANTE 20 4OTAL SYNTHESIS OF n &+ 3TRATEGIES 4ACTICS /RG 3YNTH n *ONES 4+ -ILLS 3' 2EAMER 2! !SKIN $ $ESMOND 2 6OLANTE 20 3HINKAI ) 4OTAL SYNTHESIS OF IMMUNOSUPPRESSANT n &+ * !M #HEM 3OC n *ONES 4+ 2EAMER 2! $ESMOND 2 -ILLS 3' #HEMISTRY OF TRICARBONYL HEMIKETALS AND APPLICATION OF %VANS TECHNOLOGY TO THE TOTAL SYNTHESIS OF THE IMMUNOSUPPRESSANT n &+ * !M #HEM 3OC n .AKATSUKA - 2AGAN *! 3AMMAKIA 4 3MITH $" 5EHLING $% 3CHREIBER 3, 4OTAL SYNTHESIS OF &+ AND AN &+"0 PROBE REAGENT ;# # #= &+ * !M #HEM 3OC n )RELAND 2% 'LEASON *, 'EGNAS ,$ (IGHSMITH 4+ ! TOTAL SYNTHESIS OF &+ * /RG #HEM n )RELAND 2% ,IU , 2OPER 4$ 4OTAL SYNTHESIS OF &+ 0ART #ONSTRUCTION OF THE # # FRAGMENT 4ETRAHEDRON n )RELAND 2% ,IU , 2OPER 4$ 'LEASON *, 4OTAL SYNTHESIS OF &+ 0ART #OMPLE TION OF THE SYNTHESIS 4ETRAHEDRON n 'U 2, 3IH #* 3YNTHESIS OF THE # # SEGMENT OF THE IMMUNOSUPPRESSANT &+ 4ETRAHEDRON ,ETT n *ONES !" 6ILLALOBOS ! ,INDE 2' )) $ANISHEFSKY 3* ! FORMAL SYNTHESIS OF &+ %XPLORATION OF SOME ALTERNATIVES TO MACROLACTAMIZATION * /RG #HEM n 3MITH !" ))) #HEN + 2OBINSON $* ,AAKSO ,- (ALE +* &ORMAL TOTAL SYNTHESIS OF &+ #ONCISE CONSTRUCTION OF THE # # SEGMENT VIA AN EFFECTIVE COUPLING TACTIC 4ETRAHEDRON ,ETT n "RITTAIN $%! 'RIFFITHS *ONES #- ,INDER -2 3MITH -$ -C#USKER # "ARLOW *3 !KI YAMA 2 9ASUDA + ,EY 36 4OTAL SYNTHESIS OF ANTASCOMICIN " !NGEW #HEM )NT %D n #HAKRABORTY 4+ -OHAN "+ 3TUDIES DIRECTED TOWARDS THE SYNTHESIS OF ANTASC OMICIN ! STEREOSELECTIVE SYNTHESIS OF THE # # FRAGMENT OF THE MOLECULE 4ETRAHEDRON ,ETT n #HAKRABORTY 4+ -OHAN "+ 3REEKANTH - 3TUDIES DIRECTED TOWARDS THE SYNTHESIS
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
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4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
$ESMOND 2 -ILLS 3' 6OLANTE 20 3HINKAI ) ! HIGHLY SELECTIVE METHOD FOR THE SYNTHESIS OF % α METHYL α β UNSATURATED ALDEHYDES 4ETRAHEDRON ,ETT n %VANS $! "ARTROLI * 3HIH 4, %NANTIOSELECTIVE ALDOL CONDENSATIONS %RYTHRO SELECTIVE CHIRAL ALDOL CONDENSATIONS VIA BORON ENOLATES * !M #HEM 3OC n "RAUN - $EVANT 2 2 AND 3 !CETOXY TRIPHENYLETHANOL n EFFECTIVE SYNTHETIC EQUIVALENTS OF A CHIRAL ACETATE ENOLATE 4ETRAHEDRON ,ETT n "RAUN - 'RAF 3 (ERZOG 3 2 (YDROXY TRIPHENYLETHYL ACETATE ETHANEDIOL TRIPHENYL ACETATE 2 /RG 3YN n +IMBALL $" 3ILKS ,! ))) #URRENT PROGRESS IN THE ACETATEMETHYL KETONE ALDOL REAC TION #URR /RG #HEM n %VANS $! 3JOGREN %" 7EBER !% #ONN 2% !SYMMETRIC SYNTHESIS OF ANTI β HYDROXY α AMINO ACIDS 4ETRAHEDRON ,ETT n 0OSS #3 3CHREIBER 3, 4WO DIRECTIONAL CHAIN SYNTHESIS AND TERMINUS DIFFERENTIA TION !CC #HEM 2ES n 3PIVEY !# !NDREWS ") "ROWN !$ !SYMMETRIC DESYMMETRIZATION EXPLOITING SYMMETRY TO EXPEDITE ORGANIC SYNTHESIS 2ECENT 2ES $EVELOP /RG #HEM n !SKIN $ 6OLANTE 20 2EAMER 2! 2YAN +- 3HINKAI ) ! DIASTEREOSPECIFIC NON RACEMIC SYNTHESIS OF THE # TO # SEGMENT OF &+ 4ETRAHEDRON ,ETT n 3CHREIBER 3, 3CHREIBER 43 3MITH $" 2EACTIONS THAT PROCEED WITH A COMBINATION OF ENANTIOTOPIC GROUP AND DIASTEREOTOPIC FACE SELECTIVITY CAN DELIVER PRODUCTS WITH VERY HIGH ENANTIOMERIC EXCESS EXPERIMENTAL SUPPORT OF A MATHEMATICAL MODEL * !M #HEM 3OC n "ARTLETT 0! *ERNSTEDT ++ h0HOSPHATE EXTENSIONv ! STRATAGEM FOR THE STEREOSELEC TIVE FUNCTIONALIZATION OF ACYCLIC HOMOALLYLIC ALCOHOLS * !M #HEM 3OC n 2OSSITER "% 3HARPLESS +" !SYMMETRIC EPOXIDATION OF HOMOALLYLIC ALCOHOLS 3YN THESIS OF n γ AMINO β 2 HYDROXYBUTYRIC ACID '!"/" * /RG #HEM n "ONGINI ! #ARDILLO ' /RENA - 0ORZI ' 3ANDRI 3 2EGIO AND STEREOCONTROLLED SYNTHESIS OF EPOXY ALCOHOLS AND TRIOLS FROM ALLYLIC AND HOMOALLYLIC ALCOHOLS VIA IODOCAR BONATES * /RG #HEM n "ARTLETT 0! -EADOWS *$ "ROWN %' -ORIMOTO ! *ERNSTEDT ++ #ARBONATE EXTEN SION ! VERSATILE PROCEDURE FOR FUNCTIONALIZATION OF ACYCLIC HOMOALLYLIC ALCOHOLS WITH MODERATE STEREOCONTROL * /RG #HEM n $UAN **7 3MITH !" ))) )ODINE MONOBROMIDE )"R AT LOW TEMPERATURE ENHANCED DIASTEREOSELECTIVITY IN ELECTROPHILIC CYCLIZATIONS OF HOMOALLYLIC CARBONATES * /RG #HEM n "ABINE 2% !SYMMETRIC EPOXIDATION OF DIVINYL CARBINOL A NEW APPROACH TO THE SYNTHESIS OF DIDEOXYHEXOSES 4ETRAHEDRON ,ETT n +AHN 3$ (EHRE 7* #ONFORMATIONAL ANALYSIS OF BUTEN OL A MODEL ASYMMET RIC OLEFIN 4ETRAHEDRON ,ETT n "USS !$ 7ARREN 3 3TEREOCHEMICALLY PURE % AND : ALKENES BY THE 7ITTIG (ORNER REACTION * #HEM 3OC #HEM #OMMUN n "USS !$ 7ARREN 3 CIS /LEFINS FROM THE (ORNER 7ITTIG REACTION ORIGIN AND OPTI MIZATION OF STEREOCHEMISTRY 4ETRAHEDRON ,ETT n !YREY 0- "OLTON -! "USS !$ 'REEVES . ,EVIN $ 7ALLACE 0 7ARREN 3 3TERE OCHEMICALLY CONTROLLED SYNTHESIS OF UNSATURATED ALCOHOLS BY THE (ORNER 7ITTIG REACTION * #HEM 3OC 0ERKIN 4RANS n 3CHEID ' 2UIJTER % +ONARZYCKA "ESSLER - "ORNSCHEUER 54 7ESSJOHANN ,! 3YN
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
THESIS AND RESOLUTION OF A KEY BUILDING BLOCK FOR EPOTHILONES A COMPARISON OF ASYMMET RIC SYNTHESIS CHEMICAL AND ENZYMATIC RESOLUTION 4ETRAHEDRON !SYMM n #OREY %* %RICKSON "7 /XIDATIVE HYDROLYSIS OF DITHIANE DERIVATIVES TO CARBO NYL COMPOUNDS USING . HALOSUCCINIMIDE REAGENTS * /RG #HEM n !SKIN $ *OE $ 2EAMER 2! 6OLANTE 20 3HINKAI ) %FFICIENT DEGRADATION OF &+ TO A VERSATILE SYNTHETIC INTERMEDIATE * /RG #HEM n (AMADA 9 +ATO 3 3HIOIRI 4 .EW METHODS AND REAGENTS IN ORGANIC SYNTHESIS ! SYNTHESIS OF ASCIDIACYCLAMIDE A CYTOTOXIC CYCLIC PEPTIDE FROM ASCIDIAN n DETERMINATION OF ITS ABSOLUTE CONFIGURATION 4ETRAHEDRON ,ETT n 3AKAITANI - /HFUNE 9 3ELECTIVE TRANSFORMATION OF . TERT BUTOXYCARBONYL GROUP INTO . ALKOXYCARBONYL GROUP VIA . CARBOXYLATE ION EQUIVALENT 4ETRAHEDRON ,ETT n "ALD % 3AIGO + -UKAIYAMA 4 &ACILE SYNTHESIS OF CARBOXAMIDES BY USING METHYL HALOPYRIDINIUM IODIDES AS COUPLING REAGENTS #HEM ,ETT n $ESS $" -ARTIN *# ! USEFUL ) TRIACETOXYPERIODINANE THE $ESS -ARTIN PERIO DINANE FOR THE SELECTIVE OXIDATION OF PRIMARY OR SECONDARY ALCOHOLS AND A VARIETY OF RELATED ) SPECIES * !M #HEM 3OC n 3CHREIBER 3, 3MITH $" 3TUDIES RELATING TO THE SYNTHESIS OF THE IMMUNOSUPPRES SIVE AGENT &+ SYNTHESIS OF THE CYCLOHEXYL MOIETY VIA A GROUP SELECTIVE EPOXIDATION * /RG #HEM n (ANSON 2- %POXIDE MIGRATION 0AYNE REARRANGEMENT AND RELATED REACTIONS /RG 2EACT n )RELAND 2% -UELLER 2( #LAISEN REARRANGEMENT OF ALLYL ESTERS * !M #HEM 3OC n $ANISHEFSKY 3 &UNK 2, +ERWIN *& *R #LAISEN REARRANGEMENTS OF LACTONIC SILYL ENOLATES A NEW ROUTE TO FUNCTIONALIZED CYCLOALKENES * !M #HEM 3OC n #ASTRO !-- #LAISEN REARRANGEMENT OVER THE PAST NINE DECADES #HEM 2EV n %VANS $! &U '# (OVEYDA !( 2HODIUM) CATALYZED HYDROBORATION OF OLEFINS 4HE DOCUMENTATION OF REGIO AND STEREOCHEMICAL CONTROL IN CYCLIC AND ACYCLIC SYSTEMS * !M #HEM 3OC n .OYORI 2 /HKUMA 4 +ITAMURA - 4AKAYA ( 3AYO . +UMOBAYASHI ( !KUTAGAWA 3 !SYMMETRIC HYDROGENATION OF β KETO CARBOXYLIC ESTERS ! PRACTICAL PURELY CHEMICAL ACCESS TO β HYDROXY ESTERS IN HIGH ENANTIOMERIC PURITY * !M #HEM 3OC n &RÇTER ' -ßLLER 5 'ßNTHER 7 4HE STEREOSELECTIVE α ALKYLATION OF CHIRAL β HYDROXY ESTERS AND SOME APPLICATIONS THEREOF 4ETRAHEDRON n 3EEBACH $ !EBI * 7ASMUTH $ $IASTEREOSELECTIVE α ALKYLATION OF β HYDROXYCAR BOXYLIC ESTERS THROUGH ALKOXIDE ENOLATES DIETHYL 3 2 ALLYL HYDROXYSUCCINATE FROM DIETHYL 3 n MALATE /RG 3YNTH n 2EETZ -4 +ESSELER + *UNG ! #ONCERNING THE ROLE OF ,EWIS ACIDS IN CHELATION CONTROLLED ADDITION TO CHIRAL ALKOXY ALDEHYDES 4ETRAHEDRON ,ETT n "URGESS %- 0ENTON (2 *R 4AYLOR %! 4HERMAL REACTIONS OF ALKYL . CARBOMETHOX YSULFAMATE ESTERS * /RG #HEM n 2AGAN *! .AKATSUKA - 3MITH $" 5EHLING $% 3CHREIBER 3, 3TUDIES OF THE IMMUNOSUPPRESSIVE AGENT &+ SYNTHESIS OF AN ADVANCED INTERMEDIATE * /RG #HEM n
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
'ILBERT *# 7EERASOORIYA 5 %LABORATION OF ALDEHYDES AND KETONES TO ALKYNES IMPROVED METHODOLOGY * /RG #HEM n (ART $7 3CHWARTZ * (YDROZIRCONATION /RGANIC SYNTHESIS VIA ORGANOZIRCONIUM INTERMEDIATES 3YNTHESIS AND REARRANGEMENT OF ALKYLZIRCONIUM)6 COMPLEXES AND THEIR REACTION WITH ELECTROPHILES * !M #HEM 3OC n ,ABINGER *! (ART $7 3EIBERT 7% ))) 3CHWARTZ * %LECTROPHILIC CLEAVAGE OF THE CARBON ZIRCONIUM)6 BOND #OMPARISON AND CONTRAST WITH OTHER TRANSITION METAL ALKYL SYSTEMS * !M #HEM 3OC n 3CHREIBER 3, 3AMMAKIA 4 5EHLING $% 3TUDIES RELATING TO THE SYNTHESIS OF THE IMMUNOSUPPRESSIVE AGENT &+ APPLICATION OF THE TWO DIRECTIONAL CHAIN SYNTHESIS STRATEGY TO THE PYRANOSE MOIETY * /RG #HEM n 'REENBERG 3 -OFFATT *' 2EACTIONS OF ACYLOXYISOBUTYRYL HALIDES WITH NUCLE OSIDES ) 2EACTIONS OF MODEL DIOLS AND OF URIDINE * !M #HEM 3OC n (OYE 42 0ECK $2 3WANSON 4! +INETIC LACTONIZATION OF DIMETHYL AND TETRAMETHYL HYDROXYAZELAIC ACIDS GROUND STATE CONFORMATIONAL CONTROL * !M #HEM 3OC n )RELAND 2% (IGHSMITH 4+ 'EGNAS ,$ 'LEASON *, 3YNTHESIS OF THE ACETONIDE OF DIHYDRO &+ * /RG #HEM n &ISHER -* #HOW + 6ILLALOBOS ! $ANISHEFSKY 3* /N THE REMARKABLE PROPENSITY FOR CARBON CARBON BOND CLEAVAGE REACTIONS IN THE # # REGION OF &+ * /RG #HEM n "AUMANN + /BERHAUSER " 'RASSBERGER -! (AIDL ' 3CHULZ ' 3YNTHESIS AND OXI DATIVE CLEAVAGE OF THE MAJOR EQUILIBRIUM PRODUCTS OF ASCOMYCIN AND &+ 4ETRAHEDRON ,ETT n !DAMS -! $UGGAN !* 3MOLANOFF * -EINWALD * 4OTAL SYNTHESIS OF PEDERA MIDE * !M #HEM 3OC n 4ANAKA ( +URODA ! -ARUSAWA ( (ASHIMOTO - (ATANAKA ( +INO 4 'OTO 4 /KUHARA - 0HYSICOCHEMICAL PROPERTIES OF &+ A NOVEL IMMUNOSUPPRESSANT ISOLATED FROM 3TREPTOMYCES TSUKUBAENSIS 4RANSPLANT 0 n )RELAND 2% 7IPF 0 -ILTZ 7 6ANASSE " -ETHODOLOGY FOR THE ENANTIOSELECTIVE SYNTHESIS OF ALDOLS AND OTHER DIOXYGENATED SYSTEMS * /RG #HEM n (ICKS $2 &RASER 2EID " 4HE AND # METHYL DERIVATIVES OF METHYL DIDEOXY α $ ERYTHRO HEX ENOPYRANOSOID ULOSE #AN * #HEM n (ANESSIAN 3 2ANCOURT ' #ARBOHYDRATES AS CHIRAL INTERMEDIATES IN ORGANIC SYN THESIS 4WO FUNCTIONALIZED CHEMICAL PRECURSORS COMPRISING EIGHT OF THE TEN CHIRAL CENTRES OF ERYTHRONOLIDE ! #AN * #HEM n 3UM 0 % 7EILER , 3TEREOSELECTIVE SYNTHESIS OF n α MULTISTRIATIN FROM $ GLUCOSE #AN * #HEM n )RELAND 2% -UCHMORE $# (ENGARTNER 5 . . . . 4ETRAMETHYLPHOSPHORO DIAMIDATE GROUP 5SEFUL FUNCTION FOR THE PROTECTION OR REDUCTIVE DEOXYGENATION OF ALCOHOLS AND KETONES * !M #HEM 3OC n )RELAND 2% $OW 7# 'ODFREY *$ 4HAISRIVONGS 3 4OTAL SYNTHESIS OF APHIDICO LIN AND β CHAMIGRENE * /RG #HEM n !LBRIGHT *$ 'OLDMAN , $IMETHYL SULFOXIDE ACID ANHYDRIDE MIXTURES FOR THE OXIDATION OF ALCOHOLS * !M #HEM 3OC n 2AMACHANDRAN 06 0INANE BASED VERSATILE hALLYLv BORANES !LDRICHIMICA !CTA n
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
"ROWN (# 2AMACHANDRAN 06 6ERSATILE α PINENE BASED BORANE REAGENTS FOR ASYMMETRIC SYNTHESES * /RGANOMET #HEM n -IHELICH %$ $ANIELS + %ICKHOFF $* 6ANADIUM CATALYZED EPOXIDATIONS (IGHLY STEREOSELECTIVE EPOXIDATIONS OF ACYCLIC HOMOALLYLIC ALCOHOLS PREDICTED BY A DETAILED TRANSITION STATE MODEL * !M #HEM 3OC n !NDERSON #$ 'OODMAN , "AKER "2 0OTENTIAL ANTICANCER AGENTS 6)) 3YNTHESIS AND AMMONOLYSIS OF METHYL ANHYDRO $ RIBOFURANOSIDE * !M #HEM 3OC n *ENKINS 32 7ALTON% 3YNTHESIS OF DEOXY # METHYL β $ XYLOFURANOSYL ADENINE "RANCHED CHAIN SUGAR ANALOG OF CORDYCEPIN #ARBOHYD 2ES n 4HEIS !" 4OWNSEND #! ! SIMPLE INEXPENSIVE PREPARATION OF HIGHLY PURE COPPER) BROMIDE AND ITS DIMETHYL SULFIDE COMPLEX 3YN #OMMUN n $E3HONG 0 7ALTERMIRE 2% !MMON (, ! GENERAL APPROACH TO THE STEREOSELECTIVE SYNTHESIS OF SPIROKETALS ! TOTAL SYNTHESIS OF THE PHEROMONES OF THE OLIVE FRUIT FLY AND RELATED COMPOUNDS * !M #HEM 3OC n -ORI + 3ENDA 3 0HEROMONE SYNTHESIS 3YNTHESIS OF THE ENANTIOMERS OF CIS METHYL HEXANOLIDE THE MAJOR COMPONENT OF THE SEX PHEROMONE OF THE CARPENTER BEE 4ETRAHEDRON n 2AND #, 6AN (ORN $% -OORE -7 .EGISHI % ! VERSATILE AND SELECTIVE ROUTE TO DIFUNCTIONAL TRISUBSTITUTED % ALKENE SYNTHONS VIA ZIRCONIUM CATALYZED CARBOALUMINA TION OF ALKYNES * /RG #HEM n .EGISHI % 6AN (ORN $% 9OSHIDA 4 #ONTROLLED CARBOMETALATION #ARBOMETALA TION REACTION OF ALKYNES WITH ORGANOALENE ZIRCONOCENE DERIVATIVES AS A ROUTE TO STEREO AND REGIODEFINED TRISUBSTITUTED ALKENES * !M #HEM 3OC n 7IPF 0 4RANSMETALATION REACTIONS IN ORGANOCOPPER CHEMISTRY 3YNTHESIS n #OLEMAN 23 $ANISHEFSKY 3* $EGRADATION AND MANIPULATIONS OF THE IMMUNO SUPPRESSANT &+ PREPARATION OF POTENTIAL SYNTHETIC INTERMEDIATES (ETEROCYCLES n .AKAJIMA . (ORITA + !BE 2 9ONEMITSU / -0- METHOXYBENZYL PROTECTION OF HYDROXY FUNCTIONS UNDER MILD ACIDIC CONDITIONS 4ETRAHEDRON ,ETT n (ORITA + 9OSHIOKA 4 4ANAKA 4 /IKAWA 9 9ONEMITSU / /N THE SELECTIVITY OF DEPROTECTION OF BENZYL -0- METHOXYBENZYL AND $-0- DIMETHOXYBENZYL PROTECTING GROUPS FOR HYDROXYL FUNCTIONS 4ETRAHEDRON n -C-URRY *% 3COTT 7* ! NEW METHOD OF OLEFIN SYNTHESIS #OUPLING OF LITHIUM DIALKYLCUPRATES WITH ENOL TRIFLATES 4ETRAHEDRON ,ETT n %INHORN # %INHORN * ,UCHE *, 3ONOCHEMISTRY n THE USE OF ULTRASONIC WAVES IN SYNTHETIC ORGANIC CHEMISTRY 3YNTHESIS n 0OLL 4 !BDEL (!& +ARGE 2 ,INZ ' 7EETMAN * (ELMCHEN ' . 3UBSTITUTED HYDROXYSUCCINIMIDES FROM 3 MALIC ACID AS NEW REAGENTS FOR ASYMMETRIC $IELS !LDER ADDITIONS TO ENOATES 4ETRAHEDRON ,ETT n ,UCHE *, ,ANTHANIDES IN ORGANIC CHEMISTRY 3ELECTIVE REDUCTIONS OF CONJU GATED KETONES * !M #HEM 3OC n 3UMINO 9 4OMISAKA 9 /GAWA ! #ERIUM REAGENTS IN ORGANIC SYNTHESIS -ATERIALS )NTEGRATION n %LLIOTT * (ALL $ 7ARREN 3 2EVERSED STEREOCHEMICAL CONTROL IN THE REGIOSELECTIVE
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
REDUCTION OF HINDERED DIPHENYLPHOSPHINOYL 0H0/ KETONES AND ENONES 4ETRAHEDRON ,ETT n %LLIOTT * (ALL $ 7ARREN 3 2EVERSED STEREOCHEMICAL CONTROL IN THE REGIOSELECTIVE REDUCTION OF HINDERED DIPHENYLPHOSPHINOYL 0H0/ KETONES AND ENONES 4ETRAHEDRON ,ETT n 'AREGG 0* *OHANSSON 2 /RTEGA # 3AMUELSSON " .OVEL REAGENT SYSTEM FOR CON VERTING A HYDROXY GROUP INTO AN IODO GROUP IN CARBOHYDRATES WITH INVERSION OF CONFIGU RATION 0ART * #HEM 3OC 0ERKIN 4RANS n &ßRSTNER ! #HEMISTRY OF AND WITH HIGHLY REACTIVE METALS !NGEW #HEM )NT %D n )RELAND 2% 7IPF 0 3TUDIES DIRECTED TOWARDS THE TOTAL SYNTHESIS OF &+ PREPARA TION OF A # TO # SEGMENT 4ETRAHEDRON ,ETT n 3OMERS 0+ 7ANDLESS 4* 3CHREIBER 3, 3YNTHESIS AND ANALYSIS OF "$ A HIGH AFFINITY LIGAND FOR THE IMMUNOPHILIN &+"0 * !M #HEM 3OC n 2AO !62 #HAKRABORTY 4+ 2EDDY +, 3TUDIES DIRECTED TOWARDS THE SYNTHESIS OF IMMUNOSUPPRESSIVE AGENT &+ SYNTHESIS OF THE ENTIRE BOTTOM HALF 4ETRAHEDRON ,ETT n +ATSUKI 4 -ARTIN 63 !SYMMETRIC EPOXIDATION OF ALLYLIC ALCOHOLS 4HE +ATSUKI 3HARPLESS EPOXIDATION REACTION /RG 2EACT n ,IPSHUTZ "( 7ILHELM 23 +OZLOWSKI *! 4HE CHEMISTRY OF HIGHER ORDER ORGANOCU PRATES 4ETRAHEDRON n #OREY %* "EAMES $* -IXED CUPRATE REAGENTS OF TYPE 22#U,I WHICH ALLOW SELEC TIVE GROUP TRANSFER * !M #HEM 3OC n 7OVKULICH 0- 3HANKARAN + +IEGIEL * 5SKOKOVIC -2 4OTAL SYNTHESIS OF ! * /RG #HEM n )RELAND 2% 7IPF 0 !RMSTRONG *$ ))) 3TEREOCHEMICAL CONTROL IN THE ESTER ENOLATE #LAISEN REARRANGEMENT 3TEREOSELECTIVITY IN SILYL KETENE ACETAL FORMATION * /RG #HEM n 0ÊMIES / "ÉECKVALL * % #OMBINATION OF ENZYMES AND METAL CATALYSTS ! POWERFUL APPROACH IN ASYMMETRIC CATALYSIS #HEM 2EV n "ARTLETT 0! -C1UAID ,! 4OTAL SYNTHESIS OF METHYL SHIKIMATE AND PHOS PHOSHIKIMIC ACID * !M #HEM 3OC n 'U 2, 3IH #* ! CHEMOENZYMATIC SYNTHESIS OF THE # # MOIETY OF &+ 4ETRAHEDRON ,ETT n ,INDE 2' )) %GBERTSON - #OLEMAN 23 *ONES !" $ANISHEFSKY 3* %FFICIENT PREPA RATION OF INTERMEDIATES CORRESPONDING TO # # AND # # OF &+ * /RG #HEM n (ENBEST (" 7ILSON 2!, !SPECTS OF STEREOCHEMISTRY ) 3TEREOSPECIFICITY IN FORMA TION OF EPOXIDES FROM CYCLIC ALLYLIC ALCOHOLS * #HEM 3OC n "ERNET " 6ASELLA ! &RAGMENTATION OF DEOXY HALOHEXONO ORTHOLACTONES A CONCERTED NONSTEREOSPECIFIC PROCESS (ELV #HIM !CTA n 6ILLALOBOS ! $ANISHEFSKY 3* 3TEREOSELECTIVE ROUTES TO THE # # FRAGMENT OF &+ * /RG #HEM n -ARYANOFF "% 2EITZ !" 4HE 7ITTIG OLEFINATION REACTION AND MODIFICATIONS INVOLV ING PHOSPHORYL STABILIZED CARBANIONS 3TEREOCHEMISTRY MECHANISM AND SELECTED SYN THETIC ASPECTS #HEM 2EV n %VANS $! -ORRISSEY -- $OW 2, (YDROXYL DIRECTED HYDROGENATION OF
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
HOMOALLYLIC ALCOHOLS %FFECTS OF ACHIRAL AND CHIRAL RHODIUM CATALYSTS ON STEREOCON TROL 4ETRAHEDRON ,ETT n (OFFMANN 27 !LLYLIC STRAIN AS A CONTROLLING FACTOR IN STEREOSELECTIVE TRANSFOR MATIONS #HEM 2EV n 3MITH !" ))) (ALE +* ,AAKSO ,- #HEN + 2IERA ! &+ SYNTHETIC STUDIES !N EFFICIENT ASYMMETRIC SYNTHESIS OF THE # # FRAGMENT OF &+ &2 AND &2 4ETRAHEDRON ,ETT n 3MITH !" ))) (ALE +* !N ENANTIOSELECTIVE SYNTHESIS OF THE # TO # BACK BONE OF THE POTENT IMMUNOSUPPRESSANT &+ 4ETRAHEDRON ,ETT n .ICOLAOU +# 0APAHATJIS $0 #LAREMON $! -AGOLDA 2, $OLLE 2% 4OTAL SYNTHESIS OF IONOPHORE ANTIBIOTIC 8 ! * /RG #HEM n "IERER "% 3OMERS 0+ 7ANDLESS 4* "URAKOFF 3* 3CHREIBER 3, 0ROBING IMMUNOSUP PRESSANT ACTION WITH A NONNATURAL IMMUNOPHILIN LIGAND 3CIENCE n 3TEINER *0 $AWSON 4- &OTUHI - 'LATT #% 3NOWMAN !- #OHEN . 3NYDER 3( (IGH BRAIN DENSITIES OF THE IMMUNOPHILIN &+"0 COLOCALIZED WITH CALCINEURIN .ATURE n 'OROBETS %6 -IFTAKHOV -3 6ALEEV &! 4ANDEM TRANSFORMATIONS INITIATED AND DETERMINED BY THE -ICHAEL REACTION 2USS #HEM 2EV n "ANERJEE $+ $IECKMANN CYCLIZATION AND UTILIZATION OF THE PRODUCTS IN THE SYN THESIS OF STEROIDS 0 )NDIAN !CAD 3CI ! n 'RADILLAS ! 0ÎREZ #ASTELLS * -ACROCYCLIZATION BY RING CLOSING METATHESIS IN THE TOTAL SYNTHESIS OF NATURAL PRODUCTS REACTION CONDITIONS AND LIMITATIONS !NGEW #HEM )NT %D n "ARLOW *3 $IXON $* &OSTER !# ,EY 36 2EYNOLDS $* .EW BUILDING BLOCKS FOR EFFI CIENT AND HIGHLY DIASTEREOSELECTIVE POLYOL PRODUCTION SYNTHESIS AND UTILITY OF 2 2 3 3 AND 3 3 2 2 BUTANE DIACETAL PROTECTED BUTANE TETROL DERIVATIVES * #HEM 3OC 0ERKIN 4RANS n $IXON $* &OSTER !# ,EY 36 2EYNOLDS $* 0REPARATION OF DESYMMETRIZED MESO TAR TRATE DERIVATIVES SYNTHESIS AND UTILITY OF 2 2 2 3 BUTANE DIACETAL PROTECTED DIME THYL TARTRATE * #HEM 3OC 0ERKIN 4RANS n ,EY 36 $IXON $* 'UY 24 0ALOMERO -! 0OLARA ! 2ODRIGUEZ & 3HEPPARD 4$ 3TUD IES ON THE GENERATION OF ENOLATE ANIONS FROM BUTANE DIACETAL PROTECTED GLYCOLIC ACID DERIVATIVES AND SUBSEQUENT HIGHLY DIASTEREOSELECTIVE COUPLING REACTIONS WITH ALDEHYDES AND ACID CHLORIDES /RG "IOMOL #HEM n ,EY 36 "AESCHLIN $+ $IXON $* &OSTER !# )NCE 3* 0RIEPKE (7- 2EYNOLDS $* $IACETALS ! NEW OPPORTUNITY FOR ORGANIC SYNTHESIS #HEM 2EV n "ULL *! "ALSKUS %0 (ORAN 2!* ,ANGNER - ,EY 36 3TEREOCONTROLLED TOTAL SYNTHESIS OF BENGAZOLE ! A MARINE BISOXAZOLE NATURAL PRODUCT DISPLAYING POTENT ANTIFUNGAL PROP ERTIES !NGEW #HEM )NT %D n +ECK '% 3AVIN +! #RESSMAN %.+ !BBOTT $% %FFECTS OF OLEFIN GEOMETRY ON THE STEREOCHEMISTRY OF ,EWIS ACID MEDIATED ADDITIONS OF CROTYLSTANNANES TO ALDEHYDES * /RG #HEM n +ECK '% !BBOTT $% "ODEN %0 %NHOLM %* /BSERVATIONS ON THE CHOICE OF ,EWIS ACID AND MODE OF ADDITION FOR THE ,EWIS ACID MEDIATED REACTION OF CROTYLTRI N BUTYLSTAN NANE WITH ALDEHYDES CONVENIENT AND HIGHLY SELECTIVE ACCESS TO BOTH ERYTHRO AND THREO PRODUCTS 4ETRAHEDRON ,ETT n 3CHOLL - $ING 3 ,EE #7 'RUBBS 2( 3YNTHESIS AND ACTIVITY OF A NEW GENERATION
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
OF RUTHENIUM BASED OLEFIN METATHESIS CATALYSTS COORDINATED WITH DIMESITYL DIHY DROIMIDAZOL YLIDENE LIGANDS /RG ,ETT n -AETA ( (ASHIMOTO 4 (ASEGAWA 4 3UZUKI + 'RIGNARD TYPE ADDITION OF ALKENYL AND ALKYLZIRCONOCENE CHLORIDE TO ALDEHYDE REMARKABLE CATALYTIC ACCELERATION EFFECT OF SILVER PERCHLORATE 4ETRAHEDRON ,ETT n 3UZUKI + (ASEGAWA 4 )MAI 4 -AETA ( /HBA 3 !G!S& AS SAFE ALTERNATIVE TO !G#L/ FOR GENERATING CATIONIC ZIRCONOCENE SPECIES UTILITIES IN ,EWIS ACID PROMOTED SELECTIVE # # BOND FORMING REACTIONS 4ETRAHEDRON n 2EICH (* #HOW & 0EAKE 3, 3ELENINIC ACIDS AS CATALYSTS FOR OXIDATIONS OF OLEFINS AND SULFIDES USING HYDROGEN PEROXIDE 3YNTHESIS n 9US - !RENE CATALYZED LITHIATION REACTIONS #HEM 3OC 2EV n 9US - (ERRERA 20 'UIJARRO ! /N THE MECHANISM OF ARENE CATALYZED LITHIATION THE ROLE OF ARENE DIANIONS NAPHTHALENE RADICAL ANION VERSUS NAPHTHALENE DIANION #HEM %UR * n ,EY 36 .ORMAN * 'RIFFITH 70 -ARSDEN 30 4ETRAPROPYLAMMONIUM PERRUTHENATE 0R. 2U/n 40!0 A CATALYTIC OXIDANT FOR ORGANIC SYNTHESIS 3YNTHESIS n "AL "3 #HILDERS 7% 0INNICK (7 /XIDATION OF α β UNSATURATED ALDEHYDES 4ETRA HEDRON n .ICOLAOU +# "ULGER 0' 3ARLAH $ -ETATHESIS REACTIONS IN TOTAL SYNTHESIS !NGEW #HEM )NT %D n "ATCHELOR -* 'ILLESPIE 2* 'OLEC *-# (EDGECOCK #*2 ! NOVEL APPLICATION OF THE $ESS -ARTIN REAGENT TO THE SYNTHESIS OF AN &+ ANALOGUE AND OTHER TRICARBONYL COMPOUNDS 4ETRAHEDRON ,ETT n (UGHES 0 -USSER * #ONKLIN - 2USSO 2 4HE ISOLATION SYNTHESIS AND CHARACTERIZA TION OF AN ISOMERIZATION FORM OF RAPAMYCIN 4ETRAHEDRON ,ETT n +ISHI 9 !PPLICATIONS OF NICKEL)) CHROMIUM)) MEDIATED COUPLING REACTIONS TO NATURAL PRODUCT SYNTHESES 0URE !PPL #HEM n #INTAS 0 !DDITION OF ORGANOCHROMIUM COMPOUNDS TO ALDEHYDES 4HE .OZAKI (IYAMA REACTION 3YNTHESIS n &ßRSTNER ! #ARBON #ARBON BOND FORMATION INVOLVING ORGANOCHROMIUM))) REA GENTS #HEM 2EV n 2AO !62 #HAKRABORTY 4+ 3ANKARANAYANAN $ 0URANDARE !6 3TUDIES DIRECTED TOWARDS THE SYNTHESIS OF IMMUNOSUPPRESSIVE AGENT &+ SYNTHESIS OF THE ENTIRE TOP HALF 4ETRAHEDRON ,ETT n "ARTON $(2 -C#OMBIE 37 .EW METHOD FOR THE DEOXYGENATION OF SECONDARY ALCOHOLS * #HEM 3OC 0ERKIN 4RANS n (ARTWIG 7 -ODERN METHODS FOR THE RADICAL DEOXYGENATION OF ALCOHOLS 4ETRAHE DRON n +OCH 33# #HAMBERLIN !2 %NANTIOSELECTIVE PREPARATION OF β ALKYL γ BUTYROLAC TONES FROM FUNCTIONALIZED KETENE DITHIOACETALS * /RG #HEM n .ICOLAOU +# "ERTINATO 0 0ISCOPIO !$ #HAKRABORTY 4+ -INOWA . 3TEREOSELEC TIVE CONSTRUCTION OF THE # # FRAGMENT OF RAPAMYCIN * #HEM 3OC #HEM #OMMUN n .ICOLAOU +# "ULGER 0' 3ARLAH $ 0ALLADIUM CATALYZED CROSS COUPLING REACTIONS IN TOTAL SYNTHESIS !NGEW #HEM )NT %D n -ITCHELL 4. 0ALLADIUM CATALYZED REACTIONS OF ORGANOTIN COMPOUNDS 3YNTHESIS n
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
3TILLE *+ 0ALLADIUM CATALYZED COUPLING OF ORGANOTIN REAGENTS WITH ORGANIC ELEC TROPHILES 0URE !PPL #HEM n 3TILLE *+ 4ANAKA - )NTRAMOLECULAR PALLADIUM CATALYZED CYCLIZATIONS OF ESTERS CONTAINING VINYL TRIFLATE AND VINYLSTANNANE GROUPS AT THE TERMINI SYNTHESIS OF LARGE RING LACTONES * !M #HEM 3OC n 0ATTENDEN ' 4HOM 3- 0OLYENE MACROLACTAM CONSTRUCTION USING A 3TILLE VINYL VINYL COUPLING PROTOCOL AN APPROACH TO THE ANTITUMOUR ANTIBIOTIC SUBSTANCE LEINAMY CIN 3YNLETT n +OENIG +% 7EBER 70 2EACTION OF DEUTERIUM CHLORIDE WITH CIS AND TRANS β TRI METHYLSILYLSTYRENE * !M #HEM 3OC n "LUMENKOPF 4! /VERMAN ,% 6INYLSILANE AND ALKYNYLSILANE TERMINATED CYCLIZA TION REACTIONS #HEM 2EV n 7EI ## "ERNARDO 3$ 4ENGI *0 "ORGESE * 7EIGELE - 3YNTHESIS OF CHIRAL β LACTAMS USING , ASCORBIC ACID * /RG #HEM ,UK +# 7EI ## 0REPARATION OF DERIVATIVES OF 2 BUTANETRIOL FROM L ASCORBIC ACID 3YNTHESIS n 7EE !'( -C,EOD $$ 3TUDIES ON THE 2H)) CATALYZED # ( INSERTION REACTION OF SOME DERIVATIVES OF . [ ;3 $IHYDROXYBUTYL=] α DIAZO ANILIDES SITE SELECTIVITY (ETEROCYCLES n "ASHA ! ,IPTON - 7EINREB 3- ! MILD GENERAL METHOD FOR CONVERSION OF ESTERS TO AMIDES 4ETRAHEDRON ,ETT n "ODE 3% 7OLBERG - -ßLLER - 3TEREOSELECTIVE SYNTHESIS OF DIOLS 3YNTHESIS n (OVEYDA !( %VANS $! &U '# 3UBSTRATE DIRECTABLE CHEMICAL REACTIONS #HEM 2EV n /ISHI 4 .AKATA 4 .EW ASPECTS OF STEREOSELECTIVE SYNTHESIS OF POLYOLS 3YNTHESIS n -ORI 9 3UZUKI - 3TEREOSELECTIVE SYNTHESIS OF SYN ANTI TRIOLS USING A SYN ASYMMETRIC REDUCTION A NOVEL ROUTE TO ANTI POLYOLS 4ETRAHEDRON ,ETT n -ORI 9 +UHARA - 4AKEUCHI ! 3UZUKI - 3TEREOSELECTIVE REDUCTION OF β ALKOXY KETONES A SYNTHESIS OF SYN DIOLS 4ETRAHEDRON ,ETT n -ORI 9 4AKEUCHI ! +AGEYAMA ( 3UZUKI - ! CONVERGENT GENERAL SYNTHETIC PRO TOCOL FOR SYN POLYOLS 4ETRAHEDRON ,ETT n (ECKRODT 4* -ULZER * 3YNTHESIS OF A $IELS !LDER PRECURSOR FOR THE ELISABETHIN ! SKELETON 3YNTHESIS n ,IPSHUTZ "( +OERNER - 0ARKER $! 4HIENYLCYANO COPPER LITHIUM ! LOWER ORDER STABLE hCUPRATE IN A BOTTLEv PRECURSOR TO HIGHER ORDER REAGENTS 4ETRAHEDRON ,ETT n ,AMBERT *" :HAO 9 %MBLIDGE 27 3ALVADOR ,! ,IU 8 3O *( #HELIUS %# 4HE β EFFECT OF SILICON AND RELATED MANIFESTATIONS OF σ CONJUGATION !CC #HEM 2ES n #ERNIGLIARO '* +OCIENSKI 0* ! SYNTHESIS OF n α MULTISTRIATIN * /RG #HEM n %VANS $! -ATHRE $ !SYMMETRIC SYNTHESIS OF THE ENKEPHALINASE INHIBITOR THIOR PHAN * /RG #HEM n
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
&URKERT $0 "RIMBLE -! 3YNTHESIS OF THE # # BIS SPIROACETAL DOMAIN OF SPIROLIDES " AND $ VIA ITERATIVE OXIDATIVE RADICAL CYCLIZATION /RG ,ETT n #OREY %* &UCHS 0, ! SYNTHETIC METHOD FOR FORMYL→ETHYNYL CONVERSION 2#(/→ 2#≡#( OR 2#≡#2 4ETRAHEDRON ,ETT n (ART $7 "LACKBURN 4& 3CHWARTZ * (YDROZIRCONATION ))) 3TEREOSPECIFIC AND REGI OSELECTIVE FUNCTIONALIZATION OF ALKYLACETYLENES VIA VINYLZIRCONIUM)6 INTERMEDIATES * !M #HEM 3OC n #OREY %* "AKSHI 2+ 3HIBATA 3 #HEN #0 3INGH 6+ ! STABLE AND EASILY PREPARED CATALYST FOR THE ENANTIOSELECTIVE REDUCTION OF KETONES !PPLICATIONS TO MULTISTEP SYNTHE SIS * !M #HEM 3OC n #OREY %* .EW ENANTIOSELECTIVE ROUTES TO BIOLOGICALLY INTERESTING COMPOUNDS 0URE !PPL #HEM n +IM +- 0ARK )( ! CONVENIENT HALOGENATION OF α β UNSATURATED CARBONYL COM POUNDS WITH /XONE AND HYDROHALIC ACID ("R (#L 3YNTHESIS n ,EY 36 7HITTLE !* ! CONVENIENT PREPARATION OF HALOENONES FROM ENONES USING PHENYLSELENIUM HALIDES 4ETRAHEDRON ,ETT n )TO 9 (IRAO 4 3AEGUSA 4 3YNTHESIS OF α β UNSATURATED CARBONYL COMPOUNDS BY PALLADIUM)) CATALYZED DEHYDROSILYLATION OF SILYL ENOL ETHERS * /RG #HEM n 7ICK !% &ELIX $ 3TEEN + %SCHENMOSER ! #LAISEN REARRANGEMENT OF ALLYL AND BENZYL ALCOHOLS BY . . DIMETHYLACETAMIDE ACETALS (ELV #HIM !CTA n 'RIECO 0! 'ILMAN 3 .ISHIZAWA - /RGANOSELENIUM CHEMISTRY ! FACILE ONE STEP SYNTHESIS OF ALKYL ARYL SELENIDES FROM ALCOHOLS * /RG #HEM n "LANCHETTE -! #HOY 7 $AVIS *4 %SSENFELD !0 -ASAMUNE 3 2OUSH 72 3AKAI 4 (ORNER 7ADSWORTH %MMONS REACTION 5SE OF LITHIUM CHLORIDE AND AN AMIDE FOR BASE SENSITIVE COMPOUNDS 4ETRAHEDRON ,ETT n .AKATA 4 4ANAKA 4 /ISHI 4 (IGHLY STEREOSELECTIVE SYNTHESIS OF ERYTHRO α β EPOXY ALCOHOLS BY THE REDUCTION OF α β EPOXY KETONES WITH ZINC BOROHYDRIDE 4ETRAHEDRON ,ETT .AKATA 4 4ANAKA 4 /ISHI 3TEREOSELECTIVE REDUCTION OF α HYDROXY KETONES 4ETRA HEDRON ,ETT n 6AN 2HEENEN 6 +ELLY 2# #HA $9 !N IMPROVED CATALYTIC /S/ OXIDATION OF OLEFINS TO CIS GYLCOLS USING TERTIARY AMINE OXIDES AS THE OXIDANT 4ETRAHEDRON ,ETT n 4AKAI + .ITTA + 5TIMOTO + 3IMPLE AND SELECTIVE METHOD FOR ALDEHDYES 2#(/ → % HALOALKENES 2#( #(8 #ONVERSION BY MEANS OF A HALOFORM CHROMOUS CHLORIDE SYSTEM * !M #HEM 3OC n /KAZOE 4 4AKAI + 5TIMOTO + % 3ELECTIVE OLEFINATION OF ALDEHYDES BY MEANS OF GEM DICHROMIUM REAGENTS DERIVED BY REDUCTION OF GEM DIIODOALKANES WITH CHROMIUMII CHLORIDE * !M #HEM 3OC n 7ESSJOHANN ,! 3CHEID ' 2ECENT ADVANCES IN CHROMIUM)) AND CHROMIUM))) MEDIATED ORGANIC SYNTHESIS 3YNTHESIS n #OREY %* 7OLLENBERG 2( ! NUCLEOPHILIC ETHYNYL GROUP EQUIVALENT AND ITS USE IN CONJUGATE ADDITION TO α β ENONES * !M #HEM 3OC n 3HAIR -$ 9OON 4 $ANISHEFSKY 3* ! REMARKABLE CROSS COUPLING REACTION TO CON STRUCT THE ENEDIYNE LINKAGE RELEVANT TO DYNEMICIN ! SYNTHESIS OF THE DEPROTECTED !"# SYSTEM * /RG #HEM n
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
4AKAHASHI 4 3AKAMOTO 9 9AMADA ( 5SUI 3HUJI &UKAZAWA 9 3YNTHESIS OF A DYNE MICIN ! ANALOGUE AND ITS "ERGAMAN TYPE CYCLOAROMATIZATION !NGEW #HEM )NT %D n 3HAIR -$ 9OON 4 $ANISHEFSKY 3* 4OTAL SYNTHESIS OF DYNEMICIN ! !NGEW #HEM )NT %D n #LIVE $,* "O 9 4AO 9 $AIGNEAULT 3 7U 9 * -EIGNAN ' 3YNTHESIS OF CALI CHEAMICINONE BY TWO METHODS * !M #HEM 3OC n 2OMO $ *OHNSON $$ 0LAMONDON , -IWA 4 3CHREIBER 3, 3YNTHETIC INVESTIGA TIONS OF RAPAMYCIN 3YNTHESIS OF A # # FRAGMENT * /RG #HEM n -EYER 3$ -IWA 4 .AKATSUKA - 3CHREIBER 3, 3YNTHETIC INVESTIGATIONS OF RAPAMY CIN 3YNTHESIS OF A # # FRAGMENT * /RG #HEM n %VANS $! (OVEYDA !( 3AMARIUM CATALYZED INTRAMOLECULAR TISHECHENKO REDUC TION OF β HYDROXY KETONES ! STEREOSELECTIVE APPROACH TO THE SYNTHESIS OF DIFFERENTIATED ANTI DIOL MONOESTERS * !M #HEM 3OC n 4ORMAKANGAS /0 +OSKIENEN !-0 4HE 4ISHCHENKO REACTION AND ITS MODIFICATIONS IN ORGANIC SYNTHESIS 2ECENT 2ES $EV /RG #HEM n (UCKIN 3. 7EILER , !LKYLATION OF DIANIONS OF β KETO ESTERS * !M #HEM 3OC n ,EVIN *) 4UROS % 7EINREB 3- !N ALTERNATIVE PROCEDURE FOR THE ALUMINUM MEDI ATED CONVERSTION OF ESTERS TO AMIDES 3YNTH #OMMUN n #HEN +- (ARDTMANN '% 0RASAD + 2EPIC / 3HAPIRO -* SYN $IASTEREOSELEC TIVE REDUCTION OF β HYDROXY KETONES UTILIZING ALKOXYDIALKYLBORANES 4ETRAHEDRON ,ETT n %VANS $! #LARK *3 -ETTERNICH 2 .OVACK 6* 3HEPPARD '3 $IASTEREOSELECTIVE ALDOL REACTIONS USING β KETO IMIDE DERIVED ENOLATES ! VERSATILE APPROACH TO THE ASSEM BLAGE OF POLYPROPIONATE SYSTEMS * !M #HEM 3OC n 4OMIOKA ( 4AKAI + /SHIMA + .OZAKI ( 3ELECTIVE OXIDATION OF A PRIMARY HYDROXYL IN THE PRESENCE OF SECONDARY ONE 4ETRAHEDRON ,ETT n (ASSANALY 0 $OU (*- -ETZGER * 3 !LKYLATION OF THIOXO DIHYDROIMIDAZOLE AND ITS METHYL DERIVATIVE UNDER PHASE TRANSFER CONDITIONS 3YNTHESIS n %VANS $! !NDREWS '# !LLYLIC SULFOXIDES 5SEFUL INTERMEDIATES IN ORGANIC SYN THESIS !CC #HEM 2ES n .ISHIBAYASHI 9 5EMURA 3 3ELENOXIDE ELIMINATION AND ; = SIGMATROPIC REAR RANGEMENT 4OP #URR #HEM n 3UZUKI 4 3AIMOTO ( 4OMIOKA ( /SHIMA + .OZAKI ( 2EGIO AND STEREOSELECTIVE RING OPENING OF EPOXY ALCOHOLS WITH ORGANOALUMINIUM COMPOUNDS LEADING TO DIOLS 4ETRAHEDRON ,ETT n -AIER -% 3CHÙFFLING " 3YNTHESIS OF THE CYCLOHEXYL FRAGMENT OF &+ BY INTRAMOLECULAR ENE REACTION 4ETRAHEDRON ,ETT n %IS -* 7ROBEL *% 'ANEM " -ECHANISM AND SYNTHETIC UTILITY OF BORON TRIFLUORIDE ETHERATE PROMOATED ORGANOLITHIUM ADDITIONS * !M #HEM 3OC n 9AMAGUCHI - (IRAO ) !N EFFICIENT METHOD FOR THE ALKYNYLATION OF OXIRANES USING ALKYNYL BORANES 4ETRAHEDRON ,ETT n .AJERA # 9US - $ESULFONYLATION REACTIONS RECENT DEVELOPMENTS 4ETRAHEDRON n "ALBONI ' -ARASTONI - -ERIGHI 3 "OREA 0! 4OMATIS 2 3YNTHESIS AND ACTIVITY OF PYRIDYLAMINE LIGANDS AT CENTRAL NICOTINIC RECEPTORS %UR * -ED #HEM n
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
&ISHER -* -YERS #$ *OGLAR * #HEN 3( $ANISHEFSKY 3* 3YNTHETIC STUDIES TOWARD RAPAMYCIN A SOLUTION TO A PROBLEM IN CHIRALITY MERGER THROUGH USE OF THE )RELAND REAC TION * /RG #HEM n #HEN 3( (ORVATH 2& *OGLAR * &ISHER -* $ANISHEFSKY 3* !PPLICATION OF THE )BUKA 9AMAMOTO REACTION TO A PROBLEM IN STEREOCHEMICAL COMMUNICATION A STRATEGY FOR THE STEREOSPECIFIC SYNTHESIS AND STABILIZATION OF THE TRIENE SUBSTRUCTURE OF RAPAMYCIN THROUGH SULFONE SUBSTITUTION * /RG #HEM n (AYWARD #- &ISHER -* 9OHANNES $ $ANISHEFSKY 3* !N APPLICATION OF THE 3UÇ REZ REACTION TO THE REGIOSPECIFIC AND STEREOSPECIFIC SYNTHESIS OF THE # # SEGMENT OF RAPAMYCIN 4ETRAHEDRON ,ETT n (ORVATH 2& ,INDE )) 2' (AYWARD #- *OGLAR * 9OHANNES $ $ANISHEFSKY 3* !N APPLICATION OF THE %VANS 0RASAD SYN DIOL SYNTHESIS TO A STEREOSPECIFIC SYNTHESIS OF THE # # SEGMENT OF RAPAMYCIN 4ETRAHEDRON ,ETT n 9OHANNES $ $ANISHEFSKY 3* $EGRADATION OF RAPAMYCIN RETRIEVAL OF MAJOR INTACT SUBUNITS 4ETRAHEDRON ,ETT n 9OHANNES $ -YERS #$ $ANISHEFSKY 3* $EGRADATION OF RAPAMYCIN SYNTHESIS OF A RAPAMYCIN DERIVED FRAGMENT CONTAINING THE TRICARBONYL AND TRIENE SECTORS 4ETRAHEDRON ,ETT n -ENG $ "ERTINATO 0 "ALOG ! 3U $3 +AMENECKA 4 3ORENSEN %* $ANISHEFSKY 3* 4OTAL SYNTHESES OF EPOTHILONES ! AND " * !M #HEM 3OC n .ICOLAOU +# -ONTAGNON 4 6ASSILIKOGIANNAKIS ' -ATHISON #*. 4HE TOTAL SYN THESIS OF COLEOPHOMONES " # AND $ * !M #HEM 3OC n *ULIA - 0ARIS *- 3YNTHESES WITH THE HELP OF SULFONES 6 'ENERAL METHOD OF SYN THESIS OF DOUBLE BONDS 4ETRAHEDRON ,ETT n *ULIA - 2ECENT ADVANCES IN DOUBLE BOND FORMATION 0URE !PPL #HEM n $UMEUNIER 2 -ARKO )% 4HE *ULIA REACTION )N 4 4AKEDA ED -ODERN #ARBONYL /LEFINATION 7ILEY 6#( n 'RINFELD !! #AUFIELD #% 3CHIKSUIS 2! -ATTES *& #HAN +7 !CID CATALYZED FUNC TIONALIZATION OF RAPAMYCIN 4ETRAHEDRON ,ETT n 3TEFFAN 2* +EARNEY 2- (U $# &AILLI !! 3KOTNICKI *3 3CHIKSNIS 2! -ATTES *& #HAN +7 #AUFIELD #% "ASE CATALZYED DEGRADATIONS OF RAPAMYCIN 4ETRAHEDRON ,ETT n 3EDRANI 2 *ONES ,( *UTZI %ME !- 3CHULER 7 #OTTENS 3 #LEAVAGE OF THE CYCLOHEXYL SUBUNIT OF RAPAMYCIN RESULTS IN LOSS OF IMMUNOSUPPRESSIVE ACTIVITY "IOORG -ED #HEM ,ETT n 'OULET -4 "OGER * $EGRADATIVE STUDIES ON THE TRICARBONYL CONTAINING MACROLIDE RAPAMYCIN 4ETRAHEDRON ,ETT n 3EDRANI 2 4HAI " &RANCE * #OTTENS 3 $IHYDROXYLATION OF THE TRIENE SUBUNIT OF RAPAMYCIN * /RG #HEM n ,UENGO *) 2OZAMUS ,7 (OLT $! %FFICIENT REMOVAL OF PIPECOLINATE FROM RAPAMY CIN AND &+ BY REACTION WITH N "U. #.n 4ETRAHEDRON ,ETT n ,EUNGO *) +ONIALIAN "ECK ! (OLT $! )ONIZATION OF THE # METHOXY GROUP IN RAPAMYCIN BY - LITHIUM PERCHLORATE DIETHYL ETHER 4ETRAHEDRON ,ETT n .ELSON &# 3TACHEL 3* %NG #0 3EHGAL 3. -ANIPULATION OF THE # # REGION OF RAPAMYCIN STABILITY ISSUES AND BIOLOGICAL IMPLICATIONS "IOORG -ED #HEM ,ETT n
-ATTHEW , -ADDESS -ILES . 4ACKETT AND 3TEVEN 6 ,EY
.ELSON &# 3TACHEL 3* -ATTES *& ! NOVEL RING CONTRACTION OF RAPAMYCIN 4ETRAHE DRON ,ETT n ,UENGO *) +ONIALIAN !, (OLT $! 3TUDIES ON THE CHEMISTRY OF RAPAMYCIN NOVEL TRANSFORMATIONS UNDER ,EWIS ACID CATALYSIS 4ETRAHEDRON ,ETT n 3TOTNICKI *3 +EARNEY 2- 2ING EXPANDED RAPAMYCIN DERIVATIVES 4ETRAHEDRON ,ETT n 9ANG 7 $IGITS #! (ATADA - .ARULA 3 2OZAMUS ,7 (UESTIS #- 7ONG * $ALGARNO $ (OLT $! 3ELECTIVE EPIMERIZATION OF RAPAMYCIN VIA A RETROALDOLALDOL MECHANISM MEDIATED BY TITANIUM TETRAISOPROPOXIDE /RG ,ETT n ,UENGO *) 2OZAMUS ,7 (OLT $! 3TUDIES ON SELECTIVE REDUCTIONS OF RAPAMYCIN 4ETRAHEDRON ,ETT n &ISHER -* #HOW + 6ILLALOBOS ! $ANISHEFSKY 3* /N THE REMARKABLE PROPENSITY FOR CARBON CARBON BOND CLEAVAGE REACTIONS IN THE # # REGION OF &+ * /RG #HEM n &LAHERTY " /VEREND 7' 7ILLIAMS .2 "RANCHED CHAIN SUGARS 6)) 3YNTHESIS OF $ MYCAROSE AND $ CLADINOSE * #HEM 3OC n &ERRIER 2* 5NSATURATED CARBOHYDRATES 0ART ! CARBOCYCLIC RING CLOSURE OF A HEX ENOPYRANOSIDE DERIVATIVE * #HEM 3OC 0ERKIN 4RANS ) n &ERRIER 2* $IRECT CONVERSION OF UNSATURATED HEXOPYRANOSYL COMPOUNDS TO FUNCTIONALIZED CYCLOHEXANONES 4OP #URR #HEM n -EYERS !) "ABIAK +! #AMPBELL !, #OMINS $, &LEMING -0 (ENNING 2 (EUSCHMANN - (UDSPETH *0 +ANE *- 2EIDER 0* ET AL 4OTAL SYNTHESIS OF n MAYSINE * !M #HEM 3OC n 0ATERSON ) 9EUNG +0 7ATSON # 7ARD 2! 7ALLACE 0! 4HE TOTAL SYNTHESIS OF SCYTOPHYCIN # 0ART 3TEREOCONTROLLED SYNTHESIS OF THE # # PROTECTED SECO ACID 4ETRAHEDRON n &ßRSTNER ! +ATTNIG % ,EPAGE / 4OTAL SYNTHESES OF AMPHIDINOLIDE 8 AND 9 * !M #HEM 3OC n 2EETZ -4 *UNG ! !SYMMETRIC INDUCTION IN ADDITION REACTIONS OF CHIRAL β ALKOXY ALDEHYDES EFFICIENT CHELATION CONTROL VIA ,EWIS ACIDIC TITANIUM REAGENTS * !M #HEM 3OC n 2EETZ -4 .EW SYNTHETIC METHODS #HELATE OR NONCHELATE CONTROL IN ADDI TION REACTIONS OF CHIRAL α AND β ALKOXYCARBONYL COMPOUNDS !NGEW #HEM )NT %D +IYOOKA 3 (EATHCOCK #( !CYCLIC 3TEREOSELECTION (IGH DIASTEREOFACIAL SELEC TIVITY IN THE STANNIC CHLORIDE MEDIATED REACTIONS OF ALLYLSILANES WITH CHIRAL α AND β ALKOXY ALDEHYDES 4ETRAHEDRON ,ETT n 2OUSH 72 !NDO + 0OWERS $" 0ALKOWITZ !$ (ALTERMAN 2, !SYMMETRIC SYN THESIS USING DIISOPROPYL TARTRATE MODIFIED % AND : CROTYLBORONATES PREPARATION OF THE CHIRAL CROTYLBORONATES AND REACTIONS WITH ACHIRAL ALDEHYDES * !M #HEM 3OC n 2OUSH 72 !NDO + 0OWERS $" (ALTERMAN 2, 0ALKOWITZ !$ %NANTIOSELECTIVE SYNTHESIS USING DIISOPROPYL TARTRATE MODIFIED % AND : CROTYLBORONATES REACTIONS WITH ACHIRAL ALDEHYDES 4ETRAHEDRON ,ETT n 2OUSH 72 (ALTERMAN 2, $IISPROPYL TARTRATE MODIFIED % CROTYLBORONATES HIGHLY ENANTIOSELECTIVE PROPIONATE % ENOLATE EQUIVALENTS * !M #HEM 3OC n "ARTON $(2 $OWLATSHAHI (! -OTHERWELL 7" 6ILLEMIN $ ! NEW RADICAL DECAR
4OTAL SYNTHESIS STUDIES ON MACROCYCLIC PIPECOLIC ACID NATURAL PRODUCTS
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"Y ,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES "ROAD )NSTITUTE OF (ARVARD AND -)4 #AMBRIDGE #ENTER #AMBRIDGE -! 53! LISA BROADMITEDU )NFINITY 0HARMACEUTICALS )NC -EMORIAL $RIVE #AMBRIDGE -! 53! CHARLIEJOHANNES IPICOM
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
!BSTRACT .ATURAL PRODUCTS HAVE PLAYED A CRITICAL ROLE IN THE IDENTIFICATION OF NUMEROUS MEDICINES 3YN THETIC ORGANIC CHEMISTRY AND COMBINATORIAL CHEMISTRY STRATEGIES SUCH AS DIVERSITY ORIENTED SYNTHESIS $/3 HAVE ENABLED THE SYNTHESIS OF NATURAL PRODUCT LIKE COMPOUNDS 4HE COM BINATION OF THESE APPROACHES HAS BOTH IMPROVED THE DESIRED BIOLOGICAL PROPERTIES OF NATURAL PRODUCTS AS WELL AS THE IDENTIFICATION OF NOVEL COMPOUNDS $IVERSITY CONCEPTS AND STRATEGIES TO ACCESS NOVEL COMPOUNDS INSPIRED BY NATURAL PRODUCTS WILL BE REVIEWED
)NTRODUCTION
4HE IDENTIFICATION AND DEVELOPMENT OF A DRUG CANDIDATE IS A CHALLENGING PROCESS FROM BOTH A SCIENTIFIC AND BUSINESS PERSPECTIVE &ROM A DEVELOP MENT STANDPOINT ESTABLISHING A COST EFFECTIVE SYNTHETIC ROUTE CAN BE VERY CHALLENGING ESPECIALLY IN CASES WHERE COMPLEX COMPOUNDS CANNOT BE HARVESTED AND METHODOLOGY IS REQUIRED TO INSTALL PROPER STEREOCHEMICAL RELATIONSHIPS "IOLOGICAL CONSIDERATIONS SUCH AS OPTIMIZING THE SELECTIVITY AND POTENCY OF COMPOUNDS WITH PROPER PHARMACOKINETICS AND PHARMA CODYNAMIC PROPERTIES ARE DIFFICULT TO DETERMINE AND REQUIRES LENGTHY AND COSTLY IN VIVO EXPERIMENTS &ROM A BUSINESS PERSPECTIVE ISSUES SURROUND ING INTELLECTUAL PROPERTY AND MARKETING ISSUES SUCH AS ORAL ADMINISTRATION ARE ALSO IMPORTANT 4HEREFORE THE MOST IMPORTANT AND PERHAPS INCREAS INGLY DIFFICULT CHALLENGE IS THE IDENTIFICATION OF NOVEL COMPOUNDS WITHOUT DEVELOPMENT LIABILITIES -ANY STRATEGIES TO EFFECTIVELY IDENTIFY A DRUG CAN DIDATE HAVE BEEN CONSIDERED AND MOST APPROACHES INVOLVE THE SCREENING OF COMPOUND COLLECTIONS IN CELLULAR AND BIOCHEMICAL ASSAYS 4HUS FAR AN IDEAL COMPOUND COLLECTION FOR DISCOVERING A DRUG CANDIDATE HAS NOT BEEN IDENTIFIED 4HE IDEA THAT MOST NATURAL PRODUCTS HAVE INHERENT BIOLOGI CAL PROPERTIES MAKES THEM ATTRACTIVE STARTING POINTS FOR ENGAGING CELLULAR PROCESSES AND POTENTIALLY ADDRESSES SOME OF THE DEVELOPMENTAL CONCERNS MENTIONED ABOVE ;= )NDEED IT IS WELL KNOWN THAT MANY SUCCESSFUL DRUGS HAVE BEEN DERIVED FROM OR INSPIRED BY NATURAL PRODUCTS ;= 4HIS CHAPTER WILL DISCUSS THE USE OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS $/3 TO PROVIDE NEW STARTING POINTS FOR DRUG DISCOVERY
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
"ACKGROUND 4HE RECENT EVOLUTION OF COMBINATORIAL CHEMISTRY OVER THE PAST YEARS IS A DIRECT RESPONSE TO ADVANCEMENTS IN MOLECULAR BIOLOGY WHICH HAVE ENABLED RAPID SCREENING AGAINST BIOLOGICAL TARGETS 4HE ABILITY TO SCREEN THOUSANDS OF COMPOUNDS UNVEILED A PAUCITY OF DIVERSE CHEMICAL MATTER AND DROVE THE DEVELOPMENT OF TECHNIQUES WHICH ENABLED THE PRODUCTION OF COMPOUNDS EN MASSE PROVIDING USEFUL TOOLS FOR THE DISCOVERY OF LEAD COMPOUNDS AND LEAD OPTIMIZATION .ONETHELESS COMBINATORIAL CHEMISTRY HAS BEEN CRITICIZED FOR ITS HIGH COST AND LOW SUCCESS RATE IN CREATING NEW MEDICINES 4HE PARAMETERS USED TO EVALUATE COMPOUND COLLECTIONS INCLUDE PURITY BIOLOGICAL RELEVANCE DIVERSITY AND @DRUG LIKENESS CONCEPTS FOR WHICH THERE ARE NO BROADLY ACCEPTED DEFINITIONS ;n= #ONSENSUS ON WHAT MAKES A GOOD COMPOUND COLLECTION IS CONFOUNDED BY AN OBSERVED LACK OF IMPROVE MENT IN THE RATE AT WHICH DRUGS REACH THE MARKETPLACE 4HE ATTRITION IN NEW DRUG CANDIDATES IS MAINLY A FUNCTION OF OUR LACK OF UNDERSTANDING OF SMALL MOLECULES IN VIVO AND IS HIGHLIGHTED BY AN IMPROVED AND MORE DIFFICULT CLINICAL PROCESS !DDITIONALLY MEDIA AWARENESS OF THE PROFIT DRIVEN NATURE OF THE DRUG DISCOVERY BUSINESS ;= COMBINED WITH THE UNANTICIPATED SIDE EFFECTS OF MARKETED DRUGS HAS CREATED MARKET PESSIMISM TOWARDS THE BIO TECH SECTOR AND HAS INCREASED THE COST OF DEVELOPING POTENTIAL DRUGS 4O ADDRESS THESE ISSUES THE PHARMACEUTICAL INDUSTRY IS ATTEMPTING TO GAIN A BETTER UNDERSTANDING OF THE MOLECULAR FEATURES OF COMPOUNDS THAT ARE RELEVANT TO BIOLOGY ;= AND TO UTILIZE THIS KNOWLEDGE AS EARLY AS POSSIBLE IN THE DRUG DEVELOPMENT PROCESS 2ECENT ADVANCES IN STEREOSELECTIVE NATURAL PRODUCT SYNTHESIS ;n= COUPLED WITH OUR IMPROVING COMBINATORIAL TECHNIQUES HAVE ENABLED THE INCORPORATION OF NATURAL PRODUCT LIKE FEATURES IN SYNTHETIC COMPOUNDS FOR BIOLOGICAL SCREENING ! MAJOR CHALLENGE IN USING COMBINATORIAL SYNTHETIC TOOLS TO RECREATE THE CHARACTERISTICS OF NATURAL PRODUCTS HAS BEEN HOW TO CAPTURE THE ESSENCE OF NATURAL PRODUCT DIVERSITY AND HOW IT INTERFACES WITH BIOLOGY 4OWARD THIS END COMBINATORIAL CHEMISTRY HAS EVOLVED INTO $/3 ;= A PROCESS USED TO GENERATE LIBRARIES OF MOLECULES WITH AN EMPHASIS ON STEREOCHEMICAL AND SKELETAL DIVERSITY AND DIVERGENT SYNTHETIC PATHWAYS ;n= 4HE PRODUCTS OF $/3 HAVE BEEN USED AS CHEMICAL PROBES TO STUDY CELLULAR PATHWAYS IN A PROCESS REFERRED TO AS CHEMICAL GENETICS ; =
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
4AKEN TOGETHER $/3 AND CHEMICAL GENETICS HAVE PROVIDED THE OPPORTU NITY TO PROBE THE CHEMICAL COMPLEXITY AND BIOLOGICAL DIVERSITY OF NATURE /NE OF THE DIFFICULTIES OF A DIVERSITY ORIENTED SYNTHETIC APPROACH IS TO ASCERTAIN WHAT TYPE OF CHEMICAL STRUCTURES TO SYNTHESIZE )N ORDER TO GUIDE THE SELECTION OF LIBRARY SCAFFOLDS THE NOTION OF TAKING ADVANTAGE OF PRIVILEGED STRUCTURES HAS INSPIRED ATTEMPTS TO PROVIDE A STRUCTURAL CLAS SIFICATION OF NATURAL PRODUCTS 3#/.0 ; = 4HIS APPROACH HAS BEEN COINED AS BIOLOGY ORIENTED SYNTHESIS ")/3 ;= !LTERNATE PERSPECTIVES FOR GUIDING SCAFFOLD SELECTION IN LIBRARY DESIGN SUCH AS PROTEIN STRUCTURE SIMILARITY CLUSTERING 033# HAVE ALSO BEEN PROPOSED ;= )N THIS STRATEGY PROTEINS ARE CLASSIFIED ACCORDING TO DOMAIN SIMILARITY INSTEAD OF SEQUENCE SIMILARITY AND A SCAFFOLD IS DESIGNED BASED ON THE GENERAL FEATURES OF THE DOMAIN SIMILARITIES )N THIS MANNER A DIVERSE LIBRARY CONSTRUCTED AROUND A SINGLE SCAFFOLD HAS THE POTENTIAL TO INTERACT WITH DIFFERENT CLASSES OF PROTEINS ALLOWING ACCESS TO MULTIPLE BIOLOGICAL PROCESSES WITH ONE SCAF FOLD /THER CHEMICAL TECHNOLOGIES SUCH AS RECEPTOR ASSISTED COMBINATORIAL CHEMISTRY 2!## ARE ALSO NOTEWORTHY 2!## WHICH COMBINES SYNTHESIS AND SCREENING INTO ONE STEP INCLUDES DYNAMIC COMBINATORIAL CHEMISTRY $## RECEPTOR ACCELERATED SYNTHESIS 2!3 AND PSEUDO DYNAMIC LIBRARIES P$#,S ;=
.ATURAL PRODUCTS DIVERSITY AND THE RELEVANCE TO BIOLOGY 4HERE HAVE BEEN NUMEROUS ATTEMPTS TO COMPARE DRUGS NATURAL PRODUCTS AND COMPOUNDS RESULTING FROM COMBINATORIAL CHEMISTRY IN ORDER TO IDEN TIFY PRIVILEGED SCAFFOLDS THAT HAVE BIOLOGICAL ACTIVITY ; = 4HESE STRUC TURAL COMPARISONS HAVE BEEN BROADENED TO INCLUDE CHEMICAL SPACE BIO LOGICAL SPACE AND DIVERSITY ;= /NE MAJOR CHALLENGE IN EVALUATING THESE COMPARISONS HAS BEEN IDENTIFYING A METHOD TO EMPIRICALLY MEASURE SUCH RELATIONSHIPS ;= 2EGARDLESS OF DEFINITION BIOLOGICALLY RELEVANT SPACE REP RESENTS A SMALL FRACTION OF WHAT CAN BE MADE CHEMICALLY ;= &URTHERMORE BIOLOGICALLY RELEVANT SPACE THAT CAN BE SAFELY ADMINISTERED TO HUMANS IS EVEN MORE LIMITED ;= 4WO FACTORS WHICH LIMIT THE ABILITY TO ACCURATELY EVALUATE AND CORRELATE BIOLOGICAL SYSTEMS WITH CHEMICAL DIVERSITY ARE THE QUALITY OF CHEMICAL LIBRARIES AND THE INACCURACY OF SCREENING TECHNIQUES
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
;= &OR INSTANCE IN VITRO SCREENING DATA DO NOT ADDRESS THE NOTION OF MOLECULAR CROWDING THE THEORY THAT MOLECULES BEHAVE DIFFERENTLY IN DILUTE SOLUTIONS THAN IN THE CROWDED CELLULAR ENVIRONMENT ;= %VEN IF A CON SENSUS COULD BE REACHED ON WHICH MOLECULAR FEATURES ARE MOST RELEVANT TO BIOLOGY HOW PRACTICAL WOULD IT BE TO SYNTHESIZE A COLLECTION OF MOLECULES CAPITALIZING ON THIS KNOWLEDGE 4HE CONCEPT OF THE PERFECT COMPOUND COL LECTION REMAINS ELUSIVE BECAUSE THE REGIONS OF CHEMICAL SPACE WHICH COULD BE UTILIZED ARE STILL UNDEFINED 'IVEN THAT DIVERSITY REALLY ONLY MAKE SENSE WITHIN A GIVEN FRAME OF REF ERENCE ONE MAY ARGUE THAT CHEMICAL DIVERSITY IS A CONCEPT THAT IS MORE ART THAN SCIENCE 4HERE ARE NO GENERALLY ACCEPTED CORRELATIONS TO DIFFERENCES IN STRUCTURE NOVELTY AND BIOLOGICAL ACTIVITY BETWEEN COMPOUNDS 4HIS IS A DIRECT RESULT FROM QUALITATIVE MEASUREMENTS OF CHEMICAL DIVERSITY BASED UPON COMPUTATIONAL METHODS THAT HAVE BEEN MADE WITH ADMITTEDLY ARBI TRARY SETS OF DESCRIPTORS &URTHERMORE CHEMICAL SPACE HAS BEEN COMPARED TO POTENTIALLY INACCURATE BIOLOGICAL ASSAYS !LTHOUGH DIVERSITY AND ITS RELEVANCE TO BIOLOGY IS A VERY COMPLEX ISSUE NATURAL PRODUCTS APPEAR TO CAPTURE BOTH CONCEPTS AND HAVE BEEN EXCELLENT SOURCES FOR DRUG DISCOVERY 4WO CRITICAL CHARACTERISTICS OF NATURAL PRODUCTS ARE THE NOVEL STRUCTURAL DIVER SITY AND THE STEREOCHEMICAL COMPLEXITY OF THE COMPOUND ARCHITECTURES AND THEREFORE THE THREE DIMENSIONALITY OF COMPOUNDS ISOLATED FROM NATURAL SOURCES IS OF UNDISPUTABLE IMPORTANCE TO ITS ACTIVITY AND SPECIFICITY )T IS FOR THIS REASON THAT A CHEMISTRY DRIVEN APPROACH SUCH AS $/3 THAT INCORPORATES NATURAL PRODUCT LIKE FEATURES IS A VALID APPROACH FOR DRUG DISCOVERY 4HIS HAS PROVEN TO BE A REASONABLE WAY TO GAIN ACCESS TO NEW CHEMICAL SPACE PROVIDE OPPORTUNITY TO EXPLORE BIOLOGY AND ACCESS BETTER TOOLS FOR THE UNDERSTANDING OF CELLULAR PATHWAYS ALL WITHOUT THE AID OF COMPUTATIONAL TOOLS
$/3 CONCEPTS !S HIGHLIGHTED ABOVE THE CONCEPTS OF DIVERSITY ARE PARAMOUNT IN THE DESIGN OF $/3 BASED LIBRARIES 4HE MOST CLASSIC AND STRAIGHTFORWARD DIVER SITY CONCEPT IS APPENDAGE DIVERSITY &IG !PPENDAGE DIVERSITY IS GENERATED BY DECORATING A CHEMICAL SCAFFOLD WITH DIFFERENT TYPES OF BUILDING BLOCKS TO PROVIDE PRODUCTS WHICH HAVE FUNCTIONAL AND SPATIAL DIFFERENCES EXTENDING
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
&IGURE 4RADITIONAL APPROACH TO $/3 USING DIVERGENT ! AND CONVERGENT " SYNTHESIS
&IGURE $/3 WITH BRANCHING PATHWAYS !n# TO ACCESS INCREASED DIVERSITY
BEYOND THE INITIAL SCAFFOLD 4HE RESULTING COMPOUNDS MAY THEN UNDERGO DIVERGENT ! CHEMICAL MODIFICATIONS TO GENERATE COMPLETELY NEW SCAFFOLD ARCHITECTURES REFERRED TO AS SKELETAL DIVERSITY !LTERNATELY SKELETAL DIVERSITY CAN BE OBTAINED IN A CONVERGENT " MANNER VIA A MULTICOMPONENT REACTION -#2 ;= 4HIS CONCEPT HAS MORE RECENTLY EVOLVED TO INCORPORATE ADDI TIONAL OPPORTUNITIES FOR GENERATING DIVERSITY &IG ! CORE OR MULTIPLE CORES CAN BE SYNTHESIZED IN SOLUTION AND APPENDED WITH BUILDING BLOCKS AS IN THE PREVIOUS EXAMPLE )N THIS CASE HOWEVER THE APPENDED BUILDING BLOCKS CONTAIN HANDLES WHICH ENABLE ADDITIONAL DIVERSITY GENERATING REAC
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
TIONS TO TAKE PLACE VIA ONE OR MORE BRANCHING PATHWAYS !n# 0ATH ! INCOR PORATES ADDITIONAL APPENDAGE AND FUNCTIONAL DIVERSITY THROUGH REACTIVITY OF THE HANDLES THAT WERE INTRODUCED 0ATH " PROVIDES SKELETAL DIVERSITY OF THE CORE WHILE STILL MAINTAINING THE ABILITY TO UNDERGO SUBSEQUENT 0ATH ! TYPE TRANSFORMATIONS 0ATH # PROVIDES SKELETAL DIVERSITY BY EXPLOITING STRA TEGICALLY PLACED FUNCTIONAL GROUPS TO YIELD DIFFERENTIALLY CYCLIZED PRODUCTS 7ITH APPROPRIATE DESIGN THIS PROCESS CAN CONTINUE TO MULTIPLY BY PROCEED ING DOWN PATHS ! OR # 7ITHIN THE PAST DECADE CHEMISTS HAVE CREATED A NUMBER OF NATURAL PRODUCT BASED $/3 LIBRARIES )N AGREEMENT WITH PREVIOUS REVIEWS ; = THESE LIBRARIES FALL INTO THREE MAIN CATEGORIES LIBRARIES BASED ON SPECIFIC NATURAL PRODUCTS LIBRARIES CONTAINING SUBSTRUCTURES COMMON TO SPE CIFIC CLASSES OF NATURAL PRODUCTS AND LIBRARIES WHICH CONTAIN FEATURES COMMON TO NATURAL PRODUCTS IN GENERAL 2EGARDLESS OF THE INSPIRATION OR APPROACH A $/3 LIBRARY MUST PROVIDE AN OPPORTUNITY TO GENERATE DIVERSITY ON AT LEAST ONE OF THE FOLLOWING LEVELS APPENDAGE FUNCTIONAL GROUP STEREOCHEMICAL OR SKELETAL DIVERSITY )N THE FOLLOWING SECTIONS WE WILL REVIEW SELECTED EXAMPLES OF $/3 LIBRARIES INSPIRED BY NATURAL PRODUCTS AND DISCUSS BOTH THE DIVERSITY THAT IS PRESENTED AND THE METHOD IN WHICH THE DIVERSITY WAS OBTAINED )T SHOULD BE NOTED THAT ALL DIVERSITY ASPECTS DISCUSSED ARE IMPORTANT TO BIOLOGICAL ACTIVITY AND THAT ONE TYPE OF DIVERSITY HAS NOT PROVEN TO BE MORE RELEVANT THAN ANOTHER
,IBRARIES BASED ON SPECIFIC NATURAL PRODUCTS ,IBRARIES BASED ON NATURAL PRODUCT SCAFFOLDS HAVE BEEN EXTENSIVELY REVIEWED IN THE LITERATURE ;= AND THE DESIRE TO INCORPORATE NATURAL PRODUCT LIKE FEA TURES IS GUIDED BY THE SUCCESSFUL SCREENING OF NATURAL PRODUCT MIXTURES ;= 4HROUGH THEIR DIVERSITY OF STRUCTURE AND THEIR INHERENT BIOLOGICAL ACTIVITY NATU RAL PRODUCTS HAVE ENABLED THE DISCOVERY AND DEVELOPMENT OF A VAST ARRAY OF MEDICINES ;= 2EMARKABLY NATURAL PRODUCTS REPRESENT ROUGHLY ONLY OF KNOWN CHEMICAL SPACE 4HIS IS LIKELY DUE TO THE SELECTIVITY OF THE ENZYMES INVOLVED IN GENERATING THESE MOLECULES AND OFFERS A POTENTIAL EXPLANATION FOR THE UTILITY AND UNIQUE FUNCTIONS OF NATURAL PRODUCTS 0OTENT BIOLOGICAL ACTIVITY IS A RARE PROPERTY FOR ANY ONE MOLECULE TO POSSESS ESPECIALLY FOR PROTEIN PROTEIN INTERACTIONS ;= WHERE MANY HYDROPHOBIC COMPLEMEN
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
O
OH
MeO
O
oxime/hydrazone formation
O
Br
conjugate addition
HO
N Me
N H
Mitsunobu
O
N-acylation, N-alkylation
(-)-Galanthamine
(+)-Galanthamine Scaffold
acetylcholinesterase inhibitor
N
Br
O
O S
O
OMe
N H
OH
Secramine Secretory pathway inhibitor EC50 = ~2 PM
&IGURE ! LIBRARY BASED ON THE ENANTIOMERIC SCAFFOLD OF GALANTHAMINE AN ACETYLCHOLINESTERASE INHIBITOR WAS USED TO IDENTIFY SECRAMINE A NOVEL SECRETORY PATHWAY INHIBITOR HIGHLIGHTING THE IMPORTANCE OF STE REOCHEMICAL AND APPENDAGE DIVERSITY
TARY CHARGE INTERACTIONS ARE NECESSARY 4O EXPLAIN THE PROPENSITY OF NATURAL PRODUCTS TO INCORPORATE THESE AND OTHER NECESSARY INTERACTIONS A SCREENING HYPOTHESIS FOR NATURAL PRODUCT EVOLUTION HAS BEEN PROPOSED ;= 7HILE LIBRARIES BASED ON SPECIFIC NATURAL PRODUCTS HAVE MAINLY BEEN USED TO OPTIMIZE THE KNOWN BIOLOGICAL OR PHARMACOKINETIC ACTIVITY OF THE PARENT COMPOUND SCREENING OF THESE COMPOUNDS IN UNRELATED BIOLOGICAL ASSAYS HAS ALSO LED TO THE DISCOVERY OF NEW ACTIVITIES 3UCH DISCOVERIES HIGH LIGHT THE POTENTIAL EVOLUTIONARY BIAS OF CERTAIN NATURAL PRODUCT SCAFFOLDS AS PRIVILEGED ;= OR IN A GENERAL SENSE CONTAINING THE NECESSARY FEATURES TO INTERACT IN CELLULAR PROCESSES /NE OF THE FIRST DEMONSTRATIONS OF THIS APPROACH WAS THE DISCOVERY OF SECRAMINE &IG BY 3HAIR AND CO WORKERS ;= 3ECRAMINE WAS IDENTIFIED IN THE PHENOTYPIC SCREENING OF A MEMBERED LIBRARY WHICH WAS BASED ON THE ENANTIOMERIC SCAFFOLD OF THE ALKALOID GALANTHAMINE AN ACETYL CHOLINESTERASE INHIBITOR 7HILE GALANTHAMINE ITSELF HAS NO EFFECT ON THE SECRETORY PATHWAY SECRAMINE DEMONSTRATED REMARKABLE μ- INHIBITORY ACTIVITY
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
N
NH
N
Boc
N
N
X
cytisine
N
R2
O
O
O
R1
X = Br or NO2
nicotine agonist
N
R1 = H, acyl, alkyl, sulfonamido R2 = substituted amino, H, alkenyl, aryl
CN
N Cl
O phosphatase inhibitor IC50 = ~2.0 PM (VE-PTP)
&IGURE ! LIBRARY BASED ON THE NICOTINE AGONIST CYTISINE WAS USED TO IDENTIFY AN INHIBITOR OF THE PHOSPHATASE 6% 040 THROUGH THE INTRODUCTION OF APPENDAGE DIVERSITY
4HIS GALANTHAMINE BASED LIBRARY TAKES ADVANTAGE OF APPENDAGE AND STE REOCHEMICAL DIVERSITY THROUGH A CHEMISTRY DRIVEN APPROACH !CCESS TO THE UNNATURAL ENANTIOMER PROVIDES STEREOCHEMICAL DIVERSITY AND THE GALAN THAMINE CORE PROVIDES MULTIPLE SITES FOR THE INTRODUCTION OF APPENDAGE DIVERSITY A PHENOL -ITSUNOBU SUBSTITUTION AN ENONE CONJUGATE ADDITIONOXIME OR HYDRAZONE FORMATION AND A SECONDARY AMINE . ACYLATION OR . ALKYLATION 4HE ACTIVITY OF SECRAMINE IN COMPARISON TO THE LACK OF ACTIVITY OF ITS PARENT COMPOUND DEMONSTRATES THE IMPORTANCE OF STEREOCHEMICAL AND APPENDAGE DIVERSITY IN THAT THE SAME CORE CAN POSSESS MULTIPLE BIOACTIVITIES DEPENDING ON THE CORRECT COMBINATION OF STEREOCHEM ISTRY AND SUBSTITUENTS !N ADDITIONAL EXAMPLE HIGHLIGHTING APPENDAGE DIVERSITY OF A NATURAL PRODUCT LEADING TO IDENTIFICATION OF ACTIVITY DIFFERENT THAN THE PARENT COM POUND WAS DONE BY 7ALDMANN AND CO WORKERS !S SHOWN IN &IGURE A NEW PHOSPHATASE INHIBITOR WAS DISCOVERED BY MEANS OF THE ")/3 APPROACH BASED ON THE LUPINE ALKALOID CYTISINE A NICOTINE AGONIST AS ONE OF THE CORE SCAF FOLDS ;= )N THIS LIBRARY COMPOUNDS WERE GENERATED AND SEVERAL POTENT INHIBITORS μ- OF THE TYROSINE PHOSPHATASE 6% 040 WERE IDENTIFIED 4HE MOST POTENT 6% 040 INHIBITORS WERE AT LEAST FOLD SELECTIVE FOR 6% 040 OVER OTHER PHOSPHATASES AND ARE THE FIRST INHIBITORS TO BE DESCRIBED FOR THIS ENZYME 0RIOR TO THIS EXAMPLE COMPOUNDS DERIVED FROM THE CYTISINE SCAFFOLD HAD NOT DEMONSTRATED INHIBITORY ACTIVITY TOWARDS TYROSINE PHOSPHATASES
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
O
OTIPS
Me Me
O
H O O
H
O
H
O
O
Mitsunobu biaryl coupling carbamate formation
O
Carpanone
H O
O oxime formation
O
conjugate addtion (amine, azide, thiol)
Carpanone-derived scaffold HO
OH
H
O
H O
N
O
O
O HN F
CLL-19 IC50 = ~14 PM Inhibitor of vesicular trafficking -
&IGURE ! LIBRARY BASED ON CARPANONE WAS USED TO IDENTIFY AN INHIBITOR OF VESICULAR TRAFFICKING THROUGH THE INTRODUCTION OF APPENDAGE DIVERSITY
)N A MORE RECENT EXAMPLE BY 3HAIR THE NATURAL PRODUCT CARPANONE WAS USED AS A STARTING POINT FOR THE GENERATION OF A MEMBERED $/3 LIBRARY &IG ;= 4HE AIM OF THE LIBRARY WAS TO DISCOVER MOLECULES WITH NEW BIOLOGICAL FUNCTIONS AND NOT TO IMPROVE ON THE EXISTING BIOLOGI CAL PROPERTIES OF THE PARENT NATURAL PRODUCT #ARPANONE CONTAINS A RIGID POLYCYCLIC CORE WITH FIVE CONTIGUOUS STEREOCENTERS AND ENONE AND PHENOL FUNCTIONALITIES FOR THE INTRODUCTION OF APPENDAGE DIVERSITY )N A CONVER GENT MANNER A DIMERIZATION STRATEGY INVOLVING A ; = CYCLOADDITION WAS USED TO ASSEMBLE THE CORE 4HIS EXAMPLE ILLUSTRATES HOW SYNTHETIC CHEMISTRY ENABLES THE STRATE GIC INSTALLATION OF DIVERSITY HANDLES ON A STRUCTURALLY COMPLEX NATURAL PRODUCT 3CREENING OF THIS LIBRARY IN A PHENOTYPIC ASSAY RESULTED IN THE IDENTIFICATION OF SEVERAL COMPOUNDS THAT PERTURB THE SECRETORY PATHWAY BY INHIBITING THE EXIT OF PROTEINS FROM THE 'OLGI APPARATUS ! POWERFUL ASPECT OF THIS APPROACH WAS THAT STRUCTURE ACTIVITY RELATIONSHIPS 3!2 COULD BE DETERMINED DIRECTLY FROM THE SCREENING RESULTS 4HE MOST ACTIVE OF THESE COMPOUNDS #,, BLOCKED PROTEIN TRAFFICKING AT ^ μ- WHILE CARPANONE ITSELF HAD NO EFFECT AT μ- 4HESE DATA ONCE AGAIN DEMON
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
Ph
Ph
O
R
N
CHO O
N
O
O
HN
R O
O
Ph X
Ph
H
HN
O
HN
O
N
O
O
N H
O
N-acylation
X = H or I
spirotryprostatin B
O O
amidation
X Sonogashira
Ph
Ph
O
N HO
O
O
N
O
H O
O
HN O
N
MeO
enhancer of latrunculin B EC50 = ~550 nM
&IGURE ! SPIROOXINDOLE LIBRARY INSPIRED BY SPIROTRYPOSTATIN " WAS GENERATED USING A COMBINATION OF CONVERGENT AND DIVERGENT SYNTHESIS TO PROVIDE AN ENHANCER OF THE ACTIN POLYMERIZATION INHIBITOR LATRUNCULIN "
STRATE THE IMPACT OF APPENDAGE DIVERSITY OF A NATURAL PRODUCT BASED CORE ON BIOLOGICAL ACTIVITY 3CHREIBER AND CO WORKERS SYNTHESIZED A MEMBERED LIBRARY INSPIRED BY SPIROTRYPROSTATIN " &IG A MAMMALIAN CELL CYCLE INHIBITOR ;= VIA A COMBINATION OF CONVERGENT AND DIVERGENT SYNTHESIS 4HE SPIROOXINDOLE PRODUCTS WERE SCREENED IN A YEAST CELL BASED ASSAY LEADING TO THE IDENTIFI CATION OF ENHANCERS OF LATRUNCULIN " AN ACTIN POLYMERIZATION INHIBITOR THAT INDUCES GROWTH ARREST 4HE MOST POTENT COMPOUND HAD AN %# IN THE SUBMICROMOLAR RANGE N- -EGGER HAS SHOWN THAT MODIFICATIONS OF NATURAL PRODUCTS USING APPEND AGE DIVERSITY AND STEREOCHEMICAL DIVERSITY THROUGH THE USE OF ORGANO METALLIC COMPLEXES OFFER THREE DIMENSIONAL ARCHITECTURES THAT CANNOT BE ACCESSED BY ORGANIC COMPOUNDS ALONE ; = )N ONE EXAMPLE A RUTHE NIUM COMPLEX WAS INCORPORATED INTO THE SCAFFOLD OF THE NATURAL PRODUCT STAUROSPORINE &IG IN ORDER TO GAIN NEW BINDING INTERACTIONS 4HE EFFORTS RESULTED IN THE IDENTIFICATION OF NOVEL PROTEIN KINASE INHIBITORS !LTHOUGH THE COMPOUNDS SYNTHESIZED HAVE ACTIVITY RELATED TO THE PARENT NATURAL
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
H N
N
O
O
N
H N
O
N
O
N
Ru OC
MeO NHMe Staurosporine
IC50 3nM inhibitor of GSK-3
&IGURE ! RUTHENIUM BASED COMPLEX DERIVED FROM THE NATURAL PRODUCT STAUROSPORINE WAS IDENTIFIED AS AN INHIBITOR OF THE PROTEIN KINASE '3+
PRODUCT THE CONCEPT OF ORGANOMETALLIC COMPLEXES PROVIDING ADDITIONAL OPPORTUNITY TO ACCESS DIFFERENT THREE DIMENSIONAL SPACE IS INTERESTING AND HAS NOT BEEN EXTENSIVELY EXPLORED IN MULTIPLE BIOLOGICAL ASSAYS
,IBRARIES CONTAINING SUBSTRUCTURES COMMON TO SPECIFIC CLASSES OF NATURAL PRODUCTS 3IMILAR TO NATURAL PRODUCTS THEMSELVES SUBSTRUCTURES ARE TO AN EXTENT BIOLOGICALLY VALIDATED AND IN SOME CASES MAY ALSO BE CONSIDERED PRIVILEGED STRUCTURES &OR EXAMPLE THE BIARYL MOIETY IS A STRUCTURAL MOTIF COMMON TO MANY NATURAL PRODUCTS "OTH VANCOMYCIN AND PTEROCARYANIN # &IG HAVE AXIAL DISSYMMETRIC BIARYL UNITS CONTAINED WITHIN AND MEMBERED RINGS RESPECTIVELY 3CHREIBER AND CO WORKERS WERE INSPIRED BY SUCH NATURAL PRODUCTS AND APPLIED $/3 USING A CONVERGENT CHEMISTRY DRIVEN APPROACH TO GENERATE A ^ MEMBERED LIBRARY OF BIARYL CONTAIN ING MEDIUM RINGS ;= $UE TO THE DIFFICULTY OF FORMING MEDIUM RING MACROCYCLES THE MAJORITY OF THE LIBRARY CONTAINED MEMBERED RINGS !N INTERESTING DIVERSITY FEATURE THAT WAS INTRODUCED IN THE KEY CARBON CARBON BOND FORMING PROCESS IS STEREOCHEMICAL DIVERSITY IN THE FORM OF ATROPISOMERISM 4HIS DISTINCTION IS IMPORTANT BECAUSE THE ATROPISOMERS HAD DRAMATICALLY DIFFERENT ACTIVITIES !S A RESULT OF THIS EFFORT A COMPOUND WAS IDENTIFIED THAT WAS FOUND TO BE A MODULATOR OF ZEBRAFISH CARDIOVASCULAR DEVELOPMENT ! RELATED APPROACH WAS RECENTLY REPORTED FOR THE SYNTHESIS OF BIARYL CONTAINING NINE MEMBERED RINGS ;=
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
HO HO Me O
Me
OH
O O H
HO
Cl
Cl H N
OH
OH O
OH O
O
H N
N H O
O
O
N H
H N
O
HO HO
O O
OH
O
OH
OH
Vancomycin
Pterocaryanin C
R1
O
R4
R2
H2N
H
O
R2
N Me
R1
OH
Br
R3
CHO
Br
Br
R3
O
O
HO HO
OH OH
OH
O
O
O
O
Me Me
Me
NH2
HO2C
HO
OH
HO
O
O
HO
H
OH
O
O
NH OH 2
HN
OH
O
R4
O
O
N Me
OMe
OMe modulator of zebrafish cardiovascular development
N Me
OMe
OMe No Activity
&IGURE 4HE NATURAL PRODUCTS VANCOMYCIN AND PTEROCARYANIN # SERVED AS AN INSPIRATION FOR GENERATION OF A LIBRARY OF BIARYL CONTAINING MEDIUM RINGS USING A CONVERGENT APPROACH WHICH LED TO THE IDENTIFICA TION OF A MODULATOR OF ZEBRAFISH CARDIOVASCULAR DEVELOPMENT 4HE CORRESPONDING ATROPIOSOMER WAS FOUND TO BE INACTIVE
3YNTHETIC APPROACHES THAT EMPLOY BRANCHING PATHWAYS AND THE PRIN CIPLE OF @LIBRARIES FROM LIBRARIES &IG 0ATH " HAVE PROVEN EXTREMELY USEFUL FOR THE SYNTHESIS OF COMPLEX LIBRARIES 4HIS STRATEGY WHICH WAS FIRST INTRODUCED BY (OUGHTEN ET AL FOR THE CHEMICAL TRANSFORMATION OF PEPTIDE BASED LIBRARIES ;= WAS APPLIED BY .ICOLAOU AND CO WORKERS FOR THE GEN ERATION OF A DIVERSE COLLECTION OF BENZOPYRAN BASED COMPOUNDS ;= 4HE BENZOPYRAN MOTIF IS FOUND IN NUMEROUS NATURAL PRODUCTS WHICH EXHIBIT A WIDE RANGE OF BIOLOGICAL ACTIVITIES )N A MORE RECENT STUDY BY 0ARK ET AL UNIQUE SKELETONS BASED ON THE BENZOPYRAN MOTIF WERE PREPARED FROM ONLY TWO STARTING MATERIALS &IG VIA A CHEMISTRY DRIVEN APPROACH AND NICELY DEMONSTRATES HOW IT IS POSSIBLE TO USE BRANCHING PATHWAYS TO MAXI
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
R2
R1
R1
N N N
HO
O
N R1
X
R
R
HO
O
HO
R R
O
O
R O
R
O
HO
R1
N N N R1
R
R
HO
R2 H
R1
X H
O X X H
O
R2
HO
R R
O
R2
O
O
X
O
R R
HO
O
R
R
O
R1
O
HO
N R1
R O R
O
HO
O
X H X
R2 O R
R R
R1
O
R
O X
Br
HO
R
R1
R
O
O
N R1
R O
R
N R1
R O
O
X H X
HO
HO
O
HO
O
X H X
R
R2
O
HO
R O
O
OTf
HO
R2
O
R
O X X H
O
R2
H R R
&IGURE ! @LIBRARIES FROM LIBRARIES APPROACH WAS USED TO ACCESS UNIQUE CORES WITH VARYING APPENDAGE AND SKELETAL DIVERSITY BASED ON THE BENZOPYRAN MOTIF
MIZE SKELETAL DIVERSITY ;= 3CREENING OF THESE COMPOUNDS IN AN IN VITRO CYTOTOXICITY ASSAY PRODUCED A WIDE RANGE OF )# VALUES WHICH REFLECTED THE IMPORTANCE OF SKELETAL DIVERSITY IN DETERMINING BIOLOGICAL ACTIVITY AND SPECIFICITY )NTRODUCTION OF DIVERSITY HANDLES IN THE GENERATION OF THESE SCAFFOLDS WOULD ALLOW ADDITIONAL DIVERSITY AND OFFER A NICE COMPARISON OF SKELETAL AND APPENDAGE DIVERSITY AROUND PRIVILEGED NATURAL PRODUCT LIKE STRUCTURES #HARACTERISTICS SUCH AS CONFORMATIONAL CONSTRAINT AND COMPLEX ARRAYS OF SUBSTITUTIONS AND STEREOCHEMISTRY ARE PREVALENT IN A NUMBER OF MAC ROCYCLIC POLYKETIDE NATURAL PRODUCTS !S A RESULT MACROCYCLIC STRUCTURES HAVE SERVED AS AN INSPIRATION FOR SEVERAL $/3 LIBRARIES ; n= )N ONE EXAMPLE &IG 3CHREIBER AND CO WORKERS USED MACROLIDES AND THEIR LIN EAR PRECURSORS TO STUDY THE RELATIVE INFLUENCES OF STEREOCHEMICAL AND SKEL ETAL DIVERSITY UPON BIOLOGICAL FUNCTION THROUGH MULTIDIMENSIONAL SCREEN ING ;= ! CARBOHYDRATE DERIVED LIBRARY OF MONOCYCLIC COMPOUNDS
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
OH
Me O O
O
O
O R 1O
*
OMe
* OR2 OAlloc
1) O-Capping R3X 2) Alloc removal 3)
O
O
O
RCM then cleavage
OH
O or *
*
OR2 OAlloc
R1, R2 = H, Alloc or Alloc, H
O O
HO R1
R1
*
R1 *
Cleavage
R1
OMe
*
*
HO
O
R 1O
R 3O
Monocyclic compounds Stereochemistry of carbohydrate dominant factor in biological activity
O
O R1 *
O O
Me O * OR3
*
O * R1
Bicyclic compounds Stereochemistry on macrocycle dominant factor in biological specificity
&IGURE "ICYCLIC MACROCYLES ACCESSED VIA RING CLOSING METATHESIS 2#- AND THEIR MONOCYCLIC PRECURSORS WERE USED TO PROBE THE RELATIVE INFLUENCE OF STEREOCHEMICAL AND SKELETAL DIVERSITY UPON BIOLOGICAL ACTIVITY ACROSS MULTIPLE ASSAYS
CONTAINING BOTH STEREOCHEMICAL AND APPENDAGE DIVERSITY WAS GENERATED 5SING A LIBRARY FROM LIBRARY APPROACH &IG 0ATH # AN ADDITIONAL BICYCLIC MACROCYCLES WERE GENERATED FROM THE MONOCYCLIC PRECURSORS VIA RING CLOSING METATHESIS 2#- 4HE LIBRARY WAS SCREENED IN DIFFERENT CELL BASED ASSAYS AND PRINCIPLE COMPONENT ANALYSIS DEMONSTRATED THAT THE ACTIVE MACROCYCLIC COMPOUNDS WERE MORE LIKELY TO EXHIBIT ACTIVITY IN ONLY ONE OF THE ASSAYS PROVIDING A QUANTITATIVE CONNECTION BETWEEN CONFOR MATIONAL RESTRICTION AND BIOLOGICAL SPECIFICITY (IERARCHICAL CLUSTERING ALSO REVEALED THAT THE STEREOCHEMISTRY WAS A DETERMINANT FACTOR FOR THE ACTIVITY PATTERNS FOR BOTH LIBRARIES )NTERESTINGLY THE CARBOHYDRATE STEREOCHEMIS TRY OF THE MONOCYCLIC LIBRARY AND THE STEREOCHEMISTRY OF THE MACROCYCLIC APPENDAGES FOR THE BICYCLIC LIBRARY WERE THE DETERMINANT FEATURES FOR THE ACTIVITY PATTERNS 4HIS IS AN IMPORTANT EXAMPLE OF HOW WE HAVE BEGUN TO EMPIRICALLY MEASURE AND COMPARE FEATURES OF COMPOUNDS AND CORRELATE THEM TO BIOLOGICAL ACTIVITY AND SPECIFICITY
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
&IGURE ! LIBRARY OF POLYCYCLIC COMPOUNDS CONTAINING APPENDAGE STEREOCHEMICAL AND SKELETAL DIVERSITY WAS GENERATED STARTING FROM THE NATURAL PRODUCT SHIKIMIC ACID USING A TANDEM ACYLATION DIPOLAR CYCLOADDITION
,IBRARIES CONTAINING FEATURES COMMON TO NATURAL PRODUCTS IN GENERAL )N AN EFFORT TO MIMIC NATURAL PRODUCTS IN A MORE GENERAL SENSE THERE HAVE BEEN MANY REPORTS ON LIBRARIES INCORPORATING DENSE STEREOCHEMICAL AND FUNCTIONAL DIVERSITY AROUND ONE OR MORE NOVEL MOLECULAR SKELETONS !N APPROACH SUCH AS THIS PROVIDES INCREASED OPPORTUNITY TO GENERATE TRULY NOVEL PHARMACOPHORES FOR ADDRESSING DIFFICULT BIOLOGICAL TARGETS !N EARLY EXAMPLE OF A $/3 LIBRARY WHICH CONTAINS CHARACTERISTICS REMI NISCENT OF NATURAL PRODUCTS IS THAT SHOWN IN &IGURE 3CHREIBER AND CO WORKERS SYNTHESIZED A LIBRARY OF OVER MILLION DENSELY FUNCTIONALIZED COMPOUNDS STARTING FROM SHIKIMIC ACID DERIVED EPOXYCYCLOHEXENOL ; = ! CONVERGENT TANDEM ACYLATION DIPOLAR CYCLOADDITION OF BOTH ENANTIOMERS OF EPOXYCYCLOHEXENOL WITH SUBSTITUTED NITRONE CARBOXYLIC ACIDS PROVIDED PRODUCTS WITH STEREOCHEMICAL DIVERSITY AND HANDLES FOR APPENDAGE DIVERSITY 4HESE RIGID POLYCYCLIC SCAFFOLDS THEN UNDERWENT A VARIETY OF APPENDAGE AND SKELETAL DIVERSITY GENERATING REACTIONS TO GEN ERATE BICYCLIC AND TRICYCLIC CORES &ROM THIS LIBRARY SEVERAL COMPOUNDS WERE DISCOVERED WHICH ACTIVATE THE 4'& β RESPONSIVE REPORTER GENE IN MAMMALIAN CELLS IN A CHEMICAL GENETICS ASSAY WITH THE %# OF THE MOST ACTIVE BEING μ-
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
R1 N
N
Ar O
R2 = H R
N Ar
CO2Me
R2 = N3
O
N
Ar
R
O
O
O Ph
R2
R1 = H
R1 = CH2N3
R2 = CH2N3
Bn N
Bn N
R1 = H
R1 = N3
O
R2 = H R1 = H
R
R2 = H
N
Ar
N
O
Ar Bn
N
Ar O
O
N R O Ph
CONHR
Ph
&IGURE ! LIBRARY OF SPIRO AND BICYCLIC LACTAMS WAS GENERATED FROM AZIDO ESTER INTERMEDIATES VIA FOUR BRANCHING PATHWAYS WHILE MONOCYCLIC AMIDES WERE OBTAINED VIA AMIDATION OF THE UNSUBSTITUTED ESTER CORE
2ECENTLY -ITCHELL AND 3HAW EMPLOYED $/3 FOR ACCESSING SKELETAL DIVER SITY FROM A SINGLE SOLID PHASE STARTING MATERIAL BY EXPLOITING ONLY A SINGLE FUNCTIONAL GROUP MANIPULATION ;= %MPLOYING A CLEVER DESIGN STRATEGY SKELETAL DIVERSITY WAS INTRODUCED AT A LATE STAGE THROUGH A DIVERGENT CYCLIZA TION TO PROVIDE SPIROCYCLIC OR TRICYCLIC COMPOUNDS OF AND MEMBERED RINGS &IG !PPENDAGE DIVERSITY WAS INTRODUCED THROUGH . ALKYLA TION AND . ACYLATION OF THE RESULTING LACTAMS IN A SPLITPOOL MANNER TO AFFORD THE FINAL PRODUCTS &URTHER STRUCTURAL DIVERSITY WAS ACCOMPLISHED BY DIRECTLY CONVERTING THE ACYCLIC METHYL ESTER PRECURSOR TO VARIOUS AMIDES TO PROVIDE MONOCYCLIC STRUCTURES 4HE INCORPORATION OF MONOCYCLIC FUSED AND SPIROCYCLIC PRODUCTS IN THIS MEMBERED LIBRARY ALLOWED FOR THE COV ERAGE OF A WIDE RANGE OF THREE DIMENSIONAL SPACE 0RELIMINARY SCREENING OF THIS PILOT LIBRARY LED TO THE DISCOVERY OF COMPOUNDS WHICH MODULATE THE CELLULAR PROCESS OF BOTH YEAST AND HUMAN CANCER (E,A CELLS 0ORCO ET AL HAVE RECENTLY APPLIED THE CONCEPT OF DOMAIN SHUFFLING TO SMALL MOLECULES IN PURSUIT OF COMPOUNDS WITH INCREASED BIOLOGICAL ACTIVITY ;= 4HIS STRATEGY HARNESSES KEY STRUCTURAL DOMAINS OF BIOLOGICALLY ACTIVE COMPOUNDS AND THEN SHUFFLES THEM TO PREPARE COMPLEX HYBRID STRUCTURES IN A CONVERGENT MANNER ;= 4HIS STRATEGY IS REMINISCENT OF THAT USED BY NATURE FOR POLYKETIDE BIOSYNTHESIS A PROCESS WHICH HAS BEEN EXPLOITED FOR THE COMBINATORIAL BIOSYNTHESIS OF @UNNATURAL NATURAL PRODUCTS ;= )N THIS STUDY A LIBRARY OF HYBRID OXIMES WAS CONSTRUCTED BY THE SHUFFLING OF COM
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
O N
Me
Me 12 Alkoxyamines
H2 N O
Me
Me
Me
O H N 168 hybrid domains Monomers have no activity
O
N N
N H O HO
O N
Me
Me
Me
O
H O
O
IC50 6.2 PM antiproliferative activity of A549 cells
N N
N H O HO
O 14 Aldehydes & ketones
&IGURE ! LIBRARY OF HYBRID OXIMES OBTAINED VIA DOMAIN SHUFFLING LED TO THE DISCOVERY OF A COMPOUND WITH ANTIPROLIFERATIVE ACTIVITY
PLEX ALKOXYAMINE AND CARBONYL DOMAINS &IG /NLY COMPOUNDS WERE SYNTHESIZED AND NO ADDITIONAL ELABORATION OF THE RESULTING HYBRIDS THROUGH APPENDAGE OR SKELETAL DIVERSITY REACTIONS WAS PERFORMED 3CREENING OF THIS OXIME LIBRARY AGAINST HUMAN SMALL CELL LUNG CARCI NOMA ! CELLS REVEALED A COMPOUND WITH AN )# OF μ- 3CREENING OF THE MONOMERIC DOMAINS LED TO NO ANTIPROLIFERATIVE ACTIVITY HIGHLIGHTING THE IMPORTANCE OF THE LIGATION OF TWO DISCRETE CHEMICAL DOMAINS GENERAT ING A MORE COMPLEX STRUCTURE WITH BIOLOGICAL ACTIVITY )N A RECENT PAPER 3CHREIBER TAKES ADVANTAGE OF EACH OF THE DIVERSITY PATHWAYS &IG 0ATH !n# BY USING BOTH CONVERGENT AND DIVERGENT DESIGN STRATEGIES ;= )N A CONVERGENT MANNER TWO SETS OF CAREFULLY DESIGNED CHIRAL AMINO ALCOHOLS WERE SYNTHESIZED AND THEN COMBINED TO PROVIDE DISTINCT ACYCLIC CORES THROUGH BIS AND TRIS COUPLING OF THE INDIVIDUAL AMINO ALCOHOLS &IG )N ADDITION TO THE STEREOISOMERIC CORES THE LIBRARY DESIGN INCLUDED DIFFERENTIATED HANDLES FOR FURTHER SKELETAL AND APPENDAGE DIVERSITY )N A DIVERGENT FASHION THE BIS AND TRIS COUPLED CORES USED THE SAME REACTION 2#- TO GENERATE DIFFERENT PRODUCTS ONE DIYNE CORE WAS UNREACTIVE WITH FIVE TYPES OF SKELETONS !DDITIONALLY THE RESULTING DIENES WERE FURTHER ELABORATED USING A ; = CYCLOADDITION TO PROVIDE ACCESS TO FOUR DISTINCT BI AND TRICYCLIC CORES 4HE APPENDAGE DIVERSITY AND ADDITIONAL CASCADE REACTIONS TO GENERATE MORE SKELETAL DIVERSITY HAVE NOT BEEN FULLY EXPLORED
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
Ns Bs BzO
2 diastereomers
Bs
H HO
NHBoc
HO
(S)
HO
NHBoc
BzO
H H N N
BzO
O
H NHBoc
Libraries from libraries
H NHBoc O
N Me
Me N O O Ns N N N
NHBoc
BzO
Convergent
N
BocHN
3 tri-coupled cores 12 functionalized acyclic cores
Ns
Divergent Bs
9 bis-coupled cores
H NHBs BzO
(S)
H NHBoc
H
(R)
H BzO
N
N
Bs
Ns N
H BzO
H
Ns
N
N
H
H
NHBoc
N H
BzO
NHBs
H N N Bs
(R) BzO
NHBs Bs
H N
NHBoc
H
NHBoc
N
plus enantiomer
O
Libraries from libraries
N Me Me N
O
BzO H 4 diastereomers
BzO
NHBoc H N Bs plus enantiomer
O
N
N
BzO
O H
NHBoc H
N Bs
&IGURE !N OLIGOMER BASED APPROACH FOR GENERATING SKELETAL DIVERSITY VIA CONVERGENT AND DIVERGENT SYNTHESIS
#HEMISTRY DRIVEN APPROACH TO $/3 AT )NFINITY 0ART OF THE CHALLENGE OF $/3 WITHIN THE CONTEXT OF DRUG DISCOVERY IS TO LIMIT THE LIABILITIES OF A COMPOUND COLLECTION UNDER TRADITIONAL PARAMETERS SUCH AS ,IPINSKIS RULE OF FIVE ;= AND 6EBERS ROTATABLE BONDS ;= WHILE AT THE SAME TIME HAVING A COLLECTION OF MOLECULES THAT ARE DIVERSE AND STILL REPRESENT NEW CHEMICAL SPACE !T )NFINITY WE FEEL THAT THERE ARE SUFFICIENT REASONS TO EXTEND BEYOND THE MOLECULAR WEIGHT RANGE OF DALTONS SUCH AS TARGETING SMALL MOLECULES WHICH PERTURB PROTEINnPROTEIN INTERACTIONS (OWEVER WE REMAIN MINDFUL OF KEY CHEMICAL PROPERTIES SUCH AS SOLUBIL ITY ISSUES !LOG0 AND THE EXCLUSION OF POTENTIALLY REACTIVE MOIETIES SUCH AS -ICHAEL ACCEPTORS AND PRIMARY HALIDES !DDITIONALLY THE NUMBER OF AMIDES IS LIMITED TO THREE OR FEWER WITHIN A LIBRARY PATHWAY 4HE $/3 EXAMPLES ABOVE PROVIDE A GOOD OVERVIEW OF THE DIFFERENT STRAT EGIES USED TO ACCESS COMPOUND LIBRARIES FOR THE DISCOVERY OF NEW BIOLOGI CALLY ACTIVE CHEMICAL ENTITIES AND HIGHLIGHT THE INFLUENCE OF NATURAL PROD
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
Stage 1: Idea Evaluation Scaffold Idea
Solution-Phase Feasibility
Stage 2: Diversity Establishment
Chemotype Diversity
Building Block Testing New Scaffold Direction
Stage 3: Process Chemistry Scale up of Scaffold(s)
Optimization
Stage 4: Library Production Solid-phase synthesis, cleavage & formatting
&IGURE /VERVIEW OF THE CHEMISTRY DRIVEN PROCESS EMPLOYED FOR THE GENERATION OF $/3 LIBRARIES
UCTS AND NATURAL PRODUCT LIKE FEATURES ON LIBRARY DESIGN )N MOST CASES THE BIOLOGY WAS NOT CONSIDERED A PRIORI THUS HIGHLIGHTING THAT $/3 IS A VALU ABLE APPROACH TO GENERATE CHEMICAL MATTER WITHOUT STRUCTURAL KNOWLEDGE OF THE TARGET RECEPTOR )N EACH CASE THE CIRCUMSTANCES TO REALIZE THE LIBRARIES WERE DIFFERENT BUT IN THE END WERE ALL DICTATED BY CHEMICAL FEASIBILITY /NE OF THE CHALLENGES OF $/3 IS HOW TO PRIORITIZE THE POTENTIAL NOVEL STRUCTURES THAT MIGHT BE SYNTHESIZED AND TAKE ADVANTAGE OF DIVERSITY OPPORTUNITIES !T THE VERY EARLY STAGES OF LIBRARY DESIGN THE EXACT STRUCTURES OF THE FINAL COMPOUNDS ARE DIFFICULT TO CONCEPTUALIZE UNLESS A NATURAL PRODUCT TEM PLATE IS BEING CONSIDERED 4HE FOUR STEP PROCESS WE HAVE IMPLEMENTED TO OPTIMIZE THIS DIFFICULT PROCESS IS OUTLINED IN &IGURE
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
3TAGE )DEA EVALUATION 0RIOR TO EMBARKING ON THE PRODUCTION OF A LIBRARY THE EFFORT IT TAKES TO DEFINE AND PRIORITIZE WHICH SCAFFOLDS TO SYNTHESIZE COMPUTATIONALLY OR OTHERWISE CAN BE RATHER CHALLENGING !T )NFINITY WE EMPLOY A PROCESS WHICH MAINTAINS A FOCUS ON CHEMICAL FEASIBILITY WHILE GENERATING MOLECU LAR COMPLEXITY IN THE FORM OF NOVEL CORES CONTAINING HANDLES FOR THE INTRODUCTION OF DIVERSITY .ATURAL PRODUCT SCAFFOLDS SERVE AS INSPIRATION NOT ONLY BECAUSE THEY HAVE A PROVEN TRACK RECORD OF ACCESSING BIOLOGICAL TARGETS BUT ALSO BECAUSE MANY REACTION METHODOLOGIES HAVE BEEN DEVEL OPED IN THE CONTEXT OF THE NATURAL PRODUCT TOTAL SYNTHESIS 4HE DIVERSITY OF A GIVEN LIBRARY IS DETERMINED ULTIMATELY BY CHEMICAL FEASIBILITY WITHOUT A SPECIFIC TARGET IN MIND SINCE IT IS DIFFICULT TO PREDICT BIOLOGICAL RELEVANCY OF NOVEL COMPOUNDS 7E REFER TO THIS PROCESS AS CHEMISTRY DRIVEN AND BELIEVE THAT IT OFFERS THE PATH OF LEAST RESISTANCE TO DIVERSITY IN THE CONTEXT OF ALL POTENTIALLY RELEVANT BIOLOGICAL SPACE )N ESSENCE WE APPLY A @CHEMICAL SUR VIVAL OF THE FITTEST APPROACH TO DIVERSITY IN THE CONTEXT OF THE STRUCTURAL CHARACTERISTICS OF THE FINAL COMPOUNDS 4HE BRAINSTORMING AND FEASIBILITY PORTION OF THE PROCESS IS PERHAPS THE MOST CRUCIAL FOR THE INITIATION OF A LIBRARY IDEA $URING THIS TIME KEY EXPERIMENTS ARE RUN WHICH WILL DETERMINE IF THE CHEMISTRY WILL ENABLE A PARTICULAR SCAFFOLD TO BE PURSUED FURTHER 4HE PRIORITIZATION FOR THE DIREC TION OF EACH LIBRARY DEPENDS ON THE ROBUSTNESS OF THE DIVERSITY OPTIONS !T )NFINITY WE GENERATE OUR LIBRARIES USING A COMBINATION OF SOLUTION AND SOLID PHASE CHEMISTRY TECHNIQUES )N GENERAL WE BUILD UP SCAFFOLD DIVERSITY USING SOLUTION PHASE METHODS WHILE SOLID PHASE METHODS ARE RESERVED FOR THE INTRODUCTION OF VARIOUS TYPES OF APPENDAGE DIVERSITY !LTHOUGH FEA SIBLE WE HAVE FOUND IT MORE RELIABLE AND COST EFFECTIVE TO PERFORM SOPHIS TICATED TRANSFORMATIONS SUCH AS ENANTIOSELECTIVE CATALYSIS IN SOLUTION AS OPPOSED TO ON SOLID SUPPORT 4HIS AVOIDS ATTEMPTING TO OPTIMIZE VERY SENSITIVE CHEMISTRY ON HUNDREDS OR THOUSANDS OF SUBSTRATES IMMOBILIZED ON SOLID PHASE WHERE IT IS NOT POSSIBLE TO EASILY MONITOR THE PROGRESS OF A REACTION
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
3TAGE $IVERSITY ESTABLISHMENT /NCE AVAILABLE A REPRESENTATIVE SET OF SCAFFOLDS IS LOADED ONTO SOLID SUPPORT -IMOTOPE¤ LANTERNS FOR BUILDING BLOCK TESTING AND FEASIBILITY STUDIES 4HE CORE IS LOADED THROUGH AN ALCOHOL FUNCTIONAL GROUP WHICH PROVIDES AN ADDITIONAL OPPORTUNITY FOR APPENDAGE DIVERSITY DURING SUBSEQUENT HIT FOLLOW UP &OR THE INTRODUCTION OF APPENDAGE DIVERSITY WE GENERALLY USE ROBUST WELL ESTABLISHED REACTIONS SUCH AS . ACYLATION REDUCTIVE ALKYLA TION @CLICK CHEMISTRY CONJUGATE ADDITION OXIME FORMATION -ITSU NOBU SUBSTITUTION AND PALLADIUM MEDIATED CROSS COUPLINGS )N ORDER TO TAKE ADVANTAGE OF THESE REACTIONS THE APPROPRIATE ORTHOGONALLY PROTECTED FUNCTIONAL GROUPS ON THE SCAFFOLD ARE REQUIRED /CCASIONALLY DURING SOLID PHASE STUDIES IT MAY BE FOUND THAT ACCESSING THE DESIRED DIVERSITY FOR A LIBRARY REQUIRES REVISITING THE SYNTHESIS OF THE SCAFFOLD SUCH THAT APPROPRI ATE PROTECTING GROUP CHANGES CAN BE MADE OR ALTERNATE DIVERSITYLOADING SITES INTRODUCED 4HIS ITERATIVE PROCESS OF MODIFYING SCAFFOLDS AND OPTIMIZ ING SOLID PHASE CHEMISTRY EVENTUALLY PROVIDES THE FRAMEWORK FOR LIBRARY DIVERSITY
3TAGE 0ROCESS CHEMISTRY (AVING ESTABLISHED THE POTENTIAL DIVERSITY FOR A LIBRARY THE FOCUS THEN SHIFTS TOWARDS PROCESS CHEMISTRY WHERE SCALABILITY ISSUES BECOME CRITICAL TO DRIVING THE PROCESS 5PON SCALE UP THE INITIAL SYNTHETIC ROUTE TO A SCAF FOLD OCCASIONALLY REQUIRES UNFORESEEN OPTIMIZATION WHICH MAY AFFECT THE DIVERSITY GENERATING REACTIONS THAT HAVE BEEN ESTABLISHED IN THE PREVIOUS STAGE OF THE PROCESS 4HUS IT IS NECESSARY TO BE MINDFUL OF SCALABILITY ISSUES AT THE OUTSET OF THE PROCESS 4HE AMOUNT OF EACH CORE REQUIRED CAN RANGE FROM SEVERAL GRAMS TO GRAMS DEPENDING UPON A VARIETY OF FACTORS SUCH AS THE SIZE OF A LIBRARY THE NUMBER OF CORES AND TYPES OF DIVERSITY
3TAGE ,IBRARY PRODUCTION AND ANALYSIS /NCE THE SCAFFOLDS HAVE BEEN SYNTHESIZED THE PRODUCTION STAGE OF THE PROCESS CAN BE REALIZED !T THIS POINT THE INVOLVEMENT OF INFORMATICS ANA
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
LYTICAL CHEMISTRY AND COMPOUND MANAGEMENT ARE CRUCIAL TO THE OVERALL SUCCESS OF THE PROJECT /F MAJOR IMPORTANCE IS THE ENCODING PROCESS USING 4RAN3TEMS4- RADIO FREQUENCY IDENTIFICATION TAGS 2&)$ TAGS 4HE DIVERSITY PATHWAYS THAT WERE GENERATED ARE REVIEWED AND ALL POSSIBLE PRODUCT DETER MINING PATHWAYS ARE IDENTIFIED 4HIS INFORMATION IS THEN ENUMERATED TO GENERATE THE FINAL STRUCTURES OF EACH COMPOUND BEING SYNTHESIZED 4HIS PROCESS IS PARTICULARLY EFFECTIVE BECAUSE THE 2&)$ TAG IS PHYSICALLY ATTACHED TO THE SOLID SUPPORT AND THE IDENTITY OF ANY LANTERN CAN BE DETERMINED BY SCANNING THE LANTERN AND ACCESSING THE INFORMATION IN THE DATABASE 4HIS PROVIDES A MEANS TO IMPLEMENT A WELL DESIGNED QUALITY CONTROL 1# PROCESS DURING PRODUCTION BASED ON THE FEASIBILITY STUDIES FROM 3TAGE "Y PRE ENCODING THE LIBRARY THE DATA MANAGEMENT SYSTEMS CAN BE SYN CHRONIZED WITH ANALYTICAL AND COMPOUND MANAGEMENT TO ENSURE ACCURATE TRACKING OF EACH INDIVIDUAL COMPOUND
%XAMPLE OF AN )NFINITY $/3 LIBRARY /NE OF THE INITIAL LIBRARIES GENERATED AT )NFINITY WAS INSPIRED BY THE STE REOCHEMICAL DIVERSITY OF NATURAL PRODUCTS 7E UTILIZED A CHEMISTRY DRIVEN PROCESS BY TAKING ADVANTAGE OF A WELL ESTABLISHED TANDEM DIPOLAR CYCLOADDITIONACYLATION BETWEEN NITRONE CARBOXYLIC ACIDS AND CHIRAL ALLYLIC ALCOHOLS &IG TO PROVIDE DISTINCT BICYCLIC CORES REPRESENTING A HIGH DEGREE OF STEREOCHEMICAL DIVERSITY IN THE FORM OF CONSTITUTIONAL ISOMERS )T IS IMPORTANT TO NOTE THAT THIS STEREOCHEMICAL DIVERSITY WAS INTRODUCED IN SOLUTION THEREBY ENSURING A HIGH DEGREE OF PURITY FOR THE LIBRARY &URTHER APPENDAGE AND SKELETAL DIVERSITY WAS INTRODUCED ON SOLID PHASE 4O ACCOMPLISH THESE TRANSFORMATIONS EACH OF THE CORES WAS LOADED ONTO -IMOTOPE¤ LANTERNS VIA A PRIMARY OR SECONDARY ALCOHOL BY WAY OF A SILYL ETHER LINKER &IG ;= 4O INTRODUCE APPENDAGE DIVERSITY EACH SCAF FOLD WAS SUBJECTED TO 3ONOGASHIRA REACTION CONDITIONS TO PROVIDE THE O M OR P ALKYNE SUBSTITUTED PRODUCTS !DDITIONAL SKELETAL DIVERSITY WAS INTRO DUCED IN A DIVERGENT MANNER BY CONVERSION TO THE MONOCYCLIC AMIDES VIA AMINOLYSIS OR RESERVED FOR . / BOND CLEAVAGE ! SET OF @SKIP COMPOUNDS WAS ALSO GENERATED SUCH THAT THE BICYCLIC LACTONE INTERMEDIATES COULD BE INCLUDED IN SCREENING
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
R1O *
OR2 Me
R or S
R1, R2 = Fmoc, H or H, TBS
HO H O
1) 1,3-dipolar cycloaddition 2) TBS or Fmoc removal
R1 H
O
O
I
OR3
N+ O
R2 O
H
O I
O
o,m,p
o,m,p
R-cores
S-cores
I
R1 H
N
N H
_
HO H O
R2
2 enantiomers X 2 allylic alcohols X 3 Nitrones = 12 cores R1, R2 = Me, H or H, Me
R3 = Me or H
&IGURE 3OLUTION PHASE SYNTHESIS OF THE ISOXAZOLIDINE CORES USING A TANDEM ACYLATION DIPOLAR CYCLOAD DITION AS THE KEY STEP
12 cores
Sonogashira
R1 H
O H
O
O H
R2
R4
R2
OH
O
O
NHR3
N
N H
R1
O
O
Bicyclic lactone
R4 Monocyclic amide Aminolysis
N-O bond cleavage
O R1 H
O HO
R1
R2
HO R2
OH NHR3
HN O
O
HN H
O R4
R4 Monocyclic lactone
Acyclic amide
(only one enantiomer shown)
&IGURE 3OLID PHASE DIVERSIFICATION OF THE ISOXAZOLIDINE CORES ONLY ONE ENANTIOMER SHOWN
&OLLOWING .n/ BOND CLEAVAGE THE MONOCYCLIC LACTONE INTERMEDIATES WERE SUBJECTED TO AMINOLYSIS CONDITIONS TO PROVIDE THE ACYCLIC AMIDES AND ADD APPENDAGE DIVERSITY )N TOTAL ^ ISOXAZOLIDINE AND α AMINO AMIDE COMPOUNDS WERE PREPARED 3CREENING OF THE ISOXAZOLIDINE LIBRARY IN VARIOUS ASSAYS LED TO THE DIS COVERY OF SEVERAL INHIBITORS OF THE ANTI APOPTOTIC PROTEINS "CL X, AND "CL n μ- ; = AND SEVERAL INHIBITORS OF BACTERIAL CELL GROWTH WITH AN
,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
Me
HO
HO
OH H N
O N
N
Me
(R)-core
H N
O
MW = 549 Bcl-xL inhibitor
OH
N
Me
O
H2 N
Me
H O
NH
MW = 538
Anti-bacterial compound (S)-core
&IGURE 4WO BIOLOGICALLY ACTIVE $/3 COMPOUNDS WHICH WERE IDENTIFIED IN PRIMARY SCREENING
-)# OF μGM, ;= &IG )T IS INTERESTING TO NOTE THAT ONE LIBRARY OF RELATED YET STRUCTURALLY DIVERSE COMPOUNDS PROVIDED ACTIVE COMPOUNDS IN TWO VERY DIFFERENT ASSAYS AGAIN REINFORCING THE POTENTIAL OF $/3 FOR IDEN TIFYING MOLECULES WITH DIVERSE BIOLOGICAL TARGETS -OREOVER THE IDENTIFICA TION OF μ- INHIBITORS OF "CL X, AND "CL DIRECTLY FROM PRIMARY SCREENING EFFORTS IS SIGNIFICANT CONSIDERING THE DIFFICULT NATURE OF DISRUPTING SUCH PROTEINnPROTEIN INTERACTIONS ; = 3TRUCTURALLY THE ACTIVE COMPOUNDS FOR THE "CL FAMILY AND THE ANTIBACTERIAL COMPOUNDS DIFFER IN THE STEREO CHEMISTRY OF THE CORE SCAFFOLDS ENANTIOMERIC AS WELL AS IN THE POSITION OF A SINGLE METHYL GROUP AND APPENDAGE DIVERSITY AMINE AND ALKYNE COMPONENT 4HESE DIFFERENCES REFLECT THE IMPORTANCE OF SUBSTITUTION AND STEREOCHEMISTRY ON THE OVERALL THREE DIMENSIONAL STRUCTURE OF INDIVIDUAL COMPOUNDS AND HOW THEY CORRELATE TO BIOLOGICAL ACTIVITY 4HIS OBSERVATION ALSO SUPPORTS THE PREVIOUS CONCLUSION OF PRIVILEGED STRUCTURE MOTIFS THAT ARE RELEVANT TO DIFFERENT BIOLOGICAL PROCESSES 4HE ADDITIONAL BENEFIT OF $/3 THAT IS INHERENT IN THE DESIGN OF THE LIBRARY IS BUILT IN 3!2 DATA WHICH FACILITATES THE EVALUATION OF HITS OF INTER EST &URTHERMORE CORES AND INTERMEDIATES ARE READILY ACCESSIBLE FROM THE SCALE UP PROCESS ENABLING RAPID OPTIMIZATION STUDIES )N THIS EXAMPLE MEDICINAL CHEMISTRY EFFORTS AROUND THE ACTIVE ISOXAZOLIDINE COMPOUNDS HAVE LED TO "CL FAMILY INHIBITORS WITH SUB NANOMOLAR ACTIVITY AND A COM POUND THAT WAS ACTIVE AGAINST METHICILLIN RESISTANT 3 AUREUS -23! WITH -)#S μGM, )NITIAL IN VIVO PROOF OF CONCEPT IN A MOUSE THIGH INJEC TION MODEL SHOWED EFFICACY AND NO OVERT TOXICITY FOR THE ANTI BACTERIAL COMPOUND
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
3UMMARY !S DEMONSTRATED BY THE EXAMPLES HIGHLIGHTED IN THIS CHAPTER THERE IS SIGNIFICANT POTENTIAL FOR $/3 LIBRARIES TO GENERATE NOVEL LEADS FOR DRUG DISCOVERY $/3 IS COMPLEMENTARY TO MORE TRADITIONAL METHODS SUCH AS STRUCTURE BASED DESIGN WHERE THE CRYSTAL STRUCTURE OF THE BINDING POCKET IS KNOWN AND A SPECIFIC COMPOUNDS OR A LIBRARY OF COMPOUNDS CAN BE DESIGNED 7HEN SUCH AN APPROACH IS NOT POSSIBLE $/3 IS VERY ADVANTA GEOUS PROVIDING A MEANS TO GENERATE NOVEL CHEMICAL MATTER WITH THE AIM OF ACCESSING MULTIPLE BIOLOGICAL PATHWAYS OR DIFFERENT ALLOSTERIC BINDING SITES OF KNOWN TARGETS )N ORDER TO REALIZE THE FULL POTENTIAL OF $/3 THE SCREENING OF $/3 COMPOUNDS AGAINST MULTIPLE BIOLOGICAL TARGETS IS NECES SARY )N ESSENCE THE SCREENING OF $/3 COMPOUND COLLECTIONS IS MUCH LIKE SCREENING A COLLECTION OF NATURAL PRODUCTS IN THAT IT SERVES AS A STARTING POINT FOR DIVERSE AND COMPLEX MOLECULES WHICH MAY POSSESS INTERESTING BIOLOGICAL ACTIVITY
2EFERENCES
"REINBAUER 2 6ETTER )2 7ALDMANN ( &ROM PROTEIN DOMAINS TO DRUG CANDIDATES n NATURAL PRODUCTS AS GUIDING PRINCIPLES IN THE DESIGN AND SYNTHESIS OF COMPOUND LIBRAR IES !NGEW #HEM )NT %D n .EWMAN $* #RAGG '- 3NADER +- .ATURAL PRODUCTS AS A SOURCE OF NEW DRUGS OVER THE PERIOD n * .AT 0ROD n -ARTIN 9# $IVERSE VIEWPOINTS ON COMPUTATIONAL ASPECTS OF MOLECULAR DIVERSITY * #OMB #HEM n -ARTIN 9# #RITCHLOW 2% "EYOND MERE DIVERSITY TAILORING COMBINATORIAL LIBRARIES FOR DRUG DISCOVERY * #OMB #HEM n ,IPINSKI #! ,OMBARDO & $OMINY "7 %XPERIMENTAL AND COMPUTATIONAL APPROACHES TO ESTIMATE SOLUBILITY AND PERMEABILITY IN DRUG DISCOVERY AND DEVELOPMENT SETTINGS !DV $RUG $EL 2EV n 4UDOR / (ANN - 0URSUING THE LEADLIKENESS CONCEPT IN PHARMACEUTICAL RESEARCH #URR /PIN #HEM "IOL n 2ISHTON ' .ON LEADLIKENESS AND LEADLIKENESS IN BIOCHEMICAL SCREENING $RUG $ISC 4ODAY n ,EE -, 3CHNEIDER ' 3CAFFOLD ARCHITECTURE AND PHARMACOPHORIC PROPERTIES OF NATURAL PRODUCTS AND TRADE DRUGS APPLICATION IN THE DESIGN OF NATURAL PRODUCT BASED COMBINATORIAL LIBRARIES * #OMB #HEM n $OLAN +! !MGENS ENEMIES &ORBES 3EPTEMBER (AGGARTY 3 4HE PRINCIPLE OF COMPLEMENTARITY CHEMICAL VERSUS BIOLOGICAL SPACE #URR /PIN #HEM "IOL n
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-ICKEL 3* 4OWARD A COMMERCIAL SYNTHESIS OF DISCODERMOLIDE #URR /PIN $RUG $ISC $EV n :HENG 7 3ELETSKY "- 0ALME -( ,YDON 0* 3INGER ,! #HASE #% ,EMELIN #! 3HEN 9 $AVIS ( 4REMBLAY , ET AL -ACROCYCLIC KETONE ANALOGUES OF HALICHONDRIN " "IOORG -ED #HEM ,ETT n 7ILSON 2 $ANISHEFSKY 3* 3MALL MOLECULE NATURAL PRODUCTS IN THE DISCOVERY OF THERAPEUTIC AGENTS 4HE SYNTHESIS CONNECTION * /RG #HEM n "URKE -$ 3CHREIBER 3, ! PLANNING STRATEGY FOR DIVERSITY ORIENTED SYNTHESIS !NGEW #HEM )NT %D n +UMAGAI . -UNCIPINTO ' 3CHREIBER 3, 3HORT SYNTHESIS OF SKELETALLY AND STERE OCHEMICALLY DIVERSE SMALL MOLECULES BY COUPLING 0ETASIS CONDENSATION REACTIONS TO CYCLIZATION REACTIONS !NGEW #HEM )NT %D n 7YATT %% &ERGUS 3 'AKKOWAY 72*$ "ENDER ! &OX $* 0LOWRIGHT !4 *ESSIMAN !3 7ELCH - 3PRING $2 3KELETAL DIVERSITY CONSTRUCTION VIA A BRANCHING SYNTHETIC STRATEGY #HEM #OMM n +IM 9 !RAI - !RAI 4 ,AMENZO * $EAN % 0ATTERSON . #LEMONS 0 3CHREIBER 3, 2ELATIONSHIP OF STEREOCHEMICAL AND SKELETAL DIVERSITY OF SMALL MOLECULES TO CELLULAR MEASUREMENT SPACE * !M #HEM 3OC n 3TOCKWELL "2 %XPLORING BIOLOGY WITH SMALL ORGANIC MOLECULES .ATURE n 3TOCKWELL "2 #HEMICAL GENETICS ,IGAND BASED DISCOVERY OR GENE FUNCTION .ATURE 2EV 'ENETICS n +OCH -! 3CHUFFENHAUER ! 3CHECK - 7ETZEL 3 #ASAULTA - /DERMATT ! %RTL 0 7ALD MAN ( #HARTING BIOLOGICALLY RELEVANT CHEMICAL SPACE ! STRUCTURAL CLASSIFICATION OF NATURAL PRODUCTS 0.!3 n .OREN -ULLER ! 2EIS #ORREA *R ) 0RINZ ( 2OSENBAUM # 3AXENA + 3CHWALBE (* 6EST WEBER $ #AGNA ' 3CHUNK 3 3CHWARZ / ET AL $ISCOVERY OF PROTEIN PHOSPHATASE INHIBITOR CLASSES BY BIOLOGY ORIENTED SYNTHESIS 0.!3 n $ECKER & +NOCH - 7ALDMANN ( 0ROTEIN SIMILARITY STRUCTURE CLUSTERING AND NATURAL PRODUCT STRUCTURE AS INSPIRATION SOURCES FOR DRUG DEVELOPMENT AND CHEMICAL GENOMICS #URR /PIN #HEM "IOL n "OLDT ' $ICKERSON 4 *ANDA + %MERGING CHEMICAL AND BIOLOGICAL APPROACHES FOR THE PREPARATION OF DISCOVERY LIBRARIES $RUG $ISC 4ODAY n &EHER - 3CHMIDT *- 0ROPERTY DISTRIBUTIONS DIFFERENCES BETWEEN DRUGS NATU RAL PRODUCTS AND MOLECULES FROM COMBINATORIAL CHEMISTRY * #HEM )NF #OMPU 3CI n /RTHOLAND * 'ANESAN ! .ATURAL PRODUCTS AND COMBINATORIAL CHEMISTRY "ACK TO THE FUTURE #URR /PIN #HEM "IOL n $OBSON #- #HEMICAL SPACE AND BIOLOGY .ATURE n &ERGUS 3 "ENDER ! 3PRING $ !SSESSMENT OF STRUCTURAL DIVERSITY IN COMBINATORIAL SYNTHESIS #URR /PIN #HEM "IOL n ,IPINSKI # (OPKINS ! .AVIGATING CHEMICAL SPACE FOR BIOLOGY AND MEDICINE .ATURE n -C'OVERN 3, #ASELLI % 'RIGORIEFF . 3HOICHET "+ ! COMMON MECHANISM UNDER LYING PROMISCUOUS INHIBITORS FROM VIRTUAL AND HIGHTHROUGHPUT SCREENING * -ED #HEM n %LLIS *2 -INTO ! *OIN THE CROWD .ATURE n
!PPLICATION OF NATURAL PRODUCT INSPIRED DIVERSITY ORIENTED SYNTHESIS TO DRUG DISCOVERY
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,ISA ! -ARCAURELLE AND #HARLES 7 *OHANNES
3U 1 "EELER !" ,OBKOVSKY % 0ORCO (! 0ANEK *3 3TEREOCHEMICAL DIVERSITY THROUGH CYCLODIMERIZATION 3YNTHESIS OF POLYKETIDE LIKE MACRODIOLIDES /RG ,ETT n 3CHMIDT $2 +WON / 3CHREIBER 3, -ACROLACTONES IN DIVERSITY ORIENTED SYNTHESIS 0REPARATION OF A PILOT LIBRARY AND EXPLORATION OF FACTORS CONTROLLING MACROCYLIZATION * #OMB #HEM n 4AN $3 &OLEY -! 3HAIR -$ 3CHREIBER 3, 3TEREOSELECTIVE SYNTHESIS OF OVER TWO MILLION COMPOUNDS HAVING STRUCTURAL FEATURES BOTH REMINISCENT OF NATURAL PRODUCTS AND COMPATIBLE WITH MINIATURIZED CELL BASED ASSAYS * !M #HEM 3OC n 4AN $3 &OLEY -! 3TOCKWELL "2 3HAIR -$ 3CHREIBER 3, 3YNTHESIS AND PRELIMI NARY EVALUATION OF A LIBRARY OF POLYCYCLIC SMALL MOLECULES FOR USE IN CHEMICAL GENETIC ASSAYS * !M #HEM 3OC n -ITCHELL *- 3HAW *4 ! STRUCTURALLY DIVERSE LIBRARY OF POLYCYCLIC LACTAMS RESULT ING FROM SYSTEMATIC PLACEMENT OF PROXIMAL FUNCTIONAL GROUPS !NGEW #HEM )NT %D n 3U 3 !CQUILANO $% !RUMUGASAMY * "EELER !" %ASTWOOD %, 'IGUERE *2 ,AN 0 ,EI 8 -IN '+ 9EAGER !2 ET AL #ONVERGENT SYNTHESIS OF A COMPLEX OXIME LIBRARY USING CHEMICAL DOMAIN SHUFFLING /RG ,ETT n "EELER !" 3CHAUS 3% 0ORCO *R * ! CHEMICAL LIBRARY SYNTHESIS USING CONVERGENT APPROACHES #URR /PIN #HEM "IOL n 4SOI #* +HOSLA # #OMBINATORIAL BIOSYNTHESIS OF @UNNATURAL NATURAL PRODUCTS THE POLYKETIDES EXAMPLE #HEM "IOL n 3PIEGEL $! 3CHROEDER &# $UVALL *2 3CHREIBER 3, !N OLIGOMER BASED APPROACH TO SKELETAL DIVERSITY IN SMALL MOLECULE SYNTHESIS * !M #HEM 3OC n ,IPINSKI #! ,OMBARDO & $OMINY "7 &EENEY 0* %XPERIMENTAL AND COMPUTA TIONAL APPROACHES TO ESTIMATE SOLUBILITY AND PERMEABILITY IN DRUG DISCOVERY AND DEVELOP MENT SETTINGS !DV $RUG $EL 2EV n 6EBER $& *OHNSON 32 #HENG (9 3MITH "2 7ARD +7 +OPPLE +$ -OLECULAR PROPERTIES THAT INFLUENCE THE ORAL BIOAVAILABILITY OF DRUG CANDIDATES * -ED #HEM n 4ALLARICO *! $EPEW +- 0ELISH (% 7ESTWOOD .* ,INDSLEY #7 3HAIR -$ 3CHRIEBER 3, &OLEY -! !N ALKYLSILYL TETHERED HIGH CAPACITY SOLID SUPPORT AMENABLE TO DIVERSITY ORIENTED SYNTHESIS FOR ONE BEAD ONE STOCK SOLUTION CHEMICAL GENETICS * #OMB #HEM n #ASTRO !# $ENG 7 $EPEW +- &OLEY -! &RITZ ## 'EORGES %VANGELINOS !4 'ROGAN -* (AFEEZ . (OLSON %" (OPKINS "4 ET AL #OMPOUNDS AND METHODS FOR INHIBIT ING THE INTERACTION OF "CL PROTEINS WITH BINDING PARTNERS 0#4 )NT !PPL PP 7/ 2EIBARKH - -ALIA 4* 7AGNER ' )DENTIFICATION OF INDIVIDUAL PROTEIN LIGAND ./%S IN THE LIMIT OF INTERMEDIATE EXCHANGE * "IOMOL .-2 n #HRISTENSEN "' &OLEY -! 'EORGES %VANGELINOS !4 ,UI 4 0ORTER *2 2IPKA !3 :HANG , )SOXAZOLIDINE COMPOUNDS FOR TREATMENT OF BACTERIAL INFECTIONS 0#4 )NT !PPL 7/ #ORY 3 !DAMS *- 4HE "CL FAMILY REGULATORS OF THE CELLULAR LIFE OR DEATH SWITCH .ATURE 2EV #ANCER n !RKIN - 0ROTEINnPROTEIN INTERACTIONS IN CANCER SMALL MOLECULES GOING IN FOR THE KILL #URR /PIN #HEM "IOL n
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#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS ,ESSONS FROM NATURE INSPIRING THE DESIGN OF NEW DRUGS
"Y 0ETER %RTL AND !NSGAR 3CHUFFENHAUER .OVARTIS )NSTITUTES FOR "IO-EDICAL 2ESEARCH "ASEL 3WITZERLAND PETERERTL NOVARTISCOM
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
!BSTRACT .ATURAL PRODUCTS .0S HAVE EVOLVED OVER A VERY LONG NATURAL SELECTION PROCESS TO FORM OPTIMAL INTERACTIONS WITH BIOLOGICAL MACROMOLECULES .0S ARE THEREFORE AN EXTREMELY USEFUL SOURCE OF INSPIRATION FOR THE DESIGN OF NEW DRUGS )N THE PRESENT STUDY WE REPORT THE RESULTS OF A CHEMINFORMATICS ANALYSIS OF MORE THAN .0 STRUCTURES 4HE PHYSICOCHEMICAL PROPER TIES OF .0S AND THEIR TYPICAL STRUCTURAL FEATURES ARE COMPARED TO THOSE OF BIOACTIVE MOLECULES AND AVERAGE ORGANIC MOLECULES 4HE RELATIONSHIP BETWEEN THE STRUCTURE OF .0S AND THE TYPE OF ORGANISM FROM WHICH THEY HAVE COME HAS ALSO BEEN ANALYZED 4HE AIM OF THIS STUDY WAS TO IDENTIFY THOSE PROPERTIES AND STRUCTURAL FEATURES WHICH ARE TYPICAL FOR .0S AND DISCRIMINATE THIS CLASS OF MOLECULES FROM COMMON SYNTHETIC MOLECULES WITH THE ULTIMATE GOAL BEING TO PROVIDE A GUIDE FOR THE DESIGN OF NOVEL .0 LIKE BIOACTIVE STRUCTURES (OPEFULLY THE RESULTS OF THIS ANALYSIS HELP TO ELIMINATE THE OLD MYTH ABOUT .0S AS BEING @TOO COMPLEX OR HAVING @BAD PROPERTIES AS WELL AS HELP US TO FOCUS ON THESE AREAS OF .0 STRUCTURAL SPACE WHICH ARE ESSENTIAL FOR BIOLOGICAL ACTIVITY TAKING ADVANTAGE OF THE PROCESS OF NATURAL SELECTION OVER BILLIONS OF YEARS TO GUIDE US TO NEW AND AS YET UNEXPLORED AREAS OF THE #HEMICAL 3TRUCTURE 5NIVERSE
)NTRODUCTION .ATURAL PRODUCTS .0S HAVE BEEN OPTIMIZED IN A VERY LONG NATURAL SELEC TION PROCESS FOR OPTIMAL INTERACTIONS WITH BIOLOGICAL MACROMOLECULES .0S ARE THEREFORE AN EXCELLENT SOURCE OF SUBSTRUCTURES FOR THE DESIGN OF NEW DRUGS )NDEED MANY DRUGS IN THE CURRENT PHARMACOPEIAS ARE .0S AND MANY OTHERS ARE OF .0 ORIGIN ;= )N RECENT YEARS WE CAN WITNESS A REAL EXPLOSION OF INTEREST IN USE OF .0S IN THE DRUG DISCOVERY ;= 4HIS NEW WAVE OF INTEREST MAY BE ALSO ATTRIBUTED TO THE FACT THAT SLIGHTLY EXAGGERATED EXPECTATIONS OF NEW TECHNOLOGIES WHICH HAVE BEEN INTRODUCED INTO THE DRUG DISCOVERY PROCESS A DECADE AGO HAVE NOT MATERIALIZED 4HESE TECHNOL OGIES INCLUDING COMBINATORIAL CHEMISTRY HIGH THROUGHPUT SCREENING AND VARIOUS nOMICS TECHNIQUES ALTHOUGH CONSIDERABLY IMPROVING THE EFFICIENCY OF THE WHOLE DRUG DISCOVERY PROCESS DID NOT FILL DEVELOPMENT PIPELINES OF PHARMACEUTICAL COMPANIES WITH A FLOOD OF NEW DRUG CANDIDATES .0 BASED DRUG DISCOVERY ABANDONED BY A NUMBER OF COMPANIES SOME TIME AGO AS @WITHOUT PERSPECTIVE OR @TOO COMPLEX IS AGAIN THE CENTER OF ATTENTION AS A PROMISING AND RELIABLE SOURCE OF NEW BIOACTIVE MOLECULES 3EVERAL START UPS FOCUSED ENTIRELY ON .0 BASED DRUG DISCOVERY APPEARED ;= AND TRADITIONAL PHARMACEUTICAL COMPANIES ARE INCREASING INVESTMENTS IN THEIR NATURAL PRODUCT DEPARTMENTS
0ETER %RTL AND !NSGAR 3CHUFFENHAUER
3TRUCTURES OF .0S ALSO BECOME A NEW WELCOME SOURCE OF INSPIRATION FOR THE DESIGN OF COMBINATORIAL LIBRARIES )T IS A WELL KNOWN FACT THAT THE FIRST GENERATION OF COMBINATORIAL LIBRARIES CONTAINING MOSTLY LARGE HYDROPHO BIC MOLECULES WITH MANY ROTATABLE BONDS WAS RATHER A DISAPPOINTMENT CONCERNING THEIR BIOLOGICAL ACTIVITY "UT THESE NEGATIVE RESULTS ALSO HAD A POSITIVE EFFECT #HEMISTS LEARNED THAT NOT ONLY THE AMOUNT OF MOLECULES SYNTHESIZED IS IMPORTANT BUT ALSO THEIR PROPERTIES ;= 4HIS LED TO RE EVALU ATION OF COMBICHEM DESIGN STRATEGIES INTRODUCTION OF SO CALLED @DIVERSITY ORIENTED SYNTHESIS $/3 ; = FOCUSING ON REPLACEMENT OF @CLASSICAL FLAT AROMATIC CHEMISTRY BY DIVERSE AND STRUCTURALLY COMPLEX SMALL MOLECULES !ND .0S WITH THEIR HIGH DIVERSITY ARE VERY WELL SUITED AS A SOURCE OF BIOAC TIVE SUBSTRUCTURES FOR THE DESIGN OF NEW TYPES OF COMBINATORIAL LIBRARIES ; = 4HE FACT THAT $/3 AIMS TO INCREASE THE DIVERSITY WITHIN A COMBINATO RIAL LIBRARY BY USING HIGHLY BRANCHED SYNTHESIS PATHWAYS HAS ITS ANALOGY IN NATURAL PRODUCT BIOSYNTHESIS WHERE THE PATHWAYS ARE ALSO KNOWN TO BE HIGHLY BRANCHED ;= )N LIGHT OF ALL THESE FACTS IT IS RATHER SURPRISING THAT ONLY RELATIVELY LITTLE EFFORT HAS BEEN SPENT INVESTIGATING .0S FROM THE CHEMINFORMATICS POINT OF VIEW (ENKEL ET AL ;= ANALYZED DIFFERENCES IN MOLECULAR PROPERTIES AND STRUCTURAL FEATURES SUCH AS FREQUENCIES OF DIFFERENT ATOM TYPES BETWEEN .0S AND SYNTHETIC MOLECULES AND FOUND DISTINCT DIFFERENCES BETWEEN THESE TWO GROUPS 3TAHURA ET AL ;= IDENTIFIED A SET OF DESCRIPTORS WHICH WERE ABLE TO DISTINGUISH .0S FROM SYNTHETIC MOLECULES BASED ON THEIR 3HANNON ENTROPY ,EE AND 3CHNEIDER ;= ANALYZED A SET OF .0S TO IDENTIFY WHETHER THEY CONTAIN NOVEL SCAFFOLD ARCHITECTURES FOR POTENTIAL USE IN COMBINATORIAL CHEMISTRY 3EVERAL SUCH SCAFFOLDS HAVE BEEN IDENTIFIED WHICH WERE NOT PRES ENT IN MARKETED DRUGS &EHER AND 3CHMIDT ;= COMPARED DISTRIBUTION OF VARIOUS MOLECULAR PROPERTIES AMONG .0S DRUGS AND MOLECULES ORIGINATING FROM COMBINATORIAL CHEMISTRY IDENTIFYING THE NUMBER OF CHIRAL CENTERS PRESENCE OF AROMATIC RINGS DEGREE OF SATURATION AND NUMBER OF VARIOUS HETEROATOMS BEING THE MOST IMPORTANT 7ESSJOHANN ET AL ;= STUDIED A SET OF REPRESENTATIVE MACROCYCLES OF NATURAL ORIGIN TO LEARN ABOUT STRUCTURAL FEATURES THAT ARE CHARACTERISTIC OF THIS CLASS OF MOLECULES +OCH ET AL ;= ANALYZED A LARGE DATABASE OF .0S TO STUDY .0 SCAFFOLDS BY ARRANGING THEM AS A SCAFFOLD TREE AND USED THIS INFORMATION TO NAVIGATE WITHIN THE SCAF FOLD UNIVERSE TO IDENTIFY INTERESTING REGIONS !ND RECENTLY (AUSTEDT ET AL REVIEWED RATIONAL APPROACHES TO .0 BASED DRUG DESIGN ;=
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
)N THE PRESENT STUDY WE PERFORM A CHEMINFORMATICS ANALYSIS OF A LARGE COLLECTION OF MORE THAN .0 STRUCTURES 7E ARE COMPARING PHYSI COCHEMICAL PROPERTIES OF .0S AND THEIR TYPICAL STRUCTURAL FEATURES WITH THOSE OF BIOACTIVE MOLECULES "-S AND AVERAGE ORGANIC MOLECULES /-S AND ANALYZING THE RELATIONSHIP BETWEEN THE STRUCTURE OF .0S AND THE TYPE OF ORGANISM FROM WHICH THEY HAVE COME 4HE GOAL OF THIS STUDY IS TO IDENTIFY THOSE PROPERTIES AND STRUCTURAL FEATURES WHICH ARE TYPICAL FOR .0S AND WHICH DISTINGUISH THIS CLASS OF MOLECULES FROM COMMON SYNTHETIC MOLECULES WITH THE ULTIMATE GOAL BEING TO PROVIDE GUIDE FOR THE DESIGN OF NOVEL .0 LIKE BIOACTIVE MOLECULES
0ROCESSING THE DATABASE OF NATURAL PRODUCTS !S A BASIS OF OUR STUDY THE #2# $ICTIONARY OF .ATURAL 0RODUCTS $.0 ;= IN A 3$& -$, STRUCTURE DATA FORMAT FILE WAS USED 4HIS DATABASE CONTAINS ENTRIES WITH A GIVEN CHEMICAL STRUCTURE 4HESE STRUCTURES HAVE BEEN CLEANED BY NORMALIZING CHARGES AND BY REMOVING SMALLER PARTS COUN TERIONS ETC !FTER REMOVAL OF DUPLICATES THE DATASET CONTAINED UNIQUE STRUCTURES 4HIS DATASET SERVED AS A BASIS FOR CALCULATION OF VARI OUS MOLECULAR DESCRIPTORS "EFORE THE STUDY OF STRUCTURAL FEATURES RINGS AND SUBSTITUENTS AN ADDITIONAL PROCESSING STEP WAS PERFORMED NAMELY DEGLYCOSYLATION 4HE MAIN ROLE OF SUGAR MOIETIES IN .0S IS TO AFFECT PHAR MACOKINETIC PROPERTIES OF PARENT STRUCTURES AND MAKE THEM MORE SOLUBLE ;= )N MOST CASES SUGAR UNITS DO NOT AFFECT BIOLOGICAL ACTIVITY OF AGLYCON DIRECTLY ALTHOUGH SEVERAL NOTABLE EXCEPTIONS OF THIS GENERAL RULE EXIST 4HE PRESENCE OF VARIOUS SUGAR UNITS IS THEREFORE PROBABLY THE MOST TYPICAL STRUCTURAL CHARACTERISTIC OF .0 MOLECULES !ND BECAUSE WE DID NOT WANT THIS FEATURE TO SURPASS OTHER MORE INTERESTING STRUCTURAL CHARACTERISTICS OF .0S THE SUGAR UNITS WERE REMOVED BEFORE THE ACTUAL SUBSTRUCTURE ANALYSIS 4HE DEGLYCOSYLATION STEP PRECEDING ACTUAL SUBSTRUCTURE PROCESSING PARALLELS THE STRATEGY FROM OUR EARLIER STUDY OF .0 SCAFFOLDS ;= &OR THE REMOVAL OF SUGAR UNITS WE USED A RECURSIVE DEGLYCOSYLATION PROCEDURE WRITTEN IN *AVA 4HE .0 DATABASE AFTER DEGLYCOSYLATION CONTAINED UNIQUE MOLECULES 4HE CHARACTERISTICS OF .0S HAVE BEEN COMPARED WITH THOSE OF "-S OBTAINED BY COMBINING STRUCTURES FROM THE 7ORLD $RUG )NDEX ;= AND THE -$$2 DATABASE ;= TOGETHER ABOUT UNIQUE STRUCTURES AND
0ETER %RTL AND !NSGAR 3CHUFFENHAUER
ALSO WITH A REPRESENTATIVE SET OF DRUG LIKE /-S SELECTED FROM AN IN HOUSE COLLECTION OF MORE THAN MILLION COMMERCIALLY AVAILABLE SAMPLES BY USING REPRESENTATIVE SELECTION "OTH "- AND /- DATASETS WERE NORMALIZED IN THE SAME WAY AS .0 STRUCTURES 3TRUCTURES IN THE $.0 UNFORTUNATELY DO NOT CONTAIN INFORMATION ABOUT MOLECULE STEREOCHEMISTRY !LTHOUGH THIS WOULD BE A SERIOUS DRAWBACK WHEN STUDYING THE $ STRUCTURE OF .0 MOLECULES OR THEIR INTERACTIONS WITH PROTEINS IN OUR CASE THE LACK OF STEREO INFORMATION WAS NOT SO CRITICAL SINCE WE FOCUSED MAINLY ON CALCULATED PROPERTIES WHICH DO NOT DEPEND ON STEREOCHEMISTRY AS WELL AS STEREOCHEMICALLY NON CRITICAL SUBSTRUCTURAL FEATURES 4HE STEREOCHEMISTRY HOWEVER WAS NOT COMPLETELY IGNORED IN OUR ANALYSIS &OR MOLECULES IN ALL DATASETS WE DETERMINED THE NUMBER OF POTEN TIAL STEREO CENTERS CHIRAL CARBON ATOMS WHICH PROVIDES A GOOD ASSESSMENT OF THEIR POTENTIAL STEREO VARIABILITY 4HE CHEMINFORMATICS ANALYSIS INCLUDING MOLECULE CLEANING NORMALIZA TION CALCULATION OF VARIOUS MOLECULAR PROPERTIES AND SUBSTRUCTURE ANALYSIS IDENTIFICATION OF SCAFFOLDS AND SUBSTITUENTS WAS PERFORMED WITH 0IPE LINE0ILOT ;= AND -OLINSPIRATION ;= SOFTWARE 4HE OCTANOL WATER PARTI TION COEFFICIENT LOG0 WAS CALCULATED BY THE "IO"YTE #,/'0 PROGRAM ;= !DDITIONALLY THE STRUCTURES IN THE $.0 WERE CLASSIFIED ACCORDING TO THEIR ORIGIN WHERE THIS INFORMATION WAS AVAILABLE 4HE @BIOLOGICAL SOURCE FIELD OF ALL $.0 DATABASE ENTRIES WAS MINED FOR SYSTEMATIC NAMES OF SPECIES 4HE FIRST PART OF THE SYSTEMATIC NAME DESCRIBING THE GENUS WAS LOOKED UP IN THE )4)3 )NTEGRATED 4AXONOMIC )NFORMATION 3YSTEM ;= AND THE MOL ECULES WERE CLASSIFIED ACCORDING TO THIS INFORMATION INTO THE FOUR CLASSES n PRODUCTS OF PLANTS ANIMALS FUNGI OR BACTERIA
$ISTRIBUTION OF MOLECULAR PROPERTIES AND SIMPLE STRUCTURE DESCRIPTORS 'LOBAL MOLECULAR PROPERTIES SUCH AS LOG0 POLAR SURFACE AREA 03! ;= OR THE NUMBER OF ATOMS IN THE MOLECULE ARE IMPORTANT PARAMETERS INFLUENC ING THE BIOAVAILABILITY OF MOLECULES MAINLY THEIR MEMBRANE PENETRATION #OMPARISON OF THE DISTRIBUTIONS OF THESE PROPERTIES FOR THREE LARGE COLLEC TIONS OF MOLECULES IS SHOWN IN &IGURE /NE CAN SEE THAT THE DISTRIBUTION OF
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
&IGURE #OMPARISON OF CALCULATED MOLECULAR PROPERTIES AND SIMPLE STRUCTURAL FEATURES FOR NATURAL PRODUCTS SOLID LINE BIOACTIVE MOLECULES DASHED LINE AND AVERAGE ORGANIC MOLECULES DOTTED LINE
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LOG0 03! AND THE NUMBER OF ATOMS IS FOR .0S AND "-S VERY SIMILAR 4HIS IS ON ONE SIDE A CONSEQUENCE OF A FACT THAT THE SET OF BIOACTIVE MOLECULES ITSELF CONTAINS MANY NATURAL PRODUCT AND THEIR DERIVATIVES BUT ON THE OTHER SIDE THIS ALSO SHOWS THAT PROPERTIES OF .0S ARE NOT SO @BAD OR @NON DRUGLIKE AS SOMETIMES ASSUMED $ISTRIBUTIONS OF THESE PARAMETERS FOR STANDARD /-S SHOW APPROXIMATELY THE SAME MEAN VALUES BUT THE DISTRIBUTIONS HAVE IN ALL CASES LOWER VARIABILITY 4HE NUMBER OF ROTATABLE BONDS CHARACTERIZING MOLECULE FLEXIBILITY SHOWS THAT .0 MOLECULES ARE MORE COMPACT LESS FLEX IBLE THAN MOLECULES IN OTHER SETS 4HIS IS DUE TO THE FACT THAT SKELETONS OF .0S ARE OFTEN FORMED BY COMPLEX FUSED RIGID RING SYSTEMS 7E EXAMINED ALSO THE DISTRIBUTION OF OXYGEN AND NITROGEN ATOMS AMONG THESE DATASETS (ERE ONE CAN SEE DISTINCT DIFFERENCES BETWEEN .0S AND OTHER MOLECULES .0S HAVE CONSIDERABLY LESS NITROGENS AND MORE OXYGENS THAN THE COM POUNDS FROM THE TWO OTHER SETS /-S HAVE ON AVERAGE NITROGENS AND OXYGENS WHILE AN AVERAGE "- HAS NITROGENS AND OXYGENS AND AN AVERAGE .0 MOLECULE HAS ONLY NITROGENS AND OXYGENS TAKING INTO ACCOUNT ALSO SUGAR SUBSTITUENTS 4HE NUMBER OF AROMATIC RINGS IN MOLECULE IS ALSO ONE OF THE MOST DISTINCT FEATURES SEPARATING .0S FROM OTHER CLASSES OF MOLECULES 7HILE OF .0S DO NOT CONTAIN ANY AROMATIC RING THIS FRACTION IS FOR BIOACTIVE MOLECULES AND ONLY FOR ORGANIC MOLECULES 4HE DISTRIBUTIONS OF CHIRAL CENTERS SHOW AS EXPECTED THAT .0S EXHIBIT MUCH HIGHER STEREO VARIABILITY THAN OTHER CLASSES AN AVERAGE .0 HAS STEREOCENTERS WHILE "-S HAVE AND SYNTHETIC MOLECULES ONLY STEREOCENTERS RESPECTIVELY 4HE RESULTS OF OUR ANALYSIS ARE SIMILAR TO CONCLUSIONS FROM PAPER ;= IN OUR STUDY HOWEVER WE USED A MUCH LARGER COLLECTION OF .0 MOLECULES COMPARED WITH USED IN STUDY ;= n OUR RESULTS MAY THERE FORE BE CONSIDERED MORE GENERAL
$ISTRIBUTION OF NATURAL PRODUCTS BIOACTIVE MOLECULES AND ORGANIC MOLECULES IN #HEMISTRY 3PACE )N ORDER TO VISUALIZE THE DISTRIBUTIONS OF NATURAL PRODUCTS DRUG LIKE BIOAC TIVE MOLECULES AND COMMON ORGANIC MOLECULES IN STRUCTURAL CHEMISTRY SPACE WE APPLIED A METHOD CALLED PRINCIPAL COMPONENTS ANALYSIS 0#! ;= 0#! IS A DATA PROCESSING TECHNIQUE USED TO SIMPLIFY COMPLEX MULTIDI
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
&IGURE $ISTRIBUTION OF NATURAL PRODUCTS GREEN BIOACTIVE MOLECULES BLUE AND COMMON ORGANIC MOLECULES ORANGE IN STRUCTURE SPACE FIRST TWO PRINCIPAL COMPONENTS OF THE FRAGMENT MATRIX !REAS WITH MIXED OCCUPANCY ARE GRAY
MENSIONAL DATA BY REDUCING THEIR DIMENSIONALITY $URING THE PROCESS THESE DATA ARE @SQUEEZED INTO A FEW DIMENSIONS CALLED PRINCIPAL COMPONENTS USUALLY ONLY TWO ALLOWING THE DATA TO DISPLAYED AS A TWO DIMENSIONAL MAP AND SUBSEQUENT EASY VISUAL ANALYSIS /F COURSE WHEN REDUCING DIMENSION ALITY SOME INFORMATION IS LOST BUT 0#! TYPICALLY MINIMIZES THE LOSS OF INFORMATION FOR A GIVEN NUMBER OF DIMENSIONS &OR THIS STUDY MOLECULES WERE CHARACTERIZED BY SIMPLE ONE AND TWO ATOMIC SUBSTRUCTURE FRAGMENTS THEY CONTAIN 6ERY RARE FRAGMENTS PRESENT IN LESS THAN OF MOLECULES WERE NOT CONSIDERED 4HE ORIGINAL DATA MATRIX WITH COLUMNS REPRESENTING FRAGMENT FREQUENCIES WAS NOR
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MALIZED AND THEN REDUCED INTO TWO DIMENSIONS BY THE 0#! PROCEDURE 4HE RESULTS ARE DISPLAYED IN &IGURE AS A GRID MAP 4HE GRID SQUARES EACH OF THEM CONTAINING SEVERAL THOUSAND MOLECULES ARE COLOR CODED ACCORD ING TO THE DOMINANT CLASS IE THE CLASS TO WHICH MORE THAN OF MOL ECULES IN THIS GRID BELONG )F NONE OF THE CLASSES IS DOMINANT THAT SQUARE HAS GRAY COLOR 4HE AXIS OF THE GRAPH THE TWO MOST IMPORTANT PRINCIPAL COMPONENTS REPRESENTS A COMPLEX MIX OF VARIOUS SUBSTRUCTURAL FRAGMENTS 4HE WHOLE GRAPH MAY THEREFORE BE INTERPRETED AS A SIMPLIFIED VIEW OF THE CHEMICAL STRUCTURAL SPACE 4HE MOST INTERESTING CONCLUSION WHICH CAN BE MADE BASED ON THIS ANALYSIS IS A CLEAR SEPARATION BETWEEN THE THREE CLASSES OF MOLECULES .0S GREEN OCCUPY A WELL DEFINED COMPACT REGION OF STRUCTURE SPACE WITH SEVERAL OUTLIERS TOP OF THE GRAPH CORRESPONDING TO VERY LARGE STRUCTURES MOLECULAR WEIGHT !LSO SYNTHETIC MOLECULES ORANGE OCCUPY A COMPACT PART OF STRUCTURE SPACE WELL SEPARATED FROM .0S 4HE SPACE OCCUPIED BY BIOACTIVE MOLECULES BLUE IS LESS DEFINED ONE CAN SEE THAT BIOACTIVITY IS SCATTERED OVER THE STRUCTURE SPACE ALTHOUGH IN MOST CASES @COVERED BY COMPACT AREA OF /-S OR .0S RESPECTIVELY 0ARTICULARLY INTER ESTING FOR DRUG DESIGN IS THE @GRAY REGION IN THE MIDDLE OF THE GRAPH ON THE INTERSECTION OF SPACE OCCUPIED BY .0S "-S AND /-S 4HIS IS A SPACE WHICH IS SYNTHETICALLY WELL ACCESSIBLE WITH MOLECULES CONTAINING .0 LIKE STRUCTURAL FEATURES AND ALSO POPULATED BY BIOACTIVE MOLECULES AND THERE FORE WELL SUITED AS A SOURCE OF INSPIRATION FOR THE DESIGN OF $/3 LIBRARIES OR .0 LIKE BIOACTIVE MOLECULES
2ING SYSTEMS AND SUBSTITUENTS TYPICAL FOR NATURAL PRODUCTS )N THIS PART OF THE STUDY WE ANALYZE THE SUBSTRUCTURAL FEATURES RING SYSTEMS AND SUBSTITUENTS WHICH ARE MOST TYPICAL FOR .0S AND COMPARING THEM WITH THOSE OF COMMON /-S 4HE RING SYSTEM OR SCAFFOLD IS DEFINED HERE AS A SINGLE RING OR A COLLECTION OF FUSED OR SPIRO RINGS ALSO INCLUDING EXOCYCLIC MULTIPLE BONDS ! SUBSTITUENT IS DEFINED AS A PART OF A MOLECULE THAT IS CON NECTED BY A CHEMICALLY ACTIVATED BREAKABLE NON RING SINGLE BOND TO THE REST OF THE MOLECULE ;= 4HE MAXIMUM SIZE OF SUBSTITUENTS WAS LIMITED TO NON HYDROGEN ATOMS
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
&IGURE %XAMPLE OF SEVERAL GLYCOSYLATED FORMS OF THE SAME AGLYCON
!S DISCUSSED EARLIER A DEGLYCOSYLATION STEP WAS PERFORMED BEFORE EXTRACT ING SCAFFOLDS AND SUBSTITUENTS OTHERWISE PYRANOSE AND FURANOSE RINGS AND RESPECTIVE SUBSTITUENTS WOULD BE IDENTIFIED AS THE DOMINANT .0 FEATURES )N THE DEGLYCOSYLATION STEP TO OVER SUGAR UNITS WERE REMOVED FROM MOLECULES !S AN EXAMPLE EIGHT MOLECULES FROM THE DATABASE SHAR ING THE SAME AGLYCON ARE DISPLAYED IN &IGURE 3OME AGLYCONS SHOWED VERY HIGH VARIATION IN GLYCOSYLATION PATTERNS HAVING SEVERAL HUNDREDS GLYCOSYLATED FORMS
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4HE RESULTS FROM THE SUBSTRUCTURAL ANALYSIS ARE SHOWN IN &IGURES n ! COMPARISON OF THE MOST COMMON SCAFFOLDS FOUND IN .0S &IG AND THE MOST COMMON SCAFFOLDS IDENTIFIED IN /-S &IG REVEALS CLEAR DIFFERENCES BETWEEN BOTH SETS 4HE MOST COMMON .0 RINGS CONTAIN TOGETHER OXYGEN ATOMS FOUR NITROGEN ATOMS AND NO SULFUR ATOMS SEVEN RING SYS TEMS ARE HYDROCARBONS WITHOUT ANY HETEROATOMS )N THE ORGANIC RING SET THERE ARE TOGETHER NITROGEN ATOMS SEVEN OXYGEN AND SEVEN SULFUR ATOMS AND ONLY FOUR HYDROCARBON RINGS /RGANIC MOLECULES CONTAIN ABOUT TWO TIMES AS MUCH FULLY CONJUGATED RING SYSTEMS AS .0S VERSUS !LTHOUGH THIS SIMPLE COMPARISON WAS MADE ONLY FOR THE MOST COMMON RING SYS TEMS FROM BOTH SETS IT REFLECTS QUITE WELL THE GENERAL DIFFERENCES BETWEEN THE .0S AND ORGANIC MOLECULES AND RESULTS ARE ALSO SIMILAR WHEN ANALYZING A LARGER FRACTION OF COMMON SCAFFOLDS ! COMPARISON OF THE MOST COMMON SUBSTITUENTS FOUND IN .0S &IG AND THE MOST COMMON SUBSTITUENTS IDENTIFIED IN AVERAGE /-S &IG REVEALS SOME OVERLAP BETWEEN THESE TWO SETS BUT ALSO MANY DIFFERENCES /UT OF THE SUBSTITUENTS IN BOTH SETS ARE COMMON FROM THE MOST COMMON SUBSTITUENTS ARE IN BOTH SETS -ANY ORGANIC SUBSTITUENTS ARE DERIVATIVES OF SIMPLE AROMATIC RINGS WHILE IN .0 SUBSTITUENTS ONE CAN SEE SEVERAL UNSATURATED ACYCLIC CARBOHYDRATE SIDECHAINS OF TERPENE ORIGIN SUCH AS VINYL ISOPROPENYL OR METHYL BUT ENYL 4HE RESULTS OF THIS ANALYSIS OF SCAFFOLDS AND SUBSTITUENTS MAY BE USED DIRECTLY FOR THE DESIGN OF .0 LIKE BIOACTIVE MOLECULES 4YPICAL .0 SUBSTITU ENTS OR THEIR DERIVATIVES IN THE PROPER REACTIVE STATE COMBINED WITH SIM PLER .0 SCAFFOLDS MAY BE USED AS REAGENTS IN DRUG OPTIMIZATION PROCESSES OR AS BUILDING BLOCKS IN THE DESIGN OF COMBINATORIAL LIBRARIES
$IFFERENCES AMONG NATURAL PRODUCTS ACCORDING TO THEIR ORIGIN )N THE LAST PART OF THE STUDY OF .0 MOLECULES WE IDENTIFIED RING SYSTEMS WHICH ARE TYPICAL FOR A PARTICULAR CLASS OF SOURCE ORGANISMS !S MENTIONED IN THE METHODOLOGY SECTION .0S WERE CLASSIFIED ACCORDING TO THEIR ORIGIN INTO FOUR GROUPS PRODUCTS OF PLANTS ANIMALS FUNGI AND BACTERIA 4HE GROUP OF .0S EXTRACTED FROM PLANTS IS THE LARGEST ENTRIES FOLLOWED BY .0S FROM ANIMALS BACTERIA AND FUNGI 4HIS DISTRIBUTION
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
&IGURE 4HE MOST COMMON RING SYSTEMS FOR NATURAL PRODUCTS
&IGURE 4HE MOST COMMON RING SYSTEMS FOR ORGANIC MOLECULES
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&IGURE 4HE MOST COMMON SUBSTITUENTS FOR NATURAL PRODUCTS
&IGURE 4HE MOST COMMON SUBSTITUENTS FOR ORGANIC MOLECULES
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
REFLECTS THE FACT THAT FOR A LONG TIME THE MAIN SOURCES OF NATURAL PRODUCTS WERE PLANT EXTRACTS ,ATER PRODUCTS FROM BACTERIA AND FUNGI MAINLY THANKS TO THEIR ANTIBIOTIC ACTIVITY GAINED POPULARITY 4HE VERY PROMISING AND FAST GROWING FIELD CURRENTLY IS ISOLATION OF .0S FROM MARINE INVERTEBRATES /NE REASON IS AN ENORMOUS DIVERSITY OF MARINE ECOSYSTEMS PROVIDING A SOURCE OF A LARGE NUMBER OF NOVEL DIVERSE .0 STRUCTURES !NOTHER REASON IS THE FACT THAT MARINE .0S WHEN APPLIED BY THEIR PRODUCERS AS @CHEMICAL WARFARE USUALLY DILUTES RAPIDLY IN WATER AND THEREFORE THEY NEED TO BE VERY POTENT 3EVERAL MARINE .0S HAVE BEEN MARKETED AS DRUGS RECENTLY AND SEVERAL OTH ERS ARE IN CLINICAL TRIALS ;= 4HE TYPICAL RING SYSTEMS FOR THESE FOUR CLASSES OF .0S ARE SHOWN IN &IG URES n 4HESE RINGS WERE IDENTIFIED NOT AS THE MOST FREQUENT FOR PARTICU LAR CLASS OF ORGANISMS BUT AS THE @MOST TYPICAL IE HAVING THE MAXIMUM DIFFERENCE IN FREQUENCIES BETWEEN THE SET OF .0S OF PARTICULAR ORIGIN AND OTHER .0S &OR EACH CLASS ONLY REPRESENTATIVE EXAMPLES ARE SHOWN BUT EVEN BY COMPARING THIS RELATIVELY SMALL NUMBER OF STRUCTURES ONE CAN SEE INTERESTING DIFFERENCES /NLY LIMITED NUMBER OF SCAFFOLDS ISOLATED FROM PLANTS ARE ALKALOIDS WHICH INSPIRED THE DISCOVERY OF MANY SYNTHETIC DRUG SUBSTANCES ACTING ON AMINERGIC ' PROTEIN COUPLED RECEPTORS AND FUSED CARBOCYCLES ARE MORE TYPICAL FOR THIS CLASS -ACROCYCLES ARE TYPICAL MOSTLY FOR BACTERIAL PRODUCTS BUT PRESENT ALSO IN ANIMAL AND FUNGAL METABOLITES 4HE FUNGI .0S CONTAIN SEVERAL AND MEMBERED RINGS 4HE ONLY SET CON TAINING SULFUR IS BACTERIAL METABOLITES 7E DO NOT HAVE THE SPACE TO PRESENT A MORE DETAILED ANALYSIS OF THESE DIFFERENCES THIS WILL THEREFORE BE A TOPIC OF SEPARATE STUDY
#ONCLUSIONS 7E HOPE THAT THE RESULTS OF THIS ANALYSIS WILL HELP TO ELIMINATE THE OLD MYTH ABOUT .0S AS BEING @TOO COMPLEX OR HAVING @BAD PROPERTIES /F COURSE THE STRUCTURE OF MOLECULES PRODUCED BY NATURE IS NOT SIMPLE ESPE CIALLY WHEN COMPARED WITH EASY TO PREPARE SYNTHETIC MOLECULES OR SOME FIRST GENERATION DRUGS IN SOME CASES EVEN ORIGINATED FROM AROMATIC DYES ;= 3TRUCTURAL DIVERSITY AND FEATURE RICHNESS OF .0S HOWEVER SHOULD BE TAKEN NOT AS A DRAWBACK BUT AS AN ADVANTAGE 4HE PROPER APPLICATION OF CHEMINFORMATICS TECHNIQUES CAN HELP US TO SIMPLIFY THE STRUCTURES ;=
0ETER %RTL AND !NSGAR 3CHUFFENHAUER
&IGURE 4YPICAL SCAFFOLDS OF NATURAL PRODUCTS PRODUCED BY PLANTS
&IGURE 4YPICAL SCAFFOLDS OF NATURAL PRODUCTS PRODUCED BY ANIMALS
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
&IGURE 4YPICAL SCAFFOLDS OF NATURAL PRODUCTS PRODUCED BY BACTERIA
&IGURE 4YPICAL SCAFFOLDS OF NATURAL PRODUCTS PRODUCED BY FUNGI
0ETER %RTL AND !NSGAR 3CHUFFENHAUER
AND AS ILLUSTRATED IN THE PRESENT ANALYSIS ASSIST IN FOCUSING ON THESE AREAS OF STRUCTURAL SPACE AND SUBSTRUCTURE FEATURES WHICH ARE ESSENTIAL FOR BIO LOGICAL ACTIVITY TAKING ADVANTAGE OF THE PROCESS OF NATURAL SELECTION OVER BILLIONS OF YEARS GUIDING US TO NEW AND AS YET UNEXPLORED AREAS OF THE #HEMICAL 3TRUCTURE 5NIVERSE
!CKNOWLEDGEMENTS 4HE AUTHORS WOULD LIKE TO THANK THEIR COLLEAGUES &RANK 0ETERSEN AND 3ILVIO 2OGGO FROM THE .OVARTIS .ATURAL 0RODUCTS 5NIT AS WELL AS 0ROF (ERBERT 7ALDMANN FROM THE -AX 0LANCK )NSTITUTE OF -OLECULAR 0HYSIOLOGY AT $ORTMUND AND ALSO 3TEFAN 7ETZEL 3TEFFEN 2ENNER AND -ARCUS +OCH FROM HIS GROUP FOR MANY INSPIRING DISCUSSIONS CONCERNING THE FASCINATING AREA OF NATURAL PRODUCTS 7E THANK ALSO OUR COLLEAGUE .ATHAN "ROWN FOR CRITICAL READING THE MANUSCRIPT AND HELPFUL COMMENTS
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.EWMAN $* #RAGG '- 3NADER +- .ATURAL PRODUCTS AS SOURCES OF NEW DRUGS OVER THE PERIOD n * .AT 0ROD n 2OUHI !- 2EDISCOVERING NATURAL PRODUCTS #HEM %NG .EWS n 2OUHI !- "ETTING ON NATURAL PRODUCTS FOR CURES #HEM %NG .EWS n +UBINYI ( $RUG RESEARCH MYTHS HYPE AND REALITY .ATURE 2EV $RUG $ISC n 3CHREIBER 3, 4ARGET ORIENTED AND DIVERSITY ORIENTED ORGANIC SYNTHESIS IN DRUG DISCOVERY 3CIENCE n 4AN $3 $IVERSITY ORIENTED SYNTHESIS EXPLORING THE INTERSECTIONS BETWEEN CHEM ISTRY AND BIOLOGY .ATURE #HEM "IOL n +INGSTON $ .EWMAN $ -OTHER NATURES COMBINATORIAL LIBRARIES THEIR INFLUENCE ON THE SYNTHESIS OF DRUGS #URR /PIN $RUG $ISC $EV n "REINBAUER 2 -ANGER - 3CHECK - 7ALDMANN ( .ATURAL 0RODUCT 'UIDED #OM POUND ,IBRARY $EVELOPMENT #URR -ED #HEM n &IRN 2$ *ONES #' .ATURAL PRODUCTS n A SIMPLE MODEL TO EXPLAIN CHEMICAL DIVER SITY .AT 0ROD 2EP n (ENKEL 4 "RUNNE 2- -ßLLER ( 2EICHEL & 3TATISTICAL INVESTIGATION INTO THE STRUC TURAL COMPLEMENTARITY OF NATURAL PRODUCTS AND SYNTHETIC COMPOUNDS !NGEW #HEM )NT %D n 3TAHURA &, 'ODDEN *7 8UE , "AJORATH * $ISTINGUISHING BETWEEN NATURAL PROD UCTS AND SYNTHETIC MOLECULES BY DESCRIPTOR 3HANNON ENTROPY ANALYSIS AND BINARY 13!2 CALCULATIONS * #HEM )NF #OMPUT 3CI n
#HEMINFORMATICS ANALYSIS OF NATURAL PRODUCTS
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3CIENCES AND $EPARTMENT OF -OLECULAR AND -EDICAL 'ENETICS 4ERRENCE $ONNELLY #ENTRE FOR #ELLULAR AND "IOMOLECULAR 2ESEARCH 5NIVERSITY OF 4ORONTO #OLLEGE 3TREET 4ORONTO #ANADA -3%
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
!BSTRACT $ETERMINING THE MODE OF ACTION OF BIOACTIVE COMPOUNDS INCLUDING NATURAL PRODUCTS IS A CENTRAL PROBLEM IN CHEMICAL BIOLOGY "ECAUSE MANY GENES ARE CONSERVED FROM THE YEAST 3AC CHAROMYCES CEREVISIAE TO HUMANS AND A NUMBER OF POWERFUL GENOMICS TOOLS AND METHODOLOGIES HAVE BEEN DEVELOPED FOR THIS MODEL SYSTEM YEAST IS MAKING A MAJOR CONTRIBUTION TO THE FIELD OF CHEMICAL GENETICS 4HE SET OF BARCODED YEAST DELETION MUTANTS INCLUDING THE SET OF ^ VIABLE HAPLOID AND HOMOZYGOUS DIPLOID DELETION MUTANTS AND THE COMPLETE SET OF ^ HETEROZYGOUS DELETION MUTANTS CONTAINING THE SET OF ^ ESSENTIAL GENES ARE PROVING HIGHLY INFORMATIVE FOR IDENTIFYING CHEMICAL GENETIC INTERACTIONS AND DECIPHERING COMPOUND MODE OF ACTION 'ENE DELETIONS THAT RENDER CELLS HYPERSENSITIVE TO A SPECIFIC DRUG IDENTIFY PATHWAYS THAT BUFFER THE CELL AGAINST THE TOXIC EFFECTS OF THE DRUG AND THEREBY PROVIDE CLUES ABOUT BOTH GENE AND COMPOUND FUNCTION -OREOVER COMPOUNDS THAT SHOW SIMILAR CHEMI CAL GENETIC PROFILES OFTEN PERTURB SIMILAR TARGET PATHWAYS 'ENE DOSAGE CAN BE EXPLOITED TO DISCOVER CONNECTIONS BETWEEN COMPOUNDS AND THEIR TARGETS &OR EXAMPLE HAPLOINSUFFICIENCY PROFILING OF AN ANTIFUNGAL COMPOUND IN WHICH THE SET OF ^ HETEROZYGOUS DIPLOID DELE TION MUTANTS ARE SCORED FOR HYPERSENSITIVITY TO A COMPOUND MAY IDENTIFY THE TARGET DIRECTLY #REATING DELETION MUTANT COLLECTIONS IN OTHER FUNGAL SPECIES INCLUDING THE MAJOR HUMAN FUNGAL PATHOGEN #ANDIDA ALBICANS WILL EXPAND OUR CHEMICAL GENOMICS TOOL SET ALLOWING US TO SCREEN FOR ANTIFUNGAL LEAD DRUGS DIRECTLY 4HE YEAST DELETION MUTANT COLLECTION IS ALSO BEING EXPLOITED TO MAP LARGE SCALE GENETIC INTERACTION DATA OBTAINED FROM GENOME WIDE SYNTHETIC LETHAL SCREENS AND THE INTEGRATION OF THIS DATA WITH CHEMICAL GENETIC DATA SHOULD PROVIDE A POWERFUL SYSTEM FOR LINKING COMPOUNDS TO THEIR TARGET PATHWAY %XTENSIVE APPLICATION OF CHEMICAL GENETICS IN YEAST HAS THE POTENTIAL TO DEVELOP A SMALL MOLECULE INHIBITOR FOR THE MAJORITY OF ALL ^ YEAST GENES
)NTRODUCTION &ROM THE EARLIEST ATTEMPTS TO FORMULATE THERAPEUTICS NATURAL PRODUCTS HAVE PLAYED KEY ROLES IN THE TREATMENT OF HUMAN DISEASE "ETWEEN AND OF ALL THE NEW CHEMICAL ENTITIES .#%S APPROVED DRUGS BY THE 53 &OOD AND $RUG !DMINISTRATION &$! WERE NATURAL PRODUCTS AND ANOTHER WERE DERIVED FROM NATURAL PRODUCTS ;= (ISTORICALLY BIOACTIVE COMPOUNDS OFTEN ENTERED THE CLINIC IE WERE ADMINISTERED TO PATIENTS BEFORE THEIR MODES OF ACTION WERE UNDERSTOOD 4HIS AD HOC APPROACH IS NO LONGER PURSUED AND IN CONTRAST TODAYS DRUG DISCOVERY EFFORTS SEEK TO ELUCIDATE THE COMPOUNDS MECHANISM OF ACTIVITY AND TO IDENTIFY ITS TARGET PATHWAY )T IS COMMONLY ACCEPTED THAT NATURAL PRODUCTS ARE WELL SUITED FOR MOD ULATING CELLULAR PATHWAYS BECAUSE THEY ARE THE PRODUCT OF HIGHLY SELECTIVE EVOLUTIONARY PRESSURES WHICH TAILOR THE ACTIVITY OF THE COMPOUND FOR
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MODULATION OF FUNDAMENTAL CELLULAR PATHWAYS AND THEIR CORRESPONDING PROCESSES 4HE CONSERVATION OF SIGNAL TRANSDUCTION CASCADES MEANS THAT TOXINS ORIGINALLY DESIGNED TO KILL COMPETING MICROORGANISMS MIGHT SERVE TO MODULATE OR DISRUPT SPECIFIC DISEASE RELEVANT PROCESSES IN HUMANS &OR EXAMPLE #YCLOSPORINE ! INHIBITS CALCINEURIN SIGNALING IN BOTH YEAST AND HUMANS AND FUNCTIONS AS A POTENT IMMUNOSUPPRESSANT ;= )N THE PHARMACEUTICAL INDUSTRY NATURAL PRODUCTS RESEARCH DECLINED DUR ING THE LAST DECADE ;= IN FAVOR OF APPROACHES DEEMED MORE COMPATIBLE WITH HIGH THROUGHPUT SCREENING OF SYNTHETIC COMBINATORIAL LIBRARIES (OWEVER THE LARGE SCALE CHEMICAL SYNTHESIS THAT FEEDS HIGH THROUGHPUT SCREENING REQUIRES RELATIVELY SIMPLE CHEMISTRY WHICH LIMITS BIOACTIVITY AND COMPRO MISES SPECIFICITY #ONSEQUENTLY DRUG DISCOVERY GROUPS ARE NOW EXPRESSING A RENEWED INTEREST IN NATURAL PRODUCTS -OREOVER IMPROVEMENTS IN THE ISOLATION CHARACTERIZATION AND THE SYNTHESIS OF NATURAL AND SEMI NATURAL PRODUCTS HAVE ADDED TO INTEREST IN THIS AREA ;= 4HE IDEA THAT NATURE HAS SELECTED SPECIFIC BIOACTIVE COMPOUNDS THROUGH EVOLUTION MIGHT SUGGEST THAT OUR MAJOR TASK IS TO REFINE THE STRUCTURES TO ACHIEVE HIGHER POTENCY &OR EXAMPLE .ICOLAOU AND CO WORKERS SYNTHESIZED A LIBRARY OF COMPOUNDS RELATED TO THE MARINE NATURAL PRODUCT SARCODICTYIN MANY OF THESE ANALOGS SHOWED GREATER TOXICITIES COMPARED TO THE PARENT COMPOUND ;= 4HE PRIMARY CHALLENGES DECIPHERING THE MECHANISM OF ACTIVITY OF A BIO ACTIVE COMPOUND AND IDENTIFYING ITS CELLULAR TARGET ARE FAR FROM RESOLVED AND THIS GENERAL TASK REMAINS A MAJOR HURDLE FOR MODERN DRUG DISCOVERY -ODEL ORGANISMS WITH WELL DEFINED MOLECULAR AND GENETIC METHODOLOGY OFFER A NUMBER OF TOOLS AND APPROACHES FOR TACKLING THE MODE OF ACTION PROBLEM )N PARTICULAR STUDIES WITH BAKERS YEAST 3ACCHAROMYCES CEREVISIAE HAVE ENABLED SEVERAL IMPORTANT NATURAL PRODUCT DISCOVERIES &OR EXAMPLE OUR UNDERSTANDING OF THE MECHANISM BY WHICH CAMPTOTHECIN A COM POUND DERIVED FROM THE #HINESE ORNAMENTAL TREE #AMPTOTHECA ACUMINATA INHIBITS YEAST $.! TOPOISOMERASES ) OR )) HAS BEEN INSTRUMENTAL IN ILLU MINATING EUKARYOTIC $.! SYNTHESIS AND REPAIR PATHWAYS ;= 4HE 3TREPTO MYCES METABOLITE TUNICAMYCIN HAS BEEN ANALYZED EXTENSIVELY USING YEAST REVEALING NUMEROUS ASPECTS OF THE ROLE PROTEIN GLYCOSYLATION INCLUDING PIVOTAL ROLES IN PROTEIN FOLDING AND TRAFFICKING ;= !RTEMISININ A COM POUND DERIVED FROM A WELL KNOWN PLANT IN #HINESE TRADITIONAL MEDICINE !RTEMISIA ANNUA WORMWOOD IS A SESQUITERPENE ENDOPEROXIDE WITH POTENT ANTIMALARIAL EFFECTS AND CLUES ABOUT ITS MODE OF ACTION HAVE ONLY RECENTLY
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
BEEN REVEALED BY STUDIES WITH YEAST SHOWING THAT ARTEMISININS INHIBITORY ACTIVITY IS MEDIATED BY DISRUPTING THE NORMAL FUNCTION OF MITOCHONDRIA ;= -OREOVER YEAST STRAINS HAVE BEEN ENGINEERED TO PRODUCE ARTEMISININS DRUG PRECURSOR ARTEMISINIC ACID IN HIGH TITERS WHICH MAY LEAD TO AN INEX PENSIVE AND LARGE SCALE PRODUCTION OF THIS MOLECULE ;= 3TUDIES OF YEAST MUTANTS RESISTANT TO THE MICROBIAL PRODUCT RAPAMYCIN ORIGINALLY ISOLATED AS AN ANTIFUNGAL AGENT LED TO THE DISCOVERY OF THE WELL CONSERVED 4/2 SIG NALING PATHWAY ;= 4ODAY RAPAMYCIN IS USED AS AN IMMUNOSUPPRESSANT AND IT IS THE SUBJECT OF INTENSE STUDY FOR ALTERNATIVE THERAPEUTIC USES ;= /NE OF THE MOST EFFECTIVE ANTIFUNGAL DRUGS CURRENTLY AVAILABLE IS A LIPOPEP TIDE ECHINOCANDIN CALLED CASPOFUNGIN 9EAST GENETIC SCREENS WERE CRITICAL FOR THE IDENTIFICATION OF β GLUCAN SYNTHASE ENCODED BY &+3 AND &+3 AS THE CASPOFUNGIN TARGET ;n= 4HE STEROID RELATED WORTMANNIN ORIGI NALLY FOUND IN THE FUNGUS 4ALOROMYCES WORTMANNI HAS PROVED INCREDIBLY VALUABLE FOR DISSECTING SIGNAL TRANSDUCTION PATHWAYS DESPITE THE FACT THAT THE PHARMACOLOGY OF THIS NATURAL PRODUCT REMAINS A PUZZLE BECAUSE OF THE MULTIPLICITY OF TARGETS IN BOTH MAMMALIAN AND YEAST CELLS ;= 4HUS THE CONSERVATION OF GENE PRODUCTS AND THEIR CORRESPONDING PATHWAYS FROM YEAST TO HUMANS COMBINED WITH THE NATURAL SELECTION OF BIOACTIVE COM POUNDS THAT MODULATE THESE PATHWAYS MAKES EUKARYOTIC MICROORGANISMS VALUABLE MODEL SYSTEMS FOR MODE OF ACTION STUDIES
"UDDING YEAST AS A MODEL ORGANISM 4HE BUDDING YEAST 3 CEREVISIAE REPRESENTS A CORNERSTONE IN BIOLOGICAL RESEARCH BECAUSE IT IS PROBABLY THE MOST HIGHLY CHARACTERIZED EUKARYOTIC ORGANISM 4HE POWER OF TRADITIONAL YEAST MOLECULAR GENETIC APPROACHES AND THE NEWER GENOMICS APPROACHES HAVE FACILITATED THE FUNCTIONAL ANALY SIS OF THE MAJORITY OF YEAST GENES ;= )N MOST CASES YEAST GENETICS OR FUNC TIONAL GENOMICS APPROACHES CAN BE ADAPTED FOR CHEMICAL GENETIC ANALYSIS IN WHICH AN ORGANISM IS SUBJECTED TO BOTH CHEMICAL AND GENETIC PERTURBA TIONS 4HE 3EATTLE PROJECT PIONEERED A CHEMICAL GENETIC APPROACH IN WHICH 3 CEREVISIAE WAS USED IN HIGH THROUGHPUT SCREENS DESIGNED TO IDENTIFY NOVEL CANCER THERAPEUTICS IN PARTICULAR A PANEL OF NATURAL PRODUCTS AND SYNTHETIC COMPOUNDS FROM THE .ATIONAL #ANCER )NSTITUTE COLLECTION WERE SCREENED AGAINST YEAST MUTANTS DEFECTIVE FOR $.! REPLICATION RECOMBINATION REPAIR
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OR CELL CYCLE CHECKPOINT FUNCTION ;= 4HE RATIONALE BEHIND THIS APPROACH WAS THAT SPECIFIC MUTATIONS CONTRIBUTING TO NEOPLASTIC MALIGNANCY WOULD LEAD TO TUMOR SPECIFIC COMPOUND SENSITIVITY -ORE GENERALLY YEAST HAS MANY ATTRIBUTES THAT MAKE IT ATTRACTIVE FOR CHEMICAL GENETIC ANALYSIS )N PARTICULAR IT IS A UNICELLULAR ORGANISM WITH A COMPACT GENOME OF APPROXIMATELY GENES ;= AND A LIFE CYCLE WELL SUITED TO CLASSICAL GENETIC STUDIES -OREOVER MANY CELLULAR PROCESSES ARE CONSERVED FROM YEAST TO HUMANS ESPECIALLY THE COMPONENTS OF THE PATH WAYS INVOLVED IN FUNDAMENTAL CELLULAR METABOLISM AND CELL DIVISION ;= )N FACT SEQUENCING OF YEAST ;= AND HUMAN GENOMES ; = REVEALED THAT THOUSANDS OF YEAST PROTEINS SHARE AMINO ACID SEQUENCE SIMILARITY WITH A CORRESPONDING HUMAN PROTEIN ;= MAKING YEAST AN ATTRACTIVE MODEL FOR THE STUDY OF HUMAN DISEASES ;= &OR INSTANCE THE COMPOUNDS FUMAGIL LIN AND OVALICIN WHICH ARE POTENT ANGIOGENESIS INHIBITORS ; = WERE DISCOVERED ORIGINALLY IN THE FUNGI !SPERGILLUS FUMIGATUS AND 0SEUDOROTIUM OVALIS RESPECTIVELY "OTH COMPOUNDS BIND SPECIFICALLY TO TYPE METHIO NINE AMINOPEPTIDASE -ET!0 AND INHIBIT ITS ENZYMATIC ACTIVITY THROUGH A MECHANISM CONSERVED FROM YEAST TO HUMANS ;= 4HE DEVELOPMENT OF FUNCTIONAL GENOMICS AND PROTEOMICS HAS USH ERED IN A NEW ERA OF YEAST RESEARCH 7ITH THE FIRST COMPLETELY SEQUENCED EUKARYOTIC GENOME ;= YEAST RESEARCHERS HAVE HAD A HEAD START IN THE DEVELOPMENT AND APPLICATION OF FUNCTIONAL GENOMIC METHODOLOGIES FOR SYSTEMS LEVEL BIOLOGY 4HERE ARE A NUMBER OF GENOMIC TOOLS AVAILABLE FOR GLOBAL ANALYSIS OF YEAST GENE FUNCTION AND MOST OF THESE TOOLS ARE ALSO RELEVANT TO THE STUDY OF BIOACTIVE COMPOUNDS INCLUDING GENOME WIDE TWO HYBRID CLONE SETS ; = PROTEOME CHIPS ;= GENOME WIDE MICRO ARRAYS ;= COMPREHENSIVE COLLECTIONS OF EPITOPE TAGGED YEAST STRAINS ;= AND A COMPLETE SET OF BARCODED DELETION MUTANT STRAINS ;= 4O DATE GENE EXPRESSION ; = PROTEINnPROTEIN INTERACTIONS BY YEAST TWO HYBRID ; = AND MASS SPECTROPHOTOMETRY ANALYSIS OF PROTEIN COMPLEXES ;n= PROTEIN ABUNDANCE ;= AND LOCALIZATION ;= AND GENETIC INTERAC TIONS ;n= HAVE BEEN STUDIED GENOME WIDE IN YEAST 3EVERAL DATABASES CATALOG YEAST GENES PROTEINS AND INTERACTIONS INCLUDING THE 3ACCHARO MYCES 'ENOME $ATABASE 3'$ ;= THE #OMPREHENSIVE 9EAST 'ENOME $ATABASE #9'$ AT -)03 ;= THE 'ENERAL 2EPOSITORY FOR )NTERACTION $ATASETS '2)$ ;= ALLOWING YEAST RESEARCHERS EASY ACCESS TO A WEALTH OF INFORMATION
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
3 CEREVISIAE GENE DELETION MUTANT COLLECTION 4HE 3ACCHAROMYCES GENOME DELETION PROJECT CARRIED OUT BY AN INTERNA TIONAL CONSORTIUM OF LABORATORIES CREATED DELETION MUTANTS FOR ALMOST ALL ANNOTATED YEAST /2&S ; = 4HE COLLECTION CONTAINS HETEROZYGOUS DIP LOID STRAINS CORRESPONDING TO DELETIONS IN EACH OF THE ^ YEAST GENES INCLUDING THE ^ ESSENTIAL GENES AS WELL AS A HOMOZYGOUS DIPLOID AND BOTH A -!4A AND A -!4α HAPLOID MUTANT STRAIN FOR EACH OF THE ^ NON ESSENTIAL GENES /F ALL THE NEW GENOMIC RESOURCES UNDER DEVELOPMENT AND IN USE IN 3 CEREVISIAE PERHAPS THE MOST RELEVANT TOOL FOR COMPOUND MODE OF ACTION STUDY IS THE YEAST DELETION COLLECTION BECAUSE IT ENABLES LARGE SCALE CHEMICAL GENETIC SCREENS ;= IN WHICH GENOME WIDE SETS OF MUTANTS ARE MONITORED FOR SENSITIVITY TO SPECIFIC COMPOUNDS
#HEMICAL GENETICS 4HE USE OF CHEMICAL PROBES FOR INTERROGATING MOLECULAR MECHANISMS IN LIVING ORGANISMS HAS A LONG HISTORY 7ITH THE ADVENT OF GENOMIC TECHNOLO GIES THIS PRACTICE HAS ACQUIRED A DISCIPLINE STATUS AND IS NOW REFERRED TO AS CHEMICAL GENETICS 3MALL MOLECULES ARE ATTRACTIVE PROBES FOR BIOLOGISTS BECAUSE THEY OFFER TEMPORAL CONTROL AND THE ABILITY TO MODULATE PROCESSES RAPIDLY )N YEAST COMPREHENSIVE GENOME WIDE SCREENS OF SETS OF VIABLE DELETION MUTANT STRAINS FOR DRUG SENSITIVITY @CHEMICAL GENETIC PROFILING HAVE ESTABLISHED AN INDEX OF DIAGNOSTIC CHEMICAL GENETIC INTERACTIONS WHICH ARE USEFUL FOR DEFINING BOTH GENE AND CHEMICAL FUNCTION ;= 7ITH THE SET OF ^ VIABLE DELETION MUTANTS IN HAND CREATING CHEMI CAL GENETIC PROFILES IS A RELATIVELY SIMPLE AND STRAIGHTFORWARD PROCESS AND THE USE OF THIS MUTANT COLLECTION HAS GENERATED A WEALTH OF NEW INFORMA TION ABOUT COMPOUND ACTIVITIES )NDEED SHORTLY AFTER THE RELEASE OF THE MUTANT COLLECTION A NUMBER OF GROUPS HAVE SCREENED THE SET OF ^ VIABLE DELETION MUTANTS FOR DRUG HYPERSENSITIVITY TO UNCOVER MOST OF THE PATHWAYS AND CELLULAR FUNCTIONS AFFECTED BY DRUG TREATMENT ;= /NE OF THE FIRST CHEMICAL GENETIC PROFILING PROJECTS ;= WAS AIMED AT STUDYING THE 4/2 PROTEINS IN YEAST SIGNALING MOLECULES IMPLICATED IN TRANSLATIONAL CONTROL ACTIN REORGANIZATION AND OTHER MORE OBSCURE FUNCTIONS 3IMILARLY ANOTHER GROUP ;= EXAMINED THE SET OF ^ DELETION MUTANTS FOR THOSE
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AFFECTING RESISTANCE TO THE IMMUNOSUPPRESSIVE MYCOPHENOLIC ACID -0! BY INDIVIDUALLY SCREENING STRAINS IN A WELL LIQUID FORMAT APPROXIMATELY NEW DELETION MUTANTS AFFECTING -0! RESISTANCE WERE IDENTIFIED "EN NETT ET AL SCREENED OVER HOMOZYGOUS DIPLOID MUTANTS AND IDENTIFIED NEW LOCI SENSITIVE TO IONIZING RADIATION ;= 4HESE STRAINS WERE THEN SCREENED AGAINST A NUMBER OF $.! DAMAGING AGENTS INCLUDING 56 LIGHT BLEOMYCIN --3 (5 AND CAMPTOTHECIN AND NEARLY OF THE MUTANTS WERE FOUND TO BE SENSITIVE TO AT LEAST ONE OF THE DRUGS
'ENE DOSAGE AS MEANS OF LINKING A COMPOUND TO ITS TARGET (APLOINSUFFICIENCY PROFILING WITH ESSENTIAL HETEROZYGOUS DIPLOID GENE DELETION MUTANTS (APLOINSUFFICIENCY DESCRIBES THE PHENOMENA WHEN A DIPLOID ORGANISM WITH ONLY ONE FUNCTIONAL COPY OF A GENE DOES NOT PRODUCE SUFFICIENT GENE PRODUCT TO DISPLAY A WILD TYPE PHENOTYPE (APLOINSUFFICIENCY IS RARE UNDER NORMAL CONDITIONS ;= AND THIS PHENOMENON CAN BE EXPLOITED TO GAIN INSIGHTS INTO A BIOACTIVE COMPOUNDS MODE OF ACTION ; = "ECAUSE THE MINIMAL INHIBITORY CONCENTRATION OF AN INHIBITOR IS DEPENDENT ON THE TARGET CONCENTRATION EXPRESSING HALF THE LEVEL OF AN ESSENTIAL PROTEIN WILL IN GENERAL RENDER YEAST CELLS MORE SENSITIVE TO AN INHIBITOR OF THAT PROTEIN THAN CELLS HAVING THE FULL DIPLOID COMPLEMENT ;= !N IMPORTANT COROL LARY IS THAT MUTANTS HETEROZYGOUS AT ONE ESSENTIAL LOCUS REMAIN DIPLOID AT ALL OTHERS SUCH THAT THE HYPERSENSITIVITY SHOWN BY A GIVEN HETEROZYGOTE IS SPECIFIC TO COMPOUNDS THAT INHIBIT THE REDUCED GENE PRODUCT 4HUS HETEROZYGOTES GENERALLY DISPLAY SELECTIVE HYPERSENSITIVITY TO COMPOUNDS THAT INHIBIT THEIR REDUCED PROTEIN PRODUCTS AND THEREBY LINK COMPOUNDS DIRECTLY TO THEIR TARGET PROTEINS &UNCTIONAL GENOMICS ALLOWS THE FULL POTEN TIAL OF HAPLOINSUFFICIENCY TO BE APPLIED TO DRUG SCREENING ENABLING THE SIMULTANEOUS SCREENING OF A COMPOUND AGAINST ALL CELLULAR TARGETS USING A POOLED SET OF BARCODED HETEROZYGOUS DELETION MUTANTS )N PARTICULAR PARALLEL ANALYSIS OF HETEROZYGOUS DIPLOIDS FOR COMPOUND SENSITIVITY OR HAPLOINSUFFICIENCY PROFILING IS USEFUL FOR STUDYING ESSENTIAL GENE TARGETS
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
BECAUSE COMPOUNDS THAT INHIBIT THE FUNCTION OF ESSENTIAL GENE TARGETS LEAD TO A PRONOUNCED GROWTH DEFECT EVEN AT RELATIVELY LOW CONCENTRATIONS 'IAEVER ET AL FIRST DEMONSTRATED THAT HAPLOINSUFFICIENCY PROFILING CAN BE USED TO MONITOR COMPOUND ACTIVITIES IN VIVO 3PECIFICALLY THEY IDENTI FIED THE KNOWN TARGET IN A MIXED CULTURE OF STRAINS IN THE PRESENCE OF THE DRUG TUNICAMYCIN ;= 4HIS APPROACH WAS EXTENDED BY ,UM ET AL TO ANALYZE THE ACTIVITIES OF CHEMICAL ENTITIES OF MEDICAL INTEREST IN HETERO ZYGOTES REPRESENTING ABOUT HALF OF THE YEAST GENOME ;= "Y USING A STRAIN SPECIFIC ERROR MODEL DRUG DEPENDENT GROWTH DEFECTS WERE DISTINGUISHED FROM GENERAL ONES &URTHER THEY CHOSE TWO EXAMPLES FOR IN DEPTH CHARAC TERIZATION OVEREXPRESSION OF %2' AND BIOCHEMICAL ANALYSES OF PATHWAY INTERMEDIATES INDICATED THAT %RGP MAY BE THE TARGET OF MOLSIDOMINE AND THE DRUG &5 BLOCKS R2.! PROCESSING BY THE EXOSOME ;= )N ANOTHER STUDY BY 'IAEVER ET AL CHEMICALS WERE SCREENED IN GENOME WIDE EXPERIMENTS AGAINST THE COMPLETE SET OF HETEROZYGOUS YEAST DELETION STRAINS IDENTIFYING PREVIOUSLY KNOWN INTERACTIONS ;= &OLLOWING THIS METHODOLOGY IT IS POSSIBLE IN PRINCIPLE TO TAKE ANY BIOACTIVE COM POUND THAT INHIBITS A YEAST PROTEIN AND IDENTIFY ITS TARGET &OR INSTANCE "AETZ ET AL ;= USED THE DRUG INDUCED HAPLOINSUFFICIENCY ASSAY TO DEMON STRATE THAT A SYNTHETIC ANALOG OF AN ALKALOID ISOLATED FORM MARINE SPONGES MOTUPORAMINE ;= TARGETS THE SPHINGOLIPID METABOLISM $ORER ET AL ;= IDENTIFIED THE PROTEIN KINASE -PS AS THE TARGET OF CINCREASIN WHICH IS REQUIRED FOR CHECKPOINT FUNCTION &OLLOWING THIS METHODOLOGY IT IS POS SIBLE IN PRINCIPLE TO TAKE ANY BIOACTIVE COMPOUND THAT INHIBITS AN ESSENTIAL YEAST PROTEIN AND IDENTIFY ITS TARGET ;n=
,IMITATIONS OF INDUCED HAPLOINSUFFICIENCY METHOD "ESIDES THE OBVIOUS CRITERION THAT THE DRUG AFFECTS THE GROWTH RATE OF THE CELL IN ORDER TO BE ASSAYED COMPOUNDS THAT EXERT THEIR EFFECTS THROUGH DIRECT INTERACTION WITH NON PROTEIN ELEMENTS OF THE CELL MAY NOT BE SUITABLE FOR HAPLOINSUFFICIENCY PROFILING &OR EXAMPLE THE ANTINEOPLASTIC AGENT CISPLATIN INHIBITS CELL PROLIFERATION BY COVALENTLY BINDING TO THE $.! . POSITION OF PURINES AND USING HAPLOINSUFFICIENCY PROFILING 'IAEVER AND COLLEAGUES FAILED TO IDENTIFY HETEROZYGOUS STRAINS THAT WERE HYPERSENSITIVE TO CISPLATIN )N CONTRAST CHEMICAL GENETIC PROFILING WITH THE SET OF ^
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VIABLE DIPLOID HOMOZYGOUS DELETION MUTANTS IDENTIFIED A NUMBER OF GENES THAT ARE RELEVANT TO CISPLATIN MODE OF ACTION
)DENTIFICATION OF DRUG TARGETS BY GENE DOSAGE RESISTANCE !S DESCRIBED ABOVE A POWERFUL METHOD FOR IDENTIFYING DRUG TARGETS INVOLVES MODULATION OF GENE DOSAGE 4HE PRINCIPLE OF THIS APPROACH IS THAT CELLS WITH HIGHER AMOUNTS OF A DRUG TARGET WILL TOLERATE HIGHER LEVELS OF THE DRUG IE FOR THE CASE OF METHOTREXATE IN HUMAN CELLS ;= )N YEAST A LIBRARY OF PLASMIDS THAT OVEREXPRESS GENES CAN BE SCREENED FOR THOSE THAT CONFER RESISTANCE TO THE DRUG OF INTEREST ;= 4HE MOLECULAR TARGETS OF SEVERAL KNOWN ANTIFUNGAL COMPOUNDS INCLUDING TUNICAMYCIN ;= AND FLUCON AZOLE ;= HAVE BEEN IDENTIFIED USING THIS APPROACH "UTCHER ET AL USED GENE OVEREXPRESSION IN COMBINATION WITH A MICROARRAY BASED DETECTION METHOD TO DETERMINE THE TARGET OF RAPAMYCIN ;= /NE LIMITATION OF THESE APPROACHES IS THE FACT THAT OVEREXPRESSION OF NON TARGET PROTEINS SUCH AS OTHER MEMBERS OF THE TARGET PATHWAY OR EFFLUX PUMPS CAN ALSO CONFER DRUG RESISTANCE ;= )N ADDITION IT IS POSSIBLE THAT OVEREXPRESSION OF THE TARGET GENE COMPROMISES CELLULAR FITNESS WHICH MAY PRECLUDE IT FROM DOSAGE RESISTANCE ANALYSIS ;=
,ARGE SCALE CHEMICAL GENETIC ANALYSIS 4HE YEAST DELETION COLLECTION ALLOWS COMPREHENSIVE SCORING OF GROWTH PHENOTYPES BECAUSE EVERY DELETION CONTAINS A UNIQUE BARCODE IDENTIFIER %ACH GENE DELETION MUTATION IS MARKED BY TWO UNIQUE MER NUCLEO TIDE SEQUENCES WHICH ARE FLANKED BY COMMON 0#2 PRIMER SITES ; = 4HESE @MOLECULAR BARCODES ENABLE THE IDENTIFICATION OF EACH STRAIN WITHIN A MIXED POPULATION OF MUTANTS THROUGH 0#2 BASED AMPLIFICATION OF THE BARCODES FOLLOWED BY A MICROARRAY BASED QUANTITATIVE DETECTION ;= 4HIS HIGHLY PARALLEL TECHNIQUE IS PARTICULARLY USEFUL FOR FITNESS TESTING OF LARGE NUMBERS OF MUTANTS IN SMALL VOLUMES OF MEDIA )N THIS STRATEGY STRAINS ARE POOLED AND GROWN IN PARALLEL IN LIQUID CULTURE UNDER SELECTIVE CONDITIONS FOR EXAMPLE IN THE PRESENCE OF A SMALL AMOUNT OF A GROWTH INHIBITORY COMPOUND 'ENOMIC $.! IS EXTRACTED FROM THE POOL AND THE BARCODES
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
FROM EACH STRAIN IN THE POOL ARE AMPLIFIED SIMULTANEOUSLY USING COMMON FLANKING PRIMERS THEN HYBRIDIZED TO HIGH DENSITY $.! MICROARRAYS CON TAINING OLIGONUCLEOTIDES CORRESPONDING TO THE BARCODES 5LTIMATELY THE RELATIVE ABUNDANCE OF EACH STRAIN IN THE POOL IS ASSESSED BY THE STRENGTH OF THE RESULTING SIGNALS FROM THE MICROARRAY READOUT COMPARED TO A MOCK CONTROL 5SING THIS APPROACH THE ENTIRE YEAST DELETION SET CAN BE POOLED GROWN COMPETITIVELY AND ASSESSED QUANTITATIVELY IN ONLY A FEW MILLILITERS OF GROWTH MEDIUM ;= &IG 5SING THE PARALLEL FITNESS TESTS AND BARCODE MICROARRAY ANALYSIS 'IAEVER AND COLLEAGUES PROFILED THE NEARLY COMPLETE SET OF 3 CEREVISIAE NON ESSEN TIAL DELETION STRAINS AS HOMOZYGOUS DIPLOIDS UNDER A NUMBER OF DIFFERENT CONDITIONS INCLUDING HIGH SALT SORBITOL GALACTOSE P( MINIMAL MEDIA AND NYSTATIN TREATMENT ;= ! SIMILAR METHOD WAS USED TO SCREEN THE YEAST GENOME FOR DELETION MUTANTS AFFECTED BY TWO PROTEOSOME INHIBITORS HERE STRAINS WERE IDENTIFIED AS HYPERSENSITIVE TO THE DRUGS ;= ! NUMBER OF GROUPS HAVE USED THIS STRATEGY TO SCREEN THE YEAST DELETION MUTANT COL LECTION FOR DRUG HYPERSENSITIVITY TO UNCOVER THE PATHWAYS AND CELLULAR FUNCTIONS AFFECTED BY DRUG TREATMENT ON A GLOBAL SCALE 'IAEVER ET AL ;= ALSO SCREENED THE DELETION MUTANT COLLECTION FOR SENSITIVITY TO CISPLATIN AN ANTINEOPLASTIC AGENT THAT INHIBITS CELL PROLIFERATION BY MODIFYING PURINES IDENTIFYING A NUMBER OF GENES INVOLVED IN $.! REPAIR 3IMILARLY (ANWAY AND COLLEAGUES ;= ANALYZED A COLLECTION OF YEAST DELETION STRAINS FOR 56 AND --3 SENSITIVITY AND IMPLICATED SIX GENES NOT PREVIOUSLY KNOWN TO BE INVOLVED IN $.! DAMAGE REPAIR PATHWAYS IN THE $.! DAMAGE RESPONSE 4HE COMPLETE HOMOZYGOUS DELETION COLLECTION HAS ALSO BEEN SCREENED FOR SENSITIVITY TO CISPLATIN AN ANTINEOPLASTIC AGENT THAT INHIBITS CELL PROLIFERA TION BY MODIFYING PURINES IDENTIFYING A COLLECTION OF GENES INVOLVED IN $.! SYNTHESIS AND REPAIR ;= ! CHEMICAL GENETIC INTERACTION WHERE A PARTICULAR YEAST MUTANT IS HYPERSENSITIVE TO A SPECIFIC INHIBITORY COMPOUND PROVIDES IMPORTANT CLUES TO THE MECHANISM OF DRUG ACTION 'ENE DELETIONS THAT RENDER CELLS HYPERSENSITIVE TO A SPECIFIC COMPOUND IDENTIFY PATHWAYS THAT BUFFER THE CELL AGAINST THE TOXIC EFFECTS OF THE DRUG ;= #HEMICAL GENETIC PROFILING WITH THE BARCODE APPROACH IS HIGHLY SENSITIVE BECAUSE A MODEST REDUC TION IN GROWTH RATE LEADS TO A SIGNIFICANT SIGNAL THEREFORE EVEN MODERATE SENSITIVITIES CAN BE IDENTIFIED MANY OF WHICH MAY BE RELEVANT TO DRUG MODE OF ACTION BUT MISSED IN TRADITIONAL SCREENS "ECAUSE COMPOUNDS WITH
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&IGURE "ARCODE CHEMICAL GENETIC PROFILING !N ALIQUOT OF THE POOL OF THE -!4A HAPLOID DELETION COLLECTION IS INCUBATED AT # IN THE PRESENCE OF DRUG OR SOLVENT CONTROL IN M, OF 90$ DURING GENERATIONS 'ENOMIC $.! IS EXTRACTED FROM THE POOL AND THE BARCODES FROM EACH STRAIN IN THE POOL ARE AMPLIFIED SIMULTANEOUSLY USING COMMON FLANKING PRIMERS THEN HYBRIDIZED TO HIGH DENSITY $.! MICROARRAYS CONTAINING OLIGONUCLEOTIDES COR RESPONDING TO THE BARCODES 5LTIMATELY THE RELATIVE ABUNDANCE OF EACH STRAIN IN THE POOL IS ASSESSED BY THE STRENGTH OF THE RESULTING SIGNALS FROM THE MICROARRAY READOUT COMPARED TO A MOCK CONTROL
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
SIMILAR BIOLOGICAL EFFECTS SHOULD LEAD TO SIMILAR CHEMICAL GENETIC PROFILES A COMPENDIUM OF CHEMICAL GENETIC PROFILES SHOULD PROVIDE INSIGHTS INTO THE STRUCTURAL AND FUNCTIONAL RELATIONSHIPS OF THE COMPOUNDS ! DETAILED COMPENDIUM OF CHEMICAL GENETIC INTERACTION PROFILES WILL ALSO ORGANIZE THE YEAST GENES INTO FUNCTIONAL PATHWAYS BECAUSE MUTANTS THAT ARE SENSI TIVE TO SIMILAR SETS OF COMPOUNDS HAVE HIGHLY SIMILAR PHENOTYPES AND THUS ARE LIKELY TO OCCUR WITHIN THE SAME CELLULAR PATHWAY
4HE COMPENDIUM APPROACH )DENTIFYING CELLULAR TARGETS 2ECENTLY A COMPENDIUM APPROACH WAS UNDERTAKEN BY 0ARSONS ET AL ;= WHERE SYNTHETIC COMPOUNDS AND NATURAL PRODUCTS OF WHICH ARE &$! APPROVED AND SEVEN CRUDE ANTIFUNGAL EXTRACTS DERIVED FROM DIFFERENT MARINE SPONGES AND MICROORGANISMS WERE SCREENED AGAINST THE HOMOZY GOUS MUTANT DELETION COLLECTION 4HE SIMILARITIES OF THE CHEMICAL GENETIC PROFILES WERE STATISTICALLY ANALYZED BY CLUSTERING ANALYSIS EITHER HIERARCHI CAL CLUSTERING &IG OR BY PROBABILISTIC SPARSE MATRIX FACTORIZATION 03-& ;= &IG 4HE COMPENDIUM APPROACH ALSO IDENTIFIED A LARGE SET OF MULTIDRUG RESISTANCE -$2 GENES ; = WHICH WERE REMOVED PRIOR TO CLUSTERING 4HE -$2 GENES INCLUDE %2' %2' AND %2' WHICH ENCODE GENES INVOLVED IN ERGOSTEROL BIOSYNTHESIS AS WELL AS 0$2 WHICH ENCODES A DRUG EFFLUX PUMP AND 0$2 ENCODING A TRANSCRIPTION FACTOR THAT REGULATES THE EXPRESSION OF GENES ENCODING DRUG EFFLUX PUMPS AND A RELATIVELY LARGE COLLECTION OF GENES INVOLVED IN VACUOLE TRAFFICKING AND FUNCTION WHICH SUGGESTS THAT THE VACUOLE MAY FUNCTION AS A MAJOR INTRACELLULAR SINK FOR BIOACTIVE COMPOUNDS 4HE RESULTS OF THE COMPENDIUM ANALYSIS ;= RECAPITULATED OF THE KNOWN BIOLOGY ABOUT THE DIFFERENT CHEMICAL PROBES USED INCLUDING NUMEROUS EXAMPLES OF NATURAL PRODUCTS &OR EXAMPLE THE ACTIN BINDING AGENTS LATRUN CULIN " ;= AND CYTOCHALASIN ! ;= CLUSTER TOGETHER CLUSTER TWO CELL WALL SYNTHESIS INHIBITORS STAUROSPORINE WHICH TARGETS 0ROTEIN KINASE # AND CASPOFUNGIN WHICH INHIBITS β GLUCAN SYNTHASE ;= CLUSTER TOGETHER CLUSTER CLOTRIMAZOLE AND FLUCONAZOLE CHEMICAL ANALOGS AND ANTIFUNGAL AGENTS THAT TARGET %RG ; = A PROTEIN ENCODED BY AN ESSENTIAL GENE IN THE ERGOSTEROL BIOSYNTHESIS PATHWAY CLUSTER TOGETHER CLUSTER RADICICOL AND GELDANAMYCIN ALTHOUGH STRUCTURALLY UNRELATED BOTH ACT AS HIGHLY
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&IGURE 4WO DIMENSIONAL HIERARCHICAL CLUSTERING ANALYSIS OF CHEMICAL GENETIC PROFILES CONDITIONS INCLUDING COMPOUNDS AND CRUDE EXTRACTS WERE CLUSTERED GENES ARE PLOTTED ON THE HORIZONTAL AXIS WITH THE GENE CLUSTER TREE ABOVE #OMPOUNDS ARE PLOTTED ON THE VERTICAL AXIS WITH
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
SELECTIVE INHIBITORS OF (SP FUNCTION THROUGH THEIR ABILITY TO BIND WITHIN THE !$0!40 BINDING POCKET OF THE CHAPERONE ;= FORM A UNIQUE CLUSTER CLUSTER BENOMYL ;= AND NOCODAZOLE ;= TWO MICROTUBULE POISONS ARE GROUPED CLUSTER WITHIN CLUSTER CONTAINING CLOMIPHENE FENPROPI MORPH HALOPERIDOL DYCLONINE ABIETIC ACID AND FLUFENAMIC ACID CLUSTER THREE OF THESE COMPOUNDS FENIPROPIMORPH ;= HALOPERIDOL ;= AND DYCLONINE ;= ARE ALL THOUGHT TO INHIBIT %RG FUNCTION IN YEAST #LUSTERING OF THE %RG INHIBITORS WITH ABIETIC ACID A NATURAL TERPENOID FOUND IN PINE RESIN AND FLUFENAMIC ACID A SYNTHETIC ANTI INFLAMMATORY AGENT SUGGESTS THAT ABIETIC ACID AND FLUFENAMIC ACID MAY ALSO INHIBIT ERGOSTEROL BIOSYNTHE SIS AT THE LEVEL OF %RG )NTERESTINGLY CLOTRIMAZOLE AND FLUCONAZOLE WHICH TARGET %RG A PROTEIN THAT FUNCTIONS UPSTREAM OF %RG IN THE ERGOSTEROL BIOSYNTHESIS PATHWAY CLUSTER SEPARATELY FROM THE %RG INHIBITORS REFLECT ING DIFFERENCES IN THE PROFILES BETWEEN THE TWO GROUPS OF COMPOUNDS AND SUGGESTING THAT CHEMICAL GENETIC PROFILING CAN DIFFERENTIATE BETWEEN UNIQUE PROTEIN TARGETS WITHIN THE SAME PATHWAY 4HERE ARE ADVANTAGES IN USING DIFFERENT CLUSTERING ALGORITHMS TO ANA LYZE CHEMICAL GENETIC PROFILES )N HIERARCHICAL CLUSTERING EACH MUTANT HAS A CHEMICAL SENSITIVITY SIGNATURE IN RESPONSE TO A SET OF COMPOUNDS AND MUTANTS ARE LINKED TOGETHER BASED ON A COMPARISON OF ALL COMPOUNDS 4HUS HIERARCHICAL CLUSTERING IS SATISFYING BECAUSE IT PROVIDES AN ORDERED VIEW OF THE ENTIRE DATASET )N CONTRAST PROBABILISTIC SPARSE MATRIX FACTOR IZATION 03-& ANALYSIS IS USEFUL BECAUSE THIS METHOD CAN PLACE A GIVEN CHEMICAL GENETIC PROFILE OR A SUBSET OF THE PROFILE IN MORE THAN ONE GROUP 7HEN 0-3& ANALYSIS WAS APPLIED TO THE 0ARSONS ET AL ;= COMPENDIUM DATASET IT AFFORDED A FACTOR WHERE $.! DAMAGING AGENTS IE MITOMYCIN
THE CLUSTER TREE ON THE OUTERMOST RIGHT SIDE OF THE PLOT #OMPOUNDS CLUSTERS ARE LABELED WITH NUMERALS AS REFERENCED IN THE TEXT #LUSTER THE ACTIN BINDING AGENTS LATRUNCULIN " AND CYTOCHALASIN ! #LUSTER TWO CELL WALL SYNTHESIS INHIBITORS STAUROSPORINE AND CASPOFUNGIN WHICH INHIBITS β GLUCAN SYNTHASE #LUSTER CLOTRIMAZOLE AND FLUCONAZOLE CHEMICAL ANALOGS AND ANTIFUNGAL AGENTS THAT TARGET %RG #LUSTER RADICICOL AND GELDANAMYCIN ALTHOUGH STRUCTURALLY UNRELATED BOTH ACT AS HIGHLY SELECTIVE INHIBITORS OF (SP FUNCTION THROUGH THEIR ABILITY TO BIND WITHIN THE !$0!40 BINDING POCKET OF THE CHAPERONE #LUSTER BENOMYL AND NOCODAZOLE TWO MICROTUBULE POISONS #LUSTER CLOMIPHENE FEN PROPIMORPH HALOPERIDOL DYCLONINE ABIETIC ACID AND FLUFENAMIC ACID #LUSTER MARINE CRUDE EXTRACTS FROM A SEA CUCUMBER FROM THE #OMMONWEALTH OF $OMINICA AND EXTRACT DERIVED FROM AN )NDONESIAN MARINE SPONGE AND THEIR ISOLATED BIOACTIVE PRODUCTS STICHLOROSIDE # AND THEOPALAU AMIDE RESPECTIVELY #LUSTER CELL MEMBRANE DISRUPTORS 0APUAMIDE " ALAMETHICIN CINNAMYCIN AND HIGROMYCIN " 4HIS FIGURE WAS ADAPTED FROM &IGURE IN 0ARSONS ET AL ;=
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&IGURE #LUSTERING ANALYSIS OF CHEMICALnGENETIC PROFILES BY PROBABILISTIC SPARSE MATRIX FACTORIZATION 03-& %ACH NUMBERED BLOCK ALONG THE DIAGONAL SHOWS THE DATA THAT CONTRIBUTED TO A PARTICULAR FACTOR AND DISPLAYS THE MUTANTS AND COMPOUNDS DISCOVERED BY 03-& 4HE STATISTICALLY SIGNIFICANT MUTANTS UTILIZING EACH FACTOR ARE SHOWN ALONG THE VERTICAL AXIS AND ARE SORTED ACCORDING TO THE WEIGHTING OF THE FACTOR IN EXPLAINING THE CHEMICAL SENSITIVITY PROFILE FOR THE MUTANT 4HE SUBSET OF COMPOUNDS SIGNIFICANTLY PRESENT IN EACH FACTOR ARE SHOWN ALONG THE HORIZONTAL AXIS AND ARE SORTED ACCORDING TO THEIR IMPORTANCE WITHIN THE FACTOR #OMPOUNDS AND STRAINS MAY APPEAR SEVERAL TIMES #OLOR BAR ON THE LEFT INDICATES THE SCALE OF THE NORMALIZED DATA !N EXPANDED VIEW OF SEVERAL BLOCKS ARE DETAILED ! FACTOR NUMBER " FACTOR # FACTOR $ FACTOR %ACH EXPANDED VIEW SHOWS THE EIGHT MOST IMPORTANT COMPOUNDS AND THE MOST SIGNIFICANT MUTANTS 4HIS FIGURE WAS ADAPTED FROM &IGURE IN 0ARSONS ET AL ;=
# --3 CAMPTOTHECIN CISPLATIN AND HYDROXYUREA ARE CLUSTERING IN A SINGLE FACTOR THAT ALSO INCLUDES THE ANTIBIOTIC ACTINOMYCIN A GROUPING THAT IS NOT OBSERVED BY HIERARCHICAL CLUSTERING .EVERTHELESS ACTINOMYCIN INDEED BINDS TO $.! AND INHIBITS 2.! SYNTHESIS ;= AND SO ITS MODE OF ACTION WAS REVEALED BY 0-3& CLUSTERING SPECIFICALLY )N A SECOND EXAMPLE VERRUCARIN AND NEOMYCIN SULFATE ARE TWO COMPOUNDS LINKED BY 03-& ANALYSIS AND NOT BY HIERARCHICAL CLUSTERING 6ERRUCARIN INHIBITS PROTEIN
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
SYNTHESIS IN YEAST ;= AND NEOMYCIN SULFATE IS AN AMINOGLYCOSIDE ANTIBI OTIC THAT INHIBITS PROTEIN TRANSLATION ;= AND THUS THIS CLUSTERING MAKES SENSE FUNCTIONALLY
.ATURAL PRODUCTS EXTRACTS %STABLISHING PRIORITIES IN NATURAL CHEMICAL EXTRACTS CHEMICAL ANALYSIS USING CHEMICAL GENETIC PROFILING "ECAUSE CHEMICAL GENETIC PROFILING FOCUSES ON COMPOUNDS THAT IMPAIR CELL GROWTH IT CAN ALSO BE APPLIED TO NATURAL PRODUCT EXTRACTS #ONSEQUENTLY CRUDE CHEMICAL EXTRACTS CAN ALSO BE SUBJECTED TO CHEMICAL GENETIC PROFILING AND IF THE CRUDE EXTRACT ONLY CONTAINS ONE MAJOR ANTIFUNGAL COMPOUND THEN ITS CHEMICAL GENETIC PROFILE WILL BE HIGHLY SIMILAR TO THAT OF THE PURI FIED COMPOUND ALLOWING FOR FUNCTIONAL ANALYSIS OF THE EXTRACT PRIOR TO PURIFICATION 0ARSONS ET AL STUDIED CRUDE CHEMICAL EXTRACTS FROM MARINE SPONGES WITH ANTI FUNGAL ACTIVITY AND COMPARED THEM TO THEIR ISOLATED BIO ACTIVE COMPONENTS )N THE TWO CASES EXAMINED THE ISOLATED COMPOUNDS STICHLOROSIDE # ;= AND THEOPALAUAMIDE ;= DISPLAYED CHEMICAL GENETIC PROFILES HIGHLY SIMILAR TO THOSE OF THEIR CRUDE EXTRACTS &IG #LUSTER CONFIRMING THAT THESE PURIFIED COMPOUNDS ARE LIKELY RESPONSIBLE FOR THE MAJORITY OF THE ANTIFUNGAL ACTIVITY IN THE EXTRACTS 4HIS FINDING IS PARTICU LARLY IMPORTANT AS THIS ASSAY BECOMES A POWERFUL TOOL PRIORITIZING EXTRACTS ENABLING US TO ASSESS THEIR BIOLOGICAL ACTIVITY BEFORE UNDERTAKING THE COSTLY AND TIME CONSUMING TASK OF ISOLATING AND IDENTIFYING ACTIVE COMPONENTS IN A COMPLEX CHEMICAL MIXTURE 3URPRISINGLY THE TWO DIFFERENT EXTRACTS CONTAINING STICHLOROSIDE # ;= AND THEOPALAUAMIDE ;= WHICH WERE DERIVED FROM DIFFERENT GEOGRAPHICAL LOCATIONS AND FROM DIFFERENT ORGANISMS SHOWED VERY SIMILAR CHEMICAL GENETIC PROFILES AND THEY CLUSTERED TOGETHER WITHIN THE COMPENDIUM OF PROFILES SEE #LUSTER &IG SUGGESTING THEY CONTAIN COMPOUNDS WITH SIMILAR MODES OF ACTION 4HIS RESULT IS EVEN MORE SURPRISING BECAUSE STI CHLOROSIDE # AND THEOPALAUAMIDE HAVE ENTIRELY DISSIMILAR CHEMICAL STRUC TURES .EVERTHELESS FURTHER ANALYSIS OF DRUG RESISTANT MUTANTS SUGGESTS THAT STICHLOROSIDE # AND THEOPALAUAMIDE KILL 3 CEREVISIAE CELLS THROUGH A COMMON MECHANISM BECAUSE MUTANTS THAT ARE RESISTANT TO THEOPALAUA
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MIDE SPECIFICALLY ARE ALSO RESISTANT TO STICHLOROSIDE # SPECIFICALLY 4HUS CHEMICAL GENETIC PROFILING LINKED EXTRACTS CONTAINING COMPOUNDS THAT ARE FUNCTIONALLY SIMILAR BUT CHEMICALLY DISSIMILAR
#OMPARING SYNTHETIC LETHAL GENETIC INTERACTIONS AND CHEMICAL GENETIC INTERACTIONS 3YNTHETIC 'ENETIC !RRAY 3'! ANALYSIS 4HE YEAST HAPLOID DELETION MUTANT SET HAS ALLOWED THE DEVELOPMENT OF A HIGH THROUGHPUT AND GENOME WIDE METHOD TO IDENTIFY AND STUDY GENETIC INTERACTIONS IN 3 CEREVISIAE ; = 4HIS SYSTEM REFERRED TO AS SYNTHETIC GENETIC ARRAY 3'! ANALYSIS ALLOWS THE AUTOMATED CONSTRUCTION OF DOUBLE MUTANTS THEREBY ENABLING LARGE SCALE GENETIC INTERACTION SCREENS FOR SYN THETIC LETHAL ANALYSIS AND PHENOTYPIC ENHANCEMENT 3YNTHETIC LETHAL INTER ACTIONS ARE DEFINED BY TWO GENES THAT ARE NON ESSENTIAL WHEN INDIVIDUALLY DELETED BUT ARE ESSENTIAL WHEN COMBINED AS A DOUBLE MUTANT )N 3'! A SERIES OF ROBOTIC PINNING STEPS ARE USED TO CROSS THE ORDERED ARRAY OF -!4A DELETION MUTANTS TO A -!4α STARTING STRAIN CONTAINING A QUERY MUTATION OF INTEREST &IG 4HE RESULTANT DIPLOIDS ARE SELECTED FOR AND PINNED ONTO SPORULATION MEDIUM THEN DOUBLE MUTANT MEIOTIC PROGENY ARE ISOLATED BY SELECTIVE GROWTH AND SCORED FOR FITNESS DEFECTS 4HE KEY TO THIS SYSTEM WAS THE DEVELOPMENT OF AN 3'! REPORTER -&!PR ()3 WHICH PLACES THE ()3 SELECTABLE MARKER UNDER THE CONTROL OF A -!4A SPECIFIC REPORTER AND ENABLES THE GERMINATION OF -!4A MEIOTIC PROGENY FROM A POPULATION CONTAINING BOTH -!4A AND -!4α SPORES "ECAUSE FITNESS DEFECTS OF DOUBLE MUTANTS CAN ALSO BE EXAMINED USING THE BARCODE APPROACH WITH A MICROARRAY BASED READOUT 3'! ANALYSIS CAN ALSO BE CARRIED OUT USING A POOLED POPULATION OF DELETION MUTANTS -OREOVER RATHER THAN CROSSING A MUTATION OF INTEREST INTO THE SET OF ^ VIABLE DELETION MUTANTS THE QUERY MUTATION CAN BE INTRODUCED BY TRANSFORMATION INTO HETEROZYGOUS DIPLOIDS CONTAINING THE 3'! REPORTER A METHOD REFERRED TO AS D3,!- ;= 'ENETIC INTERACTIONS ARE GENERALLY ORTHOGONAL NON OVERLAPPING WITH PROTEINnPROTEIN INTERACTIONS BUT TWO DIMENSIONAL HIERARCHICAL CLUSTERING OF THE INTERACTIONS REVEALS THAT GENES WITHIN THE SAME PATHWAY OR COMPLEX OFTEN SHOW SIMILAR PATTERNS OF
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
&IGURE 3YNTHETIC GENETIC ARRAY ANALYSIS 3YNTHETIC GENETIC ARRAY METHODOLOGY I ! -!4α STRAIN CARRYING A QUERY MUTATION BNIΔ LINKED TO A DOMINANT SELECTABLE MARKER SUCH AS THE NOURSEOTHRICIN RESISTANCE MARKER NAT-8 THAT CONFERS RESISTANCE TO THE ANTIBIOTIC NOURSEOTHRICIN AND AN -&!PR ()3 REPORTER IS CROSSED TO AN ORDERED ARRAY OF -!4A VIABLE YEAST DELETION MUTANTS EACH CARRYING A GENE DELETION MUTATION LINKED TO A KANAMYCIN RESISTANCE MARKER KAN-8 'ROWTH OF RESULTANT HETEROZYGOUS DIPLOIDS IS SELECTED FOR ON MEDIUM CONTAINING NOURSEOTHRICIN AND KANAMYCIN II 4HE HETEROZYGOUS DIPLOIDS ARE TRANSFERRED TO MEDIUM WITH REDUCED LEVELS OF CARBON AND NITROGEN TO INDUCE SPORULATION AND THE FORMATION OF HAP LOID MEIOTIC SPORE PROGENY III 3PORES ARE TRANSFERRED TO SYNTHETIC MEDIUM LACKING HISTIDINE WHICH ALLOWS FOR SELECTIVE GERMINATION OF -!4A MEIOTIC PROGENY BECAUSE THESE CELLS EXPRESS THE -&!PR ()3 REPORTER SPECIFICALLY IV 4HE -!4A MEIOTIC PROGENY ARE TRANSFERRED TO MEDIUM THAT CONTAINS BOTH NOURSEOTHRICIN AND KANAMYCIN WHICH THEN SELECTS FOR GROWTH OF DOUBLE MUTANT MEIOTIC PROGENY 4HIS FIGURE WAS ADAPTED FROM &IGURE IN 4ONG ET AL ;=
GENETIC INTERACTIONS #LUSTERING OF PREVIOUSLY UNCHARACTERIZED GENES WITH WELL STUDIED PATHWAYS ENABLES THE PREDICTION OF PRECISE MOLECULAR ROLES ! COMPLETE MAP OF SYNTHETIC GENETIC INTERACTIONS FOR YEAST IS IN THE MAKING
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AND IS EXPECTED TO IDENTIFY SETS OF COMPLEXES AND PATHWAYS THAT BUFFER EACH OTHER OR WORK TOGETHER AS BACKUP SYSTEMS
#OMPARING SYNTHETIC GENETIC AND CHEMICAL GENETIC INTERACTIONS "ECAUSE A LOSS OF FUNCTION MUTATION IN A GENE ENCODING THE TARGET OF AN INHIBITORY MODELS THE PRIMARY EFFECT OF THE COMPOUND ;= INTRODUCING SUCH A MUTATION INTO THE SET OF VIABLE MUTANTS AND SCORING THE RESULTANT DOUBLE MUTANTS FOR REDUCED FITNESS SHOULD GENERATE A GENETIC INTERAC TION PROFILE THAT RESEMBLES THE CHEMICAL GENETIC INTERACTION PROFILE OF ITS INHIBITORY COMPOUND ;= &IG 4HUS BY COMPARING THE SYNTHETIC LETHAL PROFILE OF A GENE DELETION TO THE CHEMICAL GENETIC PROFILE OF A COM POUND ONE CAN DETERMINE WHETHER THEY AFFECT SIMILAR CELLULAR PROCESSES )N A PROOF OF PRINCIPLE STUDY 0ARSONS ET AL ;= DEMONSTRATED THAT CHEMI CAL GENETIC AND GENETIC INTERACTION PROFILES OVERLAP SUBSTANTIALLY FOR SEV ERAL DIFFERENT INHIBITORY COMPOUNDS AND THEIR TARGET GENES #OMPARISON OF GENOME WIDE CHEMICAL GENETIC PROFILES WITH THE LARGE COLLECTION OF GENETIC INTERACTION SCREENS APPEARS TO BE A POWERFUL TOOL FOR DISSECTING THE PATHWAYS AND PROTEINS TARGETED BY INHIBITORY MOLECULES &OR EXAMPLE THE CHEMICAL GENETIC INTERACTION PROFILES OF CYCLOSPORIN ! AND &+ OVERLAPPED WITH ONE ANOTHER AND BOTH SHOWED SIGNIFICANT OVERLAP WITH THE GENETIC INTERACTION PROFILE FOR #." A GENE ENCODING A CRITICAL COM PONENT OF CALCINEURIN THE TARGET ACTIVITY INHIBITED BY CYCLOSPORIN ! AND &+ 3IMILARLY A SIGNIFICANT OVERLAP AMONG THE GENETIC INTERACTIONS OF A TARGET GENE AND THE CHEMICAL GENETIC INTERACTIONS OF ITS CORRESPONDING INHIBITORY COMPOUND WERE OBSERVED FOR YEAST %2' WHICH ENCODES THE TARGET OF FLUCONAZOLE AND 45" WHICH ENCODES THE TARGET OF THE MICRO TUBULE POISON BENOMYL
! CASE IN POINT 0APUAMIDE " A MARINE NATURAL PRODUCT WITH A PREVIOUSLY UNKNOWN CELLULAR TARGET 0APUAMIDE " IS A MARINE NATURAL DEPSIPEPTIDE WITH HIGH MOLECULAR WEIGHT ORIGINALLY ISOLATED FROM A MARINE SPONGE AND WITH BOTH POTENT ANTIFUN
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
&IGURE #HEMICAL GENETIC INTERACTIONS CAN BE MODELED BY SYNTHETIC GENETIC INTERACTIONS ! )N A CHEMICAL GENETIC INTERACTION AT LEFT A DELETION MUTANT LACKING THE PRODUCT OF THE DELETED GENE REPRESENTED BY A BLACK 8 IS HYPERSENSITIVE TO A NORMALLY SUBLETHAL CONCENTRATION OF A GROWTH INHIBITORY COMPOUND )N A SYNTHETIC LETHAL GENETIC INTERACTION RIGHT TWO SINGLE DELETIONS LEAD TO VIABLE MUTANTS BUT ARE INVIABLE IN A DOUBLE MUTANT COMBINATION 'ENE DELETION ALLELES THAT SHOW CHEMICAL GENETIC INTERACTIONS WITH A PARTICULAR COMPOUND SHOULD ALSO BE SYNTHETICALLY LETHAL OR SICK WITH A MUTATION IN THE COMPOUND TARGET GENE " #OMPARISON OF A CHEMICAL GENETIC PROFILE TO A COMPENDIUM OF GENETIC INTERACTION SYNTHETIC LETHAL PROFILES SHOULD IDENTIFY THE PATHWAYS AND TARGETS INHIBITED BY DRUG TREATMENT )N THIS HYPOTHETICAL FIGURE CHEMICAL GENETIC AND GENETIC INTERACTIONS ARE BOTH DESIGNATED BY RED SQUARES &OR EXAMPLE DELETION MUTANTS Δ Δ Δ AND Δ ARE HYPERSENSITIVE TO COMPOUND 8 AND A MUTATION IN QUERY GENE ! LEADS TO A FITNESS DEFECT WHEN COMBINED WITH DELETION ALLELES Δ Δ Δ AND Δ (ERE THE CHEMICAL GENETIC PROFILE OF COMPOUND 8 RESEMBLES THE GENETIC PROFILE OF GENE " THEREBY IDENTIFYING THE PRODUCT OF GENE " AS A PUTATIVE TARGET OF COMPOUND 8 4HIS FIGURE WAS ADAPTED FROM &IGURE IN 0ARSONS ET AL ;=
GAL AND ANTI ()6 ACTIVITY ;= 4HE IDENTIFICATION OF PAPUAMIDE "S CEL LULAR TARGET THROUGH THE USE OF CHEMICAL GENETIC METHODOLOGY PROVIDES A CLEAR DEMONSTRATION OF THE POWER OF COMBINING CLASSICAL GENETIC TOOLS AND GENOME WIDE APPROACHES
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#HEMICAL GENETIC PROFILING OF PAPUAMIDE " 4HE CHEMICAL GENETIC PROFILE OF PAPUAMIDE " IDENTIFIED OVER GENES MANY OF WHICH HAVE ROLES IN SECRETION AND CELL WALL BIOSYNTHESIS 4O IDEN TIFY THE TOP DELETION MUTANTS THAT WERE SPECIFICALLY HYPERSENSITIVE TO INDI VIDUAL COMPOUNDS A STRAIN SPECIFIC ERROR MODEL WAS USED AND A P VALUE WAS ASSIGNED TO EACH CHEMICAL GENETIC INTERACTION BASED ON A MODIFIED 3TU DENTS T TEST 4HIS METHOD BETTER HIGHLIGHTS CHEMICAL GENETIC INTERACTIONS SPECIFIC TO THE COMPOUND AND PUTS LESS EMPHASIS ON MORE PROMISCUOUS INTERACTIONS ;= 4HE LIST OF TOP HITS SORTED BY P VALUE WAS ENRICHED FOR GENES WITH CERTAIN 'ENE /NTOLOGY ANNOTATIONS INCLUDING VESICLE MEDIATED TRANSPORT P CELL WALL ORGANIZATION AND BIOGENESIS P E AND PROTEIN MODIFICATION P SUGGESTING THAT PAPUAMIDE " MAY AFFECT INTRACELLULAR TRANSPORT OR PERTURB SOME TARGET ON THE CELL SUR FACE TABLE #OMPARISON OF THE PAPUAMIDE " CHEMICAL GENETIC PROFILE TO THE SYNTHETIC LETHAL GENETIC INTERACTION PROFILES OF DIFFERENT QUERY GENES ;= ALSO GROUPED 0APUAMIDE " WITH GENES INVOLVED IN CELL SURFACE FUNCTION (APLOINSUFFICIENCY PROFILING ()0 OF THE SET OF THE COMPLETE SET OF HETEROZYGOTES FAILED TO IDENTIFY AN OBVIOUS CANDIDATE GENE BECAUSE NONE OF THE TOP HITS SPECIFIC TO PAPUAMIDE " WERE ESSENTIAL GENES SUGGESTING THAT THE COMPOUND EITHER DOES NOT HAVE A CLASSIC ESSENTIAL PROTEIN TARGET OR THAT ITS TARGET DOES NOT EXHIBIT HAPLOINSUFFICIENCY
)SOLATION OF PAPUAMIDE " RESISTANT MUTANTS )T IS WELL KNOWN THAT MUTATIONS THAT LEAD TO DRUG RESISTANT PHENOTYPES MAY IDENTIFY THE TARGET GENE &OR EXAMPLE MUTATIONS IN THE 4/2 KINASE GENE LEAD TO RAPAMYCIN RESISTANCE ;= )SOLATION AND COMPLEMENTATION CLONING OF A MUTANT RESISTANT TO PAPUAMIDE " IDENTIFIED THE DRUG RESISTANT STRAIN AS A CHO NULL MUTANT #(/ ENCODES A NON ESSENTIAL ENZYME REQUIRED FOR THE SYNTHESIS OF PHOSPHATIDYLSERINE 03 ;= ONE OF FOUR MAJOR PHOS PHOLIPIDS FOUND IN 3 CEREVISIAE CELL MEMBRANES )N THE EUKARYOTIC PLASMA MEMBRANE SPHINGOLIPIDS AND PHOSPHATIDYLCHOLINE ARE MAINLY LOCATED IN THE OUTER LEAFLET OF THE PLASMA MEMBRANE WHEREAS 03 AND PHOSPHATIDYL ETHANOLAMINE 0% ARE MAINLY LOCATED IN THE INNER LEAFLET 4HIS ASYMMETRY IS ESTABLISHED BY AMINOPHOSPHOLIPID TRANSLOCASES WHICH @FLIP 03 AND 0%
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
FROM THE OUTER LEAFLET TO THE INNER LEAFLET 0OTENTIAL AMINOPHOSPHOLIPID TRANSLOCASES IN YEAST INCLUDE $RS $NF AND $NF ; = CONSEQUENTLY DRSΔ DNFΔ AND DNFΔ EXPOSE MORE 03 ON THE OUTER LEAFLET OF THE CELL MEMBRANE THAN WILD TYPE CELLS AND INDEED OUR CHEMICAL GENETIC PROFILING REVEALED THAT THESE MUTANTS WERE HYPERSENSITIVE TO PAPUAMIDE "
0APUAMIDE " CHEMICAL GENETIC PROFILE CLUSTERS WITH KNOWN CELL MEMBRANE PERMEABILITY DISRUPTING AGENTS ! PLAUSIBLE MODEL FOR THE ROLE OF PAPUAMIDE " IS THAT IT BINDS PHOSPHATIDYL SERINE IN THE CELL MEMBRANE AND KILLS THE CELL BY DISRUPTING THE INTEGRITY OF THE PLASMA MEMBRANE )F THIS MODEL WERE TRUE THEN COMPOUNDS THAT DISRUPT PLASMA MEMBRANE FUNCTION SHOULD CLUSTER WITH PAPUAMIDE " IN THE COMPENDIUM OF CHEMICAL GENETIC INTERACTIONS 4O TEST THIS IDEA WE FOCUSED ON ALAMETHICIN A AMINOACID PEPTIDE FIRST ISOLATED FROM THE FUNGUS 4RICHODERMA VIRIDE ;= AND CINNAMYCIN 2O n WHICH IS A AMINOACID TETRACYCLIC PEPTIDE PRODUCED BY 3TREPTOMYCES SPECIES ;= "OTH TOXINS CAUSE CYTOTOXICITY BY DISRUPTING CELL MEMBRANE PERMEABILITY !LSO CINNAMYCIN IS A UNIQUE TOXIN IN THAT ITS RECEPTOR PHOSPHATIDYLETHANOL AMINE 0% RESIDES IN THE INNER LAYER OF THE PLASMA MEMBRANE ;= 3TRIK INGLY WHEN THESE TWO COMPOUNDS WERE PROFILED AGAINST THE WHOLE SET OF CHEMICAL GENETIC INTERACTIONS PAPUAMIDE " ALAMETHICIN AND CINNAMYCIN CLUSTERED IN A SINGLE CLUSTER #LUSTER DEMONSTRATING A UNIQUE CAPABILITY OF THIS ASSAY IN PREDICTING PHARMACOLOGICAL EFFECTS 4AKEN TOGETHER THESE LINES OF EVIDENCE SUPPORT A MECHANISM OF ACTION FOR PAPUAMIDE " IN WHICH IT COMPROMISES YEAST CELL MEMBRANE INTEGRITY THROUGH A DIRECT INTERACTION WITH 03 4O PROVIDE BIOCHEMICAL EVIDENCE FOR THIS POSSIBILITY THE EFFECT OF PAPUAMIDE " ON MARKER RELEASE FROM LIPOSOMES CONTAINING 03 OR 0% WAS ANALYZED AS PREDICTED PAPUAMIDE " SHOWED ^ FOLD MORE POTENCY AGAINST THE 03 LIPOSOMES ;=
!NTI FUNGAL DRUG DISCOVERY 3 CEREVISIAE PROVIDES A GOOD MODEL SYSTEM FOR ANTIFUNGAL DRUG DISCOVERY 4HE MOST COMMONLY USED ANTIFUNGAL COMPOUNDS INCLUDING THE AZOLES
!NDRES ,OPEZ ET AL
4ABLE 4OP SENSITIVE DELETION MUTANTS TO 0APUAMIDE " SORTED BY P VALUE /2&
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#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
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;= POLYENES ;= AND ECHINOCANDINS ;= ARE ALL ACTIVE AGAINST 3 CERE VISIAE )N MANY CASES THE GENETICS UNDERLYING THE GENE PRODUCTS TARGETED BY THESE DRUGS AND MECHANISMS OF RESISTANCE HAVE BEEN ELUCIDATED IN 3 CEREVISIAE &OR EXAMPLE 3 CEREVISIAE HAS BEEN USED TO CHARACTERIZE NEW AZOLES ;= AND DETERMINE THE MECHANISMS OF RESISTANCE TO FLUCONAZOLE IN BOTH CLINICAL ;= AND THEORETICAL ;= APPLICATIONS 3 CEREVISIAE WAS ALSO INSTRUMENTAL FOR THE IDENTIFICATION OF β GLUCAN SYNTHASE ENCODED BY &+3 AND &+3 AS THE CASPOFUNGIN TARGET THROUGH THE CHARACTERIZATION OF CASPOFUNGIN RESISTANT MUTANTS ;n= (OWEVER TECHNOLOGIES AND INFORMATION INCLUDING NEW COMPOUNDS ACTIVE AGAINST 3 CEREVISIAE HAVE AT SOME STAGE TO BE VALIDATED IN TRUE FUN GAL PATHOGENS AND EFFORTS HAVE BEGUN TO MOVE YEAST GENOMICS TOOLS AND METHODOLOGIES DIRECTLY TO SUCH PATHOGENS )NDEED ALL THAT IS REQUIRED IS AN
!NDRES ,OPEZ ET AL
ANNOTATED GENOME SEQUENCE FOR THE PATHOGEN AND SOME BASIC RECOMBI NANT METHODOLOGY #ANDIDA ALBICANS THE MAJOR HUMAN FUNGAL PATHOGEN IS PRESENTLY THE BEST DEVELOPED EXAMPLE OF THIS TREND #ANDIDA ALBICANS IS A DIPLOID WITH NO CLASSICAL GENETICS )TS GENOME IS SEQUENCED ;= AND A PREDICTED SET OF GENE ANNOTATIONS IS AVAILABLE 2ECOMBINANT TECHNOLOGIES ARE WELL ADVANCED IN THE ORGANISM AND 2OEMER ET AL ;= HAVE COMPILED AN ESSENTIAL # ALBICANS GENE SET 4O DO SO THEY DISRUPTED ONE COPY OF A GIVEN GENE WHICH GENERATED A HETEROZYGOUS DIPLOID MUTANT COLLEC TION FOR HAPLOINSUFFICIENCY DRUG PROFILING AND GENERATED A 4ET REGULAT ABLE PROMOTER FOR THE SECOND COPY IN THE DIPLOID /F THE ESSENTIAL GENES IN 3 CEREVISIAE ORTHOLOGS OF THESE WERE CLEARLY IDENTIFIABLE IN # ALBICANS AND OF SOME ORTHOLOGS DISRUPTED WERE ESSEN TIAL 4HUS HAPLOINSUFFICIENCY PROFILING WITH THE COMPLETE SET OF # ALBI CANS HETEROZYGOTE DELETION MUTANTS WILL IDENTIFY COMPOUNDS THAT TARGET ESSENTIAL GENES THAT ARE PRESENT IN BOTH # ALBICANS AND 3 CEREVISIAE AS WELL AS THOSE ESSENTIAL GENES THAT ARE SPECIFIC TO # ALBICANS WHICH INCLUDES # ALBICANS SPECIFIC GENES AND GENES THAT ARE PRESENT IN BOTH # ALBICANS AND 3 CEREVISIAE BUT ESSENTIAL UNIQUELY IN # ALBICANS #REATING A DELE TION MUTANT COLLECTION IN ANOTHER FUNGAL SPECIES EXPANDS THE POWER OF CHEMICAL GENETIC APPROACHES ENABLING US TO INTERROGATE NEW TARGET GENES AND THEIR CORRESPONDING PATHWAYS AND THEREBY EXPAND OUR ANALYSIS TO COVER MORE OF CHEMICAL SPACE 4HUS TO HARNESS HAPLOINSUFFICIENCY PROFIL ING FULLY BOTH THE 3 CEREVISIAE AND THE # ALBICANS HETEROZYGOTE DELETION MUTANT COLLECTIONS SHOULD BE SCREENED AND A SIMILAR CONCLUSION SHOULD HOLD FOR CHEMICAL GENETIC PROFILING OF THE SET OF HOMOZYGOUS VIABLE DELE TION MUTANTS
&UTURE DIRECTIONS %XPLOITING SYNTHETIC LETHAL GENETIC NETWORKS TO MAP ANTIFUNGAL COMBINATION THERAPIES !S MENTIONED PREVIOUSLY ABOVE THE 3EATTLE PROJECT PIONEERED THE IDEA OF USING YEAST AS A EUKARYOTIC MODEL FOR IDENTIFICATION OF CANCER THERAPEUTICS ;= 'IVEN THAT CANCEROUS TUMORS CONTAIN SPECIFIC MUTATIONS THAT LEAD TO GENOME INSTABILITY SYNTHETIC GENETIC ARRAY ANALYSIS ALLOWS FOR SCREENING OF MUTATIONS THAT DISPLAY SYNTHETIC LETHAL INTERACTIONS WITH YEAST MODELS OF
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
CANCER CAUSING GENETIC LESIONS 4HUS IF GENETIC INTERACTIONS LIKE NUMEROUS GENES ARE CONSERVED FROM YEAST TO HUMANS GENOME WIDE SYNTHETIC LETHAL SCREENS SHOULD BE ABLE IDENTIFY TARGETS FOR DRUGS THAT WOULD SPECIFICALLY INHIBIT PROLIFERATION OF CELLS CARRYING A CANCER CAUSING MUTATION ! DRUG SUCH AS THIS SHOULD BE LESS TOXIC THAN GENERALLY ACTING ANTI PROLIFERATIVE COMPOUNDS BECAUSE THE TARGET IS NOT REQUIRED FOR CELLULAR VIABILITY UNLESS THE CANCER CAUSING MUTATION IS PRESENT ; = ! COROLLARY OF SYNTHETIC LETHAL GENETIC ANALYSIS IS THAT A LETHAL PHE NOTYPE CAN BE INDUCED BY TARGETING TWO NON ESSENTIAL FUNCTIONS SIMUL TANEOUSLY )NDEED A CLEAR UNDERSTANDING OF THE SYNTHETIC LETHAL GENETIC NETWORK STRUCTURE SHOULD ENABLE THE DESIGN OF DRUG COMBINATIONS 4HIS MODEL GREATLY EXPANDS THE NUMBER OF POTENTIAL ANTIFUNGAL DRUG TARGETS BY LOOKING BEYOND SINGLE PROTEIN TARGETS TO COMBINATIONS OF TWO OR MORE TARGETS THAT ARE MUTUALLY REQUIRED FOR VIABILITY "ECAUSE GENETIC INTERACTIONS CAN ALSO BE MAPPED FOR ESSENTIAL GENES BY SCREENING CONDI TIONAL OR HYPOMORPHIC ALLELES ;= OUR NETWORK MODEL FOR COMBINA TIONS OF TARGETS CAN ALSO INCLUDE AN INTERACTION BETWEEN ONE ESSENTIAL AND ONE NONESSENTIAL GENE OR AN INTERACTION BETWEEN TWO ESSENTIAL GENES "ASED ON THE ESTIMATED NUMBER OF SYNTHETIC DIGENIC COMBINATIONS ;= IT SHOULD BE POSSIBLE TO IDENTIFY A FOLD WIDER REPERTOIRE OF ANTIFUNGAL DRUGS THAT ACT IN THE WAY THAT EXPLOITS A FUNDAMENTAL WEAKNESS OF CEL LULAR NETWORKS )N AN ANTIFUNGAL EXAMPLE THE NONESSENTIAL GENES #(3 ENCODING CHITIN SYNTHASE AND &+3 ENCODING A GLUCAN SYNTHASE SUBUNIT SHOW A SYNTHETIC GENETIC INTERACTION ;= !NTIFUNGAL INHIBITORS THAT WORK ON EACH OF THESE PRODUCTS ARE KNOWN THE ECHINOCANDINS EG CASPOFUNGIN FOR &KS ;= AND NIKKOMYCIN FOR #HS ;= 4HUS A COMBINATION OF AN ECHINOCANDIN AND NIKKOMYCIN MAY ACT SYNERGISTICALLY AS THEIR RESPEC TIVE TARGETS NORMALLY BUFFER EACH OTHER )NDEED IN 3 CEREVISIAE THE ECHI NOCANDIN , AND NIKKOMYCIN + HAVE EMPIRICALLY BEEN FOUND TO ACT IN THIS WAY ;= 4O FURTHER ILLUSTRATE THE POWER OF THIS APPROACH A SYNTHETIC ANALYSIS OF &+3 IN 3 CEREVISIAE ;= INDICATES THAT THERE ARE MORE GENES IN ADDITION TO #(3 THAT SHOW SYNTHETIC INTERACTIONS WITH &+3 AND WHICH WHEN MUTATED SINGLY SHOW AN ENHANCED SENSITIVITY TO THE ECHINOCANDIN CASPOFUNGIN #OMPOUNDS THAT SPECIFICALLY INHIBIT SUCH GENE PRODUCTS WOULD BE CANDIDATES FOR COMBINATION THERAPIES WITH CASPOFUNGIN
!NDRES ,OPEZ ET AL
#ONCLUSIONS #HEMICAL GENOMICS AND A SMALL MOLECULE FOR EVERY GENE PRODUCT 4HE GENERATION OF BASIC RESEARCH TOOLS IS AN IMPORTANT REASON FOR THE RESEARCH COMMUNITY TO ATTEMPT TO IDENTIFY A SMALL MOLECULE INHIBITOR FOR EVERY GENE PRODUCT AN IDEA THAT FORMS THE RATIONALE FOR THE .)( -OLECULAR ,IBRARIES )NI TIATIVE FOR HUMANS HTTPNIHROADMAPNIHGOV 4HIS CHAPTER HIGHLIGHTS THE POWER OF CHEMICAL GENETIC ANALYSIS WITH YEAST TO LINK COMPOUNDS TO THEIR TARGET PATHWAYS 4HE BASIC PRINCIPLE UNDERLYING THE CHEMICAL GENOMIC METHODOLOGIES PRESENTED HERE IS THE CONCEPT THAT INHIBITING A GENE PRODUCT WHETHER THROUGH GENETIC OR CHEMICAL MEANS IS FUNDAMENTALLY EQUIVALENT 4HE DEVELOPMENT OF LARGE COMPENDIA OF CHEMICAL GENETIC PROFILES FOR 3 CEREVISIAE WILL PROVIDE RICH FUNCTIONAL INFORMATION ABOUT THE COMPOUNDS IN THE LIBRARY AND A DATA SET FOR INTERPRETING THE MODE OF ACTION OF NOVEL COM POUNDS %XPANDING SIMILAR TOOLS ESPECIALLY THE BARCODED DELETION MUTANT COLLECTION TO OTHER FUNGAL SPECIES INCLUDING PATHOGENIC FUNGI WILL ENABLE US TO EXPLORE CHEMICAL SPACE MORE FULLY BECAUSE IT OPENS THE DOOR TO NEW GENES WHICH MEANS NEW TARGETS AND DIFFERENT WIRINGS OF GENETIC NETWORKS BOTH OF WHICH UNDERLIE THE IDENTIFICATION OF CHEMICAL GENETIC INTERACTIONS )MPORTANTLY SIMILAR CHEMICAL GENOMICS METHODS SHOULD BE POSSIBLE IN MULTICELLULAR ORGANISMS SUCH AS THE NEMATODE # ELEGANS ;= AND POTEN TIALLY HUMAN CELL LINES VIA 2.!I BASED METHODOLOGIES )NDEED THE FIRST LARGE SCALE MAPPING OF SYNTHETIC LETHAL GENETIC NETWORKS IN # ELEGANS HAS REVEALED THAT THEY SHOW A SIMILAR ARCHITECTURE TO THOSE IN YEAST ;= )N THE CONTEXT OF THE LARGER GOAL OF IDENTIFYING A SMALL MOLECULE INHIBITOR FOR EVERY PROTEIN ACCESS TO SPECIFIC BIOACTIVE COMPOUNDS BECOMES A MAJOR HURDLE ESPECIALLY FOR ACADEMIC LABS 7HILE ACADEMIC LABS HAVE DRIVEN THE DEVELOPMENT OF THESE NEW CHEMICAL GENOMIC METHODOLOGIES IN GENERAL THEY DO NOT HAVE ACCESS TO SIGNIFICANT CHEMICAL LIBRARIES SUCH AS THE VAST COLLECTIONS OF COMPOUNDS AND NATURAL PRODUCT EXTRACTS THAT HAVE BEEN ACCUMULATED BY PHARMACEUTICAL COMPANIES "ECAUSE THE PHARMACEUTICAL INDUSTRY GUARDS ITS CHEMISTRY CLOSELY ACADEMICS MUST SOMEHOW ORCHES TRATE THE ASSEMBLY OF EQUIVALENT RICH AND EXTENSIVE CHEMICAL LIBRARIES TO FULLY REALIZE THE POTENTIAL OF CHEMICAL GENOMICS #REATING RESEARCH GROUPS THAT BRING TOGETHER FUNCTIONAL GENOMICS LABORATORIES AND NATURAL PRODUCT LABS ON A RELATIVELY LARGE SCALE SHOULD ALLOW US PIONEER PROGRAMS FOR THE DEVELOPMENT OF GENOME WIDE SETS OF SPECIFIC CHEMICAL PERTURBANTS
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
!CKNOWLEDGEMENTS #" WAS SUPPORTED BY GRANTS FROM THE #ANADIAN )NSTITUTES OF (EALTH 2ESEARCH #)(2 AND 'ENOME #ANADA AND 'ENOME /NTARIO #. AND '' ARE SUPPORTED BY GRANTS FROM 4HE .)( .('2) AND THE #)(2
2EFERENCES
.EWMAN $* #RAGG '- 3NADER +- .ATURAL PRODUCTS AS SOURCES OF NEW DRUGS OVER THE PERIOD n * .AT 0ROD n ,IU * &ARMER *$ *R ,ANE 73 &RIEDMAN * 7EISSMAN ) 3CHREIBER 3, #ALCINEURIN IS A COMMON TARGET OF CYCLOPHILINnCYCLOSPORIN ! AND &+"0 &+ COMPLEXES #ELL n +OEHN &% #ARTER '4 4HE EVOLVING ROLE OF NATURAL PRODUCTS IN DRUG DISCOVERY .AT 2EV $RUG $ISCOV n #LARDY * 7ALSH # ,ESSONS FROM NATURAL MOLECULES .ATURE n .ICOLAOU +# 0FEFFERKORN *! 3OLID PHASE SYNTHESIS OF COMPLEX NATURAL PRODUCTS AND LIBRARIES THEREOF "IOPOLYMERS n 0OMMIER 9 4OPOISOMERASE ) INHIBITORS CAMPTOTHECINS AND BEYOND .AT 2EV #ANCER n +UKURUZINSKA -! "ERGH -, *ACKSON "* 0ROTEIN GLYCOSYLATION IN YEAST !NNU 2EV "IOCHEM n ,I 7 -O 7 3HEN $ 3UN , 7ANG * ,U 3 'ITSCHIER *- :HOU " 9EAST MODEL UNCOVERS DUAL ROLES OF MITOCHONDRIA IN ACTION OF ARTEMISININ 0,O3 'ENET E 2O $+ 0ARADISE %- /UELLET - &ISHER +* .EWMAN +, .DUNGU *- (O +! %ACHUS 2! (AM 43 +IRBY * ET AL 0RODUCTION OF THE ANTIMALARIAL DRUG PRECURSOR ARTEMISINIC ACID IN ENGINEERED YEAST .ATURE n #RESPO *, (ALL -. %LUCIDATING 4/2 SIGNALING AND RAPAMYCIN ACTION LESSONS FROM 3ACCHAROMYCES CEREVISIAE -ICROBIOL -OL "IOL 2EV n TABLE OF CONTENTS #HANG 3 0ERRY *$ +OSMORSKY '3 "RAUN 7% 2APAMYCIN FOR TREATMENT OF REFRAC TORY DYSTHYROID COMPRESSIVE OPTIC NEUROPATHY /PHTHAL 0LAST 2ECONSTR 3URG n $OUGLAS #- -ARRINAN *! ,I 7 +URTZ -" ! 3ACCHAROMYCES CEREVISIAE MUTANT WITH ECHINOCANDIN RESISTANT BETA $ GLUCAN SYNTHASE * "ACTERIOL n $OUGLAS #- &OOR & -ARRINAN *! -ORIN . .IELSEN *" $AHL !- -AZUR 0 "AGINSKY 7 ,I 7 EL 3HERBEINI - ET AL 4HE 3ACCHAROMYCES CEREVISIAE &+3 %4' GENE ENCODES AN INTEGRAL MEMBRANE PROTEIN WHICH IS A SUBUNIT OF BETA $ GLUCAN SYNTHASE 0ROC .ATL !CAD 3CI 53! n $OUGLAS #- $)PPOLITO *! 3HEI '* -EINZ - /NISHI * -ARRINAN *! ,I 7 !BRUZZO '+ &LATTERY ! "ARTIZAL + ET AL )DENTIFICATION OF THE &+3 GENE OF #ANDIDA ALBICANS AS THE ESSENTIAL TARGET OF BETA $ GLUCAN SYNTHASE INHIBITORS !NTIMICROB !GENTS #HEM OTHER n #ARDENAS -% 3ANFRIDSON ! #UTLER .3 (EITMAN * 3IGNAL TRANSDUCTION CASCADES AS TARGETS FOR THERAPEUTIC INTERVENTION BY NATURAL PRODUCTS 4RENDS "IOTECHNOL n
!NDRES ,OPEZ ET AL
0ENA #ASTILLO , (UGHES 42 7HY ARE THERE STILL OVER UNCHARACTERIZED YEAST GENES 'ENETICS n (ARTWELL ,( 3ZANKASI 0 2OBERTS #* -URRAY !7 &RIEND 3( )NTEGRATING GENETIC APPROACHES INTO THE DISCOVERY OF ANTICANCER DRUGS 3CIENCE n 'OFFEAU ! "ARRELL "' "USSEY ( $AVIS 27 $UJON " &ELDMANN ( 'ALIBERT & (OHEISEL *$ *ACQ # *OHNSTON - ET AL ,IFE WITH GENES 3CIENCE n "OTSTEIN $ #HERVITZ 3! #HERRY *- 9EAST AS A MODEL ORGANISM 3CIENCE n ,ANDER %3 ,INTON ,- "IRREN " .USBAUM # :ODY -# "ALDWIN * $EVON + $EWAR + $OYLE - &ITZ(UGH 7 ET AL )NITIAL SEQUENCING AND ANALYSIS OF THE HUMAN GENOME .ATURE n 6ENTER *# !DAMS -$ -YERS %7 ,I 07 -URAL 2* 3UTTON '' 3MITH (/ 9ANDELL - %VANS #! (OLT 2! ET AL 4HE SEQUENCE OF THE HUMAN GENOME 3CIENCE n (UGHES 42 9EAST AND DRUG DISCOVERY &UNCT )NTEGR 'ENOMICS n &OURY & (UMAN GENETIC DISEASES A CROSS TALK BETWEEN MAN AND YEAST 'ENE n -C #- #ALLENDER -% ,AWLIS *& *R &UMAGILLIN ( A NEW ANTIBIOTIC WITH AME BICIDAL PROPERTIES 3CIENCE n 'RIFFITH %# 3U : .IWAYAMA 3 2AMSAY #! #HANG 9( ,IU */ -OLECULAR RECOGNI TION OF ANGIOGENESIS INHIBITORS FUMAGILLIN AND OVALICIN BY METHIONINE AMINOPEPTIDASE 0ROC .ATL !CAD 3CI 53! n #ARDENAS -% #RUZ -# $EL 0OETA - #HUNG . 0ERFECT *2 (EITMAN * !NTIFUNGAL ACTIVITIES OF ANTINEOPLASTIC AGENTS 3ACCHAROMYCES CEREVISIAE AS A MODEL SYSTEM TO STUDY DRUG ACTION #LIN -ICROBIOL 2EV n )TO 4 #HIBA 4 /ZAWA 2 9OSHIDA - (ATTORI - 3AKAKI 9 ! COMPREHENSIVE TWO HYBRID ANALYSIS TO EXPLORE THE YEAST PROTEIN INTERACTOME 0ROC .ATL !CAD 3CI 53! n 5ETZ 0 'IOT , #AGNEY ' -ANSFIELD 4! *UDSON 23 +NIGHT *2 ,OCKSHON $ .ARAYAN 6 3RINIVASAN - 0OCHART 0 ET AL ! COMPREHENSIVE ANALYSIS OF PROTEINnPROTEIN INTER ACTIONS IN 3ACCHAROMYCES CEREVISIAE .ATURE n :HU ( +LEMIC *& #HANG 3 "ERTONE 0 #ASAMAYOR ! +LEMIC +' 3MITH $ 'ERSTEIN - 2EED -! 3NYDER - !NALYSIS OF YEAST PROTEIN KINASES USING PROTEIN CHIPS .AT 'ENET n (ORAK #% 3NYDER - 'LOBAL ANALYSIS OF GENE EXPRESSION IN YEAST &UNCT )NTEGR 'ENOMICS n (UH 7+ &ALVO *6 'ERKE ,# #ARROLL !3 (OWSON 27 7EISSMAN *3 /3HEA %+ 'LOBAL ANALYSIS OF PROTEIN LOCALIZATION IN BUDDING YEAST .ATURE n 'IAEVER ' #HU !- .I , #ONNELLY # 2ILES , 6ERONNEAU 3 $OW 3 ,UCAU $ANILA ! !NDERSON + !NDRE " ET AL &UNCTIONAL PROFILING OF THE 3ACCHAROMYCES CEREVISIAE GENOME .ATURE n 3PELLMAN 04 3HERLOCK ' :HANG -1 )YER 62 !NDERS + %ISEN -" "ROWN 0/ "OTSTEIN $ &UTCHER " #OMPREHENSIVE IDENTIFICATION OF CELL CYCLE REGULATED GENES OF THE YEAST 3ACCHAROMYCES CEREVISIAE BY MICROARRAY HYBRIDIZATION -OL "IOL #ELL n (UGHES 42 -ARTON -* *ONES !2 2OBERTS #* 3TOUGHTON 2 !RMOUR #$ "ENNETT (! #OFFEY % $AI ( (E 9$ ET AL &UNCTIONAL DISCOVERY VIA A COMPENDIUM OF EXPRESSION PROFILES #ELL n
#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
'AVIN !# "OSCHE - +RAUSE 2 'RANDI 0 -ARZIOCH - "AUER ! 3CHULTZ * 2ICK *- -ICHON !- #RUCIAT #- ET AL &UNCTIONAL ORGANIZATION OF THE YEAST PROTEOME BY SYSTEMATIC ANALYSIS OF PROTEIN COMPLEXES .ATURE n (O 9 'RUHLER ! (EILBUT ! "ADER '$ -OORE , !DAMS 3, -ILLAR ! 4AYLOR 0 "ENNETT + "OUTILIER + ET AL 3YSTEMATIC IDENTIFICATION OF PROTEIN COMPLEXES IN 3ACCHAROMYCES CEREVISIAE BY MASS SPECTROMETRY .ATURE n +ROGAN .* #AGNEY ' 9U ( :HONG ' 'UO 8 )GNATCHENKO ! ,I * 0U 3 $ATTA . 4IKUISIS !0 ET AL 'LOBAL LANDSCAPE OF PROTEIN COMPLEXES IN THE YEAST 3ACCHAROMYCES CEREVI SIAE .ATURE n 'AVIN !# !LOY 0 'RANDI 0 +RAUSE 2 "OESCHE - -ARZIOCH - 2AU # *ENSEN ,* "AS TUCK 3 $UMPELFELD " ET AL 0ROTEOME SURVEY REVEALS MODULARITY OF THE YEAST CELL MACHINERY .ATURE n 'HAEMMAGHAMI 3 (UH 7+ "OWER + (OWSON 27 "ELLE ! $EPHOURE . /3HEA %+ 7EISSMAN *3 'LOBAL ANALYSIS OF PROTEIN EXPRESSION IN YEAST .ATURE n 4ONG !( %VANGELISTA - 0ARSONS !" 8U ( "ADER '$ 0AGE . 2OBINSON - 2AGHIBIZA DEH 3 (OGUE #7 "USSEY ( ET AL 3YSTEMATIC GENETIC ANALYSIS WITH ORDERED ARRAYS OF YEAST DELETION MUTANTS 3CIENCE n 4ONG !( ,ESAGE ' "ADER '$ $ING ( 8U ( 8IN 8 9OUNG * "ERRIZ '& "ROST 2, #HANG - ET AL 'LOBAL MAPPING OF THE YEAST GENETIC INTERACTION NETWORK 3CIENCE n 0AN 8 9UAN $3 /OI 3, 7ANG 8 3OOKHAI -AHADEO 3 -ELUH 0 "OEKE *$ D3,!- ANALYSIS OF GENOME WIDE GENETIC INTERACTIONS IN 3ACCHAROMYCES CEREVISIAE -ETHODS n $WIGHT 33 "ALAKRISHNAN 2 #HRISTIE +2 #OSTANZO -# $OLINSKI + %NGEL 32 &EIERBACH " &ISK $' (IRSCHMAN * (ONG %, ET AL 3ACCHAROMYCES GENOME DATABASE UNDERLYING PRINCIPLES AND ORGANISATION "RIEF "IOINFORM n -EWES (7 !LBERMANN + (EUMANN + ,IEBL 3 0FEIFFER & -)03 A DATABASE FOR PROTEIN SEQUENCES HOMOLOGY DATA AND YEAST GENOME INFORMATION .UCLEIC !CIDS 2ES n "REITKREUTZ "* 3TARK # 4YERS - 4HE '2)$ THE 'ENERAL 2EPOSITORY FOR )NTERACTION $ATASETS 'ENOME "IOL 2 7INZELER %! 3HOEMAKER $$ !STROMOFF ! ,IANG ( !NDERSON + !NDRE " "ANGHAM 2 "ENITO 2 "OEKE *$ "USSEY ( ET AL &UNCTIONAL CHARACTERIZATION OF THE 3 CEREVISIAE GENOME BY GENE DELETION AND PARALLEL ANALYSIS 3CIENCE n 0ARSONS !" ,OPEZ ! 'IVONI )% 7ILLIAMS $% 'RAY #! 0ORTER * #HUA ' 3OPKO 2 "ROST 2, (O #( ET AL %XPLORING THE MODE OF ACTION OF BIOACTIVE COMPOUNDS BY CHEMI CAL GENETIC PROFILING IN YEAST #ELL n 3CHERENS " 'OFFEAU ! 4HE USES OF GENOME WIDE YEAST MUTANT COLLECTIONS 'ENOME "IOL #HAN 4& #ARVALHO * 2ILES , :HENG 8& ! CHEMICAL GENOMICS APPROACH TOWARD UNDERSTANDING THE GLOBAL FUNCTIONS OF THE TARGET OF RAPAMYCIN PROTEIN 4/2 0ROC .ATL !CAD 3CI 53! n $ESMOUCELLES # 0INSON " 3AINT -ARC # $AIGNAN &ORNIER " 3CREENING THE YEAST hDISRUPTOMEv FOR MUTANTS AFFECTING RESISTANCE TO THE IMMUNOSUPPRESSIVE DRUG MYCO PHENOLIC ACID * "IOL #HEM n "ENNETT #" ,EWIS ,+ +ARTHIKEYAN ' ,OBACHEV +3 *IN 9( 3TERLING *& 3NIPE *2 2ES
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PLEMENTARY WHOLE GENOME TECHNOLOGIES REVEAL THE CELLULAR RESPONSE TO PROTEASOME INHIBITION BY 03 0ROC .ATL !CAD 3CI 53! n (ANWAY $ #HIN *+ 8IA ' /SHIRO ' 7INZELER %! 2OMESBERG &% 0REVIOUSLY UNCHARACTERIZED GENES IN THE 56 AND --3 INDUCED $.! DAMAGE RESPONSE IN YEAST 0ROC .ATL !CAD 3CI 53! n 0ARSONS !" "ROST 2, $ING ( ,I : :HANG # 3HEIKH " "ROWN '7 +ANE 0- (UGHES 42 "OONE # )NTEGRATION OF CHEMICAL GENETIC AND GENETIC INTERACTION DATA LINKS BIOACTIVE COMPOUNDS TO CELLULAR TARGET PATHWAYS .AT "IOTECHNOL n $UECK $ -ORRIS 1$ &REY "* -ULTI WAY CLUSTERING OF MICROARRAY DATA USING PROBA BILISTIC SPARSE MATRIX FACTORIZATION "IOINFORMATICS 3UPPL In !YSCOUGH +2 3TRYKER * 0OKALA . 3ANDERS - #REWS 0 $RUBIN $' (IGH RATES OF ACTIN FILAMENT TURNOVER IN BUDDING YEAST AND ROLES FOR ACTIN IN ESTABLISHMENT AND MAINTENANCE OF CELL POLARITY REVEALED USING THE ACTIN INHIBITOR LATRUNCULIN ! * #ELL "IOL n 4ORRALBA 3 2AUDASKOSKI - 0EDREGOSA !- ,ABORDA & %FFECT OF CYTOCHALASIN ! ON APICAL GROWTH ACTIN CYTOSKELETON ORGANIZATION AND ENZYME SECRETION IN !SPERGILLUS NIDULANS -ICROBIOLOGY 0T n &ROMTLING 2! /VERVIEW OF MEDICALLY IMPORTANT ANTIFUNGAL AZOLE DERIVATIVES #LIN -ICROBIOL 2EV n 4RUAN ' %PINAT *# 2OUGEULLE # #ULLIN # 0OMPON $ #LONING AND CHARACTERIZA TION OF A YEAST CYTOCHROME B ENCODING GENE WHICH SUPPRESSES KETOCONAZOLE HYPERSEN SITIVITY IN A .!$0( 0 REDUCTASE DEFICIENT STRAIN 'ENE n 2OE 3- 0RODROMOU # /"RIEN 2 ,ADBURY *% 0IPER 07 0EARL ,( 3TRUCTURAL BASIS FOR INHIBITION OF THE (SP MOLECULAR CHAPERONE BY THE ANTITUMOR ANTIBIOTICS RADICICOL AND GELDANAMYCIN * -ED #HEM n 4HOMAS *( .EFF .& "OTSTEIN $ )SOLATION AND CHARACTERIZATION OF MUTATIONS IN THE BETA TUBULIN GENE OF 3ACCHAROMYCES CEREVISIAE 'ENETICS n +UNKEL 7 %FFECTS OF THE ANTIMICROTUBULAR CANCEROSTATIC DRUG NOCODAZOLE ON THE YEAST 3ACCHAROMYCES CEREVISIAE : !LLG -IKROBIOL n -ARCIREAU # 'UILLOTON - +ARST & )N VIVO EFFECTS OF FENPROPIMORPH ON THE YEAST 3ACCHAROMYCES CEREVISIAE AND DETERMINATION OF THE MOLECULAR BASIS OF THE ANTIFUNGAL PROPERTY !NTIMICROB !GENTS #HEMOTHER n -OEBIUS && "ERMOSER + 2EITER 2* (ANNER - 'LOSSMANN ( 9EAST STEROL # # ISOMERASE IDENTIFICATION AND CHARACTERIZATION OF A HIGH AFFINITY BINDING SITE FOR ENZYME INHIBITORS "IOCHEMISTRY n 3OBELL (- !CTINOMYCIN AND $.! TRANSCRIPTION 0ROC .ATL !CAD 3CI 53! n (ERNANDEZ & #ANNON - )NHIBITION OF PROTEIN SYNTHESIS IN 3ACCHAROMYCES CEREVI SIAE BY THE EPOXYTRICHOTHECENES TRICHODERMOL DIACETOXYSCIRPENOL AND VERRUCARIN ! 2EVERSIBILITY OF THE EFFECTS * !NTIBIOT 4OKYO n 3CHROEDER 2 7ALDSICH # 7ANK ( -ODULATION OF 2.! FUNCTION BY AMINOGLYCO SIDE ANTIBIOTICS %MBO * n +ITAGAWA ) +OBAYASHI - )MAMOTO 4 9ASUZAWA 4 +YOGOKU 9 4HE STRUCTURES OF SIX ANTIFUNGAL OLIGOGLYCOSIDES STICHLOROSIDES ! ! " " # AND # FROM THE SEA CUCUMBER 3TICHOPUS CHLORONOTUS "2!.$4 #HEM 0HARM "ULL n 3CHMIDT %7 "EWLEY #! &AULKNER $* 4HEOPALAUAMIDE A BICYCLIC GLYCOPEPTIDE
!NDRES ,OPEZ ET AL
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#HEMICAL GENETIC APPROACHES FOR EXPLORING THE MODE OF ACTION OF NATURAL PRODUCTS
* #HUA ' ,OPEZ ! ET AL %XPLORATION OF ESSENTIAL GENE FUNCTIONS VIA TITRATABLE PROMOTER ALLELES #ELL n /SMOND "# 3PECHT #! 2OBBINS 07 #HITIN SYNTHASE ))) SYNTHETIC LETHAL MUTANTS AND uSTRESS RELATEDh CHITIN SYNTHESIS THAT BYPASSES THE #3$#(3 LOCALIZATION PATHWAY 0ROC .ATL !CAD 3CI 53! n 'AUGHRAN *0 ,AI -( +IRSCH $2 3ILVERMAN 3* .IKKOMYCIN : IS A SPECIFIC INHIBITOR OF 3ACCHAROMYCES CEREVISIAE CHITIN SYNTHASE ISOZYME #HS IN VITRO AND IN VIVO * "ACTERIOL n EL 3HERBEINI - #LEMAS *! .IKKOMYCIN : SUPERSENSITIVITY OF AN ECHINOCANDIN RESISTANT MUTANT OF 3ACCHAROMYCES CEREVISIAE !NTIMICROB !GENTS #HEMOTHER n ,ESAGE ' 3DICU !- -ENARD 0 3HAPIRO * (USSEIN 3 "USSEY ( !NALYSIS OF BETA GLUCAN ASSEMBLY IN 3ACCHAROMYCES CEREVISIAE USING A SYNTHETIC INTERACTION NETWORK AND ALTERED SENSITIVITY TO CASPOFUNGIN 'ENETICS n +WOK 4# 2ICKER . &RASER 2 #HAN !7 "URNS ! 3TANLEY %& -C#OURT 0 #UTLER 32 2OY 0* ! SMALL MOLECULE SCREEN IN # ELEGANS YIELDS A NEW CALCIUM CHANNEL ANTAGONIST .ATURE n ,EHNER " #ROMBIE # 4ISCHLER * &ORTUNATO ! &RASER !' 3YSTEMATIC MAPPING OF GENETIC INTERACTIONS IN #AENORHABDITIS ELEGANS IDENTIFIES COMMON MODIFIERS OF DIVERSE SIGNALING PATHWAYS .AT 'ENET n
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%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS n PHARMACOLOGY FERMENTATION AND STRUCTURE ACTIVITY RELATIONSHIPS
"Y +ARL (EINZ !LTMANN AND +LAUS -EMMERT $EPARTMENT OF #HEMISTRY AND !PPLIED "IOSCIENCES )NSTITUTE OF 0HARMACEUTICAL 3CIENCES 3WISS &EDERAL )NSTITUTE OF 4ECHNOLOGY %4( :ßRICH 3WITZERLAND KARL HEINZALTMANN PHARMAETHZCH .OVARTIS )NSTITUTE FOR "IOMEDICAL 2ESEARCH "ASEL 3WITZERLAND
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
!BSTRACT %POTHILONES %POS ! AND " ARE NATURALLY OCCURRING MICROTUBULE STABILIZERS WHICH INHIBIT THE GROWTH OF HUMAN CANCER CELLS IN VITRO AT LOW N- OR SUB N- CONCENTRATIONS )N CONTRAST TO TAXOL PACLITAXEL 4AXOL¤ EPOTHILONES ARE ALSO ACTIVE AGAINST DIFFERENT TYPES OF MULTIDRUG RESIS TANT CANCER CELL LINES IN VITRO AND AGAINST MULTIDRUG RESISTANT TUMORS IN VIVO 4HEIR ATTRACTIVE PRECLINICAL PROFILE HAS MADE EPOTHILONES IMPORTANT LEAD STRUCTURES IN THE SEARCH FOR IMPROVED CYTOTOXIC ANTICANCER DRUGS AND %PO " %0/ PATUPILONE IS CURRENTLY UNDERGOING 0HASE ))) CLINICAL TRIALS .UMEROUS SYNTHETIC AND SEMISYNTHETIC ANALOGS HAVE BEEN PREPARED SINCE THE ABSOLUTE STEREOCHEMISTRY OF EPOTHILONES WAS FIRST DISCLOSED IN MID AND THEIR IN VITRO BIO LOGICAL ACTIVITY HAS BEEN DETERMINED !PART FROM GENERATING A WEALTH OF 3!2 INFORMATION THESE EFFORTS HAVE LED TO THE IDENTIFICATION OF AT LEAST SIX COMPOUNDS IN ADDITION TO %PO " WHICH ARE CURRENTLY AT VARIOUS STAGES OF CLINICAL EVALUATION IN HUMANS 4HE MOST ADVANCED OF THESE COM POUNDS %PO " LACTAM "-3 IXABEPILONE HAS RECENTLY OBTAINED &$! APPROVAL FOR THE TREATMENT OF METASTATIC AND ADVANCED BREAST CANCER 4HIS CHAPTER WILL FIRST PROVIDE A SUMMARY OF THE BASIC FEATURES OF THE BIOLOGICAL PROFILE OF %PO " IN VITRO AND IN VIVO 4HIS WILL BE FOLLOWED BY A REVIEW OF THE PROCESSES THAT HAVE BEEN DEVELOPED FOR THE FERMENTATIVE PRODUCTION OF %PO " 4HE MAIN PART OF THE CHAPTER WILL FOCUS ON THE MOST RELEVANT ASPECTS OF THE EPOTHILONE 3!2 WITH REGARD TO EFFECTS ON TUBULIN POLYMERIZATION IN VITRO ANTIPROLIFERATIVE ACTIVITY AND IN VIVO ANTITUMOR ACTIVITY 0ARTICULAR EMPHASIS WILL BE PLACED ON WORK CONDUCTED IN THE AUTHORS OWN LABORATORIES BUT DATA FROM OTHER GROUPS WILL ALSO BE INCLUDED )N A FINAL SECTION THE CURRENT STATUS OF THOSE EPOTHILONE ANALOGS UNDERGOING CLINICAL DEVELOPMENT WILL BE BRIEFLY DISCUSSED
)NTRODUCTION #ANCER REPRESENTS ONE OF THE MOST SEVERE HEALTH PROBLEMS WORLDWIDE WITH A TOTAL OF NEW CANCER CASES AND DEATHS FROM CANCER EXPECTED IN IN THE 53 ALONE ;= 3UBSTANTIAL PROGRESS IN OUR ABILITY TO TREAT THIS DEADLY DISEASE OR MORE ACCURATELY THIS HETEROGENEOUS GROUP OF DISEASES WILL CRITICALLY DEPEND ON THE DISCOVERY OF NEW ANTICANCER DRUGS AND THE DEVELOPMENT OF MORE EFFECTIVE CLINICAL TREATMENT STRATEGIES -UCH OF THE RECENT RESEARCH IN THESE AREAS HAS FOCUSED ON CANCER SPECIFIC MECHA NISMS AND THE CORRESPONDING MOLECULAR TARGETS WHICH MAY BE ADDRESSED EITHER BY SMALL MOLECULES OR INCREASINGLY SO BY THERAPEUTIC ANTIBODIES ;n= (OWEVER IN SPITE OF IMPRESSIVE ADVANCES IN THE DEVELOPMENT OF SIGNAL TRANSDUCTION KINASE INHIBITORS FOR CANCER TREATMENT ; = OR RECEP TOR OR LIGAND DIRECTED ANTIBODIES ; = THE SEARCH FOR IMPROVED CYTOTOXIC AGENTS ACTING ON UBIQUITOUS TARGETS SUCH AS $.! OR TUBULIN STILL CON STITUTES AN IMPORTANT PART OF MODERN ANTICANCER DRUG DISCOVERY !S THE MAJOR TYPES OF SOLID HUMAN TUMORS BREAST LUNG PROSTATE AND COLON WHICH REPRESENT THE VAST MAJORITY OF CANCER CASES TODAY ARE MULTI CAUSAL
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
IN NATURE IT IS INCREASINGLY RECOGNIZED THAT THE TREATMENT OF SOLID TUMORS WITH @MECHANISM BASED AGENTS ALONE IS UNLIKELY TO BE SUCCESSFUL )NSTEAD IMPROVED TREATMENT STRATEGIES ARE LIKELY TO INVOLVE COMBINATIONS OF EG SIGNAL TRANSDUCTION INHIBITORS WITH NEW AND BETTER CYTOTOXIC DRUGS -ICROTUBULE INHIBITORS ARE AN IMPORTANT CLASS OF ANTICANCER AGENTS ;= WHICH ARE USED IN THE CLINICAL TREATMENT OF A VARIETY OF CANCER TYPES EITHER AS SINGLE AGENTS OR AS PART OF DIFFERENT COMBINATION REGIMENS ; = -OL ECULES INTERFERING WITH MICROTUBULE FUNCTION CAN BE GROUPED INTO TWO DIS TINCT CLASSES NAMELY COMPOUNDS THAT INHIBIT THE ASSEMBLY OF TUBULIN HET ERODIMERS INTO MICROTUBULE POLYMERS @TUBULIN POLYMERIZATION INHIBITORS AND THOSE THAT STABILIZE MICROTUBULES UNDER NORMALLY DESTABILIZING CONDI TIONS @MICROTUBULE STABILIZERS ;= 4HE LATTER ALSO PROMOTE THE ASSEMBLY OF TUBULIN HETERODIMERS INTO MICROTUBULE POLYMERS 4UBULIN POLYMERIZATION INHIBITORS SUCH A VINCRISTINE AND VINBLASTINE HAVE BEEN EMPLOYED IN CANCER THERAPY FOR MORE THAN YEARS VINCRISTINE AND VINBLASTINE RECEIVED &$! APPROVAL IN AND RESPECTIVELY )N CONTRAST THE CLINICAL HISTORY OF MICROTUBULE STABILIZERS IS SIGNIFICANTLY SHORTER AND DATES BACK LESS THAN YEARS 4HE FIRST AGENT OF THIS TYPE TO BE INTRODUCED INTO CLINICAL PRACTICE WAS THE NATURAL PRODUCT TAXOL PACLITAXEL 4AXOL¤ IN &IG WHICH WAS FOLLOWED BY &$! APPROVAL FOR THE SEMISYNTHETIC TAXOL ANALOG DOCETAXEL 4AXOTERE¤ &IG IN "OTH COMPOUNDS RAPIDLY BECAME AN IMPORTANT PART OF TODAYS ARMAMENTARIUM IN THE BATTLE AGAINST CANCER WHICH MARKS THE EMERGENCE OF MICROTUBULE STABILIZING ANTICANCER DRUGS AS AN IMPORTANT MILESTONE IN THE DEVELOPMENT OF MODERN CANCER CHEMOTHERAPY ;=
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
7HILE EFFICIENT INHIBITION OF TUBULIN POLYMERIZATION CAN BE EFFECTED BY A NUMBER OF SMALL SYNTHETIC MOLECULES APART FROM COMPLEX NATURAL PRODUCTS LIKE VINCRISTINE OR VINBLASTINE ;= IT IS INTRIGUING TO NOTE THAT ALL POTENT MICROTUBULE STABILIZING AGENTS IDENTIFIED TO DATE ARE NATURAL PROD UCTS OR NATURAL PRODUCT DERIVED FOR RECENT REVIEWS SEE ;n= (ISTORI CALLY MORE THAN A DECADE PASSED AFTER THE ELUCIDATION OF TAXOLS MODE OF ACTION IN ;= BEFORE OTHER MICROTUBULE STABILIZING AGENTS WITH NON TAXOL LIKE STRUCTURES WERE DISCOVERED -OST PROMINENT AMONG THESE NEW MICROTUBULE STABILIZERS IS A GROUP OF BACTERIA DERIVED MACROLIDES THAT WERE DISCOVERED IN BY 2EICHENBACH AND (ÙFLE ;= AND HAVE BEEN TERMED @EPOTHILONES BY THEIR DISCOVERERS &IG ; = 4HE TAXOL LIKE MECHANISM OF ACTION OF THESE AGENTS IE THEIR MICROTU BULE STABILIZING PROPERTIES WAS NOT IMMEDIATELY RECOGNIZED HOWEVER AND WAS ONLY DISCOVERED YEARS LATER BY A GROUP AT -ERCK 2ESEARCH ,ABORATORIES ;= 4HE MAJOR PRODUCTS ORIGINALLY ISOLATED FROM THE MYXOBACTERIUM 3ORAN GIUM CELLULOSUM 3C ARE EPOTHILONE ! AND EPOTHILONE " %PO ! AND " BUT NUMEROUS RELATED MEMBERS OF THIS NATURAL PRODUCTS FAMILY HAVE SUB SEQUENTLY BEEN RECOVERED AS MINOR COMPONENTS FROM FERMENTATIONS OF MYXOBACTERIA ;= 4HE RELATIVE AND ABSOLUTE STEREOCHEMISTRY OF %PO " WAS DETERMINED BY (ÙFLE AND CO WORKERS IN BASED ON A COMBINATION OF 8 RAY CRYSTALLOGRAPHY AND CHEMICAL DEGRADATION STUDIES ;= 4HE AVAIL ABILITY OF THIS INFORMATION IN COMBINATION WITH THEIR AT THAT TIME UNUSUAL MECHANISM OF ACTION MADE %PO ! AND " ATTRACTIVE AND WIDELY PURSUED TAR GETS FOR TOTAL SYNTHESIS AND IN PARTICULAR TURNED THEM INTO IMPORTANT LEAD STRUCTURES FOR ANTICANCER DRUG DISCOVERY )N THIS CONTEXT AND ALTHOUGH NOT
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
REVIEWED IN THIS CHAPTER IT IS ALSO WORTH NOTING THAT A GROWING NUMBER OF ADDITIONAL NATURAL PRODUCTS HAVE BEEN RECOGNIZED OVER THE LAST FEW YEARS TO BE MICROTUBULE STABILIZERS FOR REVIEWS SEE ;n= THUS PROVIDING A WHOLE NEW SET OF DIVERSE LEAD STRUCTURES FOR ANTICANCER DRUG DISCOVERY 7HILE THEY EXERT THEIR ANTIPROLIFERATIVE ACTIVITY THROUGH INTERFERENCE WITH THE SAME MOLECULAR TARGET A MAJOR DISTINCTION BETWEEN TAXOL AND EPOTHILONES IS THE LATTERS ABILITY TO INHIBIT THE GROWTH OF MULTIDRUG RESIS TANT CANCER CELL LINES AT CONCENTRATIONS SIMILAR OR ONLY SLIGHTLY HIGHER THAN THOSE REQUIRED FOR GROWTH INHIBITION OF DRUG SENSITIVE CANCER CELLS ; n= %POTHILONES HAVE ALSO BEEN SHOWN TO BE ACTIVE IN VITRO AGAINST CANCER CELL LINES WHOSE TAXOL RESISTANCE IS MEDIATED BY SPECIFIC TUBULIN MUTATIONS ; = !T THE SAME TIME EPOTHILONES POSSESS MORE FAVORABLE BIOPHARMACEUTICAL PROPERTIES THAN TAXOL EG IMPROVED WATER SOLUBILITY ;= WHICH ENABLES THE USE OF CLINICAL FORMULATION VEHICLES LESS PROBLEM ATIC THAN #REMOPHOR¤ %, &OR A DISCUSSION OF CLINICAL SIDE EFFECTS OF 4AXOL¤ BELIEVED TO ORIGINATE IN THIS PARTICULAR FORMULATION VEHICLE SEE EG ;= %PO " AND A NUMBER OF ITS ANALOGS HAVE BEEN DEMONSTRATED TO POSSESS POTENT IN VIVO ANTITUMOR ACTIVITY AND UP TO THIS POINT AT LEAST SEVEN COM POUNDS BASED ON THE EPOTHILONE STRUCTURAL SCAFFOLD HAVE ENTERED CLINICAL EVALUATION IN HUMANS 4HESE INCLUDE %PO " ITSELF %0/ PATUPILONE DEVELOPED BY .OVARTIS %PO $ DEOXY%PO " +/3 +OSAN 2OCHE "-3 IXABEPILONE THE LACTAM ANALOG OF %PO " "-3 "-3 # AMINO %PO " "-3 !"* # DESMETHYL # METHYLSULFANYL %PO " .OVARTIS DIDEHYDRO %PO " +/3 +OSAN2OCHE AND THE FULLY SYNTHETIC ANALOG :+ %PO "AYER 3CHERING 4HESE COMPOUNDS WILL BE DISCUSSED IN MORE DETAIL BELOW !S INDICATED ABOVE EPOTHILONES HAVE BEEN AND CONTINUE TO BE ATTRAC TIVE TARGETS FOR TOTAL CHEMICAL SYNTHESIS AND NUMEROUS SYNTHESES OF %PO ! AND " HAVE BEEN PUBLISHED IN THE LITERATURE FOR REVIEWS SEE ;n= SINCE THE FIRST DISCLOSURE OF THEIR ABSOLUTE STEREOCHEMISTRY IN ;= !T THE SAME TIME THE METHODOLOGY DEVELOPED IN THE COURSE OF THOSE STUDIES HAS BEEN EXPLOITED FOR THE SYNTHESIS OF A HOST OF SYNTHETIC ANALOGS REVIEWED IN ; n = WHICH HAVE ALLOWED THE EMPIRICAL ELUCIDATION OF THE MOST IMPORTANT STRUCTURAL PARAMETERS REQUIRED FOR BIOLOGICAL ACTIV ITY 4HE CHEMISTRY DEVELOPED FOR THE PREPARATION OF SOME OF THESE ANALOGS SHOULD EVEN ALLOW THE PRODUCTION OF AMOUNTS OF MATERIAL SUFFICIENT FOR CLINICAL TRIALS ; = THUS HIGHLIGHTING THE DIFFERENCE IN STRUCTURAL COM
Epothilones as lead structures for new anticancer drugs
plexity (which is reflected in synthetic accessibility) between epothilonetype structures and taxol, for which an industrial scale synthesis is clearly out of reach. The chemistry, biology, and SAR (structure-activity-relationship) of epothilones have been extensively discussed in recent review articles [22, 25, 26, 29, 32–36] and it is not the goal of this chapter to recite all these different facets of epothilone-related research. Rather, this article will focus on some selected aspects of the fermentation, chemistry, biology, and clinical evaluation of natural epothilones and their synthetic and semisynthetic analogs, with particular emphasis on SAR work performed in our own laboratories. While this does not include any detailed discussion of the organic chemistry of epothilones, Section 3 provides an outline of the fermentation process that has been developed at Novartis for the production of Epo B, which will also be compared with processes established by other groups. In this context, some of the impressive advances in the elucidation of epothilone biosynthesis and the development of heterologous expression systems will also be briefly highlighted. However, for a more detailed discussion of this subject the reader is referred to two recent review articles [16, 38].
2
Biology and biochemistry of Epo B
2.1 Effects in vitro The basic biology and pharmacology of Epo B, which is the most potent (and, therefore, most extensively studied natural epothilone) have been reviewed in a number of previous accounts [16, 22, 25, 32, 34, 35, 39, 40]. As indicated above, the biological effects of the compound are based on its ability to bind to microtubules, thereby altering the intrinsic stability and dynamic properties of these supramolecular structures. In cell-free in vitro systems this is demonstrated by the prevention of Ca2+- or cold-induced depolymerization of preformed microtubule polymers [21] as well as the promotion of tubulin polymerization (to form microtubule-like polymers) in the absence of either microtubule associated proteins (MAPs) and/or guanosine triphosphate (GTP), at temperatures significantly below 37°C, and in the presence of Ca2+ [18, 21]. The latter phenomenon, i.e., the
279
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INDUCTION OF TUBULIN POLYMERIZATION IS FREQUENTLY USED AS A QUANTITATIVE BIOCHEMICAL READOUT FOR THE INTERACTION OF LIGANDS WITH THE TUBULINMICRO TUBULE SYSTEM %PO " IS A MORE POTENT TUBULIN POLYMERIZING AGENT THAN TAXOL WHICH IN TURN POLYMERIZES TUBULIN WITH ABOUT EQUAL EFFICIENCY AS %PO ! EG %# VALUES FOR THE POLYMERIZATION OF MICROTUBULE PROTEIN BY %PO ! %PO " AND TAXOL HAVE BEEN DETERMINED AS AND μ- RESPECTIVELY ;= )T IS IMPORTANT TO NOTE HOWEVER THAT THE EXACT MAG NITUDE OF TUBULIN POLYMERIZING EFFECTS IN VITRO ABSOLUTE AND EVEN RELA TIVE POLYMERIZATION RATES EXTENT OF TUBULIN POLYMER FORMATION STRONGLY DEPENDS ON THE ASSAY CONDITIONS EMPLOYED EG BIOLOGICAL SOURCE AND PURITY OF TUBULIN CONCENTRATION OF MICROTUBULE STABILIZING BUFFER COMPO NENTS AND REACTION TEMPERATURE ;= %POTHILONES ARE ABLE TO DISPLACE ;(= TAXOL FROM MICROTUBULES WITH EFFI CIENCIES SIMILAR OR SUPERIOR TO THOSE OF UNLABELLED TAXOL OR DOCETAXEL ; = )NHIBITION OF TAXOL BINDING BY EPOTHILONES IS OF THE COMPETITIVE TYPE WITH APPARENT +I VALUES OF μ- %PO ! AND μ- %PO " WHICH IMPLIES THAT THE MICROTUBULE BINDING SITES OF TAXOL AND %PO !" ARE LARGELY OVERLAPPING OR EVEN IDENTICAL VIDE INFRA -ORE RECENTLY THE BINDING CON STANTS OF %PO ! AND " TO STABILIZED MICROTUBULES IN VITRO AT # HAVE BEEN DETERMINED AS × -n %PO ! AND × -n %PO " USING A FLUORESCENCE BASED DISPLACEMENT ASSAY ;= -ICROTUBULE STABILIZATION BY EPOTHILONES AT LOW TEMPERATURES HAS ALSO BEEN DEMONSTRATED IN CELLS ;= BUT THE EFFECT LIKE CANCER CELL GROWTH INHIBITION VIDE INFRA IS OBSERVED AT STRIKINGLY LOWER CONCENTRATIONS THAN THOSE REQUIRED FOR THE INDUCTION OF TUBULIN POLYMERIZATION IN VITRO 4HIS APPARENT DISCREPANCY HAS BEEN RESOLVED BY CAREFUL UPTAKE EXPERIMENTS IN (E,A CELLS ; = WHICH HAVE SHOWN THAT %PO ! AND " LIKE TAXOL ;= ACCUMULATE SEVERAL HUNDRED FOLD INSIDE CELLS OVER EXTERNAL MEDIUM CON CENTRATIONS 3IMILAR FINDINGS HAVE BEEN REPORTED FOR A CLOSE ANALOG OF %PO " IN -#& CELLS ;= %ARLY EXPERIMENTS INVESTIGATING THE EFFECTS OF EPOTHILONES ON MICRO TUBULE BUNDLING IN INTACT CELLS HAD DEMONSTRATED THAT TREATMENT OF CUL TURED CELLS 2!4 (E,A (S4 (S"ST 0T+ WITH HIGH nnn - CONCENTRATIONS OF EPOTHILONES RESULTED IN THE FORMATION OF CHARACTERIS TIC EXTENSIVE MICROTUBULE BUNDLES LATERAL ASSOCIATION OF MICROTUBULES THROUGHOUT THE CYTOPLASM OF INTERPHASE CELLS ; = )N CONTRAST INTER PHASE MICROTUBULE ARRAYS WERE REPORTED TO REMAIN LARGELY UNAFFECTED AT
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
4ABLE (UMAN CANCER CELL GROWTH INHIBITION BY %PO ! AND " AND TAXOL )# VALUES ;N-= A #ELL LINE (#4 COLON
0# - ! PROSTATE LUNG
-#& BREAST
-#& !$2B C
+" CERVIX
+" B D CERVIX
%PO !
%PO "
4AXOL
A#ELLS WERE EXPOSED TO DRUGS FOR n DAYS ALLOWING FOR AT LEAST TWO POPULATION DOUBLINGS #ELL NUM
BERS WERE ESTIMATED BY QUANTIFICATION OF PROTEIN CONTENT OF FIXED CELLS BY METHYLENE BLUE STAINING $ATA FROM ;= B-ULTIDRUG RESISTANT CELL LINES C-ULTIPLE RESISTANCE MECHANISMS-$2 D0 GP OVEREXPRESSION-$2
LOWER EPOTHILONE CONCENTRATIONS nnn - WITH THE PRIMARY EFFECT OCCURRING ON CELLS ENTERING MITOSIS ;= 7HILE THE MORE RELEVANT EFFECTS OF EPOTHILONES A LOW N- CONCENTRATIONS ARE CERTAINLY ASSOCIATED WITH THE SPINDLE APPARATUS IT HAS ALSO BEEN DEMONSTRATED EMPLOYING LIVE FLUORES CENCE MICROSCOPY OF (E,A CELLS ECTOPICALLY EXPRESSING MOUSE β TUBULIN FUSED TO ENHANCED GREEN FLUORESCENT PROTEIN %'&0 THAT EVEN LOW N- CONCENTRATIONS OF %PO " LEAD TO THE BUNDLING OF INTERPHASE MICROTUBULES AFTER H OF DRUG EXPOSURE ;= )T IS GENERALLY ASSUMED THAT THE GROWTH INHIBITORY EFFECT OF EPOTHILONES AND ALSO OTHER MICROTUBULE INTERACTING AGENTS IS A CONSEQUENCE OF THE SUPPRESSION OF MICROTUBULE DYNAMICS RATHER THAN AN OVERALL INCREASE IN MICROTUBULE POLYMER MASS DUE TO MASSIVE INDUCTION OF TUBULIN POLYMER IZATION ;= 4HE CONCENTRATION DEPENDENT INHIBITION OF THE DYNAMICS OF INTERPHASE MICROTUBULES BY %PO " HAS BEEN EXPERIMENTALLY DEMONSTRATED BY +AMATH AND *ORDAN ;= BY MEANS OF TIME LAPSE MICROSCOPY IN -#& CELLS STABLY TRANSFECTED WITH '&0 GREEN FLUORESCENT PROTEIN α TUBULIN 4REATMENT OF HUMAN CANCER CELLS WITH LOW N- CONCENTRATIONS OF %PO " LEADS TO PROFOUND GROWTH INHIBITION AND CELL DEATH 4AB )N LINE WITH THE EFFECTS ON TUBULIN POLYMERIZATION IN VITRO %PO " IS A MORE POTENT ANTIP ROLIFERATIVE AGENT THAN %PO ! WHICH IN TURN IS ABOUT EQUIPOTENT WITH TAXOL %POTHILONE TREATMENT PRODUCES ABERRANT MITOTIC SPINDLES RESULTS IN CELL CYCLE ARREST IN MITOSIS AND EVENTUALLY LEADS TO APOPTOTIC CELL DEATH ; =
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7HILE IT IS OFTEN ASSUMED THAT APOPTOSIS IS A DIRECT CONSEQUENCE OF '- ARREST (ORWITZ AND CO WORKERS HAVE RECENTLY SHOWN THAT THE SITUATION IS CLEARLY MORE COMPLEX ; = 4HUS LOW CONCENTRATIONS OF %PO " AND ALSO TAXOL OR DISCODERMOLIDE PRODUCE A LARGE ANEUPLOID CELL POPULATION IN ! LUNG CARCINOMA CELLS IN THE ABSENCE OF A MITOTIC BLOCK 4HESE CELLS ARISE FROM ABERRANT MITOSIS AFTER FORMATION OF MULTIPOLAR SPINDLES ARE ARRESTED IN THE ' PHASE OF THE CELL CYCLE AND EVENTUALLY WILL UNDERGO APOPTOSIS /N THE OTHER HAND HIGHER DRUG CONCENTRATIONS LEAD TO A PROTRACTED MITOTIC BLOCK FROM WHICH THE CELLS EXIT WITHOUT DIVISION THUS FORMING TETRAPLOID ' CELLS ;= .O SUCH DIFFERENTIAL BEHAVIOR WAS OBSERVED FOR TUBULIN POLYMERIZATION INHIBITORS SUCH AS COLCHICINE NOCODAZOLE OR VINBLASTINE WHICH DO NOT GIVE RISE TO ANEUPLOID CELLS BUT ALWAYS LEAD TO MITOTIC ARREST FOLLOWED BY APOPTOSIS ;= 4HIS SUGGESTS THAT THE SUPPRESSION OF MICROTUBULE DYNAM ICS WHICH IS COMMON TO BOTH TYPES OF TUBULIN INTERACTING AGENTS CANNOT FULLY ACCOUNT FOR THE COMPLEX ARRAY OF BIOLOGICAL EFFECTS DISPLAYED BY %PO " OR OTHER MICROTUBULE STABILIZING AGENTS )N SUMMARY THE RESULTS FROM THE (ORWITZ LAB DEMONSTRATE THAT ENTRY OF CELLS INTO MITOSIS IS A FUNDAMENTAL PREREQUISITE FOR CELL KILLING BY MICROTUBULE STABILIZING AGENTS BUT THAT CELL DEATH ITSELF DOES NOT NECESSARILY REQUIRE PRIOR MITOTIC ARREST !T LOW CON CENTRATIONS OF %PO " CELLS MAY SIMPLY UNDERGO MITOTIC SLIPPAGE ABERRANT MITOSIS AND SUBSEQUENT CELL CYCLE ARREST IN ' !S FOR OTHER TYPES OF CYTOTOXIC ANTICANCER DRUGS THE CELLULAR RESPONSE TO MICROTUBULE STABILIZING AGENTS CAN BE MODULATED BY ADAPTIVE CHANGES OF THE CELL WHICH LEAD TO ACQUIRED DRUG RESISTANCE !LTERNATIVELY CELLS MAY BE INHERENTLY PROTECTED FROM THE ANTIPROLIFERATIVE EFFECTS OF CYTOTOXIC AGENTS BY A VARIETY OF MECHANISMS !S INDICATED ABOVE %PO !" IN CONTRAST TO TAXOL AND OTHER STANDARD CYTOTOXIC ANTICANCER AGENTS ARE LARGELY NON SUS CEPTIBLE TO 0 GLYCOPROTEIN 0 GP MEDIATED DRUG EFFLUX AND THUS RETAIN FULL OR ALMOST FULL ANTIPROLIFERATIVE ACTIVITY AGAINST THE CORRESPONDING MULTIDRUG RESISTANT CELL LINES IN VITRO ; = 4AB 4HIS MAY REPRESENT A DISTINCTIVE ADVANTAGE OF EPOTHILONES OVER CURRENT TAXANE BASED THERAPY PROVIDED THAT 0 GP MEDIATED DRUG RESISTANCE IS INDEED OF STRONG CLINICAL RELEVANCE 4HE LATTER QUESTION HOWEVER IS A MATTER OF SIGNIFICANT DEBATE ;= !T THE SAME TIME IT IS NOW WELL ESTABLISHED THAT CANCER CELLS CAN BECOME RESISTANT TO EPOTHILONES THROUGH ALTERNATIVE IE NON 0GP DEPENDENT MECHANISMS SUCH AS TUBULIN MUTATIONS &OR EXAMPLE 7ART MANN ET AL ;= HAVE ISOLATED AN EPOTHILONE RESISTANT SUBLINE OF THE +"
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
CERVIX CARCINOMA CELL LINE TERMED +" # IN WHICH 4HR WHICH MAPS TO THE TAXOLEPOTHILONE BINDING SITE ON TUBULIN ; = IS MUTATED TO 0RO RESULTING IN FOLD RESISTANCE TO %PO ! AND FOLD RESISTANCE TO %PO " 3IMILAR FINDINGS HAVE BEEN INDEPENDENTLY REPORTED BY 'IANNAKAKOU ET AL WHO HAVE PRODUCED AN %PO ! RESISTANT CELL LINE !! IN WHICH 4HR IS MUTATED TO )LE RATHER THAN 0RO ;= #ONSISTENT WITH THE FACT OF A SHARED BINDING SITE BETWEEN EPOTHILONES AND TAXOL BOTH !! AS WELL AS +" # CELLS ARE CROSS RESISTANT TO TAXOL ALBEIT TO VARYING DEGREES -ORE RECENTLY (E ET AL HAVE GENERATED THREE DIFFERENT %PO RESISTANT CELL LINES !" (E,A%PO! AND (E,A%PO" EACH OF WHICH IS CHARAC TERIZED BY A SPECIFIC β TUBULIN MUTATION NAMELY 'LN'LU IN !" CELLS 0RO!LA IN (E,A%PO! CELLS AND 4YR#YS IN (E,A%PO" CELLS ;= 4HE HIGHEST DEGREE OF RESISTANCE WAS ASSOCIATED WITH !%PO" CELLS WHICH WERE FOLD RESISTANT TO %PO " AND ALSO EXHIBITED MARKED CROSS RESISTANCE WITH OTHER MICROTUBULE STABILIZING AGENTS WITH THE NOTA BLE EXCEPTION OF DISCODERMOLIDE )N ADDITION THESE CELLS WERE HYPERSEN SITIVE TOWARDS TUBULIN POLYMERIZATION INHIBITORS AND THIS BEHAVIOR MAY PROVIDE A CLUE AS TO THE EFFECTS OF THE 'LN'LU MUTATION ON MICROTUBULE STABILITY VIDE INFRA 4HROUGH FURTHER SELECTION OF !" CELLS (ORWITZ AND CO WORKERS HAVE SUBSEQUENTLY GENERATED AN EVEN MORE RESISTANT CELL LINE !%PO" ;= WHICH IS CA FOLD RESISTANT TO %PO " )N ADDI TION TO THE β'LN'LU MUTATION THE %PO" LINE IS CHARACTERIZED BY MUTATIONS OF β FROM 6AL TO 0HE AND OF α α TUBULIN FROM ,EU TO -ET !S β6AL IS LOCATED AT THE END OF THE ( 3 LOOP WHICH IS ASSUMED TO BE INVOLVED IN - LOOP CONTACTS BETWEEN PROTOFILAMENTS AMINO ACID CHANGES AT THIS POSITION COULD AFFECT LATERAL CONTACTS BETWEEN PROTOFILAMENTS AND THUS LEAD TO LESS STABLE MICROTUBULES 4HIS HYPOTHESIS IS SUPPORTED BY THE FACT THAT %PO" CELLS ARE AGAIN HYPERSENSITIVE TO THE ACTION OF VINBLAS TINE BUT RATHER INTRIGUINGLY ALSO DEPEND ON LOW CONCENTRATIONS OF %PO " FOR SURVIVAL ;= 4HE ABOVE 'LN'LU MUTATION WAS ALSO IDENTIFIED BY 6ERRILLS ET AL ;= IN A HIGHLY RESISTANT SUBLINE OF THE HUMAN 4 CELL ACUTE LEUKEMIA CELL LINE ##2& #%- IN COMBINATION WITH A SECOND MUTATION AT POSITION OF β TUBULIN 4HR → !LA 4HIS CELL LINE TERMED D%PO" IS FOLD RESISTANT TO %PO $ WHICH WAS THE SELECTING AGENT AND EXHIBITS FOLD AND FOLD CROSS RESISTANCE WITH %PO " AND TAXOL RESPECTIVELY %PO $ FAILED TO INDUCE ANY MEASURABLE TUBULIN POLYMERIZATION IN CELL LYSATES PREPARED
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FROM D%PO" CELLS AT μ- COMPOUND CONCENTRATION THUS ILLUSTRATING THAT IMPAIRED GROWTH INHIBITION IS INDEED PARALLELED BY DIMINISHED EFFECTS ON TUBULIN POLYMERIZATION )N SUMMARY ALL TUBULIN MUTATIONS IDENTIFIED TO DATE IN EPOTHILONE RESISTANT CELLS ARE FOUND IN REGIONS OF THE TUBULIN STRUCTURE WHICH ARE PREDICTED TO BE IMPORTANT FOR TUBULIN POLYMERIZATION ANDOR MICROTUBULE STABILITY INCLUDING THOSE WHICH MAY ADDITIONALLY AFFECT DRUG BINDING 4HUS THESE MUTATIONS MAY NOT ONLY AFFECT DRUG TARGET INTERACTIONS BUT THEY MAY ALSO OR ALTERNATIVELY IMPAIR INTRINSIC TUBULIN FUNCTIONS IN A WAY THAT COULD RESULT IN HYPOSTABLE MICROTUBULES ; = 4HIS HYPOTHESIS WHICH HAS BEEN PREVIOUSLY PUT FORWARD BY #ABRAL ET AL BASED ON OBSERVA TIONS ON TAXOL RESISTANT #(/ CELLS ;= IS SUPPORTED BY THE FACT THAT ALL THE ABOVE CELL LINES ARE HYPERSENSITIVE TO TUBULIN DEPOLYMERIZING DRUGS SUCH AS VINCRISTINE OR COLCHICINE 4UBULIN MUTATIONS ARISING IN RESPONSE TO TAXOL TREATMENT OF HUMAN CAN CER CELL LINES DO NOT NECESSARILY LEAD TO CROSS RESISTANCE WITH EPOTHILONES ;= AND AGAIN THIS MAY ENABLE EPOTHILONE BASED TREATMENT OF 4AXOL¤ RESISTANT TUMORS (OWEVER ANY SUCH PREDICTIONS MUST BE TREATED WITH GREAT CAUTION AS THE CLINICAL SIGNIFICANCE OF INDIVIDUAL RESISTANCE MECHANISMS IDENTIFIED IN VITRO HAS NOT BEEN ESTABLISHED
)N VIVO ANTITUMOR ACTIVITY 4HE IN VIVO EFFECTS OF %PO " HAVE BEEN INVESTIGATED IN SOME DETAIL BY A GROUP AT THE 3LOAN +ETTERING #ANCER #ENTER AS WELL AS THE GROUP AT .OVARTIS 7HILE EARLY EXPERIMENTS BY THE 3LOAN +ETTERING GROUP IN XENOGRAFT MODELS OF HUMAN LEUKEMIA ##2& #%- AND ##2& #%-6", -$2 IN #" 3#)$ MICE DRUG SENSITIVE AS WELL AS MULTIDRUG RESISTANT TUMORS HAD SUGGESTED PROMISING ANTITUMOR ACTIVITY ;= THE COMPOUND IN SUBSEQUENT EXPERI MENTS WAS FOUND TO HAVE ONLY LIMITED EFFECTS ON TUMOR GROWTH IN HUMAN -8 BREAST OR 3+/6 OVARIAN TUMORS IN MICE AND TO EXHIBIT CONSIDERABLE TOXICITY 4HESE DATA LED TO THE CONCLUSION THAT %PO " MIGHT SIMPLY BE TOO TOXIC TO BECOME A CLINICALLY USEFUL ANTICANCER AGENT ;= )N CONTRAST TO THESE FINDINGS STUDIES BY THE .OVARTIS GROUP HAVE DEM ONSTRATED POTENT ANTITUMOR ACTIVITY OF %PO " IN A NUMBER OF DRUG SENSITIVE HUMAN TUMOR MODELS NUDE MICE UPON IV ADMINISTRATION ; =
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
!CTIVITY WAS OBSERVED IN MODELS ENCOMPASSING ALL FOUR MAJOR TYPES OF SOLID HUMAN TUMORS LUNG BREAST COLON PROSTATE AND WAS MANIFEST EITHER AS PROFOUND GROWTH INHIBITION STABLE DISEASE OR SIGNIFICANT TUMOR REGRESSION )N ADDITION %PO " WAS FOUND TO BE A POTENT INHIBITOR OF TUMOR GROWTH IN 0 GP OVEREXPRESSING MULTIDRUG RESISTANT HUMAN TUMOR MODELS 2EGRESSIONS WERE OBSERVED IN TWO SUCH MODELS +" CERVIX CARCINOMA ;= AND (#4 COLON CARCINOMA ;= WHERE TUMORS WERE EITHER POORLY RESPON SIVE OR COMPLETELY NON RESPONSIVE TO TREATMENT WITH 4AXOL¤ !LTHOUGH %PO " TREATMENT WAS FREQUENTLY ACCOMPANIED BY SIGNIFICANT BODY WEIGHT LOSS THERAPEUTIC EFFECTS COULD GENERALLY BE ACHIEVED AT TOLERATED DOSE LEVELS !S DEMONSTRATED IN A RECENT STUDY BY 0IETRAS ET AL THE ANTITUMOR EFFECT OF %PO " IN A MODEL OF HUMAN ANAPLASTIC THYROID CARCINOMA CAN BE POTENTIATED BY CO ADMINISTRATION WITH THE TYROSINE KINASE INHIBITOR 34) 'LEEVEC¤ WITHOUT ANY OVERT DECREASE IN TOLERABILITY ;= 4HE ENHANCED ANTITUMOR ACTIVITY OF THE COMBINATION IS PRESUMED TO BE A CONSEQUENCE OF A SELECTIVE ENHANCEMENT IN DRUG UPTAKE BY THE TUMOR DUE TO INHIBITION OF 0$'& 2 PLATELET DERIVED GROWTH FACTOR RECEPTOR BY 34) 4HE EXACT REASONS FOR THE DISPARATE RESULTS OF IN VIVO EXPERIMENTS BY THE 3LOAN +ETTERING AND THE .OVARTIS GROUPS ARE UNKNOWN AT THIS POINT )T SHOULD BE NOTED HOWEVER THAT THE DATA ARE NOT NECESSARILY INCOMPATIBLE BUT MAY SIMPLY REFLECT DIFFERENCES IN THE EXPERIMENTAL SET UPS SUCH AS TUMOR MODELS FORMULATION ANDOR DOSING REGIMENS
&ERMENTATION OF EPOTHILONES /RIGIN AND BIOSYNTHESIS /F THE VARIOUS EPOTHILONE ANALOGS THAT HAVE ENTERED CLINICAL TRIALS SO FAR VIDE SUPRA ONLY ONE IS KNOWN TO BE PRODUCED BY TOTAL SYNTHESIS NAMELY :+ %PO SAGOPILONE ;= !LL OTHER COMPOUNDS ARE EITHER PRODUCED DIRECTLY THROUGH FERMENTATION OR THROUGH SEMISYNTHESIS /BVIOUSLY A FERMENTA TION BASED PRODUCTION PROCESS FOR THE NATURAL PRODUCT STARTING MATERIAL IS ALSO REQUIRED IN THE LATTER CASE !S INDICATED IN THE )NTRODUCTION EPOTHILONES WERE FIRST ISOLATED BY (ÙFLE AND 2EICHENBACH FROM THE MYXOBACTERIUM 3ORANGIUM CELLULOSUM 3O CE WHICH HAD IN TURN BEEN RECOVERED FROM A SOIL SAMPLE COLLECTED FROM
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
THE BANKS OF THE :AMBESI 2IVER IN 3OUTH !FRICA IN ; = )N GEN ERAL EPOTHILONE BIOSYNTHESIS HAS ONLY BEEN FOUND IN A LIMITED NUMBER OF STRAINS OF THE MYXOBACTERIUM SPECIES 3ORANGIUM CELLULOSUM SUCH AS 3 3O CE OR 3 COELICOLOR -YXOBACTERIA ARE UBIQUITOUS MICROPREDATORS OR SAPROPHYTES THAT ARE FOUND IN SOIL ENVIRONMENTS 4HEY HAVE A GLIDING MEANS OF MOTILITY SHOW COMPLEX SOCIAL INTERACTIONS AND DISPLAY INTRIGUING MORPHOLOGICAL DEVELOP MENT WHICH CULMINATES IN THE PRODUCTION OF MULTICELLULAR MACROSCOPIC FRUITING BODIES AND MYXOSPORA ;= !LONG WITH CYANOBACTERIA AND THE BASIDIOMYCETES STRAINS OF THE 'RAM NEGATIVE MYXOBACTERIA HAVE EMERGED AS A RICH SOURCE OF SECONDARY METABOLITES WITH NOVEL TYPES OF MOLECULAR STRUCTURES AND AN OFTEN UNIQUE MODE OF ACTION ;= -ANY MYXOBACTERIA ARE DIFFICULT TO FERMENT HOWEVER AND THIS IS ALSO THE CASE WITH 3ORANGIUM CELLULOSUM )N ADDITION PRODUCT LEVELS IN CULTURES FOR SECONDARY METABOLITES ARE OFTEN LOW DUE TO PRODUCT DEGRADATION OR END PRODUCT INHIBITION !DSORBER RESINS EG THE NONIONIC HYDROPHOBIC POLYMERIC RESIN 8!$ ¤ 2OHM (AAS ARE OFTEN USED IN MYXOBACTERIAL CULTIVATIONS TO STABILIZE PRODUCTS AGAINST DEGRADATION AND TO AVOID FEED BACK INHIBITION ;= !DSORPTION TO RESIN BEADS ALSO FACILITATES THE RECOVERY OF PRODUCTS AS THE RESIN CAN BE SEPARATED AND THE PRODUCTS SUBSEQUENTLY ELUTED FROM THE SOLID PHASE WITH APPROPRIATE SOLVENT SYSTEMS 4HE EPOTHI LONE BINDING CAPACITY OF FRESH 8!$ HAS BEEN DETERMINED AT ABOUT MGG RESIN (OWEVER VARIOUS CONTAMINATING COMPOUNDS MAY ALSO BIND TO THE RESIN AND THIS MAY REDUCE THE BINDING CAPACITY FOR THE TARGET PRODUCT BY UP TO ;= )N ADDITION RESIN BEADS MAY CAUSE MECHANICAL DAMAGE TO LARGE SCALE EQUIPMENT SUCH AS ABRASION ON STIRRER BLADES AND BAFFLES OR IMPAIRMENT OF VALVES AND SEALS /N THE OTHER HAND THE RESIN BEADS ARE THEMSELVES AFFECTED BY IMPACT AND COLLISION WHICH CAN LEAD TO BEAD FRAC TURE AND SUBSEQUENT LOSS OF PRODUCT WHICH IS BOUND TO BEAD FRAGMENTS THAT ARE TOO SMALL TO BE RECOVERED "IOSYNTHETIC ROUTES FOR EPOTHILONES WERE FIRST PROPOSED AFTER SEQUENC ING AND ANALYZING THE BIOSYNTHETIC GENE CLUSTER OF 3ORANGIUM CELLULOSUM 3O CE WHICH INDICATED THAT A POLYKETIDE SYNTHETASE 0+3 AND A NON RIBOSOMAL PEPTIDE SYNTHETASE .203 WERE INVOLVED IN THE ASSEMBLY OF THE CARBON SKELETON AND THE THIAZOLE HETEROCYCLE OF EPOTHILONES RESPECTIVELY ; = &EEDING EXPERIMENTS WITH PRECURSORS REVEALED THAT THE INITIAL STEPS IN THE BIOSYNTHESIS CONSIST IN THE FORMATION OF THE THIAZOLE RING
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
FROM ACETATE AND CYSTEINE WHICH IS FOLLOWED BY THE CONSTRUCTION OF THE POLYKETIDE PART FROM ACETATE AND PROPIONATE UNITS PLUS ONE METHYL GROUP FROM METHIONINE ;= )T WAS ALSO DEMONSTRATED THAT THE FINAL PRODUCTS FORMED BY THE 0+3 ARE %POS # AND $ DEOXYEPOTHILONES WHILE THE # # EPOXIDE IN %PO ! AND " IS INTRODUCED SUBSEQUENTLY BY A POST 0+3 MODIFYING ENZYME NAMELY %PO #$ MONOOXYGENASE ;= ALSO REFERRED TO AS %PO+ 0 EPOXIDASE ;= 4HE LATTER ENZYME SEEMS TO BE FEEDBACK REGULATED BY ITS PRODUCTS LEADING TO INHIBITION OF THE EPOXIDATION OF %PO # AND $ ONCE %PO ! OR " ARE PRESENT IN THE CULTURE )N ADDITION %PO ! AND " CAN BE FURTHER TRANSFORMED TO %POS % AND & BY A SECOND MONOOXY GENASE %PO !" MONOOXYGENASE WHICH HYDROXYLATES THE # METHYL GROUP ON THE THIAZOLE RING ;= )T HAS BEEN SHOWN THAT BOTH FEEDBACK INHIBITION AND THE DEGRADATION OF %PO ! AND " CAN BE PREVENTED WHEN ADSORBER RESINS SUCH AS 8!$ ARE ADDED TO THE MEDIUM OF 3 CELLULOSUM CULTURES 7HILE %PO $" AND %PO #! DIFFER BY THE PRESENCE OR ABSENCE RESPEC TIVELY OF A METHYL GROUP ON # BOTH TYPES OF EPOTHILONES ARE PRODUCED BY THE SAME 0+3 4HE DIFFERENCE IN PRODUCT FORMATION SIMPLY ARISES FROM ALTERNATIVE PRECURSOR USAGE BY THE RESPECTIVE 0+3 MODULE WHICH MAY EITHER INCORPORATE AN ACETATE → %POS #! OR A PROPIONATE UNIT → %POS $" ;= 4HE %PO $%PO # RATIO AND CONSEQUENTLY ALSO THE %PO "%PO ! RATIO CAN BE INCREASED BY ADDITION OF PROPIONATE TO THE CULTURE MEDIUM ;= 7ITH THIS KNOWLEDGE AVAILABLE ADVANCED PRODUCTION PROCESSES HAVE BEEN ESTABLISHED FOR EPOTHILONE FERMENTATION BY THOSE COMPANIES THAT HAVE EPOTHILONE BASED DRUG CANDIDATES IN CLINICAL DEVELOPMENT 4HE FOL LOWING CHAPTER WILL PROVIDE AN OUTLINE OF THESE PROCESSES IN RELATION TO THOSE CLINICAL CANDIDATES WHOSE PRODUCTION IS FERMENTATION BASED EITHER DIRECTLY OR INDIRECTLY
&ERMENTATION OF %PO " 0RODUCTION OF )XABEPILONE "-3 AND "-3 )XABEPILONE "-3 AND "-3 &IG ARE SEMISYNTHETIC DERIVATIVES OF %PO " WHICH REQUIRE %PO " AS THE STARTING MATERIAL FOR CHEMI CAL TRANSFORMATION
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
)N ORDER TO PROVIDE SUFFICIENT AMOUNTS OF STARTING %PO " FOR THE CORRE SPONDING PRODUCTION PROCESSES A SIGNIFICANTLY IMPROVED PRODUCTION STRAIN WAS GENERATED BY THE "-3 GROUP THROUGH MUTAGENESIS OF THE ORIGINAL 3ORAN GIUM CELLULOSUM STRAIN 3O CE ;= 4HIS PROCESS IS LARGELY BASED ON THE PROCESS ORIGINALLY DESCRIBED BY (ÙFLE ET AL IN ;= USING ESSENTIALLY THE SAME PRODUCTION MEDIUM AND MAKING USE OF THE HYDROPHOBIC ADSORBER RESIN 8!$ FOR PRODUCT CAPTURE -AJOR IMPROVEMENTS AGAINST THE ORIGINAL PROCEDURE ARE THE FEEDING OF NUTRIENTS TO THE PRODUCTION MEDIUM IN ORDER TO PROLONG THE PRODUCTION PERIOD AND ENHANCE THE PRODUCT TITER AND THE ADDI TION OF PROPIONATE IN ORDER TO INCREASE THE %PO "%PO ! RATIO ;= 4HE PRO CESS IS PERFORMED IN , SCALE FERMENTORS 4HE HARVESTING VOLUME AFTER n DAYS IS CA , CONTAINING KG OF ADSORBER RESIN TYPICALLY LOADED WITH CA KG OF %PO " !FTER HARVESTING THE RESIN IS SEPARATED WASHED AND LOADED INTO A COLUMN !FTER ELUTION WITH -4"% METHYL TERT BUTYL ETHER %PO " IS PURIFIED BY A SEQUENCE OF CRYSTALLIZATION AND CHROMATOGRAPHY STEPS FINALLY YIELDING n KG OF %PO " CONTAINING LESS THAN OF %PO !
0RODUCTION OF 0ATUPILONE %0/ 0ATUPILONE IS THE GENERIC NAME FOR THE NATURAL PRODUCT %PO " AND AN OPTI MIZED PRODUCTION STRAIN FOR THE LARGE SCALE PRODUCTION OF %PO " WAS DERIVED IN OUR GROUP AT .OVARTIS BY MUTAGENESIS OF THE ORIGINAL STRAIN 3ORANGIUM CEL LULOSUM 3O CE )N CONTRAST TO THE PROCESS OUTLINED ABOVE NO SOLID ADSORBER RESIN SUCH AS 8!$ IS REQUIRED IN THE FERMENTATION THUS ELIMINATING THE VARIOUS PROBLEMS THAT CAN BE ASSOCIATED WITH THE USE OF SOLID RESIN BEADS IN LARGE SCALE FERMENTORS VIDE SUPRA )NSTEAD A WATER SOLUBLE COMPLEXING
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
AGENT WAS SEARCHED FOR THAT WOULD STABILIZE %PO " DURING CULTIVATION )T WAS DISCOVERED THAT CYCLODEXTRINS ARE CAPABLE TO CAPTURE EPOTHILONES AND PRO TECT THEM AGAINST DEGRADATION AT THE SAME TIME FEEDBACK INHIBITION OF THE BIOSYNTHESIS IS PREVENTED #YCLODEXTRINS ARE CYCLIC α LINKED OLIGOSAC CHARIDES OF SIX OR MORE α $ GLUCOPYRANOSE UNITS WITH A HYDROPHILIC EXTERIOR AND A RELATIVELY HYDROPHOBIC CENTRAL CAVITY IN WHICH MICROBIAL PRODUCTS SUCH AS EPOTHILONES CAN BE TRAPPED #OMPARING VARIOUS CYCLODEXTRINS OF DIFFERENT RING SIZE AND INCORPORATING DIFFERENT TYPES OF CHEMICAL MODIFICA TIONS HYDROXYPROPYL β CYCLODEXTRIN EMERGED AS THE CYCLODEXTRIN OF CHOICE AS IT GAVE THE HIGHEST %PO " TITERS ;= /NLY AFTER HARVESTING AND BIO MASS SEPARATION THE SUPERNATANT CONTAINING THE CYCLODEXTRINEPOTHILONE COMPLEX IS MIXED WITH 8!$ RESIN SO THAT THE EPOTHILONES ARE ADSORBED TO THE RESIN KG OF RESIN ARE USED TO CAPTURE THE EPOTHILONES FROM , OF HARVESTED FERMENTATION VOLUME !FTER RINSING WITH WATER %PO " IS DESORBED FROM THE RESIN WITH TOLUENE AND FURTHER PURIFIED BY REVERSED PHASE CHROMATOGRAPHY AND CRYSTALLIZATION ;=
&ERMENTATION OF +/3 +/3 IS THE +OSAN CODE NAME FOR %PO $ &IG !S INDICATED ABOVE %PO $ IS AN INTERMEDIATE IN THE BIOSYNTHETIC PATHWAY TO %PO " )N THE NATURAL PRODUCER STRAIN 3ORANGIUM CELLULOSUM 3O CE %PO $ IS A MINOR SIDE PROD UCT AS THE MAJORITY OF THIS PRECURSOR IS RAPIDLY CONVERTED TO %PO " BY THE %PO+ 0 EPOXIDASE ;= 4HEREFORE VARIOUS HOST ORGANISMS WERE TESTED FOR HETEROLOGOUS EXPRESSION OF THE EPOTHILONE GENE CLUSTER EG 3TREPTOMY CES COELICOLOR -YXOCOCCUS XANTHUS AND %SCHERICHIA COLI ;= #RITERIA FOR THE CHOICE OF A HOST ORGANISM WERE ITS EASE OF MANIPULATION ITS GROWTH VELOC ITY IE FERMENTATION THE AVAILABILITY OF THE PROPER MOLECULAR MACHINERY FOR POSTTRANSLATIONAL MODIFICATION AND A LACK OF FEEDBACK INHIBITION BY THE PRODUCT WHICH IS BEING OVERPRODUCED (ETEROLOGOUS PRODUCTION OF EPOTHILONES COULD BE DEMONSTRATED IN 3 COELICOLOR AND % COLI BOTH OF WHICH ARE BETTER CHARACTERIZED AND FASTER GROWING THAN THE ORIGINAL 3 CELLULOSUM (OWEVER THE HIGHEST PRODUCTION YIELDS OF EPOTHILONES WERE OBTAINED WHEN THE EPOTHILONE GENE CLUSTER WAS EXPRESSED IN THE MYXOBACTERIUM STRAIN -YXOCOCCUS XANTHUS ;= 7HEN A DELETION MUTANT WITH AN INACTIVE %PO+ 0 EPOXIDASE WAS USED %POS # AND $ WERE THE PRIMARY PRODUCTS WITH
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
AN %PO$%PO# RATIO OF CA 4HIS RATIO COULD BE FURTHER IMPROVED WHEN UP TO MG, PROPIONATE WERE ADDED TO THE PRODUCTION MEDIUM ;= 4HE PRODUCTION MEDIUM FURTHER CONTAINS METHYL OLEATE AS CARBON SOURCE AND 8!$ RESIN TO CAPTURE THE EPOTHILONES SECRETED BY THE CELLS &ED BATCH FERMENTATION OF THIS STRAIN IN , SCALE FERMENTORS PRODUCED %PO $ TITERS EXCEEDING MG, )N EXPERIMENTAL FERMENTATIONS ON A , SCALE EVEN HIGHER %PO $ TITERS OF UP TO MG, WERE REACHED WHEN THE CULTIVATIONS WERE PERFORMED IN SEMICONTINUOUS OR CONTINUOUS MODE ;= 0RODUCTION BATCHES ARE HARVESTED AFTER DAYS RUN TIME /N AVERAGE EACH , SCALE FERMENTATION YIELDED G OF CRYSTALLINE %PO $ AT A PURITY OF 4HE OVERALL YIELD OF SOLID PHASE EXTRACTION AND PURIFICATION BY MEANS OF DEFATTING CHRO MATOGRAPHY ACTIVATED CARBON TREATMENT AND CRYSTALLIZATION WAS ;=
%POTHILONE ANALOGS AND 3!2 STUDIES 4HE CHEMISTRY OF EPOTHILONES HAS BEEN EXTENSIVELY EXPLORED AND A WEALTH OF 3!2 INFORMATION HAS BEEN GENERATED FOR THIS FAMILY OF STRUCTURES OVER THE LAST SEVERAL YEARS BASED ON SYNTHETIC ANALOGS AND SEMISYNTHETIC DERIVATIVES -OST OF THE SYNTHETIC WORK WAS PERFORMED BY THE GROUPS OF .ICOLOAU SEE EG ;= AND $ANISHEFSKY SEE EG ;= AND TO A LESSER EXTENT ALSO THE GROUPS AT .OVARTIS !LTMANN AND COLLEAGUES NOW AT THE %4( :ßRICH ; = AND 3CHERING !' ; = 3EMISYNTHETIC WORK HAS BEEN REPORTED BY THE GROUPS AT THE FORMER @'ESELLSCHAFT FßR "IOTECHNOLOGISCHE &ORSCHUNG IN "RAUNSCHWEIG 'ERMANY '"& NOW (ELMHOLTZ #ENTRE FOR )NFECTION 2ESEARCH (:) SEE EG ; = AND "RISTOL -YERS 3QUIBB "-3 SEE EG ;= 4HE 3!2 DATA THAT HAVE EMERGED FROM THIS RESEARCH HAVE BEEN SUMMARIZED IN SEVERAL RECENT REVIEW ARTICLES ; n= )N THE FOLLOWING THE MOST SIGNIFICANT FEATURES OF THE EPOTHILONE 3!2 WILL BE DISCUSSED WITH PARTICULAR EMPHASIS ON THE WORK CONDUCTED IN OUR OWN LABORATORY IE WORK PERFORMED AT .OVARTIS AND MORE RECENTLY AT THE %4( :ßRICH
,ACTAM BASED ANALOGS 4HE REPLACEMENT OF THE LACTONE OXYGEN BY NITROGEN IE THE CONVERSION OF THE MACROLACTONE INTO A MACROLACTAM RING ; = HAS EMERGED AS ONE
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
OF THE MOST IMPORTANT SCAFFOLD MODIFICATIONS IN EPOTHILONES REPORTED SO FAR 4HE WORK ON LACTAM BASED ANALOGS WAS SPEARHEADED BY THE GROUP AT "-3 AND IT HAS LED TO THE IDENTIFICATION OF THE LACTAM ANALOG OF %PO " "-3 IXABEPILONE &IG AS A HIGHLY PROMISING ANTITUMOR AGENT WHICH HAS UNDERGONE AND STILL IS UNDERGOING EXTENSIVE CLINICAL EVALUA TION BY "-3 AND WHICH HAS RECENTLY OBTAINED &$! APPROVAL FOR CLINICAL USE IN HUMANS VIDE INFRA ,ACTAM BASED ANALOGS OF EPOTHILONES WERE CONCEIVED BY THE "-3 GROUP AS A MEANS TO STABILIZE THE LACTONE BASED NATURAL PRODUCTS AGAINST META BOLIC DEGRADATION AS IT HAD BEEN OBSERVED IN RODENT PLASMA )T IS WORTH NOTING HOWEVER THAT IN SPITE OF ITS SHORT PLASMA HALF LIFE IN RODENT SPECIES %PO " SHOWS POTENT ANTITUMOR ACTIVITY IN A VARIETY OF NUDE MOUSE HUMAN TUMOR MODELS ;= AND THE SAME IS TRUE FOR %PO $ VIDE INFRA %PO $ HAS ALSO BEEN DEMONSTRATED TO BE SIGNIFICANTLY MORE STABLE IN HUMAN THAN IN RODENT PLASMA ;= AND WE HAVE MADE SIMILAR OBSERVATIONS FOR %PO " 4HIS SPECIES DEPENDENT DIFFERENCE IN METABOLIC STABILITY IS NOT SURPRISING AND REFLECTS THE WELL KNOWN DIFFERENCE IN PLASMA ESTERASE ACTIVITY BETWEEN HUMANS AND RATS OR MICE "-3 IS A POTENT INDUCER OF TUBULIN POLYMERIZATION BUT ITS ANTIPROLIFERATIVE ACTIVITY IS CA ONE ORDER OF MAGNITUDE LOWER THAN THAT OF %PO " ; = EG )# VALUES AGAINST THE HUMAN COLON CARCINOMA CELL LINE (#4 ARE N- AND N- RESPECTIVELY FOR AND %PO " ;= -ETHYLATION OF THE LACTAM NITROGEN RESULTS IN A SUBSTANTIAL LOSS IN POTENCY ;= )N CONTRAST TO %PO " IS SIGNIFICANTLY LESS ACTIVE AGAINST THE 0 GP OVER EXPRESSING MULTIDRUG RESISTANT +" VARIANT OF THE +" CELL LINE THAN THE DRUG SENSITIVE +" PARENTAL LINE )#S OF N- AND N- AGAINST +" AND +" CELLS RESPECTIVELY ;= 4HIS INDICATES THAT THE COMPOUND IS A SUBSTRATE FOR THE 0 GP EFFLUX PUMP 3IMILAR DIFFERENCES BETWEEN 0 GP OVEREXPRESSING AND DRUG SENSITIVE HUMAN CANCER CELL LINES HAVE BEEN REPORTED FOR LACTAM BASED ANALOGS OF %PO # ;= AND $ ;= ,IKE OTHER TYPES OF MICROTUBULE INHIBITORS "-3 WAS FOUND BY "HALLA AND CO WORKERS TO INDUCE '- ARREST AND APOPTOSIS IN HUMAN CANCER CELL LINES ;= )NTERESTINGLY "HALLA AND CO WORKERS STRONGLY EMPHASIZE THAT ;"-3 INDUCED= hAPOPTOSIS OF ! ! CELLS FOLLOWS MITOTIC ARREST WHICH IS NOT ASSOCIATED WITH A MARKED INCREASE IN THE LEVELS OF SUR VIVINv SEE HOWEVER RECENT WORK BY (ORWITZ AND CO WORKERS ; =
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
"- EXHIBITS ANTITUMOR ACTIVITY SIMILAR TO THAT OF 4AXOL¤ IN 4AXOL¤ SENSITIVE TUMOR MODELS IE ! HUMAN OVARIAN CARCINOMA (#4 AND ,34 HUMAN COLON CARCINOMAS WHEN EACH DRUG IS GIVEN AT ITS OPTIMAL DOSE ;= )N SPITE OF ITS LIMITED EFFECTS AGAINST HIGHLY MULTI DRUG RESISTANT CELL LINES IN VITRO WAS ALSO SHOWN TO BE SUPERIOR TO 4AXOL¤ IN 4AXOL¤ RESISTANT TUMOR MODELS IE 0AT AND !4AX HUMAN OVARIAN CARCINOMAS 0AT HUMAN BREAST CARCINOMA 0AT HUMAN PANCREATIC CARCINOMA - MURINE SARCOMA &URTHERMORE THE COMPOUND SHOWED REMARKABLE ANTITUMOR ACTIVITY AGAINST 0AT OVARIAN AND (#4 COLON CARCINOMA XENOGRAFTS FOLLOWING ORAL ADMINISTRATION ;=
# MODIFICATIONS -ODIFICATIONS AT THE POSITION OF THE EPOTHILONE MACROCYCLE HAVE BEEN INVESTIGATED BY THE "-3 GROUP IN COLLABORATION WITH (ÙFLES GROUP AT THE '"& 4HESE STUDIES INCLUDED DEOXY DIDEHYDRO DERIVATIVES A AND B AS WELL AS DEOXY CYANO DERIVATIVES A AND B &IG "OTH TYPES OF ANA LOGS ARE READILY ACCESSIBLE THROUGH SEMI SYNTHESIS FROM %PO ! OR " ;= 1UITE REMARKABLY ANALOGS RETAIN MOST OF THE ACTIVITY OF THE PARENT NATURAL PRODUCTS WITH B EG BEING ONLY FOUR FOLD LESS POTENT THAN %PO " AGAINST THE HUMAN COLON CARCINOMA CELL LINE (#4 ;= 4HE SAME HOLDS FOR DEOXY 3 CYANO DERIVATIVE A WHICH IS ONLY TWO FOLD LESS ACTIVE THAN %PO ! )N CONTRAST THE CORRESPONDING 2 ISOMER SHOWS A SIGNIFI CANT REDUCTION IN POTENCY 7HAT HAD NOT BEEN INVESTIGATED UNTIL RECENTLY WAS THE ACTIVITY OF THE SATURATED DEOXY DERIVATIVES OF %PO ! AND " PER HAPS BECAUSE THEY WERE EXPECTED TO BE SIGNIFICANTLY LESS POTENT THAN THE α β UNSATURATED LACTONES DUE TO THE LACK OF ANY DIRECT CONFORMATIONAL CONSTRAINT ABOUT THE #n# BOND )N ORDER TO ADDRESS THIS ISSUE WE HAVE PREPARED DEOXY%PO " &IG AS PART OF A MORE COMPREHENSIVE PRO GRAM DIRECTED AT THE DEVELOPMENT OF HYPERMODIFIED EPOTHILONE ANALOGS AND ULTIMATELY OF NEW SCAFFOLDS FOR MICROTUBULE INHIBITION ;= 1UITE REMARKABLY DEOXY%PO " RETAINS POTENT BIOLOGICAL ACTIVITY WHICH IS MANIFEST IN )# VALUES FOR HUMAN CANCER CELL GROWTH INHIBITION IN THE LOW N- RANGE EG THE )# VALUES OF AGAINST THE HUMAN CERVIX CARCI NOMA CELL LINES +" AND +" ARE N- AND N- RESPECTIVELY VERSUS N- AND N- RESPECTIVELY FOR %PO " ;= 4HESE FINDINGS
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
DEMONSTRATE THAT THE PRESENCE OF A HYDROXYL GROUP IN EPOTHILONES IS NOT A CRUCIAL REQUIREMENT FOR POTENT BIOLOGICAL ACTIVITY EVEN IN THE ABSENCE OF A HARD CONFORMATIONAL CONSTRAINT ABOUT THE #n# BOND AS IT IS PRESENT IN α β UNSATURATED LACTONES
-ODIFICATIONS IN THE #n# REGION )NITIAL STRUCTURAL MODIFICATIONS IN THE #n# TRIMETHYLENE FRAGMENT ADJA CENT TO THE EPOXIDE MOIETY WERE GENERALLY FOUND TO RESULT IN DIMINISHED BIOLOGICAL ACTIVITY 4HUS EARLY WORK BY THE .ICOLAOU AND $ANISHEFSKY GROUPS SHOWED THAT RING CONTRACTION OR EXPANSION VIA THE REMOVAL OF EXIST ING OR THE INCORPORATION OF ADDITIONAL #( GROUPS IN THE #n# REGION BOTH CAUSED A SUBSTANTIAL LOSS IN BIOLOGICAL POTENCY ; = !N ALTERNA TIVE APPROACH PURSUED IN OUR LABORATORY FOR MODIFICATIONS SITUATED IN THE .ORTHERN HEMISPHERE OF EPOTHILONES WAS BASED ON AN EPOTHILONE PHARMA COPHORE MODEL WHICH HAD BEEN DERIVED FROM A COMPARATIVE ANALYSIS OF THE 8 RAY CRYSTAL STRUCTURE OF %PO " ;= WITH THOSE OF TAXOL AND DISCODERMOLIDE
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
0 &URET . 6AN #AMPENHOUT UNPUBLISHED RESULTS NO STRUCTURAL DATA FOR A TUBULIN%PO ! COMPLEX WERE AVAILABLE AT THE TIME OF THESE INVESTIGATIONS ;= "ASED ON THIS MODEL THE THREE BONDS BETWEEN ## ## AND ## WERE ALL PREDICTED TO ADOPT AN ANTI PERIPLANAR CONFORMATION IN THE BIOACTIVE CONFORMATION !S A CONSEQUENCE THE MODEL SUGGESTED THAT THE INCORPORATION OF META SUBSTITUTED PHENYL RINGS IN THE # TO # SEG MENT SUCH AS IN COMPOUNDS OR &IG SHOULD LEAD TO A STABILIZATION OF THE BIOACTIVE CONFORMATION IN THIS POTENTIALLY RATHER FLEXIBLE REGION OF NATURAL EPOTHILONES 7HILE THE SYNTHESIS AND BIOLOGICAL EVALUATION OF HAS BEEN REPORTED BY THE FORMER 3CHERING !' GROUP ;= WE HAVE RECENTLY DESCRIBED THE SYNTHESIS OF ANALOG ;= 5NFORTUNATELY AND BOTH ARE SUBSTANTIALLY LESS ACTIVE THAN %PO " OR $ ;= OR TO EXHIBIT @REDUCED ACTIVITY ;= -ORE SPECIFIC DATA ARE NOT AVAILABLE FROM ;= )N CONTRAST TO THESE DISAPPOINTING EARLY FINDINGS A NUMBER OF HIGHLY POTENT EPOTHILONE ANALOGS WITH STRUCTURAL VARIATIONS IN THE #n# TRI METHYLENE SEGMENT WERE IDENTIFIED MORE RECENTLY AND SOME OF THESE EXHIBIT HIGHLY FAVORABLE IN VIVO PHARMACOLOGICAL PROPERTIES 4HESE ANA LOGS WERE OBTAINED THROUGH A NUMBER OF DIFFERENT APPROACHES INCLUDING HETEROLOGOUS EXPRESSION OF THE MODIFIED EPOTHILONE POLYKETIDE SYNTHASES IN -YCCOCOCUS XANTHUS ;= TOTAL CHEMICAL SYNTHESIS SPEARHEADED BY THE $ANISHEFSKY GROUP ;n= OR BIOTRANSFORMATION OF %PO " ; = &OR EXAMPLE THE IN VITRO ANTIPROLIFERATIVE ACTIVITY OF EPOTHILONE &IG IS ONLY THREE TO FOUR FOLD LOWER THAN THAT OF THE PARENT COMPOUND %PO $ AGAINST THE -#& BREAST 3& GLIOMA .#) ( LUNG CANCER AND (, PROMYEOLOCYTIC LEUKEMIA CELL LINES AND THE COMPOUND IS EQUIPO
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
TENT WITH %PO " AGAINST THE HUMAN 4 CELL LEUKEMIA CELL LINES ##2- 6%- AND ##2- 6%-6", ;= 4HESE FINDINGS CORROBORATE AND EXTEND EARLIER RESULTS REPORTED BY (ÙFLE AND CO WORKERS FOR ## DEHYDRO %PO # WHICH HAD BEEN ISOLATED AS A MINOR FERMENTATION PRODUCT FROM CULTURES OF THE MYXOBACTERIUM 3 CELLULOSUM ;= 3UBSEQUENT TO THESE FINDINGS $ANISHEFSKY AND CO WORKERS HAVE SHOWN THAT THE PRESENCE OF A TRANS DOUBLE BOND BETWEEN # AND # SUCH AS IN &IG SIMILARLY RESULTS IN A MARKED INCREASE IN ANTIPROLIFERATIVE ACTIVITY OVER %PO $ THE )# VALUE OF AGAINST THE HUMAN LEUKEMIA CELL LINE ##2& #%- IS N- VERSUS N- FOR %PO $ ; = ,IKEWISE THE ## EPOXIDE CORRESPONDING TO IE &IG IS THREE TO FOUR FOLD MORE POTENT THAN %PO " ;= )N CONTRAST CIS ANALOG &IG HAS BEEN REPORTED BY 7HITE ET AL TO BE CA FOLD LESS ACTIVE THAN %PO $ AGAINST THE HUMAN CERVIX CANCER CELL LINE +" ;= .OTE THAT THE COMPOUND ASSUMED TO BE TRANS ANALOG IN ;= LATER WAS FOUND TO BE IN FACT CIS ISOMER ;= 4HESE DATA ARE IN AGREEMENT WITH RECENT SPECTROSCOPIC STUDIES ;= WHICH INDICATE THAT THE BIOACTIVE CONFORMATION OF EPOTHILONES IS CHARACTERIZED BY ANTI PERIPLANAR CONFORMATIONS ABOUT THE ## AND ## BONDS &OR
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
ANALOGS AND IT HAS ALSO BEEN SUGGESTED THAT THE PRESENCE OF A ## TRANS DOUBLE BOND FAVORS THE BIOACTIVE CONFORMATION OF THE MACROCYCLE IN THE #n# POLYKETIDE REGION ;= )N LIGHT OF THESE RECENT FINDINGS IT APPEARS LIKELY THAT THE LACK OF BIOLOGICAL ACTIVITY IN ANALOG IS RELATED TO THE INCREASE IN STERIC BULK ASSOCIATED WITH THE PRESENCE OF THE PHENYLENE MOIETY TRANS $IDEHYDRO EPOTHILONE ANALOGS AND WERE FOUND TO POS SESS MARKEDLY IMPROVED IN VIVO ANTITUMOR ACTIVITY OVER THEIR RESPECTIVE PAR ENT STRUCTURES %PO $ AND %PO " RESPECTIVELY IN A MOUSE MODEL OF HUMAN BREAST CANCER -8 &OR THIS EFFECT WAS SPECIFICALLY ASCRIBED TO A COMBINA TION OF ENHANCED ANTIPROLIFERATIVE ACTIVITY AND IMPROVED PLASMA STABILITY IN MICE ; = BUT UNFORTUNATELY THE COMPOUND IS ALSO ASSOCIATED WITH SIGNIFICANTLY ENHANCED TOXICITY ;= .EVERTHELESS AS +/3 HAS BEEN PROMOTED TO CLINICAL DEVELOPMENT STATUS AND IS CURRENTLY UNDERGOING 0HASE ) CLINICAL TRIALS )N CONTRAST TO THE CORRESPONDING # TRIFLUORO DERIVATIVE TERMED @FLUDELONE &IG EXHIBITS EXQUISITE IN VIVO ANTITUMOR ACTIVITY IN THE ABSENCE OF UNACCEPTABLE OVERT TOXICITY ; = 4HE CORRESPONDING IN VIVO INVESTIGATIONS INCLUDED MOUSE MODELS OF HUMAN BREAST -8 LUNG ! AND COLON (#4 CARCINOMAS AS WELL AS LEUKEMIA ##2& ; = AND MULTIPLE MYELOMA 20-) SUBCUTANEOUS XENOGRAFT MODEL AND DISSEMINATED #!' -- MODEL ;= 3USTAINED TUMOR REMIS SIONS @CURES WERE OBSERVED UPON TREATMENT WITH FLUDELONE IN SEVERAL EXPERIMENTS AND THE COMPOUND PROVED TO BE CLEARLY SUPERIOR OVER TAXOL BUT ALSO OVER THE NON FLUORINATED PARENT COMPOUND 3IMILAR ACTIVITY WAS OBSERVED AGAINST DRUG SENSITIVE AS WELL AS MULTIDRUG RESISTANT TUMORS WHICH WERE POORLY RESPONSIVE TO 4AXOL¤ )N ADDITION COMPARABLE EFFECTS AGAINST -8 XENOGRAFTS WERE OBSERVED AFTER IV OR ORAL ADMINISTRATION OF EQUAL DOSES OF FLUDELONE THUS INDICATING THAT THE COMPOUND HAS HIGH ORAL BIOAVAILABILITY ;= 4HE IN VITRO ANTIPROLIFERATIVE ACTIVITY OF IS COMPARA BLE WITH THAT OF %PO $ AND THE ENHANCED IN VIVO ACTIVITY OF THE COMPOUND AS FOR THE NON FLUORINATED ANALOG MAY BE A CONSEQUENCE OF IMPROVED PHARMACOKINETIC PROPERTIES INCLUDING ENHANCED METABOLIC STABILITY IN THE LIVER ;= 4HE DISCOVERY OF FLUDELONE COULD MARK A MAJOR MILESTONE IN EPOTHILONE BASED ANTICANCER DRUG DISCOVERY AND IT REPRESENTS THE PRE LIMINARY CULMINATION OF THE EXTENSIVE EFFORTS OF THE $ANISHEFSKY GROUP IN THIS AREA
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
!PART FROM THE DISCOVERY OF THE POTENT IN VIVO ACTIVITY OF WORK OF THE $ANISHEFSKY LABORATORY ON #n# MODIFICATIONS HAS ALSO SHOWN THAT THE PRESENCE OF A TRANS DOUBLE BOND BETWEEN # AND # ALLOWS THE INSERTION OF AN ADDITIONAL METHYLENE GROUP BETWEEN # AND # THUS CREATING A MEMBERED RING WITHOUT SUBSTANTIAL LOSS IN ANTIPROLIFERATIVE ACTIVITY 4HUS IN CONTRAST TO PREVIOUSLY STUDIED ANALOGS WITH INCREASED RING SIZE VIDE SUPRA ;= COMPOUND &IG IS ONLY FOUR FOLD LESS ACTIVE AGAINST THE HUMAN LEUKEMIA CELL LINE ##2& #%- THAN THE PARENT COMPOUND %PO $ ;= !S FOR OTHER MODIFICATIONS IN THE .ORTHERN PART OF THE EPOTHILONE MAC ROCYCLE THE REPLACEMENT OF # BY OXYGEN HAS BEEN SHOWN TO BE DET RIMENTAL FOR BIOLOGICAL ACTIVITY ;= WHILE THE INCORPORATION OF A FURAN MOIETY INCORPORATING # AND # APPEARS TO BE BETTER TOLERATED ;=
-ODIFICATIONS OF THE EPOXIDE MOIETY ! LARGE PART OF THE EARLY 3!2 WORK ON EPOTHILONES WAS FOCUSED ON MODIFICA TIONS OF THE EPOXIDE MOIETY AT POSITIONS OF THE MACROLACTONE RING "ASED ON THESE STUDIES IT BECAME CLEAR EARLY ON THAT THE EPOXIDE FUNCTIONAL ITY IS NOT AN INDISPENSABLE PREREQUISITE FOR EFFICIENT MICROTUBULE STABILIZA TION AND POTENT ANTIPROLIFERATIVE ACTIVITY 4HUS %PO # AND $ &IG ARE VIRTUALLY EQUIPOTENT INDUCERS OF TUBULIN POLYMERIZATION AS %PO ! AND " RESPECTIVELY 4HEY ARE ALSO POTENT INHIBITORS OF HUMAN CANCER CELL GROWTH IN VITRO ; n= ALTHOUGH THEY ARE SOMEWHAT LESS ACTIVE THAN THE CORRESPONDING PARENT EPOXIDES EG %PO $ INHIBITS THE GROWTH OF
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
THE HUMAN CERVIX CANCER CELL LINE +" AND THE LEUKEMIA CELL LINE ##2& #%- WITH )# VALUES OF N- ;= AND N- ;= RESPECTIVELY VERSUS )#S OF N- AND N- FOR %PO " 4HE REDUCED ANTIPROLIFERATIVE ACTIVITY OF %PO $ COMPARED TO %PO " MAY BE RELATED TO DIFFERENCES IN CEL LULAR UPTAKE BETWEEN THE TWO COMPOUNDS ;= !S FOR THE CORRESPONDING EPOXIDES THE DEOXY COMPOUNDS %POS # AND $ ARE POOR SUBSTRATES FOR THE 0 GP EFFLUX PUMP AND THUS ARE EQUALLY ACTIVE AGAINST DRUG SENSITIVE AND MULTIDRUG RESISTANT HUMAN CANCER CELL LINES %PO $ HAS BEEN EXTENSIVELY CHARACTERIZED IN VIVO BY THE GROUP AT THE 3LOAN +ETTERING #ANCER #ENTER %MPLOYING A SPECIFICALLY OPTIMIZED IV DOS ING REGIMEN MGKG H INFUSION QD X THE TOXICITY AND EFFICACY OF THE COMPOUND WAS SHOWN TO BE COMPARABLE TO THAT OF TAXOL WHEN TESTED AGAINST -8 BREAST CARCINOMA AND (4 COLON TUMORS ;= (OWEVER IN TWO MULTIDRUG RESISTANT MODELS -#& !DR AND ##2& #%-PACLITAXEL THE ACTIVITY OF %PO $ WAS FAR SUPERIOR 0OTENT IN VIVO ANTITUMOR ACTIVITY WAS OBSERVED IN SPITE OF THE FACT THAT %PO $ EXHIBITS A VERY SHORT HALF LIFE IN RODENT PLASMA WHICH HAS ALSO BEEN REPORTED FOR THE PARENT COMPOUND %PO " ;= VIDE SUPRA (OWEVER BOTH %PO " AND %PO $ ;= ARE SIGNIFICANTLY MORE STABLE IN HUMAN PLASMA IN VITRO THUS INDICATING THAT PLASMA STABIL ITY IS AN UNLIKELY LIMITATION FOR THERAPEUTIC APPLICATIONS OF LACTONE BASED EPOTHILONE ANALOGS IN HUMANS 4HE REPLACEMENT OF THE OXIRANE RING IN EPOTHILONES BY A CYCLOPROPANE MOIETY IS WELL TOLERATED AND NOT ASSOCIATED WITH ANY LOSS IN TUBULIN POLYM ERIZING OR ANTIPROLIFERATIVE ACTIVITY ;n= EG )#S FOR AND &IG AGAINST THE HUMAN COLON CARCINOMA CELL LINE (#4 ARE N- AND N- RESPECTIVELY ;=
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
4HE SAME IS TRUE FOR VARIOUSLY . SUBSTITUTED AZIRIDINE BASED ANALOGS ;= WHICH EXHIBIT HIGH CELLULAR POTENCY )#S FOR AND &IG AGAINST THE HUMAN COLON CARCINOMA CELL LINE (#4 ARE N- AND N- RESPECTIVELY -OREOVER REPLACEMENT OF THE EPOXIDE MOIETY BY A CYCLOPROPANE RING WAS RECENTLY SHOWN TO PRODUCE ENHANCED BINDING TO STABILIZED MICROTUBULES IN A STUDY BY "UEY ET AL ;= 4OGETHER WITH THE EARLIER DATA ON %PO # AND $ THESE FINDINGS INDICATE THAT THE OXIRANE RING SYSTEM IN EPOTHILONES SIMPLY SERVES TO STABILIZE THE PROPER BIOACTIVE CON FORMATION OF THE MACROCYCLE RATHER THAN ACTING AS A REACTIVE ELECTROPHILE OR A HYDROGEN BOND ACCEPTOR /UR OWN WORK IN THE AREA OF ## MODIFIED EPOTHILONE ANALOGS WAS INITIALLY GUIDED BY THE POTENT BIOLOGICAL ACTIVITY ASSOCIATED WITH THE DEOXYEPOTHILONE STRUCTURAL FRAMEWORK THIS APPROACH WILL BE DISCUSSED BELOW IN THE SECTION ON @AZA EPOTHILONES )N ADDITION WE HAVE INVESTI GATED A SERIES OF SEMISYNTHETIC DERIVATIVES OF %PO ! WHICH WERE OBTAINED THROUGH HYDROLYTIC RING OPENING OF THE EPOXIDE MOIETY ;= 7HILE NONE OF THE RESULTING DIOLS SHOWED ANY APPRECIABLE BIOLOGICAL ACTIVITY )#S FOR CANCER CELL GROWTH INHIBITION μ- AZIDO ALCOHOL OBTAINED THROUGH EPOXIDE RING OPENING WITH .A. &IG RATHER SURPRISINGLY PROVED TO BE SIGNIFICANTLY MORE POTENT EG )#S OF AGAINST THE HUMAN CERVIX CANCER CELL LINES +" AND +" ARE N- AND N- RESPECTIVELY ;= 4HIS INDICATES THAT THE LOSS IN ACTIVITY FOR THE ABOVE DIOLS CANNOT BE SIMPLY ASCRIBED TO INCREASED CONFORMATIONAL FLEXIBILITY (OWEVER THE INTER PRETATION OF CHANGES IN CELLULAR ACTIVITY IS NOT STRAIGHTFORWARD AS THEY MAY BE CAUSED BY A COMBINATION OF CHANGES IN TARGET AFFINITY CELLULAR PENETRA TION AND METABOLIC STABILITY )NTERESTINGLY ACETONIDES A AND A &IG
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
ARE ONLY n FOLD LESS POTENT ANTIPROLIFERATIVE AGENTS THAN %PO ! ITSELF )# VALUES AGAINST THE +" +" LINE ARE N- N- AND N- N- FOR A AND A RESPECTIVELY WHILE THE RESPECTIVE DIASTEREO ISOMERS B AND B ARE n FOLD LESS POTENT 4HESE DATA SUGGEST THAT FOR A TETRAHEDRAL GEOMETRY AT # AND # THE SIZE OF THE RING FUSED TO THE #n# SINGLE BOND CAN BE SIGNIFICANTLY INCREASED WITHOUT SUBSTANTIAL LOSS IN BIOLOGICAL POTENCY IN CONTRAST TO ANALOGS WITH A PLANAR GEOMETRY OF THE #n# BOND VIDE INFRA -OREOVER THE DATA FOR A ALSO ILLUSTRATE THAT GIVEN THE PROPER ABSOLUTE STEREOCHEMISTRY AT # AND # ACTIVITY IS RETAINED EVEN UPON MOVING FROM A CIS TO A TRANS FUSED SYSTEM VIDE INFRA !NOTHER INTRIGUING FEATURE OF THE EPOTHILONE 3!2 REVEALED DURING EARLY 3!2 STUDIES WAS THE FACT THAT EVEN ## TRANS ANALOGS OF EPOTHILONES EXHIBIT VERY POTENT TUBULIN POLYMERIZING AS WELL AS ANTIPROLIFERATIVE ACTIV ITY ; n= 4HE LITERATURE DATA AVAILABLE AT THE OUTSET OF OUR OWN WORK IN THIS AREA INDICATED THAT TRANS DEOXY%PO ! WAS ONLY SLIGHTLY LESS ACTIVE THAN DEOXY%PO ! %PO # WHEREAS IN THE " SERIES THE ACTIVITY DIFFERENCE APPEARED TO BE MORE PRONOUNCED !T THE SAME TIME TRANS %PO
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
! WAS REPORTED BY .ICOLAOU ET AL TO BE VIRTUALLY EQUIPOTENT WITH %PO ! ON AN OVARIAN ! AND A BREAST CANCER -#& CELL LINE ;= (OWEVER THE ABSOLUTE STEREOCHEMISTRY OF THE ACTIVE EPOXIDE ISOMER WAS NOT REPORTED AND THE TRANS ISOMERS WERE OBTAINED AS MINOR COMPONENTS DURING THE SYNTHESIS OF THE NATURAL CIS ISOMERS )N VIEW OF THE INTERESTING BIOLOGICAL FEATURES OF TRANS DEOXY EPOTHILONES WE EMBARKED ON THE STEREOSELECTIVE SYNTHESIS OF TRANS EPOTHILONES ! THE DETERMINATION OF THE ABSOLUTE STE REOCHEMISTRY OF THE BIOACTIVE ISOMER AND AN EXHAUSTIVE BIOLOGICAL CHAR ACTERIZATION OF THESE ANALOGS ;= 4HESE STUDIES SHOWED THAT COMPOUND &IG WHICH RETAINS THE NATURAL STEREOCHEMISTRY AT # IS A STRONG INDUCER OF TUBULIN POLYMERIZATION IN VITRO AND EXHIBITS POTENT ANTIPROLIF ERATIVE ACTIVITY WHEREAS ITS 2 2 ISOMER A IS AT LEAST FOLD LESS ACTIVE ;= 4HE AVERAGE )# VALUES ACROSS A PANEL OF SEVEN HUMAN CANCER CELL LINES HAVE BEEN REPORTED AS AND N- FOR %PO ! %PO " AND RESPECTIVELY ;= /UR FINDINGS ON THE POTENT BIOLOGICAL ACTIVITY OF THE TRANS %PO ! SCAFFOLD HAVE RECENTLY BEEN CONFIRMED AND EXPANDED BY .ICOLAOU ET AL FOR A SERIES OF HIGHLY POTENT ## CYCLOPROPANE BASED ANALOGS OF ; = )N CONTRAST ANALOGS OF TRANS %PO " ARE GENERALLY LESS POTENT THAN THE CORRESPONDING CIS ISOMERS ;=
# MODIFIED ANALOGS )N ADDITION TO THE CHANGES IN THE EPOXIDE STRUCTURE ITSELF A VARIETY OF MODIFI CATIONS OF THE METHYL GROUP IN %PO " OR $ HAVE BEEN REPORTED 4HESE STUD IES HAVE SHOWN THAT THE REPLACEMENT OF ONE HYDROGEN ATOM OF THIS METHYL GROUP BY RELATIVELY SMALL AND APOLAR SUBSTITUENTS SUCH AS & #L #( OR #(
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
&IG 2 #(& #(#L #( N #( IS WELL TOLERATED THUS PRODUCING ANALOGS WHICH ARE ONLY SLIGHTLY LESS POTENT IN VITRO THAN %PO " OR %PO $ ; = )N GENERAL ACTIVITY DECREASES WITH INCREASING SIZE OF THE # SUBSTITUENTS ; = BUT EXCEPTIONS FROM THIS GENERAL THEME HAVE BEEN REPORTED !S AN EXAMPLE # DIOXOLANYL %PO $ &IG IS MORE ACTIVE IN VITRO THAN %PO $ ;= 5NFORTUNATELY THE COMPOUND WAS FOUND TO BE SIGNIFICANTLY LESS EFFICACIOUS THAN %PO $ IN VIVO )N CONTRAST # FLUORO %PO " &IG WHICH EXHIBITS COMPARABLE IN VITRO ANTIPROLIFERATIVE ACTIVITY WITH %PO " ;= WAS DEMONSTRATED TO POSSESS SIGNIFICANTLY BETTER ANTITUMOR ACTIVITY THAN TAXOL IN A HUMAN PROSTATE XENOGRAFT MODEL WHEN BOTH COM POUNDS WERE ADMINISTERED AT EQUITOXIC DOSES ;= .O COMPARISON WITH %PO " WAS INCLUDED IN THIS WORK BUT DATA FROM OUR OWN LABORATORY INDICATE THAT THE IN VIVO PROFILE OF # FLUORO %PO " IS SIMILAR TO THAT OF %PO " ITSELF ;=
3IDE CHAIN MODIFICATIONS 4HE SECOND REGION OF THE EPOTHILONE STRUCTURE THAT HAS BEEN A FREQUENT TARGET FOR 3!2 STUDIES APART FROM THE EPOXIDE MOIETY IS THE UNSATURATED
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
HETEROCYCLE BEARING SIDE CHAIN -ODIFICATIONS OF THE SIDE CHAIN IN PARTICU LAR INVOLVING THE PENDANT HETEROCYCLE HOLD THE POTENTIAL TO MODULATE THE PHYSICOCHEMICAL AND PERHAPS ALSO THE PHARMACOKINETIC PROPERTIES OF THE NATURAL PRODUCTS 4HE CORRESPONDING 3!2 STUDIES INCLUDE MODIFICATIONS OF THE THIAZOLE RING AT THE AND POSITIONS ; n= THE REPLACEMENT OF THE THIAZOLE RING BY OTHER HETEROCYCLES ; = OR A SIMPLE PHENYL MOIETY ; = AND THE SYNTHESIS OF # DESMETHYL %PO " ; = )NVESTIGATION OF THE LATTER ANALOGS HAS SHOWN THAT THE ALLYLIC METHYL GROUP ATTACHED TO # CAN BE REMOVED WITH ONLY A MINOR CHANGE IN BIOLOGICAL ACTIVITY SEE ;= ,IKEWISE THE SUBSTITUTION OF OXYGEN FOR SULPHUR IN THE HETEROCYCLE TO PRODUCE OXAZOLE BASED EPOTHILONES DOES NOT AFFECT BIOLOGICAL POTENCY ; = 2EPLACEMENT OF THE METHYL GROUP ON THE THIAZOLE RING BY RELATIVELY SMALL SUBSTITUENTS SUCH AS #(/( #(& 3#( OR #(#( IS WELL TOLERATED WHILE MORE BULKY SUBSTITUENTS RESULT IN A SUBSTANTIAL LOSS IN POTENCY ; = /UT OF THIS LATTER FAMILY OF ANALOGS DEOXY%PO & # HYDROXY %PO $ &IG WAS REPORTED BY THE 3LOAN +ETTERING GROUP TO EXHIBIT COMPARABLE IN VIVO EFFICACY AS %PO $ ; = %MPLOYING A H CONTINUOUS IV INFUSION REGIMEN FOR BOTH COMPOUNDS WAS ALSO FOUND TO SHOW SIGNIFICANTLY SUPERIOR ANTITUMOR EFFECTS OVER "-3 IN A ##2& #%- AS WELL AS A -8 TUMOR MODEL ; = (OWEVER THESE DIFFERENCES ARE DIFFICULT TO INTERPRET AS THE H CONTINUOUS INFUSION SCHEDULE MAY BE OPTIMAL FOR %PO $ AND BUT NOT NECESSARILY FOR "-3 $UE TO ITS HIGHER WATER SOLUBILITY COMPARED WITH %PO $ MAY BE A MORE ATTRACTIVE DRUG DEVELOPMENT CANDIDATE THAN %PO $ # !MINO %PO " "-3 &IG IS UNDERGOING CLINICAL EVALUATION IN HUMANS ; = BUT ONLY LIMITED BIOLOGICAL DATA FOR THIS COMPOUND HAVE APPEARED IN THE LITERATURE SO FAR 4HUS AN )# VALUE OF N- FOR GROWTH INHIBITION OF THE HUMAN CERVIX CANCER CELL LINE +"
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
HAS BEEN REPORTED IN A PATENT APPLICATION ;= VERSUS N- FOR %PO " UNDER COMPARABLE EXPERIMENTAL CONDITIONS ;= AND THE COMPOUND WAS DEMONSTRATED TO INDUCE SUBSTANTIAL APOPTOSIS IN EARLY PASSAGE TAXOL AND PLATINUM REFRACTIVE OVARIAN CANCER CELLS /# AT A CONCENTRATION OF N- ;= 0LASMA CONCENTRATIONS OF N- OF "-3 ARE CLIN ICALLY ACHIEVABLE AT A DOSE OF MGM WHICH IS BELOW THE PHASE ) -4$ MAXIMUM TOLERATED DOSE FOR THE COMPOUND ; = "-3 EXHIBITS IMPROVED WATER SOLUBILITY OVER "-3 WHICH ENABLES THE USE OF CLINICAL FORMULATIONS NOT CONTAINING #REMOPHOR %,¤ ;= -AJOR CONTRIBUTIONS TO THE AREA OF HETEROCYCLE MODIFICATIONS IN EPOTHI LONES HAVE COME FROM THE COLLABORATIVE WORK OF THE .ICOLAOU GROUP AT 4HE 3CRIPPS 2ESEARCH )NSTITUTE 432) IN ,A *OLLA #! AND THE GROUP AT .OVARTIS !MONG OTHER THINGS THIS RESEARCH HAS SHOWN THAT PYRIDINE BASED ANALOG &IG AND METHYL SUBSTITUTED VARIANTS THEREOF ARE BASICALLY EQUIPO TENT CELL GROWTH INHIBITORS AS %PO " ;= 4AB )T IS THUS CLEAR THAT THE PRESENCE OF A MEMBERED HETEROCYCLE ATTACHED TO # IS NOT A PREREQUISITE FOR HIGHLY POTENT BIOLOGICAL ACTIVITY !T THE SAME TIME IT WAS SHOWN THAT THE OCCURRENCE OF %PO " LIKE CELLULAR ACTIVITY IN PYRIDINE BASED %PO " ANALOGS IE SUB N- )#S FOR GROWTH INHIBITION REQUIRES POSITIONING OF THE RING . ATOM ORTHO TO THE ATTACHMENT POINT OF THE LINKER BETWEEN THE HETEROCYCLE AND THE MACROCYCLIC SKELETON $IFFERENT LOCATIONS OF THE RING . ATOM SUCH AS IN AND &IG LEADS TO A SIGNIFICANT DECREASE IN CELLULAR POTENCY ;= 4AB -OREOVER THE INCORPORATION OF A SECOND NITROGEN ATOM EITHER AT THE OR THE POSITION OF THE MEMBERED RING OR &IG RESULTS IN A PRO
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
4ABLE 0YRIDINE AND PYRIMIDINE BASED ANALOGS OF %PO " )MPORTANCE OF NITROGEN POSITIONING #OMPOUND
4UBULIN POLYMERIZATIONA
)# +"n ;N-=B
A)NDUCTION OF POLYMERIZATION OF PORCINE BRAIN MICROTUBULE PROTEIN BY μ- OF TEST COMPOUND RELATIVE
TO THE EFFECT OF μ- OF %PO " WHICH GAVE MAXIMAL POLYMERIZATION OF PROTEIN INPUT B)# VALUES FOR GROWTH INHIBITION OF THE HUMAN CERVIX CARCINOMA CELL LINE +" $ATA ARE FROM
;=
FOUND DECREASE IN ANTIPROLIFERATIVE ACTIVITY EVEN WITH ONE OF THE . ATOMS IN THE OBLIGATORY POSITION ;= )N FACT THE CORRESPONDING ANALOGS ARE EVEN LESS POTENT THAN PHENYL DERIVED ANALOG &IG SEE 4ABLE ;= 4HE UNDERLYING REASONS FOR THESE DIFFERENCES HAVE NOT BEEN DETERMINED AT THIS POINT AND THEIR UNDERSTANDING WILL REQUIRE STRUCTURAL INFORMATION ON COMPLEXES BETWEEN β TUBULIN AND VARIOUS TYPES OF EPOTHILONE ANALOGS )N ADDITION STUDIES WITH DIFFERENT TYPES OF SIDE CHAIN MODIFIED ANALOGS HAVE SHOWN THAT POTENT CELLULAR ACTIVITY IS ALSO POSSIBLE FOR STRUCTURES WITH THE . ATOM IN A @NON NATURAL POSITION VIDE INFRA 4HE COLLABORATIVE WORK BETWEEN THE .ICOLAOU GROUP AT THE 432) AND THE GROUP AT .OVARTIS HAS ALSO RESULTED IN THE DISCOVERY OF DESMETHYL METHYLSULFANYL %PO " !"* &IG AS A HIGHLY PROMISING ANTITUMOR AGENT ; = WHICH HAS UNDERGONE 0HASE ) CLINICAL TRIALS SPONSORED BY .OVARTIS !"* INDUCES TUBULIN POLYMERIZATION IN VITRO WITH SLIGHTLY HIGHER POTENCY THAN %PO " OR TAXOL !T THE SAME TIME THE COMPOUND IS A MARKEDLY MORE POTENT ANTIPROLIFERATIVE AGENT THE AVERAGE )# FOR GROWTH INHIBITION ACROSS A PANEL OF DRUG SENSITIVE HUMAN CANCER CELL LINES BEING N- VER SUS N- FOR %PO " AND N- FOR TAXOL ;= 4HE COMPOUND RETAINS FULL ACTIVITY AGAINST CANCER CELLS OVEREXPRESSING THE DRUG EFFLUX PUMP 0 GP OR HARBORING TUBULIN MUTATIONS
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
4HE BINDING OF TO STABILIZED MICROTUBULES HAS BEEN EVALUATED BY "UEY ET AL ;= WHO FOUND A DIFFERENCE IN BINDING FREE ENERGY AT # OF n K*MOLE BETWEEN !"* AND %PO " CORRESPONDING TO AN INCREASE IN THE BINDING CONSTANT FROM × FOR %PO " TO × FOR !"* )NTERESTINGLY BINDING ENTHALPY IS LESS FAVORABLE FOR !"* THAN FOR %PO " AND THE INCREASE IN BINDING FREE ENERGY FOR !"* IS CAUSED BY AN INCREASE IN BINDING ENTROPY "INDING FREE ENERGY IS ALSO INCREASED FOR ## CYCLOPROPANE BASED EPOTHILONE ANALOGS AND THE ENERGETIC EFFECTS OF THE REPLACEMENT OF THE # METHYL GROUP BY A METHYLSULFANYL GROUP AND THE SUBSTITUTION OF A METHYLENE GROUP FOR THE EPOXIDE OXYGEN ARE IN FACT ADDITIVE 4HUS ANALOG &IG BINDS TO STABILIZED MICROTUBULES WITH FOLD ENHANCED AFFINITY OVER %PO " ΔΔ' # n K*MOLE ;= &URTHERMORE THIS COMPOUND IN SOME CASES HAS BEEN FOUND TO BE A MORE POTENT ANTIPROLIFERATIVE AGENT IN VITRO THAN EITHER %PO " OR !"* EG )# VALUES FOR GROWTH INHIBITION OF THE HUMAN OVARIAN CARCINOMA CELL LINE ! ARE N- N- AND N FOR %PO " AND RESPECTIVELY ; = !"* HAS DEMONSTRATED POTENT ANTITUMOR ACTIVITY ;= IN EXPER IMENTAL HUMAN TUMOR MODELS IN MICE WHERE IT PRODUCED TRANSIENT REGRES SIONS AND INHIBITION OF TUMOR GROWTH OF SLOW GROWING .#) ( LUNG ADENOCARCINOMAS AND (4 COLON TUMORS )NHIBITION OF TUMOR GROWTH WAS OBSERVED IN FAST GROWING DIFFICULT TO TREAT .#) ( LARGE CELL LUNG TUMORS &INALLY SINGLE DOSE ADMINISTRATION OF !"* RESULTED IN LONG LASTING REGRESSIONS AND CURES IN A 4AXOL¤ RESISTANT +" CERVIX CARCINOMA MODEL )N ORDER TO EXPAND THE STRUCTURAL SCOPE OF SIDE CHAIN MODIFICATIONS BASED ON MEMBERED HETEROCYCLES BEYOND SIMPLE THIAZOLE OR OXAZOLE RING SYSTEMS THE .ICOLAOU GROUP HAS ALSO INVESTIGATED %PO " ANALOGS INCOR
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
PORATING FUNCTIONALIZED IMIDAZOLE PYRAZOLE TRIAZOLE AND TETRAZOLE RINGS ANALOGS n &IG ;= 2ATHER SURPRISINGLY IMIDAZOLE CONTAINING SIDE CHAINS SUCH AS IN OR A CAUSED A SUBSTANTIAL LOSS IN ACTIVITY 4HIS IS AN INTRIGUING FINDING AS STRUCTURES OF THIS TYPE ARE PART OF THE SIDE CHAINS OF ELEUTHEROBIN OR SAR CODICTYINS WHICH ARE POTENT NATURAL PRODUCT MICROTUBULE INHIBITORS )N ORDER TO PROBE THE IMPORTANCE OF THE ELECTRON DENSITY OF SMALLER RINGS FOR BIOLOGICAL ACTIVITY .ICOLAOUS GROUP INVESTIGATED THE REPLACEMENT OF THE IMIDAZOLE MOIETY BY A TRIAZOLE OR A TETRAZOLE RING EG ANALOGS B AND RESPECTIVELY 7HILE TRIAZOLE DERIVATIVE B SHOWED NO APPRECIABLE ACTIV ITY THE TETRAZOLE BASED ANALOG IN A SURPRISING TURN OF EVENTS PROVED ALMOST EQUIPOTENT WITH %PO " IN SPITE OF THE FACT THAT EARLIER ATTEMPTS AT INCORPORATING MULTIPLE NITROGEN ATOMS INTO THE HETEROCYCLE MOIETY EG THE CHANGE FROM A PYRIDINE TO A PYRIMIDINE RING ; = HAD LED TO DECREASED ACTIVITY 4HE ACTIVITY OF COMPOUND IS PARTICULARLY REMARKABLE AS THE ANALOGOUS SUBSTITUTED THIAZOLE DERIVATIVE IS SEVERAL HUNDRED FOLD LESS ACTIVE THAN ACROSS THE SAME PANEL OF HUMAN CANCER CELL LINES (IGHLY POTENT ANTIPROLIFERATIVE ACTIVITY WAS ALSO OBSERVED FOR PYRAZOLE BASED ANA
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
LOGS SUCH AS EG #OMPOUND A WHICH INCORPORATES A . METHYL PYRAZOLE MOIETY AND WHICH MAY BE REGARDED AS A COMPLETELY ISOSTERIC ANALOG OF %PO " PROVED TO BE n FOLD MORE POTENT THAN THE LATTER DEPENDING ON THE CELL LINES INVESTIGATED EG THE )# OF A AGAINST THE HUMAN CERVIX CARCINOMA CELL LINE +" IS N- VERSUS N- FOR %PO " 7HILE PHENYL PYRAZOLE DERIVATIVE B WAS SLIGHTLY LESS POTENT THAN %PO " QUITE INTRIGUINGLY IT SHOWED ENHANCED ACTIVITY AGAINST THE %PO ! RESIS TANT !! CELL LINE AS COMPARED WITH THE PARENTAL %PO ! SENSITIVE ! LINE 4HE MOST POTENT PYRAZOLE BASED ANALOG HOWEVER PROVED TO BE THE METHYLSULFANYL DERIVATIVE C WHICH WAS FOUND TO BE SIGNIFICANTLY MORE ACTIVE THAN %PO " IN ALL CELL LINES INVESTIGATED EG THE COMPOUND SHOWS FOLD ENHANCED ACTIVITY OVER %PO " AGAINST BOTH THE HUMAN OVARIAN CAR CINOMA CELL LINE ! AND ITS %PO ! RESISTANT VARIANT !! )# VALUES OF N- AND N- RESPECTIVELY VERSUS N- AND N- FOR %PO " AND ITS ACTIVITY AGAINST THE TAXOL RESISTANT !048 LINE )# N- IS FOLD HIGHER THAN THAT OF %PO " "ASED ON THESE DATA COMPOUND C REPRESENTS THE MOST POTENT EPOTHILONE ANALOG IDENTIFIED TO DATE )N ORDER TO FURTHER PROBE THE STERIC REQUIREMENTS OF THE EPOTHILONE BIND ING POCKET AROUND THE HETEROCYCLE MOIETY A SERIES OF BICYCLIC AROMATIC THI AZOLE REPLACEMENTS WAS SYNTHESIZED AND TESTED FOR ANTIPROLIFERATIVE ACTIVITY ;= !NALOGS INCORPORATING PURINE BASED SIDE CHAINS SUCH AS A AND B &IG EXHIBIT VERY POTENT GROWTH INHIBITORY ACTIVITY WITH METH YLSULFANYL DERIVATIVE A BEING FOUR FOLD MORE POTENT THAN %PO " AGAINST BOTH THE !048 AND THE !! CELL LINES 'UIDED BY PREVIOUS DATA ON %PO # ANALOGS INCORPORATING A THIAZOL YL MOIETY AS OPPOSED TO THE NATURAL THIAZOL YL GROUP ;= OR A QUINOLINE MOIETY +# .ICOLAOU
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
ET AL UNPUBLISHED AS A THIAZOLE REPLACEMENT BENZOTHIAZOLE ANALOG &IG WAS DESIGNED AND SYNTHESIZED ;= !S FOR A THIS COMPOUND WAS FOUND TO BE MORE POTENT THAN %PO " UP TO FOUR FOLD 4AKEN TOGETHER THESE FINDINGS SUGGEST THAT THE SIDE CHAIN BINDING POCKET ON TUBULIN CAN ACCOMMODATE SIGNIFICANTLY EXPANDED HETEROCYCLES IF THE ADDITIONAL STERIC BULK IS LOCATED IN THE NORTH EASTERN QUADRANT OF THE STRUCTURE )T IS WORTH NOTING THAT THIS IS CONTRARY TO THE EFFECTS THAT HAVE BEEN OBSERVED FOR THE INCORPORATION OF BULKY SUBSTITUENTS AT THE POSITION OF THE THIAZOLE RING IN %PO " WHICH HAVE BEEN REPORTED TO CAUSE A SUBSTANTIAL LOSS IN BIOLOGICAL POTENCY ; = )N ADDITION TO ANALOGS INCORPORATING AN OLEFINIC DOUBLE BOND AS A LINKER BETWEEN THE MACROLACTONE RING AND DIFFERENT TYPES OF HETEROCYCLES WE HAVE ALSO STUDIED A NEW FAMILY OF SIDE CHAIN MODIFIED STRUCTURES WHICH ARE CHARACTERIZED BY RIGIDIFICATION OF THE ENTIRE SIDE CHAIN MANIFOLD EXEMPLIFIED FOR QUINOLINE BASED ANALOGS AND &IG ;= SEE ALSO ;= 4HE DESIGN OF THESE ANALOGS WAS GUIDED BY THE RESULTS OF .-2 STUDIES ON THE BIOACTIVE TUBULIN BOUND CONFORMATION OF EPOTHILONES WHICH INDICATED THAT THE ## DOUBLE BOND AND THE AROMATIC # . BOND WERE PRESENT IN A TRANSOID ARRANGEMENT CORRESPONDING TO A ^ # # # . TORSION ANGLE ;= )N GENERAL ANALOGS OF THIS TYPE ARE MORE POTENT INHIBITORS OF HUMAN CANCER CELL PROLIFERATION THAN THE RESPECTIVE PARENT COMPOUNDS %PO $ AND %PO " ; = &OR EXAMPLE COMPOUNDS AND INHIBIT THE GROWTH OF THE HUMAN CERVIX CARCINOMA CELL LINE +" WITH )# VALUES OF N- AND N- RESPECTIVELY VERSUS N- AND N- FOR %PO " AND %PO $ !S OBSERVED FOR OTHER ANALOGS OF THIS TYPE INCORPORATING BENZOTHIAZOLE BENZOXAZOLE OR BENZ IMIDAZOLE TYPE SIDE CHAINS THE ACTIVITY DIFFERENCE IS MORE PRONOUNCED
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
IN THE DEOXY CASE WITH BEING AN ALMOST FIVE FOLD MORE POTENT ANTIP ROLIFERATIVE AGENT THAN %PO $ ;= )NTERESTINGLY HOWEVER THE OBSERVED INCREASE IN ANTIPROLIFERATIVE ACTIVITY DOES NOT SEEM TO BE A CONSEQUENCE OF MORE EFFECTIVE INTERACTIONS WITH TUBULIN AND THIS POINT WILL BE DISCUSSED IN MORE DETAIL BELOW 3IDE CHAIN MODIFICATIONS OF THE ABOVE TYPE HAVE BEEN INDEPENDENTLY INVESTIGATED BY THE GROUPS AT 3CHERING ; = AND MORE RECENTLY AT +OSAN ;= 4HE WORK AT 3CHERING HAS LED TO THE IDENTIFICATION OF A # ALLYLATED BENZOTHIAZOLE BASED ANALOG :+ %PO SAGOPILONE &IG WHICH IS CURRENTLY UNDERGOING 0HASE )) CLINICAL TRIALS AND WHICH IS THE FIRST FULLY SYNTHETIC EPOTHILONE ANALOG TO HAVE ENTERED CLINICAL STUDIES ;= :+ %PO IS REPORTED TO BE MORE POTENT IN VITRO THAN %PO " AGAINST A VARIETY OF DRUG SENSITIVE CANCER CELL LINES AND TO RETAIN FULL ACTIVITY ALSO AGAINST MULTI DRUG RESISTANT CANCER CELLS WHILE %PO " EXHIBITS SOMEWHAT REDUCED ACTIVITY AGAINST CERTAIN TYPES OF -$2 CELL LINES ;= 4HE COMPOUND ALSO SHOWED SIGNIFICANT ANTITUMOR ACTIVITY IN A NUMBER OF MOUSE XENOGRAFT MODELS STABLE DISEASE WITHOUT INDUCING ANY PROFOUND BODY WEIGHT LOSS ;= !MONG THE FUSED HETEROAROMATIC MOIETIES THAT WE HAD INITIALLY INVESTI GATED AS SIDE CHAIN REPLACEMENTS IN EPOTHILONES THE DIMETHYL BENZIMID AZOLE GROUP STOOD OUT AS THE ONE ASSOCIATED WITH THE MOST PRONOUNCED ENHANCEMENT IN CELLULAR POTENCY ;= 7E HAVE THEREFORE FURTHER INVES TIGATED THIS MODIFICATION AS A POTENTIAL POTENCY ENHANCING ELEMENT IN COMBINATION WITH A VARIETY OF MACROCYCLE MODIFICATIONS ; = 4HESE STUDIES REVEALED THAT THE POTENCY ENHANCING EFFECT OF THE DIMETHYL BENZIMIDAZOLE MOIETY IS RATHER GENERAL IN NATURE AND EXTENDS TO THE CORRESPONDING ANALOGS OF %PO ! AND # ;= OF DEOXY%PO " ;= AND ALSO OF TRANS %PO ! ;= AND ITS DEOXY DERIVATIVE ;= &IG
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
&OR EXAMPLE COMPOUND IS ESSENTIALLY EQUIPOTENT WITH %PO " WHICH IS THE MOST POTENT NATURAL EPOTHILONE AGAINST DRUG SENSITIVE HUMAN CAN CER CELL LINES )# VALUES AGAINST THE HUMAN CERVIX CARCINOMA CELL LINE +" ARE N- AND N- FOR AND %PO " RESPECTIVELY ,IKEWISE THE MODIFICATION CAN ALMOST COMPLETELY COMPENSATE FOR THE LOSS IN CEL LULAR POTENCY CAUSED BY THE REMOVAL OF THE HYDROXYL FUNCTION IN %PO " COMPOUND )# +" N- VERSUS N- FOR %PO " ,ASTLY THE ANTIPROLIFERATIVE ACTIVITY OF COMPOUND IN SPITE OF SIGNIFICANT STRUC TURAL DEVIATIONS FROM THE ORIGINAL NATURAL PRODUCT LEADS IS STILL COMPARABLE WITH THAT OF %PO ! %PO $ OR TAXOL )# +" N- ;= 4HIS LATTER ANALOG MIGHT IN FACT BE CONSIDERED AS THE FIRST REPRESENTATIVE OF A NEW STRUCTURAL CLASS OF MICROTUBULE STABILIZERS WHOSE OVERALL PHARMA COLOGICAL PROFILE MAY BE DISTINCT FROM THAT OF %PO !" AND MORE CLOSELY RELATED ANALOGS 7HILE DIMETHYL BENZIMIDAZOLE BASED EPOTHILONE ANALOGS ARE GENERALLY MORE ACTIVE AGAINST DRUG SENSITIVE HUMAN CANCER CELL LINES THAN THE CORRESPONDING DERIVATIVES INCORPORATING THE NATURAL EPOTHILONE SIDE CHAIN AND ALSO TEND TO BE MORE WATER SOLUBLE THEY EXHIBIT SLIGHTLY INCREASED SUSCEPTIBILITY TO 0 GP MEDIATED DRUG EFFLUX EG THE )# VALUES OF COMPOUNDS AND AGAINST THE 0 GP OVEREXPRESSING HUMAN CERVIX
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
CARCINOMA CELL LINE +" ARE N- AND N- VERSUS N- AND N- RESPECTIVELY FOR THE DRUG SENSITIVE +" LINE ;= (OWEVER WE HAVE RECENTLY DEMONSTRATED THAT THE 0 GP SUSCEPTIBILITY OF BENZIMIDAZOLE DERIVED EPOTHILONE ANALOGS CAN BE OVERCOME THROUGH THE REPLACEMENT OF THE EPOXIDE OXYGEN BY A MORE LIPOPHILIC METHYLENE GROUP THUS LEADING TO SIDE CHAIN MODIFIED CYCLOPROPANE DERIVATIVES DATA NOT SHOWN ;= %POTHILONE ANALOGS INCORPORATING CONFORMATIONALLY CONSTRAINED SIDE CHAINS HAVE ALSO BEEN USED AS TEMPLATES FOR A MORE DETAILED INVESTIGATION OF THE SIGNIFICANCE OF THE POSITION OF THE SIDE CHAIN . ATOM ;= 4HE PICTURE THAT HAS EMERGED FROM THESE STUDIES WITH REGARD TO THE RELATION SHIP BETWEEN BIOLOGICAL ACTIVITY AND . POSITIONING IN THE EPOTHILONE SIDE CHAIN IS RATHER COMPLEX AND CANNOT BE FULLY RATIONALIZED AT THIS POINT 4HUS WHILE BOTH ISOMERS WITHIN COMPOUND PAIRS A AND A &IG EXHIBIT VIRTUALLY IDENTICAL EFFECTS ON TUBULIN POLYMERIZATION THEY SHOW VERY DIFFERENT ANTIPROLIFERATIVE ACTIVITIES WITH THE @NATURAL ISOMER IE THE COMPOUND WITH THE . ATOM META TO THE BOND BETWEEN THE PHE NYL MOIETY AND THE MACROLACTONE RING BEING SIGNIFICANTLY MORE POTENT FOLD ;= 4HIS SUGGESTS THE POSITION OF THE SIDE CHAIN . ATOM TO
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
BE HIGHLY RELEVANT FOR CELLULAR POTENCY BUT NOT FOR THE INTERACTIONS OF THESE ANALOGS WITH THE TUBULINMICROTUBULE SYSTEM /N THE OTHER HAND VIRTUALLY NO EFFECT OF THE LOCATION OF THE . ATOM ON BIOLOGICAL ACTIVITY WAS OBSERVED BETWEEN EPOXIDE CONTAINING ANALOGS AND A EITHER AT THE LEVEL OF TUBULIN POLYMERIZATION IN VITRO OR AT THE LEVEL OF CANCER CELL GROWTH INHIBITION ;= 7HAT SEEMS TO BE CLEAR FROM THESE DATA IS THAT FACTORS OTHER THAN TUBULIN POLYMERIZING ACTIVITY IN VITRO MUST HAVE A PRONOUNCED IMPACT ON THE CELLULAR ACTIVITY OF EPOTHILONE ANALOGS A A AND A (OWEVER FURTHER EXPERIMENTS WILL BE REQUIRED IN ORDER TO ESTABLISH A MECHANISTIC UNDERSTANDING OF THE OBSERVED DIFFERENCES IN CELLULAR POTENCY AND OF THEIR VIRTUAL DISAPPEARANCE UPON CONVERSION OF AND A TO THE CORRESPONDING EPOXIDES
!ZA EPOTHILONES AZATHILONES /F THE NUMEROUS EPOTHILONE ANALOGS REPORTED IN THE LITERATURE OVER THE LAST YEARS ONLY A FEW ARE CHARACTERIZED BY THE REPLACEMENT OF CARBON ATOMS IN THE MACROCYCLIC SKELETON BY HETEROATOMS AND MOST OF THOSE INVESTIGATED TO DATE WERE FOUND TO BE POORLY ACTIVE FOR EXAMPLES SEE ; = /VERALL HOWEVER THE POTENTIAL OF SUCH MODIFICATIONS REMAINS LARGELY UNEXPLORED WHICH IS IN SPITE OF THE FACT THAT THE REPLACEMENT OF CARBON BY HETEROATOMS IN COMPLEX STRUCTURES COULD LEAD TO IMPROVED SYNTHETIC ACCESSIBILITY AND OFFER THE POTENTIAL TO GENERATE LARGE SETS OF DIVERSE ANALOGS IN A RATHER STRAIGHTFORWARD MANNER EG THROUGH AMIDE BOND FORMATION OR REDUCTIVE AMINATION IN THE CASE OF NITROGEN )N LIGHT OF THIS FACT AND FURTHER GUIDED BY THE POTENT BIOLOGICAL ACTIVITY ASSOCI ATED WITH THE DEOXYEPOTHILONE STRUCTURAL FRAMEWORK VIDE SUPRA SOME OF OUR INITIAL WORK IN THE AREA OF EPOTHILONE MODIFICATIONS WAS DIRECTED AT THE REPLACEMENT OF THE ## OLEFINIC DOUBLE BOND IN %PO $ BY . ALKYL AMIDES OR DISUBSTITUTED HETEROCYCLES SUCH AS EG IMIDAZOLE 3TRUCTURAL UNITS OF THIS TYPE WERE HYPOTHESIZED TO ACT AS CIS # # DOUBLE BOND MIMETICS AND WERE THUS EXPECTED TO INDUCE A SIMILAR CONFORMATION OF THE MACROCYCLE AS FOR %PO $ ASSUMING A PREFERENCE OF THE # . PARTIAL DOUBLE BOND IN AMIDE BASED ANALOGS FOR A CIS CONFORMATION AND THUS SIMILAR ANTIPROLIFERATIVE ACTIVITIES AS THE PARENT DEOXYEPOTHILONES !T THE SAME TIME THESE POLAR DOUBLE BOND SUBSTITUTES WERE ASSUMED TO LEAD TO
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
IMPROVED AQUEOUS SOLUBILITY OF THE CORRESPONDING ANALOGS OVER THE VERY LIPOPHILIC %PO $ 5NFORTUNATELY NONE OF THE ANALOGS n INCLUDING . UNSUBSTITUTED SECONDARY AMIDE WHICH WOULD MIMIC A TRANS OLEFIN GEOMETRY &IG SHOWED ANY APPRECIABLE TUBULIN POLYMERIZING OR ANTI PROLIFERATIVE ACTIVITY 4HE UNDERLYING REASONS FOR THE LACK OF BIOLOGICAL ACTIVITY OF ANALOGS n HAVE NOT BEEN ELUCIDATED BUT IT SHOULD BE NOTED THAT PRELIMI NARY .-2 STUDIES WITH COMPOUND A IN $-3/WATER SHOWED A DISTINCT PREFERENCE FOR THE REQUIRED CIS CONFORMATION ABOUT THE . METHYL AMIDE BOND CISTRANS RATIO ^ /N THE OTHER HAND SUBSEQUENT DATA OBTAINED IN VARIOUS LABORATORIES ; = INCLUDING OUR OWN FOR OTHER NON AMIDE BASED STRUCTURES SUGGESTED THAT INCREASING THE STERIC BULK AT # WAS GENERALLY ASSOCIATED WITH REDUCED POTENCY )N LIGHT OF THESE FIND INGS WE DECIDED TO CONTINUE EXPLORATION OF THE POTENTIAL UTILITY OF NITROGEN INCORPORATION AT POSITION OF THE MACROCYCLE AS A FUNCTIONAL HANDLE FOR FURTHER SUBSTITUTION WITHOUT CONCOMITANT MODIFICATION OF # !T THE MOST STRAIGHTFORWARD LEVEL THIS APPROACH INVOLVED SIMPLE ACYLATION OF THE NITROGEN ATOM THUS LEADING TO AMIDE AND CARBAMATE BASED ANALOGS OF TYPE &IG WHOSE CARBONYL OXYGEN COULD POTENTIALLY ASSUME THE ROLE OF THE EPOXIDE OXYGEN IN NATURAL EPOTHILONES !NALOGS WERE TESTED FOR THEIR ABILITY TO PROMOTE IN VITRO TUBULIN POLYMERIZATION AND THEIR ANTIPROLIFERATIVE ACTIVITY WAS ASSESSED AGAINST THE HUMAN CERVIX CANCER CELL LINES +" AND +" WHICH SERVE AS REPRE SENTATIVE EXAMPLES OF DRUG SENSITIVE AND 0 GP OVEREXPRESSING MULTIDRUG RESISTANT HUMAN CANCER CELL LINES RESPECTIVELY SEE EG ; =
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
&IGURE
4ABLE )NDUCTION OF TUBULIN POLYMERIZATION AND GROWTH INHIBITION OF HUMAN CARCINOMA CELL LINES BY AZA EPOTHILONES #OMPOUND
2
4UBULIN POLYMERIZATIONA
)# +" ;N-=B
)# +" ;N-=B
A
/ TERT #(
B
/#(0H
C
/#(
D
/ ISO #(
E
#(
. $C
F
#(
G
TERT #(
H
#(
A)NDUCTION OF POLYMERIZATION OF PORCINE BRAIN MICROTUBULE PROTEIN BY μ- OF TEST COMPOUND RELATIVE TO THE EFFECT OF μ- OF %PO " WHICH GAVE MAXIMAL POLYMERIZATION OF PROTEIN INPUT B)# VALUES FOR GROWTH INHIBITION OF HUMAN CERVIX CARCINOMA CELL LINES +" AND +" $ATA ARE FROM ;= C.OT DETERMINED
!S ILLUSTRATED BY THE DATA SUMMARIZED IN 4ABLE COMPOUNDS OF TYPE WHICH WE HAVE NAMED @AZATHILONES ALTHOUGH LESS ACTIVE INHIBITORS OF CANCER CELL GROWTH THAN %PO ! OR " CAN INDEED BE POTENT ANTIPROLIF ERATIVE AGENTS ;= )NTERESTINGLY HOWEVER SOME OF THESE ANALOGS ARE SIGNIFICANTLY LESS ACTIVE AGAINST THE MULTIDRUG RESISTANT +" LINE THAN THE DRUG SENSITIVE +" PARENTAL LINE THUS INDICATING THAT COMPOUNDS
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
ARE BETTER 0 GP SUBSTRATES THAN NATURAL EPOTHILONES 4HE STRUCTURAL BASIS FOR THIS PHENOMENON IS NOT UNDERSTOOD AT THIS POINT BUT THE FINDING IS IN LINE WITH A MORE GENERAL TENDENCY FOR POLAR EPOTHILONE ANALOGS EG COM POUNDS INCORPORATING OTHER AMIDE BONDS OR ADDITIONAL HYDROXYL GROUPS TO EXHIBIT INCREASED RESISTANCE FACTORS IN THE +" +" CELL LINE PAIR IE INCREASED RATIOS OF )# +" )# +" ; = - 7ART MANN + ( !LTMANN UNPUBLISHED OBSERVATIONS 4HE MOST INTERESTING ANALOG INCLUDED IN 4ABLE IS A WHICH IS ONLY CA FOLD LESS ACTIVE AGAINST THE DRUG SENSITIVE +" LINE THAN %PO ! AND THUS ROUGHLY EQUIPO TENT WITH %PO # AND IS CHARACTERIZED BY AN ONLY MODEST RESISTANCE FACTOR OF ^ 'UIDED BY THE 3!2 DATA FOR COMPOUNDS WE HAVE ALSO INVESTIGATED ANALOG &IG WHICH INCORPORATES THE POTENTIALLY POTENCY ENHANCING DIMETHYL BENZIMIDAZOLE SIDE CHAIN VIDE SUPRA ;= !ZATHILONE WAS FOUND TO BE A HIGHLY ACTIVE ANTIPROLIFERATIVE AGENT WHICH INHIBITS THE GROWTH OF DIFFERENT TYPES OF DRUG SENSITIVE HUMAN CAN CER CELLS LINES WITH SIMILAR POTENCY AS %PO ! EG )#S OF AGAINST THE HUMAN CANCER CELL LINES ! LUNG (#4 COLON 0# - PROSTATE AND +" CERVIX ARE N- N- N- AND N- RESPECTIVELY VERSUS N- N- N- AND N- FOR %PO ! ;= ,IKEWISE PROMOTES MICROTUBULE ASSEMBLY FROM SOLUBLE TUBULIN WITH THE SAME EFFICIENCY AS %PO ! ;= 4HE REPLACEMENT OF THE NATURAL THIAZOLYL VINYL SIDE CHAIN IN A BY A DIMETHYL BENZIMIDAZOLE MOIETY THUS RESULTS IN A FOLD INCREASE IN CELLULAR POTENCY 4HIS EFFECT IS SIGNIFICANTLY HIGHER THAN WHAT HAD BEEN OBSERVED PREVIOUSLY IN THE CONTEXT OF POLYKETIDE BASED MACROCYCLES WHERE INCORPORATION OF A DIMETHYL BENZIMIDAZOLE SIDE
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
CHAIN LEADS TO A POTENCY INCREASE OF n FOLD )N LIGHT OF THE PROFOUND STRUCTURAL DIFFERENCES BETWEEN AND THE ORIGINAL NATURAL PRODUCT LEAD %PO ! THE ACTIVITY OF IS HIGHLY REMARKABLE AND THE COMPOUND MAY IN FACT BE CONSIDERED AS A FIRST REPRESENTATIVE OF A NEW STRUCTURAL CLASS OF MICROTUBULE STABILIZING AGENTS 7HILE EPOTHILONE ANALOGS BASED ON A AZA MACROLIDE CORE STILL RETAIN MANY OF THE TWO DIMENSIONAL STRUCTURAL FEATURES OF NATU RAL EPOTHILONES THEIR MACROLACTONE RING IS NO LONGER BASED ON A CONTIGUOUS POLYKETIDE BACKBONE !NALOGS OF THIS TYPE HAVE THEREFORE BEEN CLASSIFIED AS hNON NATURAL NATURAL PRODUCTSv BY !LTMANN AND CO WORKERS ;= 4HE IMPORTANCE OF THE TERT BUTYL GROUP IN FOR BIOLOGICAL ACTIVITY WAS PROBED BY ITS REPLACEMENT WITH A LESS BULKY AND LESS LIPOPHILIC ETHYL GROUP 4HE RESULTING ANALOG PROVED TO BE LESS POTENT THAN BUT )# VALUES FOR GROWTH INHIBITION OF DRUG SENSITIVE HUMAN CANCER CELL LINES ARE STILL IN THE n N- RANGE )NTRIGUINGLY THE PRESENCE OF A TRANS DOUBLE BOND BETWEEN # AND # COMPOUND A &IG PRODUCED A DRAMATIC LOSS IN BIOLOGICAL POTENCY ;= WHICH IS CONTRARY TO THE EFFECTS OF THIS MODIFICATION WITHIN THE STRUCTURAL FRAMEWORK OF NATURAL EPOTHILONES VIDE SUPRA 4HIS FINDING MAY BE INDICATIVE OF DIFFERENCES IN THE TUBULIN BOUND CONFORMATION BETWEEN NATURAL EPOTHILONES AND THE AZA MACROLIDE BASED AZATHILONES BUT FURTHER EXPERIMENTAL WORK WILL BE NECESSARY TO EITHER CONFIRM OR REFUTE THIS HYPOTHESIS 5NFORTUNATELY AND IN MARKED CONTRAST TO NATURAL EPOTHILONES THE ACTIVITY OF AS WELL AS THE CORRESPONDING ETHYL CARBAMATE AGAINST THE MULTIDRUG RESISTANT +" HUMAN CERVIX CARCINOMA CELL LINE IS SIGNIFICANTLY REDUCED WHICH INDICATES THAT THE COMPOUND IS HIGHLY SUSCEPTIBLE TO 0 GP MEDIATED DRUG EFFLUX %FFORTS ARE CURRENTLY UNDERWAY TO IMPROVE THE ACTIVITY OF AZATHILONE TYPE ANALOGS AGAINST 0 GP OVEREXPRESSING CELLS THROUGH THE INTRODUCTION OF ADDITIONAL OR ALTERNATIVE MODIFICATIONS ! THIRD TYPE OF AZA EPOTHILONES THAT WE HAVE INVESTIGATED AS PART OF OUR PROGRAM ON BACKBONE MODIFIED HETERO ANALOGS OF EPOTHILONES IS CHARAC TERIZED BY THE REPLACEMENT OF # BY NITROGEN AND THE PRESENCE OF A #. AMIDE GROUP RATHER THAN A # KETONE &IG ;= 4HESE ANALOGS WERE INSPIRED BY THE FACT THAT ONE OF THE CHARACTERISTIC FEATURES OF THE TUBULIN BOUND STRUCTURE OF %PO ! ;= IS THE PRESENCE OF A SYN PERIPLA NAR CONFORMATION ABOUT THE #n# BOND 4HE SAME GEOMETRY WOULD BE ENFORCED IN ANALOGS OF TYPE PROVIDED THAT THE AMIDE BOND BETWEEN . AND # WOULD BE PRESENT IN A CIS CONFORMATION !T THE SAME TIME
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
&IGURE
PRELIMINARY MODELING STUDIES INDICATED THAT THE PRESENCE OF A CIS AMIDE BOND IN THIS POSITION SHOULD ALLOW REPLACEMENT OF THE #n# SEGMENT BY VARIOUS TYPES OF β AMINO ACIDS WITHOUT CAUSING SIGNIFICANT DISTORTIONS IN THE BIOACTIVE CONFORMATION OF THE #n/n# SEGMENT !PART FROM THESE STRUCTURAL CONSIDERATIONS STRUCTURES OF TYPE ALSO APPEARED ATTRACTIVE FOR CHEMICAL REASONS AS THEY WOULD LEND THEMSELVES TO AN EFFICIENT COMBINA TORIAL CHEMISTRY APPROACH EMPLOYING A SINGLE ADVANCED INTERMEDIATE IE A # CARBOXYLIC ACID ENCOMPASSING THE #n# FRAGMENT EPOTHILONE NUMBERING 3O FAR ONLY A LIMITED NUMBER OF EXAMPLES OF ANALOGS OF TYPE HAVE BEEN INVESTIGATED ALL OF WHICH WERE FOUND TO LACK ANY MEANINGFUL TUBULIN POLYMERIZING OR ANTIPROLIFERATIVE ACTIVITY ;= (OWEVER A LARGER NUMBER OF THESE STRUCTURES INCORPORATING DIFFERENT TYPES OF α β AND γ AMINO ACIDS WILL NEED TO BE INVESTIGATED BEFORE ALLOWING A FINAL CON CLUSION ON THE PHARMACEUTICAL VALIDITY OF THIS MODIFICATION APPROACH "ASED ON THE CHEMISTRY DEVELOPED IN OUR LABORATORY FOR THESE COMPOUNDS ;= THE SYNTHESIS OF SUCH ADDITIONAL ANALOGS SHOULD BE A STRAIGHTFORWARD UNDERTAKING
3TRUCTURAL STUDIES AND PHARMACOPHORE MODELING )N ORDER TO GUIDE THE DESIGN OF IMPROVED AND PERHAPS STRUCTURALLY SIMPLI FIED EPOTHILONE ANALOGS THE DEVELOPMENT OF A PREDICTIVE PHARMACOPHORE MODEL FOR THESE MICROTUBULE STABILIZING AGENTS HAS BEEN A LONG STANDING GOAL IN EPOTHILONE RESEARCH %ARLY ATTEMPTS IN THIS AREA ; n= WERE GENERALLY BASED ON THE ASSUMPTION OF A COMMON TUBULIN BINDING SITE BETWEEN EPOTHILONES AND TAXOL IN CONJUNCTION WITH STRUCTURAL DATA ON
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
A COMPLEX BETWEEN TWO DIMENSIONAL TUBULIN POLYMER SHEETS AND TAXOL WHICH HAD BEEN OBTAINED BY ELECTRON CRYSTALLOGRAPHY AT ¯ RESOLUTION ;= "ASED ON THESE PREMISES 'IANNAKAKOU ET AL ;= HAVE PROPOSED A PHARMACOPHORE MODEL WHICH PLACES THE EPOXIDE OXYGEN OF EPOTHILONES IN THE SAME POSITION AS THE OXETANE OXYGEN IN TAXOL WHILE THE EPOTHILONE SIDE CHAIN IS LOCATED IN THE SAME REGION AS EITHER THE # PHENYL GROUP OR ALTERNATIVELY THE # BENZOYLOXY MOIETY OF TAXOL 4HE MODEL ALSO SUGGESTS THAT THE METHYL GROUP ATTACHED TO # IN %PO " IS INVOLVED IN HYDROPHO BIC INTERACTIONS WITH THE SIDE CHAINS OF ,EU β ,EU β ,EU β AND 0HE β AND THAT THIS MAY ACCOUNT FOR THE HIGHER ACTIVITY OF %PO " VERSUS %PO ! $IFFERENT CONCLUSIONS WITH REGARD TO THE RELATIVE POSITIONING OF TAXOL AND EPOTHILONES WITHIN THE MICROTUBULE BINDING SITE HAVE BEEN REACHED BY 7ANG ET AL ;= )N THEIR MODEL THE POSITION OF THE THIAZOLE MOIETY IN EPOTHILONES WITHIN THE MICROTUBULE BINDING POCKET MATCHES THE POSITION OF THE PHENYL GROUP OF THE # BENZAMIDO SUBSTITUENT IN TAXOL &URTHERMORE THE EPOXIDE OXYGEN IS CONCLUDED NOT TO BE INVOLVED IN INTERACTIONS WITH THE PROTEIN WHICH IS IN LINE WITH THE EXPERIMENTAL DATA DISCUSSED ABOVE FOR CYCLOPROPANE BASED EPOTHILONE ANALOGS ! MODEL SIMILAR TO THAT OF 7ANG ET AL HAS RECENTLY BEEN PROPOSED BY "OTTA AND CO WORKERS ;= !LTHOUGH THESE COMPUTATIONAL MODELS BY THEIR VERY NATURE ARE OF LIMITED ACCURACY SOME OF THEM ARE ABLE TO REPRODUCE AT LEAST PART OF THE PUBLISHED EPOTHILONE 3!2 WITH REASONABLE ACCURACY ; = (OW EVER ALL OF THESE MODELS MAY NEED TO BE REVISITED IN LIGHT OF MORE RECENT STRUCTURAL STUDIES ON THE TUBULIN BOUND CONFORMATION OF %PO ! EITHER BY .-2 SPECTROSCOPY ON A SOLUBLE β TUBULIN%PO ! COMPLEX ;= OR BY A COMBINATION OF ELECTRON CRYSTALLOGRAPHY .-2 SPECTROSCOPIC CONFORMA TIONAL ANALYSIS AND MOLECULAR MODELING OF A COMPLEX BETWEEN %PO ! AND A :N STABILIZED TWO DIMENSIONAL α β TUBULIN SHEET SOLVED AT ¯ RESO LUTION ;= 4HESE STUDIES HAVE PROVIDED COMPLETELY NEW INSIGHTS INTO THE BIOACTIVE CONFORMATION OF THE EPOTHILONE CLASS OF MICROTUBULE INHIBITORS )N PARTICULAR THE SOLID STATE STUDY BY .ETTLES ET AL ;= SERIOUSLY CALLS INTO QUESTIONS THE NOTION OF A COMMON PHARMACOPHORE BETWEEN TAXOL AND EPOTHILONES WHICH HAS BEEN A CENTRAL PROVISION IN ALL EARLIER MODELING STUDIES !CCORDING TO THE DATA PRESENTED BY .ETTLES ET AL %PO ! AND TAXOL DO INDEED OCCUPY THE SAME GROSS BINDING POCKET BUT THE ACTUAL BINDING IS MEDIATED THROUGH DIFFERENT SETS OF HYDROGEN BONDING AND HYDROPHOBIC INTERACTIONS FOR THE TWO COMPOUNDS )NTERESTINGLY THE TUBULIN BOUND
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
CONFORMATION OF %PO ! DERIVED FROM THE ELECTRON CRYSTALLOGRAPHIC DATA AT ¯ IS DIFFERENT FROM ANY OF THE ABOVE COMPUTATIONAL MODELS BUT ALSO FROM THE RECENT .-2 STRUCTURE OF SOLUBLE TUBULIN BOUND %PO ! DETER MINED BY #ARLOMAGNO ET AL BY MEANS OF MAGNETIZATION TRANSFER .-2 TECHNIQUES ;= 4HE RELATIVE VALIDITY OF THE TWO MODELS IS DIFFICULT TO ASSESS BUT IT SHOULD BE NOTED THAT ONE IMPORTANT 3!2 FEATURE OF EPOTHI LONES IS NOT WELL EXPLAINED BY THE STRUCTURE OF .ETTLES ET AL NAMELY THE FACT THAT FORMAL DEHYDRATION ACROSS THE #n# BOND PRODUCING A TRANS DOUBLE BOND IS NOT ASSOCIATED WITH ANY MAJOR LOSS IN TUBULIN POLYMER IZING OR ANTIPROLIFERATIVE ACTIVITY VIDE SUPRA 4HIS FINDING SUGGESTS THE #n# TORSION ANGLE IN THE BIOACTIVE CONFORMATION OF EPOTHILONES TO BE IN THE VICINITY OF WHICH CORRESPONDS TO THE CONFORMATION IN THE .-2 DERIVED STRUCTURE BUT IS NOT THE CONFORMATION SUGGESTED BY THE .ETTLES STRUCTURE /N THE OTHER HAND A TORSION ANGLE ABOUT THE #n# BOND IS INFERRED BY BOTH STRUCTURES THUS LOCATING THE METHYL GROUP AND THE THIAZOLE NITROGEN ON OPPOSITE SIDES OF THE #n# DOUBLE BOND )N FACT .ETTLES ET AL HAVE PROPOSED THIS NITROGEN ATOM TO BE INVOLVED IN A ( BOND WITH THE PROTONATED FORM OF β (IS 4HESE CONCLUSIONS ARE CONSISTENT WITH THE OBSERVATION THAT THE ANTIPROLIFERATIVE ACTIVITY OF PYRIDINE BASED ANALOGS OF %PO " THIAZOLE RING REPLACED BY A OR PYRIDYL MOIETY DECREASES IN THE ORDER PYRIDYL PYRIDYL ≈ PYRIDYL THUS INDICATING A REQUIREMENT FOR THE . ATOM TO BE LOCATED IN THE POSITION ORTHO TO THE ATTACHMENT POINT OF THE LINKER BETWEEN THE HETEROCYCLE AND THE MACROCYCLIC CORE VIDE SUPRA (OWEVER AS HIGHLIGHTED ABOVE IN THE DISCUSSION OF %PO " AND $ ANALOGS CONTAINING ISOMERIC BENZOTHIAZOLE OR QUINOLINE BASED SIDE CHAINS CELLULAR EFFECTS MAY NOT BE ENTIRELY REFLECTIVE OF THE STRENGTH OF LIGANDTUBULIN INTERACTIONS AND THE INTERPRETATION OF CHANGES IN CELLULAR ACTIVITY IN TERMS OF STRUCTURAL FEATURES OF A LIGANDTUBU LIN COMPLEX COULD IN FACT BE MISLEADING )T REMAINS TO BE SEEN HOW THE VARIOUS PHARMACOPHORE MODELS AND THE STRUCTURAL PROPOSALS MADE ON THE BASIS OF .-2 OR ELECTRON CRYSTALLOGRA PHY DATA WILL ACTUALLY RELATE TO THE TRUE IE DERIVED FROM ATOMIC RESOLU TION STRUCTURAL DATA IF THEY BECOME AVAILABLE BIOACTIVE CONFORMATION OF EPOTHILONES SHOULD THERE EVEN BE A SINGLE UNIQUE BIOACTIVE CONFORMA TION )N THE MEANTIME BOTH THE EXPERIMENTALLY DERIVED AS WELL AS THE PURELY COMPUTATIONAL STRUCTURES MAY SERVE AS A USEFUL BASIS FOR THE DESIGN OF NEW ANALOGS
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
%POTHILONES IN CLINICAL DEVELOPMENT !S INDICATED ABOVE THE FIRST COMPOUND OF THE EPOTHILONE CLASS OF MICROTU BULE INHIBITORS TO ENTER CLINICAL TRIALS WAS %PO " %0/ PATUPILONE 4HIS INVOLVED INITIAL TESTING IN TWO 0HASE ) STUDIES EMPLOYING EITHER A WEEKLY OR A ONCE EVERY THREE WEEKS DOSING REGIMEN ; = -4$S OF MG M AND MGM RESPECTIVELY WERE OBSERVED IN THE TWO STUDIES WITH DIARRHEA BEING THE DOSE LIMITING TOXICITY IN BOTH CASES !LL OTHER TOXICITIES OBSERVED AT -4$ WERE MILD TO MODERATE AND NO SIGNIFICANT MYELOSUPPRES SION WAS FOUND 4HE DRUG APPEARED TO BE PARTICULARLY ACTIVE IN PATIENTS WITH COLORECTAL CANCER WITH ADDITIONAL RESPONSES IN PATIENTS WITH BREAST OVARIAN AND NON SMALL CELL LUNG CANCERS AND CARCINOID TUMORS WHOSE DISEASE HAD PROGRESSED ON SEVERAL OTHER THERAPIES %PO " HAS ALSO BEEN INVESTIGATED IN A 0HASE ))) DOSE ESCALATION TRIAL IN PATIENTS WITH RELAPSED REFRACTORY OVARIAN CANCER AND RESULTS AGAIN APPEAR TO BE PROMISING ;= %PO " IS CURRENTLY UNDERGOING 0HASE ))) TRIALS ;= 3UBSEQUENT OF THE INITIATION OF CLINICAL STUDIES WITH %PO " A VARIETY OF MODIFIED ANALOGS HAVE ALSO PROCEEDED TO CLINICAL EVALUATION IN HUMANS 4HE MOST ADVANCED OF THESE COMPOUNDS IS "-3 WHICH HAS RECENTLY OBTAINED &$! APPROVAL FOR THE TREATMENT OF METASTATIC AND ADVANCED BREAST CANCER ; n= &OR RECENT REVIEWS ON THE CLINICAL PHARMACOLOGY OF THIS COMPOUND SEE ;n= SEE ALSO ;= "RIEFLY CLINI CAL TRIALS WITH "-3 WERE INITIATED IN EARLY AND A NUMBER OF OBJECTIVE RESPONSES TO SINGLE AGENT TREATMENT WITH THE COMPOUND WERE OBSERVED IN THESE STUDIES IN BREAST OVARIAN CERVICAL PROSTATE COLON LUNG AND RENAL CANCERS AS WELL AS IN SQUAMOUS CELL CANCERS OF THE HEAD AND NECK LYMPHOMA AND ANGIOSARCOMA $OSE LIMITING TOXICITIES INCLUDED FATIGUE PROLONGED NEUTROPENIA AND PERIPHERAL NEUROPATHY ;= 4HE COMPOUND WAS ALSO SHOWN TO BE ORALLY BIOAVAILABLE IN HUMANS ;= THUS CONFIRMING PREVIOUS RESULTS FROM ANIMAL STUDIES ;= VIDE SUPRA 6ERY IMPORTANTLY "-3 HAS BEEN DEMONSTRATED TO INDUCE MICROTUBULE BUNDLING IN 0"-#S PERIPHERAL BLOOD MONOCYTES OF TREATED INDIVIDUALS AND A GOOD CORRELATION WAS OBSERVED BETWEEN THE MAGNITUDE OF THIS EFFECT AND PLASMA !5#S AREA UNDER THE CURVE ;= 4HESE FINDINGS VALIDATE THE IN VITRO PHAR MACODYNAMIC FINDINGS WITH "-3 IN THE CLINICAL SETTING 0HASE )) TRIALS WITH "-3 HAVE PRODUCED OBJECTIVE RESPONSES FOR A VARIETY OF TUMOR TYPES INCLUDING TUMORS WHICH HAD BEEN REFRACTORY
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
TO TREATMENT WITH PLATINUM BASED DRUGS OR TAXANES ; = "ASED ON THESE HIGHLY PROMISING RESULTS THE COMPOUND HAS BEEN ADVANCED TO 0HASE ))) STUDIES WHICH ARE CURRENTLY ONGOING IN PARALLEL WITH A NUMBER OF 0HASE )) TRIALS INCLUDING COMBINATION STUDIES ;= 0HASE ) CLINICAL STUDIES HAVE ALSO BEEN COMPLETED WITH %PO $ AND THE COMPOUND WAS SUBSEQUENTLY ADVANCED TO 0HASE )) TRIALS SPONSORED BY +OSAN "IOSCIENCES AS +/3 ;n= !S FOR "-3 +/3 WAS DEMONSTRATED TO INDUCE MICROTUBULE BUNDLING IN PATIENT 0"-#S AND A NUMBER OF TUMOR RESPONSES HAVE BEEN NOTED IN THE 0HASE ) STUDIES ;= (OWEVER IN THE MEANTIME THE CLINICAL DEVELOPMENT OF +/3 HAS BEEN TERMINATED /THER EPOTHILONE ANALOGS THAT HAVE ENTERED CLINICAL TRIALS SUBSEQUENT TO %PO " "-3 AND %PO $ INCLUDE "-3 0HASE ) ; = # DESMETHYL # METHYLSULFANYL %PO " !"* 0HASE ) ;= DIDEHYDRO %PO " +/3 +OSAN2OCHE 0HASE ) AND THE SYNTHETIC ANALOG :+ %0/ SAGOPILONE "AYER 3CHERING 0HASE )) ;=
#ONCLUSIONS 4HE DISCOVERY BY "OLLAG ET AL THAT %PO ! AND " REPRESENT A NEW CLASS OF MICROTUBULE DEPOLYMERIZATION INHIBITORS WHICH ARE NOT SUBJECT TO 0 GP MEDIATED EFFLUX MECHANISMS TRANSFORMED THESE COMPOUNDS INTO HIGHLY INTERESTING LEAD STRUCTURES FOR THE DEVELOPMENT OF A NEW GENERATION OF 4AXOL¤ LIKE ANTICANCER DRUGS !PART FROM THEIR LOW SUSCEPTIBILITY TO 0 GP MEDIATED DRUG EFFLUX %PO ! AND " ARE MORE WATER SOLUBLE THAN TAXOL WHICH HAS ENABLED THE USE OF SIDE EFFECT FREE FORMULATION VEHICLES AT LEAST FOR SOME OF THE CLINICAL DEVELOPMENT COMPOUNDS !T THE SAME TIME AND IN CONTRAST TO MANY OTHER NATURAL PRODUCT BASED DRUG DISCOVERY EFFORTS TOTAL CHEMICAL SYNTHESIS HAS PROVEN TO BE A PERFECTLY FEASIBLE APPROACH TO ACCESS A WIDE VARIETY OF STRUCTURAL ANALOGS OF EPOTHILONES 4HIS CHAPTER HAS TRIED TO SUMMARIZE THE BASIC BIOLOGY AND PHARMACOL OGY OF %PO " AS THE MOST POTENT AND MOST WIDELY STUDIED NATURAL EPOTHI LONE )T HAS ALSO OUTLINED THE MOST IMPORTANT STRUCTURAL MODIFICATIONS THAT HAVE BEEN INVESTIGATED FOR EPOTHILONES AND THE MOST SIGNIFICANT 3!2 FEATURES THAT HAVE EMERGED FROM THESE STUDIES 4HESE EFFORTS HAVE LED TO
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
A SIZABLE NUMBER OF ANALOGS WITH ANTIPROLIFERATIVE ACTIVITIES COMPARABLE TO THAT OF %PO !" IN ADDITION MANY OTHER COMPOUNDS WITH EPOTHILONE TYPE STRUCTURES HAVE BEEN DISCOVERED TO BE HIGHLY POTENT ANTIPROLIFERA TIVE AGENTS EVEN IF THEY ARE LESS ACTIVE THAN THE CORRESPONDING PARENT EPOTHILONES )N ORDER TO ENABLE THE RATIONAL DESIGN OF IMPROVED EPOTHILONE ANALOGS DIFFERENT PHARMACOPHORE MODELS HAVE BEEN PROPOSED OVER THE PAST SEVERAL YEARS WHICH ACCOMMODATE AND RATIONALIZE AT LEAST SOME ASPECTS OF THE EPOTHILONE 3!2 4HESE COMPUTATIONAL EFFORTS HAVE RECENTLY BEEN COMPLE MENTED AND PERHAPS SUPERSEDED BY THE ELUCIDATION OF THE TUBULIN BOUND STRUCTURE OF %PO ! BY MEANS OF .-2 SPECTROSCOPY AND BY ELECTRON CRYSTAL LOGRAPHY .EVERTHELESS OUR CURRENT UNDERSTANDING OF THE INTERRELATIONSHIP BETWEEN STRUCTURE AND BIOLOGICAL ACTIVITY FOR EPOTHILONE TYPE STRUCTURES AT THE MOLECULAR LEVEL IS FAR FROM COMPLETE AND IT REMAINS TO BE SEEN HOW THE RECENT GAIN IN STRUCTURAL INFORMATION WILL IMPACT ON THE DESIGN OF IMPROVED EPOTHILONE ANALOGS 3O FAR AT LEAST SEVEN COMPOUNDS OF THE EPOTHILONE CLASS HAVE BEEN ADVANCED TO CLINICAL DEVELOPMENT 4HIS INCLUDES %PO " %0/ PATUPI LONE %PO " LACTAM "-3 IXABEPILONE # AMINO %PO " "-3 %PO $ +/3 # DESMETHYL # METHYLSULFANYL %PO " !"* DIDEHYDRO %PO " +/3 ;= AND :+ %0/ AND MORE ARE STILL LIKELY TO FOLLOW )N THIS ACCOUNT WE PROVIDED A CRITICAL REVIEW OF THE PRECLINICAL DATA AVAILABLE FOR THESE COMPOUNDS AND WE HAVE BRIEFLY SUMMA RIZED THEIR CLINICAL STATUS 7E HAVE ALSO HIGHLIGHTED THE KEY FEATURES OF THE FERMENTATION PROCESSES THAT HAVE BEEN DEVELOPED FOR THE LARGE SCALE PRO DUCTION OF %PO " AND %PO $ WHICH ARE CLINICAL CANDIDATES BY THEMSELVES AND IN THE CASE OF %PO " ALSO SERVE AS STARTING MATERIAL FOR THE PRODUCTION OF AT LEAST THREE ADDITIONAL CLINICAL DEVELOPMENT COMPOUNDS 7HILE NONE OF THESE COMPOUNDS HAS YET SUCCESSFULLY COMPLETED CLINICAL DEVELOPMENT THE AVAILABLE PUBLISHED DATA FOR THE MOST ADVANCED COMPOUNDS %0/ AND "-3 SUGGEST THAT THE HIGHLY PROMISING PRECLINICAL PROFILE OF THESE AGENTS MAY INDEED TRANSLATE INTO THERAPEUTIC UTILITY AT THE CLINICAL LEVEL IN THE VERY NEAR FUTURE )NTERESTINGLY THE CLINICAL PROFILES OF %0/ AND "-3 APPEAR TO BE DISTINCTLY DIFFERENT WHICH HIGHLIGHTS THE IMPORTANCE AND USEFULNESS OF CLINICAL TESTING EVEN OF STRUCTURALLY CLOSELY RELATED ANALOGS
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
!CKNOWLEDGEMENTS +(! WOULD LIKE TO THANK ALL FORMER COLLABORATORS AT .OVARTIS FOR THEIR HELP AND INSPIRATION OVER THE MANY YEARS )N PARTICULAR HE WANTS TO ACKNOWL EDGE THE SUPPORT BY $RS ! &LÙRSHEIMER 4 /4EILLY AND - 7ARTMANN WHO HAD WORKED WITH HIM ON THE EPOTHILONE PROGRAM AT .OVARTIS SINCE ITS VERY BEGINNING (IS SINCERE THANKS ALSO GOES TO 0ROF +# .ICOLAOU AND HIS GROUP AT THE 4HE 3CRIPPS 2ESEARCH )NSTITUTE FOR A VERY PRODUCTIVE COL LABORATION
2EFERENCES
*EMAL ! 3IEGEL 2 7ARD % -URRAY 4 8U * 3MIGAL # 4HUN -* #ANCER STATISTICS #! #ANCER * #LIN n #APDEVILLE 2 "UCHDUNGER % :IMMERMANN * -ATTER ! 'LIVEC 34) IMATINIB A RATIONALLY DEVELOPED TARGETED ANTICANCER DRUG .ATURE 2EV $RUG $ISC n 7ILHELM 3 #ARTER # ,YNCH - ,OWINGER 4 $UMAS * 3MITH 2! 3CHWARTZ " 3IMANTOV 2 +ELLEY 3 $ISCOVERY AND DEVELOPMENT OF SORAFENIB A MULTIKINASE INHIBITOR FOR TREATING CANCER .ATURE 2EV $RUG $ISC n !DAMS '0 7EINER ,- -ONOCLONAL ANTIBODY THERAPY OF CANCER .ATURE "IOTECHNOL n &ERRARA . (ILLAN +* .OVOTNY 7 "EVACIZUMAB !VASTIN A HUMANIZED ANTI 6%'& MONOCLONAL ANTIBODY FOR CANCER THERAPY "IOCHEM "IOPHYS 2ES #OMM n ,U -# !NTIMITOTIC AGENTS )N 7/ &OYE ED #ANCER CHEMOTHERAPEUTIC AGENTS !MERICAN #HEMICAL 3OCIETY 7ASHINGTON $# n -EKHAIL 4- -ARKMAN - 0ACLITAXEL IN CANCER THERAPY %XP /PIN 0HARMACOTHER n /BASAJU # (UDES '2 0ACLITAXEL AND DOCETAXEL IN PROSTATE CANCER (EMATOLOGY /NCOLOGY #LINICS OF .ORTH !MERICA n (AMEL % !NTIMITOTIC NATURAL PRODUCTS AND THEIR INTERACTION WITH TUBULIN -ED 2ES 2EV n 2OWINSKY %+ 4HE DEVELOPMENT AND CLINICAL UTILITY OF THE TAXANE CLASS OF ANTIMI CROTUBULE CHEMOTHERAPY AGENTS !NN 2EV -ED n 0RINZ ( 2ECENT ADVANCES IN THE FIELD OF TUBULIN POLYMERIZATION INHIBITORS %XP 2EV !NTICANCER 4HER n !LTMANN +( -ICROTUBULE STABILIZING AGENTS A GROWING CLASS OF IMPORTANT ANTI CANCER DRUGS #URR /PIN #HEM "IOL n -ILES $# %MERGING MICROTUBULE STABILIZING AGENTS FOR CANCER CHEMOTHERAPY !NN 2EP -ED #HEM n !LTMANN +( 'ERTSCH * !NTICANCER DRUGS FROM NATURE n NATURAL PRODUCTS AS A UNIQUE SOURCE OF NEW MICROTUBULE STABILIZING AGENTS .AT 0ROD 2EP n 3CHIFF 0" &ANT * (ORWITZ 3" 0ROMOTION OF MICROTUBULE ASSEMBLY IN VITRO BY TAXOL .ATURE n
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
(ÙFLE ' 2EICHENBACH ( %POTHILONE A MYXOBACTERIAL METABOLITE WITH PROMISING ANTITUMOR ACTIVITY )N '- #RAGG $') +INGSTON $* .EWMAN EDS !NTICANCER AGENTS FORM NATURAL PRODUCTS 4AYLOR &RANCIS "OCA 2ATON &, n 'ERTH + "EDORF . (ÙFLE ' )RSCHIK ( 2EICHENBACH ( %POTHILONS ! AND " !NTI FUNGAL AND CYTOTOXIC COMPOUNDS FROM 3ORANGIUM CELLULOSUM MYXOBACTERIA 0RODUC TION PHYSICO CHEMICAL AND BIOLOGICAL PROPERTIES * !NTIBIOTICS n SEE ALSO ' (ÙFLE . "EDORF + 'ERTH ( 2EICHENBACH 'ERMAN 0ATENT $ISCLOSURE $% #HEM !BSTR "OLLAG $- -C1UENEY 0! :HU * (ENSENS / +OUPAL , ,IESCH * 'OETZ - ,AZARIDES % 7OODS #- %POTHILONES A NEW CLASS OF MICROTUBULE STABILIZING AGENTS WITH A 4AXOL LIKE MECHANISM OF ACTION #ANCER 2ES n (ARDT )( 3TEINMETZ ( 'ERTH + 3ASSE & 2EICHENBACH ( (ÙFLE ' .EW NATURAL EPOTHILONES FROM 3ORANGIUM CELLULOSUM 3TRAINS 3O CE" AND 3O CE$ )SOLATION STRUCTURE ELUCIDATION AND 3!2 STUDIES * .AT 0ROD n (ÙFLE ' "EDORF . 3TEINMETZ ( 3CHOMBURG $ 'ERTH + 2EICHENBACH ( !NTIBI OTICS FROM GLIDING BACTERIA %POTHILONE ! AND " n NOVEL MEMBERED MACROLIDES WITH CYTOTOXIC ACTIVITY ISOLATION CRYSTAL STRUCTURE AND CONFORMATION IN SOLUTION !NGEW #HEM )NT %D n +OWALSKI 2* 'IANNAKAKOU 0 (AMEL % !CTIVITIES OF THE MICROTUBULE STABILIZING AGENTS EPOTHILONES ! AND " WITH PURIFIED TUBULIN AND IN CELLS RESISTANT TO PACLITAXEL 4AXOL * "IOL #HEM n !LTMANN + ( 7ARTMANN - /2EILLY 4 %POTHILONES AND RELATED STRUCTURES n A NEW CLASS OF MICROTUBULE INHIBITORS WITH POTENT IN VIVO ANTITUMOR ACTIVITY "IOCHIM "IOPHYS !CTA -n- 7OLFF ! 4ECHNAU ! "RANDNER ' %POTHILONE ! INDUCES APOPTOSIS IN NEUROBLAS TOMA CELLS WITH MULTIPLE MECHANISMS OF DRUG RESISTANCE )NT * /NC n 'IANNAKAKOU 0 3ACKETT $, +ANG 9+ :HAN : "UTERS *4 &OJO 4 0ORUCHYNSKY -3 0ACLITAXEL RESISTANT HUMAN OVARIAN CANCER CELLS HAVE MUTANT β TUBULINS THAT EXHIBIT IMPAIRED PACLITAXEL DRIVEN POLYMERIZATION * "IOL #HEM n .ICOLAOU +# 2OSCHANGAR & 6OURLOUMIS $ #HEMICAL BIOLOGY OF EPOTHILONES !NGEW #HEM )NT %D n (ARRIS #2 $ANISHEFSKY 3* #OMPLEX TARGET ORIENTED SYNTHESIS IN THE DRUG DISCOV ERY PROCESS A CASE HISTORY IN THE D%PO" SERIES * /RG #HEM n -ULZER * -ARTIN (* "ERGER - 0ROGRESS IN THE SYNTHESIS OF CHIRAL HETEROCYCLIC NATURAL PRODUCTS EPOTHILONE " AND TARTROLON " * (ETEROCYCL #HEM n .ICOLAOU +# 2ITZEN ! .AMOTO + 2ECENT DEVELOPMENTS IN THE CHEMISTRY BIOLOGY AND MEDICINE OF THE EPOTHILONES *#3 #HEM #OMMUN n !LTMANN +( 4HE MERGER OF AND NATURAL PRODUCT SYNTHESIS AND MEDICINAL CHEM ISTRY ON THE CHEMISTRY AND CHEMICAL BIOLOGY OF EPOTHILONES /RG "IOMOL #HEM n 7ATKINS %" #HITTIBOYINA !' *UNG *# !VERY -! 4HE EPOTHILONES AND RELATED ANALOGUES n ! REVIEW OF THEIR SYNTHESES AND ANTI CANCER ACTIVITIES #URR 0HARM $ES n 7ATKINS %" #HITTIBOYINA !' *UNG *# !VERY -! 2ECENT DEVELOPMENTS IN THE SYNTHESES OF THE EPOTHILONES AND RELATED ANALOGUES %UR * /RG #HEM n 7ARTMANN - !LTMANN +( 4HE BIOLOGY AND MEDICINAL CHEMISTRY OF EPOTHILONES #URRR -ED #HEM !NTI #ANCER !GENTS n
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
!LTMANN +( %POTHILONE " AND ITS ANALOGS n A NEW FAMILY OF ANTICANCER AGENTS -INI 2EV -ED #HEM n "ORZILLERI 2- 6ITE '$ %POTHILONES NEW TUBULIN POLYMERIZATION AGENTS IN PRE CLINICAL AND CLINICAL DEVELOPMENT $RUGS OF THE &UTURE n !LTMANN +( 2ECENT DEVELOPMENTS IN THE CHEMISTRY AND BIOLOGY OF EPOTHILONES #URR 0HARM $ES n !LTMANN +( 0FEIFFER " !RSENIYADIS 3 0RATT "! .ICOLAOU +# 4HE CHEMISTRY AND BIOLOGY OF EPOTHILONES n 4HE WHEEL KEEPS TURNING #HEM-ED#HEM n #HAPPELL -$ 3TACHEL 3* ,EE #" $ANISHEFSKY 3* /N THE TOTAL SYNTHESIS AND PRE LIMINARY BIOLOGICAL EVALUATIONS OF 2 AND 3 !ZA D%PO" A MITSUNOBU INVERSION AT # IN PRE EPOTHILONE FRAGMENTS /RG ,ETT n 7ALSH #4 /#ONNOR 3 3CHNEIDER 4, 0OLYKETIDE NONRIBOSOMAL PEPTIDE EPOTHI LONE ANTITUMOR AGENTS THE %PO! " # SUBUNITS * )ND -ICROBIOL "IOTECHNOL n (E , /RR '! (ORWITZ 3" .OVEL MOLECULES THAT INTERACT WITH MICROTUBULES AND HAVE FUNCTIONAL ACTIVITY SIMILAR TO 4AXOL $RUG $ISCOVERY 4ODAY n !LTAHA 2 &OJO 4 2EED % !BRAHAM * %POTHILONES ! NOVEL CLASS OF NON TAXANE MICROTUBULE STABILIZING AGENTS #URR 0HARM $ES n !LTMANN +( "OLD ' #ARAVATTI ' %ND . &LÙRSHEIMER ! 'UAGNANO 6 /2EILLY 4 7ART MANN - %POTHILONES AND THEIR ANALOGS n POTENTIAL NEW WEAPONS IN THE FIGHT AGAINST CANCER #HIMIA n "UEY 2- $IAZ *& !NDREU *- /"RATE ! 'IANNAKAKOU 0 .ICOLAOU +# 3ASMAL 0+ 2ITZÎN ! .AMOTO + )NTERACTIONS OF EPOTHILONE ANALOGS WITH THE PACLITAXEL BINDING SITE RELATIONSHIP BETWEEN BINDING AFFINITY MICROTUBULE STABILIZATION AND CYTOTOXICITY #HEM "IOL n *ORDAN -! 7ENDELL + 'ARDINER 3 $ERRY 7" #OPP ( 7ILSON , -ITOTIC BLOCK INDUCED IN (E,A CELLS BY LOW CONCENTRATIONS OF PACLITAXEL 4AXOL RESULTS IN ABNORMAL MITOTIC EXIST AND APOPTOTIC CELL DEATH #ANCER 2ES n ,ICHTNER 2" 2OTGERI ! "UNTE 4 "UCHMANN " (OFFMANN * 3CHWEDE 7 3KUBALLA 7 +LAR 5 3UBCELLULAR DISTRIBUTION OF EPOTHILONES IN HUMAN TUMOR CELLS 0ROC .ATL !CAD 3CI 53! n *ORDAN -! -ECHANISM OF ACTION OF ANTITUMOR DRUGS THAT INTERACT WITH MICROTU BULES AND TUBULIN #URR -ED #HEM !NTI #ANCER !GENTS n +AMATH + *ORDAN -! 3UPPRESSION OF MICROTUBULE DYNAMICS BY EPOTHILONE " IS ASSOCIATED WITH MITOTIC ARREST #ANCER 2ES n #HEN *'# (ORWITZ 3- $IFFERENTIAL MITOTIC RESPONSES TO MICROTUBULE STABILIZING AND DESTABILIZING DRUGS #ANCER 2ES n #HEN *'# 9ANG # 0(9 #AMMER - (ORWITZ 3- 'ENE EXPRESSION AND MITOTIC EXIT INDUCED BY MICROTUBULE STABILIZING DRUGS #ANCER 2ES n 'ARRAWAY ,! #HABNER " -$2 INHIBITION LESS RESISTANCE OR LESS RELEVANCE %UR * #ANCER n .ETTLES *( ,I (, #ORNETT " +RAHN *- 3NYDER *0 $OWNING +( 4HE BINDING MODE OF EPOTHILONE ! ON α β TUBULIN BY ELECTRON CRYSTALLOGRAPHY 3CIENCE n .OGALES % 7OLF 3' $OWNING +( 3TRUCTURE OF THE αβ TUBULIN DIMER BY ELECTRON CRYSTALLOGRAPHY .ATURE n 'IANNAKAKOU 0 'USSIO 2 .OGALES % $OWNING +( :AHAREVITZ $ "OLLBUCK " 0OY ' 3ACKETT $ .ICOLAOU +# &OJO 4 ! COMMON PHARMACOPHORE FOR EPOTHILONE AND
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
TAXANES MOLECULAR BASIS FOR DRUG RESISTANCE CONFERRED BY TUBULIN MUTATIONS IN HUMAN CANCER CELLS 0ROC .ATL !CAD 3CI 53! n (E , 9ANG #0 (ORWITZ 3" -UTATIONS IN β TUBULIN MAP TO DOMAINS INVOLVED IN REGULATION OF MICROTUBULE STABILITY IN EPOTHILONE RESISTANT CELL LINES -OL #ANCER 4HER n 9ANG #0( 6ERDIER 0INARD 0 7ANG & ,IPPAINE (ORVATH % (E ,& ,I $3 (ÙFLE ' /JIMA ) /RR '! (ORWITZ 3" ! HIGHLY EPOTHILONE " RESISTANT ! CELL LINE WITH MUTATIONS IN TUBULIN THAT CONFER DRUG DEPENDENCE -OL #ANCER 4HER n 6ERRILLS .- &LEMMING #, ,IU - )VERY -4 #OBON '3 .ORRIS -$ (ABER - +AVAL LARIS - -ICROTUBULE ALTERATIONS AND MUTATIONS INDUCED BY DESOXYEPOTHILONE " )MPLICATIONS FOR DRUG TARGET INTERACTIONS #HEM "IOL n #ABRAL & "ARLOW 3" 2ESISTANCE TO ANTIMITOTIC AGENTS AS GENETIC PROBES OF MICRO TUBULE STRUCTURE AND FUNCTION 0HARMACOL 4HER n 3U $3 "ALOG ! -ENG $ "ERTINATO 0 $ANISHEFSKY 3* :HENG 9( #HOU 4# (E , (ORWITZ 3" 3TRUCTURE ACTIVITY RELATIONSHIPS OF THE EPOTHILONES AND THE FIRST IN VIVO COM PARISON WITH PACLITAXEL !NGEW #HEM )NT %D n #HOU 4# :HANG 8' "ALOG ! 3U $3 -ENG $ 3AVIN + "ERTINO *2 $ANISHEFSKY 3* $ESOXYEPOTHILONE " AN EFFICACIOUS MICROTUBULE TARGETED ANTITUMOR AGENT WITH A PROM ISING IN VIVO PROFILE RELATIVE TO EPOTHILONE " 0ROC .ATL !CAD 3CI 53! n /2EILLY 4 -C3HEEHY 0-* 7ENGER & (ATTENBERGER - -ULLER - 6AXELAIRE * !LTMANN +( 7ARTMANN - %0/ EPOTHILONE " IS ACTIVE IN VIVO AGAINST EXPERIMENTAL PROSTATE TUMORS 0ROSTATE n 2OTHERMEL * 7ARTMANN - #HEN 4 (OHNEKER 4 %0/ EPOTHILONE " A PROM ISING NOVEL MICROTUBULE STABILIZER 3EMINARS IN /NCOLOGY 3UPPL n 0IETRAS + 3TUMM - (UBERT - "UCHDUNGER % 2UBIN + (ELDIN # + -C3HEEHY 0 7ART MANN - /ESTMAN ! 34) ENHANCES THE THERAPEUTIC INDEX OF EPOTHILONE " BY A TUMOR SELECTIVE INCREASE OF DRUG UPTAKE #LIN #ANCER 2ES n +LAR 5 "UCHMANN " 3CHWEDE 7 3KUBALLA 7 (OFFMANN * ,ICHTNER 2" 4OTAL SYNTHESIS AND ANTITUMOR ACTIVITY OF :+ %0/ 4HE FIRST FULLY SYNTHETIC EPOTHILONE IN CLINI CAL DEVELOPMENT !NGEW #HEM )NT %D n 2EICHENBACH ( (ÙFLE ' 0RODUCTION OF BIOACTIVE SECONDARY METABOLITES )N - $WORKIN $ +AISER EDS -YXOBACTERIA )) !MERICAN 3OCIETY FOR -ICROBIOLOGY 7ASHING TON $# n 2EICHENBACH ( (ÙFLE ' -YXOBACTERIA AS PRODUCERS OF SECONDARY METABOLITES )N 3 'RABLEY 2 4HIERICKE EDS $RUG $ISCOVERY FROM .ATURE 3PRINGER 6ERLAG "ERLIN (EIDEL BERG n 'ERTH + 3TEINMETZ ( (ÙFLE ' 2EICHENBACH ( 3TUDIES ON THE BIOSYNTHESIS OF EPOTHILONES HYDROXYLATION OF %PO ! AND " TO EPOTHILONES % AND & * !NTIBIOTICS n &RYKMAN 3 4SURUTA ( 'ALAZZO * ,ICARI 0 #HARACTERIZATION OF PRODUCT CAPTURE RESIN DURING MICROBIAL CULTIVATIONS * )ND -ICROBIOL "IOTECHNOL n -OLNAR ) 3CHUPP 4 /NO - :IRKLE 2 -ILNAMOW - .OVOK 4HOMPSON " %NGEL . 4OUPET # 3TRATMANN ! ,IGON * ET AL 4HE BIOSYNTHETIC GENE CLUSTER FOR THE MICRO TUBULE STABILIZING AGENTS EPOTHILONES ! AND " FROM 3ORANGIUM CELLULOSUM 3O CE #HEM "IOL n 4ANG , 3HAH 3 #HUNG , +ATZ , +HOSLA # *ULIEN " #LONING AND HETEROLOGOUS EXPRESSION OF THE EPOTHILONE GENE CLUSTER 3CIENCE n
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
'ERTH + 3TEINMETZ ( (ÙFLE ' 2EICHENBACH ( 3TUDIES ON THE BIOSYNTHESIS OF EPOTHILONES THE BIOSYNTHETIC ORIGIN OF THE CARBON SKELETON * !NTIBIOTICS n 'ERTH + 3TEINMETZ ( (ÙFLE ' 2EICHENBACH ( 3TUDIES ON THE BIOSYNTHESIS OF EPOTHILONES THE 0+3 AND EPOTHILONE #$ MONOOXYGENASE * !NTIBIOTICS n &RYKMAN 3 4SURUTA ( ,AU * 2EGENTIN 2 /U 3 2EEVES # #ARNEY * 3ANTI $ ,ICARI 0 -ODULATION OF EPOTHILONE ANALOG PRODUCTION THROUGH MEDIA DESIGN * )ND -ICROBIOL"IO TECHNOL n (ÙFLE ' "EDORF . 'ERTH + 2EICHENBACH ( %POTHILONES PROCESS FOR PREPARING THE SAME AND THEIR USE AS MEDICAMENTS AND AS PLANT PROTECTING AGENTS )NT 0ATENT !PPLICATION 7/ "ENIGNI $ 3TANKAVAGE 2 #HIANG 3 (OU ( %AGAN " 'U $ (OU $ -INTZMYER , 4ULLY 4 $AVIS " ET AL -ETHODS FOR THE PREPARATION ISOLATION AND PURIFICATION OF EPOTHILONE " AND X RAY CRYSTAL STRUCTURES OF EPOTHILONE " )NT 0ATENT !PPLICATION 7/ (OFMANN ( -AHNKE - -EMMERT + 0ETERSEN & 3CHUPP 4 +ßSTERS % -UTZ - &ERMENTATIVE PREPARATION PROCESS FOR AND CRYSTAL FORMS OF CYTOSTATICS )NT 0ATENT !PPLICA TION 7/ +ßSTERS % 5NTERNÉHRER ( 0ROCESS FOR THE ISOLATION AND PURIFICATION OF EPOTHILONES )NT 0ATENT !PPLICATION 7/ !RSLANIAN 2 0ARKER # 7ANG 0 -C)NTIRE * ,AU * 3TARKS # ,ICARI 0 ,ARGE SCALE ISOLATION AND CRYSTALLIZATION OF EPOTHILONE $ FROM -YXOCOCCUS XANTHUS CULTURES * .AT 0ROD n -UTKA 3 #ARNEY * ,IU 9 +ENNEDY * (ETEROLOGOUS PRODUCTION OF EPOTHILONE # AND $ IN %SCHERICHIA COLI "IOCHEMISTRY n &RYKMAN 3 4SURUTA ( ,ICARI 0 !SSESSMENT OF FED BATCH SEMICONTINUOUS AND CONTINUOUS EPOTHILONE $ PRODUCTION PROCESSES "IOTECHNOL 0ROG n +LAR 5 3KUBALLA 7 "UCHMANN " 3CHWEDE 7 "UNTE 4 (OFFMANN * ,ICHTNER 2 3YNTHESIS AND BIOLOGICAL ACTIVITY OF EPOTHILONES )N ) /JIMA '$ 6ITE + ( !LTMANN EDS !NTICANCER AGENTS n FRONTIERS IN CANCER CHEMOTHERAPY !#3 3YMPOSIUM 3ERIES !MERICAN #HEMICAL 3OCIETY 7ASHINGTON $# n 3EFKOW - +IFFE - 3CHUMMER $ (ÙFLE ' /XIDATIVE AND REDUCTIVE TRANSFORMA TIONS OF EPOTHILONE ! "IOORG -ED #HEM ,ETT n 3EFKOW - +IFFE - (ÙFLE ' $ERIVATIZATION OF THE #n# FUNCTIONAL GROUPS OF EPOTHILONES ! " AND # "IOORG -ED #HEM ,ETT n 6ITE '$ "ORZILLERI 2- +IM 3( 2EGUEIRO 2IN ! (UMPHREYS 7' ,EE &9& %POTHI LONES ! AND " 3PRINGBOARDS FOR SEMISYNTHESIS OF PROMISING ANTIMITOTIC AGENTS )N ) /JIMA '$ 6ITE + ( !LTMANN EDS !NTICANCER AGENTS n FRONTIERS IN CANCER CHEMOTHERAPY !#3 3YMPOSIUM 3ERIES !MERICAN #HEMICAL 3OCIETY 7ASHINGTON $# n "ORZILLERI 2- :HENG 8 3CHMIDT 2* *OHNSON *! +IM 3( $I-ARCO *$ &AIRCHILD #2 'OUGOUTAS *: ,EE &9& ,ONG "( ET AL ! NOVEL APPLICATION OF A 0D CATALYZED NUCLEOPHILIC SUBSTITUTION REACTION TO THE REGIO AND STEREOSELECTIVE SYNTHESIS OF LACTAM ANALOGUES OF THE EPOTHILONE NATURAL PRODUCTS * !M #HEM 3OC n ,EE &9 "ORZILLERI 2 &AIRCHILD #2 +IM 3( ,ONG "( 2EVENTOS 3UAREZ # 6ITE '$ 2OSE 7# +RAMER 2! "-3 ! NOVEL EPOTHILONE ANALOG WITH A MODE OF ACTION SIMILAR TO PACLITAXEL BUT POSSESSING SUPERIOR ANTITUMOR EFFICACY #LIN #ANCER 2ES n #HOU 4# /#ONNOR /! 4ONG 70 'UAN 9 :HANG :' 3TACHEL 3* ,EE # $ANISHEFSKY 3*
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
4HE SYNTHESIS DISCOVERY AND DEVELOPMENT OF A HIGHLY PROMISING CLASS OF MICRO TUBULE STABILIZATION AGENTS CURATIVE EFFECTS OF DESOXYEPOTHILONES " AND & AGAINST HUMAN TUMOR XENOGRAFTS IN NUDE MICE 0ROC .ATL !CAD 3CI 53! n 3CHINZER $ !LTMANN + ( 3TUHLMANN & "AUER ! 7ARTMANN - 3YNTHESIS AND BIOLOGICAL EVALUATION OF AZA EPOTHILONES #HEM"IO#HEM n 3TACHEL 3* ,EE #" 3PASSOVA - #HAPPELL -$ "ORNMANN 7' $ANISHEFSKY 3* #HOU 4 # 'UAN 9 /N THE )NTERACTIVITY OF COMPLEX SYNTHESIS AND TUMOR PHARMACOLOGY IN THE DRUG DISCOVERY PROCESS TOTAL SYNTHESIS AND COMPARATIVE IN VIVO EVALUATIONS OF THE !ZA EPOTHILONES * /RG #HEM n 9AMAGUCHI ( 0ARANAWITHANA 32 ,EE -7 (UANG : "HALLA +. 7ANG (' %POTHILONE " ANALOGUE "-3 MEDIATED CYTOTOXICITY THROUGH INDUCTION OF "AX CONFORMATIONAL CHANGE IN HUMAN BREAST CANCER CELLS #ANCER 2ES n 2EGUEIRO 2EN ! ,EAVITT + +IM 3( (ÙFLE ' +IFFE - 'OUGOUTAS *: $I-ARCO *$ ,EE &9& &AIRCHILD #2 ,ONG "( ET AL 3!2 AND P( STABILITY OF CYANO SUBSTITUTED EPOTHI LONES /RG ,ETT n #ACHOUX & )SARNO 4 7ARTMANN - !LTMANN +( 3CAFFOLDS FOR MICROTUBULE INHIBI TION THROUGH EXTENSIVE MODIFICATION OF THE EPOTHILONE TEMPLATE !NGEW #HEM )NT %D n .ICOLAOU +# 3ARABIA & .INKOVIC 3 &INLAY -2 "ODDY #.# 0ROBING THE RING SIZE OF EPOTHILONES TOTAL SYNTHESIS OF ;= ;= ;= AND ;= EPOTHILONES ! !NGEW #HEM )NT %D n &OR OTHER EPOTHILONE PHARMACOPHORE MODELS CF ; n= %ND . &URET 0 VAN #AMPENHOUT . 7ARTMANN - !LTMANN +( 4OTAL SYNTHESIS AND BIOLOGICAL EVALUATION OF A # # PHENYLENE BRIDGED ANALOG OF EPOTHILONE $ #HEM "IODIVERSITY n !RSLANIAN 2, 4ANG , "LOUGH 3 -A 7 1IU 2 ' +ATZ , #ARNEY *2 ! NEW CYTOTOXIC EPOTHILONE FROM MODIFIED POLYKETIDE SYNTHASES HETEROLOGOUSLY EXPRESSED IN -YXOCOCCUS XANTHUS * .AT 0ROD n "ISWAS + ,IN ( .JARDSON *4 #HAPPELL -$ #HOU 4# 'UAN 9 4ONG 70 (E , (ORWITZ 3" $ANISHEFSKY 3* (IGHLY CONCISE ROUTES TO EPOTHILONES THE TOTAL SYNTHESIS AND EVALUATION OF EPOTHILONE * !M #HEM 3OC n 2IVKIN ! .JARDSON *4 "ISWAS + #HOU 4# $ANISHEFSKY 3* 4OTAL SYNTHESES OF ;= AND ;=DEHYDRODESOXYEPOTHILONES " VIA A CONCISE RING CLOSING METATHESIS BASED STRATEGY CORRELATION OF RING SIZE WITH BIOLOGICAL ACTIVITY IN THE EPOTHILONE SERIES * /RG #HEM n 2IVKIN ! "ISWAS + #HOU 4# $ANISHEFSKY 3* /N THE INTRODUCTION OF A TRIFLUOR OMETHYL SUBSTITUENT IN THE EPOTHILONE SETTING CHEMICAL ISSUES RELATED TO RING FORMING OLEFIN METATHESIS AND EARLIEST BIOLOGICAL FINDINGS /RG ,ETT n 2IVKIN ! 9OSHIMURA & 'ABARDA !% #HOU 4# $ONG ( 4ONG 70 $ANISHEFSKY 3* #OMPLEX TARGET ORIENTED TOTAL SYNTHESIS IN THE DRUG DISCOVERY PROCESS THE DISCOVERY OF A HIGHLY PROMISING FAMILY OF SECOND GENERATION EPOTHILONES * !M #HEM 3OC n 9OSHIMURA & 2IVKIN ! 'ABARDA !% #HOU 4# $ONG ( 3UKENICK -OREL && 4AYLOR 2% $ANISHEFSKY 3* 3YNTHESIS AND CONFORMATIONAL ANALYSIS OF % DEHYDROEPOTHI LONE " ! SUGGESTIVE LINK BETWEEN THE CHEMISTRY AND BIOLOGY OF EPOTHILONES !NGEW #HEM )NT %D n #HOU 4# $ONG ( 2IVKIN ! 9OSHIMURA & 'ABARDA !% #HO 93 4ONG 70 $ANISHEFSKY
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
3* $ESIGN AND TOTAL SYNTHESIS OF A SUPERIOR FAMILY OF EPOTHILONE ANALOGUES WHICH ELIMINATE XENOGRAFT TUMORS TO A NONRELAPSABLE STATE !NGEW #HEM )NT %D n 2IVKIN ! #HOU 4# $ANISHEFSKY 3* /N THE REMARKABLE ANTITUMOR PROPERTIES OF FLUDELONE (OW WE GOT THERE !NGEW #HEM )NT %D n 2IVKIN ! 9OSHIMURA & 'ABARDA !% #HO 93 #HOU 4# $ONG (* $ANISHEFSKY 3* $ISCOVERY OF % DEHYDROEPOTHILONES THROUGH CHEMICAL SYNTHESIS /N THE EMER GENCE OF TRIFLUORO % DEHYDRO DESOXYEPOTHILONE " AS A PROMISING ANTI CANCER DRUG CANDIDATE * !M #HEM 3OC n 2IVKIN ! #HO 93 'ABARDA !% 9OSHIMURA & $ANISHEFSKY 3* !PPLICATION OF RING CLOSING METATHESIS REACTIONS IN THE SYNTHESIS OF EPOTHILONES * .AT 0ROD n 4ANG , ,I 2' 9ONG , +ATZ , 'ENERATION OF NOVEL EPOTHILONE ANALOGS WITH CYTO TOXIC ACTIVITY BY BIOTRANSFORMATION * !NTIBIOTICS n 3TARKS #- :HOU 9 ,IU & ,ICARI 0 )SOLATION AND CHARACTERIZATION OF NEW EPOTHI LONE ANALOGUES FROM RECOMBINANT -YXOCOCCUS XANTHUS FERMENTATIONS * .AT 0ROD n 7HITE *$ #ARTER 2' 3UNDERMANN +& 7ARTMANN - 4OTAL SYNTHESIS OF EPOTHI LONE " EPOTHILONE $ AND CIS AND TRANS DEHYDROEPOTHILONE $ * !M #HEM 3OC n 7HITE *$ #ARTER 2' 3UNDERMANN +& 7ARTMANN - 4OTAL SYNTHESIS OF EPOTHILONE " EPOTHILONE $ AND CIS AND TRANS DEHYDROEPOTHILONE $ ;%RRATUM FOR * !M #HEM 3OC n= * !M #HEM 3OC #ARLOMAGNO 4 "LOMMERS -** -EILER * *AHNKE 7 3CHUPP 4 0ETERSEN & 3CHINZER $ !LTMANN +( 'RIESINGER # 4HE HIGH RESOLUTION SOLUTION STRUCTURE OF EPOTHILONE A BOUND TO TUBULIN !N UNDERSTANDING OF THE STRUCTURE ACTIVITY RELATIONSHIPS FOR A POWER FUL CLASS OF ANTITUMOR AGENTS !NGEW #HEM )NT %D n #HOU 4# $ONG (* :HANG 8' 4ONG 70 $ANISHEFSKY 3* 4HERAPEUTIC CURE AGAINST HUMAN TUMOR XENOGRAFTS IN NUDE MICE BY A MICROTUBULE STABILIZATION AGENT FLUDELONE VIA PARENTERAL OR ORAL ROUTE #ANCER 2ES n 7U +$ #HO 93 +ATZ * 0ONOMAREV 6 #HEN +IANG 3 $ANISHEFSKY 3* -OORE -!3 )NVESTIGATION OF ANTITUMOR EFFECTS OF SYNTHETIC EPOTHILONE ANALOGS IN HUMAN MYELOMA MODELS IN VITRO AND IN VIVO 0ROC .ATL !CAD 3CI 53! n 1INTARD $ "ERTRAND 0 6IELLE 3 2AIMBAUD % 2ENARD 0 0FEIFFER " 'ESSON * 0 %NANTIOSELECTIVE SYNTHESIS OF DEHYDRO DESOXY OXA EPOTHILONE $ 3YNLETT n 3CHINZER $ "ÙHM /- !LTMANN + ( 7ARTMANN - 3YNTHESIS AND BIOLOGICAL EVALUATION OF FURANO EPOTHILONE # 3YNLETT n -ENG $ 3U $3 "ALOG ! "ERTINATO 0 3ORENSEN %* $ANISHEFSKY 3* :HENG 9( #HOU 4# (E , (ORWITZ 3" !N APPLICATION TO THE SYNTHESIS OF FULLY ACTIVE EPOTHILONE CON GENERS * !M #HEM 3OC n 3U $3 -ENG $ "ERTINATO 0 "ALOG ! 3ORENSEN %* $ANISHEFSKY 3* :HENG 9( #HOU 4# (E , (ORWITZ 3" 4OTAL SYNTHESIS OF n EPOTHILONE " AN EXTENSION OF THE 3UZUKI COUPLING METHOD AND INSIGHTS INTO STRUCTUREnACTIVITY RELATIONSHIPS OF THE EPOTHILONES !NGEW #HEM )NT %D n .ICOLAOU +# 7INSSINGER . 0ASTOR * .INKOVIC 3 3ARABIA & (E 9 6OURLOUMIS $ 9ANG : ,I 4 'IANNAKAKOU 0 ET AL 3YNTHESIS OF EPOTHILONES ! AND " IN SOLID AND SOLUTION PHASE .ATURE n .ICOLAOU +# 6OURLOUMIS $ ,I 4 0ASTOR * 7INSSINGER . (E 9 .INKOVIC 3 3ARABIA &
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
6ALLBERG ( 2OSCHANGAR & ET AL $ESIGNED EPOTHILONES COMBINATORIAL SYNTHESIS TUBULIN ASSEMBLY PROPERTIES AND CYTOTOXIC ACTION AGAINST TAXOL RESISTANT TUMOR CELLS !NGEW #HEM )NT %D n #HOU 4# :HANG 8' (ARRIS #2 +UDUK 3$ "ALOG ! 3AVIN +! "ERTINO *2 $ANISHEFSKY 3* $ESOXYEPOTHILONE " IS CURATIVE AGAINST HUMAN TUMOR XENOGRAFTS THAT ARE REFRAC TORY TO PACLITAXEL 0ROC .ATL !CAD 3CI 53! n *OHNSON * +IM 3( "IFANO - $I-ARCO * &AIRCHILD # 'OUGOUTAS * ,EE & ,ONG " 4OKAR SKI * 6ITE '$ 3YNTHESIS STRUCTURE PROOF AND BIOLOGICAL ACTIVITY OF EPOTHILONE CYCLOPROPANES /RG ,ETT n .ICOLAOU +# .AMOTO + ,I * 2ITZEN ! 5LVEN 4 3HOJI - :AHAREVITZ $ 'USSIO 2 3ACKETT $, 7ARD 2$ ET AL 3YNTHESIS AND BIOLOGICAL EVALUATION OF CYCLOPROPYL AND CYCLOBUTYL EPOTHILONES #HEM"IO#HEM n .ICOLAOU +# .AMOTO + 2ITZEN ! 5LVEN 4 3HOJI - ,I * $!MICO ' ,IOTTA $ &RENCH #4 7ARTMANN - ET AL #HEMICAL SYNTHESIS AND BIOLOGICAL EVALUATION OF CIS AND TRANS CYCLOPROPYL AND CYCLOBUTYL EPOTHILONES AND RELATED PYRIDINE SIDE CHAIN ANALOGUES * !M #HEM 3OC n 2EGUEIRO 2EN ! "ORZILLERI 2- :HENG 8 +IM 3( *OHNSON *! &AIRCHILD #2 ,EE &9 ,ONG "( 6ITE '$ 3YNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL EPOTHILONE AZIRIDINES /RG ,ETT n !LTMANN +( "OLD ' #ARAVATTI ' $ENNI $ &LÙRSHEIMER ! 3CHMIDT ! 2IHS ' 7ART MANN - 4HE TOTAL SYNTHESIS AND BIOLOGICAL ASSESSMENT OF TRANS EPOTHILONE ! (ELV #HIM !CTA n .ICOLAOU +# 2ITZEN ! .AMOTO + 2UBEN -" $IAZ & !NDREU *- 7ARTMANN - !LTMANN +( /"RATE ! 'IANNAKAKOU 0 #HEMICAL SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL EPOTHILONE " AND TRANS CYCLOPROPYL EPOTHILONE " ANALOGUES 4ETRAHEDRON n .ICOLAOU +# .INKOVIC 3 &INLAY -2 3ARABIA & ,I 4 4OTAL SYNTHESIS OF HYDROX YEPOTHILONE " AND RELATED ANALOGS *#3 #HEM #OMMUN n #HAPPELL -$ (ARRIS #2 +UDUK 3$ "ALOG ! 7U : :HANG & ,EE #" 3TACHEL 3* $AN ISHEFSKY 3* #HOU 4# ET AL 0ROBING THE 3!2 OF D%PO" VIA CHEMICAL SYNTHESIS A TOTAL SYNTHESIS EVALUATION OF # DIOXOLANYL DESOXYEPOTHILONE " * /RG #HEM n .EWMAN 2! 9ANG * &INLAY -26 #ABRAL & 6OURLOUMIS $ 3TEVENS ,# 4RONCOSO ,0 7U 8 ,OGOTHETIS #* .ICOLAOU +# ET AL !NTITUMOR EFFICACY OF FLUOROEPOTHILONE " AGAINST HUMAN PROSTATE CANCER XENOGRAFTS #ANCER #HEMOTHER 0HARMACOL n !LTMANN +( .ICOLAOU +# 7ARTMANN - /2EILLY 4 0ROC !M !SSOC #ANCER 2ES !BSTRACT (ÙFLE ' 'LASER . +IFFE - (ECHT ( * 3ASSE & 2EICHENBACH ( . OXIDATION OF EPOTHILONE ! # AND / ACYL REARRANGEMENT TO # AND # SUBSTITUTED EPOTHILONES !NGEW #HEM )NT %D n 3EFKOW - (ÙFLE ' 3UBSTITUTIONS AT THE THIAZOLE MOIETY OF EPOTHILONE (ETEROCYCLES n .ICOLAOU +# +ING .0 &INLAY -26 (E 9 2OSCHANGAR & 6OURLOUMIS $ 6ALLBERG ( 3ARABIA & .INKOVICH 3 (EPWORTH $ 4OTAL SYNTHESIS OF EPOTHILONE % AND RELATED SIDE CHAIN MODIFIED ANALOGUES VIA A 3TILLE COUPLING BASED STRATEGY "IOORG -ED #HEM n .ICOLAOU +# 3CARPELLI 2 "OLLBUCK " 7ERSCHKUN " 0EREIRA -- 7ARTMANN - !LTMANN
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
+( :AHAREVITZ $ 'USSIO 2 'IANNAKAKOU 0 #HEMICAL SYNTHESIS AND BIOLOGICAL PROPERTIES OF PYRIDINE EPOTHILONES #HEM "IOL n .ICOLAOU +# (EPWORTH $ +ING .0 &INLAY -2 3CARPELLI 2 0EREIRA -- "OLLBUCK " "IGOT ! 7ERSCHKUN " 4OTAL SYNTHESIS OF DESMETHYLEPOTHILONE " EPOTHILONE " EPOTHILONE & AND RELATED SIDE CHAIN MODIFIED EPOTHILONE " ANALOGUES #HEM %UR * n .ICOLAOU +# (EPWORTH $ &INLAY -26 0AUL +. 7ERSCHKUN " "IGOT ! 3YNTHESIS OF DESMETHYLEPOTHILONE " IMPROVED METHODOLOGY FOR THE RAPID HIGHLY SELECTIVE AND CONVERGENT CONSTRUCTION OF EPOTHILONE " AND ANALOGUES *#3 #HEM #OMM n +OLMAN ! "-3 "RISTOL -YERS 3QUIBB'"& #URR /PIN )NVESTIGATIONAL $RUGS n +AMATH !6 #HANG - ,EE & :HANG 90 -ARATHE 0( 0RECLINICAL PHARMACOKINETICS AND ORAL BIOAVAILABILITY OF "-3 A NOVEL EPOTHILONE " ANALOG #ANCER #HEMOTHER 0HARM n ,EE #" #HOU 4# :HANG 8' 7ANG :' +UDUK 3$ #HAPPELL -$ 3TACHEL 3* $ANISHEF SKY 3* 4OTAL SYNTHESIS AND ANTITUMOR ACTIVITY OF DESOXYEPOTHILONE & !N UNEXPECTED SOLVOLYSIS PROBLEM AT # MEDIATED BY REMOTE SUBSTITUTION AT # * /RG #HEM n (ÙFLE ' 'LASER . ,EIBOLD 4 3YNTHESIS AND CYTOTOXICITY OF # MODIFIED EPOTHI LONES 'ER /FFEN $% 5YAR $ 4AKIGAWA . -EKHAIL 4 'RABOWSKI $ -ARKMAN - ,EE & #ANETTA 2 0ECK 2 "UKOWSKI 2 'ANAPATHI 2 !POPTOTIC PATHWAYS OF EPOTHILONE "-3 'YNE COLOGIC /NCOLOGY n !LTMANN +( "LOMMERS -** #ARAVATTI ' &LÙRSHEIMER ! .ICOLAOU +# /2EILLY 4 3CHMIDT ! 3CHINZER $ 7ARTMANN - 3YNTHETIC AND SEMISYNTHETIC ANALOGS OF EPOTHILONES CHEMISTRY AND BIOLOGICAL ACTIVITY )N ) /JIMA '$ 6ITE +( !LTMANN EDS !NTICANCER AGENTS n FRONTIERS IN CANCER CHEMOTHERAPY !#3 3YMPOSIUM 3ERIES !MERICAN #HEMICAL 3OCIETY 7ASHINGTON $# n 7ARTMANN - ,ORETAN * 2EUTER 2 (ATTENBERGER - -ULLER - 6AXELAIRE * -AIRA 3 - &LÙRSHEIMER ! /2EILLY 4 .ICOLAOU +# ET AL 0RECLINICAL PHARMACOLOGICAL PROFILE OF !"* A NOVEL EPOTHILONE " ANALOG WITH POTENT AND PROTRACTED ANTI TUMOR ACTIVITY 0ROC !M !SSOC #ANCER 2ES !BSTRACT .ICOLAOU +# 3ASMAL 0+ 2ASSIAS ' 2EDDY -6 !LTMANN + ( 7ARTMANN - /"RATE ! 'IANNAKAKOU 0 $ESIGN SYNTHESIS AND BIOLOGICAL PROPERTIES OF HIGHLY POTENT EPOTHILONE " ANALOGUES !NGEW #HEM )NT %D n .ICOLAOU +# 0RATT "! !RSENIYADIS 3 7ARTMANN - /"RATE ! -OLECULAR DESIGN AND CHEMICAL SYNTHESIS OF A HIGHLY POTENT EPOTHILONE #HEM-ED#HEM n .ICOLAOU +# &INLAY -26 .INKOVIC 3 +ING .0 (E 9 ,I 4( 3ARABIA & 6OURLOUMIS $ 3YNTHESIS AND BIOLOGICAL PROPERTIES OF # CYCLOPROPYL EPOTHILONE ! AND RELATED EPOTHILONES #HEM "IOL n !LTMANN +( "OLD ' #ARAVATTI ' &LÙRSHEIMER ! 'UAGNANO 6 7ARTMANN - 3YNTHESIS AND BIOLOGICAL EVALUATION OF HIGHLY POTENT ANALOGUES OF EPOTHILONES " AND $ "IOORG -ED #HEM ,ETT n #ACHOUX & )SARNO 4 7ARTMANN - !LTMANN +( 4OTAL SYNTHESIS AND BIOLOGICAL ASSESSMENT OF BENZIMIDAZOLE BASED ANALOGUES OF EPOTHILONE ! !MBIVALENT EFFECTS ON CANCER CELL GROWTH INHIBITION #HEM"IO#HEM n $ONG 3$ 3UNDERMANN + 3MITH +-* 0ETRYKA * ,IU &( -YLES $# 2APID ACCESS
%POTHILONES AS LEAD STRUCTURES FOR NEW ANTICANCER DRUGS
TO EPOTHILONE ANALOGS VIA SEMISYNTHETIC DEGRADATION AND RECONSTRUCTION OF EPOTHILONE $ 4ETRAHEDRON ,ETT n #ACHOUX & )SARNO 4 7ARTMANN - !LTMANN +( 4OTAL SYNTHESIS AND BIOLOGICAL ASSESSMENT OF CYCLOPROPANE BASED EPOTHILONE ANALOGUES n MODULATION OF DRUG EFFLUX THROUGH POLARITY ADJUSTMENTS 3YNLETT n "OLD ' 7OJEIK 3 #ARAVATTI ' ,INDAUER 2 3TIERLIN # 'ERTSCH * 7ARTMANN - !LTMANN +( 3TRUCTUREnACTIVITY RELATIONSHIPS IN SIDE CHAIN MODIFIED EPOTHILONE ANALOGUES (OW IMPORTANT IS THE POSITION OF THE NITROGEN ATOM #HEM-ED#HEM n 'LUNZ 07 (E , (ORWITZ 3" #HAKRAVARTY 3 /JIMA ) #HOU 4# $ANISHEFSKY 3* 4HE SYNTHESIS AND EVALUATION OF BENZODESOXYEPOTHILONE " A HIGHLY CONVERGENT ROUTE 4ETRAHEDRON ,ETT n 3INHA 3# 3UN * 7ARTMANN - ,ERNER 2! 3YNTHESIS OF EPOTHILONE ANALOGUES BY ANTIBODY CATALYZED RESOLUTION OF THIAZOLE ALDOL SYNTHONS ON A MULTIGRAM SCALE "IOLOGI CAL CONSEQUENCES OF # ALKYLATION OF EPOTHILONES #HEM"IO#HEM n !LTMANN +( &LÙRSHEIMER ! "OLD ' #ARAVATTI ' 7ARTMANN - .ATURAL PROD UCT BASED DRUG DISCOVERY n %POTHILONES AS LEAD STRUCTURES FOR THE DEVELOPMENT OF NEW ANTICANCER AGENTS #HIMIA n &EYEN & 'ERTSCH * 7ARTMANN - !LTMANN +( $ESIGN AND SYNTHESIS OF AZA EPOTHILONES AZATHILONES n .ON NATURAL NATURAL PRODUCTS WITH POTENT ANTICANCER ACTIV ITY !NGEW #HEM )NT %D n #ACHOUX & 3CHAAL & 4EICHERT ! 7AGNER 4 !LTMANN +( 3YNTHESIS OF AZA %POTHI LONE $ !NALOGS 3YNLETT n /JIMA ) #HAKRAVARTY 3 )NOUE 4 ,IN 3 (E , (ORWITZ 3" +UDUK 3$ $ANISHEFSKY 3* ! COMMON PHARMACOPHORE FOR CYTOTOXIC NATURAL PRODUCTS THAT STABILIZE MICRO TUBULES 0ROC .ATL !CAD 3CI 53! n 7ANG - 8IA 8 +IM 9 (WANG $ *ANSEN *- "OTTA - ,IOTTA $# 3NYDER *0 ! UNIFIED AND QUANTITATIVE RECEPTOR MODEL FOR THE MICROTUBULE BINDING OF PACLITAXEL AND EPOTHILONE /RG ,ETT n -ANETTI & &ORLI 3 -ACCARI , #ORELLI & "OTTA - $ 13!2 STUDIES OF THE INTERACTION BETWEEN β TUBULIN AND MICROTUBULE STABILIZING ANTIMITOTIC AGENTS -3!! ! COMBINED PHARMACOPHORE GENERATION AND PSEUDORECEPTOR MODELING APPROACH APPLIED TO TAXANES AND EPOTHILONES )L &ARMACO n -ANETTI & -ACCARI , #ORELLI & "OTTA - $ 13!2 MODELS OF INTERACTIONS BETWEEN β TUBULIN AND MICROTUBULE STABILIZING ANTIMITOTIC AGENTS -3!! ! SURVEY ON TAXANES AND EPOTHILONES #URR 4OPICS -ED #HEM n 2UBIN %( 2OTHERMEL * 4ESFAYE & #HEN 4, (UBERT - (O 99 (SU #( /ZA !- 0HASE ) DOSE FINDING STUDY OF WEEKLY SINGLE AGENT PATUPILONE IN PATIENTS WITH ADVANCED SOLID TUMORS * #LIN /NCOL n 'ORE - +AYE 3 /ZA ! +EYZOR # 0YLE , 0ERENO 2 3KLENAR ) :AKNOEN 3 *OHRI ! * #LIN /NCOL !3#/ !NNUAL -EETING 0ROCEEDINGS 6OL .O 3 0ART ) OF )) *UNE 3UPPLEMENT !BSTRACT HTTPWWWNOVARTISONCOLOGYCOMPAGEPATUPILONEJSP ,IN . "RAKORA + 3EIDEN - "-3 "RISTOL -YERS 3QUIBB'"& #URR /PIN )NVESTIGATIONAL $RUGS n 0IVOT 8 $UFRESNE ! 6ILLANUEVA # %FFICACY AND SAFETY OF IXABEPILONE A NOVEL EPOTHILONE ANALOGUE #LINICAL "REAST #ANCER n
+ARL (EINZ !LTMANN AND +LAUS -EMMERT
,ARKIN *-' +AYE 3" %POTHILONES IN THE TREATMENT OF CANCER %XP /PIN )NVEST $RUGS n -ANI 3 -C$AID ( (AMILTON ! (OCHSTER ( #OHEN -" +HABELLE $ 'RIFFIN 4 ,EB WOHL %$ ,IEBES , -UGGIA & ET AL 0HASE ) CLINICAL AND PHARMACOKINETIC STUDY OF "-3 A NOVEL DERIVATIVE OF EPOTHILONE " IN SOLID TUMORS #LIN #ANCER 2ES n -C$AID (- -ANI 3 3HEN (* -UGGIA & 3ONNICHSEN $ (ORWITZ 3" 6ALIDATION OF THE PHARMACODYNAMICS OF "-3 AN ANALOGUE OF EPOTHILONE " DURING A 0HASE ) CLINICAL STUDY #LIN #ANCER 2ES n $ENDULURI . ,OW *! ,EE ** "ERMAN !7 7ALSHE *- 6ATAS 5 #HOW #+ 3TEINBERG 3- 9ANG 38 3WAIN 3- 0HASE )) TRIAL OF )XABEPILONE AN %POTHILONE " ANALOG IN PATIENTS WITH METASTATIC BREAST CANCER PREVIOUSLY UNTREATED WITH TAXANES * #LIN /NCOL HTTPWWWCANCERGOVCANCERTOPICSDRUGINFOFDA IXABEPILONE +OLMAN ! !CTIVITY OF EPOTHILONES #URR /PIN )NVESTIGATIONAL $RUGS n 3PRIGGS $ $UPONT * 0EZZULLI 3 ,ARKIN * #ROPP * *OHNSON 2 (ANNAH !, !!#2 .#) %/24# )NTERNATIONAL #ONFERENCE -OLECULAR 4ARGETS AND #ANCER 4HERAPEUTICS n .OVEMBER "OSTON -! 53! !BSTRACT ! 0UBLISHED AS A 3UPPLEMENT TO #LINICAL #ANCER 2ESEARCH 6OLUME )SSUE $ECEMBER (OLEN + (ANNAH ! :HOU 9 #ROPP ' *OHNSON 2 6OLKMAN * "INGER + !LBERTI $ 7ILD ING ' !!#2 .#) %/24# )NTERNATIONAL #ONFERENCE -OLECULAR 4ARGETS AND #AN CER 4HERAPEUTICS n .OVEMBER "OSTON -! 53! !BSTRACT ! 0UBLISHED AS A 3UPPLEMENT TO #LINICAL #ANCER 2ESEARCH 6OLUME )SSUE $ECEMBER HTTPWWWBIOSPACECOMNEWS?STORYASPX 3TORY)$
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(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
"Y 9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA &ERMENTATION 2ESEARCH ,ABS 0HARMACOLOGY 2ESEARCH ,ABS
$RUG $ISCOVERY 2ESEARCH !STELLAS 0HARMA )NC 4OKODAI CHOME 4SUKUBA SHI )BARAKI AND -IYUKIGAOKA 4SUKUBA SHI )BARAKI *APAN E MAIL YUHTAMASUOKA JPASTELLASCOM
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
!BSTRACT (ISTONE DEACETYLASE ($!# INHIBITORS SUCH AS TRICHOSTATIN ! AND TRAPOXIN WHICH WERE FIRST FOUND IN MICROORGANISMS POTENTLY AND SELECTIVELY INHIBIT ($!# ENZYMES 4HEY HAVE MADE A STRONG CONTRIBUTION TO RESEARCH ON ($!#S CHROMATIN CONTROL ABNORMAL EPIGENETIC CONTROL IN VARIOUS DISEASES AND THE SIGNIFICANCE OF ACETYLATION IN POSTTRANSLATIONAL MODIFICA TION 2ECENTLY ($!# INHIBITORS HAVE BEEN FOCUSED ON AS POTENTIAL DRUGS FOR THE TREATMENT OF SEVERAL DISEASES INCLUDING CANCER ALTHOUGH TRICHOSTATIN ! AND TRAPOXIN SHOW NO EFFECTS IN ANIMAL MODELS BECAUSE OF THEIR METABOLIC INSTABILITY IN VIVO #HEMICAL MODIFICATION HAS BEEN CONDUCTED IN ORDER TO OVERCOME THIS DRAWBACK 7E DISCOVERED THE MICROBIAL METABOLITES &+ ALSO KNOWN AS &2 ROMIDEPSIN DEPSIPEPTIDE .3# AND .3# $ AND 9- SPIRUCHOSTATIN ! "OTH COM POUNDS HAVE BICYCLIC STRUCTURES AND REPRESENT A NOVEL STRUCTURAL CLASS OF ($!# INHIBITOR 4HE ENZYME AND TUMOR CELL GROWTH INHIBITORY ACTIVITIES OF &+ WERE FOUND TO BE VERY POTENT )T ALSO SHOWED POTENT ($!# INHIBITORY ACTIVITY IN VIVO &+ IS THE FIRST POTENT ($!# INHIBITOR TO UNDERGO CLINICAL DEVELOPMENT AS A POTENTIAL TREATMENT FOR SOLID AND HEMATOLOGICAL CANCERS $UE TO ITS DRAMATIC EFFECT IN PATIENTS WITH REFRACTORY CUTANEOUS 4 CELL LYMPHOMA #4#, IN /CTOBER THE 53 &OOD $RUG !DMINISTRATION &$! GRANTED FAST TRACK STATUS TO &+ AS MONOTHERAPY FOR THE TREATMENT OF #4#, IN PATIENTS WHO HAVE RELAPSED FOLLOWING OR BECOME REFRACTORY TO ANOTHER SYSTEMIC THERAPY 4HUS ($!# INHIBITORS SUCH AS &+ AND 9- HAVE POTENTIAL AS TOOLS FOR LIFE SCIENCE STUDIES AND ALSO AS THERAPEUTIC AGENTS FOR VARIOUS INTRACTABLE DISEASES
)NTRODUCTION -ANY KINDS OF MEDICINAL DRUGS HAVE BEEN DEVELOPED BY SYNTHETIC ORGANIC CHEMISTRY WHILE OTHER AGENTS n ESPECIALLY ANTIBIOTICS AND ANTITUMOR AGENTS n ARE DERIVED FROM NATURAL PRODUCTS )NCLUDING METABOLIC PROD UCTS OF MICROORGANISMS SUCH AS PENICILLIN BIOLOGICAL MATERIALS SUCH AS INSULIN AND PLANT CONSTITUENTS ABOUT HALF OF THE DRUGS CURRENTLY IN CLINI CAL USE ARE OF NATURAL ORIGIN ; = )T CAN BE EXPECTED THAT EVEN IN THE ST #ENTURY METABOLIC PRODUCTS OF MICROORGANISMS WILL SERVE AS MAJOR RESOURCES FOR DRUGS !T !STELLAS WE HAVE BEEN DEVELOPING DRUGS FROM MICROBIAL METABOLITES FOR MORE THAN YEARS AND HAVE SUCCESSFULLY COM MERCIALIZED VARIOUS USEFUL PRODUCTS SUCH AS PYRROLNITRIN TACROLIMUS AND MICAFUNGIN 7E HAVE DISCOVERED TWO NOVEL CYCLIC DEPSIPEPTIDES CONTAINING A DISUL FIDE BOND &+ AND 9- &IG 4HESE WERE FOUND BY SCREENING MICROORGANISMS FOR ANTITUMOR AGENTS AND THEY REPRESENT A NOVEL STRUCTURAL CLASS OF INHIBITORS OF HISTONE DEACETYLASE ($!# AN ATTRACTIVE MOLECULAR TARGET FOR ANTITUMOR DRUGS
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
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&IGURE 3TRUCTURES OF &+ AND 9-
!CETYLATION OF $.! BOUND CORE HISTONES AND TRANSCRIPTION FACTORS IS A FUNDAMENTAL MECHANISM OF TRANSCRIPTIONAL REGULATION ;= 4HE LEVEL OF HISTONE ACETYLATION RESULTS FROM A BALANCE BETWEEN ACETYLATION AND DEACETYLATION AND THIS BALANCE IS AN IMPORTANT FACTOR IN THE REGULATION OF GENE EXPRESSION ;= 4HERE ARE TWO FAMILIES OF ENZYMES INVOLVED IN BALANCING THE STATUS OF ACETYLATION OF HISTONES HISTONE ACETYLTRANSFER ASES (!4S AND ($!#S 4O DATE HUMAN GENES THAT ENCODE PROVEN OR PUTATIVE ($!# ENZYMES HAVE BEEN IDENTIFIED ;n= #LASS ) ($!#S SHOW HOMOLOGY TO YEAST 2PD AND INCLUDE ($!#S AND #LASS )) ($!#S SHOW HOMOLOGY TO YEAST (DA AND INCLUDE ($!#S n AND #LASS ))) ($!#S SHOW HOMOLOGY TO YEAST 3IR AND INCLUDE 3)24 n 4HE MOST RECENTLY DESCRIBED #LASS )6 ($!#S COMPRISE ($!# RELATED ENZYMES 4HE ($!# INHIBITORS DESCRIBED BELOW ARE IN GENERAL INHIBITORS OF #LASS ) )) AND )6 ($!#S !BERRANT GENE EXPRESSION DUE TO ALTERED ($!# ACTIVITIES HAS BEEN ASSO CIATED WITH CANCER &OR EXAMPLE TUMOR TISSUE SPECIFIC HISTONE HYPOACETY LATION WAS OBSERVED IN SEVERAL SOLID TUMORS OF CANCER PATIENTS BUT NOT IN THEIR NORMAL TISSUES ; = 5PREGULATED EXPRESSION OF ($!# ($!# AND ($!# HAS BEEN REPORTED IN HUMAN SOLID TUMORS OF ORGANS SUCH AS THE COLON AND PROSTATE ;n= !BNORMAL RECRUITMENT OF ($!#S BY MUTANT TRANSCRIPTION FACTORS FORMED BY CHROMOSOMAL TRANSLOCATION ALSO OCCURS IN VARIOUS LEUKEMIAS ;= )T IS WELL ESTABLISHED THAT ($!# INHIBITORS CAN AFFECT SEVERAL CELLU LAR PROCESSES THAT ARE DYSREGULATED IN NEOPLASTIC CELLS ($!# INHIBITORS CAUSE GROWTH INHIBITION WITH CELLULAR DIFFERENTIATION CELL CYCLE ARREST AND
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
&IGURE /THER STRUCTURAL CLASSES OF ($!# INHIBITORS
CELL DEATH IN A BROAD SPECTRUM OF TUMOR CELLS WHILE NORMAL CELLS REMAIN RELATIVELY RESISTANT TO THESE AGENTS ;n= 4HEY ALSO INHIBIT ANGIOGENESIS ; = ($!# INHIBITORS ACTIVATE TRANSCRIPTION OF THE P7!&#IP GENE ;n= AND THIS INDUCTION IS PROBABLY DUE TO EPIGENETIC REGULATION OF THE P7!&#IP PROMOTER ASSOCIATED WITH #LASS ) ($!#S ($!# INHIBITORS CAN ALSO UPREGULATE PROAPOPTOTIC PATHWAYS AND DOWNREGULATE PROSURVIVAL PATHWAYS /THER STUDIES HAVE SHOWN THAT ($!# INHIBITORS CAN INDUCE BOTH MITOCHONDRIA MEDIATED APOPTOSIS AND CASPASE INDEPENDENT AUTOPH AGIC CELL DEATH ;n= 3EVERAL STRUCTURAL CLASSES OF ($!# INHIBITORS HAVE BEEN CHARACTERIZED INCLUDING SHORT CHAIN FATTY ACIDS SODIUM BUTYRATE PHENYL BUTYRATE AND VALPROIC ACID HYDROXAMIC ACIDS TRICHOSTATIN ! 3!(! ,"( 08$ AND #2! BENZAMIDES -3 AND #) CYCLIC PEPTIDES TRAPOXIN AND APICIDIN AND CYCLIC DEPSIPEPTIDES CONTAINING A DISULFIDE BOND &+ AND 9- ;n = &IGS 3EVERAL INHIBITORS ARE CUR RENTLY UNDERGOING CLINICAL TRIALS AND DISPLAY ANTITUMOR ACTIVITY IN PATIENTS WITH HEMATOLOGICAL AND SOLID TUMORS ;n= AND 3!(! WAS RECENTLY
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
APPROVED BY THE 53 &$! 4HEREFORE THESE OBSERVATIONS SUGGEST THAT ($!# INHIBITORS ARE VERY PROMISING AS ANTICANCER DRUGS ; = 3HORT CHAIN FATTY ACIDS SUCH AS BUTYRATE INHIBIT ($!# ACTIVITY VIA COM PETITION WITH ACETYLATED HISTONES 4HE INHIBITORY ACTIVITY OCCURS AT MILLI MOLAR CONCENTRATIONS AND THE COMPOUND HAS A SHORT HALF LIFE IN VIVO ;= 4RICHOSTATIN ! A WELL KNOWN AND HIGHLY POTENT HYDROXAMIC ACID BASED ($!# INHIBITOR WAS INITIALLY ISOLATED AS A FUNGISTATIC ANTIBIOTIC FROM MICRO BIAL PRODUCTS ;= 4HIS COMPOUND SHOWED AN EFFECT ON DIFFERENTIATION OF &RIEND LEUKEMIA CELLS AND LATER IT WAS FOUND TO ACT AS AN ($!# INHIBITOR 4HE INHIBITORY ACTIVITY OF TRICHOSTATIN ! ON ($!# OCCURS AT NANOMOLAR CONCENTRATIONS BUT ITS ACTIVITY IN CELLULAR ASSAYS IS WEAK AND NO EFFECT IS SEEN IN ANIMAL MODELS 4HIS IS PROBABLY DUE TO LOW MEMBRANE PERMEABIL ITY POOR STABILITY AND A SHORT HALF LIFE OF THE COMPOUND IN VIVO 4O CIRCUM VENT THESE DRAWBACKS MANY CHEMICALLY SYNTHESIZED ($!# INHIBITORS OF THE HYDROXAMIC ACID CLASS HAVE BEEN CREATED AND SEVERAL INHIBITORS SUCH AS 3!(! ,"( 08$ AND #2! ARE UNDERGOING CLINICAL TRI ALS ON HEMATOLOGICAL AND SOLID TUMORS "ENZAMIDE BASED ($!# INHIBITORS SUCH AS -3 AND #) INHIBIT ($!# ACTIVITY AT MICROMOLAR CONCENTRATIONS ; = 4HESE COMPOUNDS ARE ORALLY ACTIVE AND ARE UNDERGOING CLINICAL TRIALS AS WELL )T HAS BEEN POS TULATED THAT THE AMINOPHENYL GROUP IN -3 IS NECESSARY TO INHIBIT ($!# ACTIVITY BUT THE STRUCTURALLY DETAILED MECHANISM OF ITS ACTION IN ($!# INHIBITION REMAINS UNCLEAR 3EVERAL CYCLIC PEPTIDES ISOLATED FROM MICROORGANISMS HAVE BEEN REPORTED TO HAVE ($!# INHIBITORY ACTIVITY 4RAPOXIN HAS AN EPOXIDE GROUP ON THE ALIPHATIC CHAIN EXTENDING FROM THE CYCLIC TETRAPEPTIDE AND INHIBITS ($!# ACTIVITY AT NANOMOLAR CONCENTRATIONS 4RAPOXIN STRONGLY INHIBITS ($!# ACTIVITY AT THE CELLULAR LEVEL BUT IT IS NOT EFFECTIVE IN ANIMAL MODELS PROB ABLY BECAUSE OF INSTABILITY ANDOR NONSPECIFICITY ;= 3INCE THE EPOXIDE GROUP OF THIS COMPOUND IRREVERSIBLY BINDS TO ($!# ($!# PROTEINS WERE ISOLATED BY USING TRAPOXIN AS A CHEMICAL PROBE ;= 4HE DISCOVERY OF MICRO BIAL PRODUCTS SUCH AS TRAPOXIN AND TRICHOSTATIN ! HAS MADE A STRONG CONTRI BUTION TO THE PURIFICATION OF ($!# PROTEINS AND CHROMATIN RESEARCH 7E DISCOVERED THAT &+ AND 9- CYCLIC DEPSIPEPTIDES CONTAINING A DISULFIDE BOND REPRESENT ANOTHER NOVEL STRUCTURAL CLASS OF ($!# INHIBI TORS &+ IS A PRODRUG AND INHIBITS ($!# ACTIVITY AT NANOMOLAR CONCEN TRATIONS AT BOTH THE ENZYME AND CELLULAR LEVELS AND IT IS ALSO HIGHLY EFFEC
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
TIVE IN ANIMAL MODELS 4HIS ATTRACTIVE PROFILE IS DUE TO ITS UNIQUE CHEMICAL STRUCTURE THANKS TO THE UNIQUE BIOSYNTHETIC ABILITIES OF MICROORGANISMS "ELOW WE DESCRIBE THE DISCOVERY PROCESSES AND THE ACTIVITIES OF &+ AND 9-
&+ $ISCOVERY OF &+ 4HE CURE RATE FOR CANCER HAS GRADUALLY IMPROVED YEAR BY YEAR BUT CANCER HAS BEEN THE LEADING CAUSE OF DEATH IN *APAN FROM UNTIL TODAY )N *APAN ANTITUMOR AGENTS SUCH AS CISPLATIN MITOMYCIN # AND ADRIAMYCIN ARE THE MOST WIDELY USED TREATMENTS FOR CANCER 4HE DEVELOPMENT OF SUCH ANTITUMOR AGENTS STARTED IN THE EARLY S AND THE EFFORT TO FIND EVEN BETTER DRUGS CONTINUES AGGRESSIVELY TODAY THROUGHOUT THE WORLD (OW EVER SINCE THE SCREENING METHOD FOR THESE ANTITUMOR AGENTS FOCUSED ON INHIBITION OF THE GROWTH OF CARCINOMA CELLS THEIR MECHANISM OF ACTION IS MAINLY PRIMARY INHIBITION OF BIOSYNTHESIS OF CELLULAR MACROMOLECULES OR INHIBITION OF MITOSIS AS TYPIFIED BY DIRECT INHIBITION OF $.! SYNTHESIS !S A RESULT THESE AGENTS SHOW STRONG TOXICITY FOR NORMAL CELLS THAT HAVE ALSO HIGH GROWTH RATES SUCH AS BONE MARROW AND EPIDERMAL CELLS OF THE DIGESTIVE TRACT !CCORDINGLY FROM THE VIEWPOINT OF ADVERSE EFFECTS THESE ANTITUMOR AGENTS ARE NOT OPTIMAL FOR USE IN THERAPY AND DEVELOPMENT OF ANTITUMOR AGENTS HAVING A MORE BENIGN MECHANISM OF ACTION AND MUCH LESS SIDE EFFECT IS STRONGLY DESIRED 7ITH RECENT ADVANCES IN THE TECHNOLOGY OF MOLECULAR BIOLOGY REMARK ABLE PROGRESS HAS BEEN MADE IN THE STUDY OF THE MOLECULAR MECHANISMS OF TUMORIGENESIS &OR EXAMPLE IT HAS BEEN FOUND THAT ONCOGENES HAVE CLOSE RELATIONSHIPS WITH THE GENES INVOLVED IN THE VARIOUS INFORMATION TRANSDUC TION MECHANISMS NECESSARY FOR THE GROWTH OF NORMAL CELLS 4HESE GENES CAN BE CLASSIFIED INTO FIVE GROUPS STARTING UPSTREAM OF THE SIGNAL TRANSDUCTION PROCESS AS A FUNCTION OF THE STRUCTURES AND FUNCTIONS OF THEIR PRODUCTS GROWTH FACTOR RELATED GENE CLUSTER SIS FAMILY GROWTH FACTOR RECEPTOR AND PHOSPHORYLATED ENZYME RELATED GENE CLUSTER SRC FAMILY '40 BIND ING PROTEIN RELATED GENE CLUSTER RAS FAMILY HORMONE RECEPTOR RELATED GENE CLUSTER ERB! FAMILY AND INTRANUCLEAR TRANSCRIPTION FACTOR RELATED
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
GENE CLUSTER MYC FAMILY )T IS THOUGHT THAT MUTATIONS OF THE GENES COD ING FOR EACH COMPONENT OR MUTATIONS OF THE GENES RELATED TO REGULATION OF EXPRESSION CAUSE ABNORMAL SIGNAL TRANSDUCTION AND RESULT IN UNCONTROLLED GROWTH OF CELLS IE MALIGNANCY )N THE LATE S WE THOUGHT THAT SUBSTANCES WHICH SELECTIVELY CONTROL THE ACTIVITIES OF ONCOGENE PRODUCTS WOULD HAVE THE POTENTIAL AS A NEW TYPE OF ANTITUMOR AGENTS DIFFERENT FROM OTHER ANTITUMORAL AGENTS THAT ACT DIRECTLY ON $.! OR MICROTUBULES )T WAS ALSO REPORTED THAT SOME PHAR MACOLOGICAL AGENTS OF MICROBIAL ORIGIN ARE ABLE TO REVERSE THE TRANSFORMED MORPHOLOGY OF AN ONCOGENE TRANSFORMANT TO NORMAL AND IT WAS ALSO REPORTED THAT SOME SUBSTANCES SHOW ANTITUMOR POTENCY IN VIVO LIKE AZATY ROSINE !MONG THE VARIOUS ONCOGENES WE FOCUSED ON THE RAS ONCOGENE FAMILY SINCE PROSPECTIVE STUDIES HAVE BEEN CONDUCTED AND MANY CHANGES IN EXPRESSIONS IN HUMAN CANCER HAVE BEEN REPORTED 2AS ONCOGENES ARE INVOLVED IN VARIOUS SOLID TUMORS AND LEUKEMIAS AND THEIR DETECTION RATE IS SAID TO BE MORE THAN -ANY STUDIES HAVE ACCUMULATED EVIDENCE THAT RAS ONCOGENES ARE FACTORS IN THE GENERATION AND MALIGNANT ALTERATION OF HUMAN CANCERS #ONSEQUENTLY WE SEARCHED FOR NEW ANTITUMOR AGENTS HAVING A NEW ACTION MECHANISM NAMELY REVERSION OF RAS ONCOGENE TRANS FORMANTS TO THE NORMAL MORPHOLOGY AND ALSO INHIBITION OF THE GROWTH OF TUMOR CELLS 4O ESTABLISH A MALIGNANT ALTERATION MODEL INVOLVING A RAS ONCOGENE WE GENERATED TRANSFORMED CELLS BY INTRODUCING THE HUMAN (A RAS ONCO GENE INTO NORMAL CELLS 5NLIKE THE ORIGINAL NORMAL CELLS THE TRANSFORMANT ACQUIRED VARIOUS MALIGNANT PROPERTIES SUCH AS MORPHOLOGICAL CHANGES DISAPPEARANCE OF FIBRONECTIN ABILITY TO GROW IN SERUM FREE CONDITIONS COL ONY FORMING ABILITY ON SOFT AGAR MEDIUM AND TUMORIGENICITY IN NUDE MICE 7E EXPECTED THAT SUBSTANCES WHICH ACTED TO NORMALIZE VARIOUS MALIGNANT PROPERTIES WOULD INHIBIT THE ABNORMAL SIGNAL TRANSDUCTION SYSTEM CAUSED BY RAS ONCOGENES AND THUS WE SELECTED MICROSCOPIC OBSERVATION FOR REVER SAL OF MORPHOLOGICAL CHANGES AS THE SIMPLEST SCREENING METHOD !S A RESULT OF SCREENING MORE THAN STRAINS OF ACTINOMYCETES FUNGI AND BACTERIA OBTAINED FROM SOIL SAMPLES COLLECTED AT VARIOUS LOCA TIONS IN *APAN WE FOUND A NOVEL COMPOUND IN THE FERMENTATION BROTH OF BACTERIAL SAMPLE .O THAT SHOWED ACTIVITY IN REVERSING TRANSFORMED CELL MORPHOLOGY TO NORMAL 7E NAMED IT &2 &+ &IG !T THIS TIME IT WAS NOT KNOWN THAT THIS COMPOUND WAS AN ($!# INHIBITOR BUT
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
LATER IT WAS DEMONSTRATED TO HAVE BIOLOGICAL ACTIVITY SIMILAR TO THAT OF AN ($!# INHIBITOR TRICHOSTATIN ! EVEN THOUGH IT HAS A COMPLETELY DIFFERENT CHEMICAL STRUCTURE ;= &+ HAS SINCE BECOME WIDELY RECOGNIZED AS A NEW TYPE OF ($!# INHIBITOR
-ICROBIAL SOURCE AND CHEMISTRY OF &+ 3TRAIN .O THE ORGANISM PRODUCING &+ IS A BACTERIUM THAT WAS ISOLATED FROM A SOIL SAMPLE COLLECTED IN 9AMAGATA 0REFECTURE *APAN 3TRAIN .O IS A 'RAM NEGATIVE MOTILE BACTERIUM WITH A SINGLE POLAR FLAGELLUM /N THE BASIS OF ITS VARIOUS BACTERIOLOGICAL PROPERTIES WE IDENTIFIED THIS BACTERIUM AS #HROMOBACTERIUM VIOLACEUM ;= !NOTHER #HROMOBACTERIUM SP ALSO PRODUCES 9- WHICH IS THE WORLDS FIRST 'Qα INHIBITOR ;= AND IT IS ALSO NOTEWORTHY THAT THIS BACTERIUM PRODUCES SEVERAL UNIQUE SECONDARY METABOLITES
4HE STRUCTURE OF &+ % 3 3 3 2 ;: ETHYLIDENE= DIISOPROPYL OXA DITHIA TETRAAZABICYCLO; = TRICOS ENE PENTA NONE WAS DETERMINED BY A COMBINATION OF SPECTROSCOPIC CHEMICAL AND SINGLE CRYSTAL 8 RAY CRYSTALLOGRAPHIC ANALYSES ;= )N THE STRUCTURE WAS CONFIRMED BY TOTAL CHEMICAL SYNTHESIS BY ,I ET AL ;= &+ IS A UNIQUE CAGE SHAPED BICYCLIC DEPSIPEPTIDE THAT IS A MEMBERED MAC ROCYCLIC LACTONE WITH A MEMBERED RING ENCOMPASSING THE DISULFIDE BOND FORMED BETWEEN THE $ CYSTEINE AND THE THIOL GROUP OF A NOVEL ACID HYDROXY MERCAPTO HEPTENOIC ACID )T WAS LATER FOUND THAT THIS DISUL FIDE BOND PLAYS AN IMPORTANT ROLE IN THE MOLECULAR MECHANISM OF ($!# INHIBITION BY &+
!NTIMICROBIAL ACTIVITIES OF &+ &+ EXHIBITED WEAK ANTIMICROBIAL ACTIVITY AGAINST 3CHIZOSACCHAROMYCES POMBE !UREOBASIDIUM PULLULANS )&/ AND !SPERGILLUS NIGER )N CONTRAST
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
NO ANTIMICROBIAL ACTIVITY WAS SHOWN AGAINST #ANDIDA ALBICANS 3TAPHYLOCOC CUS AUREUS "ACILLUS SUBTILIS OR %SCHERICHIA COLI AT A CONCENTRATION OF μG ML ;=
($!# INHIBITORY ACTIVITIES OF &+ !S DESCRIBED ABOVE &+ WAS DISCOVERED AS A COMPOUND WHICH REVERSES (A RAS TRANSFORMED .)(4 CELLS TO NORMAL MORPHOLOGY &+ STRONGLY SUPPRESSES THE GROWTH OF TUMOR CELLS IN THE ' AND '- PHASES IN VITRO ;= AND IT ALSO STRONGLY SUPPRESSES THE GROWTH OF XENOGRAFT TUMORS IN NUDE MICE ;= !NOTHER NATURALLY OCCURRING COMPOUND WHICH WE HAVE STUDIED TRICHO STATIN ! WAS ISOLATED FROM 3TREPTOMYCES HYGROSCOPICUS 4RICHOSTATIN ! WAS ORIGINALLY DISCOVERED AS A SUBSTANCE SHOWING ANTIFUNGAL ACTIVITY AGAINST 4RICHOPHYTON ;= ,ATER IT WAS REDISCOVERED AS A SUBSTANCE INDUCING DIFFER ENTIATION OF &RIEND MURINE ERYTHROLEUKEMIA -%, CELLS ;= AND GENETIC AND BIOLOGICAL STUDIES ASCERTAINED THAT TRICHOSTATIN ! IS A SELECTIVE ($!# INHIBITOR ;= 4RICHOSTATIN ! WAS REPORTED AS A SUBSTANCE INDUCING HYPER ACETYLATION OF HISTONES CELL DIFFERENTIATION AND TRANSFORMATION ;n= &+ DIFFERS STRUCTURALLY CLEARLY FROM THE TRICHOSTATINS BUT IT SHOWED SIMI LAR ACTIVITY AND IT WAS THEN ELUCIDATED THAT ITS TARGET IS ALSO ($!#S ;= !S ALREADY MENTIONED CURRENTLY THREE CLASSES OF ($!#S ARE KNOWN 2PD LIKE (DA LIKE AND 3IR LIKE ; n= AND IT WAS REPORTED THAT &+ SHOWS STRONG INHIBITORY ACTIVITY AGAINST #LASS ) ($!#S ($!# AND ;= 4HERE HAVE BEEN NO REPORTS OF 8 RAY CRYSTALLINE STRUCTURE ANALYSIS OF THE COMBINED FORM OF &+ WITH ($!# (OWEVER FOR TRICHOSTATIN ! THERE HAS BEEN A REPORT OF 8 RAY CRYSTALLINE STRUCTURE ANALYSIS OF THE COMBINED FORM BETWEEN TRICHOSTATIN ! AND AN ($!# HOMOLOG HISTONE DEACETYLASE LIKE PROTEIN ($,0 PRODUCED BY A HYPERTHERMOPHILIC BACTE RIUM !QUIFEX AEOLICUS ;= 4RICHOSTATIN ! INSERTS ITS LONG ALIPHATIC CHAIN INTO THE TUBE LIKE POCKET OF ($,0 AND THE HYDROXAMIC ACID AT THE END BINDS TO THE ACTIVE SITE RESIDUE THROUGH A ZINC ION /N THE OTHER HAND &+ HAS A BICYCLIC STRUCTURE AND DOES NOT HAVE A LONG ALIPHATIC CHAIN LIKE TRICHOSTATIN (OWEVER IN &URUMAI ET AL REPORTED THAT &+ UNDER GOES CELLULAR REDUCTION TO A BIS THIOL DERIVATIVE THAT ACTS AS A ZINC CHELATING GROUP BINDING TO THE CATALYTIC SITE OF ($!# ;= 4HIS REDUCTION PRODUCT
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
WAS SHOWN TO BE FOLD MORE ACTIVE THAN &+ ITSELF IN THE INHIBITION OF ($!#S IN VITRO 2EDUCED FORM &+ IS EXTREMELY UNSTABLE IN SERUM WHEREAS &+ WITH THE DISULFIDE LINKAGE STILL INTACT IS STABLE IN SERUM 4HEREFORE &+ IS A NATURAL PRODRUG AND IT IS EVIDENT THAT THIS COMPOUND IS ACTIVATED BY REDUCTION AFTER UPTAKE INTO CELLS &+ SHOWS POTENT ACTIV ITY IN VIVO IN COMPARISON WITH NATURALLY OCCURRING ($!# INHIBITORS LIKE TRICHOSTATIN ! AND IT IS SUGGESTED THAT THIS PROPERTY IS A RESULT OF HIGHER MEMBRANE PERMEABILITY DUE TO STABILITY IN SERUM ANDOR HYDROPHOBIC PROPERTIES IN THE PRODRUG STATE #OMPUTER MODELING SUGGESTED THAT ONE OF THE TWO SULFHYDRYL GROUPS IN REDUCED &+ INTERACTS WITH THE ACTIVE CENTER ZINC VIA A WATER MOLECULE 4HIS WAS ALSO SUPPORTED BY THE FACT THAT THE INHIBITORY EFFECT WAS WEAKENED BY BLOCKING THE THIOL RESIDUE THROUGH METHYLATION /THER INVESTIGATORS REPORTED IN THAT THE REDUCED FORM OF &+ WAS FOUND TO HAVE A FREE 3( GROUP IN A LONG ALIPHATIC CHAIN BY A TECHNIQUE USING (0,#%3) -3 ;= MIMICKING A STRUCTURAL FEATURE OF TRICHOSTATIN ! )T IS VERY INTERESTING THAT MICROORGANISMS PRODUCE PHYSI OLOGICALLY ACTIVE SUBSTANCES IN A PRODRUG FORM &+ HAS UNIQUE PROPER TIES AS AN ($!# INHIBITOR UNLIKE THOSE OF THE OTHER NATURALLY OCCURRING ($!# INHIBITORS AND IT REPRESENTS A NEW STRUCTURAL CLASS
!NTITUMOR ACTIVITY OF &+ !S ALREADY MENTIONED ($!#S ARE CONSIDERED TO BE PROMISING TARGETS FOR CANCER CHEMOTHERAPY "ESIDES &+ CLINICAL TRIALS ARE CURRENTLY BEING CAR RIED OUT ON OTHER ($!# BLOCKERS LIKE PHENYLBUTYRATE -3 3!(! AND VALPROIC ACID !MONG THEM &+ SHOWED THE STRONGEST CYTOTOXIC EFFECTS AGAINST VARIOUS TUMOR CELL LINES SUCH AS ! 0# !$( -+. AND -#& AT LOW CONCENTRATIONS )N CONTRAST IT SHOWED NOWEAK CYTOTOXIC ITY FOR NORMAL CELLS AND A DIFFERENT CYTOTOXICITY SPECTRUM COMPARED WITH EXISTING ANTITUMOR AGENTS SUCH AS DOXORUBICIN AND MITOMYCIN # ;= 4HE ANTITUMOR EFFECTS OF &+ HAVE BEEN COMPARED WITH MITOMYCIN # CISPLATIN DOXORUBICIN AND FLUOROURACIL AGAINST SEVERAL MURINE AND HUMAN TUMORS IN MICE &+ MITOMYCIN # AND CISPLATIN EACH PROLONGED THE SURVIVAL OF 0 , AND " TUMOR BEARING MICE AFTER IP ADMINISTRA TION FOR DAYS )N VIVO &+ WAS LESS ACTIVE AGAINST 0 LEUKEMIA AND , LEUKEMIA COMPARED WITH CISPLATIN AND MITOMYCIN # BUT SHOWED
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
COMPARABLE ACTIVITY AGAINST " MELANOMA OVER A WIDER EFFECTIVE DOSE RANGE OF APPROXIMATELY n MGKG !LL THESE AGENTS PROLONGED CELL SURVIVAL ;= )N STUDIES USING VARIOUS HUMAN XENOGRAFT TUMORS IN NUDE MICE &+ GIVEN IV THREE TIMES AT DAY INTERVALS INHIBITED THE GROWTH OF ,U ,# AND 3# BY 7HEREAS MITOMYCIN # AND CISPLATIN ALMOST COMPLETELY INHIBITED THE GROWTH OF -8 &+ DID NOT )N MICE USING A NON RESISTANT 0 TUMOR MODEL AS A CONTROL &+ SHOWED STRONG ACTIVITY AGAINST MITOMYCIN CYCLOPHOSPHAMIDE VINCRISTINE AND FLUORO URACIL RESISTANT 0 TUMORS BUT WAS LESS ACTIVE AGAINST DOXORUBICIN RESIS TANT 0 TUMORS ;= 5NLIKE EXISTING ANTITUMOR AGENTS THAT DIRECTLY INHIBIT $.! SYNTHESIS IT HAS BEEN FOUND THAT THE UNIQUE CYTOTOXIC PROFILE OF &+ IS DUE TO SPECIFIC INDUCTION OF EXPRESSION AND SUPPRESSION OF VARIOUS GENES THAT ARE INVOLVED IN THE ($!# INHIBITORY MECHANISM )T WAS REPORTED THAT HYPERACETYLATION OF HISTONES BY ($!# INHIBITORS SUCH AS TRICHOSTATIN ! AND TRAPOXIN INDUCES EXPRESSION OF ONLY ABOUT OF GENES ;= BUT ($!# INHIBITORS INDUCE VARIOUS ACTIONS RELATED TO THE SUPPRESSION OF TUMOR GENERATION SUCH AS CELL CYCLE INHIBITION ;= APOPTOSIS ;= CELL DIFFERENTIATION ;= ANGIOGENESIS INHIBITION ;= AND IMMUNOMODULATION ;= )N FACT &+ CAUSES ' AND '- CELL CYCLE ARREST 4HIS IS BECAUSE &+ DOWNREGULATES CYCLIN $ AND UPREGULATES #$+ INHIBITOR P RESULTING IN INHIBITION OF #$+ ACTIVITY AND 2B PHOSPHORYLATION AND THUS ' ARREST !LSO CELLS LACKING P DID NOT UNDERGO ' ARREST CONTINUED $.! SYNTHESIS AND WERE ARRESTED IN THE '- PHASE OF THE CELL CYCLE ;= -OREOVER IN THE CASE OF APOPTOSIS CAUSED BY ($!# INHIBITORS THE FOLLOWING PHENOMENA HAVE BEEN CONFIRMED INDUC TION OF TRANSCRIPTIONAL ACTIVATION OF PROAPOPTOTIC GENES SUCH AS &AS AND "AX ;= RELEASE OF CYTOCHROME # FROM MITOCHONDRIA AND SUBSEQUENT ACTIVA TION OF CASPASE ;= AND INDUCTION OF EXPRESSION OF "CL FAMILY PROTEINS ;= )T WAS REPORTED THAT &+ INDUCES APOPTOSIS THROUGH ACTIVATION OF VARIOUS CASPASES 4.& α PRODUCTION ;= AND "CL PHOSPHORYLATION ;= &+ HAS AN ANTIANGIOGENIC EFFECT THAT IS THOUGHT TO BE CLOSELY RELATED TO ITS POTENT ANTITUMOR EFFECT )T HAS ALSO BEEN SUGGESTED THAT &+ SUP PRESSES ANGIOGENESIS BY SUPPRESSING THE EXPRESSION OF 6%'& AND B&'& IN 0# XENOGRAFTS AND BY INDUCING HISTONE ACETYLATION OF 6%'& PROMOTER REGIONS WHICH MAY SUPPRESS 6%'& GENE EXPRESSION ;= &URTHERMORE IT HAS BEEN REPORTED THAT THERE IS INVOLVEMENT OF HYPOXIA INDUCIBLE FAC TOR ()& α WHICH ACTIVATES 6%'& TRANSCRIPTION 4RICHOSTATIN ! ANOTHER
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
($!# INHIBITOR BLOCKS ANGIOGENESIS BY UPREGULATING THE EXPRESSION OF P AND 6(, IN (EP ' CELLS UNDER HYPOXIC CONDITIONS AND DOWNREGU LATING THE ()& α AND 6%'& GENES ;= &+ HAS ALSO BEEN IMPLICATED IN PROMOTING VON (IPPEL ,INDAU 6(, PROTEIN EXPRESSION INHIBITION OF ANGIOGENESIS AND HYPOXIA STIMULATED PROLIFERATION ;= AND IT HAS BEEN SHOWN TO INHIBIT ()& α INDUCTION AND ()& α ACTIVITY ;= !CCORDINGLY IT IS THOUGHT THAT IN ADDITION TO DIRECT INHIBITION OF TUMOR FORMATION BY SUP PRESSION OF GROWTH MORPHOLOGICAL DIFFERENTIATION AND INDUCTION OF APOP TOSIS &+ SUPPRESSES THE SPREAD OF TUMORS BY INHIBITING ANGIOGENESIS
/THER ACTIVITIES OF &+ )N ADDITION TO ITS DIRECT ANTITUMOR EFFECT IT HAS BEEN REPORTED THAT &+ INDIRECTLY AFFECTS THE ACTIVITY OF OTHER ANTITUMOR AGENTS &OR EXAMPLE A CONTRIBUTION OF &+ TO THE MECHANISM OF DRUG RESISTANCE WAS REVEALED 3ODIUM BUTYRATE AN ($!# INHIBITOR INDUCES EXPRESSION OF -$2 MUL TIDRUG RESISTANCE GENE IN TUMOR CELLS ;= 4HE HUMAN -$2 GENE ENCODES AN !40 BINDING CASSETTE !"# TRANSPORTER 0 GLYCOPROTEIN 0GP AND 0GP AFFECTS THE CROSS RESISTANCE OF CHEMOTHERAPEUTIC DRUGS BY DECREAS ING THE CONCENTRATIONS OF DRUGS IN CELLS VIA ENERGY DEPENDENT EFFLUX ;= 4RANSCRIPTION FACTOR .& 9 IS ALSO DEEPLY INVOLVED IN THE INCREASE IN -$2 GENE EXPRESSION INDUCED BY ($!# INHIBITORS ;= 4HE SCREENING PROJECT CONDUCTED BY THE .ATIONAL #ANCER )NSTITUTE .#) FOUND THAT &+ IS A SUBSTRATE OF 0GP )T WAS ALSO REPORTED THAT INCUBATION OF &+ WITH CANCER CELLS INDUCED EXPRESSION OF 0GP AND &+ DID NOT SHOW INHIBITORY EFFECTS ON 0GP ; = )T HAS ALSO BEEN SUGGESTED THAT 0GP IS STRONGLY INVOLVED IN TUMOR RESISTANCE TO &+ )T WAS REPORTED THAT FREQUENT &+ TREATMENTS MIGHT RESULT IN -$2 UPREGULATION AND THEREFORE TO &+ RESISTANCE AND THAT &+ REFRACTORY PATIENTS SHOULD NOT BE TREATED WITH DRUGS THAT ARE -$2 SUBSTRATES SUCH AS PACLITAXEL AND DOXORUBICIN ; = )N THIS CONNECTION IT WAS REPORTED THAT THERE WAS INDUCTION OF THE -$2 GENE AND 0GP EXPRESSION IN NORMAL AND MALIGNANT CELLS OBTAINED FROM PATIENTS AFTER &+ TREATMENT ;= AND IT CAN THUS BE ANTICIPATED THAT THE CLINICAL EFFICACY OF &+ WILL BE IMPROVED BY COADMINISTRATION OF A 0GP INHIBITOR !LSO IN THE CLINICAL APPLICATION OF &+ CAREFUL ADMINISTRATION IS WAR RANTED FOR PATIENTS WHO ARE RESISTANT TO OTHER ANTITUMOR DRUGS
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
)N ADDITION TO THE ABOVE IT WAS REPORTED THAT &+ IS EFFECTIVE IN IODINE THERAPY FOR THYROID CARCINOMA THAT IS UNABLE TO TRAP IODINE ;= 4HIS IS DUE TO INDUCTION OF .A )n SYMPORTER PROTEIN BY A LOW CONCENTRATION OF &+ RESULTING IN ) ACCUMULATION IN THE TUMOR TISSUE !CCORDINGLY IT IS ANTICI PATED THAT &+ WILL BE EFFECTIVE AS AN ADJUNCT OF RADIOIODINE THERAPY #ONTRARY TO THE REPORTS THAT &+ INDUCES EXPRESSION OF GENES RELATED TO DRUG RESISTANCE SUCH AS -$2 AND CONSEQUENTLY WEAKENS THE ANTITU MOR ACTIVITY IT HAS BEEN REPORTED THAT IT STRENGTHENS THE EFFECTS OF OTHER ANTITUMOR AGENTS &OR EXAMPLE PROTEASOME INHIBITORS AND &+ SYNERGIS TICALLY INDUCE APOPTOSIS OF HUMAN TUMOR CELLS ACCUMULATION OF P AND PHOSPHORYLATION OF P AND "IM !LSO JUDGING FROM THE FACT THAT , . ACE TYLCYSTEINE A FREE RADICAL SCAVENGER SIGNIFICANTLY SUPPRESSES CELLULAR DEATH BY THESE MECHANISMS IT CAN BE SURMISED THAT AT LEAST PARTIALLY AN INCREASE IN THE INTRACELLULAR REACTIVE OXYGEN SPECIES LEVEL AFFECTS CELLULAR DEATH ;= )T WAS REPORTED THAT COMBINED TREATMENT WITH &+ AND IMATINIB MESYL ATE A "CR !BL THYROSINE KINASE BLOCKER SIGNIFICANTLY INCREASED INDUCTION OF APOPTOSIS THAT WAS ACCOMPANIED BY ACTIVATION OF CASPASE AND CLEAVAGE OF 0!20 ;= )N ADDITION IT WAS REPORTED THAT &+ AMPLIFIED ADENOVIRUS MEDIATED TRANSGENE EXPRESSION ;= "ASED ON THAT FINDING IT CAN BE POSTU LATED THAT USE OF &+ MIGHT PERMIT US TO OVERCOME THE MAIN LIMITATION TOXICITY OF GENE THERAPY &+ HAS ALSO BEEN USED WITH GENE THERAPY IN THYROID CANCER TO ENHANCE THYROGLOBULIN PROMOTER DIRECTED EXPRESSION OF THE HERPES SIMPLEX VIRUS THYMIDINE KINASE GENE ;= )N ANOTHER GENE THERAPY APPROACH &+ INCREASED THE LEVELS OF EXPRESSION OF COXSACKIE AND ADENOVIRUS RECEPTORS AND αV INTEGRIN IN HEMATOPOIETIC CELLS THEREBY ENHANCING THE EFFICIENCY OF ADENOVIRAL INFECTION AND INCREASING TRANSGENE EXPRESSION FROM A BETA GALACTOSIDASE EXPRESSING ADENOVIRAL VECTOR ;= )T IS ANTICIPATED THAT FUTURE RESEARCH WILL ELUCIDATE THE COMPLICATED &+ MECHANISM INVOLVING VARIOUS GENES AND IDENTIFY A WIDE RANGE OF POTENTIALLY EFFECTIVE THERAPEUTIC APPLICATIONS OF &+
#LINICAL STUDIES OF &+ &+ IS THE FIRST POTENT ($!# INHIBITOR TO UNDERGO EARLY PHASE CLINI CAL DEVELOPMENT AS A POTENTIAL TREATMENT FOR SOLID AND HEMATOLOGICAL CANCERS 5NDER A JOINT DEVELOPMENT AGREEMENT WITH THE .#) A 0HASE
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
) STUDY SPONSORED BY THE .#) WAS STARTED IN 4HEN IN !PRIL WE GRANTED 'LOUCESTER 0HARMACEUTICALS A WORLDWIDE EXCLUSIVE LICENSE FOR &+ UNDER INTELLECTUAL PROPERTY RIGHTS INCLUDING PATENTS TO DEVELOP MANUFACTURE AND SELL THE DRUG 'LOUCESTER 0HARMACEUTICALS IS CURRENTLY CONDUCTING A PIVOTAL TRIAL ON THE COMPOUND AS A SINGLE AGENT IN THE TREATMENT OF CUTANEOUS 4 CELL LYMPHOMA IN PATIENTS WHO HAVE RELAPSED FOLLOWING OR BECOME REFRACTORY TO ANOTHER SYSTEMIC THERAPY 2ECENTLY AT 4HE !MERICAN 3OCIETY OF #LINICAL /NCOLOGY !3#/ MEETING IN *UNE 'LOUCESTER 0HARMACEUTICALS REPORTED AN OVERALL RESPONSE RATE OF INCLUDING A COMPLETE RESPONSE RATE AND A PARTIAL RESPONSE RATE ;= -OREOVER PATIENTS COMPLETING AT LEAST TWO CYCLES OF TREATMENT SHOWED A MEAN IMPROVEMENT IN PRURITUS 4HE TREATMENT WAS GENERALLY WELL TOLERATED )N &EBRUARY INTERIM DATA FROM A 0HASE )) TRIAL OF &+ FOR META STATIC HORMONE REFRACTORY PROSTATE CANCER WERE PRESENTED AT THE !3#/ 0ROSTATE #ANCER 3YMPOSIUM 4HE TRIAL ASSESSED THE EFFICACY AND TOLER ABILITY OF THE DRUG IN PATIENTS 4HE RADIOLOGICAL RESPONSE WAS EVALUATED IN PATIENTS /NE PATIENT ACHIEVED A MONTH PARTIAL RESPONSE AND TWO ACHIEVED STABLE DISEASE FOR MONTHS &IVE OTHER PATIENTS HAD SHOWN DIS EASE STABILIZATION BUT HAD NOT YET REACHED MONTHS 4HE PROSTATE SPECIFIC ANTIGEN 03! RESPONSE WAS EVALUATED IN PATIENTS 4WO SHOWED A OR GREATER DECREASE IN 03! WHILE ANOTHER SHOWED A DECREASE IN 03! 4HE DRUG WAS AGAIN WELL TOLERATED ;= 4OLERABILITY IS ONE OF THE FAVORABLE PROPERTIES OF &+ )T SHOWED A DRAMATIC EFFECT IN PATIENTS WITH REFRACTORY CUTANEOUS OR PERIPHERAL 4 CELL LYMPHOMA A BREAKTHROUGH FOR THIS CLASS OF ANTITUMOR AGENTS 5NDER A #OOPERATIVE 2ESEARCH AND $EVELOPMENT !GREEMENT #2!$! 'LOUCESTER AND THE .#) ARE EVALUATING &+ IN 0HASE )) TRIALS FOR RENAL CELL CARCINOMA HORMONE REFRACTORY PROSTATE CANCER MULTIPLE MYELOMA AND PANCREATIC TUMORS )N THE TREATMENT OF MOST CANCERS GREATER RESPONSES ARE OBTAINED WHEN MULTIPLE AGENTS ARE USED IN COMBINATION 4HEREFORE STUDIES OF PRACTICAL COMBINATION THERAPIES WITH &+ MAY BE CONDUCTED IN THE FUTURE -ANY PATIENTS HAVE RECEIVED &+ IN CLINICAL TRIALS TO DATE ! 0HASE ) TRIAL OF &+ IN PATIENTS WITH REFRACTORY SOLID TUMORS AND 0HASE ) AND )) STUDIES IN PATIENTS WITH LEUKEMIA AND 4 CELL LYMPHOMA SHOWED SIMILAR SIDE EFFECTS 4HE OBSERVED DOSE LIMITING TOXICITIES WERE FATIGUE NAUSEA VOMIT
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
ING THROMBOCYTOPENIA TRANSIENT NEUTROPENIA AND CARDIAC ARRHYTHMIA ; = 4RANSIENT ELECTROCARDIOGRAM ABNORMALITIES HAVE ALSO BEEN REPORTED IN CLINICAL STUDIES (OWEVER RECENTLY PUBLISHED DATA SHOW THAT THESE CHANGES ARE NOT INDICATIVE OF MYOCARDIAL DYSFUNCTION OR MYOCARDIAL DAMAGE !DDI TIONAL INVESTIGATIONS ARE NEEDED TO DETERMINE WHETHER THE OBSERVED 14C PROLONGATION HAS AN EFFECT ON THE SAFETY PROFILE OF THIS AGENT ;= !LTHOUGH CONCERNS REGARDING POTENTIAL CARDIAC EFFECTS HAVE AFFECTED ITS DEVELOPMENT THE RISK MAY NOT BE SERIOUS AS 0IEKARZ ET AL RECENTLY REPORTED ;= 4HE METABOLISM OF &+ WAS ALSO STUDIED IN A 0HASE ) TRIAL IN PATIENTS WITH REFRACTORY SOLID TUMORS THE PLASMA DISPOSITION OF &+ WAS WELL DESCRIBED BY A FIRST ORDER TWO COMPARTMENT OPEN PHARMACOKINETIC MODEL !T A DOSE OF MGM ADMINISTERED BY H IV INFUSION THE MEAN VOL UME OF DISTRIBUTION WAS ,M THE CLEARANCE WAS ,HM AND THE TERMINAL HALF LIFE WAS H 4HE MEAN MAXIMUM PLASMA CONCENTRATION RANGED FROM N- AT A DOSE OF MGM TO N- AT MGM 4HE PLASMA CONCENTRATION AND AREA UNDER THE PLASMA CONCENTRATION TIME CURVE !5# INCREASED WITH INCREASING DOSE 3ERUM TAKEN FROM PATIENTS AT VARIOUS TIME POINTS AFTER TREATMENT AT A DOSE OF MGM INDUCED CELL CYCLE ARREST OF CULTURED 0# CELLS AND HISTONE HYPERACETYLATION IN PERIPH ERAL BLOOD MONONUCLEAR CELLS CONFIRMING THAT THE LEVELS OF &+ ACHIEVED IN VIVO ARE BIOLOGICALLY RELEVANT ;=
3UMMARY OF &+ 4HE NATURAL PRODUCT &+ ALSO KNOWN AS &2 DEPSIPEPTIDE .3# AND .3# $ A POTENT ($!# INHIBITOR IS A BICYCLIC DEP SIPEPTIDE WITH A MOLECULAR WEIGHT OF THAT WAS ISOLATED FROM #HRO MOBACTERIUM VIOLACEUM )N *ULY THE )NTERNATIONAL .ONPROPRIETARY .AMES ).. BODY OF THE 7ORLD (EALTH /RGANIZATION 7(/ AND THE 5NITED 3TATES !DOPTED .AMES 53!. #OUNCIL APPROVED THE NONPROPRI ETARY GENERIC NAME @ROMIDEPSIN FOR &+ &+ WAS DISCOVERED BY &UJISAWA 0HARMACEUTICAL #O ,TD CURRENTLY !STELLAS 0HARMA )NC 3INCE IT SHOWS POTENT IN VITRO CYTOTOXICITY AGAINST VARIOUS TUMOR CELLS AND IN VIVO EFFICACY AGAINST HUMAN TUMOR XENOGRAFTS &+ IS CURRENTLY ENTERING A PIVOTAL TRIAL IN THE TREATMENT OF CUTANEOUS 4 CELL LYMPHOMA )N !PRIL !STELLAS 0HARMA )NC ENTERED INTO AN EXCLUSIVE LICENSING AGREEMENT WITH
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
'LOUCESTER 0HARMACEUTICALS FOR THE WORLDWIDE DEVELOPMENT PRODUCTION AND SALE OF &+ &+ HAS A UNIQUE STRUCTURE AND ACTIVITIES THAT ARE IMPOSSIBLE TO BE DIS COVERED THROUGH THE IMAGINATION OF A SYNTHETIC CHEMIST 4HE CURRENT MAIN APPROACH TO DRUG DISCOVERY IS @TARGET DIRECTED BUT AT THE TIME OF DISCOVERY OF &+ THE EXISTENCE OF ($!# INHIBITORS WAS STILL UNKNOWN 4HUS IT WAS NOT POSSIBLE TO USE ($!# AS A TARGET AND TARGET DIRECTED DISCOVERY OF &+ WAS IMPOSSIBLE 4HE DISCOVERY OF &+ AS A UNIQUE NATURALLY OCCURRING ANTITUMOR AGENT WAS CLEARLY A PRODUCT OF THE TRADITIONAL RANDOM SCREENING TECHNIQUE OF NATURAL PRODUCT EXTRACTS
$ISCOVERY OF 9- 9- SPIRUCHOSTATIN ! A BICYCLIC DEPSIPEPTIDE WITH A MOLECULAR WEIGHT OF WAS DISCOVERED IN THE NATURAL PRODUCT LIBRARY OF 9AMANOUCHI 0HAR MACEUTICAL #O ,TD CURRENTLY !STELLAS 0HARMA )NC AS A SUBSTANCE SHOWING TRANSFORMING GROWTH FACTOR BETA LIKE ACTIVITY ;= "RIEFLY SCREENING OF MORE THAN MICROBIAL STRAINS OBTAINED FROM VARIOUS LOCATIONS IN *APAN REVEALED TWO NOVEL COMPOUNDS IN THE FERMENTATION BROTH OF STRAIN 1 THAT INCREASED THE TRANSCRIPTIONAL ACTIVITY OF PLASMINOGEN ACTI VATOR INHIBITOR 7E NAMED THEM SPIRUCHOSTATIN ! AND " ;= 1 STRAIN IS A BACTERIUM ISOLATED FROM A SOIL SAMPLE COLLECTED IN .AGANO 0REFECTURE *APAN )T IS A 'RAM NEGATIVE MOTILE BACTERIUM WITH A SINGLE POLAR FLAGELLUM "ASED ON ITS BACTERIOLOGICAL PROPERTIES WE IDENTIFIED THIS BACTERIUM AS 0SEUDOMONAS SP 1 4HE STRUCTURE OF 9- WAS DETER MINED BY A COMBINATION OF SPECTROSCOPIC AND CHEMICAL ANALYSES ;= )N THE STRUCTURE WAS CONFIRMED BY TOTAL CHEMICAL SYNTHESIS BY 9UREK 'EORGE ! ET AL ;= 4HEY ALSO SYNTHESIZED THE 9- EPIMER AND SHOWED THAT IT HAD NO CELL GROWTH INHIBITORY ACTIVITY 3O IT BECAME APPARENT THAT THE 3 STEREOCHEMISTRY IN THE β HYDROXY ACID IN THE 9- EPIMER WAS IMPORTANT FOR ($!# INHIBITION 4HEY ALSO REVEALED THAT 9- INDUCED ACCUMULATION OF ACETYLATED HISTONE ( AND ACTIVATED THE P PROMOTER IN INTACT BREAST CANCER CELLS 4HIS SUGGESTS THAT THE TARGET MOLECULES OF 9- ARE ($!#S $ETAILED INVESTIGATIONS ON OTHER BIOLOGICAL ACTIVITIES OF 9- ARE NOW UNDERWAY
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
#ONCLUSION ($!# IS A NOVEL AND PROMISING MOLECULAR TARGET FOR HEMATOLOGICAL AND SOLID TUMOR THERAPEUTICS 7E HAVE DISCOVERED &+ AND 9- POTENT ($!# INHIBITORS AMONG MICROBIAL METABOLITES 4HEY HAVE UNUSUAL BICY CLIC STRUCTURES AND REPRESENT ANOTHER NOVEL STRUCTURAL CLASS OF ($!# INHIBI TORS &+ IS THE FIRST POTENT ($!# INHIBITOR TO UNDERGO CLINICAL DEVELOP MENT AS A POTENTIAL TREATMENT FOR SOLID AND HEMATOLOGICAL CANCERS $UE TO ITS DRAMATIC EFFECT IN PATIENTS WITH REFRACTORY #4#, IN /CTOBER THE 53 &$! GRANTED FAST TRACK STATUS TO &+ AS MONOTHERAPY FOR THE TREATMENT OF #4#, IN PATIENTS WHO HAVE RELAPSED FOLLOWING OR BECOME REFRACTORY TO ANOTHER SYSTEMIC THERAPY &+ HAS ALSO RECEIVED /RPHAN $RUG DESIGNATION BY BOTH THE &$! AND THE %UROPEAN !GENCY FOR THE %VALU ATION OF -EDICINAL 0RODUCTS %-%! AS MONOTHERAPY FOR THE TREATMENT OF #4#, ($!# INHIBITORS ISOLATED FROM MICROORGANISMS SUCH AS TRICHOSTATIN ! TRAPOXIN AND &+ ARE CONTRIBUTING GREATLY TO THE IDENTIFICATION OF ($!# ENZYMES AND THE DEVELOPMENT OF BASIC SCIENCE IN CHROMATIN RESEARCH -OREOVER &+ ALSO HAS THE POTENTIAL TO BE A HIGHLY POTENT THERAPEUTIC DRUG IN CANCER PATIENTS )T IS NOTEWORTHY THAT THEIR STRUCTURES AND PRO DUCING ORGANISMS ARE QUITE DIFFERENT INDICATING THE DIVERSITY OF MICRO BIAL BIOSYNTHETIC ABILITY IN PROVIDING MULTIPLE TEMPLATES THAT CAN BIND TO MOLECULAR TARGETS AND INHIBIT ENZYME ACTIVITY WITH GREAT POTENCY AND SELECTIVITY -ICROORGANISMS ARE SMALL BUT HIGHLY SKILLED @CHEMISTS AND HAVE TREMENDOUS POTENTIAL FOR MAKING EXCELLENT DRUGS THAT ARE COMPARABLE OR EVEN SUPERIOR TO SYNTHETIC CHEMICAL COMPOUNDS )T IS WELL KNOWN THAT NATURAL PRODUCTS ARE AN UNPARALLELED SOURCE OF MOLECULAR DIVERSITY FOR DRUG DISCOVERY %SPECIALLY THE DISCOVERY OF ANTITU MOR AGENTS HAS BEEN VERY DEPENDENT UPON SCREENING OF NATURAL PRODUCTS $ISCOVERIES OF THESE PHARMACOLOGICAL AGENTS HAVE BEEN LARGELY SUPPORTED BY *APANESE MICROBIOLOGY WHICH HAS MADE GREAT PROGRESS IN ASSOCIATION WITH THE UNIQUE DIETARY CULTURE OF *APAN )T IS OUR FIRM INTENTION TO CON TINUE TO CONTRIBUTE TO THE FUTURE DEVELOPMENT OF NEW PHARMACEUTICAL AGENTS BY APPLICATION OF OUR WORLD CLASS MICROBIOLOGICAL KNOWLEDGE AND TECHNOLOGIES WHICH HAVE BEEN ESTABLISHED BY OUR PREDECESSORS
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
2EFERENCES
"UTLER -3 .ATURAL PRODUCTS TO DRUGS NATURAL PRODUCT DERIVED COMPOUNDS IN CLINICAL TRIALS .AT 0ROD 2EP n .EWMAN $* #RAGG '- 3NADER +- .ATURAL PRODUCTS AS SOURCES OF NEW DRUGS OVER THE PERIOD n * .AT 0ROD n 0ETERSON #, ,ANIEL -! (ISTONES AND HISTONE MODIFICATIONS #URR "IOL 2n (EBBES 42 4HORNE !7 #RANE 2OBINSON # ! DIRECT LINK BETWEEN CORE HISTONE ACETYLATION AND TRANSCRIPTIONALLY ACTIVE CHROMATIN %-"/ * n (ESS 3TUMPP ( (ISTONE DEACETYLASE INHIBITORS AND CANCER FROM CELL BIOLOGY TO THE CLINIC %UR * #ELL "IOL n -ONNERET # (ISTONE DEACETYLASE INHIBITORS %UR * -ED #HEM n -INUCCI 3 0ELICCI 0' (ISTONE DEACETYLASE INHIBITORS AND THE PROMISE OF EPIGE NETIC AND MORE TREATMENTS FOR CANCER .AT 2EV #ANCER n 6OELTER -AHLKNECHT 3 (O !$ -AHLKNECHT 5 #HROMOSOMAL ORGANIZATION AND LOCALIZATION OF THE NOVEL CLASS )6 HUMAN HISTONE DEACETYLASE GENE )NT * -OL -ED n 9ASUI 7 /UE . /NO 3 -ITANI 9 )TO 2 .AKAYAMA ( (ISTONE ACETYLATION AND GASTROINTESTINAL CARCINOGENESIS !NN .9 !CAD 3CI n &RAGA -& "ALLESTAR % 6ILLAR 'AREA ! "OIX #HORNET - %SPADA * 3CHOTTA ' "ONALDI 4 (AYDON # 2OPERO 3 0ETRIE + ET AL ,OSS OF ACETYLATION AT ,YS AND TRIMETHYLATION AT ,YS OF HISTONE ( IS A COMMON HALLMARK OF HUMAN CANCER .AT 'ENET n (UANG "( ,ABAN - ,EUNG #( ,EE , ,EE #+ 3ALTO 4ELLEZ - 2AJU '# (OOI 3# )NHIBITION OF HISTONE DEACETYLASE INCREASES APOPTOSIS AND P#IP7!& EXPRESSION INDEPENDENT OF HISTONE DEACETYLASE #ELL $EATH $IFFER n :HU 0 -ARTIN % -ENGWASSER * 3CHLAG 0 *ANSSEN +0 'OTTLICHER - )NDUCTION OF ($!# EXPRESSION UPON LOSS OF !0# IN COLORECTAL TUMORIGENESIS #ANCER #ELL n 7ILSON !* "YUN $3 0OPOVA . -URRAY ," ,)TALIEN + 3OWA 9 !RANGO $ 6ELCICH ! !UGENLICHT ,( -ARIADASON *- (ISTONE DEACETYLASE ($!# AND OTHER CLASS ) ($!#S REGULATE COLON CELL MATURATION AND P EXPRESSION AND ARE DEREGULATED IN HUMAN COLON CANCER * "IOL #HEM n *OHNSTONE 27 (ISTONE DEACETYLASE INHIBITORS NOVEL DRUGS FOR THE TREATMENT OF CANCER .AT 2EV $RUG $ISCOV n 1IU , +ELSO -* (ANSEN # 7EST -, &AIRLIE $0 0ARSONS 0' !NTI TUMOUR ACTIVITY IN VITRO AND IN VIVO OF SELECTIVE DIFFERENTIATING AGENTS CONTAINING HYDROXAMATE "R * #ANCER n -ARKS 0! 2ICHON 6- -ILLER 4 +ELLY 7+ (ISTONE DEACETYLASE INHIBITORS !DV #ANCER 2ES n !TADJA 0 'AO , +WON 0 4ROGANI . 7ALKER ( (SU - 9ELESWARAPU , #HANDRAMOULI . 0EREZ , 6ERSACE 2 3ELECTIVE GROWTH INHIBITION OF TUMOR CELLS BY A NOVEL HISTONE DEACETYLASE INHIBITOR .60 ,!1 #ANCER 2ES n +IM -3 +WON (* ,EE 9- "AEK *( *ANG *% ,EE 37 -OON %* +IM (3 ,EE 3+ #HUNG (9 ET AL (ISTONE DEACETYLASES INDUCE ANGIOGENESIS BY NEGATIVE REGULATION OF TUMOR SUPPRESSOR GENES .AT -ED n
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
+WON (* +IM -3 +IM -* .AKAJIMA ( +IM +7 (ISTONE DEACETYLASE INHIBITOR &+ INHIBITS TUMOR ANGIOGENESIS )NT * #ANCER n .AKANO + -IZUNO 4 3OWA 9 /RITA 4 9OSHINO 4 /KUYAMA 9 &UJITA 4 /HTANI &UJITA . -ATSUKAWA 9 4OKINO 4 ET AL "UTYRATE ACTIVATES THE 7!&#IP GENE PROMOTER THROUGH 3P SITES IN A P NEGATIVE HUMAN COLON CANCER CELL LINE * "IOL #HEM n 3OWA 9 /RITA 4 -INAMIKAWA 3 .AKANO + -IZUNO 4 .OMURA ( 3AKAI 4 (ISTONE DEACETYLASE INHIBITOR ACTIVATES THE 7!&#IP GENE PROMOTER THROUGH THE 3P SITES "IOCHEM "IOPHYS 2ES #OMMUN n !RCHER 39 -ENG 3 3HEI ! (ODIN 2! P 7!& IS REQUIRED FOR BUTYRATE MEDIATED GROWTH INHIBITION OF HUMAN COLON CANCER CELLS 0ROC .ATL !CAD 3CI 53! n 3ATO . /HTA 4 +ITAGAWA ( +AYAHARA - .INOMIYA ) &USHIDA 3 &UJIMURA 4 .ISHIMURA ' 3HIMIZU + -IWA + &2 A NOVEL HISTONE DEACETYLASE INHIBITOR INDUCES CELL CYCLE ARREST AND SUBSEQUENT APOPTOSIS IN REFRACTORY HUMAN PANCREATIC CANCER CELLS )NT * /NCOL n 'UO & 3IGUA # 4AO * "ALI 0 'EORGE 0 ,I 9 7ITTMANN 3 -OSCINSKI , !TADJA 0 "HALLA + #OTREATMENT WITH HISTONE DEACETYLASE INHIBITOR ,!1 ENHANCES !PO , TUMOR NECROSIS FACTOR RELATED APOPTOSIS INDUCING LIGAND INDUCED DEATH INDUCING SIG NALING COMPLEX ACTIVITY AND APOPTOSIS OF HUMAN ACUTE LEUKEMIA CELLS #ANCER 2ES n 3HAO 9 'AO : -ARKS 0! *IANG 8 !POPTOTIC AND AUTOPHAGIC CELL DEATH INDUCED BY HISTONE DEACETYLASE INHIBITORS 0ROC .ATL !CAD 3CI 53! n 5NGERSTEDT *3 3OWA 9 8U 73 3HAO 9 $OKMANOVIC - 0EREZ ' .GO , (OLMGREN ! *IANG 8 -ARKS 0! 2OLE OF THIOREDOXIN IN THE RESPONSE OF NORMAL AND TRANSFORMED CELLS TO HISTONE DEACETYLASE INHIBITORS 0ROC .ATL !CAD 3CI 53! n /#ONNOR /! (EANEY -, 3CHWARTZ , 2ICHARDSON 3 7ILLIM 2 -AC'REGOR #ORTELLI " #URLY 4 -OSKOWITZ # 0ORTLOCK # (ORWITZ 3 ET AL #LINICAL EXPERIENCE WITH INTRA VENOUS AND ORAL FORMULATIONS OF THE NOVEL HISTONE DEACETYLASE INHIBITOR SUBEROYLANILIDE HYDROXAMIC ACID IN PATIENTS WITH ADVANCED HEMATOLOGIC MALIGNANCIES * #LIN /NCOL n +ELLY 7+ /#ONNOR /! +RUG ,- #HIAO *( (EANEY - #URLEY 4 -AC'REGORE #ORTELLI " 4ONG 7 3ECRIST *0 3CHWARTZ , ET AL 0HASE ) STUDY OF AN ORAL HISTONE DEACETYLASE INHIBITOR SUBEROYLANILIDE HYDROXAMIC ACID IN PATIENTS WITH ADVANCED CANCER * #LIN /NCOL n +ELLY 7+ 2ICHON 6- /#ONNOR / #URLEY 4 -AC'REGOR #URTELLI " 4ONG 7 +LANG - 3CHWARTZ , 2ICHARDSON 3 2OSA % ET AL 0HASE ) CLINICAL TRIAL OF HISTONE DEACETYLASE INHIBITOR SUBEROYLANILIDE HYDROXAMIC ACID ADMINISTERED INTRAVENOUSLY #LIN #ANCER 2ES n 0IEKARZ 2, 2OBEY 2 3ANDOR 6 "AKKE 3 7ILSON 7( $AHMOUSH , +INGMA $- 4URNER -, !LTEMUS 2 "ATES 3% )NHIBITOR OF HISTONE DEACETYLATION DEPSIPEPTIDE &2 IN THE TREATMENT OF PERIPHERAL AND CUTANEOUS 4 CELL LYMPHOMA A CASE REPORT "LOOD n 0IEKARZ 2, "ATES 3 ! REVIEW OF DEPSIPEPTIDE AND OTHER HISTONE DEACETYLASE INHIBI TORS IN CLINICAL TRIALS #URR 0HARM $ES n -ARKS 0 2IFKIND 2! 2ICHON 6- "RESLOW 2 -ILLER 4 +ELLY 7+ (ISTONE DEACETY LASES AND CANCER CAUSES AND THERAPIES .AT 2EV #ANCER n
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
4SUJI . +OBAYASHI - .AGASHIMA + 7AKISAKA 9 +OIZUMI + ! NEW ANTIFUNGAL ANTIBIOTIC TRICHOSTATIN * !NTIBIOT n 3AITO ! 9AMASHITA 4 -ARIKO 9 .OSAKA 9 4SUCHIYA + !NDO 4 3UZUKI 4 4SURUO 4 .AKANI SHI / ! SYNTHETIC INHIBITOR OF HISTONE DEACETYLASE -3 WITH MARKED IN VIVO ANTITUMOR ACTIVITY AGAINST HUMAN TUMORS 0ROC .ATL !CAD 3CI 53! n +RAKER !* -IZZEN #! (ARTL "' -IIN * !LLIS #$ -ERRIMAN 2, -ODULATION OF HISTONE ACETYLATION BY ; ACETYLAMINO . AMINO PHENYL BENZAMIDE IN (#4 COLON CARCINOMA -OL #ANCER 4HER n 4AUNTON * (ASSIG #! 3CHREIBER 3, ! MAMMALIAN HISTONE DEACETYLASE RELATED TO THE YEAST TRANSCRIPTIONAL REGULATOR 2PDP 3CIENCE n .AKAJIMA ( +IM 9" 4ERANO ( 9OSHIDA - (ORINOUCHI 3 &2 A POTENT ANTITUMOR ANTIBIOTIC IS A NOVEL HISTONE DEACETYLASE INHIBITOR %XP #ELL 2ES n 5EDA ( .AKAJIMA ( (ORI 9 &UJITA 4 .ISHIMURA - 'OTO 4 /KUHARA - &2 ! NOVEL ANTITUMOR BICYCLIC DEPSIPEPTIDE PRODUCED BY #HROMOBACTERIUM VIOLACEUM .O ) 4AXONOMY FERMENTATION ISOLATION PHYSICO CHEMICAL AND BIOLOGICAL PROPERTIES AND ANTITUMOR ACTIVITY * !NTIBIOT n 4ANIGUCHI - .AGAI + !RAO . +AWASAKI 4 3AITO 4 -ORITANI 9 4AKASAKI * (AYASHI + &UJITA 3 3UZUKI + ET AL 9- A NOVEL PLATELET AGGREGATION INHIBITOR PRO DUCED BY #HROMOBACTERIUM SP 13 * !NTIBIOT n 3HIGEMATSU . 5EDA ( 4AKASE 3 4ANAKA ( 9AMAMOTO + 4ADA 4 &2 A NOVEL ANTITUMOR BICYCLIC DEPSIPEPTIDE PRODUCED BY #HROMOBACTERIUM VIOLACEUM .O )) 3TRUCTURE ELUCIDATION * !NTIBIOT n ,I +7 7U * 8ING 7 3IMON *! 4OTAL SYNTHESIS OF THE ANTITUMOR DEPSIPEPTIDE &2 * !M #HEM 3OC n 5EDA ( .AKAJIMA ( (ORI 9 'OTO 4 /KUHARA - !CTION OF &2 A NOVEL ANTITUMOR BICYCLIC DEPSIPEPTIDE PRODUCED BY #HROMOBACTERIUM VIOLACEUM NO ON (A RAS TRANSFORMED .)(4 CELLS "IOSCI "IOTECH "IOCHEM n 5EDA ( -ANDA 4 -ATSUMOTO 3 -UKUMOTO 3 .ISHIGAKI & +AWAMURA ) 3HIMOMURA + &2 A NOVEL ANTITUMOR BICYCLIC DEPSIPEPTIDE PRODUCED BY #HROMOBACTERIUM VIOLACEUM .O ))) !NTITUMOR ACTIVITIES ON EXPERIMENTAL TUMORS IN MICE * !NTIBIOT n 9OSHIDA - .OMURA 3 "EPPU 4 %FFECTS OF TRICHOSTATINS ON DIFFERENTIATION OF MURINE ERYTHROLEUKEMIA CELLS #ANCER 2ES n 9OSHIDA - +IJIMA - !KITA - "EPPU 4 0OTENT AND SPECIFIC INHIBITION OF MAM MALIAN HISTONE DEACETYLASE BOTH IN VIVO AND IN VITRO BY TRICHOSTATIN ! * "IOL #HEM n 9OSHIDA - "EPPU 4 2EVERSIBLE ARREST OF PROLIFERATION OF RAT 9 FIBROBLASTS IN BOTH THE ' AND ' PHASES BY TRICHOSTATIN ! %XP #ELL 2ES n 9OSHIDA - (OSHIKAWA 9 +OSEKI + -ORI + "EPPU 4 3TRUCTURAL SPECIFICITY FOR BIO LOGICAL ACTIVITY OF TRICHOSTATIN ! A SPECIFIC INHIBITOR OF MAMMALIAN CELL CYCLE WITH POTENT DIFFERENTIATION INDUCING ACTIVITY IN &RIEND LEUKEMIA CELLS * !NTIBIOT n (OSHIKAWA 9 +IJIMA - 9OSHIDA - "EPPU 4 %XPRESSION OF DIFFERENTIATION RELATED MARKERS IN TERATOCARCINOMA CELLS VIA HISTONE HYPERACETYLATION BY TRICHOSTATIN ! !GRIC "IOL #HEM n 3UGITA + +OIZUMI + 9OSHIDA ( -ORPHOLOGICAL REVERSION OF SIS TRANSFORMED .)(4 CELLS BY TRICHOSTATIN ! #ANCER 2ES n
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
!LMOUZNI ' +HOCHBIN 3 $IMITROV 3 7OLFFE !0 (ISTONE ACETYLATION INFLUENCES BOTH GENE EXPRESSION AND DEVELOPMENT OF 8ENOPUS LAEVIS $EV "IOL n (OSHIKAWA 9 +WON (* 9OSHIDA - (ORINOUCHI 3 "EPPU 4 4RICHOSTATIN ! INDUCES MORPHOLOGICAL CHANGES AND GELSOLIN EXPRESSION BY INHIBITING HISTONE DEACETYLASE IN HUMAN CARCINOMA CELL LINES %XP #ELL 2ES n 'IRARDOT 6 2ABILLOUD 4 9OSHIDA - "EPPU 4 ,AWRENCE ** +HOCHBIN 3 2ELATION SHIP BETWEEN CORE HISTONE ACETYLATION AND HISTONE ( GENE ACTIVITY %UR * "IOCHEM n 3CHLAKE 4 +LEHR 7IRTH $ 9OSHIDA - "EPPU 4 "ODE * 'ENE EXPRESSION WITHIN A CHROMATIN DOMAIN THE ROLE OF CORE HISTONE HYPERACETYLATION "IOCHEMISTRY n &UTAMURA - -ONDEN 9 /KABE 4 &UJITA 9OSHIGAKI * 9OKOYAMA 3 .ISHIMURA 3 4RICHOSTATIN ! INHIBITS BOTH RAS INDUCED NEURITE OUTGROWTH OF 0# CELLS AND MORPHO LOGICAL TRANSFORMATION OF .)(4 CELLS /NCOGENE n 7ORRAD $- 4URNER "- 3CHULTZ 2- 4EMPORALLY RESTRICTED SPATIAL LOCALIZATION OF ACETYLATED ISOFORMS OF HISTONE ( AND 2.! POLYMERASE )) IN THE CELL MOUSE EMBRYO $EVELOPMENT n 6AN ,INT # %MILIANI 3 /TT - 6ERDIN % 4RANSCRIPTIONAL ACTIVATION AND CHROMA TIN REMODELING OF THE ()6 PROMOTER IN RESPONSE TO HISTONE ACETYLATION %-"/ * n 2UNDLETT 3% #ARMEN !! +OBAYASHI 2 "AVYKIN 3 4URNER "- 'RUNSTEIN - ($! AND 20$ ARE MEMBERS OF DISTINCT YEAST HISTONE DEACETYLASE COMPLEXES THAT REGULATE SILENCING AND TRANSCRIPTION 0ROC .ATL !CAD 3CI 53! n )MAI 3 !RMSTRONG #- +AEBERLEIN - 'UARENTE , 4RANSCRIPTIONAL SILENCING AND LONGEVITY PROTEIN 3IR IS AN .!$ DEPENDENT HISTONE DEACETYLASE .ATURE n 2ODRIQUEZA - !QUINO - "RUNO ) $E -ARTINO ' 4ADDEI - 'OMEZ 0ALOMA , #HEMISTRY AND BIOLOGY OF CHROMATIN REMODELING AGENTS 3TATE OF ART AND FUTURE PERSPEC TIVES OF ($!# INHIBITORS #URR -ED #HEM n &URUMAI 2 -ATSUYAMA ! +OBASHI . ,EE +( .ISHIYAMA - .AKAJIMA ( 4ANAKA ! +OMATSU 9 .ISHINO . 9OSHIDA - ET AL &+ DEPSIPEPTIDE AS A NATURAL PRODRUG THAT INHIBITS CLASS ) HISTONE DEACETYLASES #ANCER 2ES n &INNIN -3 $ONIGIAN *2 #OHEN ! 2ICHON 6- 2IFKIND 2! -ARKS 0! "RESLOW 2 0AV LETICH .0 3TRUCTURES OF A HISTONE DEACETYLASE HOMOLOGUE BOUND TO THE 43! AND 3!(! INHIBITORS .ATURE n 8IAO ** "YRD * -ARCUCCI ' 'REVER - #HAN ++ )DENTIFICATION OF THIOLS AND GLUTATHIONE CONJUGATES OF DEPSIPEPTIDE &+ &2 A NOVEL HISTONE PROTEIN DEACETYLASE INHIBITOR IN THE BLOOD 2APID #OMMUN -ASS 3PECTROM n 6AN ,INT # %MILIANI 3 6ERDIN % 4HE EXPRESSION OF A SMALL FRACTION OF CELLULAR GENES IS CHANGED IN RESPONSE TO HISTONE HYPERACETYLATION 'ENE %XPR n 1IU , "URGESS ! &AIRLIE $0 ,EONARD ( 0ARSONS 0' 'ABRIELLI "' (ISTONE DEACETY LASE INHIBITORS TRIGGER A ' CHECKPOINT IN NORMAL CELLS THAT IS DEFECTIVE IN TUMOR CELLS -OL "IOL #ELL n "ERNHARD $ !USSERLECHNER -* 4ONKO - ,OFFLER - (ARTMANN ", #SORDAS ! +OFLER 2 !POPTOSIS INDUCED BY THE HISTONE DEACETYLASE INHIBITOR SODIUM BUTYRATE IN HUMAN LEUKEMIC LYMPHOBLASTS &!3%" * n 7ARRELL 20 *R (E ,: 2ICHON 6 #ALLEJA % 0ANDOLFI 00 4HERAPEUTIC TARGETING OF
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
TRANSCRIPTION IN ACUTE PROMYELOCYTIC LEUKEMIA BY USE OF AN INHIBITOR OF HISTONE DEACETY LASE * .ATL #ANCER )NST n -AEDA 4 4OWATARI - +OSUGI ( 3AITO ( 5P REGULATION OF COSTIMULATORYADHESION MOLECULES BY HISTONE DEACETYLASE INHIBITORS IN ACUTE MYELOID LEUKEMIA CELLS "LOOD n 3ANDOR 6 3ENDEROWICZ ! -ERTINS 3 3ACKETT $ 3AUSVILLE % "LAGOSKLONNY -6 "ATES 3% 0 DEPENDENT G ARREST WITH DOWNREGULATION OF CYCLIN $ AND UPREGULATION OF CYCLIN % BY THE HISTONE DEACETYLASE INHIBITOR &2 "RITISH * #ANCER n +WON 3( !HN 3( +IM 9+ "AE '5 9OON *7 (ONG 3 ,EE (9 ,EE 97 ,EE (7 (AN *7 !PICIDIN A HISTONE DEACETYLASE INHIBITOR INDUCES APOPTOSIS AND &AS&AS LIGAND EXPRESSION IN HUMAN ACUTE PROMYELOCYTIC LEUKEMIA CELLS * "IOL #HEM n (ENDERSON # -IZZAU - 0ARONI ' -AESTRO 2 3CHNEIDER # "RANCOLINI # 2OLE OF CASPASES "ID AND P IN THE APOPTOTIC RESPONSE TRIGGERED BY HISTONE DEACETYLASE INHIBI TORS TRICHOSTATIN ! 43! AND SUBEROYLANILIDE HYDROXAMIC ACID 3!(! * "IOL #HEM n !MIN (- 3AEED 3 !LKAN 3 (ISTONE DEACETYLASE INHIBITORS INDUCE CASPASE DEPENDENT APOPTOSIS AND DOWNREGULATION OF DAXX IN ACUTE PROMYELOCYTIC LEUKAEMIA WITH T "RITISH * (AEMATOLOGY n 3UTHEESOPHON + .ISHIMURA . +OBAYASHI 9 &URUKAWA 9 +AWANO - )TOH + +ANO 9 )SHII ( &URUKAWA 9 )NVOLVEMENT OF THE TUMOR NECROSIS FACTOR 4.& 4.& RECEPTOR SYSTEM IN LEUKEMIC CELL APOPTOSIS INDUCED BY HISTONE DEACETYLASE INHIBITOR DEPSIPEPTIDE &+ * #ELLULAR 0HYSIOLOGY n 2AJGOLIKAR ' #HAN ++ 7ANG (# %FFECTS OF A NOVEL ANTITUMOR DEPSIPEPTIDE &2 ON HUMAN BREAST CANCER CELLS "REAST #ANCER 2ES 4REATMENT n 3ASAKAWA 9 .AOE 9 .OTO 4 )NOUE 4 3ASAKAWA 4 -ATSUO - -ANDA 4 -UTOH 3 !NTITUMOR EFFICACY OF &+ A NOVEL HISTONE DEACETYLASE INHIBITOR DEPENDS ON THE EFFECT ON EXPRESSION OF ANGIOGENESIS FACTORS "IOCHEM 0HARMACOLOGY n -IE ,EE 9 +IM 3( +IM (3 *IN 3ON - .AKAJIMA ( *EONG +WON ( +IM +7 )NHI BITION OF HYPOXIA INDUCED ANGIOGENESIS BY &+ A SPECIFIC HISTONE DEACETYLASE INHIBI TOR VIA SUPPRESSION OF ()& α ACTIVITY "IOCHEM "IOPHYS 2ES #OMMUN n -ORROW #3 .AKAGAWA - 'OLDSMITH -% -ADDEN -* #OWAN +( 2EVERSIBLE TRANSCRIPTIONAL ACTIVATION OF MDR BY SODIUM BUTYRATE TREATMENT OF HUMAN COLON CANCER CELLS * "IOL #HEM n 'OTTESMAN -- &OJO 4 "ATES 3% -ULTIDRUG RESISTANCE IN CANCER ROLE OF !40 DEPENDENT TRANSPORTERS .AT 2EV #ANCER n *IN 3 3COTTO +7 4RANSCRIPTIONAL REGULATION OF THE -$2 GENE BY HISTONE ACETYL TRANSFERASE AND DEACETYLASE IS MEDIATED BY .& 9 -OL #ELL "IOL n ,EE *3 0AULL + !LVAREZ - (OSE # -ONKS ! 'REVER - &OJO !4 "ATES 3% 2HOD AMINE EFFLUX PATTERNS PREDICT 0 GLYCOPROTEIN SUBSTRATES IN THE .ATIONAL #ANCER )NSTITUTE DRUG SCREEN -OL 0HARMACOL n 3CALA 3 !KHMED . 2AO 53 0AULL + ,AN ," $ICKSTEIN " ,EE *3 %LGEMEIE '( 3TEIN 7$ "ATES 3% 0 GLYCOPROTEIN SUBSTRATES AND ANTAGONISTS CLUSTER INTO TWO DISTINCT GROUPS -OL 0HARMACOL n 8IAO ** &ORAKER !" 3WAAN 07 ,IU 3 (UANG 9 $AI : #HEN * 3ADEE 7 "YRD * -ARCUCCI ' ET AL %FFLUX OF DEPSIPEPTIDE &+ &2 .3# IS MEDIATED BY 0 GLYCOPROTEIN AND MULTIDRUG RESISTANCE ASSOCIATED PROTEIN * 0HARMACOL %XP 4HER n
9UHTA -ASUOKA .OBUAKI 3HINDOH AND .ORIAKI )NAMURA
8IAO ** (UANG 9 $AI : 3ADEE 7 #HEN * ,IU 3 -ARCUCCI ' "YRD * #OVEY *- 7RIGHT * ET AL #HEMORESISTANCE TO DEPSIPEPTIDE &+ ;% 3 3 3 2 ;: ETHYLI DENE= DIISOPROPYL OXA DITHIA TETRAAZABICYCLO; = TRICOS ENE PENTANONE= IS MEDIATED BY REVERSIBLE -$2 INDUCTION IN HUMAN CANCER CELL LINES * 0HARMACOL %XP 4HER n 2OBEY 27 :HAN : 0IEKARZ 2, +AYASTHA ', &OJO 4 "ATES 3% )NCREASED -$2 EXPRESSION IN NORMAL AND MALIGNANT PERIPHERAL BLOOD MONONUCLEAR CELLS OBTAINED FROM PATIENTS RECEIVING DEPSIPEPTIDE &2 &+ .3# #LIN #ANCER 2ES n +ITAZONO - 2OBEY 2 :HAN : 3ARLIS .* 3KARULIS -# !IKOU 4 "ATES 3 &OJO 4 ,OW CONCENTRATIONS OF THE HISTONE DEACETYLASE INHIBITOR DEPSIPEPTIDE &2 INCREASE EXPRESSION OF THE .A )n SYMPORTER AND IODINE ACCUMULATION IN POORLY DIFFERENTIATED THYROID CARCINOMA CELLS * #LIN %NDOCRINOLOGY -ETAB n !DACHI - :HANG 9 :HAO 8 -INAMI 4 +AWAMURA 2 (INODA 9 )MAI + 3YN ERGISTIC EFFECT OF HISTONE DEACETYLASE INHIBITORS &+ AND M CARBOXYCINNAMIC ACID BIS HYDROXAMIDE WITH PROTEASOME INHIBITORS 03) AND 03 AGAINST GASTROINTESTINAL ADENOCARCINOMA CELLS #LIN #ANCER 2ES n +AWANO 4 (ORIGUCHI 9AMADA * )WASE 3 !KIYAMA - &URUKAWA 9 +AN 9 9AMADA ( $EPSIPEPTIDE ENHANCES IMATINIB MESYLATE INDUCED APOPTOSIS OF "CR !BL POSITIVE CELLS AND ECTOPIC EXPRESSION OF CYCLIN $ C -YC OR ACTIVE -%+ ABROGATES THIS EFFECT !NTICANCER 2ES n 4AURA + 9AMAMOTO 9 .AKAJIMA ! (ATA + 5CHINAMI ( 9ONEZAWA + (ATANO % .ISHINO . 9AMAOKA 9 )MPACT OF NOVEL HISTONE DEACETYLASE INHIBITORS #(!0 AND &2 &+ ON ADENOVIRUS MEDIATED TRANSGENE EXPRESSION * 'ENE -ED n +ITAZONO - #HUMAN 9 !IKOU 4 &OJO 4 #ONSTRUCTION OF GENE THERAPY VECTORS TARGETING THYROID CELLS ENHANCEMENT OF ACTIVITY AND SPECIFICITY WITH HISTONE DEACETYLASE INHIBITORS AND AGENTS MODULATING THE CYCLIC ADENOSINE MONOPHOSPHATE PATHWAY AND DEMONSTRATION OF ACTIVITY IN FOLLICULAR AND ANAPLASTIC THYROID CARCINOMA CELLS * #LIN %NDOCRINOLOGY -ETABOLISM n - +ITAZONO - 2AO 6+ 2OBEY 2 !IKOU 4 "ATES 3 &OJO 4 'OLDSMITH -% (ISTONE DEACETYLASE INHIBITOR &2 ENHANCES ADENOVIRUS INFECTION OF HEMATOPOIETIC CELLS "LOOD n 7HITTAKER 3 2OBAK 4 "ARAN % -C#ULLOCH 7 0RENTICE !' )NTERNATIONAL MULTI CENTER PHASE )) STUDY OF THE ($!# INHIBITOR DEPSIPEPTIDE &+ IN CUTANEOUS 4 CELL LYMPHOMA #4#, INTERIM REPORT !3#/ !NNUAL -EETING ABSTRACT -OLIFE 2 0ATTERSON 3 2IGGS # (IGANO # 3TADLER 7- $EARNALEY $ 0ARKER # -C#ULLOCH 7 3HALAUROV ! DE "ONO * ! PHASE )) STUDY EVALUATING THE ACTIVITY AND TOLERABILITY OF DEPSIPEPTIDE &+ IN PATIENTS WITH HORMONE REFRACTORY PROSTATE CANCER !3#/ 0ROSTATE #ANCER 3YMPOSIUM ABSTRACT 3ANDOR 6 "AKKE 3 2OBEY 27 +ANG -( "LAGOSKLONNY -6 "ENDER * "ROOKS 2 0IEKARZ 2, 4UCKER % &IGG 7$ ET AL 0HASE ) TRIAL OF THE HISTONE DEACETYLASE INHIBITOR DEP SIPEPTIDE &2 .3# IN PATIENTS WITH REFRACTORY NEOPLASMS #LIN #ANCER 2ES n -ARSHALL *, 2IZVI . +AUH * $AHUT 7 &IGUERA - +ANG -( &IGG 7$ 7AINER ) #HAIS SANG # ,I -: ET AL ! PHASE ) TRIAL OF DEPSIPEPTIDE &2 IN PATIENTS WITH ADVANCED CANCER * %XP 4HERAPEUTICS AND /NCOLOGY n
(ISTONE DEACETYLASE INHIBITORS FROM MICROORGANISMS THE !STELLAS EXPERIENCE
0IEKARZ 2, &RYE !2 7RIGHT ** 3TEINBERG 3- ,IEWEHR $* 2OSING $2 3ACHDEV 6 &OJO 4 "ATES 3% #ARDIAC STUDIES IN PATIENTS TREATED WITH DEPSIPEPTIDE &+ IN A PHASE )) TRIAL FOR 4 CELL LYMPHOMA #LIN #ANCER 2ES n -ASUOKA 9 .AGAI ! 3HIN 9A + &URIHATA + .AGAI + 3UZUKI + ) (AYAKAWA 9 3ETO ( 3PIRUCHOSTATINS ! AND " NOVEL GENE EXPRESSION ENHANCING SUBSTANCES PRODUCED BY 0SEUDOMONAS SP 4ETRAHEDRON ,ETT n 9UREK 'EORGE ! (ABENS & "RIMMELL - 0ACKHAM ' 'ANESAN ! 4OTAL SYNTHESIS OF SPIRUCHOSTATIN ! A POTENT HISTONE DEACETYLASE INHIBITOR * !M #HEM 3OC n
0ROGRESS IN $RUG 2ESEARCH 6OL &RANK 0ETERSEN AND 2ENÏ !MSTUTZ %DS © "IRKHÊUSER 6ERLAG "ASEL 3WITZERLAND
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
"Y 0ETER # (IESTAND -ARTIN 2AUSCH $ANIELA 0IANI -EIER AND #AROLYN ! &OSTER .OVARTIS 0HARMA !' ,ICHTSTRASSE
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!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
!BSTRACT &INGOLIMOD &49 REPRESENTS THE FIRST IN A NEW CLASS OF IMMUNE MODULATORS WHOSE TARGET IS SPHINGOSINE PHOSPHATE 30 RECEPTORS )T WAS FIRST IDENTIFIED BY RESEARCHERS AT +YOTO 5NIVERSITY AND 9OSHITOMI 0HARMACEUTICAL AS A CHEMICAL DERIVATIVE OF THE ASCOMYCETE METABO LITE )30 MYRIOCIN 5NLIKE ITS NATURAL PRODUCT PARENT &49 DOES NOT INTERFERE WITH SPHINGOLIPID BIOSYNTHESIS )NSTEAD ITS BEST CHARACTERIZED MECHANISM OF ACTION UPON IN VIVO PHOSPHORYLATION LEADING TO THE ACTIVE PRINCIPLE &49 0 IS THE RAPID AND REVERSIBLE INHIBI TION OF LYMPHOCYTE EGRESS FROM PERIPHERAL LYMPH NODES !S A CONSEQUENCE OF 30 RECEPTOR INTERNALIZATION TISSUE DAMAGING 4 CELLS CAN NOT RECIRCULATE AND INFILTRATE SITES OF INFLAMMA TION SUCH AS THE CENTRAL NERVOUS SYSTEM #.3 &URTHERMORE &49 0 MODULATION OF 30 RECEPTOR SIGNALING ALSO ENHANCES ENDOTHELIAL BARRIER FUNCTION $UE TO ITS MODE OF ACTION &49 EFFECTIVELY PREVENTS TRANSPLANT REJECTION AND IS ACTIVE IN VARIOUS AUTOIMMUNE DISEASE MODELS 4HE MOST STRIKING EFFICACY IS IN THE MULTIPLE SCLEROSIS -3 MODEL OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS WHICH HAS NOW BEEN CONFIRMED IN THE CLINIC &49 DEM ONSTRATED PROMISING RESULTS IN 0HASE )) TRIALS AND RECENTLY ENTERED 0HASE ))) IN PATIENTS WITH RELAPSING -3 %MERGING EVIDENCE SUGGESTS THAT ITS EFFICACY IN THE #.3 EXTENDS BEYOND IMMU NOMODULATION TO ENCOMPASS OTHER ASPECTS OF -3 PATHOPHYSIOLOGY INCLUDING AN INFLUENCE ON THE BLOOD BRAIN BARRIER AND GLIAL REPAIR MECHANISMS THAT COULD ULTIMATELY CONTRIBUTE TO RESTORATION OF NERVE FUNCTION &49 MAY REPRESENT A POTENT NEW THERAPEUTIC MODALITY IN -3 COMBINED WITH THE BENEFIT OF ORAL ADMINISTRATION
)NTRODUCTION -ULTIPLE SCLEROSIS -3 IS A CHRONIC INFLAMMATORY DISORDER OF THE CENTRAL NERVOUS SYSTEM #.3 AND THE MOST COMMON NEUROLOGICAL DISEASE AFFECTING YOUNG ADULTS IN THE 7ESTERN WORLD ;= )T IS TYPICALLY CHARACTERIZED BY FOCAL PLAQUES OF INFLAMMATORY DEMYELINATION IN THE WHITE MATTER OF THE BRAIN AND SPINAL CORD TOGETHER WITH DEGENERATION OF CENTRAL AXONS -OST PATIENTS BEGIN WITH A RELAPSING REMITTING 22-3 COURSE THEN THE MAJORITY WILL EVEN TUALLY ENTER A CHRONIC PROGRESSIVE PHASE WITH INCREASING CLINICAL DISABILITIES !XONAL LOSS IS THOUGHT TO BE THE UNDERLYING PROCESS RESPONSIBLE FOR IRREVERS IBLE CLINICAL DYSFUNCTION 4HE PATHOGENESIS OF TISSUE INJURY IN -3 IS COMPLEX AND INVOLVES MULTIPLE PROCESSES ;n= REFLECTED BY THE CATEGORIZATION INTO FOUR DISEASE PATTERNS AND CURRENT DIAGNOSTIC CRITERIA ; = )N GENERAL 4 CELL MEDIATED INFLAMMATION OF PUTATIVE AUTOIMMUNE ORIGIN IS REGARDED AS THE DRIVING FORCE ACCOMPANIED BY AUTOANTIBODIES DIRECTED AT MYELIN SHEATH PROTEINS /THER CONTRIBUTING FACTORS INCLUDE INCREASED BLOOD BRAIN BARRIER """ PERMEABILITY AND ABERRANT ANGIOGENESIS COUPLED WITH IMPAIRED TISSUE REPAIR &IG 4HE CLINICAL TIME COURSE AND PROGNOSIS ULTIMATELY DEPEND ON THE BALANCE OF #.3 DAMAGE AND EXTENT OF ENDOGENOUS REPAIR
0ETER # (IESTAND ET AL
&IGURE -ECHANISMS CONTRIBUTING TO THE PATHOPHYSIOLOGY OF -3
#URRENT FIRST LINE DISEASE MODIFYING THERAPIES LIKE INTERFERON BETA )&.β AND GLATIRAMER ACETATE HAVE SHOWN PARTIAL EFFICACY IN -3 SUCH AS DECREASING THE LESION LOAD MEASURED BY MAGNETIC RESONANCE IMAGING -2) AND REDUCING RELAPSE RATES BY APPROXIMATELY ;n= 3UCH DRUGS REQUIRE PARENTERAL ADMINISTRATION WITH VARIABLE ABILITY TO REDUCE DISABILITY PROGRES SION /PTIMAL EFFECTIVENESS OF FUTURE THERAPEUTIC AGENTS WILL BE DETERMINED BY THEIR ABILITY TO ACTIVELY SUPPRESS AXONAL INJURY AND CONCOMITANTLY PRO MOTE REMYELINATION THUS RESTORATION OF NEURONAL FUNCTION &URTHERMORE THE ADDED POTENTIAL OF AN ORALLY ACTIVE DRUG WOULD MOST LIKELY ENHANCE COMPLIANCE AND THEREBY CONTRIBUTE TO THE MANAGEMENT OF NEUROLOGICAL DEFICITS 4HIS REVIEW FOCUSES ON THE REMARKABLE JOURNEY OF FINGOLIMOD &49 FROM ITS NATURAL PRODUCT ROOTS AS AN ASCOMYCETE DERIVATIVE TO ITS CURRENT STATUS AS A PROMISING ORAL THERAPEUTIC FOR -3 PATIENTS
$ISCOVERY AND DEVELOPMENT OF &49 &49 AMINO ; OCTYLPHENYL ETHYL=PROPANE DIOL HYDROCHLO RIDE IS A SYNTHETIC AMINOALCOHOL LIPID THAT IS STRUCTURALLY SIMILAR TO SPHIN
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
&IGURE #HEMICAL STRUCTURES ! 3PHINGOSINE AND ITS STRUCTURAL ANALOG &49 ARE PHOSPHORYLATED BY SPHINGOSINE KINASE 3+ TO SPHINGOSINE PHOSPHATE 30 AND &49 0 RESPECTIVELY #HIRAL ANALOGS OF &49 THAT COULD NOT BE PHOSPHORYLATED SUCH AS !!, 3 WERE BIOLOGICALLY INACTIVE ; = " -YRIOCIN )30 IS ALSO IDENTICAL TO THERMOZYMOCIDIN PRODUCED BY -YCELIA STERILIA )TS STEREOCHEMISTRY WAS ELUCIDATED BY 3T *ACQUES ;= WITH TOTAL SYNTHESIS BY 9OSHIKAWA AND COLLEAGUES ;=
GOSINE A NATURALLY OCCURRING LYSOLIPID &IG ! )T IS A CHEMICAL DERIVATIVE OF )30 WHICH IS A METABOLITE ISOLATED FROM THE ASCOMYCETE )SARIA SINCLAIRII AND IS IDENTICAL TO MYRIOCIN &IG " DISCOVERED IN THE FUNGUS -YRIOCOC CUM ALBOMYCES DURING A SCREEN FOR NOVEL IMMUNOSUPPRESSANTS IN THE MIXED LYMPHOCYTE REACTION -,2 ;n= 4HE PRIMARY TARGET MEDIATING IMMUNOSUPPRESSIVE PROPERTIES OF )30 MYRIOCIN IS INHIBITION OF SERINE PALMITOYLTRANSFERASE WHICH CATALYZES SPHINGOLIPID BIOSYNTHESIS ;n= 4HE IDENTIFICATION OF &49 VIA CHEMICAL MODIFICATION OF MYRIOCIN WAS DRIVEN BY THE AIM TO GENERATE A COMPOUND WITH NOVEL BIOACTIVITY AND NEGLIGIBLE TOXICITY 4HIS WAS ACHIEVED IN UNDER THE DIRECTION OF 4ET SURO &UJITA ;= WHEREBY &49 UNLIKE MYRIOCIN DOES NOT INHIBIT SERINE PALMITOYLTRANSFERASE ; = OR INTERFERE WITH SPHINGOLIPID BIOSYNTHESIS ;= .OVARTIS LICENSED IN &49 FROM -ITSUBISHI 0HARMA FORMERLY 9OSHI TOMI 0HARMACEUTICALS AND 7ELFIDE AND IS CURRENTLY CONDUCTING PIVOTAL TRIALS IN -3
0ETER # (IESTAND ET AL
&49 MECHANISM OF ACTION &49 IS DEFINED AS A SPHINGOSINE PHOSPHATE 30 RECEPTOR MODULA TOR REPRESENTING THE FIRST OF A NEW CLASS OF IMMUNOMODULATORY AGENTS ;n= 5PON IN VIVO PHOSPHORYLATION &IG ! &49 IS CONVERTED TO ITS BIOLOGICALLY ACTIVE PHOSPHATE ESTER METABOLITE &49 0 WHICH ACTS AS A HIGH AFFINITY AGONIST FOR FOUR OF THE FIVE KNOWN ' PROTEIN COUPLED 30 RECEPTORS NAMELY 30 AND 30n WITH +D VALUES IN THE NANOMOLAR RANGE ; = 3PHINGOSINE KINASE 3+ IS THE KEY ENZYME REQUIRED FOR &49 0 FORMATION ;n= AS DEMONSTRATED IN 3+ KNOCKOUT MICE ; = 4HE FACT THAT 3+ NULL MICE BECOME LYMPHOPENIC AFTER &49 ADMINISTRATION FURTHER SUPPORTS THE CONCEPT THAT 3+ IS PRIMARILY SUFFICIENT FOR THE FUNCTIONAL ACTIVATION OF &49 ;=
$IVERGENCE FROM CONVENTIONAL IMMUNOSUPPRESSANTS 4HE MODE OF ACTION -O! FOR &49 BEARS ALMOST NO RESEMBLANCE TO HOW ITS PARENT COMPOUND WAS DISCOVERED IE MYRIOCIN MEDIATED -,2 INHIBI TION ;= ILLUSTRATING HOW SERENDIPITY CONTINUES TO INFLUENCE DRUG DISCOV ERY )NDEED IT NOT ONLY ACTS FUNDAMENTALLY DIFFERENT TO MYRIOCIN BUT ALSO TO STANDARD IMMUNOSUPPRESSIVE AGENTS SUCH AS THE CALCINEURIN INHIBITORS CYCLOSPORINE ! #S! AND TACROLIMUS AS WELL AS THE M4/2 MAMMALIAN TARGET OF RAPAMYCIN INHIBITORS SIROLIMUS AND EVEROLIMUS ;= -OST NOTA BLY &49 AT THERAPEUTICALLY RELEVANT CONCENTRATIONS DOES NOT INHIBIT THE ACTIVATION OR PROLIFERATION OF LYMPHOCYTES 4AB AND DOES NOT OVERTLY AFFECT THEIR EFFECTOR FUNCTION INCLUDING ANTIBODY RESPONSIVENESS AND 4 CELL CYTOKINE SECRETION ;n=
2EVERSIBLE MODULATION OF LYMPHOCYTE TRAFFICKING 4HE SIGNATURE FEATURE OF &49 IS ITS ABILITY TO INDUCE A RAPID REDUCTION OF CIRCULATING 4 AND " CELLS &IG ! LARGELY SPARING GRANULOCYTES AND MONOCYTES ;= -ODULATION OF LYMPHOCYTE TRAFFICKING CAN OCCUR WITHIN n H OF INTAKE AND IS REVERSIBLE UPON DRUG CESSATION AS ORIGINALLY OBSERVED BY 9OSHITOMI INVESTIGATORS IN RODENTS ;= AND SUBSEQUENTLY CONFIRMED IN
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
4ABLE !CTIVITY OF &49 VERSUS IMMUNOSUPPRESSIVE AGENTS IN HUMAN -,2A &49
-YRIOCIN
#YCLOSPORIN
4ACROLIMUS
%VEROLIMUS
A)#
N-, INDICATES THE INHIBITORY EFFECT ON 4 CELL PROLIFERATION ; =
NONHUMAN PRIMATES ; = AND HUMAN SUBJECTS ; = 4HE ENSUING BLOOD LYMPHOPENIA IS THOUGHT TO DEPEND ENTIRELY ON DOWNREGULATION OF 30 BY &49 0 ; = WHICH THEN HAMPERS THE ENTRY OF LYMPHOCYTES INTO EFFERENT LYMPHATICS WITHIN PERIPHERAL LYMPH NODES ,. AS DETAILED IN &IGURE )MPORTANTLY &49 TREATMENT DOES NOT OVERTLY INFLUENCE THE IMMUNE FUNCTION OF EFFECTOR MEMORY CELLS BUT RATHER INDUCES THE RETEN TION OF NAÔVE AND CENTRAL MEMORY CELLS ;= GIVEN THAT THEIR TRAFFICKING AND PERCOLATION RATE THROUGH THE ,. IS REGULATED BY 30
%NDOTHELIAL BARRIER FUNCTION AND VASCULAR INTEGRITY 30 IS KNOWN TO BE A POTENT ACTIVATOR OF ENDOTHELIAL CELL %# RESPONSES INCLUDING CELLULAR LOCOMOTION VASCULAR MATURATION ANGIOGENESIS AND ENHANCEMENT OF BARRIER FUNCTION ; = &49 MIMICS MULTIPLE EFFECTS OF 30 MEDIATED ENDOTHELIAL ACTIVATION ;= SUCH AS STIMULATING %# SIGNAL ING MIGRATION SURVIVAL AND DIFFERENTIATION &URTHERMORE IN VIVO EXPO SURE TO &49 ATTENUATED VASCULAR ENDOTHELIAL CELL GROWTH FACTOR 6%'& INDUCED ANGIOGENESIS AND VASCULAR PERMEABILITY ; = AS WELL AS PULMO NARY MICROVASCULAR LEAKAGE INDUCED BY ENDOTOXIN ;= %NDOTHELIAL BARRIER REGULATION IS HIGHLY DEPENDENT ON THE MOLECULAR CONFIGURATION OF SPECIAL IZED JUNCTIONS BETWEEN %# ;= 4HE REVERSAL OF CAPILLARY PERMEABILITY AND PRESERVATION OF VASCULAR INTEGRITY BY &49 IS DUE TO THE TRANSLOCATION OF ADHERENS JUNCTION PROTEINS LIKE 6% CADHERIN AND β CATENIN RESULTING IN CYTOSKELETAL REARRANGEMENTS THAT STABILIZE THE ENDOTHELIUM ; = !GO NISTIC LIGATION OF 30 IS THOUGHT TO BE THE PRIMARY MECHANISM MEDIATING &49 0 PROMOTION OF JUNCTIONAL COMPLEXES AND AUGMENTATION OF BARRIER FUNCTION ;= )N CONTRAST A RECENTLY REPORTED 30 ANTAGONIST STRONGLY ENHANCED PULMONARY CAPILLARY LEAKAGE ;=
0ETER # (IESTAND ET AL
&IGURE &49 MODULATION OF PERIPHERAL BLOOD LYMPHOCYTES DURING %!% ! 2EPRESENTATIVE TWO COLOR FLOW CYTOMETRY OF PERIPHERAL BLOOD LEUKOCYTES ON DAY POST IMMU NIZATION IN THE ,EWIS RAT MODEL OF %!%A &49 TREATMENT RESULTED IN A REDUCTION IN RELATIVE LYM PHOCYTE NUMBERS COMPARED TO VEHICLE AS SHOWN BY 4 CELLS #$ IN THE UPPER LEFT QUADRANT AND " CELLS #$2! IN THE LOWER RIGHT &ORWARD SIDE SCATTER PLOTS UPPER RIGHT SHOW A HIGHER DENSITY OF LARGE MONONUCLEAR CELLS UNDER &49 TREATMENT COMPARED TO LYMPHOCYTES GREEN " 0ROPHYLACTIC EFFECT OF &49 AND CYCLOSPORINE ! #S! ON CLINICAL DISEASE COURSE IN ACUTE MONOPHASIC %!%A "OTH COMPLETELY PREVENTED THE EMERGENCE OF NEUROLOGICAL DEFICITS DURING TREATMENT THOSE RECEIVING #S! BECAME SEVERELY PARALYZED UPON DRUG CESSATION RESULTING IN ONE DEATH o A%!% WAS INDUCED IN FEMALE ,EWIS RATS BY A μL INOCULATION OF GUINEA PIG SPINAL CORD EMULSIFIED IN COMPLETE &REUNDS ADJUVANT 4HE HOMOGENATE WAS PREPARED AS DESCRIBED ;= AND GIVEN ID AT THE DORSAL TAIL ROOT AFTER MILD ISOFLURANE SEDATION /RAL TREATMENT WITH VEHICLE MGKG &49 OR MGKG #S! STARTED AT IMMUNIZATION AND CONTINUED FOR WEEKS N PER GROUP #LINICAL GRADES WERE ASSESSED DAILY USING A TO SCALE NO PARALYSIS LOSS OF TAIL TONUS LIMB WEAKNESS OR ATAXIA COMPLETE HINDLIMB PARALYSIS "LOOD SAMPLES FROM THE RETRO ORBITAL VENOUS PLEXUS WERE IMMUNOLA BELED ON DAY AS DESCRIBED ;= AND ANALYZED BY LIVE GATE IN A &!#3 #ALIBUR FLOW CYTOMETER USING #ELL1UEST SOFTWARE
)N THE BRAIN A CONTINUOUS LAYER OF CEREBRAL VASCULAR %# IS BOUND TOGETHER BY TIGHT JUNCTIONS TO FORM THE """ SEE 3ECTION !LTHOUGH ITS EXISTENCE AS THE INTERFACE BETWEEN THE PERIPHERAL CIRCULATION AND #.3 WAS ALREADY REVEALED IN BY 0AUL %HRLICH ;= MOLECULAR MECHANISMS REGULATING THE """ CONTINUE TO CAPTIVATE INVESTIGATORS ;n= )N FACT ), β DERIVED FROM ASTROCYTES &IG " WAS RECENTLY SHOWN TO ACTIVATE A HYPOXIA RESPONSE ELEMENT THAT POTENTIATES 6%'& INDUCED """ PERMEABILITY ;=
)N VIVO ACTIVITY IN VARIOUS DISEASE MODELS &49 IS KNOWN TO BE EFFECTIVE PARTICULARLY AS PREVENTIVE TREATMENT IN DIVERSE MODELS OF TRANSPLANTATION AND AUTOIMMUNE DISEASE ; = !S
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
4ABLE )N VIVO EFFICACY OF &49 IN DISEASE MODELS $ISEASE -ODEL
3PECIES
$OSE MGKGA
2EFERENCE
!LLOGRAFT REJECTION
2AT -OUSE
n PO IP
;n= ; =
!RTHRITIS
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!STHMA
-OUSE
μG IT
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$ERMATITIS
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$IABETES
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;= ; =
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-YASTHENIA GRAVIS
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A0ROPHYLACTIC IP INTRAPERITONEAL IT INTRATRACHEAL PO ORAL
SUMMARIZED IN 4ABLE EXAMPLES INCLUDE ALLOGRAFT REJECTION ; n= ADJUVANT AND COLLAGEN INDUCED ARTHRITIS ;= ASTHMA ;= DERMATITIS ;= DIABETES MELLITUS TYPE ; n= MYASTHENIA GRAVIS ;= MYOCARDITIS ; = SYSTEMIC LUPUS ERYTHEMATOSUS ;= THYROIDITIS ;= AND UVEORETI NITIS ;= 4HE -O! FOR &49 CONFERS MULTIPLE ADVANTAGES OVER CONVENTIONAL IMMUNE SUPPRESSANTS REGARDING HOST DEFENSE GIVEN THAT IT DOES NOT IMPAIR MYELOMONOCYTIC CELL FUNCTION OR RESPONSES TO MICROBIAL CHALLENGE 0OTEN TIAL UTILITY OF &49 AS ADJUNCT THERAPY WITH IMMUNOSUPPRESSANTS LIKE #S! IS BEING EXPLORED IN DE NOVO RENAL TRANSPLANT PATIENTS ;= &49 MONOTHERAPY IS UNDER EVALUATION IN 22-3 PATIENTS SEE 3ECTION
&49 EFFICACY IN -3 MODELS 4HE MOST WIDELY USED ANIMAL MODEL FOR -3 IS EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS %!% THE ORIGINS OF WHICH DATE BACK TO THE S
0ETER # (IESTAND ET AL
;n= %!% CAN BE ACTIVELY INDUCED IN GENETICALLY SUSCEPTIBLE MAMMALS BY IMMUNIZATION WITH EITHER A HOMOGENATE OF #.3 TISSUE PURIFIED PRO TEINS DERIVED FROM MYELIN OR SYNTHETIC PEPTIDES CORRESPONDING TO SPECIFIC MYELIN EPITOPES FINAL EMULSIFICATION IS USUALLY PERFORMED IN COMPLETE &REUNDS ADJUVANT CONTAINING VARIABLE AMOUNTS OF HEAT INACTIVATED -YCO BACTERIUM TUBERCULOSIS !LTERNATIVELY %!% CAN BE PASSIVELY INDUCED BY ADOP TIVE TRANSFER OF ENCEPHALITOGENIC 4 CELLS 4HESE METHODS ELICIT AN AUTOIM MUNE PATHOPHYSIOLOGIC RESPONSE TOWARDS MYELIN SHEATH PROTEINS IN THE #.3 MEDIATED BY AUTOREACTIVE 4 CELLS THAT INFILTRATE THE BRAIN AND SPINAL CORD !LTHOUGH NO SINGLE ANIMAL MODEL MIMICS ALL FEATURES OF -3 EACH REFLECTS SPECIFIC FACETS OF THE HUMAN DISEASE 4HE ,EWIS RAT IS THE MOST COM MONLY USED MODEL FOR ACUTE %!% TYPICALLY CHARACTERIZED BY A BRIEF MONO PHASIC ATTACK WITH SPARSE OR NO DEMYELINATION &IG " OUTBRED 7ISTAR RATS DISPLAY A SIMILAR DISEASE COURSE ; = /THER RAT STRAINS SUCH AS THE $ARK !GOUTI $! AND "ROWN .ORWAY ARE HIGHLY SUSCEPTIBLE TO DEMYELIN ATION AND CHRONIC NEURODEGENERATIVE SYMPTOMS ; = AN AUTOANTIBODY RESPONSE TO MYELIN OLIGODENDROCYTE GLYCOPROTEIN -/' IMMUNIZATION IS PARTICULARLY PIVOTAL IN THE "ROWN .ORWAY RAT ;= -OUSE STRAINS SUCH AS #", 0,* AND 3*,* HAVE ALSO BEEN EXTENSIVELY INVESTIGATED FOR THIS PURPOSE ESPECIALLY ENHANCED BY THE GENERATION OF GENETICALLY ENGINEERED MUTANTS ;n=
#LINICAL PROFILE IN %!% &49 IS REMARKABLY EFFICACIOUS IN VARIOUS RODENT MODELS OF %!% ; n= 3TARTING ORAL MONOTHERAPY AT THE TIME OF ACTIVE IMMUNIZATION PREVENTIVE TREATMENT &IG " AT THE BEGINNING OF DISEASE SYMPTOMS THERAPEUTIC OR AFTER ADOPTIVE TRANSFER OF ENCEPHALITOGENIC 4 CELLS PREVENTS %!% ONSET AND REDUCES DEVELOPMENT OF NEUROLOGICAL DEFICITS IN ESTABLISHED DISEASE #LINICAL EFFICACY IN %!% WAS ACCOMPANIED BY THE PREVENTION OF DISEASE RELATED DEATH AND BODY WEIGHT LOSS IN ADDITION TO THE LACK OF #.3 INFLAMMATION AS ASSESSED BY HISTOPATHOLOGY AND M2.! LEVELS OF INFLAM MATORY PRODUCTS +ENJI #HIBA AND 9OSHITOMI COLLEAGUES WERE THE FIRST TO REPORT THAT A LYMPHODEPLETING DOSE AS LOW AS MGKG INHIBITED PARALYSIS IN THE ,EWIS RAT MODEL OF MYELIN BASIC PROTEIN INDUCED %!% ;= 0REVEN TION OF LYMPHOCYTE RECIRCULATION LIKELY CONTRIBUTES TO ITS -O! IN %!% BUT
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
&IGURE 0ROPOSED IN VIVO MECHANISM FOR &49 EFFICACY IN %!% AND -3 .AÕVE 4 CELLS CONTINUOUSLY CIRCULATE BETWEEN BLOOD AND LYMPHATIC TISSUE IN SEARCH OF FOREIGN ANTIGEN ENTERING LYMPH NODES ,. THROUGH HIGH ENDOTHELIAL VENULES AND LEAVING FROM EFFERENT LYMPHATICS VIA THE SINUS LINING ENDOTHELIUM IN A 30 DEPENDENT MANNER ; = )F ANTIGEN IS PRODUCTIVELY RECOGNIZED 4 CELLS UNDERGO CLONAL EXPANSION WITHIN THE ,. AND TRANSIENTLY DOWN MODULATE 30 WHEREBY THEY CAN NO LONGER RESPOND TO THE OBLIGATORY EGRESS SIGNAL PROVIDED BY THE SERUM LIPID 30 !CTIVATED CELLS REMAIN IN THE ,. THROUGHOUT THE PROLIFERATION PHASE UNTIL THEY UPREGULATE 30 WHICH ALLOWS THEM TO SUBSEQUENTLY EXIT IN A 3030 DEPENDENT PROCESS !FTER IN VIVO PHOSPHORYLATION &IG &49 0 ACTS AS A SUPER AGONIST AT 30 TO INDUCE ITS INTER NALIZATION INSET ! 4HIS RENDERS BOTH NAÕVE AND CENTRAL MEMORY LYMPHOCYTES UNRESPONSIVE TO 30 DEPRIVING THEM FROM THE SIGNAL TO LEAVE THE LYMPHOID COMPARTMENT !S A CONSEQUENCE POTENTIAL AUTOREACTIVE CELLS ARE UNABLE TO RECIRCULATE TO INFLAMED SITES LIKE THE #.3 BUT STILL REMAIN FUNCTIONAL WITHIN THE ,. ;= #OMPLEMENTARY TO ITS PERIPHERAL IMMUNOMODULATORY ACTION &49 MAY PRE SERVE """ INTEGRITY BY DIRECT EFFECTS ON ENDOTHELIAL CELLS %# OR MEDIATED BY 30 RECEPTOR BEARING ASTROCYTES INSET " )T COULD ADDITIONALLY ACT VIA 30 RECEPTORS EXPRESSED ON OTHER GLIAL CELLS SUCH AS MYELIN PRODUCING OLIGODENDROCYTES /,' TO PROMOTE ENDOGENOUS REPAIR MECHANISMS INSET #
A TEMPORAL MISMATCH BETWEEN CLINICAL SIGNS AND DECREASE IN PERIPHERAL LYMPHOCYTES ;= INDICATES THE INVOLVEMENT OF ADDITIONAL MECHANISMS WHICH WE HAVE PROPOSED IN &IGURE
-AGNETIC RESONANCE IMAGING .ON INVASIVE -2) CURRENTLY REPRESENTS THE GOLD STANDARD TO MEASURE QUAN TIFIABLE PARAMETERS LINKED TO THE PATHOPHYSIOLOGY OF BRAIN LESIONS IN -3 ;= $ISRUPTION OF THE """ ;n= AS DETECTED BY AN INCREASED -2) SIGNAL
0ETER # (IESTAND ET AL
IN 4 WEIGHTED IMAGES AFTER INJECTION OF GADOLINIUM 'D IS A CONSISTENT FINDING ASSOCIATED WITH NEW LESION FORMATION IN 22-3 PATIENTS ;= (IS TOLOGICALLY ENHANCEMENT OF CONTRAST AGENTS 'D $/4! 'D $40! TYPI CALLY COINCIDES WITH MACROPHAGE INFILTRATION AND PERIVENULAR INFLAMMA TION INVOLVING 4 CELLS AND MONOCYTES THIS IS SOMETIMES ACCOMPANIED BY ACTIVE DEMYELINATION ; = 1UANTITATIVE CONTRAST ENHANCED IMAGING WAS RECENTLY VALIDATED FOR THE DETECTION OF SUBTLE """ LEAKAGE WITHIN VIS IBLY NON ENHANCING LESIONS IN 22-3 ;= )N THIS !&&)2- SUBSTUDY NATALI ZUMAB DID NOT SIGNIFICANTLY LOWER THE OBSERVED LEAKAGE SUGGESTING THAT MAINTENANCE OF """ INTEGRITY IS NOT EXTENSIVELY MEDIATED BY αβ INTEGRIN AND THAT BARRIER DISRUPTION MAY OCCUR THROUGH MORE PERMANENT CHANGES IN THE VASCULAR WALL ;= ! SIMILAR INCREASE IN """ PERMEABILITY WAS OBSERVED IN %!% FIRST EXPLORED IN THE GUINEA PIG USING 'D $40! ENHANCEMENT ;= AND SUBSEQUENTLY IN THE RAT ;= 4O SIMULTANEOUSLY INVESTIGATE BY -2) THE RELATIONSHIP BETWEEN MACRO PHAGE INFILTRATION AND """ DISRUPTION CELL SPECIFIC IMAGING WAS APPLIED IN %!% MODELS USING ULTRA SMALL SUPERPARAMAGNETIC IRON OXIDE 530)/ PARTI CLES THAT LABEL PHAGOCYTIC CELLS ; n= -2) MONITORING IN THE ,EWIS RAT CONFIRMED THAT THE DEVELOPMENT OF CLINICAL DEFICITS WAS ASSOCIATED WITH INCREASED """ PERMEABILITY AND ACCUMULATION OF LABELED MACROPHAGES IN THE #.3 MEASURED BY 'D AND 530)/ SIGNAL ENHANCEMENT RESPECTIVELY ; = )N THIS %!% MODEL PROPHYLACTIC AND THERAPEUTIC TREATMENT WITH &49 PROTECTED AGAINST """ LEAKAGE 'D $/4! AND PREVENTED THE INFILTRATION OF 530)/ LABELED MACROPHAGES VERIFIED IMMUNOHISTOCHEMI CALLY BY THE LACK OF %$ POSITIVE CELLS ;= 4HE ABILITY OF &49 TO SUP PRESS MACROPHAGE INFLUX MAY HAVE IMPORTANT DOWNSTREAM IMPLICATIONS SINCE THESE CELLS PRODUCE THE PROINFLAMMATORY CYTOKINE ), WHICH CON TRIBUTES TO """ PERMEABILITY ;= &OR FUTURE INVESTIGATIONS MAGNETIZATION TRANSFER IMAGING IN A CHRONIC %!% MODEL COULD BE A SENSITIVE METHOD TO EVALUATE &49S POTENTIAL TO REVERSE TISSUE DAMAGE IN AN ESTABLISHED DIS EASE AS RECENTLY SUGGESTED BY SUBTLE CHANGES IN BENIGN -3 PATIENTS ;= AND EMERGING ANIMAL STUDIES ; =
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
&IGURE 3PHINGOLIPIDS WERE NAMED AFTER THE MYTHICAL 'REEK 3PHINX IN BY THE NEUROCHEMIST *,7 4HUDICHUM hIN COMMEMORATION OF THE MANY ENIGMAS WHICH IT PRESENTS TO THE INQUIRERv
&UNCTIONAL ELECTROPHYSIOLOGICAL STUDIES )MPAIRED NERVE IMPULSES IN -3 CAN BE QUANTIFIED BY THE AMPLITUDE AND LATENCY OF EVOKED POTENTIAL %0 RESPONSES TO SENSORY STIMULI ;= $EMY ELINATION AND AXONAL LOSS RESULT IN PROLONGED LATENCY AND DECREASED AMPLI TUDE FOR BOTH THE VISUAL AND SOMATOSENSORY %0 WAVEFORMS 3IMILAR ELECTRO PHYSIOLOGICAL ALTERATIONS OCCUR IN SOME -3 MODELS SUCH AS -/' INDUCED %!% IN THE $! RAT 5NDER THESE STRINGENT CONDITIONS &49 PROPHYLAXIS FULLY PROTECTED AGAINST DISTURBANCES IN THE NERVE CONDUCTANCE PATTERN ;A= -ORE IMPORTANTLY LATE THERAPEUTIC TREATMENT MARKEDLY REVERSED PARALYSIS AND NORMALIZED THE %0 RESPONSES CORRELATING WITH DECREASED DEMYELINATION IN THE BRAIN AND SPINAL CORD 3INCE &49 CAN GAIN ACCESS TO THE #.3 AND BE CONVERTED TO &49 0 ; = IT MAY ALSO ACT VIA GLIAL 30 RECEPTORS TO PROMOTE ENDOGENOUS REPAIR MECHANISMS THAT COMPLE MENT ITS IMMUNOMODULATORY ACTION &IG 4HE ABILITY OF &49 TO REACH ITS TARGET ORGAN #.3 LIKELY ACCOUNTS FOR THE DIFFERENTIAL EFFICACY
0ETER # (IESTAND ET AL
SEEN WITH SOME CURRENT -3 TREATMENTS ;= AND THE REMARKABLE POTENCY IN ESTABLISHED %!% COMPARED TO OTHER DISEASE MODELS 4AB
&49 ORAL MONOTHERAPY IN MULTIPLE SCLEROSIS $URING 0HASE )) STUDIES ORAL &49 SUPPRESSED INFLAMMATORY DISEASE ACTIVITY BY UP TO OVER MONTHS AS MEASURED BY -2) AND REDUCED CLINICAL RELAPSE RATES BY MORE THAN COMPARED TO PLACEBO ;= .OVAR TIS IS CURRENTLY CONDUCTING 0HASE ))) TRIALS WITH THE GOAL OF ENROLLING MORE THAN RELAPSING -3 PATIENTS WORLDWIDE ! TWO YEAR PLACEBO CON TROLLED PROGRAM &2%%$/-3 IS MEASURING REDUCTIONS IN THE FREQUENCY OF RELAPSES AND DISABILITY PROGRESSION ! ONE YEAR TRIAL 42!.&/2-3 IS COMPARING &49 WITH )&.β A !VONEX¤ 4WO YEAR DATA FROM THE EXTEN SION OF 0HASE )) SHOWED SUSTAINED BENEFITS INDICATING THAT &49 COULD BECOME ONE OF THE FIRST ORAL DISEASE MODIFYING TREATMENTS FOR -3 PATIENTS AND THUS PROVIDE AN EFFECTIVE NEW OPTION FOR THE ESTIMATED MILLION PEOPLE WORLDWIDE SUFFERING FROM THIS DISABLING NEUROLOGICAL DISEASE
2EFERENCES
&ROHMAN %- 2ACKE -+ 2AINE #3 -ULTIPLE SCLEROSIS n THE PLAQUE AND ITS PATHO GENESIS . %NGL * -ED n ,ASSMANN ( -ECHANISMS OF MULTIPLE SCLEROSIS $RUG $ISCOV 4ODAY $IS -ECH n -EINL % +RUMBHOLZ - (OHLFELD 2 " LINEAGE CELLS IN THE INFLAMMATORY CENTRAL NERVOUS SYSTEM ENVIRONMENT -IGRATION MAINTENANCE LOCAL ANTIBODY PRODUCTION AND THERAPEUTIC MODULATION !NN .EUROL n 7AXMAN 3' !XONAL CONDUCTION AND INJURY IN MULTIPLE SCLEROSIS THE ROLE OF SODIUM CHANNELS .AT 2EV .EUROSCI n #HARIL ! 9OUSRY 4! 2OVARIS - "ARKHOF & $E 3TEFANO . &AZEKAS & -ILLER $( -ONTALBAN 8 3IMON *( 0OLMAN # ET AL -2) AND THE DIAGNOSIS OF MULTIPLE SCLEROSIS EXPAND ING THE CONCEPT OF hNO BETTER EXPLANATIONh ,ANCET .EUROL n .EUHAUS / !RCHELOS ** (ARTUNG (0 )MMUNOMODULATION IN MULTIPLE SCLEROSIS FROM IMMUNOSUPPRESSION TO NEUROPROTECTION 4RENDS 0HARMACOL 3CI n (OHLFELD 2 7EKERLE ( $RUG )NSIGHT USING MONOCLONAL ANTIBODIES TO TREAT MUL TIPLE SCLEROSIS .AT #LIN 0RACT .EUROL n -ARRIE 2! 2UDICK 2! $RUG )NSIGHT INTERFERON TREATMENT IN MULTIPLE SCLEROSIS .AT #LIN 0RACT .EUROL n
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
+LUEPFEL $ "AGLI * "AKER ( #HAREST -0 +UDELSKI ! -YRIOCIN A NEW ANTIFUNGAL ANTIBIOTIC FROM -YRIOCOCCUM ALBOMYCES * !NTIBIOT n 3T *ACQUES - %LUCIDATION OF STRUCTURE AND STEREOCHEMISTRY OF MYRIOCIN ! NOVEL ANTIFUNGAL ANTIBIOTIC * /RG #HEM n &UJITA 4 )KUMOTO 4 3ASAKI 3 /KUMOTO 4 #HIBA + )MMUNOSUPPRESSANT 5NITED 3TATES 0ATENT *UNE &UJITA 4 )NOUE + 9AMAMOTO 3 )KUMOTO 4 3ASAKI 3 4OYAMA 2 #HIBA + (OSHINO 9 /KUMOTO 4 &UNGAL METABOLITES 0ART ! POTENT IMMUNOSUPPRESSIVE ACTIVITY FOUND IN )SARIA SINCLAIRII METABOLITE * !NTIBIOT n 9OSHIKAWA - 9OKOKAWA 9 /KUNO 9 -URAKAMI . 4OTAL SYNTHESIS OF A NOVEL IMMUNOSUPPRESSANT MYRIOCIN THERMOZYMOCIDIN )30 AND : MYRIOCIN #HEM 0HARM "ULL 4OKYO n +IUCHI - !DACHI + +OHARA 4 -INOGUCHI - (ANANO 4 !OKI 9 -ISHINA 4 !RITA - .AKAO . /HTSUKI - ET AL 3YNTHESIS AND IMMUNOSUPPRESSIVE ACTIVITY OF SUBSTI TUTED AMINOPROPANE DIOLS AND AMINOETHANOLS * -ED #HEM n -IYAKE 9 +OZUTSUMI 9 .AKAMURA 3 &UJITA 4 +AWASAKI 4 3ERINE PALMITOYLTRANS FERASE IS THE PRIMARY TARGET OF A SPHINGOSINE LIKE IMMUNOSUPPRESSANT )30 MYRIOCIN "IOCHEM "IOPHYS 2ES #OMMUN n #HEN *+ ,ANE 73 3CHREIBER 3, 4HE IDENTIFICATION OF MYRIOCIN BINDING PROTEINS #HEM "IOL n 3OLOMON *# 3HARMA + 7EI ,8 &UJITA 4 3HI 9& ! NOVEL ROLE FOR SPHINGOLIPID INTERMEDIATES IN ACTIVATION INDUCED CELL DEATH IN 4 CELLS #ELL $EATH $IFFER n !DACHI + +OHARA 4 .AKAO . !RITA - #HIBA + -ISHINA 4 3ASAKI 3 &UJITA 4 $ESIGN SYNTHESIS AND STRUCTURE ACTIVITY RELATIONSHIPS OF SUBSTITUTED AMINO PROPANEDIOLS DISCOVERY OF A NOVEL IMMUNOSUPPRESSANT &49 "IOORG -ED #HEM ,ETT n &UJITA 4 (IROSE 2 9ONETA - 3ASAKI 3 )NOUE + +IUCHI - (IRASE 3 #HIBA + 3AKAMOTO ( !RITA - 0OTENT IMMUNOSUPPRESSANTS !LKYL AMINOPROPANE DIOLS * -ED #HEM n -ANDALA 3 (AJDU 2 "ERGSTROM * 1UACKENBUSH % 8IE * -ILLIGAN * 4HORNTON 2 3HEI ' * #ARD $ +EOHANE # ET AL !LTERATION OF LYMPHOCYTE TRAFFICKING BY SPHINGOSINE PHOSPHATE RECEPTOR AGONISTS 3CIENCE n "RINKMANN 6 ,YNCH +2 &49 TARGETING ' PROTEIN COUPLED RECEPTORS FOR SPHINGOSINE PHOSPHATE IN TRANSPLANTATION AND AUTOIMMUNITY #URR /PIN )MMUNOL n )M $3 ,INKING #HINESE MEDICINE AND ' PROTEIN COUPLED RECEPTORS 4RENDS 0HAR MACOL 3CI n 2OSEN ( 'OETZL %* 3PHINGOSINE PHOSPHATE AND ITS RECEPTORS AN AUTOCRINE AND PARACRINE NETWORK .AT 2EV )MMUNOL n #HIBA + -ATSUYUKI ( -AEDA 9 3UGAHARA + 2OLE OF SPHINGOSINE PHOSPHATE RECEPTOR TYPE IN LYMPHOCYTE EGRESS FROM SECONDARY LYMPHOID TISSUES AND THYMUS #ELL -OL )MMUNOL n 'ARDELL 3% $UBIN !% #HUN * %MERGING MEDICINAL ROLES FOR LYSOPHOSPHOLIPID SIGNALING 4RENDS -OL -ED n 9OPP !# ,EDGERWOOD ,' /CHANDO *# "ROMBERG *3 3PHINGOSINE PHOSPHATE RECEPTOR MODULATORS A NEW CLASS OF IMMUNOSUPPRESSANTS #LIN 4RANSPLANT n "RINKMANN 6 $AVIS -$ (EISE #% !LBERT 2 #OTTENS 3 (OF 2 "RUNS # 0RIESCHL % "AUM
0ETER # (IESTAND ET AL
RUKER 4 (IESTAND 0 ET AL 4HE IMMUNE MODULATOR &49 TARGETS SPHINGOSINE PHOSPHATE RECEPTORS * "IOL #HEM n (ANESSIAN 3 #HARRON ' "ILLICH ! 'UERINI $ #ONSTRAINED AZACYCLIC ANALOGUES OF THE IMMUNOMODULATORY AGENT &49 AS MOLECULAR PROBES FOR SPHINGOSINE PHOSPHATE RECEPTORS "IOORG -ED #HEM ,ETT n "ILLICH ! "ORNANCIN & $ÎVAY 0 -ECHTCHERIAKOVA $ 5RTZ . "AUMRUKER 4 0HOS PHORYLATION OF THE IMMUNOMODULATORY DRUG &49 BY SPHINGOSINE KINASES * "IOL #HEM n 0AUGH 37 0AYNE 3' "ARBOUR 3% -ILSTIEN 3 3PIEGEL 3 4HE IMMUNOSUPPRESSANT &49 IS PHOSPHORYLATED BY SPHINGOSINE KINASE TYPE &%"3 ,ETT n 3ANCHEZ 4 %STRADA (ERNANDEZ 4 0AIK * ( 7U - 4 6ENKATARAMAN + "RINKMANN 6 #LAFFEY + (LA 4 0HOSPHORYLATION AND ACTION OF THE IMMUNOMODULATOR &49 INHIBITS VASCULAR ENDOTHELIAL CELL GROWTH FACTOR INDUCED VASCULAR PERMEABILITY * "IOL #HEM n +HAREL 9 ,EE 3 3NYDER !( 3HEASLEY /.EILL 3, -ORRIS -! 3ETIADY 9 :HU 2 :IGLER -! "URCIN 4, ,EY + ET AL 3PHINGOSINE KINASE IS REQUIRED FOR MODULATION OF LYMPHOCYTE TRAFFIC BY &49 * "IOL #HEM n :EMANN " +INZEL " -ßLLER - 2EUSCHEL 2 -ECHTCHERIAKOVA $ 5RTZ . "ORNANCIN & "AUMRUKER 4 "ILLICH ! 3PHINGOSINE KINASE TYPE IS ESSENTIAL FOR LYMPHOPENIA INDUCED BY THE IMMUNOMODULATORY DRUG &49 "LOOD n !LLENDE -, 3ASAKI 4 +AWAI ( /LIVERA ! -I 9 VAN %CHTEN $ECKERT ' (AJDU 2 2OSEN BACH - +EOHANE #! -ANDALA 3 ET AL -ICE DEFICIENT IN SPHINGOSINE KINASE ARE RENDERED LYMPHOPENIC BY &49 * "IOL #HEM n 2OSEN ( !LFONSO # 3URH #$ -C(EYZER 7ILLIAMS -' 2APID INDUCTION OF MEDUL LARY THYMOCYTE PHENOTYPIC MATURATION AND EGRESS INHIBITION BY NANOMOLAR SPHINGOSINE PHOSPHATE RECEPTOR AGONIST 0ROC .ATL !CAD 3CI 53! n "URKHART # (EUSSER # -ORRIS 2% 2AULF & 7ECKBECKER ' 7EITZ 3CHMIDT ' 7ELZENBACH + 0HARMACODYNAMICS IN THE DEVELOPMENT OF NEW IMMUNOSUPPRESSIVE DRUGS 4HER $RUG -ONIT n 0INSCHEWER $$ /CHSENBEIN !& /DERMATT " "RINKMANN 6 (ENGARTNER ( :INKERNAGEL 2- &49 IMMUNOSUPPRESSION IMPAIRS EFFECTOR 4 CELL PERIPHERAL HOMING WITH OUT AFFECTING INDUCTION EXPANSION AND MEMORY * )MMUNOL n "RINKMANN 6 #HEN 3 &ENG , 0INSCHEWER $ .IKOLOVA : (OF 2 &49 ALTERS LYMPHOCYTE HOMING AND PROTECTS ALLOGRAFTS WITHOUT INDUCING GENERAL IMMUNOSUPPRES SION 4RANSPLANT 0ROC n $IAZ 2OMERO * 6OGT ' 7ECKBECKER ' #OEXPRESSION OF #$ AND #$A ON RAT 4 CELLS IN WHOLE BLOOD A SENSITIVE MARKER FOR MONITORING 4 CELL IMMUNOSUPPRESSIVE DRUGS * )MMUNOL -ETHODS n (ABICHT ! #LARKSON -2 9ANG * (ENDERSON * "RINKMANN 6 &ERNANDES 3 *UREWICZ - 9UAN 8 3AYEGH -( .OVEL INSIGHTS INTO THE MECHANISM OF ACTION OF &49 IN A TRANSGENIC MODEL OF ALLOGRAFT REJECTION IMPLICATIONS FOR THERAPY OF CHRONIC REJECTION * )MMUNOL n +ABASHIMA + (AYNES .- 8U 9 .UTT 3, !LLENDE -, 0ROIA 2, #YSTER *' 0LASMA CELL 30 EXPRESSION DETERMINES SECONDARY LYMPHOID ORGAN RETENTION VERSUS BONE MAR ROW TROPISM * %XP -ED n #HIBA + 9ANAGAWA 9 -ASUBUCHI 9 +ATAOKA ( +AWAGUCHI 4 /HTSUKI - (OSHINO 9 &49 A NOVEL IMMUNOSUPPRESSANT INDUCES SEQUESTRATION OF CIRCULATING
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MATURE LYMPHOCYTES BY ACCELERATION OF LYMPHOCYTE HOMING IN RATS ) &49 SELECTIVELY DECREASES THE NUMBER OF CIRCULATING MATURE LYMPHOCYTES BY ACCELERATION OF LYMPHOCYTE HOMING * )MMUNOL n 1UESNIAUX 6&* -ENNINGER + +UNKLER ! 6EDRINE # "ERNHARD - (EDINGER 2 +RAUS ' 3CHUURMAN (* 4HE NOVEL IMMUNOSUPPRESSANT &49 INDUCES PERIPHERAL LYM PHODEPLETION OF BOTH 4 AND " CELLS IN CYNOMOLGUS MONKEYS WHEN GIVEN ALONE WITH #YCLOSPORINE .EORAL¤ OR WITH 2!$ 4RANSPL )MMUNOL n ,I ( -ENO 4ETANG '-, #HIBA + !RIMA . (EINING 0 *USKO 7* 0HARMACOKINET ICS AND CELL TRAFFICKING DYNAMICS OF AMINO ; OCTYLPHENYL ETHYL=PROPANE DIOL HYDROCHLORIDE &49 IN CYNOMOLGUS MONKEYS AFTER SINGLE ORAL AND INTRAVENOUS DOSES * 0HARMACOL %XP 4HER n "UDDE + 3CHMOUDER 2, .ASHAN " "RUNKHORST 2 ,ßCKER 07 -AYER 4 "ROOKMAN , .EDELMAN * 3KERJANEC ! "ÙHLER 4 ET AL 0HARMACODYNAMICS OF SINGLE DOSES OF THE NOVEL IMMUNOSUPPRESSANT &49 IN STABLE RENAL TRANSPLANT PATIENTS !M * 4RANSPLANT n +OVARIK *- 3CHMOUDER 2 "ARILLA $ 2IVIERE ' * 7ANG 9 (UNT 4 -ULTIPLE DOSE &49 TOLERABILITY PHARMACOKINETICS AND LYMPHOCYTE RESPONSES IN HEALTHY SUBJECTS * #LIN 0HARMACOL n -ATLOUBIAN - ,O #' #INAMON ' ,ESNESKI -* 8U 9 "RINKMANN 6 !LLENDE -, 0ROIA 2, #YSTER *' ,YMPHOCYTE EGRESS FROM THYMUS AND PERIPHERAL LYMPHOID ORGANS IS DEPENDENT ON 30 RECEPTOR .ATURE n ,O #' 8U 9 0ROIA 2, #YSTER *' #YCLICAL MODULATION OF SPHINGOSINE PHOS PHATE RECEPTOR SURFACE EXPRESSION DURING LYMPHOCYTE RECIRCULATION AND RELATIONSHIP TO LYMPHOID ORGAN TRANSIT * %XP -ED n (OFMANN - "RINKMANN 6 :ERWES (' &49 PREFERENTIALLY DEPLETES NAIVE 4 CELLS FROM PERIPHERAL AND LYMPHOID ORGANS )NT )MMUNOPHARMACOL n "OLTON # &LOWER 2* 4HE EFFECTS OF THE ANTI GLUCOCORTICOID 25 ON STEROID MEDIATED SUPPRESSION OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS %!% IN THE ,EWIS RAT ,IFE 3CI n -C6ERRY "* 'ARCIA *'. )N VITRO AND IN VIVO MODULATION OF VASCULAR BARRIER INTEGRITY BY SPHINGOSINE PHOSPHATE MECHANISTIC INSIGHTS #ELL 3IGNAL n -EHTA $ -ALIK !" 3IGNALING MECHANISMS REGULATING ENDOTHELIAL PERMEABILITY 0HYSIOL 2EV n "RINKMANN 6 #YSTER *' (LA 4 &49 SPHINGOSINE PHOSPHATE RECEPTOR IN THE CONTROL OF LYMPHOCYTE EGRESS AND ENDOTHELIAL BARRIER FUNCTION !M * 4RANSPLANT n ,A-ONTAGNE + ,ITTLEWOOD %VANS ! 3CHNELL # /2EILLY 4 7YDER , 3ANCHEZ 4 0ROBST " "UTLER * 7OOD ! ,IAU ' ET AL !NTAGONISM OF SPHINGOSINE PHOSPHATE RECEPTORS BY &49 INHIBITS ANGIOGENESIS AND TUMOR VASCULARIZATION #ANCER 2ES n 0ENG 8 (ASSOUN 0- 3AMMANI 3 -C6ERRY "* "URNE -* 2ABB ( 0EARSE $ 4UDER 2- 'ARCIA *'. 0ROTECTIVE EFFECTS OF SPHINGOSINE PHOSPHATE IN MURINE ENDOTOXIN INDUCED INFLAMMATORY LUNG INJURY !M * 2ESPIR #RIT #ARE -ED n $EJANA % %NDOTHELIAL CELLnCELL JUNCTIONS HAPPY TOGETHER .AT 2EV -OL #ELL "IOL n "RINKMANN 6 "AUMRUKER 4 0ULMONARY AND VASCULAR PHARMACOLOGY OF SPHINGO SINE PHOSPHATE #URR /PIN 0HARMACOL n 3ANNA -' 7ANG 3 + 'ONZALEZ #ABRERA 0* $ON ! -ARSOLAIS $ -ATHEU -0 7EI 3(
0ETER # (IESTAND ET AL
0ARKER ) *O % #HENG 7 # ET AL %NHANCEMENT OF CAPILLARY LEAKAGE AND RESTORATION OF LYMPHOCYTE EGRESS BY A CHIRAL 30 ANTAGONIST IN VIVO .AT #HEM "IOL n %HRLICH 0 $AS 3AUERSTOFF "EDàRFNISS DES /RGANISMUS %INE FARBENANALYTISCHE 3TUDIE !UGUST (IRSCHWALD "ERLIN "ALLABH 0 "RAUN ! .EDERGAARD - 4HE BLOOD BRAIN BARRIER AN OVERVIEW STRUCTURE REGULATION AND CLINICAL IMPLICATIONS .EUROBIOL $IS n (AWKINS "4 $AVIS 40 4HE BLOOD BRAIN BARRIERNEUROVASCULAR UNIT IN HEALTH AND DISEASE 0HARMACOL 2EV n !BBOTT *. 2ÙNNBÉCK , (ANSSON % !STROCYTEnENDOTHELIAL INTERACTIONS AT THE BLOODnBRAIN BARRIER .AT 2EV .EUROSCI n !RGAW !4 :HANG 9 3NYDER "* :HAO -, +OPP . ,EE 3# 2AINE #3 "ROSNAN #& *OHN '2 ), β REGULATES BLOOD BRAIN BARRIER PERMEABILITY VIA REACTIVATION OF THE HYPOXIA ANGIOGENESIS PROGRAM * )MMUNOL n #HIBA + &49 A NEW CLASS OF IMMUNOMODULATOR INHIBITS LYMPHOCYTE EGRESS FROM SECONDARY LYMPHOID TISSUES AND THYMUS BY AGONISTIC ACTIVITY AT SPHINGOSINE PHOSPHATE RECEPTORS 0HARMACOL 4HER n 3UZUKI 3 %NOSAWA 3 +AKEFUDA 4 3HINOMIYA 4 !MARI - .AOE 3 (OSHINO 9 #HIBA + ! NOVEL IMMUNOSUPPRESSANT &49 WITH A UNIQUE MECHANISM OF ACTION INDUCES LONG TERM GRAFT ACCEPTANCE IN RAT AND DOG ALLOTRANSPLANTATION 4RANSPLANTATION n 9ANAGAWA 9 3UGAHARA + +ATAOKA ( +AWAGUCHI 4 -ASUBUCHI 9 #HIBA + &49 A NOVEL IMMUNOSUPPRESSANT INDUCES SEQUESTRATION OF CIRCULATING MATURE LYMPHOCYTES BY ACCELERATION OF LYMPHOCYTE HOMING IN RATS )) &49 PROLONGS SKIN ALLOGRAFT SURVIVAL BY DECREASING 4 CELL INFILTRATION INTO GRAFTS BUT NOT CYTOKINE PRODUCTION IN VIVO * )MMUNOL n &U & (U 3 $ELEO * ,I 3 (OPF # (OOVER * 7ANG 3 "RINKMANN 6 ,AKE 0 3HI 6# ,ONG TERM ISLET GRAFT SURVIVAL IN STREPTOZOTOCIN AND AUTOIMMUNE INDUCED DIABETES MODELS BY IMMUNOSUPPRESSIVE AND POTENTIAL INSULINOTROPIC AGENT &49 4RANSPLANTA TION n 0EÖUELAS 2IVAS ' $OMÒNGUEZ 0ERLES 2 "RINKMANN 6 DEL 2IO -, -UÖOZ ,UNA ! 2AMÒ REZ 2OMERO 0 0ARRILLA 0ARICIO 0 2ODRÒGUEZ "ARBOSA *) &49 INHIBITS 4( MEDI ATED ALLOGENEIC HUMORAL IMMUNE RESPONSE 4RANSPLANT 0ROC n -ATSUURA - )MAYOSHI 4 /KUMOTO 4 %FFECT OF &49 A NOVEL IMMUNOSUPPRES SANT ON ADJUVANT AND COLLAGEN INDUCED ARTHRITIS IN RATS )NT * )MMUNOPHARMACOL n )DZKO - (AMMAR ( VAN .IMWEGEN - +OOL - -ßLLER 4 3OULLIÎ 4 7ILLART -!- (IJDRA $ (OOGSTEDEN (# ,AMBRECHT ". ,OCAL APPLICATION OF &49 TO THE LUNG ABROGATES EXPERIMENTAL ASTHMA BY ALTERING DENDRITIC CELL FUNCTION * #LIN )NVEST n +OHNO 4 4SUJI 4 (IRAYAMA + 7ATABE + -ATSUMOTO ! +OHNO 4 &UJITA 4 ! NOVEL IMMUNOMODULATOR &49 PREVENTS SPONTANEOUS DERMATITIS IN .#.GA MICE "IOL 0HARM "ULL n 9ANG : #HEN - &IALKOW ," %LLETT *$ 7U 2 "RINKMANN 6 .ADLER *, ,YNCH +2 4HE IMMUNE MODULATOR &94 PREVENTS AUTOIMMUNE DIABETES IN NONOBESE DIABETIC MICE #LIN )MMUNOL n 0OPOVIC * +OVER +, -OORE 76 4HE EFFECT OF IMMUNOMODULATORS ON PREVENTION
!SCOMYCETE DERIVATIVE TO -3 THERAPEUTIC 30 RECEPTOR MODULATOR &49
OF AUTOIMMUNE DIABETES IS STAGE DEPENDENT &49 PREVENTS DIABETES AT THREE DIFFERENT STAGES IN THE DIABETES RESISTANT BIOBREEDING RAT 0EDIATR $IABETES n -AKI 4 'OTTSCHALK 2 /GAWA . -ONACO !0 0REVENTION AND CURE OF AUTOIMMUNE DIABETES IN NONOBESE DIABETIC MICE BY CONTINUOUS ADMINISTRATION OF &49 4RANSPLAN TATION n +OHNO 4 4SUJI 4 (IRAYAMA + )WATSUKI 2 (IROSE - 7ATABE + 9OSHIKAWA ( +OHNO 4 -ATSUMOTO ! &UJITA 4 ET AL ! NOVEL IMMUNOMODULATOR &49 PREVENTS DEVEL OPMENT OF EXPERIMENTAL AUTOIMMUNE MYASTHENIA GRAVIS IN #", MICE "IOL 0HARM "ULL n +ITABAYASHI ( )SOBE - 7ATANABE . 3UZUKI * 9AZAKI 9 3EKIGUCHI - &49 PREVENTS DEVELOPMENT OF EXPERIMENTAL AUTOIMMUNE MYOCARDITIS THROUGH REDUCTION OF CIRCULATING LYMPHOCYTES * #ARDIOVASC 0HARMACOL n -IYAMOTO 4 -ATSUMORI ! (WANG -7 .ISHIO 2 )TO ( 3ASAYAMA 3 4HERAPEUTIC EFFECTS OF &49 A NEW IMMUNOSUPPRESSIVE AGENT IN A MURINE MODEL OF ACUTE VIRAL MYOCARDITIS * !M #OLL #ARDIOL n /KAZAKI ( (IRATA $ +AMIMURA 4 3ATO ( )WAMOTO - 9OSHIO 4 -ASUYAMA * &UJIMURA ! +OBAYASHI % +ANO 3 ET AL %FFECTS OF &49 IN -2, LPRLPR MICE THERAPEUTIC POTENTIAL IN SYSTEMIC LUPUS ERYTHEMATOSUS * 2HEUMATOL n (OZUMI 9 +OBAYASHI % -IYATA - &UJIMURA ! )MMUNOTHERAPY FOR EXPERIMEN TAL RAT AUTOIMMUNE THYROIDITIS USING A NOVEL IMMUNOSUPPRESSANT &49 ,IFE 3CI n +UROSE 3 )KEDA % 4OKIWA - (IKITA . -OCHIZUKI - %FFECTS OF &49 A NOVEL IMMUNOSUPPRESSANT ON EXPERIMENTAL AUTOIMMUNE UVEORETINITIS IN RATS %XP %YE 2ES n "UDDE + 3CHßTZ - 'LANDER 0 0ETERS ( 7AISER * ,IEFELDT , .EUMAYER ( ( "ÙHLER 4 &49 FINGOLIMOD IN RENAL TRANSPLANTATION #LIN 4RANSPLANT n %RCOLINI !- -ILLER 3$ -ECHANISMS OF IMMUNOPATHOLOGY IN MURINE MODELS OF CENTRAL NERVOUS SYSTEM DEMYELINATING DISEASE * )MMUNOL n 'OLD 2 ,ININGTON # ,ASSMANN ( 5NDERSTANDING PATHOGENESIS AND THERAPY OF MULTIPLE SCLEROSIS VIA ANIMAL MODELS YEARS OF MERITS AND CULPRITS IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS RESEARCH "RAIN n 3TEINMAN , :AMVIL 33 (OW TO SUCCESSFULLY APPLY ANIMAL STUDIES IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS TO RESEARCH ON MULTIPLE SCLEROSIS !NN .EUROL n "OLTON # "OREL *& #UZNER -, $AVISON !. 4URNER !- )MMUNOSUPPRESSION BY CYCLOSPORIN ! OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS * .EUROL 3CI n 4ANUMA . 3HIN 4 -ATSUMOTO 9 #HARACTERIZATION OF ACUTE VERSUS CHRONIC RELAPS ING AUTOIMMUNE ENCEPHALOMYELITIS IN $! RATS * .EUROIMMUNOL n -AIER + +UHNERT !6 4AHERI . 3ÉTTLER -" 3TORCH -+ 7ILLIAMS 3+ "ÉHR - $IEM 2 %FFECTS OF GLATIRAMER ACETATE AND INTERFERON BETA ON NEURODEGENERATION IN A MODEL OF MULTIPLE SCLEROSIS A COMPARATIVE STUDY !M * 0ATHOL n 3TEFFERL ! "REHM 5 3TORCH - ,AMBRACHT 7ASHINGTON $ "OURQUIN # 7ONIGEIT + ,ASSMANN ( ,ININGTON # -YELIN OLIGODENDROCYTE GLYCOPROTEIN INDUCES EXPERI MENTAL AUTOIMMUNE ENCEPHALOMYELITIS IN THE hRESISTANTv "ROWN .ORWAY RAT DISEASE SUSCEPTIBILITY )S DETERMINED BY -(# AND -(# LINKED EFFECTS ON THE " CELL RESPONSE * )MMUNOL n #HIBA + !DACHI + &49 $RUGS &UTURE n &UJINO - &UNESHIMA . +ITAZAWA 9 +IMURA ( !MEMIYA ( 3UZUKI 3 ,I 8 +
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!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
"Y 4HOMAS +UHN AND 9ING 7ANG 'LOBAL #HEMICAL /PERATIONS THOMASKUHN NOVARTISCOM .ATURAL 0RODUCT 5NIT .OVARTIS
)NSTITUTES FOR "IOMEDICAL 2ESEARCH .OVARTIS 0HARMA !' 0OSTFACH "ASEL 3WITZERLAND YINGWANG NOVARTISCOM
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
!BSTRACT !RTEMISININ BASED #OMBINATION 4HERAPIES !#4 ARE RECOMMENDED BY THE 7ORLD (EALTH /RGANIZATION 7(/ TO TREAT ESPECIALLY MULTIDRUG RESISTANT FORMS OF MALARIA AS CURRENTLY USED MEDICATIONS HAVE BECOME INCREASINGLY INEFFECTIVE )N THIS CHAPTER THE DISCOVERY OF ARTEMISININ FROM 4RADITIONAL #HINESE -EDICINE AND ITS FURTHER DEVELOPMENT TO !#4 ARE REVIEWED )T IS HIGHLIGHTED HOW THE COMPLEX SUPPLY CHAIN TO THE NATURALLY OCCURRING ENDO PEROXIDE ARTEMISININ REQUIRED TO PRODUCE !#4 BASED DRUGS WAS ESTABLISHED THUS ADDRESSING THE SIGNIFICANT THERAPEUTIC NEEDS AND HIGH DEMANDS FOR THE MEDICATION
4HE INFECTIOUS DISEASE MALARIA -ALARIA FROM THE )TALIAN WORDS hMALARIAv MEANING hBAD AIRv BASED ON THE EARLY BELIEF THAT THE DISEASE IS CAUSED BY BREATHING THE STALE WARM HUMID AIR AROUND SWAMPS IS CAUSED BY INFECTIONS WITH THE BLOOD PARASITE 0LAS MODIUM TRANSMITTED BY FEMALE MOSQUITOES 4HE RESEARCH ON MALARIA ITS COMPLEX INFECTION CYCLE AND THE RELATED VECTOR CONTROL HAS BEEN AWARDED FOUR .OBEL PRIZES 2ONALD 2OSS hFOR HIS WORK ON MALARIA BY WHICH HE HAS SHOWN HOW IT ENTERS THE ORGANISM AND THEREBY HAS LAID THE FOUNDA TION FOR SUCCESSFUL RESEARCH ON THIS DISEASE AND METHODS OF COMBATING ITv #HARLES ,OUIS !LPHONSE ,AVERAN hIN RECOGNITION OF HIS WORK ON THE ROLE PLAYED BY PROTOZOA IN CAUSING DISEASESv *ULIUS 7AGNER *AUREGG hFOR HIS DISCOVERY OF THE THERAPEUTIC VALUE OF MALARIA INOCULATION IN THE TREATMENT OF DEMENTIA PARALYTICAv AND 0AUL (ERMANN -ßLLER hFOR HIS DISCOVERY OF THE HIGH EFFICIENCY OF $$4 AS A CONTACT POISON AGAINST SEVERAL ARTHROPODSv ;= -ALARIA IS ASSOCIATED WITH FLU LIKE SYMPTOMS HIGH FEVER AND MAY RESULT IN COMA AND DEATH )T IS ESTIMATED THAT MILLION INFECTIONS OCCUR YEARLY IN !FRICA 3INCE EVERY SECONDS A PERSON n ESPECIALLY YOUNG CHILDREN n DIES MALARIA IS THE MOST PREVALENT TROPICAL DISEASE )N ADDI TION TO THE DEVASTATING TOLL MALARIA TAKES ON HUMAN LIFE IN TERMS OF MORBIDITY AND MORTALITY THE DISEASE HAS SUBSTANTIAL NEGATIVE IMPACT ON THE ECONOMIC DEVELOPMENT OF NATIONS IN WHICH THE DISEASE IS ENDEMIC 4HE DRAIN ON !FRICAN ECONOMIES ALONE IS ESTIMATED TO BE 53$ BILLION EACH YEAR 7(/ AND THE THREAT OF MALARIA CAN BE A SERIOUS DETER RENT TO TOURISM FURTHER HAMPERING ECONOMIC DEVELOPMENT AND GROWTH 4HE SUBSEQUENT MAP INDICATES CURRENT DISTRIBUTION OF INDIGENOUS MALARIA ACCORDING TO 7(/
4HOMAS +UHN AND 9ING 7ANG
&IGURE $ISTRIBUTION OF INDIGENOUS MALARIA ACCORDING TO 7(/ ;=
-ALARIA IS A PREVENTABLE AND TREATABLE DISEASE 0REVENTION IS BASED ON INSECTICIDE TREATED BED NETS )NDOOR 2ESIDUAL 3PRAYING )23 KILLING MOS QUITOES RESTING ON THE SPRAYED SURFACE LIKE AN INDOOR WALL AND TREATING MOSQUITOES BREEDING AREAS WITH INSECTICIDES )N MANY PARTS OF !SIA %UROPE AND THE !MERICAS MALARIA HAS BEEN BROUGHT UNDER CONTROL AND EVEN ELIMINATED (OWEVER IN !FRICA WITH INCREASING DRUG RESISTANCE AND STRUGGLING HEALTH SYSTEMS MALARIA INFEC TIONS HAVE ACTUALLY INCREASED IN THE LAST THREE DECADES 7(/ !RTEMIS ININ BASED #OMBINATION 4HERAPIES !#4 ARE THE MOST EFFECTIVE ANTIMA LARIA DRUGS CURRENTLY AVAILABLE 4HE 7(/ @2OLL "ACK -ALARIA INITIATIVE ;= PROVIDES COUNTRIES WITH GUIDELINES AND SUPPORT TO CHANGE TO !#4 4HE 'LOBAL &UND TO &IGHT !)$3 4UBERCULOSIS AND -ALARIA '&!4- ;= PROVIDES THE FINANCIAL PREREQUISITES TO IMPLEMENT THE MALARIA PROGRAMS IN THE COUNTRIES BASED ON BOTH PREVENTION AND TREATMENT 4HESE CON SORTED ACTIVITIES ARE FUNDAMENTAL FOR THE IMPROVEMENT OF THE DISEASE SITU ATION AND CREATE THE BASIS FOR FURTHER DEVELOPMENT IN THE COUNTRIES WHERE MALARIA IS ENDEMIC
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
$ISCOVERY AND DEVELOPMENT OF ARTEMISININ ARTEMETHER AND #OARTEM¤
$ISCOVERY OF OUTSTANDING ANTIMALARIAL ACTIVITY OF 1INGHAO !RTEMISIA ANNUA , BASED ON 4RADITIONAL #HINESE -EDICINE 4HE EMERGENCE AND SPREAD OF DRUG RESISTANCE REPRESENTS A CONSIDERABLE CHALLENGE TO THE CONTROL OF MALARIA 7ITH THE RISE OF RESISTANCE MANY ANTIMALARIAL DRUGS ARE BECOMING INCREASINGLY INEFFECTIVE )N THE S THE ANTIPLASMODIAL THERAPY WAS BASED MAINLY ON CHLOROQUINE DEVELOPED TOWARDS THE END OF 7ORLD 7AR )) ;= &ATALLY RESISTANCE TO CHLOROQUINE OCCURRED THROUGHOUT THE TROPICAL WORLD AND RESISTANCE TO ITS SUCCESSORS SUCH AS SULPHADOXINE PYRIMETHAMINE 30 &ANSIDAR AND -EFLOQUINE ,AR IAM HAVE DEVELOPED RAPIDLY !T THE BEGINNING OF THE S THE DRUG RESISTANCE PREVENTED CONTROL OF GLOBAL MALARIA EPIDEMICS AND DURING THE 6IETNAM 7AR !MERICAN AND 6IETNAMESE SOLDIERS WERE HEAVILY AFFECTED BY THE DISEASE 0OSSESSION OF AN ANTIMALARIAL DRUG BECAME AN IMPORTANT STRATEGIC FACTOR FOR BOTH ARMIES !MERICANS SCREENED THOUSANDS OF COMPOUNDS BUT FAILED TO FIND THE IDEAL ANTIMALARIA WONDER DRUG 6IETNAM SOUGHT HELP FROM #HINA WHICH TRIGGERED A MAJOR #HINESE INITIATIVE TO DEVELOP NEW ANTIMALARIAL DRUGS 4HE #HINESE GOVERNMENT BEGAN A SYSTEMATIC INVESTIGATION OF PLANTS USED IN 4RADITIONAL #HINESE -EDICINE 4#- WITH THE AIM OF FINDING A NEW ANTIPLASMODIAL DRUG !N EMERGENCY MILITARY PROGRAM WAS LAUNCHED IN "ECAUSE THE STARTUP DATE WAS -AY THIS PROGRAM WAS CODED AS @ INCLUDING MORE THAN SCIENTISTS FROM OVER INSTITUTES NATIONWIDE ;= -ORE THAN COMPOUNDS AND MEDICINAL PLANT EXTRACTS WERE SCREENED IN THE ASSAYS AGAINST MALARIA AT THE EARLIER STAGE OF S 4#- USED IN THE HEALTHCARE FOR THOUSANDS OF YEARS PROVIDES EXTREMELY RICH RESOURCES FOR NEW DRUG DISCOVERY -ANY MEDICINAL PLANTS WERE SCREENED AND THEIR ACTIVE PRINCIPLES HAVE BEEN ISOLATED AND IDENTIFIED SUCH AS #HANGSHAN $ICHROA FEBRIFUGA , 1INGHAO ! ANNUA , 9INGZHUA !RT ABOTRYS HEXAPETALU ,& "HAND 8IANHECAO !GRIMONIA PILOSA , $AYEAN %UCALYPTUS ROBUSTA 3M .ANTIANZU .ANDINA DOMESTICA 4 9ADANZI "RUCEA JAVANICA , -ERR ,INGSHUIANLUO 0OLYALTHIA NEMORALIS ! $# ;= 4HE OUTSTANDING ANTIMALARIAL ACTIVITY @3AMPLE WHICH WAS GENERATED BY
4HOMAS +UHN AND 9ING 7ANG
0ICTURE -EDICINAL PLANT ! ANNUA ,
ETHER EXTRACTION FROM 1INGHAO ! ANNUA , IN THE FAMILY OF !STERACEAE WAS OBSERVED ON /CTOBER ;= ! ANNUA ALSO KNOWN AS 3WEET 7ORMWOOD #HINESE 㕍⫺ 藿 PINYIN ¥ Q¥>NGH AO GROWS IN TEMPERATE CLIMATES )T IS MOST WIDESPREAD IN #HINA AND 6IETNAM ALTHOUGH IT IS ALSO FOUND AND GROWN IN %AST !FRICA THE 5NITED 3TATES 2USSIA )NDIA "RAZIL AND SOME OTHER COUNTRIES 4HE PLANT HAS FERN LIKE LEAVES BRIGHT YELLOW FLOWERS AND A CAMPHOR LIKE SCENT )T REACHES IN AVERAGE METERS HAS A SINGLE STEM WITH ALTERNATING BRANCHES CARRYING ALTERNATING LEAVES OF n CM LENGTH 7IND OR INSECTS CROSS POLLINATE THE PLANT
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
1INGHAO ! ANNUA WAS RECORDED AS A REMEDY FOR HEMORRHOIDS FOUND NOTED ON A PIECE OF SILK UNEARTHED FROM THE -AWANGDUI 4OMB OF THE 7EST (AN $YNASTY "# )N 4HE (ANDBOOK OF 0RESCRIPTIONS FOR %MERGENCY 4REAT MENTS WRITTEN BY 'E (ONG n !$ IN %AST *IN $YNASTY 1INGHAO WAS DESCRIBED FOR THE TREATMENT OF FEVER "ASED ON THE THEORY IN 4#- 1INGHAO IS COOL IN NATURE 9IN AND CAN THEREFORE BE USED TO TREAT INTERNAL HEAT CONDITIONS 9ANG )N THE FAMOUS #OMPENDIUM OF -EDICINAL (ERBS "EN CAOGANMU EDITED BY ,I 3HIZHEN IN -ING $YNASTY !$ 1INGHAO WAS A REMEDY FOR THE TREATMENT OF hHOT AND COLD DUE TO INTERMITTENT FEVER ILLNESSv AGUES !S THE FIRST PHYSICIAN IN MEDICAL HISTORY TO RECOMMEND 1INGHAO FOR THE TREATMENT OF hINTERMITTENT FEVERSv 'E (ONG WROTE THE RECIPE IN HIS BOOK hONE HANDFUL BUNCH OF 1INGHAO SOAKED IN TWO 3HENG OF WATER WRING IT OUT FOR GETTING JUICE AND DRINK IT ALLv $IFFERENT FROM THE MOST POPULAR DECOCTING WAY IN 4#- HIS RECOMMENDATION TO SOAK THE FRESH PLANT IN WATER AND TO WRING IT OUT THEREAFTER COULD BE A CRUCIAL INSTRUCTION TO GET AN EMULSION OF WATER AND PLANT JUICE CONTAINING THE ACTIVE PRINCIPLES )N THE PRELIMINARY SCREENING PROCESS 1INGHAO EXTRACTS PREPARED BY HEATING IN WATER OR ALCOHOL DID NOT SHOW SIGNIFICANT ANTIMALARIAL ACTIVITY AT ALL 4HE BREAKTHROUGH WAS ATTRIBUTED TO THE FINDING OF AN ADAPTED EXTRACTION METHOD WITH ETHER GUIDED BY THE HINT IN 'O (ONGS RECIPE DESCRIPTION ;=
$ISCOVERY OF ARTEMISININ AS AN EFFECTIVE ANTIMALARIA DRUG FROM ! ANNUA ! ANNUA CONTAINS ESSENTIAL OILS WITH AT LEAST VOLATILE COMPOUNDS AND SEVERAL NONVOLATILE SESQUITERPENES !RTEMISININ AND RELATED COMPOUNDS ARE THE SESQUITERPENES OF THE HIGHEST INTEREST DUE TO THEIR ANTIMALARIAL PROPERTIES ; = !S THE MOST PROMISING COMPOUND WITH OUTSTANDING 㕍⫺⚛ ANTIMALARIAL ACTIVITY AT LOW TOXICITY 1INGHAOSU 藿 藿 IN #HINESE OR ARTEMISININ IN %NGLISH OR OCCASIONALLY NAMED AS ARTEANNUIN WAS ISOLATED IN PURE FORM FROM ! ANNUA IN #HINA 4HE STRUCTURE ELUCIDATION OF ARTEMISININ REVEALED THE MOLECULAR FORMULA #(/ AND ITS SESQUITERPENE LACTONE SKELETON WAS READILY DETER MINED BASED ON SPECTROSCOPICAL METHODS )2 -3 AND .-2 AND ELEMENTAL
4HOMAS +UHN AND 9ING 7ANG
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ANALYSIS 4HE MAJOR DIFFICULTY WAS TO ARRANGE THE FIVE OXYGEN ATOMS IN THE CARBON MOLECULAR SKELETON SINCE ONLY ONE PROTON WAS ATTACHED TO THE CARBON BEARING AN OXYGEN ATOM )NSPIRED BY THE PEROXIDE STRUCTURE YING ZHUASU IN THE EARLY ARTEMISININ WAS CONFIRMED AS A PEROXIDE COM POUND BY SIMPLE QUALITATIVE AND QUANTITATIVE ANALYSIS )TS CRYSTAL STRUCTURE AND ABSOLUTE CONFIGURATION WERE DETERMINED BY X RAY CRYSTALLOGRAPHIC ANALYSIS ;= 4HE UNPRECEDENTED STRUCTURE OF ARTEMISININ WITH AN INTER PEROXYL KETAL ACTAL LACTONE IS QUITE UNIQUE )T WAS BROADLY DOUBTED THAT A FUNCTIONAL GROUP LIKE THE TRIOXANE RING EMBEDDED IN THE STRUCTURE OF ARTEMISININ COULD BE STABLE AT ALL 7E NOW KNOW THAT ESPECIALLY THIS UNUSUAL STRUCTURAL FEATURE OF ARTEMISININ IS THE BASIS FOR THE UNIQUE ANTIMALARIAL ACTION OF THE DRUG !RTEMISININ IS A MEMBER OF THE AMORPHANE SUB CLASS OF CADINANE SES QUITERPENES )T IS BIOSYNTHESIZED IN ! ANNUA FROM MEVALONATE OR $ GLYCER ALDEHYDE PHOSPHATE VIA DIMETHYLALLYL AND ISOPENTENYL PYROPHOSPHATES FARNESYL PYROPHOSPHATE AND AMORPHADIENE 4HE IMMEDIATE PRECURSOR IS DIHYDROARTEMISINIC ACID ARISING VIA REDUCTION OF ARTEMISINIC ACID 4HE MAX IMUM CONCENTRATIONS OF ARTEMISININ OCCUR IN THE LEAVES SMALL GREEN STEMS BUDS FLOWERS AND SEEDS WHEREAS THE MAIN STEMS AND ROOTS CONTAIN NEGLI GIBLE AMOUNTS IN ! ANNUA )T WAS ALSO REPORTED THAT ARTEMISININ OCCURS IN SOME OTHER SPECIES EG ! APIACEA AND ! LAMCEA BUT ONLY AT A MUCH LOWER LEVEL ;= 4HE ARTEMISININ BIOSYNTHESIS IS HIGHLY SENSITIVE TO A SEASONALLY DEPENDENT COMBINATION OF HUMIDITY AND DAYLIGHT LIMITING THE GEOGRAPHI CAL REGIONS FOR OPTIMAL HORTICULTURE /N STARTING TO BLOOM THE ARTEMISININ CONTENT DRAMATICALLY DECLINES !RTEMISININ FORMATION IN ! ANNUA IS USUALLY IN THE RANGE OF TO APPROXIMATELY IN WILD GROWING PLANTS ;= HOWEVER SOME CLONES PRO
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
DUCE UP TO ;= !RTEMISININ CAN ALSO BE OBTAINED FROM ARTEMISINIC ACID WHICH WAS ESTIMATED TO OCCUR AT CONCENTRATIONS AS MUCH AS FOLD HIGHER THAN ARTEMISININ ;= 4HE FIRST PATIENT TREATED WITH ARTEMISININ DURING CLINICAL TRIALS WAS A YEAR OLD PRIMARY SCHOOL PUPIL WHO SUFFERED FROM SEVERE MALARIAL SYMPTOMS )N SUBSEQUENT CLINICAL TEST SERIES WITH PATIENTS THE PARASITE OCCURRENCE DECREASED MORE THAN AFTER H WITH ARTEMISININ WHEREAS THE SAME RESULTS WITH CHLOROQUINE COULD BE ACHIEVED ONLY AFTER H ;= -OST IMPORTANTLY ARTEMISININ WAS COMPLETELY EFFECTIVE IN TREATING CHLO ROQUINE RESISTANT FALCIPARUM MALARIA AND ITS OFTEN FATAL COMPLICATION THE CEREBRAL FORM !RTEMISININ DISPLAYS A UNIQUE MECHANISM OF ACTION WHILE ITS DETAILS ARE STILL NOT YET COMPLETELY ELUCIDATED $URING THE PLASMODIAL INFECTION CYCLE THE PARASITE INVADES RED BLOOD CELLS TO DIGEST THE PROTEIN PART OF HEMO GLOBIN !S SOON AS THE HEME GROUP IS NO LONGER PROTECTED BY GLOBIN THE ACCESSIBLE IRON REACTS WITH THE PEROXIDE MOIETY OF THE ARTEMISININ FORMING HARMFUL FREE RADICALS AND ALKYLATING AGENTS DAMAGING THE PARASITIC MACRO MOLECULES AND CELL MEMBRANES ;= 4HE MECHANISM OF ARTEMISININ ACTION LED TO A NEW HYPOTHESIS OF ANTI CANCER INTERVENTION AS SOME CANCER CELLS SHOW SIGNIFICANTLY ELEVATED IRON CONCENTRATIONS 4HE POTENTIAL ANTICANCER PROPERTIES OF ARTEMISININ HAVE BEEN INVESTIGATED INDICATING A POTENTIAL BENEFIT FOR THE TREATMENT OF LEU KEMIA AND OTHER CANCER CELLS ;=
$EVELOPMENT OF A NEW GENERATION OF DRUGS DERIVED FROM ARTEMISININ 4HE COMBINATION OF AN OUTSTANDING BIOLOGICAL ACTIVITY AND AN INTRIGU ING CHEMICAL STRUCTURE INCITED SCIENTISTS TO DEVELOP TOTAL SYNTHETIC ROUTES TOWARD THIS NOVEL NATURAL PRODUCT 4HE FIRST TOTAL SYNTHESIS OF ARTEMISININ WAS REPORTED BY A RESEARCH GROUP AT (OFFMANN ,A 2OCHE ON &EBRU ARY 4HEY USED ISOPULEGOL AS THE STARTING MATERIAL AND THE SINGLET OXYGEN OXIDATION AS THE KEY STEP TO INTRODUCE THE CRITICAL PEROXY GROUP ;= "ESIDES ARTEMISININ ITSELF THE SYNTHESIS OF A SERIES OF SESQUITERPENE CONGENERS OF ARTEMISININ AND THEIR ISOMERS WERE ACCOMPLISHED IN #HINA 5P TO NOW VARIOUS SYNTHETIC ROUTES FOR THE SYNTHESIS OF ARTEMISININ HAVE
4HOMAS +UHN AND 9ING 7ANG
BEEN REPORTED 4HE PEROXIDE WAS INTRODUCED WITH PHOTOOXYDATION REAC TION IN DIFFERENT WAYS ;= OR USES THE REACTION OF VINYLSILANES WITH OZONE FOLLOWED BY REARRANGEMENT AND CYCLIZATION !LTHOUGH TOTAL SYNTHESES OF ARTEMISININ ARE NOT COMMERCIALLY VIABLE UP TO NOW THEY COULD BE USEFUL FOR THE PREPARATION OF LABELED ARTEMISININ REQUIRED FOR ANALYTICAL AND DRUG DISTRIBUTION STUDIES !RTEMISININ IS STABLE IN NEUTRAL SOLVENTS BUT SENSITIVE TO ACID AND BASE TREATMENT )N COMPARISON WITH OTHER PEROXIDES ARTEMISININ IS THERMAL STABLE EVEN AT ITS MELTING POINT # AND ONLY DECOMPOSES AFTER MELT ING !RTEMISININ TENDS TO THERMALLY INDUCE SPONTANEOUS DECOMPOSITION 5. CLASSIFICATION 0 (OWEVER IT IS POORLY SOLUBLE NOT ONLY IN WATER BUT ALSO IN OIL ;= 4HEREFORE THE OVERRIDING GOAL IN CREATING THE FIRST GENERATION OF DRUGS DERIVED FROM ARTEMISININ WAS TO IMPROVE ITS SOLUBILITY CHARACTERISTICS SO THAT THE NEW DRUGS SHOULD BE MORE EASILY FORMULATED AND MORE EFFICIENTLY DELIVERED 4HE INITIAL STUDIES ON THE STRUCTURE ACTIVITY RELATIONSHIP SHOWED THAT THE PEROXIDE GROUP OF ARTEMISININ WAS AN ESSENTIAL PART FOR ITS BIOLOGICAL ACTIVITY 4HEREFORE THE MOST ACCESSIBLE FUNCTIONALITY FOR CHEMICAL INTER VENTION IS THE LACTONE GROUP ON WHICH THE FIRST CHEMICAL MODIFICATIONS WERE STARTED $IHYDROARTEMISININ PREPARED BY SIMPLE REDUCTION OF ARTE MISININ WITH SODIUM BOROHYDRIDE ;= WAS FOUND TO BE MORE POTENT BUT WITHOUT IMPROVING THE SOLUBILITY IN WATER BY INTRODUCING A HYDROXYL GROUP INTO THE MOLECULE )T ALSO LACKED STABILITY IN VIVO (UNDREDS OF DERIVATIVES WERE PREPARED TO ATTACH A SUBSTITUENT TO THE FREE HYDROXYL GROUP INCLUDING ETHER ESTER OR CARBONATE LINKAGES ;= 4HE ETHER SUBSTITUTED DRUGS ARTEMETHER A AND ARTEETHER B ARE BOTH SOLUBLE IN SESAME OR PEANUT OIL 4HEY CAN BE ADMINISTERED BY INTRA MUSCULAR INJECTION &OR THE TREATMENT OF ADVANCED CASES OF MALARIA WATER SOLUBLE ANALOGS OF ARTEMISININ EG SODIUM ARTESUNATE ARE INJECTABLE INTRAVENOUSLY !RTEMISININ ARTEMETHER A AND SODIUM ARTESUNATE WERE APPROVED AS NEW ANTIMALARIAL DRUGS BY THE #HINESE AUTHORITIES IN n !RTEMETHER FOR INJECTION MGM, IN M, AMPOULE AND ARTESU NATE TABLET MG WERE ADDED BY 7(/ TO THE TH AND THE TH %SSENTIAL -EDICINES ,IST ;= PUBLISHED IN AND IN RESPECTIVELY -ETABOLIC INSTABILITY WAS THE MAJOR WEAKNESS FOR ARTEMETHER AND ARTE SUNATE 2APIDLY METABOLIZED TO DIHYDROARTEMISININ IN VIVO ARTEMETHER
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SHOWED QUITE SHORT HALF LIFE TIME 7ITH THE HEMI ESTER LINKAGE ARTESUNATE WAS HYDROLYZED TO DIHYDROARTEMISININ EVEN MORE RAPIDLY THAN ARTEMETHER !NALOGS WITH IMPROVED METABOLIC STABILITY WERE THE FOCUS OF SUBSEQUENT DERIVATION EFFORTS &URTHER DERIVATION STRATEGIES AIMED AT THE REMOVAL OF THE LACTONE CARBONYL GROUP INCLUDE SUBSTITUTION OF OTHER POSITIONS AND ELIMINATION OF RING " OR $ IN ARTEMISININ ! SYNTHETICALLY DERIVED LEAD COMPOUND COMPOSED ONLY BY THE TRIOXANE RING /: ALSO KNOWN AS 2"X WAS INSPIRED BY THE ENDOPEROXIDE RING SYSTEM OF ARTEMISININ 4HE COMPOUND WAS REPORTED TO BE MORE POTENT LONG LASTING AND STRUCTURALLY SIMPLER THAN THE ARTEMIS ININ AND ITS SYNTHESIS IS AMENABLE TO INDUSTRIAL SCALE UP 0HASE )) STUDIES OF THE COMPOUND BEGAN IN $ECEMBER ! FINAL PRODUCT /: IN COMBINATION WITH PIPERAQUINE COULD REACH THE MARKET BY ;=
4HOMAS +UHN AND 9ING 7ANG
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$EVELOPMENT OF !#4 n #OARTEM¤ &OLLOWING THE INCREASING RESISTANCE TO AVAILABLE AGENTS COMBINATION THERA PIES ARE BECOMING A VERY IMPORTANT STRATEGY IN ANTIMALARIAL DRUG DEVELOP MENT 4HE EXPERIENCES IN THE THERAPY OF INFECTIOUS DISEASES HAVE DEMON STRATED THAT CURE RATES ARE MOST SUCCESSFUL BY USING COMBINATIONS OF DRUGS AND THAT THIS STRATEGY PROTECTS DRUGS IN A MUTUAL FASHION 4HE USE OF SINGLE DRUGS THAT ARE EASILY PRONE TO RESISTANCE LIMITS THERAPEUTIC CHOICE AND CAN HAVE SERIOUS PUBLIC HEALTH CONSEQUENCES 4HIS IS PARTICULARLY TRUE FOR INFECTIONS LIKE MALARIA FOR WHICH A LIMITED NUMBER OF DRUGS ARE AVAILABLE 4HEREFORE COMBINATION THERAPY SHOULD NOT ONLY IMPROVE ANTIMALARIAL EFFICACY BY PROVIDING ADDITIVE OR SYNERGISTIC ANTIPARASITIC ACTIVITY BUT ALSO SLOW THE PROGRESSION OF PARASITE RESISTANCE TO THE NEW AGENTS ;= 6ARIOUS DRUG COMBINATIONS IN ANTIMALARIA THERAPY HAVE BEEN DEVEL OPED 4HOSE DRUGS PERTINENT TO THE TREATMENT OF ACUTE UNCOMPLICATED FALCIPARUM MALARIA INCLUDE CHLORPROGUANIL DAPSONE ,AP$AP DEVELOPED JOINTLY BY 'LAXO3MITH+LINE ,IVERPOOL 5NIVERSITY AND 7(/4$2 3PECIAL 0ROGRAM FOR 2ESEARCH AND 4RAINING IN 4ROPICAL $ISEASES DIHYDROARTEMIS ININPIPERAQUINE $(!001 DEVELOPED AND REGISTERED IN #HINA UNDER THE BRAND NAME !RTEKIN4- ARTESUNATE AMODIAQUINE !3 !1 AND ARTESU NATE MEFLOQUINE !3 30 WIDELY USED IN 4HAILAND AND OTHER PARTS OF THE 3OUTH %AST !SIA ARTESUNATEAYRONARIDINE !302. IN THE EARLY STAGES OF DEVELOPMENT BY -EDICINES FOR -ALARIA 6ENTURE WITH A 3OUTH +OREAN COM PANY AND 7(/4$2 ;= !RTEMETHERLUMEFANTRINE #OARTEM¤ IS THE ONLY PRE QUALIFIED FIXED DOSE !#4 )T IS A HIGHLY EFFECTIVE AND WELL TOLERATED ANTIMALARIAL THAT ACHIEVES CURE RATES OF UP TO EVEN IN AREAS OF MULTIDRUG RESISTANCE )T
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IS INDICATED FOR THE TREATMENT OF ACUTE UNCOMPLICATED FALCIPARUM MALARIA THE MOST DANGEROUS FORM OF MALARIA "OTH COMPONENTS IN THIS COMBINA TION WERE ORIGINALLY DISCOVERED AND DEVELOPED IN #HINA 4HE COMBINATION THERAPY ARTEMETHERLUMEFANTRINE WAS DISCOVERED BY THE )NSTITUTE OF -ICRO BIOLOGY AND %PIDEMIOLOGY OF THE !CADEMY OF -ILITARY -EDICAL 3CIENCES !--3 IN "EIJING ,UMEFANTRINE BELONGS TO THE FAMILY OF ARYLAMINO ALCOHOLS AND IS RELATED TO QUININE ONE OF THE FIRST AGENTS IN THE HISTORY OF CHEMOTHER APY )N THE S FLUORENEMETHANOLS WERE INVESTIGATED AND LUMEFANTRINE APPEARED AS THE CHLOROBENZYLIDENE ANALOGUE OF EARLIER EXAMPLES ,UMEFANTRINE BENFLUMETOL WAS SUCCESSFULLY DEVELOPED AND REGISTERED IN #HINA AS A SINGLE DRUG ENTITY 4HE COMBINATION OF ARTEMETHER AND LUMEFANTRINE WAS REGISTERED IN IN #HINA )N COLLABORATION WITH .OVARTIS BASED ON A LICENSE AND DEVELOPMENT AGREEMENT #OARTEMETHER WAS REGISTERED IN IN 3WITZERLAND .OVARTIS PROVIDES A THREE DAY TREATMENT REGIMEN OF #OARTEM¤ ;= FOR ADULTS AND CHILDREN AT LOW COST THROUGH THE PUBLIC MARKET #OAR TEM¤ WAS TAKEN UP ON THE %SSENTIAL -EDICINES ,IST BY THE 7(/ IN ;= AND IT WAS AGREED BETWEEN .OVARTIS AND 7(/ TO SUPPORT THE 2OLL "ACK -ALARIA PROGRAM WITH #OARTEM¤ #HINESE PARTNERS SUPPLY THE ACTIVE INGREDIENTS ARTEMETHER AND LUME FANTRINE FOR #OARTEM¤ WHICH IS PRODUCED BY .OVARTIS IN #HINA AND THE 53 #OARTEM¤ IS CURRENTLY REGISTERED IN COUNTRIES WORLDWIDE AND MORE THAN MILLION TREATMENT COURSES HAVE BEEN SUPPLIED TO THE PUBLIC SEC TOR OF MALARIA ENDEMIC DEVELOPING COUNTRIES SINCE #OARTEM¤ HAS
4HOMAS +UHN AND 9ING 7ANG
BEEN EXTENSIVELY STUDIED IN MULTICENTER CLINICAL TRIALS INVOLVING MORE THAN PATIENTS ! PEDIATRIC DOSAGE FORM DISPERSIBLE TABLET IS CURRENTLY IN DEVELOPMENT
-ARKET DEVELOPMENT THROUGH SUPPLY CHAIN ENLARGEMENT OF ! ANNUA )NTRODUCTION 4HE MAJOR CHALLENGE IN ESTABLISHING A SUPPLY CHAIN FOR PLANT DERIVED COM POUNDS IS THE TIME NEEDED FOR SEASON RELATED PLANTING CULTIVATING AND HARVESTING )N COMPARISON TO SYNTHETICALLY DERIVED COMPOUNDS THE ASSOCI ATED SPECIFIC REQUIREMENTS IN TERMS OF PLANT METABOLITE PRODUCTION AFFORD THE MANAGEMENT OF SUPPLY RISKS DIFFICULT TO CONTROL 0ARTICULARLY WEATHER CONDITIONS DURING PLANTING AND CULTIVATION DAMAGE OF THE CROP THROUGH INSECTS AND UNFORESEEABLE METEOROLOGICAL IMPACTS MAY ONLY BE ADDRESSED WITH SAFETY STOCK MARGINS ABOVE THE COMMONLY APPLIED RATIOS FOR CHEMI CALLY MANUFACTURED SUBSTANCES )F THE HARVEST FALLS UNDER A PROJECTED YIELD THE MISSING QUANTITY CAN ONLY BE RESUPPLIED DURING THE FOLLOWING VEGETA TION CYCLE USUALLY MONTHS LATER ! MARKET EXPANSION FOR AN AGRICULTURAL PRODUCT STARTING FROM AN INI TIALLY INSUFFICIENT PRODUCTION VOLUME OFTEN LEADS TO A SUBSEQUENT OVERSUP PLY !TTRACTED BY A SHORTAGE SITUATION SELLERS MARKET WITH ITS CHARACTERIS TIC HIGH PRICES ENTHUSIASTIC ENTREPRENEURS ENTER THE MARKET INVESTING IN BIOMASS AND EXTRACTION CAPACITY 4HE SCALE UP GETS BROADER THAN REQUIRED THE VOLUME IS LARGER THAN THE MARKET ACTUALLY IS ABLE TO ABSORB 7ITH THE OVERSUPPLY THE MARKET PRICE DROPS THE BUSINESS GETS LESS ATTRACTIVE PRO DUCTION IS REDUCED LEADING TO UNDERSUPPLY AGAIN 4HIS ECONOMIC CYCLE IS ALMOST IMPOSSIBLE TO CONTROL AND IT TAKES A CONSIDERABLE TIMEFRAME TO BAL ANCE SUPPLY AND DEMAND 'ENERALLY LAUNCH AND SCALE UP OF A NEW PRODUCT BEAR AN INTRINSIC UNCERTAINTY WITH REGARD TO THE NEEDED AMOUNT OF MATERIAL AND THE TIMEFRAME IN WHICH THE PRODUCED MATERIAL WILL BE TAKEN UP BY THE MARKET 4HE ECONOMIC CYCLE OF AN AGRICULTURALLY DERIVED PRODUCT AND THE ADDITIONAL AGRICULTURAL RISKS REQUIRE ADDITIONAL STRATEGIC STOCKS TO SUPPORT THE PROJECTED SUPPLY CHAIN AVOIDING A POTENTIAL SHORTAGE IN SUPPLY IN THE
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
LAUNCH PHASE $EPENDING ON THE VOLUMES SIGNIFICANT FUNDS ARE INVOLVED AND INVESTED AT RISK
-ARKET SITUATION FOR ! ANNUA IN #OARTEM¤ WAS LAUNCHED IN AND UNTIL THE TRADITIONALLY AVAIL ABLE AMOUNTS OF ! ANNUA WERE SUFFICIENT FOR DRUG SUBSTANCE PRODUCTION )N #HINA FARMERS COLLECTED THE PLANT WILD IN THE MOUNTAINOUS AREAS OF #HONGQING AND 3ICHUAN 0ROVINCE JUST BEFORE FLOWERING !FTER DRYING THE WHOLE PLANT IN THE FIELDS THE BIOMASS WAS TRANSPORTED TO COLLECTION POINTS WHERE THE LEAVES WERE SEPARATED FROM THE BRANCHES 0ACKED IN SACKS THE LEAF MATERIAL WAS FURTHER TRANSPORTED TO TRADERS WHO SOLD THE PRODUCT TO THE EXTRACTION COMPANIES 4HE REQUIRED AMOUNTS FOR THE #HINESE MARKET WERE AT THAT TIME APPROXIMATELY n TONS OF ARTEMISININ 6IETNAM WAS AN ADDITIONAL SUPPLIER PRODUCING SMALLER QUANTITIES FOR EXPORT ,OCAL PRODUC TION OCCURRED IN !FRICA AND )NDIA WHERE THE LEAVES WERE COLLECTED FOR LOCAL USE ESPECIALLY FOR MEDICINAL TEA
-ARKET SITUATION FOR ! ANNUA AFTER 3EVERAL !FRICAN COUNTRIES REACTED TO THE INCREASING RESISTANCES AGAINST TRADI TIONAL MALARIA TREATMENTS BY A SUBSTITUTION WITH !#4S IN THE MIDDLE OF 4HIS LED TO AN UNEXPECTED DEMAND FOR ARTEMISININ EXCEEDING THE SUPPLY CAPACITIES OF THE ENTIRE MARKET 4HE AVAILABLE VOLUMES FROM THE PREVIOUS YEAR COULD NOT MEET THE NEW REQUIREMENTS 4HE HARVEST VOLUMES IN WERE DETERMINED BASED ON THE DEMANDS AT THAT TIME 4HE HARVEST IN RELIED ON WILD PLANTS THE SEEDING SEASON *ANUARY FOR PLANTATION ALREADY PASSED )N THE EXPANSION OF PLANTING EFFORTS COULD BE STARTED BUT THESE ADDITIONAL AMOUNTS AFFECTED MOSTLY THE SUPPLY /NLY MINOR QUANTITIES REACHED THE MARKET IN DUE TO THE LEAD TIMES FOR HARVESTING STARTING IN *UNE 6IETNAM AND LASTING TO EARLY 3EPTEMBER #HINA AND THE LIMITED NOT YET EXPANDED EXTRACTION AND PURIFICATION CAPACITIES FOR ARTEMISININ 4HE TIGHT ARTEMISININ SUPPLY SITUATION AND THE ONSET OF COMPETITION IN BIOMASS PRODUCTION BY THE ENTRIES OF NEW SUPPLIERS INCREASED THE MARKET COMPLEXITY 4HE FARMERS HARVESTED EARLIER TO ASSURE THEIR INCOME BY BEING
4HOMAS +UHN AND 9ING 7ANG
&IGURE %NTIRE LEAD TIME TO PRODUCE THE !#4 #OARTEM¤
AMONG THE FIRST SUPPLIERS HOWEVER NEGATIVELY AFFECTING THE ARTEMISININ CONTENT IN THE BIOMASS BY MISSING THE OPTIMAL HARVEST TIME 4HE RELATED LOWER ARTEMISININ CONTENT INCREASED THE NEEDED BIOMASS VOLUME AND THE COSTS 4HE BIOMASS WAS KEPT LONGER IN STORAGE AREAS TO ARTIFICIALLY INCREASE THE SHORTAGE AND THEREFORE INCREASING THE COSTS FOR BIOMASS .EW EXTRACTION COMPANIES WITH NO OR LIMITED EXPERIENCE STARTED BUSINESS AND ENTERED THE ARTEMISININ MARKET 4HE BIOMASS SHORTAGE SITUATION FORCED THESE COMPA NIES TO OFFER HIGHER PAYMENTS TO FARMERS TO BE ABLE TO ENTER THE MARKET 4HESE COMPANIES REQUIRED MORE BIOMASS THAN EXPECTED SINCE UNSUITABLE STORAGE AND TRANSPORTATION LED TO A LOSS OF BIOMASS 3UBOPTIMAL EXTRACTION AND PURIFICATION PROCESSES ALSO LED TO POOR QUALITY OF ARTEMISININ WHICH DID NOT MEET THE PURITY CRITERIA )N THE MEANTIME ESTABLISHED TRADING ANDOR EXTRACTION COMPANIES REALIZED THEIR DECLINING MARKET INFLUENCE AND TRIED TO PROTECT THEIR MARKET STATUS BY ABSORBING AS MUCH BIOMASS AS POSSIBLE INCREASING THE BIOMASS MARKET PRICE ADDITIONALLY 4O DEVELOP ARTEMISININ AND SUBSEQUENTLY THE DRUG SUBSTANCE SUP PLY CHAIN WHILE BEING UNDER TREMENDOUS DEMAND AND TIME PRESSURE TO PRODUCE THE URGENTLY NEEDED !#4 #OARTEM¤ WAS AN EXTREMELY COMPLEX
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
TASK ESPECIALLY AGGRAVATED BY THE UNSTABLE AND VOLATILE ARTEMISININ MAR KET &ROM THE AVAILABILITY OF SEEDS TO TECHNICAL SUPPORT FOR EXTRACTION AND PURIFICATION COMPANIES THE WHOLE SUPPLY CHAIN HAD TO BE BUILT UP 4HE ENTIRE PROCESS FOR MANUFACTURING #OARTEM¤ TAKES APPROXIMATELY MONTHS FROM PLANTING OF SEEDS TO FINAL PRODUCTION OF THE MEDICINE &IG
0LANTATION EFFORTS TO MEET MARKET DEMANDS -AJOR EFFORTS WERE MADE TO COME TO A MUTUAL AGREEMENT WITH LOCAL GOVERN MENTS COMMUNITIES AND FARMERS TO CHANGE TO THIS NEW CROP AND TO ESTAB LISH LARGE CULTIVATION AREAS 0LANNING WAS HARDLY POSSIBLE AS TOO MANY KEY PARAMETERS WERE UNCLEAR 4HE QUESTIONS AS TO HOW MANY KG OF BIOMASS MAY BE HARVESTED PER HECTARE OR WHAT WOULD BE THE ARTEMISININ CONTENT ACHIEVED WITH HIGH YIELD SEEDS ON LARGER PLANTATIONS COULD NOT BE SATISFACTORILY NAR ROWED DOWN &INALLY THERE WAS NO EXPERIENCE OF HOW THE HUGE VOLUMES OF BIOMASS MAY BE DRIED AND PROCESSED 4ABLE VISUALIZES THE REQUIREMENTS BASED ON THE #OARTEM¤ DEMAND PLANNING IN THE MIDDLE OF
4ABLE $EMAND PLANNING FOR ! ANNUA AND ARTEMISININ IN 9EAR
#OARTEM¤ !RTEMISININ ;MIO AVERAGE REQUIRED ;KG= TREATMENTS=
!RTEMISININ PLANNED FOR ;KG=
!MOUNT OF 2EQUIRED AREA ;HECTARES= BIOMASS ;KG=
2EMARKS
OVERSUPPLY
OVERSUPPLY
MAX MARKET COULD SUPPLY
0LANNING FIGURES BASED ON 7(/ FORECASTS
!RTEMISININ VOLUME PLANNED DETERMINED BY 7(/ DEMAND STRATEGIC STOCKS COVERAGE OF AGRICUL TURAL RISKS !RTEMISININ CONTENT IN THE BIOMASS APPROXIMATELY WITH AN ASSUMED BIOMASS YIELD OF KG PER HECTARE
4HOMAS +UHN AND 9ING 7ANG
,OOKING AT THIS SITUATION THE SUPPORT OF LOCAL GOVERNMENTS COMMU NITIES AND FARMERS TO PLANT ! ANNUA BECAME THE CONDITIO SINE QUA NON TO ESTABLISH AN ARTEMISININ PRODUCTION ON THAT SCALE 4HE WILLINGNESS OF FARM ERS IN #HINA AND !FRICA TO SWITCH TO THE NEW CROP GENERATED AN ADDITIONAL INCOME SOURCE FOR THEM AND BECAME AN IMPORTANT ECONOMIC FACTOR FOR RURAL COMMUNITIES 4O ENCOURAGE AND ACCELERATE EXCHANGE OF CROPS AND EXPANSION OF PLAN TATION AREAS .OVARTIS SIGNED ARTEMISININ SUPPLY CONTRACTS WITH EXTRAC TION COMPANIES 4HESE COMPANIES BUILT UP NUMEROUS CONTRACTED SUPPLIER PARTNERSHIPS WITH FARMERS GUARANTEEING THE TIMELY DELIVERY OF CONSTANTLY INCREASING VOLUMES OF ! ANNUA &ARMERS RECEIVED AN ASSURED INCOME BASED ON THE SIZE OF THE ! ANNUA PLANTATION )F HOWEVER THE ARTEMISININ CONTENT EXCEEDED THE AGREED LEVEL THE PAYMENTS WERE INCREASED 4HESE MEASURES MADE THE CHANGE TO AN ! ANNUA HORTICULTURE ATTRACTIVE TO FARMERS WHO DID NOT HAVE ANY EXPERIENCE IN THE AGRICULTURAL REQUIREMENTS OF THE PLANT &OR THOSE FARMERS WHO HAD TO LEAVE THE BANKS ALONG THE 9ANGTZE 2IVER DUE TO THE 4HREE 'ORGES PROJECT EG IN THE &ENGDU #OUNTY #HONGQING #ITY THESE CONTRACTS PROVIDED A CERTAIN SECURITY TO MOVE AND START A FARM IN THEIR NEW ENVIRONMENTS 4O ASSURE THE REQUIRED BIOMASS SUPPLY THE EXTRACTION COMPANIES PRO VIDED THE ! ANNUA SEEDS FOR FREE MADE PLASTIC FOILS AVAILABLE AT NO COST TO COVER THE NURSERIES AND ORGANIZED TRAINING SEMINARS FOR FARMERS IN CO OPERATIONS WITH THE LOCAL GOVERNMENTS )N THE PLANTATIONS ! ANNUA SEEDS HAVE TO BE PLACED JUST BELOW THE SOIL SURFACE DURING *ANUARY IN #HINA )N NURSERIES THE GROWTH OF THE SMALL SEEDLINGS IS SUPPORTED BY SUFFICIENT MOISTURE AND BY LIMITED TEMPERA TURE VARIATION 7HEN THE PLANT REACHES A HEIGHT OF APPROXIMATELY CM THEY ARE TRANSFERRED TO THE CULTIVATION FIELDS IN -ARCH!PRIL BASED ON THE REQUIREMENTS OF SUFFICIENT RAIN AND A TEMPERATURE ABOVE FREEZING $URING THAT TIME SUFFICIENT WATER AND REGULAR WEEDING ARE ESSENTIAL FOR GROWTH )N THE LAST YEARS THOUSANDS OF #HINESE FARMERS DECIDED TO PLANT ! ANNUA ON THEIR FIELDS 4HE SIZE OF THE PLANTATIONS VARIES FOR EXAMPLE IN THE #HONGQING 0ROVINCE OF #HINA BETWEEN TO -5 -5 M PER FARM 4O DEVELOP A SUPPLY CHAIN FOR AN AGRICULTURALLY PRODUCED DRUG SUBSTANCE OR PRECURSOR GENERATED BY A HIGHLY FRAGMENTED AND MULTINATIONAL SUPPLIER BASIS ACCURATE AND RELIABLE INFORMATION ON THE SUPPLY STATUS IS ESSENTIAL
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
0ICTURE ! ANNUA , SEEDS
0ICTURE ! ANNUA , NURSERIES IN #HINA
4HOMAS +UHN AND 9ING 7ANG
0ICTURE ! ANNUA , PLANTATION IN #HINA
0ERSONAL CONTACTS WITH ALL INVOLVED STAKEHOLDERS n FARMERS GOVERNMENT OFFICIALS REPRESENTATIVES FROM THE EXTRACTION COMPANIES n PROVED TO BE THE MOST APPROPRIATE AND EFFECTIVE MEASURE ! DEDICATED ORGANIZATION OF BIOMASS MANAGERS TO CONSTANTLY MONITOR AND STEER THE SUPPLY PROCESS WAS IMPLEMENTED )T TURNED OUT THAT THE MOST DIFFICULT TASK WAS TO DISCOVER POS SIBLE FAILURES EARLY ENOUGH TO IMPLEMENT CORRECTIVE MEASURES AND TO ESTI MATE THEIR IMPACT ON THE HARVEST AND PROJECTED ARTEMISININ QUANTITIES 4HE BIOMASS MANAGERS VISITED VARIOUS FARMS ON A DAILY BASIS n OFTEN IN REMOTE AREAS WITH LITTLE DEVELOPED INFRASTRUCTURE 4HEY HAD TO CONTROL THE DEVELOPMENT OF THE NURSERIES )N CASE OF POTENTIAL FAILURES OR INADEQUATE TRAINING THE CORRESPONDING EXTRACTION COMPANY WAS CONTACTED AND THE SUPPORT OF LOCAL GOVERNMENT WAS ASSURED 4HE BIOMASS MANAGERS ALSO MONITORED THE TRANSFER OF THE SEEDLINGS TO THE FIELDS AND MEASURED THE SIZE OF THE PLANTATION AREA TO ASSURE THE IMPLEMENTATION OF THE ORIGINAL PLANS #ONTROL OF REGULAR WEEDING AND WATERING OF THE PLANTATION IS AN ADDITIONAL TASK TO ENSURE THE OPTIMAL GROWTH OF ! ANNUA 4HE GROWTH OF THE PLANTS WAS CAREFULLY TRACKED AND IN CASE OF NATURAL DISASTERS n MAINLY LACK OF WATER
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
THUNDERSTORMS FLOODING ETC n THE EFFECT ON THE BIOMASS VOLUME WAS ESTI MATED $URING HARVEST SEASON LATE *ULY TO EARLY 3EPTEMBER THE BIOMASS MANAGERS WERE INSTRUMENTAL ENSURING THAT THE CONTRACTED PLANT MATERIAL WAS NOT SOLD TO OTHER EXTRACTION COMPANIES 4HE HUGE AMOUNT OF BIOMASS HAD TO BE DRIED HIGH MOISTURE LEADS TO A DECOMPOSITION OF ARTEMISININ DURING STORAGE TRANSPORTED AND PROPERLY STORED 4HE ARTEMISININ CONTENT WAS ANALYZED TO AVOID SURPRISES ON LOW YIELDS IN THE DOWNSTREAM PART OF THE PROCESS &INALLY THE BIOMASS MARKET PRICE WAS RELATED TO THE ARTEMISININ PRICE !LTHOUGH THE ARTEMISININ PRICES WERE AGREED IN CONTRACTS THE SHORTAGE SITUATION LED TO A HIGHER MARKET PRICE THAN ANTICIPATED 4HE BIOMASS MANAGERS WERE A KEY SUCCESS FACTOR TO SOLVING THE PROBLEMS FACED BY .OVARTIS IN CONTRIBUTING TO THE PRODUCTION OF A LIFE SAVING DRUG PROVIDED AT COST FOR THE PUBLIC HEALTH SECTOR 4HEY NEEDED TO ESTABLISH A SEAMLESS SUPPLY CHAIN OF NUMEROUS INTERFACES OTHERWISE PROJECT FAILURES WOULD HAVE LEFT THOUSANDS OF PATIENTS WITHOUT URGENTLY NEEDED MEDICINE (OWEVER THEIR CLOSE INTERACTION WITH ALL STAKEHOLDERS AND THEIR PERMANENT PRESENCE IN THE FIELD SHAPED A STEEP LEARNING CURVE BENEFICIAL FOR UPCOMING SEASONS !LTHOUGH A LOSS OF KG BIOMASS REDUCES THE CALCULATED ARTEMISININ AMOUNT BY APPROXIMATELY ONLY KG IT WAS IMPORTANT TO SAFEGUARD THE BIOMASS AS MUCH AS POSSIBLE TO CONVINCE THE FARMERS TO STAY COMMITTED FOR THE NEXT SEASON
'EOGRAPHICAL SPLIT OF THE SUPPLY MARKET !S ARTEMISININ IS THE BASIS FOR THE LIFE SAVING DRUG #OARTEM¤ .OVARTIS ADDRESSED THE AGRICULTURAL RISKS NOT ONLY WITH STRATEGIC STOCKS BUT ALSO WITH A REGIONAL SPLIT FOR THE BIOMASS SUPPLY BASE )N ADDITION TO THE AREA ARTEMISININ USED TO COME FROM .OVARTIS WAS LOOKING TOGETHER WITH AN EXPERT TEAM AT SUITABLE LOCATIONS AND PARTNERS TO WIDEN THE SUPPLY BASE GLOBALLY 7HEN REQUIRED .OVARTIS PROVIDED SEEDS TO ENABLE THE START UP OF TRIAL PLANTATIONS 4HE BROADEST EXPERIENCE IN CULTIVATING ! ANNUA WAS FOUND IN %AST !FRICA IN 4ANZANIA +ENYA AND 5GANDA &OR SEVERAL YEARS THE CROP WAS PLANTED FOR THE LOCAL MARKET )N PARALLEL SEEDS WERE DEVELOPED IN COLLABORATION WITH -EDIPLANT A 3WISS ACADEMIC RESEARCH CENTER SPECIAL
4HOMAS +UHN AND 9ING 7ANG
0ICTURE ! ANNUA , PLANTATION WITH IRRIGATION IN +ENYA
IZED IN THE BREEDING AND CULTIVATION OF MEDICINAL AND AROMATIC PLANTS -EDIPLANT HAS BEEN WORKING ON ! ANNUA SINCE INVESTIGATING ITS COM MERCIAL CULTIVATION ;= 3UCCESSFUL TRIALS TO ADDRESS THE CLIMATE CONDITIONS IN !FRICA NEAR THE EQUATOR AND TO INCREASE THE ARTEMISININ CONTENT IN THE PLANT WERE PERFORMED 4O INITIATE LARGE SCALE ACTIVITIES IN %AST !FRICA .OVARTIS SIGNED A SUPPLY AGREEMENT WITH A LOCAL COMPANY TO FACILITATE CONTRACTS WITH FARMERS AND TO INITIATE THE CONSTRUCTION OF A NEW EXTRACTION AND PURIFICATION FACILITY 4HE LONG EXPERIENCE WITH CULTIVATION OF FLOWERS VEGETABLES AND OTHER CROPS SUPPORTED THE CHANGE TO THE NEW CROP "ESIDES SMALL PLANTATIONS LARGER AREAS UP TO HECTARES WERE PLANTED TOO 4HE COMPANY PROVIDES THE SEEDLINGS TO THE FARMERS SOMETIMES THE FARMS HAVE THEIR OWN NURSERIES )N %AST !FRICA IRRIGATION ON A LARGE SCALE IS APPLIED TO OVERCOME THE SEASONAL ITY A MORE CONSTANT FLOW OF BIOMASS IS ESTABLISHED THE AGRICULTURAL RISKS ARE REDUCED
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
%FFORTS TO IMPROVE ! ANNUA BIOMASS )MPROVING THE ARTEMISININ CONTENT IN THE PLANT BY GENERATING HIGH YIELD SEEDS WILL REMAIN A MAJOR FOCUS AND WILL AFFECT THE REQUIRED PLANTATION AREA DRYING EFFORTS TRANSPORTATION AND STORAGE !LL THESE FACTORS WILL HAVE AN IMPACT ON THE ARTEMISININ PRICE 9IELD IMPROVEMENT STRATEGIES ARE CUR RENTLY BEING INVESTIGATED IN #HINA AND IN 3WITZERLAND %XPERIENCE WITH THE NEW CROP WILL IMPROVE THE STAGES IN THE NURSERY PLANTATION AND CULTI VATION 4HE METHOD TO GENERATE SEEDLINGS FROM LITTLE BRANCHES INSTEAD OF USING SEEDS IS APPLIED SUCCESSFULLY IN !FRICA AFFECTING THE COST STRUCTURE POSITIVELY %SPECIALLY THE AVAILABILITY OF IRRIGATION FACILITIES REDUCES THE DEPENDENCE ON THE EXACT COINCIDENCE OF THE RAIN SEASON WITH THE PLANTA TION SEASON 4O ADDRESS THE DRYING ISSUES n A LARGE AMOUNT OF BIOMASS HAS TO BE DRIED AT THE SAME TIME n A DIFFERENT TYPE OF HARVEST IS PROPOSED INSTEAD OF THE DESTRUCTIVE HARVEST ONLY THE OUTER LEAVES ARE PICKED 4HE REMAINING PLANT CONTINUES TO GROW PROVIDING FURTHER @PARTIAL HARVESTS ;= 4HE PICKED LEAVES HOWEVER DRY MUCH QUICKER THAN THE WHOLE PLANT 7HETHER THIS STRATEGY YIELDS AS MUCH ARTEMISININ PER PLANT AS THE DESTRUCTIVE HARVEST HAS TO BE PROVEN YET ON A LARGER SCALE #URRENTLY THE PLANT IS NOT FACING TOO MUCH THREAT FROM ANIMALS OR INSECTS -OST INSECTS AND ANIMALS ARE REPELLED BY THE PLANT HAVING A CHARAC TERISTIC SMELL OR INSECTS DIE AFTER EATING THE PLANT )T HAS BEEN OBSERVED THAT IN PLACES WHERE INSECTS MOSTLY CATERPILLARS DO BITE THE SURFACE OF BRANCHES ADDITIONAL LEAVES DEVELOP PROVIDING MORE ARTEMISININ 4HE RESISTANCE TO INSECTS MAY CHANGE WITH LARGE PLANTATIONS OVER A LONGER PERIOD OF TIME !RTEMISININ HAS ALSO BEEN REPORTED TO BE A POTENT PLANT INHIBITOR WITH POTENTIAL AS A NATURAL HERBICIDE ; = )N -ARCH THE 7(/ MONOGRAPH ON GOOD AGRICULTURAL AND COLLECTION PRACTICES '!#0 FOR ! ANNUA , WAS PUBLISHED 4HE 7(/ GUIDELINES PRO VIDE A DETAILED DESCRIPTION OF THE CULTIVATION COLLECTION TECHNIQUES AND MEASURES REQUIRED FOR A HARVEST TO MEET QUALITY STANDARDS 4HE INFORMATION IS BASED ON RESEARCH DATA AND THE PRACTICAL EXPERIENCE OF SEVERAL COUNTRIES WHERE SUCCESSFUL CULTIVATION PRACTICES HAVE LED TO A HIGH YIELD OF GOOD QUALITY ! ANNUA
4HOMAS +UHN AND 9ING 7ANG
-ARKET DEVELOPMENT THROUGH SUPPLY CHAIN ENLARGEMENT OF ARTEMISININ
#APACITY FOR EXTRACTION AND PURIFICATION SITUATION IN !RTEMISININ HAS BEEN EXTRACTED FOR OVER YEARS IN #HINA AND 6IETNAM 4HE FOLLOWING PROCESS WAS DEVELOPED AND IS STILL BEING USED TODAY 4HE DRIED LEAVES MILLED OR UN MILLED ARE EXTRACTED SEVERAL TIMES IN REFLUX ING FRESH HEXANE OR PETROLEUM ETHER 4HE LEAVES ARE DISCHARGED FROM THE EXTRACTOR 4HE EXTRACT IS FURTHER PURIFIED BY COLUMN CHROMATOGRAPHY ON SILICA GEL !FTER ELUTION OF THE LIPOPHILIC SUBSTANCES MOSTLY WAXES ARTEMISININ IS WASHED OFF THE COLUMN BY ADDING APPROXIMATELY OF A POLAR SOLVENT LIKE ETHYL ACETATE 4HE FRACTIONS CONTAINING ARTEMISININ ARE COMBINED CONCENTRATED AND TREATED WITH CHARCOAL 4HE FILTRATE IS CON CENTRATED AND THE CRUDE ARTEMISININ IS ISOLATED BY CRYSTALLIZATION !FTER RE CRYSTALLIZATION IN ETHANOL ARTEMISININ IS OBTAINED IN HIGH PURITY 4HIS TECHNICALLY STRAIGHTFORWARD PROCESS ALLOWS THE SEPARATION OF ARTEMISININ FROM ALL THE OTHER SUBSTANCES AFFORDING A HIGH QUALITY PRODUCT 4HE AMOUNT OF LEAVES TO BE EXTRACTED IS BASED ON THE ARTEMISININ CONTENT WHICH IS DETERMINED BY PERFORMING THE PROCESS ON ANALYTICAL SCALE PRIOR TO PRODUCTION 4HE QUALITY AND YIELD VERY MUCH DEPENDS ON THE CAPABILITY TO SEPARATE THE RIGHT FRACTIONS FROM THE COLUMN ! TYPICAL YIELD OF ARTEMISININ BASED ON ITS CONTENT IN DRY LEAVES IS APPROXIMATELY 4O REDUCE RUNNING COSTS SOLVENTS ARE RECOVERED BUT THE ATTEMPTS TO REGENERATE THE SILICA GEL WERE ONLY PARTIALLY SUCCESSFUL 4HE DIFFERENT PROCESSES DEVELOPED AFTERWARDS ARE SUMMARIZED IN A REVIEW ARTICLE ;= )N ONLY A FEW COMPANIES IN #HINA AND 6IETNAM HAD THE KNOWL EDGE AND EXPERIENCE TO PERFORM THE EXTRACTION AND PURIFICATION OF ARTE MISININ 4HESE COMPANIES OPERATED ONLY IN SMALL TO MEDIUM SCALE 4HE PRODUCTION WAS MANUALLY OPERATED WITHOUT AUTOMATION CONTROL 4HE APPLIED PROCESSES WERE MAINLY BASED ON EXPERIENCE AND RUN WITH LITTLE ANALYTICAL CONTROL 4HE ESTIMATED EXTRACTION CAPACITY IN WAS APPROXIMATELY KG OF ARTEMISININ IN #HINA AND KG IN 6IET NAM /THER COUNTRIES DID NOT PROVIDE ARTEMISININ ON A COMMERCIAL SCALE
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
7ITH THE SCALE UP FOR !#4 NOT ONLY THE BIOMASS MARKET BUT ALSO THE EXTRACTION AND PURIFICATION CAPACITY HAD TO BE DEVELOPED 0ROCESS IMPROVE MENTS AND FULL USAGE OF AVAILABLE CAPACITIES WERE THE FIRST FOCUS BEFORE EXISTING CAPACITIES WERE ENLARGED AND NEW FACILITIES ESTABLISHED 4HE SUP PLY BASE WAS ENLARGED WITH SEVERAL COMPANIES 5SING A BROADER SUPPLY BASE THE RISKS COULD BE ADDRESSED BETTER NO CRITICAL SITUATION WAS FACED IF AN EXTRACTION COMPANY n FOR WHATEVER REASONS n COULD NOT FULFILL ITS CON TRACTUAL SUPPLY OBLIGATIONS $URING THE EXTRACTION AND PURIFICATION SCALE UP SEVERAL MAJOR CHALLENGES HAD TO BE ADDRESSED 4HE TRADITIONAL PROCESS IS NOT YET PERFORMED ON A LARGE SCALE 4HE INFLUENCE OF THE BIOMASS QUALITY WAS NOT INVESTIGATED THEREFORE IS STILL UNKNOWN 4HE ANALYTICAL AND '-0 SKILLS WERE LIMITED OR NOT ESTABLISHED /NLY LIMITED TECHNICAL KNOW HOW TO ADDRESS SCALE UP RELATED ISSUES WAS AVAILABLE EG SAFETY MEASURES 4HE PRODUCTION COSTS WERE ALSO NOT ADDRESSED IN THE SMALL MARKET SIZE
0ROCESS DEVELOPMENT IN ARTEMISININ PURIFICATION )N THE PROCESS DEVELOPMENT TWO MAJOR PARTS WERE INVESTIGATED INCLUDING EXTRACTION AND PURIFICATION 7ITH GROWING VOLUMES PROCESS MODIFICATIONS WERE MADE TO AVOID EXPENSIVE AND TIME CONSUMING CHROMATOGRAPHY AND CHANGES OF SOLVENTS FOR EXTRACTION WERE ALSO INVESTIGATED 4HE EXTRACTION WITH OTHER SOLVENTS LIKE METHANOL OR ETHYL ACETATE INSTEAD OF ALKANES DID NOT GIVE HIGHER EXTRACTION YIELDS BUT THE EXTRACTS WERE EVEN MORE COMPLEX AND SUBOPTIMAL FOR SUBSEQUENT SEPARATION 4HE PURIFICATION PROCESS WAS THEREFORE THE MAJOR FOCUS &IGURE SHOWS THE METABOLITE PATTERN OF THE HEXANE EXTRACT
#ONFIRMATION OF THE STRUCTURE AND ABSOLUTE CONFIGURATION OF ARTEMISININ 4HE STRUCTURE OF ARTEMISININ HAS BEEN CONFIRMED BY VARIOUS SPECTROSCOPIC METHODS INCLUDING )2 -3 AND .-2 4HE CONFORMATION OF ITS ABSOLUTE CONFIGURATION HAS ALSO BEEN MADE BY 8 RAY CRYSTALLOGRAPHIC ANALYSIS AT .OVARTIS 4HE SEVEN CHIRAL CENTERS IN ARTIMISININ ARE #2 #3 #2 #3 #2 #3 AND #2 AS INDICATED IN &IGURE
4HOMAS +UHN AND 9ING 7ANG
&IGURE (0,# CHROMATOGRAM OF CRUDE ARTEMISININ ASSAY OF RESIDUE ON EVAPORATION (0,# AREA (0,# CONDITIONS 9-# /$3 !1 20 μM ¯ × MM MLMIN ELUTION WITH (/-E#. ISOCRATIC WITH -E#. IN MIN LINEAR GRADIENT TO -E#. IN MIN FURTHER MIN ISOCRATIC WITH -E#. 56 NM
7AXES AS THE MAJOR SIDE PRODUCTS IN CRUDE ARTEMISININ SAMPLES 3CALING UP A PURIFICATION PROCESS REQUIRES ADEQUATE KNOWLEDGE ABOUT THE SIDE PRODUCTS AND THEIR SEPARATION 3YSTEMATIC SEPARATIONS OF SIDE PROD UCTS OUT OF THE CRUDE EXTRACT AND THEIR IDENTIFICATION WERE PERFORMED AT .OVARTIS 7AXES ARE A GENERAL TERM FOR THE MIXTURE OF LONG CHAIN APOLAR LIPIDS FORMING A PROTECTIVE COATING ON PLANT LEAVES AND FRUITS BUT ALSO IN ANI MALS ALGAE FUNGI AND BACTERIA 6ARIOUS MATERIALS CALLED WAXES DO NOT FORM A CHEMICALLY HOMOGENEOUS GROUP !LL WAXES ARE WATER RESISTANT MATERI ALS INCLUDING HYDROCARBONS NORMAL OR BRANCHED ALKANES AND ALKENES KETONES DIKETONES PRIMARY AND SECONDARY ALCOHOLS ALDEHYDES STEROL ESTERS ALKANOIC ACIDS TERPENES SQUALENE AND MONOESTERS WAX ESTERS ALL WITH LONG CARBON CHAINS 7AXES ARE THE MAJOR SIDE PRODUCTS TO BE REMOVED IN THE PURIFICATION PROCESS OF ARTEMISININ "ASED ON OUR ANALYSIS WITH )2 -3 AND .-2 THE
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
&IGURE !BSOLUTE CONFIGURATION OF ARTEMISININ BY 8 RAY CRYSTALLOGRAPHY OF ARTEMISININ ANALYSIS
WAXES IN CRUDE ARTEMISININ EXTRACT CONTAIN MAINLY TWO TYPES OF COMPO NENTS ALIPHATIC ALCOHOLS AND HYDROCARBONS 5NDER A MICROSCOPE THE DIFFERENCE BETWEEN THE SAMPLES WITH OR WITHOUT WAXES CAN BE CLEARLY OBSERVED &OR THE CRUDE ARTEMISININ CONTAINING WAXES QUANTIFICATION MAY BE CONVENIENTLY PERFORMED IN #$#L WITH )2 SPECTROSCOPY 4HE CALIBRATION PERFORMED WITH THE CONCENTRATION BETWEEN AND GAVE AN EXCEL LENT LINEAR CORRELATION BASED ON THE INTENSITY OF THE CARBONYL BAND AT CMn FROM ARTEMISININ WITH BASE LINE POINTS AT AND CMn $IFFERENT FROM 4,# (0,# 56 AND (0,# -3 '# &)$ IS FEASIBLE FOR THE DIRECT DETECTION OF BOTH ARTEMISININ AND WAXES IN THE QUANTIFICATION (OW EVER THE SAMPLES SHOULD BE FIRST DERIVATIZED SILYLATION 3INCE DECOMPOSI TION OF ARTEMISININ OCCURS AT # SHORT '# COLUMNS WITH THIN FILM HAVE TO BE DEVELOPED AND USED IN THE ANALYSIS 4O AVOID DECOMPOSITION IT IS ALSO NECESSARY TO PERFORM THE INJECTION DIRECTLY AT THE HEAD OF THE COLUMN ON COLUMN INJECTION IN '# CHROMATOGRAPHY
4HOMAS +UHN AND 9ING 7ANG
0ICTURE !RTEMISININ CRYSTALS WITHOUT WAXES
0ICTURE !RTEMISININ CRYSTALS WITH WAXES
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
&IGURE #ARBONYL BAND IN )2 SPECTRUM FOR QUANTIFICATION
&IGURE ,INEAR CORRELATION IN QUANTIFICATION WITH )2 9 n 8 3IGMA #ORRELATION COEFFICIENT
4HOMAS +UHN AND 9ING 7ANG
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)T WAS REPORTED THAT MORE THAN NATURAL PRODUCTS HAVE BEEN IDENTIFIED IN THE PHYTOCHEMICAL INVESTIGATIONS OF ! ANNUA WITH LEAVES STEMS FLOW ERS ROOTS AND SEEDS AS WELL AS OF ENDOPHYTES INSIDE ! ANNUA INCLUDING VARIOUS SESQUITERPENES FLAVONOIDS AND OTHER SECONDARY METABOLITES ;= &ROM THE HEXANE EXTRACT OF ! ANNUA ARTEMISININ AND A SERIES OF CLOSELY RELATED SIDE PRODUCTS HAVE BEEN IDENTIFIED BY (0,# 56 -3 ANALYSIS ON 20 COLUMN IN COMPARISON WITH AUTHENTIC COMPOUNDS 0ARTICULAR ATTENTION SHOULD BE PAID TO THE ARTEMETIN WHICH HAS VERY SIMILAR OR ALMOST EQUAL RETENTION TIME AS ARTEMISININ BUT MUCH STRONGER 56 ABSORPTION DUE TO ITS HIGHLY CONJUGATED CHROMOPHORE &IGURE SHOWS THEIR (0,# CHROMATOGRAMS SORTED BY THEIR RETENTION TIMES )N THE QUANTITATIVE (0,# 56 ANALYSIS THE DIFFERENCE AMONG THE EXTINC TION COEFFICIENTS PLAYS AN ESSENTIAL ROLE IN THE COMPOUND QUANTIFICATION )N &IGURE AND 4ABLE EXTINCTION COEFFICIENTS OF SOME SIDE PRODUCTS IN THE EXTRACT ARE SUMMARIZED AND COMPARED WITH ARTEMISININ !S CAN BE SEEN IN 4ABLE THE DIFFERENCE OF THE EXTINCTION COEFFICIENTS BETWEEN SIDE PRODUCTS AND ARTEMISININ COULD BE AS HIGH AS FOLD CHRYSO SPLENETIN NM DEPENDING ON THE STRUCTURE TYPES 3INCE THE 56 ABSORPTION OF ARTEMISININ IS QUITE WEAK THIS IS A VERY IMPORTANT FACTOR FOR ESTIMATION OF THE AMOUNT OF SIDE PRODUCTS IN (0,# ANALYSIS PARTICU LARLY FOR THOSE WITH HIGHLY CONJUGATED 56 CHROMOPHORES 4HE CONTENT OF ARTEMISINIC ACID IN ! ANNUA PLANTS WAS INDICATED TO BE n FOLD HIGHER THAN ARTEMISININ BUT WITHOUT SHOWING EXPERIMENTAL DATA ; = "ASED ON OUR ANALYSIS THIS RATIO CANNOT BE CONFIRMED !CCORDING TO OUR RESULTS WE ASSUME THAT THE INDICATION COULD BE BASED ON THE COMPARISON OF THE (0,# PEAK AREAS !S CAN BE SEEN IN 4ABLE THE DIFFERENCE OF THE EXTINCTION COEFFICIENTS BETWEEN ARTEMISININ AND ARTEMISINIC ACID IS AS HIGH AS n FOLD 4HE n FOLD HIGHER CONTENT OF ARTEMISINIC ACID WOULD NOT SUPPORT THE PROPOSAL AS POSSIBLE PRECURSOR IN THE BIOSYNTHETIC PATHWAY OF ARTEMISININ ;=
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
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4HOMAS +UHN AND 9ING 7ANG
&IGURE 2ELATED SIDE PRODUCTS (0,# CHROMATOGRAMS SORTED BY RETENTION TIME (0,# CONDITIONS 0UROSPHER 20 ENDCAPPED +M (/ -E#. (#//( IN BOTH PHASES LINEAR GRADIENT ELUTION TO IN MINS 56 NM
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!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
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)MPROVED ANALYTICAL PROCEDURE FOR ARTEMISININ 4O SUPPORT PRODUCTION SCALE UP THE ANALYTICAL METHOD HAD TO BE FURTHER IMPROVED 4HE PARTIALLY UNKNOWN QUALITY IMPACTS OF DIFFERENT BIOMASS QUALITIES AND OF MODIFIED PURIFICATION PROCESSES REQUIRED ANALYTICAL DETER MINATION OF CHANGES IN THE IMPURITY PROFILE 0URE ARTEMISININ IS ANALYZED BASED ON THE INTERNATIONALLY RECOGNIZED TESTING MONOGRAPH FROM 7(/ ;= AND THE #HINESE 0HARMACOPOEIA "OTH MONOGRAPHS DESCRIBE ANALYSIS OF RELATED SUBSTANCES IN SUM EITHER BY 4,# OR (0,# ANALYSIS 7(/ MONO GRAPH 4HE (0,# ANALYTICAL METHOD WAS IMPROVED AT .OVARTIS BY QUALIFY ING THE MAIN RELATED SUBSTANCE DEHYDRO ARTEMISININ SEE &IGURE "IOMASS WITH LOWER ARTEMISININ CONTENT OFTEN CONTAINS A HIGHER AMOUNT OF DEHYDRO ARTEMISININ 4HIS RELATED SUBSTANCE MAY NOT BE SEPARATED BY CRYSTALLIZATIONS IN PRODUCTION ONLY SILICA GEL CHROMATOGRAPHY SEPARATES EFFICIENTLY 3TUDIES ON AFLATOXIN CONTAMINATION OF ARTEMISININ WERE PERFORMED AT .OVARTIS !FLATOXINS ARE KNOWN AS SOME OF THE MOST POTENT TOXIC SUBSTANCES THAT OCCUR NATURALLY 4HEY ARE SECONDARY METABOLITES OF THE FUNGI SPECIES !SPERGILLUS FLAVUS ! PARASITICUS AND ! NOMIUS !FLATOXICOSIS IS POISONING AS A
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&IGURE (0,# CHROMATOGRAM OF ARTEMISININ SAMPLE CONTAINING DEHYDRO ARTEMISININ (0,# CONDITIONS .UCLEOSIL # +M = MM MLMIN ELUTION WITH WATER ACETONI TRILE 66 ISOCRATIC
RESULT OF INGESTION OF AFLATOXINS IN CONTAMINATED FOOD OR FEED -ORE IMPOR TANTLY AFLATOXINS BELONG TO THE STRONGEST NATURAL OCCURRING CANCEROGENIC SUBSTANCES !FLATOXIN " IS THE MOST TOXIC MEMBER OF THE FAMILY OF AFLA TOXINS )T IS FREQUENTLY FOUND TOGETHER WITH THE AFLATOXINS " ' AND '
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
!SPERGILLUS SP IS COMMON IN NATURE AND IS MOST OFTEN FOUND WHEN CROPS ARE EXPOSED TO WARM TEMPERATURE AND HIGH HUMIDITY OVER A LONGER PERIOD OF TIME !CCORDING TO h!FLATOXINVERBOTSVERORDNUNGv OF THE 'ERMAN (EALTH -INISTRY ;= PHARMACEUTICAL INGREDIENTS SHOULD CONTAIN LESS THAN μGKG AFLOTOXIN " AND μGKG OF ALL AFLATOXINS IN TOTAL .O AFLATOXIN WAS DETECTED ON ! ANNUA LEAVES EVEN IF THEY WERE HIGHLY INFECTED AND INCUBATED WITH THE AFLATOXIN PRODUCING MOULD ! PARASITICUS 4HIS RESULT SHOWS THAT EVEN IF AN AFLATOXIN PRODUCING MOULD DEVELOPS ON THE LEAVES IT DOES NOT NECESSARILY PRODUCE AFLATOXINS IN LARGE AMOUNTS ! FURTHER EXPERIMENT SHOWED THAT EVEN WITH A VERY HIGH CONCENTRATION OF AFLATOXIN SPIKE IN THE LEAVES NO AFLATOXIN IS RECOVERED IN THE HEXANE PHASE 4HE TOXIN REMAINS ADSORBED ON THE ! ANNUA LEAVES AND THEREFORE THE RISK OF AFLATOXIN CONTAMINATION OF ARTEMISININ IS EXTREMELY LOW )N OTHER WORDS EVEN IF THE CONDITIONS FOR FUNGAL GROWTH AND AFLATOXIN PRODUCTION WERE OPTIMAL AND HIGH AMOUNTS OF AFLATOXINS PRODUCED ON THE LEAVES THESE AFLATOXINS WOULD NOT BE EXTRACTED ALONG WITH ARTEMISININ 4HE ABSENCE OF AFLATOXIN CONTAMINATION WAS CONFIRMED IN THE ANALYSIS OF ARTEMISININ AND ARTEMETHER DRUG SUBSTANCE BATCHES
%FFORTS TO EXPAND CAPACITY FOR EXTRACTION AND PURIFICATION ONWARDS !S THE FIRST STEP THE EXTRACTION COMPANIES REFINED THE ANALYTICAL METHODS TO DETERMINE THE QUALITY OF THE BIOMASS )N THE PAST ONLY THE MOISTURE CONTENT WAS DETERMINED @IN THE FIELD SINCE A BETTER CONTROL OF THAT BIO MASS FEATURE REDUCES LOSSES DURING STORAGE 4HE ARTEMISININ CONTENT HAS TO BE DETERMINED NOT ONLY PRIOR TO EXTRACTION BUT ALREADY AFTER DRYING TO ENSURE QUICK AND ACCURATE PAYMENTS TO FARMERS 4HE EXTRACTION COMPA NIES USE DIFFERENT METHODS TO DETERMINE THE ARTEMISININ CONTENT IN THE BIOMASS 0RODUCTION PROCESS ON LABORATORY SCALE PROVIDES THE CONTENT BY WEIGHT OF ISOLATED ARTEMISININ %XTRACTION IN ANALYTICAL SCALE FOLLOWED BY 4,# THIN LAYER CHROMATOGRAPHY AND APPLYING REFERENCE STANDARDS PRO VIDES MORE ACCURATE RESULTS QUICKLY 4,# &)$ FLAME IONIZATION DETECTION METHOD COMBINING THE KNOWN SEPARATION TECHNIQUES OF 4,# ON PLATES WITH THE PRINCIPLES OF &)$ PROVIDES ANALYTICAL RESULTS WITH AN AUTOMATED EVALUATION
4HOMAS +UHN AND 9ING 7ANG
)N THE SECOND STEP THE PURIFICATION PROCESSES HAD TO BE REVIEWED BOTH TECHNICALLY AND ECONOMICALLY !N ALTERNATIVE PROCESS WAS PROPOSED TO REPLACE THE EXPENSIVE COLUMN CHROMATOGRAPHY BY CRYSTALLIZATIONS (OWEVER THE NEW PROCESS HAD TO BE REJECTED MAINLY DUE TO THE WAXES CO PRECIPITATING WITH ARTEMISININ DURING CRYSTALLIZATION )SSUES SUCH AS PLUGGING DURING FILTRATIONS SIGNIFICANTLY LOWER YIELDS AND A HIGHER CON TENT OF THE MAJOR RELATED SUBSTANCE DEHYDRO ARTEMISININ COULD NOT BE SOLVED 6ARIATIONS OF THE BIOMASS QUALITY FURTHER LIMITED THE SUCCESS OF THIS MODIFIED PROCESS AS WITH LOW CONCENTRATION IN THE PLANT MATERIAL THE RATIO OF ARTEMISININ TO WAXES WAS TOO LOW AND IMPACTED THE CRITICAL STEPS UNFAVORABLY &OR BIOMASS OF HIGH ARTEMISININ CONTENT A SIMILAR MODIFIED PROCESS WAS DEVELOPED 4HE BIOMASS IS MILLED AND EXTRACTED WITH COLD HEXANE 4HE ADVANTAGEOUS RATIO BETWEEN ARTEMISININ AND WAXES ALLOWS FURTHER PURIFICATION WITH CRYSTALLIZATIONS AND WASHINGS ONLY THE AMOUNT OF THE DEHYDRO ARTEMISININ IS SIGNIFICANTLY LOWER THAN IN THE LOW CONTENT BIOMASS 0RELIMINARY RESULTS WITH COUNTERCURRENT EXTRACTION TO SEPARATE ARTEMIS ININ FROM THE OTHER SUBSTANCES COULD BE A POSSIBLE NEW PROCESS HOWEVER INITIAL EFFORTS ARE NOT YET SUCCESSFUL IN COMMERCIAL SCALE 4HE EVALUATION OF ALTERNATIVE SYNTHETIC PRODUCTION PROCESSES FOR ARTE MISININ SHOWS PROMISING FIRST RESULTS BUT DOES NOT YET PROVIDE AN ALTERNA TIVE TO THE EXTRACTION OF ! ANNUA BIOMASS !T THE MOMENT NO COST COM PETITIVE TOTAL SYNTHESIS OF ARTEMISININ IS AVAILABLE 4HE ACCESS TO ARTEMISININ VIA SEMI SYNTHESIS USING ARTEMISINIC ACID AS STARTING MATERIAL COULD BECOME AN ALTERNATIVE USING % COLI AS EXPRESSION SYSTEM FOR AMORPHADIENE PRODUCTION ARTEMISINIC ACID PRECURSOR #URRENT WORK ON OPTIMIZATION OF 0 OXIDATION OF AMORPHADIENE TO ARTEMISINIC ACID LOOKS PROMISING !S INDICATED BY THE AUTHORS hALTHOUGH THE ENGINEERED YEAST IS ALREADY CAPABLE OF PRODUCING ARTEMISINIC ACID AT A SIGNIFICANTLY HIGHER SPECIFIC PRODUCTIVITY THAN ! ANNUA YIELD OPTIMIZATION AND INDUSTRIAL SCALE UP WILL BE REQUIRED TO RAISE ARTEMISINIC ACID PRODUCTION TO A LEVEL HIGH ENOUGH TO REDUCE ARTE MISININ COMBINATION THERAPIES TO SIGNIFICANTLY BELOW THEIR CURRENT PRICESv ;= 4HE SYNTHETIC TRANSFORMATION OF ARTEMISINIC ACID TO ARTEMISININ CAN NOT YET BE IMPLEMENTED IN PRODUCTION SCALE SINCE THE CURRENT LABORATORY SCALE PROVIDES AN OVERALL YIELD BETWEEN n AND THE APPLIED CHEMICAL TRANSFORMATIONS BASED ON SINGLET OXYGEN CHEMISTRY ARE NOT YET SUITABLE FOR LARGE SCALE PRODUCTION ;=
!RTEMISININ n AN INNOVATIVE CORNERSTONE FOR ANTI MALARIA THERAPY
4HE EFFORTS TO IMPROVE THE PURIFICATION PROCESS DEMONSTRATED THAT THE TRADITIONAL PROCESS PROVIDES THE HIGHEST YIELDS WITH A RELIABLE QUALITY FOR LOW CONTENT BIOMASS 4HIS PROCESS SEEMS TO BE INDEPENDENT FROM THE BIO MASS QUALITY &OR HIGH CONTENT BIOMASS A PROCESS BASED ON CRYSTALLIZATIONS IS SUFFICIENT /THER TECHNICAL SOLUTIONS LIKE SUPERCRITICAL OR COUNTERCURRENT EXTRACTIONS ARE FEASIBLE BUT RARELY APPLIED 7ITHIN THE NEXT FEW YEARS THE PRODUCTION OF ARTEMISININ BY HORTICULTURE WILL BE THE METHOD OF CHOICE 4HE DEVELOPMENT OF AN ECONOMICALLY VALUABLE ALTERNATIVE REQUIRES FURTHER EFFORTS IN BOTH RESEARCH AND DEVELOPMENT
4ECHNICAL SUPPORT FOR THE EXTRACTION COMPANIES 4HE EXTRACTION COMPANIES HANDLE SUBSTANTIAL VOLUMES OF ORGANIC SOLVENTS AND STORE HUGE AMOUNTS OF FLAMMABLE BIOMASS ! POSSIBLE ACCIDENT WOULD AFFECT PEOPLE ENVIRONMENT AND COMPANY BUSINESS 4HE CORRESPONDING HAZARDS WERE ADDRESSED WITH EQUIPMENT AND PROCESS SAFETY RISK ANALYSIS 0RECAUTIONARY MEASURES WERE IDENTIFIED AND IMPLEMENTED TO SAFEGUARD CONTINUOUS SUPPLY 4HE PRODUCTION OF ARTEMISININ AS STARTING MATERIAL FOR !#4S REQUIRES QUAL ITY STANDARDS APPLIED IN PHARMACEUTICAL INDUSTRY 'OOD -ANUFACTURING 0RAC TICES '-0 )N THIS CONTEXT THE MAIN ACTIVITIES TO SUPPORT THE EXTRACTION AND PURIFICATION COMPANIES WERE THE QUALIFICATION OF PRODUCTION AND ANALYTICAL EQUIPMENT DOCUMENTATION REGULATORY ASPECTS AND ANALYTICAL SKILLS
&INAL REMARKS
4HE NEW GENERATION OF MALARIA MEDICATION MADE ARTEMISININ AS THE FIRST NEW CHEMICAL ENTITY DISCOVERED AND DEVELOPED FROM 4RADITIONAL #HINESE -EDICINE AVAILABLE TO THE ENTIRE WORLD 4HE NECESSARY ENLARGEMENT OF THE SUPPLY CHAIN FOR ! ANNUA AFFORDED SUBSTANTIAL AGRICULTURAL AND TECHNICAL EFFORTS DURING TWO AGRICULTURAL CYCLES 4HE CLOSE COLLABORATION BETWEEN NUMEROUS FARMERS EXTRACTION COMPANIES LOCAL GOVERNMENT ORGANIZA TIONS AND THE TECHNICAL AND FINANCIAL COMMITMENT OF CUSTOMERS MADE THIS UNPRECEDENTED SCALE UP POSSIBLE 4HE HIGH MARKET NEEDS OF THE PUBLIC HEALTH INITIATIVE @2OLL "ACK -ALARIA COULD BE ADDRESSED SUCCESSFULLY
4HOMAS +UHN AND 9ING 7ANG
&OR #OARTEM¤ THE FIRST PRE QUALIFIED !#4 NOT ONLY DID THE SUPPLY CHAIN FOR ARTEMISININ NEED TO BE ENLARGED DRAMATICALLY THE PRODUCTION CAPACITY FOR THE TWO ACTIVE SUBSTANCES ARTEMETHER AND LUMEFANTRINE AND THE FINAL PHARMACEUTICAL PRODUCT HAD TO BE SUBSTANTIALLY EXPANDED TOO PARALLELED BY THE IMPROVEMENT OF THE MANUFACTURING PROCESSES ; = 7ITH ALL THESE COMBINED EFFORTS IT WAS POSSIBLE TO DELIVER OVER MILLION #OAR TEM¤ TREATMENTS TO THE !FRICAN COUNTRIES IN ORDER TO REPLACE THE INCREAS INGLY INEFFECTIVE PREVIOUS MEDICATIONS BY AN INNOVATIVE NEW TREATMENT FOR A NEGLECTED DISEASE
!CKNOWLEDGEMENTS 4HE ARTEMISININ SCALE UP AND ITS RELATED ACTIVITIES WOULD NOT HAVE BEEN POS SIBLE WITHOUT THE CONTRIBUTION OF CENTRAL AND LOCAL GOVERNMENTS OF #HINA COMPANIES AND FARMERS !--3 AND #)4)# 7E THANK %STHER 3CHMITT 0HILIPP +RASTEL +LAUS -EMMERT (ANS (OF MANN %RIC 7EBER EXTRACTION OPTIMIZATION #HRISTIAN &LEURY PURIFICATION OPTIMIZATION 3ERGE -OSS )2 ,UKAS /BERER .-2 "EATRIX 7AGNER 8 RAY #HRISTIAN 'UENAT -3 %RIC &RANCOTTE *UERGEN +UEHNOEL '# 2ENÎ /TT (0,# 0ETER "RUEGGER !FLATOXINS -ICHAEL 2OMBACH #HRISTOPH "AUER 8IANDONG ,I +URT (UANG 0URCHASING BIOMASS MANAGEMENT 'IL BERT ,UTRINGER '-0 TECHNICAL SUPPORT 2ICHARD :HANG LITERATURE 8AVIER 3IMONNET -EDIPLANT FOR THEIR CONTRIBUTIONS AND ENTHUSIASTIC SUPPORT 7E ALSO THANK 2UTH 3CHUCHTER AND +LAUS 7ENIG FOR CHALLENGING DISCUSSIONS AND IN PARTICULAR (ANSJßRG 7ETTER AND &RANK 0ETERSEN FOR THEIR HIGHLY APPRECIATED SUPPORT TO THE PROJECT
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$UKE 3 6AUGHN + #ROOM *R % %LSOHLY ( !RTEMISININ A CONSTITUENT OF ANNUAL WORMWOOD ! ANNUA IS A SELECTIVE PHYTOTOXIN 7EED 3CI n #HEN 0 ,EATHER ' +LAYMAN $ !LLELOPATHIC EFFECT OF ARTEMISININ AND ITS RELATED COMPOUNDS FROM ! ANNUA 0LANT 0HYSIOL .AMDEO ! -AHADIK + +ADAM 3 !NTIMALARIA DRUG n !RTEMISIA ANNUA 0HARMACOG NOSY -AGAZINE n 2OTH 2 !CTON . ! SIMPLE CONVERSION OF ARTEMISINIC ACID INTO ARTEMISININ * .AT 0ROD n *UNG - %L3OHLY ( #ROOM *R % 0RACTICAL CONVERSION OF ARTEMISINIC ACID INTO DES OXYARTEMISININ * /RG #HEM n #OVELLO 0 4EOH + 0OLICHUK $ 2EED $ .OWAK ' &UNCTIONAL GENOMICS AND THE BIOSYNTHESIS OF ARTEMISININ 0HYTOCHEMISTRY n WWWWHOINTMEDICINESPUBLICATIONSPHARMACOPOEIAOVERVIEWENINDEXHTML WWWBUNDESRECHTJURISDEAFLATOXINVERBOTSV 2O $ 0ARADISE % /UELLET - &ISHER + .EWMAN + .DUNGU * (O + %ACHUS 2 (AM 2 +IRBY * ET AL 0RODUCTION OF THE ANTIMALARIAL DRUG PRECURSOR ARTEMISINIC ACID IN ENGINEERED YEAST .ATURE n "OEHM - &ßNFSCHILLING 0 +RIEGER - +UESTERS % 3TRUBER & !N IMPROVED MANUFAC TURING PROCESS FOR THE ANTIMALARIA DRUG #OARTEM 0ART /RG 0ROCESS 2$ n "EUTLER 5 &ßNFSCHILLING 0 3TEINKEMPER ! !N IMPROVED MANUFACTURING PROCESS FOR THE ANTIMALARIA DRUG #OARTEM 0ART /RG 0ROCESS 2$ n
)NDEX
)NDEX !"* ACETOACETATE γ ALKYLATION 3 n ACETOXY TRIPHENYLETHANOL ACTIVATOR GENE ADSORBER RESINS AFLATOXIN ALAMETHICIN !LBRIGHT 'OODMAN OXIDATION η π ALLYLTRICARBONYLIRON LACTONE COMPLEX !LZHEIMERS DISEASE AMINOPHOSPHOLIPID TRANSLOCASE ANTASCOMICIN ANTASCOMICIN ADVANCED SYNTHESIS n ANTASCOMICIN TOTAL SYNTHESIS n !0 APICIDIN APOPTOSIS !PPEL REACTION APPENDAGE DIVERSITY ARTEMETHER !RTEMISIA ANNUA ! ANNUA BIOMASS ! ANNUA PLANTATION ARTEMISINIC ACID ARTEMISININ n ARTEMISININ BIOSYNTHESIS ARTEMISININ MARKET DEVELOPMENT ARTEMISININ PURIFICATION ARTEMISININ SUPPLY CHAIN ARTEMISININ BASED #OMBINATION 4HERAPY !#4 ASCOMYCIN ASPYRIDONE ! AND " ATROPISOMER AZA EPOTHILONE n AZATHILONE n β EFFECT BAKERS YEAST BARCODE MICROARRAY ANALYSIS "ARTON DEOXYGENATION
"ARTON LIKE FREE RADICAL DECARBOXYLATION "ARTON -C#OMBIE DEOXYGENATION BENZILIC ACID REARRANGEMENT BENZO; =DIOXIN ONE BIDIRECTIONAL SYNTHESIS BIOMASS MANAGER BIOSYNTHETIC GENE CLUSTER BIOSYNTHETIC METHODOLOGY BIS THIOL DERIVATIVE BLOOD BRAIN BARRIER "-3 SEE IXABEPILONE "-3 BORON ENOLATE "ROWN ALKOXYALLYLATION "ROWNS CROTYLATION "URGESS REAGENT BUTANEDIACETAL "$! CALCINEURIN CARBOALUMINATION CARPANONE CATECHOL PINNING TECHNIQUE ##) +" CELL LINE +" CELL LINE CELLULAR REDUCTION #HAKRABORTY SYNTHESIS n CHELATION CONTROL CHEMICAL GENETICS CHEMICAL GENETIC ANALYSIS CHEMINFORMATICS n CHEMISTRY SPACE n #HINESE 0HARMACOPOEIA CHLOROQUINE #HROMOBACTERIUM VIOLACEUM α CHYMOTRYPSIN CINNAMYCIN #OARTEM¤ COMBINATORIAL CHEMISTRY COMBINATORIAL LIBRARY CONVERGENT SYNTHESIS
)NDEX
#OREY "AKSHI 3HIBATA #"3 REDUCTION #OREY &UCHS HOMOLOGATION CUPRATE HIGHER ORDER CUTANEOUS 4 CELL LYMPHOMA #4#, CYCLODEXTRIN CYCLOPHILIN #Y0 CYCLOSPORINE ! #S! CYTISINE $ANISHEFSKY SYNTHESIS DEGLYCOSYLATION DEHYDRO ARTEMISININ DEMETHOXYRAPAMYCIN DEMYELINATION DEOXY DERIVATIVE OF %PO ! AND " DEOXY%PO & DEPSIPEPTIDE SEE ALSO &+ DESMETHYL METHYLSULFANYL %PO " SEE !"* DESYMMETRISATION $IECKMANN CONDENSATION $IECKMANN LIKE REACTION $IELS !LDER CYCLOADDITION $IELS !LDER REACTION ASYMMETRIC DIHYDROARTEMISININ DIIMIDE REDUCTION DIMETHYL CUPRATE DIMETHYL DIOXIRANE $-$/ DIMETHYL BENZIMIDAZOLE GROUP DIRECTED EPOXIDATION DISCOVERY GENOMIC GUIDED DISULFIDE BOND DITHIANE METHODOLOGY DIVERGENT SYNTHESIS DIVERSITY ORIENTED SYNTHESIS DOCETAXEL DRUG DESIGN n DRUG DISCOVERY n DYNAMIC RECRYSTALLISATION % AND : ENOLATES STEREOSELECTIVE GENERATION ENZYMATIC DESYMMETRISATION EPOTHILONES n EPOTHILONES AZIRIDINE BASED EPOTHILONES CYCLOPROPANE BASED
EPOTHILONES SIDE CHAIN MODIFICATION EPOTHILONE ! %PO ! %PO ! TUBULIN BOUND CONFORMATION β TUBULIN%PO ! COMPLEX TRANS EPO ! EPOTHILONE ANALOGS AMIDE BASED EPOTHILONE ANALOGS DIMETHYL BENZIMIDA ZOLE BASED EPOTHILONE ANALOGS PYRAZOLE BASED EPOTHILONE ANALOGS QUINOLINE BASED EPOTHILONE " %PO " %PO " ANALOGS PYRIDINE BASED DEOXY DERIVATIVE OF %PO ! AND " # DESMETHYL %PO " # !MINO %PO " SEE "-3 # FLUORO %PO " EPOTHILONE # %PO # %PO #$ MONOOXYGENASE EPOTHILONE $ %PO $ %PO+ 0 EPOXIDASE %0/ SEE 0ATUPILONE TRANS DIDEHYDRO EPOTHILONE TRANS EPO ! EPOXIDES REGIOSELECTIVE RING OPENING %SCHENMOSER #LAISEN REARRANGEMENT %SCHERICHIA COLI ESSENTIAL OILS %VANS ALDOL CONDENSATION %VANS ASYMMETRIC ALDOL REACTION %VANS 0RASAD SYN REDUCTION %VANS 4ISHCHENKO ANTI REDUCTION EVEROLIMUS EVOKED POTENTIAL EXPERIMENTAL AUTOIMMUNE ENCEPHALO MYELITIS %!% &ARINA 3COTT CATALYST SYSTEM &ELKIN !NH ANALYSIS &ERRIER REARRANGEMENT TYPE ) &ERRIER REARRANGEMENT TYPE )) FINGOLIMOD SEE &49 &INKELSTEIN REACTION &+ n &+ REDUCED FORM &+
)NDEX
&+ &+ !.!, &+"0 FLUDELONE &2 SEE &+ &RATER 3EEBACH ALKYLATION &49 n FUNGI GENOMES '- ARREST GALANTHAMINE GENE CLUSTER BIOSYNTHESIS GENE CLUSTER CRYPTIC GENOMIC MINING GEOGRAPHICAL SPLIT 'LOBAL &UND TO &IGHT !IDS 4UBERCULOSIS AND -ALARIA '!&4- GLYCOLIC ACID "$! VARIANT 'OLEC OXIDATION 'OLEC PROTOCOL GROWTH INHIBITION BY %PO " HAPLOID MUTANT STRAIN HAPLOINSUFFICIENCY (ENBEST EPOXIDATION HETERO $IELS !LDER CYCLOADDITION HIERARCHICAL CLUSTERING HISTONE DEACETYLASE ($!# n ($!# CLASS ) )6 HOMOALLYLIC EPOXIDATION (ORNER 7ADSWORTH %MMONS (7% REACTION HYBRID PRODUCTS IN BACTERIA HYDROSTANNATION PALLADIUM CATALYSED HYDROSTANNYLATION HYDROXAMIC ACID HYDROXYASCOMYCIN HYDROZIRCONATION HYPERCONJUGATION )"R ELECTROPHILIC CYCLISATION IMMUNOSUPPRESSANTS )NSTITUTE OF -ICROBIOLOGY AND %PIDEMIOLOGY OF THE !CADEMY OF -ILITARY -EDICAL 3CIENCES INTERLEUKIN ), IODOLACTONISATION
)RELAND #LAISEN REARRANGEMENT IXABEPILONE *OHNSON #LAISEN REARRANGEMENT *ONES OXIDATION *ULIA OLEFINATION +" CELL LINE +" CELL LINE 0 3 KINASE +/3 SEE EPOTHILONE $ , LIPASE !+ LIPASE 03 ,IPINSKIS RULE OF FIVE ,UCHE REDUCTION LUMEFANTRINE MACROALDOLISATION MACROETHERIFICATION MACROLACTAM RING MACROLACTAMISATION MACROLACTONISATION MAGNETIC RESONANCE IMAGING MAJOR HISTOCOMPATIBILITY COMPLEX -(# MALARIA MAMMALIAN TARGET OF RAPAMYCIN M4/2 M4/2 ACRONYMS OF MARINE SPONGE -ASAMUNE 2OUSH CONDITION -ASAMUNE 2OUSH PROTOCOL -C-URRY COUPLING FOR THE SYNTHESIS OF TRISUBSTITUTED OLEFINS -EERWEINS SALT MERIDAMYCIN METABOLIC PATHWAY SILENT METABOLITES SECONDARY α METHYL DITHIANES MICROTUBULE MICROTUBULE BUNDLING MICROTUBULE DYNAMIC MICROTUBULE INHIBITOR MICROTUBULE STABILIZER -OFFATT REAGENT
)NDEX
MOLECULAR BARCODE MOLECULAR PROPERTIES -UKAIYAMA MACROLACTAMISATION MULTICOMPONENT REACTION MULTIPLE SCLEROSIS MUTAGENESIS -YXOCOCCUS XANTHUS NATURAL PRODUCTS MARINE NATURAL PRODUCTS MICROBIAL NATURAL PRODUCTS ORIGIN OF .EGISHI CARBOALUMINATION .EURAL4- NEURODEGENERATIVE DISEASE .ICOLAOU SYNTHESIS NON %VANS ALDOL REACTION NON NATURAL NATURAL PRODUCTS NONRIBOSOMAL PEPTIDE SYNTHETASE .20 .OYORI ASYMMETRIC REDUCTION OF β KETO ESTER .OZAKI (IYAMA +ISHI .(+ COUPLING .3# SEE &+ NUCLEAR FACTOR OF ACTIVATED 4 CELLS .&!4C OCTANOL WATER PARTITION COEFFICIENT LOG0 ORGANOMETALLIC COMPLEX OZONOLYSIS SELECTIVE PACLITAXEL PAPUAMIDE " PARALLEL FITNESS TEST 0ARIKH $OERING OXIDATION 0ARKINSONS DISEASE PATHWAY SPECIFIC REGULATORY GENES EXPRESSION OF PATUPILONE 0AYNE REARRANGEMENT PENTENYL ACETALS PEPTIDE NONRIBOSOMAL PEPTIDYLPROLYL CIS TRANS ISOMERASE 00)ASE 0 GLYCOPROTEIN PHARMACOPHORE MODELING n PHOSPHATIDYLINOSITOL KINASE 0)+
PHOSPHATIDYLSERINE 0INNICK OXIDATION PLASMA STABILITY 0LASMODIUM POLAR SURFACE AREA POLYKETIDES POLYKETIDE SYNTHASE 0+3 0+3 MODULE 0+3 .203 HYBRID METABOLITE 0+3 .203 HYBRID SYNTHASE PRIVILEGED STRUCTURE PROBABILISTIC SPARSE MATRIX FACTORIZATION 03-& PRODRUG NATURAL PRODUCT INHIBITION 0ROGRAF4- 0SEUDOMONAS PTEROCARYANIN # 0URDIE METHYLATION R!$ 2APAMUNE4- RAPAMYCIN n n RAPAMYCIN DEGRADATION STUDY RAPAMYCIN DEGRADATIVE WORK RAPAMYCIN TOTAL SYNTHESIS n 2EFORMATSKY REACTION REGULATOR OF GENE CLUSTER EXPRESSION RETROALDOL RING SYSTEM RING CLOSING METATHESIS ROMIDEPSIN SEE &+ 2OUSH CROTYLATION 30 30 RECEPTOR 3ACCHAROMYCES CEREVISIAE 3ACCHAROMYCES CEREVISIAE GENE DELETION MUTANT COLLECTION 3AEGUSA OXIDATION SAGOPILONE 3ANDIMMUNE4- SCAFFOLD 3CHREIBER SYNTHESIS 3CHWARTZ REAGENT SECONDARY METABOLITES SELENO CYCLISATION
)NDEX
3EYFERTH DIAZOPHOSPHONATE REAGENT 3HARPLESS ASYMMETRIC EPOXIDATION SHORT CHAIN FATTY ACID SIGMATROPIC REARRANGEMENT SKELETAL DIVERSITY 3MITH SYNTHESIS SODIUM ARTESUNATE SOLID PHASE METHOD INTRODUCTION OF APPENDAGE DIVERSITY 3ORRANGIUM COELICOLOR 3ORRANGIUM CELLULOSUM 3O CE SOURCE ORGANISM SPHINGOSINE PHOSPHATE SPIROTRYPROSTATIN " SPIRUCHOSTATIN ! SEE 9- STAUROSPORINE STICHLOROSIDE # 3TILLE CROSS COUPLING 3TILLE REACTION 3TILL 'ENNARI OLEFINATION 3TILL 'ENNARI PHOSPHONATE 3TORK :HAO BISTRIFLUOROACETOXY IODOBENZENE PROTOCOL 3TREPTOMYCES COELICOLOR 3UÇREZ OXIDATIVE CLEAVAGE SUBSTITUENT SUBSTRUCTURAL ANALYSIS SULFENYL SULFONE SWEET WORMWOOD 3WERN OXIDATION SYNTENY SYNTHASE NONRIBOSOMAL SYNTHASE POLYKETIDE 3YNTHETIC 'ENETIC !RRAY 3'! ANALYSIS SYNTHETIC GENETIC INTERACTION SYNTHETIC LETHAL GENETIC ANALYSIS SYNTHETIC LETHAL GENETIC NETWORK TACROLIMUS 4AKAI OLEFINATION TAXOL SEE PACLITAXEL
4AXOTERE¤ TEMSIROLIMUS TETRAMETHYLAMMONIUM TRIACETOXYBORO HYDRIDE TETRAPROPYLAMMONIUM PERRUTHENATE 40!0 THEOPALAUAMIDE TOTAL SYNTHESIS UNION OF COMPLEX FRAGMENTS 4RADITIONAL #HINESE -EDICINE 4#- TRAPOXIN TRICHOSTATIN ! TRIMETHYLALUMINIUM TUBULIN MUTATION TUBULIN POLYMERIZATION INHIBITORS β TUBULIN%PO ! COMPLEX TWO DIRECTIONAL STRATEGY 5PJOHN PROCEDURE VANCOMYCIN 6ASELLA TYPE FRAGMENTATION 6EBERS ROTATABLE BONDS WAXES 7EINREB AMIDES 7ORLD (EALTH /RGANIZATION 7(/ 7(/ @%SSENTIAL -EDICINES ,IST 7(/ @2OLL BACK -ALARIA INITIATIVE 7(/ MONOGRAPH ON GOOD AGRICULTURAL AND COLLECTION PRACTICES '!#/ FOR !RTEMISIA ANNUA , XENOGRAFT MODEL 9- ZINC BOROHYDRIDE ZINCSILVER GRAPHITE :+ %0/ SEE SAGOPILONE :N"( ANTI REDUCTION
