Analytical Profiles of Drug Substances and Excipients Volume 2

Analytical Profiles of Drug Substances Volume 2

EDITORIAL BOARD

Norman W.Atwater Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. Fusari

David E. Guttman Eric H.Jensen Arthur F.Xichaelis Stephen Id. Olin Gerald J. Papariello Bernard 2. Senkowski Frederick Tishler

ACADEMIC PRESS RAPID MANUSCRIPT REPRODUCTION

Analytical Profiles of Drug Substances Volume 2 Edited by

Klaus Florey The Squibb Institute for Medical Research New Brunswick, New Jersey

Contributing Editors,

Glenn A. Brewer, Jr. Lester Chafetz Edward M.Cohen David E. Guttman

Stephen M. Olin Gerald J. Papariello Bernard Z. Senkowski Frederick Tishler

Compiled urider [he auspices of [he Phartnnceutical Analysis ant1 Control Section Academy of Phnrniac.eiitica1 Sciences

Academic Press NewYork and London 1973

1973, BY THEAMERICAN PHARMACEUTICAL ASSOCIATION COPYRIGHT ALL RIGHTS RESERVED NO PART O F THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 1wO3

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD.

24/28 Oval Road, London NW1 IDD

LIBRARY OF

CONGRESS CATALOO CARD

NUMBER:70-187259

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . .

AFFILIATIONS OF EDITORS, CONTRIBUTORS, A N D REVIEWERS FORICWORD PREFACE .

. . . . . . . . . . . . . . . . . . . . . .

vii ix xi

Ampicillin . . . . . . . . . . . . . . . . . . . . . . Eirgene I i m l i liiv

1

Chlorprot hixene . . . . . . . . . . . . . . . . . . . . Bruce C Rir& and Berriard 2.S e i i h o ~ ~ s h - i

63

Chloral Hydrate . . . . . . . . . . . . . . Johti E. Fuirbrotlier

85

Clidinium Bromide . . . . . . . . . . . . . Bruce C. Rutlj. arid Berriard Z. Senkowski

145

Dexarnethasone . . . . . . . . . . . . . Edward M. Coheri

163

Dioctyl Sodium Sulfosuccinate . . . . . . . . . . . . . . S l i t Airiija and Jerold Coireri

199

Fluorouracil . . . . . . . . . . . . . . . Bruce C. R i d y arid Berriurd Z. Serikowski

22 1

Fluphenazine Enanthate . . . . . . . . . . . . . . . . . Kbirs Florev

245

Fliiphenazine Hydrochloride . . . . . . . . . . . . . . . Klairs Florey

263

Isocarboxazid. . . . . . . . . . . . . . . . . . . . . Bruce C. Riillj, atzd Bernard Z Serikowski

295

Isopropaniide . . . . . . . . . . . . . . . . . . . . . Ralph S. Satitoro, Richurd J. Warrcn, Geriild D. Roberts, Ecl\z.urtl White, V. arid Peter P Begosli

3 I5

CONTENTS

Levallorphan Tartrate . . . . . . , . . . Bruce C. Rudy and Bernard Z. Senkowski

. . . . . . . .

339

Methyprylon . . . . . . . . . , . . . Bruce C. Rudy and Bernard Z. Senkowski

.

Phenelzine Sulfate . Robert E. Duly

. . . .

Primidone . . . . . Raymond D. Daley

.

.

.

. .

. . .

.

.

363

. . . . . . . .

.

. .

. .

3 83

. . . . . . . .

.

. . . . .

409

.

. .

. .

439

. . . .

.

467

Propiomazine Hydrochloride . . . . . . Kathleen B. Crombic and Leo F. Cullen

. .

.

.

.

Sulfamethoxazole . . . . . . . , . . . Bruce C. Rudy and Bernard Z. Senkowski

.

Sulfisoxazole . . . . . . . . . , . . . Bruce C. Rudy and Bernard Z. Senkowski

. .

.

. . . .

.

487

Triclobisonium Chloride . . . . . . . . . . . Bruce C. R u d y and Bernard Z. Senkowski

.

.

. .

.

507

. . . .

. . .

.

.

,

. . .

523

Trimethobenzamide Hydrochloride . . . . Kenneth W. Blessel, Bruce C. Rudy, and Bernard Z. Senkowski

. . .

.

. . . . .

55 1

Triflupromazine Hydrochloride Klaus Florey

ADDENDA. . . . ERRATA.. . . . CUMULATIVE INDEX

.

. . . . . . . . . . . . . . . . . . . . . . . . . . .

vi

. . . . . . . . .

. .

. . . . . .

.

. . . . . . 571 . . . . . 573 . . . . . 5 75

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

S. Ahuju, Ciba-Geigy Inc., Ardsley, New York N . W. A trvater, Searle and Co., Chicago. Illinois

P. P. Begosh, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

K. W. Blessel, Hoffmann-La Roche Inc., Nutley, New Jersey G. A . Brewer, Jr., T h e Squibb Institute for Medical Research, New Brunswick, Now Jersey

L. Clzufbtz, Warner-Lanibert Research Institute, Morris Plains, New Jersey

E: M. C'olzeti, Merck, Sharp and Dohme. Westpoint, Pennsylvania J. Colirw, Ciba-Geigy lnc., Ardsley, New York

J. P. C o / ~ c rEli , Lilly and Company, Indianapolis, Indiana

K. 5.Crornhie, Wyeth Laboratories, Philadelphia, Pennsylvania L. F. Cirlletz, Wyeth Laboratories, Philadelphia, Pennsylvania R. D. Dalc,y, Ayerst Lahoratories, Rousses Point, New York R. L'. D U / I ~Warner-Lambert . Research Institute, Morris Plains, New Jersey J. ELFuirhrotlicr, The Squibb Institute for Medical Research, Moreton, Wirral, England

K. Florcy, The Squibb Institute for Medical Research, New Brunswick, New Jersey S. A . Ft.r.suri. Parke, Davis and Company, Detroit, Michigan

D. E. Girttnian, School of Pharmacy, University of Kcntucky. Lexington, Ke ti t uc k y

vii

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

E. ivashkiv, The Squibb Institute for Medical Research, New Brunswick, New Jersey

E. H . Jensen, The Upjohn Company, Kalamazoo, Michigan

J. Kuzan, American Cyanamid, Bound Brook, New Jersey A . F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey S. M . O h ,Ayerst Laboratories, New York, New York

G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania

T. E. Ricketts, American Cyanamid, Bound Brook, New Jersey G. D.Roberts, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

B. C. Rudy, Hoffmann-La Roche Inc., Nutley, New Jersey R . S. Suritoro, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

B. Z. Senkowski, Hoffmann-La Roche Inc., Nutley, New Jersey F. Tishler, Ciba-Geigy Inc., Ardsley, New York R. J. Warrer?, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

E. White, V , Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

viii

FOREWORD The concept for gathering together and publishing pertinent information on the physical and chemical properties of various official and new drug substances had its origin with the members of the Section on Pharmaceutical Analysis and Quality Control of the Academy of Pharmaceutical Sciences. More than two years of consideration preceded the authorization of this ambitious project by the Executive Committee of the Academy in the Spring of 1970. The immediate and virtually spontaneous enlistment of the first group of contributors t o this work attested t o its importance and the wisdom of pursuing its publication. By coincidence, the delegates t o the sesquicentennial anniversary meeting of the United States Pharmacopeial Convention, Inc., in Washington, D.C. on April 8-1 0, 1970, adopted the following resolution: Whereas widespread interest has been expressed in the inclusion of additional information about physical and chemical properties of drugs recognized in the United States Pharmacopeia Be It Resolved that the Board of Trustees consider publishing in the Pharmacopeia, or in a companion publication, information on such attributes as solubilities, pH and pK values, spectra and spectrophotometric constants, and stability data, pertaining to pharmacopeial drugs.

The U.S.P.C. Board of Trustees unanimously approved the resolution in principle on June 4, 1970 and authorized the Director of Revision t o include in the U.S.P. monographs such physical-chemical information as he deemed proper and also to cooperate with the Academy of Pharmaceutical Sciences to secure the publication of other physical-chemical data. It was my privilege to be the President of the Academy during the period when Arialyticai Profiles was under consideration. I t is my unusual and unique honor as President of the Academy and Director of U.S.P. Revision to assist in the institution and dedication of this first volume. I trust that it will serve immeasurably in providing the scientific community with an authoritative source of information on the properties of many of our important drug compounds.

Jar1irar.v 19 71

Thomas J. Macek

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PREFACE Although the official compendia define a drug substance as to identity, purity, strength, and quality, they normally d o not provide other physical or chemical data, nor d o they list methods of synthesis or pathways o f physical or biological degradation and metabolism. At present such information is scattered through the scientific literature and the files of pharmaceutical laboratories. For drug substances important enough to be accorded monographs in the official compendia such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis Section, Academy of Pharmaceutical Sciences, has started a cooperative venture to compile and publish Anulyricul Profiles of Drug Substunces in a series of volumes of which this is the second. It is also planned to revise and update these profiles at suitable intervals. Our endeavor has been made possible through the encouragement we have received from many sources and through the enthusiasm and cooperative spirit of our contributors. For coining the term Analytical Profile we are indebted to Dr. James L. Johnson of the Upjohn Company. We hope that this, our contribution to the better understanding of drug characteristics, will prove to be useful. We welcome new collaborators, and we invite comment and counsel to guide the infant to maturity. Klaus Florey

xi

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T

Eiigerie Ivushkiv

1

EUGENE IVASHKIV

Table of Contents Page Description 1.1 Name:Ampicillin 1.2 Formula and Molecular Weight 1.3 Isomers 1.4 Hydrates 1.5 Salts 1.6 Appearance, Color and Odor 2. Physical Properties 2.1 Spectra 2.11 Infrared Spectra 2.12 Nuclear Magnetic Resonance Spectra 2.13 Mass Spectroscopy 2.14 Ultraviolet Absorption 2.2 Crystal Properties 2.21 Crystalline Modification of Ampicillin 2.22 X-ray Diffraction 2.23 Melting Range 2.24 Differential Thermal Analysis 2.25 Thermal Gravimetric Analysis 2.3 Solubility 2.4 Ionization Constant, pK 2.5 Optical Rotation 3. Ampicillin Stability 3.1 Modes of Penicillin Degradation 3.2 Stability of Ampicillin in Solution 3.3 Stability of Ampicillin Powders 3.4 Cupric Ion-Catalyzed Hydrolysis 4. Methods of Manufacture 4.1 Microbiological 4.2 Chemical 5. Isolation and Purification 6. Methods of Analysis 6.1 Identification Tests

1.

2

4 4 4 4 4

5 5 5

5 5 8

12 16

17 17 18 18

18 20 20 20 20 23 23 25 32 32 32 32

33 37 37 37

AMPICILLIN

Page 6.2 6.21

7. 8. 9.

O u a n t i t a t i v e Methods U l t r a v i o l e t Spectrophotom e t r i c Methods 6.22 Fluorometric Determination 6.23 Polarographic Determination 6 . 2 4 T h i n Layer Chromatography 6.25 P a p e r Chromatography 6.26 Iodometric T i t r a t i o n 6 . 2 7 Hydroxamic Acid 6 . 2 8 Amperometric T i t r a t i o n 6 . 2 9 M i c r o b i o l o g i c a l Methods P r o t e i n Binding Pharmacok i n e t i c s References

3

38 38 39 39 39 40 41 41 42 42 43 45 47

EUGENE I V A S H K I V

1.

Description

Name: Ampicillin AmpicillinL,L,jis designated by Chemical Abstracts as Q- (2-amino-2-phenylacetamido)-3,3-dimethyl-oxo-4-thia-l-azabicyclo 17.2.0-7 heptane-2-carboxylic acid. Ampicillin is also known as bb(-)-a-aminophenylacetamidd penicillanic acid, Q ( - ) -a-aminobenzylpenici llin4 and a-aminobenzylpenicillin5. 1.1

1.2

Formula and Molecular Weight

0

C)H l€12- C-N II I

I

C16H1 9N304S

349.41

Isomers The presence of a symmetric C atom in the side chain provides optical isomer6. The D-isomer, I)(-)-a-aminobenzylpenicillin, is more active than the &-isomer, L(-)-a-aminobenzylpenicillin7. The s nthesis of ampicillin epimer has been reported8 1.3

3

8,

1.4

Hydrates It has been re orted that ampicillin can exist in anhydrous sesquihydrate16 and trihydrate Austin, et al. postulate that ampicillin exists in anhydrous or trihydrate forms only. Refer to section 2.21.

4

AMPICILLIN

1.5

Salts P o t a s s i u m a n d sodium s a l t s o f a m p i c i l l i n 2 0 - 2 9 , human l y s o z me a m p i c i l l i n s a l t 3 ! 2-nitro-l,3-indandione salt3', and t h e a m p i c i l l i n s a l t of k a n a m y c 1 r - 1h ~ a~v e b e e n p r e p a r e d . 1.6

A p p e a r a n c e , C o l o r a n d Odor Ampicillin i s a free-flowing, white I t h a s an o d o r c h a r a c t e r i s t i c c r y s t a l l i n e powder. of p e n i c i l l i n s .

2.

Physical Properties

2.1

Spectra

2.11

Infrared Spectra S u b s t i t u t e d monocyclic O-lactams i n s o l u t i o n show c a r b o n y l a b s o r p t i o n w i t h i n t h e t h e B-lactam r i n g i s r a n g e 5 . 6 8 - 5 . 7 8 ~ ~ ~ When . fused t o a t h i a z o l i d i n e r i n g , the carbonyl a b s o r p t i o n occurs i n t h e range 5.62-5.651. Morin a n d c o - ~ o r k e r sh~a v~e n o t e d r o u g h c o r r e s p o n d e n c e b e t w e e n t h e f r e q u e n c i e s o f t h e f3-lactam c a r b o n y l a b s o r p t i o n and t h e b i o l o g i c a l a c t i v i t i e s of a s e r i e s of p e n i c i l l i n d e r i v a t i v e s . The i n f r a r e d s p e c t r a o f p e n i c i l l i n analogues have been discussed35. The i n f r a r e d s p e c t r u m o f a m p i c i l l i n t r i h y d r a t e was m e a s u r e d on a P e r k i n - E l m e r Model 2 1 double-beam s p e c t r o p h 0 t o m e t e r 3 ~ . The i n f r a r e d a b s o r p t i o n o f p e n i c i l l i n d e r i v a t i v e s h a s b e e n r e c o r d e d and Figure 1 and Figure 2 a r e t h e discussed37. s p e c t r a of t h e Squibb Primary Reference S u b s t a n c e s o f a m p i c i l l i n t r i h y d r a t e and anhydrous a m p i c i l l i n r e c o r d e d a s m i n e r a l o i l m u l l and p o t a s s i u m b r o m i d e p e l l e t s w i t h a P e r k i n - E l m e r Model 2 1 spectrophotometer. I n t e r p r e t a t i o n of t h e spectrum of a m p i c i l l i n t r i h y d r a t e h a s been rep ~ r t e d ~ ~ ai sn dg i v e n i n T a b l e 1. 5

F i g u r e 1.

I n f r a r e d Spectrum of Ampicillin T r i h y d r a t e Squibb Reference Standard.

FREQUENCY ( c M - ~ )

WAVELENGTH IMICRONSJ

F i g u r e 2.

I n f r a r e d S p e c t r u m o f Anhydrous A m p i c i l l i n S q u i b b Reference Standard.

EUGENE I V A S H K I V

Table I I n f r a r e d Spectrum of A m p i c i l l i n T r i h y d r a t e I R A b s o r p t i o n Band,

Interpretation

2.9 3.1 weak b a n d s 3 . 5 - 4 . 8 5.65 5.90 6 . 2 and 6 . 3 5 6.7

H20 N' H NH3+ @-lactam C = 0 Amide C = 0 + COO-, NH3 Aromatic r i n g , Amide 11, NH3+ 14.4 Monosubs t i t u t e d aromatic r i n g . I n t h e s o l i d form, a m p i c i l l i n t r i h y d r a t e e x i s t s a s t h e z w i t t e r i o n a n d t h e i n f r a r e d s p e c t r u m shows a b s o r p t i o n s t y p i c a l o f t h i s t y p e o f compound38. 2.12

N u c l e a r Magnetic Resonance S p e c t r a NMR s p e c t r a o f p e n i c i l l i n d e r i v a t i v e s i n d i f f e r e n t s o l v e n t s w e r e r e c o r d e d on a 1 0 0 MHz spectro~neter~R ~ e. c e n t l y , s t r u c t u r a l s t u d i e s w i t h l3c n u c l e a r m a g n e t i c r e s o n a n c e w e r e r e p o r t e d 4 0 €or p e n i c i l l i n s . I 3 c Chemical s h i f t a s s i g n m e n t s were made f o r t h e d i f f e r e n t c a r b o n NMR s p e c t r a i n D 2 0 s o l u t i o n s o f t h i r t e e n atoms. p e n i c i l l i n d e r i v a t i v e s h a v e b e e n r e c o r d e d and a n d Puar'' s t u d i e d NMR i n t e r ~ r e t e d ~ Cohen ~ . s p e c t r a of a m p i c i l l i n t r i h y d r a t e i n d e u t e r o d i m e t h y l s u l f o x i d e (DMSO-d6) a n d D 2 0 - D C 1 . Tetram e t h y l s i l a n e was u s e d a s a n i n t e r n a l s t a n d a r d . The V a r i a n XL-100-15 NMR s p e c t r o p h o t o m e t e r was used. I n t h e c a s e of D M S O - ~ ~B,- l a c t a m p r o t o n s c o u l d be e a s i l y d i s t i n g u i s h e d b y t h e i r c o u p l i n g pattern. The NMR s p e c t r a of t h e S g u i b b P r i mary R e f e r e n c e S u b s t a n c e a r e g i v e n i n F i g . 3 a n d F i g . 4 . The a s s i g n m e n t s a r e r e p o r t e d i n T a b l e 11. 8

9

u)

I

a

fn C .d

a, m

k

4J

$1

a

c

4

C

k E

u

.d rl rl -d -d

$ 0

rCI

5 k

a,

u

4J

a w

x m a, k

7

0-l

Irr

.rl

c

0

" e ? , , , , . , , ,

/ , , ,

Figure 4 .

, , , ,

, . , ,

,

,

/

,

, , , , , , . ,

1

, , , , I , , , ,

m

/ , , , , , , , ,

,

,

,

,

1

,

1

"

"

'

,

"

"

,

,

1

,

NMR Spectrum of Ampicillin T r i h y d r a t e in D20-DC1.

,

,

'

,

'

,

,

'

~

,

'

,

'

,

'

'

'

,

~

~

'

'

T a b l e I1 Proton-resonance

DMSO-d6

2H

3H

5H ,

1.39

1.39

5.39q

(9.0, 4.0)

5.39d

(4.0)

1.49 D20-DC1 e

1.41

4.47

6H

5.54

Lines

7H

8H

9

10

5.95b

4.84

7.36m

4.6433

5.26

7.53

>

5

1.44

6

5

c

EUGENE I V A S H K I V

2.13

Mass Spectroscopy The behavior of the penicillins in the mass spectrometer has been studied by several investigators. The fragmentation of penicillin V has been discussed by Kukolja, et a1 42. Richter and Biemann43examined penicillins G and V methyl esters and made numerous accurate mass measurements at high resolution. The nucleus fragmentation by four different routes is shown in Fig.5. Bochkarev, et al. 42 studied various penicillin derivatives substituted at the carbonyl group and showed that it was possible to deduce the nature of this substituent from the mass spectrum. The mass spectrum of ampicillin trihydrate was determined by Funke and C ~ h e n ~ ~ and is shown in Figure 6. They prepared a disilyl derivative using N,O-bis (trimethylsilyl)acid amide dissolved in pyridine. The mass spectrum exhibits the molecular ion, M+, of m/e 493 €or the disilyl derivative. The more intense M+ -15 ion at m/e 478 is due to loss of methyl radical. The m/e 421 ion corresponds to the loss of Si(CH3)2CH2 from the molecule or the mono-silyl derivative while the ion 15 amu lower at m/e 406 has lost a methyl group in addition to the silyl function. The diagnostic fragment ion at m/e 178 €or silyl derivatives of penicillins demonstrates the presence of 6-aminopenicillanic acid ( 6 - A P A ) function. In addition, the ion at m/e 106 also is diagnostic for the non-silanized phenylqlycyl derivative. The intense ion at m/e 232 is diagnostic €or all penicillin derivatives. The mass spectral assignments are summarized in Figure 7.

AMPICILLIN

F i g u r e 5.

__+

b

__+

c - - c

P e n i c i l l i n Fragmentation

RCHzCONHCH=C=O

lt.

Me2C==CHCO,Mclt' RCH2CONH

RCH~CO~HCH

H?

Me

1 N-'

/

R e p r o d u c e d w i t h p e r m i s s i o n from A d v a n c e s i n Druq R e s e a r c h , Vol. 6 , p. 2 0 0 , A c a d e m i c P r e s s , New York, 1971.

13

i EUGENE IVASHKIV RELATIVE

~

~

~

r

INTENSITY

n

-

O

c

)

-

m

-

m

l

PERCENT TOTAL IONIZATION 39

Figure 6 .

Low R e s o l u t i o n Mass Spectrum of A m p i c i l l i n Trihydrate Squibb House Standard. 14

r

n

AMPICILLIN

178

231

I

I

+

H

1

m/e

178

+

t

m/e

Fig. 7.

106

Fragmentation of Ampicillin Derivative.

IS

EUGENE IVASHKIV

2.14

U l t r a v i o l e t Absorption U l t r a v i o l e t spectra of ampicillin t r i h y d r a t e , Squibb Primary Reference Substance were r e c o r d e d on a C a r y Model 1 5 s p e c t r o p h o t o meter46 a n d E l % values calculated. The r e s u l t s a r e g i v e n i n 4aF€'e 111.

T a b l e I11 U l t r a v i o l e t Absorption of A m p i c i l l i n T r i h y d r a t e W a v e l e n g t h , nm Phosphate b u f f e r pH 5 . 3 E 1%

2 57

262

268

8.69

7.81

5.61

257

262

268

7.94

6.69

4.55

2 58

2 68

-

7.28

5.03

1 cm P h o s p h a t e b u f f e r pH 7 . 0

1% 1 cm Phosphate b u f f e r 9.5 E 1% 1 cm

With t h e i n c r e a s e o f pH o f b u f f e r s o l u t i o n El% decreases. N o p e a k a t 268 nm i n cm p H 9 . 5 b u f g e r was o b t a i n e d . Saturated methanolic ampicillin t r i h y d r a t e s o l u t i o n s p r o d u c e d maximum a b s o r p t i o n a t 2 5 8 , 262 a n d 268 nm46.

16

AMPICILLIN

2.2

Crys ta 1 Properties

Crystalline Modification of Ampicillin Ampicillin crystallizes into two forms depending on the temperature of the aqueous solution 19. Anhydrous ampicillin is obtained from aqueous solutions at temperatures above 6OoC; the trihydrate is obtained from aqueous solutions at temperatures below 5OoC. The existence of anhydrous, monohydrate, sesquihydrate and trihydrate fo-ms of ampicillin was It has been shown by x-ray dif fr a ~ t i o nthat ~ ~ ampicillin monohydra te existed. Austin et.al.19 - concluded from a study of the infrared spectra of various forms of crystalline ampicillin that only the anhydrate and trihydrate exist. They believed the other hydrates were either amorphous or partially dehydrated t r i h ~ d r a t e Crystals ~~. of anhydrous and trihydrate ampicillin were prepared. James and Hall reported crystallographic data for ampicillin trihydrate. They showed that ampicillin exists as a zwitterion with three water molecules extensively involved with ~ ~ hydrogen bonding. Grant and A l b ~ r nhave distinguished between a crystalline anhydrous form and a monohydrate, which differ in solidstate infrared spectra, density, solubility and th erma 1 stab i1ity Anhydrous amp ic i 11in wa s prepared from any form of ampicillin but reasonably rapid conversion of trihydrate required a temperature 80°-1000C19. Hydrated ampicillin was also converted into an anhydrate by heating a suspension in nitromethane or other nitrohydrocarbons. 2.21

.

17

EUGENE IVASHKIV

2.22

X-ray D i f f r a c t i o n S i n g l e - c r y s t a l x-ray d i f f r a c t i o n w a s u s e d t o d e d u c e t h e s t r u c t u r e of b e n z y l p e n i illi in^^. S e v e r a l s a l t s o f b e n z y l p e n i c i l l i n was a n a l y z e d a n d b o n d l e n g t h s determined5'. X-ray d i f f r a c t i o n a n a l y s i s o f an h y d ro u s a m p i c i l l i n a n d a m p i c i l l i n m o n o h y d r a t e were p e r f o r m e d by T.Doyne a n d r e p o r t e d 4 7 . F i g . 8 shows t h e x - r a y d i f f r a c t i o n o f a m p i c i l l i n and g i v e s t h e d v a l u e s c h a r a c t e r i s t i c o f t h i s c r y s t a l form o f a m p i c i l l i n t r i h y d r ate51. M e l t i n q Ranqe A m p i c i l l i n monohydrate melts w i t h decom o s i t i o n a t 202OC a n d s o d i u m a m p i c i l l i n a t 205°Cf4, s e s q u i h y d r a t e a n d a n h y d r o u s a m p i c i l l i n decompose a t 199-202°C16. The m e l t i n g r a n g e f o r a m p i c i l l i n t r i h y d r a t e with decomposition a t 214.5O - 215.5OC was r e p o r t e d 5 2 a n d m e l t i n g w i t h d e c o m p o s i t i o n h a s b e e n r e p o r t e d a t 202O- 204OC by another i n v e s tiga tor5?

2.23

2.24

D i f f e r e n t i a l Thermal A n a l y s i s Jacobson54 r e c o r d e d d i f f e r e n t i a l therma 1 a n a l y s i s c u r v e s o f a m p i c i l l i n tr i h y d r a te, S q u i b b P r i m a r y R e f e r e n c e S u b s t a n c e , o n a DuPont D i f f e r e n t i a l Thermal Analyzer w i t h a t e m p e r a t u r e r i s e o f 15O p e r m i n u t e . E n d o t h e r m i c steps a t 88O,92O, a n d a l a r g e e n d o t h e r m a t 100°C were observed. Some s a m p l e s of a m p i c i l l i n t r i h y d r a t e h a d a s i n g l e l a r g e e n d o t h e r m a t 125OC w h i l e Y i h e r s h a d t h e l a r g e e n d o t h e r m a t a b o u t 105O- llO°C D i f f e r e n t i a l thermal a n a l y s i s f o r t h e c o n t r o l o f It phase t r a n s f o r m a t i o n of a m p i c i l l i n i s used55. h a s b e e n f o u n d t h a t t h e r m o g r a m s were r e p r o d u c i b l e f o r a g i v e n l o t of a m p i c i l l i n t r i h y d r a t e b u t v a r i e d from lot t o l o t . The p h a s e t r a n s f o r m a t i o n of a m p i c i l l i n t r i h y d r a t e b y t h e d i f f e r e n t i a l t h e r m a l a n a l y s i s u n d e r p r e s s u r e was s t u d i e d 5 6 .

.

18

AMPICILLIN

19 0

w

c

!4

Figure 8. X-Ray Diffraction Pattern of Ampicillin Trihydrate

EUGENE I V A S H K I V

2.25

Thermal Gravimetric Analysis A DuPont Thermogravimetric Analyzer, Model 950, indicated total volatile material to be 13.8% in ampicillin trihydrate Squibb Primary Reference Substance54. The theory is 13.39%. 2.3

Solubility The solubilities of anhydrous ampicillin, ampicillin trihydrate and sodium ampicillin from two manufacturers in various solvents were determined by Marsh and we is^^^. They found a variation in the solubility of sodium ampicillin from two different manufacturers and indicated this could be due to crystalline structure. The results are reported in Table IV. Anhydrous ampicillin and ampicillin trihydrate were compared for solubility in distill d water at temperatures ranging from 7.50 to 5OoC 58, 2.4

Ionization Constant, pK

Rapson constants for 0.004 and pK Russo-Alesi66 hydrate to be = 2.66 f 0.03 2.5

and Bird5' reported ionization ampicillin to be: pK1 = 2.53 f - 7.24 f 0.02. Jacobson and calculated pK2 for ampicillin tri7.24. Hou and PooleG1 reported pK1 0.03. and pK2 = 7.24

Optical Rotation Reference

+

Ampicillin monohydrateL-d21 D (C = 1 in H20)

- - 20

Ampicillin sesquihydrateL a 4 (C = 1 in H20)

281° 14

+ 283.1° 16

-+ 209' Sodium Ampicillin/-az20 ( C = 0.2 in H20) 2o

-_

Anhydrous AmpicillinL ( C = 1 in H20)

%

+

14

287. go 16

20

Table I V S o l u b i l i t y of Amp icillin

(mg/ml)

Ampicillin Solvent

h) i

Anhydrous

Water Me t h a n o 1 Ethanol Isopropanol Isoamyl a l c o h o l Cyclohexane Benzene Petroleum E th er Isooctane Carbon T e t r a c h l o r i d e Ethyl Acetate Isoamyl A c e t a t e Acetone Methyl E t h y l Ketone Diethyl Ether Ethylene Chloride 1,4-Dioxane Chloroform

10.098 2.968 0.390 0.055 0.125 0.048 0.002 0.010 0.0 0.008 0.025 0.030 0.125 0.052 0.022 0.032 0.595 0.095

Na S a l t I*

> 20 P20 v 20 1. 13 1.902 0.075 0.022 0.025 0.022 0.032 0.035 0.105 0.518 0.178 0.022 0.060 1.375 0.118

Na S a l t 11*

T r ihydrat e

> 20 > 20

7.558 6.649 2.538

19.780 6.405

19. 300 0.0 0.0 0.0 0.0 0.0

0.058 0.048

7 20 720 0.0 0.032 1.845 0.155

0.068 0.032 0.038 0.022 0.025 0.225 0.078 8.952 2.790 0.03 0.068 2.772 0.075 continued..

.

Table IV.

Solubility of Ampicillin (mg/ml), continued Ampicillin

Solvent

13

h)

Carbon Disulfide Pyr i dine Formamide Ethylene Glycol Propylene Glycol Dime thylsul fox ide 0.1N NaOH

0.1N H C 1

Anhydrous

0.015 2.100 20 18.415

2.230 20 20 20

Na Salt I*

0.010 3.256 20 20 20 20 20 20

Na Salt I1

0.0 20 20 20 20 20 20 20

*

Trihydrate

0.022 12.131 20 19.128 4.138 20 20 20

rn C

GI

rn

z rn -

<

RI x -

<

*

Sodium ampicillin from two different manufacturers.

AMPICILLIN

The optical rotation for ampicillin tri/-&26 + 2 4 5 . 1 0 (C = 1 buffer p H 8.0) hydrate, and ampicillin anhydrous, l - ~ $+ *289O ~' ( C = 1, buffer p H 8.0) has been reported62. D ~ r s c hreported ~ ~ the optical *rjotation for ampicillin trihydrate as L-d + 249.5O to 252.7O (C = 1, buffer pH 8 . 0 p . Optical rotation in 2 1 hydrochloric acid, after dissolution of 1% ampicillin and 60 minutes of standing at 25OC is = -85. 5°C64. 3.

Ampic i 1lin Stab i 1i ty 3.1

Modes of Penicillin Degradation The main course of deterioration penicillins is hydrolysis. The course of the hydrolysis is shown in Figure 9. Dunham65 reviewed modes of penicillin degradation.

23

F i g u r e 9.

P e n i c i l l i n Degradation R-CONHCH-CH

/ RCONHCH-CH

I

/s\

I

C(CH3)z

I

NH-CHCOOH COOH

I

/s\ 1

C(CHJ,

I -CHCOO H

"/lHIH\

CO -N Pe nic ilI in

RCONHCHCHO

HOOC-CH-CH

I

1

COOH

\

\ w

P

+ RCONHCH,-

/s\

CH

C (C H3)2 I NH-CHCOOH

I

Penilloic Acid

H$J-

C(CH,), I

N-CKOOH

\ C/ I

R Penillic Acid

\

S H'

1

N

Penaldic A c i d

Penicilloic A c i d

/s\

'C(CH3)2

I

CHCOOH

RCONKH2CHO Penil loaldehyde

Penlciliamine

R e p r o d u c e d w i t h p e r m i s s i o n from " T e x t b o o k of O r g a n i c M e d i c i n a l a n d Pharmaceutical Chemistry", e d i t e d by C.O. Wilson, 0. Gisvol and R . F . D o e r g e , 5 t h E d . , 1966. L i p p i n c o t t Co., P h i l a d e l p h i a a n d T o r o n t o .

AMPICILLIN

3.2

S t a b i l i t y of Ampicillin i n Solutions S a v e l l o a n d S h a n g r a w 6 6 showed t h a t f r e e z i n g s o d i u m a m p i c i l l i n s o l u t i o n s a t -200 t o -78OC g e n e r a l l y i n c r e a s e d t h e d e g r a d a t i o n o f 1% s o l u t i o n s a n d d e c r e a s e d t h e d e g r a d a t i o n i n 25% solutions. The c o n c e n t r a t i o n of a m p i c i l l i n i n s o l u t i o n a n d t h e t y p e of v e h i c l e c o n t r o l d e g r a dation. Dextrose and mannitol a c t a s c a t a l y s t s i n t h e h y d r o l y s i s of a m p i c i l l i n . Ampicillin a t a 1% c o n c e n t r a t i o n i s i n c o m p a t i b l e w i t h d e x t r o s e Normal s a l i n e i s t h e m o s t s u i t a b l e v e h i c l e f o r intravenous administration of ampicillin. Ga 1l e 11i 6 7 c l a imed t h a t s o d i u m ampi c i 1l i n i s s t a b l e a t 5OC a n d 2 5 O C i n 1%s o d i u m c h l o r i d e s o l u t i o n s a l s o c o n t a i n i n g 5% d e x t r o s e . Jacobs et a1.,6' studied t h e s t a b i l i t y of a m p i c i l l i n i n 5% d e x t r o s e , 4.3% d e x t r o s e , 0.18% s o d i u m c h l o r i d e , 0.9% sodium c h l o r i d e , Hartmann' s s o l u t i o n a n d 1/6 M s o d i u m l a c t a t e . Ampicillin was l e a s t s t a b l e Tn s o l u t i o n s c o n t a i n i n g l a c t a t e . A m p i c i l l i n was l e a s t s t a b l e i n s o l u t i o n s c o n t a i n i n g l a c t a t e i o n , l o s i n g 40% a c t i v i t y a f t e r 4 h o u r s a n d more t h a n 60% a f t e r 24 h o u r s . Ampicillin is inactivated by sucrose, dextrose The a n d d e x t r a n s o l u t i o n s a t a l k a l i n e pH6'. s t a b i l i t y of a m p i c i l l i n i n 0.2M a c e t a t e , c i t r a t e , phosphate and l a c t a t e b u f f e r s p H 4.570 a n d i n s o l u t i o n s a t pH 6 a n d p H 6.5 was s t u d i e d 7 7 . The d e c o m p o s i t i o n r a t e o f a m p i c i l l i n i n a 1% s o l u t i o n f o l l o w s f i r s t o r d e r k i n e t i c s and o b e y s t h e A r r h e n i u s e q u a t i o n when c o m p a r e d a t 4OoC 5OoC, a n d 6OoC. T h e s e f i n d i n g s w e r e c o n f i r m e d f2 when t h e d e c o m p o s i t i o n r a t e was s t u d i e d i n t h e p r e s e n c e of H' ions. Degradation o f a m p i c i l l i n i s s e c o n d o r d e r i n t h e p r e s e n c e o f OH- i o n s . The s t a b i l i t y of a m p i c i l l i n i n a q u e o u s s o l u t i o n s The k i n e t i c s o f a t pH 4 - 9 was d e t e r m i n e d 7 3 . degradation of ampicillin i n s o l u t i o n s w e r e in-

at

EUGENE IVASHKIV

v e s t i g a t e d a t 35OC a n d c o n s t a n t i o n i c s t r e n g t h o f 0 . 5 o v e r a pH r a n g e o f 0 . 8 t o 1 0 7 4 . The o b s e r v e d r a t e s were f i r s t - o r d e r a n d s i g n i f i c a n t l y i n f l u e n c e d by g e n e r a l a c i d and g e n e r a l b a s e c a t a l y s i s . The a p p a r e n t h e a t s o f a c t i v a t i o n f o r a m p i c i l l i n d e g r a d a t i o n i n s o l u t i o n s w e r e 1 6 . 4 , 1 8 . 3 , and 9 . 2 k c a l / m o l e i n b u f f e r s o f pH 1 . 3 5 , 4 . 9 3 a n d 9.78 r e s p e c t i v e l y . The p H - r a t e p r o f i l e i n b u f f e r s o l u t i o n s showed a minimum a t a pH o f 4.85. However, a t z e r o b u f f e r c o n c e n t r a t i o n t h e maximum s t a b i l i t y was s h i f t e d t o a pH o f 5 . 8 5 . A m p i c i l l i n was s t a b l e i n s o l u t i o n s a t p H 3 - 9 when s t o r e d f o r 24 h o u r s a t 5OC a n d 25OC. A m p i c i l l i n was u n s t a b l e i n s o l u t i o n s a t pH 10 when s t o r e d a t a b o v e condition^^^. B~ndgaard~~ demonstrated a m e t a s t a b l e i n t e r m e d i a t e i n isomeration of p e n i c i l l i n t o p e n i c i l l e n i c a c i d i n a q u e o u s s o l u t i o n s . The d e g r a d a t i o n o f a m p i c i l l i n trihydrate i n solution is greatly influenced not o n l y b y pH, b u t a l s o b y t h e t y p e s of b u f f e r s a l t s 77. 2-Amino-2 ( h y d r o x y m e t h y l ) - 1 , 3 - p r o p a n e d i o l ( T r i s ) b u f f e r o f pH 7 i s h i g h l y d e l e t e r ious t o the s t a b i l i t y of ampicillin, b u t not a t C i t r a t e p r e s e n t s a converse p a t t e r n i n pH 5 . 0 . t h a t a m p i c i l l i n i s r e l a t i v e l y s t a b l e a t pH 7 b u t n o t s o a t p H 5. Phosphate i s i n t e r m e d i a t e i n a c t i o n b u t t e n d s t o resemble t h e T r i s p a t t e r n . Jacobson and Russo-Alesi7* p r e p a r e d a m p i c i l l i n t r i h y d r a t e s o l u t i o n s a t a c o n c e n t r a t i o n o f 5 mg/ml b y d i s s o l v i n g i t i n b u f f e r s o l u t i o n s o f pH 5 . 0 o r pH 7 . 0 p r e p a r e d from 0.1g s o d i u m a c e t a t e a n d a c e t i c a c i d a n d i n b u f f e r s o f pH 8 . 0 o r pH 8 . 8 p r e p a r e d . f r o m 0.1M - 2-amino-2 ( h y d r o x y m e t h y l ) - 1 , 3-propanediol and a c e t i c a c i d . Solutions w e r e k e p t a t room t e m p e r a t u r e a n d w e r e a n a l y z e d b y iodometric t i t r a t i o n . S t a b i l i t y data is given i n T a b l e V.

26

Table V Stability of Ampicillin as Function of p H

Time, hours 0.5 1 2 3 4 5 24 27 48 96

pH 5

Percentage Activity Remaining PH 7 PH 8 p H 8.8 96.0

-

-

87.3

99.4

100.6

-

-

73.3 62.0 54.0 44.0

100.6 -

100.0 I

92.0

100.3

-

-

-

0

92.0 83.

o

96.5 91

-

81.9 65.8 43.6 28.9 17.4 11.4 r

EUGENE IVASHKIV

George7’ s t u d i e d t h e s t a b i l i t y of d i l u t e a m p i c i l l i n s o l u t i o n s a t pH 5 . 8 - 6 . 0 a n d s t o r e d a t 5OC. R e s u l t s from m i c r o b i o l o g i c a l a s s a y s a r e r e p o r t e d i n Table VI. Table V I S t a b i l i t y of A m p i c i l l i n i n D i l u t e Phosphate S o l u t i o n s

Ampicillin Left, % Days 6 8 14

A m p i c i l l i n , mcg/ml

3 6 10 15 25 40

102 97 98 99 102

100

102 99 99

80 88 91 92

-

-

The s t a b i l i t y o f a m p i c i l l i n i n b u f f e r s p r e p a r e d from 0 . 1 M T r i s b u f f e r a n d a c e t i c a c i d a s a f u n c t i o n o f pH a n d t e m p e r a t u r e has b e e n S o l u t i o n s c o n t a i n i n g 5 mg per m l o f studied80. a m p i c i l l i n were made a n d s t o r e d a t room 0 t e m p e r a t u r e o r 5 C f o r 29 d a y s . Solutions w e r e t e s t e d € o r t h e r e m a i n i n g a m p i c i l l i n by t h e c o l o r i m e t r i c h y d r o x y l a m i n e method. Results a r e reported i n Table V I I .

28

Table VII stability of Ampicillin as Function of p H and Temperature Percent Activity Remaining at Room Temperature Time in Days

PH8.4

7.5 6.4 4.9 3.8 2.9 2.3 1.7 16.5 1.8 0 0 0 0

1

2 6 9 15 29

53.1 32.2 4.8 0 0 0

90.6 82.1 60.1 45.2 26.5 11.4

89.4 88.7 67.6 54.4 39.5 13.9

73.1 54.2 21.7 10.7 3.9 1

47.9 26.7 3.5 1.1 0 0

24.7 8.0 0 0 0 0

b

Percent Activity Remaininq at 5OC

L3 N

Time in Days 1 2 6 9 15 29

pH 8.4 43.9 23. 1

1 0 0 0

2.9 2.3 4.9 3.8 7.5 6.4 1.7 83.0 100 95.2 94.2 85.3 81.4 92.7

?c

95.5 86.3 91.9 89.9 86.3

z

71.2 42.5 27.7 11.9 0

87.6 78.9 73.6 63.5 48.6

93.4 94.6 93.4 85.1 79.9

92.1 82.4 71.5 58.6 39.8

81.6 64.7 51.2 33.2 13.6

67.7 39.8 23.7 9.9 1.5

The stability of ampicillin trihydrate in sodium acetate and Tris buffers as a function of time was studied8l. Data is reported in Table VIII.

r

r

Table V I I I S t a b i l i t y of A m p i c i l l i n T r i h y d r a t e i n S o d i u m A c e t a t e a n d T r i s B u f f e r s a s a F u n c t i o n of p H ( A m p i c i l l i n (2 mg/ml,

I n i t i a l pH

u O

0.1M T r i s 0.1g N a O A c 0.1M T r i s 0 . 1 M NaOAc 0.1M T r i s 0.1M - NaOAc

4.9 4.9 5.9 6.0 6.7 6.5

Hydroxylamine method)

5 Hours

24 Hours

48 Hours

120 Hours

216 Hours

Final 312 H o u r s pH

98.1 98.3

93.9 94.3

92.4 94.7

73.4 78.9

57.4 65.0

40.8 49.9

4.8 4.9

98.8 102.4 91.3 99.9

90.1 96.7 65.6 96.9

91.5 102.1 48.1 98.2

66.9 90.8 14.3 90.7

45.6 86.7 3.3 86.9

30.5 77.9 0 75.7

5.6 5.8 6.4 6.3

- - - - --

rn C

2

rn

-

< $

I E -

<

AMPICILLIN

Data have been obtained concerning the stability of ampicillin in human stool specimens. Sixtyfour to eighty-four percent of the ampicillin was recovered after 3-4 week storage of the stool-buffer blend in dry ice. A recovery cont control consisting of the sample stool-buffer blend dosed with a known amount of ampicillin was prepared f o r each sample. When a correction is made based on the recovered penicillin from the control, full recovery of ampicillin and stability through 3 weeks is obtained from the stool-buffer mixture82. When ampicillin was incubated with the fecal flora of rats and tested for antibiotic activity, only 36.7% oi the ampicillin activity was 1 e F t after 4 1 1 0 ~ ~ ~ 8 3 . G r a d r ~ i kshowed ~~ that ampicillin is unstable when injected with penicillinase into rabbits. Synergistic action of ampicillin and cloxaci llin has been demons tra ted85. Cloxacillin inhibits the microbia 1 degradation o f ampici llin. The enzymic degradation of ampicillin by extracellular and cell-bound penicillinases from an ampicillin resistant strain of Staphylococcus was also inhibited by cloxacillin. The addition of cloxacillin to solutions containing S.aureus penicillinase greatly reduced @-lactam hydrolysis of ampicillin. B-lactam ring hydrolyhydrolysis with Difco penicillinase is a basis of the spectrophotometric determination of ampicillin75. Aminoalkylcatechols have been shown to catalyze the hydrolysis of ampicillin at relatively rapid rates at neutral pH by a mechanism similar to that postulated for several hydrolytic enzymesa6. Rates of hydrolysis of ampicillin were measured in the presence of several 3,6-bis(aminoalkyl) catechols where the amino group was varied in both size and basicity. Hou and P ~ o l e ~ ~ r e p o r tthat e d ampicillin at a pH equal to the isoelectric point exists as 31

EUGENE IVASHKIV

zwitterion and in this form is most stable in aqueous solutions. The stabilization of solutions with acetone has been patented88. Kuchinskas and Levy8’ showed that ampicillin formed polymers in different molecular sizes in aqueous solutions within 1 day that were separable by filtration on columns of an acrylamide gel. Stability of Ampicillin Powders Weiss and Palmerg0 showed that ampicillin powders were stable when stored in a closed system at 43% and 81% relative humidity at room temperature for 6 weeks. Ampicillin is also stable at 35OC in the same closed system for nine weeks. Ampicillin showed little change in either moisture content or potency. 3.3

Cupric Ion-Catalyzed Hydrolysis It has been reported91,92 that the effect of Cu (11) on the penicillins was to promote their degradation to coordination complexes of Cu (11) and the corresponding penicilloic acids. Complexation was assumed to occur between Cu (11) and the intact penicillins, followed by a rate limiting hydrolysis of the complex into the corresponding penicilloic acid - Cu (11) complex, The reaction mechanism and the catalytic siteof complexation of Cu (11) with penicillin have been s tudiedg3. 3.4

4.

Methods of Manufacture 4.1

Microbiological Ampicillin can be prepared microbiologically by incubation of microbes, e.g. Pseudomonas species, Kluyvera citrophila, Rhodopseudomonas spheroides, or Micrococcus ureae with 6-aminopenicillanic acid and a-phenylglycineg4, via enzymic acylation of 6-aminopenicillanic acidg5,96 32

AMPICILLIN

or it may be synthesized by the cell-bound pen ic i 11in a cy la se of Es cher ich ia col i9 7. Factors affecting the synthesis of ampicillin by the cell-bound penicillin acylase of Escherichia coli have been studied98. Chemical Ampicillin has been synthesized by condensing 6-aminopenicillanic acid (6-APA) with a protected a-phenylglycine. The many groups used to protect the reactive primary amine of a-phenylglycine are summarized in the Table IX. Ampicillin is regenerated b y removal of the protective groups. Ampicillin can also be obtained by treating 6-APA with a-bromophenylacetyl bromide131 via azido penicillin and catalytic h y d r ~ g e n a t i o n l3 133 ~ ~ 9 134, the reaction of 6-APA with D ( - 1 -2-phenylglycine chloride135 and trime thylsiliconamine and pyr idine136. Ampici 11 in is prepared by treating a-bromobenzylpenicillin with hexamethylenetetramine137>138 and by Schiff base processl39. A flowsheet of a plant for the production of synthetic penicillins containing predetermined side chains attached to 6-APA is givenl40. 4.2

33

EUGENE IVASHKIV

Table IX Synthesis of Ampicillin Protective Group on a-Phenylglycine

Reference

2-Nitro-4-methoxyphenylsulfenyl 2,5-0xazolidinedione

99 100,101,102 103,104 110 107,108,109 105,106

-o-Nitrophenylsulfenyl

N-Carboxyanhydride E-Toluenesulfenyl N-(l-Methyl-2-phenylcarbamoylvinyl) anhydride 111 N-Formyl 112 2-Quinol-8-yl-carbonylamino 113 1-3-0xazine-2,6-dione 114 6-N-benzoyl-2-tyrosyl 115 a-Benzyloxycarbonylamino 116 2,4-Pentanedione(Acetylacetone) 117,118,119 N-Carbobenzoxy 120,121,122 @-Dicarbonyl(BzCH2Ac, Ac2CH2, ACCH2C02Et) 123,124 N (2-Hydroxy-1-naphthylmethylene ) anhydride 125 6-2-Phenylcyclohex-3-enylcarboxamido 126 Furfura1, furylacrolein,crotonaldehyde, anisaldehyde and salicylaldehyde 127 E thoxyform ic anhydride 128 Phenylsulfide (PhSH) 129 o-Acetoacetanisidine 130

34

AMPICILLIN

Production of ampicillin via methyl N( a-carboxybenzy1)-@-aminocrotonate is illustrated. T h e process for the preparation of the ted a-phenylglycine has been patented and can be summarized as follows:

i4fYf22

HCOOH

+

NaOCH3+

H2 D-

(-)-a-

phenylglycine

HCOONa

sodium methylate

+

CHCOONa t CH30H NH2 ph eny lg lyc ine sodium salt

CH3COCH2COOCHT,

H2 methyl acetoacetate

+

3s

H2°

EUGENE IVASHKIV

The patented process for the synthesis of ampicillin trih~dratell~-~’’can be outlined: ClCOOCH2CH3+

mixed anhydride

mixed anhydride

+

H2N-CHC

protected Ampicillin HC1,

+

NaOH4 H

O=C’

LHCOOH 3H20

Ampicillin trihydrate + CH3COCH2COOCH3 + N a C 1

36

AMPICILLIN

5.

Isolation and Purification Ampicillin can be purified by acid or alkaline recrystallizations. It is dissolved in a mineral acid or caustic solution and precipitated by the adjustment of pH64. Crude ampicillin is purified by dissolution in methylene dichloride containing triethylamine, filtration and precipitation with p-toluene sulfonic acid 143. Ampicillin is separated from 6-aminopenicillanic acid by the filtration of chloroform or methylene chloride containin triethylamine and evaporation of the solvent 124 . Antigenic impurities are removed from solution by gel filtrati0n~~5-148. A process is described for the isolation of ampicillin via adduct formation with aliphatic amines and precipitation with 6-amino-1,3-naphthalenedisulfonic acid149. Ampicillin was separated from dicloxacillin and cloxacillin on an ion exchange column, IRA-402 resinl50. Ampicillin was extracted with ion-pairing and adduct-forming agentsl51. Purification of ampicillin via Schiff' s bases, sulfonic acid salts, amine salts and Tergitol salts has been evaluatedl52. 6.

Methods of Analysis

6.1

Identification Tests Ampicillin was identified by infrared s p e c t r o ~ c o p y l ~ ~Chromotropic . acid, sulfuric acid, ninhydrin and potassium cupritartrate tests2 are used to identify ampicillin. Electrophoresis in an agar gel is used for separation of penicillins. Spots are detected microbiologica 1 1 ~ Thomas ~ ~and Broadbr ~ . idge155 described a method for the rapid separation and detection of mixtures of penicillins by low voltage electrophoresis. The penicillins are

EUGENE IVASHKIV

id?ntified microbiologically. Sephadex thin layer chromatography and bioautography were combined for the identif icatests for tion of a n t i b i o t i ~ s l ~ ~Identity . antibiotics sensitivity discs were developed157. Several thin layer chromatography methods for identification of penicillin are reportedl58-162. A simple and rapid polyamide chromatography of penicillins is describedl63. (See Section 6.2.4 for additional systems). Paper chromatography is used for the separation and identification of different p e n i ~ i l l i n s l ~ ~ -The ~ ~ ~Rf. values of separated samples are compared with those of standards. (See section 6.2.5 for additional methods). 6.2 6.21

Quantitative Methods Ultraviolet Spectrophotometric Methods A spectrophotometric method for the determination of ampicillin involving initial benzoylation of the side chain a-amino group is described. a-Benzamidobenzylpenicillin so formed is treated with mercuric chloride in acid solution and a-benzamidobenzylpenicillenic acid is obtained. This may be assayed spectrophotometricallyl69. Ampicillin is degraded in a buffer solution at p H 5.2 at 75OC and the absorbance is measured at 320 nm 170. Smith's method €or the spectrophotometric determination of ampicillin was adapted to the assay of ampicillin in chicken blood, bile and urinel7+ Ampicillin is dissolved in 5 g sodium hydroxide and the absorbance is measured at 279 Ampicillin is determined spectrophotometrically as its copper complexl73. Spectrophotometric and circular dichroism methods for determining the activity of ampicillin are described174. A method.based on,the degradation of.the @-lactam ring with penicillinase and formation of a 38

AMPICILLIN

copper chelate is given 175. 6.22

Fluorometric Determination A method for the fluorometric analysis of am icillin in biological fluids is describede76. Ampicillin forms a strongly fluorescent yellow product in acid solution at elevated temperatures. A fluorescent thin layer chromatography procedure for determining trace ampicillin involves the reaction of hydrolyzed penicillin with a fluorescent isothiocyanate to form the corresponding fluorescent thioureal77. 6.23

Polarographic Determination Ampicillin can be determined at p. p.m. levels by differential pulse p ~ l a r o g r a p h y l ~ ~Ampicillin . undergoes reduction at a mercury electrode and the peaks obtained can be used €or both quantitative and qualitative analyses. Peak heights are linear with concentration. 6.24

Thin Layer Chromatography A thin layer chromatographic assay of ampicillin and cloxacillin in body fluids was developed. Penicillins are separated on silica gel. Spots were obtained using a bioautographic technique177. Mixtures of synthetic penicillins are separated on a silica gel sheet and developed with butano1:ethanol:water (4:1:5) l 8 O and zones of inhibition were obtained with Bacillus subtilis ATCC6633. Mixtures of ampicillin and oxacillin were separated by thin layer chromatography on silica gel and the antibacterial activity was measured against Sarcina luteal8’. A thin layer chromatographic method for semisynthetic pen ici 11ins and tetracyclines is descr ibed18 2 . Antibiotics are separated on silica gel plates using water or p H 6.1McIlvaine’sbuffer solution

EUGENE IVASHKIV

as solvent. Biagi e t a1.183 used r e v e r s e d phase t h i n l a y e r c h r o m a t o g r a p h y on s i l i c a g e l impregn a t e d w i t h s i l i c o n e DC200 t o d e v e l o p R f v a l u e s for various penicillins. Reversed-phase t h i n l a y e r c h r o m a t o g r a p h y was u s e d t o examine t h e e f f e c t o f t h e pH o f a m o b i l e p h a s e on t h e p a r t i t i o n i n g o f v a r i o u s p e n i c i l l i n s between a p o l a r mobile phase and nonpolar s t a t i o n a r y A q u a n t i t a t i v e t h i n l a y e r chromatog r a p h i c procedure f o r measuring a m p i c i l l i n i s described. S i l i c a g e l p l a t e s impregnated w i t h s i l i c o n e f l u i d a r e used. M c I l v a n e ' s pH 4 . 1 b u f f e r c o n t a i n i n g 1 . 5 % a c e t o n e i s employed f o r development. The s p o t s a r e v i s u a l i z e d w i t h ninhydrin. A c o l o r i s e l u t e d and measured

spectrophotometrically~8~. 6.25

Paper Chromatography A paper chromatographic technique

f o r d e t e r m i n a t i o n of a m p i c i l l i n i n e p i c i l l i n Paper chromatos a m p l e s h a s b e e n developed186. grams w e r e d e v e l o p e d w i t h a w a t e r s a t u r a t e d m i x t u r e o f n - b u t a n o l and t - a m y l a l c o h o l ( 6 : l ) pH 4 . 2 M c I l T a i n e l s b u f f e r . P e n i c i l l i n s were d e t e c t e d b i o l o g i c a 11y I 8 6. R o b e r t s a n d Vah i d i l8 described a paper chromatographic procedure f o r the d e t e r m i n a t i o n of a m p i c i l l i n . Papergrams a r e developed i n n-butanol, t-amyl a l c o h o l and w a t e r (7:1:4) f o r 4 8 hours. Bioautograms of t h e s t r i p s a r e prepared using g . aureus p l a t e s . Paper chromatography of a m p i c i l l i n samples w a s c a r r i e d o u t on Whatman N o . 1 paper188. The f o l l o w i n g systems w e r e used: b u t a n o l , e t h a n o l , water(4:1:5), butanol, a c e t i c a c i d , water (12:3:5), butanol, p y r i d i n e , w a t e r (l:l:l), m e t h a n o l , p y r i d i n e , c o n c e n t r a t e d h y d r o c h l o r i c a c i d , w a t e r (160: 20:4: 3 5 ) , ammonia, p r o p a n o l , w a t e r ( 3 : 6 : 1 ) . After d e s c e n d i n g c h r o m a t o g r a p h y p a p e r s were s p r a y e d with ninhydrin or developed b i o a u t o g r a p h i c a l l y 40

AMPICILLIN

u s i n g S t a p h y l o c o c c u s a u r e u s 1 8 * . Two d i m e n t i o n a l paper chromatography f o r t h e d e t e r m i n a t i o n of a m p i c i l l i n i n u r i n e was used189. UrilgOinoculated agar p l a t e s i n p a r a l l e l s t r i p e s with 16 microbial c u l t u r e s of gram-positive, gram-negative and a c i d r e s i s t a n t microorganisms and w i t h Candida a l b i c a n s The g r o w th z o n e s a r e t h e n p a r t i a l l y c o v e r e d w i t h a f i l t e r paper s t r i p containing t h e separated a n t i b i o t i c s t o be t e s t e d . The z o n e s o f i n h i b i t i o n were m e a s u r e d a n d c o n c e n t r a t i o n o f a m p i c i l l i n calculated. 6.26

Iodometric T i t r a t i o n Alicinol91 described an iodometric m e t h o d f o r t h e d e t e r m i n a t i o n of p e n i c i l l i n s . O n l y a f t e r a l k a l i n e or p e n i c i l l i n a s e hydrolysis t h e p e n i c i l l i n s consume i o d i n e . The d i f f e r e n c e i n consumption of i o d i n e b e f o r e and a f t e r h y d r o l y s i s i s proportional to t h e q u a n t i t y of t h e a n t i b i o t i c . R u ~ s o - A l e s i ~ ~ ~ ut sh e di o d o m e t r i c t i t r a t i o n f o r e s t i m a t i o n o f a m p i c i l l i n i n f o r m u l a t i o n s . Ampic i l l i n s o l u t i o n s w e r e h y d r o l y z e d w i t h sodium h y d r o x i d e f o r 30 m i n u t e s . After acidification the i o d i n e s o l u t i o n was a d d e d . After an a d d i t i o n a l 3 0 m i n u t e s , t h e e x c e s s o f i o d i n e was t i t r a t e d w i t h A b l a n k c o n s i s t e d o f unthiosulfate solution. hydrolyzed ampicillin. The a m p i c i l l i n s t a n d a r d was t r e a t e d i n e x a c t l y t h e same m a n n e r a n d u s e d i n calculations. The h y d r o l y s i s o f a m p i c i l l i n w i t h p e n i c i l l i n a s e i n s t e a d w i t h s o d i u m h y d r o x i d e makes t h e m e t h o d more s e l e c t i v e . An a u t o m a t e d s y s t e m f o r t h e iodometric determination o f p e n i c i l l i n s using an The a c c u r a c y o f t h e AutoAnalyzer i s d e s c r i b e d l 9 3 , m e t h o d i s f r o m 1 t o 3% a n d t h e p r e c i s i o n i s 2 t o 3.4%. Hydroxamic A c i d The f o r m a t i o n o f a c o l o r e d c h e l a t e of f e r r i c i o n and t h e hydroxamic a c i d of benzyl6.27

41

EUGENE I V A S H K I V

penicillin is the basis of the colorimetric methodl94. The color produced can be extracted into butanollg5. Details of the procedure was publishedlg6. The automated methodlg7 for the determination of benzylpenicillin can be used for the determination of ampicillin. Penicillins react with hydroxylamine and ferric ion to produce a color which is measured continuously with an AutoAnalyzer. 6.28

Amperometric Titration Ampicillin is hydrolyzed to penicillamine. The SH group of penicillamine is titrated amperometrically. The author claims that the method determines only the biologically active penicillins and not the degradation productslg8. 6.29

Microbiological Methods A complete description is given for the laboratory equipment, solutions, culture diffusion and turbidimetric assays used as tests and methods for the assay of ampicillin199. Yakovlev200 determined ampicillin in blood serum by an agar-diffusion technique. Staphylococcus aureus 209P and Bacillus subtilis were used as test organisms. A modified AutoAnalyzer method of Shaw and Duncombe201 is used for the determination of ampicillin202. The inoculum was Escherichia coli 3973 grown on tryptone soya agar, prepared by the method of Dewart203. Srimshaw and Jones204 presented an automated method for the determination of ampicillin. The method produces a linear response for ampicillin over an 80-180 mcg/ml range. An automated turbidimetric system for ampicillin assay is described205.

42

AMPICILLIN

7'

O u i n n , e Bindin?06 t al.,

found t h a t a n p i c i l l i n w a s

l e s s h i g h l y bound t o serum t h a n e i t h e r b e n z y l p e n i c i l l i n o r phenoxymethylpenicillin. Serum h a d l i t t l e e f f e c t on t h e a c t i v i t y o f a m p i c i l l i n 2 0 7 . When t h e O x f o r d S t a p h y l o c o c c u s a n d a s t r a i n o f Escherichia coli w e r e used a s test organisms, t h e minimal i n h i b i t o r y c o n c e n t r a t i o n s f o r a m p i c i l l i n i n t h e p r e s e n c e o f 40% human s e r u m w e r e o n l y o n e The b i n d i n g of t u b e lower t h a n i n t h e c o n t r o l s . a m p i c i l l i n t o p r o t e i n s was i n v e s t i g a t e d 2 0 8 , P h e n o l r e d p r o v e d t o be t h e most s u i t a b l e f o r c o m p a r i s o n o f t h e b i n d i n g o f p e n i c i l l i n s t o serum I n v e s t i g a t i o n s w i t h albumin t r e a t e d proteins. with dinitrofluorobenzene proved t h a t primary r e a c t i v e amino g r o u p s o f t h e a l b u mi n m o l e c u l e s a r e r e s p o n s i b l e € o r t h e b i n d i n g of p e n i c i l l i n s . Binding of p e n i c i l l i n s , including a m p i c i l l i n , w i t h s e r u m p r o t e i n s w e r e s t u d i e d , t o o 209. P e n i c i l l i n s w e r e n o t b o u n d b y human g l o b u l i n b u t a l a r g e a m o u n t s , e x c e p t a m p i c i l l i n , was b o u n d L i p i d s o l u b i l i t y a n d serum w i t h 1%a l b u m i n . protein binding of various penicillins w e r e correlated210. Robinson and S u t h e r l a n d 2 1 1 s t u d i e d t h e b i n d i n g of p e n i c i l l i n s a n d o t h e r a n t i b i o t i c s i n serum b y u l t r a f i l t r a t i o n t e c h n i q u e . New d a t a w e r e g i v e n on t h e b i n d i n g of p e n i c i l l i n s t o t h e p r o t e i n s i n human s e r u m 2 1 2 . The e x t e n t o f b i n d i n g was d e t e r m i n e d b y d i a l y s i s , p l a t e tests and g e l f i l t r a t i o n . A new a p p r o a c h t o s t u d y i n g t h e p r o t e i n b i n d i n g of p e n i c i l l i n s h a s been reported213. G e l - f i l t r a t i o n and b o t h s t a t i c a n d c o n t i n u o u s d i a l y s i s m e t h o d s were u s e d t o examine p r o t e i n b i n d i n g o f p e n i c i l l i n s . Results showed t h a t p e n i c i l l i n s d i f f e r i n t h e d e g r e e o f binding. The r e l a t i o n s h i p b e t w e e n t h e e x t e n t of serum-protein b i n d i n g o f p e n i c i l l i n s and t h e c o r r e s p o n d i n g e f f l u e n t v o l u m e from S e p h a a e x G - 2 5 , and t h e r e l a t i o n s h i p between t h e p r o t e i n - b i n d i n g

43

EUGENE IVASHKIV

e x t e n t and t h e g e l - a f f i n i t y i s d e s c r i b e d 2 1 4 . P e n i c i l l i n s showing an i n c r e a s e i n serum b i n d i n g a l s o showed an i n c r e a s e i n t h e e f f l u e n t volume. E s t i m a t i o n a n d u s e of serum l e v e l s i n t h e e v a l u a t i o n o f t h e new p e n i c i l l i n i s p r e s e n t e d 2 1 5 . T h e d e g r e e o f b i n d i n g was e s t a b l i s h e d b y f i l t r a t i o n under n e g a t i v e p r e s s u r e through a d i a l y s i s membrane. The b i n d i n g r a t e s o f p e n i c i l l i n s t o m i x t u r e s o f a l b u m i n , e u g l o b u m i n , a n d pseudog l u b u l i n f r a c t i o n were n e a r l y t h e same a s t h o s e o f t h e whole b o v i n e serum216. The b i n d i n g r a t e o f t h e m i x t u r e o f a n y 2 o r 3 o f t h e serum p r o t e i n f r a c t i o n s i s a l w a y s l o w e r t h a n t h e sum o f t h o s e t o corresponding i n d i v i d u a l f r a c t i o n . Binding r a t e s a n d t h e modes o f b i n d i n g o f p e n i c i l l i n s w i t h v a r i o u s p r o t e i n s i n v i v o a n d i n v i t r o were i n v e s t i g a t e d by u l t r a f i l t r a t i o n and dialysis217. Kunin2I8 r e p o r t e d t h a t o n l y 23% o f a m p i c i l l i n w a s bound t o human serum. Serum p r o t e i n s b i n d a m p i c i l l i n o n l y loosely219. A m p i c i l l i n was h a r d l y a b s o r b e d on human r e d c e l l s , s l i g h t l y i n a c t i v a t e d w i t h mouse l i v e r h o m o g e n a t e s a n d was combined s l i g h t l y w i t h h o r s e serum p r o t e i n s 2 2 0 T h e e f f e c t of serum b i n d i n g on t h e d i s t r i b u t i o n of p e n i c i l l i n s i n t h e r a b b i t h a s b e e n s t u d i e d 2 2 1 . l 4 C - l a b e l e d p e n i c i l l i n s were u s e d a n d t h e i r d i s t r i b u t i o n i n b r a i n , m u s c l e , l u n g a n d h e a r t was measured and c o r r e l a t e d t o b i n d i n g . The r o l e o f a m p i c i l l i n i n a n t i b i o t i c t h e r a p y and i t s serum binding i s given222. D i f f e r e n c e s i n serum b i n d i n g among p e n i c i l l i n s e x e r t a p r o f o u n d e f f e c t upon t h e amount o f f r e e d r u g a v a i l a b l e f o r a n t i m i c r o b i a l a c t i v i t y 2 2 3 . Kunin224 f o u n d t h a t a m p i c i l l i n showed l e s s b i n d i n g t o serum p r o t e i n s t h a n d i d dicloxacillin, cloxacillin, oxacillin, nafcillin, p e n i c i l l i n s G a n d V and m e t h i c i l l i n . An a n t i microbial a c t i v i t y of p e n i c i l l i n s is increased by c o m p e t i t i v e serum b i n d i n g i n h i b i t o r s 2 2 5 . Immunochemical s t u d i e s on t h e a n t i g e n t i c b i n d i n g s 44

AMPICILLIN

o f a m p i c i l l i n a n d o t h e r p e n i c i l l i n s w e r e conducted226. T h e amount o f a m p i c i l l i n b o u n d t o t h e p r o t e i n was e s t a b l i s h e d b y d i a l y s i s a n d w i t h pchloromercuribenzoate t i t r a t i o n of t h e penicilloyl groups and by t h e formol t i t r a t i o n of p r o t e i n amino g r o u p s 2 2 7 . Pharmacokinetics Pharmacokinetics of a m p i c i l l i n t r i h y d r a t e and sodium a m p i c i l l i n f o l l o w i n g i n t r a m u s c u l a r Serum l e v e l s o f i n j e c t i o n w e r e s t u d i e d 228. a m p i c i l l i n a c t i v i t y w e r e determined. The r a t e s of a b s o r p t i o n o f t h e d r u g f r o m t h e i n t r a m u s c u l a r i n j e c t i o n s i t e s were c a l c u l a t e d . The a m p i c i l l i n s u s p e n s i o n d a t a showed t h a t a b s o r p t i o n p r o c e s s i s slower t h a n t h e e l i m i n a t i o n p r o c e s s . Ampicillin s o d i u m s o l u t i o n s a r e a b s o r b e d more e f f i c i e n t l y than ampicillin trihydrate. K l e i n e t a l . 229 g a v e three intramuscular i n j e c t i o n s of a m p i c i l l i n to e i g h t a p p a r e n t l y h e a l t h y young men. Doses o f 1 . 0 g . , a n d 0 . 5 g . w e r e g i v e n a t two-day i n t e r v a l s . A b s o r p t i o n a n d e x c r e t i o n o f i n t r a m u s c u l a r d o s e s of a m p i c i l l i n a p p e a r e d t o be somewhat d e l a y e d when Peak s e r u m compared w i t h p e n i c i l l i n s G a n d V. l e v e l s o f a m p i c i l l i n a p p e a r e d l a t e r and s i g n i f i c a n t a n t i b a c t e r i a l a c t i v i t y was also d e m o n s t r a t e d On t h e a v e r a g e , a b o u t t w i c e a s much a m p i c i l l i n was r e c o v e r e d i n t h e u r i n e a f t e r i n t r a m u s c u l a r a d m i n i s t r a t i o n t h a n when same a m o u n t s h a d b e e n given o r a l l y . f o u n d t h a t p e a k serum c o n c e n t r a t i o n a f t e r a 500 mg i n t r a m u s c u l a r d o s e of a m p i c i l l i n was a t t a i n e d w i t h i n 20-30 m i n u t e s . U r i n a r y e x c r e t i o n a m o u n t e d t o 40% o f t h e d o s e Bunn, e t a l . f o u n d within the f i r s t 8 hours. a l s o t h a t p e a k serum c o n c e n t r a t i o n o f a m p i c i l l i n was a t t a i n e d w i t h i n o n e h o u r a f t e r i n t r a m u s c u l a r i n j e c t i o n o f 5 0 0 mg, 7 5 0 mg o r 1 0 0 0 mg of ampicillin231. P r o b e n e c i d i n c r e a s e s and s u s t a i n s t h e l e v e l s of a m p i c i l l i n i n t h e s e r u m a f t e r i n t r a 8.

4s

EUGENE IVASHKIV

The muscular i n j e c t i o n of t h e a n t i b i o t i c 2 3 2 . excretion of ampicillin i n t o t h e u r i n e i s delayed, p r o l o n g e d a n d amount r e d u c e d . The c o m b i n a t i o n o f i n j e c t e d a m p i c i l l i n and o r a l probenecid h a s been used233,234,235. When a m p i c i l l i n i s g i v e n o r a l l y i n d o s e s of 250 mg, 500 mg, 750 mg, a n d 1000 mg p e a k serum c o n c e n t r a t i o n s a r e o b t a i n e d a t a b o u t 2 h o u r s a f t e r dosing236. S i g n i f i c a n t serum c o n c e n t r a t i o n s of a m p i c i l l i n a r e p r e s e n t a t s i x h o u r s of t h e d o s i n g . Doubling t h e dose d o u b l e s t h e p e a k serum c o n c e n t r a t i o n . About 30% o f a g i v e n d o s e i s e x c r e t e d i n t h e u r i n e 6-8 h o u r s . Ampicillin concentration i s n o t a f f e c t e d by food237 and a n t i b a c t e r i a l a c t i v i t y of s e r u m samples i s p r o p o r t i o n a l t o dosage. Bunn238 f o u n d t h a t 25% o f a n o r a l d o s e of a m p i c i l l i n i s e x c r e t e d i n t h e f i r s t 8 h o u r s , and t h a t t h e blood l e v e l s of a m p i c i l l i n a t 4 hours w e r e t h e same a f t e r doses of 2 5 0 , 5 0 0 , or 1000 mg. S t e w a r t e t a l . 2 3 9 showed t h a t i n h i b i t o r y l e v e l s of a m p i c i l l i n a r e a t t a i n e d a n d m a i n t a i n e d b e t w e e n o n e and one-half and s e v e n h o u r s a f t e r o r a l d o s e s With lower doses i n h i b i t o r y o f 100 mg/kg/day. c o n c e n t r a t i o n s a r e m a i n t a i n e d f o r o n e a n d onehalf t o f i v e hours, About 30-50% o f t h e t o t a l dose given over a p e r i o d of f i v e t o seven days Chromatographic b i o a s s a y s of t h e was e x c r e t e d . e x c r e t i o n product i n t h e u r i n e suggested t h a t it was l a r g e l y a m p i c i l l i n a n d n o t a m e t a b o l i t e .

46

AMPICILLIN

9.

1. 2. 3. 4. 5. 6. 7.

8.

9.

10.

11. 12. 13.

14. 15.

16. 17.

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39. 40. 41.

42. 43. 44.

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61.

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86. 87. 88.

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1 1 2 . Mestre, R . , S p a n . 3 5 5 , 3 6 1 , November 1 6 , 1 9 6 9 ; C . A . 72, 1 0 0 6 8 6 h ( 1 9 7 0 ) . 1 1 3 . Beecham G r o u p L t d . , Neth. A p p l . 6 , 6 0 3 , 0 4 4 , 6 6 , 28763m ( 1 9 6 7 ) . September 19, 1966; C.A. 114. L e p e t i t S.p.A., Belg. 622,901, J a n u a r y 15, 1963; C.A. 5 8 , 7534d ( 1 9 6 3 ) . 1 1 5 . Novo T e r a p e u t i s k L a b o r a t o r i u m A / s . , Brit. 9 8 5 , 6 5 5 , March 10, 1 9 6 5 ; C . A . 6 2 , 1 5 3 (1965). 116. Doyle, F . P . , F o s k e r , G . R . , N a y l e r , J . H . an d S m i t h H., J.Chem. S O C . 1 9 6 2 , 1 4 4 0 . 117. F o s k e r , G . R . , N a y l e r , J . H . C . an d W i l c o x , J . A . , B r i t . 9 9 1 , 5 8 6 , May 1 2 , 1 9 6 5 : C . A . 62 8368f ( 1 9 6 5 ) . 118. F o s k e r , G.R., N a y l e r , J . H . C . a n d W i l c o x , J . A . , U . S . 3,316,247, A p r i l 25, 1967. 119. Fosker, G . R . , Nayler, J . H . C . a n d Wilcox, J . A . , U.S. 3 , 3 2 5 , 4 7 9 , J u n e 1 3 , 1 9 7 6 . and Weichet, J . , Belg. 626,175, 1 2 0 . Novak, L . December 2 , 1 9 6 3 ; C . A . 61, 1 4 6 7 9 e ( 1 9 6 4 ) . 121. Chas. P f i z e r & C o . , I n c . , B r i t . 9 5 8 , 8 2 4 , May 2 7 , 1 9 6 4 ; C . A . 61, 4 3 6 1 g ( 1 9 6 4 ) . 1 2 2 . Doyle, F . P . , N a y l e r , J . H . C . and S m i t h , H . , B r i t . 8 7 3 , 0 4 9 , J u l y 1 9 , 1 9 6 1 ; C . A . 57, 7279b ( 1 9 6 2 ) . 1 2 3 . Dane, E . a n d D o c k n e r , J . , Angew. Chem. 76 ( 8 ) , 342 ( 1 9 6 4 ) . 1 2 4 . Dane, E. a n d D o c k n e r , T . , C h e m . B e r . 98 ( 3 ) , 789 ( 1 9 6 5 ) . 125. Fosher, G . R . and Nayler,J.H.c. , B r i t . 980,777, J a n u a r y 2 0 , 1 9 6 5 ; C . A . 63, 1 3 , 2 7 0 h ( 1 9 6 5 ) . 126. Smith, K l i n e and French L a b o r a t o r i e s , U.S. 3 , 1 1 7 , 1 1 9 , J a n u a r y 7 , 1 9 6 4 ; C . A . 61, 663h ( 1 9 6 4 ) . 127, U g l e s i c , A. and S e i w e r t h , R . , G e r . Offen. 1 , 9 4 0 , 5 7 1 , F e b r u a r y 1 9 , 1 9 7 0 : C . A . 72, 90451m ( 1 9 7 0 ) . s m i t h , H. a n d 1 2 8 . Doyle, F.P., N a y l e r , J . H . C . , S t o v e , E. R . , N a t u r e 191, 1 0 9 1 ( 1 9 6 1 ) . 54

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129. Luttinger, J . R . , Lein, J. and Gourevitch, A. Belg. 612,599, April 30, 1962: C.A. -958 0083f (1963). 130. Dursch, F., The Squibb Institute for Medical Research, Private Communication, September 1 1967. 131. Mestre, J.R., Span. 291,104, September 10, 1963; C.A. 61, 1869g (1964). 132. Ekstrom, B., Gromez-Revilla, A,, Mollberg,R. Thelin, H. and Sjoberg, B., Acta Chem. Scand. 19 281 (1965). 133. Sjoberg7B.O.H. and Ekstrom, B.A., Brit. 940,489,October 30, 1963: C.A. 61, 2941111 (1964). 134. Sjoberg, B.O.H. and Ekstrom, B.A., Belg. 620,519, November 14, 1962: C.A.58, 11501h (1963). 135. Johnson, D.A. and Wolfe, S., U.S.3,140,282, July 7 , 1964: C.A. 61, 8315c (1964). 136. Robinson, Ch. A. Ger. Offen. 1,960,748, July 2, 1960: C.A. 73, 45504r (1970). 137. Soulal, M.J., Brit. 975,379, November 18, 1964: C.A. 6 2 , 3890f (1965). 138. Beecham, Group Ltd. , Fr. 1,365,220, June 1964: C.A. 61, 13316f (1964). 139. Bohme, E. and Dolfini,, J.E.,The Squibb Institute for Medical Research, Privatc C3mmunication. February 17, 1969. 140. Anon. Ind. - Cher. 39 (lo), 513 (1963). 141. Farbenfab: Bayer, Brit. 986,904,March 24, 1965. 142. Farbenfab: Bayer, Ger. Offen. 1143516, August 22, 1963. 143. Koenig, H.B. and Risse, K.H., Belq. 636,975, March, 1964: C.A. 62, 8947c (1965). 144. Koenig, H.B., Ger. 1,178,434, September 1964: C.A. 61, 14476g (1964). 145. Feinberg, J.G. and Weston, R.D., Brit. 1,131,741, October 1968: C.A. 70, 14410r ~

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90, 89 (1965). 155. Thomas, A . H . a n d B r o a d b r i d g e , R . A . , A n a l y s t 95, 459 (1970). 156. Zuidweg, M . H . J . , o o s t e n d o r p , J . G . a n d BOS, C.J.K., J. C h r o m a t o g r . 42,552 (1969). 157. Wayland, L . G . a n d W e i s s , P . J . , J . P h a r m . S c i . 57, 806 (1968). 158. M c G i l v e r a y , I . J . and S t r i c k l a n d , R . D . , J . P h a r m . S c i . , 56, 77 (1967). 159. H e l l b e r g , H., J. A s s o c . of A n a l . Chem., 51, 552 (1968). 160. Weiss, P . J . , T a l i a f e r r o , B . , H u c k i n s , R. and C h a s t o n a y , R . , I b i d . 50, 1294 (1967). 161. B i a g i , G . L . , B a r b o r o , A . M . , Gamba, M.F. a n d G u e r r a , M. C . ,

J. C h r o m a t o g r . 4 1 ,

371 (1969). 162. S a c c a n i , F . ,

Boll. Chim. Farm. 56

106, 625

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(1967). 163. Wanq J e n T s e , Chou Hsun, H s i u L i n . Chun H u i a n d Chen KO S h e n q , Tai-Wan K ' o H s u e h 2 4 , 19 (1970); C . A . 74, 115942h (1971). 164. G o o d a l l , R . R . a n d L e v i , A . A . , A n a l y s t -772 277 (1947). 165. G l i s t e r , G . A . a n d G r a i n e r , A . , I b i d . 75, 310 (1950). 166. B a k e r , P . B . , D o b s o n , F. a n d M a r t i n , A . J . P . , I b i d . 75, 651 (1950). 167. S t e p h e n s , I . a n d G r a i n e r , A . , J . Pharm. Pharmacol.. 1,702 (1955). 168. Thomas, R., N a t u r e 191, 1161 (1961). 169. T u t t , D . E . a n d S c h w a r t z , M . A . , A n a l . Chem. 4 3 . 338 (1971). 170. S m i t h , J . W . G . , d e G r e y , G . E . a n d P a t e l , U . J A n a l y s t 92, 247 (1967). 171, A n g e l u c c i , L. a n d B a l d i e r i , M . , J , Pharm. P h a r m a c o l . 23, 471 (1971). 172. B u r y a k , V . P . a n d K u r i u n a y a , N . V . , F a r m . Z h , 25, 42 (1970); C . A . 74 6412h (1971). 173. D o a d r i o , A . a n d G a r c i a - M i r a s i e r r a Gomez,M., A n , R. Acad. Farm. 35, 115 (1969); C . A . -971 42363s (1969). 174. R a s m u s s e n , C . E . a n d H i g u c h i , T . , J . P h a r m . S c i . 60 1608 (1971). 175. L e p i d i , A . A . a n d N u t i , M . P . , M y c o p a t h o l . Mycol. Appl. 43, 11 (1971); C . A . 74, 146307e (1971). 176. J u s c o , W . J . , J . Pharm. S c i . , 60, 728 (1971). 177. S i n s h e i m e r , J . E . , Hong, D . D . a n d 58, 104 Burckhalter, J . H . , J.Pharm.Sci., (1969). 178. P r i n c e t o n A p p l i e d R e s e a r c h , A p p l i c a t i o n -7

-9

~

Note, AN-111.

179. T a k e o Murakawa, Y o s h i m i , W a k a i , M i n o r u N i s h i d a , R y o c h i F u j i i , M a s a t o s h i Konno, Kazuko O k a d a , S a s h i k o G o t o a n d S h o q o K u w a h a r a , J . A n t i b i o t . 2 3 , 250 (1970).

57

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180. Nishida Minoru, Murakawa Takeo, Goto Sashiko, Fujii Ryochi, Konno Masatoshi and Okada Kazuko, Nippon Kaqaku Ryooqakuai Zasshi, l7, 1973 (1969); C.A. 73, 12731j (1970). 181. Fujii Ryochi, Kondo Masatoshi, Okada Kazuko, Kumagai Michikiko, Yoshida Akio and Matsuzaki Meiki; Nippon Koqoku Ryoogakuai Zasshi, l.7, 1920 (1969); C.A. 72, 77097f (1970). 182. Sacconi, F., Boll. Chim. Farm., 106,625 (1967): C.A. 6 8 , 24523c (1968). 183. Biagi, G.L., Barbaro, A.M., Gamba M.F. and Guerra, M C., J. Chromatogr. 41, 371 (1969). 184. Biagi, G.L., Barbaro, A.M. and Guerra, M.C., J. Chromatogr., 51, 548 (1970). 185. Roberts, H., The Squibb Institute for Medical Research, Private Communication, 1972. 186. Pan, S.C., The Squibb Institute €or Medical Research, Private Communication, 1969. 187. Roberts, H.R. and Vahidi, A., The Squibb Institute for Medical Research, Private Communication, 1971. 188. Smith, J.T. and Hamilton-Miller, J.M.T., Chemotherapy l5, 368 (1970). 189. Hill, A. and Zaleski, W.A., N.Eng. J. Med., 283, 490 (1970). 190. Uri, J., Naturwissenschaften 52, 354 (1965). 191. Alicino, J., Ana1.Chem. 18,619 (1946). 192. Russo-Alesi, F.M., The Squibb Institute for Medic a 1 Research , Private Commun ica tion, 1970. 193. Bomstein, J., Evans, W.G. and Shepp, J.M., Ann. N.Y. Acad. Sci., 130, 589 (1965) 194. Boxer, G. and Everett, P., Ana1.Chem. -921 670 (1949). 195. Henstock, H., Nature, 164, 139 (1949). 196. Fed. Register 33, 4099, March 1968. 197. Niedermayer, A.O., Russo-Alesi, F.M. and 58

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201.

202. 203. 204. 205.

Shaw, W . H . C .

a n d Duncombe, R . E . ,

Analyst,

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206. O u i n n , E . L . ,

Colville, J.M., B a l l a r d , L., a n d Debnam, F . , A n t i m i c r o b . A g e n t s

J o n e s , D. C h e m o t h e r . 1962, 339 (1963). 207. R o b i n s o n , G . N . a n d S t e v e n s , S . ,

11

Br1t.Med.J.

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z,

x.

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235. M u n n i c h , D., U r i , J . a n d V a l u , G . , C h e m o t h e r a p i a , g , 226 (1964). 236. K n u d s e n , E.T., R o l i n s o n , G . N . a n d S t e v e n s , S . , B r i t . Med. J., 11, 198 (1961). 237. K l e i n , J . O . , F i n l a n d , M. a n d Wilcox, C . , Am. J . Med. S c i . 245, 5 4 4 (1963). 238. Bunn, P . A . , A n t i m i c r o b . A g e n t s C h e m o t h e r . 1962 323 (1963). 239. S t e w a r t , G . T . , C o l e s , H . M . T . , N i x o n , H . H . a n d H o l t , R . J . , B r i t . Med. J. 11, 200 (1961)

61

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CHLORPROTHIXENE

Bruce C. Rudy and Bernard Z. Senkowski

63

6. C. RUDY AND

B. Z . SENKOWSKI

INDEX Analytical P r o f i l e - Chlorprothixene

1.

Description 1.1 Name , Formula, Molecular Weight 1.2 Appearance, Color, Odor 1.3 Isomeric Forms Phys i c a l P r o p e r t i e s 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 Thermogravimetric Analysis 2.10 S o l u b i l i t y 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2 . 1 2 D i s s o c i a t i o n Constant

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug Metabolic Products

6.

Methods o f Analysis 6.1 Elemental A n a l y s i s 6.2 Phase S o l u b i l i t y Analysis 6.3 Thin Layer Chromatographic Analysis 6.4 D i r e c t S p e c t r o p h o t o m e t r i c Analysis 6.5 Colorimetric Analysis 6.6 Fluorescence Analysis 6.7 Non-Aqueous T i t r a t i o n

7.

Acknowledgements

8.

References

64

CHLORPROTHIXENE

1.

Description

Name, Formula, Mol ecul ar Weight C h l o r p r o t h i x e n e i s 2-chloro-N,N-dirnethylt h io x a n th e n e - A9 , Y - p r o p y l a m i n e . 1.1

CHCH, CH2N(CH&

&fC' C18H18ClNS

M o l e c u l a r Weight:

315.87

Appearance, C o l o r , Odor C h l o r p r o t h i x e n e i s a l i g h t yel l ow t o yel l ow c r y s t a l l i n e powder w i t h a l i g h t a m i n e - l i k e o d o r . 1.2

I s o m e r i c Forms Chlorprothixene e x i s t s as a cis- o r a t r a n s i s o m e r . The c i s - i s o m e r h a s a l s o been r e f e r r e d t o as t h e a - i s o m e r o r t F h i g h m e l t i n g i s o m e r ( 1 , Z ) . The t r a n s - i s o m e r l i k e w i s e has been r e f e r r e d t o a s t h e B-isomer o r low m e l t i n g i s o m e r . T h i s work w i l l d e a l p r i m a r i l y w i t h t h e a - i s o m e r . 1.3

3 -.

Phys i c a l P r o p e r t i e s

I n f r a r e d Spectrum The i n f r a r e d s pect r um o f bul k r e f e r e n c e s t a n d a r d c h l o r p r o t l i ~ x e n ei s shown i n F i g u r e 1 ( 3 ) . The spect rum o f a 10'" car b o n d i s u l f i d e s o l u t i o n o f c l i l o r p r o t h i x e n e ( w / v ) was measured v e r s u s car bon d i s u l f i d e i n 0 . 1 mm NaCl l i q u i d c e l l s on a P e r k i n Elmer 621 S p e c t r o p h o t o m e t e r . 2.1

The f o l l o w i n g a s s i g n m e n t s have been g i v e n t o t h e bands i n F i g u r e 1 ( 3 ) : a . C h a r a c t e r i s t i c f o r a r o m a t i c CH: 3063 cm-l b . C h a r a c t e r i s t i c f o r CH2 s t r e t c h i n g : 2939 and 2854

cmc.

'

C h a r a c t e r i s t i c f o r CHI, s t r e t c h i n g : -7816 and 2767 cm65

B. C. RUDY

i

-i

A N D B. 2. SENKOWSKI

33NVUIHSNWl K

66

0

8 0

0 0 0

9 0

e N

8 0

8

N

8 8 8 Y)

*)

*

CH LORPROTH I XE NE

d.

e.

C h a r a c t e r i s t i c f o r 4 f r e e H on benzene r i n g : 757 t o 739 cm-l C h a r a c t e r i s t i c f o r 2 f r e e H on benzene r i n g : 809 cm-l

2.2

N u c l e a r Magnetic Resonance Spectrum (NMR) The NMR s p e c t r u m shown i n F i g u r e 2 was o b t a i n e d by d i s s o l v i n g 5 2 . 2 mg o f r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e i n 0 . 5 m l o f C D C 1 3 c o n t a i n i n g t e t r a m e t h y l s i l a n e as t h e i n t e r n a l reference. The s p e c t r a l a s s i g n m e n t s are shown i n Table I ( 4 ) .

TABLE I NMR S p e c t r a l Assignments f o r C h l o r p r o t h i x e n e

Type P r o t o n s

No. o f Each Proton

Chemical S h i f t (ppm)

methyl methy 1e n e vinyl aromatic

6 4 1 7

2. 24 2.35-2.80 5.97 7.10-7.60

-

Multiplicity S

m (u) t (u) m (u)

s = s i n g l e t ; m(u) uns ym m et r i cal m u l t i p l e t ; t ( u ) = uns y mme tr ical t r i p l e t 2.3

U l t r a v i o l e t Spectrum The u l t r a v i o l e t s p e c t r u m o f c h l o r p r o t h i x e n e i n 0.1N HC1 i n t h e r e g i o n 400 t o 210 nm e x h i b i t s t h r e e maxima and t h r e e minima. The maxima a r e l o c a t e d a t 229 - 230 nm ( E = 3 . 4 x l o 4 ) , 267 - 268 nm ( E = 1 . 3 x l o 4 ) , and 323 - 324 nm (E = 2 . 8 x l o 3 ) . The minima were o b s e r v e d a t 217 nm, 254 nm, and 307 - 308 nm. The spect rum p r e s e n t e d i n F i g u r e 3 was o b t a i n e d from a r e f e r e n c e s t a n d a r d s o l u t i o n o f c h l o r p r o t h i x e n e a t a c o n c e n t r a t i o n o f 0. 602 mg p e r 100 m l o f 0.1N H C 1 ( 3 ) . 2.4

F l u o r e s c e n c e Spectrum F i g u r e 4 shows t h e e x c i t a t i o n and e m i s s i o n s p e c t r a €or r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e from 260 t o 540 nm ( 5 ) . The s p e c t r a were measured on a 0.1N HC1 s o l u t i o n o f c h l o r p r o t h i x e n e ( 0 . 5 mg/ml) u s i n g a Farand M K - 1 s p e c t r o f l u o r o m e t e r . E x c i t a t i o n a t e i t h e r 310 nm o r 352 nm produced 67

6.C. R U D Y A N D 6. Z . SENKOWSKI

68

CHLORPROTHIXENE

Figure 3 Ultraviolet Spectrum of Chlorprothixene

1.0

.8

.6

NANOMETERS

B. C. RUDY AND B. Z . SENKOWSKI

AlISN31NI

CHLORPROTHIXENE

i d e n t c a l emission s p e c t r a with a max m u m a t 401 nm. 2 5

Mass Spectrum The mass spectrum o f c h l o r p r o t h i x e n e r e f e r e n c e s t a n d a r d was o b t a i n e d u s i n g a CEC 21-110 s p e c t r o m e t e r w i t h an i o n i z i n g energy o f 70 eV (Figure 5) ( 6 ) . The low r e s o l u t i o n spectrum showed t h e f o l l o w i n g d i a g n o s t i c p e a k s : Intensity

mass (m/e) 315 313 271 257 255 2 34 221 189 58

v e r y weak v e r y weak v e r y weak weak medium - we ak weak strong medium-weak very strong

The m o l e c u l a r i o n a t m/e 315 and t h e fragment i o n s a t m/e 2 7 1 , 257, and 255 d i s p l a y t h e expected c h l o r i n e i s o t o p e peak 2 mass u n i t s h i g h e r . The b a s e peak a t m/e 58 and t h e i o n a t m/e 257 a r e both due t o c l e a v a g e b e t a t o t h e amine f u n c t i o n . The peak a t m/e 255 could be e x p l a i n e d as l o s s o f two hydrogens from m/e 257 p o s s i b l y l e a d i n g t o a r i n g - c l o s e d s t r u c t u r e . The peak a t m/e 2 2 1 i s due t o t h e l o s s o f H C 1 from m/e 257. S i n c e t h e s p e c t r a d i d n o t show any change w i t h time i t i s l i k e l y t h a t m/e 313 i s a fragment i o n d e r i v e d from m/e 315. Again, a r i n g c l o s u r e type of s t r u c t u r e i s a p o s s i b i l t y (6). 2.6

Optical Rotation C h l o r p r o t h i x e n e e x h i b i t s no o p t i c a l a c t i v i t y .

Melting Range A s h a r p m e l t i n g p o i n t i s n o t observed w i t h c h l o r p r o t h i x e n e . ' The m e l t i n g depends on t h e r a t e o f h e a t i n g . The m e l t i n g range r e p o r t e d i n NF XI11 i s 96.5' 101.5°c. 2.7

71

to

6.

C. RUDY AND 6.2. SENKOWSKI

72

CH LORPROTH I XENE

2.8

D i f f e r e n t i a l Scanni ng C a l o r i m e t r y (DSC) The DSC c u r v e €or r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e was o b t a i n e d u s i n g a P e r k i n Elmer DSC - IB C a l o r i m e t e r . With a t e m p e r a t u r e program o f 10°C/min., a m e l t i n g endotherm was o b s e r v e d s t a r t i n g a t 92.8OC (shown i n F i g u r e 6 ) and a n o t h e r endotherm s t a r t i n g a t 246OC which c o r r e s p o n d s t o t h e decom pos i t i on o f t h e c h l o r p r o t h i x e n e . The Aldf was found t o be 7 . 4 kcal / m ol e f o r t h e m e l t i n g endot h er m ( 7 ) . 2.9

Th e r m ogr avi m et r i c A n a l y s i s (TGA) The TGA performed on r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e e x h i b i t e d n o l o s s o f w ei ght when h e a t e d t o 1 0 S O c a t a h e a t i n g r a t e o f 10°C/min ( 7 ) . Solubility The s o l u b i l i t y d a t a o b t a i n e d a t 2 5 O C f o r reference standard chlorprothixene is l i s t e d i n Table I1 (8). 2.10

TABLE I 1

Ch 1o r p r o t h i x e n e

- So l u b i 1i t y

Solvent 3A a l c o h o l benzene ch 1o r 0 f o rm 95% e t h a n o l ethyl ether i sop ropano 1 me t h a n o 1 petroleum e t h e r (3Oo-6O0) water 2.11

So 1u b i 1i t y (mg/ml )

36.7 30.5 >SO0 28.2 77.6 22.0 36.5 16.9 <0.1

Crystal Properties The x - r a y powder d i f f r a c t i o n p a t t e r n o f r e f e r e n c e s t a n d a r d c h l o r p r o t h i x e n e i s p r e s e n t e d i n T a b l e I11 ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s a r e g i v e n below:

Y

u

-13a Li u)

d 2

-w

g

W xI -6

I

4 --u

-

B. C. RUDY AND B. 2.SENKOWSKI

74

CHLORPROTHIXENE

Instrumental Conditions: General E l e c t r i c Model XRD-6 Spectrogoniometer Generator: 50 KV, 12-1/2 MA Tube T a r g e t : Copper Cu KCY = 1.542 1 Radiation: 0.1' D e t e c t o r s l i t Optics: M.R. Soller s l i t 3' Beam s l i t 0.0007" N i f i l t e r 4' t a k e o f f a n g l e Scan a t 0 . 2 ' 2 0 p e r minute Goniometer: A m p l i f i e r g a i n - 16 c o a r s e , Detector : 8.7 fine Sealed proportional counter tube and DC v o l t a g e a t p l a t e a u Pulse height s e l e c t i o n EL- 5 v o l t s Eu-out Rate meter T . C . 4 2000 C/S f u l l s c a l e Recorder: Chart Speed 1 i n c h p e r 5 minutes Samples : Prepared by g r i n d i n g a t room t e m p e r a t u r e D i s s o c i a t i o n Constant The a p p a r e n t PKa f o r c h l o r p r o t h i x e n e has been determined s p e c t r o p h o t o m e t r i c a l l y t o - be 8 . 4 (10). The a p p a r e n t pKa has a l s o been determined from t h e t i t r a t i o n curve i n an i s c p r o p a n o 1 : w a t e r ( 1 : l ) mixture and found t o be 7.5 ( 1 1 ) . I n w a t e r , t h e t r i a l k y l a m i n o t y p e compounds a r e s t r o n g e r b a s e s , on t h e a v e r a g e , by 0 . 9 pK u n i t s ( 1 1 , 1 2 ) . T h e r e f o r e , t h e e s t i m a t e d PKa i n w a t e r i s 8 . 4 which i s i n good agreement w i t h t h a t found s p e c t r o p h o t o m e t r i c a l l y . 2.12

75

B. C . RUDY AND B. 2. SENKOWSKI

TABLE I11 X-ray D i f f r a c t i o n Pat t e r n o f Ch l o r p r o t h i x e n e

I/** 20 8.18 14.49 16.39 16.67 19.55 21.43 22.71 23.00 24.24 24.55 25.86 26.33 26.82 27.53 28.69 29.69 30.47 31.37 33.12 33.48 33.79 34.40 35.03 35.92 37.25 37.81 38.18 39.00 39.65 40.80 41.80 42.62 43.23 44.36 45.06 45.70 49.55 50.68

d* 10.81 6.11 5.41 5.32 4.54 4.15 3.92 3.87 3.67 3.63 3.45 3.38 3.32 3.24 3.11 3.01 2.93 2.85 2.70 2.68 2.65 2.61 2.56 2.50 2.41 2.38 2.36 2.31 2.27 2.21 2.16 2.12 2.09 2.04 2.01 1.99 1.84 1.80

76

i0

100 21 62 45 75 24 8 10 31 29 15 3 28 6 10 3 15

1 8 1 2 3 3 4 2 3

1 1 1 3 17 2 3 3 4 2 2 5

CHLORPROTHIXENE

TABLE I11 (cont.) X-ray Diffraction Pattern of Chlorprothixene 0

20

52.30 54.10

d* A 1.75 1.70

*d - (interplanar distance)

**I/

= I0

I/** I0

2 2

nX 2 Sin 0

relative intensity (based on highest intensity of 1.00)

3. Synthesis Chlorprothixene may be prepared by the reaction scheme shown in Figure 7 . 2-Chlorothioxanthone is reacted with dimethylaminopropyl chloride in the presence of magnesium to form 2-chlorothioxanthenol which is then dehydrated to give chlorprothixene (13). Stability Degradation Chlorprothixene has been shown to be stable when heated for one hour at 100°C in 0.1N HC1, 0.1N NaOH, and water. (14) When it is subjected to ultraviolet radiation or strongly basic conditions, 2 chlorothioxanthene and 2-chlorothioxanthone are formed which can be detected by TLC or paper chromatography. Also the odor of volatile aniines is apparent (15). 3.

Drug Metabolic Products Chlorprothixene is metabolized to the sulfoxide which can be either N-demethylated or further oxidized to the N oxide, Figure 8 ( 2 , l S ) . The analytical procedures for the metabolites have been described by de Silva (17). 5.

6.

Methods of Analvsis

Elemental Analysis The results from an elemental analysis of reference standard chlorprothixene is presented in Table IV (18). 6.1

77

Figure 7 Synthesis of Chlorprothixene

CH LORPROTH I XENE

Figure 8

SULFOXIDE

DESMETHYL CHLORPROTHIXENE

-

CHLORPROTHIXENE SULFOXIDE

Metabolic Products of Chlorprothixene (2)

CHLORPROTHIXENE

CHI I

CHi N

0

c ~ f b‘cH, CHLORPROTHIXENESULFOXIDE - N - O X I D E

79

-

-

B. C , RUDY AND B. Z . S E N K O W S K I

TABLE IV Elemental Analysis of Chlorprothixene E 1ement

% Theory

% Found

C H N S c1

68.45 5.74 4.43

68.36 5.72 4.45 10.26 11.36

10.15

11.22

6.2

Phase Solubility Analysis Phase solubility analysis has been carried out for chlorprothixene using either isopropanol or acetonitrile as the solvent. An example is presented in Figure 9 for the reference standard chlorprothixene along with the conditions under which the analysis was carried out (8). 6.3

Thin Layer Chromatographic Analysis (TLC) The following TLC procedure is useful for separating chlorothioxanthone and chlorothioxanthene from a-chlorprothixene (19). Using silica gel G plates and benzene:heptane (1:l) a s the solvent system, 0.1 mg of the sample in anhydrous 3A alcohol is spotted on the plate and subjected to ascending chromatography. After the solvent front has ascended 10 to 15 cm, the plate is a i r dried and sprayed with concentrated sulfuric acid. The plate is then viewed under shortwave ultraviolet radiation. The approximate Rf values are as follows: 0.0 a-chlorprothixene chlorothioxanthone 0.35 chlorothioxanthene 0.85

(orange fluorescence) (greenish fluorescence) (orange fluorescence)

The TLC procedure found useful for the separation of B-chlorprothixene and 2-chlorothioxanthenol is described below (19). A silica gel C; plate is spotted with 0.1 mg of sample and developed 10 to 15 cm using absolute ethanol: benzene:water ( ? 0 : 2 0 : 1 ) as the solvent system. Thc plate is air dried, sprayed with concentrated sulfuric acid, and viewed under shortwave ultraviolet radiation. The approximate Rf values are as follows:

Figure 9

I PHASE SOLUBILITY ANALYSIS

SAMPLE 8 CHLORPROTHIXENE SOLVENT: ISOPROBINOL SLOPE: 0 0 % EQUILIBRATION 2 0 HRS 01 25' C EXTRAPOLATED SOLUBILITY. 28.10 mg/g of SOLVENT

n

I

X

rn z rn

0 SYSTEM

25 50 75 100 COMPOSITION: mg of SAMPLE per g SOLVENT

8.

C. RUDY AND 6.2. SENKOWSKI

a-chlorprothixene 0.65 (orange fluorescence) B-chlorprothixene 0 . 5 0 (orange fluorescence) 2-chlorothioxanthenol 0 . 3 5 (reddish fluorescence) Direct Spectrophotometric Analysis The chlorprochixene content in tablets may be determined spectrophotometrically by using the following procedure. The tablets are finely ground, a portion of the powder is weighed, and then dispersed by shaking in a 1% ammonium hydroxide solution. The chlorprothixene is extraced with ethyl ether. The chlorprothixene is extracted from the ethereal solution with 0.5N HC1 and diluted to volume with 0.5N HC1. Suitable aliquots are further diluted to give a solution containing about 50 ug/ml and the absorbance measured at 324 nm. The concentration of chlorprothixene is calculated using the absorbance obtained from a solution of chlorprothixene reference standard similarly prepared and measured (5,20). 6.4

The system used with the Technicon Autoanalyzer for measuring the content uniformity of individual chlorprothixene tablets is as follows. Each tablet is crushed and allowed to stand in 5 ml of dimethylformamide for about 20 minutes. This solution is diluted with 0.1N H~SOI,until the concentration is about 50 pg/ml. The absorbance is measured at 324 nm and the amount of chlorprothixene calculated by comparison to a solution of reference standard chlorprothixene similarly prepared and measured. Colorimetric Analysis Colorimetric analysis may be accomplished by dissolving the chlorprothixene in concentrated sulfuric acid and diluting an aliquot with 50% sulfuric acid to a final concentration of approximately 0.04 mg/ml. The orange color is measured at 490 nm and the concentration obtained by means of a calibration curve (15). 6.5

Fluorescence Analysis Fluorescence analysis has proven to be a valuable method for the analysis of chlorprothixene in biological samples, A sensitivity limit of 0.010 micrograms/ml of blood has been reported when the blood is extracted and the extract dissolved in cold concentrated sulfuric acid (17). 6.6

82

CHLORPROTHIXENE

Non-Aqueous Titration The non-aqueous titration as described in the NF XI11 is the method of choice for the analysis o f b u l k chlorprothixene ( 2 0 ) . The sample is dissolved in chloroform, ethanolic methyl red indicator added, and the titration carried out with HC10, in dioxane. Each ml of 0.1N HClOk is equivalent to 31.59 mg of chlorprothixene. 6.7

7. Acknowledgements The authors wish to acknowledge the Scientific Literature Department of Hoffmann-La Roche Inc. and Dr. A. MacDonald for their assistance in the preparation of this analytical profile.

83

6. C. R U D Y A N D 6. 2. SENKOWSKI

8. References 1.

2. 3. 4. 5. 6.

7. 8. 9.

10.

11. 12. 13. 14. 15.

16.

J. D. Dunitz, H. Eser, and P. Strickler, HeZv. Chim. Acta, 4 7 , 1897 (1964) J. Raaflaub,ArzneimitteZ-Forshung, 1 7 , 1393 (1967). M. Hawrylyshyn, Hoffmann-La Roche Inc., Personal Communication. J. H. Johnson, Hoffmann-La Roche Inc., Personal Communication . J. Boatman, Hoffmann-La Roche Inc., Personal Communication. W. Benz, Hoffmann-La Roche Inc., Personal Communication. J. Donahue, Hoffmann-La Roche Inc., Personal Communication. E. MacMullan, Hoffmann-La Roche Inc., Personal Communication. R. Hagel, Hoffmann-La Roche Inc., Personal Communication. E. Lau, Hoffmann-La Roche Inc., Unpublished Data. V. Toome and G. Raymond, Hoffmann-La Roche Inc., Unpublished Data, B. Gutbezahl and E. Grunwald, J. Amer, Chem. S o c . , 75, 559 ( 1 9 5 3 ) . -

W . Kimel, Hoffmann-La Roche Inc., Unpublished Data.

I. Fischer, Hoffmann-La Roche, Unpublished Data. B. Schmidli, Hoffmann-La Roche Inc., Unpublished Data. C. L. Scheckel, Mod. Probi!. Pharmacopsychiatry, 2 , 1 (1969).

17. 18. 19.

20.

1.A.F. deSilva and L. D. D'Arconte, J. Forensic Sci., 1 4 , 184 ( 1 9 6 9 ) . F . Schzdl, Hoffmann-La Roche Inc., Personal Communication. R. Colarusso, Hoffmann-La Roche Inc., Unpublished Data. National Formulary XIII, Submitted August 25, 1 9 7 1 .

84

CHLORAL HYDRATE

J o h E. ~ Fairhrotlier

85

JOHN E . FAIRBROTHER

CONTENTS 1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2.1 Spectra 2.11 Infrared 2.12 Raman 2.13 Ultraviolet 2.14 Nuclear Magnetic Resonance 2.15 Nuclear Quadrupole Resonance 2.16 Mass Spectrum 2.17 Electron Spin Resonance 2.2 Physical Properties of the Solid 2.21 Basic Thermal Constants 2.22 Differential Thermal Analysis and Differential Scanning Calorimetry 2.23 Loss on Drying and Thermogravimetric Analysis 2.24 Optical Characteristics 2.25 X-ray Diffraction 2.26 Neutron Diffraction 2.27 Polymorphism 2.3 Solubility 2.31 Water 2.32 Water-Miscible Solvents 2 - 3 3 Water-Immiscible Solvents 2 - 3 4 Oils of Pharmaceutical Interest 2.4 Physical Properties of Solutions and Melts 2.41 Heats of Solution and Dilution 2.42 Dissociation and pH 2.43 Surface Tension 2.44 Ultrasonic Absorption 2.45 Viscosity 2.46 Cryoscopy 2.47 Refractive Index 2.48 Radiolysis Molecular Complexes 3.1 Types of Known Complexes 3.2 Structure of Complexes Synthesis and Purification ~

2.

3.

4.

86

CHLORAL HYDRATE

5.

7.

Degradation 5.1 Degradation of Solid 5.11 Volatilization 5.12 Chemical Degradation 5.2 Degradation of Solutions 5.21 Action of Light 5.22 Action of Heat 5 . 2 3 Ultrasound 5.24 Autoxidation and Polymerization 5.25 Action of Bases 5.26 Radiolysis 5.27 Microorganisms 5.28 Incompatibilities (solids and solutions ) 5.29 Interaction with Starch 5.3 Stabilization Chemical Properties 6.1 Identity Tests 6.2 Methods of Analysis 6.21 Volumetric/Titrimetric Procedures 6.22 Spectrophotometry 6.23 Ion-Exchange and Column Chromatography 6.24 Paper and Thin-Layer Chromat ography 6.25 Vapor-Phase Chromatography 6.26 Refractometry and other physical parameters 6.27 Polarography 6.28 Determination of Water 6.3 Radiolabelled Chloral Hydrate Metabolism

8.

References

6.

87

JOHN E. FAIRBROTHER

1.

Description 1.1

Name, F o r m u l a , M o l e c u l a r Weight

C h l o r a l Hydrate ( C h l o r a l i s Hydras; C h l o r a t u m H y d r a t u m ) a l s o known a s 2 , 2 , 2 - T r i c h l o r o e t h a n e - l , l - d i o l a n d t r i c h l o r o a c e t a l d e h y d e monohydrate

.

OH

c1

I

I

- c -

c1

CH

-

OH

I

c1 C2 H j C 1

1.2

3

0

Mol.Wt.

2

165.40

A p p e a r a n c e , Color, O d o r , T a s t e

Colorless c r y s t a l s with a pungent, but n o t a c r i d , o d o r , Has a p u n g e n t a n d r a t h e r b i t t e r taste. V o l a t i l i z e s s l o w l y on e x p o s u r e t o a i r . 2.

Physical Properties 2.1

Soectra 2.11

I n f r a r e d Spectrum

An u n d e r s t a n d i n g o f t h e i n f r a r e d s p e c t r u m of c h l o r a l h y d r a t e s h o u l d b e a p p r o a c h e d f r o m a s t u d y of t h e spectrum o f c h l o r a l , which h a s b e e n d e s c r i b e d i n some d e t a i l i n t h e l i t e r a t u r e 1 ~ 2 , j . C h l o r a l h y d r a t e h a s b e e n shown t o h a v e a g e m - d i o l s t r u c t u r e b y Raman s p e c t r a ? a n d by N . M . R . 5 C o m p a r i s o n of t h e i n f r a r e d ( m u l l ) s p e c t r a of t h e compound a n d of i t s d e u t e r a t e d a n a l o g 1 7 1 i n d i c a t e ; t h a t t h e t w o OH g r o u p s a r e n o t e u a l . This i s c o n f i r m e d b y s o l u t i o n s p e c t r a 1 7 , q 8 , 2 3 , 1 6 8 ~ 1 7 1a n d Ogawa23 s u g g e s t s t h a t c h l o r a l h y d r a t e e x i s t s i n s o l u t i o n i n a n e q u i l i b r i u m s t a t e , w h i c h may b e r e p r e s e n t e d as f o l l o w s : -

88

CHLORAL HYDRATE

kI

H 0

OH

\ I /C I

J1

/I\ c1 c1 c1

H

\ Yo n

L

e/\

I

H /O\

H

c1 c1 c1

H

I

ci

ci

ci

Near i n f r a r e d s t u d i e s 6 on a s o l u t i o n o f c h l o r a l h y d r a t e show t h e e x i s t e n c e o f a l a b i l e e q u i l i b r i u m between t h e gem-diol and a dimolecular 1:l c o m p l e x o f t h e a l d e h y d e a n d w a t e r . I t i s thought t h a t t h e gem-diol s t r u c t u r e r e s u l t s from t h e e l e c t r o n i c effect of t h e halogens t r a n s m i t t e d t h r o u g h t h e m o l e c u l e , r a t h e r t h a n from i n t r a molecular hydrogen bonding between t h e halogens and t h e h y d r o x y l h y d r o g e n s . The n e a r - i n f r a r e d spectrum of c h l o r a l hydrate i n chloro i c r i n (trichloronitromethane) has b e e n r e c o r d e d Infrared spectra recorded i n N u j o 1 7 , 1 7 1 a n d i n p o t a s s i u m b r o m i d e 8 , 9 , 1 0 show t h e a b s e n c e o f t h e c a r b o n y l band a t 1 7 6 5 c m - l , but s p e c t r a recorded i n s o l u t i o n (carbon tetrac h l o r i d e 7 ~ 1 0 , 1 1 , 1 6 9b e n z e n e 7 , a n d c h l o r o f o r m l 7 0 , ) show t h e f o r m a t i o n o f t h e c a r b o n y l b a n d , i n d i c a t i n g some d i s s o c i a t i o n . P i g u e t and J a c o t Guillarmod7 concluded t h a t , i n carbon t e t r a c h l o r ide solution, chloral hydrate dissociates i n t o c h l o r a l and w a t e r , e v e n t u a l l y f o r m i n g a d i s t i n c t s t a b l e hemihydrate. However, E k e b l a d l l f o u n d t h a t s o l u t i o n s c o n t a i n i n g up t o 0.l5g c h l o r a l h y d r a t e p e r 100 ml o f c a r b o n t e t r a c h l o r i d e c o u l d b e p r e pared without d i s s o c i a t i o n .

E.

89

JOHN E. FAIRBROTHER

I n t h e OH r e g i o n , d i s s o c i a t i o n c a u s e s a d e c r e a s e i n t h e OH peak e x t i n c t i o n s and t h e a p p e a r a n c e o f a new O H p e a k f r o m water a t 3720 cm-l. A s e x p e c t e d , t h e r e i s a s h i f t i n t h e OH s t r e t c h & $ frequency from t h e c r y s t a l l i n e s t a t e ) of c h l o r a l hydrate t o d i l u t e s o l u t i o n I n t h e l a t t e r c a s e , two OH p e a k s ( o f a p p r o x i m a t e l y s i m i l a r i n t e n s i t y ) a p p e a r a t 3580 a n d 3615 cmHowever, f r e s h l y p r e p a r e d s o l u t i o n s i n the concentration range t o 10-3u show no s i g n i f i c a n t v a r i a t i o n s i n t h e m o l a r e x t i n c t i o n s a t t h e t w o p e a k maxima a n d n o water or c h l o r a l peaks are observed. The i n f r a r e d s p e c t r a o f t h e S q u i b b House S t a n d a r d L o t 7 6 1 4 7 i n m i n e r o i l and i n K B r are p r e s e n t e d i n f i g u r e s 1 and 2

11?1R,5y.

.

$3 .

2.12

Raman S p e c t r u m

The Raman s p e c t r a o f b o t h c h l o r a l 3 ~ 1 3 , 1 2 221,222,223,224 and c h l o r a l h y d r a t e l 3 ~ ~ 7 ~ ~ 2'2:223,224 have been r e p o r t e d . Fonteyne223 f o u n d t h a t t h e i n c r e m e n t a l a d d i t i o n o f water t o c h l o r a l p r o d u c e d a new Raman s p e c t r u m , w h e r e a s t h e l i n e s due t o c h l o r a l d i s a p p e a r e d s l o w l y . T h e l i n e a t 1 7 6 0 cm-1, c o r r e s p o n d i n g t o t h e C F 0 displacement, i s not e n t i r e l y absent i n chloral hydrate, suggesting t h e e x i s t e n c e of an equilibrium. The most r e c e n t l y r e p o r t e d s p e c t r a a r e t h o s e of Matsushimal3. 2.13

U l t r a v i o l e t Spectrum

The U . V . s p e c t r u m o f c h l o r a l h y d r t e h a s b e e n r e c o r d e d i n a number o f s o l v e n t s . 1 ' , 2 2 5 . I n s o l u t i o n i n w a t e r or i n d i o x a n c o n t a i n i n g a few p e r c e n t o f w a t e r , t h e h y d r a t e i s s o s t a b l e t h a t no c a r b o n y l a b s o r p t i o n i s d e t e c t a b l e . However, i f t h e c r y s t a l l i n e hydrate is dissolved i n cyclohexane, it d i s s o c i a t e s s u f f i c i e n t l y t o g i v e a c o n s i d e r a b l e a b s o r p t i o n a t 290mu. 1 4 ( g i v i n g a n EO value of 3 8 . 3 ) .

90

CHLORAL HYDRATE

A spectrum of t h e U . V . absorption of a s o l u t i o n of c h l o r a l h y d r a t e i n 95% e t h a n o l (6.Og. i n 2 5 m l o f s o l u t i o n ) l 5 shows s t r o n g e n d a b s o r p t i o n a b o v e 240mu.

2.14

N u c l e a r Magneti-c R e s o n a n c e SDec t r um

T h e N.M.R. s p e c t r u m o b t a i n e d d e p e n d s on t h e p o s i t i o n of t h e c h l o r a l + . c h l o r a l hydrate

equilibrium. Chloral g i v e s a simple spectrum (CDCl3), t h e aldehyde p r o t o n a b s o r b i n g a t 6 9 . 1 . C h l o r a l h y d r a t e , however, c o n s i s t e n t l y g i v e s a p e a k a t 6 9 . 1 , p l u s o t h e r peaks whose p o s i t i o n s a r e more v a r i a b l e , for h y d r o x y l a b s o r p t i o n s a r e great18 i n f l u e n c e d b y temperature and c o n c e n t r a tion (see fig.3). A b r o a d - l i n e N.M.R. s t u d y 5 has b e e n u s e 3 t o d e f i n e the s t r u c t u r e o f c h l o r a l h y d r a t e a s a gem-diol, d i s t i n c t from t h e a l t e r n a t i v e s t r u c t u r e c o n t a i n i n g m o l e c u l a r w a t e r , a s i n gypsum. At 200Ky c h l o r a l h y d r a t e e x h i b i t s a s l i g h t t r i p l e t s t r u c t u r e due t o t h e three-spin CH(OH), group w h i c h is s u b s t a n t i a t e d b y t h e s e c o n d moment o f t h e h y d r o g e n r e s o n a n c e ( 8 . 9 + 0.5 g a u s s * ) . T h e m o l e c u l a r h y d r a t e s t r u c t u r e would g i v e a s e c o n d moment g r e a t e r t h a n 1 5 g a u s s 2 , w h e r e a s w i t h t h e g e m - d i o l s t r u c t u r e , a s e c o n d moment o f a b o u t 3 . 8 =-a s 2 would b e e x p e c t e d . The f i g u r e o b t a i n e d confirms t h e gem-diol s t r u c t u r e , b u t i t a l s o follows t h a t a a r a e nrolsortion of intermolecular b r o a d e n i n g m u s t be p r e s e n t , which c o u l d a r i s e i f t h e c r y s t a l involved e x t e n s i v e hydrogen bonding.

2.15

Nuclear Quadrupole Resonance Spectrum

The N . Q . R . s p e c t r a o f 35Cl i n c h l o r a l h y d r a t e and c h l o r a l d e u t e r a t e have been r e orded i n t h e t e m p e r a t u r e z j y g e 7’7 t o 323OK. 1 - 7 9 1 g ~ 1 9 9 2 0 ( S i m i l a r s p e c t r a of C1 h a v e a l s o b e e n r e c o r d e d 20). T h e f r e q u e n c i e s o f t h e q u a d r p e l i n e s o b t a i n e d by i n d e p e n d e n t w o r k e r s l r / , l Y y y ’ are i n

91

bI z

n

? n

v3

N

m

n

si

rn

n

Fig. 1.

I n f r a r e d spectrum o f c h l o r a l h y d r a t e , S q u i b b House S t a n d a r d ( M i n e r a l Oil M u l l ) .

CHLORAL HYDRATE

93

Fig. 2.

0

rn

I n f r a r e d spectrum of c h l o r a l hydrate, S q u i b b House S t a n d a r d ( P o t a s s i u m Bromide D i s c ) .

94 L

Fig.

3.

N.M.R. s p e c t r u m o f c h l o r a l h y d r a t e , Squibb House S t a n d a r d , in d e u t e r o a c e t o n i t r i l e .

CHLORAL HYDRATE

g o o d a g r e e m e n t , a n d show t h a t t w o OI? t h e l i n e s a r e s e p a r a t e d by o n l y a b o u t 80 K H z whereas t h e t h i r d i s a b o u t 1 . 2 5 MHz l o w e r . T h i s l a r g e r d i f f e r e n c e s u g g e s t s a d i f f e r e n t C - C 1 bonding f o r one of t h e C l a t o m s . O t h e r s t u d i e s 304 o n t h e t e m p e r a t u r e dependence o f t h e s p i n - l a t t i c e r e l a x a t i o n t i m e f o r c h l o r a l s u g g e s t t h e c r y s t a l l o g r a p h i c nonequivalency of t h e C C l 3 groups.

2.16 Mass S p e c t r u m The mass s p e c t r u m o f c h l o r a l h 5 t j r a t e h a s b e e n r e c o r d e d o n a n MS-9 i n s t r u m e n t As expected, c h l o r a l h y d r a t e undergoes thermal dehydration at t h e source temperature (2OO0C), g i v i n g t h e h i g h e s t mass d e t e c t e d a s t h e m o l e c u l a r i o n o f c h l o r a l . The f r a g m e n t a t i o n p a t t e r n f r o m t h i s s p e c t r u m , w h i c h i s g i v e n i n T a b l e 1, d i f f e r s f r o m t h a t o b t a i n e d by R e y e s e t al.25 ( w i t h a H i t a c h i P e r k i n E l m e r RMU-6D i n s t r u m e n t ) i n t h e d e t e c t i o n o f a d d i t i o n a l peaks a t m / e 118, 1 1 7 , a n d 110.

.

TABLE 1 Fragmentation p a t t e r n produced i n mass s p e c t r a l e x a m i n a t i o n o f c h l o r a l h y d r a t e m/e

146 118 117 111 110

83 82 * 47

Ion

cc13mo+

p r o d u c e d by loss f r o m m o l e c u l a r i o n of c h l o r a l of

co CHO c1

CHC12' CCl3 CC 1 2 CHGt CC12COt CC12Ht

HC 1 C l +

c c q cc1

C1

co

CHO CHO + C 1 2

* Most a b u n d a n t p e a k

Y5

t

JOHN E . FAIRBROTHER

1 slsl 9sl 8Q 7sl

6 1 4 5hl Y6l

361

2Q 1Q

6l

FTm-PFv

m ru

arara

rarur MASS/CHARGE

Fig.

4.

Mass s p e c t r u m o f c h l o r a l h y d r a t e , S q u i b b House S t a n d a r d .

96

C H L O R A L HYDRATE

2.17

E l e c t r o n Spin Resonance Spectrum

E l e c t r o n i r r a d i a t i o n i n d u c e s paramagn e t i c species i n c r y s t a l l i n e chloral hydrate w h i c h c a n b e c h a r a c t e r i z e d b y E.S.R. S p e c t r u m 2 5 . Two d i f f e r e n t i n t e n s i t y s e q u e n c e s h a v e b e e n o b s e r v e d t h o u g h t t o c o r r e s p o n d t o t h e CC13C(OH)2 and t h e C C l , C H ( O H ) 2 r a d i c a l s . 2.2

Physical Properties of t h e Solid 2.21

Basic Thermal Constants

The f o l l o w i n g h a v e b e e n r e p o r t e d : -

Heat _ _ _ _ _ a_n_d- _ F_ r_ e_ e_ _E_ n_ e_r-g y - -o -f - F- -o-r -m-a-t-i-o-n-l-l l- ( f o r t h e crystalline state) A Hf= - 1 3 2 . 2 ( k g . c a l . p e r m o l e )

State Crystalline Liquid

Temp e r a t u r e 32oc 55 t o 8 a o c

S p e c i f i c Heat 0.213 0.470

M - -e-l-t -i n_g - r-a-n- g e- _a-n-d- -b-o-i-l-i -n-g - m j n t a r e v a r i a b l e , d e p e n d i n g o n d i s s o c i a t i o n a n d , t h u s , on s u c h f a c t o r s as t h e r a t e o f h e a t i n g .

Lle&lipg-ranggs as d i f f e r e n t a s 46-47OC

5 9 - 6 0 0 ~ 113 h a v e b e e n q u o t e d i n b u t t h e f i g u r e o f 57OC g i v e n by

and

the l i t e r a t u r e , t h e Nerck I n d e x

1 1 4 a p p e a r s t o b e t h e most r e p r e s e n t a t i v e .

_ B o_i -l i-n-g- p o i n t h a s b e e n r e c o r d e d b y L a n g e l l l a s 9 6 . 3 ( 7 6 4 j d C - a n d by t h e Merck I n d e x 1 1 4 a s 98OC.

JOHN E. FAIRBROTHER

2.22

D i f f e r e n t i a l Thermal A n a l y s i s a n d D i f f e r e n t i a l Scanning Calorimetry

D . T . A . (open c o n t a i n e r ) o f t h e Squibb House S t a n d a r d o f c h l o r a l h f b r a t e l 2 g a v e a s i n g l e D.S.C.; (sealed container) endotherm a t 5 9 O C . o f a s a m p l e o f B.P. q u a l i t y c h l o r a l h y d r a t e s i m i l a r l y gave i n g l e endotherm a t 58OC. Separate with U.S.P. grade c h l o r a l hydrate experiments gave a s i n g l e , well-defined m e l t i n g endotherm a t 56 t o 5 7 O C a n d a b o i l i n g p o i n t e n d o t h e r m t h a t v a r i e d f r o m 1 0 5 t o 1 2 5 O C , d e p e n d i n g o n t h e amount encapsulated. S a m p l e s o f c h l o r a l h y d r a t e were heated t o s p e c i f i e d temperatures above t h e meltirg p o i n t , maintained f o r v a r i o u s periods a t t h e temp e r a t u r e and then allowed t o cool. Re-programming of t h e c o o l e d samples gave u n r e p r o d u c i b l e r e s u l t s t h a t f a i l e d t o show t h e p r e s e n c e o f a d i s c r e t e po l y m o r p h i c f o r m .

?”

D i l a t o m e t r i c measurements117 t h a t i n d i c a t e a phase t r a n s i t i o n o c c u r r i g a t 32OC are i n a g r e e m e n t w i t h t h e much l a t e r l l B r e p o r t o f a n e n a n t i o t r o p i c t r a n s i t i o n o c c u r r i n g a t a b o u t 32OC ( o n c o o l i n g ) ,301,302 as o b s e r v e d w i t h t h e p o l a r i p m i c r o s c o p e . T h e t r a n s i t i o n i s n o t t h e same i n t h e r e v e r s e d i r e c t i o n ( i . e . on r a i s i n g t h e temperature). D.T.A. examination of c h l o r a l h y d r a t e (room t e m p e r a t u r e t o 7 O o C ) a s r e p o r t e d b y O g a ~ ad e~t e~c t s t h e t r a n s i t i o n i n t o a h i g h - t e m p e r a t u r e form at 5 2 . 6 O C , m e l t i n g o c c u r r i n g between 55.0 and 6 4 . 5 O C . 7

2.23

Loss on D r y i n g a n d Thermogravimetric Analysis

H e a t e d i n o p e n c o n t a i n e r s f r o m room temperature t o i t s melting point, c h l o r a l hydrate d i s s o c i a t e s slowly, with v o l a t i l i z a t i o n of t h e products. The v a p o r p r e s s u r e o f c h l o r a l h y d r a t e has b e e n examined b y s e v e r a l a u t h o r s . 1 1 7 , 1 2 2 , 3 0 0 .

CHLORAL HYDRATE

I n t h e temperature range 15.65 t o 3 8 . 5 0 ~ a ~ s m o o t h v a p o r - p r e s s u r e c u r v e was o b t a i n e d ,117 r i s i n g f r o m 6 . 1 2 t o 26.90mm. a bromonaphthalene. A s e p a r a t e source122 g i v e s t h e vapor p r e s s u r e of c h l o r a l h y d r a t e a t 2OoC a s 12.7mm. Hg.

-

2.24

Optical Characteristics

C h l o r a l h y d r a t e e x i s t s i n t h e form of large monoclinic p l a t e s (hexagonal t a b u l a r c r y s t a l s ) 2 3 ~ 1 1 4118, A less s t a b l e high-temperature form, o b t a i n e d o n c r y s t a l l i z a t i o n o f t h e m e l t l g ~ 2 3 , or f r o m c h l o r o f o r m s o l u t i o n b y e v a p o r a t i o n 2 3 , e x i s t s i n a n e e d l e - i k e c r y s t a l form. D e n s i t y o f c r y s t a l l i n e s o l i d 1 * a t 20°C i s 1 . 9 0 8 .

3

2.25

X-Fiay D i f f r a c t i o n

The c r y s t a l a n d m o l e c u l a r s t r u c t u r e o f c h l o r a l h y d r a t e has been i n v e s t i g a t e d by x-ray d i f f r a c t i o n 2 3 ~ 1 1 9 ~ 1 2 O ~ 1 2 1 The ~ ~ 1 c~r.y s t a l s t r u c t u r e d i m e n s i o n s o b t a i n e d by a l l t h r e e a u t h o r s 2 3 3 119,121 a r e i n a g r e e m e n t , w i t h i n t h e l i m i t s o f e x p e r i m e n t a l e r r o r . The c r y s t a l h a b i t i s monoc l i n i c a n d b e l o n g s t o t h e p21/C s p a c e g r o u p , w i t h four molecules i n a c e l l of t h e dimensions:0

a = 11.50 t 0.03 A

0

=

6.04 t 0.02 A

c =

9.60 -t 0.03 A

b

0

The r e s u l t s o f s t u d i e 1e c u l a r and t h e s t r u c t u r e a r e l e s s c o n c l u s i v e ,1 9 Y ” Y ” r e s u l t s o r i g i n a l l y o b t a i n e d b y Kondo a n d N i t t a 2 2 w e r e f o u n d to b e i n e r r o r b y Ogawa23, who r e p e a t e d t h e w o r k a n d r e p o r t e d bond l e n g t h s a n d bond a n g l e s . From t h e s e r e s u l t s , 3gawa s u g g e s t e d t h a t t h e m o l e c u l e c o n t a i n s two b i f u r c a t e d b o n d s . However, t h e C - C l bond l e n g t h s g i v e n b y Ogawa have s i n c e b e e n d i s p u t e d b y .“(ilia and H a d j o u d i s 2 l on t h e b a s i s o f t h e i r N . Q . R . s t u d i e s , which 99

JOHN E. FAIRBROTHER

suggest t h a t o n l y one hydrogen-bonded C 1 e x i s t s . An g g r a y powder d i f f r a c t i o n p a t t e r n h a s been recorded f o r a b a t c h of C h l o r a l Hydrate U.S.P. (see Fig.5.).0gawa23 g i v e s powder-diffract i o n p a t t e r n s f o r both t h e normal hexagonal c r y s t a l p l a t e s o f c h l o r a l h y d r a t e a n d for t h e h i g h temperature needle c r y s t a l s . 2.26

Neutron D i f f r a c t i o n

Neutron d i f f r a c t i o n s t u d i e s conducted by Brown123 h a v e y i e l d e d a d d i t i o n a l d a t a a b o u t bond l e n g t h s a n d bond a n g l e s i n c h l o r a l h y d r a t e . 2.27

Polymorphism

Schi1194 r e j e c t e d t h e p o s s i b i l i t y of e n a n t i o t r o p y or t h e e x i s t e n c e o f i s o m e r i c f o r m s o f c h l o r a l h y d r a t e . However, h e was a b l e t o p r e p a r e a h e m i h y d r a t e (m.p. 9 5 O C ) r e p r o d u c i n g t h e work of Meyer a n d D u l k 1 2 4 . P i g u e t a n d J a c o t G u i l l a r m o d 7 more r e c e n t l y p r e p a r e d a "hemihydrate" t h a t gave a w e l l - d e f i n e d i n f r a r e d spect r u m d i f f e r e n t f r o m t h o s e o f c h l o r a l h y d r a t e or o f a m i x t u r e o f c h l o r a l and c h l o r a l h y d r a t e . Naveau118, r e j e c t i n g S c h i l l ' s c o n c l u s ions, presented data supporting t h e existence of a d y n a m i c i s o m e r i s m b e t w e e n two s t e r e o - i s o m e r s o f unknown c o n f i g u r a t i o n . Allenl7, i n an N.Q.R. examination of c h l o r a l h y d r a t e , n o t e d t h e n e e d t o "age" for 3 months m a t e r i a l f r e s h l y r e - c r y s t a l l i z e d from a m e l t b e f o r e i t would g i v e a s a t i s f a c t o r y s p e c t r u m . B i e d e n k a p p a n d W e i s & % n v e s t i g a t e d t h i s phenomenon more t h o r o u g h l y , r e p o r t i n g t h e e x i s t e n c e o f t w o c r y s t a l m o d i f i c a t i o n s (see s e c t i o n 2 . 1 5 ) . The l e s s s t a b l e m o d i f i c a t i o n ( 1 ) was p r o d u c e d by f r e e z i n g m e l t s or b y h e a t i n g c h l o r a l h y d r a t e t o a t least 48OC f o r 0 . 5 hour.125 Modification(1) c h a n g e d s l o w l y t o t h e more s t a b l e m o d i f i c a t i o n ( I 1 ) i n a b o u t 2-3 weeks a t room t e m p e r a t u r e , b u t no a c c e l e r a t e d t r a n s i t i o n was f o u n d e v e n a t 32OC. 100

1w

M

90

80

80

70

70

60

bO

so

50

9

a

40

40

"n

30

0

30

0

-

1

28

+

-

+

Y

?

Fig. 5.

:- a

m s

$ s%

q

?

.

A

.

?

X-ray powder-diffraction pattern of chloral hydrate, Lot T-102.

l

?

?

*

*

JOHN E. FAIRBROTHER

Ogawa23 s i m i l a r l y d e t e c t e d t h e t r a n s i t i o n i n t o a h i g h - t e m p e r a t u r e f o r m a t 52.6OC ( s e e S e c t i o n s 2.22, 2.24 and 2 . 2 5 ) . X-ray p o w d e r - d i f f r a c t i o n p a t t e r n s t a k e n a t d i f f e r e n t times a f t e r c r y s t a l l i z a t i o n o f t h e h i g h - t e m p e r a t u r e f o r m show t h a t t h i s phase changes slowly t o t h e room-temperature phase.

2.3

Solubility 2.31

S o l u b i l i t y i n Water

The s o l u b i l i t y o f c h l o r a l h y d r a t e i n water a t 2 O o C i s g i v e n by t h e B . P . ( 1 9 6 8 ) a s 1 part i n 0 . 3 p a r t s of water. However, t h e Merck Index1l4 g i v e s t h e f o l l o w i n g aqueous s o l u b i l i t i e s : Tetnperat u r e

(OC

)

0 10 20

3800 6600 10100 14300

30 40

2.32

So l u b f l i t y ( m g / m l ) 2 400

S o l u b i l i t y i n Water-Miscible Solvents

The s o l u b i l i t y f i g u r e s q u o t e d for c h l o r -

a l hydrate i n e t h a n o l d i f f e r widely. Temperature

Room Temp. 2 ooc

Volume o f E t h a n o l R e f e r e n c e (95%) required t o d i s s o l v e lg o f C h l o r a l Hydrate 1.5 m l Merck I n d e x ml B.P.196899

0.2

E q u i l i b r a t i o n of l5g of c h l o r a l h y d r a t e ( 9 5 % ) a t 25OC f o r 2 4 h o u r s r e s u l t e d i n a l i q u i d system c o n t a i n i n g about 2 t o 3 g of s o l i d chloral hydratel37. A p a r a l l e l study w i t h methanol gave similar r e s u l t s , but only about 1 g of s o l i d remainedl37. Results obtained with o t h e r w a t e r - m i s c i b l e s o l v e n t s a r e as f o l l o w s : -

with 1 m l of ethanol

CHLORAL H Y D R A T E

Solvent

Temperature

Glycerol Acetone Methyl E t h y l Ketone 2.33 Solvent

Room Temp.

-

114 c a . 1600 Freely soluble 114

-

Freely soluble 114

S o l u b i l i t y i n Water-Immiscible Solvents Temp e r a t u r e (OC)

Diethyl Ether Diethyl Ether Chloroform Chloroform (ethanol-free) Chloroform C h l o r o form (contg. 2% ethanol ) Chloroform Carbon D i sulfide Hexane Heptane n-Decane Pet. Ether

S o l u o i l i t y Refereme (mg c h l o r a l hydrate per m l solvent)

Room Temp. 20 20 25 20

25 Room Temp.

Room Temp. 25 25 25 Room Temp.

Carbon T e t r a chloride Benzene

Room Temp.

Toluene

Room Temp.

Room Temp.

103

S o l u b i l i t y Ref e r e n e (mg c h l o r a l hydrate per m l solvent) c a . 660 > 1000

114 99

155

138

380 c a . 330

137 99

525 500

137 114

11.6 44.4

114

39.2

137 137

33.0 Sparingly soluble Sparingly soluble Sparingly soluble Sparingly soluble

137 114 114

114 114

JOHN E. FAIRBROTHER

2.54 Solvent ---

Tempereture

---Tor---

Olive O i l Olive O i l C a s t o r Oil Corn O i l Liquid Pzraf f i r ) Tu r p e n t i ri e 2.4

S o l u b i l i t y i n O i l s of Pharmaceutical Interest

R O O K Temp. 25

25 25 25 Room Temp.

Pkiysi cal

R ef e-_r e n c e S o l u b i l i-t y __ ( mg c h l o r a l hydrate per m l solvent) ca.

10 14 0

114 137 137

990

137

710

8 30 13.3 Sparingly soluble

$37 114

P r o p e r t i e s of Solutions

a n d Melts 2.41

H e a t s o f S o l u t i on a n d D i l -ution

The h e a t of s o l u t i o n o f c h l o r a l h y d r a t e i r m e t h a n o l Pas b e e n d e t e r n l i n c > d mlcrc,cal o r i m e t ri c a l l y a s a b o u t - 1 . 1 2 K c a l / m o l . l o . S c h i 1 1 9 4 m e a s u r e d t h e t e n p e r a t u r e r i s e o n d i s s o l u t i o n of c h i o r a l arid m e l t e d c h l o r a l h y d r a t e i n w a t e r . In t h e same p a p e r , h e d e s c r i b e s t h e t e m p e r a t c u r e c h a n g e o b s e r v e d on d l s s o l v i n g c h l o r a l h y d r a t e i n w a t e r , a n d c o n p a r e s i t w i t h t h o s e p r o d u c e d by c h l o r a l hemihydrate (see s e c t i o n 2 . 2 ) and by c h l o r a l h y d r a t e melts t h a t had been r a p i d l y c o o l e d t o rcom t e m p e r a t u r e i m m e d i a t e l y G e f c r e dissolution. Schi11g4 suggests t h a t i n a m e l t c f c h l o r a l hydrate s e v e r a l hydrates of d i f f e r e n t s t r u c t u r e s may e x i s t . By c a l c u l a t i o n f r o m S c h i l l ' s f i g u r e s , an approx. heat o f s o l u t i o n f o r c h l o r a l h y d r a t e i r water i s - 0 . 7 8 K c a l / r r , o l . Measurements o f t h e h e a t o f d i l u t i o n o f c h l o r s l h y d r a t e a q u e o u s s o l u t i o n s h a v e b e e n made95. No N e r r s t t e m p e r a t w e c o e f f i c i e n t , i s fourid for these solutions.

104

CHLORAL HYDRATE

2.42

D i s s o c i a t i o n a n d pH

The d i s s o c i a t i o n o f c h l o r a l h y d r a t e i n cyclohexane has a l r e a d y been mentioned i n s e c t i o n 2.13. D i s s o c i a t i o n of d i l u t e aqueous s o l u t i o n s c h l o r a l h y d r a t e was r e p o r t e d i n t h e same p a p e r

yg

T h e i o n i z a t i o n c o n s t a n t , pKa, was o b t a i n e d by m e a s u r i n g t h e pH o f b u f f e r e d s o l u t i o n s o f c h l o r a l h y d r a t e g 6 . T h e mean o f 25 e x p e r i m e n t s g a v e pKa 1 0 . 0 4 , which d i f f e r s c o n s i d e r a b l y f r o m t h e v a l u e o f 11 o b t a i n e d b y E u l e r a n d E u l e r 9 7 a n d i s c l o s e r t o pK 9.77, derived i E d i r e c t l y from k i n e t i c rneasurernents98,

The d i s s o c i a t i o n o f c h l o r a l h y d r a t e i n v a r i o u s o r g a n i c s o l v e n t s (CCl4, b e n z e n e e t c . ) has been s t u d i e d by i n f r a r e d spectroscopy7 (see section 2.11). Studies of t h e near i n f r a r e d s p e c t r a 6 have a l s o been used t o c a l c u l a t e e q u i l i b r i u m and r a t e c o n s t a n t s f o r t h e d i s s o c i a t i o n o f s o l u t i o n s of c h l o r a l h y d r a t e .

2.43

Surface -___--

Tension

Teitel'baum e t a1.lo0 give a complete picture of the surface tensions obtained with aqueous s o l u t i o n s of c h l o r a l (conc. range 2 t o 1 0 0 % )w i t h i n t h e t e m p e r a t u r e r a n g e 0 t o 7 5 O C ( a t 5-OC i n t e r v a l s ) . These a u t h o r s have a l s o g i v e n a t t e n t i o n t o t h e temperature c o e f f i c i e n t , which, when p l o t t e d a g a i n s t m o l . % o f c h l o r a l , s h o w s a maximum a t 50 m o l . % , c o r r e s p o n d i n g t o t h e f o r m a t i o n o f c h l o r a l h y d r a t e , a n d t w o minima a t a p p r o x . 18 a n d 8 1 m o l . % , r e s p e c t i v e l y . F e r r o n i e t a 1 . 5 6 h a v e a l s o r e p o r t e d v a l u e s for t h e surf a c e t e n s i o n of c h l o r a l h y d r a t e s o l u t i o n s t h a t range from 59.5 f o r 1 molar t o 7 6 . 0 (dyne/cm.) a t i n f i n i t e d i l u t i o n . S u r f a c e t e n s i o n measurem e n t s made on s o l u t i o n s o f c h l o r a l h y d r a t e i n d i e t h y l e t h e r , a c e t o n e , or b e n z e n e 3 ° i n d i c a t e weak compound f o r m a t i o n i n t h e s e s y s t e m s .

JOHN E. FAIRBROTHER

The s u r f a c e t e n s i o n o f c h l o r a l h y d r a t e m e l t s 3 1 4 i n t h e t e m p e r a t u r e r a n g e 5 3 t o 100°C i s expressed ( i n dynes/cm.) by t h e e q u a t i o n u 44.6-0.110(t-53°). The c o n t a c t w e t t i n g a n g l e t 1.5O. o f i t s s o l i d phase i s 18040' Ultrasonic Absomtion

2.44

The v a r i a t i o n o f u l t r a s o n i c v e l o c i t y and a b s o r p t i o n w i t h t e m p e r a t u r e has b e e n r e c o r d e d f o r c h l o r a l h y d r a t e m e l t s ( f o r 7 MHz e m i s s i o n , v e l o c i t i e s r a n g e from 1215 m/sec. a t 5 2 O C t o The u l t r a s o n i c 1 1 2 5 m / s e c . a t 75OC ) l o 1 y 1 O 2 . a b s o r p t i o n c o e f f i c i e n t s (a / V 2 ) f o r c h l o r a l hyd r a t e , when compared w i t h t h o s e o f a m i x t u r e o f c h l o r a l a n d water a t c o r r e s p o n d i n g t e m p e r a t u r e s , show t h a t t h e e f f e c t o f d i s s o c i a t i o n b r o u g h t about by increasing temperature i s t o reduce t h e absorption values. S o l u t i o n s o f c h l o r a l i n non-aqueous s o l v e n t s 2 7 , 3 i show a b s o r p t i o n - c o e f f i c i e n t c u r v e s t h a t p a s s t h r o u g h a maximum i n t h e c a s e s o f a s s o c i a t e d s o l v e n t s s u c h as m e t h a n o l a n d e t h a n o l , corresponding t o t h e formation of a molecular compound 308. 2.45

Viscosity

The v i s c o s i t i e s o f c h l o r a l h y d r a t e melts o b t a i n e d b y u s e o f a n Ostwald Viscometer have been r e p o r t e d 1 0 1 as:-

Temperature 52 to 75

(OC

)

Shear V i s c o s i t y

(q/p)

C.S.

10.10

to 3.40

From t h e t e m p e r a t u r e c o e f f i c i e n t o f t h e s e v i s c o s i t i e s , t h e a c t i v a t i o n e n e r g y of shear v i s c o s i t y o f c h l o r a l h y d r a t e ( m e l t ) has b e e n c a l c u l ated as 1 0 . 6 Kcal/mol. The v i s c o s i t y o f s o l u t i o n s o f c h l o r a l

CHLORAL HYDRATE

i n various hydroxylated organic solvents (aliphatic alcohols, benzyl alcohol, phenols, e t c . ) , has b e e n p l o t t e d a g a i n s t c h l o r a l c o n c e n t r a t i o n s h o w i n maxim wher com ound f o r m a t i o n o c c u r s . 28,33,94,75,7%,77,7b9,b31 2.46

98,?b

C r y oscopy -

''y'cis

The f r e e z i n g - p o i n t s i o n caused l o r a l h y d r a t e i n aqueous and a l c o h o l i c s o l u t i o n s has been d e s c r i b e d .

Van R o s s e m 2 2 6 , s t u d y i n g t h e f r e e z i n g p o i n t c u r v e s of c h l o r a l and water found e v i d e n c e for t h r e e d i f f e r e n t h y d r a t e s : a " s e m i h y d r a t e " (1 m o l e o f water t o 2 m o l e s o f c h l o r a l ) , a monoh y d r a t e (1 m o l e o f e a c h ) , a n d a h e p t a h y d r a t e ( 7 m o l e s o f water t o 1 mole o f c h l o r a l ) . T h e h e p t a h y d r a t e had a s h a r p m e l t i n g p o i n t a t - 1 . 4 % . E u t e c t i c t e m p e r a t u r e s o f 5loC w i t h azobenzene and of 4 0 0 C w i t h b e n z i l have been r e p o r t e d 3 1 3 .

2.47

Refractive Index

The r e f r a c t i v e i n d e x o f a c h l o r a l h y d r a t e melt (at 6 0 . 0 O C ) rises with time94, eventually r e a c h i n g a c o n s t a n t v a l u e a t 1 . 4 8 0 7 ( i . e . m o l a r r e f r a c t i o n MR a t 6 0 O c i s 2 9 . 4 7 ) . The F e f r z t i v e index values obtained with a series of b i n a r y s y s t e m s o f c h l o r a l and v a r i o u s s o l v e n t s indi( i n c l u d i n g water, e t h a n o l , and c a t e t h e f o r m a t i o n o f a n a s s o c i a t i o n compound between c h l o r a l and e a c h s o l v e n t .

2.48

Radiolysis

Aqueous s o l u t i o n s o f c h l o r a l h y d r a t e , when i r r a d i a t e d w i t h X - r a y s , gamma r a y s , or b e t a r a y s , g i v e r i s e t o h y d r o c h l o r i c a c i d as t h e end product of a chain reaction25,I03 t o l l o .

3.

M o l e c u l a r Complexes

3.1

Types o f Known C o m p l e x es

JOHN E . FAIRBROTHER

Chloral forms a range of molecular complexes o r dducts main1 in a 1:1 ratio) with alc oho 1s 22 to 31,$9,92,53 diethy1ether,30,31 benzene, 30,j1 acetone,3°3 31 meprob amate,37 tetracycline,38 oxytetrac cline,38 acetaminophen, 299 and others32 to 36330

E.

Similarly, chloral hydrate has been shown to form c mp exes (main caffeine, betaine, lycinamide , 3 9 9 e 0 3 4 1 58,49 diazepam,50,312 ,5-dimethyl dantoin 52,307 phe a ~ e t i n , ~ 3 , 5phenazon ? tY,56 to 6 1 , glycine,1 7 4 , glucosel?6, urea53,%8 and others. 32,42,4 3,4 8,49,51,53,54,62-67 9 69 173,1753219 3.2 Structure of Complexes 8

A number of the molecular complexes of chloral and chloral hydrate have been shown to be hydrogen-bonded association compounds. The techniques employed in the examination of these complexes are summarized in Table 1. TABLE 1 Techniques used in the characterization of complexes of chloral and chloral hydrate Technique References

26,29,51,55,63,64,66,70,

Cryoscopy

71,72,73 Differential Thermal Analysis Differential Scanning Calorirne t ry Heats of Solution Refractive Index Surface Tension Viscosity Infrared Spectroscopy Raman Spectroscopy X-ray Diffraction Ultrasound Hot-stage Microscopy

62 10,83,84 10,49

74,86 30,43,56 28,33,34,75-81. 10,41,82

57 10,85,312 27,31

86

Chloral hydrate and phenazone form both 108

CHLORAL HYDRATE

(1:l) a n d (2:l) m o l e c u l a r c o m p l e x e s . The s t r u c t u r e o f t h e (1:l) c o m p l e x h a s b e e n s t u d i e d by i n f r a r e d 4 1 a n d Raman57 s p e c t r o s c o p y , i n a d d i t i o n t o a s u r f a c e - t e n s i o n 5 6 s t u d y t h a t examined b o t h complexes. The i . r . a n d Raman d a t a s u g g e s t a s t r o n g l y hydrogen-bonded complex, w i t h t h e associa t i o n a t t h e c a r b o n y l oxygen o f t n e p h e n a z o n e a n d not a t t h e n i t r o g e n . Infrared studies of t h e c o m p l e x i n c a r b o n t e t r a c h l o r i d e s o l u t i o n havi? been used t o c a l c u l a t e a s t a b i l i t y c o n s t a n t f o r t h e c o m p l e x (l:l), a n d c r y o s c o p i c m e a s u r e m e n t s on d i l u t e a q u e o u s s o l u t i o n s show t h e m o l e c u l a r compound t o b e c o m p l e t e l y d i s s o c i a t e d i n w a t e r 4 1 . T h e c r y s t a l 2nd m o l e c u l a r s t r u c t u r e o f (1:1) m o l e c u l a r c o m p l e x o f c h l o r a l h y d r a t e and bromodiazepam has b e e n d e t e r m i n e d b y x - r a y

the

diffraction studies312.

4.

S y n t h e s i s and P u r i f i c a t i o n

C h l o r a l l i y d r a t e wa's f i r s t o b t a i n e d b y L i e b i g ( 1 8 3 2 ) from t h e r e a c t i o n o f w a t e r w i t h c h l o r a l , t h e l a t t e r o b t a i n e d v i a t h e c h l o r i n a t i o n of

ethanol. I n d u s t r i a l l y , c h l o r a l i s prepared by passing chlorine i n t o cooled ethanol ( e i t h e r abs o l u t e o r 9 5 p e r c e n t ) t o form t h e h e m i a c e t a l o f t r i c h l o r o a c e t a l d e h y d e , from which c h l o r a l i s liberated by treatment w i t h s u l f u r i c a c i d . It has been found,

ev.r,

t h a t t h e herni-

a c e t a 1 may b e h y d r o 1y ze iJ.' g B ~ 2 k g b y t h e a d d i t i o n o f water, g i v i n g c h l o r a l h y d r a t e and a l c o h o l .

The o v e r - a l l r e a c t i o n may b e r e p r e s e n t e d b y the equation:E t O I l + 4Cl2 + li20 + C l , C C H ( O H ) 2 + 5 H C l 13ther p r o c e s s m o d i f i c a t i o n s i n c f u d e t h e r e p l a c e ment o f e t h a n o l w i t h e t h a n o l - a c e t i c a c i d m i x tures i n t h e c h l o r i n a t i o n step2T0, with recycling of t h e l e s s v o l a t i l e byproducts, such as c h l o r a l a c e t a t e , c h l o r a l a l c o h o l . a t e , b u t y l c h l o r a l , and 109

JOHN E. F A I R B R O T H E R

trichloroacetic acid. Crude c h l o r a l i s s e p a r a t e d by f r a c t i o n a t i o n from v o l a t i l e byproducts s u c h as e t h y l c h l o r i d e , e t h y l e t h e r , e t h y l e n e d i c h l o r i d e , and a l c o h o l , and t h e c h l o r a l r ' c u t r r i s t a k e n between 9 3 and 9 8 O C . T h i s material1g0 c o n t a i n s 2 t o 3% wateri n s o l u b l e material c o n t a i n i n g e t h y l d i c h l o r o a c e t a t e , e t h y l t r i c h l o r o a c e t a t e , and t r i c h l o r o acetal. The t e c h n i c a l c h l o r a l s e p a r a t e d from the o i l contains acetic acid (0.2 t o 0.8%), dic h l o r o a c e t a l d e h y d e ( 2 t o 5%), e t h y l d i c h l o r o a c e t a t e (about 0 . 3 % ) , e t h y l t r i c h l o r o a c e t a t e ( a b o u t 0 . 6 % ), a n d t r a c e s o f c h l o r a l a l c o h o l a t e 1 g 0 . The c h l o r a l h y d r a t e f o r m e d b y t r e a t i n g t h e c h l o r a l w i t h a n a p p r o p r i a t e q u a n t i t y o f w a t e r may c o n t a i n t r a c e s o f t h e above i m p u r i t i e s and a l s o t r i c h l o r o a c e t i c a c i d ( o x i d a t i o n p r o d u c t ) or t r i c h l o r o e t h a n o l ( r e d u c t i o n p r o d u c t ) . The c r u d e c h l o r a l h y d r a t e may t h e n b e p u r i f i e d f u r t h e r by r e c r y s t a l l i z a t i o n from a n o r g a n i c s o l v e n t s u c h as b e n z e n e . An a l t e r n a t i v e i n d u s t r i a l r o u t e t o c h l o r a l i s by t chlorination of acetaldehyde o r parald e h ~ d e 271 ? ~ ~ ~ C H CHO

3

+ 3C12-Cl

3

C C H O + 3HC1

Alternative laboratory preparations include t h e r e a c t i o n o f etli 1 f o r m a t e w i t h s u l f u r y l and t h e r e a c t i o n of c h l o r o chloride at 17OoC a c e t y l e n e w i t h sodium h y p o c h l o r i t e s o l u t i o n and s a t u r a t e d boric a c i d solution273. The p r e a r a t i m o f h i g h - p u r i t y c h l o r a l h a s b e e n a c h i e v e d 2 2 1 by h e a t i n g m e t a c h l o r a l a t 180-2OO0C.

2y2

5.

Degradation

5.1

Degradation o f S o l i d Chloral Hydrate

5.11 V o l a t i l i z a t i o n Chloral hydrate crystals,

l e f t open t o

C H L O R A L HYDRATE

t h e a i r , v o l a t i l i z e s l o w l y as c h l o r a l and water. C o n d e n s a t i o n of t h e mixed d i s s o c i a t e d v a p o r s gives back c h l o r a l hydrate?. Chloral hydrate i n sealed containers w i l l only volatilize u n t i l e q u i l i b r i u m v a p o r p r e s s u r e has b e e n a t t a i n e d 8 9 . V o l a t i l i z a t i o n may b e d e t e r m i n e d b y m e a s u r e m e n t o f w e i g h t l o s s 1 7 3 , b y G.L.C a s ~ a y * 5 ~ or by G.L.C. a s s a y o f t h e u l l a g e gases84 o f t h e container. 5.12

Chemical Degradation

Degradation of c h l o r a l hydrate occurs i n t h e p r e s e n c e o f a n e x c e s s o f free oxygen t o form phosgene, C 0 2 , and H C I , b u t only a f t e r a c o n s i d e r a b l e lag-phase (52 days)227, u n l i k e anh y d r o u s c h l o r a l , w h i c h d e c o m p o s e s much m o r e rapidly22?. Exposure of c h l o r a l h y d r a t e t o s u n l i g h t i n t h e a b s e n c e o f oxygen g e n e r a t e s no s i g n i f i c a n t l e v e l s of gaseous degradation products, even a f t e r 160 d a y ~ ~ ~ 7 . T r a c e s o f w a t e r may c a u s e t h e d e g r a d a t i o n o f c h l o r a l h y d r a t e to d i c h l o r o a c e t a l d e h y d e , t r i c h l o r o a c e t i c a c i d and h y d r o c h l o r i c a c i d ; t r a c e of a l k a l i l e a d t o t h e f o r m a t i o n o f c h l o r o f o r m a n d f o r m i c a c i d 2 1 7 91. 5.2

Degradation of Chloral Hydrate i n Solution 5.21

t A c t i o n o f L i g h-

It h a s b e e n proposed217,21? that; n e u t r a l a q u e o u s s o l u t i o n s of c h l o r a l h y d r a t e decompose by an oxidation-reduction process. 2 ClTCCII’3 ~

t

E20+Ci2CfICHO

t

IH(‘1 + C l j C C O O H

E x p o s u r e o f s o l u t i o n s t o l i g h t g r e a t l y a c c e l e r ; tes t h i s ~ I ’ O C E S S , as may Le d e m o n s t r d a t e d b y m e a s u r e -

JOHN E . FAIRBROTHER

ment of t h e f o r m a t i o n o f f r e e h y d r o c h l o r i c a c i d . 5.22

A c t i o n o f Heat

On w a r m i n g , n e u t r e l a n d s l i g h t l y a c i d i c a q u e o u s s o l u t i o n s of chloral h y d r s t e a p p e a r t o degrEde t o t r i c h l o r o a c e t i c a c i d d i c h l o r o a c e t a l dehyde, and h y d r o c h l o r i c a c i d 2 1 f . 5.23

U l t r a s o u n d ( S o n o c l e a v a g-e )

Exposure of aqueous s o l u t i o n s o f c h l o r a l h y d r a t e t o h i g h - e n e r g y u l t r > a s o u n d ( 1 M H z ; 1.8kV) has b e e n showri220 t o c a u s e a d e g r a d a t i c n o f t h e c h l o r a l h y d r z t e t h a t follows z e r o - o r d e r k i n e t i c s . The m a i n d e g r a d a t i o n p r o d u c t s a r e h y d r o c h l o r i c a c i d and d i c h l o r o a c e t a l d e h y d e .

5.24

A u t o x i d a t -__-i o n and P o l . y m e r i z a tion

A n h y d r o u s c h l o r a l h a s b e e n shown t o u n d e r g o a u t o x i d a t i o n i n a i r 2 2 7 v i a b o t k of t h e f o l l o w i n g r e a c t i o n s and p o s s i b l y o t h e r s . C13CCHO

t 0

->Cl3CCOOH

C l 3 C C H O + 02+COC12

t

C 0 2 t HCI

The a c t i o n of l i g h t a p p e a r s t o b e n e c e s s a r y for at least t h e iriitial formation of t h e peroxide c a t a l y s t . A n t i - o x i d a n t s , s u c h as h y d r o q u i n o n e ,

r e s o r c i n o l , a n d a- and 6 - n a p h t h o l s , d e c r e a s e t h e o x i d a t i o n r a t e , whereas complete p r o t e c t i o n i s g i v e n 2y a n i l i n e , d i p h e n y l a m i n e , and r e l a t e d compounds r 8 . I n a d d i t i o n t o t h e g a s e o u s p r o d u c t s of a u t o x i d a t i o n , a s o l i d p o l y m e r t h o u g h t t o b e m e t a c h l o r z l i s formed. C h l o r a l h y d r a t e does n o t a p p e a r t o u n d e r g o a u t o x i d a t i o n un s s it. f i r s t d i s s o c i a t e s t o c h l o r a l a n d water 2 3 9

.

S t r o n g sulfuric a c i d c a u s e s t h e polymeri y a t i o n of c h l o r a l t o f o r m a- a n d 8- p a r a c h l o r a l s * 3 l , wkiich h a v e b e e n shown t o b e s t e r e o i s o m e r : o f t h e c y c l i c t r i n i e r of ~ h l o r a . 1 ~ 3 ~ , ~ “lore 3 3 . dilute su 1furi c a c id p r o d u c e s t ki e amo r1i h o 11 s m e t a c h 1o r a 1

CHLORAL H Y O R A T E

231, w h ' c h may b e a n o n c y c l i c p o l y m e r o f c h l o r a l hydrate& ,

.

The a n i o n i c . p o l y m e r i z a t i o n o f c h l o r a l h a s b e e n s t u d i e d a t -78OC, w i t h B u L i a n d sodl.um rtaphthalene234 tcl 236 used as i n i t i a t o r s .

5.25 C h l o r a l h y d r a t e decomposes i n d i l u t e a q u e o u s s o l u t i o n s o f sodiuni h y d r o x i d e 9 8 . T h i s f j r s t - o r d e r r e a c t i o n i s c a t a l y s e d by t h e h y d r o x y l i o n s , by t h e c h l o r h l a t e i o n a n d b y w a t e r . It p r o c e e d s v i a a n i n t e r m e d i a t e b i v a l ent, c h l o r a l a t e i o n which decomposes f u r t k e r t o g i v e c h l o r o f o r m and t h e formate ion. F u r t h e r s t u d i e s by P f e i l e t a 1 . 2 3 7 i r i d i c a t e tflat i n t h e p r e s e n c e o f e x c e s s T a E t h e r e a c t i o n i s f i r s t o r d e r arid t h e r a t e i r r c r e a s e s a l m c , s t l i n e a r l y w i t h b a s e c o n c e n t r a t i o n . However, fcir e q u i n o l a r c o n c e n t r * a t i o n s t h e r e a c t i o n becomes secorid o r d e r 2nd w i t k l e x c e s s c h l o r a l h y d r ; t e t h e k l y d r o l y s i s o f t h e C l j C - g r o u p becomes t h e p r e dorriin;nt r e a c t i o n . The a d d j t i o n o f s o l v e n t s o f low d i e l e c t r i c c o n s t a n t (such as dioxan) i n c r e a s e s t h e r a t e of a c t i v i t y towards t h e degradation of c h l o r z l h y d r c t e t o v a r y f o r a r a n g e o f h y d r c l x i d e s 6s follows: Lj 6 Na

<

I( 4

C s < Ca < Sr> < Ba 4 T 1

T u c h e l e t a1 . 2 7 8 , 2 3 9 , 2 4 f i have a l s o e x amined t h e k i n e t i c s cf t k e d e g r a d a t i o n o f c h l o r a l h y Z r a t e i n s o l u t i o n s c o n t a j n i r . g sodium h y d r c j x i d e , b o r a x , d i s o d i u m h y d r o g e n phospl-,ate and s o d i u m p h e n o b a r b i t a l . I n a d d i t i o n , these a u t h o r s exarlined t h e s t , a b i l i z i n g e f f e c t o f c e r t a i n c o l l o j d s (gum a r c b i c , a g a r - a g a r , a n d g e l a t i n ) or] m i x e d soluticins of c h l o r a l h y d r a t e and s o d i u n phenobarbital.

JOHN

5.26

E. FAIRBROTHER

R a d i o l y s~ is -

-

Andrews a n d S h o r e l O 7 r e p o r t e d t h a t a q u e o u s s o l u t i o n s o f c h l o r a l h y d r * a t e were decomp o s e d on i r r a d i a t i o n w i t h x - r a y s ( 5 0 - 2 0 0 k v . ) t o f o r m h y d r o c h l o r i c a c i d a n d a s m a l l e r amourlt o f a w e a k 1 i o n i s e d a c i d . Many o t h e r a u t h o r ~ ~ 5 , ~t O o 3 106,188 t o 1 1 0 , 2 4 1 , 2 4 2 h a v e e x a m i n e d t h e i r r a d i a t i o n of c h l o r a l h y d r a t e s o l u t i o n s w i t h x - r a y s , gamma-rays or b e t a - r a y s . I n a l l c a s e s i t i s SUEg e s t e d t h a t t h e c h l o r a l h y d r a t e decomposes t o hydrochloric acid v i a a chain reaction. 5.27

anisms A c t i o n o f M i c r o o r g--

The d e g r a d a t j o hloral hydrate i n s o i 1s h a s b e e n e x am i n e d 2Q3y’4‘ a n d t h e r e s u l t s suggest t h a t m i c r o o r g a n i s m s p a r t i c j p a t e i n t h e d e g r a d a t i o n i n c o n j u n c t i o n w i t h chemlcal and photochemical pi-ocesses . 5.28

Incompat ib -

i l i ties

___I-

C h l o r a l h y d r z t e h a s b e e n shown t o b e c h e m i c a l 1 y i n c o m p a t i b l e w i t h many c o n p o u n d s . It i s p r o b a b l e t h a t it i s incornpati-ble w i t h a l l s u b s t a n c e s f o r m i r i g c o m p l e x e s w i t h j.t or w i t h c h l o r a l ( s e e S e c t i o n 3 . 1 , T a b l e s 1 a n d 2 ) , w i t h m o s t bases ( s e e S e c t i o n 5 . 2 5 ) a n d w i t h many compounds bearing hydroxyl groups, The f o l l o w i n g t a b 1 e l i s t s s u b s t z n c e s known t o b e i n c o m p a t i b l e w i t h c h l o r a l h y d r a t e ( o t h e r t h a n known c o m p l e x f o r m e r s ) .

114

CHLORAL HYDRATE

S u b s t a n c e s Show
I _ _ -

Caustic Alkalies Alkaline Earths Alkali C a r b o n a t e Soluble B a r b it u r a t e s Bork x D j. so d iuni Hydrogen Phosphate Piperidine Quinine S a l t s Lliuret i n Phenol M a n n i t 01

R e_-ference 245,237,98 245,246

245,246

Substance Reference Lactose Glucose Peroxides

87 176 229,230

2 4 5 , 2 3 9 , 2 4 0 Met21 A l . k y l s 2 3 4 , 2 4 9 Tannin 245

238-

I od i d e e P e r manganates

2 38 247

245 2116

245 87

245 245

* S t e a r . i c Acid *Sucrose *Corr o i l ‘Sesame 011 *PFanut O i l

88 88 a8 88 88

*Glive O i l *Castor O i l * L a n o 1i r i * C a r b c) w a x * Ce t macrogo1 1000 *Calcium Phosphate

88 88 88 88

p-Methy 1 Cyclohexanol

Menthol iso-Menthol Ijor rie ol i s 0-B o r n e o 1

248 248 248 248 248

C amp hor

245

T’hynio1

245

88 88

* Substances c a u s i n g d e g r a d a t i o n of t h e c h l o r a l h y d r F t e moiety o f a c h l o r a l h y d r a t e complex. 5.29

with Starch I n t e r a c t i o n ____ -~

C h l o r a l h y d r a t e may b e r e a c t e d w i t h s t a r c h t o form a p r o d u c t s o l u b i e i n c o l d water25] o r i c l e a r a n t i s e p t i c adhesive250 coacervate25?,

253.

5. 3

~

S t . a b j -l i z -a t i o__n -o__ f Ch l-or al H-yd rate

C h l o r a l h y d r a t e may b e p r c i t e c t e d from a u t o x i I15

JOHN E . FAIRBROTHER

d a t i o n a n d p o l y m e r i s a t i o n by t h e i n c o r p o r a t i o n o f small q u ; n t i t i e s o a n t i o x i d a n t s s u c h a s a n i l i n e or d i p h e n y l a m i n e 2 2 6 . F o l l o w i n g s i m i l a r l i n e s , c h l o r a l may b e s t a b i l i z e d b y t h e a d d i t i o n o f 0 . 2 to 1%forniamide or d j m e t h y l f 0 r r n a m i d e ~ 5 ~ .S i m i l a r l y 0 . 1 % t e t r a - a l k y l t h i u r a n l d i s u l f ' i d e or 0.1% t o 0 . 2 % a z o b i s i s o b u t y r o n i t r i l e has b e e n u s e d 2 5 5 . Chloral has a l s o been s t a b j l i z e d i n t h e presence o f t r a n s i t i o n metal p o l y m e r i z a t i o n i n i t i a t o r s s u c h a s F e , Sb or N i by t h e a d d i t i o n o f (3.05 t o 0.5% E ( o r 6 ) c a p r o l a c t a m 3 0 5 .

6.

Chemical P r o p e r t i e s

6.1

I d e n t i t y T-ests

C h l o r a l h y d r a t e may b e i d e n t i f i e d by i t s m e l t i n g p o i n t and i t s a b i l i t y t o y i e l d c h l o r o f o r m wh n r e a c t e d w i t h s o d i u m h y d r o x i d e s o l u t i o n

15,99,157.

It may b e i d e n t i f i e d by measurement o f p h y s i c a l parameters, such as i n f r a r e d spectrum12, l a s e r Ranian s p e c t r u m 2 5 7 , or G . L . C . r e t e n t i o n ti.me

195,199.

Chloral hydrate gives r a t h e r unsat sfactory conventional aldehydic d e r i ~ a t i v e s ~ 5 ~ , s u c h as o x i m e ( m . p . 56OC), s e m i c a r b a z o n e (m.p. 90°C ( d ) ) , a n d 2,4-dinit~~ophenylhydrazone( m . p . 1 3 l o C ) , b u t f o r m s many c o m p l e x e s t h a t Y2ave c l e a r l y d e f i n e d m e l t i n g p o i n t s (see S e c t i o n 3 . 1 ) . a

C h l o r a l h y d r a t e g i v e s a s t r > o n g F u j iwara c o l o r r e a c t i o n ( s e e S e c t i o n 6.22), w h i c h may b e u s e d a s a n i d e n t i t y t e ~ t ~ 9 9 , ~ 5 9S i. m i l a r l y , i t e s p o s i t i v e color t e s t s w i t h t h e d i z o - r e a g e n t t h e ir,dole r e a g e n t f o r aldehydes2%1, and t h e r e a g e n t . However, i t d o e s n o t g i v e a p o s i t i v e t e s t w i t h S c h i f f ' s r e a g e n t a n d some o t h e r a l d e h y d e r e a g e n t z 2 6 3 . A color t e s t c l a i m e d t o d i s t i n g u i s h c h l o r a l h y d r a t e from o t h e r t r i c h l o r o compounds n o t p o s s e s s i n g a c a r b o n y l g r o u p i n olves reaction with resorcinol i n d i l u t e a l k a l i .

"',

8,275.

116

CHLORAL HYDRATE

O t h e r c o l o r t e s t s employ a s r e a g e n t s c o d e i n e - s u l f u r i c acid310, t h i o b a r b i t u r i c a c i d 3 I 1 a n d 1-pl-,eny 1-3-me t hy 1- 5 - p y r a zol o n e 3 l 1 . Se Vera 1 s i t i v i t y o f l u g or l e s s h a v e spot tests with b e e n d e s c r i b e d 268,?&?.

6.2

Methods o f A n-alysis 6.21

Volumetric/Titrimetric Procedures

Crude c h l o r a l , o b t a i n e d by t h e l i q u i d phase c h l o r i n a t i o n o f e t h a n o l , has been assayed126 by a procedure i n v c l v i n g t h e volumetric measurement o f t h e c h l o r o f o r m formed a f t e r h y d r o l y s i s w i t h s t r o n g KOH s o l u t i o n .

C h l o r a l h y d r a t e i s e a s i l y decomposed by a l k a l i i n t c chloroform and a l k a l i formate. T h i s r e a c t i o n i s used s t o i c h i o m e t i * i c a l l y t o d e t e r r r i n e c h l o r a l h y d r a t e p u r i t y b y m e a s u r i n g t h e amount o f a n e x c e s s o f 1N s o d i u m h y d r c x i d e consumed in t h e r e a c t i o n ( t i t r a t i o n w i t h 1N s u l f u r i c a c i d t o p h e n 0 l ~ h t h a l ~ i n ) ~ 5 ~ 9 t9o ~ 166. ~ ~ 7A ~ l~t e ~r - 8 ~ ~ 5 ~ n a t i v e l y , t h e formate generz-ted i n t h e r e a c t i o n may be d e t e r m i n e d b y r e a c t i o n w i t h a n e x c e s s o f i o d i n e (G.lN), f ' o l l o w e d b b a c k t i t r . a t i o n w i t h 0.1N s o d i u m - t n i o s u l f a t e l 2 ~ (or h y d r a z i n e ) l g 5 o r , a l t e r n a t i v e l y , by r e a c t i o n w i t h 0.1N- p o t a s s i u m permanganate and p o t a s s i u m i o d i d e followed by t i t r a t i o n o f t h e l i b e r a t e d L o d i n e i 3 O ~ l 3 ~ .I n t h e l a t t e r p r o c e d u r e , sodium c a r b o n a t e i s used i n p l a c e o f s o d i u m h y d r o x i d e b e c a u s e i t i s less l i k e l y t o hydrolyze t h e chloroform produced. More v i g o r o u s h y d r o l y s i s of c h l o r a l hydrate w i l l convert t h e chloroform (produced i n i t i a l l y ) i n t o i o n i c c h l o r i d e , which c a n t h e n be d e t e r m i n e d by V o l h a r d t t i t r a t i o n . P r o c e d u r e s for t h i s h y d r o l y s i s h a v e b e e n d e s c r i b e d t h a t use ammoniuni p e r s u l f a t e 1 3 2 o r t h e r e f l u x o f c h l o r a l h y d r a t e w i t h a l u m i n i u m powder and a q u e o u s a c e t i c aci.dl 33. The o x i d a t i o n o f c h l o r a l h y d r a t e (to t r i c h l o r c a c e t ic a c i . d ) 1 6 7 b y a c i d - d i c h r o m a t e l - 3 4 , 117

JOHN

E. F A I R B R O T H E R

f o l l o w e d by b a c k t i t r a t i o n o f t h e e x c e s s d i c h r o mate b J i t h a s t a b i l i z e d M o h r ' s s a l t s o l u t i o n ( t o p h e n y l a n t h r a n i l i c a c i d ) , has b e e n u s e d as a q u a n t i t a t i v e procedurel34. T i t r a t i o n of c h l o r a l hydrazone w i t h s o ium m e t a v a n a d a t e s o l u t i o n has b e e n d e s c r i b e d

2.

13

O t h e r t i t r i m e t r i c methods i n c l u d e t h e potentiometricl48 t i t r a t i o n of chloral hydrate and t h e a c i d i m e t r i c l 4 9 d e t e r m i n a t i o n o f c h l o r a l , following i t s oxidation with s i l v e r oxide.

6.22

Spectrophotometry

The f o r m a t i o n o f a n i n t e n s e r e d c o l o r b y t h e a c t i o n of p y r i d i n e on c h l o r a l h y d r a t e i n t h e p r e s e n c e o f a l k a l i was f i r s t d e s c r i b e d by

F u j i w a r a l 3 9 . The r e a c t i o n i s n o t s p e c i f i c , simil a r c o l o r s b e i n g p r o d u c e d by o t h e r o r g a n i c t r i halogeno-compounds ( c h l o r o f o r m , t r i c h l o r o a c e t i c a c i d e t c . ). The c o l o r produced i s very s e n s i t i v e t o t h e r e a c t i o n c o n d i t i o n s u s e d , which h a v e t o b e c r i t i c a l l y d e f i n e d f o r t h e co!.or i n t e n s i t y t o b e

u s e d as a q u a n t i t a t i v e p a r a m e t e r . The r e a c t i o n h a s f o u n d much u s e i n t h e d e t e r m i n a t i o n o f c h l o r a l h y d r a t e i.n b i o o g i c a l t i s s u e s and f l u i d s , and a r e c e n t p a p e r lto desc r i b e s the q u a n t i t a t i v e determination of c h l o r a l h y d r a t e i n t h e r a n g e 6 t o 60pg. T h e c o n d i t i o n s o f r e a c t i o n have been studied extensively f o r the determination of c h l o r o f o r m and c a r b o n t e t r a c h l o r i d e , and t h e p r e s e n c e o f a c e t o n e i n t h e r e a c t i o n m i x t u r e was f o u n d t o i m p r o v e t h e colorl41. A number o f o t h e r m o d i f i c a t i o s o f t h e F u j iwara p r c c e d u r e have b e e n d e s c r i b e d l C 2 t o 1 4 6 .

F r i e d m a n a n d Cooper147 f o u n d t h a t t h e 118

CHLORAL HYDRATE

c h l o r a l hydrate r e a c t i o n product a l s o absorbs at 370mu, w i t h a n i n t e n s i t y t h r e e t o f o u r t i m e s t h a t of t h e a b s o r p t i o n a t 540mp. T h i s a s p e c t , and t h e u s e o f t h e r e a c t i o n t o d i f f e r e n t i a t e c h l o r a l hyd r a t e from t r i c h l o r o e t h a n o l , which g i v e s a prod440mu, have been examined uc t a b s o r b i t h o r o u g h l y 1!8,8h7. Feigll5O noted t h a t t h e c o l o r of t h e F u j i w a r a r e a c t i o n p r o d u c t i s d i s c h a r g e d by a c e t i c a c i d , and t h a t t h e a d d i t i o n o f b e n z i d i n e t h e n c a u s e s t h e f o r m a t i o n o f a v i o l e t c o l o r . H e suggests t h a t t h e products of t h e Fujiwara r e a c t i o n a r e S c h i f f bases of g l u t a c o n i c a l d e h y d e , formed by r i n g opening o f t h e p y r i d i n i u m a d d u c t s formed b e t w e e n p y r i d i n e a n d t h e p o l y h a l o g e n a t e d compounds. H y d r o l y s i s o f t h e o r i g i n a l S c h i f f bases a n d t r e a t m e n t w i t h b e n z i d i n e y i e l d s t h e same d e r i v a t i v e i r r e s p e c t i v e of t h e nature of t h e polyh a l o g e n a t e d compound. T h i s m o d i f i c a t i o n o f t h e F u j i w a r a r e a c t i o n h a s b e e n made q u a n t i t a t i v e 1 5 1 a n d a l t h o u g h i t i s u n s p e c i f i c for c h l o r a l hydrate it g i v e s a f i v e t o eight-fold increase i n s e n s i t i v i t y o v e r t h a t o f Friedman and Cooper147 and i s less s u s c e p t i b l e t o i n t e r f e r e n c e t h a n u s i n g t h e p e a k a t 370mp. S t e h w e i n a n d KUhmstedt152 d e s c r i b e a more s p e c i f i c c o l o r r e a c t i o n f o r c h l o r a l h y d r a t e which as a q u a n t i t a t i v e p r o c e d u r e h a s a s e n s i t i v i t y l i m i t of about 50pg/ml of c h l o r a l hydrate. I n t h i s r e a c t i o n c h l o r a l h y d r a t e is q u a n t i t a t i v e u c o n v e r t e d t o c h l o r a l oxime w i t h h y d r o x y l a m i n e hydrochloride. The c h l o r a l oxime i s t h e n c o n densed w i t h 2,6-diaminopyridine i n N hydrochloric a c i d t o g i v e a c h e r r y - r e d p y r i - i s a t i n d y e ( A max. 530mp). A r c h e r and H augasl77 d e v e l o p e d a c o l o r i m e t r i c p r o c e d u r e for t h e s p e c i f i c d e t e r m i n a t i o n o f c h l o r a l h y d r a t e (up t o 150pg/10ml sample s o l u t i o n ) . This procedure uses t h e c o l o r (605mu) p r o d u c e d by c h l o r a l h y d r a t e o n r e a c t i o n w i t h q u i n a l d i n e ethiodide i n a l k a l i n e (ethanolamine) isopropanol.

JOHN E . FAIRBROTHER

The s p e c i f i c i t y o f t h e p r o c e d u r e h a s b e e n w e l l e x a m i n e d l 7 7 and a w i d e r a n g e o f compounds h a v e b e n s c r e e n e d f o r i n t e r f e r e n c e i n t h e r e a c t i o n 1.g 3

.

Only c h l o r a l / c h l o r a l h y d r a t e hemia c e t a l compounds a n d m o l e c u l a r c o m p l e x e s h a v e b e e n f o u n d t o i r l t e r f e r e . The p r o c e d u r e o f f e r s good a c c u r a c y a n d p r e siderable a p p l i c a t i o n a s a s t a b i l i t y assay p r o c e d u r e f o r c h l o r s l h y drate and i t s complexes. O t h e r col.0 i m e t r i a s s y p r o c e d u r e s h a v e b e e n d e ~ c r i b e d ~t o~ 318e,298.

6.23

I o n - E x c h a n g e a n-d - Column - - - - ~Chronat ography

C h l o r a l hydrate i s not absorbed on i o n e x c h a n g e r e s i n s by iLn i o n e x c h a n g e mechanism b u t i t kas b e e n f o u n d t o b e a b s o r b e d o n a c a t i o n e x c h a n g e e s j . n (KU-1) b y a p r o c e s s o f m c i l e c u l a r s o r p t i o n 159

.

The n o n a b s o r p t i o n o f c h l o r a l h y d r a t e o n a n i o n e x c h a n g e r s has b e e n u s e d t o d e v e l o p a n assay p r o c e d u r e f o r i t i n t h e p r e s e n c e o f a c e t i c , monochloroacetic, and t r ' c h l o a c e t i c a c i d s , and sodium t r i c h l o r o a c e t a t e 189,28q.

Column c h r o m a t o g r a p h y has a l s o b e e n used t o d e t e r m i n e t h e p u r i t y cf chloral190.

6 . 2 4 ~ a p e ra n d T h i n - l a y e r Chromatoqraphy P a p e r c h r o m a t o g r a p h y has b e e n u s e d 2 y 4 t o determine t r i c h l o r o a c e t i c a c i d i n c h l o r a l hydrate. T h i n - l a y e r C h r o m a t o g r a p h y has b e e n u s e d t o s e p a r a t e c h l o r a l h y d r a t e from a m y l c h l o r i d e and d i c h l o r o e t h a n e , from admixture w i t h monochloroacetic d arid t h e a c e t a t e e s t e r o f m o n o c h l o r o e t hano 1. and from f c 1 r m a l d e h y d e 3 ~ ~ .

"'

120

C H L O R A L HYDRATE

Siniple s o l v e n t - s c r e e n i n g s t u d i e s h a v e b e e n c a r r i e d out on m i c r o c h r o m a t o p l a t e s ( K j e s e l g e l GF254 l a y e r s 2 5 0 ~t h i c k ) w j t h t h e r e s u l t s r e p o r t e d i n T a b l e s 1 a n d 2ls2, I n t h i s s t u d y 1 9 * , t h e s p o t s o f chloral h y d r a t e w e r e l o c a t e d b y spraying with a reagent (resorcinol, 5g, p o t a s s i u m h y d r o x i d e , 5g), i n a b s o l u t e e t h a n o l , 50m1, f o l l o w e d by g e n t l e w a r m i n g . C h l o r a l h y d r a t e a p p e a r s a s a pink s p o t on a w h i t e b a c k g r o u n d ( c f . r e f . 1 0 ) . TABLE 1 ~A p p r o x-i m a t e R f v. a l u e s for ch! oral S o l v e n t System

R f Value ---

C y c l o h e >a n e C a r b o n t et rt c h l @ r i d e Be n ze n e Dichloromethane C h l o r o f orni Acetone E t h a n o l ( 9 5 %) n-Fropano! Diethyl ether Ethyl acetate bIethyl e t h y l ketone n-But

0.9 1.0 1.0

1.0 1 .0 1.0

Compact C omp a c t8 S1.s p r e a d

0.9 0.9

6 no1

121

Appearance o f -- S p o t -- Compact C ompac t Compa c t S1.s p r e a d Conipac t S1.s p r e a d C ompac t S1.s p i i e a d Coii~pact Compact

9.0 0.0 0.1 0.1 0.I,

Cioxan

h y d r--a t e

JOHN E . FAIRBROTHER

TABLE 2 =roximate Rf Values f o r C h l o r a l H y d r a t e i r l Mu1.t i c o m p o n e n t S o l ve n tS ystems -. Solvent System

Ratio

R f Value

-Benzene/Methanol/ Ammonia ( 0 . 8 8 0 ) Benzene/Ethanol/ Ammonia ( G . 8 8 0 ) Methanol/A.mmonia (0.880)

Appearance of Spot

0.4

Conpac t

(48:16.5:2.5)0.8

Compact

(100:1 . 5 )

Conpact

(9O:lO:l)

0.9

The v o l a t i l i t y o f c h l o r a l c o n s i d e r a b l y reduces t h e usefulnpss of q u a n t i t z t i v e T.L.C.

6.25

V a p o r - P h a s e C h r o m a t o g r s phy (v,P.C.

,GXm

E a r l y w o r k e r s 193y194 c o n v e r t e d t h e c h l o r a l h y d r a t e t o c h l o r o f o r m by r e a c t i o n w i t h a l k a l i and t h e n s u b j e c t e d t h e chloroform t o quant i t a t i v e G.L.C. The d i r e c t i r l j e c t i o n o f c h l o r a l h y d r h t e s o l u t i o n s h a s b e e n d e s c r i b e d more r e c e n t l y b y a number o f a u t h o r s , a n d a sumriary o f t h e i r s y s tems i s g i v e n i n T a b l e 3 ( s e e a l s o r e f e r e n c e s 200 a n d 201 ) ,

Methods o f q u a n t i t a t i o n h a v e r a n g e d f r o m t h e use of s t a n d a r d p l o t s and d l r e c t comparison w i t h a s t a n d a r d , t o t h e u9e8of i n t e r n a l s t a n d a r d s , s u c h as c h l o r o b u t a n o l l 9 7 ’ and chloroform] 97. The s e n s i t i v i t y o b t a i n e d b y u s e o f a flame-ionisaticn d e t e c t o r i s only s l i g h t l y g r e a t e r t h a n t h a t cbtained with t h e modified F u j i w a r c p r o c e d u r e d e s c r i b e d by F r i e d m a n a n d ~ooperl47.

122

TABLE 3 _ -G.L..C.---(V.P.C. ) Determhation c j f Ci.ilo??al Hydrate ___

Colm Support

Chron R 60/80 (re&,) Firebrick

Colunln Stat i onmy Columri Phase Temperat urt _ _ - (OC) ~ 20% Apiezon L

100

11-12

20Z Cmbowax 20M

130

7-8

6O/eO Chromosorb W

-

h)

w

Retention Detect,or Type of Reference Det e m i n Tjme ( f i n ) System ation -

10%Apiezon L

F'ropamnied 601 80 1.5/miri f r o m 60 t o 90 2 8 h i n from 90 t o 2-60 Chromosort id 2Cs Cmbowax 20Y 100 60/8G Chi-omosorb W 20% Carbowax 20V 125

-

15% FFAF

0.6% Apiezon L

Forensic

195

Forensic

195 0

I

Metzbol i c

196

r

O

n

>

r

I

<

0

n

ca.1

-

GO/FO

Chromosorb A!: 60/80 Glass Feacis

Katherometer Electron Capt WE' F.I.D.

105

ca. 1

75

23ml a t a r h t e of 37ml/miri.

F.T.D.

Metabolic

19;

Electron Capture Electron Capture Electron Capture

Metabolic

197

Forensic

198

Metabolic

199

> ;;I

TABLE

Columu,

Support

Retention Time (min )

Detector Type of System D e t e m i n a t i c n

80

5.5 - 6

Katherometer

50

ca. 1

Electron Capt w e

Colunm Stationary Column Phase Temperatw e (OC

Column Pak T 5% Silicone 40/60 Fluid 550

-

Gas-Chrorr! Q

3%JXR

3 (cont'd)

1

Purity

Reference

12

2

Vapor Pressure

89

N

P

Poropak Q

Chrorxosorb 6O/PO

160

20% Dow-Corning S i l i c o n e 200

60

21

-

Electrcn Capture

Ka therometer

St a b i l i t y A s say for Deg-adat i o n Products

Study cf Eissociation i n Vzpor Phase

bI

91

rn n

P

R m

n

2I rn

a

7

CHLORAL HYDRATE

However, t h e u s e o f a n e l e c t r o n - c a p t u r e d e t e c t o r g r e a t l y i n c r e a s e s t h e s e n s i t i v i t y , but l i n e a r r e s p o n s e o b t a i n e d i s o n l y o v e r a snlall range. J a i n e t a l . l g 8 r p p o r t e d a working range o f 0 . 6 t o 2.5 x 1 0 - 9 c h l o r a l h y d r a t e (_i . _ e . vanl i n e a r tibove 1 . 5 x m o l e s ) . Salmon a n d Heyes89 o a i n e d s i m i l a r r e s u l t s u s i n g a P y e Unicarr: N i k 5 d e t e c t o r , s a t u r a t i o n of t h e d e t e c t o r moles being observed a t about es. wcirki ng r a n g e b e t w e e n 10-’3 a n d a b o ugivinf2amo2 t 10G . L . C . p r c c e d u r e s hhve a l s o been d e v e l oped f o r t h e d e t e r m i n a t i o n of i m u r i t i e s i n chloral hydrate1~,15,91~1~7,196,P97.

R e a c t i o n of c h l o r a l h y d r a t e h a s b e e n r e p o r t e d w i t h some of t h e n o r e common r e a g e r i t s u s e d in t h e e r e p a r a t i o n o f d e r i v a t i v e s for C.L.C. DiazomethaneL,02 g i v e s a n u n s t a b l e i n t e r m e d i a t e , C l ~ C C H ( O H ) C H = N ~ at, ,
I

0 -CE

C:

I

-OH

cc13

T1-e r e a c t i o n b e t w e e n c k l l o r o s j l a n e s and c h l o r a l c o m p l e x e s c o n t a i n i n g ni t r c g e n he . g . d i c h l o r a l - u r e a ) h a s a l s o bi.en E xarnincd2O

.

6.26

and ~ o_t h_e _ r _ p h_y -s i_c a l _____-__

Refractonetry __ parameters

N e t h o d s h a v e b e e r & s c r i b e d for t h e qum t i t 2 t i v e d e t errninat i o n o f ct-t l o r a l h y d y a t e u s i n g r e f r a c t o m e t r 1 2 0 5 anc-i s u r - f a c e t e n s i o n 1 5 5 or in(-asurenient o f s p e c i f i c g r a v i t y;O6,207.

6.27

P o l a r o g r a phy

The polarographic r e d u c t i o n o f chloral.

JOHN E . FAIRBROTHER

h y d r a t e was f i r s t r e o t e d f o r s o l u t i o n s i n 0.1N KC1 by Neiman e t a l . so$, who q u o t e d a v a l u e o f EA for c h l o r a l h y d r a t e o f - 0 . 8 ~ . F e d e r l i n 2 0 9 ~ 2 1 0 an d h i s c o - w o r k e r s r e p o r t e d a s i n g l e r e d u c t i o n wave ( E J a b o u t - 1 . 6 ~ )f o r c h l o r a l h y d r a t e i n tetramethylammonium b u f f e r , which t h e y a s c r i b e d t o a d i f f u s i o n - c o n t r o l l e d p r o c e s s invol.ving reduction of the hydrated molecule t o dichloroa c e t a l d e h y d e , w h i c h was c o n s i d e r e d t o b e nonreducible. E l v i n g a n d Bennett211,212, a f t e r a very thorough examination of t h e polarographic reduction of chloral hydrate i n a selection of ammonia, p h o s p h a t e a n d b o r a t e b u f f e r s , d i s p u t e d F e d e r l i n f s f i n d i n g s . They r e p o r t e d t h a t , i n a l l c a s e s , t h e y had f o u n d d i c h l o r o a c e t a l d e h y d e t o g i v e two k i n e t i c - c o n t r o l l e d waves ( - 1 . 0 ~a n d - 1 . 7 ~ ) . They a d v a n c e d t h e t h e o r y t h a t t h e c h l c r a l h y d r a t e wave (EJ a b o u t - 1 . 4 ~a g a i n s t S.C.F. ) r e s u l t s f r o m t h e s y n t h e s i s o f d i f f u s i o n and k i n e t i c r e d u c t i o n p r o c e s s e s . T h e i r t h e o r y i s s u p p o r t e d by p o l a r o g r a p h i c a n d c o u l o m e t r i c data showing t h e r e d u c t i o n o f c h l o r a l h y d r a t e t o d i c h l o r o a c e t a l d e h y d e h y d r a t e , wh i ch t h e n d e h y d r a t e s by a k i n e t i c p r o c e s s . The d L c h 1o r o a c e t a 1d e h y d e fo rmed r e d u c e s t o chloroacetaldehyde, then t o aceta.ldehyde, and, f i n a l l y , t o e t h y l alcohol and/or 2,3-dihydroxybutane. The c o m p l e t e r e d u c t i o n s e q u e n c e t o a c e t a l d e h y d e r e s u l t s i n t h e o n e wave, b u t t h e r e d u c t i o n of t h e a c e t a l d e h y d e p r o d u c e s a s e c o n d wave ( E J a b o u t - 1 . 7 ~ )t h a t i s o n l y c l e a r l y r e s o l v e d i n ammonia b u f f e r s . The v a r i a t i o n o f E t w i t h pH a n d w i t h t h e n a t u r e of t h e s u p p o r t i n g e l e c t r o l y t e i s su m m a ri z e d i n T a b l e s 4 a n d 5 .

126

CHLORAL HYDRATE

TABLE

4

+

-

E f f e--c t o f pH o n t h e polaro . r a p h i e b e h a v i o r 2 1 1 o f c h l o r a l h y d r at e at 2 5~ OC from -E lv i n g a ___n d B e n n e_tt) Buffer

-Dj

-

pIi

Cone.

Head

_-

fly) -__

(em.)

7.2

0.25

40

1.42

8.4

0.25

40

1.34* 1.9

9.1

1.0 1.0 1.0

80 80

1.36* 2.6 1.45 2.2 1 . 6 7 1.7

(

--

Ei

n

(v) ~~

sodiuni p h o s p h a t e

- citrate

Ammonium c h l o r i d e - ammonia Amvo n i u m c h l o r i de - arlmonia Borate-KC1 B o r a t e - KC 1

9.2

9.8

80

1.4

* In

t h e c a s e o f ammonia b u f f e r s , a s e c o n d wave i s f o u n d a t Ei a b o u t -. 1 . 6 6 ~ .

TABLE 5

E f f e c t o f s u p p o r t i n g e l e c t r n l y t e on t h e p o l a r c E p l i i c b e h a v i o r o f c h 1 o r ; l h y d r a t e a t 25OC~- ( f r c m FF1-der1i-T Solvent

-~ Water Dioxan/Water (50:50!

Supporting E 1e c t r*o 1y t e

-

pH

-

-

Cone. (mFI -)

--

-E& (V)

1.635 1.420

L i C l (0.1M) - (CH3)4NC1 TO.1M) (C€T3)4 N t 6.7

1 1 1

1.345

9.6

1

1.600

13.0

1

1.520

Li

+

(CH3 )4NOH

Use o f t h e p o l a r o g r a p h i c r e d u c t i o n a s a q u a n t i t a t i v e asstiy p ~ . o c e d u r e i s c a p a b l e o f s e l ectively determining chloral hydrate i n thp prese n c e o f dichloro- a n d c h l o r o a c e t a l d e h y d e 2 1 2 . A q u a n t i t a t i v e p o l a r o g r a h c a s s a y fcr c h l o r a l hydrate has been described3]' that L t i l -

I27

JOHN E. F A I R B R O T H E R

i z e s t h e r e d u c t i o n o f c h l o r a l oxime a t pH2 i n a H C 1 - K C 1 b u f f e r ( t w o waves a r e o b t a i n e d a t a p p r o x . EJ - 0 . 5 5 a n d - 1 . 2 O v . ) . D e t e r m i n a t i o n of Water

6.28

The water c o n t e n t o f c h l o r a l h y d r a t e has b e e n s u c c e s s f u l l y d e t e r m i n e d b y t h e Karl F i s c h e r t i t r a t i o n p r o c e d u r e 2 1 5 a n d by r e a c t i o n with calcium carbide216. 6.3

Radiolabelled C h l o r a l Hydrate

Thin-layer chromatography of c h l o r a l h y d r a t e a f t e r n e u t r o n i r r a a t i o n gave s e v e r a l r a d i o a c t i v e conponentslgi. Chloral hydrate ( l - 1 4 C ) and - ( 2 - 1 4 C ) have bee n r e a r e d by c h l o r i n a t i o n o f l a b e l l e d e t h a n o 1 2 t 4 , 3 6 6 and used t o monitor t h e degradation o f c h l o r a l hydrate i n soi1244.

-

Chloral hydrate t (3H-labelled c h l o r a l h y d r a t e ) h a s b e e n p r e p a r e d 2 6 7 by t h e W i l z b a c h t e c h n i q u e , u s i n g 8Ci of 3 H ( 1 2 m r i . ) a n d s u b j e c t i n g t o T e s l a e x c i t a t i o n for 1 5 m i n u t e s . The s p e c i f i c a c t i v i t y o f t h e p r o d u c t was 30 - 110 m C i / g .

7.

Metabolism

The h y p n o t i c p r o p e r t i e s of c h l o r a l h were f i r s t d e s c r i b e d by L i e b r e i c h ( 1 8 i s r a p i d l y a b s o r b e d from t h e stomach1 r e a c h e s a n e f f e c t i v e b l o d l e v e l w i t h i n 0 . 5 h r . ki man. Mackay a n d C o o p e r 2 8 2 s u g g e s t t h a t t h e CNS d e p r e s s i o n t h a t follows i n g e s t i o n i s m a i n l y , i f n o t e n t i r e l y , due t o i t s m e t a b o l i t e , t r i c h l o r o ethanol. C o m p a r i s o n o f t h e e f f i c a c y of c h l o r a l hydrate and q i c h l o r o e t h a n o l s u p p o r t s t h i s s u p p o s i t i o n2 5 )

.

T r i c h l o r o e t h a n o l i s produced279 by a rea c t i o n c a t a l y z e d b y t h e enzyme, a l c o h o l d i h y d r o genase2869292, which t a k e s p l a c e i n t h e l i v e r , whole b l o o d , and v a r i o u s o t h e r t i s s u e s 2 8 1 . In man, a m a j o r p r o p o r t i o n o f t h e t r i c h l o r o e t h a n o l 128

CHLORAL HYDRATE

( t r i c h l o r o e t h a n o l g l u c u r o n i d e ) , which h a s been c h a r a c t e r i z e d as ' 21,21-trichloroethyl-~-~-glu~ o s i d u r o n i c acid28g.590. A variable proportion of chloral hydrate i s o x i d i z e d i r i t h e l i v e r a n d k i d n e y by a DPN-depend e n t enzyme t o g i v e a C N S - i n a c t i v e m e t a b o l i t e , t r i c h l o r o a c e t i c a c i d 2 8 7 . T h i s i s t h e main u r i n a r y m e t a b o l i t e f o u n d i n man, b u t is m i n o r to t r i c h l o r o e t h no1 (free p l u s c o n j u g a t e d ) i n t h e d o g a n d rat185. A small p r o p o r t i o n o f t h e t r i c h l o r o e t h a n o l may a l s o b e c o n v e r t e d t o t r i c h l o r o a c e t i c a c i d 1 4 6 .

Chloral hydrate i s not d e t e c t e d i n t h e blood

or u r i n e i n m a n 1 4 6 , 1 9 7 , 1 9 8 , b u t i s f o u n d i n small amount i n t h e b l o o d o f t h e d o g , 1 4 5 r a t , 1 4 5 a n d rnouseltO. The r a t e o f m e t a b o l i s m o f c h l o r e l hyd r a t e i s s o r e p i d t h a t f r e e c h l o r a l h y d r a t e was n o t d e t e c t e d i n human b l o o d i n t h e p e r i o d 5 m i n . t o 6 h r . a f t e r t h e a d m i n i s t r a t i o n o f lg o f c h l o r a l h y d r a t e 2 9 4 ( s e n s i t i v i t y o f a n a l y t i c a l method 0 . 5 ~ 1c h l o r a l h y d r a t e / m l b l o o d ) , However, i n t h e samc.594 i n d i v i d u a l s , t r i c h l o r o e t h a n o l was s t i l l p r e s e n t i r i t h e b l o o d ( 2 0 t o 65 mg % ) 6 h r . a f t e r dose.

The d e t e r m i n a t i o n p r o c e d u r e s f o r t h e l e v e l s o f t h e m e t a b o l j t e s i n b l o o d , u r i n e , and t i s s u e have c e n t e r e d around v r i o u s m o d i f i c a t i o n s o f t h e F u j l w a r a r e a c t i o n 1fio . P r o c e d u r e s h a v e b e e n

G a s - l i q u i d chromatography has a l s o been used f o r t h e determinat,ion of c h l o r a l h y d r a t e tric h l o r o e t h a n o l , and u r o c h l o r a l i c a c i d 1 4 6 i n b l o o d , 198 u r i n e , 1 9 7 , 1 9 9 , a n d t i s s u e h o m 0 g e n & t e ~ 9 ~ , ~ 9 5 . T r i c h l o r o a c e t S c a c i d has n o t been d e t e r m i n e d d ; r e c t l y i n t h i s m a n n e r , b u t may b e d e t e r m i n e d i r i d i r 2 e c t l y a f t e r i t , s c o n v e r s i o n t,o c h l o r o f ' n r m ~ 9 7 .

JOHN E. FAIRBROTHER

U r o c h l o r a l i c a c i d is f i r s t h y d r o l y z e d t o t r i c h l o r o e t h a n o l by a c i d h y d r o l y s i s 1 9 7 , 1 9 8 , 2 9 1 or by i n c u b a t i o n w i t h B - g l u c u r o n i d a s e l 9 7 . The g l y c o n e o b t a i n e d b y t h e en zy mi c h y d r o l y s i s of u r o c h l o r a l i c a c i d h a s been i d e n t i f i e d 1 9 7 a s D - g l u c u r o n i c a c i d by T . L . C . o n a l a y e r o f S i l i c a Gel GF254, u s i n g t h e s o l v e n t s y s t e m nb u t a n o l / a c e t i c a c i d / w a t e r ( 4 : 5 : 1 ) and v i s u a l i z a t i o n by c h a r r i n g .

130

CHLORAL HYDRATE

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3,

g,

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;

118,

111,

I39

JOHN E. FAIRBROTHER

226. 227. 228. 229.

230.

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236. 237. 238. 239. 240. 241. 242. 243. 244. 245.

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Yang L. and Pin Fong Hu, J . Chin. Chem. SOC. 10, 190-193 (1943) Jensen R . B . , Butty M. and Pedersen S.B., Acta Chem. Scand. 24, 1961-1987 (1970) Nakaniwa M. , Kameoka I . , Ozaki K. , Ohashi H. and Furakawa J . , J . Polym. S c i . , P a r t B, 8-, 131-135

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Patent, Japan 3089 (Aug. 14, 1952) t o Naobiko & t o . Patent, Japan 7578 (Aug. 29, 1959) to Yusuke Sumiki and Keisuke Horizu Yu D.T. and MacMasters M . M . , St&ke 17, 75-77 (1965) Baker R.E. and MacMasters M.M., S t & G 20, 11-16

(1968)

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Patent, Ger. (East) 41,528 (Sept. 25, 1965) t o Fricke G. P a t e n t , Ger. ( E a s t ) 31,848 (Dee. 25, 1964) to Renner H.J. and Kaltenborn L. C y r , G N , Squibb P r i v a t e Comrnunicati o n Heineman P . , Univ. Ky., O f f . Res. Eng. Serv., Bull. NO. 89, 147-153 (1969) Shriner R. L. , Fuson R.C. and Curtin D. Y., "The Systematic I d e n t i f i c a t i o n of Organic Compounds" 5th ed., 1964, Wil-ey, N .Y. Deshusses J . and Desbames P . , Mitt. Lebensmitte1unters. Hyg. Bern. 52, 431-432 (1961) V i g h A. , Szasz G . , B G t n e r A. and Graczan; L.M. , Gyogyszereszet 5, 16-17 (1961) Anger V. and F i z h e r G. , M i k r o c h h Acta (4),

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1960, E l s e v i e r , Amsterdam. FeigL F. and Stark C. , Mikrochim. Acta (5-6), 9961003 (1955) 1, Pearce G.W. and Jensen J . A . , J. A g r . Food Chem. -

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Dedek

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278.

von Mering J. and Musculus O . , Chem. Ber. 8, 662-666

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408 (1882) (18751

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283. 284. 285. 286. 287. 288.

(1949)

m.

(1950) F’riedrmn R.J. and Cooper J . R . , J . Pharmacol. Exp. Ther. 1 2 , 373-376 (1960) Cooper J.R. and Friedwin P . J . , Biochem. Phamnacol. -1, 76-82 (1958) Smith J . N . and Williams R.T., Biochem. J . 56,

618-621 (1954) 289.

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and Schultze M.O.,

28,

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CHLORAL HYDRATE

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302.

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The above references attempt to be comprehensive to the end of Chemical Abstracts, Volume 74.

143

This Page Intentionally Left Blank

CLIDIN I UM BROMIDE

Brirw C R i ( ~ l jtiiicl , Bcrnurtl Z. .Sctrko\v.yki

145

B. C. R U D Y AND 6. Z . SENKOWSKI

INDEX Analytical P r o f i l e

- Clidinium

Bromide

1. D e s c r i p t i o n 1.1 1.2

2.

N a m e , Formula, Molecular Weight Appearance , C o l o r , Odor

Physical Properties

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12

I n f r a r e d Spectrum Nuclear Magnetic Resonance Spectrum U l t r a v i o l e t Spectrum F l u o r e s c e n c e Spectrum Mass Spectrum Optical Rotation M e l t i n g Range D i f f e r e n t i a l Scanning C a l o r i m e t r y Thermal G r a v i m e t r i c A n a l y s i s Solubility X-ray C r y s t a l P r o p e r t i e s D i s s o c i a t i o n Constant

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug M e t a b o l i c P r o d u c t s

6.

Methods of A n a l y s i s

6.1 6.2 6.3 6.4 6.5 6.6

7.

Elemental Analysis Phase S o l u b i l i t y A n a l y s i s Thin Layer Chromatographic A n a l y s i s Direct S p e c t r o p h o t o m e t r i c A n a l y s i s Colorimetric Analysis T i t r i m e t r i c Analysis

References

146

CLlDlNlUM BROMIDE

1. D e s c r i p t i o n 1.1

N a m e , Formula, M o l e c u l a r Weipht C l i d i n i u m bromide i s 3-hydroxy-1-methylquinuc l i d i n i u m bromide b e n z i l a t e .

M o l e c u l a r Weight:

432.36

1.2

Appearance, C o l o r , Odor C l i d i n i u m bromide o c c u r s as a w h i t e o r n e a r l y w h i t e , a l m o s t o d o r l e s s , c r y s t a l l i n e powder.

2.

Physical Properties

2.1

I n f r a r e d Spectrum ( I R ) The i n f r a r e d s p e c t r u m of c l i d i n i u m bromide i s p r e s e n t e d i n F i g u r e 1 (1). The s p e c t r u m w a s measured w i t h a Perkin-Elmer 6 2 1 S p e c t r o p h o t o m e t e r i n a K B r p e l l e t c o n t a i n i n g 1 . 7 mg o f c l i d i n i u m bromide/300 mg of K B r . T a b l e I l i s t s t h e assignments f o r t h e c h a r a c t e r i s t i c bands i n t h e I R s p e c t r u m (1). Table I I n f r a r e d Assignments f o r C l i d i n i u m Bromide Frequency (cm-')

C h a r a c t e r i s t i c of

3226 3059

OH s t r e t c h i n g v i b r a t i o n s a r o m a t i c CH s t r e t c h i n g vibration C=O s t r e t c h of t h e ester a r o m a t i c C=C s t r e t c h i n g vibration C-0-C s t r e t c h of t h e e s t e r linkage o u t of p l a n e b e n d i n g of 5 adj a c e n t 11's on b e n z e n e r i n g s

1726 1 5 9 5 and 1489 1238-1236 767 and 696

147

6 . C . RUDY AND 6 . Z . SENKOWSKI

148

CLlDlNlUM BROMIDE

2.2

N u c l e a r Magnetic Resonance Spectrum (NMR) The s p e c t r u m shown i n F i g u r e 2 was o b t a i n e d i n a J e o l c o 60 MHz NMR by d i s s o l v i n g 54.3 mg o f c l i d i n i u m bromide i n 0 . 5 m l o f DMSO-d6 c o n t a i n i n g t e t r a m e t h y l s i l a n e as a n i n t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l a s s i g n m e n t s are g i v e n in T a b l e I1 ( 2 ) . T a b l e I1 NMR Assignments f o r C l i d i n i u m Bromide

CH3 b

Protons

No. of P r o t o n s Derived from I n t e g r a t i o n

a b

5 3

C

6 1 1 10

d e f

Chemical S h i f t (PPd

1.4-2.4 3.05 3.1-4.3 5.25 6.86

7.45

Mu1t i p l i c i t y

Complex M u l t i p l e t Sing1e t Complex M u l t i p l e t Complex P l u l t i p l e t Sharp S i n g l e t Singlet

2.3

U l t r a v i o l e t Spectrum ( U V ) #en t h e W s p e c t r u m of c l i d i n i u m bromide was scanned from 350 t o 210 nm, two s h a r p maxima o c c u r r e d a t 257 nm (E = 4 . 8 x 102) and 251 nm ( E = 4 . 3 x l o 2 ) . No f u r t h e r maxima were r e a c h e d between 240 and 210 nm b u t t h e a b s o r b a n c e was s t i l l r a p i d l y i n c r e a s i n g a t 210 nm. The s p e c t r u m shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 50.735 mg of c l i d i n i u m bromide/100 m l o f water ( 3 ) .

2.4

F l u o r e s c e n c e Spectrum No f l u o r e s c e n c e w a s o b s e r v e d f o r c l i d i n i u m bromide i n water, m e t h a n o l , 0.1N H C 1 , o r 0.1N NaOH when t h e sample w a s i r r a d i a t e d w i t h polychromatic l i g h t ( 4 ) .

I49

6 . C. R U D Y AND B. Z. SENKOWSKI

150

Ia

.9

.8

7

E

CLlDlNlUM BROMIDE

Figure 3

300 NANOMETERS

3!

U l t r a v i o l e t Spectrum o f C l i d i n i u m Bromide

w

z a

0

LL

4

m .! 0 (I)

m

a

1 .C.

L

.I

250

151

B. C . R U D Y AND €3. 2. SENKOWSKI

Mass Spectrum The mass s p e c t r u m of c l i d i n i u m bromide was obt a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h a n i o n i z i r g e n e r g y o f 70 e V . The o u t p u t from t h e mass s p e c t r o m e t e r was a n a l y z e d and p r e s e n t e d i n t h e form of a b a r g r a p h , shown i n F i g u r e 4 , by a Varian 100 MS d e d i c a t e d computer s y s t e m ( 5 ) . The h i g h e s t mass o b s e r v e d a t m / e 331 i s a t h e r m a l breakdown p r o d u c t where CH3Br was l o s t from t h e p a r e n t q u a t e r n a r y ammonium compound. The b a s e peak a t m / e 1 8 3 i s due t o t h e diphenylhydroxymethyl f r a g m e n t . The r e st of t h e peaks a r e d u e t o a m u l t i t u d e of t h e r m a l breakdown p r o d u c t s r e l a t e d t o t h e p a r e n t c l i d i n i u m bromide m o l e c u l e ( 5 ) . 2.5

2.6

Optical Rotation C l i d i n i u m bromide e x h i b i t s no o p t i c a l a c t i v i t y .

2.7

M e l t i n g Range The m e l t i n g r a n g e r e p o r t e d i n t h e NF XI11 f o r c l i d i n i u m bromide i s 240° t o 244O u s i n g t h e c l a s s I p r o cedure (6).

2.8

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC s c a n € o r c l i d i n i u m bromide i s shown i n F i g u r e 5 . The e x t r a p o l a t e d o n s e t of t h e m e l t i n g endotherm o c c u r s a t 242.OoC when t h e t e m p e r a t u r e program was 10°/minUte. The AHf w a s found t o b e 9.2 k c a l / m o l e ( 7 ) . 2.9

T h e m o g r a v i m e t r i c A n a l y s i s (TGA) The TGA performed on c l i d i n i u m bromide showed no w e i g h t l o s s from a m b i e n t t o 27OoC. A s i n g l e c o n t i n u o u s w e i g h t l o s s o c c u r r e d from 270° t o 39OoC e q u i v a l e n t t o a b o u t 90% of t h e sample ( 7 ) .

2.10

Solubility The s o l u b i l i t y d a t a f o r c l i d i n i u m bromide o b t a i n a d a t 25OC a r e g i v e n i n T a b l e I11 ( 8 ) . T a b l e I11 S c l u b i l i t y of C l i d i n i u m Bromide i n D i f f e r e n t S o l v e n t s Solvent

S o l u b i l i t y (mg/ml) 9.3 0.06

3A a l c o h o l benzene chloroform

1.4 152

CLlDlNlUM BROMIDE

153

B. C. RUDY AND B. 2. SENKOWSKI

Figure 5 DSC Scan f o r C l i d i n i u m Brom ide

I

I A Hf = 9.2 k cal/mole

260

250

240 230 TEMPERATURE

154

220 OC

210

i

CLlDlNlUM BROMIDE

95% e t h a n o l ethyl ether m ethano 1 p e t r o l e u m e t h e r 30'-60' 2-propanol water

26.4
102.4 <0.05 0.6 82.7

2.11

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n o f c l i d i n i u m bromide i s p r e s e n t e d i n T a b l e I V ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s a r e g i v e n below. Instrumental Conditions: G e n e r a l E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t : Radiation :

Goniometer : Detector:

Recorder:

50 KV-12-1/2 MA Copper Cu Q = 1 . 5 4 2 2 0.10 D e t e c t o r s l i t M.R. S o l l e r s l i t 3 O Beam s l i t 0.0007 i n c h N i f i l t e r 4O t a k e o f f a n g l e Scan a t 0.2' 20 p e r m i n u t e Amplifier gain - 16 course, 8.7 f i n e Sealed proportional counter t u b e and DC v o l t a g e a t plateau P u l s e h e i g h t s e l e c t i o n EL-5 v o l t s ; EU - o u t Rate meter T . C . 4 2000 c f s f u l l scale C h a r t speed - 1 i n c h p e r 5 minutes

Samples p r e p a r e d by g r i n d i n g a t room t e m p e r a t u r e .

B. C. RUDY A N D B. 2. SENKOWSKI

Table I V

__

x-ray Powder D i f f r a c t i o n P a t t e r n of C l i d i n i u m Bromide 20 8.66 9.42 10.52 11* 10 12.36 14.26 14.70 15.55 16.18 16.82 17.20 17.72 18.26 18.80 19.18 19.56 19.94 21.14 21.64 21.96 22.40 22.72 23.13 23.38 24.10 24.76 25.50 26.35 26.92 27.48 27.61 28.25 28.27

*d ** "Io

d

(8) *

10.2 9.39 8.41 7.97 7.16 6.21 6.03 5.70 5.48 5.27 5.16 5.01 4.86 4.72 4.63 4.54 4.45 4.20 4.11 4.05 3.97 3.91 3.85 3.80 3.69 3.60 3.49 3.38 3.31 3.25 3.23 3.16 3.11

I/I

**

0 -

28 -

29.32 29.60 30.21 30.82 31.38 32.28 33.10 33.65 34.15 34.98 35.55 35.86 36.88 37.20 37.80 38.30 39.20 39.46 40.05 40.75 41.50 42.03 42.58 43.25 43.68 44.84 45.48 46.80 47.60 48.50 48.90 49.19 50.19

6 13 40 60 43 45 15 3 73 14 19 13 48 16 5 32 6

14 25 37 44 22 35 70 32 35 17 25 100 3 3 11 5

(interplanar distance)

d&* 3.05 3.02 2.96 2.90 2.85 2.77 2.71 2.66 2.63 2.57 2.53 2.50 2.44 2.42 2.38 2.35 2.30 2.28 2.25 2.21 2.18 2.15 2.12 2.09 2.07 2.02 1.99 1.94 1.91 1.88 1.86 1.85 1.82

nX 2 Sin 0

= relative intensity

(based on h i g h e s t i n t e n s i t y of 1.00) 156

=/I

-0-

**

13 11 11 17 6 10 6 24 23 11 4 7 11 6 3 6 9 8 6 3 7 2 4 2 8 15 4 3 3

11 7 4 3

CLlDlNlUM BROMIDE

3.

Synthesis C l i d i n i u m bromide i s s y n t h e s i z e d b y t h e r e a c t i o n scheme shown i n F i g u r e 6 . 3 - Q u i n u c l i d i n o l i s r e a c t e d w i t h m e t h y l b e n z i l a t e t o form t h e e s t e r . The e s t e r i s t h e n r e a c t e d w i t h methylbromide t o form t h e q u a t e r n a r y s a l t , c l i d i n i u m bromide (10). 4.

S t a b i l i t y Degradation C l i d i n i u m bromide i s s t a b l e i n aqueous s o l u t i o n between pH 2 and pH 8. I n 6N H C 1 t h e e s t e r bond g r a d u a l l y h y d r o l y z e s a t room t e m p e r a t u r e and i n a l k a l i n e media r a p i d decomposition o c c u r s (11). I n f o r m u l a t i o n s t h e d r u g h a s been found t o b e s t a b l e f o r a t l e a s t f i v e y e a r s ( 1 2 ) .

5.

Drug M e t a b o l i c P r o d u c t s C l i d i n i u m bromide i s m e t a b o l i z e d t o 1-methyl-3-hydroxyq u i n u c l i d i n i u m bromide which i s t h e m a j o r form of t h e d r u g found i n t h e u r i n e o f dog and man (11). Both t h e i n t a c t d r u g and t h e above mentioned m e t a b o l i t e a r e found i n t h e f e c e s of dog and man (11). S t u d i e s u s i n g 14C i n d i c a t e t h a t t h e d r u g i s n o t m e t a b o l i z e d by N-demethylation (11). 6.

Methods of A n a l y s i s

6.1

Elemental Analysis The r e s u l t s from t h e e l e m e n t a l a n a l y s i s a r e l i s t e d i n Table V (13). Table V E l e m e n t a l A n a l y s i s o f C l i d i n i u m Bromide Element C H N

% Theory

61.12 6.06 3.24

6.2

% Found

61.30 6.13 3.14

Phase S o l u b i l i t y Analysis P h a s e s o l u b i l i t y a n a l y s i s may b e c a r r i e d o u t u s i n g 2-propanol as t h e s o l v e n t . An example i s shown i n F i g u r e 7 which a l s o l i s t s t h e c o n d i t i o n s u n d e r which t h e analysis was carried out (8).

157

B. C. RUDY AND B. Z SENKOWSKI

I

0

I

t I

6

ti

158

Figure 7

23 2.0

1

1

1

$% +

1

PHASE

0 0

gk 1.5Z

-

0

'. lo-o n

B

z 0

0

l

l

I

1

SOLUBILITY

I

I

1

I

ANALYSIS

SAMPLE CLlDlNlUM BROMIDE SOLVENT ISOPROPANOL SLOPE 0 0 3 % EOUlLlBRATlON 20 HOURS at 2 5 O C EXTRAPOLATED SOLUBJLiTY 077 m g / g of ISOPROPANOL

..

v)

I

-

Q

U

-

-

_-

I

0

-

0.5 ..

n

0

3 -

0,

9 3

5 H m

z I-

0

C

. ,

. ,

r

I

I

L

I

I

I

1

-

, .

1 - 1

1

1

1

1

1

1

6.

C. R U D Y AND 6. 2. SENKOWSKI

6.3

T h i n Layer Chromatographic A n a l y s i s (TLC) The f o l l o w i n g TLC p r o c e d u r e is u s e f u l f o r s e p a r a t i n g c l i d i n i u m bromide from 3-hydroxy-1-methylquinuc l i d i n i u m bromide and 3 - q u i n u c l i d i n y l b e n z i l a t e ( 6 ) . On two s i l i c a g e l G p l a t e s t h a t h a v e b e e n prewashed i n t h e dev e l o p i n g s o l v e n t ( g i v e n below) and a c t i v a t e d 1 5 m i n u t e s a t 105OC, apply 2 mg of c l i d i n i u m b r o m i d e sample i n m e t h a n o l . Develop t h e p l a t e s a t l e a s t 1 0 c m i n f r e s h d e v e l o p i n g s o l v e n t made of acetone:methanol:water:concentrated HC1 Remove t h e p l a t e s , d r y a t 105' f o r 1 0 m i n u t e s , (14:4:1:1). c o o l and s p r a y w i t h t h e p o t a s s i u m i o d o p l a t i n a t e t o d e t e c t any 3 - q u i n u c l i d i n y l b e n z i l a t e (Rf 0 . 8 ) p r e s e n t and s p r a y t h e o t h e r p l a t e w i t h Modified Dragendorff r e a g e n t t o d e t e c t t h e c l i d i n i u m bromide (Rf 0 . 5 ) and any 3-hydroxy1 - m e t h y l q u i n u c l i d i n i u m bromide (Rf 0 . 4 ) p r e s e n t ( 6 ) . Direct Spectrophotometric Analysis Direct s p e c t r o p h o t o m e t r i c a n a l y s i s may b e c a r r i e d o u t i n water u s i n g t h e maximum a t 257 nm. Due t o t h e ext r e m e l y n a r r o w p e a k , however, n a r r o w s l i t w i d t h s a r e req u i r e d and g r e a t care must b e e x e r c i s e d i n l o c a t i n g t h e e x a c t maximum.

6.4

6.5

Colorimetric Analysis C l i d i n i u m bromide forms a n i o n p a i r complex w i t h thymol b l u e i n a n aqueous s o l u t i o n b u f f e r e d a t pH 8 . 8 . T h i s complex c a n b e e x t r a c t e d i n t o c h l o r o f o r m and i t s abs o r b a n c e r e a d a t t h e maximum a t a b o u t 410 nm ( 1 4 ) . 6.6

T i t r i m e t r i c Analysis The p o t e n t i o m e t r i c t i t r a t i o n w i t h p e r c h l o r i c a c i d i n d i o x a n e i s t h e method of c h o i c e t o a s s a y c l i d i n i u m bromide ( 6 ) . The s a m p l e i s d i s s o l v e d i n g l a c i a l a c e t i c a c i d , a n e x c e s s o f m e r c u r i c a c e t a t e i s a d d e d , and t h e t i t r a t i o n w i t h 0.1N HC104 i n d i o x a n e i s c a r r i e d o u t . The e q u i v a l e n c e p o i n t i s d e t e r m i n e d p o t e n t i o m e t r i c a l l y . Each m l of 0.1N HClO4 i s e q u i v a l e n t t o 43.24 mg of c l i d i n i u m bromide.

160

CLI DI N I UM BROMIDE

7.

References

10.

Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. Johnson, J . H., Hoffmann-La Roche Inc,, Personal Communication. Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. Benz, W., Hoffmann-La Roche Inc., Personal Communication. National Formulary XIII, pp. 172 and 8 1 5 ( 1 9 7 0 ) . Moros, S . , Hoffmann-La Roche Inc., Personal Communication. MacMullan, E., Hoffmann-La Roche Inc., Personal Communication. Hagel, R. B., Hoffmann-La Roche Inc., Personal Communication. Sternbach, L. H., Hoffmann-La Roche Inc., US Patent

11.

No. 2 , 6 4 8 , 6 6 7 ( 1 9 5 3 ) . Schwartz, M . A . , Koechlin, B. A . , and Postma, E.,

1.

2. 3.

4. 5. 6. 7.

8.

9.

1 2* 13.

14,

Hof fmann-La Roche Inc. , Unpublished Data. Clyburn, T., Hoffmann-La Roche Inc., Personal Communication. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. Weber, O., Billmeyer, A . , Geller, M., Venturella, V. S., and Senkowski, B. Z., Hoffmann-La Roche Inc., Unpublished Data.

161

This Page Intentionally Left Blank

DEXAMETHASONE

Edward ill. Coiitw

163

E D W A R D M . COHEN

Contents 1. D e s c r i p t i o n

1.1 N a m e , Formula, M o l e c u l a r Weight, R e g u l a t o r y S t a t u s 1 . 2 Appearance , C o l o r , Odor

2. Physical Properties 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

Crystal Properties Infrared Spectra U l t r a v i o l e t Absorbance Optical Rotation Solubility N u c l e a r M a g n e t i c Resonance S p e c t r a Thermal B e h a v i o r Mass S p e c t r a

3. S y n t h e s i s

4. Stability 5. Methods o f A n a l y s i s 5.1 Reference Standards 5 . 2 A-Ring A n a l y s i s 5 . 3 B-Ring A n a l y s i s 5 . 4 C-Ring A n a l y s i s 5 . 5 D-Ring A n a l y s i s 5.6 I n f r a r e d Analysis 5 . 7 Fluorescence Analysis 5 . 8 Mass T r a n s p o r t T e c h n i q u e s

6 . M e t a b o l i s m and P h a r m a c o k i n e t i c s 7. R e f e r e n c e s

DEXAMETHASONE

1. Description

1.1 Name, Formula, Nolecular Weight, Regulatory Status Dexamethasone (I)* is 9a-fluoro-ll6,17a,21-trihydroxy-16~-methylpregna-1,4-diene-3,2O-dione. Other names for the compound include 9a-fluoro-16a-methylprednisolone and 16a-methyl-9a-fluoroprednisolone. The blerck Index lists three other chemical names for dexamethasone. Proprietary names for dexamethasone include Decadron, Dexacortisyl, Oradexon, Hexadrol and Deronil. A number of others are listed in the monograph for dexamethasone in the Merck Index.

0

Mol. Wt.: 392.45

c 2 2 H 2 9 F05

Official monographs for dexamethasone are given in USP XVIII and B.P. 1968.

*Betamethasone is the 166-methyl configurational isomer of dexamethasone. I6S

EDWARD M. COHEN

1 . 2 Appearance, C o l o r , Odor White t o y e l l o w i s h - w h i t e ,

odorless, crystalline

powder. 2. P h y s i c a l P r o p e r t i e s

2.1 Crvstal ProDerties The o p t i c a l c r y s t a l l o g r a p h i c p r o p e r t i e s of dexamethas o n e r e p o r t e d f o r c r y s t a l s o b t a i n e d by c r y s t a l l i z a t i o n o f t h e s t e r o i d from 50% e t h a n o l are as f o l l o w s : 2 System: o r t h o r h o m b i c C r y s t a l Habit: Tabular Optic Sign: + Axial Angle: 50" O p t i c O r i e n t a t i o n : xxll a (c < a < b) yyllc zzll b

r > v strong Dispersion R e f r a c t i v e 1 n d e x e s : a ( a ) = 1.559 B (E) = 1.579 Y = 1.649 Density: .337 Molar R e f r a c t i o n : E x p e r i m e n t a l = 99.64 Calculated = 98.59

Two c r y s t a l forms of dexamethasone have been o b s e r v e d t o d a t e . The X-ray powder d i f f r a c t i o n d a t a f o r t h e two forms A and B a r e g i v e n i n T a b l e I . 3 Because of v a r i a t i o n s enc o u n t e r e d i n peak i n t e n s i t y from sample t o s a m p l e , no l i s t i n g of t h e r e l a t i v e i n t e n s i t y of t h e bands i s g i v e n . The two c r y s t a l forms have e s s e n t i a l l y t h e same s o l u b i l i t y i n water and e s s e n t i a l l y t h e same d e c o m p o s i t i o n t e m p e r a t u r e . Form B i s t h e more u s u a l l y o b s e r v e d form. No s o l v a t e s o r polymorphic m o d i f i c a t i o n s of dexarnethasone were r e p o r t e d by Mesley4 3 f o r dexamethasone c r y s t a l l i z e d from c h l o r o f o r m , a c e t o n e and e t h a n o l u s i n g X-ray d i f f r a c t i o n p a t t e r n s and s o l i d s t a t e I . R . s p e c t r a t o e v a l u a t e samples.

2 . 2 I n f r a r e d Spectrum ( I . R . S . ) Mesley r e p o r t s t h e f o l l o w i n g band a s s i g n m e n t s f o r t h e s o l i d s t a t e I.R.S . of dexamethasone ( m i n e r a l o i l m u l l ) .

I66

DEXAMETHASONE

TABLE I

X-Ray Powder Diffraction Pattern of Dexamethasone Sample - Merck Standard 88415-76 Cu-K, Radiation Form Ba

20"

db -

5.95 6.92 7.45 8.50 8.80 10.55 12.50 13.60 14.20 15.10 15.60 16.80 17.70 18.10 18.50 19.70 20.40 21.60 22.20 22.80 23.50 25.00 26.10 26.75 27.95 28.50 29.00 30.10 30.15 31.70 33.70 37.40 44.40

14.80 12.75 11.83 10.38 10.03 8.36 7.07 6.50 6.23 5.90 5.67 5.27 5.00 4.89 4.79 4.50 4.35 4.11 4.00 3.89 3.78 3.56 3.41 3.33 3.19 3.13 3.07 2.96 2.96 2.82 2.65 2.40 2.04

_c

5.70 15.48 7.95 11.10 9.90 8.92 12.45 7.10 13.50 6.55 14.45 6.12 14.95 5.91 16.10 5.49 17.65 5.02 19.25 4.60 20.00 4.43 20.60 4.30 20.90 4.24 21.00 4.22 25.00 3.56 22.30 3.98 23.00 3.86 23.65 3.76 24.00 3.70 24.70 3.60 25.20 3.53 26.30 3.38 26.70 3.33 27.20 3.27 27.30 3.26 27.70 3.21 28.25 3.15 29.10 3.06 29.65 3.01 31.10 2.87 32.25 2.77 31.70 2.82 32.10 2.78 aMerck Standard 118415-76 bCu-K, Kadiation

EDWARD M. COHEN

Wavelength ( cm.- )

Vibrational Modes

1043 1135, 1117 1090, 1056 1408, 1297, 1242 952, 930, 893, 852 830, 703

1lB-OH 17a-OH 21-OH lY4-diene-3-one

Clark lists 1655 cm.-l, 1616 cm.”, and 892 ern.-' as distinctive bands for the KBr disc I.R.S. of dexamethasone.6 Figure 1 shows the I.R.S. of Merck Standard #8415-76. Principal bands and their assignments are tabulated below. The spectrum is in substantial agreement with published I.R.S. of dexamethasone.5,6,8 Wavenumber of Assignment (cm.-’)

Vibrational Mode

3400-3675 3020, 3050 2850-3000 (severa1 bands ) 1700 1660 1620, 1600 1130-1040 (several bands) 890

OH stretch Olefinic CH stretches Aliphatic CH stretches C20 carbonyl C 3 carbonyl stretch A-ring, C=C stretch Various C-OH stretches A 1 y4-diene-3-ketone

2.3 Ultraviolet Absorbance Dexamethasone exhibits a single major absorbance band at about 240 nm. in solution attributable to the A-1,43 keto A ring chromophore. The following data have been reported in the literature: (E)

Solvent

X Maximum

cm.

Methanol Methano1 Methanol

240 nm. 238 nm. 240 nm.

355(13,920) 392(15 ,400) 380-410

168

Reference (6) (8) (9)

2.5

3

4

5

WAVELENGTH MICRONS 6 7 8 9 10

12 14

18 22 35 5 0

I00

100

80

80

60

60

k 40

40

8 w

u

z

bU -

r

U J

z U

0 4000 3500 3000

F i g u r e 1.

20

20 1

2500

2000

1700 1400 FREQUENCY (CM-' )

1100

1

1

800

500

0

200

I n f r a r e d S p e c t r u m of D e x a m e t h a s o n e ; S p l i t M u l l T e c h n i q u e F l u o r o l u b e m u l l a b o v e 1 3 3 5 crn.-l M i n e r a l o i l m u l l b e l o w 1 3 3 5 c m - I (Merck S t d . # 8 4 1 5 - 7 6 ) .

X

EDWARD M. COHEN

F i g u r e 2 shows a n u l t r a v i o l e t s c a n of dexamethasone (Merck S t d . 118415-76) i n m e t h a n o l a t a c o n c e n t r a t i o n of 0.00127%. A s i n g l e a b s o r b a n c e maximum a t 240 nm. was o b t a i n e d w i t h a n

cm.

of 393 (15,400).1°

In c o n c e n t r a t e d s u l f u r i c a c i d , t h e u l t r a v i o l e t a b s o r b a n c e s p e c t r u m o f dexamethasone undergoes a b a t h o c h r o m i c s h i f t c h a r a c t e r i s t i c of a number of s t e r o i d s . S p e c t r a l d a t a rep o r t e d f o r dexamethasone i n c l u d e : 1% Max.

cm.

262 nmSa 305 nm.a 263 nm. b

-444 - 308 422-455

Reference

11 11

6

aTwo h o u r s i n c o n c e n t r a t e d H 2 S 0 4 bNo c o n d i t i o n s s p e c i f i e d The s p e c t r a l change n o t e d f o r dexamethasone i n c o n c e n t r a t e d s u l f u r i c a c i d i s p r o b a b l y due t o t h e i n i t i a l p r o t o n a t i o n o f t h e A-1,4-3 k e t o f o l l o w e d by a coupled c h e m i c a l r e a c t i o n s i n c e t h e s p e c t r a l changes a r e a p p a r e n t l y i r r e v e r s i b l e . l 2

2.4 O D t i c a l R o t a t i o n The f o l l o w i n g s p e c i f i c r o t a t i o n s have been r e p o r t e d : 25"

IUID = +75 t o 80" (C = 1 i n d i o x a n e ) ' [u]22-240C = 86" (C = 1 i n d i o x a n e ) 8 D [ a I h 5 " = +77.5" ( C = 1 i n d i o x a n e ) l = +72 t o 80" ( C = 1 i n d i ~ x a n e ) ~ 2.5 S o l u b i l i t y The f o l l o w i n g s o l u b i l i t y d a t a a r e g i v e n i n t h e literature:

rn 0

200

250

300

350

WAVELENGTH, n m

Figure 2.

Ultraviolet Absorption Spectrum of Dexamethasone in Methanol at a Concentration of 0.00127% - 2.max. 240 nm. and E1% = 393 lcm. (Merck Std. #8415-76).

EDWARD M. COHEN

So l v e n t

Tempe ra t u r e

D i s t i l l e d Water D i s t i l l e d Water D i s t i l l e d Water Isopropyl Myris t a t e Light Mineral Oil

Ethanol Chloroform Acetone Ether E t h y l Acetate Pyridine

Solubility

Reference

37°C. 25°C. 25 " C.

1 1 . 6 mg/100 m l 8 . 4 mg/100 m l 1 0 . 0 mg/100 m l

14 15 1

37°C.

2 3 . 3 mg/100 m l

14

37°C.

0 . 0 1 mgI100 m l 1 i n 42 1 i n 165 soluble very s l i g h t l y soluble 4 . 1 mg/g

14 9 9 9

--

--

--25°C.

--

13 16

- 10%

7

2 . 6 N u c l e a r M a g n e t i c Resonance Spectrum (NMRS) The 60 MHz NMRS of a 10% s o l u t i o n of dexamethasone i n d e u t e r a t e d p y r i d i n e ( d 5 ) i s shown i n F i g u r e 3. T a b u l a t e d below are t h e p r o t o n a s s i g n m e n t s f o r t h e o b s e r v e d c h e m i c a l s h i f t s and c o u p l i n g c o n s t a n t s . 7 R e l a t i v e // of P r o t o n s

Chemical S h i f t ppm'

7. 55/doublet 6.6?/crude doublet 6.5 4 / q u a r t e t 6.43/~inglet 6.35/unresolved multiplet 5.g4/broad s i n g l e t 4.9?/AB q u a r t e t 4.73/crude doublet 1.74/singlet 1.4 / s i n g l e t 1.1 / d o u b l e t 0.88-3.80 complex overlapped s i g n a l s

-1.1

Assignments C W E f

11-OH

3.9

1.1 3.1

19.8

172

C ( 2 1)-Hz

(19) -H3 '61 8i:" 3 1 -c J A l l remaining protons

500 100 25

-I

I

cn P

I

0

z m

I

F i g u r e 3. NMR S p e c t r u m o f D e x a m e t h a s o n e i n D e u t e r a t e d P y r i d i n e ; O p e r a t i n g 51.5 C o n d i t i o n s S i g n a l : 6 0 MHz S a m p l e : Merck S t d . # 8 4 1 5 - 7 6 , m g . / . 5 c c . of CgDgN, Sweep Time: 250 s e c . S p e c t r u m A m p l i t u d e : 1 . 2 5 x 10. I n t e r n a l S t a n d a r d : T e t r a m e t h y l s i l o n e

EDWARD M. COHEN

u s e d as i n t e r n a l r e f e r e n c e 2Coupling c o n s t a n t 3Based on 29 p r o t o n s f o r sum of t o t a l s p e c t r a l i n t e g r a l 4Assignment c o n f i r m e d v i a D 2 0 exchange 'TMS

2 . 7 Thermal B e h a v i o r M e l t i n g P o i n t - The m e l t i n g p o i n t of dexamethasone i s r e p o r t e d t o depend i n p a r t on t h e r a t e o f h e a t i n g and t h e d e g r e e of powder f i n e n e s s . " Some of t h e r e p o r t e d values include: Melting Point -250" -255" 263" - s a m p l e r e c r y s t a l l i z e d from e t h e r 256-258" 262-264" - c r y s t a l s from e t h e r 2 6 8-2 72 "

Reference 13 9 17 8 1

1

D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y - The thermogram o b t a i n e d on a P e r k i n E l m e r DSC 1-B, 2 0 " l m i n u t e f o r dexamethasone (Merck S t d . #8415-76) e x h i b i t s t h e f o l l o w i n g thermal e v e n t s (Figure 4 ) : 1. 2.

S m a l l exotherm a t -254-258" Complex m e l t i n g endotherm - 258" - 267"

-

o n s e t temp.

- p e a k temp.

2 . 8 Mass S p e c t r a (MS) The low r e s o l u t i o n MS of dexamethasone h a s been described i n the l i t e r a t u r e . l e l~ 9 I m p o r t a n t f e a t u r e s of t h e spectrum i n c l u d e : 1. 2.

3.

Absence of a m o l e c u l a r i o n peak. Major p e a k a t m / e 343 (M.W. - 49) a t t r i b u t e d t o l o s s of water from t h e D-ring f o l l o w e d by c l e a v a g e of t h e C 1 8 - C 2 1 bond. The most i n t e n s e p e a k of t h e MS o c c u r s a t m / e 122 accompanied by a m a j o r p e a k a t m / e 121 a r i s i n g from c l e a v a g e of cg-c7 and C g C l 0 bonds and t r a n s f e r o f two (m/e = 1 2 2 )

174

40 5

-

U

P +-

4 'A

w 1 X W

0 l

~

280

1

l

250

l

I

1

I

I

I

200

1

I

1

1

1

150

1

1

1

1

,

100

1 in "C

Figure 4.

Thermogram of Dexamethasone (Merck S t d . #8415-76); Perkin Elmer - D . S . C . - 1 B ; 20°/min.; sealed p a n with N 2 sweep.

D.S.C.

EDWARD M, COHEN

o r one hydrogen ( m / e = 1 2 1 ) t o t h e charged fragment. The a u t h o r s 1 8 * l9 c l a i m t h a t b e t a m e t h a s o n e , t h e 166-methyl iosmer of dexamethasone, can b e u n e q u i v o c a l l y d i s t i n g u i s h e d from dexamethasone by t h e v i r t u a l a b s e n c e of t h e m / e p e a k a t 343. Formation of t h e m / e 343 d i a g n o s t i c f r a g m e n t was a t t r i b u t e d t o a f a c i l e t r a n s - e l i m i n a t i o n of water from dexamethasone It is f o l l o w e d by c l e a v a g e o f t h e a l l y l i c C z o - C z l bond. q u i t e c o n c e i v a b l e t h a t i n i t i a l e l i m i n a t i o n of water o c c u r r e d under t h e r m a l s t r e s s r a t h e r t h a n u n d e r e l e c t r o n i m p a c t alone i n t h e reported d a t a . l 8 > l 9

Attempts t o c o r r o b o r a t e t h e r e p o r t e d l i t e r a t u r e f i n d i n g s w i t h r e s p e c t t o t h e p r e s e n c e of m / e 343 as a m a j o r peak f o r dexamethasone were n o t s u c c e s s f u l i n Merck l a b o r a t o r i e s . 2 0 F i g u r e 5 shows t h e low r e s o l u t i o n MS of dexamethas o n e o b t a i n e d on an LKB-5000 mass s p e c t r o m e t e r a t 70 e V i o n i z a t i o n e n e r g y , S i g n i f i c a n t d i f f e r e n c e s between t h e s p e c t r a of dexamethasone and b e t a m e t h a s o n e w e r e found i n t h e r e l a t i v e p e a k h e i g h t a t m / e 315 and 3 4 3 , a s l i s t e d below ( i n p e r c e n t o f t h e b a s e p e a k s a t m / e 1 2 2 ) .

176

l-

a

-I W c

tx

.I

I

m /e

Figure 5.

M a s s S p e c t r u m o f D e x a m e t h a s o n e (Merck S t d . # 8 4 1 5 - 7 6 ) LKB-5000 - 7 0 e V i o n i z a t i o n e n e r g y .

E D W A R D M. C O H E N

Dexamethasone

Betamethasone

- 1% - 1% - 1% - 1%

-1% - 1% - 1%

rnfe rnfe m/e m/e mfe m/e rnfe

392 374 372 362 354 343 342 m/e 333 m / e 315 m / e 312

- 1% - 1%

-1% 15% 13%

5% 19% 6%

3% 3% 10%

12% 9%

M+ M-H20

M-HF M-CH20

M-(H20 + HF) see below 362 - HF M- (CO-CH 20H) see below 372-(CO-CH20H

+

H)

A r e a s o n a b l e i n t e r p r e t a t i o n of t h e s e two f r a g m e n t s i s b a s e d on t h e w e l l known a c i d c a t a l y z e d water e l i m i n a t i o n from 17a-hydroxy s t e r o i d s w i t h c o n c o m i t a n t m i g r a t i o n of t h e 18methyl g r o u p from C - 1 3 t o C-17. I n t h e c a s e of dexamethas o n e t h e c i s - c o n f i g u r a t i o n of t h e C-17 k e t o l and C-16-methyl groups i n t h e r e a r r a n g e d i o n rnfe 374 f a v o r s t h e d i r e c t breakdown t o rnfe 315 w h i l e t h e t r a n s - c o n f i g u r a t i o n ( b e t a methasone) a l l o w s € o r a h i g h e r p e r c e n t a g e of t h e i n t e r m e d i a t e i o n m / e 343.

-0

CH20H

d-

GH3

M@

3 3

0

I co H

-

1 L

m / e 374

178

DEXAMETHASONE

3 . Synthesis Dexamethasone can be synthesized starting from 16amethyl hydrocortisone acetate, an available intermediate of bile acid origin. 2 1 (Figure 6). While the original synthetic scheme required a selenium oxide l-dehydrogenation2*, subsequent work established that 1-dehydrogenation could be accomplished for any number of appropriate intermediates by using microorganisms of the class Schizomycetes. An entirely different synthetic scheme for dexamethasone starting from diosgenin has also been reported.2" The synthesis of both tritium labeled and carbon-14 labeled dexamethasone has been given in the literature. The following labeled material has been prepared:

1. 'H at positions 1, 2 and 4 . 2. General tritium labeling. 3. 14C in the 16-i-methyl carbon. 4 . 'H in the l6B-hydrogen. This type of labeled material provides a very sensitive handle for use as a tracer in subsequent chemical and biological studies.

4 . Stabilitv Chemical properties of the reactive mioeties of dexamethasone - A consideration of the stability information reported for dexamethasone is best viewed in connection with the molecular changes reported for dexamethasone as well as structurally related adrenocorticosteroids. A-Ring - The A' 9 43-keto grouping of prednisone acetate can undergo photocatalyzed transformations leading to a variety of compounds whose composition depend on the conditions of the experiments. 26 Two such characteristic products are shown below. Further irradiation of Compound VIII can lead to a photoinduced diene-phenol rearrangement. I t is reasonable to assume that dexamethasone can undergo similar photochemistry with the course of reaction duely influenced by the presence of the 92-fluoro group.

179

EDWARD M . COHEN

Figure 6.

CH2 O C - CH3 I ;

c=o

KC2H302

1-21-ACETATE

1

NaOCH3/GH30H

Se02

0

180

DE XAME THASONE

Vlll

VII

(A max. -233 nm. in ETOH)

(Lumiprednisone, X max. -218 nm. and 265 nm.)

Sodium bisulfite adds reversibly to the A - 1 position of dexamethasone-21-phosphate sodium forming a 1-sulfonate as shown below. 27

so’,

HSOS

1-21 PHOSPHATE S O D I U M

IX

From the data obtained, it is assumed that the active nucleophile is the dianionic sulfite ion. This reaction is typical of conjugate sulfite addition to a,@-unsaturated ketones. It should be inferred that other active nucleophilic agents may behave in a similar manner. B-Ring - The 9a-fluoro aliphatic substituent would be expected to be quite stable to hydrolytic displacement. Indeed, there are no reported instances of liberation of fluoride from the 9cu-f luoro adrecocorticosteroids in-vivo.

181

*

E D W A R D M. COHEN

C-Ring - The 11-6-hydroxyl g r o u p of s e v e r a l c r y s t a l l i n e a d r e n o c o r t i c o s t e r o i d esters undergoes a i r o x i d a t i o n , catal y z e d by b o t h t e m p e r a t u r e and l i g h t , t o form t h e c o r r e s p o n d i n g 11-one compounds. 2 9 S t r u c t u r a l r e q u i r e m e n t s f a v o r i n g t h i s t r a n s f o r m a t i o n are t h e f o r m a t i o n o f a nons t o i c h i o m e t r i c s o l v a t e which u n d e r g o e s f a c i l e d e s o l v a t i o n w i t h o u t change i n t h e X-ray d i s c e r n i b l e c r y s t a l l i n e l a t t i c e s t r u c t u r e . I t was a l s o shown t h a t 11-p-hydroxy s t e r o i d s c a n b e a i r o x i d i z e d i n s o l u t i o n u n d e r somewhat r i g o r o u s c o n d i t i o n s . I n g e n e r a l , t h e r e a c t i o n r e q u i r e s m o l e c u l a r oxygen a n d p r o d u c e s water. I t i s a c c e l e r a t e d by h e a t , g r e a t l y a c c e l e r a t e d by f r e e - r a d i c a l i n i t i a t o r s o r by U.V. l i g h t , a n d i s quenched by f r e e - r a d i c a l i n h i b i t o r s . T h e s e a u t h o r s d i d n o t r e p o r t on any f r e e C - 2 1 a l c o h o l s . D-Ring - The C-17 d i h y d r o x y a c e t a t e s i d e c h a i n , a c h a r a c t e r i s t i c s t r u c t u r a l f e a t u r e of a l l a d r e n o c o r t i c o s t e r o i d s , i s q u i t e s u s c e p t i b l e t o b o t h a e r o b i c and a n a e r o b i c t r a n s f o r m a t i o n s . The a n a e r o b i c c h a n g e s of t h e s i d e c h a i n a r e c o n s i d e r e d by Wendler as f o l l o w s :

c.0

CHOH II C-OH

X

XI

Io2

COOH

CH20H I

COOH

OH XVI

HC.0 I CHOH

0

Xlll

I CHOH

xv

XIV

182

DE XAMETHASONE

Conversion of X to the C-17 ketone (XIII) is considered as a reverse aldol reaction. Conversion of the keto-aldehyde (XIV) to the hydroxy acid (XV) is considered a benzillic acid rearrangement. The predominant reaction, under base catalyzed aerobic conditions, appears to be oxidative cleavage of the side chain to the corresponding etianic acid (XVI). Formation of C-17 ketone is only a minor reaction product.31 An alternative mode of oxidative change at the C-21 position is the formation of a glyoxal (XVII) side chain which was found to be metal catalyzed.32 This glyoxal might be expected to undergo coupled chemical reactions consistent with the reactivity of that functionality. HC=O I

c=o

OH XVI I

The D-homo rearrangement reported for 17n-hydroxy-20-keto steroids is adequately described by Wendler.3 3 Possible products of this rearrangement are shown below.

-

c=o

MAJOR

xx

H O ,CH3

MAJOR XVlll

XIX

While not specifically described in the literature, the D-homo rearrangement may be a possible occurrence for dexamethasone under certain reaction conditions by analogy with other steroids such as triamcinolone.34 183

EDWARD M. COHEN

Other possible reactions involving the C-17 side chain include acylation of the C - 2 1 hydroxyl group with available acylating agents. Prednisolone was reported to transesterify with aspirin to form prednisolone acetate in a pharmaceutical formulation matrix. 3 5 Finally, reactive reagents such as formaldehyde can attack the C-17 side chain to form the bis-methylene dioxy derivatives (BMD) 3 6 Formaldehyde is a potential impurity and/or reaction product present in such commonly used pharmaceutical adjuvants as polyethylene glycols.

.

Stability of Dexamethasone - The following reported stability information is entirely consistent with the potential reactivity discussed above. Dexamethasone solid is reported to be stable in air13 but should be protected from light.’ Solutions o f dexamethasone lose about 50% of the C - 1 7 a-ketol side chain within 6-8 minutes in the presence of a base catalyst.17 Excellent stability is shown by dexamethasone in a variety of marketed pharmaceutical dosage forms.2 Wahba has compared the thermal stability of dexamethasone in four different tablet formulations. l 7

5. Methods of Analysis 5.1 Reference Standards Both the U.S.P. and B.P. supply standard samples of dexamethasone. A highly purified sample of dexamethasone was prepared by equilibrating dexamethasone with ethyl acetate. l6 Merck Standard # 8 4 1 5 - 7 6 , which was prepared as described above, had a phase splubility analysis slope of 0.1% ? 0.1 in ethyl acetate. 5 . 2 A-Ring Analysis (without derivatization)

Ultraviolet Absorption

-

Direct measurement of the

U.V. absorbance at the X maximum - 2 4 0 nm. is an official physical measurement in both the U.S.P. and B.P. In addition, U.V. absorption has been used as an analytical measurement for dexamethasone after previous separation techniques such as liquid partition and/or chromatography have been employed to isolate dexamethasone from a pharmaceutical matrix. 9 39 Non-specif ic interference in the U . V .

184

OE XAMETHASONE

measurements may sometimes b e e l i m i n a t e d by a d i f f e r e n t i a l t e c h n i q u e i n w h i c h a s t r o n g r e d u c i n g a g e n t , s u c h as boroh y d r i d e , c o n v e r t s t h e A1y4-3 k e t o chromophore t o a s a t u r a t e d s y s t e m d e v o i d of a b s o r b a n c e a t a b o u t 240 nm.40 D e t e r m i n a t i o n of t h e U.V. a b s o r b a n c e of a n u n t r e a t e d a l i q u o t v e r s u s a r e d u c e d a l i q u o t y i e l d s i n f o r m a t i o n on t h e s t e r o i d U . V . a b s o r b a n c e i n t h e p r e s e n c e of e x t r a n e o u s absorbance. The f e a s i b i l i t y o f t h i s a p p r o a c h was r e c e n t l y d e m o n s t r a t e d f o r b e t a m e t h a ~ o n e . ~I ~ t s h o u l d be n o t e d t h a t A4-3 k e t o s t e r o i d s e x h i b i t t h e same U . V . a b s o r b a n c e b e h a v i o r a s t h e A1y4-3 k e t o s t e r o i d s and d i r e c t measurements c a n n o t d i s t i n g u i s h b e t w e e n them. The j u d i c i o u s u s e o f b o r o h y d r i d e r e d u c t i o n may e n a b l e o n e t o m e a s u r e L4-3 k e t o i m p u r i t i e s i n t h e p r e s e n c e o f A1'4-3 k e t o s t e r o i d s s u c h a s dexamethasone. N . M . R . Measurements - An N . M . R . a s s a y method f o r b o t h b u l k d r u g and f o r m u l a t i o n s b a s e d on m e a s u r i n g t h e i n t e n s i t y o f t h e r o t o n s i g n a l from t h e C - 1 p r o t o n a t a b o u t 2 . 1 Tau f o r A1y9-3 k e t o s t e r o i d s i n c l u d i n g dexamethasone w a s rec e n t l y d e s c r i b e d . 42 The t e c h n i q u e a p p e a r s t o b e c a p a b l e o f m e a s u r i n g b o t h 0-1-3 k e t o and A194-3 k e t o compounds s e p a r a t e l y o r t o g e t h e r i n t h e same s a m p l e . The r e l a t i v e l y h i g h l e v e l s of d r u g r e q u i r e d f o r a n a l y s i s (80-100 mg.1 s a m p l e ) w i l l limit a p p l i c a t i o n of t h e p r o c e d u r e as d e s cribed without appropriate modifications t o increase the a s s a y s e n s i t i v i t y by a t l e a s t two o r d e r s of m a g n i t u d e .

P o l a r o g r a p h y - The p o l a r o g r a p h i c d e t e r m i n a t i o n of dexamethasone i n t a b l e t d o s a g e Lorms i n 80% e t h a n o l , pH 4 . 2 FlcIlvaine b u f f e r has been The r e d u c t i o n s t e p e x h i b i t e d by Alr4-3 k e t o s t e r o i d u s u a l l y o c c u r s a t more a n o d i c p o t e n t i a l s , and i s e a s i l y d i s c e r n i b l e from t h e r e d u c t i o n s t e p e x h i b i t e d by t h e c o r r e s p o n d i n g C4-3 k e t o s t e r o i d s . 4Lt A-King A n a l y s i s ( w i t h d e r i v a t i z a t i o n ) Hydrazone F o r m a t i o n - I s o n i c o t i n i c a c i d h y d r a z i d e (INH) r e a c t s w i t h t h e A-ring c a r b o n y l t o p r o d u c e a y e l l o w h y d r a z o n e . 1 3 A number of s u b s t a n c e s t h a t i n t e r f e r e w i t h Recently, t h e I N H a s s a y a r e d i s c u s s e d i n R e f e r e n c e 39. T a i s h o h a s recommended t h e u s e o f INH-2HC1 r a t h e r t h a n I N H

IS5

EDWARD M. COHEN

i t s e l f as a r e a g e n t f o r t h e d e t e r m i n a t i o n of dexamethasone. 4 5 It is anticipated t h a t other carbonyl reagents w i l l likew i s e p r o v i d e a b a s i s f o r a n a l y t i c a l methodology w i t h dexamethasone. F o r i s t and Johnson d i s c u s s a number of t h e s e . 4 6

The i n t e g r i t y of t h e A-ring of s t e r o i d s i s g e n e r a l l y cons i d e r e d t o be r e l i a b l y d e t e r m i n e d by e i t h e r U.V. o r I N H a s s a y . 3 9 , 4 7 I n view of t h e r e p o r t e d p r o d u c t s of A-r ng p h o t o l y s i s of p r e d n i s o n e h a v i n g c1,B u n s a t u r a t e d k e t o n e s t r u c t u r e s ( s e e S e c t i o n 4 ) , which may i n t e r f e r e i n t h e measurement of t h e u n a l t e r e d A-ring s t e r o i d , c a u t i o n i n u s i n g t h e s e measurements s h o u l d b e e x e r c i s e d . I t i s i n t e r e s t i n g t o n o t e t h a t d a t a p r e s e n t e d i n R e f e r e n c e 47 s u g g e s t t h a t b o t h d i r e c t U . V . a n d / o r I N H a s s a y gave h i g h e r r e s u l t s f o r p h o t o l y z e d s o l u t i o n s of p r e d n i s o n e (A1y4-3 k e t o s t e r o i d ) t h a n d i d a q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h i c a s s a y of t h e same s o l u t i o n s .

5 . 3 B-Ring A n a l y s i s There a r e no l i t e r a t u r e r e p o r t s t h a t d e a l s p e c i f i c a l l y w i t h a s c e r t a i n i n g t h e i n t e g r i t y of t h e 9 a - f l u o r o s u b s t i t u e n t i n dexamethasone.

5 . 4 C-Ring A n a l y s i s While t h e r e a r e no s p e c i f i c r e p o r t s c o n c e r n i n g dexamethasone, a number of p r o c e d u r e s based on t h e r e a c t i v i t y of t h e oxygen f u n c t i o n a t C - 1 1 a r e d i s c u s s e d by F o r i s t and Johnson f o r r e l a t e d s t e r o i d s . 4 6

5 . 5 D-Ring A n a l y s i s The a - k e t o l group (CH20H-CO) p o s s e s s e s r e d u c i n g p r o p e r t i e s and i t s r e a c t i v i t y towards b l u e t e t r a z o l i u m (B.T.) i s u t i l i z e d e x t e n s i v e l y as a n a s s a y p r o c e d u r e f o r dexamethasone.39 Both t h e U.S.P. and B.P. employ t h e B.T. a s s a y f o r dexamethasone. However, t h e U.S.P. u t i l i z e s a p r i o r t h i n l a y e r c h r o m a t o g r a p h i c s e p a r a t i o n which i n c r e a s e s t h e s e l e c t i v i t y and s p e c i f i c i t y o f t h e f i n a l B.T. a s s a y measurement f o r i n t a c t dexamethasone. The p r e s e n c e o f

I86

DEXAMETHASONE

e a s i l y o x i d i z e d e x c i p i e n t s can cause i n t e r f e r e n c e i n t h e B . T . a s s a y p r o c e d u r e . A t a b u l a t i o n o f a number o f s u c h i n t e r f e r e n c e s i s g i v e n i n R e f e r e n c e 39. Both p o s i t i v e and n e g a t i v e i n t e r f e r e n c e s a r e p o s s i b l e . The f o r m e r a r e d u e t o a g e n t s w h i c h a r e o x i d i z e d u n d e r t h e c o n d i t i o n s of t h e B.T. r e a c t i o n . The l a t t e r a r e a g e n t s w h i c h c a n a l t e r t h e pH o f t h e a l k a l i n e B.T. r e a c t i o n m i x t u r e t h e r e b y d e c r e a s i n g t h e e x t e n t of c o l o r formation. E f f e c t i v e e l i m i n a t i o n of i n t e r f e r e n c e s c a n o f t e n b e a c h i e v e d by s e l e c t i v e e x t r a c t i o n and chromatographic procedures. An a u t o m a t e d B.T. a s s a y h a s b e e n d e s c r i b e d f o r dexamethasone t a b l e t s . 4 8 The 1 7 , 2 1 - d i h y d r o x y - 2 0 - k e t o g r o u p of d e x a m e t h a s o n e r e a c t s w i t h phenylhydrazine-sulfuric a c i d ( P o r t e r - S i l b e r ) r e a g e n t t o form a y e l l o w chromogen s u i t a b l e f o r a s s a y p u r p o s e s . 3 9 A s i n t h e c a s e o f t h e B.T. a s s a y , a number o f s p e c i f i c c o l o r i n t e r f e r e n c e s , which c a n a r i s e from t h e r e a c t i o n o f t h e r e a g e n t w i t h e x c i p i e n t s , may b e e l i m i n a t e d by s e l e c t i v e e x t r a c t i o n and c h r o m a t o g r a p h i c p r o c e d u r e s . A number of o t h e r a s s a y a p p r o a c h e s b a s e d on t h e r e a c t i v i t y of t h e i n t a c t C-17 s i d e c h a i n d e s c r i b e d f o r r e l a t e d stero i d s are presumably a p p l i c a b l e t o dexamethasone; These i n c l u d e : a ) 2,4-dinitrophenylhydrazine r e a g e n t 49 - t h i s r e a g e n t was f o u n d t o b e a p p r o x i m a t e l y t w i c e as s e n s i t i v e a s B . T . a s s a y f o r p r e d n i s o n e b u t i s n o t q u i t e as s e l e c t i v e a s B . T . r e a g e n t f o r i n t a c t p r e d n i s o n e i n t h e p r e s e n c e o f deg r a d a t i o n p r o d u c t s ; b ) formaldehyde e s t i m a t i o n f o l l o w i n g p e r i o d a t e o r b i s m u t h a t e o x i d a t i o n . 46

The u s e o f any o f t h e a s s a y p r o c e d u r e s d e s c r i b e d above a s a n a b s o l u t e i n d e x o f d e x a m e t h a s o n e i n t e g r i t y i n a pharmac e u t i c a l m a t r i x s h o u l d n o t b e assumed w i t h o u t i n d e p e n d e n t v e r i f i c a t i o n u t i l i z i n g h i g h r e s o l u t i o n mass t r a n s p o r t p r o c e d u r e s s u c h as t h i n l a y e r c h r o m a t o g r a p h y , b e c a u s e o f t h e complex n a t u r e of t h e c h e m i c a l c h a n g e s p o s s i b l e a t t h e C - 1 7 s i d e chain. For e x a m p l e , a TLC s e p a r a t i o n of a s a m p l e m i x t u r e f o l l o w e d b y a d e m o n s t r a t i o n of t h e a b s e n c e o f c o l o r r e s p o n s e t o a p a r t i c u l a r r e a g e n t of a l l f o r e i g n s p o t s , s a v e f o r i n t a c t d e x a m e t h a s o n e , would l e n d a s s u r a n c e as t o t h e s p e c i f i c i t y of t h a t reagent.

187

E D W A R D M. COHEN

5.6 Infrared Analysis B e l l ~ m o n t ed~e s~c r i b e d a s o l i d s t a t e I R a s s a y p r o c e d u r e (KBr m u l l ) f o r m e a s u r i n g d e x a m e t h a s o n e i n t h e p r e s e n c e of t r i a m c i n o l o n e u t i l i z i n g as a n a l y t i c a l w a v e l e n g t h s 915 cm.-' and 1140 c m . - l .

5.7 Fluorescence A n a l y s i s U n l i k e A4-3 k e t o s t e r o i d s , t h e A1,4-3 k e t o compounds do n o t e x h i b i t s i g n i f i c a n t s u l f u r i c a c i d induced f l u o r e s cence. 5 1 Recently, a f l u o r e s c e n t assay f o r c o r t i c o s t e r o i d s w a s d e s c r i b e d b a s e d on making a f l u o r e s c e n t D-ring d e r i v a t i v e . 66

5 . 8 Mass T r a n s p o r t T e c h n i q u e s Assay p r o c e d u r e s d e s c r i b e d below depend on t h e s e l e c t i v e m i g r a t i o n o f e i t h e r t h e i n t a c t m o l e c u l e o r a rec o g n i z a b l e d e r i v a t i v e o f t h e i n t a c t molecule between phases f o l l o w e d by a s p e c i f i c f u n c t i o n a l g r o u p o r n o n - s p e c i f i c q u a n t i t a t i v e measurement f o r t h e m o l e c u l e i n t h e a p p r o p r i a t e phase. Phase S o l u b i l i t y A n a l y s i s - See S e c t i o n 5.1 f o r d i s c u s s i o n of a p h a s e s o l u b i l i t y s y s t e m f o r d e x a m e t h a s o n e . L i q u i d - L i q u i d E x t r a c t i o n - S e p a r a t i o n of i n t a c t a d r e n o c o r t i c o s t e r o i d s from a c i d i c d e c o m p o s i t i o n p r o d u c t s h a s b e e n a c c o m p l i s h e d by p a r t i t i o n i n g a s a m p l e b e t w e e n a n e u t r a l o r s l i g h t l y a l k a l i n e a q u e o u s p h a s e and c h l o r o f o r m . 3 1 y 3 9 A v e r y u s e f u l d i s c u s s i o n of t h e i n t e r p r e t a t i o n o f d i f f e r e n c e s n o t e d f o r s t e r o i d c o n t e n t of s a m p l e s o b t a i n e d by B . T . , P o r t e r - S i l b e r , I N H and U . V . a s s a y i s g i v e n i n R e f e r e n c e 39. Column Chromatography - The u s e of h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y as a c o n v e n i e n t s t a b i l i t y i n d i c a t i n g a s s a y procedure f o r t h e a n a l y s i s o f c o r t i c o s t e r o i d creams and o i n t m e n t s w a s r e c e n t l y d e m o n s t r a t e d . 5 2 The a u t h o r s show a s a m p l e chromatogram o b t a i n e d f o r d e x a m e t h a s o n e u s i n g a column of 6,R l - o x y d i p r o p i o n i t r i l e on Z i p a x and 1%e t h a n o l i n hexane a s a m o b i l e p h a s e a t 500 p . s . i . ( e l u t i o n t i m e -11 m i n u t e s ) . A g e n e r a l i z e d s y s t e m f o r t h e p r e d i c t i o n o f

I88

DEXAMETHASONE

of elution curves for corticosteroids based on partition coefficients for a hexane-chloroform-dioxane-water (90-1040-5) solvent system on diatomaceous earth was also des~ r i b e d . The ~ ~ authors give data for betamethasone. A column partition system has been described by Graham which can effectively trap the corticosteroid in an acetonitrile layer on a diatomaceous earth column whi1.e interferences are removed by washing with heptane. 54 Acetonitrile and corticosteroids are then removed from the column with chloroform. The method may be modified readily for the removal of acidic, basic, and/or water soluble interferences. Data are given for both formulated and unformulated dexamethasone. Paper Chromatography - Johnson and Fowler give Rf data for dexamethasone and related steroids utilizing the following system: 5 5 Whatman No. 1 impregnated with 40% V / V formamide in methanol. Mobile Phase: Saturated solution of formamide in chloroform. Mode of Operation: Descending development for 35 cm in a saturated tank. Detection: Diphenyl styryl phenyl tetrazolium t heat = violet spots for steroids. Stationary Phase:

Compound Betamethasone Cortisone Dexamethasone Hydrocortisone Prednisolone Prednisone

Rf .16 .62 .21 .26 .15 .55

Thin Layer Chromatography (TLC) - U.S.P. XVIII utilizes TLC as both a qualitative and quantitative assay procedure for dexamethasone. l 3 N . F . XI11 employs TLC as an identity test for dexamethasone in official dosage forms. Wahba has demonstrated the utility of quantitative TLC as a stability indicating approach to the estimation of dexamethasone in aged samples of experimental tablet formulations.” There is little doubt that TLC offers the analyst

’‘

EDWARD M. COHEN

a n e x c e l l e n t h i g h l y s e l e c t i v e method, u s u a l l y w i t h o u t req u i r i n g any d e r i v a t i z a t i o n , f o r a s s e s s i n g t h e i n t e g r i t y o f dexamethasone i n common p h a r m a c e u t i c a l m a t r i c e s . Thin l a y e r chromatography c a n o f t e n f u n c t i o n as a p r i m a r y o r r e f e r r a l a s s a y p r o c e d u r e t o c o r r o b o r a t e t h e v a l i d i t y of a non-chromatographic a s s a y p r o c e d u r e . Some of t h e r e p o r t e d TLC d a t a f o r dexamethasone, i n a d d i t i o n t o t h e i n f o r m a t i o n i n t h e U.S.P. and N.F., are g i v e n i n T a b l e 11. O t h e r TLC s y s t e m s f o r dexamethasone a r e d i s c u s s e d i n R e f e r e n c e s 1 7 , 59 and 60. Of s p e c i a l i n t e r e s t i s a r e p o r t which d e s c r i b e s t h e s e p a r a t i o n of dexamethasone from b e t a m e t h a s o n e . 6 1 Gas Chromatography - S t e r o i d s p o s s e s s i n g t h e C-17 d i h y d r o x y a c e t o n e s i d e c h a i n u s u a l l y undergo m o l e c u l a r a l t e r a t i o n a f t e r a p p l i c a t i o n t o GLC columns t o y i e l d as a major product t h e corresponding 1 7 - k e t o s t e r o i d . 62 Approaches t o a t t a i n q u a n t i t a t i v e c o n v e r s i o n t o a s u i t a b l e n o n - l a b i l e d e r i v a t i v e o f s t e r o i d s r e l a t e d t o dexamethasone have been r e c e n t l y d e s c r i b e d by B a i l l i e and co-workers. 6 3 One o f t h e most s t a b l e d e r i v a t i v e s s u i t a b l e f o r g a s chromat o g r a p h y i s t h e 20-0-methyloxime-17,2l-trimethylsilyl ether of t h e a d r e n o c o r t i c o s t e r o i d . 6 . Metabolism and P h a r m a c o k i n e t i c s Normal human s u b j e c t s g i v e n 1 . 6 mcg. o f 1,2,4-3H dexamethasone o r a l l y e x c r e t e d a b o u t 15% of t h e t o t a l r a d i o a c t i v i t y i n the urine within four hours a f t e r administrat i o n . 6 4 Approximately 50% of t h e e x c r e t e d r a d i o a c t i v i t y was i n c o n j u g a t e d form, presumably a g l u c u r o n i d e and 50% o f t h e e x c r e t e d r a d i o a c t i v i t y was i n non-conjugated form. The u r i n a r y l e v e l s o f t o t a l and c o n j u g a t e d dexarnethasone i n p a t i e n t s on c h r o n i c d i p h e n y l h y d a n t o i n t h e r a p y were s i g n i f i c a n t l y i n c r e a s e d compared t o t h e l e v e l s shown by n o r m a l human s u b j e c t s . I n a n o t h e r s t u d y , t h e mean r e c o v e r y of u r i n a r y r a d i o a c t i v i t y a f t e r f o u r h o u r s and 24 h o u r s was 16% and 64%, res p e c t i v e l y € o r d o s e s of 0.5 t o 1 . 5 mg. of l a b e l e d dexamethasone a d m i n i s t e r e d i n t r a v e n o u s l y as a s o l u t i o n i n s a l i n e t o human s u b j e c t s . 6 0 The m a j o r pathway of dexamethasone metabolism, f o l l o w i n g I . V . a d m i n i s t r a t i o n , a p p e a r s t o i n v o l v e formation of p o l a r unconjugated d e r i v a t i v e s . A

190

TABLE I1

Silica Gel G a Solvent: Rf:b Reference: aA

.51d

D 1.40d

.EOd

58

58

58

C

A

1.00' 57

.;2d 58

I .i5d 58

.t5d 58

.:2d 58

.50d 58

J d

.67 58

Methylene Ch1oride:dioxane:water (100:50:50) - lower layer; Chloroform-ethanol (9:l); C = Chloroform-90% methanol (9:l); D = Cyclohexane-ethyl acetate (1:l); E = Chloroform-acetone (9 :1) ; F = Chloroform-acetone ( 4 : l ) ; G = C y c l o h e x a n e - c h l o r o f o r m - a c e t i c acid (7:2:1); H = Methylene chloride-acetone ( 4 : 1); I = Chloroform-acetic acid (9:l); J = Kethylene chloride-acetic acid (9: 1).

B

= =

bDexamethasone separated from other related steroids. Reference 58 also includes TLC data for dexamethasone on aluminum oxide and magnesium silicate stationary phases as well as useful information on detection system. C

Relative to hydrocortisone

=

1.00.

dRelative to cortisone = 1.00.

E D W A R D M. COHEN

t% of 252 m i n u t e s was n o t e d f o r t h e e l i m i n a t i o n of i n t a c t dexamethasone from plasma. I t i s a n t i c i p a t e d t h a t development of a p p r o p r i a t e s e n s i t i v e a s s a y s f o r dexamethasone w i l l e n a b l e s t u d i e s i n human subj e c t s t o be c a r r i e d out using conventional pharmaceutical dosage f o r m s , made w i t h “ c o l d ” dexamethasone, t o y i e l d a d d i t i o n a l i n f o r m a t i o n on t h e o r a l b i o a v a i l a b i l i t y o f dexamethasone. C u r r e n t a n a l y t i c a l methodology f o r dexamethas o n e i s n o t s u f f i c i e n t l y s e n s i t i v e t o a s s a y a t t h e nanogram p e r m l . l e v e l , which r e p r e s e n t s e x p e c t e d plasma c o n c e n t r a t i o n s f o l l o w i n g o r a l a d m i n i s t r a t i o n of t h e r a p e u t i c d o s e s o f dexamethasone. Techniques such as r a d i o i m m u n ~ a s s a y ~a r e l i k e l y t o p r o v i d e t h e a s s a y s e n s i t i v i t y and s e l e c t i v i t y req u i r e d f o r t h e s e s t u d i e s i n t h e p r e s e n c e of t h e normal s t e r o i d background i n b i o l o g i c a l f l u i d s .

A p r o c e d u r e f o r t h e d e t e c t i o n and i d e n t i f i c a t i o n of dexamethasone i n h o r s e u r i n e h a s been d e s c r i b e d and h a s a s e n s i t i v i t y l i m i t of 2 mcg. p e r m l . o f u r i n e t a k e n f o r

a n a l y s i s . 5’

192

DEXAMETHASONE

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20, 5 8 9

E D W A R D M. COHEN

46. F o r i s t , A. A. and J o h n s o n , J. L . i n " P h a r m a c e u t i c a l A n a l y s i s " , e d . H i g u c h i , T. and Brochmann-Hanssen, E r , I n t e r s c i e n c e , New York, 1961, pp. 71-85. 47. Hamlin, W. 4 9 , 253 ( 1 9 6 0 ) .

E.,

et &., J . &.

48. Brower, J . F . , J. (1969).

Ass.

_

L

-

60,

Super. S a n i t a , 12370h, 1962.

51. Noujaim, A . A. and J e f f e r y , D. A . ,

842

1539 (1971).

m.&.

58,

52,

O f f i c . Anal. Chem.,

&., 49. Woodson, A . L . , J. Pharm. S 50. Bellomonte, G . , (1962 ; Chem. A b s t . ,

g . Ed.,

Pharm. ASSOC.,

25, 581

Can. 2.

Pharm.

g.

5 , 26 (1970).

52. M o l l i c a , J . A. and S t r u s z , R. F . , 444 (1972). 53. Weber, D. J . , e t a l . ,

5 4 . Graham, R. E . , e t a l . ,

J. Pharm.

2.

Pharm.

g., 5,

s., 61, 689

(1972).

2. P h a r m. S c i . , 2, 1473

(1970)

55. Johnson, C. A . and Fowler, S . , J . Pharm. P h a r m a c o l . , 17T (1964).

1 6 , Supp.

56. N.F. X I I I , American Pharm. A S S O C . , Washington, 1970, pp. 208-209. 57. H o l l , A . , J . Pharm. Pharmacol.,

16,Supp.

9T ( 1 9 6 4 ) .

58. K i r s c h n e r , J. G . , "Thin-Layer Chromatography", s c i e n c e , New York, 1 9 6 7 , pp. 596-599.

Inter-

59. Moss, M. S . and R y l a n c e , H. J . , J. Pharm. P h a r m a c o l . , 18, 1 3 (1966). 60. Haque, N . ,

et al.,

2.

C l i n . Endocr.,

34, 44

(1972).

61. S c a v i n o , C . and C h i a r a m o n t i , D . , J. Pharm. B e l g . , 204 (1965).

20,

DEXAMETHASONE

6 2 . VandenHeuvel, W. J. A . i n "Theory a n d A p p l i c a t i o n s o f Gas C h r o m a t o g r a p h y in I n d u s t r y a n d M e d i c i n e " , e d . b y Kroman, H . S . a n d B e n d e r , S . R . , G r e e n e a n d S t r a t t o n , N e w York, 1 9 6 8 , p p . 120-134. 6 3 . B a i l l i e , T. A., e t a l . , A n a l . Chem., _

64. Jubiz, W.,

_

_

e t a l . , New E x . J.

_

I

30 ( 1 9 7 2 ) .

x., x,11 (1970).

6 5 . I l i d g l e y , A . K. a n d N i s w e n d e r , G . D . , S u p p l . , 1 4 7 . 320 ( 1 9 7 0 ) . 6 6 . Chayen, R., e t al.,

44, Acts

Endocrinol.

G. Biocheni., 39, 5 3 3

(1971).

This Page Intentionally Left Blank

DIOCTYL SODIUM SULFOSUCCINATE

Sut Ahuja and Jerold Cohen

Reviewed by J. Kazan and T. E. Ricketts

199

S. AHUJA AND J. COHEN

CONTENTS

7. 8.

Description 1.1 Name, Formula, Molecular Weight, Elemental Composition 1 . 2 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectrophotometry 2.2 Nuclear Magnetic Resonance 2.3 Mass Spectrometry 2 . 4 Differential Thermal Analysis 2.5 Thermogravimetric Analysis 2.6 Differential Scanning Calorimetry 2.7 Solubility 2.8 Solubilization 2.9 Effect On Surface Tension of Liquids 2.10 Crystal Properties Synthesis Stability Met abu 1ism Methods of Analysis 6 . 1 Titrimetric Analysis 6.2 Colorimetric Analysis 6 . 3 Infrared Analysis 6 . 4 Turbidimetric Analysis 6 . 5 Determination of Micellar Weight 6.6 Thin-Layer Chromatography References Acknowledgements

1.

DESCRIPTION

1.

2.

3. 4. 5.

6.

Name, Formula, Molecular Weight, Elemental Composition Chemically, dioctyl sodium sulfosuccinate is sulfosuccinic acid bis(2-ethylhexyl) ester S-sodium salt or sodium 1, 4-bis(2-ethylhexyl) sulfosuccinate o r bis(2-ethylhexyl) sodium sulfosuccinate. It is also known as sodium dioctyl sulfosuccinate; DSSj Aerosol OT; Alphasol OT; Colace; Complemix; Coprol; Dioctylal; Dioctyl-Medo Forte; Diotilan; Diovac; Disonate; Doxinate; Doxol; Dulsivac; Molatoc; Molofac; Nevax; Norval; Regutol; Softili; Solusol; Sulfimel DOS; Vatsol OT; Velmol and Waxsol (1). It has a molecular weight of 444.57 (C20H3707SNa). 1.1

200

DIOCTYL SODIUM SULFOSUCCINATE

0

// C2H5

CH2-C.

\

I

I

O-CH2-CH(CH2)3CH3

O-CH2 -CH (CH2) 3CH3

N a03S -CH-/

I

%

C2H5 0

The t h e o r e t i c a l e l e m e n t a l c o m p o s i t i o n of d i o c t y l s u l f o s u c c i n a t e is a s follows: Carbon 54.03% Hydrogen 8.39% Oxygen 25.19% Sulfur 7.21% Sodium 5. 17% 1.2

Appearance, C o l o r , Odor D i o c t y l sodium s u l f o s u c c i n a t e i s g r a y i s h t o w h i t e , w a x - l i k e , p l a s t i c s o l i d , h a v i n g a c h a r a c t e r i s t i c odor sugg e s t i v e of o c t y l a l c o h o l .

2.

PHYSICAL PROPERTIES

2.1

I n f r a r e d Spectrophotometry The i n f r a r e d s p e c t r u m ( 2 ) o f a 5% c h l o r o f o r m s o l u t i o n o f d i o c t y l sodium s u l f o s u c c i n a t e r e c o r d e d w i t h a P e r kin-Elmer I n f r a r e d S p e c t r o p h o t o m e t e r , Model 225 i s shown i n F i g u r e 1. Band a s s i g n m e n t s f o r t h e s p e c t r u m i n 5% c h l o roform s o l u t i o n a r e l i s t e d i n T a b l e I .

2.2

N u c l e a r M a g n e t i c Resonance The NMR s p e c t r u m ( 3 ) of d i o c t y l sodium s u l f o s u c c i n a t e ( F i g u r e 2 ) was r e c o r d e d i n DMSO-db on a v a r i a n A60 ~ O M H Z NMR s p e c t r o m e t e r . S t r u c t u r a l assignments f o r t h e NMR s i g n a l s a r e t a b u l a t e d i n T a b l e 11. 2.3

Mass S p e c t r o m e t r y R e l i a b l e mass s p e c t r a l d a t a ( 4 ) c o u l d n o t be obt a i n e d f o r d i o c t y l sodium s u l f o s u c c i n a t e a s t h e s p e c t r u m changes with temperature, i n d i c a t i n g thermal degradat i on.

20 1

S. AHUJA AND J. COHEN

202 rl

w

.PI

h

0

C

0

3

u

v)

I n f r a r e d Spectrum of a 5% chloroform s o l u t i o n of D i o c t y l Sodium S u l f o s u c c i n a t e 0

W

rl

H

FIGURE I.

. ..

FIGURE 2.

.. .

~~

..

Nuclear Magnetic Resonance Spectrum in DMSO-d6 of Dioctyl Sodium Sulfosuccinate

S. AHUJA AND J. COHEN

TABLE I INFRARED BAND ASSIGNMENTS FOR DIOCTYL SODIUM SULFOSUCCINATE I N 5% CHLOROFORM SOLUTION

W av enumbe r ( cm2960, 2935, 2865 1728 1465 1391, 1381 1245 1200 1048

A s s ignmen t a l i p h a t i c C H 2 ; CH3 C = O a l i p h a t i c C H 2 ; CH3

CH3 asymmetric C-0-C ( e s t e r ) asymmetric s o 3 8 symmetric C-0-C ( p o s i t i o n ) c o r r e s p o n d s t o C-O-CH2 a n d / o r symmetric S O ~ B

2.4

D i f f e r e n t i a l Thermal A n a l y s i s The t h e r m a l a n a l y s i s ( 5 ) of d i o c t y l sodium s u l f o s u c c i n a t e was c o n d u c t e d on a Du P o n t Model 900 t h e r m a l ana l y z e r i n a n i t r o g e n a t m o s p h e r e . A t h e a t i n g r a t e s of 1 0 ° C / m i n u t e and 3"C/minute, t h e thermogram ( F i g u r e 3) shows no endotherm, b u t an i r r e g u l a r c u r v e a f t e r 120°C. No t r a n s i t i o n was o h s e r v e d e x c e p t foaming of t h e sample. 2.5

Thermogravimetric A n a l y s i s A sample o f d i o c t y l sodium s u l f o s u c c i n a t e ( F i g u r e 4) l o s e s w e i g h t (5) c o n t i n u o u s l y above 35°C: 3.4% between 35°C and 152"C, a p p r o x i m a t e l y 0.5% between 152°C and 220°C. Rapid w e i g h t l o s s o c c u r s above 220"C, which amounts t o a p p r o x i m a t e l y 5 . 8 % loss up t o 248°C. A r e s i d u e o f a p p r o x i m a t e l y 15% i s l e f t a t 360°C. 2.6

D i f f e r e n t i a l Scanning Calorimetry A thermogram r u n a t lO"C/minute shows two broad endotherms ( 5 ) between 119°C and 194°C ( F i g u r e 5 ) .

2.7

Solubility D i o c t y l sodium s u l f o s u c c i n a t e i s s o l u b l e i n t h e f o l l o w i n g s o l v e n t s : carbon t e t r a c h l o r i d e , petroleum e t h e r , naphtha, xylene, d i b u t y l p h t h a l a t e , l i q u i d petrolatum, 204

DIOCTYL SODIUM SULFOSUCCINATE

F I G U R E 3.

Differential Thermal Analysis in nitrogen atmosphere of Dioctyl Sodium Sulfosuccinate

Smn

*"O

50

(00

110

1 *C

FIGURE 4.

-

wh

Z!O

2SO

lcomcmo ton CllnaMtL

300 UWlL

'

'

t '350

?nEI*ocouKcs~

400

- m 4W

Thermogravimetric Analysis of Dioctyl Sodium Sulfosuccinate 205

WO'

S. AHUJA A N D J. C O H E N

1. *C (CORRECTED FOR CHROMEL ALUMEL THERYOCOUPLESI

FIGURE 5.

Differential Scanning Calorimetry thermogram of Dioctyl Sodium Sulfosuccinate

206

DIOCTYL SODIUM SULFOSUCCINATE

TABLE I1 NMR SPECTRAL ASSIGNMENTS FOR DIOCTYL SODIUM SULFOSUCCINATE

(ppm)

Multiplicity

Number of Protons

A s s ignmen t

-H 00

c€3- c-’o 2.8-3.1

undetermined

2

I

S-CH-C:’ 0

3.35

3.6-4.1

singlet

undetermined

1.8

E20

5

S -CH -C- 0- CFl2

- lo

glycerol, pine o i l , o l e i c acid, acetone, kerosene, alcohol, benzene, d i a c e t o n e a l c o h o l , methanol, e t h a n o l , i s o p r o p a n o l , sec. b u t a n o l , methyl a c e t a t e , e t h y l a c e t a t e , amyl a l c o h o l , f u r f u r y l a l c o h o l , t e t r a h y d r o f u r f u r y l a l c oho 1, PO l y e t h y 1e n e g l y c o l , c o r n o i l , and v e g e t a b l e o i l s ( 1 ) . T a b l e 111 p r e s e n t s t h e s o l u b i l i t y o f d i o c t y l sodium s u l f o s u c c i n a t e i n s e l e c t e d s o l v e n t s . The s o l u b i l i t y f i g u r e s shown do n o t i n d i c a t e t h e l i m i t s of s o l u b i l i t y , s i n c e d i o c t y l sodium s u l f o s u c c i n a t e a p p e a r s t o be v e r y h i g h l y s o l u b l e i n most organic solvents (6).

207

TABLE I11

SOLUBILITY OF D I O C T Y L SODIUM SULFOSUCCINATE (DS S ) I N SOME ORGANIC SOLVENTS$:

Method of S o l u t i o n

Specific Gravity g/ml a t 25°C Dissolving Resulting Liquid Solution

Viscosity DSS i n S o l u t i o n Percent g/100 m l b y Weight

Of

Solution (Poises) !n

1 3

c

oc

Dissolved a t Room Temp. Heated t h e n Cooled

Carbon T e t r a c h l o r i d e Petroleum E t h e r S o l v e n t Naphtha Dibutyl Phthalate Paraffin O i l

1.55 0. 688 0.864 1.03 0.881

1.31 0.950 0.701 1.07 1.00

73.8 70.1 70.5 70.7 69.5

56.4 75.0 69.8 66.1 69.5

1.65 0.65 0.65 8.84 12.9

D

I

5

> > z 0

f0

s

rn 2

: ’ 7

R e p r i n t e d by t h e c o u r t e s y of American Cyanamid Co

DI O CT Y L SODIUM SULFOSUCCINATE

D i o c t y l sodium s u l f o s u c c i n a t e d i s s o l v e s s l o w l y i n c o l d water, b u t i t s s o l u b i l i t y i n w a t e r i n c r e a s e s w i t h an inc r e a s e i n t e m p e r a t u r e , as i n d i c a t e d i n T a b l e I V . T a b l e V p r e s e n t s t h e c o n c e n t r a t i o n of e l e c t r o l y t e s o l u t i o n i n w h i c h 1% of d i o c t y l s o d i u m s u l f o s u c c i n a t e i s s o l u b l e a t a t e m p e r a t u r e o f 25°C.

TABLE I V SOLUBILITY OF DSS I N WATER9c T e m p e r a t u r e "C

Grarns/100 r n l o f Water

25 30 40

1.5 1.8 2.3 3.0 4.0 5.5

50 60

70

R e p r i n t e d by t h e c o u r t e s y o f A m e r i c a n Cyanamid Co.

~-

~~

TABLE V SOLUBILITY OF 1% DSS I N ELECTROLYTE SOLUTIONS;': C o n c e n t r a t i o n of E l e c t r o l y t e s , % NaCl

NH4CL

0.5

0.5 2.0

3.0

:;'

,.

_LJ_ I~

(NH4)2 HP04

2 . 0<'; 3.0

:,

NaN03

N a 2 SO4

0.5 1.0

3.0

1.0

Ap p e a r a n c e of S o l u t i o n

Clear Turbid

R e p r i n t e d by t h e c o u r t e s y o f A m e r i c a n Cyanamid Co. S 1i g h t 1y t u r b i d

S. AHUJA AND J. COHEN

2.8

Solubilization Table V I presents the ( s o l v e n t ) n e c e s s a r y t o produce (w/w) i n t h e s o l v e n t and w a t e r v a l u e s r e p r e s e n t t h e amount of

amount of s o l u b i l i z i n g a g e n t 10% and 25% c l e a r s o l u t i o n s a t room t e m p e r a t u r e . The DSS and s o l v e n t t o b e added.

TABLE V I SOLVENTS CAPABLE OF SOLUBILIZING AQUEOUS SOLUTIONS OF DSS”

Solvent

S o l v e n t Required F o r 10% S o l u t i o n , % By Weight

Acetone 17.5 Amy1 a l c o h o l 5.9 (clear thick gel) Ethyl alcohol 15.0 2 - But ano 1 11.5 12.0 Bu t y 1 c a r b i t o 1$cgc Butyl c e l l u s o l v e ~ ~ ” 12.0 Diacetone alcohol 18.0 Diethylene glycol 15.0 Ethyl l a c t a t e 10.0 Furfuryl alcohol 7.4 Isopropyl alcohol 17.5 Methanol 12.5 Methyl a c e t a t e 15.0 Pine o i l Not s a t i s f a c t o r y Synosol”?; s o l v e n t 17.5 Tetrahydro f u r f u r y l 9.9 alcohol

JI.

4I .

S o l v e n t Required F o r 25% S o l u t i o n , % By Weight

12.0

-

15.0 15.0 15.0 12.0

15.0 13.0 15.0 5.0 15.0

R e p r i n t e d by t h e c o u r t e s y o f American Cyanamid Co. Trademark, C a r b i d e and Carbon Chemicals Company

D i o c t y l sodium s u l f o s u c c i n a t e c a n a c t a s a solub i l i z i n g a g e n t and c a n a l s o be a c t e d upon by o t h e r s o l u b i l i z i n g a g e n t s ( 7 ) . D i o c t y l sodium s u l f o s u c c i n a t e c a n b r i n g i n t o s o l u t i o n , i n water, m a t e r i a l s which a r e n o r m a l l y i n 210

DIOCTYL SODIUM SULFOSUCCINATE

soluble o r only sparingly soluble (e.g. organic acids, d y e s , p r o t e i n s , g e r m i c i d e s , gums, e t c . ) . Good s o l u b i l i z a t i o n c a n b e a c h i e v e d b y e n s u r i n g t h a t DSS i s u n i f o r m l y d i s t r i b u t e d throughout the bulk of p a r t i c l e s t o be dissolved, The p r o p o r t i o n o f DSS u s e d s h o u l d p r e f e r a b l y b e b e t w e e n 0.1-1.0% o f t h e m a t e r i a l t o b e d i s s o l v e d ( 6 ) . T he e x a c t However, i t mechani sm o f s o l u b i l i z a t i o n i s n o t known. apparently involves m i c e l l a r a c t i v i t y (8-11). E f f e c t On S u r f a c e T e n s i o n o f L i q u i d s DSS l o w e r s t h e s u r f a c e t e n s i o n o f l i q u i d s e . a- . w a t e r and v a r i o u s e l e c t r o l y t e s o l u t i o n s . T he e f f e c t o f DSS on s u r f a c e t e n s i o n may b e m e a s u r e d b y a v a r i e t y o f m e t h o d s ; h o w e v e r , t h e P e n d a n t Drop Method ( 1 2 ) seems t o b e most s a t i s f a c t o r y f o r m e a s u r i n g changes i n s u r f a c e t e n s i o n . The e f f e c t o f c o n c e n t r a t i o n o f DSS on s u r f a c e t e n s i o n a s d e t e r m i n e d by P e n d a n t Drop Method i s shown i n T a b l e V I I .

2.9

TABLE VII SURFACE TENSION OF DIOCTYL SODIUM SULFOSUCCINATE E Y PENDANT DROP METHOD TEMPERATURE, 2 5 ° C . SURFACE AGE, 5 SECONDS"

Concent r a t i o n DSS % S o l i d s

0 0.001 0.02 0.1 0.25 0.5 1.0 2.0

;'<

Surface Tension (dynes/cm.) 0.25% 0.5% 1.0% 2.0% Water NaCL NaCl Na2S04 Na2SOq

72.0 62.8 38.9 28.7 28.5 27.5 26.0 C 1 oudy

52.4 26.3 24.9 24.3 25.3 Cloudy Cloudy

40.1 25.3 24.8 25.2 25.5 Cloudy Cloudy

72.5 42.0 25.9 24.6 25.6 25.2 Cloudy Cloudy

72.8

41.5 26.0 25.2 25.4 25.2 Cloudy Cloudy

R e p r i n t e d b y t h e c o u r t e s y of A m e r i c a n Cyanamid Co

21 1

S . AHUJA A N D J.

COHEN

2.10

Crystal Properties X - r a y d i f f r a c t i o n a n a l y s i s ( 5 ) of d i o c t y l s o d i u m s u l f o s u c c i n a t e was c o n d u c t e d on a P h i l i p s N o r e l c o D i f f r a c t ometer U n i t ( s c i n t i l l a t i o n c o u n t e r ) , u s i n g t h e f o l l o w i n g conditions: Radiation: Cu Kol, 35 KV. 15 ma Nickel Filter: 1/2" (20) /minute Scanning Rate: 1/2 inch/minute C h a r t Speed: 1" Divergence S l i t : 0.006 inches Receiving S l i t : lo Scatter Slit: 2 seconds Time C o n s t a n t : C o u n t s , F u l l S c a l e : 1000 c p s The r e s u l t s of X-ray d i f f r a c t i o n a n a l y s i s a r e i n Table V I I I (Figure 6 ) .

TABLE V I I I X- RAY DIFFRACTION ANALYSIS

3.

Bragg A n g l e s ( d e g r e e 2 Q)

Re l a t i v e Intensity

4.29

100

SYNTHESIS

Several p a t e n t s have been issued covering t h e p r e p a r a t i o n o f DSS (13, 1 4 ) . The s y n t h e s i s of d i o c t y l s o d ium s u l f o s u c c i n a t e i s g e n e r a l l y a c c o m p l i s h e d by t h e t r e a t ethylhexanol t o proment of m a l e i c a c i d a n h y d r i d e w i t h 2 d u c e d i o c t y l m a l e a t e which i s t h e n r e a c t e d w i t h s o d i u m b i s u l f i t e u n d e r c o n d i t i o n s c o n d u c i v e t o s a t u r a t i o n of t h e o l e f i n i c bond, w i t h s i m u l t a n e o u s r e a r r a n g e m e n t o f t h e b i s u l f i t e t o t h e s u l f o n a t e s t r u c t u r e (15).

-

212

DIOCTYL SODIUM SULFOSUCCINATE

213 F I G U R E 6.

X-Ray Diffraction Analysis of Dioctyl Sodium Sulfosuccinate

S A H U J A A N D J COHEN

CH-CO

'o I

.

CHCOOC8H 1 7

2-ethylhexanol (esterificationr CHCOOC8H 1 7

CH-CO

Dioctyl ma l e a t e

Maleic a c i d anhydride

NaHS03 h ' (Additions & Rearrangement) CH-COOC8H 17 I

S03Na D i o c t y l sodium s u 1f o s u c c i n a t e Calcium, p o t a s s i u m , magnesium, aluminum h y d r o x i d e , and ammonium s a l t s of d i o c t y l s u l f o s u c c i n a t e h a v e been prep a r e d (16-20) b u t h a v e n o t found a p p l i c a t i o n s c o m p a r a b l e t o DSS.

4.

STABILITY D i o c t y l sodium s u l f o s u c c i n a t e i s a f a i r l y s t a b l e compound; a f t e r 12 weeks o f s t o r a g e a t 60°C, n o decomposiSince dioctyl sulfosuccint i o n was o b s e r v e d by TLC ( 2 1 ) . a t e i s an ester, i t can be hydrolyzed i n t h e presence of e i t h e r s t r o n g l y a c i d i c o r s t r o n g l y a l k a l i n e media. Extens i v e i n f o r m a t i o n on t h e s t a b i l i t y of DSS a s a w e t t i n g a g e n t i s a v a i l a b l e ( 6 ) . F i g u r e 7 shows t h e s t a b i l i t y o f DSS a t v a r i o u s pH v a l u e s . The s t a b i l i t y was measured by Draves T e s t which m e a s u r e s t h e w e t t i n g power i . e . r a t e of s p r e a d i n g and i m b i b i t i o n . DSS may b e used t o improve t h e p h y s i c a l s t a b i l i t y of o t h e r m a t e r i a l s s u c h a s : a) a s t a b i l i z e r f o r cocoa b u t t e r i n beverages (22) b ) i m p r o v i n g d y e u p t a k e and t h e r m a l s t a b i l i t y of p o l y a c r y l o n i t r i l e y a r n (23) c ) s t a b i l i z a t i o n and c o a g u l a t i o n o f Ag B r s o l s ( 2 4 ) d ) i m p r o v i n g t h e s t a b i l i t y of p o l y ( v i n y l a c e t a t e ) e m u l s i o n s t o f r e e z i n g and thawing(25) 214

DIOCTYL SODIUM SULFOSUCCINATE

Stable

1I

2I

3I

4I

5

i L 3

pH of Bath at 86'F.

Figure 7 .

Stability of Solutions of Dioctyl Sodium Sulfosuccinate (0.025% in solution at various pH values)"

"Reprinted by the courtesy of American Cyanamid Co.

215

S. AHUJA AND J. COHEN

5.

METABOLISM

I n human b e i n g s , d i o c t y l sodium s u l f o s u c c i n a t e i s n o t a b s o r b e d o r a l t e r e d i n t h e i n t e s t i n a l t r a c t ( 6 ) . Exp e r i m e n t s w i t h mice a p p e a r t o c o n f i r m t h i s . F u r t h e r m o r e , DSS d o e s n o t a i d i n t h e a b s o r p t i o n o f o t h e r m a t e r i a l s i n s o l u t i o n such a s i n s u l i n , h i s t a m i n e , d i a m i n o - d i p h e n y l s u l However, i t enhances t h e p e n e t r a t i o n o f s u l f a fone (26). n i l a m i d e , s u l f a t h i a z o l e , s u l f a d i a z i n e and s u l f a p y r i d i n e i n t o t h e c o r n e a , s c l e r a and aqueous humor o f human b e i n g s (27).

6.

METHODS OF ANALYSIS

Titrimetric Analysis D i o c t y l sodium s u l f o s u c c i n a t e c a n b e a s s a y e d by t i t r a t i o n a g a i n s t s t a n d a r d tebrabutylammonium i o d i d e w i t h bromphenol b l u e a s a n i n d i c a t o r , i n t h e p r e s e n c e o f c h l o roform ( 2 8 ) . DSS c a n a l s o be d e t e r m i n e d by t i t r a t i o n a g a i n s t c e t y l a l k o n i u m c h l o r i d e s o l u t i o n w i t h bromophenol b l u e as an i n d i c a t o r , i n t h e p r e s e n c e o f c h l o r o f o r m ( 2 9 ) . 6.1

6.2

Colorimetric Analysis DSS forms a complex w i t h m e t h y l g r e e n which i s s o l u b l e i n benzene and c a n be d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t 615 nm ( 6 ) . Another method of d e t e r m i n a t i o n of DSS i s b a s e d on f o r m a t i o n of t h e d i o c t y l s u l f o s u c c i n a t e s a l t of methyl e n e b l u e , e x t r a c t i o n of t h e s a l t i n t o c h l o r o f o r m , and s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n of t h e e x t r a c t e d s a l t a t 650 nm ( 3 0 ) . D e t e r m i n a t i o n of DSS by r e a c t i o n w i t h b a s i c f u c h s i n t o form a c h l o r o f o r m s o l u b l e complex, which i s measured on a c o l o r i m e t e r h a s been r e p o r t e d ( 3 1 ) . 6.3

Infrared Analysis An I R method of a n a l y s i s f o r DSS h a s been p r o posed ( 3 2 ) . T h i s method i n v o l v e s p r e c i p i t a t i o n of DSS a s i t s barium s a l t w i t h barium c h l o r i d e , f o l l o w e d by a n a l y s i s of i t s I R band a t 9 . 5 v.

6.4

Turbidimetric Analysis

A t u r b i d i m e t r i c method of a n a l y s i s based on a d d i t i o n of sodium c h l o r i d e s o l u t i o n t o a DSS s o l u t i o n w i t h 216

DlO CT Y L SODIUM SU LFOSUCCI NATE

m e a s u r e m e n t o f s u b s e q u e n t t u r b i d i t y a t 320 nm h a s b e e n reported (33). 6.5

D e t e r m i n a t i o n o f M i c e l l a r W e ight D e t e r m i n a t i o n o f m i c e l l a r w e i g h t s f o r DSS i n a nhyd r o u s and h y d r o u s h y d r o c a r b o n s o l u t i o n s h a s b e e n made b y means o f l i g h t s c a t t e r i n g ( 3 4 ) . 6.6

T h i n - l a y e r Chromatography DSS may b e c h r o m a t o g r a p h e d o n a S i l i c a G e l G p l a t e w i t h e t h y l acetate/ammonium h y d r o x i d e / e t h a n o l (50/20/20) s o l v e n t s y s t e m . D e t e c t i o n i n v o l v e s s p r a y i n g w i t h 50% s u l f u r i c a c i d f o l l o w e d by h e a t i n g t h e p l a t e a t 120°C f o r 1 hour (35). 7.

REFERENCES

1. 2.

3.

4. 5. 6. 7. 8. 9.

10.

11.

"Merck I n d e x " , 8 t h E d . , Merck & Co. , I n c . , Rahway, New J e r s e y , 1968, p. 382-383. E. G r e g o r i a n , C i b a - G e i g y C o r p o r a t i o n , A r d s l e y , N e w York, p e r s o n a l c o m m u n i c a t i o n , 1970. R. G r u l i c h , C i b a - G e i g y C o r p o r a t i o n , A r d s l e y , N e w York, p e r s o n a l c o m m u n i c a t i o n , 1 9 7 0 . J . K a r l i n e r , C i b a - G e i g y C o r p o r a t i o n , A r d s l e y , New York, p e r s o n a l c o m m u n i c a t i o n , 1970. A . P a g e , C i b a - G e i g y C o r p o r a t i o n , A r d s l e y , N e w York, p e r s o n a l c o m m u n i c a t i o n , 1970 . DSS-NF, D i o c t y l Sodium S u l f o s u c c i n a t e NF, A m e r i c a n Cyanomid Company, P e a r l R i v e r , New York, 1967 J . H . Van Ness, U.S. P a t . 3 , 1 5 1 , 9 8 6 ( M o n s a n t o C o . ) 1964. A . K i t a h a r a , T . K o b a y s h i , and T . T o r c h i b a n a , J . P h y s . Chem., 66, 363, 1 9 6 2 . A . M . S c h w a r t z , J . W . P e r r y , a n d J . Be rc h, S u r f a c e A c t i v e A g e n t s and D e t e r g e n t s , l i s t e d , I n t e r s c i e n c e P u b l i s h e r s , I n c . , N e w York, N e w York, 1958. K. S h i n o d a , T . Nakagawa, B. Tamamushi, a nd T. Isemura, C o l l o i d a l S u r f a c t a n t s . Some P h y s i c s c h e m i c a l P r o p e r t i e s , 1 s t Ed., A c a de m ic P r e s s , N e w York, N e w York, 1963. A . G . Ward, C o l l o i d s , T h e i r P r o p e r t i e s a nd A p p l i c a t i o n s , 1st Ed., I n t e r s c i e n c e P u b l i s h e r s , I n c . , New York, New York, 1 9 4 6 .

S. AHUJA AND J. COHEN

12. 13. 14. 15. 16.

17.

18. 19. 20.

21. 22. 23.

24. 25. 26.

27. 28. 29.

30. 31. 32.

33.

R. C a r y l , and W. P. E r i c , I n d . Eng. Chem., 2, 44, 1939. A . 0. J a e g e r , U . S . P a t . 2,176,423 t o American Cyanamid Co., 1939. A . 0. J a e g e r , U . S . P a t . 2,028,091 t o American Cyanamid Co., 1936. "Remington's Pharm S c i " , 1 4 t h Ed., Mack P u b l i s h i n g Company, E a s t o n , P e n n s y l v a n i a , p. 805, 1970. L . J . K l o t z , U. S . P a t . 3,035,973 (Lloyd B r o s . , I n c ) 1962. E . A V i t a l i s , U . S . P a t . 2,441,341 (American Cyanamid C o . ) , 1948. E . F. W i l l i a m s , and N . T . Woodberry, U . S . P a t . 3,002,995 (American Cyanamid C o . ) , 1961. P.M. L i s h , H . D . Bryan, and N . H . Mercer, U . S . P a t . 3,155,576 (Mead Johnson & Co.), 1964. N.H. Mercer, and H.D Bryan, U . S . P a t . 3,155,577 (Mead J o h n s o n & C o . ) , 1964. S . Ahuja, Ciba-Geigy C o r p . , A r d s l e y , New York, unpub 1i s h e d i n f o r m a t i o n , 19 70. E . B . H o t e l l i n g , F r . P a t . 1,543,204, 1968. Lonza, Ltd. (by C a m i l l e Nordmann, P a u l H a l b i g , and Roland Dagon), Swiss P a t . 396, 303, 1966. S. M . L e v i and T . K . Stepanova, K o l l o i d n Zh. 27 ( l ) , 57-63, 1965. K u n s t h a r s f a b r i e k S y n t h e s e N . V V e r f k r o n i c k 29, 108, 1956. Summary of T o x i c i t y S t u d i e s , 1963, American Cyanamid Co., t h r o u g h r e f e r e n c e 6. J. G . Bellow, and M. Gutmann, Arch. O p h t h a l . 30, 352, 1943. " N a t i o n a l Formulary", 1 2 t h Ed., Mack P u b l i s h i n g Co. E a s t o n , Penna. p. 138-139, 1965. "The U n i t e d S t a t e s Pharmacopeia", 1 8 t h Ed., Mack P u b l i s h i n g Co., Easton, P e n n a . , p . 206, 1970. J . D . F a i r i n g and F . R . S h o r t , Anal. Chem., 28, 1 1 8 2 7 , 1956. G . R. W a l l i n , Anal. Chem., 2 2 , 616, 1950. S. Ahuja, D. S p i e g e l and S . W i l d s t e i n , Ciba-Geigy C o r p o r a t i o n , A r d s l e y , N e w York, u n p u b l i s h e d i n f o r m a t i o n , 1970. G. L i n a r i ( 1 s t . Farm. B i o l . S t r o d e r , F l o r e n c e ) , B o l l . Chim. F a r . , 106, 307, 1967. C.

DIOCTYL SODIUM SULFOSUCCINATE

34. 35.

8.

S . G. F r a n k , and Z o g r a f i , J . Pharm. S c i . , 58, 993, 1969. S. Ahuja, Ciba-Geigy C o r p o r a t i o n , A r d s l e y , N e w York u n p u b l i s h e d i n f o r m a t i o n , 1970.

ACKNOWLEDGEMENTS The a u t h o r s would l i k e t o thank M r . C. B . Johnson, D r . J . Kazan and M r . T. E . R i c k e t t s of American Cyanamid Co. f o r t h e i r c o o p e r a t i o n i n r e v i e w i n g t h i s m a n u s c r i p t and Mrs. M. Kahn f o r s e c r e t a r i a l help.

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FLUOROURACIL

Brircp C. Rudy uiid Bcrnurd Z. Seiikowski

22 I

B. C. RUDY AND B. 2. SENKOWSKI

INDEX

Analytical P r o f i l e - Fluorouracil 1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2 . 2 1 Proton Spectrum 2 . 2 2 1 9 F Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 Thermogravimetric A n a l y s i s 2.10 S o l u b i l i t y 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2 . 1 2 D i s s o c i a t i o n Constant

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug Metabolic Products

6.

Methods of A n a l y s i s 6.1 Elemental A n a l y s i s 6.2 Phase S o l u b i l i t y A n a l y s i s 6.3 Thin Layer Chromatographic A n a l y s i s 6.4 Gas Liquid Chromatographic Analysis 6.5 D i r e c t S p e c t r o p h o t o m e t r i c Analysis 6.6 F l u o r i n e Analysis 6 . 6 1 O r g a n i c a l l y Bound F l u o r i n e A n a l y s i s 6.62 Free FLuori.de A n a l y s i s 6.7 Volumetric A n a l y s i s

7.

References

222

FLUOROURACI L

1.

Description 1.1

Name, Formula, Molecular Weight Fluorouracil i s 5-fluorouracil.

Molecular Weight:

C4H3FN202

130.08

Appearance, Color, Odor F l u o r o u r a c i l i s a white t o p r a c t i c a l l y w h i t e , p r a c t i c a l l y o d o r l e s s , c r y s t a l l i n e powder. 1.2

2.

Physical Prouerties 2.1

I n f r a r e d Spectrum (IR) The I R spectrum of r e f e r e n c e s t a n d a r d f l u o r o u r a c i l * i s shown i n Figure 1 ( 1 ) . The spectrum o f a mineral o i l suspension of f l u o r o u r a c i l between cesium i o d i d e d i s c s was measured on a P e r k i n Elmer 621 S p e c t r o photometer. The assignments given i n Table I t o t h e bands i n Figure 1 (1) a r e i n good agreement w i t h t h e assignments made by Brownlie ( 2 ) . TABLE I

IR S p e c t r a l Assignments f o r F l u o r o u r a c i l

Characteristic of

Frequency (cm-l)

NH s t r e t c h C=O s t r e t c h CH i n p l a n e deformation CH o u t o f p l a n e deformation

3124 1716 and 1657 1245 813

*The r e f e r e n c e s t a n d a r d f l u o r o u r a c i l r e f e r r e d t o i n t h i s work i s No. 354029. 323

6 . C. R U D Y A N D 6 . 2. SENKOWSKI

224

-8 0

-2 c5

6

T

@

w

t, z

-$!-

-E

-E 0

-8

FLUOROURACI L

2.2

N u c l e a r Magnetic Resonance Spectrum (NMR)

P r o t o n Spectrum The NMR s p e c t r u m shown i n F i g u r e 2 was o b t a i n e d by d i s s o l v i n g 5 6 . 3 mg o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l i n 0 . 5 m l o f d i m e t h y l s u l f o x i d e - dg c o n t a i n i n g t e t r a m e t h y l s i l a n e a s t h e i n t e r n a l r e f e r e n c e ( 3 ) , The s p e c t r a l a s s i g n m e n t s a r e shown i n T a b l e I1 ( 3 ) . 2.21

TABLE I 1 NMR S p e c t r a l Assignments f o r F l u o r o u r a c i l

Type P r o t o n

No. o f Each Proton

Chemical S h i f t (PPm)

C-H

1

7.80

N-H

2

11.40

Multiplicity d , ( J H - F = 6 .5 Hz)

s (b)

d= d o u b l e t ; S ( b ) = b r o a d s i n g l e t ; J = s p l i t t i n g c o n s t a n t i n Hz I 9 F Spectrum The 1YF s p e c t r u m shown i n F i g u r e 2 was o b t a i n e d w i t h a J e o l c o C-60 H L i n s t r u m e n t w i t h I 9 F module c r y s t a l m o d i f i e d t o a f r e q u e n c y o f 5 6 . 4 4 6 MHz. S e v e n t y mg o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l were d i s s o l v e d i n 0 . 5 m l o f d i m e t h y l a c e t a m i d e c o n t a i n i n g CC13F a s t h e i n t e r n a l r e f e r e n c e ( 3 ) . The s p e c t r u m c o n s i s t s o f a d o u b l e t a t -183 ppm which h a s a s p l i t t i n g c o n s t a n t ( J F - ) o f 6 . 5 Hz. The c h o i c e of CC13F as t h e i n t e r n a l r e y e r e n c e a l o n g w i t h t h e assignment o f -183 ppm i s i n a c c o r d a n c e w i t h Bovey ( 4 ) . 2.22

2.3

U l t r a v i o l e t Spectrum (UV) The UV s p e c t r u m o f f l u o r o u r a c i l i n a c e t a t e b u f f e r (pH 4 . 7 ) i n - t h e r e g i o n 350 t o 220 nm e x h i b i t s one maximum a t 266 nm ( E = 7 . 0 7 x l o 4 ) and one minimum a t 232 nm. The s p e c t r u m p r e s e n t e d i n F i g u r e 3 was o b t a i n e d u s i n g a s o l u t i o n o f 1 . 0 0 4 mg f l u o r o u r a c i l p e r 100 m l o f pH 4 . 7 a c e t a t e b u f f e r ( 5 ) .

225

Figure 2

m 0 n C 0

,

,

<

JL,, , , ,

0

-I

-181

-180

-162

-183

-w

-185

l ~ . i ' ~ " ~ l ' ' " ' " ' " ' " ' " ' " ' " ' " ' " ' " ' " ' " ' I ' ' I

m Tu

pgm I

I " I ' ' I '

I " 1 " I " I " I '

F L U O R O U R A C IL

Figure 3 Ultraviolet Spectrum o f F l u o r o u r a c i l

NANOMETERS

227

B. C. RUDY AND B. 2.SENKOWSKI

2.4

Fluorescence Spectrum Figure 4 shows t h e e x c i t a t i o n and emission s p e c t r a f o r r e f e r e n c e s t a n d a r d f l u o r o u r a c i l from 250 t o 600 nm ( 6 ) . The s p e c t r a , measured i n a methanol s o l u t i o n o f f l u o r o u r a c i l (1.0 mg/ml) u s i n g a Farrand MK-1 S p e c t r o f l u o r o m e t e r , showed one e x c i t a t i o n peak a t 315 nm and one emission peak a t 391 nm. Mass Spectrum The mass spectrum o f f l u o r o u r a c i l shown i n Figure 5 was o b t a i n e h u s i n g a CEC-21-110 mass s p e c t r o meter w i t h an i o n i z i n g energy o f 70 eV ( 7 ) . 2.5

The spectrum shows a s t r o n g molecular i o n peak a t m/e 130. The peak a t m/e 9 7 i s due t o t h e l o s s o f HNCO from t h e p a r e n t and t h e fragment a t m/e 60 i s formed by a hydrogen rearrangement g i v i n g h a l f t h e r i n g w i t h t h e f l u o r i n e s u b s t i t u e n t (C2H3FN) ( 7 ) . 2.6

Optical Rotation F l u o r o u r a c i l e x h i b i t s no o p t i c a l a c t i v i t y

2.7

Melting Range The m e l t i n g range f o r f l u o r o u r a c i l depends on t h e r a t e o f h e a t i n g . When t h e USP XVIII Class I procedure (8) i s u s e d , t h e m e l t i n g p o i n t l i e s between 280" and 284°C. 2.8

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC c u r v e shown i n Figure 6 f o r r e f e r e n c e s t a n d a r d f l u o r o u r a c i l was o b t a i n e d ;sing a P e r k i n E l m e r DSC - 1 B C a l o r i m e t e r . With a t e m p e r a t u r e program o f 1O0C/min., a m e l t i n g endotherm was observed s t a r t i n g a t 288.2OC w i t h a AHf = 6 . 7 Kcal/mole. The exotherm s t a r t i n g a t 298.5OC corresponds t o t h e decomposition o f the fluorouracil ( 9 ) . 2.9

Thermogravimetric A n a l y s i s (TGA) The TGA performed on r e f e r e n c e s t a n d a r d f l u o r o u r a c i l e x h i b i t e d no l o s s o f weight when h e a t e d from room t e m p e r a t u r e t o 1 0 5 O C a t a h e a t i n g r a t e o f lO"C/min. (9)

228

U a,

a ( d k k 3 c , M .rl

u . a a,

(I)

U

c a U m a k 0 3 4

LL

AlISN31NI

FLUOROURACIL

22Y

6.C. RUDY AND 6.2. SENKOWSKI

230

FLUOROURACI L

Figure 6

D.S . C . Curve €or F 1 uorouraci 1

I

I

I

1

I

1

I

AHf = 6.7kcal/mole

I 250

I 270

290 TEMPERATURE

23 1

310 "C

1

0.

C. RUDY AND 0 . 2. SENKOWSKI

2.10

Solubility The s o l u b i l i t y d a t a o b t a i n e d a t 25°C f o r r e f e r ence s t a n d a r d f l u o r o u r a c i l i s l i s t e d i n T a b l e 111 ( 1 0 ) . TABLE 111 Fluorouracil S o l u b i l i t y Solvent

S o l u b i l i t y (mg/ml)

3A a l c o h o l benzene chloroform 95% e t h a n o l ethyl ether isopropyl alcohol met hano 1 petroleum e t h e r (30" .60°) water

3.44 <. 1 c.1

5.54
Crystal Properties The x - r a y powder d i f f r a c t i o n p a t t e r n o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l i s p r e s e n t e d i n Table I V ( 1 1 ) . The i n s t r u m e n t a l c o n d i t i o n s a r e given below: 2.11

Instrumental Conditions: General E l e c t r i c Model XRD-6 Spectrogoniometer Generator: Tube T a r g e t : Radiation : Optics :

Goniometer: Detector:

50 KV 1 2 . 5 MA Copper 0 Cu Ka = 1.542 A 0.1" D e t e c t o r s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007" N i f i l t e r 4" t a k e o f f a n g l e Scan a t 0.2" 20 p e r minute A m p l i f i e r g a i n - 16 c o a r s e , 8.7 f i n e , Sealed proportional c o u n t e r t u b e and DC v o l t a g e a t p l a t e a u , Pulse height sel e c t i o n EL-5 v o l t s ; E u - o u t Rate meter T . C . 4 , 2000 C/S full scale 232

FLUOROURACI L

Recorder: Samples :

C h a r t s p e e d 1" p e r 5 m i n u t e s P r e p a r e d by g r i n d i n g a t room t emp e r a t u r e

TABLE IV X-ray D i f f r a c t i o n P a t t e r n o f F l u o r o u r a c i l 0

20 -

dA*

I/Io**

12.84 13.32 15.78 16.10 17.72 18.90 19.62 20.38 21.72 22.30 22.72 23.60 24.64 25.28 26.70 27.84 28.46 30.98 31.80 32.72 33.88 34.84 36.20 37.01 37.54 38.72 39.14 42.88 44.08 45.54 46.52 47.26 50.06 50.78

6.89 6.65 5.62 5.50 5.01 4.70 4.52 4.36 4.09 3.99 3.91 3.77 3.61 3.52 3.34 3.20 3.14 2.89 2.81 2.74 2.65 2.58 2.48 2.43 2.40 2.33 2.30 2.11 2.05 1.99 1.95 1.92 1.82 1.80

2 1 4 4 2 4 2 5 10 10 4 5 5 9 4 28 100 17 8 4 <1 2 2 2 <1 <1 <1 <1 <1 <1 <1 1 1 1

233

6 . C. R U D Y AND 6 . 2. SENKOWSKI

53.56 1.71 54.28 1.69 *d = ( i n t e r p l a n a r d i s t a n c e )

**I/I,

nX 2 Sin 0

<1 2

= r e l a t i v e i n t e n s i t y (based on h i g h e s t i n t e n s i t y

o f 1* 00) D i s s o c i a t i o n Constant The pKa's f o r f l u o r o u r a c i l have been determined s p e c t r o p h o t o m e t r i c a l l y t o be 8 . 0 + 0 . 1 and 13.0 + 0 . 1 ( 1 2 ) . When t h e pKa's f o r u r a c i l were defkrmined i n a s T m i l a r manner, t h e y were found t o be 9 . 4 + 0 . 1 and > 1 3 . 5 . These l a t t e r v a l u e s a r e i n good a g r e e m e n t w i t h t h e pKa's r e p o r t e d by Shugar and Fox f o r u r a c i l (13). 2.12

Synthesis F l u o r o u r a c i l may be p r e p a r e d by t h e r e a c t i o n scheme shown i n F i g u r e 7. E t h y l f l u o r o a c e t a t e i s condensed i n a C l a i s e n c o n d e n s a t i o n w i t h methyl f o r m a t e . The e t h y l sodium fluormalonaldehyde formed i s condensed w i t h 2 - e t h y l - 2 t h i o p s e u d o u r e a hydrobromide t o g i v e 2 - e t h y l m e r c a p t o - 5 f l u o r - 4 (3H) -pyrimidinone which y i e l d s f l u o r o u r a c i l on hydrolysis (14). 3.

4.

S t a b i l i t y Degradation F l u o r o u r a c i l i s s t a b l e i n s o l u t i o n s which a r e n o t s t r o n g l y b a s i c (pH l e s s t h a n 9 ) . When s u b j e c t e d t o s t r o n g l y b a s i c c o n d i t i o n s , t h e f l u o r o u r a c i l i s hydrolyzed t o u r e a , f l u o r i d e , and an aldehyde. This h y d r o l y s i s i s enhanced by i n c r e a s e d pH and t e m p e r a t u r e . Some o f t h e u r e a formed on h y d r o l y s i s r e a c t s f u r t h e r g i v i n g ammonia and C02 (15).

5.

Metabolic Products The m e t a b o l i c pathway o f f l u o r o u r a c i l i s p r e s e n t e d s c h e m a t i c a l l y i n F i g u r e 8. In man, t h e major biochemical e f f e c t o f f l u o r o u r a c i l i s t h e i n h i b i t i o n o f DNA s y n t h e s i s , s i n c e c o n c e n t r a t i o n s which i n h i b i t DNA s y n t h e s i s may s t i l l p e r m i t RNA s y n t h e s i s . F l u o r o u r a c i l i s c o n v e r t e d t o f l u o r o u r i d i n e and t h e n t o t h e mono-,di-, and t r i p h o s p h a t e s o f fluorouridine. This is then incorporated i n t o t h e f r a u d u l a n t FNA. F l u o r o u r i d i n e monophosphate i s a l s o r e duced t o f l u o r o - 2 ' - d e o x y u r i d i n e monophosphate. There i s no f u r t h e r metabolism t o t h e d i - and t r i p h o s p h a t e n u c l e o t i d e s 234

FLUOROURACIL

Figure 7

SYNTHESIS O F PLUOPOUPACIL

0

P O

0

pCHZ-E-O-C2H5

ethyl fluororcatate (1)

+

CH N30H r >

HE-o-cH~

methyl forrrte (11)

ethyl-rodiofluormaloarldehydr (111)

NH I 1 1 + H5C2-S-C’

*HBr

N ~ O - C H -I C - E - O - C ~ H ~

H-N

NI

CH30H+

‘NH

R 2-ethyl-2thiopreudourer hydrobromide (1V)

2-ethylmcrcrpto5-fluoro-4(3H)pyrlmidinoar (V)

hydrolyze,

i ’ I l

‘c-P

H-N

l

o - C \ N / C -IH

H fluorourrcil (VI)

235

Figure 8

M T A l O L I C PATHWATS

O?

?LUOlOUlACIL ( 1 6 )

a~?LuOlo-~-ALANIIIL+ U l L A + C 0 2

T f

Fo

~ - ? L U O l O - ~ - U l K I D O I l O I I O N l CACID t (I-?LUOIO-6-CUAIIIDOIlOIlONIC

t

C

0

<

5-?LUOlOUlIDI~E T l I I H O S I H A T l

DIUTDlO?LUOlOUlACIL

5-?LUOlOUlACIL

0 n

ACID

5-?LIIOlOUlIDIIIK

T

2

S-?LUOlO-Z'-DKOITUlIDINK IOHOIHOSIHAIK

t

-+

5-ILUOIOUlIDIIIL M O l l O I Y O S I H A T ~

I

D

H!!

IITIIIDTLIC ACID

DKOITUlIDlLIC ACID

T

UIIDTLIC ACID

5-FLUOlOUlIDIHE

5-FLUO102'-ULOXIUlIDIIIX

0

FLUOROURACI L

o f fluoro-2 -deoxyuridine. ‘Therefore t h e formation o f t h e f l u o r o - 2 ‘ - d e o x y u r i d i n e monophosphate i s c o n s i d e r e d t o b e the basis for the antineoplastic action of fluorouracil s i n c e i t i n h i b i t s DNA s y n t h e s i s by b l o c k i n g t h e enzyme t h y m i d y l a t e s y n t h e t a s e . I t i s t h i s enzyme t h a t c a t a l y z e s t h e methylation o f deoxyuridylic a c i d t o thymidylic a c i d (16). F l u o r o u r a c i l i s c a t a b o l i z e d i n an a n a l o g o u s manner t o u r a c i 1, f o r m i n g t h e f o l lowing d e g r a d a t i v e p r o d u c t s : d i h y d r o f 1u o r o u r a c i 1 , u - f luoro - 6 - u r e i d o p r o p i o n i c a c i d , a - f l u o r o -a - g u a n i d o p r o p i o r i i c a c i d , iy - f 1uoro-d -a1 a n i i i e , u r e a , and CO2 ( 1 6 ) . 6.

Methods o f A n a l y s i s

E l ement a1 A n a l y s i s The r e s u l t s from an e l e m e n t a l a n a l y s i s o f re f e r en c e s t a n d a r d f l u o r o u r a c i l i s p r e s e n t e d i n ‘I‘ahle V (17) * 6.1

’I’AULE

v

E 1ement a1 Aria 1y s i s o f 1-1u o r o u r a c i 1 k 1ement C 11

F

% Theory 3 b . 93 2.32 13.61

””

Found 36.94 2.50 14.57

P h a s e So 1ub i 1i t y . h a 1y s i 5 Phase s o l u b i l i t v a n a l y s i s has been c a r r i e d o u t f o r f l u o r o u r a c i l u s i n g methanol a s t h e s o l v e n t . An example i s presented i n Figure 9 f o r a t),pical l o t o f f l u o r o u r a c i l a l o n g w i t h t h e c o n d i t i o n s u n d e r which t h e a n a l y s i s was carried out (10). 6.2

T h i n Layer C h r o m a t o g r a p h i c A n a l y s i s (T’LC) The ‘I’LC o f f l u o r o u r a c i l a n d o t h e r f l u o r o p y r i m i d i n e s h a s been s t u d i e d a t l e n g t h by Mawrylyshyn, Senkowski, and W o l l i s h ( 1 8 ) . ‘The ltf v a l u e s p r e s e n t e d i n ‘I’ablc V I were o b t a i n e d i n v a r i o u s d e v e l o p i n g s o l v e n t s when 10 pgm o f f l u o r o u r a c i l was s p o t t e d on n s i l i c a g e l GI: 6.3

231

B. C. RUDY A N D 6 . 2 SENKOWSKI

238

FLUOROURACI L

plate. After development for at least 10 cm the plates were air dried and viewed under short wave ultraviolet radiation. TABLE VI Rf Values for Fluorouracil in Various Developing Solvents (18)

Solvent System

-Kf- Value

ethyl acetate:acetone :water (70: 4 0 : 10)

0.7

ethyl acetate:methanol:conc. ammonium hydroxide (75 : 25 :1)

0.4

acetone

0.8

ether

0.12

ethyl acetate

0.4

met h an01

0.8

2-propanol

0.7

ethyl acetate :methanol (80: 2 0 )

0.8

ethyl acetate:methanol (75 :25)

0.7

ethyl acetate :water (100: 1)

0.2

ethyl acetate:water ( 1 0 0 : 3 )

0.4

ethyl acetate:mcthanol :glacial acetic acid (75:25:1)

0.9

chloroform :glaci a1 acetic acid (65 :35)

0.45

Gas Liquid Chromatographic Analysis (GLC) GLC has been carried out for fluorouracil using the instrumental conditions listed below. A single peak is observed for reference standard fluorouracil after about two minutes (19). 6.4

239

6. C. RUDY AND 6. 2. SENKOWSKI

I n s t r u m e n t a l Conditions: Column : Column Packing: Column Temperature: C a r r i e r Gas: Flow Rate: Detector:

6 f t . copper t u b i n g 5% SE-30 on Chromosorb G 25OoC Nitrogen 40 cc/minute Hydrogen Flame I o n i z a t i o n

6.5

D i r e c t S p e c t r o p h o t o m e t r i c Analysis Direct spectrophotometric analysis i s c a r r i e d o u t on f l u o r o u r a c i l i n j e c t a b l e s o l u t i o n s . A volume of t h e f l u o r o u r a c i l i n j e c t i o n i s p i p e t t e d i n t o a volumetric f l a s k and d i l u t e d with pH 4 . 7 a c e t a t e b u f f e r t o o b t a i n a f i n a l c o n c e n t r a t i o n o f about 1 0 pgm/ml. The absorbance o f t h i s s o l u t i o n i s measured a t 266 nm a l o n g with t h e absorbance o f a s i m i l a r concentration of reference standard fluorouracil. From t h i s d a t a t h e c o n c e n t r a t i o n o f f l u o r o u r a c i l i n t h e injectable solution i s calculated (20). 6.6

Fluorine Analysis 6.61

O r g a n i c a l l y Bound F l u o r i n e A n a l y s i s There a r e s e v e r a l methods a v a i l a b l e t o determine t h e amount o f carbon-bonde; f l u o r i n e . One o f t h e a a r l i e r methods employed t h e Schoniger Combustion t e c h n i q u e followed by thorium n i t r a t e o r cerous c h l o r i d e t i t r a t i o n u s i n g sodium a l i z a r i n s u l f o n a t e o r murexide a s the indicator (21). With t h e advent o f good s p e c i f i c ion e l e c t r o d e s , methods were developed t o l i b e r a t e t h e bound f l u o r i n e and d i r e c t l y measure t h e f l u o r i d e c o n c e n t r a t i o n . The r e a g e n t sodium-biphenyl followed by o x i d a t i o n with hydrogen p e r o x i d e i s used t o l i b e r a t e t h e o r g a n i c a l l y bound f l u o r i n e i n f l u o r o u r a c i l . A f l u o r i d e s p e c i f i c ion e l e c trode i s used, i n conjunction with a high-ionic-strengthb u f f e r s o l u t i o n , f o r d i r e c t measurement o f t h e l i b e r a t e d fluoride (22). The l a s t method t o be p r e s e n t e d f o r t h e a n a l y s i s o f t h e carbon-bonded f l u o r i n e i n f l u o r o u r a c i l i s 19F Nuclear Magnetic Resonance s p e c t r o m e t r y ( 3 ) . A f l u o r o u r a c i l r e f e r e n c e s t a n d a r d and an i n t e r n a l s t a n d a r d , 240

FLUOROURACI L

reagent grade orthofluorobenzoi a c i d , i s dissolved i n N,N-dimethylacetamide and t h e "F s pe ct rum o b t a i n e d . From t h i s d a t a an i n t e r n a l s t a n d a r d f l u o r i n e c o n v e r s i o n f a c t o r i s o b t a i n e d as f o l l o w s : A, x C, x 14.62 x P v I = CF where A x Cb f A A

C

b f f

= height

o f orthofluorobenzoic a c i d signal

= height of reference standard fluorouracil signal = mg/ml o f r e f e r e n c e s t a n d a r d f l u o r o u r a c i l

C

= mg/ml o f o r t h o f l u o r o b e n z o i c a c i d b 14 .6 2 = t h e o r e t i c a l f l u o r i n e c o n t e n t i n f l u o r o u r a c i l

P = assay o f fluorouracil reference standard 100

=1

CF = i n t e r n a l s t a n d a r d f l u o r i n e c o n v e r s i o n f a c t o r Now a 1 9 F s p ect r um o f each f l u o r o u r a c i l sample i s o b t a i n e d and t h e % f l u o r i n e c a l c u l a t e d u s i n g t h e f o l l o w i n g f o r m u l a .

where:

As

= h e i g h t o f f l u o r o u r a c i l sample s i g n a l

C

= mg/ml o f f l u o r o u r a c i l sample

S

6 .6 2

Fr ee F l u o r i d e A n a l y s i s The d e t e r m i n a t i o n o f any f r e e f l u o r i d e p r e s e n t i n f l u o r o u r a c i l bul k s am pl es o r i n f l u o r o u r a c i l ampuls a r e c a r r i e d o u t by d i r e c t measurement u s i n g a f l u o r i d e s p e c i f i c i o n e l e c t r o d e . The measurements a r e made i n a t r i s (hydroxymethy1)amino-methane b u f f e r s o l u t i o n (pH = 7 . 5 ) . The e l e c t r o d e r e s p o n s e was found t o be l i n e a r t h r o u g h o u t t h e working r a n g e o f 0 . 0 8 t o 0 . 2 5 mg F ( - ) / 1 0 0 m l s o l u t i o n (23). 6. 7

Vo lu m et r i c A n a l y s i s F l u o r o u r a c i l may be a s s a y e d by d i s s o l v i n g an a c c u r a t e l y weighed sample i n dimethylformamide and t i t r a t i n g i t w i t h 0.1N t et r a- n- but yl - am m oni um h y d r o x i d e i n benzene t o a b l u e end p o i n t u s i n g thymol b l u e a s t h e i n d i c a t o r .

B. C . RUDY AND 6 . 2 .SENKOWSKI

Each milliliter of 0.1N tetra-n-butylammonium hydroxide is equivalent to 13.01 mg of fluorouracil (24).

242

FLUOROURACIL

7.

R efer ences 1. 2. 3. 4.

5. 6.

7. 8.

9. 10. 11.

12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22.

M. Itawrylyshyn, Hoffmann-La Roche I n c . , P e r s o n a l Communication. I . A. Brownlie, J . Chem. S o c . , 1950, 3062 ( 1 9 5 0 ) . J . H . J ohns on, Hoffmann-La Roche I n c . , P e r s o n a l Communication. F. A. Bovey, N u c l e a r Magnetic Resonance Spect r o s c o p y , Academic Press, N e w York C i t y , pp. 211214. H. K . S o k o l o f f , Hoffmann-La Roche I n c . , P e r s o n a l Commun i c a t i on. J . Boatman, Hoffmann-La Roche I n c . , P e r s o n a l Communication. W . Benz, Hoffmann-La Roche I n c . , P e r s o n a l Communication. Pharrnacopeia of t h e United S t a t e s XVIII, 935, ( 1970) . J . Donahue, Hoffmann-La Roche I n c . , P e r s o n a l Communication. E . MacMullan, Hoffmann-La Roche I n c . , P e r s o n a l Communication. R . B. H a g e l , I-loffmann-La Roche I n c . , P e r s o n a l Communication. A . Motchane, Hoffmann-La Roche I n c . , Unpublished Data. I . Shugar and J . J . Fox, Biophysica e t Biochemica Acta, 9 , 199 and 369 (1952). M. Cole, Hoffmann-La Roche I n c . , Unpublished D a t a . A . Motchane, and H. J e n n y , Hoffmann-La Roche I n c . , Unpub 1i s hed Data. E. Miller , J . Surgical Oncology, 3 ( 3 ) , 309 1971) and r e f e r e n c e s w i t h i n . A . St eyer mar k, Hoffmann-La Roche I n c . , Unpub i s h e d Data. M . fjawrylyshyn, B . Z . Senkowski, and E . G . W o l l i s h , Microchem. J . , 8 , 15 ( 1964) . F . Mahn, Hof fmazn -La Roche I n c . , P e r s o n a l Communication. Pharmacopeia of t h e United S t a t e s XVIII, 269 (1970). A . St eyer mar k, " Q u a n t i t a t i v e O r g a n i c M i c r o a n a l y s i s " 2nd E d . , Academic, N e w York, N . Y . , 1961, p p . 326332. B . C . J o n e s , J . E . Heveran, and B . Z . Senkowski,

243

6 . C. RUDY AND 13.2 . SENKOWSKI

J , Pharm. Sci., 6 0 , 1036 (1971).

E. Heveran,

23.

B . C. Jones, J .

24.

J. Pharm. S c i . , 58, 607 (1969). Pharmacopeia of z e United States XVIII, 268 (1970).

244

and B. Z . Senkowski,

FLUPHENAZINE ENANTHATE

245

KLAUS FLOREY

CONTENTS

1.

DESCRIPTION 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. PHYSICAL PROPERTIES 2.1 Infra Red Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectrum 2 . 4 Mass Spectra 2.5 pKa, pH 2.6 Boiling Point 2 .7 Differential Thermal Analysis 2.8 Solubility 2.9 Refractive Index 3. SYNTHESIS 4. STABILITY - DEGRADATION 5. DRUG METABOLISM 6. METHOD OF ANALYSIS 6.1 Elemental Analysis 6.2 Non-aqueous Titration 6.3 Spectrophotometric Analysis 6.4 Spectrofluorometric Analysis 6.5 Colorimetric Analysis 6.6 Chromatoqraphic Analysis 6.61 Paper 6.62 Thin L a y e r 6.63 Gas-Liquid 7. IDENTIFICATION AND DETERMINATION IN BODY FLUIDS AND TISSUE 8. References

246

FLUPHENAZINE ENANTHATE

1.

Description

1.1 Name, Formula M o l e c u l a r Weight Fluphenazine enanthate i s 4-LT-D-(trifluoromethyl) phenothiazin-l0-yg p r o p y g -1 p i p e r a z i n e e t h a n o l h e p t a n o a t e ( e s t e r ) : a l s o f l u p h e n a z i n e h e p t a n o a t e , SQ 1 6 , 1 4 4 : d i h y d r o c h l o r i d e s a l t : SQ 1 0 , 4 7 9

CH2-CHz-CH2-

I

Mol. W t .

2 gH 38 3N30 2

1.2

549.70

A p p e a r a n c e , Odor, C o l o r

Fluphenazine enanthate i s a pale yellow t o yellow orange, c l e a r t o s l i g h t l y t u r b i d , viscous l i q u i d with a c h a r a c t e r i s t i c odor. 2.

Physical Properties

2.1

I n f r a Red S p e c t r a

The i n f r a r e d s p e c t r a o f a s e m i - s o l i d smear and o f a c h l o r o f o r m s o l u t i o n a r e p r e s e n t e d i n f i g u r e s 1 and 2 . 2.2

N u c l e a r M a g n e t i c Resonance S p e c t r a

The NMR S p e c t r u m i s p r e s e n t e d i n f i g u r e 3. The f o l l o w i n g a s s i g n m e n t s c a n be made f o r c h e m i c a l s h i f t s of t h e s p e c t r u m of t h e f r e e b a s e2

.

247

50004

x

r N

P

00

D C rn

i

n

r 0 I]

rn

<

~

2

4

F i g u r e 1 I n f r a r e d S p e c t r a of F l u p h e n a z i n e e n a n t h a t e , Instrument: S q u i b b House S t a n d a r d b a t c h #10 s e m i s o l i d smear. Perkin-Elmer 6 2 1 . Curve # 2 9 3 7 7

FREQUENCY 0000

50004000

3000

2000 1800

2500 1

0

0

~

1400

I600 1

1

I

I

I i

O0

I

(L'I- 1 )

1200

1100

1000 950

900

I

850 M

800

750

700 I'

I

'

i

h.

I

n

r

C D I

rn

z > N z m rn

z D z

-I

I D

d I

2

3

4

5

6

7 8 9 WAVELENGTH IMlCRONSl

10

ii

12

13

F i g u r e 2 I n f r a Red S p e c t r u m o f F l u p h e n a z i n e e n a n t h a t e S q u i b b House S t a n d a r d b a t c h #10 i n c h l o r o f o r m s o l u t i o n . I n s t r u m e n t : Perkin-Elmer 621. Curve # 29377A

14

I5

w

ul 0

I

, 70

40

?,

.

.

I , . . 20

, . . . .I

I

.

,

I0

F i g u r e 3: NMR S p e c t r u m of f l u p h e n a z i n e e n a n k h a t e , S q u i b b b a t c h #10 i n CDC13. S p e c t r u m # 10450. I n s t r u m e n t : V a r i a n A-60.

, I

.

. ,

T

> 7c

c

n

r C -0

I

rn

z > N z rn

F i g u r e 4: Low R e s o l u t i o n Mass S p e c t r u m of F l u p h e n a z i n e enanthate. S q u i b b House S t a n d a r d b a t c h 1 0 . 1nstrument:MS-9.

KLAUS FLOREY

Proton

Chemical S h i f t s ( ) 6.05 8.07 7.55

5.84

8.10 8.70 9.10 3.0

2.3

triplet mu 1t i p l e t absor ptio n envelope triplet triplet mu1t i p l e t s mu 1t i p l e t aromatic

U l t r a v i o l e t Spectrum

S q u i b b House S t a n d a r d b a t c h # 7 , 3 #22272 i n e t h a n o l Wavelength (nm) 240 ( s h o u l d e r ) 261 312

252

curve

a b s o r p t i o n (EF&,) 221 625 68

2.4

Mass S p e c t r u m

The l o w r e s o l u t i o n mass s p e c t r u m is p r e s e s e n t e d i n f i g u r e 4 . The s p e c t r u m i n d i c a t e s t h e f o l l o w i n g f r a g m e n t a t i o n p a t t e r n , c o n s i s t e n t with t h a t obtained f o r fluphenazine:

KLAUSFLOREY

The s i d e c h a i n f r a g m e n t a n d i t s f u r t h e r f r a g m e n t a t i o n p r o d u c t s were a l s o found:

T2

-CH2 CH

- 1H -

~ m/e

~

~

H

1

~

c

J

o

H

171

Another prominent fragment i s t h e following:

2.5

p K a , pH.

The pKa, v a l u e was d e t e r m i n e d b y t i t r a t i o n ; t h e pKa2 v a l u e was e s t i m a t e d from t h e pH of t h e f i r s t e q u i v a l e n t p o i n t b e c a u s e f l u p h e n a z i n e e n a n t h a t e p r e c i p a t e d a t pH 8 v a l u e s s l i g h t l y above t h i s p o i n t PKa 9 ~ K a 2 Fluphenazine enanthate 3 . 5 0 ;3 . 2 9 8 . 2 ;7.7 3.90; 3.60 8.1; 7 . 8 Fluphenazine

.

A s expected t h e values a r e very similar t o t h o s e o f f l u p h e n a z i n e (see A n a l y t i c a l

P r o f i l e ) , s i n c e t h e a d d i t i o n of an ester two c a r b o n s removed from t h e t e r t i a r y amine n i t r o g e n s h o u l d h a v e a m i n i m a l e f f e ct 8 . 2.6

Boiling Point

F l u p h e n a z i n e e n a n t h a t e base i s a v i s c o u s l i q u i d a n d a t t e m p t s t o d i s t i l l it a n d t o determine a b o i l i n g p o i n t by conventional 254

FLUPHENAZINE ENANTHATE

methods h a v e b e e n u n s u c c e s s f u l 6 2.7

.

D i f f e r e n t i a l Thermal A n a l y s i s

Fluphenazine e n a n t h a t e samples s u b j e c t e d t o DTA a t a t m o s p h e r i c p r e s s u r e u n d e r N 2 a n d i n vacuo (25mm) showed n e i t h e r exothermic o r endothermic peaks up t o a t e m p e r a t u r e o f 350°C7. 2.8

Solubility

S0 l u b i 1ity gm/m 1 s o l v e n t a t 250C8

Solvent Water Ethanol Chloroform Ether 2.9

i nsoluble 41 ( V . S . ) <1 ( V . S . ) 2

R e f r a c t i v e Index

The R e f r a c t i v e I n d e x o f S q u i b b House S t a n d a r d b a t c h #7 i s d e t e r m i n e d a s : 1 . 5 4 9 5 (23OC) 3.

Synthesis Fluphenazine enanthate (I, Figure 5) h a s been prepared by r e a c t i n g f l u henazine (11) w i t h h e p t a n o y l c h l o r i d e . 6,lO

255

a,

c,

a,

c

N

.d

W

a,

m L4

h

x" u I

L

H

I M

x

9

KLAUS F L O R E Y

47

iz

N

h

V

u

256

x

? I

i?

I

u

ci N

7

m I W

x N

3

p-

util

i?? u-

Cf 0

3:

FLUPHENAZINE ENANTHATE

4.

Stability-Degradation. F l u p h e n a z i n e e n a n t h a t e , l i k e i t s p a r e n t compound f l u p h e n a z i n e h y d r o c h l o r i d e i s l i g h t sensitive. E x p o s u r e o f a 10% s o l u t i o n o f f l u phenazine enanthate i n iso-butyl methyl k e t o n e f o r o n e month r e s u l t e d i n 5% decomposition. The mechanism o f l i g h t c a t a l y z e d decomposition i s probably s i m i l a r to t h a t of fluphenazine hydrochloride(see Analytical P r o f i l e ) . Fluphenazine enanthate hydrolyzes t o f l u p h e n a z i n e i n a l k a l i n e medium.

5.

Drug M e t a b o l i c P r o d u c t The d r u g m e t a b o l i s m a n d t i s s u e d i s t r i b u t i o n o f f l u p h e n a z i n e e n a n t h a t e , l a b e l e d w i t h C1* i n t h e e t h a n o l p o r t i o n o f t h e s i d e c h a i n was studied. I n a d d i t i o n t o unchanged f l u p h e n a z i n e enanthate t w o metabolites fluphenazine (11) a n d f l u p h e n a z i n e s u l f o x i d e (111) were i d e n t i f i e d i n t h e excreta and a l l the t i s s u e s studied. However i n t h e b r a i n o n l y f l u p h e n a z i n e was f o u n d . The h y d r o l y s i s i n t h e b r a i n of fluphenazine enanthate t o fluphenazine was d e m o n s t r a t e d . 1 2 I n a l a t e r s t u d y t h e B - g l u c u r o n i d e of 7 h y d r o x y f l u p h e n a z i n e ( I V ) was i s o l a t e d from dog b i l e a f t e r a d m i n i s t r a t i o n o f l 4 C l a b e l e d fluphenazine enanthate.

6.

Method o f A n a l y s i s 6.1

Elemental Analysis Calc. C 63.37 H 6 . 98 N 7.64

Found 9 63.09 7.10 7.99

KLAUS FLOREY

6.2

Nonaqueous T i t r a t i o n

F l u p h e n a z i n e e n a n t h a t e c a n be 0 . 1 N HCl04 i n g l a c i a l a c e t i c I n d i c a t o r : c r y s t a l v i o l e t . l4 e q u i v a l e n t : c a l c . 285: Found: House S t a n d a r d ) , 6.3

t i t r a t e d with acid. Neu t r a 1i z a t i o n 271 ( S q u i b b

Spectrophotometric Analysis

The U.V. a b s o r b a n c e a t 2 6 1 nm ( s e e S e c t i o n 2 . 3 ) c a n be u s e d f o r q u a n t i t a t i v e a n a l y s i s (200 s e c t i o n 6 . 7 1 ) . 6.4

Spectrofluorometric Analysis

A l t h o u g h no d a t a a r e a v a i l a b l e , s p e c t r o f l u o r o m e t r i c a n a l y s i s of fluphenazine e n a n t h a t e s h o u l d be p o s s i b l e , s i m i l a r t o t h a t o f t h e p a r e n t compound ( s e e A n a l y t i c a l P r o f i l e on F l u p h e n a z i n e H y d r o c h l o r i d e ) . 6.5

Colorimetric Analysis

The f o l l o w i n g c o l o r t e s t s were a p p l i e d Reagent :

11

:

color:

50% S u l f u r i c a c i d Nitroprusside

red

Gibbs

Hydroxamic a c i d Bromo-cresol g r e e n Phenol r e d Phosphomolybdic a c i d

258

f a i n t grey grey yellow-orange blue red g r e e n greygreen yellow

FLUPHENAZINE ENANTHATE

6.6

Chromatographic A n a l y s i s 6.61 Paper chromatographic A n a l y s i s The f o l l o w i n g s y s t e m s h a v e been reported.

Systems:

Fluphenazine enanthate

System 112 System 2 1 2 System 3 l *

Fluphenazine sulfoxide

0.96

Fluphenazine

0.55 0.35 0.68

0.00 0.13

0.10 0.62

--

System 112:

matman paper b e n z e n e - a c e t i c a c i d - w a t e r (2:2:1)

System 2 1 2 :

m a t m a n p a p e r #3 MM 1 N sodium f o r m a t e ( a s c e n d i n g )

System 316:

Paper

Whatman#l ( e t h a n o l washed) S t a t i o n a r y p h a s e : C a s t o r o i l (2% i n ether) Developing solvent: Methanol-water (85-15) Development 16 hours time :

T h i s l a s t system can b e used f o r q u a n t i t a t i o n by l o c a t i n g t h e s p o t s u n d e r U.V. l i g h t , e l u t i o n w i t h 95% e t h a n o l and m e a s u r i n g U . V . a b s o r b a n c e a t 261 nm a g a i n s t a s t a n d a r d , l6 f o l l o w i n g t h e g e n e r a l proce-

dure. 1 7

25Y

KLAUS F L O R E Y

6.62

T h i n l a y e r chromatography:

See Table I 6.63

G a s - l i q u i d chromatography

F l u p h e n a z i n e e n a n t h a t e can be chromatog r a p h e d i s o t h e r m a l l y a t 25OoC on 8/100 mesh g l a s s b e a d s c o a t e d w i t h 0.5% of SE-30. With a r e t e n t i o n t i m e of 6-7 m i n u t e s t h e minimum d e t e c t a b l e q u a n t i t y i s 0 . 5 ~gm. R e p r o d u c i b l e q u a n t i t a t i o n was n o t a c h i e v e d - non - l i n e a r e r r a t i c response i n d i c a t e d a b s o r p t i o n of t h e enanthate with possible attendant d e g r a d a t i o n . 18 I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e s .

''

Determination by s ectrofluorometry i n r a t b r a i n and p l a s m a . Thin l a y e r ~ c h r o m a t o g r a p h y ( s e e S e c t i o n 5 ) 1 2 13.

260

Table I Thin Layer Chromatography The following systems have been reported.

Rf values

System

Fluphenaz ine en an th a te

Fluphenazine d ihydroch 1or ide

Fluphenazine sulfoxide

7-Hydroxyf luphenazine

n U

I .J

n

Benzene- ammonia dioxane (60:5 :35) 1 2 9 1 3

0.84

0.26

0.06

0.05

0.87

0.75

--

0.66

0.82

0.47

--

0.15

0.75

0.40

C h loroform-95%

ethanol-ammonia (10:85:2. 5)13 Chloroform-abs. e thano l-ammonia (80:10:1)19 Acetone-cyclohexaneammonia(50:40:1) 11

Silica gel GF thin layer plates were used. Detection and spray reagents. 11 U.V. light 50% sulfuric acid.

rn

KLAUS FLOREY

REFERENCES B. Keeler, Squibb Institute, Private Communication. M. S. Puar and G. M. Jennings, Private 2. Communication. G. A. Brewer, Private Communication. 3. 4. A. I. Cohen, Private Communications. J. N. T. Gilbert and B. J. Millard, Org. 5. Mass Spectrom. 2,17 (1969). H. L. Yale, Pri-ate communication. 6. H. Jacobson, Private Communication. 7. H. Jacobson, Private Communication. 8. H. L. Yale, A. I. Cohen and F. Sowinski, 9. J. Med. Chem. 6,347 (1963). 10. H. L. Yale and-R. C. Merrill, U. S. Patent -3,194,733 (1965). 11. J. E. Fairbrother, Private Communication. 12. A. G. Ebert and S. M. Hess, J. Pharmacol. and Exp. Therap. 148, p.412 (1965). 1 3 . J. Dreyfuss, J. J. Ross, Jr., and E. C. Schreiber, J. Pharm. Sci. 60,829(1971). 14. J. F. Alicino, Private Communication. 15. M. A. Mahju and R. P. Maickel, Biochem. Pharmacol. 2 , 2 7 0 1 (1969). 16. H. R. Roberts, Private Communication. 17. H. R. Roberts and K. Florey, J. Pharm. Sci. 51,794 (1962). 18. A. 0. Niedermayer, Private Communications.

1.

-

-

262

FLUPHENAZINE HYDROCHLORIDE

Klaus Florey

263

KLAUS FLOREY

CONTENTS DES C R I P T I ON 1.1 N a m e , F o r m u l a , M o l e c u l a r Weight I.2 A p p e a r a n c e , C o l o r , O d o r 2. PHYSICAL P R O P E R T I E S 2 . 1 I n f r a r e d Spectra 2.2 N u c l e a r Magnetic R e s o n a n c e S p e c t r a 2 . 3 U l t r a v i o l e t Spectra 2 . 4 Mass Spectra 2 . 5 pka, p H 2 . 6 Melting R a n g e 2 . 7 Differential Thermal A n a l y s i s 2.8 Thermogravimetric Analysis 2.9 Solubility 2 . 1 0 crystal Properties 3. SYNTHESIS 4 . S T A B I L I T Y - DEGRADATION 5 . DRUG METABOLIC PRODUCTS 6 . METHODS OF ANALYSIS 6.1 Elemental Analysis 6 . 2 Nonaqueous Titration 6 . 3 Polarographic A n a l y s i s 6.4 S p e c t r o f l u o r o m e t r i c A n a l y s i s 6.5 C o l o r i m e t r i c A n a l y s i s 6.6 E l e c t r o p h o r e s i s 6.7 Chromatographic Analysis 6 . 7 1 Paper 6 , 7 2 Thin-Layer 6.73 Column 6 . 7 4 Gas - L i q u i d 7 . COUNTERCURRENT SEPARATION I D E N T I F I C A T I O N AND DETERMINATION 8. I N BODY F L U I D S AND T I S S U E S 9. MISCELLANEOUS 10. REFERENCES

1.

2 64

FLUPHENAZINE H Y D R O C H L O R I D E

1.

DESCRIPTION 1.1 Name Formula, Molecular Weight Fluphenazine Hydrochloride is 4 - { 3 - [ 2 krifluoromethy1)-phenothiazine-10-yll propyl) piperazine ethanol dihydrochloride: also 4-(3-[lo -2(-trifluoromethy1)-phenothiazinylI propy1)-1piperazine-ethanol dihydrochloride; 1- (2-hydroxy ethyl)-4- [2-trifluoromethyl-lO-phenothiaz~nyl) propyl ] piperazine dihydrochloride; 10-(3'-[4"(B-hydroxy ethyl)-1"-piperazinyl] propyl-2-trifluoromethyl-phenothiazine dihydrochloride: S94; SQ 4918.

1.2 Appearance, Color, Odor White to off-white, odorless crystalline powder. PHYSICAL PROPERTIES 2.1 Infrared Spectra The infrared spectra of Squibb House Standard #48101-001, in mineral oil mull and KBr from MeOH, are presented in figures 1 and 2.1 The infrared spectrum of the free base, and a discuss ion of spectra-structure correlation of phenothiazines have been presented. 9 The spectra published by Sammul, et al.4 are in essential agreement with those presented here. 2.

265

FREQUENCY (CM-1)

x

r

t.

5 n

r 0 D rn

<

2

3

4

5

6

7 8 WAVELENGTH (MICRONS1

I0

II

12

13

is

Fig. 1 Infrared Spectrum of fluphenazine hydrochloride in mineral oil mull. Instrument: Perkin-Elmer 621

is

n

r

5 I rn

z B N 2

rn

I

< o n

0 c,

I

r 0

n

0

rn

Fig. 2 Infrared Spectrum of fluphenazine hydrochloride in KBr from methanol. Instrument: Perkin-Elmer 621

KLAUS F L O R E Y

N u c l e a r M a q n e t i c Resonance S p e c t r a The NMR s p e c t r a o f f l u p h e n a z i n e base a n d h y d r o c h l o r i d e a r e p r e s e n t e d i n f i g u r e s 3 and 4 . 5 The s p e c t r u m o f t h e f r e e base i s e s s e n t i a l l y i d e n t i c a l w i t h t h a t p u b l i s h e d p r e v i o u s l y . 3 The f o l lowing a s s i g n m e n t s c a n be made for c h e m i c a l s h i f t o f t h e s p e c t r u m of t h e f r e e base. 6 2.2

a b

6.05 8.09 7.57 6.41 6.93 2.9-3.0

C

d

e f

2.3

triplet multiplet absorption envelope triplet singlet aromatic

Ultraviolet Spectra The f o l l o w i n g U . V . d a t a h a v e been re-

corded: 1.

i n methanol’

( I n s t r u m e n t Cary 1 5 )

Amax 261 mp E i E m 646

A m a x 310 mp, E1% 69 1cm

268

Fiq. 3 NMR Spectrum of fluphenazine base in deuterochloroform. Inscrument: Varian A-60

-*+

i i

r B C v)

n

r

0

n

rn

<

-I

L

r a*

I

l . . . . , . , . , ~ . , . , , , , , , ~

, . . . . . .

I

7a

.

.

I

.

I

I

A4

30

1 1 1 , , 1 , , , , , , , , , 1 , , , , , , , , , ~

I

.(I

.

.

I

.

.

.

I

I

30

10

I

I

I

.

. . I. . . . I 0

' .

.I 0

Fig. 4 NMR Spectrum of fluphenazine hydrochloride in deuterochloroform. Instrument: Varian A-60

M ,i

FLUPHENAZINE HYDROCHLORIDE

2.3

U l t r a v i o l e t S p e c t r a Cont d . 2. i n 95% e t h a n o l 3 Amax 264 Log E 4 . 5 9 Amax 316 Log E 3 . 6 9 Amin 239 Log E 4 . 2 8 Amin 300 Log E 3 . 5 1 3. i n 50% a l c o h o l ( I n s t r u m e n t Beckman

DU) ElPPm 1c m

Xmax 259 Xmax 305

0.052 0.006 0.013 0.002

Xmin 224

Xmin 280 4.

i n methanol8 Xmax 259 Log

E

4.53

2.4

Mass S p e c t r a The l o w - r e s o l u t i o n m a s s s p e c t r u m o f f l u p h e n a z i n e base i s r e p r e s e n t e d i n f i g u r e 5 . 9 The h i g h - r e s o l u t i o n s p e c t r u m shows t h e f o l l o w i n g f r a g m e n t a t i o n p a t t e r n o f s i d e c h a i n and nucleus’, which i s i n a g r e e m e n t w i t h t h e r e s u l t s o f an independent i n v e s t i g a t i o n . 69

?zyz 307-1H

2 8 0 I157

t

H2 *CH

.+)

-CH2 I

350

27 1

Fig. 5 Low-resolution Mass Spectrum of fluphenazine.

Instrument: MS-9

FLUPHENAZINE HYDROCHLORIDE

Another prominent fragment is t h e following:

248

The s i g n i f i c a n t m a s s numbers shown a b o v e c a n be t a b u l a t e d a s f o l l o w s : Mass U n i t s D i f f . C H N 0 S F U n s a t O/E* Found- C a l c u l a t e d 437 2 2 26 3 1 1 3 10 0 0.3 419 2 2 24 3 - 1 3 10 0 -0.7 4 0 6 2 1 2 3 3 - 1 3 10.5 E -1.6 363 19 18 2 - 1 3 10.5 E -0.3 307 16 1 2 1 - 1 3 10 0 -1.1 306 1 6 11 1 - 1 3 1 0 . 5 E -0.3 280 14 9 1 - 1 3 9.5 E -0.1 E 0.5 266 1 3 7 1 - 1 3 9.5 248 14 9 1 - - 3 9.5 E -0.1 1 7 1 9 19 2 1 - - 1.5 E -0.1 E -0.7 1 5 7 8 1 7 2 1 - - 1.5 E -0.1 1 4 3 7 15 2 1 - - 1.5

"0

=

odd e l e c t r o n i o n : E

2.5

pKa;

=

even e l e c t r o n i o n

pH

The a p p a r e n t pKa of f l u p h e n a z i n e d i h y d r o c h l o r i d e i n aqueous a l c o h o l i c s o l u t i o n h a s b e e n d e t e r m i n e d as 8.05 f o r t h e s e c o n d b a s i c g r o u p . F o r t h e f i r s t b a s i c g r o u p , a v a l u e o f 3.90 w a s obt a i n e d . lo I n d e p e n d e n t l y , v a l u e s o f 7 . 8 (estimat e d ) and 3 . 6 0 , r e s p e c t i v e l y , w e r e o b t a i n e d . 7 1 The p H of a n aqueous s o l u t i o n o f t h e d i h y d r o c h l o r i d e i s 2 . 4 ; t h a t o f monohydrochloride is 273

KLAUS FLOREY

5 . 5 . 11 2.6

M e l t i n q Ranqe The f o l l o w i n g m e l t i n g p o i n t s o f t h e hydroc h l o r i d e have been r e p o r t e d : 238.5-239.5O ( R e c r y s t . from abs , e t h a n o l USP M e t h o d p ( R e c r y s c . from abs. e t h a n o l ) l2 2 31-2 33' 224-226OI3, l4 224.5-226O ( R e c r y s t . from m e t h a n o l - e t h e r ) 15 2 3 5-2 3 701 7 l7 231-232OI8 2.7

D i f f e r e n t i a l Thermal A n a l y s i s D i f f e r e n t i a l Thermal A n a l y s i s of S q u i b b House S t a n d a r d #48101-001 showed a n endotherm a t 237OC. ( I n s t r u m e n t : DuPont) 1 Thermoqravimetric A n a l y s i s Thermogravimetric a n a l y s i s o f Squibb House S t a n d a r d #48101-001 gave no w e i g h t l o s s up t o 15Ooc ( I n s t r u m e n t : DuPont) 2.8

2 9

Solubility Fluphenazine hydrochloride is hygroscopic and e x t r e m e l y s o l u b l e i n w a t e r (70% w/v a t 25OC pH 2 . 4 ) . S o l u b i l i t y i n e t h a n o l : 15% w/v a t 25OC. L e s s t h a n 2% (w/v) i n e t h e r , a c e t o n e , benzene, l i g r o i n . 11 I n s o l u b l e i n c h l o r o f o r m and e t h e r . 67 2.10 c r y s t a l P r o p e r t i e s For f o r e n s i c i d e n t i f i c a t i o n , a p i c t u r e o f f l u p h e n a z i n e d i h y d r o c h l o r i d e c r y s t a l s from 95% e t h a n o l h a s been p u b l i s h e d w i t h t h o s e o f o t h e r p h e n o t h i a z i n e s . 19' A n x-ray powder d i f f r a c t i o n p a t t e r n of fluphenazine dihydrochloride is presented i n 274

FLUPHENAZINE HYDROCHLORIDE

table l.20 S l i g h t l y d i f f e r e n t d-values ( 1 8 . 2 ; 9.07; 8 . 7 1 ; 6 . 0 7 ; 5 . 0 3 ; 4 . 8 5 ; 4 . 3 2 ; 4 . 0 2 ; 3. 7 5 ) 7 and ( 1 7 . 2 0 ; 1 1 . 5 0 ; 8 . 4 0 ; 5 . 9 0 ; 5 . 5 0 ; 5 . 4 0 ; 4 . 7 0 ; 4.50; 4 . 2 0 ; 4 . 1 0 ; 4 . 0 0 ; 3 . 9 0 ; 3 . 7 5 ; 3 . 6 0 ; 3 . 3 5 ; 3.20; 3.10; 2.85; 2.80; 2.65; 2.55; 2.45; 2.40; 2 . 2 2 ; 2 . 1 5 ) 77 w e r e a l s o r e p o r t e d . 3 . SYNTHESIS S y n t h e t i c pathways t o f l u p h e n a z i n e ( I ) a r e shown i n f i g u r e 6. The s y n t h e s i s u s u a l l y i n v o l v e s t h e a d d i t i o n of t h e propylpiperazine ethanol s i d e c h a i n t o t h e 3-trifluoromethyl-phenothiazine nuc l e u s (11). The f o l l o w i n g r o u t e s t o ( I ) h a v e b e e n reported: (1)D i r e c t a d d i t i o n o f 4 ( 3 - c h l o r o p r o y l ) -1-pipe r a z i n e e t h a n o l t o (11) t o y i e l d ( I ) .2 P ( 2 ) Formation o f i n t e r m e d i a t e 1 0 ( 3 - c h l o r o p r o p y 1 ) - 3 t r i f l u o r o m e t h y l p h e n o t h i a z i n e (111) und e r a v a r i e t y of c o n d i t i o n s , and c o n d e n s a t i o n o f (111) w i t h p i p e r a z i n e e t h a n o l t o ( I ). l2 16,l7 (3)Condensation of 3(2-trifluoromethyl-10phenothiazinyl) propylpiperazine ( I V ) with ethyle n e o x i d e 1 5 , w i t h @-bromo-ethyl a c e t a t e v i a (VI)1 3 9 14, o r w i t h 2-bromo e t h a n o l . This last method h a s a l s o been used t o p r e p a r e ( I ) , l a b e l e d w i t h 14C i n t h e two e t h a n o l c a r b o n s . 6 ( 4 )R e d u c t i o n o f 4 (f3- ( 1 0 - p h e n o t h i a z i n y l ) t h i o propionyl) piperazine ethanol with lithium aluminum h y d r i d e t o ( I ) .2 2 J

-

4 . STABILITY DEGRADATION F l u p h e n a z i n e h y d r o c h l o r i d e is h y g r o s c o p i c . When m o i s t u r e i s e x c l u d e d , t h e c r y s t a l l i n e m a t e r i a l i s s t a b l e f o r a t l e a s t 6 months a t temperat u r e s as h i g h as 5Ooc, b o t h under a i r and n i t r o gen. The compound i s p h o t o s e n s i t i v e , s i n c e t h e s u r f a c e o f c r y s t a l s exposed t o i n c a n d e s c e n t l i g h t f o r p e r i o d s o f more t h a n 4 weeks d i s c o l o r e d t o 215

KLAUS F L O R E Y

2.10 Crystal Properties

(Continued)

TABLE I X-ray Powder D i f f r a c t i o n p a t - t e r n of S q u i b b House S t a n d a r d #48101-001 d (? * i) Rel. I n t e n s i t y * * 17.65 2 0.05 0.26 0.12 9.80 8.90 0.58 (5) 8.70 0.42 6.02 0:36 5.56 5.46 0.24 0.14 5.36 5.15 0.31 4 . 8 3 _f 0 . 0 2 0.47 4.75 0.59 (4) 4.49 0.71 ( 2 ) 4. 31 0.48 4.23 0.22 4.12 0.12 4.00 0.63 (3) 3.90 1 . 0 0 (1) 3.72 u-x5 3.69 0.47 3.57 0.32 0.21 3.40 0.16 3.27 0.24 3.23 0.42 3.11 3.06 0.19 0.22 3.00 2.93 0.12 2.87 0.15 2.72 0.16 2.67 0.16 2.54 0.14 2.42 0.15 2.34 0.15 2.27 0.14 2.02 0.15 d = (interplanar distance)

m

s i AB n

Q

A-

**

2

1.539 R a d i a t i o n : ka and ka2 c o p p e r 1 Based on h i g h e s t i n t e n s i t y of 1 . 0 0 I n s t r u m e n t : Phillips 276

Curve N O . 357

I

< O m 0 D

I

r 0

a

o rn

Fig. 6

S y n t h e t i c Pathways.

KLAUS F L O R E Y

light tan. I n aqueous s o l u t i o n , f l u p h e n a z i n e hyd r o c h l o r i d e d e g r a d e s when e x p o s e d t o l i g h t . S o l u t i o n s a t 0.01% i n e t h a n o l , w a t e r , 0.1g H C 1 and 0 . 5 N H2SO4 w e r e k e p t i n t h e d a r k o r e x p o s e d t o f l u o r e s c e n t c e i l i n g l i g h t a t room t e m p e r a t u r e (see s e c t i o n 2 . 3 ) . A f t e r 1 7 days i n t h e d a r k , a l l sol u t i o n s r e t a i n e d t h e i r U.V. absorbance i n t h e Only t h e 0 . 5 g H2S04 s o l u t i o n 255-265 m p r e g i o n . In the showed a s l i g h t d e c l i n e i n a b s o r b a n c e . light-exposed samples, only t h e e t h a n o l i c solut i o n r e m a i n e d unchanged, w h i l e i n t h e r e m a i n i n g s o l u t i o n s d i s c o l o r a t i o n and t h e a p p e a r a n c e o f m u l t i p l e b a n d s a t 240 and 255 mw w e r e n o t e d . 6 4 Exposing a 0 . 0 5 % f l u p h e n a z i n e h y d r o c h l o r i d e i n 0.1g H C 1 s o l u t i o n i n p y r e x g l a s s t o s u n l i g h t c a u s e d a 40% d r o p o f a b s o r b a n c e i n 2 h o u r s . 2 3 A s t u d y o f t h e pH dependency o f t h e l i g h t i n s t a b i l i t y showed a s i m i l a r d e c l i n e i n a b s o r b a n c y from p H 0 . 3 t o 9, w h e r e a s above pH 11 t h e d e g r a d a t i o n w a s r e t a r d e d . 2 4 Again, it w a s found t h a t s o l v e n t s s u c h as m e t h a n o l , e t h a n o l , dimethylformami d e , e t h y l e n e g l y c o l , and e t h e r p r e v e n t o r r e t a r d degradation. The l i g h t - c a t a l y z e d d e g r a d a t i o n i s n o t prev e n t e d b y e x c l u s i o n o f oxygen25 and p r o c e e d s t h r o u g h f o r m a t i o n of t h e f r e e r a d i c a l ( c f 2 6 ) . The r a d i c a l c a n a l s o be formed b y a n o x i d i z i n g a g e n t , s u c h as c e r i c ammonium s u l f a t e . 26 I n phenothiazines g e n e r a l l y , t h e semiquinone r a d i c a l u n d e r g o e s d i s p r o p o r t i o n a t i o n . The p r o d u c t s o f o x i d a t i o n ar e t h e s u l f o x i d e o r t h e 3 ( o r 7 ) -hydroxy-derivatives. However, f o r m a t i o n of t h e l a t t e r is s e v e r e l y i n h i b i t e d i n 10-substituted p h e n o t h i a z i n e s . 2 6 The s e c o n d - o r d e r d e c a y r a t e c o n s t a n t of t h e s e m i q u i n o n e r a d i c a l of f l u p h e n a z i n e w a s f o u n d t o be 1100 (l/mole/min) , as meas u r e d by e l e c t r o n s p i n r e s o n a n c e . 26 The s u l f o x i d e c o n t e n t o f s e v e r a l commercial b a t c h e s o f f l u p h e n a z i n e w a s d e t e r m i n e d t o be a r o u n d 0 . 1 % b y 218

FLUPHENAZINE H Y DROCH L O R I DE

a s p e c t r o f l u o r o m e t r i c method ( e x c i t a t i o n wavel e n g t h 350 m@; f l u o r e s c e n c e w a v e l e n g t h 400 mp; see a l s o s e c t i o n 6 . 4 ) .27 The s u l f o x i d e c o n t e n t c a n a l s o be measured b y a t h i n - l a y e r method (see a l s o s e c t i o n 6.72).2 8 So f a r , t h e r e i s no e v i d e n c e f o r t h e d e g r a d a t i v e f o r m a t i o n of 7 - h y d r o x y f l u p h e n a z i ~ which h a s been i d e n t i f i e d as a m e t a b o l i c p r o d u c t (see s e c t i o n 5 ) . 5 . DRUG METABOLISM Acetylf luphenazine ( V I , Figure 6 ) was hydrolyzed e n z y m a t i c a l l y t o f l u p h e n a z i n e ( I ) i n v i t r o b y homogenates o f s m a l l i n t e s t i n a l mucosa, l i v e r , and b r a i n from e i t h e r t h e r a t o r t h e monkey.29 ( S e e a l s o s e c t i o n 7 ) . When f l u p h e n a z i n e w a s i n c u b a t e d w i t h a " m i c r o s o m a l and s o l u b l e " f r a c t i o n o f r a t l i v e r s , h y d r o x y l a t i o n on t h e p h e n o t h i a z i n e r i n g was o b s e r v e d . 3 0 The s u l f o x i d e w a s n o t det e c t e d i n t h e e x t r a c t , and it seems u n l i k e l y t h a t b i o l o g i c a l sulfoxidation precedes hydroxylation i n t h e s e s y s t e m s . 3 0 The b i o l o g i c a l d i s p o s i t i o n and m e t a b o l i c f a t e of fluphenazine-14C i n t h e dog and r h e s u s monkey w a s s t u d i e d . 7 6 When dogs w e r e t r e a t e d w i t h f l u p h e n a z i n e - 1 4 C , 7-hydroxyfluphenazine ( V I I , F i g u r e 6) w a s i from t h e u r i n e a n d i d e n t i f i e d by s y n t h e s i s . human b l o o d and u r i n e s m a l l amounts o f f l u p h e n a z i n e and i t s s u l f o x i d e , b o t h i n f r e e and c o n j u g a t e d form w e r e i d e n t i f i e d . 7 9 T i s s u e d i s t r i b u t i o n and e x c r e t i o n w e r e s t u d i e d i n t h e r a b b i t . 73 6. METHODS OF ANALYSIS 6.1 E l e m e n t a l A n a l y s i s % Found Element % Calc. R e f . 11 12 15

Wa:f

C H

51.76 5.53

F N

11.17

0

S

c1

8.23 3.13 6.28 13.89

51.72 5.62

-

8.04

-

6.05 13.66

51.73 5.86

-

8.00

-

51.68 5.42

-

-

-

-

-

-

KLAUS F L O R E Y

6 . 2 Nonaqueous T i t r a t i o n Nonaqueous t i t r a t i o n i n g l a c i a l a c e t i c a c i d w i t h 0.01g p e r c h l o r i c a c i d and Q u i n a l d i n e Red o r c r y s t a l v i o l e t i n d i c a t o r g a v e a n e u t r a l i z a t i o n e q u i v a l e n t t o 259 ( c a l c . 2 5 5 . 2 2 ) . 32

6 . 3 Polarographic Analysis The h a l f - w a v e p o t e n t i a l i n a l c o h o l i c s u l f u r i c a c i d s o l u t i o n w a s f o u n d t o be 0 . 7 1 4 ( v o l t v s . normal c a l o m e l e l e c t r o d e ) , c o m p a r a b l e t o t h a t o f o t h e r p h e n o t h i a z i n e s .8 6 . 4 Spectrofluorometric Analysis Fluphenazine hydrochloride, l i k e o t h e r phenothiazine drugs, e x h i b i t s fluorescence. This phenomenon w a s f i r s t e x p l o r e d i n 0 . 2 g s u l f u r i c a c i d . 33 The f l u o r e s c e n c e p e a k w a s f o u n d i n t h e r a n g e o f 450 t o 475 mp, s h i f t i n g t o s l i g h t l y l o w e r w a v e l e n g t h upon o x i d a t i o n w i t h p o t a s s i u m permanganate. A s l i t t l e as 0 . 0 1 t o 0 . 0 5 1-14 o f t h e p u r e s u b s t a n c e per m l . o f 0 . 2 g s u l f u r i c a c i d Due t o i n t e r f e r e n c e by o t h e r c o u l d be d e t e c t e d . substances, t h e s e n s i t i v i t y i n biological f l u i d s was l o w e r ( a b o u t 0 . 8 pg/ml f l u i d ) . T h i s method i s s u p e r i o r t o d e t e r m i n a t i o n b y U . V . , where about 4 p.g/ml a r e r e q u i r e d . I n 50% a c e t i c a c i d , t h e f o l l o w i n g d a t a w e r e o b t a i n e d , w i t h o u t o r w i t h o x i d a t i o n w i t h 30% hydrogen peroxide34:

A c t i v a t i o n maximum,m~ F l u o r e s c e n c e maximum,my Unoxidized Oxidized Unoxidized Ox i d i z e d Ref 34 Ref 35

330

-

345 3 50

460

-

380 410

I n t h e u n o x i d i z e d form, a s h a r p i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y o c c u r r e d above p H 7; i n t h e o x i d i z e d form, i n t e n s i t y w a s a b o u t e q u a l from 280

FLUPHENAZINE HYDROCHLORIDE

p H 2 t o p H 1 2 and f e l l p r e c i p i t o u s l y a t p H 1 2 . I n t h e o x i d i z e d form, as l i t t l e as 0 . 5 wg/ml c o u l d be d e t e r m i n e d q u a n t i t a t i v e l y . F l u p h e n a z i n e w a s r e l a t i v e l y l e s s f l u o r e s c e n t t h a n some o t h e r p h e n o thiazines. In concentrated s u l f u r i c acid, t h e a c t i v a t i o n maximum w a s found a t 325 m p and t h e f l u o r e s c e n c e maximum a t 500 mp. 36 The i n s t r u m e n t u s e d i n a l l i n v e s t i g a t i o n s w a s t h e Aminco-Bowman S p e c t r o f l u o r o m e t e r .

-

6.5 Colorimetric Analysis Color T e s t s f o r Identification The g e n e r a l method o f Ryan37 t o q u a n t i t a t e unoxidized phenothiazine d e r i v a t i v e s by c o l o r complex f o r m a t i o n w i t h p a l l a d i u m c h l o r i d e ( 5 0 0 550 mw m a x i m a ) h a s a l s o b e e n a d o p t e d f o r f l u p h e n a z i n e h y d r o c h l o r i d e ( s e e N . F . X I I 1 ) and c a n b e u s e d f o r s t a b i l i t y measurements. Like o t h e r phenothiazines, fluphenazine h y d r o c h l o r i d e reacts w i t h c e r i c s u l f a t e , f i r s t b y forming a red-colored semiquinone f r e e r a d i c a l (420 ml), f o l l o w e d by f o r m a t i o n o f t h e c o l o r l e s s s u l f o x i d e d e r i v a t i v e . T h i s r e a c t i o n h a s b e e n made t h e basis o f a p h o t o m e t r i c t i t r a t i o n . 3 8 The f o l l o w i n g c o l o r t e s t s h a v e been reported. ( F o r a d d i t i o n a l t e s t s , see a l s o s e c t i o n s 6 . 7 1 and 6 . 7 2 ) . FOR COLOR TESTS SEE TABLE I1

6 . 6 E l e c t r o p h o r e s is E l e c t r o p h o r e s i s w a s c a r r i e d o u t on f l u p h e n a z i n e h y d r o c h l o r i d e and s e v e r a l o t h e r phenot h i a z i n e t r a n q u i l i z e r s i n var io u s b u f f e r s prop o s e d by Werrum. 41 A Gordon-Misco a p p a r a t u s , Whatman 3MM paper 30 c m i n w i d t h , and a p o t e n t i a l d i f f e r e n c e of 500 t o 800 v w e r e u s e d . Detection o f s p o t s w a s c a r r i e d o u t w i t h a 40% s u l f u r i c a c i d

zx 1

TABLE I1 Reaqent 0.001N M e r c u r i c n i t r a t e i n h y d r o c h l o r i c a c i d 0.5% aq . ammonium m o l y b d a t e 0.5% aq. ammonium v a n a d a t e 3 7% f o r m a l d e h y d e i n c o n c . s u l f u r i c a c i d (1:20) 0.5% aq. s e l e n i o u s a c i d 1% sodium t u n g s t a t e 0.5% titanium dioxide i n conc.sulfuric acid Fuming n i t r i c a c i d , t h e n e t h a n o l i c p o t a s s i u m hydroxide (3%) 10% aq. sucrose Conc. s u l f u r i c a c i d p-dimethylaminobenzaldehyde 10% i n g l a c i a l acetic acid 1% c o b a l t a c e t a t e : 10% i s o p r o p y l a m i n e i n a c e t o n e 10% aq. c h l o r a m i n e T 1% aq. palladium c h l o r i d e a c i d i f i e d w i t h hydrochloric acid N i t r i c acid Conc. s u l f u r i c a c i d , 6% u r a n i u m n i t r a t e i n 95% e t h a n o l ( 3 : 1 7 ) 0 . 5 % ammonium v a n a d a t e i n c o n c . s u l f u r i c a c i d 4% a q . s i l v e r n i t r a t e s o l u t i o n a c i d i f i e d w i t h 0 . 1 N sulfuric acid 2% Zmmonium f e r r o u s s u l f a t e i n c o n c . sulfuric acid

as.

32

N

Color pink-violet blue-pink-violet grey-yellow 1i g h t red grey-green purple orange

R ef -

39 40 40 40 40 40 40

yellow-green grey-yellow l i g h t brown

40 40

5

l i g h t brown light blue l i g h t yellow

40 18 18

<

orange orange-brown

18 18

l i g h t orange y e l l o w brown

18 18

c r e a m wh it e

18

-

40

75

x

5

FLUPHENAZINE HYDROCHLORIDE

spray (orange color) or fluorescence under W light.42 For fluphenazine hydrochloride, anodic migration (cm) after 40 to 60 minutes was: pH :

Migration:

3.3 6.8

4.7 3.7

6.0

4.3

7.2 4.5

8.0 2.7

9.3 1.2

6.7 Chromatoqraphic Analysis 6.71 Paper Chromato-ra hic Analysis The following chromatographic systems have been reported: Solvent Systems: 1% Sodium formate 1% Sodium formate 90/n-Propanol 10 1% Sodium formate 90/1N Ammonia 10 1g Sodium formate 97/Formic acid 3 1% Sodium acetate 1% Sodium acetate 90/n-Propanol 10 10% Sodium chloride 92/n-Propanol 8 Formamide + Ammonium formate/Benzene Formamide + Ammonium formate/Cyclohexane-benzene (9:1) Petroleum (b.p. 195-220°) /EthanolAmmonia-water (60:2: 38)

Rf Ref 0.23 42 0.32 42 0.19 42 0.42 42 0.17 42 0.48 42 0.45 42 0.71 43 0.22 43 0.59 43

Ref color Detection systems : 40% H2S04 spray orange 42,43 Fluorescence under W light bluish yellow 42,43 orange 43 Dragendorff-reagent Cerium sulfate in 2g sulfuric acid red 43 1% gold (111) chloride red 43 1% dimethylaminobenzaldehyde in ethano1:chloroform (95:5)red 43 A quantitative paper chromatographic method62 can be used to measure the purity of

'83

K L A U S FLOREY

fluphenazine, following published procedures63. The paper is impregnated with castor oil ( U S P ) in ether. Developing solvent is methanol-water 85:15. Rf values of fluphenazine base approx. 0.79, fluphenazine hydrochloride 0.83. The zones are eluted from the chromatogram with 95% ethanol and the absorbances o f the eluates are determined at 261 m p in a spectrophotometer. 6.72

3

The following thin-layer chromatographic systems have been reported: FOR ABOVE SEE TABLE I11 6.73 Column (Ion-exchanqe) Chromatographic Analysis~ Ion-exchange analysis of fluphenazine hydrochloride and other phenothiazine tranquilizers on a column of Dowex sulfuric acid resin 50W-X8 was possible, but not too practical for analysis of tablets.56 6.74 Gas-Liquid Chromatoqraphic Analysis Fluphenazine was not eluted after 90 minutes at 270°, using a 210 SE-30 silicone polymer on a 80-100 mesh Gas-chrom S diatomaceous earth column57; with a 3% SE-30 on 80-100 mesh Gas-chrom Q at 25Ooc, it eluted in 4.9 minutes.28 Fluphenazine was also well separated from other tranquilizers on a 120 mesh silanized Gas-chrom P column coated with 1% Hi-Eff-8B (cyclohexanedimethanol succinate) at a temperature of 22Ooc and a retention time of 5 minutes.58 The identification of fluphenazine and other phenothiazine tranquilizers by gas chromatography of their pyrolysis products has also been reported.59

284

TABLE I11 Ads orbent Silica Gel 11

I 11 I1

I1 I' 1' 11

N

I1

m vI

Cellulose Silica Gel 11

11 11 I1

11 11

So1vent Svs tem tert. Butyl alcohol 90/1g Ammonia 10 n-Propanol 88/1g Ammonia 1 2

Ether, sat. with water 70% Methanol 85% n-Propanol n-Butanol sat. with 1s Ammonia Benzene-Dioxane-aq. Ammonia ( 6 0 : 3 5 : 5 ) Ethanol-Acetic acid-water ( 5 0 :30: 2 0 ) Methanol-butanol ( 6 0 :4 0 ) Cyclohexane-Diethylamine (9: 1) 5% aq. Ammonium sulfate sat. with Isobutanol Cyclohexane-methanol ( 5 0 : 5 0 and 2 5 : 7 5 ) Chloroform-acetone ( 5 0 : 5 0 ) Chloroform-methanol ( 5 0 : 5 0 ) chloroform-methanol (90: 10) Chloroform-acetone-methanol ( 5 0 : 2 5 : 2 5 ) Chloroform-acetone-methanol ( 2 5 : 5 0 : 2 5 ) Chloroform-acetone-methanol-ammonia ( 5 0 : 25: 25: 1)

Acetone Acetone Acetone-ammonia (100:1)

Rf 0.37 0.56 0.09 0.68 0.27 0.57 0.34 0.58 0.68 0.05

Re f 42 11

11 11 It 11

n

r

5211

C -o

1

rn 11

53

z B N z rn

1

0.03 0.36 0.03 0.59 0.44 0.42 0 , 32

54 44 44 51,44,50 45 44 44

0.54 0.06

44 44 45 44,51

0.16 0.33

0

n

0

c

1

r

0

rn

TABLE I11 Cont'd.

Adsorbent Silica Gel I1

I1 I1 I1

I1

11 11 I1

1 3

II

m

I1

rn

'1 11 '1

11 '1

*

*

* ** **

Silica Ge1,Plain Chromagram,Plain

solvent System Acetone-ammonia (95: 5 ) Acetone-methanol (50 :50) Acetone-methanol-ammonia (50 :50 :1) Methanol Methanol-ammonia ( 98 :2 ) Methanol-ammonia (98: 2 ) Chloroform-methanol-ammonia ( 8 0 : 20 : 1) Chloroform-ethanol-ammonia (80: 2 0 : 1) Acetone-benzene-ammonia (30:70:5) Butanol-pyridine-water (1:2: 1) Cyclohexane-benzene-diethylamine (75:15:10) Cyclohexane-benzene-diethylamine (75: 1 5 : 20) Methanol Acetone Benzene-ethanol-ammonia (95: 15: 5 ) Methanol 95% ethanol Chloroform + cyclohexane-diethylamine (50:40: 1 0 ) Chloroform + cyclohexane-diethylamine

Rf Ref 0.54 0.38 0.56 0.34 0.54 0.67 0.68 0.76

44

45 30 23

--

46

0.52

47

0.06

48

0.18 0.60

45 48

0.25

0.30 0.15

45 48

0.06 0.24

0.48

49

TABLE I11 C o n t d A d s o rp t i o n

Chromagram + fluor indicator Silica G e l It

*

** ***

S o l v e n t System

Rf Ref -

c h l o r o f o r m + cyclohexane-diethylamine C h l o r o f o r m - h e x a n e (1:1) 15% aq.ammonium a c e t a t e - m e t h a n o l (20:80)

p r e p a r e d w i t h 0 . 1 M KOH p r e p a r e d w i t h 0.1M NaHS03 I n t h i s s y s t e m , f l u p h e n a z i n e - s u l f o x i d e h a d a n Rf

0.40 0.48

49 50

0.75

28***

of 0.59 2

m

I

The f o l l o w i n g d e t e c t i o n s y s t e m s h a v e b e e n u s e d :

<

0

Color System F l u o r e s c e n c e u n d e r U.V. lamp 40% s u l f u r i c a c i d s p r a y 50% a q . s u l f u r i c a c i d i n e t h a n o l ( 4 : l ) Potassium iodoplatinate 5% f e r r i c c h l o r i d e , 20% p e r c h l o r i c a c i d , 50% n i t r i c a c i d (5:45:50) ( F P N ; F o r r e s t r e a g e n t ) Iodine vapors 1%p o t a s s i u m p e r m a n g a n a t e Dragendorff r eag en t

Reaction

-

a

Ref

42,44,51,30,28,50

orange 42,46,47 orange-red47,52 violet 52,54,44,51,49,28 (flesh or (pale pink 44,45,51,53,54,48 brown 54,44,48 51 brown 51,48

0 0

I

6 z0 rn

D e t e c t i o n systems used Cont' d. color System 5% p-dimethylaminobenzaldehyde i n 1 8 s s u l f u r i c acid Furf u r a l reagent 1%c e r i u m s u l f a t e i n 2 g s u l f u r i c a c i d 1%g o l d (111) - c h l o r i d e Folin-Ciocalteau reagent 1%ammonium v a n a d a t e i n 10 m l . conc. sulfuric acid 5% cinnamic aldehyde and 5% H C 1 i n e t h a n o l p-benzoquinone i n d i c h l o r o e t h a n e

R e a c tion

pink o r orange cameo red red cameo flesh cameo

-

Ref 45,47,51 51,45 47

47 45 45,48 45 55

n

r

0

n

rn

<

F LUPH E NA 2 I N E HY D R OCH LOR I DE

countercurrent Separation F l u p h e n a z i n e w a s s e p a r a t e d from a c e t y l f l u phenazine ( V I , f i g u r e 6) by countercurrent d i s t r i b u t i o n (see a l s o s e c t i o n 5 ) a c r o s s 25 t u b e s i n a s o l v e n t s y s t e m o f 0.1M a c e t a t e b u f f e r , pH 3 . 8 2 , and 1 . 5 % i s o a m y l a l c o h o l i n h e p t a n e . 2 9 7.

I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body F l u i d s and T i s s u e s Many o f t h e r e f e r e n c e s c i t e d i n p r e v i o u s sect i o n s c o n c e r n methods d e v i s e d t o i d e n t i f y a n d d e t e r m i n e f l u p h e n a z i n e h y d r o c h l o r i d e and o t h e r phenothiazines f o r pharmacological, toxicological, and f o r e n s i c p u r p o s e s ( c f . 6 0 ) . T h e s e methods c a n be c l a s s i f i e d as f o l l o w s : 8.

43,45,55,39,18,40,74,75 Color r e a c t i o n s 61,43,45,51,74,79 S e p a r a t i o n schemes 33,34,35,36,68 Spectrof luorometry 42 Electrophoresis 43,43 Paper c h r o m a t o g r a p h y (28,42-49, 51-54, 6 5 , 7 4 , T h i n - l a y e r chromatog(78,79 raphy Gas - 1 i q u i d chromat og28,57,58 raphy 65 So 1u b i 1i t y X-ray d i f f r a c t i o n b a n d s 7 M i c r o c r y s t a l l i n e iden19,72 t if i c a tion Miscellaneous The a b s o r p t i o n o f f l u p h e n a z i n e d i h y d r o c h l o r i d e and o t h e r p h e n o t h i a z i n e d e r i v a t i v e s b y s o l i d a d s o r b a n t s , s u c h a s k a o l i n , t a l c and c h a r c o a l , h a s b e e n s t u d i e d b y S o r b y e t a l . i0 The v o l u m e t r i c d e t e r m i n a t i o n o f f l u p h e n a z i n e by s t o i c h i o m e t r i c combinat i o n w i t h a n i o n i c s u r f a c e - a c t i v e a g e n t s , s u c h as sodium l a u r y l s u l f a t e and sodium d i o c t y l s u l f o s u c c i n a t e , h a s b e e n 9.

28Y

KLAUS F L O R E Y

described.66 The surface tension-lowering activity of fluphenazine and other tranquilizers h a s been studied.70 Ion-pair extraction has been described.71 REF ER ENC ES (1) G.A.Brewer, Squibb Institute, personal communication. (2) W.E Thompson, R.J.Warren, J.B.Eisdorfer and J. E.Zarembo, J.Pharm.S c i . 3 , 1819 (1965). (3) R.J.Warren, J.B.Eisdorfer,W.E ,Thompson and J.E.Zarembo, J.Pharm.Sci .55, 144 (1966). (4) O.R.Sammu1, W.L.Brannon and A.L.Hayden, J.Ass .Cffic.Anal.Chem.47, 918 (1964). (5) A.I.Cohen and G.M.Jennings, Squibb Institute, personal communication. (6) H.L.Yale, A.I.Cohen and F.Sowinski, J.Med. Chem.5, 347 (1963). (7) P.Rajeswaran and P.L.Kirk, Bull.Narcotics, 14, 19 (1962), C.A.57, 7390h (1962). (8) FKabasakalian and J.McGlotten, Anal.Chem.2, 431 (1959). (9) A. I.Cohen, Squibb Institute, personal communication. (10)D.L.Sorby, E.M.Plein and J.D.Benmaman, J. Pharm. Sci. 55, 785 (1966). (ll)H.Cords, Squibb Institute, personal communicat ion. (12)H.L.Yale and F.Sowinski, J.Amer.Chem.Soc. 82, 2039 (1960). (13)Smith, Kline and French Laboratories, Brit. Patent 829, 246 (1960), c. A. 54, 174281.1 (1960), (14)G.E.Ullyot, U.S.Patent 3,058,979 (1962), C.A. 58, 68419 (1963).

2Y0

F LUPHENAZINE HYDROCHLORIDE

( 1 5 )E . I . Anderson, G . B . B e l l i n z o n a , P . N . C r a i g , G . E . J a f f e , K . P . Janeway, C . K a i s e r , B . M . L e s t e r , E . J . N i k a w i t z , A.M. P a v l o f f , H . E . R e i f f and C . L . Z i r k l e , A r z n e i m i t t e l - F o r s c h u n g -91 2 937 (1962). ( 1 6 ) S c h e r i c o L t d . , B r i t i s h P a t e n t 833,474 (1960) C . A . 5 4 , 21,144h ( 1 9 6 0 1 . ( 1 7 )H . L . Y a l e , F . A . S o w i n s k i and J . B e r n s t e i n , U . S . P a t e n t 3 , 2 2 7 , 7 0 8 ( 1 9 6 6 ). (18)D.K.Yung and M . P e r n a r o w s k i , J . P h a r m . S c i . 52, 365 ( 1 9 6 3 ) . ( 1 9) P . R a j e s w a r a n and P . L K i r k , B u l l . N a r c o t ics , U.N.Dept. S o c i a l A f f a i r s l3, 2 1 (19611, C . A . 57, 7390h ( 1 9 6 2 ) . ( 2 0 )J . Q . O c h s and N . H . Coy, S q u i b b I n s t i t u t e , p e r s o n a l communication. (21)Michio Nakanishi, J a p a n . P a t e n t 18,512 ( 1 9 6 6 ) , C . A . 66, 3 7 , 9 3 5 ~ ( 1 9 6 7 ) . ( 2 2 ) M . Izumi and M . N a k a n i s h i , J a p a n . P a t e n t 4537 ( 1 9 6 2 ) , C . A . 58, 10,209h ( 1 9 6 3 ) . ( 2 3 )J . E . F a i r b r o t h e r , S q u i b b I n s t i t . u t e , p e r s o n a l communication. ( 2 4 )H . Kadin, S q u i b b I n s t i t u t e , p e r s o n a l communic a tion. ( 2 5 )H . Kadin, S q u i b b I n s t i t u t e , p e r s o n a l communic a tion. ( 2 6 ) T . ~ . T o z e rand L . D.Tuck, J . Pharm. S c i . 5 4 , 1169 (1964). ( 2 7 )J .E . F a i r b r o t h e r , Squibb I n s t i t u t e , p e r s o n a l communication. ( 2 8 ) Z . Kofoed, C . F a b i e r k i e w i c z and G . H . W . L u c a s , J . C h r o m a t o g r . 23, 410 ( 1 9 6 6 ) . ( 2 9 ) C . I . S m i t h and J . C . Burke, Toxic01 . Appl . P h a r m a c o l . 2, 553 ( 1 9 6 0 ) . ( 3 0 ) A . E . R o b i n s o n , J . P h a r m . P h a r m a c . l8, 1 9 ( 1 9 6 6 ) . ( 3 1 )J . D r e y f u s s and A . I . Cohen, J . Pharm. S c i . -960 826 ( 1 9 7 1 ) . ( 3 2 )J . A l i c i n o , S q u i h b I n s t i t u t e , p e r s o n a l communication.

.

79 I

KLAUS F L O R E Y

(33)T.J.Mellinger and C.E.Keeler, Anal.Chem. 35, 554 (1963). (34)J.B.Ragland and V.J.Kinross-Wright, Anal. Chem. 36, 1356 (1964). (35)S.L.Tompsett, Acta Pharmacol.Toxico1. 2 6 , 298 (1968). (36)E.A.Martin, Can. J. Chem. 44, 1783 (1966). (37)J.A.Ryan, J.Pharm.Sci. 48, 240 (1959). (38)S.P.Agarwal and M.I.Blake, J.Pharm.Sci. 48, 1011 (1969). (39)F.M.Forrest, J.S.Forrest and A.S.Mason, Amer. J.Psychiat. 116, 549 (1959). (40)P.Rajeswaran and P.L.Kirk, Bull. on Narcotics 13, No. 3 , 1 (1961), C.A. 56, 11705b (1962). (41)L.N.Werrum, H.T.Gordon and W.J.Thornburg, J. Chromatogr. 2, 125 (1960). (42)T.J.Mellinger and C. E.Keeler, J.Pharm.Sci.51, 1169 (1962). (43)K.Macek and J.Vecerkova, Pharmazie 2 0 , 605 (1965). (44)A.NoirfaliseY J.Chromatogr. l9, 68 (1965). (45)I.Zingales, J. Chromatogr. 3 l , 405 (1967). (46)T.Fuwa, T.Kido and H.Tanaka, Yakuzaigaku 25, 138 (1965), C. A. 64, 6405f (1966). (47)Y.Kiroshita, H.Koike, K.Iwashima and S.Oriyama, Nagayu Shiritzu 12, 33 (1964), C.A. 64, 19324h (1966). (48)W.W.Fike, Anal. Chem. 3 8 , 1697 (1966). (49)P.Schweda, Anal.Chem. 2, 1019 (1967), C.A. 67, 50918j (1967). (50)F.Dondzila, Squibb Institute, personal communication, (51)A.Noirfalise, J.Chromatogr. 20, 61 (1965). (52)J.Cochin and J.W.Daly, J.Pharmaco1.Exp.Ther. 139, 154 (1963). (53)J.Sunshine, Amer. J.Clin. Pathol. 40, 576 (1963). (54)A.Noirfalise and M.H.Grosjean, J.Chromatogr. 16, 236 (1964). 292

FLUPHENAZINE H Y D R O C H L O R I D E

(55)J.Jeunier, Ann. Pharm.Fr. 26, 25 (1968), C.A. 68, 117170a (1968). (56)J.W.Steele, Can.Pharm.J.Sci.Sect. 97, 91 (1964), C.A. 60, 15681d (1964). (57)M.W.Anders and G . J.Mannering, J.Chromatogr. 7, 258 (1962). (58)N.C.Cain and P.L.Kirk, Microchem. J. l2, 256 (1967). (59)C.R.Fontan, N.C.Cain and P.L.Kirk, Mikrochim. 1chnoanal.Acta 1964, 326. (GO)J.Sunshine, Handbook of Analytical Toxicology. The Chemical Rubber Co. (1969). (Gl)S.L.Tompsett, Acta Pharmacol.Toxico1. 2% 303 (1968). (62)H.R.Roberts, Squibb Institute, personal communication. (63)H.R.Roberts and K.Florey, J.Pharm.Sci. 5 l , 794 (1962). (64)R.B. Stasiak and N.H.Coy, Squibb Institute, personal communication. (65)J.L.Kiger and J.G.Kiger, Ann.Pharm.Franc. 23, 489 (1965), C.A. 6 4 , 6407e (1966). (66)L.A.Araya Ro jas, An.Fac.Quim.Farm.Univ.Chile 15, 101(1963), C.A. 62, 1 2 9 7 6 ~(1965). (67)LJacobson, Squibb Institute, personal communication. M.A.Mahju and R.P.Maicke1, Biochem.Pharmaco1. (68 18, 2701 (1969). (69 J.N.T.Gilbert and B.J.Millard, 0rg.Mass. Spectrom. 2, 17 (1969). (70 P.M.Seeman and H.S.Bialy, Biochem.Pharmaco1. 12, 1181 (1963). (71)B.A.Persson, Acta Pharm.Suec. 2, 335 (1968). (72)C.N.Andres,J.Ass .Offic.Anal.Chem. 51, 1020 (1968) and ibid. 53, 834 (1970). (73)H.Weissenstein, E.Feller and G.Schelle, Arzneimittel-Forschung l8, 685 (1968). (74)M.Pinkasova, Cesk.Farm. l7, 181 (1968).

KLAUS FLOREY

(75)B.Gothelf and A.G.Karczmar, 1nt.J.Neuropharmacol. 2, 95 (1963). (76)J. Dreyfuss, J. J.Ross, Jr. and E.C.Schreiber, J.Pharm.Sci. 60, 821 (1971). (77)A. deLeenheer and A. Heyndrickx, J.Ass. O f f ic. Anal.Chem. 54, 625 and 6 3 3 (1971). (78)C . Korczak-Fabierkiewicz, G. C imbura, J. Chromatogr. 53, 413 (1970). (79)A.Viala, J.P.Cano and A.Philippe, Ann.Pharm. Franc. 27, 511 (1969).

294

ISOCARBOXAZID

Bruce C. R u d y and Bernard Z. Senkowski

295

B. C. RUDY A N D B. 2 . S E N K O W S K I

INDEX Analytical P r o f i l e - Isocarboxazid 1.

Description 1.1 Name, Formula, M ol ecul ar Weight 1.2 Appearance, C o l o r , Odor

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F l u o r e s c e n c e Spect r um Mass Spectrum 2.5 2 .6 Optical Rotation 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a l Scanni ng C a l o r i m e t r y 2.9 Th e r m ogr avi m et r i c A n a l y s i s 2.10 S o l u b i l i t y 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2 . 1 2 Dissociation Constant

3.

Synthesis

4.

S t a b i l i t y D egr adat i on

5.

Drug M e t a b o l i c P r o d u c t s

6.

Methods o f A n a l y s i s 6.1 Ele m ent al A n a l y s i s 6.2 Phase S o l u b i l i t y A n a l y s i s 6.3 T h i n Layer Chromatographic A n a l y s i s 6.4 Colorimetric Analysis 6.41 Ammonium Molybdate React i on 6.42 T e t r a z o l i u m S a l t R eact i on 6.5 P o t e n t i o m e t r i c Sodium N i t r i z e T i t r a t i o n

7.

R ef er en c es

296

ISOCARBOXAZI D

1.

Description

Name, Formula, Mol ecul ar Weight I s o c a r b o x a z i d i s 5-methyl-3-isoxazolecarboxylic a c i d 2-benzylhydrazide. 1.1

0 -NH-CH

M ol ecul ar Weight:

C12H13N302

231.26

Appearance, C o l o r , Odor I s o c a r b o x a z i d o c c u r s as a w h i t e c r y t a l l i n e powder. I t has a s l i g h t , c h a r a c t e r i s t i c odor. 1.2

2.

Physical Properties 2.1

I n f r a r e d Spectrum (IR) The IR s p e c t r u m o f b u l k r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d was o b t a i n e d from a p e l l e t made by d i s p e r s i n g 1 . 1 mg o f i s o c a r b o x a z i d i n 300 mg o f K B r ( 1 ) . T h i s s p e c t r u m , shown i n F i g u r e 1 , was measured on a P e r k i n Elmer 621 S p e c t r o p h o t o m e t e r . A l i s t o f t h e a s s i g n m e n t s made f o r some o f t h e c h a r a c t e r i s t i c bands i s g i v e n i n T a b l e I ( 1 ) . TABLE I

C h a r a c t e r i s t i c Bands i n t h e IR Spectrum o f I s o c a r b o x a z i d Wavelength (cm-l)

Characteristic of NH s t r e t c h i n g v i b r a t i o n s O ver t one bands o f s i n g l y s u b s t i t u t e d benzene C=O s t r e t c h C=C s t r e t c h o f a r o m a t i c s Deformations o f 5 a d j a c e n t hydrogens on benzene r i n g

3269 and 3212 2000 t o 1700 1669 1593 and 1453 750 and 704

297

cd

: u

a,

a k cd

k

H

c

ccc

lo-

6.

C. RUDY AND 6. 2 . SENKOWSKI

;--

0a-a-

+-(D--

m--

e--

-

33NVlllWSNVU %

298

ISOCARBOXAZI D

2.2

N u c l e a r Magnetic Resonance Spectrum (NMR) The NMR s p e c t r u m shown i n F i g u r e 2 was o b t a i n e d by d i s s o l v i n g 5 7 . 3 mg o f reference s t a n d a r d i s o c a r b o x a z i d i n 0 . 5 m l o f C D C 1 3 c o n t a i n i n g t e t r a m e t h y l s i l a n e as t h e i n t e r n a l r e f e r e n c e . The s p e c t r a l a s s i g n m e n t s a r e shown i n Table I1 ( 2 ) .

TABLE I 1

NMK S p e c t r a l Assignments f o r I s o c a r b o x a z i d No. o f Each Type P r o t o n - CI-I

Chemical S h i f t

(PP)

Proton

Mult i p 1i c i t y

3

2.45

S

3

4.07

-MI-

1

4.85

s s(b)

=CH-

1

6.47

S

-C& -CONH-

5

7.39

S

1

9.75

s (b)

-3 -CH2 - -

S= s h a r p s i n g l e t ; S ( b ) = b r o a d s i n g l e t U l t r a v i o l e t Spectrum (UV) The UV s p e c t r u m o f i s o c a r b o x a z i d i n 2 - p r o p a n o l i n t h e r e g i o n 350 t o 210 nm e x h i b i t s n o maximamor minima b u t a s h o u l d e r o c c u r s a t 228-229 rim (~=6.6x l o 3 ) . The s p e c t r u m p r e s e n t e d i n F i g u r e 3 was o b t a i n e d from a reference standard s o l u t i o n o f isocarboxazid at a concentrat i o n o f 1 . 0 1 4 mg p e r 100 m l o f ? - p r o p a n o 1 ( 3 ) . 2.3

2.4

F l u o r e s c e n c e Spectrum S o l u t i o n s o f i s o c a r b o x a z i d i n m e t h a n o l , 0.1N H C 1 , and 0.1N NaOli d i s p l a y e d no f l u o r e s c e n c e when t h e e x c i t a t i o n e n e r g y u s e d was w h i t e l i g h t ( 4 ) .

Mass Spectrum The mass s p e c t r u m o f r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d shown i n F i g u r e 4 was o b t a i n e d u s i n g a CEC 21-110 s p e c t r o m e t e r w i t h an i o n i z i n g e n e r g y o f 70 eV ( 5 ) . T a b l e I11 l i s t s t h e d i a g n o s t i c p e a k s a p p e a r i n g i n t h e low r e s o l u t i on s p e c t r u m . 2.5

299

B. C. RUDY AND B. Z. SENKOWSKI

a .r(

N

300

ISOCARBOXAZI D

Figure 3 U l t r a v i o l e t Spectrum o f I s o c a r b o x a z i d

I.o

1

I

I

I

I

I

.8

.6 w

0

z

3 a 0

v)

m

a

.4

.2

I 0 200

2uu

,

-,L

250

300

L3W

JUU

WAVELENGTH,

30 1

nm,

6. C. R U D Y

A N D B. 2. SENKOWSKI

302

ISOCARBOXAZI D

TABLE 111

Mass (m/e) 231 216 171 154 147 127 110 106 91 2.6

Intensity

Fragment I o n s

Medium Very weak Very weak Very weak Weak Strong Medium Me d i um Very s t r o n g

Optical Rotation Isocarboxazid e x h i b i t s no o p t i c a l a c t i v i t y .

2.7

M e l t i n g Range T h e m e l t i n g r a n g e i s d e p e n d e n t on t h e h e a t i n g r a t e . Using t h e Class I m e l t i n g p r o c e d u r e o u t l i n e d i n NF X I I I , i s o c a r b o x a z i d melts between 105" and 108°C ( 6 ) . D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y (DSC) A P e r k i n Elmer DSC-1B C a l o r i m e t e r was u s e d t o o b t a i n t h e DSC c u r v e € o r r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d shown i n F i g u r e 5 . With a t e m p e r a t u r e program o f lO"C/min., a m e l t i n g endotherm was o b s e r v e d s t a r t i n g a t 103.7"C a n d a b r o a d e x o t h e r m due t o d e c o m p o s i t i o n s t a r t i n g a b o u t 150°C. The A l l f c a l c u l a t e d from t h e m e l t i n g endotherm was 7 . 4 k c a l / mole ( 7 ) . 2.8

T h e r m o g r a v i m e t r i c A n a l y s i s (TGA) The T G A p e r f o r m e d on r e f e r e n c e s t a n d a r d i s o c a r boxazid e x h i b i t e d no l o s s o f w e i g h t when h e a t e d t o 120°C a t a r a t e o f 1O"C/min. ( 7 ) . 2.9

2.10

Solubility The s o l u b i l i t v d a t a o b t a i n e d a t 25°C f o r r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d i s l i s t e d i n 'Table I V ( 8 ) .

303

-0

rd

N

.ri

X 0

e

,.c k 0

0

m H

rci 0

a,

> k 3

U U

‘a 0

v c

w,

0

9

0

0

I u

(w

0

B. C. R U D Y AND B. 2 . SENKOWSKI

304

0 Q)

0

0

0

!c

0

d -

co

0

0

03

0 0 N

Y W

3

I):

G

W

I):

5

a w

I-

ISOCARBOXAZI D

TABLE I V

Isocarboxazid - S o l u b i l i t y Solvent

S o l u b i l i t y (mg/ml)

3A alcohol benzene ch 1o r o form 95% e t h a n o l ethyl ether methanol p e t r o l e u m e t h e r (30"-60") 2 -propano1 water

39.6 44.8 348.6 46. 8 16.5 95.1 0.2 16.5 0.8

2.11

X-ray C r y s t a l P r o p e r t i e s The x - r a y powder d i f f r a c t i o n p a t t e r n o f r e f erence standard isocarboxazid i s presented i n Table V ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s are g i v e n below: Instrumental Conditions : Ge ner al E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t : Kadiation : Optics:

50 K V , 1 2 - 1 / 2 MA Copper 0 Cu Ka = 1.542 A 0. 1' D e t e c t o r s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007" N i f i l t e r 4" t a k e o f f a n g l e Scan a t 0 . 2 " 20 p e r m i nut e A m p l i f i e r g a i n - 16 c o a r s e , 8.7 fine Sealed proportional counter t u b e and DC v o l t a g e a t p l a t e a u , Pulse height selection EL - 5 volts; Eu - o u t , Rate meter T . C . 4 , 2000 C/S f u l l s c a l e C h a r t s p e e d 1"/5 m i nut es Pre a r e d by g r i n d i n g a t room t em#er a t u r e

Goniometer: Detector:

Recorder: Samples :

305

B. C. R U D Y A N D B. 2. SENKOWSKI

TABLE V

X-ray D i f f r a c t i o n P a t t e r n o f Isocarboxazid 0

20 9.100 12.440 13.460 14.860 15.240 17.640 18.440 19.040 20,700 21.260 21.720 23.240 23.480 23.920 24.960 25.780 27.300 28.660 29.060 29,680 29.780 30.160 30.280 32.480 34.520 34.800 35.780 36,540 37.400 37.940 38.120 38.760 39,340 40.920 41.420 41.860 42,320 43.080

d*A 9.7177 7.1151 6.5781 5.9614 5.8136 5.0276 4.8113 4.6610 4.2908 4.1790 4.0916 3.8273 3.7887 3,7200 3.5673 3.4557 3.2666 3.1146 3.0726 3.0099 3.0000 2.9630 2.9516 2.7565 2.5981 2.5779 2,5095 2.4590 2.4044 2.3714 2.3606 2.3231 2.2902 2.2053 2.1799 2,1580 2.1356 2,0996

306

I/Io** 41 5 9 13 9 5 4 16 44 9 7 15 18 28 100 9 3 5 6 1 2 2 3 4 7 7 2 4 1 1 1 <1 1 3 2 3 2 1

ISOCAR BOXAZI D

4 5 .480 45.880 46.480 46.860 47.980 4 9 .140 5 0 .140 5 1 .680

1. 9943 1.9778 1. 9537 1.9387 1.8960 1.8539 1.8193 1.7686

*d = ( i n t e r p l a n a r d i s t a n c e )

nX 2 Sin 0

* * I / I o = r e l a t i v e i n t e n s i t y ( bas ed on h i g h e s t i n t e n s i t y o f 1.00)

Dissociation Constant The a p p a r e n t pKa f o r i s o c a r b o x a z i d has been d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y t o be 1 0 . 4 ( 1 0 ) . 2.12

3.

Synthesis I s o c a r b o x a z i d may be p r e p a r e d by t h e r e a c t i o n scheme shown i n F i g u r e 6 . Methyl 5-methyl-3-isoxazolecarboxylate i s r e a c t e d w i t h b e n z y l h y d r a z i n e t o form i s o c a r b o x a z i d ( 1 1 ) . 4.

S t a b i l i t y D egr adat i on I s o c a r b o x a z i d i s s t a b l e when p r e s e n t i n c r y s t a l l i n e form. When h e a t e d a t 100°C i n O . 1 N H C 1 , water, and 0.1N NaOfl, h y d r o l y s i s o c c u r s . After one h o u r o f h e a t i n g , 7% of t h e i s o c a r b o x a z i d was h y d r o l y z e d i n t h e 0.1N H C l s o l u t i o n , 1 2 % i n t h e water s o l u t i o n , and 100% i n t h e 0.1N NaOH s o l u t i o n (12). 5.

Drug M e t a b o l i c P r o d u c t s The predominant pathway o f i s o c a r b o x a z i d m et abol i sm i n man i s t h e c l e a v a g e and o x i d a t i o n o f t h e benzyl m oi et y t o b e n z o i c a c i d f o l l o w e d by a c o n j u n c t i o n w i t h g l y c i n e t o g i v e h i p p u r i c a c i d ( 1 3 ) . A s econd m e t a b o l i c mechanism i s t h e h y d r o l y t i c c l e a v a g e o f i s o c a r b o x a z i d form i ng b e n z y l h y d r a z i n e . T h i s l a t t e r pathway i s more predominant i n g u i n e a p i g s t h a n i n man ( 1 4 ) . F i g u r e 7 o u t l i n e s t h e s e m e t a b o l i c pathways. b.

Methods o f A n a l y s i s 6.1

Elem ent al A n a l y s i s

307

3

a

.r-

a N .

I

z 0

-c k cd 0 0

m

m .d v)

a,

c x

c, f

Cn

f

B

N

0 .-

B. C. RUDY AND B. 2 . SENKOWSKI

'01

I

Y

&

I I

'-

2 yz\o

zI 0=u

0

0

)Y

I I

01

T I

I

z

I

I

L

z

+

6 m

I

V

?

308

0

k 3 M

3

c , 0

0

rd

n c1 0

z

I 0 0 0 c\l

-

ISOCARBOXAZID

n -

I

0

a

I

z

\ /

0

\

q==j

W

2 I-

a X

n 0

Q ‘hl

I 0 I

r I

0=0--0-0

300

I”

-#-

I

hl

e

N

a

m

a 0

0

W

z N

>-

J

I

> N

z

W

m

6 . C. RUDY AND 6 . 2 . SENKOWSKI

The r e s u l t s from an e l e m e n t a l a n a l y s i s o f r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d are p r e s e n t e d i n T a b l e IV ( 1 5 ) . TABLE VI

Elemental A n a l y s i s o f I s o c a r b o x a z i d Element

% Theory

C H N

62.33 5.67 18.17

% Found

62.22 5.66 18.20

6.2

Phase S o l u b i l i t y A n a l y s i s Phase s o l u b i l i t y a n a l y s i s h a s been c a r r i e d o u t f o r i s o c a r b o x a z i d u s i n g 2-propanol a s t h e s o l v e n t . An example i s p r e s e n t e d i n F i g u r e 8 f o r r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d a l o n g w i t h t h e c o n d i t i o n s under which t h e a n a l y s i s was performed ( 8 ) . 6.3

Thin Layer Chromatographic A n a l y s i s (TLC) The f o l l o w i n g TLC p r o c e d u r e i s u s e f u l f o r s e p a r a t i n g methyl 5-methyl-3-isoxazolecarboxylate and 1benzyl-3-methyl-5-aminopyrazole from i s o c a r b o x a z i d (6) Using s i l i c a g e l GF p l a t e s and e t h y l a c e t a t e : n-heptane (60:40) a s t h e s o l v e n t system, 1 . 0 mg o f t h e sample i n methanol i s s p o t t e d on t h e p l a t e and s u b j e c t e d t o a s c e n d i n g chromatography. After t h e s o l v e n t f r o n t has ascended a t l e a s t 1 2 cm, t h e p l a t e i s a i r d r i e d and examined under shortwave u l t r a v i o l e t l i g h t . I f any methyl 5-methyl-3i s o x a z o l e c a r b o x y l a t e i s p r e s e n t i t w i l l a p p e a r a s a dark s p o t a t about Rf 0 . 8 5 . Next s p r a y t h e p l a t e w i t h a f r e s h l y p r e p a r e d s o l u t i o n o f 10% FeC13:5% K3Fe(CN)6 ( 1 : l ) . I f any l-benzyl-3-methyl-5-aminopyrazole i s p r e s e n t i t w i l l a p p e a r a s a b l u e s p o t a t about Rf 0.25. The i s o c a r b o x a z i d produces a b l u e s p o t a t about Rf 0 . 6 .

.

6.4

Colorimetric Analysis 6.41

Ammonium Molybdate Reaction The i s o c a r b o x a z i d c o n t e n t i n t a b l e t s may be determined by t h e f o l l o w i n g p r o c e d u r e ( 6 ) . The t a b l e t s a r e f i n e l y ground, a p o r t i o n e q u i v a l e n t t o about 10 mg o f i s o c a r b o x a z i d i s weighed i n t o a 50 m l v o l u m e t r i c f l a s k and

310

Figure 8

t

1

/ PHASE SOLUBILITY ANALYSIS SAMPLE: SOLVENT:

w

c

ISOCARBOXAZID 2 - PROPANOL

su)pE: 0.0 %I EQUILIBRATION: 20 HRS. ot 25O C EXTRAPOLATED SOLUBILITY: 20.5 W/g af 2 - PROPANOL

e

I

0 0

25 SYSTEM

I 50

I 75

COMPOSITION: mg of SAMPLE per g SOLVENT

i -

I00

-

ffl

0

0

> D

m

0

8. C. RUDY AND 6. Z. SENKOWSKI

d i l u t e d t o volume w i t h a c e t o n e . A f t e r t horough m i xi ng, t h e s o l u t i o n i s c l a r i f i e d by c e n t r i f u g i n g and 5.0 m l i s p i p e t t e d i n t o a s t o p p e r e d f l a s k . To t h e f l a s k a r e a l s o added 1 . 0 m l o f water, 50.0 ml o f a c e t o n e , and 1 . 0 m l o f ammonium molybdate r e a g e n t ( 1 gm o f ammonium molybdate d i s s o l v e d i n 100 m l o f d i l u t e HC1). The f l a s k i s s t o p p e r e d and t h e s o l u t i o n mixed and t h e n a l l o w e d t o s t a n d 30 m i n u t e s w i t h o c c a s i o n a l s w i r l i n g . The abs or ba nce o f t h i s s o l u t i o n i s d e t e r m i n e d a t 420 nm a g a i n s t a b l a n k . Reference standard isocarboxazid i s prepared a t approximately t h e same c o n c e n t r a t i o n and i n a s i m i l a r manner as above. The mg o f i s o c a r b o x a z i d p e r t a b l e t i s c a l c u l a t e d by t h e following formula: (A s a m pl e) (mg s t a n d a r d ) (Av. t a b l e t w t . i n mg) ( A s t a n d a r d ) (mg (5) where 5 i s t h e d i l u t i o n f a c t o r . 6 .4 2

Tetrazolium S a l t Reaction The i s o c a r b o x a z i d c o n t e n t i n bl ood may be d eter min ed i n t h e r a n g e o f 0.5 pg/ml o f plasma by t h e f o l l o w i n g p r o c e d u r e . Add 1.5 m l o f pH 7 . 3 p h o s p h a t e b u f f e r t o 1 m l o f b l o o d and l e t s t a n d f o r 1 0 m i n u t e s . Add 4 ml o f b u t y l a c e t a t e : b u t a n o l ( 9 : l ) and 0 . 7 5 gm o f Na2S04: MgO powder (30: 1 ) . Shake f o r 30 m i nut es and t h e n c e n t r i f u g e u n t i l t h e phases s e p a r a t e . Pipet 3 m l o f t h e organic phase i n t o a t e s t t u b e , add 0 . 1 ml o f t h e t e t r a z o l i u m r e a g e n t ( 0 .1 2 5 % 3,3'-dianisole-bis-4,4'-(diphenyl) - t e t r a z o l i u m c h l o r i d e i n methanol) and 0.1 m l o f O.UL N KOH. Place i n a b a t h o f b o i l i n g w a t e r e x a c t l y one minute t h e n c o o l r a p i d l y by immersing i n c o l d w a t e r . The i s o c a r b o x a z i d h a s r e d u ced t h e t e t r a z o l i u m s a l t t o a b l u e formazan. Read t h e a bs o r b a n c e o f t h e s o l u t i o n a t 520 nm w i t h i n 30 m i n u t e s . A t t h e same t i m e r un t h e above p r o c e d u r e on a 1 ml bl ood b l a n k and a ' l m l b l o o d sample c o n t a i n i n g 5 ugm o f r e f e r e n c e s t a n d a r d i s o c a r b o x a z i d . C o r r e c t f o r t h e bl ank and use t h e r e f e r e n c e s t a n d a r d t o c a l c u l a t e an e x t i n c t i o n c o e f f i c i e n t which i s us ed t o c a l c u l a t e t h e unknowns (16,17), 6.5

P o t e n t i o m e t r i c Sodium N i t r i t e T i t r a t i o n The p o t e n t i o m e t r i c sodium n i t r i t e t i t r a t i o n as d e s c r i b e d i n t h e NF XI11 i s t h e method o f c h o i c e f o r t h e a n a l y s i s o f t h e i s o c a r b o x a z i d fiiie chemical ( 6 ) . A sample

312

ISOCARBOXAZI D

of about 700 mg is accurately weighed and dissolved in 20 m l of glacial acetic acid. Following dissolution 20 ml of I1C1 arid 40 nil o f water is added and the solution cooled to room temperature. Titrate potentiometrically with 0.1N NaN02 solution using a calomel-platinum electrode system. Each ml of 0.1N NaN02 is equivalent to 2 3 . 1 3 of isocarboxaz id.

313

B. C . RUDY AND B. 2. SENKOWSKI

7.

References

M. Hawrylyshyn, Hoffmann-La Roche Inc., Personal Communication. 2 . J. H. Johnson, Hoffmann-La Roche Inc., Personal Communication. 3. L. B. Bandong, Hoffmann-La Roche Inc., Personal Communication. 4 . J. Boatman, Hoffmann-La Roche Inc., Personal Communication. S . W. Benz, Hoffmann-La Roche Inc., Personal communication. 6 . National Formulary XIII, 378-379 ( 1 9 7 0 ) . 7 . S . Moros, Hoffmann-La Roche Inc., Personal Communication. 8 . E. MacMullan, Hoffmann-La Roche Inc., Personal Communication. 9. R. Hagel, Hoffmann-La Roche Inc., Personal Communication. 1 0 * E. Lau, Hoffmann-La Roche Inc., Unpublished Data. 11. T. Gardner and E. Wenis, Hoffmann-La Roche Inc., Unpublished Data. 1 2 . R. Colarusso, Hoffmann-La Roche Inc., Unpublished Data. 13. B. Koechlin, M. Schwartz and W. Oberhznsli, J . PharmacoZ. ExptZ. Therap. 138, 11 ( 1 9 6 2 ) . 1 4 . M. Schwartz, Proc. Soc. Exp. G l . Med. 107, 613 1.

15. 16.

17.

(1961). F . Scheidl, Hoffmann-La Roche, Personal Communica-

tion, M. Roth and J. Rieder, Biochem. Pham. 1 2 , 445 (1963). B . Koechlin, L. D'Arconte, F. Rubio and R. Engleberg, Hoffmann-La Roche Inc., Roche Product ManuaZ ( 1971) .

314

ISOPROPAMIDE

Rulph S. Suntoro, Rickurcl J. Wurrcn, Geruld D. Roberts. Edward White, V , utid Peter P. Begosh

315

RALPH S. SANTORO e t a /

CONTENTS

1.

2.

3. I+.

5. 6.

7.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor, Taste Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Thermal Analysis 2.7 Thermogravimetric Analysis 2.8 Solubility 2.9 Polarography Synthesis Stability Degradation Drug Metabolic Products Methods of Analysis 6.1 Isopropamide Chemical 6.11 Elemental Analysis 6.12 Titration of Iodide Functional Group 6.13 Non-Aqueous Titration 6.1 I+ S pectrophotometric Analy8 is 6.15 Chromatographic Analysis 6.2 Dosage Forms 6.21 Spectrophotometric Analysis 6.22 Spectrophotometric Analysis in Presence of Interferences 6.23 Colorimetric Analysis Notes and References

-

316

ISOPROPAMI DE

1.

Description 1.1

Name, Formula, Molecular Weight Isopropamide is (3-Carbamoyl-3,3-diphenylpropyl)diisopropylmethy1)amonium iodide. It is also known as Darbid; Priamid; Tyrimide; 2,2-diphenyl-4-diisopropylaminobutyrimide methiodide.

Mol. wt.:48O.443

C23H331N20

1.2

Appearance, Color, Odor, Taste A white to off-white (very pale yellow) crystalline or amorphous powder, odorless, with an extremely bitter taste. ~~

2.

Physical Properties

2.1

Infrared Spectrum Figure 1 is the infrared spectrum of isopropamide (SK+F standard SJB-4Q6-226A ) taken in a mineral oil dispersion from 4000 625 cm on a Perkin-Elmer Model 457. R. Warren assigns the following bands (cm to isopropamide:

-

-'

3530 and 3475; free NH 3300; bonded NH2 1665; amide C=O 1585 and 14%; aromatic C=C and NH2 770-710;aromatic CH out-of-plane deformation mono-substituted phenyls

317

MICRONS 2 5

3 0

.O

so

6 0

T O

8 0

P O

10

I2

I4

I6

18 1 0

15 103540 0 0

w c m

WAVENUMBER I C M ~ l l

Figure 1.

-

Isnpropamide SK+F standard SJB-4406-22&, dispersion, Instrument: Perkin-Elmer 457.

mineral oil,

ISOPROPAMIDE

2.2

Nuclear Magnetic Resonance Spectrum I a i n e d in a deutero chloroform' solution of SK+F standard SJB-4406226-A which contained about 100mg/ml and tetramethyls i l a n e as internal reference on a JEOL ModelC 60 H.The following a S S i g n ~ ~ ~(Hz) ~ t € iwere made by R. Warren:

@

0 NH2

9

@

fJ@

- C - CI - CH2

By3 CH2 N D H ( C H J ) J ~

+ @

8

@

63 8 2 ; doublet, protons a t @

177; protons a t

@ @@

244.5 ; multiplet, protons a t @ 340 and 387; protons a t @ 447; protons a t @

319

-1

-

60 M C : ~ ~480 O

I

1

420

I

360 I

300

1

240

I

180

I

120

60

0 CPS

I

I

W

F3 C

I

9

I

8

I

7

I

6

I

5

I

4

I

3

I

2

I

1

0 PP m

Figure 2.

NMR Spectrum Isopropamide, SK+F standard SJB-4406-226A in deuterochloroform tetramethylsilane internal reference, Instrument: JEOL C 60 H .

ISOPROPAMI DE

2.3 Ultraviolet Spectra The ultraviolet absorption spectrum of isopropamide (SK+F standard SJB-4406-226A ) in water is shown in Figure 3. Maxima at 265 nm (a = 0.745; € = 357.9) and 258.5 nm (a = 1.053; (= 505.9) are bands characteristic for the mono-substituted phenyl moiety (1). Figure 4 is the ultraviolet spectrum in water under conditions suitable for observing the iodide maximum absorbance at 222.5 nm (a = 37.0;E = 17,757) (2, 3 , 4 ). Beginning at 220 nm the automatic slit begins to rapidly open. Neither maximum wavelengths nor absorptivities different from N those determined in water solution were observed in C.1 hydrochloric acid and 0.1 N sodium hydroxide solutions. Figure 5 is the ultraviolet absorption spectrum of isopropamide in methylene chloride. The maximum at 245 nm (a = 33.7; f = 16,204) is due to the iodide. The automatic slit begins to rapidly open below 227.5 nm. Figure 6 is the ultraviolet absorption curve of isopropamide in water after the solution was passed through an anionic exchange resin (5) in which the iodide was converted to the chloride; this allows the monosubstituted phenyl spectrum to be more readily observed. Maxima appear at 265 nm (a = 0.721 ; f = 346.4), 258.5 nm = 411.5) (a = 0 . 9 k ; C = 451.8), and 252.5 nm (a = 0.856; Neither maximum shifts nor absorptivity differences were observed in the above spectrum when the solution pH was rendered either acidic or basic.

321

RALPH S.SANTORO e t a / .

Fig. 3

322

ISOPAOPAMIDE

Fig. 4

323

R A L P H S . SANTORO e r a /

WB-44C6-226A

Concentmtmn:

0.0338 n d m l blethylcne Chloride

cell I’ath:

1 Cm

Fig. 5

324

ISOPROPAMIDE

Fig. 6

325

RALPH

S. SANTORO era/.

2.4 Mass Spectrum The mass spectrum of SK+F 4740-5 (SK+F standard SJB-’t’t06-226-A) was obtained by direct insertion of the solid into an Hitachi Perkin-Elmer RMU-6E low resolution mass spectrometer. The results are presented in tabular form in Table I and as a bar graph in Figure 7. E. White and G. Roberts provided the following observations and assignments : The compound thermally decomposes in the instrument, and consequently no molecular ion is observed for the quaternary salt. The spectrum consists of the sum of the spectra of six thermal products which are listed in Table 11. All of the structurally significant peaks are discussed below. Compounds I, 11, and 111, tertiary amines show a molecular ion as noted in Table 11. Each of the compounds cleaves p to the amino nitrogen by losing a methyl group resulting in peaks at m/e 100, rnfe 295 and mfe 323 respectively. Compounds I1 and 111 also exhibit cleavage p to the amino nitrogen with strong peaks at rnfe 86 and mle 114 respectively. Following the loss of the methyl groups from the molecular ions of I1 and I11 there is l o s s of NH, CHO resulting in peaks at mfe 250 and m/e 278. Following the loss of the methyl group from I there is loss of propene resulting in a peak at rnfe 58 as transition is supported by a metastable at rnfe 33.6 Compounds IV, V, and VI are alkyl iodides and as noted in Table 2 a molecular ion is observed for IV and V. Although no molecular ion is observed for VI, its presence is indicated by fragment ions. The peaks at m/e 238 and mle 237 represent the loss of an iodine atom and hydrogen iodide. The ion at m / e 237 further decomposes t o m/e 236 by loss of its hydrogen atom, this transition being supported by a metastable at m/e 235.0. The probable origins of mle 193 and m/e 194 are the l o s s of NH2CH0 from mle 238 and the loss of NHCO from m/e 237. The ions at mle 178 and m/e 179 are possibly formed by loss of a methyl group from mle 193 and mle 194. Other structurally significant peaks which could have originated from more than one of the six compounds

326

ISOPROPAMIDE

MIS 15.8 16.9 19.0

P9.0

8i.a P7.R 8R.R R9.0

3m.m 31.0 3e.m

INTCN. 0 .s

MASS 79.8

INTEN.

a.p

8m.R “ 1 .G

A.5

R.5

R.S

42.6

4.7

e.9 B3.5

83.8 u4.0 R5.R 46.R

s.n 3.

s

11.7 8.9 m.4

97.G R8.R R9.m

33.c

@.a

90.A

34.a

I .9

36.1

R.9

37.R 35.0

9.n 3.4

91.n 3P.n 91.n 94.m

39.63

~m.4

y5.m

4e.e

7. A 49.7

q6.a

41.0 a8.m

43.R

94.5 5R.R

3.7 5.6

1ma.R

I

l0S.Q

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8.3

fn 9

z

20

-I

:: 0

i " ' I " ' I " '

50

Figure

90

130

I " ' I " '

170

210

I

I

I

I

I

250 290 330 370 410 450 490

7. Low resolution mass spectrum of isopropamide, SK+F standard SJB-4406-226A.

ISOPROPAMIDE

m / e 127 (I4), m / e 128 ( H I ? )

are m l e 43 [CH(CH,);?,

and m / e

,

TABLE ---

Thermal I

I1

CH,

I1

Products of I s o p r o p a m i d e

NLWCH~ )272 9?

M

+

115

M

+

310

y 3

NH2 C C CH2 CH2 N CH(CH3 I

)2

8

IV

CHj I

M

VI

M

0

329

+

+

142

n o t observed

RALPH S. SANTORO e t a /

2.5 -Melting Range

Isopropamide (SK+F standard SJB-4h6-226-A) 191.pC. without decomposition melted between 189.0 under USP conditions for class I substances (6).

-

2.6

Differential Thermal Analysis The differential thermal analysis of isopropamide (SK+F standard SJB-4406-226A ) beginning at 43O C and-with a heating rate of 20°C/min. resulted in a single major me1ting endotherm at 200' C

.

Other chemical lots tested under the same conditions showed minor endothermic transitions below the major endotherm. These minor transitions have been tentatively attributed to polymorphic forms known to exist with this compound (7).

2.7

Thermogravimetric Analysis A thermogravimetric analyeis performed on isopropamide (SK+F standard SJB-4406-2264) showed a 0.5' f 0 loss in weight complete at about 105'C. The measurement was performed under nitrogen sweep at a heating rate of Additional weight was rapidly lost as the 10'Clrnin. sample began melting 8t approximately 18pc.

330

ISOPROPAMIDE

2.8 -Solubility

The following solubilities were obtained at room temperature: Solvent ether benzene cyclohexane pH 7.5 buffer water 0.1 N hydrochloric acil 0.1 % sodium hydroxide acetzne 9 9 1 0 ethanol methylene chloride chloroform methanol

Solution -color1ess color1 ess colorless color1ess color 1ess faint yel-Jw color1ess bright yellow faint yellow brownish yellow deep brownish yellow deep brownish yellow

conc. mg / ml

0.07 0.09 0.10

20.7 21.2 23.6

23.6 24.9 109O .

330.4 >500

> 500

In addition, the following solubilities at room temperature have been reported ( 8 ): Solvent Ethyl acetate 1,1, dichloroethane isopropanol Methyl ethylketone 1 g hydrochloric acid

Conc. mdml 0.1 1*3 3.2

4.0 29.0

331

RALPH S. SANTORO et a/.

2.9

Polarography The polarogram was obtained on isopropamide (SK+F potassium nitrate with a standard SJB-kh06-28A ) in 0.1 standard calomel IdropDing mercury electrode system at a concentration of 5 x 10 -’& molar ( 9 ) . The E 112 was -0.25k V. The E 112 for iodide determined under similar conditions was [email protected] V.

7.

Synthesis The synthetic pathway to isopropamide includes the following steps (Figure 8):

2-Diisopropylaminoethyl chloride (I) is condensed with diphenylacetonitrile (11) in the presence of an alkaline condensing agent and the product 4-diisopropylamino-2,2diphenvlbutyronitrile (111) purified by distillation. By reaction with acid, the 4-diisopropyLamino-2,2diphenylbutyronitrile (111) is partially hydrolyzed to 4diisopropylamino-2,2-diphenylbutyramide (IV) which is Durified by recrystallization. Finally (3-carbamoyl-3,3-diphenylpropyl)-diisopropylmethylammonium iodide (V) is prepared by reacting 4diiso~ronylamino-2,2-diphenylbutyramide(IV) in solution with methyl iodide. The product is collected by filtration, washed, dried and assayed. IsoproDamide is covered by U . S . Patent Number 2823233. The original synthesis is described by Janssen et al. (lo). l~

-Stability - _----

Degradation

_I_--_I

The dry chemical stored in glass bottles at room

temnerature is stable for at least ? years. In aqueous eolution (1 - 5 rngtrnl) stored at room temperature the drug is stable for at least 6 months 01). Because the drug is stable, degradation products have not been observed to occur either under normal or exaggerated storage conditions.

5. g ~ u gMetabolic Products No isoDropamfde metabolic products have been reported thus far.

332

Figur.:

.!

-

SYNTHETIC PATHWAY TO ISOPROPAMIDE

NC -kH

+

C1CH2CHPi

alkaline

\HC,

/CH,

condensing agent

>

1

RALPH S.SANTORO e t a / .

6. Methods of Analysis 6.1

Isopropamide Chemical 6.11

Elemental Analysis

Element C

H N 6.12

Theory

O l a

Range Obtained

57.50 6.90

57.80

5.83

5.69

7.00

- 57.92 - 7.06 - 5.78

-Titration .

of Iodide Functional Group An accurately weighed sample (About 1 g.) is dissolved in a 5:5: 1 mixture of water:methyl alcohol: silver nitrate glacial acetic acid and titrated with 0.1 and Eosin Y as indicator until the precipitate becomes rose red. Each milliliter of 0.1 N silver nitrate is equal to 0.01269 of iodide or 0.04804 g. of isopropamide.

6.13 -_. Non Aqueous . _-.-- Titration ---- ~

~

An accurately weighed sample (about 0.75 g.) is dissolved in glacial acetic acid, mercuric acetate and methyl violet indicator added, and titrated with 0.1 N Derchloric acid in acetic acid to a blue endpoint. Ezch milliliter of 0.1 N perchloric acid is equivalent to 0.0'+804 R. of isopropamide.

6.14

-Spectrophotometric __ Analysis _~

The ultraviolet absorption spectrum obtained either by direct dilution or preliminary treatment with resin (see section 2.3) may be used for determining purity.

6.15 Chromatographic Analysis The following thin layer method may be used for the qualitative purity evaluation of isopropamide: Equilibrate a mixture of 60 ml. each of isoamyl and tert-amyl alcohols with 20 ml. of 88'1, formic acid dissolved in 100 ml. of water. Discard the aqueous layer and use the alcohol layer as the chromatographic solvent. Spot 25 and 50 micrograms of isopropamide dissolved in methanol two cm. from the edge of an 'Avicelf 334

ISOPROPAMIDE

or cellulose plate (12)~place the prepared plate in a suitable chromatographic chamber lined with filter paper saturated with the developing solvent, and allow to equilibrate for 45 minutes. Allow the solvent to rise to a line drawn across the plate 10 cm. from the origin, remove the plate, and air dry in a fume hood until solvent vapore are no longer detectable. The developed chromatogram may be visibilized under ultraviolet light (254 and 365 nm), visible light, and iodoplatinate reagent (13). The quaternary moiety has an approximate Rf of 0.8, while the iodide appears at approximately 0.2.

6.2 Dosage Forms Isopropamide is found in dosage forms both alone and in combination with primary and tertiary amines. Assay methods include ultraviolet analysis after extraction of interfering amines and treatment with an anionic exchange resin, and colorimetric analysis by ion-paring with acid dyes and extraction of the complex into an organic solvent.

S pectrophotm e t r i c Ana 1ys is The ultraviolet assay of tablets which contain only isopropamide as the active ingredient is the most direct method employed (14). Twenty tablets ( 5 mg/ tablet) are placed in a 250 ml. volumetric flask, disintegrated in about 150 ml. of water, and diluted to volume with water. The solution is filtered through Whatman No. 1 filter paper (15),W.O ml. of the filtrate percolated through an anion exchange column (16), and the column rinsed thoroughly with water. A l l eluates are collected in a 100 ml. volumetric flask and diluted to volume with water. The ultraviolet absorption spectrum of this solution is compared with that of a known standard of isopropamide under the same spectrophotometric conditi ns and the assay value per tablet calculated. 6.21

6.22 spectrophotometric Analysis in Presence of Interferences The ultraviolet assay of dosage forms which contain nther amines removable by solvent extraction is accompl shed by treating the eluate collected in 6.21 with ammonium hydroxide until basic and extracting with ether to remwe the Interfering amines (17). 1

335

RALPH S. SANTORO e t a / .

6.23

Colorimetric Analysis The s e l e c t i v e d e t e r m i n a t i o n of isopropamide . . by i o n - p a i r i n g w i t h methyl orange dye h a s been d e s c r i b e d (18). The q u a t e r n a r y amine i s i o n - p a i r e d a t pH 10.2 and e x t r a c t e d i n t o chloroform. Primary, secondary, and t e r t i a r y amines t e s t e d through t h e procedure d i d n o t cqmplex o r i n t e r f e r e w i t h t h e subsequent s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n a t 520 nm.

336

ISOPROPAMI DE

7. Notes and References XVIII, p. 825. Brode, W.R., Chemical Spectroscopy, 2nd Ed., John Wiley and Sons Inc., New York, (1947). 3. Brode, W.R., J. Am. Chem. SOC., 48, 1877, (1926). 4. Bolz, D.F., Colorimetric Determination of Nonmetals, Volume V I I I , Interscience Publishers Inc., 1947, p. 218. 5 . Amberlite IRA !+GO, chloride form, Mallinkrodt Chemical Works. 6. U . S . P . XVIII, p. 935. 7. I. B. Eisdorfer, Personal Communication. 8. Paul Janssen, Private Communication, Eupharma-NedChem Pharmaceutical Laboratories. 9. Kolthoff, I.M., and Lingane, J . J . , Polarography, Second Edition, Interscience Publishers, New York and London, 1952, p. 579. 10. Janssen et al., Arch. Intern Pharmacodyn., 103, 1955, 82. 11. Private Communication, Eupharma-Nedchem Pharmaceutical Laboratories. 12. 'Avicel'-microcrystalline cellulose American Viscose. Silica gel may cause decomposition of the compound and is to be avoided. 13. One gram of chloroplatinic acid, 10 ml. of 1; hydrochloric acid, and 5 g. of potassium iodide diluted to 250 ml. with waterasa stock solution. Refrigerate. Prepare a working solution with equal volumes of stock solution and water made 112" l o with 88" lo formic acid. 14. M. Kushner, Personal Communication. If the solution is not clear at this point it will 15 be necessary to add 5 ml. of l@ /o aluminum chloride and 2 ml. of concentrated ammonium hydroxide to the l5G ml. of water before diluting to volume. 16. Amberlite IRA LOO, chloride form. 17. P. DeLuca and W. F. Witmer, Personal Communication. 18. R. Santoro, J. Am. Pharm. ASSOC., S c i . Ed., 49, 1960, 666.

1.

U.S.P.

2.

-

-

337

RALPH S. SANTORO e t a / .

Acknowledgements The authors wish to thank Joan Rudin for her search of the literature and Patricia Brittingham for her invaluable secretarial help.

338

LEVALLORPHAN TARTRATE

Bruce C. Rudy and Berriurd Z. Senkowski

339

B. C RUDY AND B 2 . SENKOWSKI

INDEX Analytical P r o f i l e - Levallorphan T a r t r a t e 1.

Description 1.1 N a m e , F o r m u l a , M o l e c u l a r Weight 1.2 A p p e a r a n c e , C o l o r , Odor

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F l u o r e s c e n c e S p e c t r u m 2.5 Mass S p e c t r u m 2.6 Optical Rotation 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a l Scanning Cdlorimetry 2.9 Thermal G r a v i m e t r i c A n a l y s i s 2.10 S o l u b i l i t y 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2.12 D i s s o c i a t i o n C o n s t a n t

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug M e t a b o l i c P r o d u c t s

6.

Methods o f A n a l y s i s 6.1 Elemental Analysis 6.2 Phase S o l u b i l i t y Analysis 6.3 Thin Layer Chromatographic A n a l y s i s 6.4 Gas L i q u i d Chroma t o g r a p h i c A n a l y s i s 6.5 Direct S p e c t r o p h o t o m e t r i c A n a l y s i s 6.6 Colorimetric Analysis 6.7 Nephelometric Analysis 6.8 T i t r i m e t r i c Analysis

7.

References

340

LEVALLORPHAN TARTRATE

1.

Description

Name, Formula, M o l e c u l a r Weight L e v a l l o r p h a n t a r t r a t e i s (-)-17-allylmorphinan-301 t a r t r a t e (1:l). 1.1

qy

I7 CH*CH=CH,

I

HO

H2C4H406

6

C

H NO.C4H6O6 1 9 25

M o l e c u l a r Weight:

433.51

1.2

Appearance, C o l o r , Odor Levallorphan t a r t r a t e occurs as a w h i t e o r pract i c a l l y w h i t e , o d o r l e s s , c r y s t a l l i n e powder. 2.

Physical Properties

2.1

I n f r a r e d Spectrum ( I R ) The I R s p e c t r u m of l e v a l l o r p h a n t a r t r a t e i s g r e a t l y c o m p l i c a t e d by t h e p r e s e n c e o f t h e t a r t a r i c a c i d . To o b t a i n t h e I R s p e c t r u m c h a r a c t e r i s t i c of t h e a c t i v e s p e c i e s , t h e f r e e l e v a l l o r p h a n b a s e was e x t r a c t e d from a b a s i c s o l u t i o n , d r i e d , and measured a s a K B r d i s p e r s i o n ( 1 . 7 mg l e v a l l o r p h a n / 3 0 0 mg KBr) on a P e r k i n E l m e r 621 S p e c t r o p h o t o m e t e r . T h i s s p e c t r u m i s shown i n F i g u r e 1 and t h e a s s i g n m e n t s f o r t h e c h a r a c t e r i s t i c bands a r e g i v e n i n T a b l e I (1). Table I I n f r a r e d Assignments f o r L e v a l l o r p h a n Frequency (cm-1)

C h a r a c t e r i s t i c of

3580*

OH s t r e t c h

3076

Aromatic CH s t r e t c h Asymmetric and symmetric s t r e t c h of CH2 C=C s t r e t c h of v i n y l g r o u p

2924-2919 and 2851 1644

*Detectable only i n s o l u t i o n spectrum, not i n KBr p e l l e t .

341

Figure 1

Infrared Spectrum of Levallorphan

WAVELENGTH

2.5 10

3

I

4 I

I

5 I

6 1

MICRONS

7 I

8 I

9 10 l

l

12 1 I

15 1822 3550 I l l 1 m

0

n C

[7

-<

b

2 0

m N v)

m

2

x

I

I

I

I

4000 3500 3000 2500 2000

I 1700

I 1400

I 1100

FREQUENCY (CM-')

I 800

I

1

500

200

2 m 5

LEVALLORPHAN TARTRATE

1607

Aromatic s k e l e t a l v i b r a tions CH d e f o r m a t i o n s 2 CH o u t o f p l a n e b e n d i n g of v i n y l g r o u p

1454 992 and 910 2.2

N u c l e a r M a g n e t i c Resonance Spectrum (NMR) The s p e c t r a of l e v a l l o r p h a n t a r t r a t e (A) and l e v a l l o r p h a n b a s e (B) a r e shown i n F i g u r e 2. These s p e c t r a were o b t a i n e d by d i s s o l v i n g a b o u t 63 mg o f t h e a p p r o p r i a t e sample i n 0.5 m l o f DMSO-d6 c o n t a i n i n g t e t r a m e t h y l s i l a n e as a n i n t e r n a l s t a n d a r d (2). The s p e c t r a l a s s i g n m e n t s f o r t h e l e v a l l o r p h a n b a s e a r e g i v e n i n T a b l e 11. Aside from t h e a d d i t i o n a l p e a k s from t h e p r o t o n s on t h e t a r t r a t e i t s e l f , * t h e major d i f f e r e n c e between t h e b a s e and t h e t a r t r a t e s a l t o c c u r s i n t h e methylene r e g i o n . The b r o a d , n o n - d e s c r i p t bands a r e a t t r i b u t e d t o t h e change i n t h e n i t r o g e n quadrop o l e moment c a u s e d by t h e p o s i t i v e c h a r g e p r e s e n t on t h e nitrogen

.

T a b l e I1 NMR Assignments f o r L e v a l l o r p h a n Base

Type o f P r o t o n s

No. o f Protons

Chemical S h i f t ( PPm)

methylene p rotons on c a r b o n s 5,6,7, 8, and 15

10

1.00-1.95

methylene p rotons on c a r b o n 1 0 and methyne p r o t o n on c a r b o n 1 4

3

2.10-2.65

methylene p rotons on c a r b o n 1 6 and methyne p r o t o n on carbon 9

3

2.75-3.15

Multiplicity

*The t a r t r a t e h a s 2 C-H p r o t o n s which show a s h a r p s i n g l e t a t 4.10 ppm and 2 OH p l u s 2 COOH p r o t o n s which a l o n g w i t h t h e OH p r o t o n from t h e l e v a l l o r p h a n m o l e c u l e g i v e a b r o a d s i n g l e t a t 8 . 4 9 ppm. 343

B. C . RUDY AND B. 2. SENKOWSKI

Figure 2 NMR S p e c t r a of L e v a l l o r p h a n and L e v a l l o r p h a n T a r t r a t e

344

LEVALLORPHAN TARTRATE

methylene protons adjacent t o the N i n a l l y l chain

2

methylene protons a t end o f a l l y l group

2

4.95-5.25

Mu1t ip l e t

methyne p r o t o n i n a l l y l group

1

5.50-6.10

Mu1 t i p l e t

aromatic protons a t c a r b o n s 2 and 4

2

6.40-6.61

Mu1 t i p l e t

aromatic proton a t carbon 1

1

6.90

Doublet (J=8Hz)

OH p r o t o n

1

9 .oo

Broad singlet

3.10

2.3

Doublet (J=7Hz)

U l t r a v i o l e t Spectrum (UV) The UV s p e c t r a of l e v a l l o r p h a n t a r t r a t e i n water (A) and i n 1 N N a O H (B) a r e shown i n F i g u r e 3 ( 3 ) . The maxima and minima as w e l l as t h e m o l a r e x t i n c t i o n c o e f a r e l i s t e d i n T a b l e 111. The v a l u e s f i c i e n t s a t t h e imax o b t a i n e d a g r e e w e l l w i t h t h e o n e s r e p o r t e d by F a r m i l o and Genest ( 4 ) and Mu16 ( 5 ) . T a b l e I11 UV A b s o r p t i o n S p e c t r a l Data f o r L e v a l l o r p h a n T a r t r a t e

Solvent

Wavelength o f Molar Maximum (nm) A b s o r p t i v i t y

water

279

2.04

103

1N H C 1

216 278

7.25 2.08

103 103

1N N a O H

216 299

7.42 3.18

103 103

239

9.18

103

Wavelength of Minimum (nm)

245

244 268

6 . C. R U D Y A N D 8 . Z . SENKOWSKI

Figure 3 Ultraviolet Spectrum of Levallorphan Tartrate

10 4

:

Levollorphon T a r t r o l e In Woti ( I:

8

:

20,000I

Levallorphan Tarfrore ~n I N N (I:

50.0001

8

6 W

u z a at Lz

0

rn

at

a

4

2

0

210

250

300

NANOMETERS

346

350

LEVALLORPHAN TARTRATE

2.4

Fluorescence S p e c t x The e x c i t a t i o n and e m i s s i o n s p e c t r a f o r l e v a l l o r p h a n t a r t r a t e ( 1 mg/ml of m e t h a n o l ) i s shown i n F i g u r e 4 ( 6 ) . Two maxima a p p e a r i n t h e e x c i t a t i o n s p e c t r u m a t 306 nm. When 0 . 1 N H C 1 i s u s e d as t h e s o l v e n t t h e maxima o c c u r a t t h e same w a v e l e n g t h s b u t a 5 f o l d i n c r e a s e i n s e n s i t i v i t y i s obtained.

2.5

Mass Spectrum The mass s p e c t r u m o f l e v a l l o r p h a n t a r t r a t e w a s o b t a i n e d u s i n g a CEC 21-110 m a s s s p e c t r o m e t e r w i t h a n ioni z i n g e n e r g y of 7 0 eV. The o u t p u t from t h e mass s p e c t r o meter w a s a n a l y z e d and p r e s e n t e d i n t h e form of a b a r g r a p h , shown i n F i g u r e 5, by a V a r i a n 100 MS d e d i c a t e d computer s y s t e m ( 7 ) . The mass s p e c t r u m i s a s u p e r i m p o s i t i o n of t h e The s p e c t r a o f t h e l e v a l l o r p h a n b a s e and t a r t a r i c a c i d . m o l e c u l a r i o n of l e v a l l o r p h a n a t m / e 2 8 3 i s t h e s t r o n g e s t peak. The p e a k a t m / e 256 (M-27) c o r r e s p o n d s t o t h e loss of CH=CH2. The o t h e r p e a k s a t m / e 2 6 8 , 2 4 2 , 2 4 0 , and 226 a r e due t o t h e l o s s of o t h e r h y d r o c a r b o n m o i e t i e s . Since t h e p o l y c y c l i c s a t u r a t e d r i n g s y s t e m a l l o w s a m u l t i t u d e of f r a g m e n t a t i o n pathways, a n a s s i g n m e n t of t h e s e i o n s t o c e r t a i n p a r t s of t h e m o l e c u l a r s t r u c t u r e c o u l d a t b e s t t o v e r y tentative ( 7 ) . 2.6

Optical Rotation The s p e c i f i c r o t a t i o n of l e v a l l o r p h a n t a r t r a t e i n w a t e r a t 25OC i s p l o t t e d v e r s u s w a v e l e n g t h i n F i g u r e 6 ( 3 , 8 ) . The s p e c i f i c r o t a t i o n o b s e r v e d a t 5 8 9 nm w a s -39.0'. The c h a r a c t e r i s t i c p a r t s of t h e c u r v e a r e a peak a t 2 9 0 nm ( - 1 1 6 5 O ) , a t r o u g h a t 267 nm (+240°) and a peak a t 2 2 0 nm (-5854'). Zero i n t e r c e p t s o c c u r a t 2 7 0 and 264 nm ( 8 ) . The s p e c i f i c r o t a t i o n r a n g e r e p o r t e d i n t h e USP X V I I I f o r l e v a l l o r p h a n t a r t r a t e i s from - 3 7 . 0 t o -39.2' a t 5 8 9 nn- ( 9 ) .

2.7

M e l t i n g Range The m e l t i n g r a n g e r e p o r t e d i n t h e USP X V I I I f o r l e v a l l o r p h a n t a r t r a t e i s 174' t o 177O u s i n g t h e c l a s s I a procedure ( 9 ) .

2.8

D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y (DSC) The DSC c u r v e shown i n F i g u r e 7 f o r l e v a l l o r p h a n t a r t r a t e w a s o b t a i n e d u s i n g a P e r k i n E l m e r DSC-1B C a l o r i meter. With a t e m p e r a t u r e program o f 1O0C/min, a m e l t i n g

347

6 . C. RUDY AND B. 2 . SENKOWSKI

Figure 4 Fluorescence Spectrum of Levallorphan T a r t r a t e

1 Excilolion / 2 00

250

350

300

NANOMETERS

348

400

450

LEVALLORPHAN TARTRATE

349

I.

In

6.C . RUDY AND 6 . 2.SENKOWSKI

Figure 6 P l o t of S p e c i f i c R o t a t i o n v s . Wavelength of L e v a l l o r p h a n T a r t r a t e

350

LEVALLORPHAN TARTRATE

Figure 7 DSC Curve of Levallorphan Tartrate

1 A H f = 10 4 kcol/mole

I 140

150

160

TEMPERATURE

35 1

180

170 OC

190

6. C . R U D Y AND 6.

Z. SENKOWSKI

endotherm was o b s e r v e d s t a r t i n g a t 174.8' k c a l f m o l e (10)

.

w i t h a AHf = 1 0 . 4

2.9

T h e r m o g r a v i m e t r i c A n a l y s i s (TGA) The TGA s c a n r u n on l e v a l l o r p h a n t a r t r a t e showed no w e i g h t l o s s from 30' t o 217OC. One c o n t i n u o u s w e i g h t l o s s o c c u r r e d from 2 1 7 O t o 47OoC which a c c o u n t e d f o r ess e n t i a l l y a l l o f t h e sample (10). 2.10

Solubility The s o l u b i l i t y d a t a o b t a i n e d f o r l e v a l l o r p h a n t a r t r a t e a t 25OC are l i s t e d i n T a b l e I V (11). Table I V S o l u b i l i t y of L e v a l l o r p h a n T a r t r a t e i n D i f f e r e n t S o l v e n t s Solvent

S o l u b i l i t y (mgfml)

3 A alcohol benzene ch 1o r o f o r m 95% e t h a n o l ethyl ether methanol p e t r o l e u m e t h e r (30'-60') 2-propanol water

7.8 0.2 0.3 26.5 0.2 79.5 ~0.03 1.5 60.0

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of l e v a l l o r p h a n t a r t r a t e i s p r e s e n t e d i n T a b l e V ( 1 2 ) . These v a l u e s a r e v e r y s i m i l a r t o t h o s e g i v e n by F a r m i l o and Genest ( 4 ) . The i n s t r u m e n t a l c o n d i t i o n s a r e g i v e n below. 2.11

Instrument Conditions: G e n e r a l E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t : Radiation:

50KV, 12-112 MA Copper Cu K,= 1 . 5 4 2 El

352

LEVALLORPHAN TARTRATE

0.1' D e t e c t o r s l i t M.R. S o l l e r s l i t 3' B e a m s l i t 0.0007 i n c h N i f i l t e r 4O t a k e o f f a n g l e Scan a t 0.2O 28 p e r m i n u t e Amplifier g a i n - 16 c o u r s e , 8.7 f i n e Sealed proportional counter t u b e and DC v o l t a g e a t plateau P u l s e h e i g h t s e l e c t i o n EL-5 v o l t s ; EU - o u t Rate meter T.C. 4 2000 CIS f u l l s c a l e C h a r t Speed - 1 i n c h p e r 5 minutes

Optics:

Goniometer: Detector :

Recorder :

Samples p r e p a r e d by g r i n d i n g a t room t e m p e r a t u r e . Table V

X-ray Powder D i f f r a c t i o n P a t t e r n of L e v a l l o r p h a n T a r t r a t e

20

d

-

9.18 11.90 12.18 13.06 14.12 14.28 15.32 15.84 17.22 18.50 19.00 19.16 19.70 19.90 20.18 20.78 2 1 * 00 21.48

9.63 7.44 7.27 6.78 6.27 6.20 5.78 5.59 5.15 4.80 4.67 4.63 4.51 4.46 4.40 4.27 4.23 4.14

30 33 15 78 10 32

100 32 6 21 12 34 10 34 32 22 49 14

29.18 29.66 30.10 30.24 30.38 30.92 31.08 31.74 31.86 34.14 34.30 35.02 35.16 35.32 35.70 35.86 36.22 36.72 353

(1)*

3.06 3.01 2.97 2.96 2.94 2.89 2.88 2.82 2.81 2.63 2.61 2.56 2.55 2.54 2.51 2.50 2.48 2.45

I! I,**

23 10 1 2 2 10 11 14 17 1 1 11 13 12 5 5 2 5

8.C. R U D Y AND 8.2.S E N K O W S K I

21.76 23.12 23.88 24.06 24.62 24.86 25.58 26.00 26.16 26.42 26.78 27.22 27.58 27.98 28.74 28.84

*d

-

**-I/I, I

4.08 3.85 3.73 3.70 3.62 3.58 3.48 3.43 3.41 3.37 3.33 3.28 3.24 3.19 3.11 3.10

39 6 11 11 32 85 5 18 18 34 1 9 8 4

37.38 38.08 38.40 38.80 39.40 39.96 40.06 40.24 40.52 43.92 41.74 42.04 43.62 43.82 44.08 45.14

11

16

(interplanar distance) = relative intensity

2.41 2.36 2.34 2.32 2.29 2.26 2.25 2.24 2.23 2.21 2.16 2.15 2.07 2.07 2.05 2.01

1 4 6

33 1 4 6 7 4 7 1 6 3 5 13 6

nh

2 Sin 0 ( b a s e d on h i g h e s t i n t e n s i t y o f 1.00)

2.12

Dissociation Constant The pKa's a t room t e m p e r a t u r e f o r l e v a l l o r p h a n t a r t r a t e h a v e b e e n d e t e r m i n e d by a pH t i t r a t i o n w i t h sodium h y d r o x i d e t o b e 4.5 and 6.9 (13). The f i r s t pKa o b s e r v e d € o r l e v a l l o r p h a n t a r t r a t e r e s u l t s from t h e i o n i z a t i o n of t h e c a r b o x y l i c p r o t o n from t h e t a r t a r i c a c i d . The second pKa i s due t o t h e i o n i z a t i o n o f t h e p r o t o n from t h e ammonium moiety i n t h e l e v a l l o r p h a n t a r t r a t e .

3.

Synthesis L e v a l l o r p h a n t a r t r a t e may b e p r e p a r e d by t h e r e a c t i o n scheme shown i n F i g u r e 8. (+)-l-(p-hydroxybenzyl)-l,2,3,4, 5,6,7,8-octahydroisoquinoline i s r e a c t e d w i t h a l l y l b r o m i d e t o g i v e (-)-l-(p-hydroxybenzyl)-2-allyl-l,2,3,4,5,6,7,8o c t a h y d r o i s o q u i n o l i n e which i s c y c l i z e d i n p h o s p h o r i c a c i d Tartaric a c i d is then t o (-)-3-hydroxy-17-allyl-morphinan. added t o make l e v a l l o r p h a n t a r t r a t e (14).

4. S t a b i l i t y D e g r a d a t i o n Levallorphan tartrate substance is s t a b l e i n t h e p r e s e n c e o f l i g h t and oxygen from t h e a i r . I n aqueous

354

LEVALLORPHAN TARTRATE

Figure 8

SYNTHESIS OF LEVALLUWHIW TARTRATE

355

6. C . RUDY A N D 8. Z . SENKOWSKI

s o l u t i o n l e v a l l o r p h a n t a r t r a t e w a s shown t o b e s t a b l e when h e a t e d t o 45OC f o r 1 2 weeks. Only when t h e s o l u t i o n w a s s a t u r a t e d w i t h oxygen and s u b j e c t e d t o u l t r a v i o l e t r a d i a t i o n d i d s l o w d e c o m p o s i t i o n o c c u r . The l e v a l l o r p h a n tart r a t e shows no e f f e c t s from s t e r i l i z a t i o n a t 120°C f o r 20 minutes (15).

5.

Drug M e t a b o l i c P r o d u c t s The m e t a b o l i c f a t e o f l e v a l l o r p h a n t a r t r a t e i n a n i m a l s h a s been s t u d i e d by Mannering and Schanker ( 1 6 ) . Two metab o l i c p r o d u c t s were i s o l a t e d from u r i n e , f e c e s , and t h e l i v e r of mice. One o f t h e m e t a b o l i t e s w a s p o s i t i v e l y i d e n t i f i e d as t h e N-dealkylated p r o d u c t , 3-hydroxymorphinan. The o t h e r m e t a b o l i t e w a s n o t i d e n t i f i e d u n t i l r e c e n t l y when i t was shown t o b e (-)-17-allyl-3,6-dehydroxymorphinan by x-ray c r y s t a l l o g r a p h y ( 1 7 ) . The 3-hydroxymorphinan metab o l i t e w a s a l s o found i n t h e u r i n e o f g u i n e a p i g s , r a b b i t s , and dogs ( 1 6 ) . 6.

Methods o f A n a l y s i s

6.1

Elemental Analysis The r e s u l t s from t h e e l e m e n t a l a n a l y s i s o f l e v a l l o r p h a n t a r t r a t e a r e l i s t e d i n T a b l e V I (18). Table V I E l e m e n t a l A n a l y s i s of L e v a l l o r p h a n T a r t r a t e Element C H N

% Theory

63.73 7.21 3.23

% Found

63.77 7.26 3.22

6.2

Phase S o l u b i l i t y Analysis Phase s o l u b i l i t y a n a l y s i s i s c a r r i e d o u t u s i n g a b s o l u t e e t h a n o l a s t h e s o l v e n t . A t y p i c a l example i s s h m i n F i g u r e 9 which a l s o l i s t s t h e c o n d i t i o n s u n d e r which t h e a n a l y s i s was c a r r i e d o u t (11).

6.3

Thin Layer Chromatographic A n a l y s i s (TLC) S e v e r a l TLC s y s t e m s h a v e been p u b l i s h e d which c a n b e used t o s e p a r a t e l e v a l l o r p h a n b a s e from i t s m e t a b o l i t e s

356

Figure 9

1 .. 0

i

2 i

ly

SAMPLE:

LEVALLORPHAN TARTRATE

SOLVENT: SLOPE: ~~

ABSOLUTE ETHANOL

0.15%

EQL'ILIBRAT;:

20 HRS. at 25'C

1 1

PHASE SOLCBILITY ANALYSIS

~~

EXTRAPOLATED SOLUBILITY: 7 . 1 8 mg/g of ETHANOL

n

0

100 bYSTEH C O W O S I T I O N :

mg of SAHPLE p e r g SOLVENT

rn r

<

F

c

6. C. RUDY A N D B. 2. SENKOWSKI

and other similar compounds (5,191. In each case about 25 ug of sample was spotted on a Silica gel G plate (250 microns thick) and subjected to ascending chromatography. After development of at least 10 em, the plate is air-dried f o r 10 minutes, dried in an oven at 100°C f o r 15 minutes, and sprayed with iodoplatinate reagent made by adding 10 ml of a 10% solution o f platinum chloride to 250 ml of 4 % potassium iodide and diluting to 500 ml with water. The solvent systems as well as the approximate Rf of the levallorphan spot are listed in Table VII (5). Table V I I TLC Data for Levallorphan Rf of Levallorphan

Solvent System benzene:dioxane:ethanol:ammonium hydroxide (10:8:1: 1)

.98

n-butano1:conc. hydrochloric acid, saturated with water (9:l)

.73

ethanol:glacial acetic acid:water ( 6 : 3:1)

.70

ethano1:dioxane:pyridine:water (10:5: 4 : 1)

.65

n-butano1:water:glacial acetic acid ( 4 : 2 : 1 )

.64

t-amyl alcoho1:water:n-butyl ether (80:13: 7)

.44

methano1:n-butano1:water:benzene ( 1 2 : 3: 3 : 2 )

.41

Liquid Chromatographic Analysis (GLC) A GLC method for the separation and quantitation of levallorphan base in the presence of its metabolites and similar structured compounds has been reported by Mulk (5, 19). The pertinent experimental conditions as well as the retention time for levallorphan are given in Table VIII (5). 6.4

Gas

358

LEVALLORPHAN T A R T R A T E

Table V I I I GLC Method f o r L e v a l l o r p h a n as t h e F r e e Base

Co 1umn : Support: S t a t i o n a r y Phase: Detector: Temperature Injection Port: Column: Detector : Flow Rate: Q u a n t i t i e s I nj e c t e d : Retention Time:

6 f t , 3 mm i d , c o i l e d g l a s s Gas Chrom S (80-100 mesh) 2% SE-30 Radium - 226 a r g o n i o n i z a t i o n 260' 215O 215' 47 cclminute of argon 2-25 pg of l e v a l l o r p h a n b a s e i n methanol 5.72 minutes

6.5

Direct S p e c t r o p h o t o m e t r i c A n a l y s i s D i r e c t s p e c t r o p h o t o m e t r i c a n a l y s i s of l e v a l l o r p h t a r t r a t e h a s n o t b e e n found t o b e a p p l i c a b l e i f any metab o l i t e s o r s i m i l a r s t r u c t u r e d compounds are p r e s e n t . Howe v e r , i f no i n t e r f e r i n g s p e c i e s a r e p r e s e n t , t h e r e p o r t e d maximum a t 279 nm i n water and i n 1 N H C 1 o r a t 299 nm i n 1 N N a O H may b e u s e d f o r q u a n t i t a t i v e m e a s u r e m e n t s . 6.6

Colorimetric Analysis 6.61

I o n P a i r E x t r a c t i o n o f B r o m o c r e s o l Green Complex A c o l o r i m e t r i c method which t a k e s a d v a n t a g e o f t h e q u a t e r n a r y amine m o i e t y i n l e v a l l o r p h a n t a r t r a t e i s t h e e x t r a c t i o n o f t h e i o n p a i r formed w i t h b r o m o c r e s o l The l e v a l l o r p h a n t a r t r a t e i n i n j e c t d b l e s i s a s s a y e d green. b y t h i s method ( 9 ) . The complex i s formed i n an a q u e o u s 5 . 3 p h o s p h a t e b u f f e r and e x t r a c t e d i n t o c h l o r o f o r m . The a b s o r b a n c e i s r e a d a t t h e 4 2 0 nm maximum a l o n g w i t h t h e absorbance o f a reference standard levallorphan tartrate p r e p a r e d i n t h e same way. T h e s e a b s o r b a n c e v a l u e s a r e u s e d t o c a l c u l a t e t h e c o n c e n t r a t i o n of l e v a l l o r p h a n t a r t r a t c p e r ml p r e s e n t i n t h e i n j e c t a b l e .

6.62

F o l i n - C i o c a l t e u C o l o r i m e t r i c Analysis A c o l o r i m e t r i c method which t a k e s d d v m t a g e of t h e p h e n o l m o i e t y i n l e v a l l o r p h a n t a r t r a t e u t i l i z e s t l i t 35Y

6.

C. RUDY AND B . 2. SENKOWSKI

F o l i n - C i o c a l t e u r e a g e n t ( 2 0 ) . This r e a g e n t i s p r e p a r e d by by mixing 100 gm of sodium t u n g s t a t e , 25 gm of sodium molybdate, 700 m l o f water, 50 m l of 85% p h o s p h o r i c a c i d , and 100 m l of c o n c e n t r a t e d h y d r o c h l o r i c a c i d . A f t e r ref l u x i n g f o r 10 h o u r s , 150 gm o f l i t h i u m s u l f a t e , 50 m l o f w a t e r , and a few d r o p s o f l i q u i d bromine are added and t h e r e s u l t i n g s o l u t i o n b o i l e d a few m i n u t e s t o d r i v e o f f e x c e s s bromine. This s o l u t i o n i s c o o l e d , d i l u t e d t o 1000 m l and f i l t e r e d ( 2 0 ) . When t h i s r e a g e n t i s added t o l e v a l l o r p h a n t a r t r a t e i n a s l i g h t l y b a s i c s o l u t i o n (sodium c a r b o n a t e ) a b l u e c o l o r d e v e l o p s which i s measured a t t h e maximum a t a b o u t 700 nm.

6.7

Nephelometric A n a l y s i s A n e p h e l o m e t r i c method w a s d e v e l o p e d f o r t h e a n a l y s i s of l e v a l l o r p h a n t a r t r a t e i n t h e p r e s e n c e o f l e v o r p h a n o l t a r t r a t e . The method t a k e s a d v a n t a g e of t h e k i n e t i c s o f b r o m i n a t i o n of t h e a l l y 1 f u n c t i o n on l e v a l l o r phan which i n a c i d i c aqueous medium i s f a s t e r t h a n t h e b r o m i n a t i o n of t h e l e v o r p h a n o l . The b r o m i n a t i o n p r o d u c t i s i n s o l u b l e and h e n c e l e n d s i t s e l f t o a t u r b i d i m e t r i c d e t e r m i n a t i o n ( 2 1 ) . The p r o c e d u r e i n v o l v e s a d d i n g 1 mg o f l e v a l l o r p h a n t a r t r a t e ( a l o n g w i t h up t o 10 mg of l e v o r p h a n o l t a r t r a t e ) t o 5 m l of 2.5N H C 1 and 1 5 m l o f water i n a g l a s s s t o p p e r e d 25-ml g r a d u a t e d c y l i n d e r . B r i n g t o 25OC and add 3.0 m l o f 0.1N bromide-bromate s o l u t i o n , s h a k e and m a i n t a i n a t 25OC f o r e x a c t l y 45 m i n u t e s . Shake and measure t h e a b s o r b a n c e a t 640 nm. A p l o t o f a b s o r b a n c e v s c o n c e n t r a t i o n i s l i n e a r between 0 . 5 and 1 . 5 mg o f l e v a l l o r p h a n tartrate (21).

6.8

Titrimetric Analysis The p o t e n t i o m e t r i c , non aqueous t i t r a t i o n i s t h e method o f c h o i c e € o r a n a l y z i n g l e v a l l o r p h a n t a r t r a t e f i n e chemical ( 9 ) . About 250 mg o f l e v a l l o r p h a n t a r t r a t e i s d i s s o l v e d i n 100 m l of g l a c i a l a c e t i c a c i d and t i t r a t e d w i t h 0.02” p e r c h l o r i c a c i d i n d i o x a n e . Each m l o f 0.02N p e r c h l o r i c a c i d is e q u i v a l e n t t o 8 . 6 7 mg of l e v a l l o r p h a n tartrate.

360

LEVALLORPHAN TARTRATE

7.

References 1.

2. 3.

4. 5. 6.

7.

8. 9. 10.

11. 12. 13.

14. 15. 16.

17. 18. 19. 20. 21.

Hawrylyshyn, M. , Hoffmann-La Roche I n c . , P e r s o n a l Communication. J o h n s o n , J . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Bandong, L. , Hoffmann-La Roche I n c . , P e r s o n a l Communication. F a r m i l o , C . G . and G e n e s t , K . , Progress in Chemical T o x i c o l o g y , V o l . 1, Academic P r e s s , N e w York, N.Y., 1 9 6 3 , pp. 199-295. Mule, S . J . , AnaZ. Chem., 36, 1907 ( 1 9 6 4 ) . Boatman, J. , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Benz, W . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Toome, V. , Hoffmann-La Roche I n c . , P e r s o n a l Communication. United S t a t e s Pharmacopeia XVIII, 360-362 ( 1 9 7 0 ) . Moros, S . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. MacMullan, E . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. H a g e l , R . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Lau, E . and Yao, C . , Hoffmann-La Roche I n c . , Unpub 1i s h e d Data. H e l l e r b a c h , J. , Hoffmann-La Roche I n c . , Unpublished Data. H a f l i g e r , 0 . , Hoffmann-La Roche I n c . , Unpublished Data. Mannering, G . J. and S c h a n k e r , L . S . , J . Pharmacol. E x p t l . Therap., 1 2 4 , 296 ( 1 9 5 8 ) . Mohacsi, E . , B l o u n t , J . F . and Mannering, G . J . , 1 3 t h N a t i o n a l M e d i c i n a l Chemistry Symposium i n Iowa, J u n e 18-22, 1 9 7 2 . S c h e i d l , F . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. Mule', S . J . , J . Chromatog. S c i . , 10,275 ( 1 9 7 2 ) . F o l i n , 0. and C i o c a l t e u , V . , J . BioZ. &em., 73, 629 ( 1 9 2 7 ) . S c h m a l l , M., P i f e r , C.W. and W o l l i s h , E . G . , Hoffmann-La Roche I n c . , Unpublished Data.

361

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METHYPRY LON An Aiialytical Profile

363

B. C . RUDY AND B. 2. SENKOWSKI

INDEX Analytical Profile

-

Methyprylon

1.

Description 1.1 N a m e , Formula, M o l e c u l a r Weight 1.2 Appearance, C o l o r , Odor

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 N u c l e a r M a g n e t i c Resonance S p e c t r u m 2.3 U l t r a v i o l e t Spectrum 2.4 Fluorescence Spectrum Mass S p e c t r u m 2.5 2.6 Optical Rotation 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 Thermal G r a v i m e t r i c A n a l y s i s 2.10 S o l u b i l i t y 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2.12 D i s s o c i a t i o n Constant

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug M e t a b o l i c P r o d u c t s

6.

Methods o f A n a l y s i s 6.1 Elemental Analysis 6.2 Phase S o l u b i l i t y Analysis 6.3 T h i n L a y e r Chromatographic A n a l y s i s 6.4 Gas L i q u i d C h r o m a t o g r a p h i c A n a l y s i s 6.5 Direct Spectrophotometric Analysis 6.6 Colorimetric Analysis 6.7 T i t r i m e t r i c Analysis

7.

References

364

METHYPRYLON

1.

Description

1.1

N a m e , F o r m u l a , M o l e c u l a r Weight M e t h y p r y l o n i s 3,3-diethyl-5-methyl-2,4piperidinedione.

0

H M o l e c u l a r Weight:

'1 O H 1 7 No 2

183.25

1.2

A p p e a r a n c e , C o l o r , Odor M e t h y p r y l o n o c c u r s as a w h i t e t o n e a r l y w h i t e It has a s l i g h t c h a r a c t e r i s t i c odor. c r y s t a l l i n e powder. 2.

Physical Properties 2.1

I n f r a r e d Spectrum (IR) The I R s p e c t r u m o f m e t h y p r y l o n i s p r e s e n t e d i n F i g u r e 1 (1). The s p e c t r u m was m e a s u r e d w i t h a P e r k i n E l m e r 621 Spectrophotometer i n a K B r p e l l e t containing 0.5 mg o f m e t h y p r y l o n / 3 0 0 mg of K B r . The a s s i g n m e n t s f o r t h e c h a r a c t e r i s t i c bands i n t h e I R spectrum a r e l i s t e d below (1) : 3 3 4 5 cm-1 2966, 2933, a n d 2877 cm-1

NH s t r e t c h i n g v i b r a t i o n s

a l i p h a t i c CH s t r e t c h i n g vibrations C=O s t r e t c h i n g v i b r a t i o n s

1 6 9 3 and 1656 cm-l 2.2

N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r u m (NMR) The s p e c t r u m shown i n F i g u r e 2 w a s o b t a i n e d on a J e o l 60 MHz NMR by d i s s o l v i n g 5 9 . 0 mg o f m e t h y p r y l o n i n 0 . 5 m l o f CDC13 c o n t a i n i n g t e t r a m e t h y l s i l a n e a s a n i n t e r n a l The s p e c t r a l a s s i g n m e n t s a r e g i v e n i n T a b l e reference (2). I (2). In o r d e r t o make t h e s e a s s i g n m e n t s and e s t a b l i s h t h e chemical s h i f t s , extensive spin-spin decoupling experim e n t s were c a r r i e d o u t . By i r r a d i a t i n g t h e s a m p l e a t t h e 3 65

Figure 1 I n f r a r e d Spectrum of Methyprylon

WAVELENGTH

(MICRONS) 8

9

10

I

1

I

2.5

3

4

5

6

7

I

I

I

I

I

I

100

12

15

I I 1 1 1

20 I

3550 I J

m r, II C

80

0

60

m ?J

40

20

0

4000

I

I

3500

3000

1 2500

I

2000

I' 1700

I 1400

FREQUENCY

(CM-'

I 1100

I

I

I

800

500

200

METHYPRYLON

3 67

B. C. RUDY AND B. 2. SENKOWSKI

f r e q u e n c i e s 4 8 , 65, 1 1 4 , and 206 Hz, t h e s p e c t r u m w a s s i m p l i f i e d and a s s i g n m e n t s c o u l d t h e n b e made ( 2 ) . Table I

N M R Assignments f o r Methyprylon Structure

Protons a

Chemical S h i f t (ppm) 0.8

Mu1t i p 1ic i t y Triplet (JH a -H e

= 7 . 5 Hz)

b

1.14

Doublet = 7 . 0 Hz) (J %-Hd

C

Ql .90

Complex M u l t i p l e t ( J ~ e - ~ =a 7 . 5 Hz)

d

~2.50

Complex M u l t i p l e t = 6 . 8 Hz) ( JHd-Hb = 4 . 0 Hz) (JHd-He

e

3.41

Nonet ( J ~ e - ~= f7 . 0 Hz)

I H f

(JHe-Hd

= 6 . 0 Hz)

(J

= Unknown)

He-Hb f

8.00

2.3

Broad S i n g l e t

U l t r a v i o l e t Spectrum (UV) When t h e UV s p e c t r u m o f methyprylon i n 2-propanol w a s scanned between 4 0 0 a n d 220 nm, one maximum and one minimum were o b s e r v e d . The maximum i s l o c a t e d a t 294 nm ( E = 3 3 ) and t h e minimum a t 353 nm. The s p e c t r u m shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n o f 300.1 mg of methyprylon/lOO m l of 2-propanol ( 3 ) .

368

METHYPRY LON

Figure 3 U l t r a v i o l e t Spectrum of Methyprylon

.9-

.8.7.6W 0

z

U

E 0

.5-

v)

m

a

4.3-

NANOMETERS

3 69

B. C. RUDY AND B. Z. SENKOWSKI

2.4

F l u o r e s c e n c e Spectrum The e x c i t a t i o n and e m i s s i o n s p e c t r a f o r methyp r y l o n (1 mg/ml methanol) are shown i n F i g u r e 4 (4) The e x c i t a t i o n s p e c t r u m e x h i b i t s a b r o a d maximumat a b o u t 302nm and a s h o u l d e r a b o u t 239 nm. The e m i s s i o n s p e c t r u m h a s one maximum a b o u t 396 nm.

Mass Spectrum The mass s p e c t r u m of methyprylon w a s o b t a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h a n i o n i z i n g energy o f 70 eY. The o u t p u t from t h e mass s p e c t r o m e t e r was a n a l y z e d and p r e s e n t e d i n t h e form of a b a r g r a p h , shown i n F i g u r e 5 , by a Varian 100 MS d e d i c a t e d computer s y s t e m which c a l c u l a t e s r e l a t i v e peak i n t e n s i t i e s by comparing each peak t o t h e most i n t e n s e peak i n t h e s p e c t r u m ( 5 ) . The weak peak a t m / e 1 8 3 i s d u e t o t h e m o l e c u l a r i o n . The b a s e peak o c c u r s a t m / e 155 and i s due t o t h e l o s s of CO a n d / o r C2H4 from t h e p a r e n t . The remaining peaks are c o m p a t i b l e w i t h t h e known f r a g m e n t a t i o n p a t t e r n of methyprylon ( 5 ) . 2.5

2.6

Optical Rotation Methyprylon c o n t a i n s a n a s s y m e t r i c c e n t e r a t t h e 5-carbon i n t h e r i n g . However, t h e racemic m i x t u r e i s obt a i n e d from t h e s y n t h e s i s and no o p t i c a l a c t i v i t y i s exhibited

.

2.7

M e l t i n g Range The m e l t i n g r a n g e r e p o r t e d i n t h e N.F. XI11 f o r methyprylon i s 7 4 O t o 77.5O u s i n g t h e class I p r o c e d u r e (6). 2.8

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC s c a n f o r methyprylon i s shown i n F i g u r e 6. A m e l t i n g endotherm was o b s e r v e d a t 76.l0C when t h e The AHf w a s found t o t e m p e r a t u r e program w a s 10°/minute. b e 4 .I k c a l j m o l e ( 7 ) . 2.9

Thermogravimetric A n a l y s i s (TGA) The TGA performed on methyprylon e x h i b i t e d no l o s s of weight from ambient t o 115OC. One c o n t i n u o u s w e i g h t l o s s was o b s e r v e d from 115' t o 26OoC which accounted f o r e s s e n t i a l l y a l l of t h e sample ( 7 ) .

370

Figure 4 F l u o r e s c e n c e S p e c t r a of Methyprylon

z

rn -I

I

w

< -0

-4 c

W

Excitotion

200

250

300

350

I

I

I

I

400

450

500

550

NANOMETERS

B. C. R U D Y AND B. 2. SENKOWSKI

372

r’

METHYPRYLON

AH, = 4.1

I

kcal/mole

I

373

I

B. C. RUDY A N D B. 2. SENKOWSKI

2.10

Solubility The s o l u b i l i t y d a t a f o r methyprylon o b t a i n e d a t 25OC a r e p r e s e n t e d i n T a b l e I1 ( 8 ) . T a b l e I1 S o l u b i l i t y of Methyprylon i n D i f f e r e n t S o l v e n t s Solvent

S o l u b i l i t y (mg/ml)

3A a l c o h o l benzene chloroform 95% e t h a n o l ethyl ether methanol p e t r o l e u m e t h e r (30'-60') 2-propanol water

2 >5.0 x l o 2 r5.0 x 1 0 >5.0 x l o 2 >5.0 x l o 2 2 . 7 x 10' >5.0 x l o 2 1 . 7 x 10 >5.0 x l o 2 7.6 x 1 0

2.11

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of methyp r y l o n is p r e s e n t e d i n T a b l e I11 ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s a r e g i v e n below. Instrument Conditions : G e n e r a l E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t : Radiation:

Goniometer: Detector:

50 KV-12-112 MA Copper Cu & = 1.542 8 O . l o Detector s l i t M.R. S o l l e r s l i t 3O Beam s l i t 0.0007 i n c h N i f i l t e r 4 O take off angle Scan a t 0.2' 29 p e r m i n u t e Amplifier g ain - 16 co u r s e, 8.7 f i n e Sealed proportional counter t u b e and DC v o l t a g e a t plateau

3 74

M E T HY P RY L O N

Pulse height selection EL - 5 volts; EU - out Rate meter T.C.4 2000 CIS full scale Chart speed - 1 inch per 5 minutes

Recorder:

Samples prepared by grinding at room temperature. Table I11 X-ray Powder Diffraction Pattern of Methyprylon 2e -

d (2)

10.00 13.78 14.64 15.62 16.29 18.90 19.54 20.24 20.72 21.96 22.00 24.00 24.55 25.32 26.14 26.88 27.36 27.86 28.68 28.78 28.90 29.51 30.42 31.60 33.14 34.32

8.85 6.43 6.05 5.67 5.44 4.70 4.54 4.39 4.29 4.05 4.04 3.71 3.63 3.52 3.41 3.32 3.26 3.20 3.11 3.10 3.09 3.03 2.94 2.83 2.70 2.61

*d

-

**IlI0

*

**

III 0

28 -

-

13 100 53 7 50 7 3 28 6 11 11 12 4 29 17 23 2 14 3 3 3 12 18 4 6 4

35.02 35.36 35.77 36.22 38.18 38.88 39.10 40.10 40.49 40.82 41.98 42.32 42.72 43.02 43.12 44.06 44.52 44.66 44.80 45.18 46.70 48.34 48.58 50.00 50.30

d(&* 2.56 2.54 2.51 2.48 2.36 2.32 2.31 2.25 2.23 2.21 2.15 2.14 2.12 2.10 2.10 2.06 2.04 2.03 2.02 2.00 1.95 1.88 1.87 1.82 1.81

III

-0-

** 8 3 4 1 3 1 1 8 6 4 2 2 2 1 2 2 2 2 2 2 1 2 1 1 2

nh (interplanar distance) = relative intensity 2 Sin 8 (based on highest intensity of 1.00) 315

B. C. R U D Y A N D 5 . 2. SENKOWSKI

2.12 Dissociation Constant The pKa for methyprylon has been determined spectrophotometrically to be approximately 12.0 (10). 3.

Synthesis Methyprylon is prepared by the reaction scheme shown in Figure 7. 3,3-Diethyl-1,2,3,4-tetrahydro-2,4-pyridinedione, Presidon, i s reacted with formaldehyde yielding 3,3-diethyl5-hydroxymethyl-1,2,3,4-tetrahydro-2,4-pyridinedione (HMP) which is then hydrogenated using a Raney nickel catalyst to methyprylon (11).

4. Stability Degradation When methyprylon i s refluxed i n acidic, neutral, or basic aqueous solutions i n the presence of oxygen, it break down into several carboxylic acids; e.g. formic acid, acetic acid, propionic acid, and diethyl acetic acid. Traces of unidentified amines and ketones are a l s o observed (12). When pure methyprylon is stored in well-closed, light resistant containers, the substance is quite stable. 5. Drug Metabolic Products The major metabolic pathways of methyprylon i n dogs and Methyprylon as the humans is shown in Figure 8 (13-18). intact drug as well as its 5,6-dehydrogenated metabolite are found in blood (13,14), I n addition to the 5,6-dehydrogenated metabolite, several other metabolites due to oxidation are found in urine. These include the 6-oxymetabolite from the direct oxidation of methyprylon and the oxidation of the methyl group to the alcohol and then to the acid i n the 5,6-dehydrogenated metabolite (16,18). 6. Methods of Analysis 6.1

Elemental Analysis The results from the elemental analysis are listed i n Table IV (19). Table IV Elemental Analysis of Methyprylon % Found Element Theory C 65.54 65.40 H 9.35 9.46 N 7.64 7.62 316

Figure 7 S y n t h e s i s of Methyprylon

26

- 2:o 0

+

0

H-C-H N

II

CH20H

0

04

I H

I

H PR E SlDON

FORMALDEHYDE

HMP

I

H2 Raney

I cC2H5 2H5&$ O

Nickel

H N I H

H

METHYPRYLON

Figure 8 M e t a b o l i c P r o d u c t s o f Methyprylon

m 0 7J

C 0

<

A

ti

3.3- diethyl-5-methyl-1.2.3,42.4- pyridinedione

tetrohydro-

3.3-diethyl-5-methyl-2,4.6-piperidinctrione

OXIDATION

I ti 3.3- di ethyl -5- hydroxyrnethyl1,2.3,4 - t etrohydro- 2,4- pyridinedione

CO $J-O (H

0 I

ti

5.5-diethyl- 3.4,5.6-tetmhydro4.6-dioxonicotinic acid

METHYPRYLON

6.2

Phase S o l u b i l i t y A n a l y s i s P h a s e s o l u b i l i t y a n a l y s i s may b e c a r r i e d o u t u s i n g n-heptane as t h e s o l v e n t . A t y p i c a l example f o r m e t h y p r y l o n is shown i n F i g u r e 9 which a l s o l i s t s t h e cond i t i o n s u n d e r which t h e a n a l y s i s w a s c a r r i e d o u t ( 8 ) . 6.3

Thin Layer Chromatographic A n a l y s i s (TLC) A TLC s y s t e m used t o i d e n t i f y m e t h y p r y l o n from o t h e r h y p n o t i c s w a s p u b l i s h e d b y Frahm e t a l . ( 2 0 ) . T h i s s y s t e m u t i l i z e d a K i e s e l g e l C p l a t e and a d e v e l o p i n g s o l v e n t of 2-propanol:chloroform:25% ammonium h y d r o x i d e ( 9 : 9 : 2 ) . The Rf v a l u e s were v e r y s e n s i t i v e t o t h e amount of s o l v e n t s a t u r a t i o n i n t h e t a n k and t h e r e f o r e a r e f e r e n c e s t a n d a r d w a s always r u n a l o n g w i t h t h e sample f o r i d e n t i f i c a t i o n purposes. A TLC s y s t e m used t o s e p a r a t e m e t h y p r y l o n from i t s m e t a b o l i t e s and o t h e r s i m i l a r compounds employs e t h y l a c e t a t e as t h e d e v e l o p i n g s o l v e n t . The m e t h y p r y l o n s a m p l e , a p p r o x i m a t e l y 100 mcg, i s s p o t t e d o n a s i l i c a g e l G p l a t e and s u b j e c t e d t o a s c e n d i n g chromatography u n t i l t h e s o l v e n t f r o n t h a s moved a t l e a s t 15 cm. The p l a t e i s a i r d r i e d and sprayed with f r e s h l y prepared potassium f e r r i c y a n i d e (1.6 gm K3Fe(CN)6 i n 100 m l 2N KOH) and examined immediately u n d e r long-wavelength u l t r a v i o l e t r a d i a t i o n . Methyprylon h a s an Rf of 0 . 4 0 i n t h i s s y s t e m (21). 6.4

Gas-Liquid Chromatographic A n a l y s i s (GLC) The f o l l o w i n g GLC method i s u s e f u l f o r t h e s e p a r a t i o n and q u a n t i t a t i o n of m e t h y p r y l o n i n t h e p r e s e n c e o f i t s m e t a b o l i t e s and s i m i l a r s t r u c t u r e d compounds. The p e r t i n e n t e x p e r i m e n t a l c o n d i t i o n s as w e l l as t h e r e t e n t i o n t i m e f o r methyprylon are g i v e n i n T a b l e V ( 2 2 ) . Table V GLC Method f o r Methyprylon Column : Support: S t a t i o n a r y Phase: Detector:

6 f t , 1/8 i n O.D., s t a i n l e s s steel tubing D i a t o p o r t S (60-80 mesh) 8% C r a i g P o l y e s t e r S u c c i n a t e (BDS) Hydrogen Flame I o n i z a t i o n

379

Figure 9

I

I

W

>

-I

0

cn

-

?25W

I-

3 J

-

0

"

-.

- -

-

n

-"

-

I\

c

F

0 I]

0

C 0

D

PHASE

. . 15-

SOLUBILITY

ANALYSIS

-

w

< b

z 0

oc

SAMPLE : METHYPRYLON

c

Fo

-

SOLVENT : HEPTANE SLOPE : 0.39% EQUILIBRATION : 72 HOURS at 2 5 OC EXTRAPOLATED SOLUBILITY : 19.65 m g 4 of HEPTANE

I 0

25

50

I

75

-

100

?J

METHYPRY LON

Temperature OC Injection Port: Column : Detector: Flow Rate: Quantities Injected:

210

180 2 50 40 c c l m i n u t e o f N i t r o g e n 10-100 pg of m e t h y p r y l o n i n 95% e t h a n o l 11 m i n u t e s

Retention Times :

6.5

Direct Spectrophotometric Analysis D i r e c t s p e c t r o p h o t o m e t r i c a n a l y s i s i s r a r e l y used b e c a u s e of t h e v e r y l o w molar a b s o r p t i v i t y of methyprylon. However, i n t h e a b s e n c e of any i n t e r f e r i n g s p e c i e s , t h e maximum a t 294 nm ( i n 2-propanol) c o u l d b e used f o r d i r e c t spectrophotometric analysis.

6.6

Colorimetric Analysis Methyprylon u n d e r g o e s e n o l i z a t i o n i n t h e p r e s e n c e o f a l k a l i t h u s p e r m i t t i n g t h e u s e of t h e F o l i n C i o c a l t e u r e a g e n t t o form t h e b l u e c o l o r e d complex ( 2 3 ) . T h i s method i s u t i l i z e d f o r t h e d e t e r m i n a t i o n of methyprylon p r e s e n t i n b l o o d o r plasma. After appropriate extraction o r other s e p a r a t i o n , t h e F o l i n C i o c a l t e u r e a g e n t i s added t o a b a s i c s o l u t i o n of t h e m e t h y p r y l o n and t h e a b s o r b a n c e of t h e b l u e complex i s measured a t t h e maximum a b o u t 700 nm ( 1 4 ) . The s e n s i t i v i t y l i m i t i s a b o u t 5 iJg/ml of b l o o d o r p l a s m a .

6.7

T i t r i m e t r i c Analysis The p o t e n t i o m e t r i c t i t r a t i o n of a b a s i c , aqueous s o l u t i o n of m e t h y p r y l o n w i t h 0.1N p o t a s s i u m f e r r i c y a n i d e i s t h e method of c h o i c e f o r t h e a n a l y s i s of m e t h y p r y l o n i n t h e b u l k form as w e l l as i n c a p s u l e s and t a b l e t s ( 2 4 ) . The end p o i n t i s d e t e r m i n e d p o t e n t i o m e t r i c a l l y by t h e u s e o f a calomel-platinum e l e c t r o d e system. Each m l of 0.1N p o t a s sium f e r r i c y a n i d e i s e q u i v a l e n t t o 9 . 1 6 2 mg of m e t h y p r y l o n .

38 I

B. C . RUDY AND B. 2. SENKOWSKI

7. References 1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. 2. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. 3 . Rubia, L. B., Hoffmann-La Roche Inc., Personal Communication. 4. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. 5. Benz, W., Hoffmann-La Roche Inc., Personal Communication. 6. National Formulary XIII, First Supplement, p 1020 (1970). 7 . Moros, S . , Hoffmann-La Roche Inc., Personal Communication. 8. MacMullan, E., Hoffmann-La Roche Inc. , Personal Communication. 9. Hagel, R. B., Hoffmann-La Roche Inc., Personal Communication. 10. Lau, E., Hoffmann-La Roche Inc., Unpublished Data. 11. Pecherer, B., Hoffmann-La Roche Inc., Unpublished Data. 12. Frick, H., Hoffmann-La Roche Inc., Unpublished Data 13. Pellmont, B., Studer, A . , and Jurgens, R., Schweiz. Med. Wschr. , 85, 350 (1955). 14. Randall, L. O., Iliev, V., and Brandman, O., Arch. I n t . Pharmacodynamie , 106,388 (1956). 15. Bernhard, K., Just, M., Lutz, A . H., and Vuilleumier, J. P. , HeZv,. Chim. A c t a , 40, 436 (1957). 16. Pribilla, O., Archiv. f;*rT o x i k o l o g i e , 1 8 , 1 (1959). 1 7 . Boesche, J . and Schmidt, G . , Arzneim. F G s c h . , 16, 548 (1966). 18. Boesche, J., Arzneim. Forsch. , 2 , 123 (1969). 19 * Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. 20. Frahm, M., Gottesleben, A. , and Soehring, K. , Arzneim. Forsch. , 11,1008 (1961). 21. Beratti, N., Hoffmann-La Roche Inc., Unpublished Data. 22. Mahn, F., Hoffmann-La Roche Inc., Unpublished Data. 23. Folin, O., and Ciocalteu, V., J . B i o l . Chem., 73, 627 (1929). 24. National Formulary XIII, pp. 458-459 (1970).

.

382

PH ENELZINE SU LF ATE

Reviewed b y L. Chafetz

383

ROBERT E. O A L Y

CON TEN T S

Analytical Profile

1. 2.

3.

4. 5. 6.

7,

-

Phenelzine S u l f a t e

Description 1.1 N a m e , F o r m u l a , M o l e c u l a r W e i g h t 1 . 2 A p p e a r a n c e , C o l o r , Odor Ph y s i ca 1 P r o p e r t i es 2 . 1 I n f r a r e d Spectrum 2.2 N u c l e a r Maqnetic Resonance Sp ect ru m 2 . 3 Mass S p e c t r u m 2.4 Ultraviolet Spectrum 2.5 D i f f e r e n t i a l Thermal A n a l y s i s 2.6 Thermogravimetric Analysis 2.7 M e l t i n g Range 2.8 S o l u b i l i t y 2.9 X-Ray D i f f r a c t i o n Powder A n a l y s i s Synthesis Stability Metabolism Methods o f A n a l y s i s 6 . 1 Spectrophotometric Analysis 6.2 Polarography 6.3 T i t r i m e t r y 6.4 Chromatography 6 . 4 1 Gas C h r o m a t o g r a p h y 6.42 Thin Layer Chromatography 6.5 S p o t T e s t s References

384

PHENELZINE SULFATE

1.

DescriDtion

N a m e , Formula, M o l e c u l a r Weight Phenelzine S u l f a t e i s B-phenylethylI t i s a l s o known hydrazine dihydrogen s u l f a t e . as p h e n e t h y l h y d r a z i n e s u l f a t e . 1.1

A p p e a r a n c e , C o l o r , Odor White t o y e l l o w i s h w h i t e c r y s t a l l i n e powder h a v i n g a c h a r a c t e r i s t i c o d o r .

1.2

2.

Physical Properties 2.1

I n f r a r e d Spectrum The i n f r a r e d s p e c t r u m ( F i g u r e 1) of p h e n e l z i n e s u l f a t e was d e t e r m i n e d a s a K B r p e l l e t i n a P e r k i n - E l m e r model 6 2 1 s p e c t r o p h o t o m e t e r . The s p e c t r u m o b t a i n e d i s i d e n t i c a l t o t h a t o f p h e n e l z i n e s u l f a t e w h i c h a p p e a r s as S a d t l e r i n f r a r e d s p e c t r u m #24825. T h e broad band a t a b o u t 2500 c m . - ’ may b e a t t r i b u t e d t o t h e hydrazine s a l t . The b a n d s a t 758 c m . - ’ a n d 705 c m . - ’ may b e a t t r i b u t e d t o t h e m o n o s u b s t i t u t e d benzene moiety. N u c l e a r Magnetic Resonance Spectrum The NMR s p e c t r i i m o f p h e n e l z i n e s u l f a t e w a s d e t e r m i n e d i n a V a r i a n A-60 i n s t r u m e n t e m p l o y i n g DMSO a s t h e s c l v e n t ( F i g u r e 2 ) . The spectrum d i s p l a y e d s i n g l e t s a t 6 7.75 (hydrogen bonded t o n i t r o g e n ) and a t 6 7.2 (aromatic). O t h e r a b s o r p t i o n w a s c e n t e r e d around 6 3.0 ( m e t h y l e n e bonded t o m e t h y l e n e and N H ) and 6 2 . 5 (methylene bonded t o methylene and p h e n y l ) D e u t e r i u m e x c h a n g e ( F i g u r e 3 ) w a s p e r f o r m e d on t h e s a m p l e . The a b s o r p t i o n band a t 6 7 . 7 5 d i s a p p e a r e d v e r i f y i n g t h e a s s i g n m e n t o f t h i s band t o hydrogen bonded t o n i t r o g e n . 2.2

.

2.0 100

0 5000

2.5

3.0

4.0 5 . 0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

I

I

I

4000

3000

2000

1800

1700

1600

W A V E NUMBERS I N C Y

Figure 1

-

-'

1500

1400

1300

1200

I

-

11.0 12.0 13.0 14.0 15.0 I

-

I

.

-

1100

1000

.

.

.

.

I

1

1

1

1900

10.0 1

I

900

I n f r a r e d Spectrum o f P h e n e l z i n e S u l f a t e .

800

700 640

500

400

300

z 00

200

0 cpr

rn C

r

n b

--I

rn

I 8.0

I

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7.0

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3.0

I

I 2.0

I

I

1.0

F i g u r e 2 - NMR S p e c t r a of P h e n e l z i n e S u l f a t e

I

0 PPm

x

500

1

1

8.0

I

300

400

1

7.0

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I

6.0

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I 5.0

100

200

I

I

4.0

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I

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I 2.0

I

1 1.0

I

0 PPm

F i g u r e 3 - NMR S p e c t r a of P h e n e l z i n e S u l f a t e - D e u t e r i u m Exchange

PHENELZINE S UL F A T E

2.3

Mass S p e c t r u m A m a s s s p e c t r u m of p h e n e l z i n e s u l f a t e could not be obtained d e s p i t e s e v e r a l attempts. An A E I 902 w a s employed w i t h a n i o n i z i n g v o l t a g e of 7 0 ev and a t a t e m p e r a t u r e of 1 0 0 ° C . On r u n ning t h e material d i r e c t l y i n t h e probe, a spect r u m w h i c h showed l a r g e a m o u n t s of s u l f u r d i o x i d e resulted. Attempts a t running t h e f r e e base proved f r u i t l e s s a l s o . The sample w a s i n t r o d u c e d i n t o t h e mass s p e c t r o m e t e r t h r o u g h t h e h e a t e d i n l e t b u t t h e r m a l d e c o m p o s i t i o n o c c u r r e d and no spectrum w a s obtained. 2.4

U l t r a v i o l e t Spectrum R a j e s w a r a n a n d K i r k (I) r e p o r t e d h max ( E 1 8 6 2 ) of 258 nm. a n d X min o f 2 2 8 nm. i n 50% ethanol. Phenelzine s u l f a t e (0.0541% i n 50% e t h a n o l ) when s c a n n e d i n a C a r y 1 4 s p e c t r o p h o t o m e t e r b e t w e e n 350 and 2 2 0 nm. ( F i g u r e 4 ) , e x h i b i t e d t h r e e p e a k s a t 2 5 2 , 258 a n d 263 nm. w i t h A max a t 258 nm. ( a = 0 . 7 9 8 : F 1 8 6 9 ) . 2.5

D i f f e r e n t i a l Thermal Analysis A d i f f e r e n t i a l thermoqram h a s been

o b t a i n e d i n a Dupont m o d e l 9 0 0 DTA I n s t r u m e n t Two e n d o t h e r m s , o n e a t e m p l o y i n g a DSC c e l l . 134°C. ( c r y s t a l l i n e c h a n g e ) a n d t h e s e c o n d a t 1 7 2 ° C . ( m e l t i n g ) were o b s e r v e d ( F i g u r e 5 ) . H e a t i n g r a t e was 10' C . / m i n u t e . The d e t e r m i n a t i o n w a s c a r r i e d out i n a n i t r o g e n a t m o s p h e r e . A l t e r a t i o n i n t h e r a t e of h e a t i n g w o u l d t e n d t o s h i f t t h e endotherms. Thermogravimetric Analysis A thermogravimetric analysis was perf o r m e d ( F i g u r e 6 ) i n a D u p o n t m o d e l 9 5 0 TGA Instrument. The m e a s u r e m e n t was p e r f o r m e d u n d e r n i t r o g e n sweep a t a h e a t i n g r a t e o f 1 0 " C . / minute. N o w e i g h t loss w a s n o t e d u n t i l a b o u t 165" C . ( i n i t i a t i o n o f m e l t i n g ) . A f t e r t h i s p o i n t , w e i g h t w a s r a p i d l y lost. T h i s i n f o r m a t i o n t o g e t h e r w i t h t h e DTA i n d i c a t e s t h a t t h e 2.6

389

R O B E R T E. DALY

0. a

0. :

0.6

0.5

rn

*

c, Y

0.4

eq

e

0

2

0.3

pl

0.2

0.1

210

Figure 4

-

WAVELENGTH

320

W Spectrum o f Phenelzine S u l f a t e

390

0x3-

0aN3

PHENELZINE SULFATE

JV-

391

0 0

rn 0

2 3

0 '0

0

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k

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N

a r

k -!

5

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2 Y 0,

0

0 05

0 \o

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w

0

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0

ROBERT E. D A L Y

392

a 0 v,

0 v,

w

0

*0

0

m

v)

0 0

m 0 v)

w

0

0

w

2

0

1

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a v,

0

a,

c, m w

a, C

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5 k

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am:

s 2

PHENELZINE SULFATE

e n d o t h e r m a t 134O C . w a s d u e t o a n o n d e s t r u c t i v e a l t e r a t i o n i n t h e molecule (crystal change). 2.7

M e l t i n g Range The m e l t i n g r a n g e h a s b e e n r e p o r t e d ( 2 ) t o be 1 6 4 t o 1 6 8 " C . 2.8

Solubility Phenelzine s u l f a t e i s soluble i n water and p r a c t i c a l l y i n s o l u b l e i n e t h a n o l , c h l o r o f o r m and e t h e r . 2.9

X-Ray D i f f r a c t i o n Powder A n a l y s i s R a j e s w a r a n a n d K i r k (1) h a v e r e p o r t e d x-ray d i f f r a c t i o n data €or p h e n e l z i n e s u l f a t e . The powdered s a m p l e which h a d been a p p r o p r i a t e l y m o u n t e d on a r o t a r y s p e c i m e n h o l d e r , w a s s u b j e c t e d t o copper k-alpha r a d i a t i o n i n a Norelco G e i g e r C o u n t e r X-Ray S p e c t r o m e t e r . The d i f f r a c t i o n p a t t e r n s w e r e o b t a i n e d over a n a r c of 4 5 " . The i n s t r u m e n t w a s c a l i b r a t e d o v e r t h i s r a n g e , u s i n g powdered q u a r t z c r y s t a l s , a n d t h e " d " v a l u e s computed. Values are g i v e n i n T a b l e I . Table I

-

Ild" D i s t a n c e s U s i n g

C o p p e r K-Alpha Distances

3.

-

18.9, 3.24,

9.85, 2.79

&

Radiation

6.51, 5.42, 2.44

4.89,

3.90,

Synthesis

B i e l ( 3 ) h a s s y n t h e s i z e d p h e n e l z i n e by r e a c t i n g h y d r a z i n e h y d r a t e and p h e n e t h y l bromide under A y i e l d of 77% w a s o b t a i n e d . r e f l u x €or 7 h o u r s . Sacha and M a l a s n i c k i ( 4 ) condensed p h e n e t h y l c h l o r i d e w i t h hydrazine under r e f l u x i n anhydrous e t h a n o l i n a n i t r o g e n atmosphere €or 1 2 hours. T h e s o l u t i o n of t h e f r e e b a s e w h i c h w a s o b t a i n e d a f t e r removal o f h y d r a z i n e H C 1 b y f i l t r a t i o n was t r e a t e d w i t h s u l f u r i c a c i d i n anhydrous e t h a n o l

393

ROBERT E. DALY

.

(l.l/l W/W) A y i e l d of 2 1 % o f P - p h e n y l e t h y l hydrazine dihydrogen s u l f a t e w a s obtained. B i e l , e t . aZ. ( 5 ) h a s r e p o r t e d t h a t a r a l k y l a t i o n of hydrazine with primary a r a l k y l h a l i d e s a f f o r d s mono- ( a r a l k y l ) - h y d r a z i n e s i n y i e l d s of 6 0 t o 75% p r o v i d e d a t h r e e t o f i v e f o l d molar excess o f h y d r a z i n e h y d r a t e i s employed. Production of d i p h e n y l e t h y l h y d r a z i n e has been r e p o r t e d ( 6 ) as a by-product i n t h e r e a c t i o n .

0-

Reflux 7 hours

>\:2;::;:::;:

anhyd. EtOH

1

O H P - C H Z - N H - N H ~

H2S04 : E t O H

12R'/;d.

NP atm.

CH2-CH2C1 + NH~-NHP*H~O

4.

(l*l:l)

Sulfate

Stability

P h e n e l z i n e base i s e a s i l y o x i d i z e d . Decompos i t i o n o c c u r s when b a s i f i e d s o l u t i o n s a r e e x t r a c t e d , probably due t o autooxidation of t h e base. S c h l i t t , e t . aZ. ( 7 ) h a v e r e p o r t e d t h e a u t o o x i d a t i o n of p h e n e l z i n e s u l f a t e i n pH 5 . 9 p h o s p h a t e buffer. A u t o o x i d a t i o n i n pH 6 . 5 , 0 . 1 M s o d i u m c h l o r a t e a t 37' C. h a s a l s o b e e n r e p o r t e d ( 8 ) . D e c o m p o s i t i o n p r o d u c t s were n o t c h a r a c t e r i z e d . 5.

Metabolism D r a b n e r a n d Schwerd ( 9 ) i d e n t i f i e d u n c h a n g e d

394

PHENELZINE SULFATE

p h e n e l z i n e and p h e n y l a c e t y l g l u t a m i n e i n t h e u r i n e o f humans who h a d r e c e i v e d p h e n e l z i n e s u l f a t e . C l i n e s c h m i d t , e t . a l . ( 1 0 , 11) s t u d i e d t h e in v i t r o b i o t r a n s f o r m a t i o n a s w e l l as t h e in v i v o metabolism of C"-phenelzine i n rats. Phenylaceti c a c i d w a s i d e n t i f i e d as t h e m a j o r m e t a b o l i t e . F i s c h e r , e t . al. ( 1 2 ) h a v e d e s c r i b e d t h e e f f e c t o f p h e n e l z i n e on t h e e x c r e t i o n of 6phenethylamine. 6.

Methods of A n a l y s i s 6.1

Spectrophotometric Analysis

6 . 1 1 F e i g l (13) has r e p o r t e d t h e reduct i o n of l i t h i u m and s o d i u m molybdophosphotungs t a t e s o l u t i o n by h y d r a z i n e d e r i v a t i v e s . T h i s r e a c t i o n h a s b e e n a d a p t e d t o t h e d e t e r m i n a t i o n of p h e n e l z i n e s u l f a t e r a w m a t e r i a l o r t a b l e t s by Bose and V i j a y v a r g i y a (14). R e d u c t i o n i s c a r r i e d o u t i n a l k a l i n e s o l u t i o n with t h e production of a d e e p b l u e c o l o r w h i c h is m e a s u r e d a t 6 5 0 nm.

6.12 A c o l o r i m e t r i c method b a s e d on t h e formation of t h e hydrazone w i t h v a n i l l i n has been d e v i s e d ( 1 5 ) . The r e a c t i o n i s c a r r i e d o u t i n 0.5 N m e t h a n o l i c h y d r o c h l o r i c a c i d . Absorbance o f t h e y e l l o w s o l u t i o n i s r e a d a t 4 0 0 nm. S i n c e p h e n e l z i n e d e g r a d e s by o x i d a t i v e d e s t r u c t i o n of t h e h y d r a z i n e f u n c t i o n , t h i s method i s s t a b i l i t y indicating

.

Polarography The h a l f wave p o t e n t i a l versus s t a n d a r d calomel e l e c t r o d e ) of t h e a c e t o n e d e r i v a t i v e of p h e n e l z i n e w a s r e p o r t e d ( 7 ) as - 1 . 3 3 V i n pH 5 . 9 p h o s p h a t e b u f f e r ( 0 . 1 M KH~POI, : N a 2 H P O 4 * 2 H 2 O ) employing a 0 . 0 1 % g e l a t i n s o l u t i o n as a maximum s u p p r e s s o r . The d i f f u s i o n c u r r e n t c o n s t a n t ( I d ) w a s approximately 2.5. Four elect r o n s w e r e t r a n s f e r r e d f o r t h e r e d u c t i o n of t h e h y d r a z o n e d e r i v a t i v e . W a v e h e i g h t was p r o p o r t i o n a l t o c o n c e n t r a t i o n i n t h e r a n g e of 2 . 5 x lo-' t o 2.5 x M i n pH 5 . 9 p h o s p h a t e b u f f e r . 6.2

3Y.5

ROBERT E . D A L Y

6.3

Titrimetry P r o c e d u r e s b a s e d on t h e r e a c t i o n o f phenelzine s u l f a t e with iodine i n bicarbonate s o l u t i o n ( 2 ) and i n sodium h y d r o x i d e ( 1 6 ) and w i t h b r o m a t e and bromide i n a c i d s o l u t i o n ( 1 6 ) w i t h s u b s e q u e n t t i t r a t i o n of e x c e s s r e a g e n t ( i o d i n e o r bromide c o n v e r t e d t o i o d i n e ) i n a c i d s o l u t i o n w i t h t h i o s u l f a t e have been r e p o r t e d . B a s i c a l l y , t h e r e a c t i o n i n v o l v e d may be i l l u s t r a t e d a s follows:

Radecka and Nigam ( 1 7 ) have r e p o r t e d t h e d i r e c t t i t r a t i o n o f p h e n e l z i n e w i t h N-bromosuccinimide i n a c i d i c s o l u t i o n employing m e t h y l r e d a s t h e indicator. However, t h e i r r e s u l t s show a 5% p o s i t i v e b i a s . This b i a s is a t t r i b u t e d t o a l l y l i c b r o m i n a t i on by N -b r omosu c c i n i m i d e

.

6.4

Chromatography

Gas Chromatography C a r d i n i , e t . a l . (18, 1 9 ) and McMartin and S t r e e t ( 2 0 ) h a v e r e p o r t e d g a s chroma t o g r a p h i c methods which a r e r e p u t e d t o be quantitative. However, no q u a n t i t a t i v e d a t a a r e g i v e n . C o n d i t i o n s a r e r e p o r t e d i n T a b l e 11. 6.41

T h i n Layer Chromatography Q u a l i t a t i v e methods f o r t h e i d e n t i f i c a t i o n of p s y c h o t r o p i c a g e n t s have b e e n r e p o r t e d ( 2 1 , 2 2 , 2 3 , 2 4 , 2 5 ) . R f v a l u e s a r e summarized i n T a b l e 111. 6.42

6.5

Spot T e s t s A v a r i e t y of s p o t t e s t s have been re-

ported ( 2 6 ) .

T h e s e are summarized i n T a b l e 396

v.

T A B L E I1 - P a r a m e t e r s f o r G a s C h r o m a t o g r a p h y Ref. 18

19 w

c 4

20

Column g l a s s column 1 . 8 M x 2 mm.; 2 % GE-SE 3 0 on A e r o p a k 3 0 , 80100 m e s h

Column Temp.

Carrier Gas

program 70-250' a t 10.4O/

N2

Flow Rate

50 m l . / min.

Detector

FID

Detector Te m p

.

220'

C.

Injector Temp. 250'

C.

minute Q

I rn

Stainless steel 140' c o l u m n 1 . 2 M. x 2 mm.; 15%c a r b o w a x 20 M o n C h r o m o s o r b W alkalinized w i t h 5% KOH

C.

Stainless steel 140' c o l u m n 6' x 1/8" ; 2 % SE 3 0 + 0 . 1 % t r i s t e a r i n on s i l a n i z e d acid w a s h e d C h r o m o sorb W

C.

He

40 m l . / min.

FID

250'

C.

z

rn

P z

rn

m C

r

n

>

2 N2

3 0 ml./ min.

FID

ROBERT

TABLE I11

-

E. D A L Y

S u p p o r t , S o l v e n t System and Rf

V a l u e s f o r TLC of P h e n e l z i n e S u l f a t e Reference

Support

Solvent System

Rf

(X

21

silica gel G

chloroform: m e t hano 1 (8:2)

21

silica gel G

chloroform: acetone: d i e t h y lamine ( 5 : 4: 1)

88

21

silica gel G

cyclohexane: chloroform: d ie t h y l a m ine ( 4 :5 :1)

62

22

silica gel G impregnated with 0 . 1 M N aOH

hexane:benzene: diethylamine ( 7 5 : 1 5 : 10)

51

22

silica gel G impregnated with 0.1 M N aOH

methanol

72

22

silica gel G imp re gn a t ed with 0 . 1 M NaOH

acetone

75

22

silica gel G impregnated with 0 . 1 M NaHSO

methanol

41

398

100

100)

PHENELZINE SULFATE

TABLE I11 f c o n t . 1

R e f er e n ce

22

Support

Solvent System

Rf

( x 100)

silica gel G impregnated with 0 . 1 M NaHSO

95% ethanol

25

silica gel G activated a t 110" C. f o r 30 m i n u t e s

chloroform: m ethano1

37

23

silica gel G a c t i v a t e d as above

chloroform: methanol : 2 0 % ammonium hydroxide ( 2 : 1:l)

56

24

silica gel G activated at 100' C . f o r 1 hour

methanol:12 N ammon i um hydroxide (100: 1.5)

74

24

silica gel G impregnated with 0 . 1 N NaOH and activated a s above

cyclohexane: diethylamine: benzene (75: 20: 15)

44

24

s i l i c a g e l G acetone impregnated and a c t i v a t e d as above

65

24

s i l i c a g e l G chloroform: impregnated methanol and a c t i v a t e d ( 9 0 : 10) as above

75

+ ,

23

(1:l)

399

ROBERT E. DALY

TABLE I11 ( c o n t . )

Reference

Support

Solvent System

Rf

(X

100)

24

silica gel G

benzene: ethanol: 1 2 N ammonium hydroxide ( 9 5 : 15: 5 )

50

25

silica gel G imp re g n a t e d with 0 . 1 N KHSO 4

95% e t h a n o l

49

25

silica gel G impregnated w i t h 0.1N

cyclohexane: benzene : d i et h y lamine ( 7 5 : 15: 10)

52

methanol

72

acetone

75

N aOH

25

silica gel G imp r e g n a t e d with 0 . 1 N N aOH

25

silica gel G impregnated with 0.1 N N aOH

25

silica gel G imp r e g n a t e d with 0 . 1 N KHSO I,

methanol

62

25

silica gel G impregnated with 0 . 1 N NaOH

methyl acetate

53

400

PHENE LZI NE SULFATE

TABLE I11 ( c o n t . ) Reference 25

Support

Solvent System

chromedi a n-butanol: (Whatman #41) 5% c i t r i c impregnated acid w i t h 5% s o d i u m d i h y d r og e n citrate (some K2HP04 a d d e d t o prevent tailing)

Rf (x 100) 51

ROBERT E. DALY

TABLE I V

-

Reagents and Reactions

F o r TLC P r o c e d u r e s Reagent

Reaction

21

Folin-Ciocalteau's Reagent

blue violet with heating

21

5% phosphomolybdic a c i d i n e t h a n o l f o l l o w e d by exposure of t h e p l a t e t o ammonia v a p o r

blue

22

1%i o d i n e i n m e t h a n o l

brown

23

50 m l . o f 0 . 2 % ninhydrin i n methanol + 1 0 m l . of g l a c i a l a c e t i c a c i d + 2 m l . of

r o s e orange

Reference

2,4,6-trimethylpiperidine 23

1 0 % F e r r i c c h l o r i d e ; 1%

f l u o r e s c e i n : ammonia

fluorescent blue

23

S a t u r a t e d ammonium molybdate; s a t u r a t e d oxalic acid

celeste

24

Fo l i n - C i o c a l t e a u ' s Reagent

b l u i s h a t RT, b l u e when heated t o 100'

c.

24

Mandelin s R e a g e n t

pink a t RT, f l e s h a t 100' C. , f l u o r e s c e n t a f t e r heating

24

C i nn ama l d e h y d e R e a g e n t 5 m l . cinnamaldehyde + 95 m l . ethanol + 5 m l . conc. HC1

y e l l o w a t RT

402

PHENELZINE SULFATE

TABLE I V ( c o n t . )

Reference

Reagent

Reaction

24

0.25 g . p-dimethylaminobenzaldehyde + 5 g . of 85% p h o s p h o r i c a c i d + 20 m l . of water

lemon chromo after heating t o 1 0 0 ' C. f o r 1 0 min. and t h e n exposed t o ammonia v a p o r

24

Furf u r a l r e a g e n t s o l ' n A.) 1 m l . f u r f u r a l i n 99 m l . of acetone s o l ' n B . ) 4 m l . of c o n c . H 2 S 0 4 i n 96 m l . o f a c e t o n e

brown a t 1 0 0 '

25

C.

P l a t e examined u n d e r s h o r t w a v e W l i g h t and t h e n s p r a y e d w i t h t h e following sequence of r e a g e n t s : iodine

brown

Dragendorff ' s

orange

sodium n i t r i t e

d e e p brown

i odop l a t in a t e until a color develops

variously colored

403

ROBERT E. DALY

TABLE V

-

Spot Tests f o r Phenelzine S u l f a t e

Reagent

Response 0 . 5 mg.

2 0 1-14.

Froehde ' s Reagent

blue* It. b l u e

blue

Mandelin ' s R e a g e n t

green blue* brown

green faint orange

Marquis ' Reagent Mecke's R e a g e n t

r e d orange* grey

pink

R e i c k a r d ' s Reagent

It. blue

It. b l u e

F l u e c k i g e r ' s Reagent

brown

V i t a l i ' s Reagent

a. 1 b.)

Was i c k y ' s R e agen t

red o r a n g e

*

=

a f t e r warming

404

- -blue orange

---

--- --

PH E N E LZ IN E SULFATE

TABLE V I

- Composition o f Spot T e s t Reagents

Reagent

Composition

Froehde I s Reagent

0 . 5 % a q u e o u s ammonium m o 1y b d a t e

Mande l i n I s R e a g e n t

0 . 5 % a q u e o u s ammonium vanadate

Marquis

1 p a r t 37% formaldehyde i n 20 p a r t s concentrated

Reagent

sulfuric acid, freshly prepared before use Mecke ' s R e a g e n t

0 . 5 % aqueous s e l e n i o u s a c i d

(H2SeO 3 1 Reickard I s Reagent

1%a q u e o u s sodium t u n g s t a t e

F l u e c k i g e r ' s Reagent

0 . 5 % s o l u t i o n of t i t a n i u m d i o x i d e ( T i 0 2 1 i n concentrated sulfuric acid

V i t a l i s Reagent

a.) b.)

fuming n i t r i c a c i d 3% potassium hydroxide

i n ethanol Wasicky s R e a g e n t

1 0 % s o l u t i o n o f p-dimethylaminobenzaldehyde i n g l a c i a l acetic acid

305

ROBERT E. DALY

7.

References R a j e s w a r a n a n d P . L. K i r k , B u l l . N a r c o t i c s , U . N . , D e p t . S o : L 4 i l ~ f f ~ i 1r 4 s, No. I, 1 9 ( 1 9 6 2 ) .

1.

P.

2. 3. 4.

U.S.P. X V I I I . J . H . B i e l , US 3 , 0 0 0 , 9 0 3 , S e p t . 1 9 , 1 9 6 1 . A. S a c h a a n d L . M a l a s n i c k i , P o l 4 6 , 2 3 7 , O c t . 3 0 , 1 9 6 2 , C . A . 60, 4 5 4 f ( 1 9 6 3 ) . J . H . B i e l , A. E . D r u k k e r , T . H . M i t c h e l l , E . P . S p r e n g e l e r , P . A . N u h f e r , A. C . Conway and A. H o r i t a , J . A m . Chem. S o c . 8 2 , 2805 ( 1 9 5 9 ) . E . V o t o c e k a n d 0. L e m i n g e r , ColI. C z e c h . Chem. Commun. 4 , 2 7 1 ( 1 9 3 2 ) , C. A . 2 6 ,

5.

6. 7. 8. 9.

10.

11. 12. 13. 14.

15. 16. 17. 18. 19. 20.

5294 ( 1 9 3 2 ) . L . S c h l i t t , M. R i n k a n d M. von S t a c k e l b e r g , J , E Z e c t r o a n a l . Chem. 2 3 , 10 (1967). L . E . E b e r s o n a n d K . P e r s s o n , ,J. Med. Pharm. Chem. 5, 7 3 8 ( 1 9 6 2 ) . J . D r a b n e r a n d W . S c h w e r d , F r e s e n i u s ' Z. A n a l . Chem. 2 4 3 , 9 2 ( 1 9 6 8 ) . C. A . 70, 558941 ( 1 9 6 9 ) B . Van C l i n e s c h m i d t a n d A. H o r i t a , Biochem. Pharmacol. 1 8 , 1 0 1 1 (1969) Idem., i b i d . , 1021 (1969). E . F i s c h e r , B . H e l l e r , a n d A . H . Miro, A r z n e i m i t t e l - F o r s c h . 18, 1 4 8 6 ( 1 9 6 8 ) . F. F e i g l , "Spot T e s t i n O r g a n i c Analysis", E l s e v i e r Company, L o n d o n , 1 9 5 6 , p . 1 2 8 . B . C . Bose a n d R . V i j a y v a r g i y a , I n d i a n J . Pharm. 2 8 , 328 ( 1 9 6 6 ) . R. E . D a l y , u n p u b l i s h e d r e s u l t s M. M a r z a d r o a n d A. D e C a r o l i s , R e n d . I s t . S u n i t a 2 6 , 629 ( 1 9 6 3 ) . C . R a d e c k a a n d I . C. Nigam, Can. J . P h m m S c i . 2 , 17 (1966). C . C a r d i n i , V . Q u e r c i a a n d A . C a l o , :J. C h r o m a t o g . 37, 1 9 0 ( 1 9 6 8 ) . U. Q u e r c i a , C . C a r d i n i a n d A . Calo, i i ( ' l . C h i m . Farm. 1 0 7 , 3 8 3 ( 1 9 6 8 ) . C. M c M a r t i n a n d H . V . S t r e e t , J . Chromatog. 2 2 , 274 ( 1 9 6 6 ) .

.

.

406

PHENELZINE SULFATE

7.

References

21. 22. 23. 24. 25. 26.

(cont. )

E. S c h m i d , E. Hoppe, C . M e y t h a l e r a n d L. Z i c h a , A r z n e i m i t t e Z - F o r s c h , 1 3 , 9 6 9 (1963). W . W. F i k e , A n a l . Chem. 38, 1 6 9 7 ( 1 9 6 6 ) . A . A l e s s a n d r o , F. Mari a n d S . S e t t e c a s e , F arma co , Ed. Prat. 22, 437 ( 1 9 6 7 ) . I. Z i n g a l e s , J . C h r o m a t o g . 3 1 , 405 (1967). I. S u n s h i n e , W. W . F i k e a n d H . L a n d e s m a n , J . F o r e n s i c S c i . 1 1 , 428 ( 1 9 6 6 ) . P. R a j e s w a r a n a n d P . L . K i r k , B u l l . Narcotics, U.N., Dept. S o c i a l A f f a i r s 13, No. 3, 15 ( 1 9 6 1 ) .

407

This Page Intentionally Left Blank

PRIMIDONE

Raymond D. Daley

409

RAYMOND D. DALEY

CONTENTS

1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor, Taste 2.

Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2.4 Mass Spectra 2.5 Crystal Properties 2.6 Melting Points 2.7 Solubility

3.

Synthesis

4.

Stability-Degradation

5.

Drug Metabolic Products

b.

Methods of Analysis 6.I Elemental Analysis 9.2 Chromatography 6.21 Gas Chromatography 6.22 Thin Layer Chronatography 6.23 Paper Chromatography 6.3 Absorptiometric Methods 6.31 Ultraviolet Absorption 6.32 Ultraviolet Determination as Phenobarbital 6.33 Colorimetric Methods 6.4 Polarography 6.5 Titration 6.6 Other Analytical Tests

7.

References

PRIMIDONE

1.

Description

1.1 Name, Formula, Molecular Weight Primidone i s 5-ethyldihydro-5-phenyl-k,6(lH, pyrimidinedione.

5H)-

H

‘‘b N

/

H

\ /

Mol. W t .

‘1.2~ 4 N 2‘2 1.2

218.26

Appearance, Color, Odor, Taste White, odorless powder, w i t h a s l i g h t l y b i t t e r

taste. 2.

Physical P r o p e r t i e s

2.1

I n f r a r e d Spectra

I n f r a r e d s p e c t r a 3f two c r y s t a l forms of primidone (here designated as Form I and Farm 11) a r e shown i n Figures 1 and 2. The samples were prepared as mineral o i l mulls between potassium bromide p l a t e s . A Beckman Model 111-12 instrument was used. Similar s p e c t r a a r e obtained with potassium bromide d i s p e r s i o n s (1,2), b u t t h e pressing operation usually causes d i s t o r t i o n s of t h e absorption bands. Some of the absorption bands may be assigned as follows: 3200 cm’ region, bonded N-!I s t r e t c h ; 1700 cm” region, amide C=O; 1600, &$lo, 760, 700, and 520 cm-’, aromatic ring. The absorption i n t h e 2900 cm’ region and p a r t of the absorption at &60 and 1380 cm’ i s due t o .ninera.l o i l .

a

>

20 z

A

0

L

N

P 0

P r m

<

Fig. 1. Infrared spectrum of primidone, Ayerst Laboratories, Inc., house r e f e r e nc e standard P24636B, Form I, mineral oil m u l l between KBr p l a t e s .

P, w

Fig. 2.

Infrared spectrum of primidone, Form 11, mineral oil mull between KBr plates.

RAYMOND D. DALEY

2.2

Nuclear Magnetic Resonance Spectra

Figure 3 shows t h e proton magnetic resonance spectrum of primidone. This spectrum was obtained on a d6-dimethylsulfoxide solution, using a Varian HA-100D instrument, with a tetramethylsilane reference. The assignment of t h e spectrum i s shown i n Table I ( 3 ) . The resonances at 3.96 and 4.12 ppm a r e due t o the fragment NH-CH,TNH i n which t h e methylene protons have d i f f e r e n t c h e m c a l s h i f t s and thus show geminal coupling, and i n which one of t h e methylene protons (probably t h e e q u a t o r i a l ) i s coupled t o both t h e NH protons and i s thus a doublet of t r i p l e t s . The system can be described as AB$ ( 3 ) . TABLE I

Proton Magnetic Resonance Spectrum Peak Assignments (3)

PPM

Relative Intensity

0. 84

3

1.99

2

triplet quartet

2.50

-

quintet

3.35 3.96

-

4.12

1

1

7.30

€!. 64

2.3

5 2

Multiplicity

singlet doublet doublet of triplets mult i p l e t doublet

AssiRnment methyl of e t h y l group methylene of e t h y l group r e s i d u a l d5-DMSO i n solvent water i n solvent one of t h e r i n g methylene protons the other ring methylene proton phenyl group NH protons

U l t r a v i o l e t Spectra

Figure 4 shows t h e u l t r a v i o l e t absorption spectrum of primidone (Ayerst Laboratories, Inc., house The sample was dissolved i n methanol standard P a 6 3 6 B). (86.6 mg/100 m l ) and scanned VS. methanol. Scans of

414

81

'1

I

I

I

,1

,....l....I....I....I....l....I....I....,....I....,....

60

Fig. 3.

PPMWOMHz 50

40

20

Nuclear magnetic resonance spectrum of primidone in dg-dimethylsulfoldde, tetramethylsilane reference, courtesy of Dr. G. R. Bedford (3).

10

0 9

r

rn

<

Fig. 4.

Ultraviolet spectrum of primidone, Ayerst Laboratories, Inc., house reference standard P24636B, 86.6 mg/lOO ml and 10- and 100fold dilutions, in methanol, 1 cm cells.

PRIMIDONE

10-and 100-fold d i l u t i o n s of t h i s s o l u t i o n a r e a l s o shown, A Cary Model U, spectrophotometer was used. M&rna a t 264, 258, ar,d 252 nanometers have a b s o r p t i v i t i e s of 167, 216, and 183 l i t e r s per mole cm, respectively, i n reasonable agreement with previously published d a t a (1,4). There are no o t h e r maxima above 210 nanometers. The absorption bands i n t h e 260 nanometer region a r e caused by t h e phenyl group, while t h e absorption a t s h o r t e r wavelengths i s due t o a combination of t h e phenyl and a,rni.de group absorpticns. 2.4

Mass Spectra

Figure 5 shows the low r e s o l u t i o n mass spectrum of primidone. This d a t a w a s obtained on a Perkin-Elmer/ Hitachi FMU-6E mass spectrometer (3). The spectrum matches t h a t measured and i n t e r preted by Locock and Coutts ( 5 ) . The mol.ecular i o n C a H N202 i s a t m/e 21P. Loss of ethylene y i e l d s m/e 190 %l@l$202), and l o s s of an e t h y l r a d i c a l y i e l d s L,oss of CH2NH from m/e 190 y i e l d s m/e 1 W (C1#~N202). Loss of C2HkN20 from t h e molecular m/e 161 (CgHr$02). ion y i e l d s m/e 4 6 (C1oHlOO), the most abundant fragment. Loss of CO from m/e I46 y i e l d s m/e 118 (C H ~ o ) , which l o s e s H and H2 successively t o form m/e 1 7 and m/e 115 (5).

9

A t high r e s o l u t i o n , m/e 161 i s reported t o be a doublet of compositions C5H7NC2 and C1@11NO, with r e l a t i v e abundances 5 : 1 respectively; t h e l a t t e r , l e s s abundant, ion can be derived from t h e molecular i o n by loss of ~ $ 3 ~ (06). 2.5

Crystal F r o p e r t i e s

X-ray powder d i f f r a c t i o n p a t t e r n s (7,r) and i n f r a r e d s p e c t r a ( s e e Section 2.1) i n d i c a t e t h a t pricddone may c r y s t a l l i z e i n at Least two d i f f e r e n t forms. One of t h e s e (here designated Form I ) usually c r y s t a l l i z e s when a s o l u t i o n of primidone i n 95 percent ethanol i s evapcratec! a t room temperature. The second (here designated Form 11) usually forms i f primidone 417

BASE 146

P rn

120-

<

I

W LL

I ,

Fig. 5.

I1

2M'I 8 ,I1

Mass s p e c t r m of primidone, d a t a

c o u r t e s y of

Dr. G. 2. Bedford ( 3 ) .

PRIMIDONE

c r y s t a l l i z e s from hot water o r from t h e melt. The powder d i f f r a c t i o n p a t t e r n s of t h e two forms are given i n Table 11. These p a t t e r n s were obtained with a Norelco diffractorneter, using n i c k e l - f i l t e r e d copper KQ' radiation. The p a t t e r n of Form I i s t h e same as t h a t reported by Roy e t a1 (€?>,while t h e p a t t e r n of Form I1 i s s i m i l a r t o t h a t reported by Penprase and B i l e s (7). TABLE; 11

X-ray Powder D i f f r a c t i o n P a t t e r n s Form I1

Form I d

-

d

2.82

15

13 77 6.88 6.16 5.75 5. l b 4.59 4.18 4.16 3.95 3.93 3.72 3.65 3.44 3.27 3-13 3.06 2.9e 2.75 2.66

2.72 2.63 2.61 2.55 2.35 2.33 2.26

10

2.50

10.96 7.37 6.57 5.9e 5.46 4.96 4. 83

17 100 46 70 51 li.: 50

4.44

26

4.06

1

3.68

58 20

3.73 3.66 3.56 3.42 3.21 3.16 3.12 2.9s

18

15 15 20 10

11 22

5 5 7

4 4

2.25

6 5

2.19

8

419

,

1/10

u, 100 20

35 6 55 28

46 6 5 6

4

6 5

12

4 4 4

6

4

RAYMOND 0 . D A L E Y

2.6

Melting Points The following melting p o i n t s have been reported: 260-281OC 2e10c 2111-282OC 282OC 2G9OC

(15)

(11, 12, 13) (9, 10)

(16) (7)

The value 289°C was reported by t h e same i n v e s t i g a t o r s who reported the Form I1 d i f f r a c t i o n p a t t e r n ( s e e Section 2.5) ( 7 ) .

.

2 7

Solubility The s o l u b i l i t y of primidone at room temperature

i s as

fOll0wS:

Appror;imate S o l u b i l i t y . mg/ml

Solvent Methanol Ethanol (95%) Acetone Water Chloroform Ether Benzene Petroleum, e t h e r

3.

5. 6.2 2.0 0.

5

0.1 0.1 < 0.1 < 0.1

Synthesis Primidone can be prepared by: ( a ) Reductive d e s u l f u r i zat.ion of 5-ethyl-5-phenylt h i o b a r b i t u r i c a c i d w i t h Raney n i c k e l (9,12) o r z i n c and a c i d (9,21) ; ( b ) condensation of phenylethylmalondiarnide w i t h formamide (lo), o r formic o r o x a l i c acid (I.!+) ; ( c ) reduction of 2-ethoxy-5-phenyl-5ethylhexahydro-~,6-pyri11idinedione with hydrogen o r formic a c i d o r formamide o r zinc and acid (11,22); (d) hydrogenatZon of 2-methoxy-5-ethyltet rahydropyrimidi.ne-l+,6-dione (13) ; ( e ) e l e c t r o l y s i s of 420

PRIMIDONE

phenobarbital (12) or of the 2-imino or 2-cyanimino derivatives of 5-ethyl-5-phenylhexahydropyrimidine4,6-dione (15); ( f ) reduction of 2-cyanimino-5ethyl-S-phenylhemhydropyrimidine-4,6-dione w i t h zinc and acid (15).

To help the reader visualize the processes above, (a), (b), and (c) may be depicted as follows:

S

Ni 0

H

q( 0

H

0

\ H

H C -NH2

U

421

RAYMOND D. DALEY

4.

Stability-Degradation Primidone i s q u i t e s t a b l e , as might be p r e d i c t e d from t h e absence of r e a c t i v e f u n c t i o n a l groups, low s o l u b i l i t y , and high melting point. No r e p o r t s of d e g r a d a t i o n under o r d i n a r y c o n d i t i o n s were found, although i t i s metabolized ( S e c t i o n 5), and w i l l react under d r a s t i c chemical c o n d i t i o n s ( S e c t i o n 6).

5.

Drug Metabolic Products The r e p o r t e d metabolic p r o d u c t s of primidone i n v a r i o u s s p e c i e s are as follaws: Hetabolite

Phenylethylmalondiamide

Species man rabbit

rat Ph enobar-bit a l

man dog

rat

rabbit mouse p-Ilydroxyph enobarbi t a l

rabbit dog

man

p-Hydrowph e n o b a r b i t a l g l u c u r c n i de

rabbit

S t r u c t u r a l formulas f o r t h e s e rnetabol.ites are shown i n Figure 6. o.

Nethods of Analysis

6.1

Elemental k a l y s i s

PRIMIDONE

Figure 6 Metabolism of P rimidone

RAYMOND D. DALEY

Theoretical

66. ab 6.47

Carbon Hydrogen Nitrogen Ovgen

6.2

($1

l2.83 UC. 66

Found (%)(12)

66.1 6.5 12.8

----

Chromatography 6.21

Gas Chromatography

Many i n v e s t i g a t o r s have used gas chromatography t o analyze f o r primidone. Although t h e publishe work i s concerned almost e n t i r e l y with analyses f o r primidone i n plasma c r urine, many of t h e methods could be modified e a s i l y for analyses of pharmaceutical dosage forms. A number of t h e systems used f o r gas chromatography are l i s t e d i n Table 111; a d d i t i o n a l information i s given below.

Bogan and Smith ( 2 9 ) determined primidone and phenobarbital i n blood and urine by gas chromatography. The samples were adjusted t o pH 3 and extracted with chloroform. The drugs were f u r t h e r solvent p a r t i t i o n e d , and t h e f i n a l s o l u t i o n i n chloroform w a s evaporated t o a small volume before i n j e c t i n g an a l i q u o t i n t o t h e gas chromatograph.

Pippenger and G i l l e n (32) determined primidone, phenobarbital, phenylethylmalondiami.de, and a number of o t h e r anticonvulsants i n plasma, urine, and cerebrospinal f l u i d . They e x t r a c t e d t h e sample w i t h chloroform after buffering t o pH 6.8. The chloroform e x t r a c t was evaporated t o dryness and t h e residue dissolved i n a small amount of absolute e t h a n o l f o r i n j e c t i o n . Recoveries of known amounts of drugs added t o t h e body f l u i d s were 90 t o 100 percent.

424

PRIM1 DONE

S t r e e t (62) i d e n t i f i e d primidone and o t h e r drugs by i n j e c t i n g a mixture of t h e drug and N,O-bis( t r i m e t h y l s i l y l ) -acetamide i n t o t h e gas chromatograph. The s i l y l a t i n g reagent was drawn i n t o a 10 m i c r o l i t e r syringe, then t h e sample s o l u t i o n w a s drawn i n t o t h e same syringe, and t h e mixture was i n j e c t e d d i r e c t l y i n t o t h e gas chromatograph. Trimethylsilyl derivative8 were formed i n t h e column. He used 4 m i c r o l i t e r s of 10% N,Obis-( trimethylsily1)-acetamide i n acetone f o r 0.8 microl i t e r s of primidone s o l u t i o n containing 0.8 pg of t h e drug. Clarke ( 3 9 ) a l s o l i s t s a gas chromatographic system i n which t h e r e t e n t i o n t i m e f o r primidone i s 0.65 r e l a t i v e t o codeine. Evenson, Jones, and Darcey (33) e x t r a c t e d a c i d i f i e d serum with chloroform, evaporated t h e e x t r a c t , took up t h e residue i n a chloroform s o l u t i o n of dehydroisoandrosterone a c e t a t e i n t e r n a l standard, and analyzed t h e s o l u t i o n f o r both primidone and diphenylhydantoin by gas chromatography. The average recovery of primidone from serum was 98 percent. High grade chloroform was used t o avoid extraneous peaks. F k t e t a1 (34) and Kupferberg (35) found t h a t primidone could be converted t o a methylated d e r i v a t i v e i n t h e gas chromatograph by i n j e c t i n g a s o l u t i o n containing primidone and trimethylanilinium Kupferberg used t h e r e a c t i o n t o determine hydroxide. primidone, phenobarbital, and diphenylhydantoin i n plasma samples. The drugs were extracted with ethylene d i c h l o r i d e from samples buffered t o pH 7.2, f u r t h e r solvent p a r t i t i o n e d and mixed w i t h cholestane i n t e r n a l standard, and f i n a l l y mixed w i t h trimethylanilinium hydroxide s o l u t i o n and i n j e c t e d i n t o t h e gas chromatograph. Recovery of primidone from plasma o r water was only 30 percent, probably because primidone was l o s t i n t h e multiple solvent exchanges (35).

V a n Meter, Buckmaster, and Shelly (41) used primidone as an internal standard i n t h e gas chromatographic determination of phenobarbital and

425

RAYMOND D. DALEY

diphenylhydantoin i n plasma, and suggested t h a t phenob a r b i t a l could be used as an i n t e r n a l standard i n assaying primidone. Van Meter (42) recommended using mephenytoin as an i n t e r n a l standard for primidone assays. Other i n v e s t i g a t o r s have a l s o used primidone as an i n t e r n a l standard i n gas chromatography (43). Gardner-Thorpe e t a1 ( 3 6 ) determined primidone and a number of o t h e r a n t i e p i l e p t i c drugs i n rhe drugs were extracted plasma by gas chromatography. f r o m a c i d i f i e d plasma samples with chloroform and i n j e c t e d without preparing derivatives. These i n v e s t i g a t o r s observed 30 percent recovery of primidone from plasma by t h e i r technique. Proelss and Lohmann (37) developed a r a p i d gas chromatographic screening procedure for 40 drugs, including primidone, i n serum. The drugs were separated i n t o a c i d i c , n e u t r a l , and b a s i c f r a c t i o n s by solvent extraction. The t h r e e f r a c t i o n s were then analyzed by gas chromatography. Kupferberg (38) reported t h a t primidone formed a t r i m e t h y l s i l y l d e r i v a t i v e with N,O-bis( t r i m e t h y l s i l y l ) -acetamide i n pyridine. Primidone was extracted from plasma buffered t o pH 7.2, using chloroform. After s e v e r a l solvent p a r t i t i o n i n g s t e p s , t h e primidone wa8 t r e a t e d with t h e reagent for 20 minutes. The s i l y l a t e d d e r i v a t i v e was s t a b l e for s e v e r a l hours. Recovery of primidone a f t e r t h e solvent p a r t i t i o n i n g s t e p s was incomplete. Toseland, Grove, and Berry

(40) developed

a routine procedure for gas chromatographic analyses of primidone and s e v e r a l o t h e r anticonvulsant drugs i n blood. The drugs were extracted i n t o chloroform and f u r t h e r solvent p a r t i t i o n e d before chromatography. By doing double e x t r a c t i o n s , they were a b l e t o recover 58 t o 67 percent of t h e primidone; they report not being a b l e to reproduce t h e good recovery claimed by some o t h e r investigators. They preferred not t o prepare d e r i v a t i v e s , finding b e t t e r r e p r o d u c i b i l i t y i n day-to-day routine work w i t h t h e underivatized drugs.

42 6

PRIMIDONE

TABLE I11

Gas Chromatographic Systems Used f o r Primidone Analvsis Note: A l l systems l i s t e d used flame i o n i z a t i o n d e t e c t o r s . Column

6 f t . long x 118 in. O.D.

Column Temp O C

180'

stainless s t e e l t u b i n g

5% SE-30 on Aeropak 30 (100-120mesh)

6 f t . long x 4 mm I.D. g l a s s t u b i n g , 1% HIEFF-8-BP on Gas Chrom Q, 100-120 mesh

2 50°

125 crn long x 4 mm I.D. g l a s s tubing, 3.8% W-98 on D i a t o p o r t S, 80-100 mesh

215'

6 f t . long x 4 mm I.D. g l a s s tubing, 3% OV-17 on Chromosorb W (HD)

Programmed f rom 160° t o 275' at

1o0/min

3 f t . l o n g x 4 mm I.D.,

2 50'

1% CDMS ( a ) on Diatomite CQ

6 f t . long x 4 mm I.D. g l a s s tubing, 3% OV-17 on 80-100 mesh Chromosorb W (HP)

Programmed from 160' t o 250' a t 1o0/min

RAYMOND D. DALEY

Carrier

Column

Gas

T e m ~OC

N 58

225'

2 f t . long x 2 mm I.D. g l a s s tubing, 2% SP-1000 on Gas ChromW

He 50 ml/min

25 0 '

5 ft. long x 1/4 in. I.D. glass tubing, 2% SP-lo00 on Varaport

A 90 d/min

2 50"

Column

5 ft. long x 4 m I.D. g l a s s tubing, 2.5% SE-30 on 80-100 mesh Chromosorb W AWHMDS

ml/min

6 ft. long x 4 mm g l a s s tubing, 1% OV-17 on HP Chrornosorb G (80-100 mesh)

185 cm long x 3 mm I.D. 3% OV-1 on Chromosorb

,

W (HP) (80-100 mesh) 180 cm long x 4 mm I.D. g l a s s tubing, 3.5% XE-60 on Gas krom Q (100-120 mesh)

(a)

--

3

220°

He 80

240°

ml/min

ml/rnin

CDMS i s c y c l o h e m e dimethanol succinate.

428

Programmed from 190' t o 230'

PR IMI DONE

Gallagher and bumel (44)determined primidone by gas chromatography a f t e r preparing a methylated d e r i v a t i v e w i t h N,O-bis-( t r i m e t h y l s i l y 1 ) trifluoroacetamide and dimethylformamide ( 5 : l ) at room temperature. 6.22

Thin Layer Chromatography

Huisman (45) extracted primidone and o t h e r anticonvulsant drugs from serum with chloroform/methyl a c e t a t e (60:40) after adding hydrochloric a c i d and sodium chloride. The e x t r a c t was evaporated and the residue n i t r a t e d t o form t h e nitrophenyl d e r i v a t i v e of the drug. The n i t r a t e d material was chromatographed on f l u o r e s c e n t s i l i c a g e l t h i n l a y e r chromatographic p l a t e s with i s o b u t y l alcohol/chloroform/25% ammonia (72:48:L2) ; t h e developed p l a t e was d r i e d 10 minutes a t 100°C and developed a second time. The s p o t s were located under u l t r a v i o l e t illuminat i o n ; t h e n i t r a t e d primidone R was 0.76 t o 0.80, The s p o t s were scraped o f f , t h e nierophenyl d e r i v a t i v e reduced with zinc, t h e r e s u l t i n g amino group d e r i v a t i v e diazotized with n i t r o u s a c i d and coupled with a-naphthylethylenediamine, and t h e determination f i n i s h e d by measuring t h e absorbance a t 550 m. (The c o l o r i m e t r i c procedure was adapted from t h a t of D i l l e t a1 (50) f o r diphenylhydantoin. ) Fujimoto, Mason, and Murphy (24) e x t r a c t e d primidone from r a b b i t u r i n e with chloroform a f t e r adding a c e t i c acid. Chromatography was on s i l i c a g e l impregnated glass f i b e r sheet , using 2,2,4-trimethylpentae/glacial The s p o t s were detected by heating a c e t i c a c i d (5:l). t h e s h e e t a f t e r spraying with concentrated s u l f u r i c acid. The amounts of t h e drug The Rf of primidone was 0.7. were estimated from t h e s i z e and i n t e n s i t y o f t h e spots. The i n v e s t i g a t o r s reported 100 percent recovery of t h e drug from urine, and an accuracy of about 1 & primidone i n 3 t o 10 pg spots. Gardner-Thorpe, Parsonage, and T o o t h i l l (46) examined t h i n l a y e r chromatographic systems f o r primidone and 20 o t h e r drugs. The drugs were e x t r a c t e d from plasma with chloroform after a c i d i f y i n g with hydrochloric acid. The chloroform e x t r a c t w a s d r i e d and

R A Y M O N D D. D A L E Y

evaporated. The residue was dissolved and chromatographed on s i l i c a g e l plates. Nine d i f f e r e n t solvent systems and ten d e t e c t i o n systems were tested. The most s e n s i t i v e d e t e c t i o n system f o r primidone among those t e s t e d was exposure t o iodine vapor, followed by spraying with a one percent aqueous s o l u t i o n of mercurous n i t r a t e ; t h i s detected 5 pg of primidone. ( S u l f u r i c a c i d charring was not tested.) The Rf values f o r primidone i n t h e solvent systems t e s t e d were as follows: Solvent 1.

2.

3.

4. 5.

6.

7. 8.

9.

Methanol/O.P8 ammonia (38.5:1.5) (60 min e q u i l i b r a t i o n ) Di-n-butyl ether/l-butanol/acetic acid (80:40: 10) Benzene/dioxane/O. 88 ammonia (60:35: 5 ) Ethanol/acetic acid/water (50:30:20) Methanol/l-butanol (60:40) Chloroform/acetone (9O:lO) Chloroform/acetone (90:lO) 60 min e q u i l i b r a t i o n , f i l t e r paper l i n i n g tank Benzene/acetic a c i d (9O:lO) Benzene/dioxane/O. 88 ammonia (75:20: 5) Pippenger, Scott, and G i l l e n described

(47) a r a p i d semi-quantitative method f o r determining primidone and s e v e r a l o t h e r drugs i n plasma and urine. The drugs were e x t r a c t e d from a c i d i f i e d samples with chloroform. The e x t r a c t was evaporated and t h e residue dissolved i n a small amount of ethanol. Aliquots were spotted on t h r e e d i f f e r e n t s i l i c a g e l p l a t e s , along with standards. The p l a t e s were developed i n t h r e e d i f f e r e n t solvent systems and examined under s h o r t wavelength u l t r a v i o l e t illumination. The lower limit of d e t e c t i o n f o r primidone was 2 CLg. The s o l v e n t s and Rf values f o r primidone were as follows: Solvent Chloroform/acetone ( 9 : l ) Carbon tetrachloride/acetone ( 7 : 3 ) Benzene/acetone (4: 1) 430

..

19 26 11

PRIMIDONE

Yamamoto, Suzuki, and Okuo r e p o r t (48) Crg primidone by chromatographing on The Rf value s i l i c a g e l with chloroform/acetone (9:l). was 0.06 and d e t e c t i o n was with i o d i n e vapor and one percent mercurous n i t r a t e .

a s e n s i t i v i t y of one

Garrettson and Dayton (49) described a t h i n l a y e r chromatographic method i n which t h e s p o t s were scraped o f f t h e p l a t e , t h e drug e x t r a c t e d with 20 percent a l c o h o l i n e t h e r , t h e e x t r a c t evaporated, and t h e drug determined c a l o r i m e t r i c a l l y by t h e method of D i l l e t a1 (50). (See method of Huisman above, i n t h i s Section.) Their method i s about 10 times as s e n s i t i v e as t h a t of

Huisman. 6.23

Paper Chromatography

Rusiecki, Henneberg, and Ostrowska ( 2 5 ) reported a paper chromatographic s e p a r a t i o n of primidor:e and phenobarbital. The drugs were e x t r a c t e d with chloroform from a c i d i f i e d blood, urine, o r homogenized organs. The e x t r a c t s were washed with pH 7.4 phosphate b u f f e r and evaporated t o dryness. The residue was dissolved i n methanol and chromatographed on Whatman No. 1 paper impregnated with formamide. With ascending chromatography using t h e solvent system isopropyl alcohol/chloroform/25$ ammonia (45:45:10), t h e Rf f o r primidone was 0.97-0.98, w h i l e t h a t f o r phenobarbital was 0.67. Detection was by spraying w i t h mercuric chloride and e o s i n i n 95 percent ethanol. The minimum d e t e c t a b l e amount of primidone was 50 pg.

6.3

Absorptiometric Methods

6.31

U l t r a v i o l e t Absorption

The u l t r a v i o l e t absorption i n t h e 250 run region i s t h e b a s i s f o r a number of methods f o r d e t e r m n i n g primidone (1,4,17,18,39). The IJSP XVIII method (17) u s e s a 254 t o 261 nm baseline i n measuring the 257 nm peak absorbance; t h i s w i l l reduce i n t e r f e r e n c e from some u l t r a v i o l e t absorbing materials, but o t h e r s w i l l have an absorption p a t t e r n s i m i l a r t o t h a t of primidone. ‘\.e method i s t h e r e f o r e not s p e c i f i c enough f o r marly

RAYMOND D. DALEY

purposes. Furthermore, t h e a b s o r p t i v i t y i s not high, and t h i s increases t h e p r o b a b i l i t y of i n t e r f e r e n c e i f more than small amounts of u l t r a v i o l e t absorbing materials a r e present. Wysokowski and Rewerski (51) reported determination of primidone i n blood by measuring t h e absorbance of e x t r a c t s at 225 nm, but they a l s o suggested t h a t metabolites might i n t e r f e r e . 6.32

U l t r a v i o l e t Determination as Phenobarbital

Bush and Helman (16) determined primidone i n urine and blood as phenobarbital, a f t e r oxidation of t h e former w i t h dichromate. Primidone and any phenob a r b i t a l i n the samples were e x t r a c t e d with ether. The e t h e r e x t r a c t s were e x t r a c t e d with 0.1 ,M ethanolamine t o remove phenobarbital, and then evaporated t o dryness. The residue was t r e a t e d with 0.042 ,M potassium dichromate i n 9.5 ,M s u l f u r i c a c i d t o oxidize primidone t o phenob a r b i t a l . The phenobarbital formed was e x t r a c t e d i n t o ether, and t h e e t h e r washed with pH 6.2 buffer, 0.1 hydrochloric acid, and water. Finally, t h e phenobarbital formed was extracted i n t o 0.1 g ethanolamine and determined from its u l t r a v i o l e t absorption at pH 9.3, 7.3, P a r t i t i o n c o e f f i c i e n t s f o r primidone i n and 5.3. s e v e r a l solvent systems were a l s o determined.

6.33

Colorimetric Methods

Early assays f o r primidone i n blood and t i s s u e were done c o l o r i m e t r i c a l l y (52). The drug was extracted from t h e sample at pH 5.5 i n t o a mixture of methyl a c e t a t e and chloroform. The e x t r a c t was washed with pH 11.8 b u f f e r and evaporated. The residue was t r e a t e d by a method similar t o t h a t of D i l l et a1 (SO) f o r diphenylhydantoin. The metabolite phenylethylmalondiamide i n t e r f e r e s , but can be removed a f t e r conversion with n i t r o u s a c i d t o a-phenyl-n-butyric a c i d , which i s alkali-soluble (52). Primidone can be hydrolyzed i n 60 t o 80 percent s u l f u r i c acid, y i e l d i n g formaldehyde (12,52) which can be determined with chromotropic acid. The 432

PR I MI DONE

B r i t i s h Pharmacopoeia i d e n t i f i c a t i o n test f o r primidone i s t h e appearance of a pinkish-blue c o l o r on heating with chromotropic acid s o l u t i o n (53).

6.4

Polarography

Bozsai and Vastagh (54)determined primidone by polarography a f t e r n i t r a t i n g i t w i t h a s u l f u r i c - n i t r i c a c i d mixture. The phenyl group n i t r a t e s t o form t h e which i s reducible at t h e 3'-nitrophenyl d e r i v a t i v e dropping mercury electrode. The method was applied t o t a b l e t s containing primidone, as w e l l as t o t h e raw material. The observed half-wave p o t e n t i a l was -0.17 v o l t VS. the s a t u r a t e d calomel electrode.

(a),

6.5

Titration

Kalinowska e t a1 ( 5 5 ) t i t r a t e d primidone, i n an acetone-water s o l u t i o n containing sodium hydroxide, with s i l v e r n i t r a t e . The endpoint was a p e r s i s t e n t t u r b i d i t y , caused by p r e c i p i t a t i o n of t h e d i - s i l v e r salt of t h e drug ( t h e mono-silver salt was soluble).

6.6

O t h e r Analytical Tests

Barysheva ( 5 6 ) found t h a t primidone showed a blue luminescence i n t h e u l t r a v i o l e t . Cooper (57) l i s t s t h e c o l o r s formed by many drugs with p-dimethylaminobenzaldehyde; primidone forms a primrose color. Ammonia is evolved when primidone i s heated with anhydrous sodium carbonate (17). Haug and Panescu (5s) describe a rapid color test f o r d e t e c t i n g small amounts of phenob a r b i t a l or 5-phenyl-5-ethylthiobarbiturhc a c i d i n primidone. Kassau (59), Kuhnert-Brandstatter e t a1 ( 4 ) , and Penprase and Biles (7) describe microscopic t e s t s f o r primidone. Shamotienko (60) developed a color t e s t f o r primidone, i n which the same i s boiled with chloramine, copper s u l f a t e , and sodium hydrofide; primidone causes a reddish-violet color t o develop. Lobanov and Gorbunova (61) determined primidone i n t e r f e r o m e t r i c a l l y i n t a b l e t s and powders.

433

RAYMOND D. DALEY

7

.

References 1. Drug Standards 2, 101-2 (1957). 2. A. L. Hayden, 0. R. Sammul, G. B. Selzer, and J. Carol, J. Ass. Offic. Anal. Chem. 797-900 ( 1962 3. G. R. Bedford, Imperial Chemical I n d u s t r i e s Limited, personal gommunication. 4. M. Kuhnert-Brandstatter, A. Kofler, R. Hoffman, R h i , Sci. Pharm. 2,205-30 (1965). and H.-C. 5. R. A. Locock and R. T. Coutts, Org. Mass Spectrom. 2, 735-45 (1970). 6. B. R. Brewster, Imperial Chemical I n d u s t r i e s Limited, personal communication. 7. W. G. Penprase and J. A. Biles, J. Am. Pharm. ASSOC. 523-8 (195E). 8. J. Roy, M. Gadret, and C. Bregere, Bull. SOC. Pharm. Bordeaux 103,3-8 (1964). 9. W. R. &on, H. C. Carrington, and C. H. Vasey, U. S. Patent 2,576,279 (1951); C.A. 4.6, 616I.h. 10. W. R. Boon, H. C. Carrington, and C. H. Vasey, Patent 2,578,847 (1951); C. A. 46, 61621. 11. C. H. Vasey, U. S. Patent 2,676,176 (1954); C.A. 19, 11027g. 12. W. R. Boon, H. C. Carrington, N. Greenhalgh, and C. H. Vasey, J. Chem. SOC. 3263-72. 13. E. London and C. H. Vasey, Brit. Patent 710,269 (1954); C.A. 50, 2687h. 14. W. R. Boon, N. Greenhalgh, E. London, and C. H. Vasey, B r i t . Patent 709, 808 (1954); C.A. 49, 11027c 15. W. P. Seebode and C.H. Vasey, B r i t . Patent 919,703 (1963); C.A. 2,7540a. 16. M. T. Bush and E. Helnan, Life Sci. &, 403-9 ( 1965 17 Primidone monographs, United S t a t e s Pharmacopeia, 18th revision, Mack P r i n t i n g Co. , Easton, Pa., 1970, pp. 538-40. 18 V. I. Nekrasov, Aptech. Delo 12 (3), 37-41 (1966); C.A. &, 8676a. 19. V. I. Nekrasov, Tr. 1 [Pervogol Mosk. Med. Inst. 61, 257-9 (1968); C.A. 71, 94823m. 20. J. Wysokowski and W. Rewerski, Diss. Pharm. Pharmacol. 20 (4), 369-72 (1968).

u,

m,

.

.

-

434

PRIM1 DONE

21.

Trampau, Ger. (East) Patent 43, 19638g. C. H. Vasey, B r i t . Patent 710,267 (1954); C. A. I!&, l4816f. J. M. Thorp, Congr. Int. Biochirn. Resumes Commun. 3rd (Brussels) 132; C.A. 50, l4127a. J. M. F u j i m t o , W. H. Mason, and M. Murphy, J. Pharmacol. Exp. Ther. (2), 379-88 (1968); C. A. &, 48068j. W. Rusiecki, M. Henneberg, and B. Ostrowska, Diss. Pharm. Pharmacol. 18 ( 3 ) , 299-304 (1966); C.A. 66, 9578~. J. W. Huisman, Pharm. Weekbl. (27), 799-802 (1969). G. L. Plaa, J. M. Fujimoto, and C. H. Hine. J. Am. Med. ASS. 168, 1769-70 (1958). J. Bogan and H. Smith, J. Pharm. Pharmacol. 20 (11, 66-7 (1968). H.-H. Frey and I. Hahn, Arch. Int. Pharmacodyn. Ther. 128, 281-9 (1960); C.A. f?zI l0693d. J. W. Huisman, Pharm. Weekbl. (20), 573-600 (1968); C.A. 9, 427362. T. C. Butler and W. J. Waddell, Proc. SOC. Fkp. Biol. Med. 2, 544-6 (1956). C. E. Pippenger and H. W. G i l l e n , Clin. Chem. 15 (71, 582-90 (1969). M. A. Ehrenson, P. Jones, and B. Darcey, Clin. Chem. 16 ( 2 ) , 107-10 (1970). W. FGrst, H. Lauterbach, K.H. Daute, and E, Klust, Zentralbl. Pharm. loq, ( 9 ) , 907-12 (1970). H. J. Kupferberg, Clin. Chim. Acta 3, 2E3-8 (1970). C. Gardner-Thorpe, M. J. Parsonage, P. F. Smethurst, and C. T o o t h i l l , Clin. Chim. Acta 36, 223-30 (1972 ) H. F. Proelss and H. J. Lohmann, Clin. Chem. 222-8 (1971) H. J. Kupferberg, J. Pharm. Sci. 61, 284-6 (1972). E. C. C. Clarke, Ed., T s o l a t i o n and I d e n t i f i c a ion of Drugs", The Pharmaceutical Press, London, 1969. G. Knoll and L.

699 (1966); C.A.

22. 23.

24. 2 5.

37. 38.

39.

a,

m,

us

RAYMOND D. DALEY

40. 41.

42.

43. 44.

45. 46.

47. 48.

49. 50.

51. 52

53

.

Toseland J. G ve d D. J. Berry, Clin, Acta 2, '321-6~19?2~ Van Meter, H. S. Buckmaster, and Shelley, Clin. Chem. 16, 135-8 (1970). Van Meter, Clin. Chem. 460 (1971). D, Sampaon, I. Harasymiv, Bnd W. J. Hensley, Clin. ch-0 & 382-5 (1971). B. B. Gallagher and I. P. Baumel, i n "Antiepileptic Druge", D. M. Woodbury, J. K. Penry, and R. P. Schmidt, Fds., Raven P r e s s , New York, 1972, pp. 353-6. J. W. Huisman, Clin. Chim. Acta lJ, 323-8 (1966). C. Gardner-Thorpc, M. J. Parsonage, and C. T o o t h i l l , CUn. Chim. Acta 22, 39-47 (1971). C. E. Pippenger, J. E. S c o t t , and H. W. Gillen, Clin. Chem. 2,255-60 (1969). J. Yamamoto, H. Suzuki, and S. Okuo, Yakuzaigaku 3 (4), 23-6 (1965); L A . Q, 18428d. L. K. Garretson and P. G. Dayton, CUn. Pharmacol. Ther. 11, 674-9 (1970). W. A. D i l l , A. Kaeenko, L. Wolf, and A. J. Clazko, J. Pharmacol. Exp. Ther. 118, 270-9 (1956). J. Wysokawski and W. Rewerski, Dies. Pharm. PharmacOl. 20, 369-72 (1968); C.A. 70, 1031%. A. Spinks and W. S. Waring, Progr. Med. Chem. 2, 286 (1963). B r i t i s h Pharmacopeia 1968, The Pharmaceutical

x,

Press, London, p. 807. G. Bozsai and G. Vastagh, Pharm. Zentralhalle 103, 403-8 (1964); C.A. 6l, 10537h. 55. Z. E. Kalinowska, L. Podkowska, and J. Mieszczakowska, Farm. Polaka 14,329-31 (1963); C.A. 60, 10478g. 56. A. I. Barysheva, Aptech. Delo 2 (4), 78-80 (1964); C.A. &, 15157go 495-6 (1956). 57. P. Cooper, Pharm. J. lJJ, 58. U. Haug and I. Panescu, P h a m z i e l8, 340-1 ( 1963 59. E. Kasaau, Deut. Apoth. Ztg. 10 613-6 (1964). 60. G. D. Shamotienko, Farm. Zh. Kiev) 22 (4), 53-4 (1968); C.A. 70, l4468r. 61. V. I. Lobanov and L. F. Gorbunova, Farm. Zh. (Kiev) 26 (31, 38-41 (1971). 62. H. V. S t r e e t , J. Chromatog. Q, 358-66 (1969).

54.

74,

436

PRIM1 DONE

ACKNOWLEM;MENTS The writer wishes to thank Dr. B. T. Kho of Ayerst Laboratories, Inc., f o r his suggestions f o r improvement of the manuscript, and Dr. G. R. Bedford and Dr. B. R. Brewster of Imperial Chemical Industries Limited for their mass spectral and nuclear magnetic resonance information.

This Page Intentionally Left Blank

PROPIOMAZINE HYDROCHLORIDE

Kathleen B. Cvomhie mid Leo I=. Culler1

43Y

K. B. CROMBIE AND L. F. CULLEN

C0NTE;NTS

1. Description 1.1 N a m e , Formula, Molecular Weight 2.

3.

4. 5. 6. 7.

8.

1.2 Appearance, Color, Odor Physical Properties 2 . 1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 U l t r a v i o l e t Spectra 2.4 Mass Spectra 2.5 Melting Range 2.6 M f f e r e n t i a l Thermal Analysis 2.7 S o l u b i l i t y 2.8 Crystal Properties 2.9 I o n i z a t i o n Constant, pKa Synthesis S t a b i l i t y Degradation Drug Metabolic Froducts Identification Methods of Analysis 7.1 Elemental Analysis 7.2 U l t r a v i o l e t Spectrophotometric Analysis 7.3 Colorimetric Analysis 7.31 Palladium Chloride 7.32 p-Benzoquinone 7.h Polarographic Analysis 7 .5 T i t r i m e t r i c Analysis 7.51 Sodium Lauryl S u l f a t e 7.52 Ceric Sulfate 7.53 Perchloric 4cid 7.6 Chromatographic Analysis 7.61 Thin Layer Chromatography 7.62 Paper Chromatopaphy 7 -63 Gas Chromatography References

-

440

PROPIOMAZINE HYDROCHLORIDE

-

1. D e s c r i p t i o n

1.1 Name, Formula, Molecular Weight Propiomazine h y d r o c h l o r i d e i s d e s i g n a t e d by t h e f o l l o w i n g chemical names : :lo-( 2-dimethyl&nopropyl) 2 -pr opionylp hen o t h i a z i ne h d r och l o r i d e l , and p r o p i onylpromethazine hydrochloride I n t h e s u b j e c t i n d i c e s of Chemical A b s t r a c t s , t h e compound i s l i s t e d under t h e heading: 1-[lo-12-( dimethylamino) p r o p y l ] p h e n o t h i a z i n 2-yl]-1-propanone monohydrochloride. The most commonly used t r a d e name i s Largon Hydrochloride. The e m p i r i c a l formula is C20H2~N20S.ACl with a molecular weight of

-

3;.

3-76a 9 5 0 CH3 CH2-kHN (CH, ) z I

1.2

Amearance. Color. Odor

if.

Propiomazine h d r o c h l o r i d e i s a yellow, p r a c t i c a l l y o d o r l e s s powder 2.

Physical Properties

Infrared Spectra The i n f r a r e d spectrum of propiomazine hydrochloride ( N .F, Reference Standard m a t e r i a l ) , p r e s e n t e d i n Figu r e 1, h a s been r u n i n a KBr p e l l e t . The f a l l o w i n g absorpt i o n band assignments have bee made.3 V i b r a t i o n Mode Wavelenpth of Plbsorption (cm .-') CH s t r e t c h i n g 2920 2580 and 21150 NH' s t r e t c h i n g 1690 C=O s t r e k h i n g (aryl ketone ) aromatic C=C in-plane 15'90 vibration continued 2.1

.. ..

44 1

0 9

0

H E

rn

P P

D

2 0

N

rn r

z

F i g u r e 1.

I. R. S p e c t r u m of P r o p i o m a z i n e H y d r o c h l o r i d e S t a n d a r d M a t e r i a l ) - 1%KBr I n s t r u m e n t : P e r k i n E l m e r Model 2 1

(N. F.

Reference

PROPIOMAZINE HYDROCHLORIDE

2 . 1 I n f r a r e d Spectra ( c o n t i n u e d ) W b r a t i o n Mode Wavelength of Absorption (cm.-l) l460 C-CH, deformation -CH,deformation N-CH, deformation aromatic #J s t z e t c h i n p 1325 C-S s t r e t c h i n g 1055 aromatic CH out-of890 and 867 p l a n e deformation (1,2 ,h-trisubsti t v+ed benzene r i n g ) 752 aromatLc Ch out-ofp l a n e deformation (ortho-disubstituted benzene r i n g )

Nuclear Magnetic Resonance S p e c t r a The nuclear magnet.ic resonance (NMR) spectrum ( F i g u r e 2 ) was o b t a i n e d by p r e p a r i n g a s a t u r a t e d s o l u t i o n of propiomazine h y d r o c h l o r i d e ( N .F. Reference Standard m a t e r i a l ) i n deuterochloroform c o n t a i n i n g t e t r a m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d . The o n l y exchangeab1.e p r o t o n i s t h e hydrogen a s s o c i a t e d w i t h HC1. The f o lowi n g NMR p r o t o n s p e c t r a l assignments have been made. 2.2

;

Chemical S h i f t ( ppm. )

Proton

c)

- CH2-%

1.20

I!

167 2.91

CH-C& N-( CH,

3.10

I-a&CH3 N-CP,-CH

3.50 t o 4.40 4.97 7.42 and 7.75

)a

N-CHZ-CH aroma t,Z

2.3

triplet doublet singlet qu ar t.et,

---- ---

Ultraviolet Spectra Tompsett reportedAmax. at. 242 m u and 273 m u f o r propiomazine h y d r o c h l o r i d e i n 1 N h y d r o c h l o r i c a c i d . 5 Propiomazine h y d r o c h l o r i d e (N.F. Seference Standard m a t e r i a l ) when scanned between 350 mri '2nd 220 pi exhilli t e d hmax. a t 242 mu ( € 4 7 . 5 ) and 273 mu ( E - 4 5 . 2 ) . This spectrum i s shown i n F i g u r e 3.

443

0

a

z 0

rn

P P P

Figure 2.

NMR S p e c t r u m of P r o p i o m a z i n e H y d r o c h l o r i d e

(N. F. R e f e r e n c e Standard Material) Solvent: deuterochloroform, i n t e r n a l standard: tetramethylsilane I n s t r u m e n t : J e o l c o Model C - 6 0 HL

I

<

D

a 0 0

I r

Figure 3.

UV Spectrum of Propiomazine Hydrochloride (N. F. Reference Standard Material) Solvent: 1N hydrochloric acid Instrument: Cary Model 14

K. 6. CROMBIE AND L.

F. CULLEN

Mass ,Spectrum The mass spectrum of propiomazine hydrochloride (N .F. Reference Standard material) was obtained by d i r e c t 2.4

3.

i n s e r t i o n of the ample i n t o an Ms-902 double focusing mass spectrometer The ion source temperature was 20OOC. and t h e ionizing e l e c t r o n beam energy was 60 eV. The high r e s o l u t i o n d a t a was compiled and tabulated with t h e a i d of an on-line m)P-8 D i g i t a l Computer. Results a r e presented a s a bar graph (Figure 4 ) and t h e high r e s o l u t i o n mass spectrum assignments of t h e prominent ions a r e given i n Table I. Propiomazine hydrochloride gives a molecular ion of t h e free base a t m/e 340.1622. The first prominent fragment i s a t mass 269.0854 which corresponds t o t h e loss of t h e metastable i o n , N, supported by t h e fragment a t 71.0736. This metast b e ion y i e l d s a protonated f r a g ment a t mass 72.0806 which i s t h e most abundant peak i n the spectrum. Documented f i s s i o n from t h e molecular i o n of the side-chain C-C b o n d 4 t o the phenothiazine r i n g nitrogen generates t h e metastzble 10-methylene-phenothia z i n y l ion a t m/e 268.0807 which l o s e s t h e s u l f u r group t o form t h e m

ckHS

\

C OC H, C H3 i o n of m/e 236.1107 A peak a t 254.0627 i s i n d i c a t i v e of t h e charged ion 2-propionyl-phenothiazin-10-yl r e s u l t i n g from t h e cleavage of t h e e n t i r e s i d e chain attached to t h e 10-position r i n g nit.rogen. In t h e p a r t i a l fragmentation of t h e 2-substitue n t , an i o n of mass 225.0261 with the formula CUH~ONS i s observed which corresponds t o t h e loss of C2Hs from t h e fragment a t 25J-1.0627. Cleavage of t h e e n t i r e 2-substitue n t from 2-propionyl-phenothiazin-10-yl y i e l d s a charged

F,.

a t d e 197.0284.

446

F i g u r e 4.

Mass S p e c t r u m of P r o p i o m a z i n e H y d r o c h l o r i d e Standard Material) I n s t r u m e n t : AEI-Model MS 902

(N.F. R e f e r e n c e

K. B. CROMBIE AND L. F. CULLEN

TAELE I High Resolution Mass Spectrum Assignments

of Propiomazine HC1 Measured Mass

Calculated Mass

340.1622

340 .1608

281 .0891

281.0873

269.0854

269.0874

268.0807

268.0796

25’4.062 7

254.0639

236 ~ 1 0 7

236 .ii@

225.0261

225.0247

197.0284

197.0298

72.0806

72.0813

7 1 -0736

71 00734

448

PROPIOMAZINE HYDROCHLORIDE

2.5

Melting Range The fo llo win g m eltin g p o i n t temperatures ( "C.) have been obtained on propiomazine hydrochloride ( N .F. Reference Standard m a t e r i a l ) employlng t h e U .S:P .,6VIII, Class 1, conditions:9 201-201, w i t h decomposition

.

2.6

D i f f e r e n t i a l Thermal An aly s is The d i f f e r e n t i a l th ermal a n a l y s i s (DTA) curve of propiomazine hydrochloride (N .F. Reference Standard materi a l ) run from room temperature t o t h e me ltin g p o i n t exh i b i t s no e ndo th em s o r exotherms o t h e r th a n a n endotherm a t 199" 203OC. a s s o c i a t e d with t h e m e l t . 1 ° S in c e t h e compound melts with decomposition, t h i s endotherm i s n o t w e l l d e f i n e d , The DTA curve was r u n on a Dupont 900 DTA u s i n g a micro c e l l and a h e a t i n g r a t e of 5"C./rnin.

-

Solubil9 The fo llo win g approximate s o l u b i l i t y d a t a have been de t e r m i ed f o r propiomazine hydrochloride a t room temperature : ,11 water: greater th an 1 g./ml. i s o t o n i c sodium c h l o r i d e s o l u t i o n : p r e a t e r th a n lg ./ 2,7

ml

95% e t ha n o l:

100 mg./ml. chloroform: 400 mg ./ml. acetone: 10 mg./ml. benzene: less th an 0.1 mg./ml. ether: less t h a n 0.1 mg./ml.

2.8

.

Crystal P r o p e r t i e s The X-ray powder d i f f r a c t i o n p a t t e r n of propiomazine hydrochloride ( N .F. Reference Standard m a t e r i a l ) obt a i ne d with a P h i l i p s d i f f r a c t o m e t e r u s in g Cu K d . r a d i a t i o n l o i s shown i n F i g u r e 5. The ca lc lila te d d-spacings 10 f o r t h e d i f f r a c t i o n p a t t e r n shown i n Figure 5 are given i n Table 11.

449

yo

loo

Pmrrn No.: 236 W

90

m:9/I5/70

Wr.:

N.D.A

Sarapk: P m p i a n u i n HCI

VO

Sourer: N.F. Ralerence Sbmrd Molerial W

a0

TUD~

tw; cu

Sun 4 W : 1Ymin.

60

so

70

24

ma

C.P.S.: Xt@

c u d crpw Mono.: Yes 70

MA

volt: pow

@in: 1211

DMcta: Prap.

K.V.: 1.6

P e r m i Sum.: 0

Time Const.: 2

70

1

w

w

I

so

so

0

n

0

z

40

P

40

!A

F

rn

P z

0 ao

0

0

D

O

r n

a F i g u r e 5.

X-Ray D i f f r a c t i o n P a t t e r n o f P r o p i o m a z i n e H y d r o c h l o r i d e (N.F. R e f e r e n c e S t a n d a r d M a t e r i a l ) R a d i a t i o n : C u K a Instrument: Norelco P h i l i p s D i f f r a c t o m e t e r

PROPIOMAZINE HYDROCHLORIDE

TABLE I1 ttdttSpacings for Propiomazine l-@drochloride

28

d(G

6..5'

13.60 9 -03 6.76 6.02 5 .91 4.79"

9 08 13.1 1407 15.0

18 .5 19 -6.5

4.52*

2 1 .o 22.5 23.05

4.23* 3.959 3.86 3 *75 3 -67 3.62 3.39 32 7 3 -13 3 -08 2.96 2 .91 2.78 2 059

23.7

24.25

24.6 26.3

27-25;

28 .S 29 .O

30.2 30.7 32 * 2 5 3L .6 3.5.8 36.3

2.51 2 047

*Most i n t e n s e peaks

n h d=(i n t e r p l a n a r d i s t a n c e ) 2 s i n 6

45 1

K. 6. CROMBIE AND L. F. CULLEN

2.9

I o n i z a t i o n Constant, pKa (Apparent) The PKa f o r propiomazine hydrochloride h a s been determined p o t e n t i o m e t r i c a l l y togbe- 6.6 by aqueous t i t r a t i o n w i t h 0.W sodium hydroxide.

3.

S ynt he s i s Two b a s i c s y n t h e t i c r o u t e s have been r e p o r t e d f o r t h e prepa a t i o n of propiomazine hydrochloride. Farbenfabriken Bayerr2 pr e pared propiomazine by r e a c t i n g 2-dimethylaminopr opyl c h l o r i d e with 2-prapionylphenothiazine i n t h e p re sence of sodium amide as t h e condensing a g e n t , and subseq u e n t l y c onvertin g t h e b ase form t o t h e hydrochloride (See F i gur e 6 ) An a l t e ate s y n t h e s i s was developed b y E t a b l i s sements Clin-RylaB which i s based on t h e d e c a r b o x y la tio n by he a t i ng of t h e aminoalkyl ester of t h e N-carboxyphe not hi a z i ne, S p e c i f i c a l l y , 2-propionylphenothiazine i s converted i n t o i t s N-carbonyl c h l o r i d e d e r i v a t i v e by react i n g w i t h phosgene. Reaction of t h e c h l o r i d e w i t h 2dimethylamino-1-propanol y i e l d s t h e ester h y d r o c h lo r id e , which i s decarboxylated b y h e a t i n g (See F ig u r e 7).

k.

-

Stability Degradation Propiomazine h y d ro ch lo rid e i s v e r y s t a b l e i n b o t h t h e s o l i d state14 and i n b u f f e r e d aqueous s o l u t i o n s exposed t o long term a c c e l e r a t e d thermal c o n d i t i o n s which are prot e c t e d from W l i g h t and co n tain a s i t a b l e a n t i o x i d a n t system t o exclude d i s s o l v e d oxyizen.l3 It i s common for t h e phot oc a t aly zed o x i d a t i v e decomposition of s t r u c t u r a l l y r e l a t e d phenothiazine d e r i v a t i v proceed by a semiquinone free r a d i c a l mechanism. J. Kofoed et.a1?]-,*2 oxidized propiomazine t o t h e corresponding .$oxide or s u l f oxi de by r e a c t i n g t h e compound w i t h hydrogen peroxide a t 60°C. Ekposure o f aqueous s o l u t i o n s of propiomazine hyd r o c h l o r i d e t o more s e v e r e th erm al c o n d i t i o n s has r e s u l t e d i n t h e cleavage of t h e compound t o form 2-propi n y l phenot h i a z i n e as an o x i d a t i v e decomposition product .'3 A s i m i l a r cleavage r e a c t i o n was observed under a c c e l e r a t e d thermal c o n d i t i o n s f o r aqueous s o l u t i o n s of a somewhat s t r u c t u r a l l y similar phenothiazine d e r i v a t i v e , promethaz i n e hydrochloride, r e s u l t i n g i n t h e f Of lomethylphenothiazine and phenothiazine

fg-58

.

452

PROPlOMAZlNE HYDROCHLORIDE

H ~ C -CH-CH, CI

Fig. 6

'

(1)condense w i t h NaNH ( 2 ) converted t o H C 1

-- l a r b e n f a b r i k e n

Bayer Synthesis of Propiomazine H C 1

453

I

& u D?

Y

I

i?

+

8 Y

0r i

K. 6. CROMEIE AND L. F. CULLEN

a (d

s

0

F (1 f;r

P

0 0) a

454

X

u

rl

N

B 0

0

k

rl

V

PROPIOMAZINE HYDROCHLORIDE

5.

Drug Metabolic Products On i n c u b a t i o n of propiomazine w i t h a r a t l i v e r prepar a t i o n , Robinson26 c h a r a c t e r i z e d t h e predominant b i o t r a n s for m a t i on product as t h e aromatic r i n g hydroxylated metabol i t e . The metabolic product r e s u l t i n g from d e a l k y l a t i o n of t h e 1 0 - d i a l k y l m i n o a l k y l s u b s t i t u e n t , which i s a characteristic m e t a b o l i t e of most b i o l o g i c a l l y active phenot h i a z i n e d e r i v a t i v e s , was n o t d e t e c t e d i n t h i s s tu d y .

6.

Identification h o p i o m a z i n e hydrochloride can be i d e n t i f i e d b y virt u e of i t s c h a r a c t e r i s t i c I R , UV, and X-ray s p e c t r a (See The N.F. XIII' d e s c r i b e s a t e s t based 2.1, 2.3, and 2.8). on t h e f or ma tio n of t h e maleate s a l t of propiomazine and measurement of t h e c h a r a c t e r i s t i c m e l t i n g p o i n t (157' 165°C.) of t h e maleate s a l t form. A s e r i e s of microcryst a l l i n e d e r i v a t i v e 2 7 and c o l o r i d e n t i f i c a t i o n t e s t s 2 8 have been r e p o r t e d t o d e t e c t and d i f f e r e n t i a t e microgram q u a n t i t i e s of propiomazine from o t h e r s t r u c t u r a l l y r e l a t e d phe not hi a z i ne d e r i v a t i v e s .

-

7.

9

Methods of Analysis ? .1 Elemental Analysis* Element

% Theory

% Determinedlo

C H N S c1

63 -73

63 059 6.62

6.68

7 -43

7.21 8.27

8 .51 9 -40

9.48

Propiomazine HC1, N.F. Reference Standard material 7.2

U l t r a v i o l e t Spectrophotometric Analysis

The u l t r a v i o l e t a b s o r p t i o n maximum of prapiomazi ne hydrochloride a t about 240 mu. aqueous s o l u t i o n s has been u t i l i z e d f o r a s s a y purposes Determination of

ip

.

t h e a b s o r p t i v i t y value i s u s e f u l f o r a s s a y of raw material and f o r f or mu latio n s a f t e r t h e propiomazine has been i s o l a t e d . Propiomazine hydrochloride could p o s s i b l y be separ a t e d from i n t e r f e r i n g fo rmu latio n components by a n ext r a c t i o n of t h e base form w i t h o r c a n i c s o l v e n t s from a n a l k a l i n e aqueous s o l u t i o n . 455

K. 8 . CROMBIE AND L. F. CULLEN

Tompset t 5 q u a n t i t a t i v e l y determined p r opiomazine i n b i o l o e i c a l m a t e r i a l by comparing i t s absorbance t o t h a t of reference standard m a t e r i a l i n lN hydrochloric a c i d a f t e r e x t r a c t i n g t h e propiomazine from an a l k a l i n e aqueous phase with chlorof om.

7.3

Colorimetric Analysis 7.31 Palladium Chloride Modifications of t h e yRlladium-phenothiazine d e r i v a t i v e complex procedure of Ryan have been applied t o t h e q u a n t i t a t ve a n a l y s i s of propiomazine hydrochloride successfully. 1 ~ 1 $ The colorimetric procedure i s based on t h e r e a c t i o n of palladium w i t h propiomazine i n an aqueous s o l u t i o n buffered a t about pH 3 t o form a colored complex which is spectrophotometrically measured a t 465 mp. Since t h i s complex formation i s based on an e l e c t r o n t r a n s f e r from the s u l f u r moiety t o the palladium i o n s , t h e procedure provides a method t o assay propiomazine i n t h e presence of i t s corresponding s u l f oxide oxidative decompos i t i o n product.

7.32

p-Benzoquinone A color r e a c t i o n i s obtained by the a c t i o n of p-benzoquinone i n a s t r o n g l y a c i d medium o r propioma~ i n e * ~ A. s t a b l e colored species results which al-lows measurement of i t s absorption i n t h e v i s i b l e region. T h i s redox r e a c t i o n i s based on t h e formation of t h e c a t i o n i c r a d i c a l of propiomazine with p-benzoquinone a c t i n g a s an e l e c t r o n acceptor.

7.4

P o l a r o g r a p h i c :lnalysis Progiomazine hvdrochloride e x h i b i t s an anodic wave a t +O.Ov i n q c i d i c a ueous s o l u t i o n a t a concentrat i o n range of lo-' t o '10-1; using a s t a t i o n a r y platinum electrode vs. a caloinel rer'arence e l e c t r o d e , s c i t a b l e f o r a q u a n t i t a t i v e assay30, The wave corresponds t o t h e oxid a t i o n of t h e s u l f u r i n t h e phenothiazine nucleus t o t h e s u Lfoxide3I.

3

7.5

Titrirnetric Analvsis 7.51 Podium La&l SuJfgte P e l l e r i n e t . a l . J L d 3 developed a semimicro, heterogeneous p h a q i d i c aqueous-chlorof orm) ~

PROPIOMAZINE HYDROCHLORIDE

t i t r i m e t r i c procedure f o r propiomazine and r e l a t e d phenot h i a z i n e d e r i v a t i v e s a t t h e 0.0smM l e v e l employing 0.01M sodium l a u r y l s u l f a t e as the t i t r a n t and methyl yellow as t h e i n d i c a t o r . The a u t h o r s demonstrate t h a t common t a b l e t e x c i p i e n t s do n o t i n t e r f e r e i n the assay.

7.52

Ceric S u l f a t e A spectro3hotometri.c t i t r a t i o n procedure employing 0.02N c e r i c s u l f a t e as t h e t i t r a n t , used t o assay a v a r i e t y of p h en o th iazin e d e r i v a t i v e s and t h e i r dosage f o r m , can be a p p l i e d t o t h e q u a n t i t a t i v e a n a l y s i s of propiomazine hydrochloride3”. I n i t i a l l y , a c o lo r e d semi-quinone i n t e r m e d i a t e i s formed which r e p r e s e n t s t h e f i r s t s t a g e i n t h e o x id atio n . Upon l o s s of a second e l e c t r o n , t h e s o l u t i o n becomes c o l o r l e s s as a r e s u l t of t h e f or ma t i on of t h e s u l f o x i d e d e r i v a t i v e of t h e phenot h i a z i n e . This mechanism f o r t h e o x id a tio n of t h e phenot h i a z i n e under t h e con i t i o n s o f t h e t i t r a t , i o n a r e d e s c r ib e d by Merkle and Discher3 The endpoint i s a s c r i b e d when exc e s s c e r i c i o n remains i n s o l u t i o n and i s c h a r a c t e r i z e d by an i n c r e a s e i n absorbance, s p e c t r o p h o t o m e t r i c a l l y measured a t 1120 mu, which i s d i r e c t l y p r o p o r t i o n a l t o t h e c e r i c i o n c oncen tratio n . Employing a imilar t i t r i m e t r i c procedure, Chatten, Locock and Krause 3E measured t h e changes i n o p t i c a l d e n s i t y of t h e r e a c t i o n s o l u t i o n a t 270 mu, t h e wavelenpth of m a x i m u m absorbance f o r t h e i n t e r m e d i a t e semi-quinone f r e e r a d i c a l .

2.

7.53

P e r c h l o r i c Acid The non-aqueous p o t e n t i o m e t r i c t i t r a t i o n o f m i n e hydrochloride salts, in tro d u ced by P if e r - Xo llis h 3 7 and used i n t h e assay of v ario u s phenothiazine d e r i v a t i v e $ can be a p p l i e d t o t h e d eterm in atio n o f propiomazine hydroc h l o r i d e . The procedure in v o lv es i n i t i a l r e a c t i o n o f t h e m i n e hydrochloride group w i t h mercuric a c e t a t e i n an a c e t i c a c i d medium. The h a l i d e i s t i e d up as u n d is s o c ia t e d HgCl, and t h e a c e t a t e i o n l i b e r a t e d can be t i t r a t e d as a base w i t h p e r c h l o r i c a c i d . h e r c u r i c a c e t a t e i s ess e n t i a l l y u n d i s s o c i a t e d i n a c e t i c a c i d and t h e e x c e s s , t h e r e f or e , does n o t i n t e r f e r e .

457

K. B. CROMBIE AND L. F. CULLEN

7.6

Chromatographic Analysis Chromatographic met.hods can be used q u a l i t a t i v e l y f o r i d e n t i f i c a t i o n and q u a n t i t a t i v e l y f o r d e te rmin a tio n of p u r i t y and s t a b i l i t y of propiomazine hydrochloride.

7.61 Thin Layer Chromatographz The v a r i o u s e l u a n t and a d s o rb e n t systems used f o r t h i n layer chromatography of propiomazine hydroc h l o r i d e are given i n Table 111. The v i s u a l i z a t i o n te c h niques used f o r t h e d e t e c t i o n of propiomazine are a l s o in c l ude d i n Table 111.

7.62

Paper Chromatography Many paper chromatographic me t.hods have been r e p o r t e d i n t h e literature t o p e r mit the i s o l a t i o n and d e t e c t i o n of propiomazine hydrochloride. A summary of t h e a v a i l a b l e d a t a i s recorded i n Table TV.

7.63

Gas Chromatography ProDiomazine hvdrochloride has been chromatographed a t an o p e r a t i n g t e 6 e r a t u r e of 225'C. on a 2.9% SE-30 Chromosorb W column and a $ XE-60 s i l i c o n e n i t r i l e polymer on Chromosorb W Employing a 3% SEE-30 Il i a t opor t S column operated a t a temperature of 24OoC., it i s p o s s i b l e t o s e p a r a t e propiomazine hydroch3 o r i d e &rom i t s de gr a da t i on product n y l phenothiazine. Kofoed, et.al., hromatographed propiomasine, as w e l l a s i t s s u l f o x i d e , on a 3% ,SE-30 Gas Chrom Q column a t tem eratures of 210'C. and 25'0'C. Fontan, J a i n , and Kirk3 have developed an i d e n t i f i c a t i o n procedure f o r propiomazine and r e l a t e d p h e n o th ia z in e s based on examination of t h e c h a r a c t e r i s t i c g a s chromatographic p a t t e r n of i t s p y r o l y s i s products.

, 2-p~f)J~

8

458

PROPIOMAZINE HYDROCHLORIDE

Table I11 Thin Layer ChromatoRraphic Systems f o r Propiomazine HC1 Solvent System

Adsorbent

Visualization Technique

%

Reference

A

S i l i c a Gel G

A, B

0.52

39

B

S i l i c a Gel G

A* B

0.66

39 ,

C

S i l i c a Gel G

B

----

39

D

S i l i c a Gel G C,D with Fluorescent Indicator

0.77

5 , 21

E

S i l i c a Gel with 0.1N NaOH

E

0.192

LO

F

Silica Gel with 0.1N NaOH

E

F

S i l i c a Gel with 0.1N KHSO,

E

0. LO

Ll, L2

G

S i l i c a Gel w i t h 0.1N NaOH

E

H

S i l i c a Gel w i t h 0.1N NaOH o r 0 . 1 N KHSO,

B,C* D, E

I

S i l i c a Gel w i t h 0.1N

E

0.26

141, 112

KHSO, continued..

459

..

K. 8. CROMBIE AND L. F. CULLEN

Table 111 (continued) Thin Layer ChromatoRraphic Systems for Propiomazine HC1 Solvent System

Adsorbent

Visualization Technique

Reference

J

Silica Gel G with Fluo.rescent Indicator

C

h7

K

Silica Gel G

A,C,F, G,H,I*

LO

J

L

Silica Gel G with 0.1N N aOH

A,C,F,

LO, Lh

G,H$I, J

M

Silica Gel G C with Fluorescent Indicato r

N

Silica Gel

C,K,L, M

26

0

Silica Gel

C

46

P

Silica Gel

C

L6

Q

Silica Gel

C

h6

h5

Solvent Systems A

Cyclohexane:diethylamine ( 9 :1)

B Methanol: NH, OH (100:1.5) C Ch1oroform:diethylamine ( 9 : 1)

D O.!! M ammonium acetate in 80% methanol E

Cyclohexane:benzene:diethylamine (75:15:10) continued.... 460

PROPIOMAZINE HYDROCHLORIDE

Table I11 (continued) Thin Layer Chromatographic Systems for Propiomazine HC1 Solvent Systems (concluded) F

Methanol

G

Acetone

H Methyl acetate

I 95% Ethanol J

pyridine:methanol ( 5 0 : 5 0 )

K Benzene:ethanol: NH,OH (95:15:5) L Ch1oroform:methanol (90:lO) M

Benzene:acetone: NH,OH (37:7:1)

N Chloroform :ethanol:NH, OH (80 :2 0 : 1) 0 To1uene:dimethylformamide (100: 25)

P Toluene:methanol :dimethylformamide ( h 5 :30 :20)

Q Acetone :methanol:triethanolamine (100:100:3)

Visualization Techniques

A

Acid Ferric Chloride [FeClJ in 20% HC10,: 50% H N 4 ( 1*5:501

B Potassium Iodoplatinate Reagent C Ultraviolet Light D Dragendorff Reagent

continued.... 46 1

K. B. CROMBIE AND L. F. CULLEN

Table I11 (concluded) Thin Layer Chromatographic Systems for Propiomazine HC1 Visualization Techniques

(concluded)

1% I, in methanol

Folin-Ciocalteau Reagent Madelin Reagent Cinnamaldehyde Ehrlich Reagent

[p-dimethylaminobenzaldehyde in phosphoric acid acetic acid

Furfural Sulfuric Acid Spray Hydrobromic Acid Sodium Metaperiodate

-

Table

IV

Paper Chromatographic Systems for Propiomazine H C 1 Solvent System

Paper Treatment

Paper

A

Whatman No. 1

Buffered i n 5% sodium dihydrogen c it r a t e

B

Whatman No. 1

Impregnated with 10%t r i b u t y r i n i n acetone

or No. 3 P 0.

2

Visualization Technique

Ref erence

0.77

A*B*C

28, ‘7, h 8

W

C

Whatman No. 1

o r No. 3

Impregnated with lU% t r i b u t y r i n i n acetone

Solvent Systems

.. I r 0

V i s u a l i z a t i o n Technique

A

0.02 M C i t r i c Acid i n 87% n-butanol

A

U l t r a v i o l e t Light

B

Acetate b u f f e r (pH L . 6 )

B

Potassium I o d o p l a t i n a t e Reagent

C

Phosphate b u f f e r (pH 7 . L )

C

Bromocresol Green Spray

z

0,

K. B. CROMBIE AND L. F. CULLEN

8. References 1. "Merck Index," 8 t h Ed., Merck and c o o * Inc.9 Rahway, N.J. 2. I'National Formulary", 1 3 t h Ed. Mack P u b l i s h i n g Co., Easton, Pa. 3. L. J. B e l l m y , "The I n f r a r e d S p e c t r a of Complex Molecules," 2nd Ed., John Wiley and Sons, Inc., New York, N.Y., 196L. 10. B. Hofmann, Wyeth L a b o r a t o r i e s , Inc. 1 Personal Communication. 5. S. L. Tompsett, Acta PharmacoLToxicol., 6,

303 (1968).

6. K. B. Crombie and L. F. Cullen, Unpublished r e s u l t s , Wyeth L a b o r a t o r i e s , Inc. 7. S. Shrader and C. Kuhlman, Wyeth L a b o r a t o r i e s , Inc., P e r s o n a l Communication. 8. J. N. T. G i l b e r t and B. J. Millard, Org. Mass Spectrom., 2, 17 (1969). 9. ' V n i t e d S t a t e s Pharmacopeia", 1 8 t h Ed., Mack P u b l i s h i n g CQ., Easton, Pa. 10. N. DeAngelis, Wyeth L a b o r a t o r i e s , Inc., P e r s o n a l Communication. 11. J. A. Lash, Wyeth LaborRtories, Inc., Personal Comnunic a t i o n . 12. Farbenfabriken Bayer Akt.-Ges. , Brit. 800,0h9, 1 12312e (1959). Jan. 28, 1959; Chem. Abstr., z 13. E t a b l i s s e m e n t s Clin-Byla. 2.1, 176,919, A p r i l 17, 1959; Chem. Abstr., 27380b (1961). 111. F. Rosolia, Wyeth L a b o r a t o r i e s , Inc., P e r s o n a l Cornmunication. 15. J. L. Deuble, Wyeth L a b o r a t o r i e s , Inc., P e r s o n a l Communication. 16. L. Ravin, L. Kennon, J. Swintosky, J. Pharm. S c i . ,

s,

3, 760 17. 18.

(1958). S. P. Massie, Chern. Rev.,

2, 797

J. Ryan, J. Am. Pharm. Assoc.,

(195h). S c i . Ed., 2,

2/10 (1959). -- -

19.

L. Michaelis, S. Granick and M. Schubert,

J. Am. Chem. SOc.9 63, 351 (19h1). 20. A. F e l m e i s t e r and C. Discher, J. Pharm. Sci.,

464

53,

PROPIOMAZINE HYDROCHLORIDE

22. J. Kofoed, C. Fabierkiewicz and G. Lucas, J. Chromatoq., 3, 1110 (1966). 23. L. Cullen and D. Rodgers, Wyeth L a b o r a t o r i e s , Inc., P e r s o n a l Communication. T. Waaler, Pharm. Acta. Helv., 2, 168 (1960). 211. 25. R. Y m m o t o and S. Fujisawa, KonKr. Pharm. Wise., 509 (1963)26, A. E. Robinson, J. Pharm, Pharmacol., 19 (1966). 27. C. N. Andres, J, A s s . O f f i c . Anal. Chem., 2, 1020 (1965). 28. E. G. C. Clarke, I ' I s o l a t i o n and I d e n t i f i c a t i o n of Drugs," 1st Ed., The Pharmaceutical P r e s s , London, Eng., 1969. 29. J. J e u n i e r , Ann. Pharm. Fr., 5, 25 (1968). 30. R. S h u l t z , Wyeth L a b o r a t o r i e s , Inc. P e r s o n a l Communication. 31. G. S. P o r t e r , J. Pharm. Pharmacol.9 176 (1967). 32.' F. P e l l e r i n , J. G a u t i e r , and D. Demay, Ann. Pharm. Franc., 22, L95 (196L). 33. F. P e l l e r i n , J. G a u t i e r , and D. Demag, Ann. Pharm. Franc. , 20, 97 (1962). 311. S. Agarwal and M. Blake, J. Pharm. S c i . , 1011(1969). 35. F. H. Merkle and C. A. Discher, J. Pharm. Sci.,

-

18,

x,

58,

~~

53, 620 (19618). 36. L. G. Chatten, R. A. h c o c k and R. D. Krause, J. Pharm. Sci., &, 588 (1971). 37. J. F r i t z , "Acid-Base T i t r a t i o n s i n Nonaqueous Solvents", The G. F r e d e r i c k Smith Chemical Comneny, Columbus, Ohio, 1952. 38, C. R. Fontan, N. C. J a i n and P. L. Kirk, Mikrochim. Ichnoanal. Acts, 1964, 326 (1964) 576 (1961). 39. I. Sunshine, Am. J. C l i n . Pathol., LO. I. Zingales, J. Chromatom., 2, LO5 (1967). L1. I. Sunshine, W. W. Fike and H. Landesman, J. Forensic S c i . , 2,1128 (1966). 112. W. W. Fike, Anal. Chem., 38, 1697 (1966). L3. J. N. Bathish, Wyeth L a b o r a t o r i e s , Inc., P e r s o n a l Communication. IrL, J. L. Kiger and J. G. Kiger, Ann. Pharm. Franc., 23, L89 (1965).

s,

-

465

K . 8 . CROMBlE AND L. F. CULLEN

115. C. Robinson, Wyeth Laboratories, Inc. 9 Personal Communication. L6. J. Pastor and V. Valimamod, B u l l . Trav. SOC. Pharm. Lyon, 2, 312 (1965). 117. A. S. Curry and H. Powell, Nature, llh3

m,

(1951t ). @. E. Interscience 119. H. 312 (1962). 9. H. (1962).

G. C. Clarke, "Methods of Forensic Science", Publishers. Qew York, N.Y. * 1962. V, S t r e e t , Acta Pharmacol. T O X i C O l . T 21

V, S t r e e t , J. Forensic Sci. Soc.,

466

2,

118

SULFAMETHOXAZOLE

Briicc C. Rudj, and Bernard Z. Scnkowski

467

8.C. RUDY AND 6. Z. SENKOWSKI

INDEX

A n a l y t i c a l P r o f i l e - Sulfamethoxazole 1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor

2,

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F l u o r e s c e n c e Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y 2.9 Thermal G r a v i m e t r i c A n a l y s i s 2.10 S o l u b i l i t y 2 - 1 1 X-ray C r y s t a l P r o p e r t i e s 2 . 1 2 D i s s o c i a t i o n Constant

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug Metabolic Products

6.

Methods o f A n a l y s i s 6.1 Elemental A n a l y s i s 6.2 Phase S o l u b i l i t y A n a l y s i s 6.3 Thin Layer Chromatography 6.4 D i r e c t S p e c t r o p h o t o m e t r i c Analysis 6.5 Colorimetric Analysis 6.6 F l u o r i m e t r i c Analysis 6.7 T i t r i m e t r i c Analysis

7.

References

468

SULFAMETHOXAZOLE

1.

Description

Name, Formula, Mol ecul ar Weight Sulfamethoxazole i s N'- (5-methyl-3-isoxazolyl) s u l f a n i 1ami de 1.1

.

O H II

I

S-Nq I; '0

c 1 OH1 1 N 3 0 3 S

M ol ecul ar Weight :

253.28

Appearance, C o l o r , Odor Sulfamethoxazole occurs as a white t o s l i g h t l y o f f - w h i t e , p r a c t i c a l l y o d o r l e s s , c r y s t a l l i n e powder 1.2

2.

Physical Properties

I n f r a r e d Spectrum The i n f r a r e d s pect r um o f r e f e r e n c e s t a n d a r d s u l f a m e t h o x a z o l e i s p r e s e n t e d i n F i g u r e 1 ( 1 ) . The s p e c t r u m was measured w i t h a Per ki n- E l m er 621 S p e c t r o p ho to mete r i n a K B r p e l l e t c o n t a i n i n g 0 . 9 mg s u l f a methoxazole/300 mg o f K B r . T a b l e I l i s t s t h e a s s i g n m e n t s f o r t h e c h a r a c t e r i s t i c bands i n t h e i n f r a r e d s p e c t r u m ( 1 ) . 2.1

TABLE I I n f r a r e d Ass i gnnient s f o r S u l famet hoxazo 1e Frequency (cm-l) 3469 and 3379 3300 1616 1595 and 1500 1313 - 1303 1155 - 114.3 828

Characteristic of NH, s t r e t c h NIi s t r e t c h

Combination NH2 d e f o r m a t i o n and isoxazole ring s t r e t c h Aromatic C = C s t r e t c h Asymmetric SO2 s t r e t c h Symmetric SO2 s t r e t c h 'I'wo a d j a c e n t 11's on benzene r i n g

469

6. C. RUDY AND 6.2.SENKOWSKI

470

SULFAMETHOXAZOLE

N u c l e a r Magnetic Resonance Spectrum The s p e c t r u m shown i n F i g u r e 2 was o b t a i n e d on a J e o l 60 MHz NMR by d i s s o l v i n g 5 3 . 6 mg o f r e f e r e n c e s t a n d a r d s u l f a m e t h o x a z o l e i n 0 . 5 m l o f DMSO-d6 c o n t a i n i n g t e t r a m e t h y l s i l a n e a s an i n t e r n a l r e f e r e n c e ( 2 ) . The s p e c t r a l a s s i g n m e n t s are g i v e n i n T a b l e I 1 ( 2 ) . I n o r d e r t o e s t a b l i s h t h e chemical s h i f t f o r t h e s i n g l e p r o t o n (H ) on b t h e i s o x a z o l e r i n g which a p p e a r s a s a s h o u l d e r on t h e b r o a d e r p r i m a r y amine p r o t o n s peak, i t was n e c e s s a r y t o s h i f t t h e amine p r o t o n s peak t o a n o t h e r p l a c e i n t h e s p e c t r u m . T h i s was accomplished by a d d i n g 8 ~1 o f DC1 t o form t h e q u a t e r n a r y s a l t . T h i s c a u s e d t h e b r o a d s i n g l e t t o s h i f t t o 5 . 6 2 ppm and i s o l a t e d a s i n g l e t a t 6 . 1 0 ppm a s s i g n a b l e t o t h e s i n g l e p r o t o n (Hb) on t h e i s o x a z o l e r i n g (Figure 2 - I n s e r t ) . 2.2

TABLE I 1 NMR Assignments f o r S u l f a m e t h o x a z o l e

Structure

Protons

Chemical S h i f t PPm

f

11.00

Mu 1t ip 1i c i t y

s (b)

S = s h a r p s i n g l e t ; S(b) = broad s i n g l e t ; d = d o u b l e t ; J = s p l i t t i n g c o n s t a n t i n Hz 2.3

U l t r a v i o l e t Spectrum When t h e UV s p e c t r u m o f s u l f a m e t h o x a z o l e was s c a n n e d between 350 t o 215 nm, one maximum and one minimum were o b s e r v e d . The maximum i s l o c a t e d a t 256-257 nm ( ~ = 1 . 7 2x l o 4 ) and t h e minimum a t 224-225 nm. The s p e c t r u m shown i n F i g u r e 3 was o b t a i n e d from a s o l u t i o n o f 1 . 0 2 5 mg s u l f a m e t h o x a z o l e / 1 0 0 ml o f pH 7 . 5 p h o s p h a t e b u f f e r ( 3 ) . T h i s s p e c t r u m i s t h e same as ones o b t a i n e d u s i n g 0.1N NaOfI as t h e s o l v e n t ( 4 ) . 471

Figure 2 NMR Spectrum o f Sulfamethoxazole

I n s e r t : Spectrum i n Region o f 5 t o 8 ppm A f t e r A d d i t i o n o f DC1 t o Sulfamethoxazole

r

I

m 0 n C 0

SULFAMETHOXAZOLE

Figure 3 UV Spectrum of S u l famethoxazole

.8

-

0 NANOMETERS

473

6 . C. R U D Y AND B. 2. SENKOWSKI

2.4

F l u o r e s c e n c e Spectrum The e x c i t a t i o n and emission s p e c t r a f o r s u l f a methoxazole (1 mg/ml of methanol) a r e shown i n F i g u r e 4 (5). One maximum appears in t h e e x c i t a t i o n spectrum a t 314 nm and one maximum i n t h e emission spectrum a t 338 nm.

Mass Spectrum The mass spectrum of s u l f a m e t h o x a z o l e was obt a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h an i o n i z i n g energy o f 70 e v . The o u t p u t from t h e mass s p e c t r o meter was analyzed and p r e s e n t e d i n t h e form o f a b a r graph, shown i n F i g u r e 5, by a Varian 100 MS d e d i c a t e d computer system ( 6 ) . The peaks a t m/e 92, 108, 140, and , H;!N-CgHq-SO, and 156 correspond t o H ~ N - C ~ H L ,H2N-CgHq-0, H2N-C6H4-S02, r e s p e c t i v e l y . The peak m/e 189 i s due t o t h e l o s s o f SO;! from t h e p a r e n t and t h e peak a t m/e 174 i s due t o t h e l o s s o f b o t h SO2 and CH3 from t h e p a r e n t . Loss o f SO2 i s a t y p i c a l rearrangement r e a c t i o n w i t h sulfonamides. The s t r o n g M+2 peak at 255 may b e t h e 34S i s o t o p e peak, however, t h i s peak is n o t e x p e c t e d t o be s o i n t e n s e ( 6 ) . 2.5

2.6

Optical Rotation Sulfamethoxazole e x h i b i t s no o p t i c a l a c t i v i t y .

2.7

Melting Range The m e l t i n g range r e p o r t e d i n NF XI11 f o r s u l f a methoxazole i s 170' t o 173O u s i n g t h e c l a s s I p r o c e d u r e (7). 2.8

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC scan f o r sulfamethoxazole i s shown i n F i g u r e 6. A m e l t i n g endotherm i s observed a t 172' when t h e t e m p e r a t u r e program was 10°/minute. The AHf was found t o be 7.5 kcal/mole. The endotherm observed a t 204OC i s a t t r i b u t e d t o t h e decomposition o f t h e sulfamethoxazole (8). 2.9

Thermogravimetric A n a l y s i s (TGA) A thermal g r a v i m e t r i c a n a l y s i s performed on sulfamethoxazole e x h i b i t e d no l o s s o f weight when it was h e a t e d t o 105OC a t a r a t e o f 10°/min ( 8 ) . 2.10

Solubility The s o l u b i l i t y d a t a f o r sulfamethoxazole o b t a i n e d a t 25OC are given i n Table I11 (9).

474

SULFAMETHOXAZOLE

Figure 4 Fluorescence Spectrum of Sulfamethoxazole

300

400 NANOMETERS

475

B. C. RUDY AND B. 2. SENKOWSKI

I

I

I

I

I I

I I

I

1

1

I

I

I

s

I

I

01

a, rl

0

N (d

x

0

0

P

0

s

0

c?

8 0

m

3 P 0

0

r

n

0

m

0

+

0

@

0

o

0

e

AlISN31NI 3 A l l V l 3 U

476

0

m

0

w

0

-

0

a

cE

SULFAMETHOXAZOLE

Figure 6 DSC Curve f o r Sulfamethoxazole

I

I

I

I

I

I

I

I

AHf = 7 7.5 5 kcal/rnole

I

I 140

I

160

180 TEMPERATURE

477

200 OC

B. C. RUDY A N D B. Z. SENKOWSKI

TABLE I11 S o l u b i l i t y o f Sulfamethoxazole i n D i f f e r e n t S o l v e n t s Solvent

S o l u b i l i t y (mg/ml)

3A a l c o h o l benzene chloroform 95% e t h a n o l ethyl ether i s op rop an o 1 me t h ano 1 p e t r o l e u m e t h e r 30" ,60" 0.1N NaOH water

30.6 0.5

2.3 37.8 2.7 8.8 90.3 0.2 16.0 0.5

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n o f s u l f a methoxazole i s p r e s e n t e d i n Table IV ( 1 0 ) . The i n s t r u mental c o n d i t i o n s a r e given below. 2.11

Instrument C o n d i t i o n s : General E l e c t r i c Model XRD-6 Spectrogoniometer Generator: 50KV-12-1/2 MA C Tube t a r g e t : Copper Radiation: Cu Ka = 1.542 A Optics : 0.1" D e t e c t o r s l i t M.R. S o l l e r s l i t 3" Beam s l i t 0.0007 i n c h N i f i l t e r 4" t a k e o f f a n g l e Goniometer: s c a n a t 0.2' 20 p e r minute Detector: A m p l i f i e r g a i n - 16 c o a r s e , 8.7 f i n e Sealed proportional counter tube and DC v o l t a g e a t p l a t e a u P u l s e h e i g h t s e l e c t i o n EL-5 v o l t s Eu - o u t , Rate meter T . C . 4 2000 C/S f u l l s c a l e Recorder: Chart speed - 1 i n . / 5 min. Samples : Prepared by g r i n d i n g a t room temp.

47 8

SULFAMETHOXAZOLE

TABLE IV X-ray Powder D i f f r a c t i o n P a t t e r n o f S u l f a m e t h o x a z o l e 0

20

d(A) *

1/10

12.90 8.03 7.18 6.44 5.31 5.21 5.07 4.90 4.78 4.52 4.29 4.12 3.97 3.82 3.73 3.61 3.40 3.37 3.25 3.10 3.06

2 10 43 5 7 12 50 4 16 20 43 40 24 4 100 15 5 5 38 17 5

0

**

20

d(A) *

I/p

**

~

6.86 '11.02 12.32 13.74 16.70 17.03 17.48 18.12 18.56 19.62 20.68 21.56 22.39 23.28 23.86 24.68 26.20 26.44 27.40 28.76 29.22

30.06 32.22 33.37 34.50 34.92 36.42 36.95 37.68 38.25 38.80 39.52 40.71 41.70 43.32 43.68 45.10 45.80 46.38 49.00 50.72

*

d = (interplanar distance)

**

I l I 0=

2.92 2.78 2.69 2.60 2.57 2.47 2.43 2.39 2.35 2.32 2.28 2.22 2.17 2.09 2.07 2.01 1.98 1.96 1.86 1.80

2 15 5 5 8 1 4 4 1 1 3 1 4 4 3 4 2 2 3 2

nX 2 Sin 0

r e l a t i v e i n t e n s i t y ( b a s e d on h i g h e s t i n t e n s i t y o f 1.00)

2.12

D i s s o c i a t i o n Constant The pKa f o r s u l f a m e t h o x a z o l e h a s been d e t e r m i n e d s p e c t r o p h o t o i n e t r i c a l l y t o be 5 . 5 5 + 0 . 0 5 and by t i t r a t i o n o f s u l f a m e t h o x a z o l e i n an e x c e s s 07 0.1N H C 1 t o b e 5 . 6 3 + 0 . 0 3 (11). These v a l u e s a g r e e well w i t h t h e pKa v a l u e o f 5 . 6 0 a t 25'C r e p o r t e d by N a k a g a k i , Toga, a n d T e r a d a ( 1 2 ) .

419

B. C. RUDY AND 6. Z . SENKOWSKI

3.

Synthesis Sulfamethoxazole i s p r e p a r e d by t h e r e a c t i o n scheme shown i n Figure 7. 5-methyl-3-aminoisoxazole i s r e a c t e d w i t h N-acetyl -p-amino-benzene s u l f o n y l c h l o r i d e . The a c e t y l group i s t h e n c l e a v e d t o y i e l d sulfamethoxazole (11).

4.

S t a b i l i t y Degradation When 10% s o l u t i o n s o f sulfamethoxazole i n 0 . 4 N NaOH and i n water were r e f l u x e d f o r one h o u r , no decomposition was observed by t h i n - l a y e r chromatography ( 1 ) . When sulfamethoxazole i s r e f l u x e d i n 0 . 4 N H C 1 , t h e molecule f i r s t c l e a v e s t o y i e l d s u l f a n i l i c a c i d and 5-methyl-3-aminoi s o x a z o l e ( 1 , l l ) and on extended h e a t i n g i n t h e HC1 s o l u t i o n , 3 a d d i t i o n a l d i a z o t i z a b l e p r o d u c t s d e t e c t a b l e by paper chromatography a r e formed (11) Pure sulfamethoxazole was found t o be s t a b l e when s u b j e c t e d t o a l l O ° C temperat u r e f o r 5 days ( 1 1 ) .

.

Drug Metabolic Products Sulfamethoxazole i s metabolized t o i t s N4-acetyl d e r i v a t i v e which i s t h e major form found i n human u r i n e (13,14). The i n t a c t d r u g a l o n g w i t h t h r e e o t h e r m e t a b o l i t e s which have n o t been completely i d e n t i f i e d a r e a l s o p r e s e n t i n l e s s e r q u a n t i t i e s i n human u r i n e (13). In human blood t h e sulfamethoxazole e x i s t s almost e n t i r e l y as t h e i n t a c t drug ( 1 3 ) . 5.

6.

Methods o f Analysis 6.1

Elemental Analysis The r e s u l t s from t h e elemental a n a l y s i s a r e l i s t e d i n Table V ( 1 5 ) . TABLE V

Elemental Analysis o f Sulfamethoxazole E 1ement

% Theory

% Found

C H N

47.43 4.38 16.60 12.65

47.28 4.38 16.84 12.74

S

480

r. a,

k

&

L

.d

2

rn 0

Q

a,

L)

r: c x

rn

z1

x 0

O=u* I

I

+

I

SULFAMETHOXAZOLE

48 I

6.C. RUDY AND B. 2. SENKOWSKI

6.2

Phase S o l u b i l i t y A n a l y s i s Phase s o l u b i l i t y a n a l y s i s i s c a r r i e d o u t u s i n g i s o p r o p a n o l as t h e s o l v e n t . A t y p i c a l example i s shown i n Figure 8 which a l s o l i s t s t h e c o n d i t i o n s under which t h e a n a l y s i s was c a r r i e d o u t ( 9 ) . 6.3

Thin Layer Chromatographic Analysis The f o l l o w i n g TLC procedure i s u s e f u l f o r s e p a r a t i n g sulfamethoxazole from s u l f a n i l i c a c i d and s u l f a n i l a m i d e ( 1 6 ) . Using s i l i c a g e l G p l a t e s and a s o l v e n t made from a mixture o f a l c o h o l , n - h e p t a n e , c h l o r o form, and g l a c i a l a c e t i c a c i d (25:25:25:7), s p o t 0 . 1 mg o f sulfamethoxazole d i s s o l v e d i n ammoniacol methanol and s u b j e c t t o ascending chromatography. A f t e r development o f a t l e a s t 1 2 cm, t h e p l a t e i s a i r d r i e d and sprayed with Modified E h r l i c h ' s Reagent (100 mg p-dimethylaminobenzaldehyde i n 1 m l c o n c e n t r a t e d h y d r o c h l o r i c a c i d d i l u t e d t o 100 m l w i t h 3 A a l c o h o l ) . The approximate R v a l u e s are: f S u l f a n i l i c Acid S u l f a n i 1ami de S u l fame thoxazo l e

0.1 0.5 0.7

A s o l v e n t system which can be used t o s e p a r a t e sulfamethoxazole from i t s N4-acetyl m e t a b o l i t e i s c h l o r o form:heptane:alcohol ( 1 : l : l ) . Following t h e same b a s i c procedure a s above t h e Rf v a l u e f o r sulfamethoxazole i s 0.75 and f o r t h e N 2 - a c e t y l d e r i v a t i v e 0 . 4 2 ( 3 ) .

6.4

Direct Spectrophotometric Analysis Direct spectrophotometric analysis o f s u l f a methoxazole may be c a r r i e d o u t u s i n g 0.1N NaOH as t h e s o l v e n t . I n t h i s s o l v e n t B e e r ' s Law h o l d s between 0 . 2 6 ugm/ml t o 26 pgm/ml (1 x M to 1 x M) ( 1 7 ) . 6.5

Colorimetric Analysis The c o l o r i m e t r i c a n a l y s i s of sulfamethoxazole i s accomplished by d i a z o t i z a t i o n and c o u p l i n g w i t h N-(1Naph thy1 e t hylenediamine d i hydrochl o r i d e (Brat ton-Mars ha1 1 Reagent). The r e s u l t a n t r e d c o l o r i s measured a t 540 nm 08) 6.6

Fluorimetric Analysis Fluorescence a n a l y s i s has been used t o determine t h e c o n c e n t r a t i o n o f sulfamethoxazole i n blood ( 1 9 ) . For

482

Figure 8

I

P Tx.

/

w

PHASE

SOLUBILITY

I

ANALYSIS

SAMPLE SULFAMETHOXAZOLE SOLVENT: ISOPROPANOL 0.1% SLOPE: EQUILIBRATION: 20 HRS. at 25" C EXTRAPOLATED SOLUBILITY: 10.8 mg/g of SOLVENT

-.

"

0

25 SYSTEM

50

75

COMPOSITION: mg of SAMPLE per g SOLVENT

100

E. C. RUDY AND E. 2.SENKOWSKI

t h i s purpose d e t e c t a b l e l e v e l s i n t h e range o f 1 p g p e r m l o f blood a r e needed. I n o r d e r t o achieve t h i s s e n s i t i v i t y i t i s n e c e s s a r y t o make a f l u o r e s c e n t d e r i v a t i v e o f s u l f a methoxazole. One such d e r i v a t i v e i s t h e h i g h l y f l u o r e s c e n t “Dansylsul fonamide” which i s formed by t h e r e a c t i o n shown i n Figure 9 . The f l u o r e s c e n c e y i e l d o f t h i s compound i s s u f f i c i e n t t o measure about 1 pg o f sulfamethoxazole p e r m l o f blood. An even more s e n s i t i v e d e r i v a t i v e i s formed when sulfamethoxazole i s r e a c t e d w i t h 4,s-methylene dioxy phthaldehyde ( F i g u r e 9 ) . The s e n s i t i v i t y l i m i t f o r t h i s mole/ml ( 2 0 ) . d e r i v a t i v e i s from 1 x l o - * t o 1 x T i t r i m e t r i c Analysis The p o t e n t i o m e t r i c t i t r a t i o n with sodium n i t r i t e i s t h e method o f c h o i c e t o a s s a y s u l f a m e t h o x a z o l e ( 1 6 ) . The d r i e d sample i s d i s s o l v e d i n g l a c i a l a c e t i c a c i d and w a t e r ( 1 : 2 ) . H y d r o c h l o r i c a c i d i s added, t h e s o l u t i o n cooled t o 15’C, and immediately t i t r a t e d w i t h 0.1M sodium n i t r i t e . The end p o i n t i s determined p o t e n t i o m e t r i c a l l y u s i n g calomel and p l a t i n u m e l e c t r o d e s . Each m l o f 0.1M sodium n i t r i t e i s e q u i v a l e n t t o 25.33 mg of s u l f a methoxazole. 6.7

s u l f a m e t h o x a z o l e can a l s o be t i t r a t e d i n dimethylformamide w i t h 0 . 1 N L i O C H 3 u s i n g thymol b l u e a s t h e i n d i c a t o r ( 1 7 ) . The p r o t o n on t h e sulfonamide f u n c t i o n i s r e p l a c e d by t h e l i t h i u m i o n . Each m l o f 0.1N l i t h i u m methoxide i s e q u i v a l e n t t o 25.33 mg o f s u l f a m e t h o x a z o l e .

484

a k

a

,

a, M

0 N

,

m

k a,

.d

a 0

a,

F: a,

u

m a, k 3

0 4

U

Y

2

SULFAMETHOXAZOLE

I" V

0-m-0

Q

f +

Q

2

4x5

6. C. RUDY AND 6. 2. SENKOWSKI

8.

References

1. 2.

3.

4. 5. 6. 7. 8. 9. 10. 11.

12.

13. 14. 15. 16. 17. 18. 19. 20

a

Hawrylyshyn, M. Hoffmann-La Roche I n c . , P e r s o n a l Communication. Johnson, J . H . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. S o k o l o f f , H. K . , Hoffmann-La Roche I n c . , Personal Communication. F i s c h e r , I . , Hoffmann-La Roche I n c . , Personal Communication. Boatman, J . , Hoffmann-La Roche I n c . , Personal Communication. Benz, W . , Hoffmann-La Roche I n c . , P e r s o n a l Communic a t i o n . National Formulary XIII, 676 and 815, (1970). Donahue, J . , Hoffmann-La Roche I n c . , Personal Communication. MacMullan, E . , Hoffmann-La Roche I n c . , Personal Communication. Hagel, R. B . , Hoffmann-La Roche I n c . , Personal Communication. Schmidli, B . , Hoffmann-La Roche I n c . , Unpublished Data. Nakagaki, M . , Koga, N . , and Terada, H . , J . Pharm. Sac. Japan, 8 3 , 586 (1963). Koechlin, B . , and Tvron. G . , Hoffmann-La Roche I n c . Unpublished Data, Brandman, O . , and Engelberg, R . , Current Therap. Res., 2 , 364, (1960). S c h e i d i , F . , Hoffmann-La Roche I n c . , Personal Communication. National FormuZary XIII, 677, (1970). Bandong, L . , Hoffmann-La Roche I n c . , Personal Communcation. B r a t t o n , A. C . , and M a r s h a l l , F. K . , J . B i o l . Chem. 128, 537 (1939). de S i l v a , J . A . F . , and D'Arconte, L . , J . Forensic. Sci., 1 4 , 184 (1969). 85, 1049 (1965). Amano, T . , J . Pharm. Soe. Japan, -

486

SULFISOXAZOLE

Bri1c.e C. RiiL/j*arid Berriurd Z. Scnkowshi

487

B. C. R U D Y A N D B. 2. SENKOWSKI

INDEX Analytical Profile

-

Sulfisoxazole

1. Description 1.1 1.2

Name, Formula, Molecular Weight Appearance, Color, Odor

2. Physical Properties 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12

Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Fluorescence Spectrum Mass Spectrum Optical Rotation Melting Range Differential Scanning Calorimetry Thermal Gravimetric Analysis Solub ility X-ray Crystal Properties Dissociation Constant

3.

Synthesis

4.

Stability Degradation

5.

Drug Metabolic Products and Pharmacokinetics

6.

Methods of Analysis 6.1 6.2 6.3 6.4 6.5 6.6 6.7

7.

Elemental Analysis Phase Solubility Analysis Thin Layer Chromatographic Analysis Direct Spectrophotometric Analysis Colorimetric Analysis Fluorimetric Analysis Titrimetric Analysis

References

488

SULFISOXAZOLE

1.

Description

N a m e , Formula, M o l e c u l a r Weight S u l f i s o x a z o l e i s N~-(3,4-dimethyl-5-isoxazolyl) sulfanilamide. 1.1

O H

M o l e c u l a r Weight:

'1 1H13N303S

267.31

1.2

Appearance, C o l o r , Odor S u l f i s o x a z o l e o c c u r s as a w h i t e t o s l i g h t l y y e l l o w i s h , o d o r l e s s , c r y s t a l l i n e powder. 2.

Physical Properties 2.1

I n f r a r e d Spectrum The i n f r a r e d s p e c t r u m of s u l f i s o x a z o l e i s p r e s e n t e d i n F i g u r e 1 (1). The s p e c t r u m w a s measured w i t h a Perkin-Elmer 621 S p e c t r o p h o t o m e t e r i n a K B r p e l l e t cont a i n i n g 1 . 0 mg s u l f i s o x a z o l e / 3 0 0 mg o f K B r . T a b l e I l i s t s t h e a s s i g n m e n t s f o r t h e c h a r a c t e r i s t i c bands i n t h e i n f r a r e d s p e c t r u m (1). Table I I n f r a r e d Assignments f o r S u l f i s o x a z o l e Frequency (cm-1)

C h a r a c t e r i s t i c of

3485 and 3380

Asymmetric and s y m m e t r i c NH s t r e t c h NH2 d e f o r m a t i o n v i b r a t i o n s Aromatic C=C s t r e t c h Asymmetric and symmetric SO2 s t r e t c h Two a d j a c e n t H's on phenyl ring

1629 1595 and 1 5 0 3 1343 and 1160

840

489

B. C. R U D Y AND B. 2. SENKOWSKI

0

%

0

(u (u-

s

s2al

0 0

cu--

OD

0-

0

rl

0

N

rd

x

0

Q -

a--

m

d CCI

OD-

rl

1

v)

w

b-

0

-

8

0

f4 CI U

0

e

Q--

a,

a cn a a,

0 0

m--

E

f4 rd

k

w

c

H

0

2 8

e--

(u

m-

s3

0

-

m 0 0 0

*

490

SULFISOXAZOLE

2.2

N u c l e a r Magnetic Resonance Spectrum (NMR) The s p e c t r u m shown i n F i g u r e 2 w a s o b t a i n e d on a J e o l c o 60 MHz NMR by d i s s o l v i n g 6 0 . 9 mg o f s u l f i s o x a z o l e i n 0 . 5 m l of DMSO-db c o n t a i n i n g t e t r a m e t h y l s i l a n e as a n i n t e r n a l reference. The s p e c t r a l a s s i g n m e n t s a r e g i v e n i n T a b l e I1 ( 2 ) . T a b l e I1 NMR Assignments f o r S u l f i s o x a z o l e

Chemical S h i f t Fpm

Type o f P r o t o n s CH3 on no. 4 i s o x a z o l e c a r b o n CH3 on no. 3 i s o x a z o l e c a r b o n NH2 on benzene r i n g Aronatic protons ortho t o amine m o i e t y Aromatic p r o t o n s o r t h o t o sulfonamide moiety NH a d j a c e n t t o i s o x a z o l e r i n g

1.63 2.05 s6.1

Multiplicity Sharp s i n g l e t Sharp s i n g l e t Broad s i n g l e t

6.58

Doublet (J=9Hz)

7.42 '1.10.5

Doublet ( J = 9 H z ) Broad s i n g l e t

2.3

U l t r a v i o l e t Spectrum (UV) When t h e UV s p e c t r u m of s u l f i s o x a z o l e w a s s c a n n e d from 350 t o 205 nm, o n e maximum and o n e minimum were observed. The maximum i s l o c a t e d a t 253 nm ( E = 2 . 1 x l o 4 ) and t h e minimum a t 222 nm. The s p e c t r u m shown i n F i g u r e 3 w a s o b t a i n e d from a s o l u t i o n of 0 . 9 9 9 8 mg s u l f i s o x a z o l e / 100 ml of pH 7 . 5 p h o s p h a t e b u f f e r ( 3 ) . 2.4

F l u o r e s c e n c e Spectrum The e x c i t a t i o n and e m i s s i o n s p e c t r a f o r s u l f i s o x a z o l e (1 mg/ml o f m e t h a n o 1 ) a r e shown i n F i g u r e 4 ( 4 ) . One maximum a p p e a r s i n t h e e x c i t a t i o n s p e c t r u m a t 319 nm and one maximum i n t h e e m i s s i o n s p e c t r u m a t 336 nm. Mass Spectrum The mass s p e c t r a l d a t a f o r s u l f i s o x a z o l e w a s obt a i n e d u s i n g a CEC 21-110 mass s p e c t r o m e t e r w i t h an i o n i z i n g e n e r g y of 70 e v . An o n - l i n e computer, V a r i a n Data Systems 100 MS, w a s u s e d t o c a l c u l a t e t h e masses and comp a r e t h e i n t e n s i t i e s t o t h e b a s e p e a k . The mass s p e c t r u m o b t a i n e d from t h i s computer a n a l y s i s i s shown i n F i g u r e 5

2.5

3Yl

B. C. RUDY A N D B. 2. SENKOWSKI

4Y2

SULFISOXAZOLE

Figure 3

UV Spectrum of Sulfisoxazole 1.0

.0

r

.6

w

u z

8CL:

a

m

a

.4

.2

0 NANOMETERS

4Y3

B. C. RUDY AND B. 2. SENKOWSKI

494

SULF ISOXAZO LE

I

I

I

I

I

1

I

I

I 0

m

N

0 N W

*N

0

0 N

0

R

0

m

0 \

I 0

f

B 0

0

P W 0

0

495

€3.

C. RUDY AND E. Z . SENKOWSKI

( 5 ) . The peaks a t m / e 92, 108, 1 4 0 , 1 5 6 , and 1 7 2 c o r r e spond t o H2N-CgH4, H2N-C&-0, HzN-C~H~-SO, H2N-Cf,H4-S02, and H ~ N - C ~ H ~ - S O Z - N H ~r ,e s p e c t i v e l y . The peak m / e 203 i s due t o t h e l o s s o f SO2 from t h e p a r e n t and t h e peak a t rn/e 198 i s due t o t h e l o s s o f C4H7N from t h e p a r e n t . The l o s s o f SO2 is a t y p i c a l r e a r r a n g e m e n t r e a c t i o n w i t h s u l f o n amides ( 5 ) . 2.6

Optical Rotation S u l f i s o x a z o l e e x h i b i t s no o p t i c a l a c t i v i t y .

2.7

M e l t i n g Range The m e l t i n g r a n g e r e p o r t e d i n USP X V I I I f o r s u l f i s o x a z o l e is 1940 t o 199OC u s i n g t h e class I a p r o c e d u r e (6). 2.8

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The t h e r m a l p r o p e r t i e s of s u l f i s o x a z o l e i n t h e m e l t i n g r e g i o n depend g r e a t l y on t h e p r e v i o u s t h e r m a l h i s t o r y o f t h e sample. When a DSC s c a n f o r s u l f i s o x a z o l e w a s o b t a i n e d u s i n g a h e a t i n g r a t e of 10°C/minute, a m e l t i n g endotherm s t a r t i n g a t 191.6OC and a d e c o m p o s i t i o n exotherm s t a r t i n g a t 201.7OC were o b s e r v e d ( F i g u r e 6 ) . Because o f t h e sample i n s t a b i l i t y i n t h e m e l t i n g r e g i o n , t h e AHf w a s not obtained (7). 2.9

T h e r m o g r a v i m e t r i c A n a l y s i s (TGA) No w e i g h t l o s s was o b s e r v e d by TGA between ambient and 225OC when t h e sample was h e a t e d a t 10°C/minute. A w e i g h t l o s s began a b o u t 225OC and amounted t o a b o u t 60% of t h e sample w e i g h t a t 475OC ( 7 ) . 2.10

Solubility The s o l u b i l i t y d a t a f o r s u l f i s o x a z o l e o b t a i n e d a t 25OC are g i v e n i n T a b l e I11 ( 8 ) . T a b l e I11 S o l u b i l i t y of Sulfisoxazole i n Different Solvents So l u b il i t y (mg / m l )

Solvent 3A a l c o h o l benzene

18.6 2.1

496

SULFISOXAZOLE

Figure 6 DSC Curve f o r Sulfisoxazole

I

I

I

I

Exo

B. C. R U D Y A N D B. Z. SENKOWSKI

chlorof o m 95% e t h a n o l ethyl ether methanol p e t r o l e u m e t h e r 30°-60' 2-propanol water

2.8 10.5 2.5 14.9 1.4 8.6 0.2

2.11

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n of s u l f i s o x a z o l e i s p r e s e n t e d i n T a b l e I V ( 9 ) . The i n s t r u m e n t a l c o n d i t i o n s a r e g i v e n below. Instrument Conditions: G e n e r a l E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t : Radiation: optics:

50KV-12-112 MA Copper Cu =&J 1.542 8 0.1' D e t e c t o r s l i t M.R. S o l l e r s l i t 3' B e a m s l i t 0.0007 i n c h Ni f i l t e r 40 t a k e o f f a n g l e S c a n a t 0.2O 20 p e r m i n u t e Amplifier gain - 16 course, 8.7 f i n e Sealed proportional counter t u b e and DC v o l t a g e a t plateau P u l s e h e i g h t s e l e c t i o n EL-5 v o l t s ; EU - o u t Rate meter T . C . 4 2000 C I S f u l l s c a l e Chart speed - 1 i n c h p e r 5 minutes

Goniometer: Detector:

Recorder:

Samples p r e p a r e d by g r i n d i n g a t room t e m p e r a t u r e .

498

SULFISOXAZOLE

Table I V X-ray Powder D i f f r a c t i o n P a t t e r n of S u l f i s o x a z o l e

28 12.09 13.23 14.28 15.22 16.32 16.94 17.40 18.56 19.48 20.26 20.64 21.76 22.86 23.46 23.74 24.45 25.68 26.62 27.18 27.83 28.26 29.52 30.14 30.86 31.36 31.74 32.20 33.00 33.20

*d *

>k

(8)*

7.32 6.69 6.20 5.82 5.43 5.23 5.10 4.78 4.56 4.38 4.30 4.08 3.89 3.79 3.75 3.64 3.47 3.35 3.28 3.21 3.16 3.03 2.97 2.90 2.85 2.82 2.78 2.71 2.70

**

I/I 28 0 100 31 6 26 4 15 41 14 14 21 36 13 96 8 5 19 8 65 14 9 2 13 5 2 3 2 2 6 6

- (interplanar distance)

%,

2.12

d

33.76 34.32 35.00 35.26 36.04 36.52 37.82 38.06 38.44 39.16 39.78 40.62 42.12 43.56 43.94 44.66 45.30 45.90 46.40 47.02 47.99 48.76 49.00 49.70 50.14 50.73

d (2)

2.65 2.61 2.56 2.55 2.49 2.46 2.38 2.36 2.34 2.30 2.27 2.22 2.15 2.08 2.06 2.03 2.00 1.98 1.96 1.93 1.90 1.87 1.86 1.83 1.82 1.80

*

I/&)** 6 6 1 1 6 5 2 2 2 7 3 4 7 3 1

4 1 1 2 4 3 2 1 1 1 1

nX

2 Sin 0

= r e l a t i v e i n t e n s i t y ( b a s e d on h i g h e s t i n t e n s i t y

of 1.00)

D i s s o c i a t i o n Constant The pka f o r s u l f i s o x a z o l e h a s been d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y (11) and by t h e t i t r a t i o n o f

B. C. RUDY A N D B. 2. SENKOWSKI

s u l f i s o x a z o l e i n an e x c e s s of 0.1N NaOH w i t h 0.1N H C 1 t o b e 5.0 ( 1 2 , 1 3 ) .

3.

Synthesis S u l f i s o x a z o l e may b e p r e p a r e d by t h e r e a c t i o n scheme shown i n F i g u r e 7. 3,4-Dimethyl-5-aminoisoxazole is rea c t e d w i t h N-acetyl-p-aminobenzene s u l f o n y l c h l o r i d e . The a c e t y l group i s t h e n c l e a v e d t o y i e l d s u l f i s o x a z o l e (10). 4.

S t a b i l i t y Degradation When 2% s o l u t i o n s o f s u l f i s o x a z o l e are p r e p a r e d i n e t h a n o 1 : w a t e r ( 1 : l ) and e t h a n o l : O . l N NaOH (1:l) and ref l u x e d f o r one h o u r , no d e c o m p o s i t i o n was o b s e r v e d by t h i n l a y e r chromatography ( 3 ) . When a 2% s o l u t i o n of s u l f i s o x a z o l e i n e t h a n o l : O . l N H C 1 (1:l) w a s r e f l u x e d f o r one h o u r s l i g h t d e c o m p o s i t i o n t o 3,4-dimethyl-5-aminoisoxazole and s u l f a n i l a m i d e w a s observed ( 3 ) . Pure s u l f i s o x a z o l e h a s been found t o b e s t a b l e when s u b j e c t e d t o a t e m p e r a t u r e of 105OC f o r 5 days ( 3 ) . I n t a b l e t form, s u l f i s o x a z o l e i s s t a b l e f o r 5 y e a r s (14). 5.

Drug M e t a b o l i c P r o d u c t s and P h a r m a c o k i n e t i c s S u l f i s o x a z o l e i s m e t a b o l i z e d t o i t s N4-acetyl d e r i v a t i v e which i s t h e major m e t a b o l i t e found i n human u r i n e . S u l f i s o x a z o l e i s u l t i m a t e l y e l i m i n a t e d from t h e body s o l e l y by means o f u r i n a r y e x c r e t i o n w i t h a mean of 54 p e r c e n t of t h e dose e x c r e t e d as t h e ' ' f r e e " d r u g and t h e remainder as t h e N q - a c e t y l a t e d b i o t r a n s f o r m a t i o n p r o d u c t (15). A p h a r m a c o k i n e t i c p r o f i l e of s u l f i s o x a z o l e was d e t e r mined from d a t a o b t a i n e d from I.V. a d m i n i s t r a t i o n o f t h e drug i n humans. The d a t a s u g g e s t e d t h e u s e o f a two compartment open s y s t e m model ( 1 6 ) . The e x c e l l e n t agreement between t h e s i m u l a t e d and e x p e r i m e n t a l d a t a r e f l e c t s t h e r e l i a b i l i t y of t h e assumption of psuedo f i r s t o r d e r k i n e t i c s f o r a l l p r o c e s s e s (15).

6.

Methods o f A n a l y s i s

6.1

Elemental Analysis The r e s u l t s from t h e e l e m e n t a l a n a l y s i s are l i s t e d i n Table V ( 1 7 ) .

5 00

Figure 7 S y n t h e s i s o f Sulfisoxazole

s

+C H B3 C - N H o ! - NO

0

C H 3 C - N H a - C I 40 N-acetyl-p-amino-benzene sulfonyl chloride

H3C CH3 3,4-dimethy-5-

0

aminoisoxazole

5- (p-acetylsulfanilamido)

Y

H&

d imethyl-isoxazole

- 3.4-

CH3 m

C r n v)

0 X

Zl0

r rn

sulfisoxazole

8.

C. RUDY AND 5.2. SENKOWSKI

Table V Elemental A n a l y s i s of S u l f i s o x a z o l e Element C H N S

% Theory

49.43 4.90 15.72 12.00

% Found

49.32 4.81 15.74 12.07

6.2

Phase S o l u b i l i t y Analysis P h a s e s o l u b i l i t y a n a l y s i s may b e c a r r i e d o u t u s i n g e t h y l a c e t a t e a s t h e s o l v e n t . A t y p i c a l example i s shown i n F i g u r e 8 which a l s o l i s t s t h e c o n d i t i o n s under which t h e a n a l y s i s was c a r r i e d o u t ( 8 ) .

6.3

T h i n Layer Chromatographic A n a l y s i s (TLC) TLC s y s t e m s have been p u b l i s h e d which c a n b e u s e d t o s e p a r a t e s u l f i s o x a z o l e from i t s m e t a b o l i t e s and o t h e r sulfonamides. I n t h e s y s t e m r e p o r t e d by K a r p i t s c h k a (18) t h e sample i s s p o t t e d on a K i e s e l g e l G p l a t e and d e v e l o p e d i n t h e s o l v e n t , ch1oroform:n-butano1:petroleum e t h e r (1:l:l). S u l f i s o x a z o l e h a s a Rf o f 0 . 5 1 . The TLC s y s t e m r e p o r t e d by W o l l i s h e t a l . ( 1 9 ) u t i l i z e s a S i l i c a G e l G p l a t e and c h l o r o f o r m : h e p t a n e : e t h a n o l (1:1:l) a s t h e dev e l o p i n g s o l v e n t . I n t h i s s y s t e m s u l f i s o x a z o l e h a s a Rf of 0 . 7 .

6.4

D i r e c t S p e c t r o p h o t o m e t r i c Anal* Direct s p e c t r o p h o t o m e t r y i s n o t o f t e n used f o r t h e a n a l y s i s of s u l f i s o x a z o l e b e c a u s e o t h e r s u l f o n a m i d e s t e n d t o i n t e r f e r e . However, i n t h e a b s e n c e o f any i n t e r f e r e n c e s , t h e Amax a t 253 nm i n pH 7 . 5 p h o s p h a t e b u f f e r may be used f o r d i r e c t s p e c t r o p h o t o m e t r i c a n a l y s i s .

6.5

Colorimetric Analysis The c o l o r i m e t r i c a n a l y s i s o f s u l f i s o x a z o l e may be accomplished by d i a z o t i z a t i o n and c o u p l i n g w i t h N-(1naphthy1)-ethylenediamine d i h y d r o c h l o r i d e ( B r a t t o n - M a r s h a l l R e a g e n t ) . The r e s u l t a n t p u r p l e c o l o r i s measured a t a b o u t 550 nm ( 2 0 ) .

502

SU L F ISOXAZO LE

Figure 8

I-

z W

>

-I

0

. In

rn

w

I3 -I

0 fn r

rn

E

z

0 k

fn

0

a

x

0

u z

0 I3 J

0

'v 6

SAMPLE SOLVENT SLOPE

I

SULFISOXAZOLE ETHYL

ACETATE

-

0 40%

EOUlLlBRATlON

2 0 HOURS

01

EXTRAPOLATED SOLUBILITY

25OC 14 5 m g / g

of ETHYL

-

ACETATE

-

4

lo

0

SYSTEM

25 COMPOSITION

50 mg of

503

75 SAMPLE per g

I00

SOLVENT

8.

C. RUDY AND B. 2. SENKOWSKI

6.6

Fluorimetric Analysis F l u o r e s c e n c e a n a l y s i s h a s been used t o d e t e r mine t h e c o n c e n t r a t i o n of s u l f i s o x a z o l e i n plasma ( 2 1 ) . For t h i s p u r p o s e d e t e c t a b l e l e v e l s i n t h e r a n g e of 1 ug p e r m l of plasma a r e needed. I n o r d e r t o a c h i e v e t h i s s e n s i t i v i t y i t i s n e c e s s a r y t o make a f l u o r e s c e n t d e r i v a t i v e of s u l f i s o x a z o l e . One such d e r i v a t i v e i s t h e h i g h l y f l u o r e s c e n t "Dansylsulfonamide" which i s formed by t h e r e a c t i o n shown i n F i g u r e 9 . The f l u o r e s c e n c e y i e l d o f t h i s compound i s s u f f i c i e n t t o measure a b o u t 1 ug of s u l f i s o x a z o l e p e r m l of plasma. An even more s e n s i t i v e d e r i v a t i v e i s formed when s u l f i s o x a z o l e i s r e a c t e d w i t h 4,5-methylened i o x y - p h t h a l a l d e h y d e t o form a p h t h a l i m i d i n e d e r i v a t i v e ( F i g u r e 9 ) . The s e n s i t i v i t y l i m i t f o r t h i s d e r i v a t i v e is from 1 x 10-8 t o 1 x 10-10 mole/ml ( 2 2 ) .

6.7

T i t r i m e t r i c Analysis The t i t r a t i o n w i t h sodium methoxide i s t h e method of c h o i c e t o a s s a y s u l f i s o x a z o l e ( 6 ) . The sample i s d i s s o l v e d i n dimethylformamide, thymol b l u e i s added, and t h e t i t r a t i o n w i t h 0.1N NaOCH3 t o a b l u e end-point is c a r r i e d o u t . A b l a n k d e t e r m i n a t i o n i s performed and any n e c e s s a r y c o r r e c t i o n made. Each ml of 0.1N NaOCH3 i s e q u i v a l e n t t o 26.73 mg of s u l f i s o x a z o l e .

5 04

Figure 9 Fluorescence Derivatives of Sulfisoxazole

C H 0~ & N H ~ ~ - ~O ! HJ . Q \ I

N4-wetyl N(CH,),

@8 Dansyl- Reogent

CH3

metotolite

Y(CH3)2

4 N HCI c

c

o=s=o CI

lm.

H3C

\+

,

Dansyl- Sulfonomide Ex 3555/Em 485 m

phthalimidine derivative EX 330/Em 400 nm

B. C. RUDY AND B. Z. SENKOWSKI

9.

References

1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. 2 . Johnson, J.H., Hoffmann-La Roche Inc., Personal Communication. 3. Rubia, L.B., Hoffmann-La Roche Inc., Personal Communication. 4. Boatman, J., Hoffmann-La Roche Inc., Personal Communication. 5. Benz, W., Hoffmann-La Roche Inc., Personal Communication. 6. United S t a t e s Pharmacopeia XVIII, 699 and 935 (1970). 7. Moros, S., Hoffmann-La Roche Inc., Personal Communication. 8. MacMullan, E. , Hoffmann-La Roche Inc. , Personal Communication. 9. Hagel, R. B. , Hoffmann-La Roche Inc , Personal Communication. 10. Hoffer, M., Hoffmann-La Roche Inc., Personal Communication. 11. Motchane, A., Hoffmann-La Roche Inc. , Unpublished Data. 12* Rieder, J., Amzeimittel-Forsch, 13, 81 (1963). 13. Nakagaki, M., Koga, N., and Terada, H., J . Pharm. Soc. Japan, 83, 586 (1963). 14. Clyburn, T., Hoffmann-La Roche Inc., Personal Communication. 15. Kaplan, S.A., Weinfeld, R.E., Abruzzo, C.W., and Lewis, M., J . Pharm. S c i . , 61, 773 (1972). 16. Riegelman, S., Loo, J.C.K., and Rowland, M., J . Pharm. S c i . , 57, 117 (1969). 17. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. 18. Karpitschka, N., Mikrachim. Acta, 1963,157. 19. Wollish, E.G., Schmall, M., and Hawrylyshyn, M., Anal. Chem., 33, 1138 (1961). 20. Bratton, A . C . , and Marshall, F.K., J . B i o l . Chem., 128, 537 (1939). 21. deSilva, J.A.F., and D'Arconte, L., J . Forensic. S c i . , 14,184 (1969). 22. Amano, T., J . Pharm. Soc. Japan, 85, 1049 (1965).

.

506

TRICLOBISONIUM CHLORIDE

6. C. R U D Y A N D 6 . 2. SENKOWSKI

I NDE X

Analytical P r o f i l e - Triclobisonium Chloride

1.

Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, C o l o r , Odor

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 Fluorescence Spectrum 2.5 Mass Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 D i f f e r e n t i a l Scanning Calorimetry 2.9 Solubility 2.10 X-ray C r y s t a l P r o p e r t i e s

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug Metabolic Products

6.

Methods o f A n a l y s i s 6.1 Elemental A n a l y s i s 6.2 Thin Layer Chromatographic A n a l y s i s 6.3 C o l o r i m e t r i c Analysis 6.4 T i t r i m e t r i c Analysis

7.

References

508

TR I CLOBISON I UM CH LOR I DE

1.

Descriution

Name, Formula, Mol ecul ar Weight T r i c l o b i s o n i u m c h l o r i d e i s hexam et hyl enebi s [ dimet hy 1 [ 1-met hy 1- 3 - (2 , 2 ,6 - t rime t hy 1cyc l o h e x y l ) propy 11 ammonium] d i c h l o r i d e . 1.1

M ol ecul ar Weight:

C36H74C12N2

605.91

Appearance, C o l o r , Odor T r i c l o b i s o n i u m c h l o r i d e o c c u r s as a w h i t e o r n e a r l y w h i t e , p r a c t i c a l l y o d o r l e s s , c r y s t a l l i n e powder. 1.2

2.

Physical Properties 2.1

I n f r a r e d Spectrum The i n f r a r e d s pect r um o f t r i c l o b i s o n i u m c h l o r i d e i s p r e s e n t e d i n F i g u r e 1.. The s p e c t r u m was measured w i t h a P e r k in - Elme r 621 S p e c t r o p h o t o m e t e r on a K B r p e l l e t c o n t a i n i n g 1 . 5 mg t r i c l o b i s o n i u m c h l o r i d e / 3 0 0 mg o f KBr. The bands a t 2960 and 2860 cm-I a r e a t t r i b u t e d t o a l i p h a t i c C-H s t r e t c h i n g and t h e bands a t 1474 and 1382 cm-l t o C-ti deformation v i b r a t i o n s (1). N u c l e a r Magnetic Resonance Spectrum (NMR) The s p e c t r u m shown i n F i g u r e 2 was o b t a i n e d on a J e o l 60 blHz NMR by d i s s o l v i n g 5 7 . 2 mg o f t r i c l o b i s o n i u m c h l o r i d e i n 0 . 5 m l o f CDC13 c o n t a i n i n g t e t r a m e t h y l s i l a n e as an i n t e r n a l r e f e r e n c e ( 2 ) . Due t o t h e many n e a r l y e q u i v a l e n t p r o t o n s i n t h i s m o l e c u l e , t h e NMK s p e c t r u m c o n s i s t s o f b r oad bands i n t h e 0 t o 4 ppm r e g i o n . The s p e c t r a l assignments a r e given i n Table I ( 2 ) . 2.2

509

0. C. RUDY

A N D 0 . 2. S E N K O W S K I

7 (u (u-

-

aom-

0

0 0

y--

0z a-0 u)

c

0

z

*r---

I

I-

s

w--

2 ?

m--

W -J W

w-a al k cd

-

h

cu

c

H

m-

-

33NVllIWSNVU %

510

TR ICLOBlSONlUM CHLORIDE

Figure 2

NMR Spectrum of Triclobisonium C h l o r i d e

lo0

20

I20

I BC

511

B. C. RUDY AND B. 2. SENKOWSKI

Table I

NMR Assignments f o r T r i c l o b i s o n i um C h l o r i d e Type P r o t o n

Approx. Chemical S h i f t (ppm)

CH3)s on r i n g s

0. 80-0. 95

CH3's on c h a i n

1.47

CH2Is & CH's i n rings and c h a i n ( n o t a d j a c e n t t o nitrogens)

1. 05-2. 30

CH3's on n i t r o g e n s

3.30

CH2Is 6 CHIS adjacent t o nitrogens

3. 45-3. 90

U l t r a v i o l e t Spectrum (UV) T r i c l o b i s o n i u m c h l o r i d e e x h i b i t s no maxima o r minima i n an aqueous s o l u t i o n (1 mg/ml) from 700 t o 210 nm. 2.3

F l u o r e s c e n c e Spectrum T r i c l o b i s o n i u m c h l o r i d e e x h i b i t s no f l u o r e s c e n c e when i r r a d i a t e d w i t h w h i t e l i g h t . (3) 2.4

Mass Spectrum The mass s pect r um o f t r i c l o b i s o n i u m c h l o r i d e shown i n F i g u r e 3 was o b t a i n e d from a CEC 21-110 mass s p e c t r o m e t e r w i t h an i o n i z i n g e n e r g y o f 70 eV ( 4 ) .

2.5

Weak peaks were o b s e r v e d up t o m/e 616 i n t h e low r e s o l u t i o n s pect r um . S i n c e q u a t e r n a r y ammonium s a l t s are n o t s u f f i c i e n t l y v o l a t i l e , t h e mass spect rum o b t a i n e d i s p r i m a r i l y t h a t of t h e thermal degradation products. I n t h i s c a s e t h e main p r o d u c t obs er ved had a m o l e c u l a r weight o f 5 0 4, C34H68N2 by h i g h r e s o l u t i o n a n a l y s i s , which c o r r e s p o n d s t o t r i c l o b i s o n i u m c h l o r i d e minus 2 C H 3 C l ' s . The low r e s o l u t i o n s pect r um a l s o c o n t a i n e d a peak a t m/e The 518 p r o b a b l y due t o t h e l o s s o f H C 1 p l u s CH3C1. component o f m o l e c u l a r w ei ght 504 g i v e s r i s e t o t h e b a s e peak a t m/e 351 and a l s o m/e 225 v i a t h e u s u a l c l e a v a g e B t o t h e amino g r oup. The peak a t m/e 186, C11H26N2, i s p r o b a b l y a fragment from t h e c e n t e r p a r t o f t h e m ol ecul e (4) *

512

0

E 3

TRlCLOBlSONlUM CHLORIDE

513

B. C. RUDY AND B. 2. SENKOWSKI

2.6

Optical Rotation T r i c l o b i s o n i u m c h l o r i d e e x h i b i t s no o p t i c a l

activity. Melting Range T r i c l o b i s o n i u m c h l o r i d e decomposes on m e l t i n g . The decomposition range depends on t h e r a t e o f h e a t i n g . When t h e NF Class I procedure i s used ( 5 ) , t r i c l o b i s o n i u m c h l o r i d e melts w i t h decomposition between 243 and 253'C. 2.7

D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC) The DSC s c a n f o r t r i c l o b i s o n i u m c h l o r i d e , shown i n Figure 4 , was o b t a i n e d u s i n g a Perkin-Elmer DSC-1B C a l o r i m e t e r . The sample pan c o n t a i n e d a small h o l e i n t h e t o p t o allow t h e decomposition p r o d u c t s formed t o e s c a p e . With a t e m p e r a t u r e program o f 10"C/min., an endotherm was observed s t a r t i n g a t 253.4'C which i s due t o t h e melt and/ o r decomposition o f t h e molecule ( 6 ) . Due t o t h e n a t u r e o f t h i s peak no AHf was c a l c u l a t e d . 2.8

Solubility The s o l u b i l i t y d a t a f o r t r i c l o b i s o n i u m c h l o r i d e o b t a i n e d a t 25'C a r e given i n T a b l e I1 ( 7 ) . 2.9

Table I 1 S o l u b i l i t y of T r i c l o b i s o n i u m C h l o r i d e i n Different Solvents Solvent

S o l u b i l i t y (mg/ml)

3A a l c o h o l benzene ch l o r o form 95% e t h a n o l ethyl ether met hano 1 petroleum e t h e r (30'-60') 2 -propano1 water

> 5.0

x lo2

1.3 4.2 > 5.0 1.6 > 5.0 5.0

x 10 x 10; x x 10 x lo! x 10

>

514

4 . 1 x 10; 5 . 0 x 10

TRICLOBISONIUM CHLORIDE

s 8

R

0

Y W (L

3 +

a

W

(L

0 3

:$

x

0

0 U NJ

0 a, N

B. C. RUDY AND 6. 2. SENKOWSKI

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n p a t t e r n o f t r i clobisonium c h l o r i d e i s p r e s e n t e d i n Table I11 ( 8 ) . The i n s t r u m e n t a l c o n d i t i o n s are given below. 2 .1 0

Instrumental Conditions: General E l e c t r i c Model XRD-6 Spectrogoniometer Generator: Tube t a r g e t : Radiation : Optics :

50 KV-12-$ MA Copper 0 Cu K = 1.542 A 0.1" Detector s l i t M.R. S o l l e r s l i t 3' Beam s l i t 0.0007 i n c h N i f i l t e r 4" t a k e o f f a n g l e Scan a t 0.2' 20 p e r minute A m p l i f i e r g a i n - 16 c o a r s e , 8.7 fine Sealed proportional counter t u b e and DC v o l t a g e a t p 1a t eau P u l s e h e i g h t s e l e c t i o n EL-5 v o l t s ; EU - o u t Rate meter T . C . 4 2000 C/S f u l l s c a l e Chart speed - 1 i n c h / 5 min.

Goniometer Detect o r :

Recorder:

Samples p r e p a r e d by g r i n d i n g a t room t e m p e r a t u r e . TABLE I11

X-ray Powder D i f f r a c t i o n P a t t e r n Triclobisonium Chloride

20 3.98 4.44 12 .4 4 12-92 13.52 14 .7 4

0

**

d(i) *

I/I 0

22.2 19.9 7.12 6.85 6.55 6.01

39 75 19 10 25 100

20 __

23. 78 24. 46 26.18 26. 38 27. 14 28.00

5 16

d(A)* 3.74 3.64 3.40 3. 38 3.29 3.19

UI, * * 19 17 6 22 22 15

TRlCLOBlSONlUM C H L O R I D E

TABLE I11 ( c o n t . )

X-ray Powder D i f f r a c t i o n P a t t e r n T r i c l o b i sonium C h 1o r i d e 0

20

d(A) *

1/10

15.40 15.94 16.56 17.16 17.78 19.92 20. bO 21.60 22.00 23.36

5.75 5.56 5,35 5.17 4.99 4.46 4.31 4.11 4.04 3.81

6 28 6 14 67 18 4 13 19 25

0

**

20

-

~

28.62 29.42 30.56 31.04 32.10 32.76 34.96 35.74 41.16 45.12

*

d = (interplanar distance)

**

I/I,

d(A) * 3.12 3.04 2.93 2.88 2.79 2.73 2.57 2.51 2.19 2.01

1/10

**

19 28 11 15 11 13 8 8 8 8

ni 2 Sin 0

on h i g h e s t i n t e n s i t y o f 1.00)

= r e l a t i v e i n t e n s i t y (based

3.

Synthesis T r i c l o b i s o n i u m c h l o r i d e may be p r e p a r e d by t h e r e a c t i o n scheme shown i n F i g u r e 5 . A comprehensive r e v i e w o f t h e s y n t h e s i s o f s e v e r a l s y m m e t r i c a l b i s - q u a t e r n a r y amines d e r i v e d from (3-ionone has been p u b l i s h e d by T e i t e l ( 9 ) . S t a b i l i t y Degradation T r i c l o b i s o n i u m c h l o r i d e was found t o be s t a b l e t o l i g- h t and oxygen i n b o t h s l i g h t l y a c i d i c (plt 3-7) and s l i g h t l y b a s i c (pH 7-9) s o l u t i o n s . In s t r o n g l y a l k a l i n e s o l u t i o n s t h e compound decomposes t o y i e l d t e r t i a r y amines ( 1 0 ) . I n t h e cream o r o i n t m e n t , t h e t r i c l o b i s o n i u m ch1,oride i s s t a b l e f o r a minimum o f 4 y e a r s . The b u l k s u b s t a n c e i s s t a b l e when s t o r e d a t room t e m p e r a t u r e u n d e r anhydrous conditions. Decomposition o c c u r s when i t i s s u b j e c t e d t o e l e v a t e d t e m p e r a t u r e s as can be d e t e c t e d by d i s c o l o r a t i o n and o d o r o f t h e powder. 4.

Drug M e t a b o l i c P r o d u c t s T r i c l o b i s o n i u m c h l o r i d e i s an a n t i b a c t e r i a l a g e n t u s e d f o r the treatment and/or prevention o f l o c a l i n f e c t i o n s . S i n c e i t i s n o t a d m i n i s t e r e d i n t e r n a 1 1y , t r i c 1o b i s o n i um 5.

517

Figure 5 Synthesis o f Triclobisonium Chloride (9)

[email protected];0

Ammonia H (Raney Ni)

B-ionone

'

"3z CH CHNHZ

21

a 3

"3

Formic Acid Fo mialdehyde

m 0

TRlCLOElSONl UM C H L O R I D E

c h l o r i d e i s n o t m e t a b o l i z e d by t h e body. 6.

Methods o f A n a l y s i s

E 1ement a1 A n a l y s i s The r e s u l t s from t h e e l e m e n t a l a n a l y s i s a r e l i s t e d i n T a b l e IV ( 1 1 ) . 6.1

TABLE IV

Elemen tal A n a l y s i s o f T r i c l o b i s o n i u m C h l o r i d e E 1emen t C H

N c1

% Theory

71.36 12. 31 4.62 11. 70

% Found

69. 29 12. 30 4.57 11. 46

6.2

Thin Layer Chromatographic A n a l y s i s (TLC) The f o l l o w i n g TLC p r o c e d u r e may be used t o i d e n t i f y t r i c l o b i s o n i u m c h l o r i d e . Spot 500 mcg o f t r i c l o b i s o n i u m c h l o r i d e which was d i s s o l v e d i n methanol on a s i l i c a g e l G p l a t e and s u b j e c t i t t o a s c e n d i n g chromatography i n a p a p e r l i n e d , s a t u r a t e d tank using a mixture of acetone:5% KC1: benzene (80:40:1) as t h e s o l v e n t . A f t e r d e v e l o p i n g t h e p l a t e a t l e a s t 15 cm, i t i s a i r d r i e d and s p r a y e d w i t h i o d i n e - m o d i f i e d D r agendor f f r e a g e n t t o v i s u a l i z e t h e s p o t . The t r i c l o b i s o n i u m c h l o r i d e w i l l a p p e a r as a brown s p o t a bo u t Rf 0.5 ( 1 2 ) . 6.3

Colorimetric Analysis

Ion P a i r E x t r a c t i o n o f Bromocresol Green L'omDlex After d i s s o l u t i o n o f t h e t r i c l o b i s o n i u m c h l o r i d e cream, t h e t r i c l o b i s o n i u m c h l o r i d e i s complexed w i t h b r o mo c r es ol g r e e n and t h i s complex i s e x t r a c t e d i n t o a ch lo r o f o r m s o l u t i o n . The abs or bance o f t h i s s o l u t i o n , as w e l l as t h e a b s o r b a n c e o f t h e complex s i m i l a r l y p r e p a r e d from r e f e r e n c e s t a n d a r d t r i c l o b i s o n i u m , i s r e a d a t 630 nm and t h e amount o f t r i c l o b i s o n i u m c h l o r i d e p r e s e n t i n t h e cream i s c a l c u l a t e d ( 1 3 ) . 6.31

B. C. RUDY A N D B. 2. SENKOWSKI

6.32

I o n - P a i r E x t r a c t i o n o f Bromothymol Blue Comp 1ex For t h e t r i c l o b i s o n i u m c h l o r i d e ointment bromothymol b l u e i s used t o form t h e i o n - p a i r complex. The procedure i s similar t o t h a t €or t h e cream except p o l y e t h y l e n e g l y c o l 4000 and p o l y e t h y l e n e g l y c o l 400 a r e added t o t h e r e f e r e n c e s t a n d a r d t ri c l o b i s onium c h l o r i d e s o l u t i o n and a l s o t o a b l a n k . A f t e r e x t r a c t i o n o f t h e complex from a b a s i c aqueous s o l u t i o n , t h e absorbance i s r e a d a t t h e 384 nm maximum f o r t h e sample, s t a n d a r d , and t h e b l a n k . These v a l u e s a r e used t o c a l c u l a t e t h e amount o f t r i clobisonium i n t h e ointment ( 1 3 ) . 6.4

T i t r i m e t r i c Analysis The non-aqueous t i t r a t i o n a s d e s c r i b e d i n t h e NF XI11 i s t h e method o f c h o i c e f o r t h e a n a l y s i s o f bulk t r i c l o b i s o n i u m c h l o r i d e (13). An a c c u r a t e l y weighed 1 gm sample i s d i s s o l v e d i n 80 m l o f g l a c i a l a c e t i c a c i d . F i f t e e n m i l l i l i t e r s of mercuric a c e t a t e and 4 drops o f c r y s t a l v i o l e t a r e added and t h e t r i c l o b i s o n i u m c h l o r i d e i s t i t r a t e d t o a b l u e - g r e e n end-point with 0 . 1 N H C 1 0 4 i n g l a c i a l a c e t i c a c i d . A blank t i t r a t i o n i s performed and any n e c e s s a r y c o r r e c t i o n made. Each m l o f 0.1N H C 1 0 4 i s e q u i v a l e n t t o 30.30 mg o f C36H74C12N2 ( 1 3 ) .

520

TR ICLOBISON I UM CHLORIDE

7.

Keferences

1.

Hawrylyshyn, M . , Hoffmann-La Roche I n c . , P e r s o n a l Communication. 2 . J o h n s o n , J . H . , fioffmann-La Roche I n c . , P e r s o n a l Communication. 3 . Boatman, J . , Hoffmann-La Roche Inc. P e r s o n a l Communication. Benz, W . , Hoffmann-La Roche I n c . , P e r s o n a l 4. Commun i c a t i o n . 5 . National Forniulary X I I I y 815 ( 1 9 7 0 ) . 6 . bloros, S . , Hoffmann-La Koche I n c . , P e r s o n a l Communication. 7 . MacMullan, E., 1-Ioffniann-La Roche I n c . , P e r s o n a l Commun i c a t i o n . 8. H a g e l , R. B . , Hoffniann-La Koche I n c . , P e r s o n a l Communication. 9 . T e i t e l , S . , J . Med. Chem., 6 , 780 ( 1 9 6 3 ) . 1 0 . T e i t e l , S . , Iloffmann-La floche I n c . , Unpublished Data. 1 1 . S c l i e i d l , F . , Iloffmann-La Roche I n c . , P e r s o n a l Communication. 1 2 . Bandong, L . , Iloffmann-La Roche l n c . , P e r s o n a l Communication. 13. National FormuZary X I I I , 722-723 ( 1 9 7 0 ) .

52. I

This Page Intentionally Left Blank

TRIFLUPROMAZINE HYDROCHLORIDE

Kluiis Florcp

513

KLAUS F L O R E Y

1.

2.

3. 4. 5. 6,

7.

TABLE OF CONTENTS Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor. Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 pK, pH 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Thermogravimetric Analysis 2.9 Solubility 2.10 Crystal Properties Synthesis Stability - Degradation Drug Metabolic Products Methods of Analysis 6.1 Elemental Analysis 6.2 Gravimetric Analysis 6.3 Nonaqueous Titration 6.4 Electroanalysis 6.5 Titrimetric Analysis 6.6 Spectrophotometric Analysis 6.7 Spectroflurometric Analysis 6.8 Electrophoresis 6.9 Chromatographic Analysis 6.91 Paper 6.92 Thin-Layer 6.93 Gas-Liquid Identification and Determination in Body Fluids and Tissues

8.

Miscellaneous

9.

References

TR I FLUPROMAZINE HYDROCHLORIDE

1.

Description

1.1

Name, Formula, Molecular Weight Triflupromazine Hydrochloride is lO-L?-(Dimethylamino)-propy&7-2trifluromethylphenothiazine hydrochloride; also 2-firifluoromethy~ -10- ( y -dimethylaminopropyl) phenothiazine: S o 4703.

CH2-CH2-CH2-N (CH3)2

I

Cl8H19F3N2S. HC1 1.2

Mol. wt. 388.89

Appearance, Color, odor. Triflupromazine hydrochloride occurs as a white to pale tan, crystalline powder, with a slight characteristic odor.

2.

Physical Properties 2.1

Infrared Spectra The infrared spectra of Squibb Standard # 48244-003 are presented in figures 1 and 2.l The infrared spectrum of the free base and a discussion of spectra-structure

x

r

D C cn n

r

0 D

rn

<

Figure 1.

I. R. Spectrum of Trif lupromazine Hydrochloride. Squibb Standard #48241-003. Mineral oil mull. Instrument: Perkin-Elmer 21.

-I

9 n

r

5n 0 z9 N z

rn I

<

0 rn

Figure 2.

I . R . Spectrum of Triflupromazine Hydrochloride. Squibb Standard #48241-003 in KBr pellet from methanol. Instrument: Perkin-Elmer 21.

KLAUS F L O R E Y

c o r r e l a t i o n of h e n o t h i a z i n e s h a v e b e e n p r e s e n t e d . f l y 3 The s p e c t r a p u b l i s h e d b y Sammul, e t a14 a r e i n e s s e n t i a l agreement with those presented here. N u c l e a r M a g n e t i c Resonance S p e c t r u m

2.2

The NMR s p e c t r u m of t r i f l u p r o m a z i n e hydrochloride i s presented i n F i g u r e 3. 67 The N-methyl p r o t o n r e s o n a n c e s a r e s e e n a s two s i n g l e t s . The m e t h y l e n e p r o t o n r e s o n a n c e s of t h e two m e t h y l g r o u p s on t h e p r o t o n a t e d n i t r o g e n a p p e a r a s two s e t s of t r i p l e t s b e c a u s e of r o t a t i o n a l n o n - e q u i v a l e n c e . The f o l l o w i n g a s s i g n m e n t s i n t a u v a l u e s a r e consistent with the structure:

2-

CH2-CH

I

\

/

CH2-

N

NCH3 .CH3

7.35(s) 7.27 ( s )

F:Hcl

Aromatic p r o t o n s :

3.2-

2.5

z:

The NMR s p e c t r u m o f t h e f r e e b a s e h a s b e e n p r e s e n t e d and d i s c u s s e d . 528

. i #I>

Fig. 3

70

1

I 60

I

I .

.

.

,

.

,

,

,

I , , , , , 50

1 ,

,

I .o

.

, , .

I ,

I 30

1

. . . , .

I 20

.

I

, . . I .

I

I0

NMR Spectrum of Triflupromazine Hydrochloride (Squibb Batch # 48244-003 in C D C 1 3 , containing tetramethylsilane as an internal reference. Instrument: Varian A-60.

I 0

WM

,,

KLAUS FLOREY

2.3

U l t r a v i o l e t Spectrum The f o l l o w i n g U . V . recorded : 1.

i n methanol

1

d a t a h a v e been

( I n s t r u m e n t Cary 15)

max 260 nm E1% 1c m rnax 308 nm E1% 1c m 2.

92.8

3 i n 95% e t h a n o l ( I n s t r u m e n t C a r y 1 4 )

max max min min 2.4

869

258 308 224 280

nm nm nm nm

f

34,000 3,700 10,000 1,600

Mass S p e c t r u m

The low r e s o l u t i o n mass s p e c t r u m o f triflupromazine i s presented i n f i g u r e 4. The f o l l o w i n g f r a g m e n t a t i o n p a t t e r n 5 presented i n figure 5 i s consistent w i t h t h e mass s p e c t r u m a n d i n a g r e e m e n t w i t h t h e r e s u l t s o f an independent i n v e s t i g a t i o n . 6

530

4

TRIFLUPROMAZINE HYDROCHLORIDE

hlISN31NI 3 A I l V l 3 t l

531

r:

a,

m

-d N

E k ?

a ri W

k

.d

E 0

W

k

E 3 4J

u a, a m

cn

.

CnN

m o d m

.*

23 4J

.d

oa,

?4J dr:

m

4J

W E a,? ! x k

3

or: A H

d 0’1

h

.rl

0

N

7-t

m

m

sp f

N

B N

"13,

KLAUS F L O R E Y

m

d

-2

aJ

NN

x 2-u

I

u2

co

8

N aJ

Y E

\

532

TR I F LUPROMAZINE HYDROCHLORIDE

2.5

pK,

pH

The pKa o f t h e h y d r o c h l o r i d e was d e t e r m i n e d t o be a p p r o x i m a t e l y 6 . 5 7 t h e pKb o f t h e f r e e b a s e t o be 4 . 5 9 .' 8 T h e pH o f a 2% s o l u t i o n of a r e c e n t s t a n d a r d was f o u n d t o be 4 . 7 . l The pKa o f t h e f r e e b a s e was d e t e r m i n e d t o be 9 . 2 . 9 2.6

M e l t i n g Range The f o l l o w i n g m e l t i n g ( d e c o m p o s i t i o n ) ranges have been reported (OC). 173-174 ( R e c r y s t a l l i z e d from d r y chlorobenzene and a c e t o n i t r i 1 e ) l O 1 7 6- 17 7

2.7

D i f f e r e n t i a l Thermal A n a l y s i s D i f f e r e n t i a l Thermal A n a l y s i s o f S q u i b b B a t c h AR-IX-113E showed a n e n d o t h e r m a t 174OC.

2.8

Thermogravimetric Analysis Thermogravimetric a n a l y s i s of Squibb B a t c h AR-IX-113E g a v e a w e i g h t l o s s of 0 . 1 % .

533

KLAUS F L O R E Y

2.9

Solubility Triflupromazine hydrochloride is very water soluble and hygroscopic. 12 solubility in water: > l g / l ml ethanol: > l g / l ml chlorotorm: 0.6g/l ml ether: insoluble

The solubility of the free base ir water was determined to be 5

2.10

Crystal Properties For forensic identification a picture of triflupromazine crystals from 95% ethanol has been published with those of other phenothiazine~l~.

An X-ray powder diffraction pattern of triflupromazine hydrochloride is presented in table I.14

534

TRIFLUPROMAZINE HYDROCHLORIDE

Table I d

(g)*

1/100**

14.75 8.05 7.05 5.05 4.75 4.50 4.25 4.10 3.85 3.75 3.55 3.45 3.30 3.20

* **

50 50 35 60 100 25 15 80 40 40 40 10 10 15 0

d = i n t e r p l a n a r d i s t a n c e (A) B a s e d on h i g h e s t i n t e n s i t y o f 100 Cu k a r a d i a t i o n ; = 1,5418

x

The X - r a y powder d i f f r a c t i o n p a t t e r n of t h e p i c r a t e has been used f o r i d e n t i f i c a t i o n i n formulations. 14 3.

Synthesis T r i f l u p r o m a z i n e i s p r e p a r e d by a d d i t i o n o f t h e dimethylaminopropyl s i d e c h a i n to t h e o r its p h e n o t h i a z i n e n u c l e u s (11)l o , N - a c e t y l d e r i v a t i v e 6 5 (111) a n d c o n v e r s i o n of t h e b a s e t o t h e h y d r o c h l o r i d e ( I ) . D i a z o t i z a t i o n of t h e 7 - a m i n o a n a l o g u e ( I V ) a l s o y i e l d s I. 1 5

4.

S t a b i l i t y - Degradation Triflupromazine hydrochloride is hygroscopic. When m o i s t u r e i s e x c l u d e d i t i s s t a b l e i n t h e

535

KLAUS FLOREY

I I R = H I11 R = COCH3

IV

I

YH2-CH2-CHZ-N-CH3 H

CH*-CH2-CH2-N(CH3) 2 I

VIII

V

C H -CH2-CH2-N ( CH3) 1 2

VI VII Fig. 6

R = OH;R',Rlt = H = OH;R=H

R' o r R'

Synthetic and Degradative Pathways f o r Triflupromazine Hydrochloride. 536

TR I FLUPROMAZI NE HYDROCHLORIDE

crystalline state, but discolors rapid1 when exposed to ultraviolet radiation. 6 g This discoloration also occurs on U.V. radiation of aqueous solutions with spectral changes. 6 6 It is undoubtedly due to radical formation and subsequent disproportionation (see below). Thin-layer chromatographic spots on silica gel are oxidized to the sulphoxide (VIII fig. 6) when exposed to air f o r 48-72 hours. l 7 Hydrogen peroxide hastened this process. 1 7 Oxidation also proceeds through the colored semiquinone free radical (cf. 18). In phenothiazines generally these radicals undergo disproportionation. The products of oxidation are the sulfoxide or the 3-(or 7-) hydroxy derivatives. However, formation of the latter is severely inhibited in 10-substituted phenothiazines. l8 so far there is no evidence for the degradative formation of 7-hydroxy-triflupromazine (VI fig, 6 ) which has been identified as a metabolic product’’ (see Section 5). The Electron Spin Resonance spectrum of the radical has been measured and presented 18,20, 21 and was found to differ from the spectrum of the parent compound in the lack of hyper fine structure. The second order decay rate constant of the semiquinone radical of triflupromazine was found to be 870 (l/mol/min) as measured by ESR. 18

5.

Drug Metabolic Products When triflupromazine was incubated with certain liver preparation in vitro, evidence was obtained €or conversion to N-demethyltriflupromazine (V fig. 6) , 7-hydroxytriflupromazine (V~),tracesof triflupromazine 537

KLAUS FLOREY

s u l p h o x i d e ( V I I I ) and p o s s i b l 3- o r 8h y d r o x y t r i f l u p r o m a z i n e ( V I I ) . 419 6.

Methods o f A n a l y s i s 6.1

Elemental Analysis: Element

% Calc.

c18 H2 0

55.59 5.18 9.12 14.66 7.21 8.24

~~

c1

F3 N2 S

6.2

Ref.

% Found 1 0 R e f . 11

55.70 5.22

55.33 5.33

Gravimetric Analysis The p r e c i p i t a t e w i t h s i 1i c o t u n g s t i c a c i d has been used for t h e q u a n t i t a t i v e determination of triflupromazine hydrochloride i n t a b l e t s . 22

6.3

Nonaqueous T i t r a t i o n The nonaqueous t i t r a t i o n of t r i f l u p r o m a zine hydrochloride i n acetone with p e r c h l o r i c a c i d i n dioxane a s t i t r a n t and dimethyl yellow a s i n d i c a t o r h a s been described.22 I t i s s u i t a b l e f o r formulation content assays. Titration w i t h p e r c h l o r i c a c i d was employed t o determine t h e h a l f n e u t r a l i z a t i o n p o t e n t i a l s (E-1/2) w i t h a F i s h e r titrimeter i n several organic solvents. 8 The f o l l o w i n g E-1/2 v a l u e s ( i n mv) w e r e found: a c e t i c a c i d 234, a c e t o n e 455, a c e t o n i t r i l e 424, i s o p r o p a n o l 445, n i t r o m e t h a n e 314. 538

T R I F LUPROMAZI NE HYDROCHLORIDE

6.4 Electroanalysis

Controlled potential electrolysis has been applied for the coulometric quantitative determination of triflupromazine and other phenothiazines.23 Depending on the oxidation potential (anode potential with respect to S.E.C. (standard calomel electrode)) and acid strength triflupromazine was oxidized to the free radical (Ox. Pot. + 0 . 6 5 ; 12N H2SO4) or the sulfoxide (Ox. Pot. + 1.05; 1 2 N H2SO4). The electrochemistry of triflupromazine in acetonitrile has also been studied.24 Polarographic evidence has been presented to prove the complexation of of triflupromazine hydrochloride with oxygen.25 6.5

Titrimetric Analysis A titrimetric method has been reported in which triflupromazine hydrochloride was titrated visually to a colorless endpoint with ceric sulfate.26 The reaction proceeds through forming a red-colored semiquinone free radical (420 n m ) followed by formation of the colorless sulfoxide derivative. The method can be applied to pharmaceutical dosage forms.

6.6 Spectrophotometric Analysis

The ultraviolet absorption maximum at 255nm has been used for a quantitative assay of triflupromazine hydrochloride in formulations. 539

KLAUS F L O R E Y

6.7

Spectroflurometric Analysis Triflupromazine hydrochloride, l i k e o t h e r phenothiazine drugs e x h i b i t s fluorescence. T h i s phenomenon was 27 f i r s t e x p l o r e d i n 0.2N s u l f u r i c a c i d . The f l u o r e s c e n c e p e a k was f o u n d i n t h e r a n g e o f 450 t o 475 nm, s h i f t i n g t o s l i g h t l y l o w e r w a v e l e n g t h upon o x i d a t i o n w i t h potassium permanganate. A s l i t t l e a s 0 . 0 1 t o 0 . 0 5 wg o f t h e p u r e s u b s t a n c e p e r m l . o f 0.2N s u l f u r i c Due t o a c i d c o u l d be d e t e c t e d . interference by o t h e r substances, the s e n s i t i v i t y i n b i o l o g i c a l f l u i d s was l o w e r ( a b o u t 0 . 8 wg/ml o f f l u i d ) . In concentrated s u l f u r i c acid, the a c t i v a t i o n maximum was f o u n d a t 325 nm a n d t h e f l u o r e s c e n c e maximum a t 500 nm. 2 8 The l i m i t o f s e n s i t i v i t y w a s 0 . 0 5 pg/ml. I n 50% a c e t i c a c i d o x i d a t i o n t o t h e s u l p h o x i d e w i t h 30% hydrogen p e r o x i d e gave an a c t i v a t i o n maximum a t 350 m w a n d a f l u o r e s c e n c e maximum o f 410 mp.29

6.8

Electrophoresis Electrophoresis i n various buffers, p r o p o s e d b y Wernurn3' was c a r r i e d o u t on t r i f l u p r o m a z i n e h y d r o c h l o r i d e a n d several other phenothiazine t r a n q u i 11i z e r s 31 A Gordon-Misco a p p a r a t u s , Whatman 3MM p a p e r 30 c m i n width, and a p o t e n t i a l d i f f e r e n c e of Detection of 500 t o 800 v o l t was u s e d . s p o t s was c a r r i e d o u t w i t h a 40% s u l f u r i c a c i d spray (orange c o l o r ) o r

.

540

TR I F L U P R O M A Z I N E H Y D R O C H L O R I D E

f l u o r e s c e n c e u n d e r U. V. l i g h t . For t r i f l u p r o m a z i n e hydrochloride, a n o d i c m i g r a t i o n ( c m ) a f t e r 40 t o 60 m i n u t e s was: PH Migration 6.9

3.3 5.9

4.7 4.8

6.0 5.3

7.2 5.5

8.0 4.6

9.3 1.8

Chromatographic Analysis 6.91

Paper The f o l l o w i n g p a p e r c h r o m a t o g r a p h i c system have been r e p o r t e d . Rf -

Solvent systems: sodium f o r m a t e sodium f o r m a t e 9 0 / n - p r o p a n o l s o d i u m f o r m a t e 90/1N ammonia sodium f o r m a t e 97,/90% formic a c i d 3 1 N sodium a c e t a t e 1N sodium a c e t a t e 9 0 / n - p r o p a n o l 10% sodium c h l o r i d e 92/n-propanol 8 "Reverse Phase" : Paper impregnated w i t h g l y c e r o l 0.2M sodium a c e t a t e / H C l pH 4 . 5 8 1N 1N 1N 1N

10 10

10

Ref.

0.41 0.50 0.12 0.59

31 31 31 31

0.35 0.59

31 31

0 . 76

31

tributyrate,

----

32

Detection systems: 40% H2SO4 s p r a y Color F l u o r e s c e n c e u n d e r U. V. l i g h t Orange B l u i s h Yellow Iodoplatinate Dark

53 1

Ref. 31,32 31 32

KLAUS FLOREY

6.92

Absorbent

Thin-Layer The following thin-layer chromatographic systems have been reported: Solvent System

Silica G e l tert. butyl alcohol 90%/lN ammonia 10% I1 n-proianol 88%/lg ammonia 12% ether, sat. with water 70% methanol/water 30% 85% n-propanol/water 15% n-butanol sat. with 1N - ammonia benzene-dioxane-aq. ammonia ( 60:35 :5 ) 11 ethanol-acetic acid-water(50:30:20) I1 methanol-butano1(60:40) 11 cyclohexane-diethylamine ( 9 : l ) 11 chloroform-hexane(50:50) II chloroform-methanol (50:50) 11 chloroform-metha no 1 (90:10) acetone methanol-ammonia(98:2) chloroform-methanolammonia (80:20:1) cyclohexane-benzenediethylamine (75:15:10)

542

Rf

Ref.

0.36

31

0.50

31

0.48 0.22 0.22 0.72

31 31 31 31

0.95

33

0.79

33

0.40 0.76

33 34

0.48 0.86

35 35

0.54

36

0.38 0.60 0.84

36 36 19+

0.57

37

TRI FLUPROMAZINE HYDROCHLORIDE

S o l v e n t System

Absorbent Silica Gel II

*

ll* 11

,I*

*

*

I,** 11

11

I1

* ** *** + ++

*

cyclohexane-benzenediethylamine(75:15:20) methanol acetone benzene-e thanolammonia ( 9 5 : 1 5 : 5 ) methanol 95% e t h a n o l 15% a q . ammonium a c e t ate-me t h a n o 1 (20:80) n-propanol-wateracetic acid pyridine-pet.ethermethanol (1:4,5:0.1) acetone-e t h y l a c e t a tee t h a n o l 5:4:1 s a t . w i t h ammonium l a c t a t e b u f f e r pH 7.

Rf

Ref. -

0.60

36

0.52 0.48 0.74

37 37 36

0.48 0.31 0.69

37 37 17***

0.28

19++

0. 54

38

0.30

39

p r e p a r e d w i t h 0.1M KOH p r e p a r e d w i t h 0.1M NaHS03 J I n t h i s system, t r i f l u p r o m a z i n e s u l f o x i d e had a n Rf o f 0 . 5 0 . I n t h i s system, triflupromazine sulfoxide had a n Rf o f 0 . 7 0 , T r i f l u p r o m a z i n e s u l f o x i d e h a d a n Rf o f 0 . 1 0 . Generally, it is important to p r o t e c t p l a t e s from l i g h t d u r i n g s p o t t i n g a n d d ev el o p men t .

543

The f o l l o w i n g d e t e c t i o n s y s t e m s h a v e b e e n u s e d :

Syst e m F l u o r e s c e n c e u n d e r U. V. lamp 40% s u l f u r i c a c i d s p r a y Potassium i o d o p l a t i n a t e 5% f e r r i c c h l o r i d e , 20% p e r c h l o r i c acid 50% n i t r i c a c i d (5:45:50). (FPN; (Forrest reagent) Iodine 1% p o t a s s i u m p e r m a n g a n a t e Dragendorff reagent 5% p-dimethylaminobenzaldehyde i n 1 8N sulfuric acid Furfural reagent Folin-Ciocalteau reagent 1%ammonium v a n a d a t e i n 1 0 m l . c o n c . sulfuric acid 5% c i n n a m i c a l d e h y d e a n d 5% H C 1 i n ethanol S u l f u r i c acid-formaldehyde 50% a q . s u l f u r i c a c i d i n e t h a n o l ( 4 : 1)

Color R e a c tion violet orange

Ref 31,36,19,17,35 42,31 52,17

flesh Or

pink

36,34,37 37

i i

r

D

C

v)

37 cameo pink cameo

36 36 36

flesh

36,37

cameo

37 39 42,31

orange

n

r

TRI F LUPROMAZINE HYDROCHLORIDE

6.93

Gas-liquid Triflupromazine was well separated from other phenothiazine tranquilizers on a 80-100 mesh Gas-Chrom S glass column coated with 2% SE-30. Retention time relative to chlorpromaz’ne at a column . temp. of 2 0 5 O was 0.46.40 Similar results were obtained on a 60-80 mesh Diatoport copper column coated with 5% ~ ~ - 3 0 .In ~the ~ same 9 ~ system ~ triflupromazine was separated from its sulphoxide. l7 Another system used was a 100-120 mesh silanized Gas-Chrom P glass column coated with 1% Hi-Eff-8B (cyclohexanedimethanol succinate) . 4 3 The identification of triflupromazine and other phenothiazine tranquilizers by gas chromatography of their pyrolysis products has also been reported. 44

Identification and Determination in Body Fluids and Tissues Many of the references cited in previous sections concern methods devised to identify and determine triflupromazine hydrochloride and other phenothiazines for pharmacological, toxicological and forensic purposes (cf. 45). These methods can be classified as follows:

535

KLAUS FLOAEY

References Color reaction: Separation schemes: Spectrofluorometry: Electrophoresis : Paper chromatography: Thin-layer chromatography: G a s- 1 iquid

chromato-

graphy: X-ray diffraction bands : Micro crystalline identification: Photomicrography:

46,47,48,49,50,51,52,53,54 55,50,51,52 27,29,28,56 31 31,32 50,33,31,13,36,19,37,35,

39,68 17,43,69 61,15 57,58 13

8. Miscellaneous

The following parameters of triflupromazine hydrochloride and other phenathiazine have been studied: Adsorption by solids such as talc, kaolin and activated charcoal59, surface activity at the air-solution interface60361, oil-water partitioning62, photo induced interaction with a lecithin monomolecular film63, and interaction with bovine serum albumin64.

546

TRlF LUPROMAZINE HYDROCHLORIDE

9. References 1.

2.

3.

4.

5. 6.

7. 8.

9. 10.

11.

G. A. Brewer, private communication. W. E. Thompson, R. J. Warren, J, B. Eisdorfer and J. E. Zarembo, J. Pharm. Sci. 54, 1819 (1965). R. J. Warren, J. B. Eisdorfer, W. E. Thompson and J. E. Zarembo, J. Pharm. Sci. 5 5 , 144 (1966). 0. R. Sammul, W. L. Brannon and A. L. Hayden, J. Ass. Offic. Anal. Chem. 47, 918 (1964). A. I . Cohen, private communication. J. N. T. Gilbert and B. J. Millard, Org. Mass Spectrum. 2, 17 (1969). F. Russo-Alesi, private communica tion. L. G. Chatten and L. E. Harris, Anal. Chem. 34, 1495 (1962). A. L. Green, J. Pharm., Pharmacol. 9, 10 (1967). H. L. Yale, F. Sowinski and J. Bernstein, J. Am. Chem. SOC. 79, 4375 (1957). P. N. Craig, E. D. Nodiff, T. T. Lafferty and G. E. Ullyot, J. 0rg.Chem. 22, 709 (1957) H. Jacobson, private communication. P. Rajeswaran and P. L. Kirk, Bull Narcotics, 14, 19 (1962) C. A. 57, 7390h (1962). A. D e Leenheer and A. Heyndrick, J. Am. Offic. Anal. Chem. 54, 625 and 633 (1971). K. Florey and A. R. Restivo, J. Org. Chem. -323 1018 (1958). R. Valenti, private communication. 2. Kofved, C. Fabierkiewicz and G . H. W. Lucas, J. Chromatog. 2 3 , 410 (1966) and Nature 211, 147 (1966). T. N. Tozer and L. D. Tuck, J. Pharm. Sci. 54, 1169 (1964).

.

12. 13.

14. 15. 16. 17.

18.

547

KLAUS FLOREY

19. 20. 21. 22. 23. 24. 25.

A.

E.

27. 28. 29. 30.

Pharmacol.

19 (1966). D. W. S c h i e s e r a n d L. D. Tuck, J. Pharm. S c i . 2, 694 (1962). L. H. P i e t t e a n d J. S . F o r r e s t , Biochim. B i o p h y s . A c t a 57, 419 (1962). J. M i l n e , J. Pharm. S c i . 48, 117(1959). F. H. M e r k l e a n d C . A . D i s c h e r , A n a l . 36 1639 (1964). Chem. -, T . P. B i l l o n , Ann. Chim. 7, 183 (1962) C . A . 58, 11352.

-

H. F. M a r t i n , S . P r i c e a n d B. J . G u d z i n o w i c z , Biochem. B i o p h y s .

103, 26.

R o b i n s o n , J . Pharm.

18,

196 (1963).

L . G . C h a t t e n , R. A . Locock a n d R. D. K r a u s e , J. Pharm. S c i . 69,

588 (1971). T. J. M e l l i n g e r a n d C. E . K e e l e r , A n a l y t i c a l Chemistry, 35, 554 (1963). E. A. M a r t i n , Can. J. Chem. 44, 1783 (1966). S . L. T o m p s e t t , A c t a p h a r m a c o l . e t t o x i c o l . 26, 298 (1968). L. N. Werrum; H. T . Gordon a n d W.

J . T h o r n b u r g , J. Chromatog

3,

125 (1960). 31. 32.

Th. J. M e l l i n g e r a n d C . E. K e e l e r , J. Pharm. S c i . -51 9 1169 (1962). H. W. S t r e e t , A c t a p h a r m a c o l . t o x i c o l .

19, 312 (1962). J. C o c h i n a n d J . W. D a l y , J . P h a r m a c o l . Exp. T h e r . 139 154 (1963). J. S u n s h i n e , Am. J. C l i n . P a t h o l 40, 576 (1963).

I

33.

- J

34. 35. 36. 37. 38.

F. D o n d z i l a , p r i v a t e c o m m u n i c a t i o n .

I . Z i n g a l e s , J. Chromat. 31, 405(1967). W. W . F i k e , A n a l . Chem. -738 1697(1966). H. E b e r h a r d t , O . W . Lerbs a n d K. J. F r e u n d t , A r z n e i m i t t e l f o r s c h u n g 13,8 0 4 (1971). 548

TR I FLUPROMAZINE HYDROCHLORIDE

39.

2 . M a r g a s i n s k i , R. D a n i e l a k , T. Pomazanska a n d H . R a f a l o w s k a , A c t a . 5 ( 1 9 6 4 ) , C . A . -36 2 P o l o n . Pharm. 89419 ( 1 9 6 5 ) . M. W. A n d e r s a n d S . J. M a n n e r i n g , J . Chromatog. 258 ( 1 9 6 2 ) . H . F. M a r t i n , J . L . D r i s c o l l a r d B. L. G u d z i n o w i c z , A n a l . Chem. 35, 1901 (1963). B. J . G u d z i n o w i c z , J . o f G a s . C h r o m a t . 1 9 6 6 110. 7 N. C . C a i n a n d P. L. K i r k , M i c r o c h e m . J . 1 2 , 256 ( 1 9 6 7 ) . C. R. F o n t a n , N. C . J a i n a n d P. L. K i r k , M i c r o c h i m . A c t a - 19 9 6 4 3 2 6 . J. S u n s h i n e , Handbook of A n a l y t i c a l Toxicology. The C h e m i c a l R u b b e r C o . (1969). D. K. Yung a n d M. P e r n a r o w s k i , J . P h a r m . S c i . 52, 365 ( 1 9 6 3 ) . I. S . F o r r e s t a n d A . S . Mason, Am. J . P s y c h i a t . 115, 1 1 1 4 ( 1 9 5 9 ) . J. J. Heyman, M. A l m u d e v a r a n d S . M e r l i s s Am. J. P s y c h i a t . 256 ( 1 9 5 9 ) . H. K a d i n , J . Assoc. O f f i c . Agr. C h e m i s t s 44, 7 5 1 ( 1 9 6 1 ) . M. P i n k a s o v a , C e s k . Farm. 181(1968). B. G o t h e l f a n d A . G . K a r c z m a r , I n t e r n . J. N e u r o p h a r m a c o l . 2, 9 5 ( 1 9 6 3 ) . P. Haux a n d S . N a t e l s o n , Am. J . C l i n . P a t h . , 53, 77 ( 1 9 7 0 ) . J . H. Heyman, B. Bayne a n d S . M e r l i s , Am. J . P s y c h i a t . 116, 1 1 0 8 ( 1 9 6 0 ) . P . R a j e s w a r a n a n d P. L . K i r k , B u l l e t i n on N a r c o t i c s 3,No. 3 , 1 ( 1 9 6 1 ) C . A . 56, 11705b ( 1 9 6 2 ) . S . L. T o m p s e t t , A c t a p h a r m a c o l . e t . toxicol. 303 ( 1 9 6 8 ) . M . A . Mahju a n d R . P . M a i k e l , Biochem.

21,

40. 41.

42. 43. 44. 45,

46. 47. 48. 49. 50.

51. 52. 53. 54.

55. 56.

z,

-

116,

17,

2,

KLAUS FLOAEY

18,

57.

Pharmacol. 2701. C. N. A n d r e s , J. A s s . O f f i c . A n a l . Chem. 51,1020 (1968) a n d i b i d 53,834 ( 1 9 7 0 ) . C. K o r c z a k - F a b i e r k i e w i c z a n d G. H. W. L u c a s , Proc. Can. SOC. F o r e n s i c S c i . 4, 223 (1965) C . A . 63, 12972g (1965). D. L. S o r b y , E. M. P l e i n a n d J. D. Beumaman, J. Pharm. S c i . 55, 7 8 5 ( 1 9 6 6 ) . P. M. Seeman a n d H. S . B i a l y , Biochem. P h a r m a c o l . , l2, 1181 ( 1 9 6 3 ) . G. Z o g r a f i a n d I . Z a r e n d a , Biochem. P h a r m a c o l . IS, 5 9 1 ( 1 9 6 6 ) . K. S. Murthy a n d G. Z o g r a f i , J. Pharm. S c i . , 59, 1 2 8 1 ( 1 9 7 0 ) . A. F e l m e i s t e r a n d R. Schaubman, J . P h a r m . S c i . 58, 64 ( 1 9 6 9 ) . J. K r i e g l s t e i n a n d G. K u s c h i n s k y , Naunyn-Schmiedebergs A r c h , P h a r m a k o l . Exp. P a t h . 1969, 262. J. S c h w a r t z , U . S . P a t e n t 2,997,468 (1958). N. H. Coy, C. M. F a i r c h i l d , B . T . K e e l e r , p r i v a t e communication. A. I . Cohen a n d G . M. J e n n i n g s , p r i v a t e communication. C. K o r c z a k - F a b i e r k i e w i c z , C . Cimbura, J. Chromatog. 53, 413 ( 1 9 7 0 ) . B. S. F i n k l e , E. J. C h e r r y a n d D. M. T a y l o r , J. Chromatog. S c i . 9, 393 ( 1 9 7 1 ) . L

58.

59. 60. 61. 62. 63. 64.

65. 66. 67. 68. 69.

550

TRIMETHOBENZAMIDE HYDROCHLORIDE

Kenneth W . Blessel, Bruce C. R u d y , and Bernard Z. Senkowski

55 1

K. W. BLESSEL. 8 . C. RUDY, AND 8. 2. SENKOWSKI

INDEX

Analytical Profile

- Trimethobenzamide H y d r o c h l o r i d e

1.

Description 1.1 Name, Formula, M o l e c u l a r Weight 1.2 Appearance, C o l o r , Odor

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 N u c l e a r M a g n e t i c Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F l u o r e s c e n c e Spectrum Mass Spectrum 2.5 2.6 Optical Rotation 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a l Scanning C a l o r i m e t r y 2.9 Thermogravimetric A n a l y s i s 2.10 S o l u b i l i t i e s 2 . 1 1 X-ray C r y s t a l P r o p e r t i e s 2.12 D i s s o c i a t i o n C o n s t a n t

3.

Synthesis

4.

S t a b i l i t y Degradation

5.

Drug M e t a b o l i c P r o d u c t s

6.

Methods of A n a l y s i s 6.1 Elemental Analysis 6.2 Phase S o l u b i l i t y A n a l y s i s 6.3 Thin Layer Chromatographic A n a l y s i s 6.4 Direct Spectrophotometric Analysis 6.5 Fluorescence Analysis 6.6 Non-Aqueous T i t r a t i o n

7.

Acknowledgement

8.

References

552

TRIMETHOBENZAMI DE HYDROCHLORIDE

1.

Description

1.1

Name, Formula, Molecular Weight Trimethobenzamide hydrochloride is N-[p- [ 2- (dimethyl amino)ethoxy]benzyl]-3,4,5-trimethoxybenzamide monohydrochloride.

HCI /

H3C0

C21H28N205 HC1

Molecular Weight:

424.93

1.2

Appearance, Color, Odor Trimethobenzamide hydrochloride is a white crystalline powder with a slight phenolic odor. 2.

Physical Properties 2.1

Infrared Spectrum The infrared spectrum of a sample of reference standard trimethobenzamide hydrochloride is shown in Figure 1 (1). The spectrum was recorded on a sample of 1.0 mg of trimethobenzamide hydrochloride dispersed in 300 mg of KBr, using a Perkin Elmer 621 Spectrophotometer. The following assignments have been made (1): Band 3308 cm-1 2500 cm-1 region 1626 cm-l 1580 and 1496 cm-1 846 cm-l

Assignment N-H stretch Characteristic of hydrochloride of tertiary amine C=O stretch aromatic rings 2 adjacent hydrogens on phenyl ring

2.2

Nuclear Magnetic Resonance Spectrum (NMR) The NMR spectrum of a sample of trimethobenzamide hydrochloride is shown in Figure 2 (2). The spectrum was obtained by dissolving 57.2 mg in 0.5 ml of DMSO-d6 containing tetramethylsilane as the internal reference. The

553

K. W. BLESSEL, B. C. RUDY, AND B. 2. SENKOWSKI

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71:

TRIMETHOBENZAMI DE HYDROCHLORIDE

555

K. W. BLESSEL, B. C. RUDY, A N D B.

Z. SENKOWSKI

s p e c t r a l assignments a r e given i n Table I ( 2 ) .

Table I

NMX Spectral Assignments f o r Trimethobenzamide Hvdrochloride*

No.

Protons N,N-dimethyl 6-carbon of amino ethoxy group 4'-methoxy 3' and 5' methoxy

benzyl hydrogens on C1

of Each

6

2

Chemical Shift (ppm) Multiplicity Coupli,ig Constant 2.83

S

3.48

t

3.73

S

3.85

S

4.36

d

J~

-n

=

5Hz

B c r

approx. 4.3

a-carbon of amino ethoxy group C 2 and c6 hydrogens

6.95

d

C3 and C5 hydrogens

7.32

d S

c 2 ~and c6' hydrogens

2

7.31

amido hydrogen

1

approx. 9.2

hydrogen of hydrochloride

1

approx. 11.0

J ~ 6 - ~ J , J ~ =2 -8Hz ~4 J ~ 3 - ~ 6 , J ~ 4= - 8Hz ~2

t S

(broad)

*An arbitrary set of numbers was assigned to the atoms in the structure

to simplify the presentation of the data.

2.3

U l t r a v i o l e t Spectrum The u l t r a v i o l e t s p e c t r u m o f t r i m e t h o b e n z a m i d e h y d r o c h l o r i d e i s shown i n F i g u r e 3 ( 3 ) . The s p e c t r u m e x h i b i t s two maxima and one minimum i n t h e r a n g e o f 200400 nm. The maxima a r e l o c a t e d a t 212 nm ( E = 4 . 7 x l o 4 ) 556

TRIMETHOBENZAMIDE HYDROCHLORIDE

Figure 3 Ultraviolet Spectrum of Trimethobenzamide Hydrochloride

.5

.4

W 0

.3

2

a

m

E * v)

m

a

. I

NANOMETERS

551

K. W. BLESSEL, B. C. RUDY, A N D 6 . Z . SENKOWSKI

4

and 258 nm ( E = 1 . 2 x 10 ) , and t h e minimum i s a t 238-239 nm. The c o n c e n t r a t i o n of trimethobenzamide h y d r o c h l o r i d e was 0.404 mg/100 ml o f 0.1N H C 1 . 2.4

F l u o r e s c e n c e Spectrum The e x c i t a t i o n and e m i s s i o n s p e c t r a o f a sample of r e f e r e n c e s t a n d a r d t r i m e t h o b e n z a m i d e h y d r o c h l o r i d e a r e shown i n F i g u r e 4 ( 4 ) . The s p e c t r a were r e c o r d e d on methanol s o l u t i o n s of t r i m e t h o b e n z a m i d e h y d r o c h l o r i d e ( 1 mg/ml) u s i n g a Farand MK-1 r e c o r d i n g s p e c t r o f l u o r o m e t e r . E x c i t a t i o n a t 310 nm produced an e m i s s i o n s p e c t r u m h a v i n g a maximum a t 341 nm.

Mass Spectrum The low r e s o l u t i o n mass s p e c t r u m o f t r i m e t h o benzamide i s shown i n F i g u r e 5 ( 5 ) . The s p e c t r u m w a s obt a i n e d u s i n g a CEC 21-110 s p e c t r o m e t e r w i t h a n i o n i z i n g e n e r g y o f 70eV which w a s i n t e r f a c e d w i t h a V a r i a n d a t a s y s t e m 100MS. The d a t a s y s t e m a c c e p t e d t h e o u t p u t of t h e s p e c t r o m e t e r , c a l c u l a t e d t h e masses, compared t h e i r i n t e n s i t i e s w i t h t h a t o f t h e b a s e peak and p l o t t e d t h e r e l a t i v e i n t e n s i t i e s a s l i n e s of v a r i o u s h e i g h t s . The m o l e c u l a r i o n was a t m/e 388 ( f r e e b a s e ) . The two predominant c l e a v a g e p r o d u c t s o c c u r a t m/e 58, which c o r r e s p o n d s t o CH2N(CH3)2, and m / e 195, c o r r e s p o n d i n g t o t h e trimethoxybenzoyl moiety. Other c h a r a c t e r i s t i c masses a p p e a r i n g i n t h e s p e c t r u m are m / e 330, which c o r r e s p o n d s t o t h e l o s s of CH2N(CH3)2 from t h e m o l e c u l a r i o n , and m / e 317 c o r r e s p o n d i n g t o t h e l o s s o f C4H9N from t h e p a r e n t mass. The h i g h r e s o l u t i o n s p e c t r u m f u l l y c o n f i r m e d t h e r e s u l t s of t h e low r e s o l u t i o n s c a n ( 5 ) . 2.5

2.6

Optical Rotation Trimethobenzamide h y d r o c h l o r i d e e x h i b i t s no optical activity. 2.7

M e l t i n g Range The m e l t i n g r a n g e r e p o r t e d i n NF XI11 i s 187190°C when a Class I p r o c e d u r e is u s e d ( 6 ) . 2.8

D i f f e r e n t i a l Scanning Calorimetry (DSC) The DSC c u r v e f o r a sample of r e f e r e n c e s t a n d a r d trimethobenzamide h y d r o c h l o r i d e i s shown i n F i g u r e 6. The i n s t r u m e n t u s e d w a s a P e r k i n E l m e r DSC-1B u s i n g a 558

TRIMETHOBENZAMIDE HYDROCHLORIDE

Figure 4 Emission and E x c i t a t i o n S p e c t r a of Trimethobenzamide H y d r o c h l o r i d e

I

250

300

I

\

Soectrum

350 400 NANOMETERS

559

450

500

i

r

~

i

i

i

i

0

*i 0 c)

(0 0

0

% 0

" 0

n

N

0

0 N

0 N

0 N

!

>

a

?

a

u

a

?

a

0

3

'D

0

Hi-

m

K . W. BLESSEL, B. C. RUDY. A N D E. 2. S E N K O W S K I

i

560

T RI M E T HO B E NZ A M I DE HY DRO C H L OR ID E

Figure 6 DSC Curve for Trimethobenzamide Hydrochloride

I

1

I

I

A H f = I1 0 k c a l / m o l e

56 I

I

I

K. W. BLESSEL, B. C. RUDY,

A N D B. 2 . SENKOWSKI

The e x t r a p o l a t e d o n s e t of t e m p e r a t u r e program of 10°C/min. t h e m e l t i n g endotherm i s 185.8OC w i t h t h e peak a t 188.8OC. A l l t e m p e r a t u r e s are c o r r e c t e d . The AHf was c a l c u l a t e d t o b e 1 1 . 0 Kcal/mole f o r t h e m e l t i n g endotherm ( 7 ) . 2.9

Thermogravimetric A n a l y s i s (TGA) The TGA s c a n of trimethobenzamide h y d r o c h l o r i d e showed no s i g n i f i c a n t l o s s of w e i g h t up t o 23O0C a t a h e a t A c o n t i n u o u s l o s s o f w e i g h t w a s obi n g r a t e o f 10°C/min. s e r v e d between 230-5OO0C d u r i n g which range 80% of t h e sample weight was l o s t ( 7 ) . 2.10

Solubility The s o l u b i l i t y d a t a f o r a sample of r e f e r e n c e s t a n d a r d t r i n e t h o b e n z a m i d e h y d r o c h l o r i d e a t 25OC i s g i v e n i n Table I1 ( 8 ) . T a b l e I1 Solub il i t y (mg / m l )

Solvent

0.5

Petroleum E t h e r (30'-60') Diethyl Ether Water 2-Propanol 3A Alcohol Chloroform 95% E t h a n o l Benzene Methanol

0.8 >500,

1.7 14.1 19.3 53.8 1.2 139.4

2.11

X-ray C r y s t a l P r o p e r t i e s The x-ray powder d i f f r a c t i o n d a t a o b t a i n e d f o r a sample of r e f e r e n c e s t a n d a r d trimethobenzamide h y d r o c h l o r i d e a r e g i v e n i n Table I11 ( 9 ) . The o p e r a t i n g c o n d i t i o n s of t h e i n s t r u m e n t a r e g i v e n below. Instrumental Conditions: G e n e r a l E l e c t r i c Model XRD-6 S p e c t r o g o n i o m e t e r Generator: Tube t a r g e t :

50 KV, 12-112 MA Copper

5 62

TRIMETHOBENZAMIDE HYDROCHLORIDE

Cu K, = 1.542 2 Detector s l i t M.R. S o l l e r s l i t 3' B e a m s l i t 0.0007" Ni f i l t e r 4' t a k e o f f a n g l e Scan a t 0.2O 28 p e r m i nut e A m p l i f i e r g a i n - 16 c o a r s e , 8.7 f i n e Sealed proportional counter t u b e and DC v o l t a g e a t p l a t e a u P u l s e h e i g h t s e l e c t i o n EL 5 volts; Eu - Out Rate meter T . C . 4 2000 C I S f u l l s c a l e C har t s p e e d 1 i n c h p e r 5 m i nut es P r e p a r e d by g r i n d i n g a t room t empe r a t u r e

Radiation: optics:

0.1'

Goniometer: D e tec t o r :

R e co r d e r :

Samples :

T a b l e I11 I n t e r p l a n a r S p a c i n g s from Powder D i f f r a c t i o n Data f o r Trimethobenzamide H y d r o c h l o r i d e 1 2 -------a 28 dl(Ao) 1/1 28 d(Ao> I/I, 18.2 7.23 6.21 6.08 5. 86 5.64 5.33 5. 05 4.97

4.86 12.24 1 4 .2 6 14.58 15.13 15.70 1 6 .6 4 17.55 17.86 'd 2

30 6 75 35 100 19 64 12 12

31.13 31.30 31.75 32.50 34.44 35.30 35.56 36.00 36.96

= (interplanar distance)

111,

=

nh 2 Sin

2.87 2.86 2.82 2. 75 2. 60 2.54 2.52 2.49 2. 43

8 7 19 29 18 3 3 4 2

e

r e l a t i v e i n t e n s i t y ( b a s e d on h i g h e s t i n t e n s i t y o f 100)

563

K. W. BLESSEL, B. C. RUDY, A N D B. Z . SENKOWSKI

18.36 19.18 19.49 20.20 20.68 20.96 21.38 22.27 22.63 23.03 23.53 24.66 25.02 25.62 26.12 26.48 27.08 27.86 28.16 28.96 29.96 30.56

4.83 4.63 4.55 4.40 4.29 4.24 4.16 3.99 3.93 3.86 3.78 3.61 3.56 3.48 3.41 3.37 3.29 3.20 3.17 3.08 2.98 2.93

3 4 25 36 18 14 45 63 34 68 35 45 16 31 57 32 55 10 5 16 31 8

37 - 4 6 37.96 38.36 38.94 39.50 39.85 40.46 41.00 41.48 41.96 42.82 43.72 45.00 45.30 45.76 46.18 46.72 47.28 47.54 48.46 49.48 51.15

2.40 2.37 2.35 2.31 2.28 2.26 2.23 2.20 2.18 2.15 2.11 2.07 2.01 2.00 1.98 1.97 1.94 1.92 1.91 1.88 1.84 1.79

2 5 3 7 9 5 6 4 7 3 7 8 4 4 3 4 4 6 4 4 3 7

2.12

D i s s o c i a t i o n Constant The a p p a r e n t pKa o f t r i m e t h o b e n z a m i d e hydroc h l o r i d e was d e t e r m i n e d by a p o t e n t i o m e t r i c t i t r a t i o n i n The v a l u e o b t a i n e d w a s aqueous s o l u t i o n w i t h 0.02M KOH. 8 . 2 7 - 0 . 0 3 (10).

+

3.

Synthesis The r e a c t i o n s e q u e n c e shown i n F i g u r e 7 w a s u s e d t o produce 1 k - l a b e l l e d t r i m e t h o b e n z a m i d e h y d r o c h l o r i d e and i s a l s o one of t h e s y n t h e t i c r o u t e s t o produce t h i s material(l2).

4.

S t a b i l i t y Degradation Trimethobenzamide h y d r o c h l o r i d e h a s been shown t o b e s t a b l e when h e a t e d a t 100°C i n a c i d , a l k a l i n e o r n e u t r a l s o l u t i o n ( 1 2 ) . I t was a l s o found t o b e s t a b l e when s t o r e d a t room t e m p e r a t u r e € o r a p e r i o d i n e x c e s s o f f i v e y e a r s (13).

5.

Drug M e t a b o l i c P r o d u c t s A s t u d y of t h e metabolism o f trimethobenzamide hydroc h l o r i d e i n dogs h a s b e e n c a r r i e d o u t u t i l i z i n g a

5 64

TRIMETHOBENZAMI DE HYDROCHLORIDE

Figure 7 Synthesis of 1 4 C - label led Trimethobenzamide Hydrochloride

,

cn

'i-CH

2 -CH 2 -0

0

C0Cl

<

CH SOCI?

\3

/N-CH2-CH2-

CH3

CH 3

I I

I

CH 3 0 q O C H l

CH \3 CHI

565

K . W. BLESSEL, B. C. RUDY, A N D B. 2. SENKOWSKI

14C-labelled sample of the material (14). It was found that approximately 30% of the dose was excreted intact while biotransformation of part of the remainder gave the mono-N-demethylated and the N-oxide analogs of trimethobenzamide hydrochloride as shown in Figure 8 (14). 6.

Methods of Analysis

6.1

Elemental Analysis The results of an elemental analysis of a reference standard sample of trimethobenzamide hydrochloride are presented in Table IV ( 1 5 ) . Table IV Element C H

N c1

% Theory

59.36 6.88 6.59 8.34

% Found

59.28 6.80 6.66 8.34

6.2

Phase Solubility Analysis Phase solubility analyses have been carried out for trimethobenzamide hydrochloride. An example is shown in Figure 9. The solvent used was absolute ethanol and the equilibration time was 45 hours at 25OC ( 8 ) . 6.3

Thin Layer Chromatographic Analysis A thin layer chromatographic method for the

separation of trimethobenzamide hydrochloride from its possible metabolites has been developed. The adsorbent wxi silira gel G and the solvent system was ethyl acetate:ethanol: NH40H (90:10:5 ) . The solvent front is allowed to ascend for 1 5 cm The plate is then air dried and sprayed with potassium iodoplatinate detecting reagent (16). The approximate Rf values are given in Table V below.

.

Table V R -f

Compound Trimethobenzamide hydrochloride mono-N-desmethyl metabolite d i-N-desme thy1 met abo1ite 566

0.9 0.7 0.6

Figure 8 Metabolic Products of Trimethobenzamide Hydrochloride

Trimethobenzamide

N-desnothyl analog

N-oxide analog

Figure 9 c

14

1

1

1

I

1

I

I

1

I

,

I

I

1

I

7

-

-

h "

-

-

n

v

V

v

v

PHASE

SOLUBILITY

ANALYSIS

SAMPLE . T R I M E T H O B E N Z A M I D E HYDROCHLORIDE SOLVENT: ABSOLUTE ETHANOL S L O P E . 0.18 % EOUlLlBRATlON

:

m

-

<

-

4 5 H O U R S a t 25'C of

E X T R A P O L A T E D S O L U B I L I T Y : 10.96 m g / g ETHANOL

-

-

I

I

I

I

I

I

I

I

I

I

I

I

I

1

I

I

J

0 n

C 0

TRIMETHOBENZAMIDE HYDROCHLORIDE

6.4

Direct Spectrophotometric Analysis Trimethobenzamide h y d r o c h l o r i d e may b e d e t e r m i n e d d i r e c t l y i n c a p s u l e s by u t i l i z i n g t h e p r o c e d u r e which f o l lows. An amount o f c a p s u l e powder e q u i v a l e n t t o approxi m a t e l y 50 mg o f trimethobenzamide h y d r o c h l o r i d e i s weighed i n t o a 100 m l v o l u m e t r i c f l a s k . The sample i s d i s s o l v e d and d i l u t e d t o volume w i t h 0.1N H C 1 , a f t e r which i t i s f i l A 4 ml a l i q u o t o f t h e f i l t r a t e i s t r a n s f e r r e d t o a tered. 1 0 0 m l v o l u m e t r i c f l a s k and d i l u t e d t o volume w i t h 0.1N H a . The a b s o r b a n c e o f t h e s o l u t i o n i s measured a t 258 nm u s i n g The amount o f trimethobenzamide 0.1N H C 1 as a r e f e r e n c e . h y d r o c h l o r i d e i s c a l c u l a t e d by comparison t o a sample of t h e r e f e r e n c e s t a n d a r d measured i n a s i m i l a r manner ( 6 ) .

6.5

Fluorescence Analysis Trimethobenzamide h y d r o c h l o r i d e may b e d e t e r mined by way o f t h e c h a r a c t e r i s t i c f l u o r e s c e n c e of t r i methoxybenzoic a c i d which i s a p r o d u c t of h y d r o l y s i s . The h y d r o l y s i s i s maximal u s i n g 1.5N H C 1 a t 100°C f o r 1 7 h o u r s . The l i b e r a t e d t r i m e t h o x y b e n z o i c a c i d i s ext r a c t e d i n t o e t h e r , t h e e t h e r e v a p o r a t e d and t h e r e s i d u e The f l u o r e s c e n c e i s measured t a k e n up i n a pH 2 b u f f e r . a t 370 nm and 100 nm u s i n g a n a c t i v a t i n g w a v e l e n g t h o f 290 nm. The method i s a p p l i c a b l e t o t h e d e t e r m i n a t i o n o f trimethobenzamide h y d r o c h l o r i d e i n u r i n e and b l o o d a f t e r e x t r a c t i o n of t h e m a t e r i a l i n t o c h l o r o f o r m ( 1 7 ) .

6.6

Non-Aqueous T i t r a t i o n The non-aqueous t i t r a t i o n of trimethobenzamide hydrochloride with perchloric acid a s described i n NF X I 1 1 i s t h e method o f c h o i c e f o r t h e a n a l y s i s of t h e bulk material. The sample i s d i s s o l v e d i n g l a c i a l a c e t i c a c i d w i t h t h e s u b s e q u e n t a d d i t i o n of m e r c u r i c a c e t a t e T . S . and c r y s t a l v i o l e t i n d i c a t o r . The t i t r a t i o n i s performed w i t h 0 . 1 N H C 1 0 4 i n g l a c i a l a c e t i c a c i d . Each m l of O . 1 N HC104 i s e q u i v a l e n t t o 42.49 mg of thri.methobenzamide h y d m chloride (6).

7.

Acknowlegement The a u t h o r s w i s h t o acknowledge t h e R e s e a r c h Records Department of Hoffmann-La Roche I n c . f o r t h e i r h e l p i n t h e l i t e r a t u r e search f o r t h i s analytical profile.

569

K. W. BLESSEL, 6. C. RUDY, A N D 6. 2 . SENKOWSKI

8.

References 1. Hawrylyshyn, M., Hoffmann-La Roche Inc., Personal Communication. 2. Johnson, J. H., Hoffmann-La Roche Inc., Personal Communication. 3. Rudy, B., Hoffmann-La Roclie Inc., Personal Communication. 4. Boatman, J. , Hoffmann-La Roche Inc., Personal Communication. 5. Benz, W., Hoffmann-La Roche Inc., Personal Communication. 6. The NationaZ FormuZary XIII, pp. 737-740 (1970). 7. Moros, S. , Ho ffmann-La Roche Inc,, Personal Communication. 8. MacMullan, E . , Hoffmann-La Roche Inc., Personal Communication. 9. Hagel, R., Hoffmann-La Roche Inc., Personal Communication. 10. Motchane, A . , Hoffmann-La Roche Inc., Personal Communication. 11. Wineholt, R. L., Johnson, J. D., Heck, P. J. and Kaegi, H. H., Journal of LabeZZed Compounds, 5, 5 3 (1970). 12. Schmall, M., Hoffmann-La Roche Inc., Unpublished Data. 13. Clyburn, T., Hof fmann-La Roche Inc., Unpublished Data. 14. Schwartz, M. A . et al., Hoffmann-La Roche Inc., Unpublished Data. 15. Scheidl, F., Hoffmann-La Roche Inc., Personal Communication. 16. Koechlin, B., Hoffmann-La Roche Inc. , Personal Communication. 17. Koechlin, B., Hoffmann-La Roche Inc., Unpublished Data.

5 70

ADDENDA Triamcinolone: 6.6.

T i t r i m e t r i c D e t e r m i n a t i o n w i t h Lead (IV) - a c e t a t e a l s o i n t h e presence of / Triamcinolone acetonide: E. Csizer, f S . Giir6g a n d T. S z e n , M i c r o c h i m i c a A c t a 1 9 7 0 , 996.

7.

I d e n t i f i c a t i o n b y GLC: B. S . F i n k l e , E . J. C h e r r y a n d D . M. T a y l o r , J. Chromatog. S c i . 9 , 393 ( 1 9 7 1 ) . Plasma l e v e l s : M. Kusama, N. S a k a u c h i , S . Kumaoka Metab. C l i n . Exp. 2 0 , 590 ( 1 9 7 1 ) .

Triamcinolone Acetonide: 6 . 5 3 . P a r t i t i o n c o e f f i c i e n t € o r n-hexanec h l o r o f o r m - d i o x a n e w a t e r (90:10:40:5) syst e m : D . J. Weber, T . R. E n n a l s a n d H. M i t c h n e r J. Pharm. S c i . 61, 689 (1972). 7.

Plasma l e v e l s : M. Kusama, N. S a k a u c h i , S . Kumaoka Metab. C l i n . Exp. 20, 590 ( 1 9 7 1 ) .

57 1

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ERRATA A c e tohexamide V o l . 1 p. 7 F i g . Fig.

4 TGA s p e c t r u m n 3 t DTA s p e c t r u m . 5 DTA s p e c t r u m n o t TGA s p e c t r u m ,

E r y t h r omyc i n E s t o 1a t e V a l . I T?. 1 0 3 E r y t h r o m y c i n n o t E s t r o m y c i n (legenS under s t r u c t u r e )

.

Ha l o-t h a _ ne ~

p.

1 2 8 l i n e 4 : Change formlala t o : lOgl0P= 6.8513 - 1082.495 t+222.44

Leva t e r e n o 1 B i t a r t r a t e Vol.

I p.

p.

p.

1 5 3 , l i n e 8: l o n g n o t l o n e l i n e 12-14: r e v e r s e t o na = 1 . 5 3 1 2 0 . 0 0 2 nB = 1 . 5 7 7 2 0.002 ny = 1 . 6 2 0 ? 0 . 0 0 2 1 5 9 , l i n e 1 3 : d e l e t e f i r s t 1 6 9 ; also 1 5 3 ( 1 7 ) : not 1 5 3 ( 7 ) l i n e 22: d e l e t e f i r s t 169 l i n e 24-26: I n s t r u m e n t u s e d : JEOL Model IMS015SC. E i e c t r o n beam 7 5 e . v . 171, l i n e 6 : R. K. K u l l n i g

N o r t r i p t y l i n e Hydrochloride V o l . I p. 2 4 3 , l i n e 9 and 10: Bu0H-I.5%HCO2H - H 2 0

573

(12:1:7)

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CUMULATIVE INDEX Acetohexamide, 1, 1; 2, 573 Ampicillin, 2, 1 Chlorprothixene, 2,63 Chloral Hydrate, 2, 85 Chlordiazepoxide, 1, 15 Chlordiazepoxide Hydrochloride, I , 39 Clidinium Bromide, 2, 145 Cycloserine, 1, 53 Cyclothiazide, 1 , 66 Dexamethasone, 2 , 163 Diazepam, 1, 79 Dioctyl Sodium Sulfosuccinate, 2 , 199 Erythromycin Estolate, 1 , 101; 2, 573 Fluorouracil, 2 , 22 1 Fluphenazine Enanthate, 2 , 2 4 5 Fluphenazine Hydrochloride, 2, 263 Halothane, 1 , 119; 2 , 5 7 3 Isocarboxazid, 2 , 295 Isopropamide, 2 , 3 15 Levallorphan Tartrate, 2, 339 Levaterenol Bitartrate, 1, 149; 2, 573 Meperidine Hydrochloride, 1 , 175 Meprobamate, I , 209

Methyprylon, 2, 363 Nortriptyline Hydrochloride, 1 , 2 3 3 ; 2 , 573 Phenelzine Sulfate, 2, 383 Potassium Phenoxymethyl Penicillin, 1, 249 Primidone, 2, 409 Propiomazine Hydrochloride, 2 , 4 3 9 Propoxyphene Hydrochloride, 1,301 Sodium Cephalothin, I , 319 Sodium Secobarbital, 1, 343 Sulfamethoxazole, 2 , 4 6 7 Sulfisoxazole, 2, 487 Triamcinolone, 1 , 3 6 7 ; 2 , 5 7 1 Triamcinolone Acetonide, 1,397;2,571 Triamcinolone Diacetate, I , 423 Triclobisonium Chloride, 2, 507 Triflupromazine Hydrochloride, 2 , 523 Trimethobenzamide Hydrochloride, 2 , 55 1 Vinblastine Sulfate, 1 , 4 4 3 Vincristine Sulfate, 1 , 4 6 3

575

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