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Metabolism of tertiary arylaliphatic amines and formamides in rats Slatter, John Gregory 1987

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METABOLISM OF TERTIARY ARYLALIPHATIC AMINES AND FORMAMIDES IN RATS by JOHN GREGORY SLATTER BSc.(Hons.), Lakehead U n i v e r s i t y , 1977 MSc., U n i v e r s i t y of B r i t i s h Columbia, 1983 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES F a c u l t y of Pharmaceutical S c i e n c e s D i v i s i o n of Pharmaceutical Chemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA November 1987 © J o h n Gregory S l a t t e r , l 9 8 7 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 26 -y^-^J i i ABSTRACT The m e t a b o l i t e s of the b a s i c t e r t i a r y a r y l a l i p h a t i c amine N,N,a-trimethyl-7-phenylbenzenepropanamine ( R e c i p a v r i n R ) from male Wistar r a t s were c h a r a c t e r i z e d by gas chromatography-mass spectrometry (GCMS). The work was undertaken i n an attempt to determine the source of a novel m e t a b o l i t e , N-(1-methyl-3,3-d i p h e n y l p r o p y l ) formamide. The formamide m e t a b o l i t e was i s o l a t e d from the b i l e of r e c i p a v r i n dosed r a t s only a f t e r h y d r o l y s i s with the enzyme /"-glucuronidase, s u g g e s t i n g that i t arose from a g l u c u r o n i d e conjugated p r e c u r s o r . R e c i p a v r i n was chosen f o r the study based on s t r u c t u r a l s i m i l a r i t y to the n a r c o t i c a n a l g e s i c methadone which was shown to g i v e r i s e to a s i m i l a r m e t a b o l i t e , 6-formamido-4,4-diphenyl-3-heptanone. The secondary formamide was not a p l a u s i b l e c a n d i d a t e f o r a /^-glucuronidase l i b e r a t e d m e t a b o l i t e of r e c i p a v r i n , s u g g e s t i n g that a l a b i l e aglycone was r e s p o n s i b l e f o r the GCMS o b s e r v a t i o n of the formamide m e t a b o l i t e . L a b i l e isomeric compounds, a-methyl-(N-methylene)-7-phenylbenzenepropanamine N-oxide, N-(a-methyl-7-phenylbenzenpropylidene) methylamine N -oxide, and 2-(4',4'-diphenyl-but-2'-yl) o x a z i r i d i n e were s y n t h e s i z e d as p o s s i b l e p r e c u r s o r s of the formamide. N -hydroxy-a-methyl-7-phenylbenzenepropanamine, and N-hydroxy-N,a-dimethyl-7-phenylbenzenepropanamine were s y n t h e s i z e d as c a n d i d a t e s f o r l a b i l e /"-glucuronidase l i b e r a t e d aglycone p r e c u r s o r s of the n i t r o n e s . The b i l i a r y nonconjugated and conjugated m e t a b o l i t e s of r e c i p a v r i n were c h a r a c t e r i z e d i n d e t a i l . In a d d i t i o n to the formamide, 15 d i f f e r e n t m e t a b o l i t e s r e p r e s e n t i n g the N-d e a l k y l a t i o n , o x i d a t i v e deamination, N - o x i d a t i o n and phenyl r i n g o x i d a t i o n pathways were i d e n t i f i e d by GCMS. To determine i f thermal decomposition of the methylene n i t r o n e i n the GC i n l e t was r e s p o n s i b l e f o r the GCMS o b s e r v a t i o n of the formamide m e t a b o l i t e , l i q u i d chromatography-mass spectrometry (LCMS) was used t o show that the formamide and not the isomeric methylene n i t r o n e was present i n b i l e p r i o r t o GCMS a n a l y s i s . Although the s y n t h e t i c methylene n i t r o n e was shown to degrade i n the GC i n l e t to the formamide, the LCMS experiment r u l e d out the thermal g e n e r a t i o n of the b i l i a r y formamide from a n i t r o n e p r e c u r s o r . The nonconjugated and conjugated m e t a b o l i t e s of the r e c i p a v r i n m e t a b o l i t e , n o r r e c i p a v r i n were c h a r a c t e r i z e d i n d e t a i l by GCMS. Since the secondary formamide m e t a b o l i t e was observed i n the / 3-glucuronidase h y d r o l y z e d b i l e e x t r a c t , n o r r e c i p a v r i n was i m p l i c a t e d as an int e r m e d i a t e i n the b i o t r a n s f o r m a t i o n of r e c i p a v r i n to the formamide. The p o s s i b i l i t y of sol v e n t mediated f o r m y l a t i o n or f r e e r a d i c a l o x i d a t i o n of d e s a l k y l m e t a b o l i t e s to a f f o r d the formamides was r u l e d out. The methylene n i t r o n e was shown to a f f o r d the formamide m e t a b o l i t e under simulated workup c o n d i t i o n s . An a l k a l i c a t a l y z e d Beckmann rearrangement of n i t r o n e to amide was used to account f o r t h i s t r a n s f o r m a t i o n . The secondary hydroxylamine was shown t o give r i s e t o the methylene n i t r o n e under simulated workup c o n d i t i o n s . I t was concluded that the o x i d a t i o n of a ^- g l u c u r o n i d a s e l i b e r a t e d secondary hydroxylamine m e t a b o l i t e to the methylene iv nitrone followed by Beckmann rearrangement of the nitrone to the formamide was the probable source of the formamide observed by GCMS in extracts of b i l e from recipavrin dosed r a t s . The metabolism of N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide was investigated in d e t a i l to determine whether the carbinolamide, N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl) formamide was involved in the genesis of the formamide metabolite of r e c i p a v r i n . The above carbinolamide and N-(1-hydroxy-1-methyl-3,3-diphenylpropyl) formamide were i d e n t i f i e d by GCMS along with 16 other metabolites representing the metabolic pathways N-deformylation, N-dealkylation, N-oxidation and phenyl ring oxidation. The carbinolamides were not found in b i l e from re c i p a v r i n dosed rats, ruling out the p o s s i b i l i t y of a carbinolamide glucuronide precursor of the recipavrin formamide metabolite. This was the f i r s t report of the i s o l a t i o n of stable dealkylation intermediates of a high molecular weight formamide. The hepatotoxicity of the anticancer agent N-methyl formamide and the solvent dimethylformamide, suggests that the recipav r i n formamides could also be metabolized to toxic carbinolamide or glutathione related metabolites. Dr. F.S. Abbott, Research Supervisor. V T A§LE_OF_CONTENTS ABSTRACT i i TABLE OF CONTENTS v LIST OF FIGURES x v i LIST OF TABLES x x v i LIST OF ABBREVIATIONS x x v i i i ACKNOWLEDGEMENTS x x x i DEDICATION x x x i i I . INTRODUCTION 1 1. OBSERVATION OF A FORMAMIDE METABOLITE OF METHADONE 1 2. RECIPAVRIN: A STRUCTURAL ANALOGUE OF METHADONE 2 3. OVERVIEW OF PREVIOUS WORK ON RECIPAVRIN (SLATTER, 1983) 4 4. METABOLISM OF TERTIARY ARYLALIPHATIC AMINES 8 5. FORMAMIDE METABOLITES OF AMINES AND AMIDES 9 6. POSSIBLE SOURCES OF FORMAMIDE METABOLITES OF METHADONE (8) AND RECIPAVRIN (9) 11 A. Formamide a r t i f a c t s a r i s i n g from m e t a b o l i t e e x t r a c t i o n u s i n g o r g a n i c s o l v e n t s 11 B. Formamide a r t i f a c t s a r i s i n g from o x i d a t i o n of amine m e t a b o l i t e s d u r i n g sample i s o l a t i o n 13 C. Formamide m e t a b o l i t e s a r i s i n g by p e r o x i d a t i o n of i m i n i u m d e a l k y l a t i o n i n t e r m e d i a t e s 13 D. Formamide m e t a b o l i t e s a r i s i n g by d e c o m p o s i t i o n of N - o x i d i z e d m e t a b o l i t e s 16 i . H y d r o x y l a m i n e m e t a b o l i t e s as p r e c u r s o r s of n i t r o n e s 16 i i . N i t r o n e s as drug m e t a b o l i t e s 17 i i i . N i t r o n e s as c h e m i c a l p r e c u r s o r s of formamide m e t a b o l i t e s : Rearrangements of N i t r o n e s 18 a. Behrend Rearrangement 18 v i b. Martynoff Rearrangement 19 c. Beckmann Rearrangement 19 d. Photochemical rearrangements 20 E. Formamide m e t a b o l i t e s a r i s i n g from a ca r b i n o l a m i d e p r e c u r s o r 23 i . S t a b i l i t y and occurrence of car b i n o l a m i n e and ca r b i n o l a m i d e s i n t e r m e d i a t e s i n drug metabolism s t u d i e s 23 i i . Metabolism of formamides 25 i i i . Importance of metabolic s t u d i e s on a r y l a l i p h a t i c formamides 25 7. OBJECTIVES OF THE THESIS 26 I I . E X P E R I M E N T A L 28 1. CHEMICALS AND MATERIALS 28 2. ANIMAL EXPERIMENTS 31 3. PREPARATION OF TLC SPRAY REAGENTS 32 4. INSTRUMENTATION 33 A. NMR s p e c t r a 33 B. I n f r a r e d s p e c t r a 33 C. U l t r a v i o l e t s p e c t r a 33 D. M e l t i n g p o i n t s 33 E. C a p i l l a r y GCMS 34 F. D i r e c t i n s e r t i o n probe mass s p e c t r a 35 G. Packed column GCMS a n a l y s i s 36 H. L i q u i d chromatography-mass spectrometry 37 I. High r e s o l u t i o n mass spectrometry 38 5. METABOLISM EXPERIMENTS 39 A. Animal Surgery and Drug A d m i n i s t r a t i o n 39 B. B i l e sample p r e p a r a t i o n f o r GCMS 40 C. D e r i v a t i z a t i o n Methods 41 v i i 6. CHEMICAL SYNTHESES 42 A. F l a s h chromatography. 42 B. S y n t h e t i c compounds r e l a t e d to methadone 42 i . General procedure f o r meta-chloroperbenzoic a c i d (MCPBA) o x i d a t i o n s of methadone (8) and EDDP (1) 42 i i . GCMS i d e n t i f i c a t i o n of 6-N-methylformamido -4,4-diphenyl-3-heptanone (Methadone t e r t i a r y formamide (49)) 44 i i i . S y n t h e s i s of 2,3-dimethyl-5,5-diphenylcyclopent-2-enone (50) 44 C. S y n t h e t i c compounds r e l a t e d to r e c i p a v r i n 45 i . 1,1-Diphenyl-3-butanone (14) (CAS r e g i s t r y 5409-60-9) ( S l a t t e r , 1983) 45 i i . S y n/anti 1,1-diphenyl-3-butanone oxime (19) (CAS r e g i s t r y 36317-57-4)(Slatter, 1983) 45 i i i . N,N,a-trimethyl-7-phenylbenzenepropanamine h y d r o c h l o r i d e ( R e c i p a v r i n R (9)) (CAS 13957 -55-6) ( S l a t t e r , 1983) 46 i v . N,N,a-trimethyl-7-phenylbenzenepropanamine N-oxide ( r e c i p a v r i n N-oxide (53)) 47 v. a-Methyl-7-phenylbenzenepropanamine (CAS r e g i s t r y 29869-77-0) ( d i n o r r e c i p a v r i n (20)) ( S l a t t e r , 1 983) 48 v i . N-hydroxy-a-methyl-7-phenylbenzene-propanamine (primary hydroxylamine (22)) ( S l a t t e r , 1983) 48 v i i . S p e c t r a l c h a r a c t e r i z a t i o n of the h y d r o x y l -amine a u t o x i d a t i o n product (56) 50 v i i i . N,a-dimethyl-7-phenylbenzene-propanamine ( n o r r e c i p a v r i n (15)) (CAS 29869-78-1 ) ( S l a t t e r , 1983) 50 i x . N-hydroxy-N,a-dimethyl-7-phenylbenzene-propanamine (secondary hydroxylamine (17)) ( S l a t t e r , 1983) 51 x. u-Methyl-(N-methylene)-7-phenylbenzene-propanamine N-oxide (methylene n i t r o n e (24)) ( S l a t t e r , 1983) 52 v i i i x i . Cis/trans N-(a-methyl-7-phenylbenzene-propylidene) methylamine-N-oxide (methyl nitrone (44)) 53 x i i . a-Methyl-(N-methylene)-7-phenylbenzene-propanamine (polymer) (methylene imine (23) as triazane (41)) (Slatter, 1983) 54 x i i i . 2-(4',4'-Diphenyl-but-2'yl) oxaziridine (25) (Slatter , 1983) 56 xiv. N-(1-methyl-3,3-diphenylpropyl) formamide (12) ( S l a t t e r , 1983) 57 xv. Characterization of the Leuckart s p e c i f i c byproduct 4-(2,2-diphenylethyl) pyrimidine (59) 60 x v i . N-(1-methyl-3,3-diphenylpropyl) formohydroxamic acid (61) 60 x v i i . N-methyl -N-(1-methyl-3,3-diphenylpropyl) formamide (26) 61 xvi i i . N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl) formamide (carbinolamide (47)) 63 xix. N-(1-methyl-3,3-diphenylpropyl) acetamide (63) 64 xx. N-(1-methyl-3,3-diphenylpropyl) ethanimine (64) 64 x x i . 3-Carbylamino-1,1-diphenylbutane (recipavrin isocyanide (65)) 65 x x i i . N-(1-methyl-3,3-diphenylpropyl) carbamic acid methyl ester (dinorrecipavrin methylcarbamate (66)) 65 x x i i i . N-(1-methyl-3,3-diphenylpropyl) carbamic acid ethyl ester (dinorrecipavrin ethylcarbamate (67)) 66 xxiv. N-methyl -N-(1-methyl-3,3-diphenylpropyl) carbamic acid methyl ester (norrecipavrin methyl carbamate, (68)) 67 xxv. N-methyl-N-(1-methyl-3,3-diphenylpropyl) carbamic acid ethyl ester (norrecipavrin ethyl carbamate (69)) 68 D. Synthetic Compounds Related to Promethazine 68 i . I0~(2-Propynyl) phenothiazine (70) 69 ix i i . lO-(2-Propanone) phenothiazine (71) 69 i i i . 10-(2-formamidopropyl) p h e n o t h i a z i n e (72) 69 i v . Leuckart s p e c i f i c byproduct, 4-(1 -(10-p h e n o t h i a z i n y l ) methyl) p y r i m i d i n e (73) 71 v. lO-(2-Formamidopropyl) p h e n o t h i a z i n e N l 0 -oxide (75) 71 v i . 10-(N-methyl-2-formamidopropyl) p h e n o t h i a z i n e (74) 73 v i i . 10-(N,N-dimethyl-N-oxo-2-aminopropyl) p h e n o t h i a z i n e (promethazine N-oxide (76), Clement and Beckett, 1981b) 74 v i i i . C h a r a c t e r i z a t i o n of promethazine f r e e base (10) 75 E . S y n t h e t i c compounds r e l a t e d to amphetamine 76 i . a-Methyl- (N-methylene)-/3-phenylbenzene-ethanamine (78) 76 i i . 2-(3'-phenylprop-2'-yl) o x a z i r i d i n e (82) 77 i i i . N-(1-methyl-2-phenylethyl) formamide (83) (CAS r e g i s t r y 42044-69-9) 78 7. EXPERIMENTS TO DETERMINE THE SOURCE OF FORMAMIDE METABOLITES OF METHADONE AND RECIPAVRIN 79 A. Solvent r e l a t e d a r t i f a c t c o n t r o l experiments f o r methadone m e t a b o l i t e s 79 B. E f f e c t of added s y n t h e t i c secondary formamide on peak shape and r e t e n t i o n time of the r e c i p a v r i n formamide m e t a b o l i t e (12) 80 C. Experiments to e s t a b l i s h that the secondary formamide m e t a b o l i t e (12) of r e c i p a v r i n a r i s e s from a g l u c u r o n i d e p r e c u r s o r 81 i . S u l f a t a s e h y d r o l y s i s of the r e c i p a v r i n conjugated m e t a b o l i t e s 81 i i . C o n t r o l i n c u b a t i o n of r e c i p a v r i n conjugated f r a c t i o n without ^-glucuronidase enzyme 82 D. Free r a d i c a l o x i d a t i o n of r e c i p a v r i n and n o r r e c i p a v r i n as a source of the secondary formamide (12) 82 X i . Incubation of recipavrin (9) with blank b i l e under simulated workup conditions 82 i i . Incubation of norrecipavrin with blank b i l e under simulated workup conditions 83 E. E f f e c t of extraction pH on the observation of the recipavrin secondary formamide metabolite (12) 83 F. E f f e c t of immediate sample preparation and storage on the secondary formamide metabolite of r e c i p a v r i n 83 G. Solvent related a r t i f a c t control experiments for recipavrin metabolites 84 H. S o l i d phase extraction (SPE) of recipavrin metabolites 84 i . Cartridge conditioning and sample el u t i o n (J.T. Baker Ltd., 1982) 85 a. Reversed phase octadecyl (RPC 1 8) cartridges 85 b. Forward phase s i l i c a gel (FPSiC^) cartridges 85 i i . Amberlite XAD-2 column preparation 86 i i i . Preliminary s o l i d phase extraction experiments 86 a. Elution of synthetic reference compounds from RPC^ columns 86 b. RPC 1 8 test elutions using UV and TLC detection of eluates 87 i v . Fractionation of b i l e components by SPE methods 87 a. Attempted fractionation of b i l i a r y recipavrin metabolites using RPC^ columns 87 b. Preliminary cleanup and deionization of b i l e using a XAD-2 column 88 c. Separation of a l l metabolites from extremely polar b i l e constituents 88 d. Separation of nonconjugated metabolites on FPSiC>2 cartridges 89 x i I. Attempts t o i n c r e a s e secondary formamide (12) p r o d u c t i o n by the a d d i t i o n of formic a c i d , formaldehyde, or formaldehyde and hydrogen perox i d e d u r i n g sample p r e p a r a t i o n 90 J . Attempts to decrease secondary formamide (12) p r o d u c t i o n with the use of a n t i o x i d a n t s and a formaldehyde complexing agent 92 K. E f f e c t of the a n t i o x i d a n t BHT, formic a c i d and formaldehyde on d i n o r r e c i p a v r i n m e t a b o l i t e p r o f i l e s 93 L. Decomposition of r e c i p a v r i n N-oxide (53) under si m u l a t e d workup c o n d i t i o n s 93 M. GCMS a n a l y s i s and decomposition of the methylene n i t r o n e (24) 94 I I I . RESULTS AND DISCUSSION 96 1. FINAL EXPERIMENTS CONCERNING THE SYNTHESIS AND POSSIBLE SOURCES OF THE METHADONE FORMAMIDE METABOLITE 96 A. Mechanisms f o r the p e r o x i d a t i o n of EDDP to an o x a z i r i d i n e , diketone and r e l a t e d compounds 96 i . High r e s o l u t i o n mass spectrum of the methadone o x a z i r i d i n e (2) 97 i i . MCPBA o x i d a t i o n of methadone HCI 97 i i i . P e r o x i d a t i o n of EDDP f r e e base i n the presence of suspended K2C03 99 i v . Mechanism f o r the p e r o x i d a t i o n of EDDP 103 B. Solvent c o n t r o l experiments i n the i s o l a t i o n of the methadone formamide m e t a b o l i t e 107 C. C o n c l u s i o n s r e g a r d i n g the source of the methadone formamide m e t a b o l i t e 110 2. SYNTHESIS AND CHARACTERIZATION OF SYNTHETIC COMPOUNDS RELATED TO RECIPAVRIN 110 A. S y n t h e s i s and C h a r a c t e r i z a t i o n of the amines 111 B. S y n t h e s i s and C h a r a c t e r i z a t i o n of the amides 111 C. S y n t h e s i s and C h a r a c t e r i z a t i o n of oximes, hydroxylamines and t h e i r o x i d a t i o n products 122 D. S y n t h e s i s and C h a r a c t e r i z a t i o n of the r e c i p a v r i n N-oxide 127 x i i E. S y n t h e s i s and C h a r a c t e r i z a t i o n of the N i t r o n e s 127 F. S y n t h e s i s and C h a r a c t e r i z a t i o n of the methylene imine (23) (polymer) and o x a z i r i d i n e (25) 128 G. S y n t h e s i s and C h a r a c t e r i z a t i o n of the i s o c y a n i d e (65) and carbamates (66-69) 133 H. Attempted s y n t h e s i s and c h a r a c t e r i z a t i o n of the formohydroxamic a c i d (61) 134 I . S y n t h e s i s and C h a r a c t e r i z a t i o n of the promethazine r e l a t e d formamides and t h e i r o x i d a t i o n products ..137 J . S y n t h e s i s and C h a r a c t e r i z a t i o n of the o x a z i r i d i n e (82), methylene imine (78) and formamide (83) r e l a t e d to amphetamine 149 3. MASS SPECTROMETRY OF RECIPAVRIN RELATED COMPOUNDS 1 54 A. Fragmentation of the a l i p h a t i c s i d e c h a i n 154 B. Fragmentation of the s u b s t i t u t e d diphenylbutane moiety 161 4. METABOLISM OF RECIPAVRIN 163 A. B i l i a r y m e t a b o l i t e s of r e c i p a v r i n 174 i . GCMS o b s e r v a t i o n of the i n t a c t formamide 174 i i . LCMS Demonstration of the secondary formamide m e t a b o l i t e 180 i i i . O x i d a t i v e deamination 183 i v . N - d e a l k y l a t i o n 184 v. Phenyl r i n g o x i d a t i o n 187 v i . N - o x i d a t i o n 188 B. Nonconjugated b i l i a r y m e t a b o l i t e s of r e c i p a v r i n 191 C. R e c i p a v r i n metabolism c o n c l u s i o n s 191 5. METABOLISM OF NORRECIPAVRIN 194 A. Conjugated m e t a b o l i t e s 194 B. Noncon jugated m e t a b o l i t e s 201 x i i i C. Conclusions regarding norrecipavrin metabolism 201 6. METABOLISM OF DI NORRECI PAVRIN 204 A. Metabolites of dinorrecipavrin 2 0 4 7. EXPERIMENTS TO DETERMINE THE SOURCE OF THE FORMAMIDE METABOLITE 2 0 9 A. D i l u t i o n of b i l e extracts with the synthetic secondary formamide ( 1 2 ) 2 0 9 B. Attempted detection of chloroform generated a r t i f a c t s 2 1 2 C. Experiments to es t a b l i s h that the secondary formamide metabolite of recipavrin arises from a glucuronide precursor 214 i . Sulfatase hydrolysis of the conjugated metabolites 214 i i . Control incubation of the recipavrin metabolite conjugated fracti o n without 0-glucuronidase enzyme 214 D. Free r a d i c a l oxidation of recipavrin ( 9 ) and norrecipavrin ( 1 5 ) as a source of the formamide ( 1 2 ) 2 1 6 i . Incubation of recipavrin with control b i l e under simulated workup conditions 2 1 6 i i . Incubation of norrecipavrin with blank b i l e under simulated workup conditions 2 1 7 i i i . E f f e c t of extraction pH on the observation by GCMS of the recipavrin secondary formamide metabolite 2 1 8 E. S o l i d phase extraction of recipavrin metabolites 2 1 8 i . Preliminary experiments 2 1 8 a. Elution of synthetic reference compounds from RPC 1 8 columns 2 1 8 b. RPC 1 8 test elutions using UV and TLC detection of eluates 2 1 9 i i . Fractionation of b i l e components by SPE methods 2 2 0 x i v a. Attempted f r a c t i o n a t i o n of b i l i a r y r e c i p a v r i n m e t a b o l i t e s using R P C 1 8 columns 220 b. P r e l i m i n a r y cleanup and d e i o n i z a t i o n of b i l e u s i n g a XAD-2 column 221 c. F r a c t i o n a t i o n on F P S i 0 2 columns 221 d. S e p a r a t i o n of nonconjugated m e t a b o l i t e s on the F P S i 0 2 column 222 e. C o n c l u s i o n s regarding the use of SPE c a r t r i d g e s 226 F. Attempts t o i n c r e a s e secondary formamide (12) p r o d u c t i o n by the a d d i t i o n of formic a c i d , formaldehyde, or formaldehyde and hydrogen perox i d e d u r i n g sample p r e p a r a t i o n 226 i . Noncon jugated f r a c t i o n 227 i i . Conjugated f r a c t i o n 230 G. Attempts t o decrease formamide p r o d u c t i o n with the use of a n t i o x i d a n t s and a formaldehyde complexing reagent 232 8. METABOLISM OF PROMETHAZINE (10): SEARCH FOR FORMAMIDE METABOLITES 234 9. DECOMPOSITION OF N-OXIDIZED METABOLITES AS A SOURCE OF THE SECONDARY FORMAMIDE (12) ISOLATED FROM RAT BILE 242 A. Decomposition of r e c i p a v r i n N-oxide under si m u l a t e d sample workup c o n d i t i o n s 242 B. GCMS a n a l y s i s and thermal decomposition of the methylene n i t r o n e 247 C. A l k a l i c a t a l y z e d rearrangement of n i t r o n e s to amides 255 10. METABOLISM OF THE RECIPAVRIN METHYLENE NITRONE 256 11. METABOLISM OF THE RECIPAVRIN TERTIARY FORMAMIDE (26) 261 A. Mass spectrometry of a r y l a l i p h a t i c formamides and c a r b i n o l a m i d e s 270 B. Metabolism of the t e r t i a r y a r y l a l i p h a t i c formamide 278 i . N - d e f o r m y l a t i o n 278 X V i i . Phenyl r i n g o x i d a t i o n and c o n j u g a t i o n 280 i i i . N - d e a l k y l a t i o n v i a car b i n o l a m i d e s t o d e s a l k y l formamides 281 12. METABOLISM OF THE SECONDARY FORMAMIDE (12) 283 IV. SUMMARY AND CONCLUSIONS 292 V . REFERENCES 298 V I . APPENDIX 314 xvi LIST OF FIGURES Figure 1. (top) Products derived from the chemical oxidat ion of EDDP (1). (bottom) Structure of methadone (8), r ec ipavr in (9), promethazine (10), dimethylamphetamine (11), and the formamide metabolite (12) of r e c i p a v r i n . 3 Figure 2. Pre l iminary syntheses of po ten t ia l N- and alpha C - ox id ized metabolites of r ec ipavr in (9). 5 Figure 3. Superimposed LCMS ion chromatograms of the M +1 m/z 254 ion of the synthetic methylene ni trone (24), oxaz ir id ine (25) and formamide (12). 7 Figure 4. Structures of parent drug and formamide metabolites of prenylamine (27), aminopyrine (28), ca f fe ine (29), amantidine (30), mianserin (31), indecainide (32), pipemidic ac id (33), aminoanthraquinone (34), 2-naphthylamine (35), ch lor to luon (36), e t c . 10 Figure 5. Schematic showing poss ible metabolic (m) and chemical (c) transformations that could account for a secondary formamide metabolite (12) in the /^-glucuronidase hydrolyzed f rac t ion of b i l e from r e c i p a v r i n dosed r a t s . 15 Figure 6. Ionic mechanisms for the cleavage of C-O or N-0 bonds in the oxaz ir id ine r ing to a f ford ni trones or amides respec t ive ly . 16 Figure 7. Back p o l a r i z a t i o n between two canonical forms of a n i t r o n e . 18 Figure 8. Behrend rearrangement of a N-methyl n i trone to a methylene n i trone . 19 Figure 9a. Mechanism for the Beckmann rearrangement of n i trones to amides catalyzed by a c y l a t i n g agents (Lamchen, 1968). 21 Figure 9b. Mechanism for the Beckmann rearrangement of n i trones to amides catalyzed by a l k a l i (Lamchen, 1968, Zinner , 1978). 21 Figure 10. A modified Beckmann rearrangement of a hypothet ica l nitrone glucuronide (46) catalyzed by ^-glucuronidase (after Lamchen, 1968). 22 Figure 11. Free r a d i c a l mechanism for the photochemical rearrangement of a ni trone to an o x a z i r i d i n e (Lamchen, 1968). 23 F i g u r e 12. Metabolism of r e c i p a v r i n (9) to the secondary formamide (12) v i a a h y p o t h e t i c a l c a r b i n o l a m i d e pathway. F i g u r e 13. (top) Composite of high r e s o l u t i o n mass s p e c t r a l r e s u l t s f o r the methadone o x a z i r i d i n e (2) (source temperature 120o). (bottom) Mass spectrum of the methadone formamide (6) a r i s i n g from GCMS a n a l y s i s of the methadone o x a z i r i d i n e . F i g u r e 14. Mass spectrum of the t e r t i a r y formamide (49) formed by MCPBA o x i d a t i o n of methadone HCl. M + 323 absent. F i g u r e 15. P o s s i b l e r e g i o i s o m e r i c cyclopentenones (50 and 51) expected from the a l d o l condensation of the d i k e t o n e . F i g u r e 16. NMR spectrum of the cyclopentenone a l d o l product 2,3-dimethyl-5,5-diphenylcyclopent-2-enone (50) . F i g u r e 17. B a y e r - V i l l i g e r MCPBA a d d i t i o n to C=N+ bonds i n the f i r s t step of the p e r o x i d a t i o n of EDDP ( 1 ) . F i g u r e 18. P o s s i b l e mechanisms f o r the formation of the o x i d a t i o n products of EDDP that were observed by GCMS. F i g u r e 19. Mechanism f o r the formation of DDP (4) from o x i d a t i o n of the enamine tautomer of EDDP. F i g u r e 20. 400 MHz NMR spectrum of the secondary formamide (12). F i g u r e 21. 75 MHz broad band decoupled 1 3 C NMR spectrum of the secondary formamide (12). F i g u r e 22. I n f r a r e d spectrum of a n u j o l mull of the secondary formamide (12). NH s t r e t c h e s 3260 ( t r a n s ) , 3200 (shoulder, c i s ) . C=0 s t r e t c h e s 1670 ( t r a n s ) , 1650 ( c i s ) . F i g u r e 23. (top) Mass spectrum of the secondary formamide (12)(M + 253), (bottom) Mass spectrum of the secondary formamide mono TMS d e r i v a t i v e (60)(M + 325). F i g u r e 24. 270 MHz NMR spectrum of the p y r i m i d i n e byproduct (59) of formamide s y n t h e s i s . F i g u r e 25. 300 MHz 1H NMR spectrum of the t e r t i a r y formamide (26). X V I 1 1 F i g u r e 26. 75 MHz 1 3 C NMR spectrum of the t e r t i a r y formamide (26). Top. Broad band decoupled. Bottom. Attached proton t e s t . 120 F i g u r e 27. I n f r a r e d spectrum of a t h i n f i l m of the t e r t i a r y formamide (26). C=0 s t r e t c h at 1666 cm - 1. 121 F i g u r e 28. 300 MHz NMR spectrum of the primary hydroxylamine (22). 124 F i g u r e 29. I n f r a r e d spectrum of a t h i n f i l m of the primary hydroxylamine (22). 125 F i g u r e 30. Mass s p e c t r a of the u n d e r i v a t i z e d primary (22 top) and secondary (17 bottom) hydroxylamines. 126 F i g u r e 31. 300 MHz 1H NMR spectrum of the N-methyl n i t r o n e (44). 129 F i g u r e 32. 100 MHz 1 3 C NMR spectrum of the N-methyl n i t r o n e (44). 130 F i g u r e 33. Mass spectrum of the suspected methyl ether of the formohydroxamic a c i d (61). 136 F i g u r e 34. 400 MHz 1H NMR spectrum of the secondary formamide (72). 140 F i g u r e 35. 100 MHz 1 3 C NMR SFORD spectrum of the secondary formamide (72). 141 F i g u r e 36. FTIR spectrum ( f i l m ) of secondary formamide (72). 142 F i g u r e 37. 400 MHz 1H NMR spectrum of the secondary formamide N« n-oxide (75) showing only one rotamer i s p r e s e n t . 143 F i g u r e 38. 100 MHz broad band decoupled 1 3C NMR spectrum of the secondary formamide N i o ~ o x i d e (75). 144 F i g u r e 39. FTIR spectrum ( f i l m ) of the secondary formamide N l 0 ~ o x i d e (75). 145 F i g u r e 40. 400 MHz 1H NMR spectrum of the t e r t i a r y formamide (74). 146 F i g u r e 41. IR spectrum of a t h i n f i l m of the t e r t i a r y formamide (74). 147 F i g u r e 42. Mass spectrum of the t e r t i a r y formamide (74). 148 XIX F i g u r e 43. 80 MHz NMR spectrum of the amphetamine methylene imine d e r i v e d polymers (79-81). 151 F i g u r e 44. 400 MHz NMR spectrum of the amphetamine o x a z i r i d i n e (82). 152 Fi g u r e 45. 80 MHz NMR spectrum of the amphetamine secondary formamide (83). 153 F i g u r e 46. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : I. R a d i c a l s i t e i n i t i a t i o n at the s a t u r a t e d heteroatom with alpha c l e a v a g e . 156 F i g u r e 47. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : I I . Rearrangement of the gamma hydrogen to the s a t u r a t e d hetero atom with adjacent cleavage and charge r e t e n t i o n . 157 F i g u r e 48. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : I I I . R a d i c a l s i t e i n i t i a t i o n on the aromatic r i n g with a l l y l i c ( b e n z y l i c ) c l e a v a g e . 1 58 F i g u r e 49. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : IV. Rearrangement of gamma hydrogen with b e n z y l i c cleavage and l o s s of a hydrogen r a d i c a l to a f f o r d d i a g n o s t i c oxonium ions i n phenyl r i n g o x i d i z e d m e t a b o l i t e s and t h e i r d e r i v a t i v e s . 158 F i g u r e 50. Mass s p e c t r a of r e c i p a v r i n m e t a b o l i t e s i l l u s t r a t i n g the three major fragmentation schemes i n f i g u r e 46,47,48. A. N o r r e c i p a v r i n (15). B. Secondary formamide (12). 159 F i g u r e 50. Mass s p e c t r a of r e c i p a v r i n m e t a b o l i t e s i l l u s t r a t i n g the three major fragmentation schemes in f i g u r e s 46,47,48. C. ant i - d i p h e n y l b u t a n o n e oxime (19). D. R e c i p a v r i n phenol (90). 160 F i g u r e 51. General s t r u c t u r e s f o r m e t a b o l i t e s : A. S i n g l e bonded R1 s u b s t i t u e n t . B. Double bonded R1 s u b s t i t u e n t . 164 F i g u r e 52. T o t a l i o n c u r r e n t and s e l e c t e d ion chromatograms f o r 0-glucuronidase-hydrolyzed e x t r a c t s of b i l i a r y r e c i p a v r i n m e t a b o l i t e s . I . T o t a l i o n c u r r e n t f o r a l l m e t a b o l i t e s (above) and t h e i r TMS d e r i v a t i v e s (below). 167 F i g u r e 53. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms f o r 0-glucuronidase-hydrolyzed e x t r a c t s of b i l i a r y r e c i p a v r i n m e t a b o l i t e s . I I . Ion chromatogram m/z 167 showing phenyl r i n g i n t a c t m e t a b o l i t e s (above) and t h e i r TMS d e r i v a t i v e s (below). 168 XX Figure 54. Total ion current and selected ion chromatograms for 0-glucuronidase-hydrolyzed extracts of biliary recipavrin metabolites.Ill. Ion chromatogram m/z 183 showing intact phenol metabolites (above) and m/z 255 showing TMS derivatized phenols. 169 Figure 55. Total ion current and selected ion chromatograms for /J-glucuronidase-hydrolyzed extracts of biliary recipavrin metabolites.IV. Ion chromatogram m/z 213 showing intact O-methylcatechol metabolites (above) and m/z 285. 170 Figure 56. (top) Mass spectrum of the synthetic secondary formamide (12). (bottom) Mass spectrum of the underivatized secondary formamide metabolite. 176 Figure 57. (top) Chemical ionization mass spectrum of the synthetic secondary formamide (12). (bottom) Chemical ionization mass spectrum of the secondary formamide metabolite. 177 Figure 58. (top) Mass spectrum of the TMAH derivatized secondary formamide. (bottom) Mass spectrum of the unidentified TMS derivative (95). 178 Figure 59. Mass spectrum of the TMS derivative of the amphetamine secondary formamide (84). 179 Figure 60. LCMS mass spectrum of the secondary formamide (12)(top) and the methylene nitrone (24)(bottom) in the acetonitrile/ water solvent system. 181 Figure 61. Superimposed LCMS selected ion monitoring results for A. A /3-glucuronidase-hydrolyzed bile extract from a recipavrin dosed rat. B. A mixture of the synthetic methylene nitrone (24) and secondary formamide (12)(10 ng each) standards. 182 Figure 62. Total ion current and selected ion chromatograms for a nonconjugated extract of bil iary recipavrin metabolites, (top) Total ion current for a l l metabolites. (bottom) Ion chromatogram m/z 167 showing phenyl ring intact metabolites. 192 Figure 63. Metabolic pathways for recipavrin based on the metabolites observed by GCMS. 1a. Phenyl ring oxidation. 1b. Oxidation to the catechol and 3'-O-methylation. 2a. Oxidative deamination. 2b. Ketone reduction. 3. N-dealkylation. 4. N-oxidation. 193 x x i F i g u r e 64. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms f o r p'-glucuronidase-hydrolyzed e x t r a c t s of b i l i a r y n o r r e c i p a v r i n m e t a b o l i t e s . I. T o t a l i on c u r r e n t f o r a l l conjugated n o r r e c i p a v r i n m e t a b o l i t e s (above) Ion chromatogram m/z 167 (below). 195 F i g u r e 65. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms f o r p'-glucuronidase-hydrolyzed e x t r a c t s of b i l i a r y n o r r e c i p a v r i n m e t a b o l i t e s . I I . Ion chromatogram m/z 183 showing conjugated n o r r e c i p a v r i n phenol m e t a b o l i t e s (above). Ion chromatogram m/z 213 (below). 196 F i g u r e 66. (top) Mass spectrum of the u n d e r i v a t i z e d secondary formamide m e t a b o l i t e (12) of n o r r e c i p a v r i n (15). 199 F i g u r e 67. Mass spectrum of the apparent n i t r o compound O-methylcatechol m e t a b o l i t e (98) of n o r r e c i p a v r i n . 200 F i g u r e 68. (top) T o t a l ion c u r r e n t f o r the e x t r a c t of p'-glucuronidase-hydrolyzed f r a c t i o n of u r i n e from n o r r e c i p a v r i n dosed r a t s . (bottom) Mass chromatogram m/z 167 f o r the same sample. 202 F i g u r e 69. (top) Mass chromatogram m/z 167 f o r the e x t r a c t of nonconjugated m e t a b o l i t e s of u r i n e from n o r r e c i p a v r i n dosed r a t s . (bottom) Mass chromatogram m/z 167 f o r the e x t r a c t of nonconjugated m e t a b o l i t e s of b i l e . 203 F i g u r e 70. T o t a l ion c u r r e n t f o r the e x t r a c t of p'-g l u c u r o n i d a s e - h y d r o l y z e d f r a c t i o n of b i l e from d i n o r r e c i p a v r i n dosed r a t s . (bottom) Mass chromatogram m/z 167 f o r the same sample. 207 F i g u r e 71. Mass chromatogram m/z 183 (top) and 213 (bottom) f o r the e x t r a c t of p'-glucuronidase-h y d r o l y z e d f r a c t i o n of b i l e from d i n o r r e c i p a v r i n dosed r a t s . 208 F i g u r e 7 2 . ( t o p ) : Superimposed mass chromatograms of m/z 253 i n the s y n t h e t i c formamide (12)(sample 2, 30 ng, t r = l 5 . 6 8 ) , and b i l e e x t r a c t formamide (sample 1, tr=15.84). Bottom: Mass chromatograms of the m/z 253 ion i n spiked samples 3 and 4. 211 F i g u r e 73. T o t a l ion c u r r e n t and s e l e c t e d ion chromatogram f o r p'-glucuronidase-hydrolyzed c h l o r o f o r m e x t r a c t of b i l i a r y r e c i p a v r i n m e t a b o l i t e s . (top) T o t a l ion c u r r e n t f o r a l l m e t a b o l i t e s , (bottom) Ion chromatogram m/z 167. 213 F i g u r e . 74. T o t a l ion current (top) and m/z 167 ion chromatogram (bottom) for the sul fatase hydrolyzed extract of b i l e from rats dosed with r e c i p a v r i n -D 3 . Diphenylbutanone (14) is present at 13.37 min. Figure 75. T o t a l ion current (top) and mass chromatogram m/z 167 (bottom) for a p'-glucuronidase-hydrolyzed b i l i a r y r e c i p a v r i n metabolites e luted from the FPSi02 column with e thanol , hydrolyzed with ^-glucuronidase and e luted from a RPC18. F igure 76. (top) Mass spectrum of the secondary formamide metabolite of rec ipavr in eluted from the FPSi02 column with ethanol , hydrolyzed with 0-glucuronidase and e luted from a RPC18 column with 40% ethanol.(bottom) Mass spectrum of the secondary formamide. Figure 77. T o t a l ion current (top) and mass chromatogram m/z 167 (bottom) for nonconjugated b i l i a r y r e c i p a v r i n metabolites e luted from the FPSi02 column with 92/8 benzene/ethanol. Figure 78. Ion chromatogram m/z 167 of the c o n t r o l (bottom) and formaldehyde/peroxide treated (top) nonconjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s . Figure 79. Figure showing m/z 167 mass chromatograms for the contro l (top) and peroxide/ formaldehyde treated (bottom) conjugated f rac t ion of b i l e from r e c i p a v r i n dosed r a t s . Figure 80. Structures of promethazine (10), promethazine N-oxide (76), promethazine secondary formamide (72) and i t s phenol (100) and sul foxide (101) and N l 0 - o x i d e (75). Figure 81. (top) Mass spectrum of the synthet ic phenothiazine secondary formamide. (bottom) Methylene phenothiazine (m/z 212) and /3-carboline (m/z 180) resonance s t a b i l i z e d cat ions a r i s i n g from r i n g d irec ted he tero ly t i c cleavage. Figure 82. (top) Direc t inser t ion probe mass spectrum of the secondary formamide N i n - o x i d e (75). (bottom) Direc t inser t ion probe mass spectrum of the Cope e l iminat ion product 10-<2-propenylJphenothiazine (77). Figure 83. Mass chromatograms for an extract of B-glucuronidase-hydrolyzed conjugated promethazine metabolites (above) Ion chromatogram m/z 58 showing intact secondary amine metabolites (below) Ion chromatogram m/z 72. Figure 84. Total ion current for the control GCMS analysis of recipavrin N-oxide. Figure 85. (top) C a p i l l a r y GCMS t o t a l ion current of freshly synthesized and p u r i f i e d recipavrin methylene nitrone injected at low concentration onto a freshly s i l a n i z e d i n j e c t i o n port l i n e r and GC column. (bottom) Mass spectrum of the single peak. Figure 86. (top) Summary of high resolution mass spectrum of the nitrone (24) (Source temperature 120°). (bottom) Mass spectrum of the formamide. Figure 87. C a p i l l a r y GCMS of the methylene nitrone using a clean i n j e c t i o n port l i n e r . (top) Total ion current showing decomposition of 10 ug of freshly p u r i f i e d recipavrin methylene nitrone (24). Figure 88. GCMS analysis of the methylene nitrone using a clean i n j e c t i o n port l i n e r . (top) Total ion current of a sample overload (100 ug). (bottom) Close up of the t o t a l ion current showing the formamide (12, tr=21.3l) and nitrone (24, shoulder. Figure 89. C a p i l l a r y GCMS of the methylene nitrone (24) using a d i r t y injection port l i n e r . (top) Total ion current showing decomposition of 10 ug of freshly p u r i f i e d recipavrin methylene nitrone. Close up of the t o t a l ion current showing the formamide (bottom). Figure 90. Total ion current chromatogram form GCMS analysis using a clean injection port l i n e r of an extract of control b i l e spiked with 100 ug methylene nitrone (24). Figure 91. (top) Mass chromatogram m/z 167 showing phenyl ring intact metabolites of the methylene nitrone (24). (bottom) Mass chromatogram m/z 183 showing phenol metabolites of the recipavrin methylene nitrone. Figure 92. (top) Mass chromatogram m/z 213 showing O-methylcatechol metabolites of the methylene nitrone (24). (bottom) Mass spectrum of the secondary formamide (12) a r i s i n g from GCMS analysis of the nitrone metabolites. X X I V Figure 93 (top) T o t a l ion current for the BSTFA d e r i v a t i z e d b i l e extract of nitrone (24) metabol i tes . (bottom) Mass spectrum of the suspected primary hydroxylamine TMS d e r i v a t i v e (102). 260 Figure 94. Composite selected ion chromatograms used to locate metabolites in /J -glucuronidase-hydrolyzed extracts of b i l e from t e r t i a r y formamide dosed r a t s . A. Ion chromatogram m/z 167 showing phenyl r i n g intact metabolites , m/z 183. 268 Figure 95. Composite selected ion chromatograms used to locate metabolites in 0-glucuronidase-hydrolyzed extracts of b i l e from t e r t i a r y formamide dosed r a t s . B. Ion chromatogram m/z 167 showing phenyl r ing intact TMS-derivat ized metabol i tes , m/z 255 showing TMS phenols. 269 Figure 96. Mass spectra of the carbinolamide metabol i tes . (a) Carbinolamide (47). (b) Carbinolamide TMS (47a). 272 Figure 97. Proposed mass spectral fragmentations of the carbinolamide (47) and TMS der iva t ives of the carbinolamide (47a), the carbinolamide phenol (107a), and carbinolamide O-methylcatechol (110a). 273 Figure 98. Mass spectra of the carbinolamide metabol i tes . (c) Carbinolamide phenol di-TMS (107a). (d) Carbinolamide O-methylcatechol di-TMS (110a). 274 Figure 99. Proposed mass spectra l fragmentations of the carbinolamide (47) and TMS der ivat ives of the carbinolamide (47a), the carbinolamide phenol (107a), and carbinolamide O-methylcatechol (110a). Figure 100. Proposed mass spectra l fragmentations of the carbinolamide (47) and TMS der ivat ives of the carbinolamide (47a), the carbinolamide phenol (107a), and carbinolamide O-methylcatechol (110a). 275 276 Figure 101. Summary of bond cleavages in the mass spectra of the carbinolamide metabolite TMS d e r i v a t i v e s (47a, 107a and 110a). R1=TMS, R2=H or OCH3, R3= H or OTMS. 277 F i g u r e 102. M e t a b o l i c pathways f o r the t e r t i a r y formamide (26) based on the m e t a b o l i t e s observed by GCMS. a. Phenyl r i n g o x i d a t i o n , b. O x i d a t i o n to the c a t e c h o l and 3'-O-methylation. c. O x i d a t i v e deamination. d. Ketone r e d u c t i o n , e. a-Carbon o x i d a t i o n . F i g u r e 103. Mass chromatogram m/z 167 f o r the e x t r a c t of /3-glucuronidase-hydrolyzed f r a c t i o n of b i l e from secondary formamide dosed r a t s . F i g u r e 104. Mass chromatogram m/z 213 f o r the e x t r a c t of /3-glucuronidase-hydrolyzed f r a c t i o n of b i l e from secondary formamide dosed r a t s . F i g u r e 105. Mass chromatogram m/z 183 (top) and 213 (bottom) f o r the nonconjugated u r i n e f r a c t i o n from secondary formamide dosed r a t s . F i g u r e 106. (top) Mass chromatogram m/z 167 f o r the nonconjugated b i l e e x t r a c t of secondary formamide m e t a b o l i t e s Standard GC c o n d i t i o n s , (bottom) Mass chromatogram m/z 167 f o r the nonconjugated u r i n e e x t r a c t of secondary formamide m e t a b o l i t e s . F i g u r e 107.(top) Mass spectrum of the c a r b i n o l a m i d e m e t a b o l i t e (111) observed i n the conjugated and nonconjugated f r a c t i o n , (bottom) Mass spectrum of the c a r b i n o l a m i d e phenol m e t a b o l i t e (112) observed i n the conjugated and nonconjugated f r a c t i o n . F i g u r e 108. Fragmentation scheme to account f o r the M+-18 peak of the carbinolamide m e t a b o l i t e (111). LIST OF TABLES Table 1. Table showing volumes of b i l e extract and synthet ic formamide (12) used in the d i l u t i o n experiment. Table 2. Table showing volumes of reagents added to b i l e from r e c i p a v r i n dosed r a t s . Table 3. Recovery of the methadone formamide metabolite (6) using various sample i s o l a t i o n procedures. Table 4. Diagnostic masses of the d iary lmethyl ca t ion for phenyl r ing oxidized metabolites and t h e i r d e r i v a t i v e s . Table 5. Names s tructure and formulae for r e c i p a v r i n , r e c i p a v r i n metabolites and synthet ic reference compounds. Table 6. Names, s tructures and formulae for phenol and O-methyl catechol metabolites of r e c i p a v r i n . Table 7. GCMS data for phenyl r ing intact r e c i p a v r i n metabolites and t h e i r d e r i v a t i v e s . Table 8. GCMS data for phenolic metabolites of r e c i p a v r i n and t h e i r d e r i v a t i v e s . Table 9. GCMS data for O-methyl catechol metabolites of r ec ipavr in and t h e i r d e r i v a t i v e s . Table 10. Retention times and diagnost ic ion abundances for the conjugated norrec ipavr in metabol i tes . Table 11. Retention times and diagnost ic ion abundances for the conjugated d i n o r r e c i p a v r i n metabol i tes . Table 12. The e f fec t s of added synthet ic secondary formamide standard on peak height r a t i o s , peak shape and retent ion time of the formamide metabolite present in a p'-glucuronidase-hydrolyzed b i l e extract Table 13. GCMS peak areas of compounds a r i s i n g from the chemical and/or thermal degradation of r e c i p a v r i n N-oxide under various simulated workup cond i t i ons . Table 14. Names s tructures and formulae for the t e r t i a r y formamide (26) and i t s metabol i tes . xxvi i Table 15. GCMS data f o r phenyl r i n g i n t a c t m e t a b o l i t e s of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . 264 Table 16. GCMS data f o r p h e n o l i c m e t a b o l i t e s of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . 265 Table 17. GCMS data f o r O-methylcatechol m e t a b o l i t e s of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . 266 Table 17 ( c o n t i n u e d ) . GCMS data f o r O-methylcatechol m e t a b o l i t e s of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . 267 Table 18. M e t a b o l i t e s and r e t e n t i o n times f o r the secondary formamide m e t a b o l i t e s . 287 xxvi i i LIST OF ABBREVIATIONS 1. Common a b b r e v i a t i o n s Ac a c e t y l AMP amphetamine amu atomic mass u n i t APT a t t a c h e d proton t e s t Ar a r y l arom aromatic BB broad band decoupled b.p b o i l i n g p o i n t BHT b u t y l a t e d hydroxy toluene B S T F A . . . . N , 0 - b i s - ( t r i m e t h y l s i l y l ) t r i f l u o r o a c e t a m i d e CAS Chemical a b s t r a c t s s e r v i c e CI chemical i o n i z a t i o n D deuterium d doublet dd doublet of doublets DDP N , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i d i n e dimedone.5,5-dimethyl-1,3-cyclohexanedione DIP d i r e c t i n s e r t i o n probe DLl d i r e c t l i q u i d i n t r o d u c t i o n DMCS d i m e t h y l d i c h l o r o s i l a n e DMSO dimethyl s u l f o x i d e EDDP 2 - e t h y l i d e n e - 1 , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i d i n e EI e l e c t r o n impact EMDP 2 - e t h y l - 5 - m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i d i n e eq e q u i v a l e n t e q u i v . . . . e q u i v a l e n t xxix et a I.. . .et a Iia e t c e t c e t e r a EtOH et h a n o l F P S 1 O 2 . . . f o r w a r d phase s i l i c a g e l SPE c a r t r i d g e FT f o u r i e r t ransform GC gas chromatograph GCMS gas chromatography-mass spectrometry GLU g l u c u r o n i c a c i d H-bond...hydrogen bond HPLC hig h p r e s s u r e l i q u i d chromatography IEC ion exchange chromatography IR.. i n f r a red spectrometry J c o u p l i n g constant i n Hz LC . . l i q u i d chromatograph LCMS l i q u i d chromatography-mass spectrometry L i t . . . . . . l i t e r a t u r e value M + ..molecular ion m ..medium (IR), m u l t i p l e t (NMR) min......minutes m.p me l t i n g p o i n t m/z mass to charge r a t i o MC mass chromatogram MCPBA....metachloroperbenzoic a c i d MeOH methanol MOX O-methylhydroxylamine hyd r o c h l o r ide MS mass spectrometer NADP ni c o t i n a m i d e adenine d i n u c l e o t i d e phosphate NMR nucl e a r magnetic resonance spectrometry X X X NOE n u c l e a r overhauser e f f e c t pet petroleum Ph phenyl ppm p a r t s per m i l l i o n PTZ phenothiazine py p y r i d i n e q quadruplet r f TLC m o b i l i t y R P C ^ . . . .reversed phase o c t a d e c y l SPE c a r t r i d g e s s t r o n g (IR), s i n g l e t (NMR) sec...... .secondary S F O R D . . . . s i n g l e frequency o f f resonance decoupled S I M . . . . . . s e l e c t e d ion monitoring S P E . . . . . . s o l i d phase e x t r a c t i o n s t r . . . . . . s t r e t c h t . . t r i p l e t t e r t t e r t i a r y TLC t h i n l a y e r chromatography TMAH t r i m e t h y l a n i l i n i u m hydroxide TMS t e t r a m e t h y l s i l a n e t r . . r e t e n t i o n time UV u l t r a v i o l e t v v ery w weak xxxi ACKNOWLEDGEMENTS I am g r a t e f u l to the s t a f f of the mass spectrometry and high r e s o l u t i o n NMR l a b o r a t o r i e s i n the Department of Chemistry, UBC. To Ron Lee f o r a s s i s t a n c e with surgery. To Ken Jang f o r a s s i s t a n c e with s y n t h e s i s . To Roland Burton f o r a s s i s t a n c e with GCMS a n a l y s i s . To Jack Henion f o r a s s i s t a n c e with LCMS a n a l y s i s . To Dean M c N e i l l f o r f i n d i n g an e x t r a t e a c h i n g a s s i s t a n t s h i p when i t was needed. To my c o l l e a g u e s Kelem Kassahun, Sue Panesar, Ron Lee and Matt Wright f o r " h e l p f u l d i s c u s s i o n s " . To my pa r e n t s , whose f i n a n c i a l and moral support made the d i f f e r e n c e . To the Med i c a l Research C o u n c i l of Canada and U n i v e r s i t y Graduate F e l l o w s h i p Committee f o r t h e i r f i n a n c i a l support. L a s t l y , to my s u p e r v i s o r , Dr. Frank Abbott whose guidance and support made my stay i n graduate s c h o o l worthwhile and e n j o y a b l e . xxxi i DEDICATION To my p a r e n t s , Nancy and Wally S l a t t e r . 1 I . I N T R O D U C T I O N li_9§§§BYATIQN_OF_A_FORMAMIDE_ In p r e v i o u s work i n t h i s l a b o r a t o r y i t was observ e d t h a t t h e p e r c h l o r a t e s a l t of t h e major p y r r o l i d i n e m e t a b o l i t e of methadone ( ( + ) - 2 - e t h y l - 1 , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i n i u m p e r c h l o r a t e , (EDDP), 1) undergoes c h e m i c a l o x i d a t i o n t o a t h e r m o l a b i l e o x a z i r i d i n e ( 2 ) . Other o x i d a t i o n p r o d u c t s p r e s e n t i n c l u d e d a d i k e t o n e ( 3 ) , 1 , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i d o n e ((DDP), 4 ) , and 2 - e t h y l - 5 - m e t h y l - 3 , 3 - d i p h e n y l - 1 - p y r r o l i n e ((EMDP), 5 ) . The o x a z i r i d i n e (2) decomposed t o the i s o m e r i c formamide (6) d u r i n g GCMS a n a l y s i s ( f i g u r e 1) ( A b b o t t , S l a t t e r and Rang, 1986). GCMS r e s u l t s f o r the formamide (6) were i d e n t i c a l t o a n o v e l m e t a b o l i t e of methadone found i n p'-g l u c u r o n i d a s e - h y d r o l y z e d b i l e e x t r a c t s from methadone dosed r a t s ( A b b o t t , S l a t t e r , B u r t o n and Kang, 1985). The methylene n i t r o n e s t r u c t u r e (7) was o r i g i n a l l y proposed f o r the methadone m e t a b o l i t e (Kang, 1981) on the b a s i s t h a t s i m i l a r n i t r o n e s were r e p o r t e d t o a r i s e from the met a b o l i s m of a r y l a l i p h a t i c amines i n the amphetamine s e r i e s ( C o u t t s and B e c k e t t , 1977). No methylene n i t r o n e (7) was i s o l a t e d from the EDDP o x i d a t i o n m i x t u r e . The p o s s i b i l i t y t h a t the n i t r o n e was a l a b i l e p r e c u r s o r of t h e formamide m e t a b o l i t e (6) wa r r a n t e d f u r t h e r s y n t h e t i c i n v e s t i g a t i o n . The c y c l i z a t i o n of d e s a l k y l m e t a b o l i t e s of methadone (8) had been p r e v i o u s l y documented as the major c h e m i c a l change i n t h e m e t a b o l i s m of methadone i n r a t s ( P o h l a n d , et a/., 1971, S u l l i v a n , et al., 1972). The c y c l i z a t i o n was assumed t o be a 2 spontaneous a d d i t i o n of the b a s i c secondary amino group to the e t h y l ketone s i d e c h a i n of methadone with dehydration to EDDP ( 1 ) . T h i s c y c l i z a t i o n complicated attempts t o s y n t h e s i z e the n i t r o n e ( 7 ) , s i n c e open ch a i n primary and secondary hydroxylamine s y n t h e t i c i n t e r m e d i a t e s underwent the c y c l i z a t i o n r e a c t i o n common to the more b a s i c d e s a l k y l m e t a b o l i t e of methadone ( 8 ) ( S l a t t e r , 1983). Since the c y c l i z a t i o n of s y n t h e t i c i n t e r m e d i a t e s c o m p l i c a t e d the s y n t h e s i s of the p o t e n t i a l n i t r o n e p r e c u r s o r (7) of the methadone formamide (6), and because the chemical o x i d a t i o n of EDDP (1) was ap p a r e n t l y not the source of the formamide m e t a b o l i t e ( 6 ) , r e c i p a v r i n (9) was adopted as a model drug t o determine the mechanism whereby formamides are observed i n conjugated b i l i a r y e x t r a c t s from r a t s dosed with t e r t i a r y a r y l a l i p h a t i c amines. 2..RECIPAVRIN:_A _ STRUCTURAL _ ANALOGUE_OF.METHADONE R e c i p a v r i n R i s the trade name f o r (—) N,N,a-trimethyl~7-phenylbenzenepropanamine h y d r o c h l o r i d e (9, CAS R e g i s t r y 22173-83-70). T h i s drug was f i r s t s y n t h e s i z e d i n the l a t e 1940's (May and M o s e t t i g , 1948, Adamson, 1949). R e c i p a v r i n i s a b a s i c a r y l a l i p h a t i c amine (pKa=9.48) with a n t i c h o l i n e r g i c a c t i v i t y approximately equal t o imipramine or t e r o d i l i n e ( N i l v e b r a n t and Sparf, 1983) and an L D 5 0 of 80 mg/kg i n mice (Eddy, et al., 1950). The a n t i c h o l i n e r g i c p r o p e r t i e s of r e c i p a v r i n l e d to i t s marketing by A.B. Recip (Stockholm) as a spasmolytic f o r u l c e r s and s p a s t i c c o l i t i s ( U n l i s t e d Drugs, 1952), and F i g u r e 1. (top) Products d e r i v e d from the chemical o x i d a t i o n of EDDP ( 1 ) . (bottom) S t r u c t u r e of methadone ( 8 ) , r e c i p a v r i n ( 9 ) , promethazine ( 10 ) , dimethylamphetamine (11) , and the formamide m e t a b o l i t e (12) of r e c i p a v r i n . 4 l a t e r as an i n g r e d i e n t i n P r e p a r y l R i n d i c a t e d f o r v e g e t a t i v e neuroses ( U n l i s t e d Drugs, 1953) and R o b e t y l R , f o r a n x i e t y and tremor ( U n l i s t e d Drugs, 1960). R e c i p a v r i n was i n i t i a l l y s y n t h e s i z e d as an analogue of the n a r c o t i c a n a l g e s i c methadone, but was devoid of a n a l g e s i c a c t i v i t y (Eddy, et al., 1950). S t r u c t u r a l l y , the only d i f f e r e n c e was the replacement of the e t h y l ketone s i d e chain of methadone (8) with a hydrogen atom. I t i s t h i s s i m i l a r i t y t h a t has l e d to the use of r e c i p a v r i n (9) as a model compound i n drug d e r i v a t i z a t i o n s t u d i e s (Vessman, et a l . , 1970, H a r t v i g and Vessman, 1974, H a r t v i g , et al., 1972, 1976) and NMR s t u d i e s d i r e c t e d toward d i s c e r n i n g the a c t i v e conformation of methadone-related a n a l g e s i c s (Casy, 1966). R e c i p a v r i n a l s o has s t r u c t u r a l elements i n common with the a n t i h i s t a m i n e , promethazine (10) and dimethylamphetamine (11). 3i_QVERVIEW_OF_PREVigUS_WORK_ON_R^ The f i r s t s t e p i n the i n v e s t i g a t i o n of r e c i p a v r i n metabolism was to s y n t h e s i z e r e c i p a v r i n and i t s p o t e n t i a l m e t a b o l i t e s , i n p a r t i c u l a r the o x a z i r i d i n e (25), n i t r o n e (24) and formamide (12) analogues proposed on the b a s i s of the methadone s t u d i e s . The s y n t h e t i c work i s summarized i n f i g u r e 2. With r e f e r e n c e compounds i n hand i t was p o s s i b l e to show th a t the secondary formamide (12) c o u l d be observed by GCMS a n a l y s i s of e x t r a c t s of 0-glucuronidase-hydrolyzed b i l e from r e c i p a v r i n dosed r a t s . 5 1 2 1 3 V V " ^ * V 7 ' J C e K s \ / V .4 24 la. 2. 3. 4. 5. 6. 7. 25 CH^H Aid 3 b) H 30+ 3NHOH, NaBHjCN, CH3OH, pH5 CH3NH2. NaBH3CN, CH3OH, Molecular Sieve CH3ONH2, Pyridine NH2OH, MeOH, pH8 NH4AC, MeOH, NaBH3CN NaBH3CN, MeOH, pH6 CH3 I N 26 \ c / f i II 0 8. H2CO, MeOH 9. MCPBA, CH2Cl2 10. Ethyl formate 11. H2C0, C 6H 6 12. MCPBA, CHCl3i 13. TMAH, C5H5N t Figure 2. Prel iminary syntheses of po tent ia l N- and alpha C-oxidized metabolites of r ec ipavr in (9) . 6 The o x a z i r i d i n e (25) isomerized i n the GC i n l e t to the secondary formamide (12). The n i t r o n e (24) was t h e r m o l a b i l e with decomposition d u r i n g GCMS a n a l y s i s to the imine (23), oxime (19) and an u n i d e n t i f i e d compound which resembled the formamide (12) but chromatographed p o o r l y and had a d d i t i o n a l ions i n the mass spectrum. When b i l e was spi k e d with the secondary hydroxylamine (17), and worked up by sol v e n t e x t r a c t i o n , the formamide (12) was observed by GCMS. The absence of the imine i n me t a b o l i t e e x t r a c t s was taken to be evidence a g a i n s t the n i t r o n e (24) as a t h e r m o l a b i l e p r e c u r s o r of the formamide (12). . The LCMS behavior of the methylene n i t r o n e (24), formamide (12) and o x a z i r i d i n e (25) analogues of r e c i p a v r i n (9) i n aqueous methanol r e v e a l e d that the methylene n i t r o n e decomposes t o a minor extent to the secondary formamide (12) on s t a n d i n g at room temperature i n methanol s o l u t i o n ( f i g u r e 3, S l a t t e r , 1983). Thus, the n i t r o n e (24) was a l s o a p o s s i b l e chemical p r e c u r s o r of the formamide m e t a b o l i t e (12). The i n i t i a l work on r e c i p a v r i n (9) concluded that the o x a z i r i d i n e (25) and n i t r o n e (24) were both p o s s i b l e chemical p r e c u r s o r s of the formamide m e t a b o l i t e (12). There were other p o s s i b l e p r e c u r s o r s that r e q u i r e d s y n t h e s i s and c h a r a c t e r i z a t i o n of the f u l l a r r a y of r e c i p a v r i n m e t a b o l i t e s . 600«f 300. 10 2 0 3 0 M I N U T E 8 5 0 F i g u r e 3. ( t o p ) Superimposed LCMS i o n chro m a t o g r a m s of t h e M ++1 m/z 254 i o n of t h e s y n t h e t i c m e t h y l e n e n i t r o n e ( 2 4 ) , o x a z i r i d i n e (25) and formamide (12) . The formamide was a l s o p r e s e n t a s a d e c o m p o s i t i o n p r o d u c t of t h e n i t r o n e . ( B o t t o m ) LCMS mass s p e c t r a of t h e formamide (12) and n i t r o n e ( 2 4 ) . M e t h a n o l / w a t e r s o l v e n t s y s t e m . ( S l a t t e r , 1983). 4. METABOLISM OF TERTIARY ARYLALIPHATIC AMINES 8 The metabolism of t e r t i a r y amines has been reviewed by Rose and C a s t a g n o l i (1983). An important pathway i s the formation of N-oxides which are t h e r m o l a b i l e compounds commonly observed by GCMS as Cope e l i m i n a t i o n products (Cope, et al ., 1951). The t e r t i a r y amine f u n c t i o n a l group i s a l s o a p o s s i b l e s u b s t r a t e f o r N - g l u c u r o n i d a t i o n ( C a l d w e l l , 1982). The s i m i l a r i t y of r e c i p a v r i n (9) to dimethylamphetamine (11) a l l o w s the metabolic pathways of N - o x i d a t i o n , N-d e a l k y l a t i o n , o x i d a t i v e deamination and phenyl r i n g o x i d a t i o n to be a p p l i e d to the r e c i p a v r i n s e r i e s . A key d i f f e r e n c e i s that r e c i p a v r i n (molecular weight 253) i s above the minimum molecular weight f o r b i l i a r y e x c r e t i o n i n the r a t , while dimethylamphetamine (11) i s not. The metabolism of amphetamines has been reviewed by C a l d w e l l (1976). N - o x i d a t i v e metabolism of amphetamines has been reviewed by Coutts and Beckett (1977). The metabolism of dimethylamphetamine (11) has been the s u b j e c t of a short communication (Beckett and A l S a r r a j , 1972). The metabolism of a b a s i c t e r t i a r y a r y l a l i p h a t i c amine to a secondary formamide was a novel o b s e r v a t i o n which c o u l d not be a s s i g n e d to any of the common pathways of amphetamine metabolism. The c h a r a c t e r i z a t i o n of the formamide m e t a b o l i t e s of r e c i p a v r i n appeared t i m e l y as formamide m e t a b o l i t e s of other drugs began to appear i n the l i t e r a t u r e at the same time. 5. FORMAMIDE METABOLITES OF AMINES AND AMIDES 9 Formamide m e t a b o l i t e s have been d e s c r i b e d f o r prenylamine (27) (Remberg, et al . , 1977), aminopyrine (28) ( I g u c h i , et al ., 1975), c a f f e i n e (29) (Tang, et al ., 1983), amantidine (30) (Koppel and Tenczer, 1985), mianserin (31) (DeJongh, et al., 1981), i n d e c a i n i d e (32) (Lindstrom, et al., 1984), pipemidic a c i d (33) (Kurobe, et al., 1980), aminoanthraquinone (34) (Gothoskar, et al., 1979), 2-naphthylamine (35) (Boyland and Manson, 1966), c h l o r t o l u o n (36) (Muecke, et al ., 1976), N-methylbenzamide (37) (Ross, et al., 1983) and v i l o x a z i n e (38) (Case, et al., 1975) ( f i g u r e 4 ) . The formamide m e t a b o l i t e s of arylamines a r i s e by enzymatic f o r m y l a t i o n mechanisms i n v o l v i n g kynurenine formamidase (Santi and Hopsu-Havu, 1968), or i n the case of aminopyrine, have been proven to a r i s e by alpha carbon NHCHO 'CHO 1 N-CHO X 1 IJ. J -CHO NHCHO 36 H NMe 2 NMe--OEt NHCHO OSO3H Me *^CHO \ N Me N C H O ^ N > I CHO F i g u r e 4. S t r u c t u r e s of parent drug and formamide m e t a b o l i t e s of prenylamine (27), aminopyrine (28), c a f f e i n e (29), amantidine (30), mianserin (31), i n d e c a i n i d e (32), pipemidic a c i d (33), aminoanthraquinone (34), 2-naphthylamine (35), c h l o r t o l u o n (36), N,N-dimethylbenzamide (37) and v i l o x a z i n e (38). 11 6..POSSIBLE SOURCES _QF _ FORMAMI DE _METABOL ITES _OF .METHADONE _ (. 81 AND_?ECIPAVRIN_(9) There are s e v e r a l p o s s i b l e combinations of metabolic and chemical changes which c o u l d account f o r the GCMS ob s e r v a t i o n of a secondary a r y l a l i p h a t i c formamide i n m e t a b o l i t e e x t r a c t s d e r i v e d from ^ - g l u c u r o n i d a s e h y d r o l y z e d b i l e of t e r t i a r y a r y l a l i p h a t i c amine dosed r a t s . A l l center on p r e l i m i n a r y o x i d a t i o n of n i t r o g e n and carbon atoms adjacent t o n i t r o g e n . The i n t e r p l a y between chemical and metabolic changes i n the a n a l y s i s of l a b i l e N - o x i d i z e d compounds have been reviewed (Coutts and Beckett, 1977, Lindeke, 1982, H l a v i c a , 1982). The occurrence of amide m e t a b o l i t e s of arylamines from a s i m i l a r a r r a y of sources which i n c l u d e n i t r o n e s and o x a z i r i d i n e s i s under i n v e s t i g a t i o n i n another l a b o r a t o r y (Gooderham and Gorrod, 1986). Sol v e n t , amine, o x a z i r i d i n e , n i t r o n e , and amide mediated g e n e r a t i o n of the secondary formamide (12) were a l l p o s s i b l e e x p l a n a t i o n s f o r the source of the me t a b o l i t e observed by GCMS. A. Formamide a r t i f a c t s a r i s i n g from m e t a b o l i t e e x t r a c t i o n  u s i n g o r g a n i c s o l v e n t s The r e a c t i o n of s o l v e n t i m p u r i t i e s with drugs or t h e i r m e t a b o l i t e s i s a w e l l known p i t f a l l of drug m e t a b o l i t e e x t r a c t i o n (Beckett and Cowan, 1978, Chamberlain, 1985). The problem can be minimized by d e s t r o y i n g or removing the problem im p u r i t y and then d i s t i l l i n g the s o l v e n t j u s t b e f o r e use. 12 Problem s o l v e n t s such as ether or c h l o r o f o r m should be avoided, however, the v o l a t i l i t y , s o l v a t i n g power, c o s t and ease of p u r i f i c a t i o n of these s o l v e n t s make them good ch o i c e s f o r r o u t i n e m e t a b o l i t e e x t r a c t i o n . Problems a r i s e when a t r a c e m e t a b o l i t e such as a formamide c o u l d a r i s e from r e a c t i o n with s o l v e n t d e r i v e d i m p u r i t i e s . Formamides are known to a r i s e from r e a c t i o n of amines with formyl c h l o r i d e present i n chloroform ( S t i l l w e l l , et al., 1978). Carbamates can a r i s e from the r e a c t i o n of amines with phosgene i n the presence of an a l c o h o l (Wester, et al., 1981, Cone, et al., 1982). Organic peroxides such as d i e t h y l peroxide formed photochemically i n ether react with amines. The p e r o x i d a t i o n of secondary amines g i v e s r i s e t o formamides. The p e r o x i d a t i o n of iminium compounds giv e s r i s e t o l a b i l e o x a z i r i d i n e s , another p o s s i b l e source of formamide. The iminium compounds c o u l d i n t u r n a r i s e m e t a b o l i c a l l y or r e s u l t from condensation of an amine with aldehydes present i n the e x t r a c t or i n t r o d u c e d as solvent i m p u r i t i e s . A l l these r e a c t i o n s r e q u i r e a secondary amine or primary amine s u b s t r a t e . The formamide m e t a b o l i t e s of methadone and r e c i p a v r i n were d e r i v e d from the ^ - g l u c u r o n i d a s e h y d r o l y z e d f r a c t i o n . B a s i c secondary amines are not subject to g l u c u r o n i d e c o n j u g a t i o n and should not be present i n the hydrolyzed f r a c t i o n . However, the p o s s i b l e o x i d a t i o n of the secondary amine m e t a b o l i t e s was avoided by p r o t e c t i n g samples from l i g h t , keeping them on i c e , working them up as soon as p o s s i b l e a f t e r c o l l e c t i o n and by using a minimum volume of a f r e s h l y p u r i f i e d e x t r a c t i o n s o l v e n t . 1 3 B. Formamide a r t i f a c t s a r i s i n g from o x i d a t i o n of amine  m e t a b o l i t e s d u r i n g sample i s o l a t i o n The f r e e r a d i c a l o x i d a t i o n of amines by peroxides (Sayigh and U l r i c h , 1963) and by amine a u t o o x i d a t i o n i n aqueous s o l u t i o n (Beckwith et al . , 1983) are both known to a f f o r d amides from amines. A l k a l i n e c o n d i t i o n s and the presence of d i s s o l v e d oxygen are p o s s i b l e c o n t r i b u t o r s to the formation of t e r t i a r y formamide (26) and secondary formamide (12) from r e c i p a v r i n (9) and the d e s a l k y l m e t a b o l i t e n o r r e c i p a v r i n (15) r e s p e c t i v e l y . However, n e i t h e r of these amines i s l i k e l y to be present i n the conjugated f r a c t i o n . C. Formamide m e t a b o l i t e s a r i s i n g by p e r o x i d a t i o n of iminium  d e a l k y l a t i o n i n t e r m e d i a t e s Pathway a i n f i g u r e 5 d e t a i l s a p e r o x i d a t i v e route to a secondary formamide (12) mediated by an iminium compound (23) and o x a z i r i d i n e (25). Iminium compounds i n e q u i l i b r i u m with c a r b i n o l a m i n e s have been proposed as d e a l k y l a t i o n i n t e r m e d i a t e s and as e l e c t r o p h i l i c r e a c t i v e m e t a b o l i t e s of a number of d i f f e r e n t amines (Overton, et al., 1985). P e r o x i d a t i o n of iminium compounds a f f o r d s thermo and chemolabile o x a z i r i d i n e s (Emmons, 1957, Krimm, 1958). A u t o o x i d a t i o n of iminium compounds a f f o r d s i s o m e r i c o x a z i r i d i n e s and amides (Auret, et al., 1984). Thus, m e t a b o l i c a l l y generated iminium ions are p o s s i b l e s u b s t r a t e s f o r p e r o x i d a t i o n to o x a z i r i d i n e s by endogenous H 2 0 2 or organic p e r o x i d e s i n t r o d u c e d d u r i n g sample p r e p a r a t i o n . 1 4 The i s o m e r i z a t i o n of o x a z i r i d i n e s to amides i s w e l l documented (Emmons, 1957, L a t t e s , et al., 1982). Methylene o x a z i r i d i n e s a f f o r d formamides by thermal or F e 2 + mediated mechanisms. The d r i v i n g f o r c e f o r o x a z i r i d i n e rearrangements i s r e l i e f of r i n g s t r a i n . Competition between C-0 cleavage to g i v e n i t r o n e s and N-0 cleavage to g i v e amides i s determined by s u b s t i t u e n t s on n i t r o g e n and the o x a z i r i d i n e r i n g carbon. E l e c t r o n withdrawing s u b s t i t u e n t s on the r i n g carbon favor C-0 cleavage and n i t r o n e formation. A t e r t i a r y carbon s u b s t i t u e n t on n i t r o g e n s t a b i l i z e s the o x a z i r i d i n e r i n g . The i o n i c mechanisms f o r each i s o m e r i z a t i o n are shown i n f i g u r e 6. Because of the l a b i l i t y of the o x a z i r i d i n e f u n c t i o n a l group, sample a n a l y s i s c o n d i t i o n s would r e s u l t i n immediate, q u a n t i t a t i v e c o n v e r s i o n of a methylene o x a z i r i d i n e to the i s o m e r i c formamide. There i s no precedent f o r o x a z i r i d i n e s as drug m e t a b o l i t e s , although the o x a z i r i d i n e s t r u c t u r e has been proposed as an i n t e r m e d i a t e i n the metabolism of some compounds (Gorrod and Manson, 1986 (review); Manson, 1971; D o s t e r t , et al., 1985, Gooderham and Gorrod, 1986). The l a c k of a p l a u s i b l e g l u c u r o n i d e p r e c u r s o r i s the c h i e f shortcoming of t h i s pathway. Me I R= P h 2 C H C H 2 Me OH N C 4 > 42 M 4 A C 5 „ R - N O H i Me 17 c 5 A M 2 R = 0 14 R=NW 4 4 C 8 C12 O" 14 R - N + = C H 2 C 8 A \ R=NOMe R - 0 - N = C H 2 16 4 5 • \ R i n n N N n A J . I R - N H C H O 1 2 ^ Y ^ O H V O H « > - k 0 > C 0 2 H R - N H R - N H C H O 4 3 1 2 F i g u r e 5 . S c h e m a t i c showing p o s s i b l e m e t a b o l i c (m) and c h e m i c a l ( c ) t r a n s f o r m a t i o n s t h a t c o u l d accoun t f o r a s e c o n d a r y formamide m e t a b o l i t e (12) i n the ^ - g l u c u r o n i d a s e h y d r o l y z e d f r a c t i o n o f b i l e from r e c i p a v r i n dosed r a t s . LEGEND: Pa thways t o formamide ( 1 2 ) : A ; Pathway m e d i a t e d by o x i d a t i o n of s e c o n d a r y h y d r o x y l a m i n e a g l y c o n e ( 1 7 ) . A , : Pathway m e d i a t e d by f o r m a l d e h y d e c o n d e n s a t i o n w i t h p r i m a r y h y d r o x y l a m i n e a g l y c o n e ( 2 2 ) . B . Pathway m e d i a t e d by p e r o x i d a t i o n o f i m i n i u m compound ( 2 3 ) , » • • « > - « « -» . . . i M 2 Cond  ( ) . M e t a b o l i c pa thways ( M | - M ^ ) : M , . N - d e m e t h y l a t i o n . . N - o x i d a t i o n . M3. O x i d a t i v e d e a m i n a t i o n . . G l u c u r o n i d a t i o n . i t i o n s r e q u i r e d f o r c h e m i c a l changes o c c u r i n q d u r i n g ~~ * 1 *""*"" r = = ' ~ 5 5 E x c e s s C H 2 0 . i s o l a t i o n o f m e t a b o l i t e s ( C j - C n ) : C"7 250^ 0 - g l u c u r o n i d a s e . qi C H 2 0 , 0 2 / 0 H - «-2-Har r e a r r a n g e m e n t . "C7 . H 2 0 2 . C 8 . C H 2 0 / H 2 O 2 / H . Cg . Heat o r F e ' + . C ( n . OH o r a c y l a t i n g a g e n t (Beckmann r e a r r a n g e m e n t ) . C11 • H3O . C 1 2 . Behrend r e a r r a n g e m e n t . 16 H - — R + I H— C — N —R 0" C = N — + R H - C — N - R 0 H C — N - R N - R H H H F i g u r e 6. Io n i c mechanisms f o r the cleavage of C-0 or N-0 bonds i n the o x a z i r i d i n e r i n g to a f f o r d n i t r o n e s or amides r e s p e c t i v e l y . D. Formamide m e t a b o l i t e s a r i s i n g by decomposition of N- o x i d i z e d m e t a b o l i t e s Pathway B i n f i g u r e 5 shows an N - o x i d a t i v e route to the secondary formamide, wherein the tr u e m e t a b o l i t e i s a primary or secondary hydroxylamine O-glucuronide (43 and 42 r e s p e c t i v e l y ) . i . Hydroxylamine m e t a b o l i t e s as p r e c u r s o r s of n i t r o n e s Secondary a r y l a l i p h a t i c hydroxylamines are in vitro m e t a b o l i t e s of other b a s i c t e r t i a r y (Clement and Beckett, 1981, Beckett, et al., 1983) and secondary amines (Coutts and Beckett, 1977). Demonstrating the e x i s t e n c e of hydroxylamines in vivo i s not always p o s s i b l e (Beckett, et a/., 1983). The ex i s t e n c e of secondary hydroxylamine m e t a b o l i t e s as glu c u r o n i d e conjugates has been i n f e r r e d from the e x i s t e n c e of the hydroxylamine aglycones of amphetamines (Beckett and A l S a r r a j , 1972a), phenmetrazine and phendimetrazine (Beckett and Salami, 1972) and chlorphentermine (Beckett and Belanger, 1974, C a l d w e l l , et al., 1975). Other in vivo hydroxylamine 17 m e t a b o l i t e s have a l s o been r e p o r t e d f o r chlorpromazine (Beckett and E s s i e n , 1973) and phentermine (Beckett and Belanger, 1975, 1978). The reason f o r the d i f f i c u l t i e s i n c h a r a c t e r i z i n g in vivo hydroxylamines was presumed to be the ease with which the hydroxylamines undergo o x i d a t i o n , e s p e c i a l l y under the a e r o b i c and m i l d l y a l k a l i n e c o n d i t i o n s commonly employed to e x t r a c t other more b a s i c m e t a b o l i t e s (Beckett, et al., 1977). The o x i d a t i o n of the r e c i p a v r i n secondary hydroxylamine (17) under these c o n d i t i o n s c o u l d g i v e r i s e t o a mixture of N-methyl (44) and N-methylene (24) n i t r o n e s , both isomeric with the secondary formamide m e t a b o l i t e (12). A primary hydroxylamine aglycone (22) a r i s i n g from B-g l u c u r o n i d a s e h y d r o l y s i s of r e c i p a v r i n m e t a b o l i t e s c o u l d condense wi t h formaldehyde to a f f o r d the methylene n i t r o n e (24). The f a c i l e condensation of hydroxylamine m e t a b o l i t e s of amphetamines with aldehydes to a f f o r d n i t r o n e s has been c i t e d as a reason f o r the d i f f i c u l t y i n d e t e c t i n g hydroxylamine m e t a b o l i t e s i n the amphetamine s e r i e s (Beckett, et al., 1979). i i . N i t r o n e s as drug m e t a b o l i t e s The methylene n i t r o n e (24) i s analogous to n i t r o n e s d e s c r i b e d as m e t a b o l i t e s of other a r y l a l i p h a t i c amines such as methamphetamine and promethazine (10) (Beckett and Coutts, 1977, Clement and Beckett 1981). 18 C o f a c t o r dependency and g e n e r a t i o n of the amphetamine n i t r o n e from in vitro metabolism of the secondary hydroxylamine has proven that the n i t r o n e does not a r i s e s o l e l y from the o x i d a t i o n of the hydroxylamine d u r i n g i s o l a t i o n of m e t a b o l i t e s (Coutts et al., 1977). i i i . N i t r o n e s as chemical p r e c u r s o r s of formamide m e t a b o l i t e s : Rearrangements of N i t r o n e s The chemistry of n i t r o n e s has been e x t e n s i v e l y i n v e s t i g a t e d (Hamer and Macaluso, 1964, D e l p i e r r e and Lamchen, 1964, K l i e g e l , 1977, 1978, ( r e v i e w s ) ) . The d i p o l a r nature of the n i t r o n e f u n c t i o n a l group makes i t amenable to molecular rearrangement ( f i g u r e 7) There are s e v e r a l rearrangements d e s c r i b e d i n the l i t e r a t u r e which make the methylene n i t r o n e (24) an a t t r a c t i v e p r e c u r s o r i f the secondary formamide observed by GCMS i n b i l e e x t r a c t s were a c h e m i c a l l y generated a r t i f a c t . b b O + a O + O - b # H — C — N — 0 « •—- H — C = N—-0 - — - H — C — N+ i i \y\ i i H R H R H R F i g u r e 7. Back p o l a r i z a t i o n between two c a n o n i c a l forms of a n i t r o n e . a. Behrend Rearrangement The methyl n i t r o n e (44) (a k e t o - n i t r o n e ) c o u l d be converted by a 1,3-prototropic s h i f t (Behrend rearrangement, f i g u r e 8) to the methylene isomer (24, an a l d o - n i t r o n e ) i n a l k a l i n e media. (Hamer and Macaluso, 1964, Lamchen, 1968, Hei s t a n d , 1978, Smith and G l o y e r , 1975). 19 R * 6+ R \ H - c — N = C H 2 H - p N - C H 2 - = 5 ; ^ = N - M e Me O " Me 0 ~ Me O " Figure 8. Behrend rearrangement of a N-methyl nitrone to a methylene n i t rone . b. Hartynoff Rearrangement Rearrangement of the nitrones 24 and 44 under thermal condit ions should a f ford the isomeric oxime ethers 16 and 45 respec t ive ly . ( V i l l a r r e a l and Grubbs, 1978). Nitrones have also been shown to isomerize to amides when heated. Oxaz ir id ines have been proposed as intermediates in th i s react ion (Emmons, 1957, Larson, et al .,. 1970) GC phase or in t erna l surface e f fects on the s t a b i l i t y of amphetamine nitrones have been observed (Coutts, et al .,1978a). On column decomposition of the nitrone subst i tuted benzodiazepines, chlordiazepoxide and demoxepam has caused problems with GCMS analys i s (Joyce, et al . , 1984). c . Beckmann Rearrangement The Beckmann rearrangement of nitrones to amides i s catalyzed by a number of acy la t ing agents (Beckmann, 1890, 1893, 1905, 1909). Several mechanisms have been proposed. These have been reviewed and modified by Lamchen (1968) to the acy la t ion mechanism shown in f igure 9a. 20 Basic c o n d i t i o n s have a l s o converted n i t r o n e s to t h e i r i s o m e r i c amides ( B i g i a v i and M a r r i , 1934, Hamer and Macaluso, 1964, Umezawa, 1960, Zinner, 1978). T h i s rearrangement a p p l i e d t o the methylene n i t r o n e (24) would give r i s e to the formamide (12). Zinner (1978) has proposed a N-hydroxy-N,0-acetal ( n i t r o n e a l c o h o l a t e ) i n t e r m e d i a t e i n the a l k a l i c a t a l y z e d rearrangement ( f i g u r e 9b). I f the UDP-/"-Glucuronyl-transferase enzyme were i n s e r t e d i n the Beckmann rearrangement mechanism as the a c y l a t i n g agent, a n i t r o n e N,0- g l u c u r o n i d e i n t e r n a l s a l t (46), a z w i t t e r ion s i m i l a r to the N-glucuronides of t e r t i a r y amines ( C a l d w e l l , 1982) would be formed from a methylene n i t r o n e in vivo. In t h i s case the formamide (12) would be l i b e r a t e d by enzymatic h y d r o l y s i s of the n i t r o n e g l u c u r o n i d e ( f i g u r e 10). H y d r o l y s i s of the same conjugate of the N-methyl n i t r o n e (44) would a f f o r d diphenylbutanone (14). d. Photochemical rearrangements Pathways A and B i n F i g u r e 5 c o u l d be l i n k e d by a photochemical rearrangement of n i t r o n e (24) to o x a z i r i d i n e (25) (Spence, et al., 1970). The f r e e r a d i c a l mechanism shown i n f i g u r e 11 was used by Lamchen (1968) to account f o r most n i t r o n e to o x a z i r i d i n e photorearrangements. 21 H H-C=N-R 24 0>^<K5 Me H-C=N-R OAc H l H-^-N-R I Ac R = Ph 2CHCH 2CHMe X=OAc or Cl H. KC-0 I Me -AcX 0 12 H H-C-N-R }o4a Me f i g . 6 H - C - N — R 25 + AcX Figure 9a. Mechanism for the Beckmann rearrangement of nitrones to amides catalyzed by acylating agents (Lamchen, 1968). OH" OH" H I i H 0" 24 kH-0-C = N— R I H H H H 20 + 0 = C-N-R 12 H O - C T N - R + O H " H H ) H -H 20 HO—C =N —R R = Ph2CHCH2CHMe Figure 9b. Mechanism for the Beckmann rearrangement of nitrones to amides catalyzed by a l k a l i (Lamchen, 1968, Zinner, 1978). UDP I C-OH CHOH1 CHOH I Glucuronyl transferase H O - C - C H O H I C O f R = Ph 2 CHCH 2 CHMe h =GA 0 -Glu 0 -Glu = ^-Glucuronidase H l ^ + H-C=N-R O + UDP 4 6 I C-OH l GA H H - C - N - R V C -OH I GA a H H - C - N - R — Wja j 3 - G l u - ^ c - O H I GA - 6 - G l u - C - O H i GA O 12 H 25 V + tf-Glu-C-OH ^ i GA Figure 10. A modified Beckmann rearrangement of a hypothetical nitrone glucuronide (46) catalyzed by /3-glucuronidase (after Lamchen, 1968). 23 H \ 0 " H / c — F i g u r e 11. Free r a d i c a l mechanism for the photochemical rearrangement of a n i t r o n e to an o x a z i r i d i n e (Lamchen, 1968). These chemical f i n d i n g s i n d i c a t e t h a t chemical and thermal i s o m e r i z a t i o n s of the methylene n i t r o n e (24) c o u l d r e s u l t i n the o b s e r v a t i o n of a secondary formamide (12) by GCMS. E. Formamide m e t a b o l i t e s a r i s i n g from a carbinolamide  p r e c u r s o r i . S t a b i l i t y and occurrence of carbinolamine and c a r b i n o l a m i d e s i n t e r m e d i a t e s i n drug metabolism s t u d i e s The mechanism shown in f i g u r e 12 i n v o l v e s a carbinolamine mediated four e l e c t r o n o x i d a t i o n of a t e r t i a r y amine to a t e r t i a r y formamide. In non-basic t e r t i a r y amines such as N,N-d i m e t h y l a n i l i n e and t e r t i a r y N-methylamides (Ross, et al . , 1983) t h i s i s a p o s s i b i l i t y s i n c e the intermediate carbinolamine i s r e l a t i v e l y s t a b l e due to resonance e l e c t r o n d e l o c a l i z a t i o n and i s long l i v e d enough to undergo f u r t h e r o x i d a t i o n t o the c a r b i n o l a m i d e (47) or be conjugated with g l u c u r o n i c a c i d ( A l l e n , et al., 1971, McMahon and S u l l i v a n , 1965). 24 In b a s i c t e r t i a r y amines however, i t i s commonly accepted that the h i g h e l e c t r o n d e n s i t y r e s u l t s i n a spontaneous h y d r o l y s i s of the carbinolamine to the d e s a l k y l compound p l u s formaldehyde. Nonetheless, there are ex c e p t i o n s to the spontaneous h y d r o l y s i s of b a s i c carbinolamine d e a l k y l a t i o n i n t e r m e d i a t e s such as i n the metabolic o x i d a t i o n s of p y r r o l i d i n e drugs through the carbinolamine t o lactams (Hucker, 1973). The t e r t i a r y formamide (26) shown i n f i g u r e 12 i s a non-b a s i c compound, i d e a l l y s u i t e d to metabolic c a r b i n o l a m i d e formation and g l u c u r o n i d e c o n j u g a t i o n . H y d r o l y s i s of the carb i n o l a m i d e g l u c u r o n i d e (48) f o l l o w e d by decomposition of the carbinolamide aglycone (47) d u r i n g sample e x t r a c t i o n would r e s u l t i n the o b s e r v a t i o n of a secondary formamide (12). R — C H —N Me, -4e_ • ' 2 C H 3 9 2 I R — CH — N — Me -2e R - C H - N - C H 2 O H I C H , C H , 26 R= Ph2CH C H 2 47 - C H 2 0 9 ^ O ^ yC0OH 1 R - C H - f i - C H j O - / > 2 R - C H - N - H - 3 48 HH0V»H - 3 » H OH F i g u r e 12. Metabolism of r e c i p a v r i n (9) to the secondary formamide (12) v i a a h y p o t h e t i c a l c a r b i n o l a m i d e pathway. 25 i i . Metabolism of formamides R e a c t i v e m e t a b o l i t e s of the a n t i c a n c e r agent l i -me thy 1 formamide (NMF) (Pearson, et al., I987ab) and DMF ( S c a i l t e u r and Lauwerys, 1987) are known to r e s u l t in h e p a t o t o x i c i t y . Although the metabolism of these simple a l i p h a t i c formamides has been e x t e n s i v e l y i n v e s t i g a t e d ( K e s t e l l , et al., 1985, 1987, S c a i l t e u r , et al., 1984, B r i n d l e y , et a/., 1983), there are very few r e p o r t s on the metabolism of higher formamides. Borchert et al ., (1981) have s t u d i e d the demethylation r a t e of a t e r t i a r y formamide analogue of methamphetamine. Swaminathan and Bryan (1984) have s t u d i e d the metabolism of the u r i n a r y bladder carcinogen N-[4-( 5 - n i t r o - 2 - f u r y l ) - 2 - t h i a z o l y l ] formamide (FANFT). No other r e p o r t s on the metabolism of formamides with a l k y l chains l a r g e r than t - b u t y l are a v a i l a b l e . i i i . Importance of metabolic s t u d i e s on a r y l a l i p h a t i c formamides Amphetamine, methamphetamine, methylenedioxyamphetamine (MDA) and N-methyl methylenedioxyamphetamine (MMDA) are o f t e n i l l e g a l l y manufactured by the Leuckart-Wallach r e a c t i o n (Frank, 1983) and as a r e s u l t of incomplete h y d r o l y s i s , o f t e n c o n t a i n a r y l a l i p h a t i c formamides as w e l l as other i m p u r i t i e s ( L e B e l l e , et al., 1973, Kram, et al., 1977). 26 The recipavrin formamides 12 and 26 were structurally similar to the amphetamine formamides. Metabolic studies were undertaken to characterize potentially hepatotoxic compounds of importance to amphetamine abuse, and as part of a study on the source of formamide metabolites of the tertiary arylal iphatic amine recipavrin (9) . 2i_0§JECTIVES_QF_THE_THE Considering the novel nature and potential toxicity of formamide metabolites, this work set out to c lar i fy the mechanism of formamide generation with the following main objectives. 1. Studies on the source of the methadone formamide metabolite and the peroxidation of EDDP (1) were to be completed. Solvent effects on the recovery of the formamide metabolite, perturbations of the reaction conditions in the EDDP oxidation and the peroxidation of the parent drug methadone were undertaken. 2. Metabolites of recipavrin (9), especially those that may mediate the observation of a secondary formamide metabolite were to be synthesized. Improving the synthesis of the secondary formamide (12) and obtaining better analytical results for the primary hydroxylamine was also necessary. 3. The b i l i ary metabolites of recipavrin (9) were to be characterized in detai l to identify any possible precursors of the formamide metabolite (12). 27 4. The m e t a b o l i t e s of n o r r e c i p a v r i n (15) and d i n o r r e c i p a v r i n (20) were to be c h a r a c t e r i z e d i n d e t a i l to determine whether the d e s a l k y l amines were i n t e r m e d i a t e s i n the generation of the secondary formamide m e t a b o l i t e (12). 5. Formamide analogues of promethazine (10) were s y n t h e s i z e d and metabolic e x t r a c t s from promethazine dosed r a t s were screened f o r formamide m e t a b o l i t e s . 6. The p o s s i b l e chemical g e n e r a t i o n of the secondary formamide m e t a b o l i t e (12) from an isomeric o x a z i r i d i n e (25) or n i t r o n e (24) was to be i n v e s t i g a t e d . 7. The metabolism of the r e c i p a v r i n t e r t i a r y formamide (26) as an i n t e r m e d i a t e i n the c a r b i n o l a m i d e (47) mediated formation of the secondary formamide m e t a b o l i t e (12) of r e c i p a v r i n was to be i n v e s t i g a t e d . 8. The m e t a b o l i t e s of the t e r t i a r y formamide (26) and secondary formamide (12) analogues of r e c i p a v r i n were to c h a r a c t e r i z e d as of p o t e n t i a l l y t o x i c m e t a b o l i t e s of Leuckart s p e c i f i c formamides. 28 I I . EXPERIMENTAL li_Qy§MIQ ALS_AND_MATERIALS Chemicals were reagent grade and obtained from the f o l l o w i n g sources. A l d r i c h Chemical Co.(Milwaukee, Wisconsin) 1,3-Dihydronaphthalene, 4-dimethylaminopyridine, aluminum c h l o r i d e , benzalacetone, c a l c i u m c h l o r i d e , deuterochloroform ( g o l d l a b e l ) , d i e t h y l e n e g l y c o l , d i p h e n y l a c e t o n i t r i l e , e t h y l c h l o r o f o r m a t e , D-glucuronic a c i d , i m i n o d i b e n z y l , l i t h i u m aluminum h y d r i d e , methylchloroformate, n - b u t y l l i t h i u m (1.6 M i n hexane), N-methylhydroxylamine h y d r o c h l o r i d e , para-n i t r o p e r b e n z o i c a c i d , p h e n o t h i a z i n e , p r o p a r g y l bromide, sodium h y d r i d e , sodium cyanoborohydride, t e t r a h y d r o f u r a n , t r i c h l o r o m e t h y l c h l o r o f o r m a t e , t r i e t h y l a m i n e . A l l i e d Chemical (New York, N.Y.) F e r r o u s s u l f a t e , sodium a c e t a t e American S c i e n t i f i c and Chemical Co. ( S e a t t l e , Washington) Formaldehyde (38%) aqueous, h y d r o c h l o r i c a c i d , potassium hydroxide, sodium hydroxide, s u l f u r i c a c i d . A p p l i e d Science L a b o r a t o r i e s ( S t a t e C o l l e g e , Pennsylvania) Dexsil-300 on Gas Chrom Q, a m b e r l i t e XAD-2 r e s i n . 29 J.T. Baker L t d . (Phi 1ipsburg, New Jersey) F l a s h chromatography s u p p l i e s and s o l i d phase e x t r a c t i o n apparatus. BDH Chemicals (Toronto, O n t a r i o ) Acetone, a c e t o n i t r i l e , benzene, calcium carbonate, c h l o r o f o r m , e t h e r (anhydrous), ethylformate, h y d r o c h l o r i c a c i d , hydroxylamine h y d r o c h l o r i d e , l i g r o i n e (60-80°), magnesium s u l f a t e (anhydrous), pet. ether (30-60°), p y r i d i n e , sodium c h l o r i d e , sodium s u l f a t e (anhydrous), toluene. Brinkmann Instruments (Toronto, Ontario) Dragendorf's reagent. Caledon L a b o r a t o r i e s L t d . (Georgetown, Ontario) A l l Caledon p r o d u c t s were " d i s t i l l e d - i n - g l a s s " grade. A c e t o n i t r i l e , dichloromethane, e t h y l a c e t a t e , methanol (HPLC grade), water (HPLC grade). Eastman Kodak Co. (Rochester, New York) T h i n l a y e r chromatograms ( s i l i c a g e l 0.2 mm), methylamine (40% aqueous). F i s h e r S c i e n t i f i c Co. ( F a i r l a w n , New Jersey) Magnesium c h l o r i d e , sodium bicarbonate. Kabivitrum L t d . (Stockholm, Sweden) 30 N-ethyl-N,a-dimethyl-7-phenylbenzenepropanamine, Recipavrin C^ H3 (a-trideuteromethyl-N,N-dimethyl-7-phenylbenzenepropanamine), t e r o d i l i n e HC1 Linde Co. (Union Carbide, Vancouver B . C . ) Molecular sieve Type 4A Mal l inkrodt Chemicals (St . L o u i s , Missouri) Potassium carbonate (anhydrous), sodium bicarbonate, sodium s u l f a t e . Matheson L t d . (Edmonton, Alberta) Hydrogen ch lor ide gas. Merck L t d . (Rahway, New Jersey) Yellow mercuric oxide. Merck Sharpe and Dohme (Isotopes) (Montreal Que.) Deuterium oxide, Dimethylsulfoxide-Dg (DMSO-Dg) Matheson Coleman and B e l l Co. (Norward, Ohio) Dimethylsul foxide , methylacrylate , potassium carbonate. P ierce Chemical Co. (Rockford, I l l i n o i s ) N,0 - b i s - ( t r i m e t h y l s i l y l ) t r i f l u o r o a c e t a m i d e (BSTFA), 0-methylhydroxylamine HC1 (MOX), Trimethyl an i l in ium hydroxide (TMAH), a c e t o n i t r i l e ( s i l y l a t i o n grade). 31 Sigma Chemical Co. (St. Louis M i . ) Amphetamine su l fa t e , g lucurase R , su l fatase , phenolphthalein glucuronide. Supelco L t d . (Bel lafonte , Pa.) Dimethylchloros i lane . Stanchem L t d . (Winnipeg, Manitoba) Ethanol 95%. Synthesized in our laboratory (Abbott, et al . , 1979) 2 ,2 -Diphenyl -4-d imethylaminovaleroni tr i l e , ( +)-2-ethyl-1 , 5 -d imethy l -3 ,3 -d iphenylpyrro l in ium perchlorate (EDDP), methadone hydrochlor ide , 4,4-diphenyl-2,5-heptanedione (Abbott et al . , 1986). Terochem (Edmonton, Alberta) Meta-chloroperbenzoic ac id (85%). ? i _ AN IMAL EXPERIMENTS Animals and s u r g i c a l equipment were obtained from the fo l lowing s u p p l i e r s . Animal Care F a c i l i t y ( U . B . C , Vancouver, B . C . ) Wistar male rats (200 - 300g) Becton Dickinson (Rutherford, New Jersey) Yale needle 22G, 1 1/2", tubercul in syringe 1 cc 32 Cla y Adams (Parsippany, New J e r s e y ) P o l y e t h y l e n e t ubing PE 10 E t h i c o n L t d . (Peterborough, O n t a r i o ) 4-0 S i l k Permabond I n t e r n a t i o n a l D iv. (Englewood, New J e r s e y ) 910 R Adhesive 3i_PREPARATION_OF_TLC_SPRAY_R A. N a p h t h o r e s o r c i n o l spray reagent (Heyns and Ke l c h , 1953) 1,3-Dihydronaphthalene (200 mg) was d i s s o l v e d i n a s o l u t i o n of 166 ml of 95% et h a n o l and 34 ml of 85% phosphoric ac i d . B. Dragendorf's Reagent Equal volumes of Dragendorf's A and B s o l u t i o n (Brinkman Instruments, Toronto) were mixed and s t o r e d i n the r e f r i g e r a t o r u n t i l used. C. T o l l e n ' s Reagent T o l l e n ' s reagent was prepared a c c o r d i n g to Vogel (1956) and used immediately. Excess was di s p o s e d of immediately due to the e x p l o s i o n hazard. 33 4. INSTRUMENTATION A. NMR spectra NMR spectra were recorded in CDCI3 using tetramethyls i lane (TMS) as an in terna l standard on a Bruker WP-400, WP-80, or Oxford-Nicolet-270 spectrometer at the Department of Chemistry, UBC. A l l NMR data are reported as fo l lows: NMR (MHz): s h i f t in ppm from TMS ( s p l i t t i n g , coupling constant , assignment). For example, NMR (400 MHz): 1.16 (d, J=8Hz, CH 3 CH). B. . Infrared spectra Infrared spectra were recorded as l i q u i d f i lms or nujol mulls on NaCI plates using a Unicam SP-1000 spectrometer. A l l i n f r a r e d data are reported as fol lows: IR ( f i lm , so lut ion ( th ickness ) , or mul l ) : frequency in c m - 1 ( in tens i ty , assignment). For example: IR (nujol mul l ) : 1710 (s, C=0 s t r . ) . C. U l t r a v i o l e t spectra U l t r a v i o l e t spectra were recorded on a Beckmann Model 24 spectrometer. Absorbances are reported as fol lows: UV (so lvent) : Lambda max in nm, (Molar ex t inc t ion c o e f f i c i e n t , assignment). For example: UV Spectrum (methanol) 207 (18000, p i - p i * arom.) . D. Melt ing points Melt ing points were determined on a Thomas Hoover c a p i l l a r y melting point apparatus and are uncorrected. 34 E . C a p i l l a r y GCMS A l l mass spectra are reported as fol lows: GCMS (column)(capi l lary column assumed unless otherwise s p e c i f i e d ) ) : M + (% i n t e n s i t y ) ; base peak (100%); next strongest peak (% i n t e n s i t y ) ; e tc . Standard operating condit ions are assumed unless otherwise s p e c i f i e d . C a p i l l a r y GCMS was performed on a Hewlett Packard 59B7A GCMS system using a cross l inked methyls i l icone fused s i l i c a c a p i l l a r y column (HP19091-112), 25 meters by 0.32 mm i . d . (capacity approximately 250 ng per component). Under standard condi t ions , column temperature was programmed from 5 0 ° to 1 5 0 ° at 3 0 ° per minute , then at 4 ° per minute to 2 6 0 ° and then held constant for two minutes. Inject ions (1 uL) were made in s p l i t l e s s mode with helium c a r r i e r gas back pressure set at 15 p s i . In jec t ion port , interface and source temperatures were 2 5 0 ° . The solvent d iver ter was l e f t on and data a c q u i s i t i o n was delayed u n t i l 5 minutes a f ter each i n j e c t i o n . The mass range scanned was 40 to 400 AMU with a m u l t i p l i e r voltage of 2300, emission current 300 uA and ion iza t ion energy 70 eV. Modi f icat ions of standard GCMS operating condi t ions: i . Condit ion B: Standard operating condit ions with helium back pressure set at 8 p s i . 35 i i . C o n d i t i o n C : F o r p r o m e t h a z i n e r e l a t e d c o m p o u n d s , S t a n d a r d o p e r a t i n g c o n d i t i o n s , w i t h o v e n t e m p e r a t u r e p r o g r a m m e d f r o m 50° t o 200° a t 30° p e r m i n u t e a n d t h e n f r o m 200° t o 260° a t 4 ° p e r m i n u t e . H e l i u m b a c k p r e s s u r e was 15 p s i . i i i . C o n d i t i o n D : F o r p r e l i m i n a r y c h a r a c t e r i z a t i o n o f r e c i p a v r i n r e l a t e d c o m p o u n d s a n d s p i k i n g e x p e r i m e n t s a 25 M by 0 . 2 mm S E - 5 4 c o l u m n ( H e w l e t t P a c k a r d p a r t number 1 9 0 9 1 - 6 1 7 2 5 ) was u s e d u n d e r s t a n d a r d o p e r a t i n g c o n d i t i o n s . i v . C o n d i t i o n E : F o r s o l v e n t c o n t r o l e x p e r i m e n t s on m e t h a d o n e , o v e n t e m p e r a t u r e was p r o g r a m m e d f r o m 5 0 ° t o 1 5 0 ° a t 3 0 ° p e r m i n u t e , h e l d c o n s t a n t a t 1 5 0 ° f o r 5 m i n u t e s t h e n p r o g r a m m e d f r o m 1 5 0 ° t o 2 8 0 ° a t 1 0 ° p e r m i n u t e a n d h e l d c o n s t a n t a t 2 8 0 ° f o r 12 m i n u t e s . H e l i u m b a c k p r e s s u r e was 15 p s i . F . D i r e c t i n s e r t i o n p r o b e mass s p e c t r a D i r e c t i n s e r t i o n p r o b e mass s p e c t r a w e r e done on t h e H e w l e t t P a c k a r d 5987A w i t h t e m p e r a t u r e p r o g r a m m e d d e s o r p t i o n f r o m t h e p r o b e . T h e p r o b e c r u c i b l e was l o a d e d w i t h 1 u l o f a 1 u g / u l s o l u t i o n o f t h e a n a l y t e . A f t e r t h e m e t h a n o l h a d e v a p o r a t e d t h e p r o b e was i n s e r t e d a n d t h e p r o b e t e m p e r a t u r e was h e l d c o n s t a n t a t 5 0 ° f o r one m i n u t e a n d t h e n p r o g r a m m e d t o i n c r e a s e a t 3 0 ° p e r m i n u t e t o a maximum o f 3 5 0 ° . 36 G. Packed column GCMS a n a l y s i s Packed column GCMS a n a l y s i s was done on a Hewlett Packard 5700A gas chromatograph i n t e r f a c e d to a V a r i a n MAT 111 mass spectrometer v i a a v a r i a b l e s l i t s e p a r a t o r . E l e c t r o n impact mass s p e c t r a were recorded at 70 eV, ion source pressure 5x 10~ 6 t o r r , emission c u r r e n t 300 uA, and source temperature 250°. Computerized background s u b t r a c t i o n s were made and mass s p e c t r a were recorded over a scan range of 40 to 750 amu with one scan every f i v e seconds. T o t a l ion c u r r e n t (TIC) p l o t s were based on m/z 50-500. Mass chromatograms were p l o t t e d i n scan mode. The data was processed by an o n l i n e V a r i a n 620L computer system. A s i l a n i z e d g l a s s column (2m x 2mm i.d.) packed with 3% D e x s i l 300 on 80/100 mesh Chromosorb W-AW (Supelco, B e l l e f o n t e , Pennsylvania) was used with helium c a r r i e r gas (20 ml/min.). Under standard c o n d i t i o n s column temperature was programmed from 150-280° at 4° per minute and then h e l d constant f o r 8 minutes. The i n j e c t i o n p o r t , l i n e and sep a r a t o r temperatures were h e l d at 250°. Other GCMS o p e r a t i n g c o n d i t i o n s : i . C o n d i t i o n F: As above with column temperature programmed from 200 to 280° at 8° per minute. i i . C o n d i t i o n G: For amphetamine r e l a t e d compounds, column temperature was h e l d at 150° f o r 4 minutes,.then i n c r e a s e d at 4° per minute to 270° and then h e l d constant f o r 4 minutes. H. L i q u i d chromatography-mass spectrometry 37 HPLC grade a c e t o n i t r i l e and water were passed through a 0.45 u f i l t e r , degassed under water a s p i r a t o r vacuum p r i o r to use, and t r e a t e d with helium d u r i n g l i q u i d chromatography. A Hewlett Packard model 1090 l i q u i d chromatograph with a v a r i a b l e volume automatic loop i n j e c t o r , HP3392 i n t e g r a t o r , f i x e d wavelength (254 nm) d e t e c t o r and HP-85 mi c r o p r o c e s s o r was used. The LC was equipped with a 100 x 2.1 mm H y p e r s i l ODS column (5u p a r t i c l e s i z e , HP c a t a l o g u e no. 79916). Stock s o l u t i o n s were prepared i n a c e t o n i t r i l e (10 ng/ul) and passed / through a 13 mm diameter 0.45 u Alpha-450 f i l t e r (Gelman, Ann Arbour, Michigan) before i n j e c t i o n i n t o the LC. The conjugated b i l e f r a c t i o n e q u i v a l e n t to 8 ml of r a t b i l e , was blown f r e e of methanol with n i t r o g e n and r e c o n s t i t u t e d with 0.5 ml of a c e t o n i t r i l e . The sample was f i l t e r e d f r e e of s o l i d s (0.45 u Alpha-450 f i l t e r ) ; the f i l t e r was washed with 0.5 ml a c e t o n i t r i l e . The combined f i l t r a t e s were used f o r LCMS. Two u l i n j e c t i o n s were used with a flow r a t e of 200 u l / min. (column pressure=44 p s i ) and s o l v e n t r a t i o 65% water 35% a c e t o n i t r i l e , to o p t i m a l l y r e s o l v e standards and i n t e r f e r i n g peaks i n b i l e samples. The oven temperature was h e l d at 40°. A f t e r p a s s i n g through the UV d e t e c t o r , the e l u e n t e n t e r e d a sample t r a n s f e r l i n e and i n l i n e f i l t e r (HP p a r t no. 38 3150-0369) connected to a Hewlett Packard d i rec t l i q u i d in troduct ion (DLl) LCMS interface (option number 004). The s p l i t r a t i o in the DLl was determined experimentally to be 13% (26 u l / m i n . enter ing the mass spectrometer). For LCMS, the HP 5987A MS system was operated in pos i t ive chemical i on iza t ion mode with a s table source pressure of 1.3 x 10"^ t o r r , source temperature 2 5 0 ° , emission current 300 uA, repe l l er voltage 8 V, m u l t i p l i e r voltage 2400 eV and ion izat ion energy 240 eV. Sui tab le mass spectra of the secondary formamide and methylene ni trone standards were obtained with 100 ng/ul standard so lu t ion i n j e c t i o n s . Then ion monitoring of the m/z 254 (M ++1), 238 (M ++1-16), and 295 (M++1+CH3CN) ions (200 microsecond dwell time on each ion) was done on a 10 ng/ul mixture of the two standards. A blank in jec t ion was then made to ensure no carryover occurred from the standard mixture. Then the conjugated b i l e extract was analyzed by selected ion monitoring LCMS to detect the presence of the nitrone and/or formamide. I . High r e s o l u t i o n mass spectrometry High r e s o l u t i o n mass spectra were recorded on a Kratos MS 50 high performance mass spectrometer in e lectron impact mode at 70 eV with a source temperature of 1 2 0 ° . The scan range was 31-743 amu. 39 5iMETABQLISM_EXPERIMENTS A. Animal Surgery and Drug Administrat ion Male Wistar rats were fasted for 18 hours before surgery. The rats were anaesthetized with ether and the b i l e duct was cannulated with PE-10 tubing. The cannula, secured with s i l k thread and a small drop of Kodak 901 adhesive, was passed through the abdominal wall and under the skin to the back of the neck where i t was e x t e r i o r i z e d . The abdomen was closed with interrupted sutures and the rat was placed in a re s tra in t cage. F i f t een minutes af ter recovery from anaesthesia, the rat was given a subcutaneous i n j e c t i o n of rec ipavr in hydrochloride (10 mg in 0.1 ml of s t e r i l e d i s t i l l e d water). B i l e and urine were c o l l e c t e d for 18 hours. A second dose of drug was given 12 hours after the f i r s t dose. The b i l e cannula was threaded through an 18 gauge needle p i e r c i n g the rubber cap of a p l a i n Vacuta iner R tube. The c o l l e c t i o n tube was held in an ice bath and protected from l i g h t during the c o l l e c t i o n i n t e r v a l . The 18 hour b i l e sample (12-18 ml) was d i l u t e d to 20 ml, mixed wel l and worked up immediately or stored frozen at - 7 0 ° . The fol lowing drugs and doses were administered by subcutaneous (sc) or in traper i tonea l (ip) i n j e c t i o n to at l east 3 b i l e duct cannulated animals: 40 Recipavrin HC1: 10 mg/0.1 ml H 20 sc, Recipavrin 2 H 3 HC1: 10 mg/0.1 ml H 20 sc, Norrecipavrin HC1: 10 mg/0.1 ml H20 sc, Dinorrecipavrin HC1: 10 mg/0.1 ml H 20 sc, Recipavrin t e r t i a r y formamide 10 mg/0.1 ml corn o i l sc, Recipavrin secondary formamide: 10 mg/0.1 ml corn o i l sc (1 animal only), Promethazine HC1 (Phenergan R): 12.5 mg/0.1 ml H 20 sc (1 animal only), Recipavrin methylene nitrone 10 mg/0.1 ml ethanol:propylene g l y c o l 1:1 sc. (1 animal only). B. B i l e sample preparation for GCMS In 10 x 1.5 cm te f l o n capped centrifuge tubes, 2 ml aliquots of b i l e (pH 8.3) were centrifuged at 2000 rpm for 15 minutes, decanted and d i l u t e d to 4 ml with water and adjusted to pH 10 with a combination of 0.1M NaOH and a 0.12 sodium borate buffer. Nonconjugated metabolites were extracted with three, three ml aliquots of one of the following extraction solvents: 1. D i s t i l l e d in glass grade ethyl acetate, 2. D i s t i l l e d in glass grade chloroform containing 1% ethanol, 3. D i s t i l l e d in glass grade methylene ch l o r i d e , 4. Reagent grade benzene, 5. Freshly d i s t i l l e d ether. Samples were vortexed vigorously and centrifuged to separate phases. The organic phases were dried over K 2C0 3 (CHC13, CH 2C1 2) or Na 2S0 4 (ethyl acetate, benzene and ether) and then evaporated to dryness in a 38° water bath under a stream of dry nitrogen. Samples were stored at -5° and reconstituted with 20 ul of reagent grade methanol or HPLC grade a c e t o n i t r i l e just p r i o r to GCMS analysis. Samples were concentrated and rerun under column 41 overload conditions to detect minor metabolites. The aqueous remainder containing conjugated metabolites was adjusted to pH 5 with 1 M acetic acid and l y o p h i l i z e d . The residue was reconstituted in 2 ml of 0.1 M, pH 5 sodium acetate buffer, then 0.2 ml of Glucurase R was added. After a gentle mix, the sample was incubated for 18 hours at 38°. The hydrolyzed recipavrin metabolites were then extracted as outl i n e d above, using the same solvent chosen for the extraction of nonconjugated metabolites. Ethyl acetate ( d i s t i l l e d - i n - g l a s s grade) was adopted as the solvent of choice for metabolite characterization. C. D e r i v a t i z a t i o n Methods i . T r i m e t h y l s i l y l a t i o n The conjugated b i l e extract was transferred to a one ml reaction v i a l , evaporated free of methanol and d i l u t e d with 30 ul of s i l y l a t i o n grade a c e t o n i t r i l e and 30 ul of BSTFA. After heating at 60° for four hours, the sample was concentrated under a stream of nitrogen and analyzed by GCMS. i i . Methylation a. N- and O-methylation The methanolic b i l e extract was diluted with 50 ul of methelute R. The sample was concentrated under dry nitrogen and analyzed by GCMS. 42 b. O-Methylation The methanolic b i l e extract was d i l u t e d to 0.5 ml with methanol. Diazomethane, prepared from D i a z a l d R was bubbled through the solution for one minute. The sample was concentrated under nitrogen and analyzed by GCMS. §i_QHEMICAL_SYNTHESES A. Flash chromatography Using the method of S t i l l et al., (1978), a 15 x 1 cm bed of flash chromatography grade s i l i c a gel was packed dry over a 0.5 cm bed of fine glass beads. Eluent, proven by TLC to have s u f f i c i e n t p o l a r i t y to move the desired component to an rf of 0. 3-0.4 was forced through the column under pressure with nitrogen gas u n t i l no a i r bubbles remained in the packing. The samples (100 mg) were loaded dissolved in 0.5 ml of mobile phase and the column was run at 2 .ml per minute. Two ml fractions were c o l l e c t e d . The same procedure was followed using a 2.5 x 15 cm column loaded with up to 500 mg of sample and 5 ml fractio n s were c o l l e c t e d . B. Synthetic compounds related to methadone The i s o l a t i o n and chara c t e r i z a t i o n of the major oxidation products of EDDP (1) were reported previously (Slatter, 1983, Abbott, Sl a t t e r and Kang, 1986). 1. General procedure for meta-chloroperbenzoic acid (MCPBA) oxidations of methadone (8) and EDDP (1). 43 Method 1: To a s o l u t i o n of 100 mg (2.6 x 1 0 - 4 mol) of (-) EDDP p e r c h l o r a t e i n 5 ml of CHC13 at 0°, was added 100 mg (5.2 x 10" 4 mol) of MCPBA i n 5 ml of 0° CHCI3. A f t e r standing at 0° f o r 12 hours the r e a c t i o n mixture was f i l t e r e d , washed twice w i t h . c o l d 1.5 M NaOH s o l u t i o n , twice with water, and d r i e d over K2CO3 and evaporated. The yellow o i l was analyzed by packed column GCMS (3% OV-17 on 100-120 mesh Gas Chrom Q, 150-280° at 4° per min) and found to c o n t a i n EMDP (5) (tr=l8.2 min.), EDDP (1) (tr=20.0 min), diketone (3) (tr=21.8 min.), DDP (4) (tr=24.2 min.), and the o x a z i r i d i n e (2) ( e l u t e d as the formamide ( 6 ) , tr=29.5 min.). The i n d i v i d u a l components were i s o l a t e d i n pure form as d e s c r i b e d p r e v i o u s l y ( S l a t t e r , 1983, Abbott, et al., 1986). High r e s o l u t i o n mass spectrum of the o x a z i r i d i n e (2), (source temp.=120°): M + 309 (1.5, C20H23NO2); 222 (28, C-jgHigN); 56 (80, C3H6N) , remaining ions a r i s e from thermal d e g r a d a t i o n t o the formamide, 73 (100, C 3H 6NO); 253 (60, C 1 7 H 1 9 N 0 ) ; 207 ( C 1 6 H 1 5 ) , e t c . Method 2: The above procedure was repeated at one tenth s c a l e u s i n g one e q u i v a l e n t of EDDP f r e e base ( f r e s h l y e x t r a c t e d from pH 10 aqueous s o l u t i o n with chloroform) and 2 e q u i v a l e n t s of MCPBA over suspended excess K2CO3 (50 mg). Method 3: The above procedure was repeated at one t e n t h s c a l e u s i n g one e q u i v a l e n t of methadone HCI and 2 e q u i v a l e n t s of MCPBA. 44 i i . GCMS i d e n t i f i c a t i o n of 6-N-methylformamido-4,4-diphenyl-3-heptanone (Methadone t e r t i a r y formamide (49 ) ) . In methadone o x i d a t i o n mixtures and o l d nonconjugated b i l e e x t r a c t s a compound with the mass spectrum summarized below was observed. GCMS of 49: ( C o n d i t i o n F, tr=14.17 min.) M + 323 (0); 86 (100); 58 (41); 87 (40); 207 (25); 105 (20); 129 (18); 267 (18); 208 (15); 72 (14); 30 (13); 165 (9). i i i . S y n t h e s i s of 2,3-dimethyl-5,5-diphenylcyclopent-2-enone (50) The diketone (3) (4,4-diphenyl-2,5-heptanedione) was d i s s o l v e d i n EtOH and heated under r e f l u x i n an equal volume of 0.75 M e t h a n o l i c sodium hydroxide s o l u t i o n f o r three hours. The s o l u t i o n was d i l u t e d with 4 volumes of water and e x t r a c t e d w i t h e t h e r . The ether phase was washed twice with water d r i e d over Na2S0 4, and evaporated. The brown o i l was f l a s h chromatographed i n 98:2 hexane:ethyl a c e t a t e . Only one new component was pr e s e n t . I t re a c t e d q u i c k l y with i o d i n e vapour and gave mass s p e c t r a l r e s u l t s i n accord with a cyclopentenone s t r u c t u r e . The c l e a r o i l was analyzed by UV, IR and NMR and found to c o n t a i n only the t i t l e cyclopentenone and not the regi o i s o m e r 3-ethyl-4,4-diphenylcyclopent-2-enone (51). GCMS of 50: ( c o n d i t i o n F, tr= 6.6 min.): M + 262 (100); 185 (80); 247 (52); 233 (52); 219 (22); 165 (22). 45 NMR (270 MHz): 1.78 (s, CH 3 -C-C=0); 2.13 (s, C H 3 - C - C H 2 ) ; 3.32 (s, C H 2 ) ; 7.12-7.32 (m, C 6 H 5 ) . S ingle ts at 1.78 and 3.32 broadened by homoal lyl ic coupl ing . IR ( f i l m ) : 1697 (s, C=0 s t r . ) ; 1655 (s, C=C s t r . ) . UV (CH 3 CN): 316 (419, cyclopentenone R band); 240 (8,122, cyclopentenone K band); 204 (17,819, arom.); 256 (arom., b u r i e d ) . C . Synthetic compounds re la ted to rec ipavr in Some synthetic 'methodology and spectra for important synthet ic intermediates and precursors of the formamide metabolite (12) have been reported previously ( S l a t t e r , 1983) and are summarized here. Improvements and new spec tra l data are d e t a i l e d . i . 1,1-Diphenyl-3-butanone (14) (CAS r e g i s t r y 5409-60-9) ( S l a t t e r , 1983) Diphenylbutanone was synthesized using the method of Burckhal ter , et al., (1951). B . p . 125° at 0.3 mm. M . p . * 4 6 ° ( l i t . 4 6 ° ) . JH NMR: (100 MHz): 2.08 (s, C H 3 ) ; 3.22 (d, C H 2 ) ; 4.63 (t , CH); 7.15-7.4 (m, P h 2 ) . ( S l a t t e r , 1983) i i . Syn/ant i 1,1-diphenyl-3-butanone oxime (19) (CAS reg i s t ry 36317-57-4)(Slatter, 1983) Using the method of Morgan and Beckett (1975), diphenylbutanone (14) was added to a 1.1 molar excess of 46 hydroxylamine hydrochloride in pH 8.5 methanol solution(pH adjusted with 2M NaOH). The solution was s t i r r e d overnight and d i l u t e d with water, extracted with CHCI3, dried over Na2S04 and d i s t i l l e d (170° at 0.15 mm) to afford a viscous yellow o i l which NMR revealed to be a 2:1 r a t i o of anti and syn isomers. The oxime was also characterized by GCMS as syn and anti TMS ethers (52 a and b) and O-methyl ethers (16 a and b) following d e r i v a t i z a t i o n with BSTFA and TMAH respectively. The oxime methyl ether (16) was also prepared from diphenylbutanone (14) using O-methylhydroxylamine hydrochloride (MOX) reagent under s i m i l a r conditions. 1 H NMR (19) (100 MHz): Anti isomer: 1.8 (s, CH 3); 2.97 (d, CH 2); 4.35 (t, CH); 7.3 (bs (CgH 5) 2); 9.0-9.05 (s, NOH). Syn isomer: 1.57 (s, CH 3); 3.15 (d, CH 2); 4.4 (t, CH); 7.3 (bs ( C 6 H 5 ) 2 ) ; 9.0-9.5 (bs, NOH)(Slatter, 1983). i i i . N,N,a-trimethyl-7-phenylbenzenepropanamine hydrochloride (Recipavrin R (9)) (CAS 13957-55-6)(Slatter, 1983). Using the method of May and Mossetig (1948), 2-dimethylamino-4,4-diphenylvaleronitrile (4 g, (7.46 mmol) was refluxed overnight in 10 ml diethylene gl y c o l containing 4 g KOH. The dark solution was cooled, d i l u t e d with 40 ml H 20 extracted with ether, dried over Na 2S0 4, and f i l t e r e d . P r e c i p i t a t i o n from ether by the dropwise addition of ether saturated with gaseous HC1 afforded 3.66 g (88%) of beige c r y s t a l s . R e c r y s t a l l i z a t i o n twice from benzene afforded 47 white c r y s t a l s m.p. 153° ( l i t . 151-155°) (CAS 22173-83-7). IR ( f i l m , free base): 1600 (m); 1580 (w); 1500 (m); 1450 (m); 1272 (m, amine); 1138 (m);l035 (m); 1060 (m); 750 (s); 715 ( s ) . i v . N,N,a-trimethyl-7-phenylbenzenepropanamine N-oxide (recipavrin N-oxide (53)) Using the method of Craig and Purushothaman (1970), 300 mg of recipavrin hydrochloride was extracted from 5 ml of pH 12 solution with chloroform. Recipavrin free base (262 mg (1.02 mmol) in chloroform was treated with 220 mg MCPBA (1.02 mmol) in chloroform at 0-5°. After s t i r r i n g for three hours, the ice bath was removed and the solution was passed through a 3 x 2.5 cm column packed with 80-200 mesh alumina. The column was washed with chloroform and then the N-oxide was eluted with 25% methanol in chloroform. The solvent was evaporated and the chromatography was repeated. GCMS (decomposes to Cope elimination products c i s and trans 1,1-diphenyl-2-butene (54 and 55)): Cis isomer: tr=7.92 min. M+ 208 (6); 167 (100); 165 (26); 152 (18); 115 (10); 208 (6); 193 (5); 128 (4). Trans isomer: tr=8.26 min. M+ 208 (68); 115 (100); 193 (58); 178 (42); 91 (38); 178 (38); 130 (38); 165 (36). NMR (N-oxide)(80 MHz, some recipavrin present in spectrum): 1.37 (d J=6.4 Hz, CH3CH); 3.03 (d or two singlets J=1.8 Hz, "ON +(CH 3) 2); 3.55-4.10 (m, Ph 2CH); 48 3.0-3.5 (m, CHCH 3); 1.70-2.5 (m, CH 2); 7.05-7.45 (m, P h 2 ) . v. a-Methyl-7-phenylbenzenepropanamine (CAS r e g i s t r y 29869-77-0) ( d i n o r r e c i p a v r i n (20)) ( S l a t t e r , 1983) The primary amine was s y n t h e s i z e d by a c i d h y d r o l y s i s of the secondary formamide (12) f o l l o w e d by e x t r a c t i o n and d i s t i l l a t i o n of the f r e e base ( c l e a r l i q u i d , 170° at 4 mm). The r e d u c t i v e hydroxylamination of diphenylbutanone (14) with ammonium a c e t a t e and sodium cyanoborohydride i n methanol ( S l a t t e r , 1983) was abandoned. The h y d r o c h l o r i d e s a l t was p r e c i p i t a t e d with gaseous HCL (m.p. 172°, L i t . 175° ( B u r c k h a l t e r et al ., 1951)). 1H NMR (300 MHz): ( f r e e b a s e ) : 1.08 (d, CH3); 1.22 (s, NH 2); 1.75 (m, CHCH3); 2.09 (m, CH 2); 4.08, ( t , CHPh 2); 7.22 (m, P h 2 ) . v i . (—) N-hydroxy-a-methyl-7-phenylbenzenepropanamine (primary hydroxylamine (22)) ( S l a t t e r , 1983) The NMR and IR s p e c t r a l r e s u l t s r e p o r t e d p r e v i o u s l y ( S l a t t e r , 1983) f o r the hydroxylamine (22) corresponded to the a u t o x i d i z e d product (56). An improved s y n t h e t i c method i s d e t a i l e d below along with c o r r e c t s p e c t r a l r e s u l t s . Using the method of Morgan and Beckett (1975), a s t i r r e d s o l u t i o n of 0.239 g (0.001 mol) of oxime (19) i n 1.5 mL MeOH was added 63 mg (0.001 mol) sodium 49 cyanoborohydride. HC1 (2M) was then added dropwise to maintain pH 3-4 f o r 15 min. u n t i l gas e v o l u t i o n slowed. The s o l u t i o n was s t i r r e d f o r three hours, then a d j u s t e d to pH 1.5-2.0. A f t e r gas e v o l u t i o n ceased, the MeOH was evaporated and the re s i d u e was d i s s o l v e d i n water, a d j u s t e d to pH 8 with 20% K2CO3, e x t r a c t e d with ether, d r i e d over Na 2S0 4, and con c e n t r a t e d by f l a s h e v aporation at room temperature. The re s i d u e was d i s s o l v e d i n 40:60 hexane:EtOAc and f l a s h chromatographed on a 10 x 1 cm s i l i c a g e l column. A f t e r e l u t i n g the oxime (0-25 mL), the hydroxylamine was c o l l e c t e d (25-70 mL). A f t e r e v a p o r a t i o n the product (48 mg, 20%) was analyzed or used i n subsequent r e a c t i o n s immediately, s i n c e i t was r a p i d l y a u t o x i d i z e d . The product gave black spots when v i s u a l i z e d with T o l l e n ' s reagent a f t e r TLC s e p a r a t i o n . Most of the hydroxylamine decomposed d u r i n g GCMS a n a l y s i s , however a small p o r t i o n s u r v i v e d the GC s e c t o r to give a s a t i s f a c t o r y mass spectrum. The major decomposition product was the primary amine. O-TMS (57) and methyl d e r i v a t i v e s were prepared f o r GCMS a n a l y s i s . The permethyl d e r i v a t i v e (58) was i d e n t i c a l to the O-methylated secondary hydroxylamine. GCMS: ( u n d e r i v a t i z e d ( 2 2 ) ) : tr=l5.02 min.: M+, 241 (9); 60 (100); 44 (73); 165 (64); 167 (55); 208 (32); 152 (27); 91 (18). (TMS d e r i v a t i v e ) : tr= 15.5 min.: M + 313 (8); 132 (100); 44 (52); 116 (50); 75 (30); 167 (30); 118 (20); 91 (10). NMR (400 MHz): 1.09 (d, CH3); 1.9-2.0 (m, CH-jH b); 2.35-2.45 (m, CH aH b); 2.83-2.93 (m, CHCH3); 4.01-4.09 50 (t - d d , CHPh 2); 5.3-5.7 ( b r . s , NHOH); 7.1-7.3 (m, arom.). IR: ( f i l m ) : 3470-3100 (br. s, NHOH s t r . ) ; 3080-2830 (s, s e v e r a l bands); 2000-1700 (w, o v e r t o n e s ) ; 1598 (m, arom.); 1570 (w-m, arom.); 1490 ( s ) ; 1445 ( s ) ; 1365 (m); 1150 (w-m); 1095 (m); 1062 (m); 1025 (m-s); 1005 (br . m); 905 (w); 880 (w); 780 (w-m) 745, 737, ( s ) ; 700 ( s ) . v i i . S p e c t r a l c h a r a c t e r i z a t i o n of the hydroxylamine a u t o x i d a t i o n product (56). NMR (300 MHz): 1.09 (d, CH 3); 1.88-2.0 (m, C H - ^ ) ; 2.32-2.44 (m, GH aH b); 2.8-2.92 (m, CHCH 3); 3.98-4.07 (t-dd , CHPh 2); 7.1-7.3 (m, arom.). Spectrum i s i d e n t i c a l to the hydroxylamine without the NHOH resonance. IR: ( n u j o l ) 3250 (sharp m); 3000-2800 (s, n u j o l ) ; 1598 (w-m, arom.); 1570 (w, arom.); 1495-1450 (s, n u j o l ) ; 1430 (m); 1370 (m, n u j o l ) ; 1150 (w-m); 1110 (w); 1065 (m); 1020 (m-s); 935 (m); 915 (w-m); 850 (w); 835 (w-m); 780 (w-m); 750 ( s ) ; 738 ( s ) ; 702 ( s ) . v i i i . (±.) N,a-dimethyl-7-phenylbenzenepropanamine ( n o r r e c i p a v r i n (15)) (CAS 29869-78-1)(Slatter, 1983) N o r r e c i p a v r i n was s y n t h e s i z e d by r e d u c t i v e amination of diphenylbutanone (14) with methylamine and sodium cyanoborohydride i n a c i d i c MeOH using the method of Morgan and Beckett (1975) ( S l a t t e r , 1983). The h y d r o c h l o r i d e (CAS 61721-58-2) was p r e c i p i t a t e d from ether and r e c r y s t a l l i z e d once from e t h y l a c e t a t e and a c e t o n i t r i l e . The mono t r i m e t h y l s i l y l 51 d e r i v a t i v e was a l s o s y n t h e s i z e d and c h a r a c t e r i z e d by GCMS. N o r r e c i p a v r i n was a l s o o btained by a c i d h y d r o l y s i s of the t e r t i a r y formamide (26). i x . (—) N-hydroxy-N,a-dimethyl - 7-phenylbenzenepropanamine (secondary hydroxylamine ( 1 7 ) ) ( S l a t t e r , 1983) To 0.5 mL of an aqueous s o l u t i o n of 0.42 g (0.005 mol) N-methylhydroxylamine h y d r o c h l o r i d e was added 1.0 g (0.004 mol) diphenylbutanone (14) i n 25 mL MeOH. A f t e r a d j u s t i n g to pH 6, 0.35 g of sodium cyanoborohydride was added and pH 5-6 was maintained by the dropwise a d d i t i o n of 5% HCI, u n t i l the pH remained c o n s t a n t . A f t e r s t i r r i n g f o r an a d d i t i o n a l hour, the pH was lowered to one with 6N HCI, gas e v o l u t i o n was allowed to subside and the s o l u t i o n was d i l u t e d with 10 mL H 20, washed with e t h e r , a d j u s t e d t o pH 8 with 5% KOH, s a t u r a t e d with NaCl, e x t r a c t e d with e t h e r , d r i e d and evaporated to a f f o r d 0.24 g (50%) of a c l e a r o i l . Most of the hydroxylamine decomposed d u r i n g GCMS a n a l y s i s to the corresponding secondary amine. A sma l l p o r t i o n s u r v i v e d the GC to gi v e a s a t i s f a c t o r y mass spectrum. O-TMS (18) and O-methyl (57) d e r i v a t i v e s were prepared f o r GCMS a n a l y s i s . GCMS; ( u n d e r i v a t i z e d ( 1 7 ) ) : tr=l5.2 min.: M + 255 (6); 74 (100); 58 (55); 167 (45); 165 (20); 152 (10); 115 (8); 222 ( 8 ) . (TMS d e r i v a t i v e (18): tr= 15.2 min.: M + 327 (4) ; 146 (100); 58 (28); 167 (15); 132 ( 5 ) ; 73 (5); 165 (5) ; 208 ( 3 ) . (O-methyl d e r i v a t i v e ( 5 8 ) ) : tr= 13.5 min. M + 269 ( 3 ) ; 88 (100); 58 (34); 167 (16); 165 (12); 152 (6); 115 (4); 77(4). 52 1 H NMR ( 1 0 0 M H Z ) : 1.06, (d, C H 3 C H ) ; 4 . 1 ( t , CHPh 2); 2.0 (m, CH 3CH); 2.4-2.8 (m ( b u r i e d ) , C H 2); 2.56 (s, CH 3N); 7.1-7.4 (m, P h 2 ) ; 9.7 (bs, O H ) . ( S l a t t e r , 1983). IR ( n u j o l m u l l ) : 3200 (broad m, NH,OH s t r . ) ; 1950 (w); 1590 (m); 1580 (m); 1490 ( s ) ; 1450 ( s ) ; 1370 ( s ) ; 1360(m); 1220, 1190, 1160, 1130 (m); 930 (w); 910 (w); 800 (m); 780, 750, 730 (m-s); 700 ( s ) . ( S l a t t e r , 1983) x. (—) a-Methyl-(N-methylene)-7-phenylbenzenepropanamine N-oxide (methylene n i t r o n e ( 2 4 ) ) ( S l a t t e r , 1983) Using the method of Coutts et al., (1978), to benzene i n a Dean and Sta r k s apparatus was added 720 mg of the primary hydroxylamine (22) i n 10 ml benzene. The s o l u t i o n was r e f l u x e d f o r two hours and then f l a s h evaporated. The re s i d u e was f l a s h chromatographed on a 2.5 x 10 cm column using CHC1 3: EtOH (99.25:0.75) to e l u t e the yellow f o r e r u n . Then the eluent was changed to CHCl 3:MeOH:EtOH (98.25:1.0:0.75) to e l u t e the white band. T h i s f r a c t i o n was rechromatographed i n the same sol v e n t t o y i e l d 285 mg (38%) of a c l e a r o i l . GCMS: decomposes to v a r y i n g degrees i n the GC i n l e t (see d i s c u s s i o n ) . GCMS: t r , n i t r o n e (24)= 17.8 min. Mass spectrum: M + 253 (2 ) ; 165 (100); 56 (66); 91 (58); 167 (57); 115, (47); 152 (46); 193 (41); 208 (40); 73 (38); 130 (36); 179 (30); 178 (26); 236 (20); 222 (10). JH NMR (400 MHz): 1.42 (d CH3); 2.23 (dd, CHgH^CH); 2.74 (dd, CH aH bCH); 3.79 (m, CHCH 3); 5.98 (d J=8Hz, 53 CHgHb=N); 6.41 (d, CH aH b=N); 7.1-7.4 (m, arom.) (S la t ter , 1983). H e NMR: (Broad Band decoupled, s p l i t t i n g from SFORD): 19.92 (q, C H 3 ) ; 39.41 ( t , CH 2 CH); 47.74 (d, CHPh 2 ) ; 69.03 (d, CHCH 3 ) ; 122.29 (t , N=CH 2); 126.51-128.90 (8 doublets , arom.); 143.05, 144.16 (s, arom. C - C ) . ( S l a t t e r , 1983). IR ( f i l m ) : 3395 (w-m, b r . , assoc. H 2 0 ) , 3020, 2970, 2920 (w-m); 1600 (m); 1566 (m-s, n i t rone ) ; 1490 (m-s); 1450 (m-s); 1300 (m); 1065 (s, n i t rone ) ; 740, 730, (m-s, arom.); 702 (s, arom. ) (S la t t er , 1983). x i . C i s / t r a n s N-(a-methyl-7-phenylbenzenepropylidene) methylamine-N-oxide (methyl nitrone (44)) Using the method of Coutts et al., (1978), N-methylhydroxylamine hydrochloride (3.72 g, 44.6 mmol) was d i sso lved in 5 ml H 2 0 and adjusted to pH 8 with 3M NaOH. Diphenylbutanone (14) (2g, 8.9 mmol) in 150 ml benzene was added. The so lut ion was refluxed overnight in a Dean and Starks apparatus. The benzene so lut ion was f i l t e r e d and f lash evaporated. Flash chromatography of 1 g (20%) of the amber residue on a 2.5x10 cm column in 96:3:1 CHC13:MeOH:EtOH was done and the low r f component was c o l l e c t e d . Chromatography was repeated af fording 240 mg (21%) of pure n i t rone , which forms a white waxy s o l i d when pumped under high vacuum. NMR revealed a 4.3:10 r a t i o of c i s and trans isomers. 54 GCMS: tr=20.37 min . : M + 253 (6); 56 (100); 165 (82); 91 (70); 73 (50); 193 (50) 208 (47); 57 (45). Mass Spectrum (Direct in ser t i on probe): M + 253 (9); 56 (84); 165 (22); 167 (15); 57 (15); 237 (13); 152 (13); 222 (5). 1H NMR (300 MHz): Trans isomer (Major): 1.66 (s, CH 3 CH); 3.27 (d J=6 Hz, C H 2 ) ; 3.59 (d, J=0.7 Hz, N-CH3); 4.66 (t , J=6Hz, CHPh 2 ) ; 7.12-7.24 (m, 2H, arom.); 7.24-7.45 (m, 8H, arom.) . C i s (minor) isomer: 2.04 (d, J=0.75 Hz, CH 3 CH); 3.12 (d, J=5.6 Hz, C H 2 ) ; 3.28 (buried, NCH 3 ) ; 4.2 (t , J=5.8 Hz, CHPh 2 ) ; 7.12-7.45 (m, arom.) . H e NMR (100MHz): Trans isomer: 19.48 (CH 3CH); 38.81 ( C H 2 ) ; 45.80 (NCH 3 ); 47.85 (CHPh 2 ); 126.31 (para, arom.); 127.65, 128.32 (ortho, meta, arom.); 142.27 (arom. C - C ) ; 143.65 (C=N). Cis isomer: 18.39 (CH 3CH); 40.73 (CH 2 ) ; 46.60 (NCH 3 ); 48.55 (CHPh 2 ); 126.89 (para, arom.); 127.51, 128.68 (ortho, meta, arom.); 142.27 (arom C - C ) ; 145.27 (C=N). IR (nujol mul l ) : 3000 (s, n u j o l ) ; 2000-1800 (w, arom. overtones); 1660 (w); 1592 (m, arom.); 1560 (w); 1495 (m) 1470 (nujo l ) ; 1375 (nujo l ) ; 1335 (w-m); 1290 (w); 1245 (m-s); 1220 (m-s); 1160 (m-s); 1120 (w); 1040 (m); 1030 (m); 1120 (m); 935 (w-m); 920 (w-m); 790 (w-m); 740 (s); 702 (s); 692 (s) . x i i . (—) a-Methyl-(N-methylene)-7-phenylbenzenepropanamine (polymer) (methylene imine (23) as tr iazane (41) ) (S lat ter , 1983) 55 A methanolic so lut ion of d inorrec ipavr in was treated overnight with a 1.3 molar excess of 38% aqueous formaldehyde over molecular s ieve . After f i l t r a t i o n and evaporation, GCMS revealed complete conversion to a compound that chromatographed as the monomer. GCMS (packed column): tr= 2.16 min. M + (monomer (23)) 237 (4); 57 (100); 222 (18); 56 (16); 91 (9); 167 (8); 58 (8); 165 (8); 152 ( 5 ) . ( S l a t t e r , 1983) NMR (100 MHz): a r y l a l i p h a t i c resonances: 1.17 (d, J=6.4 Hz, C H 3 , 26mm=9protons, d i s t o r t e d ) ; 2.0-2.5 (m, CH a H b CHCH 3 , I9mm=6 protons); 2.75-3.1 (m, CH 2 CHCH 3 , 9mm=3 protons); 4.0-4.2 (m, CHPh 2 , 9mm=3 protons); 7.1-7.4 (97mm=30 protons); tr iazane and re lated resonances: 3.22 (s, 28mm=9 protons); 3.33 (s, 7mm=2 protons); 3.45 (s, 3mm=1 proton); 3.5 (br. s, 3mm= 1 proton); 4.28 (s, 20mm=6 protons);4.48 (d, 6mm= 2 protons); 4.62 (s, 3mm=1 proton) ( S l a t t e r , 1983). IR ( f i l m ) : 3095 (m-s); 3065, 3025, (m-s arom.); 2965 (a l iph CH s t r . ) ; 2935 (CH 3 ) ; 1598, 1583 (m-s, arom); 1490 (s) , 1448 (s) (arom.); 1378 (br. m); 1260 (w); 1235 (w); 1150 (s); 1068, 1030, 1018, 1000 (br, m); 965 (w-m); 910 (br. w-m); 780 (w); 850 (m); 738 (m-s); 704 (s, -arom. ) . (S la t ter , 1983). 56 x i i i . (—) 2 - ( 4 ' , 4 ' - D i p h e n y l - b u t - 2 ' y l ) oxaz ir id ine (25) (S lat ter , 1983) Following the modified method of Krimm (1958), 750 mg (2.88 mmol) of d inorrec ipavr in HC1 (20) in 10 ml H 2 0 was cooled to 0 ° . Aqueous formaldehyde (38%) (0.5 ml , 6.33 mmol) was added, af ter s t i r r i n g for hal f an hour. To the s t i r r e d so lut ion was added 1 g (5 mmol) MCPBA (85%) in 20 ml CHC1 3 . Af ter 2 hours , 10 ml of 1.1 M CaC0 3 was added dropwise. The chloroform phase was separated, washed three times with 1 M NaOH to hydrolyze the intermediate perester , then twice with water, d r i e d over K 2 C 0 3 and evaporated at room temperature. F lash chromatography in 9:1 pet . ether ( 3 0 - 6 0 ° ) : EtOAc afforded 164 mg (22%) of two diastereomeric oxaz ir id ines which appeared as black spots when TLC plates were developed with Dragendorf's reagent. The chromatography was repeated and the major diastereomer was i so la ted in pure form for NMR a n a l y s i s . Samples decomposed on standing to the isomeric secondary formamide (12). GCMS: Ident ica l to the secondary formamide (12). (packed column): tr=5.5 min. M + 253 (25); 73 (100); 208 (42); 167 (40); 165 (28); 193 (27); 130 (37); 181 (23); 72 (23); 115 (18); 58 (12); 44 (10). A peak c o l l e c t e d off a GC column in an ice cooled glass tube was e luted with CDC1 3 and found by NMR to be i d e n t i c a l to the secondary formamide (12) synthesized by re f lux ing the primary amine (20) in ethyl formate. Mass Spectrum (direct i n l e t ) : i d e n t i c a l to sec formamide (12) (S la t ter , 1983). 57 —H NMR; Major diastereomer: (400 MHz): 1.2 (d, C H 3 ) ; 1.86 On, CHCH 3 ); 2.21 (dd, CH a H b CH); 2.34 (dd, CH a H b CH); 3.28 (d, J=10 Hz, oxaz. C H ^ H J Q ) ; 3.82 (d, J=10 Hz, oxaz. C H a H b ) ; 4.01 ( t , CHPh 2 ) ; 7.1-7.4 ( P h 2 ) . Minor diastereomer: (100 MHz): 1.12, (d, CH 3 CH); 1.85 (m, CHCH 3 ); 2.24 (dd, CH a H b CH); 2.64 (dd, CH a H b CH); 3.46 (d, J=10 Hz, oxaz. CHgHj^); 3.95 (d, J=10 Hz, oxaz. C H a H b ) ; 4.25 (t , CHPh 2 ) ; 7.1-7.4 (m, P h 2 ) . ( S l a t t e r , 1983). -He NMR; Major diastereomer: (100 MHz): (ppm, Broad Band decoupled, s p l i t t i n g from SFORD): 19.73 (q, C H 3 ) ; 40.12 ( t , CH 2 CH); 48.23 (d, CHPh 2 ) ; 65.09 (d, CHCH 3 ); 71.97 (t or dd, oxaz. C H 2 ) ; 126.18-128.94 (d, P h 2 ) ; 144.12, 144.26 (arom. C 5 H 5 - C - C H ) ( S l a t t e r , 1983). IR ( f i l m ) : 3100-2900 (m, ser ies of bands) 1600 (m, arom.); 1585 (w, arom.); 1520 (s); 1480 (s); 1390 (m); 1250 (m-s, oxaz . ) ; 1170 (w-m); 1120 (w-m); 1065 (w-m); 1035 (w-m, oxaz . ) ; 970 (w-m); 935 (w-m, oxaz . ) ; 770 (m-s); 700 (s ) . (S la t ter ,. 1 983) x i v . (1) N-(1-methyl-3 ,3-diphenylpropyl) formamide (12) ( S l a t t e r , 1983) Because of extensive reference to {-) N-(1-methyl-3,3-diphenylpropyl) formamide (12) in th i s thes i s , for convenience, t h i s compound w i l l be re ferred to as the secondary formamide. 58 The secondary formamide (12) was synthesized by refluxing dinorrecipavrin free base in excess ethyl formate for several days using the method of Moffat et al. , ( 1962)(Slatter ,< 1983). The presence of suspended K2CO3 shortened the reaction time. R e c r y s t a l l i z a t i o n from benzene/petroleum ether (60-80°) afforded a quantitative y i e l d of white rods (m.p. 102-103°). NMR revealed a mixture of two rotamers. The secondary formamide (12) was also obtained using the method of LeBelle et al . , ( 1973) for the Leuckart-Wallach reaction of diphenylbutanone (14) in refluxing formamide. After four hours, d i l u t i o n with water, extraction with chloroform and evaporation afforded an 80% y i e l d of the secondary formamide which was separated from the less polar byproduct, 4-(2,2-diphenylethyl) pyrimidine (59) by flash chromatography in hexane : EtOAc (4:1) or by f r a c t i o n a l d i s t i l l a t i o n (170-180° at 0.3mm) (pyrimidine) and 200-205° at 0.3mm (secondary formamide (12)). The secondary formamide (12) s o l i d i f i e s into a waxy mass in the receiving f l a s k . A TMS derivative (60) was formed slowly at 80°. GCMS (12): M+ 253 (44); 73 (100); 167 (84); 165 (66); 208 (60); 44 (50); 193 (42); 181 (38). GCMS (TMS derivative (60)): M+ 325 (6); 145 (100); 73 (70); 165 (42); 167 (40); 221 (36); 152 (24); 130 (23). 59 1H NMR (400 MHz): Trans rotamer: 1.19 (d, J=6.7 Hz, CH 3); 2.0-2.1 (m, CH aCH b); 2.25-2.35 (m, J a b = 14 Hz, CHaCHfa, ); 4.02 (t , distorted, Ar 2CH and m, CH-N (buried)); 5.16 (s, broad, NH); 7.1-7.35 (arom.); 8.02-8.08 (s, HC=0). Cis rotamer: 1.24 (d, J=6.8 Hz, CH 3); 2.1-2.2 (m or ddd, CH aCH b~CH x, J a f c=l2.8 Hz, J a x = 9 Hz); 2.25-2.35 (m, buried, CH aCH b); 3.3-3.4 (m, CH-N); 4.04 (t, buried, Ar 2CH); 5.35 (s, broad, NH); 7.1-7.3 (arom.); 7.82 (d, broad, J=12 Hz, HC=0, ). H e NMR (100 MHz, Shift from BB s p l i t t i n g from SFORD): Trans rotamer: 21.07 (q, CH 3); 42.71 (t, CH2~CH); 46.40 (d, CH-CH3); 48.22 (d, Ar 2~CH); 126.19-128.89 (d, arom. CH); 144.43 (s, arom. C-R, weak); 160.24 (d, HC=0). Cis rotamer: 23.08 (q, CH 3); 43.17 (t, CH2~CH); 46.40 (overlap, d, CH2"CH); 47.86 (ArCH); 126.19-128.89 (d, arom.CH); 144.15 (s, arom. C-R); 163.76 (d, HC=0). IR ( f i l m ) : 3350-3120 (broad m-s, NH s t r . ) ; 3065 (m), 3030 (m) (arom. CH); 2970 (m, a l i p h . CH s t r . ) ; 2960 (m-s, CH 3); 2830 (m, assym. CH 3); 1950 (w), 1885 (w), 1810 (w) (arom. overtones); 1670 (m-s, c i s C=0 s t r . ) ; 1652 (s, trans C=0 s t r . ) ; 1602 (m), 1585 (w-m) (arom. C-C skel. s t r . ) ; 1548 (m-s ) , 1540, (w-m shoulder, amide I I ) ; 1495 (m), 1450 (m) (arom.); 1385 (m-s, C-N s t r . amide); 1245 (w-m, broad, amide I I I ) ; 1140 (w, CH in plane bend); 1062 (w-m), 1030 (w-m) (arom.); 1015 (w-m); 783-760 (w-m out of plane NH wag); 780 (w-m); 745 (m-s), 735 (m), 700 (s) (arom. CH bend). 60 IR (CHCI3 s o l u t i o n ) : 3439 (m, sharp, trans NH)); 3397 (cis NH); 1697 (s, trans C=0); 1685 (s, c i s C=0); 1550-1510 (m, broad, CHCI3 overlap), 1400 (m, broad) (C-N s t r . amide). xv. Characterization of the Leuckart s p e c i f i c byproduct 4-(2,2-diphenylethyl) pyrimidine (59) GCMS: M+ 260 (44); 167 (100); 165 (45); 169 (32); 152 (25); 183 (20). lH NMR (270 MHz): 3.51 (d, J=8Hz, CH 2); 4.62 (t, J=8Hz, Ar 2CH); 6.93 (d, J=5 Hz, HC5 pyrimidine); 7.2 (arom.); 8.49 (d, J=4Hz, HCg pyrimidine); 9.16 (s, HC 2 pyrimidine). IR (nujol mull): 1582 (m-s), 1577 (m) (arom.); 1550 (w-m, C=N s t r . ) ; 1495 (m, arom.); 1312 (w); 1159 (w); 1080 (w); 1032 (w, arom.); 992 (w); 959 (w), 910 (w) (arom.); 752 (m) , 739 (m), 702 (s) (arom.). x v i . ( i ) N-(1-methyl-3,3-diphenylpropyl) formohydroxamic acid (61 ) Using the method of Fishbein, et al., (1968), to 250 mg (1.04 mmol) of freshly synthesized primary hydroxylamine (22) in 2.5 ml EtOH was added dropwise a solution of 50 mg (2.1 mmol) sodium metal in 2.5 ml EtOH s u f f i c i e n t to make the solution mildly a l k a l i n e . Then 84 ul (77 mg, 1.04 mmol) ethyl formate was dissolved in 1 ml of EtOH and added to the reaction mixture. The mixture was cooled and the remaining sodium ethoxide was added. 61 A f t e r 30 minutes the s o l u t i o n was allowed to come to room temperature and s t i r r e d f o r two a d d i t i o n a l hours. A white p r e c i p i t a t e formed. The s o l u t i o n was brought to pH 4 with d i l u t e HCI and f l a s h evaporated. The res i d u e was d i s s o l v e d i n a two phase mixture of EtOAc and water. The orga n i c phase was separated and washed with water. The p o l a r product was p u r i f i e d f r e e of oxime and diphenylbutanone by f l a s h chromatography i n 2:1 hexane:EtOAc. The product was an orange red o i l . The product was d e r i v a t i z e d with TMAH f o r GCMS a n a l y s i s . GCMS (packed column, tr=5 min.): M + 283 (5); 72 (100); 208 (85); 42 (74); 167 (43); 193 (38); 130 (35); 165 (30); 74 (24); 91 (21); 115 (18); 181 (15); 252 (3); 239 (1) . IR ( f i l m ) : 3600-3000 (s br., OH); 3000-2850 ( S ) 2 0 0 - 1 8 0 0 (arom. o v e r t o n e s ) ; 1665 (s, C=0); 1600 (m); 1585 (w); 1490 (m-s ) ; 1448 (m-s); 1390 (m); 1170 (m); 1110 (w); 1075 (w); 1045 (w); 1025 (m); 1000 (w); 985 (w); 910 (w); 863 (m-s); 840 (w); 782 (w-m); 748, 735 ( s ) ; 700 ( s ) ; 680 (m). x v i i . {-) N-methyl-N-(1-methyl-3,3-diphenylpropyl) formamide (26) Because of e x t e n s i v e r e f e r e n c e to (—) N-methyl-N-(1 -methyl-3,3-diphenylpropyl) formamide i n t h i s t h e s i s , t h i s compound w i l l be r e f e r r e d to as the t e r t i a r y formamide (26). 62 The t e r t i a r y formamide was synthesized by the Leuckart-Wallach reaction of diphenylbutanone (14, 300 mg) in excess (3 mL) refluxing N-methylformamide. After 24 hours the dark brown solution was dil u t e d with water and extracted with three 25 mL aliquots of chloroform. The chloroform extract was backwashed with water, dried over Na 2S0 4 and evaporated to afford a brown l i q u i d that was analyzed by GCMS and found to be 90% t e r t i a r y formamide. The crude product was flash chromatographed twice on a 1x15 cm column using 20% EtOAc in hexane. After evaporation, 150 mg (42%) of a pale yellow o i l was analyzed by NMR and IR spectroscopy and then used for metabolism experiments. Larger quantities of the formamide could also be p u r i f i e d by d i s t i l l a t i o n (b.p. 173° at 0.04mm) yi e l d i n g a viscous yellow o i l which precipitated a pale yellow s o l i d (m.p. 85-88°) from MeOH solution when stored at -5°. 1H NMR revealed a 1.2:10 r a t i o of c i s and trans rotamers (LaPlanche and Rogers, 1964). The t e r t i a r y formamide was also obtained by on column methylation of the secondary formamide with TMAH (Sla t t e r , 1983). i-H NMR (300 MHz): Trans rotamer: 1.23 (d, J=7 Hz, CH3-CH); 2.25 (t, CH 2); 2.79 (s, NCH3); 3.37-3.50 (m, CH-CH3); 3.72-3.82 ( t , CHPh 2); 7.12-7.37 (m, Ph); 7.72 (s, CHO). Cis rotamer: 1.13 (d, J=6.5 Hz, CH3-CH); 2.25 (t (buried), CH 2); 2.62 (s, NCH3); 3,37-3.5 (m (buried), CHCH3); 3.81-3.89 (t, CHPh 2); 7.12-7.37 (m, Ph); 7.99 (s, CHO). 63 H e NMR (75 MHz): trans rotamer: 19.48 (CH3CH); 24.17 (N C H 3 ) ; 39.22 (CH 2 ) ; 47.75 (Ph 2 CH); 51.57 (CH3CH); 126.31-128.91 (arom. CH); 142.65, 144.16, (arom.C-C); 162.91 (CHO). Cis rotamer: 17.9 (CH3CH); 29.6 (NCH3); 39.22 (CH 2 (buried)) ; 45.8 (Ph 2 CH); 48.2 (CH3CH); 126.31-128.91 (arom. CH (buried)) ; 142.16, 144.16 (arom C-C (buried)) ; 162.87 (CHO) IR ( f i l m ) : 3057, 3045 (w, arom. CH); 2967 (m, a l i p h . CH); 2929, 2867 (m,CH 3 ) ; 1666 (s, C=0 s t r . ) ; 1595 (m), 1585 (w)(C-C s k e l . s t r . ) ; 1490 (w-m, CH3-N); 1446, 1425, 1403 (w-m, N-CH3, amide); 1322 (w-m), 1303 (w), 1248 (w-m), 1200 (W),1185 (w), 1155 (m); 1088, 1030 (m-s, arom. CH bend); 1060 (w-m); 920, 855, 850 (w); 790, 755 (w-m), 742 (w-m), 708 (m-s) (arom. CH bend). x v i i i . (1) N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl) formamide (carbinolamide (47)) Using the method of Nair and Francis (1980), the secondary formamide (500 mg, .02 mol) was d isso lved in 5 mL MeOH and added to a s t i r r i n g methanolic so lut ion of 324 uL (0.04 mol) of a 38% aqueous so lut ion of formaldehyde and 2 g of suspended K 2 C03» The suspension was s t i r r e d for 24 hours at room temperature, f i l t e r e d through a double thickness of f i l t e r paper and evaporated under reduced pressure. A port ion of the gelatinous p r e c i p i t a t e was disso lved in BSTFA, heated overnight at 6 0 ° and analyzed by GCMS. The carbinolamide TMS der iva t ive (62) was present as a minor componment (4%) along with the N-TMS der iva t ive of the s t a r t i n g m a t e r i a l . 64 GCMS (TMS d e r i v a t i v e (62)): tr=22.92: M + 355 (3); 85 (100); 167 (97); 265 (76); 165 (70); 144 (64); 73 (60); 103 (48). x i x . ( l ) N-(1-methyl-3 ,3-diphenylpropyl) acetamide (63) The acetamide was synthesized from d i n o r r e c i p a v r i n (20) and ace t i c anhydride (l eq. each) in dry benzene containing two equivalents of dry p y r i d i n e . The mixture was heated in a r e a c t i v i a l R overnight at 6 0 ° . GCMS: t r = l 9 . l 6 ; M + 267 (12); 87 (100); 167 (14); 44 (61); 72 (16); 167 (22); 167 (14); 86 (13); 208 (10). 1H NMR (80 MHz): Trans rotamer: 1.2 (d, CH3-CH); 1.8 (s, CH 3C=0); 2.05-2.4 (ddd, C H 2 ) ; 4.0 (m, CH-CH3); 4.0 (dd, bur i ed , CHPh 2 ) ; 5.2 (bs, NH); 7.1-7.4 (m, Ph). C i s rotamer: 1.4 (d, CH3-CH); 2.0 (s, CH 3C=0); 2.05-2.4 (ddd, C H 2 ) ; 3.8 (m, CH-CH3); 4.0 (dd, bur ied , CHPh 2 ) ; 5.2 (bs, NH); 7.1-7.4 (m, Ph) . IR ( f i l m ) : 3480 (bm, NH s t r . ) ; 1642 (s, C=0 s t r . ) ; 1550 (m); 1255 (w). xx. (—) N-(1-methyl-3 ,3-diphenylpropyl) ethanimine (64) The ethanimine was synthesized by condensation of acetaldehyde and d i n o r r e c i p a v r i n in ether s o l u t i o n . The product was character ized by GCMS only . GCMS: t f i l . 4 1 : M + 251 (8); 71 (100); 165 (46); 236 (43); 167 (30 ) ; 70 (26); 152 (24); 105 ( 2 3 ) . 65 x x i . (—) 3-Carbylamino-1,1-diphenylbutane (rec ipavr in isocyanide (65)) A mixture of 730 mg (13 mmol) of powdered KOH in 1.8 ml benzene was warmed to ref lux with s t i r r i n g in an apparatus protected with a C a C l 2 guard tube. Gradual ly , 500 mg (2.2 mmol) of d i n o r r e c i p a v r i n free base in 0.5 ml of chloroform was added. The heat was reduced and the f lask was cooled with an ice bath i f the ref lux became too vigorous. After 30 minutes ref lux the suspension was f i l t e r e d and f lash evaporated. The residue was f lash chromatographed in 20% EtOAc in hexane, a f fording 47mg (9%) of the isocyanide. GCMS (packed column, t r =2.9 min . , poss ible isomerization to the n i t r i l e ) : M + 235 (0.5); 167 (100); 132 (88); 117 (53); 165 (39) 152 (24) 193 (20); 166 (17); 168 (16). NMR (300 MHz): "1.48 (d, C H 3 ) ; 2.27 (m, C H 2 ) ; 3.40 (m, CHCH 3 ); 4.10-4.25 (m, CHPh 2 ) ; 7.25 ( P h 2 ) . IR ( f i l m ) : 3050 (m); 2150 (s, i socyanide); 1960, 1900, 1820, 1740, 1670 (w, arom overtones); 1600 (m); 1585 (m); 1500 (s); 1450 (s); 1370 (m); 1350 (m); 1270 (w); 1220 (w); 1190 (w); 1140 (m); 1100 (m); 1065 (m); 1040 (m); 1020 (m); 925 (w); 850 (w); 755 (s); 745 (s); 710 (s) . x x i i . (—) N-(1-methyl-3 ,3-diphenylpropyl) carbamic ac id methyl ester (d inorrec ipavr in methylcarbamate (66)) 66 To 75 mg (0.33 mmol) d i n o r r e c i p a v r i n f r e e base i n 10 ml benzene was added 38.4 u l (47 mg, 0.5 mmol) methyl c h l o r o f o r m a t e . A f t e r r e f l u x o v e r n i g h t , the so l v e n t was evaporated and the r e s i d u e f l a s h chromatographed. GCMS; (packed column, tr=4.5 min.): M + 283 (0.6); 167 (100); 102 (69); 208 (67); 165 (47); 130 (43); 193 (40); 168 (31); 152 (28). NMR (400 MHz): 1.05 (d J=7 Hz, CH3CH); 1.9-2.1 (m (s h a r p ) , CHgH^j); 2.13-2.3 (bm, CH aH f a); 3.54 (s, OCH3); 3.75-3.9 (bm, CHCH3); 3.9-4.0 ( t , Ph 2CH); 4.35-4.45 (bs, NH); 7.05-7.24 (m, P h 2 ) . IR ( f i l m ) : 3400 (w-m, br., NH); 3320 (m,br., NH); 3100-2850 (m); 1740-1700 ( s ) ; 1600 (w); 1540 (m-s); 1390 (m-s); 1450 (m-s); 1360 (w-m, br.) 1280 (w); 1250 (m); 1190 (w-m); 1110 (w); 1090 (w-m); 1060 (m); 910 (w); 780 (w-m); 750, 740 (m); 700 ( s ) . x x i i i . ( i ) N-(1-methyl-3,3-diphenylpropyl) carbamic a c i d e t h y l e s t e r ( d i n o r r e c i p a v r i n ethylcarbamate (67)) To 24 mg (0.11 mmol) d i n o r r e c i p a v r i n f r e e base i n 10 ml benzene was added 15.3 u l (17.3 mg, 0.16 mmol) e t h y l c h l o r o f o r m a t e . A f t e r r e f l u x o v e r n i g h t , the s o l v e n t was evaporated and the r e s i d u e f l a s h chromatographed t o give 19 mg (60 %) of the carbamate. GCMS (packed column, tr=4.9 min.): M + 297 (1 ) ; 167 (100); 208 (52); 130 (50); 165 (48); 44 (40); 31 (39); 193 (36); 152 67 (30); 116 (28); 115 (28); 179 (20). NMR (400 MHz): 1.15 (d J=6 Hz, CH3CH); 1.18-1.3 (t , C H 3 C H 2 ) ; 2.05-2.15 (m (sharp), CU^H^ ; 2.2-2.36 (bm, C H a H b ) ; 3.55-3.7 (bm, CHCH3); 4.0-4.14 (t (buried) , Ph 2 CH); 4.0-4.14 (q (over lap) , C H 2 C H 3 ) ; 4.3-4.4 (bm, NH); 7.1-7.3 (m, P h 2 ) . IR ( f i l m ) : 3400 (w-m, b r . , NH); 3320 (m,br . , NH); 3100-2850 (m); 1740-1700 (s); 1600 (w); 1540 (m-s); 1390 (m-s); 1450 (m-s); 1380 (w-m) 1330 (m); 1250 (m-s); 1210 (w-m); 1100 (m-s); 1060 (m-s); 1040 (m); 780 (w-m); 750, 740 (m); 700 (s) . xx iv . (-) N-methyl-N-(1-methyl-3,3-diphenylpropyl) carbamic ac id methyl ester (norrec ipavrin methyl carbamate, (68)) Using the method of Kapnang and Charles (1983), 66 mg of r e c i p a v r i n free base (0.26 mmol) in dry benzene was added 22 u l (0.28 mmol) methyl chloroformate. After ref lux overnight, the so lut ion was evaporated and f lash chromatographed in 85:15 hexane:EtOAc and the eluent evaporated to a f ford 62 mg (76 %) of the carbamate. GCMS (packed column tr=4.6): M + 297 (<1); 116 (100); 208 (34); 59 (22); 167 (15); 130 (12); 193 (10). IR ( f i l m , weak sample): 3400 (w-m, b r . , H 2 0 ) ; 3100-2850 (m); 1710 (s); 1600 (w): 1490 (m); 1460 (m-s); 1390 (w); 1360 (w-m, b r . ) ; 1260 (w); 1190 (w-m); 1150 (w-m); 1060 (w); 1030 (w); 750, 740 (m); 700 ( s ) . 68 xxv. (-) N-methyl-N-(1-methyl-3,3-diphenylpropyl) carbamic a c i d e thy l ester (norrec ipavrin e thy l carbamate (69)) Using the method of Kapnang and Charles (1983), 66 mg of r e c i p a v r i n free base (0.26 mmol) in dry benzene was added 26 u l (0.28 mmol) e thyl chloroformate. After ref lux overnight, the so lu t ion was evaporated and f lash chromatographed in 85:15 hexane:EtOAc and evaporated. GCMS (packed column tr= 4.8 min . ) ; M + 311 (<1); 130 (100); 58 (79); 208 (44); 29 (28); 102 (22); 86 (18); 167 (17); 193 (12). NMR (400 MHz): 1.10 (d, J=7Hz, CH3CH); 1.07 (t , C H 3 C H 2 ) ; 2.05-2.4 (m, C H ^ ) ; 2.78 (s, NCH3); 3.78-3.90 (m, CHCH3); 3.92-4.30 (t , (buried) , Ph 2 CH); 3.92-4.30 (q, (buried) , C H 2 C H 3 ) ; 7.04-7.25 (m, P h 2 ) . IR ( f i l m ) : 3100-2850 (m); 1740-1700 (s); 1600 (w); 1585 (w); 1495 (m-s); 1450 (m-s); 1410 (m-s); 1370 (m) 1330 (s); 1200 (m); 1150 (m-s); 1110 (w); 1062 (w); 1032 (w); 910 (w); 770, 750, 740 (m); 700 (s ) . D. Synthetic Compounds Related to Promethazine Reaction vessels were protected from l i g h t during a l l procedures. Solvents were dr i ed as described prev ious ly . Glassware was oven dr ied p r i o r to use. So l id s tar t ing mater ia ls were dr ied in vacuo p r i o r to use. 69 i . 10-(2-Propynyl) phenothiazine (70) The method of Clement and Beckett (1981a) was used. A 56% y i e l d of beige c r y s t a l s was obta i n e d . NMR, IR, and GCMS r e s u l t s were as r e p o r t e d i n the l i t e r a t u r e . i i . lO-(2-Propanone) phenothiazine (71) The method of Clement and Beckett (1981a) was used. M e r c u r i c s u l f a t e was prepared by the method of Newman (1960). Four g (46%) of product were recovered. NMR, IR, and GCMS r e s u l t s were as r e p o r t e d i n the l i t e r a t u r e . i i i . 10-(2-formamidopropy1) phenothiazine (72) 10-(2-Propanone) phenothiaz ine (2 g, 7.8 mmol) was r e f l u x e d i n 20 ml formamide f o r 4 hours i n the dark. The s o l u t i o n was cool e d , d i l u t e d with water and e x t r a c t e d with c h l o r o f o r m . The ch l o r o f o r m was d r i e d over K2CO3 f i l t e r e d and evaporated. The re s i d u e was d i s s o l v e d and f l a s h chromatographed on a 2.5 x 10 cm column i n chl o r o f o r m . The phe n o t h i a z i n e formamide (72) was e l u t e d l a s t (rf=0.l8 i n c h l o r o f o r m ) . Chromatography was repeated. A f t e r a short CHCI3 prerun to e l u t e a p y r i m i d i n e byproduct (73), the product was e l u t e d with CHCl 3/MeOH 98/2. E v a p o r a t i o n a f f o r d e d 600 mg (27%) of yellow c r y s t a l s . NMR r e v e a l e d a 4:1 r a t i o of c i s and tr a n s rotamers. GCMS: (tr= 9.34): M + 284 (28); 212 (100); 180 (46); 213 (16); 178 ( 8 ) ; 198 (7) ; 181 (7); 152 (4); 286 (3) ; 77 ( 2 ) . 30 eV GCMS and 70 eV d i r e c t probe mass spectrum were 70 i d e n t i c a l . On column d e r i v a t i z a t i o n with TMAH gave a peak with mass spectrum and retent ion time i d e n t i c a l to the t e r t i a r y formamide (74) described below. JH NMR; (400 MHz): Major rotamer: 1.27 (d, J=6.4 Hz, C H 3 ) ; 3.74-3.84 (dd, C H . ^ ) ; 4.08-4.17 (dd, CH a H b ) ;4 .39-4 .51 (m (sextuplet ) , CHCH 3 ); 5.50-5.70 (bs, NH); 8.10 (s, CHO); 6.92-7.00 (m, arom. C , , Cg); 7.00-7.08 (m, arom C 3 , C 7 ) 7.14-7.24 (m, arom. C 2 , C 4 , Cg, Cg); Minor rotamer: 1.32 (d, J=6.4 Hz, C H 3 ) ; 3.78-3.84 (dd, bur ied , C H a H b ) ; 3,90-4.00 (dd, over lap, C H a H f a ) ; 3.90-4.00 (m, overlap, CHCH 3 ); 5.55-5.70 (bs, bur ied , NH); 7.87 and 7.90 (d, J=12 Hz, CHO); 6.81-6.87 (d, J=8Hz, arom. Cu Cg); 6.92-7.00 (m, arom. C 3 , C 7 ) ; 7.14-7.24 (m, arom. C 2 , C 4 , Cg, Cg); H 20 at 1.64 ppm. H e NMR: (BB s h i f t (20 MHz with s p l i t t i n g from SFORD (100 MHz), 111 mg sample): Trans rotamer: 18.1 (q, C H 3 ) ; 42.7 (d, CHCH 3 ); 51.7 (t , C H 2 ) ; 161.04 (d, CHO); 138.52 (s weak, arom. C-N); 145.78 (s, weak, arom.C-S) . C i s rotamer: 19.89 (q, C H 3 ) ; 45.25 (d, CHCH 3 ); 53.75 ( t , C H 2 ) ; 163.81 (d, CHO); 145.21 (s, weak, arom. C-N); 145.35 (s, weak, arom.C-S) . Aromatic resonances for c i s and trans rotamers 115.81; 116.26; 116.75; 122.05; "122.56; 123.04; 123.53; 126.28; 126.52; 126.79; 127.10; 127.54; 127.73; • 128.03; 128.22. IR: (CHCL 3 s o l u t i o n , 0.5mm c e l l ) : 3432 (w-m, sharp, NH t r a n s ) ; 3400 (w, shoulder, NH c i s ) ; 2930 (w-m); 2870 (w-m, CHC1 3 ) ; 1692 (s, C=0 t r a n s ) ; 1681 (s, C=0 c i s ) ; 7 1 1594 (m, C H C 1 3 ) ; 1575 (m, C H C I 3 ) ; 1389 ( m ) ; 1 3 4 5 ( m ) ; 1130 (m-s , C H C I 3 ) ; 1108 ( w ) ; 1090 ( w ) ; 1051 ( w ) ; 1039 (m, s h a r p ) . I R ( f i l m , P e r k i n E l m e r F T I R ) : 3389 ( w - m , s h a r p , N H ) ; 3272 ( m , b r o a d , N H ) ; 3 0 6 0 , 2 9 7 5 , 2 9 2 9 , 2869 ( w - m ) ; 1664 ( s , 2 p e a k s , C = 0 ) ; 1592 ( m ) , 1571 ( m ) , 1538 (m, b r o a d ) , 1486 ( m ) , 1459 ( s ) , 1383 ( m - s ) , 1341 ( m - s ) , 1307 ( m - s ) , 1286 ( m ) , 1254 ( m - s ) , 1225 ( m - s ) , 1163 ( w ) , 1132 ( m ) , 1108 ( w ) , 1051 ( w ) , 1039 ( m ) , 909 ( m ) , 855 (w, b r o a d ) , 752 ( s ) , 730 ( m - s ) , 696 ( m ) . i v . L e u c k a r t s p e c i f i c b y p r o d u c t , 4 - ( 1 - ( 1 0 - p h e n o t h i a z i n y l ) m e t h y l ) p y r i m i d i n e ( 7 3 ) GCMS ( p a c k e d c o l u m n , 2 0 0 - 2 8 0 ° a t 8 ° p e r m i n u t e , t r = 9 . 6 m i n . ) M + 291 ( 1 1 ) ; 198 ( 1 0 0 ) ; 39 ( 4 4 ) ; 154 ( 1 8 ) ; 199 ( 1 7 ) ; 45 ( 1 6 ) ; 69 ( 1 2 ) . v . 1 0 - ( 2 - F o r m a m i d o p r o p y l ) p h e n o t h i a z i n e N 1 Q - o x i d e (75) U s i n g t h e m e t h o d o f C l e m e n t a n d B e c k e t t ( 1 9 8 1 b ) f o r t h e s y n t h e s i s o f p r o m e t h a z i n e N - o x i d e , t o 250 mg o f 1 0 - ( 2 -f o r m a m i d o p r o p y l ) p h e n o t h i a z i n e ( 7 2 ) i n 1.5 m l MeOH was a d d e d 1 .25 m l o f 30% ' H 2 0 2 . A f t e r 30 m i n u t e s t h e e x c e s s H 2 0 2 was d e s t r o y e d w i t h 100 mg M n 0 2 . The s o l u t i o n was f i l t e r e d , d i l u t e d w i t h w a t e r a n d e x t r a c t e d w i t h c h l o r o f o r m . The o r g a n i c p h a s e was d r i e d o v e r N a 2 S 0 4 , f i l t e r e d a n d e v a p o r a t e d , t o a f f o r d 140 mg (53%) o f a y e l l o w waxy s o l i d (mp l e s s t h a n 6 0 ° ) t h a t d e c o m p o s e d t o t h e f o r m a m i d e d u r i n g GCMS a n a l y s i s . NMR a n d I R r e v e a l e d o n l y one r o t a m e r . 72 The polar product was flash chromatographed in 97.5/2.5 CHCl3/MeOH. GCMS: Decomposes and i s desorbed slowly as the secondary formamide (72). Direct insertion probe mass spectrum: M+ 300 (3); 212 (100); 180 (46); 198 (28); 213 (16); 284 (12); 200 (12); 152 (8); 179 (8); 229 (4); 77 (2). JH NMR: 1.1 (d, J=7.2 Hz, CH 3); 4.2-4.34 (m, CHCH3); 4.59-4.645 (dd, CH aH b); 6.6 and 6.62 (bd, J=6Hz, NH); 7.66 and 7.71 (d, J=8.5 Hz, c i s CHO); 7.19-7.3 (m, arom. C 3, C 7 ) ; 7.5-7.65 (m, arom C 3, C 7, C 2, C g) 7.83-7.9 (m, arom. C 1 # C g ) ; aromatic assignments tentative. H e NMR (100MHz): 18.58 (CH 3); 46.00 (CHCH3); 50.82 (CH 2); 161.40 (CHO); 140.52 (weak, arom. C-N); 140.56 (weak, arom.C-S). Arom. CH resonances: 118.18; 118.60; 122.41; 122.51; 122.73; 127.58; 129.82; 132.54; 132.61; 132.67. IR: (CHC13 solution 0.5 mm): 3438 (w, sharp, NH); 3284 (w-m, broad); 3204 (w, shoulder); 2944, 2874 (w-m, a l i p h . ) ; 1682 (s, C=0); 1601 (m); 1590, 1580 (m); 1386 (w-m); 1368 (m); 1360 (m, shoulder); 1327 (m); 1127 (w); 1100 (w); 1068 (w); 1050 (w); 1040 (w); 1010 (s, N-0 s t r . ) . IR ( f i l m , Perkin Elmer FTIR): 3700-3200 (m, broad); 3350-3100 (m-s); 3035 (m, broad); 2972 (m); 2929 (m); 2873 (m); 1670 (s, one peak); 1585 (s); 1538 (m, broad); 1486 (m); 1461 (s); 1381 (m-s); 1361 (m-s); 1315 (w); 73 1250 (s); 1174 (w-m); 1149 (w-m); 1130 (w-m); 1098 (m); 1066 (w-m); 1047 (m-s); 1009 (s); 920 (m); 851 (m); 755 (s); 730 (s); 671 (m). v i . 10-(N-methyl-2-formamidopropyl) phenothiazine (74) 10-(2-Propanone) phenothiazine (71) (2 g, 7.8 mmol) was ref luxed in 20 ml N-methylformamide for 5 hours in the dark. The so lu t ion was cooled, poured onto 75 ml ice water and the beige p r e c i p i t a t e was extracted with chloroform. The chloroform was dr ied over K2CC>3 f i l t e r e d and evaporated to a f ford 1.7 g of crude product. The residue was d isso lved and f lash chromatographed on a 2.5 x 10 cm column, with a chloroform prerun and product e lu t ion with CHCL3/MeOH 95/5. The phenothiazine N-methyl formamide was e luted l a s t . Chromatography was repeated. NMR revealed a 2:1 r a t i o of c i s and trans rotamers. GCMS (packed column, condit ion F , tr=10.5 min . ) : M + 298 (12); 212 (100); 180 (47); 100 (28); 213 (21); 58 (12); 30 (12); 179 (10); 214 (8); 198 (8);152 (6); 299 (4). JH NMR: (400 MHz): Major rotamer: 1.27-1.32 (d J=7.2 Hz, C H 3 ) ; 2.65-2.69 (s, NCH 3 ) ; 3.90-3.96 (dd, C H ^ ) ; 3.82-3.99 (m (sextuplet) bur ied , CHCH 3 ); 6.92-6.96 (m, arom. 2 protons); 7.11-7.21 (m, arom. 6 protons); 7.72 (s, CHO). Minor rotamer: 1.22-1.27 (d J= 7.2Hz, C H 3 ) ; 2.76-2.80 (s, NCH 3 ) ; 4.02-4.12 (m, C H a ^ ) ; 4.73-4.85 (m (sextuplet ) , CHCH 3 ); 6.81-6.88 (d J=8 Hz, arom.); 6.92-6.96 (d J=8 Hz, arom.); 6.96-7.01 (d J=8 Hz, arom.); 74 7.11-7.21 (m, arom.); 8.01 (s, CHO). Overlapping c i s and t r a n s aromatic resonances make aromatic assignments u n c e r t a i n . IR ( f i l m ) : 3065 (w-m); 2970 (m); 2940 (m); 2860 (m); 1670 ( s ) ; 1592 (m-s); 1572 (m-s); 1485 (m-s); 1465 (s, broad); 1408 (m-s); 1380 (m); 1338 (m-s); 1285 (m-s); 1252 (m-s); 1218 (m-s ) ; 1160 (w); 1130 (m); 1105 (m); 1078 (m); 1038 (m); 930 (w); 860 (w); 835 (w); 750 ( s ) ; 665 (m). v i i . 10-(N,N-dimethyl-N-oxo-2-aminopropyl) phenothiazine (promethazine N-oxide (76), Clement and Beckett, 1981b) Using the method of C r a i g and Purushothaman (1970), a 1.1 molar excess of MCPBA i n i c e c h i l l e d c h l o r o f o r m was added to a c o o l e d and s t i r r e d s o l u t i o n of promethazine HCI i n ch l o r o f o r m . A f t e r s t i r r i n g f o r three hours the s o l u t i o n was passed through a bed of a l k a l i n e alumina (100-200 mesh, 20 times the combined weight of the s t a r t i n g m a t e r i a l s ) . Promethazine was washed out with CHC1 3, then the N-oxide was e l u t e d with 1/1 CHCl 3/MeOH. GCMS r e v e a l e d the Cope e l i m i n a t i o n product 10-< 2-p r o p e n y l ) p h e n o t h i a z i n e . Two minor long r e t e n t i o n time products had mass s p e c t r a l c h a r a c t e r i s t i c s of the Cope e l i m i n a t i o n product with an a d d i t i o n a l h i g h mass ion m/z 255 i n each, suggesting t h a t the aromatic N-oxide and s u l f o x i d e analogues of promethazine N-oxide were a l s o formed t o a minor extent. The product was p u r i f i e d by f l a s h chromatography i n 90/10 CHCl 3/MeOH. 75 Direc t in l e t mass spectrum: (decomposes to the Cope e l iminat ion product (77)): M + 239 (100); 237 (44); 198 (39); 223 (29); 240 (20); 224 (13). JH NMR (80 MHz): 1.38 (d J=6 Hz, CH3CH); 3.17 (d J= 1 0HZ , 0 ~ N + ( C H 3 ) 2 ) ; 3.55-3.87 (m, CH3CH); 4.15-4.53 (dd, C H a H b ) ; 5.45-5.62 and 5.64-5.77 (doublets J=4Hz each, C H a H b ) ; 7.12-8.00 (arom.); H 2 0 at 2.25-2.62 ppm. IR ( f i l m , Perkin Elmer FTIR): 3700-3100 (s, broad, H 2 0 ) ; 3070, 3030, 2982, 2960 (m-s); 2361, 2229 (w-m); 1666 (s); 1586 (s) ; 1485 (s); 1459 (s); 1364 (m); 1342 (m-s); 1301 (w); 1254 (s) ; 1172 (w); 1151 (w); 1099 (m-s); 1022 (s); 923 (w); 869 (w); 757 (s); 704 (w-m). v i i i . Character izat ion of promethazine free base (10) An in jec tab le formulation of 25 mg of promethazine HCI (PhenerganR) was adjusted to pH 10 and extracted with chloroform, dr ied over R2CO3, evaporated and character ized by GCMS, NMR and IR for comparison to compounds 72, 74, 75 and 76. GCMS (packed column, 2 0 0 - 2 8 0 ° a t 8 ° per minute, tr=6.25 min): M + 284 (0.2); 72 (100); 42 (6); 44 (5); 73 (5); 56 (4); 199 (2); 198 (2); 167 (1). NMR (80 MHz): 1.06 (d, J=6.4 Hz, CH3CH); 2.35 (s, N ( C H 3 ) 2 ) ; 2.87 -3.12 (m, CH3CH); 3.5-3.83 (dd, C H g H b ) ; 3.5-3.84 and 3.9-4.22 (dd, C H a H f a ) ; 6.75-7.3 (arom.). 76 IR: ( f i l m ) : 3050 (m); 2965, 2940, 2860, 2790, 2770 (m-s); 2000-1770 (w, arom. overtones); 1592, 1572 (s, sharp); 1500-1450 (vs, broad); 1360 (m-s); 1335 (m-s); 1305 (m); 1285 (s); 1250 (s); 1235 (s); 1182 (m); 1159 (m); 1129 (m-s); 1103 (m-s ) ; 1038 (s) ; 972 (w-m); 927 (w-m); 872, 850 (w-m); 750 (vs); 725 (m). E .Synthet ic compounds re la ted to amphetamine i . (-) a-Methyl-(N-methylene)-/3-phenylbenzeneethanamine (78) The method of Reynolds and Cossar (1971) was used. To a f lask containing 150 u l of 3N NaOH and 2 ml benzene was added a so lut ion of 275 mg (2.7 mmol) of amphetamine su l fate in 1.5 ml H 2 0 . Then 266 u l (3.3 mmol) of 37% aqueous formaldehyde was added slowly over gentle heat. The benzene layer was separated, dr i ed and evaporated, a f fording 201 mg of crude product, which was f lash chromatographed to a f ford a cloudy l i q u i d with a nutty smel l . NMR revealed a 3:2:5 mixture of 1,3,5-hexahydrotriazene (79), 1,3,5-hexahydrooxadiazene (80), and 1,3,5-hexahydrodioxazene (81). The presence of excess formaldehyde favors 1,3,5-hexahydrodioxazene formation. GCMS (packed column): tr=2.3 min . : M + (monomer (78)) 147 (0 .1); 56 (100); 91 (10); 65 (5); 57 (4); 51 (3); 132 (3); 77 (2); 54 (2) . 77 JH NMR (80 MHz): Common resonances: 2.25-2.75 (CH aH b); 2.9-3.5 (m, CH aH b and CH); 7.0-7.4 (m, 88 mm, arom.): T r i a z a n e component: 1.02 (br. d, J=6Hz, 27mm=9 protons, C H 3); 3.75 (s, I7mm=6 protons,NCH 2N); When the spectrum i s run at 60°, resonances a s s o c i a t e d with the t r i a z a n e d i s a p p e a r and are r e p l a c e d by a p a i r of s i n g l e t s at 2.77 (NCH 2N, 6 protons) and 2.85 (CH 3, 9 p r o t o n s ) . The other two components are u n a f f e c t e d by the higher temperature. Oxadiazane component: .1.22 (d, J=6Hz, I2mm=6 protons, CH3); 3.95 (s, 4mm=2 pr o t o n s , NCH 2N); 4.55 ( s, 8mm=4 protons, NCH 20). Dioxazane component: 1.25 (d, J=6 Hz, 15mm=3 protons, CH3); 4.78 ( s , 20mm=4 p r o t o n s , NCH 20); 5.22 (s, 10mm=2 protons, OCH 20). IR ( f i l m ) : 3010 (m-s); 2942 ( s ) ; 2900 ( s ) ; 2841 (m-s); 2820-2760 (m, CH 2N); 2000-1800 (w, arom . o v e r t o n e s ) ; 1607 (m, arom.); 1585 (w, arom.); 1495 (m-s, arom.); 1452 ( s , arom.); 1 402-1335 (m-s, a l i p h . ) ; 1290 (w);'1233-1080 ( m u l t i p l e bands, s, t-amine); 1185 ( s ) ; 1150 ( s ) ; 1108 ( s ) ; 1080 ( s ) ; 1040 (s, arom.); 1000 (s, arom.); 958 (m); 920 (s, e t h e r ) ; 800 (w); 744 (s, arom.); 700 (s, arom.). i i . (1) 2-(3'-phenylprop-2'-yl) o x a z i r i d i n e (82) To 300 mg (1.6 mmol) amphetamine s u l f a t e i n 2 ml H 20 was added 240 u l (3 mmol) of 38% aqueous formaldehyde. Then 575 mg (3.3 mmol) of 85% metachloroperbenzoic a c i d i n 5 ml CHCI3 was added and the 2 phase mixture was s t i r r e d f o r 2 hours. S o l i d K 2C0 3 was added u n t i l gas e v o l u t i o n ceased. The s o l u t i o n was f i l t e r e d , the chl o r o f o r m phase separated, d r i e d over K 2C0 3, 78 evaporated and f l a s h chromatographed in 97:3 hexane: EtOAc to affo r d 17 mg (6%) of a 1.23:1 r a t i o of N,C-diastereomeric oxa z i r i d i n e s . The GCMS results were i d e n t i c a l to N-formylamphetamine (83). 1 H NMR (400 MHz): (major diastereomer): 1.06 (d, J=6.4 Hz, CH 3); 2.08-2.21 (m, C H 3 C H ); 2.74-2.93 (dd, Jab=4.6 Hz, CH aCH b); 3.11-3.2 (dd, CH aCH b); 3.55-3.75 (d, Jab=10Hz, CH aH b oxaz.ring); 3.9-4.1 (d, Jab=l0 Hz, CH aH b oxaz. r i n g ) ; 7.25 (C 6H 5). (Minor diastereomer): 1.35 (d, J=6.4 Hz, C H 3 ) ; 2.1-2.2 (m, CH3CH); 2.74-2.85 (dd, CH aH b); 2.85-2.95 (dd, buried, CH aCH b); 3.12-3.26 (d, Jab=l0 Hz, CH aH b oxaz. ri n g ) ; 3.6-3.8 (d, Jab=10 Hz, CH aH b oxaz. ring); 7.25 (m, CgH 5). i i i . (-) N-(1-methyl-2-phenylethyl) formamide (83) (CAS re g i s t r y 42044-69-9) Using the method of Moffat et al ., (1962), amphetamine free base was refluxed over K 2C0 3 in ethyl formate for 2 days. The solution was f i l t e r e d and evaporated. The amber l i q u i d product was p u r i f i e d by flash chromatography in 20:80 EtOAc:petroleum ether (30-60°). NMR revealed a 3:1 r a t i o of trans and c i s rotamers. GCMS (packed column, condition G): tr=3.1 min.: M+=163 (0), 72 (100); 118 (61); 44(46); 91 (22); 117 (14); 65 (10); 119 (68). 79 GCMS (TMS d e r i v a t i v e , (84))(packed column, condit ion G): t r = 1 . 6 m i n . M + -15 , 220 (5); 144 (70); 72 (30); 75 (32); 91 (30); 118 (20). 1H NMR (80 MHz) Trans rotamer: 1.15 (d, J=6.5 Hz C H 3 ) ; 2.63-2.91 (dd, C H 2 ) ; 4.06-4.7 (m, CHCH3); 5.01-5.87 (br s, NH); 7.0-7.5 ( C g H 5 ) ; 8.1 (s, CHO). Cis rotamer: 1.27, (d, C H 3 ) ; 2.63-2.91 (dd (buried) , C H 2 ) ; 3.5-4.0 (m, CHCH 3 ); 5-6 (br . s (buried) , NH); 7.0-7.5 (m, C g H 5 ) ; 7.9 (d, J=12 Hz, C H O ) . ( l i t . values: 1.13 (d); 2.77 (m); 7.17 ( s ) . 7. EXPERIMENTS TQ_ .DETERMINE THE__ SOURCE OF FORMAMIDE METABOLITES_OF_METHAD^ A. Solvent re la ted a r t i f a c t contro l experiments for methadone  metabolites To preclude the p o s s i b i l i t y of phosgene or peroxide re la ted generation of formamides in the conjugated frac t ion of b i l e from methadone dosed r a t s , the metabolite extract ion method of Horning and M i t c h e l l (1984) was used. Extracts were reconst i tu ted with 50 u l of 5 mg/ml (250 ug) diphenylbutanone stock so lut ion just p r i o r to ana lys i s of 1 u l by GCMS. Ether and chloroform extract ions were performed with and without pretreatment of the solvent with type 4A molecular sieve ( B u r f i e l d , 1982) and d i s t i l l a t i o n just before use. This procedure removes organic peroxides from ether and phosgene from chloroform. The area of the m/z 167 ion peak common to diphenylbutanone (14) and the secondary formamide (12) 80 was integrated to estimate the r e l a t i v e amount of secondary formamide (12) present, expressed as % of diphenylbutanone. Fol lowing the standard extract ion p r o t o c o l , the fol lowing a l k a l i / s o l v e n t pa ir s were employed for the b a s i f i c a t i o n and extract ion of nonconjugated and conjugated metabolites: A. 1 M NaOH/CHCl3, Sample workup A was repeated with the f i n a l sample d i s s o l u t i o n for GCMS in EtOH rather than MeOH; B. 1 M Na0H/CH 2 Cl2; C 2 g s o l i d NH 4 C0 3 /E t0Ac; D. 2 g s o l i d N H 4 C 0 3 / d i e t h y l ether; E . 2 g s o l i d NH 4 C0 3 /benzene; F . 1 M NaOH/EtOAc, without p r i o r extract ion of nonconjugated metabolites ( a l l metabolites extracted fol lowing B-glucuronidase h y d r o l y s i s ) ; G. 1 M NaOH/CHCl 3 , without p r i o r ex trac t ion of nonconjugated metabol i tes . L a s t l y , b i l e was stored exposed to l i g h t and a i r for a period of one week to determine whether formamides could be detected in the nonconjugated f r a c t i o n . B. E f f ec t of added synthetic secondary formamide on peak shape  and retent ion time of the rec ipavr in formamide metabolite (12) The conjugated f r a c t i o n extracted from 5 ml rec ipavr in dosed rat b i l e was concentrated and 1 u l a l iquots were spiked with a l i q u o t s of a 30 ug/ml stock so lut ion of the synthetic secondary formamide (12) according to the fol lowing t a b l e . One uL a l iquot s of each sample were analyzed by GCMS without correc t ion for volume d i f f erences . 81 Table 1. Table showing volumes of b i l e extract and synthetic formamide (12) used in the d i l u t i o n experiment. Sample b i l e formamide formamide b i l e extract No. ex tr . (uL) so ln . (uL) i n j . (ng) i n j . (uL) 1 1 0 0 1 2 0 1 30 0 3 1 1 15 0.5 4 1 10 27.3 0.09 The r e l a t i v e amount of the formamide was compared to that of the rec ipavr in phenol metabol i te , by comparing the peak heights of the molecular ions (253 and 269 respect ive ly ) and the peak heights of ions corresponding to the diarylmethyl cat ion (167 and 183 r e s p e c t i v e l y ) . The concentrat ion effects on retention time and peak shape of secondary formamide (12) derived ions were a lso estimated. C. Experiments to e s t a b l i s h that the secondary formamide  metabolite (12) of r ec ipavr in a r i s e s from a glucuronide  precursor i . Sulfatase hydro lys i s of the r e c i p a v r i n conjugated metabolites B i l e (1.5 ml) from a rat dosed with r e c i p a v r i n D3 hydrochloride was extracted free of nonconjugated metabolites by the standard method. The aqueous phase was adjusted to pH 5 with 4 M HC1 and l y o p h i l i z e d . The residue was taken up in 3 ml pH 5, 0.1 M sodium acetate buffer and then hydrolyzed 82 overnight at 38° with sulfatase (0.5 mg of 21 units per mg, dissolved in 2 ml sodium acetate buffer, /3-glucurbnidase a c t i v i t y = 2.8 Fishman units per mg, Sigma product S-9754, Lot 40F-9550). The conjugated metabolites were then extracted by the standard method and analyzed by GCMS. A p a r a l l e l incubation was performed using ^-glucuronidase. i i . Control incubation of recipavrin conjugated fractio n without /3-glucuronidase enzyme. B i l e (1.5 ml) from a rat dosed with recipavrin hydrochloride was extracted free of nonconjugated metabolites under standard conditions. The aqueous phase was adjusted to pH 5 with 4 M HCI and ly o p h i l i z e d . The residue was taken up in 3 ml pH 5, 0.1 M sodium acetate buffer and then held overnight at 38° without enzyme treatment. The b i l e sample was then extracted under standard conditions and analyzed by GCMS. A p a r a l l e l incubation was performed using ^-glucuronidase. D. Free r a d i c a l oxidation of recipavrin and norrecipavrin as a  source of the secondary formamide (12) i . Incubation of recipavrin (9) with blank b i l e under simulated workup conditions a. Blank b i l e (5 ml) was spiked with 1 ml of a 1 mg/ml aqueous solut i o n of recipavrin hydrobromide, adjusted to pH 10 with 1 M sodium hydroxide, allowed to stand overnight, adjusted to pH 5, treated with /3-glucuronidase 83 f o r 36 hours at 38°, e x t r a c t e d and analyzed by GCMS by standard procedures. b. B i l e from a r e c i p a v r i n - D 3 dosed r a t was spi k e d with 10 mg of rec i p a v r i n - D Q and s t o r e d at room temperature f o r 7 days p r i o r to e x t r a c t i o n at pH 5, pH 10 and pH 12. i i . I n c ubation of n o r r e c i p a v r i n with blank b i l e under si m u l a t e d workup c o n d i t i o n s A 2 ml a l i q u o t of c o n t r o l b i l e was sp i k e d with 50 ug of n o r r e c i p a v r i n HC1 i n 50 u l propylene g l y c o l and 1.12 ug N-e t h y l r e c i p a v r i n i n 0.5 ml H2O, and c a r r i e d through standard i s o l a t i o n procedures f o r nonconjugated m e t a b o l i t e s . The e x t r a c t was examined f o r the presence of the secondary formamide. E. E f f e c t of e x t r a c t i o n pH on the o b s e r v a t i o n of the  r e c i p a v r i n secondary formamide m e t a b o l i t e (12). B i l e from r e c i p a v r i n dosed r a t s was worked up by the standard p r o t o c o l . The conjugated f r a c t i o n was e x t r a c t e d s e q u e n t i a l l y at pH 7, pH 10 and pH 12. Each organic e x t r a c t was d r i e d e v a p o r a t e d , r e c o n s t i t u t e d to a constant volume and ana l y z e d by GCMS. F. E f f e c t of immediate sample p r e p a r a t i o n and storage on the secondary formamide m e t a b o l i t e of r e c i p a v r i n B i l e from a r e c i p a v r i n dosed r a t was c o l l e c t e d d i r e c t l y i n t o an opaque tube c o n t a i n i n g a s o l u t i o n of 0.5 ml of gl u c u r a s e and 1 ml of sodium a c e t a t e b u f f e r . At the 84 end of 24 hours of b i l e c o l l e c t i o n , b i l e was adjusted to pH 5, ten ml was s p l i t into 2 f ive ml a l i q u o t s . One a l iquot sat for an a d d i t i o n a l hour at 3 8 ° , was adjusted to pH 9.8 with NaOH (1 M) and borate buffer (pH 10), extracted with 3 f ive ml a l iquots of EtOAc, d r i e d , evaporated and immediately analyzed by GCMS. The remaining a l iquot was adjusted to pH 5 and stored for two weeks at - 5 ° , thawed, treated with Glucurase R for 36 hours at 3 8 ° , adjusted to pH 10, extracted with EtOAc, d r i e d , evaporated and analyzed by GCMS. G. Solvent re lated a r t i f a c t contro l experiments for rec ipavr in  metabol i tes . i . Comparison of chloroform and EtOAc extract ion solvents on the recovery of the formamide metabolite of r e c i p a v r i n . B i l e from r e c i p a v r i n dosed rats was i so la ted using e i ther d i s t i l l e d in glass grade chloroform ( s t a b i l i z e d with 1% EtOH) or d i s t i l l e d in glass grade EtOAc. The amount of secondary formamide (12) present was estimated r e l a t i v e to other metabol i tes . H. S o l i d phase extract ion (SPE) of rec ipavr in metabolites A Baker-10 SPE manifold was used with a water aspirator vacuum. Cartr idges were ava i lab l e in 1, 3 and 6 ml s i zes . Cartr idges were loaded u n t i l adsorbed b i l e pigments were v i s i b l e to the bottom of the car tr idge packing. 85 i . Cartr idge condi t ioning and sample e lu t ion ( J . T . Baker L t d . , 1982) Baker 10 SPE cartr idges were condit ioned as fol lows: a . Reversed phase octadecyl ( R P C l 8 ) c a r t r i d g e s . To extract non polar solutes by p a r t i t i o n from aqueous s o l u t i o n , R P C 1 8 cartr idges were washed with 2 column volumes of MeOH, then two column volumes of water or buf fer , then the buffered aqueous sample was appl ied to the c a r t r i d g e . The sorbent was not allowed to run dry . The column was washed with two column volumes of water or buf fer , then allowed to a i r dry for 3 minutes, just p r i o r to sample e l u t i o n . A sample equivalent to 0.5 ml b i l e per ml of SPE adsorbent was appl ied and e luted using successive a l iquots of 1 column volume of increas ing proportions of organic solvent in water. b. Forward phase s i l i c a gel (FPSi02) c a r t r i d g e s . To extract moderately polar solutes by adsorption from organic s o l u t i o n , FPSi02 car tr idges were washed with 2 column volumes of the solvent that the solute was to be appl ied i n . The column was not allowed to run dry p r i o r to sample a p p l i c a t i o n . The sample (equivalent to 0.5 ml b i l e per ml of SPE adsorbent) was app l i ed , the column was washed with two column volumes of the d i s s o l u t i o n solvent , then a i r dr ied for three minutes. The samples were then eluted with successive one column volume a l iquots of solvents of gradual ly increasing p o l a r i t y . 86 i i . Amberlite XAD-2 column preparation XAD-2 beads were packed dry into a 1 x 15 cm glass column then washed with EtOH and then with water. The aqueous sample (10 ml of 50% aqueous b i l e or urine so lut ion adjusted to pH 8-9) was app l i ed , and allowed to percolate slowly through the column bed at 2 ml per minute. The column was washed with water u n t i l the eluent was almost c o l o r l e s s then the free and conjugated metabolites were eluted with EtOH u n t i l the eluent was almost free of yellow c o l o r a t i o n . The samples were concentrated under nitrogen and subjected to further SPE steps. i i i . Prel iminary s o l i d phase extract ion experiments a. E l u t i o n of synthetic reference compounds from R P C ^ columns Aqueous solut ions (300 ug/ml) of the synthetic standards diphenylbutanone (14) (a non polar neutral metabol i te) , d i n o r r e c i p a v r i n free base (20) (a medium p o l a r i t y basic metabolite) and the secondary formamide (12)(a medium p o l a r i t y neutra l metabolite and target compound) were prepared and one ml of each so lut ion was appl ied to separate 1 ml R P C ^ columns. The samples were eluted with successive one column volumes of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% aqueous MeOH. The a l iquots were monitored for the solutes by UV absorbance at 254 nm, using a matching concentration of aqueous MeOH as the reference s o l u t i o n . 87 b. RPC13 test e lut ions using UV and TLC detection of eluates Solut ions of d-glucuronic ac id (5 mg/ml), methadone HC1 (1 mg/ml of free base), and phenolphthalein glucuronide (5 mg/ml, turns pink in a l k a l i n e solut ion) were adjusted to pH 8 with 0.01 M NaOH and one ml of each so lut ion was appl ied to separate R P C ^ columns. Al iquots of b i l e (0.5 ml) from contro l and r e c i p a v r i n dosed rats were treated s i m i l a r l y . The columns were washed with 2 column volumes of water and then eluted with successive one column volume a l iquots of 20, 40, 60, 80, and 100% aqueous EtOH. Each eluate was spotted on 3 separate UV f luorescent s i l i c a gel TLC p la te s . Each p late was checked for adequate sample app l i ca t ion with 254 nm l i g h t and then developed by spraying with one of the fol lowing reagents: Dragendorf's reagent for basic nitrogen compounds, naphthoresorcinol reagent plus heat ( 7 0 ° for 15 minutes) for g lucuronides , and 50 % s u l f u r i c ac id in 1:1 aqueous EtOH followed by heating at 1 0 0 ° to detect a l l carbon compounds. i v . Frac t ionat ion of b i l e components by SPE methods a. Attempted f rac t ionat ion of b i l i a r y rec ipavr in metabolites using RPC 1 Q | columns. B i l e (2 ml) from a r e c i p a v r i n dosed rat was passed through a R P C l 8 column. The column was washed with one column volume of water, then with successive one column volume a l iquots of MeOH, i sopropanol , EtOH, hexane, 0.05 88 M sodium a c e t a t e i n 50% aqueous MeOH, 0 . 0 5 M sodium b o r a t e i n 5 0 % aqueous MeOH, and 0 . 0 5 % a c e t i c a c i d i n 50% aqueous MeOH. Each a l i q u o t was blown f r e e of o r g a n i c s o l v e n t under n i t r o g e n , a d j u s t e d t o pH 5 and t r e a t e d w i t h 0 . 2 5 ml ^ - g l u c u r o n i d a s e a t 38° o v e r n i g h t . The s o l u t i o n s were e x t r a c t e d a t pH 5 , then a d j u s t e d t o pH 12 and r e - e x t r a c t e d . b. P r e l i m i n a r y c l e a n u p and d e i o n i z a t i o n of b i l e u s i n g a XAD-2 column B i l e from a r e c i p a v r i n dosed r a t ( 5 ml) was d i l u t e d w i t h 5 ml H2O and passed t h r o u g h a XAD -2 column. The aqueous wash was d i s c a r d e d s i n c e g l u c u r o n i d a s e h y d r o l y s i s and e x t r a c t i o n f a i l e d t o r e v e a l s i g n i f i c a n t amounts of r e c i p a v r i n m e t a b o l i t e s . The EtOH e l u e n t was f u r t h e r p u r i f i e d by f u r t h e r SPE p r o c e d u r e s . c. S e p a r a t i o n of a l l m e t a b o l i t e s from e x t r e m e l y p o l a r b i l e c o n s t i t u e n t s The e l u e n t ( e q u i v a l e n t t o 1 ml b i l e ) was passed through a 3 ml Si02 SPE column and f o l l o w e d by a one column volume wash w i t h s u c c e s s i v e a l i q u o t s of EtOH, 60% aqueous EtOH, and then H2O. The e l u e n t s were s p o t t e d on a TLC p l a t e and v i s u a l i z e d w i t h n a p h t h o r e s o r c i n o l and heat t o l o c a t e g l u c u r o n i d e s . The e l u e n t ( e q u i v a l e n t t o 1 ml b i l e ) was passed through a 3 ml Si02 SPE column and f o l l o w e d by a one column volume wash w i t h s u c c e s s i v e a l i q u o t s of EtOH and 60% aqueous EtOH. 89 The EtOH i n each e l u a t e was evaporated under n i t r o g e n and the r e s i d u e or aqueous remainder was d i l u t e d with 200 u l of 0. 1 M pH 5 sodium acetate b u f f e r and 100 u l of g l u c u r a s e R was added. A f t e r an overnight i n c u b a t i o n at 38°, the samples were d i l u t e d with 0.7 ml of water, adjusted to above pH 9 with 25 u l 1.0 M NaOH, and a p p l i e d to a RPC^ column. The column was washed w i t h s u c c e s s i v e a l i q u o t s of a l k a l i n i z e d water, 40% aqueous EtOH, and 100% EtOH. Samples were c o n c e n t r a t e d under n i t r o g e n . The concentrated EtOH sample was ana l y z e d by GCMS. The aqueous samples were e x t r a c t e d with 3 four ml a l i q u o t s of EtOAc, d r i e d over Na 2S0 4, evaporated, r e c o n s t i t u t e d i n EtOH and a n a l y z e d by GCMS. d. S e p a r a t i o n of nonconjugated m e t a b o l i t e s on FPSi02 c a r t r i d g e s 1. To sepa r a t e the nonconjugated m e t a b o l i t e s from the crude XAD-2 EtOH e l u a t e on a s i l i c a g e l column, a benzene/ EtOH (92/8) s o l v e n t system was developed for s y n t h e t i c samples of the r e c i p a v r i n secondary formamide (12) and the r e c i p a v r i n N-methyl n i t r o n e . Both t e s t compounds are more p o l a r than any of the nonconjugated m e t a b o l i t e s , but presumably are not as p o l a r as the g l u c u r o n i d e conjugates (TLC r f n i t r o n e (44) =0.18, r f secondary formamide (12) = 0.44). T h i s e l u e n t was used to s e l e c t i v e l y e l u t e the nonconjugated f r a c t i o n i n the f o l l o w i n g p r o c e d u r e . The XAD-2 EtOH eluate equivalent to 1 ml b i l e and containing 250ug methadone free base as an in terna l standard was evaporated to dryness under ni trogen, then disso lved with 0. 8 ml EtOH and d i l u t e d slowly with 9.2 ml benzene. This f r a c t i o n was passed through a 6 ml FPSi02 c a r t r i d g e . The c a r t r i d g e was washed with 5 column volumes of 92/8 benzene/EtOH to e lute the nonconjugated metabolites . Conjugated metabolites were e luted with 5 column volumes of EtOH. The nonconjugated f r a c t i o n was concentrated under nitrogen and analyzed by GCMS. 1. Attempts to increase secondary formamide (12) production by  the add i t ion of formic a c i d , formaldehyde, or formaldehyde and  hydrogen peroxide during sample preparat ion . B i l e from a rec ipavr in dosed rat was treated as shown in the fol lowing t a b l e . Table 2. Table showing volumes of reagents added to b i l e from r e c i p a v r i n dosed r a t s . TREATMENT Reagent added A Control B 90% H 2 C 0 2 C 37% H 2CO D 30% H 2 0 2 E 30% 37% H 2 0 2 H 2CO 1. B i l e 2 ml 2 ml 2 ml 2 ml 2 ml 2. Water 180 u l 160 u l 120 u l 60 u l 60 u l 3. Treatment reagent None 20 u l 60 u l 120ul I20ul 60ul 4. Internal^ standard 1 ml 1 ml 1 ml 1 ml 1 ml 5. MeOH 2 ml 2 ml 2 ml 2 ml 2ml * In terna l standard 2.24 ug/ml N-ethy1recipavrin 91 Each sample was mixed we l l , centri fuged at 2500 rpm for 30 min, decanted free of p r e c i p i t a t e d c h o l e s t e r o l , adjusted to pH 10 - 0.1 with 1M sodium hydroxide and 1 ml of pH 10 0 . 1 2 M sodium borate buf fer , and extracted with three 4 ml a l iquots of EtOAc. The organic phase containing the nonconjugated f r a c t i o n was separated, backwashed with 1 ml water, dr i ed over sodium s u l f a t e , evaporated to dryness, reconst i tuted in 50 u l MeOH and 1 u l was analyzed by GCMS under standard operating condi t ions with a helium back pressure of 8 p s i . The aqueous phase was d i l u t e d with the backwash from the nonconjugated f r a c t i o n , adjusted to pH 5 with 4 M HC1 and 0 .1 M acet ic a c i d , frozen in l i q u i d n i trogen, l y o p h i l i z e d , d i l u t e d with 3 ml pH 5 , 0.1 M sodium acetate buffer , and incubated with 0.5 ml glucurase (gas evolved from H2O2 samples), incubated at 3 8 ° for 4 hours, treated with an a d d i t i o n a l 0 . 2 ml glucurase, and held overnight at 38 Q , centrifuged at 2500 rpm for ha l f an hour, decanted from sediment, spiked with 1 ml of 1.8 ug/ml t e r o d i l i n e hydrochlor ide , adjusted to pH 10 — 0 . 1 . Each sample was then retreated with the same volume of the same treatment reagent used in the nonconjugated f r a c t i o n . The pH was checked and readjusted to 10 i f necessary, and the samples were extracted with 4, three ml a l iquots of EtOAc. The organic phase was separated, dr i ed over sodium su l fa te , evaporated, reconst i tuted in 50 u l MeOH and 1 u l was injected into the GCMS and run under standard operating condit ions with a helium back pressure of 8 p s i . 92 J . Attempts to decrease secondary formamide (12) production  with the use of ant ioxidants and a formaldehyde complexing  agent 1-Ascorbic ac id (0.1 M) was prepared in MeOH. Butylated hydroxy toluene (BHT, 0.1 M) was prepared in MeOH. Dimedone (5,5-dimethyl-1,3-cyclohexanedione, 0.1 M) was prepared in MeOH. Samples of b i l e (1 ml) from a rec ipavr in dosed rat were d i l u t e d with 1.5 ml water and 1 ml pH 10 sodium borate buffer (0.12 M) , r e s u l t i n g in a f i n a l pH of 9 .5-9 .6 . Each sample was extracted free of nonconjugated metabolites with three 3 ml a l iquot s of EtOAc. The aqueous phase was then spiked with 0.5 ml of MeOH (control samples), or 0.5 ml of BHT or ascorbic a c i d so lut ion and allowed to stand overnight in the r e f r i g e r a t o r . Samples were adjusted to pH 5 with sodium hydroxide, frozen in l i q u i d n i trogen, l y o p h i l i z e d , reconst i tuted in 3 ml sodium acetate buffer (0.1 M, pH 5), incubated overnight with 0.2 ml glucurase at 3 8 ° , adjusted to pH 10 with sodium hydroxide and 1 ml pH 10 borate buffer , and the samples were extracted with three 3 ml a l iquots of EtOAc. The organic phase was separated, dr ied over sodium su l fa te , evaporated, reconst i tuted in 0.5 ml of a 4.5 ug/ml methanolic so lu t ion of N-e thy lrec ipavr in hydrochlor ide . The samples were evaporated to dryness reconst i tuted in 10 u l MeOH and 1 u l was in jec ted into the GCMS and run under standard operating condi t ions with a helium back pressure of 8 p s i . 93 The amount of secondary formamide (12) was estimated by the r a t i o of i n t e g r a t e d areas of the m/z 167 peak of the secondary formamide (12) (tr=21.00 min.) to that of the i n t e r n a l standard N - e t h y l r e c i p a v r i n (85)(tr=16.13 min.). T h i s v a l u e was then expressed as a percent of secondary formamide (12) i n the t r e a t e d sample r e l a t i v e to the c o n t r o l . K. E f f e c t of the a n t i o x i d a n t BHT, formic a c i d and formaldehyde  on d i n o r r e c i p a v r i n m e t a b o l i t e p r o f i l e s F o l l o w i n g /3-glucuronidase h y d r o l y s i s , the conjugated f r a c t i o n a l i q u o t s of b i l e from d i n o r r e c i p a v r i n dosed r a t s was t r e a t e d with 1 ml of methanolic 0.1 M b u t y l a t e d hydroxytoluene, or 1 ml of 0.1 M aqueous formic a c i d , or 1 ml of 0.1 M aqueous formaldehyde. Samples were a l k a l i n i z e d and the s t a n d a r d workup p r o t o c o l was continued. M e t a b o l i t e p r o f i l e s were compared by v i s u a l i n s p e c t i o n of the m/z 167 mass chromatograms and checked f o r the presence of the secondary formamide. L. Decomposition of r e c i p a v r i n N-oxide (53) under simulated  workup c o n d i t i o n s R e c i p a v r i n N-oxide was p u r i f i e d twice on an alumina column. A stock s o l u t i o n of 17.3 mg/ml in c h l o r o f o r m was prepared f o r decomposition experiments. A l i q u o t s of 50 u l (865 ug) of r e c i p a v r i n N-oxide were added to tubes c o n t a i n i n g 2 ml of one of the f o l l o w i n g s o l u t i o n s : 1. Water; 2. Sodium a c e t a t e b u f f e r , 94 0.1 M, pH 5; 3. Sodium a c e t a t e b u f f e r 0.1 M, pH 5 plus 250 u l gl u c u r a s e ; 4. Sodium bo r a t e , 0.12 M, pH 10; 5.3 M Sodium hydroxide; 6. F e r r i c c h l o r i d e , 5 mg/ml; 7. F e r r i c c h l o r i d e , 5 mg/ ml p l u s 300 u l of 30% hydrogen p e r o x i d e . The tubes were mixed w e l l and incubated overnight at 40°, allowed t o stand exposed t o l i g h t f o r 24 hours at room temperature, then a d j u s t e d to pH 11 with 3 M NaOH, e x t r a c t e d with 4 two ml a l i q u o t s of EtOAc, d r i e d over sodium s u l f a t e , evaporated to dryness and r e c o n s t i t u t e d with 20 u l MeOH for GCMS a n a l y s i s of 1 u l a l i q u o t s . The GCMS was operated under standard c o n d i t i o n s with a helium back pressure of 15 p s i . Degradation products were i d e n t i f i e d by t h e i r mass sp e c t r a when p o s s i b l e . T o t a l ion c u r r e n t peak areas of the degrad a t i o n products were compared to determine the e f f e c t s of sample treatments on the amount of each degradation product. The c o n t r o l sample was a 50 u l a l i q u o t of the N-oxide stock s o l u t i o n evaporated to dryness and r e c o n s t i t u t e d in 20 u l MeOH for GCMS a n a l y s i s of a 1 u l a l i q u o t . M. GCMS a n a l y s i s and decomposition of the methylene n i t r o n e  (24) The methylene n i t r o n e was s y n t h e s i z e d and p u r i f i e d as d e s c r i b e d e a r l i e r . Two stock s o l u t i o n s (10 and 100 ug/ul) were prepared i n EtOH f o r experiments to determine the e f f e c t s of sample l o a d i n g , chromatographic c o n d i t i o n s and the presence of b i l e c o n s t i t u e n t s on the thermal d e g r a d a t i o n of the n i t r o n e i n the GC i n l e t . 95 I n j e c t i o n port l i n e r s were c l a s s i f i e d as e i t h e r c l e a n ( b o i l e d i n n i t r i c a c i d p r i o r to use), d i r t y ( p r e v i o u s l y used f o r at l e a s t ten b i l e sample analyses and v i s i b l y l i n e d with brown m a t e r i a l ) or s i l a n i z e d (a new i n j e c t i o n port l i n e r t r e a t e d with 5% DMCS i n toluene overnight and washed to n e u t r a l i t y with MeOH). GCMS a n a l y s i s of each stock s o l u t i o n was performed on the GCMS equipped with e i t h e r the c l e a n or d i r t y i n j e c t i o n port l i n e r . On a separate o c c a s i o n the n i t r o n e was analyzed by GCMS on the s i l a n i z e d system. A l a s t experiment i n v o l v e d the a d d i t i o n of 11 mg of n i t r o n e t o 5 ml of c o n t r o l r a t b i l e f o l l o w e d by e x t r a c t i o n with EtOAc from pH 9.5 s o l u t i o n , f o l l o w e d by d r y i n g , e v a p o r a t i o n , and r e c o n s t i t u t i o n of the organ i c phase to 110 u l . One u l (approximately 100 ug) was analyzed by GCMS. 96 I I I . RESULTS AND DISCUSSION The f i r s t o b j e c t i v e of the t h e s i s was to conclude the work on the methadone formamide (6) with work on pH e f f e c t s on the o x i d a t i o n of EDDP (1) and the mechanism of the o x i d a t i o n . T h i s i n i t i a l s e c t i o n concludes with d e t a i l s of the e f f e c t of workup c o n d i t i o n s on the i s o l a t i o n of the formamide ( 6 ) . Subsequent s e c t i o n s d e s c r i b e the use of r e c i p a v r i n (9) as a model compound i n experiments concerning the source of formamide m e t a b o l i t e s of t e r t i a r y a r y l a l i p h a t i c amines. 2i_FINAL__EXPERIMENTS_CONC A. Mechanisms f o r the p e r o x i d a t i o n of EDDP to an o x a z i r i d i n e ,  diketone and r e l a t e d compounds. The p e r o x i d a t i o n of the major methadone m e t a b o l i t e (—) 2-e t h y l - 1 , 5 - d i m e t h y l - 3 , 3 - d i p h e n y l p y r r o l i n i u m p e r c h l o r a t e (EDDP, 1) with m-chloroperbenzoic a c i d (MCPBA) a f f o r d e d d i a s t e r e o m e r i c 2-(4',4'-diphenylheptan-5'-one-2'-yl) o x a z i r i d i n e ( 2 ) , 4,4-diphenyl-2,5-heptanedione (3), the known compounds 2 - e t h y l - 5 - m e t h y l - 3 , 3 - d i p h e n y l - 1 - p y r r o l i n e (EMDP, 5) and 1 ,5-dimethyl-3,3-diphenyl-'2-pyrrolidone (DDP, 4) and other minor byproducts ( F i g u r e 1 ) ( S l a t t e r , 1983, Abbott, S l a t t e r and Rang, 1986). The o x a z i r i d i n e was l a b i l e , and decomposed upon s t a n d i n g , when r e f l u x e d i n m-xylene, and i n the GC i n l e t to the i s o m e r i c formamide, 6-formamido-4,4-diphenyl-3-heptanone ( 6 ) . The GCMS c h a r a c t e r i s t i c s of the formamide were i d e n t i c a l 97 to a new metabolite of methadone isolated by solvent extraction of /3-glucuronidase-hydrolyzed rat b i l e (Abbott, S l a t t e r , Burton and Rang, 1985). Several experiments were done to r e f i n e the mechanism proposed for t h i s oxidation. i . High resolution mass spectrum of the methadone oxaziridine (2) Figure 13 shows a composite high resolution mass spectrum of the methadone oxaziridine that was recorded at the lowest possible source temperature (120°). As expected from GCMS re s u l t s , thermal decomposition to the formamide dominates the mass spectrum (figure 1b). The presence of the ion m/z 56 i s the only indication that decomposition at thi s temperature is not spontaneous. This ion (C3HgN+) corresponds to the desoxygenated iminium cation CH3~CH=N+=CH2, an alpha cleavage product which i s also common to the isomeric nitrone functional group in amphetamine related compounds (Coutts et al., 1978). i i . MCPBA oxidation of methadone HCI. When the t e r t i a r y amine, methadone HCI (8) was oxidized with MCPBA, an additional minor oxidation product with a mass spectrum in accord with the the t e r t i a r y formamide structure (49) was obtained (figure 14). This i s in accord with the re s u l t s obtained for other a l i p h a t i c formamides generated by the peroxidation of N-methylamines (Sayigh and U l r i c h , 1963). 98 >-CO z 100 -i 80 -60 -UJ > 40 UJ cc 20 0 44 < •57 56.; ::5~ 91 J72 l I t i i i I l l Jl73 P H I' "! '' '• i :•! 207 208 - - C H N O — CH N CHO — CH 129 165 1151 103 130 1 1152 193 206 222 253 252)1 I 1 • I' I I I " . 224 •mn llllll — n — 1279 'f 9 1309M + r 40 80 120 160 M/Z 200 240 280 320 M/Z F i g u r e 13. (top) Composite of high r e s o l u t i o n mass s p e c t r a l r e s u l t s f o r the methadone o x a z i r i d i n e (2) (source temperature 120°). (bottom) Mass spectrum of the methadone formamide (6) a r i s i n g from GCMS a n a l y s i s of the methadone o x a z i r i d i n e . 99 This suggested that i f post enzymatic peroxidative generation of the secondary formamide from secondary amines were occurring then i t was also l i k e l y that t e r t i a r y formamides would aris e from t e r t i a r y amines, e s p e c i a l l y since t e r t i a r y amines are more readily oxidized than secondary amines (Beckwith, et al., 1983). This was born out when b i l e from methadone dosed rats was l e f t exposed to a i r for one week at room temperature and then examined by GCMS and found to contain the t e r t i a r y formamide in the nonconjugated fracti o n (appendix). This observation of post enzymatic oxidation required that future sample preparations should be treated so as to avoid possible a r t i f a c t u a l generation of formamides in b i l e by post enzymatic oxidation. - i i i . Peroxidation of EDDP free base in the presence of suspended K2CC>3 The formamide isolated as a methadone metabolite had been isola t e d under a l k a l i n e conditions, while the oxidation of EDDP to the oxaziridine (2) was done under a c i d i c conditions. The oxidation conditions were modified to see i f EDDP produced oxaziridines by peroxidation under a l k a l i n e conditions. Oxidation of the EDDP free base in the presence of suspended K2CO3 did not afford the oxaziridine. The major products were EMDP (5) and DDP (4). The diketone (3) was cy c l i z e d to only one of two possible regioisomeric cyclopentenones, 2,3-dimethyl-5,5-diphenylcyclopent-2-enone (50). 6 P E C - 3 6 . IMI . U N J I 3 2 I S C N I 1 7 0 I B 0 K G * 1 6 7 I T E R R - 2 7 I I N T . - 241 86 58 30 M ;S»' { E S T . M A S S . I S . 276 i U 72 / CH3CH=N+(CH3)CHO 87 105 129 »1 l'" | " S 1M l«l151 I1" ,i .n l r l r iii cni Ur I l b I I * l b l i t l b l b i t * I: N-CHO i C - C H 2 - C H CH. 49 207 208 165 179 . i . lb ?b' 267 ib »b ib sb sb sb" i T . M A S S . I S . 276 lb -Figure 14. Mass spectrum of the t e r t i a r y formamide (49) formed by MCPBA oxidation of methadone HC1. M + 323 absent. 101 To c o n f i r m which of • two p o s s i b l e r e g i o i s o m e r i c cyclopentenones was produced by t h i s a l d o l condensation, the diketone was r e f l u x e d b r i e f l y i n 0.37 M e t h a n o l i c sodium hydroxide. The cyclopentenone product was de t e c t e d on the TLC p l a t e by i t s r a p i d r e a c t i o n with i o d i n e vapour. Only one regioisomer was obtained ( f i g u r e 15), with NMR ( f i g u r e 16), IR (appendix) and UV s p e c t r a i n a c c o r d with the s t r u c t u r e 2,3-dimethyl-5,5-diphenylcyclopent-2-enone (50) and not 3 - e t h y l -4,4-diphenylcyclopent-2-enone (51). NMR rev e a l e d s i n g l e t s at 1.78, 2.13 and 3.32 ppm f o r CH3CC=0, CH 3CCH 2, and CH 2 r e s p e c t i v e l y ( f i g u r e 16). H o m o a l l y l i c c o u p l i n g broadened the resonances at 1.78 and 3.32 ppm. The UV spectrum had cyclopentenone K and R bands at 240 and 316 nm r e s p e c t i v e l y . The c a l c u l a t e d v a l u e s f o r the cyclopentenone system enone p i -p i t r a n s i t i o n (K band) with a and B s u b s t i t u e n t i s 236 nm versus 224 nm f o r the s i n g l e beta s u b s t i t u e n t of the other re g i o i s o m e r . The R band n-pi i s a weak band between 310 and 330 nm and was not d i a g n o s t i c ( S i l v e r s t e i n et al., 1981). 50 51 F i g u r e 15. P o s s i b l e r e g i o i s o m e r i c cyclopentenones (50 and 51) expected from the a l d o l condensation of the d i k e t o n e . -1 F i g u r e 16. NMR spectrum of the cyclopentenone a l d o l product 2,3 dimethyl-5,5-diphenylcyclopent-2-enone (50). 103 i v . Mechanism f o r the p e r o x i d a t i o n of EDDP . I t has been e s t a b l i s h e d by M i l l i e t and co-workers (1981, 1974a, 1974b) that p e r o x i d a t i o n of N - m e t h y l p y r r o l i d i n e d e r i v a t i v e s of the a l k a l o i d conanine can a f f o r d open ch a i n k e t o - o x a z i r i d i n e s , d i c a r b o n y l compounds, c y c l i c o x a z i r i d i n i u m s a l t s , lactams, p y r r o l i n e s and r e l a t e d compounds. The s i m i l a r i t y between the a r r a y s of o x i d a t i o n products d e s c r i b e d f o r conanine and those of EDDP prompted the a p p l i c a t i o n of mechanisms d e s c r i b e d by M i l l i e t et al . (1981, 1974a, 1974b) to the EDDP o x i d a t i o n . The p e r a c i d o x i d a t i o n of imines i s thought to proceed by a two step B a y e r - V i l l i g e r type mechanism with r a t e determining oxidant a d d i t i o n to the C=N bond i n the f i r s t s t e p (Ogata and Sawaki, 1973). T h i s mechanism, a p p l i e d to EDDP i s shown i n f i g u r e 17. Ring c l o s u r e with l o s s of c h l o r o b e n z o i c a c i d f o l l o w s i n the second step to gi v e an intermediate of type a. F i g u r e 17. B a y e r - V i l l i g e r MCPBA a d d i t i o n to C=N + bonds i n the f i r s t s t e p of the p e r o x i d a t i o n of EDDP ( 1 ) . 1 04 M i l l i e t and co-workers (1974a, 1974b, 1981) have i n c o r p o r a t e d i n t e r m e d i a t e a i n t o t h e i r mechanisms of p e r o x i d a t i o n of N-methylpyrrolinium s a l t s and f r e e bases. T h e i r mechanisms, m o d i f i e d to EDDP are shown i n f i g u r e 18. They have proposed that e l i m i n a t i o n of the r i n g proton alpha to n i t r o g e n i n a (scheme 1) accounts f o r keto-imine c, t h e r m o l a b i l e C - d i s u b s t i t u t e d k e t o - o x a z i r i d i n e b and diketone (3 ) . We s p e c u l a t e that e l i m i n a t i o n of the alpha proton on the N-methyl carbon of a i n a s i m i l a r manner ( f i g u r e 18, scheme 2) accounts f o r EMDP, i n accor d with the r e s u l t s of M i l l i e t et al . (1981), as w e l l as the methylene o x a z i r i d i n e . The conanine analogues of the keto-imines c and d were observed by M i l l i e t et al. ( 1981) f o l l o w i n g p e r o x i d a t i o n s with one e q u i v a l e n t of oxid a n t . Keto-imines were not observed i n t h i s study, however they are the c o n v e n t i o n a l imine p r e c u r s o r s f o r p e r o x i d a t i o n t o o x a z i r i d i n e s b and 2 r e s p e c t i v e l y . The imine d i s presumably unstable and c o u l d form a 1 ,3,5-triazane or r e l a t e d polymers (Emmons, 1957, F a r r a r , 1968), themselves " amenable to p e r o x i d a t i o n . The i n i t i a l a d d i t i o n of MCPBA to p y r r o l i d i n e s a l t s i s a r e v e r s i b l e r e a c t i o n , u n a f f e c t e d by oxidant c o n c e n t r a t i o n ( M i l l i e t , et al., 1981). T h i s c o u l d account f o r our ob s e r v a t i o n of unreacted EDDP under a v a r i e t y of r e a c t i o n c o n d i t i o n s , i n c l u d i n g l a r g e excesses of MCPBA. Scheme 1 Scheme 2 Figure 18. Possible mechanisms for the formation of the oxidation products of EDDP that were observed by GCMS. 106 The other product of the peroxidation of EDDP, the diphenylpyrrol idone DDP (4) could ar i s e from the enamine tautomer of EDDP by known mechanisms ( M i l l i e t , et al . , 1981, Back, et a / . , 1977) (f igure 19), although the endocycl ic double bond pos i t ion i s preferred in ac id so lut ion (Hassan and Casy, 1970). . C 6 H 5 C 6 H 5 2mcpba I OH k + RCOO 0=C—R I OH 4 + RCOO + CH3CHO I O — C - R II O Figure 1 9 . Mechanism for the formation of DDP (4) from oxidation of the enamine tautomer of EDDP. 1 07 B. Solvent c o n t r o l experiments i n the i s o l a t i o n of the  methadone formamide m e t a b o l i t e To p r e c l u d e the p o s s i b i l i t y of phosgene,, peroxide and aldehyde r e l a t e d g e n e r a t i o n of formamides i n the conjugated f r a c t i o n of r a t b i l e , the m e t a b o l i t e e x t r a c t i o n method of Horning and M i t c h e l l (1984) was used. Ether and chlo r o f o r m e x t r a c t i o n s were performed with and without p r i o r treatment w i t h type 4A molecular s i e v e ( B u r f i e l d , 1982, B u r f i e l d and Smithers, 1982) and d i s t i l l a t i o n j u s t b efore use. Molecular s i e v e s remove or g a n i c p e r o x i d e s from ether and phosgene r e l a t e d i m p u r i t i e s from c h l o r o f o r m . B i l e e x t r a c t s were r e c o n s t i t u t e d with diphenylbutanone (14, Ph 2CH-CH 2-C(=0)CH 3) added as an i n t e r n a l standard j u s t p r i o r to a n a l y s i s by GCMS. The area of the m/z 167 ion peak (Ph 2CH +) which i s common to diphenylbutanone and the secondary formamide (6) was i n t e g r a t e d to estimate the r e l a t i v e amount of the formamide pres e n t . F o l l o w i n g the standard e x t r a c t i o n p r o t o c o l , or the method employing s o l i d ammonium carbonate f o r a l k a l i n i z a t i o n (Horning and M i t c h e l l , 1984), s o l v e n t / a l k a l i p a i r s were employed f o r the e x t r a c t i o n and b a s i f i c a t i o n of nonconjugated and conjugated m e t a b o l i t e s . The c h l o r o f o r m e x t r a c t e d sample was a l s o r e c o n s t i t u t e d with ethanol i n p l a c e of methanol to p r e c l u d e any c o n t r i b u t i o n of formate or formaldehyde contaminants i n methanol to the amount of the methadone formamide ( 6 ) . R e s u l t s of these sample i s o l a t i o n procedures are summarized i n t a b l e 3. 108 Table 3. Recovery of the methadone formamide metabolite (6) using various sample i s o l a t i o n procedures. B i l e f r a c t i o n ! S o l v e n t / a l k a l i / s o l v e n t used in workup?. formamide peak abund.-l %DPB A. Conj CHCl3/NaOH/MeOH 12000 1,12 A. Conj CHCl 3 5/NaOH/MeOH 13307 2.14 A. Conj CHCl 3/NaOH/EtOH 13000 -B. Conj CH 2Cl 2/NaOH/MeOH 1 3500 2.72 C . Conj EtOAc/NH 4C0 3/MeOH 12000 1.12 D. Conj Ether/NH 4 C0 3 /MeOH 1 600 0.23 D. Conj Ether 5 /NH 4 C0 3 /MeOH 2000 0.08 E . Conj C 6 H 6 /NH 4 C0 3 /MeOH 1 2000 0.75 F . A l l E10Ac/NH 4 CO 3/MeOH 1 4000 1 .62 G. A l l CHC13/NaOH/MeOH - 2.32 conj= conjugated f r a c t i o n , a l l= conjugated and nonconjugated metabolites extracted together immediately after B-glucuronidase h y d r o l y s i s . 2 . solvent=extraction solvent, / a l k a l i * 1M NaOH or s o l i d NH 4 C0 3 used to basify aqueous b i l e , /solvent= MeOH containing diphenylbutanone in terna l standard, or ethanol (EtOH) used to d isso lve b i l e extract for GCMS. 3 . Formamide m/z 167 ion abundance from the integrated areas of the m/z 167 ion chromatogram. ^ . Formamide m/z 167 ion abundance from the integrated areas of the m/z 167 ion chromatogram expressed as percent of the m/z 167 ion abundance of the standard diphenylbutanone (DPB, 5ug per i n j e c t i o n ) . 5. Solvent received no pretreatment with molecular s i eve . The re su l t s in table 3 show that regardless of solvent or p u r i f i c a t i o n method, detectable amounts of the formamide metabolite were always observed. Halogenated solvents d id r e s u l t in higher recover ies , e s p e c i a l l y when the solvent was not 109 t r e a t e d with molecular s i e v e p r i o r to use. T h i s i n d i c a t e s that a p o s s i b l e r o l e of phosgene or formyl c h l o r i d e i n formamide ge n e r a t i o n cannot be r u l e d out. The f o r m y l a t i o n of ba s i c compounds by these s o l v e n t i m p u r i t i e s i s a w e l l known p i t f a l l i n drug metabolism methodology ( S t i l l w e l l et a l . , 1978, Cone et al., 1981). However i t i s d i f f i c u l t to envisage dinormethadone, the r e q u i s i t e s u b s t r a t e i n t h i s f o r m y l a t i o n r e a c t i o n , as a component of the conjugated f r a c t i o n . I t i s g e n e r a l l y accepted t h a t any b a s i c d e s a l k y l m e t a b o l i t e of methadone spontaneously c y c l i z e s to the corresponding p y r r o l i d i n e (EDDP and EMDP f o r normethadone and dinormethadone r e s p e c t i v e l y ) ( P o h l a n d , et al., 1971). The phosgene mediated f o r m y l a t i o n of EMDP (5) and EDDP (1) was not i n v e s t i g a t e d because n e i t h e r EDDP or EMDP were components of the conjugated b i l e f r a c t i o n . The recovery of the formamide m e t a b o l i t e was lowest with d i e t h y l e t h e r , and was not a l t e r e d s i g n i f i c a n t l y by pretreatment t o remove organic p e r o x i d e s . The lower recovery may be r e l a t e d to the s o l u b i l i t y of the m e t a b o l i t e i n et h e r . T h i s s p e c u l a t i o n i s based on the requirements f o r more p o l a r c h l o r o f o r m / a l c o h o l or e t h y l a c e t a t e based s o l v e n t systems to d i s s o l v e and e l u t e r e c i p a v r i n d e r i v e d formamides or n i t r o n e s f o r chromatography on s i l i c a g e l . Exchanging r e d i s t i l l e d e t h a n o l for. methanol as the so l v e n t used to d i s s o l v e the e x t r a c t f o r i n j e c t i o n , had no e f f e c t on the abundance of the formamide i n the chlo r o f o r m 110 e x t r a c t e d b i l e . T h i s p r e c l u d e d the p o s s i b i l i t y that formate or formaldehyde i n methanol was i n v o l v e d i n the p r o d u c t i o n of the formamide m e t a b o l i t e from an u n i d e n t i f i e d p r e c u r s o r . C. C o n c l u s i o n s r e g a r d i n g the source of the methadone formamide  m e t a b o l i t e P e r o x i d a t i o n of EDDP (1) and methadone (8) has p o i n t e d to two p o s s i b l e p r e c u r s o r s of the formamide m e t a b o l i t e of methadone, namely, the o x a z i r i d i n e (2) and t e r t i a r y l i -me thy 1 formamide (49). I t was not p o s s i b l e to s y n t h e s i z e hydroxylamine and n i t r o n e p r e c u r s o r s due to c y c l i z a t i o n of int e r m e d i a t e s ( S l a t t e r , 1983). Solvent c o n t r o l experiments have shown that s o l v e n t s can c o n t r i b u t e , but are not the s o l e source of the formamide m e t a b o l i t e of methadone. T h i s c o u l d be due to chemical r e a c t i o n with u n i d e n t i f i e d p r e c u r s o r s or by d i f f e r e n t e x t r a c t i o n e f f i c i e n c i e s . 2. SYNTHESIS AND CHARACTERIZATION OF SYNTHETIC COMPOUNDS  RELATED TO RECIPAVRIN The s y n t h e s i s of p o t e n t i a l i n t e r m e d i a t e s i n the gen e r a t i o n of a formamide m e t a b o l i t e of r e c i p a v r i n was undertaken to search f o r these i n t e r m e d i a t e s by GCMS and LCMS i n b i l e e x t r a c t s . The l a b i l i t y of p o t e n t i a l i n t e r m e d i a t e s under sample i s o l a t i o n c o n d i t i o n s c o u l d a l s o be determined. The s y n t h e s i s and c h a r a c t e r i z a t i o n of the secondary hydroxylamine (17), methylene n i t r o n e (24) and o x a z i r i d i n e (25) were r e p o r t e d p r e v i o u s l y ( S l a t t e r , 1983). 111 S p e c t r a l r e s u l t s f o r a l l compounds are summarized i n the expe r i m e n t a l s e c t i o n . New spectra not i n c l u d e d i n the t e x t are pre s e n t e d i n the appendix, indexed by compound name i n the same order as the experimental s e c t i o n . A. S y n t h e s i s and C h a r a c t e r i z a t i o n of the amines The amines r e c i p a v r i n (9), n o r r e c i p a v r i n (15) and d i n o r r e c i p a v r i n (20) are a l l known compounds and gave s a t i s f a c t o r y NMR, IR, and GCMS analyses ( S l a t t e r , 1983). N o r r e c i p a v r i n and d i n o r r e c i p a v r i n both formed TMS d e r i v a t i v e s with BSTFA. N o r r e c i p a v r i n was methylated with TMAH to g i v e r e c i p a v r i n . 9 R 1=R 2=CH 3 Ph2CHCH 2CH(CH 3)NR 1R2 15 R^H, R 2=CH 3 20 R 1=R 2=H B. S y n t h e s i s and C h a r a c t e r i z a t i o n of the amides The secondary formamide (12, Ph 2CHCH 2CH(CH 3)N(H)CHO) was ob t a i n e d i n good y i e l d by r e f l u x i n g d i n o r r e c i p a v r i n (20) with e t h y l formate, or by Leuckart r e a c t i o n of diphenylbutanone (14) and formamide using the method of Moffat, et al . (1962), and Moore (1949) r e s p e c t i v e l y . 1 1 2 The proton NMR spectrum of the secondary formamide showed that two rotamers were present in a r a t i o of 2:1 (Figure 20). When the spectrum was run in DMSO-Dg at 25°, the proportion of c i s isomer decreased to 2.1:10 r e l a t i v e to the trans rotamer. The r a t i o decreased further to 1.6:10 when the spectrum was rerun in DMSO-D5 at 90° C. The position of the NH and, to a lesser extent, the CHO resonances varied between samples. The ro t a t i o n a l isomerism of the formamide functional group a r i s i n g from amide resonance-mediated r e s t r i c t e d rotation about the C-N bond has been well documented spectroscopically (LaPlanche and Rogers, 1964, Hallam and Jones, 1970) and is discussed further in the section on the promethazine formamides. The planar c i s and trans orientations described by LaPlanche and Rogers are shown below. Cis and trans nomenclature is assigned based on the r e l a t i o n between the NH and C=0 groups that are involved in hydrogen bonding. The trans rotamer predominates. In the 1 3C NMR spectrum the rotamers were also apparent (Figure 21). The infrared spectrum had d i s t i n c t c i s and trans C=0 stretches (Figure 22). The secondary' formamide reacted slowly with BSTFA at 80° to form a mono TMS derivative (60) (Figure 23). H -N [\ H H -N / I \ R H Trans (major) Cis (minor) 1 1 3 A major byproduct of the Leuckart r e a c t i o n was 4-(2,2-d i p h e n y l e t h y l ) p y r i m i d i n e (59) (Figure 24). Formamide and p y r i m i d i n e byproducts of the Leuckart r e a c t i o n of phenyl-2-propanone are important i n the f o r e n s i c i n v e s t i g a t i o n of c l a n d e s t i n e amphetamine l a b o r a t o r i e s (Kram and Kruegel, 1977, Frank, 1983) The t e r t i a r y formamide (26, Ph2CHCH2CH(CH3)N(CH3)CHO) was s y n t h e s i z e d by Leuckart r e a c t i o n of diphenylbutanone (14) and N-methyl formamide and by methylation of the secondary formamide (12) with TMAH. In the t e r t i a r y formamide, proton and 1 N M R r e s u l t s showed that the t r a n s rotamer predominates i n CDCI3 s o l u t i o n at ambient temperature (11.4% c i s ) ( f i g u r e 25, 26). NMR r e s u l t s were i n accord with those r e p o r t e d f o r the s t r u c t u r a l l y s i m i l a r formamide d e r i v a t i v e of methamphetamine ( L e b e l l e et al. 1973). C i s and tr a n s C=0 s t r e t c h e s were not r e s o l v e d i n the i n f r a r e d spectrum ( f i g u r e 27). e i N - C H O b I H - C - C H 9 - C H ej * i 12 2 rotamers dd c| b a a T60 1 "140 TZD Too ' 8 0 WO 40 R " Figure 21. 75 MHz broad band decoupled 1 3 C NMR spectrum of the secondary formamide (12). 20 Figure 22. Infrared spectrum of a nujo l mull of the secondary formamide. NH stretches 3260 ( trans) , 3200 (shoulder, c i s ) . C=0 stretches 1670 ( trans) , 1650 ( c i s ) . 1 17 Hpk fib 169? 1600-1466 •1200-1 080-RL'CIP s r c FORM 888-600-400-200-73 44-I i 77 Ph 2CHCH 2CH(CH 3)N(H)CHO 1*7 f 13d t 5 2 115 / 1 8 3 zee 1 9 3 1 8 1 1 1 20 1 60 I 1 "' ' I '00 253 flit) L100 90 80 70 -60 -50 -40 3 8 ?0 18 240 Bf'k Hb 4036 440C-H 4880-3600-3200-2S00-2400-1600^ i^'ft^ 400-ftECIP i E C FORM TMS 145 73 45 Il i i h I 91 ) 15 II .1 T 80 1 I i III ,1 l i tH Ph2CHCH2CH(CH3)N(TMS)CHO l & G 221 193 1 Jl 206 12P 160 200 i-riB 100 90 80 310 2 5 3 i -40 -16 i - i • • I • i i I • • • I • ' M ' 11 ' I 240 ?S8 320 Figure 23. (top) Mass spectrum of the secondary formamide (12)(M + 253), (bottom) Mass spectrum of the secondary formamide mono TMS der iva t ive (60)(M + 325). Figure 24. 270 MHz NMR spectrum of the pyrimidine byproduct (59) of formamide synthesis . 1 I—I 1 I I I I I l . - ' l I I I I I I I I I I I I I I I I I I t - - 1 I I I I t - ' | , L I L- j L I I L I I ' I I I 1^ 1 I I i I |—1 | | - ' | | . j | . 1 8 0 7 . 0 b . O 5.-0 4 . 0 3 . 0 2 . 0 1 .0 F i g u r e 25. 300 MHz 1H NMR spectrum of the t e r t i a r y formamide (26). B B £ C i 6 N - C H O e i b' g H - C - C H P - C H 26 J J i L„«,...,.,i. 1 1 ' 1 1 1 1 2 0 0 A P T j i i i i I i i i i I i i i i I i i i i j i i i i i I i i i i I i i i i I i i i i i i i i i I i i i i I i i i i j i i i i I i i i i 120 100 8 0 6 0 4 Cp 1 8 0 1 6 0 1 40 i i I i i i i | i i i i I 2 0 PPM 9 a e Figure 26. 75 MHz 1 3 C NMR spectrum of the t e r t i a r y formamide (26). Top: broad band decoupled. Bottom: Attached proton test (CH and CH 3 project down, C and CH? project up). M O F i g u r e 27. I n f r a r e d spectrum of a t h i n f i l m of the t e r t i a r y formamide (26). C=0 s t r e t c h at 1666 cm . 122 The secondary acetamide (63, Ph 2CHCH2CH(CH3)NHC(=0)CH 3), a p o t e n t i a l m e t a b o l i t e of r e c i p a v r i n was s y n t h e s i z e d by a c e t y l a t i o n of d i n o r r e c i p a v r i n (20) with a c e t i c anhydride. The mass s p e c t r a of the t e r t i a r y formamide (26) and the isomeric secondary acetamide are s i m i l a r , t h e i r mass s p e c t r a d i f f e r i n g only i n the presence of ions at m/z 44 (acetamide) or m/z 58 (formamide). Both ions are r e l a t e d to the base peak at m/z 87 by l o s s of the a c y l fragment. C. S y n t h e s i s and C h a r a c t e r i z a t i o n of oximes, hydroxylamines  and t h e i r o x i d a t i o n products The oxime (19, (Ph 2CHCH 2C(CH 3)=N-OH)) was s y n t h e s i z e d by condensation of diphenylbutanone (14) and hydroxylamine h y d r o c h l o r i d e ( S l a t t e r , 1983). GCMS and NMR a n a l y s i s r e v e a l e d both syn (Z) and a n t i (E) geometric isomers. The predominant a n t i (E) isomer e l u t e s at longer r e t e n t i o n time. The r a t i o of geometric isomers i s a f u n c t i o n of the bulk of each alpha-C s u b s t i t u e n t , with the l a r g e s t C=N s u b s t i t u e n t t r a n s to the N-OH bond i n the a n t i (major) isomer (Gorrod and C h r i s t o u , 1986). Mass s p e c t r a of the u n d e r i v a t i z e d oximes were s i m i l a r although the syn isomer had more in t e n s e M+-17 and a r y l a l i p h a t i c fragments but d i d not have a d e t e c t a b l e molecular i o n . The oxime formed a TMS ether d e r i v a t i v e (52) with BSTFA and was O-methylated with TMAH to g i v e the oxime ether 16 (Ph 2CHCH 2C(CH 3)=NOCH 3). 123 Ph,CH-CH 0 OH PhoCH-CH, C=N C=N / / \ CH 3 CH 3 OH Syn (Z) oxime 19 A n t i (E) oxime 19 The primary hydroxylamine (22, Ph 2CHCH 2CH(CH 3)N(H)OH) was obta i n e d by r e d u c t i o n of the oxime (19) with sodium cyanoborohydride. S a t i s f a c t o r y NMR r e s u l t s f o r the fr e e base were only o btained with f r e s h l y prepared samples ( f i g u r e 28). The NMR r e s u l t s were s i m i l a r to those of N-hydroxyamphetamine (Beckett et a/. 1975). The product a u t o x i d i z e d w i t h i n hours to the azoxy dimer (R-N=N +(0~)-R). T h i s was ev i d e n t by the l o s s of the st r o n g NH and OH s t r e t c h i n the i n f r a r e d spectrum ( f i g u r e 29) and by the disappearance of the NHOH resonance in the NMR spectrum. As a consequence of the f a c i l e a u t o o x i d a t i o n , f r e s h l y prepared hydroxylamine was used i n the s y n t h e s i s of the methylene n i t r o n e (24). The secondary hydroxylamine (17, Ph 2CH-CH 2-CH(CH 3)-N(CH 3)OH) was o x i d i z e d to the methylene n i t r o n e (24, R-N +(0~)=CH 2) by yellow mercuric oxide, i n m i l d l y a l k a l i n e s o l u t i o n and i n c o n t r o l b i l e under simulated workup c o n d i t i o n s ( S l a t t e r , 1983). The hydroxylamines both formed O-TMS d e r i v a t i v e s with BSTFA. M e t h y l a t i o n with TMAH a f f o r d e d N-methoxy-N,a-dimethyl-7-phenylbenzenepropanamine (58) from both the primary and secondary hydroxylamines. When f r e s h l y s i l a n i z e d GC a c c e s s o r i e s were used, sma l l amounts of the i n t a c t hydroxylamines s u r v i v e d the GC s e c t o r to g i v e s a t i s f a c t o r y mass s p e c t r a with base peaks Figure 28. 300 MHz NMR spectrum of the primary hydroxylamine (22). Figure 29. Infrared spectrum of a thin f i l m of the primary hydroxylamine (22). NH and OH stretch between 3600 and 3100 cm . to 126 6400-E-t.*feK-r/.2eet-3286-2460-2&e6-1213 &-4 66-1 8 - i REC1F- F'RlrtfjR OH&rUNE + VF EI UNDER 1 VPT 1 ZED sot Htm 1 6 ? 91 / ' I IS 152 2 2 Ph 2CHCH 2CH(CH 3)N(H)OH 206 193 \ .1 i i M 241 1 28 169 206 i >—' 2 4 0 1 ' ' ' 1 ' I ' 1 ' ! 1 ' 2 8 0 3 2 6 c- 6 y K » -0 2 6 CH 4 •":: ti 6-4 4 H K K -3266-2 yd 6 2 4 >?> 6-1 liClfK-121:1" 366-4 66H 6 fci.C-:>' 2Hi'K!R'v UKkf!iHC*V[ E I UHUER I VftT I 2'EH BS ».^ », if, 17 Ph 2CHCH 2CH(CH 3)N(CH 3)OH n us '=2 103 186 M 222 80 1 2 » 1 6 9 2 6 0 240 2 9 0 Figure 30. Mass spectra of the underivat ized primary (22, top) and secondary (17, bottom) hydroxylamines. 1 27 a r i s i n g by cleavage of the C-C bond adjacent to nitrogen (Figure 30). The major degradation products were the respect ive amines. This mass spectra l fragmentation and chemical i n s t a b i l i t y has been seen in the amphetamine hydroxylamines (Beckett et al. 1973, Beckett and Achar i , 1977). D. Synthesis and Character izat ion of the rec ipavr in N-oxide The N-oxide (53, Ph 2 CHCH 2 CH(CH 3 ) -N + (0~ ) (CH 3 ) 2 ) decomposed to c i s and trans 1,1-diphenylbut-2-ene Cope e l iminat ion products (54 and 55, Ph2CHCH=CHCH3) and other minor byproducts when analyzed by GCMS. The t e r t i a r y formamide (26) was present as a minor byproduct of the ox idat ion . E . Synthesis and Character izat ion of the Nitrones The methylene nitrone (24, Ph 2 CHCH 2 CH(CH 3 )-N + (0~)=CH 2 ) was obtained from the condensation of the primary hydroxylamine (22) and formaldehyde (S la t t er , 1983). Spectral r e s u l t s of 24 were comparable to that of a s imi lar amphetamine ni trone (Coutts et al. 1978). The methylene nitrone is poss ib ly a rec ipavr in metabolite which could degrade thermally or chemically to the isomeric secondary formamide (12) (see nitrone degradation s tud ie s ) . Because th i s nitrone is a poss ib le precursor of the secondary formamide, the complete synthesis and charac ter i za t ion reported previously ( S l a t t e r , 1983) are included in the experimental sec t ion . The isomeric N-methyl nitrone (44, Ph 2 CHCH 2 C(CH 3 )=N + (0" )CH 3) was obtained from condensation of N-methylhydroxylamine 128 and diphenylbutanone (14). The N-methyl n i t r o n e was i s o l a t e d as a mixture of c i s (Z) and trans (E) isomers, which c o - e l u t e d under standard GCMS c o n d i t i o n s . The geometric isomers (43% c i s ) were apparent i n the NMR s p e c t r a ( f i g u r e 31,32). F. S y n t h e s i s and C h a r a c t e r i z a t i o n of the methylene imine (23)  (polymer) and o x a z i r i d i n e (25) The o x a z i r i d i n e (25) has been c h a r a c t e r i z e d i n d e t a i l ( S l a t t e r , 1983). Because the o x a z i r i d i n e i s a p o s s i b l e p r e c u r s o r to the formamide, the complete s y n t h e s i s and s p e c t r a l r e s u l t s were i n c l u d e d i n the experimental s e c t i o n . The o x a z i r i d i n e was s y n t h e s i z e d by a m o d i f i e d method of Krimm (1958). The s y n t h e s i s i n v o l v e s the i n s i t u p e r o x i d a t i o n of an unstable methylene imine (23) d e r i v e d from the condensation of d i n o r r e c i p a v r i n (20) and formaldehyde. The condensation products of primary amines and formaldehyde are 1,3,5-triazacyclohexanes ( t r i a z a n e s ) , 1,3-dioxa-5-aza-cyclohexanes (dioxazanes) and 1-oxa-3,5-diazacyclohexanes (oxadiazanes) (Baker, et al ., 1978). A n a l y t i c a l r e s u l t s , p a r t i c u l a r l y molecular weight d e t e r m i n a t i o n s , of the condensation products are o f t e n c o n f u s i n g ( F a r r a r , 1968). The polymers d i s s o c i a t e i n the CH [ Ph 2CH-CH2-CH(CH 3)-N=CH2 ] 23 25 9 ! ! ' i Me I ; LI C H 9 - C - b j I JCH , ! Z & E 4 4 e e J vi i l l l u l t s b k I l.ltJ H n iMlLul.rfL m i l i u m • 4231 H a l i . l V 4 I C 22 I V 324 1 .613 4330 I4«'jl.i62 141 1 316 ».2«9 4403 14142.141 142 i 4 o l 231 1.614 7317 12*44.222 12a 42/1 1 107 4.221 7]<l I2VII.J2I I2U 3212 9 672 24.390 / H i I214J.412 122 4323 » 163 24.000 --7lV4 12114.433 127 3114 4 til 7.914 /•3b 12747.249 124 • 914 1 931 4.894 7473 12209.321 124 1194 4 153 17.313 I0?l>4 7771.774 ?? 1130 187 I.9U 10223 2244.793 74 9932 460 :-4.142 10/44 .'714.704 74 4742 690 2.14] 126U1 4b«3.041 41 332] o i l .2.479 12047 4HI4.bj2 42 • 347 1 672 4.199 I22J9 4411.930 44 4011 317 1.121 12712 4601.611 43 1034 2 213 10.28) T 13114 40VB.214 40 2120 922 •3.140 : 11241 1904.321 It •040 3 740 li.tot :-K l IB |y3V.342 19 4731 1 114 3.913 ; 14319 1130.6*4 It 3*40 347 1.319 : a T T ^ - I 80 150 140 130 60 40 20 0 Figure 32. 100 MHz 1 3 C NMR spectrum of the N-methyl nitrone (44) . 131 vapour phase to give a s i n g l e peak by GC. Some degree of d i s s o c i a t i o n and d i s p r o p o r t i o n a t i o n i n s o l u t i o n can r e s u l t i n i n c r e a s i n g amounts of the t r i a z a n e p l u s formaldehyde (Baker, et al., 1978). Ring NMR resonances occur as s i n g l e t s between 3.1 and 3.6 ppm (Jones, et al., 1970) c o n s i s t e n t with r a p i d r i n g r e v e r s a l and n i t r o g e n i n v e r s i o n (Bushweller, et a/., 1974). Emmons (1957) f i r s t d e s c r i b e d the i n s i t u p e r o x i d a t i o n of the t r i a z a n e compounds t o monomeric o x a z i r i d i n e s and p o s t u l a t e d t h a t the a c i d i c r e a c t i o n c o n d i t i o n s a p p a r e n t l y depolymerize the int e r m e d i a t e t r i m e r , l e a v i n g the imine a v a i l a b l e f o r p e r o x i d a t i o n . Reynolds and Cossar (1971) have shown that HCI s a l t s of hexahydrotriazanes are e q u i v a l e n t to the methylene imine i n r e a c t i v i t y towards n u c l e o p h i l e s i n Mannich r e a c t i o n s . Two e q u i v a l e n t s of MCPBA were r e q u i r e d f o r the i n s i t u o x i d a t i o n of the t r i a z a n e (41). A combination of CaCC>3 and NaOH f o r a l k a l i n i z a t i o n and p e r e s t e r h y d r o l y s i s r e s p e c t i v e l y gave the best r e s u l t s . Much lower y i e l d s r e s u l t e d from the use of K 2 C 0 3 i n p l a c e of CaC0 3. The N,C-diastereomers of o x a z i r i d i n e (25) were separated by s i l i c a g e l f l a s h chromatography. The d i a s t e r e o i s o m e r i s m a r i s e s from the high i n v e r s i o n b a r r i e r of the o x a z i r i d i n e n i t r o g e n adjacent to the asymmetric center (Montanari, et al., 1968, Biorgo and Boyd, 1973). A s i m i l a r s e p a r a t i o n has been o b t a i n e d f o r 2-(4',4'-diphenylheptan-5'-one-2'yl) o x a z i r i d i n e ( 2 ) ( A b b o t t , S l a t t e r and Rang, 1986) and 2- [ R - a - p h e n y l e t h y l ] -132 3 , 3 - d i m e t h y l o x a z i r i d i n e ( B e l z e c k i and Mostowicz, l975ab). The NMR spectrum of the o x a z i r i d i n e was s i m i l a r to that of 2-(4',4'-diphenylheptan-5'-one-2'yl) o x a z i r i d i n e (2) and to 2-t-b u t y l o x a z i r i d i n e ( C r i s t , et al., 1979). The u p f i e l d doublet (3.28 ppm and 3.46 ppm i n the major and minor diastereomers of 25 r e s p e c t i v e l y ) i s presumed to be the proton t r a n s t o the lone p a i r on n i t r o g e n (Boyd, et al., 1969, Jordan and C r i s t 1977). The i s o m e r i z a t i o n of o x a z i r i d i n e s to amides i s w e l l documented (Emmons, 1957, L a t t e s , et al., 1982). Methylene o x a z i r i d i n e s a f f o r d formamides by thermal or F e ^ + mediated mechanisms. The formamide (12) i n t h i s study was ob t a i n e d from the o x a z i r i d i n e (25) on standing at room temperature or as a r e s u l t of complete thermal degradation i n the GC i n l e t . The i d e n t i t y of the product formamide d e r i v e d from the o x a z i r i d i n e by GC a n a l y s i s was confirmed by comparison of mass s p e c t r a and NMR s p e c t r a of a peak c o l l e c t e d o f f the GC column with a u t h e n t i c formamide s y n t h e s i z e d by r e a c t i o n of d i n o r r e c i p a v r i n (20) with e t h y l formate ( S l a t t e r , 1983). The s y n t h e t i c method used here and the l a b i l i t y of the o x a z i r i d i n e product i n d i c a t e s that a l i t e r a t u r e method f o r the s y n t h e s i s of formamides from primary amines, formaldehyde and hydrogen peroxide (Seng and Ley, 1975) probably i n v o l v e s an o x a z i r i d i n e i n t e rmediate which decomposes d u r i n g d i s t i l l a t i o n or sample workup. 1 3 3 G. S y n t h e s i s and C h a r a c t e r i z a t i o n of the is o c y a n i d e (65) and carbamates (66-69) The i s o c y a n i d e ( 6 5 , Ph 2CH-CH 2~CH(CH 3 ) - N-C) and carbamates ( 6 6 - 6 9 ) were s y n t h e s i z e d as r e f e r e n c e compounds f o r s t u d i e s on p o s s i b l e formyl c h l o r i d e or phosgene mediated a r t i f a c t s that c o u l d a r i s e from c h l o r o f o r m e x t r a c t i o n of r e c i p a v r i n m e t a b o l i t e s . 6 6 R i = H , R 2 = C H 3 6 7 R T = H , R 2 = C H 2 C H 3 P h 2 C H - C H 2 - C H ( C H 3 ) - N ( R 1 ) C ( = 0 ) O R 2 6 8 R 1 = C H 3 , R 2 = C H 3 6 9 R 1 = C H 3 , R 2 = C H 2 C H 3 The i s o c y a n i d e had a strong C = N - s t r e t c h i n the i n f r a r e d at 2 1 5 0 cm - 1. The N M R spectrum had resonances corresponding to the d i p h e n y l b u t y l backbone. The i s o c y a n i d e decomposed durin g GC a n a l y s i s , however one component had the c o r r e c t molecular weight ( 2 3 5 amu) with few other d i a g n o s t i c i o n s . The e t h y l carbamate analogue of r e c i p a v r i n ( 6 9 ) has been d e s c r i b e d p r e v i o u s l y as a t e s t compound f o r d e r i v a t i z a t i o n of t e r t i a r y amines ( H a r t v i g and Vessman, 1 9 7 4 ) . S p e c t r a l data was not s u p p l i e d . The mass s p e c t r a of e t h y l and methyl carbamates of n o r r e c i p a v r i n and d i n o r r e c i p a v r i n had weak molecular ions and were dominated by the b e n z y l i c cleavage (m/z 1 6 7 ) , and fragmentation of the C-C bond of the si d e c h a i n to a f f o r d ions a t m/z 1 3 0 and 1 1 6 f o r the n o r r e c i p a v r i n e t h y l and methyl 134 carbamates (69,68) and m/z 116 and 102 f o r the d i n o r r e c i p a v r i n e t h y l and methyl carbamates (67,66) r e s p e c t i v e l y . The NMR, mass and IR s p e c t r a were i n acco r d with the carbamate s t r u c t u r e s and are i n c l u d e d i n the appendix. H. Attempted s y n t h e s i s and c h a r a c t e r i z a t i o n of the  formohydroxamic a c i d (61) The hydroxamic a c i d (61, Ph 2CH-CH 2-CH(CH 3)-N(OH)CHO) was c o n s i d e r e d b r i e f l y as a p r e c u r s o r to the formamide on the b a s i s t h a t hydroxamic a c i d m e t a b o l i t e s of aromatic amines are w e l l known and the hydroxamic a c i d f u n c t i o n a l group i s g l u c u r o n i d e conjugable. A weak p o i n t i n the hy p o t h e s i s was the mechanism whereby the hydroxamic a c i d c o u l d degrade to the formamide without a r e d u c t i o n s t e p . The hydroxamic a c i d was a l s o a p o s s i b l e p r e c u r s o r of an u n i d e n t i f i e d r e c i p a v r i n m e t a b o l i t e that had a high mass ion at m/z 251 and base peak m/z 71 at r e l a t i v e l y long r e t e n t i o n time. Dehydration of the hydroxamic a c i d (M +) i n the source would be expected to r e s u l t i n these mass s p e c t r a l c h a r a c t e r i s t i c s . The Lossen rearrangement of hydroxamic a c i d s to iso c y a n a t e s i s a s i m i l a r t r a n s f o r m a t i o n . 135 The s y n t h e s i s was attempted by t r a n s f o r m y l a t i o n of the primary hydroxylamine (22) with e t h y l formate. The r e s u l t s were not s a t i s f a c t o r y . A f t e r d e r i v a t i z a t i o n with TMAH only one minor component i n the mixture had a s a t i s f a c t o r y mass spectrum f o r the methyl ether (Ph2CH-CH2-CH(CH3)-N(OCH3)CHO. A weak molecular ion a t m/z 283 e l i m i n a t e s CH3OH to give m/z 251. Presumably cleavage of the C-C bond adjacent to n i t r o g e n i n the 251 ion a f f o r d s the base peak at m/z 72 ( f i g u r e 33). In the i n f r a r e d spectrum of the u n d e r i v a t i z e d product there was a str o n g broad OH s t r e t c h between 3600 and 3000 cm - 1, and a ca r b o n y l s t r e t c h at 1665 cm - 1. Both bands were d i a g n o s t i c f o r the hydroxamic a c i d f u n c t i o n a l group (S o c r a t e s , 1980). Since no secondary formamide or m/z 251 compound was observed when the u n d e r i v a t i z e d product was analyzed by GCMS, f u r t h e r p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of 61 was abandoned. 136 ;i«j»iio sc*# i : " A R C • sn C A L . - o I I « T . « 1135 42 44 72 OMe -I N-CHO I l252 208 H - C - C h L - C H 130 167 74 77 91 115 103 l i u J 165 152 193 181 M 252 i r;283 - * — * ii1" F i g u r e 33. Mass spectrum of the suspected methyl ether of the formohydroxamic a c i d (61). 137 I. S y n t h e s i s and C h a r a c t e r i z a t i o n of the promethazine r e l a t e d formamides and t h e i r o x i d a t i o n products. The secondary formamide (72) was s y n t h e s i z e d as a p o t e n t i a l m e t a b o l i t e of the a n t i h i s t a m i n e promethazine (10)(see s e c t i o n 8 f o r mass s p e c t r a l r e s u l t s of compounds i n t h i s s e c t i o n ) . The t e r t i a r y formamide (74) was s y n t h e s i z e d f o r use i n metabolism s t u d i e s on a r y l a l i p h a t i c formamides. CH 2-CH(CH 3)NR,R2 10 R 72 R 74 R 75 R 76 R =CH 3, R 2—CH 3 =H, R2=CHO =CH3, R2=CHO =H, R2=CHO, + N 1 0 - 0 " =CH3, R 2=CH 3, +N 1 0-0-The secondary and t e r t i a r y formamide analogues of promethazine were s y n t h e s i z e d by Leuckart r e a c t i o n of 10-(2-propanone) p h e n o t h i a z i n e with formamide and N-methyl formamide respect i v e l y . In the secondary formamide (72) c i s and trans rotamers were apparent i n the proton and 1 3 C NMR s p e c t r a ( f i g u r e 34 and 35). C i s and t r a n s s t r u c t u r e s shown below were made a c c o r d i n g to LaPlanche and Rogers (1964). The formyl resonance appeared as a broadened s i n g l e t at 8.10 ppm f o r the trans rotamer and as a doublet at 7.88 (J=12 Hz) f o r the c i s rotamer much l i k e the secondary formamide analogues of r e c i p a v r i n and methadone 138 (12 and 6 r e s p e c t i v e l y ) . The c i s isomer accounted f o r 20% of the t o t a l formamide. The l i t e r a t u r e value f o r the c i s NH-CHO proton c o u p l i n g constant of isopropylformamide i n a c i d s o l u t i o n i s 13.8 Hz. Trans NH-CHO c o u p l i n g s are approximately 2 Hz i n the lower formamides. The l a r g e r NH-CHO proton c o u p l i n g i n the c i s isomer r e f l e c t s a t r a n s r e l a t i o n s h i p between the NH and CHO protons (Laplanche and Rogers, 1964). The p a r t i a l l y overlapped NH resonances were found between 5.5 and 5.7 ppm and were broadened by the quadrupole r e l a x a t i o n of the 1^N nuc l e u s . The r e l a t i v e amount of c i s isomer i n the secondary formamides i n c r e a s e s with the bulk of the n i t r o g e n s u b s t i t u e n t . H H H R \ / \ / C—N C — N * \ // \ O R O H Trans (major) C i s (minor) The r o t a t i o n a l isomerism a r i s e s from r e s t r i c t e d r o t a t i o n about the N-C=0 bond (Laplanche and Rogers, 1964) which g i v e s r i s e to hydrogen bonded c i s dimers and t r a n s polymers. As a r e s u l t of hydrogen bonding, s p e c t r a are subj e c t to c o n c e n t r a t i o n induced s h i f t s . In the 1 3 C NMR spectrum t r a n s and c i s formyl resonances appeared at 161.04 and 163.81 ppm r e s p e c t i v e l y . In the i n f r a r e d spectrum ( f i g u r e 36), the NH (3432 and 3400 cm - 1) and C=0 (1692 and 1681 cm" 1) s t r e t c h e s were d i s t i n c t f o r each rotamer i n accord with l i t e r a t u r e IR data (Laplanche and Rogers, 1964, Hallam and Jones, 1970). A 1 39 py r i m i d i n e (73) was i d e n t i f i e d by GCMS as a major byproduct of the r e a c t i o n . P e r o x i d a t i o n of the secondary formamide (72) with hydrogen peroxide to form the N i n - o x i d e (75) r e s u l t e d i n a compound wi t h only one rotamer apparent by NMR ( f i g u r e 37 and 38). The NH resonance was d e s h i e l d e d and r e s o l v e d i n t o a broad doublet a t 6.61 ppm. The formyl proton resonance appeared as a doublet (J= 8.5 Hz) cen t e r e d at 7.85 ppm suggesting a pr e f e r e n c e f o r the c i s conformation. The l a r g e d o w n f i e l d s h i f t of the CH 2 s i d e c h a i n resonance (one peak at 4.62 ppm from separate resonances at 3.8 and 3.95 ppm) and the N-0 s t r e t c h at 1010 cm - 1 i n the i n f r a r e d spectrum support the N ^ oxide s t r u c t u r e over that of the s u l f o x i d e ( f i g u r e 39). The t e r t i a r y formamide (74) was a l s o s y n t h e s i z e d and c h a r a c t e r i z e d as a 3:1 mixture of c i s and tr a n s rotamers by NMR ( f i g u r e 40). The IR ( f i g u r e 41) and GCMS r e s u l t s are a l s o shown ( f i g u r e 42). Promethazine N-oxide (76) has been d e s c r i b e d p r e v i o u s l y and c h a r a c t e r i z e d by mass spectrometry (Clement and Beckett, 1981b). T h i s was a necessary r e f e r e n c e compound f o r metabolism s t u d i e s so i t was s y n t h e s i z e d and c h a r a c t e r i z e d by NMR, mass and IR spectrometry. Figure 34. 400 MHz 'U NMR spectrum of the secondary formamide (72) . Figure 35. 100 MHz 1 3 C NMR SFORD spectrum of the secondary formamide (72). 300 too 3171 4344 *S4* mt 7070 767) 7)54 7*01 7711 771* 7741 7**4 •021 1071* 10724 10737 10730 19731 107** 124*1 11711 nsn 13100 13117 ._L_ ma IHTfORU. IHTEHIIIf 1*21*.2*0 1*1.4013 4.190 70.044 1*1*2.091 140.3371 .171 l l . » 7 1 14130.027 140.3174 .337 14.304 13341.137 111.47]* 3.441 117.714 11141.ato 112.4110 4.113 114.411 11113.011 112.31*3 .431 11.IH •10*1.727 12*.12*1 9.112 191.4** 1111*.*]] 127.3121 .474 f.311 12147.Ml 122.72J1 >. I l l *.3*3 12)25.810 122.30*3 3.2*4 1*0.171 1211*.211 112.4104 3.217 102.43* 11*12.71* 111.3*1* 4.703 1*1.741 1H91.I12 III.1134 9.021 8*.410 7779.014 77.11*1 *.473 97.437 77*7.2M 77. I H I 3.03* 31.141 7747.12* 7*.ffl4 1.2*2 104.74* 7727.032 74.7*1* .7*0 11.141 7713.1*0 7**7.1*2 7*.*I07 7.1U IOB.33* 74.30*1 .84* 10.104 3111.2** 30.120* 4.344 141.40* 4*21.119 43.(f|* 4.40* IB*.190 11*1.147 11.3774 4.437 11*.907 21.1*4 .211* 1.310 12.729 - l . * l ? -.01*1 0.341 147. Ml e 160 140 Figure 38. 100 MHz broad band decoupled secondary formamide N^-ox ide (75). 13 C NMR spectrum of the 212 149 J . S y n t h e s i s and C h a r a c t e r i z a t i o n of the o x a z i r i d i n e (82),  methylene imine (78) and formamide (83) r e l a t e d to amphetamine The methylene imine (78, PhCH 2CH(CH 3)N=CH 2) had s i m i l a r s p e c t r a l c h a r a c t e r i s t i c s to the r e c i p a v r i n imine (23, Ph 2CHCH 2CH(CH3)N=CH 2). In the mass spectrum, the m/z 56 base peak f o r the monomer i s one mass u n i t l e s s than f o r the r e c i p a v r i n imine. T h i s i m p l i e d that alpha cleavage was dominant. In the r e c i p a v r i n imine, the a v a i l a b i l i t y of a gamma proton allowed a M c L a f f e r t y rearrangement g i v i n g a m/z 57 base peak ( S l a t t e r , 1983). In the NMR spectrum ( f i g u r e 43), the t r i a z a n e (79), oxadiazane (80), and dioxazane (81) were d i s t i n c t and present i n a r a t i o of 3:2:5. Side c h a i n resonances overlapped, with the exc e p t i o n of the CH3 doublet of each component. S i n g l e t s f o r the r i n g protons were assigned based on r a t i o s of i n t e g r a t e d peaks. When the spectrum was rerun at 60°, the methyl and t r i a z a n e r i n g resonances at 1.02 (d) and 3.75 (s) r e s p e c t i v e l y disappeared and were r e p l a c e d by s i n g l e t s at 2.85 (methyl) and 2.77 ( t r i a z a n e r i n g ) ppm. The other two components were unchanged by h e a t i n g . Although temperature induced changes i n the conformation of t r i a z a n e s have been i n v e s t i g a t e d (Bushweller et al. 1974), these r e s u l t s are not e a s i l y e x p l a i n e d . The amphetamine o x a z i r i d i n e (82) was i s o l a t e d i n very low 1 50 y i e l d from the p e r o x i d a t i o n of the t r i a z a n e (79). The product was c h a r a c t e r i z e d by NMR ( f i g u r e 44). Two d i a s t e r e o m e r s were present i n a r a t i o of 1.23:1. The o x a z i r i d i n e r i n g resonances were present as p a i r s of doublets (J=10 Hz) c e n t e r e d at 3.65 and 4.00 ppm i n the major diastereomer and 3.18 and 3.7 ppm in the minor diastereomer. The o x a z i r i d i n e decomposed on st a n d i n g and i n the GC to the isomeric secondary formamide (83). The secondary formamide (83, PhCH 2CH(CH 3)NHCHO) i s w e l l known as a Leuckart s p e c i f i c i mpurity of i l l e g a l amphetamine s y n t h e s i s (Frank, 1983). The formamide was s y n t h e s i z e d as a re f e r e n c e compound f o r comparison to the decomposition product of the isomeric o x a z i r i d i n e (82). The mass spectrum of the formamide had a base peak at m/z 72 l i k e the methadone secondary formamide but u n l i k e the r e c i p a v r i n secondary formamide which had a base peak m/z 73. T h i s r e s u l t supports the gamma proton t r a n s f e r mechanism f o r fra g m e n t a t i o n of the j r e c i p a v r i n secondary formamide (see s e c t i o n 3 ) . The amphetamine and methadone formamides (83 and 6 r e s p e c t i v e l y ) l a c k the gamma proton and presumably fragment by cleavage of the alpha C-C bond. The NMR spectrum of the formamide (83) showed c i s and tra n s rotamers and was i n accord with the r e s u l t s r e p o r t e d by Hess et al., 1976 ( f i g u r e 45). Ph R R i i j / N \ ^ a ^ N ^ a a ^ ° ^ a N N CJ O N N „' V X V X „' V X 79 80 81 R= PhCH2CHMe 80a 80 81 a W Figure 43. 80 MHz NMR spectrum of the amphetamine methylene imine derived polymers (79-81). ti p|Hii C.l T 1 * I — , 5 4 3 2 1 0 Figure 44. 400 MHz NMR spectrum of the amphetamine o x a z i r i d i n e 75 10 1] SO 10 10 5 I IS 10 5 IS 10 s Ph 8 3 4 5 NHCHO (2 rotamers) so io 10 75 10 15 SHiitmO am/cm Q i 1 i-i 1 1 1 i I I I i i i i i i i i i * • » * 5 4 1 J I Figure 45. 80 MHz NMR spectrum of the amphetamine secondary formamide (83). O ppm D ppm O 1 54 3. MASS SPECTROMETRY OF RECIPAVRIN RELATED COMPOUNDS A.Fragmentation of the a l i p h a t i c s i d e chain The c h o i c e of a major fragmentation pathway i n r e c i p a v r i n (9) and other, a r y l a l i p h a t i c compounds i s determined by charge l o c a l i z a t i o n on e i t h e r the phenyl r i n g s or the heteroatom p o r t i o n of the molecule. While pathways a r i s i n g from both p o s s i b l e i o n i z a t i o n s are always e v i d e n t , f a c t o r s such as e l e c t r o n d e n s i t y at n i t r o g e n , or whether n i t r o g e n or oxygen i s s i n g l y or doubly bonded to the side c h a i n , determine which of the two pathways predominate. G e n e r a l l y a s i n g l e bonded, basic n i t r o g e n atom f a v o r s r a d i c a l s i t e i n i t i a t i o n at N with alpha cleavage of the l a r g e s t a l k y l c h a i n (Stevenson's R u l e ) ( M c L a f f e r t y , 1980). T h i s cleavage generates base peak c a t i o n s of the general formula A ( f i g u r e 46, R 1,R 2=H or CH3). Molecular ions and a r y l a l i p h a t i c fragments (m/z 208, 193, 179, 167, 165, 152, 130, 105, 91 et c . ) are of low i n t e n s i t y as in n o r r e c i p a v r i n ( f i g u r e 50 t o p ) . When e l e c t r o n d e n s i t y decreases at the C-N s i n g l e bond, as i n the secondary formamide analogue of recipavrin,(R<=H, R2=CHO, F i g u r e 50 bottom), a protonated alpha cleavage ion (m/z 73) i s p r e s e n t as the base peak along with a l e s s i n t e n s e alpha cleavage i o n . Rearrangement of the gamma hydrogen to the amide n i t r o g e n with adjacent C-N cleavage and charge r e t e n t i o n g i v e s r i s e to the 1-methyl-2,2-diphenylcyclopropane c a t i o n 1 55 radic a l (m/z 208, figure 47) which can fragment to the d i a r y l a l i p h a t i c series of ions (m/z 208, 193, 179, 167, 165, 152, 130, 105, 91,) that have been described in work on methadone (Kang, et al. , 1979). D i a r y l a l i p h a t i c fragments in t h i s series increase in intensity with decreasing mass to a maximum at the diphenylmethine cation (m/z 167). In compounds containing nitrogen or oxygen doubly bonded to the diphenylbutane skeleton, i . e . diphenylbutanone (14, Ph2CH-CH2-C(CH3)=0) and diphenylbutanone oxime (19, Ph2CH-CH2-C(CH3)=NOH), the base peak arises by r a d i c a l s i t e i n i t i a t i o n on the aromatic ring, with a l l y l i c (benzylic) cleavage to give a resonance s t a b i l i z e d diphenylmethine cation (m/z 167) (figure 48) (Noren et al., 1985). Benzylic cleavage may compete with the McLafferty rearrangement shown in figure 49 especially in the phenyl ring oxidized d i a r y l a l k y l metabolites which form stable oxonium ions. Some alpha cleavage by radical s i t e i n i t i a t i o n at the unsaturated hetero atom occurs in diphenylbutanone and i t ' s derivatives giving a m/z 43 peak. The oxime loses an OH r a d i c a l to give M-17 ions (m/z 222). Regardless of the intensity of fragments produced by cleavage alpha to the hetero atom, the diphenylmethyl cation (Ph 2CH +) i s always present and i s the key ion in the detection of recipavrin metabolites with intact phenyl rings. R' I A r « C H - C H 0 - C H T N - R " C H 3 R I C H = N - R ' I + + A r 2 C H - C H 2 Figure 46. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : I. R a d i c a l s i t e i n i t i a t i o n at the s a t u r a t e d heteroatom with alpha cleavage. T h i s g i v e s r i s e t o base peak in compounds with b a s i c n i t r o g e n atoms. 157 F i g u r e 47. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : I I . Rearrangement of the gamma hydrogen to the s a t u r a t e d hetero atom with adjacent cleavage and charge r e t e n t i o n . T h i s g i v e s r i s e to m/z 208 cascade common to a l l phenyl r i n g i n t a c t m e t a b o l i t e s . 158 a. F i g u r e 48. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : I I I . R a d i c a l s i t e i n i t i a t i o n on the aromatic r i n g with a l l y l i c ( b e n z y l i c ) cleavage. T h i s g i v e s r i s e to base peak d i a g n o s t i c ions i n the ketones and oximes. F i g u r e 49. General mass s p e c t r a l fragmentations of d i a r y l b u t a n e s : IV. Rearrangement of gamma hydrogen with b e n z y l i c cleavage and l o s s of a hydrogen r a d i c a l to a f f o r d d i a g n o s t i c oxonium ions i n phenyl r i n g o x i d i z e d m e t a b o l i t e s and t h e i r d e r i v a t i v e s . 159 Bpk fib 14644 R E C I P B I L E C O N J ETORC 1 2 . 8 2 B i n . 14886H 12888H leeeeH 8 8 8 6 H 6 8 8 H 4888H 2 8 8 8 H 84 £8 88 188 128 I • I • I • I . I • I . I . I . 148 I • t • l&a 188 288 I . I . I . I . I . I 228 .A I . I,. 58 >• 167 208-H - C . - C H ^ C H - i - N M e I H 15 1 6 7 152 7 7 1 * 3 65 / -v H 5 138146 / .1 lii.i . I ... i . ik J . •!. > I i • i • i ~ i ' f T~ 68 88 188 128 193 2 8 8 M+ 2 3 9 j N r 118 : 188 ;98 -ee -78 -66 -G9 -48 -38 -28 18 I ' I ' I • I ' I • I • I • 1 • T 148 168 188 288 2 2 8 Bpk fib 2246 b 2488^ 2 2 8 » > 2888-1888^ 1688-1 4 8 » > 1288-l e W 888-688-488-288-RECIP BILE CONJ ETOHC 88 • i • 128 • i • • 168 ?88 2 4 8 288 1 8 . 2 8 B i n 328 73 44 • 1 6 7 / • • I • 1 8 9 / 138 / 152 8^ • 288 \ H - C T C H „ 4 C H 1 i C H r72/73 • H l I ' N - C H O 121 i n 208 12 £18 288 I ' ' ' I ' ' ' I ' 248 - 288 328 -118 -188 ^ 8 -68 -78 •*8 -58 -48 -38 : 28 -18 -8 F i g u r e 50. Mass s p e c t r a of r e c i p a v r i n m e t a b o l i t e s i l l u s t r a t i n g the three major fragmentation schemes i n f i g u r e 46,47,48. A N o r r e c i p a v r i n (15). B. Secondary formamide (12). A 'proton t r a n s f e r i s i n v o l v e d i n the formation of m/z 208 ( f i g u r e 47) R C C I P B I L E C O N J E T O f t C B p k fib 1 1 8 5 6 8 1 2 8 8 6 8 -teee**-6 8 8 8 0 -6 8 6 8 8 -4 8 8 8 8 -2 8 6 8 6 -1 4 . 8 3 min. 80 126 _i • • • • • • • 169 z e e Z4e 2 8 8 3?s 360 1 6 7 1 1 8 1 2 8 1 i f 1 Mt NOH H-C-CH^-C CH, 19 i—•—i—•—i—•—r -'—i—' i ' i 1 • • i — • — i — ' 1 * 1 * 1 ' i * i — • i * i 8 0 1 2 0 1 6 8 2 8 0 2 4 8 2 8 8 3 2 8 3 6 8 1 1 0 1 8 8 - 9 6 - 6 0 7 8 - 6 0 •56 e - 3 8 2 8 1 8 >-0 T i l e > 4 8 0 H B p k BC. ZT7J31 . 7 2 2 8 6 8 8 -2 4 0 6 6 - 1 2 8 8 8 6 -1 6 6 8 6 -1 2 8 8 6 -©00*tf 4 8 8 6 -8 8 • I . 6 1 R E C I P B I L E C O N J E T O f l C 1 1 0 C T 8 5 S U B ft»» S c a n 5 3 8 18 .65 B i l k . 1 2 8 1 6 8 . l . i 2 8 8 2 4 8 2 8 8 3 2 8 1 6 2 1 3 6 1 5 5 H - C - ^ C H 2 f C H - N M e 2 C H . 90 1 8 3 M 120' lie r i 9 6 J /. 2 2 3 2 6 9 2 5 4 / 288 >86 3 2 0 1 1 8 1 8 8 ^ 8 - 6 6 7 6 *• £e - 3 8 - 2 8 1 8 JL Figure 50. Mass spectra of r e c i p a v r i n metabolites i l l u s t r a t i n the three major fragmentation schemes in f igures 46,47,48. C anti-diphenylbutanone oxime (19). D. Recipavrin phenol (90). 161 B. Fragmentation of the subst i tuted diphenylbutane moiety Phenyl r ing metabolic oxidat ion resu l t s in s h i f t s in the masses of some of the a r y l a l i p h a t i c fragments (Benthe and Thieme,1983). Phenolic and O-methylcatechol metabolites are e a s i l y detected in under ivat ized , TMS and permethylated forms by c a l c u l a t i n g the appropriate mass increments induced by oxidat ion and d e r i v a t i z a t i o n of the diarylmethine cat ion (table 4) . The analogous peaks for the 1-methyl-2,2-diphenylcyclopropanyl cat ion r a d i c a l (m/z 208), the 2,2-diphenylcyclopropanyl cat ion (m/z 193) and a diphenylethyl cat ion (m/z 181) are a lso present in low r e l a t i v e abundance. The other ions in the d i a r y l b u t y l cascade are general ly absent, having been replaced by minor a r y l r ing substituent d i rec ted fragmentations. S e n s i t i v i t y i s greatest when the charge is l o c a l i z e d in the diphenyl region as in compounds fragmenting according to f igure 48. Nonetheless a l l r ec ipavr in re la ted compounds described here can be eas i l y located by monitoring the subst i tuted diarylmethyl c a t i o n . In phenyl r ing oxidized metabolites and the ir d e r i v a t i v e s , the f luoreny l cat ion (m/z 165) and the d iphenylethyl cat ion (m/z 181) are usual ly present as minor fragments. The permethylated catechols lose a methyl r a d i c a l to a f ford a diagnost ic ion at m/z 212. Phenyl Ring Substitution Mass Increment Major Diagnostic Ion (MDI) diarylalkyl M D I + CH2 cation radical MDI-R2R3 Other diagnostic ions R2=H,R^=H 0 167 208 181 - 208 cascade R2=H»R3-0H 16 183 224 197 165(-H20) 155(-C5 6) R2=H,R3-0TMS 88 255 296 269 165(-TMS0) -R2=H, R3=0C 30 197 238 211 165(-CH20) -R2=0CH3,R3=0H 96 213 254 223 181(-CH30H) 165 R2=0CH3,R3=0TMS 118 285 326 299 255(-CH20) 165,181,223 R2=OCH3,R3=OCH3 60 227 268 241 196(-CH30) 165,212 Ph(R 2 R 3 Ar)C + H ', Ph(R 2 R 3 Ar)CHC Ph(R 2R 3Ar)CHCH=CHCH 3 ]+-* H 2 Table ring 4. Diagnostic masses of oxidized metabolites and the diarylmethyl ca t ion the ir d e r i v a t i v e s . for phenyl 4. METABOLISM OF RECIPAVRIN 163 T h i s s e c t i o n d e t a i l s the s t r u c t u r e e l u c i d a t i o n by GCMS of the b i l i a r y m e t a b o l i t e s of r e c i p a v r i n (9) i n male Wistar r a t s . Complete c h a r a c t e r i z a t i o n of r e c i p a v r i n m e t a b o l i t e s by GCMS was r e q u i r e d t o show that the secondary formamide (12) was present i n the b i l e e x t r a c t and to c h a r a c t e r i z e any p o t e n t i a l m etabolic or chemical p r e c u r s o r s of the formamide. The systematic and t r i v i a l names, s u b s t i t u e n t s and formulas of compounds of general s t r u c t u r e A and B ( f i g u r e 51), are summarized i n Tables 5 and 6. GCMS data f o r a l l m e t a b o l i t e s , d e r i v a t i v e s and re f e r e n c e compounds are t a b u l a t e d as f o l l o w s : Table 7; M e t a b o l i t e s with i n t a c t phenyl r i n g s and t h e i r d e r i v a t i v e s . Table 8; Phenolic m e t a b o l i t e s and t h e i r d e r i v a t i v e s . Table 9; O-methylcatechol m e t a b o l i t e s and t h e i r d e r i v a t i v e s . Mass s p e c t r a of a l l m e t a b o l i t e s , d e r i v a t i v e s and re f e r e n c e compounds are in c l u d e d i n the appendix. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms used to l o c a t e r e c i p a v r i n m e t a b o l i t e s and t h e i r TMS d e r i v a t i v e s are shown i n f i g u r e s 52-55. 164 F i g u r e 51. General s t r u c t u r e s f o r m e t a b o l i t e s : A. S i n g l e bonded R v s u b s t i t u e n t . B. Double bonded R, s u b s t i t u e n t . Table 5. Names s t r u c t u r e and formulae f o r r e c i p a v r i n , r e c i p a v r i n m e t a b o l i t e s and s y n t h e t i c r e f e r e n c e compounds. Legend: A. Observed i n both conjugated and nonconjugated e x t r a c t s . B. A l s o a t e r o d i l i n e m e tabolite (Noren et al . 1985a,b). C. P o s s i b l y a s s o c i a t e d with e t h y l a c e t a t e e x t r a c t i o n . D. Sy n t h e t i c reference compound. E. TMAH d e r i v a t i v e , a l s o a r i s e s i n b i l e from prolonged storage. F. Decomposes i n GC i n l e t . Compound Number Systematic Name Trivial Name R2 R3 Empi rica Formula 9 (±) N,N, a-trimethyl-y-phenyl-benzenepropanamine A Recipavrin -N(CH3)2 H H C 1 8 H 2 3 N 15 (±) N, n-dimethyl-Y-phenyl-benzenepropanamine Norrecipavrin^ AB Di phenylbutanone (DPB) -NHCH3 H H C 1 ? H 2 1 N 14 1,1-diphenyl-3-butanone =0 H H C 1 6 H 1 6 ° \9 1,l-dipheny1-3-butanone oxime a DPB oxime =N0H (cis and trans) H H C 1 6H 1 7N0 12 (±)N-formyl-a-methyl-Y-phenyl benzenepropanamine 2° Formamide -NHCHO H H C 1 7H l gN0 B6 (±) Unidentified Unidentified H H C 1 8 H l g N 63 (±)N-acetyl-a-methyl-y-phenyl-benzenepropanami ne Acetamide1' -NHC(=0)CH3 H H C 1 8H 2 1N0 20 (±)a-methy1-y-phenyl benzenepropanami ne Dinorrecipavrin -NH2 H H C 1 6 H l g N 26 (±)N-formyl-N,a-dimethyl-Y-phenylbenzenepropanamine 3° Formamide^  -N(CH3)CH0 H H C 1 8H 2 1N0 64 (±)N-ethylidene-a-methyl -phenylbenzenepropanami ne Ethanimine0 -N=CHCH3 0-H H C 1 8 H 2 1 N 53 (±)N,N.a-trimethyl-y-phenyl benzenepropanamine-N-oxide N-oxideF -$(CH3)2 H H C 1 8H 2 3N0 Table 6. Names, s t r u c t u r e s and formulae f o r phenol and 0-methyl c a t e c h o l metabolites of r e c i p a v r i n . Legend: A. Observed in both conjugated and nonconjugated e x t r a c t s . B. A l s o a t e r o d i l i n e metabolite (Noren et al. 1985a,b). C. P o s s i b l y a s s o c i a t e d with e t h y l a cetate e x t r a c t i o n . D. S y n t h e t i c r e f e r e n c e compound. E. TMAH d e r i v a t i v e , a l s o a r i s e s i n b i l e from prolonged storage. Compound Number Systematic Name Trivial Name *1 Empirical Formula 87 88 89 (+)l-(4-hydroxyphenyl)-l-phenyl-3-butanone (±)N,a-dimethyl -y-(4-hydroxy-phenyl)-benzenepropanamine (±)l-(4-hydroxyphenyl)-l-phenyl-3-butanone oxime DPB-phenol AB Norrecipavrin phenol DPB-oxime phenol A =0 -NHCHj =N0H OH OH OH C16H16°2 C 1 ?H 2 1N0 C 1 6 H 1 7 N0 2 90 (±)N,N-a-trimethyl-Y-(4-hydroxyphenyl)-benzene-propanamine 91 (+)l-(4-hydroxy-3-methoxy-phenyl)-l-phenyl-3-butanone 9 2 (±)l-(4-hydroxy-3-methoxy-phenyl)-l-phenyl-3-butanol 93 ( + )l-(4-h'ydroxy-3-methoxy-phenyl)-l-phenyl-3-butanone oxime 94 (±)N,N,a-trimethyl-Y-(4-hydroxy-3-methoxyphenyl)-benzenepropanami ne Recipavrin phenol DPB O-methyl catechol (DPB-OMC) di phenybutanol -OMC DPB oxime-0MCA Recipavrin-OMC -N(CH3)2 -OH =N0H -N(CH3)2 OCH, OCH, OCH, OCH OH OH OH OH OH C 1 8H 2 3N0 C17H18°3 C17H20°3 C 1 7 H l g N0 3 C l g H 2 5 N0 2 0\ 167 446888-488880-368888^ 328888^ 28eee»> 248888^ 288886H i taaaa-128888-88888^ 48888-e-1 4 8 . 8 - 4 8 8 . 8 a » u . R E C I P B I L E C O N J 19a 1 4 . 8 3 E T O A C 14 18.81 1 7 . 8 7 19 1 4 . : i 15 9 11 . 9 9 i • i • i ' i T i • i ' r » i ' i • i » i • i • i • i • i ' i • i • i ' i • i 6 8 18 12 14 16 18 28 22 24 2288688^ 2866666^ 1 888868^ 1600888-1488888-1208688-; 1688888-4 8 . 9 - 4 0 8 . 0 a m i . R E C I P O V CONJ B I L E ETOfiC EXT TMJ D E R I V 1 9 . 5 9 52a 52 14-64 I 2 6 . 5 1 89a 91a F i g u r e 5 2 . T o t a l i o n c u r r e n t a n d s e l e c t e d i o n c h r o m a t o g r a m s f o r 0 - g l u c u r o n i d a s e - h y d r o l y z e d e x t r a c t s o f b i l i a r y r e c i p a v r i n m e t a b o l i t e s . I . T o t a l i o n c u r r e n t f o r a l l m e t a b o l i t e s ( a b o v e ) a n d t h e i r TMS d e r i v a t i v e s ( b e l o w ) . 168 1 6 6 . 7 - 1 6 7 . 7 Aftu . R C C I P B I L E C O N J E T O B C n e e e e -1 4 . 7 8 180000- 19a 9 8 0 8 8 -8 8 6 8 6 ; 7 0 0 0 8 -6 8 8 8 8 ^ 5 0 0 0 8 - 14 4 6 8 8 6 ^ IB . 8 1 3 0 0 0 0 -2 8 0 0 8 ; 1 8 0 0 0 -1 5 9 1 9 12 86 0-' e ' 1 • 1 ' 1 8 1 1'2 * iV 1 8 ' 2 6 ' it I . 8 . 4 . . 6 6 0 8 8 0 -£58888-6 0 8 8 0 0 -4 5 8 8 8 6 -4 0 0 6 0 6 -3 5 0 0 0 8 -3 6 0 0 0 0 -250000-2 6 0 0 0 0 -1 5 6 6 8 8 -1 8 0 0 0 6 -5 6 8 6 6 -6 1 6 6 . 7 - 1 6 7 . 7 *au . R E C I P R V C O N J B I L E E T O O C EXT THS OCRIV - 1 4 . 6 6 52 a 52 13.681 14 9 1 6 1 2 J 4 95 26.28 15a ? A 26 77 7e F i g u r e 53. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms f o r 0-glucuronidase-hydrolyzed e x t r a c t s of b i l i a r y r e c i p a v r i n m e t a b o l i t e s . i l . Ion chromatogram m/z 167 showing phenyl r i n g i n t a c t m e t a b o l i t e s (above) and t h e i r TMS d e r i v a t i v e s (below). 169 1 6 2 . 7 - 1 8 3 . 7 a.u.KeeIP I J L t C b H J ETOfiC 9888^ seee^ 788frj teee-j 4888-3888-2888-1888^ 488888-368888-328888-286660-2 5 4 . 7 - 2 5 5 . 7 a » u . R E C I P B V C O N J BILE ETOPC EXT THS DERIV. 21 . 3 7 | 89 a 87a 28.33 89 c Jl f 88 26.81 \ ta A '1 Sot O i I i | fVo • [ j i - T V ) ' i t ^ — 17 .8 18 . 8 1 9 . 8 2 8 . 8 2 1 . 8 22 ' .8 2 3 . 8 2 4 . 8 2 S . 8 2 6 . 8 F i g u r e 54. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms f o r 0-glucuronidase-hydrolyzed e x t r a c t s of b i l i a r y r e c i p a v r i n m e t a b o l i t e s . I l l . Ion chromatogram m/z 183 showing i n t a c t phenol m e t a b o l i t e s (above) and m/z 255 showing TMS d e r i v a t i z e d phenols (below). 170 2 1 2 . 7 - 2 1 3 . 7 a»u.*CCIP BILE CONJ ETOfiC uaea-leeee-9eed-oeee-7888-6888-5888-4888^ 3888; 2888; 1888-1 8 . 4 3 91 93 2 2 . 2 8 I 14 . 3 8 I • I • I • I • I • I • I • I ' T • I 8 18 12 14 16 168888-1 148888-128888-1 88888-88888-68888-48888-28868-2 8 4 . 7 - 2 8 5 . 7 a » u . R E C I P R V CONJ B I L E L TOPIC EXT TMS D E R I V 2 3 . 2 7 I 93 i a 91 a 2 8 . 1 8 22 .28 21 . 6 7 2 8 . 94a LA 93 c 2 6 . 8 1 JL 2 8 . 8 2 1 . 8 2 2 . 8 2 3 . 8 2 4 . 6 2 5 . 8 2 6 . 6 2 7 . 8 28 "I Figure 55. Tota l ion current and selected ion chromatograms for 0-glucuronidase-hydrolyzed extracts of b i l i a r y rec ipavr in metabol i tes . IV. Ion chromatogram m/z 213 showing intact 0-methylcatechol metabolites (above) and m/z 285 showing TMS der iva t i zed O-methylcatechols (below). Table 7. GCMS data f o r phenyl r i n g i n t a c t r e c i p a v r i n m e t a b o l i t e s and t h e i r d e r i v a t i v e s . Legend as i n t a b l e 5. G. TMAH d e r i v a t i v e . Compound Number Trivial Name Retention Time (Minutes) MT (* Intensity) Base Peak 100« Other Diagnostic Ions ((m/z)(t of base peak)) " l M2 H3 M4 M5 H6 9 i Recipavrin 12.42 253 ( 3) 72 167 (12) 165 ( 6) 152 ( 3) 115 ( 3) 91 ( 3) 73 ( 3) 15 Norrecipavrin* 12.02 239 ( 7) 58 167 (18) 165 (12) 152 ( 5) 208 ( 4) 103 ( 4) 193 ( 4) 15a Norrecipavrin N-THS 15.17 311 ( 5) 130 167 (19) 193 (31) 73 (25) 105 (20) 270 (13) 165 ( 7) 14 Diphenylbutanone (DPB)flB 10.79 224 (35) 167 43 (10) 103 (32) 165 (32) 181 (28) 152 (18) 77 (14) 19 DPB-ox1me (m1norA) 14.43 239 ( 0) 167 165 (57) 105 (32) 180 (24) 209 (20) 91 (20) 222 (10) 52 OPB oxime (minor)(0-THS) 13.68 311 ( 1) 167 165 (36) 75 (11) 103 (10) 220 ( 6) 296 ( 2) 152 ( 2) 19a DPB oxime* (major) 14.83 239 ( 8) 167 165 (25) 152 (15) 103 (18) 42 (12) 118 (11) 181 (11) 52a DPB-oxIme (major-0-TMS) 14.64 311 ( 8) 167 166 (60) 165 (20) 118 (20) 222 ( 8) 220 ( 8) 221 ( 8) 16 DPB-0-methylox1meG (major) 12.03 253 ( 8) 167 165 (32) 152. (18) 118 (16) 103 (12) 181 ( 8) 77 ( 5) 16a DPB-O-methyloxime (minor) 11.69 253 ( 0) 167 165 (34) 180 (18) 152 (18) 168 (12) 115 ( 6) 77 ( 6) 12 2° Formamide 18.20 253 (44) 73 167 (84) 165 (66) 208 (60) 44 (50) 193 (42) 181 (38) 8 6 Unidentified 18.78 251 ( 0) 70 167 (32) 71 (66) 165 (50) 91 (36) 208 (32) 130 (22) 95 Unidentified -TMS 20.18 323 ( 5) 144 167 (84) 165 (50) 193 (38) 251 (36) 118 (32) 91 (28) 63 Acetamlde1* 19.16 267 (12) 87 167 (14) 44 (61) 72 (16) 167 (14) 86 (13) 208 (10) 20 Dinorrecipavrin 10.78 225 ( 2) 44 208 (12) 165 ( 5) 58 ( 5) 115 ( 4) 193 ( 3) 222 ( 2) 104 01norrec1pavr1n-N-TMS 13.51 297 ( 3) 116 73 (22) 167 (16) 118 (12) 102 ( 8) 154 ( 8) 152 ( 6) 26 3° Formamide^ 19.41 267 (10) 87 86 (70) 58 (28) 72 (25) 167 (22) 165 (20) 208 (18) 64 Ethanlmlne" 11.41 251 ( 8) 71 165 (46) 236 (43) 167 (30) 70 (26) 152 (24) 105 (23) 53 Recipavrin N-ox1deF (as <=1s (minor) and trans (major) diphenyl-but-2-enes) 7.92 8.26 208 208 ( 6) (68) 167 115 165 193 (26) (58) 152 (18) 178 (42) 115 91 (10) (38) 208 ( 6) 178 (38) 193 130 ( 5) (38) 128 165 ( 4) (36) Table 8. GCMS data for phenolic metabolites of r e c i p a v r i n and the ir der iva t ive s . Legend as in table 5. G. TMAH d e r i v a t i v e . H. Diazomethane der iva t ive . I . Also a norrec ipavr in phenol d e r i v a t i v e . Retention Base Peak 0 t h e r D 1 a 9 n o s t 1 c I o n s ( ( •» / * ) ( * o f base peak)) Compound Number T r i v i a l Name i Time (Minutes) Intensi ty) loot M1 M 2 H3 M4 H5 H6 87 D Diphenylbutanone phenol 17.19 240 (18) 183 43 (12) 185 ( 8) 103 ( 6) 119 ( 5) 153 ( 5) 225 ( 2) 87a DPB phenol TMS ether 17.75 312 (13) 255 257 (22) 73 (14) 165 ( 8) 179 ( 4) 103 ( 4) 43 ( 4) 87b Diphenylbutanone G > H « O-methyl phenol 15.94 254 (16) 197 198 (16) 165 (16) 152 (10) 182 ( 6) 103 ( 4) 115 ( 3) 88 Norrecipavrin phenol 17.71 255 ( 5) 58 183 ( 7) 224 ( 6) 59 ( 5) 165 ( 2) 115 ( 2) 152 ( 2) 88a 88b Norrecipavrin phenol (N-TMS, 0-TMS) Norrecipavrin O-methyl phenolH 21.87 17.08 399 269 ( 5) (10) 130 197 255 58 (22) (28) 73 (38) 238 (24) 131 (15) 153 (20) 75 (10) 165 (14) 296 207 ( 8) (10) 58 ( 4) 222 ( 8) 89 DPB-ox1me phenol (major) 21.19 255 ( 2) 183 165 (26) 196 (16) 184 (12) 199 (10) 152 (10) 121 (10) 89a DPB-oxIme phenol-dl-TMS ether (major) 21.37 399 ( 2) 255 73 (20) 259 (18) 269 (10) 103 ( 4) 165 ( 4) 206 ( 4) B9b DPB-oxIme phenol (minor) 22.20 255 ( 0) 183 Shoulder, poor mass spectrum obtained 89c DPB-ox1me phenol-dl-TMS ether Dlphenylbutanone-O- ( r a ' n ° r ) methyloxlme-0-methyl phenol (major and minor)" 21.54 399 (14) 255 73 (56) 268 (44) 165 (24) 295 (21) 257 (20) 193 (16) 89d 17.4 17.24 283 ( 0) Good mass spectra l data not obtained due to overlapping peaks 90 Recipavrin phenol 269 ( 5) 72 183 (12) 196 ( 2) 102 ( 2) 254 ( 1) 155 ( 1) 136 ( 1) 90a Recipavrin phenol TMS ether 19.09 341 ( 6) 72 255 (12) 73 (14) 165 ( 6) 296 ( 6) 115 ( 2) 179 ( 2) 90b R e c i p a v r i n 6 * 1 phenol O-methyl 17.57 283 (10) 72 197 (22) 283 (10) 153 (10) 238 ( 8) 165 ( 8) 207 ( 6) ether Compound Table 9. GCMS data for O-methyl rec ipavr in and the ir der iva t ives . TMAH d e r i v a t i v e . H. Diazomethane norrec ipavrin phenol der iva t ive . catechol metabolites of Legend as in table 5. G. d e r i v a t i v e . I . Also a Retention Time M* (« Base Peak 0 t h e r D l « 9 n o s t 1 c 'ons ((ro/z)(i: of base peak)) Number T r i v i a l Name (Minutes) M1 M 2 M 3 1 \ H 5 H 6 91 0PB-0MCB 18 .43 270 (26) 213 43 (38) 103 (20) 152 (18) 153 (16) . 181 (12) 72 (10) 91a DPB-OMC TMS ether 20, .10 342 (23) 285 286 (24) 144 (18) 73 (14) 167 (10) 255 ( 9) 103 ( 4) 91b DPB-dimethyl catechol G , H - 18, .85 284 (32) 227 228 (18) 165 (12) 196 (11) 103 (10) 181 ( 8) 73 ( 6) 92 D1phenylbutanol-OMC 18, .95 272 (26) 213 44 (72) 182 (15) 151 ( 8) - - -93 93a DPB-oxime-OMC (major) DPB-oxime-OMC (major) di-TMS ether 22, 23 .20 .27 285 429 ( 0) ( 0) 213 285 225 (50) 286 (22) 152 (30) 73 (16) 151 103 (24) ( 6) 105 181 (16) ( 5) 91 265 (14) ( 5) 239 325 ( 6) ( 4) 93b DPB-ox1me-0MC (minor) 22. ,55 285 ( 3) 213 153 (18) 214 (12) 183 (10) 91 ( 9) 108 ( 8) 268 ( 6) 93c DPB-oxime-OMC (minor) dl-THS ether 23, .44 429 ( 0) 285 73 (52) 286 (20) 373 (16) 295 (16) 223 (14) 120 (14) 93d DIphenylbutanone-0-methyloxime-dImethyl catechol (minor)" . , 19. ,74 313 ( 6) 227 229 (14) 240 (14) 196 ( 8) 139 ( ?) 165 .( 6) 181 ( 5) 93e D1phenylbutanone-0-methyloxime-dimethyl catechol (major) G 20. ,05 313 ( 6) 227 228 (14) 196 (10) 165 (10) 72 ( 9) 241 ( 6) 181 ( 6) 93f DPB-oxime , dimethyl catechol 18. 89 299 (10) 227 228 (14) 196 ( 8) 165 ( 8) 241 ( 8) 282 ( 6) 152 ( 4) 94 Recipavrin 01'C IB. 96 299 (10) 72 213 ( 9) 152 ( 4) 58 ( 3) 254 ( 3) 115 ( 3) 75 ( 3) 94a Recipavrin OMC TMS ether 21. 16 371 (14) 72 good mass spectrum not obtained 94b Recipavrin H . dimethyl catechol ' 19. 54 313 ( 8) 72 227 (20) 237 ( 8) 165 ( 8) 91 ( 6) 115 ( 6) 152 ( 4) co A. B i l i a r y metabolites of recipavrin 174 Based on the metabolites observed by GCMS, recipavrin follows four general metabolic routes. The pathways of oxidative deamination, N-dealkylation, N-oxidation and phenyl ring oxidation are common to st r u c t u r a l l y related compounds in the amphetamine series (Caldwell, 1976, Coutts and Beckett, 1977 (reviews), Beckett and Al Sarraj, 1972), promethazine (lO)(Clement and Beckett, 1981), t e r o d i l i n e (Noren, et al., 1985a,b) and related compounds. One metabolite, the secondary formamide (12) could not be assigned to any one pathway. Experiments on the ori g i n of the formamide are discussed in section 7. i . GCMS observation of the intact formamide The secondary formamide (12) was observed by GCMS as a minor metabolite in the conjugated fraction of b i l e . The mass spectrum (figure 56) and retention time were similar to the synthetic product. The chemical ionization mass spectrum shows the M+ +1 ion at m/z 254 and associated alkane adducts M+ + C 2Hg and M+ + C3H7 from the methane reagent gas (Figure 57). The secondary formamide metabolite was methylated with TMAH to give the t e r t i a r y formamide (figure 58 (top)). Obtaining a TMS der i v a t i v e of the secondary formamide was not as straightforward. F i r s t l y the derivatization of the synthetic standard was slow and afforded an apparent mono derivative (60, Ph2CHCH2CH(CH3)N(TMS)CHO) with a retention time of 15.36 175 minutes (see figure in section 1). In the TMS derivatized b i l e extract, the underivatized formamide was not seen. A new peak (95) appeared at 20.2 minutes had a base peak at m/z 144 suggesting s i l y l a t i o n at nitrogen and an apparent molecular ion at m/z 341 (figure 58 (bottom)) which corresponds to an underivatized molecular weight of 269. The loss of TMSOH+ from m/z 341 could account for the m/z 251 peak. The mass spectrum of 95 i s in accord with de r i v a t i z a t i o n of the unidentified compound (86), since loss of TMSOH+ or H 20 in 95 and 86 respectively, affords m/z 251 from molecular ions presumed to be at m/z 341 (derivatized) and m/z 269 (underivatized). The m/z 251 ion could correspond to an isocyanate structure since alpha cleavage affords a m/z 70/71 ion doublet in the underivatized compound. The mass spectrum of un i d e n t i f i e d compound 95 (figure 58 bottom) has a base peak m/z 144 l i k e the amphetamine secondary formamide TMS derivative (84, figure 59). The reason the amphetamine formamide TMS deriva t i v e lacks the m/z 145 ion present in the mass spectrum of the synthetic recipavrin formamide TMS derivative (60, figure 23)) i s the absence of a gamma proton. 176 f i l e >.S92C RECIi l:pk flb 37S43 ? 3 JCC FOF.nun 1 SAMPLE 373B 3t1GvlwML SUB ODD ?c=n 1105 3889-1 ;'.kjt,&-44 H I N-CHO I H - C - C H 2 - C H 12 CH. X 6 7 1 3 9 152 103 L i 193 208 LA £ 5 3 2 3 8 , . . . , - ; - - [ - • : - • , - , - • - , - - , ' - ( 2 d 1&0 2gg ;"; 4 0 2 3 f) 3 £ 8  F i l e >480H Bpk Pb 2246 R E C I P B I L E CONJ ETORC 110CT85 SUB ADD S c a n 5 2 0 1 8 . 2 0 s i n . 2 4 0 6 ; 2 2 0 0 -2 8 8 8 -1 8 8 8 -1 6 0 8 ^ 1 4 8 8 -1 2 8 6 -88 I . . . I . . . I 1 2 8 168 • • • • 2 8 8 2 4 8 2 8 8 3 2 8 . . i . . . i . . . i . . . i . . . i . . . i . . . i . . . i 73 4 4 8 0 8 -6 0 0 -4 0 0 -2 0 8 -i , i 167 / 199 / 1 3 8 / 1 5 2 / I i 2 8 8 \ 1.1 I i i ii 8 8 128 ' i i e ' 2 5 3 \ 210 2 8 8 2 4 8 2 8 8 ' 3 2 8 1 1 8 1 8 8 -e* - 7 8 -68 * 8 r40 "30 "28 - 1 8 « F i g u r e 56. (top) Mass spectrum of the s y n t h e t i c secondary formamide (12). (bottom) Mass spectrum of the u n d e r i v a t i z e d secondary formamide m e t a b o l i t e . 177 y.ok P L 1 Z l ' 2oeee-1 0 0 -1 1 4*fce»-j £ 0 0 0 -H l N-CHO I ~-H NT i i i n t . - j 0t*t. • " 264 -CH„-CH 12 M++1 131 04 72 106 91 1 6 7 1*5 176 1 9 5 Li i' 2 6 9 238 226 _S2_ 120. J L 6 8 2*0 F i l e >GREG5 Bpk Ob 18816 18888-16886-" 3 8 3 E R E C I P C O N J "HETOBS 'UNDERiv c i SUS 254 J 4888-12886-16688--8886-6866-4688-2888-8---2688H 1 3 1 / I 155 1 67 / 268 197 / 248 V 294 282 294 1 280 168 288 '" 248 280 328 r--i t i . n n ) chemical i o n i z a t i o n mass spectrum of t F i g u r e 57. (top) L n e m i c f ~ x r m - d e ( 1 2 ) . (bottom Chemic i f f iUn ' ^ ' U t r S r ^ ine' secondary f o r - i m e t a b o l i t e . 1 78 800 4 00-1 00 -86. 1 IS 80 -ft ] • U r i H B H 165 152 138 167 2 1 d 208 2 1 2 SI" TttfiW H E R ] V f i l I Z E 267 C H 3 N-CHO i H-C-CH,-CH CH3 208 F i l e > 4 9 1 H RE.CIPRV CONJ BILE ETOfiC EXT THS DERIVATIVES S c a n 5 9 5 Bpk fib 36288 SUB BVC AND 28.18 »in. 88 128 168 268 248 288 326 368 i . i • i • i • i . i . t • i • i • i • i • i • i • i • i . i • i 48888-36888; 32868; 28888-24888-2868* 1688* 1286* 888* 4888- 42 88 144 16? / 251 - L a 95 268 / 341 295 . I 3E .1.1.1 356 -188 ^8 «e "78 •68 *8 •48 r38 r28 -18 128 168 288 248 288 328 368 Figure 58. (top) Mass spectrum of the TMAH derivatized secondary formamide. (bottom) Mass spectrum of the unidentified TMS derivative (95). BAKU • 14 fINT.* 8332 73 44 75 7 7 91 118 H i l l " llM it tit tU" C ..'.rt!.. 144 145 I I 1 4 ' l i t l i t I»" •iJ» \k" M*"-15| 220 ••iU 1-F i g u r e 59. Mass spectrum of the TMS d e r i v a t i v e of t h e amphetamine secondary formamide ( 8 4 ) . 180 i i . LCMS Demonstration of the secondary formamide me t a b o l i t e L i q u i d chromatography-mass spectrometry was used to determine whether the secondary formamide (12) or the th e r m a l l y u n s t a b l e isomeric methylene n i t r o n e (24) was r e s p o n s i b l e f o r the formamide observed by GCMS of B-g l u c u r o n i d a s e - h y d r o l y z e d b i l e from r e c i p a v r i n dosed r a t s . Using the d i r e c t l i q u i d i n t r o d u c t i o n (DLI) i n t e r f a c e f o r the HP-5987 mass spectrometer, s y n t h e t i c samples of the n i t r o n e and formamide were chromatographed on a HP 1090 LC equipped with a semi micro R P C ^ HPLC column. Ret e n t i o n times and mass s p e c t r a of the standards were recorded f o r 200 ng (100 ng/ul) i n scan mode ( f i g u r e 60). The instrument was then put i n s e l e c t e d ion mon i t o r i n g mode f o r the ions m/z 254 (M+1), 238 (M+1-16) and 295 (M+I+CH3CN). The l o s s of oxygen from the molecular ion (m/z 238) i s common i n the mass s p e c t r a of n i t r o n e s (Coutts et al., 1978). The standards were rerun at one t e n t h c o n c e n t r a t i o n (20 ng). A blank i n j e c t i o n showed no ca r r y o v e r from the standard i n j e c t i o n . The b i l e sample was then run under i d e n t i c a l c o n d i t i o n s . The m/z 254 ion chromatogram i n f i g u r e 61 shows that the secondary formamide (12) i s present i n the b i l e e x t r a c t . M o n i t o r i n g the base peak m/z 238 f a i l e d to r e v e a l any methylene n i t r o n e . The m/z 238 peaks at s h o r t e r r e t e n t i o n times were not i d e n t i f i e d . 1 8 1 10QH >-H CO z LU I-- 50H LU > < -I LU CC 0 F O R M A M I D E M+1 j254 H I N - C H O I H - C - C H - C H C H . 12 153 295 M+1-160 180 200 220 240 260 280 100-4 >-H CO Z ' LU r -? 50 H LU > LU NITRONE O " + N = C H 2 I M+1-16 H - C - C H Q - C H 153 C H . 2 4 161 160 I 180 238 254 M+1 200 220 240 T , ?w e ?' S m a s s s P e c t r u m of the secondary formamide U 2 M t o p ) and the methylene n i t r o n e (24) (bottom) i n the a c e t o n i t r i l e / water s o l v e n t system. 182 6 A 188 ?B8 386 488 588 688 788 888 i . i . • . • I • • • 11 • • • . I . • • • 1 1 . . • 111 • • i • • • . I. i • . I . . 1 1 1 . • • . r . • • • i • . • • l . • • • i . . • • i . • . . i . • . . i • • • i Figure 61. Superimposed LCMS selected ion monitoring results for A. A 0-glucuronidase-nydrolyzed bile extract from a recipavrin dosed rat. B. A mixture of the synthetic methylene nitrone (24) and secondary formamide (12)(10 ng each) standards. Bottom frame shows the M++1 m/z 254 of both components and the top frame shows the M++1-16 m/z 238 diagnostic ion of the nitrone. Only the formamide is present in the bile extract. 183 i i i . O x i d a t i v e deamination The mixed f u n c t i o n oxidase r e s p o n s i b l e f o r the o x i d a t i v e deamination of amphetamines was f i r s t d e s c r i b e d by Axelrod (1955). The formation of diphenylbutanone (14, Ph 2CHCH 2C(CH 3)=0) from r e c i p a v r i n (9, Ph 2CHCH 2CH(CH 3)N(CH 3) 2) i s i n d i r e c t analogy to the p r o d u c t i o n of phenylacetone from dimethylamphetamine (Beckett and A l S a r r a j , 1972). 0-m e t h y l c a t e c h o l and p h e n o l i c d e r i v a t i v e s of diphenylbutanone have been d e s c r i b e d as m e t a b o l i t e s of t e r o d i l i n e (Noren, et al . , 1985a,b). S u r p r i s i n g l y , 10-(2-propanone) phenothiazine (71) has not been observed among the in vitro m e t a b o l i t e s of promethazine (10)(Clement and Beckett, 1981) and the diketone (3) has not been observed as a m e t a b o l i t e of methadone (8)(Abbott, et al. , 1985). T h i s may i n v o l v e i n s t a b i l i t y of a key i n t e r m e d i a t e i n the case of methadone, or p r e f e r e n t i a l o x i d a t i o n of the r i n g heteroatoms i n the case of promethazine. Diphenylbutanone (14) i s present i n both conjugated and nonconjugated e x t r a c t s and i s the second most abundant m e t a b o l i t e . The mechanism of o x i d a t i v e deamination i s a matter of some c o n t r o v e r s y . I t i s l i k e l y t h a t diphenylbutanone a r i s e s from s e v e r a l p o s s i b l e mechanisms, which may i n c l u d e spontaneous h y d r o l y s i s of the alpha methine carbinolamine (Ph 2CHCH 2C0H(CH 3)N(CH 3) 2), h y d r o l y s i s of an imino c a t i o n Ph 2CHCH 2C(CH 3)=N +(CH 3) 2) a r i s i n g by d e hydration of the c a r b i n o l a m i n e or dehydrogenation of the amine or by h y d r o l y s i s of the oxime (19, Ph 2CHCH 2C(CH 3) =NOH)." Experimental evidence 184 supports a combination of two or more of the above p r e c u r s o r s ( C a l d w e l l , 1976 ( r e v i e w ) ) . The occurrence of diphenylbutanone i n the conjugated f r a c t i o n can be a t t r i b u t e d to e i t h e r the post-enzymatic a c i d h y d r o l y s i s of an oxime p r e c u r s o r d u r i n g workup or by the e x i s t e n c e of an enol s u l f a t e or g l u c u r o n i d e of diphenylbutanone. The enol s u l f a t e of phenylacetone has been c h a r a c t e r i z e d by NMR spectroscopy of amphetamine metabolic e x t r a c t s from r a b b i t s (Dring, e t . a l . , 1970). The g l u c u r a s e R used i n t h i s study was not a s u l f a t a s e f r e e p r e p a r a t i o n . The metabolic r e d u c t i o n of diphenylbutanone (14) to d i p h e n y l b u t a n o l was shown only by the presence of the 0-m e t h y l c a t e c h o l of d i p h e n y l b u t a n o l (92). No p h e n o l i c d i p h e n y l b u t a n o l , a m e t a b o l i t e of t e r o d i l i n e (Noren,et. a l . , I985a,b) or i n t a c t d i p h e n y l b u t a n o l was d e t e c t e d . No b e n z y l i c c a r b i n o l s were observed i n t h i s study, although the b e n z y l i c o x i d a t i o n of t e r o d i l i n e (Noren,1985a,b) and r e l a t e d compounds has been observed. B.enzophenone was observed and may be a metabolic or chemical o x i d a t i o n product of a d i p h e n y l c a r b i n o l m e t a b o l i t e . i v . N - d e a l k y l a t i o n R e c i p a v r i n (9, Ph 2CHCH 2CH(CH 3)N(CH 3) 2) i s d e a l k y l a t e d to n o r r e c i p a v r i n (15, Ph 2CHCH 2CH(CH 3)NHCH 3) in vivo. Small amounts of n o r r e c i p a v r i n were observed i n unhydrolyzed and hy d r o l y z e d b i l e and u r i n e e x t r a c t s by GCMS. N o r r e c i p a v r i n i s a more important nonconjugated component of the u r i n e where i t was observed along with the major components, r e c i p a v r i n and 185 r e c i p a v r i n phenol. The lack of a p l a u s i b l e g l u c u r o n i d e p r e c u r s o r to n o r r e c i p a v r i n i n d i c a t e s that i t c o u l d be c a r r i e d over from incomplete e x t r a c t i o n of the nonconjugated f r a c t i o n , or a r i s e from a chemolabile p r e c u r s o r such as a secondary hydroxylamine which c o u l d form a g l u c u r o n i d e conjugate. Trace q u a n t i t i e s of u n d e r i v a t i z e d d i n o r r e c i p a v r i n (20, Ph 2CHCH2CH(CH3)NH 2) were d e t e c t e d i n b i l e and u r i n e . D e t e c t i o n was d i f f i c u l t because of the low i n t e n s i t y of the diphenylmethyl c a t i o n and the poor d i a g n o s t i c value of the m/z 44 base peak. D i n o r r e c i p a v r i n was more e a s i l y l o c a t e d as i t s TMS d e r i v a t i v e which has a m/z 116 base peak. The low l e v e l s of d i n o r r e c i p a v r i n are i n accord with the general decrease i n r e a c t i v i t y towards N - d e a l k y l a t i o n t h a t occurs i n secondary amines r e l a t i v e t o t e r t i a r y amines. T h i s may be f u r t h e r c o m p l i c a t e d by the s t e r e o s e l e c t i v i t y that i s a f e a t u r e of secondary, but not t e r t i a r y amine d e a l k y l a t i o n (Henderson, et al. , 1974). The o b s e r v a t i o n of d i n o r r e c i p a v r i n i n the conjugated f r a c t i o n c o u l d a l s o r e s u l t from c a r r y o v e r or chemical breakdown of a N - o x i d i z e d p r e c u r s o r . D i n o r r e c i p a v r i n i s an assumed i n t e r m e d i a t e i n the o c c a s i o n a l o b s e r v a t i o n of the secondary acetamide (63, Ph 2CHCH 2CH(CH 3)NHC(=0)CH 3). The acetamide i s an a c c e p t a b l e metabolic N - a c e t y l conjugate of a primary a r y l a l i p h a t i c amine i n the r a t (Dring, e t . a l . , 1970). The N - a c e t y l compound was only seen at t r a c e l e v e l s i n e t h y l a c e t a t e e x t r a c t e d samples. 186 The ethanimine (64, Ph 2CHCH 2CH(CH3)N=CHCH 3), a s y n t h e t i c condensation product of acetaldehyde and d i n o r r e c i p a v r i n (20) o was not observed i n the b i l e e x t r a c t s . The u n i d e n t i f i e d compound (86) has a s i m i l a r mass spectrum to the ethanimine but was e l u t e d at much longer r e t e n t i o n time. The metabolic d e a l k y l a t i o n of amines i s g e n e r a l l y thought t o proceed v i a an u n s t a b l e carbinolamine i n t e r m e d i a t e , which i n the case of b a s i c amines, spontaneously d i s s o c i a t e s to the d e s a l k y l compound and an aldehyde. E x c e p t i o n s to t h i s spontaneous h y d r o l y s i s occur i n the p y r r o l i d i n e s e r i e s where the alpha carbinolamine i n t e r m e d i a t e i s f u r t h e r o x i d i z e d to a lactam (Rose and C a s t a g n o l i , 1983 (review)) or when the n i t r o g e n atom i s non b a s i c . Although an i n i t i a l four e l e c t r o n metabolic o x i d a t i o n of a b a s i c t e r t i a r y amine to a t e r t i a r y N-methyl formamide i s u n l i k e l y , the p o s s i b i l i t y of a g l u c u r o n i d e conjugated carbinolamide p r e c u r s o r of the secondary formamide was r u l e d out by s t u d y i n g the metabolism of the t e r t i a r y formamide (see l a t e r s e c t i o n ) . The d e s a l k y l m e t a b o l i t e , n o r r e c i p a v r i n was a l s o o x i d i z e d to a phenol (88). N - o x i d a t i o n of n o r r e c i p a v r i n to a secondary hydroxylamine (17, Ph 2CHCH 2CH(CH 3)N(OH)CH 3) or n i t r o n e s 24 or 44 was not observed by GCMS, probably due to the sample workup c o n d i t i o n s . The p o t e n t i a l secondary hydroxylamine and n i t r o n e involvement i n formamide p r o d u c t i o n w i l l be d i s c u s s e d i n a l a t e r s e c t i o n . 187 v. Phenyl ring oxidation Phenols and 0-methylcatechols of intact recipavrin (90 and 94) and certain compounds (87,88,89,91,92,93) in a l l three metabolic pathways were observed in b i l e by GCMS. Only norrecipavrin phenol (88) and recipavrin O-methylcatechol (94) were observed exclusively in the conjugated f r a c t i o n . The other phenols were also evident as minor components of the nonconjugated f r a c t i o n . In the amphetamine series, b i l i a r y excretion of phenyl ring oxidized metabolites i s a r e l a t i v e l y major metabolic pathway in the rat (Caldwell, et al., 1972a), but of less importance in most other species (Caldwell, et al., 1972b). Phenol formation i s thought to occur exclusively in the 4' position with subsequent 3' oxidation to the catechol. Attempts were made to detect catechol or dihydrodiol metabolites of recipavrin by monitoring for the phenylmethine-3',4'-catechol or phenylmethine-3',4'-benzenedihydrodiol moieties in the underivatized (m/z 199 and 201 respectively) and TMS derivatized (m/z 343 and 345) b i l e extracts. No metabolites with these ions were detected. The mass spectrum of an underivatized catechol metabolite of t e r o d i l i n e has been described (Noren,e< al. , 1985a,b). Therefore i n s t a b i l i t y can be ruled out as an explanation for the absence of catechol metabolites. The catechol metabolites were undoubtedly intermediates in the formation of the four 3'-O-methylcatechol metabolites (91-94) by catechol-O-methyltransferase. 188 v i . N - o x i d a t i o n Only two N - o x i d i z e d m e t a b o l i t e s of r e c i p a v r i n , the oxime (19) and N-oxide (53), were observed i n the b i l e e x t r a c t . The N-oxide decomposed d u r i n g GCMS a n a l y s i s to the Cope rearrangement.products, c i s and t r a n s 1,1-diphenylbut-2-ene (54 and 55). The most abundant m e t a b o l i t e was the oxime (19), which was observed i n conjugated and nonconjugated e x t r a c t s i n both b i l e and u r i n e . The oxime was f u r t h e r transformed to p h e n o l i c and O-methylcatechol m e t a b o l i t e s (89 and 93). Syn (Z) and a n t i (E) geometric isomers of the oxime were observed i n the e x t r a c t s . The major component occ u r r e d at long r e t e n t i o n time and corresponds to the a n t i (E) isomer. The minor isomer only a f f o r d e d a d e t e c t a b l e molecular ion as the TMS d e r i v a t i v e . The predominance of the a n t i isomer i n metabolic e x t r a c t s may be due to metabolic or chemical f a c t o r s . Beckett (1971) has i n d i c a t e d t h a t the syn isomer of phenylacetone oxime i s more s u s c e p t i b l e t o a c i d h y d r o l y s i s than the a n t i isomer. In t h i s study, b i l e was exposed to pH 5 f o r the d u r a t i o n of the g l u c u r o n i d a s e i n c u b a t i o n . T h i s c o n d i t i o n c o u l d c o n t r i b u t e to the s e l e c t i v e h y d r o l y s i s of syn diphenylbutanone oxime to diphenylbutanone. A l t e r n a t i v e l y , metabolic oxime formation may be s t e r e o s e l e c t i v e , as i t i s i n the metabolism of acetophenone imine (Gorrod and C h r i s t o u , 1986). The order of e l u t i o n of major and minor isomers i s reversed i n the phenyl r i n g o x i d i z e d oximes and t h e i r TMS d e r i v a t i v e s . The 189 mass spectrum of the u n d e r i v a t i z e d syn oxime isomer i s very s i m i l a r t o the spectrum of the u n i d e n t i f i e d m e t a b o l i t e XIII of t e r o d i l i n e (Noren, et al. ,1985b). Oxime m e t a b o l i t e s of alpha methyl t e r t i a r y amines are Well known. Dimethylamphetamine a f f o r d s phenylacetone oxime i n both f r e e and conjugated forms in vivo (Beckett and A l S a r r a j , 1972). I t i s d i f f i c u l t to determine the p r e c u r s o r to the oxime (19) in vivo s i n c e i t i s a l s o a major m e t a b o l i t e of n o r r e c i p a v r i n (15) and d i n o r r e c i p a v r i n (20)(see next s e c t i o n ) . I t i s p o s s i b l e that at l e a s t some of the oxime can be accounted f o r by o x i d a t i o n of the primary or secondary hydroxylamines (22 and 17) or n i t r o n e s (24 and 44)(Clement and Beckett, 1981, Coutts and Beckett, 1977 (review), Lindeke, 1982 ( r e v i e w ) ) . The oxime of phenylacetone g l u c u r o n i d e ( s y n t h e s i z e d with immobilized g l u c u r o n y l t r a n s f e r a s e ) has been c h a r a c t e r i z e d i n i t s i n t a c t form by mass spectrometry (Fenselau and Y e l l e t , 1986). Since oximes of t h i s type are good s u b s t r a t e s f o r g l u c u r o n y l t r a n s f e r a s e , i t i s l i k e l y that the bulk of the oxime m e t a b o l i t e of r e c i p a v r i n a r i s e s from an oxime g l u c u r o n i d e r a t h e r than by post h y d r o l y s i s o x i d a t i o n of a primary hydroxylamine g l u c u r o n i d e . Secondary a r y l a l i p h a t i c hydroxylamines (RN(OH)R') are in vitro m e t a b o l i t e s of other b a s i c t e r t i a r y (Clement and Beck e t t , 1981, Beckett, et al., 1983) and secondary amines (Coutts and Beckett, 1977). Demonstrating the e x i s t e n c e of hydroxylamines in vivo i s not always p o s s i b l e (Beckett, et 190 a/., 1983). The reason f o r the d i f f i c u l t i e s i n c h a r a c t e r i z i n g in vivo hydroxylamines was presumed t o be the ease with which the hydroxylamines undergo o x i d a t i o n , e s p e c i a l l y under the ae r o b i c and m i l d l y a l k a l i n e c o n d i t i o n s commonly employed to e x t r a c t other more b a s i c m e t a b o l i t e s (Beckett, et al., 1977) and due to the f a c i l e condensation of hydroxylamine m e t a b o l i t e s with any a v a i l a b l e aldehyde to a f f o r d n i t r o n e s (Beckett, et al., 1979). The hydroxylamines (17 and 22) were d e r i v a t i z e d with BSTFA or TMAH f o r GCMS a n a l y s i s using methods d e s c r i b e d by Beckett and A c h a r i (1977). No hydroxylamines were d e t e c t e d i n the hydrolyzed b i l e e x t r a c t s . The sample workup c o n d i t i o n s employed here are l i k e l y to have o x i d i s e d any g l u c u r o n i d a s e l i b e r a t e d primary hydroxylamine (22) to the oxime ( 1 9 ) , or allowed a condensation of the hydroxylamine with formaldehyde present in the e x t r a c t to a f f o r d a methylene n i t r o n e (24). A gl u c u r o n i d a s e l i b e r a t e d secondary hydroxylamine (17, Ph2CHCH 2CH(CH3)N(OH)CH 3) would be o x i d i z e d d u r i n g workup to a f f o r d two p o s s i b l e n i t r o n e s , 24 (Ph 2CHCH 2CH(CH 3)N +(0~)=CH 2) and 44 (Ph 2CHCH 2C(CH3)=N +(0")CH 3) both of which are isomeric with the secondary formamide (12, Ph 2CHCH 2CH(CH 3)N(H)CHO). A N-methylidene n i t r o n e has been documented as a t e r t i a r y a r y l a l i p h a t i c amine m e t a b o l i t e i n the i n v i t r o metabolism of promethazine (Clement and Beckett, 1981b), and as a m e t a b o l i t e of many secondary a r y l a l i p h a t i c amines (Coutts and Beckett, 1977, ( r e v i e w ) ) . The p o t e n t i a l of the n i t r o n e s as in t e r m e d i a t e s i n the o b s e r v a t i o n of the formamide w i l l be d i s c u s s e d i n the n i t r o n e decomposition s e c t i o n . 191 B. Nonconjugated b i l i a r y m e t a b o l i t e s of r e c i p a v r i n GCMS r e s u l t s f o r the u n d e r i v a t i z e d nonconjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s are presented i n f i g u r e 62. Nonconjugated m e t a b o l i t e s i n c l u d e r e c i p a v r i n (9); n o r r e c i p a v r i n (15); diphenylbutanone (14); diphenylbutanone oxime (19); and d e t e c t a b l e amounts of p h e n o l i c diphenylbutanone (87); diphenylbutanone oxime (89), and r e c i p a v r i n (90); and O-methylcatechol d e r i v a t i v e s of diphenylbutanone (91) and diphenylbutanone oxime (93). The formamide m e t a b o l i t e was not d e t e c t e d i n the nonconjugated f r a c t i o n . C. R e c i p a v r i n metabolism c o n c l u s i o n s In c o n c l u s i o n r e c i p a v r i n i s e x t e n s i v e l y metabolized i n the r a t v i a four major pathways, deamination, N - d e a l k y l a t i o n , N - o x i d a t i o n and phenyl r i n g o x i d a t i o n . These pathways (summarized i n f i g u r e 63) are common to most t e r t i a r y and secondary a r y l a l i p h a t i c amines. Mass s p e c t r a l evidence f o r the proposed m e t a b o l i t e s and t h e i r d e r i v a t i v e s has been presented along with s y n t h e t i c data f o r d e r i v a t i v e s of the phenyl r i n g i n t a c t m e t a b o l i t e s . One novel compound observed as a minor component of the conjugated b i l i a r y e x t r a c t has been i d e n t i f i e d by GCMS as the secondary formamide (12). P o s s i b l e o r i g i n s of formamide (12) w i l l be d i s c u s s e d i n s e c t i o n 7. 192 i l e >488D 48.8-488.8 a-u. RECIP BILE nOH ETOfiC 110CT8S 52888-48888-4 4 8 8 8 -48888-36888-32888-28888-24888-28888-16888-TIC 12.48 I9 13.58 4888-r * . 7 8 17, • 1 9 a 16.87 fl. 65 I • I • I • 1 • I ' I ' I ' I • I • ' ' I ' I ' ' ' I ' ' • ' • ' ' ' • ' 1 8 18 12 14 16 18 28 22 24 T i l e >488D 166.7-167.7 aiu.RECIP BILE UOH ETOfiC 110CT85 SMT E I P 1888-1688-1488-1 1288-1888-888-688-12 .46 9 14 18.81 54 55 & P h 2 C=0 15 12. »2 8.88 14.78 19a F i g u r e 62. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms f o r a nonconjugated e x t r a c t of b i l i a r y r e c i p a v r i n m e t a b o l i t e s , (top) T o t a l ion c u r r e n t f o r a l l m e t a b o l i t e s . (bottom) Ion chromatogram m/z 167 showing phenyl r i n g i n t a c t m e t a b o l i t e s . R 3= OH 1b R 3= OH R 2= OMe R 3=OH 1a 2b R-|= NHAc * 1a R-5= =NOH t R-|= =0 2a 2b 1ab R-,= -OH R2= - OMe R3= - OH R-,= -NHMe.. 3 R 1 = - N H 2 (H) / C H 3 C H 3 \ CH- 1a R 3= -OH 1t> R 3 = - O H R 2 = -OMe O R l 4 R ^ NHCHO R-ja «NOH i R= - N M e 2 Ri= =0 1a R3=0H 1b R 3 =0H R 2= OMe F i g u r e 63. Metabolic pathways f o r r e c i p a v r i n based on the metabolites observed by GCMS. 1a. Phenyl r i n g o x i d a t i o n . 1b. Oxidation to the c a t e c h o l and 3'-O-methylation. 2a. O x i d a t i v e deamination. 2b. Ketone r e d u c t i o n . 3. N - d e a l k y l a t i o n . 4. N-o x i d a t i o n . Dotted arrows = u n c e r t a i n o r i g i n . 1 94 5^_Metabolism_of_Norrecipavr in The metabolism of norrec ipavr in (15) was invest igated to determine whether 15 was an intermediate in the conversion of r e c i p a v r i n (9) to the secondary formamide (12). This was an experiment that was not poss ib le with methadone (8) because of the i n s t a b i l i t y of N-desalkyl methadone, a compound assumed to spontaneously c y c l i z e to the p y r r o l i d i n e EDDP (1). Based on the s i m i l a r i t y to N-methylamphetamine, norrec ipavr in is an exce l l ent candidate for N-oxidat ion to hydroxylamine and ni trone metabol i tes , and metabolism was expected to follow the pathways of N-oxidat ion , N-dea lky la t ion , phenyl r ing oxidation and oxidat ive deamination. A. Conjugated metabolites The resemblance of the t o t a l ion current ( f igure 64) for the conjugated f r a c t i o n of norrec ipavr in metabolites to that of r e c i p a v r i n i s evidence for desa lky la t ion of rec ipavr in as the major metabolic pathway. Ion monitoring for the unsubstituted (m/z 167, f igure 64) and subst i tuted (m/z 183, m/z 213, f igure 65) ions of the phenyl r ing port ion resul ted in the observation of the metabolites summarized in table 10. 195 F i l e >488H 4 8 . 8 - 4 8 8 . 8 a » u . NORECIP B I L E COMJ ETORC 110CT85 T I C 4 4 8 8 8 * 488888-3 6 8 8 8 * 3 2 8 6 8 * 2 8 8 8 8 * 249666-288888-1 6 0 0 0 0 -1 2 8 8 8 * 88888-48888-19a 14 .81 14 18 .81 Ph2C=0 8.12 97 .21 14.: 5 1 7 . 9 8 88 i • i • i 6 8 IS 88 89 2 1 - 1 7 9 3 / 9 8 t • i • i » 11 • i • i • ' 1 ' ' t 18 26 22 24 F i l e >488H 1 6 6 . 7 - 1 6 7 . 7 a » u . N O R R E C I P B I L E COHJETClRC EXT 1 1 / 1 8 / 8 5 E I P 288 1 8 8 8 8 * 9 8 8 8 * 8 8 8 6 * 7 8 8 6 * 6 8 & 8 & 5 8 8 8 * 8 8 6 * 3 8 8 6 * 2 6 8 6 * ; 1 8 8 8 * 488 • I . 6 8 8 , • I • EBB 1868 19a 14 97 19 12 i ' i ' i ' i • i ' i • i ' i ' i • ! ' i 1 i ' ; ' i 1 i 1 i ' r • i • i ' i • i 1 i • i • i • i 1 i ' i 1 i • 6 8 18 12 14 16 18 26 22 24 26 28 38 32 F i g u r e 64. T o t a l ion c u r r e n t and s e l e c t e d ion chromatograms f o r 0 - g l u c u r o n i d a s e - h y d r o l y z e d e x t r a c t s of b i l i a r y n o r r e c i p a v r i n m e t a b o l i t e s . I . T o t a l ion c u r r e n t f o r a l l conjugated n o r r e c i p a v r i n m e t a b o l i t e s (above) Ion chromatogram m/z 167 showing phenyl r i n g i n t a c t m e t a b o l i t e s (below). 196 F i l e >488H 4808-4488-4888-3688-3286-2888-2488-2088-1688-1268-888-400-1 8 2 . 7 - 1 8 3 . 7 a » u . H O R E C I P B I L E CONJ ETOPC 110CT85 SHI E I P 1 7 . 2 2 87 89 21 . 27 7 . 8 8 . 5 6 89a F i l e >488H 2 1 2 . 7 - 2 1 3 . 28600-1 8 B « e t -1 6 6 © ^ leeee-8080-4008- : 2000-a a u . N O R E C l P B I L E CONJ SNT E I P ETOPC 110CT85 1 8 . 4 3 91 1 4 . 3 3 18 92 8 . 9 6 93 2 2 . 2 0 s 2ft.58 ,98 , k 9 3 b 6 ~r~ 8 10 I 12 16 18 1 I 1 20 1 l 24 Figure 65. Tota l ion current and selected ion chromatograms for 0-glucuronidase-hydrolyzed extracts of b i l i a r y norrec ipavr in metabolites . I I . Ion chromatogram m/z 183 showing conjugated norrec ipavr in phenol metabolites (above). Ion chromatogram m/z 213 showing O-methylcatechol metabolites (below). 197 Table 10. Retention times and diagnost ic ion abundances for the conjugated norrec ipavr in metabol i tes . Phenyl r i n g Retention Metabolite Peak Height Subst i tut ion Time (diag. ion) Intact 14 97 19 19a 12 10.81 11.21 14.53 14.81 18.20 Phenol 87 17.22 88 17.80 89 21.27 O-methylcatechol 91 18.43 92 18.96 93 22.20 98 22.28 93b 22.58 Diphenylbutanone 40000 Diphenylbutanol 25000 Cis oxime 4000 Trans oxime 96000 Sec. Formamide <1000 Diphenylbutanone 4800 Norrec ipavr in 2500 Oxime 3200 Diphenylbutanone 19000 Diphenylbutanol 2000 Trans Oxime1 3000 Ni t ro Compound 1000 Cis Oxime 2000 1 The predominant oxime isomer has been assigned the trans conf igura t ion . The reason for the reversa l in the order of e lu t ion i s not c l e a r . 198 In the conjugated f r a c t i o n of b i l e , the secondary formamide (12), while not r e a d i l y apparent by ion monitoring, was d e t e c t e d and gave a s a t i s f a c t o r y mass spectrum, ( f i g u r e 66 t o p ) . Phenyl r i n g i n t a c t d i p h e n y l b u t a n o l (97) had not been d e t e c t e d among the r e c i p a v r i n m e t a b o l i t e s . I t was i d e n t i f i e d by the mass spectrum ( f i g u r e 66 bottom) wherein i t was dehydrated to diphenylbutene (55) on e l e c t r o n impact (M +=226). The diphenylbutene m/z 208 ion f o l l o w s the a r y l a l i p h a t i c cascade. The metabolic r e d u c t i o n of phenyl ketones i s w e l l documented (Smith et al . 1954) The oxime O-methylcatechol (93) was a p p a r e n t l y f u r t h e r o x i d i z e d t o a n i t r o compound (98) which was i d e n t i f i e d on the b a s i s of a molecular ion at m/z 301 ( f i g u r e 67 ( t o p ) ) . N i t r o compounds are m e t a b o l i t e s of a r y l a l i p h a t i c oximes (Matsumoto and Cho, 1982) In u r i n e , the phenyl r i n g i n t a c t conjugated m e t a b o l i t e s i n c l u d e d diphenylbutanone (14), n o r r e c i p a v r i n ' ( 1 5 ) ( 1 1 . 9 min.) and diphenylbutanone oxime (19) but at much lower c o n c e n t r a t i o n s than b i l e ( f i g u r e 68). Only t r a c e s of diphenylbutanone phenol (87) and diphenylbutanone 0-m e t h y l c a t e c h o l (91) were d e t e c t e d i n the conjugated u r i n e f r a c t i o n . D i p h e n y l b u t a n o l (97) and the formamide (12) were not d e t e c t e d i n the u r i n e . The presence of n o r r e c i p a v r i n (15) i n the conjugated f r a c t i o n was unusual ( i t was a l s o present i n r e c i p a v r i n conjugated f r a c t i o n s ) . A p o s s i b l e e x p l a n a t i o n i s t h a t i t a r i s e s from thermal decomposition of the secondary 199 F i l e >488H Bpk Rb 1693 NORECIP BILE CONJ ETOfiC 110C186 SUB RBD S c a n 519 1 8 . 2 8 Bin. Fiqure 66. (top)' Mass spectrum of the underivatized secondary formamide metabolite (12) of norrecipavrin ( 1 5 ) . T h e in tens i ty of the m/z 73 peak is enhanced by an overlapping endogenous Compound with mSjor peaks at m/z 3^ and 129 (bottom) Mass spectrum of the norrecipavrin metabolite 1,-diphenyl 3 butanol (97). 200 hydroxylamine aglycone in u n d e r i v a t i z e d samples. T h i s h y p o t h e s i s was not born out by BSTFA d e r i v a t i z a t i o n of the r e c i p a v r i n conjugated f r a c t i o n s ( S l a t t e r , 1983). Decomposition of s y n t h e t i c secondary hydroxylamine (17) i n the GC i n l e t mainly produced n o r r e c i p a v r i n . The more a c i d i c u r i n e (pH 5) may favor the s u r v i v a l of the hydroxylamine conjugate (42), whereas i n b i l e where the conjugate i s exposed to s l i g h t l y a l k a l i n e c o n d i t i o n s (pH 8.2) capable of o x i d i z i n g the aglycone br the conjugate t o a methylene n i t r o n e (24). The n i t r o n e (24) i s a p o s s i b l e source of the formamide (12) (see l a t e r s e c t i o n ) . Thus the presence of n o r r e c i p a v r i n i n the conjugated f r a c t i o n may be an i n d i r e c t i n d i c a t i o n of the N-o x i d a t i o n pathway to methylene n i t r o n e (24). F i l e > 4 8 S H B p k R b 9 9 1 8 H O R R L C I P B I L E C O H J E T O R C E X T l i / 1 8 / 8 5 S U B HDD S c a n 6 8 2 2 2 . 2 8 t i n . 8 8 1 2 8 1 6 8 I • I . I . i - • I • l _ u 2 8 8 2 4 6 -JL—. L——L_ I • I 2 8 8 • i • 3 2 8 3 6 6 - I — . 1 . I l__L 1 6 8 8 6 H 968&J ] 8 8 8 8 : 7 8 8 * 6 8 6 * 5 8 6 * 4 B 8 * 3 6 8 * 2 8 8 8 : 1 6 6 * 6 -2 1 3 1S1 91 1 1 ' 181 141 2 3 9 55 ' • ' 226 OH 98 NT 3 8 1 \ F i g u r e 67. Mass spectrum of the apparent n i t r o compound 0-met h y l c a t e c h o l m e t a b o l i t e (98) of n o r r e c i p a v r i n . B. Nonconjugated m e t a b o l i t e s 201 The major nonconjugated b i l i a r y m e t a b o l i t e was the oxime, with s m a l l amounts of diphenylbutanone (14), d i p h e n y l b u t a n o l (97), diphenylbutanone phenol (87) and diphenylbutanone 0-m e t h y l c a t e c h o l (91) ( f i g u r e 69 ( t o p ) ) . In the u r i n e e x t r a c t s , n o r r e c i p a v r i n (15) was the major component along with small amounts of diphenylbutanone (14), diphenylbutanone phenol (87) and diphenylbutanone O-methylcatechol (91) ( F i g u r e 69 (bottom)). C. C o n c l u s i o n s r e g a r d i n g n o r r e c i p a v r i n metabolism The pathways of o x i d a t i v e deamination (diphenylbutanone), N - o x i d a t i o n (oxime), and phenyl r i n g o x i d a t i o n (phenols and 0-m e t h y l c a t e c h o l s ) are common to a l l a r y l a l i p h a t i c amines ( C a l d w e l l , 1976, Coutts and Beckett, 1977). D e a l k y l a t i o n of n o r r e c i p a v r i n was not observed however, f a c i l e o x i d a t i o n of d i n o r r e c i p a v r i n (20) to the oxime (19) or hydroxylamine (22) may account f o r the absence of d i n o r r e c i p a v r i n . The t r a c e appearance of the secondary formamide (12) i n conjugated e x t r a c t s of n o r r e c i p a v r i n suggests that d e a l k y l a t i o n of r e c i p a v r i n i s the f i r s t s t e p i n the genesis of the formamide m e t a b o l i t e . The formamide was s t i l l a minor component of the conjugated b i l e f r a c t i o n o n l y . 202 F i l e >488J 4 8 . 8 - 4 8 8 . 8 a m i . **ORZClf> URINE CONJ ETOfiC EXT 11 . -18 /85 3 2 8 8 * 28888-24888-28888: 16888a 1 £ 8 8 « 8888-4888-11 .92 115 2 3 . 0 8 16 12 14 \ ' ' ' .U ' 1 ' ' 1 ' 1 ' ' ' < 16 18 26 22 24 F i l e >488J 1688-1480-1 2 8 * 1 8 8 * 8 8 * 6 8 * 4 8 * 288H 1 6 6 . 7 - 1 6 ? . ? a » u . N O R F C I P URINE CONJ SMT E I P ETOfiC EXT 1 1 / 1 8 / 8 5 19a 1 4 . 7 8 15 11 .92 14 1 8 . 8 4 s. 1 8 . 8 2 1 9 . 9 8 14 • 16 ' . 8 ' 26 ' 22 ' 24 ' Figure 68. (top) Tota l ion current for the extract of 0-glucuronidase-hydrolyzed f r a c t i o n of urine from norrec ipavr in dosed r a t s . (bottom) Mass chromatogram m/z 167 for the same sample. Standard GC condi t ions . 203 F i l e >488F 1 6 6 . 7 - 1 6 ? . 7 a » u . N O R R L C J P HON CONJ URINE ETORC 110CT81T SI1T E IP 44888-48088^ 3 6 8 6 6 : 3 2 8 8 8 : 28088^ 24000^ 2 8 0 8 8 : 16888-^ 12806^ 8888^ 4886-" 8-^  1 1 . 87 15 i • i i • i • i • i • i • i 14 16 18 -i—•—r i • i * -| 24 F i l e - >488DP 1 6 6 . 7 - 1 6 7 . 7 amu.MOE B I L E SMT E I P NON ETORC EXT 11 / -18 /85 5580-5008-4588-4008-3588-3888-2508-2006-1580-1888-586-6 14 . 7 8 19a 14 1 6 . 8 4 F i g u r e 69. (top) Mass chromatogram m/z 167 f o r the e x t r a c t of nonconjugated m e t a b o l i t e s of u r i n e from n o r r e c i p a v r i n dosed r a t s , (bottom) Mass chromatogram m/z 167 f o r the e x t r a c t of nonconjugated m e t a b o l i t e s of b i l e . 204 D i n o r r e c i p a v r i n (20) i s a t r a c e m e t a b o l i t e of r e c i p a v r i n . The metabolism of d i n o r r e c i p a v r i n was i n v e s t i g a t e d to determine whether a secondary formamide m e t a b o l i t e (12) was among the conjugated m e t a b o l i t e s . I f t h i s was the case i t c o u l d be c o n s i d e r e d an in t e r m e d i a t e i n the genesis of the r e c i p a v r i n formamide m e t a b o l i t e . A. M e t a b o l i t e s of d i n o r r e c i p a v r i n The t o t a l ion c u r r e n t and mass chromatograms 167, 183 and 213 f o r the / 3-glucuronidase-hydrolyzed f r a c t i o n of b i l e from a d i n o r r e c i p a v r i n dosed r a t are shown i n f i g u r e 70 and 71. The m e t a b o l i t e s were i d e n t i f i e d as summarized i n t a b l e 11. Tabl e 11. R e t e n t i o n times and d i a g n o s t i c ion abundances f o r the conjugated d i n o r r e c i p a v r i n m e t a b o l i t e s . Phenyl r i n g R e t e n t i o n M e t a b o l i t e Peak Height S u b s t i t u t i o n Time ( d i a g . ion) I n t a c t 14 97 19 13.56 14.16 18.17 Diphenylbutanone Diphen y l b u t a n o l Oxime 14000 2500 20000 Phenol 87 89 19.96 24.67 Diphenylbutanone Oxime 320 1800 O-Methylcatechol 91 92 98 93 21 .88 22.45 25.67 29. 12 Diphenylbutanone Diphenylbutanol N i t r o Compound Oxime 800 600 1000 1800 205 No formamide (12) was d e t e c t e d in any d i n o r r e c i p a v r i n e x t r a c t . In the nonconjugated b i l e f r a c t i o n , only diphenylbutanone (14) and diphenylbutanone oxime (19) were d e t e c t e d . TMS d e r i v a t i z a t i o n d i d not r e v e a l any primary hydroxylamine (22). The parent amine d i n o r r e c i p a v r i n was only d e t e c t e d i n the nonconjugated u r i n e f r a c t i o n . D i p h e n y l b u t a n o l (97) formed a TMS d e r i v a t i v e (tr=l5.42 min.). The mass spectrum had m/z 73/75 ion p a i r and r e t e n t i o n time change, o t h e r w i s e the mass spectrum was s i m i l a r to u n d e r i v a t i z e d d i p h e n y l b u t a n o l due to the l o s s of TMSOH+ from the molecular i o n to g i v e an intense m/z 208 cascade. Three p o s s i b i l e sources of a formamide a r t i f a c t were a l s o i n v e s t i g a t e d . These were based on known s y n t h e t i c c o n v e r s i o n s of a primary amine or hydroxylamine to a secondary formamide or n i t r o n e . Although d i n o r r e c i p a v r i n i s u n l i k e l y to e x i s t as a g l u c u r o n i d e conjugate, the f o r m y l a t i o n of d i n o r r e c i p a v r i n d u r i n g sample p r e p a r a t i o n was attempted by adding excess fo r m i c a c i d to the b i l e sample p r i o r to e x t r a c t i o n . Secondly, the c o n d e n s a t i o n of d i n o r r e c i p a v r i n with formaldehyde, f o l l o w e d by p e r o x i d a t i o n to the t h e r m o l a b i l e o x a z i r i d i n e would g i v e r i s e t o the formamide. The l a s t p o s s i b l e r e a c t i o n i n v o l v e s a hydroxylamine aglycone condensation with 206 formaldehyde to g i v e r i s e to the methylene n i t r o n e . The n i t r o n e has been shown to g i v e r i s e to the formamide under c e r t a i n GC c o n d i t i o n s and i n m i l d a l k a l i ( d i s c u s s e d l a t e r ) . These l a s t two p o s s i b i l i t i e s were t e s t e d by adding formaldehyde or the a n t i o x i d a n t BHT to the b i l e sample p r i o r to workup. Treatment of the j3-glucuronidase-hydrolyzed b i l e e x t r a c t s with BHT, formic a c i d or formaldehyde d i d not s i g n i f i c a n t l y a l t e r the m e t a b o l i t e p r o f i l e . Formate and formaldehyde d i d not g i v e r i s e to the secondary formamide. (mass chromatograms in appendix). On the b a s i s of the'metabolism of d i n o r r e c i p a v r i n and i t s treatment with two sources of formaldehyde, d i n o r r e c i p a v r i n and i t s m e t a b o l i t e s are not p r e c u r s o r s i n the generation of a secondary formamide m e t a b o l i t e of r e c i p a v r i n or n o r r e c i p a v r i n . The pathways of deamination and N - o x i d a t i o n supported by the a r r a y of d i n o r r e c i p a v r i n m e t a b o l i t e s are those expected f o r the homologue amphetamine. The r e l a t i v e amounts of conjugated m e t a b o l i t e s and b i l i a r y m e t a b o l i t e s are probably g r e a t e r i n d i n o r r e c i p a v r i n because, u n l i k e amphetamine i t i s above the minimum molecular weight (200-250 amu) f o r b i l i a r y e x c r e t i o n i n the r a t . 207 P i l e >688f 46.0-459,0 a»u. OtNORR. CONJ T I C B1LE ©PSI 1 48888-i ?8«8ft-! 88888-88888-t.8888-48888-:8868-B T i l e •&S8C: 20086^ 1 8888-1 6888-14fi88-1 2088-1 88&i 4886-2 6 8 ^ 166.7 -167 .7 &B.U .DIMORft CONJ 1 8 . 1 7 19 14 1 3 . 5 6 fclLE S P S 1 14 9 ^ r • -i r 1 8 12 97 29 .83 i-8 .97 14 2 2 24 T " 28 1 "f"1 'T 38 33. F i g u r e 70. T o t a l ion c u r r e n t f o r the e x t r a c t of B-g l u c u r o n i d a s e - h y d r o l y z e d f r a c t i o n of b i l e from d i n o r r e c i p a v r i n dosed r a t s . (bottom) Mass chromatogram m/z 167 f o r the same sample. GC c o n d i t i o n B. 208 f i l e >c.8£:C 2eee-i 800-1 686-i 488-1 280-1 000" t:«0H 69!* 4 86-•'00^  182.7-183.7 anu .DI NOKR. fOMJ Elf" 8 9 • B I L L & F - S I 20.0 21.0 22.0 22.0 24.0 ?5.8 26.0 £?'.e 28.0 29.8 38.8 31 .8 32.0 F i l e >68SC 212.7-213.7 arau .D1NORR. CONJ E I F ' 1888H 1 fc.00H i 408H 1 288H 1000^ 688-400-1 B I L E J 8 P S I 29-12 |93 : : 0 H 9 8 ZS .47 il 91 2 1 . 8 8 18 12 14 2 . 4 5 9 2 24 26 28 38 32 Figure 71. Mass chromatogram m/z 183 (top) and 213 (bottom) for the extract of 0-glucuronidase-hydrolyzed fraction of bile from dinorrecipavrin dosed rats. GC condition B. 209 7i_EXPERIMENTS__TO DETERMINE THE__SOURCE QF__THE FORMAMIDE METABOLITE A. D i l u t i o n of b i l e e x t r a c t s with the s y n t h e t i c secondary  formamide (12) E a r l y experiments demonstrating the secondary formamide i n the conjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s were o f t e n c o n f u s i n g due to peak o v e r l a p with a major a l i p h a t i c b i l e component, and with r e c i p a v r i n phenol (90) which has an i n t e r f e r i n g m/z 72 base peak. T h i s was a problem when steeper g r a d i e n t temperature programs were employed s i n c e peak o v e r l a p r e s u l t e d i n poor q u a l i t y mass s p e c t r a even with t e d i o u s manual background s u b t r a c t i o n s . The r e t e n t i o n time was a l s o a problem s i n c e a small (5-7 second) d i f f e r e n c e was observed between 'the s y n t h e t i c formamide and the b i l e e x t r a c t peak. The s p i k i n g experiment compared the i n t e n s i t i e s of two d i a g n o s t i c ions of the formamide m e t a b o l i t e (m/z 253, and 167) wit h the corre s p o n d i n g ions of the o v e r l a p p i n g m e t a b o l i t e , r e c i p a v r i n phenol (m/z 269, and 183), i n the presence of i n c r e a s i n g amounts of added s y n t h e t i c secondary formamide. The peak shape and r e t e n t i o n time of the formamide were compared from sample to sample to demonstrate c o - e l u t i o n of the metabolic and standard formamide, and the e f f e c t on r e t e n t i o n time and peak shape induced by c o - e l u t i o h with endogenous b i l e components. Table 12 shows the e f f e c t s of exogenous formamide on peak h e i g h t r a t i o s , peak shape and r e t e n t i o n time. 210 Tabl e 12. The e f f e c t s of added s y n t h e t i c secondary formamide standard on peak height r a t i o s , peak shape and r e t e n t i o n time of the formamide m e t a b o l i t e present i n a 0-glucuronidase-h y d r o l y z e d b i l e e x t r a c t Sample ng s t d 1 t r (m/z 2 5 3 ) 2 r a t i o m/z 3 r a t i o m/z 4 peak 5 number u l b i l e . r e l . to s t d . 253/269 167/183 shape 1 0/1 +0.16 0.88 1.08 sh,as 2 30/0 same - - sh,as 3 15/0.5 +0.17 2.15 2.7 ra,as 4 27.3/0.09 +0.02 6.17 7.5 ra,as 1. S y n t h e t i c formamide (ng) on column / uL b i l e on column. . R e t e n t i o n time i n minutes r e l a t i v e to the s y n t h e t i c secondary formamide (tr=15.68 min.) 3. R a t i o of peak h e i g h t s of molecular ions of the secondary formamide and r e c i p a v r i n phenol (m/z 253, m/z 269 r e s p e c t i v e l y ) . 4. R a t i o of peak h e i g h t s of d i a r y l m e t h y l c a t i o n s of the secondary formamide and r e c i p a v r i n phenol (m/z 167, m/z 183 r e s p e c t i v e l y ) . 5. Peak shape: sh= sharp, ra= ramped, as= asymmetrical. The presence of added secondary formamide (12) d i d not gi v e r i s e t o a second peak i n the sp i k e d samples. The presence of b i l e components r e s u l t e d i n a s h i f t to s l i g h t l y longer r e t e n t i o n times, an e f f e c t that was a b o l i s h e d with the lower c o n c e n t r a t i o n of b i l e e x t r a c t (sample 4, f i g u r e 72). The formamide peak was sharp i n the u n d i l u t e d b i l e and a c q u i r e d a ramped appearance as the c o n c e n t r a t i o n of formamide r e l a t i v e t o b i l e was i n c r e a s e d . The r a t i o of ions 253 and 167 i n c r e a s e d r e l a t i v e t o the corresponding ions of the other conjugated m e t a b o l i t e r e c i p a v r i n phenol.. T h i s evidence supports co-e l u t i o n of the m e t a b o l i t e and the s y n t h e t i c standard. R e t e n t i o n time d i f f e r e n c e s are a p p a r e n t l y a s s o c i a t e d with the c o - e l u t i o n of b i l e components. 21 1 1 ? 5 2 0 0 T 3 6 0 0 I n t e n s i t y ( C o u n t s ) 1 oL m / z 2 5 3 14.8 1 5 6 1 6 0 3 4 4 6 0 0 T 5 6 0 0 t r (min.) I n t e n s i t y ( C o u n t s ) 0 W I ' ' ' I m / z 2 5 3 1 4 8 . 1 5 6 1 6 0 t r (min.) I " ' ' I ' 1 • I 1 i • | • i F i g u r e 7 2 . ( t o p ) : Superimposed mass chromatograms of m/z 253 i n the s y n t h e t i c formamide (12)(sample 2, 30 ng, t r = l 5 . 6 8 ) , and b i l e e x t r a c t formamide (sample 1, t r = l 5 . 8 4 ) . Bottom: Mass chromatograms of the m/z 253 ion i n spiked samples 3 and 4 showing that a 5 f o l d decrease i n b i l e c o n c e n t r a t i o n b r i n g s the r e t e n t i o n time of the formamide peak almost back to the standard value (sample 3, tr=!5.85, sample 4, tr=15.62). 212 The sharpness of the r e c i p a v r i n phenol peak was decreased and r e t e n t i o n time was i n c r e a s e d (from 15.7 min.) by the a d d i t i o n of c o - e l u t e d secondary formamide. B. Attempted d e t e c t i o n of c h l o r o f o r m generated a r t i f a c t s The i s o c y a n i d e (65) and carbamates (66-69) were s y n t h e s i z e d as r e f e r e n c e compounds f o r the d e t e c t i o n of formyl c h l o r i d e or phosgene mediated a r t i f a c t s a r i s i n g from c h l o r o f o r m e x t r a c t i o n of r e c i p a v r i n m e t a b o l i t e s . T h i s was prompted by the o b s e r v a t i o n of both carbamate and formamide a r t i f a c t s a r i s i n g from the use of c h l o r o f o r m to e x t r a c t m e t a b o l i t e s of p e t h i d i n e ( S t i l l w e l l et al . 1978) and other compounds (Cone et al. 1981, Wester et al, 1981). I t was reasoned t h a t s i n c e the formamide a r t i f a c t s were l e s s abundant than the carbamates i n these s t u d i e s , the carbamates-should be apparent when c h l o r o f o r m was used f o r m e t a b o l i t e e x t r a c t i o n . The c o n d i t i o n s which favor formamide a r t i f a c t g eneration are c h l o r o f o r m , a l k a l i n e c o n d i t i o n s p l u s a primary (or secondary) amine p r e c u r s o r (20 or 15). The formamide c o u l d a r i s e by a d i r e c t f o r m y l a t i o n of the amine with formyl c h l o r i d e or i n d i r e c t l y a r i s e from the i s o c y a n i d e (65) (Smith, 1964). The c o n d i t i o n s f a v o r i n g carbamate a r t i f a c t g e n e r a t i o n are c h l o r o f o r m , a l k a l i n e c o n d i t i o n s , an amine p r e c u r s o r and methanol or e t h a n o l . These c o n d i t i o n s were a l l s a t i s f i e d d u r i n g b i l e sample p r e p a r a t i o n , but no evidence of carbamates was seen i n c h l o r o f o r m e x t r a c t s of r e c i p a v r i n m e t a b o l i t e s ( f i g u r e 73). 213 F i l e > 4 8 9 I 4 8 . 8 - 4 8 6 . 0 a»u. R E C I P B I L E C O H J C H C L 3 T I C 1 1 6 8 8 8 -1 6 8 6 8 8 -9 8 0 6 6 -8 6 6 0 6 -7 8 0 0 6 -6 8 8 6 8 -5 8 8 8 6 -4006©-3 8 6 6 8 -2 8 8 8 6 -1 4 . 7 7 1 8 . 8 1 l . 2 4 2 2 . 2 4 6 8 1 8 1^ 14 1 6 1 8 2 6 2 2 2 4 F i l e > 4 8 9 1 1 6 6 . 7 - 1 6 7 . ; I U . R E C I P B I L E C O N J C H C L 3 SMT E 1 P Figure 73. T o t a l ion current and selected ion chromatogram for 0-glucuronidase-hydrolyzed chloroform extract of b i l i a r y r e c i p a v r i n metabol i tes . (top) Tota l ion current for a l l metabolites , (bottom) Ion chromatogram m/z 167 showing phenyl r ing intact metabol i tes . The formamide i s present at 18.2 minutes. 214 C. Experiments to e s tab l i sh that the secondary formamide  metabolite of r e c i p a v r i n ar i ses from a glucuronide precursor i . Sulfatase hydro lys i s of the conjugated metabolites B i l e from a rat dosed with r e c i p a v r i n - D 3 was treated with sul fatase in place of /3-glucuronidase, to determine whether su l fatase contaminants in the /3-glucuronidase preparation Glucurase R contr ibuted to the observation of the secondary formamide metabol i te . The ion chromatograms shown in f igure 74 show that diphenylbutanone (14)(tr=13.37 min.) was present as a major metabolite in the su l fate conjugated f r a c t i o n . As with the phenylpropanone metabolite of amphetamines (Caldwel l , 1976) t h i s was the only su l fate metabolite of consequence. Traces of diphenylbutene (55, tr= l0 .23) , d i n o r r e c i p a v r i n (20, tr=13.78), r e c i p a v r i n (9, tr=l5.08) and diphenylbutanone oxime (19, tr=l7.61) and diphenylbutanone O-methylcatechol (91, t r= l8 .4 , m/z 213) were a lso detected. No secondary formamide (12, tr=2l .15) was detected, implying that i t ar i ses exc lus ive ly from a glucuronide precursor and not from a s u l f a t e . i i . Contro l incubation of the rec ipavr in metabolite conjugated f r a c t i o n without ^-glucuronidase enzyme. B i l e from a rat dosed with r e c i p a v r i n was extracted free of nonconjugated metabolites and treated with sodium acetate buffer (0.1 M, pH 5) at 3 8 ° overnight to determine whether non enzymatic hydro lys i s contr ibuted to the observation of the secondary formamide metabol i te . 215 F i l - 4 « . 3 - 4 5 8 . « a » u . RECIP DS BILE CONJ SULFATRSE T I C 16. 19 13 .37 19. 75 0.55 1 j ' 1 ' 1 1 1 ' J J L _ -1 ' 1 ' 1 ' 1 ' 1 ' 1 1 1 '1 1 I" U S 1 2 1 4 16 1 ? 20 ; 4 26 2 8 F i l e ?63; I 1008-^ 1 1 0 8 0 0 - i 4 9 0 0 8 -8 0 0 * 7 8 0 * 6 0 0 * 5 0 0 * 4 0 0 * 3 8 8 * 2 8 8 8 - 1 1 0 0 * * 166-7-167.7 a»u.RECIP P3 BILE CONJ SULFRTRSE 13.3? 114 18.23 7 . 2 4 A 1 9 28-81 1 0 1 2 1 4 16 I S ' 2 6 ' W ' ?A 1 2*7 T 2 8 3 F i g u r e . 74. T o t a l i o n c u r r e n t (top) and m/z 167 ion chromatogram (bottom) f o r the s u l f a t a s e h y d r o l y z e d e x t r a c t of b i l e from r a t s dosed with r e c i p a v r i n - D 3 . Diphenylbutanone (14) i s present at 13.37 min. GCMS c o n d i t i o n B. 216 T r a c e s of diphenylbutanone (14) and diphenylbutanone oxime ( 1 9 ) were observed at approximately one q u a r t e r of the c o n c e n t r a t i o n of those observed i n a p a r a l l e l i n c u b a t i o n with ^ - g l u c u r o n i d a s e . No secondary formamide m e t a b o l i t e was seen i n the non enzymatic i n c u b a t i o n thus i n d i c a t i n g that the secondary formamide a r i s e s from a g l u c u r o n i d e p r e c u r s o r . D. Free r a d i c a l o x i d a t i o n of r e c i p a v r i n (9) and n o r r e c i p a v r i n  (15) as a source of the formamide (12) The absence of formamides i n samples of r e c i p a v r i n and n o r r e c i p a v r i n used f o r metabolism s t u d i e s was confirmed by GCMS p r i o r t o a d m i n i s t r a t i o n of the drug. R e c i p a v r i n was c o n s i d e r e d as a p o t e n t i a l p r e c u r s o r of the secondary formamide i n the conjugated f r a c t i o n because t e r t i a r y a r y l a l i p h a t i c amines are known to undergo N-gl u c u r o n i d a t i o n (Caldwell,1982) and because t e r t i a r y amines a u t o x i d i z e to amides (Henbest and S t r a t f o r d , 1962). Bas i c c o n d i t i o n s and non p o l a r s o l v e n t s f a v o r the hydroperoxide mediated o x i d a t i o n (Beckwith, et al. 1983). I f r e c i p a v r i n (9) were the source of the secondary formamide (12), i t i s l i k e l y t h a t the i n i t i a l o x i d a t i o n product, the t e r t i a r y formamide (26) would a l s o be observed. i . I n c u b a t i o n of r e c i p a v r i n with c o n t r o l b i l e under simulated workup c o n d i t i o n s R e c i p a v r i n added to c o n t r o l b i l e and e x t r a c t e d at a l k a l i n e pH a f t e r s t a n d i n g at room temperature f o r one week at 217 pH 8 d i d not a f f o r d any d e t e c t a b l e secondary formamide (12). R e c i p a v r i n added t o c o n t r o l b i l e and worked up by the standard p r o t o c o l d i d not a f f o r d any secondary formamide or t e r t i a r y formamide (26) i n the nonconjugated or conjugated f r a c t i o n s . T h i s i n d i c a t e s t h a t a l k a l i n e pH or o x i d a t i o n of r e c i p a v r i n c a t a l y z e d by b i l e c o n s t i t u e n t s i s not r e s p o n s i b l e f o r the o b s e r v a t i o n of the secondary formamide m e t a b o l i t e . When b i l e from a r e c i p a v r i n dosed r a t was l e f t s t a nding at room temperature f o r s e v e r a l weeks before workup the t e r t i a r y formamide (26) was apparent by GCMS of the nonconjugated m e t a b o l i t e e x t r a c t . T h i s shows that a f r e e r a d i c a l o x i d a t i o n of r e c i p a v r i n i s p o s s i b l e , but i s an u n l i k e l y source of the secondary formamide m e t a b o l i t e . i i . I n c ubation of n o r r e c i p a v r i n with blank b i l e under simulated workup c o n d i t i o n s N o r r e c i p a v r i n (15, Ph2CHCH2CH(CH 3)N(H)CH 3), which c o u l d be o x i d i z e d i n a l k a l i n e s o l u t i o n to the secondary formamide (12, Ph 2CHCH 2CH(CH 3)N(H)CHO) was observed as a t r a c e component of the conjugated f r a c t i o n and t h e r e f o r e was a p o s s i b l e p r e c u r s o r f o r post enzymatic o x i d a t i o n to the secondary formamide observed i n r a t b i l e e x t r a c t s . When blank b i l e was sp i k e d with n o r r e c i p a v r i n and c a r r i e d through a normal i s o l a t i o n procedure no secondary formamide was d e t e c t e d by GCMS. T h i s argues a g a i n s t n o r r e c i p a v r i n as a s u b s t r a t e f o r f r e e r a d i c a l o x i d a t i o n to the secondary formamide observed i n the conjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s . 218 i i i . E f f e c t of e x t r a c t i o n pH on the o b s e r v a t i o n by GCMS of the r e c i p a v r i n secondary formamide m e t a b o l i t e . The secondary formamide m e t a b o l i t e of r e c i p a v r i n i s a non b a s i c compound and should be e x t r a c t e d from b i l e under n e u t r a l t o m i l d l y a c i d i c c o n d i t i o n s , along with the other non b a s i c m e t a b o l i t e s d e r i v e d from diphenylbutanone (14) and diphenylbutanone oxime (19). When b i l e from r e c i p a v r i n dosed r a t s was h y d r o l y z e d with ^ - g l u c u r o n i d a s e and e x t r a c t e d at pH 5 w i t h e t h y l a c e t a t e , the ketone (14) and oxime (19) were apparent, but the secondary formamide (12) was not d e t e c t e d . T h i s i m p l i e s that a l k a l i may be i n v o l v e d i n a t r a n s f o r m a t i o n of an aglycone p r e c u r s o r to the secondary formamide. E. S o l i d phase e x t r a c t i o n of r e c i p a v r i n m e t a b o l i t e s SPE experiments were undertaken to demonstrate the r e c i p a v r i n formamide m e t a b o l i t e under l e s s r i g o r o u s i s o l a t i o n c o n d i t i o n s , and to develop a f a s t e r i s o l a t i o n scheme. The c a r t r i d g e s were chosen based on l i t e r a t u r e methods f o r m e t a b o l i t e group s e p a r a t i o n s ( S j o v a l l , 1983, S j o v a l l and A x e l s o n , 1982, J.T. Baker L t d , 1982) i . P r e l i m i n a r y experiments a. E l u t i o n of s y n t h e t i c r e f e r e n c e compounds from RPCjg columns Aqueous s o l u t i o n s of the s y n t h e t i c standards diphenylbutanone (14) (a non p o l a r n e u t r a l m e t a b o l i t e ) , d i n o r r e c i p a v r i n f r e e base (20)(a medium p o l a r i t y b a s i c m e t a b o l i t e ) and the secondary formamide (12)(a medium p o l a r i t y 219 neutral metabolite and target compound) were separated on 1 ml RPC^g columns. The samples were eluted with successive one column volumes of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% aqueous methanol. U l t r a v i o l e t measurement of the car tr idge eluent revealed that e lu t ion of the three standards began with the 30% methanol, a l iquot and was maximal at 60% methanol in water. Forty percent methanol was chosen as an appropriate minimum concentration for the e lu t ion of nonconjugated metabolites on reversed phase c a r t r i d g e s . b. RPCjg test e lut ions using UV and TLC detect ion of eluates Solut ions of d-glucuronic a c i d , methadone HCI, phenolphthalein glucuronide and a l iquots of b i l e from contro l and r e c i p a v r i n dosed rats were appl ied to separate R P C ^ columns. The columns were washed with water and then eluted with successive a l iquots of 20, 40, 60, 80, and 100% aqueous e thanol . Eluates were checked for components of interes t by spott ing on th in layer chromatography plates and v i s u a l i z i n g under UV l i g h t or with a var ie ty of spray reagents. Methadone, a representative basic compound was eluted with 80% ethanol . Glucuronic ac id was eluted with the water wash. Phenolphthalein glucuronide was eluted with 20% ethanol , and a l l b i l e samples afforded naphthoresorcinol pos i t ive spots that were most intense in the 40% ethanol f r a c t i o n . The conclusions were that 80% ethanol i s required to rap id ly elute 220 nonconjugated basic metabolites , and that conjugates are most l i k e l y driven from the column with 40% ethanol , along with most other v i s i b l e (yellow) b i l e components. i i . Frac t ionat ion of b i l e components by SPE methods a . Attempted f rac t ionat ion of b i l i a r y r e c i p a v r i n metabolites using RPCjg columns. R P C ^ columns have been used for a prel iminary cleanup of glucuronide conjugates for HPLC a n a l y s i s . The glucuronides were eluted with 100% methanol (Liberato , et a / . , 1983). B i l e from a rec ipavr in dosed rat was passed through a RPCjg column, the car tr idge was eluted f i r s t with water and then with methanol. ^-glucuronidase h y d r o l y s i s , solvent extract ion and GCMS analys i s revealed no metabolites in the aqueous e luate . The methanol f r a c t i o n contained a low recovery of metabol i tes , p r i m a r i l y the oxime (19) and diphenylbutanone (14), plus small amounts of other nonconjugated metabolites . The column was s tr ipped with a var ie ty of less polar solvents and with methanolic pH 5, pH 10 and a c i d i c so lut ions yet no other r e c i p a v r i n re la ted compounds were recovered. The common endogenous b i l e const i tuents were co-e luted with the metabol i tes . Thus on the basis of s e n s i t i v i t y , p u r i t y , and metabolite group separation the f rac t ionat ion of rec ipavr in metabolites on a s ingle RP car tr idge was unsuccessful . 221 b. Pre l iminary cleanup and de ionizat ion of b i l e using a XAD-2 column Amberlite XAD-2, a weak l i p o p h i l i c r e s i n , has been used for pre l iminary cleanup and de ionizat ion of b i o l o g i c a l samples containing glucuronides . B i l e from a rec ipavr in dosed rat was passed through a XAD-2 column. The aqueous eluate was discarded since glucuronidase hydrolys i s and extract ion f a i l e d to reveal s i g n i f i c a n t amounts of rec ipavr in metabol i tes . The ethanol eluent was further p u r i f i e d by SPE to separate nonconjugated metabolites from conjugates, and to demonstrate the formamide metabol i te . c . Frac t ionat ion on FPSi02 columns The XAD-2 ethanol eluate was passed through a FPSi02 c a r t r i d g e . The car tr idge was eluted with ethanol , 60% ethanol in water, and then with water. The metabolites were detected in the ethanol wash by TLC with naphthoresorcinol detec t ion . This f r a c t i o n was blown free of ethanol , hydrolyzed with B-glucuronidase, adjusted to pH 10 and passed through a R P C ^ c a r t r i d g e . The car tr idge was washed with water, 40% ethanol and 100% ethanol . Several 0-glucuronidase-hydrolyzed metabol i tes , inc luding the secondary formamide (12, tr=17.59 m i n . ) , d i n o r r e c i p a v r i n (20, tr=l0.16 min . ) , diphenylbutanone 222 (14, tr=l0.44 min.), n o r r e c i p a v r i n (15, tr=11.56 min.), diphenylbutanone oxime (19, tr=14.24 min.), diphenylbutanone phenol (87, tr=l6.5 min.) and diphenylbutanone 0-me t h y l c a t e c h o l (91, tr=l7.8 min.) were found by GCMS i n the 40% e t h a n o l f r a c t i o n ( f i g u r e 75). The secondary formamide was a l s o c a r r i e d over i n t o the et h a n o l f r a c t i o n ( f i g u r e 76). The e x t r a c t s a l s o c o n t a i n e d endogenous components which dominated the t o t a l ion c u r r e n t . I t c o u l d be concluded that the ethanol s o l u t i o n a p p l i e d to the FPSi02 column was too p o l a r f o r m e t a b o l i t e group s e p a r a t i o n or p u r i f i c a t i o n , thus a l e s s p o l a r e l u t i n g s o l v e n t was r e q u i r e d . d. S e p a r a t i o n of noncon jugated m e t a b o l i t e s on the FPSiC>2 column To separate the nonconjugated m e t a b o l i t e s on the F P S i 0 2 column, the XAD-2 ethanol e x t r a c t was evaporated and r e c o n s t i t u t e d i n 92:8 benzene:ethanol, which was s u f f i c i e n t l y p o l a r t o j u s t move the N-methylnitrone r e f e r e n c e compound on a s i l i c a g e l TLC p l a t e ( t h i s was the most p o l a r s y n t h e t i c compound a v a i l a b l e ) . The s o l u t i o n was passed through the FPSiC>2 c a r t r i d g e and the c a r t r i d g e was e l u t e d with an a l i q u o t of e t h a n o l t o recover conjugated m e t a b o l i t e s . E v a p o r a t i o n of the 92:8 f r a c t i o n r e v e a l e d the nonconjugated m e t a b o l i t e s diphenylbutanone (14, tr=l0.42 min.), r e c i p a v r i n (9, tr=11.91 min.) and diphenylbutanone oxime (19, tr=14.29) by GCMS ( f i g u r e 77). 223 T i l e >684E 48.8-488 -8 "iron. 280000^ 1:488888-1 ?88860lH SPE RECIP PILE S)E RP48 TIC 1 600800-1 i 288888-1 1 • ! • i • ; ' i • i • i • I 1 6 8 10 12 1* 16 > 684£ 166 - , , , • I I I I , • - • ; • ! ' I 1 i • i • . • 18 20 ' 2-2 24 26 2.8 30 32 1 6 7 . 7 amu.SPE RECIP EILE S i E RP4© LIP 14000fr 12880 8-1 1 08008-1 S8W88-I fc. 0000 -1 40000-1 W088-I 10.44 114 '' I 1 • 17.< 91 17. 12 £ 2 2S. 13 i • i • i • i • i • i • •. • i • ; • : ' i ' i ' i ' i ' i • i • i • i • i • i • i • i • i • i i 8 10 12 J.4 16 18 28 22. 24 2f- 2-8 3fc 32 Figure 75. Total ion current (top) and mass chromatogram m/z 167 (bottom) for a /3-glucuronidase-hydrolyzed b i l i a r y recipavrin metabolites eluted from the FPSi0 2 column with ethanol, hydrolyzed with ^-glucuronidase and eluted from a RPCig column with 40% ethanol. The electron m u l t i p l i e r was f a i l i n g when t h i s sample was run. 224 F i l e >684E fcpk flb 72952 SPE RECIP BILE S i E RF48 1 i * .52' m in . y 8868-70800-68608-58008-48008-30888-20000-; 1 0860-o0 128 168 -1 i 1 i I i 1 i I i l_ 4* 2 H H I . 248 2«0 J . i i l _ ;26 360 I . I . i • 73 167 136 115 i i i! ii"' i..v. , iM4 u tea 193 2 S 3 128 168 pi 1 8 "I 88 98 •88 78 68 -58 -46 •38 •28 10 r—* i r—i ' T ' r 11 i' i ' i • r -;68 240 2S8 320 368 F i l e >6 84B Bpk ftb 4746 RECIP EILE SPE EXT S i E RPE 14 DEC 85 SUB Scan 493 17.59 min . 5080-4968-; 3668-' • i • i • ' • ' 12M 168 288 248 Tt7 88 73 136 193 253 -T-^—i—•—i—•—r 2 8 366 -8 8 68 -48 8 -8 -28 -48 -68 -OC4 -18 F i g u r e 76. (top) Mass spectrum of the secondary formamide. m e t a b o l i t e of r e c i p a v r i n e l u t e d from the F P S i 0 2 column with e t h a n o l , h y d r o l y z e d with ^-glucuronidase and e l u t e d from a RPC^g column with 40% ethanol.(bottom) Mass spectrum of the secondary formamide m e t a b o l i t e of r e c i p a v r i n e l u t e d from the R P C 1 8 column with 100% e t h a n o l . 225 F i l e >68?A 40.0-488.9 M U . RECIP BILE SPE EXT E1P 6060891 5588881 5888881-4 588801 408088 1 3588891-3008880-3508800-2880080-i02 •32*iC6H6 S-iETOH 31-1 H0yy08-588888-1 6 .59 o . 8 i ' ' ' 1 i 1 1 1 1 i ' • ' 1 | 1 ' ' 1 i ' • • ' l ' ' ' 1 i • ' ' 1 ! 1 1 1 ' i 1 • • • i • • • • j .8 3 .8 9.8 10.8 11.8 12.8 13.8 14.8 15.8 16.8 1 ,- . 8 F i l e - >68?fl 166 .7 -167 .7 . a»ii .RECIP BILE SPE EXT S i 02 92 -;C6K6 8 "{ETOH 31-EIP 18 8808-1 t.iByPtCV t 4 0 8 0 8 -1 20888-1 E I 0 0 8 B -3 8 8 8 8 -60088-4 9 8 8 0 -£80861 1 8 . 0 « J8 25,11 32 .48 18 12 14 16 1 Figure 77. T o t a l ion current (top) and mass chromatogram m/z 167 (bottom) for nonconjugated b i l i a r y r e c i p a v r i n metabolites eluted from the F P S i 0 2 column with 92/8 benzene/ethanol. Methadone (8) i s present as an reference compound at 18.06 min. The e lectron m u l t i p l i e r was f a i l i n g when th i s sample was run. 226 e. Conclusions regarding the use of SPE cartr idges When sample i s o l a t i o n is complicated by the requirements of metabolite group separat ion, enzymatic h y d r o l y s i s , as well as p u r i f i c a t i o n , the use of a ser ies of SPE columns resul ted in no savings in time, afforded lower y i e l d s , and required large washout volumes for complete e lu t ion without carryover . While i t was poss ib le to observe the secondary formamide metabolite by GCMS of s o l i d phase ex tract s , the time required for i s o l a t i o n and the exposure to m u l t i p l e , (a lbe i t d i f f erent ) solvents negated the u t i l i t y of SPE in a q u a l i t a t i v e inves t iga t ion of t h i s type. SPE i s more useful in automated assays for one or two well character ized components and large numbers of samples. The SPE technique would be much easier to apply to a search for drug metabolites i f a r a d i o l a b e l l e d drug was used, and car tr idge eluates were monitored for r a d i o a c t i v i t y . F . Attempts to increase secondary formamide (12) production by  the addi t ion of formic a c i d , formaldehyde, or formaldehyde and  hydrogen peroxide during sample preparat ion . To invest igate poss ib le formic ac id mediated formylation of d i n o r r e c i p a v r i n during sample preparat ion , b i l e samples were prepared in the presence of a large excess of formic a c i d . To determine whether formaldehyde i s involved in formamide 227 generation by react ion with primary hydroxylamine (22, Ph 2CHCH 2CH(CH 3)N(H)OH) ( th is would give r i s e to a nitrone precursor (24, Ph 2 CHCH 2 CH(CH 3 )N + (0")=CH 2 ) of the secondary formamide (12) during sample preparat ion) , b i l e samples were prepared in the presence of a large excess of formaldehyde. To determine whether oxidat ion of norrec ipavr in (15) to the formamide (12) or peroxidation of Sch i f f base (23, Ph2CHCH2CH(CH3)N=CH2) to the l a b i l e oxaz i r id ine (25) was o c c u r r i n g , b i l e samples were prepared in the presence of excess hydrogen peroxide. To mimic the synthesis of the o x a z i r i d i n e (25) from d inorrec ipavr in (20, Ph 2 CHCH 2 CH(CH 3 )NH 2 ) , the reagents formaldehyde and hydrogen peroxide were added together to the b i l e sample p r i o r to i s o l a t i o n and a n a l y s i s . i . Nonconjugated f rac t ion The m/z 167 mass chromatograms for the nonconjugated f rac t ions were integrated and the area representing each metabolite was compared to that of the i n t e r n a l standard N-e thy l rec ipavr in (85). This comparison was only poss ible in samples not treated with hydrogen peroxide, because H 2 0 2 oxidized the i n t e r n a l standard and r e c i p a v r i n to the corresponding N-oxides (detected by GCMS as the c i s and trans diphehylbutenes (54 and 55)) and d inorrec ipavr in (20) and norrec ipavr in (15) to diphenylbutanone (14) and diphenylbutanone oxime (19). Common degradation products for the r e c i p a v r i n metabolites and in terna l standard precluded in ter sample comparison of e f fec t s of b i l e treatments. 228 The secondary formamide (12) was not d e t e c t e d i n the c o n t r o l , formate-, formaldehyde-, or p e r o x i d e - t r e a t e d samples. I t was however r e a d i l y apparent i n the sample t r e a t e d with formaldehyde and pe r o x i d e , ( f i g u r e 78, remaining ion chromatograms i n appendix). T h i s c o u l d represent a S c h i f f b a s e / p e r o x i d a t i o n mediated formation of the t h e r m o l a b i l e o x a z i r i d i n e (25) from d i n o r r e c i p a v r i n (20) with subsequent i s o m e r i z a t i o n t o the formamide (12). T h i s i s a d i r e c t analogy t o the o x a z i r i d i n e s y n t h e s i s d i s c u s s e d e a r l i e r . Since the hydrogen peroxide o x i d i z e d a l l the amines, the ge n e r a t i o n of the formamide (12) c o u l d not be c o r r e l a t e d with the disappearance of d i n o r r e c i p a v r i n (20). The absence of the t e r t i a r y formamide (26, Ph 2CHCH2CH(CH 3)N(CH3)CHO) i n any of the samples (tr=22.3 min.) and the absence of the secondary formamide (12) i n the peroxide t r e a t e d f r a c t i o n are evidence a g a i n s t a f r e e r a d i c a l p e r o x i d a t i o n of the secondary amine n o r r e c i p a v r i n (15) to the secondary formamide (12) durin g sample p r e p a r a t i o n . Besides p r o v i n g that the o x i d a t i o n was p o s s i b l e , t h i s o b s e r v a t i o n was of l i t t l e value s i n c e the formamide m e t a b o l i t e was not present i n the nonconjugated f r a c t i o n . 229 F i l e >677E 166.7-1 67.7 a » u .RECIP HON CONJ PER0X1 IiE,'FORf1PLBEHYDE TRERTF E1P "in n 408*^ I 1 I 3688-— 1 3 2 8 * j see* 4 i 2 4 8 8 J | , 2 6 6 * I J i F i l e - >677C 166.7-167.7 a a u.RECIF HON CONJ CONTROL 21/8 / - R 6 E I P n 1 68* 120* i * • i 16.0 11.6 12.0 13.0 14.8 15.0 16.0 17.0 18.0 19.6 26.6 21.6 22.6 Figure 78. Ion chromatogram m/z 167 of the contro l (bottom) and formaldehyde/peroxide treated (top) nonconjugated f rac t ion of b i l e from rec ipavr in dosed r a t s . Shaded peaks are in terna l standard (tr=l6.6 min.) and secondary formamide (21.4 m i n . ) . Note the destruct ion of r e c i p a v r i n (tr=15.2), norrec ipavr in (tr=14.7), i n t e r n a l standard (tr=l6.6 min.) and d i n o r r e c i p a v r i n (tr=14.4) by treatment with peroxide. 230 i i . Conjugated f rac t ion The same problems encountered with the oxidat ion of the i n t e r n a l standard N-e thy lrec ipavr in (85, Ph 2CHCH2CH(CH 3)N(CH3)Et) occurred with the use of t e r o d i l i n e (99, Ph 2 CHCH 2 CH(CH 3 )N(H)t-Bu) to estimate the r e l a t i v e amounts of conjugated metabolites from sample to sample. The amount of formamide metabolite in each sample was estimated by integrat ing the area of the m/z 167 peak of the secondary formamide (12), and d i v i d i n g i t by the integrated area of the m/z 100 base peak of the in t erna l standard t e r o d i l i n e (99). The m/z 167 peak of t e r o d i l i n e could not be used because of peak overlap with the oxime metabolite (19). The r a t i o s 0.279, 0.447, and 0.433 for the c o n t r o l , formate, and formaldehyde treated samples respect ive ly indicated a small increase (approximately 1.6 fold) for both treated samples. V i s u a l inspect ion indicated no r e l a t i v e increase in the peroxide treated sample. The combination of peroxide and formaldehyde increased the amount of secondary formamide (12), and resu l ted in the appearance of the t e r t i a r y formamide (26)(f igure 79). The peak i n t e n s i t i e s were much lower in both peroxide treated samples, poss ib ly due to deact ivat ion of the /^-glucuronidase by peroxide, as these samples evolved gas upon addi t ion of the enzyme. 231 f i l e > 6 7 8 C 7 8 8 6 6 - j •i •i 6 8 6 8 6 -56000-4 0 6 0 8 -•J J 3 8 8 8 0 -: - 6 0 0 8 -F i l e 1 2 0 0 0 - j J 1 1 000H -I 1 6 0 0 0 - 1 j 9 6 0 6 -8 0 0 0 -7 0 6 8 -f . 0 0 0 - | 5 8 6 6 -4 0 0 0 -3 8 6 0 -2 8 0 8 -• 1 6 6 0 -6 ^ -[&6.7-1G7.7 i * u . R E C I P C O N J C O N T R O L E I P 17.76 13 .46 18.12 10 14 16 18 2 i . 16 £ ' 0 2 4 ; ''r 1 2 8 3 0 5 E 1 6 6 . 7 - 1 6 7 . 7 &au . R E C I P C O N J P E R O X I D E - ' F C R M P L I J E H Y D E TRFPTED E I P 17 .68 h9 13.3? 14 18.12 1 0 •ft 2& 1 5 . 5 1 4 1 6 1 8 2 8 dd i'4 2 6 2 8 3 0 F i g u r e 79. F i g u r e showing m/z 167 mass chromatograms f o r the c o n t r o l (top) and p e r o x i d e / formaldehyde t r e a t e d (bottom) conjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s . Shaded peaks are the secondary formamide (12, tr=2l.2) and t e r t i a r y formamide (26, t r = 2 3 . l 3 ) . Other major peaks are the oxime (19, tr=l7.7) and diphenylbutanone (14, tr=13.40).GC c o n d i t i o n B. 232 The area of the secondary formamide peak was measured r e l a t i v e to a l l the i n t e g r a t e d m/z 167 peaks i n each i n j e c t i o n and compared to the same r a t i o o b t a i n e d i n the c o n t r o l sample. Va l u e s of 1.29 (formate), 2.00 (formaldehyde), 0.92 (peroxide) and 12.7 (peroxide/ formaldehyde) bear out that the p e r o x i d e / formaldehyde combination can a r t i f i c i a l l y i n c r e a s e l e v e l s of the secondary formamide m e t a b o l i t e . A p o s s i b l e source of the secondary formamide (12, RNHCHO), d i n o r r e c i p a v r i n (20, RNH 2), was only d e t e c t e d i n the formate t r e a t e d sample. The r e l a t i v e amount of n o r r e c i p a v r i n (15, RNHCH3) stayed c l o s e to c o n t r o l v a l u e s (97% and 87% of c o n t r o l ) i n formate and formaldehyde t r e a t e d samples r e s p e c t i v e l y , decreased to 22% of c o n t r o l i n the p e r o x i d e -t r e a t e d sample and was not d e t e c t e d i n the peroxide/formaldehyde-treated sample. N o r r e c i p a v r i n i s a p o s s i b l e s u b s t r a t e f o r o x i d a t i v e p r o d u c t i o n of the formamide in these cases, s i n c e p e r o x i d a t i o n of N-methylamines i s known to a f f o r d formamides (Sayigh and U l r i c h , 1964). The mechanism whereby formaldehyde enhances the o x i d a t i o n i s not c l e a r . G. Attempts to decrease formamide p r o d u c t i o n with the use of  a n t i o x i d a n t s and a formaldehyde complexing reagent In s e v e r a l of the proposed pathways for the post enzymatic generation of the formamide i n the conjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s , there i s a requirement f o r an o x i d a t i o n s t e p . 233 In one pathway (A i n f i g u r e 5), the generation of the l a b i l e o x a z i r i d i n e (25) from the imine (23, RN=CH2), r e q u i r e s a p e r o x i d a t i o n s t e p . In another, the f r e e r a d i c a l o x i d a t i o n of n o r r e c i p a v r i n (15, RNHCH3) by oxygen or hydrogen peroxide would a f f o r d the formamide (12, RNHCHO). L a s t l y , the o x i d a t i o n of the secondary hydroxylamine (17, RN(OH)CH 3) to an a l k a l i or heat l a b i l e n i t r o n e (24, R-N +(0~)=CH 2) c o u l d a l s o be a pathway l e a d i n g to the secondary formamide (B in f i g u r e 5). To t h i s end, conjugated f r a c t i o n s of b i l e e x t r a c t s from r e c i p a v r i n dosed r a t s were worked up i n the presence of two a n t i o x i d a n t s , a s c o r b i c a c i d and b u t y l a t e d hydroxytoluene. In another pathway (B i n f i g u r e 5), condensation of the primary hydroxylamine (22, RNHOH) with formaldehyde a f f o r d s the l a b i l e methylene n i t r o n e (24, RN +(0~)=CH 2). To prevent t h i s step i n the workup, a formaldehyde complexing agent, dimedone (5,5-dimethyl-1,3-cyclohexanedione, 0.1 M) was added to the b i l e p r i o r to the h y d r o l y s i s of conjugates with g l u c u r a s e . Treatment with the a n t i o x i d a n t s BHT and a s c o r b i c a c i d r e s u l t e d i n s l i g h t d e c r e a s e s (60% and 80% of c o n t r o l v a l u e s r e s p e c t i v e l y ) i n the amount of the secondary formamide (12) present i n the conjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s . The f a c t t h a t the formamide was not a b o l i s h e d i s a p o i n t i n favor of a f a c i l e o x i d a t i o n , while the decrease i n the amount of the formamide suggests that chemical o x i d a t i o n i s a c o n t r i b u t i n g f a c t o r i n formamide p r o d u c t i o n . The 234 formamide was present at l eve l s c lose to i t s l i m i t of detect ion , and these decreases may not be s i g n i f i c a n t . The use of ascorbic ac id increased the amount of n o r r e c i p a v r i n (15, RNHCH3) to f ive times that of the contro l and decreased the amount of oxime (19, Ph2CHCH2C(CH3)=NOH) to one t h i r d of contro l l e v e l s . D inorrec ipavr in (20, RNH2) was not detected at the concentrations of b i l i a r y metabolites employed in th i s experiment. The addit ion of dimedone to the b i l e in an attempt to scavenge formaldehyde before i t could condense with enzymatically released d i n o r r e c i p a v r i n (20, RNH2) or primary hydroxylamine (22, RNHOH) resul ted in a s l i g h t increase in the amount of formamide recovered (136% r e l a t i v e to the contro l sample). This is the opposite resu l t expected i f formaldehyde was involved in formamide production from d i n o r r e c i p a v r i n or the primary hydroxylamine. The increase i s d i f f i c u l t to r a t i o n a l i z e and i t s s ign i f i cance i s l i m i t e d without a va l idated assay and rep l i ca ted r e s u l t s . The re su l t i n f e r s that formaldehyde a v a i l a b i l i t y was not a l i m i t i n g factor and that condensation mediated mechanisms were not major contr ibutors to the generation of the formamide metabol i te . 8i_METABOLISM__OF__PROMETH METABOLITES Two factors prompted a detour into promethazine metabolism with the hope of demonstrating yet another formamide metabol i te . 235 The f i r s t f a c t o r was the s t r u c t u r a l s i m i l a r i t y of promethazine (10) to the other a r y l a l i p h a t i c amines under i n v e s t i g a t i o n , i . e . promethazine c o n t a i n s the same i s o p r o p y l N,N,-dimethylamine s i d e c h a i n as r e c i p a v r i n (9, Ph 2CHCH2CH(CH3)N(CH 3) 2). The second f a c t o r was the p u b l i c a t i o n of the mass sp e c t r a of an u n i d e n t i f i e d in vitro m e t a b o l i t e of promethazine (Clement and B e c k e t t , 1981b). T h i s m e t a b o l i t e (X) was thought (probably c o r r e c t l y ) to be a r i n g h ydroxylated (phenol) m e t a b o l i t e of promethazine. The m e t a b o l i t e X had a mass spectrum as f o l l o w s : m/z 300 (M +,20%); 229 (29); 228 (15); 215 (17); 214 (40); 196 (25); 170 (12); 73 (80) ; 72 (100); 58 (40); 56 (40); 44 (80); 42 (60). Since the secondary formamide m e t a b o l i t e s and the parent t e r t i a r y amines are i s o b a r i c (M +=284), the m o l e c u l a r ion m/z 300 f o r t h i s compound would correspond to a p h e n o l i c m e t a b o l i t e of e i t h e r compound. The m/z 72/73 combination together with enhanced i n t e n s i t y of the higher mass ions ( u n l i k e a l l the parent t e r t i a r y amines in t h i s study which have very low abundance ions above the base peak m/z 72) suggested the compound may be the secondary formamide phenol (100), a secondary formamide s u l f o x i d e (101) or N i n - o x i d e (75) ( f i g u r e 80). 236 10 R 76 R 72 R 100 R 101 R 75 R = N(CH 3) 2, R2=H = N +(0 ) t C H 3 ) 2 , R2=H, = NHCHO, R2=H = NHCHO, R2=OH = NHCHO, R2=H, +S 5-0" = NHCHO, R2=H, +N l 0-O" Figure 80. Structures of promethazine (10), promethazine N-oxide (76), promethazine secondary formamide (72) and i t s phenol (100) and sulfoxide (101) and N l Q-oxide (75). Another metabolite (IX) (Clement and Beckett, 1981b) was assigned the promethazine N-oxide structure (76) based on the following mass spectral data: (20 eV): 300 (M +, 3); 284 (6, M+-16); 239 (9); 213 (32); 212 (8); 199 (11); 198 (4); 72 (100). This mass spectrum was similar to that which would be expected of a secondary formamide analogue of promethazine (72, M+=284, base peak m/z 72). The weak ion at m/z 300 was, however, a point against t h i s p o s s i b i l i t y . The p o s s i b i l i t y of identifying a formamide metabolite of promethazine prompted the Leuckart synthesis of the secondary formamide (72) for comparison to the l i t e r a t u r e compounds IX and X. The mass spectrum of the secondary formamide analogue of promethazine (Figure 81. (top)) was very d i f f e r e n t from the li t e r a t u r e metabolites. There was no detectable m/z 72, unlike any of the other formamides that had been studied to date. The m/z 212 base peak and the intense m/z 180 ion corresponded to the alpha cleavage products shown in figure 81 (bottom). Low eV and DIP mass spectra f a i l e d to enhance the m/z 72 ion (appendix). i 237 F i l r > 4 5 4 C 1 B p k fib 2 4 3 6 8 7 0 T V 212 NHCHO 188 152 198 44 69 7 , 8 9 127 \ ^ j 239 , 54 248 Sc an 284 9 . 3 4 t i n . r l 88 -98 J38 j-78 ^68 4?8 M+ J48 284 -\ -38 ^28 r 1 8 288 F i g u r e 81. (top) Mass spectrum of the s y n t h e t i c phenothiazine secondary formamide. (bottom) Methylene phenothiazine (m/z 212) and 0-carboline (m/z 180) resonance s t a b i l i z e d c a t i o n s a r i s i n g from r i n g d i r e c t e d h e t e r o l y t i c c l e a v a g e . 238 With perfect h inds ight , i t was apparent that the alpha h e t e r o l y t i c cleavage react ion that affords the dominant m/z 72 base peak in basic compounds l i k e promethazine was not l i k e l y to be as dominant in the less basic formamides. In the r e c i p a v r i n secondary formamide t h i s resul ted in an increase in the in tens i ty of higher mass ions, due to energet i ca l ly f eas ib le a l t e r n a t i v e fragmentations. In the promethazine formamide, alpha cleavage s t i l l occurred, but with charge l o c a l i z a t i o n on the resonance s t a b i l i z e d m/z 212 methenyl-phenothiazine cat ion instead. Two a d d i t i o n a l syntheses of promethazine analogues were done. The f i r s t involved the peroxidat ion of the secondary formamide to the N i n - o x i d e (75), poss ib ly s imi lar to metabolite X. The formamido N ^ - o x i d e (75) was thermolabile and decomposed in the GC i n l e t to the formamide (72). The d i r e c t i n l e t mass spectrum was s imi lar to the formamide (72) but had a small molecular ion at m/z 300 (f igure 82). It was c l e a r l y not s imi lar to the metabolite X described by Clement and Beckett . 239 F i l e > It IF 1 1 52880-4 8888-a4800-46888-36008-3 20 68-28866-24 888-28600-1 6886-1 2868-8688-4068-212 4 4 152 { tjf *" f ( ?7 1 2 7 I. , , TZ\ 300 4 8 8 ' 8 120 160 268 248 268 ' 3^ 1 9 8 Scan 112 .18 m n , -180 284 -68 e^e ^48 -38 -26 r i e •H-8 F i g u r e 82. (top) D i r e c t i n s e r t i o n probe mass spectrum of the secondary formamide N.Q-oxide (75). (bottom) D i r e c t i n s e r t i o n probe mass spectrum or the Cope e l i m i n a t i o n product 10-(2-p r o p e n y l ) p h e n o t h i a z i n e (77) which a r i s e s from the GCMS a n a l y s i s of the promethazine N-oxide (76). 240 The l a s t synthesis was of the promethazine N-oxide (76), which was synthesized by Clement and Beckett (1981a), but only character ized by d i r e c t in l e t low eV mass spectrometry. The N-oxide decomposes on the GC column to the Cope e l iminat ion product 10-(2-propenyl)phenothiazine. In th i s study the d i rec t i n l e t 70 eV mass spectrum afforded only the Cope e l iminat ion product ( f igure 82 (bottom). Our probe temperature condit ions must have been higher than the i n l e t temperature used by Clement and Beckett since the ir mass spectrum (metabolite IX summarized above) had abundant m/z 72 ( l ike promethazine) and a molecular ion (M+=300) corresponding to the N-oxide. It appeared that the spec tra l assignments made by Clement and Beckett were c o r r e c t . Nonetheless, we pursued the study of the in vivo metabolism of promethazine in the rat with the hope of ident i fy ing the secondary formamide (72) in 0-glucuronidase-hydrolyzed rat b i l e . Metabol i tes with intact t e r t i a r y amine and secondary amine s ide chains were detected using the m/z 72 and 58 ions respec t ive ly Figure 83. The low intens i ty of other ions in these basic compounds made assignment of s tructure to the phenothiazine port ion impossible in underivat ized samples analyzed by EI GCMS. No peaks were detected in the m/z 212 mass chromatogram corresponding to the secondary formamide analogue of promethazine. 241 File- >695I: 228* 280* 1 S66-' 168* 1 488-120* 160* 86* 68* 40* 20* 8 5 7 . 7 - 5 8 - 7 a » u . PROMETHAZINE CONJ EIP 23.33 15 PSI UG695 25-11-86 9 .54 i F i l e >695E ? e * 16 12 14 16 7 1 . 7 - 7 2 . 7 situ. PROMETHAZINE CONJ E1P 15 PSI UG695 25-11/86 288* 246* ?86* i 60* 1200 8 6 * 40* 16 12 14 16 18 34 Figure 83. Mass chromatograms for an extract of B-glucuronidase-hydrolyzed conjugated promethazine metabolites (above) Ion chromatogram m/z 58 showing intact secondary amine metabolites (below) Ion chromatogram m/z 72 showing intact t e r t i a r y amine metabolites 242 2«.DECOMPOSITION QF_N-OXIDiZED_METABOLITE SECONDARY_FQRMAMIDE_(12)_1SOLATED_FROM_RAT_BILE A. Decomposition of rec ipavr in N-oxide under simulated sample  workup condit ions N-oxides are thermolabile metabolites of a number of basic t e r t i a r y amines (Rose and Castagno l i , 1983). There are several thermal and chemical rearrangements common to N-oxides which are known to complicate the i s o l a t i o n and analys i s of t h i s compound c lass (Oae and Ogino, 1977). Before HPLC became a routine a n a l y t i c a l method, i t was common to extract a sample free of parent amine, then reduce any N-oxide metabolites to the parent amine with t i tanous c h l o r i d e and extract and quanti tate the product t e r t i a r y amine as a measure of N-oxide content (Beckett et al. 1972). It was the chemical l a b i l i t y of N-oxides and the ease with which the parent amine was oxidized to the N-oxide that resul ted in ear ly controversy as to whether N-oxides were r e a l l y metabolites or a r t i f a c t s (Anggard et al. 1975). 243 The questions of ear ly invest igators regarding the source of N-oxide metabolites of methadone and re lated compounds were s i m i l a r to those being posed for the secondary formamide metabolite in th i s study. In the case of the secondary formamide, the issue was further complicated by the lack of a r e a d i l y i d e n t i f i a b l e precursor . In th i s experiment the N-oxide (53, Ph 2 CHCH 2 CH(CH 3 )N + (0") (CH 3 ) 2 ) was considered as a possible precursor to the formamide because i t was a thermo- and chemo-l a b i l e metabolite of r e c i p a v r i n that was not e a s i l y extracted from aqueous s o l u t i o n . Although none of the rearrangements of N-oxides reported in the l i t e r a t u r e would af ford the secondary formamide metabol i te , the N-oxide was considered as a poss ible precursor to the formamide because of chemical l a b i l i t y and the s i m i l a r i t y in chemical condit ions required to generate the N-oxide and formamide funct ional groups by the oxidation of amines (Sayigh and U l r i c h , 1964). The N-oxide (53) was exposed to a var ie ty of mi ld ly a c i d i c , basic and ox id i z ing condi t ions , and then checked by GCMS to see i f any secondary formamide was produced, and to ident i fy and roughly quanti tate the degradation products. Results are summarized in table 13. 244 Table 1.3. GCMS peak areas of compounds a r i s i n g from the chemical and/or thermal degradation of rec ipavr in N-oxide under various simulated workup condi t ions . Treatment* B C D E F G H NaAc NaAc H 2 0 borate NaOH F e C l 3 F e C l 3 0-glu H 2 ° 2 * * * T o t a l ion current integrated pea.k areas 1 7.56 36.2 1.3 1 .7 1.5 7.47 12.1 0.9 1.3 2 7.82 30.6 1.8 2.5 2.7 10.9 17.0 1 .75 1 . 1 3 10.30 0.2 1.5 0.8 0.1 0.3 1 .6 6.8 6.6 4 10.68 0.2 0.3 0.2 0.2 0.6 0.7 1.6 0.0 5 11 .49 5.0 32.8 33.4 28.7 16.1 25.6 20.4 44. 1 6 1 1 .91 0.0 11.9 7.4 26.0 21.6 3.4 0.0 19.3 7 1 1 .98 22.1 36.2 44.0 25.3 34. 1 20.0 48.5 0.0 8 14.20 0.0 5.9 2.0 5.9 1 .0 0.8 0.0 1 .3 9 18.47 0.2 2.7 2.8 4.2 4.6 18.7 5.4 2.0 Treatment: A. Control ( p u r i f i e d N-oxide in MeOH). B. Sodium acetate buffer 0.1 M, pH 5. C. Sodium acetate buffer 0.1 M, pH 5 plus 250 u l glucurase. D. Water. E . Sodium borate 0.12 M pH 10. F . 3 M Sodium hydroxide. G. F e r r i c ch lor ide 5 mg/ml. H. F e r r i c c h l o r i d e 5 mg/ ml plus 300 u l of 30% hydrogen peroxide. Cpd.= decomposition product: 1. C i s 1,1-diphenyl-but-2-ene (54). 2. Trans 1,1-diphenyl-but-2-ene (55). 3. Diphenylbutanone (14). 4. d i n o r r e c i p a v r i n (20). 5. Norrec ipavr in (15). 6. U n i d e n t i f i e d . 7. Recipavrin (9). 8. Oxime (19). 9. T e r t i a r y formamide (26). A cont Cpd t r (min . ) Peak area from t o t a l ion current X 100 / t o t a l area of a l l integrated peaks in that sample. 245 Figure 84 shows the t o t a l ion current chromatogram for the contro l in j ec t ion of p u r i f i e d rec ipavr in N-oxide (53). The t o t a l ion currents for the other samples are in the appendix. The major peaks in f igure 84 correspond to the two Cope e l iminat ion products, c i s 1,1-diphenyl-but-2-ene (54, tr=7.92 min.) and trans 1,1-diphenyl-but-2-ene (55, tr=8.26 min.)(compounds 1 and 2 in table 13). The thermal Cope e l iminat ion of N-oxides i s well known (Cope et al. , 1949) and along with the thermal desoxygenation product, r e c i p a v r i n (9), the isomeric butenes (54 and 55) were expected to be the sole decomposition products. A trace of the t e r t i a r y formamide (26, tr=l8.47 min.) survived the i s o l a t i o n of the N-oxide and is a byproduct of the synthesis (Sayigh and U l r i c h , 1963). A small amount of norrec ipavr in (15) was a lso detected. The major observation was that none of the sample i s o l a t i o n methods afforded any secondary formamide (12, tr=2l.1 min). The Cope e l iminat ion products were the safest measure of how much N-oxide survived the simulated i s o l a t i o n . A c i d i c condit ions decomposed most of the N-oxide and resul ted in the formation of norrec ipavr in (15) and diphenylbutanone oxime (19). 0-glucuronidase had no ef fect on the decomposition induced by pH 5 sodium acetate . One other product which had a base peak m/z 140, and lacks m/z 165 or m/z 167 (denoting a react ion of the diphenyl portion) was a lso produced. 246 File -6S2H 48.6-458.8 a » u . REClPflVRIH N-OXIDE CONTROL TIC 888688-< -j 54 e9 55 .-•ei»6e8-26 i" I'H •'!<'!" • i " i 1 r' i i i11 i i 11 11 1 8 18 12 14 16 18 28 22 24 2b 26 38 32 Figure 84. Tota l ion current for the contro l GCMS analys i s of rec ipavr in N-oxide. A l k a l i n e condit ions E and F did not completely decompose the N-oxide (53) and produced a s imi lar amount of norrec ipavr in (15) when compared to the a c i d i c treatments. The a c i d i c ox id i z ing agent f e r r i c ch lor ide decomposed the N-oxide almost t o t a l l y , and p a r t i a l l y deaminated the norrec ipavr in to diphenylbutanone. Addi t ion of hydrogen peroxide to f e r r i c ch lor ide almost completely destroyed the N-oxide and r e c i p a v r i n . Norrec ipavrin was the major product observed by GCMS. It can be concluded that the secondary formamide metabolite does not ar i s e by exposure of the r e c i p a v r i n N-oxide to sample i s o l a t i o n procedures. It was a lso evident that quant i ta t ion of intact N-oxides precludes even mi ld ly a c i d i c or basic condit ions during sample workup and that thermo-l a b i l i t y precludes using GLC for quant i ta t ion of the N-oxide. 247 B. GCMS analys i s and thermal decomposition of the methylene  ni trone GCMS resu l t s for the methylene nitrone (24, Ph2CHCH 2CH(CH3)N+(0")=CH 2) were inconsistent from sample to sample. GCMS analys i s resul ted in the observation of diphenylbutanone oxime (19), the methylene imine monomer (23, Ph 2CHCH 2CH(CH3)N=CH 2), a compound which d i r e c t probe GCMS ana lys i s showed was the nitrone (24), and at s l i g h t l y longer retent ion time an overlapping peak with the retent ion time and mass spectrum of the secondary formamide (12) ( S l a t t e r , 1983). Analys i s of the p u r i f i e d nitrone at low concentration using a freshly s i l a n i z e d i n j e c t i o n port l i n e r and GC column showed that i t was poss ib le to analyze the ni trone (24) by GCMS without decomposit ion.( f igure 85). The high reso lut ion mass spectrum showed that the s ingle peak had peaks at m/z 56 and 236 diagnost ic of the nitrone structure (Coutts, et al. , 1978) ( f igure 86 ( top)) . The decomposition of the ni trone (24) in the GC in l e t was demonstrated to be dependent on sample loading, contamination in the i n j e c t i o n port l i n e r , and the presence of co - in jec ted blank b i l e ex trac t . Inter run comparisons of low (10 ug) and high (100 ug) sample loads were run on clean or d i r t y i n j e c t i o n port l i n e r s ( f igures 87-89). The synthet ic nitrone was a l so mixed with contro l rat b i l e and then extracted and analyzed by GCMS (f igure 86 (bottom), f igure 90). 248 r i l e >£2en 48886-44888-48888-36888-32888-28868-24888-26888-16680-12886-8686-4888-F i l e >628P. Bpk Rb 4191 4486-4868-3606-3286-2886-2488-2886-1666-1288-880-488-lit, / 48.6-758.8 a » u . RECIP EXO NITRONE STB 2&V1'86 2UG^UL TIC 17.79 11 .06 ->„ 14.636.6$-??.66 4* 28.75 6 8 16 12 14 16 18 28 T I •' I ' I' • I ' l ' l ''I ' I ' f 22 24 26 28 38 32 RECIP EXO S0 i I . NITRONE STD 25-'l/86 SUB CLP 128 J i i 168 j i 2 U C U L 280 Scan 25? 17.79 • i n . 240 165 91 / 73 / 115 185 / 138 152 193 / 179 I Ji. ii i 288 236 \ 88 128 168 M 253 286 248 i-l 18 -186 -98 -88 -78 -60 -58 -48 -38 -26 -ie Figure 85. (top) C a p i l l a r y GCMS t o t a l ion current of freshly synthesized and p u r i f i e d recipavrin methylene nitrone injected at low concentration onto a freshly s i l a n i z e d injection port l i n e r and GC column, (bottom) Mass spectrum of the single peak in 2a. 249 100i * 80 ->-CO z 60 -UJ t-2 Ul > 40 -< _t Ul CC 20 F i l e ::?1QH2 Bp*' Pt: 24 4 1 2608-24igiy-£S»8ii>-i 868-i f,aa-1 4138-1 20'"' 1 000" 688-480-280-j J 41 56 57 CHNO - - - - CHN CHO CH 73 91 115 130 J L U . 152 165 167 179 193 208 222 ;-223! 236 237 I 253 40 80 120 M/Z 160 200 240 MUHYl.ENf N i l ftOHF BILE ZP1KCH 19/-18^87 pH 10 73 ! !| ill 1 6 5 Scar. 21 . 97 i » i n ^ I - ' r :1 00 130 ne 103 M '<fa: > IB - 1 I, LJ 193 208 80 1 2 0 168 M 253 -50 I -18 2 0 0 2«0 Figure 86. (top) Summary of high resolution mass spectrum of the nitrone (24) (Source temperature 120°) Some decomposition to the methylene imine (m/z 57, 237) and formamide (m/z 73) i s apparent, (bottom) Mass spectrum of the formamide a r i s i n g from decomposition of the nitrone extracted from control b i l e (see figure 90). 250 r i l e > ? i s f i : 44866-•40666-36666-32868-23886-24880-28608-1 6606-1 2880-3000 4 666-0 - " F i l e ;710S2 Bpk Pb 1401 1 4 0 6 -!. 260-1 086 y 0 6 -460-288-56 46 .e-450.f i a » u . METHYLENE NITRONE STD !9. '18/S? pH 10 1 3 . r s 23 73 14 13 1 9 24 17.85 21.55 9 ^ * ..,.^ ,1 « 1 0 12 • • — r 16 18 ' , 1 . i • . i . 1 i 1 i ' i ' i — r 2 0 i ' i 2 4 36 METHYLENE HIT RONE ST]) 19'10. '87 P H 16 165 can t<c : l .52 » i n 91 115 152 183 138 ! c r i 4 -N=CH 2 I H - C - C H „ - C H C H , 24 t « 3 288 1 7 9 ' Mt253 \ 236 1 \ \ \ 238\ U l L - j j i 120 168 T—• •86 40 J 60 96 -8 ft -76 -6.0 •56 ^0 30 •28 •10 F i g u r e 8 7 . C a p i l l a r y GCMS o f t h e m e t h y l e n e n i t r o n e u s i n g a c l e a n i n j e c t i o n p o r t l i n e r . ( t o p ) T o t a l i o n c u r r e n t s h o w i n g d e c o m p o s i t i o n o f 10 ug o f f r e s h l y p u r i f i e d r e c i p a v r i n m e t h y l e n e n i t r o n e ( 2 4 ) t o t h e o x i m e ( 1 9 , t r = 1 7 . 8 5 m i n . ) , i m i n e ( 2 3 , t r = 1 3 . 7 5 m i n . ) , d i p h e n y l b u t a n o n e ( 1 4 , t r = 1 3 . 4 9 m i n . ) a n d t h e n i t r o n e ( 2 4 , t r = 2 l . 5 5 ) m i n . ( B o t t o m ) M a s s s p e c t r u m o f t h e n i t r o n e ( 2 4 ) . GC c o n d i t i o n B . 251 T i l e >7l8I:6 40 .8 -4S6 .R naeTMYl ENE. NITRONE SID 1 9 ' 1 6 ' 8 7 188888-166686-146886-i z e e e e ^ • 06066 : 86880-68066-4 8688-£6808-. £ 9 I'1 23 18 1 2 1 4 -1 1 6 1 8 28 F i l e -7181:6 4 8 . 8 - 4 5 8 . 0 imu . METHYLENE NITRONE STP 19 '10 E I P 40868-1 21.31 F i g u r e 88. GCMS a n a l y s i s of t h e methylene n i t r o n e u s i n g a c l e a n i n j e c t i o n p o r t l i n e r . ( t o p ) T o t a l i o n c u r r e n t of a sample o v e r l o a d (100 u g ) . (bottom) C l o s e up of the t o t a l i o n c u r r e n t showing the formamide (12, tr=21.31) and n i t r o n e (24, s h o u l d e r a t t r = 2 l . 1 l m i n . ) . GC c o n d i t i o n B. 252 Fi le > 71 PES 4©.6 -456 . f t nn<i. METHYLENE NITRONE STIi 19''16^67 DILUTE*. DIRT T I C 166666-1 . ^ 1 3.95 96668 38889-'6808-68888-^  50800-4 8880-38066-26888-1 0608: ~ !l "' " T( i\ 4 .6': 8 . 2 8 • 8 9 i 10.43 19 f 9 . e i A VI. ' 1 • ! 1 8 ! ' I 2 i . 4 t -I " I ' I ' I , _ T 460-28 .4 ' I ' 1 ' I ' ' 1 I ' 1 ' ! ' ' ' I ' ' ' 1 ' 1 ' ! ' 1 ' I ' ' 1 I ' ' : 1 . 2 2 1 . 6 2 2 . 6 2 2 . 4 2 2 . 8 Figure 89. C a p i l l a r y GCMS of the methylene nitrone (24) using a d i r t y in jec t ion port l i n e r , (top) Tota l ion current showing decomposition of 10 ug of freshly p u r i f i e d rec ipavr in methylene n i trone . Close up of the t o t a l ion current showing the formamide (12, tr=21.46) and no n i t rone . GC condit ion B. 253 F i l e > 7 i 6 i H i £20686-1 200000-! j lb0860-{ J : 4868*j , 1 1 2«0*HH J t 10008*] ~i j 86888H f .008* 40068-2 6 6 6 * 4 6 . 0 - 4 S 0 . 0 a » u . M E T H Y L E N E N I T R O N E h I L E S P I K E D 19s p H ! T I C 18.88 14.18 il 1 0 . CJ 1 i 0 .22 10 ' 1 13 14, 19^  12 21 .94 • i • r 14 lb 1 I ' I ' I • 18 20 1-— I-24 n i f n n « i ^ f 1 . 1 0 " . c u r r e n t chromatogram form GCMS a n a l y s i s using a c l e a n i n j e c t i o n port l i n e r of an e x t r a c t of c o n t r o l b i l e s p i k e d with 100 ug methylene n i t r o n e (24). GC c o n d i t i o n 254 Figure 85 shows that a s i l a n i z e d in jec t ion port l i n e r can completely abo l i sh thermal decomposition of the nitrone to the oxime and imine, g iv ing a peak with a s imi lar mass spectrum to the d i r e c t inser t ion probe analys i s of the same sample ( f igure 3) . In f igure 87, no formamide (12) was detected when 10 ug of nitrone was loaded on a GC column equipped with a clean in jec t ion port l i n e r . In f igure 88 the sample load was increased to 100 ug and the formamide began to ar i s e by decomposition in the i n j e c t i o n port l i n e r . Inject ion of the lower concentration on a column equipped with an i n j e c t i o n port l i n e r used previously for the analys i s of b i l e samples resu l ted in complete conversion to the formamide (f igure 89). C o - i n j e c t i o n of the nitrone (24) and b i l e extract on a clean l i n e r a l so resulted in complete disappearance of the ni trone (24) and appearance of the formamide (12)(figure 90). The thermal decomposition of nitrones commonly gives r i se to isomeric oxime ethers (Oae and Ogino, 1977, Lamchen, 1968). In the case of the methylene ni trone th i s i s diphenylbutanone O-methyloxime (16, Ph 2CHCH 2C(CH 3)=NOCH 3). The oxime ether (16) was synthesized for t h i s study but was never detected by GCMS in any of the ni trone decomposition s tudies . The other common thermal degradation undergone by nitrones i s desoxygenation (Lamchen, 1968). This was born out by the formation of the major decomposition product, the methylene imine (23, Ph 2CHCH 2CH(CH3)-N=CH 2) which ex i s t s as a monomer only in the gas phase (Emmons, 1957). The other major degradation product was the oxime (19, Ph 2CHCH 2C(CH 3)=NOH). Beckett et al. (1973) 255 have observed an oxime and an u n i d e n t i f i e d degradation product f o r an amphetamine methylene n i t r o n e . The u n i d e n t i f i e d product may have been the imine monomer (78, PhCH 2CH(CH3)N=CH 2) based on the decomposition of the r e c i p a v r i n n i t r o n e (24). Coutts et al. (1978) have prevented the decomposition of the methylene n i t r o n e of amphetamine i n t h e i r metabolism s t u d i e s by using a d i f f e r e n t GCMS system. These r e s u l t s have shown that methylene n i t r o n e (24) i n a b i l e e x t r a c t decomposes t o the formamide (12), imine (23) and oxime (19) d u r i n g GC a n a l y s i s . I f a n i t r o n e to formamide c o n v e r s i o n was r e s p o n s i b l e f o r the o b s e r v a t i o n of the secondary formamide m e t a b o l i t e of r e c i p a v r i n , i t was the absence of methylene imine (23) i n the GCMS r e s u l t s f o r r e c i p a v r i n m e tabolic e x t r a c t s that suggested an a l t e r n a t i v e chemical g e n e s i s of the formamide p r i o r to GC a n a l y s i s , rather than the thermal degradation demonstrated here. C. A l k a l i c a t a l y z e d rearrangement of n i t r o n e s to amides. There are s e v e r a l rearrangements d e s c r i b e d i n the l i t e r a t u r e which make the methylene n i t r o n e an a t t r a c t i v e p r e c u r s o r i f the secondary formamide observed by GCMS i n b i l e e x t r a c t s were a c h e m i c a l l y generated a r t i f a c t . These were shown i n f i g u r e s 7-10. The methyl n i t r o n e (a k e t o - n i t r o n e , R=N +(0~)CH3) c o u l d be converted to the methylene isomer (an a l d o - n i t r o n e , RN +(0~)=CH 2) by base c a t a l y s e d Behrend rearrangement (Hamer and Macaluso, 1964, Lamchen, 1968, Smith and G l o y e r , 1975, He i s t a n d , 1978). The same b a s i c c o n d i t i o n s have c o n v e r t e d other n i t r o n e s to t h e i r isomeric amides 256 ( B i g i a v i and M a r r i , 1934, Umezawa, 1960, Hamer and Macaluso, 1964, Z i n n e r , 1978). T h i s rearrangement a p p l i e d to the methylene n i t r o n e (24) would g i v e r i s e to the formamide (12). The p o t e n t i a l f o r rearrangement of the methylidene n i t r o n e (24) to the isomeric secondary formamide (12) i s s t r o n g i n the l i g h t of four o b s e r v a t i o n s : 1. The documented Beckmann rearrangement of n i t r o n e s to amides i n v a r i o u s chemical systems (Hamer and Macaluso, 1964, Lamchen, 1968, Zinn e r , 1978); 2. The a l k a l i n e workup c o n d i t i o n s that would f a v o r t h i s i s o m e r i z a t i o n ; 3. The genera t i o n of n i t r o n e and e a s i l y o x i d i z e d hydroxylamine m e t a b o l i t e s from s t r u c t u r a l l y r e l a t e d amphetamines and ph e n o t h i a z i n e s (Coutts and Beckett, 1977, Clement and Beckett, 1981b); 4. The demonstration of the formamide as a decomposition product of the methylene n i t r o n e by. LCMS ( S l a t t e r , 1983); 5. The demonstration that c o n t r o l b i l e s p i k e d with the s y n t h e t i c secondary hydroxylamine and i s o l a t e d from m i l d l y a l k a l i n e s o l u t i o n a f f o r d s the secondary formamide when analyzed by GCMS ( S l a t t e r , 1983). iQi_ METABQLISM_OF_THE_ The r e c i p a v r i n methylene n i t r o n e was ad m i n i s t e r e d to a r a t and b i l e was c o l l e c t e d f o r 24 hours. Sample p r e p a r a t i o n i n v o l v e d immediate adjustment to pH 5, 24 hour ^ - g l u c u r o n i d a s e h y d r o l y s i s , f o l l o w e d by e x t r a c t i o n of a l l m e t a b o l i t e s from pH 10 s o l u t i o n . Ion monitoring of the m/z 167 diphenylmethyl c a t i o n ( f i g u r e 91) r e v e a l e d diphenylbutanone (14, tr=13.69 min.), d i p h e n y l b u t a n o l (97, tr=14.l2 min., Ph 2CH-CH 2-CH(CH 3)OH), d i n o r r e c i p a v r i n (20, tr=14.63 min.), the oxime 257 (19, tr=l8.42 min.) and the secondary formamide (12, tr=22.l6 min.) ( f i g u r e 91). No n i t r o n e was observed by GCMS, i n accord with the r e s u l t s i n s e c t i o n 9 showing that the n i t r o n e decomposes i n the GC i n l e t to the formamide when c o - i n j e c t e d with b i l e e x t r a c t . I t would take a LC study to determine whether the n i t r o n e decomposed d u r i n g sample i s o l a t i o n or GC a n a l y s i s . T h i s was not performed. Diphenylbutanone oxime phenol (89) was d e t e c t e d at tr=25.04 min. u s i n g the m/z 183 ion ( f i g u r e 91). Compounds t e n t a t i v e l y i d e n t i f i e d as 0-methylcatechols of d i p h e n y l b u t a n o l (92, tr=22.72 min.), diphenylbutanone oxime (93, tr=26.24 min.) and 1,1-diphenyl-3-nitrobutane (98, tr=25.87 min.,) were d e t e c t e d by m o n i t o r i n g the m/z 213 ion ( f i g u r e 92). D e r i v a t i z a t i o n with BSTFA l e f t the formamide (12) i n t a c t (17.47 min.) and r e v e a l e d a compound with s i m i l a r mass spectrum t o the primary hydroxylamine TMS ether (102, tr=19.98 min), the TMS oxime (103, tr=14.82 min.) and s e v e r a l phenyl r i n g i n t a c t u n i d e n t i f i e d compounds ( f i g u r e 93). I t i s l i k e l y t h a t the primary amine ( d i n o r r e c i p a v r i n ) i n the u n d e r i v a t i z e d sample arose from decomposition of the hydroxylamine s i n c e no primary amine TMS d e r i v a t i v e (104) was d e t e c t e d . A l o g i c a l e x t e n s i o n of t h i s experiment would be to study the conjugated m e t a b o l i t e s of i . v . i n f u s e d secondary hydroxylamine. As with the r e c i p a v r i n metabolism s t u d i e s , LCMS or the t r a p p i n g of the n i t r o n e i n a s t a b l e form (Coutts et a l . , 1978) would be r e q u i r e d to demonstrate the i n t a c t n i t r o n e as a m e t a b o l i t e . 258 4 4 8&0-I 18ft4 J 6 5 . 0 - 1 6 7 . 8 antu .METHYLENE NITRONE METRE 19- ' lG/87 pH 18 EIP 48880-36886-32886-28688-24888-26866-16666-12686-8888-4888-18.42 19 14 1 3 . 6 ? 97 1 (4 . 12 12 22.16 X r-n"1' i 1 i 16 12 14 l b 18 26 22 24 I ' ! ' I"' f 1 "I ^ I i t i s 38 F i l e >718H4 182.^-183 . 7 nau .METHYLENE NITRONE METRE ly-'18-'87 pH 18 E I P 4466-4666-3 666^ 3288 2366-2486-2888^ 1 668^ 1288; 488-2S . 8 4 8 9 1 0 . 0E 2 1 . 4 7 I 1 8 1 t i p -28 ' 22 ' 24 Figure 91. (top) Mass chromatogram m/z 167 showing phenyl r ing intact metabolites of the methylene nitrone (24). (bottom) Mass chromatogram m/z 183 showing phenol metabolites of the r e c i p a v r i n methylene n i t rone . GC condit ion B. 259 r : 1 e > 710H 2406-2268-2886-1866-1666-1486-1 280-1 666-866-666-460-288-? .=u»u .METHYLENE NITRONE M E T R B 19/16/87 pH 18 EIF 2 5 . 8 2 98 ie.es 2 , 92 22 .70 21 .98 , 1 8 93 8 ' • i • i • i • i • i • i • — — f • I ' ^ ' V ^ 3 18 12 14 16 18 20 22 24 26 28 36 F i l * >?18Pi4 Hfjk fib 2749 2800-ME'.T HYLENE NITRONE ME TUB 19/18/87 pH 18 ;406-?800-1 688-1288-866-1 0 - » ? 3 : . 1 6 » i r , B -1 60 •96 165 1 15 152 1 3 0 91 Ml M i illi 80 I 1 1 '' I 126 208 181 I I li —r 1 66 253 >28 24 1 I 40 •18 I 1 , 1 I 1 , 1 I 1 1 ' I ?66 240 2S6 Figure 92. (top) Mass chromatogram m/z 213 showing 0-methylcatechol metabolites of the methylene nitrone (24). (bottom) Mass spectrum of the secondary formamide (12) a r i s i n g from GCMS analysis of the nitrone metabolites. GC condition B. 260 F P e >712E 40 6-4c-8 * a n u RECIP METHYLENE HITROHE TMS 6/2v87 15PSJ UG ' TIC 96868-36666-70006-68688-seeee-36606-26880-1 8688-14,82 102 1 3 . ?.<! 10 . 1 ^ 11.18 I—r—r -A - A . T—| -!~T 1 16 12 19.97 8 .94 24 ; F i l e '12Ii RECIP METHYLENE NITRONE TMS 6-'2''87 15PSI U£4S: Epic Sb _1 1 8 6 8 1 2668-1 8866-8686-6888-4866-2866-44 E 3DI> D'-.'C HUH l i f e 75 t j j j l . . . j , . . 111 J . - J i . 103 1 6 7 I8e 206 Scan 347 14 .82 m i n . OTMS NH I H - C - C h L - C H 102 v 222 •ia .. A , i i LA i 1 J.L 2SS 128 T 168 T 1:66 ••86 [-100 Y i M HO 313 t'-T, r ° 1+1=0 F i g u r e 93 (top) T o t a l ion c u r r e n t f o r the BSTFA d e r i v a t i z e d b i l e e x t r a c t of n i t r o n e (24) m e t a b o l i t e s . (bottom) Mass spectrum of the suspected primary hydroxylamine TMS d e r i v a t i v e (102). Standard GC c o n d i t i o n . 261 1^i_METAB0LISM_OF_THE^ The metabolism of the t e r t i a r y N-methylformamide (—) N-methyl-N-(1-methy1-3,3-diphenylpropyl)formamide (26, Ph 2CHCH2CH(CH 3)N(CH 3)CHO) was invest igated to determine whether the carbinolamide metabolite (47, f igure 12, Ph 2CHCH 2CH(CH 3)N(CH 2OH)CHO) was a poss ible precursor of the secondary formamide observed in the rec ipavr in study. Because the metabolism of higher a l i p h a t i c formamides was a v i r t u a l l y unknown area , we have character ized the metabolites of the t e r t i a r y formamide. The systematic and t r i v i a l names, subst i tuents and formulae of compounds of general s tructure A and B (f igure 51, page 153) are summarized in Table 14. GCMS data for a l l metabol i tes , TMS der iva t ives and reference compounds are tabulated as fol lows: Table 15. Metabolites with intact phenyl r i n g s ; Table 16. Phenolic metabolites; Table 17. 0-methylcatechol metabol i tes . Figure 94 and 95 show selected ion chromatograms used to locate metabol i tes . Table 14. Names s t r u c t u r e s and formulae formamide (26) and i t s m e t a b o l i t e s . f o r the t e r t i a r y COMPOUND NUMBER SYSTEMATIC NAME TRIVIAL NAME EMPIRICAL FORMULA 26 12 47 14 97 19 15 105 106 (±) N-formyl-N,a-dimethyl-Y-phenylbenzenepropanamine ( + ) N-formyl-a-methyl -y-phenyl benzenepropanamine (±) N-formyl-N-hydroxymethyl -a- methyl-y-phenylbenzene-propanamine 1 ,l-diphenyl -3-butanone (±) 1,l-diphenyl -3-butanol 1,1-diphenyl-3-butanone oxime (±)N,o- dimethyl-Y-phenyl-benzenepropanamine (±)N-formyl-N,cudimethyl -Y- (4-hydroxyphenyl) -benzene propanamine (±)N-f ormyl -a-methyl -Y- (4-hydroxyphenyl) -benzenepropanamine Tertiary formamide (3 -formamide) Secondary formamide (2 -formamide) Carbinolamide Diphenylbutanone oxime (DPB-oxime) Norrecipavrin 3 -formamide phenol 2 -formamide phenol -N(CH3)CH0 H -N(H)CH0 H CHO -N(CH20H) Diphenylbutanone (DPB) =0 Diphenylbutanol (DPB-OH) -OH =N0H NHCH, -N(CH3)CH0 H -N(H)CH0 H OH C 1 8H 2 1N0 C 1 7H l gN0 C 1 8H 2 1N0 2 C16 H16° C16 H18° C 1 6H 1 7N0 C 1 ?H 2 1N C 1 8H 2 1N0 2 OH C 1 7H l gN0 2 Table 14 (Continued). Names s t r u c t u r e s and formulae f o r t e r t i a r y formamide (26) and i t s m e t a b o l i t e s . NUMBER SYSTEMATIC NAME '07 (±)N-formy1-N-hydroxymethyl-a-methyl-Y-(4-hydroxyphenyl )-benzenepropanamine 8 7 (±)l-(4-hydroxyphenyl ) - l -phenyl-3-butanone 89 (±)1-(4-hydroxypheny1 )-1-phenyl -3-butanone oxime ' 08 (± )N-formyl - N , a-dimethyl - Y- (4-hydroXy-3-methoxyphenyl)-benzene-propanamine 109 (±)N-formyl-a-methyl -y-(4-hydroxy-3-methoxyphenyl)-benzenepropanamine 110 (±)N-formy1-N-hydroxymethy1-a-methy1 -Y-(4-hydroxy-3-methoxyphenyl )-benzenepropanami ne. 9! (±)1-(4-hydroxy-3-methoxyphenyl) -1-phenyl-3-butanone 92 (±)1-(4-hydroxy-3-methoxypheny1) -1-phenyl-3-butanol 93 (±)1-(4-hydrcxy-3-methoxypheny1) -1-phenyl-3-butanone oxime TRIVIAL NAME Carbinolamide phenol EMPRICAL FORMULA OPB-phenol DPB-oxime phenol 3 -formamide O-methylcatechol 2 -formamide O-methylcatechol Carbi nolamide O-methylcatechol Diphenylbutanone O-methylcatechol Di phenylbutanol O-methylcatechol DPB-oxime O-methyl-catechol N(CH20H)CH0 H =0 =N0H =0 -OH =N0H OH C 1 8H 2 1N0 3 OH C 1 6H 1 60 2> OH C 1 6H 1 ?N0 2 -N(CH3)CH0 0CH3 OH C 1 9 H 2 3 N 0 3 -N(H)CH0 0CH3 OH C 1 8 H 2 1 N 0 3 -N(CH20H)CH0 0CH 3 OH C i g H 2 3 N 0 4 0CH 3 OH C 1 7 H 1 8 0 3 0CH 3 OH C 1 ? H 2 0 0 3 0CH 3 OH C 1 7 H 1 9 N 0 3 CO Table 15. GCMS data f o r phenyl r i n g i n t a c t m e t a b o l i t e s of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . Legend: A. Synthetic r e f e r e n c e compound. B. TMS d e r i v a t i v e . C. TMAH d e r i v a t i v e . COMPOUND TRIVIAL NAME RETENTION Ht BASE PEAK OTHER DIAGNOSTIC IONS {{m/z) {% of base peak)) NUMBER TIME (INTENSITY) (minutes) 26 3° Formamide 19.32 267(19) 87 12 2° Formamide 18.20 253(44) 73 12a 2° Formamide N-TMSA 15.42 325( 6) 145 47 Carbinolamide 20.88 2fl3( 0) 85 47a Carbinolamide 0-TMS1 B22.92 . 355( 3) 85 14 Diphenylbutanone 10.84 224(35) 167 97 Diphenylbutanol 11.27 226( 2) 167 19 DPB-oxime (syn) 14.48 239( 0) 167 19a DPB-oxime (ant i) 14.81 239( 8) 167 103 DPB-oxime TMSB 13.64 31K 1) 167 103a (syn) „ DPB-oxime TMS 14.57 311(11) 167 16 (ant i) . DPB-O-methyl-oxime 12.03 253( 8) 167 15 (anti) Norrecipavrin 12.02 239( 7) 58 15a P Norrecipavrin 15.17 311( 5) 130 N-TMS c 9 Recipavrin 12.42 253( 3) 72 1 1 1 Unidenti f ied 12.91 251(12) 167 86(80) 165(34) 167(32) '58(30) 208(23) 193(23) 167(95) 165(86) 208(79) 193(61) 72(31) 44(29) 73(70) 165(42) 167(40) 221(36) 152(24) 130(23) 167(81) 265(75) 45(70) 165(67) 193(53) 207(34) 167(97) 265(76) 165(70) 144(64) 73(60) 103(48) 103(32) 165(32) 181(28) 152(18) 77(14) 43(10) 165(50) 208(48) 193(32) 152(28) 130(22) 115(20) 165(30) 209(32) 165(30) 152(28) 222(18) 77(14) 165(34) 152(18) 118(12) 103(10) 77( 8) 222( 6) 180(62) 165(36) 152(16) 116(12) 73( 6) 220( 6) 166(26) 152(16) 181(10) 220( 9) 103( 8) 77( 6) 165(32) 152(18) 118(16) 103(12) 181( 8) 77( 5) 167(18) 165(12) 152( 5) 208( 4) 103( 4) 193( 4) 167(19) 193(31) 73(25) 105(20) 270(13) 165( 7) 167(12) 165( 6) 15Z( 3) 115( 3) 91( 3) 73( 3) 165(48) 208(32) 152(28) 130(22) 115(15) 70( 6) CTN Table 16. GCMS data f o r phenolic m e t a b o l i t e s of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . Legend: A. S y n t h e t i c reference compound. B. TMS d e r i v a t i v e . C. TMAH d e r i v a t i v e . COMPOUND TRIVIAL NAME RETENTION Mt BASE OTHER DIAGNOSTIC IONS ((m/z) (% of base peak)) NUMBER TIME (INTENSITY) p£Atf (minutes) 105 3° Formamide phenol 25.94 283(20) 87 183(56) 58(33) 224(27) 209(22) 130(28) 181(14) 105a 3° Formamide Phenol-0-TMS 25.90 355(24) 255 296(73) 73(60) 87(58) 295(48) 269(40) 165(20) 105b 3° Formamide ^ 0-methyl phenol 24.72 297(32) ' " 197 87(82) 238(74) 211(38) 165(30) 86(36) 58(22) 106 2° Formamide phenol 25.70 - - Burled under 8 - - - -106a 2° Formam1deR phenol 0-TMS 24.71 341(15) 255 73(58) 296(22) 295(18) 281(13) 103(11) 45( 9) 106b 2° Formamide j. O-methylphenol 22.79 283(17) 197 238(22) 165(17) 211(11) 73(11) U5 ( 6) 44( 4) 107 Carbinolamide phenol - - - Decomposes to 9 - - - -107a Carbinolamide R Phenol 01-0-TMS 28.84 443 v 73 255(82) 295(72) 267(54) 85(50) 353(42) 296(36) 87 DPB-Phenol 17.19 240(18) 183 43(12) 185( 8) 103( 6) 1I9( 5) 153( 5) 225( 2) 87a D DPB-phenol 0-TMS 17.71 312(13) 255 257(22) 73(14) 165( 8) 179( 4) 103( 4) 43( 4) 87b DPB-O-methyl phenol 15.90 254(18) 197 199(12) 165(11) 153(10) 103( 6) 77( 3) 43( 3) 89 DPB-oxime phenol 21.2 255( 2) 183 165(26) 196(16) 184(12) 199(10) 152(10) 121(10) 89a DPB-oxime phenol D1-0-THSB 21.3 399( 6) 255 73(30) 256(20) 269(10) 103( 6) 268( 6) 165( 5) 89b DPB-ox1me phenol (• Dl-O-methyl ether 17.3 283( 3) 197 198(12) 211(10) 165( 9) 103( 8) 153( 8) 133( 6) Table 17. GCMS data f o r O-methylcatechol m e t a b o l i t e s of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . Legend: A. Syn t h e t i c r e f e r e n c e compound. B. TMS d e r i v a t i v e . C. TMAH d e r i v a t i v e . COMPOUND NUMBER TRIVIAL NAME RETENTION TIME (minutes) MJ (INTENSITY) BASE PEAK OTHER DIAGNOSTIC IONS ((m/z) (J of base peak)) 10B 3° Formamide -O-methylcatechol 26.70 313(46) 87 213(76) 86(39) 223(36) 58(32) 152(30) 254(29) 108a 3° Formamide O-methylcatechol TMS ether 27.67 385(53) 285 295(54) 87(42) 73(41) 286(29) 299(25) 326(16) lOBb 3° Formamide dlmethylcatechol 27.31 327(68) 227 237(80) 87(76) 242(32) 86(26) 165(26) 58(20) 109 2° Formamide O-methylcatechol 25.71 299(30) 213 44(47) 123(36) 73(25) 161(20) 239(19) 103(11) 109a 2° Formamide O-methylcatechol TMS ether 26;61' 371(33) 285 73(85) 192(58) 75(20) 286(28) 295(25) 177(20) 109b 2° Formamide -dlmethylcatechol 26.51 313(36) 227 237(27) 228(25) 165(18) 103(12) 73(12) 152(10) 1 10 Carbinolamide O-methylcatechol - - - Decomposes to 14 - - - -1 10a Carbinolamide O-methylcatechol D1-TMS ether 6 30.38 473 73 285(72) 297(46) 383(40) 103(36) 85(35) 129(32) Table 17 (continued). GCMS data for O-methylcatechol metabolites of the t e r t i a r y formamide (26) and t h e i r d e r i v a t i v e s . Compound Number 91 9 ta 91b 92 92a 92b 93 93a 93b Retention Time T r i v i a l Name (Minutes) Diphenylbutanone 18.55 O-methylcatechol Diphenylbutanone 20.01 O-methylcatec hoi TMS ether Diphenylbutanone,. 18.85 dimethylcatechol Diphenylbutanol O-methylcatechol Diphenylbutanol O-methylcatechol TMS ether 8 DPB-oxime 0- methylcatechol 0PB-0xime-OMC 01- TMS ether" 18.95 21.65 Diphenylbutanol . 19.10. dlmethylcatechol 22.2 23.24 M* (x Base Peak Intensi ty) 1 0 u * DPB-O-methyloximg 20.05 dimethylcatechol 270(26) 342(23) 284(32) 272(26) 344( 0) 286(30) 285( 2) 429( O ) 313( 4) 213 285 227 213 285 227 213 285 227 Other Diagnostic Ions ( (m/ i ) ( * of base peak)) 43(38) 103(20) 152(18) 153(16) 181(12) 72(10) 286(24) 144(18) 73(14) 167(10) 255( 9) 103( 4) 228(18) 165(12) 196(11) 103(10) 181( 8) 73( 6) 44(72) 182(15) 151(8) (poor mass spectrum) 73(38) 286(24) 75(18) 255( 8) 117( 6) 165( 4) 212(20) 181( 9) 57(12) 168(10) 253(10) 103( 8) 226(52) 152(26) 181(20) 105(16) 115(14) 76( 4) 73(12) 152( 5) 45( 5) 299( 4) 325( 3) 181( 2) 228(14) 196( 8) 165( 8) 241( 8) 282( 6) 152( 4) 268 F i g u r e 94. Composite s e l e c t e d ion chromatograms used to l o c a t e m e t a b o l i t e s i n 0-glucuronidase-hydrolyzed e x t r a c t s of b i l e from t e r t i a r y formamide dosed r a t s . A. Ion chromatogram m/z 167 showing phenyl r i n g i n t a c t m e t a b o l i t e s , m/z 183 showing phenol m e t a b o l i t e s and m/z 213 showing O-methylcatechol m e t a b o l i t e s i n the u n d e r i v a t i z e d b i l e e x t r a c t . Standard GC c o n d i t i o n s . 10 5 H 10 5H - 10^-91 . a 92 a 1 47 a 103 103a 87 a 8< 105 8a i I 93 a 106 a 109 2 108 a ^ m/z 285 107 no a a A. i m/z 255 s A A . m/z 167 14 l 1 «~ r 18 . . 22 minutes 26 30 Figure 95. Composite selected ion chromatograms used to locate metabolites in ^-glucuronidase hydrolyzed extracts of b i l e from t e r t i a r y formamide dosed ra t s . B. Ion chromatogram m/z 167 showing phenyl ring intact metabolites , m/z 255 showing TMS der iva t i zed phenols and m/z 285 showing TMS d e r i v a t i z e d 0-methylcatechols in the BSTFA der iva t i zed b i l e ex trac t . 270 A. Mass spectrometry of a r y l a l i p h a t i c formamides and  carbinolamides In the formamides (26) and (12), cleavage adjacent to nitrogen with gamma proton transfer afforded a m/z 208, 193, 179, 167, 165, 152, 130, 105, 91 a r y l a l i p h a t i c cascade (see f igure 47 for fragmentation). Phenyl r ing subst i tu t ion resu l ted in the loss of most ions in th i s cascade. However, benzyl ic cleavage, r e s u l t i n g in intense diarylmethyl cations was common to a l l metabolites and allows the detect ion of intact phenyl (m/z 167), phenol (m/z 183), TMS phenol (m/z 255), O-methylcatechol (m/z 213), and TMS O-methylcatechol (m/z 285) metabolites by monitoring the appropriate ions. Alpha cleavage was a lso important, e spec ia l l y in the formamides where base peaks ar i s e from proton transfer to the alpha cleavage product. In formyl der ivat ives of some sympathomimetic amines, the alpha cleavage product gives r i s e to the base peak cat ion ( V i l v a l a , 1979). It i s apparently the longer a l k y l chain which allows the proton transfer in the diarylformylbutanamine ser ies studied here. This resu l t s in base peak cat ion r a d i c a l s at m/z 87 and 73 in (26) and (12) r e s p e c t i v e l y . The proton transfer react ions of amides are well known (Pelah, ei al., 1963). The mass spectra l fragmentation of the underivat ized 271 carbinolamide (47) ( f igure 96) has the expected a r y l a l i p h a t i c cascade plus two important c a r b i n o l d irec ted fragmentations which resu l t in a M + - H 2 0 ion at m/z 265 and a re la ted alpha cleavage ion g iv ing the base peak ion at m/z 85. A poss ib le a lcoho l d i rec ted fragmentation a f fording a formyl a z i r i d i n i u m ion can account for the m/z 265 ion (f igure 97). The TMS ether group d i r e c t s the same fragmentation g iv ing r i s e to the m/z 265 ion in the TMS carbinolamide (47a) and the m/z 353 and 383 ions present in the mass spectra of the di-TMS carbinolamide phenol (107a) and O-methyl catechol (110a) respect ive ly ( f igure 97 and 98). The same mechanism with transfer of the d iphenylethyl moiety rather than a proton to the carb ino l oxygen i s a poss ib le explanation for the m/z 85 ion that i s common to a l l the carbinolamide derived compounds (f igure 99). A rearrangement e l iminat ion ire) mechanism of an odd e lectron ion (McLafferty, 1980) was used to account for the m/z 85 fragment, although rearrangements of th i s type commonly involve smaller migrating groups. Diphenylethanol and i t s TMS ethers lose H 2 0 or TMSOH to give ions m/z 179 (carbinolamide and carbinolamide TMS), m/z 267 (di-TMS carbinolamide phenol) and m/z 287 (di-TMS carbinolamide O-methylcatechol) (f igure 99). Rearrangement of the carb ino l proton or TMS group to the nitrogen accounts for the m/z 72 ion in the mass spectrum of the carbinolamide and m/z 144 in a l l the carbinolamide TMS d e r i v a t i v e s ( f igure 100). Other ions in the TMS der iva t i zed c a r b i n o l s can be envisaged by cleavage of bonds with or without proton transfer as summarized in f igure 101. 272 130 M / Z 220 310 100 _ >-0 - > 4 5 85 LU or 56 72 91 C H 2 - O H i N -CHO 167 1E5 130 152 H - C - C H 0 - C H C H , 47 193 179 40 207 194 110 265 J U _ J _ M / Z 180 250 Figure 96. Mass spectra of the carbinolamide metabol i tes , (a) Carbinolamide (47). (b) Carbinolamide TMS (47a). 273 47,47a m/z 265 A r = C 6 H 5 107a m/z 353 A r = C5H4OTMS 110a m/z 383 Ar = C 6H3(OTMS)(OMe) F i g u r e 97. Proposed mass s p e c t r a l fragmentations of the carbi n o l a m i d e (47) and TMS d e r i v a t i v e s of the carbinolamide (47a), the carbinolamide phenol (107a), and carb i n o l a m i d e O-meth y l c a t e c h o l (110a). A. A l c o h o l (R^H) or TMS ether (R-j =TMS) d i r e c t e d proton rearrangement/elimination to a f f o r d m/z 265 (47, 47a), m/z 353 (107a) and m/z 383 (110a). 274 100 73 >-i— co LU CH 57 H -CH2OTMS i N-CHO I -CH,-CH OCH 3 OTMS 110A 285 85 10318 129 144 207 255 297 31fe25 40 383 140 M/Z 240 340 100 _ >-1— CO 73 CH 75 57 CH 2OTMS 1 N-CHO 1 H-C-CH„-CH CH 3 107A OTMS '255 85 103 165 295 267 281 353 308 324 j 1 39E 40 140 ML 240 340 Figure 98. Mass spectra of the carbinolamide metabol i tes , (c) Carbinolamide phenol di-TMS (107a). (d) Carbinolamide 0-methylcatechol di-TMS (110a). 275 CHO Me H ^ 7 CHO m/z 85 Ph Ar lie CHO 47 47a m/z 179 107a m/z 267 110a m/z 287 Me Ph Ar RO' J K ^ H 2 H N I CHO F i g u r e 99. Proposed mass s p e c t r a l fragmentations of the car b i n o l a m i d e (47) and TMS d e r i v a t i v e s of the carbinolamide (47a), the carbinolamide phenol (107a), and carbinolamide O-met h y l c a t e c h o l (110a). B. A l c o h o l (R1=H) or TMS ether (R^TMS) d i r e c t e d diphenylethane rearrangement/elimination t o a f f o r d m/z 85 i n a l l the carb i n o l a m i d e r e l a t e d compounds. Figure 100. Proposed mass s p e c t r a l fragmentations of the carbinolamide (47) and TMS d e r i v a t i v e s of the c a r b i n o l a m i d e (47a), the carbinolamide phenol (107a), and c a r b i n o l a m i d e 0-methylcatechol (110a). C. Rearrangement of the c a r b i n o l proton (R^H) or TMS group (R^ =TMS) to n i t r o g e n to a f f o r d m/z 72 i n the carbinolamide (47) and m/z 144 i n the TMS d e r i v a t i v e s (47a, 107a and 110a). R 3 47a 193 107a 281 110 a 311 47a 1 7 ° 107a 267 .110 a 297 5 6 CH-1-CH. 8 5 CHO N CH2 47a 167 107a 255 110a 285 47a 207 _ J 107a 2 9 5 110a 325 103 47a 2 6 5 107a353 110a 3 8 3 Me t~ Si -Me 73 Me 47a 3 4 0 107a / 4 2 8 \ 110a V 4 5 8 / Figure 101. Summary of bond cleavages in the mass spectra of the carbinolamide metabolite TMS der ivat ives (47a, 107a and 110a). R1=TMS, R2=H or OCHj, R 3= H or OTMS. 278 B. Metabolism of the t e r t i a r y a r y l a l i p h a t i c formamide Based on the m e t a b o l i t e s observed by GCMS, t e r t i a r y a r y l a l i p h a t i c formamides f o l l o w three general metabolic routes ( f i g u r e 102). At the n i t r o g e n atom , N - d e a l k y l a t i o n v i a s t a b l e i n t e r m e d i a t e c a r b i n o l a m i d e s competes with N-deformylation. The phenyl r i n g s are metabolized to the corresponding phenol or 0-m e t h y l c a t e c h o l . The carbinolamide and aromatic hydroxyl groups a r e conjugated with g l u c u r o n i c a c i d . i . N -deformylation The small amounts of n o r r e c i p a v r i n (15) d e t e c t e d i n the nonconjugated u r i n e f r a c t i o n p a r t i a l l y r e p r e s e n t s the d e f o r m y l a t i o n pathway. Since n o r r e c i p a v r i n i s r a p i d l y m e t a b o l i z e d t o the oxime (19), diphenylbutanone (14) and d i p h e n y l b u t a n o l (97), the extent of d e f o r m y l a t i o n may be underestimated by q u a n t i t a t i o n of the amount of n o r r e c i p a v r i n a l o n e . I t i s not c e r t a i n whether the formamides or c a r b i n o l a m i d e s a l s o c o n t r i b u t e to the pool of oxime, diphenylbutanone, d i p h e n y l b u t a n o l and t h e i r phenyl r i n g o x i d i z e d analogues by pathways other than d e f o r m y l a t i o n . D e f o r m y l a t i o n i s a common metabolic pathway f o r the formamide f u n c t i o n a l group. N-methylamine accounts f o r 15% of the m e t a b o l i t e s of N-methylformamide ( K e s t e l l , et al., 1985), however, dimethylformamide i s a p p a r e n t l y not deformylated to dimethylamine ( S c a i l t e u r et al., 1984). The deformylated / R , " 0 H \ ' R 3 » O H J R2.OMe R3.OH Rl=NMCHO fl,=NHCH0 R 3 = O H 9 R I = N ( C H O ) C M 2 O H R2=0Me R 3 = 0 H R , - N ( C H O ) C H J OH R 3 » O H R,= N H C H O R,«N (CH0)CH20H e *ZO H C — C M 2 — C 5 * M — C H 3 / — *3" R , « O H • • O OMe OH I \ C H 3 R 3 = 0 H R 2 a O M e R 3 " O H - R i NHMe R , » » N O H R 3 - OH R2= O M e R 3 = O H F i g u r e 102. Metabolic pathways f o r the t e r t i a r y formamide (26) based on the metabolites observed by GCMS. a. Phenyl r i n g o x i d a t i o n , b. Oxidation to the c a t e c h o l and 3'-O-methylation. c. O x i d a t i v e deamination. d. Ketone r e d u c t i o n , e. a-Carbon o x i d a t i o n to the carbinolamide. f . N - o x i d a t i o n . g. N-de f o r m y l a t i o n . h. H y d r o l y s i s . Dotted arrows = u n c e r t a i n o r i g i n . KD 280 m e t a b o l i t e ANFT i s r a p i d l y produced by the carcinogen FANFT (Swaminathan and Bryan, 1984), and kynurenine formamidase a c t i v a t e s the d i f o r m y l a n t i m a l a r i a l prodrug 4,4'-d i f o r m a m i d o d i p h e n y l s u l f o n e to the corresponding diamine (Chiou et al, 1971). Although enzymatic d e f o r m y l a t i o n r e a c t i o n s are w e l l known (Shinohara and I s h i g u r o , 1970), d e f o r m y l a t i o n c o u l d a l s o be a r e s u l t of metabolic o x i d a t i o n of the formamide to an u n s t a b l e carbamic a c i d with subsequent d e c a r b o x y l a t i o n ( K e s t e l l , et al., 1985). i i . Phenyl r i n g o x i d a t i o n and c o n j u g a t i o n The phenol and O-methylcatechol m e t a b o l i t e s were found by m o n i t o r i n g the a p p r o p r i a t e ion f o r the d i a r y l m e t h y l moiety (m/z 183, 213 ( u n d e r i v a t i z e d ) , m/z 255, 285 (TMS)). The phenols and O-methylcatechols were expected based on s i m i l a r b i o t r a n s f o r m a t i o n products of methadone (Kang,et a/.,1979), r e c i p a v r i n , t e r o d i l i n e (Noren, et al., 1985a,b) and r e l a t e d a r y l a l i p h a t i c amines. M e t a b o l i t e s 12, 14, 19, 15, 87, 89, 91, 92 and 93 were a l s o m e t a b o l i t e s of r e c i p a v r i n . An attempt was made to d e t e c t c a t e c h o l or d i h y d r o d i o l m e t a b o l i t e s as di-TMS d e r i v a t i v e s by m o n i t o r i n g the m/z 343 and 345 i o n s . Only one compound was d e t e c t e d (tr=31.57 min.) with base peak m/z 343 and m/z 87, 58 s u g g e s t i n g that the m e t a b o l i t e was the t e r t i a r y formamide c a t e c h o l or the 4,4'-diphenol. 281 i i i . N - d e a l k y l a t i o n v i a c a r b i n o l a m i d e s to desalkylformamides A n a l y s i s of the conjugated f r a c t i o n of b i l e by GCMS re v e a l e d t h a t the secondary formamide (12) was a major component. However, GCMS a n a l y s i s of the TMS d e r i v a t i z e d b i l e e x t r a c t showed that the major m e t a b o l i t e was the carbinolamide (47) which decomposed i n the GC i n l e t to the d e s a l k y l compound (12) d u r i n g the a n a l y s i s of the u n d e r i v a t i z e d b i l e e x t r a c t . The s t r u c t u r e of the carbinolamide was confirmed with a low y i e l d s y n t h e s i s i n which the a d d i t i o n of the secondary formamide to formaldehyde was fol l o w e d by d e r i v a t i z a t i o n with BSTFA. Analogous di-TMS p h e n o l i c and O-methylcatechol c a r b i n o l a m i d e s (107a, 110a) were observed at long r e t e n t i o n time i n the BSTFA d e r i v a t i z e d e x t r a c t s of / 3-glucuronidase-h y d r o l y z e d b i l e . I t was not known whether these are phenyl r i n g - o x i d i z e d m e t a b o l i t e s of the carbinolamide or alpha carbon o x i d i z e d m e t a b o l i t e s of the t e r t i a r y formamide phenol and 0-m e t h y l c a t e c h o l . The phenyl r i n g - i n t a c t compound e l u t e d at 12.91 minutes i s a l s o a m e t a b o l i t e of the secondary formamide. I t has been t e n t a t i v e l y i d e n t i f i e d as the alpha-methine carbinolamide (111). The mass spectrum i s d i s c u s s e d i n the s e c t i o n on secondary formamide metabolism. 282 M e t a b o l i c N - d e a l k y l a t i o n of t e r t i a r y amines proceeds v i a u n s t a b l e c a r b i n o l a m i n e intermediates (Rose and C a s t a g n o l i , (review) 1983). When the e l e c t r o n d e n s i t y at n i t r o g e n i s decreased by such f a c t o r s as amide resonance or aromatic s u b s t i t u e n t s the s t a b i l i t y of m e t a b o l i c a l l y - d e r i v e d c a r b i n o l a m i d e s or carbinolamines i s i n c r e a s e d s u f f i c i e n t to a l l o w m e t a b o l i c c o n j u g a t i o n with g l u c u r o n i c a c i d . In e a r l y s t u d i e s , the e x i s t e n c e of the carbinolamide d e a l k y l a t i o n i n t e r m e d i a t e was i n f e r r e d from the presence of the d e s a l k y l m e t a b o l i t e i n /3-glucuronidase-hydrolyzed f r a c t i o n s (McMahon and S u l l i v a n , 1965, S u l l i v a n , et al . , 1968, A l l e n , et al . , 1971). L a t e r c a r b i n o l a m i d e m e t a b o l i t e s were i s o l a t e d and c h a r a c t e r i z e d i n i n t a c t form by HPLC and as TMS d e r i v a t i v e s by GCMS and GLC (Ross, et al., 1983). Carbinolamide m e t a b o l i t e s of dimethylformamide ( S c a i l t e u r , et al., 1984) and N-methylformamide ( K e s t e l l , et al ., 1987) have been d e s c r i b e d . Reference m e t a b o l i t e s can be s y n t h e s i z e d by the a d d i t i o n of the d e s a l k y l compound to formaldehyde (Ross, et al . , 1983). C a r b i n o l a m i d e s have been proposed to be the a c t i v e s p e c i e s i n the a c t i v i t y of a v a r i e t y of nonbasic a n t i c a n c e r agents (Soloway, et al ., 1983). However, N-hydroxymethyl formamide was not found to be the a c t i v e s p e c i e s in the a n t i c a n c e r a c t i o n of N-methylformamide (Cooksey, et al . , 1983), and i n t e r e s t i n t o x i c or a c t i v e m e t a b o l i t e s of NMF i s c u r r e n t l y focused on g l u t a t h i o n e r e l a t e d adducts ( K e s t e l l , et al ., 1986). In c o n c l u s i o n , the t e r t i a r y formamide (26) was 283 metabolized in the rat v i a three major pathways. N-deformylation with oxidat ion of the product amine (15) gave r i s e to oxime (19), diphenylbutanone (14), diphenylbutanol (97) and the phenyl r ing oxidized metabolites 87, 89, 91, 92 and 93. N-dealkylat ion v i a a stable carbinolamide (47), carbinolamide phenol (107) arid carbinolamide O-methylcatechol (110), gave r i s e to secondary formamide (12) and i t s phenol and O-methylcatechol analogues (106) and (109). L a s t l y , the parent formamide (26) gave r i s e to a phenol and O-methyl-c a t e c h o l . A l l pathways were common to the N-formyl or a r y l a l i p h a t i c moiet ies . The absence of any TMS-carbinolamide (47a) in der iva t i zed r e c i p a v r i n metabolite extracts suggests that the carbinolamide pathway i s not the source of the formamide metabolite of r e c i p a v r i n . 12-_METABOLISM_OF_THE_SECON^ The metabolism of the secondary formamide (12, Ph 2CHCH 2CH(CH 3)N(H)CHO) was invest igated to determine whether i t was an intermediate in the production of a number of metabolites of the t e r t i a r y formamide, notably, those involved in the N-oxidat ion , N-dea lky la t ion , deformylation and oxidat ive deamination pathways. The t o t a l ion current and mass chromatograms shown in f igure 103-106 show that the secondary formamide (12) i s biotransformed to a number of metabolites common to the t e r t i a r y formamide (26). The metabolites observed in b i l e and urine are l i s t e d in table 18. 284 F i l e :: 627P 212.7-213.7 amy .i'ECF ORM METRES EI IE CONJ BCLIIC 888-^  7 00 608-58 ft-80-500-£88-l 08-91 9 8 109 112 i ' '' 1 i • i * p if *' i M ' i ' " f r A T l j r- f '* i _ y 12 14 16 IS 28 22 24 26 28 F i g u r e 103. Mass chromatogram m/z 167 f o r the e x t r a c t of B-g l u c u r o n i d a s e - h y d r o l y z e d f r a c t i o n of b i l e from secondary formamide dosed r a t s . Standard GC c o n d i t i o n s . F 1 1 £- 166.7-16?. . 7 amu .SECFORM METSES RILE CONJ tCLUC 3-'2--ft6 L i P 768' 6 ft-PP8-488-S66-286-3 88-14 111 19 1 0 12 14 16 18 20 22 24 F i g u r e 104. Mass chromatogram m/z 213 f o r the e x t r a c t of B-g l u c u r o n i d a s e - h y d r o l y z e d f r a c t i o n of b i l e from secondary formamide dosed r a t s . Standard GC c o n d i t i o n s . 285 6586-6800-5E08-5686-4 5 0 0 -4000-?.£88-3688 2 5 ea-se ee-1 5 8 6 -; 866-5 8 0 -'F. J 8 2 . 7 - t 8 3 . 7 A » . i . S £ C F O R M U R I N E N O N B P S I E I F ' t i l e > 9886-8686-,7868-6886-5866-4666 3668-1:000-1 88W-112 28 [31 8 7 2<S .6£> l ; i | • i • | • 1 1 P 1 ! P 12 14 16 18 20 38 .1*1 4106 I 1 I ' I 24 26 I ' I • I ' I 38 32 ' E 212.7-213.7 atu .SEC: F O R M URINE N O N 8 P S I . . . • ;. E I P Z9.20 1113 91 21 .75 Figure 105. Mass chromatogram m/z 183 (top) and 213 (bottom) for the nonconjugated urine f rac t ion from secondary formamide dosed r a t s . GC condit ion B. 286 F " i l * >esr.T. E280-4 888-4 400-A a Ci c*-: 3600-2880-2400-2000-1 600" i 200-880-480-166.7-167.? i B t u . S E C fORH MEilPBi: B1L.EN0N CONJ 3 ' 2 / 8 6 E I F 19a 111 19 S 1 0 . 0 % 88 8-1 4000-j 3 0 0 1 808-1 1 1 . 0 1 2 . 0 1 3 . 8 1 4 . 0 i S.6 1 6 . 0 1 7 . 0 1 6 6 . 7 - 1 6 7 . 7 i SEC FO|tM URINE WON" 8PSI E1P 13.68 14 ?. a . 8 19 .0 10.-'8 6 111 12 i • ; 1 i 1 '. 1 i • i 14 1 C 18 I ' l l I'8 22 L'4 26 Figure 106. (top) Mass chromatogram m/z 167 for the nonconjugated b i l e extract of secondary formamide metabolites Standard GC condi t ions , (bottom) Mass chromatogram m/z 167 for the nonconjugated urine extract of secondary formamide metabol i tes . GC condit ion B. 287 Table 18. M e t a b o l i t e s and r e t e n t i o n times f o r the secondary formamide m e t a b o l i t e s . Phenyl r i n g S u b s t i t u t i o n Retent ion Time M e t a b o l i t e Diphenylbutanone Carbinolamide* ** Oxime Sec. formamide *** Diphenylbutanone ie ie ie Carbinolamide *** Sec. formamide *** I n t a c t 14 11 1 19 12 Phenol 87 1 12 106 O-methylcatechol 91 93 98 113 109 * Present i n nonconjugated u r i n e e x t r a c t and conjugated b i l e e x t r a c t . R e t e n t i o n times from 15 p s i i n j e c t i o n ( r e t e n t i o n times i n b r a c k e t s from 8 p s i i n j e c t i o n ) . 11.10 (13.68) 12.90 (15.84) 14.60 (21 .49) (20.66) (28.34) (30.14) 18.30 (21.75) (23.00) 21 .82 24.80 (29.20) 25.40 (30.26) Diphenylbutanone _ *** Oxime N i t r o compound Carbinolamide Sec. formamide* **** ** Present i n both b i l e f r a c t i o n s . ***. Observed i n the nonconjugated u r i n e e x t r a c t o n l y , r e t e n t i o n time longer due to 8 p s i He back p r e s s u r e . **** Present i n the conjugated b i l e e x t r a c t o n l y . 288 The compound e l u t i n g at 12.91 minutes ( f i g u r e 106) has mass s p e c t r a l c h a r a c t e r i s t i c s i n accord with the carbinolamide s t r u c t u r e (111) shown i n f i g u r e 108. The c a r b i n o l a m i d e (111) i s a s t a b l e d e a l k y l a t i o n i n t e rmediate i s o l a t e d both before and a f t e r /^-glucuronidase h y d r o l y s i s and i s probably h y d r o l y z e d to diphenylbutanone and formamide. The high mass ion at m/z 251 suggests that the c a r b i n o l a m i d e , (111, 3-formamido-3-hydroxy-1,1-diphenylbutane (M +=269)) dehydrates on e l e c t r o n impact to a f f o r d the M+-H20 ion at m/z 251. The mechanism proposed f o r the t e r t i a r y c a r b i n o l a m i d e dehydration ( f i g u r e 99) can be m o d i f i e d to account f o r the m/z 251 ion as shown in f i g u r e 108. The presence of a weak m/z 70 ion cannot be r a t i o n a l i z e d by the same mechanism as that g i v i n g r i s e to the m/z 85 peak of the t e r t i a r y formamide c a r b i n o l a m i d e . The carbinolamide (111) was a l s o a m e t a b o l i t e of the t e r t i a r y formamide (26). T h i s experiment showed that the secondary formamide i s an i n t e r m e d i a t e i n the b i o t r a n s f o r m a t i o n of the t e r t i a r y formamide (26) to c a r b i n o l a m i d e (111). The c a r b i n o l a m i d e phenol (112) had few d i a g n o s t i c ions and was i d e n t i f i e d based on r e t e n t i o n time and weak ions at m/z 267 (M +-18) and m/z 70. The carbinolamide O-methylcatechol (113) had no d i a g n o s t i c ions other than the m/z 213 base peak and r e q u i r e s c o n f i r m a t i o n by TMS d e r i v a t i z a t i o n . I t i s probable that d e f o r m y l a t i o n of 12 to d i n o r r e c i p a v r i n (20) a l s o o c c u r s , with f u r t h e r metabolism to the oxime (19) and diphenylbutanone (14). No d i n o r r e c i p a v r i n was d e t e c t e d . 289 F i l e >62Sn Epk Ht. 4 828 5288-4 888-4 4 8 8-4886-3668-3280 2888-2488-2888-11".0&-i 289-o00-4 80-42 EC F0RM HETfiBS BTLVETN0H C O N J SUB ftDI' 128 160 _1_ >O0 _ J ,_ Vc an 12.91 240 167 H i N - C H O I H - C ~ C H O - C - 0 H C H 3 111 M + 269 IS2 130 77 7© 103 115 ilil i — i — r 1 28 288 193 179 168 ?00 > 1 8 min . L r-1 1 0 f ^•100 [•68 t-(• J-78 L I. t6.8 2S1 1 — i — r _ 240 r '28 1 8 F i l e >63 fcpk Hb 3 4808-3688-3288^ 2380-2488-2088-j 680-1 200-888-7E 968 48£ 44 ? E C F O R M U R I N E N O H SPSI iUI: PlUi I»VC rtHIi 168 I 183 288 H i N - C H O i H - C - C H „ - C - O H C H , M + 285 112 169 71 93 99 122. 144 1 68 Scar. 64 3 29.34 m in . 24# ... .1 . . . 1 224 288 " " ! ;4 8 -1 1 8 -180 K-0 F t*i8 E ^30 i-28 aw t F i g u r e 107.(top) Mass spectrum of the carbinolamide m e t a b o l i t e (111) observed i n the conjugated and nonconjugated f r a c t i o n , (bottom) Mass spectrum of the carbinolamide phenol m e t a b o l i t e (112) observed i n the conjugated and nonconjugated f r a c t i o n . 290 Figure 1 0 8 . Fragmentation scheme to account for the M - 1 8 peak of the carbinolamide metabolite ( 1 1 1 ) . Ph OH Me Ph ' ^ ^ N H C H O (111) Ph Ph + O H 2 NHCHO - H 2 0 Me ] ' m/z 251 P h N / P h / V ^ N H C H O 291 I t can be concluded that o x i d a t i v e deamination, N-o x i d a t i o n , N - d e a l k y l a t i o n and phenyl r i n g o x i d a t i o n are the major pathways of metabolism of the secondary formamide (12). N-deformylation to d i n o r r e c i p a v r i n (20) may be a r e q u i s i t e s t e p i n the g e n e r a t i o n of ketone and oxime m e t a b o l i t e s , but can only be i n f e r r e d . Diphenylbutanone (14) was a l s o the expected decomposition product of the c a r b i n o l a m i d e (111). Conjugation appears to be l e s s important than with r e c i p a v r i n (9) and t e r t i a r y formamide (26) metabolism. Compounds c o n t a i n i n g the N-methylformamide f u n c t i o n a l group such as the Leuckart s p e c i f i c byproducts of amphetamine s y n t h e s i s are probable sources of h e p a t o t o x i c N-methylformamide. As a compound c l a s s , N-methylformamide i s the only compound a v a i l a b l e f o r comparison of metabolic products. N-methylformamide i s N-deformylated, N - d e a l k y l a t e d by s t a b l e c a r b i n o l a m i d e i n t e r m e d i a t e s (Gescher, et al., 1^83), and forms g l u t a t h i o n e adducts which are c u r r e n t l y thought to mediate the h e p a t o t o x i c e f f e c t s of t h i s a n t i c a n c e r agent ( K e s t e l l et al. 1986, 1987). 292 IV. SUMMARY AND CONCLUSIONS 1 . INVESTIGATION OF POSSIBLE CHEMICAL SOURCES OF A SECONDARY  FORMAMIDE (6) METABOLITE OF METHADONE (8) A . A mechanism was proposed for the oxidat ion of EDDP (1) with MCPBA to account for products (2 ,3 ,4 ,5 ,6 ,7) i d e n t i f i e d by GCMS. B. The a l d o l condensation of 4,4-diphenyl-2,5-heptanedione (3) r e g i o s p e c i f i c a l l y produced 2 ,3-dimethyl -5 ,5-diphenylcyclopent-2-enone (50). C . Solvent contaminants were not a major source of the secondary formamide metabolite (6). 2. METABOLISM OF THE TERTIARY ARYLALIPHATIC AMINE RECIPAVRIN  (9) A . The /' n vi vo b i l i a r y metabolites of the t e r t i a r y a r y l a l i p h a t i c amine N , N , a - t r imethyl-7-phenylbenzenepropanamine (Rec ipavr in R ) from Wistar rats were character ized by GCMS. B. Nonconjugated metabolites included r e c i p a v r i n (9), n o r r e c i p a v r i n (15), diphenylbutanone (14), diphenylbutanone oxime (19), detectable amounts of phenolic diphenylbutanone (87), diphenylbutanone oxime (89), and rec ipavr in (90), and 0-methylcatechol der iva t ives of diphenylbutanone (91) and diphenylbutanone oxime (18). C . Fol lowing B -glucuronidase hydro lys i s and extract ion from pH 10 s o l u t i o n , diphenylbutanone (14), diphenylbutanone oxime 293 (19), an u n i d e n t i f i e d compound (86), d i n o r r e c i p a v r i n (20), n o r r e c i p a v r i n (15), r e c i p a v r i n (9), n o r r e c i p a v r i n phenol (88), the phenols 87, 89 and 90, di p h e n y l b u t a n o l O-methylcatechol (92), r e c i p a v r i n O-methylcatechol (94), the O-methylcatechols 91 and 18 and a secondary formamide (12) were i d e n t i f i e d by GCMS. D. To determine whether thermal i s o m e r i z a t i o n of the methylene n i t r o n e i n the GC i n l e t was the source of the formamide, LCMS r e s u l t s f o r b i l e e x t r a c t s were compared to those of s y n t h e t i c standards 12 and 24 to show that the secondary formamide (12) and not the isom e r i c n i t r o n e (24) was present i n the b i l e e x t r a c t p r i o r t o GCMS a n a l y s i s . E. V a r i o u s p u r i f i e d e x t r a c t i o n s o l v e n t s were employed i n sample workup. The formamide was present r e g a r d l e s s of sol v e n t used. F. M e t a b o l i t e s i s o l a t e d a f t e r / 3-glucuronidase h y d r o l y s i s were c h a r a c t e r i z e d by GCMS i n t h e i r i n t a c t form, as t r i m e t h y l s i l y l (TMS) d e r i v a t i v e s , or f o l l o w i n g d e r i v a t i z a t i o n with t r i m e t h y l a n i l i n i u m hydroxide (TMAH), i n an attempt to s t a b i l i z e and de t e c t l a b i l e p r e c u r s o r s of the n i t r o n e and formamide such as the hydroxylamines (17 and 22). No hydroxylamines were d e t e c t e d . 3 METABOLISM OF TERTIARY AMINE (9) TO SECONDARY FORMAMIDE (12) A. P o t e n t i a l chemical p r e c u r s o r s of the secondary formamide m e t a b o l i t e (12) of r e c i p a v r i n (9) th a t were r u l e d out: 294 i . A pathway from r e c i p a v r i n (9) to t e r t i a r y formamide (26) to ca r b i n o l a m i d e (47) to carbinolamide g l u c u r o n i d e (48) was r u l e d out based on the absence of the t e r t i a r y formamide i n nonconjugated b i l e e x t r a c t s and by the absence of ca r b i n o l a m i d e (47) i n 0-glucuronidase-hydrolyzed b i l e e x t r a c t s . i i . A u t o x i d a t i o n of n o r r e c i p a v r i n (15) to formamide (12) was r u l e d out by adding n o r r e c i p a v r i n to c o n t r o l b i l e and then f o l l o w i n g normal e x t r a c t i o n procedures. GCMS f a i l e d to demonstrate the formamide (12). i i i . F o r m y l a t i o n of d i n o r r e c i p a v r i n (20) to formamide (12) was r u l e d out by s t u d y i n g the e f f e c t s of added formaldehyde or formic a c i d on b i l e e x t r a c t s from d i n o r r e c i p a v r i n dosed r a t s . No formamide (12) was de t e c t e d by GCMS. i v . Formaldehyde condensation with / 3-glucuronidase l i b e r a t e d primary hydroxylamine (22) was r u l e d out by t r e a t i n g the b i l e e x t r a c t with excess formaldehyde d u r i n g h y d r o l y s i s and e x t r a c t i o n . No change i n the amount of formamide (12) was de t e c t e d by GCMS. v. Chloroform and s o l v e n t r e l a t e d g e n e r a t i o n of formamide (12) was r u l e d out by the use of a l t e r n a t i v e s o l v e n t s for m e t a b o l i t e e x t r a c t i o n , by the use of s o l i d phase e x t r a c t i o n methods, and by the absence i n b i l e e x t r a c t s of carbamate r e l a t e d a r t i f a c t s which a r i s e by s i m i l a r mechanisms. v i . The secondary formamide (12) was not a m e t a b o l i t e of 295 d i n o r r e c i p a v r i n (20), thus r u l i n g out the primary amine (20) and i t s m e t a b o l i t e s as metabolic i n t e r m e d i a t e s . B. P o t e n t i a l sources of the formamide: i . N o r r e c i p a v r i n was demonstrated to be a metabolic i n t e r m e d i a t e i n the co n v e r s i o n of r e c i p a v r i n to the formamide (12) by st u d y i n g the metabolism of n o r r e c i p a v r i n . The secondary formamide (12) was found i n the conjugated f r a c t i o n of b i l e from n o r r e c i p a v r i n dosed r a t s , suggesting that r e c i p a v r i n i s d e a l k y l a t e d p r i o r to formamide me t a b o l i t e g e n e r a t i o n . i i . I t was p o s s i b l e to i n c r e a s e the r e l a t i v e amount of the secondary formamide observed by GCMS by t r e a t i n g the conjugated f r a c t i o n of b i l e from r e c i p a v r i n dosed r a t s with a combination of formaldehyde and hydrogen pe r o x i d e . Thus p e r o x i d a t i o n of an imine i n t e r m e d i a t e i s a p o s s i b l e source of the formamide m e t a b o l i t e (12), but a p l a u s i b l e g l u c u r o n i d e p r e c u r s o r f o r the imine or amine s u b s t r a t e cannot be l o g i c a l l y proposed. i i i . The formamide (12) was de t e c t e d by GCMS i n e x t r a c t s of b i l e from r a t s a d m i n i s t e r e d the n i t r o n e (24). i v . The n i t r o n e (24) was shown to degrade i n the GC i n l e t to the formamide (12), e s p e c i a l l y when i n j e c t e d at high c o n c e n t r a t i o n s or c o - i n j e c t e d with b i l e components. v. The n i t r o n e (24) had been shown by LCMS to degrade at room temperature t o the formamide (12) ( S l a t t e r , 1983). The a l k a l i 296 c a t a l y z e d Beckmann rearrangement of n i t r o n e s to amides was used t o account f o r t h i s o b s e r v a t i o n . v i . The n i t r o n e (24) had been shown to a r i s e by o x i d a t i o n of the secondary hydroxylamine (17) under simulated b i l e workup c o n d i t i o n s ( S l a t t e r , 1983). Hydroxylamine m e t a b o l i t e s of s t r u c t u r a l l y r e l a t e d a r y l a l i p h a t i c amines are known to be gl u c u r o n i d e conjugated. C. The aval I abl e evi dence from the study of the recipavrin formamide metabolite favors the metabolic sequence: Recipavrin (9), norrecipavrin (15), secondary hydroxylamine (17), secondary hydroxylamine glucuronide (42). Upon ^-glucuronidase hydrolysis, the unstable hydroxylamine (17) could be oxidized to nitrone (24) and then converted to formamide (12) either by Beckmann rearrangement during isolation or by isomerization in t he GC i nlet . 4. METABOLISM OF ARYLALIPHATIC FORMAMIDES. A. The in vivo b i l i a r y and u r i n a r y m e t a b o l i t e s of the t e r t i a r y a r y l a l i p h a t i c formamide (—)-N-methyl-N-(1-methyl-3,3-d i p h e n y l p r o p y l ) formamide (26) from male w i s t a r r a t s have been c h a r a c t e r i z e d by GCMS. i . In u r i n e , nonconjugated m e t a b o l i t e s i n c l u d e d a ketone (14) and secondary amine (15). ^-Glucuronidase treatment l i b e r a t e d the ketone (14), a secondary a l c o h o l (97), a keto-oxime (19), a c a r b i n o l a m i d e (47), and i t s decomposition product, the secondary formamide (12), p h e n o l i c analogues of the ketone (87), oxime (89), and t e r t i a r y formamide (105) and 0-297 methylcatechol analogues of the ketone (91), a secondary a l c o h o l (92), the oxime (93), secondary formamide (109) and t e r t i a r y formamide (108). i i . In b i l e , compounds 12,19,26,47,87,89,91 and 97 were present as noncon jugated metabolites . /3-Glucuron idase l i b e r a t e d a phenol analogue of the secondary formamide (106) and a carbinolamide (111). A l l of the previous ly l i s t e d compounds except the secondary amine (15) were also detected. i i i . T r i m e t h y l s i l y l a t i o n of the conjugated b i l e f r a c t i o n revealed two add i t iona l compounds, 107 and 110 which were der ived from phenolic and O-methylcatechol analogues of the carbinolamide (47). B. The in vivo b i l i a r y and urinary metabolites of the secondary a r y l a l i p h a t i c formamide ( - ) -N-(1-methyl -3 ,3-diphenylpropyl ) formamide (12) have been character ized by GCMS in male wistar r a t s . i . Secondary formamide (12) was metabolized mainly by deformylat ion to norrec ipavr in (15). The carbinolamide (111) was charac ter i zed in 0 -glucuronidase-hydrolyzed b i l e ex trac t s . C. 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