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UBC Theses and Dissertations

The metabolic profiling of clobazam in rats Borel, Anthony Gerard Francis 1990

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THE METABOLIC PROFILING OF CLOBAZAM IN RATS by ANTHONY GERARD FRANCIS BOREL B.Sc. (Hons.)/ U n i v e r s i t y of Guelph, 1985 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Fa c u l t y of Pharmaceutical Sciences 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 t o the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1990 (C) ANTHONY BOREL, 1990 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. Faculty ;P p^ei)tNMiXC( of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada D a t e April 27, 1990 DE-6 (2/88) i i ABSTRACT Clobazam (CLBZ) i s a 1 , 5 - b e n z o d i a z e p i n e w i t h p o t e n t a n t i c o n v u l s a n t a c t i v i t y . The m e t a b o l i s m o f t h i s d r u g was i n v e s t i g a t e d i n t h e r a t and in vitro w i t h t h e a i d o f s t a b l e i s o t o p e - l a b e l l e d a n a l o g u e s and gas chromatography-mass s p e c t r o s c o p y (GCMS). P e n t a d e u t e r i o p h e n y l CLBZ [ 2Hs]CLBZ was s y n t h e s i z e d i n e s s e n t i a l l y q u a n t i t a t i v e i s o t o p i c p u r i t y , and c h a r a c t e r i z e d by ^H-NMR and GCMS. Of t h e f i v e s t e p s i n v o l v e d i n t h e s y n t h e s i s o f [ 2Hs]CLBZ, t h e most s u s c e p t i b l e t o d e u t e r i u m exchange was t h e n u c l e o p h i l i c s u b s t i t u t i o n o f 2,4-d i c h l o r o n i t r o b e n z e n e by a n i l i n e - d 7 t o form N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l amine 18. In t h i s s t e p , t h e i s o t o p i c i m p u r i t y a n i l i n e - 2 , 3 , 4 , 5 , - d s i n t r o d u c e d p r o t o n s from n i t r o g e n i n t o t h e ortho and para p o s i t i o n s o f t h e d e u t e r i o p h e n y l r i n g o f 18. Phenol and c a t e c h o l m e t a b o l i t e s o f CLBZ and N-de s m e t h y l c l o b a z a m (DMC) were s y n t h e s i z e d a c c o r d i n g t o t h e method used f o r [ 2 Hs]CLBZ u s i n g 4-m e t h o x y a n i l i n e and 3 , 4 - d i m e t h o x y a n i l i n e as s t a r t i n g m a t e r i a l s . The methyl e t h e r p r o t e c t i n g group was w e l l - s u i t e d f o r t h e s y n t h e t i c p r o c e d u r e and was c l e a n l y removed under m i l d c o n d i t i o n s w i t h BBr3 t o a f f o r d t h e p h e n o l s and c a t e c h o l s i n good y i e l d . The O-methylated c a t e c h o l s o f CLBZ and DMC were e n z y m a t i c a l l y s y n t h e s i z e d from t h e c a t e c h o l a n a l o g u e s o f CLBZ and DMC u s i n g r a t l i v e r c y t o s o l as a s o u r c e o f c a t e c h o l 0 - m e t h y l t r a n s f e r a s e (COMT) and S-a d e n o s y l - L - m e t h i o n i n e as t h e methyl donor. A meta/para 0 - m e t h y l a t i o n r a t i o o f 2 was o b t a i n e d from t h e CLBZ c a t e c h o l . C o n d e n s a t i o n o f fo r m a l d e h y d e w i t h DMC c a t a l y z e d by K2CO3 produced a compound whose GCMS ( E I ) s p e c t r a l p r o p e r t i e s were c o n s i s t e n t w i t h t h e c a r b i n o l a m i d e , however, t h i s compound c o u l d n ot be i s o l a t e d because o f i t s i i i m a r g i n a l s t a b i l i t y . A major p r o d u c t f o r t h i s r e a c t i o n was 3-hydroxymethyl DMC w h i c h was c h a r a c t e r i z e d by ^-NMR and LCMS. Formaldehyde c o n d e n s a t i o n c a t a l y z e d by KOH a l s o a f f o r d e d 3-hydroxymethyl DMC. Whether f o r m a l d e h y d e a d d i t i o n a t t h e 3 - p o s i t i o n o c c u r s as a k i n e t i c p r o d u c t o r whether i t a r i s e s as a r e s u l t o f an e q u i l i b r i u m p r o c e s s subsequent t o N - h y d r o x y m e t h y l a t i o n r e m a i n s t o be r e s o l v e d . R a t s were a d m i n i s t e r e d CLBZ:[ 2H5]CLBZ as an a p p r o x i m a t e 50:50 m i x t u r e . I s o t o p e s h i f t s d e t e c t e d by GCMS a l l o w e d t h e f o l l o w i n g CLBZ m e t a b o l i t e s t o be i d e n t i f i e d . In b i l e , d e c r e a s i n g l e v e l s o f 4'-hydroxy CLBZ, 4'-hydroxy DMC, O-methylated CLBZ c a t e c h o l s , 4'-hydroxy-3'-methoxy DMC and 3 ' , 4 ' - d i h y d r o x y CLBZ appeared as both g l u c u r o n i d e and s u l f a t e c o n j u g a t e s . In u r i n e d e c r e a s i n g l e v e l s o f t h e f o l l o w i n g s u l f a t e c o n j u g a t e s were o b s e r v e d : 4'-hydroxy-3'-methoxy CLBZ, 4'-hydroxy CLBZ, 4'-hydroxy DMC and 3 ' , 4 ' - d i h y d r o x y CLBZ. In b i l e , t h e g l u c u r o n i d e s were predominant o v e r t h e s u l f a t e s , whereas i n u r i n e o n l y s u l f a t e c o n j u g a t e s were d e t e c t e d . There was no d e t e c t a b l e e v i d e n c e o f u n c o n j u g a t e d CLBZ m e t a b o l i t e s i n b i l e o r u r i n e . As t h e b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e , 3'-hydroxy-4'-methoxy CLBZ c o n s t i t u t e d <2 % o f t h e O-methylated CLBZ c a t e c h o l s , whereas i n b i l e , t h e s u l f a t e c o n j u g a t e o f t h i s m e t a b o l i t e c o n s t i t u t e d 30 %. One p o s s i b l e e x p l a n a t i o n f o r t h e h i g h e r l e v e l s o f t h i s m e t a b o l i t e as a b i l i a r y s u l f a t e c o u l d be t h e low r e g i o s p e c i f i c m e t h y l a t i o n o f d i h y d r o x y CLBZ by COMT combined w i t h a r y l s u l f a t a s e a c t i v i t y p r e s e n t i n t h e k i d n e y . N e i t h e r t h e CLBZ c a r b i n o l a m i d e n or t h e N-hydroxy DMC (h y d r o x a m i c a c i d ) was p r e s e n t i n d e t e c t a b l e l e v e l s i n t h e c o n j u g a t e d o r u n c o n j u g a t e d f r a c t i o n s o f b i l e o r u r i n e . I s o t o p e e f f e c t s i n t h e m e t a b o l i s m o f CLBZ and DMC i n t h e r a t were m a r g i n a l f o r phenyl r i n g o x i d a t i o n as measured f o r phenol m e t a b o l i t e s i v ( k H / k D =1.103 ± .090) and f o r c a t e c h o l s (kL|/kn. = 1.088 ± .207). The m e t a b o l i s m o f CLBZ and DMC in vitro was examined u s i n g c o n t r o l and p h e n o b a r b i t a l - i n d u c e d r a t l i v e r microsomes. CLBZ m e t a b o l i s m r e s u l t e d i n N - d e m e t h y l a t i o n t o DMC and phenyl r i n g h y d r o x y l a t i o n a t t h e 4 ' - p o s i t i o n . There was no d e t e c t a b l e e v i d e n c e f o r t h 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 . I n d u c t i o n appeared t o i n c r e a s e N - d e m e t h y l a t i o n compared t o phenyl r i n g h y d r o x y l a t i o n . DMC was m e t a b o l i z e d t o 4'-hydroxy DMC w i t h no d e t e c t a b l e p r o d u c t i o n o f t h e hydroxamic a c i d . The i s o t o p e e f f e c t a s s o c i a t e d w i t h CLBZ N - d e m e t h y l a t i o n was d e t e r m i n e d by i n c u b a t i n g an a p p r o x i m a t e 50:50 m i x t u r e o f p e n t a d e u t e r i o p h e n y l C L B Z : t r i d e u t e r i o m e t h y l CLBZ w i t h c o n t r o l and p h e n o b a r b i t a l - i n d u c e d r a t l i v e r microsomes. In t h i s way, t h e s o u r c e o f DMC d e r i v e d from d e m e t h y l a t i o n ([ 2H5]DMC) and d e d e u t e r i o m e t h y l a t i o n (DMC) c o u l d be d e t e r m i n e d . The v a l u e s o f ku| /krj, d e t e r m i n e d by p r o t i o / d e u t e r i o p r o d u c t r a t i o s , were c a l c u l a t e d a t 3.65 ± 0.27 (n = 3) and 2.80 ± 0.16 (n = 4) f o r c o n t r o l and i n d u c e d microsomes r e s p e c t i v e l y . V TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS v LIST OF FIGURES v i i i LIST OF SCHEMES x i i i LIST OF TABLES xv LIST OF ABBREVIATIONS x v i ACKNOWLEDGEMENTS x i x DEDICATION xx I INTRODUCTION 1 1. THE MECHANISM OF ACTION OF CLOBAZAM 2 2. THE THERAPEUTIC STATUS OF CLOBAZAM 4 (A) A n t i c o n v u l s a n t E f f i c a c y o f CLBZ 4 (B) R e s t r i c t i o n s t o CLBZ Therapy 5 (C) N-desmethylclobazam: The A c t i v e CLBZ M e t a b o l i t e 6 (D) The P h a r m a c o k i n e t i c s o f CLBZ 7 (E) Drug I n t e r a c t i o n s w i t h CLBZ 8 (F) A n a l y t i c a l T e c h n i q u e s used i n The M o n i t o r i n g o f CLBZ and DMC 8 3. THE METABOLISM OF CLOBAZAM 9 (A) The N - d e m e t h y l a t i o n o f CLBZ 13 (B) The N - o x i d a t i o n o f DMC 17 (C) The A r o m a t i c H y d r o x y l a t i o n o f CLBZ 21 4. THE OBJECTIVES OF THE THESIS 25 I I EXPERIMENTAL 26 1. CHEMICALS AND MATERIALS 26 2. ANIMALS AND SURGICAL EQUIPMENT 28 v i 3. ACCESSORY EQUIPMENT 29 4. INSTRUMENTATION 29 (A) NMR S p e c t r o s c o p y 29 (B) Mass S p e c t r o s c o p y 30 (C) I n f r a r e d S p e c t r o s c o p y 33 (D) M e l t i n g P o i n t 33 (E) U l t r a v i o l e t S p e c t r o s c o p y 33 (F) High P r e s s u r e L i q u i d Chromatography 33 (G) L i q u i d Chromatography-Mass S p e c t r o s c o p y 34 5. METABOLISM EXPERIMENTS 34 (A) Dosage Regimen 34 (B) S u r g e r y and C o l l e c t i o n o f U r i n e and B i l e 35 (C) P r e p a r a t i o n o f B i l e and U r i n e f o r GCMS 36 (D) P r e p a r a t i o n and A n a l y s i s o f L i v e r F r a c t i o n s 36 (E) M i c r o s o m a l I n c u b a t i o n s 38 6. CHEMICAL SYNTHESES 40 (A) Medium p r e s s u r e l i q u i d ( F l a s h ) chromatography 40 (B) S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l c l o b a z a m ( [ 2 H 5 ] C L B Z ) 40 (C) S y n t h e s i s o f T r i d e u t e r i o m e t h y l c l o b a z a m ( [ 2 H 3 ] C L B Z ) 45 (D) S y n t h e s i s o f R i n g - O x i d i z e d M e t a b o l i t e s o f CLBZ 46 (E) S y n t h e s i s o f N-hydroxymethyl CLBZ ( c a r b i n o l a m i d e ) 56 (F) A t t e m p t e d S y n t h e s e s o f N-hydroxy DMC (hy d r o x a m i c a c i d ) 59 I I I RESULTS AND DISCUSSION 62 1. THE SYNTHESIS OF DEUTERATED ANALOGUES OF CLBZ AND CLBZ METABOLITES 62 (A) The S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l CLBZ 62 (B) The S y n t h e s i s o f T r i d e u t e r i o m e t h y l CLBZ 84 (C) The S y n t h e s i s o f Phenol and C a t e c h o l M e t a b o l i t e s o f CLBZ 86 (D) GCMS A n a l y s i s o f CLBZ and I t s M e t a b o l i t e s 91 (E) S y n t h e s i s o f N-hydroxymethyl CLBZ ( c a r b i n o l a m i d e ) 104 (F) A t t e m p t e d S y n t h e s e s o f N-hydroxy DMC (hy d r o x a m i c a c i d ) 123 2. THE METABOLISM OF CLBZ 127 v i i (A) Phenol and C a t e c h o l M e t a b o l i t e s o f CLBZ i n Rat B i l e and U r i n e 127 (B) M e t a b o l i c F o r m a t i o n o f The P u t a t i v e C a r b i n o l a m i d e 146 (C) M e t a b o l i c F o r m a t i o n o f The P u t a t i v e N-hydroxy DMC (h y d r o x a m i c a c i d ) 148 (D) The I n f l u e n c e o f Cytochrome P-450 I n d u c t i o n on CLBZ M e t a b o l i s m 149 (E) The D e u t e r i u m I s o t o p e E f f e c t a s s o c i a t e d w i t h CLBZ N-d e m e t h y l a t i o n 153 IV SUMMARY AND CONCLUSIONS 154 V REFERENCES 156 VI APPENDIX 174 v i i i LIST OF FIGURES F i g u r e 1. 400 MHz !H-NMR o f [ 2 H 5 ] C L B Z . 66 F i g u r e 2. E l e c t r o n impact mass s p e c t r a o f (A) CLBZ and (B) [ 2 H 5 ] C L B Z . 67 F i g u r e 3. 400 MHz !H-NMR s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a - d e u t e r i o p h e n y l a m i n e 18, and (B) N-(5-c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o - p h e n y l a m i n e 3 7 . 69 F i g u r e 4. E l e c t r o n impact mass s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18, and (B) N-(5-c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o p h e n y l a m i n e 3 7 . 70 F i g u r e 5. 75 MHz broad-band d e c o u p l e d ^C-NMR sp e c t r u m o f N-( 5 - c h l o r o - 2 - n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18. 71 F i g u r e 6. The f o r m a t i o n o f a 6-membered r i n g between t h e n i t r o g roup and amine p r o t o n o f t h e phenyl amine 18 r e s u l t s i n N-H d e s h i e l d i n g and d i m i n i s h e d D2O exchange. 74 F i g u r e 7. Thermal rearrangement o f t h e carbamoyl a c e t a t e 19 t o th e phenylamine 18 r e s u l t s i n i d e n t i c a l r e t e n t i o n t i m e s i n t h e TIC p l o t s o f (A) 19 and (B) 18. 75 F i g u r e 8. Thermal r e a r r a n g e m e n t o f t h e carbamoyl a c e t a t e 19 t o th e phenylamine 18 r e s u l t s i n i d e n t i c a l GC ( E I ) mass s p e c t r a o f (A) 19 and (B) 18. 76 F i g u r e 9. 300 MHz *H-NMR o f t h e carbamoyl a c e t a t e 19. 78 F i g u r e 10. The f o r m a t i o n o f r e s o n a n c e s p e c i e s o f 19 w h i c h r e d u c e t h e a c i d i t y o f t h e methylene p r o t o n s and so i n h i b i t k e t o - e n o l t a u t o m e r i s m . 79 F i g u r e 11. TIC p l o t s o f (A) [ 2H 5]DMC and (B) [ 2H 5]DMC-TMS show t h e improved chromatography o b t a i n e d w i t h TMS-d e r i v a t i z a t i o n . 82 F i g u r e 12. GCMS ( E I ) s p e c t r a o f (A) [ 2H 5]DMC and (B) [ 2H 5]DMC-i x TMS show t h e improvement i n mass s p e c t r o s c o p y o b t a i n e d w i t h T M S - d e r i v a t i z a t i o n . 83 F i g u r e 13. (A) 400 MHz !H-NMR and (B) GCMS ( E I ) s p e c t r a o f [ 2 H 3 ] C L B Z ( 2 2 ) . 85 F i g u r e 14. Mass chromatogram o f CLBZ 0-methyl c a t e c h o l i s o m e r s a t M +-, m/z 418. 90 F i g u r e 15. TIC p l o t s o f (A) T M S - d e r i v a t i z e d m e t a b o l i t e s t a n d a r d s 26, 28, 32 and 35 a f t e r r e c o v e r y from s p i k e d c o n t r o l b i l e . (B) 1 5 - f o l d d i l u t i o n o f (A) w i t h EtOAc. 94 F i g u r e 16. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-DMC (26) showing t h e p r e s e n c e o f M + -a t m/z 446. 95 F i g u r e 17. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-CLBZ (28) showing t h e p r e s e n c e o f M +* a t m/z 388. 95 F i g u r e 18. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3',4'-dihydroxy-DMC (32) showing t h e p r e s e n c e o f M +* a t m/z 534. 96 F i g u r e 19. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3',4'-d i h y d r o x y CLBZ (35) showing t h e p r e s e n c e o f M +' a t m/z 476. 96 F i g u r e 20. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy-CLBZ (36a) showing t h e p r e s e n c e o f M+* a t m/z 418. 97 F i g u r e 21. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3'-hydroxy-4'-methoxy-CLBZ (36b) showing t h e p r e s e n c e o f M +' a t m/z 418. 97 F i g u r e 22. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) showing t h e p r e s e n c e o f M+-a t m/z 476. 98 F i g u r e 23. HPLC o f c r u d e p r o d u c t , (A) f r e s h l y p r e p a r e d and X (B) a f t e r o v e r n i g h t s t o r a g e a t 4°C. 108 F i g u r e 24. (A) LCMS chromatogram o f t h e c r u d e p r o d u c t a f t e r o v e r n i g h t s t o r a g e a t 4 4C shows t h e appearance o f 45, 46 and 47 a t tR 4.68, 6.68 and 7.37 min r e s p e c t i v e l y . (B) LCMS ( C I ) spec t r u m o f 46 shows t h e appearance o f MH + and [M H - H 2 0 ] + a t m/z 317 and 299 r e s p e c t i v e l y . (C) LCMS ( C I ) spec t r u m o f 47 (DMC) shows t h e appearance o f MH4 + a t m/z 287 and 304 r e s p e c t i v e l y . 109 F i g u r e 25. (A) GCMS ( C o n d i t i o n , b*) TIC p l o t o f f r e s h l y p r e p a r e d , T M S - d e r i v a t i z e d c r u d e p r o d u c t . P u t a t i v e c a r b i n o l a m i d e - T M S appears a t tR 17.33 min. (B) GCMS ( E I ) mass spec t r u m o f a p p a r e n t c a r b i n o l a m i d e -TMS shows t h e p r e s e n c e o f M+- a t m/z 388. 110 F i g u r e 26. (A) GCMS ( C o n d i t i o n , b*) TIC p l o t o f p u r i f i e d 46 d e r i v a t i z e d w i t h TMS. (B) GCMS ( E I ) spec t r u m o f 46 shows t h e p r e s e n c e o f M + > a t m/z 370. 112 F i g u r e 27. 400 MHz ^-NMR spect r u m o f 47. S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e o f DMC. 115 F i g u r e 28. 400 MHz *H-NMR sp e c t r u m o f 46- S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e 3-hydroxymethyl DMC. 116 F i g u r e 29. P a r t i a l 400 MHz *H-NMR s p e c t r a o f (A) compound 40 and (B) compound 46 i n DMS0-d5 r e v e a l t h e o c c u r r e n c e o f m e t h i n e , methylene and h y d r o x y l p r o t o n s as (A) t r i p l e t s i n 40 and as (B) a t r i p l e t , d o u b l e t and broad s i n g l e t r e s p e c t i v e l y i n 46. 119 F i g u r e 30. P a r t i a l 400 MHz ^H-NMR s p e c t r a o f (A) compound 40 and (B) compound 46 i n DMSO-ds w i t h D2O show c h e m i c a l i d e n t i t y f o r t h e 3-hydroxymethyl f u n c t i o n . 120 F i g u r e 31. (A) GCMS ( C o n d i t i o n , d*) TIC p l o t o f p u r i f i e d 40. (B) GCMS ( E I ) spec t r u m o f 40 shows t h e p r e s e n c e o f M+- a t m/z 370. 121 F i g u r e 32. 400 MHz ^-NMR o f t h e M o V I complex ( 4 2 ) . 125 F i g u r e 33. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h ^ - g l u c u r o n i d a s e . F i g u r e 34. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . F i g u r e 35. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f u r i n e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . F i g u r e 36. P r o f i l e o f c o n j u g a t e d CLBZ m e t a b o l i t e s i n t h e Sprague Dawley r a t d e r i v e d from i n d i v i d u a l l y p o o l e d b i l e and u r i n e samples. F i g u r e 37. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy CLBZ (28) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+- and [ 2H4]M +* a t m/z 388 and 392 r e s p e c t i v e l y . F i g u r e 38. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy DMC (26) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2H4]M +- a t m/z 446 and 450 r e s p e c t i v e l y . F i g u r e 39. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy CLBZ (36a) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+* and [ 2H3]M +- a t m/z 418 and 421 r e s p e c t i v e l y . F i g u r e 40. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) i s o l a t e d f rom b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+* and [ 2 H 3 ] M + - a t m/z 476 and 479 r e s p e c t i v e l y . F i g u r e 41. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 3 ' , 4 ' - d i h y d r o x y CLBZ (35) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+* and [ 2H3]M +- a t m/z 476 and 479 r e s p e c t i v e l y . x i i F i g u r e 42. Mass chromatograms (m/z 418) o f T M S - d e r i v a t i z e d EtOAc e x t r a c t s o f b i l e and u r i n e showing t h e p r e s e n c e o f 0 - m e t h y l a t e d c a t e c h o l s o f CLBZ (36a+b) as (A) a b i l i a r y g l u c u r o n i d e , (B) a b i l i a r y s u l f a t e and (C) a u r i n a r y s u l f a t e . 143 F i g u r e 43. Comparison o f peak a s s i g n m e n t s o f t h e CLBZ 0-m e t h y l a t e d i s o m e r s (36a+b) formed (A) i n v i t r o and (B) as a b i l i a r y g l u c u r o n i d e . 144 F i g u r e 44. Mass chromatograms a t (A) m/z 418 and (B) m/z 421 show t h a t t h e peaks o f t h e CLBZ 0 - m e t h y l a t e d c a t e c h o l s (36a+b) o c c u r r i n g as t h e b i l i a r y s u l f a t e s a r e i s o t o p i c a l l y r e l a t e d . 145 F i g u r e 45. (A) SDS-PAGE (Laemmli e t a l . , 1970), and (B) immunoblot (Towbin e t a l . , 1979) o f l i v e r m i c r o s o m a l p r o t e i n o f Sprague Dawley r a t s . F i g u r e 46. SIM p l o t s o f microsomal CLBZ m e t a b o l i t e s . 151 152 LIST OF SCHEMES Scheme I . P u t a t i v e m e t a b o l i c pathways o f clobazam i n man, monkey and r a t . Scheme I I . Mechanisms o f p o t e n t i a l methyl t e r m i n u s t o x i f i c a t i o n o f clobazam. Scheme I I I . Mechanisms o f p o t e n t i a l phenyl t e r m i n u s t o x i f i c a t i o n o f clobazam. Scheme IV. P o s s i b l e mechanism f o r t h e o x i d a t i v e N-d e m e t h y l a t i o n o f N,N-dimethylamine by cytochome P-450. Scheme V. P o s s i b l e mechanisms f o r t h e P-450 c a t a l y z e d o x i d a t i o n o f amides. Scheme V I . M e t a b o l i c pathways i n t h e h e p a t o c a r c i n o g e n i c a c t i v a t i o n o f AAF. Scheme V I I . Pathways i n v o l v e d i n a r o m a t i c o x i d a t i o n . Scheme V I I I . F o r m a t i o n o f r e a c t i v e i n t e r m e d i a t e s from 2-h y d r o x y e s t r o g e n . Scheme IX. The s y n t h e s i s o f [^HsJCLBZ. Scheme X. Mechanism f o r t h e s e l e c t i v e i n t r o d u c t i o n o f p r o t o n s i n t o t h e d e u t e r i o p h e n y l n u c l e u s o f N - ( 5 - c h l o 2 - n i t r o p h e n y l ) - d e u t e r i o p h e n y l a m i n e 37. Scheme X I . Thermal r e a r r a n g e m e n t o f 19 t o form 18 by /J-p r o t o n a b s t r a c t i o n and e l i m i n a t i o n o f t h e n e u t r a l k e t e n e . Scheme X I I . K e t o - e n o l t a u t o m e r i s m o f 19 showing s t r o n g e q u i l i b r i u m d i s p l a c e m e n t t o t h e k e t o form. Scheme X I I I . S y n t h e s i s o f p h e n o l i c and c a t e c h o l i c m e t a b o l i t e s o f CLBZ . x i v Scheme XIV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n r e t e n t i o n o f b e n z o d i a z e p i n e n i t r o g e n . 99 Scheme XV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n l o s s o f t h e (A) N-phenyl and (B) N-methyl m o i e t i e s . 100 Scheme X V I . Proposed f r a g m e n t a t i o n o f 0-methyl c a t e c h o l s . 101 Scheme X V I I . Proposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds r e s u l t i n g i n t h e l o s s o f a methyl r a d i c a l . 101 Scheme X V I I I . Proposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds 20, 32 and 33 r e s u l t i n g i n l o s s o f t h e N-phenyl r i n g . 102 Scheme XIX. E q u l i b r i u m c o n d e n s a t i o n between an amide and fo r m a l d e h y d e . 104 Scheme XX. The c o n d e n s a t i o n o f form a l d e h y d e w i t h DMC i l l u s t r a t e s a p r o t o t y p e b a s e - c a t a l y z e d f o r m a t i o n o f a c a r b i n o l a m i d e . 104 Scheme X X I . (A) F r a g m e n t a t i o n o f T M S - d e r i v a t i z e d 39 (m/z 388) shows t h e m i g r a t i o n o f TMS from t h e hyd r o x y m e t h y l t o t h e e n o l oxygen c e n t r e . (B) F r a g m e n t a t i o n o f m/z 388 r e s u l t i n g i n l o s s o f a methyl r a d i c a l . I l l Scheme X X I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC (50) by o x i d a t i o n o f DMC-TMS. 123 Scheme X X I I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC by r i n g c l o s u r e o f t h e a r y l h y d r o x y l a m i n e . 126 L I S T O F T A B L E S T a b l e 1. GCMS d a t a f o r s y n t h e t i c C L B Z - r e l a t e d compounds. T a b l e 2. GCMS d a t a f o r Phase I CLBZ m e t a b o l i t e s t y p i f i e d by b i l i a r y g l u c u r o n i d e s . M e t a b o l i t e s were e x t r a c t e d from b i l e a f t e r h y d r o l y s i s w i t h ^ - g l u c u r o n i d a s e and d e r i v a t i z e d w i t h TMS. x v i LIST OF ABBREVIATIONS amu a t o m i c mass u n i t s APT a t t a c h e d p r o t o n t e s t asymm asymmetric s t r e t c h ( i n f r a r e d ) BB broad band bp b o i l i n g p o i n t bs broad s i n g l e t BSA b o v i n e serum a l b u m i n °C d e g r e e s C e l c i u s ca c i r c a ( a p p r o x i m a t e l y ) CBZ carbamazepine CLBZ clob a z a m COMT c a t e c h o l 0 - m e t h y l t r a n s f e r a s e D d e u t e r i u m d d e u t e r i u m ( n o m e n c l a t u r e ) , d o u b l e t (NMR) dd d o u b l e t o f d o u b l e t s DMC N-desmethylclobazam DMF N,N-dimethylformamide DMSO d i m e t h y l s u l f o x i d e EI e l e c t r o n impact e q u i v e q u i v a l e n t e t a l . e t alia EtOH e t h a n o l f o d t r i s ( 6 , 6 , 7 , 7 , 8 , 8 , 8 - h e p t a f l u o r o - 2 , 2 - d i m e t h y l - 3 , 5 -o c a t a n e d i o n a t o ) FT F o u r i e r t r a n s f o r m g gram (mass), g r a v i t a t i o n a l a c c e l e r a t i o n , 9.8 m/s ( c e n t r i f u g a l a c c e l e r a t i o n ) GC gas chromatography GCMS gas chromatography-mass s p e c t r o s c o p y h hours HPLC h i g h p r e s s u r e l i q u i d chromatography Hz H e r t z i . e . /tf est ( t h a t i s ) i . p . i n t r a p e r i t o n e a l ( l y ) x v i i i b i d . ibidem ( i n t h e same p l a c e ) IR i n f r a r e d J c o u p l i n g c o n s t a n t i n Hz kg k i l o g r a m LC l i q u i d chromatography m m u l t i p l e t M + m o l e c u l a r i o n m/z mass t o c h a r g e r a t i o MeOH methanol mg m i l l i g r a m MHz mega H e r t z min m i n u t e s mL m i l l i l i t r e mp m e l t i n g p o i n t MS mass s p e c t r u m MSTFA N - m e t h y l - N - 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 NADH n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e ( r e d u c e d ) NADPH n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e phosphate ( r e d u c e d ) NMR n u c l e a r m a g n e t i c r e s o n a n c e P-450 cytochrome P-450 p-TsOH p - t o l u e n e s u f o n a t e PAGE p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s PB p h e n o b a r b i t a l p e t p e t r o l e u m PH p h e n y t o i n ppm p a r t s p e r m i l l i o n p s i pounds p e r square i n c h Rf r e l a t i v e m o b i l i t y by TLC s s i n g l e t SAM S - a d e n o s y l - L - m e t h i o n i n e SDS sodium d o d e c y l s u l f a t e SIM s e l e c t e d i o n m o n i t o r i n g s t r s t r o n g symm symmetric s t r e t c h ( i n f r a r e d ) t t r i p l e t TIC t o t a l i o n c u r r e n t TLC t h i n l a y e r chromatography TMS t e t r a m e t h y l s i l a n e (NMR), t r i m e t h y l s i l y l ( d e r i v a t i v e ) t R r e t e n t i o n t i m e v volume x i x ACKNOWLEDGEMENTS I would l i k e t o than k my s u p e r v i s o r , Dr. Frank A b b o t t f o r h i s k i n d s u p p o r t and g u i d a n c e t h r o u g h o u t t h i s p r o j e c t . I am a l s o g r a t e f u l t o t h e s t a f f o f t h e NMR and Mass S p e c t r o m e t r y F a c i l i t i e s o f The C h e m i s t r y Department, U.B.C. f o r t h e i r a s s i s t a n c e . To Sue P a n e s a r f o r a s s i s t a n c e w i t h m i c r osomal p r e p a r a t i o n s and g e l e l e c t r o p h o r e s i s . To Ron Lee and Abdul M u t l i b f o r t h e i r a s s i s t a n c e i n s u r g e r y . To Rol a n d B u r t o n f o r a s s i s t a n c e w i t h GCMS a n a l y s i s . To my a b l e c o - w o r k e r s Kelem Kassahun, Ron Lee, Abdul M u t l i b , Sue Pan e s a r and J i a o j i a o Zheng f o r t h e i r t o l e r a n c e and generous exchange o f i d e a s . To t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada and B e r l e x L a b o r a t o r i e s I n c . f o r t h e i r f i n a n c i a l s u p p o r t . To Dean M c N e i l l f o r t h e p r o v i s i o n o f a t e a c h i n g a s s i s t a n t s h i p . To Hoechst Canada I n c . f o r t h e s u p p l y o f CLBZ and DMC s t a n d a r d s . And f i n a l l y , t o my f a m i l y ( t h e hard c o r e ) f o r " b e i n g b e h i n d me a l l t h e way". XX DEDICATION TO MY FAMILY... THANKS FOR THE SUPPORT. 1 I INTRODUCTION Clobazam (CLBZ) i s a 1 , 5 - b e n z o d i a z e p i n e w h i c h was o r i g i n a l l y s y n t h e s i z e d by R o s s i e t a l . ( 1 9 6 9 ) , and f o u n d t o have p o t e n t a n t i c o n v u l s a n t a c t i v i t y ( G a s t a u t and Low, 1979). A t p r e s e n t CLBZ i s marketed i n 50 c o u n t r i e s as an a n t i e p i l e p t i c , however, i t s c l i n i c a l a p p l i c a t i o n i n Canada i s s t i l l i n v e s t i g a t i o n a l ( P o r t e r , 1989). The f o c u s o f t h i s t h e s i s i s t h e m e t a b o l i s m o f CLBZ, and i n o r d e r t o p r o v i d e a framework w i t h i n w h i c h t h e r e s e a r c h can be a p p r o p r i a t e l y p e r c e i v e d , t h i s i n t r o d u c t i o n s e e ks t o p r o v i d e some background c o n c e r n i n g t h i s d r u g . F i r s t l y , t h e mechanism whereby CLBZ e x e r t s i t s a n t i c o n v u l s a n t a c t i o n w i l l be examined. S e c o n d l y , t h e t h e r a p e u t i c s t a t u s o f CLBZ w i l l be r e v i e w e d w i t h r e g a r d t o (A) a n t i c o n v u l s a n t e f f i c a c y , (B) r e s t r i c t i o n s a s s o c i a t e d w i t h i t s u s e , (C) i t s a c t i v e N-desmethyl m e t a b o l i t e , (D) p h a r m a c o k i n e t i c s , (E) i n t e r a c t i o n w i t h o t h e r a n t i c o n v u l s a n t m e d i c a t i o n and (F) t h e a n a l y t i c a l t e c h n i q u e s used t o m o n i t o r t h e p a r e n t d r u g and i t s N-desmethyl m e t a b o l i t e . T h i r d l y , t h e m e t a b o l i s m o f CLBZ w i l l be r e v i e w e d w i t h r e g a r d t o t h e mechanisms i n v o l v e d i n i t s (A) N - d e m e t h y l a t i o n , (B) p o t e n t i a l N - o x i d a t i o n and (C) a r o m a t i c h y d r o x y l a t i o n . CLOBAZAM 2 1. THE MECHANISM OF ACTION OF CLOBAZAM. The a n t i c o n v u l s a n t a c t i o n o f b e n z o d i a z e p i n e s (BZs) i s e x e r t e d i n r e g i o n s o f t h e b r a i n l i k e t h e s u b s t a n t i a n i g r a and a r e a tempestas ( G a l e , 1989) by t h e p o t e n t i a t i o n o f n e u r o n a l t r a n s m i s s i o n i n v o l v i n g t h e i n h i b i t o r y t r a n s m i t t e r - y - a m i n o b u t y r i c a c i d (GABA) (M a c d o n a l d , 1989; P o r t e r , 1989; DeLorenzo, 1988). The a c t i v i t y o f GABA i s f o r t h e most p a r t , m o d ulated p o s t s y n a p t i c a l l y t h r o u g h an o l i g o m e r i c complex c o n s i s t i n g o f BZ, GABA and b a r b i t u r a t e r e c e p t o r s a d j a c e n t t o a c h l o r i d e i o n o p h o r e ( O l s e n , 1981). The a c t i v i t y o f l i g a n d s a t t h e i r r e s p e c t i v e r e c e p t o r s on t h e complex r e g u l a t e s t h e o p e n i n g o f t h e c h l o r i d e c h a n n e l , t h e l e v e l o f c h l o r i d e i o n i n f l u x , and c o n s e q u e n t l y t h e s t a t e o f n e u r o n a l i n h i b i t i o n . In t h e c a s e o f BZs, r e c e p t o r b i n d i n g i n c r e a s e s t h e f r e q u e n c y o f t h e c h l o r i d e c h a n n e l b e i n g g a t e d i n t h e open s t a t e and i n t h i s way CNS i n h i b i t i o n i s enhanced (Twyman e t a l . , 1989). The a n t i c o n v u l s a n t r e s p o n s e e l i c i t e d by t h e b i n d i n g o f BZs t o t h e i r r e c e p t o r r e p r e s e n t s o n l y a p a r t o f a co n t i n u u m o f a c t i v i t y evoked by BZ r e c e p t o r l i g a n d s ( G a r d n e r , 1988). Indeed, t h e r e i s a s p e c t r u m o f a n t a g o n i s t s t o " i n v e r s e a g o n i s t s " t o t h e BZ r e c e p t o r w h i c h e l i c i t from n u l l t o c o n v u l s a n t r e s p o n s e s r e s p e c t i v e l y . A t h r e e - s t a t e model was p r o p o s e d t o a c c o u n t f o r t h i s complex s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p ( P o l e e t a l . , 1982). In t h i s model, t h e BZ r e c e p t o r was vie w e d as a c o u p l i n g u n i t between t h e GABA r e c e p t o r and t h e c h l o r i d e c h a n n e l . Thus, a g o n i s t s , a n t a g o n i s t s and " i n v e r s e a g o n i s t s " r e g u l a t e t h e s t a t e o f BZ r e c e p t o r c o u p l i n g which i s r e f l e c t e d i n c h l o r i d e t r a n s p o r t b e i n g enhanced, u n a f f e c t e d o r d i m i n i s h e d r e s p e c t i v e l y . T h i s s c e n a r i o was p e r c e i v e d by E h l e r t e t a l . (1983) as an a l l o s t e r i c r e g u l a t i o n o f t h e GABA r e c e p t o r (and 3 hence c h l o r i d e c h a n n e l - o p e n i n g ) by l i g a n d s t o t h e BZ r e c e p t o r w i t h a g o n i s t s and " i n v e r s e a g o n i s t s " a c t i n g as p o s i t i v e and n e g a t i v e m o d i f i e r s r e s p e c t i v e l y . S e v e r a l r e p o r t s s u p p o r t t h e c o n c e p t o f an a l l o s t e r i c model ( S i e g h a r t , 1985; M a r t i n e t a l . , 1983; C h i u and Ro s e n b e r g , 1983), which a c c o u n t s f o r most o f t h e complex a c t i o n s o f l i g a n d s t h a t b i n d t o t h e BZ r e c e p t o r . S t u d i e s on t h e i n f l u e n c e o f s t r u c t u r e on t h e p h a r m a c o l o g i c a l a c t i v i t y o f t h e BZs have f o c u s s e d on t h e 1,4-type o f w h i c h diazepam i s o f t e n c o n s i d e r e d a p r o t o t y p e . CLBZ, on t h e o t h e r hand, i s s t r u c t u r a l l y d i f f e r e n t from diazepam i n two i m p o r t a n t a s p e c t s : ( i ) CLBZ p r e s e n t s a 1 , 5 - d i s p o s i t i o n o f N-atoms i n t h e d i a z e p i n e h e t e r o c y c l e as opposed t o a 1,4- i n d i a z e p a m , and ( i i ) CLBZ p o s s e s s e s a carboxamide f u n c t i o n a t p o s i t i o n 4 as opposed t o t h e more b a s i c i m i n e i n diazepam (Kuch, 1979). CLBZ does p o s s e s s t h e m o l e c u l a r d e t e r m i n a n t s r e q u i r e d f o r BZ a c t i v i t y , namely: ( i ) a benzo group f u s e d t o a 7-membered l a c t a m r i n g i n which t h e c a r b o n y l 0-4 o f CLBZ can be c o n s i d e r e d b i o i s o s t e r i c w i t h t h e i m i n e N-4 o f d i a z e p a m , ( i i ) p h e n y l s u b s t i t u t i o n a t C-5, ( i i i ) m e t hyl s u b s t i t u t i o n a t N - l , and ( i v ) an e l e c t r o n - w i t h d r a w i n g h y d r o p h o b i c s u b s t i t u e n t a t p o s i t i o n 7 ( S t e r n b a c h , 1 9 7 3 ) . In t h i s r e g a r d , CLOBAZAM DIAZEPAM 4 SAR models used t o a c c o u n t f o r t h e a c t i v i t y o f 1,4-BZs s h o u l d by and l a r g e accommodate CLBZ. 2. THE THERAPEUTIC STATUS OF CLOBAZAM. (A) A n t i c o n v u l s a n t E f f i c a c y o f CLBZ. The f i r s t t h e r a p e u t i c a p p l i c a t i o n f o r CLBZ was i n t h e t r e a t m e n t o f a n x i e t y ( R o b e r t s o n , 1986), but t h i s d r u g was s u b s e q u e n t l y found t o be an e f f e c t i v e a n t i c o n v u l s a n t ( G a s t a u t and Low, 1979). In t h e i r t r e a t m e n t o f 103 p a t i e n t s w i t h p r i m a r y g e n e r a l i z e d , s e c o n d a r y g e n e r a l i z e d and p a r t i a l e p i l e p s i e s G a s t a u t and Low (1979) o b s e r v e d an u n p r e c e d e n t e d a n t i c o n v u l s a n t c o n t r o l i n 74 p a t i e n t s (72 % ) , o f whom 29 (28 %) were r e n d e r e d s e i z u r e -f r e e . S e v e r a l o t h e r c l i n i c a l i n v e s t i g a t i o n s a t t e s t t o t h e e f f e c t i v e n e s s o f CLBZ i n a n t i c o n v u l s a n t t h e r a p y ( S c o t t and M o f f e t t , 1988; Munn e t a l . , 1988; C a l l a g h a n and G o g g i n , 1988; K i l p a t r i c k e t a l . , 1 987), e s p e c i a l l y i n t h e t r e a t m e n t o f s e c o n d a r y g e n e r a l i z e d e p i l e p s y ( G a s t a u t , 1981b), complex p a r t i a l s e i z u r e s (Koeppen e t a l . , 1987), and s t a r t l e - i n d u c e d and i n f a n t i l e spasms ( F a r r e l l , 1986). A p a r t from i t s broad a n t i c o n v u l s a n t s p e c t r u m , CLBZ o f f e r s a d d i t i o n a l t h e r a p e u t i c b e n e f i t s i n c l u d i n g a r a p i d o n s e t o f a c t i o n , o r a l e f f e c t i v e n e s s i n c h r o n i c e p i l e p s y and a h i g h t h e r a p e u t i c i n d e x ( G a s t a u t , 1981a). The a n t i c o n v u l s a n t e f f e c t i v e n e s s o f CLBZ has a l s o been d e m o n s t r a t e d i n d i f f e r e n t a nimal s p e c i e s . In t h e c a s e o f a n i m a l s , t h e c o n d i t i o n s o f g r and mal and p e t i t mal g e n e r a l i z e d e p i l e p s y have been mimicked w i t h t h e use o f k i n d l i n g and p e n t y l e n e t e t r a z o l e i n f u s i o n r e s p e c t i v e l y ( G a s t a u t , 1981b). The s u c c e s s f u l use o f CLBZ has been r e p o r t e d i n t h e s e i z u r e 5 c o n t r o l o f k i n d l e d r a t s (Young e t a l . , 1988; V a j d a e t a l . , 1987), and p e n t y l e n e t e t r a z o l e i n f u s e d mice ( F e e l y e t a l . , 1986; Gent e t a l . , 1985; Ha i g h e t a l . , 1984). (B) R e s t r i c t i o n s t o CLBZ Therapy. R e l a t i v e l y m i l d s i d e e f f e c t s o f s e d a t i o n , m u s c l e weakness and a t a x i a were o b s e r v e d by G a s t a u t and Low (1979) i n t h e i r t r e a t m e n t o f p a t i e n t s w i t h CLBZ. A ma j o r drawback, however, was t h e development o f t o l e r a n c e i n 33 % o f t h e i r p a t i e n t p o p u l a t i o n . The development o f t o l e r a n c e t e n d s t o r e p r e s e n t a common d e n o m i n a t o r among c l i n i c a l i n v e s t i g a t i o n s , however, t h e o c c u r r e n c e o f t h i s phenomenon i s v a r i a b l e . Of t h e 22 open s t u d i e s r e v i e w e d by R o b e r t s o n ( 1 9 8 6 ) , t h e i n c i d e n c e o f t o l e r a n c e r a n g e d from 0 t o 86 %, w i t h a mean o c c u r r e n c e o f 36 %. S c o t t and M o f f e t t (1988) r e p o r t e d " g r e a t b e n e f i t " f o r 13 (43 %) o f 30 p a t i e n t s w i t h c h r o n i c i n t r a c t a b l e e p i l e p s y f o r up t o 3 y e a r s , F e e l y and G i b s o n (1984) r e p o r t e d c o m p l e t e a v o i d a n c e o f t o l e r a n c e w i t h i n t e r m i t t e n t CLBZ t h e r a p y i n c a t e m e n i a l e p i l e p s y , and F a r r e l l (1986) o b s e r v e d a r e t r i e v a l o f a n t i c o n v u l s a n t c o n t r o l w i t h an i n c r e a s e d dosage i n p e d i a t r i c p a t i e n t s who had d e v e l o p e d p a r t i a l t o l e r a n c e . On t h e o t h e r hand, K i l p a t r i c k e t a l . (1987) o b s e r v e d r a p i d l o s s o f a n t i c o n v u l s a n t p o t e n c y i n 8 o f 11 p a t i e n t s (72 % ) , and Munn e t a l . ( 1 9 8 8 ) , u n l i k e F a r r e l l ( 1 9 8 6 ) , were u n a b l e t o r e s t o r e t h e l o s s o f a n t i c o n v u l s a n t a c t i v i t y by i n c r e a s i n g t h e dosage w i t h i n t h e i r p e d i a t r i c g r o u p . The mechanism by wh i c h t o l e r a n c e t o BZs o c c u r s r e m a i n s u n c l e a r . S e v e r a l w o r k e r s ( F e e l y e t a l . , 1989; H a i g h and F e e l y , 1988; B o a s t and G e r h a r d t , 1987) have p e r c e i v e d t h i s phenomenon t o o c c u r a t a c e l l u l a r l e v e l w h e r e i n a f u l l a g o n i s t t o t h e BZ r e c e p t o r p r e c i p i t a t e s t h e gamut o f 6 r e c e p t o r a s s o c i a t e d a c t i v i t y , namely, t h e a n t i c o n v u l s a n t r e s p o n s e , t o l e r a n c e and a s s o c i a t e d s i d e e f f e c t s , whereas an a p p r o p r i a t e p a r t i a l a g o n i s t t a r g e t s t h e r e c e p t o r more s e l e c t i v e l y f o r b e n e f i c i a l a c t i v i t y . In summary, t h e development o f t o l e r a n c e appears t o be an i d i o s y n c r a t i c phenomenon w i t h i n t h e p a t i e n t p o p u l a t i o n w i t h an u n p r e d i c t a b l e t i m e o f o c c u r r e n c e . (C) N - d e s m e t h y l clobazam: The A c t i v e CLBZ M e t a b o l i t e . The p r i m a r y m e t a b o l i c r o u t e f o r CLBZ i n man and mice i s N-d e m e t h y l a t i o n t o N-desmethylclobazam (DMC) ( G a r a t t i n i , 1985; D a v i e s , 1985) w h i c h a l t h o u g h p h a r m a c o l o g i c a l l y a c t i v e , i s 2.4- t o 7 - f o l d l e s s p o t e n t t h a n t h e p a r e n t d r u g (Koeppen, 1985; F i e l d i n g and Hoffmann, 1979). D e s p i t e i t s l o w e r a n t i c o n v u l s a n t p o t e n c y , DMC o f f e r s s p e c i f i c a dvantages o v e r CLBZ. ( i ) DMC i s more s e l e c t i v e i n i t s a c t i o n t h a n CLBZ, a f f o r d i n g an enhanced d i f f e r e n t i a t i o n between a n t i c o n v u l s a n t a c t i v i t y and s e d a t i v e s i d e - e f f e c t ( F i e l d i n g and Hoffman, 1979). ( i i ) B e t t e r c o r r e l a t i o n e x i s t s f o r DMC between plasma ( H a i g h e t a l . , 1984) and b r a i n ( C a c c i a e t a l . , 1980a + b) l e v e l s i n mice and a c t i v i t y a g a i n s t p e n t y l e n e t e t r a z o l e i n d u c e d s e i z u r e s , w h i c h i s i n a c c o r d a n c e w i t h c l i n i c a l f i n d i n g s w h i c h d e m o n s t r a t e s u p e r i o r a n t i c o n v u l s a n t a c t i v i t y b e i n g d e r i v e d w i t h more r a p i d m e t a b o l i s m o f CLBZ t o DMC (G o g g i n and C a l l a g h a n , 1985). ( i i i ) The development o f t o l e r a n c e i n mice i s much l e s s pronounced w i t h DMC ( H a i g h e t a l . , 1987). In f a c t , i t seems l i k e l y t h a t a c u t e t o l e r a n c e t o CLBZ i s l a r g e l y p r e c i p i t a t e d by t h e p a r e n t d r u g ( F e e l y e t a l . , 1986). ( i v ) The s t e a d y - s t a t e h a l f - l i f e o f DMC i n monotherapy (42 h) ( P u l l a r e t a l . , 1987) i s l o n g e r t h a n t h a t o f CLBZ (34 h) ( G r e e n b l a t t e t a l . , 1981) wh i c h s u g g e s t s t h a t t h e m e t a b o l i t e c o u l d 7 p r o v i d e more e f f e c t i v e a n t i c o n v u l s a n t c o n t r o l i n c h o n i c t h e r a p y . (D) The P h a r m a c o k i n e t i c s o f CLBZ. The p h a r m a c o k i n e t i c s o f CLBZ i s s u b j e c t t o c o n s i d e r a b l e i n t e r - s u b j e c t v a r i a b i l i t y w h i c h c o u l d be i n f l u e n c e d by age, s e x , and c o n c o m i t a n t m e d i c a t i o n (Bun e t a l . , 1985; G r e e n b l a t t e t a l . , 1981; T e d e s c h i e t a l . , 1981). CLBZ i s r a p i d l y a b s o r b e d a f t e r o r a l d o s i n g and peak plasma c o n c e n t r a t i o n s a r e u s u a l l y a c h i e v e d i n 0.5 t o 2.5 h ( G r e e n b l a t t e t a l . , 1981), b u t c o u l d be as l o n g as 4 h (Rupp e t a l . , 1 9 7 9 ) . G r e e n b l a t t e t a l . (1981) s t u d i e d t h e i n f l u e n c e o f age and sex on t h e p h a r m a c o k i n e t i c s o f CLBZ a f t e r a 20 mg o r a l dose. CLBZ was bound t o plasma t o t h e e x t e n t o f 85 % to 91.4 %, and b i n d i n g t o plasma p r o t e i n d e m o n s t r a t e d no c o r r e l a t i o n w i t h age o r s e x . On t h e o t h e r hand, b o t h age and sex i n f l u e n c e d d i s t r i b u t i o n (Vd) wh i c h was h i g h e r i n women and i n c r e a s e d w i t h age i n bo t h s e x e s . T h i s r e l a t i o n s h i p was c o n s i d e r e d a r e f l e c t i o n o f t h e i n c r e a s e d c o n t r i b u t i o n to t o t a l body w e i g h t o f f a t ( i n women and i n t h e e l d e r l y ) i n t o w h i c h the l i p o p h i l i c CLBZ c o u l d be d i s t r i b u t e d t h e r e b y i n c r e a s i n g Vd. Age and sex a l s o i n f l u e n c e d e l i m i n a t i o n and c l e a r a n c e . Whereas i n young s u b j e c t s t h e l a r g e e l i m i n a t i o n h a l f - l i f e i n women (18 - 46 h) compared t o men (11 - 23 h) was a r e f l e c t i o n o f t h e g r e a t e r d i s t r i b u t i o n (Vd) i n women, t h e h a l f -l i f e i n o l d e r males (29 - 77 h) was s i g n i f i c a n t l y l o n g e r t h a n i n t h e i r young c o u n t e r p a r t s (11 - 23 h ) . More e x t e n s i v e m e t a b o l i s m i n t h e younger s u b j e c t s c o u l d p r o b a b l y a c c o u n t f o r t h i s p a t t e r n ( T e d e s c h i e t a l . , 1981). 8 (E) Drug I n t e r a c t i o n s w i t h CLBZ. I n t e r a c t i o n w i t h drugs c o n c o m i t a n t l y used i n a n t i c o n v u l s a n t t h e r a p y a l s o i n f l u e n c e s t h e p h a r m a c o k i n e t i c s o f CLBZ. Thus, Cano e t a l . (1981) o b s e r v e d a d r a m a t i c d e c r e a s e i n CLBZ plasma l e v e l s w i t h a c o r r e s p o n d i n g e l e v a t i o n i n DMC when carbamazepine (CBZ) o r p h e n y t o i n (PH) were c o n c o m i t a n t l y a d m i n i s t e r e d . T h i s phenomenon was c o n s i d e r e d t o be t h e r e s u l t o f i n d u c t i o n o f CLBZ m e t a b o l i s m . However, e l e v a t e d DMC l e v e l s a l s o o c c u r r e d w i t h c o n c o m i t a n t CBZ o r PH a d m i n i s t r a t i o n when DMC was used as t h e p a r e n t d r u g ( P u l l a r e t a l . , 1987). T h e r e f o r e , i t i s a p p a r e n t t h a t t h e i n d u c t i o n o f CLBZ m e t a b o l i s m a c c o u n t s o n l y i n p a r t f o r t h e i n c r e a s e o f DMC plasma l e v e l s , and o t h e r mechanisms c o u l d be o p e r a t i v e . Drug i n t e r a c t i o n a l s o e x i s t s between CLBZ and v a l p r o a t e (VPA) (Cocks e t a l . , 1985). A d d i t i o n o f CLBZ t o t h e t h e r a p y o f e p i l e p t i c p a t i e n t s s t a b i l i z e d on VPA r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n VPA plasma l e v e l s . To a c c o u n t f o r t h i s , i t was p r o p o s e d t h a t VPA m e t a b o l i s m c o u l d be i n h i b i t e d by CLBZ. (F) A n a l y t i c a l T e c h n i q u e s used i n The M o n i t o r i n g o f CLBZ and DMC. A n a l y t i c a l t e c h n i q u e s used i n t h e t h e r a p e u t i c m o n i t o r i n g o f CLBZ and DMC i n c l u d e HPLC ( D u s c i e t a l . , 1987; Tom a s i n i e t a l . , 1985; B r a c h e t -L i e r m a i n e t a l . , 1982), and GC w i t h n i t r o g e n - s p e c i f i c (Pena, 1986), e l e c t r o n - c a p t u r e (EC) (Badcock and Z o a n e t t i , 1987; Douse, 1984; R i v a e t a l . , 1981) and mass s p e c t r o s c o p i c ( D r o u e t - C o a s s o l o e t a l . , 1989) d e t e c t i o n . Each o f t h e r e p o r t e d methods was s u f f i c i e n t l y s e n s i t i v e t o d e t e c t CLBZ and DMC l e v e l s a t t h e i r u s u a l t h e r a p e u t i c serum l e v e l s o f 50 - 300 ng/mL and 800 - 4000 ng/mL r e s p e c t i v e l y . However, GC w i t h EC d e t e c t i o n a f f o r d e d t h e 9 h i g h e s t s e n s i t i v i t y , and l e v e l s o f d e t e c t i o n o f 2 ng/mL and 4 ng/mL f o r CLBZ and DMC r e s p e c t i v e l y have been a c h i e v e d ( B a d c o c k and Z o a n e t t i , 1987). 3. THE METABOLISM OF CLOBAZAM. There a r e few r e p o r t s i n t h e l i t e r a t u r e c o n c e r n i n g t h e m e t a b o l i s m o f t h e 1,5-BZs, and o f t h e s e , t h e m e t a b o l i c p r o f i l i n g o f CLBZ ( V o l z e t a l . , 1979) (Scheme I ) and t r i f l u b a z a m ( A l t o n e t a l . , 1975a + b) i n man and i n d i f f e r e n t animal s p e c i e s a f f o r d t h e most e l a b o r a t e a c c o u n t s . Because o f t h e c arboxamide f u n c t i o n a t t h e 4 , 5 - p o s i t i o n , CLBZ i s not s u s c e p t i b l e t o 3 - h y d r o x y l a t i o n o r r i n g o p e n i n g l i k e diazepam ( C a l d w e l l , 1985; Kuch 1979). I n s t e a d , m e t a b o l i s m o c c u r s w i t h t h e f o r m a t i o n o f N-d e s m e t h y l c l o b a z a m (DMC) and t h e 4 ' - h y d r o x y , 4'-hydroxy-3'-methoxy and 3 ' , 4 ' - d i h y d r o d i o l m e t a b o l i t e s o f b o t h CLBZ and DMC ( V o l z e t a l . , 1979) (Scheme I ) . A l t h o u g h m e t a b o l i c p r o c e s s e s g e n e r a l l y d e t o x i f y x e n o b i o t i c s , i t i s now w e l l known t h a t m e t a b o l i s m ca n p a r a d o x i c a l l y p o t e n t i a t e t h e t o x i c i t y o f t h e p a r e n t compound. D e s c r i b e d i n Scheme I a r e p o t e n t i a l mechanisms f o r CLBZ t o x i f i c a t i o n o c c u r r i n g e i t h e r a t t h e m e t h y l t e r m i n u s (Scheme I I ) o r TRIFLUBAZAM 10 p h e n y l t e r m i n u s (Scheme I I I ) . In t h i s r e g a r d , t h e f o l l o w i n g b i o t r a n s f o r m a t i o n s w i l l be examined f r o m a t o x i c o l o g i c a l p e r s p e c t i v e : (A) N - d e m e t h y l a t i o n (Scheme I I ) , (B) N - o x i d a t i o n (Scheme I I ) , and (C) A r o m a t i c h y d r o x y l a t i o n (Scheme I I I ) . OH OCH3 OH OCH. Scheme I . P u t a t i v e m e t a b o l i c pathways o f c l o b a z a m i n man, monkey and r a t ( V o l z e t a l . , 1979). 11 Scheme I I . Mechanisms o f p o t e n t i a l methyl t e r m i n u s t o x i f i c a t i o n o f clobazam. A: P-450 o x i d a t i o n t o c a r b i n o l a m i d e ; B: A m i n o m e t h y l a t i o n o f i n t r a c e l l u l a r n u c l e o p h i l e s ; C: R e l e a s e o f t o x i c f o r m a l d e h y d e ; D: P-450 o x i d a t i o n t o hydroxamic a c i d ; E: B i n d i n g t o i n t r a c e l l u l a r n u c l e o p h i l e s e.g. g l u t a t h i o n e (GSH). 12 B i n d i n g t o l i v e r p r o t e i n s OH OH Scheme I I I . Mechanisms o f p o t e n t i a l phenyl t e r m i n u s t o x i f i c a t i o n o f c l o b a z a m . F: P-450 o x i d a t i o n t o arene e p o x i d e ; G: B i n d i n g t o i n t r a c e l l u l a r n u c l e o p h i l e s e.g. g l u t a t h i o n e (GSH); H: O x i d a t i o n t o c a t e c h o l ; I : O x i d a t i o n t o r a d i c a l s e m i q u i n o n e ; J : B i n d i n g t o l i v e r p r o t e i n s ; K: D e t o x i f i c a t i o n by O - m e t h y l a t i o n . 13 Each o f t h e b i o t r a n s f o r m a t i o n p r o c e s s e s i n v o l v i n g N - d e m e t h y l a t i o n , N-o x i d a t i o n and a r o m a t i c h y d r o x y l a t i o n i s c a t a l y z e d by t h e cytochome P-450 enzymes ( W i s l o c k i e t a l . , 1985); however, t h e u n d e r l y i n g mechanism whereby t h e s e a p p a r e n t l y d i f f e r e n t b i o t r a n s f o r m a t i o n s o c c u r can be summarized i n seven s t e p s ( H a l l , 1986; G u e n g e r i c h and L i e b l e r , 1985): ( i ) f o r m a t i o n o f t h e P - 4 5 0 - s u b s t r a t e complex, ( i i ) f i r s t one e l e c t r o n r e d u c t i o n i n v o l v i n g NADPH-P-450 r e d u c t a s e , ( i i i ) b i n d i n g o f m o l e c u l a r O2 , ( i v ) second one e l e c t r o n r e d u c t i o n i n v o l v i n g NADPH-P-450 r e d u c t a s e , (v) a c c e p t a n c e o f 2 H + w i t h c o n c o m i t a n t d i s s o c i a t i o n o f H2O, ( v i ) i n s e r t i o n o f oxygen i n t o t h e s u b s t r a t e and ( v i i ) r e l e a s e o f t h e p r o d u c t from t h e enzyme. A l t h o u g h P-450 c a t a l y s i s can be g e n e r a l l y p e r c e i v e d i n t h i s way, t h e d i v e r s i t y i n r e a c t i o n t y p e s a r i s e s due t o i d e n t i t y o f t h e r e d u c i n g e q u i v a l e n t ( e . g . e", H*) and r e a r r a n g e m e n t o f t h e p r o d u c t m o l e c u l e . (A) The N - d e m e t h v l a t i o n o f CLBZ. Many o f t h e m e c h a n i s t i c s t u d i e s on t h e P-450 c a t a l y z e d N-d e a l k y l a t i o n s f o c u s on s e c ondary and t e r t i a r y amines ( L i n d e k e and Cho, 1982; W o l f , 1982; W i s l o c k i e t a l . , 1980), however, t h e N - d e a l k y l a t i o n o f amides s h o u l d p r o c e e d i n l i k e f a s h i o n . The N - d e m e t h y l a t i o n o f N,N-d i m e t h y l a m i n e proceeds v i a a-carbon d e p r o t o n a t i o n as shown i n Scheme IV ( H o l l e n b e r g e t a l . , 1985; Miwa e t a l . , 1983). The mechanism i n v o l v e s an i n i t i a l o n e - e l e c t r o n removal from t h e s u b s t r a t e (-e~) t o t h e aminium c a t i o n r a d i c a l , w hich i n t u r n undergoes p r o t o n a b s t r a c t i o n (-H+) t o t h e c a r b o n -c e n t r e d r a d i c a l . Subsequent r e c o m b i n a t i o n w i t h an enzyme-bound h y d r o x y l r a d i c a l (+0H*) g i v e s r i s e t o t h e u n s t a b l e c a r b i n o l a m i n e which e l i m i n a t e s f o r m a l d e h y d e t o a f f o r d t h e d e m e t h y l a t e d p r o d u c t . 14 -e C H 3 R-N-CH3 C H 3 R-N-CH3 OH-H R-N-CH3 ' f C H 2 Q C H 3 R-N-CH2OH Scheme IV. P o s s i b l e mechanism f o r t h e o x i d a t i v e N - d e m e t h y l a t i o n o f N,N-d i m e t h y l a m i n e by cytochome P-450 ( H o l l e n b e r g e t a l . , 1985; Miwa e t a l . , 1983). The N-hydroxymethyl ( 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 w h i c h o c c u r i n t h e d e m e t h y l a t i o n o f N-methylamides v a r y i n t h e i r s t a b i l i t y . F o r example, N-h y d r o x y m e t h y l - N - m e t h y l b e n z a m i d e ( I ) decomposed r e a d i l y t o t h e N-desmethyl compound, whereas N-hydroxymethylbenzamide (2) was s u f f i c i e n t l y s t a b l e t o be i d e n t i f i e d (Ross e t a l . , 1983). O O 1 2 15 Many a c c o u n t s i n t h e l i t e r a t u r e a t t e s t t o t h e s t a b i l i t y o f t h e c a r b i n o l a m i d e s . F o r example, t h e c a r b i n o l a m i d e s o f bambuterol (3) ( L i n d b e r g e t a l . , 1989), N-methylbenzamide (2) (Ross e t a l . , 1983) and monuron (4) (Ross e t a l . , 1982) were formed in vitro, and ( t h e c a r b i n o l a m i d e s o f ) r e c i p a v r i n t e r t i a r y formamide (5) ( S l a t t e r e t a l . , 1989), Zolpidem (6) ( V a j t a e t a l . , 1 988), monuron (4) (Ross e t a l . , 1982) and d i p h e n a m i d (7) (McMahon and S u l l i v a n , 1965) o c c u r r e d in vivo either as t h e f r e e s p e c i e s o r t r a p p e d as a c o n j u g a t e . Me x Me N Me' V C H 2 O H 6 Two s t r u c t u r a l f e a t u r e s appear t o s t a b i l i z e N-hydroxymethyl i compounds: ( i ) a c a r b o n y l group a d j a c e n t t o t h e N-hydroxymethyl f u n c t i o n (Ross e t a l . , 1982) and ( i i ) t h e l o c a t i o n o f t h e h y d r o x y m e t h y l b e a r i n g n i t r o g e n i n a c o n j u g a t e d system ( G o r r o d and Temple, 1976). McMahon and S u l l i v a n (1965) o b s e r v e d t h a t c a r b i n o l a m i d e s were more s t a b l e t h a n t h e i r 16 c a r b i n o l a m i n e c o u n t e r p a r t s . Ross e t a l . (1983) have s u g g e s t e d t h a t t h e c a r b o n y l group r e d u c e s t h e e l e c t r o n d e n s i t y a t t h e h y d r o x y m e t h y l n i t r o g e n and t h e r e b y m i l i t a t e s a g a i n s t f o r m a l d e h y d e d i s s o c i a t i o n . T h i s r a t i o n a l e was e x t e n d e d t o a c c o u n t f o r t h e enhanced s t a b i l i t y o f N-( h y d r o x y m e t h y l ) b e n z a m i d e (2) o v e r N - ( h y d r o x y m e t h y l ) - N - m e t h y l b e n z a m i d e ( I ) , c l a i m i n g t h a t t h e e l e c t r o n - d o n a t i n g e f f e c t o f t h e methyl m o i e t y a n t a g o n i z e d t h e i n f l u e n c e o f t h e c a r b o n y l group. G o r r o d and Temple (1976) o b s e r v e d t h a t t h e i n c l u s i o n o f n i t r o g e n i n t h e c o n j u g a t e d system o f N-m e t h y l c a r b a z o l e (8) l o w e r e d t h e pKa o f t h e h e t e r o a t o m , and t h e r e b y a c c o u n t e d f o r t h e enhanced s t a b i l i t y c o n f e r r e d on t h e N-hydroxymethyl compound ( 9 ) . 8 9 The c a r b i n o l a m i d e (10) formed from CLBZ p o s s e s s e s a c a r b o n y l and a phenyl group a d j a c e n t t o t h e h y d r o x y m e t h y l b e a r i n g n i t r o g e n , both o f w h i c h c o u l d enhance t h e s t a b i l i t y o f t h i s compound by r e s o n a n c e d e l o c a l i z a t i o n o f n - e l e c t r o n s . On t h i s b a s i s t h e p r o b a b i l i t y o f i s o l a t i n g a 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 i n t h e f o r m a t i o n o f DMC appeared t o be q u i t e h i g h . 17 10 CLBZ c a r b i n o l a m i d e (10) showing p o s s i b l e r e s o n a n c e f o r m s . The i n v e s t i g a t i o n o f a p o t e n t i a l CLBZ c a r b i n o l a m i d e i s t o x i c o l o g i c a l l y r e l e v a n t f o r two r e a s o n s : ( i ) N-hydroxymethyl i n t e r m e d i a t e s a c t as t r a n s p o r t forms o f t o x i c f o r m a l d e h y d e ( K i n g s t o n , 1965), and ( i i ) t h e y a r e c a p a b l e o f a m i n o m e t h y l a t i o n o f i n t r a c e l l u l a r n u c l e o p h i l e s l i k e g l u t a t h i o n e , p r o t e i n s and n u c l e i c a c i d s (Soloway e t a l . , 1983). (B) The N - o x i d a t i o n o f DMC. T e r t i a r y amides a r e not m e t a b o l i z e d by N - o x i d a t i o n . The l o n e p a i r o f e l e c t r o n s on n i t r o g e n a r e n o t a v a i l a b l e f o r N-0 bond f o r m a t i o n , b u t a r e i n s t e a d d e l o c a l i z e d by t h e c a r b o n y l g r o u p (Damani, 1982). However, subsequent t o t h e N - d e m e t h y l a t i o n o f CLBZ, i t i s c o n c e i v a b l e t h a t t h e se c o n d a r y amide f u n c t i o n o f DMC c o u l d undergo f u r t h e r o x i d a t i o n by P-450 t o N-hydroxy DMC (hy d r o x a m i c a c i d ) ( N e l s o n , 1985) (Scheme V ) . The mechanism whereby amides a r e o x i d i z e d i s u n c e r t a i n , and d e p e n d i n g on t h e P-450 isoenzyme i n v o l v e d one o f s e v e r a l pathways c o u l d be o p e r a t i v e . Some o f t h e s e i n c l u d e : ( i ) s i n g l e t - t y p e oxene r e a c t i o n w i t h t h e i m i n o l t a u t o m e r t o for m an o x a z i r a n e w h i c h e x i s t s i n e q u i l i b r i u m w i t h t h e hyd r o x a m i c a c i d ; ( i i ) s e q u e n t i a l e l e c t r o n and p r o t o n a b s t r a c t i o n s f o l l o w e d by h y d r o x y l 18 r a d i c a l t r a n s f e r t o t h e ami d y l r a d i c a l ; and ( i i i ) h ydrogen atom a b s t r a c t i o n f o l l o w e d by h y d r o x y l r a d i c a l t r a n s f e r . O - F e -HOH + R OH R OH HOO (i) _N=C- -N-C- * -N-C- *- -N-C-HO HO + O HOO . . . . 1 11 -pr 1 11 _u + • » OH 1 " (n _ N -C- -N-C- -H-^-N-C- — - N - C -H O O HO O ( i i i ) -N-C- -N-C- -N-C-Scheme V. P o s s i b l e mechanisms f o r t h e P-450 c a t a l y z e d o x i d a t i o n o f amides ( N e l s o n , 1985). Cramer e t a l . (1960) were t h e f i r s t t o r e p o r t t h e m e t a b o l i c N-o x i d a t i o n o f amides w i t h t h e d i s c o v e r y o f N - h y d r o x y - N - a c e t y l a m i n o f l u o r e n e (N-OH AAF) (12) as a g l u c u r o n i d a t e d u r i n a r y m e t a b o l i t e o f N-a c e t y l a m i n o f l u o r e n e (AAF) (11) i n r a t s . AAF i s a p o t e n t h e p a t o c a r c i n o g e n , and o x i d a t i o n t o t h e hydroxamic a c i d (N-OH AAF) i s t h o u g h t t o be t h e mechanism o f t o x i c o l o g i c a l a c t i v a t i o n (Scheme V I ) (Hanna and Banks, 1985). However, t h e f o r m a t i o n N-OH AAF r e p r e s e n t s o n l y a p r e r e q u i s i t e s t e p i n t h e b i o a c t i v a t i o n o f AAF, and u l t i m a t e t o x i f i c a t i o n o f N-OH AAF i s mediated by phase I I f o r m a t i o n o f t h e s u l f a t e e s t e r (13), a p o t e n t e l e c t r o p h i l e (Van den Goorbergh e t a l . , 1987), w h i c h r e a c t s n o n - s p e c i f i c a l l y w i t h m e t h i o n y l 19 r e s i d u e s t o form S-adducts w i t h h e p a t i c p r o t e i n s (Hanna and Banks, 1985) 8/—f"S—V H 7^"~\-^ / V N - C O C H J € 5 4 11 Binding to hepatic proteins a c t i v a t i o n N-hydroxylation <Cyt.P-«50) OH <tT//\^ //-N_C0CH3 12 sulfotransferase ^OSOsH PAPS \ / ) - ( \ / ) - N - C 0 C H 3 *" V, / M \ />"N-COCHj 13 Scheme V I . M e t a b o l i c pathways i n t h e h e p a t o c a r c i n o g e n i c a c t i v a t i o n o f AAF (Hanna and Banks, 1985). OeBaun e t a l . (1968) o b s e r v e d a c l o s e p a r a l l e l between t h e l e v e l o f s u l f o t r a n s f e r a s e a c t i v i t y i n d i f f e r e n t r o d e n t s p e c i e s and t h e i r s u s c e p t i b i l i t y t o c a r c i n o g e n e s i s w i t h N-OH AAF, and p r o p o s e d t h a t s u l f a t e e s t e r f o r m a t i o n gave r i s e t o t h e u l t i m a t e t o x i f i c a t i o n o f N-OH AAF. Meerman and M u l d e r (1981) s u b s t a n t i a t e d t h i s h y p o t h e s i s when t h e y found t h a t p r e t r e a t m e n t o f r a t s w i t h t h e s u l f o t r a n s f e r a s e i n h i b i t o r p e n t a c h l o r o p h e n o l c o m p l e t e l y a b o l i s h e d t h e c a r c i n o g e n i c e f f e c t s o f N-OH AAF. G l u t a t h i o n e (GSH) p l a y s an i m p o r t a n t r o l e as an i n t r a c e l l u l a r n u c l e o p h i l e by b i n d i n g t o s e v e r a l x e n o b i o t i c e l e c t r o p h i l e s . The n i t r e n i u m c a t i o n g e n e r a t e d by t h e d e c o m p o s i t i o n o f N-OH AAF i s no e x c e p t i o n (van den Goorbergh e t a l . , 1985), and two b i l i a r y GSH c o n j u g a t e s i n r a t s dosed w i t h N-OH AAF were i d e n t i f i e d , namely, 1- and 3 - ( g l u t a t h i o n - S - y l ) AAF (Meerman e t a l . , 1982). D e s p i t e t h e o c c u r r e n c e o f t h e s e c o n j u g a t e s , GSH a f f o r d s 20 l i t t l e p r o t e c t i o n i n t h e d e t o x i f i c a t i o n o f N-OH AAF because o f i t s i n a b i l i t y t o e f f e c t i v e l y compete f o r t h e t r a p p i n g o f t h e n i t r e n i u m i o n (van den Goorbergh e t a l . , 1985). Indeed, when r a t s were p r e t r e a t e d w i t h d i e t h y l m a l e a t e (DEM) t o d e p l e t e h e p a t i c and r e n a l GSH, e x c r e t i o n o f t h e GSH c o n j u g a t e s was d e c r e a s e d by 60 % y e t , no significant increase in the covalent binding of N-OH AAF to hepatic and renal macromolecules was observed (Meerman and T i j d e n s , 1985). I n s t e a d , i t was s u g g e s t e d o t h e r d e t o x i c a n t s l i k e NADH and a s c o r b i c a c i d c o u l d have f a c i l i t a t e d t h e d e t o x i f i c a t i o n o f t h e n i t r e n i u m i o n i n t h e absence o f GSH. U n l i k e t h e p o l y c y c l i c a r o m a t i c s ( e . g . A A F ) , a c e t a n i l i d e s a r e l e s s r e a d i l y p r e d i s p o s e d t o N-hydroxy1 a t i o n (Damani, 1 9 8 2 ) , n e v e r t h e l e s s , t h e i r b i o t r a n s f o r m a t i o n i s t o x i c o l o g i c a l l y r e l e v a n t ( M u l d e r e t a l . , 1977; H i nson and M i t c h e l l , 1976). M u l d e r e t a l . (1977) have s p e c u l a t e d t h a t N-h y d r o x y p h e n a c e t i n (14) c o u l d be r e s p o n s i b l e f o r t h e h e p a t o t o x i c and n e p h o t o x i c a c t i v i t y o f t h e p a r e n t d r u g ( 1 5 ) . The s u l f a t e c o n j u g a t e o f 14, because o f i t s h i g h r e a c t i v i t y , was c o n s i d e r e d t h e t o x i c s p e c i e s i n v o l v e d i n c o v a l e n t b i n d i n g i n t h e l i v e r , whereas t h e g l u c u r o n i d e c o n j u g a t e b e i n g l e s s r e a c t i v e a c t e d as a t r a n s p o r t f o r m o f 14 whose a c t i v i t y was e x e r t e d a f i e l d i n t h e k i d n e y and b l a d d e r where g l u c u r o n i d e s a r e c o n c e n t r a t e d . C l o s e s i m i l a r i t y e x i s t s between t h e s e c o n d a r y amide c h e m i s t r y o f p h e n a c e t i n (15) and DMC w h i c h c o u l d s i g n i f y t h e p o s s i b i l i t y f o r DMC t o be o x i d i z e d , l i k e 15, t o a h ydroxamic a c i d . o 21 (C) The A r o m a t i c H y d r o x y ! a t i o n o f CLBZ. The cytochome P-450 me d i a t e d c a t a l y s i s o f a r o m a t i c h y d r o x y l a t i o n , t o a l a r g e e x t e n t , i n v o l v e s t h e d i r e c t a d d i t i o n o f oxygen a c r o s s t h e C-C a r o m a t i c bond via an o x e n o i d t y p e mechanism t o a f f o r d t h e arene e p o x i d e (Scheme V I I ) ( T r a g e r , 1980). The f o r m a t i o n o f a r e n e e p o x i d e s i n a r o m a t i c o x i d a t i o n i s now w e l l e s t a b l i s h e d , and a l t e r n a t i v e m e t a b o l i c pathways f o r t h i s i n t e r m e d i a t e e x i s t . The e p o x i d e c a n e i t h e r (A) s p o n t a n e o u s l y r e a r r a n g e t o t h e p h e n o l , o r (B) i s b i o t r a n s f o r m e d s e q u e n t i a l l y t o t h e d i h y d r o d i o l and c a t e c h o l ( K a l f , 1987). Scheme V I I . Pathways i n v o l v e d i n a r o m a t i c o x i d a t i o n . The r a t e - d e t e r m i n i n g s t e p i n a r o m a t i c h y d r o x y l a t i o n i s t h e h e t e r o l y t i c c l e a v a g e o f t h e C-0 bond o f t h e a r e n e e p o x i d e t o form t h e c a r b o c a t i o n ( T r a g e r , 1980). The c a r b o c a t i o n i n t e r m e d i a t e c a n , i n t u r n , e i t h e r f o r m t h e phenol d i r e c t l y w i t h t h e l o s s o f X+ o r i s o m e r i z e t o t h e k e t o t a u t o m e r o f t h e phenol w i t h a 1 , 2 - m i g r a t i o n o f X", known as t h e NIH s h i f t , b e f o r e r e a r r a n g i n g t o t h e f i n a l p r o d u c t . G e n e r a l l y , s u b s t i t u e n t e f f e c t s d e t e r m i n e t h e e x t e n t t o which l a b e l i s r e t a i n e d , w i t h r e t e n t i o n b e i n g f a v o u r e d by e l e c t r o n w i t h d r a w i n g g r o u p s . A p a r t from t h e i r n o n - s p e c i f i c rearrangement t o p h e n o l , arene e p o x i d e s a r e h y d r o l y z e d by e p o x i d e h y d r o l a s e t o t h e trans d i h y d r o d i o l (Oesch, 1972), w h i c h can i n t u r n be c o n v e r t e d by d i h y d r o d i o l dehydrogenase t o c a t e c h o l ( K a l f , 1987; B i l l i n g s , 1985). Arene e p o x i d e s a r e r e a c t i v e e l e c t r o p h i l e s ( J e r i n a and D a l y , 1974) w h i c h have been i m p l i c a t e d i n c o v a l e n t b i n d i n g t o c e l l u l a r n u c l e o p h i l e s ( R i l e y e t a l . , 1989; Gerson e t a l . , 1983). However, r e p o r t s i n t h e l i t e r a t u r e s u p p o r t t h e v i e w t h a t o t h e r r e a c t i v e s p e c i e s d i s t a l f rom t h e e p o x i d e and more i n t i m a t e l y r e l a t e d t o t h e s e c o n d a r y phenol m e t a b o l i t e c o u l d a l s o be i n v o l v e d i n c o v a l e n t b i n d i n g . F o r example, Tunek e t a l . (1978) o b s e r v e d no s i g n i f i c a n t r e a c t i o n between benzene e p o x i d e and microsomal p r o t e i n compared t o t h e p h e n o l , w h i c h prompted them t o c o n s i d e r t h e i n v o l v e m e n t o f a quinone i n t e r m e d i a t e . T h i s h y p o t h e s i s appears t o h o l d t r u e , b e i n g s u b s t a n t i a t e d by t h e c h e m i c a l c h a r a c t e r i z a t i o n o f t h e GSH c o n j u g a t e o f p - h y d r o x y p h e n o l formed m i c r o s o m a l l y from phenol (Tunek e t a l . , 1980), and t h e GSH c o n j u g a t e o f p h e n y l - p - h y d r o x y p h e n o l (Nakagawa and Tayama, 1989) as a b i l i a r y m e t a b o l i t e i n r a t s dosed w i t h o - p h e n y l p h e n o l . A l t h o u g h t h e a c t i o n o f e p o x i d e h y d r o l a s e d e t o x i f i e s arene e p o x i d e s t o t h e d i h y d r o d i o l , subsequent dehydrogenase a c t i v i t y on t h i s i n n ocuous s u b s t r a t e t o form t h e c a t e c h o l c o u l d be p e r c e i v e d as a t o x i f i c a t i o n pathway. The d i r e c t h y d r o x y l a t i o n o f 2-hydroxy e s t r o g e n s (2-HE, 16) t o c a t e c h o l s i s t h o u g h t t o be r e s p o n s i b l e f o r t h e c o v a l e n t b i n d i n g o f t h e l a t t e r t o p r o t e i n s ( P u r b a e t a l . , 1987). The u l t i m a t e s p e c i e s i n v o l v e d i n 23 b i n d i n g , however, i s c o n j e c t u r a l ( I r o n s and Sawahata, 1985) and c o u l d i n v o l v e a r a d i c a l s e miquinone (Marks and He c k e r , 1969) o r an epoxyphenol i n t e r m e d i a t e (Numazawa and Nambara, 1977) (Scheme V I I I ) . Two d e t o x i f i c a t i o n pathways a v a i l a b l e t o 2-HE appear t o be o p e r a t i v e . GSH f u l f i l s i t s r o l e as a d e t o x i c a n t by b i n d i n g t o t h e e l e c t r o p h i l i c i n t e r m e d i a t e s (Maggs e t a l . , 1983); t h u s , 1-, and 4 - g l u t a t h i o n - S - y l 2-h y d r o x y - e s t r a d i o l a d d u c t s o c c u r r e d as b i l i a r y m e t a b o l i t e s i n r a t s dosed w i t h e s t r a d i o l ( E l c e and H a r r i s , 1971) (Scheme V I I I ) . C a t e c h o l 0-m e t h y l t r a n s f e r a s e (COMT), on t h e o t h e r hand, m e t h y l a t e s t h e c a t e c h o l and t h e r e b y a b o l i s h e s f u r t h e r o x i d a t i o n t o e l e c t r o p h i l i c s p e c i e s . Thus, Haaf e t a l . (1987) o b s e r v e d a 70 % r e d u c t i o n o f c o v a l e n t b i n d i n g o f e s t r a d i o l t o hamster l i v e r microsomes when t h e i n c u b a t i o n m i x t u r e was e n r i c h e d w i t h COMT and S - a d e n o s y l - L - m e t h i o n i n e (SAM). The i m p o r t a n c e o f COMT i n t h e d e t o x i f i c a t i o n o f 2-hydroxy e s t r a d i o l ( L i e t a l . , 1989; Haaf e t a l . , 1987; S t r a m e n t i n o l i e t a l . , 1979) c o u l d i n t u r n , s i g n i f y t h e r e l e v a n c e o f 0-m e t h y l a t i o n as a c r u c i a l d e t o x i f i c a t i o n pathway f o r t h e c a t e c h o l m e t a b o l i t e s o f CLBZ. 24 Scheme V I I I . F o r m a t i o n o f r e a c t i v e i n t e r m e d i a t e s f r o m 2-hydroxy e s t r o g e n . Pathway (A) l e a d s t o t h e s e m i q u i n o n e and (B) t o t h e e p o x y p h e n o l . D e t o x i f i c a t i o n mechanisms i n v o l v e (1) GSH b i n d i n g and (2) COMT 0-m e t h y l a t i o n . 25 4. THE OBJECTIVES OF THE THESIS. S e v e r a l m e t a b o l i t e s o f CLBZ have been r e p o r t e d , a l b e i t u n s u b s t a n t i a t e d by p u b l i s h e d s p e c t r a l e v i d e n c e ( V o l z e t a l . , 1979). In a d d i t i o n , CLBZ c o u l d a l s o be b i o t r a n s f o r m e d i n t o r e a c t i v e c a r b i n o l a m i d e o r h y d r o x a m i c a c i d m e t a b o l i t e s . In o r d e r t o c h a r a c t e r i z e t h e m e t a b o l i t e s o f CLBZ w h i c h would i n c l u d e t h e i d e n t i f i c a t i o n and v e r i f i c a t i o n o f t r a c e and p o t e n t i a l l y r e a c t i v e s u b s t a n c e s , t h e f o l l o w i n g o b j e c t i v e s were p r o p o s e d . ( i ) To s y n t h e s i z e p e n t a d e u t e r i o p h e n y l CLBZ f o r t h e a d m i n i s t r a t i o n t o r a t s t o i d e n t i f y CLBZ m e t a b o l i t e s i n b i l e and u r i n e by u s i n g i s o t o p e c l u s t e r a n a l y s i s and GCMS. ( i i ) To p r o v i d e s p e c t r a l e v i d e n c e f o r t h e 4'-hydroxy, 4'-hydroxy-3'-methoxy and 3 ' , 4 ' - d i h y d r o d i o l d e r i v a t i v e s o f b o t h CLBZ and DMC. ( i i i ) To d e t e r m i n e whether N-hydroxymethyl CLBZ and/or i t s c o n j u g a t e s e x i s t as m e t a b o l i t e s o f CLBZ. ( i v ) To d e t e r m i n e whether N-hydroxy DMC a nd/or i t s c o n j u g a t e s e x i s t as m e t a b o l i t e s o f CLBZ. 26 I I EXPERIMENTAL 1. CHEMICALS AND MATERIALS. C h e m i c a l s were r e a g e n t g r a d e and p u r c h a s e d f r o m t h e f o l l o w i n g s o u r c e s : A l d r i c h C h e m i cal Co. ( M i l w a u k e e , W i s c o n s i n ) 2 , 4 - d i c h l o r o n i t r o b e n z e n e , 4 - a m i n o v e r a t r o l e ( 3 , 4 - d i m e t h o x y a n i l i n e ) , a c e t i c a n h y d r i d e , boron t r i b r o m i d e , c a l c i u m c h l o r i d e ( a n h y d r o u s ) , CDC!3, DC1 (37 wt. % s o l u t i o n i n D2O, 99 atom % D ) , d e u t e r i u m o x i d e (99.8 atom % D), f o r m a l d e h y d e (37 % s o l u t i o n ) , h y d r o c h l o r i c a c i d , i r o n powder, methyl i o d i d e , n i t r o b e n z e n e - d s (99 atom % D), p-a n i s i d i n e ( 4 - m e t h o x y a n i l i n e ) , p o t a s s i u m h y d r i d e (35 wt. % ) , t r i m e t h y l s i l y l i o d i d e , z i n c d u s t . A l l i e d C h e m i c al (New Y o r k , New Y o r k ) sodium a c e t a t e . Anachemia C h e m i c a l s L t d . ( M o n t r e a l , Quebec) e t h y l a c e t a t e ( d i s t i l l e d i n g l a s s ) . BDH I n c . ( V a n c o u v e r , B.C.) m e t h a n o l , e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA), c h l o r o f o r m , benzene, e t h y l e t h e r , p e t r o l e u m e t h e r , magnesium c h l o r i d e ( h e x a h y d r a t e ) , p o t a s s i u m c h l o r i d e , sodium s u l f a t e ( a n h y d r o u s ) , s i l i c a g e l 60 (230 - 400 mesh), p h e n o b a r b i t o n e sodium, p o t a s s i u m h y d r o x i d e , p o t a s s i u m b i c a r b o n a t e , sodium c h l o r i d e , sodium hydrogen o r t h o p h o s p h a t e , sodium d i h y d r o g e n o r t h o p h o s p h a t e . 27 B i o - R a d L a b o r a t o r i e s (Richmond, C a l i f o r n i a ) a c r y l a m i d e N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e ( B I S ) 0 sodium d o d e c y l s u l f a t e (SDS) B o e h r i n g e r Mannheim ( M o n t r e a l , Quebec) ^ - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e , r e d u c e d (NADPH) ^ - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e , r e d u c e d (NADH) b o v i n e serum al b u m i n (BSA). Cambridge I s o t o p e L a b o r a t o r i e s (Woburn, MA) d i m e t h y l - d ^ s u l f o x i d e . 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 J e r s e y ) q u i n o l i n e . H o e f e r S c i e n t i f i c I n s t r u m e n t s (San F r a n c i s c o , C a l i f o r n i a ) n i t r o c e l l u l o s e membrane. J.T.B a k e r ( P h i l ! i p s b u r g , New J e r s e y ) s i l i c a g e l (40 n p a r t i c l e d i a m e t e r ) . L i n d e Co. ( U n i o n C a r b i d e , Vancouver, B.C.) n i t r o g e n g a s . M a l l i n c k r o d t Chemical Works L t d . ( M o n t r e a l , Quebec) a c e t i c a c i d ( g l a c i a l ) . Matheson, Coleman and B e l l (Norwood, O h i o ) p o t a s s i u m c a r b o n a t e . 28 MSD I s o t o p e s ( M o n t r e a l , Quebec) methyl-03 i o d i d e . P i e r c e C h e m ical Co. ( R o c k f o r d , I l l i n o i s ) N - m e t h y l - N - t r i m e t h y l s i l y l - t r i f 1 u o r o a c e t a m i d e (MSTFA). Sigma Chemical Co. ( S t . L o u i s , M i s s o u r i ) d i m e t h y l s u l f o x i d e , g l u c u r a s e ( 6 - 4 8 8 2 ) , s u l f a t a s e ( S - 9 7 5 4 ) , S-a d e n o s y l - L - m e t h i o n i n e ( p - t o l u e n e s u f o n a t e s a l t ) , p r o p y l e n e g l y c o l , s u c r o s e , T r i s . H C l , u r e t h a n e . Stanchem L t d . ( W i n n i p e g , M a n i t o b a ) e t h a n o l (95 % ) . S u p e l c o ( O a k v i l l e , O n t a r i o ) 3 % D e x s i l 300 on 100/120 mesh S u p e l c o p o r t . Whatman L t d . ( M a i d s t o n e , K e n t , England) s i l i c a g e l p l a t e s (AL S I L G/UV). 2. ANIMALS AND SURGICAL EQUIPMENT. Animal C a r e F a c i l i t y (U.B.C., Vancouver, B.C.) Sprague Dawley male r a t s . F o r l i v e r m icrosomal p r e p a r a t i o n s : 200-225 g F o r in vivo d r u g m e t a b o l i s m : 250-275 g Be c t o n D i c k i n s o n ( R u t h e r f o r d , New J e r s e y ) V a c u t a i n e r tube 6430, Y a l e n e e d l e 19G 1 1/2"; 23G 1", 25G 1", t u b e r c u l i n s y r i n g e 1 c c . 29 C o e v a l , I n c . ( S t . J o s e p h , I l l i n o i s ) C orn cob g r a n u l e s E t h i c o n , I n c . ( S o m e r v i l l e , New J e r s e y ) 000 S i l k t h r e a d P u r i n a M i l l s I n c . , ( S t . L o u i s , M i s s o u r i ) P u r i n a L a b o r a t o r y Chow 5001 3. ACCESSORY EQUIPMENT. Beckman Model J2-21 c e n t r i f u g e w i t h JA-20 r o t o r Beckman Model L8-60M u l t r a c e n t r i f u g e w i t h 50.2 T i r o t o r Hakke SWB20 w a t e r - b a t h / s h a k e r H o e f e r TE 52 T r a n s p h o r u n i t w i t h power l i d P o t t e r - E l v e h j e m t i s s u e g r i n d e r . 4. INSTRUMENTATION. (A) NMR S p e c t r o s c o p y . A l l NMR s p e c t r o s c o p y was pe r f o r m e d i n t h e Department o f C h e m i s t r y , U n i v e r s i t y o f B r i t i s h C o l u m b i a . ^H-NMR s p e c t r o s c o p y was r e c o r d e d on e i t h e r a B r u k e r WH-400 (400 MHz) o r V a r i a n XL-300 (300 MHz) i n s t r u m e n t . 1 3C-NMR s p e c t r o s c o p y was r e c o r d e d on e i t h e r a V a r i a n XL-300 (75 MHz) o r a B r u k e r AC-200 (75 MHz) i n s t r u m e n t w i t h broad-band d e c o u p l i n g on t h e ^H- f r e q u e n c y , and an A t t a c h e d P r o t o n T e s t . 2H-NMR s p e c t r a were t a k e n i n CHCI3 s o l u t i o n on a B r u k e r WH-400 (84.68 MHz) i n s t r u m e n t and c h e m i c a l 30 s h i f t s measured r e l a t i v e t o an e x t e r n a l CDCI3 s t a n d a r d . A l l 1H-NMR and 1 ^  C-NMR s p e c t r a were d e t e r m i n e d i n CDCI3 s o l u t i o n , u n l e s s o t h e r w i s e s t a t e d , and t h e c h e m i c a l s h i f t s a r e i n p a r t s p e r m i l l i o n r e l a t i v e t o t e t r a m e t h y l s i l a n e e i t h e r as an i n t e r n a l o r e x t e r n a l s t a n d a r d . NMR s p e c t r a a r e r e c o r d e d as f o l l o w s : NMR S ( s p l i t t i n g t y p e , c o u p l i n g c o n s t a n t , i n t e g r a l , a s s i g n m e n t ) , e.g. ^ H-NMR 6 6.74 (dd, J = 10 Hz and 2 Hz, 1H, H-4). (B) Mass S p e c t r o s c o p y . ( i ) C a p i l l a r y GCMS. Gen e r a l o p e r a t i n g c o n d i t i o n s a p p l y t o a l l c a p i l l a r y GCMS a n a l y s e s , whereas s p e c i f i c c o n d i t i o n s a r e d e s c r i b e d as ( a ) , ( b ) , ( c ) and ( d ) . G e n e r a l o p e r a t i n g c o n d i t i o n s . C a p i l l a r y GCMS was performed on an HP 5987A GCMS system f i t t e d w i t h an HP-1 25 m x 0.32 mm i . d . x 0.52 / i c a p i l l a r y column w i t h a C r o s s l i n k e d M e t h y l S i l i c o n e Gum s t a t i o n a r y phase. The e l e c t r o n impact mode o f i o n i z a t i o n was used w i t h an en e r g y o f 70 eV, e m i s s i o n c u r r e n t o f 300 /iA and m u l t i p l i e r v o l t a g e o f 2400 eV. Temperature zones were as f o l l o w s . I n j e c t i o n p o r t : 240°C, i n t e r f a c e oven: 240°C, GC i n t e r f a c e p r o b e : 250°C and i o n s o u r c e : 240°C. The He c a r r i e r gas was s e t a t a f l o w r a t e o f 1 ml/min w i t h a back p r e s s u r e o f 10 p s i . I n j e c t i o n s were p e r f o r m e d i n t h e s p l i t l e s s mode. 31 S p e c i f i c o p e r a t i n g c o n d i t i o n s . (a) 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 b i l i a r y CLBZ m e t a b o l i t e s : Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 30*C/min from 100°C t o 200°C, where i t was h e l d f o r 3 min, t h e n i n c r e a s e d a t 8°C/min t o 280°C, where i t was h e l d f o r 12 min and t h e r e a f t e r a p o s t - r u n t e m p e r a t u r e o f 290°C h e l d f o r 2 min. T o t a l r un t i m e was 28.33 min. S c a n n i n g i n t h e l i n e a r mode commenced a f t e r 4 min and was o v e r t h e range o f 70 - 500 amu. E l e c t r o n m u l t i p l i e r v o l t a g e was 2,800 eV. GCMS s e n s i t i v i t y was o p t i m a l d u r i n g t h e p r e l i m i n a r y m e t a b o l i t e s t u d i e s . The mass s p e c t r a o b t a i n e d under t h e s e c o n d i t i o n s were o f t h e b e s t q u a l i t y and a r e used i n t h e F i g u r e s 28 t o 32. (b) R o u t i n e c h a r a c t e r i z a t i o n o f CLBZ m e t a b o l i t e s : Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 30°C/min from 150°C t o 200°C, where i t was h e l d f o r 3 min, t h e n i n c r e a s e d a t 8°C/min t o 280°C, where i t was h e l d f o r 12 min and t h e r e a f t e r a p o s t - r u n t e m p e r a t u r e o f 290°C h e l d f o r 3 min. T o t a l r un t i m e was 26.67 min. Sc a n n i n g i n t h e l i n e a r mode commenced a f t e r 12 min and was o v e r a range o f 70 - 550 amu. E l e c t r o n m u l t i p l i e r v o l t a g e was 2,400 eV. (c ) S e l e c t e d i o n m o n i t o r i n g f o r CLBZ and i t s m e t a b o l i t e s : Oven c o n d i t i o n s were programmed a c c o r d i n g t o p r o c e d u r e ( b ) . Ions scanned a r e s p e c i f i e d . Dwell t i m e was 100 m s e c / i o n . 32 (d) A n a l y s i s o f s y n t h e t i c compounds: Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 30°C/min from 100°C t o 280°C, where i t was h e l d f o r 10 min and t h e r e a f t e r a p o s t - r u n t e m p e r a t u r e o f 290°C h e l d f o r 2 min. T o t a l run t i m e was 16 min. S c a n n i n g i n t h e l i n e a r mode commenced a f t e r 2.5 min and was o v e r t h e range o f 70 - 550 amu. E l e c t r o n m u l t i p l i e r v o l t a g e was 2,400 eV. ( i i ) Packed column GCMS. Packed column GCMS was performed on a V a r i a n MAT-111 MS i n t e r f a c e d t o an HP 5700A GC f i t t e d w i t h a 1.8 m x 2 mm i . d . g l a s s column packed w i t h 3 % D e x s i l 300 on 100/120 mesh S u p e l c o p o r t . Data were r e c o r d e d u s i n g a P a c k a r d B e l l computer (IBM AT c l o n e ) and a program d e v e l o p e d i n o u r l a b o r a t o r y . Mass s p e c t r a were r e c o r d e d from m/z 14 t o m/z 750 a t 12 sca n s p e r m i n u t e . T o t a l i o n c u r r e n t (TIC) p l o t s were based on m/z 50 -500. The e l e c t r o n impact mode o f i o n i z a t i o n was used w i t h an energy o f 70 eV and e m i s s i o n c u r r e n t o f 300 /iA. I n j e c t i o n p o r t , l i n e and s e p a r a t o r t e m p e r a t u r e s were 250"C. The He c a r r i e r gas was s e t a t a f l o w r a t e o f 25 mL/min. I n j e c t i o n s were p e r f o r m e d i n t h e s p l i t l e s s mode. Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 32°C/min from 150°C t o 300°C, where i t was h e l d f o r 16 min. Mass s p e c t r a were r e c o r d e d as f o l l o w s : GCMS: r e t e n t i o n t i m e , P a r e n t i o n : ( R e l a t i v e i n t e n s i t y , % ) , O t h e r i o n ( s ) : m/z ( % ) , e t c . e.g. MS: M + - ( 3 5 C 1 ) : m/z 305 (100 % ) , M +' ( 3 7 C 1 ) : m/z 307 (34 % ) , [ M - 1 7 ] + * : m/z 288 (43 % ) , C 6 D 5 + ' : m/z 82 (48 % ) . 33 (C) I n f r a r e d S p e c t r o s c o p y . IR s p e c t r a were r e c o r d e d on a M i c h e l son BOMEM MB-100 FT-S p e c t r o m e t e r . Samples were a n a l y z e d e i t h e r as a s o l u t i o n i n CCI4 i n NaCl c e l l s o f 0.5 mm p a t h l e n g t h o r as a N u j o l m u l l on NaCl p l a t e s . IR s p e c t r a were r e c o r d e d as f o l l o w s : IR (CCI4 s o l u t i o n / N u j o l m u l l ) f r e q u e n c y i n cm"* ( i n t e n s i t y , a s s i g n m e n t ) , e.g. IR (CCI4 s o l u t i o n ) 1745 ( s , e s t e r c a r b o n y l g r o u p ) , 1693 ( s , amide c a r b o n y l g r o u p ) , 1534 ( s , N O 2 )asymmetric s t r e t c h , 1351 cm"* ( s , N02)symmetric s t r e t c h . (D) M e l t i n g P o i n t . M e l t i n g p o i n t s were d e t e r m i n e d i n open c a p i l l a r y t u b e s on a Thomas Hoover C a p i l l a r y m e l t i n g p o i n t a p p a r a t u s ( P h i l a d e l p h i a , PA) and a r e u n c o r r e c t e d . (E) U l t r a v i o l e t S p e c t r o s c o p y . UV s p e c t r o s c o p y was pe r f o r m e d on an HP Diode A r r a y 8452A S p e c t r o p h o t o m e t e r i n t e r f a c e d t o an HP V e c t r a Computer. Samples were a n a l y z e d i n q u a r t z c e l l s o f p a t h l e n g t h 1 cm. (F) High P r e s s u r e L i q u i d Chromatography. HPLC a n a l y s i s was done on an HP 1050 S e r i e s System c o n s i s t i n g o f Q u a t e r n a r y Pump and M u l t i p l e Wavelength D e t e c t o r (254 nm) f i t t e d w i t h a H y p e r s i l ODS 5 /1, 200 cm x 4.6 mm i . d . column. A l l chromatography was 34 p e r f o r m e d i s o c r a t i c a l l y . Flow r a t e s were c o n t r o l l e d a t 1 mL/min and m o b i l e phase c o m p o s i t i o n s a r e s p e c i f i e d . (G) L i q u i d Chromatography-Mass S p e c t r o s c o p y . LCMS a n a l y s i s was performed on a K r a t o s MS-80 mass s p e c t r o m e t e r c o n s i s t i n g o f a Waters pump and a V e s t e c t h e r m o s p r a y i n t e r f a c e . The system was f i t t e d w i t h an ODS 200 cm x 4.6 mm i . d . column c o n n e c t e d t o a Rheodyne i n j e c t o r . Ammonium a c e t a t e (1 M) was used t o f a c i l i t a t e p o s t -column i o n i z a t i o n . Temperature s e t t i n g s were as f o l l o w s : p r o b e , 114°C; v a p o r , 172°C; b l o c k , 219°C and j e t , 249°C. The c o m p o s i t i o n o f t h e m o b i l e phase was 50:50 (v / v ) Me0H:H20. 5. METABOLISM EXPERIMENTS. (A) Dosage Regimen. CLBZ:[H5]CLBZ was p r e p a r e d as an a p p r o x i m a t e 50:50 (w/w) m i x t u r e , w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f 53.2:46.8 f o r mass chromatograms a t m/z 300 and 305 r e s p e c t i v e l y , i . e . CLBZ/[ 2H5]CLBZ = 1.137. The m i x t u r e was d i s s o l v e d i n p r o p y l e n e g l y c o l (10 mg/mL) and a d m i n i s t e r e d i . p . a t 10 mg/kg t o t h r e e r a t s e v e r y 6 h f o r 6 d o s e s . One o t h e r animal was a d m i n i s t e r e d t h e v e h i c l e ( p r o p y l e n e g l y c o l c o n t r o l ) . 35 (B) S u r g e r y and C o l l e c t i o n o f U r i n e and B i l e . A n i m a l s were housed i n s t a i n l e s s s t e e l m e t a b o l i c cages and were a l l o w e d a c c e s s ad libitum t o fo o d and w a t e r . U r i n e c o l l e c t i o n commenced a t t h e f i r s t dose and c o n t i n u e d u n t i l t h e t i m e o f s a c r i f i c e . C o l l e c t e d u r i n e was p e r i o d i c a l l y removed and s t o r e d a t - 2 0 T u n t i l work-up. A f t e r t h e f i f t h dose o f CLBZ:[ 2 Hs]CLBZ, r a t s were a n e s t h e t i z e d w i t h u r e t h a n e (1.2 g/kg) wh i c h was a d m i n i s t e r e d i . p . as a s o l u t i o n i n d i s t i l l e d w a t e r (0.4 g/mL). A f t e r a n e s t h e s i a was i n d u c e d ( c a 30 min) an i n c i s i o n o f 3 cm was made on t h e v e n t r a l s u r f a c e o f t h e animal l e f t o f t h e abdominal m i d - l i n e toward t h e diaphragm. The i n t e s t i n e s were d i s p l a c e d , t h e p o r t i o n o f t h e b i l e d u c t a t t h e j u n c t i o n w i t h t h e duodenum, l o c a t e d and s u r r o u n d i n g t i s s u e g e n t l y removed w i t h c o t t o n w o o l . A p u n c t u r e was made i n t o t h e duodenum w i t h a 25G1 n e e d l e t o a c c e s s t h e b i l e d u c t . Through t h i s a p e r t u r e a b e v e l l e d c a n n u l a o f PE-10 t u b i n g was i n s e r t e d 2 cm i n t o t h e b i l e d u c t , l i g a t e d a t t h e duodenal j u n c t i o n w i t h s i l k t h r e a d , and e x t e r i o r i z e d t h r o u g h t h e l o w e r abdominal w a l l g u i d e d by t h e bore o f an e x t e r n a l l y i n s e r t e d 19G1 1/2 n e e d l e . The d i s p l a c e d i n t e s t i n e s were r e l o c a t e d i n t o t h e abdominal c a v i t y and t h e i n c i s i o n c l o s e d w i t h i n t e r r u p t e d s u t u r e s . B i l e was c o l l e c t e d i n s c i n t i l l a t i o n v i a l s , and u r i n e , a t t h a t t i m e , c o l l e c t e d i n u n s t o p p e r e d V a c u t a i n e r ^ t u b e s s e c u r e d t o t h e u r e t h r a o f t h e animal w i t h t a p e . B i l e and u r i n e were p e r i o d i c a l l y e mptied f o r s t o r a g e a t -20°C u n t i l work-up. B i l e was c o l l e c t e d f o r 18 h a t which p o i n t t h e a n i m a l , w h i c h was s t i l l u nder a n e s t h e s i a , was s a c r i f i c e d by d e c a p i t a t i o n . 36 (C) P r e p a r a t i o n o f B i l e and U r i n e f o r GCMS. B i l e (2.5 mL) and u r i n e (5 mL) samples were e x t r a c t e d (x 4) w i t h an e q u a l volume o f EtOAc t o remove u n c o n j u g a t e d m e t a b o l i t e s . The o r g a n i c e x t r a c t was s t o r e d a t 4°C o v e r anhydrous Na2S04 u n t i l f u r t h e r p r e p a r a t i o n . The aqueous f r a c t i o n s o f b i l e (pH 9.0) and u r i n e (pH 7.1) were a d j u s t e d t o pH 5.0 w i t h sodium a c e t a t e b u f f e r , e n r i c h e d w i t h e i t h e r g l u c u r a s e (1,500 U) o r s u l f a t a s e (60 U) and i n c u b a t e d w i t h g e n t l e s h a k i n g i n a w a t e r b a t h a t 37°C f o r 20 h. The d e c o n j u g a t e d m e t a b o l i t e s were e x t r a c t e d w i t h an equal volume o f EtOAc (x 4) and t h e o r g a n i c e x t r a c t s d r i e d o v e r anhydrous Na2S04 o v e r n i g h t a t 4°C. The d r i e d o r g a n i c e x t r a c t s o f t h e u n c o n j u g a t e d and d e c o n j u g a t e d m e t a b o l i t e s were c o n c e n t r a t e d t o d r y n e s s i n a w a t e r b a t h a t ca 30°C under a s t r e a m o f n i t r o g e n . F o r d e r i v a t i z a t i o n , t h e e x t r a c t s were r e c o n s t i t u t e d i n d r y EtOAc (25 / i L ) , MSTFA (25 /xL) added, and t h e m i x t u r e h e a t e d a t 45°C f o r 90 min. I n j e c t i o n volume f o r GCMS: 3 /xL. (D) P r e p a r a t i o n and A n a l y s i s o f L i v e r F r a c t i o n s . A n i m a l s were housed i n s t a i n l e s s s t e e l cages w i t h c o r n cob b e d d i n g , and p r o v i d e d a c c e s s ad libitum t o f o o d and w a t e r u n t i l t h e t i m e o f s a c r i f i c e . R e g u l a r 12 h c y c l e s o f l i g h t and d a r k were p r o v i d e d . F o u r a n i m a l s were i n t r a p e r i t o n e a l l y a d m i n i s t e r e d p h e n o b a r b i t o n e sodium (26 mg/mL) a t 78 mg/kg-body w e i g h t i n an i s o t o n i c s a l i n e s o l u t i o n once d a i l y f o r 4 d a y s . Four o t h e r a n i m a l s were a d m i n i s t e r e d t h e v e h i c l e ( s a l i n e c o n t r o l ) . A n i m a l s were s a c r i f i c e d by d e c a p i t a t i o n . L i v e r s were i m m e d i a t e l y removed, s h e a r e d w i t h a s c i s s o r s and homogenized i n i c e - c o l d 37 T r i s - H C l (0.05 M)/KC1 (1.15 %) b u f f e r (pH 7.5) w i t h a P o t t e r - E l v e h j e m t i s s u e homogenizer. The homogenate was c e n t r i f u g e d a t 9,000 g f o r 20 min a t 5°C and f i l t e r e d t h r o u g h cheese c l o t h . The r e s i d u e was d i s c a r d e d , and t h e s u p e r n a t a n t (S9 f r a c t i o n ) c e n t r i f u g e d a t 105,000 g f o r 60 min a t 5°C. The c y t o s o l i c f r a c t i o n was r e c o v e r e d as t h e s u p e r n a t a n t and s t o r e d a t -80°C u n t i l r e q u i r e d , w h i l e t h e microsomal p e l l e t was res u s p e n d e d i n EDTA (10 mM)/KCl (1.15 %) b u f f e r (pH 7.4). T h i s homogenate was a g a i n c e n t r i f u g e d a t 105,000 g f o r 60 min a t 5°C. The s u p e r n a t a n t was d i s c a r d e d and t h e microsomal p e l l e t , a f t e r r e s u s p e n s i o n i n an a p p r o x i m a t e l y equal volume o f 0.25 M s u c r o s e s o l u t i o n , s t o r e d a t -80°C u n t i l r e q u i r e d . Microsomes were d i l u t e d 1:20 i n N a 2 P 0 4 (0.1 M, pH 7 . 4 ) / g l y c e r o l (20 %)/EDTA (0.1 mM) f o r d e t e r m i n a t i o n o f cytochrome P-450 c o n t e n t u s i n g t h e method o f Omura and Sa t o ( 1 9 6 4 ) . Cytochrome P-450 l e v e l s were 18.9 nmol/mL and 33.9 nmol/mL f o r c o n t r o l and i n d u c e d microsomes r e s p e c t i v e l y . Cytochrome P-450 a s s a y s were performed a t t h e tim e t h e microsomes were t o be used. P r o t e i n c o n c e n t r a t i o n s o f microsomal and c y t o s o l i c f r a c t i o n s were p e r f o r m e d a c c o r d i n g t o t h e method o f Lowry e t a l . (1951) u s i n g BSA as s t a n d a r d . P r o t e i n c o n c e n t r a t i o n s f o r t h e d i f f e r e n t f r a c t i o n s were as f o l l o w s : c o n t r o l m i c r o s o m a l : 20.8 mg/mL, c o n t r o l c y t o s o l i c : 16.1 mg/mL, i n d u c e d m i c r o s o m a l : 18.6 mg/mL and i n d u c e d c y t o s o l i c : 20.4 mg/mL. Gel e l e c t r o p h o r e s i s was performed a c c o r d i n g t o t h e method o f Laemmli ( 1 9 7 0 ) , u s i n g 7.5 % sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE). A p p r o x i m a t e l y 10 ug o f microsomal p r o t e i n was l o a d e d on t o each w e l l . The i m m u n o b l o t t i n g p r o c e d u r e o f Towbin e t a l . (1979) was used f o r t h e t r a n s f e r o f s e p a r a t e d microsomal p r o t e i n from t h e g e l t o a n i t r o c e l l u l o s e membrane. Development o f t h e b l o t was a c c o m p l i s h e d w i t h 38 t h e s t e p w i s e use o f r a b b i t a n t i - P - 4 5 0 b/e p o l y c l o n a l a n t i b o d y as t h e p r i m a r y a n t i b o d y , and p e r o x i d a s e c o n j u g a t e d g o a t a n t i - r a b b i t as t h e s e c o n d a r y a n t i b o d y a t a d i l u t i o n o f 1:25,000. (E) M i c r o s o m a l I n c u b a t i o n s . ( i ) G e n e r a l i n c u b a t i o n p r o c e d u r e . A l l cytochrome P-450 i n c u b a t i o n s i n v o l v e d t h e use o f b o t h c o n t r o l and P B - i n d u c e d microsomes, and were pe r f o r m e d a c c o r d i n g t o t h e method o f R e t t i e e t a l . ( 1 9 8 8 ) . I n t o a c u l t u r e tube were p l a c e d sodium phosphate b u f f e r (0.2M, pH 7.4): 1 mL, M g C l 2 (0.3 M): 50 xxL, NADPH (0.1 M): 50 /xL, NADH (0.1 M): 50 /xL, microsomal cytochrome P-450 (10 nmol) and d i s t i l l e d w a t e r t o a f f o r d a f i n a l volume o f 2.1 mL. The enzyme was a l l o w e d t o i n c u b a t e f o r 3 min, a f t e r w h i c h s u b s t r a t e (2 /xmol) was added i n a 10 % m e t h a n o l i c s u s p e n s i o n . The r e a c t i o n was t e r m i n a t e d by f r e e z i n g t h e m i x t u r e i n an i c e - b a t h . A f t e r 5 min t h e r e a c t i o n m i x t u r e was e x t r a c t e d w i t h an e q u a l volume o f EtOAc (x 4 ) . C e n t r i f u g a t i o n was i n v a r i a b l y r e q u i r e d t o s e p a r a t e t h e e m u l s i o n . The o r g a n i c e x t r a c t was d r i e d o v e r anhydrous Na2S04, c o n c e n t r a t e d t o d r y n e s s under a s t r e a m o f N 2 , r e c o n s t i t u t e d i n 25 /xL o f EtOAc and d e r i v a t i z e d w i t h 25 /xL MSTFA a t 45°C f o r 90 min. I n j e c t i o n volume: 3 /xL. ( i i ) S p e c i f i c i n c u b a t i o n c o n d i t i o n s . (a) M i c r o s o m a l m e t a b o l i s m o f CLBZ t o i n v e s t i g a t e c a r b i n o l a m i d e  f o r m a t i o n . The s u b s t r a t e , CLBZ:[ 2H5]CLBZ, was p r e p a r e d as a m i x t u r e w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f 37.8:62.2 f o r mass chromatograms a t m/z 300 and 305 r e s p e c t i v e l y , i .e . C L B Z / [ 2 r l s ] C L B Z = 0.608. I n c u b a t i o n t i m e s were 15 min and 60 min. (b) M i c r o s o m a l m e t a b o l i s m o f DMC t o i n v e s t i g a t e h ydroxamic a c i d  f o r m a t i o n . The s u b s t r a t e , DMC:[ 2H5]DMC, was p r e p a r e d as a m i x t u r e w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f i . e . 45.0:55.0 f o r t h e mass chromatograms o f t h e T M S - d e r i v a t i v e s a t m/z 358 and 363 r e s p e c t i v e l y , i . e . DMC/[ 2H5JDMC = 0.818. I n c u b a t i o n t i m e s were p e r f o r m e d o v e r 15 and 60 min. ( c ) D e t e r m i n a t i o n o f N - d e m e t h v l a t i o n i n t e r m o l e c u l a r i s o t o p e e f f e c t . [ 2 H 5 ] C L B Z : [ 2 H 3 ] C L B Z was p r e p a r e d as an a p p r o x i m a t e 50:50 (w/w) m i x t u r e , w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f 54.1:45.9 f o r mass chromatograms a t m/z 305 and 303 r e s p e c t i v e l y , i . e . [ 2H5]CLBZ/[ 2H3]CLBZ = 1.178. I n c u b a t i o n s were performed o v e r 15, 30, 45 and 60 min. 40 6. CHEMICAL SYNTHESES. (A) Medium p r e s s u r e l i q u i d ( F l a s h ) chromatography. P u r i f i c a t i o n s i n v o l v i n g medium p r e s s u r e column chromatography were p e r f o r m e d a c c o r d i n g t o t h e method o f S t i l l e t a l . (1978) on s i l i c a columns o f d i a m e t e r s 1.5 cm, 2.5 cm o r 5.5 cm, and v a r i a b l e l e n g t h d e p e n d i n g on t h e amount o f m a t e r i a l t o be c h romatographed. M o b i l e phases were p r e p a r e d i n o r d e r t o a f f o r d an Rf o f 0.3 - 0.5 f o r t h e a n a l y t e on TLC. I f improved r e s o l u t i o n was n e c e s s a r y , t h e p o l a r i t y o f t h e m o b i l e phase was i n c r e a s e d i n a g r a d i e n t f a s h i o n d u r i n g e l u t i o n . (B) S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l c l o b a z a m ( T ^ I C L B Z ) . A n i l i n e - d y ( 1 7 ) . I n t o a t h r e e - n e c k e d f l a s k e q u i p p e d w i t h a m e c h a n i c a l s t i r r e r and a d r o p p i n g f u n n e l f i l l e d w i t h DC1 (15 mL, 16.5 mmol) were p l a c e d nitrobenzene - d s (20.2 g, 15.8 mmol), D2O (50 mL) and i r o n powder (24.2 g, 46.9 mmol). The r e a c t i o n f l a s k was f l u s h e d w i t h N 2 and s e a l e d under t h e s l i g h t p o s i t i v e p r e s s u r e o f a N 2 f i l l e d b a l l o o n a t t a c h e d t o a r e f l u x c o n d e n s e r . To t h e s t i r r e d m i x t u r e was added DC1 a t a r a t e t o m a i n t a i n t h e t e m p e r a t u r e below 80°C. When t h e a d d i t i o n o f DC1 was c o m p l e t e t h e m i x t u r e was s t i r r e d a t 80°C f o r 12 h. The r e s u l t i n g m i x t u r e was c o o l e d i n an i c e - b a t h and t h e pH a d j u s t e d t o 12 w i t h NaOD w h i l e m a i n t a i n i n g t h e r e a c t i o n m i x t u r e under N 2 . Under a N 2 atmosphere s o l i d s were removed by f i l t r a t i o n and t h e f i l t r a t e appeared as a 41 y e l l o w i s h - g r e e n l i q u i d w i t h a suspended p a l e brown o i l . KC1 ( c a . 10 g) was added t o t h e f i l t r a t e t o " s a l t o u t " aqueous a n i l i n e w h i c h was e x t r a c t e d w i t h e t h e r and c o n c e n t r a t e d in vacuuo t o a r u s t c o l o u r e d o i l . F r a c t i o n a l d i s t i l l a t i o n gave 12.7 g (80 %) o f ani l i n e - d z as a s l i g h t l y v i s c o u s c o l o u r l e s s l i q u i d : bp 28 - 30"C (0.4 t o r r ) . 2H-NMR S 3.47 ( s , 1.80D*, -ND 2), 6.60 ( s , 2D, ortho), 6.78 ( s , ID, para), 7.17 ( s , 2D, meta). (*N,N-D2 i s o t o p i c p u r i t y c a l c u l a t e d a t 90 % from i n t e g r a l ) . N - ( 5 - C h l o r o - 2 - n i t r o p h e n v l ) - p e n t a d e u t e r i o p h e n y l a m i n e ( 1 8 ) . The p e n t a d e u t e r i o p h e n y l a m i n e 18 was s y n t h e s i z e d by u s i n g a p p r o p r i a t e m o d i f i c a t i o n s t o t h e p r o c e d u r e o f Werner and Wagner ( 1 9 7 8 ) . Dry d i c h l o r o n i t r o b e n z e n e (2.23 g, 11.6 mmol) was d i s s o l v e d i n 17 (4.60 g, 3.6 e q u i v ) t o a f f o r d an orange s o l u t i o n . The s t i r r e d s o l u t i o n was heat e d a t 170 - 180°C under r e f l u x i n a N 2 atmosphere f o r 8.5 h and b e f o r e c o o l i n g , quenched under N 2 w i t h c o n t i n u e d s t i r r i n g by c a u t i o u s a d d i t i o n o f c a . 10 mL o f d r y benzene. The r e s u l t i n g b l a c k s o l u t i o n was a l l o w e d t o r e a c h room t e m p e r a t u r e and f u r t h e r d i l u t e d w i t h an a d d i t i o n a l 30 mL d r y benzene. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (15 cm x 5.5 cm) u s i n g t h e f o l l o w i n g g r a d i e n t e l u t i o n : benzene (100 mL); 2:1 ( v / v ) p e t r o l e u m e t h e r : e t h e r (240 mL); 3:2 ( v / v ) p e t r o l e u m e t h e r : e t h e r (625 mL). The p r o d u c t - c o n t a i n i n g f r a c t i o n s were p o o l e d and s o l v e n t removed in vacuuo t o a f f o r d 2.69 g (91 %) o f 18 as b r i g h t orange n e e d l e l i k e c r y s t a l s : mp 109 - HO'C. Rf 0.79, m o b i l e phase, 2:1 ( v / v ) p e t r o l e u m e t h e r : E t O A c . !H-NMR S 6.74 (dd, J = 10 Hz and 2 Hz, 1H, H-4), 7.16 ( d , J = 2 Hz, 1H, 42 H-6), 7.27 - 7.31 (m, 0.23H*, H-2'/H-6' and H-4'), 8.18 ( d , J = 10 Hz, 1H, H-3), 9.52 ( b s , 1H, N-H #). ( i n t e g r a t i o n i n d i c a t e d t h a t 8 % p r o t o n a t i o n o f t h e ortho and para p o s i t i o n s had t a k e n p l a c e . ^Does not exchange w i t h D2O) . 1 3C-NMR 8 114.99 ( C - 4 ) , 117.73 ( C - 6 ) , 124.26 ( t , C-2'/C-6'), 125.77 ( t , C-4'), 127.97 ( C - 3 ) , 129.35 ( t , C-3'/C-5'), 131.32 ( C - l ' ) , 137.59 ( C - l ) , 142.34 ( C - 5 ) , 143.75 ( C - 2 ) . GCMS ( d ) : t R , 5.35 min, M +'( 3 5C1): m/z 253 (100 % ) , M + - ( 3 7 C 1 ) : m/z 255 (37 % ) . IR (CCI4 s o l u t i o n ) 3345 (N-H), 1487 (N0 2)asymm, 1310 cm" 1 (N0 2)symm. E t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n v l ) - N - p e n t a d e u t e r i o p h e n y l carbamoyl a c e t a t e H D . . To a s t i r r e d s o l u t i o n o f 18 (2.76 g, 10.9 mmol) i n c a . 25 mL d r y benzene was added e t h y l m a l o n y l c h l o r i d e (4.12 g, 27.3 mmol) and t h e r e s u l t i n g s o l u t i o n h e a t e d a t 75 - 80°C under r e f l u x f o r 20 h. The m i x t u r e was washed w i t h s a t u r a t e d NaHC03, d r i e d , and c o n c e n t r a t e d t o a p a l e brown o i l . The c r u d e p r o d u c t was chromatographed on a s i l i c a column (15. cm x 5.5 cm) u s i n g t h e f o l l o w i n g g r a d i e n t : benzene (100 mL); 2:1 ( v / v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (450 mL); 3:2 (v/ v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (250 mL). P r o d u c t - c o n t a i n i n g f r a c t i o n s were p o o l e d and c o n c e n t r a t e d t o a f f o r d 3.31 g (85 %) o f 19 as p a l e y e l l o w c r y s t a l s : mp 90 - 92°C. Rf 0.49, m o b i l e phase, 2:1 ( v / v ) p e t r o l e u m e t h e r : E t 0 A c . ^-NMR 6 1.26 ( t , 3H, -CH 2CH 3), 3.40 ( s , 2H, -C(=0)CH 2C=0), 4.17 ( q , 2H, -0CH 2CH 3), 7.21 ( d , J = 1 Hz, 1H, H-6), 7.35 ( d d , J = 9 Hz and 1 Hz, 1H, H-4), 7.93 ( d , J = 9 Hz, 1H, H-3). 43 GCMS ( d ) : t R , 5.34 min, [M-114] + - ( 3 5C1): m/z 253 (100 % ) , [M-114]" 1"' ( 3 7 C 1 ) : m/z 255 (37 % ) . I R (CCI4 s o l u t i o n ) 1745 ( e s t e r c a r b o n y l g r o u p ) , 1693 (amide c a r b o n y l g r o u p ) , 1534 (N02)asymm, 1351 cm" 1 (N02)symm. P e n t a d e u t e r i o p h e n y l N-desmethvlclobazam ( 2 0 ) . To a s t i r r e d s u s p e n s i o n o f 19 (2.0 g, 5.45 mmol) i n 17 mL e t h a n o l and c o n c e n t r a t e d HCl (10 mL, 110 mmol) was c a u t i o u s l y added Zn d u s t (1.74 g, 26.6 mmol) o v e r 30 min w h i l e m a i n t a i n i n g t h e t e m p e r a t u r e between 25 - 30°C. The m i x t u r e was s t i r r e d a t 30°C f o r an a d d i t i o n a l 2 h t o a f f o r d 20 as a w h i t e p r e c i p i t a t e . The p r o d u c t was removed, t h e s u p e r n a t a n t t r e a t e d w i t h a d d i t i o n a l Zn (0.58 g, 8.9 mmol) as p r e v i o u s l y d e s c r i b e d , and s t i r r i n g c o n t i n u e d o v e r n i g h t a t 30°C t o a f f o r d a second c r o p o f 20. The combined p r o d u c t was washed w i t h c o l d 3:2 ( v / v ) ac e t o n e : w a t e r and d r i e d in vacuuo t o a f f o r d 0.56 g (35 %) o f 20 as an amorphous w h i t e s o l i d . ! H - N M R 8 3.55 ( s , 2H, C H 2 ) , 6.93 ( d , J = 3 Hz, 1H, H-6), 7.11 ( d , J = 9 Hz, 1H, H-9), 7.21 (dd, J = 9 Hz and 3 Hz, 1H, H-8), 8.21 ( b s , 1H, N-H). GCMS ( d ) : t R , 8.64 min, M + - ( 3 5 C 1 ) : m/z 291 (92 % ) , M + - ( 3 7 C 1 ) : m/z 293 (30 % ) , [M-42] + - ( 3 5 C l ) : m/z 249 (84 % ) , C 6 D 5 + - : m/z 100 (41 % ) . TMS d e r i v a t i v e : t R , 7.34 min, M + - ( 3 5 C 1 ) : m/z 363 (26 % ) , M + - ( 3 7 C 1 ) m/z 365 (10 % ) , [ M - 1 5 ] + - m/z 348 (20 % ) , [ M - 1 2 4 ] + - m/z 239 (46 % ) , [ M - 1 3 8 ] + - m/z 225 (100 % ) . I R ( N u j o l m u l l ) 3176 (N-H), 1691 and 1673 cm" 1 (amide c a r b o n y l g r o u p s ) . 44 P e n t a d e u t e r i o p h e n y l clobazam ( 2 1 ) . M e t h y l a t i o n o f 20 was per f o r m e d a c c o r d i n g t o t h e method o f I s e l e and L u t t r i n g h a u s ( 1 9 7 1 ) . To an i c e - c o o l e d (15°C) s o l u t i o n o f 20 (491 mg, 1.68 mmol) and p u l v e r i z e d KOH (114 mg, 2.05 mmol) i n 6 mL DMSO was added CH3I (193 ZJL, 3.06 mmol) w i t h s t i r r i n g . The r e a c t i o n was m a i n t a i n e d a t t h i s t e m p e r a t u r e f o r 30 min a f t e r w h i c h i t was a l l o w e d t o r e a c h room t e m p e r a t u r e w i t h c o n t i n u e d s t i r r i n g f o r an a d d i t i o n a l 90 min. The r e a c t i o n m i x t u r e was d i l u t e d w i t h 300 mL e t h y l a c e t a t e , washed w i t h a s a t u r a t e d NaCl s o l u t i o n , d r i e d o v e r anhydrous Na2S04, and c o n c e n t r a t e d t o a y e l l o w o i l . The crude p r o d u c t was d i s s o l v e d i n ca 10 mL benzene and chromatographed on a s i l i c a column (15 cm x 2.5 cm) u s i n g 210 mL o f 20:1 ( v / v ) CHCl3:Me0H. D e s p i t e s e v e r a l m o d i f i c a t i o n s t o t h e c h r o m a t o g r a p h i c c o n d i t i o n s , u n r e a c t e d 20 c o u l d n ot be s e p a r a t e d from 21 i n i t s l a t t e r f r a c t i o n s . Hence, t h e m i x t u r e was c o n c e n t r a t e d and r e -chromatographed. F r a c t i o n s c o n t a i n i n g o n l y 21 were c o n c e n t r a t e d t o a p a l e y e l l o w o i l and t h e p r o d u c t r e c r y s t a l l i z e d from e t h y l a c e t a t e / p e t r o l e u m e t h e r t o a f f o r d 188 mg (37 %) o f 21 i n q u a n t i t a t i v e i s o t o p i c p u r i t y as w h i t e c r y s t a l s : mp 182 - 185°C. *H-NMR (CDCI3) 6 3.50 ( d , J = 10 Hz, 1H, H-30*), 3.51 ( s , 3H, N-CH3), 3.56 ( d , J = 10 Hz, 1H, H-3a*), 6.93 ( d , J = 3 Hz, H-6), 7.25 (dd, J = 10 Hz and 3 Hz, 1H, H-8), 7.31 ( d , J = 10 Hz, 1H, H-9). (*Assignments c o n s i s t e n t w i t h t h e c o n f o r m a t i o n a l s t r u c t u r e p r o p o s e d by A v e r s a e t a l . ( 1 9 8 0 ) . !H-NMR (C 6D6) 5 2.81 ( d , J = 12 Hz, 1H, H-3/3), 2.95 ( s , 3H, N-CH 3), 3.46 ( d , J = 12 Hz, 1H, H-3a), 6.38 ( d , J = 9 Hz, 1H, H-9), 6.74 (dd, J = 9 Hz and 3 Hz, 1H, H-8), 6.79 ( d , J = 3 Hz, H-6). 45 MS: M +'( 3 5C1): m/z 305 (100 % ) , M +- ( 3 7 C 1 ) : m/z 307 (34 % ) , [ M - 1 7 ] + - : m/z 288 (43 % ) , CeD5+-: m/z 82 (48 % ) . IR (CCI4 s o l u t i o n ) 1711 and 1686 cm"* (amide c a r b o n y l g r o u p s ) . HPLC: The r e c r y s t a l l i z e d 21 was a n a l y z e d by HPLC u s i n g a m o b i l e phase o f 60:40 ( v / v ) MeOH:H20 and found t o have a r e t e n t i o n t i m e o f 6.17 min and p u r i t y o f >98 %. (C) S y n t h e s i s o f T r i d e u t e r i o m e t h v l c l o b a z a m ( T ^ I C L B Z ) (22). I2M3lCLBZ (22) was s y n t h e s i z e d from DMC (101 mg, 0.35 mmol) and CD3I u s i n g t h e approach used f o r 21. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 cm x 1.25 cm) u s i n g a m o b i l e phase o f 20:1 ( v / v ) Me0H:CHCl3 (60 mL). E a r l y f r a c t i o n s c o n t a i n i n g pure 22 were c o n c e n t r a t e d t o a y e l l o w o i l wh i c h was c r y s t a l l i z e d from EtOAc:pet e t h e r t o a f f o r d 34 mg (32 %) o f w h i t e n e e d l e l i k e c r y s t a l s . The y i e l d on t h i s s y n t h e s i s c o u l d have been improved i f t h e l a t t e r f r a c t i o n s c o n t a i n i n g 22 and c o n t a m i n a t i n g DMC were r e c h r o m a t o g r a p h e d , however, no a t t e m p t was made t o do so. JH-NMR 6 3.52 ( d , J = 12 Hz, 1H, H-30), 3.58 ( d , J = 12 Hz, 1H, H-3a), 6.94 ( d , J = 3 Hz, H-6), 7.20 (dd, J = 8 Hz and 2 Hz, 2H, H-2', H-6'), 7.24 (dd, J = 10 Hz and 2 Hz, 1H, H-8), 7.31 ( d , J = 10 Hz, 1H, H-9), 7.36 (m, 1H, H-4'), 7.43 (m, 2H, H-3', H-5'). MS: M + ' ( 3 5 C 1 ) : m/z 303 (100 % ) , M+- ( 3 7 C 1 ) : m/z 305 (34 % ) , [M-17] + *: m/z 286 (51 % ) , [ M - 6 0 ] + - : m/z 243 (22 % ) , [ M - 1 1 9 ] + ' : m/z 184 (23 % ) . 46 (D) S y n t h e s i s o f R i n g - O x i d i z e d M e t a b o l i t e s o f CLBZ. ( i ) C h e m i c al S y n t h e s e s . N - ( 5 - C h 1 o r o - 2 - n i t r o p h e n y l ) - 4 - m e t h o x y p h e n y l a m i n e ( 2 3 ) . 4-Methoxyphenylamine (11.38 g, 92.5 mmol) and 2 , 4 - d i c h l o r o -n i t r o b e n z e n e (4.96 g, 25.8 mmol) were f u s e d , and h e l d under r e f l u x a t 170 - 180°C f o r 9 h. The r e s u l t i n g s o l u t i o n was d i l u t e d w h i l e h ot w i t h benzene and t h e c r u d e p r o d u c t chromatographed on a s i l i c a column (15 cm x 5.5 cm) u s i n g 1:1 ( v / v ) pet e t h e r : e t h e r (800 mL). The p u r i f i e d 23 was c o n c e n t r a t e d in vacuuo t o a f f o r d 6.20 g (86 %) o f r u s t y brown c r y s t a l s . *H-NMR 8 3.85 ( s , 3H, -0CH 3), 6.69 ( d d , J = 8 Hz and 2 Hz, 1H, H-4), 6.96 ( d , J = 2 Hz, 1H, H-6), 7.05 ( d , J = 8 Hz, 2H, H-2', 6 ' ) , 7.20 ( d , J = 8 Hz, 2H, H-3', 5 ' ) , 8.16 ( d , J = 8 Hz, 1H, H-3), 9.43 ( b s , 1H, N-* * H ). ( Does not exchange w i t h D2O). GCMS ( d ) : t R , 7.00 min, M + - ( 3 5 C 1 ) : m/z 278 (100 % ) , M + ' ( 3 7 C 1 ) : m/z 280 (31 % ) , [ M - 1 5 ] + - ( 3 5 C 1 ) : m/z 263 (44 % ) , [ M - 1 5 ] + - ( 3 7 C 1 ) : m/z 265 (14 % ) . E t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n v 1 ) - N - ( 4 - m e t h o x y p h e n v 1 ) c a r b a m o y l a c e t a t e 1241. The carbamoyl a c e t a t e 24 was s y n t h e s i z e d u s i n g 23 (4.20 g, 14.4 mmol) and e t h y l m a l o n y l c h l o r i d e (4.32 g, 28.6 mmol) f o l l o w i n g t h e method used f o r t h e s y n t h e s i s o f 19. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (20 cm x 5.5 cm) u s i n g t h e f o l l o w i n g g r a d i e n t : 2:1 ( v / v ) p e t ether-.EtOAc (900 mL); 3:2 ( v / v ) p e t e t h e r : E t 0 A c 47 (550 mL). P u r i f i e d 24 was c o n c e n t r a t e d t o a p a l e y e l l o w o i l w h i c h was c r y s t a l l i z e d f r o m 1:1 ( v / v ) p e t e t h e r : e t h e r t o a f f o r d 4.89 g (87 %) o f p a l e y e l l o w c u b e s . !H-NMR 6 1.27 ( t , 3H, -CH 2CH 3), 3.38 ( s , 2H, C(=0)CH 2C(=0), 3.85 ( s , 3H, -0C H 3 ) , 4.17 ( q , 2H, CH 2 C H 3 ) , 6.98 ( d , J = 7 Hz, 2H, H-2', H-6'), 7.21 ( d , J = 1 Hz, 1H, H-6), 7.34 (dd, J = 7 Hz and 1 Hz, 1H, H-4), 7.45 ( d , J = 7 Hz, 2H, H-3', H-5'), 7.92 ( d , J = 7 Hz, 1H, H-3). GCMS ( d ) : t R , 7.00 min, [ M - 1 1 4 ] + - ( 3 5 C 1 ) : m/z 278 (100 % ) , [M-114] + - ( 3 7 C 1 ) : m/z 280 (31 % ) , [M-114-15] +- ( 3 5C1): m/z 263 (44 % ) , [M-1 1 4 - 1 5 ] + ' ( 3 7 C 1 ) : m/z 265 (14 % ) . 4'-Methoxy N-desmethvlclobazam ( 2 5 ) . The DMC analogue 25 was s y n t h e s i z e d a c c o r d i n g t o t h e method used f o r 20 by u s i n g 24 (2.01 g, 5.12 mmol) as t h e s t a r t i n g m a t e r i a l . The p r o d u c t o c c u r r e d as 620 mg (38 %) o f w h i t e amorphous s o l i d . !H-NMR 6 3.52 ( s , 2H, -CH 2-), 3.84 ( s , 3H, OCH3), 6.96 (dd, J = 9 Hz and 3 Hz, 3H, H-6 and H-2', 6 ' ) , 7.07 ( d , J = 9 Hz, 1H, H-9), 7.12 ( d , J = 9 Hz, 2H, H-3', 5'), 7.19 (dd, = 9 Hz and 3 Hz, 1H, H-8), 7.77 ( b s , 1H, N-H). GCMS ( d ) , TMS d e r i v a t i v e : t R , 8.85 min, M+- ( 3 5C1): m/z 388 (36 % ) , M+-( 3 7C1) m/z 390 (14 % ) , [ M - 1 5 ] + - m/z 373 (15 % ) , [ M - 1 4 9 ] + - m/z 239 (34 % ) , [ M - 1 6 3 ] + - m/z 225 (100 % ) . 4'-Hydroxy N-desmethvlclobazam ( 2 6 ) . The s y n t h e s i s o f 26 was c a r r i e d o u t by s u s p e n d i n g 25 i n d r y CHCI3 48 under N 2 i n a septum s e a l e d f l a s k . The s u s p e n s i o n was c o o l e d i n an a c e t o n e / d r y i c e bath ( c a . -40°C) and w h i l e s t i r r i n g BBr3 (252 mg, 1.00 mmol) added by s y r i n g e . S h o r t l y t h e r e a f t e r , t h e s o l v e n t began t o f r e e z e and so t h e f l a s k was t e m p o r a r i l y w i t h d r a w n from t h e b a t h . P e r i o d i c w i t h d r a w a l from t h e ba t h was c o n t i n u e d f o r t h e f o l l o w i n g 5 min t o e n s u r e t h a t t h e s o l v e n t remained l i q u i d . A f t e r t h a t t i m e t h e m i x t u r e was a l l o w e d t o r e a c h room t e m p e r a t u r e and s t i r r i n g c o n t i n u e d f o r 5 h. TLC o f t h e r e a c t i o n i n 10:1 ( v / v ) CHCl3:Me0H showed an e s s e n t i a l l y q u a n t i t a t i v e c o n v e r s i o n o f 25 ( R f 0.52) t o a more p o l a r p r o d u c t ( R f , 0.37). The r e a c t i o n was quenched w i t h t h e d r o p w i s e a d d i t i o n o f MeOH, c o n c e n t r a t e d i n vacuuo, r e d i s s o l v e d i n CHCI3 (70 mL), washed w i t h aqueous NaCl s o l u t i o n , and d r i e d o v e r anhydrous Na 2S04. The o r g a n i c s o l u t i o n was c o n c e n t r a t e d i n vacuuo, r e d i s s o l v e d i n c a 2 mL CHCI3 w i t h a few d r o p s o f MeOH and chromatographed on a s i l i c a column (14 cm x 1.25 cm) u s i n g a m o b i l e phase o f 10:1 ( v / v ) CHCl3:Me0H. The p u r i f i e d 26 was c o n c e n t r a t e d i n vacuuo t o a f f o r d 10 mg (26 %) o f amorphous w h i t e s o l i d . JH-NMR (DMS0-d 6) 6 3.35 ( b u r i e d i n H 20, - C H 2 - ) , 6.85 ( d , J = 9 Hz, 2H, H-2', H-6'), 7.06 ( d , J = 9 Hz, 2H, H-3', H-5'), 7.25 - 7.40 (m, 3H, H-6, H-8, H-9), 9.77 ( s , 1H, 4'-0H), 10.68 ( s , 1H, N-H). GCMS ( d ) , T M S - d e r i v a t i v e : 12.45 min, M + - ( 3 5 C 1 ) : m/z 446 (71 % ) , [M-1 5 ] + - : m/z 431 (27 % ) , [ M - 4 3 ] + - : m/z 403 (10 % ) , [ M - 1 1 5 ] + - : m/z 331 (11 % ) , [ M - 2 0 7 ] + - : m/z 239 (39 % ) , [ M - 2 2 1 ] + - : m/z 225 (100 % ) . 4'-Methoxy clobazam ( 2 7 ) . The s y n t h e s i s o f 27 from 25 (250.9 mg, 0.79 mmol) and CH3I (90 ul, 1.44 mmol) was a c c o r d i n g t o t h e method used f o r 21. No c h r o m a t o g r a p h i c 4 9 p u r i f i c a t i o n was n e c e s s a r y a f t e r work-up because o f t h e e s s e n t i a l l y q u a n t i t a t i v e f o r m a t i o n o f 27. I n s t e a d , t h e c r u d e p r o d u c t was r e c r y s t a l l i z e d from EtOAc:pet e t h e r t o a f f o r d 180.6 mg (69 %) o f 27 as w h i t e c r y s t a l s . !H-NMR S 3.47 ( d , J = 12 Hz, 1H, H-3/3), 3.50 ( s , 3H, N-CH 3), 3.55 ( d , J = 12 Hz, 1H, H-3a), 3.83 ( s , 3H, - 0 C H 3 ) , 6.94 ( d , J = 10 Hz, 2H, H-2', H-6'), 7.11 ( d , J = 10 Hz, 2H, H-3', H-5'), 7.23 (dd, J = 8 Hz and 2.5 Hz, 1H, H-8), 7.26 ( d , J = 2.5 Hz, 1H, H-6), 7.28 ( d , J - 8 Hz, 1H, H-9 ) . GCMS ( d ) : t R , 10.11 min, M + - ( 3 5 C 1 ) : m/z 330 (100 % ) , M+- ( 3 7 C 1 ) : m/z 332 (32 % ) , [ M - 1 7 ] + - : m/z 313 (32 % ) , [ M - 5 7 ] + " : m/z 273 (50 % ) , [M-149] +«: m/z 181 (22 % ) . 4'-Hydroxy clob a z a m ( 2 8 ) . The f o l l o w i n g i s an improved d e m e t h y l a t i o n p r o c e d u r e o v e r t h a t used f o r 26. In d r y CHCI3 27 (51.3 mg, 0.155 mmol) was d i s s o l v e d , t h e f l a s k f l u s h e d w i t h N2, and s e a l e d w i t h a r u b b e r septum. The s o l u t i o n was c o o l e d i n an i c e / s a l t b a t h (-5°C) and w h i l e s t i r r i n g BBr3 (236 mg, 0.94 mmol) was added by s y r i n g e . The r e s u l t i n g s u s p e n s i o n was a l l o w e d t o r e a c h room t e m p e r a t u r e and s t i r r i n g c o n t i n u e d f o r an a d d i t i o n a l 5 h. A t t h a t t i m e TLC a n a l y s i s o f t h e r e a c t i o n m i x t u r e i n 10:1 CHCl3:MeOH showed t h a t t h e r e was an e s s e n t i a l l y q u a n t i t a t i v e c o n v e r s i o n o f 27 ( R f , 0.77) t o a more p o l a r p r o d u c t ( R f , 0.50). The r e a c t i o n was quenched w i t h t h e d r o p w i s e a d d i t i o n o f H2O, s t i r r e d f o r an a d d i t i o n a l hour, t a k e n up i n t o CHCI3 (70 mL), washed w i t h aqueous NaCl s o l u t i o n , and d r i e d o v e r anhydrous Na2S04. The o r g a n i c s o l u t i o n was c o n c e n t r a t e d in vacuuo t o 50 a f f o r d a p u r p l e gum whi c h was d i s s o l v e d i n c a 2 mL EtOAc and t o a v o i d c r y s t a l l i z a t i o n i m m e d i a t e l y chromatographed on a s i l i c a column (14 x 1.5 cm) w i t h t h e f o l l o w i n g m o b i l e phase: EtOAc (2 mL), 10:1 ( v / v ) CHCl3:MeOH (45 mL). P u r i f i e d 28, c o n t a i n i n g 27 as a min o r (<5 %) c o n t a m i n a n t , was c o n c e n t r a t e d in vacuuo t o a f f o r d 44.7 mg (91 %) o f w h i t e c r y s t a l s . !H-NMR 6 3.49 ( d , J = 12 Hz, 1H, H-3/5), 3.50 ( s , 3H, N-CH 3), 3.57 ( d , J = 12 Hz, 1H, H-3a), 6.06 ( b s , 1H, 3'-0H), 6.82 ( d , J = 10 Hz, 2H, H-2', H-6'), 6.97 ( d , J = 2.5 Hz, 1H, H-6), 7.01 ( d , J = 10 Hz, 2H, H-3', H-5' ) , 7.24 (dd, J = 12 Hz and 2.5 Hz, 1H, H-8), 7.30 ( d , J = 12 Hz, 1H, H-9), 7.30 ( d , J = 12 Hz, 1H, H-9). C o n t a m i n a t i n g peaks: 4.14, q, EtOAc; 3.85, s, 4'-methoxy CLBZ (<5 % ) . GCMS ( d ) , T M S - d e r i v a t i v e : t R , 15.65 min, M + - ( 3 5 C 1 ) : m/z 388 (100 % ) , [M-1 5 ] + - : m/z 373 (22 % ) , [ M - 1 8 ] + - : m/z 371 (21 % ) , [ M - 4 2 ] + - : m/z 346 (19 % ) , [ M - 4 5 ] + - : m/z 343 (50 % ) , [ M - 5 7 ] + < : m/z 331 (29 % ) , [M-207J+-: m/z 181 (17 % ) , [M-235]"1"-: m/z 153 (13 % ) . N - ( 5 - C h l o r o - 2 - n i t r o p h e n y 1 ) - 3 . 4 - d i m e t h o x y p h e n v 1 a m i n e ( 2 9 ) . The s y n t h e s i s o f 29 from 2 , 4 - d i c h l o r o n i t r o b e n z e n e (4.99 g, 26 mmol) and 3 , 4 - d i m e t h o x y a n i l i n e (13.90 g, 91 mmol) was a c c o r d i n g t o t h e p r o c e d u r e used f o r 23. The cr u d e p r o d u c t was t a k e n up i n ca 80 mL benzene, and t h e s o l u t i o n d i v i d e d i n t o 2 equal p o r t i o n s . Each p o r t i o n was chromatographed on a s i l i c a column (19 cm x 5.5 cm) w i t h t h e f o l l o w i n g g r a d i e n t e l u t i o n : 1:2 ( v / v ) p e t r o l e u m e t h e r : e t h e r (300 mL); 2:7 ( v / v ) p e t r o l e u m e t h e r : e t h e r (450 mL); e t h e r (100 mL). On bo t h o c c a s i o n s t h a t t h e cr u d e 29 was chromatographed some o f t h e p r o d u c t came o u t o f s o l u t i o n and was r e t a i n e d by t h e column; however, no att e m p t was 51 made t o r e c o v e r t h i s p o r t i o n . P u r i f i e d 29 was c o n c e n t r a t e d in vacuuo t o produce f i n e n e e d l e l i k e r u s t c o l o u r e d c r y s t a l s i n 69 % (5.56 g) y i e l d . *H-NMR 6 3.90 ( s , 3H, 4'-0CH 3), 3.94 ( s , 3H, 3'-0CH 3), 6.70 (dd, J = 9 Hz and 2 Hz, 1H, H-6'), 6.79 ( d , J = 2 Hz, 1H, H-2'), 6.87 ( d d , J = 9 Hz and 3 Hz, 1H, H-4), 6.96 ( d , J = 9 Hz, 1H, H-5'), 7.01 ( d , J = 3 Hz, 1H, H-6), 8.16 ( d , J = 9 Hz, 1H, H-3), 9.44 ( b s , 1H, N-H*). (*Does not exchange w i t h D2O). GCMS ( d ) : t R , 7.90 min; M ' + ( 3 5 C 1 ) : m/z 308 (90 % ) , M' + ( 3 7 C 1 ) : m/z 310 (31 % ) , [ M - 1 5 ] - + ( 3 5 C 1 ) : m/z 293 (100 % ) , [ M - 1 5 ] - + ( 3 7 C 1 ) : m/z 293 (35 % ) . E t h y l N - ( 5 - c h 1 o r o - 2 - n i t r o p h e n y 1 ) - N - ( 3 , 4 - d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e 1301. In t h e f o l l o w i n g p r o c e d u r e , a p p r o p r i a t e m o d i f i c a t i o n s t o t h e method used f o r t h e s y n t h e s i s o f 19 were n e c e s s a r y . E t h y l m a l o n y l c h l o r i d e (3.14 mL, 24.5 mmol) and 29 (3.02 g, 9.79 mmol) were d i s s o l v e d i n d r y benzene (50 mL) and t h e r e s u l t i n g s o l u t i o n r e f l u x e d f o r 22 h a t 75 - 85°C. The c r u d e p r o d u c t w h i c h o c c u r r e d as a brown s o l u t i o n was e x t r a c t e d w i t h s a t u r a t e d NaHC0 3, d r i e d o v e r Na 2S04 and c o n c e n t r a t e d i n vacuuo t o a brown o i l . The l a t t e r was d i s s o l v e d i n c a 10 mL CHC1 3 and chromatographed on a s i l i c a column (18 cm x 2.5 cm) w i t h t h e f o l l o w i n g g r a d i e n t e l u t i o n : benzene (40 mL), 1:1 ( v / v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (80 mL); 2:3 (v/ v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (100 mL). P u r i f i e d f r a c t i o n s o f 30 were c o n c e n t r a t e d i n vacuuo t o p a l e y e l l o w c r y s t a l s i n 96 % (3.97 g) y i e l d . ^H-NMR 6 1.27 ( t , 3H, -CH 2CH 3), 3.39 ( s , 2H, -(C=0)CH 2(C=0)-), 3.88 ( s , 3H, 4'-0CH 3), 3.92 ( s , 3H, 3'-0CH 3), 4.16 ( q , 2H, 0 C H 2 C H 3 ) , 6.91 ( d , J = 52 7 Hz, H-5'), 7.07 ( d , J = 2 Hz, 1H, H-2'), 7.08 (dd, J = 7 Hz and 2 Hz, 1H, H-6'), 7.23 ( d , J = 2.5 Hz, 1H, H-6), 7.35 (dd, J = 9 Hz and 2.5 Hz, 1H, H-4), 7.91 ( d , J = 9 Hz, H-3). GCMS ( d ) : tR, 7.83 min; [M-114] * + ( 3 5 C l ) : m/z 308 (98 % ) , [M-114]- + ( 3 7 C 1 ) : m/z 310 (33 % ) , [M-114-15]- + ( 3 5 C 1 ) : m/z 293 (100 % ) , [M-114-1 5 ] ' + ( 3 7 C 1 ) : m/z 293 (33 % ) . 3',4'-Dimethoxy N - d e s m e t h v l c l o b a z a m ( 3 1 ) . The s y n t h e s i s o f 31 from 30, (2.11 g, 4.99 mmol) was a c c o r d i n g t o t h e method used f o r 20, by t r e a t m e n t w i t h Zn powder (1.6 g, 24.9 mmol) and c o n c e n t r a t e d HCl (10 mL, 110 mmol). TLC a n a l y s i s i n 15:1 ( v / v ) CHCl3:MeOH a f t e r 30 h showed t h e p r e s e n c e o f a p r o d u c t a t R f 0.37 w i t h much u n r e a c t e d s t a r t i n g m a t e r i a l a t Rf 0.84. F u r t h e r m o r e , t h e p r o d u c t d i d n ot p r e c i p i t a t e from t h e r e a c t i o n m i x t u r e l i k e 20 and 25 but was s o l u b l e . The r e a c t i o n was t r e a t e d w i t h a d d i t i o n a l HCl (3 mL., 33 mmol) and Zn (0.82 g, 12.7 mmol) and s t i r r i n g c o n t i n u e d f o r a n o t h e r 24 h. TLC a n a l y s i s a f t e r 24 h showed t h a t no f u r t h e r r e a c t i o n had o c c u r r e d and s t i r r i n g was d i s c o n t i n u e d . The r e a c t i o n m i x t u r e was c o n c e n t r a t e d in vacuuo, t a k e n up i n t o CHCI3, backwashed w i t h H2O, and d r i e d o v e r anhydrous Na2S04. The c r u d e p r o d u c t was c o n c e n t r a t e d in vacuuo and chromatographed on a s i l i c a column (20 cm x 2.5 cm) u s i n g a m o b i l e phase o f 12:1 ( v / v ) CHCl3:Me0H (130 mL). P u r i f i e d 31 was c o n c e n t r a t e d in vacuuo t o an o i l w h i c h was p r e c i p i t a t e d from CHCl3:pet e t h e r t o a f f o r d 101 mg (6 %) o f an amorphous w h i t e s o l i d . ^H-NMR 6 3.53 ( s , 2H, C H 2 ) , 3.83 ( s , 3H, 4'-0CH 3), 3.90 ( s , 3H, 3'-0 C H 3 ) , 6.69 ( d , J = 2.5 Hz, 1H, H-2'), 6.76 (dd, J = 9 Hz and 2.5 Hz, H-53 6 ' ) , 6.90 (d J = 9 Hz, 1H, H-5'), 6.97 ( d , J = 1.5 Hz, 1H, H-6), 7.07 (m, 2H, H-8, H-9) 9.54 ( b s , 1H, N-H). GCMS ( d ) , TMS d e r i v a t i v e : t R, 9.57 min, M +' ( 3 5 C 1 ) : m/z 418 (91 % ) , M+-( 3 7 C 1 ) m/z 420 (35 % ) , [ M - 1 5 ] + - m/z 403 (28 % ) , [ M - 1 7 9 ] + - m/z 239 (32 % ) , [ M - 1 9 3 ] + - m/z 225 (100 % ) . 3 ' , 4 ' - D i h v d r o x y N-desmethylclobazam ( 3 2 ) . The s y n t h e s i s o f 32, l i k e 28, was a c c o m p l i s h e d by t h e t r e a t m e n t o f 31 (51.6 mg, 0.15 mmol) w i t h BBY3 (140 * i L , 1.49 mmol). The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 x 1.5 cm) u s i n g 10:1 (v / v ) CHCl3*.MeOH (55 mL). P u r i f i e d 32 was c o n c e n t r a t e d in vacuuo t o a f f o r d 45 mg (96 %) o f an amorphous w h i t e s o l i d . *H-NMR (DMS0-d 6), 8 3.35 ( b u r i e d i n H 20, C H 2 ) , 6.45 ( d d , 1H, J = 8.5 Hz and 2.5 Hz, H-6'), 6.58 ( d , J = 2.5 Hz, 1H, H-2'), 6.78 ( d , J = 8.5 Hz, 1H, H-5'), 6.88 ( d , J = 2.5 Hz, 1H, H-6), 7.23 ( d , J = 8.5 Hz, 1H, H-9), 7.30 (dd, J = 8.5 Hz and 2.5 Hz, 1H, H-8), 9.40 ( s , 2H, 3'-0H, 4'-0H), 10.52 ( s , 1H, N-H). GCMS ( d ) , T M S - d e r i v a t i v e : 13.90 min, M + ' ( 3 5 C 1 ) : m/z 534 (100 % ) , [M-15]+': m/z 519 (29 % ) , [ M - 2 9 5 ] + - : m/z 239 (19 % ) , [ M - 3 0 9 ] + ' : m/z 225 (50 %). 3'.4'-Dimethoxv clobazam ( 3 4 ) . The s y n t h e s i s o f 34> l i k e 21, was a c c o m p l i s h e d by t r e a t m e n t o f 31 (50.1 mg, 0.14 mmol) w i t h K0H (13.2 mg, 0.24 mmol) i n d r y DMSO. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 cm x 1.5 cm) 54 u s i n g 25:1 ( v / v ) CHCl3:MeOH (52 mL). P u r i f i e d 34 was c o n c e n t r a t e d in vacuuo t o a f f o r d 42 mg (81 %) o f f a i n t l y p u r p l e c r y s t a l s . !H-NMR 6 3.41 ( d , J = 12 Hz, 1H, H-3/3), 3.44 ( s , 3H, N-CH 3), 3.49 ( d , J = 12 Hz, 1H, H-3a), 3.80 ( s , 3H, 4'-0CH 3), 3.85 ( s , 3H, 4'-0CH 3), 6.64 (dd, J = 10 Hz and 2 Hz, 1H, H-6'), 6.68 ( d , J = 2 Hz, 1H, H-2'), 6.83 ( d , J = 10 Hz, 1H, H-5'), 6.92 ( d , J = 3 Hz, 1H, H-6), 7.18 (dd, J = 10 Hz and 3 Hz, 1H, H-8). GCMS ( d ) : t R , 11.65 min, M + ' ( 3 5 C 1 ) : m/z 360 (100 % ) , [ M - 1 7 ] + * : m/z 343 (16 % ) , [ M - 5 7 ] + - : m/z 303 (26 % ) , [ M - 1 7 9 ] + - : m/z 181 (24 % ) . 3 ' . 4 ' - D i h v d r o x v clobazam ( 3 5 ) . The s y n t h e s i s o f 35 was a c c o m p l i s h e d by t r e a t m e n t o f 34 (41.2 mg, 0.115 mmol) w i t h B B r 3 (108 /xL, 1.15 mmol). The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 cm x 1.5 cm) w i t h 25:1 ( v / v ) CHCl 3:MeOH (78 mL). The p u r i f i e d p r o d u c t was c o n c e n t r a t e d in vacuuo t o a f f o r d 33.5 mg (82 %) o f w h i t e c r y s t a l s . !H-NMR 6 3.50 ( d , J = 12 Hz, 1H, H-3/3), 3.52 ( s , 3H, N-CH 3), 3.57 ( d , J = 12 Hz, 1H, H-3a), 6.28 ( b s , 1H, 4'-0H), 6.44 (dd, J = 8 Hz and 2 Hz, 1H, H-6'), 6.69 ( d , J = 2 Hz, 1H, H-2'), 6.77 ( d , J = 8 Hz, 1H, H-5'), 7.03 ( d / b s , J = 3 Hz, 2H, H-6/3'-0H), 7.25 ( d d , J = 10 Hz and 3 Hz, 1H, H-8), 7.29 ( d , J = 10 Hz, 1H, H-9). GCMS ( d ) , T M S - d e r i v a t i v e : t R , 18.11 min, M + - ( 3 5 C 1 ) : m/z 476 (100 % ) , [M-1 5 ] + ' : m/z 461 (24 % ) , [ M - 4 5 ] + - : m/z 431 (73 % ) , [ M - 5 7 ] + " . m/z 419 (52 % ) , [ M - 1 3 3 ] + - : m/z 343 (14 % ) . 5 5 ( i i ) In vitro s y n t h e s e s . 4'-Hydroxy-3'-methoxy N-desmethvlclobazam ( 3 3 ) . The O-methylated c a t e c h o l o f DMC (33) was s y n t h e s i z e d from 32 u s i n g t h e method o f A l t o n e t a l . ( 1 9 7 5 a ) . Rat l i v e r c y t o s o l (as a s o u r c e o f COMT), phosphate b u f f e r (0.50 M, pH 7.9, 2 mL), M g C l 2 (60 mM, 330 iii) and SAM.p-TsOH (0.50 /xmol i n 300 ul v/v/v H 20:95 7, Et0H:5 % H C l , 2:2:1) were mixed i n a c u l t u r e t u b e , and p r e i n c u b a t e d a t 37°C w i t h s h a k i n g f o r 3 min i n a w a t e r - b a t h . The s u b s t r a t e 32 (0.52 /zmol i n 150 tiL MeOH) was t h e n added, and t h e i n c u b a t i o n c o n d u c t e d f o r 80 min. The r e a c t i o n was t e r m i n a t e d by f r e e z i n g t h e m i x t u r e i n an i c e - b a t h . A f t e r 5 min t h e r e a c t i o n m i x t u r e was e x t r a c t e d w i t h an equal volume o f EtOAc (x 4 ) . The o r g a n i c e x t r a c t was d r i e d o v e r anhydrous Na 2S04, c o n c e n t r a t e d t o d r y n e s s under a s t r e a m o f N 2 , r e c o n s t i t u t e d t o 25 IJL i n EtOAc and d e r i v a t i z e d w i t h 25 fil MSTFA a t 45*C f o r 90 min. I n j e c t i o n volume: 3 Hi. GCMS ( b ) , T M S - d e r i v a t i v e : t R , 18.42 min, M + ' ( 3 5 C 1 ) : m/z 476 (58 % ) , [M-15] + -: m/z 461 (17 % ) , [ M - 3 0 ] + - : m/z 446 (8 % ) , [ M - 2 3 7 ] + - : m/z 239 (18 % ) , [M-251J+-: m/z 225 (48 % ) . O - methylated c a t e c h o l s o f cloba z a m (36a and 3 6 b ) . The meta- 36a and para- 36b 0-methyl a t e d c a t e c h o l s o f CLBZ were s y n t h e s i z e d from 35 u s i n g t h e p r o c e d u r e d e s c r i b e d f o r 33. GCMS ( b ) , T M S - d e r i v a t i v e : m e t a - i s o m e r , t R , 23.49 min, para-isomer t R , 22.80 min, M + - ( 3 5 C 1 ) : m / z 418 (100 % ) , [M-30] + ': m/z 388 (20 % ) , [M-4 5 ] + - : m/z 373 (40 % ) , [ M - 7 2 ] + - : m/z 346 (42 % ) , [ M - 2 3 7 ] + * : m/z 181 (48 %). (E) S y n t h e s i s o f N-hydroxymethyl CLBZ ( c a r b i n o l a m i d e ) ( 3 9 ) . ( i ) K2CO3 and 37 % f o r m a l d e h y d e . U s i n g t h e approach o f N a i r and F r a n c i s ( 1 9 8 0 ) , DMC (80.4 mg, 0.28 mmol) and K2CO3 (26.2 mg, 0.19 mmol) were s t i r r e d i n 37 % aqueous f o r m a l d e h y d e a t room t e m p e r a t u r e f o r 24 h. A f t e r t h a t t i m e , TLC a n a l y s i s i n 15:1 ( v / v ) CHCl3:Me0H r e v e a l e d t h e p r e s e n c e o f t h r e e s p o t s a t Rf 0.20, 0.29 and 0.36 w i t h a p p a r e n t c o r r e s p o n d i n g r e l a t i v e i n t e n s i t i e s -45 %, =45 % and =5 %. A minor amount o f u n r e a c t e d s t a r t i n g m a t e r i a l ( R f 0.41) was a l s o p r e s e n t . The r e a c t i o n was d i l u t e d i n H2O, e x t r a c t e d w i t h EtOAc and d r i e d o v e r anhydrous Na 2S04. A l i q u o t s o f t h e cr u d e p r o d u c t were removed f o r GCMS and HPLC a n a l y s i s . The r e m a i n d e r was c o n c e n t r a t e d in vacuuo t o an o i l w h i c h was chromatographed on a s i l i c a column (14 cm x 1.25 cm) u s i n g a 15:1 ( v / v ) Me0H:H20 (50 mL). F l a s h chromatography d i d not s e p a r a t e t h e components o f t h e m i x t u r e and so p r e p a r a t i v e TLC was n e c e s s a r y . The c r u d e p r o d u c t was c o n c e n t r a t e d , and chromatographed on t h r e e s i l i c a p l a t e s (20 cm X 20 cm) u s i n g 15:1 (v/ v ) Me0H:H 20 as t h e m o b i l e phase. Three major bands were removed from t h e p l a t e s and t h e s i l i c a e x t r a c t e d w i t h EtOAc (5 mL x 2) and 1:1 ( v / v ) CHCI3: MeOH (5 mL x 2 ) . The e x t r a c t s were c o n c e n t r a t e d under a stre a m o f N 2 and t h e r e a f t e r in vacuuo. A n a l y t i c a l TLC o f t h e f r a c t i o n s showed pure compounds a t Rf 0.20, 0.30 and 0.43 (DMC). 57 (a) A n a l y s i s o f cr u d e p r o d u c t . HPLC, 50:50 ( v / v ) Me0H:H20: ( i ) f r e s h l y p r e p a r e d p r o d u c t , t R 7.85 min (19.5 % ) , 8.57 min (28.4 % ) , 9.61 min (29.4 % ) , 10.68 min (22.6 % ) ; ( i i ) a f t e r o v e r n i g h t s t o r a g e a t 4°C, tR 7.67 min (19.1 % ) , 8.35 min (58.6 % ) , 10.40 min (22.3 % ) . LCMS, 50:50 ( v / v ) MeOH:H20 ( i ) LCMS o f f r e s h l y p r e p a r e d p r o d u c t c o u l d n o t be p e r f o r m e d ; ( i i ) a f t e r o v e r n i g h t s t o r a g e a t 4°C, tR 4.68 min, scan 94, poor mass s p e c t r u m o b t a i n e d ; 6.68 min, scan 130, MH +: m/z 317 (100 % ) , [ M H-H 20] +: m/z 299 (55 % ) ; 7.37 min, scan 145, MH +: m/z 287 (100 % ) , MNH 4 +: m/z 304 (40 % ) . GCMS ( b ) , T M S - d e r i v a t i v e : t R , 17.33 min, M + - ( 3 5 C 1 ) : m / z 388 (20 % ) , [M-1 5 ] + - : m/z 373 (24 % ) , [M-30]"1"': m/z 358 (6 % ) , [M-149 ] + ': m/z 239 (10 % ) , [ M - 1 6 3 ] + - : m/z 225 (20 % ) , CH 20-TMS +-: m/z 103 (35 % ) . (b) A n a l y s i s o f t h e p u r i f i e d f r a c t i o n s . Rf 0.20 ( 4 5 ) . 2H-NMR (DMS0-d6), 5 6.64 ( d , J = 2 Hz, 1H, H-6), 7.18 (dd, J = 9 Hz and 1 Hz, 2H, H-2', H-6'), 7.25 ( d , J = 9 Hz, 1H, H-9), 7.30 (d d , J = 9 Hz and 2 Hz, 1H, H-8), 7.42 (m, 1H, H-4'), 7.50 (m, 2H, H-3', H-5'), 10.89 ( s , 1H, N-H). U n a s s i g n e d : 3.75 ( b s , 1H), 3.84 ( b s , 1H), 4.58 ( b s , 1H*). *Exchanges w i t h D 20. Rf 0.30 (46). *H-NMR (DMS0-d6), 8 3.42 ( t , 1H, -CHCH 2), 3.94 ( d , J = 6 Hz, 2H, CHCH 20H), 4.56 ( b s , 1H, -CH 20H), 6.88 ( d , J = 2 Hz, 1H, H-6), 7.20 (dd, J = 9 Hz and 1 Hz, 2H, H-2', H-6'), 7.32 ( d , J = 9 Hz, 1H, H-9), 7.37 58 ( d d , J = 9 Hz and 2 Hz, 1H, H-8), 7.40 (m, 1H, H-4'), 7.48 (m, 2H, H-3', H-5'), 10.80 ( s , 1H, N-H). C o n t a m i n a t i n g peaks: 3.31 ( b s , H 2 0 ) , 2.51 (m, DMSO). GCMS ( b ) , T M S - d e r i v a t i v e : t R , 14.39 min, M + - ( 3 5 C 1 ) : m/z 370 (12 % ) , [M-1 5 ] + - : m/z 355 (8 % ) , [ M - 9 7 ] + - : m/z 301 (10 % ) , [M-119 ] + ' : m/z 251 (34 % ) , [M-145]"*"-: m/z 225 (46 % ) , TMS +": m/z 73 (100 % ) , . Rf 0.43 (DMC) ( 4 7 ) . *H-NMR (DMS0-d6), 6 3.20 ( b s , 0.6H, H-30*), 3.41 ( b s , 0.8H, H-30, H-3a), 3.68 ( b s , 0.6H, H-3a*), 6.82 ( d , J = 3 Hz, 1H, H-6), 7.25 (dd, J = 8 Hz and 1 Hz, 2H, H-2', H-6'), 7.30 ( d , J = 9 Hz, 1H, H-9), 7.36 (dd, J = 9 Hz and 3 Hz, 1H, H-8), 7.42 (m, 1H, H-4'), 7.50 (m, 2H, H-3', H-5'), 10.70 ( s , 1H, N-H). *The c h e m i c a l s h i f t s a t 3.20 and 3.68 ppm d e m o n s t r a t e t h e p a r t i a l l y d i a s t e r e o m e r i c c h a r a c t e r o f H-3. (See R e s u l t s and D i s c u s s i o n ) . ( i i ) KOH and p a r a f o r m a l d e h y d e . The f o l l o w i n g d e s c r i b e s t h e s y n t h e s i s o f 40. I n t o a s t i r r e d s o l u t i o n o f DMC (80.5 mg, 0.28 mmol) and p u l v e r i z e d KOH (24.3 mg, 0.43 mmol), m a i n t a i n e d a t 20°C, was p l a c e d p a r a f o r m a l d e h y d e (18.9 mg, 0.63 mmol). A f t e r 20 min t h e r e a c t i o n was a l l o w e d t o r e a c h room t e m p e r a t u r e w i t h s t i r r i n g c o n t i n u e d f o r an a d d i t i o n a l 2 h. A t t h a t t i m e TLC a n a l y s i s i n 20:1 ( v / v ) CHCl3:Me0H r e v e a l e d t h e p r e s e n c e o f one major p r o d u c t (-95 %) a t Rf 0.17 and u n r e a c t e d s t a r t i n g m a t e r i a l a t Rf 0.34. The r e a c t i o n was d i l u t e d t o c a . 80 mL w i t h EtOAc, washed w i t h aqueous NaCl (2 x 20 mL) and H 20 (2 x 20 mL), d r i e d o v e r anhydrous Na 2S04 and 59 c o n c e n t r a t e d in vacuuo t o a f f o r d 17 mg o f 40, a w h i t e amorphous s o l i d . *H-NMR (DMS0-d 6), 8 3.43 ( t , 1H, -CHCH 2), 3.94 ( t , 2H, CHCH 20H), 4.56 ( t , 1H, -CH 20H), 6.90 ( d , J = 2 Hz, 1H, H-6), 7.20 ( d d , J = 9 Hz and 1 Hz, 2H, H-2', H-6'), 7.32 ( d , J = 9 Hz, 1H, H-9), 7.37 (dd, J = 9 Hz and 2 Hz, 1H, H-8), 7.40 (m, 1H, H-4'), 7.48 (m, 2H, H-3', H-5'), 10.77 ( s , 1H, N-H). C o n t a m i n a t i n g peaks: 4.07 ( q , Et O A c ) , 3.33 ( b s , H 2 0 ) , 2.51 (m, DMSO). *H-NMR (DMS0-d6), D 20 exchange. 8 3.94 ( d , J = 6Hz, 2H, CHCH 20H), 4.56 and 10.77 ppm (e x c h a n g e d ) . Benzo and phenyl c h e m i c a l s h i f t s unchanged. GCMS ( d ) , T M S - d e r i v a t i v e : t R , 7.88 min, M + ' ( 3 5 C 1 ) : m/z 370 (34 % ) , [M-1 5 ] + - : m/z 35 (24 % ) , [ M - 9 7 ] + ' : m/z 301 (24 % ) , [M-119 ] + > : m/z 251 (76 % ) , [ M - 1 4 5 ] + - : m/z 225 (100 % ) , TMS+-: m/z 73 (68 % ) , (F) A t t e m p t e d S y n t h e s e s o f N-hydroxy DMC (hy d r o x a m i c a c i d ) ( 5 0 ) . ( i ) O x i d a t i o n o f DMC-TMS (Mat! i n e t a l . . 1979). (a) S y n t h e s i s o f ( b i s ( N , N - d i m e t h y l f o r m a m i d o ) o x o d i p e r o x o m o l y b d e n u m ( V I ) 1421-M o l y b d i c a c i d (2.00 g, 0.014 mol) was s t i r r e d i n 30 % H 2 0 2 a t 35°C f o r 15 min t o a f f o r d a y e l l o w s o l u t i o n . The r e a c t i o n m i x t u r e was c o o l e d t o 15"C, DMF (2.34 mL, 0.30 mol) added, and s t i r r i n g c o n t i n u e d f o r a n o t h e r 20 min. The r e a c t i o n was c o n c e n t r a t e d in vacuuo a t 30°C and t h e r e s i d u e washed w i t h e t h e r ( 2 x 3 mL), MeOH ( 2 x 3 mL) and e t h e r a g a i n ( 2 x 3 mL). The washed r e s i d u e was d r i e d in vacuuo o v e r phosphorus p e n t a o x i d e t o a f f o r d 1.79 g (45 %) o f an amorphous y e l l o w 60 s o l i d . !H-NMR ( D 2 0 ) S 2.89 ( b s * , 3H, N-CH3), 3.05 ( b s * , 3H, N-CH3), 7.96 ( b s , 1H, N-H). * S o l v e n t - a s s o c i a t e d peak b r o a d e n i n g . C o n t a m i n a t i n g peak: 3.38 ( b s , CH3OH). L i t . 6 2.9 ( s , 3H, N-CH3), 3.0 ( s , 3H, N-CH3), 7.9 ( b s , 1H, N-H). (b) S y n t h e s i s o f DMC-TMS ( 4 8 ) . DMC (10.5 mg, .037 mmol) was suspended i n d r y CHCI3, t h e r e a c t i o n v i a l purged w i t h N 2 and s e a l e d . MSTFA (90 ill, 0.46 mmol) was added by s y r i n g e , and t h e r e a c t i o n s t i r r e d a t 45 °C f o r 90 min. (c ) A t t e m p t e d s y n t h e s i s o f t h e Molybdenum hydroxamate complex ( 4 9 ) . To t h e c r u d e DMC-TMS, was added 42 (234 mg, 0.69 mmol), t h e v i a l s e a l e d under N 2 and s t i r r i n g c o n t i n u e d a t room t e m p e r a t u r e . A f t e r 48 h, no r e a c t i o n was o b s e r v e d , and so t h e s y n t h e s i s was d i s c o n t i n u e d . ( i i ) R i n g c l o s u r e o f t h e hydroxy!amine. (a) A t t e m p t e d s y n t h e s i s o f t h e hydroxy!amine ( 5 2 ) . A c c o r d i n g t o t h e method o f Ro n d e s t v e d t and Johnson ( 1 9 7 7 ) , e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - p h e n y l c a r b a m o y l a c e t a t e (51) (40.6 mg, 0.112 mmol) and 10 % Pd/C (3 mg) were suspended i n 50:50 ( v / v ) THF:95 % EtOH (3 mL). The m i x t u r e was warmed t o 35°C and w h i l e s t i r r i n g , h y d r a z i n e monohydrate (190 ill, 1.33 mmol) s l o w l y added i n 50 /zL 61 t e m p e r a t u r e w i t h c o n t i n u e d s t i r r i n g . TLC a n a l y s i s o f t h e r e a c t i o n i n 3:2 ( v / v ) p e t e t h e r : E t O A c a f t e r 40 h r e v e a l e d t h a t no r e a c t i o n had o c c u r r e d , and so s y n t h e s i s was d i s c o n t i n u e d . 62 I I I R E S U L T S AND D I S C U S S I O N T h i s s e c t i o n w i l l be d i v i d e d i n t o t h e a r e a s o f (1) s y n t h e s i s and (2) m e t a b o l i s m . In (1) t h e s y n t h e s i s o f d e u t e r a t e d CLBZ a n a l o g u e s and CLBZ m e t a b o l i t e s w i l l be p r e s e n t e d , and i n (2) t h e m e t a b o l i t e p r o f i l e o f CLBZ i n t h e r a t w i l l be c h a r a c t e r i z e d . 1. THE SYNTHESIS OF DEUTERATED ANALOGUES OF CLBZ AND CLBZ METABOLITES. (A) The S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l CLBZ. The use o f s t a b l e i s o t o p e l a b e l l e d d rugs t o probe t h e mechanisms o f b i o t r a n s f o r m a t i o n p r o c e s s e s i s w e l l - r e c o g n i z e d , and t h e s u b j e c t has been e x t e n s i v e l y r e v i e w e d (Vandenheuvel, 1987; B a i l l i e , 1981). When combined w i t h GCMS, s t a b l e i s o t o p e l a b e l l i n g f a c i l i t a t e s i d e n t i f i c a t i o n o f d r u g -r e l a t e d s p e c i e s i n a m e t a b o l i t e pool t h r o u g h i s o t o p e c l u s t e r s g e n e r a t e d by t h e l a b e l l e d and u n l a b e l l e d a n a l o g u e s . Such an approach was c o n s i d e r e d t o be i d e a l f o r o u r i n v e s t i g a t i o n o f CLBZ m e t a b o l i t e s i n t h e r a t . C u r s o r y a p p r a i s a l o f t h e c h e m i c a l s t r u c t u r e o f CLBZ r e v e a l s s e v e r a l p o s s i b l e s i t e s f o r d e u t e r i u m l a b e l l i n g , namely, t h e N-methyl, C3-methylene and t h e benzo and phenyl r i n g s . The s y n t h e s i s o f t r i d e u t e r i o m e t h y l CLBZ has been r e p o r t e d ( P o u p a e r t e t a l . , 1988); however, s i n c e t h e d e m e t h y l a t i o n o f CLBZ i s a f a c i l e m e t a b o l i c p r o c e s s ( V o l z e t a l . , 1979), such an an a l o g u e would be i n a p p r o p r i a t e f o r t h e i d e n t i f i c a t i o n o f l a b e l l e d m e t a b o l i t e s . The l o c a t i o n o f C-3 i n a b - d i c a r b o n y l system r e n d e r s t h e methylene p r o t o n s a c i d i c ( March, 1985a), and prone t o exchange under b a s i c c o n d i t i o n s . Thus, a 63 m e t h y l e n e - l a b e l l e d a n alogue was an u n s u i t a b l e m e t a b o l i t e p r o b e , and l a b e l l i n g a t t h e a r o m a t i c p o s i t i o n s a f f o r d e d t h e most v i a b l e a l t e r n a t i v e . F a c i l e a pproaches t o t h e de u t e r i u m - e x c h a n g e o f a r o m a t i c systems have been r e p o r t e d , e.g. C6U6 and a l k y l a l u m i n i u m d i c h l o r i d e ( G a r n e t t e t a l . , 1 9 7 2 ) , and BF3 and D2O ( L a r s e n and Chang, 1978). However, n e i t h e r approach was c o n s i d e r e d a p p l i c a b l e t o t h e l a b e l l i n g o f CLBZ f o r t h e f o l l o w i n g r e a s o n s : ( i ) N i t r o g e n - c o n t a i n i n g a r o m a t i c s f a i l t o exchange w i t h C^DQ, and d e a c t i v a t e t h e a l u m i n i u m c a t a l y s t t h r o u g h complex f o r m a t i o n , and ( i i ) t h e s l o w and i n c o m p l e t e exchange t h a t o c c u r s w i t h a r o m a t i c s and BF3.D2O, i s l i k e l y t o r e s u l t i n a m i x t u r e o f d e u t e r a t e d CLBZ a n a l o g u e s . In t h i s r e g a r d , p e n t a d e u t e r i o p h e n y l CLBZ ( [ 2 Hs]CLBZ), appeared t o be t h e most a p p r o p r i a t e c a n d i d a t e f o r a s e l e c t i v e l y d e u t e r a t e d CLBZ a n a l o g u e . D e u t e r i u m i n t h e 5-phenyl r i n g i s n o t r e a d i l y s u s c e p t i b l e t o exchange, p r e s e n t s no e x p e c t e d major i s o t o p e e f f e c t s , and even w i t h t h e f o r m a t i o n o f c a t e c h o l m e t a b o l i t e s a t l e a s t t h r e e 2 H - l a b e l s s h o u l d be r e t a i n e d . F u r t h e r m o r e , t h e p r e s e n c e o f 2 H - l a b e l s a t a l l f i v e p o s i t i o n s o f t h e phenyl r i n g e n s u r e s t h a t a l l u n s u b s t i t u t e d p o s i t i o n s w i l l be l a b e l l e d subsequent t o m e t a b o l i s m d e s p i t e t h e o c c u r r e n c e o f an arene e p o x i d e - a s s o c i a t e d NIH s h i f t ( N e l s o n and Burk e , 1979; N e l s o n and P o w e l l , 1979). The s y n t h e s i s o f [^ HsJCLBZ ( o u t l i n e d i n Scheme IX) was a c c o m p l i s h e d i n e s s e n t i a l l y q u a n t i t a t i v e i s o t o p i c y i e l d , as d e t e r m i n e d by *H-NMR ( F i g u r e 1) and mass s p e c t r a ( F i g u r e 2 ) , w i t h a p p r o p r i a t e m o d i f i c a t i o n s t o t h e p r o c e d u r e s o f Werner and Wagner (1978) and R o s s i e t a l . , ( 1 9 6 9 ) . In t h e f i r s t s t e p , r e d u c t i o n o f nitrobenzene - d s (99 atom % D) a f f o r d e d a n i l i n e - d y 17 i n good y i e l d w i t h an N,N-D2 i s o t o p i c p u r i t y o f 90 % as d e t e r m i n e d by 2H-NMR s p e c t r o s c o p y . GCMS a n a l y s i s was u n s u i t a b l e f o r t h e a c c u r a t e d e t e r m i n a t i o n o f N - d e u t e r a t i o n because t h e l a b i l e d e u t e r i u m atoms on 64 n i t r o g e n were e x t e n s i v e l y exchanged on t h e GC pack e d column p r i o r t o mass s p e c t r a l d e t e c t i o n . Thus, [ M - 2 ] + * a t m/z 98 ap p e a r e d as t h e most i n t e n s e h i g h mass i o n i n t h e mass s p e c t r u m o f 17 f o l l o w i n g GCMS a n a l y s i s ( A p p e n d i x ) . Scheme I X . The s y n t h e s i s o f pHsJCLBZ. A: DC1, Fe B: 2 , 4 - d i c h l o r o n i t r o b e n z e n e , 170°C C: e t h y l m a l o n y l c h l o r i d e / d r y benzene D: Zn/HCl E: KOH, DMSO/CH3I 65 N u c l e o p h i l i c s u b s t i t u t i o n o f 2 , 4 - d i c h l o r o n i t r o b e n z e n e by 17 o c c u r r e d e x c l u s i v e l y ortho t o t h e n i t r o group t o a f f o r d 18 i n h i g h y i e l d . The h i g h r e g i o s p e c i f i c i t y ( f o r ortho s u b s t i t u t i o n ) c l e a r l y d e m o n s t r a t e s t h e r e q u i r e m e n t f o r t h e e l e c t r o n w i t h d r a w i n g i n f l u e n c e o f t h e n i t r o group t o be e x e r t e d t h r o u g h both r e s o n a n c e and f i e l d e f f e c t s (March, 1985c) i n o r d e r t o reduce t h e e l e c t r o n d e n s i t y a t t h e 2 - p o s i t i o n and t h e r e b y f a c i l i t a t e n u c l e o p h i l i c a t t a c k (March, 1985b). The *H-NMR spect r u m o f 18 shown i n F i g u r e 3A r e v e a l e d t h a t r e s o n a n c e a t t h e c h e m i c a l s h i f t o f t h e meta p r o t o n s o f t h e u n s u b s t i t u t e d phenyl r i n g (H-3'/H-5', 7.48 ppm) was a b s e n t , a l t h o u g h weak s i g n a l s appeared i n t h e r e g i o n c h a r a c t e r i s t i c o f t h e ortho/para p r o t o n s (H-2'/6', H-4', 7.27 -7.31). Thus, 2 H - l a b e l s a t t h e meta p o s i t i o n were c o m p l e t e l y r e t a i n e d w i t h m i n o r exchange o c c u r r i n g a t t h e ortho/para p o s i t i o n s . I n t e g r a t i o n o f t h e ortho/para p r o t o n s was used t o c a l c u l a t e t h e i s o t o p i c p u r i t y o f 18 a t 92 %. Mass ( F i g u r e 4A) and ^3C-NMR ( F i g u r e 5) s p e c t r a c o n f i r m e d t h e i d e n t i t y o f th e p r o d u c t . 66 F i g u r e 1. 400 MHz *H-NMR o f [ 2 H 5 ] C L B Z . f K N M N u u u a N »> a A 00 N »> a a 0 0 a a a a a 0 £ 0 a a a a a a Q s a a a a a a a till I I I. I. I I I I I I I I I t I Relative Intensity (%) H N N N a N a eg a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a o H a a R e l a t i v e i n t e n s i t y (%) 68 A l i k e l y s o u r c e o f t h e p r o t o n s b e i n g i n c o r p o r a t e d i n t o t h e d e u t e r i o p h e n y l r i n g o f 18 c o u l d have been t h e N-H group o f a n i l i n e -2,3,4,5,6-d5 w h i c h o c c u r r e d as an i s o t o p i c i m p u r i t y i n a n i l i n e - d 7 - Indeed, s u b s t a n t i a l i n t r o d u c t i o n o f p r o t o n s a t t h e ortho/para p o s i t i o n s o f t h e d e u t e r i o p h e n y l r i n g o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o p h e n y l a m i n e 37 o c c u r r e d when aniline-2,3,4,5,6 - d s was used as t h e n u c l e o p h i l e (Scheme X, F i g u r e 3 B ) . In o r d e r t o a s s i g n t h e e x a c t l o c a t i o n o f d e u t e r i u m exchange, t h e ortho/para s i g n a l s were r e s o l v e d w i t h 0.4 e q u i v a l e n t o f E u ( f o d ) 3 , and b o t h ortho (7.29 ppm) and para (7.26 ppm) p o s i t i o n s were found t o be i n v o l v e d ( F i g u r e 3B, I n s e t ) . The mass s p e c t r u m o f 37 ( F i g u r e 4B) p r e s e n t e d h i g h mass i o n s a t m/z 253, 252, 251 and 250 w h i c h d e m o n s t r a t e d t h a t i n c o m p l e t e exchange o f t h r e e d e u t e r i o p h e n y l l a b e l s had o c c u r r e d t o a f f o r d a m i x t u r e o f [ ^ 5 ] , [ 2 H 4 ] , [ 2 H 3 ] and [ 2 H 2 l a n a l o g u e s . Harada and T i t a n i (1936) r e p o r t e d t h e t h e r m a l r e a r r a n g e m e n t o f C6H5NH2D"1" t o i t s ortho and para r i n g d e u t e r a t e d a n a l o g u e s . O k a z a k i and Okumura (1961) proposed t h a t above 150°C and below t h e m e l t i n g p o i n t , t h e p r o t o n s o f t h e NH3 + group o f a n i l i n i u m s a l t s were i n t r a m o l e c u l a r l y t r a n s f e r r e d a l o n g t h e w - e l e c t r o n c l o u d t o t h e c a r b o n atoms o f h i g h e r e l e c t r o n d e n s i t y . Thus, t h e ortho and para p o s i t i o n s a r e s e l e c t i v e l y t a r g e t e d f o r p r o t o n exchange. In an a n a l o g o u s f a s h i o n , aniline-2,3,4,5, - d s c o u l d be r e a r r a n g e d t o i t s r i n g p r o t o n a t e d a n a l o g u e s t h r o u g h i t s a n i l i n i u m i o n as shown i n Scheme X. 69 F i g u r e 3. 400 MHz H-NMR s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) p e n t a -d e u t e r i o p h e n y l a m i n e 18, and (B) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o -p h e n y l a m i n e 37 showing s u b s t a n t i a l i n t r o d u c t i o n o f p r o t o n s a t t h e ortho and para p o s i t i o n s o f t h e d e u t e r i o p h e n y l r i n g . R e s o l u t i o n o f t h e ortho/para p r o t o n s w i t h 0.4 e q u i v a l e n t E u ( f o d ) 3 shows t h a t exchange o c c u r r e d a t both ortho and para p o s i t i o n s ( I n s e t ) . 70 253 S4 30 •ni .ill 172 143 1X34 i i i i i i i i i l iLll i i Jlu .mil, l|, ,.titiiB|. „ill 207218 i.. •I..n1. i 23S Ivf + 252 B S3 170 8 1 142 20S 218 235 MAM J l . j M m/z F i g u r e 4. E l e c t r o n impact mass s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18 showing h i g h i s o t o p i c p u r i t y w i t h M +-, m/z 253, and (B) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o p h e n y l a m i n e 37 showing t h a t i t o c c u r r e d as a m i x t u r e o f [ 2 H 5 ] ( M + * , m/z 2 5 3 ) , [ 2H4] ( M + > , m/z 2 5 2 ) , [ 2 H 3 ] (M+-, m/z 251) and [ 2 H 2 ] ( M + - , m/z 250) a n a l o g u e s . 71 3 6 PPH F i g u r e 5. 75 MHz broad-band d e c o u p l e d 1 3C-NMR s p e c t r u m o f N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18 showing t h e p r e s e n c e o f d e u t e r i u m a t t h e ortho, para and meta p o s i t i o n s o f t h e p h e n y l n u c l e u s w i t h t h e a p p e a r a n c e o f t r i p l e t s a t 124.26, 125.77 and 129.35 ppm r e s p e c t i v e l y . 72 Scheme X. Mechanism f o r t h e s e l e c t i v e i n t r o d u c t i o n o f p r o t o n s i n t o t h e d e u t e r i o p h e n y l n u c l e u s o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - d e u t e r i o p h e n y l a m i n e 37. (A) N u c l e o p h i l i c a t t a c k , (B) P r o t o n a b s t r a c t i o n and d e p a r t u r e o f C I " , (C) Thermal rearrangement o f a n i l i n i u m c a t i o n , x = 2, 3, 4, 5, (D) P r o t o n a b s t r a c t i o n t o form r e a r r a n g e d a n i l i n e n u c l e o p h i l e . The s u b s t i t u t i o n o f 2 , 4 - d i c h l o r o n i t r o b e n z e n e by a n i l i n e - 2 , 3 , 4 , 5 , 6 - d 5 p r o c e e d s via a 2- s t e p S^jAr mechanism ( M a r c h , 1985b). In t h e f i r s t s t e p a n i l i n e a c t s as a n u c l e o p h i l e a t t a c k i n g t h e e l e c t r o n d e f i c i e n t 2 - p o s i t i o n o f t h e s u b s t r a t e t o a f f o r d a t e t r a h e d r a l i n t e r m e d i a t e . In t h e second s t e p t h e C I " l e a v i n g group d e p a r t s and a p r o t o n i s a b s t r a c t e d by a n i l i n e f u n c t i o n i n g as a base. A t t h e r e a c t i o n t e m p e r a t u r e o f 170°C t h e r e s u l t i n g a n i l i n i u m c a t i o n r e a r r a n g e s w i t h t h e N - p r o t o n s b e i n g i n t r o d u c e d a t t h e 73 ortho and para p o s i t i o n s o f t h e phenyl r i n g . The r e s u l t i n g c o n j u g a t e base o f t h e r e a r r a n g e d a n i l i n i u m c a t i o n i n t u r n , p a r t i c i p a t e s as a n u c l e o p h i l e t o a f f o r d 37. GCMS a n a l y s i s o f t h e r e a c t i o n m i x t u r e a f t e r t h e f o r m a t i o n o f 37 r e v e a l e d t h a t N-D exchanges w i t h N-H (as d i s c u s s e d above) i n t h a t t h e e x c e s s a n i l i n e was no l o n g e r f u l l y d e u t e r a t e d i n t h e phenyl r i n g but i n s t e a d c o n s i s t e d o f a m i x t u r e o f -ds ( M + * , m/z 98, 45 % ) , -d4 ( M +*, m/z 97, 1 0 0 % ) , -d3 ( M + - , m/z 96, 75 % ) , and -d2 ( M + - , m/z 95, 20 %) a n a l o g u e s . On t h e o t h e r hand, when a n i l i n e - d 7 was used i n t h e s y n t h e s i s o f 18 t h e p e n t a d e u t e r i o p h e n y l n u c l e u s remained e s s e n t i a l l y i n t a c t because o f t h e absence o f a p r o t o n s o u r c e w i t h which exchange c o u l d o c c u r . GCMS: a n i l i n e -d6 ( M + - , m/z 99, 2 % ) , -d5 (M+-, m/z 98, 100 % ) , -d4 ( M + - , m/z 97, 25 % ) , and -d3 ( M + - , m/z 96, 3 % ) . Because t h e rearrangement o f t h e a n i l i n i u m c a t i o n o c c u r s above l S C C (Scheme X ) , c a r e f u l a t t e n t i o n t o t h e e x c l u s i o n o f p o t e n t i a l p r o t o n s o u r c e s was o n l y r e q u i r e d a t e l e v a t e d t e m p e r a t u r e s . Thus, a f t e r t h e r e a c t i o n was a d j u s t e d t o room t e m p e r a t u r e , t h e work-up o f 18 c o u l d be c o n t i n u e d under ambient c o n d i t i o n s w i t h o u t t h e p o s s i b i l i t y o f d e u t e r i u m exchange. F u r t h e r m o r e , t h e subsequent s t e p s (C -> E, Scheme IX) i n t h e s y n t h e s i s o f [ 2Hs]CLBZ do n o t r e s u l t i n t h e l o s s o f 2 H - l a b e l s . A l t h o u g h t h e in situ d e u t e r i u m r e a r r a n g e m e n t i n t h e f o r m a t i o n o f 18 i s one o f i t s more s a l i e n t f e a t u r e s , t h e r e i s y e t a n o t h e r n o t e w o r t h y s t r u c t u r a l p r o p e r t y , namely t h e pronounced d e s h i e l d i n g and absence o f D 20 e x c h a n g e a b i l i t y a s s o c i a t e d w i t h t h e amine p r o t o n . I n t r a m o l e c u l a r H-bonding w i t h t h e n i t r o group i n a s t r u c t u r e d 6-membered r i n g ( F i g u r e 6) can acc o u n t f o r b o t h phenomena. E l e c t r o s t a t i c i n t e r a c t i o n w i t h t h e n i t r o group r e d u c e s t h e e l e c t r o n d e n s i t y a t N-H and d e s h i e l d s t h e p r o t o n f r o m i t s normal c h e m i c a l s h i f t (=3 - 5 ppm) ( S i l v e r s t e i n e t a l . , 1981) t o 9.52 ppm, and t h e 74 f o r m a t i o n o f a 6-membered r i n g s t a b i l i z e s t h e p r o t o n t h e r e b y m i n i m i z i n g D£0 e x c h a n g e a b i l i t y . F i g u r e 6. The f o r m a t i o n o f a 6-membered r i n g between t h e n i t r o group and amine p r o t o n o f t h e phenylamine 18 r e s u l t s i n N-H d e s h i e l d i n g and d i m i n i s h e d D2O exchange. Treatment o f 18 w i t h e t h y l m a l o n y l c h l o r i d e a f f o r d e d t h e carbamoyl a c e t a t e 19 i n good y i e l d . Two n o t e w o r t h y c h e m i c a l p r o p e r t i e s o f t h e carbamoyl a c e t a t e 19 were o b s e r v e d under GCMS and NMR a n a l y s e s : ( i ) th e r m a l r e a r r a n g e m e n t o f 19 o c c u r s d u r i n g GCMS a n a l y s i s t o a f f o r d 18 (Scheme X I ) , and ( i i ) t h e /3 - d i c a r b o n y l s y s t e m o f 19 i s n o t r e a d i l y p r e d i s p o s e d t o k e t o - e n o l t a u t o m e r i s m (Scheme X I I ) . TLC w i t h a m o b i l e phase o f p e t e t h e r : E t O A c ( v / v ) 2:1 p r o v i d e d an e f f e c t i v e probe i n t o t h e s y n t h e s i s o f 19 ( R f , 0.49) from 18 ( R f , 0.79), because o f t h e d i f f e r e n c e i n p o l a r i t y o f t h e two compounds. However, by GCMS bo t h 19 and 18 appeared t o have i d e n t i c a l r e t e n t i o n t i m e s ( F i g u r e 7) and mass s p e c t r a ( F i g u r e 8 ) . Because o f t h e s u b s t a n t i a l d i f f e r e n c e i n t h e i r p o l a r i t y i t was u n l i k e l y t h a t 19 and 18 would have i d e n t i c a l r e t e n t i o n t i m e s . A p l a u s i b l e e x p l a n a t i o n i s t h a t 19 undergoes t h e r m a l r e a r r a n g e m e n t t o 18 a t t h e i n j e c t i o n p o r t (240°C) by ^ - p r o t o n a b s t r a c t i o n and e l i m i n a t i o n o f t h e n e u t r a l ketene (Scheme X I ) . o II 7 5 F i l e >RB198 60.6-488.6 amu. d5-PCNN TIC 6888608 5566868 5866668 4566666 4066666 3586068 3088808-2586808 2800608 1500000-1000800-500000 0 S.3S 3.97 • !•• t, ••I^Tr r^Tr.-, -s jTT, ,-, |h< p n i l . i i i i n i i i i i i i i i i i i i 3.0 4.0 5.0 6.0 7.0 8.6 9.0 10.0 11.0 12.0 13.0 14.0 15.6 16.0 L01 F i l e >AB 2200000 2000888 1800000-1600000-1400000 1206888 1800008 800000-686008 400000 280000 0 195 60.0-408.0 arou. d5-PCHfl TIC 5.34 B ^BMEg9»949 11 .99 ,,,,,|,,,,|,,,,,,,,,|,,,,,,,,,, I . • • . , 3.8 4.0 5.8 6.0 7.0 8.0 9.8 10.0 11.0 12.0 13.0 14.0 15.0 16.0 F i g u r e 7. Thermal rearrangement o f t h e car b a m o y l a c e t a t e 19 t o t h e phen y l a m i n e 18 r e s u l t s i n i d e n t i c a l r e t e n t i o n t i m e s i n t h e TIC p l o t s o f (A) 19 and (B) 18. 76 F i l e >RB198 Bpk flb 786117 dS-PCNM CLP Scan 241 5.33 min. 800000-700000-600000^ 500000-400000-^  300000-200000^ 100000-82 \ <iill.M r-88 86 OEt 172 171 189119 • / 143 142 y 100 128 148 —T 16JT 218 287 195 • / IL J 236 180 208 220 248 F i l e >0B195 Bpk fib 394946 d5-PCNfi 400080-369000-320088^ 280000-248888-200000-160000-120000-80000-40080-B 8 2 1 4 3 144 |j 1 7 3 \ 86 \ / If/ 1 9 S / 189118 jr J | ; •—1—•—1—1—1—1—1—•—1—1—1—•—1—•—i—1—1—•—1—<—1—• 1 • 1 ' 88 180 128 148 166 180 200 CLP 172 218 287 / Scan 235 5.33 min. 236 253 liL ill... -J r l l 0 j-iee |90 r?0 |68 |se J48 r38 J20 j-10 220 248 260 F i g u r e 8. Thermal r e a r r a n g e m e n t o f t h e carbamoyl a c e t a t e 19 t o t h e phen y l a m i n e 18 r e s u l t s i n i d e n t i c a l GC ( E I ) mass s p e c t r a o f (A) 19 and (B) 18. 77 Scheme X I . Thermal rearrangement o f 19 t o f o r m 18 by 0-proton a b s t r a c t i o n and e l i m i n a t i o n o f t h e n e u t r a l k e t e n e . Ample d r i v i n g f o r c e f o r such a r e a r r a n g e m e n t e x i s t s i n t h e a c i d i t y o f t h e meth y l e n e p r o t o n s and t h e l o s s o f t h e s t a b l e c o n j u g a t e d k e t e n e (38) t o form 18. The absence o f an o l e f i n i c p r o t o n (=5 - 6 ppm) and e n o l p r o t o n (-15.0 ppm) ( S i l v e r s t e i n e t a l , 1981) i n t h e lH NMR s p e c t r u m o f 19 ( F i g u r e 9) r e v e a l e d t h a t t h e l a t t e r does not r e a d i l y t a u t o m e r i z e (Scheme X I I ) . Resonance forms w h i c h a r i s e due t o back d o n a t i o n o f t h e l o n e p a i r s o f e l e c t r o n s on t h e amide n i t r o g e n and e s t e r oxygen s e r v e t o reduc e t h e a c i d i t y o f t h e methylene p r o t o n s and hence r e s t r i c t e n o l f o r m a t i o n ( F i g u r e 1 0 ) . H-3 ci 2 ^ N 0 2 0 N' "OEt 19 -C(=0)CH 2C=0) -0CH 2 CH 3 f ) \ JL •CH 2 CH 3 LJ 1111111111111111111111111111 1111>111' 111111111111111 i i i [ 1 1 1 1 1 1 1 i 11 11 6 5 4 3 2 Figure 9. 300 MHz *H-NMR of the carbamoyl acetate 19. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 CO 79 CI Scheme X I I . K e t o - e n o l t a u t o m e r i s m o f 19 showing s t r o n g e q u i l i b r i u m d i s p l a c e m e n t t o t h e k e t o form. F i g u r e 10. The f o r m a t i o n o f r e s o n a n c e s p e c i e s o f 19 w h i c h r e d u c e t h e a c i d i t y o f t h e methylene p r o t o n s and so i n h i b i t k e t o - e n o l t a u t o m e r i s m . In t h e t r e a t m e n t o f 19 w i t h Z n / H C l , c o n c o m i t a n t r e d u c t i o n o f t h e n i t r o group t o an amine and d e e s t e r i f i c a t i o n t o t h e c a r b o x y l i c a c i d r e s u l t e d i n r i n g c l o s u r e t o [ 2H5]DMC ( 2 0 ) . Because o f i t s r e l a t i v e h i g h p o l a r i t y , 20 chromatographed p o o r l y on t h e m e t h y l s i l i c o n e column. TMS d e r i v a t i z a t i o n o f 20 d r a m a t i c a l l y improved i t s c h r o m a t o g r a p h i c ( F i g u r e 11) and mass s p e c t r a l ( F i g u r e 12) p r o p e r t i e s . A l t h o u g h t h e TMS d e r i v a t i v e i s d i a g r a m a t i c a l l y r e p r e s e n t e d as b e i n g f u n c t i o n a l i z e d a t t h e s e c o n d a r y amide N - p o s i t i o n , t h i s does not p r e c l u d e t h e l i k e l i h o o d o f e n o l i c 0-OEt 80 d e r i v a t i z a t i o n . No d i s t i n c t i o n c o u l d be made between t h e two i s o m e r s , s i m p l y because a l l t h e f r a g m e n t s w h i c h were a s s i g n e d a r e e q u a l l y d i a g n o s t i c o f t h e two for m s . F u r t h e r m o r e , t h e moderate s t a b i l i t y o f t h i s d e r i v a t i v e d i d n o t a l l o w p u r i f i c a t i o n f o r NMR a n a l y s i s . The p o s s i b l e m i g r a t i o n o f t h e TMS group from N- t o O-centres on amides due t o k e t o - e n o l t a u t o m e r i s m has been p r e v i o u s l y d e s c r i b e d f o r h i p p u r i c a c i d (Goodman and Markey, 1981). Lower mass f r a g m e n t s a t m/z 225 and m/z 239 w h i c h appeared i n TMS-d e r i v a t i z e d 20 were c h a r a c t e r i s t i c o f a l l T M S - d e r i v a t i z e d D M C - r e l a t e d m e t a b o l i t e s . Proposed pathways w h i c h a c c o u n t f o r t h e s e f ragment i o n s w i l l be d i s c u s s e d i n t h e s e c t i o n on m e t a b o l i t e s y n t h e s i s . The f i n a l s t e p i n t h e s y n t h e s i s o f 21 i n v o l v e d t h e m e t h y l a t i o n o f 20, u s i n g t h e method o f I s e l e and L u t t r i n g h a u s ( 1 9 7 1 ) . S e l e c t i v e m e t h y l a t i o n o f t h e s e c o n d a r y amide o f 20 i n t h e p r e s e n c e o f t h e m e t h y l e n e group, r e q u i r e d c a r e f u l c o n t r o l o f t h e number o f e q u i v a l e n t s o f base. Use o f 1.2 e q u i v a l e n t s o f KOH and e x c e s s CH3I a f f o r d e d [ 2Hs]CLBZ i n 37 % y i e l d and v i r t u a l l y no e v i d e n c e o f t h e d i m e t h y l compound 3-methyl[ 2H5JCLBZ. In t h e *H-NMR spect r u m o f [ 2Hs]CLBZ t h e m e t h y l e n e p r o t o n s appeared as a d o u b l e t o f d o u b l e t s w i t h H-3/J and H-3a a t 3.50 and 3.56 ppm r e s p e c t i v e l y ( F i g u r e 1 ) . These a s s i g n m e n t s a r e c o n s i s t e n t w i t h 21 e x i s t i n g i n a pseudo-boat c y c l o h e p t a d i e n e - 1 i k e c o n f r o m a t i o n w i t h H-3/J p s e u d o a x i a l l y d i r e c t e d i n t o t h e s h i e l d i n g r e g i o n o f t h e benzo r i n g ( A v e r s a e t a l . , 1980). The absence o f phenyl r i n g r e s o n a n c e due t o d e u t e r i u m - l a b e l l i n g a l l o w e d t h e c h e m i c a l s h i f t s o f t h e benzo r i n g t o be d i a g n o s t i c a l l y a s s i g n e d w i t h H-6, H-8 and H-9 p r e s e n t a t 6.93, 7.25 and 7.31 ppm r e s p e c t i v e l y . I t i s n o t e w o r t h y t h a t H-6 and H-9 a r e d i a m e t r i c a l l y d i s p o s e d about H-8 w i t h H-6, d e s p i t e i t s p r o x i m i t y t o t h e e l e c t r o n w i t h d r a w i n g CI g r o u p , b e i n g u p f i e l d f r o m H-9. T h i s s u g g e s t s t h a t t h e e l e c t r o n d o n a t i n g i n f l u e n c e o f t h e N-phenyl m o i e t y 81 ortho t o H-6 o u t w e i g h s t h a t o f t h e N-methyl group ortho t o H-9. In t h e GCMS ( E I ) s p e c t r u m o f 21, [M-0H] +- (m/z 288) and m/z 181 a r e d i a g n o s t i c ( F i g u r e 2 ) . The d e t a i l s o f t h e s e and o t h e r t y p i c a l C L B Z - r e l a t e d f r a g m e n t a t i o n s w i l l be d i s c u s s e d i n t h e s e c t i o n on m e t a b o l i t e s y n t h e s i s . HPLC a n a l y s i s r e v e a l e d t h a t [ 2Hs]CLBZ was >98 % p u r e , and chromatographed ( t R 6.17 min) s l i g h t l y i n f r o n t o f an a u t h e n t i c s t a n d a r d o f CLBZ ( t R 6.32 m i n ) . The d i f f e r e n c e i n t h e r e t e n t i o n t i m e s between compounds and t h e i r d e u t e r a t e d a n a l o g u e s on r e v e r s e phase HPLC has ample p r e c e d e n c e i n t h e l i t e r a t u r e (Honma e t a l . , 1987). 82 ••"-le >RB2S3 70.0-5S0.B aau. dS-DMC T I C F i g u r e 11. TIC p l o t s o f (A) [ 2H 5]DMC and (B) [ 2H 5]DMC-TMS show t h e improved chromatography o b t a i n e d w i t h T M S - d e r i v a t i z a t i o n . 83 3 6 6 6 6 -3 2 8 0 0 -2 8 8 6 0 : 2 4 0 8 0 : 2 0 0 0 0 -1 2 8 0 0 ^ 8 0 0 8 -4 0 0 0 -8 2 . . a 1 6 8 1 8 5 1 1 8 1 2 4 N \ 1 .LILJT^ I««^ H.J(LL>WFIVJLI «• • • i • • • i 8 8 1 2 6 1 6 8 2 8 0 2 4 0 2 8 0 3 2 0 3 6 0 F i g u r e 12. GCMS ( E I ) s p e c t r a o f (A) [2Hs]DMC and (B) [2Hs]DMC-TMS show t h e i n c r e a s e d mass s p e c t r a l f r a g m e n t a t i o n o b t a i n e d w i t h T M S - d e r i v a t i z a t i o n . 84 (B) The S y n t h e s i s o f T r i d e u t e r i o m e t h y l CLBZ. T r i d e u t e r i o m e t h y l CLBZ ( [ 2 H 3 ] C L B Z , 22) was p r e v i o u s l y s y n t h e s i z e d by P o u p a e r t e t a l . (1988) i n o r d e r t o i n v e s t i g a t e t h e d e u t e r i u m i s o t o p e e f f e c t on t h e p h a r m a c o l o g i c a l a c t i o n o f CLBZ. A l t h o u g h a p r o l o n g e d d u r a t i o n o f a c t i o n was o b s e r v e d , no (k^ /kg) v a l u e s f o r t h e i s o t o p e e f f e c t were r e p o r t e d . In o r d e r t o d e r i v e t h e kn/kn, v a l u e f o r t h i s i s o t o p e e f f e c t in vitro, t r i d e u t e r i o m e t h y l CLBZ was s y n t h e s i z e d by t r i d e u t e r i o m e t h y l a t i o n o f DMC w i t h C D 3 I ( I s e l e and L u t t r i n g h a u s , 1971). *H-NMR ( F i g u r e 13A) r e v e a l e d t h e d i a s t e r e o m e r i c n a t u r e o f t h e H-3/J (3.52 ppm) and H-3a p r o t o n s (3.58 ppm), p r e v i o u s l y d e s c r i b e d f o r [ 2Hs]CLBZ. In c o n t r a s t t o t h e l a t t e r compound, t h e r e s o n a n c e c h a r a c t e r i s t i c o f t h e N-methyl p r o t o n s was ab s e n t i n [ 2H3]CLBZ t h e r e b y d e m o n s t r a t i n g t h e h i g h i s o t o p i c p u r i t y o f t h e N - d e u t e r i o m e t h y l g r o u p . F u r t h e r m o r e , r e s o n a n c e a t 7.20 (H-2', H-6'), 7.36 (H-4') and 7.43 (H-3', H-5') c h a r a c t e r i s t i c o f t h e phenyl p r o t o n s o f [ 2H3]CLBZ a f f o r d e d a c o n v e n i e n t c o m p a r i s o n t o p e n t a d e u t e r i o p h e n y l CLBZ ( i n which t h i s r e s o n a n c e was a b s e n t ) . The mass s p e c t r u m o f [ 2H3]CLBZ s u b s t a n t i a t e d t h e ^-NMR d a t a w i t h M +* a t m/z 303 and v i r t u a l l y no e v i d e n c e f o r t h e p r o t i o a n a l o g u e . C h a r a c t e r i s t i c f r a g m e n t s a t [M-0H] +* m/z 286 and m/z 184 ( t h e d e u t e r i o a n a l o g u e o f m/z 181 d e s c r i b e d f o r [ 2H5]CLBZ) w i l l be d i s c u s s e d l a t e r . 85 F i l e : Bpk Rl 10000- 95 127 ilii<ilinUh,..„ti.,.illJill([„^ !l(n, 86 120 ' ' i 1 ' 1 l ' ' ' I 160 200 240 280 F i g u r e 13. (A) 400 MHz *H-NMR and (B) GCMS ( E I ) s p e c t r a o f [ 2H3]CLBZ (22) 86 (C) The S y n t h e s i s o f Phenol and C a t e c h o l M e t a b o l i t e s o f CLBZ. ( i ) C h e m i c a l S y n t h e s i s o f P h e n o l s and C a t e c h o l s . The phenol and c a t e c h o l CLBZ m e t a b o l i t e s were s y n t h e s i z e d a c c o r d i n g t o t h e p r o c e d u r e used f o r [ 2Hs]CLBZ by u s i n g 4 - m e t h o x y a n i l i n e ( p - a n i s i d i n e ) and 3 , 4 - d i m e t h o x y a n i l i n e ( 4 - a m i n o v e r a t r o l e ) r e s p e c t i v e l y as s t a r t i n g m a t e r i a l s (Scheme X I I I ) . The s t a r t i n g m a t e r i a l s w h i c h were c o m m e r c i a l l y a v a i l a b l e a f f o r d e d t h e 0-methyl e t h e r as t h e p r o t e c t i n g group f o r t h e h y d r o x y l f u n c t i o n a l i t y . M e t h y l e t h e r s a r e r e s i s t a n t t o h e a t , and a r e s t a b l e i n t h e p r e s e n c e o f Zn/HCl and KOH (Greene, 1981). These p r o p e r t i e s were p a r t i c u l a r l y r e l e v a n t w i t h r e g a r d t o o u r s y n t h e t i c p r o c e d u r e (see Scheme X I I I ) . In o u r e x p e r i e n c e t h e 0-methyl e t h e r was f ound t o be an i d e a l p r o t e c t i n g group t h r o u g h o u t t h e s y n t h e t i c scheme, however, s e v e r a l a t t e m p t s were made a t d e p r o t e c t i o n b e f o r e t h i s p r o c e d u r e was s u c c e s s f u l l y a c c o m p l i s h e d . High y i e l d s have been r e p o r t e d f o r t h e d e m e t h y l a t i o n o f methoxybenzene (Jung and L y s t e r , 1977) and o f g a l l o p a m i l ( M u t l i b and N e l s o n , i n p r e s s ) u s i n g t r i m e t h y l s i l y l i o d i d e (TMSI) a t room t e m p e r a t u r e w i t h s t i r r i n g f o r no more th a n 48 h r . Under s i m i l a r c o n d i t i o n s 4'-methoxy-DMC (25) and 4'-methoxy-CLBZ (27) c o u l d n o t be d e m e t h y l a t e d , even a f t e r s t i r r i n g f o r 4 d a y s . Minamikawa and B r o s s i (1978) r e p o r t e d t h e r e s i s t a n c e o f sesamol methyl e t h e r and m e t h y l e n e d i o x y - 3 - m e t h o x y b e n z e n e t o d e m e t h y l a t i o n w i t h TMSI under m i l d c o n d i t i o n s but a c c o m p l i s h e d d e p r o t e c t i o n i n good y i e l d w i t h t h e same r e a g e n t a t 180*C f o r 70 min i n anhydrous q u i n o l i n e . Both 25 and 27 were h e l d a t 180°C f o r 6 h r w i t h 13 and 6.6 e q u i v a l e n t TMSI r e s p e c t i v e l y . 87 Scheme X I I I . S y n t h e s i s o f p h e n o l i c and c a t e c h o l i c m e t a b o l i t e s o f CLBZ. Legend f o r c h e m i c a l s t r u c t u r e s : No- E 2 3 - 2 8 H 29 - 31,34 0CH3 32, 35 OH A: 2 , 4 - d i c h l o r o n i t r o b e n z e n e , 170°C B: e t h y l m a l o n y l c h l o r i d e / d r y benzene C: Zn/HCl D: KOH/DMSO, CH3I E: B B r 3 / d r y CHCI3, H 20 F: r a t l i v e r c y t o s o l , SAM-pTsOH, pH 7.9, MgCl2 88 (The r a t i o n a l e b e h i n d u s i n g t w i c e t h e number o f e q u i v a l e n t s o f TMSI f o r 25 t h a n f o r 27 was due t o t h e f a c t t h a t 25 consumes one e x t r a e q u i v a l e n t i n t h e f o r m a t i o n o f an amide N - s i l y l e t h e r ) . M a r g i n a l d e m e t h y l a t i o n o f 25 a f f o r d e d 26 i n low y i e l d (<9 % ) , and no d e m e t h y l a t i o n o f 27 o c c u r r e d . S u c c e s s f u l d e p r o t e c t i o n o f 25 and 27 was f i n a l l y a c c o m p l i s h e d w i t h BBr3 i n anhydrous CHCI3 a t room t e m p e r a t u r e (McOmie e t a l . , 1968). A h i g h y i e l d was o b t a i n e d f o r 28, but l o s s o f p r o d u c t compromised t h e y i e l d o f 26. The l a t t e r a pproach was a l s o used t o a f f o r d 32 and 35 i n h i g h y i e l d from 31 and 34 r e s p e c t i v e l y . The r e a c t i o n o f methyl e t h e r s w i t h TMSI ( S c h m i d t , 1981; Jung and L y s t e r , 1977) and BBr3 (McOmie e t a l . , 1968) i s t h o u g h t t o pr o c e e d via n u c l e o p h i l i c a t t a c k o f t h e e t h e r oxygen a t t h e S i - and B - c e n t r e s r e s p e c t i v e l y t o a f f o r d t h e c o r r e s p o n d i n g oxonium i o n s 43 and 44- The e l e c t r o n - w i t h d r a w i n g i n f l u e n c e o f bromine r e s u l t s i n t h e B - c e n t r e o f BBr3 b e i n g more e l e c t r o n d e f i c i e n t and c o u l d be r e s p o n s i b l e f o r t h e h i g h e r r e a c t i v i t y o f t h i s r e a g e n t w i t h e t h e r s compared t o TMSI ( V i c k e r y e t a l . , 1979). T h i s would a c c o u n t f o r t h e s u p e r i o r d e m e t h y l a t i o n o f 25 and 27 w i t h BBr3 compared t o TMSI. 43 44 89 ( i i ) In Vitro S y n t h e s i s o f C a t e c h o l O-Methvl E t h e r s . The c a t e c h o l 0-methyl m e t a b o l i t e s 33 and 36a+b were s y n t h e s i z e d e n z y m a t i c a l l y f r o m 32 and 35 r e s p e c t i v e l y by u s i n g S - a d e n o s y l - L - m e t h i o n i n e ( p - t o l u e n e s u f o n a t e s a l t ) as t h e methyl d o n o r , and r a t l i v e r c y t o s o l i c c a t e c h o l 0-methyl t r a n s f e r a s e (COMT) ( A l t o n 1975a). Rat l i v e r c y t o s o l i s a good s o u r c e o f COMT ( G u l d b e r g and Marsden, 1975). E n r i c h m e n t w i t h M g 2 + was n e c e s s a r y because o f t h e e n z y m a t i c r e q u i r e m e n t f o r a d i v a l e n t c a t i o n , and o p t i m a l a c t i v i t y i s a s s o c i a t e d w i t h M g 2 + ( J e f f e r y and R o t h , 1987). Because t h e me t a - O - m e t h y l a t e d i s o m e r s o f 33 and 36 were c o n s i d e r e d t h e b i o l o g i c a l l y r e l e v a n t s p e c i e s , i t was t a c i t l y assumed t h a t COMT would c o n f e r r e g i o s p e c i f i c O - m e t h y l a t i o n t o t h e i r r e s p e c t i v e c a t e c h o l s . T h i s was th o u g h t t o have been t h e case when GCMS a n a l y s i s o f t h e TMS d e r i v a t i v e s o f 33 and 36a+b s u g g e s t e d t h e p r e s e n c e o f one p r o d u c t . However, upon c l o s e r s c r u t i n y o f t h e l i t e r a t u r e , i t was fo u n d t h a t COMT c a t a l y s i s in vitro r e s u l t s i n d i v e r s e m i x t u r e s o f meta- and para-O-methyl d e r i v a t i v e s , w i t h c o m p o s i t i o n d e p e n d i n g on s u b s t i t u e n t e f f e c t s on t h e s u b s t r a t e , pH o f t h e i n c u b a t i o n medium and c o n c e n t r a t i o n o f M g 2 + ( C r e v e l i n g e t a l . , 1972). Re-e x a m i n a t i o n o f 36a+b by GCMS r e v e a l e d t h a t , i n f a c t , b o t h meta- and para-O-methyl i s o m e r s were p r e s e n t ( F i g u r e 1 4 ) . S i m i l a r r e - e x a m i n a t i o n o f 33 f o r an i s o m e r i c p r o d u c t m i x t u r e was n o t p o s s i b l e because t h e TMS d e r i v a t i v e had decomposed. A subsequent a t t e m p t t o s y n t h e s i z e 33 was u n s u c c e s s f u l and s o , a n a l y t i c a l i n t e r e s t s were f o c u s s e d on 36a+b. In o r d e r t o a s s i g n t h e r e t e n t i o n t i m e s f o r t h e meta and para i s o m e r s , t h e r a t i o o f peak a r e a s was compared t o t h e p r o d u c t r a t i o o f a COMT s u b s t r a t e (41) ( C r e v e l i n g e t a l . , 1972) w h i c h was anal o g o u s t o 35. The meta:para p r o d u c t r a t i o o b t a i n e d f o r 41, 72:28, a l l o w e d t h e meta ( t R 23.49 min, 68 %) and p a r a ( t R 22.80 min, 32 90 %) i s o m e r s t o be a s s i g n e d t o 36a and 36b r e s p e c t i v e l y . O OH 41 F i l e >PB294 417.7-418.7 amu.4' 0H-30Me-CLBZ-TMS SMT ADC EIP 23.49 meta ' i I v i i i | i i t i i r*i i i j i i i H s r i i T I i t T-f - i ' f T n ^ T ^ i i f •) i i i v | i i i i | 13.0 14.8 IS. 016.817.0 18.019.8 20.021.0 22.0 23.8 24.0 25.0 26.0 F i g u r e 14. Mass chromatogram o f t h e T M S - d e r i v a t i v e s o f CLBZ 0-methyl c a t e c h o l i s o m e r s a t M + , » m/z 418. Meta i s o m e r ( 3 6 a ) : t R 23.49 min, para i s o m e r ( 3 6 b ) : 22.80 min. 91 (D) GCMS A n a l y s i s o f CLBZ and I t s M e t a b o l i t e s . In o r d e r t o d e t e r m i n e t h e r e t e n t i o n t i m e s o f t h e b i o l o g i c a l l y - d e r i v e d m e t a b o l i t e s , c o n t r o l b i l e was s p i k e d w i t h 4'-hydroxy CLBZ ( 2 8 ) , 4'-hydroxy DMC ( 2 6 ) , 3 ' , 4 ' - d i h y d r o x y CLBZ (35) and 3 ' , 4 ' - d i h y d r o x y DMC ( 3 2 ) . The b i l e was e x t r a c t e d and p r e p a r e d f o r GCMS i n a s i m i l a r f a s h i o n t o t h e b i l e o f t h e dosed r a t s . TIC p l o t s f o r t h e T M S - d e r i v a t i z e d s t a n d a r d s a r e p r e s e n t e d i n F i g u r e 15. Because o f t h e l i m i t e d q u a n t i t i e s o f t h e 0-methyl c a t e c h o l a n a l o g u e s o f CLBZ (36a+b) and DMC ( 3 3 ) , t h i s e x p e r i m e n t c o u l d n ot be per f o r m e d w i t h t h e s e samples. Mass s p e c t r a o f t h e T M S - d e r i v a t i z e d s t a n d a r d s a r e p r e s e n t e d i n F i g u r e s 16 - 22, and p o s s i b l e f r a g m e n t a t i o n mechanisms p r o p o s e d i n Schemes XIV - X V I I I . GCMS d a t a a r e summarized i n T a b l e 1. There a r e l i m i t e d r e p o r t s i n t h e l i t e r a t u r e d ocumenting t h e mass s p e c t r o s c o p y o f CLBZ and DMC ( R i v a e t a l . , 1981; C a c c i a e t a l . , 1979) and t h e i r 4'-hydroxy m e t a b o l i t e s ( D r o u e t - C o a s s o l o e t a l . , 1989). V o l z e t a l . (1979) r e p o r t e d s e v e r a l m e t a b o l i t e s o f CLBZ b u t p r o v i d e d no s u b s t a n t i a t i n g s p e c t r a l e v i d e n c e . To o u r knowledge, t h e f o l l o w i n g r e p r e s e n t s t h e f i r s t d e t a i l e d a c c o u n t o f t h e mass s p e c t r o s c o p y o f CLBZ and i t s m e t a b o l i t e s . ( i ) Mass s p e c t r o s c o p y o f CLBZ. The a v a i l a b i l i t y o f CLBZ, and i t s d e u t e r a t e d a n a l o g u e s [2Hs] CLBZ (21) and [ 2h"3] CLBZ (22) w i t h d e u t e r i u m p r e s e n t i n t h e phenyl and methyl c e n t r e s r e s p e c t i v e l y a l l o w e d s e v e r a l f r a g m e n t s o f CLBZ t o be r e a d i l y e l u c i d a t e d . F r a g m e n t a t i o n s were p e r c e i v e d as a r i s i n g from b o t h k e t o and en o l t a u t o m e r s o f t h e l a c t a m (Scheme X I V ) . When t h e e n o l t a u t o m e r was 9 2 invoked i t was apparent that loss of OH* gave r i se to the highest mass fragments for 21 ([M-0H] +*, m/z 288) and 22 ([M-0H] +*, m/z 286). Subsequent loss of CO, charac te r i s t i c of lactams (Budzikiewicz et a l . , 1967) afforded ions m/z 260 and m/z 258 for (21) and (22) respect ive ly . It i s noteworthy that th is cascade proceeds with the prel iminary loss of OH*, and not CO as evidenced by the v i r tua l absence of [M-C0]* + at m/z 277 and 275 in the mass spectra of (21) and (22) respect ive ly . The loss of neutral carbenes i s cha rac te r i s t i c of aromatic amides (Budzikiewicz et a l . , 1967) and could account for the loss of H2=C=0 with concomitant r ing closure to afford [M-42] + *. The loss of isocyanate from semicarbazones has been described (Budzikiewicz et a l . , 1967). In l i k e fashion, th is fragmentation was extrapolated to the lactam of CLBZ to account for the loss of phenylisocyanate and methylisocyanate (Scheme XV). Loss of phenyl isocyanate gave r i se to the ions m/z 181 and 184 for 2_1 and 22 respect ive ly . Correspondingly, loss of methylisocyanate afforded ions m/z 248 and 243. ( i i ) Mass spectroscopy of CLBZ metabol i tes. The mass spectral fragmentation cha rac te r i s t i c of CLBZ was extrapolated to CLBZ metabolites with appropriate increments made for -OTMS, di-OTMS and -OTMS-OMe subst i tuted fragments (Scheme XIV). Fragmentation pathways did not allow the TMS-derivatized 0-methyl catechols 36a+b to be d is t ingu ished. TMS-directed loss of the methyl rad ica l from both the M +* and [M-H2CC0]+* ions gave r i se to prominent high mass ions for 28, 35 and 36a+b (Scheme XIV). In add i t ion , loss of C 2 He from M +* and [M-H 2 CC0] + * of 36a+b gave r i se to the major ions m/z 388 and 346 respect ively 93 (Scheme X V I ) . The l o s s o f Si(013)4 f r o m di-TMS c a t e c h o l s t o form a 5-membered s i l y l o x y r i n g (Goodman and Markey, 1981) i s an a l o g o u s t o t h e l o s s o f C2H6 from 36a+b shown i n Scheme X V I . However, t h e f o r m a t i o n o f t h i s f r a gment w i t h t h e di-TMS c a t e c h o l s o f CLBZ was n o t a p p a r e n t . ( i i i ) Mass s p e c t r o s c o p y o f T M S - d e r i v a t i z e d N-desmethvl m e t a b o l i t e s . Whether T M S - d e r i v a t i z a t i o n o f amides o c c u r s a t t h e k e t o n i t r o g e n o r t h e e n o l h y d r o x y l t h r o u g h t a u t o m e r i s m i s u n c l e a r (Goodman and Markey, 1981). F o r c o n v e n i e n c e , t h e N-TMS d e r i v a t i v e s a r e r e p r e s e n t e d . D i a g n o s t i c f r a g m e n t a t i o n o f t h e N-TMS d e r i v a t i v e s i n v o l v e d l o s s o f th e methyl r a d i c a l (Scheme X V I I ) , and l o s s o f t h e ph e n y l m o i e t y (Scheme X V I I I ) . Loss o f t h e p h e n y l i s o c y a n a t e w h i c h was p r e v i o u s l y d i s c u s s e d g i v e s r i s e t o m/z 239. The i o n m/z 225 was a m a j o r f r a g m e n t f o r 20, 26, 32 and 33 and p r o b a b l y a r o s e from t h e l o s s o f a n e u t r a l l a c t a m (Scheme X V I I I ) . The d i a g n o s t i c l o s s o f C2H6, d e s c r i b e d f o r M +* and [M-H 2CC0] +- o f 36a±b, a l s o o c c u r r e d w i t h 33 t o a f f o r d t h e i o n m/z 446 (Scheme X V I ) . 94 f i l e >RB313 70.0 17 2000000 1800800 1608000-1400080-1200088^ 1000000-800008-600000 400008-200808 0--550.0 amu. METflB-STB-BILE-SPIKE EIP 98 26, 16 .88 21 .36 ' i • ' • • i ' ' • ' i • 1 ' ' l • ' 1 ' l • • • • i • • • • i • • • • i • • • • i • • • • i • • 17.0 18.0 19.8 20.0 21.8 22.0 23.0 24.0 25.0 26.0 f i l e >RG313 70.0-558.0 amu. METfiB-STD-BILE-SPIKE TIC 15 .56 F i g u r e 15. TIC p l o t s o f (A) T M S - d e r i v a t i z e d m e t a b o l i t e s t a n d a r d s 26, 28, 32 and 35 a f t e r r e c o v e r y from s p i k e d c o n t r o l b i l e . (B) 1 5 - f o l d d i l u t i o n o f (A) w i t h EtOAc. 9 5 F i l e >AB313 Bpk Ab 121760 130O0O-120000-110000-100000-98888-80888-70088-60008-50000-40000-38800- 75 20008-1 10000-e-1 METflB-SID-BILE-SPIKE CLP 22S 193 13S 197 88 120 168 208 17 431 331 356 389 446 / Sean 83 ,89 M i n . pi 10 i l e a :90 I 8 0 i70 :60 :S0 :40 r30 rZO rl0 •^ 0 240 280 328 360 480 440 F i g u r e 16. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-DMC (26) showing t h e p r e s e n c e o f M +* a t m/z 446. F i l e >AB313 Bpk Ab 56520 60000-55000-50000-45000-4000O-35008-30008-25000-20000-15000-10000- ^76 500O-0-L A 8Q METAB—STD—BILE-SPIKE CLP 179 153 151 343  76 117 \ „„„ 303 35 B L ™ ( I ll 2 1 7 / I jl .ijLiu<ji.llit.li JlLk i 1, .iL-li AifaiLjIt J.it.^c.itL. JVt^/iL ,JL L Soan 21.19 » 388 371 128 168 200 248 280 328 360 400 275 ii n . c-110 00 r90 r80 r70 E*8 i50 i40 r30 r20 O <"-0 F i g u r e 17. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-CLBZ (28) showing t h e p r e s e n c e o f M + - a t m/z 388. 96 F i l e >AB313 Bpk Ab 52280 55800-50888-45800-40000 35000-38800-25000-20000-15000-10000 5000 0 METAB-STD-BILE-SPIKE CLP 99 * i i Y j - J i - d l L 225 2 6 7 " 6 p9 389*?* 4 4 4 4 9 3 I 2 5 0 3 0 0 3 5 8 4 8 0 4 5 8 5 8 8 485 4  108 158 288 Scan 177 19.51 n i n . 534 r l l 0 rl88 |90 :88 r7 0 :68 !se :48 r3 0 |2G 4 3 < [ i i e -0 F i g u r e 18. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 3',4'-dihydroxy-DMC (32) showing t h e p r e s e n c e o f M+* a t m/z 534. F i l e >AB313 Bpk Ak 47688 52000-1 48000 44888 40000 36800 32888-28000 24000-20808 16800-12000-8000-4080-0-92 1 3 7 1 5 3 _ IIJI.^1 • ' i t i l lni '^l i METAB-STD-BILE-SPIKE CLP 1 7 9 267 303 L .*c^A . j i«^-wx^i^ jtLtJt. A. .... Scan 428 23.81 M i n . 476 188 1 5 8 288 258 3 8 8 3 4 3 4 3 1 ' 3 5 8 4 8 8 4 5 ' a ' ' r l l 0 J100 r98 is 8 70 r*8 ise 8 r3 0 :28 8 F i g u r e 19. EI mass s p e c t r u m o f t h e TMS d e r i v a t i v e o f 3 ' , 4 ' - d i h y d r o x y CLBZ (35) showing t h e p r e s e n c e o f M+* a t m/z 476. F i l e >flG294 Bpk Rb 10895 3' ,4'-0H,0Me-CLBZ CLP S c a n 452 23.70 m i n . 12600-1 11000-10000-9000-8000-7000-6000-5000-4000-3000-2000-1000-0-73 418 O - S i -346 373 166 / 153 117 lllill'ii'qi.kllL T—'—1—'—I—* 209 303 3 ; 8 245 282 \ lllltjliL>,,ili,itt<MltUll 80 i —1 — i — 1 — i — ' — i — ' — i — ' — i — ' — i — ' — r 120 160 200 240 280 l ink i I "i—1—i—'—]—>—i—"—r 320 360 400 r-110 ;108 r90 |80 r70 7&0 |50 r46 :30 r20 Ll0 F i g u r e 20. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3' methoxy-CLBZ (36a) showing t h e p r e s e n c e o f M +* a t m/z 418. F ; i l e >flG294 flb 7087 3' ,4'-0H,0Me-CLBZ CLP 7000H 6000-1 5000-1 40004 3000-1 2000-1 1000-1 73 166 / 418 \ 147 117 \ 20; \ 209 •IlLff 303 318 281 { / ill u^^tiLyLil.ti 80 120 160 ' 280 240' ' 280' ' 320" ' 368' ' '40V F i g u r e 21. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3'-hydroxy-4' methoxy-CLBZ (36b) showing t h e p r e s e n c e o f M +* a t m/z 418. S c a n 414 23.05 m i n . HO j-100 r90 J80 r70 J60 5^0 f40 30 720 :10 98 F i l e >AB291 Bpk Ab 13430 -MeO—DMC—TMS CLP Scan 18 . 42 14000-12000-10000 8000 6000 4000 2000 0-* 73 225 135 ^137 197^ 476 \ CH3O 239 / 266 296 1 / S 361 418 446 100 150 200 2S0 300 198 M i n . pi 10 h-00 i90 jso r?0 r60 :S0 ?40 "-3 0 ^0 ri0 ^0 350 400 450 F i g u r e 22. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) showing t h e pr e s e n c e o f M +* a t m/z 476. 99 Scheme XIV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n r e t e n t i o n o f b e n z o d i a z e p i n e n i t r o g e n . Legend f o r c h e m i c a l s t r u c t u r e s a p p l i e s t o Schemes XIV t o X V I I I . Loss o f t h e methyl r a d i c a l shown f o r 36a i s r e p r e s e n t a t i v e o f 36b. No. R l R 2 R3 R 4 M+- (m/z) 21 2 H 2 H 2 H CH 3 305 22 H H H CD 3 303 28 H OTMS H CH 3 388 35 OTMS OTMS H CH 3 476 36a 0CH3 OTMS H CH 3 418 36b OTMS OCH3 H CH 3 418 100 B Scheme XV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n l o s s o f t h e (A) N-phenyl and (B) N-methyl m o i e t i e s . Loss o f t h e p h e n y l i s o c y a n a t e i n A gave r i s e t o m/z 181 f o r 21, 28, 35, 36a and 36b, and m/z 184 f o r 22. (B) Loss o f methyl i s o c y a n a t e gave r i s e t o t h e c o r r e s p o n d i n g compound and fragment p a i r s , i . e . (Compound No., [M-R 4 N C 0 ] + ' ) . 21, m/z 248; 22, m/z 243; 28, m/z 331; 35, m/z 419; 36a and 36b. m/z 361. 101 Scheme X V I . Proposed f r a g m e n t a t i o n o f O-methyl c a t e c h o l s . Ions u n d e r g o i n g t h i s f r a g m e n t a t i o n were M+- o f 36a+b (m/z, 4 1 8 ) , t h e i r d a u g h t e r i o n s [M-H 2CCO] +- (m/z 376) and M+- o f 33 (m/z 4 7 6 ) . L o s s o f C 2 H 6 from t h e s e p r o g e n i t o r i o n s a f f o r d e d t h e c o r r e s p o n d i n g f r a g m e n t s m/z 388, 346 and 446. Scheme X V I I . Proposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds r e s u l t i n g i n t h e l o s s o f a methyl r a d i c a l . I o n s u n d e r g o i n g t h i s f r a g m e n t a t i o n , 20 (m/z 2 6 3 ) , 32 (m/z 534) and 33 (m/z 476) a f f o r d e d i o n s m/z 348, 519 and 461 r e s p e c t i v e l y . 102 A B R 2 Scheme X V I I I . P roposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds 20, 32 and 33 r e s u l t i n g i n l o s s o f t h e N-phenyl r i n g . (A) Loss o f t h e p h e n y l i s o c y n a t e gave r i s e t o t h e i o n m/z 239 and (B) l o s s o f t h e l a c t a m gave r i s e t o t h e major i o n m/z 225. Table 1. GCMS data for synthetic CLBZ-related compounds. Compound (No.)a tR (min) M+'(m/z, %) Diagnostic ions (m/z, %) [2H53DHC (20) 13.80 363 (26) 225 (100) 239 (46) 82 (42) 348 (21) [2H5]CLBZ (2J.) 15.97 305 (100) 288 (44) 260 (44) 181 (18) 248 (16) [2H3]CLBZ (22) 15.99 303 (100) 258 (94) 261 (76) 286 (52) 184 (22) 4'-OH DMC (26) C 17.98 446 (70) 225 (100) 239 (40) 431 (26) 403 (14) 389 (12) 4'-OH CLBZ (28) C 21.17 388 (100) 343 (75) 181 (33) 331 (42) 153 (32) 3',4'-<OH)2 DMC (32) C 19.54 534 (100) 225 (70) 519 (30) 239 (22) 4'-0H#3'-0Me DMC (33) 18.42 476 (58) 225 (48) 239 (18) 461 (17) 446 (8) 3',4'-<OH)2 CLBZ (35) C 23.83 476 (100) 461 (30) 343 (28) 431 (26) 419 (20) 4'-OH,3'-OMe CLBZ (36a) 23.49 418 (100) 346 (42) 373 (40) 181 (22) 388 (20) 3'-OH,4'-OMe CLBZ (36b) 22.80 418 (72) 346 (42) 373 (40) 181 (22) 388 (20) carbinolamide (39) 17.33 388 (12) 373 (24) 343 (24) 225 (20) 239 (10) 358 (6) a: Compounds 20, 26-39 were TMS-derivatized. b: Retention times were obtained according to GCMS Condition (b). c: Retention times obtained when recovered from spiked bile, d: % = Relative intensity of ion. 104 (E) S y n t h e s i s o f N-hvdroxvmethyl CLBZ ( c a r b i n o l a m i d e ) . The f o r m a t i o n o f c a r b i n o l a m i d e s by t h e c o n d e n s a t i o n o f amides and fo r m a l d e h y d e i s an e q u i l i b r i u m p r o c e s s (Scheme XIX) i n wh i c h t h e p r o d u c t i s f a v o u r e d by low t e m p e r a t u r e s , low c o n c e n t r a t i o n s o f aqueous a c i d o r base, and h i g h f o r m a l d e h y d e c o n c e n t r a t i o n (Zaugg and M a r t i n , 1965). Both a c i d i c and b a s i c c o n d i t i o n s c a t a l y z e t h e r e a c t i o n . B a s i c c o n d i t i o n s a f f o r d t h e c a r b i n o l a m i d e as t h e f i n a l p r o d u c t , whereas a c i d i c c o n d i t i o n s promote d i m e r i z a t i o n o f t h e formed c a r b i n o l a m i d e t o t h e c o r r e s p o n d i n g e t h e r (R-CONHCH2)20 o r m e t h y l e n e b i s a m i d e (R-C0NH) 2CH2 ( N a i r and F r a n c i s , 1980; Zaugg and M a r t i n , 1965). Under base c a t a l y s i s , t h e g e n e r a t e d amide i o n , R-C(=NH)0" » R-C(=0)NH", i n i t i a t e s n u c l e o p h i l i c a t t a c k a t t h e c a r b o n c e n t r e o f f o r m a l d e h y d e (March, 1985d) as e x e m p l i f i e d i n Scheme XX. RC0NH 2 + CH 20 ^ S-RC0NHCH 20H Scheme XIX. E q u l i b r i u m c o n d e n s a t i o n between an amide and f o r m a l d e h y d e . Scheme XX. The c o n d e n s a t i o n o f f o r m a l d e h y d e w i t h DMC i l l u s t r a t e s a p r o t o t y p e b a s e - c a t a l y z e d f o r m a t i o n o f a c a r b i n o l a m i d e . 105 B a s e - c a t a l y z e d c o n d e n s a t i o n has been used i n t h e s y n t h e s i s o f s e v e r a l c a r b i n o l amides ( K e d d e r i s e t a l . , 1989; S l a t t e r e t a l . , 1989; S l a t t e r , 1987; V a r i a e t a l . , 1984; N a i r and F r a n c i s , 1980), w i t h t h e y i e l d d e p e n d i n g t o some e x t e n t , on t h e c o n d i t i o n s used. F o r example, t r e a t m e n t o f t h e s e c o n d a r y formamide o f r e c i p a v r i n w i t h 37 % f o r m a l d e h y d e i n t h e p r e s e n c e o f K2CO3 (0.5 e q u i v ) a f f o r d e d t h e c a r b i n o l a m i d e i n 4 % y i e l d ( S l a t t e r , 1987). A l t e r n a t i v e l y , when t h e amide i o n was g e n e r a t e d w i t h KH i n d r y THF, c o n d e n s a t i o n w i t h gaseous formaldehyde r e s u l t e d i n a 90 % c o n v e r s i o n t o t h e c a r b i n o l a m i d e ( S l a t t e r e t a l . , 1989). Two o f t h e approachs used t o s y n t h e s i z e t h e CLBZ c a r b i n o l a m i d e a r e d e s c r i b e d . The c o n d e n s a t i o n o f f o r m a l d e h y d e w i t h DMC u s i n g ( i ) K2CO3 and 37 % f o r m a l d e h y d e ( N a i r and F r a n c i s , 1980) and ( i i ) KOH and p a r a f o r m a l d e h y d e . ( i ) S y n t h e s i s o f t h e c a r b i n o l a m i d e u s i n g K 2C0 3 and 37 % f o r m a l d e h y d e ( N a i r  and F r a n c i s . 19801. DMC was s t i r r e d w i t h 37 % f o r m a l d e h y d e i n t h e p r e s e n c e o f K2CO3 f o r 24 h a t room t e m p e r a t u r e . A f t e r t h e r e a c t i o n was t e r m i n a t e d , a n a l y t i c a l TLC w i t h 15:1 ( v / v ) CHCl3:MeOH r e v e a l e d t h a t t h e r e a c t i o n m i x t u r e c o n s i s t e d o f t h r e e p r o d u c t s a t Rf 0.20, 0.29 and 0.36 w i t h c o r r e s p o n d i n g r e l a t i v e u.v. i n t e n s i t i e s 45 %, 45 % and 5 %. There was a l s o a m i n o r amount o f s t a r t i n g m a t e r i a l a t Rf 0.41. A p r e l i m i n a r y a t t e m p t t o p u r i f y t h e s e components i n v o l v e d t h e use o f f l a s h c h r o m a t o g r a p h y ; however, i n c o m p l e t e r e s o l u t i o n o f t h e r e s u l t i n g f r a c t i o n s n e c e s s i t a t e d t h e use o f p r e p a r a t i v e TLC. P r e p a r a t i v e TLC w i t h 15:1 (v / v ) CHCl3:Me0H was used t o s e p a r a t e t h r e e components 45 ( R f , 0.20), 46 ( R f , 0.30) and 47 ( R f , 0.43) r e s p e c t i v e l y i n 106 h i g h p u r i t y . (a) HPLC a n a l y s i s . HPLC a n a l y s i s o f t h e f r e s h l y p r e p a r e d c r u d e p r o d u c t i n 50:50 ( v / v ) MeOH:H20 showed f o u r compounds a t tR 7.85, 8.57, 9.61 and 10.68 min w i t h i n t e n s i t i e s 19.5, 28.4, 29.4 and 22.6 % r e s p e c t i v e l y ( F i g u r e 2 3 ) . Repeat i n j e c t i o n s a f f o r d e d r e p r o d u c i b l e HPLC p r o f i l e s . The p r o d u c t was s t o r e d a t 4°C, b u t subsequent HPLC a n a l y s i s showed t h e p r e s e n c e o f o n l y three compounds a t t R 7.67, 8.35 and 10.40 min w i t h i n t e n s i t i e s 19.1, 58.6 and 22.3 % r e s p e c t i v e l y ( F i g u r e 2 3 ) . The e v i d e n c e s u g g e s t s t h a t t h e compound a t tR 9.61 min may have decomposed i n t o t h e o t h e r component o f t h e m i x t u r e a t tR 8.57 min as j u d g e d by t h e i n c r e a s e o f t h e peak a r e a o f t h e l a t t e r w i t h c o n c o m i t a n t d i s a p p e a r a n c e o f t h e f o r m e r . S i n c e t h e compound a t tR 9.61 min decomposed b e f o r e LCMS a n a l y s i s , i t s c h e m i c a l i d e n t i t y c o u l d not be c h a r a c t e r i z e d . (b) LCMS a n a l y s i s . The LCMS r e s u l t s o f t h e cr u d e p r o d u c t a f t e r o v e r n i g h t s t o r a g e r e v e a l e d t h e p r e s e n c e o f t h r e e compounds ( F i g u r e 2 4 ) . The compound a t tR 4.68 min (45) gave a poor mass spec t r u m and a f f o r d e d l i t t l e s t r u c t u r a l i n f o r m a t i o n . The LCMS ( C I ) spectrum o f t h e compound w i t h t R 6.68 min (46) c o n t a i n e d i o n s MH +, m/z 317 and [MH-H20] +, m/z 299. These i o n s s u g g e s t e d t h a t a hy d r o x y m e t h y l group was added t o DMC i n t h e r e a c t i o n w i t h f o r m a l d e h y d e , b u t p r o v i d e d no i n f o r m a t i o n about t h e p o s i t i o n o f a d d i t i o n . The s t a r t i n g m a t e r i a l DMC (47) (MH +, m/z 287; [MNH4] +, m/z 304) was p r e s e n t 107 a t t R 7.37 min. ( c l GCMS A n a l y s i s . The GCMS (TIC) p l o t o f an a l i q u o t o f t h e T M S - d e r i v a t i z e d f r e s h l y p r e p a r e d c r u d e p r o d u c t ( F i g u r e 25) r e v e a l e d a compound a t tR 17.33 min w i t h M +* a t m/z 388. Mass s p e c t r a l e v i d e n c e s u g g e s t e d t h a t t h i s compound c o u l d be t h e T M S - d e r i v a t i v e o f t h e c a r b i n o l a m i d e (39) (Scheme X X I , T a b l e 1 ) . The pr e s e n c e o f i o n [M-15] + * a t m/z 373 and TMS + a t m/z 73 s u b s t a n t i a t e d t h e i d e n t i t y o f t h e compound as a T M S - d e r i v a t i v e . The i o n CH20-TMS+ a t m/z 103 i s c h a r a c t e r i s t i c o f T M S - d e r i v a t i z e d p r i m a r y a l c o h o l s (Goodman and Markey, 1981), and i s c o n s i s t e n t w i t h t h e c a r b i n o l a m i d e s t r u c t u r e . The i o n ([M-3 0 ] + * a t m/z 358 was c o n s i s t e n t w i t h t h e m i g r a t i o n o f t h e TMS group from t h e hydroxyme t h y l oxygen t o t h e c a r b o n y l oxygen accompanied by l o s s o f CH2O. F r a g m e n t a t i o n o f t h i s t y p e w h i c h r e s u l t s i n t h e e l e c t r o p h i l i c a t t a c k o f S i on remote e l e c t r o n dense c e n t r e s has been d e s c r i b e d ( S e i b l , 1988). R e p o r t s ' i n t h e l i t e r a t u r e documenting t h e f o r m a t i o n o f [ M - 3 0 ] + * f o r T M S - d e r i v a t i z e d c a r b i n o l a m i d e s a r e d i v i d e d . Some w o r k e r s have o b s e r v e d t h i s f r a g m e n t a t i o n ( S l a t t e r e t a l . , 1989; A l t o n e t a l . , 1975b), whereas o t h e r s have n o t ( V a j t a e t a l . , 1988). Loss o f CH2O g i v e s r i s e t o t h e en o l 0-TMS d e r i v a t i v e o f DMC ( [ M - 3 0 ] + * , m/z 358) whi c h i n t u r n c o u l d f ragment t o a f f o r d t h e i o n s [M-30-1 5 ] + - , m/z 343, [M-30-119]" 1"-, m/z 239 and [M-30-133]" 1"-, m/z 225 c o n s i s t e n t w i t h pathways shown i n Schemes XV I I and X V I I I . A f t e r TLC p u r i f i c a t i o n , t h e GCMS ( E I ) s p e c t r a o f t h e T M S - d e r i v a t i z e d 45 and 46 were o b t a i n e d . The EI mass s p e c t r u m o f 45, l i k e t h e LCMS ( C I ) mass s p e c t r u m y i e l d e d l i t t l e s t r u c t u r a l i n f o r m a t i o n . T M S - d e r i v a t i z e d 46 appeared a t tR 14.39 min i n t h e GCMS (TIC) p l o t , and r e v e a l e d M+- a t m/z 108 370 i n t h e EI mass spectrum ( F i g u r e 2 6 ) . O t h e r d i a g n o s t i c i o n s i n c l u d e d [ M - 1 5 ] + - , m/z 355 and [M-145]" 1"-, m/z 225. A l t h o u g h LCMS d a t a on 46 (MH +, m/z 317) r e v e a l e d t h a t i t was a h y d r o x y m e t h y l a t e d d e r i v a t i v e o f DMC, GCMS e v i d e n c e s u g g e s t e d t h a t t h i s compound ( M + * , m/z 370) was not t h e p u t a t i v e c a r b i n o l a m i d e o b t a i n e d i n t h e c r u d e p r o d u c t ( M + * , m/z 388, F i g u r e 25 B ) . Thus, -"H-NMR s p e c t r o s c o p y was employed t o f u r t h e r probe t h e s t r u c t u r e o f t h e s e compounds. F i g u r e 23. HPLC o f c r u d e p r o d u c t , (A) f r e s h l y p r e p a r e d and (B) a f t e r o v e r n i g h t s t o r a g e a t 4°C. M o b i l e phase 50:50 ( v / v ) Me0H:H20. Chromatograms s u g g e s t t h a t t h e compound a t tp» 9.61 min i n (A) upon s t o r a g e , may have decomposed i n t o t h e compound a t t R 8.57 min. 109 DISPBK.I4S CTIC-89900, 100X-37219] EI 100 SB SB IB 60 58 «0 3B — ] — i — i — i — r 20* I 1 1 ' zzs 275 MH" 3B4 MNH^ _ r r _ r " 300 3S0 F i g u r e 24. (A) LCMS chromatogram o f t h e c r u d e p r o d u c t a f t e r o v e r n i g h t s t o r a g e a t 4°C shows t h e appearance o f 45, 46 and 47 a t t R 4.68, 6.68 and 7.37 min r e s p e c t i v e l y . (B) LCMS ( C I ) s p e c t r u m o f 46 shows t h e appearance o f MH + and [MH-H 20] + a t m/z 317 and 299 r e s p e c t i v e l y . (C) LCMS ( C I ) s p e c t r u m o f 47 (DMC) shows t h e appearance o f MH + and MNH4 + a t m/z 287 and 304 r e s p e c t i v e l y . 110 688000 558088-588080 458880-400000-350009-388080-i ; 250088-208000" 158080-108800-58008 0 13.93 IS .78 18 15 . IS .04 39 17.3: A. 'i 16.< Z 8 . y3 • - . - \ ;« .17.1 JUU..L. • • i • • • • i• • • • i • • •• i • • • • i • • • • i • • • • i • • • • i • • • • i • • • • i • • • r i • • • • i • 1 3 . 0 1 4 . 0 1 5 . 0 1 6 . 8 1 7 . 0 1 8 . 8 1 9 . 0 2 0 . 8 2 1 . 0 2 2 . 8 2 3 . 0 2 4 . 0 2 5 . 0 2 6 . 0 Scan 304 17.27 min . 14800-12800-1 0080-.aeee-6080-4000-2000-73 B 103 163 193 239 301 ;16 00 lO CO t i i era oran -1 0 0 790 r78 760 -f~0 4^0 373 739 388 fM+413 516 ^28 r 18 . . , . . . . . . . .•1 (A(h A C tA F i g u r e 25. (A) GCMS ( C o n d i t i o n , b ) TIC p l o t o f f r e s h l y p r e p a r e d , TMS-d e r i v a t i z e d c r u d e p r o d u c t . P u t a t i v e c a r b i n o l a m i d e - T M S appears a t t R 17.33 min. (B) GCMS ( E I ) mass spectrum o f a p p a r e n t c a r b i n o l a m i d e - T M S shows t h e p r e s e n c e o f M+* a t m/z 388 (*See E x p e r i m e n t a l f o r d e t a i l s ) . I l l A Scheme X X I . (A) F r a g m e n t a t i o n o f T M S - d e r i v a t i z e d 39 (m/z 388) shows t h e m i g r a t i o n o f TMS from t h e h y d r o x y m e t h y l t o t h e e n o l oxygen c e n t r e ( S e i b l , 1988) w i t h t h e c o n c o m i t a n t l o s s o f CH2O t o a f f o r d t h e i o n [ M - 3 0 ] + * , m/z 358. (B) F r a g m e n t a t i o n o f m/z 388 r e s u l t i n g i n l o s s o f a methyl r a d i c a l t o a f f o r d m/z 373. 112 fiile >R2962 70.0-580.0 anu. CfiRBINOLOMIDE-2-TMS 1 TIC -<?00000-808000-700000 600000-580880 460000-380886 200000 108000 0-- 11.90 14.39 1 16.15 43 i ] i i i / | i 1 ! i i | i i i i | i i i i | i i i i | i I'T •"> f v . .•• f >-r. • 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.8 20.6 21 .0 22.0 23.0 24.8 25.8 f i l e >R2962 Bpk fib 120392 CfiRBIN0LflMIDE-2-TMS 130000-120000-110000-100000-90000-88008-70000-60000-50000-4W000-30000-20080-10000-0-73 225 251 100 301 327 80 288 X \ - 1 Z 4 179 \ i 273 \ \ 328 \ / j 155 / \ I ' I \y 1 I • • • I • •' I ' ' • I • • • I ' • • I ' • ' I ' ~ I 1 ' ' I • • • I • '" I • • J j • • • l " • • I ' • Scan 219 14.31 min. riie :100 r98 jee :78 6^0 r50 H0 -30 20 18 370 120 168 200 240 280 320 3co F i g u r e 26. (A) GCMS ( C o n d i t i o n , b ) TIC p l o t o f p u r i f i e d 46 d e r i v a t i z e d w i t h TMS. (B) GCMS ( E I ) spect r u m o f 46 shows t h e p r e s e n c e o f M+- a t m/z 370. *See E x p e r i m e n t a l f o r d e t a i l s . 113 (d) NMR a n a l y s i s . Because t h e T L C - p u r i f i e d components 45, 46 and 47 were p o o r l y s o l u b l e i n CDCI3, DMSO-ds was used as an a l t e r n a t i v e NMR s o l v e n t . Complete d i s s o l u t i o n o f 45, 46 and 47 i n t h i s s o l v e n t a l l o w e d w e l l - d e f i n e d *H-NMR s p e c t r a t o be o b t a i n e d . In t h e *-H-NMR sp e c t r u m o f 47 (DMC) ( F i g u r e 2 7 ) , t h e amide p r o t o n appeared a t 10.70 ppm whi c h was l o w e r d o w n f i e l d t h a n t h e amide p r o t o n o f [2Hs]DMC (20) i n CDCI3 (N-H, 8.21 ppm) ( A p p e n d i x ) . The d e s h i e l d i n g o f t h e amide p r o t o n o f 47 was t h e r e s u l t o f s t r o n g H-bonding w i t h DMSO (Chapman and K i n g , 1964). U p f i e l d r e s o n a n c e d i s p l a y e d by 47 i n DMSO i s a l s o n o t e w o r t h y . Resonance f o r t h e C-3 p r o t o n s o f 47 i n DMSO appeared as broad s i g n a l s a t 3.20 and 3.68 ppm, s y m m e t r i c a l l y d i s p l a c e d about t h e c h e m i c a l s h i f t 3.41 ppm. In CDCI3, t h e C-3 p r o t o n s o f [ 2 H s]DMC (20) appeared as a s i n g l e t (2H, 3.55 ppm) ( F i g u r e 27, I n s e t ) , because r a p i d e q u i l i b r a t i o n between t h e pseudo-boat c o n f o r m e r s ( o f (20)) r e n d e r s t h e (C-3) p r o t o n s e q u i v a l e n t . However, i n DMSO i t a p p ears t h a t t h i s e q u i l i b r a t i o n o c c u r s l e s s r a p i d l y on t h e NMR t i m e s c a l e w i t h t h e a c q u i s i t i o n o f p a r t i a l d i a s t e r e o m e r i c c h a r a c t e r f o r H-3/J (3.20 ppm) and H-3o: (3.68 ppm). The d i a s t e r o m e r i c d i s p o s i t i o n o f H-3/5 and H-3a i n t h e b e n z o d i a z e p i n e r i n g was p r e v i o u s l y d i s c u s s e d f o r [ 2Hs]CLBZ ( 2 1 ) . The c h e m i c a l s h i f t s o f 46 f o r t h e amide N-H, benzo and phenyl p r o t o n s ( F i g u r e 28) were s i m i l a r t o 47 (DMC). However, r e s o n a n c e i n t h e u p f i e l d r e g i o n r e v e a l e d t h a t t h e C-3 c e n t r e was c h e m i c a l l y m o d i f i e d by r e a c t i o n w i t h f o r m a l d e h y d e . The p r e s e n c e o f a t r i p l e t , d o u b l e t and broad s i n g l e t ( e x c h a n g e a b l e w i t h D2O) a t 3.42, 3.94 and 4.56 ppm r e s p e c t i v e l y was c o n s i s t e n t w i t h h y d r o x y m e t h y l a t i o n t a k i n g p l a c e a t t h e 3 - p o s i t i o n o f DMC. The -^ H-NMR sp e c t r u m o f 45 (A p p e n d i x ) was s i m i l a r t o t h a t o f 47 f o r 114 t h e a r o m a t i c p r o t o n s and o f f e r e d no d i a g n o s t i c s t r u c t u r a l i n f o r m a t i o n i n t h e u p f i e l d r e g i o n . SHHPLt M : t 2 i ' B.BUCl 082!, 7-5 H-9 r ' • • • • . • 1 1 I • ' I ' ' I : •—. — — — ' — ' r — ^ — - r — • _ r - r - ^ - -6-"- 8.8 7.3 7.a 6.5 6.Z 5.5 S .2 t.'s 4*0 3^ 5 P?.1 F i g u r e 27. 400 MHz *H-NMR spectrum o f 47. S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e o f DMC. I n s e t shows t h e e q u i v a l e n c e f o r C -3 p r o t o n s o f [ £H5]DMC (20) i n CDCI3 . "•"Contaminating EtOAc. H-3', H-5' 1 H-2', H-6' , , ^ r - T T - T - r i ; i i . . i i i - p - | i , , , . . i i, , I , . . , , | i , , , . •, , . • I I . • - r - P T- -T—r - .—r-r—•-•r—T- -T—T-t—r- *" T ~ ' !l « !«.» 9.5 9.8 S.v a.B 7.5 I 7 . » 6 .5 S.Z 5.5 5 . a c. 5 *.« 3'5 3»0 I p.=.« F i g u r e 28. 400 MHz 'H-NMR spectrum o f 46. S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e 3-hydroxymethyl DMC. H H a* 117 (e) Summary o f a n a l y t i c a l d a t a . * In summary, t h e s e d a t a s u g g e s t t h a t a compound w i t h mass s p e c t r a l p r o p e r t i e s c o n s i s t e n t w i t h t h e c a r b i n o l a m i d e was p r e s e n t i n t h e f r e s h l y p r e p a r e d c r u d e p r o d u c t , but c o u l d n ot be i s o l a t e d because o f i t s m a r g i n a l s t a b i l i t y . The moderate s t a b i l i t y o f s e v e r a l c a r b i n o l a m i d e s has been r e p o r t e d ( V a j t a e t a l . , 1988; Ross e t a l . , 1983; A l t o n e t a l . , 1975b; McMahon and S u l l i v a n , 1965). The compound d e t e c t e d by HPLC which decomposed on s t o r a g e was not c h a r a c t e r i z e d . The s t r u c t u r e o f t h e compound 46 has been e l u c i d a t e d by LCMS and *H-NMR s p e c t r o s c o p y . LCMS r e v e a l e d t h e e x i s t e n c e o f a hydroxym e t h y l a t e d DMC d e r i v a t i v e w i t h MH + a t m/z 317 and [MH-H20] + a t m/z 299. The ^ H-NMR sp e c t r u m r e v e a l e d t h e p r e s e n c e o f amide, m e t h i n e , methylene and h y d r o x y l p r o t o n s w h i c h i s c o n s i s t e n t w i t h DMC b e i n g h y d r o x y m e t h y l a t e d a t p o s i t i o n - 3 . A t p r e s e n t , GCMS ( E I ) d a t a does not appear t o s u p p o r t t h e e v i d e n c e o b t a i n e d by LCMS and NMR, because a p a r e n t i o n o f m/z 370 i s not c o n s i s t e n t w i t h t h e p r e s e n c e o f two d e r i v a t i z a b l e c e n t r e s i n 46, namely t h e amide and h y d r o x y l p r o t o n s . The f u t u r e use o f d i r e c t probe and tandem mass s p e c t r o s c o p i c t e c h n i q u e s , w h i c h were not i m m e d i a t e l y a v a i l a b l e , s h o u l d p r o v i d e some e x p l a n a t i o n f o r t h i s s p e c t r a l phenomenon. ( i i ) S y n t h e s i s o f t h e c a r b i n o l a m i d e u s i n g KOH and p a r a f o r m a l d e h y d e . A n o t h e r approach t o t h e b a s e - c a t a l y z e d c o n d e n s a t i o n o f form a l d e h y d e w i t h DMC i n v o l v e d t h e use o f KOH as base and p a r a f o r m a l d e h y d e , as t h e s o u r c e o f f o r m a l d e h y d e , i n DMSO s o l u t i o n . In t h e ^ H-NMR spect r u m o f t h e p r o d u c t ( 4 0 ) , t h e s i m i l a r i t y o f r e s o n a n c e t o DMC i n t h e a r o m a t i c r e g i o n , 118 and t h e o c c u r r e n c e o f u p f i e l d c h e m i c a l s h i f t s a t 3.43 ( t , 1H, -CHCH2), 3.94 ( t , 2H, CHCH 20H), 4.56 ( t , 1H, -CH 20H) ( F i g u r e 29 A) were c o n s i s t e n t w i t h t h e s t r u c t u r e 3-hydroxymethyl DMC. The s p l i t t i n g o f t h e met h y l e n e and h y d r o x y l p r o t o n s i n t o t r i p l e t s c o u l d be a c c o u n t e d f o r by t h e red u c e d r a t e o f h y d r o x y l p r o t o n exchage i n DMSO due t o hydrogen b o n d i n g w i t h t h e s o l v e n t (Chapman and K i n g , 1964). A l t h o u g h a s i m i l a r s t r u c t u r e was prop o s e d f o r 46, s p l i t t i n g o f t h e h y d r o x y l p r o t o n was not o b s e r v e d ( F i g u r e 29 B ) . One p o s s i b l e e x p l a n a t i o n c o u l d be t h e p r e s e n c e o f t r a c e amounts o f a c i d w h i c h c o u l d c a t a l y z e h y d r o x y l p r o t o n exchange (Chapman and K i n g , 1964). In t h e pr e s e n c e o f D 20, t h e s i m i l a r i t y o f t h e u p f i e l d c h e m i c a l s h i f t s o f 40 and 46 ( F i g u r e 30) l e n d s s u p p o r t t o t h e c h e m i c a l i d e n t i t y o f t h e s e two compounds. T h i s h y p o t h e s i s was s u b s t a n t i a t e d by t h e s i m i l a r i t y o f t h e GCMS ( E I ) spe c t r u m o f 40 ( F i g u r e 31) t o t h a t o f 46 ( F i g u r e 2 6 ) . i 119 F i g u r e 29. P a r t i a l 400 MHz AH-NMR spectra o f (A) compound 40 and (B) compound 46 i n DMSO-ds reveal t h e occurrence o f methine, methylene and hydroxyl protons as (A) triplets i n 40 and as (B) a tr iplet, doublet and b r o a d singlet respectively i n 46. The full NMR spectrum o f 40 i s shown i n t h e A p p e n d i x . Contaminating EtOAc. 120 CHCH 20H J CHCH 20H -CHCH2 -CHCH2 4 . 5 4. a 3.a A. Z F i g u r e 30. P a r t i a l 400 MHz *H-NMR spectra of (A) compound 40 and (B) compound 46 in DMSO-ds with D20 show chemical identity for the 3-hydroxymethyl function. Contaminating EtOAc. 121 F i l e >R225C 260088-240086-.20000-200000-180080-160000-140086-120060-100080-80000-60000-40060-26000-60.0-556.0 amu. N-HYDROXYNETHYL-DMC TIC 7.88 l TMS-DTVE CR-LINK V 9.45 . . l e >R225C N-HYDROXYMETHYL-DMC Bpk Rb 28544 TMS-BTVE CR-LINK MESI Scan 313 7.87 min. 22006-28080-18000-16000-14800-12006-10000-8600-6000-4080-2888-eJJ 73 225 155 188 1 2 4 1 7 9 \ 1 9 7 251 \ 273 \ 3 8 1 \ 370 \ 327 316| 80 128 160 280 248 i • • ' i 280 r-110 ~-\00 r90 J60 i-70 f60 rS0 J40 -30 r 2 0 -10 I I • I I I I I , I 320 368 F i g u r e 3 1 . (A) GCMS ( C o n d i t i o n , d*) TIC p l o t o f p u r i f i e d 40. (B) GCMS ( E I ) s p e c t r u m o f 40 shows t h e p r e s e n c e o f M +- a t m/z 370. *See E x p e r i m e n t a l f o r d e t a i l s . 122 ( i i i ) C a r b i n o l a m i d e s y n t h e s i s i n p e r s p e c t i v e . I t i s a p p a r e n t t h a t t h e s y n t h e s i s o f t h e CLBZ c a r b i n o l a m i d e o f f e r s a u n i q u e s y n t h e t i c c h a l l e n g e , i n t h a t u n l i k e t h e s y n t h e s e s o f o t h e r c a r b i n o l a m i d e s f r o m t h e i r N-desmethyl d e r i v a t i v e s ( K e d d e r i s e t a l . , 1989; S l a t t e r e t a l . , 1989), t h e r e appears t o be a c o m p e t i t i o n between two a l t e r n a t i v e c e n t r e s on t h e m o l e c u l e f o r f o r m a l d e h y d e a d d i t i o n , namely t h e amide and methylene g r o u p s . I t i s unusual t h a t t h e c o n d e n s a t i o n o f fo r m a l d e h y d e w i t h DMC when c a t a l y z e d by both K2CO3 and KOH s h o u l d r e s u l t i n t h e f o r m a t i o n o f 3-hydroxymethyl DMC as opposed t o N-hydroxymethyl DMC ( c a r b i n o l a m i d e ) . In t h e s y n t h e s i s o f [ 2Hs]CLBZ and [ 2H3]CLBZ t h e s e c o n d a r y amide p r o t o n o f t h e desmethyl compound d i s p l a y e d a h i g h e r a c i d i t y t h a n t h e meth y l e n e p r o t o n s and was s e l e c t i v e l y a l k y l a t e d o v e r t h e methylene group by b a s e - c a t a l y z e d t r e a t m e n t w i t h w i t h CH3I and CD3I r e s p e c t i v e l y . Whether f o r m a l d e h y d e a d d i t i o n a t t h e 3 - p o s i t i o n o c c u r s as a k i n e t i c p r o d u c t o r whether i t a r i s e s as a r e s u l t o f an e q u i l i b r i u m p r o c e s s subsequent t o N-h y d r o x y m e t h y l a t i o n r e m a i n s t o be r e s o l v e d . F u r t h e r m o r e , d e s p i t e t h e p r e s e n c e o f s t r u c t u r e s w h i c h s h o u l d e x e r t a s t a b i l i z i n g i n f l u e n c e on N-hydroxymethyl CLBZ, namely, a phenyl r i n g ( G o r r o d and Temple, 1976) and a c a r b o n y l group (Ross e t a l . , 1983; McMahon and S u l l i v a n , 1965) a d j a c e n t t o t h e hy d r o x y m e t h y l b e a r i n g n i t r o g e n t h e p u t a t i v e c a r b i n o l a m i d e was m a r g i n a l l y s t a b l e . 123 (F) A t t e m p t e d S y n t h e s e s o f N-hvdroxv DMC ( h y d r o x a m i c a c i c ) ( 5 0 ) . Two approaches were used i n an a t t e m p t t o s y n t h e s i z e t h e hydroxamic a c i d ( 5 0 ) : ( i ) M o V I p e r o x i d e o x i d a t i o n o f DMC-TMS (Scheme X X I I ) , and ( i i ) b a s e - c a t a l y z e d r i n g - c l o s u r e o f t h e a r y l h y d r o x y 1 amine carbamoyl a c e t a t e (52) (Scheme X X I I I ) . Scheme X X I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC (50) by o x i d a t i o n o f DMC-TMS. A: MSTFA, 45°C B: bis(N,N-diDMF)oxodiperoxomolybdenum ( V I ) (42) C: EDTA 124 ( i ) Mo-^ p e r o x i d e o x i d a t i o n o f DMC-TMS. Trea t m e n t o f m o l y b d i c a c i d w i t h H2O2 and N,N-dimethylformamide (DMF) a f f o r d e d bis(N,N-diDMF)oxodiperoxomolybdenum ( V I ) (42) i n 45 % y i e l d . The -^ H-NMR s p e c t r u m o f 42 i n D2O ( F i g u r e 32) was c o n s i s t e n t w i t h i t s s t r u c t u r e . Resonance a t 2.89 (N-CH3) and 3.05 (N-CH3) ppm s u g g e s t e d t h e p r e s e n c e o f two r o t a m e r s o f DMF. *H-NMR a n a l y s i s a l s o i n d i c a t e d t h e p r e s e n c e o f c o n t a m i n a t i n g MeOH, whi c h c o u l d n o t be removed d e s p i t e t h e washi n g s o f 42 w i t h e t h e r , and ext e n d e d d r y i n g in vacuuo o v e r P2O5. The p r o b l e m a t i c r e t e n t i o n o f MeOH by 42 was n o t r e p o r t e d by M a t l i n e t . a l . (1 9 7 9 ) . T r e a t m e n t o f DMC w i t h MSTFA a f f o r d e d t h e T M S - d e r i v a t i v e (48) w h i c h was t r e a t e d w i t h o u t p u r i f i c a t i o n w i t h 42. S t i r r i n g o f t h e m i x t u r e f o r up t o 48 h gave no r e a c t i o n . C o n t a m i n a t i n g MeOH i n 42 c o u l d have h y d r o l y z e d 48 and p r e v e n t e d i t s o x i d a t i o n t o t h e hydroxamate-Mo complex ( 4 9 ) . o 1 M o 1 o ^ r o M e ?x  1 > - C - H M e ' 42 1 N-Me I N-Me O i Mo i Me v n > - C - H Me' F i g u r e 32. 400 MHz XH-NMR o f t h e M o v l complex ( 4 2 ) . 126 ( i i ) R i n g c l o s u r e o f t h e hydroxy!amine carbamoyl a c e t a t e . An a l t e r n a t i v e approach t o t h e hyd r o x a m i c a c i d was sought i n t h e b a s e - c a t a l y z e d h y d r o l y s i s o f h y d r o x y l a m i n e carbamoyl a c e t a t e (52) t o t h e c a r b o x y l i c a c i d w i t h c o n c o m i t a n t r i n g c l o s u r e t o t h e hydroxamate (50) (Scheme X X I I I ) . Treatment o f c h l o r o n i t r o a r e n e s w i t h h y d r a z i n e - P d has been used t o produce t h e h y d r o x y l a m i n e i n l e s s t h a n 3 h r w i t h m i n o r amounts o f c o n t a m i n a t i n g phenylamine ( R o n d e s t v e d t and J o h n s o n , 1977). However, t r e a t m e n t o f 51 under s i m i l a r c o n d i t i o n s f o r 40 h r a f f o r d e d no d e t e c t a b l e r e d u c t i o n . S h o u l d t h e r e d u c t i o n o f 51 t o 52 be a c c o m p l i s h e d , base-c a t a l y z e d r i n g c l o s u r e c o u l d y e t a f f o r d t h e h y d r o x a m i c a c i d ( 5 0 ) . The r e d u c t i o n o f 51 w i t h Zn and NH4CI t o a f f o r d 52 (Kamm, 1941) i s a p o s s i b i l i t y w h i c h c o u l d be examined. Scheme X X I I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC by r i n g c l o s u r e o f t h e a r y l h y d r o x y l a m i ne. A: Pd-C, h y d r a z i n e monohydrate B: sodium m e t h o x i d e 127 2. THE METABOLISM OF CLBZ. In. t h i s s e c t i o n t h e 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 o f CLBZ i n Sprague Dawley r a t s w i l l be c h a r a c t e r i z e d . C L B Z - d e r i v e d m e t a b o l i t e s were i d e n t i f i e d by c o m p a r i s o n o f GCMS p r o p e r t i e s t o s y n t h e t i c s t a n d a r d s , and by t h e p r e s e n c e o f i s o t o p e peak s h i f t s o f l a b e l l e d m e t a b o l i t e s from t h e i r u n l a b e l l e d a n a l o g u e s . Mass chromatograms shown i n t h e Ap p e n d i x i d e n t i f y CLBZ m e t a b o l i t e s by t h e p r e s e n c e o f i s o t o p i c a l l y - r e l a t e d p e a k s . (A) Phenol and C a t e c h o l M e t a b o l i t e s o f CLBZ i n Rat B i l e and U r i n e . The m e t a b o l i t e p i c t u r e f o r CLBZ was d e r i v e d from i n d i v i d u a l l y p o o l e d b i l e and u r i n e samples which were o b t a i n e d under t h e f o l l o w i n g c o n d i t i o n s . U r i n e c o l l e c t i o n commenced a f t e r t h e f i r s t dose o f CLBZ:[ 2H5]CLBZ and c o n t i n u e d t h r o u g h o u t t h e d o s i n g regimen ( o f one dose e v e r y 6 h f o r 6 do s e s ) u n t i l t h e t i m e o f s a c r i f i c e . B i l e was c o l l e c t e d s h o r t l y b e f o r e t h e s i x t h d o se, and c o n t i n u e d t h e r e a f t e r u n t i l t h e t i m e o f s a c r i f i c e a p p r o x i m a t e l y 18 h l a t e r . B i l e and u r i n e samples were e x t r a c t e d w i t h EtOAc b e f o r e and a f t e r s e p a r a t e enzyme h y d r o l y s e s w i t h ^ - g l u c u r o n i d a s e and a r y l s u l f a t a s e . E x t r a c t s were T M S - d e r i v a t i z e d w i t h MSTFA, and a n a l y z e d by GCMS. T h i s p r o c e d u r e a l l o w e d CLBZ m e t a b o l i t e s o f t h e u n c o n j u g a t e d f r a c t i o n , as w e l l as g l u c u r o n i d e and s u l f a t e m e t a b o l i t e s o f t h e c o n j u g a t e d f r a c t i o n t o be d i s t i n g u i s h e d . In r e p o r t i n g t h i s d a t a t h e f o l l o w i n g a s s u m p t i o n s were made: ( i ) a s i m i l a r e x t r a c t i o n r a t i o f o r each CLBZ m e t a b o l i t e from b i l e and u r i n e , and ( i i ) a s i m i l a r GCMS r e s p o n s e f o r each T M S - d e r i v a t i z e d m e t a b o l i t e . L e v e l s o f CLBZ m e t a b o l i t e s were s e m i - q u a n t i t a t i v e l y compared u s i n g t h e a r e a under M +* f o r t h e T M S - d e r i v a t i z e d m e t a b o l i t e . 128 There was no d e t e c t a b l e e v i d e n c e o f u n c o n j u g a t e d CLBZ m e t a b o l i t e s i n b i l e o r u r i n e . TIC p l o t s o f c o n j u g a t e d CLBZ m e t a b o l i t e s i n b i l e and u r i n e a r e shown i n F i g u r e s 33 - 35, and r e l a t i v e m e t a b o l i t e l e v e l s a r e compared i n F i g u r e 36. In b i l e d e c r e a s i n g l e v e l s o f t h e f o l l o w i n g m e t a b o l i t e s were d e t e c t e d as g l u c u r o n i d e and s u l f a t e c o n j u g a t e s : 4'-hydroxy CLBZ ( 2 8 ) . 4'-h y d r o x y DMC ( 2 6 ) , 4'-hydroxy-3'-methoxy CLBZ (36a) + 3'-hydroxy-4'-methoxy CLBZ ( 3 6 b ) , 4'-hydroxy-3'-methoxy DMC (33) and 3 ' , 4 ' - d i h y d r o x y CLBZ ( 3 5 ) -In u r i n e t h e o r d e r o f d e c r e a s e f o r s u l f a t e c o n j u g a t e s was 4'-hydroxy-3'-methoxy CLBZ ( 3 6 a ) . 4'-hydroxy CLBZ ( 2 8 ) , 4'-hydroxy DMC ( 2 6 ) , 3',4'-d i h y d r o x y CLBZ ( 3 5 ) . In b i l e , t h e g l u c u r o n i d e s were predominant o v e r t h e s u l f a t e s , whereas i n u r i n e o n l y s u l f a t e c o n j u g a t e s were d e t e c t e d . 129 'le >AB81 60.8-558.0 amu. CLBZ-BILE-GLUC-TMS EIP 788888-4 15.77 688008-500080-480080-308088 288888-188800 8 2%i.%8 • < t » i i « i « i • i • 11 • 111111111 i 111 | i 111 | i 1111 11 111 i 1111 • • i • • ' • i 15.0 16.8 17.8 18.8 19.8 28.0 21.8 22.8 23.8 24.8 25.8 26.0 F i g u r e 33. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h ^ - g l u c u r o n i d a s e . 26: 4'-hydroxy DMC; 28: 4'-hydroxy CLBZ; 33: 4'-hydroxy-3'-methoxy DMC; 35: 3 ' , 4 ' - d i h y d r o x y CLBZ; 36a: 4'-hydroxy-s' -methoxy CLBZ; 36b: 3'-hydroxy-4'-methoxy CLBZ. 130 f i l e >RB02 68.0-558.8 amu. CLBZ-BILE-SULF-TMS EIP 21 .87 28 21 .56 22.91 22.34 24.12 36. i i i i - J i — l i i | i i i i |—r—i—i—r -1• i i i i—j—«—i—i—i"i i—i—i—i | i i i — f | i—i "11 i | i J. 16.0 17.0 18.0 19.0 20.0 21 .0 22.0 23.0 24.0 25.8 26.0 F i g u r e 34. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . 26: 4'-h y d r o x y DMC; 28: 4'-hydroxy CLBZ; 36a: 4'-hydroxy-3'-methoxy CLBZ; 36b: 3'-hydroxy-4'-methoxy CLBZ. 131 r i l e >fiB84 6 0 . 8 - 5 5 8 . 8 amu. CLBZ-URIN-SULF-TMS EIP 228088 280880-188888^ 16Q000 : 148888-1 1 128888-1J 108888-88888-60000-480O0-28888 : 17.79 .93 18.73 18.49^^ 23 .18 28.feei.56 3 6 a < l 3 ' 4 7 8 • | —> •—r-'—•—•—>—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 17 .8 18 .0 19.8 2 8 . 8 2 1 . 8 2 2 . 8 2 3 . 8 2 4 . 0 25 .8 26 .8 F i g u r e 35. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f u r i n e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . 26: 4'-hydroxy DMC; 28: 4'-hydroxy CLBZ; 35: 3 ' , 4 ' - d i h y d r o x y CLBZ; 36a: 4'-hydroxy-3'-methoxy CLBZ. 132 c <L> o c _o > O CD 110 1 0 0 -90 80 + 70 60 + 50 40 3 0 -2 0 -1 0 -0 0 28 26 28: 4'-OH CLBZ 26: 4'-OH DMC 36a: 4'-OH,3'-Olvle CLBZ 36b: 3'-OH,4'-OMe CLBZ 33: 4,-OH,3'-OMe DMC 35: 3',4'-(OH)2 CLBZ • • Bile glucuronide VZZ Bile sulfate I I Urine sulfate 1 36a+b 33 Metabolite icf n 35 F i g u r e 36. P r o f i l e o f c o n j u g a t e d CLBZ m e t a b o l i t e s i n t h e Sprague Dawley r a t d e r i v e d from i n d i v i d u a l l y p o o l e d b i l e and u r i n e samples. U r i n e was c o l l e c t e d a f t e r t h e f i r s t dose o f drug t h r o u g h o u t t h e dosage regimen u n t i l t h e t i m e o f s a c r i f i c e . B i l e was c o l l e c t e d a f t e r t h e f i f t h dose u n t i l t he t i m e o f s a c r i f i c e . A f t e r t r e a t m e n t w i t h / 3 - g l u c u r o n i d a s e o r a r y l s u l f a t a s e , b i l e and u r i n e samples were e x t r a c t e d w i t h EtOAc, T M S - d e r i v a t i z e d and a n a l y z e d by GCMS. R e l a t i v e amounts o f CLBZ m e t a b o l i t e s a r e based on mass chromatogram peak a r e a s f o r M +* o f t h e TMS d e r i v a t i v e . A s i m i l a r e x t r a c t i o n r a t i o and GCMS r e s p o n s e i s assumed f o r a l l m e t a b o l i t e s . 133 ( i ) G l u c u r o n i d a t i o n vs. S u l f a t i o n . The f o r m a t i o n o f g l u c u r o n i d e and s u l f a t e e s t e r s i s c a t a l y z e d by UDP-g l u c u r o n y l t r a n s f e r a s e ( B u r c h e l l and C o u g h t r i e , 1989) and s u l f o t r a n s f e r a s e ( J a k o b y e t a l . , 1984) r e s p e c t i v e l y , w i t h both h a v i n g o v e r l a p p i n g s u b s t r a t e s p e c i f i c i t i e s . S e v e r a l f a c t o r s g o v e r n t h e b a l a n c e between g l u c u r o n i d a t i o n and s u l f a t i o n p r o c e s s e s in vivo. These i n c l u d e : ( i ) s p e c i e s ( M u l d e r , 1984), ( i i ) dosage ( C a l d w e l l , 1980), ( i i i ) t h e p a r t i t i o n i n g o f t h e s u b s t r a t e i n t h e c e l l u l a r compartments ( M u l d e r , 1984) and ( i v ) t h e s u b s t r a t e s p e c i f i c i t y o f t h e c o n j u g a t i n g enzyme ( M u l d e r and Hagedoorn, 1974). A l t h o u g h t h e r e i s a p r o p e n s i t y t o e i t h e r g l u c u r o n i d a t e o r s u l f a t e among animal s p e c i e s , both c o n j u g a t i o n e v e n t s a r e e f f i c i e n t l y a c c o m p l i s h e d i n t h e r a t ( M u l d e r , 1984). T h i s v i e w g a i n s s u p p o r t w i t h t h e o c c u r r e n c e o f CLBZ m e t a b o l i t e s as both g l u c u r o n i d e and s u l f a t e c o n j u g a t e s . The dosage l e v e l o f a s u b s t r a t e i n f l u e n c e s t h e e x t e n t t o w h i c h each c o n j u g a t i o n pathway i s i n v o l v e d . Thus, a t low doses o f p h e n o l s , s u l f a t i o n t e n d s t o p r e d o m i n a t e , whereas a t h i g h e r d o s e s , g l u c u r o n i d a t i o n i n c r e a s e s ( C a l d w e l l , 1980). Two r e a s o n s c o u l d a c c o u n t f o r t h i s s h i f t i n m e t a b o l i s m ( M u l d e r , 1984). ( i ) S u l f o t r a n s f e r a s e s u s u a l l y have l o w e r Km v a l u e s t h a n U D P - g l u c u r o n y l t r a n s f e r a s e s f o r t h e same s u b s t r a t e s , and ( i i ) t h e c a p a c i t y f o r s u l f o t r a n s f e r a s e t o c o n j u g a t e i s more r e a d i l y compromised by d e p l e t i o n o f i t s c o - s u b s t r a t e . I t i s not known whether t h e dose o f CLBZ a d m i n i s t e r e d was s u f f i c i e n t t o produce l e v e l s o f phase I m e t a b o l i t e s t h a t c o u l d s a t u r a t e t h e s u l f a t i o n pathway i n t h e r a t . I f t h i s were t h e c a s e , a h i g h e r s u l f a t i o n p a t t e r n c o u l d emerge a t l o w e r doses o f t h e d r u g . The n a t u r e o f t h e c o n j u g a t e formed, t o a l a r g e e x t e n t , d e t e r m i n e s t h e 134 e x c r e t o r y pathway i n t o w h i c h i t i s c h a n n e l e d ( C a l d w e l l , 1980). B i l i a r y e x c r e t i o n o f a drug depends on ( i ) an a p p r o p r i a t e h y d r o p h i l i c / 1 i p o p h i l i c b a l a n c e , ( i i ) t h e p r e s e n c e o f an a n i o n i c f u n c t i o n , and ( i i i ) a minimum r e q u i r e m e n t f o r m o l e c u l a r w e i g h t which i s dependent on s p e c i e s ( P o w e l l and O l a v e s e n , 1981; P o w e l l , 1981). S t e r o i d c o n j u g a t e s a r e more r e a d i l y e x c r e t e d as g l u c u r o n i d e s i n t h e b i l e ( M a t s u i , 1982; B a i l l i e e t a l . , 1975; Honma and Nambara, 1974), whereas s u l f a t e c o n j u g a t e s a r e g e n e r a l l y e x c r e t e d i n t h e u r i n e ( C a l d w e l l , 1980) by a c t i v e t r a n s p o r t ( M u l d e r , 1984). Phase I m e t a b o l i t e s o f CLBZ a r e r e l a t i v e l y p o l a r and t h e i r m o l e c u l a r w e i g h t s (MW, 302 - 336) a r e i n t e r m e d i a t e i n t h e r e q u i r e m e n t f o r b i l i a r y g l u c u r o n i d e e x c r e t i o n i n t h e r a t (MW > 325 ± 5 0 ) , ( M i l l b u r n e t a l . , 1967). C o n s i s t e n t w i t h t h e s e c h e m i c a l p r o p e r t i e s , h y d r o x y l a t e d CLBZ m e t a b o l i t e s were a b l e t o a c c e s s b o t h b i l i a r y and u r i n a r y e x c r e t o r y pathways. M e t a b o l i t e s o c c u r r e d p r e d o m i n a n t l y i n t h e b i l e as g l u c u r o n i d e s and were d e t e c t e d e x c l u s i v e l y as s u l f a t e s i n t h e u r i n e . T h i s p a t t e r n i s i n a c c o r d a n c e w i t h t h e r e s u l t s o f V o l z e t a l . (1979) who r e p o r t e d t h e predominance o f s u l f a t e s i n t h e u r i n e f o r CLBZ m e t a b o l i t e s . However, i t i s not known how t h e s e w o r k e r s c o u l d have d i s t i n g u i s h e d from which c o n j u g a t e t h e h y d r o l y z e d m e t a b o l i t e s o r i g i n a t e d , because m e t a b o l i t e s were e x t r a c t e d a f t e r t r e a t m e n t w i t h a fi-g l u c u r o n i d a s e / a r y l s u l f a t a s e m i x t u r e . ( i i ) Mass S p e c t r a l Data f o r Phase I M e t a b o l i t e s o f CLBZ i s o l a t e d from b i l e  and u r i n e . GCMS ( E I ) s p e c t r a f o r d e c o n j u g a t e d CLBZ m e t a b o l i t e s , t y p i f i e d by t h e b i l i a r y g l u c u r o n i d e s , a r e p r e s e n t e d i n F i g u r e s 37 - 41. GCMS d a t a a r e summarized i n T a b l e 2. 135 Fll« >AB16 Bpk Ab 479939 520000-48QO0Q-44Q0O0-400000-^73 36Q8QQ-320000-28Q0OO-240000-200000-160000-120000-60000-40000-o- - r - l — r - r - r -CLBZ-BILE-GLUC-THS CLP 153 181 135 388 19 S o n 690 .19 M i n . 388 t.392 343 217 239 27 > 3 '3 I 320 y LJ 371 80 p l l O i i o o r90 :«o r70 ^60 iso :40 i-30 £ 0 rlO 120 160 200 240 280 320 360 400 F i g u r e 37. GCMS ( C o n d i t i o n , a ) ( E I ) s p e c t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydr o x y CLBZ (28) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+- and [ 2 H 4 ] M + * a t m/z 388 and 392 r e s p e c t i v e l y . ( D e s c r i b e d i n t h e E x p e r i m e n t a l ) . F i l e >AB16 Bpk Ab 934278 CLBZ-BILE-CLUC-THS CLP l e o e o o o -900000 800000 780O0O-600000 s e o o o o 400OOO-388880-208000-l e o o o o o-* 73 22S 103 147 187 eo 1 Scan 53 4 6.42 H I D . . 4 4 6 .450 446 : l l 267 299 331 361 40S 405 I 280 24Q 288 p i l e r t o o r90 :80 r-70 isa r30 SO rlO 320 360 4QO 440 F i g u r e 38. GCMS ( C o n d i t i o n , a ) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy DMC (26) i s o l a t e d f r o m b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2 H 4 ] M + * a t m/z 446 and 450 r e s p e c t i v e l y ( * D e s c r i b e d i n t h e E x p e r i m e n t a l ) . 136 Fil« > « B 1 « ^ B p k i « 4 4 4 9 z g o a a o -i8oasa-K s e o a -1 2 0 0 0 0 -l a a e a e -s e a a a -« e a e a : 4 O Q O Q -2 0 0 0 0 -73 CLBZ-B1LE-CLUC-TMS CLP 167 "<W<«a ," 129 3 1—I—• 1 1—i • 1—' IT—'—I ' 1—'—i '—r—" 8 0 1 2 0 1 6 0 2 0 0 2 4 0 346 299 268 3ZO „ S o n L«1 2Q.71 , 418 776 t i n . r l l O r i o o i90 :60 r70 i6Q iso r*o r30 E20 :1Q 36Q 4 0 O F i g u r e 39. GCMS ( C o n d i t i o n , a ) ( E I ) s p e c t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy CLBZ (36a) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +- and [ 2H3JM +" a t m/z 418 and 421 r e s p e c t i v e l y ( * D e s c r i b e d i n t h e E x p e r i m e n t a l ) . F i l * >AB16 Bpk A I, 24806S 2 6 0 0 0 0 -2 4 0 0 0 0 -2 2 0 0 0 0 -Z O O O O O -i s o o o o -1 6 0 0 0 0 -1 4 Q O O O 1 2 0 0 0 0 I O O Q O O -eaooe-6 0 0 Q 0 -4 0 0 0 0 2 0 0 0 0 -0 - * 73 1 2 9 1 4 7 | *" If 2 8 1 f *s 4 Z 1 ^ CLBZ-BILC-CLUC-TKS CLP 2 2 S \ 299 1 9 7 359 361 Scan 593 17.SO M i n . ^ 4 7 6 1.479 l e a 158 2 0 0 2 S O 3SO 111 'JW 476 446 U 4SO r l l O OO r90 i « e •70 rCO i50 a r3 8 O F i g u r e 40. GCMS ( C o n d i t i o n , a ) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) i s o l a t e d f r o m b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2 H 3 ] M + * a t m/z 476 and 479 r e s p e c t i v e l y ( * D e s c r i b e d i n t h e E x p e r i m e n t a l ) . 137 Fil« >AB1C CLBZ-BILE-CLUC-IHS Scan 795 Bpk Ab 2X4337 CLP 21.05 M i n . 100 ISO 200 2SQ 300 3S0 4O0 4SO F i g u r e 41. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 3 ' , 4 ' - d i h y d r o x y CLBZ (35) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2H3]M +* a t m/z 476 and 479 r e s p e c t i v e l y ( * D e s c r i b e d i n t h e E x p e r i m e n t a l ) . Table 2. GCMS data for Phase I CLBZ metabolites typified by biliary glucuronides. Metabolites were extracted from bile after hydrolysis with jjj-glucuronidase and derivatized with TMS. Compound (No.)a t. (min) M+*(m/z, X) Diagnostic ions (m/z, X) c 4'-OH DMC (26) 17.91 446 (48) 225 (100) 239 (24) 431 (14) 4'-OH [2H4]DMC (26') 17.86 450 (24) 225 (100) 239 (24) 435 (9) 4'-OH CLBZ (28) 21.37 388(100) 343 (56) 181 (35) 331 (30) 153 (28) 4'-OH I2H41CLBZ (28') 21.30 392 (64) 347 (45) 181 (35) 335 (21) 153 (28) 4'-0H,3'-0Me DMC (33) 19.13 476 (16) 225 (20) 239 (8) 461 (5) 446 (5) 4'-0H,3'-0Me 19.11 479 (14) 225 (20) 239 (8) 464 (4) [2H3 DMC(W') 3',4'-(0H)2 CLBZ (35) 23.50 476 (26) 461 (6) 343 (6) 431 (6) 419 (5) 3',4'-(OH)2 23.47 479 (21) 464 (5) 346 (4) 434 (4) 422 (4) [2H3]CLBZ (35') 4'-0H,3'-0Me CLBZ (36a) 23.22 418 (78) 346 (26) 373 (25) 388 (16) 401 (8) 4'-0H,3'-OMe 23.17 421 (72) 349 (18) 376 (14) 391 (14) 404 (9) t2H3]CLBZ(36a') 3'-OH,4'-OMe CLBZ (36b) 22.44 418 (10)d 3'-0H,4'-OMe 22.37 - 421 (7) d t2H3]CLBZ (36b') a: The compound number for the deuterio analogues is indicated by the primed ('). b: Retention times were obtained according to GCMS Condition (b) described in the Experimental. H c: X = Relative intensity of ion for the combined protio/deuterio spectrum (See Figures 28-31). u CO d: Only M * could be diagnostically assigned in this spectrum. 139 Loss o f one d e u t e r i u m d u r i n g b i o t r a n s f o r m a t i o n a f f o r d e d an i s o t o p e peak s h i f t o f f o u r mass u n i t s i n t h e GCMS ( E I ) s p e c t r a o f t h e TMS-d e r i v a t i z e d b i l e and u r i n e e x t r a c t s , and r e a d i l y a l l o w e d phenol m e t a b o l i t e s t o be i d e n t i f i e d . Comparison o f t h e GCMS p r o p e r t i e s o f t h e l a t t e r t o s y n t h e t i c s t a n d a r d s c o n f i r m e d t h a t m o n o h y d r o x y l a t i o n had o c c u r r e d a t t h e 4 ' - p o s i t i o n o f CLBZ and DMC. I t s h o u l d be no t e d t h a t c y tochrome P-450 can a l s o c a t a l y z e p h e n y l m o n o o x i d a t i o n s a t t h e meta (Korzekwa e t a l . , 1989) and ortho p o s i t i o n s ( J e r i n a e t a l . , 1971). In s p i t e o f t h i s , para h y d r o x y l a t i o n i s l i k e l y t o p r e d o m i n a t e , as borne o u t by t h e v a r i e t y o f s u b s t r a t e s t h a t a r e m o n o h y d r o x y l a t e d i n t h i s f a s h i o n e.g. amphetamine, N-methylamphetamine, diazepam, n o r e p h e d r i n e and p h e n f o r m i n ( T e s t a and J e n n e r , 1976). 3 ' , 4 ' - D i h y d r o x y CLBZ (35) was a minor c o n j u g a t e d m e t a b o l i t e i n b i l e and u r i n e . There was no e v i d e n c e f o r t h e desmethyl c a t e c h o l 3',4'-d i h y d r o x y DMC ( 3 2 ) , p r o b a b l y because t h i s m e t a b o l i t e was p r e s e n t a t l e v e l s below t h e l i m i t o f d e t e c t i o n . 4'-Hydroxy-3'-methoxy CLBZ (36a) was t h e major c o n j u g a t e d u r i n a r y m e t a b o l i t e and was a l s o s i g n i f i c a n t i n t h e c o n j u g a t e d f r a c t i o n o f b i l e . 4'-Hydroxy-3'-methoxy DMC (33) was s i g n i f i c a n t i n t h e c o n j u g a t e d f r a c t i o n o f b i l e and a b s e n t i n u r i i i e . The low l e v e l o f d i h y d r o x y CLBZ (35) and t h e u n d e t e c t a b l e l e v e l o f d i h y d r o x y DMC (32) compared t o t h e i r O - methylated d e r i v a t i v e s speak f o r t h e h i g h h e p a t i c COMT a c t i v i t y p r e s e n t i n t h e r a t ( G u l d b e r g and Marsden, 1975). C o n s i s t e n t w i t h t h e s p e c i f i c i t y o f COMT f o r m e t a - O - m e t h y l a t i o n o f c a t e c h o l a m i n e s in vivo ( G u l d b e r g and Marsden, 1975), t h e meta (36a):para (36b) r a t i o s o f O-methylated CLBZ c a t e c h o l s p r e s e n t as t h e b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e were 98:2 and 100:0 r e s p e c t i v e l y , as 140 d e t e r m i n e d from mass chromatograms o f t h e i r m o l e c u l a r i o n s ( F i g u r e 3 3 ) . In o r d e r t o v e r i f y t h e peak a s s i g n m e n t s f o r t h e O-methylated CLBZ c a t e c h o l s , t h e b i l i a r y g l u c u r o n i d e {meta:para, 98:2) was s p i k e d w i t h t h e m i x t u r e o f CLBZ O-methylated c a t e c h o l s formed in vitro (meta:para, 68:32). The r e t e n t i o n t i m e s f o r t h e O-methylated c a t e c h o l s formed (A) in vitro, (B) as t h e b i l i a r y g l u c u r o n i d e ( u n s p i k e d ) , and (C) as t h e b i l i a r y g l u c u r o n i d e ( s p i k e d ) were c o n s i s t e n t , and t h e l a t t e r a f f o r d e d a metaipara r a t i o o f 74:16 ( F i g u r e 4 3 ) . In t h i s way, t h e peak a s s i g n m e n t s f o r t h e O-methylated c a t e c h o l s f o r t h e b i l i a r y g l u c u r o n i d e , and i n f e r e n t i a l l y t h e b i l i a r y s u l f a t e and u r i n a r y s u l f a t e were c o n f i r m e d . In c o n t r a s t t o t h e e s s e n t i a l l y q u a n t i t a t i v e f o r m a t i o n o f meta-O-m e t h y l a t e d CLBZ c a t e c h o l s as t h e b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e , t h e b i l i a r y s u l f a t e d i s c l o s e d an unus u a l meta (36a) -.para (36b) r a t i o o f 70:30. Repeat i n j e c t i o n s o f t h e b i l e and u r i n e samples v e r i f i e d t h e meta ( 3 6 a ) : p a r a (36b) r a t i o o f t h e b i l i a r y s u l f a t e . In a d d i t i o n , t h e a p p r o x i m a t e l y e q u a l meta (36a) -.para (36b) peak a r e a r a t i o s i n mass chromatograms a t m/z 418, and f o r t h e d e u t e r a t e d a n a l o g u e s a t m/z 421 e n s u r e d t h a t t h e peaks were m e t a b o l i t e - r e l a t e d ( F i g u r e 4 4 ) . P a r a - O - m e t h y l a t e d c a t e c h o l s a r e uncommon m e t a b o l i t e s in vivo which u s u a l l y o c c u r as t h e minor i s o m e r compared t o t h e meta-O-methylated d e r i v a t i v e . F o r example t h e p a r a - O - m e t h y l a t e d i s o m e r s o f v a n y l e t h y l amine (O'Gorman e t a l . , 1970) and 3 , 4 - d i h y d r o x y a c e t o p h e n o n e ( D a l y e t a l . , 1960) c o n s t i t u t e d 10 % and 25 % o f t h e O-methylated c a t e c h o l s r e s p e c t i v e l y . The r a t i o o f t h e O-methylated c a t e c h o l s o f CLBZ, however, was u n i q u e i n t h a t a d i f f e r e n t metaipara r a t i o o c c u r r e d w i t h i n t h e same m e t a b o l i t e pool f o r d i f f e r e n t c o n j u g a t e s . In the light of the available data, a h y p o t h e s i s i s pr o p o s e d t o r a t i o n a l i z e t h e e s s e n t i a l l y q u a n t i t a t i v e o c c u r r e n c e o f 4'-141 hydroxy-3'-methoxy CLBZ as a b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e , and th e appearance o f 3'-hydroxy-4'-methoxy CLBZ (36b) as a b i l i a r y s u l f a t e . COMT c a t a l y z e d O - m e t h y l a t i o n o f c a t e c h o l s i s much more r e g i o s p e c i f i c f o r t h e meta i s o m e r in vivo t h a n in vitro ( C r e v e l i n g e t a l . , 1972). Whether t h i s r e g i o s p e c i f i c i t y i s c o n t r o l l e d d u r i n g t h e O - m e t h y l a t i o n p r o c e s s by COMT i n i t s c e l l u l a r e n v i r o n m e n t , o r whether i t a r i s e s from subsequent e n z y m a t i c a c t i v i t y ( A x e l r o d , 1956) i s u n c l e a r . S h o u l d t h e meta ( 3 6 a ) \ p a r a (36b) r a t i o o b t a i n e d i n t h e COMT c a t a l z y e d O - m e t h y l a t i o n o f t h e d i h y d r o x y s u b s t r a t e (35) in vitro (68:32) be r e f l e c t e d t o some e x t e n t in vivo, t h e n i t i s p o s s i b l e t h a t b o t h meta- (36a) and p a r a - O - m e t h y l a t e d (36b) d e r i v a t i v e s c o u l d s e r v e as s u b s t r a t e s f o r phase I I enzymes. C o n s i s t e n t w i t h such a s c e n a r i o , a h i g h e r s u b s t r a t e " s p e c i f i c i t y o f UDP-g l u c u r o n y l t r a n s f e r a s e f o r t h e meta i s o m e r (36a) t h a n t h e para i s o m e r ( 3 6 b ) , would a l l o w t h e l a t t e r t o be more r e a d i l y c o n j u g a t e d as a s u l f a t e . The s p e c i f i c i t i e s d e s c r i b e d f o r phase I I c o n j u g a t i o n a r e n o t u n l i k e l y , because t h e f o r m a t i o n o f a s u l f a t e e s t e r a t t h e 3 ' - p o s i t i o n f o r t h e para-O-methyl a t e d i s o m e r (36b) s h o u l d be s t e r i c a l l y more f a v o u r a b l e t h a n t h e f o r m a t i o n o f a g l u c u r o n i d e . S u l f a t e e s t e r s a r e more h y d r o p h i l i c t h a n g l u c u r o n i d e s and as su c h , t h e f o r m e r a r e p r e d o m i n a n t l y d e s t i n e d f o r t h e k i d n e y and subsequent u r i n a r y e x c r e t i o n . Second t o t h e l i v e r , t h e k i d n e y p o s s e s s e s t h e h i g h e s t t i s s u e l e v e l s o f a r y l s u l f a t a s e C i n t h e r a t ( D o l l y e t a l . , 1971). A l t h o u g h e s t r o g e n s u l f a t e s a r e p r o b a b l y t h e n a t u r a l s u b s t r a t e s f o r t h i s enzyme, o t h e r x e n o b i o t i c s u l f a t e s a r e a l s o d e c o n j u g a t e d w i t h v a r y i n g s p e c i f i c i t i e s (Kung e t a l . , 1988; Kawano and Aikaw a , 1987; Zuckerman and Hagerman, 1969). H y d r o l y s i s o f t h e s u l f a t e e s t e r o f t h e p a r a - O - m e t h y l a t e d i s o m e r (36b) by a r y l s u l f a t a s e C c o u l d a f f o r d t h e u n c o n j u g a t e d s p e c i e s . The l a t t e r , b e i n g 142 more l i p o p h i l i c c o u l d be more r e a d i l y r e a b s o r b e d i n t h e k i d n e y t u b u l e s . T h i s would a c c o u n t f o r t h e absence o f t h e p a r a - i s o m e r (36b) i n t h e u r i n e , and i t s p r e s e n c e i n b i l e . The p o s s i b i l i t y o f 3 , 4 - d i h y d r o d i o l CLBZ f o r m a t i o n was i n v e s t i g a t e d i n t h e r a t , inasmuch as d i h y d r o d i o l s a r e p r e c u r s o r s t o c a t e c h o l s ( K a l f , 1987; B i l l i n g s , 1985). Mass chromatograms f o r t h e i s o t o p e c l u s t e r m/z 478/483 c o r r e s p o n d i n g t o t h e T M S - d e r i v a t i v e o f 3 , 4 - d i h y d r o d i o l CLBZ r e v e a l e d t h e absence o f d e t e c t a b l e l e v e l s o f t h i s m e t a b o l i t e i n t h e e x t r a c t s o f enzyme-h y d r o l y z e d b i l e and u r i n e . In c o n t r a s t t o o u r o b s e r v a t i o n s , V o l z e t a l . (1979) and A l t o n e t a l . (1975a) r e p o r t e d t h e o c c u r r e n c e o f t h e d i h y d r o d i o l m e t a b o l i t e s o f CLBZ and t r i f l u b a z a m r e s p e c t i v e l y i n t h e r a t , but t h e s e w o r k e r s were u n a b l e t o d e t e c t t h e c o r r e s p o n d i n g c a t e c h o l s . No p l a u s i b l e e x p l a n a t i o n c o u l d be p r o v i d e d f o r t h e d i s c r e p a n c y between o u r r e s u l t s and t h o s e i n t h e l i t e r a t u r e . F u r t h e r m o r e , i t i s u n l i k e l y t h a t i n o u r hands, a c o n j e c t u r a l CLBZ d i h y d r o d i o l would undergo spontaneous d e h y d r o g e n a t i o n t o t h e c a t e c h o l under work-up o r GCMS c o n d i t i o n s . 143 i l e >OB01 4 1 7 . 7 - 4 1 3 . 7 amu.CLBZ-BILE-GLUC-TMS SMT ADC E I P 1600-1 1200-800-400-2 3 . 2 2 >2.4> 36a 12.0 13.0 14.0 15.0 16.0 17 .018.0 19.0 2 0 . 0 2V. 6 2 2 . 0 2 3 .024.0 2 5 . 0 2 6 . 0 36bM F i l e >OB02 4 1 7 . 7 - 4 1 8 . 7 a m u . C L B Z - B I L E - S U L F - T M S SMT ADC E I P 400-3 0 0 2 0 0 : 1 0 8 : 0 2 2 . 8 9 B 22.32 36b. 36a 13.0 14.0 15.0 16.0 17.0 18.8 19.0 2 0 . 0 2 1 . 8 2 2 . 0 2 3 . 0 2 4 . 0 25.0 2 6 . 0 F i l e >OB04 4 1 7 . 7 - 4 1 8 . 7 amu.CLBZ-URIH-SULF-TMS SMT ADC E I P 6Q0-409-~1 23 .12 : c 36a 13.27 I 1 1 '• 1 i ' • • i > 1, 12.0 13.0 14.015.0 16.6 17.0 13.0 19.020.0 21 .022.023.024.025.026.0 F i g u r e 42. Mass chromatograms (m/z 418) o f T M S - d e r i v a t i z e d EtOAc e x t r a c t s o f b i l e and u r i n e showing t h e p r e s e n c e o f O - m e t h y l a t e d c a t e c h o l s o f CLBZ (36a+b) as (A) a b i l i a r y g l u c u r o n i d e , (B) a b i l i a r y s u l f a t e and (C) a u r i n a r y s u l f a t e . 144 F i l e >RB294 417.7-418.7 amu.4'0H-30Me-CLBZ-TMS SMT ADC EIP 23.49 13.0 14.0 1S.0 16.0 17.0 18.0 19.0 20.0 21 .0 22.0 23.0 24.0 25.0 26.0 r i l e >RB0lO 417.7-418.7 amu.ABOl NERT SHT RBC EIP 680 see 400 380 208 106-e B 23.31 36a 18.4 36b22-«<jf . . . 13.0 14.0 1S.0 16.6 17.6 18.8 19 .8 20 .0 21 . G 22 .6 23.8 24.8 25 .8 26 .8 e >RG012 417.7-418.7 an,u .RBOlO'CLBZOH-OHe RB812 (REPEAT) SMT RDC EIP •< 23.53 500 400 300-280 100 8 r2.Sfl 36a 13.814.8 15.8 16.8 17.8 18.0 19.0 20.0 21 .0 22.0 23.0 24.8 25.0 26.0 F i g u r e 43. Comparison o f peak a s s i g n m e n t s o f t h e CLBZ O-methylated isomers (36a+b) formed (A) in vitro and (B) as a b i l i a r y g l u c u r o n i d e . (C) S p i k i n g o f sample-B w i t h sample-A s u g g e s t s t h a t b o t h meta ( 3 6 a , t R , 23.53 min) and para (36b. t R , 22.80 min) i s o m e r s a r e p r e s e n t as a b i l i a r y g l u c u r o n i d e j u d g i n g f r o m t h e i n c r e a s e i n peak a r e a o f t h e min o r m e t a b o l i t e from (B) t o ( C ) . 145 F i l e >AB02 386"j 288-; 108 : 8-4 1 7 . 7 - 4 1 8 . 7 a m u . C L B Z - B I L E - S U L F - T M S SMT ABC E IP 2 2 . 8 9 22.32 36b L36a 1 T I 'I | lITt | T 1 1 1'J ' T T T 1 J 1 I I 1 [' M » I] T i l 1 p T Tl "p'TI I | I 1"l 1 |T 1 VV^ I I 1 l " | 1 'VI'l'j'T I'M ] 1 3 . 8 1 4 . 0 15.8 1 6 . 8 17.0 18.0 19 .0 2 0 . 8 2 1 . 8 2 2 . 8 23 .8 24 .0 2 5 . 0 26 .0 F i l e >flB82 386-280-188-4 2 8 . 7 - 4 2 1 . 7 a m u . C L B Z - B I L E - S U L F - T M S SMT ADC E IP B 22 .86 22.21 36a T"i i i i t11 i | " n , T T | i r r f T - r , n i « | t i i T | i T i T | T \ I I \ i" i r r i ' i ' T H T p i ' t r r j i' t"i ~i "y i i i Imi | i i i i | 1 3 . 0 1 4 . 8 15.8 16.0 17.8 18.8 19 .0 2 8 . 0 2 1 . 8 2 2 . 8 23 .8 24 .8 25 .8 26 .8 F i g u r e 44. Mass chromatograms a t (A) m/z 418 and (B) m/z 421 show t h a t t h e peaks o f t h e CLBZ O-methylated c a t e c h o l s (36a+b) o c c u r r i n g as t h e b i l i a r y s u l f a t e s a r e i s o t o p i c a l l y r e l a t e d . 146 ( i i i ) I s o t o p e E f f e c t s i n Phenyl R i n g O x i d a t i o n . I n t e r m o l e c u l a r i s o t o p e e f f e c t s , c a l c u l a t e d by p r o t i o / d e u t e r i o p r o d u c t r a t i o s , were m a r g i n a l f o r phenyl r i n g o x i d a t i o n t o p h e n o l s ( k ^ / k p =1.103 ± .090) and c a t e c h o l s ( k ^ / k p = 1.088 ± .207). These r e s u l t s a r e i n a c c o r d w i t h t h e o b s e r v a t i o n s o f Baba e t a l . (1986) and H o s k i n s and Farmer (1982) who r e p o r t e d modest i s o t o p e e f f e c t s f o r t h e p h e n y l h y d r o x y l a t i o n o f e p h e d r i n e and d i p h e n y l h y d a n t o i n r e s p e c t i v e l y . Our k^/kg v a l u e s were a l s o c o n s i s t e n t w i t h t h a t r e p o r t e d by Tunek e t a l . (1978) ( k ^ / k p = 1.10 ± .13) f o r t h e in vitro o x i d a t i o n o f benzene t o p h e n o l . The r a t e - d e t e r m i n i n g s t e p i n t h e f o r m a t i o n o f p h e n o l s i s t h e h e t e r o l y t i c c l e a v a g e o f t h e C-0 bond o f t h e arene e p o x i d e i n t e r m e d i a t e t o a c a r b o c a t i o n ( T r a g e r , 1980), and as such phen y l r i n g h y d r o x y l a t i o n i s g e n e r a l l y n o t s u s c e p t i b l e t o an i s o t o p e e f f e c t . C a t e c h o l s a r e a l s o formed from t h e arene e p o x i d e , t h r o u g h t h e s e q u e n t i a l a c t i v i t y o f e p o x i d e h y d r o l a s e (Oesch, 1972) and d i h y d r o d i o l dehydrogenase ( K a l f , 1987; B i l l i n g s , 1 985). Thus, i t i s u n l i k e l y f o r c a t e c h o l f o r m a t i o n t o be a s s o c i a t e d w i t h an i s o t o p e e f f e c t . The m i n o r i s o t o p e e f f e c t a s s o c i a t e d w i t h [ 2H5]CLBZ d e u t e r i o p h e n y l o x i d a t i o n augurs w e l l f o r t h e s u i t a b i l i t y o f t h i s a n a l o g u e f o r m e t a b o l i s m s t u d i e s u s i n g GCMS. No p e r t u r b a t i o n o f CLBZ m e t a b o l i s m i s l i k e l y t o o c c u r as a r e s u l t o f m e t a b o l i c s w i t c h i n g due t o i s o t o p e e f f e c t s ( F o s t e r , 1984). (B) M e t a b o l i c F o r m a t i o n o f The P u t a t i v e C a r b i n o l a m i d e . The o c c u r r e n c e o f c a r b i n o l a m i d e s as s t a b l e N - d e m e t h y l a t i o n i n t e r m e d i a t e s has been r e p o r t e d ( S l a t t e r e t a l . , 1989; V a j t a e t a l . , 1988). 147 In o r d e r t o i n v e s t i g a t e t h e p o s s i b l e m e t a b o l i c f o r m a t i o n o f t h e CLBZ c a r b i n o l a m i d e i n t h e r a t , t h e i s o t o p e c l u s t e r m/z 388/393 c o r r e s p o n d i n g t o t h e T M S - d e r i v a t i v e o f t h e CLBZ c a r b i n o l a m i d e was m o n i t o r e d i n t h e c o n j u g a t e d and u n c o n j u g a t e d f r a c t i o n s o f b i l e and u r i n e . There was no d e t e c t a b l e e v i d e n c e f o r t h e CLBZ c a r b i n o l a m i d e . P r e v i o u s w o r k e r s have shown t h a t c o n j u g a t i o n s t a b i l i z e s t h e c a r b i n o l a m i d e s o f monuron (Ross e t a l . , 1982) and d i p h e n a m i d (McMahon and S u l l i v a n , 1965) as d e m o n s t r a t e d by t h e appearance o f t h e N-demethylated s p e c i e s i n t h e c o n j u g a t e d u r i n a r y f r a c t i o n s o f a n i m a l s . S i n c e t h e c a r b i n o l a m i d e o f CLBZ was not d e t e c t e d , we i n v e s t i g a t e d t h e p o s s i b i l i t y o f DMC a r i s i n g from t h e c o n j u g a t e d f r a c t i o n s o f b i l e and u r i n e as i n d i r e c t e v i d e n c e f o r t h e e x i s t e n c e o f t h e c a r b i n o l a m i d e m e t a b o l i t e . Hence, t h e i s o t o p e c l u s t e r m/z 358/363 f o r DMC-TMS was m o n i t o r e d , inasmuch as d e f o r m y l a t i o n t o DMC c o u l d have o c c u r r e d c o n c o m i t a n t l y w i t h d e c o n j u g a t i o n . No d e t e c t a b l e e v i d e n c e f o r t h i s i s o t o p e c l u s t e r was fo u n d . The 4'-hydroxy c a r b i n o l a m i d e o f t r i f l u b a z a m was i d e n t i f i e d as a u r i n a r y m e t a b o l i t e i n t h e r a t ( A l t o n e t a l . , 1975b); t h e r e f o r e , m o n i t o r i n g f o r t h e an a l o g o u s CLBZ m e t a b o l i t e a t m/z 476/480 was performed f o r t h e c o n j u g a t e d f r a c t i o n s o f u r i n e and b i l e . L i k e t h e c a r b i n o l a m i d e , t h e 4'-hyd r o x y a n a l o g u e o f t h e c a r b i n o l a m i d e c o u l d n o t be d e t e c t e d . R e p o r t s i n t h e l i t e r a t u r e documenting t h e f o r m a t i o n o f c a r b i n o l a m i d e s in vitro ( L i n d b e r g e t a l . , 1989; K e d d e r i s e t a l . , 1989), prompted us t o i n v e s t i g a t e t h e p o s s i b l e f o r m a t i o n o f t h e c a r b i n o l a m i d e by microsomal o x i d a t i o n o f CLBZ. A 50:50 m i x t u r e o f [ 2H5]CLBZ:CLBZ was i n c u b a t e d w i t h c o n t r o l and PB-i n d u c e d microsomes and GCMS a n a l y s i s o f t h e T M S - d e r i v a t i z e d e x t r a c t r e v e a l e d t h a t d e m e t h y l a t i o n t o DMC (m/z 358/363) had o c c u r r e d w i t h v i r t u a l l y no e v i d e n c e f o r a 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 (m/z 388/393). 148 I f t h e c a r b i n o l a m i d e i s a s u f f i c i e n t l y s t a b l e i n t e r m e d i a t e t o s e r v e as a s u b s t r a t e f o r c o n j u g a t i o n , i t c o u l d be t r a p p e d in vivo by t h i s p r o c e s s . Indeed, r e p o r t s i n t h e l i t e r a t u r e prompt f u r t h e r i n v e s t i g a t i o n i n t o t h e f o r m a t i o n o f t h e CLBZ c a r b i n o l a m i d e in vivo. F i r s t l y , compared t o t h e mouse ( C a c c i a e t a l . , 1980a+b) and man ( V o l z e t a l . , 1979), N-d e a l k y l a t i o n o f CLBZ does not p r o c e e d as r e a d i l y i n t h e r a t . T h e r e f o r e , i t i s p o s s i b l e t h a t t h e r a t may not be a s u i t a b l e model t o examine t h e f o r m a t i o n o f t h e c a r b i n o l a m i d e o f CLBZ. S e c o n d l y , V o l z e t a l . (1979) o b s e r v e d t h e appearance o f DMC as a s i g n i f i c a n t u r i n a r y m e t a b o l i t e i n man. However, t h e e x p e r i m e n t a l p r o c e d u r e was n o t s u f f i c i e n t l y e x p l i c i t t o d e t e r m i n e whether t h i s m e t a b o l i t e a r o s e b e f o r e o r a f t e r d e c o n j u g a t i o n w i t h a r y l s u l f a t a s e / / J - g l u c u r o n i d a s e . Were DMC t o a r i s e from t h e c o n j u g a t e d f r a c t i o n , t h i s c o u l d be c o n s i d e r e d i n d i r e c t e v i d e n c e f o r t h e e x i s t e n c e o f t h e c a r b i n o l a m i d e . (CI M e t a b o l i c F o r m a t i o n o f The P u t a t i v e N-hvdroxv DMC ( h v d r o x a m i c a c i d ) . S e l e c t e d i o n m o n i t o r i n g f o r t h e i s o t o p e c l u s t e r m/z 374/379 c o r r e s p o n d i n g t o M +* o f t h e TMS d e r i v a t i v e o f t h e h y d r o x a m i c a c i d d i d not p r o v i d e any e v i d e n c e f o r t h i s m e t a b o l i t e i n b i l e and u r i n e . The p o s s i b i l i t y o f N - h y d r o x y l a t i o n o f DMC t o t h e h ydroxamic a c i d was i n v e s t i g a t e d in vitro by i n c u b a t i n g a m i x t u r e o f DMC:[ 2H5]DMC w i t h c o n t r o l and P B - i n d u c e d microsomes. S i m i l a r t o t h e r e s u l t s o b t a i n e d in vivo, no e v i d e n c e f o r o x i d a t i o n a t t h e s e c o n d a r y n i t r o g e n t o form t h e hydroxamic a c i d was o b t a i n e d . A l t h o u g h , N - h y d r o x y l a t i o n o f t h e s e c o n d a r y amide a c e t y l a m i n o f l u o r e n e (AAF) t o t h e h ydroxamic a c i d N-hydroxy AAF i s a s i g n i f i c a n t m e t a b o l i c 149 pathway i n t h e r a t (Cramer e t a l . , 1960) and in vitro ( I b a n e z e t a l . , 1987; As t r o m e t a l . , 1 986), t h i s b i o t r a n s f o r m a t i o n p r o c e e d s l e s s r e a d i l y among t h e a c e t y l a n i l i d e s (Damani, 1982). The absence o f N - h y d r o x y l a t i o n o f 3'-h y d r o x y a c e t a n i l i d e i n t h e mouse and in vitro (Rashed e t a l . , 1989) s u p p o r t s t h i s o b s e r v a t i o n . On t h e o t h e r hand, t h e microsomal N - h y d r o x y l a t i o n o f p h e n a c e t i n ( H i n s o n and M i t c h e l l , 1976) and p - c h l o r o a c e t a n i l i d e ( H i n s o n e t a l . , 1975) has been d e m o n s t r a t e d in vitro w i t h hamster microsomes. I t i s n o t e w o r t h y t h a t t h e hamster i s a more e f f i c i e n t N - h y d r o x y l a t o r o f AAF t h a n t h e r a t ( A s t r o m e t a l . , 1986). Thus, t h e u n d e t e c t a b l e f o r m a t i o n o f N-hy d r o x y DMC i n t h e r a t c o u l d be ( i ) a r e f l e c t i o n o f t h e t r e n d d i s p l a y e d among a c e t y l a n i l i d e s and/or ( i i ) a r e s u l t o f t h e i n c a p a c i t y o f t h e r a t t o a c c o m p l i s h t h i s b i o t r a n s f o r m a t i o n . (D) The I n f l u e n c e o f Cytochrome P-450 I n d u c t i o n on CLBZ M e t a b o l i s m . The microsomes from u n t r e a t e d r a t s and PB-i n d u c e d r a t s were a n a l y z e d by g e l e l e c t r o p h o r e s i s and i m m u n o b l o t t i n g ( F i g u r e 4 5 ) . PB was r e s p o n s i b l e f o r t h e i n d u c t i o n o f cytochrome P-450b as r e v e a l e d by c o m p a r i s o n t o t h e cyto c h r o m e P-450 s t a n d a r d on t h e g e l , and r e a c t i o n o f t h e t r a n s f e r r e d p r o t e i n when c h a l l e n g e d w i t h a p o l y c l o n a l a n t i b o d y f o r cytochrome P-450b+e. I n c u b a t i o n o f an a p p r o x i m a t e 50:50 m i x t u r e o f CLBZ:[ 2 Hs]CLBZ w i t h c o n t r o l and P B - i n d u c e d microsomes r e v e a l e d t h a t N - d e m e t h y l a t i o n was i n c r e a s e d w i t h PB t r e a t m e n t , whereas phenyl h y d r o x y l a t i o n d i d not appear t o be a f f e c t e d t o t h e same e x t e n t ( F i g u r e 4 6 ) . These r e s u l t s s u g g e s t t h a t cytochrome P-450b i s one o f t h e cytochrome P-450 enzymes w h i c h c a t a l y z e t h e N - d e m e t h y l a t i o n o f CLBZ. In t h i s r e g a r d , i t i s unus u a l t h a t Cano e t a l . (1981) o b s e r v e d no s i g n i f i c a n t i n c r e a s e i n 150 N - d e m e t h y l a t i o n i n p a t i e n t s who were c o n c o m i t a n t l y a d m i n i s t e r e d PB and CLBZ, whereas a pronounced i n c r e a s e ( i n N - d e m e t h y l a t i o n ) o c c u r r e d when ca r b a m a z e p i n e (CBZ) and/or p h e n y t o i n (PH) were c o - a d m i n i s t e r e d w i t h CLBZ. P a n e s a r and c o - w o r k e r s (1989) have d e m o n s t r a t e d t h a t CBZ i s a t b e s t , as e f f e c t i v e an i n d u c e r o f cytochrome P-450b as PB. Were t h e i n d u c t i o n o f CLBZ d e m e t h y l a t i o n by CBZ r e s p o n s i b l e f o r t h e e l e v a t e d plasma l e v e l o f DMC o b s e r v e d by Cano e t a l . ( 1 9 8 1 ) , t h e n PB c o - a d m i n i s t r a t i o n s h o u l d have a f f o r d e d a comparable degree o f DMC f o r m a t i o n . F u r t h e r m o r e , P u l l a r e t a l . (1987) o b s e r v e d e l e v a t e d DMC plasma l e v e l s w i t h c o n c o m i t a n t CBZ o r PH a d m i n i s t r a t i o n when DMC was used as the parent drug. C o n s i d e r e d a l t o g e t h e r , t h e s e r e s u l t s s u g g e s t , as P u l l a r e t a l . (1987) have p r o p o s e d , t h a t i n d u c t i o n o f CLBZ d e m e t h y l a t i o n i s not s o l e l y r e s p o n s i b l e f o r t h e e l e v a t i o n o f plasma DMC when CBZ and/or PH a r e c o - a d m i n i s t e r e d w i t h CLBZ. 151 A B est ssi =3: ttSt Ml j O t U B I Y f § 8 B B 4M» ^ . ... jatft 1 2 3 4 5 1 2 3 4 5 F i g u r e 45. (A) SDS-PAGE (Laemmli e t a l . , 1970), and (B) immunoblot (Towbin e t a l . , 1979) o f l i v e r m i c r o somal p r o t e i n o f Sprague Dawley r a t s . Lane a s s i g n m e n t s a r e as f o l l o w s . 1: P B - i n d u c e d s t a n d a r d , 2: 3-m e t h y l c h o l a n t h r e n e (3-MC)-induced, 3: 3-M C - c o n t r o l ( c o r n o i l ) , 4: PB-i n d u c e d and 5: P B - c o n t r o l ( s a l i n e ) . 152 F i l * >AF1 24O00Q -220000 -200000 -lflOOQO -1«0000 -140000 -12QOOO -laaaag -e o o o e -e o o a o -40000-20000-9 9 9 9 . o - o . a 13.014.0 13.a l « . O 17.016.0X9.020.021.0 22.0 F i l * >AF4 9999.0 - O . O 2O0OO0 ~ l e a o o o 1600Q0 ~ 14O0OQ Z 120000 2_ laofloa -~ • o o o a -~ t e e e a : 40000 : i 20000: B 19.93 20^03 12 . O 13.014.015.0 16.017 . O 18.019.020.0 21.O 220000 -200000 -l e o e o o -i £ o a a o -14OOO0 -120000 -l a a o o o -e o o a a -6OOOO-40000-2OOOO-12 . / rt« 7 3 17.03 - A 13.0 1 4 . a i 3 . « l C . e i 7 . 0 1 S . 0 19.Q20.Q21.0 • i • >AFS 220OOO-2O0OOO l a o a o a 140000 -140000 i z a a o a l e o o o a S0O0O c o o o o 4OOO02: 20000 9999 . O-0 . O » « » A— Ft I* >AFJ) 220000 z a a a a o • o o o o c o a o o 40000 20000 1 2 . 0 1 3 ' . a i V . o IS'.O o ' i i r ' . o l i e " . o i i ' . « 20'.0 21'.o IS. C7 \ E 11 O i 7 IK 1 39 k s 19.96 70 I -. 1 3 . a 14. a i s . a i « . 0 1 7 . 0 i a . a 1 9 . 0 2 0 . 0 2 1 . 0 F i g u r e 46. SIM p l o t s o f microsomal CLBZ m e t a b o l i t e s . CLBZ, t R , 15.7 min; DMC-TMS, t R 13.6 min; 4'-hydroxy DMC, t R 17.0 min and 4'-hydroxy CLBZ t R 19.9 min. A: CLBZ, no microsomes, 60 min i n c u b a t i o n ; B: c o n t r o l microsomes 15 min i n c u b a t i o n ; C: c o n t r o l microsomes; 60 min i n c u b a t i o n ; D: PB in d u c e d microsomes, 15 min i n c u b a t i o n ; E: PB i n d u c e d m i c r o s o m e s , 60 min i n c u b a t i o n . 153 (E) The D e u t e r i u m I s o t o p e E f f e c t a s s o c i a t e d w i t h CLBZ N - d e m e t h y l a t i o n . P o u p a e r t e t a l . (1988) o b s e r v e d t h a t when mice were t r e a t e d w i t h t r i d e u t e r i o m e t h y l CLBZ ( [ 2 H 3 ] C L B Z ) , an e x t e n d e d d u r a t i o n o f a n t i c o n v u l s a n t a c t i v i t y was d e r i v e d compared t o CLBZ. They c o n c l u d e d t h a t t h i s phenomenon was a t t r i b u t a b l e t o a p r o l o n g e d a c t i o n o f t h e p a r e n t d r u g w h i c h was caused by an i s o t o p e - a s s o c i a t e d r e d u c t i o n i n N - d e m e t h y l a t i o n . In o r d e r t o i n v e s t i g a t e t h e i s o t o p e e f f e c t a s s o c i a t e d w i t h CLBZ N-d e m e t h y l a t i o n in vitro, an a p p r o x i m a t e 50:50 m i x t u r e o f p e n t a d e u t e r i o p h e n y l C L B Z : t r i d e u t e r i o m e t h y l CLBZ was i n c u b a t e d w i t h c o n t r o l and P B-induced r a t l i v e r microsomes. By t h i s a p p r o a c h , t h e s o u r c e o f DMC d e r i v e d from d e m e t h y l a t i o n ([ 2H5JDMC) and d e d e u t e r i o m e t h y l a t i o n (DMC) c o u l d be d e t e r m i n e d , k /^kp v a l u e s , d e t e r m i n e d by p r o d u c t r a t i o s , were c a l c u l a t e d a t 3.65 ± 0.27 (n = 3) and 2.80 ± 0.16 (n = 4) f o r c o n t r o l and i n d u c e d microsomes r e s p e c t i v e l y . These v a l u e s , a r e c o n s i s t e n t w i t h t h e d e p r o t o n a t i o n o f an aminium r a d i c a l (k^Ap, < 3.6) (Miwa e t a l . , 1983). Hence, t h e cytochrome P-450 c a t a l y z e d d e m e t h y l a t i o n o f CLBZ s h o u l d p r o c e e d via t h e f o l l o w i n g pathway: e l e c t r o n r e m o v a l , f o l l o w e d by p r o t o n a b s t r a c t i o n , o x y g e n a t i o n t o t h e c a r b i n o l a m i d e , and d e f o r m y l a t i o n t o DMC. 154 IV SUMMARY AND CONCLUSIONS. 1. ['H 5]CLBZ was s y n t h e s i z e d i n e s s e n t i a l l y q u a n t i t a t i v e i s o t o p i c and c h e m i c a l p u r i t y . The most s u s c e p t i b l e s t e p t o d e u t e r i u m exchange i n t h e s y n t h e s i s o f t h i s compound was t h e n u c l e o p h i l i c s u b s t i t u t i o n o f 2,4-d i c h l o r o n i t r o b e n z e n e by a n i l i n e - d 7 t o form N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18. In t h i s s t e p c o n t a m i n a t i n g a n i l i n e - 2 , 3 , 4 , 5 , 6 - d 5 i n t r o d u c e d p r o t o n s i n t o t h e ortho and p a r a p o s i t i o n s o f 18. A mechanism a c c o u n t i n g f o r t h i s r e a r r a n g e m e n t has been p r o p o s e d . 2. 4'-Hydroxy CLBZ ( 2 8 ) , 4'-hydroxy DMC ( 2 6 ) , 3 , 4 - d i h y d r o x y CLBZ (35) and 3 , 4 - d i h y d r o x y DMC (32) were c h e m i c a l l y s y n t h e s i z e d as CLBZ m e t a b o l i t e s t a n d a r d s . The 0-methyl e t h e r was a s u i t a b l e p h e n o l i c p r o t e c t i n g group w h i c h was e a s i l y removed i n good y i e l d w i t h BBr3. The 0 - m e t h y l a t e d c a t e c h o l s o f CLBZ (36a+b) and DMC (33) were e n z y m a t i c a l l y s y n t h e s i z e d by u s i n g d i h y d r o x y CLBZ (35) and d i h y d r o x y DMC (32) r e s p e c t i v e l y as s u b s t r a t e s , l i v e r c y t o s o l as a s o u r c e o f c a t e c h o l O - m e t h y l t r a n s f e r a s e and S - a d e n o s y l - L - m e t h i o n i n e as t h e methyl donor. The 0 - m e t h y l a t e d c a t e c h o l s o f CLBZ appeared as a m i x t u r e w i t h a meta (36a):para (36b) r a t i o o f 68:32. 3. T r e a t m e n t o f DMC w i t h formaldehyde i n t h e p r e s e n c e o f K2CO3 p r o d u c e d a compound whose GCMS ( E I ) s p e c t r a l p r o p e r t i e s were c o n s i s t e n t w i t h t h e c a r b i n o l a m i d e ( 3 9 ) . U n f o r t u n a t e l y , t h i s compound c o u l d n o t be p u r i f i e d f o r *H-NMR a n a l y s i s because o f i t s m a r g i n a l s t a b i l i t y . A major p r o d u c t i n t h i s r e a c t i o n was 3-hydroxymethyl DMC (46) which was c h a r a c t e r i z e d by *H-NMR and LCMS. Formaldehyde c o n d e n s a t i o n c a t a l y z e d by KOH a l s o a f f o r d e d 3-hy d r o x y m e t h y l DMC ( 4 0 ) . Whether formaldehyde a d d i t i o n a t t h e 3 - p o s i t i o n o c c u r s as a k i n e t i c p r o d u c t o r whether i t a r i s e s as t h e r e s u l t o f an 155 i s o m e r i z a t i o n subsequent t o N - h y d r o x y m e t h y l a t i o n r e m a i n s t o be r e s o l v e d . 4. GCMS i d e n t i f i c a t i o n o f i s o t o p e c l u s t e r s a l l o w e d t h e f o l l o w i n g c o n j u g a t e d m e t a b o l i t e s o f CLBZ t o be i d e n t i f i e d : 4'-hydroxy CLBZ ( 2 8 ) , 4'-hy d r o x y DMC ( 2 6 ) , 3 ' , 4 ' - d i h y d r o x y CLBZ ( 3 5 ) , 4'-hydroxy-3'-methoxy DMC ( 3 3 ) , 4'-hydroxy-3'-methoxy CLBZ ( 3 6 a ) , and 3'-hydroxy-4'-methoxy CLBZ ( 3 6 b ) . The l a t t e r m e t a b o l i t e was o n l y s i g n i f i c a n t as a b i l i a r y s u l f a t e where i t c o n s t i t u t e d 30 % o f t h e 0-methyl c a t e c h o l a n a l o g u e s o f CLBZ. A mechanism f o r t h i s n o n - u n i f o r m d i s p o s i t i o n o f i s o m e r s has been p r o p o s e d . N e i t h e r t h e c a r b i n o l a m i d e o f CLBZ, n o r t h e h y d r o x a m i c a c i d were d e t e c t a b l e m e t a b o l i t e s o f CLBZ i n t h e r a t . 5. The m e t a b o l i s m o f CLBZ and DMC by c o n t r o l and PB - i n d u c e d microsomes was examined. CLBZ was m e t a b o l i z e d t o DMC, 4'-hydroxy CLBZ and 4'-hydroxy DMC. There was no e v i d e n c e f o r t h 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 i n N-d e m e t h y l a t i o n . P B - i n d u c t i o n appeared t o i n c r e a s e N - d e m e t h y l a t i o n o v e r r i n g h y d r o x y l a t i o n . DMC was m e t a b o l i z e d t o 4'-hydroxy DMC. There was no e v i d e n c e f o r N - o x i a t i o n t o t h e hydroxamic a c i d . 6. In t h e r a t , d e u t e r i u m i s o t o p e e f f e c t s were m a r g i n a l f o r phenyl r i n g o x i d a t i o n t o p h e n o l s (ki t /kg =1.103 ± .090) and c a t e c h o l s (ku/kn, = 1.088 ± .207). 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GCMS ( E I ) spec t r u m o f a n i l i n e - d 7 (17) shows [ M - 2 ] + * as most i n t e n s e h i g h mass i o n because o f e x t e n s i v e d e u t e r i u m exchange d u r i n g GC. 1.4. 84.68 MHz 2H-NMR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a -d e u t e r i o p h e n y l a m i n e (18) shows t h e p r e s e n c e o f d e u t e r i u m a t t h e ortho/para (7.32 ppm) and meta (7.50 ppm) p o s i t i o n s o f t h e phenyl r i n g . 1.5. FT-IR s p e c t r u m o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a d e u t e r i o p h e n y l a m i n e (CCI4 s o l u t i o n ) ( 1 8 ) . 1.6. FT-IR s p e c t r u m o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - p e n t a d e u t e r i o -phenyl carbamoyl a c e t a t e (CCI4 s o l u t i o n ) ( 1 9 ) . 1.7. 400 MHz ^H-NMR spectrum o f p e n t a d e u t e r i o p h e n y l N-desmethylclobazam ( 2 0 ) . + E t 0 A c . 1.8. FT-IR s p e c t r u m o f p e n t a d e u t e r i o p h e n y l N - d e s m e t h y l c l o b a z a m ( N u j o l m u l l ) ( 2 0 ) . 1.9. FT-IR s p e c t r u m o f p e n t a d e u t e r i o p h e n y l c l o b a z a m (CCI4 s o l u t i o n ) ( 2 1 ) . 1.10. 400 MHz ^H-NMR spect r u m o f p e n t a d e u t e r i o p h e n y l CLBZ (21) i n CQDQ. 1.11. 400 MHz *H-NMR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 4 ' - m e t h o x y -phenylamine ( 2 3 ) . 1.12. 300 MHz !H-NMR spect r u m o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 -m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 2 4 ) . 1.13. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 4 ' - m e t h o x y -p h e n y l a m i n e (23) and (B) e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 -m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 2 4 ) . 1.14. 400 MHz *H-NMR spectrum o f 4'-methoxy-N-desmethylclobazam ( 2 5 ) . 1.15. 400 MHz *H-NMR spectrum o f 4'- h y d r o x y - N - d e s m e t h y l c l o b a z a m (26) i n DMS0-d 6. 1.16. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 4'-methoxy-N-desmethyl clobazam (25) and (B) 4'-methoxyclobazam ( 2 7 ) . 175 1.17. 400 MHz XH-NMR spect r u m o f 4'-methoxyclobazam ( 2 7 ) . 1.18. 400 MHz *H-NMR spect r u m o f 4'- h y d r o x y c l o b a z a m ( 2 8 ) . C o n t a m i n a t i n g p e a k s : 4.14 ( q , E t O A c ) , 3.85 ( s , 4'-methoxy CLBZ (<5 % ) ) . 1.19. 400 MHz *H-NMR spect r u m o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' -d i m e t h o x y p h e n y l a m i n e ( 2 9 ) . 1.20. 400 MHz *H-NMR spect r u m o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 3 , 4 -d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . 1.21. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' -d i m e t h o x y p h e n y l a m i n e (29) and (B) e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) -e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 3 , 4 -d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . 1.22. 400 MHz ^H-NMR spect r u m o f 3',4'-d i m e t h o x y - N - d e s m e t h y l c l o b a z a m ( 3 1 ) . 1.23. 400 MHz *H-NMR spectrum o f 3' , 4 ' - d i h y d r o x y - N - d e s m e t h y l c l o b a z a m (32) i n DMS0-d6-1.24. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 3',4'-dimethoxy-N-desmethyl c l o b a z a m (31) and (B) 3',4 ' - d i m e t h o x y c l o b a z a m ( 3 4 ) . 1.25. 400 MHz ^H-NMR spectrum o f 3',4'-di m e t h o x y c l o b a z a m ( 3 4 ) . 1.26. 400 MHz !H-NMR sp e c t r u m o f 3 ' , 4 ' - d i h y d r o x y c l o b a z a m ( 3 5 ) . 1.27. 400 MHz *H-NMR spect r u m o f compound ( 4 0 ) . 1.28. 400 MHz ^H-NMR spect r u m o f compound ( 4 5 ) . 2. SPECTRA RELATED TO THE METABOLISM OF CLBZ IN THE RAT. 2.1. Mass chromatograms a t m/z 388 (M +-) and 392 ( [ 2 H 4 ] M + * ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f / f - 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 t y p i f y t h e d e t e c t i o n o f 4'-hydroxy CLBZ (28) a t t R 21.37 and t R 21.30 min r e s p e c t i v e l y . The absence o f t h e i s o t o p i c a l l y r e l a t e d m/z 388/393 shows t h a t a c a r b i n o l a m i d e was n o t d e t e c t a b l e . 2.2. Mass chromatograms a t m/z 446 ( M + > ) and 450 ( [ 2 H 4 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f / 3 - 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 t y p i f y t h e d e t e c t i o n o f 4'-hydroxy DMC (26) a t t R 17.91 and t R 17.86 min r e s p e c t i v e l y . 2.3. Mass chromatograms a t m/z 476 (M +-) and 479 ( [ 2 H 3 ] M + ' ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f / 3 - 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 t y p i f y 176 t h e d e t e c t i o n o f 4'-hydroxy-3'-methoxy DMC (33) a t tR 19.13 and tR 19.17 min r e s p e c t i v e l y , and d i h y d r o x y CLBZ (35) a t t R 23.50 and tR 23.47 min r e s p e c t i v e l y . Mass chromatograms a t m/z 418 (M +-) and 421 ([ 2H"3]M +-) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f / J - 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 show t h e p r e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t tR 23.22 and tR 23.17 min r e s p e c t i v e l y , and 3'-hydroxy-4'-methoxy CLBZ (36b) a t tR 22.44 and t R 22.37 min r e s p e c t i v e l y . Mass chromatograms a t m/z 418 (M +-) and 421 ( [ 2 H 3 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f a r y l s u l f a t a s e - h y d r o l y z e d u r i n e show t h e p r e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t tR 23.12 and tR 23.08 min r e s p e c t i v e l y . 177 178 S A M P L E N 0 2 3 4 7 A B O R E L S A M P L E I D A B 1 7 6 / M T A T — | — i — ; — i — i — j — i — I — I — I — | — ! — ; — I — ; — j — : — i — I — i — [ ~ — — i — i ' j i i i I ] : i i 145 140 135 130 125 120 115 P P M 1.2. 75 MHz 1 3C-NMR sp e c t r u m o f a n i l i n e - a j ( 17) shows t h e p r e s e n c e o f d e u t e r i u m a t t h e C-3, C-4 and C-2 p o s i t i o n s w i t h t h e appearance o f t r i p l e t s . 98 71 28 42 32 33 49 34 37 76 82 88 1.3. GCMS (EI ) spectrum o f a n i l i n e - d y (17) shows [M-2]+* as most i n t e n s e h i g h mass i o n because o f e x t e n s i v e d e u t e r i u m exchange d u r i n g GC. 180 1.4. 84.68 MHz ^H-NMR spect r u m o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a -d e u t e r i o p h e n y l a m i n e (18) shows t h e p r e s e n c e o f d e u t e r i u m a t t h e ortho/para (7.32 ppm) and meta (7.50 ppm) p o s i t i o n s o f t h e phenyl r i n g . 1.5. FT-IR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a d e u t e r i o p h e n y l a m i n e (CCI4 s o l u t i o n ) ( 1 8 ) . 1 . 6 . FT-IR spectrum o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - p e n t a d e u t e r i o - p h e n y l carbamoyl a c e t a t e (CCI4 s o l u t i o n ) ( 1 9 ) . H CO to 183 Hz 5.000 Hz 2,500 4 .000 2 .000 x 8 JL 1.7. 400 MHz *H-NMR spectrum o f p e n t a d e u t e r i o p h e n y l N-desmethylclobazam ( 2 0 ) . + E t O A c . 5 4 3 1.11. 400 MHz *H-NMR spectrum of N-(5-chloro-2-nitrophenyl)-4 '-methoxy-phenylamine (23). J Ju^LJ VARI AN X L - 3 0 0 STANDARO 1H OBSERVE F.XP8 PULSE SEQUENCE STD 1 H DATE 2 1 - 0 6 - 8 9 SOL VENT F I L E CDCL 3 H : 0 l>7 A C Q U I S I T I O N DEC 4 VT TN 1 . 750 DN 1 . 750 SW 4 0 0 0 . 0 DO 350 . 3 A r 3. 752 OW NNN NP J 0 0 1 6 DLP 14 PW 35 0 P i 0 P R O C E S S I N G 01 0 SE 3. 183 02 0 LB 0. 100 TO 300 FN 65536 NT 500 MATH F CT 100 PW90 46. 0 D I S P L A Y SS 0 SP - 6 0 . 1 IL y WP 2 6 9 9 . 5 IN Y VS 300 OP r SC 100 HS NN wc t s RFL RFP TH INS 400 120 5 4 9 . 3 0 20 1 . 000 r \ 1 1 1 1 1 1 1 1 1 I I I | 1 I I I | I I I I | I I I I [ I M I | I I M | 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 7 6 5 '''' 4 3 2 1.12. 300 MHz *H-NMR spectrum o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 -methoxyphenyl)carbamoyl a c e t a t e ( 2 4 ) . 03 03 f i l e >RB141 Bpk fib 20848 4-Otle CNPB Scan 284 7.88 min. 75 92 / 123 / 111 1,,,,^ 111,1,1 i l l ..i.lnn,, I ill,. . mill, 1 3 9 / 182 ^ \ 188 II, ..III •illlll.l ,.M\ 216 \ 217 278 2 6 3 \ 244 I V 88 128 168 288 240 288 . l i e >RB147 Bpk Rb 2056 2200-2800-1800-1600-1488-1288-1009-888-680-408-0-1' 75 B 92 4-OMe PCMM 123 III I, II 111 / ,1 III hlin 154 281 152 182 \ 216 Scan 285 7.02 min. 278 263 244 / »17 88 120 168 288 248 288 13. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 4 ' - m e t h o x y p h enylamine (23) and (B) e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 2 4 ) . 1.15. 400 MHz *H-NMR spectrum o f 4'-hydroxy-N-desmethylclobazam (26) i n DMS0-d 6. 192 f i l e >0B232 Bpk fib 37808 40000-^  36000-32000-28000-24000-20000^ 16800^ 12000^ 8000^ 75 / 4000^ | 1 0-^  L i 80 4*-0Me-DMC-TMS CLP 225 121 \ 135 167 \ 197 178 y 273 298 331 347 M I { 120 160 200 240 280 - I -320 Scan 390 8.79 min. r110 400 r90 r80 r70 J60 ^0 388 : \ |40 r30 r20 jie 360 ...le >RB233 Bpk fib 5504 -MeO-CLBZ Scan 468 10.11 min. 6080-5500-5000-4500-4000-3580-3000-2580-2000-1500-1000-500-J i 92 \ B 121 108 I LHmllml 153 \ 111, J l 217 181 V 230 / 167 / Ji • III Hill J 0,11 247 80 120 160 200 240 285 273 / i i 330 313 280 328 110 j-100 f90 770 r60 rS0 r40 •30 E20 10 -^3 1.16. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 4'-methoxy-N-desmethyl c l obazam (25) and (B) 4'-methoxyclobazam ( 2 7 ) . 194 1.18. 400 MHz H-NMR spectrum o f 4 ' - h y d r o x y c l o b a z a m ( 2 8 ) . C o n t a m i n a t i n g peaks: 4.14 ( q , EtOAc), 3.85 ( s , 4'-methoxy CLBZ (<5 % ) ) . 1.19. 400 MHz *H-NMR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' -dimethoxyphenylamine ( 2 9 ) . — — r — — , 1 — i r 5 4 3 2 1 1.20. 400 MHz *H-NMR spectrum o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N -( 3 , 4 - d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . 197 f i l e >0B24S Bpk fib 49624 52800-48000-44880-40000-36000-32000-28000-24800-20000-16000-12080-8008-4000-0-3,4-ditleO-CNPR 79 / .Inl.j... U l 107 / 140 183 \ 188 07 y I 153 X*/" Scan 334 7.84 min. 293 204 231 / III. All 274 \ 246 111. . . . H i . \\L 308 \ 110 100 9^0 6^0 70 ^0 J50 r48 •30 20 -10 80 128 160 200 240 280 . l i e >RB248 Bpk Rb 40736 44000-40000-36000-32080-28000-2400frj 20000-16080-12000^ 8009-4080^ 8-3,4-ditleO-PCNM B. 79 / •It. . H i . .1 140 183 ' 153 168 / J l 1^1... j\i Jnlll..Jillll,JlJ.illn,,,,, 111 107 y 204 / LU Scan 331 7.79 min. 293 308 231 274 \ 246 I... ..Iii. .ln,L 111- .. 118 100 9^0 i«0 r70 |60 j€8 r48 r30 r28 10 80 120 160 200 240 280 1.21. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' d i m e t h o x y p h e n y l a m i n e (29) and (B) e t h y l N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) - e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 3 , 4 -d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . SBMPLf N0:6BS6 fl.BORH 1 I 1 1 ' ' I ' ' ' ' I ' 1 ' ' I 1 1 ' 1 r 1 ' 1 ' I 1 ' 1 1 I ' ' ' ' I i i . . I I , , , , , , . . . ! m - ' j i a J.'J -s.'i D.a 7.7i c . ' i F . a s . i b.a ^ . i a . a 3.r> J.B 1.23. 400 MHz H-NMR spectrum o f 3 ' , 4 ' - d i h y d r o x y - N - d e s m e t h y l c l o b a z a m (32) i n DMS0-d 6. 200 . i le >RB250 Bpk Ob 5368 5500-5000-4500-4000-3500-3000-2500-2008- 75 / 1508-1000-500- i 0- L,r J 3' ,4'-(0Me)2-DMC-TMS CLP 225 I 124 179 197 / 239 X 2 8 5 37 80 120 160 200 240 280 320 360 303 361 375 418 \ Scan 434 9.52 min. 110 :100 J90 r80 '-79 |60 rse J40 r30 -20 18 400 f i l e >AB288 Bpk Ob 8001 3,4-diMeOCLBZz CLP Scan 555 1 .60 min. 8380-1 7000-^  6000-1 5800H 4000-^  3000H 2000-1 79 1800-1 B 151 / 117 \ 138 \ 4n,.ljulitliiit.iHii. 80 120 315 \ 1 8 1 » 8 9 260 24S llli 217 Mill 277 l u l l 360 319 160 200 240 280 320 110 100 r90 r80 •70 r60 rS0 r48 •30 20 10 CO 360 1.24. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 3' ,4'-dimethoxy-N-d e s m e t h y l c l o b a z a m (3_i) and (B) 3 ' , 4 ' - d i m e t h o x y c l o b a z a m ( 3 4 ) . 1.25. 400 MHz *H-NMR spectrum o f 3',4'-dimethoxyclobazam ( 3 4 ) . 202 100 so H z 5 . 0 0 0 H i 2 . 5 0 0 4 . 0 0 0 3 . 0 0 0 1.26. 400 MHz *H-NMR spectrum o f 3 ' , 4 ' - d i h y d r o x y c l o b a z a m ( 3 5 ) . 205 F i l e >RB81 387.7-333.7 amu.CLBZ-BILE-GLUC-TMS-SMT ADC EIP 4800-3000-2080-1000-21 .37 15.75 23^0? 2 I - " 1 " ! , i f | 12.013.014.015.016.017.818.819.8 20.821.822.823.024.025.026.8 F i l e >RB01 2S00^ 2888 1500: 1880-| See-s' 391 .7-392.7 amu.CLBZ-BILE-GLUC-TMS SMT RDC EIP • •A. /A. . . 21 .30 12.8 1 3 . 8 1 4.0 1 5 . 0 1 6.6 1 7 . 8 1 8.0 19* .81 2 0 . 0 2 l'.'eI22.G123:".'024.0125.0 26.8 F i l e >RB81 888-600-400-200-392 .7-393 .7 amu.CLBZ-BILE-GLUC-TMS SMT RDC EIP 17.87 12.75 i l .38 23.17 22.91 JL 1 2 . 0 1 3 . 8 i V.'o i 5 . 0 i 6 . 0 i 7 . 0 i 8 . 0 i 9.0' 2 0 . 0 2i'.'e 2 2 . 0 2 3 .'024 .'0 25 .'e 26. e 2.1. Mass chromatograms a t m/z 388 (M +-) and 392 ( [ 2 H 4 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f 0 - 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 t y p i f y t h e d e t e c t i o n o f 4'-hydroxy CLBZ (28) a t t R 21.37 and t R 21.30 min r e s p e c t i v e l y . The absence o f t h e i s o t o p i c a l l y r e l a t e d m/z 388/393 shows t h a t a c a r b i n o l a m i d e was n o t d e t e c t a b l e . 206 J 17-91 1 5 . 9 2 1 5 . 0 9 K-r><y?-; ~ 4 1 3 . 0 1 4 . 0 1 5 . 0 1 6 . 0 1 7 . 8 1 8 . 6 1 9 . 0 2 8 . 6 2 1 . 6 2 2 . 6 2 3 . 6 2 4 . 6 2 5 . 0 2 6 . 6 I J 1 17-86 1 5 . 1 2 2 6 . 3 9 1 9 . 1 1 1 3 . 6 1 4 . 0 1 5 . 6 1 6 . 6 1 7 . 6 1 3 . 8 1 9 . 6 2 6 . 6 2 1 . 6 2 2 . 6 2 3 . G 2 4 . 6 2 5 . 6 2 6 . 6 2.2. Mass chromatograms a t m/z 446 (M +-) and 450 ( [ 2 H 4 ] M + * ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f / J - 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 t y p i f y t h e d e t e c t i o n o f 4'-hydroxy DMC (26) a t t R 17.91 and t R 17.86 min r e s p e c t i v e l y . 207 19-13 18 . 4 0 rT"1 2 3 . 5 0 . 0 2 2 . 0 2 3 . 8 2 4 . 8 2 5 . 0 2 6 . 0 1 3 . G 1 4 . 6 1 5 . 0 1 6 . 0 i ; 18 . 0 19 . 8 2 6 . 0 21 19-17 2 3 . 4 7 1 3 . 8 1 4 . 0 1 5 . 0 1 6 . 0 1 7 . 0 1 8 . 0 1 9 . 0 2 0 . 6 2 1 . O 2 2 . 0 2 3 . 0 2 4 . 0 2 5 . 0 2 6 . 0 2.3. Mass chromatograms a t m/z 476 (M +-) and 479 ( [ 2 H 3 ] M + ' ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f 0 - 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 t y p i f y t h e d e t e c t i o n o f 4'-hydroxy-3'-methoxy DMC (33) a t t R 19.13 and t R 19.17 min r e s p e c t i v e l y , and d i h y d r o x y CLBZ (35) a t t R 23.50 and tR 23.47 min r e s p e c t i v e l y . 208 F i l e >f»B81 1666-i zoo-see-480 41; .7-418.7 amu.CLBZ-BILE-GLUC-TMS SMT ABC EIP 23 .22 12.013.014.015. i i 11 • * ' i • I 1 1 ' ' ' ' I * ' ' ' ' ' ' ' ' * ' ' ' ' S16.017.0l8.019.O20.821.02i 623 .024.625.626 .0 F i l e >fiB81 1600-888 608-460 280 426 .7-421 .7 amu.CLBZ-BILE-GLUC-TMS SMT PLC EIP 0-141^ .421 4 17.8419.84 18.41 23.17 48 12.81 3.0 14.0 15.0 16 .8 i 7.018.0 19.020.0 2l '.'o 22 .023 .824.8 25 .8 26.8 2.4. Mass chromatograms a t m/z 418 (M +-) and 421 ( [ 2 H 3 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f / J - 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 show t h e p r e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t t R 23.22 and tR 23.17 min r e s p e c t i v e l y , and 3'-hydroxy-4'-methoxy CLBZ (36b) a t tR 22.44 and t R 22.37 min r e s p e c t i v e l y . 209 F i l e >flB04 417.7-418.7 amu.CLBZ-URIN-SULF-TMS SMT ADC EIP 800-600-400-200-0 12 13 .27 i I 23.12 0 13.0 14.015.0 16.6 17.6 13.0 19.0 20.021.0 22.0 23.0 24.0 25.0 26.0 F i l e >AB04 420.7-421.7 amu.CLBZ-URIN-SULF-TMS SMT ODC EIP 500-480-300 200-100 23.08 IZ.'e 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.6 2l'.0 22.0 23.0 24.0 25.8 26.0 2.5. Mass chromatograms a t m/z 418 (M +-) and 421 ( [ 2 H 3 ] M + > ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f a r y l s u l f a t a s e - h y d r o l y z e d u r i n e show t h e pr e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t t R 23.12 and tR 23.08 min r e s p e c t i v e l y . 

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