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Studies on the metabolism of tocainide in humans Kwok, David W. K. 1987

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STUDIES ON THE METABOLISM OF TOCAINIDE IN HUMANS BY DAVID W. K. KWOK B.Sc. (Pharm), The U n i v e r s i t y of B r i t i s h Columbia, 1984 A THESIS SUBMITTED AS PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n The F a c u l t y of Graduate S t u d i e s F a c u l t y of Pharmaceutical S c i e n c e s D i v i s i o n of Pharmaceutical Chemistry. We accept t h i s t h e s i s as conforming to the r e q u i r e d standard The U n i v e r s i t y of B r i t i s h Columbia _ A p r i l 1987 ( c ) David W.K. Kwok In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmaceutical Chemistry The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e A p r i l 30,1987. DE-6(3/81) A b s t r a c t T o c a i n l d e carbamoyl e s t e r g l u c u r o n l d e (TOCG) (R-NHCO.O-GA) R Is a major metabolite of t o c a i n l d e (Tonocard ). The s t r u c t u r e of TOCG was f i r s t proposed by E l v i n <35) based on the s t r u c t u r e of 3-<2,6-xylyl)-5-methylhydantoln, a base h y d r o l y z e d product of TOCG i n u r i n e . Due to the presence of two carb o n y l groups on the hydantoln r i n g , TOCG was proposed to a r r i v e from a novel metabolic pathway i n v o l v i n g the a d d i t i o n of carbon d i o x i d e to the term i n a l n i t r o g e n of t o c a i n l d e f o l l o w e d by g l u c u r o n i c a c i d c o n j u g a t i o n . With the i n i t i a l i n t e n t i o n of c a r r y i n g out a b i o a v a i l a b i l i t y study of t o c a i n l d e using a de u t e r a t e d pseudoracemic sample, the s t e r e o s p e c i f i c s y n t h e s i s of R<->- and S<+ ) - t r l d e u t e r a t e d t o c a i n l d e was attempted through two s y n t h e t i c approaches. T h i s t h e s i s d e s c r i b e s a chemical r e a c t i o n between t o c a i n l d e and urea, a second pathway which leads to the formation of 3-<2,6-xylyl)-5-methylhydantoin through a t o c a i n l d e urelde i n t e r m e d i a t e . With t h i s o b s e r v a t i o n , a t o c a i n l d e N-urelde g l u c u r o n l d e s t r u c t u r e <R-NHCO.NH-GA) was proposed f o r TOCG in support of the theory that an i n vivo r e a c t i o n between t o c a i n l d e and urea may have r e s u l t e d a t o c a i n l d e N-ureide which can be f u r t h e r conjugated with g l u c u r o n i c a c i d . Attempts were made to a s s i g n the c o r r e c t s t r u c t u r e of TOCG by I d e n t i f i c a t i o n of the t h e o r e t i c a l t o c a i n l d e carbamic a c i d (based on E l v l n ' s proposed s t r u c t u r e ) or the t o c a i n l d e N-ureide intermediate in ur i n e . i i T h i s t h e s i s a l s o d e s c r i b e s the p r e p a r a t i v e HPLC i s o l a t i o n and the s t r u c t u r a l c h a r a c t e r i z a t i o n of t h i s novel g l u c u r o n i c a c i d c onjugate. Evidence obtained as proof f o r the I d e n t i t y of TOCG as a conjugate was obtained from a c i d h y d r o l y s i s , b a s i c h y d r o l y s i s , b e t a - g l u c u r o n l d a s e h y d r o l y s i s , with or without the presence of sac c h r o - 1 , 4 - l a c t o n e , and a n a p h t h o r e s o r c l n o l c o l o r t e s t . S t r u c t u r a l evidence f o r the carbamoyl e s t e r l i n k a g e of TOCG was obtained from proton-NMR and FAB a n a l y s i s . The 400-MHz proton NMR data of the I s o l a t e d g l u c u r o n i d e p r o v i d e d p a r t i a l evidence f o r the Intact s t r u c t u r e of TOCG. In FAB a n a l y s i s , the [M+ll Ion adduct at m/z 413, IM+Nal a t m/z 435, and [M-H+2Nal at m/z 457 have pr o v i d e d p o s i t i v e evidence f o r the molecular Ion of TOCG at m/z 412 In f a v o r of the carbamoyl e s t e r s t r u c t u r e . In a d d i t i o n to the h y d r o l y s i s of TOCG at pH > 12 to the hydantoin, t h i s hydantoln was found to a l s o undergo spontaneous f i r s t - o r d e r h y d r o l y s i s at pH > 12. To assay the l e v e l s of TOCG In u r i n e as the hydantoin, a s e t of a c c u r a t e l y timed c a l i b r a t i o n samples were employed i n an assay p r o t o c o l to take Into account the spontaneous h y d r o l y s i s of the hydantoin. Based on t h i s a n a l y t i c a l approach, the l e v e l s of TOCG were determined in three s u b j e c t s both a f t e r an IV and o r a l dose of 200 mg t o c a l n l d e HC1. The u r i n a r y e x c r e t i o n h a l f - l i v e s of TOCG of 13.86 hours and 13.33 hours, a f t e r an IV and o r a l dose r e s p e c t i v e l y , were found to agree with l i t e r a t u r e v a l u e s . i i i Table of Contents Page A b s t r a c t i i Table of Contents iv L i s t of T a b l e s x L i s t of F i g u r e s x i i L i s t of S t r u c t u r e s xvii L i s t of Schemes xvi i i L i s t of P h a r m a c o k i n e t i c E q u a t i o n s xx Symbols and A b b r e v i a t i o n s xxi Acknowledgement xxi i i INTRODUCTION 1 A . B r i e f Overview of T o c a i n l d e 2 B . E l e c t r o p h y s i o l o g i c a l P r o p e r t i e s 2 C . Pharmacodynamics 3 1. C l i n i c a l E f f i c a c y 3 2. E f f i c a c y in R e f r a c t o r y A r r h y t h m i a s 3 3. P r o p h y l a x i s A n t i a r r h y t h m i c Therapy 3 D. P h a r m a c o k i n e t i c s and Pharmacodynamics 4 1. B i o a v a i l a b i l i t y 4 2. D i s p o s i t i o n K i n e t i c s 4 3. Dose-response Pharmacodynamics 4 4. Serum P r o t e i n B i n d i n g 5 5. The E f f e c t s on K i n e t i c s from M e t a b o l i c I n d u c t i o n 6 E . S t e r e o s e l e c t i v i t y In Drug Metabol i sm 6 i v Table of Contents (Contd) Page F . Metabo l i sm 9 1. O b s e r v a t i o n of a Major Novel M e t a b o l i t e of T o c a l n l d e n 2. A n a l y s i s of G l u c u r o n i c A c i d Conjugates 15 3. S p e c u l a t i v e S t r u c t u r e s of the Novel T o c a l n l d e G l u c u r o n l d e 17 a . N-carbamoyla t i on \-j b . Carbonate E q u i l i b r i u m 19 c . T o c a l n l d e - u r e a Adduct F o r m a t i o n 21 1. U r e a - a d d u c t F o r m a t i o n 21 i i . Urea Condensa t ion wi th T o c a l n l d e 22 4. Drug G l u c u r o n i d a t i o n 23 a . A c y l - O - g l u c u r o n l d e s ( E s t e r G l u c u r o n l d e s ) 24 b . N - g l u c u r o n i d e s 24 G . T o x i c o l o g y 25 H . A g r a n u l o c y t o s i s 26 EXPERIMENTAL 29 A . Chemica l s and M a t e r i a l s 30 B . I n s t r u m e n t a t i o n 32 1. F a s t Atom Bombardment Mass Spec trometry 32 2. Gas Chromatography Mass Spec trometry 32 a . V a r l a n M a g n e t i c - s e c t o r GCMS 32 b . H e w l e t t - P a c k a r d Quadrupole GCMS 33 3. P r o t o n N u c l e a r Magnet i c Resonance 34 4. Gas Chromatography 34 5. L i q u i d Chromatography 34 6 . I n f a r e d S p e c t r o s c o p y 34 v Table of Contents (Contd) Page 7. Melt ing Point Determination 3 4 C. Synthesis of 3' , 4 ' , 5 ' - t 2 H]~ -2 -amino -2 ' , 6 ' -propioxyl Idide 3 5 1. Synthesis of S ( + > - 3 ' , 4 ' , 5 ' - [ 2 H I 3 - t o c a l n l d e 3 5 2. Synthesis of 3 , 4 , 5 - [ 2 H ] „ - 2 , 6 - d 1 methylani1ine 3 6 o D. Attempted Synthesis of l-[ H ] „ - 2 - a m i n o - 2 * , 6 ' -p rop ioxy l ld lde 3 8 1. Synthesis of N-carbobenzyloxy-(+)-D 3 -a lanine 3 8 E. Synthesis of Potent ia l Tocainlde Metaboli tes 3 9 1. Synthesis of 2 -e than imIno-2 ' ,6 ' -p rop ioxy l id ide 3 9 2. Synthesis of 3 - (2 ,6 -xy ly l ) -5 -methylhydanto in 4 0 3. Synthesis of 3 - (2 ,4 ,6 -xy ly l ) -5 -methy lhydanto in 4 1 a . Synthesis of 2 - a m l n o - 2 ' , 4 * , 6 ' - p r o p l o x y l i d i d e 4 1 b. Reaction with Trlchloromethyl Chloroformate 4 1 F. I d e n t i f i c a t i o n of Potent ia l Tocainlde Metaboli tes 4 2 1. Permethylation 4 2 a . Preparat ion of Dry Dimethylsulfoxide (DMSO) 4 3 b. Generation of Sodium Methylsulf lnylmethide Carbanion (DMSO) Sodium 4 3 c . Permethylation 4 3 2. Attempted I d e n t i f i c a t i o n of Permethylated Urinary Tocainlde Metaboli tes 4 4 a . I so la t ion of Tocainide-carbamoyl Ester Glucuronlde 4 5 b. Acid and Enzyme Hydrolys is 4 6 c . L iqu id Chromatographic Analys is of the Hydantoln Derived from the Glucuronic Acid Conjugate of Tocainlde 4 7 v i Table of Contents (Contd) Page d . Gas Chromatography/Mass S p e c t r o m e t r y A n a l y s i s of P e r m e t h y l a t e d T o c a l n l d e Carbamoyl G l u c u r o n i d e 47 3. Attempted I d e n t i f i c a t i o n of U r i n a r y T o c a l n l d e M e t a b o l i t e s by L y o p h i l i z a t i o n and GCMS A n a l y s i s 48 4. Attempted I s o l a t i o n and I d e n t i f i c a t i o n of T o c a l n l d e N - c a r b a m i c A c i d o r N - u r e l d e In termedia te 49 5. Attempted I s o l a t i o n and I d e n t i f i c a t i o n of U r i n a r y M e t a b o l i t e s by F l a s h Chromatography 50 . G . R e a c t i o n of T o c a i n i d e w i t h Urea 51 1. E f f e c t of Temperature and R e a c t i o n D u r a t i o n 51 2. E f f e c t of pH 52 3. Attempted S y n t h e s i s of T o c a l n l d e - N - u r e i d e -g l u c u r o n l d e 52 4. Attempted S y n t h e s i s of 2 , 6 - D i m e t h y l a n i 1 i n e - N -g l u c u r o n i d e 53 H . I s o l a t i o n and S t r u c t u r a l E l u c i d a t i o n of T o c a i n i d e G l u c u r o n i d e 53 1. P r e p a r a t i v e HPLC I s o l a t i o n of T o c a i n i d e Carbamoyl E s t e r G l u c u r o n i d e 54 a . A c i d H y d r o l y s i s w i t h H y d r o c h l o r i c A c i d 56 b . B e t a - g l u c u r o n l d a s e Enzyme H y d r o l y s i s 56 c . N a p h t h o r e s o r c i n o l C o l o r R e a c t i o n 57 d . Sodium Hydroxide H y d r o l y s i s 57 I . P h a r m a c o k i n e t i c s of T o c a l n l d e Carbamoyl E s t e r G l u c u r o n i d e 57 1. H y d r o l y s i s K i n e t i c s of T o c a l n l d e G l u c u r o n i d e In Sodium Hydroxide 58 2. H y d r o l y s i s of 3 - < 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o l n 58 3. H y d r o l y s i s of 3 - ( 2 ,4 , 6 - x y l y l > - 5 - m e t h y l h y d a n t o i n 58 4. 3 - < 2 , 6 - X y l y l ) - 5 - m e t h y l h y d a n t o I n C a l i b r a t i o n Curve 58 v i i Table of Contents (Contd) Page 5 . I n t e r - a s s a y and I n t r a - a s s a y V a r i a b i l i t y 5 9 6 . E x t r a c t i o n E f f i c i e n c i e s of T o c a i n i d e and the H y d a n t o i n in to 5 mL of Methylene C h l o r i d e 6 0 7. A n a l y s i s of T o c a i n i d e G l u c u r o n i d e in K i n e t i c S t u d i e s 6 1 RESULTS AND DISCUSSION 6 2 A . S y n t h e t i c pathways f o r 3 ' , 4 ' , 5 ' - t r l d e u t e r a t e d T o c a i n i d e 6 3 1 . S y n t h e s i s of 3 ' , 4 ' , 5 ' - [ 2 H ] 3 - 2 - a m i n o - 2 ' , 6 ' -p r o p i o x y l i d i d e 6 3 a . 2 , 6 - D l m e t h y l a n l 1 l n e D e u t e r a t l o n 64 b . R e a c t i o n w i t h C a r b o b e n z y l o x y l - a l a n i n e 7 1 2 2 . Attempted S y n t h e s i s of 1 - [ H 1 3 - 2 - a m i n o - 2 ' , 6 ' -p r o p i o x y l i d i d e 73 B . P o t e n t i a l T o c a i n i d e M e t a b o l i t e s 7 7 1 . 2 - E t h a n i m i n e - 2 ' , 6 ' - p r o p 1 o x y l I d i d e 77 2 . 3 - ( 2 , 6 - X y l y l ) - 5 - m e t h y l h y d a n t o l n 77 3 . 3 - ( 2 , 4 , 6 - X y l y l ) - 5 - m e t h y l h y d a n t o i n 8 4 C . T o c a l n l d e M e t a b o l i t e s 8 9 1 . D e r i v a t i z a t i o n Techniques f o r G l u c u r o n i d e s 8 9 2 . I d e n t i f i c a t i o n of T o c a l n l d e G l u c u r o n i d e by TLC I s o l a t i o n and C H ^ I / P e r m e t h y l a t i o n 9 0 3 . Attempted I d e n t i f i c a t i o n of T o c a i n i d e M e t a b o l i t e s by L y o p h i l l z a t i o n F o l l o w e d by GCMS ( P e r m e t h y l a t i o n ) 9 6 4 . Attempted I d e n t i f i c a t i o n of M e t a b o l i c I n t e r m e d i a t e s of T o c a i n i d e 9 7 5 . I d e n t i f i c a t i o n of T o c a i n i d e G l u c u r o n i d e by F l a s h Chromatography and C H ^ I / C D g l P e r m e t h y l a t i o n 1 0 6 6 . T o c a l n l d e - u r e a R e a c t i o n 1 1 1 7. S y n t h e s i s of N - g l u c u r o n l d e s 1 1 3 v i i i Table of Contents (Contd) Page D. HPLC I s o l a t i o n and S t r u c t u r a l E l u c i d a t i o n of T o c a i n l d e G l u c u r o n i d e 116 1. P r e p a r a t i v e HPLC I s o l a t i o n 116 2. EI Mass S p e c t r a l A n a l y s i s of the T o c a i n l d e G l u c u r o n l d e 122 3. 400 MHz P r o t o n N u c l e a r Magnet ic Resonance Experiment 122 4. F a s t Atom Bombardment Mass S p e c t r o m e t r y A n a l y s i s 128 E . P h a r m a c o k i n e t i c s of T o c a i n l d e G l u c u r o n l d e 133 1. T o c a i n l d e G l u c u r o n i d e B a s i c H y d r o l y s i s K i n e t i c s 133 2. H y d r o l y s i s of 3 - < 2 , 4 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o l n 136 3. C a l i b r a t i o n Curve Data 139 4. The E l i m i n a t i o n K i n e t i c s of T o c a i n l d e G l u c u r o n i d e 145 SUMMARY AND CONCLUSIONS 155 References 157 Appendix 1 164 Appendix 2 165 i x L i s t of T a b l e s Page 1. Examples of v a r i o u s f u n c t i o n a l groups known to g ive r i s e to N - g l u c u r o n i c a c i d conjugate in man. 24 2. The Rf of u r i n a r y components s e p a r a t e d on a KC-18F 20x20 cm TLC p l a t e . 45 3. The Rf three u r i n a r y components s e p a r a t e d on a KC-18F TLC p l a t e d u r i n g a second p u r i f i c a t i o n . 46 4. Summarized d a t a from the 80-MHz-t-'-H] -NMR spectrum of D - 2 , 6 - d i m e t h y l a n i l i n e . 68 5. Summarized d a t a from the 400-MHz-C 1 H]-NMR spectrum of 3 - < 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 83 6. Summarized d a t a from the 80-MHz p r o t o n NMR spectrum of 3 - < 2 , 4 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 87 7. The samples o b t a i n e d from the e x p e r i m e n t a l p r o t o c o l used f o r i d e n t i f y i n g t o c a i n l d e g l u c u r o n l d e i n t e r m e d i a t e s . 103 8. The R e a c t i o n of T o c a i n l d e wi th Urea a t D i f f e r e n t T e m p e r a t u r e s . I l l 9 . The R e a c t i o n of T o c a i n l d e w i t h Urea at D i f f e r e n t pH. 112 10. Summarized d a t a from the 400-MHz p r o t o n NMR spectrum of t o c a i n l d e . 125 11. Summarized d a t a from the 400-MHz p r o t o n NMR spectrum of the I s o l a t e d t o c a l n i d e carbamoyl e s t e r g l u c u r o n i d e . 127 12. C a l i b r a t i o n curve d a t a f o r t o c a i n l d e and the h y d a n t o i n u s i n g e t i d o c a i n e and the 4 ' - m e t h y l h y d a n t o i n i n t e r n a l s t a n d a r d s . 142 13. I n t r a - a s s a y v a r i a b i l i t y of t o c a l n i d e and the h y d a n t o i n a t 6, 15, 20 ug/mL c o n c e n t r a t i o n . 143 14. I n t e r - a s s a y v a r i a b i l i t y of t o c a i n l d e and the h y d a n t o i n a t 6, 15, 20 ug/mL c o n c e n t r a t i o n . 143 15. E x t r a c t i o n e f f i c i e n c e s of t o c a i n l d e and the h y d a n t o i n in methylene c h l o r i d e . 144 x L i s t of T a b l e s (Contd) Page 16. U r i n a r y e x c r e t i o n r a t e s of t o c a l n i d e and the h y d a n t o i n a f t e r a 200 mg IV dose of t o c a i n l d e HC1 i n a h e a l t h y human v o l u n t e e r . 146 17. U r i n a r y e x c r e t i o n r a t e s of t o c a i n l d e and the h y d a n t o i n a f t e r a 200 mg o r a l dose of t o c a i n l d e HC1 in a h e a l t h y human v o l u n t e e r . 147 18. The summarized p h a r m a c o k i n e t i c d a t a f o r t o c a i n l d e and the h y d a n t o i n In three h e a l t h y human v o l u n t e e r s a f t e r both an IV and o r a l dose . 148 x i L i s t of Figures Page 1. The metabolism of tocainide in humans. 10 2. The metabolism of tocainide in r a t s . 14 3. The total- ion-chromatogram of crude deuterated 2,6-dimethylani l i ne product. 65 4. The mass spectrum of crude deuterated 2,6-dimethylani l ine at scan 51. 65 5. The mass spectrum of crude deuterated 2,6-dimethylani l ine at scan 61. °6 6. The mass spectrum of unlabel led 2,6-dime thy lan i1 ine . 66 7. The total- ion-chromatogram of d i s t i l l e d D3-2,6-dimethylani1 ine. 69 8. The mass spectrum of d i s t i l l e d D3-2,6-dimethylani1ine. 69 9. 80-MHz-NMR Spectrum of D -2 ,6-dimethylani1 ine <CDC1 ) . 70 10. 80-MHz-NMR Spectrum of D -2 ,6-dimethylani1 ine a f te r D 0 exchange. 70 11. The infared spectrum of synthet ic tocainide (mul l ) . 72 12. The total- ion-chromatogram of synthet ic toca in ide . 74 13. The EI mass spectrum of synthet ic t o c a i n i d e . 74 14. The infared spectrum of N-carbobenzy1oxy-alanine (mul l ) . 76 15. The total- ion-chromatogram of synthet ic 2-ethanimine t o c a i n i d e . 78 16. The EI mass spectrum of 2-ethanimine t o c a i n i d e . 78 17. The infared spectrum of tocainide ethanimine (mul l ) . 79 18. The infared spectrum of 3 - (2 ,6 -xy ly l ) -5 -methylhydantoin (mul l ) . 80 19. Total-ion-chromatogram of synthet ic 3 - ( 2 , 6 - x y l y l ) - 5 -methylhydantoin. 82 x i i L i s t of F i g u r e s (Contd) Page 20. The EI mass spectrum of the s y n t h e t i c 3 - ( 2 , 6 - x y 1 y 1 ) -5 - m e t h y l h y d a n t o i n . 83 21. 400-MHz-[ H1-NMR Spectrum of the s y n t h e t i c 3 - ( 2 , 6 -x y l y l ) - 5 - m e t h y l h y d a n t o i n (CDCl ) . 83 22. The i n f a r e d spectrum of 3 - ( 2 , 4 , 6 - x y 1 y 1 ) - 5 -m e t h y l h y d a n t o i n ( m u l l ) . 85 23. T o t a l - i o n - c h r o m a t o g r a m of the s y n t h e t i c 3 - ( 2 , 4 , 6 -x y l y l ) - 5 - m e t h y l h y d a n t o i n . 86 24. The EI mass spectrum of the s y n t h e t i c 3 - ( 2 , 4 , 6 - x y 1 y 1 ) -5 - m e t h y l h y d a n t o i n . 86 25. The 80-MHz-[ H]-NMR spectrum of the s y n t h e t i c 3 - ( 2 , 4 , 6 -x y l y l ) - 5 - m e t h y l h y d a n t o i n (CDCl ) . 88 26. The CI mass spectrum of the T M S - d e r i v a t i v e of g l u c o s e . 91 27. The CI mass spectrum of the T M S - d e r i v a t i v e of p -n i t r o p h e n o l g l u c u r o n i d e . 91 28. T o t a l - i o n - c h r o m a t o g r a m of p e r m e t h y l a t e d XAD/crude u r i n e e x t r a c t i s o l a t e d band lb sample . 92 29. Mass spectrum of p e r m e t h y l a t e d t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e ( e l e c t r o n impact i o n i z a t i o n ) . 93 30. F r a g m e n t a t i o n p a t t e r n of the proposed t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e . 93 31. T o t a l - i o n - c h r o m a t o g r a m of p e r m e t h y l a t e d p a r a -n i t r o p h e n o l g l u c u r o n i d e . 95 32. The CI mass spectrum of the p e r m e t h y l a t e d p a r a n i t r o p h e n o l g l u c u r o n i d e . 95 33. The t o t a l - i o n - c h r o m a t o g r a m of p e r m e t h y l a t e d t o c a i n i d e . 100 34. The EI mass spectrum of p e r m e t h y l a t e d t o c a i n i d e . 100 35. The EI mass spectrum of p e r m e t h y l a t e d 2 ,6 -d i m e t h y l a n i 1 i n e . 101 36. The t o t a l - i o n - c h r o m a t o g r a m of p e r m e t h y l a t e d l a c t o x y l i d i d e . 102 x i i i L i s t o f F igures (Contd) Page 37. The EI mass spectrum of the permethylated l a c t o x y l i d ide. 102 38. The total- ion-chromatogram of the permethylated 3-(2 ,6 -xy ly l ) -5 -methy lhydanto in . 104 39. The EI mass spectrum of the permethylated 3- (2,6-xyly l ) -5-methylhydantoIn. 104 40. Total- ion-chromatogram of permethylated ur ine . 108 41. The EI spectrum of permethylated tocainide carbamoyl es ter glucuronide ( C H 3 I ) . 108 42. The EI fragmentation pattern of permethylated tocain ide carbamoyl ester g lucuronide. 109 43. The EI mass spectrum of permethylated tocainide carbamoyl ester glucuronide (CD3D. 110 44. T o t a l - 1on-chromatogram of permethylated toca ln lde -N-ure ide . 114 45. The EI mass spectrum of permethylated toca ln lde -N -ure ide . 114 46. The total-1on-chromatogram of the permethylated 2,6-d imethylani1 ine-N-glucuronide. 115 47. The EI mass spectrum of permethylated 2,6-d imethylani l ine-N-g lucuronIde . 115 48. Preparat ive HPLC chromatogram of crude ur ine . 118 49. The UV absorbance of the 2 mL f rac t ions c o l l e c t e d from preparat ive HPLC. 118 50. Preparat ive HPLC chromatogram of recyc led tocalnlde TOCG conta in ing f r a c t i o n . 120 51. Preparat ive HPLC chromatogram of recyc led TOCG In sodium phosphate s a l t . 120 52. Preparat ive HPLC chromatogram of recycled TOCG In sodium c h l o r i d e . 121 53. 400-MHz-[ 1Hl-NMR Spectrum of tocalnlde (D,-DMSO). 123 x i v L i s t of Figures (Contd) Page 54. 400-MHz-[ 1 H1-NMR Spectrum of t o c a i n l d e a f t e r D 9 0 exchange <D 6~DMSO). • 123 55. The 400-MHz p r o t o n NMR spectrum of g l u c u r o n i c a c i d . 124 56. 400-MHz-[ lHl -NMR Spectrum of t o c a i n l d e carbamoyl e s t e r g l u c u r n o i d e (D -DMSO). 126 6 57. 400-MHz- t lHl -NMR Spectrum of t o c a i n i d e carbamoyl e s t e r g l u c u r o n l d e <8 X i n t e n s 1 t y / D , - D M S O ) . 126 o 58. The f a s t atom bombardment spectrum of t o c a i n l d e carbamoyl e s t e r g l u c u r o n l d e in g l y c e r o l matr ix w i th sodium c h l o r i d e . 129 59. The f a s t atom bombardment spectrum of t o c a i n i d e carbamoyl e s t e r g l u c u r o n l d e in t h l o g l y c e r o l matr ix w i t h sodium c h l o r i d e . 129 60. The f r a g m e n t a t i o n p a t t e r n of t o c a i n l d e carbamoyl e s t e r g l u c u r o n i d e In f a s t atom bombardment mass s p e c t r o m e t r y . 130 61. The f a s t atom bombardment spectrum of t o c a l n i d e carbamoyl e s t e r g l u c u r o n l d e in t h i o g l y c e r o l matr ix w i th low mass ion m o n i t o r i n g . 131 62. The h y d r o l y s i s p r o f i l e of t o c a l n i d e g l u c u r o n i d e and the h y d a n t o i n in the presence of sodium h y d r o x i d e . 134 63. S e m i - l o g p l o t of the t o c a l n i d e g l u c u r o n l d e and the h y d a n t o i n h y d r o l y s i s k i n e t i c s in v a r i o u s c o n c e n t r a t i o n s of sodium h y d r o x i d e . 135 64. S e m i - l o g p l o t of the h y d r o l y s i s of 3 - < 2 , 6 - x y l y l ) - 5 -methylhydantoIn In v a r i o u s c o n c e n t r a t i o n s of sodium h y d r o x i d e . 137 65. The h y d r o l y s i s of t o c a i n i d e g l u c u r o n i d e and the 4 ' -m e t h y l h y d a n t o i n in the presence of 0.5 M sodium h y d r o x i d e . 138 xv L i s t of F i g u r e s (Contd) Page 66. T h e o r e t i c a l p r o f i l e s of the 3 - ( 2 , 6 - x y l y l ) - 5 -m e t h y l h y d a n t o i n h y d r o l y s i s u s i n g both e t i d o c a i n e and 3 - ( 2 , 4 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n as i n t e r n a l s t a n d a r d . A: Peak a r e a - t i m e p r o f i l e s of the h y d a n t o i n and the 4 ' - m e t h y l h y d a n t o i n . B: The peak a r e a r a t i o of the h y d a n t o i n to the 4 * - m e t h y l h y d a n t o i n ( I . S . ) . C: Peak a r e a - t i m e p r o f i l e s of e t i d o c a i n e and the h y d a n t o i n . D: The peak a r e a r a t i o of the h y d a n t o i n to e t i d o c a i n e ( I . S . ) in a t h e o r e t i c a l TOCG h y d r o l y s i s p r o f i l e . 140 67. The s e m i - l o g p l o t of the u r i n a r y e x c r e t i o n p r o f i l e of t o c a i n i d e (a) and the h y d a n t o i n (b) a f t e r r e c e i v i n g a 200 mg IV dose of t o c a i n i d e . 150 68. The s e m i - l o g p l o t of the u r i n a r y e x c r e t i o n p r o f i l e of t o c a i n i d e (a) and the h y d a n t o i n (b) a f t e r r e c e i v i n g a 200 mg o r a l dose of t o c a i n i d e . 152 x v i L i s t of Structures Page I. Toca i n ide. 2 II. L idoca ine. 2 I l l . Tocainide carbamoyl ester g lucuronide. 10 IV. Lac toxy l id ide. 10 V. Tocainide Oxime. 10 VI . 3- (2,6 -Xyly l ) -5 -methylhydanto in 10 VII . Tocainide carbamic a c i d . 12 VIII . Tocainide aldehyde adduct. 14 IX. Oxidative deaminated product. 14 X. 4'-Hydroxytoca in ide 13 XI . Glucuronic ac id conjugate of l a c t o x y l i dide . 13 XII . 2 ,6-dime thylani1ine EI ion fragment at m/z 95. 67 XIII . 2 ,6 -dimethylani1ine EI ion fragment at m/z 80. 67 XIV. 2 ,6-dime thylani1 ine EI ion fragment at m/z 124. 67 XV. 2 ,6 -dimethylani1ine EI ion fragment at m/z 109. 67 x v i i L i s t of Schemes Page 1. V a r i o u s ev idence f o r the presence of a nove l t o c a l n i d e carbamoyl e s t e r g l u c u r o n l d e . 11 2. The proposed m e t a b o l i c pathway l e a d i n g to the f o r m a t i o n of t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e . 12 3. Pathways of ammonia metabo l i sm. ASA, a r g i n i n o s u c c i n l c a c i d ; OMP, b r o t l d i n e monophosphate; IMP, u r i d i n e monophosphate. 18 4. The t h e o r e t i c a l e q u i l i b r i u m r e a c t i o n s of t o c a i n i d e carbamate . 19 5. The mechanisms of carbamic a c i d c y c l l z a t i o n to 3 - < 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o l n . 20 6. The Isomeric t r a n s f o r m a t i o n of u r e a . 21 7. The mechanisms of t o c a l n l d e - u r e I d e f o r m a t i o n . 22 8. The mechanisms of t o c a l n l d e - u r e i d e c y c l l z a t i o n to 3 - ( 2 , 6 - x y l y l > - 5 - m e t h y l h y d a n t o i n . 23 9. Attempted i d e n t i f i c a t i o n of p e r m e t h y l a t e d u r i n a r y t o c a i n l d e m e t a b o l i t e s . 44 10. Attempted i d e n t i f i c a t i o n of u r i n a r y t o c a i n l d e m e t a b o l i t e s by 1yoph11lzat1 on and GCMS a n a l y s i s . 48 11. Attempted I s o l a t i o n and i d e n t i f i c a t i o n of t o c a i n l d e N - c a r b a m i c a c i d or N - u r e i d e i n t e r m e d i a t e . 49 12. Attempted i s o l a t i o n and I d e n t i f i c a t i o n of u r i n a r y m e t a b o l i t e s by f l a s h chromatography . 50 13. The s y n t h e t i c pathway f o r t r i d e u t e r a t e d t o c a i n l d e . 63 14. The s y n t h e s i s of N - c a r b o b e n z y l o x y a l a n i n e . 7 5 15. The t h e o r e t i c a l t o c a i n l d e i n t e r m e d i a t e s d e r i v e d from the h y d r o l y s i s of the t o c a l n i d e g l u c u r o n l d e . 99 16. The e x p e r i m e n t a l s teps i n v o l v e d in i s o l a t i n g the t o c a i n l d e g l u c u r o n i d e . 117 17. Schemat ic r e p r e s e n t a t i o n of the k i n e t i c s of TOCG h y d r o l y s i s In the presence of NaOH. 133 x v i i i L i s t of Schemes Page 18. Schemat ic r e p r e s e n t a t i o n of the e l i m i n a t i o n of t o c a i n i d e in humans. 151 x i x L i s t o f P h a r m a c o k i n e t i c E q u a t i o n s Log d X u = Log k X ° - K E (t .,) dt~ 2303 m l d dX -K Terminal slope of the plot Log u vs. t . , = E ST M I D ^ T 3 T J 3 Y-intercept of plot Log d X u vs. t . , = Log k X,,0  r r b j ^ — mid & e B where k g = urinary excretion rate of tocainide. Xg°= dose of tocainide. Log ^ u = Log k k.X ° - k (t . ,) & -rr— & mu f B __mu_ mid Q U k~ K l 2.303 mu - E the slope of the above equation as obtained by feathering = -k & mu 2.303 assuming K £ Log dM = Log k k. X ° - K„ (t . ,) & u 6 mu f B E mid dt "Tc - K_ 2.303 mu E Y-intercept of plot log dM vs. t . , = Log k k^X^ r r & u m l c j & mu f B o dt k K Z mu - E where k = urinary excretion rate of TOCG mu J Xg° = dose of tocainide i n body K„ = t o t a l elimination of tocainide E X 0 0 = X ° k u B e K E M k r = r a t i o of dose excreted as metabolite u f TT— i n urine. (F)dose K £ xx Symbols and A b b r e v i a t i o n s ACN a c e t o n i t r i l e BSTFA BIs( t r lmethyls I ly l ) t r i f1uoroacetarnide CBZC1 carbobenzyloxyl ch lor ide C D C I 3 deuterated chloroform CD3I deuterated methyl iodide CH 3I methyl iodide CI chemical ion iza t ion CNS centra l nervous system C.V. % c o e f f i c i e n t of va r ia t ion D5-DMSO deuterated dimethyl su l fox ide DMSO dimethyl sul foxide D2O deuterium oxide D2SO4 deuterated su lphur ic ac id EI e lec t ron Impact Ionizat ion FAB fast atom bombardment GA g lucuronic ac id GC gas chromatography GCMS gas chromatography Interfaced with mass spectrome try HBr hydrogen bromide HC1 hydrochlor ic ac id HFBA heptaf1uorobutyic anhydride HPLC high performance l i q u i d chromatography HYD 3-<2,6-xylyl ) -5-methylhydantoin IR infared spectroscopy IV intravenous xx i 8ymbols and Abbreviations (Contd) J kmu M + m/z M u ° ° N a H 2 P 0 4 NMR ODS ppm PTFE t e f l o n PVC R f RP TCF TIC TLC TMS TOC TOCG t 1/2 UV Xu oo c o u p l i n g c o n s t a n t e l i m i n a t i o n r a t e c o n s t a n t ( t o c a i n l d e ) u r i n a r y e x c r e t i o n r a t e c o n s t a n t ( t o c a l n i d e ) f o r m a t i o n r a t e c o n s t a n t (TOCG) u r i n a r y e x c r e t i o n r a t e c o n s t a n t (TOCG) m o l e c u l a r ion mass to charge r a t i o t o t a l amount of TOCG e x c r e t e d in u r i n e sodium phosphate monobasic n u c l e a r magnetic resonance o c t a d e c y l s 1 lane p a r t s per m i l l i o n p o l y t e t r a f l u o r o e t h y l e n e t e f l o n premature v e n t r i c u l a r c o n t r a c t l n r e t e n t i o n f a c t o r r e v e r s e - p h a s e t r i c h l o r o m e t h y l c h l o r o f o r m a t e t o t a l - 1 o n - c h r o m a t o g r a m t h i n l a y e r chromatography t r i methyls i 1 y l tocaInIde t o c a i n l d e carbamoyl e s t e r g l u c u r o n l d e h a l f - 1 I f e u l t r a v l o l e t t o t a l amount of t o c a l n i d e e x c r e t e d In u r i n e x x x i Acknowledgements I would l i k e to express my a p p r e c i a t i o n to my t h e s i s s u p e r v i s o r , Dr. K e i t h McErlane, f o r h i s k i n d encouragement and support d u r i n g the course of these s t u d i e s . The h e l p f u l d i s c u s s i o n s and c r i t i c i s m s from the other members of my r e s e a r c h Committee, Dr. Frank Abbott, Dr. James Axelson, Dr. Helen Burt, Dr. Charles Kerr and Dr. John S i n c l a i r (Chairman), are a l s o g r a t e f u l l y acknowledged. Many thanks to Roland Burton f o r h i s help with GCMS ins t r u m e n t a t i o n , and to Greg S l a t t e r f o r h i s advice on NMR i n t e r p r e t a t i o n . The a s s i s t a n c e from the s t a f f at the NMR and mass spectrometry f a c i l i t i e s in the Department of Chemistry i s a l s o a p p r e c i a t e d . S p e c i a l thanks to Kathleen Cheng f o r the p r o d u c t i o n of t h i s t h e s i s . T h i s work would not have been p o s s i b l e without the Research Trainee S c h o l a r s h i p provided by the B.C. Heart Foundation, and a grant from the B.C. Hea l t h Care Research Foundation. L a s t l y , I s i n c e r e l y thank my parents f o r t h e i r understanding and encouragement throughout the years of my academic s t u d i e s . x x i i i 1 INTRODUCTION 1 A. B r i e f Overview of T o c a i n i d e T o c a i n i d e , 2-amino-2',6'-propionoxylidide (I) i s a new a n t i a r r h y t h m i c agent developed by A s t r a P h a r m a c e u t i c a l s . I t i s marketed in Canada as the h y d r o c h l o r i d e s a l t (Tonocard ) in 400 mg and 600 mg t a b l e t s . The usual recommended dosing regimen i s 400 mg to 600 mg every 8 hours up to 800 mg every 8 hours i f necessary. At present, l i d o c a i n e ( I I ) i s the agent of choice f o r the treatment of acute myocardial i n f a r c t i o n . However, l i d o c a i n e e x h i b i t s f i r s t - p a s s metabolism in the l i v e r , thus r e n d e r i n g i t s o r a l b i o a v a i l a b i l i t y h i g h l y v a r i a b l e . U n l i k e l i d o c a i n e , t o c a i n i d e i s a primary amine analog which o f f e r s the advantage of both o r a l and intravenous routes of a d m i n i s t r a t i o n . B. E l e c t r o p h y s i o l o g i c a l P r o p e r t i e s A c c o r d i n g to Vaughan W i l l i a m ' s c l a s s i f i c a t i o n of a n t i a r r h y t h m i c drugs, c l a s s I compounds such as q u i n i d i n e , procainamide, disopyramide, phenytoin and l i d o c a i n e , a l l possess a membrane-stabilizing a c t i o n which i n t e r f e r e s with sodium i n f l u x , thereby d e c r e a s i n g the r a t e of r i s e in phase zero of the c a r d i a c a c t i o n p o t e n t i a l ( 1 ) . In a d d i t i o n to t h i s slowing of the d e p o l a r i z a t i o n r a t e , t o c a i n i d e can a l s o decrease impulse (I) (II) 2 e x c i t a b i l i t y and a u t o m a t i c i t y in i s o l a t e d dog P u r k i n j e f i b e r s ( 2 ) . These e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s a l l c o n t r i b u t e to the a n t i a r r h y t h m i c a c t i v i t y of t o c a i n i d e ; C. Pharmacodvnamics 1 • C l i n i c a l E f f i c a c y The pre-marketing c l i n i c a l experience with t o c a i n i d e in the U n i t e d S t a t e s can be summarized as f o l l o w s . McDevitt and co-workers f i r s t r e p o r t e d a 60 % r e d u c t i o n i n premature v e n t r i c u l a r c o n t r a c t i o n s (PVCs) in 5 out of 7 p a t i e n t s r e c e i v i n g more than 100 mg o r a l t o c a i n i d e ( 3 ) . Winkle and co-workers (4) subsequently demonstrated a 91 % + 10 % r e d u c t i o n i n PVCs in 11 out of 15 p a t i e n t s with o r a l t o c a i n i d e therapy. Woosley and co-workers (5) l a t e r documented over a 75 % s u p p r e s s i o n of PVCs in 8 out of 12 p a t i e n t s with v e n t r i c u l a r e c t o p i c a c t i v i t y . 2. E f f i c a c y i n R e f r a c t o r y Arrhythmias To c a i n i d e was found to be e f f e c t i v e in s u p p r e s s i n g v e n t r i c u l a r e c t o p i c a c t i v i t y in 15 out of 19 p a t i e n t s r e f r a c t o r y to c o n v e n t i o n a l therapy with p r o p r a n o l o l , q u i n i d i n e or procainamide <6). S i m i l a r f i n d i n g s were r e p o r t e d by Ryan, W. ( 7 ) , H a f f a j e e , C . I . ( 8 ) , and Moloney, J.D. ( 9 ) . 3. P r o p h y l a c t i c A n t i a r r h y t h m i c Therapy D o u b l e - b l i n d placebo s t u d i e s were c a r r i e d out to evaluate the u s e f u l n e s s of t o c a i n i d e in the p r o p h y l a c t i c therapy of v e n t r i c u l a r arrhythmias a f t e r myocardial i n f a r c t i o n (10-12). The r e s u l t s of these s t u d i e s showed.some p r e l i m i n a r y b e n e f i c i a l e f f e c t s of t o c a i n i d e in a b o l i s h i n g v e n t r i c u l a r t a c h y c a r d i a in p a t i e n t s with acute myocardial i n f a r c t i o n , but f u r t h e r study in 3 t h i s area would be r e q u i r e d . At present, t o c a i n i d e i s proven to be an e f f e c t i v e a n t i a r r h y t h m i c agent. D. Pharmacokinetics and Pharmacodynamics 1. B i o a v a i l a b i 1 i t v In h e a l t h y human v o l u n t e e r s , o r a l t o c a i n i d e h y d r o c h l o r i d e was well absorbed with 90 to 100 % b i o a v a i l a b i l i t y (13). A f t e r a 400 mg dose of t o c a i n i d e h y d r o c h l o r i d e , peak plasma l e v e l s of 1.82 ug/mL were achieved in 1 to 2 hours in f a s t i n g s u b j e c t s . A d m i n i s t r a t i o n of the drug with food decreased peak l e v e l s and delayed the r a t e of a b s o r p t i o n , but the o v e r a l l b i o a v a i l a b i l i t y (13) was not a f f e c t e d . 2. D i s p o s i t i o n K i n e t i c s F o l l o w i n g zero-order i n f u s i o n of 300 mg of t o c a i n i d e over a 30 minute p e r i o d , the peak plasma l e v e l was found to be 2.5 ug/mL. The b i e x p o n e n t i a l plasma-drug c o n c e n t r a t i o n p r o f i l e was c h a r a c t e r i z e d by a r a p i d d i s t r i b u t i o n phase and a slower t e r m i n a l e l i m i n a t i o n phase, with h a l f - l i v e s of 0.18 hour and 11.5 hours r e s p e c t i v e l y (13). The apparent volume of d i s t r i b u t i o n at steady s t a t e was found to vary between 1.46 L/Kg to 2.9 L/Kg in he a l t h y s u b j e c t (13,14). At steady s t a t e , 30 to 40 % of an ad m i n i s t e r e d dose was ex c r e t e d unchanged in the uri n e with an average r e n a l c l e a r a n c e of 70 mL/min (14,15). 3. Dose-response Pharmacodynamics For t o c a i n i d e , the plasma-drug c o n c e n t r a t i o n - a n t i a r r h y t h m i c response r e l a t i o n s h i p was found to e x h i b i t a l a r g e degree of i n t e r s u b j e c t v a r i a b i l i t y (16). The r e p o r t e d e f f e c t i v e t h e r a p e u t i c plasma c o n c e n t r a t i o n s of t o c a i n i d e v a r i e d among 4 s e v e r a l s t u d i e s (3,15,16), but the accepted e f f e c t i v e range i s c o n s i d e r e d to be from 4 to 10 ug/mL. An average s t e a d y - s t a t e p l a s m a - t o c a i n i d e c o n c e n t r a t i o n of 1.7 ug/mL was reached a f t e r an o r a l dosage regimen of 100 mg every 8 hours (15). V e n t r i c u l a r e c t o p i c beats were suppressed i n 70 % of the p a t i e n t s t a k i n g t o c a i n i d e 400 mg o r a l l y every 8 hours with an average plasma c o n c e n t r a t i o n of 6 ug/mL. Doses g r e a t e r than 800 mg every 8 hours were seldom t o l e r a t e d due to adverse CNS e f f e c t s ( 3 ) . 4. Serum P r o t e i n B i n d i n g L a l k a (13) f i r s t r e p o r t e d that t o c a i n i d e was 50 % bound to plasma p r o t e i n s at c l i n i c a l l y e f f e c t i v e c o n c e n t r a t i o n s . E l v i n (18) l a t e r r e p o r t e d a b i n d i n g of only 4 to 12 % among 10 h e a l t h y human v o l u n t e e r s . In trauma p a t i e n t s with e l e v a t e d a l p h a - 1 - a c i d g l y c o p r o t e i n s , the percentage of drug bound was found to range from 10 to 20 %. Sedman (18) l a t e r examined the s t e r e o s e l e c t i v e b i n d i n g of t o c a i n i d e enantiomers. The extent of b i n d i n g of the S ( + ) - t o c a i n i d e enantiomer was s l i g h t l y g r e a t e r than that of the R(-)-enantiomer. Between the t h e r a p e u t i c c o n c e n t r a t i o n range of 4-12 ug/mL, there were no s i g n i f i c a n t changes i n the f r a c t i o n bound f o r the two enantiomers, suggesting dose-independent b i n d i n g . N e v e r t h e l e s s , the high f r e e f r a c t i o n of t o c a i n i d e in plasma i s not l i k e l y to c r e a t e c l i n i c a l l y s i g n i f i c a n t b i n d i n g displacement i n t e r a c t i o n s . 5. The E f f e c t s of M e t a b o l i c I n d u c t i o n on K i n e t i c s Animal models were used to examine the e f f e c t s of metabolic Induction and i n h i b i t i o n on the d i s p o s i t i o n of t o c a i n i d e (19,20). In the r a t , with p h e n o b a r b i t a l pre treatment, 5 a s i g n i f i c a n t r e d u c t i o n i n the area under the t o c a i n i d e - p l a s m a c o n c e n t r a t i o n versus time curve was observed a f t e r an intravenous dose. Pretreatment with SKF 525A, a known i n h i b i t o r of the enzyme r e s p o n s i b l e f o r g l u c u r o n l d e c o n j u g a t i o n , was found to impair the e l i m i n a t i o n of t o c a i n i d e as r e f l e c t e d by a r e d u c t i o n in c l e a r a n c e <19>. However, s t u d i e s in man f a i l e d to d e t e c t any a l t e r a t i o n i n the percent of unchanged drug recovered in the urin e a f t e r pretreatment with p h e n o b a r b i t a l , s a l i c y l a m I d e , or c l o f i b r a t e (21). P h e n o b a r b i t a l i s a known enzyme inducer whereas s a l i c y l a m l d e Is a potent I n h i b i t o r of the g l u c u r o n l d a t i o n of some drugs. C l o f i b r a t e i s known to form m e t a b o l i t e s t h a t may be c o m p e t i t i v e i n h i b i t o r s of the g l u c u r o n i c a c i d c o n j u g a t i o n pathway. However, none of these agents have a f f e c t e d the u r i n a r y e x c r e t i o n of the g l u c u r o n i c a c i d conjugate of t o c a i n i d e i n man. E. S t e r e o s e l e c t i v i t y in Drug Metabolism T o c a i n i d e i s a c h i r a l compound which can e x i s t as two o p t i c a l l y a c t i v e enantiomers, R ( - ) - and S ( + ) - t o c a i n i d e . I t i s well r e c o g n i z e d that drug a b s o r p t i o n , d i s t r i b u t i o n , metabolism and e x c r e t i o n may a l l e x h i b i t s t e r e o s e l e c t i v i t y or s t e r e o -s p e c i f i c i t y , p a r t i c u l a r l y so when the process i n v o l v e s enzyme transformat i on. To c a i n i d e i s marketed as a racemic mixture. In the l i t e r a t u r e , there are numerous examples of an s t e r e o s e l e c t i v e drug d i s t r i b u t i o n and metabolism. An ext e n s i v e review on ste r e o c h e m i c a l drug d i s p o s i t i o n was p u b l i s h e d by Jenner and Testa ( 2 3 ) . For i n s t a n c e , the enantiomeric d i f f e r e n c e s i n d i s t r i b u t i o n and metabolism of p r o p r a n o l o l have been well 6 s t u d i e d . In humans, racemic p r o p r a n o l o l was shown to have a lower c l e a r a n c e than that of the v a s o i n a c t i v e R(+)-enantiomer (24). S t e r e o s e l e c t i v i t y i n aromatic h y d r o x y l a t i o n , s i d e c h a i n h y d r o x y l a t i o n , and g l u c u r o n i d a t i o n were a l s o r e p o r t e d f o r p r o p r a n o l o l (25,26). Furthermore, s t e r e o s e l e c t i v i t y in the b i o a v a i l a b i l i t y of p r o p r a n o l o l was a l s o r e p o r t e d (25). The R(+)-enantiomer was found to have a lower o r a l b i o a v a i l a b i l i t y than the p h a r m a c o l o g i c a l l y more a c t i v e S(-)-enantiomer. A l s o , s t e r e o s e l e c t i v e b i n d i n g of p r o p r a n o l o l to a l p h a - l - a c i d g l y c o p r o t e i n was r e p o r t e d (27). The S ( - ) - p r o p r a n o l o l was found to have a longer plasma h a l f - l i f e than the racemate in the r a b b i t (28). R e c e n t l y , i t was d i s c o v e r e d that the v a s o a c t i v e S(-)-enantiomer of p r o p r a n o l o l has g r e a t e r accumulation in the heart and b r a i n of the r a t (29). These s t u d i e s have e s t a b l i s h e d the s t e r e o s e l e c t i v e d i f f e r e n c e s i n the d i s p o s i t i o n of the two enantiomers of p r o p r a n o l o l i n both humans and experimental an i mals. In l i g h t of a p o s s i b l e s t e r e o s e l e c t i v e r o l e i n the d i s p o s i t i o n of t o c a i n i d e , Gal and co-workers f i r s t s t u d i e d the d i s p o s i t i o n of ( R , S ) - t o c a i n i d e i n r a t s and mice (30). S(+)-t o c a i n i d e was r e p o r t e d in r a t urine at h i g h e r l e v e l s than the (R)-antipode. However, i n the mouse model, t h i s o b s e r v a t i o n was r e v e r s e d . The enantiomers of t o c a i n i d e have been s u b j e c t e d to pharmacological e v a l u a t i o n . The R(-)-enantiomer was found to be three times more potent than the S(+)-isomer i n the c h l o r o f o r m -induced arrhythmic model in the mouse (31), but the d i f f e r e n c e in a n t i a r r h y t h m i c a c t i v i t i e s between the two enantiomers was s m a l l e r 7 i n coronary-1 igated dogs ( 3 D . McErlane and P i l l a i <32) f i r s t r e p o r t e d the s t e r e o s e l e c t i v e d i s p o s i t i o n of t o c a i n i d e i n man. A s i m i l a r study i n t o the s t e r e o s e l e c t i v e d i s p o s i t i o n of t o c a i n i d e in man r e v e a l e d an e l i m i n a t i o n h a l f - l i f e f o r R ( - ) - t o c a i n i d e and S ( + ) - t o c a i n i d e to be 10.0 and 16.7 hours r e s p e c t i v e l y (33). The h a l f - l i f e f o r t o c a i n i d e carbamoyl g l u c u r o n i d e was found to be 10.3 hours su g g e s t i n g that t h i s g l u c u r o n i c a c i d conjugate may have p r e f e r a b l y formed from the R ( - ) - t o c a i n i d e . The s t e r e o s e l e c t i v e d i s p o s i t i o n of t o c a i n i d e enantiomers into s a l i v a was r e p o r t e d by P i l l a i et ai.<34>. The s a l i v a l e v e l s of the i n d i v i d u a l enantiomers were measured a f t e r an intravenous dose of t o c a i n i d e . The s t e r e o s e l e c t i v e d i s p o s i t i o n p r o f i l e s of the enantiomers in s a l i v a were compared with those in plasma and u r i n e . F o l l o w i n g an o r a l dose of 200 mg t o c a i n i d e h y d r o c h l o r i d e , the mean enantiomer c o n c e n t r a t i o n r a t i o of S<+>-tocainide/R(->-t o c a i n i d e i n the plasma was 1.52 at 24 hours. S i m i l a r e n a n t i o m e r i c e x c r e t i o n p r o f i l e s were observed in the u r i n e . However, in s a l i v a , the enantiomer c o n c e n t r a t i o n r a t i o was d i f f e r e n t in that the R(-)-enantiomer c o n c e n t r a t i o n was higher than t h a t of the S(+)-enantiomer, and moreover, the l e v e l s of both enantiomers were higher than those found in the c o r r e s p o n d i n g plasma samples. The authors suggested that a s t e r e o s e l e c t i v e pH-independent e x c r e t i o n process was r e s p o n s i b l e f o r the r a p i d d i s t r i b u t i o n of the R(-)-enantiomer i n t o the s a l i va. 8 In humans, 28 to 55 % of an o r a l dose of t o c a i n i d e was e x c r e t e d unchanged in the u r i n e i n 24 hours (35,36). I t was suggested that another 15 to 30 % of the dose was metabolized v i a a novel pathway i n v o l v i n g the a d d i t i o n of carbon d i o x i d e to the primary amine n i t r o g e n , f o l l o w e d by c o n j u g a t i o n with g l u c u r o n i c a c i d to form t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e ( I I I ) (35). Another 1 to 2 % of the dose was i d e n t i f i e d as a deaminated m e t a b o l i t e , l a c t o x y l i d i d e ( I V ) , which c o u l d undergo f u r t h e r metabolism (36). A t r a c e m e t a b o l i t e , t o c a i n i d e oxime (V) has a l s o been i d e n t i f i e d (37). The major m e t a b o l i t e , t o c a i n i d e carbamoyl-0-p-D-glucuronide was r e p o r t e d to c y c l i z e at pH > 12 to form 3-(2,6-xylyl)-5-methy1hydantoin (VI) (35,37) as shown in F i g u r e 1. However, in a d d i t i o n to the c o n j u g a t i o n pathway, t h i s hydantoin metabolite was a l s o r e p o r t e d to form v i a a carbamic a c i d - u r e a intermediate as a metabonate in r a t s (38). 9 10 1• O b s e r v a t i o n of a Maior Novel M e t a b o l i t e of T o c a i n i d e In 1979, E l v i n and co-workers (35) i d e n t i f i e d a novel g l u c u r o n i c a c i d conjugate of t o c a i n i d e . The b a s i s f o r e l u c i d a t i n g the s t r u c t u r e of t h i s conjugated metabolite i s d e s c r i b e d in Scheme 1. A: A c i d h y d r o l y s i s 1 M HC1/100°C. B: Beta Glucuronidase h y d r o l y s i s . C: Basic h y d r o l y s i s 1 M NaOH (pH 12). D: Heat (100°C) . The Hovel Tocainlde Metabolite Tocainlde Hydantoin Scheme 1 : V a r i o u s e v i d e n c e f o r the p r e s e n c e o f a n o v e l  t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e . A c i d h y d r o l y s i s (HC1) of urine samples produced f r e e t o c a i n i d e , probably r e l e a s e d from a conjugated m e t a b o l i t e . H y d r o l y s i s experiments with b e t a - g l u c u r o n i d a s e produced s i m i l a r amounts of free t o c a i n i d e r e l e a s e d l i k e l y from a g l u c u r o n i c a c i d conj ugate. 11 The i n i t i a l i n t e r p r e t a t i o n of these data was to hypothesize a N - g l u c u r o n i c a c i d conjugate of t o c a i n i d e . However, the i n s t a b i l i t y of t h i s N-glucuronide i n a l k a l i n e s o l u t i o n , forming a h y d r o l y z e d product 3 - ( 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n , suggested that the s t r u c t u r e of t h i s novel metabolite c o n t a i n e d a carbonyl on the primary amine p o s s i b l y as a carbamoyl e s t e r l i n kage with g l u c u r o n i c a c i d . Based on the proposed carbamoyl e s t e r conjugated s t r u c t u r e ( I I I ) , t o c a i n i d e must f i r s t be presented as a carbamic a c i d intermediate (VII) as shown i n Scheme 2. T h i s carbamic a c i d intermediate has not yet been i s o l a t e d nor i d e n t i f i e d . Tocalnlde (I) Tocanlnlde Carbamic Acid (VII) 3-(2,6-xylyl)-5-nethylhvdantoln (VI) A C B Uridine dlphoiphate/glucuronlc acid. HaOH. Scheme 2 : The proposed metabolic pathway leading to the formation of  tocainide carbamoyl ester glucuronideT 12 F o l l o w i n g t h i s i n i t i a l f i n d i n g , an e x t e n s i v e i n v e s t i g a t i o n was undertaken with e i g h t s p e c i e s of animals to e s t a b l i s h an animal model f o r the t o x i c o l o g i c a l e v a l u a t i o n of t o c a i n i d e . However, the amount of t o c a i n i d e carbamoyl g l u c u r o n i d e e x c r e t e d i n the u r i n e , expressed as the percent of dose, was found to range from the lowest of < 1 % in the g e r b i l to the h i g h e s t of 13 % in the r a b b i t (39). These f i g u r e s are c o n s i d e r e d to be low compared to 30 to 40 % in humans. In humans, the e l i m i n a t i o n h a l f - l i v e s f o r t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e and l a c t o x y l i d i d e were r e p o r t e d to be 13.0 hours and 29.1 hours r e s p e c t i v e l y <36). Although the 1 a c t o x y l i d i d e was known to accumulate d u r i n g c h r o n i c therapy, both t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e and the l a c t o x y l i d i d e were shown not to possess a n t i a r r h y t h m i c a c t i v i t y . In a d d i t i o n , the two m e t a b o l i t e s were shown not to possess c a r d i a c nor CNS t o x i c e f f e c t s (36). From an animal study (37), four major me t a b o l i t e s of t o c a i n i d e were i d e n t i f i e d , namely the 2,6-dimethylani1ine (IV) from amide h y d r o l y s i s , an aldehyde adduct ( V I I I ) , an o x i d a t i v e deaminated metabolite ( I X ) , and the novel carbamoyl e s t e r g l u c u r o n i c a c i d conjugate of t o c a i n i d e ( I I I ) , as shown i n Fi g u r e 2. OI) (XI) 13 2,6-dimethylaniline (IV) Aldehyde Adduct (VIII) Oxidative Deaminated (IX) Tocalnlde Carbamoyl  Ester Glucuronide ( I I I ) Figure 2 : The metabolism of tocainide in rats. Three of these m e t a b o l i t e s : the a n i l i n e , the ethanimine and the d i k e t o n e , were not found in human u r i n e . In a d d i t i o n to the i d e n t i f i e d m e t a b o l i t e s i n human u r i n e , we suspected the presence of a 4'-hy d r o x y p r o p i o n o x y l i d i d e (X) metabolite and p o s s i b l y an 14 e t h e r g l u c u r o n i c a c i d conjugated metabolite (XI) being formed from the l a c t o x y l i d i d e . 2. A n a l y s i s of G l u c u r o n i c A c i d Conjugates It i s g e n e r a l l y r e c o g n i z e d that g l u c u r o n i d a t i o n i s a s t e r e o s e l e c t i v e p r o c e s s . To date, only one study has used an i n d i r e c t b e t a - g l u c u r o n i d a s e enzyme h y d r o l y t i c method to assay the k i n e t i c p r o f i l e of the t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e (35). The d i s p o s i t i o n h a l f - l i f e of t h i s g l u c u r o n i d e was found to be comparable to that of the R-(-)-enantiomer, s u g g e s t i n g that the g l u c u r o n i d e may have p r e f e r e n t i a l l y formed from the R-(-)-enantiomer of t o c a i n i d e (35). However, a n a l y s i s of the enantiomeric composition of the i n t a c t g l u c u r o n i d e of t o c a i n i d e has not been r e p o r t e d . The q u a n t i t a t i v e a n a l y s i s of g l u c u r o n i d e s u s u a l l y i n v o l v e s a c i d or enzyme h y d r o l y s i s by b e t a - g l u c u r o n i d a s e , f o l l o w e d by a n a l y s i s of the l i b e r a t e d aglycone. T h i s a n a l y t i c a l approach i s , however, s u b j e c t to a number of drawbacks: 1. The h y d r o l y t i c r e a c t i o n may not be s p e c i f i c f o r g l u c u r o n i c a c i d conjugates; i t u s u a l l y r e q u i r e s v e r i f i c a t i o n by the use of a c o m p e t i t i v e enzyme i n h i b i t o r , s a c c h a r i c a c i d 1,4-lactone. 2. The r a t e and extent of the h y d r o l y t i c r e a c t i o n i s dependent on the source of the enzyme, thus g i v i n g poor r e p r o d u c i b i l i t y of data between experiments. 3. The procedure i s time consuming (up to 24 hours i n c u b a t i o n ) and does not d i s t i n g u i s h between mono-and d i - g l u c u r o n i d e s . 15 4. The procedure may lack day to day r e p r o d u c i b i 1 1 i t y due to a p o s s i b l e v a r i a t i o n in enzyme a c t i v i t y with s t o r a g e . Due to the l i m i t a t i o n of an enzyme h y d r o l y t i c assay, i t was necessary to develop an assay f o r the d i r e c t r e s o l u t i o n of the d i a s t e r o m e r i c e s t e r g l u c u r o n i d e of t o c a i n i d e , and to examine the d i s p o s i t i o n p r o f i l e of t h i s g l u c u r o n i d e . The metabolism and s t e r e o s e l e c t i v e d i s p o s i t i o n of t o c a i n i d e may be e f f e c t i v e l y s t u d i e d with the use of a de u t e r a t e d pseudoracemic dose of t o c a i n i d e (equal p r o p o r t i o n s of deut e r a t e d S(+)-enantiomer and u n l a b e l l e d R(-)-enantiomer). Plasma, urine and s a l i v a samples obtained from such a study in human s u b j e c t s can then be used to study p o s s i b l e metabolic c h i r a l i n v e r s i o n , in vivo r a c e m i z a t i o n of the i n t a c t drug, metabolic s u b s t r a t e s t e r e o s e l e c t i v i t y and s t e r e o s e l e c t i v i t y in drug t r a n s p o r t i n t o s a l i v a . Furthermore, the use of pseudoracemic dosing of t o c a i n i d e w i l l a s s i s t the i d e n t i f i c a t i o n of drug m e t a b o l i t e s . Hence, the i n i t i a l aims of t h i s p r o j e c t were t h r e e - f o l d : 1. S y n t h e s i s of e n a n t i o m e r i c a l 1 y pure R ( - ) , or S(+>-3 ' , 4 * , 5 * - t r i d e u t e r a t e d t o c a i n i d e f o r b i o a v a i l a b i l i t y measurements and metabolite i s o l a t i o n / i d e n t i f i c a t i o n u s i ng the d e u t e r i u m - l a b e l l e d compound. 2. Develop necessary a n a l y t i c a l techniques to separate enantiomeric or d i a s t e r e o m e r i c m e t a b o l i t e s of t o c a i n i d e and to examine t h e i r k i n e t i c p r o f i l e . 3. I s o l a t e the t o c a i n i d e g l u c u r o n i d e and e l u c i d a t e the i n t a c t s t r u c t u r e of t h i s novel m e t a b o l i t e . 16 3. S p e c u l a t i v e S t r u c t u r e s of the Novel T o c a i n i d e Glucuronide The s t r u c t u r e of 3-(2,6-xy1y1)-5-methylhydantoin (VI) i n d i c a t e d t h at the formation of the novel g l u c u r o n i d e i n v o l v e d the i n c o r p o r a t i o n of a carbonyl group onto the primary amine n i t r o g e n of t o c a i n i d e f o l l o w e d by g l u c u r o n i c a c i d c o n j u g a t i o n . The true i d e n t i t y of t h i s carbamoyl intermediate has not been e s t a b l i shed. T h e o r e t i c a l l y , the t e r m i n a l primary amine n i t r o g e n can be carbamoylated through a number of biochemical pathways, a. N-carbamovlation High l e v e l s of N-carbamoy1-beta-alanine and N-carbamoylaspartate were found in the urine of p a t i e n t s with o r n i t h i n e carbamoyl t r a n s f e r a s e d e f i c i e n c y (40). Scheme 3 o u t l i n e s the r o l e of carbamoylphosphate in the metabolism of urea. T o c a i n i d e possesses an a l a n i n e moiety with a primary amine and can t h e o r e t i c a l l y be N-carbamoy1ated by the enzyme carbamoylphosphate followed by g l u c u r o n i c a c i d c o n j u g a t i o n . 17 Carbamoyl Phosphate Dihydrouracil Camosine 1 t| N-carbamoyl B-alanine B-alanine Scheme 3 : Pathways o f ammonia metabolism. ASA, a r g i n i n o s u c c i n i c a c i d ; OMP, o r o t i d i n e monophosphate; IMP, u r id ine monophosphate. 18 b. Carbonate E q u i l i b r i u m Carbon d i o x i d e i s present i n the body as b i c a r b o n a t e s , carbonates and d i s s o l v e d carbon d i o x i d e gas. The formation of carbonates has been s t u d i e d i n human red c e l l s and plasma as well as animal muscle and b r a i n t i s s u e s (41). Carbon d i o x i d e can r e a c t with the u n i o n i z e d amino group of amino a c i d s to form carbonates as shown in Scheme 4: ToC-NH2 • C0 2*=± ToC-NHCOCf • H + , kfi - I ™ ^ 2 ^ ^ . [ToC-NH2l [H } a [ToC-NH3+] T 0 C - N H 3 ToC-NH2 + H ; k where k £ • carbamate equilibrium constant. kfl » ionization constant of the amino group. Scheme 4 : The t h e o r e t i c a l e q u i l i b r i u m react ions o f toca in ide carbamate. 19 T h i s carbon d i o x i d e - c a r b o n a t e e q u i l i b r i u m r e a c t i o n has been s t u d i e d i n human plasma (41) with g l y c i n e and g l y c y l g l y c i n e (pKa of 8.0 f o r both compounds). T o c a i n i d e , having a pKa of 7.8, may t h e o r e t i c a l l y combine with carbon d i o x i d e depending on the pH and carbon d i o x i d e c o n c e n t r a t i o n of the m e t a b o l i z i n g t i s s u e . In agreement with t h i s theory, Venkataramanan and Axelson (38) demonstrated the formation of a small amount of the hydantoin (VI) when an aqueous s o l u t i o n of t o c a i n i d e was incubated at 100 °C f o r 1 hour with s o l i d carbon d i o x i d e , p o s s i b i l y through the formation of carbamic a c i d intermediate f o l l o w e d by c y c l i z a t i o n with the x y l i d i d e n i t r o g e n as shown in Scheme 5: CH3 0 Scheme 5 : The mechanisms o f carbamic ac id c y c l i z a t i o n to 3- (2 ,6 -xy1y1) -5-methylhydantoin. 20 C . T o c a i n i d e - u r e a Adduct Formation Venkataramanan et al. (38) f i r s t observed the degradation of t o c a i n i d e in the presence of urea in urine samples. However, no attempts were made to i d e n t i f y the d e g r a d a t i o n p r o ducts. Our p r e l i m i n a r y r e s u l t s from u r e a - t o c a i n i d e h y d r o l y s i s experiments r e v e a l e d the formation of the hydantoin (VI) i n the presence of 8.0 M urea at 100 °C. The f o l l o w i n g s e c t i o n s w i l l d e s c r i b e the two p o s s i b l e chemical pathways which can l e a d to the formation of t h i s hydantoin through a r e a c t i o n of t o c a i n i d e with urea, i . Urea-adduct Formation An aqueous s o l u t i o n of urea can undergo i s o m e r i c t r a n s f o r m a t i o n to ammonium cyanate as shown in Scheme 6: 0 H + NH2 - C - NH2 * NH4 w ^ 0 - C = N Scheme 6 : The i s o m e r i c t r a n s f o r m a t i o n o f u r e a . T h i s spontaneously r e v e r s i b l e r e a c t i o n was s t u d i e d by Dirnhuber & Schutz, 1948 (42). I t was found that e q u i l i b r i u m i s reached w i t h i n 30 minutes at 100 °C, while at body temperature (38 ° C ) , e q u i l i b r i u m i s reached a f t e r s e v e r a l days. A l s o , in d i l u t e d urea s o l u t i o n , the r e a c t i o n thermodynamical1y favours the d i s s o c i a t e d cyanate s p e c i e s . Based on t h i s r e a c t i o n , ammonium cyanate can break down at a c i d i c u r i n a r y pH g i v i n g r i s e to carbon d i o x i d e . The r e s u l t a n t carbon d i o x i d e can combine with the amino group of t o c a i n i d e to 21 form the carbamic a c i d intermediate as d e s c r i b e d , i i • Urea Condensation with T o c a i n i d e A l t e r n a t e l y , t o c a i n i d e can condense with urea to form a carbamate intermediate as proposed in Scheme 7: 0 I N H - C - C H - N t ^ C H , HN, ^ C-0 t o c a i n i d e urea H N H - C - C H - N I I A CH3H [ N ^ " 0 1 1 0 l N H - C - C H - N H - C - N H 2 + CR3 NHi t o c a i n i d e u r e i d e Scheme 7 : The mechanisms of tocainide-ureide formation. 22 T h i s t o c a i n i d e ureide was found to undergo c y c l i z a t i o n under b a s i c c o n d i t i o n s to form ( V I ) . The mechanisms f o r the t o c a i n i d e ureide c y c l i z a t i o n are shown i n Scheme 8. + IH3 Scheme 8 : The mechanisms o f t o c a i n i d e - u r e i d e c y c l i z a t i o n to  3 - (2 ,6 -xy ly l ) -5 -methy1 hydantoin . 4. Drug G l u c u r o n i d a t i o n The metabolic pathway and the types of x e n o b i o t i c s g i v i n g r i s e to g l u c u r o n i c a c i d conjugates have been e x t e n s i v e l y reviewed (43,44). According to the aforementioned metabolic 23 pathways l e a d i n g to the formation of a phase I t o c a i n i d e metabolite intermediate f o l l o w e d by GA c o n j u g a t i o n , the i n t a c t s t r u c t u r e of TOCG can e i t h e r be a N-ureide g l u c u r o n i d e , (-NHCO.NH-GA), or a carbamoyl e s t e r g l u c u r o n i d e , (-NHCO.O-GA). a. Acvl-O-Glucuronides ( E s t e r G l u c u r o n i d e s ) These conjugates are known to be unstable a t a l k a l i n e c o n d i t i o n s . The aglycone can be a r y l , primary, secondary or t e r t i a r y a l i p h a t i c or h e t e r o c y c l i c compounds as in s a l i c y l i c a c i d , indomethacin, iodopanoic a c i d , t r i m e t h y l a c e t i c a c i d and n i c o t i n i c a c i d r e s p e c t i v e l y (43). The benzoate and s a l i c y l a t e e s t e r g l u c u r o n i d e s are known to hydrolyze completely when incubated with a pH 12 b u f f e r a t room temperature f o r 5 to 30 minutes (45). b. N-alucuron ides The x e n o b i o t i c s g i v i n g r i s e to N - g l u c u r o n i c a c i d conjugates are summarized in Table 1: T a b l e 1 : Examples o f v a r i o u s f u n c t i o n a l groups known t o g i v e  r i s e t o N - g l u c u r o n l c a c i d c o n j u g a t e i n man. F u n c t i o n a l Groups Examples A r o m a t i c amine A z a h e t e r o c y c l e Carbamate Sulphonamide H y d r o x y l a m i n e - N -T e r t i a r y a l i p h a t i c U r e a a n i l i n e s u l p h i s o x a z o l e meprobamate s u l p h a d i m e t h o x i n e N - h y d r o x y - 2 - a c e t a m i d o f l u o r e n e C y p r o h e p t a d i l e D u l c i n The carbamate g l u c u r o n i d e s (-NHC0.0-GA) are more s t a b l e than the amine conjugates (-NH-GA) at an a c i d i c pH. C e r t a i n 2 4 N-glucuornides have been formed spontaneously in v i t r o through condensation of an aglycone with g l u c u r o n i c a c i d at p h y s i o l o g i c a l pH and temperature. Meprobamate-N- (46), urethane-N- (47), and many arylamine-N- (48) and alky l a m i n e - N - g l u c u r o n i d e s (49) have been s y n t h e s i z e d in v i t r o u s i n g a simple admixture procedure. The theme of t h i s t h e s i s w i l l c e n t r e on i s o l a t i o n of the t o c a i n i d e g l u c u r o n i d e and on e l u c i d a t i o n of the s t r u c t u r e of a t h e o r e t i c a l l y p o s s i b l e N- ureide g l u c u r o n i d e (-NHCO.NH-GA) or the proposed carbamoyl e s t e r g l u c u r o n i d e (-NHCO.O-GA). Attempts w i l l a l s o be made to i d e n t i f y p o s s i b l e new conjugated or non-conjugated m e t a b o l i t e s . G. ToyicQJogy At the onset of t h i s p r o j e c t , t o x i c o l o g i c a l s t u d i e s had not r e v e a l e d any major t o x i c e f f e c t s from t o c a i n i d e nor from i t s two major m e t a b o l i t e s i n humans. A review r e p o r t i n d i c a t e d that the common adverse e f f e c t s of t o c a i n i d e were g a s t r o i n t e s t i n a l i n nature. Other adverse e f f e c t s i n c l u d e d s p o r a d i c cases of lupus erythematosus (50) and i n t e r s t i t i a l pneumonitis (51) and the r e c e n t l y r e p o r t e d cases of bloo d d y s c r a s i a s (52). In e a r l y 1985, r e p o r t s were r e l e a s e d from A s t r a Pharmaceuticals summarizing the p o s s i b l e h a e m atological t o x i c i t i e s which have r e s u l t e d from the use of t o c a i n i d e world wide. The f i r s t r e p o r t was prepared i n January 1984. At the time, 40 cases of haematological d i s o r d e r s ( l e u c o p e n i a and a g r a n u l o c y t o s i s ) were r e p o r t e d from a 25,000 to 30,000 p a t i e n t p o p u l a t i o n ( i n c i d e n c e of 1:750). In November 1984, with the approval of t o c a i n i d e in the U n i t e d S t a t e s and i n over 20 other c o u n t r i e s , the number of cases of 25 t o c a i n i d e r e l a t e d haematological t o x i c i t i e s reached a t o t a l of 125. In l i g h t of the i n c r e a s i n g incidence of blood d y s c r a s i a s , i t was important to study the metabolism of t o c a i n i d e with r e f e r e n c e to the t o x i c i t y of i t s m e t a b o l i t e s . As a r e s u l t of the hig h incidence of a g r a n u l o c y t o s i s t o x i c i t i e s , A s t r a Pharmaceuticals withdrew t o c a i n i d e from general use thus p r o h i b i t i n g the use of t h i s drug i n h e a l t h y human v o l u n t e e r s . Due to the sudden change i n drug r e g u l a t i o n and the withdrawal of support from A s t r a Pharmaceuticals to continue b a s i c r e s e a r c h on t o c a i n i d e , the i n i t i a l aims of t h i s p r o j e c t have s i n c e been r e v i s e d as f o l l o w s : 1. S y n t h e s i s of R(-) or S<+>-3',4',5'-t r i d e u t e r a t e d t o c a i n i d e . 2. Attempt i d e n t i f i c a t i o n of new m e t a b o l i t e s of t o c a i n i d e from human urine samples c o l l e c t e d d u r i n g p r e v i o u s pharmacokinetic s t u d i e s . 3. I s o l a t i o n of the i n t a c t t o c a i n i d e g l u c u r o n i d e and e l u c i d a t i o n of the i n t a c t s t r u c t u r e of t h i s m e t abolite . 4. L a r g e - s c a l e i s o l a t i o n of the t o c a i n i d e g l u c u r o n i d e f o r a g r a n u l o c y t o s i s t o x i c i t y s t u d i e s . H. Aaranulocvtos i s On the market today, numerous drugs are a s s o c i a t e d with g r a n u l o c y t o p e n i a . Phenothiazines are widely used a n t i p s y c h o t i c agents t h a t can cause r e v e r s i b l e g r a n u l o c y t o p e n i a (53). A n t i t h y r o i d medications, p r o p y l t h i o u r a c i l and methimazole can 26 cause g r a n u l o c y t o p e n i a i n 0.3 % to 0.6 % of p a t i e n t s (54). The sulfonamides were r e p o r t e d to be a g r a n u l o c y t o g e n i c through an immunological mechanism (54). The a n a l g e s i c and a n t i -inflammatory drug aminopyrine was r e p o r t e d to cause g r a n u l o c y t o p e n i a (54,55). Phenytoin was a l s o r e p o r t e d to cause a 2 % incidence of g r a n u l o c y t o p e n i a (53). The p e n c i l l i n s and c e p h a l o s p o r i n s were a l s o o c c a s i o n a l l y connected with g r a n u l o c y t o p e n i a i n longterm, high-dose therapy (54). In a l l cases of drug-induced g r a n u l o c y t o p e n i a and neu t r o p e n i a , the e t i o l o g y of the d i s e a s e can be e x p l a i n e d by three general mechanisms (55): 1. The g e n e r a t i o n of r e a c t i v e t o x i c m e t a b o l i t e s of the s t r u c t u r e R-N. 2. I n t e r a c t i o n of the a c t i v e m e t a b o l i t e s with n e u t r o p h i l s and macromolecules. 3. Formation of a n t i b o d i e s to n e u t r o p h i l s . The metabolism of aminopyrine can serve as an example to i l l u s t r a t e the g e n e r a t i o n of t o x i c m e t a b o l i t e s . Aminopyrine i s an enzyme inducer which can induce cytochrome P-450, cytochrome P-448 monooxygenases, and g l u c u r o n y l t r a n s f e r a s e . Aminopyrine was p o s t u l a t e d to be metabolized through a two-step monooxygenase r e a c t i o n f o l l o w e d by g l u c u r o n y l c o n j u g a t i o n to y i e l d a r e a c t i v e R-N m e t a b o l i t e . T h i s metabolic pathway, i n v o l v i n g endogenous monooxygenase enzymes f o l l o w e d by a c o n j u g a t i o n r e a c t i o n with e i t h e r g l u c u r o n y l t r a n s f e r a s e or s u l f o n t r a n s f e r a s e , i s b e l i e v e d to be common to phenylbutazone, chlorpromazine, promazine, and chl o r a m p h e n i c o l . Because of high chemical r e a c t i v i t y , the 27 g l u c u r o n i c a c i d and sulphate conjugates of these drugs are l a b i l e molecules which can produce the r e a c t i v e e l e c t r o p h i 1 i c s t r u c t u r e R-N. T h i s s p e c i e s of r e a c t i v e metabolite R-N generated from a g r a n u l o c y t o t i c drugs i s capable of i n t e r a c t i n g with n u c l e o p h i l e s of p r o t e i n molecules, thereby e l i c i t i n g an immune response with the formation of a n t i b o d i e s . The R-N r e a c t i v e molecules can a l s o r e a c t with the n u c l e o p h i l e s of DNA and RNA d i s r u p t i n g DNA r e p l i c a t i o n and p r o t e i n s y n t h e s i s . Bone marrow c e l l s , being one of the most r e a c t i v e c e l l s undergoing r e p l i c a t i o n in the body, are l i k e l y to s u f f e r the most d e t r i m e n t a l e f f e c t s . With our knowledge of the r e a c t i v e nature of the carbamoyl e s t e r g l u c u r o n i d e of t o c a i n i d e at b a s i c pH, and the r e c e n t r e p o r t s of a g r a n u l o c y t o s i s in the l i t e r a t u r e , we propose to examine whether the r e p o r t e d a g r a n u l o c y t o s i s t o x i c i t y of t o c a i n i d e was e l i c i t e d by the t o c a i n i d e g l u c u r o n i d e . 28 EXPERIMENTAL A. Chemicals and M a t e r i a l s Chemicals are reagent grade ( u n l e s s s p e c i f i e d ) . The f o l l o w i n g s u p p l i e r s are l i s t e d a l p h a b e t i c a l l y : 1 . A l d r i c h Chemical Co. (Milwaukee, Wisconsin, USA) D-alanine, benzyl chloroformate ( c a r b o b e n z y l o x y l c h l o r i d e ) , 2,6-dimethylani1ine, N - c a r b o b e n z y l o x y l -D-al a n i n e , T r i c h l o r o m e t h y l c h l o r o f o r m a t e , 2,4,6-trimethy1 a n i 1 i n e , c a l c i u m h y d r i d e , sodium hydride (50% in mineral o i l ) , sodium g l u c u r o n i c a c i d , phosphoric a c i d , n a p h t h o r e s o r c i n o l (1,3-d ihydroxynaphthalene) 2. A n a l y t i c a l I n t e r n a t i o n a l Inc. (Harbor c i t y , CA, USA) Bond-elute C-18 e x t r a c t i o n columns 3. A p p l i e d Science L a b o r a t o r i e s . (State C o l l e g e , P e n n s y l v a n i a , USA) S i l a r - l O c l i q u i d phase 4. A r i s t a r ^ BDH Chemical. (Poole, England) Sodium hydroxide ( a n a l y t i c a l grade) 5. A s t r a Pharmaceuticals Canada L t d . ( M i s s i s s a u g a , O n t a r i o , Canada) Toca i n ide 6. Beckman Instruments Inc. ( B e r k e l e y , CA, USA) C-18 ODS 5um 4.6mm x 25cm HPLC column 7. BDH Chemicals. (Vancouver, B.C., Canada) HPLC grade s o l v e n t s : methanol, water, a c e t o n i t r i l e , hexane, 2-propanol, methylene c h l o r i d e . Reagent grade: c h l o r o f o r m , anhydrous sodium s u l f a t e , e t h e r , ethanol-100, t o l u e n e , d i m e t h y l s u l f o x i d e , sodium phosphate monobasic, a c e t i c a c i d 8. BrownLee Labs Inc. (Santa C l a r a , CA, USA) Ramnin 0.45 urn o n - l i n e f i l t e r , ramnin C-18 guard column 9. Chromatographic S p e c i a l t i e s . ( B r o c k v i l l e , Ont., Canada) Chromosorb W-HP 3 0 10. Eastman Organic Chemicals. (Rochester, N.Y., USA) D i c y c l o h e x y l c a r b o d i i m i d e 11. F i s h e r S c i e n t i f i c Co. ( F a i r Lawn, New J e r s e y , USA) Methyl i o d i d e , 30-32% hydrogen bromide in a c e t i c a c i d 12. J.T. Baker L t d . (Phi 1ipsburg, New J e r s e y , USA) S i l i c a g e l ( f l a s h chromatography) 13. Mandel S c i e n t i f i c Company. (Nisku, A l b e r t a , Canada) XAD-2 r e s i n , SE-30 (bonded) 0.25um x 50m fused s i l i c a c a p i l l a r y column, C h i r a s i l - V a l R 0.25um x 50m fused s i l i c a c a p i l l a r y column 14. Merk Sharp & Dohme. ( i s o t o p e s ) (Montreal, Quebec, Canada) Deuterated methyl i o d i d e , D -DMSO 15. Parr Pressure Reaction Apparatus. (Moline, I l l i n o i s , USA) V a r i a c pressure r e a c t i o n v e s s e l 16. P i e r c e Chemical Co. (Rockford, I l l i n o i s , USA) P y r i d i n e , BSTFA 17. Sigma Chemical Co. ( S t . L o u i s , M i s s o u r i , USA) HFBA, bet a - g l u c u r o n i d a s e (glucurase ), s a c c r o - 1 , 4 - l a c t o n e , a c e t i c anhydride, deuterium oxide (99.8 atom% D), d e u t e r a t e d c h l o r o f o r m (99.8% atom% D), d e u t e r a t e d s u l p h u r i c a c i d (99+ atom% D), L - a l a n i n e , 18. Supelco L t d . ( B e l l a f o n t e , Pennsylvania, USA) Dexsil-300 l i q u i d phase 19. Union Carbide Canada L t d . (Toronto, Ont., Canada) Helium, n i t r o g e n , hydrogen, medical a i r 20. Whatman Inc. ( C l i f t o n , NJ., USA) P a r t i s i l - 1 0 magnum-9 p r e p a r a t i v e column, 20 x 20cm KC-18F t h i n l a y e r p l a t e s 3 1 B . InstrumentatIon 1. Fast Atom Bombardment A l l FAB S p e c t r a were recorded at the mass spectrometry f a c i l i t y in the Department of Chemistry, U n i v e r s i t y of B r i t i s h Columbia. Samples were analyzed e i t h e r in g l y c e r o l or t h i o g l y c e r o l matrix with a Kr a t o s / A e l Ms9 instrument. The a c c e l e r a t i n g voltage and the FAB were at 6 KV and 7 KV r e s p e c t i v e l y . C o l l i s i o n gas was Xenon and the samples were analyzed on a copper probe t i p . 2. Gas Chromatography Mass Spectrometry GCMS a n a l y s i s was c a r r i e d out using e i t h e r the V a r i a n M a g n e t i c - s e c t o r or the Hewlett-packard quadrupole instrument, a. V a r i a n M a g n e t i c - s e c t o r GCMS A Hewlett-packard 5700A Gas Chromatograph was i n t e r f a c e d to a V a r i a n M a t - I l l Mass Spectrometer. The e l e c t r o n - i m p a c t s p e c t r a were recorded on a V a r i a n 620L computer and p l o t t e d on a c h a r t -r e c o r d e r . The f i l a m e n t c u r r e n t was 300 uA with an e l e c t r o n beam energy of 70 eV, ion source pressure at 8 X 10"° t o r r , and scan from a c y c l e of 15 to 500 mass u n i t s every 5 seconds. T o t a l - i o n -chromatograms were obtained from a p l o t of m/z 50 to 500. The gas chromatograph was operated in the s p l i t l e s s mode with GC i n l e t , i n j e c t i o n p o r t and se p a r a t o r temperature a l l at 250 °C. The f o l l o w i n g column c o n d i t i o n s were employed: 1. A 3 % S i l a r 10C l i q u i d phase was coated on chromosorb W-HP before packing i n t o a 2 mm i n t e r n a l diameter X 2 m g l a s s column. The oven temperature 32 was programmed from 150 to 270 C at 8 C/min. The column helium flow r a t e was 20 mL/min. 2. A 3 % Dexsil-300 l i q u i d phase was coated on chromosorb W-HP before packing i n t o a 2 mm i n t e r n a l diameter X 2 m g l a s s column. The oven temperature was programmed from 150 to 300 °C at 8 °C/min. The column helium flow r a t e was 20 mL/min. b. Hewlett-Packard Quadrupole GCMS An HP 5880A gas chromatograph was i n t e r f a c e d with an 5987A mass spectrometer. E l e c t r o n impact s p e c t r a were obtained with a e l e c t r o n beam energy at 70 eV and emission c u r r e n t at 300 uA. TIC were obtained from a p l o t of m/z 120 to m/z 800. Ion source temperature was at 240 °C. Chemical i o n i z a t i o n was c a r r i e d out using methane as the reagent gas with a source pressure of 1.45 x 10"^ Torr and em i s s i o n c u r r e n t of 300 uA. D i r e c t probe experiments were c a r r i e d out e i t h e r in the EI or CI mode. The gas chromatograph was operated in the s p l i t mode with i n l e t temperature at 250 °C. The f o l l o w i n g chromatographic columns were employed: 1. A c a p i l l a r y SE-30 (bonded-phase), 0.25 mm X 50 m fused- s i l i c a (HP) was used with an oven temperature program from 40 to 300 °C at 30 °C/min. The column helium flow r a t e was 1 mL/min. p 2. A C a p i l l a r y C h i r a s i l - V a l , 0.25 mm X 50 m fused-s i l i c a column was used with an oven temperature program from 40 to 190 °C at 30 °C/min. Column helium flow r a t e was 1 mL/min. 33 3. Proton Nuclear Magnetic Resonance The 80 MHz s p e c t r a were recorded on a Bruker WP-80 or a V a r i a n XL-100 spectrometer. The 400 MHz experiments were c a r r i e d out usi n g a Bruker WP-400 spectrometer. A l l s p e c t r a were recorded at the NMR f a c i l i t y i n the Department of Chemistry. 4. Gas Chromatography Routine gas chromatographic a n a l y s i s was c a r r i e d out using e i t h e r a Hewlett-Packard 5880A or 5830 GC with a flame i o n i z a t i o n 6 3 or e l e c t r o n - c a p t u r e detectorC N i ) . The chromatographic columns used were as o u t l i n e d f o r the quadrupole GCMS experiments. The flame i o n i z a t i o n d e t e c t o r and the e l e c t r o n capture d e t e c t o r were operated at 275 °C and 300 °C r e s p e c t i v e l y . 5. L i q u i d Chromatography L i q u i d chromatographic a n a l y s i s was c a r r i e d out using a Hewlett-packard 1082B L i q u i d Chromatograph, with an Al t e x V a r i a b l e wavelength d e t e c t o r and a 7000 U l t r o r a c automatic f r a c t i o n c o l l e c t o r . A C-18RP P a r t i s i l - 1 0 , magnum-9 p r e p a r a t i v e column was used with a R a i n i n 0.45 urn o n - l i n e f i l t e r and a R a i n i n C-18 guard column. Flow r a t e s were ranging from 2.3 to 3.5 mL/min with a column pressure between 13,790 KPa (2000 p s i ) to 17,237 KPa (2500 p s i ) . 6 . I n f a r e d Spectroscopy IR s p e c t r a were obtained with sodium c h l o r i d e d i s k s as a n u j o l mull using an Unicam SP-1000 spectrometer. 7. M e l t i n g P o i n t Determination M e l t i n g P o i n t s were determined 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 apparatus and were u n c o r r e c t e d . 3 4 C. S y n t h e s i s of 3*,4'.5'-C 2H] 3-2-amino-2'.6'-p r o p j o x y l jclj^e The s y n t h e s i s f o l l o w e d a modified method r e p o r t e d by Byrnes (31). The s y n t h e t i c steps were s i m i l a r to those u t i l i z e d f o r p eptide s y n t h e s i s (62). The procedure i n v o l v e s the use of N-carbobenzyloxyl-D or L - a l a n i n e and 3, 4 , 5 - t r i d e u t e r a t e d - 2 , 6 -d i m e t h y l a n i l i n e i n the presence of d i c y c l o h e x y l c a r b o d i i m i d e to form N-carbobenzyloxyl t o c a i n i d e . The carbobenzyloxy1 group was then removed by H B r / a c e t i c a c i d y i e l d i n g t o c a i n i d e hydrogen bromide s a l t . 1. S y n t h e s i s of S(+)-3*. 4 '.5'-C 2H13-tocainide. I n i t i a l attempts to s y n t h e s i z e the t r i d e u t e r a t e d t o c a i n i d e were c a r r i e d out using non-deuterated 2,6-dimethylani1ine and commercially a v a i l a b l e N - c a r b o b e n z y loxyl-D-alanine. N-Carbobenzyloxy-D-alanine (5.0 g, 0.0224 mole) and 2 , 6 - d i m e t h y l a n i l i n e (2.72 g, 0.0224 mole) were d i s s o l v e d i n 20 mL of methylene c h l o r i d e . D i c y c l o h e x y l u r e a was f i l t e r e d o f f with s u c t i o n and the f i l t r a t e was d r i e d under vacuum to y i e l d 3.08g of N-carbobenzyloxyl t o c a i n i d e . A s o l u t i o n of 30 to 32 % hydrogen bromide i n a c e t i c a c i d was added in s u f f i c i e n t q u a n t i t i e s to d i s s o l v e the t o c a i n i d e d e r i v a t i v e f o l l o w e d by vacuum removal of excess hydrogen bromide at room temperature. Then ether was added in small q u a n t i t i e s f o r the c r y s t a l l i z a t i o n of t o c a i n i d e hydrogen bromide s a l t . The e t h e r e a l s o l u t i o n was l e f t at 0 °C ov e r n i g h t to y i e l d 2.83 g of t o c a i n i d e hydrogen bromide s a l t (0.0147 mole, 65 % y i e l d ) . The hydrogen bromide s a l t was removed by e x t r a c t i o n of 3 5 t o c a i n i d e base in methylene c h l o r i d e , d r i e d under n i t r o g e n , r e c o n s t i t u t e d with ethanol-100 and a c i d i f i e d with e t h a n o l / h y d r o c h l o r i c a c i d s o l u t i o n . The s a l t was c r y s t a l l i z e d e t h e r at 0 °C overn i g h t to y i e l d 0.36 g t o c a i n i d e h y d r o c h l o r i d e s a l t . Gas Chromatography ( C h i r a s i 1 - V a l column); S ( + ) - t o c a i n i d e (61 % ) , R ( - ) - t o c a i n i d e (39 %) M e l t i n g P o i n t : 266 °C un c o r r e c t e d ( r e f : 264.5 - 265.5 °C) (31). Inf a r e d ( N u i o l m u l l ) : 3300 cm _ 1(NH s t r e t c h ) , 1540(C=O s t r e t c h ) , 1660(C=C s t r e t c h ) , 1150, 1090, 970, 780 ( t r i - s u b s t i t u t e d v i c i n a l a r o m a t i c ) . Mass Spectrum (GCMS): m/z 192 (M + 5 % ) , 44(100), 121(8), 105(5), 91(3). 2. S y n t h e s i s of 3 , 4 , 5 - [ 2 H l o - 2 , 6 - d i m e t h v l a n i 1 i n e D e u t e r a t i o n of 2,6-dimethylani1ine was c a r r i e d out using a modified p e r d e u t e r a t i o n technique r e p o r t e d by F r i s c h k o r n (51). A sample of 2,6-dimethylani1ine (2 mL, 0.016 mole) was added to 2 mL of (0.1 mole) f o l l o w e d by a s u f f i c i e n t amount of D2SO4 to form the a n i l i n e s u l f a t e s a l t . The mixture was r e f l u x e d in a s i n g l e condenser apparatus f o r 2 hours before the r e s i d u a l was removed by r o t a t o r y e v a p o r a t i o n over a water bath at 100 °C. The dry a n i l i n e s a l t was r e c o n s t i t u t e d with 5 mL of D20/D2SO^ (pH 1) and heated i n a V a r i a c ^ r e a c t i o n v e s s e l f o r 18 hours at 250 °C with 4137 KPa (600 PSI) 36 c o l d n i t r o g e n . The excess pressure i n the r e a c t i o n v e s s e l was r e l e a s e d p e r i o d i c a l l y d u r i n g the experiment to maintain the pressure below 13790 KPa (2000 PS I ) . A f t e r h e a t i n g , the r e a c t i o n mixture was b a s i f i e d to pH 10 with 1.0 M sodium hydroxide, e x t r a c t e d with c h l o r o f o r m , d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d and evaporated to dryness at room temperature, y i e l d i n g 1.8 mL (90 %) of a dark brown l i q u i d . Mass Spectrum (TIC ) : unresolved 3 major peaks: SCAN 51: (1) m/z 126 (M + 100 % ) , 100(95), 80(35). SCAN 61: (2) m/z 125 (M + 100 % ) , 109(55), 110(55), 80(20). The r e a c t i o n product in c h l o r o f o r m was then washed 3 times with 2 mL p o r t i o n s of water ( f o r exchange of the amine deuterium atoms) before i t was d r i e d over anhydrous sodium s u l f a t e , f i l t e r e d , and evaporated to dryness over n i t r o g e n . The r e a c t i o n product was then p u r i f i e d by d i s t i l l a t i o n over an o i l bath. The f r a c t i o n c o l l e c t e d at 218 °C was a l i g h t brown l i q u i d (1.1 mL, chemical y i e l d 50 % ) . Mass Spectrum ( T I C ) : s i n g u l a r component: SCAN 48: (1) m/z 124 (M + 100 % ) , 109(60), 80(20). 80 MHz-C1Hl-NMR ( C D C I 3 ) : S ppm, 2.2s(6H, -CH3), 3.5s(2H, -NH 2), 6.65s(lH, p a r a - a r o m a t i c ) , 6.95s(2H, meta-aromatic). 37 D. Attempted S y n t h e s i s of 1-C H 3 3 - 2-amino - 2 * . 6 * - p r o p i o x y l i d i d e Due to damage of the V a r i a c r e a c t i o n v e s s e l , the o r i g i n a l 9 s y n t h e t i c route f o r s y n t h e s i z i n g 3 ' , 4 ' , 5 ' - [ ^ H l 3 - t o c a i n i d e was abandoned in f a v o r of an a l t e r n a t i v e method f o r the s y n t h e s i s of 1- 1 H ] 3~2-amino-2',6'-propioxy1idide. T h i s approach i n v o l v e s the s y n t h e s i s of N-carbobenzy1oxy-<D)-[2H^-alanine from commerically 2 a v a i l a b l e [ *"H 1 3 - a l a n ine and carbobenzy1oxy c h l o r i d e (CBZCL) 2 followed by r e a c t i o n with 2 , 6 , - d i m e t h y l a n i l i n e to form l - [ H^-2- amino-2*,6'-propioxy1idide as d e s c r i b e d p r e v i o u s l y . I. S y n t h e s i s of N-carbobenzyloxy-(+)-Do-alanine I n i t i a l attempts were c a r r i e d out using u n l a b e l l e d D-alanine. D-alanine (2.62 g, 0.0294 mole) was d i s s o l v e d in 200 mL of s a t u r a t e d sodium bicar b o n a t e s o l u t i o n with s t i r r i n g . Carbobenzy1oxy c h l o r i d e (14.18 mL, 0.0294 mole) was added with s t i r r i n g and under ice c o o l i n g . The r e a c t i o n mixture was s t i r r e d under ice u n t i l a l l CBZC1 was d i s s o l v e d (6.0 h o u r s ) . The r e a c t i o n was terminated by the dropwise a d d i t i o n of c o n c e n t r a t e d h y d r o c h l o r i c a c i d u n t i l the s o l u t i o n i s a c i d i c to l i t m u s paper. The white p r e c i p i t a t e of N-CBZ-alanine was f i l t e r e d and washed with 0.01 M h y d r o c h l o r i c a c i d before being vacuum-dried at room temperature, y i e l d i n g 5.7 g of product (87.9 h chemical y i e l d ) . 38 M e l t i n g P o i n t : 71 - 72 °C <ref. 79.5 - 80 °C, Sigma Chemical Co.). o A f t e r c r y s t a l l i z a t i o n i n c h l o r o f o r m / e t h e r at 0 C f o r 24 hours, the r e a c t i o n product y i e l d e d 3.2 g white powder (55.8 % ) . M e l t i n g P o i n t : 79 °C. ( r e f . 79.5 - 80 °C, Sigma Chemical Co.) I n f r a r e d ( N u i o l m u l l ) : 1700 cm - 1(C=0 s t r e t c h ) , 3000(0-H s t r e t c h ) , 3350CN-H s t r e t c h ) , 1540(N-H s t r e t c h ) , 1380C0-H s t r e t c h ) . E. S y n t h e s i s of P o t e n t i a l T o c a i n i d e M e t a b o l i t e s 1. S y n t h e s i s of 2- E t h a n i m i n o - 2 ' , 6 ' - p r o p i o x y l i d i d e - fi T o c a i n i d e base (1.0 mg, 5.2 x 10 mole) was added to 1 mL (2.3 x 10 mole) a c e t y l aldehyde at room temperature in a 5 mL r e a c t i o n v i a l . The r e a c t i o n proceeded at room temperature f o r 15 minutes before excess aldehyde was removed by d r y i n g under n i t r o g e n . The r e a c t i o n product was r e c o n s t i t u t e d with methanol f o r GCMS a n a l y s i s . I n f r a r e d ( N u i o l m u l l ) : 1680 cm~l(C=0 s t r e t c h ) , 3300(N-H s t r e t c h ) , 1670(C-N s t r e t c h ) , 700, 730 and 1 0 3 0 ( t r i - s u b s t i t u t e d v i c i n a l a r o m a t i c ) . Mass Spectrum (GCMS): m/z 218 (M+10 % ) , 71(100), 56(35), 44(15), 190(10). 39 2. S y n t h e s i s of 3-(2.6-Xvlvl)-5-methvlhvdantoin S y n t h e s i s of t h i s hydantoin i n v o l v e d the formation of a r e a c t i v e isocynate d e r i v a t i v e f o l l o w e d by i n t e r n a l c y c l i z a t i o n to form the hydantoin r i n g . S y n t h e s i s of the isocyanate d e r i v a t i v e of t o c a i n i d e was c a r r i e d out using a phosgene s u b s t i t u t e , t r i c h l o r o m e t h y l chloroformate (TCF), as d e s c r i b e d by Ozaki (58) and K u r i t a (59). To a stock s o l u t i o n of f r e s h TCF (0.1 mole/200 mL) i n sodium d r i e d toluene, t o c a i n i d e base (1.0 g) and 0.1 g of a c t i v e carbon was added in small q u a n t i t i e s with s t i r r i n g and gradual h e a t i n g from 50 to 80 °C over a 30 minute p e r i o d . Another 0.6 mL of TCF was added dropwise to the r e a c t i o n mixture f o l l o w e d by h e a t i n g from 80 to 100 °C over 2 hours. The white p r e c i p i t a t e was f i l t e r e d o f f and the f i l t r a t e was d r i e d under vacuum at room temperature, y i e l d i n g 1.5 g of white c r y s t a l s . The r e a c t i o n product was r e c r y s t a l 1 i z e d i n chl o r o f o r m at 0 °C f o r 24 hours, y i e l d i n g 0.5 g of the pure hydantoin. T h i s r e c r y s t a l 1 i z e d product was analyzed by IR, GCMS and NMR. M e l t i n g P o i n t ; 138 - 140 °C ( r e f . 138 - 142.5 °C) (31). I n f r a r e d (Nuiol m u l l ) : 3300 cm - 1(N-H s t r e t c h ) , 2300(N-C=0 s t r e t c h ) , 1795(C=0 s t r e t c h ) , 1700(C=0 s t r e t c h ) . Mass Spectrum (GCMS): m/z 218 (M + 100 % ) , 147(90), 44(72), 119(30), 105(28), 132(25), 113(20), 203(10), 176(5). 40 400 MHz-[ H]-NMR ( C D C I 3 ) ; t> ppm 1.54s(3H, 5-CH3), 2.18s(3H, xylidine-CH3>, 2.22s(3H, x y l i d i n e - C H 3 ) , 4.24dq(J=8Hz, 1H, 5-CH), 6.56s(lH, l-NH), 7.1 to 7.3s(3H, aromatic-H). 3. S y n t h e s i s of 3 -(2.4,6-Xvlyl)-5-methvlhvdantoin The s y n t h e t i c procedures were s i m i l a r to those d e s c r i b e d f o r the 2 , 6 - x y l y l analog. However, i n i t i a l attempts were made to sy n t h e s i z e 2-amino-2',4',6'-propioxy1 id i d e (4'-methyl analog of t o c a i n i d e ) . a S y n t h e s i s of 2-Amino-2*.4*.6'-propioxvlidide The s y n t h e t i c steps were s i m i l a r to those d e s c r i b e d f o r the 2',6'-propioxylidide analog. N-carbobenzyloxy1-alanine (1.0 g, 0.0488 mole) was added to 6.05 g (0.0443 mole) of 2,4,6-trime thy1 a n i 1 i n e in 60 mL of dry methylene c h l o r i d e . D i c y c l o h e x y l c a r b o d i i m i d e (9.23 g, 0.0448 mole) was added s l o w l y under s t i r r i n g and the r e a c t i o n mixture was l e f t to stand at room temperature f o r 1 hour. The mixture was f i l t e r e d and the f i l t r a t e was d r i e d under vacuum. A s o l u t i o n of 30 to 32 % hydrogen bromide in a c e t i c a c i d was added, and the hydrogen bromide s a l t of the 4'-methyltocainide analog was recovered and r e c r y s t a l 1 i z e d by the a d d i t i o n of et h e r y i e l d i n g 5.61 g of hydrogen bromide s a l t . b. R e a c t i o n with T r i c h l o r o m e t h v l Chloroformate The 4*-methyltocainide hydrogen bromide s a l t (2.0 g, 0.0097 mole) was e x t r a c t e d i n base with methylene c h l o r i d e , d r i e d with anhydrous sodium s u l f a t e , and evaporated to dryness under a stream of n i t r o g e n . The base was d i s s o l v e d in 30 mL dry toluene, 41 f o l l o w e d by dropwise a d d i t i o n of TCF (1.16 mL, 0.0097 mole) with a g i t a t i o n and gradual h e a t i n g from room temperature to 80 °C as d e s c r i b e d . The r e a c t i o n mixture was then f i l t e r e d , evaporated to dryness, and r e c o n s t i t u t e d i n c h l o r o f o r m f o r r e c r y s t a l 1 i z a t i o n . The c h l o r o f o r m s o l u t i o n y i e l d e d 0.83 g of 3 - ( 2 , 4 , 6 - x y l y l ) - 5 -methylhydanto i n . Me 11 i ng P o i n t : 137.5 - 139 °C I n f r a r e d ( N u i o l m u l l ) : 3300 cm - 1(N-H s t r e t c h ) , 2300(N-C=0 s t r e t c h ) , 1795(C=0 s t r e t c h ) , 1700(C=0) s t r e t c h ) . Mass Spectrum (GCMS): m/z 232 (M+ 100 % ) , 161(100), 44(40), 146(35), 119(25), 217(10). 80 MHz-C^-Hl-NMR ( CDCl 3 ) : S ppm. 1.52s(3H, 5 - C H 3 ) , 2.12s(3H, x y l i d i n e - C H 3 ) , 2.12s(3H, x y l i d i n e - C H 3 ) , 2.32s(3H, 4 ' - x y l i d i n e - C H 3 ) , 4.25dq(J=8 Hz, 1H, 5 - C H 3 ) , 6.15bs(lH, 1-NH), 6.9s(2H, aromatic-H). F. I d e n t i f i c a t i o n of P o t e n t i a l T o c a i n i d e M e t a b o l i t e s 1. P e r m e t h v l a t l o n The procedure i n v o l v e d g e n e r a t i o n of f r e s h sodium methyl s u 1 f i n y 1 methide carbanion as r e a c t i o n c a t a l y s t / s o l v e n t followed by a d d i t i o n of CH^I as o u t l i n e d by Thompson and Des i d e r i o (61). 42 a- P r e p a r a t i o n of Dry D i m e t h v l s u l f o x i d e (DMSO) Because of the hygr o s c o p i c nature of DMSO, i t was d i s t i l l e d f r e s h with c a l c i u m hydride before each p e r m e t h y l a t i o n experiment. DMSO (20 mL) was added to 2.0 g of c a l c i u m hydride i n a round bottom f l a s k and d i s t i l l e d under vacuum at 70 °C u n t i l 15 mL of dry DMSO was c o l l e c t e d . b. G e n e r a t i o n of Sodium M e t h y l s u l f i n v l m e t h i d e Carbanion  (DMSO) Sodium The s a l t was prepared with sodium h y d r i d e . Sodium hydride (250 mg, 50 % d i s p e r s e d in mineral o i l ) , an amount e q u i v a l e n t to 5 moles of sodium h y d r i d e , was washed r a p i d l y three times with a t o t a l of 3 mL anhydrous e t h e r . The washed sodium hydride was t r a n s f e r r e d q u i c k l y to a round bottom f l a s k c o n t a i n i n g 5 mL of dry DMSO. The grey c o l o r suspension was then heated g e n t l y under n i t r o g e n u n t i l the formation of hydrogen ceased. The r e s u l t i n g s t r a w - c o l o r suspension was allowed to s e t t l e g i v i n g a c l e a r s o l u t i o n . The f i l t e r e d supernatant was s t o r e d under n i t r o g e n at -20 °C. c. Permethylat ion An a l i q u o t of 200 uL of i s o l a t e d m e tabolite sample i n methanol (as d i s c u s s e d i n the f o l l o w i n g experimental s e c t i o n s ) was d r i e d under n i t r o g e n . The res i d u e was then r e c o n s t i t u t e d with 50 uL of dry DMSO under n i t r o g e n i n a 5 mL screw-capped r e a c t i o n v i a l . DMSO sodium carbanion (20 uL) was added, s t i r r e d in a vortex mixer, and l e t stand f o r 15 minutes at room temperature. Subsequently, 1.2 uL of methyl io d i d e was added and the mixture was s t i r r e d as b e f o r e . The r e a c t i o n was allowed to 43 proceed at room temperature f o r 1 hour before i t was terminated by the a d d i t i o n of 1 mL of water. The permethylated g l u c u r o n i d e was e x t r a c t e d with 1 mL of ch l o r o f o r m and back-washed three times with 1 mL of water. The c h l o r o f o r m e x t r a c t was then con c e n t r a t e d under n i t r o g e n before GCMS a n a l y s i s . 2. Attempted I d e n t i f i c a t i o n of Permethylated U r i n a r y T o c a i n i d e  M e t a b o l i t e s Scheme 9 o u t l i n e s the p r o t o c o l used i n s c r e e n i n g both conjugated and non-conjugated m e t a b o l i t e s . PH 3 P H 12 •(tract with •ethylene Chi or id* u r i n e (100 mL). I PH 3 •stract with aiethylene chloride XAD r e s i n 20 f r a c t i o n s e a c h c o n t a i n i n g 3 mL m e t h a n o l i c e l u e n t I c o n c e n t r a t e w i t h n i t r o g e n HPLC r e v e r s e - p h a s e C - 1 B B a s i f y t o PH 13 ( b a s i c h y d r o l y s i s ) 2 . 3 . 1 TLC E n z y n e h y d r o l y s i s r e v e r s e - p h a s e C - 1 8 F OP I A c i d h y d r o l y s i s V i s u a l i z a t i o n unde r UV OR N a p h t h o r e s o r c i n o l r e a g e n t ac C h i r a s i l - V a l c a p i l l a r y c o l u m n i r a l l 1, 2 , and 3 g i v e p o s i t i v e OR I F o n l y 3 g i v e s p o s i t i v e r e s u l t . I D e r i v a t i z a t i o n f o r B C / M S • e t h y l a t i o n w i t h d i azome thane • • t h y l a t i o n w i t h atethy 1 i o d i d e / d i a t e t h y l s u l f o x i d e a n i o n t r i a t e t h y l s i l y a t i o n w i t h BSTFA S C / R S Scheme 9 : Attempted i d e n t i f i c a t i o n o f permethylated u r i n a r y t o c a i n i d e m e t a b o l i t e s . 44 a. I s o l a t i o n of Tocainide-carbamoyl E s t e r Glucuronide T o t a l urine (1 to 10 h o u r s ) , c o l l e c t e d from a human vo l u n t e e r a f t e r a 200 mg o r a l dose of t o c a i n i d e , was pooled. A 2 cm x 20 cm XAD-2 r e s i n column was used i n i t i a l l y to t r a p u r i n a r y m e t a b o l i t e s which were subsequently e l u t e d with methanol. The crude methanolic sample thus c o l l e c t e d from the XAD a d s o r p t i o n r e s i n was a p p l i e d as a narrow band onto a 20 x 20 cm KC18F TLC p l a t e and the p l a t e was e l u t e d with a 60/40 methanol/water s o l v e n t system. A f t e r 15 cm of e l u t i o n , the p l a t e was d i v i d e d into four f r a c t i o n s . The f o l l o w i n g Table 2 o u t l i n e d the Rf values of the 13 bands separated from the crude e x t r a c t . Various bands on the TLC were i s o l a t e d together as a p l a t e f r a c t i o n , and thus four p l a t e p l a t e f r a c t i o n s were obtained as ou11ined be 1ow. Table 2 : The Rf o f u r inary components separated on a KC-18F 20x20 cm  TLC p l a t e . p l a t * f r a c t i o n 1 S 3 « O. 64 • -•1 • . 74 • . 70 • • • 4 • .61 • .67 • .63 • . 60 • . 4 3 • .36 • . 3 4 o.3o The samples, from each p l a t e f r a c t i o n were agai n i n d i v i d u a l l y e v a l u a t e d chromatographical1y on 20 x 20 cm KC18F p l a t e s u s i n g a 50/50 methanol/water s o l v e n t system. Band 1 of p l a t e f r a c t i o n 1, obtained from the 60/40 methanol/water s o l v e n t system, was r e s o l v e d into bands l a and lb by the 50/50 methanol/ 45 water s o l v e n t system. These two bands were i s o l a t e d and l a b e l l e d band l a and lb r e s p e c t i v e l y . The Rf values of the band l a and lb are l i s t e d in Table 3. Table 3 : The Rf three ur inary components separated on a KC-18F  TLC p la te dur ing a second p u r i f i c a t i o n . Band la Band lb Band 2 Voliriteer 2 Band 1 0.84 0.69 0.06 The r e s t of the 11 bands in p l a t e f r a c t i o n s 1 to 4 were a l s o i s o l a t e d . b. A c i d and Enzyme H y d r o l y s i s A p o r t i o n of each of the samples i s o l a t e d from the TLC p l a t e s was h y d r o l y z e d with h y d r o c h l o r i c a c i d or b e t a -g l u c u r o n i d a s e enzyme. For a c i d h y d r o l y s i s , 0.5 mL of 1.0 M h y d r o c h l o r i c a c i d was added to the band f r a c t i o n s and than heated at 100°C f o r 30 minutes. A f t e r treatment, the samples were b a s i f i e d , e x t r a c t e d with methylene c h l o r i d e , and d e r i v a t i z e d with 20 uL of HFBA in hexane at 55°C f o r 30 minutes before analyzed with a gas chromatograph equipped with a 25 m x 0.31 mm i d . C h i r a s i 1 - V a l ^ c a p i l l a r y column. Samples i s o l a t e d from the band f r a c t i o n s were a l s o h y d r o l y z e d with 0.5 mL of the beta-g l u c u r o n i d a s e - g l u c u r a s e ^ i n the presence of 1.0 mL 46 of a c e t a t e b u f f e r at pH 5 i n P T F E - l i n e d screw-capped g l a s s tubes. The tubes were incubated in a water bath at 37°C f o r 12 hours. A f t e r h y d r o l y s i s , the samples were b a s i f i e d , e x t r a c t e d , d e r i v a t i z e d with HFBA, and analyzed by GC as d e s c r i b e d . Band lb y i e l d e d t o c a i n i d e a f t e r both a c i d and enzymatic h y d r o l y s i s i n d i c a t i n g t h at the f r a c t i o n c o n t a i n e d a g l u c u r o n i c a c i d conjugate of t o c a i n i d e . c. L i q u i d Chromatographic A n a l y s i s of the Hydantoin  Derived from the G l u c u r o n i c A c i d Conjugate of T o c a i n i d e Band f r a c t i o n s i s o l a t e d from reverse-phase TLC p l a t e s were s u b j e c t e d to pH 12 a l k a l i n e c o n d i t i o n by the a d d i t i o n of 1.0 N sodium hydroxide s o l u t i o n . Upon b a s i f i c a t i o n to pH 12, t o c a i n i d e carbamoyl gl u c u r o n i d e has been shown to c y c l i z e to 3 - ( 2 , 6 - x y l y l ) -5-methylhydantoin which can be monitored by HPLC and compared with the s y n t h e s i z e d m a t e r i a l . A f t e r b a s i f i c a t i o n , the samples were e x t r a c t e d into methylene c h l o r i d e and the s o l v e n t c o n c e n t r a t e d under n i t r o g e n at 55 °C. L i q u i d chromatographic a n a l y s i s was c a r r i e d out on a 5 urn C-18 ODS (25 cm x 4.6 mm) HPLC column with a 25 % a c e t o n i t r i l e in 0.05 M potassium phosphate s o l v e n t system. d. Gas Chromatography/Mass Spectrometry A n a l y s i s of Permethvlated T o c a i n i d e Carbamoyl Glucuronide The suspected t o c a i n i d e g l u c u r o n i d e was permethylated with methyl iodide i n dry d i m e t h y l s u l f o x i d e (DMSO) in the presence of m e t h y l s u l f i n y l m e t h i d e c a rbanion as d e s c r i b e d p r e v i o u s l y . 47 3. Attempted I d e n t i f i c a t i o n of U r i n a r y T o c a i n i d e M e t a b o l i t e s bv L v o o h i 1 i z a t i o n and GCMS A n a l y s i s Urine samples were c o l l e c t e d from a h e a l t h y v o l u n t e e r between 2 to 16 hours a f t e r an o r a l dose of 200 mg t o c a i n i d e HC1. The f o l l o w i n g Scheme 10 o u t l i n e s the experimental p r o t o c o l . 2-16 hours pooled urine pH 12 (1.0M NaOH) Unadjusted pH 3 (1.0M HCL) —1 f| 1 L y o p h i l l z a t i o n I L y o p h i l l z a t i o n Extracted Extracted Extracted Methylene Chloride 1 Methylene Chloride Methylene Chloride 1 Pennethylation (CH3I/DMS0) 1 1 GC/MS (S i l a r - l O C Column) Scheme 10 : Attempted identi f icat ion of urinary tocainide metabolites  by lyophil izat ion and GCMS analysis . 48 4. Attempted I s o l a t i o n and I d e n t i f i c a t i o n of T o c a i n i d e  N-carbamic A c i d or N-ureide Intermediate Urine samples from a h e a l t h y human s u b j e c t were c o l l e c t e d between 1 to 20 hours a f t e r an o r a l dose of 200 mg t o c a i n i d e HC1. Scheme 11, as shown below, o u t l i n e d the experimental d e s i g n that was used in an attempt to i s o l a t e and i d e n t i f y the t o c a i n i d e metabolite i n t e r m e d i a t e s . (10 mL) 1-20 hours pooled u r i n e I C-18 Bond-elute s o l i d e x t r a c t i o n (MeOH) Beta-i glucuronidase Acid Hydro Lysis Hydrolysis Extracted Extracted Extracted Extracted Extracted Extracted pH 3 pH 12 P H 3 I ,H 12 pH 3 pH 12 Permethylation DMS0 NatCH^I)-GCMS Scheme 11 : Attempted isolat ion and identi f icat ion of tocainide  N-carbamic acid or N-ureide intermediate. 49 5. Attempted I s o l a t i o n and I d e n t i f i c a t i o n of U r i n a r y  M e t a b o l i t e s bv F l a s h Chromatography A volume of 500 mL of u r i n e , c o l l e c t e d from 1 to 7 hours a f t e r an o r a l dose of 200 mg of t o c a i n i d e in a human s u b j e c t was used. As shown in Scheme 12, the pooled urine sample was f i r s t c o n c e n t r a t e d on a XAD-2 a b s o r p t i o n r e s i n column as d e s c r i b e d using a c e t o n i t r i l e as an e l u a n t . 500 mL pooled u r i n e I XAD-2 r e s i n column ( 2x35 cm ) I Eluted with a c e t o n i t r i l e I r o t a t o r y evaporated under vaccuni • TLC : 1. KC-18F si l i c a ' c o l u m n 2. Alumina 3. S i l i c a Scheme 12 : Attempted isolat ion and identi f icat ion of urinary metabolites  by flash chromatography. A 6-inch s i l i c a g e l column was dry-packed i n a 1 cm i d . g l a s s c y l i n d e r with 0.5 cm s i l i c a sand forming a s e a l on the column ends. A 30/20/50 hexane/2-propanol/methanol combination s o l v e n t was f l u s h e d through the packing with 137 KPa (20 PSI) n i t r o g e n at 1 mL/min u n t i l a l l gas bubbles were e l u t e d . A volume of 5.0 mL of the XAD-2 u r i n a r y a c e t o n i t r i l e e x t r a c t was d e l i v e r e d on top of the column and then e l u t e d with the d e s c r i b e d s o l v e n t 50 system at 1 mL/min c o l l e c t i n g 1 mL f r a c t i o n s . An a l i q u o t of each f r a c t i o n was s p o t t e d on a s i l - U V ^ t j ^ 2 x 5 cm TLC p l a t e and the p l a t e was e l u t e d with 30/20/50 Hexane/2-propanol/ methanol. F r a c t i o n s 4 and 5, 6 to 11, and 12 to 16 were found to correspond to 3 d i s t i n c t Rf values, and these v a r i o u s f r a c t i o n s were pooled into three samples. The s o l v e n t i n each sample was evaporated to dryness and the r e s i d u e was d e r i v a t i z i e d with CH^I or CD^I i n the presence of DMSO sodium, f o l l o w e d by GCMS a n a l y s i s . G. R e a c t i o n of To c a i n i d e with Urea Due to a p r e l i m i n a r y o b s e r v a t i o n that t o c a i n i d e r e a c t s with urea to form a hydantoin s t r u c t u r e , experiments were c a r r i e d out to study the r e a c t i o n at d i f f e r e n t pH, temperature, and r e a c t i o n t i me . 1. The E f f e c t of Temperature and Reaction D u r a t i o n T o c a i n i d e base (50 mg) was added to 1 mL of 8.0 M urea s o l u t i o n . The s o l u t i o n was then heated i n a block heater at 100°C f o r 1 hour, 9 hours or 13 hours, or a l t e r n a t i v e l y at 60°C f o r 3 hours. The samples were subsequently b a s i f i e d by the a d d i t i o n of 200 uL of 1.0 M sodium hydroxide and then e x t r a c t e d with 5 mL methylene c h l o r i d e . The s o l v e n t was d r i e d over anhydrous sodium s u l f a t e before being c o n c e n t r a t e d over a stream of n i t r o g e n f o r GCMS a n a l y s i s . In a d d i t i o n , a 500 mg sample of t o c a i n i d e was added to a f r e s h l y c o l l e c t e d d rug-free urine sample and was e i t h e r l e f t at room temperature or heated at 100 °C f o r 1 hour or 9 hours. For r e f e r e n c e , t o c a i n i d e was added to d i s t i l l e d d e i o n i z e d water and heated at 100 °C f o r 13 hours. 51 2. The E f f e c t of D H Into three separate tubes, t o c a i n i d e base (100) mg was d i s s o l v e d i n 1 mL of 8.0 M urea s o l u t i o n f o l l o w e d by pH adjustment to 2.3, 7.8 or 12.5 with 0.1 M h y d r o c h l o r i c a c i d or with 0.1 M sodium hydroxide. The samples were heated at 60 °C f o r 6 hours. The samples were then d i r e c t l y s p o t t e d on a 5 x 20 cm RPC18F p l a t e and e l u t e d with 80/20 methanol/water. F o l l o w i n g e l u t i o n on the TLC p l a t e and v i s u a l i d e n t i f i c a t i o n under UV at 254 nm, the three r e a c t i o n mixtures were then i s o l a t e d s e p a r a t e l y by Bond-elut reverse phase s o l i d e x t r a c t i o n columns followed by p e r m e t h y l a t i o n with CH^I i n sodium DMSO and GCMS a n a l y s i s as d e s c r i b e d p r e v i o u s l y . TLC (KC-18F); 80/20 methanol/water: t o c a i n i d e Rf = 0.45, hydantoin Rf = 0.75, N-ureide t o c a i n i d e Rf = 0.60. Mass Spectrum (GCMS): permethylated N-ure i d e - t o c a i n i d e , m/z 191 ( M + t r a c e ) , 72(100), 129(45), 191(5). 3. Attempt S y n t h e s i s of T o c a i n i d e - N - u r e i d e - q l u c u r o n i d e T o c a i n i d e base (400 mg) and 300 mg sodium g l u c u r o n i c a c i d were d i s s o l v e d in 1.0 mL of 8 M urea. The s o l u t i o n was heated at o R 50 C f o r e i t h e r 1 or 2 hours f o l l o w e d by Bond-elut s o l i d e x t r a c t i o n i s o l a t i o n and pe r m e t h y l a t i o n with CHgl/Na DMSO as d e s c r i b e d p r e v i o u s l y . In another experiment, t o c a i n i d e was f i r s t heated at 50 °C f o r 1 hour i n the presence of urea p r i o r to the a d d i t i o n of sodium g l u c u r o n i c a c i d . The r e a c t i o n mixture was 52 then l e f t at room temperature o v e r n i g h t before s o l i d phase e x t r a c t i o n and pe r m e t h y l a t i o n with CH^I/Na DMSO as d e s c r i b e d . 4. Attempt S y n t h e s i s of 2.6-Dimethvlani1ine- N-gl ucuron ide 2 , 6 - D i m e t h y l a n i l i ne (2.5 mL, 1.02 mole) was added to 3 mL 2:1 ace tone:water c o n t a i n i n g 0.5 g sodium g l u c u r o n i c a c i d . The r e a c t i o n mixture was a d j u s t e d to pH 5 with 1.0 M h y d r o c h l o r i c a c i d and mixed on a r o t a t i n g rack o v e r n i g h t . The products were sp o t t e d on a 2 x 5 cm RPC-18F TLC p l a t e and e l u t e d using 40/60 methanol/water. F o l l o w i n g TLC v i s u a l i z a t i o n under UV at 254 nm the product was i s o l a t e d by s o l i d phase e x t r a c t i o n and permethylated with CH^I/Na DMSO, fo l l o w e d by GCMS a n a l y s i s . TLC (KC-18F): 40/60 methanol/water: 2,6-dimethylani1ine Rf = 0.25, N-glucuronide Rf = 0.45. Mass Spectrum (GCMS): permethylated N-glucuronide. m/z 353 (m+ 8 % ) , 103(100), 219(90), 134(72), 75(68), 85(40). H. I s o l a t i o n and S t r u c t u r a l E l u c i d a t i o n of T o c a i n i d e  Glucuron ide Urine samples were c o l l e c t e d from a human s u b j e c t who had r e c e i v e d an intravenous dose of 200 mg of t o c a i n i d e h y d r o c h l o r i d e . An a l i q u o t of 100 mL of the 14th hour urine c o l l e c t i o n was con c e n t r a t e d under vacuum to y i e l d 5.9 mL of a yellow r e s i d u e . T h i s r e s i d u e was ad j u s t e d to pH 3 with 0.1 M phosphoric a c i d and e x t r a c t e d with 10 mL of hexane p r i o r 53 to e v a l u a t i o n by p r e p a r a t i v e HPLC. In another experiment, 100 mL of the 7th hour urine from the same s u b j e c t was c o n c e n t r a t e d by a z e o t r o p i c e v a p o r a t i o n at room temperature with 10% ACN under vacuum to y i e l d 10 mL of a yellow r e s i d u e . The r e s i d u e was e x t r a c t e d 3 times with 10 mL methylene c h l o r i d e at pH 7 f o l l o w e d by 3 r e p e t i t i v e e x t r a c t i o n s with 10 mL of hexane at pH 3. The e x t r a c t e d samples were then f i l t e r e d through a 0.45 urn s y r i n g e f i l t e r before p r e p a r a t i v e HPLC i s o l at i on. 1. P r e p a r a t i v e HPLC I s o l a t i o n of T o c a i n i d e Carbamoyl E s t e r  Glucuron ide I n i t i a l s e l e c t i o n of s o l v e n t systems using reverse-phase TLC i n d i c a t e d a high p r o p o r t i o n of water in the mobile phase was r e q u i r e d f o r the e l u t i o n of t o c a i n i d e g l u c u r o n i d e . Through a s y s t e m a t i c approach, 10 % a c e t o n i t r i l e i n 0.01 M sodium phosphate (monobasic) b u f f e r (pH 3) s o l v e n t system d e l i v e r e d at 3 mL/min, was found to o f f e r reasonable s e p a r a t i o n and r e t e n t i o n of u r i n a r y components. The column e l u a n t was monitored by UV at 220, 225, or 266 nm. E i g h t y 2.0 mL f r a c t i o n s were c o l l e c t e d at the d e t e c t o r o u t l e t with an automatic f r a c t i o n c o l l e c t o r . The absorbance of each f r a c t i o n was measured by a UV spectrophotometer at the c o r r e s p o n d i n g d e t e c t i o n wavelength that was used to monitor the column e l u a n t . F r a c t i o n s c o n t a i n i n g major peaks were pooled and analyzed by base, a c i d and enzyme h y d r o l y s i s as well as by a n a p h t h o r e s o r e i n o l c o l o r t e s t , as w i l l be d e t a i l e d i n l a t e r s e c t i o n s . T h i r t y - s i x i n j e c t i o n s of the a z e o t r o p i c a l 1 y prepared 54 sample were made with a 200 uL sample i n j e c t i o n loop using a 10 % a c e t o n i t r i l e in 0.01 M sodium phosphate (monobasic) b u f f e r at pH 3 mobile phase. F r a c t i o n 4, which was i d e n t i f i e d to c o n t a i n the t o c a i n i d e g l u c u r o n i d e , was pooled and c o n c e n t r a t e d under vacuum. A second s o l v e n t system c o n s i s t i n g of 3 % a c e t o n i t r i l e i n 0.01 M sodium phosphate (monobasic) at pH 7 was used to r e - p u r i f y the c o l l e c t e d f r a c t i o n 4 sample. The column el u a n t was again c o l l e c t e d in f r a c t i o n s and t e s t e d f o r the presence of TOCG as d e s c r i b e d . Based on the volume of u r i n e , c o n t a i n i n g 67 ug/mL of the hydantoin, that was used f o r t h i s 2-step i s o l a t i o n , an estimate of 10 mg of pure t o c a i n i d e g l u c u r o n i d e i n sodium phosphate (monobasic) s a l t (63 mg) c o u l d be i s o l a t e d . The sodium phosphate s a l t c o n t a i n e d in the e l u a t e was removed by r e c y c l i n g the sample in 3 % a c e t o n i t r i l e in 0.3 % a c e t i c a c i d ( a d j u s t e d to pH 7 with 0.1 M sodium h y d r o x i d e ) . The TOCG-containing f r a c t i o n was c o n c e n t r a t e d under vacuum, and the sodium a c e t a t e s a l t was removed by a c i d i f y i n g the sample to pH 3 with d i l u t e h y d r o c h l o r i c a c i d f o l l o w e d by e v a p o r a t i o n of the free a c i d under vacuum at room temperature. The r e s u l t e d TOCG was i s o l a t e d in NaCl. UV Spectrum: * max 202.5 nm (A max t o c a i n i d e = 202.5 in 3 % a c e t o n i t r i l e in 0.01M sodium phosphate, pH 7 ). 55 Mass Spectrum (GCMS); (BSTFA/pyridine d e r i v a t i z a t i o n leads to decomposition of TOCG) m/z 218 (M + 100 %) . ( a c e t i c anhydride/diazomethane d e r i v a t i z a t i o n leads to decomposition of TOCG) m/z 218 (M + 100 %>. 400-MHz-[1-NMR (D^-DMSO): S ppm 1.38d(3H,I - C H 3 ) , 2 . 1 1 s ( 3 H , x y l i d i n e - C H 3 ) , 3.69d(glucuronic a c i d ) , 3.92d(glucuronic a c i d ) , 4.25t(1H,2-CH3), 5 . 7 7 s ( g l u c u r o n i c a c i d ) , 7.06s(3H,xylidine-H), 9 . 3 9 s ( l H , x y l i d i n e - N H ) . Fast Atom Bombardment; G l y c e r o l : m/z 413 (M + 1), 435(M + Na), 457(M -H+2Na). T h i o g l y c e r o l : m/z 413 (M + 1), 435(M + Na), 457(M -H+2Na). a. A c i d H y d r o l y s i s of TOCG with H y d r o c h l o r i c A c i d Each pooled f r a c t i o n , c o l l e c t e d from the p r e p a r a t i v e HPLC, was c o n c e n t r a t e d to 5 mL under vacuum by a z e o t r o p i c e v a p o r a t i o n with a c e t o n i t r i l e . To each c o n c e n t r a t e d f r a c t i o n , 1.0 mL of co n c e n t r a t e d h y d r o c h l o r i c a c i d was added and the sample was then heated at 100 °C f o r 1 hour, f o l l o w i n g which, i t was b a s i f i e d with 5.0 M sodium hydroxide, e x t r a c t e d with 2 mL of methylene c h l o r i d e , c o n c e n t r a t e d under n i t r o g e n and analyzed f o r the presence of t o c a i n i d e by GC with a SE-30 c a p i l l a r y column. b. B e t a - q l u c u r o n i d a s e Enzyme H y d r o l y s i s Each pooled f r a c t i o n c o l l e c t e d from the p r e p a r a t i v e HPLC was c o n c e n t r a t e d by a z e o t r o p i c e v a p o r a t i o n as d e s c r i b e d , followed by adjustment to pH 5 with 0.1 M sodium hydroxide or 0.1 M 56 h y d r o c h l o r i c a c i d , and h y d r o l y z e d by the a d d i t i o n of 500 uL of bovine b e t a - g l u c u r o n i d a s e enzyme in ac e t a t e b u f f e r at pH 5. The h y d r o l y s i s was c a r r i e d out at 37°C f o r 12 hours, f o l l o w e d by b a s i f i c a t i o n with sodium hydroxide and e x t r a c t i o n with methylene c h l o r i d e . The o r g a n i c s o l v e n t was evaporated to dryness, r e c o n s t i t u t e d with methanol and analysed by GC f o r the presence of t o c a i n i d e as d e s c r i b e d above. Each f r a c t i o n c o l l e c t e d from the l i q u i d chromatograph was a l s o t r e a t e d in a s i m i l a r f a s h i o n with the bovine b e t a - g l u c u r o n i d a s e enzyme, but with the a d d i t i o n of 2 ug of sa c c r o - 1 , 4 - l a c t o n e as an enzyme i n h i b i t o r . c. N a p h t h o r e s o r c i n o l C o l o r R e a c t i o n A f t e r c o n c e n t r a t i o n under vacuum, each HPLC f r a c t i o n was spotted on a 2 x 5 cm RPC-18F TLC p l a t e and sprayed with a 2 % n a p h t h o r e s o r c i n o l reagent in phosphoric a c i d / e t h a n o l . The p l a t e s were then heated at 100°C f o r 10 minutes. A blue c o l o r a t i o n of the r e s u l t i n g spots i n d i c a t e d the presence of a g l u c u r o n i c a c i d mo i e t y . d. Sodium hydroxide H y d r o l y s i s Each c o n c e n t r a t e d HPLC f r a c t i o n was b a s i f i e d to pH 12 with a 2.0 M sodium hydroxide s o l u t i o n . The sample was then e x t r a c t e d with methylene c h l o r i d e . The o r g a n i c l a y e r was t r a n s f e r r e d to a c l e a n tube, evaporated, and r e c o n s t i t u t e d with methanol p r i o r to GC a n a l y s i s f o r the presence of 3-(2,6-xy1yl)-5-methylhydantoin. I. Pharmacokinetics of T o c a l n l d e Carbamoyl E s t e r Glucuron ide The q u a n t i t a t i o n of t o c a i n i d e g l u c u r o n i d e i n uri n e was c a r r i e d out using a modified i n d i r e c t method as d e s c r i b e d by 57 Hoffmann (60). The procedure i n v o l v e d the h y d r o l y s i s of t o c a i n i d e g l u c u r o n i d e in urine at pH > 12 forming 3-(2,6-xy1y1)-5-methylhydantoin. Q u a n t i t a t i o n of the hydantoin d e r i v a t i v e can prov i d e an e s t i m a t i o n f o r the t o c a i n i d e g l u c u r o n i d e . 1. H y d r o l y s i s K i n e t i c s of To c a i n i d e Glucuronide i n Sodium Hydroxide Into 7 tubes, 0.4 mL of urine c o l l e c t e d at the 10th hour from a human s u b j e c t who had r e c e i v e d 200 mg of t o c a i n i d e o r a l l y , and 0.1 mL of the i n t e r n a l standard e t i d o c a i n e base (1.0 ug/mL) were added to 0.5 mL of 2.0 M, 1.0 M or 0.5 M sodium hydroxide with mixing. The r e a c t i o n product was e x t r a c t e d at 0.5, 1, 2, 4, 6, 10 and 20 minutes by shaking with 5.0 mL of methylene c h l o r i d e f o r 1 minute. The methylene c h l o r i d e was then d r i e d over anhydrous sodium s u l f a t e , evaporated to dryness and r e c o n s t i t u t e d with 20 uL of methanol f o r GC a n a l y s i s using a bonded phase SE-30 c a p i l l a r y column. The h y d r o l y s i s of t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e , as r e f l e c t e d by the formation of the hydantoin, was p l o t t e d as the peak h e i g h t r a t i o of the hydantoin to that of e t i d o c a i n e , over the timed i n t e r v a l s 1.5, 2, 3, 5, 7, 11 and 21 minutes. 2. H v d r o l v s i s of 3-(2.6-Xvlvl)-5-methylhvdantoin Into 7 tubes, 0.1 mL of an aqueous hydantoin s o l u t i o n (15 ug/mL) and 0.1 mL of e t i d o c a i n e i n t e r n a l standard (10 ug/mL) s o l u t i o n were added to 0.2 mL of 2.0 M, 0.2 M or 0.1 M sodium hydroxide. The h y d r o l y s i s samples were e x t r a c t e d at a c c u r a t e l y timed i n t e r v a l s from 0.5 to 40 minutes by shaking with 5.0 mL methylene c h l o r i d e f o r 1 minute. The s o l v e n t was d r i e d , 58 evaporated to dryness, and r e c o n s t i t u t e d with 2.0 uL methanol f o r GC a n a l y s i s . 3. H v d r o l v s i s of 3-<2,4,6-Xvlvl)-5-methvlhvdantoin The p r o t o c o l was the same as d e s c r i b e d f o r the 2 , 6 - x y l y l analog. 4. 3-(2,6-Xvlvl)-5-methvlhydantoin C a l i b r a t i o n Curve A l l c a l i b r a t i o n samples were prepared in d u p l i c a t e . Into 6 separate tubes, 100 uL of 1, 3, 6, 10, 15 and 20 ug/100 uL of aqueous t o c a i n i d e base and hydantoin standard s o l u t i o n were added to 100 uL of 10 ug/100 uL e t i d o c a i n e base i n t e r n a l standard s o l u t i o n . Into each sample, 100 uL of 4'-methylhydantoin i n t e r n a l standard (10 ug/100 mL) and 300 uL of blank urine were a l s o added. An a l i q u o t of 700 uL of 1.0 M sodium hydroxide was then added to each tube. The tubes were mixed on a vortex mixer f o r 10 minutes at room temperature, f o l l o w e d by e x t r a c t i o n with 5 mL methylene c h l o r i d e f o r 1 minute. The o r g a n i c l a y e r was t r a n s f e r r e d to a c l e a n tube and evaporated to dryness under -n i t r o g e n . Each sample was r e c o n s t i t u t e d with 20 uL of methanol p r i o r to GC a n a l y s i s . C a l i b r a t i o n samples were s t o r e d at 4 °C and a random sample.was used to monitor d a i l y instrumental v a r i a t i o n . A new c a l i b r a t i o n curve was c o n s t r u c t e d i f peak area r a t i o s were found to d e v i a t e more than 10 % between d a i l y a n a l y s i s. 5. I n t e r - a s s a v and I n t r a - a s s a v V a r i a b i l i t y D u p l i c a t e c a l i b r a t i o n samples of 6, 15 and 20 ug were each analyzed i n d u p l i c a t e . The average peak area r a t i o ± C.V. % was used to determine i n t e r - and i n t r a - a s s a y sample v a r i a t i o n . 59 A c o e f f i c i e n t of v a r i a t i o n of l e s s than 10 % was c o n s i d e r e d a c c e p t a b l e . 6. E x t r a c t i o n E f f i c i e n c i e s of To c a i n i d e and the Hydantoin  i n t o 5 mL of Methylene C h l o r i d e Samples of 1 mg/mL of t o c a i n i d e h y d r o c h l o r i d e and e t i d o c a i n e base were f i r s t prepared in methanol. The e x t r a c t i o n e f f i c i e n c y of t o c a i n i d e and the hydantoin i n methylene c h l o r i d e was ev a l u a t e d in a c o n c e n t r a t i o n range of 6 to 20 ug/mL. Into 3 separate tubes, 100 uL of each of t o c a i n i d e h y d r o c h l o r i d e and the hydantoin, e q u i v a l e n t to 6, 10 and 20 mg of t o c a i n i d e base and the hydantoin r e s p e c t i v e l y , was added to 300 uL of blank u r i n e . An a l i q u o t of 500 uL of 1.0 M sodium hydroxide was then added to each tube immediately f o l l o w e d by e x t r a c t i o n with 5 mL of methylene c h l o r i d e f o r 1 minute. The o r g a n i c l a y e r s were then t r a n s f e r r e d to c l e a n tubes, evaporated to dryness under n i t r o g e n and r e c o n s t i t u t e d with 100 uL of e t i d o c a i n e as an e x t e r n a l standard (1 ug/uL i n methanol) p r i o r to GC a n a l y s i s . In another 3 separate tubes, 100 uL of each of t o c a i n i d e base and the hydantoin i n 6, 10 and 20 ug/lOOuL methanol was added to 100 uL of e t i d o c a i n e (the same methanol i n t e r n a l standard s o l u t i o n ) . The samples were c o n c e n t r a t e d under n i t r o g e n f o l l o w e d by GC a n a l y s i s . The peak area r a t i o s between t o c a i n i d e and the hydantoin to that of e t i d o c a i n e i n the e x t r a c t e d samples were compared with the cor r e s p o n d i n g peak area r a t i o s from the methanol samples. The percent d i f f e r e n c e i n the peak area r a t i o s between the two s e t s of samples pr o v i d e d the e x t r a c t i o n e f f i c i e n c i e s of t o c a i n i d e and the hydantoin, assuming minimal 60 breakdown of the hydantoin d u r i n g the shor t e x t r a c t i o n p e r i o d . 7. A n a l y s i s of Toc a i n i d e Glucuronide i n K i n e t i c S t u d i e s Urine samples were c o l l e c t e d into p o l y e t h y l e n e bags d u r i n g p r e v i o u s pharmacokinetic s t u d i e s in three f a s t e d s u b j e c t s who had r e c e i v e d e i t h e r a 200 mg o r a l or slow (20 minutes) intravenous dose of t o c a i n i d e h y d r o c h l o r i d e . Urine samples were c o l l e c t e d h o u r l y f o r the f i r s t 10 hours and as o f t e n as r e q u i r e d t h e r e a f t e r f o r up to 96 hours. Urine volumes and pH were recorded immediately a f t e r c o l l e c t i o n . The samples were s t o r e d at -10 °C and were thawed into 10 mL screw-capped tubes, b a s i f i e d with sodium hydroxide, e x t r a c t e d with methylene c h l o r i d e and analysed in the same manner as d e s c r i b e d f o r h a n d l i n g of the c a l i b r a t i o n samples. A l l work-up procedures and GC analyses were done on a da i1y bas i s. 61 3 RESULTS AND DISCUSSION A. S y n t h e t i c Pathways f o r 3 * , 4 * . 5 * - T r i d e u t e r a t e d T o c a i n i d e The s y n t h e t i c procedures f o r t o c a i n i d e as d e s c r i b e d by Byrnes (31) are s i m i l a r to those used f o r peptide s y n t h e s i s . Since the 3', 4 ' , 5 ' - p o s i t i o n s on the x y l i d i d e r i n g of t o c a i n i d e have not been r e p o r t e d to undergo metabolism, i n i t i a l attempts were aimed at exchanging these protons with deuterium. 1. S y n t h e s i s of 3*.4*•5'.-[ 2H1 3-2-Amino-2*.6 * - p r o p i o x v l i d i d e The s y n t h e t i c approach as shown in. Scheme 13 was modified from a method r e p o r t e d by Byrnes (31): D ,"3 « 3 + CH3 Dicyclohexylurea N-carbobenzyloxyl Tocainide A: Dicyclohexylcarbodiimide/CH,C1, (25°C, 1 hour). B: F i l t e r , vaccum dry (HBr/acetlc A c i d ) . (removed) CH3 Tocainide HBr Scheme 13. The synthetic pathway for trideuterated tocainide. 63 a. 2,6-Dimethvlani1ine D e u t e r a t i o n I n i t i a l d i s c u s s i o n w i l l d e s c r i b e the d e u t e r a t i o n of the 3,4,5- p o s i t i o n s of 2,6-dimethylani1ine. The procedures f o l l o w e d a p r e d e u t e r a t i o n method f o r l a b e l l i n g t o l u i d i n e s and x y l i d i n e s as d e s c r i b e d by F r i s c h k o r n (57). The r e a c t i o n mechanism i s t y p i c a l l y an e l e c t r o p h i 1 i c aromatic s u b s t i t u t i o n (62). For 2,6-d i m e t h y l a n i l ine , the amine i s a s t r o n g a c t i v a t i n g group d i r e c t i n g ortho- and p a r a - s u b s t i t u t i o n , while the 2,6-methyl groups are only weak a c t i v a t o r s . P r e l i m i n a r y attempts at r e f l u x i n g the a n i l i n e with D2O/D2SO4 y i e l d e d 70 % pure 4-[ 2Hl-2,6-dime t h y l a n i 1 i n e . The F r i s c h k o r n method, however, r e q u i r e d h e a t i n g at 250 °C in the presence of D 2 O / D 2 S O 4 . To prevent v a p o r i z a t i o n of D2O at 250 °C, a 4137 KPa (600 PSI ) 15 pressure was r e q u i r e d . To achieve t h i s c o n d i t i o n , a V a r i a c r e a c t i o n v e s s e l was p r e s s u r i z e d at room temperature to 4137 KPa (600 PSI) with n i t r o g e n p r i o r to h e a t i n g at 250 °C. Mass S p e c t r a : Mass s p e c t r a l a n a l y s i s of the crude r e a c t i o n product r e v e a l e d a mixture of components with molecular ions m/z 126 and m/z 125 (see F i g u r e s 3 to 5 ) . A f t e r exchange of the amino deuteriums with hydrogens, the l o s s of one mass un i t from m/z 126 to m/z 125 suggested t h i s ion c o n t a i n e d the amino group. However, the ion fragment at and m/z 80 was unchanged, thus sugge s t i n g that the m/z 80 fragment d i d not c o n t a i n the amino group (see S t r u c t u r e s x i i to xv) . A mass spectrum of u n l a b e l l e d 2,6-dimethylani1ine was included in F i g u r e 6 f o r comparsion. 64 0 J J 100 Scan F i g u r e 3 : T o t a l - i o n - c h r o m a t o g r a m o f crude d e u t e r a t e d 2 , 6 - d i m e t h y l a n i l i n e p r o d u c t . 126 110 95 " * — * — * — 4 t % » r - * — * at * -F i g u r e 4 . The mass spectrum 0 f CruriP d ^ t e r m - P d m/z 65 125 109 80 94 m/z F i g u r e 5 : The mass spectrum o f crude d e u t e r a t e d  2 , 6 - d i m e t h y l a n i l i n e a t scan 61. 121 106 77 Figure 6 : The mass spectrum of un labe l led 2 ,6 -d imethy lan i l ine-.-66 67 The mass s p e c t r a l data of the d i s t i l l e d product r e v e a l e d a molecular ion of m/z 124 as w e l l as the co r r e s p o n d i n g ion fragments which i n d i c a t e d d e u t e r a t i o n at 3,4,5- r i n g p o s i t i o n s (as shown i n F i g u r e s 7 and 8, a l s o see S t r u c t u r e s x i i to xv) A f t e r p u r i f i c a t i o n by d i s t i l l a t i o n , the TIC r e v e a l e d a s i n g l e component with m/z 124 (M + d 3 100 % ) , 109 (M + -CH3 d 3 60), 95 (M + -NH-CH3 d 3 12), 80 (M + 2C2H6 d3 20) 80 MHz-[1H]-NMR: As shown in F i g u r e s 9 and 10, the proton NMR s p e c t r a of the deu t e r a t e d product i n d i c a t e d only t r a c e i n t e n s i t y of s i g n a l s from the aromatic protons at around 7.00 ppm. The proton assignments are summarized on Table 4. T a b l e A : Summarized da ta from the 80-MHz-[*H]-NMR spectrum o f D , - 2 , 6 - d i m e t h y l a n i l i n e . S h i f t (ppm) Decoupling I n t e g r a t i o n (mm) Proton(s) Assignment TMS (0.0) 2.20 s 113 6HU9/H) x y l i d i n e - CH, 3.5 8 6 2H x y l i d i n e - NH, 6.65 bs 2 2H aromatic - meta 6.95 bs 5 1H aromatic - para 68 scan F i g u r e 7 The t o t a l - i o n - c h r o m a t o g r a m o f d i s t i l l e d D o - 2 , 6 - d i m e t h y l a n i l i n e . 124 109 "m/z F i g u r e 8 : The mass spec trum o f d i s t i l l e d D , - 2 , 6 - d i m e t h y l a n i l i n e . 69 70 A s p l i t t i n g of the s i g n a l at 2.12 ppm suggested that a small p o r t i o n of the protons on the "CH^ group might have been exchanged with deuterium. The i n t e g r a t i o n at 2.2 ppm r e v e a l e d 113 u n i t s / 6 protons or 19 u n i t s / p r o t o n . In the same r e s p e c t , the chemical s h i f t at 3.5 ppm <2H, NH 2) was i n t e g r a t e d f o r 4 u n i t s / p r o t o n which suggested t h a t 78.9 % of the amino protons had exchanged. The meta- and para-aromatic protons at 6.65 ppm(2H, Ar-H) and 6.95 ppm(lH, Ar-H) were i n t e g r a t e d f o r 1 u n i t / p r o t o n and 5 u n i t s / p r o t o n , suggesting a deuterium l a b e l l i n g p u r i t y of 94.8 h and 73.7 % r e s p e c t i v e l y , b. R e a c t i o n with C a r b o b e n z v l o x v l - a l a n i n e With the s u c c e s s f u l s y n t h e s i s of the d e u t e r a t e d - a n i 1 i n e , s y n t h e s i s of t r i d e u t e r a t e d t o c a i n i d e f o l l o w e d the method by Byrnes (31). D i c y c l o h e x y l c a r b o d i i m i d e has been used widely f o r the s y n t h e s i s of p e p t i d e s and many de h y d r a t i o n r e a c t i o n s (63) i n v o l v i n g an amino and a c a r b o x y l i c a c i d group. The commonly c i t e d by-product of these r e a c t i o n s i s the i n s o l u b l e d i c y c l o h e x y l u r e a which can be e a s i l y f i l t e r e d and removed. The r e a c t i o n product a f t e r r e c r y s t a l 1 i z a t i o n y i e l d e d 0.36 g t o c a i n i d e h y d r o c h l o r i c a c i d . I n f r a r e d ( N u i o l M u l l ) : The i n f r a r e d spectrum of the s y n t h e t i c compound as shown in F i g u r e 11 was superimposable to that obtained from r e f e r e n c e t o c a i n i d e . A prominent a b s o r p t i o n a t 3300 cm~^ i n d i c a t e d HN-s t r e t c h i n g and C=0 s t r e t c h i n g at 1670 cm~l. 71 F i g u r e 11 : The i n f a r e d spectrum of s y n t h e t i c t o c a i n i d e (mull) Mass Spectrum (GCMS): A n a l y s i s was c a r r i e d out using a 3 % s i l a r - l O C packed column. The TIC and Mass Spectrum are shown in F i g u r e s 12 and 13. The m/z 44 base peaks i n d i c a t e d a cleavage between the carbonyl and the c h i r a l carbon. Fragmentation between the C-N bond of the x y l i d i n e n i t r o g e n gave r i s e to m/z 106, while the fragment at m/z 121 was r e s u l t e d from cleavage between the n i t r o g e n and the C-3 c a r b o n y l . Only 3 to 5 % of m/z 192 molecular ion was observed. The Mass Spectrum with i t s fragmentation p a t t e r n agreed with data obtained from r e f e r e n c e t o c a i n i d e . ? 2. Attempted S y n t h e s i s of 1-C H13-2-Amino-2*,6 * - p r o p i o x y l i d i d e T h i s s y n t h e t i c approach p r o v i d e d an a l t e r n a t i v e method of l a b e l l i n g three deuterium atoms on the c h i r a l methyl group of t o c a i n i d e using commerically a v a i l a b l e D ^ - a l a n i n e . U n f o r t u n a t e l y , d u r i n g p i l o t attempts to s y n t h e s i z e the D 2 - a n i l i n e , the V a r i a c ^ r e a c t i o n v e s s e l was damaged. Th e r e f o r e , t h i s a l t e r n a t i v e s y n t h e t i c approach was designed and was found to s a t i s f y the requirements of s y n t h e s i z i n g t r i d e u t e r a t e d t o c a i n i d e . Since c a r b o b e n z y l o x y l - D ^ - a l a n i n e is not commerically a v a i l a b l e , i n i t i a l experiments were c a r r i e d out to s y n t h e s i z e the c a r b o b e n z y l o x y - a l a n i n e using u n l a b e l l e d m a t e r i a l as d e p i c t e d i n Scheme 14. The r e a c t i o n y i e l d e d 5.7 g (87.9 % chemical y i e l d ) of the crude CBZ-alanine. R e c r y s t a l 1 i z a t i o n of the product in c h l o r o f o r m / e t h e r o f f e r e d a f i n a l y i e l d of 3.2 g (55.8 % ) . In 73 50 100 scan F i g u r e 12 : The t o t a l - i o n - c h r o m a t o g r a m o f  s y n t h e t i c t o c a i n i d e . 44 N H f C - C H - N H j A H 44 121 121 ll 7 7 9 1 J W » - W tr tr «r itr £ 1 192 * * r * r *r i,--^--^-^ * fr- i l l / Z F i g u r e 13 : The EI mass spectrum o f s y n t h e t i c t o c a i n i d e 74 accordance to t h i s s y n t h e t i c approach, the o v e r a l l t h e o r e t i c a l chemical y i e l d f o r s y n t h e s i s of t h i s t r i d e u t e r a t e d t o c a i n i d e from D3~alanine would o f f e r approximately 6 % chemical y i e l d . I n f r a r e d ( N u i o l Mull) ; As shown in Fig u r e 14, the a b s o r p t i o n at 3350 cm"^ from the NH s t r e t c h of the amide was pr e s e n t . The broad -OH s t r e t c h i n g v i b r a t i o n from the c a r b o x y l i c a c i d give r i s e to the broad s i g n a l at 3000 cm"-'-, o v e r l a p i n g the n u j o l -CH s t r e t c h . The two carbonyl s t r e t c h i n g a b s o r p t i o n s overlapped at 1700 cm--'-. Absorptions at 700 c m _ l and 750 c m _ l i n d i c a t e d the -CH r o c k i n g s i g n a l s from the monosubstituted benzene r i n g . o I CH2- 0-C-C1 + H2N-CH-00OK CBZCl Alanine 1 . N-CBZ-alanine A: Saturate HaHCO,/ice c o o l i n g (6 hours). B: 0.1 M HC1 (PH 3). Scheme 14 The synthesis of N-carbobenzvloxvalamnp 75 76 B. P o t e n t i a l T o c a i n i d e M e t a b o l i t e s 1. Z-Ethanimino-Z'.6 * - p r o p i o x v l i d i d e T h i s ethanimine was i d e n t i f i e d by Venkataramanan as a t o c a i n i d e metabolite in r a t s (37). Since t h i s metabolite has not been i d e n t i f i e d i n humans, i t was s y n t h e s i z e d as a r e f e r e n c e compound f o r subsequent metabolism s t u d i e s . The TIC and mass spectrum are shown in F i g u r e s 15 and 16 r e s p e c t i v e l y . I n f r a r e d (Nuiol M u l l ) : As shown in F i g u r e 17, a b s o r p t i o n at 3300 cm~l i n d i c a t e d the presence of N-H s t r e t c h i n g v i b r a t i o n s . The C=0 and C-N s t r e t c h i n g probably o c c u r r e d at 1680 and 1670 cm~l r e s p e c t i v e l y . The aromatic a b s o r p t i o n s o c c u r r e d at 700 and 730 cm - 1. Mass Spectrum (GCMS): The mass spectrum of t h i s ethanimine metabolite as shown in F i g u r e 16 agreed with p u b l i s h e d data (37). 2. 3-(2,6-Xvlvl)-5-methylhydantoin The s y n t h e t i c procedure r e p o r t e d by E l v i n (35) was m o d i f i e d . T o c a i n i d e base was used in place of a l a n i n e and 2,6-dime t h y l a n i 1 i n e . A phosgene s u b s t i t u t e , t r i c h l o r o m e t h y l c h l o r o f o r m a t e , was used in sodium d r i e d t o l u e n e . The r e s u l t i n g product gave a s i n g l e peak on the TIC from GCMS a n a l y s i s . I n f r a r e d (Nuiol M u l l ) : As shown in F i g u r e 18, a b s o r p t i o n at 3310 cm~l i n d i c a t e d the c y c l i c amide N-H s t r e t c h i n g . The 2300 cm~l(W) a b s o r p t i o n 77 100 J 1 1 1 1_ 200 scan Figure 15. The total-ion-chromatoqram of synthetic 2-ethanimine tocainide. 71 56 JLUL 190 120 i l . 4 I . I. « 3 I O I 120- 1 9 0 -218 L 50 100 ... 1 ,. 1 150 200 m/z Figure 16. The EI mass spectrum of 2-ethanimine tocainide. 78 Figure 17 : The infared spectrum of tocainide ethanimine (mull). 79 80 was due to the isocyanate N-C=0 s t r e t c h i n g . Both 1795 cm~l and 1700 cm~l were from the hydantoin r i n g 0=0 s t r e t c h i n g . These a b s o r p t i o n values were in agreement with p u b l i s h e d data (35). Mass Spectrum (GCMS): As shown in F i g u r e s 19 and 20, the hydantoin M + at m/z 218 with the base peak at m/z 147, and the co r r e s p o n d i n g fragmentation were i d e n t i c a l to E l v i n ' s p u b l i s h e d spectrum (35). 400 MHz-[XH]-NMR ( C D C I 3 ) : Table 5 o u t l i n e d the proton assignments. As shown i n Figu r e 21, the 400 MHz spectrum r e v e a l e d a doublet at 1.54 ppm due to s p l i t t i n g of the s i g n a l from the asymmetric C-5 methyl group pro t o n s . F u r t h e r d o w n f i e l d , at s h i f t 2.18 and 2.22 ppm, were the s i g n a l s of the x y l i d i d e methyl protons. With molecular modelling, the conformation of the hydantoin r i n g in r e l a t i o n to the x y l i d i n e r i n g was such that the c a r b o n y l s were c a u s i n g s t e r i c hindrance to one of the x y l i d i n e -CH^ groups, thus r e n d e r i n g the two methyl groups non-equivalent. T h i s i n t e r p r e t a t i o n proved to be h e l p f u l in determining the conformation of the t o c a i n i d e g l u c u r o n i d e which w i l l be d i s c u s s e d in l a t e r s e c t i o n s . The C-5 proton, at s h i f t 4.25 ppm formed a qu a r t e t due to a small quadrupole c o u p l i n g e f f e c t of the n i t r o g e n at r i n g - 1 - p o s i t i o n . The proton on the amide n i t r o g e n was s h i f t e d d o w n f i e l d to 6.58 ppm in t o a broad s i n g l e t , again due to the quadrupole e f f e c t of the n i t r o g e n atom. The aromatic protons were found between 7.1 and 7.3 ppm. The s i g n a l at 1.63 ppm was l i k e l y due to r e s i d u a l water present in the sample. 81 • I _ l I 1 1_ 100 200 scan F i g u r e 19 •. T o t a l - i o n - c h r o m a t o g r a m o f the s y n t h e t i c  3 - ( 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 218 147 44 119 105 JlLLj J 2Q3 I , J L , 50 100 150 200 m/z F i g u r e 20 : The EI mass spectrum of the s y n t h e t i c  3 - ( 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 82 J L A - r JL 5~ 4 ~ 3 2 1 e 21 : 400 MHz-^ "H-NMR spectrum of the synthetic * 3- (2,6-xylyl)-5-methylhydantoin (CDCU). Table 5 : Summarized data from the 400-MHz-[H]-NMR _ "spectrum of 3 - ( 2 , 6-xylvl)-S-methylhydantom, 7.1 -7.3 2.18 S h i f t (ppm) Coupling I n t e g r a t i o n (mm) Proton(s) Assignment (TMS) 0.0 5 - CH, 1.54d J . 6 Hz 60 3H(20mm/H) 2.18s* 63 3H x y l i d i n e - CH 3 2.22s* 63 3H x y l i d i n e - CH 3 4.25dq J - 8 Hz 19 1H 5 - CH 6.61 bs 20 1H 1 - NH 7.1 - 7.3 61 3H x y l i d i n e - H Note * non-equivalent (H) due to r i n g conformational hinderance. 83 3. 3 -(2,4,6-Xvlvl)-5-methylhydantoin The s y n t h e s i s of 3-(2,4,6-xy1y1)-5-methylhydantoin was c a r r i e d out using a 2-step approach. 2-amino-2',4*,6'-p r o p i o x y l i d i d e was f i r s t s y n t h e s i z e d from 2,4,6-trime thy1 a n i 1 i n e and N-CBZ-alanine using the procedures as d e s c r i b e d f o r the s y n t h e s i s of t o c a i n i d e . T h i s 2',4',6*-methy1 analog of t o c a i n i d e was then r e a c t e d with TCF to form the 3-(2,4,6-xy1y1)-5-methylhydantoin analog using the procedure as d e s c r i b e d f o r the 3-(2,4-xylyl)-5-methylhydanto i n . I n f r a r e d ( N u i o l M u l l ) : As shown in Fig u r e 22, the a b s o r p t i o n at 3200 cm-^" i n d i c a t e d amide N-H s t r e t c h i n g . The 1720 cm"^ and 1770 cm"-*-a b s o r p t i o n s were probably from r i n g C-0 s t r e t c h i n g . Furthermore, 700 cm"-'- and 750 cm--'- were i n d i c a t i v e of the aromatic a b s o r p t i o n . Mass Spectrum (GCMS)(silar-1 PC): The r e c r y s t a l 1 i z e d product appeared as a s i n g l e peak in the TIC as shown in F i g u r e s 23. The mass spectrum i n Fig u r e 24 i n d i c a t e d t hat a molecular ion at m/z 232 was observed as the base peak. Fragments at m/z at 161, 144, 146, 119 and 217 were found to agree with the c o r r e s p o n d i n g fragmentation as d e s c r i b e d f o r the 2 , 6 - x y l y l hydantoin analog. 8 4 Figure 22 : The infared spectrum of 3-(2,U ,6-xylyl)-5-methyl- hydantoin (mull). 85 / 100 200 scan F i g u r e 23 : T o t a l - i o n - c h r o m a t o g r a m o f the s y n t h e t i c 3 - ( 2 , 4 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 232 160 44 146 119 i i 50 100 217 150 200 m/z F i g u r e 24 : The EI mass spectrum o f the s y n t h e t i c 3 - ( 2 , 4 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 86 80 MHz-[ H1-NMR: As shown in Fi g u r e 25, the 80 MHz spectrum r e v e a l e d s i m i l a r proton s h i f t c h a r a c t e r i s t i c s as d e s c r i b e d f o r the 2,6-xyl y l - 5 - m e t h y l h y d a n t o i n analog. Table 6 o u t l i n e s the proton assignments. Table 6. Summarized data from the 80-MHz proton NMR spectrum of 3-(2.4.6-xv1v1)-5-methv1hvdantoin. Shift (ppm) Coupling Integration (mm) Proton(s) Assi gnment (TMS) 0.00 1.54 d J=6 Hz 60 3H 5-CH3 Z.18s 63 3H xylidine - CH 3 2.20s 63 3H xylidine - CH 3 2.3 2s 63 3H xyli d ine-CH, 4.25 dq J=8 Hz 19 1H 5-CH J 6.15bs 20 1H 1-NH 6.90s 40 2H xylidine - H 8 7 I I I I I 1 1 1 1 1 ' ! 1 6 5 4 3 2 1 PPm The 80-MHz-[HI-NMR spectrum of the synthetic "3- (2, 4 , 6-xylyl) -5-methylhydantoin (CDCl^). 88 C. T o c a i n i d e M e t a b o l i t e s 1. D e r i v a t i z a t i o n Techniques f o r Glucuronides A p e r m e t h y l a t i o n method has been d e s c r i b e d by Thompson and D e s i d e r i o (61) f o r the a n a l y s i s of i n t a c t e ther g l u c u r o n i d e s . T h i s permethy1 a t i o n procedure, using dime t h y 1 s u l f i n y 1 methide sodium (DMSO -) and methyl iodide ( C H ^ I ^ i s a u s e f u l technique f o r the a n a l y s i s of s a c c h a r i d e s , o l i g o p e p t i d e s and u r o n i c a c i d r e s i d u e s i n p o l y s a c c h a r i d e s (64,65). Various d e r i v a t i z a t i o n techniques have been p u b l i s h e d f o r GC or GCMS a n a l y s i s of i n t a c t g l u c u r o n i d e s . Fenselau (65) and Johnson reviewed these techniques f o r use with the a p p r o p r i a t e f u n c t i o n a l groups on the aglycone, as well as on the g l u c u r o n i c a c i d moiety. The types of d e r i v a t i z a t i o n reagents used f o r GC a n a l y s i s of g l u c u r o n i d e s a r e : 1. A c e t i c anhydride/methanesulfonic a c i d / d i azome thane. 2. T r i f 1 u o r o a c e t i c a n h y d r i d e / p y r i d i n e / d iazome thane. 3. Methyl Iodide/DMSO" ( p e r m e t h y l a t i o n ) . 4. B i s ( 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 r n i d e (BSTFA). 5. BSTFA/diazomethane. 6. T r i - S i l - Z R . I n i t i a l d e r i v a t i z a t i o n experiments of raw urine samples with T r i - S i l - Z , BSTFA and BSTFA with diazomethane d i d not y i e l d u s e f u l i n f o r m a t i o n f o r v a r i o u s known metab o l i t e s of t o c a i n i d e . However, the use of r e f e r e n c e compounds, P-nitr o p h e n o l g l u c u r o n i d e and D-glucose, in these r e a c t i o n s to monitor the d e r i v a t i z a t i o n c o n d i t i o n s proved the u s e f u l n e s s of these d e r i v a t i z a t i o n 89 techniques to o b t a i n molecular weight i n f o r m a t i o n f o r compounds of high molecular weight. The mass s p e c t r a of these r e f e r e n c e compounds are shown in F i g u r e s 26 and 27. With these data from the d e r i v a t i z e d sugar and P-nitr o p h e n o l g l u c u r o n i d e , we s p e c u l a t e d that the carbamoyl- or u r e i d o -g l u c u r o n i d e of t o c a i n i d e were probably h y d r o l y z e d e i t h e r d u r i n g r e a c t i o n or in the GC i n l e t . 2. I d e n t i f i c a t i o n of T o c a i n i d e Glucuronide by TLC  I s o l a t i o n and CHoI/Permethylation Band lb sample, ob t a i n e d a f t e r a 2-step TLC i s o l a t i o n , r e v e a l e d a s u b s t a n t i a l q u a n t i t y of f r e e t o c a i n i d e a f t e r a c i d h y d r o l y s i s . Enzyme h y d r o l y s i s of the same two bands r e v e a l e d a s l i g h t l y reduced q u a n t i t y of free t o c a i n i d e compared to that from a c i d h y d r o l y s i s . The presence of free t o c a i n i d e in the sample a f t e r enzyme h y d r o l y s i s i n d i c a t e d that the sample co n t a i n e d a g l u c u r o n i c a c i d conjugate of t o c a i n i d e . A f t e r the i s o l a t e d band lb was a d j u s t e d to pH 13 with sodium hydroxide, the sample was analyzed by re verse-phase HPLC using a mobile phase of 25 % a c e t o n i t r i t e in 0.05 M potassium a c e t a t e . T h i s sample r e v e a l e d the presence of 3-<2,6-xy1yl)-5-methy1hydantoin having an i d e n t i c a l r e t e n t i o n time to that of the s y n t h e t i c standard. The presence of t o c a i n i d e a f t e r b e t a - g l u c u r o n i d a s e h y d r o l y s i s , and the presence of the hydantoin d e r i v a t i v e a f t e r base h y d r o l y s i s suggest that the sample c o n t a i n e d a g l u c u r o n i c a c i d conjugate of t o c a i n i d e . In an attempt to examine the i n t a c t s t r u c t u r e of t h i s g l u c u r o n i d e , band lb was permethylated with 90 153 • • ' • • 6866-5566-4533-4oea-3533-3eee-2366-ie.ee-i8ee-583-141 3i i £3 / 2ee .. i . , 7 I O S I L GLUCOSE 25f> 388 356 • • ' • • 191 / ie.e 243 \ 361 271 2F;9 319 J L J i i L i i i ^ i . . L . 458 435 1: ee L'66 258 366 353 393 497 525 1 \ . i 436 453 5 6 « ^e -66 :73 e -33 26 13 F i g u r e 26 : The CI mass spectrum o f T M S - d e r i v a t i v e o f g l u c o s e 6 3 8 8 8 3 -5 5 8 8 8 3 -588888-456666-4 68836-358663-388666-? & 8 6 6 8 -286666-158888-188666-56686-8-169 : « 0 . i • . T f c l S J L PNPG 333 483 5 3 6 688 . . i . . 788 • • ' • • 37S 212 \ ?4e ?S4 333 46! 498 53T -1 £ l . . .1 676 D63 366 488 563 666 766 166 -98 -66 •76 •66 -56 -46 36 f-28 16 •-3 F i g u r e 27 : The CI mass spectrum o f T M S - d e r i v a t i v e o f p- n i t r o p h e n o l g l u c u r o n i d e . 91 1266866-1 n a c i a c t o - i 666666-468686-;eeeee-.77 1.4 4.f. &.2 .fc. 6.6 6.4 t-.P 7.2 .".t> 6.6 P . 4 & . & " l l , l u l « Figure 28 : Total-ion-chromatogram of permethylated XAD/crude urine extract i s o l a t e d band lb sample. >c an 1 4 4 56 166 i?6 i . . . . i . . . . i 44666-48666-36eee-32686-2£;66<>-24666^ 2&66en 1 6666-12866-2P.Pi • • • • • :-5e , i . . . . i , 366 3S6 466 i . . . . i . . . . i . . . . i , 98 165 4866H I 56 156 JL 2 W l / 1 177 / '.32 / 362 334 166 1S8 266 2&6 366 356 '(""!• • • • | ' 486 n e -166 -96 -66 -76 -66 6 -46 -36 -26 16 -6 Figure 29 : Mass spectrum of permethylated tocainide carbamoyl ester glucuronide (electron impact ionization). 92 methyl iodide in dry DMSO as d e s c r i b e d . The TIC and the mass spectrum of the permethylated g l u c u r o n i d e are shown i n F i g u r e s and 29. The p o s s i b l e mass ion fragments r e s u l t e d from the permethylated i n t a c t t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e are o u t l i n e d in Fig u r e 30. 93 Although the molecular ion of the t o c a i n i d e carbamoyl g l u c u r o n i d e was not observed, the m/z 277 ion was p r e s e n t . T h i s ion was most l i k e l y formed v i a the cleavage of the amide linkage at the s i t e of c o n j u g a t i o n . The m/z 334 ion may have been r e s u l t e d from the cleavage of the carbonyl bonding w i t h i n the t o c a i n i d e moiety. The m/z 232 ion was probably r e s u l t e d v i a cleavage of the g l u c o s i d i c bond accompanied by proton t r a n s f e r from the permethylated g l u c u r o n i d e moiety to the aglycone. The spectrum c o n t a i n i n g the mass ions m/z 232, 201, 169, 141, 161, 101 and 75 suggested that the permethylated sample was a g l u c u r o n i d e . These ions, r e s u l t e d from the cleavage of the g l u c u r o n i c a c i d moiety, are d i a g n o s t i c f o r the i d e n t i f i c a t i o n of permethylated g l u c u r o n i c a c i d . During mass s p e c t r a l a n a l y s i s , p - n i t r o p h e n o l g l u c u r o n i d e was used as a r e f e r e n c e compound to con f i r m our r e s u l t s with p u b l i s h e d data. For the permethylated p - n i t r o p h e n o l g l u c u r o n i d e , the mass s p e c t r a l a n a l y s i s was c a r r i e d out using the CI mode with methane as the reagent gas. The mass spectrum and t o t a l - i o n chromatogram are shown on F i g u r e s 31 and 32. The s t r i k i n g f e a t u r e s which were common to the s p e c t r a of these compounds were the p a i r of ions m/z 201 and 101. One of these two ions was present i n every spectrum. These ions have proved to be the most d i a g n o s t i c ions in i d e n t i f y i n g permethylated g l u c u r o n i d e s (65-68). 94 j 3686-12868-11866-9 8 8 6 -6 6 6 8 -• 7 6 6 6 -6 B P P -5 6 6 6 -3 8 6 8 -1666-6-/ 6 . 6 c:.6 9 . 6 1 0 . 6 F i g u r e 31 : T o t a l - i o n - c h r o m a t o g r a m o f p e r m e t h y l a t e d p a r a - n i t r o p h e n o l g l u c u r o n i d e . 126 1 6 8 i 3 6 6 - j H 326-1 246H 268-j - i 166-1 1 2 86-46-11 189 134 I ! 2 6 i 177 | ?48 ? P P 126 ~ T ~ " J76 '~ r r ' ' • ' T 24 6 528 ' 3 6 P ?66 ?96 r - , - r ^ -286 34 1 ' • 1 • ' • I 326 S c a n 336 4 P P 1661 (-96 f76 | I 1 j U . I L-;6 4 66 i v -26 366 I ' " I *' 468 F i g u r e 32 : The CI mass spectrum o f the p e r m e t h y l a t e d  p a r a - n i t r o p h e n o l g l u c u r o n i d e . 95 The molecular ion m/z 372 of the permethylated p - n i t r o p h e n o l g l u c u r o n i d e was pr e s e n t . The m/z 400 ion was formed from the C2H5 adduct. The m/z 340 ion was probably r e s u l t e d from the cleavage of a methoxy group, and m/z 308 was probably r e s u l t e d from the cleavage of a CH-OH group. Again the fragment at m/z 201 was present along with i o n s . a t m/z 169 and 232. A l l these three ions must have r e s u l t e d from the fragmentation of the permethylated g l u c u r o n i c a c i d moiety. As p o i n t e d out by Thompson (66), the M + ions of permethylated g l u c u r o n i d e s are not always present or d e t e c t e d by GCMS tec h n i q u e s . The peaks of m/z 277, 334 and 248 were mostly l i k e l y r e s u l t e d from the cleavages of the i n t a c t carbamoyl g l u c u r o n i d e . These ion fragments were of important d i a g n o s t i c value f o r the i d e n t i f i c a t i o n of permethylated t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e . The m/z 105 ion was a prominent ion from deamination of the x y l i d i d e p o r t i o n of t o c a i n i d e . T h i s l a t t e r ion fragment, together with the former ones d i s c u s s e d , suggested the g l u c u r o n i d e was a t o c a i n i d e carbamoyl e s t e r g l u c u r o n i d e . 3. Attempted I d e n t i f i c a t i o n of Toca i n i d e M e t a b o l i t e s by  L v o o h i 1 i z a t i o n Followed by GCMS (Perm e t h y l a t i o n ) The purpose of t h i s experimental p r o t o c o l was to i d e n t i f y conjugated m e t a b o l i t e s which were s u f f i c i e n t l y p o l a r to prevent s o l v e n t e x t r a c t i o n . From the TIC of the permethylated blank u r i n e sample, h i p p u r i c a c i d was i d e n t i f i e d as i t s methyl e s t e r . From the u n d e r i v a t i z e d l y o p h i l i z e d sample, a molecular ion at m/z 218 was i d e n t i f i e d to be the 3-(2,6-xy1yl)-5-methylhydantoin. No f u r t h e r u s e f u l data were ob t a i n e d d u r i n g 96 t h i s experiment. 4. Attempted I d e n t i f i c a t i o n of Met a b o l i c Intermediates of  Toca in ide A c c o r d i n g to e a r l i e r d i s c u s s i o n s , formation of the t o c a i n i d e g l u c u r o n i d e c o u l d take place through a carbamoyl or ureido i n t e r m e d i a t e . T h i s s e c t i o n d e s c r i b e s the attempted i d e n t i f i c a t i o n of these i n t e r m e d i a t e s from human u r i n e . As o u t l i n e d p r e v i o u s l y in Scheme 11, bet a - g l u c u r o n i d a s e enzyme was used to produce the i o n i z e d carbamic a c i d or urea-adduct i n t e r m e d i a t e . F o l l o w i n g h y d r o l y s i s , a c i d i c e x t r a c t i o n with methylene c h l o r i d e , and pe r m e t h y l a t i o n , the o b s e r v a t i o n of the carbamic a c i d intermediate by GCMS should be t h e o r e t i c a l l y p o s s i b l e . Conversely, a f t e r b a s i c e x t r a c t i o n with methylene c h l o r i d e and pe r m e t h y l a t i o n , the o b s e r v a t i o n of the urea-adduct intermediate should be p o s s i b l e . L a t e r s e c t i o n s w i l l d i s c u s s the s y n t h e s i s of t h i s urea-adduct and the GCMS data of t h i s permethylated h y p o t h e t i c a l i n t e r m e d i a t e . In s t u d y i n g the formation of unstable intermediates using enzyme h y d r o l y s i s , the s i t e of the enzymatic cleavage must be co n s i d e r e d . E i s e n b e r g , F. J r . (69) s t u d i e d the cleavage of benzoyl g l u c u r o n i c a c i d and methyl g l u c o s i d u r o n i c a c i d i n o x y g e n - l a b e l l e d a c e t a t e b u f f e r . From the d i s t r i b u t i o n of 0-^ i n the h y d r o l y z e d products, i t was i n f e r r e d that i n both s u b s t r a t e s the g l u c u r o n o s y l - 0 bond was c l e a v e d . V a r i a t i o n s i n pH from 4 to 6 had no e f f e c t on the isotope d i s t r i b u t i o n in the enzymatic r e a c t i o n . Non-enzymatic h y d r o l y s i s of both s u b s t r a t e s in st r o n g a c i d s a l s o r e s u l t e d in glucuronosyl-O-cleavage (69). 9 7 The i m p l i c a t i o n s of these f i n d i n g s , as they r e f l e c t on the p o s s i b l e i d e n t i f i c a t i o n of the t o c a i n i d e metabolic i n t e r m e d i a t e s , are o u t l i n e d in Scheme 15. With the p o s s i b l e enzymatic and non-enzymatic cleavages i n mind, i f the s p l i t t i n g occurs at the C-0 or the C-N bond, then e i t h e r the carbamoyl e s t e r or the ureido g l u c u r o n i d e would give r i s e to the same hyd r o l y z e d product. During t h i s experiment, the mass s p e c t r a of the permethylated t o c a i n i d e , p a r a - n i t r o p h e n o l g l u c u r o n i d e , 2,6-d i m e t h y l a n i l ine , l a c t o x y l i d i d e and the hydantoin were obtained as r e f e r e n c e s p e c t r a f o r p o s i t i v e i d e n t i f i c a t i o n . In F i g u r e s 33 and 34, the molecular ion of permethylated t o c a i n i d e at m/z 244 suggested a p e r m e t h y l a t i o n of 3 "CH^ groups, r e p l a c i n g 2 hydrogens from the primary amine n i t r o g e n and 1 hydrogen from the amide n i t r o g e n . The base peak at m/z 72 corresponded to the cleavage between C-2 and C-3 bond of permethylated t o c a i n i d e . In F i g u r e 35, the mass spectrum of the permethylated 2,6-d i m e t h y l a n i l i ne r e v e a l e d a s i m i l a r fragmentation p a t t e r n as was obtained from e a r l i e r d e u t e r a t i o n experiments. The molecular ion at m/z 149 i n d i c a t e d an a d d i t i o n of 2 "CH^ groups on the a n i l i n e n i t r o g e n . The permethylated l a c t o x y l i d i d e molecular i o n , as shown in F i g u r e s 36 and 37, was observed with a molecular ion a t m/z 221. The presence of m/z 134, as found in the spectrum of permethylated t o c a i n i d e , suggested a permethylated x y l i d i n e moiety. In a d d i t i o n , m/z 206 suggested that a [M-153 + ion was l o s t due to the cleavage of a -CH2 on the permethylated l a c t i c 98 A: Beta-glucuronidase enzyme h y d r o l y s i s . B: Basic h y d r o l y s i s (PH 12). Scheme 15. The t h e o r e t i c a l t o c a i n i d e intermediates der ived from the  h y d r o l y s i s o f the t o c a i n i d e g lucuron ide . 99 105 244 • II i . 100 200 m/ z F i g u r e 34 : The EI mass spectrum o f the p e r m e t h y l a t e d t o c a i n i d e . 100 149 154 138 125 L _ i y , I i J . 1 i' , [ i L_J. 100 150 200 m/z Figure 35 : The EI mass spectrum of the permethylated  2,6-dimethylaniline. 101 / 100 200 300 scan F i g u r e 36 : The t o t a l - i o n - c h r o m a t o g r a m o f the p e r r o e t h v l a t P H l a c t o x y l i d i d e .  F i g u r e 37 88 192 134 221 100 150 200 m/ z The EI mass spectrum o f the p e r m e t h y l a t e d  l a c t o x y l i d i d e . 102 a c i d moiety. The TIC and the mass spectrum of the permethylated 3-(2,6-x y l y l ) - 5 - m e t h y l h y d a n t o i n , as shown in F i g u r e s 38 and 39, c o u l d not be i n t e r p r e t e d . The TIC r e v e a l e d two symmetrical peaks of equal i n t e n s i t y and a mass s p e c t r a l fragmentation p a t t e r n which suggested the presence of a p a i r of d i a s t e r e o i s o m e r s . The base peak of m/z 135 may have r e s u l t e d from the permethylated x y l i d i n e , and the m/z 162 ion may have been due to the N-carboxyl fragment ( x y l i d i n e - N C H 3.CO-). However, an ion at m/z 282 d i d not correspond to any h y p o t h e t i c a l s t r u c t u r e . For ease of i n t e r p r e t a t i o n of the f o l l o w i n g data in t h i s s e c t i o n , d i s c u s s i o n of the experimental r e s u l t s w i l l f o l l o w the c o r r e s p o n d i n g numbering as shown in Table 7. T a b l e 7. The samples obtained from the experimental protocol used fo r  i d e n t i f y i n g t o c a i n i d e g lucuronide in te rmed ia tes . Sample Experimental (pooled urine sample) 1 c Extraced by RPC-18 elute-bond Column 2 Extracted at PH 12 3 Extracted at PH 3 k Enzyme hydrolyzed, extracted at PH 3 5 Enzyme hydrolyzed, extracted at PH 12 6 Acid hydrolyzed 7 Basic extracted PH 9, underivatized 8 Acidic extracted PH 3 9 Blank urine Note: A l l samples were permethylated with CH,I(CD,I)/ DMSO Na except Sample 7 and 8. 1 0 3 100 200 300 scan F i g u r e 38 : The t o t a l - i o n chromatogram o f the p e r m e t h y l a t e d  3- ( 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 135 72 Jul u J. U IJ 106 i . i i 50 100 152 235 282 150 200 250 m/z F i g u r e 39 : The EI mass spectrum o f the p e r m e t h y l a t e d  3 - ( 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . 104 Sample 1 ( e x t r a c t e d by RP-18 B o n d - e l u t e K column) and sample 9 (blank u r i n e ) r e v e a l e d a major component with a base peak at m/z 105. T h i s compound was i d e n t i f i e d to be R permethylated h i p p u r i c a c i d when compared with the S a d l t e r (70) r e f e r e n c e spectrum. C a f f e i n e was a l s o i d e n t i f i e d from i t s base peak at m/z 194. From sample 1, n e i t h e r t o c a i n i d e nor i t s m e t a b o l i t e s c o u l d be i d e n t i f i e d , p o s s i b i l y due to lack of s e n s i t i v i t y . The TIC from Sample 2 r e v e a l e d two components, both with molecular ion at m/z 135 and a base peak at m/z 88. These two components have a l s o been observed with i d e n t i c a l r e t e n t i o n times to those from the permethylated hydantoin sample. Acomponent at Scan 136 was found to c o n t a i n the m/z 162 i o n . T h i s was suspected to be the xy1idide-NCH3.CO- fragment, as observed from the permethylated hydantoin spectrum. However, the i d e n t i t y of t h i s compound c o u l d not be c o n c u l s i v e l y determined. The a c i d i c - e x t r a c t e d , and permethylated Sample 3, was found to c o n t a i n c a f f e i n e as the only i d e n t i f i a b l e component. Both Samples 4 and 5 a g a i n were found to c o n t a i n h i p p u r i c a c i d methyl e s t e r , with a molecular ion at m/z 206, and c a f f e i n e , with a molecular ion at m/z 194. Attempts to i d e n t i f y the t o c a i n i d e - u r e a , or the t o c a i n i d e - c a r b a m i c a c i d i n t e r m e d i a t e , were not s u c c e s s f u l . In the a c i d h y d r o l y z e d Sample 6, h i p p u r i c a c i d methyl e s t e r was p r e s e n t . Again, n e i t h e r of the t o c a i n i d e conjugated in t e r m e d i a t e s were observed. 105 In the u n d e r i v a t i z e d Sample 7, t o c a i n i d e and the hydantoin were i d e n t i f i e d with molecular ions m/z 192 and m/z 218, r e s p e c t i v e l y . C a f f e i n e with a molecular ion at m/z 194 was a l s o observed. The u n d e r i v a t i z e d , a c i d - e x t r a c t e d Sample 8, r e v e a l e d c a f f e i n e as the only i d e n t i f i a b l e compound. 5. I d e n t i f i c a t i o n of To c a i n i d e Glucuronide by F l a s h  Chromatography and C H 0 I / C D 3 I Permethylation I n i t i a l attempts to separate the c o l o r e d u r i n a r y components from drug m e t a b o l i t e s by the use of d i f f e r e n t chromatographic methods l e d to the use of f l a s h chromatography. This method has been shown to provide quick s e p a r a t i o n s of non-polar compounds and i s widely used i n the p u r i f i c a t i o n of s y n t h e t i c products. I n i t i a l chromatographic e v a l u a t i o n of the A c e t o n i t r i l e - XAD urine e x t r a c t with three TLC p l a t e s : KC-18F, alumina and s i l i c a g e l , r e v e a l e d that s i l i c a g e l separated the p o l a r u r i n a r y components, hence, f l a s h chromatography was c a r r i e d out using a dry-packed s i l i c a g e l column. The crude ACN-XAD e x t r a c t was e l u t e d from t h i s column as d e s c r i b e d in the Experimental S e c t i o n . The crude ACN-XAD e x t r a c t , p r i o r to e v a l u a t i o n by f l a s h chromatography, r e v e a l e d a prominent m/z 232 with a fragmentation p a t t e r n s u g g e s t i n g that the permethylated hydantoin was prese n t . No other s i g n i f i c a n t ions were i d e n t i f i e d in t h i s the sample. A f t e r f r a c t i o n 4 and 5 were c o l l e c t e d from the s i l i c a g el column, pooled and permethylated as d e c r i b e d , the r e s u l t i n g mass ion m/z 105 was found to be present in two peaks i n the TIC. A molecular ion at m/z 206 from one peak i n d i c a t e d the presence of h i p p u r i c a c i d . Another t o t a l ion peak with a molecular ion at 106 m/z 221 i n d i c a t e d the presence of the l a c t o x y l i d i d e . The use of CD3I d u r i n g another p e r m e t h y l a t i o n experiment confirmed the s t r u c t u r e s of these compounds. The TIC and mass spectrum of the permethylated, pooled f r a c t i o n s 6 to 12 are shown in F i g u r e s 40 and 41. At Scan 66 and 99, c a f f e i n e and h i p p u r i c a c i d were i d e n t i f i e d r e s p e c t i v e l y . At Scan 142, the base peak at m/z 147, with a molecular ion m/z 218, was i d e n t i f i e d to be the hydantoin. L a s t l y , at Scan 182, as shown in Figure 41, a base peak of m/z 98, with h i g h e s t mass d e t e c t e d at m/z 334, was found to be i d e n t i c a l to the proposed fragmentation p a t t e r n of the permethylated t o c a i n i d e e s t e r g l u c u r o n i d e as d e s c r i b e d in e a r l i e r d i s c u s s i o n s . P e r m e t h y l a t i o n experiments with CD3I confirmed the presence of 6 r e p l a c a b l e hydrogens on the t o c a i n i d e g l u c u r o n i d e a c c o r d i n g to the carbamate s t r u c t u r e . As shown in F i g u r e 42, the s t r u c t u r e of the t o c a i n i d e g l u c u r o n i d e o f f e r e d 4 r e p l a c a b l e hydrogens from the hydroxyl groups of g l u c u r o n i c a c i d moiety, p l u s two amide hydrogens from t o c a i n i d e . The CD3I permethylated sample i s shown in Figure 43. The fragment at m/z 334 suggested cleavage between the C2 and C 3 carbon of t o c a i n i d e c o n t a i n i n g 5 r e p l a c a b l e hydrogens. CD3I p e r m e t h y l a t i o n r e v e a l e d a corresponding 15 mass u n i t increase to m/z 349, s u g g e s t i n g the l a b e l l i n g of 5 methyl groups. Again, the ion fragment at m/z 277 from the (N-CO) cleavage c o n t a i n i n g 4 r e p l a c e a b l e hydrogens, showed a c o r r e s p o n d i n g 12 mass u n i t increase to m/z 288. The ion fragment at m/z 105 from the x y l i d i n e p o r t i o n of t o c a i n i d e c o n t a i n e d no r e p l a c e a b l e hydrogens, d i d not show any change in mass a f t e r p e r m e t h y l a t i o n 107 i _ J u l u F i g u r e 40 100 200 300 scan T o t a l - i o n - c h r o m a t o g r a m o f the pp.rmethvlated u r i n e . 46 ......J,....::. I i. If i Jll ll I, , illi 1,1 II -k + a Ji * 277 98 7? i II ul 1 0 5 156 143 116 201 172 . I I , i l l , .1, . i 4 lit Th-i n / Z 232 248 302 334 J * '* ^...^.1 dr * A r - ' - i r s » i t * r s r * > <kr • * r A- m/z F i g u r e 41 : The E I spectrum o f p e r m e t h y l a t e d t o c a i n i d e  carbamoyl e s t e r t o c a i n i d e (CH^ I). 108 F i g u r e 42. The EI fragmentation pat tern o f permethylated toca in ide carbamoyl  e s t e r g lucuron ide . 109 101 52 75 -..Jit:..- JJlJ jl.!!!]]!.:. .!# !.J|...:.,|,! Ll^ fl,...!..! 108 142 152 Ij i I! 213 m / z 288 244 2 5 7 .1 .d * 314 I 349 i d * > ar A- * r * r ar 4r sr ar ar ar * »>•• m / z F i g u r e 43. The EI mass spectrum of permethylated tocainide carbamoyl ester  glucuronide (CD^I). 110 with C D 3 I . L a s t l y , the permethylated g l u c u r o n i c a c i d moiety, with ion fragment m/z 232 c o n t a i n i n g 4 r e p l a c e a b l e hydrogens, showed an increase of 12 mass u n i t s to m/z 248, cor r e s p o n d i n g to the 4 methyl groups l a b e l l e d . The absence of a molecular ion from these experiments, as d e s c r i b e d b e f o r e , was probably due to the i n s t a b i l i t y of t h i s g l u c u r o n i d e under GC c o n d i t i o n s . I t should be noted that the samples were a l s o analyzed in the CI mode without o b s e r v i n g the molecular ion of the permethylated TOCG. Other f r a c t i o n s c o l l e c t e d from the s i l i c a - g e l column d i d not r e v e a l any a d d i t i o n a l u s e f u l i n f o r m a t i o n . 6. T o c a i n i d e - u r e a Reaction The r e a c t i o n between t o c a i n i d e and urea was examined at d i f f e r e n t urea c o n c e n t r a t i o n s and at d i f f e r e n t temperatures. The experimental r e s u l t s are summarized i n Table 8. Table 8 : The Reaction of Tocainide with Urea at Different Temperatures Tenp. Time GOB Data Tocainide (m/Z 192) Hydantoin (m/Z 21£ Tocainide (base) Urea (8.0 M) 100°C 1 hr. 9 hrs. + + + + 60°C 13 hrs. 3 hrs, • Blank Urine 25°C 100°C 10 hrs, 1 hr. + <*> 100°C 9 hrs. + Distilled Water 100°C 13 hrs, -Mexilitine (base) Urea Distilled 100°C 13 hrs, Mexilitine Methanimine WZ Water 100°C 13 hrs, Mexilitine I l l In f r e s h , drug-free urine at room temperature (25 C), t o c a i n i d e d i d not r e a c t with urea to any measurable e x t e n t . Thus, the hydantoin was not l i k e l y to a r i s e as an a r t i f a c t formed d u r i n g sample s t o r a g e . In the presence of 8 M urea at 100 °C f o r 13 hours, almost q u a n t i t a t i v e c o n v e r s i o n of t o c a i n i d e to the hydantoin was observed. In a d d i t i o n , when t o c a i n i d e was heated at 100 °C f o r 1 hour in f r e s h drug-free u r i n e , the hydantoin c o u l d a l s o be d e t e c t e d . As shown in Table 9, formation of the hydantoin favored n e u t r a l to a l k a l i n e c o n d i t i o n s . At a c i d i c pH, the i o n i z e d t e r m i n a l amine of t o c a i n i d e probably r e p e l l e d the e q u a l l y charged amino group of urea p r e v e n t i n g condensation. T a b l e 9 J The Reaction of Tocainide With Urea at Different pH f* (80/20: Methanol/H20) PH Tocainide Hydantoin Suspected Tocainide-N-Ureide 2.5 0.45 0.75 _ 7.8 0.45 0.75 0.6 12.5 0.45 0.75 0.6 Note: * KC-18 TLC. 112 f From these experiments, the formation of the hydantoin was r e p e a t e d l y demonstrated. T h i s hydantoin was thought to be evol v e d through a t o c a i n i d e ureide i n t e r m e d i a t e . Subsequently, p e r m e t h y l a t i o n of the r e a c t i o n mixture, which c o n t a i n e d t h i s i n t e r m e d i a t e , r e v e a l e d the presence of the t o c a i n i d e - N - u r e i d e as shown in Fi g u r e 44. The molecular ion of the permethylated t o c a i n i d e - N - u r e i d e at m/z 291 was observed with t r a c e i n t e n s i t y . As shown in Figure 45, a base peak at m/z 72 i n d i c a t e d the permethylated (-CO.NCH^CH^-) fragment, whereas m/z 129 corresponded to the permethylated C 2~ C 3 c l e a v a 9 e product of the t o c a i n i d e - N - u r e i d e . The i d e n t i f i c a t i o n of t h i s t o c a i n i d e - N - u r e i d e suggested that such an intermediate can occur at near n e u t r a l pH. 7. S y n t h e s i s of N-glucuronides The attempted chemical s y n t h e s i s of t o c a i n i d e - N - u r e i d e g l u c u r o n i d e f o l l o w i n g the procedures of A r i t a (46), Tsakamoto (47), I s h i d a t e (48) and T a k i t a n i (49) was not s u c c e s s f u l , as evidenced by p e r m e t h y l a t i o n f o l l o w e d by GCMS a n a l y s i s . These methods have p r e v i o u s l y been shown to be s u i t a b l e f o r N-glucuronide condensation ( s e c t i o n 4b). Experiments with 2,6-dimethylani1ine using the p u b l i s h e d procedures of A r i t a (46) y i e l d e d the permethylated 2,6-dimethy1 ani 1 i n e - N - g l u c u r o n i d e molecular ion at m/z 353 as shown in F i g u r e s 46 and 47. However, t h i s permethylated a n i l i n e g l u c u r o n i d e was not observed in any permethylated urine samples from v o l u n t e e r s who had ingested t o c a i n i d e . 113 100 200 300 scan F i g u r e 44 : T o t a l - i o n - c h r o m a t o g r a m o f the p e r m e t h y l a t e d t o c a i n i d e - N - u r e i d e . 72 a L 129 AX 191 291 100 150 200 250 'm/ z 50 F i g u r e 45 : The EI mass spectrum o f the p e r m e t h y l a t e d t o c a i n i d e - N - u r e i d e . 114 F i g u r e 46 100 200 scan The t o t a l - i o n - c h r o m a t o g r a m o f the p e r m e t h y l a t e d  2 , 6 - d i m e t h y l a n i l i n e - N - g l u c u r o n i d e . 103 76 uuLJi 135 1 1 219 353 50 00 150 200 250 300 350 m/ z F i g u r e 47 : The EI mass spectrum of the p e r m e t h y l a t e d 2 , 6 - d i r n e t h y l a n i l i n e - N - g l u c u r o n i d e . 115 D. HPLC I s o l a t i o n and S t r u c t u r a l E l u c i d a t i o n of To c a i n i d e  Glucuron ide Thus f a r , from p r e v i o u l y d i s c u s s e d mass s p e c t r a l data, the s t r u c t u r e of TOCG was e s t a b l i s h e d based on the mass fragments from the permethylated TOCG. The i n t a c t molecular s t r u c t u r e of TOCG was not observed. T h e r e f o r e , p r e p a r a t i v e HPLC was employed to i s o l a t e l a r g e r q u a n t i t i e s of t h i s conjugate f o r NMR and FAB s p e c t r a l measurements. Scheme 16 summarizes the experimental steps i n v o l v e d in the i s o l a t i o n procedure. 1. P r e p a r a t i v e HPLC I s o l a t i o n To concentrate urine samples f o r p r e p a r a t i v e HPLC, 10 % a c e t o n i t r i l e was added f o r a z e o t r o p i c e v a p o r a t i o n of the urine sample under vacuum. The s o l v e n t admixture o f f e r e d reasonable time (2 hours) to concentrate 100 mL urine to 10 mL, compared to l y o p h i l i z a t i o n which r e q u i r e d 12 hours f o r the same volume. Chromatographic e v a l u a t i o n of the co n c e n t r a t e d urine sample was c a r r i e d out using a mobile phase of 10 % a c e t o n i t r i l e in 0.01 M sodium phosphate b u f f e r , a d j u s t e d to pH 3 with phosphoric a c i d . D e t e c t i o n was at UV 226 nm and column e l u a n t was c o l l e c t e d in 2 mL f r a c t i o n s . Figure 48 and 49 show the chromatogram at 226 nm and the UV absorbance of each i n d i v i d u a l f r a c t i o n at 226 nm, r e s p e c t i v e l y . F r a c t i o n 4 was found to y i e l d the hydantoin a f t e r a l k a l i n e h y d r o l y s i s . T e s t i n g of f r a c t i o n 4 by n a p h t h o r e s o r c i n o l r e a c t i o n on a TLC p l a t e r e v e a l e d a blue c o l o r , i n d i c a t i n g the presence of a g l u c u r o n i c moiety. Subsequent to t h i s f i n d i n g , f r a c t i o n 4 was c o l l e c t e d over 116 (16 hr) RE urine (100 ml) XAD-2 resin azeotrophic evaporation in vacuo with ACN 1 I adjusted to PH 3 extracted with extracted with CH2C12 at PH 7 CH 2 C 1 2 extracted with hexane at PH 3 preparative HPLC, partisil-10 ODS-3, 10 uir(9 arr. id-, x 25 cm) J, 1st separation 10 i ACN in 0.01 M NaK.,PO, at PH 3 (2.5-3 mL/min.) i Fractions collection, 2 mL —j j j — j I basic naphthoresorcinol acid beta-glucjror.idase 4- hydrolysis testing hydrolysis hydrolysis (with/vitho-j:) Collected fraction T T * sacchro-l.t-lactor J, 2nd separation 3 Z ACN in 0.01 K NaK ,PC at P H 7 I Fractions collection, 2 mL I Collected fraction containing ToCG Azeotrophic evaporation J-Estimated yield: 1 mg glucuronide/86 rag NaH^ PO^  > FAB 3rd separation 3 ! ACN in 0.3 2 acetic acid at PH 7 I Collected ToCC in Na acetate J. adjust to PH 3 (HC1) evaporation in vacuo I ToCG in NaCl * FAB NMR Scheme 16 : The experimental steps involved in i s o l a t i n g the  t o c a i n i d e g lucuron ide . 117 Column: Whatmann Partisil-10 Magnum -9, 9mm id. x 25 cm UV detection: 226 nm 10Z ACN in 0.01 M Na^PO^ (PH 3) flow rate: 2.7 mL/min 6 12 18 24 30 36 42 Figure 48 : Preparative HPLC chromatogram of crude urine. m m , 2 mL f r a c t i o n s UV a b s o r b a n c e s : 225 nm 2 1 f r a c t i o n F i g u r e 49 : UV absorbance o f 2 mL f r a c t i o n s c o l l e c t e d  from p r e p a r a t i v e HPLC. 118 36 r e p e t i t i v e a n a l y s e s . To f u r t h e r r e s o l v e the u r i n a r y components w i t h i n t h i s c o l l e c t e d f r a c t i o n , a mobile phase composition of 3 % a c e t o n i t r i l e in 0.01 M sodium phosphate b u f f e r , a d j u s t e d to pH 7 with sodium hydroxide, was used. The r e s u l t i n g chromatogram using UV d e t e c t i o n at 220 nm i s shown on F i g u r e 50. F r a c t i o n 4 was found to give p o s i t i v e r e s u l t s with b a s i c , a c i d i c and enzymatic h y d r o l y s i s , as well as showing p o s i t i v e n a p h t h o r e s o r c i n o l c o l o r t e s t . Furthermore, the UV spectrum of f r a c t i o n 4 was found to be i d e n t i c a l to that obtained from t o c a i n i d e with a X max at 205 nm. Subsequently, f r a c t i o n 4 was c o l l e c t e d over 42 i n j e c t i o n s , c o n c e n t r a t e d by a z e o t r o p i c e v a p o r a t i o n , and rechromatographed as shown in F i g u r e 51, showing 91 % p u r i t y . A f t e r e v a p o r a t i o n to dryness under vacuum, the sample y i e l d e d 62 mg of t o c a i n i d e glucuronide/sodiurn phosphate s a l t . I n i t i a l GC assay of the urine sample estimated a c o n c e n t r a t i o n of 67 ug/mL (30 mole/mL) of the hydantoin. According to the 100 mL urine used f o r i s o l a t i o n , 12.3 mg (30 umoles) of t o c a i n i d e g l u c u r o n i d e c o u l d t h e o r e t i c a l l y be i s o l a t e d . To f u r t h e r remove the phosphate s a l t , the s a l t sample was r e c o n s t i t u t e d in water and chromatographed using 10 % a c e t o n t r i l e i n 0.03 % a c e t i c a c i d , a d j u s t e d to pH 4.3 using 1.0 M sodium hydroxide. UV d e t e c t i o n was at 225 nm as shown in F i g u r e 52. The r e s u l t i n g conjugate was i s o l a t e d in sodium ac e t a t e s a l t . The acetate was removed as i t s f r e e a c i d by 119 Column: Whatmann Partisil-10 Magnum -9, 9mm id. x 25 cm UV detection: 220 run 3Z ACN in 0.01 M Nar^PO^ (PH 7) flow rate: 2.7 mL/min min. Figure 50 : Preparative HPLC chromatogram of recycled  TOCG containing f r a c t i o n . 10 15 min. Figure 51 : Preparative HPLC chromatogram of recycled  TOCG i n sodium phosphate saTt~. 120 ft Column: Whatmann P a r t i s i l - 1 0 Magnum-9, 9mm i d . x 25 cm UV detection : 225 nm 10% ACN i n 0.3% acetic acid (PH 4.3) flow rate: 3.0 mL/min 10 20 m m . Figure 52 : Preparative HPLC chromatogram of recycled TOCG i n sodium chloride s a l t . 121 a d j u s t i n g the pH to 2.8 with 1.0 M h y d r o c h l o r i c a c i d and eva p o r a t i n g under vacuum. This sample was s t o r e d i d e s i c c a t e d at -10 °C. 2. EI Mass S p e c t r a l A n a l y s i s of the Toca i n i d e Glucuronide Attempts to e l u c i d a t e the s t r u c t u r e of the i s o l a t e d g l u c u r o n i d e by GCMS d e r i v a t i z a t i o n methods were not s u c c e s s f u l . 3. 400 MHz Proton Nuclear Magnetic Resonance Experiment The i n t e r p r e t a t i o n of the t o c a i n i d e g l u c u r o n i d e NMR spectrum was s i m p l i f i e d with the a i d of the r e f e r e n c e s p e c t r a of t o c a i n i d e and g l u c u r o n i c a c i d obtained i n the same s o l v e n t . Figure 53 and 54 show the 400 MHz proton s p e c t r a of t o c a i n i d e before and a f t e r deuterium exchange. Fi g u r e 55 shows the spectrum of g l u c u r o n i c a c i d . Table 10 summarizes the proton assignments of the t o c a i n i d e spectrum. F i g u r e 56 and 57 show the 400 MHz proton NMR spectrum of the i s o l a t e d t o c a i n i d e g l u c u r o n i d e in D^-DMSO. The proton-NMR assignments are summarized in Table 11. The doublet s i g n a l observed at 1.38 ppm was i n t e g r a t e d f o r 3 protons and i t s chemical s h i f t was found to correspond to the c h i r a l methyl group of t o c a i n i d e (1.54 ppm). The s i n g l e t at 2.11 ppm was i n t e g r a t e d f o r 6 methyl groups, a c c o u n t i n g f o r the two x y l i d i n e CH^ groups. Chemical s h i f t at 3.69 ppm, 3.92 ppm, and 5.77 ppm were found to correspond to the s i g n a l s obtained from the r e f e r e n c e g l u c u r o n i c a c i d spectrum. Furthermore, the s i g n a l s at 7.06 ppm and 9.39 ppm were assigned as the aromatic protons of the x y l i d i n e r i n g and the xy l i d i n e - N H group, r e s p e c t i v e l y . 122 ppm F i g u r e 53 : 400-MHz H-NMR spectrum of t o c a i n i d e (D.-DMSO) " — . ' A ppm F i g u r e 54 : 400-MHz-1H-NMR spectrum of t o c a i n i d e a f t e r D^ O exchange (D^-DMSO). 123 124 T a b l e 10 Summarized data from the 400-MHz proton NMR spectrum of t o c a i n i d e . S h i f t (ppm) Coupling I n t e g r a t i o n (mm) Proton(s) Assignment TMS (0.0) TMS 1.54d J - 7.2 Hz 36 3H(12mm/H) 1 - CH 3 2.16s 72 6H x y l i d i n e - CH 3 2.51s DMSO 3.30s HjO 4.12bm 12 1H 2 - CH (due to quadrapol broadening by -N 7.10s 35 3H x y l i d i n e - H 8.25 bm 32 2H exchange with D 20 (2-amino-NHj) 9.85bs 10 1H exchange with D 20 ( x y l i d i n e -NH) 125 1 J A 1 . ** 9 8 7 6 i . . J * L J J, , — ( • ' ] F i g u r e 56 : 400 MHz-^H-NMR spectrum o f t o c a i n i d e carbamoyl  e s t e r g l u c u r o n i d e (D^-DMSO). 1 JL 7 6 5 4 3 2 1 1 F i g u r e 57 400 MHz- H-NMR spectrum o f t o c a i n i d e carbamoyl  e s t e r g l u c u r o n i d e ( 8 X i n t e n s i t y / D^-DMSO). P 126 T a b l e 11 Summarized data from the 400-MHz proton NMR spectrum of the  isolated tocainide carbamoyl ester glucuronide. OH H Shift (ppm) Coupling Integration (mm) Proton(s) Assignment 1 .36d J • 6.6 Hz 26 3H(8mm/H) 1 - CH3 (Tocainide) 2 . l i s 56 6H(8mm/H) xylidine - CH3 2 .51 DMSO 3 .34 H20 3.68 > 3 3.7l' i .69d J = 9 Hz 16 2 3 55 Glucuronic acid^'53 3 .92d J = 9 Hz trace 2 3 99 Glucuronic a c i d ^ ' ^ U .25t 7 1H 2 - CH (Tocainide) 5 .30d J = 8 Hz 16 5 .77s 10 2 Glucuronic acid 5.72 7 .06bs 27 3H xylidine - H 7 .63d J = 7.2 Hz 9 7 .77bs 3 9 .39bs 8 1H xylidine - NH Note: 1. Singlet, indicating straight chain conformation. 2. Clin Chem 26/9: 1323-1335 (1980) (Wu, et. al.). 127 These proton NMR s i g n a l s from the i s o l a t e d g l u c u r o n i d e have p a r t i a l l y suggested the presence of a g l u c u r o n i c a c i d conjugate of t o c a i n i d e . However, the anomeric proton s i g n a l was not determined. Moreover, as shown in Figure 55, the r e f e r e n c e g l u c u r o n i c a c i d spectrum c o n t a i n e d many broad s i n g l e t s from 6 to 11 ppm which were absent in the r e f e r e n c e g l u c u r o n i c spectrum. One p l a u s i b l e e x p l a n a t i o n f o r the l o s s of the g l u c u r o n i c a c i d s i g n a l s was the i n s u f f i c i e n t c o n c e n t r a t i o n of TOCG in the sample submitted f o r a n a l y s i s . 4. Fast Atom Bombardment A n a l y s i s The i s o l a t e d t o c a i n i d e g l u c u r o n i d e in sodium c h l o r i d e submitted f o r FAB a n a l y s i s in both g l y c e r o l and t h i o g l y c e r o l m a t r i c e s . F i g u r e s 58 and 59 show the FAB s p e c t r a of the conjugate in g l y c e r o l and t h i o g l y c e r o l , r e s p e c t i v e l y . In both s p e c t r a , a prominent i n t e n s i t y of the 457 m/z ion adduct was observed, suggesting an a d d i t i o n of 2 sodium ions onto the [m-H] + molecular ion of 411 m/Z. The e q u a l l y intense ion at 435 m/z from the two s p e c t r a suggested the presence of the tM+Na] + adduct. In the s p e c t r a , the 1M+1]+ adduct at 413 m/Z was a l s o observed. As shown i n Figure 60, the fragmentation p a t t e r n of the conjugate would l i k e l y to give r i s e to the s u c c e s s i v e mass ions a t m/Z 235, 236, 237, and 239. These ion fragments were of d i a g n o s t i c value in determining the carbamoyl e s t e r versus the ureide s t r u c t u r e . As shown in Figure 61, when the mass a n a l y z e r was ad j u s t e d to observe the low mass ions, the fragment ion at m/z 221 was 128 F i g u r e 58 : The fast atom bombardment spectrum of toca in ide carbamoyl e s t e r glucuronide in g lycero l matrix with sodium c h l o r i d e . (237, +2Na) 412 m/Z 193 (239, +2Na) F i g u r e 60 The fragmentation pattern of tocainide carbamoyl ester glucuronide  in fast atom bombardment mass spectrometry. 130 237 239 CO 189 221 iiHi.iii hi i i i 235 \ 295 297 247 279 327 403 N F i g u r e 61 : The f a s t atom bombardment spectrum of t o c a i n i d e carbamoyl e s t e r  g l u c u r o n i d e i n t h i o g l y c e r o l m a t r i x w i t h low mass i o n m o n i t o r i n g . found to correspond to the e s t e r g l u c u r o n i c moiety (-C0.0-GA). Ions at m/z 237 and m/z 239 were found to correspond to the 2Na ion adduct of t o c a i n i d e (toc-H+2Na) and of g l u c u r o n i c a c i d (0-GA+2Na) r e s p e c t i v e l y . C o r r e s p o n d i n g l y , m/z 295 and m/z 297 were probably a r r i v e d from the (2Na+NacL) ion adducts. These FAB fragmentation ions from the i s o l a t e d TOCG have pro v i d e d d e f i n i t i v e evidence f o r a carbamoyl e s t e r s t r u c t u r e f o r TOCG. In a d d i t i o n , the o b s e r v a t i o n of the i n t a c t molecular ion at m/z 412 with i t s c h a r a c t e r i s t i c ion adducts at m/z 435, and m/z 457 agreed with the carbamoyl e s t e r s t r u c t u r e of molecular weight 412.395 g/mole. 132 E. Pharmacokinetics of T o c a i n i d e Glucuronide To f o l l o w the k i n e t i c p r o f i l e of t o c a i n i d e g l u c u r o n i d e in u r i n e , an i n d i r e c t b a s i c h y d r o l y s i s assay was used as d e s c r i b e d by Hoffmann (60). The method r e q u i r e d i n i t i a l h y d r o l y s i s of the t o c a i n i d e g l u c u r o n i d e to i t s hydantoin at pH 12, f o l l o w e d by q u a n t i t a t i o n of the hydantoin. 1. T o c a i n i d e Glucuronide B a s i c H y d r o l y s i s K i n e t i c s The h y d r o l y s i s - t i m e p r o f i l e of TOCG in a urine sample obtained from a v o l u n t e e r who had been gi v e n 200mg t o c a i n i d e o r a l l y i s shown in Fig u r e 62. From the semi-log p l o t , as shown in Figu r e 63, i t can be i n f e r r e d that the h y d r o l y s i s of t o c a i n i d e g l u c u r o n i d e followed the r e a c t i o n Scheme 17 below. Both k-^  and k 2 are f i r s t - o r d e r r a t e c o n s t a n t s . 1 K 2 ToCG > h y d a n t o i n > d e g r a d a t i o n product Scheme 17 Schematic r e p r e s e n t a t i o n o f the k i n e t i c s of ToCG h y d r o l y s i s  i n the presence of NaOH. 133 c o C5 Cy Cr 0.50 M NaoH 0.25 M NaoH 10 15 — i — 20 — i — 25 — i — 30 m i n u t e F i g u r e 62 The hydrolysis profile of tocainide glucuronide and the hydantoin in the presence of sodium hydroxide. 134 • 0.5 M NaoH O 0.25 M NaoH D 0.05 M NaoH F i g u r e 63 Semi- log p lo t o f the t o c a i n i d e glucuronide and the hydantoin h y d r o l y s i s k i n e t i c s in var ious concentrat ions o f sodium hydroxide. 135 During the i n i t i a l 7 minutes of h y d r o l y s i s , i t was evident that the amount of the hydantoin was i n c r e a s i n g , thereby i n d i c a t i n g a p o s i t i v e k-^  . However, as the amount of t o c a i n i d e g l u c u r o n i d e was d e p l e t e d , and as k-^  approached zero, the h y d r o l y s i s of the hydantoin became apparent. To demonstrate that V<2 r e p r e s e n t e d the d e g r a d a t i o n r a t e constant of the hydantoin, a s y n t h e t i c sample, h y d r o l y z e d as d e s c r i b e d , r e v e a l e d an i d e n t i c a l \<2 value compared to that obtained from urine samples. H y d r o l y s i s experiments of the s y n t h e t i c hydantoin in 1.0 M, 0.1 M and 0.05 M NaOH r e v e a l e d a p a r a l l e l p s e u d o - f i r s t -order d e g r a d a t i o n k i n e t i c s , with r a t e c o n s t a n t s ranging from 0.0122 to 0.0241 min as shown in F i g u r e 64. 2. H y d r o l y s i s of 3-<2,4.6-Xvlvl)-5-methvlhvdantoin According to Hoffmann's p u b l i s h e d assay, i f the i n t e r n a l standard 3-<2,4,6-xylyl)-5-methylhydantoin (4'-methylhydantoin) followed the same p s e u d o - f i r s t - o r d e r h y d r o l y s i s k i n e t i c s as the TOCG d e r i v e d hydantoin, then the change in r e l a t i v e amounts of the metabolite hydantoin versus the 4'-methylhydantoin, as r e f l e c t e d by the peak height r a t i o , would r e v e a l l i t t l e change with time. However, Hoffmann was not aware of the h y d r o l y t i c nature of the hydantoin i n base. To demonstrate t h i s r e l a t i o n s h i p , the 3-<2,4,6-xylyl)-5-methylhydantoin was s y n t h e s i z e d and s u b j e c t e d to the same h y d r o l y s i s experiment as d e s c r i b e d , but using e t i d o c a i n e as an i n t e r n a l standard. The r e s u l t s are a l s o shown i n F i g u r e 65. The 4'-methylhydantoin was found to e x h i b i t p s e u d o - f i r s t - o r d e r d e g r a d a t i o n k i n e t i c s with a r a t e constant of 0.0122 min ^ . 136 o o 1.0 M NaoH 0.10 M NaoH 0.05 M NaoH o c c c cr o c CC u cc o I-J* cc Cv a. 10 15 20 25 —i— 30 n i n u t e s F i g u r e 64 Semi- log p lo t o f the h y d r o l y s i s o f 3- f2.6-xv1v1)-5-methv1hvdantoin i n var ious concentra t ions o f sodium hydrox ide r 137 •jk '-methylhydantoin/etidocaine + hydantoin/A'-methylhydantoin 1 1 — — 1 — — — • 1 1 1 1 1 U 8 12 16 20 24 28 32 m i n u t e s F i g u r e 65 The hydrolysis of tocainide olucuronide and the 4'-methylhydantoin in the presence of 0.5M sodium hydroxide. 138 s i m i l a r to those obtained from the m e t a b o l i t e - d e r i v e d hydantoin. The peak h e i g h t r a t i o of the hydantoin/4*-methylhydantoin obtained from the urine of a v o l u n t e e r who had been given t o c a i n i d e , r e v e a l e d an i n i t i a l hydantoin formation phase, at which k^ i s very much g r e a t e r than k2« As k^ approached zero, the peak height r a t i o of the hydantoin to the 4'-methylhydantoin i n t e r n a l standard approached a constant as shown in F i g u r e 65. In essence, the peak height r a t i o of the hydantoin/4'-methylhydantoin over time should t h e o r e t i c a l l y experience a gradual d e c l i n e depending on the r a t e of h y d r o l y s i s of the two compounds. As shown in F i g u r e 66A, the p a r a l l e l f i r s t - o r d e r d e c l i n e of the hydantoin and 4'-methylhydantoin would r e f l e c t a very gradual f a l l in peak area r a t i o of the hydantoin depending on the steepness of the f i r s t - o r d e r d e c l i n e as d e p i c t e d in Figure 66B. 3. C a l i b r a t i o n Curve Data E t i d o c a i n e was found to be s t a b l e at pH 12 and was used as an i n t e r n a l standard. To maintain r e p r o d u c i b i l i t y and to assay the l e v e l s of the hydantoin i n the presence of i t s spontaneous h y d r o l y s i s , an a c c u r a t e l y timed assay p r o t o c a l was r e q u i r e d . Since urine samples c o n t a i n e d v a r y i n g amounts of t o c a i n i d e g l u c u r o n i d e , a predetermined h y d r o l y s i s time had to be choosen f o r both the c a l i b r a t i o n curve and the k i n e t i c urine samples. As shown in F i g u r e 66D, at any time > t , the q u a n t i t y of hydantoin i n a u r i n e sample can be approximated by an i d e n t i c a l l y timed c a l i b r a t i o n sample, as d e p i c t e d in Figure 66C. Since at time 139 Figure 66 Theoretical p r o f i l e s of the 3-(2,6-xylyl)-5-methylhydantoin hydrolysis using both etidocaine  and 3-(2,4,6-xylyl)-5-methylhydantoin as  int e r n a l standards. A: Peak area-time p r o f i l e of  the hydantoin and the 4'-methylhydantoin. B: The  peak area r a t i o of the hydantoin to the 4'-methyl- lydantoin. C: Peak area-time p r o f i l e s of etidocaine  and the hydantoin. D: The peak area r a t i o of the  hydantoin to etidocaine. 140 zero, i n a urine sample, u n l i k e a c a l i b r a t i o n sample, only a small p o r t i o n of the t h e o r e t i c a l amount of hydantoin was s u b j e c t e d to h y d r o l y s i s , t h i s a c c u r a t e l y timed p r o t o c o l p rovided a c l o s e approximation f o r the l e v e l s of hydantoin in u r i n e . If the h y d r o l y s i s of the hydantoin was not accounted f o r by the use of an a c c u r a t e l y timed c a l i b r a t i o n samples, then the l e v e l s of TOCG in the u r i n e , as r e f l e c t e d by the l e v e l s of the hydantoin, c o u l l d not be c o r r e c t l y determined. C a l i b r a t i o n data are t a b u l a t e d in Table 12. The assay included the use of 4'-methylhydantoin as a second i n t e r n a l standard to q u a n t i t a t e the amount of hydantoin i n the u r i n e . L i n e a r r e g r e s s i o n of the 3 c a l i b r a t i o n curves r e v e a l e d a c o e f f i c i e n t of d e t e r m i n a t i o n of 0.9974, 0.9997 and 0.9943 f o r t o c a i n i d e / e t i d o c a i n e , h y d a n t o i n / e t i d o c a i n e , and hydantoin/4'-methylhydantoin r e s p e c t i v e l y . I n t e r - and i n t r a - a s s a y v a r i a b i l i t y were determined using the c a l i b r a t i o n samples. The data are t a b u l a t e d in Tables 13 and 14. For t o c a i n i d e , i n t r a - a s s a y v a r i a t i o n s were expressed as % C.V. between d u p l i c a t e d e t e r m i n a t i o n s of 6 samples with c o n c e n t r a t i o n s r a n g i n g from 6 to 20 ug/mL. The % C.V. was found to range from 0.05 % to 1.76 %. I n t e r - a s s a y v a r i a t i o n s expressed as % C.V. from 3 samples in the same c o n c e n t r a t i o n range were found to range from 0.04 % and 1.75 %. For the hydantoin, i n t r a - a s s a y v a r i a b i l i t y was higher from 0.13 to 0.05 %, whereas i n t e r - a s s a y v a r i a b i l i t y was ranged from 1.94 % to 8.59 %. This higher v a r i a b i l i t y i n the hydantoin assay of the hydantoin was probably due to the time dependent v a r i a t i o n in the q u a n t i t y of the 141 T a b l e 12 C a l i b r a t i o n curve data f o r toca in ide and the hydantoin using  e t idoca ine and the 4 ' -methylhydantoin in te rna l s tandards . Concentration^ 2 Tocainide 2 Hydantoin 3 Hydantoin (ug/mL) (avg.+S.D.) (avg.+S.D.) (avg.+S.D.) 1 0.064+0.005 0.047+0.010 0.067+0.011 3 0.226+0.009 0.166+0.022 0.251+0.008 6 0.477+0.014 0.318+0.015 0.496+0.006 10 0.877+0.014 0.580+0.011 0.840+0.010 15 1.385+0.002 0.963+0.008 1.299+0.005 20 1.927+0.049 1.358+0.065 1.746+0.001 r2=0.9974 r2=0.9997 r2=0.9943 1. n=2 2. peak area r a t i o using etidocaine (int e r n a l standard) 3. peak area r a t i o using 4'-methylhydantoin (inte r n a l standard) 142 Table 13 Intra-assay v a r i a b i l i t y of tocainide and the hydantoin  at 6, 15, 20 ug/mL concentrations. Sample Tocainide"'' C . V . 7 o 2 Hydantoin''' C .V. (ug) (avg.+S.D.) (avg.+S.D.) 6 0.472+0.007 1 .49 0.319+0.016 5 .05 6 0.481+0.008 1 .76 0.339+0.015 4 .42 15 1.385+0.001 0 .05 0.964+0.007 0 .72 15 1.385+0.002 0 .16 0.957+0.001 0 .13 20 1.946+0.022 1 .12 1.397+0.009 0 .68 20 1.898+0.003 0 .16 1.359+0.066 4 .80 1. peak area r a t i o using lOug etidocaine (int e r n a l standard) 2. n=2 Table 14 Intra-assay v a r i a b i l i t y of tocainide and the hydantoin  at 6, 15, 20 ug/mL concentrations"! Sample Tocainide"'" C . V . 7 o 2 Hydantoin"'" C .V. (ug) (avg.+S.D.) (avg.+S.D.) 6 0.476+0.006 1 .28 0.329+0.014 4 .38 15 1.385+0.001 0 .04 0.960+0.083 8 .59 20 1.922+0.034 1 .75 1.378+0.027 1 .94 143 hydantoin metabolite d u r i n g h y d r o l y s i s . The e x t r a c t i o n e f f i c i e n c i e s of 6, 10 and 20 ug/ml of t o c a i n i d e and the hydantoin in 5 mL methylene c h l o r i d e , as o u t l i n e d in the experimental s e c t i o n , were determined to range from 77.8% to 81.2 % f o r t o c a i n i d e and 56.6% to 64.6 % f o r the hydantoin. T a b u l a t i o n of the data i s shown in Table 15. C o n c e n t r a t i o n (ug/mL) % r e c o v e r y t o c a i n i d e base hydanto i n 20 10 6 77.8 80. 1 81.2 63.6 64.6 56.6 1 in 5 mL of methylene c h l o r i d e . 2 d u p l i c a t e d e t e r m i n a t i o n s . Table 15 Extraction efficiences of tocainide and the hydantoin in  methylene chloride. 144 4. The E l i m i n a t i o n K i n e t i c s of T o c a i n i d e Glucuronide The k i n e t i c s of t o c a i n i d e and the hydantoin in u r i n e , a f t e r an IV and o r a l dose of 200 ug t o c a i n i d e , were examined in three h e a l t h y human v o l u n t e e r s . The u r i n a r y e x c r e t i o n r a t e s of t o c a i n i d e and the hydantoin a f t e r an IV and o r a l dose from one of the three s u b j e c t s are shown in Table 16 and 17. The u r i n a r y e x c r e t i o n r a t e data from the two other s u b j e c t s are shown in Appendixes 1 and 2. The pharmacokinetic data from the three s u b j e c t s are summarized in Table 18. The semi-log u r i n a r y e x c r e t i o n r a t e s of t o c a i n i d e and the hydantoin were p l o t t e d a g a i n s t mid-time of c o l l e c t i o n using the one-compartment Wagner-Nelson^ k i n e t i c s •D program on an Apple ^ II p l u s computer. The e l i m i n a t i o n r a t e c o n s t a n t s (K^,) of t o c a i n i d e from the s u b j e c t s , c a l c u l a t e d from the t e r m i n a l slope of the e x c r e t i o n r a t e p l o t s using Equation 1, were found to range from 0.037 to 0.059 hr ^ a f t e r an intravenous dose. These values were comparable to the range of 0.036 to 0.057 hr ^ a f t e r an o r a l dose su g g e s t i n g f h a t the net r a t e s of o v e r a l l e l i m i n a t i o n of t o c a i n i d e between o r a l and intravenous routes of a d m i n i s t r a t i o n are s i m i l a r (assuming 100 % b i o a v a i l a b i l i t y and with equal d o s e s ) . Log - Log V B ° - ^ W Terminal slope of the p l o t Log dX. dT u vs. t. mid ...Equat i on 1 145 Table 1 6 Urinary excretion rates of tocainide and the hydantoin after  a 200 mg IV dose of tocainide HCL in a healthy human volunteer. urine dtoc ug/mL dHYD hours vol (mL) Toe2 uR/mL dt HYD2 dt HYD3 0 1 203 6.56 1331.68 8.21 1666.63 8.18 2 521 3.91 2037.11 5.45 2839.45 5.87 3 135 8.19 1105.65 14.29 1929.15 16.01 5 96 12.31 590.88 38.32 1839.26 36.99 7 93 23.63 1098.80 67.91 3157.82 61.78 10 146 28.23 1373.86 44.71 2175.89 41.92 14 175 44.55 1949.06 59.82 2617.13 49.84 48 40 6.08 243.20 5.46 218.40 4.34 53 198 2.41 95.44 5.69 225.32 4.49 59 180 7.61 228.30 5.68 170.40 4.35 62 101 6.96 234.32 4.86 163.62 3.53 71 302 3.97 133.22 2.30 77.18 1.83 72 56 2.72 152.32 1.60 89.60 1.31 76 202 1.38 69.69 1.55 78.28 1.24 82 345 1.67 96.03 1.41 81.08 1.15 87 378 1.86 140.62 - - -95 270 1.46 49.28 - - -96 148 - - - -Note: 1. Averaged value from duplicate determinations. 2. Using etidocaine as internal standard. 3. Using 4'-methylhydantoin as internal standard. 1 4 6 t Table 17 Urinary e x c r e t i o n r a t e s of t o c a i n i d e and the hydantoin  a f t e r a 200 me, o r a l dose of t o c a i n i d e HCL i n a healthy human volunteer. hours ur ine v o l (mL) (ug/mL) 1 t o c a i n i d e ^ dToc dt (ug/mL) 1 HYD2 dHYD dt (ug/mL) HYD" 0 1 44 52.35 2303 40 50.63 2227 .72 46.97 2 264 12.87 3397. 68 15.34 4049 .76 14.72 3 132 17.38 2360. 16 17.84 2354 .88 22.17 5 68 50.01 1700. 34 77.55 2636 .70 67.64 7 106 28.60 1515 80 55.35 2933 .55 51.34 10 130 36.73 1591. 63 46.10 1997 .67 47.68 14.5 140 37.90 1179. 11 47.49 1477 .47 43.03 23.5 336 37.79 1410. 83 38.19 1425 .76 43.03 24.5 50 15.48 774. 0 17.43 871 .50 13.27 28 190 5.14 279. 03 14.11 765 .97 13.29 32 212 11.87 629. 11 16.34 866 .02 12.72 38 222 13.37 494. 69 14.54 537 .98 10.86 47 246 16.85 460. 57 10.04 274 .43 9.11 48 34 5.35 181. 90 5.39 183 .26 4.76 52 158 2.08 82. 15 4.32 170 .64 -58 220 7.52 275. 73 5.13 188 .10 4.12 62 130 7.26 235. 95 3.91 127 .08 3.23 72 294 4.14 121. 72 2.54 74 68 2.12 76 183 - - -83 233 3.51 116. 83 2.53 84 .21 -Note: 1. Average value from d u p l i c a t e samples. 2. Using e t i d o c a i n e as i n t e r n a l standard. 3. Using 4'-methylhydantoin as i n t e r n a l standard. 147 Table 18 The summarized pharmacokinetic data for tocainlde and the hydantoin In three healthy human volunteers after both an IV and oral dose. IV SR ORAL IV CK ORAL IV RE ORAL AVERAGE IV (n-3) ORAL KE (hr - 1 ) 0.057 0.059 0.036 0 044 0.037 0.037 0 .044 0.047 t 1/2 12.25 11.84 19.27 15 78 18.73 18.75 15 .75 14.85 Kmu (hr - 1 ) 0.055 0.054 0.043 0 047 0.052 0.055 0 .050 0.052 t 1/2 12.63 12.72 15.93 14 63 13.36 12.57 13 .86 13.33 Xy- 1 (mg) 195.A3 52.51 49,57 24 23 24.87 53.8 _4 4 M u - 2 (mg) 91.24 40.58 33.15 10 92 37.87 61.81 _4 _4 Mu-3 (mg) 93.35 39.45 39.11 9 91 34.50 54.39 _4 _4 k e (hr' 1) 0.028 5 0.014 5 0.010 _5 0 .0172 .5 k f (hr - 1 ) _6 _5 0.0089 5 0.0061 _5 0 .00757 _5 1 dose excreted intact 47.97 5 31.37 5 25.68 _5 X dose excreted as ToCG _6 _5 24.72 .5 16.49 _5 Note: 1. May represent underestimation of true due to incomplete urine collection. 2. Using etidocaine as I.S. (may represent underestimation due to incomplete urine collection). 3. Using 4-methylhydantoin as I.S. (may represent underestimation due to incomplete urine collection). 4. Cannot be determined due to incomplete urine collection. 5. Cannot be determined due to lack of absolute bioavailability data. 6. Cannot be determined due to k < KE. 7. n=2. rau The e l i m i n a t i o n h a l f - l i v e s of t o c a i n i d e among the three s u b j e c t s averaged 15.75 hours ( o r a l ) and 14.85 hours ( i n t r a v e n o u s ) , both of which are in agreement with l i t e r a t u r e r e p o r t e d h a l f - l i v e s (33). The u r i n a r y e x c r e t i o n r a t e constants of t o c a i n i d e (ke) from the three s u b j e c t s a f t e r an IV dose, as determined by an e x t r a p o l a t i o n p l o t as shown in Figure 67A using Equation 2, were averaged to be 0.0172 hour"''". Due to incomplete ur i n e c o l l e c t i o n from a l l three s u b j e c t s , the e x t r a p o l a t i o n method based on equation 2 i s the only reasonable estimate f o r the u r i n a r y e x c r e t i o n r a t e constant ( k e ) . In a d d i t i o n , due to the lack of o r a l b i o a v a i l a b i l i t y data, the e x t r a p o l a t i o n method cannot estimate the e x c r e t i o n r a t e c o n s t a n t s a f t e r the o r a l dose s. dX o Y - i n t e r c e p t o f p l o t Log u v s . t ^ = Log k £ X g dt where k g = u r i n a r y e x c r e t i o n r a t e o f t o c a i n i d e . X g ° = dose o f t o c a i n i d e . Equation 2 149 log _ > C | dt F i i i i i L^-i—^—*—ITI. . 1 — J — J — L i — i mid F i g u r e a: Ur inary e x c re t ion rate p r o f i l e o f t o c a i n i d e . log _ i u HYD | dt >—1 mid F i g u r e Urinary excre t ion rate p r o f i l e o f hydantoin, K — ^ ^ h L . . i . . i i - i . L > J • I i .1 J I I I F i g u r e c: U r i n a r y PH. F i g u r e 67 The semi - loq p l o t o f the ur inary e x c re t ion p r o f i l e o f t o c a i n i d e (a)  and the hydantoin (b) a f t e r r e c e i v i n g a 200mg IV dose o f t o c a i n i d e . 150 ka Tocain ide m Ur inary t o c a i n i d e , Tocain ide Glucuronide Other metabol i tes Scheme 18 Schematic representa t ion o f the e l i m i n a t i o n o f toca in ide in  humans. A c c o r d i n g to Scheme 18 shown above, the e l i m i n a t i o n of t o c a i n i d e can be broken down into v a r i o u s pharmacokinetic parameters d e s c r i b e d by the corres p o n d i n g f i r s t - o r d e r r a t e c o n s t a n t s . The u r i n a r y e x c r e t i o n r a t e c o n s t a n t s (kmu) f o r the g l u c u r o n i d e , as c a l c u l a t e d by the method of f e a t h e r i n g , as shown in Figure 67B and 68B using Equation 3, were averaged to be -1 -1 0.050 hour ( o r a l ) and 0.052 hour ( i n t r a v e n o u s ) . The averaged u r i n a r y e x c r e t i o n h a l f - l i v e s f o r the gl u c u r o n i d e from the three s u b j e c t s were c a l c u l a t e d to be 13.86 and 13.33 hours a f t e r r e c e i v i n g an o r a l and an intravenous dose r e s p e c t i v e l y , and are i n agreement with p u b l i s h e d l i t e r a t u r e values (33). Log u = Log k k.X ° - k (t . ,) mu f B mu mid a t F ~ KT 2.303 mu - E the slope of the above equation as obtained by feathering = -k 2.303 assuming K £ Equation 3 151 F i g u r e a : Ur inary exc re t ion rate p r o f i l e o f t o c a i n i d e . !• •! •'»—i-i—•—i—<-i—•—i—. i i i , i . • i i i . i • F i g u r e c: U r i n a r y PH F i g u r e 68 The s e m i - l o q p l o t o f the ur inary exc re t ion p r o f i l e o f t o c a i n i d e (a)  and the hydantoin (b) a f t e r r e c e i v i n g a 200mq ora l dose o f t o c a i n i d e . 152 The formation r a t e c o n s t a n t s ( k f ) of the t o c a i n i d e g l u c u r o n i d e from the three s u b j e c t s were again estimated by e x t r a p o l a t i o n using Equation 4. Log dM^ = Log k m u k f X B ° - K £ ( t m i d ) dt ~Tc - K„ 2.303 mu E Y - i n t e r c e p t of p l o t l o g dM u v s . t m i d = Log k m u k f X B ° di~ k KT mu - E where k = u r i n a r y e x c r e t i o n r a t e of TOCG mu J Xg° = dose of t o c a i n i d e i n body = t o t a l e l i m i n a t i o n of t o c a i n i d e E .... Equat i on 4 The formation r a t e c o n s t a n t s of the g l u c u r o n i d e from the three s u b j e c t s were averaged to 0.0075 hour~l a f t e r an IV dose. Due to the lack of absolute b i o a v a i l a b i l i t y data from the three s u b j e c t s , kf cannot be estimated by the e x t r a p o l a t i o n method a f t e r r e c e i v i n g an o r a l dose. The t o t a l amount of t o c a i n i d e e x c r e t e d i n the urine o o (Xu ) a f t e r an IV dose, as c a l c u l a t e d by Equation 5 , was found to range from 95.93 mg (47.97 % dose) to 51.35 mg (25.68 % dose) among the three s u b j e c t s . X 0 0 - X ° k ... Equat i on 5 153 The percent of dose e x c r e t e d in the urine as t o c a i n i d e g l u c u r o n i d e was c a l c u l a t e d by the r a t i o kf/K^, as shown in Equation 6. r a t i o o f dose e x c r e t e d as m e t a b o l i t e i n u r i n e . Equation 6 The t o t a l amount of the glu c u r o n i d e expressed as % dose was c a l c u l a t e d to 24.72 % and 16.49 % in two s u b j e c t s . In summary, the pharmacokinetic data c a l c u l a t e d from the three h e a l t h y human v o l u n t e e r s are in agreement with p u b l i s h e d l i t e r a t u r e v a l u e s . 154 SUMMARY AND CONCLUSIONS The o r a l b i o a v a i l a b i l i t y of t o c a i n i d e can be c o n v e n i e n t l y determined by the use of a s i n g l e pseudo-racemic dose. With the pre v i o u s o b j e c t i v e f o r such a study, the s t e r e o s p e c i f i c s y n t h e s i s of R(->- and S ( + ) - t r i d e u t e r a t e d t o c a i n i d e was designed through two approaches. To c a i n i d e carbamoyl e s t e r g l u c u r o n i d e was known to c y c l i z e to 3 - ( 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n at pH > 12. Th i s t h e s i s d e s c r i b e d a novel r e a c t i o n between t o c a i n i d e and urea, a second pathway which l e d to the formation of 3-<2,6-xy1y1)-5-methylhydantoin through a t o c a i n i d e N-ureide i n t e r m e d i a t e . T h i s f i n d i n g l e d to the s p e c u l a t i o n of an a l t e r n a t i v e t o c a i n i d e - N -ureide g l u c u r o n i d e s t r u c t u r e f o r TOCG. However, s t r u c t u r a l evidence f o r the carbamoyl e s t e r c o n j u g a t i o n s t r u c t u r e was obtained from p e r m e t h y l a t i o n experiments ( C ^ I / C D ^ I ) and GCMS a n a l y s i s . Furthermore, a p r e p a r a t i v e HPLC assay was developed to i s o l a t e TOCG f o r NMR and FAB s p e c t r a l measurements. The 400-MHz proton NMR data of the i s o l a t e d g l u c u r o n i d e provided only p a r t i a l evidence f o r the i n t a c t s t r u c t u r e of TOCG. However, FAB a n a l y s i s of the sample in both g l y c e r o l and t h i o g l y c e r o l r e v e a l e d [M+l], [M+Na1 and [M-H+2Na3 ion adducts at m/z 413, 435 and 457 r e s p e c t i v e l y , i n d i c a t i n g the i n t a c t molecular ion of TOCG at m/z 412. The l e v e l s of TOCG i n urine can be c o n v e n i e n t l y assayed as the hydantoin a f t e r h y d r o l y s i s of TOCG at pH > 12. However, the hydantoin was found to a l s o undergo f i r s t - o r d e r h y d r o l y s i s at pH > 12 c o n d i t i o n s . To account f o r the spontaneous h y d r o l y s i s of 155 the hydantoin, a set of a c c u r a t e l y timed c a l i b r a t i o n sample used in order to approximate the true l e v e l s of TOCG in u r i n e . Based on t h i s a n a l y t i c a l scheme, the l e v e l s of TOCG i n urine were examined in three s u b j e c t s a f t e r IV and o r a l d o s i n g of 200 mg t o c a i n i d e h y d r o c h l o r i d e . The u r i n a r y e x c r e t i o n h a l f - l i v e s of TOCG were found to be 13.86 hours and 13.33 hours a f t e r an IV and o r a l dose r e s p e c t i v e l y . These data were in agreement with l i t e r a t u r e v a l u e s . These s t u d i e s have c o n c l u s i v e l y e s t a b l i s h e d the s t r u c t u r a l i d e n t i t y of TOCG as a carbamoyl e s t e r g l u c u r o n i d e . In a d d i t i o n , a novel chemical r e a c t i o n between t o c a i n i d e and urea was observed to give r i s e to a ureide i n t e r m e d i a t e . T h i s urea condensation r e a c t i o n has not been r e p o r t e d in drug metabolism. 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R e f e r e n c e H NMR S p e c t r u m . 163 APPENDIX 1 IV Oj_t dToC dHYD dToC dHYD hours dt dt hours dt dt 1 4071.04 1517.33 1 845.05 646.46 2 2275.38 1423.92 2 - -3 3630.96 2700.69 3 - -5 2084.13 1696.41 5 - -7 2383.54 1589.66 7 - -9 1693.74 1059.68 9 2110.81 1402.30 11 1068.69 1198.91 10 - -12.5 498.04 - 16 - -13.5 506.27 639.11 20 1424.20 388.04 19 157.62 1047.77 24 - -21 1208.67 471.26 30 491.92 341.46 24 506.81 337.59 32 275.62 160.25 27 1681.42 606.70 35 278.83 159.42 28 1427.28 -• 37 463.11 208.71 30 505.12 283.52 43 369.29 218.13 32.5 688.53 279.36 45 442.37 170.97 37 1085.19 515.04 48 278.73 160.61 43 529.08 249.48 54 224.08 -46 425.80 186.77 57 211.72 117.34 53 - - 61 - -58 - - 64 356.24 -63 - - 66 186.32 -68 - - 69 135.56 -72 - - 72 54.51 55.06 78 89.79 69.62 61 29.57 -82 - - 83 - -86 - -96 - -Appendix 1 : The urinary excretion rates of tocainide and  the hydantoin a f t e r a 200mg or a l and IV dose  of tocainide HC1 i n a healthy volunteer CK. 164 APPENDIX 2 IV pRAL dToC dHYD dToC dHYD hours dt dt hours dt dt 1 1082.43 1328.04 1 2 - - 2 4107.88 4623.46 3 1638.20 1273.64 3 5377.42 5056.92 5 4363.81 2447.44 5 3455.85 3090.57 7 3194.67 2183.09 7 336.73 254.41 10 39469.54 19853.81 10 5664.46 3614.07 15.5 2177.26 951.77 15.5 - -23 1998.95 1033.72 23 1428.36 902.41 24 2716.40 1299.98 26 291.55 361.90 28 223.92 191.00 30 507.58 416.91 30 157.24 62.29 31.5 806.87 644.47 32 1528.67 637.25 36 - -35 7182.43 312.76 38 - -39 377.92 271.74 44 - -48 - - 48 - -50 - - 50 - -54 - - 52 - -58 267.29 171.83 56 225.23 171.05 62 150.54 98.66 61 174.59 144.19 71 - - 64 130.83 106.54 72 - - 72 137.25 114.54 79 - - 76 - -84 - - 82 - -85 - - 87 - -92 - - 94 - -96 - - 96 - -Appendix 2 : The u r i n a r y e x c r e t i o n r a t e s o f t o c a i n i d e and  the h y d a n t o i n a f t e r a 200mg o r a l and IV dose  of t o c a i n i d e HC1 i n a h e a l t h y v o l u n t e e r R E . 165 

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