STUDIES ON THE METABOLISM OF IN TOCAINIDE HUMANS BY DAVID W. K. KWOK B.Sc. ( P h a r m ) , The U n i v e r s i t y of B r i t i s h Columbia, A THESIS SUBMITTED AS PARTIAL FULFILLMENT OF THE REQUIREMENTS MASTER FOR THE DEGREE OF OF SCIENCE in The F a c u l t y o f G r a d u a t e F a c u l t y of Pharmaceutical D i v i s i o n of Pharmaceutical Studies Sciences Chemistry. We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o the r e q u i r e d s t a n d a r d The University of B r i t i s h Columbia _ April 1987 ( c ) D a v i d W.K. Kwok 1984 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 P h a r m a c e u t i c a l Chemistry The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e DE-6(3/81) April 30,1987. Abstract Tocainlde carbamoyl Is a major m e t a b o l i t e ester glucuronlde of tocainlde (Tonocard ). (R-NHCO.O-GA) The s t r u c t u r e of TOCG was f i r s t 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 , a base h y d r o l y z e d TOCG in urine. proposed (TOCG) R by E l v i n <35) b a s e d on t h e s t r u c t u r e Due t o t h e p r e s e n c e h y d a n t o l n r i n g , TOCG was p r o p o s e d o f two c a r b o n y l to a r r i v e m e t a b o l i c pathway i n v o l v i n g the a d d i t i o n terminal of tocainlde nitrogen followed product of g r o u p s on t h e from a n o v e l of carbon dioxide by g l u c u r o n i c t o the acid conjugation. With the i n i t i a l study of tocainlde stereospecific tocainlde This and i n t e n t i o n of c a r r y i n g using a deuterated synthesis was a t t e m p t e d through 3-<2,6-xylyl)-5-methylhydantoin glucuronlde support can made the structure that an i n vivo the c o r r e c t theoretical tocainlde structure) a tocainlde reaction ur i n e . i i N-urelde f o r TOCG i n acid. N-ureide which Attempts were o f TOCG by I d e n t i f i c a t i o n o f carbamic a c i d o r the t o c a i n l d e urelde between a tocainlde with glucuronic structure tocainlde t o the f o r m a t i o n of <R-NHCO.NH-GA) was p r o p o s e d be f u r t h e r c o n j u g a t e d proposed between through a t o c a i n l d e a n d u r e a may have r e s u l t e d to assign approaches. reaction leads this observation, of the theory tocainlde two s y n t h e t i c a chemical u r e a , a s e c o n d pathway w h i c h With p s e u d o r a c e m i c sample, t h e o f R<->- a n d S < + ) - t r l d e u t e r a t e d thesis describes intermediate. out a b i o a v a i l a b i l i t y (based N-ureide on E l v l n ' s intermediate in This and thesis also the s t r u c t u r a l conjugate. hydrolysis, presence test. evidence from hydrolysis, o f TOCG basic w i t h o r without the f o r the carbamoyl partial evidence for the molecular carbamoyl ester first-order this t o the h y d r o l y s i s hydantoln hydrolysis on t h i s a t m/z o f TOCG. 413, IM+Nal a t evidence 412 In f a v o r o f t h e a t pH > 12. to also 200 mg t o c a l n l d e i n an a s s a y protocol approach, The u r i n a r y found the l e v e l s t o take o f TOCG calibration Into account of the h y d a n t o i n . and 13.33 h o u r s , were spontaneous a s e t of a c c u r a t e l y timed analytical HC1. undergo To a s s a y i n three s u b j e c t s both a f t e r respectively, structure o f TOCG a t pH > 12 t o t h e was f o u n d spontaneous h y d r o l y s i s 13.86 h o u r s glucuronide 457 have p r o v i d e d p o s i t i v e Ion o f TOCG a t m/z s a m p l e s were e m p l o y e d determined Ion a d d u c t a t m/z In u r i n e a s the h y d a n t o i n , Based linkage of structure. In a d d i t i o n hydantoin, data of the I s o l a t e d f o r the Intact t h e [M+ll 435, and [M-H+2Nal ester proton-NMR and FAB a n a l y s i s . 400-MHz p r o t o n NMR m/z of acid novel g l u c u r o n i c a c i d o f s a c 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 In FAB a n a l y s i s , the from isolation f o r the I d e n t i t y beta-glucuronldase hydrolysis, TOCG was o b t a i n e d provided of t h i s o b t a i n e d as p r o o f was o b t a i n e d Structural The characterization Evidence as a c o n j u g a t e d e s c r i b e s t h e p r e p a r a t i v e HPLC iii o f TOCG were an IV a n d o r a l excretion half-lives after to agree the l e v e l s with an IV a n d o r a l literature dose o f o f TOCG dose values. Table of Contents Page Abstract Table ii of Contents iv List of Tables List of Figures List of Structures List of Schemes List of Pharmacokinetic Symbols x xii xvii xviii Equations xx and A b b r e v i a t i o n s xxi xxiii Acknowledgement INTRODUCTION 1 A. Brief B. Electrophysiological C. Pharmacodynamics 3 1. Clinical Efficacy 3 2. Efficacy in R e f r a c t o r y 3. Prophylaxis D. E. Overview of Tocainlde 2 Properties 2 Arrhythmias 3 A n t i a r r h y t h m i c Therapy 3 and P h a r m a c o d y n a m i c s 4 Pharmacokinetics 1. Bioavailability 4 2. Disposition 4 3. Dose-response 4. Serum P r o t e i n B i n d i n g 5. The E f f e c t s Kinetics Pharmacodynamics 5 on K i n e t i c s Stereoselectivity 4 from M e t a b o l i c In Drug M e t a b o l i s m iv Induction 6 6 Table of Contents (Contd) Page F. Metabolism 9 1. Observation of 2. Analysis Glucuronic Acid 3. Speculative Glucuronlde Structures of Metabolite of Tocalnlde Conjugates the Novel n 15 Tocalnlde 17 a. N - c a r b a m o y l a t i on \-j b. Carbonate 19 c. Tocalnlde-urea 1. ii. 4. of a Major Novel Equilibrium Adduct F o r m a t i o n Urea-adduct Formation Urea Condensation with Tocalnlde Drug G l u c u r o n i d a t i o n 21 21 22 23 a. Acyl-O-glucuronldes b. N-glucuronides (Ester Glucuronldes) 24 24 G. Toxicology 25 H. Agranulocytosis 26 EXPERIMENTAL 29 A. Chemicals and M a t e r i a l s B. Instrumentation 30 32 1. Fast Atom Bombardment Mass S p e c t r o m e t r y 2. Gas C h r o m a t o g r a p h y Mass S p e c t r o m e t r y 32 a. Varlan Magnetic-sector 32 b. Hewlett-Packard GCMS Q u a d r u p o l e GCMS Magnetic 33 3. Proton Nuclear 4. Gas C h r o m a t o g r a p h y 34 5. Liquid Chromatography 34 6. Infared Spectroscopy 34 v Resonance 32 34 Table of Contents (Contd) Page 7. C. M e l t i n g P o i n t Determination 34 S y n t h e s i s of 3' , 4 ' , 5 ' - t H ] ~ - 2 - a m i n o - 2 ' 2 ,6'- propioxylIdide D. 35 1. S y n t h e s i s of S ( + > - 3 ' , 4 ' , 5 ' - [ H I - t o c a l n l d e 35 2. S y n t h e s i s of 3 , 4 , 5 - [ H ] „ - 2 , 6 - d 1 methylani1ine 36 2 3 2 Attempted S y n t h e s i s of l-[ o H]„-2-amino-2*,6'- propioxylldlde 1. E. F. 38 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 - ( + ) - D - a l a n i n e 3 S y n t h e s i s of P o t e n t i a l Tocainlde Metabolites 38 39 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 39 2. S y n t h e 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 i n 40 3. S y n t h e 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 41 a. 41 S y n t h e s i s of 2 - a m l n o - 2 ' , 4 * , 6 ' - p r o p l o x y l i d i d e b. R e a c t i o n with T r l c h l o r o m e t h y l Chloroformate 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 l d e M e t a b o l i t e s 1. Permethylation 2. 41 42 42 a. P r e p a r a t i o n of Dry D i m e t h y l s u l f o x i d e (DMSO) 43 b. Generation of Sodium M e t h y l s u l f l n y l m e t h i d e Carbanion (DMSO) Sodium 43 c. Permethylation 43 Attempted I d e n t i f i c a t i o n Tocainlde Metabolites a. of Permethylated U r i n a r y 44 I s o l a t i o n of T o c a i n i d e - c a r b a m o y l E s t e r Glucuronlde 45 b. A c i d and Enzyme H y d r o l y s i s 46 c. L i q u i d Chromatographic A n a l y s i s of the Hydantoln Derived from the G l u c u r o n i c A c i d Conjugate of Tocainlde 47 vi Table of Contents (Contd) Page d. 3. 4. 5. G. Tocalnlde 49 A t t e m p t e d I s o l a t i o n and I d e n t i f i c a t i o n M e t a b o l i t e s by F l a s h C h r o m a t o g r a p h y Urinary of Tocainide with Effect of Temperature 2. Effect of pH 3. Attempted S y n t h e s i s glucuronlde of Attempted S y n t h e s i s glucuronide of Preparative of 50. Urea 51 and R e a c t i o n Duration 51 52 Tocalnlde-N-ureide52 2,6-Dimethylani1ine-N53 I s o l a t i o n and S t r u c t u r a l Glucuronide Elucidation of Tocainide 53 HPLC I s o l a t i o n of Tocainide Carbamoyl Glucuronide 54 a. Acid Hydrolysis with b. Beta-glucuronldase c. Naphthoresorcinol Hydrochloric Acid Hydrolysis Kinetics 56 Enzyme H y d r o l y s i s 56 Color Reaction d. Sodium H y d r o x i d e H y d r o l y s i s 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 Glucuronide 1. 47 A t t e m p t e d 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 N-carbamic Acid o r N - u r e l d e Intermediate Ester I. of 48 1. 1. Spectrometry A n a l y s i s Carbamoyl G l u c u r o n i d e 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 Reaction 4. H. Gas C h r o m a t o g r a p h y / M a s s Permethylated Tocalnlde of Tocalnlde 57 57 Ester Glucuronide 57 In Sodium H y d r o x i d e 58 2. Hydrolysis of 3-<2,6-xylyl)-5-methylhydantoln 58 3. Hydrolysis of 3-(2,4,6-xylyl>-5-methylhydantoin 58 4. 3-<2,6-Xylyl)-5-methylhydantoIn vii C a l i b r a t i o n Curve 58 Table of Contents (Contd) Page 5. Inter-assay 6. E x t r a c t i o n E f f i c i e n c i e s o f T o c a i n i d e and the H y d a n t o i n i n t o 5 mL o f M e t h y l e n e C h l o r i d e 60 Analysis 61 7. of and Intra-assay Tocainide Variability Glucuronide in K i n e t i c Studies RESULTS AND DISCUSSION A. Synthetic 1. pathways Synthesis 62 3 ' , 4 ' , 5 ' - t r l d e u t e r a t e d Tocainide for B. C. 63 3',4',5'-[ H]3-2-amino-2',6'- of 2 propioxylidide 2. 59 63 a. 2,6-Dlmethylanl1lne Deuteratlon 64 b. Reaction 71 with Carbobenzyloxyl-alanine Attempted S y n t h e s i s propioxylidide Potential Tocainide of 1-[ 2 H13-2-amino-2',6'73 Metabolites 77 1. 2 - E t h a n i m i n e - 2 ' , 6 ' - p r o p 1oxylIdide 77 2. 3-(2,6-Xylyl)-5-methylhydantoln 77 3. 3-(2,4,6-Xylyl)-5-methylhydantoin 84 Tocalnlde Metabolites 89 1. Derivatization 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 90 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 Metabolites 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 ) 96 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 97 3. 4. 5. Techniques for of Glucuronides Metabolic 89 Intermediates I d e n t i f i c a t i o n o f 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 C h r o m a t o g r a p h y and C H ^ I / C D g l P e r m e t h y l a t i o n 106 6. Tocalnlde-urea 111 7. Synthesis of Reaction N-glucuronldes viii 113 Table of Contents (Contd) Page D. HPLC I s o l a t i o n Glucuronide Preparative 2. E I Mass S p e c t r a l Glucuronlde 4. of Tocainlde HPLC I s o l a t i o n Analysis Atom Bombardment Pharmacokinetics of of the Magnetic Tocainlde Resonance 122 Mass S p e c t r o m e t r y Tocainlde 1. Tocainlde Glucuronide 2. Hydrolysis 3. C a l i b r a t i o n Curve Data 4. The E l i m i n a t i o n K i n e t i c s of 116 122 400 MHz P r o t o n N u c l e a r Experiment Fast Elucidation 116 1. 3. E. and S t r u c t u r a l Analysis Glucuronlde Basic Hydrolysis 128 133 Kinetics 133 3-<2,4,6-xylyl)-5-methylhydantoln 136 139 of Tocainlde Glucuronide 145 SUMMARY AND CONCLUSIONS 155 References 157 Appendix 1 164 Appendix 2 165 ix List of Tables Page 1. 2. 3. 4. 5. 6. 7. 8. E x a m p l e s of v a r i o u s f u n c t i o n a l g r o u p s known t o r i s e t o N - g l u c u r o n i c a c i d c o n j u g a t e i n man. The Rf of u r i n a r y components 20x20 cm TLC p l a t e . separated give 24 on a K C - 1 8 F 45 The Rf t h r e e u r i n a r y components s e p a r a t e d on a K C - 1 8 F TLC p l a t e d u r i n g a s e c o n d p u r i f i c a t i o n . 46 Summarized d a t a from the 80-MHz-t-'-H] -NMR s p e c t r u m of D - 2 , 6 - d i m e t h y l a n i l i n e . 68 Summarized d a t a from the 4 0 0 - M H z - C H ] - N M R 3-<2,6-xylyl)-5-methylhydantoin. 1 spectrum of 83 Summarized d a t a from the 80-MHz p r o t o n NMR s p e c t r u m 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 . The s a m p l e s o b t a i n e d used f o r i d e n t i f y i n g intermediates. from the e x p e r i m e n t a l tocainlde glucuronlde protocol 103 The R e a c t i o n o f Temperatures. T o c a i n l d e with Urea at 9. The R e a c t i o n o f T o c a i n l d e with Urea at 10. Summarized d a t a of t o c a i n l d e . from the Summarized d a t a o f the I s o l a t e d glucuronide. from the 400-MHz p r o t o n NMR s p e c t r u m t o c a l n i d e carbamoyl e s t e r 11. 12. 13. 14. 15. 87 Different Ill Different pH. 112 400-MHz p r o t o n NMR s p e c t r u m 125 127 C a l i b r a t i o n c u r v e 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 standards. 142 I n t r a - a s s a y v a r i a b i l i t y o f 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 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 a t 6, 15, 20 ug/mL c o n c e n t r a t i o n . Extraction efficiences in methylene c h l o r i d e . of tocainlde and the hydantoin 143 and the hydantoin 144 x List of Tables (Contd) Page 16. 17. 18. 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 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 o f 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 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 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 o f 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 . 147 The s u m m a r i z e d 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 t h r e e h e a l t h y human v o l u n t e e r s a f t e r b o t h an IV and o r a l d o s e . 148 xi List of F i g u r e s Page 1. The metabolism of t o c a i n i d e in humans. 10 2. The metabolism of t o c a i n i d e in r a t s . 14 3. The t o t a l - i o n - c h r o m a t o g r a m of crude deuterated d i m e t h y l a n i l i ne p r o d u c t . 4. The mass spectrum of crude deuterated d i m e t h y l a n i l ine at scan 51. 2,6- 5. The mass spectrum of crude deuterated d i m e t h y l a n i l ine at scan 61. 2,6- 6. The mass spectrum of u n l a b e l l e d 2,6-dime t h y l a n i 1 i n e . 66 7. The t o t a l - i o n - c h r o m a t o g r a m of d i s t i l l e d dimethylani1ine. 69 8. The mass spectrum of d i s t i l l e d dimethylani1ine. 9. 80-MHz-NMR Spectrum of D <CDC1 ) . 10. 2,6- 65 °6 D3-2,6- D3-2,6- 69 -2,6-dimethylani1ine 80-MHz-NMR Spectrum of D - 2 , 6 - d i m e t h y l a n i 1 i n e 65 70 after D 0 exchange. 70 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). 12. The t o t a l - i o n - c h r o m a t o g r a m of s y n t h e t i c t o c a i n i d e . 74 13. The EI 74 14. 15. The i n f a r e d spectrum of N-carbobenzy1oxy-alanine ( m u l l ) . The t o t a l - i o n - c h r o m a t o g r a m of s y n t h e t i c 2-ethanimine tocainide. 78 16. The EI 78 17. The i n f a r e d spectrum of t o c a i n i d e ethanimine 18. The i n f a r e d spectrum of (mull). 19. Total-ion-chromatogram of s y n t h e t i c methylhydantoin. mass spectrum of s y n t h e t i c t o c a i n i d e . mass spectrum of 2-ethanimine xii tocainide. (mull). 3-(2,6-xylyl)-5-methylhydantoin 3-(2,6-xylyl)-5- 72 76 79 80 82 List of Figures (Contd) Page 20. 21. 22. 23. 24. 25. The EI mass s p e c t r u m o f 5-methylhydantoin. the synthetic 3-(2,6-xy1y1)83 4 0 0 - M H z - [ H1-NMR S p e c t r u m of the s y n t h e t i c x y l y l ) - 5 - m e t h y l h y d a n t o i n (CDCl ) . The i n f a r e d s p e c t r u m of methylhydantoin (mull). of 83 3-(2,4,6-xy1y1)-585 T o t a l - i o n - c h r o m a t o g r a m of xylyl)-5-methylhydantoin. The EI mass s p e c t r u m 5-methylhydantoin. 3-(2,6- the synthetic 3-(2,4,686 the synthetic 3-(2,4,6-xy1y1)86 The 8 0 - M H z - [ H]-NMR s p e c t r u m of the x y l y l ) - 5 - m e t h y l h y d a n t o i n (CDCl ) . synthetic 3-(2,4,688 26. The CI mass s p e c t r u m of the TMS-derivative of glucose. 27. The CI mass s p e c t r u m of the nitrophenol glucuronide. TMS-derivative of p- 28. 29. 30. 31. 32. 91 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 u r i n e e x t r a c t i s o l a t e d band l b s a m p l e . XAD/crude 92 Mass s p e c t r u m of p e r m e t h y l a t e d t o c a i n i d e c a r b a m o y l 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 F r a g m e n t a t i o n p a t t e r n of the carbamoyl e s t e r g l u c u r o n i d e . 93 T o t a l - i o n - c h r o m a t o g r a m of nitrophenol glucuronide. proposed tocainide permethylated The CI mass s p e c t r u m of the nitrophenol glucuronide. para95 permethylated para 95 33. The t o t a l - i o n - c h r o m a t o g r a m 34. The EI mass s p e c t r u m of permethylated 35. The EI mass s p e c t r u m dimethylani1ine. of permethylated 36. 91 of permethylated tocainide. tocainide. 100 100 2,6101 The t o t a l - i o n - c h r o m a t o g r a m l a c t o x y l i d ide. xiii of permethylated 102 L i s t o f Figures (Contd) Page 37. 38. 39. The EI mass spectrum of the l a c t o x y l i d ide. permethylated 102 The t o t a l - i o n - c h r o m a t o g r a m of the permethylated (2,6-xylyl)-5-methylhydantoin. The EI mass spectrum of the permethylated xylyl)-5-methylhydantoIn. 3104 3-(2,6104 40. T o t a l - i o n - c h r o m a t o g r a m of permethylated urine. 41. The EI spectrum of permethylated ester glucuronide ( C H 3 I ) . 42. The EI fragmentation p a t t e r 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 . 109 43. The EI mass spectrum of permethylated carbamoyl e s t e r g l u c u r o n i d e ( C D 3 D . 110 44. T o t a l - 1on-chromatogram of permethylated ureide. 45. The EI mass spectrum of permethylated ureide. 46. The total-1on-chromatogram of the permethylated dimethylani1ine-N-glucuronide. 47. The EI tocainide mass spectrum of permethylated 108 carbamoyl tocainide tocalnlde-N- tocalnlde-N2,6- 108 114 114 115 2,6- dimethylaniline-N-glucuronIde. 115 48. Preparative 118 49. The UV absorbance of the 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. P r e p a r a t i v e HPLC chromatogram of r e c y c l e d t o c a l n l d e TOCG c o n t a i n i n g f r a c t i o n . 120 51. P r e p a r a t i v e HPLC chromatogram of r e c y c l e d TOCG sodium phosphate s a l t . In 120 52. P r e p a r a t i v e HPLC chromatogram of r e c y c l e d TOCG sodium c h l o r i d e . In 53. 400-MHz-[ Hl-NMR Spectrum of t o c a l n l d e 50. HPLC chromatogram of crude u r i n e . 1 xiv (D,-DMSO). 118 121 123 L i s t o f F i g u r e s (Contd) Page 54. 400-MHz-[ H1-NMR Spectrum of e x c h a n g e <D ~DMSO). 1 tocainlde 6 55. The 400-MHz p r o t o n NMR s p e c t r u m 56. 400-MHz-[lHl-NMR Spectrum of e s t e r g l u c u r n o i d e (D - D M S O ) . of after D 0 • 9 123 glucuronic acid. tocainlde carbamoyl 126 6 57. 58. 59. 60. 61. 62. 63. 64. 65. 124 4 0 0 - M H z - t l H l - N M R S p e c t r u m of t o c a i n i d e c a r b a m o y l 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 ) . o The f a s t atom bombardment s p e c t r u m 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 matrix with sodium c h l o r i d e . 126 129 The f a s t atom bombardment s p e c t r u m 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 l d e in t h l o g l y c e r o l matrix w i t h sodium c h l o r i d e . 129 The f r a g m e n t a t i o n ester glucuronide spectrometry. 130 pattern In f a s t of t o c a i n l d e carbamoyl atom bombardment mass The f a s t atom bombardment s p e c t r u m 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 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 . 131 The h y d r o l y s i s p r o f i l e o f 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 i n the p r e s e n c e o f sodium h y d r o x i d e . 134 S e m i - l o g p l o t of the hydantoin hydrolysis of sodium h y d r o x i d e . 135 t o c a l n i d e g l u c u r o n l d e and the k i n e t i c s in various concentrations S e m i - l o g p l o t o f the 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 I n In v a r i o u s c o n c e n t r a t i o n s o f sodium hydroxide. 137 The h y d r o l y s i s o f t o c a i n i d e g l u c u r o n i d e and the m e t h y l h y d a n t o i n i n the p r e s e n c e o f 0 . 5 M s o d i u m hydroxide. 138 xv 4'- List of Figures (Contd) Page 66. 67. 68. T h e o r e t i c a l p r o f i l e s o f the 3 - ( 2 , 6 - x y l y l ) - 5 methylhydantoin h y d r o l y s i s using 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 standard. A : Peak a r e a - t i m e p r o f i l e s o f 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 t o t h e 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 o f 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 t o e t i d o c a i n e ( I . S . ) i n a t h e o r e t i c a l TOCG h y d r o l y s i s profile. 140 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 o f t o c a i n i d e . 150 The s e m i - l o g p l o t o f 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 xvi List of S t r u c t u r e s Page I. Toca i n i d e . 2 II. L idoca i n e . 2 Ill. 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 . 10 IV. Lactoxylid ide. 10 V. T o c a i n i d e Oxime. 10 VI . 3 - ( 2 , 6 - X y l y l ) - 5 - m e t h y l h y d a n t o in 10 VII . T o c a i n i d e carbamic a c i d . 12 VIII . T o c a i n i d e aldehyde a d d u c t . 14 IX. O x i d a t i v e deaminated p r o d u c t . 14 X. 4 ' - H y d r o x y t o c a in ide 13 XI . G l u c u r o n i c a c i d conjugate of l a c t o x y l i dide . 13 XII . 2,6-dime t h y l a n i 1 i n e EI ion fragment a t m/z 95. 67 XIII . 2,6-dimethylani1ine EI ion fragment a t m/z 80. 67 XIV. 2,6-dime t h y l a n i 1 i n e EI ion fragment a t m/z 124. 67 XV. 2,6-dimethylani1ine EI ion fragment at m/z 109. 67 xvii List of Schemes Page 1. 2. 3. 4. 5. 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 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 . The p r o p o s e d m e t a b o l i c f o r m a t i o n of t o c a i n i d e 18 The t h e o r e t i c a l carbamate. 19 equilibrium reactions The mechanisms 8. The mechanisms o f t o c a l n l d e - u r e i d e 3-(2,6-xylyl>-5-methylhydantoin. 12. of tocainide The mechanisms o f c a r b a m i c a c i d c y c l l z a t i o n 3-<2,6-xylyl)-5-methylhydantoln. 7. 11. Isomeric t r a n s f o r m a t i o n of of of 20 urea. tocalnlde-ureIde Attempted i d e n t i f i c a t i o n tocainlde metabolites. to 21 formation. 22 cycllzation permethylated urinary 44 A t t e m p t e d 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 1 y o p h 1 1 l z a t 1 on and GCMS a n a l y s i s . 48 A t t e m p t e d I s o l a t i o n and i d e n t i f i c a t i o n o f N-carbamic a c i d or N-ureide i n t e r m e d i a t e . tocainlde 49 A t t e m p t e d i s o l a t i o n and I d e n t i f i c a t i o n m e t a b o l i t e s by f l a s h c h r o m a t o g r a p h y . urinary for of trideuterated 50 The s y n t h e t i c pathway 14. The s y n t h e s i s of 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 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 . 17. to 23 13. 16. 12 Pathways o f ammonia m e t a b o l i s m . A S A , 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. The 10. 11 pathway l e a d i n g t o the carbamoyl e s t e r g l u c u r o n i d e . 6. 9. novel tocainlde. N-carbobenzyloxyalanine. The e x p e r i m e n t a l s t e p s tocainlde glucuronide. involved in 63 75 isolating from 99 the 117 S c h e m a t i c 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 h y d r o l y s i s In the p r e s e n c e of NaOH. xviii of TOCG 133 List of Schemes Page 18. Schematic tocainide representation i n humans. xix of the elimination of 151 List Log u dt~ = d X of Pharmacokinetic Log k X ° - K Equations E ( t .,) 2303 m Terminal slope of the p l o t Log l dX d ST Y-intercept of p l o t Log u v s . t . , j ^ — mid d X r where k g r -K u vs. t . , = b M = I E ^T3TJ3 D Log k X,, e B 0 & = u r i n a r y e x c r e t i o n r a t e of t o c a i n i d e . Xg°= dose of t o c a i n i d e . Log & ^ u = Log k k.X ° mu f B k~ Kl mu - E & -rr— Q U - k ( t . ,) __mu_ mid 2.303 the slope of the above equation as obtained by f e a t h e r i n g = -k mu 2.303 & assuming Log & dM dt K £ = Log k k. X ° mu f B "Tc - K_ mu E u - 6 Y - i n t e r c e pr t of pr l o t l o&g dM dt u K„ ( t . ,) E mid 2.303 o v s . t j. , = Log k k^X^ & mu f B m l c K Z mu - E k 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 K„ E X u 0 0 = = 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 X ° k B e E K M u (F)dose k = r a t i o o f dose e x c r e t e d f TT— i n urine. K r £ xx as m e t a b o l i t e Symbols and A b b r e v i a t i o n s ACN acetonitrile BSTFA BIs(trlmethyls CBZC1 carbobenzyloxyl c h l o r i d e CDCI3 deuterated chloroform CD3I deuterated methyl CH I methyl CI chemical CNS central 3 C.V. % Ilyl)trif1uoroacetarnide iodide iodide ionization nervous system coefficient of variation D5-DMSO deuterated dimethyl sulfoxide DMSO dimethyl D2O deuterium D2SO4 deuterated EI electron FAB f a s t atom bombardment GA glucuronic acid GC gas chromatography GCMS gas chromatography sulfoxide oxide sulphuric acid Impact Ionization I n t e r f a c e d with mass spectrome t r y HBr hydrogen bromide HC1 hydrochloric acid HFBA heptaf1uorobutyic HPLC high performance HYD 3-<2,6-xylyl)-5-methylhydantoin IR infared spectroscopy IV intravenous xx i anhydride l i q u i d chromatography 8ymbols and A b b r e v i a t i o n s J coupling constant elimination rate constant urinary excretion (tocalnide) formation kmu urinary M molecular + m/z mass t o Mu°° total NaH P0 (Contd) rate rate constant excretion rate (tocainlde) constant (TOCG) constant ion charge ratio amount o f TOCG e x c r e t e d sodium phosphate monobasic NMR nuclear resonance ODS o c t a d e c y l s 1 lane ppm parts 2 PTFE PVC R f 4 teflon magnetic in urine per m i l l i o n polytetrafluoroethylene teflon premature ventricular contractln retention factor RP reverse-phase TCF trichloromethyl TIC total-1on-chromatogram TLC thin TMS t r i methyls i 1 y l TOC tocaInIde TOCG tocainlde t half-1Ife 1/2 (TOCG) chloroformate l a y e r chromatography carbamoyl e s t e r UV ultravlolet Xu oo t o t a l amount o f urine xxxi tocalnide glucuronlde excreted In Acknowledgements I would supervisor, support like to express Dr. K e i t h McErlane, d u r i n g the c o u r s e The helpful my a p p r e c i a t i o n f o r h i s kind of these discussions (Chairman), and c r i t i c i s m s gratefully to Roland and t o G r e g interpretation. The a s s i s t a n c e also appreciated. production This Trainee grant of t h i s Abbott, facilities Special Slatter from the s t a f f i n the Department thanks Sinclair to Kathleen a t t h e NMR and of Chemistry i s Cheng f o r the thesis. t h e B.C. H e a l t h Care I sincerely encouragement D r . James f o r h i s a d v i c e on NMR without S c h o l a r s h i p p r o v i d e d by the B.C. H e a r t from the other f o r h i s h e l p w i t h GCMS work would n o t have been p o s s i b l e Lastly, and from acknowledged. Burton instrumentation, mass s p e c t r o m e t r y e n c o u r a g e m e n t and B u r t , D r . C h a r l e s K e r r and D r . J o h n are a l s o Many t h a n k s thesis studies. members o f my r e s e a r c h Committee, D r . F r a n k A x e l s o n , Dr. Helen t o my thank throughout the Research Foundation, Research Foundation. my p a r e n t s for their understanding t h e y e a r s o f my a c a d e m i c xxiii and a studies. 1 INTRODUCTION 1 A. Brief Overview Tocainide, antiarrhythmic marketed 400 is mg 400 and mg 600 agent mg t o 600 developed exhibits every of acute first-pass 8 hours myocardial highly tocainide i s a primary both and (Tonocard u s u a l recommended d o s i n g metabolism bioavailability oral The (I) is a up t o 800 mg every ( I I ) i s the a g e n t infarction. i n the variable. liver, thus Unlike antiarrhythmic procainamide, of c h o i c e f o r lidocaine rendering i t s the advantage of administration. Properties t o Vaughan W i l l i a m ' s c l a s s i f i c a t i o n drugs, class disopyramide, I compounds s u c h as p h e n y t o i n and a membrane-stabilizing action which t h e r e b y d e c r e a s i n g the rate cardiac action In a d d i t i o n potential rate, (1). tocainide 2 of quinidine, lidocaine, interferes influx, the d e p o l a r i z a t i o n regimen (II) Electrophysiological According ) in lidocaine, amine a n a l o g w h i c h o f f e r s i n t r a v e n o u s r o u t e s of It is 8 hours i f However, (I) B. new Astra Pharmaceuticals. the h y d r o c h l o r i d e s a l t tablets. mg by At p r e s e n t , l i d o c a i n e treatment oral 2-amino-2',6'-propionoxylidide i n Canada as necessary. the of T o c a i n i d e of r i s e can a l s o with sodium i n phase to t h i s a l l possess z e r o of s l o w i n g of decrease impulse the excitability and fibers 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 (2). contribute C. to the The United antiarrhythmic States more t h a n 100 15 be reported mg oral (5) of 12 with later tocainide; 2. E f f i c a c y in Refractory ventricular ectopic to c o n v e n t i o n a l procainamide Ryan, W. ventricular patients of 10 t o be Similar of placebo tocainide with acute (4) i n PVCs 11 % reduction therapy. o v e r a 75 receiving co-workers Woosley % suppression in and of PVCs activity. e f f e c t i v e in i n 15 out of suppressing 19 patients quinidine f i n d i n g s were r e p o r t e d ( 8 ) , and or by M o l o n e y , J.D. i n the were c a r r i e d out prophylactic a f t e r myocardial studies refractory (9). Therapy studies tocainide these and ventricular ectopic activity arrhythmias r e s u l t s of % + Winkle tocainide Antiarrhythmic Double-blind usefulness (3). the Arrhythmias (7), Haffajee,C.I. Prophylactic effects 7 patients of in and i n 5 out therapy with propranolol, <6). McDevitt (PVCs) with found follows. tocainide i n premature documented patients was as with % reduction oral 8 out Tocainide experience tocainide in The of a 60 d e m o n s t r a t e d a 91 patients co-workers summarized contractions subsequently of can first ventricular the activity Purkinje Efficacy pre-marketing c l i n i c a l co-workers 3. i n i s o l a t e d dog Pharmacodvnamics 1• Clinical out automaticity to therapy infarction evaluate of (10-12). showed.some p r e l i m i n a r y b e n e f i c i a l in a b o l i s h i n g myocardial ventricular tachycardia infarction, 3 but in f u r t h e r study in this be area would be r e q u i r e d . an e f f e c t i v e At p r e s e n t , antiarrhythmic tocainide i s proven to agent. D. P h a r m a c o k i n e t i c s and P h a r m a c o d y n a m i c s 1. B i o a v a i l a b i 1 i t v In h e a l t h y was w e l l a human v o l u n t e e r s , absorbed with 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 , Administration delayed the r a t e food of a b s o r p t i o n , (13). peak p l a s m a in fasting decreased peak After l e v e l s of subjects. levels but the o v e r a l l ( 1 3 ) was n o t a f f e c t e d . zero-order minute p e r i o d , 2.5 ug/mL. i n f u s i o n o f 300 mg o f t o c a i n i d e o v e r a t h e peak p l a s m a The b i e x p o n e n t i a l level was f o u n d terminal e l i m i n a t i o n phase, with hours r e s p e c t i v e l y (13). steady subject administered average renal dose response At steady was e x c r e t e d clearance Dose-response For (13,14). between of d i s t r i b u t i o n a t 1.46 L/Kg t o 2.9 L/Kg i n s t a t e , 30 t o 40 % o f a n i n the u r i n e with an (14,15). Pharmacodynamics t o c a i n i d e , the plasma-drug r e l a t i o n s h i p was f o u n d intersubject o f 0.18 h o u r and 11.5 volume unchanged o f 70 mL/min profile phase and a s l o w e r half-lives The a p p a r e n t s t a t e was f o u n d t o v a r y healthy t o be plasma-drug c o n c e n t r a t i o n 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 3. hydrochloride Disposition Kinetics Following 30 i n 1 to 2 hours of the drug with bioavailability 2. tocainide 90 t o 100 % b i o a v a i l a b i l i t y 1.82 ug/mL were a c h i e v e d and oral variability (16). concentration-antiarrhythmic to e x h i b i t a l a r g e degree of The r e p o r t e d 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 4 effective of t o c a i n i d e v a r i e d among several studies considered (3,15,16), but the a c c e p t e d t o be from plasma-tocainide oral dosage ectopic 4 t o 10 ug/mL. concentration tocainide 400 mg o r a l l y concentration o f 6 ug/mL. h o u r s were s e l d o m 4. Serum P r o t e i n Lalka every 10 h e a l t h y found binding than significant 5. 800 mg e v e r y 8 CNS e f f e c t s ( 3 ) . concentrations. of only bound binding. i n plasma binding 4 t o 12 % among I n trauma p a t i e n t s w i t h and i n h i b i t i o n tocainide (19,20). were no greater significant f o r t h e two e n a n t i o m e r s , i s not l i k e l y Induction The e x t e n t o f Between t h e t h e r a p e u t i c Nevertheless, models were used examined the e n a n t i o m e r was s l i g h t l y displacement elevated o f d r u g bound was Sedman ( 1 8 ) l a t e r o f 4-12 ug/mL, t h e r e The E f f e c t s o f M e t a b o l i c Animal than of t o c a i n i d e enantiomers. i n the f r a c t i o n tocainide an a v e r a g e t o c a i n i d e was 50 % bound t o effective 10 t o 20 %. binding range dose-independent of that of the R ( - ) - e n a n t i o m e r . concentration changes plasma a binding of the S ( + ) - t o c a i n i d e that 8 hours with reported reported from stereoselective Ventricular taking g l y c o p r o t e i n s , the percentage t o range (15). Binding human v o l u n t e e r s . alpha-1-acid steady-state i n 70 % o f t h e p a t i e n t s t o l e r a t e d due t o a d v e r s e (13) f i r s t (18) l a t e r 8 hours Doses g r e a t e r plasma p r o t e i n s a t c l i n i c a l l y Elvin An a v e r a g e range i s o f 1.7 ug/mL was r e a c h e d a f t e r an r e g i m e n o f 100 mg e v e r y b e a t s were s u p p r e s s e d effective the h i g h to create free suggesting fraction clinically interactions. Induction on K i n e t i c s t o examine the e f f e c t s of metabolic on t h e d i s p o s i t i o n o f In the r a t , with 5 phenobarbital pre t r e a t m e n t , a significant reduction concentration versus i n the a r e a time c u r v e tocainide-plasma was o b s e r v e d a f t e r dose. Pretreatment enzyme responsible 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 in clearance alteration urine with under the f o r glucuronlde <19>. after pretreatment o f some d r u g s . competitive i n man with Is a p o t e n t inhibitors t o form of the g l u c u r o n i c a c i d c o n j u g a t e well and i n Drug is a chiral i n the salicylamIde, or inducer metabolites of the g l u c u r o n i c a c i d excretion optically reduction I n h i b i t o r o f the g l u c u r o n l d a t i o n However, none o f t h e s e Tocainide found to t o d e t e c t any i s a known enzyme C l o f i b r a t e i s known Stereoselectivity of the by a failed phenobarbital, pathway. E. was intravenous o f unchanged d r u g r e c o v e r e d (21). Phenobarbital whereas s a l i c y l a m l d e inhibitor conjugation, However, s t u d i e s i n the percent clofibrate be SKF 525A, a known an that conjugation a g e n t s have a f f e c t e d t h e u r i n a r y of t o c a i n i d e i n man. Metabolism compound w h i c h c a n e x i s t a s two a c t i v e e n a n t i o m e r s , R ( - ) - and S ( + ) - t o c a i n i d e . recognized that drug a b s o r p t i o n , distribution, 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 specificity, particularly so when t h e p r o c e s s It is metabolism or stereo- involves enzyme t r a n s f o r m a t i on. Tocainide literature, i s marketed as a r a c e m i c there drug d i s t r i b u t i o n stereochemical Testa distribution mixture. In t h e a r e numerous e x a m p l e s o f an s t e r e o s e l e c t i v e and m e t a b o l i s m . drug d i s p o s i t i o n (23). For instance, may An e x t e n s i v e was p u b l i s h e d the enantiomeric and m e t a b o l i s m o f p r o p r a n o l o l 6 r e v i e w on by J e n n e r a n d differences in have b e e n well studied. In humans, r a c e m i c p r o p r a n o l o l lower c l e a r a n c e (24). than propranolol and of R ( + ) - e n a n t i o m e r was the propranolol found (28). was longer S(-)-enantiomer and i t was of b r a i n of (27). The rat (29). stereoselective differences enantiomers of p r o p r a n o l o l for in the (25). The bioavailability to a l p h a - l - a c i d S(-)-propranolol that has chain S(-)-enantiomer. t h a n the discovered propranolol the lower o r a l of p r o p r a n o l o l plasma h a l f - l i f e Recently, also reported more a c t i v e reported side stereoselectivity was a R(+)-enantiomer were a l s o r e p o r t e d t o have a stereoselective binding t o have a the vasoinactive Furthermore, pharmacologically glycoprotein heart the glucuronidation (25,26). bioavailability Also, of shown t o have S t e r e o s e l e c t i v i t y in aromatic hydroxylation, hydroxylation, than that was racemate the greater These i n the was i n the found rabbit vasoactive accumulation studies have the established d i s p o s i t i o n of i n b o t h humans and in the two experimental an i m a l s . In light of a p o s s i b l e stereoselective role disposition of t o c a i n i d e , Gal disposition of (R,S)-tocainide tocainide was reported However, reversed. enantiomers pharmacological three times induced more p o t e n t antiarrhythmic i n r a t s and i n the of evaluation. arrhythmic co-workers in r a t urine (R)-antipode. The and than model activities first mice at higher the studied (30). levels mouse model, t h i s tocainide in the S(+)-isomer between 7 the than have b e e n s u b j e c t e d R ( - ) - e n a n t i o m e r was mouse S(+)- (31), two i n the but the observation The i n the the the was to found to be chloroformdifference e n a n t i o m e r s was in smaller in coronary-1 igated McErlane disposition and of 10.0 and tocainide Pillai first i n man. carbamoyl that preferably formed glucuronide from the stereoselective i n d i v i d u a l enantiomers the enantiomers urine. the tocainide. i n the an different in that than that of the the rapid be 10.3 may The an to be for hours have enantiomers of 200 with mg saliva levels 24 those hydrochloride, hours. Similar i n the The the s a l i va. 8 found authors pH-independent e x c r e t i o n d i s t r i b u t i o n of was m o r e o v e r , the those in was higher levels of the suggested p r o c e s s was R(-)-enantiomer urine. ratio R(-)-enantiomer concentration than and S<+>-tocainide/R(->- enantiomer concentration S ( + ) - e n a n t i o m e r , and intravenous in plasma tocainide r a t i o of at into d i s p o s i t i o n p r o f i l e s of p r o f i l e s were o b s e r v e d plasma samples. stereoselective to ai.<34>. 1.52 b o t h e n a n t i o m e r s were h i g h e r corresponding half-life were measured a f t e r an dose p l a s m a was i n s a l i v a , the the S(+)-tocainide tocainide stereoselective oral enantiomeric excretion the found into revealed a c i d conjugate mean e n a n t i o m e r c o n c e n t r a t i o n However, The i n s a l i v a were compared Following tocainide for was et Pillai The and (33). d i s p o s i t i o n of of of stereoselective R(-)-tocainide. was dose by the i n man for R(-)-tocainide saliva the tocainide this glucuronic reported reported A s i m i l a r study hours r e s p e c t i v e l y suggesting The <32) d i s p o s i t i o n of half-life 16.7 (3D. tocainide stereoselective elimination dogs that a responsible into the I n humans, 28 excreted t o 55 unchanged suggested that another pathway primary amine n i t r o g e n , acid form Another metabolism also been urine i n 24 to % of the 15 t o c a i n i d e carbamoyl the dose lactoxylidide (36). was by 1. hours However, identified metabolite was acid-urea intermediate as It as with a major m e t a b o l i t e , was reported (VI) also reported a metabonate 9 to via the (35). deaminated further (V) has tocainide to c y c l i z e a t pH (35,37) as conjugation to was glucuronic (III) undergo The was metabolized t o c a i n i d e oxime i n a d d i t i o n t o the hydantoin (35,36). dose was ( I V ) , which c o u l d (37). tocainide conjugation A trace metabolite, identified of ester glucuronide 3-(2,6-xylyl)-5-methy1hydantoin Figure dose a d d i t i o n of carbon d i o x i d e followed carbamoyl-0-p-D-glucuronide form 30 i n v o l v i n g the 1 t o 2 % of metabolite, oral i n the a novel to % o f an > shown pathway, form via a in r a t s (38). 12 to in this carbamic 10 1• O b s e r v a t i o n In 1979, glucuronic Elvin acid elucidating described of a Maior the and Novel co-workers conjugate (35) of t o c a i n i d e . s t r u c t u r e of i n Scheme M e t a b o l i t e of this Tocainide identified The conjugated a novel basis for metabolite is 1. A: B: C: A c i d h y d r o l y s i s 1 M HC1/100°C. Beta G l u c u r o n i d a s e 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 1 M NaOH (pH 12). D: Heat ( 1 0 0 ° C ) . The Hovel T o c a i n l d e M e t a b o l i t e Tocainlde Hydantoin Scheme 1 : V a r i o u s e v i d e n c e f o r t h e 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 . Acid tocainide, h y d r o l y s i s (HC1) probably free u r i n e samples p r o d u c e d r e l e a s e d from H y d r o l y s i s experiments amounts o f of a conjugated with beta-glucuronidase tocainide released likely conj ugate. 11 from free metabolite. produced similar a glucuronic acid The initial interpretation of these a N-glucuronic acid conjugate instability N-glucuronide hydrolyzed that on of t h i s product the s t r u c t u r e the p r i m a r y glucuronic Based (III), d a t a was of t o c a i n i d e . to hypothesize However, t h e in alkaline solution, 3-(2,6-xylyl)-5-methylhydantoin, of t h i s novel amine p o s s i b l y forming suggested metabolite contained a carbonyl as a carbamoyl ester linkage with acid. on t h e p r o p o s e d tocainide must first carbamoyl ester conjugated structure be p r e s e n t e d a s a c a r b a m i c intermediate ( V I I ) a s shown i n Scheme 2. intermediate has n o t y e t b e e n i s o l a t e d nor T h i s carbamic acid acid identified. Tocalnlde ( I ) Tocanlnlde Carbamic Acid (VII) 3-(2,6-xylyl)-5-nethylhvdantoln (VI) A B C Uridine dlphoiphate/glucuronlc a c i d . HaOH. Scheme 2 : The proposed metabolic pathway leading to the formation of tocainide carbamoyl ester glucuronideT 12 a Following was this initial undertaken with eight animal model However, t h e amount from of t o c a i n i d e expressed the lowest 13 % i n the r a b b i t compared lactoxylidide respectively during glucuronide These carbamoyl t o be Although chronic activity. were study identified, from amide h y d r o l y s i s , I n humans, ester excreted found to both n o r CNS (37), four metabolite glucuronic a c i d conjugate t o be low glucuronide and hours was known t o tocainide carbamoyl t h e two m e t a b o l i t e s were toxic effects (36). major m e t a b o l i t e s of adduct ( I X ) , and t h e n o v e l of t o c a i n i d e ( V I I I ) , an carbamoyl (III), (XI) (IV) oxidative ester a s shown i n 2. 13 ester not to possess namely t h e 2 , 6 - d i m e t h y l a n i 1 i n e OI) of the e l i m i n a t i o n 13.0 h o u r s and 29.1 In a d d i t i o n , an a l d e h y d e deaminated Figure tocainide. o f d o s e , was the 1 a c t o x y l i d i d e therapy, shown n o t t o p o s s e s s c a r d i a c From an a n i m a l of glucuronide and t h e l a c t o x y l i d i d e were shown antiarrhythmic tocainide t o e s t a b l i s h an f i g u r e s are considered i n humans. were r e p o r t e d <36). carbamoyl investigation of < 1 % i n the g e r b i l to the h i g h e s t for tocainide accumulate of animals as t h e p e r c e n t (39). t o 30 t o 40 % half-lives species 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 i n the u r i n e , range f i n d i n g , an e x t e n s i v e 2 , 6 - d i m e t h y l a n i l i n e (IV) Aldehyde Adduct (VIII) O x i d a t i v e Deaminated ( I X ) T o c a l n l d e Carbamoyl E s t e r Glucuronide ( I I I ) Figure 2 : The metabolism of tocainide in r a t s . Three the diketone, identified of of these m e t a b o l i t e s : the a n i l i n e , were n o t f o u n d metabolites i n human u r i n e . the ethanimine In a d d i t i o n i n human u r i n e , we s u s p e c t e d a 4'-hydroxypropionoxylidide 14 (X) m e t a b o l i t e and t o the the presence and p o s s i b l y an ether from 2. glucuronic acid the indirect profile glucuronide found the the The To disposition not of may of the liberated of 2. The the The However, a n a l y s i s glucuronide of on procedure usually involves by not conjugates; the extent the use by approach i s , of i s time specific for i t usually requires enzyme 1,4-lactone. the hydrolytic o f the of d a t a reaction is enzyme, t h u s between consuming does not di-glucuronides. be of a c o m p e t i t i v e source 15 followed This a n a l y t i c a l r e a c t i o n may i n c u b a t i o n ) and and formed drawbacks: poor r e p r o d u c i b i l i t y 3. glucuronide R-(-)-enantiomer, intact saccharic acid r a t e and dependent this beta-glucuronidase, acid verification inhibitor, assay have p r e f e r e n t i a l l y aglycone. hydrolytic glucuronic to an reported. however, s u b j e c t t o a number of 1. The used ester 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 of has method tocainide (35). composition been study half-life t o t h a t of the glucuronide R-(-)-enantiomer has o n l y one t o c a i n i d e carbamoyl comparable t h a t the date, o r enzyme h y d r o l y s i s by analysis formed that glucuronidation is a enzyme h y d r o l y t i c of the The enantiomeric tocainide acid (35). t o be suggesting from process. beta-glucuronidase kinetic was (XI) being Conjugates is generally recognized stereoselective the metabolite lactoxylidide. A n a l y s i s of G l u c u r o n i c A c i d It of conjugated (up distinguish giving experiments. to 24 hours between mono- 4. The p r o c e d u r e due may l a c k day t o day r e p r o d u c i b i 1 1 i t y to a p o s s i b l e v a r i a t i o n i n enzyme a c t i v i t y with storage. Due to the l i m i t a t i o n necessary to develop diasteromeric disposition The o f an enzyme h y d r o l y t i c an a s s a y f o r the d i r e c t ester glucuronide profile of t h i s metabolism resolution 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 p s e u d o r a c e m i c dose of t o c a i n i d e (equal t h e use o f a can then vivo be used to study racemization stereoselectivity saliva. tocainide p r o p o r t i o n s of deuterated such a study possible metabolic of the i n t a c t drug, 1. S y n t h e s i s aims o f t h i s inversion, in substrate transport p r o j e c t were into the d e u t e r i u m - l a b e l l e d 2. D e v e l o p n e c e s s a r y enantiomeric analytical three-fold: p u r e R ( - ) , o r S(+>- isolation/identification compound. techniques or diastereomeric and t o examine their s t r u c t u r e of t h i s novel to separate m e t a b o l i t e s of kinetic the t o c a i n i d e g l u c u r o n i d e 16 metabolites. tocainide for bioavailability measurements a n d m e t a b o l i t e tocainide of drug of enantiomerical1y 3',4*,5*-trideuterated intact chiral i n drug the i d e n t i f i c a t i o n Hence, t h e i n i t i a l 3. I s o l a t e urine t h e use o f p s e u d o r a c e m i c d o s i n g o f assist using Plasma, i n human s u b j e c t s metabolic and s t e r e o s e l e c t i v i t y Furthermore, will from of tocainide deuterated S ( + ) - e n a n t i o m e r and u n l a b e l l e d R ( - ) - e n a n t i o m e r ) . samples o b t a i n e d o f the glucuronide. s t u d i e d with saliva i t was o f t o c a i n i d e , and t o examine t h e may be e f f e c t i v e l y and assay, profile. and e l u c i d a t e t h e metabolite. 3. Speculative The indicated the Structures s t r u c t u r e of that the of the of formation The identity true tocainide of Tocainide Glucuronide 3-(2,6-xy1y1)-5-methylhydantoin of the i n c o r p o r a t i o n of a c a r b o n y l nitrogen Novel followed this novel group onto by glucuronide the involved primary glucuronic acid carbamoyl (VI) intermediate amine conjugation. has not been e s t a b l i shed. Theoretically, carbamoylated a. the terminal p r i m a r y amine n i t r o g e n c a n t h r o u g h a number o f b i o c h e m i c a l pathways, N-carbamovlation High levels of carbamoylaspartate ornithine outlines were carbamoyl the role N - c a r b a m o y 1 - b e t a - a l a n i n e and found i n the urine N- of p a t i e n t s transferase d e f i c i e n c y (40). of carbamoylphosphate i n the with Scheme metabolism 3 of urea. Tocainide amine and can p o s s e s s e s an theoretically carbamoylphosphate followed alanine moiety with a be N - c a r b a m o y 1 a t e d by by glucuronic acid 17 primary the enzyme conjugation. be Carbamoyl Phosphate Dihydrouracil 1 N-carbamoyl B-alanine Camosine t| B-alanine Scheme 3 : Pathways o f ammonia m e t a b o l i s m . 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 i d i n e monophosphate. 18 b. Carbonate Equilibrium Carbon d i o x i d e i s present c a r b o n a t e s and d i s s o l v e d c a r b o n a t e s has b e e n as carbon dioxide studied a n i m a l muscle and b r a i n react with the u n i o n i z e d c a r b o n a t e s a s shown i n t h e body T0C-NH3 tissues amino i n Scheme where k k ToC-NH + H 2 ; k (41). + , k fi - ™ I and p l a s m a a s acids well can t o form ^ 2 ^ ^ [ToC-NH l [H } 2 a [ToC-NH ] + 3 • carbamate equilibrium constant. fl » ionization constant of the amino group. : The t h e o r e t i c a l f o r m a t i o n of 4: £ Scheme 4 The Carbon d i o x i d e g r o u p o f amino 2 . gas. bicarbonates, i n human r e d c e l l s ToC-NH • C 0 * = ± ToC-NHCOCf • H 2 as equilibrium reactions of tocainide 19 carbamate. This studied (pKa carbon i n human p l a s m a ( 4 1 ) w i t h o f 8.0 f o r b o t h may t h e o r e t i c a l l y and dioxide-carbonate carbon compounds). combine g l y c i n e and g l y c y l g l y c i n e Tocainide, having carbon demonstrated this theory, the f o r m a t i o n of a small ( V I ) when an aqueous s o l u t i o n incubated a t 100 ° C f o r 1 h o u r w i t h possibily through followed by c y c l i z a t i o n with tissue. Venkataramanan and A x e l s o n hydantoin the formation a pKa o f 7.8, d i o x i d e d e p e n d i n g on t h e pH 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 In a g r e e m e n t w i t h (38) with e q u i l i b r i u m r e a c t i o n h a s been amount o f t h e o f t o c a i n i d e was solid carbon of carbamic a c i d the x y l i d i d e dioxide, intermediate n i t r o g e n a s shown i n Scheme 5: CH3 0 Scheme 5 : The mechanisms o f carbamic 5-methylhydantoin. 20 acid cyclization to 3-(2,6-xy1y1)- C. T o c a i n i d e - u r e a Adduct Formation V e n k a t a r a m a n a n e t al. of tocainide no attempts preliminary revealed i n the of urea possible from 100 °C. The chemical Urea-adduct following a reaction aqueous s o l u t i o n Scheme 6 30 the d i s s o c i a t e d acidic urinary pH carbon d i o x i d e can * undergo as shown NH w 4 t o the f o r m a t i o n of with urea, isomeric i n Scheme ^ 0 - C= at 100 6: N was found studied that after several by equilibrium °C, w h i l e a t body the r e a c t i o n is temperature days. thermodynamical1y Also, in favours species. reaction, giving reaction I t was is reached cyanate this 2 (42). minutes solution, on d e s c r i b e the + reversible 1948 °C), equilibrium Based lead of : 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 . & Schutz, within H - C - NH 2 spontaneously urea will of t o c a i n i d e t o ammonium c y a n a t e NH diluted experiments i n the presence sections of urea can 0 (38 Our Formation transformation reached (VI) pathways w h i c h c a n through However, the d e g r a d a t i o n p r o d u c t s . f o r m a t i o n of the h y d a n t o i n hydantoin Dirnhuber i n u r i n e samples. urea-tocainide hydrolysis the This the d e g r a d a t i o n results two An observed identify M urea at i. presence first were made t o 8.0 this (38) rise combine ammonium c y a n a t e to carbon can dioxide. w i t h the amino g r o u p 21 break The down a t resultant of t o c a i n i d e to form i i• the carbamic a c i d Urea Condensation with Tocainide Alternately, carbamate i n t e r m e d i a t e as d e s c r i b e d , tocainide c a n condense i n t e r m e d i a t e as proposed w i t h u r e a t o form a i n Scheme 0 I 7: HN, ^ C-0 NH-C-CH-Nt^ CH, tocainide urea H NH- C-CH-N " 0 A CH3H [ N ^ I I 1 1 0 l NH-C-CH-NH-C-NH 2 + NHi CR3 tocainide ureide Scheme 7 : The mechanisms o f tocainide-ureide formation. 22 This basic tocainide conditions ureide ureide t o form cyclization was found (VI). a r e shown t o undergo c y c l i z a t i o n The mechanisms i n Scheme Scheme 8 : The mechanisms o f t o c a i n i d e - u r e i d e 3-(2,6-xylyl)-5-methy1 hydantoin. rise f o r the t o c a i n i d e 8. + 4. D r u g IH3 cyclization to Glucuronidation The m e t a b o l i c pathway and t h e t y p e s o f x e n o b i o t i c s to g l u c u r o n i c acid reviewed under (43,44). giving c o n j u g a t e s have been e x t e n s i v e l y According t o the a f o r e m e n t i o n e d 23 metabolic pathways l e a d i n g t o the f o r m a t i o n metabolite structure intermediate be a N - u r e i d e (-NHCO.NH-GA), o r a c a r b a m o y l These c o n j u g a t e s conditions. tertiary acid, indomethacin, nicotinic ester acid glucuronides incubated with minutes ( 4 5 ) . b. N-alucuron ides acid, (43). secondary rise t r i m e t h y l a c e t i c a c i d and The b e n z o a t e and s a l i c y l a t e completely to N-glucuronic i n Table or compounds a s i n s a l i c y l i c a r e known t o h y d r o l y z e a r e summarized Groups A r o m a t i c amine Azaheterocycle Carbamate Sulphonamide Hydroxylamine-NTertiary aliphatic Urea than iodopanoic primary, when f o r 5 to acid 1: : E x a m p l e s o f v a r i o u s f u n c t i o n a l g r o u p s 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. Functional The or h e t e r o c y c l i c xenobiotics giving conjugates Table 1 c a n be a r y l , at alkaline a pH 12 b u f f e r a t room t e m p e r a t u r e 30 The (-NHCO.O-GA). Glucuronides) a r e known t o be u n s t a b l e respectively the i n t a c t glucuronide, ester glucuronide, (Ester The a g l y c o n e aliphatic I tocainide f o l l o w e d by GA c o n j u g a t i o n , o f TOCG c a n e i t h e r a. A c v l - O - G l u c u r o n i d e s o f a phase carbamate Examples aniline sulphisoxazole meprobamate sulphadimethoxine N-hydroxy-2-acetamidofluorene Cyproheptadile Dulcin glucuronides t h e amine c o n j u g a t e s (-NHC0.0-GA) a r e more s t a b l e (-NH-GA) a t a n a c i d i c 24 pH. Certain N-glucuornides condensation pH have been f o r m e d s p o n t a n e o u s l y of an aglycone and temperature. many a r y l a m i n e - N - with g l u c u r o n i c a c i d (48) and a l k y l a m i n e - N - g l u c u r o n i d e s theme o f t h i s tocainide also possible carbamoyl admixture ester glucuronide possible ( 4 7 ) , and ( 4 9 ) have procedure. c e n t r e on i s o l a t i o n of the of the s t r u c t u r e N- u r e i d e g l u c u r o n i d e be made t o i d e n t i f y non-conjugated G. will g l u c u r o n i d e and on e l u c i d a t i o n theoretically proposed thesis through at physiological Meprobamate-N- ( 4 6 ) , u r e t h a n e - N - b e e n s y n t h e s i z e d in v i t r o u s i n g a s i m p l e The in v i t r o of a (-NHCO.NH-GA) o r t h e (-NHCO.O-GA). Attempts will new c o n j u g a t e d o r metabolites. ToyicQJogy At revealed the onset of t h i s a n y major major m e t a b o l i t e s common a d v e r s e toxic project, effects i n humans. effects toxicological from A review of tocainide adverse erythematosus (50) and i n t e r s t i t i a l reports possible use reported cases haematological world indicated included sporadic cases pneumonitis Astra Pharmaceuticals toxicities wide. 1984. A t t h e t i m e , 1984, over with 20 o t h e r c o u n t r i e s , of lupus summarizing w h i c h have r e s u l t e d the from t h e The f i r s t r e p o r t was p r e p a r e d i n 40 c a s e s of haematological d i s o r d e r s p o p u l a t i o n ( i n c i d e n c e o f 1:750). the a p p r o v a l that the I n e a r l y 1985, ( l e u c o p e n i a a n d 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 30,000 p a t i e n t i t s two (51) and the of blood d y s c r a s i a s (52). were r e l e a s e d from of tocainide January effects report n o r from were g a s t r o i n t e s t i n a l i n nature. Other recently tocainide s t u d i e s had n o t of tocainide I n November i n the U n i t e d S t a t e s and t h e number o f c a s e s o f 25 a 25,000 t o in tocainide 125. it related In l i g h t was As use a result thus to study the metabolism t o the t o x i c i t y toxicities, toxicities reached a t o t a l of of the i n c r e a s i n g i n c i d e n c e o f b l o o d d y s c r a s i a s , important reference haematological of i t s metabolites. of the h i g h incidence of a g r a n u l o c y t o s i s Astra Pharmaceuticals prohibiting of t o c a i n i d e with withdrew t h e use o f t h i s drug t o c a i n i d e from in healthy general human volunteers. Due t o t h e sudden change withdrawal of support basic research have i n drug r e g u l a t i o n and the from A s t r a P h a r m a c e u t i c a l s on t o c a i n i d e , t h e i n i t i a l to continue aims o f t h i s p r o j e c t s i n c e been r e v i s e d a s f o l l o w s : 1. S y n t h e s i s o f R ( - ) o r S<+>-3',4',5'- trideuterated 2. Attempt identification tocainide previous 3. tocainide. Isolation o f new m e t a b o l i t e s o f from human u r i n e pharmacokinetic of the i n t a c t elucidation samples c o l l e c t e d during studies. t o c a i n i d e g l u c u r o n i d e and of the i n t a c t s t r u c t u r e of t h i s metabolite . 4. L a r g e - s c a l e isolation of the t o c a i n i d e for agranulocytosis toxicity glucuronide studies. H. A a r a n u l o c v t o s i s On t h e market granulocytopenia. agents today, Phenothiazines t h a t c a n cause Antithyroid numerous d r u g s a r e a s s o c i a t e d reversible medications, are widely used a n t i p s y c h o t i c granulocytopenia (53). propylthiouracil 26 with and m e t h i m a z o l e c a n cause granulocytopenia sulfonamides were r e p o r t e d immunological inflammatory mechanism drug granulocytopenia a i n 0.3 % t o 0.6 t o be a g r a n u l o c y t o g e n i c (54). aminopyrine (54,55). The a n a l g e s i c and was reported Phenytoin 2 % i n c i d e n c e of g r a n u l o c y t o p e n i a cephalosporins In a l l c a s e s three general i n longterm, (53). the e t i o l o g y and with granulocytopenia and c a n be e x p l a i n e d by structure of r e a c t i v e t o x i c neutrophils 3. F o r m a t i o n The m e t a b o l i s m of and metabolites with macromolecules. of a n t i b o d i e s to n e u t r o p h i l s . of aminopyrine the g e n e r a t i o n inducer metabolites R-N. 2. I n t e r a c t i o n o f t h e a c t i v e enzyme The p e n c i l l i n s mechanisms ( 5 5 ) : the an to cause therapy (54). o f the d i s e a s e 1. The g e n e r a t i o n illustrate an anti- was a l s o r e p o r t e d high-dose of drug-induced through The to cause were a l s o o c c a s i o n a l l y c o n n e c t e d granulocytopenia neutropenia, % of p a t i e n t s ( 5 4 ) . of t o x i c which can induce can serve a s an example t o metabolites. Aminopyrine i s c y t o c h r o m e 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 postulated a two-step reaction R-N t o be m e t a b o l i z e d 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 metabolite. monooxygenase either to through This metabolic enzymes glucuronyl monooxygenase to y i e l d involving f o l l o w e d by a c o n j u g a t i o n a reactive endogenous r e a c t i o n with 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 be common t o p h e n y l b u t a z o n e , chloramphenicol. pathway, was Because chlorpromazine, of high chemical 27 p r o m a z i n e , and reactivity, the glucuronic molecules a c i d and which can sulphate produce conjugates the of these drugs are reactive electrophi1ic labile structure R-N. This species agranulocytotic of p r o t e i n the with are the drugs of the replication of likely to of glucuronide of reported the reactive tocainide i n the The of generated an reactive and RNA at of basic literature, the pH, we toxicity glucuronide. 28 nucleophiles disrupting the propose of being With one body, our ester recent reports t o examine tocainide also DNA i n the effects. carbamoyl and with m o l e c u l e s can Bone marrow c e l l s , most d e t r i m e n t a l nature from immune r e s p o n s e undergoing r e p l i c a t i o n agranulocytosis tocainide DNA R-N i n t e r a c t i n g with R-N synthesis. s u f f e r the the of eliciting antibodies. protein agranulocytosis by thereby nucleophiles and metabolite is capable most r e a c t i v e c e l l s knowledge the reactive molecules, formation react of was of whether elicited EXPERIMENTAL A. Chemicals and Chemicals following 1. Materials are reagent grade suppliers Aldrich are Chemical listed Co. (unless specified). The alphabetically: (Milwaukee, Wisconsin, USA) D-alanine, benzyl chloroformate (carbobenzyloxyl c h l o r i d e ) , 2,6-dimethylani1ine, N-carbobenzyloxylD-alanine, Trichloromethyl chloroformate, 2 , 4 , 6 - t r i m e t h y 1 a n i 1 i n e , c a l c i u m h y d r i d e , sodium h y d r i d e ( 5 0 % i n m i n e r a l o i l ) , sodium g l u c u r o n i c a c i d , phosphoric acid, naphthoresorcinol (1,3-d i h y d r o x y n a p h t h a l e n e ) 2. Analytical International Bond-elute 3. Applied C-18 Inc. (Harbor c i t y , extraction Science Laboratories. CA, USA) columns (State College, Pennsylvania, USA) Silar-lOc 4. Aristar^ BDH liquid Chemical. Sodium h y d r o x i d e 5. Astra phase (Poole, England) (analytical grade) P h a r m a c e u t i c a l s Canada L t d . ( M i s s i s s a u g a , O n t a r i o , Canada) T o c a i n ide 6. Beckman I n s t r u m e n t s C-18 ODS 7. BDH 8. BrownLee L a b s 5um USA) 4.6mm x 25cm HPLC c o l u m n C h e m i c a l s . ( V a n c o u v e r , B.C., Canada) HPLC g r a d e s o l v e n t s : m e t h a n o l , w a t e r , a c e t o n i t r i l e , hexane, 2 - p r o p a n o l , methylene c h l o r i d e . R e a g e n t g r a d e : c h l o r o f o r m , a n h y d r o u s sodium s u l f a t e , ether, ethanol-100, toluene, dimethylsulfoxide, sodium p h o s p h a t e m o n o b a s i c , a c e t i c a c i d Inc. (Santa C l a r a , Ramnin 0.45 9. I n c . ( B e r k e l e y , CA, Chromatographic Chromosorb urn o n - l i n e Specialties. W-HP 30 CA, filter, USA) ramnin (Brockville, C-18 Ont., guard column Canada) 10. Eastman O r g a n i c Chemicals. (Rochester, N.Y., USA) Dicyclohexylcarbodiimide 11. Fisher Scientific Methyl 12. 13. J.T. Co. iodide, (Fair Lawn, New 30-32% h y d r o g e n b r o m i d e Baker L t d . ( P h i 1 i p s b u r g , New Silica (flash Mandel gel Scientific Jersey, Jersey, USA) in a c e t i c acid USA) chromatography) Company. ( N i s k u , A l b e r t a , Canada) XAD-2 r e s i n , SE-30 ( b o n d e d ) 0.25um x 50m f u s e d 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 0.25um x 50m f u s e d s i l i c a c a p i l l a r y column R 14. Merk S h a r p & Dohme. ( i s o t o p e s ) ( M o n t r e a l , Quebec, Deuterated 15. Parr Pressure Variac 16. Reaction pressure P i e r c e Chemical Pyridine, 17. methyl Co. iodide, D -DMSO Apparatus. reaction (Rockford, (Moline, Illinois, vessel Illinois, USA) BSTFA Sigma C h e m i c a l Co. (St. Louis, Missouri, USA) HFBA, b e t a - g l u c u r o n i d a s e ( g l u c u r a s e ), saccro-1,4-lactone, a c e t i c anhydride, d e u t e r i u m o x i d e (99.8 atom% D ) , d e u t e r a t e d c h l o r o f o r m ( 9 9 . 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 Ltd. (Bellafonte, Dexsil-300 19. Union Carbide liquid Pennsylvania, USA) phase Canada L t d . ( T o r o n t o , Ont., Canada) Helium, n i t r o g e n , hydrogen, medical a i r 20. Whatman I n c . (Clifton, 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 c o l u m n , 20 x 20cm KC-18F t h i n l a y e r p l a t e s 31 Canada) USA) B. InstrumentatIon 1. F a s t Atom All Bombardment FAB S p e c t r a were r e c o r d e d facility i n the Department a t t h e mass of Chemistry, spectrometry University of B r i t i s h Columbia. Samples were a n a l y z e d matrix with voltage a Kratos/Ael either in glycerol Ms9 i n s t r u m e n t . or t h i o g l y c e r o l The a c c e l e r a t i n g a n d t h e FAB were a t 6 KV and 7 KV r e s p e c t i v e l y . Collision g a s was Xenon a n d t h e s a m p l e s were a n a l y z e d on a copper probe t i p . 2. Gas C h r o m a t o g r a p h y Mass GCMS a n a l y s i s Magnetic-sector was c a r r i e d A Hewlett-packard a Varian Mat-Ill recorder. energy from instrument, The e l e c t r o n - i m p a c t on a V a r i a n 620L c o m p u t e r and p l o t t e d spectra on a c h a r t - The f i l a m e n t c u r r e n t was 300 uA w i t h a n e l e c t r o n a cycle pressure o f 15 t o 500 mass obtained units from a t 8 X 10"° t o r r , every a plot g a s c h r o m a t o g r a p h was o p e r a t e d GC i n l e t , 250 ° C . quadrupole 5700A Gas C h r o m a t o g r a p h was i n t e r f a c e d t o Mass S p e c t r o m e t e r . c h r o m a t o g r a m s were with the V a r i a n GCMS o f 70 eV, i o n s o u r c e The out using e i t h e r or the Hewlett-packard a. V a r i a n M a g n e t i c - s e c t o r were r e c o r d e d Spectrometry injection 5 seconds. A 3 % Silar Total-ion- i n the s p l i t l e s s p o r t and s e p a r a t o r temperature 10C l i q u i d c h r o m o s o r b W-HP b e f o r e diameter and s c a n o f m/z 50 t o 500. The f o l l o w i n g c o l u m n c o n d i t i o n s were 1. mode all at employed: phase was c o a t e d on packing X 2 m g l a s s column. 32 beam i n t o a 2 mm The oven internal temperature was programmed The 2. from column h e l i u m 150 t o 270 flow r a t e A 3 % Dexsil-300 l i q u i d chromosorb W-HP phase The column h e l i u m b. H e w l e t t - P a c k a r d Quadrupole electron TIC Electron beam e n e r g y flow r a t e using was temperature °C/min. was 20 mL/min. interfaced spectra w i t h an 5987A were o b t a i n e d w i t h a a t 70 eV and e m i s s i o n c u r r e n t o f m/z was a t 240 ° C . C h e m i c a l 120 t o m/z ionization a t 300 uA. 800. Ion source was c a r r i e d o u t methane a s t h e r e a g e n t g a s w i t h a s o u r c e p r e s s u r e o f 1.45 x 10"^ T o r r and e m i s s i o n c u r r e n t experiments The inlet The oven internal GCMS impact were o b t a i n e d from a p l o t temperature i n t o a 2 mm 150 t o 300 °C a t 8 An HP 5880A gas c h r o m a t o g r a p h mass s p e c t r o m e t e r . was c o a t e d on before packing from C/min. was 20 mL/min. d i a m e t e r X 2 m g l a s s column. was programmed C at 8 were c a r r i e d out e i t h e r gas chromatograph temperature c o l u m n s were 1. probe i n t h e EI o r CI mode. was o p e r a t e d a t 250 ° C . i n the s p l i t The f o l l o w i n g mode w i t h chromatographic employed: A capillary fused- The SE-30 ( b o n d e d - p h a s e ) , silica temperature 2. o f 300 uA. D i r e c t (HP) was used w i t h an program column h e l i u m A Capillary silica program from c o l u m n was used Column h e l i u m was 33 oven w i t h an oven was °C/min. 1 mL/min. , 0.25 mm 40 t o 190 °C a t 30 flow rate X 50 m 40 t o 300 °C a t 30 flow r a t e p Chirasil-Val from 0.25 mm X 50 m f u s e d temperature °C/min. 1 mL/min. 3. P r o t o n The Nuclear Magnetic 80 MHz s p e c t r a were r e c o r d e d XL-100 s p e c t r o m e t e r . using the 4. Resonance a Bruker NMR on a B r u k e r The 400 MHz e x p e r i m e n t s WP-400 s p e c t r o m e t e r . facility i n the Department WP-80 o r a V a r i a n were c a r r i e d o u t A l l s p e c t r a were r e c o r d e d a t of Chemistry. Gas C h r o m a t o g r a p h y Routine either gas chromatographic a Hewlett-Packard analysis was c a r r i e d out using 5880A o r 5830 GC w i t h a flame ionization 6 3 or e l e c t r o n - c a p t u r e d e t e c t o r C columns used The flame were a s o u t l i n e d ionization were o p e r a t e d 5. Liquid chromatographic f o r the quadrupole d e t e c t o r and t h e e l e c t r o n GCMS experiments. capture detector a t 275 °C a n d 300 °C r e s p e c t i v e l y . Chromatography Liquid chromatographic Hewlett-packard wavelength fraction collector. c o l u m n was used guard analysis 1082B L i q u i d Variable C-18 N i ) . The was c a r r i e d out using a Chromatograph, w i t h an A l t e x d e t e c t o r and a 7000 U l t r o r a c A C-18RP P a r t i s i l - 1 0 , automatic magnum-9 p r e p a r a t i v e w i t h a R a i n i n 0.45 urn o n - l i n e f i l t e r column. Flow r a t e s were r a n g i n g mL/min w i t h a c o l u m n p r e s s u r e between from and a R a i n i n 2.3 t o 3.5 13,790 KPa (2000 p s i ) t o 17,237 KPa (2500 p s i ) . 6. Infared IR nujol Spectroscopy s p e c t r a were o b t a i n e d w i t h mull u s i n g an Unicam SP-1000 7. M e l t i n g P o i n t Melting capillary sodium c h l o r i d e d i s k s as a spectrometer. Determination P o i n t s were d e t e r m i n e d melting point apparatus 34 on a Thomas-Hoover^ and were uncorrected. C. S y n t h e s i s o f 3 * , 4 ' . 5 ' - C H ] - 2 - a m i n o - 2 ' . 6 ' 2 3 p r o p j o x y l jclj^e The Byrnes for s y n t h e s i s f o l l o w e d a m o d i f i e d method r e p o r t e d by (31). peptide The s y n t h e t i c s y n t h e s i s ( 6 2 ) . The p r o c e d u r e carbobenzyloxyl-D dimethylaniline form s t e p s were s i m i l a r i n the presence tocainide. The c a r b o b e n z y l o x y 1 yielding Initial hydrogen 2 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 out u s i n g n o n - d e u t e r a t e d commercially available 2,6-dimethylaniline methylene suction was d r i e d N-carbobenzyloxyl tocainide. bromide acid the t o c a i n i d e A solution was a d d e d derivative in small q u a n t i t i e s overnight salt. to y i e l d (0.0147 mole, 65 % i n 20 mL o f f with u n d e r vacuum t o y i e l d 3.08g o f o f 30 t o 32 % h y d r o g e n in sufficient q u a n t i t i e s to f o l l o w e d by vacuum r e m o v a l h y d r o g e n b r o m i d e a t room t e m p e r a t u r e . hydrogen bromide and ( 5 . 0 g, 0.0224 mole) and 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 and the f i l t r a t e dissolve 2,6-dimethylani1ine (2.72 g, 0.0224 mole) were d i s s o l v e d chloride. in acetic tocainide N-carbobenzyloxyl-D-alanine. N-Carbobenzyloxy-D-alanine The was S(+)-3*.4'.5'-C H13-tocainide. were c a r r i e d added tocainide group salt. 1. S y n t h e s i s o f excess i n v o l v e s t h e use o f N- 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 t h e n removed by H B r / a c e t i c a c i d of utilized o r 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 - N-carbobenzyloxyl bromide to those Then e t h e r f o r the c r y s t a l l i z a t i o n The e t h e r e a l s o l u t i o n 2.83 g o f t o c a i n i d e was of t o c a i n i d e was l e f t hydrogen bromide a t 0 °C salt yield). hydrogen bromide salt was removed by e x t r a c t i o n o f 35 of tocainide base reconstituted i n methylene with under ethanol-100 and a c i d i f i e d ethanol/hydrochloric ether chloride, dried acid a t 0 °C o v e r n i g h t solution. to y i e l d nitrogen, with The s a l t was c r y s t a l l i z e d 0.36 g t o c a i n i d e hydrochloride sal t. Gas C h r o m a t o g r a p h y (Chirasi1-Val S(+)-tocainide (61 R(-)-tocainide (39 %) Melting % ) , Point: 266 ° C u n c o r r e c t e d Infared (Nuiol stretch), _ 1 1660(C=C s t r e t c h ) , vicinal ( r e f : 264.5 - 265.5 ° C ) ( 3 1 ) . mull): 3300 c m ( N H 1540(C=O 1150, 1090, 970, 780 ( t r i - s u b s t i t u t e d (GCMS): m/z 192 ( M 2. S y n t h e s i s 5 % ) , 44(100), + 121(8), of 2,6-dimethylani1ine perdeuteration A sample of 2,6-dimethylani1ine residual bath technique (0.1 mole) f o l l o w e d the a n i l i n e in a single was c a r r i e d reported (2 mL, 0.016 mole) was added t o by a s u f f i c i e n t sulfate salt. condenser apparatus The d r y a n i l i n e 5 mL o f D20/D2SO^ vessel salt (pH 1) a n d h e a t e d amount o f The m i x t u r e was f o r 2 h o u r s b e f o r e the over a water was r e c o n s t i t u t e d w i t h i n a Variac f o r 18 h o u r s a t 250 ° C w i t h 36 out using a by F r i s c h k o r n ( 5 1 ) . was removed by r o t a t o r y e v a p o r a t i o n a t 100 ° C . reaction 91(3). 2 modified D2SO4 t o form 105(5), 3,4,5-[ Hlo-2,6-dimethvlani1ine of Deuteration refluxed stretch), aromatic). Mass S p e c t r u m 2 mL o f column); ^ 4137 KPa ( 6 0 0 P S I ) cold nitrogen. The e x c e s s released periodically pressure below mixture filtered d u r i n g the experiment t o pH 10 w i t h with chloroform, d r i e d and e v a p o r a t e d 1.8 mL (90 %) o f a d a r k Mass S p e c t r u m unresolved over anhydrous sodium a t room t e m p e r a t u r e , peaks: 100 % ) , 1 0 0 ( 9 5 ) , 80(35). SCAN 61: ( 2 ) m/z 125 ( M + 100 % ) , 1 0 9 ( 5 5 ) , 110(55), reaction product reaction o i l bath. liquid i t was d r i e d and e v a p o r a t e d singular sodium 124 ( M collected + by d i s t i l l a t i o n a t 218 °C was a l i g h t 50 % ) . 100 % ) , 1 0 9 ( 6 0 ) , 80(20). (CDCI3): S ppm, 2.2s(6H, - C H 3 ) , 3.5s(2H, -NH ), p a r a - a r o m a t i c ) , 6.95s(2H, 37 deuterium over n i t r o g e n . was t h e n p u r i f i e d yield 3 times sulfate, component: MHz-C Hl-NMR 6.65s(lH, o f t h e amine (TIC): SCAN 48: ( 1 ) m/z 1 over anhydrous The f r a c t i o n Mass S p e c t r u m ( f o r exchange to dryness product (1.1 mL, c h e m i c a l 80(20). i n c h l o r o f o r m was t h e n washed 2 mL p o r t i o n s o f w a t e r The yielding liquid. + filtered, sulfate, (TIC): 3 major atoms) b e f o r e 80 hydroxide, 126 ( M with was h e a t i n g , the r e a c t i o n 1.0 M s o d i u m to dryness brown vessel t o m a i n t a i n the SCAN 51: ( 1 ) m/z The an i n the r e a c t i o n 13790 KPa (2000 P S I ) . A f t e r was b a s i f i e d extracted pressure 2 meta-aromatic). over brown D. Attempted S y n t h e s i s 1-C H 3 3 - 2 - a m i n o - 2 * . 6 of *- propioxylidide Due t o damage o f the Variac r e a c t i o n v e s s e l , the original 9 for synthesizing 3',4',5'-[^Hl3-tocainide synthetic route abandoned i n f a v o r o f an alternative method 1- 1 H ] 3 ~ 2 - a m i n o - 2 ' , 6 ' - p r o p i o x y 1 i d i d e . available by r e a c t i o n with I. S y n t h e s i s Initial D-alanine. mL of attempts D-alanine and was hydrochloric The with under ice u n t i l reaction white 0.01 the were c a r r i e d (2.62 g, out form l-[ H^- a l l CBZC1 was terminated by acid the until the The with before stirring. added r e a c t i o n mixture is acidic was being g of product was hours). dropwise a d d i t i o n of solution 38 d i s s o l v e d in 0.0294 mole) was of N-CBZ-alanine 5.7 solution d i s s o l v e d (6.0 M hydrochloric acid yielding using unlabelled 0.0294 mole) was ice c o o l i n g . precipitate temperature, to N-carbobenzyloxy-(+)-Do-alanine of s a t u r a t e d sodium b i c a r b o n a t e stirring involves as d e s c r i b e d p r e v i o u s l y . C a r b o b e n z y 1 o x y c h l o r i d e (14.18 mL, under This approach 2,6,-dimethylaniline 2- a m i n o - 2 * , 6 ' - p r o p i o x y 1 i d i d e 200 s y n t h e s i s of o f N - c a r b o b e n z y 1 o x y - < D ) - [ 2 H ^ - a l a n i n e from c o m m e r i c a l l y 2 [ *"H 1 3 - a l a n ine and c a r b o b e n z y 1 o x y c h l o r i d e (CBZCL) 2 synthesis followed f o r the was to stirred The concentrated litmus filtered with and paper. washed v a c u u m - d r i e d a t room (87.9 h chemical yield). Melting Point: 71 - 72 °C < r e f . 79.5 - 80 ° C , Sigma C h e m i c a l C o . ) . o After 24 h o u r s , crystallization the r e a c t i o n in chloroform/ether at 0 product yielded 3.2 g white C for powder (55.8 % ) . Melting Point: 79 °C. ( r e f . 79.5 Infrared (Nuiol - 80 °C, Sigma C h e m i c a l Co.) mull): 1700 c m ( C = 0 s t r e t c h ) , 3000(0-H s t r e t c h ) , - 1 3350CN-H s t r e t c h ) , 1540(N-H E. S y n t h e s i s of P o t e n t i a l stretch), 1380C0-H s t r e t c h ) . Tocainide Metabolites 1. S y n t h e s i s o f 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 - Tocainide 1 mL base ( 2 . 3 x 10 mole) a c e t y l a 5 mL r e a c t i o n for 15 m i n u t e s nitrogen. for ( 1 . 0 mg, vial. 5.2 x 10 aldehyde The r e a c t i o n p r o d u c t was mole) was a t room proceeded before excess aldehyde The r e a c t i o n fi was added temperature a t room reconstituted with (Nuiol mull): 1680 cm~l(C=0 s t r e t c h ) , 3300(N-H stretch), 1670(C-N s t r e t c h ) , 700, 730 and 1030(tri-substituted vicinal aromatic). Mass S p e c t r u m m/z 218 (GCMS): (M+10 % ) , 71(100), 56(35), 39 44(15), 190(10). in temperature removed by d r y i n g GCMS a n a l y s i s . Infrared to under methanol 2. S y n t h e s i s o f Synthesis reactive to form the h y d a n t o i n internal cyclization S y n t h e s i s of the i s o c y a n a t e trichloromethyl toluene, tocainide o u t u s i n g a phosgene chloroformate ( T C F ) , as d e s c r i b e d by of f r e s h base TCF (0.1 mole/200 mL) in small q u a n t i t i e s was added d r o p w i s e from with stirring yielding yielding was r e c r y s t a l 1 i z e d 0.5 g o f the pure Another 0.6 heating mL o f precipitate under vacuum a t room crystals. The reaction i n c h l o r o f o r m a t 0 ° C f o r 24 hydantoin. product was a n a l y z e d by IR, GCMS and Melting Point; This (Nuiol NMR. mull): 3300 c m ( N - H s t r e t c h ) , -1 1795(C=0 s t r e t c h ) , Mass S p e c t r u m m/z 218 ( M 132(25), + 2300(N-C=0 1700(C=0 stretch), stretch). (GCMS): 100 % ) , 1 4 7 ( 9 0 ) , 113(20), 203(10), 44(72), 176(5). 40 hours, recrystal1ized 138 - 140 ° C ( r e f . 138 - 142.5 ° C ) ( 3 1 ) . Infrared carbon f o l l o w e d by The w h i t e was d r i e d 1.5 g o f w h i t e and g r a d u a l mixture 2 hours. o f f and t h e f i l t r a t e temperature, product t o the r e a c t i o n 80 t o 100 °C o v e r filtered i n sodium ( 1 . 0 g) and 0.1 g o f a c t i v e 50 t o 80 °C o v e r a 30 minute p e r i o d . heating was f o l l o w e d by was c a r r i e d a stock s o l u t i o n was a d d e d TCF ring. i n v o l v e d the f o r m a t i o n of a ( 5 8 ) and K u r i t a ( 5 9 ) . To from hydantoin of t o c a i n i d e substitute, dried of t h i s isocynate d e r i v a t i v e derivative Ozaki 3-(2.6-Xvlvl)-5-methvlhvdantoin 119(30), 105(28), 400 (CDCI3); MHz-[ H]-NMR 1.54s(3H, 5 - C H 3 ) , t> ppm 2.18s(3H, x y l i d i n e - C H 3 > , 2.22s(3H, x y l i d i n e - C H ) , 3 6.56s(lH, 3. l-NH), 1H, 5 - C H ) , 4.24dq(J=8Hz, 7.1 t o 7 . 3 s ( 3 H , aromatic-H). S y n t h e s i s of 3-(2.4,6-Xvlyl)-5-methvlhvdantoin The the synthetic 2,6-xylyl synthesize p r o c e d u r e s were s i m i l a r analog. However, initial t o those d e s c r i b e d f o r attempts 2-amino-2',4',6'-propioxy1 idide were made t o (4'-methyl analog of tocainide). a Synthesis of 2-Amino-2*.4*.6'-propioxvlidide The synthetic s t e p s were s i m i l a r 2',6'-propioxylidide analog. 0.0488 mole) was added t o those d e s c r i b e d f o r the N-carbobenzyloxy1-alanine t o 6.05 g (0.0443 mole) o f 2,4,6- t r i m e thy1 a n i 1 i n e i n 60 mL o f d r y m e t h y l e n e Dicyclohexylcarbodiimide under stirring temperature f o r 1 hour. was d r i e d hydrogen bromide bromide salt mixture The m i x t u r e vacuum. in acetic acid was l e f t was f i l t e r e d A solution was t o s t a n d a t room and the o f 30 t o 32 % added, and the hydrogen o f t h e 4 ' - m e t h y l t o c a i n i d e a n a l o g was r e c o v e r e d and recrystal1ized hydrogen under chloride. ( 9 . 2 3 g , 0.0448 mole) was added s l o w l y and t h e r e a c t i o n filtrate ( 1 . 0 g, by t h e a d d i t i o n bromide of e t h e r y i e l d i n g 5.61 g o f salt. b. R e a c t i o n w i t h T r i c h l o r o m e t h v l C h l o r o f o r m a t e The 4 * - m e t h y l t o c a i n i d e hydrogen 0.0097 mole) was e x t r a c t e d with anhydrous stream sodium of n i t r o g e n . i n base sulfate, The base bromide salt w i t h methylene and e v a p o r a t e d was d i s s o l v e d 41 ( 2 . 0 g, chloride, to dryness dried under a i n 30 mL d r y t o l u e n e , followed by agitation dropwise a d d i t i o n and described. dryness, The gradual The and heating reaction solution TCF (1.16 mL, 0.0097 mole) w i t h from room t e m p e r a t u r e mixture reconstituted chloroform of was then in chloroform yielded 0.83 to filtered, 80 °C as evaporated for r e c r y s t a l 1 i z a t i o n . g of 3-(2,4,6-xylyl)-5- methylhydanto i n . Me 11 i ng 137.5 Point: - Infrared 139 °C (Nuiol mull): 3300 c m ( N - H stretch), - 1 1795(C=0 s t r e t c h ) , Mass S p e c t r u m m/z 232 (M+ 2300(N-C=0 s t r e t c h ) , 1700(C=0) s t r e t c h ) . (GCMS): 100 % ) , 161(100), 44(40), 146(35), 119(25), 217(10). 80 ( CDCl 3 ) : MHz-C^-Hl-NMR 1.52s(3H, 5 - C H 3 ) , S ppm. 2.12s(3H, x y l i d i n e - C H ) , 3 4.25dq(J=8 Hz, 6.9s(2H, 1H, 2.32s(3H, 5-CH3), I d e n t i f i c a t i o n of 1. Permethvlatlon procedure addition Des i d e r i o 4'-xylidine-CH ), 3 6.15bs(lH, Potential involved of CH^I as Tocainide generation s u 1 f i n y 1 m e t h i d e c a r b a n i o n as by xylidine-CH3), 1-NH), aromatic-H). F. The 2.12s(3H, reaction outlined (61). 42 by of Metabolites fresh sodium catalyst/solvent Thompson and methyl followed to 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 Because fresh with of the h y g r o s c o p i c calcium hydride DMSO ( 2 0 mL) was added bottom flask nature before (DMSO) o f DMSO, i t was d i s t i l l e d each p e r m e t h y l a t i o n t o 2.0 g o f c a l c i u m h y d r i d e and d i s t i l l e d experiment. ina under vacuum a t 70 ° C u n t i l round 15 mL o f d r y DMSO was c o l l e c t e d . b. G e n e r a t i o n o f 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 (250 salt was p r e p a r e d mg, 50 % d i s p e r s e d with sodium h y d r i d e . i n mineral oil), o f 3 mL a n h y d r o u s e t h e r . transferred dry DMSO. nitrogen quickly The g r e y until straw-color solution. t o a round color suspension The f i l t e r e d three times with a The washed s o d i u m h y d r i d e was bottom suspension the formation hydride a n amount e q u i v a l e n t t o 5 moles o f s o d i u m h y d r i d e , was washed r a p i d l y total Sodium flask c o n t a i n i n g 5 mL o f was t h e n heated of hydrogen ceased. was a l l o w e d to s e t t l e supernatant gently under The r e s u l t i n g giving a clear was s t o r e d u n d e r n i t r o g e n a t -20 °C. c. Permethylat ion An aliquot methanol was dried o f 200 uL o f i s o l a t e d (asdiscussed under n i t r o g e n . vial. in a vortex The r e s i d u e was t h e n temperature. the mixture DMSO sodium c a r b a n i o n mixer, and l e t s t a n d Subsequently, was s t i r r e d sample i n i n the f o l l o w i n g experimental w i t h 50 uL o f d r y DMSO u n d e r n i t r o g e n reaction metabolite sections) reconstituted i n a 5 mL s c r e w - c a p p e d (20 u L ) was a d d e d , stirred f o r 15 m i n u t e s a t room 1.2 uL o f m e t h y l i o d i d e was added and as b e f o r e . 43 The r e a c t i o n was a l l o w e d t o p r o c e e d a t room t e m p e r a t u r e f o r 1 h o u r b e f o r e by the a d d i t i o n was e x t r a c t e d with o f 1 mL o f w a t e r . with under n i t r o g e n 2. A t t e m p t e d The p e r m e t h y l a t e d g l u c u r o n i d e 1 mL o f c h l o r o f o r m 1 mL o f w a t e r . The c h l o r o f o r m before i t was t e r m i n a t e d a n d back-washed extract three was t h e n c o n c e n t r a t e d GCMS a n a l y s i s . Identification of Permethylated Urinary Tocainide Metabolites Scheme 9 o u t l i n e s conjugated the p r o t o c o l used in screening both and n o n - c o n j u g a t e d m e t a b o l i t e s . PH 3 PH 12 u r i n e (100 mL). • ( t r a c t with •ethylene Chi or i d * I • s t r a c t with aiethylene chloride PH 3 XAD r e s i n 20 f r a c t i o n s each c o n t a i n i n g I concentrate 3 mL m e t h a n o l i c with eluent nitrogen 2. HPLC reverse-phase C-1B TLC reverse-phase C-18F I B a s i f y t o PH 13 (basic hydrolysis) Vi sual i z a t i o n u n d e r UV OR Naphthoresorcinol reagent 3. 1 Enzyne h y d r o l y s i s OP Acid hydrolysis ac C h i r a s i l - V a l c a p i l l a r y column i r a l l 1, 2 , a n d 3 g i v e p o s i t i v e OR IF 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 BC/MS • e t h y l a t i o n w i t h d i azomethane • • 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 t r i a t e t h y l s i l y a t i o n w i t h BSTFA anion SC/RS Scheme 9 : A t t e m p t e d tocainide times identification metabolites. 44 of permethylated urinary a. I s o l a t i o n of Total Tocainide-carbamoyl urine (1 volunteer after a 2 cm cm x 20 urinary The crude KC18F TLC plate the Rf the four bands on the TLC ou11ined and be : The TLC thus were plate initially eluted The 15 to with XAD band onto a 20 with a cm of methanol. x 20 cm 60/40 e l u t i o n , the following from A trap the the i s o l a t e d together plate pooled. Table 2 crude as f r a c t i o n s were a plate outlined extract. plate obtained as Rf o f u r i n a r y components s e p a r a t e d on a KC-18F 20x20 cm plate. •. • .36 •.34 o.3o • . 60 •.43 70 from e a c h p l a t e individually evaluated plates a f r a c t i o n were chromatographical1y 50/50 m e t h a n o l / w a t e r f r a c t i o n 1, was « 3 • .67 • .63 • ••4 • .61 samples, using fraction S O. 64 • -•1 • .74 system, After was 1ow. plat* plate eluted bands s e p a r a t e d four 1 The was fractions. 13 used a narrow system. the tocainide, subsequently as plate of fraction, of t h u s c o l l e c t e d from applied solvent into values Various and Glucuronide c o l l e c t e d from a human o r a l dose sample r e s i n was divided mg hours), w h i c h were methanolic was 10 XAD-2 r e s i n c o l u m n was methanol/water 2 200 metabolites adsorption Table to Ester obtained resolved from i n t o bands 45 the on solvent 20 again x 20 system. cm Band 60/40 m e t h a n o l / w a t e r l a and l b by the KC18F 1 of solvent 50/50 m e t h a n o l / water s o l v e n t band are l a and listed system. These two lb r e s p e c t i v e l y . in Table bands were The Rf i s o l a t e d and values of the band labelled l a and lb 3. T a b l e 3 : The Rf t h r e e 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 . Band l a Voliriteer 2 Band 1 The rest of the 11 bands Band l b 0.84 Band 2 0.69 in plate 0.06 fractions 1 to 4 were also isolated. b. Acid and Enzyme H y d r o l y s i s A portion plates was hydrolyzed glucuronidase hydrochloric at 100°C uL with a gas with acid was added hydrochloric glucuronidase acid hydrolysis, t o the band or 0.5 55°C f o r 30 column. hydrolyzed with the a 25 - g l u c u r a s e ^ i n the 46 0.5 TLC betamL of 1.0 samples and M than heated were derivatized minutes b e f o r e Samples with the f r a c t i o n s and treatment, chromatograph equipped were a l s o i s o l a t e d from methylene c h l o r i d e , i n hexane a t Chirasi1-Val^ capillary fractions samples minutes. A f t e r e x t r a c t e d with o f HFBA the enzyme. F o r a c i d f o r 30 basified, 20 o f e a c h of m x 0.31 with analyzed mm i s o l a t e d from mL of the beta- presence of 1.0 mL id. the band of a c e t a t e The b u f f e r at t u b e s were After yielded conjugate c. the in PTFE-lined that of analyzed Band subjected from the Glucuronic the t o pH 12 solution. synthesized were e x t r a c t e d of Acid the has by been be monitored material. i n t o methylene c h l o r i d e a 5 urn C-18 25 the on by Tocainide 55 % acetonitrile and °C. t o pH the ODS of 12, to were 1.0 N tocainide 3-(2,6-xylyl)- HPLC and Liquid i n 0.05 plates addition After basification, c a r r i e d out compared the samples solvent chromatographic (25 cm M potassium x 4.6 mm) HPLC phosphate system. Chromatography/Mass S p e c t r o m e t r y Permethvlated The Tocainide suspected methyl i o d i d e of lb acid TLC shown t o c y c l i z e analysis Gas Band hydrolysis of Upon b a s i f i c a t i o n at column w i t h a hours. Hydantoin Conjugate under n i t r o g e n d. described. a glucuronic concentrated solvent as enzymatic alkaline condition glucuronide was 12 extracted, f r a c t i o n s i s o l a t e d from r e v e r s e - p h a s e 5 - m e t h y l h y d a n t o i n which can with GC for tubes. tocainide. sodium h y d r o x i d e carbamoyl by f r a c t i o n contained L i q u i d Chromatographic A n a l y s i s Derived 37°C s a m p l e s were b a s i f i e d , a f t e r b o t h a c i d and the screw-capped g l a s s in a water bath a t w i t h HFBA, and tocainide indicating 5 incubated hydrolysis, derivatized pH in dry Carbamoyl tocainide methylsulfinylmethide carbanion 47 as of Glucuronide glucuronide dimethylsulfoxide Analysis was permethylated (DMSO) i n the described with presence previously. 3. A t t e m p t e d I d e n t i f i c a t i o n of Urinary Metabolites Urine between HC1. bv L v o o h i 1 i z a t i o n samples Tocainide a n d GCMS were c o l l e c t e d from a h e a l t h y 2 t o 16 h o u r s a f t e r an o r a l dose The f o l l o w i n g Scheme Analysis 10 o u t l i n e s volunteer o f 200 mg tocainide the experimental 2-16 hours pooled u r i n e pH 12 (1.0M NaOH) —1 Unadjusted f | I Lyophillzation Extracted Methylene C h l o r i d e 1 1 pH 3 (1.0M HCL) Lyophillzation Extracted Methylene C h l o r i d e Extracted Methylene C h l o r i d e 1 Pennethylation (CH I/DMS0) 3 1 1 ( S i l a r -GC/MS l O C Column) Scheme 10 : Attempted i d e n t i f i c a t i o n of urinary tocainide metabolites 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 protocol. 4. A t t e m p t e d Isolation N-carbamic A c i d Urine between HC1. samples or N-ureide that tocainide human s u b j e c t an o r a l dose 11, a s shown b e l o w , o u t l i n e d was u s e d metabolite of Tocainide Intermediate from a h e a l t h y 1 t o 20 h o u r s a f t e r Scheme design and I d e n t i f i c a t i o n i n an a t t e m p t were o f 200 mg tocainide the experimental to i s o l a t e a n d i d e n t i f y the intermediates. (10 mL) 1-20 I C-18 Bond-elute hours pooled u r i n e s o l i d e x t r a c t i o n (MeOH) Beta-i glucuronidase Hydro Lysis Extracted pH 3 collected Extracted pH 12 Extracted H 3 P I Permethylation Extracted ,H 12 Acid Hydrolysis Extracted pH 3 Extracted pH 12 DMS0 NatCH^I)- GCMS Scheme 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 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 o f U r i n a r y Metabolites bv F l a s h Chromatography A volume o f 500 mL o f u r i n e , after an o r a l dose used. As shown concentrated using c o l l e c t e d from o f 200 mg o f t o c a i n i d e i n Scheme 12, t h e p o o l e d on a XAD-2 a b s o r p t i o n acetonitrile 1 t o 7 hours i n a human s u b j e c t urine sample r e s i n column as was was first described a s an e l u a n t . 500 mL p o o l e d 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 • silica'column TLC : 1. KC-18F 2. Alumina 3. S i l i c a Scheme 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 urinary metabolites by flash chromatography. A 6-inch glass silica c y l i n d e r with column ends. solvent nitrogen g e l c o l u m n was d r y - p a c k e d 0.5 cm s i l i c a sand i n a 1 cm i d . forming a seal A 30/20/50 h e x a n e / 2 - p r o p a n o l / m e t h a n o l was f l u s h e d through a t 1 mL/min u n t i l the packing with on t h e combination 137 KPa ( 2 0 P S I ) a l l g a s b u b b l e s were e l u t e d . of 5.0 mL o f t h e XAD-2 u r i n a r y acetonitrile on t o p o f the column and then e l u t e d 50 with extract A volume was d e l i v e r e d the d e s c r i b e d solvent system a t 1 mL/min c o l l e c t i n g was s p o t t e d on a s i l - U V ^ t j ^ fraction plate was e l u t e d Fractions to with dryness 30/20/50 H e x a n e / 2 - p r o p a n o l / Rf v a l u e s , a n d t h e s e three samples. The s o l v e n t o f DMSO s o d i u m , Due to a preliminary u r e a t o form study a hydantoin the r e a c t i o n found sample and t h e to correspond were pooled was e v a p o r a t e d t o w i t h CH^I o r CD^I i n 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 o f T o c a i n i d e w i t h to i n each of each methanol. various fractions a n d t h e r e s i d u e was d e r i v a t i z i e d the p r e s e n c e An a l i q u o t 2 x 5 cm TLC p l a t e 4 a n d 5, 6 t o 11, a n d 12 t o 16 were 3 distinct into 1 mL f r a c t i o n s . Urea observation that structure, at different tocainide experiments reacts with were c a r r i e d o u t pH, t e m p e r a t u r e , and r e a c t i o n t i me . 1. The E f f e c t o f Temperature Tocainide solution. 100°C 60°C the base addition o r 13 h o u r s , The s a m p l e s were sulfate o f n i t r o g e n f o r GCMS a n a l y s i s . sample of tocainide was added a n d was e i t h e r 100 °C f o r 1 h o u r to d i s t i l l e d and then The s o l v e n t In a d d i t i o n , to a f r e s h l y left or 9 hours. b a s i f i e d by was d r i e d before b e i n g c o n c e n t r a t e d over a stream added or a l t e r n a t i v e l y a t subsequently w i t h 5 mL m e t h y l e n e c h l o r i d e . sample i n a block heater a t o f 200 uL o f 1.0 M s o d i u m h y d r o x i d e o v e r a n h y d r o u s sodium urine t o 1 mL o f 8.0 M u r e a was t h e n h e a t e d 9 hours f o r 3 hours. extracted ( 5 0 mg) was added The s o l u t i o n f o r 1 hour, and R e a c t i o n D u r a t i o n collected a t room t e m p e r a t u r e a 500 mg drug-free or heated a t F o r r e f e r e n c e , t o c a i n i d e was d e i o n i z e d water and heated hours. 51 a t 100 °C f o r 13 2. The E f f e c t Into three separate dissolved for tubes, tocainide i n 1 mL o f 8.0 M u r e a s o l u t i o n adjustment with of D H 6 hours. 20 cm RPC18F The s a m p l e s were plate elution and e l u t e d then d i r e c t l y w i t h 80/20 on t h e TLC p l a t e s e p a r a t e l y by B o n d - e l u t extraction columns acid or a t 60 ° C s p o t t e d on a 5 x methanol/water. and v i s u a l identification mixtures r e v e r s e phase were then solid f o l l o w e d by p e r m e t h y l a t i o n w i t h CH^I sodium DMSO a n d GCMS a n a l y s i s TLC f o l l o w e d by pH The s a m p l e s were h e a t e d u n d e r UV a t 254 nm, t h e t h r e e r e a c t i o n isolated ( 1 0 0 ) mg was t o 2.3, 7.8 o r 12.5 w i t h 0.1 M h y d r o c h l o r i c 0.1 M s o d i u m h y d r o x i d e . Following base in as d e s c r i b e d p r e v i o u s l y . (KC-18F); 80/20 m e t h a n o l / w a t e r : t o c a i n i d e Rf = 0.45, h y d a n t o i n Rf = 0.75, N-ureide Mass S p e c t r u m 3. A t t e m p t + N-ure i d e - t o c a i n i d e , trace), were d i s s o l v e d base described heated 191(5). ( 4 0 0 mg) a n d 300 mg s o d i u m g l u c u r o n i c a c i d 1 or 2 hours isolation previously. The s o l u t i o n f o l l o w e d by B o n d - e l u t was h e a t e d a t R solid a n d p e r m e t h y l a t i o n w i t h CHgl/Na DMSO a s In a n o t h e r a t 50 °C f o r 1 h o u r addition 129(45), i n 1.0 mL o f 8 M u r e a . o C for either extraction 72(100), Synthesis of Tocainide-N-ureide-qlucuronide Tocainide 50 Rf = 0.60. (GCMS): permethylated m/z 191 ( M tocainide experiment, i n the presence o f sodium g l u c u r o n i c a c i d . 52 tocainide of urea p r i o r The r e a c t i o n was f i r s t to the m i x t u r e was then left a t room t e m p e r a t u r e overnight before s o l i d phase extraction a n d p e r m e t h y l a t i o n w i t h CH^I/Na DMSO a s d e s c r i b e d . 4. A t t e m p t S y n t h e s i s of 2.6-Dimethvlani1ine- N - g l u c u r o n ide 2 , 6 - D i m e t h y l a n i l i ne 2:1 ace t o n e : w a t e r reaction acid mixture and mixed spotted was a d j u s t e d t o pH 5 w i t h rack permethylated by s o l i d The p r o d u c t s were and e l u t e d F o l l o w i n g TLC v i s u a l i z a t i o n was i s o l a t e d The 1.0 M h y d r o c h l o r i c overnight. on a 2 x 5 cm RPC-18F TLC p l a t e the p r o d u c t t o 3 mL 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 . on a r o t a t i n g methanol/water. TLC ( 2 . 5 mL, 1.02 mole) was a d d e d using 40/60 u n d e r UV a t 254 nm phase e x t r a c t i o n a n d w i t h CH^I/Na DMSO, f o l l o w e d by GCMS a n a l y s i s . (KC-18F): 40/60 m e t h a n o l / w a t e r : 2,6-dimethylani1ine N-glucuronide Mass S p e c t r u m Rf = 0.25, Rf = 0.45. (GCMS): permethylated N-glucuronide. m/z 353 (m+ 8 % ) , 1 0 3 ( 1 0 0 ) , 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 Elucidation of Tocainide G l u c u r o n ide Urine had s a m p l e s were c o l l e c t e d received a n i n t r a v e n o u s dose hydrochloride. collection yellow An a l i q u o t from a human s u b j e c t who o f 200 mg o f t o c a i n i d e o f 100 mL o f t h e 14th h o u r was c o n c e n t r a t e d u n d e r vacuum residue. phosphoric acid to y i e l d urine 5.9 mL o f a T h i s r e s i d u e was a d j u s t e d t o pH 3 w i t h 0.1 M and e x t r a c t e d w i t h 53 10 mL o f hexane prior to e v a l u a t i o n by preparative HPLC. In a n o t h e r e x p e r i m e n t , same s u b j e c t temperature was with residue. The chloride a t pH concentrated 10% ACN residue 7 o f hexane a t pH t h r o u g h a 0.45 100 was The by of the extracted by filter hour u r i n e evaporation yield 10 3 times with 3 repetitive extracted urn s y r i n g e 7th azeotropic u n d e r vacuum t o followed 3. mL mL 10 at mL the room of a yellow methylene e x t r a c t i o n s with s a m p l e s were t h e n before from 10 mL filtered preparative HPLC i s o l a t 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 Ester G l u c u r o n ide Initial indicated required s e l e c t i o n of solvent proportion of f o r the e l u t i o n of tocainide glucuronide. approach, (pH was reasonable to o f f e r components. 266 nm. detector The 2.0 o u t l e t with absorbance of each used to mL an the m o n i t o r the major p e a k s were p o o l e d hydrolysis be as well as d e t a i l e d in l a t e r Thirty-six solvent by i n 0.01 phase M sodium and 3 mL/min, urinary at 220, f r a c t i o n s were c o l l e c t e d at the automatic was fraction measured by corresponding collector. a The d e t e c t i o n wavelength Fractions and b a s e , a c i d and by a naphthoresoreinol the enzyme color test, 54 azeotropical1y that containing as sections. i n j e c t i o n s of 225, UV column e l u a n t . analyzed a phosphate r e t e n t i o n of m o n i t o r e d by TLC was Through system d e l i v e r e d at separation was mobile UV fraction spectrophotometer at was 3) column e l u a n t Eighty i n the 10 % a c e t o n i t r i l e (monobasic) b u f f e r found water reverse-phase a high systematic or systems u s i n g prepared will sample were made w i t h acetonitrile pH i n 0.01 M sodium 3 mobile phase. the re-purify phosphate the c o l l e c t e d collected as d e s c r i b e d . estimate The Based with dilute acid under UV for this 2-step tocainide glucuronide t h e sample contained i n sodium isolated. i n t h e e l u a t e was 0.1 M sodium was c o n c e n t r a t e d under was removed by a c i d i f y i n g hydrochloric acid was i s o l a t i o n , an in 3 % acetonitrile ( a d j u s t e d t o pH 7 w i t h salt eluant o f u r i n e , c o n t a i n i n g 67 ug/mL salt acetate of 3 % a c e t o n i t r i l e The column sodium fraction under and t e s t e d f o r the p r e s e n c e o f TOCG t h a t was u s e d removed by r e c y c l i n g isolated 4 sample. on t h e volume phosphate to c o n t a i n and c o n c e n t r a t e d (63 mg) c o u l d be TOCG-containing u s i n g a 10 % (monobasic) b u f f e r a t (monobasic) s a l t acid loop ( m o n o b a s i c ) a t pH 7 was u s e d t o fraction o f 10 mg o f pure phosphate sodium was p o o l e d in fractions the h y d a n t o i n , acetic phosphate A second s o l v e n t system c o n s i s t i n g i n 0.01 M sodium again injection F r a c t i o n 4, w h i c h was i d e n t i f i e d tocainide glucuronide, vacuum. of a 200 uL sample i n 0.3 % hydroxide). vacuum, and the t h e sample f o l l o w e d by e v a p o r a t i o n vacuum a t room t e m p e r a t u r e . The The r e s u l t e d t o pH 3 of the f r e e TOCG was in NaCl. Spectrum: * max 202.5 nm 0.01M sodium ( A max t o c a i n i d e = 202.5 p h o s p h a t e , pH 7 ) . 55 in 3 % acetonitrile in Mass S p e c t r u m (GCMS); (BSTFA/pyridine m/z 218 ( M (acetic anhydride/diazomethane d e r i v a t i z a t i o n 218 ( M ppm 100 + (D^-DMSO): 1.38d(3H,I-CH3), acid), 2.11s(3H,xylidine-CH ), 3 3.92d(glucuronic 4.25t(1H,2-CH3), 5 . 7 7 s ( g l u c u r o n i c 7.06s(3H,xylidine-H), Atom leads to %>. 3.69d(glucuronic Fast o f TOCG) o f TOCG) 400-MHz-[1-NMR S leads to decomposition 100 %) . + decomposition m/z derivatization acid), acid), 9.39s(lH,xylidine-NH). Bombardment; Glycerol: m/z Thioglycerol: 413 (M + 1 ) , 435(M + N a ) , 457(M m/z -H+2Na). 413 (M + 1 ) , 435(M + N a ) , 457(M -H+2Na). a . A c i d H y d r o l y s i s o f TOCG w i t h H y d r o c h l o r i c A c i d Each p o o l e d was c o n c e n t r a t e d fraction, collected heated with To e a c h c o n c e n t r a t e d hydrochloric acid a t 100 °C f o r 1 h o u r , 5.0 M sodium h y d r o x i d e , chloride, presence concentrated fraction, 1.0 mL o f was added and t h e sample f o l l o w i n g which, extracted with i t was was 2 mL o f m e t h y l e n e f o r the o f t o c a i n i d e by GC w i t h a SE-30 c a p i l l a r y column. Each pooled concentrated then basified under n i t r o g e n and a n a l y z e d b. B e t a - q l u c u r o n i d a s e adjustment t h e p r e p a r a t i v e HPLC, t o 5 mL u n d e r 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 . concentrated from Enzyme H y d r o l y s i s fraction collected from the p r e p a r a t i v e HPLC was by a z e o t r o p i c e v a p o r a t i o n a s d e s c r i b e d , f o l l o w e d by t o pH 5 w i t h 0.1 M sodium h y d r o x i d e 56 o r 0.1 M hydrochloric bovine acid, beta-glucuronidase hydrolysis was c a r r i e d basification chloride. with o u t a t 37°C f o r 12 h o u r s , sodium h y d r o x i d e with methanol liquid with and a n a l y s e d c. N a p h t h o r e s o r c i n o l After f o r the fraction heated resulting reagent from fashion the a d d i t i o n inhibitor. Reaction in phosphoric indicated fraction and s p r a y e d A blue the presence was with a 2 % acid/ethanol. a t 100°C f o r 10 m i n u t e s . spots presence collected enzyme, b u t w i t h on a 2 x 5 cm RPC-18F TLC p l a t e were t h e n by GC a s an enzyme methylene to dryness, c o n c e n t r a t i o n u n d e r vacuum, e a c h HPLC naphthoresorcinol the Color with treated in a similar beta-glucuronidase 2 ug o f s a c c r o - 1 , 4 - l a c t o n e spotted Each The f o l l o w e d by and e x t r a c t i o n c h r o m a t o g r a p h was a l s o the bovine o f 500 uL o f i n a c e t a t e b u f f e r a t pH 5. t o c a i n i d e as d e s c r i b e d a b o v e . the of enzyme by t h e a d d i t i o n The o r g a n i c s o l v e n t was e v a p o r a t e d reconstituted of and h y d r o l y z e d The p l a t e s c o l o r a t i o n of of a g l u c u r o n i c a c i d mo i e t y . d. Sodium h y d r o x i d e Hydrolysis Each c o n c e n t r a t e d 2.0 M s o d i u m h y d r o x i d e with clean methylene tube, GC a n a l y s i s HPLC solution. chloride. evaporated, was b a s i f i e d The sample The o r g a n i c l a y e r and r e c o n s t i t u t e d f o r the presence I. Pharmacokinetics fraction of was was with t o pH 12 w i t h a then e x t r a c t e d transferred methanol p r i o r to 3-(2,6-xy1yl)-5-methylhydantoin. of T o c a l n l d e Carbamoyl Ester G l u c u r o n ide The carried 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 out u s i n g a m o d i f i e d indirect 57 to a in urine was method a s d e s c r i b e d by Hoffmann (60). tocainide The p r o c e d u r e glucuronide 5-methylhydantoin. provide involved the h y d r o l y s i s of i n u r i n e a t pH > 12 f o r m i n g 3-(2,6-xy1y1)- Q u a n t i t a t i o n of the h y d a n t o i n d e r i v a t i v e c a n an e s t i m a t i o n f o r t h e 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 Tocainide Glucuronide i n Sodium Hydroxide Into 7 tubes, 0.4 mL o f u r i n e c o l l e c t e d a human s u b j e c t who had r e c e i v e d 0.1 mL o f t h e i n t e r n a l added mixing. The r e a c t i o n 10 a n d 20 m i n u t e s for 1 minute. product sulfate, column. glucuronide, plotted evaporated over was t h e n d r i e d to dryness f o r GC a n a l y s i s The h y d r o l y s i s as r e f l e c t e d the timed with was e x t r a c t e d a t 0.5, 1, 2, 4, 6, chloride over and r e c o n s t i t u t e d u s i n g a bonded phase of tocainide carbamoyl SE-30 ester by t h e f o r m a t i o n o f t h e h y d a n t o i n , was a s t h e peak h e i g h t r a t i o etidocaine, ( 1 . 0 ug/mL) were by s h a k i n g w i t h 5.0 mL o f m e t h y l e n e 20 uL o f methanol capillary base from o r a l l y , and 1.0 M o r 0.5 M s o d i u m h y d r o x i d e The m e t h y l e n e c h l o r i d e anhydrous sodium with 200 mg o f t o c a i n i d e standard etidocaine t o 0.5 mL o f 2.0 M, a t t h e 10th h o u r of the h y d a n t o i n intervals to that of 1.5, 2, 3, 5, 7, 11 a n d 21 minutes. 2. H v d r o l v s i s o f 3 - ( 2 . 6 - X v l v l ) - 5 - m e t h y l h v d a n t o i n Into 7 tubes, 0.1 mL o f a n aqueous h y d a n t o i n (15 ug/mL) a n d 0.1 mL o f e t i d o c a i n e solution were a d d e d hydroxide. timed methylene from chloride standard (10 ug/mL) t o 0.2 mL o f 2.0 M, 0.2 M o r 0.1 M s o d i u m The h y d r o l y s i s intervals internal solution s a m p l e s were e x t r a c t e d a t a c c u r a t e l y 0.5 t o 40 m i n u t e s f o r 1 minute. 58 by s h a k i n g w i t h 5.0 mL The s o l v e n t was d r i e d , evaporated GC t o d r y n e s s , and reconstituted with 2.0 uL methanol for analysis. 3. H v d r o l v s i s o f The 3-<2,4,6-Xvlvl)-5-methvlhvdantoin protocol was the same as d e s c r i b e d f o r the 2,6-xylyl analog. 4. 3-(2,6-Xvlvl)-5-methvlhydantoin C a l i b r a t i o n All 6 calibration separate tubes, aqueous t o c a i n i d e to 100 uL of solution. internal also nitrogen. prior 1, 3, 6, 10, hydantoin sample, 100 uL (10 ug/100 mL) o f 700 tube. a t room The chloride sample was analysis. base of 1.0 M sodium h y d r o x i d e tubes of blank u r i n e were was were mixed on a v o r t e x mixer f o l l o w e d by with The extraction organic layer evaporated to dryness with 20 uL of s a m p l e s were s t o r e d to monitor curve added standard of variation. A new calibration ratios found t o d e v i a t e more t h a n were internal were of 4'-methylhydantoin reconstituted used ug/100 uL uL Calibration a random sample.was 20 Into 300 uL and 15 and standard s o l u t i o n f o r 1 minute. tube in d u p l i c a t e . and temperature, to a c l e a n Each t o GC of base and aliquot to each methylene transferred and An 10 m i n u t e s 5 mL uL 10 ug/100 uL e t i d o c a i n e standard t h e n added for 100 Into each added. s a m p l e s were p r e p a r e d Curve daily was was under methanol at 4 °C instrumental c o n s t r u c t e d i f peak 10 % between area daily a n a l y s i s. 5. I n t e r - a s s a v and Duplicate Intra-assav V a r i a b i l i t y calibration analyzed in duplicate. was to determine used s a m p l e s o f 6, The inter- average and 59 15 and 20 peak a r e a r a t i o intra-assay sample ug were ± C.V. each % variation. 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 considered acceptable. 6. E x t r a c t i o n into Efficiencies 5 mL o f M e t h y l e n e of Tocainide Chloride Samples o f 1 mg/mL o f t o c a i n i d e base of were first tocainide evaluated prepared and t h e h y d a n t o i n tubes, hydrochloride i n methanol. in a concentration 3 separate and t h e H y d a n t o i n i n methylene c h l o r i d e range was o f 6 t o 20 ug/mL. Into hydrochloride hydantoin, and t h e h y d a n t o i n r e s p e c t i v e l y , was added t o 300 uL o f b l a n k An a l i q u o t then added o f 500 uL o f 1.0 M s o d i u m h y d r o x i d e to each tube immediately 5 mL o f m e t h y l e n e c h l o r i d e then transferred 10 and 20 mg o f t o c a i n i d e and the urine. t o 6, etidocaine The e x t r a c t i o n e f f i c i e n c y 100 uL o f e a c h o f t o c a i n i d e equivalent and to clean followed f o r 1 minute. was by e x t r a c t i o n The o r g a n i c tubes, evaporated base layers to dryness and r e c o n s t i t u t e d with external standard (1 ug/uL i n m e t h a n o l ) p r i o r t o GC a n a l y s i s . another 3 separate tubes, uL The hydantoin i n 6, of e t i d o c a i n e 100 uL o f e a c h o f t o c a i n i d e 10 a n d 20 ug/lOOuL methanol ( t h e same methanol s a m p l e s were c o n c e n t r a t e d analysis. hydantoin under n i t r o g e n followed The peak a r e a r a t i o s between t o c a i n i d e to that compared with methanol samples. of e t i d o c a i n e the c o r r e s p o n d i n g The p e r c e n t between t h e two s e t s efficiencies i n the e x t r a c t e d In base and t o 100 solution). by GC and the samples were peak a r e a r a t i o s from t h e difference o f samples p r o v i d e d of t o c a i n i d e a s an was added internal standard were under nitrogen the 100 uL o f e t i d o c a i n e with i n t h e peak a r e a the e x t r a c t i o n and t h e h y d a n t o i n , 60 ratios assuming minimal breakdown o f t h e h y d a n t o i n 7. A n a l y s i s o f T o c a i n i d e Urine samples during Glucuronide were c o l l e c t e d previous pharmacokinetic studies received either oral dose f o r the f i r s t up t o 96 h o u r s . immediately and were sodium in a 200 mg after thawed hydroxide, the same manner samples. polyethylene in three or slow Studies during f a s t e d s u b j e c t s who (20 m i n u t e s ) Urine bags samples had intravenous were collected 10 h o u r s and a s o f t e n as r e q u i r e d t h e r e a f t e r Urine volumes collection. into in Kinetic into of t o c a i n i d e h y d r o c h l o r i d e . hourly for the s h o r t e x t r a c t i o n p e r i o d . 10 mL and pH were The s a m p l e s screw-capped e x t r a c t e d with as d e s c r i b e d A l l work-up p r o c e d u r e s da i 1 y b a s i s. 61 were tubes, methylene recorded s t o r e d a t -10 °C basified c h l o r i d e and for handling with analysed o f the c a l i b r a t i o n and GC a n a l y s e s were done on a 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 The Byrnes Since s y n t h e t i c procedures (31) a r e s i m i l a r have n o t been r e p o r t e d used f o rpeptide on t h e x y l i d i d e ring t o undergo metabolism, were a i m e d a t e x c h a n g i n g 1. S y n t h e s i s f o r t o c a i n i d e a s d e s c r i b e d by to those the 3 ' , 4 ' , 5 ' - p o s i t i o n s Tocainide these protons with synthesis. of t o c a i n i d e initial attempts deuterium. o f 3*.4*•5'.-[ H1 -2-Amino-2*.6 * 2 3 propioxvlidide The s y n t h e t i c a p p r o a c h a s shown from a method r e p o r t e d by B y r n e s in. Scheme 13 was m o d i f i e d (31): D ,"3 «3 + CH3 N-carbobenzyloxyl Tocainide Dicyclohexylurea (removed) A: D i c y c l o h e x y l c a r b o d i i m i d e / C H , C 1 , (25°C, 1 h o u r ) . B: F i l t e r , vaccum dry ( H B r / a c e t l c Acid). CH3 T o c a i n i d e HBr Scheme 13. The synthetic pathway for trideuterated t o c a i n i d e . 63 a. 2,6-Dimethvlani1ine Initial Deuteration discussion 3,4,5- p o s i t i o n s of a method predeuteration will describe 2,6-dimethylani1ine. for labelling described by Frischkorn typically an e l e c t r o p h i 1 i c aromatic d i m e t h y l a n i l ine , the orthoonly and (57). amine The w i t h D2O/D2SO4 y i e l d e d dime t h y l a n i 1 i n e . The heating at i n the prevent vaporization 250 °C 70 Frischkorn at followed xylidines mechanism as is For 2,6- a c t i v a t i n g group d i r e c t i n g the 2,6-methyl g r o u p s attempts at of the procedures refluxing the 2 D2O/D2SO4. °C, are 4-[ Hl-2,6- method, however, 250 of s u b s t i t u t i o n (62). % pure presence D2O of The reaction while Preliminary deuteration t o l u i d i n e s and is a strong para-substitution, weak a c t i v a t o r s . aniline the a 4137 required To KPa (600 PSI ) 15 pressure was reaction vessel (600 PSI) Mass required. was To pressurized with nitrogen this at condition, a room t e m p e r a t u r e p r i o r to h e a t i n g at 250 Variac to 4137 KPa °C. Spectra: Mass s p e c t r a l a n a l y s i s revealed a mixture ions 126 of achieve m/z the unit and of the crude reaction of components w i t h m/z 125 (see Figures molecular 3 to 5 ) . amino d e u t e r i u m s w i t h h y d r o g e n s , from m/z amino g r o u p . 126 to m/z 125 However, the suggested ion was unchanged, thus s u g g e s t i n g not contain the amino g r o u p A mass s p e c t r u m of in Figure comparsion. 6 for 64 the this After l o s s of exchange one mass ion c o n t a i n e d fragment a t and m/z that m/z fragment (see unlabelled product the Structures 80 x i i to 2,6-dimethylani1ine the 80 did xv) . was included J 100 0 Figure 3 : T o t a l - i o n - c h r o m a t o g r a m of crude 2,6-dimethylaniline product. J Scan deuterated 126 110 95 " * — * — * — 4 Figure 4 . The mass s p e c t r u m 65 0 f C ruriP t%»r-*—* d^term-Pd at * - m/z 125 109 80 94 m/z Figure 5 : The mass s p e c t r u m o f c r u d e 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 at scan 61. 121 106 77 Figure 6 : The mass spectrum o f 2 , 6 - d i m e t h y l a n i l ine-.- 66 unlabelled 67 The mass s p e c t r a l a molecular i o n o f m/z fragments which (as shown After 95 ( M + of the d i s t i l l e d -NH-CH3 d 3 revealed d e u t e r a t i o n a t 3,4,5- r i n g i n F i g u r e s 7 and 8, a l s o 124 ( M + d 1 2 ) , 80 ( M t h e TIC r e v e a l e d a 100 % ) , 109 ( M 3 positions see S t r u c t u r e s x i i t o xv) by d i s t i l l a t i o n , w i t h m/z product 124 a s w e l l a s t h e c o r r e s p o n d i n g i o n indicated purification component data + -CH3 d 3 single 60), 2C2H6 d3 20) + 80 MHz-[ H]-NMR: 1 As shown i n F i g u r e s 9 and 10, the p r o t o n deuterated from product the a r o m a t i c indicated protons a s s i g n m e n t s a r e summarized Table A only trace on T a b l e Decoupling TMS (0.0) 2.20 3.5 6.65 6.95 s 8 bs bs 68 of s i g n a l s The p r o t o n 4. f r o m t h e 80-MHz-[*H]D,-2,6-dimethylaniline. I n t e g r a t i o n (mm) 113 6 2 5 s p e c t r a o f the intensity a t a r o u n d 7.00 ppm. : Summarized d a t a NMR s p e c t r u m o f S h i f t (ppm) NMR Proton(s) 6HU9/H) 2H 2H 1H Assignment xylidine xylidine aromatic aromatic - CH, NH, meta para scan Figure 7 The t o t a l - i o n - c h r o m a t o g r a m o f distilled Do-2,6-dimethylaniline. 124 109 "m/z Figure 8 : T h e mass s p e c t r u m o f distilled 69 D,-2,6-dimethylaniline. 70 A splitting portion of exchanged the of the protons s i g n a l at on the with deuterium. 113 u n i t s / 6 protons the chemical shift units/proton or at 3.5 ppm which s u g g e s t e d meta- and Ar-H) p p m ( l H , Ar-H) and 5 units/proton, h and 94.8 b. 6.95 73.7 Reaction With synthesis Byrnes the of cited an <2H, NH ) In was 2 78.9 2.2 the the were suggesting integrated a deuterium for synthesis of tocainide the of peptides amino and followed of and has 6.65 4 had ppm(2H, unit/proton purity many d e h y d r a t i o n reactions be i s the easily method of by been used w i d e l y reactions a c a r b o x y l i c a c i d group. these The for (63) commonly insoluble filtered and removed. 0.36 g The tocainide Mull): i n f r a r e d spectrum stretching for acid. (Nuiol tocainide. respect, 1 the after recrystal1ization yielded product 11 same deuterated-ani1ine, reaction Figure revealed labelling which can The ppm amino p r o t o n s dicyclohexylurea Infrared small Carbobenzvloxvl-alanine successful by-product hydrochloric a been integrated % of Dicyclohexylcarbodiimide synthesis involving i n t e g r a t i o n at that that g r o u p might have para-aromatic protons at trideuterated (31). suggested % respectively, with the ppm units/proton. e x c h a n g e d . The and "CH^ The 19 2.12 was of superimposable A prominent and the C=0 synthetic to that absorption s t r e t c h i n g at 71 at 1670 compound as obtained 3300 cm~^ cm~l. from shown in reference indicated HN- Figure 11 : The i n f a r e d spectrum of synthetic tocainide (mull) Mass S p e c t r u m (GCMS): A n a l y s i s was The TIC m/z 44 base p e a k s and the c h i r a l the xylidine at m/z the and carried 121 C-3 out using a 3 % s i l a r - l O C Mass S p e c t r u m a r e indicated carbon. shown a cleavage Fragmentation n i t r o g e n gave r i s e was resulted carbonyl. in Figures from Only packed column. 12 and 13. between the between the t o m/z 106, cleavage the between the 3 t o 5 % of m/z 192 carbonyl C-N while The bond of fragment n i t r o g e n and molecular ion was observed. The Mass S p e c t r u m w i t h data obtained from i t s fragmentation p a t t e r n agreed reference with tocainide. ? 2. A t t e m p t e d S y n t h e s i s of 1-C H13-2-Amino-2*,6 * - propioxylidide This synthetic approach labelling three deuterium tocainide using commerically Unfortunately, D2 aniline, - Therefore, was found to atoms on during p i l o t this alternative to s a t i s f y the attempts The vessel was crude as d e p i c t e d i n Scheme CBZ-alanine. yielded was the designed 5.7 a and of s y n t h e s i z i n g experiments using is were c a r r i e d out unlabelled 14. g (87.9 Recrystal1ization chloroform/ether offered g r o u p of Since carbobenzyloxyl-D^-alanine initial of damaged. s y n t h e t i c approach available, reaction method D^-alanine. 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 material methyl to s y n t h e s i z e requirements tocainide. commerically the c h i r a l available the V a r i a c ^ r e a c t i o n trideuterated not p r o v i d e d an a l t e r n a t i v e final 73 yield % chemical y i e l d ) of o f the p r o d u c t o f 3.2 g (55.8 the in %). In 50 F i g u r e 12 scan 100 : The t o t a l - i o n - c h r o m a t o g r a m synthetic tocainide. of 44 NHf C - C H - N H j AH 44 121 121 192 l 7 W » - W F i g u r e 13 7 9 tr tr 1 «r itr J £ 1 * * r * r *r : The E I mass s p e c t r u m o f 74 i,--^--^-^ synthetic * fr- ill/ Z tocainide accordance chemical to this yield D3~alanine Infrared As (Nuiol vibration signal The 1700 offer the o v e r a l l trideuterated approximately 6 % chemical o f t h e amide was p r e s e n t . from the c a r b o x y l i c two c a r b o n y l s t r e t c h i n g - indicated benzene tocainide from yield. 14, t h e a b s o r p t i o n a t 3350 cm"^ from t h e acid The b r o a d give r i s e a t 3000 cm"-'-, o v e r l a p i n g t h e n u j o l cm-'-. theoretical Mull) ; in Figure NH s t r e t c h approach, f o r s y n t h e s i s of t h i s would shown synthetic -CH -OH stretching t o the broad stretch. absorptions overlapped at A b s o r p t i o n s a t 700 c m l a n d 750 c m l _ t h e -CH r o c k i n g s i g n a l s _ from the m o n o s u b s t i t u t e d ring. o I CH - 0-C-C1 + 2 H N-CH-00OK 2 CBZCl Alanine 1. N-CBZ-alanine A: B: Scheme 14 S a t u r a t e HaHCO,/ice c o o l i n g (6 h o u r s ) . 0.1 M HC1 (PH 3 ) . The synthesis of N-carbobenzvloxvalamnp 75 76 B. P o t e n t i a l Tocainide 1. Z-Ethanimino-Z'.6 * - p r o p i o x v l i d i d e This ethanimine tocainide been was metabolite identified Metabolites i n humans, f o r subsequent spectrum are Infrared (Nuiol Mull): shown presence shown stretching 730 synthesized studies. 15 and absorption The aromatic at as The a has not reference TIC and mass 16 r e s p e c t i v e l y . at 3300 c m ~ l stretching vibrations. occurred metabolite 1680 The and absorptions C=0 1670 i n d i c a t e d the and C-N cm~l occurred a t 700 and - 1 (GCMS): mass s p e c t r u m Figure 2. this a cm . Mass S p e c t r u m The Since metabolism 17, probably respectively. i t was in F i g u r e s in Figure of N-H by V e n k a t a r a m a n a n as in r a t s (37). compound As identified 16 a g r e e d of this with ethanimine published data metabolite as shown i n (37). 3-(2,6-Xvlvl)-5-methylhydantoin The s y n t h e t i c procedure modified. Tocainide 2,6-dime t h y l a n i 1 i n e . chloroformate, product Infrared As base was used A phosgene used gave a s i n g l e by Elvin in place (35) of a l a n i n e and substitute, trichloromethyl i n sodium d r i e d t o l u e n e . peak on was the TIC The resulting from GCMS a n a l y s i s . (Nuiol Mull): shown cyclic was reported in Figure amide N-H 18, absorption stretching. 77 The at 3310 cm~l i n d i c a t e d the 2300 cm~l(W) a b s o r p t i o n 100 F i g u r e 15. 200 J 1 1 1 1_ scan The total-ion-chromatoqram of synthetic 2-ethanimine tocainide. «3 71 120- 190 I O 1 9 I 0 - 218 120 56 L JLUL 50 il. 100 F i g u r e 16. 4 I . I. 150 ... 1200,. 1 m/z The EI mass spectrum of 2-ethanimine tocainide. 78 Figure 17 : The i n f a r e d spectrum o f t o c a i n i d e ethanimine 79 (mull). 80 was due t o t h e i s o c y a n a t e N-C=0 s t r e t c h i n g . 1700 c m ~ l were absorption values Mass S p e c t r u m As shown with from the h y d a n t o i n were ring i n agreement Both 1795 c m ~ l and 0=0 s t r e t c h i n g . with These published data (35). (GCMS): in Figures t h e base 19 a n d 20, t h e h y d a n t o i n peak a t m/z fragmentation were M a t m/z 218 + 147, and t h e c o r r e s p o n d i n g identical to E l v i n ' s published spectrum ( 3 5 ) . 400 MHz-[ H]-NMR (CDCI3): X Table Figure 5 outlined 21, t h e 400 MHz s p e c t r u m due t o s p l i t t i n g group p r o t o n s . of the s i g n a l modelling, steric ring hindrance rendering conformation later quartet at from proved methyl ring The p r o t o n at s h i f t 4.25 ppm coupling effect into a broad singlet, again were between 7.1 a n d 7.3 ppm. The s i g n a l water p r e s e n t 81 be d i s c u s s e d formed a of the n i t r o g e n on t h e amide n i t r o g e n was o f t h e n i t r o g e n atom. due t o r e s i d u a l thus This which w i l l effect likely causing i n d e t e r m i n i n g the quadrupole found molecular i n r e l a t i o n to -CH^ g r o u p s , groups non-equivalent. t o be h e l p f u l t o 6.58 ppm With t h a t t h e c a r b o n y l s were The C-5 p r o t o n , ring-1-position. C-5 m e t h y l 2.18 and 2.22 ppm, protons. o f the h y d a n t o i n due t o a s m a l l q u a d r u p o l e downfield at s h i f t of the t o c a i n i d e g l u c u r o n i d e sections. a t 1.54 ppm the asymmetric t o one o f t h e x y l i d i n e t h e two m e t h y l interpretation in was s u c h As shown i n revealed a doublet of the x y l i d i d e the c o n f o r m a t i o n xylidine assignments. Further downfield, were t h e s i g n a l s the the p r o t o n shifted due t o t h e The a r o m a t i c protons a t 1.63 ppm was i n the sample. • I _l Figure 19 I 1 1_ 100 scan 200 •. T o t a l - i o n - c h r o m a t o g r a m o f the synthetic 3-(2,6-xylyl)-5-methylhydantoin. 218 147 44 119 105 2Q3 JlLLj 50 Figure 20 : 100 J I ,JL, 150 The E I mass s p e c t r u m o f the 200 synthetic 3-(2,6-xylyl)-5-methylhydantoin. 82 m/z J L A JL - r 5~ e 21 : * 4 ~ 3 2 400 MHz-^"H-NMR spectrum 1 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 Table 5 (CDCU). : Summarized data from the 400-MHz-[H]-NMR _ "spectrum of 3 - ( 2 , 6 - x y l v l ) - S - m e t h y l h y d a n t o m , 7.1 -7.3 2.18 S h i f t (ppm) (TMS) 0.0 1.54d 2.18s* 2.22s* 4.25dq 6.61 bs Coupling J . 6 Hz J - 8 Hz 7.1 - 7.3 Note * non-equivalent I n t e g r a t i o n (mm) Proton(s) 3H(20mm/H) 3H 3H 1H 1H 3H 60 63 63 19 20 61 (H) due t o r i n g c o n f o r m a t i o n a l 83 Assignment 5 - CH, x y l i d i n e - CH x y l i d i n e - CH 3 3 5 - CH 1 - NH xylidine - H hinderance. 3. 3-(2,4,6-Xvlvl)-5-methylhydantoin The carried s y n t h e s i s of 3-(2,4,6-xy1y1)-5-methylhydantoin out using a 2-step propioxylidide and N-CBZ-alanine synthesis was was f i r s t approach. s y n t h e s i z e d from analog 2 , 4 , 6 - t r i m e thy1 a n i 1 i n e as d e s c r i b e d f o r the T h i s 2',4',6*-methy1 a n a l o g t h e n r e a c t e d w i t h TCF t o form methylhydantoin 2-amino-2',4*,6'- u s i n g the procedures of t o c a i n i d e . was of t o c a i n i d e the 3-(2,4,6-xy1y1)-5- u s i n g the procedure a s d e s c r i b e d f o r the 3-(2,4-xylyl)-5-methylhydanto in. Infrared As (Nuiol shown Mull): i n F i g u r e 22, t h e a b s o r p t i o n a t 3200 cm ^" - amide N-H s t r e t c h i n g . absorptions The 1720 cm"^ a n d 1770 cm"-*- were p r o b a b l y Furthermore, indicated from ring C-0 700 cm"-'- a n d 750 cm-'- were - stretching. indicative of the aromatic absorption. Mass S p e c t r u m ( G C M S ) ( s i l a r - 1 PC): The recrystal1ized in t h e T I C a s shown Figure 24 i n d i c a t e d observed 119 product a s t h e base a n d 217 were fragmentation appeared i n F i g u r e s 23. that a molecular peak. found as a s i n g l e peak The mass s p e c t r u m i n i o n a t m/z 232 was F r a g m e n t s a t m/z a t 161, 144, 146, t o agree with the c o r r e s p o n d i n g as d e s c r i b e d f o r the 2 , 6 - x y l y l analog. 84 hydantoin F i g u r e 22 : The i n f a r e d spectrum of 3-(2,U , 6 - x y l y l ) - 5 - m e t h y l hydantoin ( m u l l ) . 85 / 100 F i g u r e 23 : 200 scan Total-ion-chromatogram of the synthetic 3-(2,4,6-xylyl)-5-methylhydantoin. 232 160 44 146 119 217 i i 50 Figure 24 : 100 The E I mass 150 spectrum of 200 the m/z synthetic 3-(2,4,6-xylyl)-5-methylhydantoin. 86 80 MHz-[ H1-NMR: As shown proton in Figure shift 25, t h e 80 MHz s p e c t r u m characteristics xylyl-5-methylhydantoin proton as d e s c r i b e d analog. Table revealed f o r the similar 2,6- 6 o u t l i n e s the 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 60 3H 5-CH3 Z.18s 1.54 d 63 3H 2.20s 63 3H 2.3 2s 63 3H xylidine - CH xylidine - CH 19 1H 4.25 d q J=6 Hz J=8 Hz xyli d ine-CH, 5-CH J 6.15bs 20 1H 1-NH 6.90s 40 2H xylidine - H 87 3 3 I I I 6 5 I I 4 1 1 3 1 1 2 1 ' ! 1 The 80-MHz-[HI-NMR spectrum of the s y n t h e t i c "3- (2, 4 , 6 - x y l y l ) -5-methylhydantoin (CDCl^). 88 1 PPm C. Tocainide 1. Derivatization Metabolites Techniques A permethylation Desiderio (61) sodium for (DMSO ) and the procedure, a n a l y s i s of residues Johnson reviewed functional acid analysis intact these g r o u p s on moiety. The ether (CH^I^is a useful types techniques glucuronides. f o r use aglycone, as with appropriate w e l l as the use the on the reagents GC and glucuronic used for GC are: acid/ Trif1uoroacetic anhydride/pyridine/ d iazome t h a n e . 3. Methyl 4. Bis(trimethylsilyl)-trif1uoroacetarnide 5. BSTFA/diazomethane. 6. Tri-Sil-Z . information for (65) 2. Tri-Sil-Z, acid Fenselau 1. A c e t i c a n h y d r i d e / m e t h a n e s u l f o n i c d i azome t h a n e . Initial uronic have been p u b l i s h e d of d e r i v a t i z a t i o n of g l u c u r o n i d e s technique (64,65). techniques the and glucuronides. 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 derivatization o r GCMS a n a l y s i s of intact Thompson u s i n g dime t h y 1 s u l f i n y 1 m e t h i d e iodide in polysaccharides Various been d e s c r i b e d by a n a l y s i s of methyl - Glucuronides method has f o r the T h i s permethy1 a t i o n for Iodide/DMSO" ( p e r m e t h y l a t i o n ) . (BSTFA). R derivatization BSTFA and experiments BSTFA w i t h of raw diazomethane f o r v a r i o u s known m e t a b o l i t e s of r e f e r e n c e D-glucose, in these conditions proved of compounds, P - n i t r o p h e n o l r e a c t i o n s to monitor the usefulness 89 of these the urine d i d not samples yield tocainide. glucuronide useful However, and derivatization derivatization with techniques of high to obtain molecular these glucuronide, glucuronide reaction o r i n t h e GC Band using of Tocainide Enzyme reduced after quantity This synthetic during phase band two bands r e v e a l e d of free tocainide indicated that was a n a l y z e d to that i n the t h e sample t o pH 13 with by r e v e r s e - p h a s e HPLC i n 0.05 M p o t a s s i u m the p r e s e n c e an i d e n t i c a l a of t o c a i n i d e . l b was a d j u s t e d revealed acid of 3-<2,6-xy1yl)-5- r e t e n t i o n time t o t h a t of standard. presence of t o c a i n i d e a f t e r and t h e p r e s e n c e conjugate of f r e e t o c a i n i d e a f t e r o f 25 % a c e t o n i t r i t e sample isolation, o f f r e e t o c a i n i d e compared the sample base h y d r o l y s i s s u g g e s t structure either by TLC a 2 - s t e p TLC a c i d conjugate the i s o l a t e d hydrolysis, acid after The p r e s e n c e a glucuronic a mobile The hydrolyzed h y d r o l y s i s o f t h e same methy1hydantoin having the the c a r b a m o y l - or u r e i d o - Glucuronide enzyme h y d r o l y s i s hydroxide, acetate. that P-nitrophenol inlet. from a c i d h y d r o l y s i s . sodium 26 and 27. a substantial quantity After reference t h e d e r i v a t i z e d s u g a r and l b sample, o b t a i n e d contained f o r compounds and C H o I / P e r m e t h y l a t i o n hydrolysis. sample information o f t o c a i n i d e were p r o b a b l y Isolation slightly from speculated 2. I d e n t i f i c a t i o n revealed in Figures data we weight w e i g h t . The mass s p e c t r a o f t h e s e compounds a r e shown With molecular that beta-glucuronidase o f the h y d a n t o i n t h e sample contained o f t o c a i n i d e . In an a t t e m p t of t h i s glucuronide, 90 band derivative after a glucuronic t o examine the l b was p e r m e t h y l a t e d intact with 7IOSIL 153 68665566- 2ee GLUCOSE 25f> .. i . , •• '•• 388 356 458 • •' • • 1:ee 361 141 ^e -66 45334oea- :73 35333eee- e 2366i ie.ee- £3 / i8ee- 435 / \ 2F;9 3i J L J i i L i i i ^ i . . Figure 26 L'66 26 271 243 583- ie.e -33 191 258 319 L. 353 366 525 393 497 i 1 436 453 56« : The C I mass s p e c t r u m o f T M S - d e r i v a t i v e TfclSJL :«0 of glucose PNPG 536 483 333 . i • . 13 \ . 638883- 788 688 . .i .. • • ' • • 37S 166 558883588888- -98 456666- -66 4 68836- •76 358663- •66 388666- -56 ?&8668- -46 28666636 15888818866656686- 169 212 \ ?4e 46! ?S4 333 498 -1 8D63 F i g u r e 27 676 366 488 563 16 53T £ l ...1 666 : The C I mass s p e c t r u m o f T M S - d e r i v a t i v e nitrophenol glucuronide. 91 f-28 •-3 766 o f p- 1266866- 1 naciacto-i 666666.77 468686- ;eeeee- 1.4 Figure 4.f. &.2 .fc. 6.6 6.4 28 : Total-ion-chromatogram urine t-.P 7.2 .".t> 6.6 P.4 &. & " l o f permethylated XAD/crude e x t r a c t i s o l a t e d band l b sample. >c a n 1 4 4 56 166 44666- 98 2P.Pi ••• •• i?6 i.... i . . . . i :-5e , i . . . . i, 165 366 3S6 i . . . . 466 i . . . . i . . . . i , n e -166 48666156 36eee- -96 2Wl -66 / 326862£;66<>- -76 177 / 24666^ -66 2&66en 6 1 6666- -46 12866- -36 -26 '.32 4866H I JL 56 166 1 1S8 362 / 266 2&6 366 16 334 356 '(""!• • • • | ' 486 F i g u r e 29 : Mass spectrum o f permethylated t o c a i n i d e ester glucuronide carbamoyl ( e l e c t r o n impact i o n i z a t i o n ) . 92 -6 l , l u l « methyl i o d i d e spectrum and i n d r y DMSO a s d e s c r i b e d . o f the p e r m e t h y l a t e d glucuronide The T I C and t h e mass a r e shown in Figures 29. The possible permethylated outlined mass intact in Figure ion fragments r e s u l t e d tocainide carbamoyl 30. 93 ester from t h e glucuronide are Although glucuronide likely the s i t e resulted cleavage from formed the cleavage moiety. 141, The m/z 334 i o n may have of the c a r b o n y l of the g l u c o s i d i c spectrum o f t h e amide bond a c c o m p a n i e d glucuronide moiety c o n t a i n i n g t h e mass 161, 101 a n d 75 s u g g e s t e d linkage been resulted v i a by p r o t o n transfer to the a g l y c o n e . i o n s m/z 232, 201, 169, t h a t the p e r m e t h y l a t e d These glucuronic moiety, a r e 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 p e r m e t h y l a t e d During glucuronic mass s p e c t r a l used as a r e f e r e n c e published the data. mass s p e c t r a l analysis, F o r the p e r m e t h y l a t e d a n a l y s i s was c a r r i e d gas. compounds were t h e p a i r these two i o n s was p r e s e n t p-nitrophenol out u s i n g glucuronides was with glucuronide, t h e CI mode with The mass s p e c t r u m a n d t o t a l - i o n 31 and 32. to the s p e c t r a of o f i o n s m/z 201 a n d 101. i n every spectrum. t o be t h e most d i a g n o s t i c i o n s permethylated of the glucuronide our r e s u l t s f e a t u r e s w h i c h were common these proved p-nitrophenol to confirm c h r o m a t o g r a m a r e shown on F i g u r e s striking the cleavage acid. compound methane a s t h e r e a g e n t The from sample was a glucuronide. acid ions, r e s u l t e d This bonding w i t h i n the The m/z 232 i o n was p r o b a b l y the p e r m e t h y l a t e d The t h e m/z 277 i o n was p r e s e n t . v i a the cleavage of c o n j u g a t i o n . from tocainide i o n of the t o c a i n i d e carbamoyl was n o t o b s e r v e d , i o n was most at the m o l e c u l a r (65-68). 94 These in identifying One o f i o n s have j 36861286811866- 98866668• 76666BPP- / 5666- 3868- 166666 Figure 31 : Total-ion-chromatogram para-nitrophenol of 10.6 9.6 c:.6 .6 permethylated glucuronide. Scan 126 ?48 168 i 366-j 528 ?PP 36P 336 4PP ' 26i H 1661 326-1 (-96 177 | 246H f76 | I 1 j 268-j -i 166-1 U. 12 ?96 ?66 86- L-;6 4 66 189 134 i v 34 1 -26 46-11 I ! ' ' • ' r 126 Figure 32 : ~ T ~ " J76 ' ~ r T The CI mass s p e c t r u m para-nitrophenol 95 r-,-r^- 24 6 of 286 the ' • 1•' • I 326 366 permethylated glucuronide. I ' " I *' 468 I The molecular glucuronide was p r e s e n t . C2H5 a d d u c t . cleavage from i o n m/z 372 o f t h e p e r m e t h y l a t e d three o f a CH-OH g r o u p . with glucuronic acid likely glucuronide. resulted These from glucuronide. fragment, the glucuronide conjugated urine From (66), the M + ions of was a t o c a i n i d e c a r b a m o y l Followed of t h i s extraction. diagnostic t o c a i n i d e carbamoyl i o n from This latter suggested ester glucuronide. o f T o c a i n i d e M e t a b o l i t e s by by GCMS (Permethylation) experimental p r o t o c o l was t o i d e n t i f y p o l a r to prevent From t h e T I C o f t h e p e r m e t h y l a t e d sample, h i p p u r i c a c i d was i d e n t i f i e d 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 m/z 218 was i d e n t i f i e d carbamoyl ones d i s c u s s e d , m e t a b o l i t e s w h i c h were s u f f i c i e n t l y methylhydantoin. or detected of the i n t a c t of permethylated the former Identification purpose of the moiety. p o r t i o n of t o c a i n i d e . together with Lvoohi1ization All the f r a g m e n t a t i o n The m/z 105 i o n was a p r o m i n e n t of the x y l i d i d e ion resulted the fragment a t i o n f r a g m e n t s were o f i m p o r t a n t ester 3. A t t e m p t e d from the c l e a v a g e s f o r the i d e n t i f i c a t i o n solvent from t h e The p e a k s o f m/z 277, 334 a n d 248 were value deamination Again g l u c u r o n i d e s a r e not always present GCMS t e c h n i q u e s . The resulted i o n s . a t m/z 169 a n d 232. p o i n t e d o u t by Thompson permethylated mostly along i o n s must have r e s u l t e d permethylated by from t h e o f a methoxy g r o u p , a n d m/z 308 was p r o b a b l y m/z 201 was p r e s e n t As The m/z 400 i o n was f o r m e d The m/z 340 i o n was p r o b a b l y the cleavage these p-nitrophenol blank a s i t s methyl sample, a m o l e c u l a r ester. ion a t t o be t h e 3 - ( 2 , 6 - x y 1 y l ) - 5 - No f u r t h e r useful 96 d a t a were o b t a i n e d during this 4. experiment. Attempted Identification of Metabolic Intermediates of T o c a i n ide According glucuronide to e a r l i e r could take d i s c u s s i o n s , formation place through a carbamoyl of the t o c a i n i d e 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 of these As intermediates from human u r i n e . o u t l i n e d p r e v i o u s l y i n Scheme enzyme was used urea-adduct extraction observation t o produce intermediate. with the i o n i z e d methylene c h l o r i d e , intermediate p o s s i b l e . Conversely, discuss this intermediate should the s y n t h e s i s of t h i s permethylated In s t u d y i n g after benzoyl Eisenberg, acetate the hydrolyzed the glucuronosyl-0 6 h a d no e f f e c t reaction. acids the o b s e r v a t i o n of the products, a n d t h e GCMS d a t a o f intermediate. of unstable intermediates of the enzymatic and m e t h y l cleavage using must be buffer. glucosiduronic acid in From t h e d i s t r i b u t i o n i t was i n f e r r e d bond was c l e a v e d . that Non-enzymatic h y d r o l y s i s of both substrates 4 to i n the enzymatic substrates in glucuronosyl-O-cleavage 97 i n both o f 0-^ i n V a r i a t i o n s i n pH from on t h e i s o t o p e d i s t r i b u t i o n also resulted will F. J r . ( 6 9 ) s t u d i e d t h e c l e a v a g e o f glucuronic acid oxygen-labelled b a s i c e x t r a c t i o n with urea-adduct the f o r m a t i o n by GCMS s h o u l d be be p o s s i b l e . L a t e r s e c t i o n s hypothetical enzyme h y d r o l y s i s , t h e s i t e considered. carbamic a c i d or and p e r m e t h y l a t i o n , the methylene c h l o r i d e and p e r m e t h y l a t i o n , urea-adduct 11, b e t a - g l u c u r o n i d a s e Following hydrolysis, acidic of the carbamic a c i d theoretically identification (69). in strong The implications possible are identification outlined With mind, rise of the i n Scheme tocainide metabolic intermediates, and they r e f l e c t non-enzymatic o c c u r s a t the ester or t o the same h y d r o l y z e d During this permethylated as enzymatic splitting the c a r b a m o y l findings, tocainide, the C-0 o r the the mass s p e c t r a identification. for positive molecular a p e r m e t h y l a t i o n of hydrogens from the p r i m a r y cleavage The between C-2 In F i g u r e 35, d i m e t h y l a n i l i ne obtained at m/z base from 149 and the 3 "CH^ indicated groups, peak a t m/z C-3 72 replacing addition 244 2 1 hydrogen to from tocainide. 2,6- f r a g m e n t a t i o n p a t t e r n as of 2 "CH^ The groups the the permethylated d e u t e r a t i o n experiments. as 33 a t m/z corresponded of the 2,6- In F i g u r e s bond o f p e r m e t h y l a t e d mass s p e c t r u m an then were o b t a i n e d tocainide amine n i t r o g e n and revealed a similar earlier in the the h y d a n t o i n i o n of p e r m e t h y l a t e d suggested amide n i t r o g e n . bond, para-nitrophenol glucuronide, reference the C-N of and 34, cleavages u r e i d o g l u c u r o n i d e would g i v e 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 product. experiment, spectra on 15. the p o s s i b l e i f the either of these was molecular on ion the aniline nitrogen. The Figures The permethylated 36 and presence permethylated 37, o f m/z was lactoxylidide observed 134, tocainide, moiety. In a d d i t i o n , lost t o the c l e a v a g e due m/z as suggested i n the o f a -CH2 spectrum on that the a shown i n i o n a t m/z a permethylated suggested 98 i o n , as with a molecular found 206 molecular 221. of xylidine [M-153 + permethylated ion was lactic A: B: 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 s i s . 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 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 o f the t o c a i n i d e g l u c u r o n i d e . 99 105 244 • II i . Figure 34 : m/ z 200 100 The E I mass s p e c t r u m o f tocainide. 100 the permethylated 149 154 138 125 L_iy ,I i J . 1 i' , 100 F i g u r e 35 [ 150 i L_J. 200 m/z : The EI mass spectrum of the permethylated 2,6-dimethylaniline. 101 / 100 Figure 36 : 200 300 The t o t a l - i o n - c h r o m a t o g r a m lactoxylidide. scan of the perroethvlatPH 88 192 221 134 100 Figure 37 150 200 The E I mass s p e c t r u m o f t h e lactoxylidide. 102 m/ z permethylated acid moiety. The TIC and the mass s p e c t r u m xylyl)-5-methylhydantoin, not be equal interpreted. intensity suggested peak o f m/z xylidine, fragment not the 135 and section, the 7. m/z in Figures 38 and the peaks k 5 6 7 8 9 Note: of p a t t e r n which The base permethylated have been due However, an to ion at the m/z interpretation the as of the experimental shown following results in Table data N-carboxyl 282 will in did the 7. Experimental (pooled urine sample) c Extraced by RPC-18 elute-bond Column Extracted at PH 12 Extracted at PH 3 Enzyme hydrolyzed, extracted at PH 3 Enzyme hydrolyzed, extracted at PH 12 Acid hydrolyzed Basic extracted PH 9, underivatized Acidic extracted PH 3 Blank urine A l l samples were permethylated with CH,I(CD,I)/ DMSO Na except Sample 7 and 8. 103 this follow 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 i d e n t i f y i n g tocainide glucuronide intermediates. 1 2 3 could hypothetical structure. numbering Sample 3-(2,6- 39, symmetrical fragmentation from i o n may NCH3.CO-). permethylated of d i a s t e r e o i s o m e r s . resulted 162 the r e v e a l e d two of a p a i r have t o any of TIC shown mass s p e c t r a l d i s c u s s i o n of corresponding Table may (xylidine- ease a presence correspond For and The as of used for 100 Figure 38 200 300 scan : The t o t a l - i o n c h r o m a t o g r a m o f t h e 3- ( 2 , 6 - x y l y l ) - 5 - m e t h y l h y d a n t o i n . permethylated 135 72 152 106 235 Jul Figure 39 u J. U IJ 50 i.i 100 282 i 150 200 250 m/z : The E I mass s p e c t r u m o f t h e p e r m e t h y l a t e d 3-(2,6-xylyl)-5-methylhydantoin. 104 Sample sample 1 (extracted by RP-18 Bond-elute column) K 9 ( b l a n k u r i n e ) r e v e a l e d a major component peak a t m/z 105. T h i s compound was identified to and w i t h a base be R permethylated hippuric acid (70) r e f e r e n c e s p e c t r u m . base peak a t m/z 194. m e t a b o l i t e s c o u l d be when compared w i t h Caffeine was From sample identified, also Sadlter identified 1, n e i t h e r possibily the from i t s tocainide due to lack nor i t s of sensitivity. The TIC molecular from Sample i o n a t m/z 135 components have a l s o to those Scan from 136 was suspected from of found t o be methyl tocainide-urea, the be with a molecular o r the 194. two times as at was observed identity determined. Sample 3, was found component. to c o n t a i n h i p p u r i c i o n a t m/z Attempts retention However, the identifiable found These This fragment, spectrum. 5 a g a i n were i o n a t m/z ion. permethylated only with sample. A c o m p o n e n t conculsively and 88. identical 162 both to tocainide-carbamic acid 206, and identify caffeine, the i n t e r m e d i a t e , were successful . In the was as 4 and ester, with a molecular not hydantoin acidic-extracted, Samples m/z xy1idide-NCH3.CO- to c o n t a i n c a f f e i n e Both with hydantoin t o c o n t a i n the the components, a base peak a t m/z the p e r m e t h y l a t e d compound c o u l d not The acid and been o b s e r v e d the p e r m e t h y l a t e d this 2 r e v e a l e d two acid present. intermediates h y d r o l y z e d Sample Again, were neither o f the observed. 105 6, hippuric tocainide acid methyl conjugated ester In were t h e u n d e r i v a t i z e d Sample identified respectively. observed. caffeine 5. with molecular i o n s m/z as the o n l y Initial drug identifiable attempts Permethylation C H 0 I / C D 3 I to separate i s widely Initial used quick three components, h e n c e , dry-packed from The silica this crude flash chromatography. TLC p l a t e s : No o t h e r After The c r u d e ion m/z 105 was f o u n d A molecular of hippuric acid. m/z 232 w i t h a hydantoin identified in this Another total 106 from fragmentation was p r e s e n t . the sample. the s i l i c a g e l as d e c r i b e d , the r e s u l t i n g t o be p r e s e n t i o n a t m/z 206 from Section. t o e v a l u a t i o n by f l a s h 4 and 5 were c o l l e c t e d and p e r m e t h y l a t e d out u s i n g a ACN-XAD e x t r a c t was t h a t the p e r m e t h y l a t e d i o n s were and s i l i c a i n the E x p e r i m e n t a l prior - XAD the p o l a r u r i n a r y c h r o m a t o g r a p h y was c a r r i e d ACN-XAD e x t r a c t , fraction column, p o o l e d KC-18F, a l u m i n a column as d e s c r i b e d significant compounds of s y n t h e t i c products. g e l separated g e l column. suggesting T h i s method has e v a l u a t i o n of the A c e t o n i t r i l e chromatography, r e v e a l e d a prominent pattern chromatographic s e p a r a t i o n s of non-polar chromatographic revealed that s i l i c a eluted t h e c o l o r e d u r i n a r y components i n the p u r i f i c a t i o n e x t r a c t with 8, r e v e a l e d by F l a s h m e t a b o l i t e s by t h e use o f d i f f e r e n t been shown t o p r o v i d e gel, 194 was a l s o compound. of Tocainide Glucuronide methods l e d t o t h e use o f f l a s h urine i o n a t m/z 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 Chromatography and and 192 a n d m/z 218, Caffeine with a molecular Identification from 7, t o c a i n i d e a n d t h e h y d a n t o i n i n two p e a k s one peak mass i n the TIC. indicated the presence i o n peak w i t h a m o l e c u l a r ion at m/z 221 CD3I indicated during another structures The 6 to was 142, the m/z 41, 334, mass s p e c t r u m shown t o be the found hydantoin. t o be glucuronide offered in e a r l i e r shown in Figure CD3I Again, containing mass u n i t the m/z 334 of the and respectively. At highest t o the 42, moiety, suggested pooled At S c a n 66 at Scan i o n m/z 182, 218, as shown mass d e t e c t e d proposed tocainide ester discussions. Permethylation presence of 6 r e p l a c a b l e the t o the s t r u c t u r e of p l u s two sample from the i s shown cleavage carbamate the t o c a i n i d e hydroxyl amide h y d r o g e n s from in Figure between the revealed a corresponding 349, suggesting ion fragment at m/z the 277 4 r e p l a c e a b l e hydrogens, i n c r e a s e t o m/z xylidine use C2 43. and 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. t o m/z the the CD3I p e r m e t h y l a t e d permethylation increase with 4 r e p l a c a b l e hydrogens groups of g l u c u r o n i c a c i d of 41. tocainide glucuronide according As at 98, permethylated CD3I c o n f i r m e d structure. The Lastly, identical p a t t e r n of the with C3 carbon i n F i g u r e s 40 and with a molecular experiments fragment permethylated, 147, as d e s c r i b e d The the base peak a t m/z the The confirmed identified glucuronide tocainide. experiment were was h y d r o g e n s on of a base peak of m/z fragmentation lactoxylidide. hippuric acid identified in Figure of the compounds. 12 a r e and presence permethylation these and caffeine Scan at of TIC fractions 99, the p o r t i o n of hydrogens, d i d not 288. The labelling from the change 107 unit of 5 methyl groups. (N-CO) c l e a v a g e showed a c o r r e s p o n d i n g ion fragment tocainide contained show any 15 mass a t m/z 105 12 from no r e p l a c e a b l e i n mass a f t e r permethylation i _ J ul u 100 Figure 200 Total-ion-chromatogram of 40 300 the scan pp.rmethvlated urine. 98 46 105 156 7 ? 201 143 116 172 ......J,....::. i. If i Jll ll I, , illi -k + a Ji I 1,1 i IIII * ul .II , ill, .1, .i 4 lit Th- in/Z 277 232 J F i g u r e 41 : * 248 '* ^...^.1 334 302 dr * Ar-'-ir s » i t * r The E I s p e c t r u m o f p e r m e t h y l a t e d carbamoyl ester tocainide 108 sr*> <kr • * r tocainide (CH^ I ) . A- m/z Figure 42. The EI f r a g m e n t a t i o n ester glucuronide. pattern 109 o f permethylated tocainide carbamoyl 101 108 52 142 75 213 152 Ij i I! -..Jit:..- JJlJ jl.!!!]]!.:. .!# !.J|...:.,|,! Ll^fl,...!..! m/z 288 244 .1 Figure .d 43. 2 57 349 314 * i d * > ar I A- *r *r ar 4r sr ar ar ar * »>•• m/z The EI mass spectrum of permethylated tocainide carbamoyl glucuronide (CD^I). 110 ester with CD3I. Lastly, with i o n fragment the permethylated 4 methyl The groups absence described of a molecular molecular 6. analyzed fractions any a d d i t i o n a l Tocainide-urea The reaction different urea experimental Table 8 : these experiments, due t o t h e i n s t a b i l i t y I t should be n o t e d i n t h e CI mode w i t h o u t i o n of the permethylated Other reveal i o n from under GC c o n d i t i o n s . s a m p l e s were a l s o t o m/z 248, c o r r e s p o n d i n g t o labelled. b e f o r e , was p r o b a b l y glucuronide moiety, 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 h y d r o g e n s , showed a n i n c r e a s e o f 12 mass u n i t s the glucuronic acid collected useful from as of this that the o b s e r v i n g the TOCG. the s i l i c a - g e l column d i d n o t information. Reaction between t o c a i n i d e a n d u r e a was e x a m i n e d a t c o n c e n t r a t i o n s and a t d i f f e r e n t results a r e summarized i n Table temperatures. 8. The Reaction of Tocainide with Urea at Different Tenp. The Time Temperatures GOB Data Tocainide (m/Z 192) Hydantoin (m/Z 21£ Tocainide (base) Urea (8.0 M) 100°C 60°C Blank Urine hr. hrs. hrs. hrs, + + + + • 25°C 100°C 100°C 10 hrs, 1 hr. 9 hrs. Distilled Water 100°C 13 hrs, Urea 100°C Mexilitine 13 hrs, Methanimine WZ Distilled Water 100°C 13 hrs, Mexilitine <*> Mexilitine (base) 1 9 13 3 Ill + + - In fresh, tocainide Thus, drug-free d i d not the react hydantoin u r i n e a t room t e m p e r a t u r e with was urea not likely formed d u r i n g sample storage. 100 ° C f o r 13 h o u r s , almost to hydantoin was observed. at f o r 1 hour the heated 100 ° C hydantoin As could also shown neutral in Table to a l k a l i n e terminal amine amino g r o u p of Table be t o any measurable to a r i s e In the artifact of 8 M urea urine, 9, f o r m a t i o n of the conditions. At a c i d i c probably urea p r e v e n t i n g hydantoin pH, repelled the the Note: * KC-18 the 2 Hydantoin 0.45 0.45 0.45 0.75 0.75 0.75 Suspected Tocainide-N-Ureide _ 0.6 0.6 TLC. f 112 favored equally condensation. * (80/20: Methanol/H 0) Tocainide 2.5 7.8 12.5 was ionized 9 J The Reaction of Tocainide With Urea at Different pH PH tocainide when t o c a i n i d e drug-free at detected. of t o c a i n i d e f C), extent. c o n v e r s i o n of In a d d i t i o n , in fresh as an presence quantitative (25 charged From these repeatedly evolved demonstrated. through permethylation intermediate, shown experiments, corresponded mixture, r e v e a l e d the presence The m o l e c u l a r t o the p e r m e t h y l a t e d of the t o c a i n i d e - N - u r e i d e as i o n of the p e r m e t h y l a t e d with C 2~ 3 C c l e a v suggested that near 7. Synthesis of N-glucuronides 9 a The i d e n t i f i c a t i o n at trace intensity. e product of of t h i s such an i n t e r m e d i a t e c a n o c c u r n e u t r a l pH. The attempted glucuronide Tsakamoto analysis. chemical following as e v i d e n c e d These procedures of A r i t a ( 4 8 ) and T a k i t a n i condensation with of A r i t a ( 4 9 ) was n o t been shown t o be s u i t a b l e (section 4b). 2,6-dimethylani1ine (46) y i e l d e d u s i n g the p u b l i s h e d the p e r m e t h y l a t e d 2,6-dimethy1 a n i 1 i n e - N - g l u c u r o n i d e m o l e c u l a r i n F i g u r e s 46 and 47. glucuronide from (46), 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 methods have p r e v i o u s l y N-glucuronide Experiments 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 the procedures (47), Ishidate successful, samples this (-CO.NCH^CH^-) f r a g m e n t , whereas m/z 129 tocainide-N-ureide. shown Subsequently, which c o n t a i n e d a t m/z 291 was o b s e r v e d tocainide-N-ureide for intermediate. t o be i n F i g u r e 45, a base peak a t m/z 72 i n d i c a t e d t h e permethylated the ureide of the r e a c t i o n i n F i g u r e 44. shown T h i s h y d a n t o i n was t h o u g h t a tocainide tocainide-N-ureide As t h e f o r m a t i o n o f t h e h y d a n t o i n was However, t h i s was n o t o b s e r v e d i o n a t m/z 353 a s permethylated i n any p e r m e t h y l a t e d urine v o l u n t e e r s who h a d i n g e s t e d t o c a i n i d e . 113 aniline 100 F i g u r e 44 : 200 300 Total-ion-chromatogram of the scan permethylated tocainide-N-ureide. 72 129 191 291 AX a L 50 F i g u r e 45 : 100 150 The E I mass s p e c t r u m o f tocainide-N-ureide. 114 200 the 250 'm/ z permethylated 100 Figure 46 200 scan The t o t a l - i o n - c h r o m a t o g r a m of the permethylated 2,6-dimethylaniline-N-glucuronide. 103 219 135 76 uuLJi 50 F i g u r e 47 : 00 1 353 1 150 200 The E I mass s p e c t r u m o f 250 the 300 350 permethylated 2,6-dirnethylaniline-N-glucuronide. 115 m/ z 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 o f T o c a i n i d e G l u c u r o n ide Thus f a r , from p r e v i o u l y d i s c u s s e d the s t r u c t u r e o f TOCG was e s t a b l i s h e d b a s e d fragments from structure o f TOCG was n o t o b s e r v e d . was e m p l o y e d NMR the p e r m e t h y l a t e d to isolate the on t h e mass The i n t a c t Therefore, molecular preparative l a r g e r q u a n t i t i e s of t h i s 16 summarizes isolation conjugate HPLC for To the e x p e r i m e n t a l HPLC concentrate urine samples was added sample vacuum. The s o l v e n t time lyophilization Chromatographic e v a l u a t i o n a mobile 0.01 M sodium p h o s p h a t e acid. Detection 2 mL Figure nm and the UV a b s o r b a n c e respectively. phase 10 % o f the u r i n e offered reasonable t o 10 mL, compared f o r t h e same volume. urine sample o f 10 % a c e t o n i t r i l e i n t o pH 3 w i t h 226 nm and column e l u a n t phosphoric was collected 48 and 49 show t h e c h r o m a t o g r a m a t 226 of each F r a c t i o n 4 was Testing individual found hydrolysis. reaction on a TLC p l a t e r e v e a l e d presence of a g l u c u r o n i c to t h i s 12 h o u r s HPLC, of the c o n c e n t r a t e d alkaline Subsequent admixture buffer, adjusted was a t UV fractions. evaporation 100 mL u r i n e which r e q u i r e d out u s i n g for preparative for azeotropic (2 h o u r s ) t o c o n c e n t r a t e was c a r r i e d involved in Isolation acetonitrile under steps procedure. 1. P r e p a r a t i v e in TOCG. data, and FAB s p e c t r a l measurements. Scheme to mass s p e c t r a l to y i e l d of f r a c t i o n a blue fraction a t 226 the h y d a n t o i n 4 by color, nm, after naphthoresorcinol i n d i c a t i n g the moiety. finding, 116 fraction 4 was c o l l e c t e d over (16 hr) RE urine (100 ml) XAD-2 resin azeotrophic evaporation in vacuo with ACN 1 I adjusted to PH 3 extracted with CH C1 2 extracted with CH C1 at PH 7 2 2 2 extracted with hexane at PH 3 preparative HPLC, partisil-10 ODS-3, 10 uir(9 arr. id-, x 25 cm) 1st separation J, 10 i ACN in 0.01 M NaK.,PO, at PH 3 (2.5-3 mL/min.) i Fractions collection, 2 mL — j I 4- j Collected fraction T J, 2nd separation 3 Z ACN in 0.01 j basic naphthoresorcinol hydrolysis testing K NaK,PC at T PH 7 I Fractions collection, 2 mL I Collected fraction containing ToCG Azeotrophic evaporation JEstimated 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 e x p e r i m e n t a l tocainide steps i n v o l v e d i n glucuronide. 117 isolating the — j acid beta-glucjror.idase hydrolysis hydrolysis (with/vitho-j:) * sacchro-l.t-lactor Column: Whatmann P a r t i s i l - 1 0 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 Figure 48 : Preparative HPLC chromatogram of crude urine. 42 mm, 2 mL f r a c t i o n s UV fraction 2 1 F i g u r e 49 : absorbances: UV a b s o r b a n c e o f from p r e p a r a t i v e 2 mL f r a c t i o n s HPLC. 118 collected 225 nm 36 repetitive To analyses. further resolve collected fraction, acetonitrile with sodium using was UV i n 0.01 enzymatic 4 was found with concentrated a X by concentration According (30 to at the u m o l e s ) of mL 7 Fraction 4 and to the that UV spectrum obtained of from nm. 91 collected over and % purity. sample 42 injections, rechromatographed After evaporation y i e l d e d 62 mg of as to tocainide salt. urine ug/mL (30 100 50. basic, acidic Furthermore, phosphate 67 t o pH chromatogram Figure evaporation, the 3 % showing p o s i t i v e 4 was showing of resulting with identical fraction assay of as 205 vacuum, the GC The of this buffer, adjusted i s shown on well to be max 51, glucuronide/sodiurn Initial nm azeotropic in Figure under used. color test. Subsequently, dryness 220 h y d r o l y s i s , as tocainide shown was positive results naphthoresorcinol fraction phase c o m p o s i t i o n M sodium phosphate hydroxide, to g i v e u r i n a r y components w i t h i n a mobile d e t e c t i o n at found the urine sample mole/mL) of used for tocainide glucuronide estimated the a hydantoin. isolation, 12.3 mg could theoretically salt, the be i solated. To f u r t h e r remove reconstituted acetontrile i n w a t e r and i n 0.03 sodium hydroxide. Figure 52. acetate The salt. the chromatographed UV d e t e c t i o n was acetate conjugate was 119 at was 225 salt using % a c e t i c acid, adjusted resulting The phosphate t o pH nm as isolated removed as 10 sample was % 4.3 using shown in i n sodium i t s free acid by 1.0 M Column: Whatmann Partisil-10 Magnum -9, 9mm i d . x 25 cm UV detection: 220 run 3Z ACN in 0.01 M Nar^PO^ (PH 7) flow rate: 2.7 mL/min min. F i g u r e 50 : P r e p a r a t i v e HPLC chromatogram o f r e c y c l e d TOCG c o n t a i n i n g f r a c t i o n . 10 15 min. F i g u r e 51 : P r e p a r a t i v e HPLC chromatogram o f r e c y c l e d TOCG i n sodium phosphate saTt~. 120 ft Column: Whatmann P a r t i s i l - 1 0 Magnum-9, 9mm UV d e t e c t i o n i d . x 25 cm : 225 nm 10% ACN i n 0.3% a c e t i c a c i d (PH 4.3) flow r a t e : 3.0 mL/min 10 Figure 20 mm. 52 : P r e p a r a t i v e HPLC chromatogram o f r e c y c l e d TOCG i n sodium c h l o r i d e s a l t . 121 adjusting t h e pH t o 2.8 w i t h evaporating under vacuum. 1.0 M h y d r o c h l o r i c a c i d and This sample was s t o r e d i desiccated a t -10 ° C . 2. EI Mass S p e c t r a l A n a l y s i s o f t h e T o c a i n i d e Attempts glucuronide 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 by GCMS d e r i v a t i z a t i o n 3. 400 MHz P r o t o n The Nuclear interpretation was simplified and glucuronic acid 54 show acid. Table Experiment of the t o c a i n i d e g l u c u r o n i d e the a i d o f the r e f e r e n c e obtained exchange. tocainide i n t h e same Figure solvent. 55 shows t h e s p e c t r u m 10 summarizes t h e p r o t o n assignments 56 and 57 show the i s o l a t e d t h e 400 MHz p r o t o n tocainide glucuronide NMR Figure 53 and and a f t e r of g l u c u r o n i c of the i n Table 11. chemical shift to correspond The p r o t o n - The d o u b l e t a t 1.38 ppm was i n t e g r a t e d f o r 3 p r o t o n s was found spectrum i n D^-DMSO. observed and t o the c h i r a l signal its methyl group t o c a i n i d e (1.54 ppm). The singlet accounting a t 2.11 ppm was i n t e g r a t e d f o r 6 m e t h y l f o r t h e two x y l i d i n e CH^ g r o u p s . 3.69 ppm, 3.92 ppm, and 5.77 ppm were f o u n d signals obtained Furthermore, as spectrum spectra of tocainide s p e c t r a of t o c a i n i d e before NMR a s s i g n m e n t s a r e summarized of NMR spectrum. Figure of with methods were n o t s u c c e s s f u l . M a g n e t i c Resonance t h e 400 MHz p r o t o n deuterium Glucuronide protons shift at to correspond the r e f e r e n c e g l u c u r o n i c a c i d the s i g n a l s the a r o m a t i c group, from Chemical respectively. 122 ring t o the spectrum. a t 7.06 ppm and 9.39 ppm were of the x y l i d i n e groups, assigned and t h e x y l i d i n e - N H ppm Figure 53 : 400-MHz H-NMR s p e c t r u m o f t o c a i n i d e " — (D.-DMSO) . ' A ppm Figure 54 400-MHz- H-NMR s p e c t r u m 1 : e x c h a n g e (D^-DMSO). 123 of tocainide after D^O 124 Table 10 Summarized data from the 400-MHz p r o t o n NMR spectrum of tocainide. Shift (ppm) Coupling Integration (mm) Proton(s) TMS (0.0) 1.54d 2.16s Assignment TMS J - 7.2 Hz 36 3H(12mm/H) 72 6H 1 - CH xylidine - CH 2.51s DMSO 3.30s HjO 3 3 4.12bm 12 1H 2 - CH (due t o q u a d r a p o l b r o a d e n i n g by -N 7.10s 35 3H xylidine 8.25 bm 32 2H exchange w i t h D 0 (2-amino-NHj) 9.85bs 10 1H exchange w i t h D 0 ( x y l i d i n e -NH) 125 - H 2 2 1 J A 9 8 1 . ** 6 7 ,—(•'] . i . LJ J J, * F i g u r e 56 : 400 MHz-^H-NMR s p e c t r u m o f ester glucuronide 1 7 Figure 57 400 glucuronide carbamoyl (D^-DMSO). JL 5 4 1 MHz- H-NMR s p e c t r u m o f ester 6 tocainide 3 tocainide ( 8 X intensity 126 2 / 1 carbamoyl D^-DMSO). P T a b l e 11 Summarized data from the 400-MHz proton NMR spectrum o f the i s o l a t e d tocainide carbamoyl ester glucuronide. OH Shift (ppm) 1 .36d 2. l i s 2.51 3.34 3.68 3.7l' Coupling J • 6.6 Hz Integration (mm) 26 56 DMSO H0 Assignment 3H(8mm/H) 6H(8mm/H) 1 - CH (Tocainide) xylidine - CH 1H 3 55 Glucuronic a c i d ^ ' 5 3 2 3 99 Glucuronic a c i d ^ ' ^ 2 - CH (Tocainide) 3H Glucuronic acid 5.72 xylidine - H 1H xylidine - NH 3 3 2 J = 9 Hz 16 3.92d J = 9 Hz trace U .25t 5 .30d 5 .77s 7.06bs 7.63d 7.77bs 9.39bs Proton(s) 2 .69d >3 i H J = 8 Hz J = 7.2 Hz 7 16 10 27 9 3 8 2 Note: 1. Singlet, indicating straight chain conformation. 2. Clin Chem 26/9: 1323-1335 (1980) (Wu, et. a l . ) . 127 These partially of proton NMR suggested s i g n a l s from the presence glucuronic ppm a c i d spectrum w h i c h were a b s e n t acid the s i g n a l s was sample 4. F a s t submitted isolated submitted conjugate [m-H] 435 m/z tM+Na] from shown m/Z As spectrum. concentration o f TOCG i n i n sodium c h l o r i d e i n b o t h g l y c e r o l and t h i o g l y c e r o l intensity o f the 457 m/z In t h e s p e c t r a , ion adduct intense the presence the 1M+1] + In b o t h was i o n s o n t o the The e q u a l l y suggested of the respectively. an a d d i t i o n o f 2 sodium t h e two s p e c t r a in Figure would 60, t h e f r a g m e n t a t i o n likely to give rise 235, 236, 237, and 239. diagnostic ureide 6 to adduct ion at o f the a t 413 m/Z observed. conjugate at from Analysis i o n o f 411 m/Z. adduct. + was a l s o As suggesting molecular + glucuronic 58 and 59 show t h e FAB s p e c t r a a prominent observed, singlets f o r the l o s s o f t h e g l u c u r o n i c i n g l y c e r o l and t h i o g l y c e r o l , spectra, s i g n a l was n o t many b r o a d tocainide glucuronide Figures conjugate for analysis. f o r FAB a n a l y s i s matrices. have 55, t h e r e f e r e n c e i n the r e f e r e n c e the i n s u f f i c i e n t Atom Bombardment The in Figure contained One p l a u s i b l e e x p l a n a t i o n glucuronide of a g l u c u r o n i c a c i d t o c a i n i d e . However, the a n o m e r i c p r o t o n d e t e r m i n e d . M o r e o v e r , as shown 11 the i s o l a t e d value in determining pattern of the to the s u c c e s s i v e These i o n fragments the c a r b a m o y l ester mass ions were o f versus the structure. shown to observe in Figure the low mass 61, when the mass a n a l y z e r i o n s , the fragment 128 was i o n a t m/z adjusted 221 was Figure 58 : The f a s t atom bombardment spectrum o f t o c a i n i d e carbamoyl g l u c u r o n i d e i n g l y c e r o l m a t r i x w i t h sodium c h l o r i d e . ester (237, +2Na) 412 m/Z 193 (239, +2Na) Figure 60 The fragmentation pattern of tocainide carbamoyl ester glucuronide in fast atom bombardment mass spectrometry. 130 237 239 CO 295 297 247 189 221 279 327 235 403 \ N iiHi.iii hiiii Figure 61 : The f a s t atom bombardment s p e c t r u m o f t o c a i n i d e c a r b a m o y l 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 Ions to correspond a t m/z ion adduct to the e s t e r 237 and m/z of t o c a i n i d e 239 were g l u c u r o n i c moiety found to correspond C o r r e s p o n d i n g l y , m/z were p r o b a b l y a r r i v e d t h e (2Na+NacL) provided FAB TOCG. In a d d i t i o n , m/z 412 w i t h 457 a g r e e d from fragmentation definitive evidence i o n s from the i s o l a t e d ester the o b s e r v a t i o n o f t h e i n t a c t the carbamoyl 412.395 g/mole. 132 ion adducts ester 295 and m/z 297 ion adducts. f o r a carbamoyl i t scharacteristic with t o the 2Na (toc-H+2Na) and o f 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 . These (-C0.0-GA). structure for molecular a t m/z structure TOCG have 435, and of m o l e c u l a r ion at m/z weight E. Pharmacokinetics To urine, by follow the k i n e t i c an i n d i r e c t Hoffmann tocainide of Tocainide glucuronide to i t s hydantoin 1. T o c a i n i d e Glucuronide from a v o l u n t e e r orally i s shown in Figure Figure 63, i t c a n be followed k 2 are f i r s t - o r d e r Basic who Scheme 17 Schematic in a t pH h y d r o l y s i s o f the 12, f o l l o w e d in a urine by that sample 200mg t o c a i n i d e 62. From t h e s e m i - l o g inferred plot, a s shown i n the h y d r o l y s i s of t o c a i n i d e the r e a c t i o n Scheme rate 17 b e l o w . B o t h k-^ and constants. hydantoin 2 > representation the presence o f TOCG had been g i v e n K > initial Hydrolysis Kinetics profile 1 ToCG in hydantoin. hydrolysis-time glucuronide of t o c a i n i d e g l u c u r o n i d e ( 6 0 ) . The method r e q u i r e d of the obtained profile b a s i c h y d r o l y s i s a s s a y was u s e d a s d e s c r i b e d quantitation The Glucuronide o f NaOH. 133 degradation o f the k i n e t i c s product o f ToCG h y d r o l y s i s c o C5 Cy 0.50 M NaoH 0.25 M NaoH Cr 10 Figure 62 15 —i— —i— —i— 20 25 30 minute The hydrolysis p r o f i l e of tocainide glucuronide and the hydantoin in the presence of sodium hydroxide. 134 • O D Figure 63 S e m i - l o g p l o t o f the t o c a i n i d e hydrolysis k i n e t i c s in various 135 0.5 M NaoH 0.25 M NaoH 0.05 M NaoH g l u c u r o n i d e and the hydantoin c o n c e n t r a t i o n s o f sodium h y d r o x i d e . During that the the amount of indicating the a positive glucuronide hydrolysis was of 7 m i n u t e s of h y d r o l y s i s , i t was hydantoin k-^ . depleted, the V<2 r e p r e s e n t e d a initial and as degradation \<2 v a l u e compared M, order 0.1 M and 0.05 degradation According the kinetics, as with by of To shown in Figure then hydantoin the the the versus peak h e i g h t hydantoin demonstrate methylhydantoin internal was urine in ranging from i f the internal (4'-methylhydantoin) the ratio, not amounts of as would r e v e a l change aware of results found with are the the little hydrolytic t o the same u s i n g e t i d o c a i n e as a l s o shown in Figure 65. to e x h i b i t p s e u d o - f i r s t - o r d e r a rate constant 136 3-<2,4,6-xylyl)-5- subjected as d e s c r i b e d , but The in r e l a t i v e the 4'-methylhydantoin, relationship, s y n t h e s i z e d and 4 ' - m e t h y l h y d a n t o i n was kinetics change as in base. this experiment standard. degradation an 64. t o Hoffmann's p u b l i s h e d a s s a y , However, Hoffmann was hydrolysis hydantoin, 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 metabolite that 3-<2,4.6-Xvlvl)-5-methvlhvdantoin TOCG d e r i v e d h y d a n t o i n , nature from rate constants the time. the s y n t h e t i c hydantoin followed with of to t h a t o b t a i n e d 3-<2,4,6-xylyl)-5-methylhydantoin reflected the described, revealed standard the zero, To d e m o n s t r a t e rate constant as of t o c a i n i d e 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 - 0.0122 t o 0.0241 min 2. H y d r o l y s i s of amount k-^ a p p r o a c h e d s a m p l e s . H y d r o l y s i s e x p e r i m e n t s of 1.0 the became a p p a r e n t . s y n t h e t i c sample, h y d r o l y z e d identical i n c r e a s i n g , thereby However, as hydantoin the was evident o f 0.0122 min ^ . an The 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 IJ* cc Cv a. 10 Figure 64 15 20 25 —i— 30 ninutes S e m i - l o g p l o t o f the h y d r o l y s i s o f 3-f2.6-xv1v1)-5-methv1hvdantoin i n v a r i o u s c o n c e n t r a t i o n s o f sodium h y d r o x i d e r 137 •jk '-methylhydantoin/etidocaine + hydantoin/A'-methylhydantoin 1 1 — — U 1——— 8 • 12 1 16 1 20 1 24 1 1 28 32 minutes Figure 65 The hydrolysis of tocainide olucuronide and the 4'-methylhydantoin in the presence o f 0.5M sodium hydroxide. 138 similar The to those o b t a i n e d from peak h e i g h t r a t i o obtained from tocainide, w h i c h k^ zero, the o f the r e v e a l e d an in Figure initial gradual depending decline of As the Figure steepness internal levels to the a constant as hydantoin/4'- theoretically the r a t e experience of h y d r o l y s i s the p a r a l l e l 4'-methylhydantoin i n peak a r e a r a t i o o f the o f the first-order o f the two first-order o f the h y d a n t o i n decline a would r e f l e c t a depending as d e p i c t e d i n Curve was Data found To t o be an a c c u r a t e l y stable a t pH 12 and maintain r e p r o d u c i b i l i t y i n the p r e s e n c e timed assay of protocal a predetermined the c a l i b r a t i o n i n F i g u r e 66D, i n a u r i n e sample calibration curve a t any c a n be hydrolysis and time time was had the k i n e t i c by an used to assay required. tocainide to be choosen u r i n e samples. hydantoin identically sample, as d e p i c t e d i n F i g u r e 66C. as spontaneous > t , the q u a n t i t y o f approximated 139 was and its u r i n e s a m p l e s c o n t a i n e d v a r y i n g amounts o f both shown on o f the h y d a n t o i n glucuronide, for should i n F i g u r e 66A, standard. hydrolysis, Since approached 66B. Etidocaine the time shown fall 3. C a l i b r a t i o n an k^ standard approached the h y d a n t o i n and very gradual As the peak h e i g h t r a t i o over compounds. been g i v e n f o r m a t i o n phase, at o f the h y d a n t o i n internal methylhydantoin on hydantoin had 65. essence, decline hydantoin/4*-methylhydantoin i s v e r y much g r e a t e r t h a n k2« 4'-methylhydantoin In metabolite-derived hydantoin. u r i n e o f a v o l u n t e e r who the peak h e i g h t r a t i o shown the Since at timed time As F i g u r e 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 methylhydantoin 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-xylyl)-5-methylhydantoin as i n t 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 t o the 4'-methyll y d a n t o i n . C: Peak area-time 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 area r a t i o of the hydantoin to e t i d o c a i n e . 140 zero, i n a u r i n e sample, small portion subjected a close to h y d r o l y s i s , approximation calibration as r e f l e c t e d n o t be c o r r e c t l y Calibration samples, t o q u a n t i t a t e t h e amount I n t e r - and calibration For 0.05 from 0.9997 intra-assay variability and 0.9943 f o r hydantoin/4'- The d a t a a r e t a b u l a t e d i n T a b l e s intra-assay variations were e x p r e s s e d u s i n g the 13 and 14. as % C.V. d e t e r m i n a t i o n s of 6 samples w i t h c o n c e n t r a t i o n s was %. I n t e r - a s s a y v a r i a t i o n s from % and 1.75 %. found were F o r the h y d a n t o i n , was h i g h e r from 0.13 t o 0.05 variability was r a n g e d from 1.94 % t o 8.59 variability i n the h y d a n t o i n a s s a y dependent variation 141 t o range expressed variability t o t h e time internal were d e t e r m i n e d i n t h e same c o n c e n t r a t i o n range due assay curves revealed a 3 samples 0.04 The i n the u r i n e . h y d a n t o i n / e t i d o c a i n e , and 6 t o 20 ug/mL. The % C.V. % t o 1.76 the l e v e l s of respectively. between d u p l i c a t e ranging If f o r by t h e use 12. of h y d a n t o i n o f the 3 c a l i b r a t i o n samples. tocainide, in urine. as a second o f d e t e r m i n a t i o n o f 0.9974, methylhydantoin provided determined. standard tocainide/etidocaine, was of the h y d a n t o i n , data are t a b u l a t e d i n Table regression only a protocol then by t h e l e v e l s t h e use o f 4 ' - m e t h y l h y d a n t o i n coefficient timed of h y d a n t o i n included Linear of h y d a n t o i n accurately f o r the l e v e l s timed i n the u r i n e , coulld this amount sample, o f t h e h y d a n t o i n was n o t a c c o u n t e d an a c c u r a t e l y TOCG a calibration of the t h e o r e t i c a l the h y d r o l y s i s of unlike from as % C.V. found from t o range intra-assay %, whereas %. inter-assay This higher o f the h y d a n t o i n was probably i n t h e q u a n t i t y o f the T a b l e 12 C a l i b r a t i o n curve data f o r t o c a i n i d e and the hydantoin 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 . (ug/mL) Tocainide (avg.+S.D.) Hydantoin (avg.+S.D.) 3 Hydantoin (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 r =0.9974 r =0.9997 r =0.9943 Concentration^ 2 2 2 2 2 1. n=2 2. peak area r a t i o u s i n g e t i d o c a i n e ( i n t e r n a l standard) 3. peak area r a t i o u s i n g 4'-methylhydantoin ( i n t e r n a l standard) 142 Table Sample 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 i n i d e and the hydantoin at 6, 15, 20 ug/mL c o n c e n t r a t i o n s . Tocainide"'' C.V.7o 2 Hydantoin''' C .V. (ug) (avg.+S.D.) 6 6 0.472+0.007 0.481+0.008 1 .49 1 .76 0.319+0.016 0.339+0.015 5 .05 4 .42 15 15 1.385+0.001 1.385+0.002 0 .05 0 .16 0.964+0.007 0.957+0.001 0 .72 0 .13 20 20 1.946+0.022 1.898+0.003 1 .12 0 .16 1.397+0.009 1.359+0.066 0 .68 4 .80 (avg.+S.D.) 1. peak area r a t i o u s i n g lOug e t i d o c a i n e ( i n t e r n a l 2. n=2 Table Sample standard) 14 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 i n i d e and the hydantoin at 6, 15, 20 ug/mL concentrations"! Tocainide"'" C.V.7o 2 Hydantoin"'" C .V. (ug) (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 (avg.+S.D.) 143 hydantoin The metabolite extraction tocainide outlined from during hydrolysis. efficiencies and the h y d a n t o i n i n the e x p e r i m e n t a l o f 6, 10 a n d 20 ug/ml o f i n 5 mL m e t h y l e n e c h l o r i d e , a s section, 77.8% t o 81.2 % f o r t o c a i n i d e hydantoin. Concentration t o range a n d 56.6% t o 64.6 % f o r t h e T a b u l a t i o n of the data (ug/mL) were d e t e r m i n e d i s shown % i n T a b l e 15. recovery tocainide base hydanto i n 20 77.8 63.6 10 80. 1 64.6 6 81.2 56.6 1 i n 5 mL o f m e t h y l e n e c h l o r i d e . 2 duplicate determinations. T a b l e 15 Extraction efficiences of tocainide and the hydantoin in methylene chloride. 144 4. The Elimination The an IV k i n e t i c s of and healthy K i n e t i c s of tocainide o r a l dose of 200 ug human v o l u n t e e r s . tocainide and the subjects three excretion rate Appendixes The the tocainide are The shown from the collection hydantoin urinary two and 16 other in urine, were e x a m i n e d excretion IV in Table Glucuronide rates o r a l dose and 17. subjects in three of from The are after one of urinary shown in 2. in Table and the tocainide, pharmacokinetic data summarized and h y d a n t o i n a f t e r an data 1 and Tocainide the using 18. The the semi-log hydantoin the from three subjects urinary excretion were p l o t t e d a g a i n s t one-compartment are rates mid-time Wagner-Nelson^ of of kinetics •D program on The calculated plots 0.059 h r to II p l u s from the 1, fhat of the were net 100 rates - Log of intravenous of to r a n g e These V B ° excretion and of administration with equal dX. dT u v s . t.mid . . . E q u a t i on 145 oral W Log to were ^ a f t e r an of the from 0.037 values routes - ^ T e r m i n a l slope of the p l o t the from overall elimination % bioavailability doses). Log tocainide slope found dose. of 0.036 t o 0.057 h r between o r a l and s i m i l a r (assuming (K^,) terminal intravenous range computer. constants Equation the suggesting tocainide are using ^ a f t e r an comparable dose Apple ^ elimination rate subjects, rate an 1 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. hours urine vol (mL) 0 1 2 3 5 7 10 14 48 53 59 62 71 72 76 82 87 95 96 203 521 135 96 93 146 175 40 198 180 101 302 56 202 345 378 270 148 Toe uR/mL 2 6.56 3.91 8.19 12.31 23.63 28.23 44.55 6.08 2.41 7.61 6.96 3.97 2.72 1.38 1.67 1.86 1.46 - dtoc dt ug/mL HYD dHYD dt HYD 1331.68 2037.11 1105.65 590.88 1098.80 1373.86 1949.06 243.20 95.44 228.30 234.32 133.22 152.32 69.69 96.03 140.62 49.28 8.21 5.45 14.29 38.32 67.91 44.71 59.82 5.46 5.69 5.68 4.86 2.30 1.60 1.55 1.41 1666.63 2839.45 1929.15 1839.26 3157.82 2175.89 2617.13 218.40 225.32 170.40 163.62 77.18 89.60 78.28 81.08 8.18 5.87 16.01 36.99 61.78 41.92 49.84 4.34 4.49 4.35 3.53 1.83 1.31 1.24 1.15 2 - - Note: 1. Averaged value from duplicate determinations. 2. Using etidocaine as internal standard. 3. Using 4'-methylhydantoin as internal standard. 146 - - 3 - t Table 17 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 t h e h y d a n t o i n after a 200 me, o r a l dose o f t o c a i n i d e HCL i n a h e a l t h y human v o l u n t e e r . ur ine hours vol (mL) (ug/mL) 1 tocainide^ dToc (ug/mL) dt HYD 1 2 dHYD (ug/mL) dt 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 15.48 774. 0 17.43 871 .50 13.27 50 24.5 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 4.12 58 220 7.52 275. 73 5.13 188 .10 62 130 7.26 235. 95 3.91 127 .08 3.23 121. 72 2.54 74 68 2.12 116. 83 2.53 84 .21 - 72 294 4.14 76 183 - 233 83 Note: 1. 2. 3. - 3.51 Average v a l u e from d u p l i c a t e samples. U s i n g e t i d o c a i n e as i n t e r n a l s t a n d a r d . U s i n g 4 ' - 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 . 147 Table 18 The summarized pharmacokinetic data for tocainlde and the hydantoin In three healthy human volunteers after both an IV and oral dose. SR KE ( h r ) -1 t 1/2 Kmu ( h r ) t 1/2 RE ORAL IV ORAL IV 0.057 0.059 0.036 0 044 0.037 12.25 -1 CK IV 0.055 11.84 0.054 19.27 15 78 0.043 0 047 18.73 0.052 ORAL 0.037 18.75 0.055 12.63 12.72 15.93 14 63 13.36 12.57 AVERAGE (n-3) IV ORAL 0 .044 0.047 15 .75 14.85 0.052 0 .050 13 .86 _4 13.33 4 Xy- 1 (mg) 195.A3 52.51 49,57 24 23 24.87 53.8 M - 2 (mg) 91.24 40.58 33.15 10 92 37.87 61.81 _4 _4 Mu- (mg) 93.35 39.45 5 39.11 9 91 5 34.50 54.39 _5 _4 _4 u 3 k e (hr' ) k f (hr ) 1 -1 1 dose excreted intact X dose excreted as ToCG Note: 1. 2. 3. 4. 5. 6. 7. 0.028 _6 47.97 _6 _5 0.014 0.0089 5 0.010 0.0061 _5 5 31.37 5 25.68 _5 _5 24.72 .5 16.49 _5 .5 0 .0172 0 .0075 7 _5 May represent underestimation of true due to incomplete urine collection. Using etidocaine as I.S. (may represent underestimation due to incomplete urine collection). Using 4-methylhydantoin as I.S. (may represent underestimation due to incomplete urine collection). Cannot be determined due to incomplete urine collection. Cannot be determined due to lack of absolute bioavailability data. Cannot be determined due to k < KE. n=2. rau The elimination half-lives averaged 15.75 h o u r s of tocainide among t h e t h r e e ( o r a l ) a n d 14.85 h o u r s o f w h i c h a r e i n agreement with literature subjects (intravenous), both reported half-lives (33). The from urinary the t h r e e extrapolation averaged plot excretion lack cannot after a s shown from a l l t h r e e on e q u a t i o n urinary the subjects rate of o r a l estimate constants in Figure 67A u s i n g reasonable rate (ke). constant bioavailability estimate method f o r the I n a d d i t i o n , due t o data, rate the e x t r a p o l a t i o n constants after of plot Log dX u vs. t ^ = Log k X g o £ dt where k g = urinary Xg°= dose o f excretion rate of tocainide. tocainide. Equation 2 149 method the o r a l dose s. Y-intercept 2, were urine the e x t r a p o l a t i o n 2 i s the only by an Equation Due t o i n c o m p l e t e subjects, the e x c r e t i o n of t o c a i n i d e (ke) an IV d o s e , a s d e t e r m i n e d t o be 0.0172 hour"''". collection based excretion log _ > C dt | F i Figure log _i HYD dt u i i a: i i .1—J—J—Li—i L^-i—^—*—ITI. mid Urinary excretion rate p r o f i l e mid 1 Urinary excretion rate p r o f i l e Figure K L. .i. of hydantoin, — ^ ^ h . i Figure 67 tocainide. | >— Figure of i - i. c: L Urinary > J • Ii .1 J I I I PH. The s e m i - l o q p l o t o f t h e u r i n a r y e x c r e t i o n p r o f i l e o f 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 200mg IV dose o f t o c a i n i d e . 150 Urinary ka Tocainide Tocainide Glucuronide m Scheme 18 tocainide t o Scheme parameters d e s c r i b e d averaged the are various -1 Equation ( o r a l ) and 0.052 h o u r r e c e i v i n g an o r a l i n agreement with and an published u = Log k a t first-order rate (kmu) f o r the -1 k.X ° mu f B F~ KT mu - E (intravenous). The f o r the g l u c u r o n i d e 13.86 and intravenous literature - shown t o be k mu 2.303 the slope of the above equation f e a t h e r i n g = -k dose from 13.33 h o u r s r e s p e c t i v e l y , and values (33). ( t . ,) mid as o b t a i n e d by 2.303 assuming K in pharmacokinetic 3, were a v e r a g e d s u b j e c t s were c a l c u l a t e d t o be Log of tocainide as c a l c u l a t e d by t h e method o f f e a t h e r i n g , as urinary excretion half-lives three after into by t h e c o r r e s p o n d i n g 67B and 68B u s i n g 0.050 h o u r o f the e l i m i n a t i o n 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 glucuronide, in Figure metabolites 18 shown a b o v e , t h e e l i m i n a t i o n o f c a n be b r o k e n down constants. Other Schematic r e p r e s e n t a t i o n humans. According tocainide, £ Equation 151 3 Figure a: !• •! •'»—i-i—•—i—<-i—•—i—. Figure Figure 68 c: Urinary excretion rate p r o f i l e o f i i i , i . • Urinary i i i . i tocainide. • PH The s e m i - l o q p l o t o f t h e u r i n a r y e x c r e t i o n p r o f i l e o f 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 200mq o r a l dose o f t o c a i n i d e . 152 The formation glucuronide from extrapolation Log dM^ the using = Log dt k m u ~Tc Y-intercept rate constants three subjects Equation k X ° f - B of p l o t the tocainide were a g a i n estimated K (t £ m i d ) 2.303 l o g dM vs. u t m i = Log d di~ k m u k X ° f mu Xg° = urinary excretion rate = dose o f tocainide i n body J = total B k mu where k by 4. - K„ E mu ( k f ) of e l i m i n a t i o n of of KT E - TOCG tocainide E .... The three Due subjects to the subjects, after formation lack kf of a b s o l u t e cannot total constants were a v e r a g e d r e c e i v i n g an The rate be the glucuronide 0.0075 h o u r ~ l bioavailability estimated oral amount to of by the E q u a t i on from after data an from extrapolation 4 the IV dose. the three method dose. of tocainide excreted i n the urine oo (Xu found ) after an to r a n g e d o s e ) among the IV from d o s e , as c a l c u l a t e d by 95.93 mg three Equation (47.97 % d o s e ) t o 51.35 5 , mg was (25.68 subjects. X 0 0 - X ° k ... 153 E q u a t i on 5 % The percent glucuronide Equation o f dose excreted was c a l c u l a t e d i n the urine as t o c a i n i d e by t h e r a t i o kf/K^, a s shown i n 6. r a t i o o f dose in urine. e x c r e t e d as metabolite Equation The total calculated amount healthy literature expressed a s % dose was t o 24.72 % and 16.49 % i n two s u b j e c t s . In summary, three of the g l u c u r o n i d e the p h a r m a c o k i n e t i c data calculated human v o l u n t e e r s a r e i n agreement values. 154 6 with from t h e published SUMMARY AND CONCLUSIONS The oral bioavailability determined previous of objective f o r such a study, carbamoyl a novel pathway w h i c h ester finding ureide l e d t o the s p e c u l a t i o n glucuronide structure obtained from p e r m e t h y l a t i o n analysis. through isolate only partial analysis revealed N-ureide f o r TOCG. ester evidence [M+l], This tocainide-N- However, s t r u c t u r a l conjugation experiments s t r u c t u r e was ( C ^ I / C D ^ I ) and GCMS HPLC a s s a y was d e v e l o p e d measurements. f o r the i n t a c t s t r u c t u r e The provided o f TOCG. However, i n b o t h g l y c e r o l and t h i o g l y c e r o l [M+Na1 a n d [M-H+2Na3 457 r e s p e c t i v e l y , intermediate. of the i s o l a t e d g l u c u r o n i d e o f t h e sample thesis and u r e a , a s e c o n d o f an a l t e r n a t i v e TOCG f o r NMR a n d FAB s p e c t r a l NMR d a t a This o f 3-<2,6-xy1y1)-5- Furthermore, a preparative 400-MHz p r o t o n i o n a d d u c t s a t m/z 413, 435 i n d i c a t i n g the i n t a c t molecular i o n o f TOCG m/z 412. The l e v e l s o f TOCG i n urine hydantoin a f t e r hydrolysis hydantoin pH synthesis was known t o c y c l i z e a t pH > 12. through a t o c a i n i d e f o r the carbamoyl the was d e s i g n e d r e a c t i o n between t o c a i n i d e evidence at glucuronide l e d t o the f o r m a t i o n methylhydantoin and W i t h the the s t e r e o s p e c i f i c tocainide 3-(2,6-xylyl)-5-methylhydantoin described FAB conveniently approaches. Tocainide to c a n be by t h e use o f a s i n g l e p s e u d o - r a c e m i c d o s e . R(->- a n d S ( + ) - t r i d e u t e r a t e d two to of t o c a i n i d e was f o u n d > 12 c o n d i t i o n s . to also c a n be c o n v e n i e n t l y o f TOCG a t pH > 12. undergo To a c c o u n t 155 first-order a s s a y e d as However, t h e hydrolysis at f o r the spontaneous h y d r o l y s i s of the hydantoin, in order on this a s e t of a c c u r a t e l y to approximate analytical examined i n three subjects hydrochloride. TOCG were found oral respectively. literature These identity novel reaction t o be after o f TOCG o f TOCG IV and o r a l sample in urine. i n urine dosing used Based were o f 200 mg 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 o f 13.86 h o u r s and 13.33 h o u r s a f t e r These d a t a were i n agreement an IV and with values. 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 t h e s t r u c t u r a l o f TOCG a s a c a r b a m o y l chemical to g i v e levels scheme, t h e l e v e l s tocainide dose the t r u e timed c a l i b r a t i o n rise ester glucuronide. r e a c t i o n between t o c a i n i d e and u r e a to a u r e i d e intermediate. h a s n o t been r e p o r t e d s u c h a r e a c t i o n between u r e a i n drug and o t h e r remains p o s s i b l e . 156 This urea In a d d i t i o n , a was condensation metabolism. primary observed amine Nevertheless, xenobiotics REFERENCES 1. Vanughan W i l l i a m s , E.M. 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Reference H NMR S p e c t r u m . 163 - Beta APPENDIX 1 hours 1 2 3 5 7 9 11 12.5 13.5 19 21 24 27 28 30 32.5 37 43 46 53 58 63 68 72 78 82 86 96 IV dToC dt 4071.04 2275.38 3630.96 2084.13 2383.54 1693.74 1068.69 498.04 506.27 157.62 1208.67 506.81 1681.42 1427.28 505.12 688.53 1085.19 529.08 425.80 - dHYD dt 1517.33 1423.92 2700.69 1696.41 1589.66 1059.68 1198.91 639.11 1047.77 471.26 337.59 606.70 -• 283.52 279.36 515.04 249.48 186.77 - 89.79 69.62 - - hours 1 2 3 5 7 9 10 16 20 24 30 32 35 37 43 45 48 54 57 61 64 66 69 72 61 83 Oj_t dToC dt dHYD dt 845.05 646.46 - - 2110.81 1424.20 1402.30 - 388.04 - - 491.92 275.62 278.83 463.11 369.29 442.37 278.73 224.08 211.72 341.46 160.25 159.42 208.71 218.13 170.97 160.61 - - 356.24 186.32 135.56 54.51 29.57 - - 117.34 55.06 - Appendix 1 : 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 CK. 164 APPENDIX 2 IV hours 1 2 3 5 7 10 15.5 23 24 28 30 32 35 39 48 50 54 58 62 71 72 79 84 85 92 96 dToC dt dHYD dt 1082.43 1328.04 - - 1638.20 4363.81 3194.67 39469.54 2177.26 1998.95 2716.40 223.92 157.24 1528.67 7182.43 377.92 1273.64 2447.44 2183.09 19853.81 951.77 1033.72 1299.98 191.00 62.29 637.25 312.76 271.74 - 267.29 150.54 171.83 98.66 - - Appendix - - 2 hours 1 2 3 5 7 10 15.5 23 26 30 31.5 36 38 44 48 50 52 56 61 64 72 76 82 87 94 96 pRAL dToC dt dHYD dt 4107.88 5377.42 3455.85 336.73 5664.46 4623.46 5056.92 3090.57 254.41 3614.07 1428.36 291.55 507.58 806.87 902.41 361.90 416.91 644.47 - - - - 225.23 174.59 130.83 137.25 171.05 144.19 106.54 114.54 - - : 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 a n d t h e h y d a n t o i n a f t e r a 200mg o r a l a n d IV d o s e o f 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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Studies on the metabolism of tocainide in humans Kwok, David W. K. 1987
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Title | Studies on the metabolism of tocainide in humans |
Creator |
Kwok, David W. K. |
Publisher | University of British Columbia |
Date Issued | 1987 |
Description | Tocainide carbamoyl ester glucuronlde (TOCG) (R-NHCO.O-GA) is a major metabolite of tocainide (TonocardR). The structure of TOCG was first proposed by Elvin (35) based on the structure of 3-(2,6-xylyl)-5-methylhydantoln, a base hydrolyzed product of TOCG in urine. Due to the presence of two carbonyl groups on the hydantoin ring, TOCG was proposed to arrive from a novel metabolic pathway involving the addition of carbon dioxide to the terminal nitrogen of tocainlde followed by glucuronic acid conjugation. With the initial intention of carrying out a bioavailability study of tocainide using a deuterated pseudoracemic sample, the stereospecific synthesis of R(-)- and S(+)-trideuterated tocainide was attempted through two synthetic approaches. This thesis describes a chemical reaction between tocainide and urea, a second pathway which leads to the formation of 3-(2,6-xylyl)-5-methylhydantoin through a tocainide ureide intermediate. With this observation, a tocainide N-ureide glucuronide structure (R-NHCO.NH-GA) was proposed for TOCG in support of the theory that an in vivo reaction between tocainide and urea may have resulted a tocainide N-ureide which can be further conjugated with glucuronic acid. Attempts were made to assign the correct structure of TOCG by identification of the theoretical tocainide carbamic acid (based on Elvln's proposed structure) or the tocainide N-ureide intermediate in urine. This thesis also describes the preparative HPLC isolation and the structural characterization of this novel glucuronic acid conjugate. Evidence obtained as proof for the identity of TOCG as a conjugate was obtained from acid hydrolysis, basic hydrolysis, beta-glucuronidase hydrolysis, with or without the presence of sacchro-1,4-lactone, and a naphthoresorcinol color test. Structural evidence for the carbamoyl ester linkage of TOCG was obtained from proton-NMR and FAB analysis. The 400-MHz proton NMR data of the isolated glucuronide provided partial evidence for the intact structure of TOCG. In FAB analysis, the [M+1] ion adduct at m/z 413, [M+Na] at m/z 435, and [M-H+2Na] at m/z 457 have provided positive evidence for the molecular ion of TOCG at m/z 412 in favor of the carbamoyl ester structure. In addition to the hydrolysis of TOCG at pH > 12 to the hydantoin, this hydantoin was found to also undergo spontaneous first-order hydrolysis at pH > 12. To assay the levels of TOCG in urine as the hydantoin, a set of accurately timed calibration samples were employed in an assay protocol to take Into account the spontaneous hydrolysis of the hydantoin. Based on this analytical approach, the levels of TOCG were determined in three subjects both after an IV and oral dose of 200 mg tocainide HC1. The urinary excretion half-lives of TOCG of 13.86 hours and 13.33 hours, after an IV and oral dose respectively, were found to agree with literature values. |
Subject |
Anti-Arrhythmia Agents Myocardial depressants Glucuronates -- metabolism |
Genre |
Thesis/Dissertation |
Type |
Text |
Language | eng |
Date Available | 2010-07-14 |
Provider | Vancouver : University of British Columbia Library |
Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
DOI | 10.14288/1.0096967 |
URI | http://hdl.handle.net/2429/26428 |
Degree |
Master of Science - MSc |
Program |
Pharmaceutical Sciences |
Affiliation |
Pharmaceutical Sciences, Faculty of |
Degree Grantor | University of British Columbia |
Campus |
UBCV |
Scholarly Level | Graduate |
Aggregated Source Repository | DSpace |
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