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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.  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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