Stereoselective metabolism and pharmacokinetics of ifosfamide in the rat.

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type o f computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Kgher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor Ml 48106-1346 USA 313/761-4700 800/521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. STEREOSELECTIVE METABOLISM AND PHARMACOKINETICS OF IFOSFAMIDE IN THE RAT BY JINGHUA WANG A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Pharmaceutical Sciences) May 1995 Copyright 1995 Jinghua Wang Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 9621731 UMI Microform 9621731 Copyright 1996, by UMI Company. All rights reserved. This microform edition Is protected against unauthorized copying under Title 17, United States Code. 300 North Zeeb Road Ann Arbor, MI 48103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90007 This dissertation, written by .............................Jinghua Wang......................... under the direction of h..is. Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of re quirements for the degree of DOCTOR OF PHILOSOPHY C . . ----X Dean of Graduate Studies Date DISSERTATION COMMITTEE ........ . / Chairperson .................................. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To the Memories of My Loving Parents Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A C K N O W L E D G M E N T S I would like to express my sincere gratitude to my mentor, Dr. Kenneth K. Chan, whose guidance is the major contribution to the completion of this dissertation project. His insights surely will shape my future academic career as well as my personal life. I am very grateful to Dr. Eric J. Lien, my committee chair, for his understanding and generosity. My thanks also goes to Drs. Wei-Chiang Shen and Colin Paul Spears, my committee members, for their valuable advice. Finally, I thank Drs. Philip Hong, Amy Srigritsanapol, Jenny Zheng, and Ms. Ya-chun Nieh for their technical assistance. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS CONTENTS PAGE DEDICATION............................................................................................................il ACKNOWLEDGMENTS.......................................................................................... iii LIST OF TABLES......................................................................................................vil! LIST OF FIGURES................................................................................................... xi LIST OF SCHEMES................................................................................................ xlx CHAPTER 1 INTRODUCTION..............................................................................1 1.1 SPECIFIC AIMS....................................................................................2 1.2 ANTICANCER PROPERTY AND CHEMISTRY OF IFOSFAMIDE.........................................................................................3 1.3 METABOLISM OF IFOSFAMIDE........................................................5 1.4 PHARMACOKINETICS OF IFOSFAMIDE........................................ 9 1.5 MECHANISM OF ANTICANCER ACTION OF IFOSFAMIDE......................................................................................... 11 1.6 CLINICAL PHARMACOLOGY OF IFOSFAMIDE..............................12 1.7 CHIRALITY: IMPACT ON THE METABOLISM, PHARMACOKINETICS AND BIOLOGICAL EFFECTS....................13 1.8 STUDY STRATEGY.............................................................................17 CHAPTER 2 DESIGN AND ASYMMETRIC SYNTHESIS OF UNLABELED AND STRATEGICALLY DEUTERIUM- LABELED ENANTIOMERS OF IFOSFAMIDE..............................23 2.1 ABSTRACT.......................................................................................... 24 2.2 INTRODUCTION................................................................................ 25 2.3 RATIONAL DESIGN OF DEUTERIUM-LABELED ENANTIOMERS OF IFOSFAMIDE SUITABLE FOR PSEUDORACEMATE EXPERIMENTS.............................................. 26 2.4 METHODS AND PROCEDURES.......................................................27 2.4.1 Synthesis of unlabeled enantlomers of Ifosfamlde...............28 2.4.2 Synthesis of 2-chloro-2,2-dldeuterioethylamlne hydrochloride.......................................................................... 32 2.4.3 Synthesis of enantlomers of 6,6,2',2'-tetradeuterlo- Ifosfamlde................................................................................34 2.5 RESULTS AND DISCUSSION........................................................... 38 CHAPTER 3 IDENTIFICATION OF NEW METABOLITES OF IFOSFAMIDE IN RAT URINE USING ION CLUSTER TECHNIQUE....................................................................................50 3.1 ABSTRACT........................................................................................... 51 3.2 INTRODUCTION.................................................................................52 3.3 MATERIALS AND METHODS............................................................. 53 3.3.1 MATERIALS...........................................................................53 3.3.1.1 Chemicals and reagents........................................ 53 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.3.1.2 Surgical Instrument and supplies..............54 3.3.2 METHODS............................................................................54 3.3.2.1 Animal surgery.......................................................54 3 3.2.2 Metabolic studies...................................................55 3 3.2.3 Sample extraction and dehvatization....................55 3.3.2.4 GC/MS analysis....................................................56 3.3.3 SYNTHESIS OF NEW AND KNOWN METABOLITES OF IFOSFAMIDE AND (±)- IFOSFAMIDE-D4 ....................................................................57 3.3.3.1 Synthesis of alcoifosfamide................................... 57 3.3.3 2 Synthesis of 4-hydroxy N3-dechloroethyl ifosfamlde.................................................................58 3.3.3.3 Synthesis of N-dechloroethyl iphosphoramide mustard.........................................59 3.3.3.4 Synthesis of N2,N3-didechloroethyl ifosfamlde.................................................................60 3.3.3.5 Synthesis of 4-hydroxy ifosfamlde and 4- ketoifosf amide..........................................................61 3.3.3.6 Synthesis of N2-dechioroethyl ifosfamlde.............64 3.3.3 7 Synthesis of N3-dechloroethyl ifosfamlde.............6 6 3.3.3 8 Synthesis of iphosphoramide mustard..................67 3.3.3 9 Synthesis of (±)-ifosfamide-d4 ............................... 6 8 3.4 RESULTS..............................................................................................70 3.4.1 A Ico ifosfamlde....................................................................... 70 3.4.2 4-hydroxy ifosfamlde............................................................ 71 3.4.3 4-hydroxy N2-dechioroethyi ifosfamlde and 4- hydroxy N3-dechloroethyl ifosfamlde................................. 72 3.4.4 N-dechloroethyl iphosphoramide mustard........................... 74 3.4.5 N2,N3-didechloroethyl ifosfamlde........................................ 75 3.5 DISCUSSION...................................................................................... 76 CHAPTER 4 STEREOSELECTIVE METABOLISM OF THE ENANTIOMERS OF IFOSFAMIDE IN THE RAT.......................... 124 4.1 ABSTRAOT......................................................................................... 125 4.2 INTRODUCTION................................................................................126 4.3 MATERIALS AND METHODS............................................................127 4.3.1 MATERIALS...........................................................................127 4.3.1.1 Chemicals and reagents.........................................127 4.3.1.2 Surgical instrument and supplies........................... 127 4.3.1.3 Instrumentation.........................................................128 4.3.2 METHODS............................................................................. 128 4.3.2.1 Animal surgery.........................................................128 4.3.2.2 Metabolic studies.................................................... 129 4.3 2.3 Sample extraction and derivatization......................129 4.3.2.4 GC/MS analysis.................................................... 130 4.4 RESULTS.............................................................................................130 4.4.1 GC/MS CHARACTERIZATION OF IFOSFAMIDE, 4-HYDROXY IFOSFAMIDE, N2- DECHLOROETHYL IFOSFAMIDE, N3- DECHLOROETHYL IFOSFAMIDE, ALCOIFOSFAMIDE, AND IPHOSPHORAMIDE MUSTARD...............................................................................130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.4.2 ENANTIOMERIC RATIOS OF IFOSFAMIDE, 4- HYDROXY IFOSFAMIDE, N2-DECHL0R0ETHYL IFOSFAMIDE, N3-DECHL0R0ETHYL IFOSFAMIDE, ALCOIFOSFAMIDE, AND TOTAL IPHOSPHORAMIDE MUSTARD IN RAT URINE................133 4.5 DISCUSSION.....................................................................................135 CHAPTER 5 ENANTIOSELECTIVE PHARMACOKINETIC DISPOSITION OF IFOSFAMIDE IN THE RAT........................... 168 5.1 ABSTRACT........................................................................................169 5.2 INTRODUCTION..............................................................................170 5.3 MATERIALS AND METHODS.......................................................... 173 5.3.1 MATERIALS..........................................................................173 5.3.1.1 Chemicals and reagents........................................ 173 5.3.1.2 Surgical instrument and supplies...........................174 5.3.1.3 Instrumentation........................................................175 5.3.2 METHODS............................................................................175 5.3.2.1 Animalcannulation..................................................175 5.3.2.2 Phaimacokineticstudies........................................ 176 5.3.2.3 Sample extraction and derivatization.....................176 5.3.2 4 GC/MS analysis..................................................177 5.3.2.5 Recoveries of ifosfamide, ifosfamide-d4 , N2-dechloroethyl ifosfamide and N3- dechloroethyl ifosfamide from extraction.................178 5.3.2.6 Recovery of cyanohydrin of 4-hydroxy ifosfamide from extraction....................................... 179 5.3.2.7 Data analysis.........................................................180 5.3.3 SYNTHESIS OF DEUTERIUM-LABELED STANDARDS AND INTERNAL STANDARDS FOR GC/MS ASSAY..................................................................... 181 5.3.3.1 Synthesis of N3-dechloroethyl 4,4,5,5,6,6,2',2'-octadeuterio-ifosfamide and 4,4,5,5,6,6,2',2'-octadeuterio-ifosfamide.................181 5.3.3.2 Synthesis of N3-dechloroethyl 4,4,5,5,6,6- hexadeuterio-ifosfamide and 4,4,5,5,6,6- hexadeuterio-ifosfamide.........................................184 5.3.3.3 Synthesis of 4-hydroperoxy 6,6,2',2',2",2"- hexadeuterio-ifosfamide.........................................186 5.3.3.4 Synthesis of N2-dechloroethyl 6,6,1',T,2',2'-hexadeuterio-ifosfamide....................188 5.3.3.5 Synthesis of N2-dechloroethyl T,T,2',2'- tetradeuterio-ifosfamide.......................................... 190 5.3.3.6 Synthesis of 2',2'-dideuterio- iphosphoramide mustard........................................ 192 5 3.3.7 Synthesis of T,1',2',2',1",1",2",2"- octadeuterio-iphosphoramide mustard...................194 5.4 RESULTS............................................................................................. 196 5.4.1 VALIDATION OF ANALYTICAL METHODS..................... 196 5.4.1.1 Analysis of pseudoracemate of ifosfamide........... 196 5.4.1.2 Analysis of the derived metabolite of ifosfamide - 4-hydroxy ifosfamide........................196 5.4.1.3 Analysis of the derived metabolite of ifosfamide - N2-dechloroethyl ifosfamide..............197 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.4.1.4 Analysis of the derived metabolite of ifosfamide - N3-dechioroethyl ifosfamide.............198 5.4.1.5 Analysis of the derived metaboiite of ifosfamide -- iphosphoramide mustard.................. 198 5.4.2 PHARMACOKINETICS OF PARENT DRUGS AND THEIR RESPECTIVE METABOLITES FOLLOWING INTRAVENOUS ADMINISTRATION OF PSEUDORACEMATE OF IFOSFAMIDE..................... 199 5.4.2.1 Pharmacokineticsof ifosfamide................................ 199 5.4.2.2 Metabolite pharmacokinetics of 4-hydroxy ifosfamide................................................................200 5.4.2.5 Metabolite pharmacokineticsof iphosphoramide mustard........................................ 201 5.4.2.4 Metabolite pharmacokineticsof N2- dechioroethyl ifosfamide......................................... 202 5.4.2.5 Metaboiite pharmacokinetics of N3- dechloroethyl ifosfamide......................................... 202 5.5 DISCUSSION..................................................................................... 203 CHAPTER 6 SUMMARY AND FUTURE PERSPECTIVES....................................284 REFERENCES.........................................................................................................291 VII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES TABLE Table 4.1 Table 4.2 Table 4.3 Table 5.1 Table 5.2 Table 5.3 Table 5.4 Table 5.5 Table 5.6 Table 5.7 PAGE The selected ions and the respective retention times of derivatives of enantiomeric ifosfamides and their metabolites monitored...............................................................................................141 Isotopic composition of Ifosfamide and five metabolites in urine of rats following administration of a 1:1 mixture of deuterium-labeled and unlabeled ifosfamide of the same enantiomeric form (control).................................................................... 142 IsotopIc composition of Ifosfamide and five metabolites in urine of rats following administration of pseudoracemates................. 143 Ions selected for monitoring and the respective retention times of derivatives of enantiomeric ifosfamides, their metabolites and Internal standards...................................................... 212 Plasma concentration-tlme data of S-lfosf amide and R- ifosfamide-d4 (Rat 1 and Rat 2) foilowing iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg.................................................................................. 213 Plasma concentration-tlme data of S-lfosfamide and R- ifosfamide-d4 (Rat 3), and S-ifosfamide-d4 and R-lfosfamlde (Rat 4) following iv administration of a 1:1 mixture of S- ifosfamide and R-ifosfamide-d4 (Rat 3) or S-ifosfamide-d4 and R-lfosfamide (Rat 4)at a totai dose of 40 mg/kg.................. .214 Plasma concentration-time data of S-ifosfamide and R- ifosfamide-d4 (Rat 5 and Rat 6) foilowing iv administration of a 1:1 mixture of S-ifosfamlde and R-ifosfamide-d4 at a total dose of 40 mg/kg....................................................................... .215 Plasma concentratlon-time data of 4-hydroxyifosfamide, 4- hydroxyifosfamlde-d4 in Rat 1 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a totai dose of 40 mg/kg........................................................................ .216 Plasma concentration-tlme data of 4-hydroxy ifosfamide, 4- hydroxy lfosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-dg in Rat 2 following iv administra tion of a 1:1 mixture of S-ifosfamlde and R-lfosfamlde-d4 at a total dose of 40 mg/kg............................................................... .217 Plasma concentration-tlme data of 4-hydroxy ifosfamide, 4- hydroxy ifosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-dg in Rat 3 foliowing iv administra tion of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................................................... .218 VIII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.8 Plasma conœntration-time data of 4-hydroxy ifosfamide, 4- hydroxy ifosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-dg in Rat 4 following iv administra tion of a 1:1 mixture of S-ifosfamide-d^ and R-ifosfamide at a total dose of 40 mg/kg............................................................... .219 Table 5.9 Plasma concentration-time data of 4-hydroxy ifosfamide, 4- hydroxy ifosfamide-d^, iphosphoramide mustard, and iphosphoramide mustard-da in Rat 5 following iv administra tion of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d^. at a total dose of 40 mg/kg............................................................... .220 Table 5.10 Plasma concentratlon-time data of 4-hydroxy ifosfamide, 4- hydroxy ifosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-d2 in Rat 6 following iv administra tion of a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................................................... .221 Table 5.11 Plasma concentration-time data of N2-dechloroethyl ifosfa mide, N2-dechloroethyl ifosfamide-d2 , N3-dechloroethyl ifosfamide, and N3-dechloroethyl ifosfamide-d4 in Rat 1 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .222 Table 5.12 Plasma concentratlon-time data of N2-dechloroethyl ifosfa mide, N2-dechloroethyl ifosfamide-d2 , N3-dechloroethyl ifosfamide, and N3-dechloroethyl ifosfamide-d4 in Rat 2 following Iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .223 Table 5.13 Plasma concentration-time data of N2-dechloroethyl ifosfa mide, N2-dechloroethyl ifosfamide-d2 , N3-dechloroethyl ifosfamide, and N3-dechloroethyl ifosfamide-d4 in Rat 3 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .224 Table 5.14 Plasma concentratlon-time data of N2-dechloroethyl ifosfa mide, N2-dechloroethyl ifosf amide-d2 , N3-dechioroethyl ifosfamide, and N3-dechloroethyl ifosfamide-d4 in Rat 4 following iv administration of a 1:1 mixture of S-ifosfamide-d4 and R-ifosfamIde at a total dose of 40 mg/kg........................... .225 T able 5.15 Plasma concentration-time data of N2-dechloroethyl ifosfa mide, N2-dechloroethyl ifosfamide-da, N3-dechloroethyl ifosfamide, and N3-dechloroethyl ifosfamide-d4 in Rat 5 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .226 Table 5.16 Plasma concentration-tlme data of N2-dechloroethyl ifosfa mide, N2-dechloroethyl ifosfamide-d2 , N3-dechloroethyl ifosfamlde, and N3-dechloroethyl ifosfamide-d4 in Rat 6 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .227 IX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.17 Relevant pharmacokinetic parameters of ifosfamide from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 orS-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg.....................................................................................................228 Tabie 5.18 Relevant pharmacokinetic parameters of 4-hydroxyifosfamide from rats treated with a 1:1 mixture of S-ifosfamide and R- ifosfamide-d4 orS-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg.................................................................................. 230 Table 5.19 Relevant pharmacokinetic parameters of iphosphoramide mustard from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 orS-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg......................................................................231 Tabie 5.20 Relevant pharmacokinetic parameters of N2-dechloroethyl ifosfamide from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 or S-ifosfamide-d4 and R-ifosfamide at a totai iv dose of 40 mg/kg......................................................................232 Tabie 5.21 Relevant pharmacokinetic parameters of N3-dechioroethyl ifosfamide from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 orS-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg......................................................................233 Tabie 5.22 Stereoselective metabolism of ifosfamide as manifested by piasma AUC ratios...............................................................................234 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES FIGURE PAGE Figure 1.1 Chemical structures of oxazaphosphorines..........................................20 Figure 1.2 Chemical structures of enantlomers of ifosfamide................................. 21 Figure2.1 The numbering system of ifosfamide....................................................43 Figure2.2 IVIetabolic tracing by using 6,6,2',2'-tetradeuterio-ifosfamlde................44 Figure 3.1 Total and selected ion GC/CIMS chromatograms of the derivatized CHgClg extract residue from urine of a rat receiving (±)-ifosfamide. A) Total ion; B) Selected ion m/z387 for alcoifosfamide; C) Selected ion at /t7 /z 4 1 2 forA- hydroxyifosfamide; D) Selected ion at m/z 422 for 4-hydroxy N2-dechloroethyl ifosfamide or 4-hydroxy N3-dechloroethyl ifosfamide............................................................................................. 80 Figure 3.2 Mass spectrum of derivatized alcoifosf amide detected in the urinary extract from a rat given (±)-ifosfamide.....................................81 Figure 3.3 Ion cluster mass spectrum of derivatized alcoifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (±)-ifosfamide and (±)-ifosfamide-d4 ....................................82 Figure 3.4 Ion cluster mass spectrum of derivatized alcoifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 ................................. 83 Figure 3.5 Ion cluster mass spectrum of derivatized alcoifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (R)-ifosfamide and (S)-ifosfamide-d4 ..................................84 Figure 3.6 Mass spectrum of derivatized authentic alcoifosfamide...................... 85 Figure3.7 Mass spectrum of derivatized authentic cyanohydrin of 4- hydroxy ifosfamide...............................................................................86 Figure3.8 Mass spectrum of derivatized cyanohydrin of 4-hydroxy ifosfamide obtained from the urinary extract from a rat given (±)-ifosfamide........................................................................................87 Figure3.9 Mass spectrum of derivatized ^^C-cyanohydrin of 4-hydroxy ifosfamide obtained from the urinary extract from a rat given (±)-ifosfamide........................................................................................88 Figures.10 Ion cluster mass spectrum of derivatized cyanohydrin of 4- hydroxy ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (±)-ifosfamide and (±)-ifosfamide-d4 ..........89 XI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.11 Ion cluster mass spectrum of derivatized cyanohydrin of 4- hydroxy ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide- d4........................................................................................................... 90 Figure3.12 Ion cluster mass spectrum of derivatized cyanohydrin of 4- hydroxy ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (R)-ifosfamide and (S)-ifosfamide- d4........................................................................................................... 91 Figure3.13 Mass spectrum of the silylated 4-hydroxy N2-dechloroethyl or N3-dechloroethyl ifosfamide cyanohydrins obtained from the urinary extract from a rat given (±)-ifosfamide...............................92 Figure3.14 Mass spectrum of the silylated 4-hydroxy N2-dechloroethyi or N3-dechloroethyl ifosfamide ""^C-cyanohydrins obtained from the urinary extract from a rat given (±)-ifosfamide........................93 Figure3.15 Ion cluster mass spectrum of the silylated 4-hydroxy N2- dechloroethyl or N3-dechloroethyl ifosfamide cyanohydrins detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 ..................................94 Figure3.16 Ion cluster mass spectrum of the silylated 4-hydroxy N2- dechloroethyl or N3-dechloroethyl ifosfamide cyanohydrins detected in the urinary extract from a rat receiving a 1 ;1 mixture of (R)-ifosfamide and (S)-ifosfamide-d4 ..................................95 Figure3.17 Mass spectrum of derivatized authentic cyanohydrin of 4- hydroxy N3-dechloroethyl ifosfamide................................................. 96 Figure3.18 Total and selected ion GC/CIMS chromatograms of the silylated extract obtained from solid phase extraction of urine sample of a rat receiving ifosfamide. A) Total ion; B) Selected ion at /77/z339for N-dechloroethyl iphosphoramide mustard: C) Selected ion at m/z353 for N2,N3-didechloroethyl ifosfamide..............................................................................................97 Figure3.19 Mass spectrum of the silylated N-dechloroethyl iphosphoramide mustard obtained from the urinary extract from a rat given (±)-ifosfamide.............................................. .98 Figure3.20 Mass spectrum of the silylated N-dechloroethyl iphosphoramide mustard obtained from the urinary extract from a rat given a 1:1 mixture of (±)-ifosfamide and (±)- ifosfamide-d4 ......................................................................... .99 Figure3.21 Ion cluster mass spectrum of the silylated N-dechloroethyl iphosphoramide mustard detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)- ifosfamide-d4 .........................................................................................100 XII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.22 ion cluster mass spectrum of the silylated 4-hydroxy N2- dechloroethyl or M3-dechloroethyl ifosfamide cyanohydrins detected in the urinary extract from a rat receiving a 1:1 mixture of (R)-ifosfamide and (S)-ifosfamide-d4 ..................................101 Figure 3.23 Mass spectrum of derivatized authentic N-dechloroethyl iphosphoramide mustard......................................................................102 Figure 3.24 Mass spectrum of the silylated N2,N3-didechioroethyl ifosfamide obtained from the urinary extract from a rat given (±)-ifosfamide.........................................................................................103 Figure3.25 Ion cluster mass spectrum of the silylated N2,N3- didechloroethyl ifosfamide obtained from the urinary extract from a rat given a 1:1 mixture of (±)-ifosfamide and (±)- ifosfamide-d4 .........................................................................................104 Figure3.26 Ion cluster mass spectrum of the silylated N2,N3- didechloroethyl ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)- ifosfamide-d4 .........................................................................................105 Figure3.27 Ion cluster mass spectrum of the silylated N2,N3- didechloroethyl ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (R)-ifosfamide and (S)- ifosfamide-d4 .........................................................................................106 Figure3.28 Mass spectrum of derivatized authentic N2,N3- didechloroethyl ifosfamide....................................................................107 Figure 4.1 Chemical structures of control pairs and pseudoracemates of ifosfamide..............................................................................................144 Figure4.2 Total ion GC/CIMS chromatogram of the derivatized CH2CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 .................................................................................. 145 Figure4.3 Selected ion [m/z225 for (S)-ifosfamide-do and m/z229 for (R)-ifosfamide-d4 ) GC/CIMS chromatograms of the derivatized CH2CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 . Insets show those from rat urine blank.....................................................................146 Figure 4.4 Figure 4.5 Figure 4.6 Mass spectrum of the derivatized (S)-ifosfamide and (R)- ifosfamide-d4 from CH2 CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 ...................... .147 Selected ion (m/z235 for N2-dechloroethyl ifosfamide and m/z237 for N2-dechloroethyl ifosfamide-d2 ) GC/CIMS chromatograms of the derivatized CH2CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)- ifosfamide-d4 . Insets show those from rat urine blank.......... Mass spectrum of the derivatized N2-dech!oroethyl ifosfamide and N2-dechioroethyl ifosfamide-d2 from CH2CI2 .148 XIII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 ................................................................................ 149 Figure4.7 Selected ion {m/z235 for N3-dechioroetfiyl ifosfamide and m/z239forN3-dechioroethyl ifosfamide-d4 ) GC/CIMS ctiromatograms of tfie derivatized CH2 CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)- ifosfamide-d4 - insets stiow tfiose from rat urine blank...................... 150 Figure4.8 Mass spectrum of the derivatized N3-dechioroethyi ifosfamide and N3-dechloroethyi ifosfamide-d4 from CH2 Ci2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 .................................................................................151 Figure4.9 Selected ion {m/z387 for alcoifosfamide and m/z391 for alcoifosfamide-d4 ) GC/CIMS chromatograms of the derivatized CH2 CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 . Insets show those from rat urine blank.....................................................................152 Figure 4.10 Mass spectrum of the derivatized alcoifosfamide and aicoifosfamide-d4 from CH2CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 ..............................153 Figure4.11 Selected ion (m/z412 for 4-hydroxy ifosfamide and m/z416 for 4-hydroxy ifosf am ide-d4 ) GC/CIMS chromatograms of the derivatized CH2CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 . Insets show those from rat urine blank.....................................................................154 Figure4.12 Mass spectrum of the derivatized 4-hydroxy ifosfamide and 4-hydroxy ifosfamide-d4 from CH2 CI2 extract residue from urine of a rat receiving (S)-ifosfamide and (R)-ifosfamide-d4 ............. 155 Figure 4.13 Total ion GC/CIMS chromatogram of the derivatized solid phase extract residue from urine of a rat receiving (S)- ifosfamide and (R)-ifosfamide-d4 ......................................................... 156 Figure 4.14 Selected ion {m/z329 for iphosphoramide mustard and m/z 333 for iphosphoramide mustard-d2) GC/CIMS chromatograms of the derivatized solid phase extract residue from urine of a rat receiving (S)-ifosfamide and (R)- ifosfamide-d4 - Insets show those from rat urine blank....................... 157 Figure4.15 Mass spectrum of the derivatized iphosphoramide mustard and iphosphoramide mustard-d2 from solid phase extract residue from urine of a rat receiving (S)-ifosfamide and (R)- ifosfamide-d4 ........................................................................................ 158 Figure5.1 Chemical structures of two pseudoracemates of ifosfamide.............235 Figure5.2 Chemical structures of unlabeled and deuterium-labeled compounds; ifosfamide, ifosf amide-d4 , ifosfamide-dg, 4- hydroxy ifosfamide, 4-hydroxy ifosfamide-dg, N2- xiv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure5.5 Figure5.6 Figures.? Figure 5.8 FigureS.9 dechloroethyl ifosfamide, N2-dech!oroethyiifosfamide-d4, N3- dechloroethyi ifosfamide, N3-dechloroethyl ifosfamide-dg, iphosphoramide mustard, iphosphoramide mustard-da, iphosphoramide mustard-dg....................................................... .236 FigureS.3 Figure 5.4 A representative total ion GC/CIMS chromatogram of the derivatized plasma CH2CI2 extract from a rat treated iv with a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 at a total dose of 40 mg/kg......................................................................... .238 Representative GC/CIMS selected ion chromatograms for ifosfamide (m/z225), ifosfamide-d4 (m/z229), and ifosfamide- dg (m/z233). a) blank extract containing IF-dg as the intemal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 at a total dose of 40 mg/kg......................................................................... .239 Representative GC/CIMS selected ion chromatograms for 4- hydroxy ifosfamide (m/z412), 4-hydroxy ifosfamide-d4 (m/z 416), and 4-hydroxy ifosfamide-dg (m/z420). a) blank extract containing 4-hydroxy ifosfamide-dg as the intemal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 at a total dose of 40 mg/kg........................................................................ .241 Representative GC/CIMS selected ion chromatograms selected for N2-dechloroethyl ifosfamide (m/z235), N2- dechloroethyl ifosfamide-d2 (m/z237), and N2-dechloroethyl ifosfamide-d4 (m/z239). a) blank extract containing N2- dechloroethyl ifosfamide-d4 as the internal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of (S)- ifosfamide and (R)-ifosfamide-d4 at a total dose of 40 mg/kg... .243 Representative GC/CIMS selected ion chromatograms selected forN3-dechloroethyl ifosfamide (m/z235), N2- dechloroethyl ifosfamide-d4 (m/z239), and N2-dechloroethyl ifosfamide-dg (m/z243). a) blank extract containing N3- dechloroethyl ifosfamide-dg as the internal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of (S)- ifosfamide and (R)-ifosfamide-d4 at a total dose of 40 mg/kg... .245 A representative total ion GC/CIMS chromatogram of the derivatized plasma solid phase extract from a rat treated iv with a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 at a total dose of 40 mg/kg.......................................................... .247 Representative GC/CIMS selected ion chromatograms for iphosphoramide mustard (m/z329), iphosphoramide mustard-d2 (m/z333), and iphosphoramide mustard-dg (m/z 337). a) blank extract containing iphosphoramide mustard-dg as the internal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of (S)-ifosfamide and (R)- ifosfamide-d4 at a total dose of 40 mg/kg.................................. .248 XV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.10 A representative standard curve of S-ifosfamide in rat plasma.................................................................................................. 250 Figure 5.11 A representative standard curve of R-ifosfamide-d4 in rat plasma.................................................................................................. 251 Figure 5.12 A representative standard curve of 4-fiydroxy ifosfamide in rat plasma............................................................................................. 252 Figure 5.13 A representative standard eu n/e of N2-dechloroetfiyl ifosfamide in rat plasma........................................................................253 Figure5.14 A representative standard curve of N3-decfiloroethyl ifosfamide in rat plasma........................................................................254 Figure 5.15 A representative standard curve of ipfiosphoramide mustard in rat plasma......................................................................................... 255 Figure 5.16 A representative standard curve of ipfiosphoramide mustard- dg in rat plasma.................................................................................... 256 Figure5.17 Plasma concentration-time profiles of S-ifosfamide (*), and its derived metabolites 4-hydroxy ifosfamide (a), N2- dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (H) in Rat 1 following iv administration of a 1:1 mixture of 8- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............... 257 Figure 5.18 Plasma concentration-time profiles of R-ifosfamide-d4 (+) and its derived metabolites 4-hydroxyifosfamide-d4 (o), N2- dechloroethyl ifosfamide-dg (A), and N3-dechloroethyl ifosfamide-d4 (c$ in Rat 1 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................................................................................... 258 Figure 5.19 Plasma concentration-time profiles of S-ifosfamide (*) and its derived metabolites 4-hydroxy ifosfamide (®), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (h) in Rat 2 following iv administration of a 1:1 mixture of S-ifosfamide and R- ifosfamide-d4 at a total dose of 40 mg/kg............................................259 Figure5.20 Plasma concentration-time profiles of R-ifosfamide-d4 (+) and its derived metabolites 4-hydroxy ifosfamide-d4 (o), iphosphoramide mustard-dg (0), N2-dechloroethyl ifosfamide- dg (A), and N3-dechloroethyl ifosfamide-d4 (û) in Rat 2 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg........................................260 Figure 5.21 Plasma concentration-time profiles of S-ifosfamide (*) and its derived metabolites 4-hydroxy ifosfamide (®), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (b) in Rat 3 following iv xvi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. administration of a 1:1 mixture of S-ifosfamide and R- ifosfamide-d4 at a total dose of 40 mg/kg................... .261 Figure 5.22 Plasma concentration-time profiiesof R-ifosfamide-d4 (+) and its derived metabolites 4-hydroxy ifosfamide-d4 (o), iphosphoramide mustard-dg (0), N2-dechloroethyl ifosfamide- dg (A), and N3-dechloroethyl ifosfamide-d4 (□) in Rat 3 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .262 Figure 5.23 Plasma concentration-time profiles of S-ifosfamide (*) and ifs derived metabolites 4-hydroxy ifosfamide («), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (h) in Rat 4 following iv administration of a 1 ;1 mixture of S-ifosfamide and R- ifosfamide-d4 at a total dose of 40 mg/kg................................... .263 Figure 5.24 Plasma concentration-time profiies of R-ifosfamide-d4 (+) and its derived metabolites 4-hydroxy ifosfamide-d4 (o), iphosphoramide mustard-dg (0), N2-dechloroethyl ifosfamide- dg (A), and N3-dechloroethyl ifosfamide-d4 (□) in Rat 4 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .264 Figure 5.25 Plasma concentration-time profiles of S-ifosfamide (* ) and its derived metabolites 4-hydroxy ifosfamide (•), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide ( h) in Rat 5 following iv administration of a 1:1 mixture of S-ifosfamide and R- ifosfamide-d4 at a totai dose of 40 mg/kg................................... .265 Figure 5.26 Piasma concentration-time profiles of R-ifosfamide-d4 (+) and ifs derived metabolites 4-hydroxy ifosfamide-d4 (o), iphosphoramide mustard-dg (0), N2-dechloroethyl ifosfamide- dg (A), and N3-dechloroethyl ifosfamide-d4 (□) in Rat 5 foliowing iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .266 Figure5.27 Plasma concentration-time profiles of S-ifosfamide (* ) and its derived metabolites 4-hydroxy ifosfamide (o), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (h) in Rat 6 following iv administration of a 1:1 mixture of S-ifosfamide and R- ifosfamide-d4 at a total dose of 40 mg/kg................................... .267 Figure 5.28 Plasma concentration-time profiles of R-ifosfamide-d4 (+) and its derived metabolites 4-hydroxy ifosfamide-d4 (o), iphosphoramide mustard-dg (0), N2-dechloroethyl ifosfamide- dg (A), and N3-dechloroethyl ifosfamide-d4 (□) in Rat 6 foilowing iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg............................... .268 XVII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 6.1 Chemical structure of 2-ethyienimine-3-(2-chloroethyi)tetra- hydro-1,3,2-oxazaphosphorine 2-oxide.............................................290 XVIII Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF SCHEMES SCHEME PAGE Scheme 1.1 Metabolic pathways of ifosfamide.....................................................22 Scheme 2.1 Route of synthesis of (S)-(-)-ifosfamide............................................45 Scheme 2.2 Route of synthesis of (R)-(+)-ifosfamide...........................................46 Scheme 2.3 Route of synthesis of 2-chloro-2,2-dideuterioethylamlne hydrochloride.....................................................................................47 Scheme 2.4 Route of synthesis of (S)-(-)-6,6,2',2'-tetradeuterio- ifosfamide..........................................................................................48 Scheme 2.5 Route of synthesis of (R)-(+)-6,6,2',2'-tetradeuterio- ifosfamlde..........................................................................................49 Scheme 3.1 The updated metabolic pathways of ifosfamide...............................108 Scheme 3.2 Route of synthesis of alcoifosfamide.................................................109 Scheme 3.3 Route of synthesis of 4-hydroxy N3-dechloroethyl ifosfamide.......................................................................................... 110 Scheme 3.4 Route of synthesis of N-dechloroethyl iphosphoramide mustard.............................................................................................. 111 Scheme 3.5 Route of synthesis of N2, N3-didechloroethyl ifosfamide.................112 Scheme 3.6 Route of synthesis of 4-hydroxy ifosfamide and 4- ketoifosfamide....................................................................................113 Scheme 3.7 Route of synthesis of N2-dechloroethyl ifosfamide..........................114 Scheme 3.8 Route of synthesis of N3-dechloroethyl ifosfamide......................... 115 Scheme 3.9 Route of synthesis of Iphosphoramide mustard...............................116 Scheme 3.10 Route of synthesis of 1 ',T,2',2'-tetradeuterioifosfamide................... 117 Scheme 3.11 The formation of ion m/z387 from alcoifosfamide (MSTFA: methylsilyltrifluoroacetamide)............................................................ 118 Scheme 3.12 The formation of ion m/z412 from 4-hydroxy ifosfamide (MSTFA; methylsilyltrifluoroacetamide)............................................119 Scheme 3.13 The formation of ion m/z 422 from 4-hydroxy N2-dechloro- ethyl ifosfamide and/or 4-hydroxy N3-dechloroethyl ifos famide (MSTFA: methylsilyltrifluoroacetamide).................................120 XIX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Scheme 3.14 Differentiation of 4-hydroxy N2-dechloroethyl ifosfamide and 4-hydroxy N3-dechloroethyl ifosfamide by deuterium labeling/mass spectrometry.............................................................. 121 Scheme 3.15 The formation of ion m/z 339 from N-dechloroethyl iphos phoramide mustard (MSTFA; methylsilyltrifluoroacetamide)............ 122 Scheme 3.16 The formation of ion m/z353 from N2,N3-dideuterio- ifosfamide (MSTFA: methylsilyltrifluoroacetamide)...........................123 Scheme 4.1 Flow chart of stereoselective metabolic studies of ifosfamide...........................................................................................159 Scheme 4.2 Flow chart of GC/MS assay for (S)-ifosfamide, (R)- ifosfamide, N2-dechloroethyl ifosfamide, N3-dechloroethyl ifosfamide, 4-hydroxy ifosfamide, and alcoifosfamide......................160 Scheme 4.3 Flow chart of GC/MS assay for iphosphoramide mustard................161 Scheme 4.4 Derivatization scheme of ifosfamide and ifosfamide-d4 . (MSTFA: methylsilyltrifluoroacetamide)............................................ 162 Scheme 4.5 Derivatization scheme of N2-dechloroethyl ifosfamide-do and N2-dechloroethyl ifosfamide-d2 . (MSTFA: methylsilyltrifluoro-acetamide)...........................................................163 Scheme 4.6 Derivatization scheme of N3-dechloroethyl Ifosfamide and N3-dechloroethyl ifosfamide-d4. (MSTFA: methylsilyltiifiuoro-acetamide)...........................................................164 Scheme 4.7 Derivatization scheme of alcoifosfamide and alcoifosfamide- d4 . (MSTFA: methylsilyltrifluoroacetamide)...................................... 165 Scheme 4.8 Derivatization scheme of 4-hydroxy ifosfamide and 4- hydroxy ifosfamide-d4 . (MSTFA: methylsilyltrifluoroacetamide)............................................................. 166 Scheme 4.9 Derivatization scheme of iphosphoramide mustard and iphosphoramide mustard-dg. (BSTFA: N,0-bis- (trimethylsilyl)trifluoroacetamide: MSTFA: methylsilyltrifluoroacetamide).............................................................167 Scheme 5.1 Flow chart of stereoselective pharmacokinetic studies of ifosfamide.......................................................................................... 269 Scheme 5.2 Flow chart of GC/MS assay for (S)-ifosfamide, (R)- ifosfamide-d4 , N2-dechloroethyl ifosfamide, N3- dechloroethyi ifosfamide, and 4-hydroxy ifosfamide........................270 Scheme 5.3 Flow chart of GC/MS assay for iphosphoramide mustard...............271 Scheme 5.4 Derivatization scheme of ifosfamide, ifosf am ide-d4 , and internal standard ifosfamide-dg. (MSTFA: methylsilyltrif luoroace-tamide).......................................................... 272 XX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Scheme 5.5 Derivatization scheme of 4-hydroxy ifosfamide, 4-hydroxy ifosfamide-d4 , and intemal standard 4-hydroxy ifosfamide- de- (MSTFA: methyisiiyltrifluoroacetamide)......................................273 Scheme 5.6 Derivatization scheme of N2-dechloroethyl ifosfamide, N2- dechioroethyi ifosfamide-d2, and intemal standard N2- dechloroethyiifosfamide-d^. (MSTFA: methyisiiyltrifluoroacetamide).............................................................274 Scheme 5.7 Derivatization scheme of N3-dechloroethyi ifosfamide, N3- dechloroethyl ifosfamide-d^, and intemal standard N3- dechloroethyl ifosfamide-de. (MSTFA: methylsilyltri- fiuoroacetamide)................................................................................ 275 Scheme 5.8 Derivatization scheme of iphosphoramide mustard, iphos phoramide mustard-dg, and internal standard iphos phoramide mustard-ds. (BSTFA: N,0-bis-(trimethyl- siiyl)trifluoroacetamide; MSTFA: methylsilyl- tiifiuoroacetamide)..............................................................................276 Scheme 5.9 Route of synthesis of ifosfamide-da and N3-dechloroethyl ifosfamide-dg..................................................................................... 277 Scheme 5.10 Route of synthesis of ifosfamide-dg and N3-dechioroethyl ifosfamide-dg..................................................................................... 278 Scheme 5.11 Route of synthesis of 4-hydroxyifosfamide-dg.................................279 Scheme 5.12 Route of synthesis of N2-dechloroethyl ifosfamide-dg.................. 280 Scheme 5.13 Route of synthesis of N2-dechloroethyl ifosfamide-d4 .................. 281 Scheme 5.14 Route of synthesis of iphosphoramide mustard-dg....................... 282 Scheme 5.15 Route of synthesis of iphosphoramide mustard-dg....................... 283 XXI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. C H A P T E R 1 INTRODUCTION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1 S P E C IF IC A IM S This dissertation project focuses on the stereochemical metabolism and pharmacokinetics of ifosfamide (IF), an important alkylating anticancer agent. IF possesses an asymmetric phosphorus atom and is therefore a chiral drug. IF itself is inactive and requires metabolic activation to ilicit its antitumor activity. IF also undergoes extensive metabolic transformation, and these metabolic pathways may display stereoselectivity. The possible stereoselective metabolism provides the basis of a potentially stereoselective pharmacokinetic disposition, which may give rise to different pharmacological and toxicological effects of their enantiomers. This information once obtained may justify the use of the eutomer, an enantiomer with higher antitumor activity and less side effects in preference to the racemic mixture which is currently used in the clinic. To accomplish this goal, stable isotopically labeled pseudoracemates employing gas chromatography-mass spectrometry (GC/MS) methodology are selected for this investigation. At the same time, the labeled compounds may be used for identification of potential, new metabolites by ion cluster analysis. Thus, the specific aims of this study are; 1. to synthesize enantiomeric IF and strategically deuterium-labeled IF enantiomers as pseudoracemates; 2. to identify new metabolites of IF in the urine of rats receiving IF using ion cluster technique; 3. to synthesize known and new metabolites of IF for metabolic and pharmacokinetic studies including 4-hydroxy ifosfamide (4-OHIF), N2- dechloroethyl ifosfamide (N2D) and N3-dechloroethyl ifosfamide (N3D), 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. iphosphoramide mustard (IPM), alcoifosfamide (alcolF), 4-hydroxy N3- dechloroethyl ifosfamide (4-OHN3D), N-dechioroethyi iphosphoramide mustard (NDIPM), and N2,N3-didechloroethyl ifosfamide (N2N3D); 4. to study in vivo stereoselective metabolism of IF in the rat; 5. to synthesize deuterium-labeled IF and its metabolites as the internal standards for the GC/MS quantitation; 6. to establish quantitative GC/MS assay methods for IF and its metabolites in bioiogical fluids; 7. to investigate the stereoselective metaboiism and metaboiites pharmacokinetics in the rat. 1.2 ANTICANCER PROPERTY AND CHEMISTRY OF IF Cancer is defined as a group of diseases characterized by uncontrolled cellular growth with local tissue invasion or systemic spread of the disease or both. The standard modalities used in treatment of cancer include surgery, radiation, chemotherapy, and immunotherapy. Although surgery and radiation therapy may be effective in the treatment of localized tumors, they are of limited value in treatment of disseminated cancers. Unfortunately, many patients with cancers eventualiy develop metastasis. Therefore, the use of chemotherapy is necessary. To date, approximately 40 anticancer agents are commercially available, each of which can be used alone or in combination. Many of these agents are classified as alkylating agents. In fact, alkylating agents were the first group of drugs systematically developed against neoplastic diseases. A landmark in the 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. development of alkylating agents was a report in 1964 demonstrating that nitrogen mustard [NM, bis-(2-chloroethyl)-methyl-amine], the prototype of alkylating agents, produced an objective remission in a patient with malignant iymphoma (1 ). However, despite its significant activity, the agent was not widely accepted in the clinic due to its severe toxicity. Nevertheless, NM served as the lead compound in the subsequent development of several classical alkylating agents. I F [2-(2-chloroethyl)amino-3-(2-chloroethyl)tetrahydro-2H- f,3,2-oxazaphos- phorine 2-oxide], together with its structural isomer cyclophosphamide (CP, 2-[bis- (2-chloroethyl)amino]tetrahydro-2H-f,3,2-oxazaphosphorine 2-oxide), is a member of the second generation alkylating agents called oxazaphosphorines (Figure 1.1). They are the products developed from NM based on the principle of prodrugs, which are chemical entities requiring in vivo activation to exert their antitumor effects. In 1954, Friedman and Seligman (2) synthesized a group of N- phosphorylated nor-nitrogen mustard [NNM, bis-(2-chloroethyl)-amine] derivatives, including phosphoramide mustard (PM), with the intention of : 1) to reduce the reactivity of NM by attachment of an electron-withdrawing group on the nitrogen atom and 2) to produce selectivity in tumor cells based on Gomeh's assumption that there is higher phosphoramidase activity, an enzyme catalyzing the hydrolysis of the P-N bond, in certain tumor cells (3). The cleavage of the P-N bond in N-phosphorylated nor-nitrogen mustards would release NNM. Shortly after, Amold et al. synthesized a series of cyclic nitrogen mustard phosphamide esters classified as oxazaphosphorines, including CP (4,5,6). These drugs, designed as the latent form of NNM, are chemically stable and pharmacologically inactive. Although Gomeh's assumption was later proven to be questionable, CP was introduced in the clinic in 1960's and remains to be one of the most widely 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. used anticancer drugs today, interestingly, PM was later found to be the ultimate alkylating metabolite of CP. IF was later synthesized as an analog of CP by relocating one of 2-chloroethyl moieties on the exo nitrogen atom onto the endo nitrogen atom on the 1,3,2- oxazaphosphorine ring. This seemingly slight change brings significant differences in terms of their metabolism, spectrum of anticancer activity, and side effects (7). 1.3 METABOLISM OF IF Apparently due to stabilization by the oxazaphosphorine ring, IF and CP are not able to alkylate biological macromolecules directly. They undergo in vivo biotransformation to generate active species to exert their pharmacological effects. While metabolism of CP has been weil documented, that of IF has not been studied extensively. One reason was probably due to the structural similarity between IF and CP, so that one assumes that their metabolism are similar. While this is partially true, major difference in metabolism between IF and CP exists. The metabolic activation leading to the formation of the alkylating species IPM is similar to that of CP, which generates PM. It is generally believed that IF exerts its in vivo cytotoxicity by this metabolic route. IF is oxidized enzymatically to its in vitro active form 4-hydroxyifosfamide (4-OHIF). The enzymes catalyzing this reaction are mixed-function cytochrome P450 monooxygenases, and the liver is believed to be the major organ to carry out this reaction. Little work has been devoted to identify the isozymes for this crucial metabolic reaction until recently, possibly because of the complexity of the cytochrome P450 system. Wax et ai. (8) used 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chemical reagents and P450-form selective inhibitory antibody and found that the constitutively expressed P450 enzymes belonging to the subfamily 2C (forms 2C11 /2C6) contribute significantly to the activation of CP and IF in uninduced rat liver microsomes, while the phenobarbital-inducible P450 2B1 is a major catalyst of their activation in the phenobarbital-induced microsomes. They also demonstrated that dexamethasone-inducible family P450 3A also contributes significantly to the activation of IF but not CP. Similar work has been conducted in their laboratory using human liver microsomes (9). They found substantial individual variation (4-9 fold) in rate of hydroxylation of CP and IF by a panel of 12 human liver microsomes, and a significant correlation has been observed between the activation of these two drugs. It was revealed that 4-hydroxylation of these drugs was best fitted by a 2-component Michaelis-Menton model composed of low Km (0.031-0.133 for CP and 0.007-0.086 mM for IF) and high Km (3.2-7.0 mM for CP and 5.5-8.1 mM for IF). This study established that several human liver P450s can activate these oxazaphosphorines. CYP2B preferentially activates CP, while CYP3A is important to the activation of IF, and two other CYP2 enzymes may contribute to some of the observed inter-individual variations In metabolism and clinical pharmacokinetics of these anticancer drugs. Extrahepatic metabolism of IF and CP has not been explored; possible metabolism in the central nervous system is of interest since it has been found to possess appreciable cytochrome P450 activities (10). An understanding of the CNS metabolism of these compounds will provide some insights into the neurotoxicity observed in patients given IF. 4-OHIF produced by the aforementioned bioactivation readily isomerizes to its open-chain tautomer, aldoifosfamide (aldolF) and converts to its hydrates. The 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. existence of these species was demonstrated by Conners et al., who used mass spectrometer to identify the trapped compound, 4-ethyloxy ifosfamide in In vitro rat liver microsomal metabolic study (11). However, these species are very labile, and the in wVo characterization has been achieved indirectly by identification of their degradation products IPM and acrolein. Indeed, this has been the most common method to quantify ievels of these compounds in plasma and urine. For example, fluorometric determination of 4-OHiF/aldo-IF was developed based on the measurement of the generated acrolein by the reaction between acrolein and m-aminophenol. Allen at al. demonstrated that the in vitro rat microsomal metabolism of IF followed Michaelis-Menten kinetics. The Vmaxforthe activation of IF was the same as that of CP, 5.4 pmol/g of liver/hour; however, the Km for the activation of IF was 19.4 mM, almost five times of that for CP. The difference in Km values could be explained based on the structural difference of IF and CP. The author suggested that the bulky chloroethyl group on the endo nitrogen might prevent close association of this substrate with a binding site on the P450 enzyme surface (1 2 ). Carboxyphosphamide (carboxyCP) is the major metabolite of CP in vivo. However, its congener carboxy ifosf amide (carboxyl F) was found to be a minor component in the urine of patients receiving IF. Carboxyl F was also found in the urine of rabbits receiving IF subcutaneously along with two side-chain oxidation products and considerable amount of unchanged IF (13). When synthetic 4-OHIF was given to the rabbit subcutaneously, carboxyl F was found to be the principal urinary metabolite(13). In a separate study in the dog, IF was found to be metabolized rapidly. Only a small amount of unchanged IF appeared in the urine, along with carboxylF and 4-ketoifosfamide (4-ketolF) (14). CarboxyiF 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. presumably results from the oxidation of aldolF by aldehyde dehydrogenase, a polymorphic enzyme catalyzing the simiiar reaction for aidoCP (15). Aicoifosfamide (alcolF) was identified in blood of mice and in the urine of dogs receiving iF (16). The bio-transformations of 4-OHIF to the inactive metabolites 4- ketoiF and alcolF might reiate to the fate of IF in vivo, since these pathways may constitute a mechanism of detoxification. Iphosphoramide mustard (IPM), a metabolite of IF both in vitro and in vivo (16,17), is released by spontaneous p- elimination from aldolF. Acrolein, the causative factor of urotoxicity for oxazaphosphorines is the co-product of this reaction (18,19). In contrast to CP, the side-chain oxidation contributes a much greater extent to the metabolism of IF and several metabolites have been identified (13,16,20-24). The first step of the side-chain metabolism is cataiyzed by liver cytochrome P450 enzymes, which are probably different from the isozymes for the ring oxidation. Supports for this argument are observations that phénobarbital induced side-chain metabolism of IF but did not change the ring activation (12,25,26). The proposed products T and 1 "-hydroxy ifosfamide have not been detected in biological system due to their chemical instability; however, their subsequent degradation products have been detected or isoiated. Chloroacetaldehyde (CAA) is a common product from both of the side-chain oxidations, and is a potential metabolite responsible for the observed CNS toxicity in patients receiving IF (22). CAA has been directly detected in plasma of patients receiving IF (22), and isolated In urine in the forms of S-carboxymethylcysteine and thiodiacetic acid, known products of metabolism of chloroacetate (20). N3-dechloroethyl ifosfamide (N3D) and N2- dechloroethyi ifosfamide (N2D) were isolated from the in vitro microsomal Incubation. These metabolites were also detected in plasma and urine of animais 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and patients given IF. When N-dechioroethyiation of CP was compared with that of IF using rat liver microsomes (27), it was found that the maximal rate of N- dechloroethylation of IF is about three times that of CP (0.95 versus 0.38 nmol product/mg protein/min) in uninduced rat microsomes. Different patterns of inhibition on the metabolic activation pathways of IF and CP by SFK 525-A and tamoxifen were observed, suggesting the participation of different isozyme(s) in the dechloroethylation of these two oxazaphosphorines. Scheme 1 shows the current metabolic pathways of IF. 1.4 PHARMACOKINETICS OF IF The pharmacokinetics of IF has been investigated in man since the early 1970s. Allen etal. (28) found that the half-life of IF was schedule dependent. Following iv administration of large singe doses (3.8-5.0 g/m^), plasma pharmacokinetic data obtained were best fitted by a two-compartment open model which gave a terminal half-life of 16 hrs. When divided daily doses of IF (1.6-2.4 g/m2 ) were used, plasma pharmacokinetic data showed a one-compartment model behavior with a half-life of 6.9 hrs (28), resembling that of CP. After infusion of ■'^C IF at 5 g/m2 over 45 min in patients, the urinary recovery of radioactivity was 81.6%, of which 61.6% was the unchanged drug (29). Smaller divided doses of IF (1.6-2.4 g/m2 daily for three days) decreased the urinary excretion of the unchanged IF to 20% (28). A non-linear pharmacokinetic model including a term for Michaelis-Menten transformation revealed that IF had a large central-compartment volume, representing plasma and well-perfused organ tissues, while the metabolites had a small distribution space of 2 .1 liters and were excreted directly 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. into urine (29). Unchanged IF was found in cerebrospinal fluid (CSF) at 38% and 49% of plasma drug levels in 2 patients, but only negligible amounts of metabolites were detected (29). However, more resent results showed the definitive existence of 4-OHiF and aikyiating activity in CSF (30, 31 ). By using a fluorometric detection method, CSF 4-OHiF level in rhesus monkeys was determined as high as 13% of that in piasma, following an IF infusion of 1 g/m^. Subsequent studies using different doses and dose schedules showed a biexponential decay of the plasma level of IF, with a terminal half-life in the range of 4.1 -6.9 hrs. The total clearance was in the range of 2.7-4.7 l/h, and the volume of distribution 0.34-0.63 I/kg . The nonlinear kinetics has not been confirmed (32, 33, 34, 35, 36), however. Orai absorption of IF was rapid and peak piasma concentrations were attained within 1 hourfoliowing administration at 1 and 2 g/m^ oral doses (37). Linear kinetics was observed in patients receiving doses up to 5 g/m^ both oraily or intravenously (32). The bioavailability of oral and subcutaneous administrations was found nearly 1 0 0 % in several different studies (32,37,38). While the reproducibility of steady state plasma concentrations in the same subject couid be reached, the inter-individual differences in the plasma profile of IF were considerable, presumably due to the individual difference in the cytochrome P450 system (possibly pharmacogenetic difference). Continuous administration of IF decreases the terminal hale-iife of piasma IF level as a result of the auto-induction of this enzyme system (35,39,40). The effects of route and fractionation of dose on IF metabolism in patients have been the foci of recent investigations (34, 41, 42). It has been shown that there was no significant difference between the terminal half-life, area under curve and renal clearance of the parent drug IF with respect to the route of administration. 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, oral administration was shown to result in higher urinary levels of metaboiites of IF. The maximal plasma 4-OHIF levels in patients were 2.3 fold higher when IF was given orally than that when IF was given intravenously. Similar results were observed for the metabolite CAA following the same dose regimen (41). 1.5 MECHANISM OF ANTICANCER ACTION OF IF The antitumor activity of IF is manifested following metabolic activation. IPM is believed to be the ultimate alkylating species. Although the specific mechanism of action has not be reported in the literature, it is assumed that it may undergo transformation to form highly reactive aziridinium ions. These ions can then form covalent bonds with electron-rich sites on macromolecules, e.g. nucleic acids and proteins. It is known that certain positions on the DMA molecule are particularly susceptible to alkylation, e.g. the N7 position on guanine. Since IPM has two alkylating side chains, it can alkylate two adjacent nucleotide bases, forming cross links. This process of cross-linking is believed to be responsible for much of the cellular toxicity of IPM. There are three possible outcomes of alkylation. First, the DNA template or strand being replicated may be misread or mispaired during DNA synthesis. Second, cross-linking may prevent DNA strands from unwinding, therefore hindering the replication process. Third, alkylation may produce either singer- or double-strand breaks on the DNA. Any of these actions may result in the inhibition of DNA, RNA, or protein synthesis, leading to cell death. This type of action is cell cycle nonspecific and it has the greatest impact on rapidly dividing cells such as cancer cells. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-OHIF could also play an important role In these processes. Similar to PM, IPM is a highly polar compound. It is anticipated that this compound may have difficulty passing across lipoprotein membranes. On the other hand, Its precursor 4-OHIF, being a more lipophilic compound can readily penetrate the biological membranes and can therefore act as the transport form of IPM. Once this species enters cells, IPM may be liberated spontaneously to exert Its alkylation on macromolecules. The structural difference between CP and IF may also affect drug resistance. The spatial distance between two alkylating sites are different for these two drugs. The repair enzyme which is capable of mending damages caused by PM may have little activity to repair the cross-links caused by IPM. This offers an explanation to the observation that there is little cross-resistance between these two drugs. 1.6 CLINICAL PHARMACOLOGY OF IF IF was first under clinically evaluation as an anticancer drug in the 1970s, and now it is used as part of the first-line therapeutic regimens In approximately half of all children presented with newly diagnosed cancers in Germany (42). Comparing to CP, IF is less myelosuppressive and the dose-limiting toxicity is hemorrhagic cystitis. The urotoxicity associated with IF is greatly reduced by co-administration of uroprotectors, a class of organic thiols such as N-acetylcysteine and mesna (sodium mercaptoethyl sulfonate). It has been demonstrated that the concomitant administration of mesna did not change the metabolism and pharmacokinetics of IF (43). This is due to the rapid oxidation of mesna into dimesna once It enters the 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. biological system. When the dimer is excreted in the glomeruli of the kidneys, about one-third of it is retransformed into mesna by glutathione reductase. The free thiol-containing mesna then neutralizes the reactive urodamaging species acroiein by a simple Michael addition. Thus, the definitive role in modern chemotherapy of IF is established. The antitumor activity spectrum of IF differs from that of CP (44). In patients with relapsed or refractory disseminated nonseminomatous testicular cancer, a salvage regimen of IF/mesna, cisplatin and either etoposide or vinblastine produced complete response in approximately 25% of patients. As a component of both induction and salvage regimens, IF/mesna treatment has produced favorable response rates in small cell lung cancer, pediatric solid tumor, non- Hodgkin's and Hodgkin's lymphoma, and ovarian cancer, induction chemotherapy with IF and mesna has been encouraging in non-small cell lung cancer, adult soft- tissue sarcoma, and as neoadjuvant therapy in advanced cervical cancer. As salvage therapy, IF/mesna containing regimens have a palliative role in advanced breast cancer and advanced cervical cancer. Furthermore, IF/mesna can elicit responses in patients refractory to numerous other antineoplastic drugs, including its prototype CP (44), possibly because of different spectrum of drug resistance (45). 1.7 CHIRALITY: ITS IMPACT ON METABOLISM, PHARMACOKINETICS AND BIOLOGICAL EFFECTS Recent advances in chemical synthesis and analytical techniques prompted metabolic, pharmacokinetic, pharmacological, and toxicological investigations on 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. enantiomers of drugs which were used in a racemic form originally. It has now become clear that the enantiomers of a chiral drug should be regarded as two different chemical entities, since they may differ in pharmacoiogical action, potency, and pharmacokinetics (46). Furthermore, interactions between two enantiomers have also been demonstrated in some cases (47). IF, like other oxazaphosphorine nitrogen mustards, possesses a chiral phosphorus center (Figure 1.2) and requires metaboiic activation. As mentioned previously, IF undergoes extensive in vivo metabolism, most of the steps involve enzymic catalysis. These metabolic reactions may be stereoselective, resulting in difference in potency and pharmacokinetic behaviors. In addition, the side-chain oxidation which leads to the formation of the neurotoxic species CAA may also be stereoselective. In fact, metabolic, pharmacokinetic, and pharmacological studies on the enantiomers of IF have been the foci of recent investigations (48). Kusnierczyk et ai. (49) reported the results of the in vivo activities of the enantiomers of IF against transplanted mouse tumor models: LI 210 and P388 lymphoid leukemias, Lewis lung carcinoma, and mouse mammary carcinoma 160 MAC. A higher therapeutic index was observed in all three tumor models except LI 210 when (S)-(-)-IF was administrated. The toxicity of this enantiomer was also found to be higher. However, Blaschke et ai. (50) did not observe a significant difference among (R)- (-)- and (S)-(-)-IF and racemic IF in acute toxicity and activity against L5222 leukemia in rats. A recent study by Masurel et ai. (51 ) showed that there was no statistically significant difference between the efficacy of the enantiomers and racemic IF against childhood rhabdmyosarcoma (HxRh28) In xenografts in immune- 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. deprived female CBA/CaJ mice. Simiiar result was found in another toxicity study conducted in non-tumor-bearing female CBA/CaJ mice. Several studies investigating the metabolism of enantiomers of IF have been reported and stereoselectivity was noted. Misiura etal. (23) using 3ip n m r with a chiral shifting agent Eu(tfc)3 determined the composition of urinary metabolites from two patients receiving racemic IF. They found that unchanged IF was enriched in (+) while N2D in (-) enantiomer. The enrichment in (-i-)-IF was oniy 1.22 and 1.33, respectively, in these two patients; however, the amount of N2D produced from (S)-(-)-IF was 2.7 and 6.7-fold higher than those from (R)-(-i-)-IF, respectively. N3D, the most abundant urinary metabolite, was also produced stereoselectively. In one patient, the (R) isomer was slightly enriched (R/S ratio 1.22:1), but in another patient, the (S) isomer was enriched (S/R ratio 2.6:1). The opposite data between these two patients might result from different disease states of the patients. Blaschke et ai. (52) chromatographically resolved the enantiomers of ^H-labeled IF and gave the enantiomers intraperitoneally to two separate groups of female NMRI mice. Levels of the parent drug and its metabolite in both blood and urine were monitored. In blood, a higher peak concentration of IF was found in mice treated with (S)-(-)-IF than in those treated with the R isomer (S/R ratio 1.7:1). Interestingly, blood levels of IPM generated from the 8 enantiomer were not significantly different from those obtained from the R enantiomer, while peak level of carboxylF were higher in mice treated with (S)-(- )IF (S/R ratio 1.7:1). Blood levels of carboxylF were of the highest among those of the metabolites, followed by 4-OHIF and IPM. Low 4-KetolF levels were found in blood, and only formed from (S)-(-)-IF. This observation disputes the previous contention of rapid equilibrium between 4-OH-IF and aldo-IF. If 4-OHIF under 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. goes rapid interconversion with the open-chain tautomer aldolF, then the asymmetry of the phosphorus atom would be lost and 4-ketolF formation could not have been stereoselective. It was also found that urinary level of unchanged IF from mice treated with (R)-(-)-IF was higher compared with those from mice treated with S-lsomer (R/S ratio 1.3:1). However, no significant difference In the urinary levels of 4-OHIF and IPM was found In these groups of mice. SIde-chaIn metabolism showed significant stereoselectivity so that N2D was produced to a greater extent from (R)-(+)-lF (R/S ratio 1.9:1) and N3D was formed to a greater extent from (S)-(-)-IF (S/R ratio 3.3:1). Recently, Boos et al. (53) studied the cumulative urinary excretion of N2D and N3D In 14 children receiving IF In combination regimens with IF doses ranged from 400-3000 mg/m^. They found that the total cumulative urinary excretion of IF and Its Inactive dechloroethyl metabolites ranged from 27% to 50% of the total dose. Between 14% and 34% of the dose was detected as IF, 9% to 29% as N3D, and 2% to 8 % as N2D. In all patients, slightly higher (R)-(+)-IF than the S Isomer was excreted (53-61% of total IF) and N2D was enriched In (-) enantiomer. Different stereoselectivity was observed regarding the formation of N3D. (S)-N3D was higher In six of fourteen patients whereas the R Isomer was enriched In the other eight patients. In a separate study, Walner etal. (54) found a significantly higher clearance for (S)-(-)- IF (1 .94 l/h/m^) relative to the R Isomer (1.65 l/h/m^) In pediatric patients receiving racemic IF administered as a 15 mln Infusion at 1.6 g/m^ for five days. Masural etal. (51 ) separately Incubated racemic and enantiomeric IFs with mice hepatic microsomes obtained from normal female CBA/CaJ mice. They observed no statistically significant difference In the calculated kinetic parameters, Vmax and Km, for the production of aldolF and IPM. They also conducted a pharmacokinetic 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. study in mice, and concluded that there was no difference in biood clearance for the two enantiomers, both deciined with a haif iife of 6.9 min. Thus, it is obvious that there are stereoselective processes on the in vivo metaboiism, pharmacokinetics, and pharmacology of IF at least in some species. There is possibiy a species difference in stereoselectivity, which was aiso observed in the case of CP (55). Mice may not be a suitable model for stereoselective study since data from mice differed from that of human (51 ). Little metabolite pharmacokinetic information of IF has been reported and this information is important since IF metabolites are responsibie for the therapeutic effect and toxicity. According to the theory of stereopharmacokinetics (56), pharmacokinetic parameters may be classified according to three leveis of organization in the body and the hybrid character of parameters increases with the ievel of organization that they represent. Parameters such as total body clearance are of the highest degree of hybrid character and are the least sensitive parameters reflecting the stereoselectivity of moiecular processes, it vaiidates our endeavor to study the individual metabolite profiiesfoliowing IF administration. 1.8 STUDY STRATEGY Selecting a suitabie methodology is crucial to a well-planned study. There are several methods to investigate the stereoselective metaboiism and pharmacokinetics of a chiral drug. The conventional way to study differential metaboiism of enantiomers of a drug is to give the individual enantiomers separately to different subjects or the same subject in different occasions. This 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. method has shortcomings and the outcome often suffers from considerable inter individual variation and even intra-individual variation due to enzyme auto induction as in the case of oxazaphosphorines. In such a case, the result would be valid only when a large population of subject is used. Administration of a racemic mixture of a chiral drug followed by separation and detection of individual enantiomer by a chiral separation method {e.g. a chiral column) is another method for stereoselectivity study, such as the one used by Blaschke etal. (52). However, when many metabolites exist, total separation of the enantiomers of the parent drug and their metabolites would be difficult to achieve on a single column. Alternatively, enantiomeric drugs and their chiral metabolites could first be derivatized to form diastereomers prior to achiral column separation. But the similar problem would be encountered. These two methods also encounter a major drawback; these techniques cannot differentiate formation of 4-OHIF and IPM from the enantiomers of IF since the former is an achiral molecule, and the later equilibrates with its open-chain tautomer aldolF, which is also an achiral molecule. Phosphorus-31 NMR has been used to study the metabolism of IF and the stereoselective metabolism of the enantiomers of IF (23). This technique allows simultaneous identification and quantitation of the major chiral metabolites since they contain phosphorus in their structures. Differentiation of the enantiomers of the parent drug and its chiral metabolites is made possible by using a chiral shifting agent. Since ^tpNMR is capable of detecting phosphorus-containing species directly in unprocessed body fluid, the problems associated with extraction, recovery, and chemical derivatization are avoided. However, the sensitivity of this technique is intrinsically lower compared to many other detection 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. methods. Moreover, this method is also incapable of differentiating 4-OHIF/aidolF and IPM derived from the enantiomers of IF. Additionally, the extended period required for measurement also makes it difficult for detecting labile metabolites. A pseudoracemate (58) consists of a 1:1 mixture of a unlabeled enantiomer of a chiral drug and a stable isotopically labeled {e.g. deuterium and carbon-13) enantiomer with the opposite configuration, resembling the racemate of a chiral drug. The individual isomers only differ in mass, besides optical activity. The stable isotope labels on one of the enantiomers allow a chemical discrimination of the enantiomers without an elaborate resolution. Additionally, the labels can be used for the tracing of the enantiomers of parent drug and all of their derived metabolites, with a potential of finding new, labile, difficult-to Isolate, trace metabolites. This method also offers an unique advantage in analyzing achiral metabolites derived from a chiral drug by virtue of the labels. Moreover, using pseudoracemate in vivo, enantiomeric interaction if exists would be preserved. The structural elucidation, quantitation, and mass discrimination are all readily achievable by GC/MS. Meanwhile, the mass spectrometer detector assures the ultra sensitivity of the assay. This methodology has been successful in several studies, including warfarin (58) and CP (59,60). Thus, it is also chosen for our investigation. 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Oxazaphosphorines 4 3 C N 6 1 Cl rx rv '— N—\ \ — 0 \jH-v '— C l cyclophosphamide ifosfamide Figure 1 .1 Chemical strucutres of oxazaphosphorines. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o 0 ,a Cl :nh X I (R)-(+)-IFOSFAMIDE (S)-(-)-IFOSFAMIDE Figure 1.2 Chemical structures of enantiomeric ifosfamides. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ D O Q. C g Q. ■ D CD C/) C/) 8 ■ D CD 3. 3 " CD CD ■ D O Q. C a O 3 "O o CD Q. ■D CD C/) C/) — HO “ N o afcoW \ / / dehydrogenase A ^ 0 NHR 4-ketolF cytochrome cytochrome âifiUlE c c N o X O NHR C M ÿ O NH- c R > f O NHa HO -Cl ^O H ^R HN Q d d e h yd a \ / / dehïtJrogenase A ^ -O NHR carboxvlF U asrolgln H NHR HO— NHR 1£M r T / A O NHR M3fi aldolF alcpIP ( R = CHzCHzCI ) Scheme 1 .1 Metabolic pathways of ifosfamide. CHAPTER 2 DESIGN AND ASYMMETRIC SYNTHESIS OF UNLABELED AND STRATEGICALLY DEUTERIUM-LABELED ENANTIOMERS OF IFOSFAMIDE 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.1 A B S T R A C T IF, an oxazaphosphorine antineoplastic agent, possesses a chiral phosphorus center which may have influence on its metabolism. To investigate the potentially stereoselective metabolism of IF by pseudoracemate and mass spectrometric methodology, the synthesis of R(+)- and S(-)-6,6-dideuterio-3-(2- chloro-ethyl)-2-[(2-chloro-2,2-dideuterioethyl) amino] tetrahydro- f,3,2-oxazaphos- phorine 2-oxide and their unlabeled counterparts was designed. The labeled locations would minimize potential isotope effect involved in metabolism. An established synthetic approach by Pankiewicz et al. was used for the stereo synthesis using prepared deuterium-labeled synthons. Michael reaction of ethyl acrylate with R- or S-a-methylbenzyl amine afforded the ethyl N-(R)- or (S)-a- methylbenzyl-3-amino-propionate which, upon reduction by lithium aluminum deuteride, gave R- or S-N-a-methylbenzyl-2,2-dideuterio-3-amino-propan-1-ol. This labeled alcohol, when condensed with POCI3 followed by treatment with 2- chloro-2,2-dideuterioethylamine, yielded the diastereomeric mixture of 3-a- methylbenzyl-6,6-dideuterio-2-[(2-chloro-2,2-dideuterioethyi)amino]tetrahydro-f,3, 2-oxazaphosphorine 2-oxide. Resolution of the mixture followed by hydro- genolysis gave the unsubstituted oxazaphosphorine ring compound. Acylation with chloroacetyl chloride followed by hydroboration afforded the final deuterium- labeled IFenantiomer. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2 Introduction It has now become clear that the enantiomers of a chiral molecule may display major differences in metabolism, pharmacokinetics, and pharmacological effects (46). IF, a widely used alkylating agent and a structural isomer of another important drug CP, possesses an asymmetric phosphorus atom, therefore existing in enantiomers (4,5,6,45). The clinically used form of IF is a racemic mixture. IF itself is not cytotoxic but requires hepatic activation to exert both therapeutic and side effects (7,45). The foremost important metabolic pathway is 4-hydroxylation and generates 4-OHIF which may play an important role in the transport across cells (11-13). Subsequent ring opening and cleavage of this metabolite generates IPM which is considered to be the ultimate intracellular alkylating metabolite (16,17). Dealkylation on the side chains generates CAA which has been implicated in the involvement in neurotoxicity observed in patients receiving IF therapy (21,22). These various metabolic pathways may display stereoselectivity. To investigate this possibility we resort to the use of pseudoracemate coupled to GC/MS which has been considered to be an elegant methodology (58-60). To further probe into these various key metabolic pathways simultaneously and to monitor the pharmacokinetics of the enantiomers of IF and their metabolites while at the same time minimizing the kinetic isotope effects, synthesis of deuterium labeled enantiomers of IF with labels at the strategic positions was designed. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3 RATIONAL DESIGN OF DEUTERIUM-LABELED ENANTIOMERS OF IFOSFAMIDE SUITABLE FOR PSEUDORACEMATE EXPERIMENT Design of appropriate deuterium-labeled enantiomers for pseudoracemate experiment use is critical to obtain unbiased metabolic and pharmacokinetic results. Several aspects for designing these compounds must be considered (58). Firstly, the labeled compounds must possess physicochemical properties similar to the unlabeled molecules in order to have nearly Identical behaviors such as distribution, extraction, and derivatization. For that reason, minimal number of deuterium labels should be incorporated to the labeled molecules, yet still allowing adequate mass discrimination between the labeled and unlabeled compounds. Secondly, the labeled compounds should have very similar metabolic behavior with minimal metabolic isotope effect. Clearly, all known metabolic transformations should be taken into consideration when the label positions are selected. Thirdly, the labels should also be retained in the major metabolites so that these metabolites could be traced by mass spectre metric method. Finally, the designed compounds should be synthetically achievable with reasonable efforts. IF molecule contains two chlorine atoms, which give rise to a cluster of isotope peaks at M, M+2, and M+4 in an approximate ratio of 9:6:1 under chemical ionization mass spectrometry. Such cluster ions may interfere with ions from deuterium labeling. For example, compound labeled with 4 deuterium atoms will interfere with M+4 from 3 7c i2 alone. However, when IF was derivatized with silylating agent (MSTFA) and processed through GC/CIMS, the IF derivative was converted to a dehydrochiorinated cyclic compound and one chlorine was lost during that process. Therefore, in this case four deuterium atoms were adequate 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for the labeled enantiomer since the increase in mass unit was 4, which was 2 mass units higher than the s^CI isotopic ion of the unlabeled dehydrochiorinated IF. Seven carbon atoms on the molecule of IF could be potentially labeled with deuterium atoms (Figure 2.1). However, certain positions are not suitable for deuterium labeling because they are sites for metabolic transformation and possible isotope effect arisen may complicate data interpretation. Among them are C4, C l', C l", and C5, the first three involve in enzymatic hydroxylation and the last involves in the p-eliminationof4-OHIF/aldolFto form IPM (45). Therefore C6 is chosen as one of the labeling sites. The other site selected was C2' because of synthetic feasibility. Therefore, R(+)- and S(-)-6,6-dideuterio-3-(2-chioroethyl)- 2-[(2-chioro-2,2-dideuterioethyl)amino]tetrahydro-f,3,2 -oxazaphosphorine 2- oxide were designed for use in the pseudoracemate-mass spectrometric studies. As shown in Scheme 2.1, metabolites including 4-OHIF, alcolF, N2D, N3D and IPM could be traced by the deuterium labels. 2.4 Methods and Procedures All melting points were measured on a Kofler hot stage and are uncorrected. CH2 CI2 , THF, (CH2 C H2CI)2 were purchased from E. M. Science (Gibbstown, N.J.), distilled and dried by conventional methods before use. All chemicals were purchased from Aldrich Chemical Incorporation (Milwaukee, Wl). Mass spectra were obtained on a Hewlett-Packard 5985A quadrupoie mass spectrometer under chemical ionization (Cl) mode using ammonia as the reagent gas. 1HNMR spectra were recorded with a Bruker NR-250 FT NMR spectrometer. Optical activity measurements were made with a Perkin-Elmer 241 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. photopolarimeter. Silica gei (70-230 mesh) was used for open column chromatography and all TLC were performed on silica gel plates. 2.4.1 Synthesis of S-lfosfamide and R-lfosfamide S-IF and R-IF were synthesized using S- and R- (-)-a-methylbenzylamine as the respective starting materials (Scheme 2.2 and 2,3). Ethyl N-(S)'a-methylbenzyi-3-amino-proplonate and Its R enantiomer. To a solution of ethyl acrylate (11.65 g, 116.5 mmol) in absolute ethyl alcohol (50 ml) was added a solution of S-(-)-a-methylbenzylamine (14.10g, 116.5 mmol) in the same solvent (50 ml) at room temperature. The mixture was refluxed for 24 hrs. Ethanol was then removed by rotary evaporation under reduced pressure. The remaining oily residue (22.62 g) was distilled under vacuum (136°C/0.65 mm Hg, oil bath 150-160°C) to give the product as a colorless liquid (15.88 g, 61.7%); Rf 0.72 (ethyl acetate): NMR (CDCI3) ô 1.25 (t, J=6 . 8 Hz, 3H, -O-CHp-CH.g). 1.34 (d, J=6 . 6 Hz, 3H, -CH-ÇH3), 1.72 (bs, 1H, -NH-), 2.46(t, J=6.5 Hz, -C(0)- ÇH2-), 2.70 (m, 2 H, -NH-ÇH2-), 3.77 (q, J=6 . 6 Hz, 1 H, -ÇH-CH3), 4.13 (q, J=6 . 8 Hz, ) -O-ÇH2-CH3), 7.22-7.40 (m, 5H, 5 x -Ph-H); MS (Cl) m/z2 2 2 (MH+). Similarly, starting with R-(+)-a-methyl benzylamine, ethyl N-(R)-a- methylbenzyl-3-amino-propionate was obtained in 63.4 % yieid, with same TLC and NMR data. N-(S)-a-methyibenzyl-3"amino-propan-1-ol and its R enantiomer. To a cooled (-78°C) suspension of lithium aluminum hydride (1.20 g, 31.67 mmol) in THF (50 ml) was added dropwise a solution of ethyl N-(S)-a-methylbenzyl-3- amino-propionate (7.00 g, 31.67 mmol) in THF (50 ml). The reaction mixture was brought to ambient temperature (22°C) and the stirring was continued for 1 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. additional hr. Thereafter, 10 ml of water was added to decompose the excessive hydride. The precipitate was removed by filtration and washed with THF (3 x 50 ml). The filtrate and washings were combined and the solvent was removed In vacuo to give the product as a pale yellow liquid (5.63 g, 99.3%); Rf 0.13 (ethyl acetate); NMR (CDCI3) 81.36 (d, J=6 . 6 Hz, -CH-ÇH3 ). 1.40-1.92 (m, 3H, -NH-, -CH 2-ÇH 2 -CH 2 -), 2.62-2.83 (m, 2H, -NH-CH?-). 3.63-3.92 (m, 4H, -OH, -ÇH- CH3 . -ÇH2 -OH), 7.23-7.42 (m, 5H, 5 x -Ph-H); MS (Cl) m/z 180 (MH+). This product was used directly for the subsequent stage without further purification. Similarly, starting with ethyl N-(R)-a-methylbenzy!-3-amino-propionate, N- (R)-a-methylbenzyl-3-amlno-propan-1-ol was obtained in 99.5%, with same TLC, NMR, and MS data. (S)-{2 -chloroethyl)am ino-3 -[(S)-a-methylben2 yl]tetratiydro-Y,3 > 2 -oxa- zaphosphorine 2-oxide and its R enantiomer. To a cooled (-78°C) solution of N-[(S)-a-methylbenzyl]-3-amino-propan-1-ol (5.63 g, 31.45 mmol) and triethyiamine (6.35 g, 62.90 mmol) in CH2 CI2 (50 mi) was added dropwise oxyphosphorus chloride (4.26 g, 31.45 mmol) In CH2CI2 (20 ml). The reaction mixture was stirred for 1 hr at -78°C, and then for another hr at ambient temperature. The progress of reaction was monitored by TLC and MS. Once the starting material disappeared, chloroethylamlne hydrochloride (3.8 g, 33 mmol) was added to the reaction mixture. While maintaining at 0°C, to the reaction mixture was added dropwise a solution of triethyiamine (6 . 6 g, 6 6 mmol) in 2 0 ml of C H2CI2 . The reaction mixture was stirred at room temperature for 1 hr, then it was heated to reflux for 20 hrs. Triethyiamine hydrochloride formed was removed by filtration and the filtrate was washed with distilled water (50 ml x 3). The organic phase was dried over anhydrous sodium sulfate for 5 hrs. After filtration, the 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. organic solvent in the filtrate was removed in vacuo. The residue was crystallized in acetone. Colorless crystalline product (2.23 g) was obtained: m.p. 115-117°C; RfO.62 (CH2 Cl2 -acetone 1:1); NMR (CDGI3) ô 1.53 (d, J=6 .6 , -CH-ÇH3 ), 1.60- 1.75 (m, 2H, G5-H), 2.71-2.88 (m, 1 H. NH), 2.91-3.12 (m, 2H, G4-H), 3.25-3.42 (m, 2H, -ÇH2 -GH2 -GI), 3.67 (t. J=5.7 Hz, -CH,-GH,-Gh. 4.08-4.33 (m, 2H, G6 - H), 4.85-5.02 (m, 1 H, -ÇH-GH3), 7.20-7.58 (m, 5H. Ph-H); MS (Gl) m/z 303 (MH+). The mother liquor was concentrated and loaded onto a silica gel column ( 1 0 0 g) and eluted with GH2 Cl2 -acetone (1 0 :1 ) to give second crop of product (1.17 g). The overall yield from the amino alcohol was 46.3%. In an analogous manner, (R)-(2-chloroethyl)amino-3-[(R)-a-methylbenzyl]- tetrahydro-1,3,2-oxazaphosphorine 2-oxide was obtained (47.5%): m.p. 115- 116°G with spectroscopic data identical to those of the 8 8 enantiomer. (R)-2-(2-ch!oroethyl)aminotetrahydro-t,3,2-oxazaphosphorine 2 -oxide (R-N3D) and its S enantiom er (S-N3D). (S)-(2-chloroethyl)amino-3-[(S)-a- methylbenzyl]tetrahydro-1,3,2-oxazaphosphorine 2-oxide (4.73 g, 15.66 mmol) was dissolved in GH2GI2 (50 ml). After addition of freshly hexane washed 1 0 % palladium on activated charcoal (1 .0 g), the mixture was hydrogenated at room temperature for 20 hrs under normal pressure. The progress of reaction was followed by TLG. When the reaction completed, the catalyst was removed by filtration. GH2 GI2 was removed by evaporation under reduced pressure. The residue (2.74 g) was purified by recrystallization in acetone to give R-N3D as colorless needles (2.30 g). The mother liquor was subjected to silica gel column chromatography with CH2Gl2 :ethanol (2 2 :1 ) as the eluant to give an additional amount of the product (0.25 g). The overall yield was 82.2%: m.p. 108-110°G; Rf 0.18 (GH2 Gl2-acetone 1:3); NMR (acetone-de) d 1.60-1.88 (m, 2 H, G5-H), 3.12- 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.32 (m, 4H, C4-H, 3.62 (t, J=6 . 6 Hz, C2'-H). 3.68-4.18 (m. 2H, 2 x NH), 4.18-4.28 (m, 2H, C5-H); MS m /z^Q9 (MH+). H ydrogenolysis of (R)-(2-chloroethyl)amino-3-[(R)-a-methylbenzyl]tetra- hydro-7,3,2-oxazaphosphorine 2-oxide was performed similarly and S-N3D (81.2%, m.p. 108-111°C) was obtained with spectroscopic data identical to those of the S enantiomer. (S)-(2-chloroethylamino)-3-chloroacetyltetrahydro-f,3,2-oxazaphos- phorlne 2-oxide and Its R enantiomer. To a solution of R-N3D (2.50 g, 12.6 mmol) in THF (25 ml) was added dropwise chloroacetyl chloride (1.41 g, 12.6 mmol) at -78°C. The reaction mixture was stirred for 1 hr. The temperature was raised to room temperature and the stirring continued for 10 additional hrs. The acétylation was followed by TLC. When the starting material disappeared, the solvent was evaporated. The residue was chromatographed on a short silica gel column (20 g) with CH2 Cl2 -acetone as the eluant. The product (2.29 g, 66.3%) was obtained as a clean thick oil which solidified under storage in a freezer. Crystallization in acetone-ethyl ether afforded colorless crystals: m.p. 85-86°C; Rf 0.72 (CH2 Cl2 -acetone 1:1); NMR (CDCI3) Ô 1.92-2.30 (m, 2H, C5-H), 3.25-3.40 (m, 3H, C4-H, CT-H), 3.59-3.80 (m, 3H, NH, C2'-H), 4.24-4.56 (m, 3H, C4-H, C6 -H), 4.52 (d, J=14.4 Hz, C2"-H), 4.71 (d, J=14.4 Hz, C2"-H); MS (Cl) m/z 275 (MH+). In a similar manner, (R)-(2-chloroethylamino)-3-chloroacetyltetrahydro-1,3,2- oxazaphosphorine 2-oxide was obtained: m.p. 85-87°C with spectroscopic data identical to those of the 8 enantiomer. (S)-2-(2-chioroethyl)amino-3-(2-chloroethyl)tetrahydro-1,3,2-oxaza- phosphorine 2-oxide (S-IF) and its enantiom er (R-iF). To a cooled (-78°C) 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. solution of (S)-(2-chloroethylamino)-3-chioroacetyltetrahydro- 7,3,2-oxazaphos- phorine 2-oxlde (2.29 g, 8.36 mmoi) in THF (50 ml) was added 1M solution of diborane in THF (33.44 ml). The reaction mixture was stirred at ambient temperature for 1 hr. After the reduction was complete, 5 ml of water was added. The organic solvent in the mixture was removed In vacuo and CH2CI2 (50 ml) was added. The content was washed with water ( 1 0 m ix 3). The organic phase was combined and dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed In vacuo. The crude material (3.25 g) was purified by silica gel chromatography with CH2Cl2 :acetone:methanol (80:6:1) as the eluant. S-IF (1.17 g) was obtained in 53.9 % yield: m.p. 64-65°C (ethyl ether); Rf 0.33 (CH2Cl2 -acetone 1 :1 ); NMR (CDCI3) ô 1.86-2.05 (m, 2 H, C5-H), 3.12-3.70 m , 11H, NH, C4-H, 2x -CH2 CH2CI), 4.18-4.45 (m, 2H, C6 -H); MS (Cl) m/z 261 (MH+); [a]25p -38.9° (c 0.42 MeOH). R-IF was obtained in similar yield from (R)-(2-chloroethylamino)-3- chloroacetyltetrahydro-f,3,2-oxazaphosphorine 2-oxide: m.p. 64-65°C; +39.0° (c 0.45, MeOH) with spectroscopic data identical to those of S-IF. 2.4.2 Synthesis of 2 -chloro-2 ,2 -dideuterio-ethylamfne hydrochloride The synthetic scheme of 2-chloro-2,2-dideuterio-ethy!amine hydrochloride is shown in Scheme 2.4. Ethyl N-triphenylmethyl glycine ester. To a suspension of ethyl glycine ester hydrochloride (15.00 g, 107.53 mmol) and triphenylmethyl chloride (30.00 g, 107.53 mmol) inCH 2 Cl2 (200 ml) was added triethyiamine (21.61g, 215.06 mmol) in 50 ml CH2 CI2 at 0°C. After the completion of addition, the mixture was heated to reflux for 20 hrs. The precipitated triethyiamine hydrochloride was removed by 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. filtration and ttie filtrate was washed with water (50 ml x 3). The organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent In the filtrate was evaporated In vacuo to give the crude product (37.00 g, 99.7%), which was crystallized in CH2 Cl2-acetone (1:1) to give fine cubic crystals ( 35.20g): m.p. 100-105°C (softening): Rf 0.58 (CH2 CI2 ); NMR (CDCI3 ) ô 1.78 (t, J=7.2 Hz, 3H, -CH 3 ), 2 . 4 5 (bs, NH), 4.05 (q, J=7.2 Hz, 2H, -CH2 -). 7.15-7.50 (m, 15H, Ph-H); MS (Cl) m/z346 (MH+). N-triphenylmethyl 2,2-dideuterio-ethanolamine. To a cooled (-78°C) suspension of lithium aiuminum deuteride (3.04 g, 72.40 mol) in THF (50 ml), was added slowly a solution of ethyl N-triphenylmethyl glycine ester (50.00 g, 144.80 mmoi) in THF (100 ml). The temperature was then raised to room temperature whiie stirring was maintained for 2 hrs. Water (10 ml) was added to destroy the remaining hydride. The slurry material was extracted with THF (50 ml x 3). The organic extract was dried over anhydrous sodium sulfate for 2 hrs. After filtration, the solvent in the filtrate was removed in vacuo to give the crude product (46.41 g, 100 %): m.p. 53-60°C (softening); Rf 0.06 (CH2 CI2 ): NMR (CDCI3 ) d 1 . 6 8 (bs, 2H, -OH, -NH-), 2.34 (s, 2 H, -CH2-), 7.14-7.48 (m, 15H, -Ph-H); MS (Cl) m/z 306. This product was used for the subsequent stage without further purification. 2,2-Dideuterio-ethanolamine hydrochloride. To a solution of N-trityl-2,2- dideuterio-ethanolamine (46.41 g, 152.16 mmoi) in methanol (200 ml) was added 37% hydrochloric acid (12 ml). The mixture was refluxed for20 hrs. Methanol was removed by rotary evaporation. Water (50 ml) was added to the residue, and the lipophilic by-products were removed with CH2 CI2 (50 mi x 3) extraction. The resulting aqueous solution was treated with activated charcoal. After filtration, the solvent in the filtrate was evaporated In vacuo to give the crude alcohol amine salt. 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The product was recrystallized In ethyl alcohol to give colorless crystals (14.78 g, 97.6%): m.p. 82-84°C (lit. 84-86°C for unlabeled compound): Rf 0.09 (acetone- methanol 1:1); MS (Cl) m/z 64 (MH+of free amine). 2-Chloro-2,2-dideuterio-ethylamine hydrochloride. To a cooled (0°C) suspension of 2,2-dideuterio-ethanolamine hydrochloride (7.45 g, 75.63 mmol) in 1,2-dichloroethane (50 ml) was added dropwise thionyl chloride (18.00 g, 151.27 mol). After the completion of addition, the reaction mixture was stirred at room temperature for 0.5 hr, and then it was heated gradually to 50-60°C. The chlorination was monitored by TLC. The precipitated product (8.82 g, 99.7%) was collected by filtration and washed with CH2CI2 and acetone. Recrystallization in ethyl alcohol gave colorless crystals: m.p. 141-143°C (lit. 143- 146°Cfor unlabeled compound); Rf 0.58 (acetone-methanol 1:1); MS m/z82 (MH+ of the free amine). 2.4.3 Synthesis of S-IF-d# and R-IF-d^ The synthetic schemes of S-IP-d4 and R-IF-d4 are shown in Scheme 2.5 and 2.6. N-(S)-a-Methylbenzyl-3-amino-1,1-dideuterio-propan-1-ol and its R enantiomer. To a cooled (-78°C) suspension of lithium aluminum deuteride (0.76 g, 18.10 mmoi) in THF (50 ml) was added dropwise a solution of ethyl N-(S)-a- methylbenzyl-3-amino-propionate (4.00 g, 18.10 mmol) in THF (10 ml). The temperature of the reaction mixture was then raised to ambient temperature (22°C ) and the stirring was continued for 1 hr. Thereafter, water (0.8 g) in THF (50 ml) was added to decompose the excessive hydride. Precipitated solid was removed by filtration and washed with THF (50 ml x 3). The filtrate and washings were 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. combined and concentrated in vacuo to give N-(S)-a-methylbenzyl-3-amino-1,1 - dideuterio-propan-1-ol as a pale yellow liquid (3.19 g, 97.6%): Rf 0.13 (ettiyl acetate): NMR (CDCI3) ô 1.36 (d, J=6 . 6 Hz, -CH-ÇH3), 1.40-1.92 (m, 3H, -NH-, -CH9-CH9-CH9-). 2.60-2.85 (m, 2H, -NH-CH9-). 3.73 (q, J=6 . 6 Hz, -ÇH-CH3), 7.20-7.42 (m, 5H, 5 X -Ph-H); MS (Cl) m/z ^ 80 (MH+). The product was used directly in the subsequent stage without further purification. Starting with ethyl N-(R)-a-methylbenzyl-3-amino-propionate, and in an analogous procedure, N-(R)-a-methylbenzyl-3-amino-1,1-dideuterio-propan-1-ol was obtained in 99.1%, with spectroscopic data identical to those of the S- enantiomer. (S)-(2-chloro-2,2-dideuterio-ethyl)amino-3-[(S)-a-methylbenzyl]-6,6- dideuterio-tetrahydro-f,3,2-oxazaphosphorlne 2-oxide and its RR enantiomer. To a cooled (-78°C) solution of N-(S)-a-methylbenzyl-3-amino-1,1- dideuterio-propan-1 -ol (3.19 g, 17.62 mmol) and triethyiamine (3.56 g, 35.24 mmol) in CH2 CI2 (50 ml) was added dropwise a solution of oxyphosphorus chloride (2.70 g, 17.62 mmol) in CH2CI2 (20 ml). The reaction mixture was stirred for 1 hr at -78°C, and then another hr at room temperature. The reaction was monitored by TLC. When the starting material disappeared, 2-chloro-2,2-dideuterio-ethylamine hydrochloride (2.08 g, 17.62 mmol) was added to the reaction mixture, then triethyiamine (3.56 g, 35.24 mmol) in 20 ml of CH2 CI2 was added dropwise. Stirring was continued at room temperature for 1 hr, then the reaction mixture was heated to reflux for 24 hrs. Triethyiamine hydrochloride formed was removed by suction filtration and the filtrate was washed with distilled water (20 ml x 3). The organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was evaporated In vacuo. The residue (6.01 g) was purified 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by silica gel chromatography with CH2 Cl2 -acetone as the eluant to give (S)-(2- chloro-2,2-dideuterio-ethyl)amino-3-[(S)-a-methylbenzyl]-6,6-clideuterio-tetrahydro - 7,3,2-oxazaphosphorine 2-oxide as colorless crystals (2.16 g, 40.1%): m.p. 114- 116°C; Rf 0.62 (CH2 Cl2 -acetone 1:1): NMR (CDCb) 61.53 (d, J=6 .6 , -CH-ÇH3 ), 1.61-1.80 (m,2H, C5-H), 2.69-2.85 (m, 1H, NH), 2.90-3.10 (m, 2H, C4-H), 3.20- 3.40(m,2H, -ÇH2 -CH2 -CI). 4.87-5.02 (m, IN . -ÇH-CH3 ), 7.22-7.58 (m, 5H, Ph- H); MS (Cl) m/z307 (MH+). In an analogous manner, (R)-(2-chloro-2,2-dideuterio-ethyl)amino-3-[(R)-a- methylbenzyl]-6 ,6 -dideuterio-tetrahydro- 7,3,2-oxazaphosphorine 2-oxide was prepared similarly (43.5%): m.p. 113-116°C; with spectroscopic data identical to those of the 8 8 enantiomer. (R)-2-(2-Chloro-2,2-dideuterio-ethyl)amlno-5,5-dideuterio-tetrahydro- 7,3,2-oxazaphosphorlne 2-oxlde (R-N3 D-d4 ) and its S enantiomer (S-N3D- d4 ). (8)-(2-Chloro-2,2-dideuterio-ethyl)amino-3-[(8)-a-methylbenzyl]-6,6-dideu- terlotetrahydro-7,3,2-oxazaphosphorine 2-oxide (2.16 g, 7.06 mmol) was dissolved in GH2CI2 (50 ml). After addition of freshly hexane washed 10% palladium on activated charcoal (1 . 0 g), the solution was hydrogenated at room temperature for 48 hrs under normal pressure. The progress of reaction was followed by TLC. When the reaction was completed, the catalyst was removed by filtration, the solvent was removed in vacuo. The residue (1.25 g) was purified by crystallization in acetone to give R-N3 D-d4 as colorless needles (0.98 g), the mother liquor was concentrated and chromatographed on a silica gel column with C h 2Cl2-acetone (4:1) as the eluant to give an additional amount of the product (0.20 g). R-N3 D-d4 was obtained in 75.5% yield: m.p. 107-109°C; Rf 0.18 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (CHaCla-acetone 1:3); NMR (acetone-de) ô 1.58-1.85 (m, 2H, C5-H), 3.10-3.28 (m, 4H, C4-H, CT-H), 3.60-4.08 (m, 2H, 2x NH); MS m/z203 (MH+). Hydrogenolysis of (R)-(2-chloro-2,2-dideuterio-ethyi)amlno-3-[(R)-a-methyi- benzyl]-6,6-dideuterio-tetrahydro-1,3,2-oxazaphosphorine 2-oxide was performed similarly and 2(S)-2-(2-cfiloro-2,2-dideuterio-ethyl)amino-5,5-dideuterio-tetrahydro- 7,3,2-oxazaphosphorine 2 -oxide (80.2%, m.p. 107-108°C) was obtained. The spectroscopic data were identical to those of R-N3 D-d4 . (S)-{2"Chloro-2,2-dideuter[o-ethylamino)-3-chloroacetyl-6,6-dideuterio- tetrahydro-1,3,2-oxazaphosphorine 2-oxide and its R enantiomer. To a solution of R-N3D-d4 (0.93 g, 4.60 mmol) in THF (20 ml), was added dropwise chloroacetyl chloride (0.52 g, 4.60 mmol) at 0°C. The reaction mixture was stirred for 5 hrs at room temperature. The acétylation was followed by TLC. When the starting material disappeared, THF was evaporated. The residue was redissolved in 20 ml of CH2 CI2 and washed with distilled water (10 ml x 2). The organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed in vacuo. The residue was purified on a short silica gel column with C H2 Cl2-acetone (4:1 ) as the eluant to afford the product as colorless crystals(1.05 g, 82.3%): m.p. 84-86°C (acetone-ethyl ether); Rf 0.72 (CH2 Cl2-acetone 1:1); NMR (CDCI3) Ô 1.90-2.20 (m, 2H C5-H), 3.25-3.39 (m, 3H, C4-H, CT-H), 3.40- 3.70 (m, 1H, NH), 4.42-4.58 (m, 1H, C4-H), 4.50 (d, J=14.4 Hz, C2"-H), 4.71 (d, J=14.4 Hz, C2"-H); MS (Cl) m/z279(MH+). Sim ilarly, (R)-(2-chloro-2,2-dideuterio-ethylamino)-3-chloroacetyl-6,6-dldeu- teriotetrahydro-7,3,2-oxazaphosphorine 2-oxide was obtained: m.p. 85-86°C. The spectroscopic data were identical to those of the S enantiomer. 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (S)-2-{2-chloro-2,2-dideuterjo-ethyl)amino-3-(2-chloroethyi)-6,6- d id e u te rio -te tra h y d ro -3,2-oxazaphosphorine 2-oxide (S-IF-d 4 ) and its R enantiom er (R-IF-d4 ). To a cooled (-78°C) solution of (S)-(2-chloro-2,2- dideuterio-ethylamino)-3-chloroacetyl-6,6-dideuterio-tetrahydro-7,3,2-oxazaphos- phorine 2-oxide (1.00 g, 3.60 mmol) in THF (10 ml) was added a 1M solution of diborane in THF (14 ml, 4:1 excess). Ttie reaction mixture was stirred at ambient temperature for 1 fir. After tfie reduction was completed (TLC), 1.5 ml of distilled water in 10 ml of THF was added to decompose tfie remaining diborane. The solvent in the mixture was evaporated in vacuo and the residue was dissolved in CHgClg. After washing with water (10 ml), the organic phase was dried over anhydrous sodium sulfate and concentrated. The crude material (1.12 g) was purified by silica gel chromatography with CH2 Cl2 ’acetone:methanol (80:6:1 ) as the eluant. S-IF-d4 (0.58 g) was obtained in 61.1 % yield: m.p. 63-64°C (ethyl ether): Rf 0.33 (CH2 Cl2 -acetone 1:1); NMR (CDCI3 ) ô 1.86-2.05 (m, 2H, C5-H), 3.10-3.60 m, 7H, NH, C4-H, Cl'-H, CT'-H), 3.66 (t, J=6.4 Hz, C"-H); MS (Cl) m/z265 (MH+); [ajZSg-ss.So (c 0.35, MeOH). R-IF-d4 was obtained in a simiiar yield from 2(R)-(2-chloro-2,2-dideuterio- ethylamino)-3-chloroacetyl-6,6-dideuterio-tetrahydro-7,3,2-oxazaphos-phorine 2- oxide: m.p. 63-64°C; +38.9° (c 0.5. MeOH) with spectroscopic data identical to those of S-IP-d4 . 2.5 RESULTS AND DISCUSSION The enantiomers of IF could be obtained in two different ways. The resolution of commercially available racemate was achieved by Blaschke et al. (61 ) using 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. liquid chromatography with a chiral stationary phase. Racemic IF has also been resolved by forming diastereomeric Pt(ll) complexes which may be separated and then converted back to R- and S-IF (62). Since the recemate of labeled IF is not avaiiable commerciaily, we resorted to stereospecific synthesis of the iabeled and unlabeled enantiomers of IF. The stereospecific synthesis of the enantiomeric IFs was first reported in 1978 foilowing that of CP by Kinas et al. (62). Using the enantiomers of a- methylbenzylamine as the resolving agents, they initially attempted using a synthetic route based upon the column separation of two diastereomers with the introduced resolving group residing on the exocyciic nitrogen of IF molecule. However, the separation of these diastereomers and especially the subsequent hydrogenation removal of the resolving group were found very difficult and synthesis of these enantiomers in preparative scale was almost Impossible (63,64). Pankiewicz et al. developed an alternative approach basis on the preparation, separation and hydrogenation of diastereomers with the resolving group residing on the endocyclic nitrogen (65). In their approach, N-(S)-a- or N- (R)-a-Methylbenzyl-3-amino-propan-1-ol was firstly obtained by the reaction of (R)-a- or (S)-a-methylbenzylamine with 3-chloroethyl-propan-1-ol. Reaction of this amino alcohol with phosphoryl chloride afforded a mixture of diastereomers of 2-chloro-3-[(R)- and (S)-a-methylbenzyl]tetrahydro-f,3,2-oxazaphosphorine 2 - oxide in a ratio of 4:1. After condensed with aziridine (ethylenimine), this mixture gave a mixture of phosphoroethylenimides in the same ratio. The predominant diastereomer (SS or RR) could be crystallized and separated. The mother liquor was treated with anhydrous hydrogen chloride to give a mixture of diastereomers of 2 -chloroethyl derivatives, which could be separated by silica column 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chromatography. The availability of (S)-2-(2-chloroethyl)-3-[(S)-a-methyi- benzyi]tetrahyc!ro-f,3,2-oxazaphosphorine 2 -oxide and (R)-2-(2-chloroethyl)-3- [(R)-a-methylbenzyi]tetrahydro-f,3,2-oxazaphosphorine 2 -oxides was the key to the asymmetric synthesis. Hydrogenolysis of these compounds in ethanol for 2-4 days readily removed the methylbenzyl group on the endocyclic nitrogen and provided the enantiom ers of 2-(2-chloroethyl)amino-tetrahydro-2H-f,3,2- oxazaphosphorine 2-oxide with a yield of 73%. Upon acylation with 2- chloroacetyl chloride and subsequent reduction with diborane in THF, these enantiomers gave the enantiomeric IFs. The absolute configuration of the enantiomers of IF has been established by means of both stereochemical correlation with the enantiomers of cyclophosphamide and direct X-ray crystallography (65). In order to adapt these synthetic routes to the present synthesis of labeled enantiomeric IFs, several modifications were made. The use of 2-chloroethylamine instead of ethylenimine was preferred since it was easier to synthesize the labeled synthon of 2 -chloro-2 ,2 -dideuterioethylamine than that of 2 ,2 - dideuterioethylenimine. Labeled N-(R)- and (S)-a-methylbenzyl-3-amino-propan- 1 -ol was also synthesized with two deuterium atoms incorporated into appropriate positions of the molecule. In our modified procedures for the synthesis of (S)-IF, (S)-a- methylbenzylamine was used as the resolving agent. Michael addition of this amine with ethyl acrylate in methyl alcohol under reflux condition for 2 days led to the formation of ethyl N-(S)-a-methylbenzyl-3-amino-propionate, which was purified by vacuum distillation. This compound served as the common precursor for both the unlabeled and labeled S enantiomer of IF. Reduction of methyl N-(S)- 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a-methylbenzyl-3-amino-propionate with lithium aluminum hydride (LAH) in THF gave the corresponding alcohol, N-(S)-a-methylbenzyl-3-amino-propan-1-ol, which was used for the subsequent stage without further purification. The reduction was carried out in anhydrous THF at room temperature. No major side product was detected by TLC and MS. Condensation of N-(S)-a-methylbenzyl- 3-amino-propan-1-ol with oxyphosphorus chloride formed the oxazaphosphorine frame with a mixture of diastereomers of (8 )- and (R)-2-chloro-3-[(S)-a- methylbenzyl]tetrahydro-2H-f,3,2-oxazaphosphorine 2-oxide. The reaction was carried out in dichloromethane at low temperature (-78°C), and it was found that low temperature favored the stereoselective formation of the desired 8 8 diastereomer. 2-Chloroethylamine hydrochloride was added directly to the above reaction mixture to give the diastereomeric mixture, (8 )- and (R)-2 - (chloroethylamino)-3-[(8)-a-methylbenzyl]tetrahydro-2H-f,3,2-oxazaphosphorine 2-oxide. The predominant diastereomer ( 8 8 form) appeared in solid state and was separated from the other diastereomer (8 R form) by silica gei open column chromatography. The 8 R diastereomer appeared in liquid form, which was found difficult to purify and was therefore discarded. The removal of the a-methylbenzyl group from (8)-2-(chloroethylamino)-3-[(8)-a-methyl-benzyl]tetrahydro-7,3,2- oxazaphosphorine 2-oxide by hydrogenation to form R enantiomer of 2-(2- chloroethyiamino)tetrahydro-7,3,2 oxazaphosphorine 2-oxide was carried out in dichloromethane. It was noticed that degradation products were detected when ethyl alcohol was used as according to the literature procedure (65). Additionally, for complete reaction, it was found beneficial to wash the catalyst palladium on charcoal with hexane immediately before use. The reason for this effect is not known, possibly due to the renewal of the surface of the catalyst. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The enantiomer of 2-(2-chioroethylamino)tetrahydro-7,3,2 oxazaphosphorine 2-oxide was an important intermediate for the synthesis of the enantiomer of IF. Reaction of the R enantiomer with an equal molar amount of chloroacetyl chloride in THF at room temperature gave the corresponding (S)-2-(chloroethylamino)-3- chioroacetyltetrahydro-7,3,2-oxazaphosphorine 2-oxide. This process was regiospecific. Only the desired product was detected by TLC. There was no evidence of acylation on the exocyciic nitrogen nor diacyiation. Subsequent reduction required the use of excess amount of diborane for complete reaction. Synthesis of 2,2-Dideuterio-2-chloroethylamine hydrochloride as a synthon for the deuterium labeled enantiomeric IFs was accomplished by an indirect method. Direct reduction of ethyl glycine ester with lithium aluminum deuteride was not successful due to poor extraction efficiency. This might be due to a tight binding of the product with the inorganic salt. Thus, ethyl glycine ester hydrochloride was treated with triphenylmethyl chloride in dichloromethane to give the highly lipophilic compound ethyl N-triphenylmethyl-glycine ester, which was readily reduced by lithium aluminum deuteride at room temperature. The product was readily isolated by CH 2 CI2 extraction in excellent yield (100%). The triphenylmethyl group was removed by concentrated hydrochloric acid (37%) in acetone under reflux condition to give 2 ,2 -dideuterio-ethanolamine, which was readily crystallized in ethyl alcohol. Chlorination of this alcohol with thionyl chloride in 1 ,2-dichloroethane gave 2,2- dideuterio-2-chloroethylamine (Scheme 2.4), which precipitated when the reaction was compieted. The reaction was almost quantitative and the purity of the alcohoi was found to be the major determinant to the completion of this reaction. 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N H CI Figure 2 .1 The numbering system of ifosfamide. 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c Y < / NH, N2D X c V • o' \ h X N30 O N H t Y < / NH V » / 4-OHIF \ HO - ' \ h C l AicolF IPM Rgure 2.2 Metabolic tracing by 6,6,2',2'-tetradeuterio-ifosfamide. 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. z /Œ 0 = Q .^ \ 0.= O O^/^C I Ü /C °= V ^ o O ,o .= o o-^/^o: o ; \ _ C L = 0 O ^l^X. \z ]3 _ p I t £ r z - c o / 0) •o E I C O i2 z -£c OI A o £ _ J j= z —I " 1 0 . f i Q.= 0 O l a : o / , ) 3 I £ C»-pio II c I I 0 1 ir C M 0) I 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. \ Q.=0 Ltr Ol c l o IG a \ X2-Œ O / cl C2-Œ » 4 O 4 * Z— 4"i|^ 0 =a^\ I / = °= V k ° = v l Ol cl II c 0) T3 E I c 0 1 c « 0 1 ir (M (\i i 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ o O Q. C g Q. "D CD C/) C/Î 8 ■ D HaNCHaCOOCaHs^HCI ------------ TrNHCHaCOOCgHg — CHaCIa, r.l. THF, r.l. TrNHCHaCDaOH - ^ 5 != TrNHCHaCDaCI (CHaCI)a 3 . 3" CD "O O ■ D CD So^CHa" H^NCHaCDaCHC, CD "O O Q. C a o 1rs CD Û. Scheme 2.3 Route of synthesis of 2-chloro-2,2-dideuterioethylamine hydrochloride. (/) (/) °=Vk . 4 1 1 = 0 Ü la: > 4 1 u ^0.=O O / = x z- û : > 4 1 S z-c = 4 O o mO S ‘ i o 'h ° S ‘ i >=o II c 0) 1 * 0 ) 0 1 0) E I C M . C M « 3 (0 "S I i "S % S c v i I 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. i ü _ /Œ 0 =IL^\ .-R S X z ^û-= 0 _ I u ^û-=0 O '"/ Œ .- R XZ-Œ ■ 4 i - % ü K X z /= °= V k . 4 i I 0) 3 E I a I ; C V J C M C D (O 2jz-c = 4 + z — J 'H ltL X O i o.d!^ f f U*-T-"X II c 0 1 I 0 1 ir L O c\i (U I 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3 IDENTIFICATION OF NEW METABOLITES OF IFOSFAMIDE IN RAT URINE USING ION CLUSTER TECHNIQUE 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.1 A B S T R A C T The metabolism of the anticancer drug IF was investigated in Sprague-Dawley rats. Along with four known metaboiites, nameiy, N2D, N3D, alcolF, and IPM, four new urinary metabolites were identified utilizing combined techniques of chemical modification/derivatization, capillary GC/CIMS (ammonia), deuterium-iabeiing/ion cluster analysis, and chemical synthesis. Secondary metabolites of N2D and N3D formed from 4-hydroxylation, i.e., 4-hydroxy N2-dechloroethyl ifosfamide (4- 0HN2D) and 4-hydroxyl N3-dechloroethyl ifosfamide (4-OHN3D), respectively, and their subsequent decomposed product, N-dechloroethyl iphosphoramide mustard (NDIPM), were identified. Secondary dealkylation pathways of N2D and N3D were also demonstrated through the characterization of N2,3-didechloroethyl ifosfamide (N2N3D). In addition, the key active metabolite of IF, 4-OHIF, was characterized as a cyanohydrin adduct for the first time. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2 INTRODUCTION IF, a structural isomer of the oxazaphosphorine CP (4,5), has been found to possess significant activity against human and experimental malignancies such as nonseminomatous testicular cancer, small-cell lung cancer, pediatric solid tumors, non-Hodgkin's and Hodgkin's lymphomas, and ovarian cancer (7,66,67). Like CP, IF is a prodrug that requires in vivo enzymatic activation to be cytotoxic (4,5,12,45). Hydroxylation mediated by the hepatic cytochrome P450 system at the C4 position of the oxazaphosphorine ring leads to the formation of 4-OHIF, the most important metabolite for the anticancer activity of this drug. This species spontaneously equilibrates with its open-chain tautomer, aide IF. Further oxidation of these species by alcohol oxidase and aldehyde dehydrogenase gives 4-ketolF and carboxylF, respectively. AldolF is reduced by aldehyde reductase to alcolF, and at the same time undergoes spontaneous decomposition to form IPM, the purported ultimate intracellular alkylating metabolite (14,16) with the elimination of acrolein, the causative agent for the urotoxicity of oxazaphosphorines (18). Unlike CP, N-dealkylation of IF occurs to a much greater extent, leading to the formation of monoalkylating species, N2D and N3D (13,20,21,23,24), with the release of the co-product, CAA, the metabolite implicated in the neurotoxicity observed in patients receiving IF therapy (22). Although 4-OHIF and aldolF are believed to be the most important metabolites of IF, neither of them has been unequivocally identified as a metabolite because of their chemical instability. The metabolic pathway of IF is shown in Scheme 3.1. The previously unknown metabolism of N2D and N3D is derived from the results reported herein. The metabolic 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pathways of N3D are similar to those of N2D and Is therefore omitted from the scheme. Conventional metabolite identification techniques involve isolation and purification of the metabolite from biological excreta, followed by the usual spectroscopic analyses, such as infrared (IR), nuclear magnetic resonance (NMR), ultraviolet (UV), and mass spectrometry (MS), and are tedious and time- consuming. Using a 1:1 mixture of stable isotopically labeled and unlabeled drugs, tentative identification of metabolites can rapidly be achieved on the basis of observed ion doublets or ion clusters using electron impact (El) (68,69) and chemical ionization (Cl) (70,71) mass spectrometry on either crude extracts or partially purified extracts. Subsequent structural identification of a metabolite can be accomplished by loss or retention of specific isotope labels, derivatization, and by chemical synthesis. This approach has been successfully applied to a number of drugs, including nortriptyline (6 8 ), (+)-propoxyphene (69), warfarin (70) and cyclophosphamide (71). Using stable isotope labeling, chemical modification, ion cluster analysis, and GC/CiMS, we identified four new metabolites of IF. 3.3 MATERIALS AND METHODS 3.3.1 Materials 3.3.1.1 Chemicals and reagents (+)- and (-)-IF, (+)- and (-)-(2 ',2 ',6 ,6 -2 H4 )IF [(+) and (-)-IF-d4] were synthesized in this laboratory (Chapter 2). (±)-IF was provided by Drug Synthesis and Chemistry Branch, the Nationai Cancer Institute. (±)-[1',T,2',2'- 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 H4 ]-IF [(±)-IF-d4 ], 4-OHIF, N2D, N3D, and IPM were all synthesized in this laboratory (Section 3.4.1 ). Ail HPLC grade organic solvents were purchased from E.M. Science (Gibbstown, NJ). N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), N,0-bis(trimethy!silyl)trifluoroacetamide (B S TFA ), and N- trimethylsilylimidazole (TMSI) were purchased from Pierce (Rockford, IL). C18 reversed-phase resin was obtained from Analytichem International (Harbor City, CA). 3.3.1.2 Surgical instrument and supplies Surgical instruments were purchased from Roboz Surgical Instrument Co., Inc. (Washington, DC). Monoject hypodermic needles and Monoject sterile disposable tuberculin syringe without needle were obtained from Western Medical Supply (Arcadia, CA). Surgical sutures (size 3 and 4) were purchased from George Tieman & Company (Plainview, NY). Curity gauze sponges were obtained from Kendall Company (Boston, MA). Intramedic polyethylene 50 tubings (PE 50, 0.58 mm I.D. and 0.965 mm O.D.) were purchased from Fisher Scientific (Tustin, CA). Sodium chloride injection (9%), USP was obtained from Western Medical Supply (Arcadia, CA) as was heparin sodium injection USP 1000 units/ml. Isopropyl alcohol was obtained from Fisher Scientific (Pittsburgh, PA). 3.3.2 Methods 3.3.2.1 Animal surgery - jugular vein cannuiation Animal experiments were carried out according to a protocol approved by the Animal Use Review Committee at the Ohio State University. Four male Sprague- Dawley rats (Harlan, Indianapolis, IN) weighing between 250-300 g were used in 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. this study. The jugular vein of each rat was cannulated under ethyl ether anesthesia. The fur around the right side of the neck was shaved. The skin was sterilized with 75% isopropyi alcohol solution. A 2-3 cm-long incision was made at the exposed region, and the neck muscle was gently separated to expose the right jugular vein. After clear separation of the vein, a small nick was made and a 10-cm-beveled-cut PE 50 tubing was inserted and pushed into the right atrium. The muscle and the skin were closed with stitches and the cannula was exteriorized under the skin to the back of the neck. The blood flow was reexamined and the tubing filled with heparinized saline solution (200 lU/ml). The animal was allowed to recover for 2 hrs before dosing. 3.3.2 2 Metabolic studies After animals became completely conscious, drugs dissolved in normal saline (1 ml) at the total dose of 40 mg/kg were injected into the animals through the jugular vein cannula. One animal was given (±)-IF only, one given a 1:1 mixture of (±)-IF and (±)-IF-d4 , one given a pseudoracemate consisting of (+)-IF-d4 and (-)- IF, and one given a pseudoracemate consisting of the opposite composition. The cannula was washed three times with 0.3 ml each of 0.9% sodium chloride solution. Rat chow (Teklad, Indianapolis, IN) and water were given ad libitum. Urine was collected continuously for 24 hrs into a container immersed in ice, and immediately stored at-76°C until analysis. 3 3.2.3 Sample extraction and derivatization Rat urine was divided into two portions and each portion was screened for possible new metabolites, using different methods as described below. For the 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. analysis of CH2 Cl2 -extractab!e metabolites (e.g., 4-OHIF and 4-OHN2D), 50 mg of KCN or K^scN was added to 0.5 ml of urine from each rat. The mixture was allowed to stand at room temperature for 30 min. Dichloromethane (CH2 CI2 , 5 ml) was added and the mixture was shaken for 15 min in a horizontal shaker (Eberbath, Ann Harbor, Ml). The organic phase was separated after centrifugation and evaporated under a stream of N2 . The residue was derivatized with 35 [J MSTFA for 60 min at 120°C. An aliquot (0.5 pJ) of the mixture was analyzed by G C/MS. For the assay of more polar components (e.g., NDIPM, and N2N3D), solid- phase extraction was used. Rat urine (0.5 ml) was loaded onto a disposable Poly-prep column (Bio-Rad, Richmond, CA) containing 400 mg of C18 reversed- phase resin, and the resin was washed with 0.5 ml of cold saline followed by centrifugation at 200 x g for 20 min to remove water. The mini-column was then eluted with 1 ml of methanol, which was collected and evaporated under a stream of N2 at room temperature. The residue was derivatized with 35 pi of a mixture of BSTFA and TMSI (5:1 ) at 120°C for 60 min. An aliquot (0.5 pi) of the mixture was analyzed by G C/MS. 3.3 2.4 GC/MS analysis A Finnigan ITS40 Ion Trap mass spectrometer (FInnigan MAT, San Jose, CA) coupled to a 3300/3400 Varian gas chromatograph (Walnut City, CA) was used for the analysis. An A200S autosampler with a capillary splltless injector was purchased from Finnigan MAT. Temperatures of the injection port, transfer line, and source were maintained at 220,260 and 230°C, respectively. The analysis was carried out using chemical ionization mode with ammonia as the reagent gas. A 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DB-5,30-m X 0.25-mm I.D. capillary column bonded with a 0.25-um film thickness of 5% methyisilicone (J&W Scientific, Folsom, CA) was used for the separation. The oven temperature was programmed at 150°C for 2 min and increased to 190°C at a rate of 5°C/min, and then to 250°C at a rate of 15°C/min. The final temperature was held for 3 additional min. 3.3.3 Synthesis of new and know metabolites of ifosfamide and (±)-ifos- famlde-d4 3.3.3.1 Synthesis of alcoifosfamide The synthesis of alcoifosfamide was accomplished through an intermediate, 3- benzyloxylpropyl N,N'-bis(2-chloroethyl)phosphorodiamidate, which was prepared via a one-pot reaction starting with oxyphosphorus, O-benzyl-1,3- propane-dioi and 2-chloroethylamine hydrochloride (Scheme 3.2). 3-Benzy foxy I propyl N,N'-bis(2-chloroethyl)phosphorodiamidate. Into a cooled (-78°C) solution of oxyphosphorus chloride (1.35 g, 10.00 mmol) in CHgClg ( 2 0 mi) was added dropwise a solution of O-benzyi-1 ,3-propane-diol (1 .6 6 g, 1 0 . 0 0 mmol) and triethylamine (1 .0 1 g, 1 0 . 0 0 mmol) in the same solvent (10 ml), while vigorous stirring was maintained. The progress of reaction was followed by TLC. When all the starting material disappeared (in 10 min), 2- chloroethylamine monohydrochloride (3.32 g, 20.00 mmoi) was added into the reaction mixture, followed by a solution of triethylamine (4.04 g, 40.00 mmol) in CH2 CI2 (5 ml). The mixture was stirred at room temperature for 1 hr. The solvent in the reaction mixture was removed in vacuo, and the residue was extracted with acetone. The extract was concentrated to give a pale yellow oil (4.04 g), which was purified by silica column chromatography (CH2Cl2 -acetone 2 :1 ) to give a 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. colorless oil product (3.42 g, 92.7%): Rf 0.47 (CHaCla-acetone 1:1); NMR (CDCI3 ) Ô 1.96 (p, J=6.5 Hz, 2 H, -CH,-CH,-CH,-). 3.02-3.40 (m, 6 H, 2 x -NH- CH 2 -), 3.52-3.66 (m, 6 H, 2 x -CH2-CI, -CH2-0 Bn), 4.12 (q, J=6.5 Hz, 2H, -CH2- O-P), 7.28-7.42 (m, 5H, Ph-H). AlcolF. Into a solution of benzyloxypropyl phosphorodiamidate (0.70 g, 1.90 mmol) was added freshly prepared hexane washed 10% Pd/C (0.50 g), and the mixture was hydrogenated at room temperature. The reaction was monitored by TLC. When the starting material disappears (2 hrs), the catalyst was removed b y filtration. Concentration of the filtrate in vacuo afforded the crude product (0.62 g), which was purified by column chromatography (CH2 Cl2 -acetone 1 :1 ) to give alcolF, a colorless oil (0.38 g, 68.4%): Rf 0.44 (CH2 Cl2 -acetone 1:3); NMR (CDCI3 ) Ô 1.76 (bs, 1 H, OH), 1.85 (p, J=5.6 Hz, 2 H, -CH^-CHp-CH?-). 3.08- 3.40 (m, 6 H, 2 X -NH-CH2 -), 3.64 (t. J=5.6 Hz, 4H, 2 x -CH2 CI), 3.75 (t, J=5.6 Hz, 2H, -CHp-OH), 4.10-4.24 (m, 2H, -CH2-O-P). The assigned structure was consistent with GC/MS analysis. 3.3.3.2 Synthesis of 4-hydroxy N3-dechloroethyl ifosfamide The synthesis of 4-OHN3D was conducted according to the procedure of G. Perterefa/. for the synthesis of 4-hydroperoxy ifosfamide (72) and is depicted in Scheme 3.3. N3D (0.10 g, 0.50 mmol) was dissolved in aqueous acetone (1:1,10 ml). After addition of 30% HgOg (1 ml), the solution was ozonized at 0°C for 2 hrs. Acetone was removed in vacuo at room temperature. The isolation of the product was found to be difficult and the resulting mixture was directly used as the source of 4-OOHN3D. The chemical identity was confirmed by GC/MS analysis. To a culture tube containing 100 ml of the resulting mixture was added 1 M 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NaaSaOa solution (100 ml) followed by 1M KCN (250 ml). The mixture was allowed to stand at room temperature for 30 min. CH2 Ci2 (5 ml) was added to extract the cyanohydrin adduct of 4-OHN3D. The extract was evaporated under a stream of N2 and the residue derivatized with fVISTFA (40 ml) at 120°C for 1 hr. An aliquot was subjected to GC/MS analysis. A major peak at m/z 422 (no Cl) was observed, which is consistent with the assigned structure tri-(trimethylsilyi)- dechlorinated cyanohydrin adduct of 4-OHN3D. 3.S.3.3 Synthesis of N-dechloroethyl iphosphoramide mustard Benzyl N-diphenylm ethyl-N'-2-chloroethylphosphorodiamidate. To a cooled (-78°C) solution of oxyphosphorus chioride (1.53 g, 10.00 mmol) in CHaCia ( 2 0 mi) was added 1 0 ml of benzyl alcohol (1.08 g, 1 0 . 0 0 mmol) and triethylamine (1.01 g, 10.00 mmol) in CH2CI2 . The mixture was stirred for 15 min, then added a solution of diphenylmethylamine (1.83 g, 10.00 mmol) and triethylamine (1 .0 1 g, 1 0 . 0 0 mmoi) in CH2CI2 (5 ml). After vigorous stirring for 2 hrs at ambient temperature, 2 -chloroethylamine monohydrochloride (1.16 g, 1 0 . 0 0 mmoi) was added into the reaction mixture, followed by triethylamine (2 . 0 2 g, 2 0 . 0 0 mmol) in CH2CI2 (5 ml). The stirring was maintained overnight. The reaction mixture was washed with H2 O (20 ml x 4) to remove triethylamine chloride. The CH2 CI2 layer was dried over anhydrous Na2S O4 and the filtrate concentrated in vacuo to give a thick oii (4.52 g), which was purified by coiumn chromatography (CH2 Cl2-acetone 20:1 ) to give the diamidate as a colorless oil (3.24 g, 78.3%): Rf 0.28 (CH2 Cl2 -acetone 20:1); NMR (CDCI3) 6 2.75-2.95 (m, 1 H, NH), 3.00-3.18 (m, 2H, -NH-CH,-). 3.30-3.46 (m, 3H, NH, -CH2 CI), 4.75-5.07 (m, 2H, -CH2 -O-), 5.42-5.54 (m, 1H, -CH-NH-). 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N-(2-Chloroethyl) phosphorodiamidic acid (NDIPM). The diamidate (0.83 g, 2.00 mmol) was dissolved in MeOH (40 ml), and the solution was hydrogenated under normal pressure at room temperature with 10% Pd/C (0.40 g) and Pd(0 H)2/C (0.40 g) as catalysts. The reaction was followed by TLC. When the starting material disappeared (1 hr), the catalysts were removed by filtration. The solvent in the filtrate was removed in vacuo. Diethyl ether (5 ml) was added to the residue, resulting in the formation of crystalline material. The crystals were collected by filtration and washed with ethanol to give NDIPM (0.25 g, 78.9%, m.p. 103-105°C). The chemical identity was confirmed by GC/MS analysis. The synthetic scheme of NDIPM is shown in Scheme 3.4. 3.3.S.4 Synthesis of N2,N3-didechloroethyl ifosfamide N2N3D was prepared in two steps (Scheme 3.5). To a stirred solution of oxyphosphorus chloride (1.53 g, 10.00 mmol) in EtzO (40 ml) at -78°C was added a mixture of 3-amino-propan-1-ol (0.75 g, 10.00 mmol) and triethylamine (2.02 g, 20.00 mmol) in EtaO (5 ml). The stirring was maintained for 2 hrs at ambient temperature. The precipitated triethylamine hydrochloride was removed by filtration and the filtrate was used directly in the subsequent step. Ammonia was bubbled into the rigorously stirred solution for 1 hr at room temperature. The precipitated ammonium chloride was removed by filtration. The filtrate was concentrated in vacuo \o give N2N3D as a white solid (0.48 g, 35.3%): m.p. 105- 108°C; Rf 0.18 (acetone): NMR (acetone-de) ô 1.54-1.86 (m, 2H, C5-H), 3.14- 3.62 (m, 5H, NH, NH2 . C4-H), 4.08-4.30 (m, 2H, G6 -H); The chemical identity was further confirmed by GC/MS analysis. The MSTFA derivatized sample gave a 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. peak with ion at m/z 353, consistent with the assigned structure, N,N,N'-tri- (trimethyisiiyl) N2 N3 D. 3.S.3.5 Synthesis of 4-hydroxy Ifosfamide and 4-ketoifosfamide 4-OHIF and 4-ketolF were synthesized following the procedure of Takamizawa et al. (73,74). 0-3-Butenyl N,N'-bis(2-chloroethyl)phosphoro- diamidate was prepared in 70% yield by a one-pot reaction of oxyphosphorus chioride with 3-buten-1-ol and 2-chioroethyiamine hydrochloride in CHgClg at low temperature. Ozonolysis of the obtained phosphorodiamidate in aqueous acetone, followed by treatment with 30% hydrogen peroxide (H2 O 2). afforded 4- hydroperoxy ifosfamide (4-OOHIF) in about 30% yield. Treatment of the peroxide with triethyi phosphite and ferrous sulfate gave 4-OHIF and 4-ketolF in good yield. The synthetic scheme of 4-OHiF and 4-ketolF is shown in Scheme 3.6. 0-(3-butenyl)-N,N'-bis(2-ch[oroethyl)phosphorodiamidate. To a stirred solution of oxyphosphorus chloride (7.68 g, 50.0 mmol) in CH2CI2 (50 mi) was added dropwise a solution of 3-buten-1-ol (3.61 g, 50.0 mmol) and triethylamine (5.06 g, 50.0 mmoi) in CH2CI2 (10 ml) at -76°C. The mixture was stirred for 30 min at ambient temperature. 2 -Chloroethyiamine hydrochloride (1 1.6 g, 1 0 0 mmol) was added into the mixture, followed by a solution of triethylamine (20.24 g, 200.0 mmoi) in CH2CI2 (10 ml). After the reaction mixture was stirred for 1 additional hr at 0°C, the precipitated triethylamine hydrochloride was removed by filtration. The filtrate was washed by distilled water (50 ml x 3), dried over anhydrous sodium sulfate, and concentrated in vacuo to give a yellow oil (13.70 g, 99.6%). Column purification with ethyl acetate as the eluant gave 0-(3-butenyl)-N,N'-bis(2- 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chloroethyOphosphorodiamidate as a colorless oil: Rf 0.41 (CH2 Cl2 -acetone 1:1); NMR (CDCI3 ) Ô 2.47 (2H, q, J=6.7 Hz, -CH,-CH=CH,). 280-3.45 (6 H, m, 2 x -NH-CH2 -), 3.63 (t, J=6.4 Hz, 4H, -CH2-CI), 4.06 (2H, q, J=6 . 8 Hz, -O-CH9-). 5.08-5.23 (2H, m, -CH=CH,I. 5.70-5.88 (1H, m, -CH=CHç>). 4-OOHIF. To a Stirred solution of the phosphorodiamidate (5.76 g, 20.9 mmol) In aqueous acetone (1:1,60 ml) was bubbled with O3 at a flow rate of 3.5 ml/mln (90v, 90w) for 1.5 hr at 0°C, then 30% H2O 2 (5 ml) was added to the ozonized solution. After standing at 4°C for 3 days, acetone in the reaction mixture was removed in vacuo and the resulting aqueous residue was extracted with CH2Ci2 (50 ml X 4). The organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated in vacuo to give a coiorless oil (5.45 g), which crystallized by addition of a mixture of acetone (0.5 ml) and ethyl ether (5 ml). After standing at -70°C overnight, the crystals were collected by filtration and washed with cooled (0°C) ethyl ether to give 4-OOHIF (0.78 g). The mother liquor was concentrated in vacuo. The resulting oil was redissolved In aqueous acetone (1:1, 30 ml), 30 % H2 O 2 (5 ml) was then added to the mixture. After standing at 4°C for 2 days, acetone was removed in vacuo, and the remaining aqueous solution was extracted with CH2CI2 (50 ml x 5). Second crop of 4- OOHIF (0.55 g) was obtained by a similar treatment. The resulting mother liquor was once again treated similarly to give the third crop (0.44 g). The total amount of product was 1.77 g (29.1%). Recrystallization of the product from methanol gave colorless prisms, m.p. 114-115°C (dec. lit. 113-114°C); Rf 0.74 (CH2Cl2-acetone 3:1): NMR (acetone-de) 52.08-2.30 (2 H, m, C5-H), 3.15-3.60 (4H, m, CT-H, C"- H), 3.63 (t, J=6 . 6 Hz, 2H, C2'-H), 3.84 (t, J=7.6 Hz, 2H, C2"-H),4.03-4.20 (IN, m. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. C6 -H), 4.22-4.40 (m, 1H, NH), 4.45-4.60 (m. 1H, C6 -H), 5.06 [1H, d-t, J(P, C4- H)=19.4 Hz, J(C4-H, C5-H)=2.7 Hz, C4-H], 10.99 (1H, s, OOH). 4-OHIF. To a stirred suspension of 4-OOHIF (0.293 g, 1 mmoi) in CH2CI2 in an ice-water bath was added excess amount of triethyl phosphite (0.2 g). After stirring for 1 0 min, the suspension turned into a clear solution from which fine needles gradually precipitated, and the entire reaction mixture turned into a white cake in 30 min. After washing with ethyl ether, the solid was collected by filtration to give 4-OHIF as a white solid (0.204 g, 73.8%). Recrystallization from acetone- ethyl ether gave coloriess needles: m.p. 75-76°C (dec. lit 74-75°C); Rf 0.62 (CH 2Cl2 -acetone3 :1 ): NMR (acetone-de) ô 1.80-2.00 (m, 2H, C4-H), 3.12-3.58 (m, 4H, Cr-H, C1"-H), 2.60-2.90 (bs, 1H, NH), 3.60 (t, J=6 . 6 Hz, 2H, C2'-H), 3.81 (t, J=7.6 Hz, C2"-H), 4.00-4.18 (m, 1H, C6 -H), 4.50-4.66 (m, 1H, C6 -H), 5.02 [d-t, J(P, C4-H)=15.6 Hz, J(C5-H, C4-H)=3.2 Hz], 04-H. The chemical identity was further confirmed by its conversion into the cyanohydrin adduct, which was derivatized with MSTFA to give a ion peak at m/z 412 under GC/MS, in accord with the assigned structure. 4-KetoiP. To a stirred suspension of 4-OOHIF (0.293 g, 1 mmol) in CH2CI2 (10 ml) was added a solution of Fe2 S 0 4 * 7 H2 0 (0.556 g, 2 mmol) in H2 O (20 ml) and the mixture was stirred vigorously for 1 hr at room temperature. The organic phase, after separation, was washed with H2O ( 1 0 ml x 2 ) and dried over anhydrous sodium sulfate. Removal of the solvent in vacuo gave 4-ketolF as a white solid (0.258 g, 93.5%): m.p. 111-113°C (ethanol): Rf 0.67 (GH2Cl2 -acetone 1:1); NMR (CDCI3 ) ô 2.65-2.95 (m, 2H, G5-H), 3.22-3.38 (m, 2H, GT-H), 3.50- 4.08 (m, 7H, NH, C2"-H, C1"-H, C2"-H), 4.18-4.59 (m, 2H, G6 -H); MS (El) m/e 274 (M+), 225 ([M-GH2GI]+, 100%). 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.S.3.6 Synthesis of N2-dechloroethyl Ifosfamide Methyl N-2-hyd roxy I ethy l-3-a m I no propionate. To a solution of methyl acrylate (8.61 g, 100.0 mmol) in THF (150 ml), was added ethanolamine (6.11 g, 100 mmol). The mixture was stirred at room temperature for 2 days. The solvent in the reaction mixture was evaporated in vacuo to give a colorless oil (14.71 g, 99.9%): Rf 0.07 (CHaCla-acetone 1:1); NMR. (CDCI3 ) Ô2.26 (bs, 1H, OH), 2.52 (t, J=6.3 Hz, 2H, C2-H), 2.79 (t, J=5.2 Hz, 2H, Cl'-H), 2.92 (t, J=6.3 Hz, C3-H), 3.57-3.62 (m, 6 H, NH, C2'-H, -OCH3 ). The product was used for the subsequent step without further purification. Methyl N-2-chloroethyl-3-amino-proplonate. To a cooied (0°C) solution of methyl N-2-hydroxylethyl-3-amino-propionate (5.00 g, 34.0 mmol) In 1,2- dichloroethane (20 ml), was added dropwise thionyl chloride (8.03 g, 6 8 mmol). Temperature of the mixture was raised gradually to 60°C in 30 min. The reaction was followed by TLC. Once the reaction was completed, dichloroethane and the excess thionyl chloride were removed In vacuo. H2 O (20 mi) was added to dissoive the residue and the mixture was washed with CHaCia (50 ml x 3). The organic phase was discarded. The remaining aqueous solution was neutralized to pH 9-10 using saturated NaOH solution, and was then extracted with CH2CI2 (50 ml X 4). The organic phase was washed with H2 O 2 (10 ml), and dried over anhydrous Na2 S O4 , followed by evaporation in vacuo to give the product as a pale yellow oil (5.03 g, 89.3%): Rf 0.57 (CH2 Gl2-acetone 1:1); NMR (CDCI3 ) ô 2.70 (t, J=6.5 Hz, 2H, C2-H), 3.07 (t, J=6.5 Hz, 2H, C3-H), 3.11 (t, J=6.0 Hz, 2H, CT-H), 3.67-3.80 (m, 6 H, NH, C2 '-H, -OCH3 ). N-2-Chloroethyl-3-amino-propan-1 -ol. Methyl N-2-chloro-ethyl-3-amino- propionate (1.655 g, 10.00 mmol) was dissolved in THF (20 ml), and the resultant 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. solution was added dropwise to a cooled (-76°C) suspension of lithium aluminum hydride (0.285 g, 7.50 mmol) in THF (20 ml). The mixture was stirred at room temperature for one hr. The progress of the reaction was followed by TLC (CH2CIg-acetone-methanol 1:1:1). Once the starting material disappeared, H2O 2 (0.5 ml) in THF (20 ml) was added to destroy the remaining hydride. After filtration, the solid was extracted with CH2CI2 . The filtrate and extract were combined and dried over anhydrous sodium sulfate. The volume of the organic phase was reduced in vacuo to about 10 ml at a temperature below 25°C. 2-Chloro-3-(2-chioroethyl)-tetrahydro-1,3,2-oxazaphosphor!ne 2 -oxide. The CH2 CI2 solution containing N2-chloroethyl-3-amino-propan-1-ol was added dropwise to a cooled (-76°C) solution of oxyphosphorus chloride (1.54 g, 10.00 mmol) in CH2 CI2 ( 1 0 ml). A solution of triethylamine (2 . 0 2 g, 2 0 . 0 0 mmol) in GH2 CI2 (5 ml) was then dropped into the mixture. The reaction mixture was stirred for 1 hr at room temperature. The solvent in the reaction mixture was removed In vacuo and the residue was extracted using anhydrous ethyl ether. The extract was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed in vacuo to give a colorless oil (1.75 g), which was purified by silica column chromatography with ethyl ether as the eluant to give 2-chloro-3-(2-chloro- ethyl)-tetrahydro-f,3,2-oxazaphosphorine 2-oxide as a pale oil (0.78 g, 34% starting from the ester): NMR (CDGI3 ) Ô 1.60-1.95 (m, 2H, G5-H), 3.10-3.80 (m, 6 H, G4-H, -GH2GH2 GI), 4.10-4.63 (m, 2H, G6 -H). 2-Amino-3-(2-chloro-ethyl)-tetrahydro-^,3,2K}xazaphosphorine 2 -oxide (N2D). To a solution of 2-chloro-3-(2-chloro-ethyl)-tetrahydro-7,3,2-oxaza- phosphorine 2-oxide (0.78 g, 3.58 mmol) in ethyl ether (50 ml) was bubbled with anhydrous ammonia at room temperature. After 8 hrs, the starting material could be 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. detected by TLC. Acetone (200 ml) was added to the mixture and precipitated ammonium chloride was removed by filtration. The filtrate was dried over anhydrous sodium sulfate and after filtration, the solvent in the filtrate was removed in vacuo to give a colorless solid (0.77 g), which was purified by silica gei column separation using CHaCla-acetone (1:1) as the eiuant to give N2D (0.57 g, 80.3%). m.p. 102-103°C; Rf 0.22 (CHgClg-acetone 1:3); NMR (acetone-de) ô 1.72-1.98 (m, 2H, C5-H), 3.12-3.35 (m, 4H, C4-H, CT-H), 3.55-3.80 (m, 4H, NH2 , C2'-H), 4.12-4.26 (m, 2H, C6-H). The chemical identity of the product was also confirmed by GC/MS analysis. The synthetic scheme of N2D is shown in Scheme 3.7. 3.3.3 7 Synthesis of N3-dechioroethyl ifosfamide 2-(2-C hloroethyl)am ino-tetrahydro-f,3,2-oxazaphosphorine 2-oxide (N3D). To a cooied (-78°C) solution of oxyphosphorus chloride (1.53 g, 10.00 mmol) in anhydrous CH2 CI2 (20 mi) was added slowly a mixture of 1 -amino- propan-3-oi (0.75 g, 10.00 mmol) and triethylamine (2.02 g, 20.00 mmoi) in CH2CI2 (5 ml). Stirring was maintained for 1 hr at room temperature. 2-Chloroethylamine hydrochloride (1.68 g, 14.44 mmoi) was then added into the reaction mixture, followed by triethylamine (2.02 g, 20.00 mmol) in CH2CI2 (5 ml). The reaction mixture was stirred for 2 additional hrs at room temperature. The solvent in the reaction mixture was removed by in vacuo. The residue was extracted with hot acetone (50°C, 20 ml x 4). Concentration of the extract afforded a semi-soiid (2.52 g), which was purified by column chromatography using CH2Cl2/EtOH (15:1) as the eiuant to give N3D (0.92 g, 46.5% starting from amine alcohol): m.p. 107- 108°C; Rf 0.18 (CH2 Cl2 -acetone 1:3); NMR (acetone-de) ô 1.58-1.88 (m, 2H, C5- 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. H), 3.12-3.34 (m, 4H, C4-H, CT-H), 3.62 (t, J=6.7 Hz, 2H, G2'-H), 3.71 (bs, 1H, NH), 3.97 (bs, 1H, NH), 4.18-4.30 (m, 2H, C6-H). The chemical identity was confirmed by GC/MS analysis after its derivatization with MSTFA. The synthetic scheme of N3D is shown in Scheme 3.4. 3.3 3.8 Synthesis of iphosphoramide mustard Phenyl N,N'-bis-(2-chloroethyl) diamidophosphate. To a cooied (0°C) solution of phenyl dichlorophosphate (1.82 g, 8.63 mmoi) in GHaCig was added 2- chioroethyiamine hydrochloride (2.00g, 17.24 mmoi). Triethylamine (3.48 g, 34.52 mmoi) in CHgCia (10 mi) was then added dropwise into the stirred suspension. The stirring was continued at room temperature for 1 hr. Acetone (200 mi) was added to the mixture and the precipitated triethylamine chioride was removed by filtration. The organic phase was concentrated in vacuo to give a light yellow oii (3.11 g). The crude material was purified by silica gei coiumn chromatography using CH2Ci2-acetone (5:1) as the eiuant to give a colorless oii (2.36 g, 92.2%): Rf 0.65 (CHaCia-acetone 1:1); NMR (CDCis) 53.16-3.42 (m, 6H, 2 x -NH-CHa-), 3.54-3.66 (m, 4H, 2 x -CHa-Ci), 7.10-7.38 (m, 5H, Ph-H). MS (Ci) m/z2Q7 (MH+). N,N-bis-(2-chloroethyl) diamidophosphoric acid (IPM). To the solution of phenyl N,N'-bis-(2-chioroethyi) diamidophosphate (0.51 g, 1.72 mmoi) in CHaCia (20 mi) was added 10% PtOa (0.20 g). The mixture was hydrogenated at mom temperature under normal pressure. The progress of hydrogenation was monitored by TLC. When the starting material disappeared (5 hrs), the catalyst and the precipitated product was collected by filtration and washed with CHaCia. The product was dissolved in methanol and separated from the catalyst by filtration. Removal of methanol in the filtrate In vacuo afforded iphosphoramide 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mustard (0.35g, 92.2%) as tiny crystals, m.p. 118-119°C ( lit. 106-107°C). GC/MS analysis confirmed its chemical identity by comparison with an authentic sample provided by Chemistry and Drug Synthesis Branch, the National Cancer Institute. The synthetic scheme of IPM is show in Scheme 3.9. 3.3.3 9 Synthesis of (±)-lfosfamide-d4 2-(2-Chloro-1,1,2,2-tetradeuterio-ethyl)am!no-tetrahydro-7,d,2-oxaza- phosphorine 2-oxide. To a cooied (-76°C) solution of oxyphosphorus chloride (1.53 g, 10.00 mmol) in anhydrous CH2CI2 (20 ml) was added slowly a mixture of 1 -amino-propan-3-ol (0.75 g, 10.00 mmol) and triethylamine (2.02 g, 20.00 mmol) in CH2CI2 (5 ml). The reaction mixture was stirred for 1 hr at room temperature. 2- Chloro-1,1,2,2-tetradeuterio-ethyiamine hydrochloride (1.20 g, 10.00 mmol) was then added to the reaction mixture, followed by a solution of triethylamine (2.02 g, 20.00 mmol) in CH2CI2 (5 ml). The reaction mixture was stirred for 2 additional hrs at room temperature. The solvent was removed by rotary evaporation in vacuo. The residue was extracted with hot acetone (50°C, 20 ml x 4). Concentration of the extract afforded a semi-solid (2.31 g), which was purified by silica gel coiumn chromatography using CH2 Cl2/EtOH (15:1) as the eiuant to give N3D-d4 as colorless crystals (0.78 g, 39.6%): m.p. 105-107°C; Rf 0.18 (CH2Cl2 -acetone 1:3). The chemical identity was confirmed by GC/MS analysis after its deri vatization with MSTFA. 2-(2-Chloro-1,1,2,2-tetradeuterio-ethyl)amino-3-chloroacetyl-tetrahy- dro-f,3,2-oxazaphosphorlne 2-oxide. To a cooled (0°C) solution of N3D-d4 (0.78 g, 3.86 mmol) in THF (25 mi) was added a soiution of chloroacetyl chloride 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (1.30 g, 11.58 mmol, 1:3 excess) in THF (5 ml). The reaction was monitored by TLC. Once the reaction was completed, the volume of the solution was reduced to 10 ml. Water (10 ml) was then added and the mixture was extracted with CH2 CI2 (50 ml X 4). The organic phase was dried over anhydrous sodium suifate. After filtration, the solvent in the filtrate was removed in vacuo to give a coiorless thin oil (1.41 g), which was chromatographed on a short silica column with 0 H2 C 1 2- acetone (4:1) as the eluant to give a colorless oil (0.82 g, 76.0%): Rf 0.72 (CH2Cl2 -acetone 1:1). 2-(2-Chloro-1,1,2,2-tetradeuterlo-ethyl)amino-3-chloroethyl-tetrahydro -•1,3,2-oxazaphosphorlne 2-oxide [(±)-IF-d4 ]. To a cooled (-78°C) solution of 1 M BH3 in THF (17.7 ml, 6:1 excess) was added dropwise a solution of acetylated N3 D-d4 (0.82 g, 2.95 mmol) in THF (10 ml). After the reaction mixture was stirred for 1 hr at ambient temperature, TLC showed the complete disappearance of the starting material. H2O 2 ( 1 0 ml) was then added dropwise to destroy the remaining BH3 . After the removal of THF In vacuo, the resultant residue was extracted with CH2 CI2 (20 ml X 4). The organic phase was dried over sodium sulfate. After filtration, the solvent in the filtrate was removed in vacuo to give a colorless oil (1.34 g), which was purified by column chromatography with CH2 Cl2-acetone- methanol (40:3:1) as the eluant to give a colorless oil (0.44 g, 56.1%): Rf 0.33 (CH2 Cl2-acetone 1:1). (±)-IF-d4 crystallized during storage in a freezer (-70°C). The chemical identity was confirmed by GC/MS analysis comparing with authentic unlabeled ifosfamide. The synthetic scheme of (±)-IF-d4 is shown in Scheme 3.10. 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.4 RESULTS 3.4.1 alcoifosfamide The GC/MS chromatogram of the derivatized urinary CHgClg extract obtained from a rat given (±)-IF is shown in Figure 3.1 A. The ion chromatograms with specific ions are shown in Figure 3.1 B-D. As shown, an ion at m/z 387 with the retention time of 14.30 min was observed (Figure 3.1 B) and this ion was absent in the blank urine (Figure 3.1 B inset). The mass spectrum for this component (Figure 3.2) shows a strong ion at m/z 387 and a small ion at m/z 389, the intensity of which is consistent with one chlorine atom. Based on this information, the structure of trimethyisiiyi dehydrochlorinated alcolF was tentatively assigned. Scheme 3.11 illustrates the formation of this compound from alcolF. When the urine sample obtained from a rat treated with a 1 ;1 mixture of (±)-IF and (±)-IF-d4 was analyzed, an additional set of ions at m/z 391/393 at the same retention time was seen (Figure 3.3). The mass difference between the corresponding ions in these two sets of ions was 4, indicating that these metabolites were derived from the labeled and non-labeled parent drugs. Similar results were found from urine samples obtained from rats treated with pseudoracemates (+)-IF-d4/(-)-IF or (-)-IF-d4/(+)-IF (Figure 3.4,3.5). The definitive identification of alcolF as a metabolite of IF in the rat was accomplished from the analysis of the authentic compound. When the synthetic alcolF was derivatized under identical conditions and the derivative analyzed by GC/MS, It gave an Ion set at m/z387/389 representing MH+ and the chlorine isotope peak of the assigned trimethyisiiyi dechlorinated alcolF, respectively, approximately at the expected ratio and at the same retention time (Figure 3.6). 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.4.2 4-hydroxy ifosfamide 4-OHIF has been postulated as the primary metabolite of IF (67); however, direct evidence for its existence has not appeared In the literature. Because its presence in urine was anticipated, to facilitate its identification, an authentic 4-OHIF was first synthesized as described previously (13). Then this synthetic compound was characterized using a methodology similar to that of Fenselau et al. (75) for the identification of 4-hydroxy cyclophosphamide (4-OHCP) as a metabolite of CP. When the authentic 4-OHIF was treated with potassium cyanide (KCN) followed by extraction and derivatization with MSTFA, it gave a single component with a retention time of 16.0 min with ions at m/z 412/414 upon GC/MS analysis (Figure 3.7). The mass spectral data were consistent with the formation of a cyclic trimethylsilyiated derivative (Scheme 3.12) similar to that of 4- OHCP (75,76). Once authentic 4-OHIF had been characterized by GC/MS, its possible presence in the urine sample from a rat treated with (±)-IF was examined using the analytic approach similar to that for the authentic compound. After KCN treatment, extraction, and derivatization, a set of Ions at m/z 412/414 was detected at the same retention time as the authentic compound under identical condition (Figure 3.1 C and 3.8). No ion at m/z 412 was detected at that retention time in blank rat urine (Figure 3.1 C inset). To ascertain that the observed component was indeed derived from KCN trapping, the experiment was repeated with K^%N. The resultant product gave a component at 16.0 min, but with a mass unit shifted from 412/414 to 413/415 as expected (Figure 3.9). Ion cluster analysis was also conducted using urine sample from a rat treated with a 1:1 mixture of (±)- IF and (±)-IF-d4 . The ammonia Cl mass spectrum for the derivatized extract from the urine is shown in Figure 3.10. A set of ion cluster were seen at m/z412/414 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and at m / z 416/418, consistent with 4-OHIF generated from these precursors. Similar results were obtained in other samples from rats treated with a 1:1 mixture of (+)-IP-d4 and (-)-IF, and a 1:1 mixture of (-)-IF-d4 and (+)-IF (Figure 3.11, 3.12). Thus, 4-OHIF as a metabolite of IF was unequivocally established. 3.4.2.S 4-hydroxy N2-dechloroethyl ifosfamide and 4-hydroxy N3-dechlo- roethyl ifosfamide Since N2D and N3D have been previously found to be two of the major metabolites of IF, sequential 4-hydroxylation of these metabolites might be possible. Thus, rat urine was screened for the possible presence of 4-OHN2D and 4-OHN3D, using an experimental approach similar to that for the identification of 4-OHIF. When a sample obtained from a rat treated with (±)-IF was processed through KCN treatment followed by extraction, silylation, and GC/MS analysis, a component was eluted at the retention time of 14.0 min with an ion at m / z 422 (Figure 3.1 D). The mass spectrum (Figure 3.13) showed no chlorine present in the molecule. The structure of cyclic trimethyisiiyi dechlorinated derivative of 4- 0HN2D and/or 4-OHN3D was proposed (Scheme 3.13). Replacement of KCN by K^^CN resulted in a single mass unit shift from m / z 422 to m / z 423 in the component of interest (Figure 3.14). These data indicated that this component contained an aldehydic group similar to that of 4-OHIF. When a 1:1 mixture consisting of (+)-IF-d4 and (-)-IF was given to a rat, the urine was subjected to treatment with KCN as before. As expected, the GC/MS mass spectrum of the silylated extract gave ion doublets at m / z 422 and 424 at the same retention time as before, indicating that this component was a metabolite derived from the parent 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. drug (Figure 3.15). A similar result was obtained In the urine sample from a rat treated with a 1:1 mixture of (-)-IF-d4 and (+)-IF (Figure 3.16). The final Identity of this metabolite was confirmed by GC/MS analysis of the synthetic 4-OHN3D. The authentic sample was treated with KCN followed by silylation as before. GC/MS analysis revealed that the trimethylsilyiated dehydrochlorinated cyanohydrin adduct of 4-OHN3D eluted at the retention time identical to and with a mass spectrum virtually Identical to those observed for the component In the urinary extracts, thus confirming the structure as 4-OHN3D (Figure 3.17). However, both 4-OHN2D and 4-OHN3D would lead to the same derivative (stereochemistry not Implied) following this same treatment (Scheme 3.14, R=H). It was therefore not possible to distinguish between the two structural Isomers on the basis of the authentic sample. Thus, the remaining task was to determine the exact location of the chloroethyl side chain. Differentiation between the two structures could be accomplished by specific deuterium labeling as shown In Scheme 3.14 (R=D). Following administration of [6,6,2',2'-2H4]IF, the metabolite N2D and subsequent 4-OHN2D would retain only two deuterium atoms. The silylated cyanohydrin adduct for the latter would generate the expected cyclic structure with only two deuterium atoms {m/z 424). On the other hand, N3-dealkylatlon would give rise to N3D and subsequent 4- 0HN3D with retention of all four deuterium atoms. The silylated cyanohydrin adduct under the GC/MS condition would generate the expected cyclic structure with retention of four deuterium atoms {m/z 426). After careful examination of the Ion cluster In the mass spectrum obtained from the urine of a rat treated with a 1:1 mixture of IF and [2',6-^H4]IF, an Ion at m/z 426 was discerned. However, an Ion at m/z 424 was also found but with higher Intensity (Figure 3.15,3.16). Therefore, 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. it was concluded that both primary metabolites N2D and N3D were further hydroxylated by cytochrome P450 mixed-function oxidases to form the secondary metabolites 4-OHN2D and 4-OHN3D, respectively, with the preponderance of the former. This contention was further investigated by giving a rat a 1:1 mixture of (±)-1F and (±)-[1'1',2',2'-^H4]IF. GC/MS analysis of the urinary extract revealed the presence of a trace amount of the d4 analog (representing 4-OHN3D, Figure 17). These data confirmed that 4-OHN2D, in comparison with 4-OHN3D, was the major secondary metabolite of IF. 3 4.2.4 N-dechloroethyl iphosphoramide mustard The identification of 4-0HN2D and 4-OHN3D prompted the search for its decomposition product/metaboiite, NDIPM. A urine sample obtained from a rat treated with (±)-IF was extracted by C-18 reversed-phase resin and the residue was derivatized with silylating agents (BSTFA:TMSI, 5:1). The derivatized sample was subjected to GC/MS analysis. A component with m/z 339 was eluted at the retention time of 6.2 min (Figure 1.188), which was absent from the blank rat urine sample following the same treatment (Figure 3.188 inset). The mass unit is consistent with the protonated moiecular ion of trimethylsilyiated dechlorinated NDIPM (Figure 3.19). Scheme 3.15 shows the formation of this compound from NDIPM. Further confirmation was obtained from the anaiysis of a urine sample obtained from a rat injected with of a 1:1 mixture of (±)-IF and (±)- [1'1',2',2'-2|H4]iF. GC/MS analysis showed a major component with an intensive ion at m/z 339 and a weak ion at m/z 343 (Figure 3.20). The ion at m/e 339 represented the NDIPM derived from the metabolite N2D, and the ion at m/z 343 derived from N3D-d4. The reiative abundance of these two peaks was consistent 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with the finding that 4-OHN2D, not 4-OHN3D, was the major secondary hydroxyiated metabolite. Samples from rats treated with a 1:1 mixture of (+)-IF- d4 /(-)-IF or (-)-IF-d4 /(+)-IF were processed and analyzed in the same manner. An ion at m/z 339 was observed as the major peak in these cases, representing NDIPM derived from N2D and from N2D-d2. A small peak at m/e 341 represented the existence of deuterated NDIPM (NDIPM-dg), which was derived from N3D-d4 (Figure 3.21, 3.22). The final identification was furnished by the synthetic authentic NDIPM. Under similar treatment, the authentic sample produced a major component at the identical retention time with a nearly identical mass spectrum (Figure 3.23). 3.4.2 5 N2,N3-dldechloroethyl ifosfamide The observation of the secondary hydroxylation pathways also suggested that sequential dechloroethylation of N2D and N3D might exist. Thus, the presence of N2N3D was examined in the urine samples from all rats. If such a metabolite existed, the silylated metabolite would exhibit an ion at m/z 353 under the present GC/MS conditions (Scheme 3.16). While there was no such ion found in the blank urine (Figure 3.180 inset), a component with an ion at m/z 353 was indeed observed in the urine samples from rats treated either with IF alone (Figure 3.180 and 3.24) or with a 1 ;1 mixture of (±)-IF and (±)-IF-d4 (Figure 3.25). No ion cluster was expected to be found in the latter case because of the complete loss of deuterium atoms. Since the four deuterium atoms in the molecule of (±)-IF-d4 were all in the N2 side chain, they were totally lost during the transformation to N2N3D. However, with [6,6,2',2'-2H4]IF, since two of the four deuterium atoms were on the oxazaphosphorine ring, they should be retained in the molecule of 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N2N3D. Indeed, an ion doublet at m/z353/355 was found in the mass spectrum of a derlvatized urine extract from rat treated with 1:1 mixture of (+)-IF-d4/(-)-IF or (- )-IF-d4/(+)-IF (Figure 3.26 and 3.27). The final confirmation was achieved by the chemical synthesis of N2N3D. The tri-(trimethylsilyl) derivative of N2N3D was eluted at the identical retention time as the metabolite, with an almost identical mass spectrum (Scheme 3.28). 3.5 DISCUSSION The versatility of deuterium labeling and MS in metabolite identification was demonstrated in this study. Labile, difficult-to-isolate metabolites, and those present in very small amounts could be readily identified by applying properly labeled drug after suitable chemical modification. In addition to unchanged IF and its known metabolites (IPM, N2D, and N3D), four new metabolites in the urine of rats injected with IF were identified. AlcolF has been detected in the blood of mice and in the urine of dogs (16). A recent report described the detection of alcolF in human urine (77). However, this is the first report of the presence of alcolF in rat urine. The activation of IF to 4-OHIF has been postulated to be parallel to the pathway of CP, since the metabolic successors (e.g., IPM, carboxylF, and 4- ketolF) of 4-OHIF/aldolF have already been identified as metabolites of IF. However, the definite existence of such metabolites has yet to be proven. Manz et al. (78), using ion-pair extraction and fast atom bombardment mass spectrometry (FABMS), identified and quantitated the conjugates of activated cyclophosphamide and ifosfamide with mesna (sodium mercaptoethyl sulfonate) in 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. rat and human urine. The authors suggested that these conjugates be derived from the respective 4-hydroxy metabolites {e.g. 4-OHCP and 4-OHIF) by displacement of the hydroxy group with mesna. In a separate study, Conners at a/. (11) identified 4-ethyloxy ifosfamide as the trapped 4-OHIF in an in vivo rat liver microsomal metabolism experiment using mass spectrometry. However, all these conjugates could also have been derived from addition of mesna or ethanol across the double bond between N3 and C4 of a possible imino intermediate (75). The identification of the cyanohydtin adduct of aldolF from the urine of rats treated with IF reported here provided more consistent and direct proof for the identification of 4-OHIF/aldolF as metabolites of IF. Nevertheless, the position of the postulated equilibrium between 4-OHIF and aldolF could not be ascertained. In an experiment investigating the stereoselective metabolism of IF in mice, Blaschke and Widey (52) found that 4-ketolF was formed almost entirely from (-)-IF, thus refuting the previously suggested rapid equilibrium between 4-OHIF and aldolF. While 4-OHIF is a chiral molecule, ring opening to aldolF results in the loss of chirality because of the existence of two Identical 2-chloroethylamino groups attached to the phosphorus atom. Thus, a rapid tautomeric equilibrium between 4- OHIF and aldolF would result in racemization, which would made the subsequent stereoselective oxidation to 4-ketolF impossible. The observed highly stereo selective oxidation to 4-ketolF would indicate that the subsequent oxidation occurred at a rate faster than the ring closure from the aldo tautomer. A fluorometric method (79,80) has been widely used for the quantitation of 4- OHIF and this method was based on the reaction of the released acrolein from 4- OHIF with m-aminophenoi to form fluoresent 7-hydroxyquinoline. This method is 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. proven here to be nonspecific since acrolein would also be formed from other 4- hydroxy metabolites such as 4-OHN2D and 4-OHN3D. Gilard etal. (77) used P-31 NMR to study the urinary metaboiite excretion of IF in patients receiving IF therapy. 4-OHIF and IPM were not detected because of chemical instability of the metabolites and intrinsic low sensitivity of the method, however, two degradation products of N2N3D but not itself were detected. These two species could be artifacts spontaneously formed during urine collection or sample processing and analysis, since 15-24 hours were generally required for the recording of signals in their approach. The contribution of secondary metabolism of these metabolites on the anticancer activity of ifosfamide is not expected to be significant. The end product of this pathway, NDIPM, appeared to be significantly lower than IPM in the urine of rats receiving IF, although it was also detected in rat plasma. In addition, this metaboiite is a monofuctional alkylating agent, which, in general, is unlikely to possess significant anticancer activity. In a preliminary cytotoxicity study conducted in this laboratory, NDIPM showed weak activity against L1210 cells. Although the etiology of the neurotoxicity observed in patients receiving IF has not been well established, evidence from electroencephalograms supports a metabolic or toxic etiology (81,82). Concomitant to the dealkylation pathways of IF, the presumed toxicophore CAA could also be derived from further metabolism of the two most abundant metabolites, N2D and N3D. CAA generated from hepatic and/or first-pass metabolism could be transported to the central nervous system (CNS) to exert its toxic effects, possibly by the formation of a Schiff's base with one of several essential CNS amino-containing neurotransmitters. The transport itself could be mediated by the association/dissociation process of this 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reactive Chemical with endogenous macromolecules (e.fir., albumins) as its carriers; alternatively, CAA could be generated on-site in the CNS, because it has been reported that the CNS also possesses a certain cytochrome P450 activity (10). Both of these two processes are possible. Further investigation to elucidate the mechanism of toxicity is necessary. In conclusion, our results showed that IF metabolism is more complex than that of its congener, CP (Scheme 3.1). In addition to the common activation pathway (4-hydroxylation), IF undergoes extensive side chain oxidation, leading to the formation of dealkylation metabolites (N2D and N3D) along with CAA. Furthermore, these dealkylated metabolites can be sequentially hydroxylated and/or dealkylated. The enzymes and isoenzymes involved in these reactions have not been fully characterized at this time. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 8 % 4-OHIF alcolF N3D TOT 387 13:23 a 16:48 15:08 1.68^ 16:48 422 15:88 16:48 15:88 8 : 2 8 13:28 6 : 4 8 18:08 11:48 Retention time (min) Figure 3.1 Total and selected ion GC/CIMS chromatograms of the derivatized CH2CI2 extract residue from urine of a rat receiving (±)-ifosfamide. A) Total ion; B) Selected ion m/z387 for alcoifosfamide; C) Seiected ion at m/z 412 for 4-hydroxyifosfamide; D) Selected ion at m/z 422 for 4- hydroxy N2-dechloroethyl ifosfamide or 4-hydroxy N3-dechloroethy! ifosfamide. 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I B B X 387 I I I • Cl I TMSO f 1 : 6 TMS M H * 387 389 |i)li| ; i / i | . i/il 1J 1 1 ' |T -r f n / z Figure3.2 Mass spectrum of derivatized alcoifosfamide detected in tfie urinary extract from a rat given (±)-ifosfamide. 81 Reproduced witti permission of ttie copyrigiit owner. Furttier reproduction proiiibited wittiout permission. 391 1 8 0 % g TMSO Cl 387 TMS M H * 387 D TMS M H * 391 393 T ' n - -f ï 208 223 Figure 3.3 ion cluster mass spectrum of derivatized alcoifosfamide detected in the urinary extract from a rat receiving a 1 ;1 mixture of (±)-ifosfamide and (±)-ifosfamide-d4 . 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. t est e 391 MSO TMSO 389 c s 387 ) TM S M H * 391 TMS M H * 387 e .Ë 393 I 460 426 440 288 228 248 288 288 388 328 340 368 m / z Figure 3.4 Ion cluster mass spectrum of derivatized alcoifosfamide detected in the urinary extract from a rat receiving a 1 ;1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % I .5 ■ I I TMSO M H * 387 D TMS M H * 391 T -I'f ' I ' I i-l^r^ -i'^r T T I ‘ 1 «-T 2Z8 248 268 288 388 387 389 391 i " I I I ] 340 368 388 393 ' T i-l-r-rr-r r 408 428 448 Figure 3.5 Ion cluster mass spectrum of derivatized alcoifosfamide detected In the urinary extract from a rat receiving a 1:1 mixture of (R)-lfosfamlde and (S)-lfosfamlde-d4 . 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 387 1 8 8 X £ b ç TMS M H * 387 / 389 i l n ,iii, 1 1 1 1 l ' i ' m r - ^ ' - - - 388 408 420 448 - r r ^ 288 T+T 223 248 268 288 388 323 348 368 m/t Figure 3.6 Mass spectrum of derivatized authentic alcoifosfamide. 85 Reproduced w«h permission of the copyrigh. owner. Further reproduction prohibited whhout permission. 188% I OTMS MH+412 412 / 414 388 488 428 448 288 228 248 268 288 328 m/i 348 368 Figure 3.7 Mass spectrum of derivatized authentic cyanohydrin of 4-hydroxy ifosfamide. 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % NC O T M S r ^ C o TMS MH*412 ü i k k 412 4 M - / 414 • I > 1 * — ] I I I I I * I : I > I l"l' *' t ^ ~J ' i * ( ^ i ^ I ' 1 ^ I * ' I ^ * * Ï * * 0 ^ * 280 220 240 260 280 300 328 340 360 388 408 428 440 m/i Figure3.8 Mass spectrum of derivatized cyanohydrin of 4-hydroxy ifosfamide obtained from the urinary extract from a rat given (±)-ifosfamide. 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IBBX I N "C OTMS MH+413 i|ii 1 I p ■ ■ ■ I « I 388 413 415 / I ‘ 1 *1 I " I I I , ' T ' I ' I 368 388 488 428 288 228 248 268 288 328 m/z - i - p r 348 Figure3.9 Mass spectrum of derivatized ‘'^c-cyanohydrin of 4-fiydroxy ifosfamide obtained from the urinary extract from a rat given (+)- ifosfamide. 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % I NC OTMS NO OTMS M H * 416 M H *412 416 418 ’f 'j' l V ‘ | ' I ‘ i ‘ i ' I ‘ > ' i 288 228 248 268 288 388 32 8 348 368 388 488 428 448 m / z Figure 3.10 Ion cluster mass spectrum of derivatized cyanohydrin of 4-hydroxy ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (±)-ifosfamide and (±)-ifosfamide-d4 . 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 0 % 416 NC OTMS IM S M H *412 NC OTMS 414 412- \ M H * 416 288 220 248 268 288 388 4iy> , I / 418 rn * 328 348 m/s 368 "T-f 388 480 428 448 Figure 3.11 Ion cluster mass spectrum of derivatized cyanohydrin of 4-hydroxy ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VBBx I NC O T M S r '^ l y S TMS MH* 412 NC OTMS M H * 416 I If I ' I ' I ‘ I ' I ‘ I ‘ I ' I ' I ' I ' ) ' 1 ' I ' I ' I I I' I 'I" 288 228 249 2 6 8 2 8 8 3 8 0 3 2 8 3 4 6 3 6 8 3 8 8 408 428 m/t ■ U j4 418 I ngure3.12 Ion cluster mass spectrum of derivatized cyanohydrin of 4-hydroxy ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (R)-ifosfamide and (S)-ifosfamide-d4. 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % I NC OTMS TMS MH+ 422 I ' f 't I i'- ‘ v ~ h " ' I ‘ I ' l ' i ' n " ' ! n - n - ' T ' I" »'!' 288 228 248 268 288 388 328 348 368 388 488 428 448 m / z J i 4 2 2 M l '‘ ‘h I ' 1 ■f P-T Figure 3.13 Mass spectrum of the siiyiated 4-hydroxy N2-dechioroethyi or N3- dechloroethyl ifosfamide cyanohydrins obtained from the urinary extract from a rat given (±)-ifosfamide. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % I I 'l*! j-i-v f 423 N’ ^C OTMS TMS MH* 423 'I ti. T-r-r-f 448 288 228 248 T 268 288 388 328 m / z 348 368 388 488 428 Figure3.14 Mass spectrum of the siiyiated 4-hydroxy N2-dechloroethyl or N3- dechloroethyl ifosfamide i^C-cyanohydrins obtained from the urinary extract from a rat given (±)-ifosfamide. 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 8 % J 422 OTMS IM S NC Q N OTMS TMS NC OTMS TM S MH + 422 M H + 4 2 4 |i.i 424 M H * 426 288 r 228 248 Tn^ 260 288 388 ' I '"I > r 328 348 m/z Figure 3.15 Ion cluster mass spectrum of the siiyiated 4-hydroxy N2-dechloro- ethyl or N3-dechloroethyl ifosfamide cyanohydrins detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 - 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0 8 % 422 1 O TM S TMS NC O t / M H + 4 2 2 PTM S TMS O N OTMS M H * 426 M H + 4 2 4 I ’ I ' I ■ '1 ' I 1 * * 1 * 1 I’I 'n ■ m / z Figure 3.16 ion cluster mass spectrum of the siiyiated 4-hydroxy N2-dechioro- ethyi or N3-dechioroethyi ifosfamide cyanohydrins detected in the urinary extract from a rat receiving a 1 ;1 mixture of (R)-ifosfamide and (S)-ifosfamide-d4. 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % 422 OTMS TMS O N - M H + 4 2 2 T 'T '' I ' I I I ' f "''! " '" ! ' 'I •’ "I I I ' ' I' ’ I 'I i ~ l I I '"I I 'I i ' | T-|-i [-1 T l T i I'T I' 2 8 8 228 248 268 2 8 8 388 323 348 3 6 8 3 8 8 488 428 4 4 8 m / z Figure 3.17 Mass spectrum of derivatized auttientic cyanohydrin of 4-hydroxy N3-dechioroethyl ifosfamide. 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 488% lO T - 2 ? s IS : ) .2 1 § 3 3 9 -j 1 ■s O S 3 5 3 - IPM / NDIPM 5 :8 8 6 : 4 8 5 :8 8 6 : 4 8 rT-rvT'i I I I . I I c 5 : 8 8 ’’'I ' ' ' ^ r - TT I I , ."I I I I I ry'11-T-.'i I I I I I I I . 6 : 4 8 8 :2 8 1 8 :8 8 1 1 :4 8 ' 1 1 I'l I " " I " " r ' 1 3 :2 8 1 5 :8 8 1 6 :4 8 Retention time (min) Figure 3.18 Total and selected ion GC/CIMS chromatograms of the siiyiated extract obtained from solid phase extraction of urine sample of a rat receiving ifosfamide. A) Total ion; B) Selected ion at m/z 339 for N- dechloroethyl iphosphoramide mustard; C) Selected ion at m/z 353 for N2,N3-didechloroethyl ifosfamide. 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 0 % TMS I N. > o TMSO N -^ TMS MH*339 T < y > i 339 • ’~r"'~T ■ I 480 428 T-f 288 228 248 268 288 ■ m 388 328 n t/t T ' I ' I ‘ r 348 368 388 r 448 Figure 3.19 Mass spectrum of the siiyiated N-dechioroethy! iphosphoramide mustard obtained from the urinary extract from a rat given (+)- ifosfamide. 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 B 8 X 339 S I TMS TMS TMSO N TMSO N — \ I TMS MH* 343 TMS 343 2 8 8 2 2 8 2 4 8 2 6 8 2 8 8 3 8 8 3 2 8 3 4 8 3 6 8 3 8 8 4 8 8 4 2 8 4 4 8 m/s Figure 3.20 Mass spectrum of the siiyiated N-dechioroethy! iphosphoramide mustard obtained from the urinary extract from a rat given a 1:1 mixture of (±)-ifosfamide and (±)-ifosfamide-d4 . 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % I A TMS A TMSO N- I TMS MH* 339 ,ii. ■ ,1 , 339 341 TMS TMSO N - ^ I D TMS M H * 341 I ' i 1 i'i't'7 I" 11 ' I 1*1^! 1 Y "I "I T I I |" T * i" rj I I I I' z e e 2 2 8 248 268 288 3 8 8 328 348 368 3 8 8 4 8 8 4 2 8 448 ni/z Figure 3.21 ion cluster mass spectrum of the siiyiated N-dechioroethyi iphospho ramide mustard detected in the urinary extract from a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4. 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10BX t TMS I N. TMSO X J N* I TMS MH+339 339 TMS v S A L - d TMSO N - ^ I 0 TMS M H * 341 ,| I «i^T-r-rY^ I I r I I’ l ' I 'T ' I ' I ' I ' I'n l 341 2 8 8 2 2 9 2 4 9 2 6 8 2 8 8 æ 8 3 2 8 3 4 8 n t/i 3 6 8 3 8 8 4 9 8 4 2 9 4 4 8 Rgure3.22 Ion cluster mass spectrum of the silylated 4-hydroxy N2-dechloro- ethyl or N3-dechloroethyl ifosfamide cyanohydrins detected in the urinary extract from a rat receiving a 1:1 mixture of (R)-ifosfamide and (S)-ifosfamide-d4. 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 180X I TMS Y] TMSO N -^ TMS MH*339 339 ' I ' I ' I ’’ "1 i"| 'I' I'I‘I*|-i-'|'i'[ I'l I I V'l ‘r^'« r-7 -i I "I ;'f '| I I I I I |"' i I I I'i'i I 2 8 8 2 Z 8 Z i a Z & 8 Z 8 8 3 6 9 3 Z a 3 4 B 3 Ê 8 3 8 8 4 0 0 4 2 8 4 4 a ttt/Z Figure 3.23 Mass spectrum of derivatized authentic N-dechloroethyi iphosphoramide mustard. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % I I 353 C TMS y 0 N — TMS / TMS MH+353 I i-r f l - fr T - f I zee 228 248 I ■ f n - n - ’- 268 288 388 328 348 mà J ^ I ' i • » ' J ' I * j • i ’ I • I 366 388 488 428 448 Figure 3.24 Mass spectrum of the silylated N2,N3-didechioroethyl ifosfamide obtained from the urinary extract from a rat given (±)-ifosfamide. 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % 353 f £ TMS / c x ^— 0 N — TMS TMS MH+353 - - "T 1 1 U ,1 1 \ 1" S Ij' . ili , . „ -i"r, m * , r-l- 288 228 248 268 288 328 343 368 388 488 428 440 m / z Figure3.25 Ion cluster mass spectrum of the silylated N2,N3-didechloroethyl ifosfamide obtained from the urinary extract from a rat given a 1:1 mixture of (±)-ifosfamide and (±)-ifosfamide-d4 . 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % I C TMS V " O N —TMS I 355 TMS MH* 353 353 J \ i j i TMS r y 0 - ) — O N —TMS ° TMS MH+355 I ■ 1 ' t ‘ I ‘ I ‘ I ' i 'T ^'i ‘’“ f f ‘i 'T '*-! I I ' l | ‘i r-v 1-i-i‘r-f I i"i"|- < -r 288 228 248 268 288 388 328 348 368 388 488 428 448 ni/z Figure 3.26 ion cluster mass spectrum of the silylated N2,N3-didechloroethyl ifosfamide detected in the urinary extract from a rat receiving a 1 ;1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TMS O N — TMS TMS N— TMS MH+355 M H*353 288 228 248 268 288 388 328 348 368 388 408 428 448 m /l Figure 3.27 Ion cluster mass spectrum of the silylated N2,N3-didechloroethyl ifosfamide detected in the urinary extract from a rat receiving a 1:1 mixture of (R)-ifosfamide and (S)-lfosfamide-d4 . 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IBBX 353 r TMS / N O X O N "“ TMS TMS M H*353 2 1 3 2 2 9 2 4 2 2 S 6 2 S S 2 3 7 3 2 4 3 3 8 I I I I | - i " I r ' f i T * i y 2 2 8 2 4 8 2 6 8 2 8 8 r '-T-rr?'I i*i"‘ r f 7 T |-v p - rr- [- 3 8 8 3 2 8 3 4 8 3 6 8 3 8 8 4 f f i m / z 3 7 7 3 B 9 4 8 9 4 2 1 4 3 5 2 8 8 I ' M 4 2 8 ' I ' 4 4 8 Figure 3.28 Mass spectrum of derivatized authentic N2,N3-didechioroethyl ifosfamide. 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V X ) 0= 0. X “> < V . J V i * * ' y V V , A x 0 = V V o J X 2 ■ v < ; v < Ï a c e 1 8 I C O C 3 . 0 Ô 1 E T3 I Q ) m 0 1 È 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. g I 8 C O 0 1 s c 0 B 3 ir C M C Ô 0} 1 ■ s w 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. . 0 X ) E Î 2 t 0 € § 1 0 C f l m 1 0 a 3 IT « C O 1 s 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. s "2 I E < D S I E S - o I I V 0 1 0 1 (T : E I 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V G V Q ) 2 I >» # 2 0 € ! z 1 0 1 I 0 B 3 IT in C Ô E 1 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lb W % S E I 4" 1 (D I I I - o I o S c CO CP I 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 u i X . X u O X o d X ° ; < : u a s •D E Î o > % I o x: Ü 0 ) 3 z 0 1 £ I 'o B 3 Œ 0 E 0 ) 1 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. u d V u (D •g E I t 0 ) p o 0 1 0 1 £ ! 0 B 3 ir 0 0 CÔ m 1 € w 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V I 1 E a > S I t (0 E .9- 0 1 f 0 •§ Œ 05 tri 0 5 1 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. i U 0 ° o , Û ' V V U I O X s E I § I g CM O « M 0 ) o O J o ce CO (U E 0 ) I 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HO NH MSTFA TMSO 0 / TMS AlcolP MH*387 Scheme 3.11 The formation of ion m/z387 from alcoifosfamide. (MSTFA: methyisilyltrifluoroacetamide) 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-OHIF OTMS KCN NC OH MSTFA M HM 12 Scheme 3.12 The formation of ion m/z 412 from 4-hydroxy ifosfamide. (MSTFA; methyisilyltrifluoroacetamide) 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N2D \ O H 1)KCN 2) MSTFA NH O M HM 22 N3D Scheme 3.13 The formation of ion m/z422 from 4-hydroxy N2-dechloroethyi ifosfamide and/or 4-hydroxy N3-dechloroethyi ifosfamide. (MSTFA: methyisilyltrifluoroacetamide) 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lO -M i r t i I 'a.'' ’ ; a I 1 a I j " 1-' i 1 s ' a I $ 1 0 / ‘ X / -4 S -^ z î M ‘ 1 ; | h ■ s J 2s 0 U - B i / / Q. 1 \° î < ■ II £ 0 ) S o € ? C O } ■ o c m s 1 C O o 0 ) o s z c 0 ) ? C sl z t T j- 0 ■l 1 ê Q ■ s f c d 0 ) i (J ) C D l i E E Î I 5 3 3 0 ) "O 0 ) s I o 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TMS ■ ^ y / M STPA V ] -Cl TMS M H * 339 Scheme 3.15 The formation of ion m/z339 from N-dechloroethyl Iphosphoramide mustard. (f^STFA; methyisilyltrifluoroacetamide) 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TMS NH p ------------------------------------------------------------------ -------- NH O y MSTFA / \ / / /\ \ A o NH; '------ O N----- TMS / TMS MH* 353 Scheme 3.16 The formation of ion m/z353 from N2,N3-dideuterioifosfamide. (MSTFA ; methyisilyltrifluoroacetamide) 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. C H A PTER 4 STEREOSELECTIVE METABOLISM OF THE ENANTIOMERS OF IFOSFAMIDE IN THE RAT 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.1 A B S T R A C T The titled study was performed using strategically deuterium iabeied enantiomericifosfamides(iFs), R-(+)- and S-(-)-6,6-dideuterio-3-(2-chloroethyl)-2- [{2-chioro-2,2-dideuterioethyl)amino]tetrahydro-7,5,2-oxazaphosphcrine 2-oxide (respectively, R- and S-IF-d4) synthesized in this iaboratory. These iabeied positions would minimize the potential isotope effect invoived in metabolism and serve as tracking probes for metabolic pathways of IF. When pseudoracemates consisting of a 1:1 mixture of R-iF-d4 /S-IF or R-iF/S-iF-d4 were given to male Sprague-Dawley rats at 40 mg/kg i.v., urinary metaboiites were extracted, derivatized, and monitored by GC/MS under ammonia chemical ionization condition. Products resuited from ring and side chain N-dealkylations (N2D and N3D), 0-4 hydroxylation alone (4-OHiF), C-4 hydroxylation with subsequent reduction (aicoiF), and cleavage to iPM were among the major metabolites identified, based on their GC-mass spectra and retention time data when comparing with those of authentic samples. Control experiments utilizing a 1:1 mixture of R-iF and R-IF-d4 and a 1:1 mixture of S-IF-d4 and R-IF to correct for any isotope effect were performed. Significant enantiomeric selectivity for the side chain dealkylation, 0-4 hydroxylation, and surprisingly IPM formation was observed. Low enantiomeric selectivity for the ring dealkylation, however, was found. These results may have implication in stereoselective antitumor activity and toxicity of IF. 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 .2 IN T R O D U C T IO N It has now become clear that the enantiomers of a chiral molecule may display major differences in metabolism, pharmacokinetics, and pharmacological effects (46,83). IF, a widely used alkylating agent and a structural isomer of another important drug CP, possesses an asymmetric phosphorus atom, therefore existing in enantiomeric (R and S) forms (4-7). The clinically used IF is a racemic mixture. IF Itself is not cytotoxic but requires hepatic activation to exert both therapeutic activity and side effects (45). The foremost important metabolic pathway is hydroxylation at the endocyclic C-4 position, generating 4-0HIF. Possibly similar to its congener 4-OHCP, 4-OHIF may also be a transport form of an active metabolite across cells (12,84). Subsequent ring opening and cleavage of 4- OHIF forms IPM, the purported ultimate intracellular alkylating metabolite of IF (14,16,85). Dealkylation on the exo and endo nitrogen atoms generates N2D and N3D (13,20-24) with the concomitant formation of CAA, which has been implicated in the involvement in the observed neurotoxicity in patients receiving IF therapy (22,86). These metabolic pathways may display stereoselectivity and the study of this possibility constitutes the focus of the present report. To facilitate this investigation, pseudoracemates consisting of a 1 :1 mixture of labeled and unlabeled enantiomers with opposite configurations were used (58). When administered in vivo, the fate of each antipode can be simultaneously monitored by a discriminatory GC/iyiS methodology. 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 .3 M A T E R IA L S A N D M E TH O D S 4.3.1 Materials 4.3.1.1 Chemicals and reagents R- and 8 - ifosfamide (8 - and R-IF), R- and 8 -ifosfamide-d4 (R- and 8 - IF-d4 ), 4-OHIF, N2D, N3D, alcolF, and IPM were all synthesized in this laboratory (Chapter 2 and 3). Chemical structures of pseudoracemates and two control pairs are shown in Figure 4.1. Racemic IF was provided by Drug 8 ynthesis and Chemistry Branch, the National Cancer Institute (Bethesda, MD). All organic solvents were of HPLC grade and purchased from E. M. 8 cience (Gibbstown, NJ) and used without further purification. N-Methyl-N-trimethylsilyl-trifluoro- acetamide (M8 TFA), N,0-bis(trimethylsilyl)trifluoroacetamide (B8 TFA), and N- trimethylsilylimidazole (TM8 I) were purchased from Pierce (Rockford, IL). C18 reversed-phase resin was obtained from Analytichem International (Harbor City, CA). 4.3.1.2 Surgical Instrument and supplies Surgical instruments were purchased from Roboz Surgical Instrument Co., Inc. (Washington, DC). Monoject hypodermic needles and Monoject sterile disposable tuberculin syringes without needle were obtained from Western Medical Supply (Arcadia, CA). Surgical sutures (size 3 and 4) were purchased from George Tieman & Company (Plainview, NY). Curity gauze sponges were obtained from Kendall Company (Boston, MA). Intramedic nonradiopaque polyethylene 50 tubings (PE 50, 0.58 mm I.D. and 0.965 mm O.D.) were purchased from Fisher Scientific (Tustin, CA). 0.9% Sodium chloride injection, USP was obtained from 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Western Medical Supply (Arcadia, CA) as was heparin sodium injection USP 1000 units/ml. isopropyl alcohol was obtained from Fisher Scientific (Pittsburgh, PA). 4.3.1.3 Instrumentation A iTS40 Ion Trap mass spectrometer directly coupled to a 3300/3400 Varian gas chromatograph (Walnut Creek, CA) and CTC A200S autosampler with a capillary splltless injector were purchased from Finnigan MAT (San Jose, CA). Refrigerated centrifuge "Accupsin FR®" was obtained from Beckman Instrument, inc. (Palo Alto, CA). 4.3.2 Methods 4.3.2.1 Animal surgery - jugular vein cannulation Animal experiments were carried out according to a protocol approved by the Animal Use Review Committee at the Ohio State University. Six male Sprague- Dawley rats (Harlan, Indianapolis, iN) weighing between 250-300 g were used in this study. Two rats (rat 3 and rat 4) were given a pseudoracemate consisting of R-iF-d4 and S-IF; another two rats (rat 5 and rat 6 ) were given a pseudoracemate consisting of S-iP-d4 and R-IF. Another two rats were used as controls and each was given a 1 ;1 mixture of R-iF-d4 and R-iF (rat 1 ) and of S-IF-d4 and S-iF (rat 2). Rat chow (Teklad, indianapoiis, IN) and water were given ad libitum. The jugular vein of the rat was cannulated under diethyl ether anesthesia (Chapter 3). The animai was ailowed to recover for two hours before dosing. 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.2.2 Metabolic study After the rat became completely conscious, 1 ml of a saline solution of the appropriate drug mixture at the total dose of 40 mg/kg was injected to the animal through the jugular vein cannula. The cannula was then washed with 3 x 0.3 ml of 0.9% sodium chloride solution. Urine sample was collected continuously for 24 hours, frozen with solid CO2 upon collection, and stored at -76°C until GC/MS analysis (Scheme 4.1 ). 4.3.2.S Sample extraction and derivatization For the determination of enantiomeric ratios of unchanged IF, 4-OHIF, N2D, N3D and alcolF, 50 mg of KCN was at first added to 0.5 ml of urine from each rat to trap 4-OHIF into its cyanohydrin adduct. The mixture was allowed to stand at room temperature for 30 min. Dichloromethane (5 ml) was added and the mixture was shaken for 15 min In a horizontal shaker (Eberbath, Ann Harbor, Ml). The organic phase was separated after centrifugation, and dried under a stream of N2 . The residue was derivatlzed with 35 ni of MSTFA for 60 min at 120°C. An aliquot (1 pi) of the mixture was analyzed by GC/MS (Scheme 4.2). For the determination of the ratio of IPM generated from each enantiomeric IF, 0.5 ml of rat urine was placed onto a disposable Poly-prep column (Bio-Rad, Richmond, CA) containing 400 mg of 018 reversed-phase resin. The resin was washed with 0.5 ml of cold saline (0-5°C) followed by centrifugation at 200 x g for 20 min to remove water. The mini-column was then eluted with 1 ml of anhydrous methanol, and the methanol fraction was collected. Methanol was removed under a stream of N2 at room temperature. During the process of elution and evaporation, 4-OHIF, originally existing in urine, was totally converted to IPM. 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The residue was derivatlzed with 35 jx! of a mixture of BSTFA and IM S! (5:1 ; v/v) at 120°C for 60 min. A aliquot (0.5 \i\) of the mixture was analyzed by GC/MS (Scheme 4.3). 4.3.2.4 GC/MS Analysis The temperatures of the injection port, transfer line, and source were maintained at 220,260 and 230°C, respectively. The analysis was carried out on chemical ionization mode using ammonia as the reagent gas. A 30 m x 0.25 mm I.D. capillary column bonded with a 0.25 um film thickness of 5% methylsilicone (DB-5 column, J&W Scientific, Folsom, CA) was used for the separation. For the analysis of IF, 4-OHIF, N2D, N3D and alcolF and their labeled analogs, the temperature of the oven was programmed at 150°C for 2 min and increased to 190°C at a rate of 5°C/min, and then to 250°C at a rate of 15°C/min. The final temperature was held for 3 additional min. The same GC temperature program described above was used for the analysis of total IPM and its labeled dg analog. 4.4 RESULTS 4.4.1 GC/MS characterization of pseudoracemic IF and metabolites To initiate the stereochemical study, 4-OHIF, alcolF, IPM, N2D and N3D were used along with IF to characterize for their GC/MS properties including retention time and mass spectrum data. They were derivatized by appropriate siiylating agents prior to GC/MS analysis to reduce their polarity and to increase the stability. Then ions for the major fragments from each of the authentic compounds and their respective retention times were determined (Table I). Urine samples 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. obtained from the control and treated animals were processed according to the appropriate extraction procedures as described previously. Unchanged IF, 4- OHIF, N2D, N3D, and alcolF were extracted from urine by dichloromethane after KCN treatment. The derivatized residue was analyzed by GC/MS using the condition previously described. Ions specific for each chemical entity was also selected for monitoring. A representative GC/MS total ion chromatogram of the derivatized urinary CH2 CI2 extract obtained from a rat given a 1:1 mixture of S- IF/R-IF-d4 is shown in Figure 4.2. Upon derivatization with MSTFA followed by GC/MS analysis, the unlabeled and the deuterium-labeled IFs were detected as their dehydrochlorinated derivatives at m/z 225 (MH+) and m/z 229 (MH+) (Figure 4.4), respectively, with the retention time of 10.2 min (Figure 4.3). Scheme 4.4 illustrates the formation of these compounds from pseudoracemic IF upon MSTFA treatment. As shown in Figure 4.3 inset, no interference ions in the corresponding retention time region were found in the blank urine sample. N2D generated from the metabolism of unlabeled IF and N2 D-d2 from the deuterium-labeled IF were detected as their trimethylsilyl dehydrochlorinated derivatives at m/z 235 (MH+) and m/z 237 (MH+) (Figure 4.6), respectively, with the retention time of 10.0 min (Figure 4.5). Scheme 4.5 illustrates the formation of these compounds from N2D and N2 D-d2 derived from psudoracemic IF upon MSTFA treatment. As shown in Figure 4.5 inset, no interference ions in the corresponding retention time region were found in the blank urine sample. The N3-dealkyiated metabolites N3D generated from unlabeled IF and NSD-d^ from the deuterium-labeled IF were monitored also as their trimethylsilyl dehydrochlorinated derivatives (Scheme 4.6) and were detected at m/z 235 (MH+) and at m/z 239 (MH+) (Figure 4.8), respectively, with the retention time of 6.9 min (Figure 4.7). As shown in Figure 4.7 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. inset, no interference ions in the corresponding retention time region were found in the blank urine sample. AlcolF derived from unlabeled IF and alcolF-d4 derived from deuterium -labeled IF were detected as their trimethylsilylated dehydrochlorinated derivatives (Scheme 4.7) at m/z 387 (MH+) and at m/z 391 (MFI+) (Figure 4.10), respectively, with the retention time of 14.60 min (Figure 4.9). As shown in Figure 4.9 inset, no interference ions in the corresponding retention time region were found in the blank urine sample. For the assay of 4-OHIF, it was necessary to first trap this species as a stable intermediate because of its chemical instability. Thus, the added KCN reacted with the open-chain tautomeric form of 4-OHiF, aldolF, to form the stable cyanohydrin adduct, shifting the equilibrium to the entire aldehyde form. This procedure was similar to the detection and analysis of 4-OHCP (75,76,87). Derivatization with MSTFA converted the cyanohydrin adduct of 4-OHIF and its labeled analog into trimethylsilylated dechlorinated derivatives (Scheme 4.8) which were detected at m/z 412 (MH+) and at m/z 416 (MH+) (Figure 4.12) with the retention time of 16.4 min (Figure 4.11). No interference ions in the corresponding retention time region were found in the blank urine sample (Scheme 4.11 inset). For the assay of total IPM, a solid-phase extraction procedure was used. It was previously found that IPM could not be completely derivatized by MSTFA but was fully converted to the silyl derivative by the use of BSTFA-TMSi (5:1 ; v/v) (85). Representative total and selected ion GC/MS chromatograms of the derivatized extracts from a rat given S-IF/R-IF-d4 are shown in Figure 4.13 and 4.14. After separation on a GC column, the trimethylsilyl derivatives of dechlorinated IPM and its da analog (Scheme 4.9) were detected by MS at m/z 329 (MH+) and m/z331 (MH+) (Figure 4.15), respectively, with the retention time 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of 9.8 min (Figure 4.14). As shown in Figure 4.14 inset, no interference ions in the corresponding retention time region were found in the blank urine sample. Since 4- OHIF is converted to IPM during sample processing, 4-OHIF was assayed as IPM. Thus, the measured IPM levels in this study represented the total IPM, i.e., the actual IPM plus IPM generated from 4-OHIF. Table 4.1 summarizes the ions selected and the retention times for each of the components monitored. The identities of the ion peaks of IF and its metabolites were confirmed by comparing their retention times and mass spectra with those of authentic samples. 4.4.2 Enantiomeric ratios of IF, 4-OHiF, N2D, N3D, alcolF and total IPM in rat urine The enantiomeric ratios of IF and its metabolites were determined by dividing the areas of the appropriate ion peak derived from one enantiomer (e.g. the area of ion at m/z 412 for 4-OHIF derived from S-IF) by that from the other enantiomer (e.g. the area of ion at m/z 416 for 4 -OHIF-d4 derived from R-IF-d4 ) for each chemical entity. Table 4.2 shows the results from the two control experiments. When a 1:1 mixture of R-IF-d4 and R-IF or S-IF-d4 and S-IF was administered to a rat, enantiomeric ratios of IF and all its metabolites were not significantly different from unity (ranging from 0.91 to 1.11), indicating that no appreciable deuterium isotope effect due to metabolism was observed on the present labeled positions. in the two rats given a pseudoracemate composed of R-IF-d4 and S-IF, the parent drug recovered from the 24 hr urine displayed no enantiomeric enrichment and gave an average enantiomeric ratio of 1.08 (Table 4.3). 133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For 4-OHIF, an average R/S ratio of 1.53 was obtained, clearly indicating that the P450 enzyme system preferred the R configuration of IF. However, whether or not the hydroxylation also exhibited substrate-product enantiomeric selectivity cannot be ascertained because of the ring opening tautomerism of 4-OHIF to aldolF which lost the asymmetry bearing the hydroxy moiety. This enantiomeric preference was preserved in two subsequent metabolic pathways, cleavage to IPM and reduction to alcolF. The mean R/S ratios of the metabolites were 1.51+0.03 and 1.57+0.01, respectively. Stereoselective metabolism of IF was also observed in the N-dealkylation pathways. While the dealkylation on the endo nitrogen (N3) only displayed a small preference for the R configuration (R/S ratio 1.13±0.10), that on the exo nitrogen (N2) showed a pronounced preference for the opposite (S) configuration (R/S ratio 0.33±0.02). Similar results were obtained in two rats given the opposite pseudoracemates (S-IF-dVR-IF) at the same dose (Table 4.3). The R form of ifosfamide was slightly enriched in the urine (R/S 1.13+0.03), possibly due to inter-subject variation. While the exact values of the R/S ratios of metabolite composition were not the same, the general trend of the above results regarding stereoselective metabolism of this drug was retained, with the exception that in one of the rats, N3-dealkylation preferred S-IF slight over the R configuration (R/S ratio 0.92+0.12). This could be due to the individual metabolic difference, a phenomenon also observed in human studies (23,53). 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 .5 D IS C U S S IO N It Is of interest to investigate the stereoselective metabolism of oxazaphosphorines since these drugs possess chiral phosphorus atoms rather than carbons as are the cases for many drugs. Furthermore, these drugs are themselves inactive and require metabolic activation to exert their antitumor activity and toxicity. Owing to the potential stereoselectivity of their metabolic processes, it is entirely possible that the enantiomers may exhibit difference in pharmacologic activity and/or toxicity. Thus, the stereoselective metabolic information may help to select a better enantiomer (eutomer) for clinical use. it was demonstrated that metabolism of the enantiomers of CP and its intermediates was stereoselective in the mouse, rat, and rabbit, and that there were marked species differences in the extent and preference of the stereoselectivity (60,89). Since the optical activity of enantiomers of IF is much higher than that of CP (ao 39.2° vs. 2.3°), it would be of interest to find out if the degree of stereochemical difference in metabolism between the enantiomers of IF is also greater than that of CP. The use of pseudoracemates in the study of stereoselective metabolism offers several advantages. First, the pseudoracemic mixture mimics the use of race mate in the clinical situation but the label can be used for tracking the fate of the individual enantiomers. Secondly, the enantiomeric interaction if exists will be preserved (47). Thirdly, the concomitant administration of the enantiomers will avoid the generation of inter-subject variability in separate studies. Fourthly, this design will greatly reduce the number of animals necessary to accomplish the same study objectives and may generate better quality of data. 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The study of stereoselective metabolism using pseudoracemate requires a proper labeling at positions not affected by metabolism to avoid isotope effects (58). For IF, labeling at the C4, C5, C l' and 01" positions would not be unsuitable since all these sites are involved in biotransformations (45). Therefore, enantiomers strategically labeled with deuterium atoms at the 06 and 0 2 ' positions were selected and synthesized for this study. Additionally, the 02' labels may be potentially useful In tracking the fate of the side chain, since the metabolic product (OAA) may be involved In side effect and toxicity of IF, whereas the labels at 0-6 may be useful in following the fate of the ring moiety. In this study. It was assumed that neither of the enantiomers undergoes inversion of configuration in vivo. Although no direct evidence is available for IF, NMR study using optically active shift reagents show that there was no such racémisation for its structural isomer, OP, during in vitro metabolism (88,89). In order to probe into the stereoselective pathways, three important metabolic pathways were examined: the activation pathway leading to the formation of the primary active metabolite 4-OHIF, Its subsequent cleavage to form IPM, and the reduction of 4-OHIF to form alcolF. The enantiomeric ratio of each of these metabolic products was measured. Additionally, two dealkylation pathways leading to the toxic metabolite CAA were also examined by measuring the enantiomeric ratios of N2D and N3D derived from IF enantiomers. Data from the control experiments using a 1:1 mixture of deuterium-labeled and unlabeled IF of the same enantiomeric form (both S or both R) showed minimal isotope effect, which ensured the proper interpretation of data from pseudoracemates. 136 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stereoselective metabolism of IF in all three metabolic pathways were demonstrated in the present study. The hydroxylation pathway at C-4 preferred the R configuration and this preference was preserved through the subsequent decomposition to IPM. This finding is consistent with the contention that the decomposition of 4-OHIF to IPM is non-enzymatic. The enantiomeric ratio of the derived alcolF was also nearly the same as that of their precursors aldolF/4-OHIF, indicating no substrate stereoselectivity for the equilibrium between 4-OHIF and aldolF. Unlike CP, IF undergoes extensive side chain dealkylation. Both of its 2- chloroethyl moieties could be lost metabolically, where different patterns of stereoselectivity were observed. While N3 dealkylation showed slight preference for R configuration, N2 dealkylation displayed strong preference for the S configuration. Interestingly, the enantiomeric ratio of the parent drug excreted in urine was found not significantly different from unity even though significant stereoselective metabolism was demonstrated. This fact illustrates the significance in probing into stereoselectivity of metabolite formation even in the absence of enantiomeric enrichment of the parent drug (56). Previous stereoselective metabolic studies with IF showed a substantial species difference (23,52,53). Blaschke etal. (52) administered the enantiomers of ®H-labeled IF intraperitoneally to female NMRI mice. They found that urinary excretion of the unchanged IF was higher in mice treated with R-IF compared that with S-IF (1.3:1), while no difference in urinary levels of 4-OHIF and IPM were found. They also found significant stereoselective side-chain dealkylations. N2D was generated preferentially from R-IF (1.9:1), while N3D was produced to a 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. greater extent from S-IF (3.3:1). However, these results differed from those in human. Using ^ipNMR with a chiral shifting agent Eu(tfc)3 Misiura et al. (23) determined the urine enantiomeric composition of IF and its two metabolites (N2D and N3D) in two patients given racemic ifosfamide. R-IF was found to be slightly enriched (1.28:1); however, N2D was preferentially formed from S-IF (4.7:1). The stereoselectivity for N3D was not clear-cut: in one patients, N3D was generated stereoselectively from R-IF (2.57:1), while in the other patient, it showed a slight preference for the S-isomer (1.22:1). It was not possible to analyze 4-OHIF and IPM due to a technical problem. Similar result was obtained in a separate study when the urinary excretion of N2D and N3D in 14 pediatric patients was examined (53). The results from our study resemble those in the human but differ from those of mice. Several techniques in the pseudoracemate-GC/MS methodology has proven useful in this study. It was also found advantageous to use chemical ionization (Cl) over electron impact ionization (El) in the MS analysis. In a study on the differential metabolism of enantiomeric CP by Cox et al. using pseudo- racemate/EIMS (60), the theoretically preferred enantiomeric CP with deuterium labels on the C2' and C2" to minimize isotope effect could not be used since the terminal -CD2CI was lost under electron impact condition to give a predominant peak at [M-CDaCI]'*'. Thus, labeled enantiomers with deuterium on the Cl ' and C l " (a to the exo nitrogen atom) had to be used in their study and significant isotope effect due to N-dealkylation has greatly compromised their data interpretation. On the other hand, the CIMS in the present study allows the use of labeling at the 2 ' position of the chloroethyl side chain because no such cleavage was found. 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chemical modification of the IF derivatives prior to MS has also facilitated the enantiomeric analysis. For example, dehydrochlorination of these derivatives either during derivatization or on the GO column eliminated one chlorine atom. This elimination allows the use of the d^/do pseudoracemic pairs and the direct estimation of R/S ratio for the parent drug and metabolites without any correction for isotopic contribution for 37ci-containing ions. On the other hand, the use of an alternate analog with more deuterium atoms might impart a potential isotope effect and their synthesis would have been more difficult. This advantage covered IF, 4- OHIF, N2D, N3D and alcolF. However, in the case of IPM, the enantiomeric ratio as estimated by ions at m/z 329 and at m/z 331 stiii required correction of the isotopic contribution from 3^01 for ion at m/z 329. This complication arose due to the loss of two deuterium on the oxazaphosphorine ring during p-eiimination to form acrolein and IPM from 4-OHIF. The mass difference between two sources of iPM from IF pseudoracemate was no longer 4 but 2 which necessitate the isotope correction from chlorine. The observed stereoselective metabolism of IF might have clinical and pharmaceutical implications. Our data clearly showed that the R form of IF was more readily activated by the cytochrome P450 mixed-function monooxygenases to form 4-OHIF. On the other hand, S-IF unden/vent N2-dealkylation to a greater extent than R-IF, producing a higher amount of the co-product, CAA, the metabolite implicated in the observed neurotoxicity in patients receiving IF therapy. In fact, oral use of IF was abandoned because of a higher incidence of neurotoxicity (22,90). Thus, the overall benefit of using R-iF in place of racemic IF is considerable. While for economic reason, the use of a racemic drug is understandable; however, for compelling advantages such as an increase in 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. efficacy and reduction in toxicity, i.e., an substantial increase in ttie therapeutic index, the use of a pure enantiomer may be justified. 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4 .1 The selected ions and the respective retention times of derivatives of enantiomeric ifosfamides and their metabolites monitored. Chemical entity Selected ion {m/z) Retention time (min) IF S- or R-IF 225 1 0 . 2 0 S- or R-IF-d4 229 10.17 N2D N2D 235 10.07 N2D-d2 237 10.05 N3D N3D 235 6 . 8 8 N3D-d4 239 6.85 IPM IPM 329 9.82 IPM-d2 333 P C I) 9.80 4-OHIF 4-OHIF 412 16.43 4-OHIF-d4 416 16.42 alcolF alcolF 387 14.62 ak)cllF-d4 391 14.60 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.2 Isotopic composition of ifosfamide and five metabolites in urine of rats following administration of a 1 :1 mixture of deuterium-labeled and un labeled ifosfamide of the same enantiomeric form (control). Drug/metabolite Ratio do-da or do:d4 excreted Rat 1 Rat 2 IF 1 .1 1 1.03 4-OHIF 1.03 1 .0 1 aloolF 1.07 0.97 IPM 1 .0 2 1 .0 1 N2D 0.91 1.04 N3D 0.96 1 .0 0 Rats were each given a pseudoracemate consisting of R-IF/R-IF-d^ (rat 1) or S-IF/S-IF-d^ (rat 2), each at a total dose of 40 mg/kg iv. 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ o I c 8 O . ■o CD C/) W o ' 3 2, 5" CD 8 ■ o v < ë ' i 3 CD C p. CD ■ D O Q. C a O 3 T 3 O CD Q. Table 4.3 Isotopic composition of ifosfamide and five metabolites In urine of rats following administration of pseudoracemates. Drug/metabolites excreted Ratio d2 :do or d4 :do Rat 3 Rat 4 A Ratio doida or do:d4 Rat 5 Rat 6 A IF 1.04 1.12 1.08 1.15 1.10 1.13 4-OHIF 1.59 1.47 1.53 1.79 1.51 1.65 alcolF 1.53 1.48 1.51 1.71 1.50 1.61 IPM 1.58 1.56 1.57 1.61 1.52 1.57 N2D 0.35 0.31 0.33 0.44 0.35 0.40 N3D 1.23 1.03 1.13 1.03 0.80 0.92 Rats were each given a pseudoracemate consisting of R-IP-d^/S-IP (rat 3 and 4) or R-IP /S-IP-d4 (rat 5 and 6), respectively, each at 40 mg/kg total dose iv. "O CD C/) o' 3 u. a 0 = 0 . o c / { o c o = ■ a u L u. I J - u. u . ir Ô ) w £ E 55 C V J o a C Q i I 0 1 IX ! s C V J I I I I I I 0 1 Ï Î 1 0 g 1 . 1 E S E 3 IT 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N 2 D 4-OHIF alcolF g N30 3 :2 8 6 : 4 8 13:28 16:48 Retention time (min) Figure4.2 Tolal ion GC/CIMS chromatogram of the derivatized CHgClg extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . 145 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22% 225 5 23/ 229 18:08 16:48 28 1 0 :8 0 Retention time (min) 13:28 Figure4.3 Selected ion [m/z225 for (S)-ifosfamide and m/z 229 for (R)- ifosfamide-d^] GC/CIMS chromatograms of the derivatized CH2CI2 extract residue from urine of a rat receiving a 1 ;1 mixture of (S)- ifosfamide and (R)-ifosfamide-d4. Insets show those from rat blank urine. 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 108% c o I 229 225 C X ' M H + 225 4 D M H * 229 l~'‘ I '' I " T ‘ I i ' T ‘ " r i " | '" Y -, , 1 - , 't ' i ' i -, n ' i f |- T -| - r T T -T - i - i - i'T - r 'i i | 200 220 240 260 200 300 320 340 360 380 400 420 440 460 /tl/z Rgure4.4 Mass spectrum of the derivatized (S)-ifosfamide and (R)-ifosfamide- d4. from CH2 CI2 extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . 147 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32% 18 .'88 18:88 1 3 :2 8 3 : 2 8 6 : 4 8 1 8 :8 8 Retention time (min) 1 6 : 4 8 Figure 4.5 Selected ion (m/z235 for N2-dechloroethyl ifosfamide and m/z237 for N2-decfiioroettiyi ifosfamide-dg) GC/CIMS cfiromatograms of tfie derivatized CH2CI2 extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . Insets stiow those from rat blank urine. 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1B0% 235 c o « TMS 235 237 o M H + 237 TMS I I t ' 'I~ ' 'i "r ' T ‘" ï ' “i '' i"' T-i- r f - i-r i - r ^- r 200 220 210 260 280 300 320 310 360 300 4Q0 120 110 tn /z 160 Figure 4.6 Mass spectrum of the derivatized N2-dechloroethyl ifosfamide and N2-dechioroethyl ifosfamide-dg from CH2 CI2 extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)- ifosfamide-d4 . 149 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.13% 235 î e c 3.13 .> I 239 l > u u l ! ... A » A iw Vu. _ i 6 :4 8 6:40 ,,M ( Ij l'il \ k u . '| k A .t. o'i k r w / M f r , , . , . ', . y w i 20 6:40 1 0 :0 0 Rétention time (min) 13:20 16:40 Figure4.7 Selectetj Ion {m/z235 for N3-dechioroethyi ifosfamide and m/z239 for N3-dechloroethyf ifosfamide-d^) GC/CIMS cliromatograms of the derivatized CH2 CI2 extract residue from urine of a rat receiving a 1 ;1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . Insets sfiow those from rat blank urine. 150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 108% 239 235 T M S TMS I a § I 200 220 240 260 283 360 323 340 360 330 403 423 440 460 tn /z Figure 4.8 Mass spectrum of the derivatized N3-dechloroethyl ifosfamide and N3-dechloroethyl ifosfamide-d4 from CH2 CI2 extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)- ifosfamide-d4 . 151 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8.31% 387 g 8.41/ I 3 91 r 1 3 :2 8 ■If' 1 I .'.■ 'I I 1 I.< 1» I II u M l , II 1 w I , 1 I " I Ik i l IW wv.v A J iV -s -;' 1 3 :2 8 A, Di . ■ . m iK J ll» .! T // ,.A lA .ll,H .A 6 :4 8 3 : 2 8 1 8 :8 0 Retention time (min) 13:28 16:40 Figure 4.9 Selected ion (m/z387 for alcoifosfamide and m/z391 foraicoifos- famlde-d4 ) GC/CIMS chromatograms of the derivatized CH2 CI2 extract residue from urine of a rat receiving a 1:1 mixture of (S)- ifosfamide and (R)-ifosfamide-d4. Insets show those from rat blank urine. 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % TMSO TM Sq 391 t o I TMS M H + 3 S7 D TMS 387 200 220 240 260 230 300 320 340 360 ' 303 430 420 440 460 nt/z Figure 4.10 Mass spectrum of the derivatized alco ifosfamide and alco ifosfamide- d4 from CH2CI2 extract residue from urine of a rat receiving a 1 ;1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . 153 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.32% 412 g 1.78%“ 416 I 13:28 16:40 U ilil hlH < 1 1 ih'U 13:20 16:40 iv a ^ I j, > y i U A « I h hi'd L iti — i; y 3:28 6:48 18:88 Retention finie (min) 13:20 I 16:40 Figure4.11 Selected ion (m /z4l2 for4-hydroxy ifosfamide and /7i/z416for4- hydroxy ifosfamide-d4) GC/CIMS chromatograms of the derivatized CH2CI2 extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . Insets show those from rat blank urine. 154 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 8 % NC OTHS G r T M S MH'*’ 412 ilJà NC pTMS T M S *' M H * 416 Ml 416 412 280 220 248 268 288 380 328 348 368 388 488 420 448 468 m/z Figure 4.12 Mass spectrum of the derivatized 4-hydroxy ifosfamide and 4- hydroxy ifosfamide-d4 from CH2CI2 extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4 . 155 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1B8% IPM Î c § 5 3 : 2 8 1 3 :2 8 Retention time (min) Figure 4.13 Total ion GC/CIMS chromatogram of the derivatized solid phase extract residue from urine of a rat receiving a 1 ;1 mixture of (8)- ifosfamide and (R)-ifosfamide-d4 . 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 . 36% 3 2 9 î c 1 8 :8 8 1.38%" 3 3 3 IL U 1 8 :0 8 1 8 :0 8 13:28 1 6 :4 8 Retention time (min) Figure4.14 Selected ion (/7Vz329 for iphosphoramide mustard and m/z333ior iphosptioramide mustard-dg) GC/CIMS chromatograms of the derivatized solid phase extract residue from urine of a rat receiving a 1:1 mixture of (S)-ifosfamide and (R)-ifosfamide-d4. Insets show those from rat blank urine. 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 188% 331 e o 329 TMSO 208 220 240 260 I I I I I r 288 380 320 - Ç TMS C l I TMS M H + 329 M H + 331 333 ‘ I ‘ I ‘ I ' 340 368 T-hr n " 380 400 I' I I 'I ‘ " p "I 420 440 460 «✓r Figure 4.15 Mass spectrum of the derivatized iphosphoramide mustard and iphosphoramide mustard-d2 from solid phase extract residue from urine of a rat receiving a 1 ;1 mixture of (S)-ifosfamide and (R)- ifosfamide-d4 . 158 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. jugular vein cannulation and i.v. bolus IF total dose 40 mg/kg urine collection 0-24 hr GC-MS assay for IPM GC-MS assay for (S)-IF, (R)-IF, N2D, N3D and 4-OHIF Scheme 4.1 Flow chart of stereoselective metabolic studies of ifosfamide. 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. urine sample Ng to dryness extraction with CH2CI2 trapping with KCN 30 min at r.t. derivatization with MSTFA 120°C/60 min GC-MS (Finnlgan ITS40) column: DB-5, capillary temp: 150-250% Scheme 4.2 Flow chart of GC/MS assay for (S)-ifosfamlde, (R)-ifosfamide, N2- dechloroethyl ifosfamide, N3-dech!oroethyl ifosfamide, 4-hydroxy ifosfamide, and alcoifosfamide. 160 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. urine sample C l 8 solid phase extraction MeOH elution O Ns to dryness O derivatization with BSTFA/TMSI (5:1) 120°C/60 min Finnigan ITS 40 column: DB-5 capillary temp: 150-250°C Scheme 4.3 Flow chart of GC/MS assay for iphosphoramide mustard. 161 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. yCl q — O NH-v \ — (y \ V -q 1 / IF MH* 225 M STFA IF d . MH* 229 Scheme 5.4 Derivatization of ifosfamide and ifosfamide-d#. (MSTFA: methyi- siiyitrifiuoroacetamide) 162 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MSTFA C ? '‘IM S N2D MH+ 235 N 0 O NH. 0 D MSTFA O p—N o ' \ N2D dz MH* 237 Scheme 4.5 Derivatization of N2-dechloroethyl ifosfamide and N2-dechloroethyl ifosfamide-d2 - (MSTFA; methylsiiyltrifiuoraacetamide) 163 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TMS / “ W MSTFA /—\ P \ A \ A NH-n- 0 '— Cl K N3D MH* 235 TMS NHO D MSTFA D N3D-d4 MH* 239 Scheme 4.6 Derivatization of N3-dechloroethyl ifosfamide and N3-dechloroethyl ifosfamide-d4 . (MSTFA: methylsilyltrifluoroacetamide) 164 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TMSO MSTFA r " AlcoiF MH* 387 0 NH AlcolF-d^ TMSO MSTFA MH* 387 Scheme 4.7 Derivatization of alcoifosfamide and alcoifosfamide-d^. (MSTFA: methylsilyltrifluoroacetamide) 165 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 3 CD ■ D O Q. C g Q. "O CD C/) e n o" 3 O 8 "O 3 CD HO \ - o \ h A D - \ - C " V > 0 . KCN PM /— f"» H M - i V ° V h i h m ^,o d A NH-v dA - " ' MSTFA NC O T M S r^ Q. N TMS® MH* 412 4-OHIF 3 3 " CD "O O Q. C a O 3 ■D O CD Q. HO 4-OHIF-d, • i H / ----- / H N p D V o \ h : v KCN C oty— f - D / h n ^ o d A / ' N H -A ) A r « MSTFA NC OTMS T M S *- MH" 416 "O CD C/) C/) g Scheme 4.8 Derivatization of 4-hydroxy ifosfamide and 4-hydroxy ifosfamide-d4 . (ti/iSTFA : methylsilyltrifluoroacetamide) % . r P h o ' \ h r Cl -Cl B STFA /TM SI (5:1) X : ] TMSO N I TMS IPM MH* 329 0 ° Y - c i % nh- / p HO \ h - -Cl B STFA /TM SI ' ( 5 ÏÏ) Cl K J TMSO N I TMS IP M -d, MH* 331 I.P. 333 Scheme4.9 Derivatization of iphosphoramide mustard and iphosphoramide mustard-dg. [BSTFA: N,0-bis-(trimethylsiiyl)trifluoro-acetamide; MSTFA ; methylsilyltrifluoroacetamide] 167 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5 ENANTIOSELECTIVE PHARMACOKINETIC DISPOSITION OF IFOSFAMIDE IN THE RAT 168 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.1 A B S TR A C T Pseudoracemates consisting of 1:1 mixture of R-IF-d^. and S-IF or thie reversed composition were given to Sprague-Dawley rats at 40 mg/kg iv and plasma concentrations of enantiomeric IFs and tfieir metabolites including 4- OHIF, IPM, N2D, and N3D were analyzed using a sensitive and specific GC/CIMS method. For the analysis of each species a third deuterium-labeled analog was synthesized and used as the internal standard to facilitate the analysis. Plasma concentration-time data of the parent compounds and their metabolites were fitted by suitable compartmental models to obtain pharmacokinetic parameters. The result showed R-IF was eliminated from blood stream slightly faster than S-IF, with a lower value of AUC (R/S ratio 0.86±0.04). Significant enantiomeric selectivity on C-4 hydroxylation, and IPM formation was observed. The values of AUC of 4-OHIF and IPM generated from R-IF were significantly higher than those from S-IF (R/S ratio 1.62±0.27 and 1.46+0.27, respectively). On the other hand, N2D was derived stereoseiectively from S-IF. The AUC value of N2D generated from R-IF was significantly lower than that from S-IF (R/S ratio G.29±0.Q4). The enantiomeric selectivity for ring dealkylation, however, was not significant. Since N-dealkylations of IF generate a potentially neurotoxic side product (CAA) while 4-hydroxylation is considered the necessary activation process, the observed enantioselective pharmacokinetic disposition of IF may have pharmaceutical and clinical implication in favor of the use of the eutomer - R-IF. 169 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.2 INTRODUCTION It has long been recognized that stereoselectivity plays a fundamental role in life processes (46,91 -94). In fact, all four types of basic biological materials, i.e., nucleotide, protein, polysaccharide and lipid are chiral. Thus, it is not surprising that a stereoselectivity process is involved when a chiral drug interacts with one of these four types of molecules. More than 40% of drugs clinically used are in racemic form. Much of the previous investigation on the metabolism and pharmacokinetics of those chiral drugs were considered as "scientific nonsense" (93,94) because in these works, the enantiomers of a chiral drug were regarded as the same. Consequently, results obtained from the composite outcome of the two enantiomers were regarded erroneously as one. The reason was due to a lack of proper methodology and the differentiation of the enantiomers on the metabolism and pharmacokinetics of a chiral drug demands specific analytical method. With the advances of stereoselective analytical methodology and the improvement of asymmetric synthesis, knowledge on stereoselectivity on biological processes is increasing. It is now well recognized that the stereoisomers of a chiral molecule may display major differences in metabolism, pharmacokinetics, and pharmacological and/or toxic effects (46,83,94). This includes a number of anticancer drugs which are used as racemic mixtures. IF, an oxazaphosphorine alkylating agent (4-7), has an established role in cancer chemotherapy (7,66,67). It possesses an asymmetric phosphorus atom, therefore existing in enantiomers. Major metabolic pathways of IF have been elucidated (45). The most important metabolic pathway is 4-hydroxylation generating 4-OHIF, which may readily partition across cells (12,84). Subsequent 170 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ring opening and p-elimination of this metabolite generates IPM, which has been considered as the ultimate intracellular alkylating metabolite (14,16,85). Dealkylation of the 2-cfiloroethyl chains generates N2D and N3D, along with CAA which has been implicated in ttie neurotoxicity observed in patients receiving IF therapy (22,86). These metabolic pathways may display stereoselectivity. However, the majority of previous metabolic and pharmacokinetic studies utilized racemic IF without consideration of the possible differences of the two enantiomers (95-98). This may be less important if there were no metabolic and pharmacological differences between (S)-(-)-IF and (R)-(+)-IF. Kusnierczyk etal. (49) reported the synthesis of enantiomeric IFs and evaluated their in vivo antitumor activity against four transplanted tumor mice models; LI 210 and P388 lymphoid leukemias, Lewis lung cancer carcinoma, and mouse mammary carcinoma 16C MAC. They observed a higher therapeutic index against ail tumors except L I210 when (S)-(-)-IF was administered. A substantial species difference on the stereoselective metabolism of IF has been noticed (23,51 -53). Recently, a systematic study was conducted by Masurel et al. (51) to study the efficacy, toxicity, pharmacokinetics, and the in vitro metabolism of the enantiomers of IF in mice. The in vivo efficacy investigation was conducted in a childhood rhabdomyosarcoma (HxRh28) maintained as a xenograft in immune-deprived female GBA/CaJ mice and toxicity and pharmacokinetic studies conducted in normal female CBA/CaJ mice. The in vitro studies investigated on the activation pathway by the determination of two metabolites, 4-OHIF and IPM. No statistically significant differences were found between R-IF, S-IF, and racemic IF. However, these results were inconsistent with those in human (23,53). 171 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It was concluded that mouse may not be an appropriate model for human efficacy, toxicity, metabolic and pharmacokinetic studies of IF (51,94), possibly because the cytochrome P450 enzymes responsible for the metabolism of IF in mouse are largely different from those in human. Therefore, we resorted to use the rat as the model system. Studies on the stereoselective metabolism of the enantiomers of IF was conducted in Sprague-Dawley rats using a pseudoracemate/mass spectrometry methodology (Chapter 4). The enantio meric ratios of the parent drugs and their derived metabolites including 4-OHIF, alcolF, IPM, N3D, and N2D in rat urine were determined, it was found that the activation pathway preferred R-IF, generating higher levels of 4-OHIF, IPM and alco IF than those from S-IF. Different patterns of stereoselectivity on N- dealkylation were found. While N2-dealkylatlon displayed strong preference for the S configuration, N3-dealkylation showed slight selectivity favoring the R configuration. These results resemble those from human studies (23,53), constituting the rationale for the stereoselective pharmacokinetic studies reported herein. Several analytical techniques have been applied to quantitate IF and its metabolites, including gas liquid chromatography (97,99-103), high performance liquid chromatography (21,104,105), thin layer chromatography (106), alkylating activity assay using N-nitrobenzylpyridine (NBP) (51), total radioactivity assay after administration of ^H or ^'^C-labeled IF (51), and GC/MS (107). However, many of these methods are non-specific or with low sensitivity. A recently reported ^''PNMR method (24) allows simultaneous identification of all phosphorylated metabolites, its intrinsic sensitivity is low, limiting its application to pharmacokinetic studies. A sensitive fluorometric method (79,80) using 4- 172 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aminophenol has been developed to measure the unstable active metabolite 4- OHIF in plasma and urine based on the assay of acrolein released. This method has shown to be nonspecific, since acrolein can also be generated from secondary metabolites of IF such as 4-OHN2D and 4-OHN3D (Chapter 3). In spite of various analytical methods developed, few complete pharmacokinetic profiles of IF and its metabolites have been reported (91). This may attribute to potential problems in applying these methods in biological system. Therefore, the development of better quantitative methods for IF and its metabolites is of paramount importance and constitutes a major focus of our research project in this laboratory. Thus, the methodology of pseudoracemate coupled to gas chromatography and mass spectrometry (GC/MS) has first been developed. Using this method with respective deuterium labeled internal standards, enantiomeric IFs and their metabolites including 4-OHIF, N2D, N3D and IPM can be simultaneously detected and quantitated. 5.3 MATERIALS AND METHODS 5.3.1 Materials 5.3.1.1 Chemicals and reagents Pseudoracemate component R- and S-ifosfamide (R- and S-IF), R- and S- 6,6,2',2'-tetradeuterio-ifosfamide (R- and S-IF-d4 ) were strategically designed and synthesized in this laboratory (Chapter 2). The chemical structures of the two pseudoracemates are shown in Figure 5.1. Racemic Ifosfamide [(±)-IFj was provided by Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, the National Cancer Institute. Other standard compounds and 173 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. internal standards 4-OHIF, N2D, N3D, IPM, 4,4,5,5,6,6-hexadeuterlo-ifosfamide (IF-de), 4-hydroperoxy 6,6,2',2',2",2"-hexadeuterlo-ifosfamide (4-OOHIF-d6), N2- dechloroethyl 1',1',2',2'-tetradeuterio-lfosfamide (N2D-d4), N3-dechloroethyl 4,4,6,6,1',r,2',2'-octadeuterlo-ifosfamlde (N3D-ds), and 2',2'-dideuterio- Iphosphoramide mustard (IPM-d2 ), and 2',2',2",2"-tetradeuterlo-iphosphoramide mustard (IPM-ds) were all synthesized in this laboratory (Chapter 2 and Section 5.3.3 in this Chapter). The internal standard 4-hydroxy 6,6,2',2',2",2"- hexadeuterio-ifosfamide (4-OHIF-d6) was prepared by the reduction of 4- hydroperoxy 6,6,2',2',2",2"-hexadeuterio-ifosfamide (4-OOHIF-d6) with sodium thiosulfate immediately before use. The structures of these compounds are shown in Figure 5.2. All synthetic reagents were obtained from Aldrich Chemical Company (Minneapolis, MN) and all HPLC grade organic solvents were purchased from Fisher Scientific (Pittsburgh, PA). N-methyl-N- trimethylsilyltrifluoroacetamide (MSTFA), N, 0-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and N-trimethyl-silylimidazole (TMSI) were purchased from Pierce (Rockford, IL). C-18 reversed-phase resin was obtained from Analytlchem International (Harbor City, CA). 5.3.1.2 Surgical instrument and supplies Surgical instruments were purchased from Roboz Surgical Instrument Co., Inc. (Washington, DC). Monoject hypodermic needles and Monoject sterile disposable tuberculin syringe without needle were obtained from Western Medical Supply (Arcadia, CA). Surgical sutures (size 3 and 4) were purchased from George Tieman & Company (Plainview, NY). Curity gauze sponges were obtained from Kendall Company (Boston, MA). Intramedic polyethylene 50 174 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tubings (PE 50, 0.58 mm I.D. and 0.965 mm O.D.) were purchased from Fisher Scientific (Tustin, CA). Sodium chloride injection (9%), USP was obtained from Western Medical Supply (Arcadia, CA) as was heparin sodium injection USP 1000 units/ml. Isopropyl alcohol was obtained from Fisher Scientific (Pittsburgh, PA). 5.3.1.3 Instrumentation A ITS40 Ion Trap mass spectrometer directly coupled to a 3300/3400 Varian gas chromatograph (Walnut Creek, CA) and CTC A200S autosampler with a capillary splitless injector were purchased from Finnigan MAT (San Jose, CA). Fused-silica capillary columns (30m x 0.25 mm I.D.), DB-5, coated with a 0.25- micron thick film of methylsilicone plus 5% phenyl methylsilicone, were purchased from J&W Scientific (Folsom, CA). Refrigerated centrifuge "Accupsin FR®" was obtained from Beckman Instrument, Inc. (Palo Alto, CA). 5.3.2 Methods 5.3.2.1 Animal surgery - jugular vein cannulation Animal experiments were carried out according to a protocol approved by the Animal Use Review Committee at the Ohio State University. Six male Sprague- Dawley rats (Harlan, Indianapolis, IN) were used in this study. Five animals (Rat 1-3,5,6) were given pseudoracemate consisting of R-IF-d4 and S-IF, and one (Rat 4) was given pseudoracemate consisting of S-IF-d4 and R-IF. Rat chow (Tekland, Indianapolis, IN) and water were given ad libitum. The jugular vein of each rat was cannulated under ethyl ether anesthesia (Chapter 3). The animal was allowed to recover for 2 hrs before dosing. 175 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.3 2.2 Pharmacokinetic study After the animal became completely conscious, Pseudoracemate dissolved in normal saline (1 ml) at the total dose of 40 mg/kg (20 mg/kg for each component) was injected into the animal through the jugular vein cannula. The cannula was washed three times with 0.3 ml each of 0.9% sodium chloride solution. Blood samples (0.2-0.6 ml) were collected in heparinized plastic tubes at the following predetermined time schedule: 0, 5, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420 min. After collection of each sample, the same volume of saline was injected into the rat to replace the lost fluid. Plasma was obtained from each sample by centrifugation at 200 x g and 4°C for 2 min. The plasma was obtained and immediately stored at -76° until analysis (Scheme 5.1). 5.3.2.3 Extraction and derivatlzatlon Each plasma sample was thawed at 0-5°C and divided into two portions. One (50-150 pi) for the analysis of IF, 4-OHIF/aldolF, N2D, N3D was immediately placed in a culture tube (16x100 mm) containing 200 pil of 1.5 M, pH 8 KCN solution and 1 jxg of IF-de, 200 ng each of N2D-d4, N3D-ds and 3 pig of 4-OHIF- dg. The samples were allowed to stand at room temperature for 30 min, and then 5 ml of CH2 CI2 was added. The mixture was shaken for 15 min in a horizontal shaker (Eberbath, Ann Harbor, Ml). The organic phase was separated after centrifugation, and evaporated under a stream of nitrogen. The residue was derivatized with 35 p .1 of MSTFA for 1 hr at 120°C and an aliquot of the derivatized sample was analyzed by G C/MS under ammonia chemical ionization (Scheme 5.2-5.6). 176 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A procedure developed in this laboratory was used for the analysis of total IPM and its deuterium-labeled analog (Scheme 5.7). The other portion of the plasma sample (50-150 ( j . 1 ) was placed in another culture tube containing 1 [xg of IPM-da as the internal standard, and the mixture was loaded onto a disposable Poly-prep column (Bio-Rad, Richmond, CA) containing 400 mg of C-18 reversed- phase resin. The column was washed with 0.5 ml of ice-cooled saline followed by centrifugation at 200 x g for 20 min to remove water. The column was then eluted with 1 ml of methanol, and the methanol fraction was collected and evaporated under a gentle stream of nitrogen to dryness at room temperature. During the process, 4-OHIF, originally existing in plasma, was degraded totally to IPM. The residue was derivatized with 35 fxl of a mixture of BSTFA and TMSI (5:1, v/v) at 120°C for 1 hr (Scheme 5.8) and an aliquot of the derivatized sample was analyzed by GC/MS. S.3.2.4 GC/MS analysis The temperatures of the injection port, transfer line, and source were maintained at 220, 260 and 230°C, respectively. The analysis was carried out under chemical ionization condition with ammonium as the reagent gas and the emission current was set at 10 pamp. Helium was used as the carrier gas with a head pressure set at 15 psi. For the analysis of IF, 4-OHIF, N2D and N3D and their deuterium-labeled analogs, the temperature of the oven was programmed at 150°C for 2 min and increased to 190°C at a rate of 5°C/min, and then to 250°C at a rate of 15°C/min. The final temperature was held for 3 additional min. The retention times of the dehydrochlorinated IF and its d4 and de (internal standard) analogs 177 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were found to be 8.62, 8.57, 8.55 min, respectively. The retention times of the trimethyisiiyiated derivatives of the dehydrochiorinated cyanohydrins of 4-OHIF and its d4 and dg (internai standard) analogs were 15.25, 15.23, 15.22 min, respectively. The retention times of the trimethyisiiyiated derivatives of the dehydrochorinated N2D and its 62 and d4 (internai standard) analogs were 8.58, 8.57, 8.55 min, respectively. The retention times of the trimethyisiiyiated derivatives of the dehydrochorinated N3D and its d4 and ds (internal standard) analogs were 5.70, 5.67, 5.63 min, respectively. Respective ions were selected and monitored; for IF and its labeled analogs, m/z 225, 229, 233; for 4-OHiF and its labeled analogs, m/z 412,416, 420; for N2D and its labeled analogs, m/z 235, 237, 239; for N3D and its labeled analogs, 235,239, 243. For the analysis of total iPM and its labeled analog, the same GC temperature program was used. The trimethylsilyi derivatives of the dechlorinated IPM and its da and da analogs were eluted at 9.82, 9.80, 9.78 min. Ions at m/z 329, 331 and 337 were selectively monitored. Table 5.1 summarized the selected ion and retention time for each chemical entity under GC/MS analysis. 5 3.2.5 Recoveries of ifosfamlde, ifosfamlde-d4, N2-dechloroethyl ifos- famide and NS-dechloroethyl Ifosfamlde from extraction A methanolic solution containing 500 ng each of S-IF, R-IF-d4 and 100 ng each of N2D and N3D placed in a culture tube (16 x 100 mm) was evaporated to dryness under a stream of nitrogen. Two hundred [x l of rat blank plasma was added to each tube and the content was extracted with 5 ml of CHgClg. After separation, to the organic phase was added 1 pg of IF-de, 200 ng each of N2D- 178 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. d4 and N3D-ds and the solvent was evaporated under a stream of nitrogen. The residue was derivatized with 35 [xi of MSTFA and an aliquot was analyzed by GC/MS. As a control, a methanolic solution containing 500 ng each of S-IF and R-IF- d4 , 1 |xg of IF-de, 100 ng each of N2D, N3D, 200 ng each of N2D-d4, N3D-dg was evaporated. The residue was derivatized with 35 jd of MSTFA and analyzed by GC/MS. The recovery for each compound (IF, lF-d4 , N2D and N3D) was calculated by dividing the area ratio of sample ion over the respective internal standard ion from the extraction group to that from the non-extraction group. 5.3.2.6 Recovery of the cyanohydrin adduct of 4-OHIF from extraction Since the analysis of 4-OHlF is through its conversion to the cyanohydrin adduct, the recovery of the cyanohydrin adduct of 4-OHlF was conducted instead of the parent compound as described below. A. Into a culture tube containing 1 pg of 4-OHIF immersed in an ice-bath was added 200 pi of 1.5 M, pH 8, KCN solution and 200 pi rat plasma. The mixture was allowed to stand at room temperature for 30 min and then extracted with 5 ml of CH2CI2 . The extract was kept in a -76°C freezer. B. To prepare the internal standard the cyanohydrin adduct of 4-OHlF-d6,1 pg of 4-OHlF-d6 was placed into a culture tube, followed by an addition of 200 pi of 1.5 ,M , pH 8, KCN solution. The mixture was allowed to stand for 30 min, and then lyophilized. Into the residue was added the CH2CI2 extract from Step A. The solvent was evaporated under a stream of 179 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nitrogen and the residue was derivatized with 35 (x l of MSTFA. An aliquot of the derivatized mixture was analyzed by GC/MS. 0. The control was processed by procedures similar to those in Step B. into a culture tube containing 1 [xg each of 4-OHIF and 4-OHIF-d6 was added 200 fx l of 1.5 M, pH 8, KCN solution. After standing at room temperature for 30 min, the mixture was lyophilized. The residue was derivatized with MSTFA, and an aliquot of the solution was analyzed by GC/MS. The recovery of the cyanohydrin adduct of 4-OHIF was calculated by dividing the area ratio of 4-OHIF ion over that of its internal standard from the extraction group by that from the control group. 5 3.2.7 Data analysis Regressional analysis and pharmacokinetic model fitting were accomplished using a RSTRIP Program (MicroMath Scientific Software, Salt Lake City, UT) on an IBM PC. A weighting factor of 1/y2 was used in most of the fitting. An appropriate compartment model was selected based on the smallest values of standard error (SE), the weighted sum of square (WSS), and the correlation coefficient. In addition, the selection of the preferred compartment model was based on the Akaike's Information Criteria (A 1C ) and the model which gave the smallest A 1 C was chosen. The Akaike's Information Criteria (AlC) is shown as follows: AlC =nlnWSS + 2P (1) where n = number of observation WSS = weighted sum of squares P = number of parameters to be estimated 180 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The difference in mean values of appropriate parameters between two sets of paired data was analyzed by paired t-test, respectively, at the significant level of a=0.05. Other pharmacokinetic parameters such as total drug clearance (CLj), mean resident time (MRT) and steady-state volume of distribution (Vdgg) are calculated as follows: CLy = Dosep/AUCp (2) MRT = AUMC/AUC (3) Vdgg = MRT X CLy (4) where AUG = area under the curve, and AUMC = area under the first moment curve 5.3.3 Synthesis of deuterium-labeled standard and Internal standards for GC/MS assay S.3.3.1 Synthesis of N3-dechloroethyl 4,4,5,5,6,6,2',2'-octadeuterio-ifosfa- mide and 4,4,5,5,6,6,2',2’-octadeuterio-ifosfamide Ethyl 2,2-dideuteriocyanoacetate. A mixture of ethyl cyanoacetate (10.0 g) and deuterium oxide (10.0 g) was heated to reflux for 15 min. The reaction mixture was then extracted with CH2CI2 (50 ml x 2). The extract was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated in vacuo to give a colorless oil (9.2 g). The same procedure was repeated for 4 more times to get complete isotope exchange. The final recovery was 7.3 g (73.0 %). NMR (CDCI3) Ô 1.33 (t, J = 7.1 Hz, 3H, -CH3): 4.29 (q, J = 7.1 Hz, 2H, -CH2-). 1-Amino-1,1,2,2,3,3-hexadeuterio-propan-3-oi. To a cooled (-78°C) sus pension of lithium aluminum deuteride (2.0 g) in anhydrous THF (30 ml) was 181 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. added dropwise a solution of ethyl 2,2-dideuterio-cyanoacetate (3.45 g) In THF (10 ml). The mixture was stirred for 1 hr at ambient temperature and the temperature was then raised to reflux. The reaction mixture was stirred for 10 additional hrs. A saturated solution of sodium suifate (5 ml) was added slowly to the cooled (0°C) mixture to quench the reaction. The solid material was collected by filtration and extracted with THF (200 ml) using a Soxhlet apparatus for 2 days. The filtrate and the extract were combined and concentrated to give a yellow oil (1.79 g, 73.6 %). NMR (CDCI3 ) ô 1.65 (bs, 3H, OH, NH2 ). 2-Chloro-4,4,5,5,6,6-hexadeuterio-tetrahydro-ï,3,2-oxazaphosphorine 2- oxide. To a cooled (-78°C) solution of oxyphosphorus chloride (1.97 g, 12.84 mmol) in anhydrous CH2 CI2 (40 ml) was added slowly a mixture of 1-amino- 1,1,2,2,3,3-hexadeuterio-propan-3-ol (1.04 g, 12.84 mmol) and triethylamine (2.59 g, 25.68 mmol) in CH2Ci2 (20 mi). The reaction mixture was stirred for 4 hrs at room temperature. Anhydrous ethyl ether (50 ml) was then added to precipitate the co-product triethylamine hydrochloride. The soiuble material was collected by filtration and concentrated to give the oxazaphosphorine monochloride product as a yellow oil (1.48 g), which was used for the subsequent reaction without further purification. 2-(2-Chloro-2,2-dideuterio-ethyl)amino-4,4,5,5,6,6-hexadeuteriotetrahy- dro-Ï,3,2-oxazaphosphorine 2-oxide (NSD-ds). To a cooled (0°C) solution of the oxazaphosphorine monochloride (1.48 g) in anhydrous CH2CI2 (40 ml) was added previously synthesized 2,2-dideuterio-2-chloroethylamine (1.51 g, 12.84 mmol). Triethylamine (2.59 g, 25.68 mmol) in CH2Ci2 (10 ml) was then added dropwise to the suspension. The reaction mixture was stirred for 2 hrs at room temperature. The solvent was removed by rotary evaporation in vacuo. The 182 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. residue was extracted with hot acetone (20 ml x 4). Concentration of the extract afforded a semi-solid (2.94 g). Silica gel column purification using CHgClg/EtOH (15:1 ) as the eluant gave N3D-ds as colorless crystals (0.59 g, 22.3% from amine alcohol): mp 107-109°C; Rf 0.18 (CHgClg-acetone 1:3). The chemical identity of N3D-dg was confirmed by GC/MS analysis after Its derivatlzatlon with MSTFA. 2-(2-Chioro-2,2-dldeuterio-ethyl)amino-3-chloroacetyl-4,4,5,5,6,6-hexa- deuterio-tetrahydro-7,3,2-oxazaphosphorine 2-oxide (N3-chioroacetyl N3D- dg). To a cooled (0°C) solution of N3D-dg (0.28 g, 1.35 mmol) in anhydrous THF (20 ml) was added a solution of chloroacetyl chloride (0.30 g, 2.70 mmol, 2:1 excess) In THF (10 ml). The reaction was followed by TLC. Once the acylation was completed, the volume of the solution was reduced in vacuo to 10 ml. Water (10 ml) was added and the mixture was extracted with CH2 CI2 (50 ml x 4). The CH2 CI2 extract was dried over anhydrous sodium sulfate. After filtration, the filtrate was evaporated to afford N3-chloroacetyl NOD-dg as a colorless oil (0.92 g). Purification on a silica gel column with CH2Cl2 -acetone (4:1) as the eluant gave a colorless oil (0.3 g, 77.7%): Rf 0.72 (CH2 Cl2 -acetone 1:1). 2-(2-Ch[oro-2,2-dideuterlo-ethy[)amino-3-chloroethyi-4,4,5,5,6,6-hexa- deuterlo-tetrahydro-7,3,2-oxazaphosphorine 2-oxide (IF-dg). Into a cooled (- 78°C) solution of 1 M BHg in anhydrous THF (6.36 ml, 1:6 excess) was added dropwise N3-chloroacetyl N3D-dg (0.30 g, 1.06 mmol) in THF (10 ml). The reaction mixture was stirred for 1 hr. Water (0.5 ml) in THF (10 ml) was then added to destroy the remaining BHg. After the removal of THF in vacuo, the residue was extracted with CH2CI2 (20 ml x 4). The organic phase was dried over anhydrous sodium sulfate. After filtration, the fiitrate was concentrated to give a colorless oil (0.31 g), which was chromatographed using CH2 Cl2 -acetone- 183 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. methanol (40:3:1) as the eluant to give IF-dg (0.17 g, 60.0%): Rf 0.33 (CH2 CI2- acetone 1:1). The chemical Identity of IF-dg was confirmed by GC/MS analysis based on the GC retention data and MS analysis by comparing with IF. The synthetic scheme of IF-dg and N3D-dg is shown in Scheme 5.9. 5.3.3 2 Synthesis of N3-dechloroethyi 4,4,5,5,6,6-hexadeuterlo-ifosfamide and 4,4,5,5,6,6-hexadeuterio-ifosfamide 2-Chloro-4,4,5,5,6,6-hexadeuterio-tetrahydro-7,3,2-oxazaphosphorine 2- oxide. Into a cooled (-78°C) solution of oxyphosphorus chloride (2.21 g, 14.44 mmol) in anhydrous CH2CI2 (20 ml) was added slowly a mixture of 1-amino- 1,1,2,2,3,3-hexadeuterio-propan-3-ol (1.17 g, 14.44 mmol) and triethylamine (2.92 g, 28.88 mmol) in CH2CI2 (5 ml). The reaction mixture was stirred for 1 hr at room temperature. Anhydrous ethyl ether (50 ml) was added to precipitate the co-product triethylamine hydrochloride. The solid was removed by filtration and the filtrate was evaporated to give the oxazaphosphorine monochloride product as a yellow oil, which was used for the next stage without further purification. 2-(2-ChIoroethyl)amino-4,4,5,5,6,6-hexadeuterio-tetrahydro- 1,3,2-oxaza- phosphorine 2-oxide (N3D-dg). To a CH2CI2 solution (20 ml) of the oxazaphosphorine monochloride was added 2-chloroethylamine hydrochloride (1.68 g, 14.44 mmol). Triethylamine (2.92 g, 28.88 mmol) in CH2 CI2 (5 ml) was then added dropwise to the suspension. The reaction mixture was stirred for 2 hrs at room temperature. The solvent was removed by rotary evaporation in vacuo. The residue was extracted with hot acetone (20 ml x 4). Evaporation of the solvent in the extract afforded a semi-solid (2.52 g), which was chromatographed on a silica gel column using CH2 Cl2/EtOH (15:1) as the eluant 184 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to give N3D-de (0.58 g, 23.1% from the amine alcohol): mp 105-107°C: Rf 0.18 (CHaCia-acetone 1:3). The chemical identity of N3D-dg was confirmed by GC/MS analysis. 2-(2-Chloroethyl)amino-3-chloroacetyl-4,4,5,5,6,6-hexadeuterio-tetrahy- dro-1,3,2-oxazaphosphor!ne 2-oxide (N3-chloroacetyl N3D-de). To a cooled (0°C) solution of N3D-d6 (0.48 g, 2.35 mmol) in THF (20 ml) was added a solution of chloroacetyl chloride (0.79 g, 7.05 mmol, 3:1 excess) in THF (5 ml). The reaction was monitored by TLC. Once the acylation was completed, the volume of the solution was reduced in vacuo to 10 ml. Water (10 ml) was then added and the mixture was extracted with CH2CI2 (50 ml x 4). The CH2CI2 extract was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to give N3-chloroacetyi NSD-dg as a colorless oil (0.92 g). Column purification (silica gel) with CH2 Ci2-acetone (4:1 ) as the elutant gave a colorless oil (0.56 g, 85.0%): Rf 0.72 (CH2 Cl2 -acetone 1:1 ). 2-(2-Chloroethyl)amino-3-chloroethyl-4,4,5,5,6,6-hexadeuterio-tetrahy- dro-1,3,2-oxazaphosphorine 2-oxlde (IF-dg). Into a cooled (-78°C) solution of 1 M BH3 in anhydrous THF (12 ml, 6:1 excess) was added dropwise N3- chloroacetyi N3D-de (0.56 g, 2.00 mmol) in THF ( 1 0 ml). The reaction mixture was stirred for 1 hr at ambient temperature. TLC showed complete disappearance of the starting material. Water (10 ml) was then added to destroy the remaining BH3 . After the removal of THF in vacuo, the resulting mixture was extracted with CH2CI2 (20 ml x 4). The organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was evaporated to give a colorless oil (1.00 g). Column chromatography (silica gel) using CH^Ck-acetone- methanol (40:3:1) as the eluant gave IF-dg as a colorless oil (0.29 g, 54.7%): Rf 185 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.33 (CHaCla-acetone 1 :1 ), which crystallized during storage in a freezer (-76°C). The chemical identity was confirmed by GC/MS anaiysis comparing with authentic IF. The synthetic scheme of IF-de and N3D-de is shown in Scheme 5.10. S.3.3.3 Synthesis of 4-hydroperoxy 6,6,2',2',2",2"-hexadeuterlo-lfosfamide 4-OOHIF-de was synthesized following the procedure of Takamizawa et al. (13) using deuterium iabeled synthons. Briefly, 0 -1 ,1 -dideuterio-3-butenyl N,N'- bis(2-chloro-2,2-dideuterio-ethyl)phosphorodiamidate was prepared in 70% yield by a one-pot reaction of oxyphosphorus chloride with synthesized 1 ,1 -dideuterio- 3-buten-1-ol and 2-chloro-2,2-dideuterio-ethylamine hydrochloride in CH2 CI2 . Ozonolysis of the obtained deuterated phosphorodiamidate in aqueous acetone, followed by treatment with 30% hydrogen peroxide, afforded 4-OOHIF-d6 in 35% yield (Scheme 5.11). 1,1-Dideuterio-3-buten-1-oi. To a cooled (-76°C) suspension of lithium aiuminum deuteride (1.16 g, 27.56 mmol) in anhydrous THF (30 ml) was added slowly a solution of vinyl acetic acid (4.74 g, 55.12 mmol) in THF (10 ml). The mixture was heated to reflux for 5 hrs. Water (3 ml) was added dropwise to destroy the remaining deuteride while the reaction flask was immersed in an ice- bath. The solid materiai was collected by suction filtration, and was extracted with THF for two days using a Sohxiet extractor. The filtrate and the extract was combined and dried over anhydrous sodium sulfate. Removal of THF gave 1,1- dideuterio-3-buten-1-ol as a light brown oil (2.05 g, 50.2%): Rf 0.84 (CH2 CI2- acetone 1:1): NMR (CDCI3 ) ô 1.55 (bs, 1H, OH) 2.31 (d, J=6.7 Hz, C2-H), 5.08- 186 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.20 (m, 2H, C4-H), 5.70-5.88 (m, 1H, C3-H). The crude product was used for the subsequent step without further purification. 0-{1,1-dicleuterlo-3-butenyl)-N,N'-bis(2-chioro-2,2-dideuterio-ethyl)phos- phorodiamidate. To a stirred solution of oxyphosphorus chloride (1.04 g, 6.76 mmol) in CH2 CI2 (20 ml) was added dropwise a solution of 1,1-dideuterio-3- buten-1-ol (0.5 g, 6.76 mmol) and triethylamine (0.68 g, 6.76 mmol) in CH2 CI2 (5 ml) at -76°C. The reaction mixture was stirred for 1.5 hr. 2-Chloro-2,2-dideuterio- ethylamine hydrochloride (1.60 g, 13.52 mmol) was added to the reaction mixture, followed by triethylamine (2.73 g, 27.04 mmol) in CH2 CI2 (5 ml). The reaction mixture was stirred for 2 additional hrs at room temperature. The precipitated triethylamine hydrochloride was removed by filtration. The filtrate was washed with distilled water (10 ml x 3) and the organic phase was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was evaporated to give a yellow oil (1.48g, 75.9%). Column purification (silica gel) with CH 2 Cl2 -acetone (5:1) as the eluant gave the product (0.63 g, 32.3%) as a colorless oil: Rf 0.41 (CH2 Cl2-acetone 1:1); NMR (CDCI3 ) ô 2.42 (d, J=6.7 Hz, 2H), 2.94-3.08 (m, 2H, 2 x NH), 3.18-3.33 (m, 4H, 2 x NH-ÇH2-), 5.08-5.20 (m, 2 H, -CH=ÇH2 ), 5.72-5.90 (m, 1 H, -CH=CH2). 4-OOHIF-dG A stirred solution of the phosphorodiamidate (0.43 g, 1.53 mmol) in aqueous acetone (1:1, 30 ml) was bubbled with O 3 at a flow rate of 3.5 ml/min (90v, 90w) for 1 hr at 0°G. Hydrogen peroxide (30%, 0.5 ml) was then added to the ozonized solution. After standing at 4°C for 3 days, acetone in the reaction mixture was removed in vacuo and the resulting aqueous residue was extracted with CH 2 CI2 (50 ml x 4). The combined extract was dried over anhydrous sodium sulfate. After filtration, the filtrate was evaporated in vacuo to 187 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. give a colorless oil (0.3 g), which crystallized by addition of acetone (0.3 ml) and ethyl ether (3 ml). After standing at -70°C overnight, the crystals were collected by filtration and washed with cooied (0 °C) ethyl ether to give A-OOHIF-de as a white soiid (0.08g). The mother liquor was concentrated and the resulting oil was redissolved in aqueous acetone (1:1, 30 ml). Hydrogen peroxide (30%, 0.5 ml) was added to the mixture. After standing at 4°C for 2 days, acetone was removed in vacuo and the remaining aqueous solution was extracted with CH2 CI2 (50 ml X 5) and the second crop of A-OOHIF-de (0.07g) was obtained by a similar treatment. The remaining mother liquor was treated similarly and the third crop (0 .0 1 g) was obtained. The overall yield was 35.0%. Rf 0.74 (CH2 CI2 - acetone 3:1). The chemical identity was confirmed by GC/MS analysis by comparison with 4-OOHIF. S.3.3.4 Synthesis of N2-dechloroethyi 6,6,1',1',2',2'-hexadeuterlo-lfcsfamide Methyl N-1,1,2,2-tetradeuter[o-2-hydroxylethyl-3-amino propionate. Into a solution of methyl acrylate (1.33 g, 15.38 mmol) in THF (15 ml) was added 1,1,2,2-tetradeuterio-ethanolamine (1.00 g, 15.38 mmol), the mixture was stirred at room temperature for 2 days. The reaction was followed by TLC (CH2 CI2 - acetone 1:1). When the reaction was completed, THF was removed in vacuo to give a colorless oil (2.32 g, 99.6%) Rf 0.07 (CH2Cl2 -acetone 1:1). The product was used for the subsequent stage without purification. Methyl N-2-chloro-1,1,2,2-tetradeuterioethyl-3-amino-propionate. To a cooled (0°G) solution of methyl N-1,1,2,2-tetradeuterio-2-hydroxylethyl-3-amino propionate (2.32 g, 15.36 mmol) in GH2 CI2 (10 mi) was added dropwise thionyi chloride (3.63 g, 30.72 mmol). The reaction mixture was heated gradually to 188 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60°C in 30 min. The progress of the reaction was monitored by TLC. Once the reaction was completed, water ( 2 0 ml) was added while the reaction flask was immersed in an ice-bath. The resultant mixture was washed with CH2 CI2 (20 ml X 3) and the washings were discarded. The aqueous solution was neutralized to pH 9-10 using saturated NaOH solution and was then extracted with GH2 Ci2 (20 ml X 4). The combined extract was washed with water (10 ml), and dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was evaporated in vacuo to give the product as a pale yellow oil (2.00 g, 76.8%): Rf 0.57 (GH2 Cl2-acetone 1:1). N-2-Chloro-1,1,2,2-tetradeuterloethyl 3-amino-1,1-dideuterio-propan-1- 01. To a cooled (-76°G) suspension of lithium aluminum deuteride (0.5 g, 23.60 mmol) in THF (20 ml) was added dropwise a solution of methyl N-2-chloro- 1,1,2,2-tetradeuterioethyl-3-amino-propionate (2.00g, 11.80 mmol) in THF (5 ml). The reaction mixture was stirred at room temperature for 1 hr. The reduction was followed by TLG. Once the starting material completely disappeared, water (1 ml) In THF (10 mi) was added to destroy the remaining deuteride. After filtration, the insolubie material was extracted with GH2GI2 . The filtrate and extract were combined and dried over anhydrous sodium sulfate. After filtration, the voiume of the filtrate was reduced in vacuo to about 10 ml at a temperature below 25°G. 2-Chloro-3-(2-chloro-1,1,2,2-tetradeuterio-ethyl)-5,5-dldeuterio-tetrahy- dro-2H-f,3,2-oxazaphosphorine 2-oxide. To a cooied (-76°G) solution of oxyphosphorus chloride (1.81 g, 11.80 mmol) in GH2GI2 ( 1 0 ml) was added the CH2 GI2 solution of N-2 -chloro-1 ,1 ,2 ,2 -tetradeuterioethyl 3-amino-1,1-dideuterio- propan-1 -ol. A solution of triethylamine (2.51 g) in GH2GI2 (5 ml) was then added dropwise to the mixture. The reaction mixture was stirred for 1 hr at room 189 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. temperature. The solvent was removed in vacuo and the residue extracted with anhydrous ethyl ether. The extract was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed to give a colorless oil (1.27 g), which was purified by silica gel column chromatography using ethyl ether as the elutant. 2-Amino-3-(2-chloro-1,1,2,2-tetradeuterio-ethyl)-5,5-dldeuterio-tetrahy- dro-f,3,2-oxazaphosphorine 2 -oxide (N2D-de). To a ethyl ether solution of the oxazaphosphorine chloride obtained from the previous step was bubbled with anhydrous ammonia at room temperature. After 2 hrs, the starting material was completely consumed as shown by TLC. Acetone (200 ml) was then added into the mixture and precipitated ammonium chloride was removed by filtration. The filtrate was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed to give colorless crystals (0.63 g, 26.3% from amine ester): mp 101-103°C; Rf 0.22 (CHgClz-acetone 1:3). The chemical identity of the product was confirmed by TLG and GC/MS analysis comparing data from authentic N2D. The synthetic scheme of N2D-de is shown in Scheme 5.12. 5.3.3.S Synthesis of N2-dechloroethyl 1',1',2',2'-tetradeuter!o-ifosfamide Methyl N-1,1,2,2-tetradeuterio-2-hydroxyiethy[-3-am!no propionate. To a solution of methyl acrylate (0.66 g, 7.69 mmol) in THF (10 ml) was added 1,1,2,2- tetradeuterio-ethanolamine (0.50 g, 7.69 mmol). The mixture was stirred at room temperature for 2 days. The solvent was removed in vacuo to give a colorless oil (1.16 g, 100%): Rf 0.07 (CHaCla-acetone 1:1). The product was used for the subsequent step without purification. 190 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Methyl N-2-chloro-1,1,2,2-tetradeuterioethyl-3-am!no-propionate. Into a cooled (0 °C) solution of methyl N-1,1,2,2-tetradeuterio-2-hydroxylethyl-3-amino propionate (1.16 g, 7.68 mmol) In CH2 CI2 (10 ml) was added dropwise thionyi chloride (1.82 g, 15.36 mmol). The mixture was heated gradually to 60°C. The reaction was followed by TLC. Once the reaction was completed, water (20 ml) was added to destroy the remaining thionyi chloride. The mixture was extracted with CH2 CI2 (20 ml X 4) and the extract was discarded. The aqueous solution was neutralized to pH 9-10 using saturated NaOH solution. The mixture was then extracted with GH2 CI2 ( 2 0 ml x 4). The combined organic phase was washed with water (10 ml) and dried over anhydrous sodium sulfate. After filtration, the solvent in the fiitrate was removed in vacuo to give the product as a pale yeliow oil (1.20 g, 92.2%): Rf 0.57 (CH2Cl2 -acetone 1:1). N-2-chloro-1,1,2,2-tetradeuterioethyl 3-amino-propan-1-ol. To a cooled (- 76°C) suspension of lithium aluminum deuteride (0.27 g, 7.08 mmol) in THF (20 ml) was added dropwise a solution of methyl N-2-chloro-1,1,2,2- tetradeuterioethyl-3-amino-propionate (1.20 g, 7.08 mmol), in THF (5 ml). The mixture was stirred at room temperature for 1 hr. Water (1 mi) in THF (10 ml) was added to destroy the remaining hydride. After filtration, the solid materiai was extracted with CH2CI2 . The filtrate and extract were combined and dried over anhydrous sodium sulfate. After filtration, the volume of the filtrate was reduced to about 10 ml in vacuo. The resulting solution was used directly for the subsequent step. 2-Chloro-3-(2-chloro-1,1,2,2-tetradeuterioethyl)-tetrahydro- 1,3,2-oxaza- phosphorine 2-oxlde. To a cooled (-76°C) solution of oxyphosphorus chloride (1.08 g, 7.08 mmoi) in CH2 CI2 (10 ml) was added the solution containing N-2- 191 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chloro-1,1,2,2-tetradeuterio-ethyl 3-amino-propan-1-ol obtained from the previous step. A solution of triethylamine (1 .43 g, 14.16 mmol) in CHgClg (5 ml) was then added to the mixture. The reaction mixture was stirred for 1 hr at room temperature. The solvent was removed in vacuo and the residue was extracted with anhydrous ethyl ether. The extract was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed to give a colorless oil (1.23 g), which was purified by silica gel column chromatography using ethyl ether as the eluant. 2-Amino-3-(2-chloro-1,1,2,2-tetradeuterloethyl)-tetrahydro-2H-ï,3,2-oxa- zaphosphorine 2-oxide (N2 D-d4). To the ethyl ether solution of the oxazaphosphorine chloride directly obtained from the previous step was bubbled with anhydrous ammonia at room temperature. After 3 hrs, when no more starting material could be detected by TLC, acetone (200 ml) was added to the mixture. Precipitated ammonium chloride was removed by filtration. The filtrate was dried over anhydrous sodium sulfate. After filtration, the solvent in the filtrate was removed to give colorless crystals (0.28 g, 19.6% from amine ester): mp 101-103°C; Rf 0.22 (CHaOla-acetone 1:3). The chemical identity of the product was confirmed by TLC and GC/MS analysis by comparing data from authentic N2D. The synthetic scheme of N2 D-d4 is shown in Scheme 5.13. S.3.3.6 Synthesis of 2',2'-dideuterio-iphosphoramide mustard Phenyl N-(2 -chloroethyi)-amidophosphoryi chloride. To a cooled (0°C) solution of phenyl dichlorophosphate (2 .1 1 g, 1 0 . 0 0 mmol) in CH2CI2 ( 2 0 ml) was added 2-chloro-ethylamine hydrochloride (1.16 g, 10.00 mmoi). Triethylamine 192 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (2 . 0 2 g, 2 0 . 0 0 mmol) in CH2CI2 ( 1 0 ml) was then added dropwise into the stirred suspension. The stirring was continued at room temperature for 1 hr. Anhydrous ethyl ether ( 1 0 0 ml) was added to the mixture and precipitated triethylamine chloride was removed by filtration. The filtrate was concentrated in vacuo to give a light yeliow oil (2.85 g). This crude material was purified on a silica gel column using CH2Ci2 as the elutant to give a colorless oil (2.44 g, 96.2%). Phenyl N-(2-chloroethyl) N'-(2-chloro-2,2-dideuterioethyl) diam ido- phosphate. Into a solution of phenyl N-(2 -chloroethyl)-amidophosphoryl chloride (1.67 g, 6.57 mmol) in CH2CI2 (20 ml) was added 2-chloro-2,2- dideuterioethyiamine (0.78 g, 6.57 mmol), followed by a solution of triethylamine (1.33 g, 13.14 mmol) in CH2 CI2 (5 ml) at room temperature. The reaction was completed in 0.5 hr as shown by TLC. Acetone (100 mi) was added into the reaction mixture to precipitate triethylamine hydrochloride, which was removed by filtration. The filtrate was evaporated in vacuo to give a pale yellow oil (2.40 g) which was purified using silica gel chromatography (CH2Ci2 -acetone 6 :1 ) to give a colorless oil (1.77 g, 90.2%): Rf 0.65 (CH2CI2-acetone 1:1); NMR (CDCI3 ) ô 3.17-3.45 (m, 6 H, 2x -NH-CH2-), 3.61 (t, J=5.7 Hz, 2 H, -GH2 CI), 7.12-7.43 (m, 5H, Ph-H). N-{2-chlora-ethyl) N’-(2-chloro-2,2-dicleuterio-ethyl) diamidophosphoric acid (IPM-d2). The phenyl phosphate (0.43 g, 1.44 mmol) was dissolved in CH2 CI2 (50 mi). After addition of 10% Pd/C (0.14 g), the mixture was hydrogenated at room temperature under normal pressure. The progress of hydrogenation was monitored by TLC. When the starting material disappeared (4 hrs), the precipitated product along with the catalyst was collected by filtration and washed with CH2 CI2 and acetone. Methanol was used to dissolve the 193 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. product and the catalyst was removed by filtration. Removal of methanol in the filtrate in vacuo at temperature below 25°C afforded crystalline IPIVI-dg (0.28 g, 87.5%); mp 115-116°C. GC/MS analysis confirmed its chemical identity. The synthetic scheme of IPM-da is shown in Scheme 5.14. S.3.3.7 Synthesis of 1',1',2',2',1",1",2",2"-octadeuterio-iphosphoram ide mustard 2-Chloro-1,1,2,2-tetradeuterio-ethylamlne hydrochloride. To a cooled (- 7600) 1 M HOI ethereal solution (20 ml) was added dropwise a solution of 1,1,2,2-tetradeuterio-ethanolamine hydrochloride (1.0 g, 15.38 mmol) in anhydrous ethyl ether (5 ml) while vigorous stirring was maintained. The amine alcohol hydrochloride salt was obtained as a white powder when the remaining HOI and ethyl ether were removed by rotary evaporation In vacuo. The salt was suspended in 1,2-dichloroethane (10 ml). Into the cooled (0°C) suspension was added thionyi chloride (5.49 g, 46.14 mmol). The reaction mixture was stirred for 0.5 hr at room temperature, and was then heated gradually to 50-60°C. The progress of the chlorination was followed by TLG. The precipitated product was collected by filtration and washed with CH2CI2 and acetone. Recrystallization in ethyl alcohol gave 2 -chloro-1 ,1 ,2 ,2 -tetradeuterio-ethylamine hydrochloride as colorless crystals (1.76 g, 95.3%); mp 136-138°C (softening, 143-146°C for the unlabeled compound); Rf 0.58 (acetone-methanol 1:1); MS m/z 84 (MH+ of the free amine). Phenyl N,N'-bls-(2-chloro-1,1,2,2-tetradeuterio-ethyl) diamidophosphate. To a cooled (0 °C) solution of phenyl dichlorophosphate (0 . 8 8 g, 4.17 mmol) in CH2 CI2 (15 ml) was added 2-chloro-1,1 ,2 ,2 -tetradeuterio-ethylam ine 194 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hydrochloride (1.0 g, 8.34 mmol). Triethylamine (1.68 g, 16.68 mmol) in CH2CI2 (10 ml) was added dropwise into the stirred suspension. The reaction mixture was stirred at room temperature for 1 hr. Acetone (200 ml) was then added to the mixture and precipitated triethylamine chloride was removed by filtration. The solvent in the filtrate was removed in vacuo to give a light yellow oil (1.68 g). The crude material was purified by siiica gel column chromatography eluted with CH2 Ci2 -acetone (5:1) to give a colorless oil (1.28 g, 1 0 0 %): Rf 0.65 (CH2 CI2- acetone 1:1): NMR (CDCI3 ) ô 3.07-3.48 (bs, 2 H, 2 x NH), 7.12-7.40 (m, 5H, Ph- H). N,N'-bis-(2'Chloro-1,1,2,2-tetradeuterio-ethyl) diamidophosphoric acid (IPM-ds). The phenyl phosphate (1.28 g) was dissolved in CH2Ci2 (50 ml). After addition of 10% Pd/C (0.5 g), the mixture was hydrogenated at room temperature under normal pressure. The progress of the hydrogenation was followed by TLG. When the starting materiai disappeared (4.5 hrs), the catalyst along with the product was collected by filtration and washed with GH2 CI2 . Methanol (40 ml) was used to dissolve the product and the catalyst was then removed by filtration. Removal of the solvent in the fiitrate in vacuo afforded crystalline IPM-dg (0.93 g, 96.9%): mp 114-115°G. GG/MS analysis confirmed its chemical identity. The synthetic scheme of IPM-ds is shown in Scheme 5.15. 195 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.4 RESULTS 5.4.1 Validation of analytical methods 5.4.1.1 Analysis of pseudoracemate of ifosfamide Representative GC/MS chromatograms of the selected ions at m/z 225 (IF), 229 (IP-d4 ), 233 (IF-de, internal standard) from the derivatized extract of a rat blank plasma sample and a plasma sample obtained from a rat given a pseudoracemate of IF (R-IF-d4/S-IF) are shown in Figure 5.4. As shown, no interference in ion regions at m/z 225, 229, and 233 was found in the rat blank plasma. Representative standard curves of S-IF and R-IF-d4 in rat plasma are shown in Figure 5.10 and 5.11. As shown, linear relationship between peak area ratios of S-IF or R-IF-d4 to their internal standard IF-dg and the amount of IF was found from 50 to 2000 ng monitored with correlation coefficients (r^) of 0.999 and 1.000, respectively. The within-run coefficients of variation were 3.7% (n=8) and 3.5% (n=8) at the level of 500 ng. The between-run coefficients of variation of the assay were 10.0% and 10.4% (n=6). The extraction recoveries of S-IF and R-IF- d4 from rat plasma were 92.4% and 93.1%, respectively. 5.4.1.2 Analysis of the derived metabolite of ifosfamide — 4-hydroxy ifos famide Representative GC/MS chromatograms of the selected ions at m/z 412 (4- OHIF), 416 (4-OHIF-d4), 420 (4-OHIF-dg, internal standard) from the derivatized extract of a rat blank plasma sample and a plasma sample obtained from a rat given a pseudoracemate of IF (R-IF-d4/S-IF) are shown in Figure 5.5. As shown, 196 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. no interference in ion regions at m/z 412,416, and 420 was found in the rat blank plasma. A representative standard curve of 4-OHIF in rat plasma Is shown in Figure 5.12. As shown, linear relationship between peak area ratio of 4-OHIF to Its internal standard 4 -0 HIF-dg and the amount of 4-OHIF was found from 50 to 2000 ng monitored with a correlation coefficient (r^) of 0.997. The within-run coefficient of variation was 1.1% (n=8 ) at the level of 500 ng. The between-run coefficient of variation of the assay was 8.1% (n=6 ). The extraction recovery of the cyanohydrin adduct from rat plasma was 52.5%. 5.4.1.3 Analysis of the derived metabolite of Ifosfamide - N2-dechloroethyl ifosfamide Representative GC/MS chromatograms of the selected ions at m/z 235 (N2D), 237 (N2 D-d2 ), 239 (N2 D-d4 , internal standard) from the derivatized extract of a rat blank plasma sample and a plasma sample obtained from a rat given a pseudoracemate of IF (R-IF-d^S-IF) are shown in Figure 5.6. As shown, no interference in ion regions at m/z 235, 237, and 239 was found in the rat blank plasma. A representative standard cun/e of N2D in rat plasma is shown in Figure 5.13. Linear relationship between peak area ratio of N2D to its internal standard N2D- d4 and the amount of N2D was found from 10 to 400 ng monitored with a correlation coefficient (r^) of 0.9992. The within-run coefficient of variation was 1.7% (n=8 ) at the level of 100 ng. The between-run coefficient of variation of the assay was 10.3% (n=6 ). The extraction recovery of N2D from rat plasma was 60.9%. 197 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.4.1.4 Analysis of the derived metabolite of ifosfamide - N3-dechloroethyl ifosfamide Representative GC/MS ctiromatograms of the selected ions at m/z 235 (N3D), 239 (N3 D-d4 ), 243 (N3D-da, internal standard) from the derivatized extract of a rat blank plasma sample and a plasma sample obtained from a rat given a pseudoracemate of IF (R-IF-d4/S-IF) are shown in Figure 5.7. As shown, no interference in the interested region at m/z 235, 239, and 243 was found in the rat plasma blank. A representative standard curve of N3D in rat plasma is shown in Figure 5.14. As shown, linear relationship between peak area ratio of N3D to its internal standard N3D-dg and the amount of N3D was found from 10 to 400 ng monitored with a correlation coefficient (r^) of 0.997. The within-run coefficient of variation was 4.4% (n=8 ) at the level of 100 ng. The between-run coefficient of variation of the assay were 11.7% (n=6 ). The extraction recovery of N3D from rat plasma was 66.5%. 5.4.1.5 Analysis of the derived metabolite of ifosfamlde - iphosphoramlde mustard Representative GC/MS chromatograms of the selected ions at m/z 329 (IPM), 333 (IPM-dg), 337 (IPM-ds, internal standard) from the derivatized extract of a rat blank plasma sample and a plasma sample obtained from a rat given a pseudoracemate of IF (R-IF-d4 /S-IF) are shown in Figure 5.8. As shown, no interference in ion regions at m/z 329, 333, 337 was found in the rat blank plasma. 198 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Representative standard curves of IPM and IPM-dg in rat plasma are shown in Figure 5.115 and 5.16. As shown, linear relationship between peak area ratios of IPM and IPM-da to their internal standard IPM-ds and the amount of IPM and IPM-dz was found from 50 to 2000 ng monitored with correlation coefficients (r^) of 0.997 and 0.996, respectively. 5.4.2 Pharmacokinetics of the enantiomers of ifosfamide and the derived metabolites in the rat following iv administration of pseudoracemate of ifosfamide Plasma concentration-time data of the enantiomers of IF and the derived metabolites, 4-OHIF, N2D, N3D, and IPM following iv administration of 1:1 mixture of respective pseudoracemate at a total dose of 40 mg/kg are shown in Tables 5.2 to 5.16. The respective plasma concentration-time profiles of the enantiomers and their derived metabolites are displayed in Figures 5.17 to 5.28. 5.4.2.1 Pharmacokinetics of (S)- and (R)-ifosfamide The pharmacokinetic parameters of enantiomeric IFs and their respective mean values are shown in Table 17. After intravenous administration of IF pseudoracemates at a total dose of 40 mg/kg to six rats, plasma concentration-time data of the S-enantiomer exhibited monoexponential profiles with an average elimination half-life (ti/2 ) of 37.82 min (range 31.66 - 44.68 min) and could therefore be described by one-compartment kinetics. The mean total clearance (CLj) was computed to be 5.02 ± 1.38) ml/min and the mean volume of distribution to be 265.79 ± 47.68 ml. The mean 199 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. initial concentration (Co) was 23.63 ± 4.34 pg/ml and the mean area under concentration (AUG) value was 1297.66 ± 372.04 min-^ig/ml. Similarly, plasma concentration-time data of the R-enantiomer also exhibited monoexponentional declines in all rats with a mean elimination half-life (t-ig) of 34.21 min (range 30.26 - 41.45 min) following intravenous administration of IF pseudoracemates. The mean total clearance (CLy) was 5.86 ± 1.48 ml/min and the mean volume of distribution 284.10 ± 58.85 ml. The mean Co was 22.27 ± 4.2 |xg/ml and the area under the curve (ADC) 1109.83 ± 329.44 mimpg/ml. The mean relevant pharmacokinetic parameters derived from the S-IF were compared to those derived from the R-IF and no significant difference was found on the volume of distribution. However, small but statistically significant difference (p < 0.05) were found on ADC value and total clearance. The ADC value of the S enantiomer was slightly larger than that of the R enantiomer, while R isomer was cleared from the blood stream slightly faster. 5.4.2.2 Metabolite pharmacokinetics of 4-hydroxy ifosfamide As shown, after iv administration of the respective pseudoracemate, plasma profiles of the derived metabolite 4-OHIF displayed both the ascending and descending phase in three of the rats (Rat 2,3,6), while In the other three (Rat 1,4,5), only the descending phase was observed. This could be caused by the inter-subject variation. Plasma profiles of this metabolite was best-fitted by a one-compartment metabolite model and the relevant pharmacokinetic parameters were computed (Table 5.18). 4-OHIF and 4 -OHIF-d4 were eliminated from blood stream with half-lives of 44.96 and 39.30 min, values comparable to that of their respective parent compounds. This could be 200 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. explained as partially due to the well-known phenomenon of a flip-flop model for drug metabolite. Significantly higher levels of 4-OHIF derived from R-IF than from S-IF were found in all six rats. The mean peak concentration (Tmax) of this metabolite derived from R-IF was 1.97 ± 0.77 pil/ml, while that derived from S-IF, 1.13 + 0.43 |jJ/ml. The mean AUC values of 4-OHIF derived from S-IF and R-IF were 130.92 ± 27.90 min»p.g/ml and 81.89 ± 18.87 min*pg/ml, respectively. S.4.2.3 Metabolite pharmacokinetics of iphosphoramide mustard IPM levels In Rat 1 were not analyzed because of inadequate sample volumes. Therefore, pharmacokinetic parameter calculations were based on data from five rats. As shown, both the ascending phase and the descending phase were observed in ail five rats. The plasma profiles of this metabolite were best-fitted by a one-compartment metabolite model In two rats (Rat 2,6), and by a two-compartment model in the remaining three rats. The relevant pharmaco kinetic parameters are shown in Table 5.19. Compared to the parent drugs S-IF and R-IF, IPM displayed a longer elimination phase with half-lives of 82.12 min (range 68.92 - 98.17 min) and 87.01 min (range 60.02 - 113.69 min) when derived from 8 - and R-IF, respectively. Similar to those of its metabolic precursor 4-OHIF, IPM levels derived from R- IF in all six rats were higher than those derived from the S-enantiomer. The mean peak concentration of this metabolite derived from R-IF was 1.51 ± 0.38 pl/ml and appeared between 12.16 and 71.58 min, whiie that derived from the S- enantiomer was 0.97 ± 0.23 pl/ml and appeared between 5.70 and 57.64 min. The mean AUC values of IPM derived from S-IF and R-IF were 230.01 ± 83.22 201 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. miiTng/ml and 156.06 ± 52.92 mitT[ig/mi, respectively, also Indicating significantly more IPM derived from R-IF than from S-IF. 5.4.2.4 Metabolite pharmacokinetics of N2-dechloroethyl ifosfamide Following iv administration of the respective pseudoracemate, both the ascending phase and the descending phase were observed in the plasma profiles of the derived metabolite N2D in all rats. The plasma profiles of this metabolite were best-fitted by a one-compartment metabolite model and the relevant pharmacokinetic parameters were computed as shown in Table 5.20. Compared to the parent enantiomers, N2D displayed a longer elimination phase with half-lives of 163.19 min (range 121.43 - 227.23 min ) and 140.51 min (range 127.01 - 175.17 min) when derived from S- and R-IF, respectively. In contrary to 4-OHIF and IPM, the plasma levels of N2D derived from S-IF were much higher than those derived from the R-enantiomer in all six rats. The mean peak concentration of this metabolite derived from S-IF was 1.31 ± 0.31 (xl/ml and appeared between 73.66 and 116.41 min, while that derived from the R-enantiomer was 0.48 ± 0.05 pl/ml and appeared between 35.48 and 91.58 min. The mean AUC values of N2D derived from S-IF and R-IF were 494.09 ± 92.12 min*jxg/ml and 140.46 ±21.73 min-pg/ml, respectively. 5.4.2.5 Metabolite pharmacokinetics of N3-dechloroethy! ifosfamide The plasma profiles of N3D derived from the respective enantiomers of IF displayed both the ascending phase and the descending phase in all rats. The plasma profiles of this metabolite were best-fitted by a one-compartment metabolite modei and the relevant pharmacokinetic parameters were computed 202 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. as shown (Table 5.21). Similar to N2D, N3D displayed a long elimination phase with half-lives of 193.47 min (range 159.53 - 204.03 min) and 164.34 min (range 129.90 - 213.20 min) when derived from 8 - and R-IF, respectively. In two rats (Rat 1,2), N3D plasma levels derived from R-IF were higher than those derived from the S-enantiomer; however, the opposite result was found in the rest four rats. The mean peak concentration of this metabolite derived from the S-enantiomer was 0.50 ± 0.10 p,l/ml and appeared between 44.67 and 153.25 min, while that derived from the R enantiomer was 0.63 ± 0 .1 6 [xl/ml and appeared between 36.91 and 107.87 min. The mean AUC value of N3D derived from S-IF 191.01 ± 27.82 min*txg/ml, which was not statistically different from that of N3D derived from R-IF, 209.39 ± 55.69 min'pg/ml (p=0.49). Table 5.22 shows the R/S ratios of AUG values between R-IF and S-IF and their respective metabolites and the results from statistical analysis. 5.5 Discussion The use of a suitable methodological approach is crucial to a well-planned study. There are two major methods for the investigation of stereoselective metabolism and pharmacokinetics of a chiral drug. A conventional method involves the administration of the individual enantiomers separately to two different groups of subjects or the same group of subjects at different occasions. This method has inherent shortcomings in that it requires a large population of subjects to overcome inter-subject or intra-subject variations. Additionally, this method loses potential enantiomer-enantiomer interaction as in the case of racemic drug since only one enantiomer at a time is 203 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. studied (47). Therefore, this study design does not mimic the actuai stereoselective metabolism and pharmacokinetics of a racemate. A more frequentiy used method requires administration of a racemic drug to a subject. The plasma or urinary ieveis of the enantiomers are anaiyzed foliowing separation of enantiomers on a chirai GC or HPLC column. However, this method cannot be used in our study. First, there are totally ten compounds, including enantiomeric IPs and four metabolites derived from each of them, need to be quantitated, it would be difficult to separate all these compounds on one column. Secondly, some of the metabolites are achirai aithough they are generated from stereoselective metaboiism. Therefore, these metaboiites could not be differentiated by this method; thus it would be virtually impossible to probe stereoselective metabolism for these metabolites by this method. Alternatively, before column separation, the sample containing the racemic drug and its chiral metabolites are derivatized with a chirai reagent to form diastereomers. Since diastereomers possess different physicochemical pro perties, they can be separated by chromatographic methods. However, this approach is also not suitable in this study for the same reasons described above. A recent method appeared in the literature involving the use of ^tpNMR for the detection of phosphorus-containing chirai drugs, including IF (23). The differentiation of the enantiomers of the parent drug and their chiral metabolites was made possible by using a chiral shifting agent, for example, tris-(trifluoro- methylhydroxyimethiene)-d-camphorate-europium(lll). Since ^ipNMR enables the direct measurement of phosphorus containing compounds in intact body fluid, the problems associated with extraction, recovery, and chemical derivatization 204 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. are avoided. However, the sensitivity of this techniques is intrinsically lower compared to other detection methods, making its application to pharmacokinetic study virtually impossible. Again, this method does not allow the differentiation of IPM and 4-OHIF/aldolF derived from their enantiomeric precursors because they are achiral. Additionally, the extended period required for measurement may not be applicable for labile metabolites. All of these difficulties mentioned above could be circumvented by a method using pseudoracemate and GC/MS. A pseudoracemate (58) mimics the racemate of a chiral drug; therefore, enantiomeric interaction between (R)-IF and (S)-IF if exists would be preserved. The stable isotope labels on one of the enantiomer allow a chemical discrimination of the enantiomers without an eiaborate resoiution method. The enantiomers of the parent drug and their metabolite 4-OHIF, N3D, N2D, and IPM could be anaiyzed by GC/MS. The use of selected ions for quantitation allows specific analysis of the parent pseudoracemate and their metabolites even when some of the compounds are eluted closely. This method offers one unique advantage in analyzing achiral metabolites generated from their enantiomeric precursors (IPM and 4- OHIF/aldolF) by virtue of the labels. Additionally, the mass spectrometer detector provides high sensitivity for the assay. While several analytical methods for IF have been published, few for its metabolites are available. Because of the chemical instability of some of the metabolites (e.g. IPM and 4-OHIF/aldolF), analytical procedures without the use of isotopically labeled internal standard, especially those Involving labor-intensive manipulations, could suffer from inherent loss and produce less reliable and more scattered data. The present GC/MS analytical method for IF and its metabolites 205 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4-OHIF, N3D, N2D, and IPM utilized corresponding stable isotopically labeled analogs as the internal standards, which compensate procedural losses, providing a distinct advantage. Selecting a good internal standard is crucial for the GC/MS assay. It is commonly recognized that labeled analogs serve as the best internal standards. Generally, for GC/MS analytical methodology using a quadrupole instrument, an analog of the analyte with different retention time or with similar retention time but with different mass may be a suitable internal standard. Thus, CP-dg has been successfully used as the internal standard for the GC/MS assay for CP and alcoCP (87). However, with the Ion Trap instrument, when different structures than the analyte were used as internal standards, the results were found less satisfactory, even though the chemical structures and the chromatographic behaviors were similar. Subsequently, it was found necessary to employ the deuterium-labeled analog as the internal standard for each of the analyte and the results obtained were superior. However, several unexpected problems have been encountered and special considerations were given as follows. For the analysis of IF, IF-dg was first synthesized as the potential internal standard. It was soon found that IF-dg was not suitable because It interfered with the analysis of the metabolite N2 D. Using the present GC/MS conditions, derivatized IF and N2D were eluted at the same retention time. Dehydrochlorinated IF-dg gave the molecular ion at m/z 233 under GC/CIMS condition. Since this ion contains one Cl atom, the s^ci isotopic peak at m/z 235 overlaps with the Ion of derivatized N2D, which also has a molecular ion at m/z 235. To solve this problem, IF-dg was synthesized and used as the internal 206 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. standard for IF. However, the molecular ion of derivatized iP-de {m/z 231) could not be used since it overiapped withi the ^^Cl isotopic signai of IP-d4 , which was a component of the pseudoracemate. Thus, the s^CI isotopic peak of the derivatized IP-de, which appeared at m/z 233, was used as the reference ion for the internal standard. Deuterium-labeled compounds have slight different physicochemical properties compared with their unlabeled analogs. For example, slight differences in retention time were observed between the labeled and unlabeled analogs. Therefore, unlabeled IP and IP-d4 were quantitated using different standard curves to avoid any potential variation risen from that matter. Problem was also encountered in selecting an appropriate internal standard for the analysis of N2 D. Initially, N2D-de was synthesized and used as the internal standard for N2D assay. It was later found that the fragmentation of the molecular ion {m/z 241 ) of the derivatized N2D occurred, generating an ion at m ^ 225. This ion interfered with the analysis of unlabeled IP, the ion selected for which was also at m/z 225. Therefore, N2 D-d4 was synthesized and the molecular ion {m/z 239) of its derivative was found to be suitable. 4-OHIP-d6 was synthesized and used as the internal standard for the analysis of 4-OHIP. The moiecular ion for its siiyi cyanohydrin adduct is at m/z 418. 4- 0 HIP-d4 was derived in v/Vofrom IP-d4 and its silyl derivative of the cyanohydrin adduct gave a molecular ion at m/z 416. Since this ion contains a Cl atom, its 37CI isotope gave an ion at m/^ 418. Therefore, the s^CI isotopic ion of the derivatized 4-OHIF-d6 at m/z 420 couid be used as the reference ion and 4- OHIP-de was successfully used as the internal standard. 207 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IPM-d4 was previously used as the internal standard for the pharmacokinetic and stability study of IPM (85). However, this labeled compound was found not suitable as the internal standard in this study since IPM-da is metabolically generated from the pseudoracemate component IP-d4 . The isotopic ion of the derivatized IPM-dg, m /z333 would overlap with the molecular ion of IPM-d4 (m/z 333). Therefore, IPM-ds was synthesized and used as the internal standard for the analysis of IPM and IPM-dg. The proper use of s^CI isotopic ion also played an important role in the analysis of IPM in this study. The molecuiar ion (m/z 331 ) of the derivatized IPM- dg could not be used directly for its quantitation, since ion m/z 331 is also derived from the ^^Cl isotope of derivatized IPM (m/z 329). To circumvent this problem, the isotopic ion of the molecular ion of the derivatized IPM-da at m/z 333 needed to be used as the reference ion for IPM-dg. For this reason, IPM-da was synthesized and used as the standard compound. Thus, the intricacy of this GC/MS method for the analysis of the complex mixture of metabolites with different labels relied heavily on the proper selection of labeled internal standards and the suitable ions. This attested to the power of the stable isotope-GC/MS technology for the analysis of a complex mixture. The current analytical method covers the parent drug IF and four of its metabolites. This method has been used to examine simultaneously all three metabolic pathways responsible for the antitumor and toxicological effects. With all five internal standards available, this method is specific and sensitive. With the use of a low cost mass detectore (MSD), this assay method can be applied to support routine clinical pharmacokinetic studies of IF. 208 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The stereoselective metabolism of enantiomeric IF has been demonstrated by the determination of the R/S ratios of the unchanged enantiomeric IPs and their respective metabolites in rat urine (Chapter 4). To further investigate the stereoselectivity on the pharmacokinetic disposition, three metabolic pathways (ring oxidation, ring N-dealkylation, and side chain N-dealkylation) were examined by measuring plasma levels of IF, 4-OHIF, N2D and N3D. The stereoselectivity on the activation pathway of IF was examined by study the generation of 4-OHIF and the ultimate alkylating metabolite iPM. The result confirmed the observation obtained from the urinary study that the activation pathway preferred R-IF. The similarity on the values of R/S ratio of 4-OHIF and IPM also suggested that the subsequent formation of IPM be a simple chemical process. The pharmacokinetic data showed iPM derived from enantiomeric IF declined slowly in plasma with half-lives of 82.12 and 87.01 min. It has been reported that when IPM was directly administered to the rat, it was eiiminated rapidiy with a short half-life of 12.7 min (85). This difference in t i /2 can be explained on the basis of a well known flip-flop phenomenon for drug metabolite and the existence of a diffusional barrier for polar metabolites (108,109). It has long been a controversy regarding whether 4-OHCP/4-QHIF or PM/IPM are the most important metabolites responsible for the antitumor activity of CP/IF. Some investigators favor 4-OHCP/4-OHIF because these moiecuies are relatively lipophilic compared to PM/IPM; therefore, 4-OHGP/4-OHIF can readily diffuse across biologically membranes (110,111). On the other hand, some investigators believe PM/IPM are more important, since with longer exposure time (4 hr), PM/IPM are highly toxic to LI 210 and CCRF-GEM cel is (16,85). Our 209 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. data showed that the peak plasma levels of 4-OHIF and IPM were comparable, suggesting both maybe important for the antitumor activity. According to the plasma profiles of 4-OHIF and IPM, it is possible that the antitumor activity of IF could be separated into two phases. The initial phase probably involves both 4- OHIF and IPM, while the later phase only involves IPM since 4-OHIF is eliminated with a relative short half-life. While no selectivity on the N3-dealkylation was observed, significant stereoselectivity on the N2-dealkylation was found. Since N2D was selectively generated from the S enantiomer. It is also expected that the co-product, CAA, is produced stereoselectively from the same enantiomer. This finding is also consistent with the result from the urinary study in general (Chapter 4). Both N2D and N3D have long half-lives. Since they are both monofunctional alkylating agents, they are not expected to contribute significant antitumor activity. However, N2D and N3D have been shown to undergo further N- dealkylation (Chapter 3). CAA generated from these pathways may also contribute to the observed CNS toxicity in patients receiving IF (22,86). Recently, Wainer etal. (112) reported the results of the efficacy and toxicity studies using purified enantiomers of IF in F344 rats. The R enantiomer was found to possess higher myelotoxicity. The pharmacokinetic analysis confirmed that R-IF was metabolized to a greater extent than S-IF. Our results were consistent with their finding, and provided a metabolite pharmacokinetic explanation. In conclusion, an efficient and specific methodology has been developed for the investigation of stereoselectivity on the metabolism and pharmacokinetics of the enantiomers of IF. Our data showed that after Intravenous dosing, R-IF was 210 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. more readily activated by cytochrome P450 enzymes to higher levels of 4-OHIF and IPM. On the other hand, S-IF underwent N2-dealkylation to a greater extent, expectedly leading to higher plasma levels of CAA, the metabolite implicated as the species responsible for the observed neurotoxicity in patients receiving IF therapy (22,86). Since the averaged R/S ratio of the AUC values of 4-OHIF and IPM is 1.54, and that of N2D is 0.29, it is approximately estimated that the ratio of the therapeutic index of R-IF and S-IF is 5.3:1 (1.54 x 1/0.29). The potential of the use of the R-IF (the eutomer) alone instead of racemic IF in human chemotherapy is worthy of exploring. 211 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.1 ions selected for monitoring and the respective retention times of derivatives of enantiomeric ifosfamides, their metabolites and internal standards. Chemical entity Selected ion (m/z) Retention time (min) IF S -or R-IF 225 8.62 S- or R-IF-d4 229 8.57 IF-de (IS) 233 (3701) 8.55 N2D N2D 235 8.58 N2D-d2 237 8.57 N2D-d4 (IS) 239 8.55 N3D N3D 235 5.70 N 3 D -d 4 239 5.67 N3D-da (IS) 243 5.63 IPM IPM 329 8.55 IPM-dg 333 (37CI) 8.53 IPM-ds (IS) 337 8.48 4-OHIF 4-OHIF 412 15.25 4-OHlF-d4 416 15.23 4-OHIF-ds (IS) 420 (37CI) 15.22 212 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.2 Plasma concentration-time data of S-ifosfamide and R-ifosfamide-d^ (Rat 1 and Rat 2) following iv administration of a 1:1 mixture of S- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. R ati Rat 2 Time S-IF R-IF-d4 Time S-IF R-IF-d4 (min) (pg/ml) (pg/ml) (min) (pg/ml) (pg/ml) 0 0 5 31.12 32.35 6 27.62 26.14 1 0 18.84 17.54 15 22.38 21.16 2 0 15.14 14.59 30 17.98 16.50 45 7.70 7.01 47 14.37 12.64 60 6.30 5.86 60 1 2 . 2 0 10.67 90 3.48 2.97 90 9.14 7.34 1 2 0 1.83 1.59 1 2 0 5.78 4.51 150 1 .0 2 0.84 150 3.71 2.70 180 0.62 0.49 180 2.18 1.61 2 1 0 0 . 2 2 0.27 2 1 0 1.34 0.94 240 0.05 0 . 1 0 240 0.70 0.48 300 0.26 0.18 213 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.3 Plasma concentration-time data of S-ifosfamide and R-ifosfamide-d4 (Rat 3), and S-ifosfamide-d4 and R-ifosfamide (Rat 4) following iv administration a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 (Rat 3) or S-ifosfamide-d4 and R-ifosfamide (Rat 3) at a total dose of 40 mg/kg. Rats Rat 4 Time (min) S-iF (Md/ml) R-IF-d4 (fig/ml) Time (min) S-!F-d4 (ug/ml) R-IF (pg/ml) 0 0 6 20.48 21.03 5 18.11 17.77 15 17.68 17.33 15 15.62 14.65 30 13.87 12.72 30 1 2 . 1 2 10.45 45 11.36 9.35 47 9.64 7.60 60 7.90 6.25 60 8 .0 1 6.30 90 4.28 3.25 90 4.23 2.75 1 2 0 2.25 1.63 1 2 0 2.65 1.59 150 1.30 0.80 150 2.14 1.27 180 0.62 0.38 180 1.25 0.59 2 1 0 0.36 0 .2 1 2 1 0 1.08 0.39 240 0 . 1 0 0.03 214 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.4 Plasma concentration-time data of S-ifosfamide and R-ifosfamlde-d4 (Rat 5 and Rat 6 ) following iv administration of a 1 ;1 mixture of S- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. Rats Rate Time (min) S-IF (Mfl/mi) R-IF-d4 (pg/ml) Time (min) S-IF (pg/ml) R-!F-d4 (fig/mi) 0 0 5 2 0 . 6 6 19.25 5 17.41 17.65 15 14.17 13.06 15 15.94 15.64 30 10.54 9.39 30 12.95 12.43 47 6.50 5.30 47 11.61 10.32 1 2 0 2 . 2 0 1.64 60 8.64 8 . 2 2 150 0.81 0.67 90 4.59 3.60 180 0.51 0.30 1 2 0 2.65 1.89 2 1 0 0.17 150 1.48 1.18 180 1 .0 0 0.46 2 1 0 0.94 0.31 240 0.40 0.28 215 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T able 5.5 Plasma concentration-time data of 4-hydroxy ifosfamide, 4-hydroxy ifosfamide-d4 in Rat 1 following iv administration of a 1 ;1 mixture of S- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. Time (min) 4-OHIF (pg/ml) 4-OHIF-d4 (pg/ml) 0 5 1.10 1.76 10 0.93 1.39 20 1.07 1.56 30 0.63 0.95 45 0.68 1.15 60 0.54 0.88 90 0.40 0.55 120 0.30 0.41 150 0.19 0.19 180 0.12 0.16 216 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.6 Plasma concentration-time data of 4-hydroxy ifosfamide, 4-hydroxy ifosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-dg in Rat 2 following iv administration of a 1:1 mixture of S- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. Time (min) 4-OHIF (pg/ml) 4-OHIF-d4 (pg/ml) IPM (MS/ml) IPM-dg (M Q /m l) 0 6 0.69 1.67 0.12 1.13 15 0.72 1.38 0.53 1.24 30 1.03 1.64 0.73 1.20 47 0.71 1.50 0.89 1.12 60 0.62 1.04 1.45 2.20 90 0.42 0.78 1.09 1.79 120 0.15 0.40 1.12 1.50 150 0.30 0.36 0.71 1.02 180 0.09 0.17 0.72 0.98 210 0.07 0.22 0.52 0.57 240 0.54 0.72 300 0.21 0.35 360 0.13 0.32 420 0.10 0.20 217 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.7 Plasma concentration-time data of 4-hydroxy ifosfamide, 4-hydroxy ifosîamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-da in Rat 3 following iv administration of a 1 ;1 mixture of S- ifcsfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. Time (min) 4-OHIF (ixg/mi) 4-OHIF-d4 (MQ/ml) IPM (pg/ml) IPM-da (pg/mi) 0 6 0.44 1.05 0.86 1.28 15 0.53 1.05 1.18 2.09 30 0.58 0.95 0.99 1.75 45 0.36 0.68 1.08 1.70 60 0.45 0.67 0.93 1.46 90 0.22 0.31 0.73 1.10 120 0.13 0.18 0.49 0.74 150 0.09 0.17 0.41 0.61 180 0.05 0.10 0.44 0.69 210 0.43 0.62 240 0.41 0.59 300 0.34 0.53 360 0.27 0.41 218 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.8 Plasma concentration-time data of 4-hydroxy ifosfamide, 4-hydroxy ifosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-d2 in Rat 4 following iv administration of a 1:1 mixture of S- ifosfamide-d4 and R-ifosfamide at a total dose of 40 mg/kg. Time (min) 4-OHIF (pg/ml) 4-0HIF-d4 (ng/ml) IPM (pg/ml) IPM-da (pg/mi) 0 5 2.00 1.25 15 1.92 1.22 0.20 0.20 30 1.29 1.00 0.87 0.42 45 0.67 0.60 1.27 0.75 60 0.70 0.60 1.04 0.64 90 0.52 0.44 0.78 0.57 120 0.36 0.31 0.68 0.47 150 0.19 0.19 0.51 0.48 180 0.13 0.16 0.39 0.35 210 0.46 0.43 240 0.55 0.38 300 0.40 0.29 360 0.35 0.23 420 0.32 0.22 219 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.9 Plasma concentration-time data of 4-hydroxy ifosfamide, 4-hydroxy ifosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-da in Rat 5 following iv administration of a 1:1 mixture of S- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. Time 4-OHIF 4-QH!F-d4 1P M IPM-da (min) (M g/m i) (pg/ml) (MS/ml) (pg/ml) 0 5 1.79 3.56 15 1.22 2.61 1.54 1.94 30 1.16 2.09 2.98 2.83 47 0.59 1.00 - - 60 0.45 0.69 - 0.82 90 0.30 0.48 0.43 0.63 120 0.17 0.30 0.35 - 150 0.07 0.12 0.33 0.48 180 0.06 0.37 0.52 210 0.03 - - 240 - - 300 - - 360 0.18 0.41 220 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.10 Plasma concentration-time data of 4-hydroxy ifosfamide, 4-hydroxy ifosfamide-d4 , iphosphoramide mustard, and iphosphoramide mustard-da in Rat 6 following iv administration of a 1:1 mixture of S- ifosfamide and R-ifosfamide-d^. at a total dose of 40 mg/kg. Time (min) 4-OHIF (Mg/ml) 4-OHlF-d4 (Mg/ml) 1 P M (pg/ml) iPM-da (pg/ml) 0 5 0.77 1.41 0.12 0.34 15 1.13 1.78 0.71 1.26 30 0.83 1.47 1.21 1.78 45 0.79 1.31 1.00 1.62 60 0.50 1.15 1.17 1.64 90 0.31 0.48 0.90 1.27 120 0.26 0.32 0.55 0.85 150 0.19 0.26 0.44 0.53 182 0.59 0.72 210 0.40 0.49 240 0.26 0.33 300 0.14 0.15 360 0.09 0.08 221 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.11 Plasma concentration-time data of N2-dechloroettiyl ifosfamide, N2- dechloroethyl ifosfamide-ds, N3-dectiloraettiyl ifosfamide, and N3- dechioroethyi iîosfamide-d4 in Rat 1 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. Time (min) N2D (pg/ml) N2D-d2 (ng/ml) N3D (pg/ml) N3D-d4 (M Q/m l) 0 5 0.48 0.28 0.30 0.33 10 0.75 0.42 0.27 0.46 20 1.08 0.50 0.46 0.59 45 0.91 0.44 0.42 0.64 60 0.88 0.39 0.36 0.58 90 1.17 0.42 0.42 0.61 120 1.07 0.38 0.41 0.54 150 0.96 0.30 0.31 0.47 180 0.88 0.27 0.29 0.40 210 0.77 0.27 0.23 0.37 240 0.67 0.21 0.20 0.31 300 0.58 0.16 0.17 0.23 360 0.50 0.14 0.21 0.26 420 0.42 0.11 0.21 222 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.12 Plasma concentration-time data of N2-dechioroethyl Ifosfamide, N2- dechioroetfiyi ifosfamide-da. N3-dechioroettiy! Ifosfamide, and N3- dectiioroettiyl lfosfamide-d4 In Rat 2 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d^ at a total dose of 40 mg/kg. Time (min) N2D (pg/ml) N2D-d2 (MO/ml) N3D (pg/ml) N3D-d4 (ng/ml) 0 6 0.45 0.15 0.06 0.30 15 0.70 0.26 0.07 0.36 30 1.11 0.42 0.27 0.60 47 1.36 0.46 0.26 0.76 60 1.43 0.49 0.33 0.80 90 1.83 0.60 0.50 0.91 120 1.75 0.53 0.42 0.87 150 1.84 0.51 0.44 0.82 180 1.75 0.48 0.73 0.98 210 1.46 0.37 0.49 0.66 240 1.35 0.33 0.42 0.69 300 0.99 0.27 0.27 0.47 360 0.80 0.18 0.25 0.35 420 0.66 0.13 0.21 0.29 223 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.13 Plasma concentration-time data of N2 -dechioroethyi ifosfamide, N2- decfiioroethyiifosfamide-da, N3 -dectiioroettiyi ifosfamide, and N3- dechioroettiyi ifosfamide-d4 in Rat 3 following iv administration of a 1 ;i mixture of S-ifosfamide and R-ifosfamide-d^. at a total dose of 40 mg/kg. Time (min) N2D (ng/mi) N2D-ds (pg/ml) N3D (MS/mi) N3D-d4 (M 0 /mi) 0 6 0.40 0.18 0.18 0.25 15 0.79 0.33 0.35 0.41 30 1.32 0.53 0.47 0.55 45 1.36 0.47 0.56 0.63 60 0.53 0.49 0.55 0.62 90 0.64 0.50 0.54 0.57 1 2 0 1.51 0.41 0.50 0.48 150 1.52 0.39 0.44 0.41 180 1.17 0.27 0.34 0.31 2 1 0 1.08 0.26 0.29 0.28 240 0.95 0 .2 1 0.25 0.27 300 0 . 6 6 0.14 0.23 0 . 2 0 360 0.54 0 . 1 0 0.19 0.17 420 0.54 0.09 0 .2 1 0.16 224 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.14 Plasma concentration-time data of N2-dechioroethyl ifosfamide, N2- dechloroethylifosfamide-d2 , N3-dechioraethyl ifosfamide, ànd N3- dechloroethyl ifosfamide-d^. in Rat 4 following iv administration of a 1:1 mixture of S-ifosfamide-d4 and R-ifosfamide at a total dose of 40 m g4<g. Time (min) N2D (M Q /m l) N2D-da (M Q /m l) N3D (M Q /m l) N3D-d4 (M Q /m l) 0 5 0.28 0.60 0.49 0.39 15 0.40 0.79 0.64 0.60 30 0.40 0.90 0.69 0.66 45 0.42 1.04 0.69 0.61 60 0.64 1.20 0.72 0.68 90 0.44 1.21 0.68 0.64 150 0.39 1.25 0.58 0.58 180 0.35 1.25 0.51 0.55 210 0.38 1.04 0.46 0.50 240 0.24 0.93 0.36 0.37 300 0.19 0.67 0.27 0.31 360 0.13 0.56 0.21 0.25 420 0.11 0.44 0.17 0.18 225 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.15 Plasma œncentratlon-time data of N2-dechloroethyl ifosfamide, N2- dechloroethyi ifosfamide-d2 , N3-dechloroethyl ifosfamide, and N3- dechloroethyi ifosfamide-d^. in Rat 5 following iv administration of a 1:1 mixture of S-iîosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. Tirre (min) N2D (pg/ml) N2D”d2 (pg/ml) N3D (pg/ml) N3D-d4 (pg/ml) 0 5 0.87 0.46 0.47 0.42 15 0.84 0.38 0.44 0.38 30 0.99 0.47 0.47 0.43 47 1.02 0.47 0.43 0.42 60 - 0.47 0.59 0.50 90 1.03 0.41 0.52 0.44 120 1.15 0.44 0.44 0.41 150 1.22 0.38 0.46 0.45 180 1.09 0.32 0.43 0.40 210 1.01 0.33 0.33 0.31 240 0.92 0.25 0.34 0.30 300 0.70 0.18 0.27 0.21 360 0.57 0.15 0.22 0.18 420 0.46 0.13 0.19 0.16 226 Reproduced witti permission of ttie copyrigfit owner. Furtfier reproduction profiibited witfiout permission. Table 5.16 Plasma concentration-time data of N2-dechloroethyl ifosfamide, N2- dechloroethyl ifosfamide-dg, N3-dech!oroethyl ifosfamide, and N3- dechloroettiyl ifosfamide-d^ in Rat 6 following iv administration of a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 ng/kg. Time (min) N2D (pg/ml) N2D-d2 (pg/ml) N3D (pg/ml) N3D-d4 (pg/ml) 0 5 0.30 0.10 0.08 0.09 15 0.62 0.24 0.20 0.22 30 0.70 0.30 0.36 0.43 45 0.85 0.37 0.44 0.45 60 0.87 0.42 0.48 0.52 90 1.00 0.39 0.46 0.45 120 1.10 0.34 0.44 0.49 150 0.99 0.28 0.37 0.42 180 0.90 0.28 0.44 0.48 210 0.71 0.26 0.31 0.32 240 0.77 0.24 0.30 0.30 300 0.58 0.13 0.22 0.24 360 0.49 0.12 0.26 0.22 420 0.34 0.07 0.18 0.13 227 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5 .1 7 Relevant pharmacokinetic parameters of S- and R-ifosfamide from rats treated with a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 or S-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg. Rat Parent Dose Weight k Co No. drug _ . ( m g ) _ (kg) (min-’ ) (nd/ml) 1 S-IF 8.00 0.400 0.0218 24.26 R-IF-d4 8.02 0.0214 21.72 2 S-IF 6.01 0.295 0.0155 31.32 RWFd, 6.01 0.0167 29.30 3 S-IF 5.97 0.295 0.0200 25.15 R-IF-d, 5.97 0.0229 24.95 4 S-IF-d4 5.81 0.290 0.0165 20.07 R-IF 5.81 0.0189 18.06 5 S-IF 5.36 0.270 0.0219 20.61 R-IF^4 5.38 0.0223 18.00 6 S-IF 5.84 0.290 0.0164 20.41 R-IF-d4 5.84 0.0208 21.60 Mean ±SD 6.17 0.31 0.0187 23.63 (derived from S-IF) ±0.93 ±0.05 ±0.0029 ±4.34 Mean ± SD 6.17 0.0205 22.27 (derived from R-IF) ±0.93 ±0.0023 ±4.32 k= ascending rate constant 228 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.17 Relevant pharmacokinetic parameters of S- and R-ifosfamide from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 or S-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg (continued). Rat Parent tl/2 AUC CLt Vdss No. drug (mm) (min*uq/ml) (ml/tnin) (ml) 1 S-IF 31.86 1114.8 7.18 329.76 R-IF-d4 32.40 1015.3 7.90 369.24 2 S-IF 44.68 2018.8 2.98 191.89 R4Fd4 41.45 1752.4 3.43 205.12 3 S-IF 34.41 1248.3 4.78 237.38 R-IF-d4 30.26 1088.8 5.48 239.28 4 S-IF-d4 42.00 1216.3 4.78 289.49 R-IF 36.65 954.88 6.08 321.71 5 S-IF 31.66 941.46 5.69 260.07 R-IF-d4 31.08 807.29 6.66 298.89 6 S-IF 42.32 1246.3 4.69 286.13 R4F-d4 33.39 1040.3 5.61 270.37 Maan ± SO 37.82 1297.66 5.02 265.79 (derived from S-IF) ±4.32 ±372.04 ±1.38 ±47.68 Mean ± SO 34.21 1109.83 5.86 284.10 (derived from R-IF) ±4.19 ±329.44 ±1.48 ±58.85 p-valuB 1.10-3* 8.5e-4* 0.10 ' denotes statistically significant difference 229 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T able 5.18 Relevant pharmacokinetic parameters of 4-hydroxy ifosfamide from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 or S-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg. Rat f4etabolite X 2 ' i 5,X 2 C m ax fmax AUC No. (min-f) (min-1) (min) (uqAnI) (min) (min-ua/ml) 1 4-OHIF - 0.0129 53.60 1.11 0 95.69 4-OHIF-d4 - 0.0163 42.44 1.66 0 135.69 2 4-OHIF 0.0150 0.0153 45.33 0.86 18.94 77.31 4-OHlF-d4 0.0378 0.0168 41.21 1.59 15.26 159.79 3 4-OHIF 0.0689 0.0163 42.56 0.54 20.11 50.23 4-OH!F-d4 1.0043 0.0149 46.43 1.16 8.29 83.14 4 4-OHIF-d4 - 0.0123 56.34 1.27 0 105.19 4-OHIF - 0.0158 43.88 2.23 0 141.17 5 4-OHIF - 0.0215 32.22 1.83 0 84.94 4-OHIF-d4 - 0.0224 30.93 3.39 0 150.78 6 40HIF 0.2911 0.0174 39.69 1.14 12.10 77.99 4-OHlF-d4 0.1338 0.0224 30.90 1.81 14.31 114.96 Mean±SD 0.0160 44.96 1.13 8.52 81.89 (derived from S-IF) ±0.0033 ±8.95 ±0.43 ±9.73 ±18.87 Mean ± SD 0.0181 39.30 1.97 6.31 130.92 (derived from R-IF) ±0.0034 ±6.72 ±0.77 ±7.31 ±27.90 P - value 2.00-3* Xj = ascending rate constant; Xg = descending rate constant; t,^j^ = terminai half-life * denotes statistically significant difference 2 3 g Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T able 5.19 Relevant pharmacokinetic parameters of iphosphoramide mustard from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide- d4 or S-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg. Rat Metabolite ^2 C m ax 1 " m a x AUC No. (min-1) (min-1) (min) fuq/ml) (m in) (min-ua/mi) 2 IPM 0.0231 0.0085 81.22 1.12 71.58 235.23 IPM-da 0.0429 0.0061 113.69 1.55 57.64 351.31 3 IPM 0.5211 0.0071 98.17 1.19 12.16 178.87 IPM-dg 1.3327 0.0082 84.69 2.11 8.74 266.35 4 IPM-dg 0.0233 0.0101 68.90 0.64 70.33 121.29 IPM 0.0670 0.0089 78.28 1.09 47.62 167.17 5 IPM 0.5727 0.0078 88.97 1.06 12.27 144.18 IPM-d2 2.2355 0.0071 98.19 1.54 5.70 222.80 6 IPM 0.2576 0.0094 73.36 0.84 26.58 100.71 IPM-da 0.1066 0.0115 60.21 1.25 32.57 142.44 Mean ± SD 0.2796 0.0086 82.12 0.97 38.58 156.06 (derived from S-IF) ±0.2628 ±0.0012 ±11.80 ±0.23 ±30.13 ±52.92 Mean ± SD 0.7569 0.0084 87.01 1.51 30.45 230.01 (derived fro m R-IF) ±0.9908 ±0.0021 ±20.23 ±0.38 ±23.04 ±83.22 P- value 5.8e-3* X, = ascending rate constant: Xg = descending rate constant: t,^j^ = terminal half-life * denotes statistically significant difference 231 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T able 5.20 Relevant pharmacokinetic parameters of N2-dechioroethyl ifosfamide from rats treated with a 1:1 mixture of S-ifosfamide and R-ifosfamide- d4 or S-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg. Rat Metabolite Xa h/iM C m ax Tm ax AUC No. (m in-1) (m in-1) (m in) (uq/ml) (min) (min-ua/ml) 1 N2D 0.0293 0.0030 227.23 1.06 73.66 447.47 N2D-da 0.0741 0.0040 175.17 0.47 35.48 138.11 2 N2D 0.0124 0.0050 137.93 1.78 116.41 655.10 N2D-da 0.0180 0.0054 127.82 0.56 91.58 173.30 3 N2D 0.0301 0.0041 170.09 1.60 76.10 537.16 N2D-da 0.0456 0.0055 125.46 0.52 52.13 125.81 4 N2D-dz 0.0100 0.0057 121.43 1.27 107.26 460.69 N2D 0.021 0.0052 132.92 0.48 69.08 144.48 5 N2D 0.0091 0.0046 149.85 1.14 99.90 474.91 N2D-ds 0.0175 0.0045 154.66 0.46 48.84 151.13 6 N2D 0.0185 0.0040 172.63 1.03 97.94 389.22 N2D-dz 0.0283 0.0055 127.01 0.40 70.72 109.94 Mean ± SD 0.0182 0.0044 163.19 1.31 95.21 494.09 (derived from S-IF) ±0.0095 ±0.0009 ±36.87 ±0.31 ±17.05 ±92.12 Mean ± SD 0.0341 0.0050 140.51 0.48 61.31 140.46 (derived from R-IF) ±0.0222 ±0.0006 ±20.14 ±0.05 ±19.86 ±21.73 p . value 9.6e-5* = ascending rate constant; Xg = descending rate constant; t„2j^ = terminal half-life ' denotes statistically significant difference 232 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.21 Relevant pharmacokinetic parameters of N3-dechloroethyl ifosfamide from rats treated a 1:1 mixture of S-ifosfamide and R-ifosfamide-d^ or S-ifosfamide-d4 and R-ifosfamide at a total iv dose of 40 mg/kg. Rat Metaboiite K z Cmax 1"m ax AUC No. (min-1) (min-1) (min) (uq/ml) (min) (min-uq/ml) 1 N3D 0.0529 0.0034 204.03 0.41 44.67 145.34 N3D-d4 0.0876 0.0033 213.20 0.63 36.91 219.78 2 N3D 0.0101 0.0040 174.77 0.42 153.25 195.23 N3D-d4 0.0130 0.0053 129.90 0.89 107.87 310.99 3 N3D 0.0609 0.0034 2 0 2 .8 8 0.53 50.50 184.21 N3D-d4 0.0650 0.0042 165.96 0.58 44.37 168.95 4 N3D-d4 0.0210 0.0043 159.53 0.68 69.78 231.70 N3D 0.0252 0.0048 145.70 0.73 58.77 219.47 5 N3D 0.0129 0.0037 189.44 0.49 58.51 199.13 N3D-d4 0.0150 0.0041 170.86 0.46 63.03 170.28 6 N3D 0.0344 0.0030 230.14 0.45 73.17 190.44 N3D-d4 0.0273 0.0043 160.40 0.51 80.18 166.84 Mean ± SD 0.0320 0.0036 193.47 0.50 75.82 191.01 (derived from S-IF) ±0.0211 ±0.0005 ±24.75 ± 0 .1 0 ±39.87 ±27.82 Mean ± SD 0.0389 0.0043 164.34 0.63 65.79 209.39 (derived from R-IF) ±0.0304 ±0.0007 ±28.22 ±0.16 ±25.79 ±55.69 P- value 0.49 = ascending rata constant; Ig = descending rate constant; t,g x: = terminal half-life 233 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5.22 Stereoselective metabolism of ifosfamide as manifested by plasma AUC ratios. R/S ratio of AUC values between R-IF and S-IF and their resoective derived metabolites Rat No. IF 4-OHIF IPM N2D N3D 1 0.91 1.42 ■ 0.31 1.51 2 0.87 2.07 1.49 0.26 1.59 3 0.87 1.66 1.49 0.23 0.92 4 0.79 1.34 1.38 0.31 0.95 5 0.86 1.78 1.55 0.32 0.86 6 0.83 1.47 1.41 0.28 0.88 Ave. ± SD 0.86±0.04 1.62±0.27 1.46±0.07 0.29±0.04 1.12±0.34 P-vaiue <0.05’ <0.05’ <0.05’ <0.05’ 0.49 Rats were given a 1:1 mixture of S-IF and R-IF-d.* (Rat 1-3.5.6) ora 1:1 mixture of S-IF-d^ and R-IF* do (Rat 4), res-pectiveiy. all at 40 mg/kg total iv dose • denotes statistically significant difference 234 Reproduced witfi permission of tfie copyrigfit owner. Furtfier reproduction profiibited witfiout permission. ? 0 (R )-(+ )-6 ,6 ,2 ',2 '-te tra d e u te rio - (S )-(-)-ifo s fa m id e ifosfam ide jf J - O D 'C l I Cl -Cl {R )-{+ )-lfo s fa m id e (S )-(-)-6 ,6 ,2 ',2 '-!e tra d e u te rio - ifosfam ide Figure 5.1 Chemical structures of two pseudoracemates of Ifosfamide. 235 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rgure5.2 Chemical structures of unlabeled and deuterium-labeled compounds: Ifosfamide, ifosfamide-d4 , ifosfamide-de. 4-hydroxy ifosfamide, 4- hydroxy ifosfamide-de, N2-dechioroethyl ifosfamide, N2- dechloroethyl ifosfamide-d4, N3-dechloroethyl ifosfamide, N3- dechloroethyl ifosfamide-de, iphosphoramide mustard, iphosphoramide mustard-da, iphosphoramide mustard-de- 236 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a ; IF \ / / V H O V 4-OHIF V O C N2D N M , N2D-d« W X O N H - N30 \ A A IPM V A N O N M > IPM-d, rf • V ” IPM-d. 237 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8 9 * I I N3D 5 :0 8 IF T / .L ^ u L l W vlwW 4-OHIF I -T —. 6 :4 8 3 :2 8 1 8 :0 8 1 1 :4 8 1 3 :2 8 Retention time (min) 1 5 :0 8 y T 1 6 :4 8 FigureS.S A representative total Ion GC/CIMS chromatogram of the derivatized plasma CH2 CI2 extract from a rat treated Iv with a 1:1 mixture of S- Ifosfamide and R-lfosfamlde-d4 at a total dose of 40 mg/kg. 238 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure5.4 Representative GC/CIMS selected Ion chromatograms for Ifosfamide (m ^225), ifosfamlde-d4 (m/z229) and Ifosfamide-de {m/z233). a) blank extract containing Ifosfamlde-dg as the Internal standard, and b) sample extract from a rat treated Iv with a 1:1 mixture of S-lfosfamlde and R-lfosfamlde-d4 at a total dose of 40 mg/kg. 239 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2ZS 2 2 9 2 3 3 6 :4 8 8:28 13:28 1 6 : 4 8 18:80 Retention time (min) 225 229 S 0 . 7 9 - 2 3 3 683 18:80 Retention time (min) 588 8:28 788 11:48 13:28 240 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 5.5 Representative GC/CiMS selected ion chromatograms for 4-hydroxy ifosfamide {m/z 412), 4-hydroxy ifosfamide-d4 {m/z416), and 4- hydroxy ifosfamide-de (m/z420). a) blank extract containing 4- hydroxy ifosfamide-de as the internal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of S-ifosfamide and R- ifosfamide-d4 at a total dose of 40 mg/kg. 241 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 . 15% ■= 7.98- 5 :8 8 6:48 8:28 18:88 11:48 13:28 15:88 16:48 Retention time (min) 2.35% 412 S 5.28% r a e 416 _2 8 .7 % g & 428 b. À 1 - 1 ^; ' ' ...................j M I ., L .. ...................................................... j 11 111 I j I . j , I ., , . . I . j 1. j . I . j 5:88 6:48 8:28 18:88 11:48 13:28 15:88 16:48 Retention time (min) 242 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure5.6 Representative GC/CIMS selected ion ctiromatograms selected for N2-dectiloroethyl ifosfamide {m/z235), N2-dechloroethyl ifosfamide- da (/T7/Z237), and N2-dechloroettiyl ifosfamide-d4 (m/z239). a) blank extract containing N2-dechloroethyl ifosfamide-d4 as the internal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 243 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 .47% 235 S ^ 1 T I ' f T r ? ' I f ^ ^ ................................................... ' ' " " ' " .................... 1 r " " ' r-.'fi-i'-i , i W . i p " , f Retention time (min) 4.86% 235 1.4 I I } ............. ' ' ^ ' 1 1 1 1 1 1 1 1 1 n i 1 1 ............., p , , 1 m t I ..................................... ....... 1 1 p - j : J ;» > 0 r W ‘ V o > < T K » p r 7 1 1 ^ r - i T . ^ S ? i ' 1 i r p . - i . I I Retention time (min) 244 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rgure5.7 Representative GC/CiMS selected ion chromatograms for N3- dechloroethyl ifosfamide {m/z235), N3-dech!oroethyi ifosfamide-d4 (m /^239), and N3-dechloroethyl ifosfamide-da {m/z243). a) blank extract containing N3-dechloroethyi ifosfamide-da as the internal standard, and b) sample extract from a rat treated iv with a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 r r ^ g . 245 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.47% 235- w w ys«t=p« 8:28 18:88 11:48 13:28 15:88 1 6 :4 8 i - I 5:88 6:48 Retention time (min) 2 3 5 1.34 s a a § 239 S 2 4 3 'rryT V jH ‘']iVT^ v^^7yr> 1 3 :2 8 1 5 :8 8 8 8:28 1 8 :8 8 1 1 :4 8 Retention time (min) 246 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o IPM g a B II S s 18:88 5:88 6:48 8:28 Retention time (min) RgureS.8 A representative total ion GC/CiMS chromatogram of the derivatized plasma solid phase extract from a rat treated iv with a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 247 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rgure5.9 Representative GC/CiMS selected ion chromatograms for iphosphoramide mustard (m/z329), iphosphoramide mustard-d2 (m/z 333), and iphosphoramide mustard-dg (m/z 337). a) blank extract containing iphosphoramide mustard-dg as the internal standard, and b) sample extract from a rat treated iv with a 1:1 mixture of S- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 248 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ D O Q. C g Q. ■ D CD C/) o' 3 O 8 ( O ' Relative total ion intensity 3. 3 " CD CD ■ D O Q. C a o 3 ■ D O CD Q. "D CD ( /) (/) ? I g (0 s cr 0 > Ê O S s È Relative total ion intensity s. c a a. 1 2 .0 10.0 - m 8 8 0 « I - I 4 .0 2.0 - 0.0 y - -0.040 + 4.506X r‘2 = 0.999 .la 0.0 0.5 • 0 !I S' 1.0 1.5 2.0 Amt S-IF-do in ng 2.5 Figure 5.10 A representative standard curve of S-ifosfamide in rat plasma. 250 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12.0 10.0 - 1 " j C M I o 6.0 - 2 m 2 4.0 < 2.0 - 0.0 y - 0.134 + 4.050X r*2 = 1.000 X 0.0 0.5 1.0 1.5 2.0 Amt R-IF-d4 in pg 2.5 Figure 5.11 A representative standard curve of R-ifosfamlde-d4 In rat plasma. 251 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6.0 5.0 -J § 4.0 — .2 3.0 2 S 2.0 1.0 0.0 y « -0.049 + 2.929X r‘ 2 = 0.997 0.0 A 1----------r 0.5 1.0 1.5 Amt 4-OHlF in p .g 2.0 Figure 5.12 A representative standard curve of 4-hydroxy ifosfamide In rat plasma. 252 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.0 3.0 ~ I o 2.0 ■f .3 y » -0.034 + 7.607X r‘ 2 = ■ 0.997 1.0 - 0.0 0.0 0.1 0.2 0.3 Amt N2D in I — ,------- 1 1 0.4 0.5 Figure 5.13 A representative standard curve of N2-decliloroettiyi ifosfamide in rat plasma. 253 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.5 0.003 + 4.737% r‘ 2 = 0.997 2.0 I R o a y 1 (0 2 < 0.5 0.0 0.5 0.4 0.3 0.2 0.1 0.0 Amt N3D in F«ure5.14 A representative standard curve of N3-dechioroethyi ifosfamide in rat plasma. 254 R eprod u ced with perm ission of the copyright owner. Further reproduction prohibited without permission. 2.5 0.997 -0.036 + 1.496X 2.0 h» I n o 1 g < 0.5 0.0 1.4 0.0 0.2 0.4 1.2 0.6 0.8 1.0 Am t IPM-do in ng Figures. 15 A representative standard curve of iphosphoramide mustard in rat plasma. 255 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.5 -0.009 + 1.330X 0.996 i n n 1.0 k 0.5 0 0.0 0.2 0.4 0.6 0.8 Amt IPIW-d2 in Figure 5.16 A representative standard curve of iphosphoramide mustard-d2 in rat plasma. 256 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o a K - lff------ c_ cz a U 500 400 100 Z O O Time in m ln 300 Figure5.17 Piasmaconcentratlon-timeprofllesofS-ifosfamide(*) and its deri ved metabolites 4-hydroxy ifosfamide (®), N2-dechloroethy! ifos famide (A), and N3-dechioroethyi ifosfamide (h) in Rat 1 following iv administration of a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 257 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o 3 C — 4 C o — I o.. 1 D D 200 Time in m ln 500 300 400 Figure 5.18 Plasma concentration-time profiles of R-ifosfamide-d4 (+) and its deri ved metabolites 4-hydroxy ifosfamide-d4 (o), N2-dechloroethyl ifos- famide-da (A), and N3-dechloroethyl ifosfamide-d4 (d) in Rat 1 following iv administration of a 1:1 mixture of S-ifosfamide and R- ifosfamide-d4 at a total dose of 40 mg/kg. 258 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o - A.._, C '* —— ^ 7 "a " '■ » ■ u 100 200 300 Time in m in 400 500 Figures. 19 Plasma concentration-time profiles of S-ifosfamide (*) and its derived metabolites 4-hydroxy ifosfamide («), iphosphoramide mustard (♦), N2-dechioroethyi ifosfamide-do (A), and N3-dechloroethyl ifosfamide- do M in Rat 2 foilowing iv administration of a 1:1 mixture of S- ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 259 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. g lO’ -r â r :::::;;« r ü 10-' 4 200 300 Time in m in Figure 5.20 Plasma concentration-time profiles of R-ifosfamide-cl4 (+) and ifs deri ved metabolites 4-liydroxy ifosfamide-d^ (o), iphosphoramide mus- tard-da (0), N2-dechioroethyi ifosfamide-da (A), and N3-dechioroethyl ifosfamide-d4 (d) in Rat 2 following iv administration of a 1 ;1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 260 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c a S L_ t-* C H a CJ 1 0 0 200 300 400 500 Time in m in Figure 5.21 Plasma œncentration-time profiles of S-ifosfamide (* ) and its derived metabolites 4-hydroxy ifosfamide (e), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (iQ in Rat 3 following iv administration of a 1:1 mixture of S-ifosfamide and R-itostamide-d4 at a total dose of 40 mg/kg. 261 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c a L_ •■ 0 -. § 10-' C a CJ 100 0 200 300 400 500 Time in m in Figure 5.22 Plasma concentration-time profiles of R-ifosfamide-d4 (+) and ifs deri ved metabolites 4-hydroxy ifosfamide-d^ (o), iphosphoramide mus- taiti-da (0), N2-dechioroethyi ifosfamide-dg (A), and N3-dechioroethyl ifosfamide-d4 (t^ in Rat 3 foilowing iv administration of a 1 ;1 mixture of S-itosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 262 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. X o i Z3 C ■H C a 1 D D 200 300 Time in m in 400 500 Figure 5.23 Plasma concentration-time profiles of S-ifosfamide (*) and its derived metabolites 4-hydroxy ifosfamide (e), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (B $ in Rat 4 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 263 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c o ■Q-. a U 200 300 400 500 1 D D 0 Time in m in Figure 5.24 Plasma concentration-time profiles of R-ifosfamide-d^ (+) and its deri ved metabolites 4-hydroxy ifosfamide-d^ (o), iphosphoramide mus- t a it i- d 2 (0), N2-dechloroethyl ifosfamide-da (A), and N3-dechioroethyl ifosfamide-d4 (i^ in Rat 4 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 264 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. "-H E m 3 L_ cz g O CJ 100 500 200 Time in fnin 300 400 Rgure5.25 Plasma concentration-time profiles of S-ifosfamide (*) and ifs derived metabolites 4-hydroxy ifosfamide (9), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide (h) in Rat 5 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 265 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -H H \ O 3 C a ^4 ro £_ I n -' a O 400 100 200 300 Time in m in 500 Figure 5.26 Plasma œncentration-time profiles of R-ifosfamide-d4 (+) and its deri ved metabolites 4-hydroxy ifosfamide-d^ (o), iphosphoramide mus- tard-da (0), N2-dechloroethyi ifosfamide-da (A), and N3-dechloroethyl ifosfamide-d4 (q) in Rat 5 following iv administration of a 1:1 mixture of S-ifosfamide and R-lfosfamide-d4 at a total dose of 40 mg/kg. 266 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cr o i L_ 10-2 4 0 100 200 300 400 500 Time in m in Figure 5.27 Plasma concentration-time profiles of S-ifosfamide (*) and its derived metabolites 4-hydroxy ifosfamide («), iphosphoramide mustard (♦), N2-dechloroethyl ifosfamide (A), and N3-dechloroethyl ifosfamide in Rat 6 foilowing iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 267 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 'É \ OI 3 C U ^-4 % L_ t-* C Ë a CJ 0 1 D D Z O O 300 400 500 Time in m in Figure5.28 Plasma concentration-time profiles of R-ifosfamide-d4 (+) and its deri ved metabolites 4-hydroxy ifosfamide-d4 (o), iphosphoramide mus- tard-dz (0), N2-dechloroethyl ifosfamide-dz (A), and N3-dechloroethyl ifosfamide-d4 (Û ) in Rat 6 following iv administration of a 1:1 mixture of S-ifosfamide and R-ifosfamide-d4 at a total dose of 40 mg/kg. 268 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. maie Sprague-Dawley rat jugular vein cannuiation and i.v. bolus total dose 40 mg/kg, (S)-IP/(R)-IP-d4 1:1 or (S)-iP-d4/(R)-IP sampling at 0,5,15,30, 45,60, 90,120, 150,180, 210, 240, 300, 360 and 420 min centrifugation 200 x g x 2 min at 4°C 0 plasma I GC-MS assay for IPM GC-MS assay for (S)-IF, (R)-IF, N2D, N3D and 4-OHIF Scheme 5.1 Flow chart of stereoselective pharmacokinetic studies of ifosfamide. 269 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. trapping with KCN 30 min at r.t. plasma + IS's (IF-dg, N2D-d, N3D-dg and 4-OHIF-d6) extraction with CHgClg Ng to dryness derivatization with MSTFA 120°C/60 min GC-MS (Finnigan ITS40) column; DB-5, capillary temp: 150-250°C Scheme 5.2 Flow chart of GO/MS assay for (S)-ifosfamide, (R)-tfosfamide, N2- dechloroethyl ifosfamide, N3-dechloroethyl ifosfamide, and 4-hydroxy ifosfamide. 270 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. plasma + IS (IPM-ds) O C IS solid phase extraction MeOH elution Ng to dryness o derivatization with BSTFA/TMSI (5:1) 120°C/60 min GC-MS (Finnigan ITS40) column: DB-5, capillary temp; 150-250°C Scheme 5.3 Flow chart of GO/MS assay for iphosphoramide mustard. 271 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MSTFA 'n h ^ ^ — o' V-c, l7 IF MH* 225 • N / > M S T F A / ~ \ f A -------------- < A ° T " : v » v „ ^ D D IF-ds MH* 229 Cl Cl D D, V ° MSTFA V ° O NH-\ D ' J ^ q \ j \ - c i \ 7 IP-dg (I.S.) MH* 231 I.P. 233 I S.: Internal standard; I.P.: isotopIc peak Scheme 5.4 Derivatization scheme of ifosfamide, ifosfamide-d4 , and the internal standard ifosfamide-de- (MSTFA: methylsiiyitrifluoroacetamide) 272 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. f t ' % E % J r ° r i‘ E ° t ° . . j ° u Q b < r : > U r ' t » . A ‘ : A “ r t ' E - o L X "sLn iX o A A î ! : > U r ' _ o f t ' ? < % A ' a o ° Â - ° "vL o L! X Q A A = § I 1 1 I I I 1 f s E I il 0 ( U E 1 V) c I 'C Q tn in 0) I 1 2 8 (O ■9 e u 2 1 £ 2 JC 2 7 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CI c o . N / M STFA N 2 D M H + 2 3 5 C l • O V "STF* . / V-N D D N 2 D -d z M H * 2 3 7 ° i - f o " x ï C N O d M S T F A / — N p D /L ------ CV'- N 2 D -d « (I.S .) M H * 2 3 9 Scheme 5.6 Derivatization scheme of N2-dechloroethyi ifosfamide, N2- dechloroethyl ifosfamide-dg, and the internai standard N2- dechloroethyiifosfamide-d4 . (fvfSTFA: methylsiiyitrifluoroacetamide) 274 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TMS 0 NH— v '— 0 ' — Cl K N 3D MH* 235 MSTFA 0 NH-v N3D-d4 - ? MH*239 NHO N H MSTFA N3D-da (I.S.) D TMS Scheme 5.7 Derivatization scheme of N3-dechloroethyl ifosfamide, N3- dechloroethyi ifosfamide-d4 , and the internal standard N3- dechloroethyiifosfamide-da. (MSTFA: methylsiiyitrifluoroacetamide) 275 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 , NH V HO \ h r Cl -a B STFA /TM SI ( 5 ÏÏ) % C l 1 TMSO' TMS IPM MH* 329 V " HO \ i H - y '— a IPM dz B STFA /TM SI (5 ÏÏ7 TMSO N I rt‘ > 0 TMS MH* 331 I.P. 333 D °Y -c i HO'W J - ^ D ' - T 0 Cl IPM -da (I.S.) B STFA /TM SI “ (5M) TMSO TMS MH* 337 Scheme 5.8 Derivatization scheme of iphosphoramide mustard, iphosphoramide mustand-da, and the internal standard iphosphoramide mustard-ds- (BSTFA: N,0-bis-(trimethy!silyl)trifluoroacetamide; fVlSTFA: methyi- silyltrifluoroacetamide) 276 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. X ^ g •O Q O / \ Q Û a ■ o Q ) S I Î O O 03 "O C O "O c (0 CO ? 03 3 E p o ■ % I 0 a 3 £ T O ) in 03 1 o œ 277 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. > < ' V r u a. ■ o a a. I C O V £ E I -5 I 1 C O "O d ) “ 2 E I p 0 1 f " o 5 C U 3 I I C O 278 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. > < z o z o o <D ? I I o f 0 1 Œ i n 0 ) E I 279 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L I . f g I ê t 0 ) P o 0 ) *o C N J z 0 1 £ I o B 3 0 oc CM in 0 ) 1 S 280 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I 0 • O S ? < u ■ g E I t 0) o o CD "O Cvl 0 .£2 % £ 1 o B 3 c CO in 0 } E < u to 281 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9 1 E (D 3 E ç o I Q . 0 1 o ■ § æ in d ) E I 282 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GO GO I Ï ± ? 1 E 0 ) 2 I f .9- 0 1 o ■ § E U Î in J 283 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. C H A P T E R 6 SUMMARY AND FUTURE PERSPECTIVES 284 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The principal objective of this research project was to study the stereoselective metabolism and pharmacokinetics of the enantiomers of IF In the rat using pseudoracemates and GC/MS methodology. The enantiomers of unlabeled IF and strategically deuterium-labeled IF were obtained by asymmetric synthesis, where R- and S-a-methylbenzyl amine were served as the resolving agents. Known and potential metabolites of IF were also synthesized to facilitate the study. Using isotope labeling, GC/MS, and ion cluster technique, four new metabolites of IF were identified In rat urine. The stereoselective metabolism of the enantiomers of IF was studied In Sprague-Dawley rats by measuring the enantiomeric ratios of unchanged IF and its metabolite 4-0HIF, IPM, alcolF, N2D, and N3D in urine following administration of pseudoracemates. A semi-quantitative GC/MS method was developed for this purpose. Potential isotope effect of the deuterium labels on IF metabolism was examined by control experiments using the enantiomers of unlabeled IF and deuterium-labeled IF with the same configuration. The enantioselectlve pharmacokinetic disposition of IF was also investigated in Sprague-Dawley rats using pseudoracemates. A GC/MS method was developed for the quantitation of the enantiomers of IF and their metabolites 4- OHIF, IPM, N2D, and N3D. To overcome problems of sample loss associated with stability of labile metabolites during sample manipulation, deuterium-iabeled analogs for each of these species were synthesized and used in the assay method as the internal standards. The plasma profiles of the enantiomers of IF and their metabolites were fitted by suitable compartmental models and pharmacokinetic parameters were computed. Results from each of the enantiomers were compared. 285 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In summary, the major work and finding presented in this dissertation are as follows. 1 ) Unlabeled and strategically deuterium-labeled enantiomers of IF were synthesized. The optical purity of these compounds was demonstrated by measuring the optical activities. These compounds were used to constitute pseudoracemates. 2) IF metabolites 4-OHIF, IPM, N2D, N3D, and alcolF were synthesized. 3) Four new metabolites namely 4-OHN2D, 4-OHN3D, N2N3D, and NDIPM were identified in rat urine. The identities of the metabolites were confirmed by authentic compounds synthesized. Thus, these results revealed that IF undergoes metabolism more complex than CP. Additionally, the most important metabolite of IF, 4-OHIF, was identified as the cyanohydrin adduct for the first time. 4) The metabolic study of IF revealed that all three major biotransformation pathways displayed stereoselectivity. The activation pathway of IF leading to the formation of 4-OHIF preferred the R configuration (R/S ratio 1.53±0.04). This preference was preserved in two subsequent steps, cleavage to IPM (R/S ratio 1.51±0.03) and reduction to alcolF (R/S ratio 1.57+0.01 ). The dealkylation on the endo nitrogen only showed a small preference for the R configuration (R/S ratio 1.13±0.10), however, that on the exo nitrogen displayed a pronounce preference for the S configuration (R/S ratio 0.33+0.02). 286 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5) Minimal deuterium isotope effect on the metaboiism of iF with the present labeled positions was observed, ensuring ttie interpretation of results using such pseudoracemates. 6) Deuterium iabeled compounds including IF-ds, IF-de, IF-d4 , N2D-d6, N2D- de, N3D-ds, N3D-de, 4-OHIF-d6, IPM-dg, and IPM-ds were synthesized as the standard and internal standards for the GC/MS assay. 7) A sensitive and specific GC/MS assay method was developed for the analysis of IF, IF-d4 , 4-OHIF, 4-OHIF-d4, IPM, IPM-da, N2D, N2D-da, N3D, and N3D-d4 in rat plasma. 8) Following iv administration of pseudoracemates, small but statistically significant differences in the area under the concentration-time curve (AUC, R/S ratio 0.86±0.04) and total clearance (CLj, R/S ratio 1.17) between the enantiomers of IF were detected. However, no significant difference was found on the volume of distribution (Vdss, R/S ratio 1.07). 9) Higher plasma levels of 4-OHIF derived from R-IF were detected than those from S-IF, suggesting that the 4-hydroxyiation prefer the R configuration of IF. The R/S ratio of AUC of 4-OHiF was 1.62±0.27. 10) Similarly, the plasma levels of IPM derived from R-IF were higher than those derived from S-IF. The R/S ratio of IPM was 1.46±0.07. 287 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11) Significant stereoselectivity was observed on the N2-dealkylation. In contrary to 4-OHIF and IPM, N2D derived from S-IF displayed much higher plasma concentrations than those derived from R-IF. The R/S ratio of N2D was 0.29±0.04. 12) N3-dealkylation did not display stereoselectivity. The plasma levels of this metabolite derived from R-IF were higher than those derived from S-IF in two rats while the opposite results were obtained from the rest four rats. The overall R/S ratio was 1.12±0.34 (p=0.49). In the future, several research areas extended from the present study can be explored as described below. 1 ) The stereoselective metabolism of IF can be further investigated using in vitro system to examine cytochrome P450 isozymes responsible for these stereoselective processes. 2) The developed pseudoracemate-GC/MS method can be used on human subjects to obtained more clinically relevant data. 3) With the developed GC/MS method, systemic clinical pharmacokinetics of IF and its metabolites 4-OHIF, IPM, N2D, and N3D can be readily investigated. 288 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4) The developed deuterium-labeling and GC/MS method can be modified to conduct bioavaiiabiiity study in experimental animals and human. 5) During the metabolite identification study, several additional unknown metabolites were detected. However, the present GC/MS method was unable to definitively identify these species due to interference. Other method such as HPLC/MS may be useful for further metabolic studies. 6) Compared to CP, IF undergoes more complex metabolism but appears to be less activated through the 4-hydroxylation pathway. Significant amount of CAA, a potential neurotoxic metabolite, is generated through N2- and N3- dealkylation of IF and secondary dealkylation of N2D and N3D. It seems logic to develop IF analogs such as 2-ethylenimine-3-(2-chloroethyl)tetrahydro-f,3,2- oxazaphosphorine 2-oxide (Figure 6.1) which may not produce CAA in vivo, yet preserve its unique antitumor activity. 289 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 6.1 Structure of 2-ethylenimine-3-(2-chloroethyl)tetrahydro-1,3,2- oxazaptiosphorine 2-oxide. 290 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES 291 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. Goodman, L.S., Wintrobe, M.M., and Dameshek, W. Nitrogen mustard therapy, use of methyl-bis(p-chloroethyl)amino hydrochloride for Hodgkin's disease, lyphomsarcoma, and certain allied and miscellaneous disorders. J. Am. Med. Assoc., 1132:126-32,1946. 2. Friedman, O.M. and Seligman, A.M. Preparation of N-phosphorylated derivatives of bis-p-chloroethylamine. J. Am. Chem. Soc., 76:655-658,1954. 3. Gomori,G. Histochemical demonstration of sites of phosphamidase activity. Proa. Soc. Exp. Bio. Med., 69:407-409,1948. 4. Arnold, H., Bourseaux, P., and Brock, N. Chemotherapy action of a cyclic nitrogen mustard phosphamide ester (B 518-Asta). Nature, 181:931,1958. 5. Arnold H., Bourseaux, P., and Brock, N. Liber beziehungen zwischen chemischer konstitution und cancerotocischer wirkung in der reihe der phosphamidester des bis-(p-chlorathyl)-amins. Arzneim.-Forsch., 11:143- 158,1961. 6. Asta-Werke A.-G. Process for the production of new N-substituted amides and esteramide of phosphoric and thiophosphoric acid. Brit, patent No. 1,188,159,15 Apr, 1970. Ger. Appl. 11 Jul 1966. 7. Schoenike, S.E. and Dana, W.J. IP and mesna, Clin. Pharm., 9:179- 191,1990. 8. Webber, G.P. and Waxman, D.J. Activation of the anticancaer drug ifoasfamide by rat liver microsomal P450 enzymes. Blochem. Pharmacol., 45:1685-1694,1993. 9. Chang, T.K.H., Webber, G.P., Crespi, C.L., and Waxman, D.J. Differential activation of cyclophosphoramide and ifosfamide by cytochromes P450 2B and 3A in human liver microsomes. Cancer Res., 53:5629-5637,1993. 10. Jezequel, S.G. Central nervous system penetration of drugs: importance of physicochemical properties. Progress Drug Met., 13:141 -178,1992. 11. Conners, T.A., Cox, P.J., Parmer, P.B., Poster, A.B., and Jarman, M. Some studies on the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide. Blochem. Pharm., 23:115-129, 1974. 12. Allen, L.M. and Creaven, P.J. In vitro activation of ifosfamide (NSC- 109724), a new oxazaphosphorine, by rat liver microsomes. Cancer Chemo. Rep., 56:603-610,1972. 13 Takamizawa, A., Iwata, T., and Matsumoto, S. Studies on the urinary metabolites of isophosphamide and its activated species in rabbits. Chem. Pharm. Bull., 25:2900-2909,1977. 14. Hill, D.L., Laster, W.R. Jr, Kirk, M.C., Dareer, S.E., and Struck, R.P. Metabolism of iphosphamide [2-(2-chloroethylamino)-3-(2-chloroethyl)tetra- hydro-2R-1,3,2-oxazaphosphorine 2-oxide] and production of a toxic iphosphamide metabolite. Cancer Res., 33:1016-1022,1973. 292 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15. Hadidi, A.F.A., Coulter, G.E.A., and Idle, J.R. Phenotypically deficient urinary elimination of cyclophospliamide after cyclophosphamide administration to cancer patients. Cancer fîes., 48:5167-5171,1988. 16. Struck, R.F., Dykes, D.J., Corbett T.H., Suling W.J., and Trader M.W. Iphosphoramide mustard, a metabolite of iphosphamide with activity against murine tumours comparable to cyclophosphamide. Br. J. Cancer, 47:15-26, 1983. 17. Bryant, B.M., Jarman, M., Baker, M.H., Smith, I.E., and Smyth, J.F. Q uantification by gas chromatography of N,N'-di(2-chloroethyl)- phosphorodiamidic acid In the plasma of patients receiving isophosphamide. Cancer Res., 40:4734-4738,1980. 18. Brock, N., Stekar, J., Pohl, J., Niemeyer, U ., and Scheffler, G. Acrolein, the causative factor of urotoxic side-effects of cyclophosphamide, ifosfamide, trofosfamide and sulofosfamide. Azneim.-Forsch., 29:659-661,1979. 19. Alarcon, R.A., Meienhofer, J., and Atherton, R. Ifosfamide as a new acrolein- producing antineoplastic isomer of cyclophosphamide. Cancer Res., 32:2519-2523,1972. 20. Norpoth, K. Studies on the metabolism of isophosphamide (NSC-109724) in man. Cancer Treat. Rep., 60:437-442,1976. 21 Goren, M.P. Determination of urinary 2- and 3-dechloroethylated metabolites of ifosfamide by high-performance liquid chromatography. J. Chromatogr., 570:351-359,1991. 22. Goren, M.P., Wright, R.K., Pratt, C.B., and Pell, F.E. Dechloroethylation of ifosfamide and neurotoxicity. Lancet, 2:1219-1220,1986. 23. Misiura, K., Okruszek, A., Pankiewicz, K., Stec, W.J., Crownicki, Z., and Utreacka, B. Stereospecific synthesis of chiral metabolites of ifosfamide and thelrdetermination in the urine. J. Med. Chem., 26:674-679,1983. 24. Martino, R., Crasnler, P., Chouini-Lalanne, N., Gllard, V., Niemeyer, U., De Forni, M., and Malet-Martino, M. A new approach to the study of ifosfamide metabolism by the analysis of human body fluids with 3ip nclear magnetic resonance spectroscopy. J. Pharmaco. Exp. Then, 260:1133-1144,1992. 25. Clarke, L, and Waxman, D.J. Oxidative metabolism of cyclophosphamide: identification of the hepatic monooxygenase catalysts of drug activation. Cancer Res., 49:2344-2350,1989. 26. Anthony, L.B., Garrow, G.C., Davenport, M.C., Struck, R.F., and Hande, K.R. Metabolic routes of ifosfsamide in a isolated perfused rat liver model. Proc. Am. Assoc. CanerRes. 33:404,1992. 27 Ruzicka, J.A. and Ruenitz, P.O. Cytochrome P450-mediated N-dechloro- ethylation of CP and IF in the rat. Drug Metab. dispos., 20:770-772,1992. 293 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28. Allen, L.M., Creaven, P.J., and Nelson, R.L. Studies on human pharmacokinetics of isophosphamide (NSC109724). Cancer Chemo. Rep., 60:451-458,1976. 29. Creaven, P.J., Allen, L.M., Alford, D.A., and Cohen, M.H. Clinical pharmacology of isophosphamide. Clin. Pharmacol. Then, 16:77-86,1974. 30. Arndt, C.A.S., Balls, P.M., McCully, C.L., Colvin, O.M., and Poplack, D.G. Cerebrospinal fluid penetration of active cyclophosphamide and ifosfamide in rhesus monkeys. Cancer Res., 48:2113-2115,1988. 31. Ninane, J., Baurain, R., De Kraker, J., Ferster, A., Trouet, A., and Cornu, G. Alkylating activity in serum, urine, and CSF following Ifosfamide in children. Cancer Chemo. Pharmacol., 24:82-86,1989. 32. Cemy, T., Margison, J.M., Thatcher, N., and Wilkinson, P.M. Bioavailability of ifosfamide in patients with bronchial carcinima. Cancer Chem. Pharmcol., 18:261-264,1986. 33. Lewis, L.D., Fitzgerald, D.L., Mohan, P., and Rogers, H.J. Fractionated ifosfamide therapy produces a time-dependent increase in ifosfamide metabolism. Bri. J. Clin. Pharmacol., 30:725-732,1990. 34. Lewis, L.D., Fitzgerald, D.L., Mohan, P., Thatcher, N., and Harper, P.G. The pharmacokinetics of ifosfamide given as short intravenous infusions in cancer patients. Clin. Pharmacoi., 31:77-82,1991. 35. Lind, M., Cerny, T., Margison, J., Thatvher, N., and Wilkinson, P.M. Decreased half-life of ifosfamide after daily oral administration. Bri. J. Cancer, 54:213,1986. 36. Lind, M.J., Margison, J.M., Cerny, T., Thatcher, N„ and Wilkinson, P.M. Comparative pharmacokinetics and alkylating activity of fractionated intravenous and oral ifosfamife in patients with brochogenic carcinoma. Cancer Res., 49:753-757,1989. 37. Wagner, T.and Drings, P. Pharmacokinetics and bioavailability of oral ifosfamide. Arzeim.-Forsch., 36:878-880,1986. 38. Cerny, T., Graf, A., Rohner, P., and Zeugin, T. Bubcutaneous continuous infusion of ifosfamide and cyclophosphamide in ambulatory cancer patients: bioavailability and feasibility. J. Cancer Res. Clin. Oncol., 117:8129-134, 1991. 39. Pearcey, R., Calvert, R., and Mehta, A. Disposition of ifosfamide in patients receiving ifosfamide infusion therapy for the treatment of cervical carcinoma. Cancer Chemo. Pharmacoi, 22:353-355,1988. 40. Lind, M., Roberts, H.L., Thatcher, N., and Idle, J.R. The effect of route of administration and fractionation of dose on the metabolism of ifosfamide. Cancer Chemo. Pharmacoi, 26:105-111,1990. 294 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41. Kurowsky, V., Cerny, T.T., Kupfer, A., and Wagner, T. Metabolism and pharmacokinetics of oral and intravenous ifosfamide. J. Cancer Res. Clin. Onco/., 117:3117,1991. 42. Jurgens, H., Treuner, J., Winkler, K., Gobel, U. Ifosfamide in pediatric malignancies. Semin. Oncoi. 16 [SuppI 3], 46-50,1989. 43. Bryant, B.M., Jarman, M.J., Ford, H.T., and Smith, I.E. Prevention of isophosphoramide-induced urothelial toxicity with 2-mercaptoethane sulphonate sodium (mesnum) in patients with advanced carcinoma. Lancet, 22:657-659,1980. 44. Dechant, K.L., Brogden, R.N., Pilkington, T., and Faulds, D. Ifosfamide/mesna, a review of its antineoplastic activity, pharmacokinetic properties and therapeutic efficacy in cancer. Drug, 42:428-467,1991. 45. Sladek, N.E. Metabolism of oxazaphosphrines. Pharmacoi. Then, 37:30A- 355,1988. 46. Ariens, E.J. Stereochemistry: A source of problems in medicinal chemistry. Part 1. Med. Res. Review, 6:451-466,1986. 47. Kroemer, FI.K., Fischer, C., Meese, C.O., Eichelbaum, M. Enantio- mer/enantiomer interaction of (S)- and (R)-propafenone for cytochrome P450IID6-catalyzed 5-hydroxylation: invitro evaluation of the mechanism. Mol. Pharmacol. ,40:135-142,1991. 48. Tjaden, U.R. and De Bruijn, E.A. Chromatographic analysis of anticaner drugs. J. Chromatogr., 531:235-294,1990. 49. Kusnierczyk, H., Radzikowski, 0., Paprocka, M., Budzynski, W., Rak, J., Kinas, R., Misiura, K., and Stec, W. Antitumor activity of optical isomers of cyclophosphamide, ifosfamide and trofosfamide as compared to clinically used racemates. J. Immunopharmacol., 8,455-480,1986. 50. Blaschke, V.G., Hilgard, P., Maibaum, J., Niemeyer, U., Pohl, J. Preparative trennung der ifosfamid-enantiomere und ihre pharmakologisch-toxikologische untersuchung. ArzeIm.-Forsch., 36:1493-1495,1986. 51. Masural, D., Houghton, P.J., Young, C.L., and Wainer, I.W. Efficacy, toxicity, pharmacokinetics, and in vitro metabolism of the enantiomers of ifosfamide in mice. Cancer Res., 50:252-255,1990. 52. Blaschke, G. and Widey, W. Metabolismus der enantiomere des zytostatifums ifosfamid. Arzneim.-Forsch., 39:223-226,1989. 53. Boos, J., Welslau, U., Ritter, J., Blaschke, G., and Schellong, G. Urinary excretion of the enantiomers of ifosfamide and its inactive metabolites in children. Cancer Chemo. Pharmacoi., 28:455-460,1991. 54 Wainer, I.W., Stewart, C.F., Young, C., Masurel, D., and Krank, H. Clinical pharmacokinetics of the enantiomers of ifosfamide (IFF) in pediatric patients. Proc. Am. din. Oncol., 7:72,1988. 295 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 55. Farmer, P.B. Enantiomers of cyclophosphamide and iphosphamide. Biochem. Pha/maco/., 37:145-148,1988. 56. Levy, R.H. and Bobby, A.V. Stereoselectivity in pharmacokinetics: a general theory. Pharm. Res., 8:551 -556,1991. 57. Hutt, A.J. Enantiospecific analytical methodology: application in drug metabolism and pharmacokinetics. Progress Drug Met., 12:257-361,1990. 58. Trager, W.F. Biotransformation and excretion : quantitative studies. J. Clin. Pharmacol., 26:443-446,1986. 59 Cox, P.J., Farmer, P.B., Foster, A.B., Griggs, L.J., and Jarman, M.J. Application of deuterium iabelling mass spectrometry in a study of the metabolism of the enantiomers of cyclophosphamide. Biomed. Mass Spec., 4:371-375,1977. 60. Cox, P.J., Farmer, P.B., Jarman, M., Kinas. R.W., and Stec, W.J. Stereoselectivity in the metabolism of the enantiomers of cyclophosphamide in mice, rats, and rabbits. Drug Metab. Dispos., 6:617-622,1978. 61. Blaschke, G and Mailbaum J. Chromatographic resolutions. XIV. Optical resolution of the racemic anticancer drug ifosfamide and other chiral oxazaphosphorines. J. Chromatogr., 366:329-334,1986. 62. Wroblewski, A.E., Socol, S.M., Okruszek, A., and Verkade, J.G. Optical resolution of the anticancer agents isophosphoramide and triphosphamide by means of disatereomeric platinum (II) complexes. Inorg. Chem., 19:3713- 3719,1980. 63. Kinas, R.W., Pankiewcz, K., Stec, W.J., Farmer, P.B., Foster, A.B., and Jarman, M. The synthesis of the optical isomers of 2-(2-chloroethylamino)- 3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide (isophos phamide). Bull. Acad. Pol. Sc/., 26:39-42,1987. 64. Ludeman, S.M., Bariett, D.L., and Zon, G. Stereospecific synthesis of (R)- and (S)-isophosphamide. J. Org. Che/r?., 44:1163-1166,1979. 65. Pankiewicz, K., Kinas, R., Stec, W.J., Foster, A.B., Jarman, M., and Van Maanen, J.M.S. Synthesis and absolute configuration assignment of enantiomeric forms of ifosfamide, sulfosphamide and trofosphamide. J. Am. Chem. Soc., 101:7712-7718,1979. 66. Sarosy, G. Ifosfamide-pharmacologic overview. Semin. Oncol., 161: 2-8, 1989 67. Wagner, I . Ifosfamide clinical pharmacokinetics. Clin. Phanvacokinetics, 26:439-56,1994. 68. Knapp, D.R., Gaffney, I.E., and McMahon, R.E. Use of isotope mixtures as a labeling technique in drug metabolism studies. Blochem. Pharmacol., 21:425-429,1972. 296 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 69. McMahon, R.E., Sullivan, H.R., Due, S.L., and Marshall, F.J. The metabolite pattern of d-propoxyphene in man. The use of heavy isotopes in drug disposition studies. LlfeScL, 12:463-473,1973. 70. Pohl, L.R., Nelson, S.D., Garland, W.A., and Trager, W.F. The rapid identification of a new metabolite of warfarin via a chemical ionization mass spectrometry ion doublet technique. Biomed. MassSpectr., 2:23-30,1975. 71. Chan K.K., Hong, 8-0., Watson, E., and Deng, S-K. identification of new metabolites of phosohoramide mustard and nor-nitrogen mustards and cyclophosphamide in rat using ion cluster techniques. Biomed. Environ. Mass Spectr., 13:145-154,1986. 72. Peter, G., Wagner, T., and Hohorst, H.-J. Studies on 4- hydroperoxycyciophosphamide (NSC-181815): a simple preparation method and its application for the synthesis of a new class of "activated" sulfur-containing cyclophosphamide (NSC-26271) derivatives. Cancer Treat. Rep., 60:429-435,1976. 73. Takamizawa, A., Matsumoto, S., Iwata, T., and Makino, I. Synthesis, stereochemistry and antitumor activity of 4-hydroperoxyisophosphamide (NSC-227114) and reiated compounds. Chem. Pharm. Buil, 25:1877-1891, 1977. 74. Takamizawa, A., Matsumoto, S., Iwata, T., Tochino, Y., Katagiri, K., Yamaguchi, K., and Shiratori, 0. Synthesis and metaboiic behavior of the suggested active species of isophosphamide having cytostatic activity. J. Med. Chem., 17:12371239,1974. 75. Fenselau, C., Kan, M.N., Rao, S.S., Myles, A., Friedman, O.M., and Colwin, M. Identification of aldophosphamide as a metabolite of cyclophosphamide in vitro and in vivo in humans. Cancer Res., 37:2538-2543,1977. 76. Hong, P.S., and Chan, K.K. Analysis of 4-hydroxycyclophosphamide by gas chromatography-mass spectrometry in plasma. J. Chromatogr., 495:131-138,1989. 77. Gilard, V., Malet-Martino, M.C., de Forni, M., Niemeyer, U., Ader, J.C., and Martino, R. Determination of the urinary excretion of ifosfamide and its phosphorated metabolites by phosphorus-31 nuciear magnetic resonance spectroscopy. CancerChemo. Pharmacoi., 31:387-394,1993. 78. Manz, I., Dietrich, I., Przybylski, M., Niemeyer, U., Pohl, J., Hilgard, P., and Brock, N. Identification and quantification of metaboiite conjugates of activated cyclophosphamide and ifosfamide with mesna in urine by ion-pair extraction and fast atom bombardment mass spectrometry. Biomed. Mass. Spectr., 12:545-553,1985. 79. Wagner, T., Heydrich, D., Jork, T., Voelcker, G., and Hohorst, H.-J. Comparative study on human pharmacokinetics of activated ifosfamide and cyclophosphamide by a modified fluorometric test. J. Cancer Res. Clin. Oncoi., 100:95-104,1981. 297 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80. Ikeuchi, I. and Amano, T. Fluorometric determination of 4-hydroxyifosfamide in blood and urine. Chem. Pharm. Bull., 33:2416-2420,1985. 81. Pratt, C.B., Green, A.A., Horowitz, M.E., Meyer, W.H., Etcubanas, E., Douglas, E., Hayes, F.A., Thompson, E., Wllimas, J., IgarashI, M., and Kovnar, E. Central nervous system toxicity following the treatment of pediatric patients with ifosfamide/mesna. J. Clin. Onco/., 4:1253-1261,1986. 82. Meanwell, C.A., Blake, A.E., Latief, T.N., Blackledge, G., Mould, J.J., Blake, D.R., Shaw, I.e., Honigsberger, L., Sponer, D., Williams, A.C., and Jones, S.R. Encephalopathy associated with ifosfamide/mesna therapy. Lancet, 1:406-407,1985. 83. Ariens, E.J., Wuis, E.W., and Veringa, E.J. Stereoseiectivity of bioactive xenobiotics. A pre-Pasteur attitude in medicinal chemistry, pharmacokinetics and clinical pharmacology. Blochem. Pharmacol. 37:9-18,1988 84. Hilton, J. Role of aldehyde dehydrogenase In cyclophosphamide-resistant L I210 leukemia. Cancer Res., 44:5156-5160,1984. 85. Zheng, J.J., Chan, K.K., and Muggia, F. Preciinical pharmacokinetics and stability of isophosphoramide mustard. Cancer Chemother. Pharmacol., 33:391-398,1994. 86. Shaw, I.e., Graham, M.I., and McLean, A.E.M. 2-Chloroacetyaidehyde, a metabolite of cyclophosphamide in the rat. Xenobiotica, 13:433-437.1983. 87. Hong, P.S., Srigritsanapol, A., and Chan, K.K. Pharmacokinetics of 4- hydroxycyclophosphamide and metaboiites in the rat. Drug Metab. Dispo., 19:1-7,1991. 88. Tsui, P.P., Brandt, J.A., and Zon, G. Effects of enantiomeric homogeneity on the in vitro metaboiism and In vivo anticancer activity of (+)- and (-)- cyciophosphamide. Biochem. Pharmacol., 28:367-374,1979. 89. Zon, G. Cyclophosphamide stereochemistry. I. Synthesis and configu- rationai stability of diastereomeric chclophosphamide derivatives. Tetrahedron Letters, 36:3139-3142,1975. 90. Kaijser, G.P., Beijnen, J.H., Bult, A., and Underberg, W.J.M. Ifosfamide metabolism and pharmacokinetics. Anf/cancerf?es., 14:517-532,1994. 91 Drayer, D.E. Pharmacodynamic and pharmacokinetic differences between drug enantiomers in humans: an overview. Clin Pharmacol. Therap., 40:125- 133,1986. 92 Testa, B. and Jenner, P. Stereochemical methodology. In: Drug fate and metabolism. Methods and techniques. Vol. 2, Eds.:E.R. Garret and J.L. Hirtz, Marcel Dekker, New York, pp99-108,1978. 93 Ariens, E.J. Stereochemistry, a basis for sophisticated nonsense in pharmacokinetics and clinical pharmacology. Eur.J. Clin. Pharmacol., 26:663- 668,1984 298 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 94 Wainer, I. W. and GranviI.C.P. Stereoselective separations of chiral anticancer drugs and their application to pharmacodynamic and pharmacokinetic studies. Ther.drugmonit., 15:570-575,1993. 95 Lind, M.J., Margison, J.M., Cerny, T. Thatcher, N., and Wilkinson, P.M. The effect of age on the pharmacokinetics of ifosfamide. Bri. J. Cancer, 30:140- 143,1990. 96 Boddy, A.V., Yule, S.M., Wyllie, R., Price, L., Pearson, A.D., and Idle, J.R. Pharmacokinetics and metabolism of ifosfamide asministered as a continuous infusion in children. Cancer Res., 53:3758-3764,1993. 97 Kurowski, V. and Wagner, T. Comparative Pharmacokinetics of ifosfamide, 4-hydroxyifosfamide, chloroacetaldehyde, and 2- and 3-dechloroethyl ifosfamide in patients on fractionated intravenous ifosfamide therapy. Cancer Chemo. Pharmacol., 33:36-42,1993. 98 Hartley, J.M., Hansen, L., Harland, S.j., Nicholson, P.W., Pasini, P., and Souhami, R.L. Metabolism of ifosfamide during a 3 day infusion. Bri. J. Cancer, 69:931-936,1994.. 99 Allen, L.M. and Creaven, P.J. Gas chromatographic method for the determination of plasma isophosphamide(NSC-109724). Cancer Treat. Rep. Part 1,56:721-723,1972. 100 Holdiness, M.R. and Morgan, L.R. Jr. Electron-capture gas chromatographic analysis of ifosfamide in human plasma and urine. J. Chromatogr., 275:432- 435,1983. 101 Kaijser,G.P., Beijnen,J.H., Bult, A., Wiese, G., Kraker, J.D., and Underberg, W.J.M. Gas chromatographic determination of ifosfamide in microvolumes of urine and plasma. J. chromatogr., 571:121 -131,1991. 102 Kaijser, G.P.,Beijnen, J.H.,Bult, A.,Wiese, G., Krakerk, J.D., Keizer, H.J., and Underberg, W.J.M. Gas chromatographic determination of 2-and 3- dechloroethylifosfamide in pllasma and urine. J. chromatogr., 583:175-182, 1992. 103 Blaschke, G, and Koch, U. Gaschromatographische bestimmung und enantiomertrennug von ifosfamid sowie der metaboliten 2- und 3- deschlorethyl ifosfamid. Arch.Phanm., 319:1052-1054,1986. 104 Margison, J.M., Cerny,T., Thatcher, N., and Wilkinson, P.M. A simple quantitative HPLC assay for ifosfamide in biological fluids. Biomed. Chrom., 1:101-103,1986. 105 Burton,.L.C., James, C.A. Rapid method for the determination of ifosfamide and cyclophospham ide in plasma by high-perform ance liquid chromatography with solid-phase extraction. J. Chromatogr, 431:450-454, 1988. 106 Boddy, A.V.,Idle, J.R. Combined thin-layer chromatography-densitometry for the quantification of ifosfamide and its principal metabolites in urine, cerebrospinal fluid and piasma. J. Chromatogr, 575:137-142,1992. 299 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 107 Lambrechts, H., Gheuens, E.O., Gauwenberghe, K.A.V., Pattyn, G.G.O., Osterom, A.T.V., Bruijn, E.A.D., and Leclercq, P.A. Determination of ifosfamide by gas chromatography-mass spectrometry. Anal. Chimica Acta, 247:229-233,1991. 108 Gibaldi, M. and Perrier, D. Pharmacokinetics, 2nd edn. Marcel dekker. New York, pp 17-19,1982. 109 Pang, K.S., Cherry, W.F., Terrell, J.A., and Ulm, E.H. Disposition of enalapril and its diacid metaboiite, enaiaprilat, in a perfused rat liver preparation: presence of adiffusional barrier for enaiaprilat into hepatocytes. Drug Metab. Dispos., 12:309-313,1983. 110 Powers, J.F. and Sladek, N.E. Cytotoxic activity relative to 4-hydroxy cyciophosphamide and phosphoramide mustard concentrations in the plasma of cyclophosphamide-treated rats. Cancer Res., 43:1101-1106, 1983. 111 Sladek, N.E., Powers, J.F., and G rage, G.M. Half-life of oxazaphosphorines in biological fluids. Drug Metab. Dispos., 12:553-559,1984. 112 Wainer,I.W., Granvil,C.P., Wang I , and Batist,G. Efficacy and toxicity of ifosfamide stereoisomere in an in vivo rat mammary carcinoma model. Cancer Res., 54,4393-4397,1994 300 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Wang, Jinghua (author)

Core Title Stereoselective metabolism and pharmacokinetics of ifosfamide in the rat.

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Chemistry, pharmaceutical,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-489258

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Rights Wang, Jinghua

Type texts

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