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Design, synthesis, biological testing and QSAR analysis of new Schiff bases of N-hydroxysemicarbazide as inhibitors of tumor cells

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Design, synthesis, biological testing and QSAR analysis of new Schiff bases of N-hydroxysemicarbazide as inhibitors of tumor cells

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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, som e thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality o ff this reproduction is dependent upon the quality off 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 U M I a complete manuscript and there are missing pages, these w ill be noted. Also, if unauthorized copyright material had to be removed, a note w ill 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. ProQuest Information and Learning 300 North Zeeb Road, Arm Arbor, M l 48106-1346 USA 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. DESIGN, SYNTHESIS, BIOLOGICAL TESTING AND QSAR ANALYSIS OF NEW SCHIFF BASES OF N-HYDROXYSEMICARBAZIDE AS INHIBITORS OF TUMOR CELLS by Shijun Ren 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) August 2001 Copyright 2001 Shijun Ren Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U M I Number 3065839 Copyright 2001 by Ren, Shijun All rights reserved. UMI’ U M I Microform 3065839 Copyright 2002 by ProQuest Information and Learning Company. A ll rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, M l 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY MRK LOS ANGELES. CALIFORNIA 90007 This dissertation, written by Shijun Ren u nder the direction of fciJL Dissertation C o m mit tee, 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 O ttn of Graduate Studies DISSERTATION COMMITTEE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To my parents, my wife, my daughter and those from whom I have learned. ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS DC I M i I wish to acknowledge my most sincere gratitude to my advisor. Professor Eric J. Lien, whose invaluable advice and constant moral support encouraged me to pursue and complete my graduate studies. I am extremely thankful for his expert technical guidance, encouragement and nice personality. I also wish to thank Dr. Zoltan A. Tokes and Csaba Csipke of the Department of Biochemistry and Molecular Biology, the Norris Comprehensive Cancer Center, Keck School of Medicine USC, for their guidance and help for the cytotoxicity assays. In addition, I wish to thank my committee members, Drs. Ian Haworth, Robert Koda and Curtis Okamoto for their helpful suggestions and support throughout the course of my studies. I also wish to thank Dr. Chou-Shin Hsu, his family and H & L Charitable Foundation for generous support throughout my stay at USC. Without their support, I could not have completed my graduate studies. Finally, I would like to thank Mrs. Linda L. Lien, Dr. Yun Yen, Dr. Bingsen Zhou, Dr. Arima Das, Dr. Hua Gao, Dr. Rubin Wang, Dr. Kenichi Komatsu, Dr. Charles E. McKenna, Dr. Wei-Chiang Shen, Yegor Zyrianov, Patricia Bonaz-Krause, Allen L. Lee, Alex Fu, Sandy Huang and Jennica Zaro for their help and support throughout my stay at USC. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS DEDICATION............................................................................................ ii ACKNOWLEDGEMENTS........................................................................ iii LIST OF TABLES...................................................................................... viii LIST OF FIGURES.................................................................................... xi ABSTRACT............................................................................................... xvi CHAPTER I. INTRODUCTION...............................................................................~ 1 1. Physiological Role of Ribonucleotide Reductase.................................... 1 2. History of Ribonucleotide Reductases.................................................... 1 3. Classification of Ribonucleotide Reductases.......................................... 6 4. Comparison of Ribonucleotide Reductases from Aerobic E. coli and from Mammalian Sources...................................................................... 9 A. Aerobic E. coli ribonucleotide reductase (AERR)..................... 9 B. Mammalian ribonucleotide reductase (MRR)............................ 10 5. Catalytic Mechanism of Ribonucleotide Reductase................................ 16 6. Allosteric and Genetic Regulation of Ribonucleotide Reductase 18 A. Allosteric regulation................................................................. 18 B. Genetic regulation..................................................................... 18 7. Ribonucleotide Reductase Expression in Tumor C ells........................... 19 8. Ribonucleotide Reductase Inhibitors as Antitumor/Antiviral A gents 20 A. Hydroxyguanidine and its derivatives........................................ 20 B. Thiosemicarbazones................................................................... 20 C. Hydroxyurea.............................................................................. 22 D. Other ribonucleotide reductase inhibitors................................... 23 E. Recognition of the essential pharmacophore.............................. 24 D. RATIONALE..................................................................................... 30 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HL CHEMISTRY................................................................................. .... 33 1. Methods and M aterials......................................................................... 33 A. Design of Schiff bases of hydroxysemicarbazide............................. 33 B. Synthesis of hydroxysemicarbazide and its Schiff bases................... 33 (1). Synthetic scheme....................................................................... 33 (2). Synthetic procedure................................................................... 36 (a). Synthesis of N-hydroxy phenyl carbamate......................... 36 (b). Synthesis of hydroxysemicarbazide.................................... 36 (c). Synthesis of Schiff bases of hydroxysemicarbazide 37 -<3-Trifluoromethylbenzylidene)-4-hydroxysemicarbazide (RW L1) 37 -(2-Hydroxy-3,5-dichlorobenzylidene)-4-hydroxysemicarbazide (RWL2) 37 -(2-Hydroxy-5-bromobenzyIidene)-4-hydroxysemicarbazide (RW L3).... 37 -(2-Hydroxy-33-dibromobenzytidene)-4-hydroxysemicarbazide (RWL4) 38 -(2-Hydroxy-3-methoxy-5-bromobenzylidene)-4-hydroxysemicarbazide (RWL5)................................................................................................... 38 -(3-Iodobenzylidene)-4-hydroxysemicarbazide (RW L6).......................... 39 -(2-Hydroxy-3,5-diiodobenzylidene)-4-hydroxysemicarbazide (RW L7).. 39 -(4-Cyanobenzylidene)-4-hydroxysemicarbazide (RW L8)....................... 39 -(4-Dimethyiaminobenzylidene)-4-hydroxysemicarbazide (RW L9) 40 -(2-Hydroxy-3,5-dinitrobenzylidene)-4-hydroxysemicarbazide (RWL10) 40 -(3-Nitrobenzylidene)-4-hydioxysemicarbazide (RWL11 )....................... 40 -<3-Methoxybenzylidene)-4-hydroxysemicarbazide (RW L12)................. 41 -(4-Methoxybenzylidene)-4-hydroxysemicarbazide (RW L13)................. 41 -(2,5-DimethoxybenzylideneM-hydroxysemicarbazide (RW L14).......... 42 -<2-Hydroxy-4-methoxybenzylidene)-4-hydroxysemicarbazide (RWL15) 42 -(2-Hydroxy-4,6-dimethoxybenzylidene)-4-hydroxysemicarbazide (RWL16)................................................................................................. 42 -(4-benzyloxybenzyiidene)-4-hydroxysemicarbazide (RW L17).............. 43 -(4-phenylbenzylidene)-4-hydroxysemicarbazide (RW L18).................... 43 -(2,4-dihydroxybenzylidene)-4-hydroxysemicarbazide (RW L19)............ 44 -(4-acetamidobenzylidene)-4-hydroxysemicarbazide (RW L20)............... 44 -(23,4-trihydroxybenzylidene)-4-hydroxyseniicarbazide (RW L21)........ 44 -(2-pyridylmethylene)-4-hydroxysemicarbazide (RWL23)...................... 45 -[2-<6-methylpyridyl)methylenej-4-hydroxysemicarbazide (RW L24)..... 45 -[5-(4-methylimidazolyl)methylene]-4-hydroxysemicarbazide (RW L26). 46 -[2-(5-nitrothienyl)methylene]-4-hydroxysemicarbazide (RW L27)......... 46 -(3-indolylmethylene)-4-hydroxysemicarbazide (RWL28)...................... 46 -[3-(6,8-dichloro-4-oxo-4H-l-benzopyran)methylene]-4- hydroxysemicarbazide (RWL31)............................................................ 47 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 -[3-(6-isopropyI-4-oxo-4H-1 -benzopyran)methylene]-4- hydroxysemicarbazide (RW L32)........................................................... 47 1 -(1,4-benzodioxan-6-ylmethyIene}-4-hydroxysemicarbazide (RW L33)... 48 1 -[9-( 10-methyl an thryl)methylene]-4-hydroxysemicarbazide (RW L35)... 48 1 -[9-< 10-chloroanthryl)methylene]-4-hydroxyseinicarbazide (RW L36).... 48 C. Structure identification and determination of physicochemical properties............................................................................................... 49 (1). Melting point measurement............................................... 49 (2). Elemental analysis............................................................. 49 (3). Assessment of relative hydiophobicity and purity by thin-layer chromatographic (TLC) study........................................... 49 (4). Infrared (IR) absorption spectra......................................... 50 (5). !H and l3 C nuclear magnetic resonance (NMR) spectra........... 50 (6). Mass spectra (M S )................................................................... 51 (7). Measurement of partition coefficient Gog P) and ionization constants (pK a)........................................................................ 51 (8). Stability study in phosphate buffers.................................... 54 2. Results and Discussion......................................................................... 55 (1). Melting points.......................................................................... 55 (2). Elemental analysis................................................................... 55 (3). Assessment of relative hydrophobicity and purity by thin-layer chromatographic (TLC) study................................................. 56 (4). Infrared (IR) spectra................................................................. 56 (5). Nuclear magnetic resonance (NMR) spectra............................ 74 (6). Mass spectra (M S )................................................................... 92 (7). Measurement of partition coefficient Gog P) and ionization constants (pK a)........................................................................ 100 (8). Stability study in phosphate buffers.......................................... 100 (9). Reaction mechanism of Schiff base formation......................... 101 IV. BIOLOGICAL EVALUATION..................................................... 107 1. An Overview of the Cytotoxicity Assay................................................ 107 2. Experimental Procedures.................................................................... 110 A. Inhibition of leukemia suspension cells......................................... 110 (1). Mouse leukemia L1210 cells..................................................... 110 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a. Cell culture............................................................................. 110 b. Determination of IC5 0 values................................................. 110 (2). Human leukemia CCRF-CEM cells............................................ 116 a. Cell culture.............................................................................. 116 b. Determination of IC jo values.................................................. 116 B. Inhibition of solid tumor cells (B16, CHO, HT29 and ZR7S) 117 (1). Cell culture.................................................................................. 117 (2). Measurement of % inhibition at 50 pM concentration............... 117 (3). Determination of IC5 0 values...................................................... 119 C. Selective toxicity against tumor cells vs. 3T3 Swiss mouse embryo fibroblasts......................................................................................... 122 D. Inhibition of hydroxyurea-resistant KB cells................................... 122 3. Results and Discussion.......................................................................... 123 A. Inhibition of leukemia suspension cells.............................................. 123 (1). Mouse leukemia L1210 c e lls...................................................... 123 (2). Human leukemia CCRF-CEM cells............................................. 172 B. Inhibition of solid tumor cell lines (B16, CHO, HT29 and Z R 75)..... 172 (1). % Inhibition at 50 pM concentration.......................................... 172 (2). IC 50 values against the solid tumor cell lines.............................. 173 C. Selective toxicity against tumor cells vs. 3T3 Swiss mouse embryo fibroblasts........................................................................................... 193 D. Inhibition of hydroxyurea-resistant KB cells..................................... 203 (1). IC 50 values against hydroxyurea-resistant KB cells..................... 203 (2). Mechanism of hydroxyurea resistance......................................... 203 E. Discussion........................................................................................... 206 V. QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP - 213 1. Methods.............................................................................................. 213 2. Results and Discussion...................................................................... 216 A. RWL1 to RW L36.......................................................................... 216 B. RWL1 to RW L21........................................................................... 235 VL CONCLUSIONS............................................................................... 241 REFERENCES.................................................................................... 244 APPENDICES...................................................................................- 256 A. The IR spectra............................................................................... 257 B. The mass spectra........................................................................... 290 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table No. Caption Page No. 1.1 Different characteristics of the three classes of ribonucleotide reductases...................................................................................... 8 1.2 Allosteric regulation of ribonucleotide reductase........................ 25 1.3 In vitro cytotoxicity and in vivo toxicity of the representative Schiff bases of AHG.Ts reported previously................................. 26 m . 1 The recrystallization solvents, yields and melting points of the 31 Schiff bases of hydroxysemicarbazide synthesized...................... 57 in.2 Elemental analyses of the 31 newly synthesized Schiff bases of hydroxysemicarbazide.................................................................. 61 HI.3 The Rf values of hydroxysemicarbazide, semicarbazide, hydroxyurea and Schiff bases of hydroxysemicarbazide 64 m.4 Infrared absorption frequencies (in cm'1 ) .................................... 68 ffl.5 Proton chemical shifts, multiplicity and coupling constants of the newly synthesized compounds...................................................... 76 m.6 Carbon-13 NMR chemical shifts of the Schiff bases of hydroxysemicarbazide.................................................................. 84 ffl.7 Mass spectral fragmentation and fragment ions of RW L5,12,18, 26,28,35,36 .............................................................................. 93 m.8 The concentrations and UV absorbances of RWL1 used in derivation of its standard curves in phosphate buffer solutions saturated with 1-octanol.............................................................. 102 m.9 The concentrations and UV absorbances of RWL1 used in derivation of its standard curves in 1-octanol saturated with the phosphate buffers.......................................................................... 102 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m.10 m il m.12 IV. 1 IV .2 IV .3 IV .4 IV.5 IV .6 IV.7 IV.8 IV.9 IV.10 The UV absorption maxima ( L i ) , absorption intensities (E1 % lc m ), apparent partition coefficient Gog P’) and pKa values of RWL1 ... 104 The substituent constants (n) of =NNHCONHOH and - CH=NNHCONHOH derived from the measured log P of RWL1 and other n constants................................................................... 105 The concentrations and UV absorbances of RWL1 used in the stability study.............................................................................. 100 Inhibition of mouse leukemia L1210 cells by Schiff bases of hydroxysemicarbazide, and reference compounds (hydroxyurea, semicarbazide, and hydroxysemicarbazide).............................. 124 The inhibitory activities of RWL4, RWL21, RWL35 and hydroxyurea against human leukemia CCRF-CEM cells 174 The % inhibition of the selected Schiff bases of hydroxysemicarbazide against solid tumor cell lines at 50 pM concentration............................................................................... 180 Inhibition of B16 (mouse melanoma) cells by RWL35 and hydroxyurea.................................................................................. 181 Inhibition of CHO (Chinese hamster ovary carcinoma) cells by RWL35 and hydroxyurea.............................................................. 182 Inhibition of HT29 (human colon adenocarcinoma) cells by RWL35 and hydroxyurea.............................................................. 182 Inhibition of ZR75 (human breast carcinoma) cells by RWL35 and hydroxyurea.................................................................................... 184 In vitro toxicity of RWL35 and hydroxyurea against 3T3 fibroblasts....................................................................................... 194 Comparison of in vitro selectivity of RWL35 and hydroxyurea 197 The inhibitory activities of the 31 Schiff'bases of hydroxysemicarbazide against cancer and non-cancer cell lines .... 198 IX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV. 1 1 The inhibitory activities of RWL2, RWL35 and hydroxyurea against the wild type (KB-W), hydroxyurea-resistant (KB-HUR) and gemcitabine-resistant (KB-GR) KB tumor c ells.................. 209 IV. 12 Comparison of the inhibitory activities (IC50) of the Schiff bases of hydroxysemicarbazide with the clinically used anticancer drugs against different tumor cell lines.................................................... 211 V. 1 The inhibitory activities and physicochemical properties of hydroxyurea (HU), semicarbazide (SC), hydroxysemicarbazide (HSC), and Schiff bases of HSC analyzed in the regression analysis............................................................................................ 227 V.2 The squared correlation matrix (R2 ) of the physicochemical parameters used in the regression analysis (n = 3 3 )...................... 231 V.3 The inhibitory activities and physicochemical properties of the phenyl-containing Schiff bases of hydroxysemicarbazide (RWL1- RWL21) used in the regression analysis...................................... 237 V.4 The squared correlation matrix (R2 ) of the physicochemical parameters used in the regression analysis for RWL1 to RWL20 (n = 2 0 )............................................................................................... 240 x Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure No. Caption Plage No. 1 .1 DNA synthesis in mammalian cells via the de novo and the salvage pathways...................................................................... 4 1.2 Enzymatic reduction of ribonucleotides..................................... 5 1.3 Schematic diagram of E. coli ribonucleotide reductase 13 1.4 3-D structure of R| subunit of E coli ribonucleotide reductase.. 14 1.5 The R2 subunits of E. coli and mouse ribonucleotide reductases 15 1.6 The catalytic mechanism of E. coli ribonucleotide reductase ... 17 1.7 Thiosemicarbazones with antitumor activity............................. 28 1.8 The structural similarity of various N-OH derivatives with antitumor activity.................................................................. 29 m . 1 Design of Schiff bases of hydroxysemicarbazide................... 34 m.2 Synthetic scheme of Schiff bases of hydroxysemicarbazide .... 35 ffl.3 The major mass spectral fragmentation pathway of Schiff bases of hydroxysemicarbazide.............................................. 92 ffl.4 Standard curves used for determining the log P’ values of RWL1....................................................................................... 103 IV. 1 Structures of tetrazoliums (MTT, XTT and MTS), formazans produced by viable cells, and electron coupling reagents (PMS andPES)................................................................................... 109 IV.2 Schematic layout of a 96-well plate used in the IC50 determination............................................................................ 114 IV.3 Dose response curves of RWL14 and RWL19 against L1210 cells obtained by the MTS/PES colorimetric assay.............. 115 xi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV .4 The different procedures used in the IC5 0 determinations of the Schiff bases of hydroxysemicarbazide for suspension (L1210 and CCRF-CEM leukemia cell lines) and adherent (B16, CHO, HT29 and ZR7S solid tumor cell lines) cell cultures................ 121 IV.5.(1) Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against L1210 cells....................... 139 IV.5.(2) Dose response curve (A) and probit transformed dose response curve (B) of hydroxysemicarbazide against LI 210 cells 140 IV.5.(3) Dose response curve (A) and probit transformed dose response curve (B) of RWL1 against L1210 cells................................... 141 IV.5.(4) Dose response curve (A) and probit transformed dose response curve (B) of RWL2 against L1210 cells................................... 142 IV.5.(5) Dose response curve (A) and probit transformed dose response curve (B) of RWL3 against L1210 cells................................... 143 IV5.(6) Dose response curve (A) and probit transformed dose response curve (B) of RWL4 against L1210 cells................................... 144 rv.5.(7) Dose response curve (A) and probit transformed dose response curve (B) of RWL5 against L1210 cells................................... 145 IV.5.(8) Dose response curve (A) and probit transformed dose response curve (B) of RWL6 against L1210 cells................................... 146 IV.5.(9) Dose response curve (A) and probit transformed dose response curve (B) of RWL7 against L1210 cells................................... 147 IV.5.( 10) Dose response curve (A) and probit transformed dose response curve (B) of RWL8 against L1210 cells................................... 148 IV.5.( 11) Dose response curve (A) and probit transformed dose response curve (B) of RWL9 against L1210 cells................................... 149 IV.5.(12) Dose response curve (A) and probit transformed dose response curve (B) of RWL10 against L1210 cells................................. 150 xii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV.5.(13) Dose response curve (A) and probit transformed dose response curve (B) of RWL11 against L1210 c ells.............................. 151 IV.5.(14) Dose response curve (A) and probit transformed dose response curve (B) of RWL12 against L1210 c ells.............................. 152 IV.5.(15) Dose response curve (A) and probit transformed dose response curve (B) of RWL13 against L1210 c ells.............................. 153 IV.5.(16) Dose response curve (A) and probit transformed dose response curve (B) of RWL14 against L1210 c ells.............................. 154 IV.5.(17) Dose response curve (A) and probit transformed dose response curve (B) of RWL15 against L1210 c ells.............................. 155 IV.5.( 18) Dose response curve (A) and probit transformed dose response curve (B) of RWL16 against L1210 c ells.............................. 156 IV.5.( 19) Dose response curve (A) and probit transformed dose response curve (B) of RWL17 against L1210 c ells.............................. 157 IV.5.(20) Dose response curve (A) and probit transformed dose response curve (B) of RWL18 against L1210 c ells.............................. 158 rv.5.(21) Dose response curve (A) and probit transformed dose response curve (B) of RWL19 against L1210 c e lls.............................. 159 IV.5.(22) Dose response curve (A) and probit transformed dose response curve (B) of RWL20 against L1210 c ells.............................. 160 IV.5.(23) Dose response curve (A) and probit transformed dose response curve (B) of RWL21 against L1210 c ells.............................. 161 IV.5.(24) Dose response curve (A) and probit transformed dose response curve (B) of RWL23 against L1210 c e lls.............................. 162 IV.5.(25) Dose response curve (A) and probit transformed dose response curve (B) of RWL24 against L1210 c e lls.............................. 163 IV.5.(26) Dose response curve (A) and probit transformed dose response curve (B) of RWL26 against L1210 c e lls.............................. 164 xiii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV.5.(27) Dose response curve (A) and probit transformed dose response curve (B) of RWL27 against L1210 c ells.............................. 165 IV.5.(28) Dose response curve (A) and probit transformed dose response curve (B) of RWL28 against L1210 c ells.............................. 166 IV.5.(29) Dose response curve (A) and probit transformed dose response curve (B) of RWL31 against L1210 cells.............................. 167 IV.5.(30) Dose response curve (A) and probit transformed dose response curve (B) of RWL32 against L1210 c ells.............................. 168 IV.5.(31) Dose response curve (A) and probit transformed dose response curve (B) of RWL33 against L1210 c ells.............................. 169 IV.5.(32) Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against L1210 c ells.............................. 170 IV.5.(33) Dose response curve (A) and probit transformed dose response curve (B) of RWL36 against L1210 cells.............................. 171 IV.6.(1) Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against CCRF-CEM cells............... 176 IV.6.(2) Dose response curve (A) and probit transformed dose response curve (B) of RWL4 against CCRF-CEM c e lls...................... 177 IV.6.(3) Dose response curve (A) and probit transformed dose response curve (B) of RWL21 against CCRF-CEM c ells....................... 178 FV.6.(4) Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against CCRF-CEM cells....................... 179 IV.7.(1) Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against B16 c e lls.......................... 185 IV.7.(2) Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against B16 cells................................... 186 IV.8.(1) Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against CHO c e lls........................... 187 xiv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV.8.(2) Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against CHO cells................................... 188 IV.9.(1) Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against HT29 cells........................... 189 rv.9.(2) Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against HT29 cells............................... 190 IV. 10.(1) Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against ZR75 cells........................... 191 IV. 10.(2) Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against ZR75 cells................................... 192 IV. 11.(1) Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against 3T3 fibroblasts.................... 195 IV. 1 1 .(2) Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against 3T3 fibroblasts............................ 1% IV. 12. Dose response curves of RWL2 (A) and RWL35 (B) against the wild type (KB-W), hydroxyurea-resistant (KB-HUR) and gemcitabine-resistant (KB-GR) KB tumor c e lls...................... 210 V. 1 A plot of log 1/ICso (after correcting for differences in CMR and I) vs. Clog P for the compounds analyzed (Eq. 4, n = 33, R = 0.798).................................................................................. 232 V.2 A plot of log 1/ICso (after correcting for differences in CMR and I) vs. Clog P for the compounds analyzed (Eq. 7, n = 31, R = 0.977).................................................................................. 233 V.3 A plot of calculated log 1/ICso vs. observed log 1/ICso for the compounds analyzed (Eq. 5, n = 30, R = 0.979)...................... 234 V.4 The general structure of the phenyl-containing Schiff bases of hydroxysemicarbazide.............................................................. 240 xv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Ribonucleotide reductase (RR) is a rate-limiting enzyme involved in de novo DNA synthesis, and is considered to be a potential target for cancer chemotherapy. Among all RR inhibitors investigated, only hydroxyurea (HU) is currently used for the treatment of various cancers, but it has several drawbacks. To obtain more potent and selective RR inhibitors, thirty-one Schiff bases of hydroxysemicarbazide were synthesized. Their molecular structures and purity were established by NMR, IR and MS spectra, and by elemental analyses. The cytotoxicities of the 31 compounds were first evaluated against murine leukemia L1210 cells using the MTS/PES colorimetric assay. Seventeen of the 31 compounds exhibited higher cytotoxicities than HU against the L1210 cells. Six compounds with IC5 0 values in the micromolar range (2.7 - 7.2 pM) were found to be 1 1 to 30-fold more potent than HU (IC5 0 = 82 pM). The active compounds were further tested against human leukemia CCRF- CEM cells and four solid tumor cells. Among these, three compounds (RWL-4, 21, 35) inhibited the CCRF-CEM cells with IC 5 0 values ranging from 2.7 to 7.0 pM. RWL35 [l-(9-[10-methylanthryl]methylene)-4-hydroxysemicarbazide] was the strongest inhibitor, and showed 74 to 692-fold activity, compared to HU, against the solid tumor cells tested. RWL35 exhibited more favorable selectivity (8-187 fold) than HU, and had no cross-resistance with HU and gemcitabine, two known RR inhibitors. xvi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The log P and pKa values of a model compound, RWL1 [l-{3- trifluoromethylbenzylidene)-4-hydroxysemicarbazide], were measured by the shake-flask method, and then the Hansch-Fujita it constant of the functional group -CH=NNHONHOH was derived for the calculation of log P of other congeners. Besides the essential pharmacophore (-NHCONHOH), among the physicochemical parameters examined in the QSAR analysis, hydrophobicity (log P), molecular size/polarizability (MR) and the indicator variable (I) for o-oxygen function turned out to be the important determinants of the antitumor activities observed. Six of the title compounds have been shown to be remarkable inhibitors of various tumor cells including the HU-resistant cells. The most active compounds merit additional investigations for further development as anticancer drugs. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER L INTRODUCTION 1 .1. Physiological Role o f Ribonucleotide Reductase In all procaryotic and eucaryotic cells DNA synthesis requires a continuous and balanced supply of the four deoxyribonucleoside triphosphates (dNTPs). The formation of dNTPs can be through either the de novo pathway (-90%) utilizing ribonucleotides as precursors, or the salvage pathway (-10%) using preformed deoxyribonucleotides (see Figure 1.1.)- However, due to the limited pool size of deoxyribonucleotides in mammalian cells, rapid synthesis of dNTPs is necessary for proper cell replication. The de novo synthesis of dNTPs depends on the rate limiting enzyme ribonucleotide reductase (RR) (see Figure L2.). 1.2. History of Ribonucleotide Reductase The history of ribonucleotide reductases (RRs) was full of many surprises. The fust surprise was their existence. The discovery of the first RR in Escherichia coli (Reichard et al., 1961) was met with considerable skepticism since organic chemistry then did not recognize a reaction whereby a carbon-bound OH-group could be replaced directly by a hydrogen. The second surprise was that contrary to the prevailing belief the enzyme did not contain vitamin B 12 (Holmgren et al., 196S; Jordan and Reichard, 1998). A few years after the discovery of the E. coli reductase a second RR was discovered in Lactobacillus leichm annii (Blakley and Barker, 1964). This enzyme required adenosylcobalamin, which led researchers to believe 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that RRs were Bi2 enzymes and that their malfunction was the cause of defective DNA synthesis in pernicious anemia, a disease that could be cured by administration of vitamin Bn. When the £ coli RR was shown not to require vitamin B n this was the first sign indicating more than one class of RRs. The £ coli RR became the prototype of class I enzymes, to which all eucaryotic RRs belong, whereas the L leichmannii enzyme became the prototype for a large group of microbial class II RRs that depended on adenosylcobalamin. Originally it looked as if each of the four ribonucleotides required a specific RR. It soon became clear that only a single enzyme was involved, with allosteric effects directing its substrate specificity (Larsson and Reichard, 1966). Then came the biggest surprise of all: an organic free radical (Ehrenberg and Reichard, 1972), identified as tyrosyl-122 (Sjoberg et al., 1978), forms part of the polypeptide structure of one of the subunits of the £ coli enzyme and it is required for activity. The radical was surprisingly stable and survived for the two weeks it took to purify the enzyme. This highly unexpected result had no precedent. The discovery of a second RR in anaerobically grown £ coli (Fontecave et al., 1989) came as no surprise, as the generation of the tyrosyl radical of class I enzymes requires oxygen for its formation. The anaerobic enzyme is also a protein radical, with the unpaired electron located on a glycyl residue (Gly 681) at the COOH-terminal region of the polypeptide chain (Sun et al., 19%). It contains an iron-sulfur cluster in a separate subunit and uses this cluster and S- 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. adenosylmethionine to generate the glycyl radical (Ollagnier et al., 1996). The anaerobic E. coli RR is the prototype for class m RRs, whose genes have been found in other anaerobically grown eubacteria, in phage T4, and in archaebacteria (Fleischmann et al., 199S; Young et al., 1994; Bult et al., 19%). A big surprise was the discovery in Salmonella typhimurium of an additional class I enzyme that has an unknown physiological role (Jordan et al., 1994a, 1994b). The importance of this discovery also lies in the subsequent realization that class I RRs of many other bacteria resemble the Salmonella typhimurium RR in structure and properties. Therefore, two subgroups of class I RR came into being: one (class la) has the original E coli enzyme as the prototype, and also includes the RR enzymes from eucaryotes, viruses and a limited number of eubacteria; the second (class lb) has the Salmonella typhimurium enzyme as its prototype and is found exclusively in eubacteria (not in eucaryotes, viruses, and archaebacteria). The multiplicity of RRs may seem bewildering. At first sight the members of the three different classes show little resemblance. Their amino acid sequences are different, and they use different mechanisms to generate their protein radicals. However, they catalyze the same free radical chemistry at the nucleotide level, and all employ amino acid free radicals. A second common denominator is the way in which they regulate their substrate specificity by allosteric effects. This suggests that they share a common tertiary structure, at least in part, in spite of large differences in the primary structures. 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Nucleoside kinases The de novo pathway (-90% ) DNA polymerase DNA RNA Ribonucleotide Reductase (RR) dTMP dUMP NDP kinase kinases kinases dNDP L dNMP Purines and pyrimidines biosynthesis Preformed deoxyribonucleosides dATP<->dADP dGTP<->dGDP dCTP<->dCDP dUDP dTTP <->dTD P The salvage pathway (-10% ) Figure 1.1. DNA synthesis in mammalian cells via the de novo and the salvage pathways (adapted from Lien, 1987a). p p —I base HO OH base HO H Ribonucleotide reductase r : SH Thioredoxin /glutathione reductases NADP+ NADPH + H* Figure 1 2 . Enzymatic reduction of ribonucleotides (PP = pyrophosphate; base = adenine, cytosine, guanine or uridine; R = thioredoxin or glutaredoxin). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L 3. Classification o f Ribonucleotide Reductases RRs can be grouped into three major classes (Jordan and Reichard, 1998), based on the mechanisms they use for radical generation and on their structural differences. Class I can be subdivided further into two subclasses, la and lb, based mainly on differences in allosteric regulation but also on involvement of auxiliary protein. An overview of the different characteristics of the three classes of RRs is summarized in Table 1.1. Class I enzymes consist of homodimeric proteins, Ri ( ( X 2), coded by nrdA gene, and R2 (fc), coded by nrdB (Fontecave et al., 1992). The large a chain harbors the catalytic site and allosteric sites. The small 0 chain contains an oxygen-linked diferric center and a stable tyrosyl free radical. The RR of aerobically grown E. coli is the prototype of class la enzymes and has been extensively characterized. Class lb, presenting exclusively in eubacteria, has the enzyme from Salmonella typhimurium as its prototype. A distinguishing feature of all known lb enzymes is that they lack approximately S O amino-terminal residues of the la enzymes and are not inhibited by dATP (Eliasson et al., 1996). A second characteristic feature is that lb operons code for an additional auxiliary protein (NrdI) of unknown function and for a specific external electron donor (NrdH) (Jordan et al., 1996, 1997a). Class la RRs have been found in eucaryotes, viruses and a limited number of eubacteria, while class lb RRs present only in eubacteria. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lactobacillus leichmannii RR is the prototype for a large group of microbial class II RRs which have a simpler structure (a or O f e ) (Panagou et al., 1972; Tsai and Hogenkamp, 1980). Class n enzymes, presenting in L Leichm annii (Blakley and Barker, 1964) and also in other eubacteria and archaebacteria (Reichard, 1993a), use adenosylcobalamin as the radical generator. The anaerobic E. coli RR is the prototype for class m RRs (Fontecave et al., 1989; Reichard, 1993b), whose genes have been found in other anaerobically grown eubacteria, in phage T4, and in archaebacteria. Gass m enzymes are ( X 2P 2 heterotetramers (Ollagnier et al., 1996). The a polypeptide, coded by nrdD gene, contains a stable glycyl radical (Sun et al., 1996). The 3 polypeptide chain, coded by nrdG gene, contains an iron-sulfur cluster that together with S-adenosylmethionine can generate the glycyl radical (Ollagnier et al., 19%). Ribonucleoside diphosphates are the substrates for class I enzymes, whereas the class ID enzymes investigated so far use ribonucleoside triphosphates (Fontecave et al., 1989; Young et al., 1994). The prototype class I I L leichm annii enzyme also uses ribonucleoside triphosphates (Panagou et al., 1972), but other later discovered members of this class use ribonucleoside diphosphates (Jordan et al., 1997b; Tsai and Hogenkamp, 1980; Tauer and Benner, 1997; Riera et al., 1997). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1.1. Different characteristics of the three classes of ribonucleotide reductases (Adapted from Jordan and Reichard, 1998). Class la Class lb Class II Class III Oxygen dependence aerobic aerobic aerobic/anaerobic anaerobic Structure ( X 2P 2 C I 2P2 a(a 2) C X 2P2 Genes nrdA, nrdB nrdE, nrdF not named nrdD, nrdG Radical tyrosyl tyrosyl adenosylcobalamin glycyl Metal site Fe-O-Fe Fe-O-Fe, Mn-O-Mn Co Fe-S Substrate NDP NDP NDP/NTP NTP Reductant thioredoxin glutaredoxin NrdH-redoxin glutaredoxin thioredoxin formate Allosteric sites 2 1 1 2 dATP inhibition Yes No No Yes Occurrence eucaryotes, eubacteria, bacteriophages, viruses eubacteria archaebacteria, eubacteria, bacteriophages, archaebacteria, eubacteria, bacteriophages, Prototype aerobic E. coli (AERR) mouse (MRR) Salmonella typltimurium and Corynebacterium ammoniagenes Lactobacillus leichmannii anaerobic E. coli o o 1.4. Comparison o f Ribonucleotide Reductases from Aerobic £ coli and from Mammalian Sources Both the aerobically grown E. coli RR (AERR) and mammalian RRs (MRR) belong to class la enzymes, while the anaerobic E coli RR belongs to class III enzymes. Until a few years ago, studies on MRRs were greatly restricted by the low concentration and instability. During recent years, technical problems concerning the study of MRRs have been overcome and studies are proceeding gradually. However, the knowledge of RR still refers mainly to the AERR. MRRs share several characteristics with the AERR, but some important differences have recently been identified. This is important for designing an anti-RR agent specifically targeting MRR enzymes. In this dissertation, I only concentrate on the RRs from aerobic E. coli and from mammalian sources. 1 .4. A. Aerobic £ coti ribonucleotide reductase (AERR) The AERR holoenzyme is composed of two rather easily dissociable, separately inactive subunits, an R| subunit (171 kD) and an R2 subunit (87 kD) (Reichard, 1993a) (see Figure L3.). High-resolution structures of several forms of the aerobic E. coli Ri and R2 subunits, a plausible model for the Ri:R2 holoenzyme are now available (Uhlin and Eklund, 1994, 1996; Nordlund et al., 1990; Nordlund and Eklund, 1993; Aberg et al., 1993; Logan et al., 1996; Kauppi et al., 1996). 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Ri subunit is a large homodinier (az). Each a polypeptide has one activity site, one substrate specificity site, and only one kind of catalytic substrate- binding site that contains a critical pair of SH groups and can handle all four ribonucleoside diphosphate substrates. The catalytic site has been identified from a GDP-R) complex (Eriksson et al., 1997). It is located deep inside each promoter in the center of a cleft between the N-terminal and barrel domains. C225 and C462 form the redox-active cysteine pair involved in the reduction of the ribose. A third C439 is responsible for the transient thiol radical required for catalysis. A second redox pair (C754/C759) at the C-terminal end receives the electrons from the external hydrogen donor (Aberg et al., 1989; Mao et al., 1992; Licht et al., 1996). The structures of the two allosteric sites (activity site and substrate specificity site) were established from complexes between Ri and effectors (the ATP analog AMPPNP and cflTP) (Eriksson et al., 1997). The activity site was found to be in a cleft at the tip of the N terminus of Ri. The substrate specificity site is at the interface between the two subunits of Ri (see Figures L3. and L4.). The R2 subunit can be considered as a metalloprotein coenzyme consisting of two identical fc polypeptide chains (43 kD), each of which contains a non-heme iron center and a stable tyrosyl free radical (Tyr 122) (Reichard, 1993a). 1.4. B. Mammalian Ribonucleotide Reductase (MRR) In mammalian systems, RR also consists of two subunits, Ri (MW of mouse Ri: 168 kD) and R2 (MW of mouse R2: 90 kD) (Thelander et al., 1980). The 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mammalian Rt is a homodimer ( C C 2), and also contains two allosteric sites and one catalytic site. The mammalian R2 @2) also contains two iron/radical centers using tyrosyl radicals in the reaction. The common features between the MRR and AERR are shown in Table LI. Studies during 1980s have demonstrated that four of the six best studied MRRs are different from the AERR (Whitfield and Youdale, 1989). The comparisons of RRs from aerobic E. coli and from mammalian sources have been summarized in a tabular form by Lien and others [Lien, 1987a, 1987b; Chan, 198S; T'ang, 1984). The data up to 1987 were covered in the above stated comparisons. The mammalian (mouse and calf thymus) Ri and R2 subunits were purified to homogeneity by different groups (Thelander et al., 1980, 198S; Caras et al., 1985; Salem et al., 1993; Davis et al., 1994). The study by Uhlin and Eklund (1994) suggested that the mammalian Ri subunit had the same overall 3-D structure as that of aerobic E. coli. Although aerobic E. coli R2 in many respects serves as a model for R2S in higher organisms, a number o f significant differences have been observed (see Figure L5.). First, the amino acid sequence identity between the E coli and mouse R2S is only about 25% (Thelander and Graslund, 1994). Unlike the E coli R2, the mouse R2 is completely devoid of P-strands. The amino ends of R2 differ considerably in length for the AERR and the mouse RR. The mammalian sequences are about 50 residues longer than those of the aerobic E coli R2 . In mouse R2, the C- terminal seven residues are essential for subunit interactions, while the C-terminal 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. peptide with the same function in the aerobic £ coli R2 is much longer (Kauppi et al., 1996). The most striking difference between the aerobic £ coli and mouse R2 proteins concerns the stability of the iron center. Unlike the aerobic £ coli R2, the free radical-iron center in mouse R2 is labile and the protein loses 50% of its iron after 30 minutes at 37°C (Mann et al., 1991; Nyholm et al., 1993a, 1993b). Therefore, it has to be continuously regenerated in vivo in a reaction that requires ferrous iron and oxygen. Another significant difference is the sensitivity to radical scavengers. Hydroxyurea reduces the bee radical about 3 times faster in the mouse R2 than in the aerobic £ coli R2 (Nyholm et al., 1993a, 1993b). The mouse R2 is also much more sensitive towards bulkier, hydrophobic radical scavengers, which indicates a more open structure around the iron/radical center in the mouse protein (Potsch et al., 1995). The similar results have been obtained by Kauppi et al. (1996). The radical/iron site in the mouse R2 subunit is less shielded and communicates via a narrow hydrophobic channel with the solvent, which explains its greater susceptibility to radical scavengers and metal chelators (e.g. hydroxyurea). In aerobic £ coli R2 , only the tyrosyl radical is destroyed by hydroxyurea whereas the iron center is left intact (Karlsson et al., 1992). In mouse R2, however, both the tyrosyl radical and the iron center are reduced by hydroxyurea (Nyholm et al., 1993a, 1993b) 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Allosteric sites SpecHldty ATPdATP (2x85.7 kD) Substrate binding catalytic site HU S B -H S C 2V * •) Ft r * M * / R2 y (2 x 43.4 Figure 1.3. Schematic diagram of E. coli ribonucleotide reductase. R| subunit contains the catalytic site with its active thiols and two allosteric sites, one regulating the activity of the enzyme, the other its substrate specificity. R2 subunit, with its two diferric Fe (III) centers and two tyrosyl radicals, provides the protein radical required for the reaction (adapted from Reichard, 1993a). Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Activity sites Specificity sites PPN P Substrate binding sites Figure 1.4. 3-D structure of R| subunit of E coli ribonucleotide reductase (adapted from Jordan and Reichard, 1998). Iron centers Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure I.S. T h e R * subunits o f E . coli a n d m o u se ribonucleotide reductases (adapted f r o m Kauppi e t al., 1996). 1.5. Catalytic Mechanism of Ribonucleotide Reductase C ass la enzymes (e.g. £ coli and mouse RRs) receive the elections required for the reduction of ribose from thioredoxins and glutaredoxins with redox-active thiols (Holmgren and Bjomstedt, 1995; Holmgren and Aslund, 1995). Ultimately, the reducing power comes from NADPH, which reduces the cystines of the redoxins either via a specific thioredoxin reductase or via glutathione and glutathione reductase (see Figure L2.). The reaction sequence proposed by Stubbe’s group (Stubbe, 1990; Mao et al., 1992) shows a generic radical mechanism for class I enzymes, with details taken from the £ coli class la enzyme (see Figure I.6.). The reaction is initiated by binding of the substrate (ribonucleoside diphosphate) into the catalytic site of the reduced enzyme. This leads to a transfer of the radical function from Y122 (Tyr 122) of the R2 subunit to C439 (Cys 439) of the Ri subunit, generating a thiol radical. The radical initiates the reduction of the ribonucleotide by abstracting the 3’-H atom, thereby generating a substrate radical (step 1). Radical formation facilitates the leaving of the protonated OH-group at C-2’ position (step 2). A substrate cation radical is subsequently reduced by the redox-active cysteine pair C225 and C462 (step 3). Finally the H atom stored at C439 is returned to C-3’ (step 4) with regeneration of the thiol radical at C439 (step 5). E441 (Glu441) and N437 (Asn437) stabilize the interaction between enzyme and substrate by H-bonding to the oxygens at C-3’ and C-2’, respectively. 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. base PPO HO C462 C439 C225 E441 N437 PPO base Hb HS -H,0 SHa C462 NHj C439 C225 E441 N437 PPO base SHa C462 C439 C225 E441 N437 PPO base base PPO PPO base Ha. Hb HO SHa SHa C462 C462 C462 C439 C439 C439 C225 C225 C225 E441 E441 E441 N437 N437 N437 Figure 1.6. The catalytic mechanism of E. coli ribonucleotide reductase (adapted from Stubbe, 1990; Mao et al., 1992). 1.6. Allosteric and Genetic Regulations of Ribonucleotide Reductase 1 .6. A. Allosteric regulation Most class la RRs are regulated allosterically by binding either ATP (activating) or dATP (inhibitory) to the activity site of Ri subunit (Brown and Reichard, 1969). Class la RRs are also endowed with an additional and unique allosteric mechanism that regulates their substrate specificity by binding of end products (dATP, dGTP and dTTP) to the substrate specificity site of Ri subunit. The allosteric sites communicate with the catalytic substrate-binding site and also affect each other. Binding of (d)ATP to the specificity site induces reduction of CDP and UDP, dGTP induces reduction of ADP, and dTTP induces reduction of GDP (see Table L2.). These control mechanisms ensure adequate supplies of different deoxyribonucleotides needed for the de novo DNA synthesis. 1 .6. B. Genetic regulation RR plays a central role in the regulation of the pool sizes of the four dNTPs required for DNA synthesis. The small dNTP pools suffice in mammalian cells for only a few minutes of DNA replication and must therefore be renewed continuously during S phase. Cells that are not in S phase also synthesize dNTPs de novo, but at a much slower rate (Bianchi et al., 1997). To satisfy the changing demands for dNTPs the synthesis of RR is tightly adapted to the cell cycle. The genes for mouse Ri and Ri subunits are regulated separately and are located on separate chromosomes, nrdA (R|) on chromosome 7, nrdB (R2 ) on 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chromosome 12. Activity of mouse RR is cell cycle dependent, very low during G(/Gi and high in S phase. Ri subunit can be found in proliferating cells during the whole cell cycle, while R2 subunit can only be found in G |-S phase (Eriksson et al., 1984). Ri has a longer half-life of 18-24 h as compared to the brief half-life of R2 (3- 4 h). Evidences show that R2 is the rate-limiting subunit in each cell cycle phase (Nocentini, 1996). RR activity regulation may also be obtained by controlling the amount of R2 tyrosyl radicals and irons. 1 .7. Ribonucleotide Reductase Expression in Tumor C ells Although RR activity is high in normally proliferating cells, activity increases even further in tumor cells with similar growth rates (Weber, 1983). RR activity has also been correlated with the degree of malignant transformation as reviewed by Wright (1989). In the past few years these studies have been confirmed by other interesting findings. RR activity as well as Rt and R2 mRNA expression is increased following contact with tumor promoters (e.g. TP A, TGF-P) (Choy et al., 1989; Hurta and Wright, 1992). These findings clearly indicate that RR plays a fundamental role in the critical early events involved in tumor promotion, and the RR activity is tightly linked to the neoplastic expression state. Therefore, RR is one of the choice enzymes for designing key enzyme-targeted chemotherapy. 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.8. Ribonucleotide Reductase Inhibitors as Antitmnor/Antiviral Agents 1 .8. A. Hydroxyguanidine and its derivatives Several different classes of compounds have been shown to be relatively specific inhibitors of RR. These compounds can be classified into seven classes (Nocentini, 1996). Among all RR inhibitors investigated, Schiff bases of aminohydroxyguanidine tosylate (AHG.Ts) have been extensively studied by Lien’s group. So far, 54 different Schiff bases of AHG.Ts have been synthesized and tested for cytotoxic/antiviral activities (lien, 1987a, 1987b, 1993; Lien et al., 2001; Tai et al., 1984; T’ang et al., 1985; Wang et al., 1990,1991; Koneru et al., 1993; Hui et al., 1994; Das et al., 1999; Cory et al., 1985; Weckbecker et al., 1987; 1988a; 1988b). Most of them have been found to be more active than both hydroxyguanidine sulfate and hydroxyurea. Submicromolar I C 50 values have been observed against various tumor cell lines for several compounds. Compound ATL14 showed in vivo activity against murine P388 leukemia. The studies by Lien’s group have shown that the cytotoxicities of Schiff bases of AHG.Ts correlate significantly with RR inhibition. The ratios of in vivo LD50 to in vitro IC50 range from 32 to 338 for the four Schiff bases of AHG.Ts (see Table L3.). 1 .8. B. Huosemicarbazones Thiosemicarbazone (H2NNHCSNH2) derivatives have been extensively studied as RR inhibitors and antitumor agents. Brockman et al. (1956) first reported that 2-formylpyridine thiosemicarbazone (PT) increased the life span of mice bearing 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L1210 leukemia (see Figure L7 for the structure). Further development of this compound was curtailed because of its relatively low therapeutic index. Even though 5-hydroxy-2-formyIpyridine thiosemicarbazone (5-HP) was found to be very active in animal models, it had very low potency in humans and showed gastrointestinal toxicity and myelosuppression (Agarwal and Sartorelli, 1975; Moore and Sartorelli, 1984). Its poor activity in human has been attributed to its extremely short half-life. A few years later, French and Blanz (1966) synthesized 1 -formylisoquinoline thiosemicarbazone (IQ-1) and a variety of other a-(N>heterocyclic carboxaldehyde thiosemicarbazones. Members of this class have been shown to have antitumor activity against a wide spectrum of transplanted rodent neoplasm. However, IQ-1 was extremely insoluble and could not be formulated for parenteral use (Agarwal and Sartorelli, 1978). In 1995, twelve substituted isoquinoline- 1 -carboxaldehyde thiosemicarbazones have been synthesized and evaluated for antitumor activity in mice bearing L1210 leukemia by Liu et al. (1995). The most active compounds were 4-aminoisoquinoline-l-carboxaldehyde thiosemicarbazone and 4- (methylamino)isoquinoline- 1 -carboxaldehyde thiosemicarbazone, which both produced optimum %T/C (the percentage increase in median survival time of treated tumor-bearing mice over control tumor-bearing mice) values of 177 against L1210 leukemia in mice. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Among a series of pyridine-2-carboxaldehyde thiosemicarbazones synthesized, triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) has been studied both in vitro and in vivo, and demonstrated clinical potential as an antitumor agent (Finch et al., 2000). 1 .8. C. Hydroxyurea Of all RR inhibitors investigated, only hydroxyurea (HU, Hydrea®) (H2NCONHOH) was marketed and is currently used as an anticancer drug in the treatment of melanoma, resistant chronic myelocytic leukemia, and recurrent, metastatic, or inoperable ovarian cancer. HU is also used in the local control of primary squamous cell (epidermoid) carcinomas of the head and neck (Medical Economics Company, 1999). The line of research on HU has gained new momentum in the wake of success stories about combination therapy in cancer and AIDS treatment (Navarra and Preziosi, 1999). In 1998, HU (Droxia®) was marketed for use in the treatment of sickle cell anemia (Anonymous, 1998). HU inhibits HIV replication in vitro (Lori et al., 1994). Recently, it has been used in the treatment of AIDS in combination with didanosine, showing no viral rebound for 1 year after 1 year’s treatment (Vila et al., 1997). Moreover, HU has been shown to be an immune stimulating modulator recently (Lori, 1999). The mechanism by which HU produces its cytotoxic effect has been extensively studied. HU causes an immediate inhibition of DNA synthesis by acting as an RR inhibitor. It has been reported that HU inhibits the enzyme activity by one- 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. electron transfer from HU to the tyrosyl radical of R2 subunit, and by destabilizing the iron center (Lassmarm and Liermann, 1989; Lassmann et al., 1992). Because of its mechanism of action, HU principally affects cells actively synthesizing DNA at S-phase. HU crosses the intestinal wall and cell membrane by passive diffusion (Evered and Selhi, 1972; Morgan et al., 1986). In patients with cancer, HU is 79% available to the systemic circulation following oral administration (Tracewell et al., 199S). The oral LD3 0 of HU is 7330 mg/kg in mice and 5780 mg/kg in rats, given as a single dose (Medical Economics Company, 1999). The phase I clinical trial by Gandhi et al. (1998) has established 27 g/m2 of HU infused over 24 hours as the maximum-tolerated dose. However, HU has several disadvantages such as a short half-life (1.9-3.9 h), the necessity of using high dosage (80 mg/kg every third day or 20-30 mg/kg daily) and the rapid development of resistance (Gwilt and Tracewell, 1998; Zhou et al., 1995; Medical Economics Company, 1999). 1 .8. D. Other ribonucleotide reductase inhibitors RR inhibition has been achieved by peptidomimetics and nonpeptides derived from the amino sequence of RR subunits. The studies by Cohen et al. (1986) and Liuzzi et al. (1994) have shown that the C terminus of R2 subunit is essential for the formation of active R1-R2 holoenzyme, and the formation can be inhibited by synthetic peptides with an amino acid sequence corresponding to the C terminus of R2. A potent pepddomimetic inhibitor of herpes virus RR with antiviral activity in vivo has been derived from the C terminus of R2 subunit of herpes simplex virus RR 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Liuzzi et al., 1994). RR inhibitors have also been designed as Ri allosteric modulators (e.g. nucleoside analogs), agents reacting with Ri cysteine residues, metal chelators, and thioltransferase inhibitors. So far, only three nucleoside analogs cladribine, fludarabine, gemcitabine are being studied in phase II clinical trials (Nocentini, 1996). 1.8. E. Recognition o f the essential pharmacophore HU, hydroxyguanidine, thiosemicarbazone, substituted hydroxamic acid, and their derivatives have been shown to have antitumor and/or antiviral activities (Lien. 1987a; Cory and Cory, 1989; Sartorelli and Agrawal, 1976). Based on the previous studies, the essential pharmacophore [-C(=X)NHOH, X = O or NH] for the antitumor/antiviral activity has been identified (Lien, 1993) (see Figure I.8.). 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T able 1.2. Allosteric regulation of ribonucleotide reductase. Substrate Allosteric effectors Activity site Substrate specificity site positive negative positive negative CDP ATP dATP ATP dGTP UDP ATP dATP ATP dTTP, dGTP GDP ATP dATP dTTP dGTP ADP ATP dATP dGTP dATP 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1.3. In vitro cytotoxicity and in vivo toxicity of the representative Schiff bases of AHG.Ts reported previously. Ar-CH=NNHC(=NH)NHOH.CH3 C6 H4 0 3 SH Compound/Ar cytotoxicity or toxicity cell lines or animals LD30/IC50 ref. Hydroxyurea H2 NC(=0)NH0H IC5 0 = 60.6 pM LD50 = 7330 mg/kg = 96371.3 pM L1210 mice 1590 Tai et al., 1984 Hydroxyguanidine sulfate H2 NC(=NH)NH0H.H2 S 04 IC50 = 59.5 pM L1210 Tai et al., 1984 ATL25 f T V ^ l NSC376463 CH= IC50 — 0.8 |iM IC50= 1.1 pM IC30 = 3.3 pM MCF-7 CCRF-CEM L1210 T’ang et al., 1985; Lien et al., 2001 ATL14 Br OH NSC371168 i ) - a " Br At 100 mg/kg (190 pM), T/C = 140% (the percentage increase in median survival time of treated tumor-bearing mice over control tumor-bearing mice) P388-bearing mice T’ang et al., 1985 £ Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1.3. (continued) Ar cytotoxicity or toxicity cell lines or animals LDj o /ICj o ref. LT1 NSC355188 IC50= 10.9 nM LD50 = 315 mg/kg = 896.4 nM L1210 C57 B1/6J mice 82 Tai et al., 1984 LT4 NSC355190 , ^ 0 - C H = IC50= 12.5 iiM LDj o = 190 mg/kg = 398.9 *iM L1210 C57 B1/6J mice 32 Tai et al., 1984 ATL13 NSC371167 N C —^ C H = IC50 = 9.4 nM LDS0= 156.5 mg/kg = 416.8 nM L1210 Swiss-webster mice 44 T’ang et al., 1985 ATL19 NSC376466 jHp o z n IC50 ~ 2.7 LD50 = 322.2 mg/kg = 911.7 nM L1210 Swiss-webster mice 338 T ’ang et al., 1985 s I I 'CH=N— N H -C -N H , (PT) 2-Formytpyridine thiosemicarbazone 7T CH=N-I -NH-C-NHj (5-HP) 5-Hydroxy-2-fonnyipyridme thiosemicarbazooe C C i N XH =N —NH-C-NHj (3-AP) 3-Amino- 2-formyipyridine thiosemicarbazone S CH=N-NH-C-NH, (IQ-D 1-Fonnylisoquinoline thiosemicarbazone NH, S CH=N-NH-C-NH, 4-Amino-1 -formy fasoqumolme thiosemicarbazone 8 CH=N-NH-C-NHj 4-Metfaylanmo-1 -formy lisoquino line thiosemicarbazone Figure L7. Thiosemicarbazones with anti tumor activity. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ? i HjNt C -N -O H j Hydroxyurea NH I I HjN-’ -C — N— OH ‘ H Hydroxyguamdine at-c h = n 4 n - c - n h2 j H i T hiosem icarbazooe 1 1 ArfC-N-OH H 10 ! ii i Ar-CH=N-NTC-N-OH H H I J Ar-CH =N -N +C-N -O H Hi H Substituted hydroxamc add Schiff base of hydro x y anukarbddc Schiff base of anmohydroxyguanidine Figure L8. The structural similarity of various N-OH derivatives with antitumor activity. The essential pharmacophores are highlighted by frames. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IL RATIONALE Ribonucleotide reductase (RR) is a key enzyme, which catalyzes the reduction of ribonucleotides to deoxyribonucleotides, and thus provides die building blocks for DNA replication in all dividing cells. Therefore it serves as a promising target for designing anticancer drugs. Mammalian RR is sensitive to radical scavengers and metal chelators, therefore, the compounds possessing free radical scavenging and/or chelating properties are expected to be potential RR inhibitors. Of all RR inhibitors investigated, only hydroxyurea (HU) is currently used as an anticancer drug for the treatment of various cancers. HU inhibits RR enzyme by one-electron transfer from HU to the tyrosyl radical, and by destabilizing the iron center, and thus inhibits DNA synthesis at S-phase. Therapeutic application of HU has several disadvantages such as a short half-life (1.9 - 3.9 h), the necessity of using high dosage (80 mg/kg every third day or 20-30 mg/kg daily) and the rapid development of resistance. A new series of Schiff bases of hydroxysemicarbazide (HSC) designed in this study retains the essential pharmacophore (-NHCONHOH) which possesses free radical scavenging property as shown by HU, but their hydrophobic, electronic and steric properties were diversified by adding various auxopharmacores. Because the results obtained from the previous structure-activity relationship studies on the Schiff bases of aminohydroxyguanidine tosylate (AHG.Ts) may or may not directly be applicable to the new Schiff bases of HSC, the auxopharmacores used in this study not only include the “good” ones as shown in the Schiff bases of AHG.Ts, but also 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. include the “bad” ones as well as the ones not used in the Schiff bases of AHG.Ts. The new series of Schiff bases of HSC contain the essential pharmacophore - NHCONHOH, they are expected to have higher cytotoxicity than HU against tumor cells and may have acceptable or even better pharmacodynamic and pharmacokinetic profiles. The lymphocytic mouse leukemia cell line L1210 has been used extensively for routine screening programs of chemical agents and natural products for cytotoxic activity. The cells exhibit a double time of 8-10 hours and grow as stationary suspension culture. The cell line was chosen for primary screening because of their sensitivity to a broad spectrum of antitumor agents and its fast growth rate. Based on the primary screening results obtained from the L1210 cells, the most active Schiff bases of HSC were further tested against a human leukemia cell line, CCRR-CEM, because leukemia is one of the clinical indications of HU. HU is clinically used in the treatment of melanoma and ovarian cancer. Therefore, B-16 (mouse melanoma) and CHO (Chinese hamster ovary carcinoma) solid tumor ceil lines were further selected for testing of the most active compounds. In order to test whether the compounds are active against other solid tumors with high incidences in human, ZR-75 (human breast carcinoma), and HT-29 (human colon adenocarcinoma) were also chosen in in vitro studies. Quantitative structure-activity relationship (QSAR) analysis was carried out, in order to see whether the optimal compound has been made or not, and what binding forces would be involved in the drug-enzyme interaction. The QSAR study 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. will provide a useful guideline for further structural modification and development of the second generation of HSC derivatives. Literature search indicates that there are no reports on the antitumor activity of the Schiff bases of HSC proposed. It is considered worthy of synthesizing and testing the target compounds. It is expected that good anticancer drug candidates or leads will be identified, and some o f these compounds may be developed for the treatment of melanoma, chronic myelocytic leukemia and other cancers in the future. At present, cancer is still a deadly disease all over the w orld This study may advance the new anticancer drug research and produce additional armament in fighting the war against cancer. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER HI. CHEMISTRY T3U DLL Methods and M atm ab IR IA . Design o f Schiff bases o f hydroxysemkarhazide Because of the disadvantages of hydroxyurea, there is plenty of room for improvement of its anti tumor activity. Based on the structure of hydroxyurea, a general structure containing the pharmacophore (-NHCONHOH) and an auxopharmacore (Ar-CH=) was designed. Various Ar- groups that include monocyclic, bicyclic, and tricyclic moieties were systematically selected. The Ar- groups have a wide range of hydrophobic, electronic, and steric properties (see Figure m .1 ). DLLS. Synthesis of hydroxysemkarbazide and its Schiff bases Synthetic scheme Schiff bases of hydroxysemicarbazide were synthesized by a dehydration reaction of hydroxysemicarbazide with different aldehydes. All aldehydes were purchased from Aldrich and other chemical companies. Hydroxysemicarbazide, which was not commercially available, was synthesized in two steps using modified procedures of Grobner and Muller (1974), and Steinberg and Bolger (1956) (see Figure DL2). 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. auxopharmacore pharmacophore At = P - R2 C C r Wide ranges of physicochemical properties of whole molecules: Hydrophobicity: Clog P -0.95 to 4.11 Electronic properties: dipole moment 1.50 to 8.90 D Molecular size: molecular weight 168.16 to 446.97 Figure m.1. Design of Schiff bases of hydroxysemicarbazide. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o (liquid) ft K ,C03 O—C—a + H-jNOH-HCl----- :-------« * ^ j ether/ water (solid) NHOH+ HQ + H jN N H ^O ethanol O -0” (very soluble in ethanol) ff H ^ - N — C -N -O H ‘ H H (slightly soluble in ethanol) + Ar-CHO methanol H ,0 + Ai— C = N -N — C -N -O H 2 H H H Figure III.2. Synthetic scheme of Schiff bases o f hydroxysemicarbazide. 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 111.13.(2). Synthetic procedure III.lJJ.(2).(a). Synthesis o f N-hydroxy phenyl carbam ate To a solution of 12.7 g (0.183 mole) of hydroxyanhne hydrochloride (MW = 69.49) in 350 ml of ether and 5 ml of water, 253 g (0.183 mole) of potassium carbonate (MW = 138.21) was added. The mixture formed was used immediately in the next step. Under stirring, 21.4 g (0.137 mole) of phenyl chloroformate (MW = 156.57) in 50 ml of ether was gradually added to the mixture in 15 minutes. The resulting mixture was stirred at room temperature for 3 hours and then the precipitate was filtered and washed with ether. The filtrate was dried by addition of anhydrous MgS04 overnight After removal of MgS04 by filtration, the filtrate was stilled to recycle the solvent ether, and then concentrated in vacuo to dryness. A white crystalline product (18.6 g) was obtained by recrystallization in 20 ml of benzene (m.p. 105-107 °C, yield = 89%, reported m.p. 105-107 °C, Grobner and Muller, 1974). U I.13.(2).(b). Synthesis of hydroxysemicarbazide To a solution of 153 g (0.1 mole) of N-hydroxy phenyl carbamate (MW = 153.14) in 100 ml of ethanol, 7.0 g (0.14 mole) of hydrazine monohydrate (MW = 50.06) in 50 ml of ethanol was added, and mixed thoroughly. The mixture was undisturbed for 3 hours, and then white shiny crystals appeared. The product hydroxysemicarbazide (7.8 g) was obtained after filtration (m.p. 129-131 °C, yield = 86%, reported m.p. 126 °C, Grobner and Muller, 1974). 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m .l.B .2 .(c). Synthesis o f Schiff bases o f hydi' oxyrtcmicarfaaride l-(3>TrifhioroniethyibenzylideneM ^iydroxysem icarbazide (RWL1) A mixture of 5.36 g (0.0308 mole) of 3-trifluorobenzaIdehyde (MW = 174.12) and 2.81 g (0.0308 mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in methanol : ethyl acetate : chloroform (M:E:C, 2 : 3 : 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 5.47 g of white shiny crystals (m.p. 205-207 °C, yield = 72 %). l-(2-Hydroxy-3,5-dichlorobeiizylidene)-4-hydroxysem icarbazide (RWL2) A mixture of 3.82 g (0.02 mole) of 3,5-dichlorosalicylaldehyde (MW = 191.02) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 150 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to obtain 4.22 g of cream-white crystals (m.p. 204-206 °C, yield = 80 %). l-(2-Hydroxy-5-bromobenzylidene)-4-hydroxysetnicarbazkie (RWL3) A mixture of 4.02 g (0.02 mole) of 5-bromosalicylaldehyde (MW = 201.03) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.79 g of cream-white crystals (decomposition at 225-227 °C, yield = 69 %). l-(2-Hydroxy-3? 5-dibromobaizylkiene)-4-hydroxysenikart>azide (RWL4) A mixture of 4.20 g (0.015 mole) of 3,5-dibromosalicylaldehyde (MW = 279.93) and 1.37 g (0.015 mole) of hydroxysemicarbazide (MW = 91.07) in 150 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.53 g of cream-white crystals (tn.p. 210-212 °C, yield = 67 %). l-(2-Hydroxy-3-methoxy-5-bromobenzyIidene)-4-hydroxyseniicJU-bazide (RWL5) A mixture of 4.62 g (0.02 mole) of 3-methoxy-5-bromosalicylaldehyde (MW = 231.06) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3:4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 4.02 g of grayish crystals (m.p. 200-202 °C, yield = 6 6 %). 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MS-IodobenzytktereM-hydroxysemicarbaade (RWL6) A mixture of 4.64 g (0.02 mole) of 3-iodobenzaldehyde (MW = 232.02) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW - 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 5.18 g of grayish crystals (m.p. 210-212 °C, yield = 79 %). 1 -(2-Hydroxy-3,5-dnodobenzylidene)-4-hydroxyseniicarbazkle (RWL7) A mixture of 7.48 g (0.02 mole) of 3,5-diiodosalicylaldehyde (MW = 373.92) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 150 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 6.62 g of light yellow crystals (m.p. 205-208 °C, yield = 74 %). l-(4-CyanobenzyUdeneH-hydroxysenucarbazide (RWL8) A mixture of 2.62 g (0.02 mole) of 4-cyanobenzaIdehyde (MW = 131.13) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M £:C (2:3: 4), the mixture was concentrated to 5-10 ml in vacuo. The 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. precipitate was collected by filtration and recrystallized in methanol to give 3.09 g of white shiny crystals (m.p. 186-188 °C, yield = 72 %). 1 -(4-Dimethylaminobenzy Udene)-4-hydroxyseinicarbazide (RWL9) A mixture of 2.98 g (0.02 mole) of 4-dimethylaminobenzaldehyde (MW = 149.19) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 :4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.51 g of yellow shiny crystals (m.p. 159-162 °C, yield = 79 %). l-(2-Hydroxy-3,5-dinitrobenzylidene)-4-hydroxyseinicarbazide (RWL10) A mixture of 3.70 g (0.017 mole) of 3,5-dinitrosalicylaldehyde (MW = 212.12) and 1.59 g (0.017 mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 :4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.90 g of yellow shiny crystals (m.p. 171-173 °C, yield = 79 %). 1 -(3-Nitrobenzylidetie)-4-hydroxysemicarbazide (RWL11) A mixture of 4.53 g (0.03 mole) of 3-nitrobenzaldehyde (MW = 151.12) and 2.73 g (0.03 mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. methanol was nefluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 4.71 g of light yellow crystals (m.p. 213-215 °C, yield = 70 %). 1 -(3-Methoxybenzy HdeneM-hydroxysemkarhazide (RWL12) A mixture of 4.08 g (0.03 mole) of 3-methoxybenzaldehyde (MW = 136.13) and 2.73 g (0.03 mole) o f hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3:4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 4.01 g of white crystals (m.p. 175-178 °C, yield = 64 %). 1 -(4-M etboxybenzylidene)-4-hydroxysemicarbazide (RWL13) A mixture of 4.08 g (0.03 mole) of 4-methoxybenzaldehyde (MW = 136.13) and 2.73 g (0.03 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 :4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.80 g of white crystals (m.p. 164-166 °C, yield = 61 %). 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 -(2^DimethoxybenzytMene)-4-hydroxy8eiiucarbazide (RWL14) A mixture of 3.32 g (0.02 mole) of 2,5-dimethoxybenzaldehyde (MW = 166.18) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3:4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 2.96 g of cream-white crystals (m.p. 150-153 °C, yield = 62 %). l-(2-Hydroxy-4-methoxybenzylidene)-4-hydroxyseinicarbazide (RWL15) A mixture of 3.04 g (0.02 mole) of 4-methoxysalicylaldehyde (MW = 152.15) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.48 g of light pink crystals (m.p. 155-157 °C, yield = 77 %). 1 -(2-Hydroxy-4,6-dimethoxy benzyfidene)-4-hydroxysemicarbaz»de (RWL16) A mixture of 3.08 g (0.017 mole) of 4,6-dimethoxysalicylaldehyde (MW = 182.18) and 1.54 g (0.017 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3:4), the mixture was concentrated to 5-10 ml in vacuo. The 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. precipitate was collected by filtration and recrystallized in methanol to give 3.41 g of cream-white crystals (decomposition at 169-171 °C, yield = 79 %). 1 -<4-benzyloxy benzyfidene)-4-hydroxysem icarbazide (RWL17) A mixture o f 531 g (0.025 mole) of 4-benzyloxybenzaldehyde (MW = 212.25) and 2.27 g (0.025moie) of hydroxysemicarbazide (MW = 91.07) in 150 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 10-20 ml in vacuo. The precipitate was collected after filtration and recrystallized in methanol to give 6.10 g of cream-white crystals (m.p. 155-157 °C, yield = 8 6 %). l-(4-phenyIbenzylideiie)-4-hydroxysem icarbazide (RWL18) A mixture o f 3.64 g (0.02 mole) of 4-phenylbenzaldehyde (4- biphenylcarboxaldehyde, MW = 182.22) and 1.82 g (0.02 mole) of hydroxy semicarbazide (MW 91.07) in 150 ml of anhydrous methanol was refluxed for 24 hours. The mixture was concentrated to 10-20 ml in vacuo. After checking for product formation via TLC in M:E:C (2:3: 4), the precipitate was collected by filtration and recrystallized in methanol to give 3.72 g of cream-white crystals (m.p. 158-160 °C, yield = 73 % ). 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 -(2,4-dihydroxy benzylkiei»e)-4-hydroxysemicarbazkie (RWL19) A mixture of 2.76 g (0.02 mole) of 2,4-dihydroxybenzaldehyde (MW = 138.12) and 1.82 g (0.02mole) of hydroxysemicarbazide (MW = 91.07) in S O ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 5-10 ml in vacuo and placed in a flask for overnight The precipitate was collected by filtration and recrystallized in ethanol to give 2.62 g of light pink crystals (m.p. 179-181 °C, yield = 62%). l-(4-acetamidobenzylidene)-4-hydroxyseinicarbazide (RWL20) A mixture of 2.45 g (0.015 mole) of 4-acetamidobenzaldehyde (MW = 163.18) and 1.37 g (0.015mole) of hydroxysemicarbazide (MW = 91.07) in 100 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in ethanol to give 2.10 g of cream white crystals (m.p. 205-207 °C, yield = 64 %, reported m.p. 215 °C, Grobner and Muller, 1974). l-(23,4-trihydroxybenzy lideneH-hydroxysem icarbazide (RWL21) A mixture of 3.08 g (0.02 mole) of 2,3,4-trihydroxybenzaldehyde (MW = 154.12) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. formation via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 5-10 ml in vacuo and placed in a flask overnight The precipitate was collected by filtration and recrystallized in ethanol to give 3.98 g of cream-white crystals (m.p. 182-185 °C, yield = 8 8 %). 1 -(3-pyridy lmethy lene)-4-hydroxy semkarbazide (RW L23) To a solution of 2.73 g (0.03 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol, 3.21 g (0.03 mole) of 2-pyridinecarboxaldehyde (MW = 107.11) was added dropwise. The reaction mixture was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected after filtration and recrystallized in methanol to give 3.80 g of pale white crystals (m.p. 166-168 °C, yield = 70%). 1 -[2-(6-methylpyridyl)methylene]-4-hydroxysemicarbazide (RWL24) A mixture of 2.73 g (0.03 mole) of hydroxysemicarbazide (MW = 91.07) and 3.63 g (0.03 mole) of 6-methyl-2-pyridinecarboxaldehyde (MW = 121.14) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3:4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected after filtration and recrystallized in methanol to give 4.01 g of pale white crystals (m.p. 164-166 °C, yield = 69 %). 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. l-[5-(4-methylimklazolyl)nrethylene]-4-hydroxysemicarbazkle (RWL26) A mixture of 3.30 g (0.03 mole) of 4-methyl-5-imidazoIecarboxaldehyde (MW - 110.12) and 2.73 g (0.03 mole) of hydroxysemicarbazide (MW = 91.07) in S O ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 4.20 g of white shiny crystals (m.p. 184-186 °C, yield = 76 %). l-[2-(5-nitrothienyl)methyleiie]-4-hydroxyseniicarbazkle (RW L27) A mixture of 3.14 g (0.02 mole) of S-nitro-2-thiophenecarboxaldehyde (MW = 157.15) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.51 g of yellow shiny crystals (m.p. 185-187 °C, yield = 76 %). l-(3-indolylmethylene)-4-liydroxysemicarbazide (RWL28) A mixture of 2.18 g (0.015 mole) of indole-3-carboxaldehyde (MW = 145.16) and 137 g (0.015 mole) of hydroxysemicarbazide (MW = 91.07) in 50 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 5-10 ml in 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 2.08 g of cream-white crystals (m.p. 150-152 °C, yield = 64 %). l-[3-(64^tehloro-4-oxo-4H -l-benzopyran)niethylene}-4-hydroxysem icartazide (RWL31) A mixture of 4.86 g (0.02 mole) of 6,8-dichloro-3-fonnylchromone (MW = 243.05) and 1.82 g (0.02 mole) of hydroxysemicarbazide (MW = 91.07) in 150 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3: 4), the mixture was concentrated to 10-15 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 4.40 g of pale yellow crystals (m.p. 184-186 °C, yield = 70 %). 1 -[3-(6-isopropy l-4-oxo-4H-1 -benzopyran)methy ieiie]-4-hydroxysem icarbazide (RWL32) A mixture of 2.78 g (0.013 mole) of 6-isopropyl-3-formylchromone (MW = 216.24) and 1.17 g (0.013 mole) of hydroxysemicarbazide (MW = 91.07) in 150 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3:4), the mixture was concentrated to 10-15 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.09 g of white shiny crystals (m.p. 177-179 °C, yield = 83 %). 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. l-(l,4-benzodioxan-6-ytmethylenc)-4-hydroxyseniiairbazide (RWL33) A mixture of 4 3 7 g (0.027 mole) of 1,4-benzodioxan-6-carboxaldehyde (MW = 164.16) and 2.42 g (0.027 mole) of hydroxysemicarbazide (MW = 91.07) in ISO ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2:3 :4), the mixture was concentrated to 5-10 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 4.98 g of cream-white crystals (m.p. 167-169 °C, yield = 79 %). l-[9-(10-rnethylanthryl)methyleiie]-4-hydroxysemicarbazide (RWL35) A mixture of 2.7S g (0.012 mole) of 10-methylanthracene-9-carboxaldehyde (MW = 220.27) and 1.13 g (0.012 mole) of hydroxysemicarbazide (MW = 91.07) in 200 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 : 4), the mixture was concentrated to 10- 15 ml in vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 3.30 g o f yellow crystals (m.p. > 250 °C, yield = 90 %). 1 -[9-( 10-chloroanthryl)methyiene]-4-hydroxysemicarbazkie (RWL36) A mixture of 2.37 g (0.0098 mole) of 1 0-chloro-9-anthraldehyde (MW = 240.69) and 0.89 g (0.0098 mole) of hydroxysemicarbazide (MW = 91.07) in 200 ml of anhydrous methanol was refluxed for 24 hours. After checking for product formation via TLC in M:E:C (2 : 3 :4), the mixture was concentrated to 10-15 ml in 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vacuo. The precipitate was collected by filtration and recrystallized in methanol to give 2.60 g of yellow crystals (decomposition at 210 °C, yield = 84 %). m .l.G . Structure identification and determination o f physicochemical properties IIL l.C (l). Melting point measurement The melting points of the well-dried compounds were determined in melting point capillary tubes with a MEL-TEMP melting point apparatus and were corrected. m .l.C .(2 ). Elemental analysis All samples were dried in a desiccator with P2O5 under vacuum for at least 72 hours before sent out for analysis. All elemental analyses were performed by Galbraith Laboratories, Knoxville, Tennessee. m .l.C .(3 ) Assessment o f relative hydrophobidty and purity by thin-layer chromatography (TLO The TLC study was carried out on polyester backed, pre-coated silica gel chromatographic plates with layer thickness of 250 micrometer, particle size of 5-17 micrometer, and pore size of 60 A. The TLC plates were purchased from Aldrich Chemical Company, Inc. (Cat No. Z12277-7), and were run in methanol : ethyl acetate : chloroform (CH3OH : CH3CO2C2H5: CHCI3, M:E:C) (2:3:4 v/v/v) as the eluent The chromatograms were developed in an iodine chamber and their Rf values 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. determined as the following: Rf = dj/do, where dj = the distance o f the spot center from the initial point of application, and do = the distance of the solvent front from the initial point of application. The Rf values were converted to Rm values by die following equation: R m = log (1/Rf - 1) (Bate-Smith and Westall, 1950; Boyce and Miborrow, 1965; Lien, 1994). Rm is related to hydrophobicity Gog P) by the following equation: Rm = a • log P + b. The Rm values were used in the subsequent QSAR analysis (see Chapter V). ffl.l.C .(4 ). Infrared absorption spectra The infrared spectra were obtained on a Beckman IR-4210 spectrometer. All the samples were thoroughly dried in a desiccator with P2O5 under vacuum for at least 24 hours. The samples were prepared as KBr pellets and used immediately. IIL 1.C (5). * H and UC Nuclear magnetic resonance (NMR) spectra The !H- and l3C-NMR spectra were measured on a Bruker AMX-500 MHz FT NMR spectrometer at the Department of Chemistry of USC. All the samples were dried in a desiccator with P2O5 and dissolved in dimethyl sulfoxide-tfe (tfe- DMSO) using 5 mm NMR tubes. The NMR spectra were processed by using Nuts 2D Version 4.27 NMR Data Processing Program (Acom NMR Inc., 1994). Some compounds were further characterized by HH COSY (RWL1 and 11) and CH correlated NMR spectroscopy (RWL1,6,7,11,12,14). 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m .l.G ( 6 ). Mass spectra (MS) Another physical method that is used in the elucidation of chemical structures is mass spectrometry (MS). Unlike IR, NMR, and UV spectroscopy, MS does not involve the absorption of electromagnetic radiation but operates on a completely different principle. As the name implies, MS is used to determine the molecular weight and the structure of an unknown compound by analyzing the fragment ions in the spectrum. To further establish the correct structures of RWL-5, 12, 18, 26, 28, 35, 36 (the compounds with the elemental analyses beyond 0.4% limit without considering solvent of crystallization), MS spectra were obtained from Mass Spectrometry Laboratory of University of California Riverside, using DCI (Desorption Chemical Ionization) method on a VG 7070 High Resolution Mass Spectrometer. HL1.C.(7). Measurements o f partition coefficient (log P) and ionization constants (pKa) Physicochemical properties such as partition coefficient (log P) and ionization constant (pKa) are important parameters for interpreting the forces involved in the interaction between the RR enzyme and the title compounds in the subsequent QSAR analysis. Therefore, measurement of log P and pKa values of one representative compound RWL1 was performed using the partition method with two immiscible solvents (1 -octanol/phosphate buffer). The advantage of this method is that log P and pKa can simultaneously be measured. After the log P value was 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. measured, the Hansch-Fujita substituent constants (it) of -CH=NNHCONHOH and =NNHCONHOH were derived for the first time, and then can further be used for calculating log P values of other structurally related compounds by taking J ji. Because phosphate buffer gave the closer log P values to those obtained from 1- octanol/water than acetate and bicarbonate buffer systems, it is the most suitable buffer system for measuring true (or corrected) partition coefficients of acidic and neutral compounds (Wang and Lien, 1980). RWL1 was first dissolved in methanol (2mg/ml). To 38.4 ml of phosphate buffers (pH 2.2, 7.3, 11.4) saturated with l-octanol, 1.6 ml of RWL1 methanol solution and 40 ml of l-octanol saturated with phosphate buffers were added. The mixtures were shaken for 4 hours and centrifuged 3 minutes at 1000 RPM (D E C Clinical Centrifuge, International Equipment Company). The concentrations of RWL1 and its charged species in phosphate buffer, and those in l-octanol phase were measured with a Hitachi U-2000 UV spectrophotometer at Xmax. The standard curves were obtained for l-octanol, pH2, pH7 and pH ll buffer systems, and then used for measurement of RWL1 concentrations in l-octanol, pH2, pH7 and pH 11 buffer systems, respectively. The true (or corrected) partition coefficient (P) of the neutral molecular species were determined at pH 7.2 where the neutral form is predominated. The P value was calculated as following: 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. --- ► HA ------ ► H+ x A " l-octanol phosphate buffer F = [HA]o/[HA]pb The apparent partition coefficient (F ) or distribution coefficient (D) was similarly determined at pH values of 2.2 and 11.4 near to where the pKa values were suspected to be. The F value was calculated as following: F = [HA]</([HA]pb + [A' Ip b ) where [HA] 0 is the concentration of RWL1 neutral form in l-octanol phase, [HA],* the concentration of RWL1 neutral form in phosphate buffer, and [A ] the concentration of RWL1 charged form. The pKa values were calculated by the following equations derived from the buffer equation. At pH 11, pKa (acid) = pH - log (P • [D ]^[D ]0 - 1) [for (N)-OH group] At pH 2, pKa (base) = pH + log (P ■ [D]pt/[D]0 - 1) (for the imino -N = nitrogen) The equations were derived as following: For acids: a=l/[l+ 1 0 (p K a _ p H )] P = P7(l- a) = F- [1 + lO(p H " p K a > ] = [D]J[D]pb • [1 + lO(p H - p K * ) ] P • [Dlpbf[DJ0 = 1 + 10(pH _pK a) P [D ]p b /[D]o- l = l0(p H - p K *) 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Log (P * [D]pbt[D]0 - 1 ) = pH - pKa pKa = pH - log (P • [D]pb/[D]0 - 1) For bases: a=l/[l+ 1 0 (p H ' p l C a ) ] P = P /(l- a ) = P- [1 + io< P K a -PH > ] = [DJMDJpt - [1 + IO ^ -p ^ ] P • [D]pbl[D]„ = 1 + iO(p K a - p H ) Log (P • [Dipt![DJ0 - 1) = pKa - pH pKa = pH + log (P • [D]p b l[D]0 - 1) Where a is the degree of ionization, [D]a the concentration of neutral form of an acid/base in l-octanol, and [DJpb ( = [HA],* + [A"],*) the concentration of neutral and ionized forms of an acid/base in phosphate buffer. m .l.C (8 ). Stability study in phosphate buffers In order to know the remaining percentage of the newly synthesized compounds after 3-day period of cell culture, the stability of one representative compound RWL1 was studied in pH3, pH7 and pH ll phosphate buffers at room temperature up to 72 hours. The concentrations of RWL1 were determined using the Hitachi U-2000 UV spectrophotometer. The rate of a first-order reaction (k) and the half-life (ti/2) values of RWL1 were estimated by the following equations: k = -2303 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. x slope, t |/2 = 0.693/k, using a plot of log (a-x) as a function of time (t) [log (a-*) - log a - kt/2.303], where a = initial concentration, a-x = remaining concentration at a time point IIL2. Results and Discussion All the 31 Schiff bases of hydroxysemicarbazide were synthesized with relatively high yields (61-90%). The purity of the newly synthesized compounds was determined by single spot on TLC plate, sharp melting points and elemental analyses. The correct structures of the target compounds were further validated by IR, MS, 'H- and 13C-NMR spectra. All results are shown in Tables IIL1 to I1L6 and Appendices A and B. HL2.(1). M elting points The corrected melting points, recrystallization solvents and yields of the target compounds are presented in Table DLL m JL(2). Elemental analysis Elemental analyses on C, H, and N were performed by Galbraith Laboratories, and the results are listed in Table IIL2. For each compound, the first row values are the calculated values and the second row values in parentheses are experimental values. All results are within ±0.4% of calculated values except the % C and/or % N values of RWL5, RWL12, RWL18, RWL26, RWL28, RWL35 and 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RWL36. For the seven compounds, the experimental values of % C and/or % N are 0.5 to 0.96% lower than the calculated values, most likely due to incomplete combustion during the analyses, since TLC, IR and NMR all confirmed the purity of the compounds with the expected structures. IIL2.(3). Assessment o f relative hydrophobkity and purity by thin-layer chromatographic (TLC) study The Rf values of the target compounds are shown in Table D U . The Rf values of the compounds reflect their hydrophobicity. The higher the Rf value of a compound the more hydrophobic it is. The Rm values converted from Rf values were used as a hydrophobic parameter in the following QSAR analysis. IIL2.(4). Infrared (IR) spectra The characteristic absorptions of IR spectra of the target compounds are summarized in Table IIL4. The IR spectra are presented in Appendix A. The characteristic absorptions are used to identify the common fragment - C=NNHCONHOH. All compounds show similar absorption frequencies of C=N at 1690-1650 cm'1 , and -NH, -OH and -C = 0 stretching of -ONNHCONHOH at 3390-3300 cm'1 , 3280-3180 cm'1 , and 1710-1650 cm '1 , respectively. Besides the common absorption features, each compound shows characteristic absorption frequencies of specific functional groups (see Table JBA for details). 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table DLL The recrystallization solvents, yields and melting points of die 31 Schiff bases of hydroxysemicarbazide synthesized. Ar-CH=NNHCONHOH code At recry. yield, % mp, *C solvent RWL1 \ _ 7 ~ ~ CH= MeOH 72 205-207 C F / RWL2 MeOH 80 204-206 OH OH RWL3 < v /)— CH= MeOH 69 225-227 (dec.) RWL4 y ^ ~ CH= MeOH 67 210-212 Br OH RWL5 MeOH 6 6 200-202 H,CO OH RWL6 MeOH 79 210-212 I . OH RWL7 (f V -C H = MeOH 74 205-208 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table DLL (continued) code Ar recry. yield, % mp, * C solvent RWL9 RWL11 RWL12 RWL13 RWL14 RWL15 RWL8 ||C H s MeOH 72 186-188 MeOH 79 159-162 RWL10 O /l OH H3 CO RWL16 CHs ° \ , CH= OCH, MeOH 79 171-173 MeOH 70 213-215 MeOH 64 175-178 MeOH 61 164-166 MeOH 62 150-153 MeOH 77 155-157 MeOH 79 169-171 (dec.) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HI. 1. (continued) code Ar recry. yield, % mp, °C solvent RWL17 MeOH 8 6 155-157 RWL18 V / O H RWL19 RWL20° h o _ O m 5 h * HsC- 2 - {j - Q - c H = MeOH 73 158-160 EtOH 62 179-181 EtOH 64 205-207 HQ OH RWL21 RWL23 h ° - 0 - c h = - C H = a EtOH 8 8 182-185 MeOH 70 166-168 RWL24 RWL26 RWL27 HjC ti CH= V C MeOH 69 164-166 MeOH 76 184-186 MeOH 76 185-187 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table m .l. (continued) code Ar recry. yield, % mp, ° C solvent RWL28 RWL31 RWL32 CH MeOH MeOH 64 150-152 70 184-186 83 177-179 RWL33 RWL35 RWL36 H= MeOH 79 167-169 MeOH 90 >250 MeOH 84 210 (dec.) A known compound (Groebner and Muller, 1974). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table III.2. Elemental analyses of the 31 newly synthesized Schiff bases of hydroxysemicarbazide. code formulas MW elemental analysis ‘ C H N RWL1 C9H8N3O2F3 247.18 43.73“ “ (43.94)“ * * 3.26 (3.24) 17.00 (17.14) RWL2 CgHTChNaOs 264.07 36.39 (36.39) 2.67 (2.74) 15.91 (15.87) RWL3 CgHgBrNsCb 274.08 35.06 (35.40) 2.94 (2.96) 15.33 (14.96) RWL4 CgH 7Br2N303 352.98 27.22 (27.48) 2 . 0 0 (2.09) 11.90 (11.78) RWL5 CqHioBrNsC^ + 0.2 (CH3OH) 304.11 35.59 (35.98) 3.51 (3.49) 13.53 (13.32) RWL6 CgHglNsO: 305.08 31.50 (31.52) 2.64 (2.65) 13.77 (13.75) RWL7 CgH7l2N303 446.97 21.50 (21.75) 1.58 (1.77) 9.40 (9.40) RWL8 C9H10N 4O3 +1.0 (H20 ) 204.19 48.65 (48.72) 4.54 (4.61) 25.21 (25.23) RWL9 C 10H14N4O2 222.25 54.04 (54.26) 6.35 (6.49) 25.21 (25.25) RWL10 Cs Ht Ns O; 285.17 33.69 (33.93) 2.47 (2.52) 24.56 (24.67) RWL11 C gH gN ^ 224.18 42.86 (43.04) 3.60 (3.75) 24.99 (24.70) 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IIL2. (continued) code Form ulas MW Elem ental analysis C H N RWL12 C9H 11N3O3 + 0.135 (CH3OH) 209.21 5138 (50.98) 5.45 (5.44) 19.68 (20.07) RWL13 C9H 11N3O3 209.21 51.67 (51.90) 530 (5.35) 20.09 (20.19) RWL14 C 10H 13N3O4 239.23 50.21 (50.00) 5.48 (5.59) 17.57 (17.56) RWL15 C9H 11N3O4 225.20 48.00 (48.04) 4.92 (5.00) 18.66 (18.55) RWL16 C 10H 13N3O5 255.23 47.06 (47.16) 5.13 (5.23) 16.46 (16.47) RWL17 C15H 15N3O3 285.30 63.15 (62.80) 5.30 (5.30) 14.73 (14.93) RWL18 C,4HI3N302 + 0.2 (CH3OH) 255.28 65.18 (64.91) 5.32 (5.36) 16.06 (16.22) RWL19 C8H9N3O4 211.18 45.50 (45.49) 4.30 (4.35) 19.90 (19.88) RWL20 C 10H 12N4O3 236.23 50.84 (50.51) 5.12 (5.40) 23.72 (23.39) RWL21 Q H 9N3O5 227.18 4230 (42.14) 3.99 (4.06) 18.50 (18.44) RWL23 C7H8N402 180.17 46.67 (46.39) 4.48 (4.79) 31.10 (30.98) RWL24 C8H 10N4O2 194.19 49.48 (49.08) 5.19 (5.26) 28.85 (28.80) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table m.2. (continued) code Form ulas MW Elem ental analysis C H N RWL26 0 * ^ 5 0 2 + 0.08 (CH3OH) + 0.1 (H2O) 183.17 38.94 (38.66) 5.12 (5.05) 3734 (37.73) RWL27 C^HglSLOsS + 1.0(H2O) 230.20 29.03 (29.05) 3.25 (3.26) 2237 (2239) RWL28 C 10H10N4O2 + 0.2 (CH3OH) 218.22 55.54 (54.31) 4.85 (4.95) 24.94 (25.18) RWL31 CnH 7a 2N304 316.10 41.80 (41.98) 2.23 (2.42) 13.29 (12.91) RWL32 CI4H 15N3 0 4 289.29 58.13 (56.69) 5.23 (5.48) 14.53 (14.13) RWL33 CI0H11N3O4 237.22 50.63 (50.39) 4.67 (4.73) 17.71 (17.46) RWL35 C,7H15N302 + 0.2 (CH3OH) 29333 68.92 (68.82) 5.31 (5.25) 14.02 (14.18) RWL36 C16H 12C1N30 2 + 0.3 (CH3OH) 313.74 60.55 (60.86) 4.11 (4.47) 12.99 (12.47) “ All analyses elemental were conducted at Galbraith Laboratories; c a U d Calculated values; e x p t Experimental values, all results were within ± 0.4% of calculated values with considering solvent of crystallization. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table D li. The Rf values of hydroxysemicarbazide, semicarbazide, hydroxyurea and Schiff bases of hydroxysemicarbazide. Ar-CH=NNHCONHOH code Ar Rr values in CHsOHrCHsCOzCzHs: CHCh (2 A 4 v/v/v) R -* RWL1 Q * - C P j v 0.72 -0.410 RWL2 0 - c H= Cl OH 0.70 -0.368 RWL3 Q - c t e OH Brs 0.72 -0.410 RWL4 M CH“ Br OH 0.68 -0.327 RWL5 y ^ c H = H3 CO OH 0.64 -0.250 RWL6 ^ ^ - C H = 1 'v 0.70 -0.368 RWL7 Q h c h = 1 OH 0.69 -0347 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table m.3. (continued) code Ar Rf values in C H jO H i C H jC O i C A : CHCb (2 3 :4 v A v A r ) RWL8 NC H= RWL9 (C RWL10 O /l OH RWL11 RWL12 RWL13 RWL14 Of t H= H,CO H ,C O - ^ ^ - C H = RWL15 RWL16 0.65 0.68 0.47 0.66 0.69 0.69 0.72 0.70 0.73 -0.269 -0.327 0.052 -0.288 -0.347 -0347 -0.410 -0.368 -0.432 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1 1 1 . 3 . (continued) code Ar Rr values in Cl^OHtCUjCOiCjHs: CHCb (2 3 :4 vAr/v) R . RWL17 P h - C H j O - ^ ^ - C H s 0.68 -0327 RWL18 0.73 -0.432 RWL19 h ° - 0 M 5 h = 0.62 -0.213 RWL20 : 0.53 -0.052 RWL21 I I X •6 o X I X 0.05 1.279 RWL23 ___ ,CH= a 0.40 0.176 RWL24 H ,C ^ C ^ C H = 0.46 0.070 RWL26 0.27 0.432 RWL27 i i X o 0.65 -0.269 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.3. (continued) code Ar Rf values in CUjOHsCUjCOiCiHs: CHCh (2;3:4 v/v/v) ,C H RWL28 0.58 -0.140 RWL31 CH RWL32 U J RWL33 RWL35 RWL36 Hydroxysemi- carbazide H2 NNHC(=0)NHOH Semi- carbazide H2NNHC(=0)NH Hydroxyurea H2NC(=0)NH0H 0.37 0.27 0.70 0.74 0.72 0.14 0.14 0.45 0 . 2 3 1 0.432 -0.368 -0.454 -0.410 0.788 0.788 0.087 a Rm = log(l/R f- l ) 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table III.4. Infrared absorption frequencies (in cm '1 ). Common absorptions compound vN-H vO-H vtp-H vC-H (aliphatic) vC=N vC=0 6N-H Characteristic absorptions RWL1 3430 (m) 3200 (s,b) 3120 (s.b) 2980 (m,b) 1660 (s) 1705 (s) 1530 (s) 750-650 (m, 1 ,3-sbr) 1360-1100 (s, CF,) RWL2 3360 (m) 3240 (m,b) 3120 (m,b) 2990 (m,b) 1665 (s) 1695 (s) 1540 (m) 870 (w, 1,2, 3 ,5-sbr) 800-700 (w, C-CI) RWL3 3360 (s) 3220 (m,b) 3110(m,b) 2980(m) 1660 (s) 1695 (s) 1525(m) 870-800 (w, 1 , 2, 5-sbr) RWL4 3350 (s) 3220 (m,b) 3120(m,b) 3000 (m,b) 1670 (s) 1700 (s) 1525 (s) 870 (w, 1 ,2 ,3 ,5-sbr) RWL5 3360 (m) 3220 (m,b) 3120 (m,b) 2980 (m,b) 1670 (s) 1695 (s) 1525 (s) 1105 (m, OCHj) RWL6 3380 (m) 3200 (m,b) 3110(m,b) 2980 (m,b) 1670 (s) 1700 (s) 1525 (s) 860-700 (w, 1,3-sbr) RWL7 3340 (s) 3190(s,b) 3050 (m,b) 2930 (w,b) 1675 (s, vC=N & vC=0 overlapping) 1540 (m) 860 (m, 1 ,2 ,3 ,5-sbr) 3420 (m, bonded NH) Table III.4. (continued) compound RWL8 RWL9 RWL10 RWLU RWL12 Common absorptions v N-H vO-H vtp-H vC-H vC=N vC=0 8N-H Characteristic (aliphatic) absorptions 3340 (m) 3250(w.b) 3120 (w,b) 2970 (m,b) 1675(s,vC=N& 1550 (m) 3570 (m, bonded OH) vC=0 overlapping) 2230 (s, C«N) 3330 (m) 3190 (m,b) 3090 (s.b) 2950 (m,b) 1680 (s) 1610 (s) 1520 (m) 1360 [s, N(CH2 )2 ] 3390 (m) 3260 (m.b) 3100 (s) 2870 (w.b) 1615 (s) 1640 (s) 1530 (s) 1530 (s. N02 . overlapping with 8N-H), 1345 (s, N02 ) 3390 (m) 3190 (m.b) 3120 (m.b) 2980 (m,b) 1660 (s) 1705 (s) 1525 (s) 1525 (s, NOj, overlapping with 8N-H), 1350 (s, N02 ) 3340 (m) 3280-3220 3U0(m,b) 2970 (m.b) 1670 (s) 1690 (s) 1525 (m) 2840 (w). 1275 (s), 1140 (m,b) (s), 1050 (m), OCH2 ; 805- 750 (w, 1,3-sbr) Table III.4. (continued) compound RWL13 RWL14 RWL15 RWL16 Common absorptions v N-H vO-H v < p -H vC-H vC=N vC=0 8N-H Characteristic absorptions (aliphatic) 3330 (m) 3200-3150 3080 (s,b) 2960 (s.b) 1615 (m) 1670 (s) 1500 (s) 2840 (m, OCH,), 1250 (s, (m,b) OCHj), 1130 (s, OCH,), 1035 (s, OCH,) 3420 (m) 3320 (s.b) 3120 (m.b) 2950 (m,b) 1675(s,vC=N& 1535 (s) 2840 (m, OCH,), 1225 (s. vC=0 overlapping) OCH,), 1140 (w, OCH,), 1045 (s, OCH,), 830-800 (m, 1,3,5- sbr) 3300 (s) 3230 (s,b) 3080 (s.b) 3000-2900 1630(s,vC=N& 1565 (s) 2850 (m, OCH,), 1250 (s, (m.b) vC=0 overlapping) OCH,), 1140 (s, OCH,), 1030 (m, OCH,) 3350 (m) 3230 (m,b) 3110 (w.b) 2990-2960 1610 (s) 1635 (s) 1575 (s) 2860 (m, OCH,). 1215 (s, (w.b) OCH,), 1115 (s, OCH,), 1050 (w. OCH,) Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table III.4. (continued) Common absorptions compound v N-H vO-H v < p -H vC-H vC=N vC=0 8N-H (aliphatic) RWL17 3330 (m) 3210 (m,b) 3110(m,b) 2970 (m,b) 1685(s,vC=N& 1505 (s) vC=0 overlapping) RWL18 3310 (m) 3230 (m,b) 3070 (w,b) 2940 (w,b) 1650(s,vC=N& 1555 (s) vC=0 overlapping) RWL19 3320 (s) 3280 (s.b) 3130 (s,b) 2920 (m.b) 1630 (s) 1655 (s) 1575 (s) RWL20 3340 (s) 3200-3160 3070 (m,b) 2920 (m,b) 1640 (s) 1680 (s) 1535 (s) (m,b) RWL21 3320 3280 (s,b) Not obvious, overlapping 1630 (s) 1665 (s) 1565 (s) (m,b) 3490 (s) with vN-H & vO-H Characteristic absorptions 2870 (w, b. -OCHr ), 1225 (m, -OCHj-), 1115 (w, - OCHr ), 1000 (m, -OCHr) 850-800 (m, 1 , 3 ,5-sbr) 1335 (s, -C(=0)CHj] Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table III.4. (continued) Common absorptions compound v N-H vO-H vtp-H vC-H vC=N vC=0 8N-H (aliphatic) Characteristic absorptions RWL23 3270 (m) 3150 (s,b) 3070 (s.b) 2860 (s.b) 1650 (s) 1685 (s) 1545 (s) RWL24 3390 3520 (s) 3200-3050 2980 (s) 1640 (s) 1675 (s) RWL26 (m,b) (s,b) 3240 (s,b, vN-H & 3100 (m,b) 2920 (w.b) 1605 (m) 1635 (s) vN-H overlapping) 1520 (s) 2820 (s, b, CH,) 1450 (s, CH,) 1560 (s) 2860 (w, b, CH,) RWL27 3310 (s) 3560 (m) 3110(m) 2970-2900 1680(s,vC=N& 1560 (m) 1520 (m, NO,). 1325 (s. (m,b) vC=0, overlapping) NO*), 730 (m, thiophene) RWL28 3400 (s) 3250 (s.b) 3120 (m.b) 2980-2940 1655 (s) 1690 (s) 1540 (s) 1625 (s), 1585 (w). 1450 (m.b) (s), indole ring Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1 1 1 .4 . (continued) Common absorptions Characteristic absorptions compound v N-H vO-H vtp-H vC-H vC=N vC=0 8N-H (aliphatic) RWL31 3300 (m,b) 3190(s,b) 3075 (s.b) 2920 (m,b) 1640 (s) 1670 (s) 1555 (s) 1150(s,C-O) RWL32 3260 (m) 3210 (m,b) 3080 (w.b) 2975 (m) 1620 (s) 1640 (s) 1545 (s) 2880 (w.b.CHj), 1355 (m, -C(CHj)2 ], 1135 (m, C-O) RWL33 3310 (s) 3180 (s.b) 3090 (s.b) 2980 (s) 1680 (s. vC=N& 1580 (s) 1125 (s, C-O-C) vC=0 overlapping) *070 (s, -CHj-O-) RWL35 3310 (m) 3220 (s.b) 3060 (m.b) 2980 (w) 1660 (s. vC=N& 1530 (s) 2890 (w. b. CH,) vC=0 overlapping) RWL36 3240 (s.b. vN-H & vO- 3080 (m.b) 2980 (m) 1660 (s) 1685 (s) 1555 (s) H overlapping) HSC 3340 (s,b) 3200 (s.b). 3080 1620 (s) 1675 (s) 1520 (s) (s.b), 2880 (s.b) Note: s = strong, m = medium, w = weak, b = broad, sbr = substituted benzene ring, HSC = hydroxysemicarbazide. HL2.(5). Nuclear magnetic resonance (NMR) spectra The chemical shifts, splitting patterns and the number of protons represented are summarized in Table IIL5. All the spectra contain the peaks of -O H . -NHb, - NHc protons from -CH =NNHcCONHbOH, group at 9.4,10.5, 8.5 ppm, respectively, and the d^-DMSO peaks (undeuterated) at 2.49 ppm. The methylene group (-CH =) of -CH=NNHCONHOH in each compound gives an absorption around 8.2 ppm as a singlet. The protons from the aromatic rings have absorptions ranging from 6 ppm to 8.5 ppm. The individual compound can be identified by the presence of certain peaks representing specific substituents on the aromatic rings. For example, phenolic OH gives an absorption at 12 ppm, -OCH 3 at 3.8 ppm, -N(CH3 > 2 at 2.9 ppm, -CH 2O - at 5.1 ppm, and -CO CH 3 at 2.0 ppm. The chemical shifts and the total number of carbon atoms shown by the l3 C NMR spectra are summarized in Table IIL6. All spectra contain the peaks of -C =0 from -CH=NNHCONHOH at 157 ppm, the peaks of -CH= at 140 ppm, and the tfc- DMSO peaks at 39.50 ppm. The chemical shifts of aromatic carbons give a wide range of absorptions (90-160 ppm) due to the different electromagnetic effects of the substituents on the aromatic ring systems. The individual carbon attached cm the aromatic rings can be identified by the presence of a specific peak. The carbons of methyl groups (-CH3) on different ring systems have different chemical shifts. For example, the carbon of -CH 3 on an anthryl ring has an absorption at 14.3 ppm, the carbon of -CH3 on a phenyl ring has an absorption at 23.9 ppm, while the carbon of - CH3 on an imidazolyl ring gives a peak at 8 . 8 ppm. The isopropyl group, - 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CH(CH3)2, has two peaks at 32.9 ppm [-CH(CH3)2] and 23.7 ppm [-CH(CH3)2], respectively (the italic and bold C indicates the carbon atom assigned). Methoxy groups (-OCH3) give absorptions at 55-56 ppm, -NHCOCH3 at 24.0 ppm, - NHCOCH3- at 168.5 ppm, -CH2O- at 693 ppm, -CN at 118.8 ppm, -N(CH3>2 at 39.8 ppm, and CF3 at 124.1 ppm. The purity and identity of an individual compound can be determined by counting the total number of carbon atoms detected by the 13C- NMR spectrum. Some compounds were further characterized by HH COSY NMR (RWL1 and RWL11) and CH correlated NMR spectroscopy (RWL 1, 6 , 7, 11, 12, 14). In general, the NMR spectra of the compounds provide no evidence for the presence of a mixture of Z/E isomers. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HL5. Proton chemical shifts, multiplicity and coupling constants of the newly synthesized compounds. A r-CH ^N N H cCO N H bO H , Code At Proton 5 (ppm) Multi. J (Hz) No. of protons RWL1 5 .__ . « a 9.48 s 1 b 10.46 s 1 Vr c 8.53 s 1 / s * F 3 C d 8.05 s 1 2 8 . 1 0 s 1 4 7.64 d 7.1 1 5 7.59 t 7.3 1 6 7.92 d 7.5 1 RWL2 C l a 9.44 s 1 b 1 1 . 1 0 s 1 c 8.87 s 1 /j 2\ d 8.32 s 1 a oh 2-OH 1 2 . 0 2 s 1 4 \ 7.57 d 2.5 1 6 J 7.49 d 2 . 6 1 RWL3 B r a 9.45 s 1 b 1 0 . 6 8 s 1 c 8 . 6 6 s 1 j d 8 . 2 2 s 1 O H 2-OH 10.78 s 1 3 6.81 d 9.0 1 4 730 dd 8.7,2.6 1 6 7.87 d 2.5 1 RWL4 B r a 9.41 s 1 b 11.16 s 1 c 8.91 s 1 2\ d 831 s 1 B r O H 2-OH 12.32 s 1 4 i 7.72 d 2.4 1 6 J 7.67 d 2 . 1 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table III.5 (continued) Code Ar Proton 5 (ppm) Mu ItL J (Hz) No. of protons RWL5 B r a 9.43 s 1 \»_■ b 10.66 s 1 4 V " c 8.65 s 1 /3 2\ d 8.24 s 1 c h 3 o o h 2-OH 10.25 s 1 3-OCH3 3.81 s 4 7.06 d 22 1 6 7.50 d 2.1 1 RWL6 • j----.• a 9.54 s 1 • r v - b 10.51 s 1 c 8.57 s 1 /* i d 7.88 s 1 2 8.19 t 1.6 1 4 7.68 ddd 7.7,1.7,1.0 1 5 7.17 t 7.8 1 6 7.62 dt 1 3 , 1.3 1 RWL7 i a 9.37 s 1 V— V * b 11.17 s 1 — c 8.93 s 1 /J 2\ d 8.24 s 1 1 O H 2-OH 12.66 s 1 4 7.96 d 1.6 1 6 7.70 d 2.0 1 RWL8 * __« a 9.60 s 1 NC~~^ — b 10.70 s 1 3 * c 8.65 s 1 d 7.98 s 1 2,6 I 7.81 d 8.3 2 3,5 J 7.89 d 8.4 2 RWL9 * _ _ • a 9.10 s 1 b 10.11 s 1 3 2 c 8.51 s 1 d 7.85 s 1 2,6 7.49 d 9.0 2 3,5 6.68 d 8.4 2 4-N(CH3 )2 2.92 s 6 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table in.5 (continued) Code At Proton 5 (ppm) Mul ti. J (Hz) No. of protons RWL10 O j N a 9.61 s 1 b 1134 s 1 *\ /i— c 8.74 s (overlap) 1 / j a\ 2-OH 8.49 s (overlap) 1 0 ^ O H d 8.49 s 1 4 8.75 d 2 3 1 6 J 8.68 d 2.8 1 RWL11 i__. « a 9.65 s 1 b 10.67 s 1 c 8.63 s 1 /» 2 O j N d 8.06 s 1 2 8.56 t 2.0 1 4 8.15 ddd 8.1,23,1.6 1 5 7.66 t 8.0 1 6 8.11 dt 7.9,1.1 1 RWL12 a 9.44 s 1 b 10.42 s 1 c 8.57 s 1 / 1 c h j O d 7.91 s 1 2 7.34 t 1.5 1 3 -OCH3 3.78 s 4 6.90 ddd 8.035,1.0 1 5 7.28 t 8.0 1 6 7.18 dt 7.4,1.1 1 RWL13 * _ _ . a 9.27 s 1 b 10.29 s 1 3 \ / 1 ' 2 c 838 s 1 3 * d 7.92 s 1 2,6 7.63 d 8.2 2 3,5 6.93 d 9.3 2 4 -OCH3 3.76 s 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table III.5 (continued) Code Ar Proton 5 Multi. J(H z) No. of (ppm) prot a 930 s b 10.29 s c 8.41 s d 8.26 s 2^-OCH3 T3.74 s 1.3.76 s 3 6.97 d 10.3 4 6.90 dd 9 .1 ,3 3 6 7.66 d 2.8 a 9.14 s b 10.60 s c 8.76 s d 8.29 s 2-OH 11.28 s 3 6.46 d 2.3 4-OCH3 3.73 s 5 6.44 dd 5.2, 2.3 6 7.33 d 8.4 a 9.03 s b 10.76 s c 8.79 s d 8.67 d 2-OH 12.27 s 6.07 d 2.3 5 j 6.09 d 2 2 4,6-OCH3 f 3.74 s 3 L 3.78 s 3 a 9.26 s 1 b 10.29 s 1 c 8.54 s 1 d 7.90 s 1 -CH2O- 5.12 s 2 2,6 7.63 d 8.7 2 3,5 7.02 d 8.8 2 2’,6’ 7.44 dd 7.1,1.4 2 3’,5’ 738 td 7.4,1.4 2 4 ’ 732 tt 7.4,1.4 1 RWL14 OCH, RWL15 RWL16 O CH , RWL17 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table M.5 (continued) Code Ar Proton 5 (ppm) Multi. J (Hz) No. of protons RWL18 a 9.42 s 1 s a $ « b 10.50 s 1 . / w v \ t y \ - c 8.64 s 1 3 2 3 2 d 8.03 s 1 23,5,6 1 7.68 d 8.2 4 2’,6’ J 7.79 d 83 2 3’3 ’ 7.45 td 7.7,1.8 2 4’ 736 tt 73,1.1 1 RWL19 •_ _ .• a 9.09 s 1 HOJ n L b 10.52 s 1 \ = 6 c 8.74 s 1 O H d 8.25 s 1 2-OH I 9.78 s 1 4-OH f 11.12 s 1 3 6.27 d 2.3 1 5 6.29 dd 8 3 , 2.0 1 6 7.21 d 8.6 1 RWL20 a 9.26 s 1 * _ _ • b 10.34 s 1 CH/JONH-2 ^ \ — c 8.56 s 1 d 7.90 s 1 2,6 1 7.59 d 8.7 2 3 3 J 7.60 d 9.0 2 -COCH3 2.04 s 3 4-NHCO- 10.04 s 1 RWL21 s • a 9.07 s 1 ho^ A - b 1038 s 1 c 8.76 s 1 / 3 2 \ H O O H d 8.04 s 1 5 6.67 d 8.7 1 6 634 d 8.6 1 2-OH ' 835 s 1 3-OH ► 9.29 s 1 4-O H , 11.16 s 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HI-5 (continued) Code Ar Proton 5 Multi. J (Hz) (ppm) a 9.55 s b 10.63 s c 8.69 s d 7.99 s 2 8.82 d 33 4 8.14 dt 8 .2 , 1 . 6 5 7.38 dd 83,4.9 6 8.50 dd 5.2, 1.8 a 9.52 s b 10.71 s c 8.69 s d 7.96 s n 7.88 d 7.8 5 J 7.16 d 7.8 4 7.66 t 7.7 6 -CH3 2.43 s a b c d 8.03 s 1-imi.NH 11.90 s 2 6.89 d 4 -CH3 1.42 s a 9.47 s b 10.94 s c 8.75 s d 8.22 s 3 7.39 d 43 4 8.05 d 4.0 No. of protons RWL23 : a RWL24 h3 c n RWL26 C H 3 ' K t H RWL27 XX 1 1 1 3 1 1 1 1 1 1 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HL5 (continued) Code At Proton 5 Muhi. J (Hz) (ppm) a 8.93 s b 10.12 s c 8.61 s d 8.24 s 1-iiuLNH 11.44 s 2 7.67 s 4 \ 7.41 d 8.0 7 J 8.25 d 8.1 5 1 7.10 td 7.4,13 6 J 7.17 td 7.8,13 a 9.52 s b 10.62 s c 9.09 s d 7.98 s 2 8.61 s 5 1 7.96 d 2.3 7 J 8.18 d 2.4 a 9.48 s b 10.53 s c 8.97 s d 8.05 s 2 8.58 d 1.0 5 7.90 d 2.0 6-CH(Cff3 )2 f 1.22 s I 1.24 s 6-C//(CH3 )2 3.04 sep 7.0 7 7.72 dd 8.7,2.0 8 7.62 d 8.7 a 9.32 s b 1038 s c 8.53 s d 7.83 s 23 4.24 s 5 7.27 d 1.7 7 7.11 d 8.5 8 6.84 d 8.5 No. of protons RWL28 RWL31 C l RWL32 (CHjJjHC RWL33 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.5 (continued) Code Ar Proton 5 Multi. (PPrc)________ J (Hz) No. of protons RWL35 RWL36 a b c d Ar-H IO-CH3 9.27 10.80 9.14 8.80 7.58 8.38 838 3.07 9.26 10.89 9.23 8.85 8.44 8.64 7.67 7.73 s s s s m m m s s s s s d d td td 8.3 8.7 1.2 7.5 1 1 1 1 4 2 2 3 1 1 1 1 2 2 2 2 Notes: s = singlet, d = doublet, t = triplet, sep = septet, m = multiplet. 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table m .6. I 3 C NMR chemical shifts of the Schiff bases of hydroxysemicarbazide. Ar-CbH=NNHC,ONHOH Code RWL1 At RWL2 RWL3 O H O H RWL4 B r Carbon 5 (ppm) a 156.96 b 139.96 1 135.90 2 122.65 3 129.69 4 125.17 5 129.47 6 130.68 -c f 3 124.07 a 156.70 b 141.90 1 1 121.87 3 J 121.44 2 151.45 4 129.20 5 122.92 6 127.22 a 156.92 b 139.30 1 110.58 155.48 3 118.26 4 132.51 5 122.29 6 129.52 a 156.70 b 142.45 1 121.91 2 152.92 3 111.22 4 134.54 5 11037 6 131.01 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.6 (continued) Code At carbon 8 (PPm) RWL5 RWL6 RWL8 O H RWL7 I a 156.91 b 13929 1 121.91 2 145.22 3 148.95 4 115.07 5 11025 6 121.04 -o c h 3 56.21 a 156.77 b 139.51 1 137.01 2 134.33 3 95.17 4 137.45 5 130.56 6 126.50 a 156.72 b 142.99 1 121.03 2 155.98 3 81.86 4 145.51 5 87.67 6 137.82 a 156.63 b 13933 1 139.29 2,6 127.24 3,5 132.46 4 110.94 -CN 118.82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HI.6 (continued) Code At Carbon 5 (ppm) a 157.42 b 142.74 1 122.28 2,6 127.90 3,5 111.75 4 150.97 -N(CH3 )2 39.81 a 156.63 b 140.19 1 123.54 2 156.49 3 1 137.01 5 J 137.85 4 121.26 6 127.64 a 156.70 b 139.00 1 136.69 2 120.56 3 148.30 4 \ 123.27 5 J 130.01 6 132.98 a 156.92 b 141.23 1 136.14 2 110.48 3 159.50 4 1 115.56 6 J 119.83 5 129.58 -OCH3 55.17 RWL9 (CHjJjN RWL10 om RWL11 O jN RWL12 CH.O Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.6 (continued) Code Ar Carbon 8 (ppm) a 15733 b 141.69 1 127.40 2,6 12836 3,5 114.12 4 16031 -OCH3 55.25 a 155.39 b 135.73 1 123.18 2 T 151.68 5 J 153.40 3 113.00 4 116.67 6 109.93 -OCH3 55.63,56.12 a 157.26 b 144.45 1 112.38 2 157.26 3 101.15 4 16135 5 106.09 6 130.31 -OCH3 55.20 a 157.28 b 142.59 1 100.94 2 159.03 3 93.80 4 162.29 5 90.25 6 160.24 -OCH3 55.32,55.84 RWL13 RWL14 O C H 3 RWL15 •_ _ . ch^ A O H RWL16 O CH , Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HI.6 (continued) Code Ar Carbon 5 (ppm) RWL17 a 157.14 i—! « __ k . b 141.36 \ = / r H 20 \ = A " l' 1 12735 1 2 * 1 * 2,6 127.67 2’,6’ > 127.82 3’,5’ 128.13 4’ J 12838 3,5 114.88 4 159.19 r 136.83 -c h 2 o - 69.26 RWL18 \__‘ * __6 a 157.11 \ b 141.21 \ 1 . 2 1,1’,4 133.92,139.50,140.69 3 2 9 * 2(6)3(5),2’(6’) 126.55,127.05,128.99 4’ 127.40 3’, 5’ 126.34 RWL19 a 157.43 b 145.00 > = f 1 111.16 \ O H 2 158.74 3 102.70 4 159.95 5 107.46 6 130.56 RWL20 * __• a 157.20 CHjCONH-^ b 14136 \ = / 1 12938 3 2 2,6 127.24 3,5 118.71 4 140.18 -NHCOCH3 23.97 -NHCO- 168.47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.6 (continued) Code At Carbon S (PPm> RWL21 * • H O O H b 1 2 4 3 5 6 } 157.50 146.42 111.53 146.94 148.00 132.79 107.52 120.42 RWL23 or N a b 2 6 3 4 5 } 157.08 138.64 149.88 148.34 130.73 133.39 123.84 RWL24 H 3 C N a 157.43 b 142.27 2,6 153.16,156.81 3,5 116.84,122.91 4 136.57 -CH3 23.88 RWL26 .CH, H a 157.98 b 134.28 2 131.50 4,5 129.69,130.85 -CH3 8.83 RWL27 XX O jN a b 2 5 3 4 156.27 135.31 150.01 147.51 127.84 130.26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.6 (continued) Code At Carbon 8 (ppm) RWL28 RWL31 C l RWL32 (CH j JjHC a 157.85 b 140.06 2 129.08 3,7 111.66 4 1 120.19 5 r 122.14 6 J 122.47 7 111.81 8 136.97 9 124.26 a 156.55 b 131.81 2 154.20 3 111.18 4 173.39 5 123.28 6 130.01 7 133.68 8 123.96 9 150.24 10 125.41 a 156.71 b 146.18 2 154.05 3 118.51 4 174.83 5,7 121.54,121.58 6 146.17 8 118.55 9 154.23 10 123.12 -CH(CH3 )2 23.67 -CH(CH3 > 2 32.92 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.6 (continued) Code At Carbon 8 (ppm) RWL33 i I : • ' o ' 5 a b 2 3 5,7,8 6 9,10 157.17 14132 64.02,64.25 114.82,117.14,120.48 128.24 143.57,144.55 RWL35 a b Ar-CH Ar-C -CH3 { { 157.43 142.39 124.95,125.17, 125.45,125.86 124.63,129.07, 129.19,132.15 14.25 RWL36 a b Ar-CH Ar-C { i 157.32 141.36 124.43,125.76, 127.02,127.47 126.60,127.79, 128.88, 129.70 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DL2.(6). Mass spectra (MS) The mass spectra of RWL5,12,18,26,28,35,36 are given in Appendix B. The fragmentation products of the compounds are shown in Table m .7 and the major fragmentation pathway is summarized in Figure m . 3. Six out of the seven compounds studied by mass spectrometry have the expected molecular weights. One compound (RWL26) does not show the expected mass, but it follows the same fragmentation pathway as the other six compounds, and forms explainable fragment ions. 8 + -oh 8 h A r —C=N—N — C— N—OH ----------------------------A r —0= N —N —C — N H H H H H H -CONH -N -NH + A r —CHj A r-C =N + A r-C = N -N H Figure D IJ. The major mass spectral fragmentation pathway of Schiff bases of hydroxysemicarbazide. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.7. Mass spectral fragmentation and fragment ions of RW L5,12,18,26, 28,35,36. code m/e fragment ions relative abundance, % RWL5 304 290 :=N— N— C-N-OH O O H 23.6 (molecular + ion) h3 c :=N-N-C-N-OH + O O H 48.1 288 245 •C=N O H 0 f + -N -N -C -N H + 40.2 Hjp C=N~NH O H O H jC 59.5 230 :=N O O H H ,C 100 (base peak) 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table ffl.7. (continued) code m/e fragment ions relative ____________ abundance, % RWL12 210 194 192 i - n- c- n— oh 40.9 (molecular ion) «3P i - c- n— oh 100 (base peak) 33.0 P Hfi 151 72.1 I LIU H O 136 y _ r % =N-NH 57.4 H O 121 24.3 H O Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HL 7. (continued) code m/e fragment ions relative abundance, % RWL18 256 4.8 (molecular ion 240 46.6 197 P " - 0 ~ S = "-NH’ 100 (base peak) 182 36.1 167 0 - 0 * 29.6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table EL 7. (continued) code m/e fragment ions relative ___________ ______ ____ abundance, % RWL26 C H j 167 DC. 187 k . A , I I + 5.7 (molecular N C = N — N— C— N— OH - H H H H ion?) (183)' DC O I I 'C = N — N— C— NH H H H / C H > ii i f ° 1 5 1 V ^ c = n - n - ! ! * 1 0 0 (base peak) H H H D C *25 ^ Z S = n - n h * 5 7 0 H H ,<*3 110 T l || 60.9 g c _ . H H 97 i[ 35.6 The expected mass. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HI. 7. (continued) code m/e fragment ions relative abundance, % RWL28 219 ^ ^ 7] —C=N— N — C~N— O H * ^ Jj H H H H 15.5 (molecular ion) 203 O p - r ' i r ^ * * H 15.9 160 Q ^ - ^ n- nh' N H 1 0 0 (base peak) 145 Q y ^ n ' N H 46.6 130 H 55.7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HI. 7. (continued) code m/e fragment ions relative abundance, % RWL35 294 N — N — C-N— O H H H S.l (molecular ion) N — N H 1 0 0 (base peak) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HI. 7. (continued) code m/e fragment ions relative abundance, % RWL36 314 N — N — C-N-O H 20.5 (molecular ion) 298 C-NH H 22.5 255 1 0 0 (base peak) 240 225 65.4 58.1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OL2.(7). M easurement o f partition coefficient (log P) and ionization constant (pKa) The concentrations and absorptions of RWL1 used in derivation of standard curves for RWL1 in phosphate buffers and in 1-octanol are summarized in Tables IIL8andIIL9. The standard curves are shown in Figure HL4. The log P and pKa values of RWL1 were measured for the first time. RWL1 was found to have a log P value of 1.64, and pKa values of 1.87 and 10.16. RWL1 is an amphoteric compound with a basic group (=N-, pKa = 1.87) and an acidic group (-OH, pKa = 10.16) (see Table m .10). The Hansch-Fujita substituent it constants of =NNHCONHOH and - CH=NNHCONHOH were derived from the measured log P value of RWL1 and the it values of other groups (method 1). They were further cross validated by method 2, in which the it values of related substituents were used (Hansch et al., 1995; Lien, 1994) (see Table 00.11). 111.2.(8). Stability study in phosphate buffers The preliminary stability study demonstrated that there was no change in absorbance in 72 hours at pH 3 and room temperature. At higher pH some changes in absorbance were observed. RWL1 remained 97.3% intact at pH7, and 78.7% at pHl 1. The half-lives of RWL1 were estimated to be 1851.1 hours and 208.0 hours in pH7 and pHl 1 phosphate buffers, respectively, using the first-order kinetic [log (a-x) = log a - kt/2.303] (see Table 10.12). The data indicate that RWL1 might remain relatively stable in the cell culture media in 3-day period of cell culture. In 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. addition, the hydrolysis of RWL1 was found to be pH-dependent, namely when pH increased, RWL1 became less stable. Therefore, use of low pH environment in formulation of structurally related compounds may improve the stability of the compounds. mj.(9). Reaction mechanism o f Schiff base formation Reaction of an aldehyde (or ketone) with a primary amine gives a Schiff base. The reaction mechanism of Schiff base formation is called the SN2 (substitution nucleophilic bimolecular) mechanism. First, the amine (nucleophile) reacts with the aldehyde to give an unstable addition compound called a carbinolamine (Step 1). The carbinolamine loses water by acid-catalyzed pathways (Step 2). Typically the dehydration of the carbinolamine is the rate-determining step of Schiff base formation. The acid concentration can not be too high because the amine is a basic compound. If the amine is protonated, Step 1 is pulled to the left, and carbinolamine formation can not occur. Therefore many Schiff base syntheses are best carried out at mildly acidic pH (see Steps 1 and 2). R1\ ^ ^ C = 0 + FLN-R2 H ^ R2—N-<p—O H R1 H H I I Step 1 carbinolamine R2—N -C -O -H H R1 carbinolamine R2—N=C + OH H ' R 1 Step 2 R2— N=CX + HzO R1 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table D D L S . The concentrations and UV absorbances of RWL1 used in derivation of its standard curves in phosphate buffer solutions saturated with 1-octanol (before partitioning). concentration of RWL1 (fig/ml) 2 4 1 0 2 0 pH2.2 wavelength (nm) 279.0 278.8 279.4 m 2 absorbance 0.03 0.075 0269 0.699 pH7.3 wavelength (nm) 279.5 280.1 279.8 278.6 absorbance 0.185 0.364 0.878 1.644 pH11.4 wavelength (nm) 289.0 282.1 282.0 280.9 absorbance 0 . 1 0 0 0257 0.630 1.312 Note: all values are the averages of two measurements. Table EEL9. The concentrations and UV absorbances of RWL1 used in derivation of its standard curve in 1-octanol saturated with the phosphate buffers (before partitioning). Concentration of RWL1 (pg/ml) 2 5 1 0 2 0 40 1-octanol wavelength (nm) 286.5 286.7 285.8 285.4 287.0 absorbance 0.164 0.323 0.791 1.484 1.906 Note: all values are the averages of two measurements. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.8 1.6 1.4 8 1.2 c s w ■ pH 7.3 buffer 6 0.8 I 0.6 0.4 0.2 A pH 11.4 buffer • 1-octanol 10 0 20 30 concentration (ug/ml) Figure IIL4. Standard curves used for determining the log P * values of RWL1 (pH 2.2 buffer y = 0.0376x - 0.0703, R2 = 0.9918; pH 73 buffer y = 0.0808x + 0.0398, R2 = 0.9990; pH 11.4 buffer y = 0.0667x - 0.0260, R2 = 0.9995; 1-octanol: y = 0.0749x - 0.0020, R2 = 0.9954). 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table QL10. The UV absorption maxima (Xun), absorption intensities (EI % > lc m ), apparent partition coefficient (log P*) and pKa values of RWL1. PH (nm) glfe.lcm P * Log P1 1-octanol buffer 1-octanol buffer pKa 2 . 2 0 286 274 801 269 24.34 ±0.46 1.39 ±0 . 0 1 1.87 ±0.03 (base) 7.26 286 280 791 878 43.33 ±1.49 1.64 ±0 .0 2 “ 11.4 285 283 794 630 1.07 ±0.23 0.03 ±0.09 10.16 ±0.05 (acid) Note: all values are the averages of three measurements. “ Because ionization is negligible (< 0.1 %) at pH 7.26, log P * = log P. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 111. 11. The substituent constants (ft) of =NNHCONHOH and - CH=NNHCONHOH derived from the measured log P of RWL1 and other ft constants. C F : The structure of RWL1 Method 1 Method 2 I t (NVOH = lo g P H2NCONHOH - I t -NHCONH2 = -1.80-(-1.30) = -0.50 It=NNHCONHOH It=NNHCONHOH = lo g P RWL1 - I t -C6H5 * I t -CF3 - I t -CH= = I t ^H=NNHCONH2 - It-C H = + I t (N>OH = 1.64-1.96 - 0.88 - 0.33 = -0.86 - 0.33+ (-0.50) = -1.53 = -1.69 I t -CHzNNHCONHOH I t -CH=NNHCONHOH = lo g P RWLI " I t -C6H5 - I t -CF3 = It -CH=NNHCONH2 + I t (N>OH = 1.64-1.96 -0.88 = -0.86+ (-0.50) = -1 . 2 0 = -1.36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table HL12. The concentrations and UV absorbances used in the stability study of RWL1. Concentration of RWLl(ng/mI) 2 4 1 0 2 0 A t— »(run) 279.0 278.8 279.4 279.2 Abs (0 h) 0.030 0.075 0.269 0.699 Ptt* Abs (72 h) 0.031 0.073 0.268 0.728 Remaining % 101.7 96.7 99.6 104.1 Average remaining % 100.5±3.1 n = 4 A m u (nm) 279.5 280.1 279.8 278.6 Abs (0 h) 0.185 0364 0.878 1.644 Pw/ Abs (72 h) 0.176 0356 0.856 1.623 Remaining % 95.4 97.7 97.5 98.8 Average remaining % 97.311.4 n =4 X w — t (run) 289.0 282.1 282.0 280.9 Abs (0 h) 0 . 1 0 0 0.257 0.630 1.312 Abs (72 h) 0 . 1 1 2 0.208 0.508 0.976 PHI 1 Remaining % 80.9 80.7 74.4 Average remaining % 78.713.7 n = 3 Note: all values are the averages of two measurements; Abs = absorbance. 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV. BIOLOGICAL EVALUATION IV .l. An Overview o f the Cytotoxicity Assay A number of biological assays have been developed to measure cytotoxicity of antitumor agents and viability/proliferation of cells. Most commonly used are the uptake of [^thymidine into cellular DNA and the cleavage of tetrazolium inner salts such as MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H- tetrazolium hydroxide}, and MTS [3-(4,5-dimethylthiazoI-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (see Figure IV.l for the structures). Although [^thym idine is still commonly used because of its sensitivity and reliability, the main disadvantage is the radiation hazard to laboratory personnel and the environment. Therefore, practical non-radioactive alternatives are preferred. The MTT-microculture tetrazolium assay was first introduced to the in vitro assessment of drug effects on cell growth by Alley et al. (1986). The MTT micro titration assay is based on the conversion of a tetrazolium salt into a colored formazan product by mitochondrial activity of viable cells. The amount of formazan produced by dehydrogenase enzymes is directly proportional to the number of living cells in the culture and can be measured colorimetrically (Promega Corp., 2000). However, there are several inherent disadvantages of this assay, including the inefficient metabolism of MTT by some human cell lines, and a laborious and error- 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prone solubilization of the formazan crystals by dimethyl sulfoxide (DMSO) prior to colorimetric measurement (Scudiero et al., 1988). Two alternative tetrazolium salts XTT and MTS, which form water-soluble formazans, have been developed (Scudiero et al., 1988; Barltrop et al., 1991). These methods require the addition of an intermediate electron acceptor, namely phenazine methosulfate (PMS) and phenazine ethosulfate (PES, see Figure IV.l for the structures), to accelerate their bioreduction and the production of their formazans. An important advantage of the recently marketed CellTiter 96® Aqueous MTS/PES One Solution Reagent (Product of Promega Corporation) over the previous tetrazolium salt MTT is the production of water-soluble formazan which eliminates the need for DMSO solubilization. Its advantages over XTT/PMS, which also yields a water-soluble formazan product, include the rapidity of color development, and the improved storage stability (Buttke et al., 1993). As compared with MTS/PMS (MTS and PMS solutions are supplied separately), the MTS/PES solution has an improved convenience because it combines a tetrazolium MTS and an electron-coupling reagent PES in one solution. The use of PES as the electron-coupling reagent allows storage of the mixture for several months (Riss and Moravec, 19%). Therefore, CellTiter 96® Aqueous MTS/PES One Solution (Promega Corp.) was chosen and used in the following cytotoxicity assay. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tetrazolium salt (light yellow) Formazan (dark brown) th N Viable cells V c h . CH, MTT Water-insoluble j r - \ ff N - n ^ ^ S O ^ H Viable cells 7 " SQ,H ► 0 - tr ?-I-N=N - 0 - N o ! C H j O ^ ^ N O , CHjO Water-soluble Viable cells NH C H . MTS Water-soluble C K , rx s c h . PES CH, PM S Figure IV.l. Structures of tetrazoliums (MTT, XTT and MTS), formazans produced by viable cells, and electron coupling reagents (PMS and PES). 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV 2 Experimental Procedures IV 1A . Inhibition o f leukem ia suspension cells IV.2~A.(1). Mouse leukem ia L1210 cells IV lA .(l)a . Cell culture The lymphocytic mouse leukemia cell line LI 210 was obtained from Cell Culture Core Facility of Cancer Research Laboratory at the University of Southern California (CRL-USC). They were maintained in a 5% CO2 humidified atmosphere at 37 °C in RPMI-1640 medium (CRL-USC) containing penicillin (170 lU/ml), streptomycin (170 pg/ml) (Mediatech-Cellgro®) and 10% fetal bovine serum (FBS) (Omega Scientific, Inc.). The cell culture was passaged every 3 days in an initial density of 4 x 10 4 cells/ml. IV.2~A.(l).b. Determination o f ICj* values IC jo (50% inhibitory concentration) of the newly synthesized compounds against L1210 cells were determined using previously described MTS/PES microculture tetrazolium assay (see section IV.l) in duplicate in 96-well microtiter plates (Costar Brand Tissue Culture 96-well plate) except for RWL14 and RWL19. The experimental procedures were the following: 110 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. Each test compound and hydroxyurea (positive control) was initially solubilized in 100% DMSO and further diluted into RPMI-1640 medium to obtain 6 gradual concentrations ranging from 10' 3 to 1 0 ~ * M. The final concentration of DMSO in the cell cultures was less than 1% (v/v) (< 0.25% except the first concentration) to avoid the DMSO cytotoxicity. 2. The dilutions of the test compounds were added to column 2 (the highest concentration) to column 7 (the lowest concentration) and column 9 to column 1 1 in triplicate (see Figure IV.2 for the layout). 3. One hundred pi of RPMI-1640 medium was dispensed to columns 1 and 12 which were used for the blanks to record the background absorbance of the mixture of 100 pi of RPMI-1640 medium and 20 pi of MTS/PES colorimetric reagent (added in Step 7), and plate bottom itself. Column 8 containing 50 pi of the cell suspension and the same concentration of DMSO in 50 pi of RMPI-1640 medium was used for the negative control to account for any solvent effects on cell growth. Hydroxyurea (Aldrich Chemical Company) was used as a positive control. 4. Rapidly growing L1210 cells were harvested and the cell number was counted by using a Coulter Counter (Coulter Electronics, Inc.). After determining the cell viability by trypan blue exclusion test (0.4% Trypan Blue Stain, Sigma Chemical Company), the cells were resuspended to a final concentration of 1 x 1 0 s cells/ml in RPMI-1640 medium. Ill Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. The cell suspension (SO pi) was dispensed into all wells of the plate, except columns 1 and 12. The final volume in each well was 100 pi total. 6 . The plate was incubated for 65 hours at 37 °C in a 5% CO2 humidified atmosphere. 7. Twenty pl/well of MTS/PES CellTiter 96® Aqueous One Solution Reagent was added to each well. 8 . The plate was incubated for 3 hours at 37°C in a 5% CO2 humidified atmosphere. 9. The absorbance at 490nm was recorded using a Microplate Reader (Dynatech Laboratories, Revelation version 3.04,1996). The corrected absorbances at 490nm (Y axis) versus concentrations of the test compounds (X axis) was plotted as dose-response curves. The inhibition of L1210 cell growth at different concentrations was expressed as a percent of growth of the negative control. After the % inhibitions were converted into probits, IC50 values were calculated from the regression equations by substituting a probit value of 5 to represent 50% inhibition. IC50 values of RWL14 and RWL19 could not be titrated by MTS/PES tetrazolium assay, due to the risen absorbances at 490nm at high concentrations (500 pM to 1000 pM, see Figure IV J ). This risen absorbance resulted in a non-sigmoid dose response curve. Consequently, the probit transformed dose response curves were not straight lines. 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The following experiments were alternatively performed to determinate the IC50 values of RWL 14 and RWL19. RWL 14 and RWL19 were initially solubilized in DMSO, and diluted into RMPI 1640 medium containing penicillin (170 lU/ml), streptomycin (170 pg/ml) (Mediatech-Cellgro®) and 10% fetal bovine serum (FBS) (Omega Scientific, Inc.). The final concentration of DMSO in the cell cultures were less than 1% . Six dilutions of different concentrations (1 ml) and cell suspension (1 ml) containing a cell density of 7 x 10 4 cells/ml were pipetted into one well of a 24- well plate in duplicate. Negative controls containing only DMSO at identical dilutions were also prepared in the same manner. The plate was incubated in a 5% CO2 humidified atmosphere at 37°C for 65 hours. After 65 hours, cells in each well was diluted 10 folds with phosphate buffer saline (PBS) and counted using a Coulter Counter. The counts were corrected for the dilution and the Coulter Counter, and then the IC50 value was determined by probit transformation. 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 A b c c c c c c n c c c b B b c c c c c c n c c c b C b c c c c c c n c c c b D b c c c c c c n c c c b E b c c c c c c n c c c b F b c c c c c c n c c c b a b c c c c c c n c c c b H b c c c c c c n c c c b Figure IV.2, Schematic layout of a 96-well plate used in the IC jo determination [b = blank (containing 100 pi of RPMI-1640 medium and 20 pi of MTS/PES); n = negative control (containing S O pi of cell suspension + DMSO in 50 pi of RPMI-1640 medium + 20 pi of MTS/PES); c = wells with drug solutions (containing 50 pi of cell suspension + DMSO and test compound in 50 pi of RPMI-1640 medium + 20 pi of MTS/PES)]. Absorbance (490nm) Absorbance (490nm) 1.6 RWL14 12 0.8 0.4 02 0.0 -6.0 -3.0 -2.5 - 2.0 -5.5 -4.0 Log concentration (M ) 2.0 1.8 1.6 1.4 12 1.0 0.8 0.6 0.4 02 0.0 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 Log concentration (M ) RWL19 Figure IV3. Dose response curves of RWL14 and RWL19 against L1210 cells obtained by the MTS/PES colorimetric assay. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV 2 A .(2 ). Hum an leukemia C C R F-C EM cells IV.2~A.(2).a. Cell culture The lymphoblastic human leukemia cell line CCRF-CEM was obtained from the Cell Culture Core Facility of the Norris Comprehensive Cancer Center at the University of Southern California (CRL-USC). They were maintained in a 5% COi humidified atmosphere at 37 °C in RPMI-1640 medium (CRL-USC) containing penicillin (170 IU/ml), streptomycin (170 pg/ml) (Mediatech-Cellgro®) and 10% fetal bovine serum (FBS) (Omega Scientific, Inc.). The cell culture was passaged every 3 days in an initial density of 1 x 105 cells/ml. IV lA .(2).b . Determination of IQ * values Of all the compounds tested, RWL-2,4, 7, 21, 35 and 36 are the most potent with micromolar IC5 0 values against L1210 cells. According to the chemical structures, the six compounds can be divided into three groups. RWL-2, 4 and 7 containing a 2-OH-3,5-dihalgen-substituted aromatic ring consist of the first group. RWL21 with a 2,3,4-OH-substituted aromatic ring is the second category. RWL-35 and 36 belong to the third group because both of them have substituted anthracene moieties. Three compounds (RWL4, RWL21 and RW135) representing each of the three groups were further tested against human leukemia CCRF-CEM cells. The 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. experimental procedures were the same as those used for L1210 cells except a different initial cell density of 1.2 xlO5 cells/ml used for CCRF-CEM cells due to slow growth rate. W2&. Inhibition o f solid tumor cells (B id, CHO, HT29 and ZR75) IV ^ B .(l). Cell culture The B16 (mouse melanoma), CHO (Chinese Hamster Ovary Carcinoma), HT29 (human colon adenocarcinoma) and ZR7S (human breast carcinoma) adherent cells were obtained from Cell Culture Core Facility of Cancer Research Laboratory at the University of Southern California (CRL-USC). They were maintained in a 5% CO2 humidified atmosphere at 37 °C in DMEM medium (CRL-USC) containing penicillin (170 IU/ml), streptomycin (170 fig/ml) (Mediatech-Cellgro®) and 10% fetal bovine serum (FBS) (Omega Scientific, Inc.). The cell cultures were passaged by trypsinization every 7 days or when confluence was reached. IV.2JL(2). Measurement of % inhibition at 50 jiM concentration Eighteen compounds ranging from most, middle, and least active in inhibition of L1210 cells were further evaluated against 4 different adherent cell lines, namely B16 (mouse melanoma), CHO (Chinese Hamster Ovary Carcinoma), 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HT29 (human colon adenocarcinoma) and ZR75 (human breast carcinoma) cells. The experimental procedures were as the following: 1. Trypsinize a subconfluent monolayer culture and collect cells in DMEM medium containing serum. 2. Resuspend cells in growth medium and count cells. 3. Dilute cells to 1 - 8 x 10 4 cells/ml (1 x 10 4 cells/ml for B16 and HT29 cells, 8.0 x 1 0 4 cells/ml for CHO cells, 1 . 2 x 10 4 cells/ml for ZR75 cells). 4. Add 50 pi of cell suspension into each well of the central 10 columns of a flat- bottomed 96-well plate, starting with column 2 and ending with column 11. Add 100 ill of DMEM medium to the eight wells in columns 1 and 12. The two columns will be used to blank the plate reader. 5. Put plates in a 5% C02 humidified atmosphere at 37°C for 2 days such that cells are in the exponential phase of growth at the time of drug addition. 6 . Dissolve the compounds in DMSO and dilute into DMEM medium to give a concentration of 50 pM for each test drug. The final concentration of DMSO in cell cultures is less than 0.5%. 7. Add 50 pi of the compound dilutions to columns 2 to 10. Only four wells are needed for each compound such that rows A-D can be used for one compound and rows E-H for a second compound. Feed the cells in the eight well in column 11 with 50 pi of fresh DMEM medium containing the same concentration of DMSO as negative control. 8 . Incubate in a 5% CO2 humidified atmosphere at 37°C for 65 hours. 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9. Add 20 |il of MTS/PES reagent into each well of the plate and incubate for 3 hours. 10. Record the optical density at 490nm using a Microplate Reader. IVJLB.(3). Determination of IC5* values The IC50 values of the most active compound RWL35 and reference compound hydroxyurea were determined utilizing a similar procedure as described in section IV.2.B.(2). 1. Trypsinize a subconfluent monolayer culture and collect cells in DMEM medium containing serum. 2. Resuspend cells in growth medium and count cells. 3. Dilute cells to 1 - 8 x 10 4 cells/ml (1 x 10 4 cells/ml for B16 and HT29 cells, 8.0 x 10 4 cells/ml for CHO cells, 1.2 x 104 cells/ml for ZR75 cells). 4. Add 50 |il of cell suspension into each well of the central 10 columns of a flat- bottomed 96-well plate, starting with column 2 and ending with column 11. Add 100 |il of DMEM medium to the eight wells in columns 1 and 12. The two columns will be used to blank the plate reader. 5. Put plates in a 5% CO2 humidified atmosphere at 37°C for 2 days such that cells are in the exponential phase of growth at the time of drug addition. 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 . Dissolve the compounds in DMSO and dilute into DMEM medium to give 6 gradual concentrations. The final concentration of DMSO in cell cultures is less than 1%. 7. Add SO ill of the dilutions to columns 2 to 10. Only two to four wells are needed for each concentration such that rows A-C can be used for one concentration, rows D-E for a second concentration and F-H for a third concentration. Feed the cells in the eight well in column 8 with 50 pi of fresh DMEM medium containing the same concentration of DMSO as negative control (see Figure IV-2 for the layout). 8 . Incubate in a 5% CO2 humidified atmosphere at 37°C for 65 hours. 9. Add 20 pi of MTS/PES reagent into each well of the plate and incubate for 3 hours. 10. Record the optical density at 490nm using a Microplate Reader. Different procedures were used in the IC50 determinations of the test compounds for suspension (L1210 and CCRF-CEM) and adherent (B16, CHO, HT29 and ZR75) cell cultures (see Figure IV.4). In the case of suspension cell culture, 50 pi of drug solution and 50 pi of cell suspension were added into one well of a microtitration plate simultaneously, while for adherent cell cultures 50 pi of drug solution was dispensed into the plate after 48-hour incubation of the adherent cells. The total of incubation time is 5 days for adherent cell cultures, and only 3 days for suspension cell cultures. 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Adherent cultures (B16, CHO.HT29 and ZR7S cells) suspension cultures (LI210 and CCRF-CEM cells) incubate for 3 hours incubate for 63 hours add SO pi of drug solution preincubate for 48 hours add 30 pi of cell suspension drug solutions (10 -1 0 M) add 20 pi of MTS/PES reagent add S O pi of drug solution + 30 pi of cell suspension record the absorbance at 490nm using 96-well plate ELISA reader Figure IV.4. The different procedures used in the IC $o determinations of the Schiff bases of hydroxysemicarbazide for suspension (L1210 and CCRF-CEM leukemia cell lines) and adherent (B16, CHO, HT29 and ZR73 solid tumor cell lines) cell cultures. IV J.C Selective toxicity against tumor cells vs. 3T3 m ouse embryo fibroblasts The 3T3 ceils were derived from Swiss mouse embryo fibroblasts. To evaluate the selectivity of the target compounds against cancer cells and non-cancer cells, the 3T3 fibroblasts were chosen as an in vitro model in this study. Since the 3T3 fibroblasts were adherent cells in nature, the experimental procedures used were the same as those for the four solid tumor cell lines (B16, CHO, HT29 and ZR75) except a different initial cell density of 2 x 1 0 4. IV.2J). Inhibition o f hydroxyurea-resistant KB cells Human oropharyngeal carcinoma KB cells (American Type Culture Collection) were cultured with 5% CO2 at 37°C in RPMI1640 supplemented with 10% FBS. Hydroxyurea (HU) and gemcitabine resistant clones were sequentially selected in a stepwise manner in the presence of HU (1 mM) and gemcitabine ( 8 fxM ), respectively. The resistant cells were maintained in a drug-free medium for 3 days prior to designative study. The IC50 values of RWL-2 and 35 against the wild type (KB-W), HU- resistant (KB-HUR) and gemcitabine-resistant (KB-GR) KB tumor cells were determined by using methylene blue assay in collaboration with Dr. Yen Yun’ group of the City of Hope National Medical Center. The cells in logarithmic growth were plated at a density of 5 x 104 cells/ml in a 24-well plate. After 72-hour incubation at indicated drug concentrations, methylene blue assays were subsequently performed, and the IC50 values were determined by interpolation of plotted data to show the concentration of 50% cell death. 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV J . Results and Discussion IV J X Inhibition o f leukemia suspension cell cultures IV JA .(1). Mouse leukemia L1210 cells The ICso values of 31 new Schiff bases of hydroxysemicarbazide and three reference compounds, hydroxyurea, semicarbazide and hydroxysemicarbazide, were first determined against L1210 cells. The drug concentrations, % inhibition, probit values, squared correlation coefficients (R2 ) and the ICso values obtained from each individual determination are summarized in Table IV.1. The average ICso values of multiple determinations are given in the last column of Table IV .l. The dose response curves and probit transformed dose response curves of the 31 Schiff bases tested, and three reference compounds are shown in Figures IV.5.(1) to IV,5.(33). The ICso values of the compounds tested against L1210 cells range from 2.7 pM to 944.1 pM. Six most promising compounds with the ICso values in a micromolar range are RWL-2, 4, 7, 21, 35, and 36. They are 1 1 to 30-fold more potent than hydroxyurea. It is interesting to note that RWL27 with a nitro- substituted thienyl group has an ICso value of 10.6 pM. RWL-10, 20, 24, and 26 with ICso values greater than 800 pM are the least active of the 31 compounds. The ranking order of the IC5 0 values of semicarbazide (H2NNHCONH2, ICso > 2192.2 pM), hydroxysemicarbazide (H2NNHCONHOH, IC5 0 = 281.6 pM), and hydroxyurea (H2NCONHOH, IC5 0 = 82.0 pM) reveals that (N)-OH function is essential for the antitumor activities observed. 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.l. Inhibition of mouse leukemia L1210 cells by the Schiff bases of hydroxysemicarbazide, and reference compounds (hydroxyurea, semicarbazide, and hydroxysemicarbazide). Compound Cone. * (xlO^M) % r Probit ICso (xlO-6 M) IC50 ± s-d.d (xlCT 6 M) Hydroxy urea 2472.00 99.99 8.719 1 49430 93.60 6.522 98.86 50.50 5.013 19.77 12.40 3.845 3.95 0 . 1 0 1.910 2 2483.00 99.99 8.719 496.50 94.80 6.626 99.30 66.60 5.429 19.86 8 . 2 0 3.608 3.97 0 . 1 0 1.910 3 2511.00 99.99 8.719 502.10 95.10 6.655 100.40 37.50 4.681 20.08 7.50 3.561 4.01 0 . 1 0 1.910 4 2511.00 99.99 8.719 502.10 95.20 6.665 100.40 47.40 4.935 20.08 15.60 3.989 4.01 0 . 1 0 1.910 219230 46.40 1 876.88 NT 350.75 NI 2 2192.20 38.20 876.88 NI 350.75 NI 0.990 81.04 82.0 ± 6 . 0 n = 4 0.995 77.31 0.989 90.73 0.988 78.93 Semicar bazide >2192.20 >2192.20 >2192.2 n = 2 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.l. (continued) Compound Cone. (xlO-6 M) % I Probit R2 ICso (xlO*6 M) ICso i s.d. (xlO- 6 M) Hydroxy- semicar- bazide 495.20 61.50 5.292 0.983 304.66 281.6±21.4 1 198.10 43.20 4.829 n = 3 79.23 10.80 3.763 31.69 0.80 2.591 12.68 0.10 1.910 1.27 NI 2 1070.60 59.20 0.997 277.71 428.24 85.00 6.036 171.30 11.30 3.789 68.52 0.10 1.910 27.41 NI 3 1070.60 61.50 1.000 262.34 428.24 86.80 6.117 171.30 16.80 4.038 68.52 0.10 1.910 27.41 NI RWL1 146.05 87.30 6.141 0.920 41.39 39.5 ± 2.7 1 43.82 82.10 5.919 n = 2 13.14 4.00 3.249 3.94 0.10 1.910 1.18 NI 2 146.05 87.10 6.131 0.937 37.63 43.82 79.90 5.838 13.14 7.50 3.561 3.94 0.40 2.348 1.18 0.10 1.910 RWL2 54.72 99.10 7.366 0.964 6.11 6.510.6 1 16.42 96.40 6.799 n = 2 4.93 21.40 4.207 1.48 6.00 3.445 0.44 0.10 1.910 0.04 NI 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.l. (continued) Compound Cone. (xlO- 6 M) % I Probit R2 IC50 (xlO"6 M) ICso ± s .< L (xlO- 6 M) RWL2 (contd.) 54.72 98.30 7.125 0.920 6.97 2 16.42 97.20 6.911 4.93 10.00 3.718 1.48 4.60 3.315 0.44 0.10 1.910 0.04 NI RWL3 147.04 95.90 6.739 0.953 9.13 10.1 ± 1.0 1 44.11 85.90 6.076 n = 3 13.23 84.60 6.019 1.19 2.60 3.057 0.12 0.10 1.910 2 109.10 91.20 6.353 0.947 11.15 43.64 92.30 6.426 17.46 79.60 5.827 6.98 15.90 4.001 2.79 5.50 3.402 0.28 0.10 1.910 3 109.10 93.40 6.506 0.936 10.02 43.64 95.50 6.695 17.46 81.40 5.893 6.98 13.60 3.902 2.79 7.40 3.553 0.28 0.10 1.910 RWL4 54.96 99.54 7.605 0.956 6.02 7.2 ± 1.7 1 16.49 94.20 6.572 n = 2 4.95 15.60 3.989 1.48 10.80 3.763 0.45 0.10 1.910 0.04 NI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.l. (continued) Compound Cone. (xlO ^M ) % I Probit R2 IC50 (xlO*6 M) IC50 ± s.d. (xlC 6 M) RWL4 (contd.) 2 54.96 98.81 7.260 0.932 8.39 16.49 94.30 6.581 4.95 8.40 3.621 1.48 0.40 2348 0.45 0 . 1 0 1.910 0.04 NI RWL5 115.42 95.80 6.728 0.880 33.66 37.7 ± 5.7 1 34.63 26.70 4.378 a I I K l 10.39 5.10 3.365 3.12 6.30 3.470 0.94 0 . 1 0 1.910 0.09 NI 2 115.42 95.20 6.665 0.967 41.77 34.63 28.20 4.420 10.39 1.60 2.856 3.12 0 . 1 0 1.910 0.94 NI 0.09 NI RWL6 256.00 80.00 5.842 0.974 82.91 80.4 ± 3.5 1 128.00 73.90 5.640 n = 2 64.00 43.40 4.834 32.00 4.40 3.294 16.00 3.80 3.226 1.60 0 . 1 0 1.910 2 256.00 78.20 5.779 0.981 77.90 128.00 76.10 5.710 64.00 48.80 4.970 32.00 17.50 4.065 16.00 8.30 3.615 1.60 0 . 1 0 1.910 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.l. (continued) Compound Cone. (xlO* M) % I Probit R2 IC50 (xlO^M ) ICso ± s.d. (xlO*6 M) RWL7 108.28 96.80 0.968 5.73 4.7 ±1.5 1 32.48 99.99 8.719 n = 2 9.75 76.50 5.723 2.92 2.90 3.104 0.88 0.10 1.910 0.09 2 108.28 97.40 0.868 3.64 32.48 99.99 8.719 9.75 73.00 5.613 2.92 13.20 3.883 0.88 12.60 3.855 0.09 0.10 1.910 RWL8 465.78 85.60 6.063 0.988 209.16 208.5 ± 1.0 1 139.73 23.40 4.274 n = 2 41.92 4.00 3.249 12.58 0.10 1.910 3.77 NI 0.38 NI 2 465.78 84.20 6.003 0.970 207.76 139.73 21.70 4.218 41.92 7.80 3.581 12.58 0.30 2.252 3.77 7.60 0.38 0.10 RWL9 1013.00 76.00 5.706 0.983 415.20 446.3 ±44.0 1 405.20 59.00 5.228 n = 2 162.08 17.00 4.046 64.83 3.20 3.148 25.93 0.10 1.910 2 1013.00 77.80 5.766 0.962 477.39 405.20 53.60 5.090 162.08 10.10 3.724 64.83 0.10 1.910 25.93 NI 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.l. (continued) Compound Cone. (xlO-6 M) % I Probit R2 ICso (xlO-6 M) IC50 ± s.d. (xlO* M) RWL10 1037.60 57.20 5.182 0.932 862.02 820.6 ±73.1 1 518.80 24.60 4313 1 1 c 259.40 0 . 1 0 1.910 129.70 NI 64.85 NI 2 1189.10 77.60 0.924 902.38 356.73 1.70 107.02 0 . 1 0 32.11 NI 9.63 NI 3 1060.10 76.60 5.726 0.967 756.63 530.10 28.30 4.426 265.00 0 . 1 0 1.910 132.50 NI 66.26 NI 33.13 NI 4 1060.10 73.90 5.640 0.949 761.27 530.10 31.80 4.527 265.00 0 . 1 0 1.910 132.50 NI 66.26 NI 33.13 NI RWL11 432.24 54.90 5.123 0.994 337.02 399.3 ± 88.1 1 216.10 44.60 4.864 n = 2 108.10 2 1 . 0 0 4.194 54.03 11.60 3.805 27.02 4.10 3.261 2.70 0 . 1 0 1.910 2 432.24 45.50 4.887 0.990 461.65 216.10 37.50 4.681 108.10 16.60 4.030 54.03 9.60 3.695 27.02 6 . 2 0 3.462 2.70 0 . 1 0 1.910 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. % I Probit R2 ICso IC50 ± s.d. (xlff* M) (xlO- 6 M) (xlO- 6 M) RWL12 1020.00 90.10 6.287 0.990 369.85 368.8 ± 1.5 1 408.00 59.70 5.246 n = 2 163.20 9.90 3.713 65.28 1.70 2.880 26.11 0.10 1.910 2 1020.00 90.10 6.287 0.991 367.77 408.00 58.40 5.212 163.20 14.80 3.955 65.28 0.80 2.591 26.11 0.10 1.910 RWL13 995.70 84.80 6.028 0.983 325.95 316.7 ±13.1 1 298.71 47.80 4.945 n = 2 149.36 23.50 4.278 74.68 9.40 3.684 22.40 0.10 1.910 6.72 NI 2.02 NI 2 995.70 86.30 6.094 0.962 307.50 298.71 42.00 4.798 14936 33.50 4.574 74.68 11.60 3.805 22.40 0.10 1.910 6.72 NI 2.02 NI RWL14 652.51 96.90 6.866 0.931 33.58 30.1 ±5.0 1 195.75 96.10 6.762 n = 2 58.73 85.90 6.076 17.62 9.50 3.689 5.29 9.00 3.659 0.53 0.10 1.910 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1 . (continued) Compound Cone. (xlO*6 M) % I Probit R2 ICso (xlO- 6 M) ICso ± s.d. (xlO*M) RWL14 (contd.) 652.51 97.30 6.927 0.943 26.56 195.75 96.60 6.825 58.73 88.90 6.221 17.62 18.90 4.118 5.29 16.70 4.034 0.53 0.10 1.910 RWL15 248.70 85.20 6.045 0.980 61.93 60.2 ± 2.4 1 74.61 68.20 5.473 n = 2 22.38 21.70 4.218 6.71 1.00 2.674 2.01 0.10 1.910 0.60 NI 2 248.70 83.00 5.954 0.975 58.56 74.61 68.10 5.471 22.38 14.90 3.959 6.71 9.40 3.684 2.01 0.50 2.424 0.60 0.10 1.910 RWL16 993.60 50.50 0.942 92.02 91.4 ±0.9 1 298.08 92.10 6.412 n = 2 149.04 60.50 5.266 74.52 23.20 4.268 22.36 10.70 3.757 6.71 4.30 3.283 2.01 0.10 1.910 2 993.60 51.80 0.998 90.73 298.08 92.30 6.426 149.04 74.60 5.662 74.52 35.10 4.617 22.36 5.50 3.402 6.71 0.10 1.910 2.01 NI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. % I Probit R5 ICso ICso ± s.d. (xlO-6 M) (xlO-6 M) (xlO* M) RWL17 98.18 73.30 5.622 0.904 55.13 48.1 ±9.9 1 29.45 20.10 4.162 n = 2 8.84 8.70 3.641 2.65 7.70 3.575 0.80 0.10 1.910 0.24 NI 2 98.18 77.10 5.742 0.938 41.12 29.45 34.30 4.596 8.84 25.70 4.347 2.65 0.90 2.634 0.80 0.24 0.10 1.910 RWL18 96.25 84.80 6.028 0.970 37.09 42.7 ± 7.9 1 28.88 31.70 4.524 n = 2 8.66 12.50 3.850 2.60 0.10 1.910 0.78 NI 0.23 NI 2 96.25 81.20 5.885 0.949 48.24 28.88 28.60 4.435 8.66 8.40 3.621 2.60 2.00 2.946 0.78 0.23 0.10 1.910 RWL19 1055.50 96.70 6.838 0.957 139.47 130.8 ± 12.3 1 316.65 91.40 6.366 n = 2 95.00 20.10 4.162 28.50 10.20 3.730 8.55 0.10 1.910 0.86 NI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. (xlO^M ) % I Probit R1 ICso (xlO^M ) IC50 ± s.d. (xlO^M ) RWL19 1055.50 96.80 6.852 0 . 8 8 8 122.03 (contd.) 2 316.65 92.50 6.440 95.00 16.90 4.042 28.50 5.10 3.365 8.55 3.80 3.226 0 . 8 6 0 . 1 0 1.910 RWL20 1031.20 60.10 5.256 0.947 999.73 944.1 ±78.7 1 515.60 7.60 3.568 n = 2 257.80 3.80 3.226 128.90 0 . 1 0 1.910 64.45 NI 32.23 NI 2 1031.20 62.90 5.329 0.963 888.47 515.60 12.50 3.850 257.80 5.60 3.411 128.90 0 . 1 0 1.910 64.45 NI 32.23 NI RWL21 51.59 99.45 7.543 0.917 2.32 2.72 ±0.6 1 10.32 99.45 7.543 n =4 2.06 16.80 4.038 0.41 0.50 3.402 0.08 0 . 1 0 1.910 2 51.59 99.63 0.927 2.18 10.32 99.99 8.719 2.06 11.80 3.815 0.41 0 . 2 0 2 . 1 2 2 0.08 0 . 1 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. % I Probit R5 ICso ICso ± s.d. (xlO- 6 M) (xlO- 6 M) (xlO*6 M) RWL21 55.02 96.90 0.963 2.96 (contd.) 3 22.01 100.40 8.80 99.99 8.719 3.52 45.70 4.892 1.41 2.30 3.005 0.14 0.10 4 55.02 101.40 0.987 3.44 22.01 101.00 8.80 99.99 8.719 3.52 36.00 4.642 1.41 0.10 1.910 0.14 NI RWL23 1001.00 81.70 5.904 0.970 557.89 607.0 ±69.5 1 500.50 44.80 4.869 n = 2 250.30 11.70 3.810 125.10 3.70 3.213 62.56 1.70 2.880 25.02 0.10 1.910 10.01 NI 2 1001.00 79.40 5.820 0.993 656.15 500.50 33.60 4.577 250.30 1.90 2.925 125.10 0.10 1.910 62.56 NI 25.02 NI 10.01 NI RWL24 2008.90 75.40 5.687 0.998 981.74 891.0 ± 128.3 1 602.67 32.10 4.535 n = 2 180.80 4.70 3.325 54.24 0.10 1.910 16.27 NI 1.63 NI 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. (xlO"6 M) % I Probit R2 ICso (xlO"6 M) ICso ± s.d (xlO- 6 M) RWL24 2008.90 77.20 5.745 0.967 800.24 (contd) 2 602.67 32.70 4.552 180.80 14.40 3.938 5424 5.60 3.411 16.27 0.10 1.910 1.63 NI RWL26 1061.90 66.50 5.426 0.937 934.78 901.4 ±47.2 1 318.57 6.50 3.486 n = 2 95.57 2.60 3.057 28.67 0.10 1.910 8.60 NI 0.86 NI 2 1004.50 77.40 5.752 0.771 868.03 301.35 6.70 3.502 90.41 3.00 3.119 27.12 6.20 3.462 8.14 0.10 1.910 2.44 NI RWL27 256.63 99.99 8.719 0.948 10.40 10.6 ±0.3 1 76.99 99.85 7.968 n = 2 23.10 94.90 6.635 6.93 9.80 3.707 2.08 2.90 3.104 0.21 NI 2 256.63 96.20 0.990 10.86 76.99 99.99 8.719 23.10 94.30 6.581 6.93 10.10 3.724 2.08 0.20 2.122 0.21 NI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. %I Probit R2 ICso IC50 — s.d. (xlff6 M) (xlO-6 M) (xlO-6 M) RWL28 976.50 61.90 5.303 0.901 450.27 476.6 ±37.3 1 292.95 51.50 5.038 n = 2 87.89 12.60 3.855 26.37 0.10 1.910 7.91 NI 2.37 NI 2 976.50 54.10 5.103 0.859 502.95 292.95 50.30 5.008 87.89 14.40 3.938 26.37 0.10 1.910 7.91 NI 2.37 NI RWL31 433.09 99.99 8.719 0.973 26.36 28.2 ± 2.6 1 129.93 98.73 7.235 n = 2 38.98 46.60 4.915 11.69 10.50 3.746 3.51 1.60 2.856 0.35 NI 2 433.09 99.99 8.719 0.993 30.01 129.93 97.50 6.960 38.98 55.90 5.148 11.69 17.30 4.058 3.51 0.10 1.910 0.35 NI RWL32 131.40 88.90 6.221 0.943 32.51 32.2 ± 0.4 1 39.42 41.90 4.796 n = 2 11.83 22.70 4.251 3.55 10.90 3.768 1.06 0.10 1.910 0.32 NI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. (xlO"6 M) % I Probit R2 ICso (xlO-6 M) IC5o±s.d. (xlO"6 M) RWL32 131.40 88.50 6.200 0.969 31.92 (contd.) 39.42 47.50 4.937 11.83 15.70 3.993 3.55 11.90 3.820 1.06 13.00 032 0.10 1.910 RWL33 504.60 85.30 6.049 0.965 181.48 178.3 ±4.5 1 151.38 39.60 4.736 n = 2 45.41 930 3.678 13.62 4.50 3.305 4.09 0.30 2.252 1.23 0.10 1.910 2 504.60 82.90 5.950 0.946 175.13 151.38 37.00 4.668 45.41 10.40 3.741 13.62 8.20 3.608 4.09 0.10 1.910 1.23 NI RWL35 44.66 101.40 8.719 0.933 4.40 4.4 ±0.0 1 13.40 86.80 6.117 n = 2 4.02 13.10 3.878 1.21 10.90 3.768 0.36 0.10 1.910 0.11 NI 2 44.66 103.10 8.719 0.958 4.45 13.40 92.90 6.468 4.02 14.60 3.946 1.21 3.90 3.238 0.36 0.10 1.910 0.11 NI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV. 1. (continued) Compound Cone. (xlO-6 M) % I Probit R2 ICso (xlO"6 M) ICso±s.d. (xlO- 6 M) RWL36 53.71 102 JO 8.719 0.933 5.20 5.4 ±0.4 1 16.11 88.60 6.206 n =2 4.83 12.80 3.864 1.45 11.80 3.815 0.44 0.10 1.910 0.13 NI 2 53.71 100.60 8.719 0.958 5.70 16.11 88.40 6.195 4.83 16.10 4.010 1.45 2.40 3.023 0.44 0.10 1.910 0.13 NI Notes:a Molar concentrations;b % inhibition;c squared correlation coefficients;d An average 50% inhibitory concentration of different determinations ± standard deviation;e NI = no inhibition. 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.6 I 1.4 § 1 -2 S 1.0 8 0.8 3 0.6 s 0.4 a 0.2 -6.0 -5.5 -5.0 -4.5 -4 .0 -3.5 -3.0 -2.5 -2.0 Log concentration (M ) 9.0 y = 2.3800x+ 14.7860 R2 = 0.9951 8.0 7.0 6.0 a 2 5.0 Q . 4.0 3.0 2.0 -4.0 -3.0 - 2.0 -6.0 -5.0 Log concentration (M ) Figure IV.5.(1). Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.0 E c o a > ® o c m - Q w o a > n < 0.0 0.8 0.6 0.4 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M) -3.5 -3.0 6.0 y = 2.2624x+ 12.955 R2 = 0.983 ^ 5.0 4.0 Q . 3.0 2.0 -5.0 -2.0 -6.0 -4.0 -3.0 Log concentration (M) Figure IV.5.(2). Dose response curve (A) and probit transformed dose response curve (8) of hydroxysemicarbazide against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ~ 1.4 g 1 2 1.0 8 0.8 I 0.6 S 0.4 .Q < 0 2 0.0 -6.0 -5.0 -5.5 -4.5 -4.0 -3.5 Log concentration (M ) 7.0 i y = 2.2 8 2 1 x + 15.0970 R2 = 0.9366 6.0 5.0 | 4.0 Q _ 3.0 2.0 -7.0 -6.0 -4.0 -5.0 -3.0 Log concentration (M) Figure IV.5.(3). Dose response curve (A) and probit transformed dose response curve (B) of RWL1 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o O ) © o c CO € o C O n < -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M ) 8.0 y = 2.7283x + 19.226 F P = 0.9643 . 7.0 6.0 5.0 4.0 3.0 2.0 -6.0 -7.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(4). Dose response curve (A) and probit transformed dose response curve (B) of RWL2 against L1210 cells. 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.6 E c s 8 0.8 •e o.6 o 5 0.4 < 0.2 0.0 -3.5 -6.5 -6.0 -5.0 -4.5 -4.0 -7.0 -5.5 Log concentration (M ) 7.0 y = 2.1209x+ 15^04 R2 = 0.9465 6.0 5.0 C L 3.0 2.0 -7.0 -4.0 -3.0 -6 .0 -5.0 Log concentrati on (M) Figure IV.5.(5). Dose response curve (A) and probit transformed dose response curve (B) of RWL3 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o 0 3 3 , ® o c 0 .a w o to .a < -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 Log concentration (M ) -4.0 8.0 y = 2.856x+19.497 R 2 = 0.9316 7.0 6.0 _ 5.0 .o o 4.0 3.0 2.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(6). Dose response curve (A) and probit transformed dose response curve (B) of RWL4 against L1210 cells. 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 .0 E c o O ) ® o c C D n 0 .8 0 .6 8 0.4 < 0 .2 0.0 J -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M) 7.0 y=3.0273x + 18.257 R 2 = 0.9674 j 6.0 5.0 C L 3.0 2.0 1.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(7). Dose response curve (A) and probit transformed dose response curve (B) of RWL5 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.6 E c o 0 > 8 0.8 | 0.6 S 0.4 £ 1 < 0 2 - 0.0 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M) 7.0 y = 1.8698x + 12.682 Ff = 0.9811 * 6.0 5.0 £ 3 2 4.0 Q . 3.0 2.0 1.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(8). Dose response curve (A) and probit transformed dose response curve (B) of RWL6 against L1210 cells. 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ? 2 .0 c 1.8 8 1-6 § 1.4 C D O c C O A W 0 .6 o 0.4 - & 0 .2 < 0 .0 -L- -7.5 -3.5 -4.5 -6.5 -5.5 Log concentration (M) 9.0 8.0 7.0 6.0 4.0 3.0 y = 4.4075X + 28.103 R2 = 0.9684 2.0 -3.0 -7.0 -6.0 -5.0 -4.0 Log concentration (M) Figure IV.5.(9). Dose response curve (A) and probit transformed dose response curve (B) of RWL7 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 .8 E c o o > Tf < D 1 .0 - c 0.8 _ 0 .6 8 0.4 - < 0 .2 0.0 J CO € 7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M ) y = 2.5786X + 14.488 R F = 0.9882 6.0 5.0 .o o w a. 3.0 2.0 1.0 -6.0 -5.0 -4.0 -3.0 -2.0 Log concentration (M ) Figure IV.5.(10). Dose response curve (A) and probit transformed dose response curve (B) of RWL8 against L1210 cells. 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 2 2.0 E c o 0 3 © O C (0 -O w 0.8 8 0.6 - § 0.4 - 0 2 - 0.0 -L- -5.0 -3.0 -2.5 -4.5 -4.0 -3.5 Log concentration (M ) 7.0 n y = 2.4307x+ 1322 R2 = 0.9833 6.0 5.0 o 4.0 3.0 - 2.0 -6.0 -5.0 -4.0 -2.0 -3.0 Log concentration (M ) Figure IV.5.(11). Dose response curve (A) and probit transformed dose response curve (B) of RWL9 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.0 E c o 05 8 1.0 § 0.8 1 0.6 2 0.4 < 0.2 0.0 -3.5 -3.0 -2.5 -4.0 Log concentration (M ) 6.0 y = 6.3378x + 24.781 R 2 = 0.9673 i 5.0 4.0 0. 3.0 2.0 -4.0 -3.0 -2.0 -5.0 Log concentration (M ) Figure IV.5.(12). Dose response curve (A) and probit transformed dose response curve (B) of RWL10 against L1210 cells. 150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 .8 < 0 .2 0 .0 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M ) 6.0 y= 1.4987X+ 10.204 R2 = 0.994 4 5.0 4.0 2 a. 3.0 2.0 -5.0 - 2.0 -6.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(13). Dose response curve (A) and probit transformed dose response curve (B) of RWL11 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 . 0 i E c o o> © o c (0 -D o (0 n < 0.8 0.6 0.4 0.2 0.0 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 Log concentration (M ) 7.0 y =2.8587x + 14.818 R P = 0.9912 6.0 5.0 4.0 Q. 3.0 2.0 1.0 -4.0 -3.0 -5.0 -2.0 Log concentration (M ) Figure IV.5.(14). Dose response curve (A) and probit transformed dose response curve (B) of RWL12 against L1210 cells. 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.8 E c o 0> ® o c (S n 0.8 0.6 - 8 0.4- £ 0.2 - 0.0 J -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 Log concentration (M) 7.0 6.0 5.0 a 2 4.0 Q . 3.0 y =2.4515x + 13.548 R P = 0.983 2.0 -5.0 -4.0 -3.0 - 2.0 Log concentration (M ) Figure rV.5.(15). Dose response curve (A) and probit transformed dose response curve (B) of RWL13 against L1210 cells. 153 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100000 2 80000 I 60000 1 40000 2 20000 0 8.0 7.0 6.0 .t; 5.0 -Q 2 Q- 4.0 3.0 2.0 1.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration ( M ) y = 1.7566x + 12.859 R P = 0.9305 .0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(16). Dose response curve (A) and probit transformed dose response curve (B) of RWL14 against L1210 cells. 154 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o 05 © w.w c 0.6 (0 | 0.4 J § 0.2 < 0 .0 1 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 Log concentration (M ) 7 y = 1.6194x + 11.854 RP= 0.9745 6 5 4 3 2 1 -6.0 -3.0 -7.0 -5.0 -4.0 Log concentration (M ) Figure IV.5.(17). Dose response curve (A) and probit transformed dose response curve (B) of RWL15 against L1210 cells. 155 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.8 E c o 0 5 O 0 c (0 ■ e 0.6 1 1 0.4 < 0 2 0.0 - 0.8 -6.0 -5.5 -5.0 -4.5 -4.0 -3 .5 -3.0 Log concentration (M ) 7.0 y = 2.7304x+16.037 R2 = 0.9975 6.0 5.0 4.0 o. 3.0 2.0 -5.0 -6.0 -4.0 -2.0 -3.0 Log concentration (M ) Figure rv.5.(18). Dose response curve (A) and probit transformed dose response curve (B) of RWL16 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o 03 ® O C (0 n o CO n < 1.4 1.2 1.0 0.8 0 .6 0.4 0 .2 0.0 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M ) 6 .0 -i y= 1.4941x + 11.553 R2 = 0.9384 5.0 4.0 n 2 a. 3.0 2.0 1.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(19). Dose response curve (A) and probit transformed dose response curve (B) of RWL17 against L1210 cells. 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 .2 1 .0 E c o O) _ _ S 0.8 a > o c CD n o CO .Q < 0 .6 0.4 0 .2 0 .0 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M) -3.5 -3.0 6.0 y = 1.4588x +11.297 R 2 = 0.9487 5.0 4.0 - Q 2 CL 3.0 2.0 1.0 -7.0 -6.0 -3.0 -5.0 -4.0 Log concentration (M ) Figure IV.5.(20). Dose response curve (A) and probit transformed dose response curve (B) of RWL18 against L12I0 cells. 158 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120000 g 100000 g 80000 o 60000 o 40000 Z 20000 0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -25 L o g concentration (M ) 7.0 6.0 5.0 Q . 4.0 3.0 y = 1.9752x + 12.73 R 2 = 0.888 2.0 -6.0 -5.0 -4.0 -3.0 -2.0 Log concentration (M) Figure IV.5.(21). Dose response curve (A) and probit transformed dose response curve (B) of RWL19 against L1210 cells. 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M ) -2.5 6.0 i y = 3.5535x + 15.843 R 2 = 0.9628 1 5.0 4.0 Q . 3.0 2.0 1.0 -2.0 -5.0 -3.0 -4.0 Log concentration (M ) Figure IV.5.(22). Dose response curve (A) and probit transformed dose response curve (B) of RWL20 against L1210 cells. 160 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.0 E 16 g 1-4 S 12 8 i.o I 0.8 o ~ * C O < 0.4 02 0.0 -7.0 -6.0 -7.5 -6.5 -5.5 -4.5 -4.0 -5.0 Log concentration (M ) 8.0 y = 2.2041x+ 17.418 R 2 = 0.9166 7.0 6.0 - 5.0 n S * 4.0 3.0 2.0 1 .0 -8.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(23). Dose response curve (A) and probit transformed dose response curve (B) of RWL21 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o O) CD O c CO n k_ 0 .6 o 0 .4 & 0 -2 < 0 .0 - I -5.0 -4 .5 -4.0 -3 .5 -3.0 -2.5 Log concentration (M ) 6.0 i y = 2.3993x + 12.806 R F = 0.9698 J 4.0 X I 8 a. 2.0 -5.0 -3.0 -4.0 -2.0 Log concentration (M ) Figure IV.5.(24). Dose response curve (A) and probit transformed dose response curve (B) of RWL23 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 O c 0 n w 0 .8 _ 0 .6 8 0 4 2 0.2 < 0 .0 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 Log concentration (M) 6.0 y= 1.6853x+10.219 R2 = 0.9669 J 5.0 4.0 - 2.0 1.0 -5.0 -4.0 -2.0 -3.0 Log concentration (M ) Figure IV.5.(25). Dose response curve (A) and probit transformed dose response curve (B) of RWL24 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.0 E 1.6 - 8 1.4 - fl) S 12. - g 1-0 - § 0.8 - | 0.6 - 8 0.4 - < 0 2 0.0 - ■ -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 Log concentration (M ) 6.0 y = 2. 0995X + 11.36 R 2 = 0.9369 5.0 4.0 Q . 3.0 2.0 1.0 -5.0 -4.0 -3.0 -2.0 Log concentration (M ) Figure IV.5.(26). Dose response curve (A) and probit transformed dose response curve (B) of RWL26 against LI 210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.6 E c o 0 3 0 0-8 c 0.6 ■ e 0.4 1 0.2 < 0.0 -0.2 7.0 -6.5 -6.0 -5.5 -5 .0 -4.5 -4 .0 -3.5 -3.0 Log concentration (M ) 9.0 y= 2.9625x +19.762 ff = 0.9481 8.0 7.0 6.0 2 a. 5.0 4.0 3.0 2.0 -3.0 -6.0 -5.0 -4.0 L og concentration (M ) Figure IV.5.(27). Dose response curve (A) and probit transformed dose response curve (B) of RWL27 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4 E c o 0 5 © O c < 0 0.8 1 0.6 < 0.4 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2 .5 -2.0 Log concentration (M ) 6.0 5.0 4.0 . o 2 Q . 3.0 y = 2 . 1 7 3 x + 1 2 . 2 7 2 FP = 0 . 9 0 1 4 2.0 -3.0 -5.0 -4.0 -2.0 L o g c o n c e n t r a t io n ( M ) Figure IV.5.(28). Dose response curve (A) and probit transformed dose response curve (B) of RWL28 against L1210 cells. 166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 . 2 - I 2.0 5 0.6 £ 0 .4 - < 0.2 -7.0 -6.5 -6 .0 -5.5 -5 .0 -4.5 -4 .0 -3 .5 -3 .0 Log concentration (M ) 9.0 8.0 7.0 6.0 •Q 2 Q. 4.0 3.0 y = 3.1596x + 19.29 F F = 0.9929 2.0 1.0 -6.0 -5.0 -3.0 -2.0 -4.0 Log concentration (M ) Figure IV.5.(29). Dose response curve (A) and probit transformed dose response curve (B) of RWL31 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4 | 1.2 i 1 0 ^ 0 .8 0 c < 0 1 0-4 i | 0 .2 0 .0 0 .6 7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M ) 7.0 y = 1.5456x + 11.949 R P = 0.9692 6.0 5.0 2 4.0 3.0 2.0 1.0 -3.0 -6.0 -4.0 -7.0 -5.0 Log concentration (M ) Figure IV.5.(30). Dose response curve (A) and probit transformed dose response curve (B) of RWL32 against LI 210 cells. 168 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4 ? 1* o 1.0 0) — 0.8 0 c 0.6 O S 1 0.4 to § 0 2 0.0 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 Log concentration (M ) 7.0 y = 1.5586x + 10.831 R P = 0.9654 4.0 Q . 3.0 2.0 -4.0 -5.0 -3.0 -7.0 -6.0 Log concentration (M ) Figure IV.5.(31). Dose response curve (A) and probit transformed dose response curve (B) of RWL33 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -7.0 -6.5 -6.0 -5.5 -5.0 Log concentration (M ) -4.5 -4.0 9.0 y = 3.2224X + 22.244 FF = 0.9575 8.0 7.0 6.0 n S 5.0 Q . 4.0 3.0 2.0 1.0 -8.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.5.(32). Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4 12 0 1.0 S 0.8 g 0.6 < 5 0.4 1 0 2 £ o.o < -0 2 E c -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M ) 9.0 y = 3.2113x + 21.841 R 2 = 0.9577 8.0 7.0 6.0 5.0 Q . 4.0 3.0 2.0 -7.0 -6.0 -ao -5.0 -4.0 L o g concentration ( M ) Figure IV.5.(33). Dose response curve (A) and probit transformed dose response curve (B) of RWL36 against L1210 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IVJA.(2). Human leukemia CCRF-CEM cells After all the newly synthesized compounds were tested against the rapidly growing L1210 cells (doubling time = 8 -1 2 hours), three most active compounds (RWL4, RWL21 and RWL35) were further evaluated in human leukemia CCRF- CEM cell line (doubling time = 24 hours). The IQo values, % inhibition at different concentrations, and probit values of the three compounds and hydroxyurea (positive control) are shown in Table IV.2. The dose response curves and probit transformed dose response curves of RWL-4, 21, and 35 as well as hydroxyurea are given in Figures IV.6.(1) to IV.6.(4). The results demonstrate that the inhibitory activities of the three compounds against the human CCRF-CEM cells are comparable to those against the mouse L1210 cells, and they are 10 to 27-fold more potent than hydroxyurea. For rapid screening, it is easier to use the L1210 cells in stead of the CCRF-CEM cells. IVJ.B. Inhibition of solid tumor ceil lines (B16, CHO, HT29 and ZR75) IVJ.B.(1). % inhibition at 50 pM concentration Seventeen compounds (RWL-1, 4, 7, 9, 10, 12,15, 18-21, 24, 26-28, 32, and 35) and reference compound hydroxyurea were also tested against four solid tumor cell lines (B16, CHO, HT29 and ZR74). The inhibitory activities of the 17 compounds expressed as % inhibition of negative control at 50 pM concentration are summarized in Table IVJ. The inhibitory activities showed a parallel trend 172 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with those obtained from the L1210 cell line. In general, among the compounds tested at 50 pM concentration, RWL35 exhibited the highest inhibitions (92.5-100%) against the solid tumor cell lines, followed by RWL27, RWL4 and RWL7 with more than 50% inhibitions. Other compounds had only marginal or no inhibition. IVJ.B.(2). ICso values against the solid tumor cell lines The most potent compound RWL35 and hydroxyurea were further evaluated against B16. CHO, HT29 and ZR75 adherent cells. Their ICso values are listed in Tables IV.4 to IV.7. The dose response curves and probit transformed dose response curves of RWL35 and hydroxyurea are shown in Figures IV.7.(1) to TV.10.(2). The micromolar/supramicromolar ICso values of RWL35 suggest that RWL35 is active not only against the suspension cell cultures, but also against the adherent cell cultures. In contrast, hydroxyurea is almost inactive against CHO, HT29 and ZR75 adherent cells, and mildly active against mouse melanoma B16 cells. The ICso ratios of hydroxyurea over RWL35 ranged from 74 to 692, indicating that RWL35 may have therapeutic applications in solid tumors, such as breast cancer. It must be noted that RWL21 is active against leukemia cell lines (L1210 and CCRF-CEM) with the ICso values in micromolar range, but inactive against the solid tumor cell lines tested in this study. 173 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV 2 The inhibitory activities of RWL4, RWL21, RWL35 and hydroxyurea against human leukemia CCRF-CEM cells. Compound Cone. * (xlO- 6 M) % I b Probit R2c ICso (xlO- 6 M) ICso±s.d.d (xlO- 6 M) Hydroxy urea 933.50 90.10 6.287 0.967 66.62 732 ±9.3 1 280.10 86.60 6.108 n =2 84.02 73.70 5.634 25.20 7.56 7.00 3.524 0.76 0.10 1.910 2 933.50 90.10 6.287 0.955 79.74 280.10 87.10 6.131 84.02 73.10 5.616 25.20 14.50 3.942 7.56 3.00 3.119 0.76 0.10 1.910 RWL4 49.58 97.40 6.943 0.941 9.35 7.0 ± 3.4 1 14.87 93.30 6.499 n =2 4.46 12.20 3.835 1.34 0.10 1.910 0.40 NI 0.04 NI 2 49.58 97.70 6.995 0.909 4.54 14.87 93.60 6.522 4.46 20.70 4.183 1.34 14.90 3.959 0.40 4.10 3.261 0.04 NI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV .2. (continued) Compound Cone. (xlO"6 M) % I Probit R* ICso (xlO-6 M) ICso± s.d. (xlO- 6 M) RWL21 66.03 96.30 0.963 3.24 3.2 ±0.5 I 19.81 99.99 8.719 n =2 5.94 67.00 5.440 1.78 3.50 3.188 0.53 0.10 1.910 0.05 NI 2 66.03 97.20 0.854 2.90 19.81 99.99 8.719 5.94 58.50 5.215 1.78 6.30 3.470 0.53 4.40 3.294 0.05 NI RWL35 23.70 99.99 8.719 0.919 2.89 2.7±0.2 1 7.11 78.40 5.786 n =2 2.13 7.20 3.539 0.64 2.20 2.986 0.19 0.10 1.910 0.02 NI 2 23.70 99.99 8.719 0.945 2.56 7.11 83.90 5.990 2.13 13.60 3.902 0.64 3.90 3.238 0.19 0.10 1.910 0.02 NI Notes:a Molar concentrations; b % inhibition;c squared correlation coefficients;d An average 50% inhibitory concentration of different determinations ± standard deviation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.8 I 14 i 1 2 ® 1-0 | 0.8 C D | 0.6 I 0.4 0.2 0.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 Log concentration (M ) 7.0 y=1.5684x+11.428 R2 = 0.955 . ' 6.0 5.0 4.0 o. 3.0 2.0 7.0 -6.0 -5.0 -4.0 -3.0 -2.0 Log concentration (M) Figure IV.6.(1). Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against CCRF-CEM cells. 176 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E 14 I © o | 0.6 § 0.4 -Q < 0.2 0.0 -0.2 - -6.5 0.8 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M ) 8.0 y = 3.3974x + 22.086 R P = 0.9412 7.0 6.0 5.0 .o 9 “■ 4.0 3.0 2.0 1.0 -7.0 -5.0 -3.0 - 6.0 -4.0 Log concentration ( M ) Figure IV.6.(2). Dose response curve (A) and probit transformed dose response curve (B) of RWL4 against CCRF-CEM cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.8 _ 1.6 I 14 0 -jo o > S 1.0 g 0.8 n 0.6 1 0.4 5 0.2 < 0.0 - 0.2 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M ) 9.00 8.00 7.00 6.00 o 5.00 o. 4.00 3.00 2.00 1.00 -7.00 -6.00 -5.00 -4.00 -3.00 Log concentration (M ) Figure IV.6.(3). Dose response curve (A) and probit transformed dose response curve (B) of RWL21 against CCRF-CEM cells. y = 4.3373x + 28.615 R 2 = 0.9625 178 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 .0 1 1 4 ' 2 1-2 - o 10- g 0.8 - | 0.6 g 0.4- £ 02 -02 -I -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M) 9.0 y = 3.1311x + 22.508 ff = 0.9449 8.0 7.0 6.0 n 2 5.0 0. 4.0 3.0 2.0 1.0 -8.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.6.(4). Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against CCRF-CEM cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV J . The % inhibition of the selected Schiff bases of h ydrox ysemicarbazi de against the solid tumor cell lines at S O jiM concentration (NI = no inhibition). compound % inhibition at 50 JiM B16 CHO HT29 ZR75 HU 43.5 13.8 NI 6.1 RWL1 53.9 57.2 10.0 36.6 RWL4 87.1 52.2 67.3 85.2 RWL7 82.9 61.9 79.8 85.9 RWL9 26.1 8.8 NI 3.3 RWL10 17.1 10.6 NI NI RWL12 23.7 13.1 NI 8.6 RWL15 7.9 15.5 NI 7.7 RWL18 53.3 55.3 8.8 40.1 RWL19 14.9 15.7 11.6 16.5 RWL20 25.5 NI NI 6.8 RWL21 15.4 NI NI NI RWL24 18.5 9.0 NI NI RWL26 16.3 7.9 NI 6.0 RWL27 93.8 66.2 94.0 87.4 RWL28 27.1 10.2 NI 7.9 RWL32 46.5 69.9 NI 70.1 RWL35 100 100 92.5 97.5 180 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.4. Inhibition of B16 (mouse melanoma) cells by RWL35 and hydroxyurea. Compound Cone. * (xlO* M) % Ib Probit R2c ICso (x 10* M) IC so±s.d.d (x 10* M) Hydroxy 2035.20 71.70 5.574 0.985 481.18 497.2 ± 22.6 urea 1 814.08 63.00 5.332 I I a 325.60 43.30 4.831 130.30 24.90 4.322 52.10 18.10 4.088 5.21 4.60 3.315 2 2035.20 71.10 5.556 0.986 513.13 814.08 61.40 5.290 325.60 43.10 4.826 130.30 23.90 4.291 52.10 16.70 4.034 5.21 12.60 RWL35 51.82 96.80 6.852 0.963 6.55 6.7 ±0.2 1 20.73 87.80 6.165 n = 2 8.29 55.30 5.133 3.316 14.60 3.946 1.33 6.40 3.478 0.13 0.30 2.252 2 51.82 96.90 6.866 0.982 6.83 20.73 88.00 6.180 8.29 55.40 5.136 3.316 17.20 4.054 1.33 9.10 3.665 0.13 5.60 Notes:a Molar concentrations;b % inhibition;c squared correlation coefficients;d An average 50% inhibitory concentration of different determinations ± standard deviation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.5. Inhibition of CHO (Chinese hamster ovary carcinoma) cells by RWL35 and hydroxyurea. Compound Cone. * (xlO*M) % Ib Probit R2c ICso (x 10* M) IC5o ± s.d .d (x 10* M) Hydroxy 1880.00 43.30 4.831 0.947 1464.22 1463.0 ± 1.8 urea 1 752.00 42.90 4.821 9 I I 300.80 35.90 4.639 120.30 23.30 4.271 48.13 9.20 3.672 4.81 1.50 2.830 2 1880.00 45.80 4.895 0.867 1461.73 752.00 45.10 4.874 300.80 40.00 4.747 120.30 23.40 4.274 48.13 11.40 3.795 4.81 0.10 RWL35 64.43 98.84 7.270 0.989 12.26 12.0 ± 0.4 1 25.77 83.50 5.974 I I C 10.31 30.20 4.481 4.12 7.10 3.532 1.65 0.90 2.634 0.16 0.10 2 64.43 98.81 7.260 0.994 11.76 25.77 85.30 6.050 10.31 34.60 4.604 4.12 8.20 3.608 1.65 0.90 2.634 0.16 0.10 1.910 Notes:* Molar concentrations;b % inhibition;c squared correlation coefficients;d An average 50% inhibitory concentration of different determinations ± standard deviation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.6. Inhibition of HT29 (human colon adenocarcinoma) cells by RWL35 and hydroxyurea. Compound Cone.a (xlO* M) % Ib Probit R5c IC50 (x 10* M) ICso±s.d.d (x 10* M) Hydroxy 2035.20 49.20 4.980 0.971 1917.28 1888.9 ± urea 40.1 I 814.08 30.90 4.501 n = 2 325.60 19.30 4.133 130.30 4.70 3.325 52.10 5.21 2 2035.20 50.30 5.008 0.986 1860.54 814.08 30.60 4.493 325.60 16.30 4.018 130.30 3.90 3.238 52.10 0.10 5.21 RWL35 51.82 96.80 6.852 0.963 6.55 6.7 ±0.2 1 20.73 87.80 6.165 n = 2 8.29 55.30 5.133 3.32 14.60 3.946 1.33 6.40 3.478 0.13 0.30 2.252 2 51.82 96.90 6.866 0.982 6.84 20.73 88.00 6.175 8.29 55.40 5.136 3.32 17.20 4.054 1.33 9.10 3.665 0.13 5.60 Notes:a Molar concentrations;b % inhibition;c squared correlation coefficients;d An average 50% inhibitory concentration of different determinations ± standard deviation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.7. Inhibition of ZR75 (human breast carcinoma) cells by RWL35 and hydroxyurea. Compound Cone.1 %Ib Probit R2c ICso ICso± s.d.d (xlO"6 M) (x Iff6 M) (x 10"6 M) HU 2035.20 47.10 4.927 0.950 1869.20 n = 1 814.08 25.00 4.326 325.63 12.60 3.855 130.25 0.30 2.252 52.10 0.10 1.910 5.21 RWL35 53.18 92.30 6.762 0.994 2.86 2.7 ± 0.2 1 21.27 96.10 6.011 n = 2 8.51 84.40 5.264 3.40 60.40 4.248 1.36 22.60 0.14 10.40 2 53.18 92.40 6.685 0.981 2.52 21.27 95.40 5.931 8.51 82.40 5.169 3.40 56.70 4.238 1.36 22.30 3.057 0.14 2.60 Notes:1 Molar concentrations;b % inhibition;c squared correlation coefficients;d An average 50% inhibitory concentration of different determinations ± standard deviation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. _ 0.6 g 0.4 < 0.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2 .< Log concentration (M ) 6.0 y =0.9015x + 7.9909 R 2 = 0.9849 5.0 Q . 3.0 2 .0 J -4.0 -3.0 -6.0 -5.0 - 2.0 Log concentration (M ) Figure rv.7.(l). Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against B16 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 02 -7.0 -6.5 -6.0 -5.5 -5.0 Log concentration (M ) -4.5 -4 .C 8.0 y = 1.8386X+ 14.531 W = 0.9628 7.0 6.0 5.0 °- 4.0 3.0 2.0 8.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M ) Figure IV.7.(2). Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against B16 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o 0> ® o c C O € o C O -Q < 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 L og concentration (M ) -2.5 -2.0 6.0 y = 0.8339X + 7.3636 R F = 0.9473 5.0 n 2 a_ 3.0 2.0 -4.0 -3.0 - 2.0 - 6.0 -5.0 Log concentration (M ) Figure IV.8.(1). Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against CHO cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.0 1.8 g 1.0 2 0 8 I 0.6 £ 0.4 < 0.2 0.0 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M) 8.0 y =2.9382x +19.484 R 2 = 0.9938 7.0 6.0 -Q 2 Q . 4.0 3.0 2.0 -5.0 - 6.0 -4.0 -7.0 -3.0 Log concentration (M ) Figure IV.8. (2). Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against CHO cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o o> © o 0.8 0.6 0 .4 - 0.2 0.0 c < 8 € o C O £3 < 5 .5 -5.0 -4.5 -4.0 -3.5 -3.0 -2 .5 -2.0 Log concentration (M ) 6.0 y = 1.4538x + 8.9694 F F = 0.9861 5.0 Q . 3.0 2.0 -5.0 -4.0 - 2.0 -3.0 Log concentration (M ) Figure IV.9.(1). Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against HT29 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 Log concentration (M ) 8.0 y = 1 .8 3 8 6 x + 14.531 R2 = 0.9628 6.0 .a o Q - 4.0 3.0 2.0 1.0 -8.0 -7.0 -6.0 -5.0 -4.0 -3.0 Log concentration (M) Figure IV.9.(2). Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against HT29 cells. 190 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c o a > < D o c < 0 A h - o C O .Q < -4.5 -4.0 -3.5 -3.0 Log concentration (M ) -2.5 -2. 6.0 y=2.0375x + 10.559 = 0.9501 5.0 4.0 . Q 2 CL 3.0 2.0 1.0 -5.0 -4.0 -3.0 - 2.0 Log concentration (M ) Figure IV. 10.(1). Dose response curve (A) and probit transformed dose response curve (6) of hydroxyurea against ZR75 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. E c s s § € o (0 A < 1.4 1.0 0.8 0.6 0.4 02 0.0 -6.0 -7.0 -6.5 -4.5 -4.0 -5.5 -5.0 Log concentration (M ) 7.0 y = 1.674x +14.371 f f = 0.981 6.0 5.0 Q. 4.0 3.0 2.0 -7.0 - 8.0 - 6.0 -4.0 -5.0 Log concentration (M ) Figure IV. 10.(2). Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against ZR75 cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IVJ.C. Selective toxicity against tumor cells vs. 3T3 Swiss mouse embryo fibroblasts The in vitro toxicides (expressed as TCso, 50% toxic concentration) of RWL35 and hydroxyurea are summarized in Table IV.8. The dose response curves and probit transformed dose response curves are plotted in Figures IV. 11.(1) and IV.11.(2). RWL35 has selectivity indices (TCso against 3T3 fibroblasts/ICso against tumor cells) ranging from 1.3 to 5.6, while hydroxyurea shows similar inhibitory activities against the non-cancer 3T3 fibroblasts and the two leukemia cell lines. Interestingly, hydroxyurea is more potent against the 3T3 fibroblasts than all solid tumor cell lines tested. Overall, RWL35 has more favorable selectivity indices than hydroxyurea, which is currently used for the treatment of various cancers (see Table IV.9). This implies that RWL35 may have a low toxicity in vivo, and merits further in vivo animal study to explore the potential for human clinical trials. The inhibitory activities (ICso or TCso) of the newly synthesized compounds and hydroxyurea against both the cancer cell lines (L1210, CCRF-CEM, B16, CHO, HT29, ZR75) and the non-cancer cell line (3T3 embryo fibroblasts) are summarized in Table IV.10 for comparison. 193 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.8. In vitro toxicity of RWL35 and hydroxyurea against 3T3 fibroblasts. Compound Cone. * (xio-6 M) % lb Probit R2c TCso (xIO-6 M) TCso±s.d.d (xlO-6 M) HU 846.70 88.20 0.964 52.06 54.6 ± 3.5 1 338.70 89.40 6.248 n = 2 135.50 84.00 5.995 54.19 67.20 5.445 21.68 25.50 4.341 2.17 0.10 1.910 2 846.70 86.60 0.963 57.07 338.70 87.20 6.136 135.50 81.90 5.912 54.19 65.20 5.391 21.68 21.40 4.207 2.17 0.10 1.910 RWL35 65.46 98.62 7.203 0.994 15.77 15.2 ±0.8 1 26.18 76.30 5.716 n = 2 10.47 25.20 4.332 4.19 1.50 2.830 1.68 0.10 1.910 0.17 NI 2 65.46 97.70 6.995 0.998 14.70 26.18 76.60 5.726 10.47 29.60 4.464 4.19 6.30 3.470 1.68 0.20 2.122 0.17 NI Notes:* Molar concentrations;b % inhibition;c squared correlation coefficients;d An average 50% toxic concentration of different determinations ± standard deviation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. — 12 I 10 ~ 0 . 8 o c c o ■ e 8 o-4 . Q < 02 0.6 0.0 -4.5 - 6.0 -5.5 -5.0 -4.0 -3.5 -3.0 Log concentration (M ) 7.0 y =2.0635x +13.839 R 2 = 0.9643 6.0 5.0 Q . 3.0 2.0 1.0 -7.0 -6.0 -5.0 -4.0 -3.0 -2.0 Log concentration (M ) Figure IV. 11.(1). Dose response curve (A) and probit transformed dose response curve (B) of hydroxyurea against 3T3 fibroblasts. 195 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.4 E < § 1 - 0 a > 2 - 0.8 a > o § 0.6 € S 0.4 .a < 0 2 0.0 -7.0 - 6.0 -5.5 -4.5 -6.5 -5.0 -4.0 Log concentration (M ) 7.0 y=3.0164x + 19.577 F ¥ = 0.9983 6.0 5.0 Q . 4.0 3.0 2.0 -5.0 - 6.0 -7.0 -4.0 -3.0 Log concentration Figure IV. 11 .(2). Dose response curve (A) and probit transformed dose response curve (B) of RWL35 against 3T3 fibroblasts. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table IV.9. Comparison of in vitro selectivity of RWL35 and hydroxyurea. Compound L1210 Selectivity index (SI = CCRF-CEM B16 CHO T C 50 3T3 non-cancer ccIIj/ICso cancer cclk) HT29 ZR75 KB-W KB-HUR KB-GR hydroxyurea 0.7 0.7 0.1 0.04 0.03 0.03 0.1 0.004 0.02 RWL35 12.4 5.6 2.3 1.3 2.3 5.6 2.1 5.4 2.3 Relative selectivity 17.7 8.0 23.0 32.5 76.7 186.7 21.0 135.0 115.0 index (RSI = SIrwus/SIhu) Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table IV.10. The inhibitory activities of the 31 Schiff bases of hydroxysemicarbazide against cancer and non-cancer cell lines. Name IC50 (x 10‘6 M) or (% inhibition at 5 x 10' 5 M) TC50 (x 10 6 M) 3T3 (non-cancer cells) L1210 CCRF-CEM B16 CHO HT29 ZR75 Hydroxy urea 82.016.0 n = 4 73.219.3 497.2122.6 n = 2 n = 2 1463.011.8 n = 2 1888.9140.1 n = 2 1869.2 n = 1 54.613.5 n = 2 Semicar- bazide >2192.2 n = 2 (43.5%) (13.8%) (NI)* (6.1%) Hydroxy semicar bazide 2 8 1 .6 1 2 1 .4 n = 3 RWLl 39.512.7 n = 2 (53.9%) (57.2%) (10.0%) (36.6%) RWL2 6.510.6 n = 2 RWL3 10.111.0 n = 2 Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table IV. 10. (continued) Name 1C5 0 (x 10* M) or (% inhibition at 5 x 10 s M) TCso (x 10 6 M) 3T3 (non-cancer cells) L1210 CCRF-CEM B16 CHO HT29 ZR75 RWL4 7.2±1.7 n = 2 7.013.4 (87.1%) (52.2%) (67.3%) n = 2 (85.2%) RWL5 37.7±5.7 n = 2 RWL6 80.4±3.5 n = 2 * RWL7 4.711.5 n = 2 (82.9%) (61.9%) (79.8%) (85.9%) RWL8 208.511.0 n = 2 RWL9 446.3144.0 n = 2 (26.1%) (8.8%) (NI) (3.3%) RWL10 820.6173.1 n = 4 (17.1%) (10.6%) (NI) (NI) v 8 Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table IV. 10. (continued) Name IC50 (x 1 0 6 M) or (% inhibition at 5 x 10 s M) TC5 0 (x 10 6 M) 3T3 LI 210 CCRF-CEM B16 CHO HT29 ZR75 (non-cancer cells) RWLll 399.3±88.1 n = 2 RWL12 368.811.5 n = 2 (23.7%) (13.1%) (NI) (8.6%) RWL13 316.7113.1 n = 2 RWL14 30.115.0 n = 2 RWL15 60.212.4 n = 2 (7.9%) (15.5%) (NI) (7.7%) RWL16 91.410.9 n = 2 RWL17 48.119.9 n = 2 Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table IV. 10. (continued) Name IC50 (x 10'6 M) or (% inhibition at 5 x 10' 5 M) TCso (x 10^ M) 3T3 L12I0 CCRF-CEM B16 CHO HT29 ZR75 (non-cancer cells) RWL18 42.717.9 n = 2 (53.3%) (55.3%) (8 .8 %) (40.1%) RWL19 130.8112.3 n = 2 (14.9%) (15.7%) ( 1 1 .6 %) (16.5%) RWL20 944.1178.7 n = 2 (25.5%) (NI) (NI) (6 .8 %) RWL21 2.710.6 n = 2 3.210.5 (15.4%) n = 2 (NI) (NI) (NI) RWL23 607.0169.5 n = 2 RWL24 891.01128.3 n = 2 (18.5%) (9.0%) (NI) (NI) RWL26 901.4147.2 n = 2 (16.3%) (7.9%) (NI) (6 .0 %) £ Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table IV. 10. (continued) Name IC50 (x 1 0 * M) or (% inhibition at 5 x 1 0 s M) TCjo (x 1 0 * M) 3T3 (non-cancer cells) L1210 CCRF-CEM B16 CHO HT29 ZR75 RWL27 I0.6±0.3 n = 2 (93.8%) (6 6 .2 %) (94.0%) (87.4%) RWL28 476.6137.3 n = 2 (27.1%) ( 1 0 .2 %) (NI) (7.9%) RWL31 28.212.6 n = 2 RWL32 32.210.4 n = 2 (46.5%) (69.9%) (NI) (70.1%) RWL33 178.314.5 n = 2 RWL35 4.410.0 n = 2 2.710.2 6.710.2 n = 2 n = 2 ( 1 0 0 %) 12.010.4 n = 2 ( 1 0 0 %) 6.710.2 n = 2 (92.5%) 2.710.2 n = 2 (97.5%) 15.210.8 n = 2 RWL36 5.410.4 n = 2 1 No inhibition. IVJJ). Inhibition of hydroxyurea-resistant KB cells IV JJ).(l). IC59 values against hydroxyurea-resistant KB cells The inhibitory activities of RWL-2, 35 and hydroxyurea (HU) against the wild type (KB-W), HU-resistant (KB-HUR) and gemcitabine-resistant (KB-GR) KB tumor cells are shown in Table IV.11. Among the three compounds tested RWL35 is the most potent inhibitor of KB-HUR and KB-GR resistant tumor cells, with the IC50 values of 2.8 x lO "6 M and 6.5 x 10-6 M, respectively, followed by RWL2. HU is inactive against the two resistant cell lines with the IC50 of 1.25 x 10'2 M and 2.36 x 1 0 * 3 M, respectively (see Figure IV.12). The results indicate that in cell cultures RWL35 and RWL2 have no cross-resistance to HU and gemcitabine, two RR inhibitors known to target the different subunits of the same enzyme. IVJ.D.(2). Mechanism o f hydroxyurea resistance The resistance of tumor cells to anticancer drugs remains a major cause of treatment failure in cancer patients. The resistance mechanisms of tumor cells to anticancer drugs may include decreased drug accumulation (decreased drug uptake and/or increased drug efflux), altered intracellular drug distribution, increased detoxification, diminished drug-target interaction and etc. HU specifically inhibits DNA synthesis by interacting with the R2 subunit of the RR enzyme. One of its drawbacks in the therapeutic applications is the development of resistance. To study this problem, the molecular and cellular characterizations of HU-resistant human KB cells and HU-resistant Chinese hamster 203 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ovary carcinoma cells have been carried out by Yen’s group (Yen et al., 1994; Zhou et al., 1995) and by Tagger and Wright (1988). The investigation of the HU-resistant phenotype revealed an R2 subunit gene amplification, increased levels of R2 mRNA and protein in the HU-resistant KB cells. After passage of the HU-resistant KB cells in the absence of HU, the cells lost their resistance to HU. This reversion was due to the return of RR to the level comparable to that of the wild (parental) KB cells (Yen et al., 1994). Another study by the same group demonstrated that overexpression of the R2 mRNA and protein (but not those of Rl) was sufficient to induce resistance to HU in the transfected KB cells, and the resistance was greatly affected by the amount of the R2 subunit in the KB cells (Zhou et al., 1995). Tagger and Wright (1988) have conducted a similar investigation using two Chinese hamster ovary carcinoma cell lines exhibiting either low or relatively high resistance to HU. Both HU-resistant cell lines had increased levels of RR activity. The elevation in the enzyme activity was due to an increase in the R2 subunit. The study with R2 cDNA indicated that the two HU-resistant cell lines possessed elevated levels of R2 mRNA and R2 gene copy number. So far, three reports are available about the mechanism responsible for the uptake of HU by mammalian cells. The three reports demonstrated that HU crossed the mucosa of the rat small intestine and cell membranes of the different cell types by passive diffusion (Evered and Selhi, 1972; Morgan et al., 1986; Tagger et al., 1987). The active transport was not observed in these investigations. At present, no 204 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. direct evidence of involvement of p-glycoprotein (p-gp) efflux pump and multidrug resistance (MDR) in the HU-resistance is available. The finding of Tagger and Wright (1988) suggested that the Chinese hamster ovary carcinoma cells exhibiting high resistance to HU might not share the MDR phenotype, because the HU-resistant cells were not cross-resistant to colchicine or puromycin, two different anticancer drugs. In another study by Christen et al. (1992), a nonlethal concentration of HU accelerated the rate of loss of vinblastine resistance in human epidermoid carcinoma KB VI cells. The KB VI cells exposed to HU for the time required to complete 12 cell doublings accumulated more vinblastine than the control cells grown in the absence of HU. Studies thus far reported that HU could induce the loss of unstably amplified genes, and enhanced the sensitivity of tumor cells to other anticancer drugs. Use of HU to alter drug resistance of human tumor cells is being investigated by several groups (Berg and Von Hoff, 1995). RWL35 showed no cross-resistance to HU in the HU-resistant KB cells. This may be due to increased cellular uptake of RWL35 and increased intracellular drug concentration. As indicated in Chapter V, RWL35 has a considerably higher lipophilicity (log P = 3.90) than HU (log P = -1.80). The higher lipophilicity may result in the increased RWL35 uptake due to the increased membrane penetration. In addition, since the mammalian R2 subunit has a hydrophobic channel as an entrance to the tyrosyl fiee radical, RWL35 may have a stronger binding to the channel by 205 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hydrophobic interaction than HU, and thus results in inactivation of the RR enzyme in the HU-resistant KB cells at the experimental condition. E. Discussion In summary, in a variety of cancer and non-cancer cell lines, as well as HU and gemcitabine resistant cancer cells, RWL35 has stronger inhibitory effects and more favorable selectivity indices than HU. At the present time, HU is the first-line chemotherapeutic agent for the treatment of chronic myelogenous leukemia. Clinic data have clearly shown the superiority of HU over busulfan (Lill and Koeffler, 2001). However, HU has several disadvantages such as a short half-life, low potency, and rapid occurrence of resistance. These disadvantages are associated with its physicochemical properties, e.g. very high hydrophilicity (log P = -1.80) and small molecular size (MW = 76). In this study the SB-HSCs retaining the essential pharmacophore of -NHCONHOH have more favorable physicochemical properties, namely increased lipophilicity and molecular size for the most active compounds. The compounds may have better pharmacokinetic profiles such as longer half-lives and higher RR inhibitory activities. RWL35 is 74 to 692-fold more potent than HU against the four solid tumor cell lines, indicating that RWL35 may have additional potential therapeutic applications in solid tumors, such as melanoma, breast and colon cancers. A comparison of the cytotoxicities (IC50) of the Schiff bases of hydroxysemicarbazide with the clinically used anticancer drugs against different tumor cell lines is shown in Table IV. 12. The anticancer drugs selected in the comparison are the first line 206 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chemotherapeutic agents or commonly used chemotherapeutic agents in the treatment of leukemia, melanoma, and colonic, breast and ovarian cancers. RWL21 and RWL35 are superior to busulfan and HU in anti-leukemia activity. RWL35 is more active than dacarbazine, carmustine, HU, carboplatin and cyclophosphamide, but less active than cisplatin, paclitaxel, doxorubicin and methotrexate, against the solid tumor cells (melanoma, breast and ovary). The cytotoxicity of RWL35 against human colon adenocarcinoma HT29 cells is equivalent to that of 5-fluorouracil. Compounds RWL35 and RWL2 have no cross-resistance with HU and gemcitabine in vitro. This implies that RWL35 may have potential clinical applications in the HU-resistant cancers. All compounds tested show varied sensitivities toward the different tumor cells used. The most sensitive tumor ceil lines are the leukemia cell lines (L1210 and CCRF-CEM), followed by B16. The least sensitive tumor cell line is HT29. Compound RWL21 with 2,3,4-trihydroxy groups has the strong inhibitions against L1210 and CCRF-CEM leukemia cells (IC50 = 2.7 x lO "6 and 3.2 x K T 6 M, respectively), but not against solid tumor cell lines. It has been reported that resveratrol (3,5,4’-triphydroxystiIbene) and p- propoxyphenol are active RR inhibitors with the IC50 values of 1.0 x 10"* M and 3.0 x 10"4 M (Fontecave et al., 1998; Potsch et al., 1994). Compound RWL21 combines the structural features of resveratrol and p-propoxyphenol as well as HU, resulting in a higher activity than what was predicted from the regression equation against the leukemia L1210 cells. 207 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MTS/PES-MTA colorimetric assay has been used in proliferation and cytotoxicity experiments widely. An important advantage of the assay over the previous tetrazolium salt MT1 assay is the production of water-soluble formazan which eliminates the DMSO solubilization. MTS/PES yields colored formazan rapidly in cell culture, and have the improved storage stability (Buttke, et al., 1993; Barltrop et al., 1991; Riss and Moravec, 19%; Promega Corp., 1999). The IC5 0 of HU observed by this method (7.3 x 10'5 M) is comparable to the value reported by NCI (5.4 x 10‘ 5 M) against the same cell line (CCRF-CEM) (URL: http://dtp.nci.nih.gov). However, when the assay is used to evaluate colored compounds such as compound RWL10, the background absorbance from compound itself must be subtracted from the overall absorbance. The MTS/PES colorimetric assay can only detect the cell viability, but can not elucidate the molecular mechanisms of the antitumor activities. Additional molecular and cellular level investigations (e.g. enzyme inhibition study, DNA and RR protein expression) are needed to disclose the molecular mechanisms of the compounds as inhibitors of tumor cells. There is still a great interest in the development of RR inhibitors as anticancer and antiviral drugs. The results show for the first time that, at least in vitro, SB-HSCs are remarkable inhibitors of various tumor cells including HU- resistant cells. The data suggest that some of the SB-HSCs merit further in vivo antitumor and toxicity testing, and enzyme inhibition study in the future development. 208 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.ll. The inhibitory activities of RWL2, RWL35 and hydroxyurea against the wild type (KB-W), hydroxyurea-resistant (KB-HUR) and gemcitabine-resistant (KB-GR) KB tumor cells. cells IC50 (x 10* M) HU RWL2 RWL35 KB-W 430 18.0 7.4 KB-HUR 12500 16.0 2.8 KB-GR 2360 28.0 6.5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.5 A.RWL-2 5 *1.5 3 CO KB-Wt KB-HURs KB-Gem o O 0.5 200 0.32 1.6 8 40 0 Drug Concentration (pM) 2 5 2 1 5 1 0 5 0 0 0 5 2 1 .6 8 4 0 2 0 0 D ru g G t n s r t r a n f y M ) Figure IV.12. Dose response curves of RWL2 (A) and RWL35 (B) against the wild type (KB-W), hydroxyurea-resistant (KB-HUR) and gemcitabine-resistant (KB-GR) KB tumor cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.12. Comparison of the inhibitory activities (IC50) of the Schiff bases of hydroxysemicarbazide with the clinically used anticancer drugs against different tumor cell lines. Cancer Drugs Tumor cell lines ICsaOiM) Busulfan CCRF-CEM (human leukemia) 157.0 (NCI)* hydroxyurea CCRF-CEM 53.8 (NCI)a leukemia hydroxyurea CCRF-CEM 73.2 (this study)1 * RWL21 CCRF-CEM 3.2 (this study )b RWL35 CCRF-CEM 2.7 (this study)b Dacarbazine human melanomac 31.6 (NCI)* Carmustine human melanomac 63.1 (NCI)* melanoma Cisplatin human melanomac 1.6 (NCI)* hydroxyurea human melanomac 631.0 (NCI)* hydroxyurea B-16 (murine melanoma) 497.2 (this study)b RWL35 B-16 6.7 (this study)5 Cisplatin human ovaryc 4.0 (NCIT ovary Carboplatin human ovaryc 100 (NCI)* Paclitaxel human ovaryc 0.019 (NCI)* RWL35 CHO (Chinese hamster ovary carcinoma) 12.0 (this study)5 211 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table IV.12.(condnued) Cancer Drugs Tumor ceD lines IC5# (pM) colon 5-Fiuorouracil HT-29 (human colon adenocarcinoma) 6.0 (NCD* RWL35 HT-29 6.7 (this study)b Doxorubicin (Adriamycin) MCF-7 (human breast adenocarcinoma) 0.013 (NCD* Paclitaxel MCF-7 0.002 (NCD* breast Cyclophosphamide MCF-7 250.0 (NCD* Cisplatin MCF-7 2.9 (NCD* Methotrexate MCF-7 0.041 (NCD* 5-Fluorouracil MCF-7 1.7 (NCD* RWL35 ZR-75 (human breast carcinoma) 2.7 (this study)6 Notes: * From the NCI website http://dtp.nci.nih.gov:b From this study;c The average IC50 values against a panel of human tumor cell lines. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V. QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP In order to ascertain whether the optimal compound has been made and what factors are involved in the pharmacokinetic and pharmacodynamic processes of the newly synthesized compounds, quantitative structure-activity relationship (QSAR) analysis was carried o u t The QSAR study may provide useful guidance for further structural modification and development of second generation of hydroxysemicarbazide derivatives and other ribonucleotide reductase inhibitors. V .l. Methods The inhibitory activities of the three reference compounds (hydroxyurea, semicarbazide, hydroxysemicarbazide) and 31 new Schiff bases of hydroxysemicarbazide against the mouse leukemia L1210 cells were given in Chapter IV of this dissertation. The physicochemical parameters used in the QSAR analysis included hydrophobic Gog P, it, Rm), steric (MR, E*) and electronic (o, ji) parameters. Log P, representing hydrophobicity, refers to the logarithm of partition coefficient of the neutral form of a given compound between 1-octanol and water (Hansch and Leo, 1995), as shown by equation (I): 213 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HAo 1-octanol water Log P = log ([HA]o/[HA]w ) (I) Where [HA]0 and [HA]W are the concentrations of the neutral form of the compound in 1-octanol and water, respectively. In 1964 Fujita and Hansch (1964) proposed that log P was an additive property and could be calculated by taking the sum of the log P of the parent molecule and the n of the substituent, as in equations (II) and (III). Log P s u b s t i t u t e d m o l e c u le = & + log P p a r e n t m o l e c u l e (II) For example: Log Pckc^ich, = log P c 6 h6 + J t o t , + = 2.13 + 0.56 + 0.71 = 3.40 (calculated) (ffl) measured log P = 3.33 Hansch-Fujita substituent constant (it) for any substituent X was thereafter defined and calculated as follows: Jtx = log PY -x - log PY - h (IV) in which Y is the parent structure. For example: JLcH j = log P c H jC g H j - log Pc6 H j = 2.69-2.13 = 0.56 (V) 214 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Another hydrophobic parameter Rm was converted from the Rf obtained by the thin-layer chromatography (TLC) experiment (see Chapter HI; Bate-Smith and Westall, 1950; Boyce and Miborrow, 1965; Lien, 1994), as shown by equation (VI); Rm = log (1/Rf — 1) (VI) Rm is related to log P by equation (VII); R m = a log P + b (VII) Molar refractivity (MR) was defined by Lorentz-Lorenz equation: MR = [(n2 - l)/(n2 + 2)](MW/d) (Vm) In this expression, n represents the refractive index, MW is the molecular weight, and d is the density of a compound. Theoretically, MR is related to London dispersion forces as follows: MR = 47tNa/3 (IX) where N is Avogadro’s number, t i is 3.142, and a is the polarizability of the substance (Martin, 1978; Hansch and Leo, 1995). Taft steric constant (E*) was defined as the logarithm of the relative rate of the acid-catalyzed hydrolysis of substituted methyl ester (XC0 2 Me) compared to that of methyl acetate (MeCChMe): Esx = log k x c o jM c — log kifcco^ or Es* = log ( k x c O j I f c / k if a C Q jM e ) (X) k x c o jM . is the rate constant for hydrolysis of substituted ester, and kM ^ tlr is that for hydrolysis of methyl acetate. Hence, methyl is the reference group for E * values (Taft, 1956). 215 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hammett electronic parameters a (e.g. Co, c m , c p) and calculated dipole moment (p) were not significantly correlated with the inhibitory activities (log 1/ICso) against L1210 cells, therefore, they were not discussed here. In this QSAR analysis the log I/IC5 0 values were correlated with different physicochemical properties using non-weighted least square regression. The latest CQSAR program of BioByte Corp. (2001) was utilized to calculate Clog P (calculated log P) and CMR (calculated MR), and to derive all regression equations. Slog P was calculated by taking the sum of the n o f -CH=NNHCONHOH derived from the experimental log P value of RWL1 (see Chapter HI) and other n constants (Hansch et al., 1995; Lien, 1994). Other parameters such as E * , n were taken from the CQSAR program. An indicator variable I o f 1 was used for the oxygen- containing group (e.g. -OH. -OCH3, keto) at the ortho position of the Ar aromatic rings. V.2. Results and Discussion V.2.A. RWL1 to RWL36 The inhibitory activities (IC50 and log I/IC 50) and physicochemical properties of the 31 Schiff bases of hydroxysemicarbazide and 2 reference compounds (hydroxyurea, hydroxysemicarbazide) are summarized in Table V .l. The IC50 values range from 2.7 x 1 0 -6 M (log I/IC50 = 5.57) to 944.1 x l C T 6 M Gog I/IC50 = 3.03), Clog P from -2.91 to 4.11, and CMR from 1.57 to 8.91. Another reference 216 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. compound semicarbazide (H2NNHCONH2) was not included in the QSAR analysis because it lacked the common -OH group as compared to other compounds. The quantitative relationship between the structure (represented by the physicochemical properties) and the activity (represented by log I/IC 5 0) can be described by the following stepwise regression equations. (1) Log I/IC50 = 0.269 (0.165) Clog P + 3.866 (0.293) N = 33. R = 0.510. R2 = 0.260, Q2 = 0.158. s = 0.683, Fu , = 10.91. p < 0.005 (2) Log I/IC50 = 0.255 (0.146) Clog P + 0.665 (0.437) 1 + 3.618 (0.307) N = 33, R = 0.662, R2 = 0.438, Q2 = 0.288, s = 0.605, F2 j< , = 11.67, p < 0.0005: F, jo = 9.46, p < 0.001 (3) Log I/IC50 = 0.092 (0.158) Clog P + 0.097 (0.057) (Clog P)2 + 0.850 (0.392) I + 3.403 (0.293) N = 33, R = 0.777, R2 = 0.604, Q2 = 0.456, s = 0.517, F3 jq = 14.75, p < 0.0005; F, js = 12.19, p < 0.005 (4) Log I/IC50 = 0.313 (0.323) Clog P + 0.091 (0.056) (Clog P)2 + 0.950 (0.404) I - 0.229 (0.293) CMR + 4.549 (1.499) N = 33, R = 0.798, R2 = 0.637, Q2 = 0.458, s = 0.503, F4 j 8 = 12.29, p < 0.0005; F ij> 8 = 2.55, p < 0.25 217 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (5) Log l/IC st = 0.465 (0.114) Clog P + 0.088 (0.019) (Clog P)2 +1.012 (0.137) I - 0329 (0.100) CMR + 4423 (0508) N = 30, R = 0 579, R2 = 0458, Q2 = 0429, s = 0.102, = 14235, p < 0.0005, deleted outliers RWL-21,27,10 with deviations >2s. (6) Log I/IC5 0 = 0.434 (0.187) Clog P + 0.075 (0.046) (Clog P)2 + 1.059 (0374) I - 0.314 (0.206) CMR + 5.000 (1.056) N = 33, R = 0.839, R2 = 0.704, Q2 = 0.590, s = 0.455, F4J8 = 16.64, p < 0.0005, the log D values were used for the ionized compounds (RWL-2,4 ,7 , 10,26). (7) Log I/IC5 0 = 0.492 (0.072) Clog P + 0.087 (0.018) (Clog P)2 + 1.047 (0.145) I - 0.350 (0.078) CMR + 5.032 (0.401) N = 31, R = 0.977, R2 = 0.954, Q2 = 0.932, s = 0.171, F4J6 = 135.18, p < 0.0005, the log D values were used for the ionized compounds (RWL-2,4, 7, 10,26), and RWL-21,27 were deleted as outliers with deviations > 2s. The statistical parameters describing the regression are N, the number of data points upon which the equation is based; R, the correlation coefficient; Q2 , a measure of the predictive power of the equation (also called cross-validated R2 ); and s, the standard deviation from the regression. The level of significance of single- or multiple-variable equations, and that of addition of a single variable were examined 218 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by overall and stepwise F test, respectively. Probability of being a chance correlation is designated as p. The numbers in parentheses are 95% confidence intervals of each coefficient in the equations. When Clog P was replaced with Slog P in the regression analysis, equations 8 to 12 were obtained. (8) Log l/ICso = 0.273 (0.163) Slog P + 3.950 (0.264) N = 33, R = 0.521, R2 = 0.271, Q2 = 0.170, s = 0.678, F, j , = 11.55, p < 0.005 (9) Log l/ICso = 0.270 (0.141) Slog P + 0.699 (0.424) I + 3.677 (0.283) N = 33, R = 0.684, R2 = 0.468, Q2 = 0.323, s = 0.588, F^o = 13.66, p < 0.0005; F! jo = 1149, p < 0.005 (10) Log l/ICso = 0.164 (0.140) Slog P + 0.090 (0.056) (Slog P)2 + 0.857 (0384) I + 3.450(0.285) N = 33, R = 0.784, R2 = 0.614, Q2 = 0.473, s = 0.510, F3 j 9 = 15.38, p < 0.0005; F,j9 = 10.95, p < 0.005 (11) Log l/ICso = 0.400 (0.275) Slog P + 0.085 (0.054) (Slog P)2 + 1.005 (0.396) I - 0.255 (0.259) CMR + 4.794 (1.394) N = 33, R = 0.814, R2 = 0.663, Q2 = 0.493, s = 0.485, F4 jg = 13.76, p < 0.0005; Fijg = 4.05, p < 0.10 219 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (12) Log 1/IC* = (1503 (0.112) Slog P + 0.084 (0.02®) (Slog V? + 1.039 (0.148) I - 0313 (0.101) CMR + 4385 (0342) N = 30, R = 0 3 7 4 , R2 = 0352, Q2 = 0324, s = 0.174, F ^ = 123.03, p < 0.0005, deleted outliers RWL-21,27,10 with deviations > 2s. (13) Log l/ICso = 0.473 (0.174) Slog D + 0.067 (0.042) (Slog D)2 + 1.074 (0384) I - 0.302 (0.202) CMR + 5.085 (1.059) N = 33, R = 0.833, R2 = 0.693, Q2 = 0.549, s = 0.463, F4 3 = 15.82; p = 0.0005, the log D values were used for the ionized compounds (RWL-2,4, 7, 10,26). (14) Log l/ICso = 0.543 (0.068) Slog D + 0.082 (0.017) (Slog D)2 + 1.074 (0.153) I - 0.344 (0.078) CMR + 5.154 (0.410) N = 31, R = 0.975, R2 = 0.950, Q2 = 0.929, s = 0.178, F4J6 = 124.23, p < 0.0005, the log D values were used for the ionized compounds (RWL-2,4 ,7 , 10,26), RWL-21,27 were deleted as outliers with deviations > 2s. Non-weighted least square regression analysis of the data reveals three parameters to be the best predictors of the inhibitory activities of the newly synthesized compounds against L1210 cells. These parameters are Clog P (or Slog P), CMR and an indicator variable I. 1) Clog P (or Slog P), a measure of the hydrophobicity of a given compound, makes a positive contribution to the inhibitory 220 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activities of the compounds tested. The inhibitory activities (log l/ICso) nonlinearly depend on Gog P (or Slog P). It is worth to note that the coefficients of G og P (or Slog P) and (Gog P)2 [or (Slog P)2 ] are both positive. This is different from conventional parabolic or reversed parabolic dependence of biological activities cm hydrophobicity. 2) CMR, a measure of molecular size and polarizability, makes a negative contribution to the inhibitory activities against L1210 cells, suggesting that as CMR increases, the inhibitory activity decreases after correcting the contribution of hydrophobicity (G og P or Slog P). 3) I, representing the presence (I = 1) or absence (I = 0) of an oxygen-containing group (e.g. -O H , -OCH3, keto) at ortho position of the Ar aromatic rings, makes positive contribution to the inhibitory activities. This indicates that addition of an oxygen-containing substituent at the ortho position is beneficial to the inhibitory activity. By the best two equations (Eqs. 5 and 12), 95.8% and 95.2% of the variances in the data can be accounted for. RWL-10, 21, 27 were the statistical outliers with deviations > 2s. Upon deletion of the outliers, better equations with higher R values of 0.979 and 0.976, and lower s values of 0.162 and 0.174 were obtained (Eq. 5 and Eq. 12, respectively), revealing that RWL-10, 21, and 27 might have different limiting steps. Besides the common pharmacophore of -NNHCONHOH, the three phenolic OH groups of RWL21 may act as additional free radical scavengers, and/or chelate the Fe(III) ions which are required for the RR enzyme activity. At this point it is hard to explain the outlier behavior of RWL27 which contains a 5-nitrothienyl as Ar group. Further study is needed to clarify the point 221 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The outlier behavior (observed log I/IC 5 0 < calculated log l/ICso, deviation = -1.128) of RWL10 is probably resulted from the ionization of the phenolic OH group. The phenolic OH group with a pKa of 3.99 (BioByte Corp., 2001) is about 99.96% ionized at the physiological condition (pH = 7.4) due to the strong electron- withdrawing effect of 3,5-dinitro groups. The degrees of ionization of RWL-2,4,7, and 26 range from 26-56 %. The distribution coefficients Gog D) of these ionized compounds were calculated according to the relationship of log D and log P (Lien, 1994), and then used in the regression analysis. RWL10 with a deviation of -0.056 was no longer an outlier in equations 7 and 14, due to the considerable difference between log P (0.73) and log D (-2.68) (see Figures V .l and V2). However, the use of the log D values of RWL-2, 4, 7, and 26 did not significantly improve the correlation, simply because only 0.13 to 0.37 log unit differences were observed between the log D and log P values for the four compounds (see Table V .l). It is interesting to note that hydroxyurea and hydroxysemicarbazide fit equations 5 and 12 very well, suggesting that hydroxyurea, hydroxysemicarbazide and Schiff bases of hydroxysemicarbazide have the same rate limiting step and mechanism as inhibitors of tumor cells. Here the QSAR analysis provides an insight for the mechanism of action of the compounds tested against tumor cells. The squared correlation matrix of the physicochemical properties used in the regression analysis is shown in Table V.2. From Table V.2, one can see that Gog P (or Slog P) and CMR are highly interdependent, and the other parameters have no 222 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. covariance with each other. A plot of calculated log I/IC 5 0 vs. observed log l/ICso using Eq. 5 are shown in Figure V J. When R m , a hydrophobic parameter converted from Rf obtained by the TLC experiment (see Chapter IE, eluent = methanol : ethyl acetate : chloroform, 2:3:4, v/v/v), was used in the regression analysis, equations 15 and 16 were observed. From the decreased R2 , Q2 and increased s values as compared with equations 5 and 1 2 , it is obvious that R m is not as good a descriptor to correlate with the inhibitory activities Gog I/IC5 0) as Clog P (or Slog P). (15) Log l/ICso = -0 748 (0.477) CMR + 0.078 (0.041) (CMR) 2 - 1.152 (0.659) Rm + 1.895 (0.829) Rm 2 + 0.666 (0.364) I + 4.894 (1.350) N = 33, R = 0.840, R2 = 0.706, Q2 = 0.507, s = 0.462, F5 .2 7 = 12.94, p < 0.0005 (16) Log I/IC50 = -0.804 (0.313) CMR + 0.084 (0.027) (CMR)2 - 1.083 (0.451) R m + 1.756 (0.564) R m 2 + 0.901 (0.251) I + 4.956 (0.879) N = 30, R = 0.938, R2 = 0.880, Q2 = 0.739, s = 0.298, FS m = 35.12, p < 0.0005, deleted outliers RW L-27,16,10 with deviations > 2s. The antitumor activities Gog l/ICso) were not significantly correlated with log MW, dipole moment (ji) and the maximum hydrogen bonding capacity (H j> ) (Lien and Gao, 1995), as shown by equations 17-19. 223 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (17) Log I/IC5 0 = -25.767 (30.397) log MW + 6.150 (6.802) (log MW)2 + 0.619 (0.782) 1-0.121 (0.165) p - 0.061 (0.156) Hb + 31.500 (33.107) N = 33, R = 0.694, R2 = 0.482, Q2 = 0.090, s = 0.613, F5J7 = 5.02, p < 0.005 (18) Log l/ICso = -23.881 (19.726) log MW + 5.674 (4.416) (log MW)2 + 1.074 (0.539) I + 0.030 (0.121) p - 0.215 (0.124) Hb + 30.906 (21.372) N = 30, R = 0.876, R2 = 0.767, Q2 = 0.616, s = 0.390, FSM = 15.81, p < 0.0005 (19) Log l/ICso = -23.698 (19.380) log MW + 5.643 (4.340) (log MW)2 + 1.065 (0.529) I - 0.212 (0.122) H b + 30.718 (20.999) N = 30, R = 0.874, R2 = 0.765, Q2 = 0.633, s = 0.384, F S ja = 20.30, p < 0.0005 It has been reported that Rm is related to log P by the following equation: Rm = a log P + b (Bate-Smith and Westall, 1950; Boyce and Miborrow, 1965; Lien, 1994). The relationship was examined in this study (see equations 20,21). (20) Clog P = -2.362 (1.061) Rm + 0.707 (0.436) n = 33, R = 0.628, R2 = 0.395, Q2 = 0.252, s = 1.172, FU 1 = 2023, p < 0.0005 224 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (21) Slog P = -2347 (1.049) R* + 0.212 (0.431) n = 33, R = 0.631, R2 = 0398, Q2 = 0.263, s = 1.158, Fu , = 20.46, p < 0.0005 Because of the limits inherent in the mathematical formula R m = log (1/Rf - 1), Rm ranges only from -1.996 to +1.996 for all possible compounds. This makes it less sensitive than the direct measurement of log P. From equations 20 and 21, one can see that in terms of hydrophobicity Rm is not as good as log P. Nevertheless, for many compounds it is much easier and more economical to measure Rm than log P. The Clog P values (calculated by using the CQSAR program) for the 31 Schiff bases of hydroxysemicarbazide were highly correlated to Slog P (calculated by taking the sum of the 7 1 of -CH=NNHCONHOH derived from the experimental log P value of RWL1 and other it constants), with a mean difference of 033 (Gog P - Slog P), as shown by equation 22: (22) Clog P = 0.990 (0.047) Slog P + 0.328 (0.078) n = 31, R = 0.992, R2 = 0.985, Q2 = 0.982, s = 0.187, F ,^ = 1860.70, p < 0.0005 The entire set of the Schiff bases of hydroxysemicarbazide can be divided into two groups according to the antitumor activities. The first group includes 14 compounds less active than HU (ICso > 82 pM), and the second group consists of 17 225 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. compounds more active than HU (IC50 < 82 jiM). A careful comparison reveals that the first group of compounds (the average log P = 0.45) are more hydrophilic than the second group (the average log P = 1.89). Eleven out of the 14 compounds in the first group do not carry an ortho oxygen-containing group. In contrast, 10 out of the 17 compounds in the second group contain an ortho oxygen-containing group. Although the first group of compounds are more lipophilic than HU, but they have much higher MR values (5.75 vs. 1.57), which counterbalances the increases in the log P values, and makes the first group of compounds less active than HU. Overall, it seems that the lipophilicity makes the most important contribution to the anti tumor activity, following by the indicator variable (I). MR only counterbalances the lipophilicity, and is least critical among the three parameters. These points revealed by the comparison are consistent with the regression equations presented before. The QSAR analysis indicates that the optimal compound has not been synthesized in the study. More active compounds (the structures not shown) with the calculated IC5 0 values < 0.02 pM have been predicted from the regression equations, and should be synthesized and tested in the future study. 226 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table V .l. The inhibitory activities and physicochemical properties of hydroxyurea (HU), semicarbazide (SC), hydroxysemicarbazide (HSC), and Schiff bases of HSC analyzed in the regression analysis. code ICS o±s.d." Iogl/IC S O (M) Dev! c io g F Slog I*1 CMR* 1 * Log MW p(D )r Hb * (xlO ^M ) Obsd Calcd1 ’ *HU 82.016.0 4.086 3.856 0.230 (60.6)h s c j >2192.2 HSC 281.6121.4 3.550 3.680 -0.130 RWL1 39.512.7 4.403 4.332 0.071 RWL2 6.510.6 5.187 5.092 0.095 RWL3 10.111.0 4.996 4.905 0.091 RWL4 7.211.7 5.143 5.191 -0.048 RWL5 37.715.7 4.424 4.536 -0.112 RWL6 80.413.5 4.095 4.271 -0.176 -1.80' -1.80' 1.57 0 1.88 3.31 10 -2.75* -2.751 1.78 -2.91 -3.26 1.94 0 1.96 1.70 12 1.94 1.64k 5.55 0 2.39 1.48 10 1.88 (1.75)' O O O S 6.17 1 2.42 3.71 13 1.55 1.16 5.97 1 2.44 4.33 13 2.23 (2.03)' 1.97 (1.77)' 6.75 1 2.55 4.03 13 1.32 1.01 6.59 1 2.48 5.17 15 2.18 1.91 6.34 0 2.48 2.85 10 8 ■ o o 3 _C "5 C o u > _o JD e s X x CO - — r- cs cs 2 W a w A 3.32 1.74 4.38 6.91 3.69 o o CO CO 5.18 3.23 3.41 L o g MW 2.65 2.31 2.35 2.46 2.35 2.32 2.32 2.38 2.35 - - ' o o - © o o - - CMR o 0 0 5.52 6.33 6.41 5.65 5.66 5.66 6.27 5.81 O m 00 C t 2.39 :2.19)' 610 0.94 -0.47 -3.88)1 o o o 0.74 0.74 0.72 0.07 3 3 ft. 00 e Q Q '> * . V S 0 0 CS ^ cs 0.49 1.22 0.73 -2.68)1 o oo o 0.97 0.97 1.06 0.49 ► Q in u 5 M 5 K > c U c 1 e s U •o H XI e » ■ g u 0 0 ■vj- o t— C S O ' o C O d o o o © vn in O ’ cs C O m vn C O CO oo © es 00 in CO vs CO vri C O CO in o o + 1 5 £ r - m •H 0 0 CO o v s " cs 5 0 0 IQ « N 5 5 - o T 9 Tt C_^ r ; cs VS 00 o eo m CO r— C v VO C v o I — 1 o d ■ o ■ o • T f O ' eo O ' C v eo eo vs c v in co vo C v m eo vo vo O V O VO o- m o ■ * J - r ~ C M CO O v o CO ov cs cs • v r in es CO CO v C —. IO —■ o o o o ■ H C O O v CV CO - H o o o o vo CO 2 £ » S 2 £ es CO +i r- 2 CO CO o ir i +j © CO cs’ -H e s © VO VO 228 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Q A £ S e o 5 S - M £ Cfl C£ O □ > a u «c B £ e •o u 8 •o tt £ e p ~ C M © vO Ov o Ov © 0 0 wo CO CO C M CO CO ■ v p v o •vp ■ V p CO CO C M C M C M C M - © o - CO ■ v i w o w o TP CO v o' 0 0 w o w o o C M TP C M p- 0 0 w o d C M * C M o 1 C M W O v p OV o o C M o c m " C M o • o o 3 w o ■ v p 8 C M c o p ~ d 1 o ' © 1 o ' • r - 0 0 o CO c^ o w o © w o © • V p ' ■ V p ■ v p ■o u 3 C o o > « j X ) C S H u < = « ■ e e w cs C v 0 0 r- — C M r - n CM — O vo co C M o v o 8 . C M — o co vo C M O o © o I T ) w o w o C M o o o o c o E VO OV — r - ~ r-: t- CO o o o r - o 5 o * Ov vo o CO wo ov wo OS C-; Vp wo C v C M vo C M vO CO C M C M C M © o o ov C M C M P - V O w o ■ v p ’ wo - 0 . 2 0 -0.95 C M CO 0.13 8 o C M VO U O O ^ v 0 0 C M X C M W O 8 o ' C M V O o o C M o VO 0 0 0 0 r ~ 'p C M 3 C M CO CO CO CO Ov CO © . •v p Ov s ■ v p Ov 00 CO ■ V p ’ Ov o v -H 0 0 ■^- o C O ■ vp o v -H P - C M CO o o o o CO CO C M + s o o o ' CO o v 13 £ es wo wo C M v o o W O CO wo v o s s +i C M s o v r - C M CO w o Ov 1 o vo c o C M o wo o CO c o 0 0 C M 1 o v o o w o 3 CO C M p ^ ? ■ V C o Ov CO wo E V O p - wo o v o v CO CO w o r» Ov CO s vo d E p^ C M 229 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table V .l. (continued) code IC 50 /y !«•* \/f\ _ log 1/ICso (M) Dev. C logP SlogP CMR I Log MW M D) H„ 1U l ¥ i ) obsd. calcd. RWL28 476.6±37.3 3.322 3.477 -0.155 1.04 0.71 6.16 0 2.34 4.10 12 RWL31 28.2±2.6 4.550 4.530 0.020 1.72 1.38 7.50 1 2.50 1.57 14 RWL32 32.210.4 4.492 4.398 0.094 1.72 1.38 7.90 1 2.46 2.99 14 RWL33 178.314.5 3.749 3.461 0.288 0.98 0.58 6.09 0 2.38 4.42 14 RWL35 4.410.0 5.357 5.158 0.199 3.90 3.64 8.88 0 2.47 3.96 10 RWL36 5.410.4 5.268 5.394 -0.126 4.11 3.84 8.91 0 2.50 4.26 10 " T h e a v e ra g e IC 5 0 ;h C a lc u la te d fro m E q . 5. r C a lc u la te d b y u sin g th e C Q S A R p ro g ra m o f B io B y tc (2001); rf C a lc u la te d b y ta k in g th e su m o f th e n o f -C H = N N H C O N H O H d e riv e d fro m th e e x p e rim e n ta l lo g P v a lu e o f R W L 1 a n d o th e r n c o n s ta n ts ; ' an in d ic a to r v a ria b le in d ic a tin g th e p re se n c e (I = 1 ) o r a b se n c e (I = 0) o f an o x y g e n -c o n ta in in g su b stitu e n t at th e o rth o p o sitio n o f th e A r a ro m a tic rin g s; f C a lc u la te d b y u sin g H y p c rC h e m p ro g ra m ; * H y d ro g e n b o n d in g c a p a c ity (L ie n a n d G a o , 1995); h F ro m T ai e t al. 1984; 1 M e a su re d lo g P v a lu e s o b ta in e d fro m th e C Q S A R p ro g ra m ; ^ N o t in c lu d e d in th e re g re ssio n a n a ly s is ; * E x p e rim e n ta l lo g P v a lu e fro m th is stu d y ; 1 C a lc u la te d d istrib u tio n c o e ffic ie n ts (lo g D); m S ta tis tic a l o u tlie rs, e x c lu d e d fro m E q s . 5 a n d 12; " C a lc u la te d fro m E q . 7. Table V.2. The squared correlation matrix (R2 ) o f the physicochemical parameters used in the regression analysis for hydroxyurea, hydroxysemicarbazide, and Schiff bases of hydroxysemicarbazide (n = 33). a o g p SlogP CMR I Log MW R m It ClogP 1.000 0.983 0.809 0.004 0.652 0.395 0.030 0.092 SlogP 1.000 0.744 0.000 0.593 0.397 0.019 0.134 CMR 1.000 0.046 0.792 0.169 0.030 0.000 I 1.000 0.176 0.009 0.022 0.483 Log MW 1.000 0.164 0.032 0.035 Rm 1.000 0.028 0.125 It 1.000 0.024 Hb 1.000 231 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 o 1 , co C M S • . cc §S O ) o 4 3 2 1 0 - 1 HSC ♦ RWL21 ♦ HU ♦ RWL27 ♦ ♦ V ♦ RWL10 - 2 - 1 0 1 2 3 Clog P Figure V. 1. A plot of log I/IC 50 (after correcting for differences in CMR and I) vs. Clog P for the compounds analyzed (Eq. 4, n = 33, R = 0.798). 232 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. + — C M §8 9 ui i ■ c c © s 1 0 * O O o «" 1 0 o> 9 o o 4 3 2 RWL10 HSC 1 HU ♦ ♦ 0 1 -1 -4 -3 -2 -1 0 1 2 3 Clog P Figure V.2. A plot of log 1/ICso (after correcting for differences in CMR and I) vs. Clog P for the compounds analyzed (Eq. 7, n = 31, R = 0.977). The log D values of the ionized compounds (RWL-2,4,7,10 and 26) were used in the equation. RWL-21 and 27 were deleted as outliers with the deviations > 2s. 233 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. " O © o a o, o to O 9 6 5 4 2 2 3 4 5 6 log 1/IC50 (observed) Figure V J. A plot of calculated log 1/ICso vs. observed log I/IC 5 0 values using Eq. 5 (n = 30, R = 0.979). 234 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V.2JJ. RWL1 to RWL21 RWL1 to RWL21 contain a substituted phenyl moiety in common (see Figure V.4 for the general structure). As a subgroup, the following QSAR analysis was further conducted to elucidate the physicochemical requirements at different positions of the phenyl ring. The substituent constants and inhibitory activities Gog I/IC 5 0 ) of RWL1 to RWL21 are summarized in Table V 3. The physicochemical parameters used in equations 23 to 25 are it.5, Hansch-Fujita substituent it constant at position 5; Sitsub, the sum of the it at positions 2 to 6 ; E,^, Taft steric parameter at position 2; XMRjab, the sum of the MR of substituents at positions 2 to 6 (see Figure V.4). The squared correlation matrix (R2 ) of these parameters in Table V.4 indicates that the physicochemical parameters used in the regression analysis have no covariance with each other. (23) Log I/IC5 0 = 1-335 (0.526) il5 + 3.846 (0.237) N = 20, R = 0.782, R2 = 0.612, Q 2 = 0.538, s = 0.449, F u 8 = 28.37, p < 0.0005 (24) Log I/IC5 0 = 0.563 (0.145) In** -1.751 (0.498) + 3.536 (0.197) N = 20, R = 0.924, R2 = 0.854, Q 2 = 0.794, s = 0.283, F2 .1 7 = 49.82, p < 0.0005 235 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (25) Log 1/ICst = 0.739 (0.161) S X * - 1.921 (0.411) - 0.313 (0.199) XMR,ab + 3.990 (0329) N = 20, R = 0.956, R2 = 0.914, Q2 = 0.846, s = 0.224, Fw« = 56.70, p < 0.0005; Fu< = 11.12, p < 0.005 The inhibitory activities (log I/IC50) of the compounds analyzed are positively dependent on Snmb, and negatively dependent on E,^ and ZM Rsah- SJtsub is the most important contributor to the inhibitory activities, followed by E * . 2. and ZM R sub- This implies more hydrophobic substituents at positions 2-6 will improve the inhibitory activities. Simultaneously, a larger group (more negative Ej value) at position 2 and smaller groups Gower SMRnb) on other positions are required to make more active compounds. 236 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table V.3. The inhibitory activities and physicochemical properties of the phenyl-containing Schiff bases of hydroxysemicarbazide (RWL1 to RWL21) used in the regression analysis (see Figure V.4 for the general structure). code Substituent IC5 0 “ (xlO* M) log I/IC 50 (M) Obsd" Calcdb Dev.c Es-2d 7t-5d 5Xubd lM R rob d RWL1 3 -CF3 39.5 4.403 4.387 0.016 0.00 0.00 0.88 0.81 RWL2 2-OH-3.5-C1 6.5 5.187 5.102 0.085 -0.55 0.71 0.75 1.59 RWL3 2-OH-5-Br 10.1 4.996 4.755 0.241 -0.55 0.86 0.19 1.37 RWL4 2-OH-3,5-Br 7.2 5.143 5.145 -0.002 -0.55 0.86 1.05 2.16 RWL5 2-OH-3-OCH3 -5-Br 37.7 4.424 4.526 -0.102 -0.55 0.86 0.17 2.06 RWL6 3-1 80.4 4.095 4.285 -0.190 0.00 0.00 1.12 1.70 RWL7 2-OH-3.5-I 4.7 5.328 5.212 0.116 -0.55 1.12 1.57 3.17 RWL8 4-CN 208.5 3.681 3.274 0.407 0.00 0.00 -0.57 0.94 RWL9 4-N(CH3 )2 446.3 3.350 3.539 -0.189 0.00 0.00 0.18 1.86 T able V.3. (continued) cod e Substituent IC 50 logl/ICso(M ) D e v E ,.2 r t . 3 2 Xub SM RW b •o 'a U ■ o 0 9 A o N O 0 0 s ON O 1 C O P M 00 P M P M O PM O 1 O 1 O 1 O 1 0 0 PM s 8 8 O 1 0 0 © m in s 8 8 O * © 0 0 -0.469 -0.057 6610- eero- m in m N O in • n J- P M C O N O PM CO NO C O C O C O CO 0 0 r- C M NO O O O C O * o © C M 00 ON ON to c o c o ON ON c o 0 0 0 0 NO c o N O CO 0 Z 1 \ n co 1 X o C M O z I CO X u 0 1 m s d P M o ■ N j " X 8 ■ in C M * O n N O CO O O O o c o CO O N O CO O N C M PM ■ N T ■ N f i n C M CO CO TP Tf’ — 1 C M s s r- C M C M « n r- o N O N O 0 0 C M N O O N m m m in in « n © 1 9 d 1 CO 0 0 o o C M o 0 0 r- P M PM 00 P M mm mm *n • Nt • 4 r • nj- O n 00 0 CO O CO CO PM X u o 238 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission. Table V.3. (continued) code substituent IC 5 0 (xlO"* M) log I/IC 50 (M) Dev. E » -2 n. 5 £ftfub Z M R n ib Obsd Calcd RWL19 2,4-OH 130.8 3.883 3.813 0.070 -0.55 0.00 -1.34 0.77 RWL20 4-NHCOCHj 944.1 3.025 2.709 0.316 0.00 0.00 -0.97 1.80 RWL21* 2,3,4-OH 2.7 5.565 3.261* 2.304* -0.55 0.00 -2.01 0.95 Notes:a The inhibitory activities against mouse leukemia L1210 cells;b Calculated from Eq. 25. c Deviation (observed log 1/ICjo - calculated log I/IC 5 0) ;d Obtained from the CQSAR program of BioByte Corp. (2001);c A statistical outlier, excluded from Eqs. 23 to 25. K v o Table V A The squared correlation matrix (R2 ) of die physicochemical parameters used in the regression analysis for RWL1 to RWL20 (n = 20). E * - 2 S t l j a i ) Jt-J S ^ ^ R j n b E j . 2 1.000 0.050 0.263 0.002 1.000 0.168 0.448 t t - 5 1.000 0.107 S M R j a b 1.000 meta ortho para 4 N -C -N -O H H H m eta ortho Figure V A The general structure of the phenyl-containing Schiff bases of hydroxysemicarbazide. 240 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER VI. CONCLUSIONS Ribonucleotide reductase (RR) is a rate-limiting enzyme in de novo DNA synthesis, and is considered to be a potential target for cancer chemotherapy. To overcome the drawbacks of hydroxyurea (HU, H 2NCONHOH), a known RR inhibitor, thirty-one Schiff bases of hydroxysemicarbazide (Ar- CH=NNHCONHOH) were designed and synthesized. Their chemical structures and purity were established by NMR, E R and MS spectra, and by TLC and elemental analyses. The antitumor activities of the 31 Schiff bases of hydroxysemicarbazide were first tested against murine leukemia L1210 cells using the MTS/PES microculture tetrazolium assay in 96-well plates. Seventeen of the 31 compounds exhibited higher antitumor activities than HU against the L1210 cells. Six compounds with IC50 values in the micromolar range (2.7 - 7.2 pM) were found to be 1 1 to 30-fold more potent than HU (IC50 = 82 pM). Based on the results obtained from the L1210 cell line, three active compounds were further tested against a human leukemia cell line (CCRF-CEM), seventeen tested against four solid tumor cell lines (B16, CHO, HT29, ZR75), and one tested against a non-cancer cell line (3T3 Swiss mouse embryo fibroblasts). Among these, RWL-4, 21, 35 inhibited the CCRF-CEM cells with IC5 0 values ranging hnom 2.7 to 7.0 pM. RWL35 was the strongest inhibitor, and showed 74 241 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to 692-fold activity, compared to HU, against the solid tumor cell lines tested. The in vitro relative selectivity study demonstrated that RWL35 had more favorable selectivity (8-187 fold) than HU against the cancer cells. RWL35 had no cross-resistance with HU and gemcitabine, two known RR inhibitors acting at the different sites of the same reductase enzyme. From the results of the stability study in phosphate buffers at room temperature, there was no change in the UV absorbance in 72 hours at pH 3 for RWL1. At 25°C 97.3% of RWL1 remained at pH7, and 78.7% remained at pH 11 at the end of 72 hours. The partition coefficient (log P) and ionization constants (pKa) of a model compound, RWL1, were measured by the shake-flask method, and then the Hansch-Fujita n constant of the functional group -CH=NNHCONHOH was derived for the calculation of log P of other congeners. Besides the essential pharmacophore (-NHCONHOH), among the various physicochemical parameters examined in the QSAR analysis, hydrophobicity (log P), molecular size/polarizability (MR) and the indicator variable (I) for an oxygen-containing group at the ortho position turned out to be the important determinants of the antitumor activities observed. The antitumor activities positively depend on log P and I, and negatively depend on MR. The QSAR analysis indicates that the optimal compound has not been synthesized in this study. More active compounds with the calculated IC5 0 values < 0.02 pM can be predicted by the 242 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regression equations. The findings obtained from the QSAR analysis can be used as useful guidelines for further structural modification and development of the second generation of hydroxysemicarbazide derivatives as anticancer drugs. From the chemical structure point of view, the HU-based Schiff bases of hydroxysemicarbazide most likely target the same enzyme as HU. This is further supported by the QSAR analysis in which HU fits the regression equations very well. The results of this study showed for the first time that, at least in vitro, six Schiff bases of hydroxysemicarbazide were remarkable inhibitors of the different tumor cells including the HU-resistant tumor cells. The six compounds may have potential therapeutic applications in the treatment of leukemia, solid cancers (e.g. melanoma and breast cancer), and HU-resistant cancers. The results obtained from the in vitro selectivity study imply that the compounds may have low toxicity and favorable selectivity in vivo. The most active compounds merit further in vivo anti tumor and toxicity testing, as well as enzyme inhibition study for further development as anticancer drugs. 243 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES Aberg A, Hahne S, Karlsson M, Larsson A, Ormo M, Ahgrcn A and Sjobeig A. (1989) Evidence for two different classes of redox-active cysteines in ribonucleotide reductase of Escherichia coli. J. B iol Chem. 264:12249-12252. Aberg A, Nordlund P and Eklund H. (1993) Unusual clustering of carboxyl side chains in the core of iron-free ribonucleotide reductase. Nature 361: 276-278. Acorn NMR Inc. 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(1994) Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine. Cancer Res. 54: 3686-3691. Young P, Ohman M, Xu MQ, Shub DA and Sjoberg BM. (1994) Intron-containing T4 bacteriophage gene sunY encodes an anaerobic ribonucleotide reductase. 7. Biol. Chem. 269:20229-20232. Zhou BS, Hsu NY, Pan BC, Doroshow JH and Yen Y. (1995) Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M l is sufficient to increase resistance to hydroxyurea in transfected cells. Cancer Res. 55:1328-1333. 255 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendices Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix A. The IR Spectra Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. r ? a l l s I l e a ■ 9 258 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-l. T h e infrared sp ectru m o f RWL1 . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-2. T h e in frared spectrum o f RWL2. % 8 s s i § « 0 * S » " * m v u 1 * 0 0 * Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-3. The infrared spectrum o fR W L 3. NOirowiHve* 261 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -4. The infrared spectrum of R W L 4 . 262 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -5. T h e infrared spectrum o f RWL5. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-6. T h e infrared spectrum o f RWL6. NOlfStwfMVU U O M 264 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-7. The infrared spectrum of RW L7. m w J B P Wfw » n IM 3IM 265 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -8. T h e infrared spectrum o f RWL8. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -9. T h e infrared sp ectru m o f RWL9. i H O itS iw tN v e i I N C M 267 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-1 0 . T h e in frared spectrum o f RWL10. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-ll. T h e infrared spectrum ofRWL11. HOOtnfNTU IM M l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-12. T h e infrared spectrum o f RWL12. I a 270 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -13. T h e infrared sp ectru m o f RWL13. 271 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright o - I i rf 273 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-16. T h e infrared spectrum o f RWL16. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-1 7 . T h e infrared spectrum o f RWL17. MOKfmfwvu 275 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-18. T h e in frared sp ectru m o f RWL18. M e m m f » r * u i M O S t f 276 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -19, T h e in frared spectrum o f RWL19. S ^ .J Iil^ fji.^ S SSSSSieg^r:^ n r - ; ^ i 7 r i % i s ^ j r j j f c ^ r : : - ^ r " W i -,-,' r t ' ' T ^ ’:5“ ^ ^ ^ m i t a r j r t ; : ^ * K * i»; ^ ^ i ..» ^ f r ^ 5 ? V - t t r e - i i . ■ iiiij:-'::.r* t:8 ‘? . ^1 ."- .-f— . ; i , , , : t 5 . . . ,.', ^ '- ^ 1 * * * 1 . i.rrj n •yi, : . ’"»^^7 ^ 7 _r .f T » ' _j“"*'"""' '' "I * * - 1 " p * * ■ " “'• jiT l'S L ’ I l M : : . . . . I . : : ''.T : ' : ; ; ; : r.z i t : ■ ; ■ m ^ii iJ':iii,::n”t'!i :';ri i: 'i"ii t t ; "Tit; :::tv ;■ ;• • • » • .- . • ■ • — ^ ! ! i i IM .: i i ! ; a ; ” i ; r ^ : - - i J : ^ i _ , : ; . . . j : . ; L . j :.;.;i.: ; . . i : : : . . - i . - .. v . . 1 , : . . . . . ilii:il[;i;il:f e ''< ;^ ^ i " - ' .;” ,j!!;i; ■ ■ ; ;■;.; I l p P p O L iliil 1 ^ 1 1 ; l h ^ f T : ; 1 .niljiu illJ-:!.1.^!..!;;..!!;--— n« jg..=- ng 3 r.p T ^:-':.': , j l g ^ : ! : ! ! ," " ■ .r i 'i r |; : T |: ! ,T rr!’ r:r:!'> 'i''" -i -crisr^ si : : ! L h . .( J ,.; ,,,,, " '■ T ip * - s - ;:1 '-:-sg i;; y i p s p ; ;^ sg iiii:Ti..i Jifi! L L " ^ i 4 ~ i 3 S 3 J ^ i i d l O i L J s i i liT:E!STT;Si*:3S3!jaS3SiS bnpuBtanW HEfaBfl t e a i g r a a » W 8 » riag i ao!5!Si:ip;GiJil^=ni^S5i: ; s3 B i p a H ! " 8 8 3 9 3 8 9 S — N O rtsn n w v u msoi 277 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-20. T h e in frared spectrum o f RWL20. N O H O w t M v e t H O M Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -21. T h e infrared spectrum o f RWL21. 2 ii m r ™ i. i j m , i r; m i 3 2 H K S 3 0 s^ i^ a e ^igiiT iim T iB ^ w p s P 5 f iS iS E iim L SEiifflssioiiaaaisi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-22. T h e in frared spectru m o f RWL23. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [ ~ p i rx«r| E J : r ^rifl |r h. h P m r rr % r U - = k r'l‘r t* r • t e ' a j n ^ m m s a s m :.~,: :;:^!H ZS3SgasiS Psl'j-iirisf^sSESgSSEl ^ ' - r = lEg:aa5'HE: -U ’ - \ ‘: 'M . ' " ' h**-1 * »H.:»«tiBfl r ^ . - w i . n ’■ « ' ' n :.- * * ? ! .. rv4 :” ^ 3 3 R IB S 9 9 ^ s S S S lS M Iia m m m z & m x a s m B m irii!!S K iii.iia s^ M S B H a a " 8 8 3 S 3 2 9 3 R 2 WOHW1NTI1 I H P t t 281 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-24. T h e infrared sp ectru m o f RWL26. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -2S. T h e in frared sp ectru m o f RWL27. t l l t e l l l l i ; ^ sif liSaniiji ? jji 1 2 iiliS s K -I if ^ ills !S .;iB H !iiS u j:jd !!!^ ^ : ^ ii3 ^ :D JE :J& ifc ^ J & s n j C ! i i ! u n ^ s n i i ! ; H : !^ n :L ^ ii:i!rr::T in 3 ^ .1 r^ iflS iE IIM i u l i i U j J i i i L u l l l i i i u J 'i i .i i i i l i - .'i j - l i l J ! : £ i ! ] ! i ! J l S ! ! « ii;!i^i®!l;ii!l!IU!M=SK^a33ia 1 5 I 1 I I mpFTppjr^priit g m f l r e a a i E g ia B fe a a m m s ^ s s s m m m B s s m 1 J 283 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-26. T h e infrared spectrum o f RWL28. £ i i n ; i i n a s i S 3 s a a E 3 i 3 i i i i i i L S s i i i s i a i a i a i i b e s s i s i g 8 I 7 J i s p | g | | | | | : p ;2 :tr.::r::;'^ - -’a n u .:::!-;-.:n :”:" J^*f-"jj 8 5 S ■ :u;.::.: :11:::::::;i;: :::l: l :.. H j ! ' r ; : : i i , . I j j j L Z ’ .'J j: itilSc l i i L ^ r J j S S B S O ' : ; ; : j - : ; " ; ; ; ! i - r ' j;: i v ; s r . - , ; : - '; . " V | \ Bl iii^IlSiSlaiiiSiiiisiaaagli v i i i i i s r a s a B i i B g S^iliii!i;121!i;:!.;siE3Sa31il s r?i:iiS ;S g2S gisasE 2 ^ ig ^ ^ i^ iliiip ijjijsagaeaig s i i i i ^ ^ a s ^ i M B a a i E s €:i • _ • • < iississ gaaaiis ^ p p i p s s M s s i S s B i a s a M m | | | g | i | | | | | | | | | | | | | | I E B S E i » l ! ! i i l l S l l E i 8 1 s § s a s s e s s « * NORimtMVit M M i 2 < 9 O s 284 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-27. T h e infrared spectrum o f RWL31. X u ^fL?!2lI131Sl4" 2 = ^ l l |l S S S p ^ — , : ; r : ; : u . ' : : : : : - : ) : : . ; ,! - ii :; * . : ! . ( ■ ! i : : l u . . l ; . . j s . — • ■ ■ - * ■ ~ J - ” :” v:-: - : } -a . ^ -r*a air- :--'' . ~ : v '~ :^ ::::: ! : ! \r ::! _ j3 a ! ilx = ': H ;i ; 1 : ~ i ‘HJ ■ tin in i: pir ’T :r » r ^ a “ 35d3!!Lb^^iiii2:S y i;;:5 ^ !::!ili:ii;;iiS £ E Jk : : rJ'-'VC^S, = : s H > ! H : ; : , . i } 'T i : , j! ! i r '3 ; : : .: h - ^ ^ s^J= 'ipW lBrT^lp^T? $ cs cn o E E « * o e o. C O •o fi £ .E o -C H cs ,:" ^ Z l a i i a a s « I i g ^ | i | | i i E i a | | | S S ^ ^ i i l l i i i i i i i e i ^ a n i i s i a a i i r s r s s s a a f s i i r • • t 33r™si«ans»* P I l l l l f ’ PISB E I ilfesel -E___ "8 8 3 s s a ■ o m iT C N n i Man 285 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M Om nwVM vu iM O O tf Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-29. The infrared spectrum ofR W L 33. ag ji s i i i i 3 i c s j 3 i i a a a s i s a s is ii3 3 E .! 53Z!isgam H Sia f O F i i n s i s i a s n a i a i s j H a S i i s i s i 3 B s a B « a i a S l i i l l ^ I I I S l K I O l S l i ^'His^siasaaaaiina « S E ii;e is ii5 S fiS H ia 2 S 3 3 5 H if S £ S 2 S B » H fla iia i ■i. S:!U.lr*l^St== — ,.i£ 3 £25 287 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-30. The infrared spectrum of RW L35. S ^ ^ S S ; ^ S S i l 2 i a H l l M I t e ^ S i S i J J £ ? I £ S M M « a I21S ;ir ~13&5BB*M 11 I s i ^ i ^ i a E i f l i a a a a M a t e ^ l E ^ S I S l i l i m a S i l i ^ j i M i i s a n f i n i i i a @ i ^ e i ^ 2 a s s a a n i i i a s V W M S s aasp"'1 -:*'!'" |i5 5 h .X 3 ^ ir :i: ■ ~r"" ; rrr nrr- C 1B 3IIE 3 ', ‘ ' V - : 1 ~ 3 i * i : )P F' i U r .! SniiS^iSw w ^s ;:i • : " r • ;: 'C ^ - v .t .:: I:-::-::,::-:--:;-:"- ••yjsi-’T T ! .. : ; .r: * £ £ , ; : • 1 3 :;: .,..ni:K ^i>3i— ^ j..-a-iij*i 1 ,1 :1 1 ; r - ".s . : :: n n w . - n - J . — - .■ ■ ■ ■ ' ~ ' u . ■ jj j $ m i | i 1 8 ;ilir-::"::H:i:j.:\:.:u: . .'u . :'' I 3 Ui-J-i1 — :::::::: .-. . — - ■ - 4 - _ CT2r? rrH n i:r:i:iT - ••-••";• 1 = *tg .JJiULj.iir.i.':::;.;:-.:-: .. _ _ ...., ' . nn .r; :U*:.S EJ 32 . , T S TA.-JI 9B S ^ ' k .L:,,: 33iJ:¥*5B3B ?I i i f f i l i l l i i ii ii! i S ^ a l l l k l n B E S B U r a H H n s n s s n x a f l a M :i!!i:» isi2 H M .jiiaiaa« i ~ 8 S 2 S 8 S e s g 288 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A -3 1 . The infrared spectrum of R W L 36. sisasaseiisasssiniiiiss s & m a s i g R r a s K K 'i i i S : 3!E3S3*SSS23!a2aBDm3BSt: 3 £ ^ iiis s a a n ra ra ii s k s ^ ^ s s s e u a i i s ^ s s ^ S @ K 3S B H I I I H H £ 3H I ^£^Ei^iaaiHiM2jas3; ^ip^|||aueiB aB |||fl if® ^ ililiiiJ S i^ :iii s SS:g3^S3SSSE3B5 J f i i - . . ^ j j L r ' S s i S S a S S r - - im i O Si.-SJ^3'3asaS3B::^iSi. jiiiiEii^nri3d3^aas3Si::::.:isii. llils : 3Si!K»i* lyifiSiiiaSgSSi":' W M S E I S S ^ S S S S S S ^ S S S ! t o f ^ 8 I ? ~ r i s I 5 r - -afs^p^ . “ a s e ^ - S i ; w z s z m ^ m a i s u m 83 I ;3 « £ :i3 • — gnq - I Z Z a a F < r . ; • r ,: LESSEE £3 9 2 ^ p s p 3 i ? s p m s : i 3 M « 5 S j a a B : . "8 8 3 s « a s NQOTWINTIt IMOIU 11 289 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A-32. T h e in frared sp ectru m o f hydroxysemicarbazide. Appendix B. The Mass Spectra Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O g n co m so n f t w n A -o ■ C O n ZX o o o ov N - 0 0 CM © in CM n> CM m CM 'O m " ® r—< ' v o o\ m C M in n CM m o m c o c o in in o in m n in o m r- so o in o v o m m 291 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B - l . The mass spectmm of R W L 5 . S & n o\ co v o in -d -C D n - < o •n ZX OX C M o N 0 > -C O m VO 0 > 00' CO in v o m o in in o m m ^ in m o v o v o in GO 292 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B - 2 . The mass spectrum of R W L 1 2 . o m o -m ZX z x o m in c « ov -in o J © m in o in o in o C N C N * -♦ fH m o n n in o © © m o v o v o in m o m m c o 293 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B -3 . T h e m ass spectrum ofRWL18. % o n n ci m r* c m m m n n °= ? ci ZX c < o o o -0 0 6 0 I © O in - m in in o > d(rrrp r o in m in m in m m m 294 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B -4 . T h e m ass spectrum o f RWL26. N WWWMWMM&lWUUWMfiattWMWWMM in*-»r-»csao**om*-ic^roco^aio«Hr,* c n tn m © o n n n n ZX -G O M Z X d a* -ci C l n o C l -«0 m « * v fH to a\ to m o to o in in in ^ to to in m rH 295 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B-5. T h e m ass spectrum o f RWL28. rt c o m c * n n ci ct w in in o n ro n n o in Z X d in m m C C l nn ci in r> ( N C l m o< C l o o o t in in o as as in in c o m m m ci ci 296 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B - 6 . The mass spectrum of R W L 3 5 . n n n n c i C l O Cl Cl Cl in ci o ci Z X ci o C l o C O at -C O . n M to re in N O in re w o to O ' -CO o IO in n n o m in m o m c o q o r- o m o in to « n m * o in o c t 297 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B -7 . The mass spectrum of R W L 3 6 .

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Asset Metadata

Creator Ren, Shijun (author)

Core Title Design, synthesis, biological testing and QSAR analysis of new Schiff bases of N-hydroxysemicarbazide as inhibitors of tumor cells

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

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

Tag chemistry, pharmaceutical,health sciences, oncology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Lien, Eric J. (committee chair), Haworth, Ian S. (committee member), Koda, Robert T. (committee member), Okamoto, Curtis T. (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-202705

Unique identifier UC11338979

Identifier 3065839.pdf (filename),usctheses-c16-202705 (legacy record id)

Legacy Identifier 3065839.pdf

Dmrecord 202705

Document Type Dissertation

Rights Ren, Shijun

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA