Triazaspiroalkenes as potential inhibitors of dihydrofolate reductase 2

TABLE OF CONTENTS TABLE OF CONTENTS PAGE i SUMMARY iv LIST OF TABLES vii LIST OF FIGURES ix LIST OF SCHEMES xi LIST OF ABBREVIATIONS xii INTRODUCTION CHAPTER 1. Introduction 1.1 The Overall Drug Discovery Process 1.2 Anticancer Drug Discovery 1.3 High Throughput Screening in Drug Discovery 1.3.1 The Magic Triangle of High Throughput Screening 1.3.2 High Throughput Screening for Enzyme Assay 1.3.3 High throughput Screening for Dihydrofolate Reductase (DHFR) Inhibitors 1.4 Dihydrofolate Reductase as Therapeutic Target 1.4.1 The Folate Pathway 1.4.2 Dihydrofolate Reductase 1.4.2.1 Human DHFR 1.4.2.2 Escherichia coli DHFR 1.4.2.3 Lactobacillus casei DHFR 1.4.3 DHFR Inhibitors as Therapeutics Agents 1.4.3.1 DHFR Inhibitors as Anticancer Agents 1.4.3.2 Classical DHFR Inhibitors 1.4.3.3 Non-Classical DHFR Inhibitors 1.4.3.4 Triazines 1.4.3.5 Diamino-triazaspiroalkenes 1.5 Synthesis of Triazaspiroalkenes 1.6 Biological Evaluation 1.6.1 DHFR Inhibition Assay 1.6.2 Antiproliferative Activity by MTT Assay 1.6.3 Cell Cycle Analysis by Flow Cytometry 1 4 10 12 CHAPTER 2. Rational and Objectives 49 15 15 18 18 20 22 24 25 29 33 34 36 40 44 44 45 47 i RESULTS AND DISCUSSION CHAPTER 3. High Throughput Screening for the Identification of Potential Murine DHFR Inhibitors 3.1 Method Development 3.2 Validation 3.3 Application of New HTS Method 3.4 Conclusion 57 CHAPTER 4. Diamino-3’- or 4’-(Substituted Phenyl)-Triazaspiroalkene Hydrochloride (P-I) 4.1 The Synthesis of Library P-I 4.2 Biological Evaluation 4.2.1 Inhibitory Activity of Library P-I against the Recombinant Human DHFR 4.2.2 Antiproliferative Activity of Library P-I against A549 and MDA-MB-231 4.3 Conclusion 71 CHAPTER 5. Diamino-3’- or 4’-(Prop-2-ynyloxy)phenyl-Triazaspiroalkene Hydrochloride (P-II) 5.1 The Synthesis of Library P-II 5.2 Biological Evaluation 5.2.1 Inhibitory Activity of Library P-II against the Recombinant Human DHFR 5.2.2 Antiproliferative Activity of Library P-II against A549 and MDA-MB-231 5.3 Conclusion 85 CHAPTER 6. Diamino- 3’- or 4’-((1-Phenylalkyl-1H-1,2,3-triazol-4yl)methoxyphenyl)-Triazaspiroalkene Hydrochloride (P-III) 6.1 The Synthesis of Library P-III 6.2 Biological Evaluation 6.2.1 Inhibitory Activity of Library P-III against Recombinant Human DHFR 6.2.2 Antiproliferative Activity of Library P-III against A549 and MDA-MB-231 6.3 Conclusion 93 CHAPTER 7. Diamino-3’- or 4’-(Phenylalkyloxy)phenyl-Triazaspiroalkene hydrochloride (P-IV) 7.1 The Synthesis of Library P-IV 7.2 Biological Evaluation 7.2.1 Inhibitory Activity of Library P-IV against Recombinant Human DHFR 7.2.2 Antiproliferative Activity of Library P-IV against A549 and MDA-MB-231 7.2.3 Cellular Selectivity Study of P-IV 7.2.4 Cell Cycle Analysis 58 64 68 70 72 74 74 78 83 86 88 88 89 91 96 99 99 102 106 107 108 112 112 114 118 120 ii 7.3 7.2.4.1 Cell Cycle Analysis of A549 Post Compound Treatment 7.2.4.1.1 Methotrexate (MTX) 7.2.4.1.2 Library P-IV 7.2.4.2 Cell Cycle Analysis of MDA-MB-231 Post Compound Treatment 7.2.4.2.1 Library P-IV Conclusion 122 122 125 132 132 139 CONCLUSION AND FUTURE WORK CHAPTER 8. Conclusion and Future Work 8.1 Conclusion 8.2 Future Work 142 142 145 MATERIALS AND METHODS CHAPTER 9. Materials and Methods 9.1 High Throughput Screening 9.2 Chemistry 9.2.1 Materials and Equipments 9.2.2 General Procedure for the Synthesis of Diamino-3’- or 4’(Substituted Phenyl)-Triazaspiroalkene Hydrochloride (P-I) 9.2.3 General Procedure for the Synthesis of Diamino-3’- or 4’(Prop-2-ynyloxy)phenyl)-Triazaspiroalkene Hydrochloride (P-II) 9.2.4 General Procedure for the Synthesis of Diamino- 3’- or 4’((1-Phenylalkyl-1H-1,2,3-triazol-4-yl)methoxyphenyl)Triazaspiroalkene Hydrochloride (P-III) 9.2.5 General Procedure for the Synthesis of Diamino-3’- or 4’(phenylalkyloxy)phenyl)-triazaspiroalkene hydrochloride (P-IV) 9.3 DHFR Enzyme Assay 9.4 Antiproliferative Assay 9.4.1 Cryopreservation and Storage of Cell Lines 9.4.2 Cell Lines and Cell Culture 9.4.3 Drug and Reagents 9.4.4 MTT Assay 9.5 Cell Cycle Analysis by Flow Cytometry 9.5.1 Fixation of Cells 9.5.2 Staining of Cells 9.5.3 Flow Cytometry 9.5.3.1 Cell Cycle Analysis of A549 Post Compound Treatment 9.5.3.2 Cell Cycle Analysis of MDA-MB-231 Post Compound Treatment 147 147 148 148 148 BIBLIOGRAPHY 196 166 169 177 186 187 187 188 188 189 189 189 190 190 191 193 iii SUMMARY High throughput screening (HTS) has been adopted in the screening for dihydrofolate reductase (DHFR) inhibitors in 96-well plate. Maintaining sensitivity and being able to measure real time kinetic of the assay are some challenges to be overcome in further miniaturizing of the assay. It was hypothesised that the UV spectrophotometric DHFR assay can be adopted to 384-well microplate to render HTS of potential DHFR inhibitors with sufficient sensitivity and selectivity. A sensitive and cost-effective HTS for rapid identification of murine DHFR inhibitors in 384-well microplate was developed. The absorbance of NADPH at two time points was measured as an indicator of the DHFR activity instead of the continuous monitoring of the rate of decrease of NADPH in real time. This method allowed more flexibility in the detection process. This is the first report of the miniaturization of DHFR assay into a 384-well microplate, using a final volume of 50 µL as the reaction mixture. Diamino-phenyl-triazaspiroalkenes were found to be biologically active yet were less studied in the past. It was hypothesized that diamino-phenyl-triazaspiroalkenes of a particular ring size, when appropriately substituted, would possess DHFR inhibitory activity as well as anticancer activity. The objectives of this second section were to synthesise four libraries of diamino-phenyl-triazaspiroalkenes and to investigate their activities against recombinant human DHFR and two cancer cell lines, namely the human lung cancer A549 and the human breast adenocarcinoma MDA-MB-231. A library (P-I) of fifty-three diamino-(substituted-phenyl)-triazaspiroalkenes was synthesised by one-pot three-component synthesis from differently substituted anilines, cyanoguanidine and ketones. Library P-I showed to some extent of rhDHFR inhibitory activity and antiproliferative activity against the cancer cell lines. iv Six diamino-(prop-2-ynyloxy)phenyltriazaspiroalkenes (P-II) were synthesised from prop-2-ynyloxy aniline, cyanoguanidine and ketones in one-pot reaction. Four compounds showed moderate rhDHFR inhibitory activity and antiproliferative activity against A549. A linear regression correlation (R2= 0.8651) between the antiproliferative activity and the rhDHFR inhibitory activity suggested that the mechanism of antiproliferative activity of P-II against A549 could occur via the DHFR inhibition. A library of seventeen diamino-((1-phenylalkyl-1H-1,2,3-triazol-4-yl)methoxy)phenyltriazaspiroalkenes (P-III) was synthesised using click chemistry where a triazole was formed from the Cu-(I) catalysed 1,3-polar addition of azide onto acetylene. P-III demonstrated dramatically improved rhDHFR inhibitory activity, particularly, all 2,4-diamino-5-phenyl-1,3,5-triazaspiro[5.5]undeca-1,3-dienes of PIII were able to inhibit rhDHFR. Triazole possesses two nitrogen atoms which are capable of forming hydrogen bonds with Asn64 in rhDHFR; thus providing strategic interaction points for the compound to the active site of rhDHFR. With the overall enhancement of flexibility and lipophilicity, diamino- (phenylalkyloxy)phenyl-triazaspiroalkene (P-IV) showed excellent antiproliferative activity against both A549 and MDA-MB-231 cell lines. Compounds with longer side chain showed the best antiproliferative activity, suggesting that lipophilicity was crucial for the penetration of cell membrane. 2,4-Diamino-5-phenyl-1,3,5triazaspiro[5.5]undeca-1,3-dienes of P-IV did not inhibit rhDHFR. Cell cycle analysis suggested that diamino(phenylalkyloxy)phenyl-triazaspiro[5.5]undeca-1,3-dienes caused G1 phase arrest of the A549 cell cycle; meanwhile, S phase arrest was observed for MDA-MB-231 cell cycle. This observation proposed that there might be other biological target for diaminophenyltriazaspiro[5.5]undeca-1,3-dienes. v In conclusion, the objective of the development of a miniaturised HTS in 384-well microplate for the identification of murine DHFR inhibitors was achieved. The hypothesis that diamino-phenyl-triazaspiroalkenes of a particular ring size when appropriately substituted in the phenyl ring would possess DHFR inhibitory activity as well as anticancer activity was proven to be true. Keywords: Triazaspiroalkenes, high throughput screening, dihydrofolate reductase inhibitors, antiproliferative activity, cell cycle analysis. vi LIST OF TABLES TABLE PAGE Comparative costs of screening compounds in 96-well or 9600-well plates. (Based on a 100,000-member compound library) Comparison of small but focused libraries vs. large but random libraries Inhibitors of DHFR identified in the libraries by Wyss et al. 13 In vitro DHFR inhibitory activity of diaminotriazaspiroalkenes (44) 37 Inhibitory activity of WR99210 analogue against bovine DHFR 39 Reaction condition of conventional DHFR assay vs. initial DHFR assay in 96-well microplate 59 Reaction condition of newly developed HTS in 384-well microplate compared to the reported HTS in 96-well plate by Zolli-Juran et al. 61 Reagents comparison of screening compounds in one 384-well microplate to four 96-well microplates 62 HTS screening results against murine DHFR vs. reported bovine DHFR inhibitory activity of H-I, H-II and MTX 65 10 Murine DHFR inhibitory activity of MTX and TMP from HTS screening. 68 11 Murine DHFR inhibitory activity of library H-IV from HTS screening 69 12 Yields of library P-I 73 13 Recombinant Human DHFR inhibitory activity of library P-I 75 14 Percentage of cell viability of A549 and MDA-MB-231 after treated with library P-I 80 15 Yields and melting points of library P-II. 88 16 Recombinant human DHFR inhibitory activity of P-II 88 17 Percentage of cell viability of A549 and MDA-MB-231 after treated with library P-II 90 18 Yields and melting points of library P-III 99 19 Recombinant human DHFR inhibitory activity of library P-III 100 vii 20 Percentage of cell viability of A549 and MDA-MB-231 after treated with library P-III 103 21 Yields and melting points of library of P-IV 111 22 Recombinant human DHFR inhibitory activity of library P-IV 112 23 Percentage of cell viability of A549 and MDA-MB-231 after treated with library P-IV 116 24 Cellular selectivity study of P-IV 119 25 Cell cycle analysis of A549 after treatment with MTX (Higher Concentration) 191 26 Cell cycle analysis of A549 after treatment with MTX (Lower concentration) 191 27 Cell cycle analysis of A549 after treatment with P-IV-01 191 28 Cell cycle analysis of A549 after treatment with P-IV-03 192 29 Cell cycle analysis of A549 after treatment with P-IV-04 192 30 Cell cycle analysis of A549 after treatment with P-IV-06 192 31 Cell cycle analysis of A549 after treatment with P-IV-07 193 32 Cell cycle analysis of A549 after treatment with P-IV-09 193 33 Cell cycle analysis of MDA-MB-231 after treatment with P-IV-01 193 34 Cell cycle analysis of MDA-MB-231 after treatment with P-IV-03 194 35 Cell cycle analysis of MDA-MB-231 after treatment with P-IV-04 194 36 Cell cycle analysis of MDA-MB-231 after treatment with P-IV-06 194 37 Cell cycle analysis of MDA-MB-231 after treatment with P-IV-07 195 38 Cell cycle analysis of MDA-MB-231 after treatment with P-IV-09 195 viii LIST OF FIGURES FIGURE PAGE Three main stages in the development of a new drug The magic triangle of HTS 10 Structures of the folate family of cofactors 15 Interference of the folate pathways by MTX, TMP and PYR. 17 Crystal structure of recombinant human DHFR with folate (with permission PDB ID = 1dhf) 18 Structure and numbering of folate 19 Crystal structure of E. coli DHFR with folate (with permission PDB ID =1rg7) 20 Structure and numbering of methotrexate (MTX) 21 Crystal structure of L. casei DHFR with MTX (with permission PDB ID =3dhr) 22 10 Schematic representation of hydrogen bonding between DHFR and the pteridine ring of (a) DHF and (b) MTX 23 11 Classical representation of various cell cycle phases 47 12 (a) Schematic representation of relationship between DNA content and cell position in the cell cycle. (b) Theoretical DNA content distribution histogram. 48 13 Structural revolution of the DHFR inhibitors 50 14 Newly designed diamino-phenyl-triazaspiroalkenes 54 15 Representation of pathlength of mL cuvette, 96-well microplate and 384-well microplate 59 16 Measurement of the absorbance of NADPH at different time points, i.e., (i) before initiation and (ii) after initiation by enzyme 61 17 Schematic representation of the newly developed HTS for DHFR assay 63 18 Schematic representation of cluster of murine DHFR inhibitors Vs noninhibitors 67 ix 19 A549 antiproliferative activity versus rhDHFR inhibitory activity of PII 91 20 1,4-Disubstituted 1,2,3-triazole shares similar electronic features with the amide connector 102 21 DNA content distribution histogram of untreated A549 cells (M1= subG1, M2= G1, M3= S, M4= G2) 122 22 Cell cycle profiles of A549 after treatment with MTX (a) Higher concentration (b) Lower concentration. 123 23 Histogram representation of DNA content of A549 treated with MTX analysed by cell phases (a) Higher concentration (b) Lower concentration 124 24 Cell cycle profiles of A549 after treatment with (a) P-IV-01 (b) P-IV03 (c) P-IV-04 (d) P-IV-06 127 25 Histogram representation of DNA content of A549 treated with (a) P-IV-01 (b) P-IV-03 (c) P-IV-04 (d) P-IV-06 129 26 Cell cycle profiles of A549 after treatment with (a) P-IV-07 (b) P-IV09 130 27 Histogram representation of DNA content of A549 treated with (a) PIV-07 and (b) P-IV-09 131 28 DNA content distribution histogram of untreated MDA-MB-231 cells (M1= subG1, M2= G1, M3= S, M4= G2). 132 29 Cell cycle profiles of MDA-MB-231 after treatment with (a) P-IV-01 (b) P-IV-03 (c) P-IV-04 (d) P-IV-06 (e) P-IV-07 (f) P-IV-09 133 30 Histogram representation of DNA content of MDA-MB-231 treated with (a) P-IV-01 (b) P-IV-03 (c) P-IV-04 (d) P-IV-06 (e) P-IV-07 and (f) P-IV-09 136 x LIST OF SCHEMES SCHEME PAGE One-pot three-component synthesis of diamino-phenyltriazaspiroalkenes 40 Two-component synthesis of diamino-triazaspiroalkenes 41 Triethyl orthoacetate is employed as water scavenger in the modified two-component synthesis 42 Synthesis of triazaspiroalkenes from 1,3,5-triazapenta-1,3-dienes catalysed by TiCl4 42 Synthesis of triazaspiro compound from 1,4-dimethylurazole 43 One pot three-component synthesis of 2-amino-9-alkyl-5-cyano-1,3,9triazaspiro[5.5]undeca-2-en-4-one 43 Mitochondrial dehydrogenase-catalysed reduction of MTT to MTT formazan 46 Three-component synthesis of library P-I 72 The synthesis of library P-II 86 10 Preparation of prop-2-ynyl aniline 87 11 Copper (I)-catalysed synthesis of triazole from azide and acetylene 94 12 Possible catalytic cycle for the copper (I)-catalysed triazole synthesis 95 13 The synthesis of library P-III (Method A) 96 14 The synthesis of library P-III (Method B) 97 15 The synthesis of library P-IV 108 16 The preparation of phenylalkyloxy aniline 109 17 Proposed synthesis of diaminotriazaspiroalkene 60 146 xi LIST OF ABBREVIATIONS A549 Human lung adenocarcinoma epithelial cell line Abs Absorbance ADME Adsorption, distribution, metabolism, excretion AICAR 5-Aminoimidazole-4-carboxamide ribonucleotide AMT Aminopterin ANOVA Analysis of variance Boc Tert-Butyl carbamate CO2 Carbon dioxide CYC Cycloguanil d Doublet dATP Deoxyadenosine triphosphate dGTP Deoxyguanosine triphosphate DHF 7,8-Dihydrofolate DHFR Dihydrofolate reductase DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dTMP Deoxythymidylate dTTP Deoxythymidine triphosphate dUMP Deoxyuridylate EDTA Ethylenediaminetetraacetic acid ESI-MS Electrospray ionization mass spectrometry FDA The Food and Drug Administration FPGS Folylpolyglutamate synthase GAR Glycinamide ribonucleotide xii HCl Hydrochloric acid HTS High throughput screening Hz Hertz IC50 Concentration of inhibitor that causes 50% of inhibiton J Coupling constant LV Leucovorin m Multiplet MDA-MB-231 Human breast adenocarcinoma cell line MRC-5 Normal human fetal lung fibroblast MRP Multidrug-resistance related protein MTT 3-(4,5-Dimethylthiazol-2yl)2,5-diphenyltetrazolium bromide MTX Methotrexate MTXGlun MTX polyglutamates NADPH Reduced form of nicotinamide-adenine dinucleotide phosphate NDA New Drug Application NMR Nuclear magnetic resonance PABA Para aminobenzoic acid PBS Phosphate buffer saline PDX 10-Propargyl-10-deazaaminopterin PI Propidium iodide PYR Pyrimethamine q Quartet QSAR Quantitative structure-activity relationship RFC Reduced folate carrier rhDHFR Recombinant human DHFR xiii RNase Ribonuclease rpm Round per minute RPMI Roswell Park Memorial Institute s Single SHMT Serine hydroxymethyl transferase t Triple TFA Trifluoroacetic acid THF 5,6,7,8-Tetrahydrofolate TLC Thin layer chromatography TMP Trimethoprim TS Thymidylate synthase TZT Baker’s triazinate UV Ultraviolet xiv [...]...LIST OF SCHEMES SCHEME PAGE 1 One-pot three-component synthesis of diamino-phenyltriazaspiroalkenes 40 2 Two-component synthesis of diamino -triazaspiroalkenes 41 3 Triethyl orthoacetate is employed as water scavenger in the modified two-component synthesis 42 4 Synthesis of triazaspiroalkenes from 1,3,5-triazapenta-1,3-dienes catalysed by TiCl4 42 5 Synthesis of triazaspiro compound from... pot three-component synthesis of 2- amino-9-alkyl-5-cyano-1,3,9triazaspiro[5.5]undeca -2- en-4-one 43 7 Mitochondrial dehydrogenase-catalysed reduction of MTT to MTT formazan 46 8 Three-component synthesis of library P-I 72 9 The synthesis of library P-II 86 10 Preparation of prop -2- ynyl aniline 87 11 Copper (I)-catalysed synthesis of triazole from azide and acetylene 94 12 Possible catalytic cycle for... Analysis of variance Boc Tert-Butyl carbamate CO2 Carbon dioxide CYC Cycloguanil d Doublet dATP Deoxyadenosine triphosphate dGTP Deoxyguanosine triphosphate DHF 7,8 -Dihydrofolate DHFR Dihydrofolate reductase DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dTMP Deoxythymidylate dTTP Deoxythymidine triphosphate dUMP Deoxyuridylate EDTA Ethylenediaminetetraacetic acid ESI-MS Electrospray ionization mass... Folylpolyglutamate synthase GAR Glycinamide ribonucleotide xii HCl Hydrochloric acid HTS High throughput screening Hz Hertz IC50 Concentration of inhibitor that causes 50% of inhibiton J Coupling constant LV Leucovorin m Multiplet MDA-MB -23 1 Human breast adenocarcinoma cell line MRC-5 Normal human fetal lung fibroblast MRP Multidrug-resistance related protein MTT 3-(4,5-Dimethylthiazol-2yl )2, 5-diphenyltetrazolium... Possible catalytic cycle for the copper (I)-catalysed triazole synthesis 95 13 The synthesis of library P-III (Method A) 96 14 The synthesis of library P-III (Method B) 97 15 The synthesis of library P-IV 108 16 The preparation of phenylalkyloxy aniline 109 17 Proposed synthesis of diaminotriazaspiroalkene 60 146 xi LIST OF ABBREVIATIONS A549 Human lung adenocarcinoma epithelial cell line Abs Absorbance ADME... NADPH Reduced form of nicotinamide-adenine dinucleotide phosphate NDA New Drug Application NMR Nuclear magnetic resonance PABA Para aminobenzoic acid PBS Phosphate buffer saline PDX 10-Propargyl-10-deazaaminopterin PI Propidium iodide PYR Pyrimethamine q Quartet QSAR Quantitative structure-activity relationship RFC Reduced folate carrier rhDHFR Recombinant human DHFR xiii RNase Ribonuclease rpm Round per... human DHFR xiii RNase Ribonuclease rpm Round per minute RPMI Roswell Park Memorial Institute s Single SHMT Serine hydroxymethyl transferase t Triple TFA Trifluoroacetic acid THF 5,6,7,8-Tetrahydrofolate TLC Thin layer chromatography TMP Trimethoprim TS Thymidylate synthase TZT Baker’s triazinate UV Ultraviolet xiv . 15 1.4 .2 Dihydrofolate Reductase 18 1.4 .2. 1 Human DHFR 18 1.4 .2. 2 Escherichia coli DHFR 20 1.4 .2. 3 Lactobacillus casei DHFR 22 1.4.3 DHFR Inhibitors as Therapeutics. Methotrexate (MTX) 122 7 .2. 4.1 .2 Library P-IV 125 7 .2. 4 .2 Cell Cycle Analysis of MDA-MB -23 1 Post Compound Treatment 1 32 7 .2. 4 .2. 1 Library P-IV 1 32 7.3 Conclusion 139. Throughput Screening for Enzyme Assay 10 1.3.3 High throughput Screening for Dihydrofolate Reductase (DHFR) Inhibitors 12 1.4 Dihydrofolate Reductase as Therapeutic Target 15 1.4.1