Design, Synthesis, and Evaluation of Inhibitors of Steroid Sulfatase
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Design, synthesis, and evaluation of inhibitors of steroid sulfatase by Yaser Abdel-Hady Mostafa Abdel-Karem A thesis Presented to the University of Waterloo in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Chemistry Waterloo, Ontario, Canada, 2014 © Yaser Abdel-Hady Mostafa Abdel-Karem 2014 i Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by examiners. I understand that my thesis may be made electronically available to public. ii Abstract Steroid sulfatase (STS) catalyzes the desulfation of biologically inactive sulfated steroids to yield biologically active desulfated steroids and is currently being examined as a target for therapeutic intervention for the treatment of breast and other steroid-dependent cancers. A series of 17-arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ol were prepared and evaluated as inhibitors of STS. Introducing n-alkyl groups into the 4´-position of the 17- benzenesulfonamide derivative resulted in an increase in potency with the n-butyl derivative exhibiting the best potency with an IC50 of 26 nM. A further increase in carbon units (to n- pentyl) resulted in a decrease in potency. Branching of the 4´-n-propyl group resulted in a decrease in potency while branching of the 4´-n-butyl group (to a tert-butyl group) resulted in a slight increase in potency (IC50 = 18 nM). Studies with 17-benzenesulfonamides substituted at the 3´- and 4´-positions with small electron donating and electron withdrawing groups revealed the 3´-bromo and 3´-trifluoromethyl derivatives to be excellent inhibitors with IC50‘s of 30 and 23 nM respectively. The 17-2´-naphthalenesulfonamide was also an excellent inhibitor (IC50 = 20 nM) while the 17-4´-phenylbenzenesulfonamide derivative was the most potent inhibitor with an IC50 of 9 nM. Kinetic studies with 3´-bromo derivative revealed it to be a non-competitive inhibitor and so these types of inhibitors might be capable of binding at the active site and also at a secondary site outside the active site. The amide analogs of some of these compounds were found not to be as potent inhibitors as the sulfonamides. Introducing a nitro group or fluorine atom into the 4-position of the 17-arylsulfonamide inhibitors resulted in an increase in potency. Some of these compounds are the most potent reversible STS inhibitors ever reported with apparent Ki‘s as low as 1 nM. 3-O-Sulfation of these compounds did not significantly alter their potency. It is not known if 3-O-sulfated derivatives were acting as iii inhibitors or reversible suicide inhibitors. Docking studies were performed on selected inhibitors to gain insight into how they might interact with STS. Selected 17-arylsulfonamide inhibitors were sent to the NCI (USA) for in vitro screening with a panel of 60 human tumor cell lines (NCI-60 panel). Almost all of the compounds exhibited GI50‘s in the 1 to 10 M range with all 60 cell lines and so were only moderately potent in terms of their ability to inhibit the growth. None of the compounds stood out in terms of their ability to inhibit the growth of any breast cancer, prostate cancer or any other cancer cell line studied. The thiadiazolidinedione group was proposed as a sulfate mimic for obtaining STS inhibitors. A new approach to the synthesis of 3-aminoestrone was achieved as part of an attempt to prepare the thiadiazolidinedione target. 3-O-Sulfamoylation of one of the 17- arylsulfonamide inhibitors was attempted using a variety of reaction conditions but was unsuccessful. iv Acknowledgements I would like to thank my supervisor professor Scott D. Taylor for his guidance, inspiration, and continuous support throughout my graduate studies especially at thesis writing stage and his great care about getting the thesis in a perfect shape, really thank you so much Prof. Taylor. I also would like to thank my advisory committee, Professor Michael J. Chong for his continuous support especially with spectral identification problems, Professor Gary Dmitrienko, and Professor Richard Manderville for their valuable guidance and help during my graduate program. I also would like to thank Mrs. Jan Venne for help with NMR use. I also would like to thank Dr. Praveen Nekkar at Faculty of Pharmacy, University of Waterloo for his help in conducting molecular modelling studies in his lab. I would like to have very special thanks for Ahmed Y. Desoky my Egyptian brother who helped me from the first moment of arrival to Canada to overcome all the difficulties I faced here in Canada . I also thank Samy Al-Mohamady my Egyptian lab mate for good company during these years. I also would like to thank all former and current members of the Taylor group especially Joshua Williams, Duncan Li, Magda Karski, and Chuda Lohani. I am grateful to Guelph-Waterloo Centre for Graduate Work in Chemistry (GWC2) for its financial support during my PhD study. v To My Parents, My Lovely Wife Heba, My Angel Malak and Mr. Mohammed vi Table of Contents Author Declaration ii Abstract iii Acknowledgments v Dedications vi Table of Contents vii List of Figures x List of Tables xiii List of Schemes xiv List of Abbreviations xv Chapter 1 – Steroid Sulfatase: Function, Localization, Structure, Mechanism, and Inhibitors 1.1 Introduction 1 1.1.1 Sulfatases 1 1.2 Steroid Sulfatase (STS) 2 1.2.1 Substrates and Physiological function 2 1.2.2 Post Translation Modification and Catalytic Mechanism 4 1.2.3 STS and Breast Cancer 8 1.3 Current Therapies for Treating Hormone-Dependent Breast Cancer 10 1.4 STS Inhibitors 12 1.4.1 Non-sulfamate Steroid-based Inhibitors 12 1.4.1.1 Early Studies and Substrate Analogs 12 1.4.1.2 Estradiol-Based Inhibitors 16 1.4.1.3 E1S-based Suicide Inhibitors (SIs) 20 1.4.2 Steroidal Sulfamate-Based Inhibitors 22 1.4.2.1 Estrone-3-O-Sulfamate (EMATE) 22 1.4.2.2 Modification of A-ring of EMATE 25 1.4.2.3 Modification of D-ring of EMATE 26 1.4.3 Non-Steroidal Sulfamate-based STS Inhibitors 37 1.4.3.1 Monocyclic Aryl Sulfamates 37 1.4.3.2 Bicyclic and Polycyclic Aryl Sulfamates 40 1.4.4 Non-Steroidal, Non-Sulfamate-based STS Inhibitors 43 1.5 Clinical Studies with STS Inhibitors 46 1.6 Thesis Objectives and Overview 47 Chapter 2 – Purification of Steroid Sulfatase 2.1 Introduction 49 2.1.1 Why Purify STS? 49 2.2 Objectives 50 2.3 Results and Discussion 50 2.3.1 Placental Purification of STS 50 2.3.2 Evaluation of Protein Concentration, Specific Activity, Molecular 53 vii Weight of STS 2.4 Conclusion 54 2.5 Experimental 55 2.5.1 Materials 55 2.5.2 Methods 55 2.5.2.1 Activity Assay 55 2.5.2.2 Homogenization and Chromatography 56 2.5.2.3 Determination of Protein Concentration 57 Chapter 3 – 17-Arylsulfonamides of 17-Aminoestra-1,3,5(10)-trien-3-ol as Highly Potent Inhibitors of Steroid Sulfatase. 3.1 Introduction 58 3.2 Objectives 59 3.3 Results and Discussion 60 3.3.1 Studies with model sulfonates and sulfonamides 60 3.3.2 Inhibition of STS with 17β-sulfonamides 66 3.3.3 Studies with 17β-amides of E1 75 3.3.4 X-ray Crystallography of Compound 3.33 77 3.3.5 Molecular Modelling Studies 79 3.4 Conclusions and Future Work 85 3.5 Experimental 86 3.5.1 General 86 3.5.2 Syntheses 87 3.5.3 Inhibition Studies 129 3.5.3.1 General 129 3.5.3.2 Determination of IC50s 129 3.5.3.3 Determination of Ki and αKi values for compound 3.28 130 3.5.4 Molecular Modeling (Docking) Experiments 131 3.5.5 X-ray Crystallography of Compound 3.33. 131 Chapter 4 – Steroid Sulfatase Inhibitors: A-Ring Modification of 17-arylsulfonamide E1 Derivatives. 4.1 Introduction 133 4.2 Objectives 134 4.3 Results and Discussion 134 4.3.1 Studies with 4-fluoro-17-E2 (compound 4.2) 134 4.3.2 2- and 4-Nitro-17-arylsulfonamide E1 derivatives as STS 135 inhibitors 4.3.3 2- and 4-Bromo-17-arylsulfonamide E1 derivatives as STS 142 inhibitors 4.3.4 A 4-fluoro-17-arylsulfonamide E1 derivative as an STS inhibitor 146 4.3.5 Inhibition Studies with a 4-Formyl-17-arylsulfonamide derivative 153 of E1 4.3.6 Inhibition studies with 3-O-sulfated-17-arylsulfonamide 155 viii derivatives of E1 4.3.7 Molecular Modelling (Docking) Studies 158 4.3.8 Anti-proliferative effect of the 17-arylsulfonamide E1 derivatives 162 with the NCI-60 panel 4.4 Conclusions and Future Work 163 4.5 Experimental 166 4.5.1 General 166 4.5.2 Syntheses 167 4.5.3 Inhibition Studies 199 4.5.3.1 General 199 4.5.3.2 Determination of IC 4.9, 4.10, 4.13-4.16, 4.27, 4.28, 199 50 4.51-4.54 4.5.3.3 Determination of IC for Tight-Binding Inhibitors, 200 50 compounds 4.12, 4.13, 4.52, and 4.58 4.5.3.4 Determination of Ki and Ki of compound 4.2 200 4.5.3.5 Examining Time- and/or Concentration-dependent 201 Inhibition of STS with compound 4.2 4.5.3.6 Dialysis Experiment for compound 4.53 202 4.5.4 Molecular Modeling (Docking) Experiments 202 4.5.5 Methodology of the In Vitro Cancer Screening 202 4.5.5.1 General Methods 202 4.5.5.3 The In-Vitro Testing Results Data Sheet 203 Chapter 5 - Towards the Synthesis of an E1S Analog Bearing a Thiadiazolidinedione Sulfate Mimic and a 3-O-Sulfamate of a 17-Arylsulfonamide STS Inhibitor 5.1 Introduction 205 5.2 Objectives 207 5.3 Results and Discussion 208 5.3.1 Towards the synthesis of 5.2 208 5.3.2 Attempts to prepare a sulfamate derivative of a 17β- 214 arylsulfonamide inhibitor 5.4 Conclusion and Future Work 218 5.5 Experimental 219 5.5.1 General 219 5.5.2 Syntheses 219 References 227 Appendix A Supplementary Figures and Tables for Chapter 3 240 Appendix B Supplementary Figures and Tables for Chapter 4 265 Appendix C Supplementary Data for X-ray Crystallographic Data for 277 Compound 3.33 ix List of Figures Chapter 1 Figures Fig.