University of Bath PHD Novel inhibitors of steroidogenesis for the treatment of hormone-dependent breast cancer Fischer, Delphine S. Award date: 2004 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 08. Oct. 2021 NOVEL INHIBITORS OF STEROIDOGENESIS FOR THE TREATMENT OF HORMONE-DEPENDENT BREAST CANCER submitted by Delphine S. Fischer for the degree of PhD of the University of Bath 2004 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the prior written consent of the author. This thesis may not be consulted, photocopied or lent to other libraries without the permission of the author for three years from the date of acceptance of the thesis. UMI Number: U602172 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U602172 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ij-0 2 9 JUL 2034 Abstract Inhibition of steroidogenic enzymes, which are involved in the biosynthesis of active estrogens, represents an attractive approach to treating hormone-dependent breast cancer. Synthetic routes to novel steroid-based inhibitors of two key enzymes, steroid sulfatase (STS) and 17 P-hydroxysteroid dehydrogenase type 1 (17P-HSD type 1), are described. Biological evaluation of the derivatives in vitro indicated that most of the compounds synthesised were active against the selected targets to varying degrees. Particular attention was given to assessing the estrogenicity of potential inhibitors, since agonist activity at the estrogen receptor is undesired in the treatment of estrogen-dependent pathologies. Modifications to the D-ring of estrone-3-O-sulfamate EMATE, a potent STS inhibitor, successfully afforded a 3-sulfamoyloxy-16,17-sec<?-estra-l,3,5(10)-triene- 16,17-imide template, from which a series of A-alkylated analogues were prepared. The TV-propyl and A-pyridin-3-ylmethyl derivatives were 18 times more potent than EMATE in vitro, with IC 5 0 S of 1 nM in placental microsomes. In vivo, these compounds inhibited 99% of rat liver sulfatase activity at an oral dose of 10 mg/kg. Importantly, they were also devoid of estrogenic activity. The SAR for the derivatives synthesised is discussed and a QSAR was established. Substrate-based design of 17P-HSD type 1 inhibitors resulted in the identification of several lead compounds, mostly D-ring modified estrogens. The potential of 16- alkylidenes derivatives of estradiol as enzyme-generated irreversible inhibitors was examined. Small modifications to the estra-l,3,5(10)-triene nucleus were investigated. Both studies were assisted by molecular modelling, using the crystal structure of the enzyme. D-Ring fused heterocyclic derivatives of estrone were prepared and an SAR was built around N-alkylated 16,17-fused pyrazoles. The N- ethoxymethyl derivative emerged as a potent inhibitor of 17p-HSD type 1 in vitro without being estrogenic. A preliminary study focused on the design of dual inhibitors of STS and 17P-HSD type 1 is also reported here. The synthetic work was supported by single crystal X-ray analyses. Acknowledgements I wish to thank my supervisor Professor B. V. L. Potter for offering me the opportunity to carry out this research project, and for his guidance and enthusiasm throughout its course. I would like also to extend my thanks to Dr. L. W. L. Woo and Dr. N. Vicker for their encouragement and helpful advice. I thank Sterix Ltd. for financial support. I wish to extend my grateful appreciation to Professor M. J. Reed and Dr. A. Purohit at the Department of Endocrinology and Metabolic Medicine, Imperial College School of Medicine, St. Mary’s Hospital, London as well as to Dr. L. Wood and Dr. G. Packham at the Cancer Research UK Oncology Unit, Southampton General Hospital, Southampton for biological evaluation of the compounds synthesised. I am grateful to J. J. Robinson for carrying out molecular modelling studies. I would like to thank Dr. S. J. Black, R. R. Hartell and D. Wood for recording NMR spectra, Dr. M. F. Mahon, Department of Chemistry, University of Bath for X-ray crystallographic studies and A. C. Smith for HPLC analyses. Dr. S. J. Black is additionally thanked for helpful suggestions. I extend my thanks to the staff of microanalysis and mass spectrometry at the University of Bath. My thanks are also due to all my colleagues for their help, encouragement and proof­ reading of this thesis. Special thanks are due to Dr. C. Bubert for his practical guidance in the laboratory and for sharing his knowledge of organic synthesis. Dr. N. Vicker is additionally thanked for constructive criticism of the manuscript. I especially wish to express my gratitude to Dr. G. Wagner for comprehensive proof­ reading of this thesis, and for support and encouragements through difficult times. Finally, I wish to thank my family and Russell for their consistent moral guidance throughout this course. Publications Some of the work described in this thesis has appeared in the following publications: Fischer, D. S.; Woo, L. W. L.; Mahon, M. F.; Purohit, A.; Reed, M. J. and Potter, B. V. L. D-Ring modified estrone derivatives as novel potent inhibitors of steroid sulfatase. Bioorg. Med. Chem. 2003, 77, 1685-1700 Fischer, D. S.; Chander, S. K.; Woo, L. W. L.; Fenton, J. C.; Purohit, A.; Reed, M. J. and Potter, B. V. L. Novel D-ring modified steroid derivatives as potent, non estrogenic, steroid sulfatase inhibitors with in vivo activity. J. Steroid Biochem. Mol. Biol. 2003, 84, 343-349 iv Abbreviations A Angstrom ( 1 O' 8 cm) Ac acetyl app apparent (spectral) aq. aqueous Ar aryl Bn benzyl br broad (spectral) Bu butyl rBu tert-butyl °C degrees Celsius ca. approximately CA carbonic anhydrase cm centimetre(s) COUM ATE 4-methylcoumarin-7 - 0-sulfamate 5 chemical shift d doublet (spectral) DBD DNA binding domain DCM dichloromethane DEPT distortionless enhancement by polarisation transfer DMA dimethylacetamide DMF Af,Af-dimethylformamide DMSO dimethylsulfoxide El estrone E2 estradiol E3 estriol e.g. for example EMATE estrone-3-O-sulfamate eq. equivalent ER estrogen receptor Et ethyl f femto FAB fast atom bombardment (mass spectrometry) G gram(s) h hour(s) HBD hormone binding domain HDBC hormone-dependent breast cancer HMBC heteronuclear multiple bond correlation HPLC high-performance liquid chromatography HRMS high resolution mass spectroscopy HRT hormone-replacement therapy 17p-HSD 17P-hydroxysteroid dehydrogenase Hz Hertz IC5o concentration causing 50% inhibition IPA isopropyl alcohol IR infrared J coupling constant (spectral) Kg kilogram(s) Ki inhibition constant Km Michaelis-Menten constant L litre(s) LBD ligand binding domain lit. literature ji micro m milli, multiplet (spectral) M moles per litre, molecular ion (mass spectrometry) mJz mass to charge ratio Me methyl MHz megaHertz min minute(s) mol mole(s) mp melting point MS mass spectrometry v spectral number (infrared spectroscopy) n nano NAD(P)H nicotinamide adenine dinucleotide (phosphate) nd not determined NMR nuclear magnetic resonance NOE nuclear Overhauser effect pH logarithm of the concentration of H+ p/sfa logarithm of the acidity constant p.o. per oral ppm parts per million q quartet (spectral) QSAR quantitative structure-activity relationship rbc(s) red blood cell(s) rt room temperature Rf retention factor Rt retention time s singlet SAR structure-activity relationship s.c. subcutaneous S.D. standard deviation STS steroid sulfatase t triplet (spectral) TBAF tetrabutylammonium fluoride THF tetrahydrofuran TLC thin-layer chromatography TMS tetramethylsilane vs. versus wt. weight Trivial/Approved names of steroid Androstenedione 4-Androstene-3,7-dione Cholesterol 5-Cholesten-3|3-ol Dehydroepiandrosterone 5-Androstene-3 p-ol-17-one Estradiol Estra-1,3,5 (10)-triene-3,17 p-ol Estriol Estra-1 ,3,5 (10)-triene-3,16a, 17 P-triol Estrone 3-Hydroxy-estra-l ,3,5( 10)-triene- 17-one Progesterone 4-Pregnene-3,20-dione