Synthetic Modifications of Estrogens and Androgens Somdatta Deb University of Helsinki Faculty of Science Department of Chemistry Laboratory of Organic Chemistry Finland ACADEMIC DISSERTATION To be presented with the permission of the Faculty of Scienec of the University of Helsinki for public criticism in Auditorium A110, Department of Chemistry on June 23rd, 2011, at 12 o’clock. Helsinki 2011 Supervisor Professor Kristiina Wähälä Laboratory of Organic Chemistry Department of Chemistry University of Helsinki Finland Reviewers Professor Erkki Kolehmainen Department of Chemistry University of Jyväskyla Finland Docent Salme Koskimies Adjunct. Professor University of Helsinki Opponent Professor Maria Christina das Neves Oliveira Unidade de Ciências Químicas e Radiofarmacêuticas Portugal ISBN 978-952-92-9174-8 (paperback) ISBN 978-952-10-7051-8 (PDF) Helsinki 2011 Unigrafia Acknowledgements This thesis work was carried out at the Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki. I would like to express my deep gratitude to my supervisor Prof. Kristiina Wähälä for introducing me to the fascinating field of steroid chemistry and for providing the facilities to do this work. I warmly thank emeritus professor Tapio Hase for his helpful, invaluable scientific advice during my work. Prof. Matti J. Tikkananen and his group are gratefully acknowledged for pleasant collaboration. My warmest thanks are due to all personnel in the Laboratory of Organic Chemistry, specially the members (former and present) of Phyto-Syn group for their support. They have been always there when needed. In particular I would like to thank Gudrun Silvennoinen for technical assistance and friendship. I am thankful to Dr. Ullastiina Hakala for introducing me to the field of ionic liquid. Thank you Lea for being such a good friend. I am also grateful to Dr. Sami Heikkinen for his expertise in NMR spectroscopy and assistance. I extend my thanks to Dr. Jorma Matikainen for advising me in mass spectrometry. I also thank Maiju Soikkeli for technical assistance. Special thanks are owed to Dr. Jorma Koskimies and Dr. Leena Kaisalo for their generous help in any matter. Professor Erkki Kolehmainen and Docent Salme Koskimies are warmly acknowledged for reviewing the manuscript of this thesis. They gave their comments remarkably fast. I would like to take this opportunity to thank my friends and former teachers in India who induces passion for research in me. My sincerest appreciation goes to my parents for encouragement and versatile support. I am deeply indebted to my late mother for sowing logical thinking seed in me. I thank my husband Suvendu Mitra for his patience, love and encouragement during these years. I am grateful to him for listening and understanding during the most difficult time. Helsinki, May 2011 Somdatta Deb CONTENTS Abstract............................................................................................................................................... 4 Abbreviations..................................................................................................................................... 6 1. INTRODUCTION ........................................................................................................................ 8 2. LITERATURE REVIEW.........................................................................................................14 2.1 C-7 substituted estrogen.........................................................................................................................14 2.1.1 Synthesis of C-7 alkyl substituted estrogen by converting other steroids to estrogen ...................................................................................................................................................................... 15 2.1.2 Direct addition of substituent to C-7 of estrane skeleton ............................................... 19 2.1.3 Synthesis of 7-hydroxyestrone and estradiol...................................................................... 26 2.2 Studies on 8-isoestrogens.......................................................................................................................28 2.3 Immunoassay ..............................................................................................................................................30 2.4 Fatty acid esters of steroids ..................................................................................................................33 3. AIMS OF STUDY ......................................................................................................................38 4. RESULTS AND DISCUSSION ...............................................................................................39 4.1 Synthesis of estriol ....................................................................................................................................39 4.2 Protection of hydroxyl groups of estradiol and estriol...............................................................42 4.3 Oxidation at C-6 of estradiol and estriol.........................................................................................44 4.4 Alkylation at C-7........................................................................................................................................47 4.5 Hydroxylation and etherification at C-7..........................................................................................54 4.6 Synthesis of 8-isoestradiol derivative................................................................................................57 4.7 Selective debenzylation ...........................................................................................................................59 4.8 Synthesis of steroid fatty acid esters..................................................................................................65 5. CONCLUSION ...........................................................................................................................69 6. EXPERIMENTAL .....................................................................................................................70 6.1 General methods........................................................................................................................................70 6.2 Synthesis of estriol ....................................................................................................................................70 6.2.1 Synthesis of 3-acetoxyestra-1,3,5(10)16-tetraene-17-one (111) ................................. 70 6.2.2 Synthesis of 3,17β-diacetoxyestra-1,3,5(10)16-tetraene (114).................................... 71 6.2.3 Synthesis of 16α,17α-epoxy-estra-1,3,5(10)triene-3,17β-diol diacetate (116)...... 71 6.2.4 Reduction of 116 with LAH (normal addition)................................................................. 72 6.2.5 Reduction of 78 with LAH (inverse addition) ................................................................... 72 6.2.6 Synthesis of estriol (3) ............................................................................................................... 72 6.3 Protection of estriol..................................................................................................................................74 6.3.1 Attempted protection of estriol as triisopropylsilyl ether (117) with imidazole and TIPSCl........................................................................................................................................................ 74 6.3.2 Protection of estriol as tert-butyl ether (118) with isobutylene and Amberlyst-1574 6.3.3 Synthesis of estra-1,3,5(10)-triene-3,16α,17β-triol-3,16,17-tris(methoxymethyl) ether (122)................................................................................................................................................. 76 6.4 C-6 oxidation of estradiol......................................................................................................................76 6.4.1 Synthesis of estra-1,3,5(10)-triene-3,17β-diol diacetate (123) .................................... 76 6.4.2 Synthesis of 6-oxo-estra-1,3,5(10)-triene-3,17β-diol diacetate (124) (oxidation with CrO3/ 3,5-dimethyl pyrazole) ................................................................................................... 77 6.4.3 Synthesis of estra-1,3,5(10)-triene-3,17β-diol dibenzoate (125)................................. 77 6.4.4 Synthesis of 6-oxo-estra-1,3,5(10)-triene-3,17β-diol dibenzoate (126) (oxidation with K2S2O8/CuSO4.5H2O) ................................................................................................................. 78 6.4.5 Synthesis of estra-1,3,5(10)-triene-3,17β-diol-3,17-di-tert-butyl ether (127) ........ 78 1 6.4.6 Synthesis of 6-hydroxy-estra-1,3,5(10)-triene-3,17β-diol-3,17di-tert-butyl ether (130)............................................................................................................................................................ 79 6.4.7 Oxidation of 130 to 6-oxo-estra-1,3,5(10)-triene-3,17β-diol-3,17di-tert-butyl ether (129)............................................................................................................................................................ 80 6.5 Oxidation of estriol...................................................................................................................................80 6.5.1 Oxidation of 131