Labeling and Synthesis of Estrogens and Their Metabolites Paula Kiuru University of Helsinki Faculty of Science Department of Chemistry Laboratory of Organic Chemistry P.O. Box 55, 00014 University of Helsinki, Finland ACADEMIC DISSERTATION To be presented with the permission of the Faculty of Science of the University of Helsinki, for public criticism in Auditorium A110 of the Department of Chemistry, A. I. Virtasen Aukio 1, Helsinki, on June 18th, 2005 at 12 o'clock noon Helsinki 2005 ISBN 952-91-8812-9 (paperback) ISBN 952-10-2507-7 (PDF) Helsinki 2005 Valopaino Oy. 1 ABSTRACT 3 ACKNOWLEDGMENTS 4 LIST OF ORIGINAL PUBLICATIONS 5 LIST OF ABBREVIATIONS 6 1. INTRODUCTION 7 1.1 Nomenclature of estrogens 8 1.2 Estrogen biosynthesis 10 1.3 Estrogen metabolism and cancer 10 1.3.1 Estrogen metabolism 11 1.3.2 Ratio of 2-hydroxylation and 16α-hydroxylation 12 1.3.3 4-Hydroxyestrogens and cancer 12 1.3.4 2-Methoxyestradiol 13 1.4 Structural and quantitative analysis of estrogens 13 1.4.1 Structural elucidation 13 1.4.2 Analytical techniques 15 1.4.2.1 GC/MS 16 1.4.2.2 LC/MS 17 1.4.2.3 Immunoassays 18 1.4.3 Deuterium labeled internal standards for GC/MS and LC/MS 19 1.4.4 Isotopic purity 20 1.5 Labeling of estrogens with isotopes of hydrogen 20 1.5.1 Deuterium-labeling 21 1.5.1.1 Mineral acid catalysts 21 1.5.1.2 CF3COOD as deuterating reagent 22 1.5.1.3 Base-catalyzed deuterations 24 1.5.1.4 Transition metal-catalyzed deuterations 25 1.5.1.5 Deuteration without catalyst 27 1.5.1.6 Halogen-deuterium exchange 27 1.5.1.7 Multistep labelings 28 1.5.1.8 Summary of deuterations 30 1.5.2 Enhancement of deuteration 30 1.5.2.1 Microwave irradiation 30 1.5.2.2 Ultrasound 31 1.5.3 Tritium labeling 32 1.6 Deuteration estrogen fatty acid esters 34 1.7 Synthesis of 2-methoxyestradiol 35 1.7.1 Halogenation 35 1.7.2 Nitration of estrogens 37 1.7.3 Formylation 38 1.7.4 Fries rearrangement 39 1.7.5 Other syntheses of 2-methoxyestradiol 39 1.7.6 Synthesis of 4-methoxyestrone 40 1.8 Synthesis of 2- and 4-hydroxyestrogens 41 2. AIMS OF THE STUDY 43 2 3. RESULTS AND DISCUSSION 44 3.1 Deuteration of estrogens 44 3.1.1 Calculation of isotopic purity 44 3.1.2 Deuteration of estrone with CF3COOD in microwave conditions 45 3.1.3 Deuteration of metabolites of estrogen 2- and 4-hydroxylation routes with CF3COOD 45 3.1.4 Deuteration with other acid catalysts 46 3.1.5 NaOD as catalyst 47 3.2 Synthesis of deuterated estrane-3,17-diones 48 3.3 Synthesis of deuterated estrogen fatty acid esters 49 3.4 Synthesis of 2-methoxyestradiol 50 3.5 Use of labeled estrogens in biological studies 51 4. CONCLUSIONS 54 5. EXPERIMENTAL 55 5.1 General methods 55 5.2 Deuterations of estrone 55 5.2.1 DCl in MeOD 55 5.2.2 D2SO4 in MeOD 56 5.2.3 NaOD in MW 56 5.2.4 NaOD 56 5.3 Deuterations of estrone 2- and 4-metabolites 56 5.3.1 General procedure for CF3COOD deuteration 56 5.3.2 General procedure for LiAlD4 reduction 57 6. REFERENCES 58 3 ABSTRACT Estrogens are endogenous hormones, which exert diverse biological effects on mammals. The metabolites that form when they undergo oxidative metabolism can influence cancer formation by preventing or promoting tumor formation. The most potentially useful estrogen metabolite in the prevention of cancer is 2-methoxyestradiol, which has shown to prevent tumor growth in various cell lines. It is currently under clinical trial. Hydroxyestrone metabolites, in contrast, can form adducts with DNA and thus induce cancer formation. In this study, a new synthesis route, in four steps, was developed for the estrogen metabolite 2-methoxyestradiol. The metabolite 2-hydroxyestradiol was obtained using a modified synthesis strategy. Because estrogen metabolites have divergent biological effects, it is important to quantify their occurence in biological samples. Isotopically labeled estrogens are required as internal standards in the quantitation of estrogens by the isotope dilution GC/MS SIM analysis method. A new deuterium- labeling method (CF3COOD), with high isotopic purity was developed for estrone and other oxoestrogen metabolites. The corresponding deuterated estradiols were synthesized using a LiAlD4 reduction method. Deuteration under microwave irradiation was investigated and found to to reduce the reaction times significantly as compared with traditional heating. Deuteration of estrone with Pd/C and D2 in EtOAc was shown to cause reduction of the aromatic ring of the estrone. The product was d8- deuterated estrane-3,17-dione, the first estrane-3,17-dione product to be identified in metal-catalyzed hydrogenation of estrone, which normally produces estrane-3,17-diols. Estrogen fatty acid esters are considered to function in a hormonal storage capacity in lipoproteins and they inhibit in vitro lipoprotein oxidation. Five deuterated estrone fatty acid esters (palmitate, stearate, oleate, linolate, and linolenate) and ten deuterated estradiol mono- and diesters were synthesized for the first time. The new microwave irradiation technique was utilized in their synthesis. 4 ACKNOWLEDGMENTS The experimental work of this thesis was carried out at the Laboratory of Organic Chemistry of the University of Helsinki. I am most grateful to my supervisor, Prof. Kristiina Wähälä for introducing me the interesting field of steroids and for her support and criticism during the years of this work. I want to thank Emer. Prof. Tapio Hase for helpful scientific advice during my work. My warmest thanks to Emer. Prof. Herman Adlercreutz for valuable collaboration and thanks to Prof. Matti J. Tikkanen and his group for collaboration in the fatty acid project. I want to thank all present and former members of Phyto-Syn group. Work has never been boring in our group and I have enjoyed the discussions during the long lasting coffee breaks. Especially thanks to Sirpa, Auli and Barbara. Thanks to Antti and Sampo for endless patience in technical problems. I am grateful to all students who have worked in the estrogen project, especially Ullastiina and Maarika. Also thanks to all personnel of Organic Chemistry Laboratory. Thanks to Tiina for friendship during all these years. I wish to thank Dr. Jorma Matikainen for running the mass spectra, Seppo Kaltia for recording the 2D NMR data and Dr. Jorma Koskimies for theoretical calculations. And I wish to thank Terhi Hakala for assistance in radiolabeling. I am grateful to Prof. Harri Lönnberg and Dr. Sirpa Rasku for reviewing the manuscript of this thesis and for their helpful comments. I am grateful to Dr. Kathleen Ahonen for revising the language of this manuscript. My warmest thanks to Tuomas for understanding and patience during these years. I want to thank the former Graduate School of Steroid Research for inspiring meetings. Financial support from the Alfred Kordelin, Emil Aaltonen, and Magnus Ehrnrooth foundations, Finnish Cultural foundation, University of Helsinki and the Graduate School of Bioorganic Chemistry is gratefully acknowledged. Helsinki May 2005 Paula Kiuru 5 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original articles, which are referred to in the text by their Roman numerals (I-VI): I. Kiuru, P. S., and Wähälä, K. Expedient microwave deuteration of estrone in CF3COOD. Tetrahedron Lett. 2002, 43, 3411-3412. II. Kiuru, P. S., and Wähälä K. Short synthesis of 2-methoxyestradiol and 2-hydroxyestradiol. Steroids, 2003, 68, 373-375. III. Wähälä, K., Kiuru, P., Kaltia, S., Koskimies, J., and Hase T. The hydrogenation and deuteration of estrone. Synlett, 1997, 460. IV. Adlercreutz, H., Kiuru, P., Rasku, S., Wähälä, K., and Fotsis, T. An isotope dilution gas chromatographic – mass spectrometric method for the simultaneous assay of estrogens and phytoestrogens in urine. J. Steroid Biochem. Molec. Biol. 2004, 92, 399-411. V. Vihma, V., Adlercreutz, H., Tiitinen, A., Kiuru, P., Wähälä, K., and Tikkanen, M. J. Quantitative determination of estradiol fatty acid esters in human pregnancy serum and ovarian follicular fluid. Clin. Chem. 2001, 47, 1256-1262. VI. Kiuru, P. S., and Wähälä, K. Synthesis of deuterated estrogen fatty acid esters. Steroids 2005 (submitted) 6 LIST OF ABBREVIATIONS Ac acetyl APCI atmospheric pressure chemical ionization CI chemical ionization COSY correlated spectroscopy CYP cytochrome P450 DAD diode array detector DEAE diethylaminoethyl DMAP 4-(dimethylamino)pyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxide EC electron capture EI electron impact EIA enzyme immunoassay ELISA enzyme-linked immunosorbent assay ESI electrospray ionization EtOAc ethyl acetate FIA fluoroimmunoassay GC gas chromatography HFB heptafluorobutyryl HMDS hexamethyldisilazane HMPA hexamethylphosphoramide HPLC high-performance liquid chromatography HRT hormone replacement therapy ID isotope dilution ip isotopic purity IR infrared IS internal standard LC liquid chromatography MCPBA m-chloroperbenzoic acid Me methyl MOM methoxymethyl MS mass spectrometry MW microwave NBS N-bromosuccinimide NI negative ion NMR nuclear magnetic resonance PFB pentafluorobenzoyl Ph phenyl RIA radioimmunoassay SIM selected ion monitoring SRM single reaction monitoring TR-FIA time-resolved fluoroimmunoassay THF tetrahydrofuran THP tetrahydropyranyl TLC thin layer chromatography TMCS trimethylchlorosilane UV ultraviolet 7 1. INTRODUCTION Estrogens are female sex hormones, belonging to a group of steroids, and they are responsible for the sexual characteristics of the female.1 They also have effects on bone, cardiovascular system, brain, and skin. The main human estrogens are estrone (E1) 1, estradiol (E2) 2, and estriol (E3) 3 (Fig. 1). Biologically the most active and abundant estrogen is estradiol 2. The estrogen actions occur through the binding of the estrogens to estrogen receptors. The estrogen receptors α and β — the latter found by Gustafsson’s group2 in 1996 — are present in various tissues.