Design, chemical synthesis, and in vitro biological evaluation of simplified estradiolÀadenosine hybrids as inhibitors of 17b-hydroxysteroid dehydrogenase type 1 Marie Be ´ rube ´ and Donald Poirier Abstract: A series of estradiol (E2) derivatives were designed to interact with both the substrate- and the cofactor-binding sites of 17b-hydroxysteroid dehydrogenase type 1 (17b-HSD1). These analogues of potent E2Àadenosine hybrid inhibitor EM-1745, where the adenosine moiety was replaced by a more stable benzene derivative, were synthesized from estrone using alkene cross-metathesis and Sonogashira coupling reactions as key steps. In vitro biological evaluation of these ste- roid derivatives revealed that a spacer of 13 methylenes, between the 16b-position of E2 and the adenosine mimic bearing a carboxylic acid group, gave the best inhibition of 17b-HSD1. Key words: 17b-hydroxysteroid dehydrogenase, enzyme, inhibitor, steroid, metathesis, Sonogashira coupling. Re ´sume ´: Plusieurs de ´rive ´s de l’estradiol (E 2 ) ont e ´te ´ pre ´pare ´s pour interagir avec le site de liaison du substrat et le site de liaison du cofacteur de la 17b-hydroxyste ´roı ¨de de ´shydroge ´nase type 1 (17b-HSD1). Ces analogues du puissant inhibi- teur EM-1745, un hybride E 2 Àade ´nosine dont la partie ade ´nosine a e ´te ´ remplace ´e par un de ´rive ´ benzylique plus stable, ont e ´te ´ obtenus a ` partir de l’estrone en utilisant la me ´tathe `se de Grubbs et le couplage de Sonogashira comme e ´tapes cle ´. L’e ´valuation biologique in vitro de ces de ´rive ´s ste ´roı ¨diens a montre ´ qu’une chaı ˆne ayant 13 groupes me ´thyle `nes, entre la position 16b du ste ´roı ¨de E 2 et le noyau benzylique porteur d’un acide carboxylique, provoquait la meilleure inhibition de l’enzyme 17b-HSD1. Mots-cle ´s : 17b-hydroxyste ´roı ¨de de ´shydroge ´nase, enzyme, inhibiteur, ste ´roı ¨de, me ´tathe `se, couplage de Sonogashira. Introduction 17b-Hydroxysteroid dehydrogenase type 1 (17b-HSD1) or human estradiol dehydrogenase [E.C.1.1.1.62] was partially purified from human placenta by Langer and Engel in 1958. 1 It is a 327-amino-acids protein that exists as a homo- dimer. This enzyme preferentially catalyzes the transforma- tion of estrone (E 1 ) into estradiol (E 2 ), the most potent estrogen, using NAD(P)H as cofactor (Fig. 1), 2À7 but also catalyzes the reduction of dehydroepiandrosterone (DHEA) into 5-androstene-3b,17bdiol (D 5 -diol). 4 17b-HSD1 activities have been found in many human tissues such as placenta, ovary, endometrium, liver, and breast. 8 In peripheral tissues such as the breast, enzyme substrates come from circulating DHEA-sulfate (DHEAS) or E 1 -sulfate (E 1 S), which are con- verted into DHEA and E 1 by steroid sulfatase. 9À11 E 2 and D 5 -diol have, respectively, high and weak affinity for the estrogen receptor (ER). The resulting receptorÀestro- gen complex produces estrogenic effects. Thus, in estro- gen-dependent breast cancer, activation of ER by E 2 is crucial for the stimulation of growth of tumour cells. In fact, E 2 concentration has been found to be significantly higher in breast tumours than in normal breast tissue. 12,13 Furthermore, the intratumoral E 2 levels are high in postme- nopausal patients with very low serum E 2 levels; a study aimed at explaining this revealed that the expression of 17b-HSD1 and the E 2 /E 1 ratio are higher in breast cancer tissues of postmenopausal patients than in those of preme- nopausal ones. 14 Such results suggest that the accumulation of E 2 in breast cancer tissue is mainly caused by the intra- crine activity of 17b-HSD1. In fact, because E 2 increases breast-cancer-cell proliferation, inhibition of 17b-HSD1 ac- tivity constitutes a good way to reduce tumour estrogen lev- els, thus promoting tumour regression. Consequently, the steroidogenic enzyme 17b-HSD1 is an interesting therapeu- tic target for the treatment of estrogen-sensitive diseases such as breast cancer. The design of potent 17b-HSD1 inhibitors has been at- tempted during the past decades, but progress was not sig- nificant enough to justify their use in the treatment of estrogen-sensitive diseases. 15À17 However, interesting clini- Received 6 March 2009. Accepted 5 May 2009. Published on the NRC Research Press Web site at canjchem.nrc.ca on 30 July 2009. This article is dedicated to Dr. Robert H. Burnell, a retired professor from the Department of Chemistry at Laval University (Que ´bec City, Canada) who passed away in November of 2008, for his creative contribution to the field of natural product synthesis and his inspiring mentorship. M. Be ´rube ´ and D. Poirier. 1 Laboratory of Medicinal Chemistry, CHUQ (CHUL)-Research Center and Laval University, Que ´bec City, QC G1V 4G2, Canada. 1 Corresponding author (e-mail: donald.poirier@crchul.ulaval.ca). 1180 Can. J. Chem. 87: 1180–1199 (2009) doi:10.1139/V09-083 Published by NRC Research Press