Article Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/jmc
Targeted Endocrine Therapy: Design, Synthesis, and Proof-ofPrinciple of 17β-Hydroxysteroid Dehydrogenase Type 2 Inhibitors in Bone Fracture Healing
Downloaded via UNIV OF GOTHENBURG on January 16, 2019 at 02:59:10 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Ahmed S. Abdelsamie,†,‡,○ Steven Herath,§,○ Yannik Biskupek,§ Carsten Börger,∥ Lorenz Siebenbürger,∥ Mohamed Salah,⊥ Claudia Scheuer,# Sandrine Marchais-Oberwinkler,¶ Martin Frotscher,⊥ Tim Pohlemann,§ Michael D. Menger,# Rolf W. Hartmann,⊥,∇ Matthias W. Laschke,# and Chris J. van Koppen*,†,⊥ †
ElexoPharm GmbH, Im Stadtwald, Building A1.2, 66123 Saarbrücken, Germany Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, 12622 Cairo, Egypt § Department of Trauma, Hand and Reconstructive Surgery, and #Institute of Clinical & Experimental Surgery, Saarland University, 66421 Homburg/Saar, Germany ∥ PharmBioTec GmbH, 66123 Saarbrücken, Germany ⊥ Department of Pharmaceutical and Medicinal Chemistry, Saarland University, 66123 Saarbrücken, Germany ¶ Institute of Pharmaceutical Chemistry, Philipps-University, 35032 Marburg, Germany ∇ Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), 66123 Saarbrücken, Germany ‡
S Supporting Information *
ABSTRACT: Current therapies of steroid hormone-dependent diseases predominantly alter steroid hormone concentrations (or their actions) in plasma, in target and nontarget tissues alike, rather than in target organs only. Targeted therapy through the inhibition of steroidogenic enzymes may pose an attractive alternative with much less side effects. Here, we describe the design of a nanomolar potent 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) inhibitor (compound 15) and successful targeted intracrine therapy in a mouse bone fracture model. Blockade of 17β-HSD2 in bone is thought to increase intracellular estradiol (E2) and testosterone (T), which thereby inhibits bone resorption by osteoclasts and stimulates bone formation by osteoblasts, respectively. Administration of compound 15 in the mouse fracture model strongly increases the mechanical stability of the healing fractured bone because of a larger periosteal callus with newly formed bone without changing the plasma E2 and T concentrations. Steroidogenic 17β-HSD2 inhibition thus enables targeted intracrine therapy.
■
to treat patients with benign prostatic hyperplasia,2 11β-HSD1 inhibitors,3 and 17β-HSD1 inhibitors in an endometrial hyperplasia animal model.4 In the present study, we have developed novel nanomolar potent 17β-HSD2 inhibitors and successfully performed a proof-of-principle study in a mouse bone fracture healing model. Approximately 10% of the patients with bone fractures suffer from serious nonunion or delayed/impaired bone fracture healing.5 Inadequate bone healing is not only difficult to treat, with the therapeutic options being very limited,6−8 but the demand for novel drugs with fewer side effects is also increasing rapidly because of the relatively high prevalence of bone fractures, the anticipated
INTRODUCTION Current therapies of steroid hormone-dependent diseases, like osteoporosis and endometriosis, involve primarily treatment with drugs that alter the steroid hormone concentrations (or their actions) in plasma, in target and nontarget tissues alike, rather than in the target organs only. As a consequence, these treatments most often are accompanied with highly undesirable side effects.1 Local intracrine inhibition of steroidogenic enzymes, affecting the intracellular concentrations in the target organs only, represents an attractive, alternative therapeutic paradigm with much less side effects and disturbances of the hypothalamus−pituitary organ axes. Unfortunately, successful proof-of-principle studies with steroidogenic enzyme inhibitors, which do not change systemic hormone concentrations, have been rather infrequent. These include 5α-reductase inhibitors © XXXX American Chemical Society
Received: September 25, 2018
A
DOI: 10.1021/acs.jmedchem.8b01493 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Article
human and murine 17β-HSD2 (h + m17β-HSD2), resulting in a small library of 16 compounds. As a starting point, the nonsubstituted scaffold structure (compound A) was chosen. Previous investigations revealed that the low metabolic stability of the BSHs was primarily due to phase II biotransformation of the phenolic OH group, which is essential for activity.19 Therefore, stage 1 of the design strategy, aiming at the improvement of metabolic stability, consisted in the introduction of small substituents on the hydroxyphenyl moiety (ring A) of compound A. These groups could protect the OH functionality from biotransformation by the electron withdrawal effect and/or steric hindrance (Chart 1, compounds 1−8). Compound 8 showed an enhanced metabolic stability and was chosen as a starting point for the optimization of the substitution pattern of the D-ring (stage 2 of the design strategy). Only such groups were selected to be introduced that were likely to maintain potency toward both human and mouse 17β-HSD2.18 We aimed for a slight (three- to fourfold) selectivity over the h17β-HSD1 enzyme (Chart 2, 9− 16). On the one hand, a highly selective 17β-HSD2 inhibitor would induce an undesirable increase in intracellular E2 in tissues that express similar levels of 17β-HSD2 and 17β-HSD1 and prone to E2-dependent proliferation (i.e., breast20,21 and endometrium22), whereas on the other hand, a nonselective 17β-HSD2/1 inhibitor would likely affect the role of 17βHSD1 in regulating the endometrium cyclicity in women of childbearing age.22 The starting point for the syntheses of compounds 1−16 was a Friedel−Crafts reaction of 2-bromothiophene with the appropriate benzoyl chloride, which in case of the chlorinated 3b and 4b had to be prepared from the corresponding benzoic acids. The obtained intermediates 1b−8b were subjected to Suzuki cross-coupling reactions with 3-aminophenylboronic acid to afford anilines 1a−8a. The latter were reacted with the sulfonamides using the appropriately substituted sulfonic acid chloride, giving direct access to compound 1. Ether cleavage using BBr3 in dichloromethane yielded the final compounds 2−16 (Scheme 1). The introduction of an electron-donating methyl group on ring A (Table 1, 1) led to a twofold decrease in inhibitory potency toward h17β-HSD2 compared to lead A. In contrast, the presence of an electron-withdrawing fluorine or chlorine atom in the same position strongly increased the activity; see 1 versus 2 and 3. The beneficial effect of electron-withdrawing substituents on the inhibitory activity was also apparent for 4−8, in agreement with the observations made recently for the BSHs lacking the sulfonamide moiety.22 Metabolic stability was determined for 3, 7, and 8 as these compounds displayed selectivity over 17βHSD1 and nanomolar potency toward m17β-HSD2. The trifluoro substitution pattern of 8 resulted in an improved metabolic stability and was therefore maintained in the subsequent optimization of ring D. All eight compounds of this second series (9−16) showed a strong inhibition of human and mouse 17β-HSD2 as well as a low activity toward m17βHSD1. Compounds 14 and 15 were especially interesting as they displayed moderate h17β-HSD2 selectivity, which was aimed at, as well as improved metabolic stability. Because of their favorable potency, selectivity, and metabolic stability properties, 14 and 15 were selected for testing for potential cytotoxicity. Both 14 and 15 were not toxic in the MTT assay with HEK293 cells (i.e., 90% in vitro during 24 h. This corresponds to the fairly high metabolic stability measured in mouse liver S9 fraction (t1/2 of 46 min). Next, to analyze bone fracture repair, C57BL/6 male mice received 15 subcutaneously once daily (50 mg/kg) or vehicle (0.5% gelatine/5% mannitol in water) for 14 or 28 days, starting immediately following fracturing of the femur under anesthesia. Bending stiffness of fractured bones, relative to the contralateral unfractured bones in the same animal, is the upmost important functional recovery parameter at the site of the fracture in bone fracture healing.16,24,25 After 14 and 28 days, the bending stiffness of the fractured femurs of the
viability over 66 h) up to concentrations that were approximately 11 000-fold over the respective h17β-HSD2 IC50 value. We also checked whether 14 and 15 do not bind to ERs at relevant concentrations to avoid E2 being blocked from activating ER receptors in bone. Compound 15 displayed negligible ERα and ERβ binding, whereas 14 showed a higher ER binding (i.e., 28 and 21% inhibition of E2 binding in the presence of a 1000-fold excess of 15 over E2 vs 64 and 49% inhibition in the presence of 1000-fold excess of 14, respectively). Compound 15 was able to inhibit endogenously expressed 17β-HSD2 in the cellular assay (human MDA-MB 231 cells) with an IC50 of 2.4 nM, which is very similar to the IC50 of 2.0 nM in the 17β-HSD2 cell-free assay (Table 1). This indicates that compound 15 readily enters cells at pharmacologically relevant concentrations. We thus selected 15 for further profiling and investigated in vitro whether 15 does not induce the expression of hepatic CYP450 enzymes, which otherwise may reduce the plasma concentrations of 15 during repetitive dosing in the bone fracture healing study. At 3.16 μM, 15 did neither activate the aryl hydrocarbon receptor, constitutive androstane receptor, nor the pregnane X receptor (
