Research paper
Synthesis of 17β-hydroxysteroid dehydrogenase type 10 steroidal inhibitors: Selectivity, metabolic stability and enhanced potency

https://doi.org/10.1016/j.ejmech.2020.112909Get rights and content

Highlights

  • 128 N-substituted 3β-piperazinylmethyl-3α–OH–5α-androstane derivatives were prepared.

  • They inhibited the oxidation of estradiol to estrone by 17β-HSD10.

  • Compound D-3,7 is the best inhibitor with IC50 value of 0.14 μM.

  • D-ring modifications increased metabolic stability.

  • D-ring modifications increased selectivity of inhibition (17β-HSD10 vs 17β-HSD3).

Abstract

17beta-Hydroxysteroid dehydrogenase type 10 (17β-HSD10) is the only mitochondrial member of 17β-HSD family. This enzyme can oxidize estradiol (E2) into estrone (E1), thus reducing concentration of this neuroprotective steroid. Since 17β-HSD10 possesses properties that suggest a possible role in Alzheimer’s disease, its inhibition appears to be a therapeutic strategy. After we identified the androsterone (ADT) derivative 1 as a first steroidal inhibitor of 17β-HSD10, new analogs were synthesized to increase the metabolic stability, to improve the selectivity of inhibition over 17β-HSD3 and to optimize the inhibitory potency. From six D-ring derivatives of 1 (17-Cdouble bondO), two compounds (17β-H/17α-OH and 17β-OH/17α-Ctriple bondCH) were more metabolically stable and did not inhibit the 17β-HSD3. Moreover, solid phase synthesis was used to extend the molecular diversity on the 3β-piperazinylmethyl group of the steroid base core. Eight over 120 new derivatives were more potent inhibitors than 1 for the transformation of E2 to E1, with the 4-(4-trifluoromethyl-3-methoxybenzyl)piperazin-1-ylmethyl-ADT (D-3,7) being 16 times more potent (IC50 = 0.14 μM). Finally, D-ring modification of D-3,7 provided 17β-OH/17α-Ctriple bondCH derivative 25 and 17β-H/17α-OH derivative 26, which were more potent inhibitor than 1 (1.8 and 2.4 times, respectively).

Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common cause of dementia [1]. About 50 million people are living with dementia worldwide and it is estimated that this prevalence will almost double every 20 years [2]. AD cause the gradual loss of important neuronal functions as language, motor functions, but especially memory. These are clinical symptoms, but neuropathologically, AD is characterized by the brain atrophy and the abundance of extracellular amyloid beta (Aβ) plaques [1]. Human 17β-hydroxysteroid dehydrogenase (17β-HSD) is a family of oxidoreductases that catalyzes the interconversions of ketones and alcohols. These enzymes transform functional groups at position 17 of the steroid backbone depending on their substrate specificity [3]. Some 17β-HSDs have thus key roles in up or down regulation of androgens, estrogens and neurosteroids, making them interesting therapeutic targets in many diseases [[3], [4], [5]].

Mitochondrial 17β-HSD type 10 (17β-HSD10), a member of 17β-HSD family, possesses properties that suggest a possible role in AD [6,7]. One of these properties is its capacity to transform estradiol (E2) into estrone (E1). In fact, E2 is one substrate among others of 17β-HSD10 (Km = 14–43 μM, Fig. 1) [[7], [8], [9], [10]]. The E2 concentration in brains of AD patients being reduced, the inhibition of 17β-HSD10 could help to restore E2 levels and promote its neuroprotective effects. 17β-HSD10 is also known for the formation of a high affinity neurotoxic complex with Aβ-42, the peptide responsible of the formation of Aβ plaque in the brain. In mitochondria, Aβ-42 can bind to 17β-HSD10, disrupts the oxidoreductase function of the enzyme and cau`ses dysfunction and neuronal cell death [11]. Furthermore, several studies showed that inhibiting this neurotoxic complex formation is a promising therapeutic approach [6,7,[12], [13], [14]]. It is also important to note that higher concentrations of 17β-HSD10 are found in the brain of AD patients [7,15]. Consequently, the development of 17β-HSD10 inhibitors offer the opportunity to validate the role of 17β-HSD10 in AD and provide a new therapeutic approach against this disease.

There are few known 17β-HSD10 inhibitors [[16], [17], [18]]. One of them is AG18051, an irreversible inhibitor with a pyrimidine core identified by Kissinger et al. [19]. Benzothiazole and frentizole derivatives were also synthesized and evaluated as potent inhibitors of 17β-HSD10 [[20], [21], [22], [23], [24], [25], [26], [27], [28]]. None of these inhibitor, including AG18051, were however based on a steroid scaffold. In fact, our research group reported compound 1 (Fig. 2) as the first steroidal inhibitor of 17β-HSD10 [29,30]. This 5α-androstane-3α,17β-diol derivative inhibited (IC50 = 0.55 μM) the transformation of E2 to E1 by HEK-293[17β-HSD10] cells, but it also inhibited 17β-HSD3, an enzyme that catalyzes the reduction of 4-androstene-3,17-dione (4-dione) into testosterone (T). Since a low level of androstane (T or 5α-dihydrotestosterone) is associated with an increased risk of AD [31], and considering the expression of 17β-HSD3 in hippocampus [32], the type 10/type 3 selectivity of compound 1 needed to be improved. Furthermore, a first round of metabolic stability assay also showed that compound 1 was weakly stable in the conditions of the assay using human liver microsomes, highlighting a potential issue toward its translation into a drug.

Three different strategies (Fig. 2) were tested to improve the biological activities of 17β-HSD10 steroidal inhibitor 1. In the first one, D-ring derivatives of 1 (modifications at C16 or C17) were synthesized using classical chemistry in solution to increase its metabolic stability and type 10/type 3 selectivity. In the second strategy, libraries of amide, sulfonamide, urea, thiourea and amine derivatives of 1 (side chain modifications) were generated by parallel solid phase-synthesis to increase its inhibitor potency and selectivity. Finally, in the third strategy, two hybrid compounds were synthesized to combine the results of strategies 1 and 2.

Section snippets

Chemical synthesis of D-ring derivatives 27 (first strategy)

The lead compound 1 was first synthesized from epi-androsterone (epi-ADT; 12) or dihydrotestosterone (DHT; 15a) using the conditions previously published [33], and then used as starting material for the synthesis of 24 (Scheme 1). The C-17 carbonyl of 1 underwent a stereoselective reduction using sodium borohydride to give 17β-OH derivative 2. The compound 3 (17β-OH/17α-CH3) was synthesized through a Grignard reaction with methyl magnesium iodide. Compound 4b (17β-OH/17α-Ctriple bondCH) was synthesized

Discussion

Optimization of 17β-HSD10 lead inhibitor 1 has been addressed by using three successive SAR strategies. We first performed six D-ring modifications to improve metabolic stability and/or selectivity of action over 17β-HSD3 by synthesizing compounds 27 using classic chemistry in solution. Secondly, we prepared 120 3β-piperazinyl-methyl-ADT derivatives (libraries A-D members represented by general structures 811) by parallel solid-phase synthesis in order to increase the level of inhibition of

Conclusion

We synthesized 128 compounds (3α-hydroxy-5α-androstane derivatives with D-ring modification or/and N-substituted 3β-piperazinylmethyl side chain) and tested their ability to inhibit the transformation (oxidation) of E2 to E1 by 17β-HSD10, a mitochondrial enzyme suspected to play a role in AD. Two D-ring modifications (17β-OH/17α-CCH and 17β-H/17α-OH) made it possible to increase the metabolic stability of lead compound 1 while making these inhibitors selective for 17β-HSD10 over the 17β-HSD3.

General

Chemical reagents were purchased from Sigma-Aldrich (Saint-Louis, MI, USA), Matrix Innovation (Québec, QC, Canada), Alfa Aesar (Wood Hill, MA, USA) and AstaTech (Bristol, PA, USA), Enamine Building Blocks (Cincinnati, OH, USA), Platte Valley Scientifics (Gothenburg, NE, USA), Aldlab Chemicals (Woburn, MA, USA) and LabNetwork Compounds (Cambridge, MA, USA). The glycerol polymer bound with a loading of 1.0 mmol/g was supplied by Sigma-Aldrich. Anhydrous dichloromethane (DCM), diethyl ether (Et2

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

  • RM, JR and DP have ownership interests on patent applications and patents related to 17β-HSD inhibitors. SB declares no conflict of interest.

Acknowledgments

This work was supported by a seed grant from Merck Sharpe & Dome – Faculté de médecine (Université Laval) and a financial support from Mitacs Inc (Montréal, QC, Canada). Sophie Boutin would like to thank the foundation of CHU de Québec (Endocrinology and Nephrology Unit) and the Faculty of Medicine of Université Laval for two fellowships. The authors would like to thank IPSEN INNOVATION (France) for providing LNCaP[17β-HSD3] cells. Our thanks also to Dr. Martin Perreault, for its participation

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