Research paperSynthesis of 17β-hydroxysteroid dehydrogenase type 10 steroidal inhibitors: Selectivity, metabolic stability and enhanced potency
Graphical abstract
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 2–7 (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 2–4 (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-CCH) 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 2–7 using classic chemistry in solution. Secondly, we prepared 120 3β-piperazinyl-methyl-ADT derivatives (libraries A-D members represented by general structures 8–11) 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
References (48)
- et al.
17β-Hydroxysteroid dehydrogenase (17β-HSDs) as therapeutic targets: protein structure, functions, and recent progress in inhibitor development
J. Steroid Biochem. Mol. Biol.
(2011) - et al.
Roles of 17β-hydroxysteroid dehydrogenase type 10 in neurodegenerative disorders
J. Steroid Biochem. Mol. Biol.
(2014) - et al.
Hydroxysteroid (17β) dehydrogenase X in human health and disease
Mol. Cell. Endocrinol.
(2011) - et al.
In vitro assay development and HTS of small-molecule human ABAD/17b-HSD10 inhibitors as therapeutics an Alzheimer”s disease
SLAS Discov.
(2017) - et al.
Crystal structure of human ABAD/HSD10 with a bound inhibitor: implications for design of Alzheimer’s disease therapeutics
J. Mol. Biol.
(2004) - et al.
Design, synthesis and in vitro evaluation of benzothiazole-based ureas as potential ABAD/17β-HSD10 modulators for Alzheimer’s disease treatment
Bioorg. Med. Chem. Lett.
(2016) - et al.
Synthesis and evaluation of frentizole-based indolyl thiourea analogues as MAO/ABAD inhibitors for Alzheimer’s disease treatment
Bioorg. Med. Chem.
(2017) - et al.
Identification of steroidal derivatives inhibiting the transformations of allopregnanolone and estradiol by 17β-hydroxysteroid dehydrogenase type 10
Bioorg. Med. Chem. Lett.
(2018) - et al.
Comparison of sex-steroid synthesis between neonatal and adult rat hippocampus
Biochem. Biophys. Res. Commun.
(2009) - et al.
Development of 3-substituted-androsterone derivatives as potent inhibitors of 17β-hydroxysteroid dehydrogenase type 3
Bioorg. Med. Chem.
(2011)
Chemical synthesis, cytotoxicity, selectivity and bioavailability of 5α-androstante-3α,17β-diol derivatives
Bioorg. Med. Chem.
Chemical synthesis, NMR analysis and evaluation on a cancer xenograft model (HL-60) of the aminosteroid derivative RM-133
Steroids
D-ring allyl derivatives of 17β- and 17α-estradiols: Chemical synthesis and 13C NMR data
Steroids
Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings
Adv. Drug Deliv. Rev.
Lead- and drug-like compounds: the rule of five revolution
Drug Discov. Today
Alzheimer’s and Parkinson’s diseases: the prion concept in relation to assembled Aβ, tau, and α-synuclein
Science
World Alzheimer Report 2015: the Global Impact of Dementia
Design and validation of specific inhibitors of 17β-hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis
Endocr. Relat. Cancer
17β-Hydroxysteroid dehydrogenase inhibitors: a patent review
Expert Opin. Ther. Pat.
Friend or enemy? Review of 17β-HSD10 and its role in human health or disease
J. Nerochem.
Roles of mitochondrial 17β-hydroxysteroid dehydrogenase type 10 in Alzheimer’s disease
J. Alzheimers Dis.
Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Aβ-induced cytotoxicity
J. Biol. Chem.
ABAD directly links Abeta to mitochondrial toxicity in Alzheimer’s disease
Science
Inhibitition of the mitochondial enzyme ABAD restores the amyloid-β-mediated deregulation of estradiol
PLoS One
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