Developing contraceptive and anticancer agents RAR ALPHA antagonists and pironetin analogs.

Abstract

Despite much interest in the male contraceptive pill, none has been approved yet. Gene knockout studies have validated retinoic acid receptor α (RARα) as a target for male contraceptive discovery. Male RARα knockout mice are sterile due to impaired spermatogenesis but show no observable phenotypic abnormalities. This indicates that inhibiting RARα function could produce a contraceptive effect without an inherent risk for side effects. Therefore, we aimed to develop a selective RARα antagonist as a safe, effective, and reversible male contraceptive agent with no off-target effects on RARβ and RARγ. We utilized the structural differences between RARα, β, and γ ligand-binding domains to achieve RARα selectivity. Additionally, the structural differences between RARα bound to the agonist and the antagonist facilitated the design of full antagonists. We also explored modification on many RAR ligand scaffolds, employing structural information obtained from ligand-bound crystal structures of RARs. Over 130 compounds were synthesized and evaluated for RARα antagonist activity and selectivity using a β-lactamase reporter assay. Through structural modifications, we were able to increase compound potency more than 20-fold. We also obtained several antagonists with high antagonistic potency for RARα and excellent selectivity over RARβ and RARγ. Once fully characterized for in vitro and in vivo PK properties, potent and selective antagonists could serve as preclinical candidates for male contraceptive discovery.Compared to non-covalent RAR antagonists, covalent antagonists could offer a longer duration of action at a lower dose due to their longer on-target residence time. Therefore, we developed several covalent RARα antagonists by targeting the nucleophilic C235 residue in the RARα ligand binding pocket with a novel 5-hydroxydihydrofuran-2(3H)-one warhead. By leveraging the structural difference between RARα and RARβγ, we prepared one pan-RAR antagonist and one RARα selective antagonist. Both compounds demonstrated reversible covalent binding with RARα and potent time-dependent antagonism in a cell-based assay. The RARα-selective covalent antagonist could serve as a promising candidate for in vivo evaluation. We also prepared synthetic analogs of the natural product pironetin for anticancer application. Pironetin, a polyketide natural product, has shown anticancer effects by binding to α-tubulin and inhibiting tubulin polymerization. Despite the excellent in vitro cytotoxicity of pironetin against many cancer cell lines, it was not effective in an in vivo tumor model due to poor PK/PD properties and off-target toxicities. Our group previously identified major metabolites of pironetin, and we designed new analogs to block the site of metabolism by replacing the metabolically susceptible exocyclic double bond with phenyl and 4-fluorophenyl groups. We synthesized three analogs using 18-step stereoselective reactions and evaluated their cytotoxic potency using several ovarian cancer cell lines. Two analogs showed cytotoxicity comparable to pironetin yet lacked metabolic stability. concurrently, we also observed a linear trend between clogP and instability in liver microsomes, indicating that increasing the clogP of the analogs increased their metabolic susceptibility.