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Pharmacokinetic properties of our first-generation HIF-2α antagonist PT2385, including modest solubility, resulted in a high recommended phase 2 dose (RP2D) of 800 mg BID and motivated the pursuit of novel scaffolds which could improve solubility and formulation parameters with the goal of improved pharmacokinetics. Herein we disclose our successful efforts to identify such HIF-2α antagonists through an optimization strategy characterized by: (1) increasing the fraction of sp3 hybridized carbons (Fsp3), (2) replacing the aromatic portion of the indane core with pyridine heterocycles, and (3) improving a putative Olp→π*Ar interaction, an underutilized electrostatic contact in medicinal chemistry. These efforts emphasize the importance of employing multiple strategies in parameter optimization. In isolation, modifications to areas (1) and (2) improved solubility, but with the compromise of reduced potency. In area (3), understanding the importance of an Olp→π*Ar interaction, as documented through a wealth of crystal structures and retrospective calculations, proved essential in guiding SAR and identifying the trifluoromethyl group as a suitable replacement of the sulfone. Only by combining these three strategies could inhibitors with substantially improved solubility and comparable potency be discovered. Finally, the overall improvement in pharmacokinetic properties of the newly identified inhibitors is highlighted through a battery of ADME and in vivo data, including use of pharmacodynamic biomarkers indicative of HIF-2α antagonism. [ABSTRACT FROM AUTHOR]

Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.