Harnessing coumarin privilege to achieve potency and selectivity: hypoxia-targeted alkyl coumarin-tethered phenyl quinazolinones as dual hCA IX/HSP90α inhibitors with chemosensitizing activity

Abstract

Hypoxic tumor regions remain challenging to treat in breast cancer therapy due to extracellular acidification, chemoresistance, and other adaptive mechanisms promoted by the coordinated actions of tumor-associated carbonic anhydrase IX (hCA IX), heat shock protein 90 alpha (HSP90α), and other proteins. Inspired by the privileged role of coumarins as selective hCA IX inhibitors and as HSP90α modulators, together with the ATP-mimetic and anticancer properties of phenyl quinazolinones, a series of alkyl coumarin-tethered phenyl quinazolinones (4a–l) were rationally designed and synthesized as hypoxia-targeted dual hCA IX/HSP90α inhibitors. 1H NMR, 13C NMR-DEPTQ, HRMS, elemental analysis, and HPLC unequivocally established the structures of the synthesized compounds. The synthesized hybrids exhibited potent, highly selective inhibition of hCA IX (in the nanomolar range), while displaying negligible activity against the off-target isoforms hCA I and II. Compound 4b emerged as the lead derivative, exhibiting a KI value of 51.7 nM against hCA IX together with exceptional selectivity (>1934-fold over hCA I and hCA II). Remarkably, 4b also exhibited potent HSP90α inhibitory activity (IC50 = 13.21 nM), comparable to that of ganetespib. Furthermore, 4b preferentially suppressed the proliferation of hypoxic MCF-7 and MDA-MB-231 breast cancer cells, sensitized them to doxorubicin, and induced p53-mediated mitochondrial apoptosis. Molecular docking and molecular dynamics simulations against hCA IX and HSP90α, along with in silico ADME and toxicity studies, further supported the favorable binding behavior, stability, and druglike characteristics of the lead compound.