Discovery of new carbonic anhydrase IX inhibitors as anticancer agents by toning the hydrophobic and hydrophilic rims of the active site to encounter the dual-tail approach

Abstract

The hydrophobic and the hydrophilic rims in the active site of human carbonic anhydrase IX (hCA IX) which as well contains a zinc ion as part of the catalytic core, were simultaneously matched to design and synthesize potent and selective inhibitors using a dual-tail approach. Seventeen new compounds, 5a-q, were designed to have the benzenesulfonamide moiety as a zinc binding group. In addition, N-substituted hydrazone and N-phenyl fragments were chosen as the hydrophilic and hydrophobic parts, respectively to achieve favorable interactions with the corresponding halves of the active site. All synthesized compounds successfully suppressed the CA IX, with IC₅₀ values in nanomolar range from 13.3 to 259 nM. Compounds, 5h, 5c, 5m, 5e, and 5k were the top-five compounds efficiently inhibited the tumor-related CA IX isoform in the low nanomolar range (K_I = 13.3, 22.6, 25.8, 26.9 and 27.2 nM, respectively). The target compounds 5a-q developed remarkable selectivity toward the tumor-associated isoforms (hCA IX and XII) over the off-target isoforms (hCA I and II). Furthermore, they were assessed for their anti-proliferative activity, according to US-NCI protocol, against a panel of fifty-nine cancer cell lines. Compounds 5d, 5k and 5o were passed the criteria for activity and scheduled automatically for evaluation at five concentrations with 10-fold dilutions. Compound 5k exhibited significant in vitro anticancer activity with GI₅₀-MID; 8.68 μM compared to compounds 5d and 5o with GI₅₀-MID; 25.76 μM and 34.97 μM respectively. The most selective compounds 5h and 5k were further screened for their in vitro cytotoxic activity against SK-MEL-5, HCC-2998 and RXF 393 cancer cell lines under hypoxic conditions. Furthermore, 5k was screened for cell cycle disturbance, apoptosis induction and intracellular reactive oxygen species (ROS) production in SK-MEL-5 cancer cells. Finally, molecular docking studies were performed to gain insights for the plausible binding interactions and affinities for selected compounds within hCA IX active site.

Keywords: Anticancer; CA inhibitors; Carbonic anhydrase; Dual-tail approach; Selectivity; Sulfonamides.