Given the excellent synthetic accessibility score and biological profile emerged by both chalcone scaffolds and sulfonamide moieties, two novel series of chalcone-sulfonamide hybrids, designated as 3a-i and 6a-e, were synthesized and characterized using NMR spectroscopy (1H and 13C). The designated compounds were assessed for their in vitro cytotoxic effects on a panel of four of the most common cancers: breast carcinoma (MCF-7), colorectal carcinoma (HCT-116), cervical carcinoma (HeLa), and lung cancer cells (A549). Most of the evaluated hybrids demonstrated comparable anticancer efficacy compared to the reference drug, SAHA, across the four cancer cell lines. Hybrids 6a and 6e demonstrated remarkable efficacy against the four cancer cell lines, exhibiting IC50 values that ranged from sub-micromolar to single digit micromolar (0.60 ± 0.03 to 2.54 ± 0.14 μM). Mechanistically, both unsubstituted and bromo hybrids 6a and 6e demonstrated significant inhibition potency related to the reference drug SAHA against HDAC bio targets 1, 2, 3, 4, 6, and 8 with IC50 values ranging between 100 and 900 nM. Additionally, compounds 6a and 6e were found to be potent carbonic anhydrase inhibitors against the tested hCA isoforms, with inhibition constant (KIs) values mainly reaching a low nanomolar range (50.76-298.71 nM) compared to acetazolamide reference, beside their remarkable tubulin polymerization potential. As expected, after 48 h., compounds 6a and 6e successfully increased the protein levels of Caspase-9 in the MCF-7 cells to 14.973 ± 0.66 ng/mL and 13.57 ± 1.08 ng/mL, respectively, compared to the control (2.67 ± 0.13 ng/mL). The flow cytometry analysis in MCF-7 cells demonstrated that hybrid 6e effectively arrested the cell cycle at the G2/M phase and induced apoptosis in a dose-dependent manner through mitochondrial apoptotic activation mechanisms. The apoptotic mechanistic evaluation has been attributed to accumulation of reactive oxygen species (ROS), inhibition of PARP-1, elevate Bax/Bcl-2 ratio, and activation of caspases-7, and 9. Furthermore, the docking results aligned with the biological screening indicating higher affinity of the chalcone-sulfonamide hybrid 6e against HDAC2, CA-IX and tubulin biotargets, serving it as promising multi-targeted inhibitor candidate for cancer therapy. Using a standard atomistic 100 ns dynamic simulation analysis, the stability of the complex formed between compound 6e and the HDAC2, CA-IX, and tubulin active sites was examined. Furthermore, the ADMET properties of 6e fulfilled Pfizer's criteria, demonstrating an optimal pharmacokinetic and safety profile.
