SLC-0111

Hypoxia, a characteristic feature of solid tumors, develops as a result of excessive cell proliferation and rapid tumor growth exceeding the oxygen supply, and can result in angiogenesis activation, increased invasiveness, aggressiveness, and metastasis, leading to improved tumor survival and suppression of anticancer drug therapeutic impact. SLC-0111, a ureido benzenesulfonamide, is a selective human carbonic anhydrase (hCA) IX inhibitor in clinical trials for the treatment of hypoxic malignancies. Herein, we describe the design and synthesis of novel 6-arylpyridines 8a-l and 9a-d as structural analogues of SLC-0111, in the aim of exploring new selective inhibitors for the cancer-associated hCA IX isoform. The para-fluorophenyl tail in SLC-0111 was replaced by the privileged 6-arylpyridine motif. Moreover, both ortho- and meta-sulfonamide regioisomers, as well as an ethylene extended analogous were developed. All 6-arylpyridine-based SLC-0111 analogues were screened in vitro for their inhibitory potential against a panel of hCAs (hCA I, II, IV and IX isoforms) using stopped-flow CO₂ hydrase assay. In addition, the anticancer activity was firstly explored against a panel of 57 cancer cell lines at the USA NCI-Developmental Therapeutic Program. Compound 8g emerged as the best anti-proliferative candidate with mean GI% value equals 44. Accordingly, a cell viability assay (MTS) for 8g was applied on colorectal HCT-116 and HT-29 cancer cell lines as well as on the healthy HUVEC cells. Thereafter, Annexin V-FITC apoptosis detection, cell cycle, TUNEL, and qRT-PCR, colony formation, and wound healing assays were applied to gain mechanistic insights and to understand the behavior of colorectal cancer cells upon the treatment of compound 8g. Also, a molecular docking analysis was conducted to provide in silico insights into the reported hCA IX inhibitory activity and selectivity.

Sulfonamides constitute an important class of classical carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Herein we have accomplished the conjugation of biotin with an ample number of sulfonamide motifs with the aim of testing them in vitro as inhibitors of the human carbonic anhydrase (hCA) isoforms I and II (cytosolic isozymes), as well as hCA IX and XII (transmembrane, tumor-associated enzymes). Most of these newly synthesized compounds exhibited interesting inhibition profiles, with activities in the nanomolar range. The presence of a 4-F-C₆H₄ moiety, also found in SLC-0111, afforded an excellent selectivity towards the tumor-associated hypoxia-induced hCA isoform XII with an inhibition constant (K_I) of 4.5 nM. The 2-naphthyl derivative was the most potent inhibitor against hCA IX (K_I = 6.2 nM), 4-fold stronger than AAZ (K_I = 25 nM) with very good selectivity. Some compounds were chosen for antiproliferative activity testing against a panel of 3 human tumor cell lines, one compound showing anti-proliferative activity on glioblastoma, triple-negative breast cancer, and pancreatic carcinoma cell lines.