Synthesis of indomethacin thiadiazole urea derivatives and determination of GSTA4 inhibition and cytotoxic activity against colorectal carcinoma

Twelve new indomethacine-thiadiazole-urea derivatives were synthesized and evaluated for their inhibitory effects on Glutathione S-transferase Alpha 4 (GSTA4). Among these, compound 3a showed the most potent inhibitory activity with an IC₅₀ value of 3.12 μM against GSTA4. The enzyme inhibition mechanisms of the synthesized derivatives were elucidated through kinetic parameter analysis. Compounds 3a and 3b were identified as competitive inhibitors with calculated K_i values of 2.38 μM and 2.40 μM, respectively, confirming their strong affinity for the active site. The cytotoxicity of target compounds was further assessed on colorectal carcinoma (HT-29) and colorectal adenocarcinoma (DLD-1) cell lines. Based on the cytotoxicity results, compound 3e (IC₅₀ = 7.64 μM) exhibited the highest cytotoxic activity against DLD-1 cells, followed closely by compound 3f (IC₅₀ = 7.87 μM). Conversely, compound 3f demonstrated the strongest cytotoxic effect on HT-29 cells with an IC₅₀ value of 44.25 μM. The potential impact of the synthesized compounds on healthy cells was also evaluated using the CCD-986Sk cell line. Molecular docking studies demonstrated that compound 3b, acting as an uncompetitive inhibitor with -10.239 kcal/mol docking score, binds within the GSTA4 enzyme-substrate complex (PDB ID: 1GUL), and molecular modelling further highlighted its potential binding interactions stabilizing the complex. Also, compounds 3a and 3g demonstrated the most favourable binding affinities, with docking scores of -14.308 and -13.142 kcal/mol, respectively. Indomethacin showed a docking score of -12.162 kcal/mol against GSTA4.

Cytotoxicity; GSTA4 inhibition; Indomethacin urea; Molecular docking; Thiadiazole ring.