Novel 3,4-diaminothieno[2,3-b]thiophene-2,5-dicarbohydrazide-based scaffolds as EGFR, EGFR, and tubulin polymerization inhibitors with anti-proliferative activity.

Using 3,4-diaminothieno[2,3-b]thiophene-2,5-dicarbohydrazide 5, new scaffolds were created that inhibit EGFR, EGFR protein kinases, and tubulin polymerization. Target compounds were evaluated for anti-proliferative efficacy toward A431, H1975, and A549 cells via the MTT technique. The results indicated that compounds 7a, 7b, and 9 exhibited the most significant anti-proliferative activity, exhibiting IC values ranging from 4.64 to 9.45 μM, surpassing the potency of erlotinib, gefitinib, and osimertinib. Compound 7a inhibited EGFR and EGFR kinases via IC values of 24.6 and 28.1 nM, respectively. The outcomes exceeded those of osimertinib and gefitinib, which had IC values of 58.1, 8.1, 17.8, and 373.5 nM. Derivative 7a showed significant anti-proliferative effects on HCT116 and T47D cells, via IC values of 3.12 and 4.12 μM, respectively. Analog 7a selectively suppressed HEK293 cells and hampered tubulin polymerization through IC values of 5.1 μM, comparable with CA-4 (IC = 4.3 μM). The synthetic analogs (7a, 7b, and 9) dock at the EGFR, EGFR, and tubulin active binding sites, matching their in vitro data. Analogs 7a and 7b, like osimertinib and neratinib, can create an irreversible covalent binding with Cys797 in the EGFR ATP cleft, potentially treating mutations. MD simulations by the iMODs server evaluated protein-7a complex stability. In silico ADME analysis of prospective EGFR inhibitors 7a and 9 utilizing the egg-boiled technique showed favorable lipophilicity, GIT absorption, and BBB permeability. Thus, our proposed compounds 7a and 9 showed promising anti-proliferative properties, targeting EGFR and EGFR kinases and inhibiting tubulin.