Impact of trifluoromethyl Ugi adducts as anticancer agents: EGFR inhibition, apoptosis induction and miRNA up-regulation

A series of novel bis-amide Ugi adducts with a Y-shaped configuration imitating the thematic feature of fourth-generation EGFR inhibitors was designed and synthesized <i>via</i> a one-step Ugi four-component reaction. All the synthesized Ugi adducts were evaluated for their anti-proliferative efficacy against MDA-MB-231 human breast cancer and A549 non-small cell lung cancer cell lines. Their selectivity was assessed using normal human lung epithelial cells (BEAS-2B). The Ugi adduct 5 stood up as the study hit concerning cytotoxic efficacy and selectivity. It was 1.42- and 1.20-fold more potent than 5-FU and cisplatin, respectively, against the MDA-MB-231 cell line, and 1.82- and 1.62-fold more potent than 5-FU and cisplatin, respectively, against the A549 cell line. Additionally, compound 5 demonstrated the most prominent selectivity against MDA-MB-231 and A549 cells (SI = 7 and 11.7, respectively); it was 14 to 20.9-fold safer than 5-FU and cisplatin. Accordingly, it was subjected to further enzymatic and mechanistic studies. The Ugi adduct 5 showed excellent sub-micromolar potency (IC₅₀ = 0.19 μM) against human mutant EGFR^{T790M/C797S/L858R} enzyme equipotent with that of osimertinib (IC₅₀ = 0.1 μM). It exhibited a promising IC₅₀ value of 2.1 nM against the EGFR^WT enzyme, comparable to erlotinib (IC₅₀ = 1.3 nM). In comparison to the untreated control, the Ugi adduct 5 caused a decrease in the expression of the cancer initiation, angiogenic, and metastatic markers (c-Myc, CD-44, CD-133, VEGF, and TGF) to 0.36, 0.5, 0.7, 0.5, and 0.4-fold, respectively, in MDA-MB-231 cells. Regarding A549 cells, the exposure to compound 5 showed a 0.41, 0.64, 0.58, 0.71, and 0.69-fold reduction in the expression of c-Myc, CD-44, CD-133, VEGF, and TGF markers compared to the untreated control. Compound 5 markedly elevated miRNA-132 and miRNA-200c expressions in the MDA-MB-231 cell line by 3.8 and 3.1-fold, while in A549 cells, compound 5 demonstrated enhancement of miRNA-132 and miRNA-200c expression by 2.4 and 1.9-fold changes compared to that of the control. It promoted apoptosis induction <i>via</i> caspase 3/9 activation (1.8 and 2.3-folds) in the A549 cell line. The molecular docking interpretations of the most potent Ugi adduct 5 in EGFR^{L858R/T790M/C797S} (PDB ID: 6LUB) and the wild-type EGFR^WT (PDB ID: 1M17) enzymes are aligned with and explain its potential to dually inhibit EGFR^{L858R/T790M/C797S} and EGFR^WT tyrosine kinases.