Synthesis, characterization, anti-proliferative, and apoptotic activity of a novel quinazoline-containing 1,2,3-triazole toward three human cancer cells

Abstract

A novel quinazoline-containing 1,2,3-triazole (4-TCPA) was synthesized to target VEGFR2 signaling for cancer treatment. Although current VEGFR2 inhibitors exhibit strong anticancer activity, their clinical use is limited due to severe side effects. Structural analysis of effective VEGFR2 inhibitors guided the design of 4-TCPA, ensuring the retention of Erlotinib's essential pharmacophoric features for optimal receptor binding. The compound was tested for its anticancer efficacy against A549 (lung cancer), MCF7 (breast cancer), K562 (leukemia), and human normal fibroblast HFF2 cells. 4-TCPA demonstrated inhibitory activity in nearly all assays, with IC₅₀ concentrations of 35.70 μM for A549, 19.50 μM for MCF7, 5.95 μM for K562, and 135.2 μM for HFF2. Further in vitro biological evaluations examined its mechanistic impact on EGFR, mTOR, Akt, MAPK, and PIK3CA. Apoptotic activity was assessed through Annexin V/PI double staining and caspase-3/7 activation, confirming its ability to induce both early and late apoptosis. Additionally, real-time quantitative PCR (qRT-PCR) was conducted to evaluate the expression levels of Akt, mTOR, MAPK, PIK3CA, EGFR, and VEGFR2. Results showed that 4-TCPA down-regulates these targets in a time-dependent manner, triggering apoptosis across all tested cancer cell lines. The study suggests that 4-TCPA may serve as a promising anticancer agent due to its targeted VEGFR2 inhibition and apoptotic induction, warranting further investigation. These findings support the potential of 4-TCPA as an effective treatment strategy for various cancers.

Keywords: Apoptosis; Cancer cell; Quinazoline; VEGFR2.

Fig. 1

Reaction conditions and reagents: (a) chloroacetyl chloride 2 (1.2 equiv.), Et₃N (1.2 equiv.), acetone, r.t., overnight; (b) NaN₃ 4 (1.5 equiv.), DMF, r.t., overnight; (c) propargyl amine 7 (1.3 equiv.), Et₃N (1.3 equiv.), DMF, 80 °C, 12h; (d) CuSO₄.5H₂O (0.3 equiv.), sodium ascorbate (0.3 equiv.), DMF, r.t., overnight.

Fig. 2

The cytotoxicity induction of 4-TCPA and Erlotinib (as a positive control) on A549 (a), MCF7 (b), K562 (c) and HFF2 (d) cell lines. The chemical was applied to the cells at different concentrations (0–200 µg/mL) during 72 h. The data reflect the mean plus standard deviation (SD) of three separate studies. By contrasting each group with its control, values of *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were discovered.

Fig. 3

A549, MCF7, and K562 cells treated with 4-TCPA and Erlotinib (as a positive control) were subjected to a quantitative annexin V/PI double staining test to measure apoptosis. (A) 4-TCPA caused apoptosis in A549 (a,b,c), MCF7 (d,e,f), and K562 (g,h,i) cells in a dose- and time-dependent manner, according to flow cytometric analysis in. (B) The graph shows the statistical quantification of data A549 (a), MCF7 (b) and K562 (c) following 4-TCPA treatment. Apoptosis analysis using flow cytometry revealed a time- and dose-dependent increase in early and late apoptotic cells. The data reflect the mean plus standard deviation (SD) of three separate studies. Comparing each group to its control resulted in ***p < ****p < 0.0001.

Fig. 4

Assessment of caspase 3/7 enzymatic activities. The activation of caspase 3/7 was evaluated in A549 (a), MCF7 (b) and K562 (c) cell lines after exposure to the 4-TCPA at indicated time point (72h), as explained in the material and method. Plots represent caspase 3/7 enzymatic activity (mU/mL) of the treated cells relative to the untreated groups and positive control (Erlotinib). The results are expressed as mean ± SD (n = 3) (****p < 0.0001 (A549), **p < 0.0013 (MCF7), **p < 0.0023 (K562)) versus positive control.

Fig. 5

Measurement of mRNA expression levels following 4-TCPA exposure. At the designated time, 4-TCPA with an IC₅₀ concentration was treated with A549 (a), MCF7 (b), K562 (c), cells. qRT-PCR was used to assess changes in the transcript levels of Akt, mTOR, MAPK, PIK3CA, EGFR and VEGFR2. Plots represent Akt, mTOR, MAPK, PIK3CA, EGFR and VEGFR2 expression levels of the treated cells relative to the control (B2M). The findings are presented as the mean SD (n = 3); (****p < 0.0001 (A549), (MCF7) and (K562) VEGFR2) versus control.