Design, Synthesis, and Anti-Cervical Cancer and Reversal of Tumor Multidrug Resistance Activity of Novel Nitrogen-Containing Heterocyclic Chalcone Derivatives

Abstract

This study involved the design and synthesis of 21 new nitrogen-containing heterocyclic chalcone derivatives utilizing the active substructure splicing principle, with glycyrrhiza chalcone serving as the lead compound. The targets of these derivatives were VEGFR-2 and P-gp, and their efficacy against cervical cancer was evaluated. Following preliminary conformational analysis, compound 6f ((E)-1-(2-hydroxy-5-((4-hydroxypiperidin-1-yl)methyl)-4-methoxyphenyl)-3-(4-((4-methylpiperidin-1-yl)methyl)phenyl)prop-2-en-1-one) exhibited significant antiproliferative activity against human cervical cancer cells (HeLa and SiHa) with IC₅₀ values of 6.52 ± 0.42 and 7.88 ± 0.52 μM, respectively, when compared to other compounds and positive control drugs. Additionally, this compound demonstrated lower toxicity towards human normal cervical epithelial cells (H8). Subsequent investigations have demonstrated that 6f exerts an inhibitory impact on VEGFR-2, as evidenced by its ability to impede the phosphorylation of p-VEGFR-2, p-PI3K, and p-Akt proteins in HeLa cells. This, in turn, results in the suppression of cell proliferation and the induction of both early and late apoptosis in a concentration-dependent manner. Furthermore, 6f significantly curtails the invasion and migration of HeLa cells. In addition, 6f had an IC₅₀ of 7.74 ± 0.36 μM against human cervical cancer cisplatin-resistant HeLa/DDP cells and a resistance index (RI) of 1.19, compared to 7.36 for cisplatin HeLa cells. The combination of 6f and cisplatin resulted in a significant reduction in cisplatin resistance in HeLa/DDP cells. Molecular docking analyses revealed that 6f exhibited binding free energies of -9.074 and -9.823 kcal·mol^-1 to VEGFR-2 and P-gp targets, respectively, and formed hydrogen bonding forces. These findings suggest that 6f has potential as an anti-cervical cancer agent and may reverse cisplatin-resistant activity in cervical cancer. The introduction of the 4-hydroxy piperidine and 4-methyl piperidine rings may contribute to its efficacy, and its mechanism of action may involve dual inhibition of VEGFR-2 and P-gp targets.

Keywords: anti-cervical cancer activity; azacyclic; cisplatin resistance; glycyrrhiza chalcone; molecular docking.

Figure 1

Chalcone derivatives with inhibitory effects on VEGFR-2 and P-gp targets. The marked blue groups represent the α,β-unsaturated aryl ketones of chalcone; Green represents the morpholine ring, and red represents the piperazine ring.

Figure 2

Hybridization modification of chalcone with VEGFR-2 inhibitor sorafenib. The marked blue groups represent the α,β-unsaturated aryl ketones of chalcone; Green represents the urea group of sorafenib, and red represents the N-methylpicolinamide ring.

Figure 3

Structure of the potential compound synthesized in the previous phase. The red area represents the p-dimethylamine group of chalcone derivatives and the blue font represents the hydroxyl group.

Scheme 1

Synthesis of intermediates 1–3 (Intermediates 1 represents 1-(2,4-dihydroxyphenyl)ethan-1-one, 2 represents 1-(2-hydroxy-4-methoxyphenyl)ethan-1-one, and 3 represents 1-(5-(chloromethyl)-2-hydroxy-4 methoxyphenyl)ethan-1-one). (a) ZnCl₂, CH₃COOH, 110–115 °C, 3 h; (b) CH₃COCH₃, K₂CO₃, 50 °C, 6 h; (c) CH₃COOH, r.t, 6 h.

Scheme 2

Synthesis of intermediates 4a–4f (Intermediates 4a represents 1-(2-hydroxy-5-((4-hydroxypiperidin-1-yl)methyl)-4-methoxyphenyl)ethan-1-one, 4b represents 1-(2-hydroxy-4-methoxy-5-((4- methylpiperazin-1-yl)methyl)phenyl)ethan-1-one, 4c represents 1-(2-hydroxy-4-methoxy-5-((4-methoxypiperidin-1-yl)methyl)phenyl)ethan-1-one, 4d represents 1-(2-hydroxy-4-methoxy-5-((4-methylpiperidin-1-yl)methyl)phenyl)ethan-1-one, 4e represents 1-(2-hydroxy-4-methoxy-5-(morpholinomethyl) phenyl)ethan-1-one, 4f represents 1-(2-hydroxy-4-methoxy-5-(pyrrolidin-1-ylmethyl)phenyl)ethan-1-one). (d) CH₂Cl₂, triethylamine, 45 °C, 6 h, reflux.

Scheme 3

Synthesis of intermediates 5a–5f (Intermediates 5a represents 4-((4-hydroxypiperidin-1-yl)methyl)benzaldehyde, 5b represents 4-((4-methylpiperazin-1-yl)methyl)benzaldehyde, 5c represents 4-((4-methoxypiperidin-1-yl)methyl)benzaldehyde, 5d represents 4-((4-methylpiperidin-1-yl)methyl)benzaldehyde, 5e represents 4-(morpholinomethyl)benzaldehyde, 5f represents 4-(pyrrolidin-1-ylmethyl)benzaldehyde). (e) CH₂Cl₂ or THF, triethylamine, 45 °C, 6 h, reflux.

Scheme 4

Synthesis of chalcones 6a–6u (Represents 21 chalcone derivatives containing different substituted aza rings, See Section 3.2.6 Synthesis of Target Compounds for specific names. The red areas represent the different nitrogen-containing heterocyclic substituents). (f) CH₃CH₂OH, 20% KOH, 60 °C, 8 h.

Scheme 5

(A) Immunoblotting of HUVEC cells treated with the indicated concentrations of 6f and sorafenib for 24 h; (B) quantification of proteins in HUVEC cells by 6f in the bar graph. All results performed above are presented as mean ± SD from three independent experiments. * p < 0.05, compared to the control group.

Scheme 6

(A) Immunoblotting of HeLa cells treated with the indicated concentrations of 6f and sorafenib for 24 h; (B) quantification of proteins in HeLa cells by 6f in the bar graph. All results performed above are presented as mean ± SD from three independent experiments. * p < 0.05, compared to the control group.

Scheme 7

(A) Compound 6f induced the apoptosis of Hela cells, red represents the number of cells, Q1: The cells in this area are necrotic cells. There may also be a few late apoptotic cells in it, and even mechanically damaged cells are included. Q2: The cells in this region are late apoptotic cells. Q3: The cells in this region are early apoptotic cells.Q4: Cells in this region are live cells. (B) quantitative analysis of the proapoptotic effect of 6f in the bar graph on HeLa cells; (C) immunoblotting of HeLa cells treated with the indicated concentrations of 6f and sorafenib for 24 h; (D) quantification of proapoptotic proteins in HeLa cells by 6f in the bar graph. All results performed above are presented as mean ± SD from three independent experiments. * p < 0.05, compared to the control group.

Scheme 8

(A,B) HeLa cells were treated with indicated concentrations of 6f for 24 h and stained with 0.1% crystal violet solution, and quantitative analysis of the number of migrated cells in transwell migration assay; (C,D) the effect of 6f in transwell invasion assay. The HeLa cells that underwent migration and invasion were stained blue-purple with 0.1% crystalline violet. Hela cell morphology was examined under an inverted microscope (magnification: ×100). beiAll results performed above are presented as mean ± SD from three independent experiments. * p < 0.05, compared to the control group.

Scheme 9

(A) Compound 6f blocked the P-gp of HeLa/DDP cells; (B) quantification of P-gp protein expression in HeLa cells by 6f in the bar graph; (C) compound 6f blocked the p-VEGFR-2 of HeLa/DDP cells; (D) quantification of p-VEGFR-2 protein expression in HeLa cells by 6f in the bar graph; all results performed above are presented as mean ± SD from three independent experiments. * p < 0.05, compared to the control group.

Scheme 10

(A1,A2,B1,B2) Molecular docking of chalcone with VEGFR-2 and P-gp proteins; (C1,C2,D1,D2) molecular docking of 6f with VEGFR-2 and P-gp proteins; (E1,E2,F1,F2) molecular docking of 6k with VEGFR-2 and P-gp proteins; (G1,G2) molecular docking of sorafenib with VEGFR-2 proteins; (H1,H2) molecular docking of verapamil with P-gp proteins.