Identification of new benzimidazole-triazole hybrids as anticancer agents: multi-target recognition, in vitro and in silico studies

Figures & data

Figure 1. Reported 2-substituted benzimidazoles as multi-target enzyme inhibitors.

Figure 2. Reported 1,2,3-triazole scaffolds as multi-target enzyme inhibitors.

Figure 3. Rational design of the newly synthesised benzimidazole-triazole hybrids.

Scheme 1. Reagents and conditions: (a) DMSO, stirring, rt, 15 h, (b) EAA, K₂CO₃, DMSO, stirring, rt, overnight, (c) NH₂NH₂.H₂O, EtOH, stirring, rt, overnight, (d) THF, stirring, rt, (e) EtOH, AcOH, reflux

Table 1. IC₅₀ values (µM) of target compounds 5a-h and 6a-g against four cancer cell lines in comparison with Dox.

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Comp. no.	R / Z	X	In vitro Cytotoxicity IC50 (µM)^a
			HepG-2	HCT-116	MCF-7	HeLa
5a	Cl / –	S	7.68 ± 0.5	8.34 ± 0.6	6.81 ± 0.4	3.87 ± 0.2
5b	Cl / –	O	53.60 ± 2.9	37.69 ± 2.6	21.61 ± 1.9	46.20 ± 2.7
5c	H / Phenyl	O	67.42 ± 3.5	59.23 ± 3.3	45.35 ± 2.9	27.63 ± 2.0
5d	H / –	S	57.23 ± 3.2	41.38 ± 2.8	30.78 ± 2.3	12.39 ± 0.9
5e	NO2 / –	O	25.03 ± 1.9	32.75 ± 2.4	16.57 ± 1.4	19.14 ± 1.4
5f	CH3 / –	S	72.65 ± 3.8	68.45 ± 3.7	64.12 ± 3.3	58.15 ± 3.1
5g	OCH3 / –	S	13.59 ± 1.0	18.67 ± 1.5	9.39 ± 0.8	8.70 ± 0.6
5h	H / –	O	42.14 ± 2.7	54.81 ± 3.0	38.02 ± 2.6	31.56 ± 2.3
6a	4-Cl	–	47.04 ± 2.8	28.18 ± 2.5	39.52 ± 2.6	35.33 ± 2.5
6b	H	–	84.46 ± 4.1	75.09 ± 3.8	57.56 ± 3.3	61.23 ± 3.4
6c	3-Br	–	56.28 ± 3.2	63.60 ± 3.3	44.81 ± 2.9	48.79 ± 2.8
6d	3-NO2	–	71.50 ± 3.6	36.45 ± 2.7	48.25 ± 3.0	52.16 ± 3.1
6e	3,4-(OCH3)2	–	33.62 ± 2.3	19.69 ± 1.7	23.73 ± 1.9	29.07 ± 2.3
6f	4-N(CH3)2	–	18.31 ± 1.5	11.72 ± 0.9	14.69 ± 1.1	22.75 ± 1.9
6g	4-OH, 3-OCH3	–	10.92 ± 0.8	3.34 ± 0.4	7.11 ± 0.5	9.84 ± 0.7
Dox^b	–	–	4.50 ± 0.2	5.23 ± 0.3	4.17 ± 0.2	5.57 ± 0.4

^aIC₅₀ value is the concentration of compound that inhibits 50% of the cancer cell growth after 48 h of drug exposure as obtained from MTT assay. Each value was shown as mean ± SD of three independent experiments. IC₅₀, (µM): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), and above 100 (non-cytotoxic).

^bDox: Doxorubicin, used as reference drug. Bolded values represent the most potent anticancer derivatives.

Table 2. In vitro cytotoxic study of the most active compounds 5a and 6g against WI-38 cell line and their selectivity indexes.

Comp.	Normal cells IC50 (µM) WI-38	SI^a
		HepG-2	HCT-116	MCF-7	HeLa
5a	37.16 ± 2.4	4.73	4.46	5.46	9.60
6g	43.28 ± 2.6	4.2	12.95	6.09	4.39
Dox	6.72 ± 0.5	1.49	1.28	1.61	1.2

^aSI: Selectivity Index = IC₅₀ for normal cells/IC₅₀ for cancer cells.

Table 3. In vitro EGFR-2, VEGFR-2, and Topo II inhibitory effects of the synthesised compounds 5a and 6g compared to reference drugs.

Compound	EGFR IC50 (mean ± SD μM)	VEGFR-2 IC50 (mean ± SD μM)	Topo II IC50 (mean ± SD μM)
5a	0.086 ± 0.009	0.107 ± 0.005	2.52 ± 0.15
6g	0.131 ± 0.005	0.229 ± 0.01	8.37 ± 0.49
Gefitinib	0.052 ± 0.003	–	–
Sorafenib	–	0.048 ± 0.002	–
Dox	–	–	3.62 ± 0.21

Table 4. DNA binding assay results (IC₅₀, µM) of compounds 5a and 6g.

DNA-active compounds	DNA/methyl green (IC50 µM)
5a	33.17 ± 1.7
6g	42.03 ± 2.1
Dox	31.54 ± 1.5

IC₅₀ values represent the concentration (mean ± SD, n = 3–5 separate determinations) required for a 50% decrease in the initial absorbance of the DNA/methyl green solution.

Figure 4. Flow cytometry analysis of DNA ploidy in HepG-2 cells after treatment with compounds 5a and 6g.

Table 5. Effect of compounds 5a and 6g on the cell cycle distribution in HepG-2 cells.

Comp. no.	Cell cycle distribution (%)
	G0-G1	S	G2/M	Pre-G1
5a/HepG-2	36.93	49.06	14.01	44.49
6g/HepG-2	38.91	27.28	33.81	36.28
Control	44.82	36.59	18.59	2.17

Figure 5. Effect of compounds 5a and 6g on the percentage of Annexin V-FITC-positive staining in HepG-2 cells. The cells were treated with DMSO as a control, 5a and 6g for 24 h. Q1: Necrotic cells, Q2: Late apoptosis, Q3: Live cells, Q4: early apoptosis.

Figure 6. Early, late, total apoptosis, and necrosis induced by compounds 5a and 6g in HepG-2 cells compared to control.

Table 6. Apoptosis and necrosis induction analysis in HepG-2 cells after treatment with compounds 5a and 6g.

	Apoptosis			Necrosis
Compound	Total	Early	Late
5a/HepG-2	37.37	12.24	25.13	7.12
6g/HepG-2	30.87	11.02	19.85	5.41
Control	0.39	0.37	0.12	1.68

Table 7. The binding energy of compound 5a and 6g in comparison to the co-crystallised ligand for each prospective target.

No.	Compound	Topo II	VEGFR-2	EGFR
1	5a	−36.4	−34.1	−28.6
2	6g	−33	−27.8	−22.4
3	Topo II co-crystallised ligand	−45	–	–
4	VEGFR-2 co-crystallised ligand	–	−43.9	–
5	EGFR co-crystallised ligand	–	–	−27.8

Figure 7. (a) 2D interaction of compound 5a with Topo II active site (PDB code: 1ZXM). (b) Aligned conformation of compound 5a (Green) with co-crystallised ligand (Salmon) inside Topo II.

Figure 8. (a) 2D interaction of compound 6g with Topo II active site (PDB code: 1ZXM). (b) Aligned conformation of compound 6g (Red) with co-crystallised ligand (Salmon) inside Topo II.

Figure 9. (a) 2D interaction of compound 5a with VEGFR-2 active site (PDB code: 2OH4). (b) Aligned conformation of compound 5a (Green) with co-crystallised ligand (Cyan) inside VEGFR-2.

Figure 10. (a) 2D interaction of compound 6g with VEGFR-2 active site (PDB code: 2OH4). (b) Aligned conformation of compound 6g (Red) with co-crystallised ligand (Cyan) inside VEGFR-2.

Figure 11. (a) 2D interaction of compound 5a with EGFR active site (PDB code: 2J6M). (b) Aligned conformation of compound 5a (Green) with co-crystallised ligand (Magenta) inside EGFR.

Figure 12. (a) 2D interaction of compound 6g with EGFR active site (PDB code: 2J6M). (b) Aligned conformation of compound 6g (Red) with co-crystallised ligand (Magenta) inside EGFR active site.

Table 8. Calculated Lipinski’s rule of five for compound 5a, 6g, Dox, Sorafenib, and Gefitinib.

Compound	Log P	TPSA	MW	nHBA	nHBD	nRB	nVs
5a	2.7959	181.354	440.920	6	4	4	0
6g	1.9892	170.761	405.418	8	3	6	0
Dox	0.0013	222.081	543.525	12	6	5	3
Sorafenib	5.5497	185.111	464.831	4	3	5	1
Gefitinib	4.2756	184.642	446.910	7	1	8	0

Log P: octanol-water partition coefficient; TPSA: topological surface area; MW: molecular weight; nHBA: number of hydrogen bond acceptors; nHBD: number of hydrogen bond donors; nRB: number of rotatable bonds; nVs: number of violations of Lipinski’s rule.

Figure 13. Bioavailability radar plot for compounds 5a, 6g, Dox, Sorafenib, and Gefitinib.