Novel benzimidazole derivatives as anti-cervical cancer agents of potential multi-targeting kinase inhibitory activity

2.1 Synthesis

The synthetic approaches of the targeted benzimidazole compounds were described in Schemes 1-3. The key starting compound 1-(6-benzoyl-2-(3,4-dimethoxyphenyl)-1H-benzo[d]imidazol-1-yl)propan-2-one (1) was prepared by the reaction of o-phenylenediamine A with 3,4-dimethoxybenzaldeyde to give the corresponding (2-(3,4-dimethoxyphenyl)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone derivative B which was alkylated with chloroacetone in dry acetone in the presence of potassium carbonate as a basic medium to give the target starting precursor 1. Compound 1 was allowed to react with different appropriate aromatic aldehydes in ethyl alcohol in the presence of a catalytic amount of piperidine as a basic medium to give the corresponding chalcone derivatives 2a-2e. In order to construct the Schiff bases 3a-3f, the starting 1 was heated with various aromatic amines in ethanol containing a few amounts of acetic acid at the refluxing temperature (Scheme 1).

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Scheme 1

Synthesis of phenylbuten-2-one-benzimidazole and substituted phenyl iminopropyl – benzimidazole derivatives (2a-e and 3a-f).

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Scheme 2

Synthesis of hydrazine-(thio)carboxamide-benzimidazole derivatives (4–8).

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Scheme 3

Synthesis of benzimidazole derivatives conjugated with 2,5-dioxopyrrolidine, 2,5-dioxopyrroline, 1,3-dioxoisoindoline, thiazoline and thiazolidine rings (9–13).

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Also, compound 1 was allowed to react with hydrazine hydrate in absolute ethanol to give the corresponding hydrazone derivative 4, while its reaction with p-toluic hydrazide and 2-furoic acid hydrazide led to the formation of the corresponding hydrazone derivatives 5 and 6 (Scheme 2). When the acetyl compound 1 was condensed with semicarbazide hydrochloride in absolute ethanol in the presence of sodium acetate afforded the corresponding semicarbazone derivative 7, while condensation of 1 with thiosemicarbazide in ethanol containing a few drops of concentrated hydrochloric acid furnished the corresponding thiosemicarbazone derivative 8.

The thiosemicarbazone analogue 8 was utilized as a useful intermediate for further preparation of different benzimidazole-heterocyclic compounds. Thus, condensation of 8 with different acid anhydrides such as succinic acid anhydride, maleic acid anhydride and phthalic acid anhydride in acetic acid led to the formation of 2,5-dioxopyrrolidine, 2,5-dioxopyrroline and 1,3-dioxoisoindoline derivatives 9, 10 and 11, respectively. Also, cyclization of the 8 with haloketones such as chloroacetone and ethyl bromoacetate accomplished the thiazoline and thiazolidinone derivatives 12 and 13, respectively (Scheme 3).

2.2 In vitro anticancer activity

The present investigation deals with construction of new derivatives bearing 2-(3,4-dimethoxyphenyl)-1-substituted-benzo[d]imidazol-6-yl)(phenyl)methanone scaffold 2–13 for anti-cervical cancer evaluation. Accordingly, the new derivatives were subjected to MTT assay against Hela cell line using Doxorubicin as a reference drug (Thavamani et al., 2013; Prasetyaningrum et al., 2018) as represented in Fig. 3. The results were tabulated in Table 1. It has been detected that the 4-(dimethylamino)phenyl)but-3-en-2-one derivative 2c, the thiazolylsulfonyl derivative 3f, the 2,5-dioxopyrrolidine derivative 9, the 4-methylthiazoline derivative 12 and thiazolidin-4-one 13 exhibited the most potent cytotoxic activity that was higher than that gained by the reference drug (IC_{50 2c}; 1.84, IC_{50 3f}; 1.91; IC_{50 9}; 1.62; IC_{50 12}; 1.71; IC_{50 13}; 1.44; IC_{50 Dox;} 2.05 µM). A slight decrease in the potency was detected by the 4-nitrophenyl-iminopropyl derivative 3c of IC₅₀ 3.04 µM. It could be noted that the increase in the number of nitrogen atoms in addition to the incorporation of thiazole rings or pyrrolidine moiety contributed to more cytotoxic potency. This result comes in agreement with many researches which investigated the unique anticancer activity of different clinically applied anticancer drugs and compounds bearing thiazole and pyrrolidine scaffolds (Sharma et al., 2020; Smolobochkin, et al., 2019). A detectable drop-in the cytotoxic activity of the rest of the compounds was observed inducing IC₅₀ values ranging from 6.20 − 28.12 µM. One of the most important drawbacks of the anticancer drugs is the deterioration of the normal cells. Accordingly, the cytotoxic activity of the potent tested compounds (2c, 3f, 9, 12 and 13) against cervical cancer cell (Hela) was also examined against the human normal fibroblast cells (WI-38) using Doxorubicin as a reference drug, and their IC₅₀ values were determined by the MTT assay. The IC₅₀ doses of all tested compounds against the normal cells were higher than their IC₅₀ values against the Hela cells (Table 1). The most potent cytotoxic candidates 9 and 13 exhibited significant cytotoxic activity against Hela cell line and noteworthy safety profile against the normal cells.

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Fig. 3

Anti-cervical cancer evaluation of compounds 2–13 against Hela cell line compared with Doxorubicin.

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Table 1 Cytotoxic activity of the synthesized benzimidazoles 2-13 on Hela cell line and normal cell line WI-38.

Cpd No.	R/Ar	Hela IC50 (µM)	WI-38 IC50 (µM)
2a	H	16.08 ± 0.74	–
2b	3,4-(OCH3)2	22.87 ± 0.61	–
2c	4-N(CH3)2	1.84 ± 0.08	12.45 ± 1.41
2d	4-NO2	12.53 ± 0.27	–
2e	4-F	6.20 ± 0.18	–
3a	-CO-CH3	7.29 ± 0.25	–
3b	-COOC2H5	6.53 ± 0.47	–
3c	-NO2	3.04 ± 0.07	–
3d	-Br	11.07 ± 0.53	–
3e	-SO2-NH2	8.51 ± 0.33	–
3f	-SO2-NH-thiazolyl	1.91 ± 0.13	13.62 ± 1.08
4	-NH2	15.96 ± 0.62	–
5	-CO-p-tolyl	28.12 ± 0.11	–
6	-CO-2-furan	11.67 ± 0.46	–
7	-CO-NH2	16.29 ± 0.67	–
8	-CS-NH2	8.99 ± 0.29	–
9	2,5-dioxopyrrolidine	1.62 ± 0.16	57.38 ± 2.66
10	2,5-dioxopyrroline	6.99 ± 0.42	–
11	1,3-dioxoisoindoline	6.23 ± 0.18	–
12	4-methylthiazoline	1.71 ± 0.09	25.11 ± 1.94
13	thiazolidin-4-one	1.44 ± 0.06	77.34 ± 3.61
Doxorubicin		2.05 ± 0.03	79.7 ± 0.07

2.3 Evaluation of EGFR/HER2 inhibitory activity

The compounds showing promising cytotoxic potency 2c, 3f, 9, 12 and 13 were further evaluated for their dual EGFR/HER2 inhibitory activity referring to Erlotinib (Table 2, Fig. 4). The 2,5-dioxopyrrolidine derivative 9 and the thiazolidin-4-one 13 represented the most promising potential EGFR inhibiting activity but slightly less than that of the reference Erlotinib (IC_{50 9}; 0.157, IC_{50 13}; 0.109 µM, IC_{50 Erlotinib}; 0.079 µM) and are more potent than that of the reference Nazartinib (IC_{50 Nazartinib}; 0.16 µM). Further decrease in the suppression effect was investigated by the compounds 2c, 3f and 12 of IC₅₀ values; 0.214, 0.342, 0.305 µM, respectively, which might be contributed to improper fitting in the EGFR active site. On the other hand, all of the examined compounds exhibited significant inhibition against HER2 enzyme. Compound 13 produced 6.5 folds more potency than Erlotinib producing IC₅₀; 0.19 µM, IC_{50 Erlotinib}; 1.23 µM. Furthermore, compound 9 represented 5 folds more inhibition potency of HER2 than the reference drug of IC₅₀; 0.25 µM. Compounds 12, 3f and 2c were more potent suppressors against HER2 than Erlotinib by 5–3 folds, respectively. Scrutinizing the resultant data, the examined benzimidazole derivatives are more selective towards HER2 than EGFR enzyme and the most potent cytotoxic candidates 9 and 13 induced dual EGFR/HER2 inhibitory activity.

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Fig. 4

EGFR/HER2 inhibitory effects of compounds 2c, 3f, 9, 12 and 13 compared with Erlotinib.

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2.4 In vitro kinase inhibition assay

Since it has been reported that the receptor tyrosine kinases (RTKs); EGFR, vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor (PDGFR) participate significantly in regulating tumor cell proliferation, differentiation, survival, angiogenesis and apoptosis, they were successfully considered as attractive targets for anticancer therapies, particularly for producing multi-target anticancer drugs of improved therapeutic effectiveness (Li et al., 2011; Knight et al., 2010). Additionally, they also work in a synergistic pattern in regulating tumor response to anticancer RTK inhibitor drugs. Hence, multi-target RTK drugs that simultaneously inhibit EGFR, VEGFR-2 and PDGFR-β give substantially optimized anticancer therapeutic effect than drugs that inhibit individual RTKs (Wang et al., 2019). Accordingly, the promising cytotoxic compounds 2c, 3f, 9, 12 and 13 were subjected to kinase assay against PDGFR-β and VEGFR-2. As shown in Table 2, both compounds 9 and 13 exhibited promising inhibitory activity against both kinases (Fig. 5). Interestingly, the thiazoline compound 13 appeared about 3.8 times more potent than the reference drug Erlotinib against PDGFR-β (IC₅₀; 21.61 µM, IC_{50 Erlotinib}; 83.11 µM) and about 1.8 times more potent than Erlotinib against VEGFR-2 (IC₅₀; 69.62 µM, IC_{50 Erlotinib}; 124.7 µM). Furthermore, the 2,5-dioxopyrrolidine derivative 9 showed good inhibitory activities against PDGFR-β (IC₅₀; 76.09 µM, IC_{50 Erlotinib}; 83.11 µM) and against VEGFR-2 (IC₅₀; 123.27 µM, IC_{50 Erlotinib}; 124.7 µM). Further decrease in the suppression effects were investigated by the compounds 2c, 3f and 12 of IC₅₀ values against PDGFR-β; 95.31, 89.11, 85.11 µM, respectively, and IC₅₀ values against VEGFR-2; 143.44, 138.15, 133.54 µM, respectively.

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Fig. 5

PDGFR-β and VEGFR-2 inhibitory effects of compounds 2c, 3f, 9, 12 and 13 compared with Erlotinib.

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It could be noted that the potent anti-cancer activity of compound 13 against Hela cells comes as a companion to its significant multi-kinase suppression activity against the receptor tyrosine kinases (RTKs); EGFR, HER-2, PDGFR-β and VEGFR-2.

2.5 Cell cycle analysis and detection of apoptosis

The most promising compounds 9 and 13 were selected for further investigation to demonstrate their effects on the progression of cell cycle and apoptosis induction in Hela cell line. Exposure of Hela cells to both compounds 9 and 13 at their IC₅₀ concentrations 1.62 and 1.44 µM for 24 h alongside induction of apoptosis were examined. Cells Exposure to both compounds led an interference with the normal cell cycle distribution. Both compounds 9 and 13 produced a pronounced increment in the cell percentages at pre-G1 phase by 13 and 27 folds, respectively and at G2/M phase by 7 and 10 folds, respectively compared to the untreated Hela cells (Table 3 and Fig. 6). Cumulation of cells in pre-G1 phase might be contributed to degradation of the genetic materials indicating a probable role the tested derivatives in inducing apoptosis in the cancer cells. Furthermore, cumulation of the cells at G2/M phase indicted cell cycle disruption at this stage preventing its mitotic stage.

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Fig. 6

Cell Cycle analysis of compounds 9 and 13.

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2.6 Apoptosis detection by annexin V-FITC assay

Apoptosis occurs due to the autonomous and organized destruction of genes-controlled cells (He et al., 2009). In addition, multiple external factors influence its startup. Thus, it was of interest to investigate the relationship between apoptosis of Hela cells and the compounds 9 and 13. The induction of apoptosis of the latter compounds was examined utilizing annexin V-FITC and propidium iodide (PI) double staining flow cytometry analysis (Cao et al., 2020; Yang et al., 2020). Based on the obtained results presented in Table 4 and Fig. 7, both derivatives effectively induced cell apoptosis at their IC₅₀ concentrations of 1.62, 1.44 µM. Treatment of Hela cells with compounds 9 and 13 for 24 h led to 8.47% and 18.99% of apoptotic cells (late apoptosis), respectively, compared to 0.18% of apoptotic cells in the untreated control. Also, there was an increase in the early apoptosis from 0.47% (control) to 2.81% and 5.44% cause by 9 and 13, respectively with necrosis percent of 1.89 and 2.61% vs. 1.23% produced by DMSO control. It has been noted that the late apoptotic percentages caused by both examined derivatives were higher than that of the early phase which makes it more challenging to recover the apoptotic cells to safe ones. These results revealed that compounds 9 and 13 inhibited cell growth through cell apoptosis induction.

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Fig. 7

Apoptotic activity of benzimidazole compounds 9 and 13.

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2.7 Molecular docking study

In order to better rationalize how the compounds 2c, 3f, 9, 12 and 13 contributed to the HER2 kinase inhibitory activities, molecular docking simulation studies were carried. The HER2 kinase domain complexed with a ligand was downloaded from the protein databank (PDB ID: 3RCD, http://www.rcsb.org). Protein preparation wizard of Schrödinger Suite (Sastry et al., 2013, Schrödinger, 2018) was used to correct the protein structural file by correcting bond orders, adding any missing hydrogen atoms, and completing unresolved side chains or loops.

The ligands showed good fitting in the active site of HER2 with Glide docking scores in proportion to their biological activities. In general, the docking poses of the ligands demonstrated several good interactions with the DFG region, catalytic loop, activation loop, G-rich loop, phosphate binding loop and α-C helix of HER2 as shown in Table 5. Compound 2c was docked well in the ligand binding pocket with a Glide docking score of −8.03 kcal/mol. It shows strong hydrogen bonding and cation-π contacts with Lys 753. It has hydrophobic and electrostatic interactions with the surrounding amino acids in the binding site. Compound 3f showed a docking score of −9.1 kcal/mol (Fig. 8), and a strong hydrogen bonding with Gly 881 in the activation loop. The sulphonyl substituent shifted the structure to some extent inside the binding site. Other hydrophobic and polar contacted are observed with the kinase structural motifs. Compounds 9 (Fig. 9) and 12 (Fig. 10) have docking score of −9.4 and −9.3 kcal/mol, respectively. Both compounds demonstrated strong π-π contacts with Phe 864 in the DFG regions, and multiple polar/nonpolar contacts with the surrounding residues. Compound 13 exhibited strong fitting in the binding site with a Glide score of −9.7 kcal/mol. The compound is strongly interacting with Asn 850 of the catalytic loop, and Asp 863 of the DFG region via hydrogen bonding. It shows π-π contacts with Phe 864 of the DFG region (Fig. 11). It could be noted that the most potent HER2 inhibitors 9 and 13 showed the highest negative energy score of −9.4, −9.7 kcal/mol, respectively, with higher predicted binding affinity than the co-crystalized ligand Erlotinib which showed a docking score of −5.8 kcal/mol but lower binding affinity than Lapatinib (docking score = -12 kcal/mol) (Fig. 12).

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Fig. 8

2D diagram (A) and 3D representation (B) of compound 3f showing its interaction with the HER2 kinase active site.

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Fig. 9

2D diagram (A) and 3D representation (B) of compound 9 showing its interaction with the HER2 kinase active site.

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Fig. 10

2D diagram (A) and 3D representation (B) of compound 12 showing its interaction with the HER2 kinase active site.

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Fig. 11

2D diagram (A) and 3D representation (B) of compound 13 showing its interaction with the HER2 kinase active site.

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Fig. 12

2D diagram (A) and 3D representation (B) of Lapatinib showing its interaction with the HER2 kinase active site.

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4.1 Synthesis

The instruments utilized in measuring the melting points, spectral data (IR, Mass, ¹H NMR and ¹³C NMR) and elemental analysis are cited in details in the Supplementary material.

4.2 Biological evaluation