Design, synthesis, and analysis of antiproliferative and apoptosis-inducing activities of nitrile derivatives containing a benzofuran scaffold: EGFR inhibition assay and molecular modelling study

Abstract New cyanobenzofurans derivatives 2–12 were synthesised, and their antiproliferative activity was examined compared to doxorubicin and Afatinib (IC50 = 4.17–8.87 and 5.5–11.2 µM, respectively). Compounds 2 and 8 exhibited broad-spectrum activity against HePG2 (IC50 = 16.08–23.67 µM), HCT-116 (IC50 = 8.81–13.85 µM), and MCF-7 (IC50 = 8.36–17.28 µM) cell lines. Compounds 2, 3, 8, 10, and 11 were tested as EGFR-TK inhibitors to demonstrate their possible anti-tumour mechanism compared to gefitinib (IC50 = 0.90 µM). Compounds 2, 3, 10, and 11 displayed significant EGFR TK inhibitory activity with IC50 of 0.81–1.12 µM. Compounds 3 and 11 induced apoptosis at the Pre-G phase and cell cycle arrest at the G2/M phase. They also increased the level of caspase-3 by 5.7- and 7.3-fold, respectively. The molecular docking analysis of compounds 2, 3, 10, and 11 indicated that they could bind to the active site of EGFR TK.

Considering the above results, in this work, a series of benzofuran scaffolds 2-12 ( Figure 2) was synthesised based on bioisosteric modifications of compounds shown in Figures 1 and 2. In the prepared derivatives, the benzofuran core was linked with alkylnitrile or nicotinonitrile moieties. The anti-cancer activity of the designed compounds was analysed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The most active compounds were also evaluated against the target EGFR TK. Moreover, the induction of apoptosis and the effects of the most active derivatives on the caspase-3 level were assessed using a flow cytometry technique. The cell cycle activity was also detected for the most potent compounds to determine the possible cell cycle stage at which the new derivatives could suppress the growth of cancer cells. Lastly, molecular modelling was conducted to explore the plausible binding modes of the most promising derivatives in the binding site of EGFR.

Chemistry
The synthetic pathway adopted to prepare the novel series of benzofuran-incorporating nitrile derivatives is shown in Scheme 1. Knoevenagel condensation of 2-acetyl benzofuran (1) in an ethanolic solution of malononitrile afforded 2-(1-(benzofuran-2yl)ethylidene)malononitrile (2), which reacted with N,N-dimethylformamide dimethylacetal (DMFDMA) to give (E)-2-(1-(benzofuran-   (3). The subsequent condensation reaction with different primary amines yielded 4-(benzofuran-2-yl)-2-(substituted)-nicotinonitriles 4-12. The structure of compound 2 was confirmed by infra-red (IR) spectroscopy, which showed absorption bands at 2222 cm À1 (CN) and 1572 cm À1 (C¼C). Additionally, the disappearance of a band at 1680 cm À1 was detected (C¼O). The 13 C nuclear magnetic resonance (NMR) spectrum revealed the disappearance of carbon signals at 186 ppm (C¼O) as well as the appearance of two peaks at 113.24 and 113.60 ppm, which were attributed to two nitrile groups. Moreover, the presence of the methyl group of the ethylidene moiety (CH 3 -C¼C) was confirmed by the singlet peaks at 2.63 and 19.38 ppm in the 1 H NMR and 13 C NMR spectra, respectively. The structure of compound 2 was verified by elemental analysis and mass spectrometry, which showed a molecular ion peak (M þ ) at m/z 208. The NMR spectrum of compound 3 was characterised by the absence of the methyl signal of the ethylidene moiety (CH 3 -C¼C) at 2.63 and 19.38 ppm. In addition, two singlet peaks corresponding to the dimethylamino group (N(CH 3 ) 2 ) were observed at 3.09 and 3.23 ppm in the 1 H NMR spectrum as well as at 37.86 and 45.77 ppm in the 13 C NMR spectrum. The structures of compounds 4-12 were confirmed by IR, 1 H NMR, 13 C NMR, and mass spectrometry data. The IR spectra showed absorption signals at 3343-3370 and 2209-2219 cm À1 due to the presence of (NH) and (CN) groups, respectively. Moreover, the peaks corresponding to the dimethylamino group (N(CH 3 ) 2 ) at 3.09 and 3.23 ppm in the 1 H NMR spectra as well as at 37.86 and 45.77 ppm in the 13 C NMR spectra of compounds 4-12 disappeared. However, the signals ascribed to the (NH) group were detected at 6.66-7.18 ppm. The presence of the aliphatic residue was confirmed by the peaks at 0.90-4.56 and 13.49-59.28 ppm in the 1 H NMR and 13 C NMR spectra, respectively.

Biological screening
2.2.1. In vitro antiproliferative activity and structure activity relationship (SAR) The antiproliferative activity of the newly synthesised compounds 2-12 against five human cancer cell lines, including hepatocellular carcinoma (HePG2), colorectal carcinoma (HCT-116), human breast adenocarcinoma (MCF-7), human prostate carcinoma (PC3), cervical carcinoma (HeLa), and normal cell (WI38) was evaluated by an MTT assay employing a previously described procedure 47,48 . Doxorubicin (DOX) and Afatinib were used as positive control. The antiproliferative activity of the tested compounds is summarised in Table 1 and  , respectively. Introduction of a 4-methoxyl moiety in compound 11 afforded derivative 12, which displayed weak antiproliferative activity. The cytotoxic activity of the new compounds was also examined against normal W138 fibroblast cell to study the safety of the newly synthesised compounds, using (MTT) colorimetric assay ( Table 1). The tested compounds did not display cytotoxicity towards W138 cells (IC 50 values of 59.49-204.00 mM) compared to doxorubicin (IC 50 values of 55.29 mM).

EGFR TK inhibition assay
The most active derivatives, that is, 2, 3, 8, 10, and 11, were subjected to the EGFR TK inhibition assay 7,10,23-25 . The results revealed that several of the tested compounds were promising EGFR TK inhibitors (Table 2). It was evident that compounds 2, 3, 10, and 11 exhibited strong inhibitory activities against EGFR (IC 50 values of 1.09, 0.93, 1.12, and 0.81, respectively). Notably, this activity was comparable to that of the reference drug gefitinib (IC 50 value of 0.90 mM). It was observed that all of the tested derivatives showed 50% inhibition against EGFR of less than 1.2 mM, except compound 8, which was found to be the least effective EGFR inhibitor (IC 50 ¼ 4.24 mM). It was determined that compounds incorporating a phenethyl moiety, such as derivative 11, displayed higher inhibitory activity against EGFR than the corresponding compounds containing a benzyl fragment (e.g. 10) or a propanol group (e.g. 8).

Caspase-3 assay and induction of apoptosis
Caspases are critical mediators of programmed cell death, that is, apoptosis 49 . Caspase-3 is important in processes involving dissociation of the cell and the formation of the apoptotic element; therefore, it is regarded as one of the best biochemical hallmarks of apoptosis 49 . This, to examine the apoptotic activity of compounds 3 and 11, the level of caspase-3 was measured after treating the HCT-116 and MCF-7 cells with 3 and 11, respectively ( Table 3). The concentration of active caspase-3 was measured using the ELISA technique 49 . In addition, the fluorescence density produced by the tested compounds is illustrated in Figure 4. Interestingly, compound 3 significantly induced apoptosis in HCT-116 cells after 24 h of treatment. The level of caspase-3 increased 5.7-fold compared to the control. Moreover, a considerable 7.3fold increase in the caspase-3 level was detected following the treatment of the MCF-7 cells with compound 11. The bioluminescent intensities of caspase-3 indicated the apoptotic activity of compounds 3 and 11.

Cell cycle arrest analysis and detection of apoptosis
The cell cycle is a sequence of growth and development steps that lead to DNA replication and cell division. It consists of four distinct phases: the G1 phase, S phase (synthesis), G2 phase, and M phase 23,24,[50][51][52] . Apoptosis, that is, programmed cell death, is considered an important target of the most anti-tumour agents, resulting in G2/M arrest 23,24,50-52 . Our promising derivatives 3 and 11 were subjected to cell cycle analysis and an apoptotic assay to investigate their roles in the cell cycle progression of HCT-116 and MCF-7 cells, respectively. To better characterise the mode of cell death induced by the tested compounds, following treatment of the HCT-116 and MCF-7 cells with compounds 3 and 11 at a concentration of 10 mM for 24 h, respectively, the cells were stained with propidium iodide (PI). The DNA contents were measured by flow cytometry (Tables 4 and 5; Figures 5-8). Compared with the control, which was treated with DMSO, following treatment of HCT-116 and MCF-7 cells with compounds 3 and 11, the cell proportion at the S phase decreased to 20.91% and 21.36%, respectively. In addition, compounds 3 and 11 increased the cell proportion at the G2/M phase to 12.62% and 15.28%, respectively, compared to the control cells (4.19% and 3.66%, correspondingly). These results indicated that the cells were arrested at the G2/M phase. Furthermore, the pre-G1 population was detected following treatment with compounds 3 and 11 (13.06% and 16.25% compared to 0.39% and 0.55% in the control cells, respectively). Moreover, annexin-5/PI staining 23,24,52 was performed for   Table 5, and Figure 8 suggest an increase in the early apoptosis from 0.16% (control sample in DMSO) to 5.95% for compound 3. In contrast, derivative 11 showed an increase in the early apoptosis to 6.89%. Compounds 3 and 11 increased the late apoptosis from 0.11% (DMSO) to 5.79% and 8.13%, respectively. It was also evident that 3 and 11 preferentially activated the apoptotic pathway rather than the necrotic pathway. This induced action was the result of the cell cycle arrest at the G2/M phase.

Molecular modelling study
Molecular modelling is a tool used to inspect bioactive molecules within a putative binding site of a particular enzyme or receptor [53][54][55][56][57] . It can also be employed for studying the molecular structure and structural activity relationship of different molecules 6,58,59 . In this study, the MOE 2008.10 software obtained from the Chemical Computing Group Inc. (Montreal, QC, Canada) was used for the docking protocol. The docked compounds and the co-bound inhibitor were docked into the putative binding site of the protein to generate an appropriate binding orientation. Molecular docking of the most active compounds 2, 3, 10, and 11 was conducted to explore their binding modes and interactions with the constitutive amino acids in the active site of EGFR. Molecular operating environment (MOE) software version 2008.10 was used for the analysis (Figure 9). The crystal structure of the EGFR TK receptor in complex with erlotinib was obtained from the RCSB protein data bank (PDB ID: 1M17) and was utilised to establish the starting docking model of EGFR TK 60 . The quinazoline core of the erlotinib inhibitor exhibited a hydrogen bond with Met769 60 . Erlotinib was subjected to one run of docking into the binding site to verify and validate the docking process. Docking of 2, 3, 10, and 11 revealed that all compounds fit into the enzyme active site almost at the same position as erlotinib (Figure 9). One of the nitrile groups of compound 2 (S ¼ À8.53 kcal/mol) is bound to the active site of EGFR TK through hydrogen bonding with the vital amino acid Met769. The second nitrile moiety interacted with the amino acid residues Thr766 and Cys751 through water-mediated hydrogen bonding.
Moreover, the benzofuran core is connected to Gly772 by cation-H interactions. Derivative 3 (S ¼ À9.38 kcal/mol) interacted with residues Met769 and Gly772 via hydrogen bonding using its two nitrile moieties. The benzofuran ring of 3 bound to Thr766 by water-mediated arene-H interactions. The nitrile moiety in compound 10 (S ¼ À9.61 kcal/mol) connected to Met769 by hydrogen bonding, while the benzofuran ring interacted with amino acid residues Thr766 and Cys751 via water-mediated cation-p bonding. In addition, the benzyl fragment is bound to residue Leu694 by cation-p interactions. The best docking score was achieved for compound 11 (À10.38 kcal/mol). 11 bound to the active site of EGFR TK through two hydrogen bonds to the critical amino acid Met769 using the N atom of the nitrile moiety and the N atom of phenethylamine. Lastly, the benzofuran core bound to Thr766 by water-mediated arene-H interactions (Figure 9).

Conclusion
To develop potent anti-tumour agents, a series of cyanobenzofuran hybrids were designed and synthesised in this work. The in vitro antiproliferative activity of the prepared compounds was evaluated for HePG2, HCT-116, MCF-7, PC3, and HeLa cancer cell  lines. The biological results revealed that compounds 2, 3, 10, and 11 exhibited a broad spectrum antiproliferative activity against selected cell lines. Moreover, the most active derivatives were further evaluated for their inhibitory activity against EGFR kinase. Compounds 3 and 11 displayed significant EGFR TK inhibitory activity (IC 50 0.93 and 0.81 mM, respectively) even compared to the reference drug gefitinib (IC 50 0.9 mM). Compounds 2 and 10 also showed good EGFR TK inhibitory activity (IC 50 1.09 and 1.12 mM, respectively). The apoptosis assay and cell cycle analysis results demonstrated that derivatives 3 and 11 induced apoptosis of cancer cells and arrested the cell cycle at the G2/M phase. In addition, 3 and 11 led to an increase in caspase-3 by 5.7-and 7.3-fold, respectively. These outcomes indicated that the potent pro-apoptotic activity of compounds 3 and 11 was a result of the induction of the intrinsic apoptotic pathway rather than the necrotic pathway. Compared to erlotinib, the molecular docking analysis of the most active compounds 2, 3, 10, and 11 showed good fitting and suitable interactions with the key amino residues in the binding site of the EGFR kinase. The presence of the cyano group in the compounds enabled hydrogen bonding interactions with the Met769 amino acid. Additionally, the benzofuran moiety exhibited van der Waals interactions with the EGFR binding site. Based on these findings, it can be concluded that derivatives 3 and 11 are promising scaffolds for further modification and optimisation to obtain potent and selective anti-tumour agents with EGFR inhibitory activity.

Chemistry
All melting points ( C) were recorded using a Fisher-John melting point apparatus and were uncorrected. The IR spectra were determined for KBr discs on a Thermo Fischer Scientific Nicolet IS10 spectrometer (wavenumber in cm À1 ) at the Faculty of Pharmacy, Mansoura University, Egypt. The 1 H NMR spectra were obtained in DMSO-d 6

Antiproliferative screening
The in vitro antiproliferative activity of all synthesised compounds was evaluated by an MTT assay according to the reported method 47,48 .

Epidermal growth factor inhibition assay
EGFR enzyme assay was conducted as described in our previous reports [23][24][25] .

Caspase-3 assay
Sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine the level of active human caspase-3 as previously reported and according to the manufacturer's instructions 49 .

Docking study
The molecular modelling calculations and docking studies were performed using the MOE 61 software version 2008.10 (Chemical Computing Group Inc., Montreal, Quebec, Canada). The X-ray crystallographic structure of EGFR with erlotinib was obtained from the RCSB protein data bank (PDB ID: 1m17).