Synthesis and biological evaluation of 2-styrylquinolines as antitumour agents and EGFR kinase inhibitors: molecular docking study

Abstract A new series of 4,6-disubstituted 2-(4-(dimethylamino)styryl)quinoline 4a,b–9a,b was synthesized by the reaction of 2-(4-(dimethylamino)styryl)-6-substituted quinoline-4-carboxylic acids 3a,b with thiosemicarbazide, p-hydroxybenzaldehyde, ethylcyanoacetate, and 2,4-pentandione. In addition, the antitumour activity of all synthesized compounds 3a,b–9a,b was studied via MTT assay against two cancer cell lines (HepG2 and HCT116). Furthermore, epidermal growth factor receptor (EGFR) inhibition, using the most potent antitumour compounds, 3a, 3b, 4a, 4b, and 8a, was evaluated. The interpretation of the results showed clearly that the derivatives 3a, 4a, and 4b exhibited the highest antitumour activities against the tested cell lines HepG2 and HCT116 with IC50 range of 7.7–14.2 µg/ml, in comparison with the reference drugs 5-fluorouracil (IC50 = 7.9 and 5.3 µg/ml, respectively) and afatinib (IC50 = 5.4 and 11.4 µg/ml, respectively). In vitro EGFR screening showed that compounds 3a, 3b, 4a, 4b, and 8a exhibited moderate inhibition towards EGFR with IC50 values at micromolar levels (IC50 range of 16.01–1.11 µM) compared with the reference drugs sorafenib (IC50 = 1.14 µM) and erlotinib (IC50 = 0.1 µM). Molecular docking was performed to study the mode of interaction of compounds 3a and 4b with EGFR kinase.

The above-mentioned results encouraged us in designing and synthesizing a series of SQs, which are anticipated to be as potent as structurally related quinazoline bioisosteres. The antitumour activity of the target compounds was evaluated using two tumour cell lines, namely, human hepato-cellular carcinoma cell line (HepG2) and human colorectal carcinoma cell line (HCT116) [45][46][47][48] . In addition, some compounds were evaluated for their inhibitory activity against the EGFR tyrosine kinase enzyme. Moreover, a molecular docking method was used to study the putative binding mode of the target molecules into the receptor pocket of EGFR kinase 1,8-9,12,30 .

Chemistry
Melting points ( C) were recorded using Stuart melting point apparatus and were uncorrected. IR spectra were recorded on a Mattson 5000 FT-IR spectrometer (in cm À1 ) (Mattson Instruments, Cambridge, UK) using KBr disk at the Faculty of Pharmacy, Mansoura University. 1 H-NMR and 13 C NMR spectra were recorded on a Bruker Avance spectrometer (400 MHz) in DMSO-d 6 at Georgia State University, Atlanta, GA. The chemical shifts in ppm are expressed in d units, using tetramethylsilane as an internal standard, and coupling constants in Hz. Mass spectrum analyses were performed on Thermo Fisher Scientific (Waltham, MA, USA) SID GC GC/MS, DSQ II in the Faculty of Science, Mansoura University. Reaction times were determined using a TLC technique on silica gel plates 60 F 245 E. Merck, and the spots were visualised by U.V. (366, 245 nm). Biological screening was conducted at the Pharmacognosy Department, Faculty of Pharmacy, Mansoura University. Compound 3a was prepared according to its previous report 49 .

General method for synthesis of compounds 6a,b and 7a,b
A mixture of the acid chloride 5 (10 mmol), 4-hydroxybenzaldehyde (1.22 g, 10 mmol) or phenyl hydrazine (1.08 g, 10 mmol) and potassium carbonate (1.38 g, 10 mmol) in dimethylformamide (25 ml) was heated at 90 C for 24 h. After cooling, the reaction mixture was poured into ice water. The obtained solid was filtered, washed with water, dried, and crystallised from ethanol to give pure products.

Antitumour activity using MTT assay
The designed compounds were evaluated for their in vitro antitumour effect using the standard MTT method against two human tumour cell lines, namely, HepG2 and HCT116 [45][46][47][48] . The quantitative evaluation of the cytotoxicity was performed using tetrazolium salt MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide) assay. The cytotoxic activity was expressed as the concentration of the compound that caused 50% growth inhibition (IC 50, mean ± SEM) compared with the growth of untreated cells.

EGFR kinase inhibition assay
EGFR kinase activity was determined via enzyme-linked immunosorbent assay (ELISA) in 96-well plates 50 . The EGFR kinase activity for each compound were expressed as IC 50 values. Data were represented as mean ± SD from three independent experiments, and differences between groups were considered statistically significant at p < .05.

Docking methodology
All modelling experiments were conducted with MOE programs running on PC computer (MOE 2008.10, Chemical Computing Group, Inc., Montreal, QC, Canada) [51][52][53][54] . Starting coordinates of the X-ray crystal structure of the EGFR enzyme in complex with erlotinib (PDB code 1M17) were obtained from the RCSB Protein Data Bank 29 .

Antitumour activity
The antitumour activity of the designed compounds 3a,b-9a,b and both reference drugs 5-fluorouracil (5-FU) and afatinib against HepG2 and HCT116 cell lines is shown in Table 1 and Figure  2

EGFR inhibitory activity
The mechanism of antitumour activity of the target compounds was studied using ELISA-based EGFR-TK assay 50 . Five compounds with the highest antitumour activities were evaluated against EGFR kinase activity assays with sorafenib and erlotinib as the reference drugs. IC 50 values of the tested compounds were calculated and are listed in Table 2, where compounds 3a (IC 50 ¼ 2.23 mM) and 4b (IC 50 ¼ 1.11 mM) showed the highest inhibitory activity against EGFR, compared to the other tested compounds. The activities of compounds 3b (IC 50 ¼ 8.01 mM), 4a (IC 50 ¼ 8.78 mM), and 8a (IC 50 ¼ 16.01 mM) against EGFR were found to be weakly active comparable to those of sorafenib (IC 50 ¼ 1.14 mM) and erlotinib (IC 50 ¼ 0.1 mM). Based on these results, we can conclude that EGFR-TK inhibitory activity of the target compounds is correlated to their antitumour activities against HepG2 and HCT116.

Molecular docking study
The inhibitory activities of compounds 3a and 4b on EGFR kinase prompted us to carry out molecular docking into the putative binding site of EGFR kinase. Both compounds 3a and 4b were flexibly docked into the active site of EGFR kinase along with the reference inhibitor erlotinib (PDB code: 1M17) 29 . All docking calculations were performed using MOE 2008.10 software 7b, 51-54 . The interaction energies of compounds 3a and 4b and erlotinib, docked into the active site of EGFR, were -18.54, -20.89, and  -29.01 kcal/mol, respectively (Table 2 and Figure 3). The molecular docking results of the most active compound 4b demonstrated a hydrophobic interaction of the quinoline ring with surrounding amino acids, such as Val 702 , Leu 694 , and Leu 820 . The substituent group at C-4 of the quinoline ring is the main moiety affecting the binding mode of compound 4b in both activation and catalytic loops, where a 2-aminothiadiazole ring uniquely formed trifurcated hydrogen bonds with the distinctive residue Met 769 , Gln 767 , and Thr 766 . Moreover, the 2-styryl fragment of compound 4b was firmly extended to the backbone, similar to the 6,7-dialkoxy moiety of erlotinib, augmenting the recognition and the overall inhibitory activity (Figure 3, lower left panel).
Compound 3a binds in a similar manner to compound 4b, where hydrophobic interaction is clearly observed among amino acid residues Val 702 , Leu 694 , and Leu 820 and quinoline core. It was found that the carboxylic group at position-4 of the quinoline core was clearly recognised, with hydrogen bonding to the amino acid residue Gln 767 , while this carboxylic group was shifted away from the distinctive amino acid residue Met 769 . Moreover, the bromine atom at position-6 formed bifurcated hydrogen bonds with the amino acid residue Met 742 and Glu 738 (Figure 3, lower right panel). It is clear that the results of the molecular docking can be used to design novel quinoline derivatives with potential antitumour activity and binding to EGFR kinase.

Conclusions
Novel 4,6-disubstituted 2-SQ derivatives 3a,b-9a,b have been synthesized, and their antitumour activity and EGFR inhibition have been evaluated. Among the tested compounds, 3a and 4a,b (IC 50 ffi 7.7-9.8 mg/ml) were identified as the most potent antitumour agents against HepG2 and HCT116 cancer cell lines, with activity comparable to that of 5-FU (IC 50 ffi 5.37-7.9 mg/ml) and afatinib (IC 50 ffi 5.4 -11.4 mg/ml). Moreover, compound 3b exhibited strong antitumour activities against HepG2 and HCT116 cancer cell lines with IC 50 values of 17.2 and 14.8 mg/ml, respectively. Compounds 3a and 4b have moderate inhibitory activity on EGFR with IC 50 values of 2.23 and 1.22 mM, respectively. Accordingly, both compounds 3a and 4b are expected to exert their antitumour activity through inhibition of EGFR. A molecular docking study was conducted for compounds 3a and 4b and the putative binding site of EGFR kinase, which revealed a binding mode similar to that of the reference inhibitor erlotinib.