Synthesis, anticancer and apoptosis-inducing activities of quinazoline-isatin conjugates: epidermal growth factor receptor-tyrosine kinase assay and molecular docking studies.

A new series of quinazolinone compounds 16-34 incorporating isatin moieties was synthesized. The antitumor efficacy of the compounds against MDA-MB-231, a breast cancer cell line, and LOVO, a colon cancer cell line, was assessed. Compounds 20, 21, 22, 23, 25, 27, 28, 29, 30, 31, 32, 33, and 34 displayed potent antitumor activity against MDA-MB-231 and LOVO cells (IC50: 10.38-38.67 μM and 9.91-15.77 μM, respectively); the comparative IC50 values for 5-fluorouracil and erlotinib in these cells lines were 70.28 μM, 22.24 μM and 15.23 μM, 25.31 μM respectively. The EGFR-TK assay and induction of apoptosis for compound 31 were investigated to assess its potential cytotoxic activity as a representative example of the novel synthesized compounds. At a concentration of 10 μM, compound 31 exhibited efficient inhibitory effect against EGFR-TK and induced apoptosis in MDA-MB-231 cells. Furthermore, a molecular docking study for compound 31 and erlotinib was performed to verify the binding mode toward the EGFR kinase enzyme, and showed a similar interaction as that with erlotinib alone. Graphical Abstract: Compound 31 showed potent antitumor activity and efficient inhibitory effect against EGFR-TK and induced apoptosis of MDA-MB-231 cells at a concentration of 10 μM.


Introduction
Cancer is one of the most worldwide dangerous health problems and is one of the leading causes of death 1 . Many of the current anticancer agents are highly toxic and nonspecific, so the production of innovative, safe, and selective anticancer molecules is an important goal for the medicinal chemistry researchers. The quinazolinone scaffold is a vital structure in medicinal chemistry  .
Anilinoquinazolines, such as gefitinib 23,24 and erlotinib 25 , have been established as EGFR kinase inhibitors for the treatment of breast cancer (Figure 1). The 3-phenethylquinazoline derivative (I) has broad spectrum antitumor activity with a mean GI 50 value of 3.16 lM, in addition to EGFR-TK inhibitory activity 11 (Figure 1).
Additionally, isatin derivatives exhibit broad spectrum biological effects such as anticancer activity 26 . A 5-fluoro-3-substituted isatin analog (Sunitinib) was approved by the FDA for the treatment of renal carcinoma and gastrointestinal stromal tumors 27,28 (Figure 1). Methyl 3-(1-(4-bromobenzyl)-2,3-dioxoindolin-5-yl)acrylate showed broad spectrum anticancer activity and a weak cytotoxic effect in normal human cells 29 . A series of indolinone hydrazides, including 2-(6-oxo-1,6-dihydropyrimidin-4-yl)-N 0 -(2oxoindolin-3-ylidene)acetohydrazide (II) and 2-(4-fluoro-3-hydroxyphenyl)-N 0 -(2-oxoindolin-3-ylidene)acetohydrazide (III), were reported as potent anticancer agents with IC 50 values of 5.99 and 0.054 lM, respectively 30 ( Figure 1). As an attempt to develop effective cytotoxic agents, we synthesized hybrids of quinazoline conjugated to 5-substituted isatin that contained an acylhydrazone moiety and evaluated their cytotoxic activity. Additionally, the EGFR-TK assay and apoptosis induction were investigated for the most active compound, as a representative example of the novel synthesized compounds, to identify their potential cytotoxic activity. A molecular docking study was conducted to verify the structural requirements of the antitumor activity of the target molecules and to support the results of binding of the active compounds to EGFR 31 .

WST-1 cell proliferation assay
The cell proliferation assay was conducted according to a previously reported method 32 .

Immunofluorescence microscopy
The EGFR immunofluorescence assay was conducted according to a previously reported method 33 .

Apoptosis assay
Vybrant apoptosis assay kit (Annexin-V, APC conjugate; Molecular Probes TM ) was used to evaluate cell viability in accordance with the manufacturer's recommendation 33 .

Cell proliferation inhibition assay
The in vitro antitumor activity of compounds 16-34 against the human breast cancer cell line, MDA-MB-231, and the colon cancer cell line, LOVO, was determined by WST-1 assay 32 using 5-FU and erlotinib as a reference drugs, and IC 50 was calculated for each cell line (Table 1). In the present study, the active compounds exhibited a characteristic selectivity potential in addition to broadspectrum antitumor activity.

GFR tyrosine kinase enzyme inhibition assay
The enzyme activity assay of the most active compound 31 toward the MDA-MB-231 breast cancer cell line was selected as representative example of the compounds and administered at a single concentration (10 lM) against EGFR-TK to investigate the mechanism of action of the newly synthesized compounds 33 . The immunofluorescence staining of EGFR in MDA-MB-231 cells treated with compound 31 at 10 lM indicated a good selectivity of compound 31 to EGFR-TK, as shown by inhibition of the level of EGFR on the cell membrane as well as in the nucleus (Figure 2).

Molecular docking results
The antitumor activities of the weakly active compound 28 and the highly active compound 31 in MDA-MB-23 cells, which highly express epidermal growth factor receptor (EGFR) 7,10,11,15,19,22 and the binding activity of compound 31 with EGFR, encouraged us to conduct molecular docking simulations of the binding site of the EGFR kinase. Compounds 28 and 31 were docked into the receptor active site of EGFR along with their inhibitor erlotinib (Tarceva TM ) (PDB code: 1M17) 35 . All calculations were performed using MOE 2008.10 software 34 . The docking study of the most active compound 31 revealed that the quinazoline ring typically overlaid the corresponding ring of erlotinib without clashing with the surrounding amino acids. The substituted linkage at the C-2 hybrid of the binding of compound 31 in both the activation and catalytic loops where N1 was uniquely bound with the distinctive residue Met 769 . A semicarbazide nitrogen atoms was recognized via hydrogen bonding with Leu 768 , while the second semicarbazide nitrogen atom performed hydrophilic interaction by cross interaction with Pro 717 through the water molecule in the pocket. The two adjacent conserved amino acids Leu 768 and Met 769 firmly held the backbone of compound 31, which augmented the recognition and the overall inhibition activity (Figure 4).
In contrast, compound 28 was bound in different manner, which dramatically lowered the overall complementarity. Although N1 was clearly recognized with hydrogen bonding to the distinctive residue Met 769 , N3 was buried away from the surrounding amino acids owing to the rigidity of the connected phenyl group. However, the semicarbazide linkage enriched the hydrophilic interaction by cross interaction with Pro 717 through the water molecule in the pocket ( Figure 5).

Conclusions
A new series of quinazolinone-isatin conjugates 16-34, which strongly inhibited growth in the MDA-MB-231 breast cancer cell line and LOVO colon cancer cell line, was synthesized. Compounds