VEGFR-2 inhibitors and apoptosis inducers: synthesis and molecular design of new benzo[g]quinazolin bearing benzenesulfonamide moiety

Abstract Two series of novel 4-(2-(2-(2-(substituted) hydrazinyl)-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 5–17 and 4-(2-(2-(substituted-1H-pyrazol-1-yl)-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 18–24 were synthesised from the starting material 4-(2-(2-hydrazinyl-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 5, to be evaluated for their inhibitory activity towards VEGFR-2. The target compounds 5–24, were screened for their cytotoxic activity against MCF-7 breast cancer cell line and the percentage inhibition against VEGFR-2. Compounds 9, 20, 22 and 23, showed excellent VEGFR-2 inhibitory activity with IC50 ranging from 0.64 to 1.04 µm. Being the most potent, compound 9 was evaluated for its apoptotic inducer effect by studying the effect on caspase-3, it was found to increase its level. Compound 9 boosted the level of Bax and reduced the level of BCl2, compared to the control. Cell cycle analysis was conducted, compound 9 showed cell cycle arrest at G2/M phase. Moreover, mild cytotoxic effect (IC50 = 29.41 µm, respectively) in normal breast cells MCF-12 A, was observed when treated with the same compound. Finally, a molecular docking study was performed to investigate the possible binding interaction inside the active site of the VEGFR-2 enzyme.


Introduction
Tyrosine kinases are responsible for the phosphorylation of tyrosine residues in proteins. This phosphorylation leads to changing the protein's function 1 . They are considered an important member involved in cell signalling pathways 2 . Mutations can cause some tyrosine kinases to become continuously active, leading to the development of cancer 3 . Vascular endothelial growth factor (VEGF) is an important signalling protein involved in both vasculogenesis and angiogenesis 4 . It was found to enhance the microvascular permeability thus, promoting endothelial cell mitogenesis and cell migration 5 . VEGF is up-regulated in many tumours due to an imbalance between proangiogenic and anti-angiogenic factors 6 . They consist of three subtypes, which are VEGFR-1 (Flt-1), VEGFR-2 (KDR) and VEGFR-3 (Flt-4) 7 . Selectivity of kinase inhibitors is difficult to predict based on chemical structure and sequence. VEGFR-2 can be divided into three subtypes, kinase I inhibitors, which interact with the ATP-binding site by one to three hydrogen bonds, mimic that formed by ATP 8 . An example of this type is sunitinib ( Figure 1) 9 , which demonstrated competitive inhibition to ATP. On the contrary, type II indirectly competes with ATP by occupying the hydrophobic pocket adjacent to the ATP binding site. Some type II inhibitors are able to form hydrogen bonds to the ATP binding site. However, this is not necessary for activity 10 . An example of type II inhibitors is sorafenib ( Figure 1) that acts by blocking the phosphorylation of VEGFR by using its hydrophobic pocket 11 . The third class is known as covalent inhibitors, they covalently bind to cysteine at specific sites of the kinase allowing the inhibitor to block the binding of ATP to the kinase 12 . An example of these inhibitors is Vatalanib (Figure 1) 13 . Vandetanib, the reference drug used in this study is a dual inhibitor towards VEGFR-2 and EGFR, approved in 2011, it was found to disrupt the angiogenesis process and starve tumours of nutrients. It is an example of an extended-spectrum agent 14 .
Once activated, VEGFR-2 undergoes autophosphorylation, triggering signalling pathways leading to endothelial cell proliferation and subsequent tumour angiogenesis 15,16 . Folkman 17 proposed that tumour growth and metastasis are angiogenesis-dependent, and hence, blocking angiogenesis could be a strategy to hinder tumour growth.
Breast cancer (BC) is the most common malignancy and the leading cause of cancer death in women worldwide 18 . VEGFR-2 inhibitors were reported in treating BC due to their high safety profile [19][20][21] . But a combination therapy with other chemotherapy or radiotherapy was reported to maximise the therapeutic effect 22 .
In order to design our targeted compounds, the essential requirements for VEGFR-2 receptor bearing quinoline inhibitor (PDB ID: 3U6J) were studied [23][24][25] (  replaced by benzo[g]quinazoline in our target structures and forms the same interactions. (ii) Substituted anilino group in position 4 of the quinoline interacting as an H-bond donor by its NH with Glu885 and through hydrophobic interaction by its substituted phenyl moiety with the hydrophobic back pocket lined with the hydrophobic side chains of Ile 888, Ile 892, Leu 1019 and Ile 1049. In our target compounds, the substituted anilino group was replaced with NH 2 of the sulfonamide group and was found to form H-bond with Glu 885, while, the benzo[g]quinazolin fits inside the hydrophobic pocket. (iii) Hydrogen bond acceptor as nitrogen lone pair or oxygen atom attached to position 4 of quinoline via benzyl or phenyl moiety, which interacts with the backbone NH of Cys 919 in the hinge region of the enzyme. In our target compounds, the oxygen atom in the sulfonamide group binds with Cys 919.
In this respect, we designed novel compounds based on benzo[g]quinazoline core and sulfonamide moiety, these derivatives were subjected to in vitro cytotoxic evaluation against MCF-7, followed by VEGFR-2 inhibitory profile. Molecular docking was performed in the active site of VEGFR-2, to determine their binding mode and their ability to satisfy the pharmacophoric features required to induce the desired inhibition. Moreover, VEGFR-2 inhibition in cancer cells was found to trigger apoptosis which synergistically augments the antitumour effect 26,27 . So, the apoptotic effect of the most potent compound was discussed in comparison to vandetanib, through the inhibition of the caspase-3 enzyme, detection of BAX and BCl2 levels, and cell cycle analysis. Also, the cytotoxicity of the most potent compound against normal breast cell line was investigated.

Materials and methods
Melting points were determined in an open capillary on a Gallen Kamp melting point apparatus (Sanyo Gallen Kamp, Leicestershire, UK). Precoated aluminium sheets Silica gel Merck 60 F254 were used for thin layer chromatography and were visualised by UV lamp (Merck, Darmstadt, Germany). The developing solvent system was chloroform/methanol 7:3. IR spectra (KBr disc) were recorded using an FT-IR spectrophotometer (PerkinElmer), OH, USA. 1 H-NMR spectra were scanned on an NMR spectrophotometer (Bruker AXS Inc., Flawil, Switzerland), operating at 500 MHz for 1 H-and 125.76 MHz for 13 C. Chemical shifts are expressed in d-values (ppm) relative to trimethylsilyl group as an internal standard, using DMSOd 6 as a solvent. Elemental analyses were done on a model 2400 CHNSO analyzer (PerkinElmer). All the values were within ±0.4% of the theoretical values. All reagents used were of AR grades.

VEGFR-2 assay
VEGFR-2 activity and IC 50 of the selected compounds were determined in MCF-7 cells. Cells were cultured using the above-mentioned methods. The kinase activity of VEGFR-2 was measured by the use of a phosphotyrosine antibody with the Alpha Screen system (PerkinElmer) according to manufacturer's instructions. The tested compounds at final concentrations ranging from 0 to 100 mg/ml and enzyme were incubated for 5 min at room temperature. The reactions were quenched by the addition of 25 ml of 100 mm EDTA. The plate was incubated in the dark overnight and then read by ELISA Reader (PerkinElmer). Percent inhibition was calculated by the comparison of compounds treated to control incubations, and the data were compared with Vandetanib as a standard VEGFR-2 inhibitor.

Effect on active caspase-3
To determine the effect of the synthesised compounds on apoptosis, the active caspase-3 level was measured by using Quantikine-Human active Caspase-3 Immunoassay (R&D Systems, Inc. Minneapolis, MN) according to the manufacturer protocol. Cells were obtained from American Type Culture Collection, then were grown in Roswell Park Memorial Institute (RPMI) 1640 containing 10% foetal bovine serum at 37 C, stimulated with the compounds to be tested for caspase3, and lysed with Cell Extraction Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay, the optical density of each well was determined within 30 min using a microplate reader set at 450 nm to determine the human active caspase-3 content.

Effect on BAX and bcl-2 levels
Cells were grown in RPMI 1640 containing 10% foetal bovine serum at 37 C, stimulated with the compounds to be tested for Bax, and lysed with cell extraction buffer. This lysate was diluted in the standard diluent buffer over the range of the assay and measured for human active Bax and BCl2 content according to the reported method 29 .

Analysis of cell cycle distribution
To determine the effect of compounds 9 and vandetanib on the cell cycle distribution MCF-7 cell line; cell cycle analysis was performed using the CycleTEST TM PLUS DNA Reagent Kit (Becton Dickinson Immunocytometry Systems, San Jose, CA). Control cells with known DNA content (PBMCs) were used as a reference point for determining the DI (DNA Index) for the test samples. The cells were stained with propidium iodide stain following the procedure provided by the kit and then run on the DNA cytometer. Cell cycle distribution was calculated using CELLQUEST software (Becton Dickinson Immunocytometry Systems).

Molecular docking
All the molecular modelling studies were carried out using MOE, 10.2008 software (North Buona, Singapore). Energy minimisations were performed with a root mean standard deviation (RMSD) gradient of 0.05 kcal mol À1 Å À1 with an MMFF94X force field and the partial charges were calculated. The protein data bank file (PDB: 3U6J) was selected for this purpose. The file contains VEGFR-2 cocrystallised with a N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide, obtained from protein data bank. The enzyme was prepared for docking studies: (i) Water molecules were ignored; (ii) hydrogen atoms were added to the enzyme; (iii) MOE Alpha Site Finder was used for the active sites search in the enzyme and (iv) removal of the co-crystallised ligand and docking of the new targeted structures.

Apoptosis studies
From the above results, most of the synthesised compounds proved to have VEGFR-2 inhibitory activity. As mentioned before, the inhibition of VEGFR-2 enzyme can lead to the induction of apoptosis 26,27 . Therefore, the ability of the most potent compound 9 to induce the apoptosis cascade will be investigated.

Activation of caspase-3
Activation of caspases plays a key role in the initiation and execution of the apoptotic process 31 . Caspase 3 is initiated by the death cascade. It is activated by the upstream of caspase 8 and 9. So, it acts as a convergence point in different signalling pathways 32,33 .
The effect of compound 9 on caspase 3 was evaluated and compared to vandetanib as a reference drug. It showed an increase in the level of active caspase 3 by 7-folds, compared to the control cells. While vandetanib induced caspase 3 approximately by 10 times (Table 2, Figure 3).

Effects on bcl-2 family proteins
The B-cell lymphoma protein 2 (Bcl-2) family plays a key role in tumour progression or inhibition of intrinsic apoptotic pathway triggered by mitochondrial dysfunction 34 . The Bcl2 protein inhibits apoptosis (anti-apoptotic) while Bax stimulates it (proapoptotic). Thus, the balance between these two different opposing proteins regulates the cell fate 35,36 . Increments in the Bax/Bcl2 ratio trigger the release of mitochondrial cytochrome C into the cytosol which in turn potentiates a cascade of caspases that ultimately leads to activation of caspase 3; the apoptosis executioner 37 . In this study, MCF-7 cells were treated with the IC 50 of compound 9 and their effect on the expression levels of Bcl2, and Bax were determined as illustrated in Table 3 and Figure 4. As shown by the results, compound 9 boosted the level of the proapoptotic protein; Bax to 230.8 pg/ml compared to the control (13.68 pg/ml). Moreover, compound 9 markedly reduced the levels of the anti-apoptotic proteins Bcl2 to 0.561 ng/ml compared to the control (1.953 ng/ml). While vandetanib boosted the level of Bax to 397.9 pg/ml and reduced the level of the anti-apoptotic proteins Bcl2 to 0.948 ng/ml. Collectively, these findings proved that both compound 9 and vandetanib markedly increased Bax level and downregulated Bcl2 proved undoubtedly their proapoptotic effect.

Cell cycle analysis
In general, the anticancer agents abort the growth and proliferation of cancerous cells by arresting cell division at various checkpoints. These checkpoints present at G1/S phase, S-phase and G2/M phases 38 . Treatment of the cancer cells with anticancer agents can determine at which phase apoptosis occurs in the cell cycle. In the current study, MCF-7 cells were treated with compound 9 at its IC 50 (0.10 mm). The obtained data (Figures 5 and 6(A)) obviously indicate that compound 9 arrested the cell cycle at a G2/M phase when compared to the untreated control (24.17% Table 3. Effect of compound 9 on the expression of the gene of some apoptosis key markers.     (Figure 6(B)). Parallel to these findings, the cell population in G1 and S phases decrease after treatment (38.44 and 24.38% versus 22.51 and 29.32%, respectively) in the case of compound 9. While in the case of vandetanib, the cell population in G1 and S phases markedly decreases after treatment to (22.51% and 26.32%, respectively). These results reveal that in MCF-7 cells, cell cycle arrest occurs in the G2/M phase in case of compound 9 and vandetanib.

Cytotoxicity test
The cytotoxic effects of compound 9 and vandetanib were tested on normal breast cells MCF-12 A using sulforhodamine B assay 39 . Both compounds showed mild cytotoxic effect with an IC 50 of 29.41 and 22.06 mm, respectively (Figure 7). This result indicates      the selectivity of compounds 9 and vandetanib for tumour BC cells and their relative safety for normal breast cells.
funding of this research through the Research Group Project No. RGP-302.