New benzoxazole derivatives as potential VEGFR-2 inhibitors and apoptosis inducers: design, synthesis, anti-proliferative evaluation, flowcytometric analysis, and in silico studies

Abstract A new series of benzoxazole derivatives were designed and synthesised to have the main essential pharmacophoric features of VEGFR-2 inhibitors. Cytotoxic activities were evaluated for all derivatives against two human cancer cell lines, MCF-7 and HepG2. Also, the effect of the most cytotoxic derivatives on VEGFR-2 protein concentration was assessed by ELISA. Compounds 14o, 14l, and 14b showed the highest activities with VEGFR-2 protein concentrations of 586.3, 636.2, and 705.7 pg/ml, respectively. Additionally, the anti-angiogenic property of compound 14b against human umbilical vascular endothelial cell (HUVEC) was performed using a wound healing migration assay. Compound 14b reduced proliferation and migratory potential of HUVEC cells. Furthermore, compound 14b was subjected to further biological investigations including cell cycle and apoptosis analyses. Compound 14b arrested the HepG2 cell growth at the Pre-G1 phase and induced apoptosis by 16.52%, compared to 0.67% in the control (HepG2) cells. The effect of apoptosis was buttressed by a 4.8-fold increase in caspase-3 level compared to the control cells. Besides, different in silico docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.


Introduction
Angiogenesis, a complex process of new blood vessel creation, is crucial for cell development and reproduction 1,2 . Considering the similar function in cancerous cells, uncontrolled or abnormal angiogenesis has been linked to tumour progression and metastasis 3 . Therefore, finding efficient anti-angiogenesis agents could be considered as a hopeful approach for cancer treatment 4 .
Growth factors, including vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), control angiogenesis [5][6][7] . Three main vascular endothelial growth factor receptor subtypes are well-defined namely, VEGFR-1, VEGFR-2, and VEGFR-3 8 . These receptors are the key players' intermediates in controlling tumour angiogenesis and in the development of new blood vessel networks essential to supply nutrition and oxygen for tumour growth 9 . Among the three VEGFRs subtypes, VEGFR-2 plays the most critical role in promoting tumour angiogenesis 10 . Following its activation by VEGF, VEGFR-2 initiates downstream signal transduction via dimerisation and then autophosphorylation of tyrosine receptor. These signalling pathways result in tumour angiogenesis 11 . Thus, hindering the VEGF/VEGFR-2 signalling pathway or reducing its response by tyrosine kinases inhibitors (TKIs) is a supreme significant target in anti-angiogenesis therapy against cancer 12 . Over the last decades, several small molecules have been approved for obstructing this critical pathway in angiogenesis 13,14 . Development of tumour resistance to the effect of the current clinically used small-molecule TKIs opens the door for the investigation of the effectiveness of new chemotypes.

Rationale of molecular design
In view of the above-mentioned pharmacophoric requirements and depending on our ongoing project to develop novel cytotoxic small molecules based on various chemotypes, it is considered of interest to begin a research work directed towards the design of a new series of anti-angiogenic VEGFR-2 inhibitors. A cross-hybridisation approach between different pharmacophoric elements of the well-known TKIs was the idea of the current study. The approach applied for designing the new target compounds is demonstrated in Figure 2.
Herein, and while conserving the carboxamide moiety of sunitinib to serve as a hydrogen-bonding donor/acceptor moiety, a molecular replacement of the indolinylidene core of sunitinib by a benzoxazole core was performed in the hope of testing the effect of the introduction of another heteroatom to the aromatic scaffold in binding with the hinge region of ATP binding site.
Additionally, the fluorine atom in sunitinib was also replaced either by hydrogen, methyl, or chlorine bioisosteres. On the other side, the terminal phenyl ring of sorafenib was kept playing its key role of occupying the allosteric lipophilic pocket. Contrariwise, the 4-chlorine atom and the 3-trifluoro methyl group of sorafenib were replaced by different substituents as represented in the target compounds. Regarding the central aromatic linker moiety, we found that our previously reported linker of compound 3 25 could give the opportunity to the designed compounds to be oriented into the DFG motif and allosteric binding site as well ( Figure 2).
The wide diversity of modifications enabled us to study the SAR of the designed candidates as potent anti-proliferative agents with potential VEGFR-2 inhibitory effects. To confirm such a design, in silico molecular docking studies of the designed compounds were performed against the prospective biological target (VEGFR-2).
The potassium salts 9a-c were heated in dry DMF with the formerly prepared derivatives 13a-e to afford the final target compounds 14a-o, (Scheme 3).

Biological testing
2.2.1. Breast cancer and hepatocellular carcinoma, in vitro antiproliferative activities The anti-proliferative activities of the newly synthesised compounds were assessed in vitro against two human cancer cell lines namely, breast cancer (MCF-7) and hepatocellular carcinoma (HepG2) cell lines, using the standard MTT method 35 . The tested cell lines were chosen carefully depending on their VEGF overexpression. Sorafenib, the potent VEGFR-2 inhibitor drug, was coassayed as a positive control. The cytotoxicity results were demonstrated in Table 1. A general observation of the obtained results revealed that all the newly synthesised members had high inhibitory activities towards the two cancer cell lines with IC 50 values ranging from 4.054 ± 0.17 to 32.53 ± 1.97 mM for MCF-7 and from 3.22 ± 0.13 to 32.11 ± 2.09 mM for HepG2.
With reference to their cytotoxic activity, it was noticed that counterparts incorporating 5-chlorobenzo[d]oxazole moiety were slightly more advantageous than the unsubstituted benzo[d]oxazole analogs. However, the 5-methylbenzo[d]oxazole-containing derivatives displayed less potent inhibitory activity against the tested cell lines.
Lastly in this regard, concerning 5-methylbenzo[d]oxazole containing derivatives, it was found that compound 14i displayed the strongest anti-proliferative effect against HepG2 cell line (IC 50 ¼ 3.22 ± 0.13 mM) compared to the reference drug, sorafenib. Compound 14i, moreover, showed a strong effect with respect to MCF-7 cells with IC 50 of 6.94 ± 0.22 mM. It is also noteworthy that member 14l, 2,5-dichloro phenyl, strongly inhibited the MCF-7 and HepG2 proliferation with IC 50 values of 6.87 ± 0.23 and 6.70 ± 0.47 mM, respectively. Other modifications of the terminal phenyl ring did not increase the cytotoxic activity with increasing the IC 50 range (7.01 ± 0.52 to 22.05 ± 1.79 mM) comparing the other derivatives ( Figure 3).

Wound healing assay
The compound 14b reduced human umbilical vascular endothelial cell (HUVEC) proliferation and migratory potential.    One of the hallmarks of angiogenesis is cell migration, which happens in the earlier stages of the angiogenic cascade. A wound-healing assay was performed to investigate the migratory effect of compound 14b. Compound 14b and sorafenib extremely reduced the HUVECs migration potential exhibiting deeply reduced wound healing patterns after 72 h. Wound closure (%) was significantly lower in the compound 14b group (47.2 ± 2.88) and sorafenib group (39.8 ± 1.9) when compared to the control group (95.86 ± 4.51) ( Figure 5).

Cell cycle analysis
Compound 14b which demonstrated remarkable cytotoxic potency and significant inhibitory effect against VEGFR-2 was nominated for further cellular mechanistic study. This involved study of its impact on cell cycle progression and induction of apoptosis in HepG2 cells.
The cell cycle process was analysed after exposure of HepG2 cells to 14b with a concentration of 4.61 mM for 48 h. Flow cytometry data 36 revealed that the percentage of cells arrested at the Pre-G1 phase increased from 1.49% (in control cells) to 24.59% (in 14b) treated cells. In addition, the percentage of HepG2 cells mild increased at the S phase from 35.21 to 37.26%. Such findings revealed that compound 14b arrested the HepG2 cell growth mostly at the Pre-G1 phase (Table 3 and Figure 6).

Apoptosis analysis
To quantify the apoptosis triggered by 14b, Annexin-V/propidium iodide (PI) staining assay was conducted 37 . In such a procedure, compound 14b at a concentration of 4.61 mM was applied on HepG2 cells for 48 h. As shown in Table 4 and Figure 7, the apoptotic effect of 14b in HepG2 cells was about twenty-four times more than observed in control cells. In detail, compound 14b induced apoptosis by 16.52%, compared to 0.67% in the control cells.

Caspase-3 determination
To investigate the effect of the synthesised compounds on caspase-3 level, the most promising member 14b was applied on the most sensitive cells (HepG2) at a concentration of 4.61 mM for 48 h. The results revealed that compound 14b produced a significant increase in the level of caspase-3 (4.8-fold) compared to the control (HepG2) cells ( Figure 8).

Docking study
A docking study was carried out in the hope of getting an insight into the mode of interaction of the synthesised compounds to their biomolecular targets [38][39][40] . Thus, VEGFR-2 kinase domain crystal structure PDB ID: 2OH4 in complex with a benzimidazole-urea inhibitor was adopted for the current study. After protonation and preparation of the protein, the validity of the used docking protocol was checked by redocking of the bound benzimidazole-urea inhibitor. The redocking validation step successfully regenerated the experimental binding pattern of the co-crystallized ligand with high efficiency. The docking pose reproduced the key interactions accomplished by the co-crystallized ligand in the active site via binding with Cys917 in the hinge region, Asp1044 of the DFG motif, and Glu883 in the a-C helix. The reproduced binding mode in addition to the small RMSD (0.71 Å) between the docked pose and the co-crystallized ligand proved the effectiveness of the adopted protocol for the planned docking study (Figure 9). Sorafenib, a potent VEGFR-2 inhibitor used in the experimental in vitro assays, was used as a reference in the docking study as  well. Sorafenib interacted by its urea NH groups with the carboxylate side chain of Glu883 in the a-C helix through H-bond interactions. While the urea carbonyl group was involved in an H-bond interaction with the NH group of Asp1044 of the DFG motif. On the hinge region, sorafenib was found to interact by an H-bond with Cys917. Sorafenib interacted, furthermore, via several hydrophobic interactions with the hydrophobic pocked formed by Lys886, Val897, Ile886, Phe1045, and Cys917 ( Figure 10). Investigation of the docking results revealed that the synthesised compounds were able to identify the VEGFR-2 kinase ATP binding site and interact with key amino acids thereof in a manner like that of sorafenib. The studied compounds all occupied the same orientation of sorafenib in the VEGFR-2 kinase active pocket. Thus, as displayed in Figures 11-13, the benzoxazole moieties of the designed compounds 14b, 14n, and 14l were oriented towards the hinge region of the active site forming an Hbond between their nitrogen and Cys917 residue. On the other side, the benzamide scaffolds of the titled compounds were accommodated in the pocket central area, the gate area, interacting via one H-bond with the carboxylate side chain of Glu883 and another one with the NH moiety of Asp1044 of the conserved DFG motif in VEGFR-2. However, the orientation of the later moieties allowed the compounds' hydrophobic substituents to fit in the hydrophobic allosteric pocket in the active site permitting these hydrophobic substituents to interact with hydrophobic side chains of Ile886, Leu887, Ile890, Val896, Val897, Leu1017, and Ile1042 residues lining the back pocket of VEGFR-2.

In silico ADME analysis
Results of ADME analysis were illustrated in the Supplementary Data.

Toxicity studies
The toxicity profiles of all the tested compounds were examined. This involves using seven constructed toxicity models (illustrated in Table 5) utilising Discovery studio 4.0 software (Supplementary Data).

Conclusion
A new series of benzoxazole derivatives was designed hoping to discover novel VEGFR-2 inhibitor agents. Fifteen compounds were synthesised and tested in vitro for their anti-proliferative activities against two human cancer cell lines, MCF-7 and HepG2. The

In vitro anti-proliferative activity
The anti-proliferative activity of all tested compounds was performed on MCF-7 and HepG2 cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 35,[41][42][43][44] . The MTT assay is based on the capability of living cells to reduce the yellow product MTT to a blue product, formazan, by a reduction reaction occurring in the mitochondria. Briefly, in MTT assay, 5000 cells/ well were plated in a 96-well plate and allowed to grow 24 h, then treated with Roswell Park Memorial Institute (RPMI) 1640 media that contain increased concentrations (0, 0.1, 1, 10, 100, and 1000 mM) of tested compounds. Each experiment was carried out in triplicate. Then media were removed and 100 mL of MTT was added to each well and incubated for 4 h. The formed formazan crystals were solubilised by adding 100 mL of dimethyl sulfoxide (DMSO) solution and absorbance was measured at 570 nm using ELISA microplate reader (Epoc-2 C micro-plate reader, Bio Tek, VT, USA). The IC 50 values [the concentration required for 50% inhibition of cell viability] were calculated and the results are expressed as the relative percentage of the control cells (100% of cell viability).

In vitro VEGFR-2 protein concentration assay
The in vitro assessment of VEGFR-2 protein concentration after exposure of HepG-2 cells to the most cytotoxic candidates was carried out using Enzyme-Linked Immunosorbent Assay (ELISA) kit (Cat. NO. EK0544) (AVIVA System Biology, USA) according to manufacturer instructions 45 .

Wound healing assay
Allow 10 min for the 24-well plate with CytoSelect TM Wound Healing Inserts to warm up at room temperature before applying 500 ml of HUVECs cell suspension (1.0 Â 10 6 ) in media containing 10% foetal bovine serum (FBS) to each well. In a cell culture incubator, incubate the cells until they form a monolayer. Remove the implant from the well gently to begin the wound healing assay. Aspirate the media from the wells slowly and discard it.
To eliminate dead cells and debris, wash wells with the medium. Finally, fill wells with medium to keep cells hydrated, and examine them under a light microscope. The wells were subsequently filled with media containing the indicated concentrations of compound 14b or sorafenib for 72 h. A light microscope is used to monitor the wound closure. Calculate the percentage of cells that have closed into the wound field 46 .

Analysis of the cell cycle distribution phases
The propidium iodide staining followed by flow cytometric analysis was conducted according to the cell cycle kit (PN C03551) and previously published works 43,47 to investigate the effect of compound 14b on the cell cycle phases. In Brief, HepG2 cells were allowed to grow in 25 cm 3 flask until reach 70-80% confluence, then treated with compound 14b for 48 h. Then the cells were harvested and fixed. The cells were centrifuged at 2000 rpm for 5 min then, the supernatant was aspirated. The pellet of fixed cells was resuspended in a 0.5 ml cell cycle kit, vortexed, and incubated at 25 C for 15 min. Finally, DNA was stained with 50 mg/ml propidium iodide for 30 min. Flow cytometric analysis of cell cycle performed on a COULTER V R EPICS V R XL TM Flow Cytometer (USA).

Annexin V-FITC apoptosis assay
For the detection of apoptosis in treated cells, Annexin V-FITCapoptosis detection kit (PN IM3546) was used, followed by flow cytometric analysis according to manufacturer protocol. In this assay, HepG-2 cells were allowed to grow in a 25 cm 3 flask until 70-80% confluence. Then HepG-2 cells were treated with compound 14b for 48 h followed by a wash in PBS and suspended in 1Â binding buffer. To 100 mL of the cell suspensions, 1 mL of annexin V-FITC solution and 5 mL of dissolved PI were added and incubated for 15 min in the dark. Then 400 mL of ice-cold 1Â binding buffer was added and mixed gently. The flow cytometric analysis for the percentage of apoptotic cells was performed on a COULTER V R EPICS V R XL TM Flow Cytometer (USA) 42,48 .

Caspase-3 determination
The effect of compound 14b on Caspase-3 level was assessed using ELISA kit (Catalog # KHO1091) according to manufacturer instructions.