Design, synthesis, docking, ADMET studies, and anticancer evaluation of new 3-methylquinoxaline derivatives as VEGFR-2 inhibitors and apoptosis inducers

Abstract Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a critical role in cancer angiogenesis. Inhibition of VEGFR-2 activity proved effective suppression of tumour propagation. Accordingly, two series of new 3-methylquinoxaline derivatives have been designed and synthesised as VEGFR-2 inhibitors. The synthesised derivatives were evaluated in vitro for their cytotoxic activities against MCF-7and HepG2 cell lines. In addition, the VEGFR-2 inhibitory activities of the target compounds were estimated to indicate the potential mechanism of their cytotoxicity. To a great extent, the results of VEGFR-2 inhibition were highly correlated with that of cytotoxicity. Compound 27a was the most potent VEGFR-2 inhibitor with IC50 of 3.2 nM very close to positive control sorafenib (IC50 = 3.12 nM). Such compound exhibited a strong cytotoxic effect against MCF-7 and HepG2, respectively with IC50 of 7.7 and 4.5 µM in comparison to sorafenib (IC50 = 3.51 and 2.17 µM). In addition, compounds 28, 30f, 30i, and 31b exhibited excellent VEGFR-2 inhibition activities (IC50 range from 4.2 to 6.1 nM) with promising cytotoxic activity. Cell cycle progression and apoptosis induction were investigated for the most active member 27a. Also, the effect of 27a on the level of caspase-3, caspase-9, and BAX/Bcl-2 ratio was determined. Molecular docking studies were implemented to interpret the binding mode of the target compounds with the VEGFR-2 pocket. Furthermore, toxicity and ADMET calculations were performed for the synthesised compounds to study their pharmacokinetic profiles


Introduction
According to WHO reports, cancer is considered the second major cause of death 1 . By 2030, the incidence of cancer deaths will reach thirteen million worldwide 2 . Although the high advances in the diagnosis and treatment of cancer diseases, the survival of patients remains poor due to the widespread adverse effects of anticancer agents 3 . So that, the discovery of new, effective, selective, and less toxic anticancer agents remains one of the most urgent needs 4 .
Angiogenesis process plays an important role in the growth and regeneration of tissues. Such a role is crucial to prevent ischaemic necrosis and facilitate the survival of the damaged tissues 5 . During the normal state, angiogenesis is controlled by some protein kinases (PKs), which comprise VEGFRs, FGFRs, and EGFRs 6 . PKs can be deregulated under pathological conditions, producing a disturbance in angiogenesis process. This leads to an increasing in the rate of cell division, creating tumour disease 7 .
VEGFRs and their specific agonist (VEGF) are overexpressed in many human tumours, especially solid tumours as gliomas and carcinomas 8 . Therefore, VEGFRs are considered as one the most important regulators of angiogenesis and consequently tumour growth 9 . VEGFRs family comprises three subtypes including VEGFR-1, VEGFR-2, and VEGFR-3 10 . VEGFR-1 controls embryonic vasculogenesis 11 . VEGFR-2 regulates both embryonic vasculogenesis and tumour angiogenesis 12 . On the other hand, VEGFR-3 is responsible for lymphangiogenesis 13 . So that, VEGFR-2 is now the foremost target for antiangiogenic therapy, and its blocking is a relevant approach for the discovery of new drugs against angiogenesis-dependent malignancies 14 . VEGFR-2 inhibitors demonstrated effective suppression of tumour progression. ATP binding site is the main target of most VEGFR-2 inhibitors 15 .
The crystal structures of VEGFR-2 revealed the presence of many pockets at the ATP binding site. (i) Hinge region which locates on the front side and comprises two key amino acid residues (Cys919 and Glu917). These residues participate in H-bond interactions with the adenine ring of ATP. (ii) Gatekeeper region which separates between the hinge region and DFG-motif region. (iii) DFG-motif region which locates on the backside and contains two key amino acids (Glu885 and Asp1046). For maximum activity, VEGFR-2 inhibitors have to bind with these two residues via H-bonds. (iv) Allosteric hydrophobic region which consists of much hydrophobic amino acid residues [16][17][18][19] .
VEGFR-2 inhibitors can be classified into three types. Type I inhibitors (ATP competitive inhibitors), e.g. sunitinib 1 can bind to the adenine binding region of the ATP binding site 15 . Type II inhibitors, e.g. sorafenib 2 induce inactive activation of DFG-out confirmation of activation loop. Such type can bind additionally the allosteric hydrophobic pocket of ATP 20 . Type III inhibitors, e.g. vatalanib 3 can form covalent interaction with cysteine amino acid residue at ATP binding site 21,22 .
Many drugs targeting VEGFR-2 have been approved for clinical use in the treatment of different types of cancers ( Figure 1). Sorafenib 2 is a potent VEGFR-2 inhibitor 23 . It is mainly used in the treatment of advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC) 24 . Regorafenib 4, a fluoro derivative of sorafenib, has been developed to inhibits VEGFR1-3 25 . Tivozanib 5, a VEGFR-2 inhibitor, has been approved by the FDA in March 2021 for the treatment of RCC [26][27][28] . Sunitinib 1 is a VEGFR-2 kinase inhibitor that was approved for the treatment of RCC and of gastrointestinal stromal tumours (GIST) 29 .
Binding mode sorafenib, a representative example of VEGFR-2 inhibitors, against VEGFR-2 active site (PDB: 4ASD) was reported in many publications. The heterocyclic (N-methylpicolinamide) moiety is buried in the hinge region forming two H-bonds with Cys919 through the N atom of the pyridine ring and NH group of acetamide moiety. In addition, the urea moiety binds to the receptor at the DFG motif through various H-bonding interactions with Glu885 and Asp1046. The terminal phenyl ring occupies the allosteric site forming hydrophobic interactions with the hydrophobic pocket created by the DFG flipping out [30][31][32] .
Studying the SAR of VEGFR-2 inhibitors and analysing the binding mode of sorafenib revealed that the majority of potent and selective VEGFR-2 inhibitors have four common pharmacophoric features that facilitate their fitting with the active binding   [33][34][35] . The first feature is a flat heteroaromatic ring system (ahead) incorporating at least one H-bond acceptor to interact with the crucial amino acid residue Cys919 in the hinge region 30,31 . A second feature is a spacer group that gives the inhibitor enough length 36,37 , making the third feature (pharmacophore) near at DFG motif to bind with two crucial residues (Glu883 and Asp1044) 37,38 . A pharmacophore is a functional group that comprises at least one H-bond acceptor (HBA) and one Hbond donor (HBD) group (typically amide or urea) 31,38 . The fourth feature is a terminal hydrophobic moiety that can occupy the created allosteric hydrophobic binding site 16,39 .
Apoptosis as a form of cellular suicide is one of the important mechanisms by which anticancer agents can affect cancer cells 40 . Apoptosis is regulated by several mediators 41 . Among these are caspases, specifically caspase-3 and caspase-9 42,43 . Caspase-3 protease is a major mediator of apoptosis catalysing the cleavage of many vital cellular proteins leading to cell death 44 . Caspase-9 activates other executioner caspases as caspase-3, -6, and -7 initiating apoptosis as they cleave several other cellular targets 45 . In addition, the Bcl-2 family proteins are key regulators of apoptosis. This family comprised pro-apoptotic proteins as BAX that promote cell death and anti-apoptotic proteins as Bcl-2 which suppress cell death 46,47 . The balance between pro-apoptotic and anti-apoptotic proteins (BAX/Bcl-2 ratio) regulates cell fate 48 . Current evidence suggested that inhibition of angiogenesis, anti-angiogenic therapies have been shown to increase apoptosis in tumour cells 49 . VEGFR-2 inhibitors were found to induce and accelerate apoptosis in cancer cells which synergistically potentiates their antitumor effect 30,[49][50][51] .
In our previous work, we developed [1,2,4]triazolo [4,3-a]quinoxaline containing derivatives as VEGFR-2 inhibitors. Compounds 8 and 9 were the most potent candidates exhibiting an excellent VEGFR-2 inhibitory activity with a promising cytotoxic efficacy against breast and hepatocellular carcinoma 63,64 . In continuation of our work 63-65 aimed at synthesising new anticancer agents targeting VEGFR-2 inhibition, new quinoxaline derivatives were designed and synthesised. The synthesised compounds were evaluated for their anti-proliferative activity. In addition, VEGFR-2 inhibitory activities were estimated for all compounds to hint at the potential mechanism of their cytotoxicity. Furthermore, deep investigations were performed on the most active member to assess its effect on apoptotic (caspase-3, caspase-9, and BAX) and anti-apoptotic (Bcl-2) mediators.

Chemistry
To reach the designed compounds, four Schemes 1-4 were adopted. The synthesis was initiated by the reaction of ophenylenediamine 9 with sodium pyruvate 10 in glacial acetic acid according to the reported procedure to furnish 3-methylquinoxalin-2(1H)-one 11 67 . This starting material 11 was subjected to subsequent treatment with potassium hydroxide to produce the corresponding potassium salt 12 67 . Chlorination of compound 11 was achieved using phosphorous oxychloride to afford 2-chloro-3methylquinoxaline 13 68 . Refluxing the latter with thiourea in absolute ethanol resulted in 3-methylquinoxaline-2-thiol 14 69,70 which was underwent heating with alcoholic potassium hydroxide to provide the corresponding potassium salt 15 (Scheme 1).
Synthesis of the final target compounds was illustrated in Schemes 3, 4. The 3-methylquinoxalin-2(1H)-one derivative (compounds 27a-f, 28, and 29) were obtained by heating of potassium salt 12 in dry DMF and a catalytic amount of KI with the previously prepared intermediates 26a,d,g-j, 24 b, and 25, respectively (Scheme 3).
IR spectra of compounds 27a-f and 30a-i exhibited the presence of characteristic stretching bands at 3428-3215 cm À1 corresponding to NH groups. Furthermore, the NMR spectra of compounds 27a-f and 30a-i supported their assigned structures. 1 H NMR charts of these compounds revealed the appearance of two singlet signals around d 10.5 ppm attributed to the introduced two amidic NH protons. It also demonstrated increased integration of the aromatic protons corresponding to the additional phenyl ring. A characteristic up-field singlet signal equivalent to CH 2 of acetamide moiety was detected at about d 4.30 ppm. Additionally, a singlet signal of CH 3 group of 3-methylquinoxalin-2(1H)-one moiety appeared around d 2.49 ppm. 13 C NMR spectra displayed the presence of two peaks at the aliphatic region attributed to the CH 2 and CH 3 groups around d 40.0 and 22.5 ppm, respectively. 1 H NMR spectra of compounds 28, 29, 31a,b, and 32 demonstrated the presence of the amidic protons of hydrazide moiety at a range of d 10.02-12.71 ppm. Additionally, 13 C NMR spectrum of compounds 31b as an example of these compounds revealed the appearance of two peaks at d 35.45 and 22.18 ppm corresponding to CH 2 and CH 3 groups, respectively. Mass spectroscopic analysis displayed a base peak at 517.0 (m/z) corresponding to the exact mass of the compound 31b.

In vitro anti-proliferative activity
In vitro cytotoxic activities of the synthesised compounds were evaluated against two different cancer cell lines; breast cancer (MCF-7) and hepatocellular carcinoma (HepG2) using standard MTT colorimetric assay as described by Mosmann 78,79 . Sorafenib was used as a reference cytotoxic drug. The results of cytotoxicity were expressed as growth inhibitory concentration (IC 50 ) values and summarised in Table 1.  General investigation of the cytotoxicity results clarified that the examined compounds had greater anti-proliferative activities against HepG2 than MCF-7 cells. In particular, compound 27a was found to be the most potent derivative. Such compound showed strong anti-proliferative activities against MCF-7 and HepG2 cancer cell lines with IC 50 values of 7.7 and 4.5 mM, respectively. These values were close to that of the sorafenib (IC 50   and 32 showed weak activities against only HepG2 with IC 50 values ranging from 40.8 to 43.7 mM. In the contrast, these compounds were inactive against the MCF-7 cell line. Comparing to the lead compounds 7 (IC 50 ¼ 7.2 and 4.1 mM) and 8 (IC 50 ¼ 4.4 and 3.3 mM), it was found that the most active compounds 27a (IC 50 ¼ 7.2 and 4.1 mM) was slightly less active than these compounds against MCF-7 and HepG2, respectively.

In vitro kinase inhibition assay
The newly prepared compounds have been further assayed for their inhibitory activity towards sorafenib's crucial target (VEGFR-2). The results were reported as 50% inhibition concentration values (IC 50 , expressed as nM) in comparison to sorafenib as a reference drug (Table 1).
To a great extent, the reported results were in good agreement with that of cytotoxicity. This may clarify the possible mechanism of cytotoxic action for the designed compounds. Most of the tested compounds exhibited excellent, moderate, to weak VEGFR-2 inhibitory activities with IC 50 values ranging from 3.2 to 38.9 nM, compared to positive control sorafenib (IC 50 ¼ 3.12 nM).
Among them, compound 27a was the most potent VEGFR-2 inhibitor with an IC 50  Comparing to the lead compounds 7 (IC 50 ¼ 3.4 nM) and 8 (IC 50 ¼ 3.2 nM), it was found that the most active compounds 27a (IC 50 ¼ 3.2 nM) had comparable VEGFR-2 inhibitory activity with these lead compounds.

Cytotoxicity against primary rat hepatocytes (normal hepatic cells)
It is of high importance for anticancer agents to have minimum side effects on normal cells. To assess the selectivity of the synthesised compounds against cancer cells over normal ones, the cytotoxicity of the most active anti-proliferative compounds 27a and 30f was evaluated in vitro against primary rat hepatocytes using sorafenib as reference 80 . The two compounds 27a and 30f showed cytotoxic activity against cancer HepG2 cell line (4.5-fold and 1.5-fold, respectively) more than cytotoxic activity against normal hepatic cells in comparison to sorafenib (8-fold) ( Table 1).

Structure-activity relationship
It was noticed that the synthesised compounds were more effective against HepG2 than the MCF-7 cell line ( Figure 3). The obtained data from VEGFR-2 inhibition was highly matched with that of cytotoxicity. So that, the SAR can be built on the results of cytotoxicity and/or VEGFR-2 inhibition. SAR of the newly synthesised compounds was studied along with the pharmacophoric features outlined in the rationale of molecular design. Firstly, the effect of the flat heteroaromatic ring on biological activity was examined. Comparing the cytotoxicity and VEGFR-2 inhibitory activities of the synthesised compounds incorporating 3-methylquinoxalin-2(1H)one moiety (compounds 27a-f, 28, and 29) with that incorporating 3-methylquinoxaline-2-thiol moiety (compounds 30a-i, 31a,b, and 32), it was found that 3-methylquinoxalin-2(1H)one moiety was more valuable than methylquinoxaline-2-thiol nucleus.
In addition, the impact of pharmacophore (HBD/HBA) moiety was explored. Regarding 3-methylquinoxalin-2(1H)one derivatives, it was found that compounds comprising the amide group (compounds 27a-f) were more active than that containing diamide group (compound 28), which in turn was more active than that with hydrazide moiety (compound 29). Concerning for 3-methylquinoxaline-2-thiol series, despite maintaining the same order of activity, it was found that compounds bearing amide (compounds 30a-i), diamide (compounds 31a,b), and hydrazide (compound 32) groups showed decreased activity than that incorporating 3methylquinoxalin-2(1H)one nucleus.
Furthermore, the structure of the terminal hydrophobic tail gave wide varieties of biological activity. In cornering methylquinoxalin-2(1H)one derivatives, it was noticed that hydrophobic aliphatic moiety was critical for activity as found in the most potent member 27a containing tertiary butyl tail.
For the hydrophobic aromatic tail, the heterocyclic ring (compound 27f) displayed high activity than non-heteroaromatic ones (compounds 27a-d). Then, the effect of the substitution on the aromatic rings was examined. it was observed that the cytotoxicity and VEGFR-2 inhibitory activities were highly fluctuated by substitution with electron-withdrawing (EWG) and electron-donating (EDG)groups. The unsubstituted phenyl ring (compound 27b) markedly decreased the biological activity. Substitution with EDG as a 2,6-dimethoxy group (compound 27c) increased the cytotoxicity. Changing the substitutions to be 2,6-dimethyl groups (compound 27d) markedly decreased the activity. Additionally, it was found that the cytotoxicity decreased upon substitution with the electron-withdrawing group as in compound 27e.
Investigating the activity of compounds 30c, 30d, and 30e demonstrated that, insertion of carbon bridge between the phenyl moiety (hydrophobic tail) and the pharmacophore moiety produced an increase in activity with higher priority for a one-carbon bridge (compound 30c) over than two-carbon one (compound 30d).

Cell cycle analysis
Tissue homeostasis is tightly controlled by the balance between cell proliferation and cell death 81 . This creates a great link between the cell cycle and apoptosis 82 . Therefore manipulation of the cell cycle efficiently affected apoptotic response 83 .
Flow cytometry analysis as described by Wand et al. 84,85 was used to investigate the effect of the most active compound 27a on the cell cycle progression and apoptosis induction utilising HepG2 cell line. HepG2 cells were treated with 4.5 mM (IC 50 of compound 27a) for 24 h, then analysed for its effect on cell cycle distribution. The cell cycle parameters of the incubated cells were compared with untreated control cells (Table 2 and Figure 4).
The data of flow cytometric analysis revealed the presence of massive accumulation of the treated HepG2 cells at the G2/M (34.14%, 3.5-fold) compared to control cells (9.62%). In addition, a slight difference was observed at the S phase in the percentages of the treated cells (26.96%) and control ones (29.32%). On other    hand, the percentage of HepG2 cells increased in the sub G1 phase from 1.13% (control cell) to 1.24% (treated cell). Also, it decreased at the G1 phase from 59.93% (control cell) to 37.66% (treated cell). Such outcomes indicated that compound 27a had a high ability to hinder cell cycle progression of HepG2 cells at the G2/M phase.

Detection of apoptosis
As compound 27a produced a high accumulation of HepG2 cells at the G2/M phase, such compound was further investigated for its apoptotic effect using Annexin V and PI double staining assay 86 . In such a procedure, HepG2 cells were treated with compound 27a at a concentration of 4.5 mM and incubated for 24 h. The results revealed that compound 27a induced an increase of HepG2 cells at early (40.47%) and late (0.35%) stages of apoptosis by about five times more than the untreated cells (8.52 and 0.14%, respectively) ( Table 3 and Figure 5).

Effects on the levels of active caspase-3 and caspase-9
To analyse the effect of the most active compound 27a on protein expression levels of caspase-3 and caspase-9, Western blot analysis was utilised 87 . In this test, HepG2 cells were treated with compound 27a at its cytotoxic concentration (4.5 mM) for 24 h. The results displayed a marked increase in the level of caspase-3 (2.5-fold) and caspase-9 (3.43-fold) compared to the control cells (Table 4 and Figure 6). Such findings are consistent with previous reports declared that VEGFR-2 inhibitors can up-regulate both caspase-3 and caspase-9 to induce apoptosis 88,89 .

Effects on the levels of Bcl-2 family and BAX/Bcl-2 ratio
In this study, BAX and Bcl-2 expression levels in the HepG2 cell line after the treatment with compound 27a were determined by quantitative Western blotting. The results revealed that compound 27a significantly affected the apoptosis pathway in HepG2 cells as it increased the BAX level by 2.6-fold compared to the control  cell. Also, it decreased the Bcl-2 level by 2-fold less than the control cells. In addition, the BAX/Bcl-2 ratio was increased 5-fold in the treated cells compared to the control cell (Table 4 and Figure  6). Meanwhile, compound 27a increased BAX/Bcl-2 ratio, it could trigger apoptosis in the experienced cells 90,91 .

Molecular docking studies
Computational docking studies were carried out against two VEGFR-2 crystal structures (PDB ID; 2OH4 and 4ASD) 92 . Docking studies were performed to explore the binding mode of the synthesised compounds against the ATP binding pocket of VEGFR-2.
The docking experiments were carried out using MOE.14 software.
Sorafenib was used as a reference molecule. The binding free energies (DG) of the tested ligands and sorafenib against each protein were presented in Table 5.
At first, the co-crystallised ligands of each protein were redocked into the active pockets of VEGFR-2. The resulted RMSD values between the original co-crystallised ligands and the redocked ones were 1.04 and 1.15 Å. Such values approved the validation of docking processes (Figures 7 and 8).
Docking of sorafenib as a reference compound was performed to compare its binding mode with those of the target compounds. The proposed binding mode of the docked sorafenib was the same in the two pockets. The urea moiety bound the receptor through three hydrogen bonding interactions with the crucial amino acids; Glu883 (Glu885) and Asp1044 (Asp1046). Moreover, the N-methylpicolinamide moiety occupied the hinge region, Figure 5. Flow cytometric analysis of apoptosis in HepG2 cells exposed to compound 27a. Table 4. Effect of compound 27a on the levels of active caspases-3, active caspases-9, BAX, and Bcl-2 proteins in HepG2 cells treated for 24 h.
The results of docking studies against VEGFR-2 (PDB ID; 2OH4) showed that the tested ligands have binding modes similar to that of sorafenib against the VEGFR-2 active pocket. From each series, the most cytotoxic compound was selected to analyse its biding mode against the active pocket. Compound 27a as a representative example of 3-methylquinoxalin-2(1H)one series revealed an affinity value of À25.71 kcal/mol. The pharmacophore (amide) moiety bound to the key amino acids Asp1046 and Glu388, where the NH group formed a hydrogen bond with Glu388 while the C¼O group formed another hydrogen bond with Asp1046. Four hydrophobic interactions took place between the phenyl ring (linker) and the amino acid residues Val897, Cys1043, Val914, and Lys866 in the linker region. Furthermore, the 3-methylquinoxalin-2(1H)one moiety occupied the hinge region and was involved in five hydrophobic interactions with Leu838, Leu1033, Phe1045, and Phe916. The tert-butyl moiety occupied the allosteric pocket ( Figure 11).
The proposed binding mode of 30f as an example of 3-methylquinoxaline-2-thiol series against VEGFR-2 (PDB ID; 2OH4) was like that of sorafenib. The docking score of such a compound was À23.22 kcal/mol. Compound 30f interacted with the key amino acid Asp1046 via its hydrogen bond acceptor (C¼O) group of amide moiety, while the hydrogen bond donor (NH) group interacted with the carboxylate moiety of Glu388. Three hydrophobic interactions were observed between the spacer phenyl ring and amino acid residues Lys866, Val897, and Val914. In addition, 3methylquinoxaline-2-thiol moiety formed seven hydrophobic interactions with Phe1045, Val846, Leu838, Phe916, and Leu1033. Finally, the terminal 2-tolyl moiety occupied the allosteric biding site forming two hydrophobic interactions with Ile868 ( Figure 12).
The proposed binding mode of 28 against VEGFR-2 (PDB ID; 4ASD) was like that of sorafenib. The docking score of such a compound was À29.46 kcal/mol. Compound 28 interacted with Cys1045 via its hydrogen bond acceptor (C¼O) group of amide moiety, while the hydrogen bond donor (NH) group interacted with the carboxylate moiety of Glu885. Two hydrophobic interactions were observed between the spacer phenyl ring and amino acid residues Lys868, Phe1047. In addition, 3-methylquinoxaline moiety formed one hydrogen bond with the key amino acid Cys919 and one hydrophobic interaction with Leu840. Finally, the terminal p-nitrophenyl moiety occupied the allosteric biding site forming an extra hydrogen bond with Cys1024 ( Figure 13).

In silico ADMET study
To predict the pharmacokinetics properties of the newly synthesised compounds, computer-aided ADME studies were performed using Discovery Studio 4.0 software. Sorafenib was used as a reference drug. These studies include the assessment of certain parameters as blood-brain barrier (BBB) penetration, absorption level, aqueous solubility, CYP2D6 binding, and plasma protein binding. Predictions of ADME properties for the studied compounds were listed in Supplementary Data).  The results showed that compounds 30a-f have medium BBB diffusion levels while the rest of the compounds showed low to very low levels. Consequently, the CNS side effects were anticipated to be minimal for the majority of the synthesised compounds. Regarding, aqueous solubility, compounds 27a-c, 27e, 28, and 29 exhibited good levels, while compounds 27d, 27f, 30a-i, 31a,b, and 32 showed poor levels. With respect to absorption parameter, all compounds demonstrated good absorption levels except compounds 27e showed moderate absorption level and compounds 28, 30h, and 31b which showed poor to very poor intestinal absorption levels. Moreover, the effect on cytochrome P450 2D6 was investigated. The results showed that all the tested compounds were non-inhibitors of CYP2D6. Consequently, hepatotoxicity is not expected upon their administration. The plasma protein binding model displayed that compounds 27a, 28, 30a-c, 30g,h, 31a,b, and 32 were anticipated to bind plasma protein <90%. On the other hand, compounds 27b-f, 29, 30d-f, and 30i were expected to bind plasma protein more than 90% (Figure 14).

In silico toxicity study
The toxicity profile of the synthesised compounds was determined based on the validated and constructed models in Discovery studio 4.0 software 93,94 . This includes the prediction of certain parameters as FDA rodent carcinogenicity, carcinogenic potency TD 50 , rat maximum tolerated dose, developmental toxicity potential, rat oral LD 50 , rat chronic lowest observed adverse effect level (LOAEL), ocular irritancy, and skin irritancy. Regarding the FDA rodent carcinogenicity model compounds 27a-f, 28, 30b, 30g, and 30i were forecasted to be non-carcinogenic. For carcinogenic potency, TD 50 mouse model compounds 27a,b, 29, 30a, 30c-f, 30h, i, 31a,b, and 32 showed TD 50 values ranging from 15,600 to 225,175 mg/kg body weight, which is higher than sorafenib (14,244 mg/kg body weight), while compounds 27c-f, 28, 30b, and 30g showed carcinogenic potency TD 50 lower than that of sorafenib. With respect to the rat maximum tolerated dose model (MTD), compounds 29, 30e,f, 30i, 31a, and 32 demonstrated maximum tolerated dose with a range of 0.096 to 0.194 g/kg body weight, which is higher than that of sorafenib (0.089 g/kg body weight). In Addition, all compounds were predicted to be non-toxic against the developmental toxicity potential model except compounds 27f, 30g, and 30i. For the rat oral LD 50 model, all compounds revealed oral LD 50 values in a range of 1.523 to 19.408 g/kg body weight which is higher than that of sorafenib (0.823 g/kg body weight). For the rat chronic LOAEL model, the examined members displayed LOAEL values ranging from 0.036 to 0.376 g/kg body weight. These values are higher than sorafenib (0.005 g/kg body weight). Additionally, all the tested compounds were predicted to be mild irritants against the ocular irritancy model and non-irritant against the skin

Conclusion
New twenty quinoxaline derivatives were designed and synthesised as anticancer agents with VEGFR-2 inhibitory activity. The synthesised compounds were evaluated in vitro for their antiproliferative activities against breast cancer (MCF-7) and hepatocellular carcinoma (HepG2). Compound 27a was the most potent derivative revealing strong anti-proliferative activity against MCF-7 and HepG2 cell lines with IC 50 values of 7.7 and 4.5 mM, respectively, comparing to sorafenib (IC 50 ¼ 3.51 and 2.17 mM, respectively). In addition, compounds 28 (IC 50 ¼ 17.2 and 11.7 mM), 30f (IC 50 ¼ 18.1 and 10.7 mM), 30i (IC 50 ¼ 17.2 and 12.7 mM), and 31b (IC 50 ¼ 19.2 and 13.7 mM) demonstrated promising cytotoxicity against MCF-7 and HepG2, respectively. The results of the VEGFR-2 enzyme assay were highly correlated with that of cytotoxicity, where the most potent antiproliferative derivatives exhibited good VEGFR-2 inhibitory effects. Compounds 27a was the most potent VEGFR-2 inhibitor with an IC 50 value of 3.2 nM in comparison to sorafenib (IC 50 ¼ 3.12 nM). SAR revealed that 3-methylquinoxalin-2(1H)one moiety was more efficient than 3-methylquinoxaline-2-thiol moiety as a heterocyclic ring. The amide moiety was more advantageous as a pharmacophore than the corresponding diamide and hydrazide moieties. In addition,  the tert-butyl moiety was the most effective hydrophobic tail. Cell cycle analysis of compounds 27a revealed that such compound can arrest HepG2 cell growth at G2/M phase by 3.5-fold greater than control cell. Moreover, compound 27a induced apoptosis in HepG2 cells by five times more than the control cells. Furthermore, it increased the level of caspase-3 and caspase-9 by 2.5-folds and 3.43-fold, respectively. Also, compound 27a showed an increase in the BAX level (2.6-fold), decrease in the Bcl-2 level (2-fold), and elevation of BAX/Bcl-2 ratio (5-fold) for the control cell. The results of docking studies revealed that the proposed binding modes of the designed compounds were similar to that of sorafenib. Finally, this work presents compound 27a as a lead candidate that can be further optimised for the synthesis of a promising VEGFR 2 inhibitor.
4.1.1. General procedure for the synthesis of target compounds 27a-f, 28, and 29 A mixture of potassium salt 12 (0.5 g, 2 mmol) and appropriate intermediates 26a,d,g-j, 24b, and 25 (1 mmol) in dry DMF (15 ml) with the presence of potassium iodide (0.2 g, 1.2 mmol), was heated over a water bath for 3 h. The reaction mixture was then cooled, poured into ice-cooled water (150 ml) with continuous stirring. The solid separated was filtered, washed with water several times, then dried and crystallised from the proper solvent to afford the final target compounds 27a-f, 28, and 29, respectively.

In vitro anti-proliferative activity
The in vitro antiproliferative activities were assessed using the MTT assay protocol 78,79,95 . As shown in Supplementary Data.

In vitro VEGFR-2 enzyme inhibition assay
The synthesised compounds were estimated for their in vitro inhibition on human VEGFR-2 in HepG2 cell line; using ELISA kit 96 as described in Supplementary Data.

Flow cytometry analysis for cell cycle
Cell cycle analysis for the most potent candidate 27a has been carried out through Flow cytometric analysis as described in Supplementary Data 84,85 .

Flow cytometry analysis for apoptosis
Apoptosis analysis for compound 27a in HepG2 cells was carried out using Annexin V-FITC as shown in Supplementary Data 30,86 .