Design, Synthesis, Molecular Modeling, and Anticancer Evaluation of New VEGFR-2 Inhibitors Based on the Indolin-2-One Scaffold

A new series of indoline-2-one derivatives was designed and synthesized based on the essential pharmacophoric features of VEGFR-2 inhibitors. Anti-proliferative activities were assessed for all derivatives against breast (MCF-7) and liver (HepG2) cancer cell lines, using sunitinib as a reference agent. The most potent anti-proliferative derivatives were evaluated for their VEGFR-2 inhibition activity. The effects of the most potent inhibitor, 17a, on cell cycle, apoptosis, and expression of apoptotic markers (caspase-3&-9, BAX, and Bcl-2) were studied. Molecular modeling studies, such as docking simulations, physicochemical properties prediction, and pharmacokinetic profiling were performed. The results revealed that derivatives 5b, 10e, 10g, 15a, and 17a exhibited potent anticancer activities with IC50 values from 0.74–4.62 µM against MCF-7 cell line (sunitinib IC50 = 4.77 µM) and from 1.13–8.81 µM against HepG2 cell line (sunitinib IC50 = 2.23 µM). Furthermore, these compounds displayed potent VEGFR-2 inhibitory activities with IC50 values of 0.160, 0.358, 0.087, 0.180, and 0.078 µM, respectively (sunitinib IC50 = 0.139 µM). Cell cycle analysis demonstrated the ability of 17a to induce a cell cycle arrest of the HepG2 cells at the S phase and increase the total apoptosis by 3.5-fold. Moreover, 17a upregulated the expression levels of apoptotic markers caspase-3 and -9 by 6.9-fold and 3.7-fold, respectively. In addition, 17a increased the expression level of BAX by 2.7-fold while decreasing the expression level of Bcl-2 by 1.9-fold. The molecular docking simulations displayed enhanced binding interactions and similar placement as sunitinib inside the active pocket of VEGFR-2. The molecular modeling calculations showed that all the test compounds were in accordance with Lipinski and Veber rules for oral bioavailability and had promising drug-likeness behavior.


Introduction
The treatment of cancerous diseases represents an exceptionally challenging battle for medicinal chemists to develop potent and safe chemotherapeutic agents [1][2][3][4]. These efforts mainly aim to identify and target the various biochemical processes involved in the progression and metastasis of tumors [5,6]. Angiogenesis, a complex process that involves the growth of new blood vessels from preexisting vasculature, is essential for normal organ growth and wound healing [7,8]. However, its imbalance is involved in the pathogenesis of different disorders, including cancer, psoriasis, multiple sclerosis, diabetic neuropathy, and rheumatoid arthritis [9][10][11][12][13].

Rationale and Design of the Work
The reported virtual screening and pharmacophore modeling studies revealed that most VEGFR-2 inhibitors contain four essential pharmacophoric features [42][43][44][45]. The reported features are: (1) a flat heteroaromatic ring contains hydrogen bond acceptor center that interacts with the key Glu917 and Cys919 in the ATP binding domain (hinge region) through hydrogen bond formation [43]; (2) a central aromatic system that occupies the linker region [46]; (3) a hydrogen bond acceptor and hydrogen bond donor moiety (HBA-HBD) that fits into the DFG domain [47]; (4) a terminal hydrophobic tail placed inside the hydrophobic allosteric pocket ( Figure 2) [48,49].

Rationale and Design of the Work
The reported virtual screening and pharmacophore modeling studies revealed that most VEGFR-2 inhibitors contain four essential pharmacophoric features [42][43][44][45]. The reported features are: (1) a flat heteroaromatic ring contains hydrogen bond acceptor center that interacts with the key Glu917 and Cys919 in the ATP binding domain (hinge region) through hydrogen bond formation [43]; (2) a central aromatic system that occupies the linker region [46]; (3) a hydrogen bond acceptor and hydrogen bond donor moiety (HBA-HBD) that fits into the DFG domain [47]; (4) a terminal hydrophobic tail placed inside the hydrophobic allosteric pocket ( Figure 2) [48,49].  According to the previous findings and based on our previous work on anticancer agents generally and kinase inhibitors specifically [50][51][52], we reported the design of a new series of VEGFR-2 inhibitors based on the indoline-2-one scaffold [43] to attain more potent anticancer agents. Different bioisosteric modifications, including replacement, extension, ring closure, and expansion, were applied at the four essential pharmacophoric features to generate our candidate compounds, as illustrated in (Figure 3). The privileged indolin-2-one scaffold is regarded as one of the most promising heteroaromatic pharmacophore moieties that bind to the hinge region in the ATP active pocket of VEGFR-2 [53,54]. Therefore, the indolin-2-one nucleus was selected as the heterocyclic aromatic ring system for our target compounds. Secondly, the phenylimino moiety was chosen as the central aromatic system that occupies the linker region. Such a linker The privileged indolin-2-one scaffold is regarded as one of the most promising heteroaromatic pharmacophore moieties that bind to the hinge region in the ATP active pocket of VEGFR-2 [53,54]. Therefore, the indolin-2-one nucleus was selected as the heterocyclic aromatic ring system for our target compounds. Secondly, the phenylimino moiety was chosen as the central aromatic system that occupies the linker region. Such a linker was selected to extend the available aromatic surface for interaction through ring expansion of the pyrrole ring of sunitinib to benzene and offer a possible new site for interaction through the imino nitrogen atom. The third pharmacophoric HBA-HBD feature was realized using various moieties; ester (compounds 7a,b), thiosemicarbazide (compounds 5a,b and 17a,b), hydrazide (compounds 10a-g and 14a,b), N-(2,5-dioxopyrrolidin-1-yl)amide (compounds 12a,b), and oxadiazole ring (compounds 15a,b). Different HBA-HBD moieties were chosen to offer a wide selection of hydrogen-bond-rich groups with different geometries and variable metabolic stabilities. Lastly, the terminal hydrophobic tail was varied to be either aliphatic chains (compounds 5a,b, 7a,b, and 14a,b) or aromatic heterocyclic rings (compounds 12a,b, and  15a,b), or a substituted benzene ring with different hydrophobic, electronic, and topological groups (compounds 10a-g and 17a,b).
Therefore, in the current study and guided by preliminary docking studies, a new series of VEGFR-2 inhibitors based on the indolin-2-one scaffold was designed and synthesized in an endeavor to obtain potent anticancer agents. The growth inhibition activities for all the prepared compounds against the MCF-7 and HepG2 cancer cell lines were evaluated using sunitinib as the reference agent. The most potent anti-proliferative derivatives were tested for their VEGFR-2 inhibition activity. The molecular docking simulations were accomplished to predict the affinity and binding properties with VEGFR-2. Further biological investigations for the most potent inhibitor, 17a, on cell cycle, apoptosis, and expression of caspase-3&-9, BAX, and Bcl-2, were assessed to gain a better understanding of its apoptotic activity. Finally, in silico physicochemical properties and pharmacokinetic profiling were calculated to ensure the drug-likeness ability of the designed compounds.

Chemistry
The preparation methodologies adopted to synthesize the target compounds 3a,b-17a,b are outlined in Schemes 1-3. The structures of the final compounds were supported by various spectral and elemental analyses. The final compounds were obtained as a mixture of E and Z isomers, and the spectral data were reported for the major isomer. The preparation of the target benzhydrazidehydrazones 10a-g is described in Scheme 2. The intermediate Schiff base esters 7a,b were prepared by condensation of isatins 1a,b with benzocaine 6 in ethanol under acetic acid catalysis [57]. The hydrazinolysis of esters 7a,b with hydrazine afforded the benzohydrazide derivatives 8a,b in excellent 92-93% yield. The target hydrazones 10a-g were prepared by condensation of hydrazides 8a,b with different benzaldehyde derivatives 9a-g using catalytic glacial acetic acid [58].
The synthetic procedures followed for preparation of the target compounds 12a,b- The synthesis of the designed N-methylthiosemicarbazides 5a,b is outlined in Scheme 1. First, benzoic acid derivatives 3a,b were obtained by condensing isatins 1a,b with 4-aminobenzoic acid 2 in refluxing ethanol and catalytic glacial acetic acid [55]. Next, acids 3a,b were subjected to an EDC/HOBt assisted coupling with 4-methylthiosemicarbazide 4 to afford the desired N-methylthiosemicarbazides 5a,b in 78-82 % yield [56].
The preparation of the target benzhydrazidehydrazones 10a-g is described in Scheme 2. The intermediate Schiff base esters 7a,b were prepared by condensation of isatins 1a,b with benzocaine 6 in ethanol under acetic acid catalysis [57]. The hydrazinolysis of esters 7a,b with hydrazine afforded the benzohydrazide derivatives 8a,b in excellent 92-93% yield. The target hydrazones 10a-g were prepared by condensation of hydrazides 8a,b with different benzaldehyde derivatives 9a-g using catalytic glacial acetic acid [58].
The synthetic procedures followed for preparation of the target compounds 12a,b-17a,b are shown in Scheme 3. The cyclic imides 12a,b were synthesized by cyclodehydration of hydrazides 8a,b with phthalic anhydride 11 in glacial acetic acid under sonication at 50 • C [59]. Treatment of hydrazides 8a,b with ethyl acetoacetate 13 under the acetic acid catalysis afforded esters 14a,b in excellent 86-88% yield [60].
Next, hydrazides 8a,b were subjected to cyclization conditions with carbon disulfide and potassium hydroxide followed by acidification to furnish the oxadiazole derivatives 15a,b in 82-86% yield [61]. The N-phenylthiosemicarbazides 17a,b were prepared by reaction of benzhydrazides 8a,b with phenyl isothiocyanate 16 in refluxing ethanol for eight hours.

In Vitro Cytotoxic Activity Assay
The cytotoxic activities against breast MCF-7 and liver HepG2 cancer cell lines for all the synthesized compounds 3a,b-17a,b were evaluated by MTT-based cytotoxicity assay [62]. Sunitinib was selected as a reference standard in this study, and the results are presented as IC 50 in Table 1.
Generally, the fluorinated derivatives showed better cytotoxic activity than their unsubstituted counterparts, except for 8b, 14b, 15b, and 17b derivatives. Remarkably, it was observed that the lack of hydrophobic tail pharmacophoric feature in compounds 3a,b and 8a,b resulted in a significant drop in the cytotoxic activity, which clearly indicates the importance of this feature for efficient binding with the allosteric hydrophobic pocket within VEGFR-2. Additionally, among the series of benzohydrazidehydrazones 10a-g, compound 10g, which has the highest iLOG P (2.93), emerged as the most potent derivative against the MCF-7, which demonstrates the importance of hydrophobic interactions and the efficacy of the hydrazide group as the HBA-HBD moiety for VEGFR-2 inhibitory activity. The use of bulky cyclic phthalimide moiety as the hydrophobic tail in compounds 12a,b decreased the cytotoxic activity, which may be attributed to steric bulkiness and inability of the HBA-HBD group to freely rotate for efficient binding inside VEGFR-2. The cytotoxicity test results were promising to evaluate the most potent derivatives for in vitro VEGFR-2 inhibition.
Incorporating the pharmacophoric HBA-HBD moiety within an oxadiazole ring resulted in derivatives 15a,b, which showed promising cytotoxic activities. Oxadiazole 15a was two times more potent than sunitinib against the MCF-7 cell line and exhibited close potency against the HepG2 cell line. Additionally, utilizing the thiosemicarbazide group as the HBA-HBD moiety proved to be very efficient for cytotoxic activity. The N-methyl derivative 5b was the second most potent against the MCF-7 cell line, and the N-phenyl derivative 17a was the most potent against the HepG2 cell line. Table 1. Anti-proliferative activities of compounds 3a,b-17a,b against the MCF-7 and HepG2 cell lines.

Cell Cycle Analysis
Compound 17a showed potent cytotoxic and VEGFR-2 inhibition activities, and was selected to explore its activity on the cell cycle distribution and cell proliferation of HepG2 cells. The HepG2 cells were exposed to 17a (1.13 µM equal to its anti-proliferative IC 50 ) for 24 h, and cell cycle progression was monitored by flow cytometry; the results are reported in Table 3 [64]. The obtained data revealed that compound 17a decreased the distribution at the G0-G1 phase (42.91%) and the G2/M phase (9.07%) compared with the control (49.02 and 17.31%, respectively). In addition, the percentage of cell population increased at the S phase by 1.43-fold more than the control. These findings revealed that compound 17a induced arrest of the cell cycle of the HepG2 cells at the S phase ( Figure 4). Sorafenib was reported to induce a cell cycle arrest at the S phase and G2/M phase in HepG2 liver cancer cells [65]. The obtained data revealed that compound 17a decreased the distribution at the G0-G1 phase (42.91%) and the G2/M phase (9.07%) compared with the control (49.02 and 17.31%, respectively). In addition, the percentage of cell population increased at the S phase by 1.43-fold more than the control. These findings revealed that compound 17a induced arrest of the cell cycle of the HepG2 cells at the S phase ( Figure 4). Sorafenib was reported to induce a cell cycle arrest at the S phase and G2/M phase in HepG2 liver cancer cells [65].

Apoptosis Analysis
The HepG2 cells were treated with 17a (1.13 µM for 24 h), and the apoptotic effect was determined using Annexin V-FITC/PI assay. The results (Table 4) demonstrated that 17a enhanced total apoptosis by 24-fold compared to the control (46.38 % and 1.91 %, respectively). Additionally, 17a increased the percentage of early apoptosis compared with

Apoptosis Analysis
The HepG2 cells were treated with 17a (1.13 µM for 24 h), and the apoptotic effect was determined using Annexin V-FITC/PI assay. The results (Table 4) demonstrated that 17a enhanced total apoptosis by 24-fold compared to the control (46.38% and 1.91%, respectively). Additionally, 17a increased the percentage of early apoptosis compared with the control HepG2 cells (33.86% and 0.63%, respectively). Moreover, it increased the percentage of late apoptotic cells by 74-fold more than the control cells (from 0.17% to 7.99%). In addition, 17a enhanced the necrosis percentage 4-fold more than the control. These details suggested that compound 17a could induce the apoptotic mechanism of programed cell death in the HepG2 cell line ( Figure 5).

Caspase-3 and -9 Expression Assay
The expression of the apoptotic markers (caspase-3&-9) in the HepG2 cells treated with 17a was studied to investigate the signal transduction pathway for its apoptotic activity.
The gene expression fold change of caspase-3 and -9 in the HepG2 cells treated with 1.13 µM of compound 17a for 24 h was determined using quantitative real-time PCR analysis. The obtained results (Table 5) revealed that 17a elevated the gene expression of caspase-3 by 6.9-fold and caspase-9 by 3.7-fold more than the control HepG2 cells. The obtained data suggested that the caspase transduction pathway is involved in the apoptotic effect of compound 17a ( Figure 6).

Caspase-3 and -9 Expression Assay
The expression of the apoptotic markers (caspase-3&-9) in the HepG2 cells treated with 17a was studied to investigate the signal transduction pathway for its apoptotic activity.
The gene expression fold change of caspase-3 and -9 in the HepG2 cells treated with 1.13 µM of compound 17a for 24 h was determined using quantitative real-time PCR analysis. The obtained results (Table 5) revealed that 17a elevated the gene expression of caspase-3 by 6.9-fold and caspase-9 by 3.7-fold more than the control HepG2 cells. The obtained data suggested that the caspase transduction pathway is involved in the apoptotic effect of compound 17a (Figure 6). 1.13 µM of compound 17a for 24 h was determined using quantitative real-time PCR ana ysis. The obtained results (Table 5) revealed that 17a elevated the gene expression o caspase-3 by 6.9-fold and caspase-9 by 3.7-fold more than the control HepG2 cells. Th obtained data suggested that the caspase transduction pathway is involved in the apop totic effect of compound 17a ( Figure 6).

BAX and Bcl-2 Expression Assay
The apoptotic BAX and anti-apoptotic Bcl-2 proteins play critical roles in caspaseindependent apoptosis [66]. The ratio of the two proteins indicates the liability of a cell to be subjected to mitochondrial apoptosis [67].
The expression of BAX and Bcl-2 were evaluated in the HepG2 cells after treatment with 1.13 µM of compound 17a for 24 h. The Western blotting technique was utilized to determine the levels of Bax and Bcl-2 proteins and estimate the Bax/Bcl-2 ratio.
The results (Table 6) showed that 17a increased the expression of BAX by 2.7-fold more than the control cells. Moreover, it exhibited a pronounced decline in the expression level of Bcl-2 by 1.9-fold in comparison with the control. Additionally, compound 17a enhanced the BAX/Bcl-2 ratio by 5-fold. These outcomes signified that compound 17a induced mitochondrial apoptosis in the HepG2 cells (Figure 7). The apoptotic BAX and anti-apoptotic Bcl-2 proteins play critical roles in caspaseindependent apoptosis [66]. The ratio of the two proteins indicates the liability of a cell to be subjected to mitochondrial apoptosis [67].
The expression of BAX and Bcl-2 were evaluated in the HepG2 cells after treatment with 1.13 µM of compound 17a for 24 h. The Western blotting technique was utilized to determine the levels of Bax and Bcl-2 proteins and estimate the Bax/Bcl-2 ratio.
The results (Table 6) showed that 17a increased the expression of BAX by 2.7-fold more than the control cells. Moreover, it exhibited a pronounced decline in the expression level of Bcl-2 by 1.9-fold in comparison with the control. Additionally, compound 17a enhanced the BAX/Bcl-2 ratio by 5-fold. These outcomes signified that compound 17a induced mitochondrial apoptosis in the HepG2 cells (Figure 7).

Docking Study
Molecular docking is the most utilized virtual drug design technique when the 3D structure of the target protein is available [68]. Simulations were carried out to predict the

Docking Study
Molecular docking is the most utilized virtual drug design technique when the 3D structure of the target protein is available [68]. Simulations were carried out to predict the affinity and investigate the potential binding patterns of derivatives 3a,b-17a,b within the active pocket of VEGFR-2. A docking study was performed on VEGFR-2 tyrosine kinase co-crystallized with sunitinib (PDB: 4AGD) [69] using MOE 2020.09 computational software [70]. First, validation of the molecular docking protocol was established by redocking sunitinib in the ATP binding domain of the VEGFR-2 active pocket. Reproduction of the same binding interactions and orientation inside the active site as the co-crystallized ligand demonstrated that the applied docking setup was appropriate for the study. This was also confirmed by the small RMSD obtained (0.6797 Å) between the native ligand and the re-docked one.
Sunitinib achieved a docking score of −16.5974 kcal/mol and was interacted by the NH and C=O groups of its indolin-2-one with the Glu917 and Cys919 of hinge residues, respectively. Additionally, it showed multiple hydrophobic interactions with Leu840, Ala866, Val916, Phe918, and Leu1035 ( Figure 8).
The docking simulation of 5b in the ATP binding pocket of VEGFR-2 ( Figure 9) showed that 5b was fitted in the hinge region and the docking score was −19.0360 kcal/mol. Compound 5b formed two hydrogen bond interactions by the NH and CO groups of its indolin-2-one scaffold with Glu917 and Cys919 hinge residues, respectively. Additionally, several hydrophobic and Van der Waals interactions were observed with Leu840, Val848, Ala866, Val916, Phe918, Leu1035, and Phe 1047.
The docking pose of compound 10g into the ATP binding domain of VEGFR-2 ( Figure 9) showed two hydrogen bond interactions: isatin NH of the ligand with Glu917 and isatin C=O with Cys919. Additionally, several Van der Waals and hydrophobic contacts were observed with Val848, Ala866, Val916, Phe918, Leu1035, and Phe 1047. These interactions were reflected in the docking score of 10g (−21.2368 kcal/mol) and supported the obtained in vitro activity results.
The binding mode of compound 15a (docking score −17.2599 kcal/mol) inside VEGFR-2 is shown in Figure 10. It displayed two hydrogen bond interactions by the NH and C=O groups of its isatin ring with Glu917 and Cys919 residues, respectively. Furthermore, a dipole interaction was observed between the S atom of the oxadiazole ring and Lys920 residue. Moreover, various Van der Waals and hydrophobic contacts were observed with Leu840, Val848, Ala866, Val916, Phe918, Leu1035, and Phe 1047.
Considering the binding mode of compound 17a into the active pocket of VEGFR-2 (docking score −20.1061 kcal/mol), it formed two hydrogen bonds by the NH and C=O groups of its indolinone nucleus with Glu917 and Cys919, respectively ( Figure 10). Additionally, an arene cation interaction was noticed between the terminal benzene ring and Lys838 residue. In addition, many hydrophobic and Van der Waals interactions were regarded with Leu840, Val848, Ala866, Val899, Val916, Phe918, Leu1035, and Phe 1047. The molecular docking study revealed the ability of the tested compound to interact with the key amino acids in the ATP active site of VEGFR-2. The binding interactions and energy binding scores are in agreement with the obtained experimental in vitro anticancer and VEGFR-2 kinase inhibition activities for these compounds. The docking simulation of 5b in the ATP binding pocket of VEGFR-2 (Figu showed that 5b was fitted in the hinge region and the docking score was −19 kcal/mol. Compound 5b formed two hydrogen bond interactions by the NH an groups of its indolin-2-one scaffold with Glu917 and Cys919 hinge residues, respect Additionally, several hydrophobic and Van der Waals interactions were observed Leu840, Val848, Ala866, Val916, Phe918, Leu1035, and Phe 1047. The binding mode of compound 15a (docking score −17.2599 kcal/mol) inside VEGFR-2 is shown in Figure 10. It displayed two hydrogen bond interactions by the NH and C=O groups of its isatin ring with Glu917 and Cys919 residues, respectively. Furthermore, a dipole interaction was observed between the S atom of the oxadiazole ring and Lys920 residue. Moreover, various Van der Waals and hydrophobic contacts were observed with Leu840, Val848, Ala866, Val916, Phe918, Leu1035, and Phe 1047. Considering the binding mode of compound 17a into the active pocket of VEGFR-2 (docking score −20.1061 kcal/mol), it formed two hydrogen bonds by the NH and C=O groups of its indolinone nucleus with Glu917 and Cys919, respectively ( Figure 10). Additionally, an arene cation interaction was noticed between the terminal benzene ring and Lys838 residue. In addition, many hydrophobic and Van der Waals interactions were regarded with Leu840, Val848, Ala866, Val899, Val916, Phe918, Leu1035, and Phe 1047. The molecular docking study revealed the ability of the tested compound to interact with the key amino acids in the ATP active site of VEGFR-2. The binding interactions and energy binding scores are in agreement with the obtained experimental in vitro anticancer and VEGFR-2 kinase inhibition activities for these compounds.

Physicochemical Properties and Drug-Likeness Predictions
The estimation of physicochemical parameters and drug-likeness profiles for the designed molecules is a crucial step for the development process of new drug candidates. Several active drug candidates were rejected during clinical trials because of their poor pharmacokinetics. Accordingly, in silico tools were developed to predict physicochemical parameters and drug-likeness profiles at an early stage of drug design to save time, reduce cost, and increase chances of success [71]. The oral bioavailability of the target compounds 3a,b-17a,b were evaluated based on drug-likeness parameters of the Lipinski and the Veber rules.
The Lipinski rule of five is considered one of the highly influential rules in pharmacokinetic drug design. The Lipinski rule states that orally active molecules should have three of the following properties: molecular weight (MW) is less than 500, octanol/water partition coefficient (iLOG P) is less than five, and H-bond acceptors (HAs) and donors (HDs) are not more than ten and five, respectively [72]. The Veber rule states that compounds with a total polar surface area (TPSA) less than 140 Å 2 and rotatable bonds (RBs) less than 10 were found to have better oral bioavailability [73].
The physicochemical properties of VEGFR-2 inhibitors 3a,b-17a,b were calculated using the SwissADME online tool [74], and the key parameters are summarized in Table 7. The physicochemical properties data revealed that the MWs of the designed compounds were in the range from 266.25 to 465.27 g/mol, with 3a as the lowest and 10g as the highest, respectively. The iLOG P values varied between 0.78 and 2.93, with 8b as lowest and 10g as highest, respectively. The HDs and HAs of the molecules have been found in the range from 2 to 4 and 3 to 7, respectively. The number of RBs of the tested compounds was between 2 and 8. Compounds 7a,b had the lowest TPSA with a value of 67.76 Å 2 , while compounds 10a and 10c had the highest TPSA with a value of 128.74 Å 2 . The obtained data demonstrated that all the target compounds 3a,b-17a,b were in accordance with the Lipinski and Veber rules.
Additionally, two important pharmacokinetic parameters, passive GI absorption (HIA) and brain accessibility (BBB), of compounds 3a,b-17a,b were predicted and graphically depicted by the Brain Or IntestinaL EstimateD permeation method (BOILED-Egg). The BOILED-Egg method predicts pharmacokinetic parameters by computing the lipophilicity (WLOG P) against the TPSA, as shown in Figure 11. The molecules located in the white region have a high probability of passive gastrointestinal absorption, while the molecules located in the yellow region (yolk) have a high probability of brain access. Moreover, the blue points represent molecules predicted to be actively effluxed by the P-glycoprotein (PGP+), while the red points represent molecules that are not predicted to be actively effluxed by the P-glycoprotein (PGP−) [75]. Additionally, two important pharmacokinetic parameters, passive GI absorption (HIA) and brain accessibility (BBB), of compounds 3a,b-17a,b were predicted and graphically depicted by the Brain Or IntestinaL EstimateD permeation method (BOILED-Egg). The BOILED-Egg method predicts pharmacokinetic parameters by computing the lipophilicity (WLOG P) against the TPSA, as shown in Figure 11. The molecules located in the white region have a high probability of passive gastrointestinal absorption, while the molecules located in the yellow region (yolk) have a high probability of brain access. Moreover, the blue points represent molecules predicted to be actively effluxed by the P-glycoprotein (PGP+), while the red points represent molecules that are not predicted to be actively effluxed by the P-glycoprotein (PGP−) [75]. The results revealed that all the tested derivatives were suggested to have good intestinal absorption and bioavailability according to the Lipinski and Veber estimated parameters. Compounds 7a,b were predicted to display high blood-brain permeability and gastrointestinal absorption. All the tested compounds, with the exception of 5a,b and 8b, were predicted to be not subjected to active efflux by P-glycoprotein (red dot). Compounds 5b, 10e, 10g, 15a, and 17a could be potential candidates for drug discovery because of their potent cytotoxic activity and their good drug-likeness and pharmacokinetic properties.

Chemistry
All commercially purchased chemicals were used as received. Experiments were conducted under nitrogen or argon. Thin-layer chromatography (TLC) was performed on Merck TLC silica gel 60 F254 pre-coated aluminum sheets. Melting points were determined using Stuart electrothermal melting point apparatus and were uncorrected. Infrared spectra were recorded as KBr discs on a Thermo-912AO683 FT-IR spectrophotometer and were reported in frequency of absorption (cm -1 ). NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer (Supplemental Figures 3-19). Chemical shift (δ) values were reported in parts per million (ppm) relative to internal standard tetramethylsilane at δ 0.00 ppm. Coupling constants (J) were reported in Hertz. The spin multiplicity was abbreviated as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Elemental analysis was The results revealed that all the tested derivatives were suggested to have good intestinal absorption and bioavailability according to the Lipinski and Veber estimated parameters. Compounds 7a,b were predicted to display high blood-brain permeability and gastrointestinal absorption. All the tested compounds, with the exception of 5a,b and 8b, were predicted to be not subjected to active efflux by P-glycoprotein (red dot). Compounds 5b, 10e, 10g, 15a, and 17a could be potential candidates for drug discovery because of their potent cytotoxic activity and their good drug-likeness and pharmacokinetic properties.

Chemistry
All commercially purchased chemicals were used as received. Experiments were conducted under nitrogen or argon. Thin-layer chromatography (TLC) was performed on Merck TLC silica gel 60 F 254 pre-coated aluminum sheets. Melting points were determined using Stuart electrothermal melting point apparatus and were uncorrected. Infrared spectra were recorded as KBr discs on a Thermo-912AO683 FT-IR spectrophotometer and were reported in frequency of absorption (cm −1 ). NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer (Supplementary Figures S3-S19). Chemical shift (δ) values were reported in parts per million (ppm) relative to internal standard tetramethylsilane at δ 0.00 ppm. Coupling constants (J) were reported in Hertz. The spin multiplicity was abbreviated as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Elemental analysis was performed on AnalysenSysteme GmbH-D-63452-HANAU apparatus and a Perkin Elmer 2400 CHN elemental analyzer. The values of elemental analyses were within ± 0.4% of the theoretical values.
3.1.5. General Procedure for Synthesis of N -(4-Substitutedbenzylidene)-4-((5-substituted-2oxoindolin-3-ylidene)amino)benzohydrazide 10a-g Benzaldehyde derivatives 9a-g (1.8 mmol) were added to a solution of hydrazides 8a,b (1.8 mmol) in absolute ethanol (10 mL). Next, 0.3 mL of glacial acetic acid was added, and the reaction mixture was heated under reflux for 6 h. After cooling, the precipitate was collected, and the crude precipitate was recrystallized from methanol to give hydrazones 10a-g.  13  The anticancer activities of the target compounds 3a,b-17a,b were quantitatively assessed using the MTT protocol against the MCF-7 and HepG2 cell lines, as described in the Supplementary Materials.

Cell Cycle Analysis
The liver HepG2 cells were treated with 1.13 µM of 17a, and the effect on the cell cycle distribution was evaluated by flow cytometric analysis, as described in the Supplementary Materials.

Apoptosis Analysis
The apoptotic ability of compound 17a to the HepG2 cells was assessed using Annexin V-FITC/PI dual staining by flow cytometric analysis, as shown in the Supplementary Materials.

Caspase-3 and -9 Expression Assay
The expression of caspase-3 and -9 in the liver HepG2 cells treated with 1.13 µM of 17a was determined using quantitative real-time PCR analysis, as presented in the Supplementary Materials.

BAX and Bcl-2 Expression Assay
The expression of apoptotic BAX and antiapoptotic Bcl-2 proteins in the liver HepG2 cells treated with 1.13 µM of 17a was determined using Western blot analysis, as described in the Supplementary Materials.

Molecular Docking Study
The molecular docking simulation studies were performed on a Dell precision T3600 workstation with Intel Xeon ® CPU-1650.0 @ 3.20 GHz with Windows 7 operating system using Molecular Operating Environment software (MOE 2020.09) [70]; the docking protocol is described in the Supplementary Materials.

Physicochemical Properties and Drug Likeness Predictions
The physicochemical parameters of drug-likeness and the pharmacokinetic properties, such as gastrointestinal absorption, brain permeability, and P-glycoprotein efflux, were estimated using the SwissADME online tool (http://www.swissadme.ch/ access date 5 March 2022) for calculations [74].

Conclusions
In this investigation, a series of indoline-2-one derivatives 3a,b-17a,b were designed and synthesized based on the pharmacophore model of the reported VEGFR-2 inhibitors.
The results showed that compounds 5b, 10e, 10g, 15a, and 17a exhibited the most potent anticancer activities with IC 50 values from 0.74-4.62 µM against the breast MCF-7 cancer cell line (sunitinib IC 50 = 4.77 µM) and from 1.13-8.80 µM against the liver HepG2 cancer cell line (sunitinib IC 50 = 2.23 µM). Furthermore, these members displayed potent VEGFR-2 kinase inhibitory activities (IC 50 from 0.078-0.358 µM) compared with sunitinib (IC 50 = 0.139 µM). Moreover, the cell cycle of HepG2 cells was blocked by compound 17a at the S phase, and the total apoptosis was enhanced by 24-fold. In addition, compound 17a increased the expression of caspase-3, -9, and BAX by 6.88, 3.703, and 2.69-fold, and decreased the expression of Bcl-2 by 1.88 fold. The docking simulations demonstrated that the prepared compounds have similar interactions and orientation to sunitinib inside VEGFR-2. Finally, the molecular modeling studies showed that all the target compounds are not violating Lipinski and Veber rules for oral bioavailability and have promising drug-likeness behavior.