New antiproliferative 3-substituted oxindoles inhibiting EGFR/VEGFR-2 and tubulin polymerization

New 3-substituted oxindole derivatives were designed and synthesized as antiproliferative agents. The antiproliferative activity of compounds 6a–j was evaluated against 60 NCI cell lines. Among these tested compounds, compounds 6f and 6g showed remarkable antiproliferative activity, specifically against leukemia and breast cancer cell lines. Compound 6f was the most promising antiproliferative agent against MCF-7 (human breast cancer) with an IC50 value of 14.77 µM compared to 5-fluorouracil (5FU) (IC50 = 2.02 µM). Notably, compound 6f hampered receptor tyrosine EGFR fundamentally with an IC50 value of 1.38 µM, compared to the reference sunitinib with an IC50 value of 0.08 µM. Moreover, compound 6f afforded anti-tubulin polymerization activity with an IC50 value of 7.99 µM as an outstanding observable activity compared with the reference combretastatin A4 with an IC50 value of 2.64 µM. In silico molecular-docking results of compound 6f in the ATP-binding site of EGFR agreed with the in vitro results. Besides, the investigation of the physicochemical properties of compound 6f via the egg-boiled method clarified good lipophilicity, GIT absorption, and blood–brain barrier penetration properties. Supplementary Information The online version contains supplementary material available at 10.1007/s11030-023-10603-z.


Introduction
Cancer represents the utmost challenging disease in authority for the highest mortality rate worldwide, subsequently cardiovascular diseases [1][2][3][4].Breast cancer is a principle cause of higher cancer mortality among women globally.It was accounted that about 14% of 23% of the total breast cancer cases died [5].Numerous research studies claimed that the upregulation of various protein kinases (PKs) results in the development of metastatic breast cancers such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) [6][7][8].
Additionally, EGFR represents the furthermost critical member among the serine/threonine protein kinases.It promotes the propagation of signal transduction cascades responsible for cellular proliferation, migration, adhesion, angiogenesis, and apoptosis [18][19][20].Therefore, genetic mutations affecting EGFR activity stand among the main causes of the progression of numerous human cancers [21][22][23].In the last few years, there have been many EGFR TKIs (tyrosine kinase inhibitors) approved for the treatment of NSCLC (non-small cell lung cancer), including indolebased FDA-approved EGFR inhibitors such as torceranib (III) (Fig. 1) [24].Notably, it was found that EGFR and/ or its ligand TGF-α showed higher countenance levels that were predominantly assessed in most of the preclinical breast cancer cases and were characterized by bleak prognosis, higher resistance to various approved cytotoxic drugs (e.g., doxorubicin, 5-fluorouracil, vinblastine, and cisplatin), and being unresponsive to hormonal therapy [25].For instance, indole-pyrimidine-based derivative IV displayed circumvention activities with IC 50 values of 0.094, 0.099, and 0.595 µM against EGFR (T790M), EGFR (L858R), and c-MET, respectively (Fig. 1) [26].Moreover, indol-3-acrylamide derivative V showed highly promising antiproliferative activities.It afforded a potent hampering activity with about 22-folds selectivity versus EGFR L858R/T790M over EGFR WT  kinase with IC 50 values of 1.7 and 37 nM, respectively (Fig. 1) [27].
Dual-EGFR/tubulin polymerization inhibition represents a unique chemotherapeutic technique to restrict the unlimited proliferation of versatile cancer cell types [65,66].Therefore, designing dual-EGFR/tubulin polymerization inhibitors as antiproliferative agents is an obvious and attractive approach.For instance, 2-oxindole-based scaffold XIII acts as an efficient dual-EGFR and tubulin assembly inhibitor with IC 50 values of 0.19 μg/ml and 1.66 μM; compared with positive references, gefitinib and CA-4 with IC 50 of 0.057 μg/ml and 0.42 µM, respectively (Fig. 3) [67].

Rationale
Notably, designed compounds (6a-j) within this research work fulfills these previously mentioned four key pharmacophoric features to act as promising dual-EGFR/VEGFR-2 antagonists (oxindole ring acting as hydrophobic aromatic tail, benzylidene group acting as central spacer moiety, acetyl-oxy moiety acting as hydrogen bond acceptor and/ or donor group, and substituted phenyl ring as hydrophobic aromatic head) (Fig. 4).Also, these scaffolds achieve the presence of three rings (A, B, and C) with the seven previously mentioned pharmacophoric requirements to act as efficient tubulin polymerization antagonists (substituted phenyl ring acting as ring A, phenoxy acetyl moiety as ring B linker, and oxindole ring as ring C) (Fig. 4).Therefore, based on these previously mentioned research findings, our new 3-substituted indole derivatives (6a-j) were designed and synthesized as dual-EGFR/VEGFR-2 and tubulin polymerization inhibitors to get optimum antiproliferative activity.

Chemistry
The outline for synthesizing target compounds 6a-j is illustrated in Scheme 1. Alkylated p-hydroxy benzaldehyde derivatives 4a-c were obtained via S N 2 substitution reaction on various phenacyl bromide derivatives 2a-c using p-hydroxy benzaldehyde's nucleophilic hydroxyl group.The alkylated p-hydroxy benzaldehyde derivatives 4ac underwent a base-catalyzed Knoevenagel condensation reaction with the oxindole derivatives 5a-e to afford compounds 6a-j in a good yield [68]. NMR data supported the success of the used route. 1 H NMR of all target compounds showed characteristic singlet signals at δ: 7.58-7.92ppm for the benzylic protons; besides, NH protons were noticed as singlet signals at δ: 10.36-10.74ppm.Additional singlet signals were also All the aromatic protons and carbons belonging to the oxindole, phenacyl, and phenyl scaffolds were reliable with the proposed structures of the compounds.The ESI/MS data of compounds 6a-j appeared at their expected values via LC/MS.
Among the synthesized compounds 6a-j, compounds 6d and 6j were obtained as mixtures of the E and Z-diastereomers in different ratios, as identified with their vinylicproton and 2′ and 6′-benzylidene Hs chemical shifts (ppm) of E and Z in H NMR. The assignment of the E/Z-diastereomers was confirmed by 1 H NMR, where the vinylic protons resonated slightly deshielded in the Z-diastereomer compared to the E-diastereomer due to the influence of the carbonyl group at position 2 of the oxindole ring.Additionally, 2′ and 6′ ortho-benzylidene protons were found to be more upfield shifted in the E-diastereomers for the same reason.For example, through the 1 H NMR spectra of compound 6d, the chemical shifts of the major Z-diastereomer revealed that the signals of vinylicH at 7.77 ppm, H 4 of oxindole ring at 8.47 ppm, and H2′,6′ at 7.63-7.60ppm, while those of the minor E-diastereomer resonated at 7.66, 8.41, and 7.40-7.36ppm, respectively.We calculated the ratio of between E and Z-diastereomers for each compound depending on the total value of the integration of the vinylic-H in both for each compound.For instance, we suppose that the integration of the vinylic-H in both E and Z-diastereomers that is equal to 1 represents 100% of the existence of both E and Z-diastereomers, therefore, if the more upfield-shifted vinylic-H integration is equal to 0.3 that means the ratio of the E-diastereomer is equal to 30%, and also the more downfield-shifted vinylic-H integration is equal to 0.7 that means the ratio of the Z-diastereomer is equal to 70% [69].

Cytotoxic assays
One-dose assay of compounds 6a-j against sixty NCI cancer cell lines All novel scaffolds 6a-j were carefully selected by the National Cancer Institute (NCI), Bethesda, USA, to elucidate their cytotoxic activity versus 60 human cancer cell lines at 10 µM as a single-dose concentration after a 48 h incubation period.In relation, compounds 6c and 6h showed moderate antiproliferative activity with cell growth inhibition % (GI %) of 41.17 and 43.66 against SNB-75 cells (CNS cancer cell line) and NCI/ADR-RES (ovarian cancer cell line), respectively.Moreover, compound 6i exhibited remarkable antiproliferative activity with a GI% of 62.14 against HOP-92 (non-small cell lung cancer cell line).Regarding compound 6f, it displayed highly promising antiproliferative activities versus UO-31, K-562, MOLT-4, SR, A549/ATCC, NCI-H23, SK-MEL-5, LOX IMVI, MCF-7, and T-47D cancer cell lines with GI% values of 51.52, 72.2, Based on the results mentioned earlier about the NCI antiproliferative activities of compounds 6a-j, we could conclude that (Fig. 5).
1-The presence of Br substitution at the para position of phenacyl moiety of the synthesized 3-substituted oxindole derivatives was favorable over the unsubstituted ring, which in turn was preferred over the methoxy group in the same position for affording potent antiproliferative activity against various NCI cancer cell lines, including MCF-7 (e.g., 6f, and 6g).2-The unsubstituted oxindole ring was preferred over substituted ones.3-Regarding the order of substitution on position, five of the oxindole ring was H > Cl > F > OCH 3 .4-In relation, position six of the oxindole ring being unsubstituted was more preferred rather than being occupied by chlorine atom for optimum antiproliferative activity versus various NCI cancer cell lines, including MCF-7 Cytotoxicity versus MCF-7 cell line Compounds 6f and 6g showed the uppermost observed antiproliferative activity versus the human breast cancer MCF-7 cell lines, so they were selected to detect the concentration required to hamper 50% of cellular growth (IC 50 ) versus the MCF-7 cell lines compared to 5-fluorouracil (5FU) using the MTT assay [70].
Compound 6f showed the highest considerable antiproliferative activity, specifically against MCF-7 cell lines, with a growth inhibition percentage (GI%) of 72.61%, affording an IC 50 value of 14.77 μM; however, compound 6g displayed a GI% value of 70.03%, exhibiting an IC 50 value of 25.35 μM, compared to the reference standard 5FU with an IC 50 of 2.02 μM (as shown in Table 1).

Margin of safety assessment upon detection of cytotoxicity on human skin fibroblasts (HSF) cells
Depending on the outcome pointed out, compound 6f displayed higher cytotoxicity than compound 6g on MCF-7 cells.The safety profile of compound 6f and the reference drug 5FU were investigated against MCF-7 and HSF cells (Human Skin Fibroblasts).This compound 6f exhibited remarkable cancer selectivity (IC 50 = 14.77µM for MCF-7, IC 50 = 19.45µM for HSF; selectivity index (SI) = 1.31) comparable to the reference drug 5FU with selectivity index (SI) value of 1.70 (as shown in Table 1).These results showed that compound 6f had a relatively good safety profile on the HSF normal cells than 5FU.
Fig. 5 Structure-activity relationship of the designed compounds 6aj for more potent antiproliferative activity

Cell cycle analysis
To detect the possible mechanism displayed by compound 6f to afford its antiproliferative activity, the effect of 6f on cell cycle progression in MCF-7 cell lines was performed via DNA flow cytometric analysis.The obtained data for compound 6f are shown in Fig. 6, displaying great cell cycle profile disruption compared with untreated MCF-7 cells.Treatment of MCF-7 cells with compound 6f increased the percentage of accumulation of cells at Freq G1 (from 40.68 to 51.30%) combined with an increase in the proportion at the Freq S phase and a decrease in Freq G2/M phase, indicating that compound 6f arrested cell cycle fundamentally at G0-G1phase.

RTK kinases' circumvention
Due to the fundamental role of RTKs in cancer pathogenesis, we investigated the inhibitory efficacy of compounds 6f on three types of RTKs (EGFR, VEGFR-2 (KDR), and PDGFR_ẞ receptors using the kinase kit assay protocol [71].The outcomes illustrated that compound 6f fundamentally inhibited EGFR, VEGFR-2, and PDGFR_ẞ with an IC 50 of 1.38 ± 0.008, 5.75 ± 0.011, and 3.18 ± 0.007 µM, respectively, compared to sunitinib as a reference (IC 50 of 0.08 ± 0.005, 0.33 ± 0.006, and 0.18 ± 0.003 µM, respectively).These results, as mentioned above, demonstrated that compound 6f principally circumvented EGFR with the lowest IC 50 value detected for this scaffold; therefore, EGFR represents the most crucial target among the tested kinases' series (as shown in Table 2).

Tubulin polymerization inhibition assays
To examine the tubulin polymerization hampering activity for the desired scaffolds and legitimize the design, the most potent compound, 6f, was selected to investigate the circumvention of the tubulin polymerization process using combretastatin A4 (CA-4) as a positive control.The attained outcomes (as shown in Table 2) reflected that compound 6f

In silico studies
The in silico docking study was carried out to explain the bindings modes of all synthesized compounds (6a-j) inside the binding sites of human VEGFR-2, EGFR, and tubulin (PDB IDs: 1YWN, 4HJO, and 5LYJ, respectively).
As shown in Table 3, compounds 6a-j showed convergent binding modes with scores ranging from −7.6 to −8.9 kcal/ mol upon fitting into the VEGFR-2's binding site.Compounds 6a-e showed the same binding interactions inside the VEGFR-2's binding site, forming a common H-bond with Glu883 and Lys-866 in addition to two common hydrophobic interactions with Val-914 and Val-846.Compound 6b was the best-scoring compound in this series and the whole set of compounds with a binding score of −8.9 kcal/ mol (Fig. 7A).Compounds 6f-j showed a different common binding mode, where they established two common H-bonds with Asn-1031 and Lys-866, along with three hydrophobic interactions with Leu-1033, Val-914, and Leu-838.Both 6f and 6g were the top-scoring compounds in this series with scores of -8.6 and -8.5 kcal/mol, respectively (Fig. 7B).Compound 6f was the most active compound in the in vitro enzyme assay, and hence, its binding mode inside the VEGFR-2's binding site might be the best binding mode in terms of stability and inhibitory activity.All the best-scoring compounds shared two hydrophobic interactions with the reference inhibitors (i.e., with Leu-1033 and Leu-838).
Regarding EGFR, compounds 6a-j showed comparable binding modes inside the enzyme's binding site, with docking scores ranging from −8.0 to −10.6 kcal/mol (Table 3).Compounds 6a-e exhibited almost the same binding modes and interactions, where they established several hydrophobic interactions with Leu-834, Leu-820, Lys-721, and Val-702.The remaining compounds, 6f-j, showed a slightly different orientation making most of them able to form two H-bonds with Thr-766 and Lys-721, particularly compounds 6f and 6g (Fig. 8A).In addition, they established five common hydrophobic interactions with Leu-820, Cys-773, Leu-753, Met-742, and Val-702.The co-crystalized inhibitor (i.e., Erlotinib) together with another reference inhibitor (i.e., sunitinib) was able to establish the same hydrophobic interactions along with single H-bond with either Met-769 or Lys-721 (Fig. 8B-C).It was reported that lapatinib interacted with the inactive kinase due to the bulkiness of [(3-fluorobenzyl)oxy] moiety on its aniline side ring.This bulky moiety could not interact with the small-sized ATPbinding pocket, so it became able to interact with the allosteric-binding site, which opened through the displacement of the αC helix in the inactive conformation [72].Similarly, the bulkiness of the phenacyl-phenoxy moiety of compound 6f was unable to fit inside a narrow small-sized ATP-binding site; therefore, compound 6f interacted selectively with the inactive EGFR conformation but not with the active conformation.As a result, compound 6f was the most active derivative against EGFR among the synthesized series of compounds, indicating that its binding mode, which was convergent to the reference inhibitors, is the most enzyme inhibition-relevant one [73].Finally, docking of compounds 6a-j inside the tubulin's colchicine-binding site showed that compound 6f was the only derivative that was able to perfectly align with the cocrystalized inhibitor Combretastatin A4 with the highest docking score (-10.2 kcal/mol; Table 3) sharing the same hydrophobic interactions, in addition, two extra H-bonds with Asn-258 and Val-238 (Fig. 9).The binding mode of compound 6f explains its superior in vitro tubulin polymerization inhibition in comparison with the other derivatives.

Physicochemical, ADME, and pharmacokinetic characteristics forecast
The purchasing free easily entered Swiss ADME website supplied by the Swiss Institute of Bioinformatics (SIB) stands among the furthermost practical computational approaches to offer a worldwide assessment of the pharmacokinetics characterization and prospects of the drug-likeness property of small scaffolds [74].This was carried out to ensure that the novel synthesized scaffolds are hopeful members of physiological potency and pharmacokinetic properties.The most active antiproliferative 3-substituted oxindolebased compounds (6f and 6g) revealed a predicted logP o/w value of 4.52 and 5.18, respectively, with good hydrophilicity and good GIT absorption and blood-brain barrier (BBB) penetration.Figure 10 demonstrates a BOILED-Egg graph of the WLOGP vs. TPSA (Topological Polar Surface Area) for the submitted scaffolds 6f and 6g [75].The submitted scaffolds are placed with BBB permeability in the human intestinal absorption (HIA) sector.Furthermore, this diagram illustrates that the installed scaffolds were not P-glycoprotein substrates (PGP-) and not susceptible to the efflux framework influenced by like this kind of transporters, which is applied by many carcinogenic cell lines as an impedance technique.
The prophesied good GIT absorption of the installed scaffolds 6f and 6g is owing to their idyllic physicochemical characteristics positioned within the appropriate physicochemical characters area for oral bioavailability.Figure 11 displays the bioavailability radar chart for scaffolds 6f and 6g.The radar plotting consists of six axes for six vital properties of oral bioavailability: saturation (INSATU), flexibility (FLEX), lipophilicity (LIPO), size (SIZE), polarity (POLAR), and solubility (INSOLU) [75].The pink area is considered the range of the ideal property values.The red lines represent scaffolds 6f (Fig. 11A) and 6g (Fig. 11B), whose forecasted properties are almost entirely comprised in the pink area, demonstrating their good prophesied oral bioavailability.The Swiss ADME online website exhibited that the 3-substituted oxindole-based scaffolds 6f and 6g fulfill drug-resemblance properties well-defined by the top pharmaceutical companies: Lipinski's (Pfizer) [76], Ghose's (Amgen) [77], and Veber's (GSK) filters [78].This signposts that the novel synthesized scaffolds own auspicious pharmacokinetic characters and respectable drug-resemblance characteristics.

Conclusions
The current research work was employed to synthesize a novel series of 3-substituted oxindole derivatives 6a-j as antiproliferative agents aiming to target multiple cellular targets concurrently.The newly synthesized hybrids 6a-j  respectively.Critically, it was found that compound 6f more potent than 6g in MTT assay against MCF-7.6f can trigger cytotoxicity versus the MCF-7 cell line with an IC 50 value of 14.77 µM.This cytotoxic activity is accompanied by its ability to potently circumvent various tyrosine kinases with good IC 50 values, including EGFR, VEGFR-2, and PDGFR-ẞ.Additionally, compound 6f can circumvent tubulin polymerization with an IC 50 value of 7.99 µM compared with CA-4 with an IC 50 value of 2.63 µM, resulting in a mitotic division disturbance.Moreover, via the cell cycle arrest analysis, it was detected that compound 6f could trigger cell cycle arrest at the G0-G1phase.The molecular-docking simulation into the active site of VEGFR-2 revealed that the newly designed hybrids 6a-j showed higher binding free energies toward VEGFR-2 enzyme than the native ligand, erlotinib, and the reference sunitinib.In relation, fitting into the EGFR-binding site revealed that the newly designed hybrids 6a-j displayed higher binding free energies toward EGFR enzyme than the native ligand, erlotinib, and the reference sunitinib, depicting the most active compounds, 6f, and 6g.Eventually, fitting into the tubulin-binding site elucidated that compound 6f had the highest binding energy score among the synthesized compounds toward tubulin enzyme compared to the reference combretastatin A4, which was in good agreement with the in vitro results.Based on the biological data, the acceptable physicochemical and pharmacokinetic characteristics, and the good agreement with the molecular-docking study results propose that compound 6f can be utilized as an efficient lead compound in designing and developing novel therapeutic agents for fighting MCF-7 breast cancer.

Chemistry
All chemicals, including solid compounds and organic solvents utilized to produce the desired scaffolds, were of the commercial mark and bought from Sigma Aldrich, Alfa Aesar, and Pio-chem companies for pharmaceutical and chemical materials and handled without additional purification.Reaction monitoring was carried out using precoated TLC plates (Kiesslgel 60 F254, Merck), and spot detection was performed via UV lamb at 254 nm as a wavelength value.Measurement of melting points was carried out on Stuart Electro-Thermal apparatus and recorded without further correction. 1 HNMR was done on a JOEL JNM-ECA400 ( 1 H: 400 MHz) spectrophotometer at Zagazig University, Egypt. 13CNMR has performed on a JOEL JNMECA400 ( 13 C: 100 MHz) spectrophotometer at Zagazig University, Egypt.Chemical shifts were measured in parts per million (ppm) relative to standard TMS using DMSO (δ: 2.5) as a deuterated solvent, and the coupling constant (J) was measured in Hertz (Hz).Multiplicity was nominated as s singlet; d doublet; t triplet; q quartet; p pentet; dd doublet of doublet; and m for multiplet.Elemental micro-analysis was performed at the regional center for mycology and biotechnology, Al-Azhar University, Cairo, Egypt.LC/MS data were carried out on the LC/MS/MS (Agilent 1260 Infinity II (USA) with 6420 Triple Quad LC/MS detector) at Nawah, Mokatam, Egypt.

General procedures for the synthesis of target compounds 2a-c
A mixture of acetophenone or acetophenone derivatives (1 mmol), N-bromo succinimide (1 mmol), and p-toluene sulfonic acid (1 mmol) in acetonitrile (50 ml) were refluxed for 24 h.The solvent was removed under reduced pressure.The obtained residue was recrystallized from acetonitrile to afford the target compounds 2a-c.

General procedures for the synthesis of target compounds 4a-c
A mixture of phenacyl bromide (1 mmol), p-hydroxy benzaldehyde (122 mg, 1 mmol), and potassium carbonate (276 mg, 2 mmol) in acetonitrile (50 ml) was refluxed for 24 h.The reaction mixture is cooled to room temperature, diluted with water, and then extracted three times with ethyl acetate.After drying the organic layer with anhydrous MgSO 4 , the product was obtained as yellow powder via evaporation of the ethyl acetate layer.The obtained residue was recrystallized from acetonitrile to afford the target compounds 4a-c [82,83].

General procedures for the synthesis of target compounds 6a-j
A mixture of compounds 4a-c (1 mmol) and 2-oxindole or oxindole derivatives 5a-e (1 mmol) in absolute ethanol (50 ml) and piperidine (85 mg, 1 mmol) was refluxed for 24 h.Compounds 6a-j were formed on hot, filtrated, washed several times with hot ethanol, and dried in a vacuum oven to afford compounds 6a-j [68].

Margin of safety assessment upon detection of cytotoxicity on human skin fibroblasts (HSF) cells
Cell viability was assessed via SRB assay.Aliquots of 100 µl cell suspension (5 × 10 3 cells) were in 96-well plates and incubated in a complete media for 24 h.Cells were treated with another aliquot of 100 µl media containing compound 6f at various concentrations (up to IC 50 value against MCF-7 is equal to 19.43 µM).After 72 h of drug exposure, cells were fixed by replacing media with 150 µl of 10% TCA and incubated at 4 °C for 1 h.The TCA solution was removed, and the cells were washed 5 times with distilled water.Aliquots of 7 µl SRB solution (0.4 w/v) were added and incubated in a dark place at room temperature for 10 min.Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight.Then, 150 µl of TRIS (10 mM) was added to dissolve the protein-bound SRB stain; the absorbance was measured at 540 nm using a BMG LAB TECH®-FLUO star Omega microplate reader (Ortenberg, Germany) [85,86].

Cell cycle arrest analysis
MCF-7 Cells were placed in a six-well plate at 1 × 10 5 concentration of cells/well and then kept for incubation for 24 h.MCF-7 Cells were treated with compound 6f (19.43 μM) for 24 h.Cells were then collected and fixed for 12 h using icecold 70% ethanol at 4 °C.Ethanol was then removed, and cells were washed with cold phosphate buffer saline (PBS, 0.5 ml) and preserved for 30 min at 37 °C.The cells were stained for 30 min with propidium iodide in the dark.A flow cytometer was used to detect DNA content [87].

RTK kinases' circumvention
The receptor tyrosine kinases, namely EGFR, VEGFR-2, and PDGFR_ẞ Kinases Assay Kits, are designed to measure EGFR, VEGFR-2, and PDGFR_ẞ Kinases' activities for screening and profiling applications using Kinase-Glo® MAX as a detection reagent [70].The EGFR, VEGFR-2, and PDGFR_ẞ Kinases Assay Kits come in a convenient 96-well format, with enough purified recombinant EGFR enzyme, EGFR substrate, ATP, and kinase assay buffer for 100 enzyme reactions.The master mixture (25 µl per well) was equipped out of Kinase assay buffer (6 µl), ATP (500 µM, 1 µl), PTK substrate (50x, 1 µl), and water (17 µl) and transferred into each well.The inhibitor solution was made by dissolving compound 6f in a "liquid labeled" "inhibitor buffer."An inhibitor solution (5 µl) was added to each well, labeled "Test Inhibitor."For "Positive Control" and "Blank," add 5 µl inhibitor buffer).EGFR, VEGFR-2, and PDGFR_ẞ Kinases were softened on ice.Its tube was spun briefly to recover its full content of the tube.These enzymes were diluted with 1 × Kinase assay buffer to 1 ng/ µl.The reaction was initiated by adding 20 µl of diluted EGFR, VEGFR-2, and PDGFR_ẞ enzymes to the wells designated "Positive Control" and "Test Inhibitor Control."The plate was incubated at 30 °C for 40 min.After the 40-min reaction, the Kinase-Glo Max (50 µl) reagent was added to each well.The plate was covered with aluminum foil and incubated at room temperature for 15 min, and the luminescence was measured using the microplate reader.

Docking studies
The new designed compounds (i.e., 6a-j) were drawn and docked in the active site of VEGFR-2, EGFR, and tubulin enzymes (PDB IDs: 1YWN, 4HJO, and 5LYJ, respectively) using Auto Dock Vina software.4.2 program as reported in the literature.

Fig. 6
Fig. 6 Cell cycle arrest analysis of A compound 6f (cells treated with IC 50 of compound 6f), and B DMSO as control

Fig. 7
Fig. 7 Binding modes of compounds 6b (A), 6f, and 6g (B; brick red-and cyan-colored structures) together with two reference inhibitors, Sunitinib and Erlotinib (C and D, respectively) inside the binding site of the kinase domain of human VEGFR (PDB ID: 1YWN)

Fig. 8 Fig. 9
Fig. 8 Binding modes of compounds 6f and 6g (A; brick red-and cyan-colored structures, respectively) together with two reference inhibitors, Sunitinib and Erlotinib (B and C, respectively) inside the binding site of the kinase domain of human EGFR (PDB ID: 4HJO)

Table 2 The
value of 7.99 ± 0.49 µM, compared to the reference CA-4 (IC 50 = 2.64 ± 0.16 µM).Consequently, one of the proposed mechanisms for affording the antiproliferative activity of compound 6f is the circumvention of tubulin Polymerization.

Table 3
Molecular modeling results for the synthesized compounds

6a-j, sunitinib, and erlotinib at
the active binding site of VEGFR-2 protein kinase, EGFR protein kinase, and tubulin enzyme (PDB