New benzimidazole‐oxadiazole derivatives as potent VEGFR‐2 inhibitors: Synthesis, anticancer evaluation, and docking study

We report herein, the design and synthesis of benzimidazole‐oxadiazole derivatives as new inhibitors for vascular endothelial growth factor receptor‐2 (VEGFR‐2). The designed members were assessed for their in vitro anticancer activity against three cancer cell lines and two normal cell lines; A549, MCF‐7, PANC‐1, hTERT‐HPNE and CCD‐19Lu. Compounds 4c and 4d were found to be the most effective compounds against three cancer cell lines. Compounds 4c and 4d were then tested for their in vitro VEGFR‐2 inhibitory activity, safety profiles, and selectivity indices using the normal hTERT‐HPNE and CCD‐19Lu cell lines. It was determined that compound 4c was the most effective and safe member of the produced chemical family. Vascular endothelial growth factor A (VEGFA) immunolocalizations of compounds 4c and 4d were evaluated relative to control by VEGFA immunofluorescence staining. Compounds 4c and 4d inhibited VEGFR‐2 enzyme with half‐maximal inhibitory concentration values of 0.475 ± 0.021 and 0.618 ± 0.028 µM, respectively. Molecular docking of the target compounds was carried out in the active site of VEGFR‐2 (Protein Data Bank: 4ASD).

The regulation of vital cellular functions as growth, differentiation, survival, metabolism, and proliferation is greatly influenced by these proteins (El-Metwally et al., 2023).Angiogenesis is essential for the survival and spread of tumor cells, which is a primary cause of cancerrelated deaths.As a result, the importance of angiogenesis inhibition in cancer treatment is growing (Ghannam et al., 2023).Signal transduction networks initiated by vascular endothelial growth factor A/vascular endothelial growth factor receptor-2 (VEGFA/VEGFR2), the most prominent ligand-receptor complex in the VEGF system, leads to endothelial cell proliferation, migration, survival, and new vessel formation involved in angiogenesis (Aparna et al., 2023).VEGFR-2 is a well-known member of the receptor tyrosine kinases family that directly controls the angiogenesis, cell adhesion, migration, and proliferation of endothelial cells (Eissa et al., 2023b;Elkady, El-Dardir, et al., 2023).
Benzimidazole moiety plays a critical role in a number of kinase inhibitors that are undergoing clinical trials, such as dovitinib (phase III), AT-9283 (phase II), and lifirafenib (phase I).In fact, this core has been regarded as a special segment for blocking ATP-dependent kinases.In addition, benzimidazole is regarded as a bioisostere of purine bases that are found naturally, making it suitable for substituting the ATP adenine base in the adenine pocket (Elkady, Abuelkhir, et al., 2023).
In this study, building on our earlier studies and attempts to create new heterocyclic compounds with diverse bioactive scaffolds that have effective anticancer properties, the current study relies on the hybridization of oxadiazole and benzimidazole scaffolds, two wellknown physiologically active entities (Acar Çevik et al., 2020(Acar Çevik et al., , 2023)).All the target compounds were synthesized and their antitumor activity against the human lung adenocarcinoma cell line (A549), the human breast cancer cell line (MCF-7), and the human pancreas cell line (PANC-1) were evaluated in vitro, comparing with cisplatin as a positive control drug.A molecular docking study was conducted to determine how the produced chemicals bound to the VEGFR-2 tyrosine kinase.

| Chemistry
In this study, nine new compounds bearing the benzimidazoleoxadiazole structure were synthesized and their structures were elucidated by spectroscopic methods ( 1 H nuclear magnetic resonance [NMR], 13 C NMR, and high-resolution mass spectrometry [HRMS]).
Synthesis of the target compounds is outlined in Figure 2.
In the first step of the synthesis studies, 4-substituted benzaldehyde and sodium metabisulfide were reacted in dimethylformamide F I G U R E 1 Food and Drug Administrationapproved vascular endothelial growth factor receptor-2 inhibitors.
under microwave irradiation, and as a result of the condensation reaction of the resulting benzaldehyde sodium metabisulfite adduct and 3,4-diamino benzoate under microwave irradiation, compound 1 derivative was obtained.In the next step, compound 1 was treated with hydrazine hydrate under microwave irradiation to obtain The synthesized hydrazide derivative (2) was dissolved in ethanol, NaOH, and CS 2 were added and the oxadiazole ring was obtained.In the last step, compound 3 and phenacylbromide derivatives were reacted in acetone to reach the target products.At the end of the reaction, acetone was evaporated and the precipitated product was washed with water.

| Anticancer activity
To determine the half-maximal inhibitory concentration (IC 50 ) values of the compounds identified, the MTT test was run on two different normal cell lines (hTERT-HPNE, CCD-19Lu) and three distinct cancer cell lines (PANC-1, A549, MCF-7).The findings of the cytotoxicity are displayed in Table 1.Cisplatin was used as the reference drug.
Compounds 4c and 4d were found to be more effective than the reference drug with IC 50 values of 5.079 and 3.608 µM against the PANC-1 cell line.Again, compounds 4c and 4d were found to be more effective than the reference drug against the A549 cell line.
When the effects of the compounds against the MCF-7 cell line were F I G U R E 2 Synthesis procedure for obtaining target compounds (4a-4ı).evaluated, compound 4c and compound 4d were found to be the most effective compounds in the series.
Benzimidazole and oxadiazole rings are shared by all of the compounds, as can be seen by looking at their structures.The compounds were derived from the ortho, meta, and para positions of the phenyl ring on the oxadiazole side.When the structure−activity relationships are examined, it is seen that the chlorine and fluorine substituents at the para position of the phenyl ring increases the activity.

| Selectivity index (SI)
By comparing the compounds' cytotoxic effect against tumor cell lines and normal cell lines, the drug safety parameter for the anticancer activity of the compounds was evaluated, thereby confirming the cytoprotective qualities of the compounds.The normal human lung (CCD-19Lu) and normal human pancreas (hTERT-HPNE) lines were used as a control in this study.Table 2 shows the calculated SI for the tested compounds by scaling its IC 50 value against various tumor cell lines and IC 50 value against a normal cell line.When the SI value of a compound is >10 then it is considered a selective anticancer agent (Taghour et al., 2022).
The maximum selectivity index value was recorded for compound 4c (19.11) against PANC-1 cell line.Thus, it was determined that compound 4c was the most effective and safe member of the produced chemical family.

| VEGFA immunofluorescent staining
An endogenous angiogenic growth factor called VEGFA primarily promotes the migration and proliferation of vascular endothelial cells (Wu et al., 2024).Fluorescent staining degrees of MCF7 cancer cell line marked with anti-VEGFA antibody and immunofluorescence technique were compared under light microscopy.
Accordingly, compounds 4c and 4d were applied to the cell lines and kept for 24 h.VEGFA immunolocalization of compounds 4c and 4d in MCF7 breast cancer cells was compared with control.
Accordingly, while the highest VEGFA immunolocalization was observed in the control group, less VEGFA immunolocalization was observed in the compounds 4c and 4d.Five different areas were selected for each group.All cell nuclei seen in selected areas are compared with those stained VEGFA positive.When evaluated as a percentage, much less VEGFA immunolocalization was observed for compounds 4c and 4d compared to the control.VEGFA immunofluorescent staining images of compounds 4c and 4d are shown in Figure 3.

| Assessment of VEGFR-2 inhibition
Compounds 4c and 4d were evaluated for their VEGFR-2 inhibitory activity.Sorafenib (the reference drug) produced IC 50 value of Table 3.According to the results obtained, compounds 4c and 4d showed VEGFR-2 inhibition effects close to the reference drug sorafenib, with values of 0.475 ± 0.021 and 0.618 ± 0.028 μM, respectively.

| Molecular docking
In the presented molecular docking study, Figure 4 illustrates the binding pose of compounds 4c and 4d to the VEGFR-2 active site.
Both compounds show substantial interaction with the active site of VEGFR-2, with compound 4c demonstrating a slightly stronger binding affinity based on the docking scores.The presence of multiple hydrogen bonds with distances within the optimal range for strong hydrogen bonding is indicative of a robust interaction with the receptor.
The π-cation interaction observed for compound 4d is a distinctive aspect that could contribute to its selective binding properties.In comparison to sorafenib with a docking score of −12.09 kcal/mol, compounds 4c and 4d exhibit competitive binding affinities.Sorafenib's binding is characterized by a network of hydrophobic interactions and hydrogen bonds, along with a halogen bond to I1044 (2.99 Å), underscoring its strong and specific interaction with VEGFR-2 (Anwer et al., 2023).
Finally, sorafenib is redocked to the VEGFR-2 active site for validation, and low RMSD is measured as 0.11 Å between the natural pose and docking pose (El-Gaby et al., 2024; Mohamed et al., 2024).
) or Merck Chemicals (Merck KGaA).Melting points of the obtained compounds were determined by MP90 digital melting point apparatus (Mettler Toledo) and were uncorrected. 1H NMR and 13 C NMR spectra of the synthesized compounds were performed by a Bruker 300 and 75 MHz digital FT-NMR spectrometer (Bruker Bioscience) in dimethyl sulphoxide (DMSO)-d 6 , respectively.Splitting patterns were designated as follows: s: singlet; d: doublet; t: triplet; m: multiplet in the NMR spectra.Coupling constants (J) were reported as Hertz.All reactions were monitored by thinlayer chromatography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA).

F I G U R E 3
Abbreviation: IC 50 , half-maximal inhibitory concentration.
T A B L E 2 a SI = cytotoxicity against hTERT-HPNE cells/cytotoxicity against PANC-1 cell line.b SI = cytotoxicity against CCD-19Lu cells/cytotoxicity against A549 cell line.