1. INTRODUCTION
Cancer is characterized by the uncontrolled proliferation of abnormal cells that invade surrounding tissues and metastasize to distant organs [1]. According to the World Health Organization, it is the second leading cause of death worldwide, responsible for one in six deaths [2]. This global burden underscores the urgent need for novel anticancer agents with greater potency and reduced side effects.
1,3,4-Oxadiazole is a five-membered heterocyclic ring containing one oxygen and two nitrogen atoms at positions 1, 3, and 4, respectively [3]. It serves as an ideal scaffold for innovative drug development owing to its thermodynamic stability, ease of synthesis, and distinctive bioisosteric characteristics [4]. The oxadiazole nucleus acts as a bioisostere of amides and esters, enhancing receptor binding, metabolic stability, and pharmacokinetic behavior. With its broad pharmacological potential and diverse biological activities, 1,3,4-oxadiazole has become a privileged structure in modern medicinal chemistry [5].
Vascular endothelial growth factor receptor II (VEGFR-II) is a validated therapeutic target in oncology because of its central role in angiogenesis, the process by which tumors establish new blood vessels to sustain growth and metastasis [6]. Overexpression of VEGFR-II correlates with increased tumor vascularization and poor prognosis in breast and ovarian cancers [7]. Therefore, VEGFR-II inhibition represents a promising strategy for developing anti-angiogenic and anticancer therapies [8].
In this study, we designed and synthesized a novel series of 1,3,4-oxadiazole derivatives incorporating the bis (2-chloroethyl)amino pharmacophore, a well-known alkylating group with established cytotoxic potential [9]. The design rationale was to merge the hydrogen-bond-accepting and π-stacking capacity of the oxadiazole ring, which is ideal for anchoring within the Adenosine triphosphate-binding pocket of VEGFR-II, with the electrophilic bis (2-chloroethyl) amino moiety capable of interacting covalently or semi-covalently with nucleophilic residues such as cysteine or lysine. This dual interaction pattern was expected to improve VEGFR-II binding affinity and inhibitory persistence compared with conventional reversible inhibitors such as sorafenib. The present work, therefore, reports the synthesis, structural characterization, molecular docking, Absorption, Distribution, Metabolism, and Excretion prediction, in vitro cytotoxicity, molecular dynamics (MD) simulation, and anti-angiogenic evaluation of these novel hybrids, aiming to assess their potential as new VEGFR-II inhibitor scaffolds.
2. MATERIALS AND METHODS
Oxadiazole derivatives were synthesised using different aromatic carboxylic acids as starting materials. The required chemicals were procured from Sigma-Aldrich Chemicals Private Limited, 12, Bommasandra Jigani Link Road, Industrial Area, Anekal Taluk, Bangalore, Karnataka, India. Structural characterization was done by techniques such as IR spectroscopy (BRUKER, model Alpha II), H1 nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz Bruker Advanced II spectrometer using tetra methyl silane as internal standard and Dimethyl sulfoxide as solvent. Mass spectroscopy was done in Agilent-infinity 1290. All spectral studies were carried out in Green Med Labs, Chennai.
We employed grid-based ligand docking with the energetics tool of the Schrodinger Suite-Maestro 12.5 for molecular docking studies to predict ligand-receptor interactions. MD simulations were performed using GROMACS. ADME studies of the synthesised compounds were done with the SwissADME web-based tool. In vitro anticancer evaluation was done by MTT assay. Anti-angiogenic activity was evaluated using the chorioallantoic membrane (CAM) assay.
2.1. Chemistry
The synthetic pathway for the preparation of the target 1,3,4-oxadiazole derivatives is outlined in Scheme 1. The synthesis proceeded through four consecutive steps: (i) formation of benzohydrazide, (ii) cyclization to 5-aryl-1,3,4-oxadiazole-2-thiol, (iii) thioesterification with chloroacetyl chloride, and (iv) final amination with bis(2-chloroethyl)amine to yield the target compounds (1D–8D).
2.1.1. Procedure for synthesis of benzohydrazide
Substituted aromatic acids (1.0?g, ~8.0?mmol) were dissolved in ethanol (20?ml), and concentrated sulfuric acid (1?ml) was added dropwise with stirring. The reaction mixtures were refluxed for 3?hours to afford the corresponding ethyl esters. Upon completion, the mixtures were cooled and neutralized with saturated sodium bicarbonate solution. The precipitated products were filtered and washed with cold ethanol–water (1:1) to yield pure esters as white solids. The resulting esters (0.5?g, ~3.3?mmol) were then refluxed with excess hydrazine hydrate (1.0?g, ~10?mmol) in ethanol (50?ml) for 2?hours. After completion, the solvents were removed under reduced pressure, and the crude products were extracted with ethyl acetate and water. The organic layers were dried over anhydrous sodium sulphate and concentrated to obtain the corresponding aromatic benzohydrazide derivative [10].
2.1.2. Synthesis of 5-Aryl-1,3,4-oxadiazole-2-thiol
Aromatic benzohydrazide derivatives (1.0?g, ~7.3?mmol) were dissolved in absolute ethanol (12?ml) in a 250?ml round-bottom flask. To this solution, potassium hydroxide (0.42?g, 7.3?mmol) was added, and stirring was continued until complete dissolution. Carbon disulfide (2.32?ml, 29?mmol) was then added dropwise, and the reaction mixture was refluxed with vigorous stirring for 6?hours. The evolution of hydrogen sulphide gas was observed during the reaction, indicating progress.
Upon completion, the reaction mixture was cooled, diluted with distilled water, and acidified to pH?2–3 using concentrated hydrochloric acid. The precipitated products were collected by filtration, washed thoroughly with water, and purified by recrystallization from ethanol to yield the desired 5-aryl-1,3,4-oxadiazole-2-thiol derivatives [11].
2.1.3. Synthesis of S-(5-aryl-1,3,4-oxadiazol-2-yl) 2-chloroethanethioate
In a 50?ml round-bottom flask, the corresponding 5-aryl-1,3,4-oxadiazole-2-thiol derivative (0.2?g, ~1.1?mmol) was dissolved in 10?ml of dry Dimethylformamide. Potassium carbonate (0.775?g, 5.6?mmol) was added as a base, and the mixture was stirred at room temperature for 30 minutes to ensure deprotonation. Chloroacetyl chloride (0.506?g, 4.4?mmol) was then added dropwise, and stirring was continued for an additional 3?hours. The progress of the reaction was monitored using thin-layer chromatography (TLC) on silica gel 60 F254 plates. Upon completion, the reaction mixture was poured into ice-cold water to induce precipitation. The resulting solid was filtered, washed with water, and dried under vacuum to yield the corresponding S-(5-aryl-1,3,4-oxadiazol-2-yl) 2-chloroethanethioate derivative [12].
2.1.4. Synthesis of S-(5-aryl-1,3,4-oxadiazol-2-yl) 2-(bis(2-chloroethyl)amino) ethanethioate derivatives
In a round-bottom flask, the appropriate S-(5-aryl-1,3,4-oxadiazol-2-yl) 2-chloroethanethioate derivative (0.0007?mol, ~0.2?g) was dissolved in ethanol, and potassium hydroxide (0.094?g, 0.002?mol) was added as a base. To this mixture, bis(2-chloroethyl)amine (0.00015?mol, 0.280?ml) was added dropwise under continuous stirring. The reaction was maintained at room temperature for 48 hours. Reaction completion was monitored using TLC on silica gel 60 F254 plates. Spots were visualized under UV light at 254 nm. The main solvent systems used are hexane: ethyl acetate (Hex:EtOAc) for 1D, 2D, 3D, 4D, 6D, and 7D, and Dichloromethane: methanol (DCM:MeOH) for 5D and 8D with base ammonium hydroxide. The solvent systems and Rf values are summarized in Table 1.
Table 1. TLC data of final oxadiazole derivatives (1D–8D) along with observed Rf values.
| Compound | Solvent (v/v) | Additive | Rf value |
|---|---|---|---|
| 1D | Hex:EtOAc 7:3 | — | 0.50 ± 0.05 |
| 2D | Hex:EtOAc 7:3 | — | 0.58 ± 0.05 |
| 3D | Hex:EtOAc 6:4 | — | 0.43 ± 0.05 |
| 4D | Hex:EtOAc 7:3 | — | 0.52 ± 0.05 |
| 5D | DCM:MeOH 95:5 | +0.5% NH4OH | 0.48 ± 0.07 |
| 6D | Hex:EtOAc 7:3 | — | 0.62 ± 0.05 |
| 7D | Hex:EtOAc 7:3 | — | 0.61 ± 0.05 |
| 8D | DCM:MeOH 95:5 | +0.5% NH4OH | 0.45 ± 0.07 |
All TLCs were performed on silica gel 60 F254 plates and visualized under UV 254 nm. Rf values are reported as mean ± SD. Additives were used for basic compounds to reduce tailing.
Upon completion, distilled water was added, resulting in the precipitation of the target S-(5-aryl-1,3,4-oxadiazol-2-yl) 2-(bis(2-chloroethyl) amino) ethanethioate derivatives. The solids were filtered, washed with water, and dried. The overall synthesis of compounds (1D–8D) proceeded efficiently across all substituents. Stepwise percentage yields for each reaction stage are presented in Table 2.
Table 2. Percentage yields of intermediates and final compounds obtained in each step of the synthesis.
| Starting acid | Step 1 – Hydrazide (%) | Step 2 – Thiol (%) | Step 3 – Thioester (%) | Step 4 – Final (%) |
|---|---|---|---|---|
| Benzoic acid | 91.3 | 47.7 | 96.6 | 67.6 |
| 4-Methyl benzoic acid | 96.9 | 44.2 | 81.4 | 65.1 |
| 4-Methoxy benzoic acid | 87.6 | 40.8 | 86.4 | 62.4 |
| 4-Fluoro benzoic acid | 94.4 | 43.3 | 80.2 | 64.4 |
| Pyridine-3-carboxylic acid | 79.6 | 47.4 | 71.3 | 67.5 |
| 4-Chloro benzoic acid | 88.5 | 41.6 | 63.1 | 61.8 |
| 2-Chloro benzoic acid | 83.2 | 39.2 | 88.4 | 61.8 |
| 4-Amino benzoic acid (PABA) | 96.3 | 44.0 | 71.0 | 64.9 |
The structural elucidation of the formed derivatives was performed using spectroscopic methods Fourier-transform infrared spectroscopy, mass spectrometry (FTIR, NMR, and MS).
2.2. In silico analysis
The in-silico analysis done on the synthesized molecules was molecular docking, ADME studies, and MD simulation.
2.2.1. Docking study
Molecular docking was performed using the Glide module of the Schrödinger Suite (Maestro v12.5) to predict the binding affinities and interactions of the synthesized oxadiazole derivatives with the VEGFR-2 kinase domain. The X-ray crystal structure of VEGFR-2 complexed with a 2-anilino-5-aryl-oxazole inhibitor (PDB ID: 1Y6A, resolution 2.0 Å) was obtained from the RCSB Protein Data Bank. This structure was selected because it represents the biologically active conformation of VEGFR-2 and includes the canonical ATP-binding cleft.
The protein was prepared using the Protein Preparation Wizard, which corrected bond orders, assigned proper protonation states, optimized hydrogen bonding, and minimized the structure with the OPLS4 force field to relieve steric clashes. The potential binding sites were characterized using the SiteMap tool, which identified a deep hydrophobic pocket surrounded by hydrogen-bond donor (HBD) and acceptor regions corresponding to the hinge region (Cys917–Glu915) and adjacent hydrophobic residues (Val914, Leu838, and Phe1045). Based on this analysis, the receptor grid was centered on the coordinates of the co-crystallized ligand to encompass the hinge and hydrophobic back pocket that define the ATP-binding site.
All synthesized 1,3,4-oxadiazole derivatives (1D–8D) were drawn using ChemSketch, converted to three-dimensional structures, and energy-minimized with LigPrep under the OPLS4 force field. Multiple low-energy conformations and ionization states were generated at physiological pH (7.0 ± 0.5). Docking was carried out using Glide Extra Precision (XP) mode, and the resulting GlideScore values were used to estimate relative binding affinities. Sorafenib, a clinically validated multikinase VEGFR-2 inhibitor, was docked under identical conditions for comparative analysis.
The docking protocol was validated by re-docking the native co-crystallized ligand from 1Y6A, which reproduced the experimental binding orientation and conserved the key hinge-region hydrogen bonds with Cys917 and Glu915, confirming the reliability of the docking parameters (Figs. 1–3). Visualization and analysis of hydrogen-bonding, halogen, and hydrophobic interactions were performed using the 2D Ligand Interaction Diagram and Pose Viewer modules of Glide [13].
 | Figure 1. 2D ligand–protein interaction diagram of VEGFR-2 kinase domain (PDB ID: 1Y6A) showing key interactions between the docked compound and active-site residues. [Click here to view] |
 | Figure 2. 2D interaction diagram of VEGFR-2 kinase domain (PDB ID: 1Y6A) after redocking, illustrating the binding mode of the ligand within the active site. [Click here to view] |
 | Figure 3. 3D interaction diagram of VEGFR-2 kinase domain (PDB ID: 1Y6A) showing the docked ligand within the active site, confirming proper orientation with the sitemap region. The ligand (green) fits well within the hydrophobic pocket (yellow surface) and forms key hydrogen bond interactions (red and blue surfaces) with surrounding amino acid residues, validating its stable binding conformation and complementarity with the receptor’s active site topology. [Click here to view] |
2.2.2. ADME and physico-chemical properties
ADME and physicochemical properties of the synthesized ligands were determined using Swiss ADME software, which is a free, web-based tool developed by the Swiss Institute of Bioinformatics that helps predict the pharmacokinetic properties of small molecules, especially their ADME characteristics [14]. The ADME properties evaluated are Gastrointestinal absorption, BBB permeability, glycoprotein substrate identification, and Cytochrome P450 inhibition. The physicochemical properties assessed include molecular weight, lipophilicity (LogP), topological polar surface area (TPSA), number of HBD and acceptors (HBA), and rotatable bonds. All these parameters play a role in predicting oral bioavailability. Additionally, drug likeness was assessed using Lipinski’s Rule of 5 and the Veber rule, along with bioavailability score and boiled-egg model prediction for passive absorption.
2.2.3. MD Simulation studies
Based on the in vitro biological evaluation, four compounds (1D, 2D, 3D, and 4D) were selected for MD simulation studies. The MD simulation studies were conducted using the GROMACS package for 100 ns, employing the AMBER99SB-ILDN force field [15]. The system was solvated with TIP3P water, energy minimized, equilibrated under onstant Number of particles, Volume, Temperature / Constant Number of particles, Pressure, Temperature conditions, and analysed for stability indicators such as Root Mean Square Deviation, Root Mean Square Fluctuation, radius of gyration (Rg), hydrogen bonding, and potential energy fluctuations. The simulation results were visualized using PyMol and plotted using MATPLOTLIB to evaluate system stability, compactness, flexibility, and ligand-protein interactions over time [16].
2.3. In vitro antiproliferative evaluation by MTT assay
The antiproliferative activity of the synthesized compounds was evaluated using the MTT assay on human cancer cell lines: MCF-7 (breast adenocarcinoma), PA-1 (ovarian teratocarcinoma), and A-549 (lung carcinoma). HEK293 (human embryonic kidney 293) cells were used as a representative normal human cell line to evaluate the cytotoxic selectivity of the synthesized compounds. Cells were obtained from the National Centre for Cell Science, Pune, and maintained in appropriate culture medium (DMEM or RPMI-1640, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a humidified incubator with 5% CO2. Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and allowed to adhere overnight. The next day, cells were treated with various concentrations (6.5, 12, 25, 50, and 100 µg/ml) of the test compounds. Sorafenib was used as the standard reference drug. After 48 hours of incubation, 20 µl of MTT solution [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well, followed by incubation for 4 hours at 37°C. Subsequently, the medium was carefully removed, and 100 µl of DMSO was added to each well to solubilize the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. Percentage cell viability was calculated [17].
All experiments were performed in triplicate and repeated at least three times. Results are expressed as mean ± SD. IC50values were determined using non-linear regression analysis (four-parameter logistic model) in GraphPad Prism 9. Statistical significance of differences between groups was evaluated using one-way ANOVA followed by Tukey’s post hoc test, with p < 0.05 considered statistically significant.
2.4. Chick embryo CAM assay
CAM assay is a method to study angiogenesis. As the derivatives are VEGFR II inhibitors, this method was used to analyse their anti-angiogenic properties. The derivative that showed the best activity in the MTT assay (compound 4D) was selected for the CAM assay. Fertilized leghorn chicken eggs were selected. Fertilized eggs were incubated horizontally at 37°C and 70% humidity for 7 days. On Day 7, a small circular window was made in the eggshell under sterile conditions to expose the CAM. The inner shell membrane was gently removed to expose blood vessels. The test compound, 4D, at two concentrations (10 µg and 25 µg/disc), was dissolved in PBS and applied onto sterile filter paper discs (5 mm in diameter). The filter paper disc was placed on the CAM membrane near the major vessels. Sorafenib (5 µg/disc), a known VEGFR-II inhibitor, was used as the positive control, and PBS served as the normal (vehicle) control. The eggs were resealed and incubated for another 72 hours. On the 10th day, eggs were reopened. The CAM was examined under a stereomicroscope, and the number of blood vessels surrounding the disc was counted [18]. No separate sham control was included, as PBS (vehicle) is non-toxic and routinely used as a standard solvent in CAM assays. Since experimentation was terminated before day 14, in accordance with Organisation for Economic Co-operation and Development-aligned Hen’s Egg Test – Chorioallantoic Membrane guidelines, this model is considered an alternative to animal testing and is exempt from animal ethics committee approval requirements. The CAM assay was performed once per group as a preliminary qualitative screening to visualize angiogenic inhibition. Therefore, vessel counts represent single representative observations without statistical replication.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The 1,3,4 oxadiazole derivatives (1D-8D) were synthesised following the scheme (Fig. 4). The synthesis was accomplished through a five-step reaction sequence starting from various substituted aromatic carboxylic acids. In the first step, esterification of the aromatic acids was carried out using ethanol under reflux to yield the corresponding ethyl esters. These esters were then treated with hydrazine hydrate to obtain the respective aromatic hydrazides. In the third step, the hydrazides were dissolved in alcoholic potassium hydroxide and reacted with carbon disulfide to form 5-aryl-1,3,4-oxadiazole-2-thiol derivatives.
 | Figure 4. Synthetic scheme. [Click here to view] |
The fourth step involved the reaction of the resulting thiol intermediates with chloroacetyl chloride in the presence of potassium carbonate to afford S-(5-aryl-1,3,4-oxadiazol-2-yl) 2-chloroethanethioates. Finally, the synthesized chloroethanethioates were dissolved in ethanol, and potassium hydroxide was added, followed by the dropwise addition of bis(2-chloroethyl)amine (0.00015 mol, 0.280 ml) with continuous stirring at room temperature for 48 hours to yield the final aryl derivatives.
The progress of each step was monitored by TLC. Structural confirmation of the synthesized compounds was carried out using spectroscopic techniques, including FTIR, NMR, and MS.
3.2. Spectral data
S-(5-phenyl-1,3,4-oxadiazol-2-yl) [bis(2-chloroethyl)amino]ethanethioate-1D: Creamy white solid, m.p. 122.?7–123.2?. IR (cm−1): 2,934 (Ar C–H), 1,610 (C=O, thioester; conjugated/shifted), 1,572 (C=N, oxadiazole), 1,508, 1,448 (Ar C=C), 1,295, 1,155, 1,060 (C–O), 10,24 (C–N), 858, 771, 751, 696 (oop Ar/C–Cl), H1NMR(400 MHz, DMSO-d6, delta ppm): DMSO-d6) δ 7.89–7.57 (m, 5H, Ar-H), 3.95–3.92 (m, 4H, CH2CH2Cl), 3.44–3.36 (m, 2H, O=C–CH2), 2.51–2.50 (m, 4H, N–CH2–CH2). Total = 15 H; Mass: high-resolution mass spectra (HRMS) (TOF-ESI+): m/z [M+H]+ calcd for C13H16Cl2N4O2S, 361.0371; found, 361.0369.
S-[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl] [bis(2-chloroethyl)amino]ethanethioate-2D: Pale yellow solid,mp 127.3?–128.5?. IR (cm−1): 3,084 (Ar C–H), 2,934, 2,874 (aliphatic C–H), 1,617 (C=O, thioester), 1,512 (C=N, 1,3,4-oxadiazole), 1,452, 1,378 (Ar C=C / CH3 bend), 1,295, 1,180, 1,072, 1,044 (C–O/C–N), 1019 (C–N), 831, 749, 695 (Ar-H oop / C–Cl), H1NMR(400 MHz, DMSO-d6, delta ppm): δ 8.161–7.61 (m, 4H, Ar-H), 4.224–4.033 (m, 4H, CH2CH2Cl), 3.996–3.739 (m, 2H, O=C–CH2), 3.572–3.146 (m, 4H, N–CH2–CH2), 2.971–2.496 (m, 3H, Ar–CH3). Total = 17 H;Mass: HRMS (TOF-ESI+): m/z [M+H]+ calcd for C14H18Cl2N4O2S, 375.0527; found, 375.0524.
S-[5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl] [bis(2-chloroethyl)amino] ethanethioate-3D :light brownish solid mp 129.3?–131.3?. IR (cm−1): 3,047 (Ar C–H), 2,958, 2,874 (aliphatic C–H), 1,614 (C=O, thioester), 1,520 (C=N, oxadiazole ring), 1,499 (Ar-ring stretch), 1,251 (Ar–O–CH3, C–O),1,007(C-O stretching, ring oxygen), 834 (p-disubstituted Ar C–H oop), 726, 692 (C–Cl / fingerprint), H1NMR(400 MHz, DMSO-d6) δ 7.90–7.11 (m, 4H, Ar-H), 4.00–3.81 (m, 3H, OCH3), 3.796–3.750 (m, 4H, CH2CH2Cl), 3.683–3.384 (m, 2H, O=C–CH2), 3.162–2.667 (m, 4H, N–CH2–CH2). Total = 17 H; Mass: HRMS (TOF-ESI+): m/z [M+H]+ calcd for C14H18Cl2N4O3S, 391.0476; found, 391.0472.
S-[5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl] [bis(2-chloroethyl)amino] ethanethioate-4D: yellow solid mp. 125.4?–127.2?. IR (cm−1) : 3046 (w, Ar C–H),2941 (aliphatic C–H), 1,601 (C=O, thioester; conjugated), 1,499 (C=N, oxadiazole ring), 1,072 (Ar–F). H1NMR(400 MHz, DMSO-d6) δ 7.965–7.416 (m, 4H, Ar-H), 3.960–3.928 (m, 4H, CH2CH2Cl), 3.454–3.358 (m, 2H, O=C–CH2), 2.518–2.501 (m, 4H, N–CH2–CH2). Total = 14 H.; Mass: HRMS (TOF-ESI+): m/z [M+H]+ calcd for C13H15Cl2FN4O2S, 379.0276; found, 379.0273.
S-[5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl] [bis(2-chloroethyl)amino]ethanethioate-5D: yellow solid.mp 127.8?–130.2?. IR (cm−1): 3048 (Ar C–H), 2934 (aliphatic C–H), 1,661 (C=O, thioester, s), 1,595(-C=N stretching pyridine ring), 1,508(C=N, oxadiazole ring), 1,243 (C–N, pyridyl), 1,158(C-O stretching ring oxygen), H1NMR(400MHz, DMSO-d6) δ 8.112–7.411 (m, 4H, Ar-H), 3.983–3.993 (m, 4H, CH2CH2Cl), 3.637 (s, 2H, O=C–CH2), 3.018–3.001 (m, 4H, N–CH2–CH2). Total = 14 H ; Mass : HRMS (TOF-ESI+): m/z [M+H]+ calcd for C12H14Cl2N4O2S, 361.0214; found, 361.0210.
S-[5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl] [bis(2-chloroethyl)amino]ethanethioate-6D: pale yellow solid mp,96.2?–97.5?. IR (cm−1): 3,106 (w, Ar C–H), 2995 (aliphatic C–H), 1,602 (C=O, thioester), 1,589 (C=N, oxadiazole ring), 1,535, 1,466 (aromatic ring modes), 1,187(C-N stretching),729 (C-Cl stretching); H1NMR(400 MHz, DMSO-d6) δ 7.965–7.416 (m, 4H, Ar-H), 3.974–3.897 (m, 4H, CH2CH2Cl), 3.733–3.596 (m, 2H, O=C–CH2), 3.083–3.015 (m, 4H, N–CH2–CH2). Total = 14 H; Mass: HRMS (TOF-ESI+): m/z [M+H]+ calcd for C13H15Cl3N4O2S, 393.0085; found, 393.0081.
S-[5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl] [bis(2-chloroethyl)amino]ethanethioate-7D: pale yellow solid,mp 85.9?–86.1? IR (cm−1):3,116, 3,029 (aromatic C=H), 1,562 (C=O,thioester), 1,589 (C=N,oxadiazole ring), 1,254(C–N stretching),692 (C-Cl stretching); H1NMR(400 MHz, DMSO-d6) δ 7.978–7.537 (m, 4H, Ar-H), 3.951–3.920 (m, 4H, CH2CH2Cl), 3.704–3.638 (m, 2H, O=C–CH2), 3.214–3.014 (m, 4H, N–CH2–CH2). Total = 14 H; Mass : HRMS (TOF-ESI+): m/z [M+H]+ calcd for C13H15Cl3N4O2S, 393.0085; found, 393.0083.
S-[5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl] [bis(2-chloroethyl)amino]ethanethioate-8D: pale brown solid mp .143.8?–146.1?. IR (cm−1): 3,326(-NH2 stretching, Primary amine), 3,057 (aromatic C=H), 2,959, 2,936 (aliphatic C–H stretch), 1,536 (C=N, oxadiazole ring), 1,257 (C–N stretching), 845 (aromatic C–H out-of-plane), 766 (C-Cl stretching); H1NMR(400MHz, DMSO-d6) δ 7.978–7.573 (m, 4H, Ar-H), 4.011–3.994 (m, 4H, CH2CH2Cl), 3.839 (s, 2H, O=C–CH2), 3.013 (m, 4H, N–CH2–CH2). NH2 not clearly observed (exchange-broadened in DMSO-d6). Total observed = 14 H; molecule contains 16 H including NH2; Mass: HRMS (TOF-ESI+): m/z [M+H]+ calcd for C13H17Cl2N5O2S, 376.0479; found, 376.0476.
Due to partial overlap and exchange broadening in DMSO-d6, reliable J values could not be extracted; therefore, H1NMR data are reported as apparent multiplets with integrations and assignments.
HRMS were recorded on a TOF-ESI+ instrument. The observed m/z values for [M+H]+ ions matched the calculated molecular weights of the synthesized compounds, confirming molecular integrity.
3.3. Molecular docking and validation of docking protocol
To ensure the reliability of the docking setup, the co-crystallized ligand of VEGFR-2 (PDB ID: 1Y6A) was re-docked into the prepared receptor using Glide. The reproduced pose closely matched the crystallographic orientation, conserving the characteristic hinge-region hydrogen bonds with Cys917 and Glu915, and hydrophobic contacts with Val914, Leu838, and Phe1045.
The 2D interaction diagrams (Figs. 1 and 2) clearly show that the key polar and hydrophobic interactions of the crystal and redocked poses are nearly identical. In contrast, the 3D SiteMap-based visualization (Fig. 3) confirms that the defined docking grid encompassed the validated ATP-binding cleft. These results confirm that the grid parameters and scoring function were reliable for docking of the synthesized oxadiazole derivatives.
The eight oxadiazole derivatives were docked with target 1Y6A; among the best docking scores was shown by compound 4D, with a docking score of −8.158, 1D showed a docking score of −8.087, and the standard drug sorafenib showed a docking score of −5.62. Although sorafenib displayed a less favorable docking score, it was chosen as a comparator because it is a clinically validated VEGFR inhibitor.
Although compound 4D exhibited a more favorable docking score than Sorafenib, its in vitro cytotoxicity was comparatively moderate. This discrepancy arises from factors beyond static binding affinity. Docking simulations represent an idealized, rigid interaction, whereas biological systems involve receptor flexibility, solvation effects, and cellular processes such as permeability and metabolism. Sorafenib, as a clinically optimized multikinase inhibitor, benefits from superior physicochemical and pharmacokinetic properties, enhancing cellular uptake and multi-target inhibition, which results in higher cytotoxicity despite a weaker docking score. In contrast, 4D forms stable hydrogen bonds and hydrophobic contacts within the VEGFR-2 binding pocket, but limited bioavailability or conformational adaptability inside the cell may reduce its potency. These observations underscore the importance of integrating molecular docking with MD simulations and biological assays to obtain a realistic assessment of ligand efficacy. The primary interactions observed in docking were hydrogen bonds and halogen bonds, which contributed to ligand stabilization within the binding pocket. Detailed docking results are summarized in Table 3, with 2D interaction diagrams presented in Figures 5–7.
 | Figure 5. Interaction between compound 1D and 1Y6A. [Click here to view] |
 | Figure 6. Interaction between compound 4D and 1Y6A. [Click here to view] |
 | Figure 7. Interaction between compound sorafenib and 1Y6A. [Click here to view] |
Table 3. Ligand target interaction.
| Sl No | Compound | G score | Type of interaction | H-bond and AA residue |
|---|---|---|---|---|
| 1 | 1D | −8.087 | H- bond and Halogen bond | Hydrogen bonding between CYS 917 A: and the Nitrogen of oxadiazole ring Halogen bond between ARG 850 and chlorine |
| 2 | 2D | −6.812 | H- bond | Hydrogen bonding between CYS 917 A: and N4 of oxadiazole ring |
| 3 | 3D | −7.801 | H- bond | Hydrogen bonding between LYS 866: and O of C=O |
| 4 | 4D | −8.158 | H- bond | CYS 917 A: and the Nitrogen of oxadiazole ring |
| 5 | 5D | −5.841 | Halogen bond | Halogen bond between LYS 866:and Chlorine |
| 6 | 6D | −5.129 | Halogen bond | Halogen bond between Water molecule and Chlorine |
| 7 | 7D | −5.072 | H-bond | Hydrogen bonding between CYS 917 A: and the Nitrogen of Oxadiazole |
| 8 | 8D | −5.007 | H-bond | Hydrogen bonding between CYS 917 A: and the Nitrogen of Oxadiazole |
| 9 | Sorafenib | −5.6 21 | H- bond | Hydrogen bonding between CYS 917 A: and the Nitrogen of urea linker |
3.4. ADME properties
The physicochemical and pharmacokinetic parameters of the synthesized compounds were evaluated using the SwissADME platform. The analysis helps to understand the drug likeness and oral bioavailability. The properties assessed were GI absorption permeability, P-gp substrate identification, and CYP inhibition; none of the derivatives were predicted to be P-gp substrates, suggesting a reduced likelihood of efflux-mediated resistance. However, all compounds showed inhibitory potential toward multiple CYP isoforms, indicating a possible risk of drug–drug interactions that warrants further in vitro validation. Based on the in silico predictions, the synthesized derivatives demonstrated lower logP values and higher polarity than sorafenib, which suggests a potential for better aqueous solubility. The moderate logP, balanced TPSA, and acceptable numbers of HBD and acceptors further support a favorable polarity that may facilitate solubility and permeability. It is, however, acknowledged that these observations are derived from computational predictions, and experimental solubility or stability studies were not performed as part of the current work. Future studies will therefore focus on experimental evaluation to validate these predicted solubility and stability properties. The data are provided in Table 4.
Table 4. ADMET properties of compounds 1D–8D.
| Parameters | 1D | 2D | 3D | 4D | 5D | 6D | 7D | 8D | Sorafenib |
|---|---|---|---|---|---|---|---|---|---|
| Molecular weight (g/mol) | 360.26 | 374.29 | 390.28 | 378.25 | 361.25 | 394.70 | 394.70 | 375.27 | 464.82 |
| LogP (WLOGP) | 3.13 | 3.44 | 3.14 | 3.69 | 2.53 | 3.79 | 3.79 | 2.73 | 6.32 |
| TPSA (Ų) | 84.53 | 84.53 | 93.76 | 84.53 | 97.42 | 84.53 | 84.53 | 110.55 | 92.35 |
| HBD | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 3 |
| HBA | 5 | 5 | 5 | 6 | 6 | 5 | 5 | 5 | 7 |
| Rotatable bonds | 9 | 9 | 10 | 9 | 9 | 9 | 9 | 9 | 9 |
| GI absorption | High | High | High | High | High | High | High | High | Low |
| BBB permeability | NO | NO | NO | NO | NO | NO | NO | NO | NO |
| P-gp substrate | NO | NO | NO | NO | NO | NO | NO | NO | NO |
| CYP inhibition | CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP3A4 | CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4 |
| Bioavailability score | 0.55 | 0.55 | 0.55 | 0.55 | 0.55 | 0.55 | 0.55 | 0.55 | |
| Lipinski violations | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Veber rule | Yes | Yes | Yes | Yes | Yes | Yes | Yes | Yes |
3.5. MD simulation studies
Based on the in vitro biological evaluation, four compounds (1D, 2D, 3D, and 4D) were selected for MD simulation studies. The simulations were performed for 100 ns using the GROMACS package with the AMBER99SB-ILDN force field, employing a TIP3P water model. The systems were energy-minimized, equilibrated under NVT/NPT conditions, and analysed for RMSD, RMSF, Rg, hydrogen-bonding, and potential energy fluctuations to assess system stability. Four complexes were generated: Complex A: VEGFR II-Benzoic Acid Derivative (1D); Complex B: VEGFR II-4-Methoxy Benzoic Acid Derivative (3D); Complex C: VEGFR II-4-Fluoro Benzoic Acid Derivative (4D); and Complex D: VEGFR II-4-Methyl Benzoic Acid Derivative (2D).
The results highlight significant differences in stability, ligand interactions, and structural behavior among the complexes compared with the control Sorafenib. The protein Cα-RMSD values indicated that Complex D displayed the greatest structural stability, maintaining a low and steady deviation of 0.40–0.50 nm, while Complex C showed similar stable behavior (0.40–0.50 nm). Complex A demonstrated moderate fluctuations (0.50–0.60 nm), whereas Complex B showed the highest deviation (0.60–0.70 nm), indicating reduced stability. A similar trend was observed in ligand RMSD, where Complexes C and D exhibited the lowest deviation (0.15–0.20 nm), confirming stable ligand positioning in the binding pocket. In contrast, Complex B exhibited the highest ligand deviation (≈approximately 0.30 nm), indicating weaker binding.
RMSF analysis revealed region-specific flexibility variations. It gave these findings: Complex D displayed the lowest residue fluctuations, indicating a rigid and stable protein conformation, followed by Complex C, while Complex B exhibited the highest RMSF values, indicating increased flexibility and a less stable protein–ligand environment. This instability in Complex B may be attributed to the steric influence of the methoxy substituent, which likely limited strong and persistent interactions within the VEGFR-2 active site.
Additionally, the average Rg values further confirmed overall structural compactness. Compact Rg profiles (≈2.10–2.14 nm) were observed for Complexes A, C, and D, indicating tighter folding compared to Complex B. Complex D maintained 1–2 persistent hydrogen bonds throughout the simulation, while Complex C showed moderate and relatively stable H-bond interactions. Complex A exhibited intermittent hydrogen bonding, whereas Complex B formed negligible H-bonds, further confirming the difference in stability. Collectively, these findings suggest that Complex D and Complex C achieve the most stable and favorable ligand–protein interactions, followed by Complex A, whereas Complex B exhibits comparatively weak and unstable binding. Overall, the MD results clearly demonstrate that Complex D is the most stable system, followed by Complex C and Complex A, whereas Complex B is the least stable (Figs. 8–16). The data are given in Tables 5 and 6.
 | Figure 8. MD simulation analysis of protein-ligand complexes over 100 ns. [Click here to view] |
 | Figure 9. Residual interaction analysis of VEGFR2 kinase-ligand complex A at 0, 50, and 100 ns. [Click here to view] |
 | Figure 10. Residual interaction analysis of VEGFR2 kinase-ligand complex B at 0, 50, and 100 ns. [Click here to view] |
 | Figure 11. Residual interaction analysis of VEGFR2 kinase-ligand complex C at 0, 50, and 100 ns. [Click here to view] |
 | Figure 12. Residual interaction analysis of VEGFR2 kinase-ligand complex D at 0, 50, and 100 ns. [Click here to view] |
 | Figure 13. Superposition of ligand conformations during MD simulations (complex A). [Click here to view] |
 | Figure 14. Superposition of ligand conformations during MD simulations (complex B). [Click here to view] |
 | Figure 15. Superposition of ligand conformations during MD simulations (complex C). [Click here to view] |
 | Figure 16. Superposition of ligand conformations during MD simulations (complex D). [Click here to view] |
Table 5. Comparative analysis of MD simulation metrics for selected complexes.
| Complex | Potential energy (kJ/mol) | Rg (nm) | H-bond behavior | Ligand RMSD (nm) | Protein RMSD (nm) | RMSF observation | Remarks |
|---|---|---|---|---|---|---|---|
| A | −990,000 to −996,000 | 2.10 | Intermittent | 0.2–0.25 | 0.5–0.6 | High at 920–950, 1000–1020 | Moderate stability & fluctuation |
| B | −990,000 to −996,000 | 2.17 | Negligible | 0.3 | 0.6–0.7 | Highest RMSF values | Most flexible, less stable |
| C | −990,000 to −996,000 | 2.12–2.14 | Moderate | 0.15–0.2 | 0.4–0.5 | Moderate fluctuation | Stable structure & conformation |
| D | −990,000 to −996,000 | 2.12–2.14 | 1–2 constant | 0.15–0.2 | 0.4–0.5 | Lowest RMSF values | Most stable & rigid |
Table 6. Molecular interactions and stabilizing forces in ligand–target complex.
| Sl no | Parameter | Details |
|---|---|---|
| 1 | Critical residues | C919, L1035, N923, F918, F921, V916, E850, D1046 |
| 2 | Hydrogen bonds | C919, L1035, N923 formed strong polar interactions |
| 3 | Hydrophobic interactions | V916, V899, F921 contributed via van der Waals and π-π stacking |
| 4 | Electrostatic interactions | E850 and D1046 provided charged stabilization |
| 5 | Ligand stability | Ligand maintained position with minimal deviation, indicating stable binding |
3.6. In vitro anti-proliferative evaluation by MTT assay
The MTT assay was performed to assess the in vitro antiproliferative activity of the synthesized oxadiazole derivatives against three human cancer cell lines—MCF-7 (breast), PA-1 (ovarian), and A-549 (lung)—as well as normal human HEK-293 cells to evaluate selectivity. As summarized in Table 7, compound 4D exhibited the highest cytotoxic effect among the tested derivatives, showing IC50values of 22.94 µg/ml (PA-1), 29.48 µg/ml (MCF-7), and 39.56 µg/ml (A-549). In contrast, the reference drug sorafenib demonstrated greater potency, with IC50values of 7.39 µg/ml (PA-1), 9.95 µg/ml (MCF-7), and 6.10 µg/ml (A-549), consistent with its clinically established antiproliferative activity. To assess selectivity, the compounds were also tested on HEK293 normal human cells. All synthesized derivatives showed IC50values >100 µg/ml in HEK293, indicating minimal cytotoxicity, whereas sorafenib displayed an IC50of ~99 µg/ml. The calculated selectivity index (SI = IC50Normal / IC50Cancer) revealed that 4D possessed the most favorable selectivity profile (SI >3), followed by 1D, 2D, and 6D (SI >2). By contrast, 5D, 7D, and 8D demonstrated limited selectivity (SI ≤1.5). All experiments were performed in triplicate, and results are expressed as mean ± SD. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test, with p < 0.05 considered significant compared to vehicle control and sorafenib. The inclusion of 0.1% DMSO vehicle control confirmed that the solvent did not affect cell viability (~100% viability across all lines). Collectively, these results indicate that compound 4D represents a promising lead scaffold for further structural optimization toward selective VEGFR-2 inhibition and anticancer potential (Fig. 17).
 | Figure 17. % Cell Viability MTT assay. [Click here to view] |
Table 7. Cytotoxicity of synthesized compounds in cancer (MCF-7, PA-1, A-549) and normal (HEK293) cell lines by MTT assay.
| Compound | % Viability (MCF-7) @100 µg/ml | IC50(MCF-7) (µg/ml) | % Viability (PA-1) @100 µg/ml | IC50(PA-1) (µg/ml) | % Viability (A-549) @100 µg/ml | IC50(A-549) (µg/ml) | % Viability (Normal) @100 µg/ml | IC50(Normal) (µg/ml) | SI |
|---|---|---|---|---|---|---|---|---|---|
| Vehicle (0.1% DMSO) | ~100 ± 0.5 | - | ~100 ± 0.5 | - | ~100 ± 0.5 | - | ~100 ± 0.5 | - | - |
| 1D | 36.60 ± 0.64 | 52.49 | 36.34 ± 1.18 | 45.91 | 30.02 ± 1.13 | 43.51 | ~87–96 | >100 | >2 |
| 2D | 40.66 ± 0.90 | 40.59 | 38.08 ± 1.38 | 55.61 | 37.28 ± 0.55 | 58.36 | ~93–97 | >100 | >2 |
| 3D | 41.10 ± 0.82 | 64.96 | 42.59 ± 1.28 | 62.19 | 29.15 ± 1.16 | 54.00 | ~92–101 | >100 | >1.5 |
| 4D | 30.36 ± 0.49 | 29.48 | 27.10 ± 1.50 | 22.94 | 28.78 ± 0.34 | 39.56 | ~83–92 | >100 | >3 |
| 5D | 41.31 ± 0.47 | 74.04 | 66.59 ± 0.48 | 62.19 | 52.38 ± 0.18 | 106.85 | ~95–100 | >100 | >1 |
| 6D | 30.47 ± 0.42 | 45.46 | 45.63 ± 0.85 | 42.94 | 43.98 ± 0.81 | 73.24 | ~88–98 | >100 | >2 |
| 7D | 36.88 ± 0.20 | 58.33 | 44.99 ± 1.00 | 45.91 | 44.41 ± 1.31 | 72.97 | ~93–113 | >100 | >1.5 |
| 8D | 40.85 ± 1.02 | 67.64 | 46.47 ± 1.45 | 55.61 | 52.02 ± 0.49 | 96.14 | ~80–102 | ~82 | ~1 |
| Sorafenib | 22.04 ± 0.61 | 9.95 | 17.79 ± 0.58 | 7.39 | 21.27 ± 0.03 | 6.10 | ~98–101 | ~99 | ~10 |
Data are presented as mean ± SD (n = 3). Vehicle: 0.1% DMSO. SI = IC50(Normal) / IC50(Cancer). *p < 0.05, **p < 0.01 versus vehicle (One-way ANOVA with Tukey’s test). *p < 0.05 and **p < 0.01 compared with vehicle control (one-way ANOVA followed by Tukey’s post hoc test).
3.7. Chick embryo CAM assay
As compound 4D had shown the best antiproliferative activity in the MTT assay, it was selected for the CAM assay to evaluate its anti-angiogenic potential. The treatment with derivative 4D showed a significant reduction in the number of newly formed blood vessels on the CAM (fig. 18). The data obtained are given in Table 8. The CAM assay indicates anti-angiogenic potential of 4D, further validation through molecular assays, such as Western blotting for phosphorylated VEGFR-II, is required to confirm the mechanism.
 | Figure 18. CAM assay (a) 4D (25 µg/disc); (b) 4D (5 µg/disc); (c) normal control; (d) Sorafenib (5 µg/disc). [Click here to view] |
Table 8. CAM assay % inhibition of angiogenesis.
| Sl No | Group | Avg number of vessels before treatment | Avg number of vessels after treatment | % Inhibition |
|---|---|---|---|---|
| 1 | Normal control | 9.3 | 10.3 | - |
| 2 | Sorafenib (5 µg/ disc) | 9.6 | 2.6 | 72.9 |
| 3 | 4D– Low dose (5 µg/disc) | 10.6 | 6.6 | 37.7 |
| 4 | 4D – High dose (25 µg/disc) | 10.6 | 3.5 | 66.9 |
4. CONCLUSION
In this study, a novel series of 1,3,4-oxadiazole derivatives (1D-8D) was successfully synthesised, and structures were characterized using IR, Mass, and H1NMR spectroscopy. The 1,3,4-oxadiazole scaffold served as a promising core for the development of potential anti-cancer agents. Molecular docking studies demonstrated a favorable binding affinity of the synthesized compounds towards VEGFR II, a key target in angiogenesis and cancer progression. Among the derivatives, compound 4D exhibited the most notable biological profile, showing cytotoxic activity across multiple cancer cell lines with acceptable SI against normal cells, as well as anti-angiogenic activity in the CAM assay. MD simulations further confirmed stable interactions of 4D with VEGFR II. While these findings highlight 4D as a potential lead for further development, the conclusions remain preliminary. In vivo toxicity, pharmacokinetic studies, and mechanistic validation (e.g., VEGFR-II phosphorylation assays) will be essential to substantiate its therapeutic promise.
Plasma stability and pharmacokinetic data for the derivative 4D were not included in this study, as our work primarily aimed to establish in vitro anti-cancer and anti-angiogenic potential. Subsequent studies are planned to address these aspects, including plasma stability assays and in vivo pharmacokinetic profiling, to better define the drug-likeness and translational potential of compound 4D.
5. ACKNOWLEDGMENTS
The authors thank Dayananda Sagar University, Bangalore, for providing support throughout the preparation of the manuscript.
6. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
7. FINANCIAL SUPPORT
There is no funding to report.
8. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
10. DATA AVAILABILITY
All data generated and analyzed are included in this research article.
11. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
12. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
13. SUPPLEMENTARY MATERIAL
The supplementary material can be accessed at the journal’s website: Link here [https://japsonline.com/admin/php/uploadss/4738_pdf.pdf].
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