Synthesis, Biological Evaluation, and in Silico Studies of Novel Triazole- and Oxadiazole-Based Acetamides as Urease and α-Glucosidase Inhibitors

Summary

A series of triazole- and oxadiazole-based acetamide derivatives were synthesized and evaluated as urease and α-glucosidase inhibitors. Compound 8c showed the strongest urease inhibition, while 9e outperformed acarbose against α-glucosidase. BSA binding and in silico studies supported their drug-like properties, identifying both compounds as promising lead candidates.

Abstract

This study investigated the therapeutic potential of novel 1,2,4-triazole- and 1,3,4-oxadiazole-based acetamide derivatives as dual-target inhibitors of urease and α-glucosidase, two enzymes implicated in gastrointestinal disorders and type 2 diabetes, respectively. An integrated approach combining multistep organic synthesis, in vitro biological evaluation, molecular docking, in silico ADME prediction, and density functional theory (DFT) analysis was employed to identify potent lead compounds with favorable pharmacokinetic properties. Starting from benzoic acid, a series of 1,2,4-triazole derivatives (8a–c) and 1,3,4-oxadiazole derivatives (9d–f) bearing substituted acetamide moieties were successfully synthesized. All compounds exhibited inhibitory activity against both target enzymes. Compound 8c was the most potent urease inhibitor (IC₅₀ = 6.14 ± 1.06 µM), while compound 9e showed the strongest α-glucosidase inhibition (IC₅₀ = 27.29 ± 0.41 µM), surpassing the reference inhibitor acarbose (IC₅₀ = 38.25 ± 0.12 µM). Computational analyses supported the experimental findings: ADME predictions indicated favorable drug-like properties, and molecular docking demonstrated strong binding interactions, with compound 8a exhibiting the highest affinity for α-glucosidase (−7.165 kcal/mol) and compound 9e showing the strongest binding to urease (−7.30 kcal/mol). DFT calculations further correlated biological activity with electronic properties, revealing relatively small HOMO–LUMO energy gaps for the most active compounds, 8c (0.14831 a.u.) and 9e (0.14302 a.u.). Collectively, these findings identify compounds 8c and 9e as promising lead scaffolds for the development of next-generation inhibitors of urease and α-glucosidase.

Introduction

Heterocyclic moieties have been previously reported in many existing drugs, and heterocycles represent an important class of enzyme inhibitors used in the treatment of metabolic and infectious diseases¹․ Among heterocycles‚ 1‚3‚4-oxadiazoles are one of the most studied scaffolds in drug discovery and have been‌ reported as‌ antiviral‚ antibacterial‚ anticancer‚ hypotensive‚ neuroprotectant‚ anticonvulsant and antitubercular agents^2,3,4,5․ Similarly‚ 1‚2‚4-triazole derivatives have also shown‌ anticonvulsant‚ antibacterial‚ antiviral‚ antitubercular‚ antidiabetic‚ anti-inflammatory‚ antiproliferative‚ antioxidant‚ antimalarial‚ and urease‌ inhibitory activities^6,7․ Acetamide-containing compounds possess a strong pharmacological profile and have been used as synthetic intermediates in heterocyclic chemistry as well as antidiabetic, anticoagulant, and chemosensitizing agents, making them attractive lead structures for drug discovery^8,9․

Significant efforts have been directed toward the development of multitarget inhibitors of α-glucosidase and urease. α-Glucosidase inhibition delays carbohydrate digestion and glucose absorption, thereby lowering postprandial blood glucose levels in patients with type 2 diabetes mellitus. Current inhibitors such as‌ acarbose are effective but are associated with gastrointestinal side effects‚ which creates a need for structurally diverse inhibitors^10,11․ Urease breaks‌ down urea into‌ ammonia and carbon dioxide․ The reaction enables the bacterium Helicobacter pylori‚ which causes ulcers and makes‌ the host more susceptible to gastric cancer‚‌ to survive in the stomach and therefore urease is an important target in anti-ulcer drug development․ A therapeutic goal is the development of‌ new inhibitors for both‌ enzymes^9,12․

However‚ despite the available research‌ studies on 1‚2‚4-triazole and 1‚3‚4-oxadiazole scaffolds‚ very few studies have been reported related to the acetamide derivatives of 1‚2‚4-triazole and 1‚3‚4-oxadiazole containing a thioether moiety as dual α-glucosidase and urease inhibitors¹³․ Their protein-binding behavior and computational characterization have also received limited attention in this context․ Herein, we proposed that merging the triazole or oxadiazole‌ and substituted acetamide fragments into the same molecular framework would yield a satisfactory enzyme inhibitory profile with acceptable physicochemical properties․

To test this hypothesis, a series of 1,2,4-triazole-based acetamide derivatives (8a–c) and 1,3,4-oxadiazole-based acetamide derivatives (9d–f) were synthesized and evaluated for their inhibitory activity against α-glucosidase and urease. The binding interactions of the most active compounds (8a, 8c, and 9e) with bovine serum albumin (BSA) were investigated using fluorescence spectroscopy, binding constant determination, and thermodynamic analysis. In addition, molecular docking, ADME prediction, and density functional theory (DFT) calculations were performed to investigate their binding modes, pharmacokinetic properties, and electronic characteristics.

Protocol

All reagents were of analytical grade and used without further purification. Reaction progress was monitored by thin-layer chromatography (TLC) on silica gel using n-hexane/ethyl acetate (7:3–6:4, v/v), with spots visualized under ultraviolet light. Unless otherwise stated, all reactions were performed under ambient atmosphere. The synthesized compounds were characterized by Fourier transform infrared (FTIR), ¹H NMR, and ¹³C NMR spectroscopy. The ¹H and ¹³C NMR spectra were recorded in CDCl₃ at 600 MHz and 150 MHz, respectively, and chemical shifts were reported in ppm relative to the residual solvent signal. The synthetic pathway is depicted in Figure 1. The complete physicochemical characterization data for all synthesized compounds are provided in Supplementary File 1.

Synthesis of ethyl benzoate (1)

Benzoic acid (50 g, 0.41 mol) was placed in a 500 mL round-bottom flask, and absolute ethanol (150 mL) was added. Concentrated sulfuric acid (6 mL) was added dropwise as a catalyst under continuous magnetic stirring. The reaction mixture was heated under reflux at 78 °C for 4–6 h using a water-cooled condenser. Reaction progress was monitored by TLC. Upon completion, the mixture was cooled to room temperature and neutralized with saturated aqueous sodium carbonate until the pH reached 7–8. The reaction mixture was transferred to a separatory funnel, and the organic layer was collected, washed with distilled water, and dried over anhydrous sodium sulfate. After filtration, ethyl benzoate (1) was used directly in the subsequent step without further purification (Figure 1).

Synthesis of benzohydrazide (2)

Ethyl benzoate (1; 0.41 mol) was dissolved in absolute ethanol (100 mL), and hydrazine hydrate (80%, 25 mL, excess) was added under continuous stirring. The reaction mixture was heated under reflux at 78 °C for 4–5 h under air using a water-cooled condenser. Reaction progress was monitored by TLC. Upon completion, the white crystalline precipitate of benzohydrazide (2) was collected by filtration, washed with cold ethanol, dried to constant weight at room temperature, and used in subsequent synthetic steps (Figure 1).

Synthesis of 5-phenyl-1,3,4-oxadiazole-2-thiol (3)

Benzohydrazide (2; 10 g, 0.073 mol) and potassium hydroxide (4.12 g, 0.073 mol) were placed in a 250 mL round-bottom flask containing ethanol (30 mL). The mixture was heated under reflux at 78 °C for 10 min using a water-cooled condenser. Carbon disulfide (CS₂; 5 mL, 0.083 mol) was then added, and reflux was continued for an additional 3–4 h. Reaction progress was monitored by TLC. Upon completion, the reaction mixture was transferred to distilled water and heated with continuous stirring. Dilute hydrochloric acid was added dropwise until the pH reached 5, resulting in precipitation of compound (3) (Figure 1). The precipitate was collected by filtration, washed thoroughly with cold distilled water, dried to constant weight, and weighed to determine the yield. The compound was characterized by FTIR, ¹H NMR, and ¹³C NMR spectroscopy.

Synthesis of 4,5-diphenyl-4H-1,2,4-triazole-3-thiol (4)

Benzohydrazide (2; 5 g, 0.037 mol) and phenylisothiocyanate (4 mL, 0.037 mol) were placed in a 100 mL round-bottom flask fitted with a water-cooled condenser. The reaction mixture was heated under reflux at 78 °C until formation of the thiosemicarbazide intermediate was complete, as confirmed by TLC using n-hexane/ethyl acetate (7:3, v/v). Potassium hydroxide (2 g, 0.036 mol), dissolved in ethanol (20 mL), was then added, and reflux was continued until formation of compound (4), as confirmed by TLC. The reaction mixture was transferred to distilled water, heated with continuous stirring, and acidified with dilute hydrochloric acid to pH 5 to precipitate compound (4) (Figure 1). The precipitate was collected by filtration, washed thoroughly with cold distilled water, dried to constant weight, and characterized by FTIR, ¹H NMR, and ¹³C NMR spectroscopy.

Synthesis of bromoacetamide intermediates 7(a–f)

Each substituted aniline derivative 5(a–f) (1 equiv.) was dissolved in chloroform and cooled in an ice bath to maintain the reaction temperature at 0–5 °C. Bromoacetyl bromide (6, ≥98%, 1 equiv.) was added dropwise with continuous stirring. During the reaction, 18% aqueous sodium carbonate solution was added as needed to maintain the reaction pH between 9 and 10 by neutralizing the hydrobromic acid generated. After the addition of bromoacetyl bromide was complete, the reaction mixture was allowed to warm to room temperature and stirred until TLC confirmed complete consumption of the starting material. The resulting bromoacetamide intermediates 7(a–f) were collected by filtration, washed with cold distilled water, dried to constant weight, and used directly in the subsequent synthetic step without further purification (Figure 1).

Synthesis of triazole-based acetamide derivatives 8(a–c)

Triazole derivative (4; 0.5 g, 0.002 mol) was dissolved in N, N-dimethylformamide (10 mL) in a 50 mL round-bottom flask. The appropriate bromoacetamide intermediate (7a, 7b, or 7c; 0.002 mol, 1 equiv.) was added with continuous stirring, followed by sodium hydride (0.05 g, 0.002 mol) to promote S-alkylation. The reaction mixture was stirred at room temperature for 24–48 h, with reaction progress monitored by TLC. Upon completion, the reaction mixture was poured into ice-cold water to induce precipitation. The precipitate was collected by filtration, washed thoroughly with cold water, dried, and characterized by FTIR, ¹H NMR, and ¹³C NMR spectroscopy (Figure 1).

Synthesis of oxadiazole-based acetamide derivatives 9(d–f)

Oxadiazole derivative (3; 0.5 g, 0.002 mol) was dissolved in N, N-dimethylformamide (10 mL) in a 50 mL round-bottom flask. The corresponding bromoacetamide intermediate, 7d, 7e, or 7f (0.002 mol, 1 equiv.), was added, followed by sodium hydride (0.05 g, 0.002 mol) to facilitate S-alkylation. The reaction mixture was stirred at room temperature for 24–48 h, with progress monitored by TLC. Upon completion, the mixture was poured into ice-cold water to precipitate the product. The precipitate was collected by filtration, washed thoroughly with cold water, dried, and characterized by FTIR, ¹H NMR, and ¹³C NMR spectroscopy (Figure 1).

α-Glucosidase inhibition assay

The α-glucosidase inhibitory activity was evaluated by mixing 50 µL of the test sample solution with 100 µL of α-glucosidase solution (0.35 U/mL) prepared in phosphate buffer (pH 6.8). The reaction mixture was incubated at 37 °C for 10 min, then 100 µL of 1.5 mM p-nitrophenyl-α-D-glucopyranoside was added as the substrate. After further incubation at 37 °C for 20 min, the reaction was terminated by adding 1 mL of 1 M sodium carbonate (Na₂CO₃). The absorbance was measured at 400 nm¹⁴. Acarbose was used as the positive control. The reaction mixture without the test compound served as the negative control, whereas the reaction mixture without the enzyme was used as the blank.

All experiments were performed in triplicate, and the results are expressed as the mean ± standard deviation (SD). The percentage inhibition of α-glucosidase activity was calculated using the following equation(1)¹⁴:

     (1)

where A_s represents the absorbance of the test sample solution, A_b is the absorbance of the reagent blank (without α-glucosidase), and A₀ is the absorbance of the negative control (without the sample). The IC₅₀ values were determined by testing compound concentrations ranging from 3.125 to 100 µM. Nonlinear regression analysis was performed using standard graphing software.

Urease inhibition assay

Urease inhibitory activity was determined using a modified Berthelot method. The reaction mixture consisted of 300 µL of the test sample solution, 200 µL of jack bean urease solution, and 200 µL of 25 mM urea. The mixture was incubated in a water bath at 37 °C for 30 min. After incubation, 500 µL of a color-developing solution containing 10 g/L phenol and 50 g/L sodium nitroprusside, followed by 500 µL of a second solution containing 5% (v/v) sodium hypochlorite and 5 g/L sodium hydroxide, was added. The reaction mixture was then incubated for an additional 15–30 min to allow color development.

The absorbance of the solution was measured at 625 nm against a reagent blank¹⁵. Thiourea was used as the positive control, the reaction mixture without the test compound as the negative control, and the mixture without the urease enzyme as the blank. All experiments were performed in triplicate (n = 3), and results are expressed as mean values ± standard deviation (S.D.). The percentage of inhibition was calculated using the following mathematical formula (2)¹⁵:

    (2)

where A₀ is the absorbance of the control (without inhibitor) and A_s is the absorbance of the test sample (with inhibitor). Graphing and non-linear regression analysis to determine IC₅₀ values were performed using standard data plotting software.

BSA binding assay

Binding interactions between bovine serum albumin (BSA) and the synthesized compounds were investigated using fluorescence quenching spectroscopy. A 10 µM BSA stock solution was prepared in 20 mM phosphate buffer (pH 7.4). For each titration experiment, 1.0 mL of the BSA solution was mixed with 2.0 mL of phosphate buffer in a quartz cuvette. The solution was titrated by successive additions of 5 µL aliquots of the test compound (1.5 mM in methanol). Fluorescence emission spectra were recorded over the wavelength range of 300–400 nm following excitation at 295 nm, with the maximum emission observed at approximately 336 nm. Fluorescence measurements were performed using a fluorescence spectrophotometer equipped with standard fluorescence acquisition software, with excitation and emission slit widths set to 10 nm and 2.5 nm, respectively. To determine the thermodynamic parameters of binding, including the Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS), the titration experiments were repeated at 298, 308, and 313 K.

To investigate the fluorescence quenching mechanism, the experimental data were analyzed using the Stern–Volmer equation (Equation 3)¹⁶:

     (3)

where F₀ and F represent the fluorescence intensities of BSA in the absence and presence of the quencher, respectively; K_q is the bimolecular quenching rate constant; τ₀ is the average lifetime of BSA in the absence of a quencher; K_SV is the Stern-Volmer quenching constant; and [Q] is the concentration of the quencher. The binding constant (K_b) and the number of binding sites (n) were subsequently determined by plotting the double logarithm according to Equation (4)¹⁶:

     (4)

Furthermore, the standard Gibbs free energy change (ΔG°), standard enthalpy change (ΔH°), and standard entropy change (ΔS°) governing the binding interaction were calculated using the Van't Hoff and Gibbs-Helmholtz relationships expressed in Equations (5) and (6)¹⁷:

ΔG^∘ = -RT ln K_b    (5)

     (6)

where R is the universal gas constant and T is the absolute temperature in Kelvin.

Molecular docking

All molecular modeling and docking simulations were performed using the Schrödinger Suite (Maestro interface), with docking performed using the Glide module. The crystal structures of α-glucosidase (PDB ID: 5NN8) and urease (PDB ID: 4H9M) were retrieved from the RCSB Protein Data Bank. Protein structures were prepared using the Protein Preparation Wizard by removing co-crystallized ligands, water molecules, and nonessential heteroatoms, adding missing residues and loops, assigning correct bond orders, and adding polar hydrogen atoms. Protonation and tautomeric states were assigned at physiological pH (7.0 ± 2.0) using the Epik module. The prepared protein structures were subsequently subjected to restrained energy minimization with the OPLS force field to relieve steric clashes.

The two-dimensional structures of compounds 3, 4, 8(a–c), and 9(d–f), together with the reference inhibitors acarbose and thiourea, were prepared using the LigPrep module. Bond orders, stereochemistry, and ionization states at physiological pH were optimized, and the resulting three-dimensional ligand conformations were energy-minimized using the OPLS force field.

Receptor grids were generated using the Receptor Grid Generation tool, with the grid boxes centered on the cocrystallized native ligands to define the active sites. Grid box dimensions were set to 25 × 25 × 25 Å for both proteins. The urease grid was centered at x = −38.76, y = −44.14, and z = 66.88, whereas the α-glucosidase grid was centered at x = 21.14, y = −7.56, and z = 24.37.

Docking calculations were performed using the extra-precision (XP) mode of Glide with flexible ligand sampling. Multiple binding poses were generated for each ligand, and the pose with the lowest Glide docking score was selected for further analysis. The docking protocol was validated by redocking the cocrystallized ligands into their respective receptor grids. Root-mean-square deviation (RMSD) values of ≤2.0 Å confirmed the reliability and reproducibility of the docking protocol.

The binding conformations of the docked complexes were visualized using PyMOL (version 3.1)¹⁸. Protein–ligand interactions, including hydrogen bonds, hydrophobic contacts, and electrostatic interactions, were analyzed using Discovery Studio Visualizer¹⁹.

Density functional theory (DFT) analysis

All density functional theory (DFT) calculations were performed using Gaussian 09, and the optimized molecular geometries and frontier molecular orbitals were visualized using GaussView 5. The synthesized triazole- and oxadiazole-based acetamide derivatives, 8(a–c) and 9(d–f), were subjected to geometry optimization in the aqueous phase using the Becke three-parameter Lee–Yang–Parr (B3LYP) hybrid functional in conjunction with the 6-311+G(d,p) basis set²⁰. Solvent effects were incorporated using the Conductor-like Polarizable Continuum Model (CPCM)²¹. Vibrational frequency calculations were subsequently performed at the same level of theory to confirm that all optimized structures corresponded to true local minima on the potential energy surface, as indicated by the absence of imaginary frequencies.

To understand the electronic transitions and kinetic stability of the synthesized compounds, the energies of the highest occupied molecular orbital (E_HOMO) and the lowest unoccupied molecular orbital (E_LUMO) were calculated. The frontier molecular orbital energy gap (ΔE = E_LUMO - E_HOMO) was determined for each derivative to assess its chemical reactivity and kinetic stability, with a smaller energy gap indicating higher reactivity.

Furthermore, global chemical reactivity descriptors—including chemical hardness (η), softness (S), chemical potential (μ), electronegativity (X), and the electrophilicity index (w)—were computed from the frontier orbital energies using the following standard operational equations²²:

     (7)

     (8)

     (9)

      (10)

The computed electronic properties and global descriptors were systematically compared across the series to establish structure-activity relationships (SARs) that reflect their observed α-glucosidase and urease inhibitory profiles.

Additionally, molecular electrostatic potential (MESP) maps were generated and visualized using VESTA to map the charge distribution across the optimized molecular frameworks. The MESP surfaces allowed for the identification of electrophilic (electron-deficient, nucleophilic attack favored) and nucleophilic (electron-rich, electrophilic attack favored) regions. This electrostatic potential distribution was ultimately correlated with the specific non-covalent binding interactions, such as hydrogen bonding and electrostatic contacts, observed in the molecular docking simulations.

ADME analysis

To evaluate the drug-likeness and biopharmaceutical properties of the synthesized triazole- and oxadiazole-based acetamide derivatives 8(a–c) and 9(d–f), an in silico absorption, distribution, metabolism, and excretion (ADME) analysis was performed. The simplified molecular-input line-entry system (SMILES) representations of all synthesized compounds, together with the reference inhibitors acarbose and thiourea, were generated using a molecular modeling software package and submitted to the SwissADME web server²³.

The calculated physicochemical parameters included molecular weight (MW), lipophilicity (logP), topological polar surface area (TPSA), the number of hydrogen bond donors (HBD), the number of hydrogen bond acceptors (HBA), and the number of rotatable bonds (RB).

Pharmacokinetic properties were evaluated by predicting gastrointestinal (GI) absorption and blood–brain barrier (BBB) permeability. In addition, the compounds were assessed for compliance with Lipinski's rule of five and other established drug-likeness criteria to estimate their oral bioavailability. Finally, the synthetic accessibility (SA) score was calculated for each compound on a scale ranging from 1 (easy to synthesize) to 10 (very difficult to synthesize). The predicted ADME and drug-likeness profiles of the synthesized compounds were compared with those of the reference inhibitors to evaluate their potential as lead candidates for drug development.

Results

Chemistry and characterization of synthesized compounds

The target triazole- and oxadiazole-based acetamide derivatives were synthesized according to the synthetic pathway shown in Figure 1. The desired compounds were obtained in good to excellent yields (69%–87%) as white to yellow crystalline solids. Their purity was confirmed by TLC, melting point determination, and spectroscopic analyses. Representative NMR spectra confirming the characteristic functional groups of the synthesized compounds are shown in Figure 2. The spectra exhibited the expected absorption bands corresponding to the amide carbonyl (C=O), N–H, C=N, C–S, and aromatic C–H functional groups, supporting successful synthesis of the target derivatives.

Representative ¹H NMR spectra are presented in Figure 3. The spectra showed characteristic resonances for the amide NH proton, aromatic protons, methylene linker, and methyl substituents, which were consistent with the proposed structures. Representative ¹³C NMR spectra are shown in Figure 4. The observed carbon resonances confirmed the presence of carbonyl, aromatic, methylene, and heterocyclic carbons expected for the synthesized derivatives. Representative compounds included 8a (87% yield), 8c (approximately 80%–85% yield), and 9e (69% yield). Complete physicochemical characterization data and NMR spectra for all synthesized compounds are provided in Supplementary File 1.

α-Glucosidase inhibition

The α-glucosidase inhibitory activities of the synthesized compounds are summarized in Table 1. All compounds inhibited α-glucosidase to varying extents. Compound 9e exhibited the highest inhibitory activity, with an IC₅₀ value of 27.29 ± 0.41 µM, followed by compound 8a (33.15 ± 0.07 µM) and compound 8c (40.93 ± 0.59 µM). Compounds 8b, 9d, and 9f showed comparatively weaker inhibition, with IC₅₀ values of 108.19 ± 0.18 µM, 108.10 ± 0.03 µM, and 94.44 ± 0.25 µM, respectively. Compound 9e exhibited greater inhibitory potency than the reference inhibitor acarbose (IC₅₀ = 38.25 ± 0.12 µM), whereas compound 8a showed comparable activity. The concentration-dependent inhibition profiles are presented in Figure 5.

Urease inhibition potential

The urease inhibitory activities of the synthesized compounds are summarized in Table 1. Compound 8c exhibited the strongest urease inhibition, with an IC₅₀ value of 6.14 ± 1.06 µM, followed by compounds 9e (8.14 ± 0.71 µM) and 8a (12.25 ± 0.42 µM). These compounds demonstrated greater inhibitory activity than the reference inhibitor thiourea (IC₅₀ = 21.25 ± 0.15 µM). Compounds 8b, 9d, and 9f were less active, with IC₅₀ values ranging from 82.15 to 103.05 µM. The corresponding concentration-dependent inhibition curves are shown in Figure 5.

BSA binding

The fluorescence quenching parameters are summarized in Table 2. For compounds 8a and 9e, the Stern–Volmer constant (Ksv) increased with increasing temperature, whereas the quenching constant (Kq) exceeded the maximum diffusion-controlled limit (2 × 10¹⁰ M⁻¹ s⁻¹), indicating a mixed static and dynamic quenching mechanism. In contrast, compound 8c exhibited decreasing Ksv values with increasing temperature, while maintaining Kq values above the diffusion-controlled limit, indicating predominantly static quenching. Thermodynamic analysis showed negative ΔH and ΔS values for compound 8a, suggesting that hydrogen bonding and van der Waals interactions predominated during binding. Positive ΔH and ΔS values obtained for compounds 8c and 9e indicated that hydrophobic interactions were the major driving force. Binding affinity analysis performed at 298 K demonstrated that compound 8a exhibited the highest binding constant (Kb = 3.92 × 10⁵ M⁻¹), followed by compound 8c (1.54 × 10⁴ M⁻¹) and compound 9e (6.78 × 10¹M⁻¹). The fluorescence spectra and Stern–Volmer plots are presented in Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, and Figure 11.

Docking analysis

The molecular docking results are summarized in Table 3. The docking protocol was validated by redocking the co-crystallized ligands into their respective binding sites, yielding RMSD values ≤2.0 Å, thereby confirming the reliability of the docking protocol (Supplementary Figure 1).

Based on the biological activity and docking scores, compounds 8a, 8c, and 9e were selected for detailed interaction analysis. Representative docking poses within the α-glucosidase active site are presented in Figure 12A–D. Compound 8a exhibited the highest docking score (−7.165 kcal/mol), followed by compounds 8c (−6.80 kcal/mol) and 9e. These compounds established hydrogen-bond interactions with key catalytic residues, including D282, D404, D616, R600, S523, and H674.

Representative docking poses within the urease active site are shown in Figure 12E–H. Compound 8c established hydrogen bonds with A636, Q635, and R439, whereas compound 9e formed an extensive hydrogen-bonding network involving E493, D494, H593, R609, and A636. Compound 8a also demonstrated favorable interactions with catalytic-site residues. Overall, the docking analysis supported the experimental inhibition results.

DFT studies

The calculated quantum chemical descriptors are summarized in Table 4. The frontier molecular orbital distributions and molecular electrostatic potential maps of the reference compounds are presented in Figure 13, whereas the corresponding analyses for the synthesized compounds are shown in Figure 14 and Figure 15.

Among the synthesized compounds, 9e exhibited the smallest HOMO–LUMO energy gap (0.14302 a.u.), followed by 8c (0.14831 a.u.) and 8a (0.15788 a.u.). The molecular electrostatic potential maps identified electron-rich and electron-deficient regions that may contribute to intermolecular interactions. The calculated global reactivity descriptors, including ionization potential, electron affinity, electronegativity, chemical potential, hardness, softness, and electrophilicity index, are listed in Table 4. Compounds of the 9-series exhibited lower HOMO–LUMO gaps and higher electrophilicity than the corresponding 8-series derivatives.

ADME analysis

The predicted pharmacokinetic properties are summarized in Table 5. All synthesized compounds satisfied the major drug-likeness criteria. The 9-series derivatives showed no Lipinski violations, whereas each compound in the 8-series exhibited one violation. All compounds were predicted to possess high gastrointestinal absorption, no blood–brain barrier permeability, and acceptable hydrogen-bond donor and acceptor counts. Bioavailability scores were consistently predicted to be 0.55. Synthetic accessibility scores ranged from 5.70 to 6.39, indicating moderate synthetic complexity.

DATA AVAILABILITY:

The datasets and supporting materials generated during this study have been deposited in the Zenodo repository and are publicly available at https://doi.org/10.5281/zenodo.21238952.

Figure 1: Synthetic route for the preparation of triazole- and oxadiazole-based acetamide derivatives. Reaction scheme illustrating the synthesis of intermediates and final compounds 8(a–c) and 9(d–f). Please click here to view a larger version of this figure.

Figure 2: NMR spectra of compound 8a. (A) ¹H NMR spectrum of compound 8a. (B) ¹³C NMR spectrum of compound 8a. Please click here to view a larger version of this figure.

Figure 3: NMR spectra of compound 8c. (A) ¹H NMR spectrum of compound 8c. (B) ¹³C NMR spectrum of compound 8c. Please click here to view a larger version of this figure.

Figure 4: NMR spectra of compound 9e. (A) ¹H NMR spectrum of compound 9e. (B) ¹³C NMR spectrum of compound 9e. Please click here to view a larger version of this figure.

Figure 5: Inhibitory activities of the synthesized compounds against α-glucosidase and urease. (A) Percentage inhibition of α-glucosidase and urease by compounds 3, 4, 8(a–c), and 9(d–f) compared with the reference inhibitors acarbose and thiourea, respectively. (B) IC₅₀ values of the same compounds against α-glucosidase and urease. Data are presented as mean ± SD (n = 3). Statistical significance was determined relative to the corresponding reference inhibitor (p < 0.05, p < 0.01, p < 0.001, p < 0.0001; ns = not significant). Please click here to view a larger version of this figure.

Figure 6: Fluorescence quenching analysis of bovine serum albumin (BSA) by compound 8a. (A) Fluorescence emission spectra of BSA (3.33 × 10⁻⁶ M) following incremental addition of compound 8a at 298 K. (B) Stern–Volmer plots obtained at 298, 308, and 313 K. (C) Double-logarithmic plot of log[(F₀ − F)/F] versus log Q. Please click here to view a larger version of this figure.

Figure 7: Fluorescence quenching analysis of bovine serum albumin (BSA) by compound 8b. (A) Fluorescence emission spectra of BSA (3.33 × 10⁻⁶ M) following incremental addition of compound 8b at 298 K. (B) Stern–Volmer plots obtained at 298, 308, and 313 K. (C) Double-logarithmic plot of log[(F₀ − F)/F] versus log Q. Please click here to view a larger version of this figure.

Figure 8: Fluorescence quenching analysis of bovine serum albumin (BSA) by compound 8c. (A) Fluorescence emission spectra of BSA (3.33 × 10⁻⁶ M) following incremental addition of compound 8c at 298 K. (B) Stern–Volmer plots obtained at 298, 308, and 313 K. (C) Double-logarithmic plot of log[(F₀ − F)/F] versus log Q. Please click here to view a larger version of this figure.

Figure 9: Fluorescence quenching analysis of bovine serum albumin (BSA) by compound 9d. (A) Fluorescence emission spectra of BSA (3.33 × 10⁻⁶ M) following incremental addition of compound 9d at 298 K. (B) Stern–Volmer plots obtained at 298, 308, and 313 K. (C) Double-logarithmic plot of log[(F₀ − F)/F] versus log Q. Please click here to view a larger version of this figure.

Figure 10: Fluorescence quenching analysis of bovine serum albumin (BSA) by compound 9e. (A) Fluorescence emission spectra of BSA (3.33 × 10⁻⁶ M) following incremental addition of compound 9e at 298 K. (B) Stern–Volmer plots obtained at 298, 308, and 313 K. (C) Double-logarithmic plot of log[(F₀ − F)/F] versus log Q. Please click here to view a larger version of this figure.

Figure 11: Fluorescence quenching analysis of bovine serum albumin (BSA) by compound 9f. (A) Fluorescence emission spectra of BSA (3.33 × 10⁻⁶ M) following incremental addition of compound 9f at 298 K. (B) Stern–Volmer plots obtained at 298, 308, and 313 K. (C) Double-logarithmic plot of log[(F₀ − F)/F] versus log Q. Please click here to view a larger version of this figure.

Figure 12: Binding modes of the reference inhibitors and selected synthesized compounds within the α-glucosidase and urease active sites. (A) Binding pose of acarbose within the α-glucosidase active site. (B) Binding pose of compound 8a. (C) Binding pose of compound 8c. (D) Binding pose of compound 9e. (E) Binding pose of thiourea within the urease active site. (F) Binding pose of compound 8a. (G) Binding pose of compound 8c. (H) Binding pose of compound 9e. Hydrogen bonds are represented by yellow dashed lines. Please click here to view a larger version of this figure.

Figure 13: Frontier molecular orbitals and molecular electrostatic potential (MEP) maps of the reference inhibitors. (A) Acarbose. (B) Thiourea. The highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), and MEP surfaces are shown. Please click here to view a larger version of this figure.

Figure 14: Frontier molecular orbitals and molecular electrostatic potential (MEP) maps of the triazole derivatives. (A) Compound 8a. (B) Compound 8b. (C) Compound 8c. The HOMO, LUMO, and MEP surfaces are shown for each compound. Please click here to view a larger version of this figure.

Figure 15: Frontier molecular orbitals and molecular electrostatic potential (MEP) maps of the oxadiazole derivatives. (A) Compound 9d. (B) Compound 9e. (C) Compound 9f. The HOMO, LUMO, and MEP surfaces are shown for each compound. Please click here to view a larger version of this figure.

Codes	Urease		α-Glucosidase
	Inhibition (%) at 0.5 mM	IC50 (µM)	Inhibition (%) at 0.5 mM	IC50 (µM)
3	67.54 ± 1.03	41.51 ± 0.01	63.09 ± 0.25	79.12 ± 0.43
4	62.51 ± 0.42	45.14 ± 0.14	65.26 ± 0.55	70.28 ± 0.42
8a	95.22 ± 1.55	12.25 ± 0.42	95.01 ± 0.57	33.15±0.07
8b	62.64 ± 1.01	82.15 ±1.48	38.02 ± 1.71	108.19 ± 0.18
8c	99.39 ± 0.27	06.14 ± 1.06	90.51 ± 0.01	40.93 ± 0.59
9d	47.51 ± 1.01	103.05 ± 0.54	39.08 ± 0.25	108.10 ± 0.03
9e	98.57 ± 0.04	08.14 ± 0.71	91.55 ± 0.34	27.29 ± 0.41
9f	58.07 ± 1.68	85.15 ± 0.12	43.15 ± 0.42	94.44 ± 0.25
Thiourea	96.24 ± 0.16	21.25 ± 0.15	-	-
Acarbose	-	-	92.23 ± 0.14	38.25 ± 0.12

Table 1: Inhibitory activities of the synthesized compounds against α-glucosidase and urease. IC₅₀ values and percentage inhibition of compounds 8(a–c) and 9(d–f) compared with the reference inhibitors acarbose and thiourea. Data are presented as mean ± SD (n = 3). Abbreviations: IC₅₀ = half-maximal inhibitory concentration; SD = standard deviation.

Compound		298K				308K		313K
	N	Kb (M-1)	ΔG	ΔH	ΔS (J/mol.K)	N	Kb (M-1)	N	Kb (M-1)
			(kJ/mol)	(kJ/mol)
8a	1.28	3.92 x105	-32.55	-218.28	-623.24	1.12	6.49 x104	0.85	4.33 x103
8b	1.31	6.49 x105	-34.24	-306.6	-913.96	1.1	6.95 x104	0.66	1.07 x103
9d	1.47	2.41 x106	-36.32	-204.1	-563.01	1.17	1.44 x105	1.04	4.84 x104
9e	0.62	6.78 x101	-10.32	243.16	850.61	0.81	1.32 x103	0.91	7.93 x103
9f	1.11	4.69 x104	-26.35	178.13	686.21	1.26	2.99 x105	1.41	1.67 x106
8c	0.91	1.54 x104	-23.98	341.31	1225.82	1.36	1.53 x105	1.6	1.10 x107

Table 2: Binding constants and thermodynamic parameters for the interactions of selected compounds with bovine serum albumin (BSA). Binding constant (Kb), number of binding sites (n), Stern–Volmer quenching constant (Ksv), bimolecular quenching rate constant (Kq), Gibbs free energy (ΔG), enthalpy change (ΔH), and entropy change (ΔS) for compounds 8a, 8c, and 9e determined at 298, 308, and 313 K. Abbreviations: BSA = bovine serum albumin; Kb = binding constant; n = number of binding sites; Ksv = Stern–Volmer quenching constant; Kq = bimolecular quenching rate constant; ΔG = Gibbs free energy; ΔH = enthalpy change; ΔS = entropy change.

Compounds	Alpha Glucosidase (kcal/mol)	Urease (kcal/mol)
Acarbose	−6.90	-
Thiourea	-	−5.80
3	−4.35	−4.50
4	−4.10	−4.30
8a	−7.165	−7.624
8b	−5.20	−5.45
8c	−6.80	−7.10
9d	−5.10	−5.35
9e	−6.70	−7.30
9f	−5.00	−5.25

Table 3: Molecular docking scores of the synthesized compounds against α-glucosidase and urease. Docking scores (kcal/mol) of compounds 8(a–c) and 9(d–f), together with the reference inhibitors acarbose and thiourea, against α-glucosidase and urease. Abbreviations: kcal/mol = kilocalories per mole.

Ligand	Dipole moment (Debye)	HOMO	LUMO	Energy	Ionization	Electron affinity (eV)	Electro- negativity χ (eV)	Electro- chemical potential μ (eV)	Hardness η (eV)	Softness	Electro- philicity
		(a.u.)	(a.u.)	Gap (ΔEGap)	Potential (eV)					S (eV)	ω (eV)
Acarbose	8.1124	-0.1597	-0.0336	0.12612	4.345	0.913	2.629	-2.629	1.716	0.291	2.015
Thiourea	7.619	-0.2193	-0.0179	0.20135	5.967	0.488	3.228	-3.228	2.739	0.182	1.903
8a	8.8025	-0.2044	-0.0466	0.15788	5.566	1.266	3.416	-3.416	2.15	0.233	2.71
8b	8.2451	-0.2033	-0.0467	0.15659	5.534	1.27	3.402	-3.402	2.132	0.234	2.72
8c	8.319	-0.2039	-0.0555	0.14831	5.55	1.511	3.53	-3.53	2.02	0.247	3.08
9d	3.8264	-0.2065	-0.0621	0.14432	5.619	1.691	3.655	-3.655	1.964	0.255	3.4
9e	4.0969	-0.2052	-0.0621	0.14302	5.584	1.691	3.637	-3.637	1.947	0.257	3.4
9f	4.298	-0.2059	-0.0627	0.14324	5.604	1.704	3.654	-3.654	1.95	0.256	3.42

Table 4: Density functional theory (DFT) descriptors of the synthesized compounds. Calculated quantum chemical descriptors of compounds 8(a–c) and 9(d–f), together with the reference compounds acarbose and thiourea, including HOMO and LUMO energies, HOMO–LUMO energy gap (ΔE), ionization potential (IP), electron affinity (EA), electronegativity (χ), chemical potential (µ), chemical hardness (η), chemical softness (S), dipole moment (D), and electrophilicity index (ω). Abbreviations: DFT = density functional theory; HOMO = highest occupied molecular orbital; LUMO = lowest unoccupied molecular orbital; ΔE = HOMO–LUMO energy gap; IP = ionization potential; EA = electron affinity; χ = electronegativity; µ = chemical potential; η = chemical hardness; S = chemical softness; D = dipole moment; ω = electrophilicity index.

Compound	HA	RB	HBA	HBD	TPSA (Ų)	iLOGP	Consensus LogP	GI Absorption	BBB Permeability	Lipinski Violations	Bioavailability Score	SA Score
8a	30	7	5	4	84.86	4.41	4.71	High	No	1	0.55	6.32
8b	30	7	5	4	84.06	4.41	4.62	High	No	1	0.55	6.34
8c	30	7	5	4	84.26	4.41	4.11	High	No	1	0.55	6.39
9d	24	6	5	4	90.95	3.08	2.75	High	No	0	0.55	5.71
9e	24	6	5	4	90.82	3.08	2.27	High	No	0	0.55	5.7
9f	24	6	5	4	90.85	3.08	2.77	High	No	0	0.55	5.73

Table 5: Predicted absorption, distribution, metabolism, and excretion (ADME) properties of the synthesized compounds. Predicted physicochemical, pharmacokinetic, and drug-likeness properties of compounds 8(a–c) and 9(d–f) were generated using the SwissADME web server. Abbreviations: ADME = absorption, distribution, metabolism, and excretion; MW = molecular weight; LogP = octanol/water partition coefficient; TPSA = topological polar surface area; RB = rotatable bonds; HBA = hydrogen bond acceptor; HBD = hydrogen bond donor; GI = gastrointestinal; BBB = blood–brain barrier; SA = synthetic accessibility.

Supplementary Figure 1: Docking validation through redocking of native ligands. (A) Superimposition of the co-crystallized ligand (reference pose) and redocked acarbose within the active site of α-glucosidase (PDB ID: 5NN8), demonstrating close alignment of binding conformations. (B) Binding pose of thiourea within the active site of urease (PDB ID: 4H9M), used for docking validation of the urease system, showing preservation of key interactions within the catalytic pocket.Please click here to download this file.

Supplementary File 1: Physicochemical characterization data and complete ¹H and ¹³C NMR spectra of compounds 3, 4, 8(a–c), and 9(d–f).Please click here to download this file.

Discussion

This study synthesized a series of triazole- and oxadiazole-based acetamide derivatives and evaluated their structural, biological, computational, and pharmacokinetic properties. Spectroscopic characterization by HRMS, elemental analysis, ¹H NMR, and ¹³C NMR confirmed the successful synthesis of the target molecules. The observed chemical shifts, coupling patterns, and carbon resonances were consistent with the proposed structures. Characteristic amide NH signals, methylene resonances, aromatic proton distributions, and quaternary carbon signals further confirmed the successful incorporation of the substituted acetamide moieties into both the triazole and oxadiazole scaffolds. The generally high synthetic yields obtained for these derivatives demonstrate the efficiency of the synthetic route and suggest that these heterocyclic frameworks are amenable to further structural modification.

A clear influence of the substituent pattern on the urease inhibitory activity was observed. Compound 8c, bearing a 2,6-dimethyl substituent, exhibited the highest urease inhibitory activity, followed by compounds 8a (2,3-dimethyl) and 9e (2,5-dimethyl), identifying these molecules as promising lead candidates. A similar trend was observed against α-glucosidase, with compound 9e displaying the strongest inhibition, whereas compounds 8a and 8c also demonstrated considerable activity. Because compounds 8a and 8c differ only in the positions of the methyl substituents on the aromatic ring, whereas compound 9e differs in both substitution pattern and heterocyclic scaffold, these findings suggest that both methyl-substitution pattern and scaffold architecture contribute to biological activity within this series. The superior activity of compound 8c may reflect a more favorable orientation within the enzyme active site, whereas the strong activity of compound 9e suggests an additional contribution from the oxadiazole scaffold together with the 2,5-dimethyl substitution pattern. These observations are consistent with previous reports indicating that steric effects arising from phenyl substitution can influence active-site binding in triazole- and oxadiazole-based enzyme inhibitors^24,25,26,27. Nevertheless, establishing a definitive structure–activity relationship will require evaluation of a larger series of regioisomeric derivatives.

Analysis of the interactions between the most active compounds and bovine serum albumin (BSA) by fluorescence quenching and thermodynamic analysis demonstrated that compounds 8a and 9e followed a mixed static/dynamic quenching mechanism, whereas compound 8c exhibited predominantly static quenching. Thermodynamic parameters indicated that binding of compound 8a was primarily driven by hydrogen bonding and van der Waals interactions, whereas hydrophobic interactions predominated in the binding of compounds 8c and 9e. Compound 8a exhibited the strongest affinity for BSA, with a binding constant of 3.92 × 10⁵ M⁻¹ and a favorable negative Gibbs free energy of binding. These findings indicate stable protein–ligand complex formation and suggest the potential for albumin-mediated transport under physiological conditions.

The experimental findings were further supported by molecular docking studies, which demonstrated that compounds 8a, 8c, and 9e adopted favorable binding conformations within the active sites of both α-glucosidase and urease. These compounds formed hydrogen bonds with key residues, including D282, A284, D404, R600, D616, and H674 in α-glucosidase and A636, Q635, R439, and A440 in urease. Similar interactions have previously been reported for structurally related enzyme inhibitors^28,29. The greatest discrepancy between docking score and experimental activity was observed for compound 8a, which exhibited the most favorable docking score against α-glucosidase (−7.165 kcal/mol), whereas compound 9e showed the greatest in vitro inhibitory potency (IC₅₀ = 27.29 µM). This difference is not unexpected because docking scores estimate ligand binding within a relatively rigid protein structure and do not account for factors such as ligand solubility, conformational flexibility, desolvation, protein dynamics, or other solution-phase effects that influence experimentally determined IC₅₀ values. Consequently, differences between predicted binding affinity and measured biological activity represent a recognized limitation of molecular docking rather than a contradiction between computational and experimental results. Overall, the docking and enzyme inhibition data consistently identify compounds 8a, 8c, and 9e as the most promising derivatives in this series.

The electronic properties of the most active compounds were further investigated by frontier molecular orbital (FMO) and molecular electrostatic potential (MEP) analyses. Compounds with lower HOMO–LUMO energy gaps generally exhibited greater electronic reactivity, consistent with previous DFT studies of structurally related triazole- and oxadiazole-based derivatives³⁰. Among the synthesized compounds, 8a, 8c, and 9e exhibited relatively small HOMO–LUMO energy gaps, indicating favorable electron-transfer characteristics. Their MEP maps revealed distinct electron-rich and electron-deficient regions, with compounds 8c and 9e displaying particularly pronounced nucleophilic regions that may contribute to their favorable enzyme interactions and biological activities. Global reactivity descriptors further supported the experimental and computational findings. Compound 9e exhibited a favorable combination of ionization potential, electron affinity, softness, and electrophilicity index, whereas compounds 8a and 8c showed comparatively high softness values that are consistent with their strong inhibitory activities. The greater electrophilicity and softness observed for the 9-series, particularly compound 9e, may contribute to stronger interactions with catalytic residues and enhanced biological activity.

In silico ADME profiling of compounds 8(a–c) and 9(d–f) predicted favorable pharmacokinetic characteristics, including high gastrointestinal absorption, acceptable drug-likeness, and satisfactory oral bioavailability. No major ADME liabilities were identified within the evaluated parameters. Collectively, the in vitro biological data, BSA-binding analysis, molecular docking, DFT calculations, and in silico ADME predictions support continued investigation of these triazole- and oxadiazole-based acetamide derivatives as dual urease and α-glucosidase inhibitors.

Taken together, these findings identify compounds 8a, 8c, and 9e as promising lead candidates for developing triazole- and oxadiazole-based acetamide derivatives targeting urease and α-glucosidase. The combined spectroscopic, biological, BSA-binding, molecular docking, DFT, and ADME data provide a strong foundation for future studies. Further investigations should include molecular dynamics simulations to evaluate protein–ligand stability over time, enzyme kinetic studies to determine the mechanism of inhibition, and extension of the BSA-binding analysis to the remaining derivatives. Finally, in vivo studies will be necessary to validate the promising in vitro and in silico findings and to further assess the therapeutic potential of this compound series.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1-Naphthylamine	Merck	106148	Aromatic amine used for preparation of substituted analogs
2,5-Dimethylaniline	Sigma-Aldrich	D146706	Aromatic amine used for synthesis of substituted derivatives
3,4-Dimethylaniline	Alfa Aesar	A15416	Aromatic amine used for preparation of structural analogs
Benzoic Acid	Sigma-Aldrich	242381	Starting material for synthesis of benzohydrazide intermediate
Bromoacetyl Bromide	Sigma-Aldrich	B17601	Electrophilic reagent used in synthesis of bromoacetamide intermediates
Carbon Disulfide	Sigma-Aldrich	289116	Key reagent for cyclization to oxadiazole ring system
Chloroform	Sigma-Aldrich	C2432	Solvent used in extraction and purification processes
Dimethylformamide (DMF)	Sigma-Aldrich	227056	Polar aprotic solvent used in derivatization and substitution reactions
Diphenylamine	Sigma-Aldrich	D23802	Aromatic amine used in synthesis of derivative compounds
Epik (2022-1)	Schrödinger, LLC	N/A (software)	Used to generate ligand ionization states at pH 7.0; RRID: N/A
Ethanol	Sigma-Aldrich	E7023	Reaction solvent for esterification and hydrazide formation
Ethyl Acetate	Sigma-Aldrich	270989	Polar mobile phase component in TLC analysis and extraction
Gaussian 09, Revision D.01 (Gaussian 09W)	Gaussian, Inc.	N/A (software)	Software used for DFT calculations (HOMO-LUMO, MESP, global reactivity descriptors); RRID:SCR_014897
GaussView 5	Gaussian, Inc., Wallingford, CT, USA	N/A (software)	Used for visualization of optimized structures and orbital energies; RRID: N/A
Glide (2022-1)	Schrödinger, LLC	N/A (software)	Docking software used for receptor grid generation and extra-precision (XP) molecular docking; RRID:SCR_000187
GraphPad Prism, version 9	GraphPad Software, Boston, MA, USA	N/A (software)	Used for IC50 regression analysis and statistical evaluation of enzyme inhibition data; RRID:SCR_002798
Hydrazine	Sigma-Aldrich	225819	Reagent used for conversion of ester to benzohydrazide
L(+)-Glutamine	Sigma-Aldrich	G3126	Reagent used in biological evaluation studies
LigPrep (2022-1)	Schrödinger, LLC	N/A (software)	Used for ligand structure optimization and 3D conformer generation; RRID:SCR_016746
Lithium Hydride	Sigma-Aldrich	201049	Catalyst used for activation of thiol group in substitution reactions
Maestro (v13.2, Schrödinger Release 2022-1)	Schrödinger, LLC	N/A (software)	Molecular modeling suite used for protein/ligand structure preparation and visualization; RRID:SCR_016748
Methanol	Sigma-Aldrich	34860	Solvent used in purification and biological assay preparations
n-Hexane	Merck	104391	Non-polar mobile phase component in TLC analysis
OPLS-2005 / OPLS-AA	Schrödinger, LLC	N/A (force field)	Force field used for energy minimization of protein and ligand structures; RRID: N/A
Phenyl Isothiocyanate	Merck	807028	Reagent used in synthesis of triazole derivatives
Protein Preparation Wizard (2022-1)	Schrödinger, LLC	N/A (software)	Used for receptor structure refinement prior to docking; RRID:SCR_016749
Sodium Hydroxide	Sigma-Aldrich	221465	Base used for pH adjustment and reaction neutralization
Sulfuric Acid	BDH	10276	Acid catalyst used in esterification step under reflux conditions
Trimethylamine	Riedel-de Haen	22129	Organic base used during synthesis of substituted derivatives
VESTA, version 3.5.8	Momma & Izumi (developers); freely distributed	N/A (software)	Used for visualization of molecular electrostatic potential (MESP) and HOMO-LUMO surface maps; RRID: N/A