1. INTRODUCTION:

Type 2 diabetes is a metabolic disorder caused by insulin resistance and thereby impaired insulin secretion, which often results in hyperglycemia.^1,2 Patients with chronic hyperglycemia suffer from the ever-growing incidence of neuropathy, retinopathy, and cardiovascular diseases, especially coronary heart disease.^3,4 Reports have documented that the inhibition of the carbohydrate-hydrolyzing enzyme α-glucosidase in the small intestines can control postprandial hyperglycemia.^5,6

In contrast to most of the antidiabetic drugs like sulfonylureas and dipeptidyl peptidase-4 (DDP4) inhibitors which generally act via increased insulin secretion or sensitivity⁷, α-glucosidase inhibitors competitively inhibit α-glucosidase and delay postprandial absorption of glucose from the gastrointestinal tract.^8,9 The International Diabetes Federation (IDF) recommends and supports the use of α-glucosidase inhibitors as first-line drugs in patients with uncontrolled diabetes for the management of postprandial hyperglycemia, either in monotherapy or in combination therapy.¹⁰ Acarbose, miglitol, and voglibose are some FDA-approved α-glucosidase inhibitors that maintain glucose levels in the bloodstream, reduce the risk of cardiovascular diseases, and thereby increase the life expectancy of patients with type 2 diabetes mellitus (T2DM).

However, these drugs have their limitations, which include abdominal distension, flatulence, meteorism, and possibly diarrhoea. Therefore, it is imperative to explore other scaffolds for the generation of α-glucosidase inhibitors.¹¹ Imidazole, despite being a small chemical entity, is a privileged moiety with the possibility of versatile chemical functionalization and complexity in its structure.^12,13 Interestingly, 2,4,5-trisubstituted imidazoles have drawn a lot of attention due to their exceptional biological actions, including platelet aggregation inhibitor,^14,15 non-steroidal anti-inflammator,¹⁶ antiproliferative agents,^17-19 anti-microbial,²⁰antimycobacterial, and anti-inflammatory analgesic activities. Encouraged by these diverse activities reported for 2,4,5-triarylated imidazoles, our research group has recently synthesised a new library of compounds incorporating 2,4,5-triphenyl imidazoles (TSIs) (Figure 1) by utilising a novel L-pipecolic acid-catalysed highly efficient synthetic approach²¹(Figure 1). Reports have documented that benzimidazole analogues serve as potential inhibitors of α-glucosidase enzymes. Considering this, we speculated that our synthesised 2,4,5-triphenyl imidazole analogues, TSIs, could better ameliorate T2DM via a plausible concurrent interaction with α-glucosidase. To elucidate the anti-hyperglycemic activity, in vitro activity was carried out with glucosidase moleculer target. Consequently, in silico molecular docking of these synthesised TSIs to the target α-glucosidase (PDB: 3WY1) was done to analyse their interaction with the assigned target.

The present study is an endeavour to provide new insights into the inhibitory mechanistic and computational attributes of α-glucosidase to advocate the ameliorative potential of novel imidazole derivatives against hyperglycemia.

Figure 1: Structures of synthesized 2,4,5-trisubstituted imidazole (R= OH,CH₃, OCH₃, F,Cl, NO₂,)

2. MATERIALS AND METHODS:

2.1. Chemicals:

α-glucosidase (Saccharomyces cerevisiae) and 3,5-di-nitro salicylic acid (DNS) were procured from Sigma-Aldrich, Bangalore. p-nitro-phenyl-α-D-glucopyranoside (p-NPG), sodium carbonate (Na₂CO₃), sodium dihydrogen phosphate (NaH₂PO₄), and di-sodium hydrogen phosphate (Na₂HPO₄) were purchased from Hi-Media, Mumbai.

2.2. In vitro α-glucosidase inhibitory activity:

α-glucosidase inhibitory activity was conducted as per the standard procedure with minor modifications.²² In a 96-well plate, a reaction mixture containing 50μL phosphate buffer (100mM, pH = 6.8), 10μL α-glucosidase (1U/mL), and 20μL of varying concentrations of TSIs (0, 100, 200, 300, 400, 500, and 1000μg/mL) was pre-incubated at 37°C for 15min. Then, 20μL of P-NPG (5mM) was added as a substrate and incubated further at 37°C for 20min. The reaction was finished by adding 50μL of Na₂CO₃ (0.1M). The absorbance of the released p-nitrophenol was measured at 405nm using an ELISA Microplate (iMark Bio Rad) reader. Acarbose at varying concentrations (0–1000 µg/mL) was used as a positive control. Control was also set up in parallel, and the experiments were performed in triplicate. The results were expressed as percentage inhibition, which was calculated using the formula:

Inhibitory activity (%) = (1 − As/Ac) ×100. Where As is the absorbance in the presence of the test substance and Ac is the absorbance of the control.

2.3. Statistical Analysis:

All the measurements were done in triplicate, and the results are expressed in terms of the mean±standard deviation. The percentage inhibition and IC₅₀ values were calculated using GraphPad Prism 5 version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA.) statistical software.

2.4 Preparation of Ligand and Protein:

Molecular docking was conducted to evaluate the precise binding affinity of ligands on the assigned protein targets and their subsequent assessment of docking poses. The 3-D structure of human liposomal acid α-glucosidase (PDB: 3WYI) was retrieved and downloaded in PDB format from the database of the Protein Data Bank (https://www.rcsb.org/). The previous ligands bonded to the selected proteins and molecules of water were detached.^23,24 The chemistry of proteins was corrected for the missing hydrogen before they were optimized for energy. Employing Autodock version 4.2.6 and Biovia Discovery Studio, the protein receptor was enriched and modified. After the water molecules were eliminated and the x, y, and z coordinates were set to 1, -18, 17, and -4.42 with a radius of 1.000 Å, the amino acid residues were provided with polar hydrogen atoms. The PDBQT format of the protein was produced, and the Auto Grid program was used to impute it.²⁴ The structures of synthesized ligands were optimized in 3D PDB format by Avogadro program 1.2.0n and were converted to PDBQT files (pdbqt format) by correcting the torsion angles using Autodock (Ligand options) for docking with different receptors through Autodock Vina 1.1.2.^25,26

2.5 Docking Methodology:

The fourteen optimised ligands from the TSIs series were docked into the active binding pocket of the receptor downloaded from the Protein Data Bank.^27,28 The crystal structure of alpha-glucosidase (PDB:3WY1) (Table 1) was selected as a target for in-silico molecular modeling. Grid points were generated surrounding the active site of the target, which was bound with the available co-crystallised ligand wherever available. The co-crystallised molecule has been taken as a reference to assign the binding site for the ligand X-ray group. All the previously synthesised compounds (TSIs) and the reference drug acarbose were docked individually against the generated grid to examine the binding pattern and energy of ligands with all three assigned targets.^29,30The software-generated grid was initially validated by docking the reference ligand, acarbose, against the assigned targets. A flexible docking module was incorporated in AutodockVina 1.1.2.

The results were evaluated using Discovery Studio Visualizer.³¹ The programmes were used with default docking parameters.

2.6 SWISS-ADME:

The Swiss ADME software web tool was utilize to evaluate the individual ADME characteristics of the synthesized compounds.³² The drug-like properties, Pharmacokinetics and physicochemical properties were studied and Brain Or IntestinaL EstimateD permeation method (BOILED EGG)was employed to know the permeation of the synthesized molecules (Figure 5).

3 RESULTS AND DISCUSSION:

3.1 In- vitro α-glucosidase inhibition assay:

In the current studies, all the synthesised imidazole derivatives (3a–3n) were evaluated for α-glucosidase inhibitory activity using acarbose as a standard inhibitor. The substitutions were mainly introduced in the phenyl ring present at the second position of the imidazole moiety. The 3e was found to have three OH (electron-donating groups) present at 2, 3, and 4 positions. Compounds 3a, 3b, and 3f also had OH and OCH₃ substitutions at the 2nd, 3^rd, 4^th, and 5^th positions, which increased the glucosidase inhibitory activity. The replacement of the OH group with halogens and other electron-withdrawing groups like F, Cl, and NO₂ at the same positions somehow reduces the activity in compounds 3c, 3d, 3g, 3j, 3k, and 3m. Those molecules having substitution of electron withdrawing as well as electron donating group compounds 3h, 3i, 3l, and 3n showed promising results (Figure 2).

Amongst all the synthesised compounds, 3e was the most active, showing 49.18±2.0 α-glucosidase percentage inhibition at 0.5mM and having an IC50 value of 4.3μg/ml (Table 1, Figure 4). Apart from this, two other compounds, 3a and 3b, were also found to have reasonable inhibition, having an IC50 value of 4.8 μg/ml and 5.3μg/ml, respectively.

Figure 2: α-gluosidase inhibition assay of 3a –3n synthesized compound using Acarbose as standard drug.

3.2 Molecular Modelling:

The novel compounds 3a–3n were docked to analyse the possibility of interaction with the amino acid of the protein α-glucosidase (PDB ID: 3WY1). All the molecules were docked in the same binding pockets. The derivatives of imidazole showed comparable binding energy when compared with the reference compound, acarbose (Table 1). Standard drug aacarbose showed pi-alkyl and hydrogen bond interactions with GLY228, ASP333, and ARG400 (Figure 3). Similarly, most of the TSIs showed a common binding pattern with the amino acids of the targeted protein. The phenyl ring present at the 4^th and 5^th positions of the imidazole showed pi-alkyl and pi-anion kinds of interactions with GLY228, PHE297, ASP333, VAL334, HIS332, PHE297, and ARG400 amino acids. The imidazole ring created a pi-anion interaction with, and its NH fragment was involved in a hydrogen bond with ASP333. The third phenyl moiety in position 2 (3a–3n) created a pi-alkyl, pi-anion interaction with VAL334, HIS332, PHE297, and ARG400. (Figure1). Variation in the binding energy may be due to the varied substituent attached to the phenyl ring present at the 2^nd position of imidazole scaffold (Figure 4).

Figure 3: Visualisation image of a-glucosidase docked with standard drug –Acarbose

Figure 4: Visualisation image of compound 3e docked in the receptor.

Table 1: In silico prediction results of compounds 2,4,5-trisubstituted imidazole’s (3a-n)

Sr, No.	Sample Code Product	IC50 Value (μg/ml)	% Inhibition (0.5 mM	Binding Affinity (Kcal/Mol)against α glucosidase
1.	Acarbose	4.0	54.08 ± 2.07	-8.2
2.	3a	4.8	46.29 ± 2.09	-8
3.	3b	5.3	43.18 ± 1.09	-7.9
4.	3c	11	40.20± 2.11	-7.8
5.	3d	15.4	40.8± 2.10	-8.2
6.	3e	4.3	49.18± 1.17	-8
7.	3f	6.4	40.17± 2.11	-7.5
8.	3g	13.2	41.12± 2.17	-7.9
9.	3h	11.4	41.11± 1.09	-7.8
10.	3i	10.9	41.12± 2.21	-8.1
11.	3j	14.1	40.12± 1.02	-8.2
12.	3k	15.6	40.01± 2.02	-8.2
13.	3l	10.5	42.16± 2.01	-8.1
14.	3m	20.9	40.10± 1.09	-7.8
15.	3n	10.2	40.10± 2.10	-7.4

3.3 ADME Profile:

The result showed the drug-like characteristics of 3e the most potent compound of the series, with no violation of the Lipinski rule. This is corroborated by the radar plot. Furthermore, the BOILED EGG model demonstrated that the molecule was present in the gastrointestinal region (Figure 5).

Figure 5: ADME prediction a) Radar plot, b) Boiled egg model

4. CONCLUSION:

Amongst the synthesised compounds, Compound 3e was found to be the most active, with an IC50 value of 4.3 μg/mL as compared to the standard drug acarbose of 4.0 μg/mL. Further, the findings of in silico molecular docking with the assigned target demonstrated comparable binding affinity with that of the reference drug Acarbose. Importantly, the study suggested that most TSIs showed the formation of a stable binding complex with the binding pockets of the glucosidase enzyme. The compounds were also found to possess the required ADME characteristic. This study has prompted us to further explore these molecules In vivo. There is a significant role of these molecules in inhibition, and they can be a promising lead to developing more non-glucosidic inhibitors for diabetes.

5. LIST OF ABBREVIATIONS:

MCRs = multi-component reactions, T2DM = Type 2 Diabetes Mellitus, PDB = Protein Data Bank,TSIs = trisubstituted imidazoles

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest, financial or otherwise.

7. ACKNOWLEDGEMENTS:

Authors acknowledge Amity Institute of Pharmacy, Amity University for providing the research facilities to carry out this work.

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Received on 23.07.2024 Revised on 13.12.2024

Accepted on 24.02.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):3262-3267.

DOI: 10.52711/0974-360X.2025.00470

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

The results were evaluated using Discovery Studio Visualizer.31 The programmes were used with default docking parameters.

The results were evaluated using Discovery Studio Visualizer.³¹ The programmes were used with default docking parameters.