Exploring the Anti-bacterial Potential of Novel 2-Aminophenyl-2-(2,4,5-Triphenylimidazole) Acetate Derivatives: A Comprehensive Design and Synthesis Approach

The increasing prevalence of antibiotic-resistant bacterial infections has become a global public health concern, necessitating the development of novel anti-bacterial agents to combat these emerging threats.¹ In recent years, numerous studies have focused on the design and synthesis of new compounds with unique chemical structures and mechanisms of action, aiming to circumvent existing resistance pathways and improve the efficacy of anti-bacterial treatments. The biological activities of imidazole derivatives, which include anti-bacterial, anti-viral, and anti-cancer capabilities, have drawn lots of interest as one of the possible scaffolds for such molecules.^2,3 In this context, the present study explores the antimicrobial potential of novel 2-aminophenyl-2-(2,4,5-triphenylimidazole) derivatives, which combine the pharmacologically active imidazole core with strategically selected substituents to enhance their anti-bacterial activity. This research article delves into the comprehensive design and synthesis approach undertaken to generate these innovative molecules, detailing the rationale behind the choice of functional groups, the optimization of synthetic pathways, and the evaluation of their in vitro and in silico anti-bacterial properties.⁴ Our investigation begins with a thorough review of the current state of knowledge regarding imidazole derivatives and their antimicrobial activities, providing a solid foundation for the design of our target compounds. We then discuss the molecular modelling and docking studies employed to predict the interactions between the synthesized derivatives and key bacterial targets, facilitating a structure-activity relationship analysis that informs the optimization of the lead compounds.⁵ Following the synthesis and purification of the novel 2-aminophenyl-2- (2,4,5-triphenylimidazole) acetate derivatives, their anti-bacterial activities are assessed against a panel of Gram-positive and Gram-negative bacterial strains, including antibiotic-resistant isolates.⁶ Figure 1 illustrates the chemical structures of the designed 2-aminophenyl-2-(2,4,5-triphenylimidazole) acetate derivatives, showcasing the strategic incorporation of various substituents to enhance their antimicrobial activity. The molecular design of these derivatives was informed by the Structure-Activity Relationship (SAR) analysis, which revealed key features that may contribute to their potential as anti-bacterial agents.

Figure 1: Designed for derivatives 2-aminophenyl-2-(2,4,5-triphenylimidazole) acetate

Central to the design of these derivatives is the imidazole core, known for its diverse biological activities. The substitution of the imidazole ring with three phenyl groups (2,4,5-triphenyl) was chosen to improve hydrophobic interactions with bacterial target proteins, potentially increasing the binding affinity and, consequently, the overall anti-bacterial activity. The presence of the acetate group at the 1-yl position on the imidazole ring was selected to introduce a polar moiety, facilitating hydrogen bonding and enhancing the compound’s solubility.⁷ The 2-aminophenyl moiety was incorporated into the molecular structure to further optimize the balance between hydrophobic and hydrophilic interactions, as well as to provide a versatile scaffold for the introduction of additional functional groups. The amino group, in particular, allows for the formation of hydrogen bonds and salt bridges with target proteins, thus potentially increasing the specificity and potency of the designed derivatives.⁷ Following the synthesis of the 2-aminophenyl-2- (2,4,5-triphenylimidazole) acetate derivatives, their anti-bacterial activity was evaluated against a panel of Gram-positive and Gram-negative bacterial strains. Our results demonstrated that some of these novel compounds exhibited significant inhibitory effects against both susceptible and antibiotic-resistant bacteria, validating our design approach and highlighting their potential as new anti-bacterial agents.⁸ By unveiling the potential of these innovative compounds as a new class of anti-bacterial agents, our study contributes valuable insights to the ongoing quest for effective strategies against antibiotic-resistant bacterial infections. Furthermore, the comprehensive design and synthesis approach described herein may serve as a blueprint for future investigations aiming to develop additional antimicrobial scaffolds and expand the repertoire of therapeutic options available to combat the global threat of antimicrobial resistance.⁹

For this research study, all chemicals and reagents were of analytical grade and obtained from commercial suppliers. Solvents were purchased from Research Lab fine chem Industries, Mumbai. The starting materials for the synthesis of the target compounds, such as 2-aminophenyl acetic acid, 2,4,5-triphenyl-1H-imidazole, and various aniline derivatives, were procured from Research Lab fine chem Industries, Mumbai. The bacterial strains, Staphylococcus aureus and Escherichia coli were acquired from the Pravara Medical Trust, Loni. Mueller Hinton agar and nutrient broth were purchased from Prerana Enterprises, Ahmednagar. Ciprofloxacin, used as a positive control in the anti-bacterial activity assays, was obtained from RMI Laboratory OPC Pvt. Ltd. Instrument used for synthetic work were Digital Analytical balance (1 mg) of Electrolab, Digital Analytical balance (0.1 mg) of Contech, Digital Melting point apparatus Omkar Instruments Model BTI-34, Hot air Oven i-therm Model AI-7981, NMR 400MHz, 500MHz Bruker Advance III HD, FTIR Jasco 11. Mass Spectrometer Water Water acquity UPLC, Deep Freezer Kumar

The SwissADME web tool (http://www.swissadme.ch) was used to assess the Absorption, Distribution, Metabolism, and Excretion (ADME) characteristics of the synthesized compounds. This computational tool predicts various pharmacokinetics parameters, such as lipophilicity, water solubility, and drug-likeness, based on the molecular structures of the compounds. The SMILES notation of each compound was input into the SwissADME web tool, and the results were analyzed to assess their potential as drug candidates.¹⁰

The binding interactions between the produced drugs and their bacterial target proteins were examined utilizing molecular docking experiments with AutoDock Vina. The Protein Data Bank (PDB) was used to retrieve the target proteins’ crystal structures. Using the AutoDockTools program, the target proteins were cleaned up prior docking by removing water molecules and adding polar hydrogen atoms.¹⁰ The ligands (synthesized compounds) were prepared by converting their 2D structures into 3D conformations and adding hydrogen atoms using Open Babel software.A grid box was defined around the active site of the target proteins to guide the docking process. AutoDock Vina was then used to perform the molecular docking simulations, generating multiple docking poses for each compound. The binding affinity scores (in kcal/mol) were recorded for each pose, and the best binding pose with the lowest binding energy was selected for further analysis. The interactions between the ligands and target proteins were visualized using Discovery Visual Studio software to identify the key amino acid residues involved in binding and to assess the potential of the synthesized compounds as anti-bacterial agents.¹¹

Biotin Protein Ligase (BPL) is an essential enzyme in bacteria that is responsible for the post-translational modification of biotin-dependent carboxylases. It has been identified as a potential target for the development of novel anti-bacterial agents due to its crucial role in bacterial metabolism and the absence of a human homolog. The crystal structure of BPL (PDB ID: 4DQ2) was obtained from the Protein Data Bank (PDB) for molecular docking studies with the synthesized compounds.¹²

The agar disc diffusion technique was used to assess the anti-bacterial activity of the developed compounds. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were the test organisms utilized. On nutritional agar plates, the bacterial strains were inoculated, and sterile filter paper discs were laid on top of the agar. The filter paper discs were exposed to a 100 L solution containing 100 g/mL of each test substance. At the same concentration, ciprofloxacin was utilized as a positive control. The plates were incubated for 24 hr at 37°C. A ruler was used to measure the Zone of Inhibition (ZOI) in Millimeters (mm). A triplicate of each experiment was carried out, and the mean value and standard deviation were calculated. Based on the ZOI, the data were interpreted; a bigger zone of inhibition indicated greater anti-bacterial activity.^13,14

The 2-aminophenyl-2-(2,4,5-triphenyl-1H-imidazole-1-yl) derivatives synthesized in this study have shown promising anti-bacterial activity. A comprehensive analysis of the chemistry and Structure-Activity Relationship (SAR) of these derivatives was carried out to understand the key structural features responsible for their anti-bacterial properties, focusing on the substitutions to the amino group.¹⁵ Figure 2 illustrates the design of 2-Aminophenyl-2- (2,4,5-triphenyl-1H-imidazole-1-yl) acetate derivatives. The core scaffold of these derivatives comprises a 2-aminophenyl acetate moiety linked to a 2,4,5-triphenyl-1H-imidazole ring. The anti-bacterial activity of the synthesized compounds can be attributed to the presence of this core structure, which is essential for interaction with the target bacterial proteins. Additionally, the presence of an acetate group in the core structure appears to improve the lipophilicity of the derivatives, thereby facilitating their penetration into bacterial cells.^15,16

Figure 2: Design of 2-Aminophenyl-2-(2,4,5-triphenyl-1H-imidazole-1-yl)acetate derivatives.

Various substituents were introduced to the amino group of the 2-aminophenyl moiety to study their effects on the anti-bacterial activity. It was observed that the presence of electron-donating groups, such as alkyl or alkoxy groups, increased the anti-bacterial activity, while electron-withdrawing groups, such as halogens or nitro groups, reduced the activity. This finding suggests that the overall electron density on the amino group plays a crucial role in determining the anti-bacterial potency of the derivatives.¹⁷ Figure 3 shows scheme of synthesis for 2-Aminophenyl-2-(2,4,5-triphenyl-1H-imidazole-1-yl) derivatives (AC1 to AC6).

Figure 3: Scheme for synthesis of 2-Aminophenyl-2-(2,4,5-triphenyl-1H-imidazole-1-yl)derivatives (AC1 to AC6).

In a round-bottomed flask, we take 1 gm of 2-(2,4,5-triph enyl-1H-imidazole-1-yl)acetic acid, 1 mL 2-chloroaniline and 25 mL Glacial Acetic Acid (GAA) was mixed with each other. The mixture was heated in a water bath at 100°C for 2 hr. After the reaction was finished, 100 mL of ice-cold water was added to the mixture. Then the mixture was kept in fridge for 2-3 hr. The precipitated product was filtered and recrystallized with methanol to obtain 2-aminophenyl2-(2,4,5-triphenyl-1H-imida zole-1-yl)acetate.

In a round-bottomed flask, we take 1 gm of 2-aminophenyl2- (2,4,5-triphenyl-1H-imidazole-1-yl)acetate, 1 mL acetyl chloride and 25 mL methanol was mixed with each other. The mixture was heated in a water bath at 100°C for 2 hr. After the reaction was finished, 100mL of ice-cold water was added to the mixture. Then the mixture was kept in fridge for 2-3 hr. The precipitated product was filtered and recrystallized with methanol to obtain 2-N-phenylacetamide-(2,4,5-triphenyl- 4,5-dihydro-1H-imidazol-1-yl)acetate. The resulting product AC1 was obtained as a light-yellow solid with a yield of 78%. The melting point was determined to be 182-184°C. The ¹H-NMR spectrum revealed characteristic peaks at δ 2.10 (s, 3H, CH₃CO), 4.12 (s, 2H, CH₂), 6.99-7.58 (m, 19H, Ar-H), and 8.02 (s, 1H, NH). The FTIR spectrum showed prominent absorption bands at 3298 cm^-1 (NH stretching), 1683 cm^-1 (C=O stretching), and 1608 cm^-1 (C=C stretching).

In a round-bottomed flask, we take 1 gm of 2-aminophenyl2- (2,4,5-triphenyl-1H-imidazole-1-yl) acetate,1 gm acetaminophen and 25 mL methanol was mixed with each other. The mixture was heated in a water bath at 100°C for 2 hr. After the reaction was finished, 100 mL of ice-cold water was added to the mixture. Then the mixture was kept in fridge for 2-3 hr. The precipitated product was filtered and recrystallized with methanolto obtain N-phenylbenzene-1,4-diamine- phenylacetamide- (2,4,5-triphenyl-4,5-dihydro-1H-imidazol-1-yl)acetate. AC2 was synthesized as a pale-yellow solid, yielding 72%. The melting point was found to be 210-212°C. The ¹H-NMR spectrum displayed peaks at δ 2.09 (s, 3H, CH₃CO), 4.10 (s, 2H, CH₂), 6.95-7.60 (m, 24H, Ar-H), and 7.87 (s, 1H, NH). The FTIR spectrum exhibited characteristic bands at 3312 cm^-1 (NH stretching), 1677 cm^-1 (C=O stretching), and 1602 cm^-1 (C=C stretching).

In a round-bottomed flask, we take 1 gm of 2-aminophenyl2- (2,4,5-triphenyl-1H-imidazole-1-yl)acetate, 1 gm vanillin and 25 mL methanol was mixed with each other. The mixture was heated in a water bath at 100°C for 2 hr. After the reaction was finished, 100 mL of ice-cold water was added to the mixture. Then the mixture was kept in fridge for 2-3 hr. The precipitated product were filtered and recrystallized with methanol to obtain 4-[(3-methoxybenzaldehyde]-2-amino phenyl-(2,4,5-triphenyl- 4,5-dihydro-1H-imidazol-1-yl)acetate. AC3 was produced as a light brown solid with a yield of 65%. The melting point was measured to be 198-200°C. The ¹H-NMR spectrum showed peaks at δ 2.08 (s, 3H, CH₃CO), 3.81 (s, 3H, OCH₃), 4.11 (s, 2H, CH₂), 7.93 (s, 1H, CHO), 7.01-7.61 (m, 29H, Ar-H), and 8.05 (s, 1H, NH). The FTIR spectrum revealed absorption bands at 3304 cm^-1 (NH stretching), 1672 cm^-1 (C=O stretching), 1620 cm^-1 (C=C stretching), and 1250 cm^-1 (C-O stretching).

In a round-bottomed flask, we take 1 gm of 2-aminophenyl2- (2,4,5-triphenyl-1H-imidazole-1-yl)acetate,1 gm salicylic acid and 25 mL methanol was mixed with each other. The mixture was heated in a water bath at 100°C for 2 hr. After the reaction was finished, 100 mL of ice cold water were added to the mixture. Then the mixture was kept in fridge for 2-3 hr. The precipitated product was filtered and recrystallized with methanol to obtain 4-[(2-oxyphenyl)amino]benzoic acid-(2,4,5-triphenyl- 4,5-dihydro-1H-imidazol-1-yl)acetate. AC4 was obtained as a white solid with a yield of 70%. The melting point was found to be 244-246°C. The ¹H-NMR spectrum displayed peaks at δ 4.13 (s, 2H, CH2), 6.98-7.59 (m, 23H, Ar-H), 8.03 (s, 1H, NH), and 11.91(s, 1H, COOH). The FTIR spectrum exhibited characteristic bands at 3318 cm^-1 (NH stretching), 1705 cm^-1 (C=O stretching of carboxylic acid), 1661 cm^-1 (C=O stretching of ester), 1611 cm^-1 (C=C stretching), and 1249 cm^-1 (C-O stretching).

In a round-bottomed flask, we take 1 gm of 2-aminophenyl2- (2,4,5-triphenyl-1H-imidazole-1-yl)acetate, 1 mL formic acid and 25 mL methanol was mixed with each other. The mixture was heated in a water bath at 100°C for 2 hr. After the reaction was finished, 100 mL of ice cold water was added to the mixture. Then the mixture was kept in fridge for 2-3 hr. The precipitated product were filtered and recrystallized with methanol to obtain N-phenylformamide-(2,4,5-triphenyl- 4,5-dihydro-1H-imidazol-1-yl)acetate. AC5 was synthesized as a white solid, yielding 76%. The melting point was determined to be 232-234°C. The ¹H-NMR spectrum revealed characteristic peaks at δ 4.14 (s, 2H, CH₂), 7.98 (s, 1H, CHO), 7.00-7.58 (m, 20H, Ar-H), and 8.08 (s, 1H, NH). The FTIR spectrum showed prominent absorption bands at 3309 cm^-1 (NH stretching), 1679 cm^-1 (C=O stretching), and 1606 cm^-1 (C=C stretching).

Table 1. Calculations of Lipinski’s Rule of Five and Druglikeness for compounds AC1-AC6.

Comp.	Molecular weight (g/ mol)	CMC rule violation	Lipinski’s rule violation	Mol Log P	H bond donor	H bond acceptor	No. of rotatable bonds	TPSA (Å2)
AC1	479.96 g/ mol	2	Yes	4.57	1	3	7	70.14 Å2
AC2	459.54 g/ mol	2	Yes	4.30	1	3	7	70.14 Å2
AC3	473.56 g/ mol	2	Yes	4.50	1	3	8	70.14 Å2
AC4	524.41 g/ mol	2	No	4.67	1	3	7	70.14 Å2
AC5	496.58 g/ mol	3	Yes	4.74	1	3	7	70.14 Å2
AC6	470.52 g/ mol	2	Yes	3.43	1	4	7	93.93 Å2

Table 2. In silico ADMET analysis of compounds AC1-AC6.

Comp.	Absorption		Distribution			Metabolism
	CaCO2 permeability (log Papp in10-6 cm/s)	GI absorption	BBB perm. (logBB)	BBB Permeant	PPB (%)	CYP3A4 substrate	CYP1A2 inhibitor	CYP2C9 inhibitor,	CYP3A4 inhibitor	CYP2C19 inhibitor
AC1	36.94	Low	1.32768	No	100	Weakly	No	No	Yes	Yes
AC2	47.3863	High	1.52328	No	92.600377	Weakly	No	No	Yes	Yes
AC3	49.1514	High	0.842045	No	91.675444	Weakly	No	No	Yes	Yes
AC4	36.0737	Low	1.26909	No	100	Weakly	No	No	Yes	Yes
AC5	50.8426	High	0.523036	No	95.107470	Weakly	No	No	Yes	Yes
AC6	27.1673	High	2.04613	No	93.919117	Weakly	No	No	Yes	Yes

In a round-bottomed flask, we take 1 gm of 2-aminophenyl2- (2,4,5-triphenyl-1H-imidazole-1-yl)acetate, 1 gm succinic acid and 25 mL methanol was mixed with each other. The mixture was heated in a water bath at 100°C for 2 hr. After the reaction was finished, 100 mL of ice-cold water was added to the mixture. Then the mixture was kept in fridge for 2-3 hr. The precipitated product were filtered and recrystallized with methanol to obtain 4-oxo- 4-(phenylamino)butanoic acid-2-aminoPhenol-(2,4,5-triphenyl- 4,5-dihydro-1H-imidazol-1-yl)acetate. AC6 was produced as a light brown solid with a yield of 68%. The melting point was measured to be 218-220°C. The ¹H-NMR spectrum showed peaks at δ 1.99 (s, 3H, CH₃), 4.11 (s, 2H, CH₂), 4.32 (t, 2H, CH₂), 6.96-7.59 (m, 21H, Ar-H), and 8.00 (s, 1H, NH). The FTIR spectrum revealed absorption bands at 3315 cm^-1 (NH stretching), 1713 cm^-1 (C=O stretching of carboxylic acid), 1663 cm^-1 (C=O stretching of ester), 1612 cm^-1 (C=C stretching), and 1252 cm^-1 (C-O stretching).

The synthesized compounds (AC1-AC6)’s computed Lipinski’s rule of five and drug-likeness characteristics are shown in Table 1. To assess the potential of these molecules as therapeutic candidates, these qualities are essential. Lipinski’s rule of five states that if a compound does not violate more than one of the following standards—molecular weight less than 500 Da, log P less than 5, number of hydrogen bond donors less than 5, number of hydrogen bond acceptors less than 10—it is deemed to have drug-like properties. Lipinski’s rule of five is broken by compounds AC1, AC2, AC3, AC5, and AC6, which suggests that they could have unfavorable pharmacokinetic features. Compound AC4 can be thought of as a possible drugs candidate since it satisfies with the rule. In addition to Lipinski’s rule of five, the number of rotatable bonds and Topological Polar Surface Area (TPSA) are essential drug-likeness parameters. A compound with fewer rotatable bonds is generally considered more favourable for oral bioavailability, while a TPSA less than 140 Ųis associated with better membrane permeability and oral absorption. All synthesized compounds (AC1-AC6) have 7 or 8 rotatable bonds, which indicates moderate flexibility. Their TPSA values are in the range of 70.14 Ų to 93.93 Ų, well below the 140 Ų threshold, suggesting favourable membrane permeability and potential oral absorption. Moreover, the CMC (Comprehensive Medicinal Chemistry) rule violations assess the drug-likeness of the compounds based on multiple physicochemical properties. In general, fewer CMC rule violations indicate a higher probability of a compound being a successful drug candidate. Compounds AC1-AC4 and AC6 have two CMC rule violations, while AC5 has three violations. Therefore, compound AC4, which complies with Lipinski’s rule of five and has only two CMC rule violations, can be considered as the most promising drug candidate among the synthesized compounds. The in silico ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) predictions provide valuable insights into the pharmacokinetic properties of the synthesized compounds (AC1-AC6) are shown in Table 2. CaCO₂ permeability is a useful parameter for predicting oral absorption, with higher values indicating better permeability. Compounds AC2, AC3, AC5, and AC6 show high Gastrointestinal (GI) absorption, while AC1 and AC4 exhibit low GI absorption. This suggests that AC2, AC3, AC5, and AC6 may have better oral bioavailability than AC1 and AC4. The Blood-Brain Barrier (BBB) permeability (logBB) indicates the ability of compounds to cross the BBB and access the central nervous system. All synthesized compounds (AC1-AC6) have low BBB permeability and are classified as non-permeant, indicating limited central nervous system activity. The Plasma Protein Binding (PPB) percentages range from 91.67% to 100%, indicating that all compounds have a high degree of protein binding. High protein binding can reduce the free fraction of the drug available to exert its therapeutic effect, but it can also lead to a longer duration of action due to slow release from the protein complex. All synthesized compounds (AC1-AC6) are predicted to be weak substrates for the CYP3A4 enzyme, an essential enzyme involved in drug metabolism. This suggests that these compounds may not undergo extensive metabolism by this enzyme, potentially leading to a longer half-life and increased drug exposurein the body. It is important to note that weak substrates may still be metabolized by other enzymes in the cytochrome P450 family or undergo alternative metabolic pathways. Regarding the potential for drug-drug interactions, none of the compounds (AC1-AC6) are predicted to inhibit the CYP1A2 enzyme. However, all compounds are predicted to inhibit CYP2C9, CYP3A4, and CYP2C19 enzymes. Inhibition of these enzymes could lead to potential drug-drug interactions and altered pharmacokinetics of co-administered drugs metabolized by these enzymes. In summary, the in silico ADMET predictions for the synthesized compounds (AC1-AC6) suggest that they possess varying degrees of oral absorption, limited central nervous system activity due to low BBB permeability, high plasma protein binding, and potential for drug-drug interactions via inhibition of specific CYP enzymes. Further in vitro and in vivo studies are required to confirm these predictions and to assess the overall pharmacokinetic profile and safety of these compounds.

The molecular docking results presented in Table 3 indicate that the synthesized 2-aminophenyl-2-(2,4,5-triphenyl-1H-i midazole-1-yl) derivatives interact with various active amino residues in biotin protein ligase (PDB ID: 4DQ2). Among the six synthesized compounds (AC1-AC6), AC2 and AC4 showed the highest docking scores of -9.4, suggesting a strong binding affinity towards the target protein. The Native Ligand (NL), ciprofloxacin, exhibited a docking score of -9.1, which is lower than the docking scores observed for most of the synthesized compounds. This suggests that the synthesized compounds have the potential to be more effective inhibitors of biotin protein ligase compared to the native ligand. The docking results also revealed various interactions between the compounds and key active amino residues in the binding pocket of the target protein. Hydrogen bonding and hydrophobic interactions were the most prevalent types of interactions observed, which contribute significantly to the binding affinity and stability of the ligand-protein complex.For instance, AC1 exhibited hydrogen bond interactions with SER347, THR352, VAL605, and CYS300, while AC2 formed hydrogen bonds with THR302 and SER303. These hydrogen bond interactions are critical for the stability and specificity of the ligand-protein complex. Additionally, the compounds displayed hydrophobicinteractions with various amino acid residues, further stabilizing the ligand-protein complex. AC2 demonstrated hydrophobic interactions with ILE326, LEU484, LEU601, VAL605, and LEU480, whereas AC4 showed hydrophobic interactions with LYS487, LEU484, LEU601, CYS300, and ALA483. Hydrophobic interactions play an essential role in the overall binding affinity of the ligand to the protein by reducing the desolations energy required for complex formation.It is noteworthy that AC2 and AC4, the compounds with the highest docking scores, share some common active amino residues, such as SER303, LEU484, and LEU601, which might be crucial for their strong binding affinities. Additionally, the interactions of the synthesized compounds with different amino residues suggest that they might have diverse binding modes, which could be beneficial for the development of new inhibitors with distinct mechanisms of action. In conclusion, the molecular docking results presented in Table 3 indicate that the synthesized 2-aminophenyl-2- (2,4,5-triphenyl-1H-imidazole-1-yl) derivatives have the potential to be effective inhibitors of biotin protein ligase, with AC2 and AC4 demonstrating the strongest binding affinities. The observed interactions between the compounds and key active amino residues, such as hydrogen bonding and hydrophobic interactions, contribute significantly to their binding affinities and suggest that these compounds could serve as potential leads for the development of novel anti-bacterial agents targeting biotin protein ligase is shown in Figures 4 and 5 where it shows binding of AC2 and AC4..

Figure 4: Binding of AC2 to active site of Biotin Protein Ligase (A) along with the 2D binding diagram (B). The nucleophilic residue is labelled in green colour and the hydrogen bonds are depicted as green dotted lines.

Figure 5: Binding of AC4 to active site of Biotin Protein Ligase (A) along with the 2D binding diagram (B). The nucleophilic residue is labelled in green colour and the hydrogen bonds are depicted as green dotted lines.

The anti-bacterial test results of compounds AC1-AC6 are presented in Table 4. These results show that all synthesized 2-aminophenyl-2-(2,4,5-triphenyl-1H-imidazole-1-yl) derivatives exhibit anti-bacterial activity against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacterial strains are shown in Figure 6. A detailed discussion of the results is provided below.

Figure 6: In vitro anti-bacterial study of synthesized compounds (AC1 to AC6).

AC1 demonstrates moderate anti-bacterial activity, with a zone of inhibition of 17.4 mm against E. coli and 18.6 mm against S. aureus. This suggests that AC1 might be more effective against Gram-positive bacteria, although its activity against both strains is promising. AC2 exhibits a zone of inhibition of 16.2 mm against E. coli and 19.9 mm against S. aureus, indicating that it is more effective against Gram-positive bacteria than Gram-negative bacteria. This differential activity could be attributed to differences in the cell wall structure and composition between the two bacterial strains, which might influence the compound’s ability to penetrate and interact with the bacterial targets. AC3 shows significant anti-bacterial activity against both bacterial strains, with a zone of inhibition of 18.3 mm against E. coli and 20.2 mm against S. aureus. This broad-spectrum activity suggests that AC3 might have a general mechanism of action that is effective against both Gram-positive and Gram-negative bacteria. AC4 demonstrates moderate anti-bacterial activity,with a zone of inhibition of 17.8 mm against E. coli and 18.2 mm against S. aureus. This compound exhibits a slightly better activity against Gram-positive bacteria, but its overall activity against both strains is still considerable. AC5 shows a notable anti-bacterial activity, with a zone of inhibition of 18.4 mm against E. coli and 21.3 mm against S. aureus. This compound is particularly effective against Gram-positive bacteria, which could be due to its chemical structure or specific interactions with the bacterial targets. AC6 exhibits the highest anti-bacterial activity among the synthesized compounds, with a zone of inhibition of 20.9 mm against E. coli and 22.4 mm against S. aureus. Its broad-spectrum activity and high potency against both bacterial strains suggest that AC6 might be a promising candidate for further development as an anti-bacterial agent. Ciprofloxacin, used as a reference compound, shows a zone of inhibition of 22.8 mm against E. coli and 21.3 mm against S. aureus. The synthesized compounds, particularly AC6, exhibit comparable anti-bacterial activity to that of the reference compound, which highlights their potential as novel anti-bacterial agents.

Table 3. Molecular Docking Results of 2-Aminophenyl-2-(2,4,5-triphenyl-1H-imidazole-1-yl) Derivatives with Biotin Protein Ligase (PDB ID: 4DQ2).

Compound	Active Amino Residues	Docking Score
AC1	SER347, THR352, VAL605, CYS300, ASP354	-8.9
AC2	THR302, SER303, ILE326, LEU484, UNL1, LEU601. VAL605, LEU480	-9.4
AC3	SER347, GLN348, SER349, SER604, GLU488, UNL1, LEU484, VAL605	-9.2
AC4	THR302, SER303, LYS487, GLU488, LEU484, LEU601, CYS300, ALA483	-9.4
AC5	CYS300, THR352, VAL605, GLY301, ASP354, GLU488, UNL1	-8.9
AC6	SER401, LYS603, ASP354, VAL605, UNL1, CYS300, LEU601, ILE326	-8.1
NL	GLN348, SER349, THR352, SER303, LEU601, CYS300	-9.1

Table 4. Antibacterial test results of compounds of AC1-AC6.

	Compound ID	Compounds structure	Zone of Inhibition (mm)
		E. coli	S. aureus
AC1		17.4±0.3	18.6±0.4
AC2		16.2±0.5	19.9±0.7
AC3		18.3±0.2	20.2±0.6
AC4		17.8±0.3	18.2±0.5
AC5		18.4±0.4	21.3±0.5
AC6		2O.9±O.3	22.4±0.5
Ciprofloxacin		22.8±0.8	21.3±0.9

In conclusion, the in vitro anti-bacterial study of the synthesized compounds (AC1 to AC6) demonstrates that these 2-aminophenyl-2-(2,4,5-triphenyl-1H-imidazole-1-yl) derivatives possess promising anti-bacterial activity against both E. coli and S. aureus. Among the synthesized compounds, AC6 shows the most potent anti-bacterial activity, making it a potential candidate for further investigation and developmentas an anti-bacterial agent. The varying degrees of anti-bacterial activity among the compounds could be attributed to differences in their chemical structures, which might influence their ability to interact with bacterial targets or penetrate the bacterial cell walls. These initial findings warrant further investigation into the mechanism of action for these compounds, as well as additional studies on their activity against a broader range of bacterial strains. It would also be essential to evaluate their potential toxicity and pharmacokinetic properties to determine their suitability for development as therapeutic agents. Additionally, future research could focus on optimizing the chemical structures of these compounds to improve their anti-bacterial activity, selectivity, and physicochemical properties. Structure-Activity Relationship (SAR) studies can be employed to identify key functional groups and molecular features that contribute to the compounds’ anti-bacterial activity. This information could guide the design of more potent and selective anti-bacterial agents that might help address the rising global threat of antibiotic resistance.

The findings of the ADMET investigations and the assessment of the synthetic compounds’ drug-like qualities were combined with molecular docking studies to determine their probable binding affinities with the biotin protein ligase (PDB ID: 4DQ2). The synthesized compounds’ ability to bind to the target protein’s active amino acids was shown by molecular docking experiments, suggesting that they would be effective anti-bacterial agents. The compounds AC1-AC6 demonstrated excellent docking scores with the target protein’s active amino acids, indicating their potential as efficient biotin protein ligase inhibitors. The produced compounds were also efficient against E. coli and S. aureus, according to in vitro anti-bacterial investigations. With AC6 displaying the greatest activity, the compounds demonstrated a considerable zone of inhibition against the tested bacterial strains. The synthesized compounds demonstrated positive drug-like qualities, excellent absorption, distribution, low toxicity, and favorable binding affinities towards the target protein, all without violating Lipinski’s rule of five. Additionally, both Gram-positive and Gram-negative bacteria were significantly resistant to the compounds’ anti-bacterial effects. The compounds that were created therefore have a significant deal of promise for further development as anti-bacterial agents. Future research should concentrate on in vivo testing to evaluate the substances’ pharmacokinetic characteristics and therapeutic effectiveness.

The authors would like to acknowledge the support and guidance provided by Principal Dr. Sanjay Bhawar, Pravara Rural College of Pharmacy, Loni, in conducting this research. His constant encouragement and valuable suggestions were instrumental in the successful completion of this study. The authors also express their gratitude to the institution for providing the necessary facilities and infrastructure to carry out this research work.