PD-Nanocatalyst Mediated Synthesis and Molecular Docking Studies of Benzimidazole Analogs as Antimicrobial Agents

Dielectric volumetric heating is utilized by microwave-assisted organic synthesis to generate heat. Because of the uniform heat distribution, this method produces rapid and quite selective reactions. Chemical reactions are stimulated by microwave dielectric heating, which tries to make utilization of the medium's capacity to transform electromagnetic radiation into heat. This occurs when, as a result of microwave (MW) irradiation, the dipoles or ions present in the reaction mixture align in an applied electric field [1]. In the reaction vessel, the MW irradiation can readily transmit through the walls and couple with the reaction mixture's molecules and ions. As a result, the mere possibility of the reaction mixture boiling is reduced. Reaction vials ought to be transparent to electromagnetic waves because dielectric heating can only take place within an absorbing material that transforms MW energy into heat [2]. MW reactions accomplish much quicker, under milder and eco-friendly process parameters, produce greater synthetic yields, consume moderate energy, and are selective or occasionally demonstrate different selectivity. MW has indeed been extensively utilized in the disciplines of synthetic organic and inorganic chemistry [3]. Benzimidazole is a heterocyclic aromatic organic compound that consists of the fusion of benzene and imidazole having multifold applications in pharmaceutical industries. N-ribosyl-dimethyl benzimidazole is a well-known benzimidazole compound that serves as an axial ligand for cobalt in vitamin B12 [4]. Benzimidazole was first synthesized in 1872 by Hoebrecker [5]. Over many years of resolute research, a wide variety of benzimidazole and its derivatives have been described for their importance in medicinal chemistry enclosing a broad spectrum of biological activities such as anti-parasitic [6], anti-ulcer [7], anti-cancer [8], anti-psychotics [9], anti-inflammatory [10], anti-bacterial [11], anti-fungal [12][13],     anti-oxidant [12], anti-Alzheimer [14], anti-HIV [15], and anti-malarial [16]. Numerous biochemical and pharmaceutical investigations have demonstrated the efficacy of benzimidazole compounds against different types of bacteria [17].  Public health is gravely threatened by infectious diseases, due to the frequent usage of antibacterial and antifungal medications, resistance has been developed to these substances leading to an increase in microbial infections [18]. Antimicrobial resistance causes more than 700,000 deaths worldwide each year, and by 2050, it's predicted it could result in more than 10 million fatalities [19]. The benzimidazole ring system structurally resembles the heterocyclic bases found in the structure of nucleic acids, they can demonstrate their antibacterial activity by competing with them, which prevents bacteria from synthesizing nucleic acids and proteins [20]. The last ten years have seen a rise in the prevalence of fungal infections as the leading cause of morbidity and mortality, especially in immunocompromised patients undergoing cancer chemotherapy or organ transplantation, as well as AIDS patients [21]. In addition to having antibacterial properties, benzimidazoles also have antifungal properties. They are well-known for their notable capacity to effectively control a wide range of fungi-related illnesses [22,23] by competitively inhibiting the enzyme lanosterol 14-?-demethylase (CYP51), which is a crucial component for fungal sterol biosynthesis and fungal microtubular activity [23,24]. Several clinically relevant gram-positive strains staphylococci, enterococci, and streptococci, have emerged as a major worldwide health concern. The development of new antimicrobial drugs is one strategy for combating this problem, while the proper use of commercially present antibiotics is another [23]. Preferably, the chemical properties of the new agents should be distinctly different from those of the current agents [25]. Based on these results, the goal of the research was to synthesize the benzimidazole-based scaffolds under MW parameters and to examine their antibacterial and antifungal properties.

2. MATERIALS AND METHODS

The reagents and solvents were purchased from Sigma-Aldrich Chemicals private limited. Thin-layer chromatography (TLC) was utilized to monitor the reaction using 0.25 mm Merck 60 F 254 silica plates and various solvent systems, the TLC plate was visualized using UV light. The compounds were purified using Biotage Isolera flash chromatography.  Chemi-line melting point apparatus was used to record the melting points of the compounds. The IR (KBr) spectra were obtained using Agilent Cary 630 FTIR spectrophotometer. The nuclear magnetic resonance (NMR) analysis was performed using Agilent VNMRS-400 MHz-NMR and Bruker spectrophotometer in dimethylsulphoxide (DMSO) solvent. Further, mass spectral analysis was carried out using a VG70-70H spectrometer.

2.1. Chemistry

The synthetic route of the title compounds 6a-d is depicted in Scheme 1. The           N-benzyl-2-nitroaniline 3 was synthesized via microwave irradiation between 1-fluoro-2-nitrobenzene 1 and benzylamine 2 using sodium acetate and nano-[Fe₃O₄–dopamine–Pd] nano-catalyst.

Further, reduction of the nitro group of compound 3 using nano SmFeO₃, potassium hydroxide in propane-2-ol furnished N-benzyl-1,2-diaminobenzene 4. Finally, the title compounds N-benzyl-2-phenyl-1H-benzo[d]imidazoles 6a-d were procured as a result of the cyclization of compound 4 with substituted aldehydes 5a-d in DMF.

2.2.1.        General procedure for the synthesis of N-benzyl-2-nitroaniline 3:

Synthesis of magnetic nano-ferrites

Upon dissolving FeSO₄.7H₂O (3.5 g) and Fe₂(SO₄)₃ (4 g) in 100 ml of water, a slow addition of 25% ammonium hydroxide solution was done to lower the pH of the mixture down to 10. At 60 ^oC, the reaction mixture is stirred for over one hour. After being isolated magnetically, the precipitated nanoparticles were washed with water until their pH attained 7, and they were then vacuum-dried for 2 hours at 60 ?. Then, additional chemical alteration was undertaken using this magnetic nano-ferrite (Fe₃O₄).

Surface functionalization of nano-ferrites

In 25 ml of water, 2 g of nano-Fe3O4 was sonicated for 30 mins. Upon adding 2 g of dissolved dopamine hydrochloride in 5 ml of water, the mixture was sonicated for                   2 hours. After utilizing acetone to precipitate the amine-functionalized nanomaterial, it was collected via centrifugation and vacuum-dried for 2 hours at 60 ^oC.

Synthesis of [Fe₃O₄–dopamine–Pd] catalyst

1 g of dopamine-functionalized nano-ferrite was stirred into 25 ml of methanol. After adding 200 mg of PdCl₂ progressively to the suspension, the mixture is stirred for 10 min. Then, drop by drop, 25% ammonium hydroxide was added to the medium to raise its pH to 9. For 24 hours, the solution was mixed at room temperature. An external magnet was utilized to isolate the nano-[Fe₃O₄-dopamine-Pd] catalyst, which was washed utilizing acetone and vacuum-dried for 2 hours at 60 °C.

Water (5 ml) and glycerol (5 mmol) were mixed with a mixture of 1-fluoro-2-nitrobenzene 1 (1 mmol), benzylamine 2 (1.5 mmol), sodium acetate (2 mmol), and [Fe₃O₄–dopamine–Pd] catalyst (25 mg, 2.4 mol percent). For 10 min, the reaction mixture was irradiated to microwave radiation, and TLC (1:9 hexane and ethyl acetate) was employed to monitor the reaction's progression. The pure product N-benzyl-2-nitroaniline 3 was achieved by simple column chromatography.

N-Benzyl-2-nitroaniline 3: Yield: 89 %. Mp. 120-122 ^oC. IR (KBr, cm^-1): 3130-3300 (NH). ¹H NMR (DMSO): δ 4.64 (s, 2H, CH₂), 6.65-8.66 (m, 9H, Ar-H), 8.68 (s, 1H, NH). LC-MS m/z 229 (M+1). Anal. Calcd. for C₁₃H₁₂N₂O₂ (228): C, 68.41; H, 5.30; N, 12.27. Found:       C, 68.37; H, 5.22; N, 12.11 %.

2.2.2. General procedure for the synthesis of N-benzyl-1,2-diaminobenzene 4:

A nano-sized SmFeO₃ catalyst (50 mg, 5 mol percent) was added to a solution of N-benzyl-2 nitroaniline 3 (5 mmol), potassium hydroxide (5 mmol), and propane-2-ol (15 ml). Subsequently, the reaction mixture was placed in a microwave oven set at 2.45 GHz with a maximum power of 900 W. It was subjected to 20 % power (180 W) for 8 mins, and TLC was used to track the reaction's progress. The mixture was allowed to cool to room temperature after the reduction was finished, and then the organic layer was recovered utilizing 3 × 10 ml of ethyl acetate, dried over sodium sulphate, and evaporated under reducing pressure. Column chromatography was employed to generate pure product 4. (carbon tetrachloride-ethyl acetate).

N-Benzyl-1,2-diaminobenzene 4: Yield: 92 %. Mp. 117-119 ^oC. IR (KBr, cm^-1): 3130-3300 (NH), 3300-3490 (NH₂). ¹H NMR (DMSO): δ 4.28 (s, 2H, NH₂), 4.53 (s, 2H, CH₂), 5.09       (s, 1H, NH), 6.31-7.37 (m, 9H, Ar-H). LC-MS m/z 199 (M+1). Anal. Calcd. for C₁₃H₁₄N₂ (198): C, 78.75; H, 7.12; N, 14.13. Found: C, 78.69; H, 7.05; N, 14.02 %.

2.2.3. General procedure for the synthesis of N-benzyl-2-phenyl-1H-benzo[d]imidazoles 6a-d:

N-Benzyl-1,2-diaminobenzene 4 (1 mol, 1eq), and substituted aldehydes 5a-d            (1 mol, 1 eq) were dissolved in wet DMF (9 ml DMF and 1 ml H₂O) and the resulting reaction mixture was stirred at 80 ⁰C. The reaction progress was monitored by TLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude product obtained was purified by flash chromatography to afford the corresponding benzimidazoles 6a-d.

N-Benzyl-2-phenyl-1H-benzo[d]imidazole 6a: Yield: 87 %. Mp. 103-105 ⁰C. IR (KBr,         cm^-1):1622 (C=N). ¹H NMR (DMSO): δ 5.53 (s, 2H, CH₂), 6.98-7.75 (m, 14H, Ar-H).        LC-MS m/z 285 (M+1). Anal. Calcd. for C₂₀H₁₆N₂ (284): C, 84.48; H, 5.67; N, 9.85. Found:             C, 84.41; H, 5.61; N, 9.78 %.

N-Benzyl-2-(2-methoxyphenyl)-1H-benzo[d]imidazole 6b: Yield: 92 %. Mp. 111-113 ⁰C.    IR (KBr, cm^-1): 1622 (C=N). ¹H NMR (DMSO): δ 3.71 (s, 3H, OCH₃), 5.27 (s, 2H, CH₂), 6.95-7.68 (m, 13H, Ar-H). LC-MS m/z 315 (M+1). Anal. Calcd. for C₂₁H₁₈N₂O (314):         C, 80.23; H, 5.77; N, 8.91. Found: C, 80.16; H, 5.71; N, 8.83 %.

N-Benzyl-2-(2,4-dimethoxyphenyl)-1H-benzo[d]imidazole 6c: Yield: 89 %. Mp. 123-125 ⁰C. IR (KBr, cm^-1): 1622 (C=N). ¹H NMR (DMSO): δ 3.69 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 5.26 (s, 2H, CH₂), 6.66-7.66 (m, 12H, Ar-H). LC-MS m/z 345 (M+1). Anal. Calcd. for C₂₂H₂₀N₂O₂ (344): C, 76.72; H, 5.85; N, 8.13. Found: C, 76.65; H, 5.79; N, 8.07 %.

N-Benzyl-2-(4-fluorophenyl)-1H-benzo[d]imidazole 6d: Yield: 89 %. Mp. 115-117 ⁰C.        IR (KBr, cm^-1): 1622 (C=N). ¹H NMR (DMSO): δ 5.58 (s, 2H, CH₂), 6.98-7.79 (m, 13H,             Ar-H). LC-MS m/z 303 (M+1). Anal. Calcd. for C₂₀H₁₅N₂F (302): C, 79.45; H, 5.00; N, 9.27. Found: C, 79.37; H, 4.95; N, 9.12 %.

2.3. Biological studies

The following gram-positive bacteria (Micrococcus luteus, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus aureus) and against gram-negative bacteria (Enterobacter aerogenes, Salmonella typhimurium, Salmonella paratype-B, Proteus vulgaris, and Klebsiella pneumonia) were employed in the investigations. Additionally, fungal strains including Botrytis cinerea, Candida albicans, Malassezia pachydermatis, and Candida krusei were also used. All cultures were obtained from the Department of Microbiology, Manasagangothri, Mysuru. 

2.3.1. Preparation of inoculums

Cells were obtained from the Department of Microbiology at Manasagangothri, Mysuru, and cultivated in Mueller Hinton Broth (MHB) (Himedia) for a period of 24 hours at 37 ° C to generate bacterial inoculums. The first cell counts of around 104 CFU/ml were achieved by diluting these cell suspensions with sterile MHB. The fungi were cultivated using Sabouraud Dextrose Agar (SDA) slants for 10 days at a temperature of 28 °C. After that, the spores were extracted and homogenised utilizing sterile double-distilled water.

2.3.2. Disc diffusion assay

The compounds 6a-d were assessed for their antibacterial and antifungal properties using the disc diffusion method. For each Petri plate, about 20 ml of sterile Mueller Hinton Agar (MHA) were employed. After being swabbed on top of the hardened media, the test cultures were given ten minutes to dry. Subsequently, the test was conducted at a density of 1000 µg/disc. The loaded discs were positioned on the medium's surface and left to diffuse for 30 minutes at room temperature. The appropriate solvent was used to prepare the negative control. For bacterial and fungal strains, respectively, positive controls of streptomycin and ketoconazole (10 µg/disc) were employed. Every plate was incubated for 24 hours to screen for bacterial strains.[26].

2.3.3. Minimum inhibitory concentration (MIC)

The MIC assay for the antibacterial and antifungal properties of the title compounds 6a–d was computed employing standard protocol (NCCLS, 2002).[27,28]. For the antimicrobial assay, the compounds 6a-d were dissolved in 2 % DMSO and diluted to generate consecutive two-fold dilutions at the requisite concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.62 µg/ml. The second stage comprised inoculating each well in 96-well plates using 100 µl of inoculum after samples had been introduced to each medium. The antifungal drugs fluconazole and ketoconazole as well as the antibacterial drugs streptomycin and ciprofloxacin functioned as positive controls for the assays. The plates were then incubated for 48 hours at 27 ? for fungi and for 24 hours at 37 °C for bacteria. The minimum concentration of the compound needed to minimize the test cultures on the agar plate from developing noticeably was determined as the minimum inhibitory concentration (MIC) for microbiological strains.

2.4. Molecular Docking

The RCSB PDB database (https://www.rcsb.org/) provided the crystal structures of                       S. aureus and C. albicans (PDB IDs: 1HSK/ 5V5Z). Using AutoDock 4.2, the protein was prepared according to the prior work. [29]. Based on the available literature, the binding site was predicted [30,31]. The grid box of size 40×40×40 ų was built (x = 179.77 Å, y = 149.01 Å, and z= 163.65 Å and x= -47.73 Å, y= -13.42 Å, and z= 22.98 Å) that covers the inhibitor binding pocket for both the proteins. The protein and the ligands 6a-d Structures were produced using AutoDock Tools 1.5.6 for docking investigations, as per our earlier research [32,33]. Water and heteroatoms were taken out of the macromolecules during purification, except polar hydrogen for stabilization. Kollmann-united and Gasteiger charges were used to reduce the energy of the protein and ligands structures. After parameter optimization, an Auto Dock 4 atom type was given to every atom. To aid docking experiments, the generated protein and ligands structures were acquired in PDBQT format. ACD Chem Sketch was used to draw and refine the 2D chemical structures in 3D. By using Auto Dock 4.2 and adhering to established procedures that have been previously documented, the ligands were prepared for docking [34,35].

Using the command-line-based programme Auto Dock Vina 1.1.2, ligands 6a-d protein complexes were virtually screened. The score function of each ligand conformation was examined, and the Broyden-Fletcher-Goldfarb-Shanno (BGFS) approach was used to perturb and place ligands within the target sites [36,37]. Because ligand synthesis produces a large number of torsions, ligands were treated as flexible throughout the docking investigations while proteins were treated as stiff. Moreover, ten degrees of freedom were granted to the first binding pose of the ligand molecules with an atomic position root-mean-square deviation (RMSD) of zero. Additionally, the largest binding affinities were seen in these positions, suggesting potentially stronger interactions. The molecular docking simulation was visualized using an open-source graphical programme called Biovia Discovery Studio Visualizer 2021.

2.4.1. Molecular dynamics simulation

The docked protein complexes in the molecular dynamics simulation, chosen ligand 6b, and standards streptomycin and ketoconazole were used. The molecular dynamics simulation was carried out using the GROMACS-2018.1 software suite, which is renowned for its capacity to precisely estimate nonbonded interactions [38]. The ligand topologies were generated utilizing the SwissParam (https://www.Swiss param.ch/) assistance, and the assessment proceeded utilizing the CHARMM36 force field. Similar to this, the protein's CHARMM36 force field was assigned using the pdb2gmx module. The next step involves a 5,000-step, steepest descent approach to vacuum energy minimization. The simulation box included each protein complex, which was placed there while keeping a distance of ten units from the box's boundaries. The TIP3P water model contained the solvent, and counter-ions Na⁺ and Cl^- were injected to keep the salt concentration at 0.15 M. The simulation was run for intervals of 100 ns at a temperature of 310 K and a pressure of 1 bar. The parameters of the ligand hydrogen bond as well as root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA) were used in the trajectory analysis. With the aid of the graphical user interface (GUI) application XMGRACE, the results of the molecular dynamics simulation were represented [36,39].

2.4.2. Binding free energy calculations

To compute the binding free energy, Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) was employed on the findings of the 6a-d complexes and standards molecular dynamics simulations. The GROMACS 2018.1 trajectory, the MmPbSaStat.py script, and the g_mmpbsa software were implemented to achieve the results [40,41]. Three crucial variables were taken into account while calculating the binding free energy: molecular mechanical energy, polar solvation energy, and apolar solvation energy. These variables account for the affinity between ligand 6b and proteins. The binding free energy was determined by the g_mmpbsa tool using molecular dynamics trajectories from the previous 50 ns, and the changes in free energy (G) were computed over a time interval of 1000 frames.

3. RESULTS AND DISCUSSION

3.1 Structural activity relationship

The benzimidazole compounds that have been generated were evaluated for antibacterial and antifungal activities. The phenyl ring that links to the benzimidazole ring is substituted in the synthesized compounds 6a-d played a pivotal role in displaying diverse antimicrobial activity. The benzimidazole scaffolds have shown significant in vitro antibacterial activity.  The results of the zone of inhibition of the gram-positive strains revealed that the compound 6d with electron-withdrawing fluoro group exhibited potent activity among the series. Followed by, compound 6b with an electron-releasing methoxy group displayed a better activity, but the compound 6c with two electron-releasing methoxy groups and compound 6a without substitution manifested a weaker activity. Conversely, the same trend was followed with gram-negative strains. In fungal strains, the activity for compound 6d showed a significant zone of inhibition in comparison to the rest of the compounds. 

In MIC determination for bacterial strains, compound 6d effectively inhibited        gram-positive and gram-negative strains. In addition, compound 6d exhibited more potent activity against the strain P.vulgaris, than the standard streptomycin. Further, compound 6b also showed moderate activity against bacterial strains among the series. The MICs obtained for fungal strains, compound 6d with electron-withdrawing fluoro group effectively inhibited the growth of fungal strains. Interestingly, compound 6d manifested more potent activity against the strain B. cinerea, than the standards ketoconazole and fluconazole. Additionally, compound 6b exhibited a modest level of efficacy against a range of bacteria. The generated benzimidazole derivative 6d with electron-withdrawing fluorine substitution therefore prevailed in terms of activity for suppressing both bacterial and fungal strains, according to the examination of the data.

3.2. Chemistry

The IR, NMR, and mass spectral information were used to characterize the title compounds 6a-d and were synthesized in accordance with Scheme 1. The compound N-benzyl-2-nitroaniline 3 was synthesized using 1-fluoro-2-nitrobenzene 1 and benzylamine 2. The IR data of compound 3 displayed a band at 3130-3300 cm^-1 corresponding -NH group. Further, the NMR data of compound 3 showed a singlet peak at δ 4.64 ascribing two protons of the -CH₂ group, a multiplet peak at a range of  δ 6.65-8.66 ascrbing the presence of nine aromatic protons, and another singlet peak at δ 8.68 for the -NH proton. Also, the mass spectral data with m/z 229 as the M+1 peak supported the formation of compound 3. Then, the compound 3 was reduced via nano-catalysis to achieve N-benzyl-1,2-diaminobenzene 4. Compound 4 revealed the presence of IR absorption bands ranging from 3130-3300 and 3300-3490 cm^-1 corresponding to -NH and -NH₂ groups, correspondingly. The ¹H NMR spectrum displayed a singlet peak at δ 4.28 for two protons of the -NH₂ group, two singlets at δ 4.53 and 5.09 for two protons of the -CH₂ group, and one amide proton. Additionally, a multiplet peak at δ 6.31-7.37 was displayed for nine aromatic protons elicudating the formation of compound 4. Also, the mass spectral data revealed a stable M+1 peak at m/z 199 supporting the formation of compound 4. The final compounds 6a-d were synthesized by the cyclization of compound 4 with substituted aldehydes 5a-d. The representative compound 6a from the series 6a-d, revealed the IR band at 1622 cm^-1 corresponding to the C=N groups of benzimidazole. Further, the ¹H NMR spectrum revealed the presence of two -CH₂ protons at δ 5.53 and multiplet peak ranging at δ 6.98-7.75 corresponded to fourteen aromatic protons. Additionally, a M+1 peak at m/z 285 appeared in the mass spectrum, which indicates the formation of the final compound 6a.

3.3. Biological studies

Using standard streptomycin, all compounds 6a–d were assessed for their in vitro antibacterial activity against gram-positive and gram-negative bacterial strains. Standard ketoconazole was used to assess the compounds 6a–d in vitro antifungal activity. Additionally, measurements of the minimum inhibitory concentration (MIC) for compounds 6a–d were carried out against bacterial studies using the standards streptomycin and ciprofloxacin, and the standards fluconazole and ketoconazole were referred for evaluation of fungal strains inhibitory activity.

3.3.1. In vitro antibacterial activity of compounds 6a-d

According to Table 1, the analytical findings of compounds 6a–d demonstrated the zone of inhibition's substantial activity for both gram-positive and gram-negative bacteria.            Gram-positive bacterial strains included M. luteus, S. aureus (MRSA), and S. aureus.           For instance, in the strain M. luteus, compound 6d (23 mm) exhibited the highest zone of inhibition among the series, also, the activity was comparable to the standard streptomycin (24 mm). Further, the compound 6b (21 mm), showed moderate activity, while, the compounds 6a (17 mm) and 6c (19 mm) displayed comparatively lower activity. In the strain S. aureus (MRSA), compound 6d (22 mm) displayed a large extent of the zone of inhibition and the activity was comparable to that of the standard streptomycin (23 mm), followed by the compound 6b (21 mm) but the compounds 6a and 6c showed lesser inhibitions (16 mm). Additionally, compound 6b (24 mm) covered a better zone of inhibition against the strain      S. aureus, amongst the series, and exhibited comparable activity with the standard streptomycin (25 mm). Interestingly, the compounds 6a and 6d exhibited similar zone of inhibition (21 mm), whereas, compound 6c (20 mm) showed weaker activity.

Table 1. In vitro antibacterial activity of compounds 6a-d

The analysis results of compounds 6a-d showed significant activity of zone of inhibition against the gram-negative bacterial strains such as E. aerogenes, S. typhimurium,               S. paratype-B, P. vulgaris, and K. pneumonia. Further, in the strain E. aerogenes,       compound 6b (22 mm) exposed a larger zone of inhibition in contrast to the other synthesized compounds, also, the activity was comparable to the standard streptomycin (27 mm). The activity of compounds 6c (15 mm) and 6d (16 mm) was noticeably lower. Moreover, compound 6d (25 mm) showed the highest zone of inhibition against the strain                       S. typhimurium, than the other compounds in the series, also, the activity leveled the standard streptomycin (26 mm) and compound 6c (19 mm) showed declined inhibitory activity. In the strain S. paratype-B, compound 6d (17 mm) showed a moderate zone of inhibition, and also, the activity was lower than that of the standard streptomycin (25 mm) and compounds 6a     (12 mm) and 6b (10 mm) demonstrated lower inhibitory activity. Conversely, compound 6c (19 mm) exhibited a passable zone of inhibition against the strain P. vulgaris, and also, showed close-by activity to the standard streptomycin (21 mm), and compounds 6b (15 mm) and 6d (11 mm) displayed reduced activity. In the strain K. pneumonia, compound 6b          (22 mm) manifested the highest zone of inhibition among the other compounds, also, the inhibitory activity concerning the standard streptomycin (24 mm) and compound 6a (16 mm) showed comparatively decreased activity. The compound 6b was observed to have potent antimicrobial activity among the series.

3.3.2. In vitro antifungal activity of compounds 6a-d

Besides, the in vitro antifungal activity against B. cinerea, C. albicans,                       M. pachydermatis, and C. krusei, strains using ketoconazole as a standard reference was examined. In strain B. cinerea, compounds 6a and 6d exhibited excellent zone of inhibition (16 mm) and their activities were comparable to the standard ketoconazole (17 mm), followed by the compounds 6b (15 mm) and 6c (14 mm) displayed lower activities. Interestingly, compounds 6a (23 mm) and 6d (23 mm) exhibited the highest zone of inhibition followed by the compound 6b (22 mm) against the strain C. albicans, also, the activity was comparable to the standard ketoconazole (24 mm), whereas, compound              6c (21 mm) manifested a lower activity in comparison. In strain M. pachydermatis, compound 6d (24 mm) showed the larger zone of inhibition, concerning the standard ketoconazole (25 mm), and compound 6c (16 mm) displayed the least activity among all. Moreover, compound 6d (18 mm) exhibited the highest zone of inhibition against the strain C. krusei, about the standard ketoconazole (19 mm) followed by compounds 6a (16 mm),    6b (16 mm), and 6c (15 mm).

Table 2. In vitro antifungal activity of compounds 6a-d

3.3.3. MIC studies of compounds 6a-d against tested bacterial strains

All the title compounds 6a-d were screened for in vitro antimicrobial activity against gram-positive and gram-negative strains. To control the sensitivity of the test organisms, the MICs of standards streptomycin and ciprofloxacin were determined simultaneously in the experiment. The MIC values of the test compounds 6a-d and the standard drugs are presented in Table 3. According to the findings, compound 6d (15.62 ?g/ml) had considerable activity against M. luteus when compared to the standard streptomycin (6.25 ?g/ml) and lowest in comparison to ciprofloxacin (<0.78 ?g/ml), conversely, the compound 6c displayed low activity. Further, the compound 6a (31.25 µg/ml) demonstrated better effectiveness concerning streptomycin and ciprofloxacin against S. aureus (MRSA). Further, when compared to the standards streptomycin (6.25 µg/ml) and ciprofloxacin (<0.78 µg/ml), compounds 6b and 6c exhibited the same MIC value of 15.62 µg/ml with modest efficacy against S. aureus. Interestingly, compounds 6a, 6b and 6d remarkably displayed the same MIC value of 15.62 µg/ml and better efficacy than the standards streptomycin and ciprofloxacin against E. aerogenes strain. Surprisingly, compound 6d (15.62 µg/ml) displayed excellent activity compared to streptomycin and ciprofloxacin against the               S. typhimurium strain. Besides, compound 6d (15.62 µg/ml) demonstrated lesser efficacy compared to the standards streptomycin and ciprofloxacin against S. paratype-B. The compound 6d (15.62 µg/ml) displayed better activity than the reference to streptomycin which showed no inhibition and lesser activity than ciprofloxacin against P. vulgaris. In addition, compounds 6b, 6c, and 6d (15.62 µg/ml) manifested decreased activity in contrast to the standard streptomycin (6.25 µg/ml) and ciprofloxacin (<0.78 µg/ml) against                 K. pneumonia strain.

Table 3. MIC (?g/ml) of compounds 6a-d against tested bacterial strains

ni =  no inhibition

3.3.4. MIC studies of compounds 6a-d against tested fungal strains

All compounds 6a-d were tested for their antifungal efficacy against various fungal strains using fluconazole and ketoconazole as positive controls as shown in Table 4. The compound 6d (15.62 µg/ml) performed the best from the series, against the B. cinerea, strain but it was less active than the standard ketoconazole (25 µg/ml) and better than fluconazole (with no inhibition). Interestingly, the activity of compounds 6a, 6b, and 6d against                C. albicans, the activity was identical (31.25 µg/ml), comparable to that of standard ketoconazole with MIC of 25 µg/ml, but less potent than fluconazole (MIC >100 µg/ml). Again, the activity of compound 6d (15.62 µg/ml) against M. pachydermatis, the strain was comparable to that of standard ketoconazole (MIC: 15 µg/ml), although it was less potent than fluconazole (MIC: 12.5 µg/ml). At last, when tested against C. krusei, compounds 6b and 6d recorded the same MIC value of 15.62 µg/ml exhibiting activity comparable to that of the standard ketoconazole (15 µg/ml), but less effective than fluconazole (12.5 µg/ml).

Table 4.  MIC (µg/ml) of compounds 6a-d against tested fungal strains

ni =  no inhibition.

3.4. Molecular docking simulation

3.4.1. Molecular docking studies

Utilizing the AutoDockVina program, the mechanism of interaction between compounds 6a–d and the proteins was examined (S. aureus and C. albicans). Table 5 displays the binding affinity, the total number of intramolecular contacts, and the results of the complexes' virtual screening. The highest docked score (most negative binding energy) of -9.3 and -9.7 kcal/mol was showed by compound 6b against S. aureus and C. albicans, respectively. The standards streptomycin and ketoconazole exhibited the lowest docked score of -8.9 and -9.0 kcal/mol, respectively against the target proteins.

Table 5: Virtual screening of compounds along with target proteins S. aureus and   C. albicans (PDB ID: 1HSK and 5V5Z)

Concordant to the Kumar et al. [42] study, the active site of S. aureus located at the interface of domain A contains conserved residues around the substrate-binding site, and the compound 6b was found to be bound within the active site. The docked structure was visualized to show that 6b interacted with important amino acid residues of proteins, such as arg188, arg225, and gly153 via hydrogen bonding, one electrostatic bond was formed with arg188, and hydrophobic bonds of pi-alkyl and pi-alkyl T shaped were formed with met150, ala152, pro141 and phe274. Comparatively, streptomycin formed only 3 non-bounded interactions. The hydrogen bonds were formed via arg188, gln229, and his271, also, the unfavorable donor-donor bonds were formed via ser238 and arg225 (Figure 1). Based on these findings, it is possible to conclude that the interaction level of ligand 6b was higher than that of streptomycin.

It was found that the ligand in the C. albicans was bound inside the binding area where the co-crystal ligand occupied. According to Gómez-García et al. [43] research, hydrogen-bound catalytic residues provide a potent inhibiting effect which could considerably reduce activity. Significant protein residues comprising amino acids have been stimulated by the ligand 6b, such as tyr127 and ser383 via hydrogen bonding, one pi-sigma bond was formed with leu384, and hydrophobic bonds of alkyl and pi-alkyl formed with leu381, tyr127, leu384 and met314. Whereas ketoconazole-protein formed a total of five hydrophobic bonds with tyr127, leu381, cys472, val312, and phe477. Figure 2 depicts the binding interaction between ligand 6b and ketoconazole with C. albicans. The interaction results indicated that ligand 6b was superior to ketoconazole

3.4.2. Molecular dynamics simulation

Though docking was used to understand the interactions between compound 6b with target proteins (S. aureus and C. albicans) along with control streptomycin and ketoconazole, it was unclear how the substance and proteins interacted differently, particularly in terms of the complexes' stability and protein structural flexibility. In order to verify the docking result of compound 6b with target proteins, a dynamic simulation was carried out by utilizing the trajectories in relation to the solvent accessible surface area (SASA), ligand hydrogen bonds (H-bond number), radius of gyration (Rg), mean square deviation (RMSD), and root mean square fluctuations (RMSF).

Table 6: Molecular dynamics values of ligand 6b and streptomycin complexes along with   S. aureus

Table 6 provides the data following the trajectories investigated by S. aureus with compounds 6a-d, and Figure 3 illustrates the graphs. RMSD was implemented to assess the conformational stability of the complex at a particular time. According to the investigation, the protein is found to be at par stable for around 15 ns throughout the simulation, however the ligand 6b and control compounds reach stability between 30 and 73 ns. There is a minor rising variation approximately ~70 ns and enhances stability across. Given that the lower the RMSD, greater the degree to which stable a system is, the plot demonstrates that ligand 6b possesses an average RMSD that is reduced than that of streptomycin control. Additionally, since ligand 6b remained inside the inhibitor binding site for the entire length of the simulation, the 6b complex demonstrated the best stability.

However, the RMSF figure demonstrates that the control complex varies more than the 6b complex. The ligand 6b complex's varying residues fell between 0.1 and 0.5 nm in the RMSF range. When attached to compounds, the target protein indicates little variability and equivalent secondary structural stability, as indicated by the plot evaluation. In contrast, RMSF graphs of conventional streptomycin revealed larger swings, suggesting instability within the inhibitor binding site.

Rg plot analysis was done to assess how the structure of the proteins altered during complex formation. Throughout the simulation, the Rg values of the streptomycin complex and 6b remained rather constant, varying at about 2 nm. This suggests that the binding site had less of an impact on the structures. To forecast the conformational change in the binding area, the SASA plot was assessed. Both ligand 6b and conventional streptomycin's SASA varied between 135 and 145 nm². Ultimately, an analysis of the hydrogen bond between ligand 6b and protein was conducted in order to comprehend the structural agreement. The plot suggests that the complex may have undergone structural modification, and the analysis also revealed that, in comparison to the standard, ligand 6b formed more H-bonds with protein over the 100 ns simulation, suggesting how the 6b complex seemed more stable.

Table 7: Molecular dynamics values of 6b and ketoconazole complexes along with       C. albicans

The apo-protein and the 6b complex in the instance of C. albicans were determined to be between the bands of 0.20–0.25 nm, according to the RMSD plots. Contrary to the ketoconazole complex, which was shown to have a range of 0.20-0.35 nm, 6b was found to remain steady throughout the simulation with no variation. Both the 6b and ketoconazole complexes, as well as the apo-protein atoms, showed similar, varying behavior in the RMSF study. To further show the structural compactness of the resulting structure, Rg and SASA graphs were analyzed.

According to the Rg study, the protein and ligand 6b were discovered to be within the same 2.31 nm range. Likewise, the SASA value was found to be comparable and to follow a similar pattern. Ketoconazole and ligand 6b SASA plot varied within the same 210–218 nm² range. In addition, the H-bond was analyzed to detect structural re-agreement, and conformational alterations to the complex were clearly visible. Ketoconazole, however, generated less hydrogen bond than 6b in terms of ligand hydrogen bonding interactions. The simulation is illustrated in Figure 4, and the values of the trajectories for C. albicans are shown in Table 7.

3.4.3. Binding free energy calculations

It has been shown that the most widely used technique to estimate free binding energies is MM/PBSA.

Table 8: Binding free energy values of target proteins complexed with 6b and their respective standards