Synthesis, Molecular Docking, Molecular Dynamic Simulation Studies, and Antitubercular Activity Evaluation of Substituted Benzimidazole Derivatives

Tuberculosis, also known as TB, is a widespread bacterial infection that remains a significant global health issue. This study focuses on conducting a thorough investigation into the synthesis, evaluation of anti-Tb activity, molecular docking, and molecular dynamic simulation of substituted benzimidazole derivatives. A series of twelve substituted benzimidazole derivatives (1–12) were successfully synthesized, employing a scaffold consisting of electron-withdrawing and electron-donating groups. The newly synthesized compounds were defined by their FTIR, 1H NMR, and mass spectra. The microplate Alamar blue assay (MABA) was used to evaluate the antimycobacterial activity of the synthesized compound against Mycobacterium tuberculosis (Mtb). Compounds 7 (MIC = 0.8 g/mL) and 8 (MIC = 0.8 g/mL) demonstrated exceptional potential to inhibit M. tuberculosis compared to the standard drug (isoniazid). In addition, the synthesized compounds were docked with the Mtb KasA protein (PDB ID: 6P9K), and the results of molecular docking and molecular dynamic simulation confirmed the experimental results, as compounds 7 and 8 exhibited the highest binding energy of −7.36 and −7.17 kcal/mol, respectively. The simulation results such as the RMSD value, RMSF value, radius of gyration, and hydrogen bond analysis illustrated the optimum potential of compounds 7 and 8 to inhibit the M. tuberculosis strain. Hydrogen bond analysis suggested that compound 7 has greater stability and affinity towards the KasA protein compared to compound 8. Moreover, both compounds (7 and 8) were safe for acute inhalation and cutaneous sensitization. These two compounds have the potential to be potent M. tuberculosis inhibitors.


Introduction
Te infectious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb) [1].It is the 13 th leading cause of death globally, accounting for 1.6 million fatalities in 2022 alone [2].Te antimicrobial agents such as frst-line drugs (such as isoniazid, rifampin, and pyrazinamide) and second-line drugs (such as aminoglycosides and fuoroquinolones) are one of the primary strategies for treating tuberculosis [3].Te emergence of drug-resistant Mtb strains has posed a signifcant obstacle to the treatment of the disease.Resistance to anti-TB medications is a signifcant concern in the treatment of tuberculosis.Te situation has become more complex and costly due to the emergence of multidrug-resistant (MDR) tuberculosis, posing additional challenges to therapy.Terefore, new medicines with a low potential for resistance must be developed [4].
In synthetic chemistry, two prevalent strategies for drug design involve the repurposing of an existing parent molecule to enhance its activity, as well as the development and synthesis of a novel therapeutic molecule that has potential activity.Te frst strategy has a high success rate, and the benzimidazole scafold (Figure 1, Bz) may be the best candidate to modify into prospective anti-TB agents from this strategy [5,6].Benzimidazoles are heterocyclic compounds containing an atom of nitrogen in the ring system.Tey have a wide variety of biological actions, including antiinfammatory, antitumor, antibacterial, and antiparasitic efects [7].Te ability of substituted benzimidazoles to inhibit the synthesis of mycolic acids, a key component of the cell wall of M. tuberculosis, is one of their primary mechanisms of action.Mycolic acids are essential for the survival and persistence of Mtb, and their synthesis is mediated by the mycolic acid synthase group of enzymes.Specifcally, KasA, which is responsible for the elongation of the fatty acid moiety, facilitates the synthesis of mycolic acid.Moreover, KasA and KasB add two carbons to acyl-AcpM (mycobacterial acyl carrier protein, AcpM) to synthesize ketoacyl-AcpM (Figure 2) although KasA is superior to KasB due to its independence from cultural requirements (nature of genetic disruption growth media).Terefore, KasA protein is chosen over KasB for molecular docking investigations [9].
Substituted benzimidazoles are believed to compromise the bacterial cell wall and cause its mortality.Benzimidazoles may also destroy bacterial cells by competing with purines and inhibiting the formation of nucleic acids and proteins, according to a second hypothesis regarding their mode of action.Moreover, it has been reported that these compounds are potent topoisomerase inhibitors [10].Developing novel agents with minimal risk of resistance is crucial for the prevention of TB.Te chemical space of benzimidazole with aromatic substitution greatly enhances the binding afnity to the novel tubercular receptor.Te basic benzimidazole moiety shows signifcant interactive property but to stabilize the interaction, substitution on C2 position is necessary (Figure 1, compound Bz1).Tis combination results the new antitubercular compound along with stability [11].Te substitution of substituted aryl methananimine ring on C2 position of benzimidazole moiety (Figure 1, compound Bz2 and Bz3) reports the excellence antibacterial activity and their molecular shows the DNA gyrase inhibition as their major mode of action [12].Te antitubercular action of synthesized 1,2-disubstituted benzimidazole-5-carboxylic acid derivatives is predicted by the 3D QSAR (quantitative structure activity relationship) model, which also provides a MIC value of 0.0975 μM (Bz4 and Bz5).Compounds (Bz6, Bz7, and Bz8) with a cyclohexylethyl substituent at the C2 position and a halogen atom or methyl group at the benzimidazole ring can increase the compound's overall antitubercular efcacy [13,14].Several studies also indicate that the electron-withdrawing group can serve as a promising substitution in benzimidazole scafolds to boost biological activity [15].Moreover, addition of heterocyclic and aromatic substitution on benzimidazole leads to structural fexibility and target specifcity [16].
Te goal of this research is to synthesize a series of substituted benzimidazole compounds and assess their antituberculosis activity.Molecular modelling was used to provide insight into the binding mechanisms at molecular level.Te toxicity study is also conducted to determine the safety range of synthesized compounds.Tese compounds have the potential to enhance the treatment of tuberculosis and resolve the problem of drug-resistant M. tuberculosis strains.

Reagents and Instruments.
All the solvent and reagents used in the experiment were bought from a commercial source (analytical grade, IR grade).Shimadzu-5400 FTIR (Fourier's transform infrared) spectroscopy was used to record the IR spectra, while GC-MS (gas chromatographymass spectroscopy) (from SAIF IIT-Madrs, India) was used to record the mass spectra (for basic molecule only).With a Bruker Advance-400 MH Z spectrometer, 1 H NMR (proton nuclear magnetic resonance) was recorded (solutions in DMSO or CDCl 3 ).Te chemical shift values were given in, parts per million (ppm), about the internal standard.To check the completion of reactions, 0.2 mm silica gel 60 F254 precoated (E-Merck) plates were used for thin-layer chromatographic (TLC) studies.Open glass capillaries were used to calculate the uncorrected melting points (±2 °C).

Molecular Docking.
Te synthesized benzimidazole compounds were drawn in Chem Draw in structural data fle (SDF) format and subjected to ligand preparation using the ligand prep module of the Schrodinger suite.Using default conditions at pH 7.0 ± 2.0, the ligand preparation preserves chirality and generates at least fve low-energy stereoisomers per ligand [21,22].Using Maestro's protein preparation wizard, the crystal structure of M. tuberculosis (Mtb) KasA in complex with DG167 (PDB ID: 6P9K) was generated.Tis included the addition and optimization of hydrogen bonds, the elimination of atomic collisions, the addition of formal charges to the hetero groups, and the fnal optimization at neutral pH.Using the OPLS-3e (optimized potential for liquid simulations-3e) force feld, the structure was then reduced.A three-dimensional receptor interaction grid was constructed at the center of the bound ligand (DG167).Finally, docking was accomplished using the SP docking technique [23,24].
Where, R = Substituted Aniline / Aromatic Aldehyde substitution  [25] of promising compounds 7 and 8. To solvate the systems, a simple point charge (SPC) water model was utilized, and a suitable pair of ions, such as sodium and chloride, were used to establish a neutral environment.
Using the Desmond System Builder salt concentration panel, 0.15 M NaCl was added to the physiological system.Tis solvated and neutral system was subjected to unrestricted energy reduction using the steepest descent criterion in the OPLS3e force feld to resolve steric conficts [26].As the same parameters were used for the systems analyzed in this research, previous research may contain additional information regarding the MD (simulation box type, thermostat, barometer values, calculations of short-and longrange interactions, etc.) [27].

Results and Discussion
3.1.Chemistry.It is essential to evaluate the chemical stability, reactivity, and solubility profle, as well as the biological activity, of a substituted benzimidazole using a variety of techniques to determine how it could be used as a therapeutic agent or for other purposes.Te substitution of electron-donating or electron-withdrawing groups on benzimidazole can signifcantly alter its biological activity.Te chemical stability, reactivity, and/or solubility of the molecule may be impacted by substitutions at the diferent position on ring, which may then have an impact on the molecule's capacity to bind to a particular target or traverse a particular biological membrane, among other properties.Te presence of electron-withdrawing groups, such as chlorine or nitro, can increase the stability and hydrophobicity of a molecule.Tis can make the molecule more selective for a specifc target and increase its potency by decreasing its metabolism or elimination rate.Te electronwithdrawing groups may also enhance the lipophilicity of the molecule, thereby facilitating its passage through cell membranes.Tis can be advantageous for drug molecules, enhancing their therapeutic efcacy [29,30].
In contrast, electron-donating groups such as amino or hydroxyl groups can decrease the molecule's stability and increase its hydrophilicity.Tis can reduce the selectivity and potency of the molecule by accelerating its metabolism or elimination from the body, and it may also have difculty traversing cell membranes [31,32].Benzimidazole's biological activity can also be afected by the position of the substituent group.A substituent group at the C2 position of the ring can interact with the nitrogen atoms at positions 1 and 3 and may have a greater infuence on the molecule's chemical properties than a substituent group at a diferent position [14].

Evaluation of Anti
Depending on where the substitution occurs on the benzene ring, a molecule's activity may change.Te charge on the moiety is stabilised in compounds 7 and 8 due to the presence of electron-donating and electronwithdrawing groups in the appropriate places (Figure 4), which may account for their enhanced activity.Both 2′ and 4′ (7) or 3′ and 4′ (8) (Figure 5) were shown to be hotspots of activity.Te MIC for compounds 11 and 12 is 6.25 μg/mL because they include a strong electrondonating group (-NH 2 ) and a mild electron-donating group (-CH 3 ) in the benzene ring.Te -CHO substitution at position 4′ (2) on the benzene ring, on the other hand, completely abolishes activity (Figure 5).Te binding afnity of the compounds to their target may change as a result of this spatial variation.
Several substituted benzimidazoles have been evaluated in preclinical studies for their anti-TB activity.Veena et al. developed 1a (Figure 6), one of the most promising trisubstituted benzimidazoles that have shown promise to suppress the proliferation of Mtb.It is a derivative of benzimidazole moiety whose potency (MIC � 0.28 μM) and pharmacokinetic properties have been modifed to obtained better therapeutic outcomes [33].Similarly, 1b, which was developed by Jiménez-Juárez et al., is among the most promising compounds.In in vitro and in vivo investigations, the disubstituted benzimidazole 1b exhibited potent activity against Mtb.It is extremely selective for Mtb and has a low resistance potential.In animal studies, it has also demonstrated adequate oral bioavailability and a favorable safety profle [34].Yoon et al. reported that compounds 1c and 1d showed excellent potency against Mtb-H37RV with MIC of <0.2 μM [35].In addition, several substituted benzimidazoles have demonstrated favorable oral bioavailability and safety profles based on in silico studies [36].

6
Advances in Pharmacological and Pharmaceutical Sciences

Toxicity Prediction.
In the development of new pharmaceuticals, the relationship between molecular structure, biological activity, and toxicity is of paramount importance.Te presence of specifc functional groups, rigidity of the structure, atomic arrangement, and interatomic radii infuence the toxicity of the molecule [37].Te expected results were denoted by a negative sign (−) for nonharmful compounds and a positive sign (+) for toxic compounds [38].
Te functional groups contributed to the toxicity characteristics were also anticipated (Table 2).Te structural color green denotes the functional group that contributes to the nontoxic characteristics, whereas the structural color brown denotes the functional group that contributes to the toxic characteristics.Te compounds 7 and 8 were found to be nontoxic for acute inhalation and sensitization of the epidermis.Te chlorine atom and amine group (-NH 2 ) were responsible for toxicity, as shown in Figure 7.

Molecular Docking Analysis. Molecular docking studies
have been carried out to inquire into the binding mechanism and binding energy of the synthesized compounds and to reveal the ligand-protein interactions behind the observed KasA selectivity [39].Te validation of the molecular docking method was conducted using the redocking approach, employing the cocrystallized ligand (GSK DG167) bound to the Mtb KasA protein.Tis involved docking the cocrystallized ligand within the binding pocket of the Mtb KasA protein and comparing the resulting docked pose with the crystal structure pose through the calculation of the rootmean-square deviation (RMSD) value, which was found to be 1.34 Å.As a general criterion, a docking method can be deemed valid if the RMSD value is ≤ 2.0 Å. Figure 8 illustrates that the docked pose nearly perfectly overlapped with the crystal orientation of the Mtb KasA protein.Tis strongly suggests the validity of our docking method.Te binding afnity and mode of binding at the binding site of Mtb KasA (PDB ID: 6P9K) were investigated to gain a clearer understanding of the biological activities by which the synthesized benzimidazole analogs induced their efcacy [40].Mtb KasA was chosen as the docking target because its crystal structure with a cocrystallized benzimidazole inhibitor was readily available [41].In addition, it was reported that the benzimidazole-based inhibitors GSK DG167 and their analogs were investigated as KasA inhibitors.
Benzimidazole-based inhibitors have demonstrated promising activity against this target, indicating that KasA is a validated drug target for Mtb.It was discovered that the nitrogen atom and bridge amine group on the imidazole moiety are advantageous, as are the substituted amine groups on the side ring connected to the benzimidazole ring.Compounds 7 and 8 had the highest docking scores of −7.368 and −7.173 kcal/mol, respectively.In Table 3, the binding scores of all compounds are listed.
As shown in  9. Te secondary amino group in compound 8 creates a hydrogen bond with the carbonyl of Glu 199 (bond distance: 2.01 Å).Te 3-amino phenyl group also forms bivalent hydrogen bonds with Gly 117 and Glu 120.A similar docking scores and binding afnities of compounds 7 and 8 with DG167 indicate that the synthesized compounds have the potential to inhibit Mtb KasA efciently.

Molecular Dynamics Simulation Study.
Te utilization of MD modeling has emerged as a prominent and indispensable tool in the feld of protein-ligand research, enabling scientists to delve into the intricate nuances of protein structural stability and the intricate molecular patterns of interaction [42].Te conformational sampling of the ligand, protein backbone, and side-chain atoms are achieved through the implementation of a suitably extensive MD simulation [43].Contrary to what one might expect, this work of writing provides a deep understanding of the complex dynamics of the solvated system by illuminating the many nonbonded interactions that occur inside it and the consequent energetics.Te extraordinary fexibility of binding sites and the profound infuence of loop fexibility are just two examples of the complex mechanisms that this topic involves.By circumventing the constraints inherent in docking studies, this approach efectively eludes the limitations previously encountered, thereby ofering precise estimations of binding afnity [44].To better comprehend the binding process and the complex's stability over time, MD analyses of the target Advances in Pharmacological and Pharmaceutical Sciences   8 Advances in Pharmacological and Pharmaceutical Sciences protein-ligand complex were conducted.Using a variety of quantitative metrics, including root mean square deviation (RMSD), root-mean-square fuctuation (RMSF), radius of gyration (RGyration), and protein-ligand H bond interactions (Table 4), the dynamic behavior of the entire simulated system was exhaustively investigated.By analyzing the root-mean-square deviation (RMSD) of the protein alpha carbon atoms about simulation time, the overall structural variations and conformational stability of each compound were determined.Te RMSD indicates how the structure of the protein's atoms changed during the MD simulation.Low RMSD is a reliable indicator of a more stable system, as demonstrated by the simulation results.A wide range of oscillations in the RMSD graph, on the other hand, indicates that ligand binding to the target protein structure is unstable [45].
After the initial equilibration phase, the RMSD values of both the 7-KasA and 8-KasA complexes were stable within a range of 3 Å.Te maximum RMSD deviation observed for the 7-KasA complex was 2.89 Å, while the maximum RMSD deviation for the 8-KasA complex was 2.86 Å. Tese results indicate that compounds 7 and 8 were able to bind and interact with KasA stably.Te Apo KasA protein, on the other hand, exhibited a greater maximal fuctuation with a maximum RMSD deviation of 3.36 Å. Tis suggests that the Apo KasA protein underwent moderate structural changes and fuctuations during simulations in the absence of compounds 7 or 8 (Figure 10).Te average RMSD values of 2.33 Å for the 7-KasA complex and 2.42 Å for the 8-KasA complex further support the stability of the complexes.Te marginally lower average RMSD for the 7-KasA complex compared to the 8-KasA complex suggests that it may have attained a slightly more stable conformation.Overall, these results suggest that compounds 7 and 8 interacted efectively with KasA and formed stable complexes, as exemplifed by the relatively low RMSD values and acceptable variations observed during simulations.
Te assessment of root-mean-square fuctuation (RMSF) ofers a comprehensive portrayal of the dynamic tendencies exhibited by individual residues within the protein backbone.Tis evaluation considers their specifc placement and participation in the interaction with a designated ligand.Trough the meticulous examination of protein structures, this comprehensive analysis unveils the remarkable ability to discern regions of adaptability and fexibility.A discernibly elevated RMSF value serves as an indicator of an augmented degree of atomic fuctuation within the atomic Cα coordinates of the protein, in contrast to its customary position as observed in the MD simulations [46].Te examination of the complexes revealed a striking similarity in the overall pattern of residue fuctuations.Tis remarkable consistency was vividly illustrated by the RMS plot, which showcased the nearly identical nature of these fuctuations.Except for a limited number of residues situated in the loop regions and C-terminal ends, which are distantly positioned from the site where the ligand binds, the majority of complexes displayed a moderate level of variation.Notably, key residues interacting with compounds  11).Te low fexibility of these residues confrms their ability to form stable interactions with the promising compounds 7 and 8 compared to other residues in the KasA protein binding site.
Te radius of gyration (RGyr) analysis was performed to investigate the compactness and folding pattern of the Apo KasA protein, as well as the ligand-bound complexes with compounds 7 and 8. Te RGyr values for the Apo KasA protein, 7-KasA complex, and 8-KasA complex are presented in Table 4. Te results indicate that all three systems exhibited similar RGyr patterns, suggesting that the presence of ligands did not induce signifcant structural switching in the KasA protein.Tis observation further supports the stability of the protein-ligand complexes.Te diference between maximum and minimum RGyr values observed was 0.23 Å for the Apo KasA protein, 0.23 Å for the 7-KasA complex, and 0.26 Å for the 8-KasA complex (Figure 12).Tese fndings suggest that there were minimal diferences in the overall compactness and folding patterns among the systems.Figure 11   Advances in Pharmacological and Pharmaceutical Sciences    Hydrogen-bond analysis plays a crucial role MD simulations for understanding molecular interactions and their dynamics.Hydrogen bonds are formed when a hydrogen atom interacts with an electronegative atom.Tey contribute to the stability and specifcity of protein-ligand complexes and can afect binding afnities and conformational changes [47].Te 7-KasA complex and 8-KasA complex were analyzed for hydrogen bonds in the current study.Te minimum number of hydrogen bonds detected in the 7-KasA complex was 1, while the maximum was 3, with an average of 2.69.Similarly, the minimal and maximum numbers of hydrogen bonds for the 8-KasA complex were 1 and 3, respectively, with an average of 1.72 (Figure 13).Te analysis of hydrogen bonds reveals the presence of hydrogen bonds in both complexes, indicating the possibility of stabilizing interactions between the ligands and the KasA protein.Te average number of hydrogen bonds provides an estimate of the overall frequency and strength of these interactions.Te higher number of average hydrogen bonds in the 7-KasA complex (2.69), compared to the 8-KasA complex (1.72), suggests that compound 7 forms more hydrogen bonds with the KasA protein.Tis may indicate stronger and more extensive interactions between compound 7 and the protein, which may contribute to its increased binding afnity and site stability.
Te molecular mechanics/generalized born surface area (MM/GBSA) method serves as a pivotal tool in elucidating the energetics underlying molecular interactions postmolecular dynamics (MDs) simulations.Its signifcance lies in its ability to provide a detailed and comprehensive understanding of the binding free-energy landscape, ofering valuable insights into the stability and afnity of molecular complexes.It enables a quantitative assessment of the binding afnity between molecules by decomposing the overall binding free energy into individual contributions, such as van der Waals, electrostatic, and solvation energies [48].Te fnal 10 nanoseconds snapshot of the simulation trajectory was utilized for the binding free-energy analysis.Te MM/GBSA analysis provides comprehensive insights into the binding free energies of the 7-KasA complex and the 8-KasA complex (Supplementary fle, Table S1 and S2).Both complexes exhibit favorable overall binding energies, with ΔG bind values of −57.889 kcal/mol and −48.591 kcal/mol, respectively.Electrostatic interactions (ΔG bind Coulomb) contribute signifcantly to the favorable binding energies in both cases, with values of −22.234 kcal/mol and −18.007 kcal/mol, respectively.Moreover, hydrogen bonding interactions (ΔG bind H bond) also play a crucial role, contributing −1.782 kcal/mol and −2.397 kcal/mol to the binding energies of the 7-KasA complex and the 8-KasA complex, respectively.In addition, lipophilic interactions (ΔG bind Lipo) further stabilize the complexes, with values of −15.88 kcal/mol and −13.01 kcal/mol, respectively.However, the positive contribution of solvation energy (ΔG Residue number

RGyr (Å)
Radius of Gyration   bind Solv GB) suggests unfavorable solvation efects upon ligand binding, with values of 24.035 kcal/mol and 22.429 kcal/mol for 7-KasA complex and the 8-KasA complex, respectively (Figure 14).Although covalent interactions (ΔG bind covalent) play a minor role, their negligible contribution indicates the absence of signifcant covalent bonding.Overall, these fndings underscore the stable nature of both complexes, emphasizing the importance of electrostatic, hydrogen bonding, and lipophilic interactions in mediating ligand-receptor binding.

Conclusion
Te purpose of this investigation was to conduct a comprehensive analysis of the antimycobacterial properties of a series of recently developed benzimidazole derivatives, specifcally twelve compounds denoted as 1-12.In addition, the study aimed to assess the intricate molecular interactions between these compounds and the KasA protein.Te synthesized compounds underwent characterization using various spectral techniques, and their efcacy was investigated through in silico analysis.Te remarkable inhibitory activity against the M. tuberculosis H37RV strain, with a MIC value of 0.8 μg/mL, was observed in the chloro aniline-substituted benzimidazole derivatives, specifcally compounds 7 and 8.Moreover, a comprehensive investigation involving molecular docking and MD simulation analysis was conducted to validate the interaction between the KasA protein and the ligand.Te results of this study revealed distinct hydrogen bonding patterns between the ligand and the protein, further supporting the binding affnity between the two entities.It is worth noting that compounds 7 and 8 exhibited the most favorable binding scores, measuring at −7.368 and −7.173 kcal/mol, respectively.Upon careful examination of the in vitro and in silico studies, one can confdently surmise that compounds 7 and 8 possess the inherent capability to emerge as the most promising inhibitors of Mtb.

Supplementary Materials
Table S1: MM-GBSA-binding free energies components for the 7-KasA complex obtained from molecular dynamics trajectories.Table S2: MM-GBSA-binding free energies components for the 8-KasA complex obtained from molecular dynamics trajectories.

Figure 6 :
Figure 6: Some published promising anti-TB compounds.Blue color indicates the benzimidazole moiety.

Figure 8 :
Figure 8: Validation of the molecular docking protocol employed using before docking (green) and after glide SP docking (orange) pose of cocrystallized ligand (GSK DG167).

Figure 10 :
Figure 10: Time-dependent RMSD of Cα atoms of Apo KasA protein and in complex with compounds 7 and 8.

Figure 11 :
Figure 11: RMSF of individual amino acids of Cα atoms of Apo KasA protein and in complex with compounds 7 and 8.

Figure 12 :
Figure 12: Time-dependent radius of gyration (RGyr) of KasA protein and in complex with compounds 7 and 8.

Figure 13 :
Figure 13: Time-dependent hydrogen bond analysis of KasA protein in complex with compounds 7 and 8.
Figure S6: GCMS data with library search results for compound 1.
-TB Activity.Minimum inhibitory concentration (MIC) values pertain to the efcacy of various compounds against M. tuberculosis isolates.Te MIC values are measured in micrograms per milliliter (μg/mL) and indicate the lowest concentration of a compound that can inhibit bacterial growth.Compounds 7 and 8 have MIC values of 0.8 μg/mL, which are lower than the MIC values of standard drug isoniazid (MIC � 1.6 μg/mL), pyrazinamide (MIC � 3.12 μg/mL), and ethambutol (MIC � 1.6 μg/mL).Tis indicates that compounds 7 and 8 may be more efective against Mtb than the conventional drugs isoniazid, pyrazinamide, and ethambutol.Compounds 7 and 8 have MIC values of 0.8 μg/mL, which is equivalent to the MIC values of the conventional antibiotic rifampicin and streptomycin (MIC � 0.8 μg/mL).Te result indicates that compounds 7 and 8 are equally efective against the tested microorganisms such as rifampicin and streptomycin.In contrast, compound 2 has a MIC of 50 μg/mL, which is signifcantly higher than the MICs of all fve conventional medications.Tis indicates that compound 2 is less efcacious than standard anti-TB medications.Compounds 4, 5, 10, 11, and 12 have MIC values of 6.26 μg/mL, which is greater than the MIC values of the standard antibiotic isoniazid (MIC � 1.6 μg/mL), ethambutol (MIC � 1.6 μg/mL), rifampicin (MIC � 0.8 μg/mL), and streptomycin (MIC � 0.8 μg/mL).Te compounds 3, 6, and 9 have similar MIC values as that of isoniazid, i.e., 1.6 μg/mL.Tis indicates that compounds 3, 6, and 9 are equally efcacious against Mtb as isoniazid.All the information is presented in Table

Table 3
, the synthesized compounds have a docking score between −5.149 and −7.541 kcal/mol when compared to the other cocrystallized ligand, DG167 (−7.101 kcal/mol).All the synthesized compounds (1-12) were found to ft snugly into the active site of the enzyme at sites near DG167, which is composed of the amino acids Glu 199, Glu 200, Pro 201, Ile 202, Glu 203, Pro 206, Phe 239, and Ile 347.MIC values of 0.8 μg/mL indicate that compounds 7 and 8 exhibit promising inhibition in vitro.Te docking scores of −7.368 and −7.173 kcal/mol for compounds 7 and 8 indicate that they can bind to the active site of KasA with signifcant binding afnity.Te nitrogen of the p-amino phenyl ring and the benzimidazole moiety of compound 7 engage in hydrogen bond interactions at 2.31 and 2.20 Å, respectively, as depicted in Figure

Table 1 :
Te MIC value of synthesized compounds.

Table 4 :
Te minimum, maximum, and average values of diferent parameters, RMSD, RMSF, RGyr, and hydrogen bonding of the studied complexes.