Insights into in-vitro studies and molecular modelling of the antimicrobial efficiency of 4-chlorobenzaldehyde and 4-methoxybenzaldehyde derivatives

Abstract Owing to the significant gap in the knowledge and understanding of the mechanisms of antimicrobial action and the development of resistance, the optimization of antimicrobial therapies therefore becomes a necessity. It is on this note, that this study seeks to both experimentally and theoretically investigate the antimicrobial efficiency of two synthesized compounds namely; 1-((4-methoxyphenyl) (morpholino)methyl)thiourea (MR1) and diethyl 4-(4-chlorophenyl)-2,6-diphenyl-1,4-dihydropyridine-3,5-dicarboxylate (HRC). Utilizing the density functional theory (DFT), the compounds were optimized at ωB97XD/6-31++G(2d, 2p) level of theory. This provided a clear explanation for their distinct reactivity and stability potentials. More so, the natural bond orbital (NBO) analysis confirmed strong intra and intermolecular interactions, which agreed with the calculated reactivity parameters and density of states (DOS). Upon assessing the antimicrobial efficacy of the synthesized compounds, it was found that they exhibited lower activity against Enterobacter and A. niger, but considerable activity against Moraxella. In contrast, they showed higher activity against B. subtilis and Trichophyton, indicating that the compounds are more effective against gram-positive bacteria than gram-negative ones. Hence, it can be asserted that the synthesized compounds have superior antifungal action than antibacterial activity. A fascinating aspect of the data is that they show interactions that are incredibly insightful, totally correlating with the simulations of both molecular docking and molecular dynamics. Therefore, the alignment between experimental findings and computational simulations strengthens the validity of the study’s conclusions, emphasizing the significance of the synthesized compounds in the context of optimizing antimicrobial therapies. Communicated by Ramaswamy H. Sarma


Introduction
The ability of a substance or treatment to successfully eradicate or prevent the growth of microorganisms, such as bacteria, viruses, fungus, or parasites, is known as antimicrobial efficiency (Aati et al., 2022;Islam et al., 2022;Liu et al., 2022; Van et al., 2022).The minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) of the substance or therapy, which denotes the lowest concentration necessary to prevent or kill the bacterium, is often evaluated in order to quantify antimicrobial efficiency (Premjit et al., 2022;Shah et al., 2022;Stein et al., 2023;Zhai et al., 2022).Contextual factors that influence antimicrobial agent's effectiveness include the type of microbe, the application method, the concentration and duration of exposure, and the existence of any potential interactions or resistance mechanisms (Shen et al., 2022).The right antimicrobial agent and delivery technique must be chosen depending on the intended usage and the precise microorganisms targeted in order to obtain the highest antibacterial efficacy (Caciandone et al., 2022;Eltaweil et al., 2022;Keawpeng et al., 2022;Rezi� c et al., 2022).Antibiotics, disinfectants, antiseptics, and preservatives are typical antimicrobial agents utilized in industrial and medical contexts.Typically, antibiotics are used to treat bacterial infections; however, the effectiveness of an antibiotic depends on the type of bacteria being treated and how susceptible they are to the antibiotic (Sophia et al., 2022).The effectiveness of these compounds depends on the type of microorganism and the preservative's concentration (Dey et al., 2022).
Additionally, with the rise of multidrug-resistant bacteria, there is an urgent need to develop new antimicrobial agents with higher efficacy and fewer side effects.One approach to this problem is to use computational techniques to investigate the antimicrobial efficiency of various compounds against different bacterial and fungal pathogens (Chandwani et al., 2022).Overall, the effectiveness of antimicrobial agents depends on a variety of factors, and selecting an appropriate agent and delivery method requires careful consideration of the intended use and the specific microorganisms targeted (Babu et al., 2023;Sklyar et al., 2023;Xie et al., 2023).By understanding the principles of antimicrobial efficiency, we can better prevent and treat infections and promote public health.Therefore, the investigation of the antimicrobial efficiency of compounds against these pathogens using DFT, molecular docking, and molecular dynamics simulation can provide valuable insights into the mechanisms of action of antimicrobial agents and aid in the development of more effective drugs (Cao et al., 2023).By identifying the specific interactions between antimicrobial compounds and bacterial and cells, researchers can design compounds that are more selective and effective against these pathogens, while minimizing toxicity to human cells (Shalaby et al., 2023).In recent years scientists have employed Density Functional Theory (DFT) which is a quantum mechanical method that allows for the calculation of electronic properties of molecules (Meng et al., 2023;Slassi et al., 2023).
The need for this study is driven by the growing concern over antimicrobial resistance, which poses a serious threat to public health.Hence, the comprehensive aim of this study is to evaluate the antimicrobial effectiveness of two compounds, namely 1-((4-methoxyphenyl)(morpholino)methyl) thiourea and diethyl 4-(4-chlorophenyl)-2,6-diphenyl-1,4-dihydropyridine-3,5dicarboxylate, through in-vitro studies and molecular modelling.Also, the study seeks to determine the antibacterial and antifungal properties of these compounds against various strains of bacteria and fungi.Additionally, the study aims to use molecular modelling techniques to understand the reactivity and stability of these compounds and to ultimately provide insights into the potential of these compounds as antimicrobial agents for future therapeutic use.And as such, the synthesis, characterization and identification of MR1 and HRC have been fully accounted for employing the FT-IR, 1 H and 13 C-NMR with their respective mass spectra analysis.These synthesized compounds were further examined utilizing the frontier molecular orbital (FMO) and density of state (DOS) to corroborate the reactivity and stability potentials of the compounds optimized at the DFT/xB97XD/6-31þþG(2d, 2p) level of theory.This was further substantiated by the evaluation of their respective inter and intra molecular interactions utilizing the natural bond orbital (NBO).Additionally, following standard microbiological techniques, several bacterial and fungal pathogens were isolated and identified as significant causes of infections in humans, notably Enterobacter, B. subtilis, Moraxella, A. niger, and Trichophyton.After which the antimicrobial potentials of the synthesized compounds were analyzed against the bacterial isolates (in-vitro investigation).Note: Enterobacter is a gram-negative bacterium commonly found in hospital settings and can cause infections in immunocompromised patients (Davin-Regli & Pag� es, 2015).B. subtilis is a gram-positive bacterium found in soil and can cause infections in humans (Kov� acs, 2019).Moraxella catarrhalis is a gram-negative bacterium commonly found in the respiratory tract and can cause infections such as conjunctivitis and otitis media (Verduin et al., 2002).A. niger is a filamentous fungus commonly found in soil and can cause infections in immunocompromised patients (Schuster et al., 2002).Trichophyton is a dermatophyte fungus that can cause skin and nail infections in humans (Gr€ aser et al., 1999).Summarily, molecular docking computational technique that predicts the binding affinity and orientation of a ligand to its receptor was incorporated in this study.Additionally, molecular dynamics simulation which is a technique that allows for the investigation of the stability and dynamics of antimicrobial compounds in complex biological environments, such as bacterial cells were equally investigated.The results of this study are expected to provide valuable insights into the potential use of these compounds as antimicrobial agents.The study may also contribute to the development of new strategies for combating antimicrobial resistance, which is a critical public health issue.Furthermore, the molecular modelling aspect of the study may provide new insights into the design of more effective antimicrobial agents in the future.
The reaction mixture was taken in a RB flask and kept over a magnetic stirrer and stirred at cold condition for 4 hrs.The product separated out was filtered and washed several times with water and dried over vacuum.See Scheme 1.

The choice of bacterial and fungal species
The choice of bacterial and fungal species in this study is based on their relevance in testing the antimicrobial efficiency of the two compounds, 1-((4-methoxyphenyl) (morpholino)methyl)thiourea and diethyl 4-(4-chlorophenyl)-2,6diphenyl-1,4-dihydropyridine-3,5-dicarboxylate.Enterobacter, B. subtilis, and Moraxella are commonly used bacterial strains in antimicrobial studies due to their ability to cause infections in humans and their susceptibility to various antimicrobial agents.These bacterial species also have different structures and characteristics, which make them suitable for testing the broad-spectrum antimicrobial activity of the compounds.As for the fungal strains, A. niger is commonly used in antimicrobial studies due to its high resistance to antimicrobial agents and its ability to produce various enzymes, while Trichophyton is a common fungal pathogen that causes skin infections in humans.Thus, these two fungal strains were chosen to test the antifungal activity of the compounds.Overall, the choice of these bacterial and fungal strains in the study is aimed at providing insights into the in vitro antimicrobial activity of the two compounds, as well as using molecular modelling to gain a better understanding of the molecular interactions between the compounds and the microbial cells.

Microorganisms and culture media
Bacterial cultures were obtained from Eumic analytical Lab and Research Institute, Tiruchirappalli, Tamilnadu, India.Bacterial strains were maintained on Nutrient agar slants (Hi media) at 4 � C. Three bacterial strains viz., Enterobacter, B. subtilis and Moraxella and two fungi strains viz., A. niger and Trichophyton were tested against HRC employing Oflaxacin and Amphotericin B as positive standards for bacteria and fungi strains, respectively.

Inoculum preparation
Bacterial cultures were sub cultured in liquid medium (Nutrient broth) at 37 � C for 8 h and further used for the test (10 5 -10 6 CFU/mL).These suspensions were prepared immediately before the test was carried out.The ingredients were added into the distilled water and boiled until the medium dissolve completely and the same was sterilized by autoclaving at 15 lb psi pressure (121 � C) for about 15 min.The nutrient broth was prepared by the same composition without agar.

Details of computation
The geometrical structures of the compounds analyzed in this study were optimized at the DFT/xB97XD/6-31þþG(2d, 2p) level of theory using Gaussian 16 (Frisch et al., 2016) and GaussView (Dennington et al., 2001), as illustrated in Figure 1 and Table S1 of the supporting information.This optimization process motivated a subsequent vibrational spectroscopic investigation in order to assign the fundamental bands observed in the FTIR analysis.The physical and chemical properties of the compounds were also screened using computational calculations based on Density Functional Theory (DFT), including a natural bond orbital (NBO) analysis to determine the redistribution of electron density (ED) in various bonding, antibonding, and E(2) energies.Molecular geometry, Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) energies, and Molecular Electrostatic Potential (MEP) analysis were conducted to provide insight into charge transfer within the molecule.The overlapping of molecular orbitals (PDOS) was investigated by combining Total Density of States (TDOS) and Partial Density of States (PDOS) using multiwfn 3.7 (Lu & Chen, 2012).HOMO-LUMO visualization was carried out using Chemcraft.The results of this study provide a comprehensive analysis of the vibrational and electronic properties of the compounds, combining both theoretical and experimental data.

The choice for target receptors and molecular docking protocol
With careful consideration given to their connotations with B. subtilis, A. niger, Enterobacter, Moraxella catarrhalis, and Trichophyton, the target receptors with PDB IDs: 7AQC, 1KUM, 3SU9, 4IMM, and 4ZHS employed in this study were downloaded from the protein data bank (www.RCSBPDB.org) on the following basis: B. subtilis (PDB: 7AQC) The selection of 7AQC, which depicts the structure of the B. subtilis RQC complex, was based on the study's findings that Hsp15 homolog stabilizes the P-site tRNA conformation.The P-site tRNA is a transfer RNA molecule that is bound to the ribosome during translation and carries the peptide chain growing on the ribosome.In the presence of a stalled ribosome, the RQC complex recognizes the stalled nascent polypeptide and recruits Hsp15 homolog, which stabilizes the P-site tRNA conformation, thereby allowing the stalled nascent polypeptide to be degraded.The cryoelectron microscopy images obtained by Filbeck et al. (2021) revealed that the B. subtilis RQC complex is a large and flexible structure composed of several components, including Hsp15 homolog, which binds to the ribosome's large subunit near the peptidyl transferase center.This binding stabilizes the Psite tRNA conformation, which is necessary for the efficient degradation of stalled nascent polypeptides.Furthermore, the study also revealed that the B. subtilis RQC complex undergoes conformational changes during the process of Ala tail synthesis, which is a modification of the nascent polypeptide that occurs during translation.These findings provide insights into the mechanism of the RQC system and highlight the importance of Hsp15 homolog in the proper functioning of the RQC complex.In conclusion, the study by Filbeck et al. (2021) provides important insights into the structure and function of the B. subtilis RQC complex, which is important for maintaining the fidelity of protein synthesis in the cell.The use of 7AQC in the current study allows for the investigation of the potential effects of the compounds on this complex, providing insights into their antimicrobial activity and potential therapeutic applications.

Aspergillus niger (PDB: 1KUM)
The study by Sorimachi et al. (1996) investigates the structure of the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger (PDB: 1KUM) using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing.The researchers found that the SBD structure does not undergo significant conformational changes upon ligand binding, indicating that the binding process is mainly driven by electrostatic and van der Waals interactions.This is in contrast to other studies that have shown significant conformational changes in protein structures upon ligand binding.Also, the use of the SBD structure from Aspergillus niger in the study is important because it is a well-characterized system for studying starch binding and hydrolysis.The use of NMR spectroscopy and simulated annealing allows for the investigation of the dynamic properties of the protein structure and the interactions with ligands at the atomic level.Overall, the study by Sorimachi et al. (1996) provides important insights into the structure and function of the SBD of glucoamylase 1 from Aspergillus niger, which is important for understanding the mechanisms of starch digestion in fungi and other organisms.The findings have implications for the development of novel therapies for metabolic disorders and highlight the importance of studying protein-ligand interactions at the atomic level.
Enterobacter (PDB: 3SU9) (https://www.rcsb.org/structure/3SU9) The crystal structure of MurA (PDB: 3SU9) has been determined, revealing important insights into the mechanism of inhibition by fosfomycin.The structure of MurA consists of two domains, with the active site located in a cleft between them.Fosfomycin binds to the active site of MurA, where it forms a covalent bond with the enzyme's cysteine residue.This blocks the enzyme's ability to catalyze the reaction that it is responsible for, ultimately leading to the disruption of the bacterial cell wall and the death of the cell.The relevance of the MurA receptor in the context of Enterobacter is particularly significant because Enterobacteriaceae are becoming increasingly resistant to antibiotics.In recent years, there has been a rise in the prevalence of Enterobacteriaceae strains that produce extended-spectrum b-lactamases (ESBLs) and carbapenemases, which confer resistance to many classes of antibiotics.This has led to an urgent need for new antibiotics that can effectively target these bacteria.While fosfomycin remains an effective treatment for some Enterobacteriaceae infections, resistance to this antibiotic is also emerging.Resistance to fosfomycin can arise through mutations in the MurA enzyme that prevent the antibiotic from binding to the active site.Other resistance mechanisms include the production of enzymes that can degrade or modify the antibiotic.In conclusion, the discovery of the crystal structure of MurA and its inhibition by fosfomycin has provided important insights into the mechanism of action of this antibiotic.However, the emergence of resistance to fosfomycin highlights the ongoing need for the development of new antibiotics that can effectively target Enterobacteriaceae and other bacterial pathogens.

Trichophyton rubrum (PDB: 4ZHS)
The study conducted by Li et al. (2016) determined the crystal structure of aspartate semialdehyde dehydrogenase (ASADH) from Trichophyton rubrum and provided insights into the categorization of ASADHs into dimeric and tetrameric enzymes with different orientations for NADP binding.This is significant because ASADH is a key enzyme in the biosynthesis of essential amino acids and a potential target for developing antifungal drugs.The crystal structure of ASADH from T. rubrum revealed that the enzyme exists in two distinct conformations: a dimeric form and a tetrameric form.The dimeric form of ASADH has a single active site and binds NADP in a manner similar to that of other NADP-dependent dehydrogenases.In contrast, the tetrameric form of ASADH has two active sites and binds NADP in a different orientation, with the coenzyme interacting with residues from both subunits.The authors also found that the tetrameric form of ASADH is more prevalent in fungal species, while the dimeric form is found in bacteria and plants.This suggests that the tetrameric form of ASADH may be a more important target for developing antifungal drugs.The crystal structure of ASADH from T. rubrum also revealed several key amino acid residues that are involved in substrate binding and catalysis.These residues could serve as targets for designing specific inhibitors that selectively target fungal ASADHs and prevent their activity, leading to the inhibition of fungal growth.Overall, this study provides important insights into the structure and function of ASADHs and offers a framework for developing new antifungal drugs that target this enzyme.Further research is needed to identify specific inhibitors that can selectively target fungal ASADHs and to determine their potential as antifungal agents.
Moraxella catarrhalis (PDB: 4IMM) (https://www.rcsb.org/structure/4IMM) The crystal structure of BamB has provided important insights into the mechanism of the Bam complex.For example, the structure has revealed that the hydrophobic groove in BamB is important for stabilizing the BamA subunit and for maintaining the integrity of the Bam complex.In addition, the structure has shown that BamB interacts with other subunits of the Bam complex, such as BamC and BamD, and that these interactions are important for the proper function of the Bam complex.The crystal structure of BamB has also been used to design small molecules that can inhibit the function of the Bam complex.These small molecules have potential as antibacterial agents, as they can disrupt the biogenesis of outer membrane proteins in gramnegative bacteria and thus make these bacteria more vulnerable to antibiotics.In summary, the crystal structure of BamB from Moraxella catarrhalis (PDB: 4IMM) has provided important insights into the mechanism of the Bam complex and has potential applications in the development of new antibiotics.
Summarily, these proteins were meticulously prepared with the help of the Biovia discovery studio (Zala et al., 2023), followed by the synthesized compounds being appropriately docked with the target receptors with the help of the Autodock vina.Also, with aid of PyMol and the Biovia discovery studio visualizers, the results of the docking experiments were primarily reported on the abundance of hydrogen bonds, van der Waals interactions, electrostatic interactions, and entropy changes upon the binding of the ligands to the receptors.

Molecular dynamic (MD) simulation
The MD studies were performed for the promising compound HRC and MR1 in with different protein targets 1KUM, 7AQC, 3SUQ, 4IMM, 4ZHS using the Schr€ odinger Desmond 2021-1 MD simulation programme, installed on a Z2 TWR G4 workstation with the configuration Ubuntu 18.04.3LTS 64-bit, Intel Core i7-9700 and NVIDIA Quadro graphics processing unit (Kikiowo et al., 2022;Sudevan et al., 2022).The protein-ligand complex systems were solvated using a simple point charge (SPC) water model, and an adequate pairing of ions like sodium and chloride were employed to develop the neutral environment, and a 0.15 M NaCl salt concentration correlating to the physiological system was assigned using the Desmond System Builder panel.Using a fixed parameter of the OPLS3e force field, this solvated and neutral system was subjected to unconstrained energy minimization using the steepest descent criterion in order to eliminate steric conflicts.The resultant system underwent a short 100 ps isothermal-isobaric or NPT ensemble equilibrium circumstance at constant pressure and temperature.The temperature was maintained at 300 K for 100 ps during NPT equilibration using a 'Nose-Hoover chain thermostat'.The Martyna-Tobias-Klein barostat was used to maintain the pressure at 1.0315 bar during the NPT equilibration.The smooth particle mesh Ewald approach was used to evaluate long-range electrostatic interactions with a tolerance of 1e-09, while the short-range Van der Waals and Coulomb interactions were estimated with a cut-off radius of 9.0 Å.The simulation was run for 100 ns, and trajectory snapshots were taken at intervals of 100 ps.Desmond's Simulation Interaction Diagram (SID) was used to analyze 1000 MD trajectories and determine the stability and binding orientation of the ligand (Patel et al., 2023;Puri et al., 2023).
The ligand binding free energies for the simulated complex were calculated using molecular mechanics generalized Born/solvent accessibility (MM-GBSA) over a 100 ns time period using the thermal_mmgbsa.pypython script provided by Schrodinger.This script takes a Desmond trajectory and divides it into individual snapshots.On each frame, the MM-GBSA calculations are performed, and the average computed binding energy and standard deviation are then output.

Vibrational analysis (FT-IR)
Fourier-transform infrared spectroscopy (FT-IR) is a very useful analytical technique whose importance cannot be over emphasized.It is used to compare and detect frequencies of compounds with a great speed, high sensitivity and calibration (Agwamba et al., 2022a;Benjamin et al., 2022c).The advantage of FT -IR spectroscopy on detecting the frequencies of the organic compounds is based on the fact that the atomic masses and force constant (bonding strength) are different across the chemical groups and component atoms present in the molecules which results to the appearance of different adsorption bands/frequencies on the spectrograph at different points which again corresponds to different functional groups as present in such a molecule (Asogwa et al., 2022;Eno et al., 2022).In The organic drug molecules in this studies, HRC exist as a non-linear compound which has 61 atoms and 177 vibrational modes while MR1 consist of 38 atoms with 108 vibrational modes.IR spec has an intrinsic potential to convert a mid-IR-irradiated IR-active molecule to radiate unique and complex vibrational motions which can be obtained by simply redesigning it to produce normal simple vibrations as seen in the equation: 3 N-5 for linear and 3 N-6 for non-linear molecules, respectively, where N denotes the number of atoms present in the compound (Agwamba et al., 2022c;Makhlouf et al., 2023).The theoretical waveband determination was done by employing the density functional theory at the DFT/xB97XD/6-31þþG(2d, 2p) level of theory) level of theory while the frequencies corresponding to the functional groups of the various bands obtained from the experimental analysis is shown in Table 1.As shown in the experimental IR spectra analysis (as shown in Figures S1 and S2 of the supporting information), notable vibrational regions were observed for the organic compounds in studies.Below is a detailed analysis on the theoretical vibrations present in the studied drug compounds.

C-H Vibrations
the C-H vibrations is considered to have the highest frequency of adsorption in IR spectra.The experimental FT-IR C-H stretching was observed to peak at 2852 cm À 1 in HRC while the theoretical wavelength was observed at 3100 cm À 1 , CH stretching due to methine group in MR1 was observed to be 2960 cm À 1 in the experimental.On the theoretical vibrations, the frequencies observed was 2990.82 cm À 1 and 3144.36 cm À 1 for MR1 and HRC, respectively.C-H aromatic stretch in HRC was complemented with a wavelength of 3065 cm À 1 in the theoretical observation while bending vibration was experimentally observed at 1367 cm À 1 and the theoretically calculated peaks at 1374,1378.75,1382.36cm À 1 .Meanwhile, theoretical aromatic C-H vibrations was also observed for MR1 at 2958.92 to 3240.78 cm À 1 .Peak at 2834 cm À 1 was observed in MR1 for CH stretching of -OCH 3 as observed from the experimental FT-IR.the theoretical frequency was observed at 3162.18, 3091.16,3027.35cm À 1 .These observed vibrations are in accordance with previously reported literatures.

C ¼ C Vibrations
Coming from the baseline of already reported studies, the C ¼ C vibrations observed in this studies are in good agreement with previous reported studies.In HRC, the experimental waveband observed was 1592 cm À 1 and 1511 cm À 1 for MR1.In the theoretical calculations, wavelength observed for HRC was peaked at 1738.13, 1712.40,1677.09 and 1669.74cm À 1 while peaks observed for MR1 was 1691.79, and 1652.75 cm À 1 .

C-N Vibrations
C-N vibrations had been previously reported to occur in the ranges of 1200 cm À 1 to 1350 cm À 1 .In this studies, the carbon to nitrogen stretching observed herein were basically symmetric and occurred in the experimental analysis in MR1 at 1395 cm À 1 , on the theoretical analysis C-N vibrations was noticed at 1390.01 cm À 1 , and 1400.33 cm À 1 .

C ¼ S Vibrations
Experimental waveband observed for C ¼ S vibrations in MR1 was seen at 1121 cm À 1 while theoretical wavelengths was observed in 1136.53 cm À 1 , 1266.68 cm À 1 , and 1294.59cm À 1 .This in accordance with wavebands previously reported ranging from 1025 cm À 1 to 1225 cm À 1 .

N-H Vibrations
Waveband at 3348 cm À 1 was experimentally observed for HRC and also 3343.24 cm À 1 and 1605 cm À 1 for NH stretching and bending of MR1 molecule.In the DFT theoretical calculations, NH stretching was observed for HRC at the wavelength of 3667.78 cm À 1 and in MR1, 3645.98 cm À 1 and 1650.81cm À 1 was observed for NH stretching and bending vibrations, respectively.

Other vibrations
Other vibrations as well as functional groups as observed for the studied organic drug molecules includes C-Cl stretching which was experimentally observed at 753 cm À 1 in HRC and was theoretically observed at 1126.02 cm À 1 , symmetric C-O-C stretching of MR1 was experimentally observed at 1026 cm À 1 and theoretically observed at 1174.97 cm À 1 .C ¼ O vibration in HRC was experimentally noticed at 1730 cm À 1 while it was theoretically peaked at the wavelengths of 1790.59,1779.52, and 1738.13 cm -1.Also, NH and CH bending vibrations was observed at 1581.10 cm À 1 for MR1 drug.Substantially, the demonstration of multiple theoretical frequencies for key functional groups, such as vsCH, vsC ¼ C, basymC-H, vsC ¼ O, vs-OCH3, vsC ¼ C, vsC ¼ S, and vsC-N, further reinforces the scientific validity of the DFT-FTIR analysis.The fact that these calculated frequencies fall within the expected ranges validates the experimental frequencies, providing strong evidence that the DFT-FTIR method accurately captures the vibrational behavior of these functional groups.The consistency between theoretical and experimental values indicates the reliability of the calculations and supports the conclusion that the DFT-FTIR analysis provides an accurate representation of the molecular vibrations.While the vsC-Cl frequency calculation in the HRC compound showed a more pronounced difference between experimental (753 cm À 1 ) and calculated (1126.02cm À 1 ) values, it is important to recognize that slight deviations are expected in both experimental and theoretical approaches.Experimental measurements can be influenced by various factors, such as instrument calibration, sample preparation, and environmental conditions, while theoretical calculations rely on approximations and assumptions in the computational model.The fact that the differences between theoretical and experimental values in the table generally fall within an acceptable range indicates that the DFT-FTIR analysis provides a reasonable estimation of the vibrational behavior of the synthesized compounds.To provide additional support and references for our findings, we have included relevant and verifiable in these literatures (Agwamba et al., 2022a(Agwamba et al., , 2022c;;Asogwa et al., 2022;Benjamin et al., 2022c;Eno et al., 2022;Makhlouf et al., 2023).In conclusion, the multiple theoretical frequencies within calculated ranges, the close agreement between theoretical and experimental values for various functional groups, and the inclusion of relevant literature references collectively provide robust scientific evidence for the accuracy and reliability of the DFT-FTIR analysis in predicting the vibrational characteristics of the studied compounds.

1 H and 13 C-NMR
According to the experimental data, the peaks at 9.8 and 7.8 ppm shows the presence of NH 2 and NH protons, respectively, as substantiated by the theoretical calculations at 9.9 and 9.5 pp, respectively (see Figures S3a-S4b of the supporting information).The signals ranges from 7.3 to 6.9 ppm are due to aromatic protons.Methine proton shows a peak at 5.6 ppm.A peak appeared at 3.86 ppm indicates the presence of -OCH 3 protons.CH 2 protons of Morpholine are attributed by the signals from 3.7 to 3.4 ppm, these are all in agreement with the theoretical calculations.Furthermore, the 13 C-NMR of MR1 is shown in Figures S4a-S4b of the supporting information.Thiocarbonyl carbon of the compound is indicated by a signal at 191 ppm for the experimental calculations while 66.7 ppm was accounted for the theoretical calculations.Aromatic carbons exhibited the signal ranges from 164 to 113 ppm while the theoretical 13 C-NMR.Ranged from 130.6 to 159.2.A peak appeared at 66 ppm shows the presence of methine carbon.The peaks obtained from 61 to 55 ppm are assigned to the carbons of Morpholine.The Mass spectrum of MR1 is given in Figure S5 of the supporting information.The molecular ion peak appeared at m/z 281 matches with the exact molecular mass of the compound.
1 H-NMR spectrum of HRC has been shown in Figure S3-S6.The experimental aromatic protons of Chloro phenyl and phenyl ring are indicated by the signals range from 8.1 to 7.8 and 7.7 to 7.2 ppm, respectively, whereas the theoretical 1 H-NMR calculates a range from 9.1 to 7.2 and 7.7 to 7.2, respectively.A peak appeared at 5.1 ppm indicates the presence of NH proton.Methine proton shows a peak at 4.1 ppm.CH 2 and CH 3 protons of ester group show peaks at 3.7 and 0.73, respectively which is in agreement with the theoretical calculations of 4.7 and 4.0, respectively (see Figures S6a-S6b of the supporting information).Additionally, the 13 C-NMR of the compound HRC has been presented in the.Carbonyl carbon of ester shows a peak at 167.49 ppm for the experimental calculations while the theoretical calculations predicted 144.2 ppm.The peaks ranging from 136 to 127 indicate the aromatic carbon.CH 2 and CH 3 carbons of ester are indicated by peaks at 60.8 and 13.37 ppm, respectively.A peak at 42.41 ppm is due to methine carbon (Figures S7a-S7b of the supporting information).Figure S8 represents the mass spectra of the compound HR2.The peak appearing at m/z 488 confirms the calculated molecular mass of the compound.The intense peak at m/z 486 is the base peak.

Reactivity and stability descriptors
The frontier molecular orbital is comprised of the HOMO (highest occupied molecular orbital) which serves as the Lewis electron donor and secondly the LUMO (lowest occupied molecular orbital) which acts as the Lewis electron acceptor (Agwamba et al., 2022b;Benjamin et al., 2022a;Louis et al., 2022).The difference between the HOMO and the LUMO gives the energy gap of a molecule or complex.The energy gap gives us insight on the reactivity and conductivity as well as kinetic stability which is determined by the ease in HOMO to LUMO electron transfer (Apebende et al., 2022;Inah et al., 2023).Small HOMO-LUMO energy gap entails high reactivity of a studied compound and the opposite is the case for a compound with high energy gap calculated in this studies using the DFT/xB97XD/6-31þþG(2d, 2p) level of theory.The HOMO are related by ionization potential (IP), while the LUMO is related by electron affinity (EA) of the drug molecules.Global quantum reactivity parameters proposed by Koopsman were employed to further gain knowledge on the reactivity of the drug molecules these parameters included the IP, EA, global hardness (g), global softness (S), chemical potential (l), electronegativity (✗), fermi energy level (E FL ) and electrophilicity index which were calculated using Eqs.( 1)-( 8) and obtained values recorded in Table 2.
The obtained values from the analysis of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) provide significant insights into the reactivity and stability of the studied compounds.The concentration of electrons in the HOMO and the electron donation to the LUMO indicate the electron flow within the molecules.In this study, it is observed that HRC exhibits a HOMO-LUMO energy gap (DE) of 8.089 eV, while MR1 shows a slightly higher value of 8.359 eV.This suggests that MR1 is more chemically stable compared to HRC, as indicated by the lower DE value.Moreover, the values of global hardness (g) and softness reflect the stability and reactivity of the compounds, respectively.HRC demonstrates the highest global hardness value of 0.124 eV, indicating its greater stability.On the other hand, MR1 has a slightly lower value of 0.120 eV, suggesting its relatively higher reactivity.Chemical potential, represented by the symbol l, also relates to reactivity.In this study, HRC exhibits higher l values, further supporting its increased reactivity.Additionally, electronegativity (✗) signifies a molecule's ability to accept electrons from interacting species, while the Fermi energy level (EFL) represents the midpoint of the HOMO-LUMO energy gap.The low values of EFL provide valuable insights into the reactivity of compounds.Overall, the reactivity parameters analyzed in this study, including HOMO-LUMO energy gap, global hardness, chemical potential, electronegativity, and Fermi energy level, support the observation that both organic drug molecules exhibit reactivity.However, HRC shows a higher electron flow and greater reactivity, while MR1 displays higher chemical stability.Figure 2 in the study illustrates the HOMO-LUMO isosurfaces, visually representing the distribution of electronic densities within the molecules, further supporting the analysis and discussion of their reactivity.

Natural bonding orbital (NBO)
To fully understand in details, the intra and intermolecular charge transfer within a molecule as well as the delocalization of pi electron between the Lewis donor orbital to the Lewis acceptor electron orbital, the natural bond orbital (NBO) analysis was introduced into this studies (Apebende et al., 2023;Gharbi et al., 2023;Qader et al., 2022).High stabilization energy (E (2) ) will lead to strong interactions between orbitals which will in turn lead to a high stabilization and high delocalization of a studied molecule.Employing the DFT/xB97XD/6-31þþG(2d, 2p) computational level of theory, the energetic stabilization which could be due to a forward transition from donor to acceptor orbital s can be estimated using the second order perturbation theory of the fock matrix through Eq. ( 9) and results are recorded on Table 5.
Where qi signifies the donor orbital occupancy, Ei and Ej signifies the orbital energies of the donor and acceptor NBO orbitals.
According to Table 3, there were very high stabilizations energy in HRC with energy values of 30780.49kcal/mol, 39580.44 kcal/mol, 97839.56kcal/mol, 38858.95kcal/mol and 33429.13kcal/mol while the major orbital contributions was in the r !r � forward transition.The perturbation energies recorded for MR1 on the other hand showed a lower energy values with the highest energy of 4375.14 kcal/mol with the r !r � having a major contribution.This is to say that HRC molecule as a stronger electron transfer stability between its molecules when compared to MR1.The natural bond orbital (NBO) analysis as seen herein is in correspondence with the FMO analysis.

Density of states (DOS) evaluation
The density of states which includes the total density of states (TDOS), partial density of states (PDOS) and the overlap density of state (OPDOS) is used to account for the number of states matching with a unit energy and represented by atoms fragments (Muthu et al., 2013).The density of states can be used to represent the molecular distribution of orbitals and the strengths of atom fragments of the studied organic structures and contributions to chemical bonding (Paulraj & Muthu, 2013).The different curves on the maps indicates the number of molecular orbitals present at various quantum state.The pictorial visualization of the plot is given in Figure 3.The analysis of the highest occupied molecular orbital (HOMO) and lowest occupied molecular orbital (LUMO) provides essential insights into the conductivity and electronic transport reactivity of molecules.In our study, examining the TDOS and PDOS orbitals revealed significant findings regarding the contributions of different atomic fragments to the electronic structure of the compounds.For HRC, it was observed that hydrogen (H) atom fragments had a prominent contribution from the HOMO to the LUMO orbital.As the plot transitioned to the OPDOS orbital, carbon (C) atom fragments exhibited a greater contribution to the overall molecule, although hydrogen (H) atoms remained dominant throughout the plot.This observation suggests that HRC is characterized by a significant presence of hydrogen (H) fragments, particularly in the energy range from À 14.0 eV to À 12.5 eV.Additionally, there was a minimal contribution within the energy range from À 13.5 eV to À 3.0 eV.The Fermi energy level for HRC was determined to be À 8.15 eV, indicating the midpoint of the HOMO-LUMO energy gap.In the case of MR1, the major orbital contribution in the HOMO orbital came from hydrogen (H) fragments.However, as the plot transitioned across the Fermi level to the OPDOS orbital, oxygen (O) atom fragments became dominant in the LUMO orbital.This finding indicates that MR1 exhibits a different distribution of orbital contributions compared to HRC.The Fermi energy level for MR1 was determined to be À 8.11 eV, which provides insight into its electronic reactivity.Overall, the analysis of the HOMO and LUMO orbitals for both HRC and MR1 provides valuable information about the electronic structure and contributions of different atomic fragments.These insights contribute to our understanding of the conductive properties and electronic transport reactivity of the compounds, enhancing our knowledge of their potential applications in various fields.

Antimicrobial evaluation of MR1
The zone of inhibition analysis presented in Table 4 and Figure 4a and b provides valuable insights into the antimicrobial activity of the compound MR1.The results indicate that MR1 exhibits low activity against Enterobacter and A. niger, as evidenced by the small zone of inhibition observed.However, it demonstrates moderate activity against Moraxella, suggesting some effectiveness against this microorganism.On the other hand, MR1 shows high potency against B. subtilis and Trichophyton, as indicated by the larger zones of inhibition observed in these cases.It is noteworthy that the maximum activity is observed at a concentration of 100 mL.This concentration seems to be most effective in inhibiting the growth of the tested microorganisms.Furthermore, the findings reveal that MR1 exhibits potent activity against gram-positive bacteria while showing relatively less activity against gram-negative bacteria and fungal strains.These observations highlight the selective antimicrobial action of MR1, indicating its efficacy against specific types of microorganisms.The compound's higher potency against gram-positive bacteria suggests its potential as an antibacterial agent targeting this particular group.However, its lower activity against gram-negative bacteria and fungal strains indicates a limited spectrum of activity in relation to these types of microorganisms.

Antimicrobial evaluation of HRC
The antimicrobial evaluation results presented in Table 5 and Figure 5a and b provide valuable insights into the activity of the compound HRC against different microorganisms.The findings indicate that HRC exhibits low activity against Enterobacter and A. niger, as evidenced by the smaller zones of inhibition observed.However, it demonstrates considerable activity against Moraxella, suggesting its effectiveness in inhibiting the growth of this particular microorganism.On the other hand, HRC shows higher activity against B. subtilis and Trichophyton, as indicated by the larger zones of inhibition observed in these cases.It is noteworthy that HRC exhibits greater activity against B. subtilis and Trichophyton than the standard drug.This indicates the potential of HRC as a more effective antimicrobial agent specifically targeting these microorganisms.Furthermore, the results demonstrate that HRC exhibits a higher degree of activity against grampositive bacteria compared to gram-negative bacteria.This suggests that HRC is more effective in inhibiting the growth of gram-positive bacteria, highlighting its potential as an antibacterial agent against this particular group.Overall, the findings support the notion that HRC possesses excellent antifungal activity while demonstrating relatively less antibacterial activity.These observations suggest that HRC may be a promising candidate for the development of antifungal agents and further investigations into its specific mechanism of action against fungal strains are warranted.

Molecular docking simulation
To further understand the suitability of the studied Antimicrobial evaluation, molecular docking studies has been performed on the structures of the studied compounds and the bioactivity score detected based on the interaction with the selected receptor proteins (Abraham et al., 2019;Benjamin et al., 2022b;Nicely et al., 2022;Patel et al., 2017;Rahuman et al., 2020;Upadhyay et al., 2023).It's worth noting that hydrogen bonds and amino acids can be used in the design of drugs.For example, hydrophilic amino acids can be used to promote the solubility of a drug molecule, while hydrophobic amino acids can be used to increase its stability and reduce its susceptibility to degradation (Upadhyay et al., 2023).
Hydrogen bonds can also be used to facilitate the formation of supramolecular complexes between a drug molecule and a carrier molecule, which can enhance its delivery and uptake by cells (Gannouni et al., 2023;Udoikono et al., 2022;Vala et al., 2021).Interestingly, the obtained results depict very insightful interactions, thus agreeing completely with in-vitro examinations earlier discussed.To begin with, both MR1 and HRC is observed to calculate very significant binding affinities with the bacterial and fungal target receptors.Notably, HRC@7AQC and MR1@7AQC predicted very high binding affinities of À 9.0 kcal/mol, À 8.3 kcal/mol, respectively.This was evident with the significant hydrogen bond interactions embedded as the following amino ARG H:10, SER H:76, ARG H:76, GLY A:59.With no doubt, this investigation suggests that both synthesized compounds possess efficient potentials towards the inhibition of B. subtilis.This was followed by the remarkable interactions seen to occur at MR1@41MM and HRC@4IMM with binding affinities of À 7.4 kcal/mol and À 6.1 kcal/mol, respectively, built on the following amino acids: LYS A:291, LYS A:201, ASN A:370, LEU A:154, ASP A:74, and MET A:199.This investigation implies that Moraxella catarrhalis is greatly susceptible to both synthesized compounds, thus optimizing the pharmacokinetics of the compounds by controlling its solubility and bioavailability.Additionally, HRC@4ZHS and MR1@4ZHS revealed very intriguing conventional hydrogen bonds (ARG A:53, PRO A:190, TYR A:189, ALA A:17, GLY A:186, LYS A:211, LYS A:111) with relatively similar binding affinities of À 5.2 kcal/mol and À 5.5 kcal/mol, respectively.Contrarily, Enterobacter and A. niger target receptors revealed very minimally low binding affinities ranging from À 2.1 kcal/mol to À 4.3 kcal/mol.However, key amino acid residues such as GLU A:597, VAL A:611 ARG A:596 TYR A:598, GLY A:539, VAL A:565, ILE A:549, and VAL A:565 were recovered from the interactions.
Summarily, the antimicrobial efficiency of the studied compounds accounts that the isolated, characterized and identified microorganisms can greatly be inhibiting them.Thus suggesting that the drug designers can further modify the synthesized compounds to enhance its affinity and selectivity for the target receptor (Table 6 and Figure 6).

Molecular dynamic simulation
MD simulations are an important aspect of the investigation because they validate the stability of the studied compounds in the target receptor's binding region.To get a more realistic model of the interaction patterns between promising compounds HRC and MR1 and the protein targets, the docked complexes were solvated in an explicit solvent (SPC water model) in a PBC box for a 100 ns simulation.The Auto-Dock protein-ligand docking computation was used to rigidly examine the crystal structure of the protein targets.
Following HRC and MR1 binding, the time-evolutionary structural changes in protein targets were examined using RMSD analysis.Figure 7 shows the temporal evolution change in the Ca-RMSD of all atoms to illustrate the conformational stability of the compound HRC and MR1-protein complex.The RMSD measures both structural variation and protein stability.The degree of fluctuation in RMSD is inversely related to a complex's stability: the less variation, the better the stability (Halder et al., 2022;Haque et al., 2022;Mathew et al., 2022).All trajectories were significantly equilibrated throughout the simulation phase except 4imm Complex, as can be displayed.Figure 7 shows the RMSD of synthesized compounds HRC and MR1, and it was observed that the complex was stable for the simulation period.A little drift was noticed, but it stayed constant throughout the simulation duration.There were no significant differences in the simulation trajectory against the protein in the presence of studied compound HRC and MR1.Values of each trajectory analyzing parameter in terms of maximum, minimum, and average values for the RMSD of 1KUM, 7AQC, 3SUQ, 4IMM, 4ZHS backbone bound with HRC and MR1 profiles are given in Table 7. Stable geometry HRC of the assessed protein is also visible to correlate the RMSD analysis.Moderate deviation of the backbone was found when bound with HRC and MR1 ligands in complex with 4imm protein.The average of 4imm protein bound with HRC and MR1 has been found to be 4.06 and 4.04 Å, respectively.These moderate values imply that the 4imm protein is less stable than other complexes.while no noticeable deviation of the 1KUM, 7AQC, 3SUQ, 4ZHS proteins backbone was found when bound with proposed HRC and MR1.It is also worth noting that the maximum RMSD across all frames was 4.38, indicating that not a single frame had substantial deviation throughout the MD simulation.The low average and consistent fluctuation of RMSD sufficiently supported the stability of each complex in dynamic situations.The root-mean-square fluctuation (RMSF) metric is extensively used in MD simulations to quantitatively analyze protein folding, binding, and stability in biomolecular systems.It provides valuable insights into the stability and conformational changes of proteins or macromolecules over time, while also highlighting local structural differences.RMSF quantifies the average deviation of atom positions from their mean positions throughout an MD simulation, enabling the identification of regions with high flexibility or mobility, thereby enhancing our understanding of dynamic behavior in the system.Regions with high RMSF values are associated with flexible loops, dynamic domains, or solvent-exposed regions, indicating significant atom or residue movements.In contrast, low RMSF values indicate reduced fluctuations and higher stability, commonly found in structural cores, secondary structures, or densely packed regions.Such low RMSF values suggest stiffer and more stable protein regions, crucial for maintaining overall structural integrity (Girase et al., 2023;Jagatap et al., 2023;Sayed et al., 2023).
Among the studied complexes, both HRC-1kum and MR1-1kum complexes exhibit similar RMSF patterns, suggesting moderate fluctuations with moderate flexibility in their structures, as evidenced by minimum values of 0.47 Å and 0.52 Å, maximum values of 2.78 Å and 2.94 Å, and average values of 1.29 Å and 1.26 Å, respectively.The HRC-7aqc Complex demonstrates the lowest minimum RMSF value of 0.25 Å, indicating a relatively rigid structure, while the MR1-aqc complex exhibits an even lower minimum RMSF value of 0.19 Å, indicating higher stability and reduced flexibility.Both complexes show comparable average RMSF values to the previous complexes, suggesting moderate overall fluctuations.The HRC-3suq Complex displays relatively higher average RMSF values (0.90 Å), indicating increased flexibility compared to previous complexes, while the MR1-3suq Complex exhibits a higher maximum RMSF value (5.60 Å), indicating the presence of highly flexible regions and increased overall flexibility.Notably, the N-terminal residues ranging from Asp159 to Val163 show higher fluctuations in these complexes.The HRC-4imm Complex displays the lowest average RMSF value (0.83 Å) among all the studied complexes, while the MR1-4imm Complex exhibits slightly higher fluctuations but still maintains relatively low average RMSF values.In both 4imm complexes, the loop region residues ranging from Thr232 to Asp249 exhibit higher RMSF values compared to other parts of the protein.The 4zhs complexes demonstrate comparable average RMSF values of around 0.96 Å and 0.97 Å, respectively, suggesting moderate fluctuations and flexibility in their structures.Notably, higher fluctuations are observed in the C-terminal and residues ranging from Tyr26 to Glu29, which are not involved in ligand contacts (Figure 8).Due to the low RMSF values observed in the interacting residues indicates stability for the proposed HRC and MR1 within the binding cavities of 1KUM, 7AQC, 3SUQ, and 4ZHS proteins.
Hydrogen bond analysis is valuable in quantifying the strength and specificity of interactions between ligands and proteins.By identifying and analyzing hydrogen bonds formed between the ligand and the protein, researchers can evaluate the binding affinity and predict the stability of the ligand-protein complex.This information is particularly crucial in drug discovery and design, as it assists in identifying potential binding sites and optimizing ligand-protein interactions.In the case of the HRC and MR1 proteins in the binding cavities of 1KUM, 7AQC, 3SUQ, and 4ZHS proteins, hydrogen bond analysis provides insights into their respective affinities.Among the complexes analyzed, the HRC-3suq complex exhibits the highest average number of hydrogen bonds (1.4) in its binding cavity.This suggests a relatively stronger affinity between the HRC and 3SUQ complex compared to the other complexes.The HRC-1kum, HRC-7aqc, HRC-4imm, and HRC-4zhs complexes display similar average hydrogen bond values ranging from 0.7 to 0.8.These values indicate a moderate affinity between the respective proteins and compound HRC in their binding cavities.On the other hand, the complexes MR1-3suq, MR1-4imm, and MR1-4zhs exhibit lower average hydrogen bond values ranging from 0.1 to 0.6.These results imply that the compound MR1 and targeted proteins have a lower affinity in their respective binding cavities.The hydrogen bond analysis revealed that the HRC-3SUQ Complex exhibited the maximum number of hydrogen bonds.This finding suggests a strong potential for hydrogen bonding interactions between the ligand and the protein in the binding cavity of the HRC-3suq Complex.The presence of a higher number of hydrogen bonds indicates a more favorable binding environment and can contribute to the stability of the ligand-protein complex.This observation further validates the binding affinity and stability of the ligand in the HRC-3suq Complex during the MD simulation.
The MMGBSA (Molecular Mechanics/Generalized Born Surface Area) analysis provides information about the binding free energy (DG Binding) and various energy components such as Coulomb, H-bond, Lipo, Solv_GB, and vdW for the complexes studied.A more negative DG Binding value indicates a higher affinity between the ligand and the protein (Khan et al., 2023;Saranya et al., 2019).Among the HRC-1kum and MR1-1kum complexes, both exhibit similar DG Binding values of approximately À 51.7 kcal/mol and À 51.3 kcal/mol, respectively.These values suggest a strong binding affinity between the ligand and the respective proteins.The energy components, including Coulomb, H-bond, Lipo, Solv_GB, and vdW, contribute to the overall binding affinity.For the HRC-7aqc and MR1-aqc complexes, the DG Binding values are approximately À 49.5 kcal/mol and À 48.9 kcal/mol, respectively.These values indicate a relatively high binding affinity, although slightly lower compared to the HRC-1kum and MR1-1kum complexes.The HRC-3suq complex shows a DG Binding value of approximately À 48.0 kcal/mol, while the MR1-3suq complex has a lower DG Binding value of approximately À 37.6 kcal/mol.These results suggest that the binding affinity is stronger in the HRC-3suq complex compared to the MR1-3suq complex.The HRC-4imm and MR1-4imm complexes exhibit DG Binding values of approximately À 48.1 kcal/mol and À 43.5 kcal/mol, respectively (Table 8).These values indicate a favorable binding affinity, with the HRC-4imm complex showing a slightly higher affinity compared to the MR1-4imm complex.Among the HRC-4zhs and MR1-4zhs complexes, the DG Binding values are approximately À 47.6 kcal/mol and À 43.0 kcal/mol, respectively.These results suggest a significant binding affinity in both complexes, with a slightly higher affinity observed in the HRC-4zhs complex.In summary, based on the DG Binding values, the complexes with higher negative values indicate a stronger binding affinity.From the obtained data, the HRC-1kum complex shows a relatively higher binding affinity compared to the other complexes studied.The MMGBSA analysis of the complexes revealed that the binding energy, as indicated by the DG Binding values, was influenced by different energy components.Specifically, the solvation energy had a negative impact on the overall binding affinity.On the other hand, the Coulomb (electrostatic) and van der Waals energies had favorable impacts on the binding affinity.

Figure 3 .
Figure 3. Graphical representation of the density of state (DOS).

Figure 4 .
Figure 4. (a, b) Analysis of the zone of inhibition of MR1.

Figure 5 .
Figure 5. (a, b) Analysis of the zone of inhibition of HRC.

Figure 6 .
Figure 6.(a-c) 2D and 3D representation of the molecular docking results.

Figure 7 .
Figure 7. RMSD, RMSF and Hydrogen bond analysis plot of promising compound HRC and MR1 with different protein targets 1KUM, 7AQC, and 3SUQ vs. time of the simulation.

Figure 8 .
Figure 8. RMSD, RMSF and Hydrogen bond analysis plot of promising compound HRC and MR1 with different protein targets 4IMM and 4ZHS vs. time of the simulation.

Table 1 .
Analysis of the experimental and experimental vibrational frequencies.

Table 5 .
Antimicrobial evaluation of HRC.

Table 3 .
Natural bond orbital analysis.

Table 6 .
Analysis of the molecular docking results.

Table 7 .
Minimum, maximum, and average RMSD analysis of studied complexes.

Table 8 .
Prime MM-GBSA energies in kcal/mol for HRC and MR1 binding at the active site of the 1KUM, 7AQC, 3SUQ, and 4ZHS proteins.