Design, synthesis, molecular docking, molecular dynamic simulation, and MMGBSA analysis of 7-O-substituted 5-hydroxy flavone derivatives

Abstract A series of chrysin derivatives were designed, synthesized, and evaluated for their antibacterial activity against four different bacterial strains. We have synthesized new propyl-substituted and butyl-substituted chrysin-piperazine derivatives, which show marvellous inhibition against E. coli and S. aureus. The free hydroxyl group at the C-5 position of chrysin improved therapeutic efficacy in vivo and was a beneficial formulation for chemotherapy. All synthesized compounds were confirmed by various spectroscopic techniques such as IR, NMR, HPLC, and mass spectrometry. The compounds exhibited moderate to good inhibition, and their structure–activity relationship (SAR) has also been illustrated. Among the synthesised compounds, compounds 4 and 10 were the most active against S. pyogenes and E. coli, with 12.5 g/mL MICs; additionally, compound 12 exhibits significant activity on both the S. aureus and E. coli stains. Based on the promising activity profile and docking score of compound 12, it was selected for 100 ns MD simulation and post-dynamic binding free energy analysis within the active sites of S. aureus TyrRS (PDB ID: 1JIJ) and E. coli DNA GyrB (PDB ID: 6YD9) to investigate the stability of molecular contacts and to establish how the newly synthesized inhibitors fit together in the most stable conformations. Communicated by Ramaswamy H. Sarma

Many N-heterocycles have played a significant role in medicinal chemistry in the past decades (Girase et al., 2021;Patel et al., 2020).Nowadays, piperazine-based compounds have also attracted increasing attention due to their versatile and eminent biological and pharmacological profiles, such as antibacterial (Foley et al., 2014), antipsychotic (Cao et al., 2018;Moussa et al., 2010), antifungal (Ji et al., 2019), anti-Alzheimer (He et al., 2017), antitumor (Chen et al., 2010), anti-inflammatory (Migliore et al., 2016) and antidiabetic (Rathi et al., 2016;Tamayo et al., 2015).Two opposing nitrogen-containing piperazines improve drug pharmacokinetic features due to their appropriate pKa values (Lacivita et al., 2009).This nitrogen site is necessary to enhance water solubility and is key in bioavailability (Maia et al., 2012).Further, the piperazine analog shows distinct conformations such as a chair, boat, twist-boat, and half-boat (Lee et al., 2009).Several piperazine derivatives of flavones have diverse biological activities such as antitumor, antioxidant, antimicrobial, and anti-inflammatory reported in publications (Hatnapure et al., 2012;Zhang et al., 2021).Even though many chemotherapeutic drugs are available in the market, due to drug resistance development, the emergence of new diseases, and the improvement in therapeutic properties of old drugs, there is always a need for new, novel drugs of a broad spectrum (Patel et al., 2023).Chrysin and piperazine derivatives are useful pharmacophores with potential therapeutics (Patel & Kumari, 2022a).With this perspective, various chrysinbased piperazine derivatives were designed, synthesized, and in vitro evaluated against various microbes.In continuation of our previous work (Patel et al., 2016) here we synthesized two new butyl-substituted Chrysin-piperazine compounds and evaluated their antibacterial activities.We have witnessed significant findings that we can employ in developing new therapeutics.Recent studies have shown theoretical and experimental studies of chrysin-piperazine moieties.The theoretical data has contributed significantly for synthesizing more active and effective compounds (Bilgic¸li et al., 2020;Genc ¸ Bilgic¸li et al., 2020;Zala et al., 2022aZala et al., , 2022b)).Molecular docking is widely used, which gives an idea about the biochemical interactions of compounds before the experimental processes.7-O substituted chrysin derivatives were reported to be more effective than chrysin itself (Bhowmik et al., 2022).Thus, this study is based on the 7-O substitution of chrysin via piperazine to further develop the potential antibacterial drug.The structure-activity relationship indicates that the substitution pattern should be chosen to ensure a varied lipophilic and electronic environment that could influence the activity of targeted molecules.In vitro testing revealed significant antibacterial activity against S. pyogenes and E. coli for compound 4 with p-methoxy substitution and compound 10 with N-benzyl substitution.When the number of alkyl chain carbons in N-benzyl derivatives is increased, activity increases against three bacterial pathogens.Activity against E. coli declines with the addition of more halogens to the phenyl ring.Although in accordance with the in-silico analysis, compound 12 demonstrated the best interaction with the active sites of S. aureus TyrRS and E. coli DNA GyrB (PDB ID: 1JIJ and 6YD9, respectively), Table 1.
The 1 H NMR spectra of the synthesized compounds (3-12) were obtained in a Dimethyl sulfoxide (DMSO)-d 6 solution.The free OH gave a singlet peak at d 12.78-12.81ppm.All aromatic hydrogen gave a peak in the range of d 6.36-8.33ppm.The characteristics of the H-CO protons of propoxy or butoxy chain observed d 4.09-4.24ppm.Hydrogen of the piperazine ring was manifested in the range of d 2.24-3.55ppm. 13C NMR spectra of carbonyl carbon of the chrysin ring resonated in the range of d 182.32-182.57ppm and practically it was observed at �183 ppm.In addition, aromatic carbon resonated between d 166.44-93.61ppm, and aliphatic carbon resonated between d 75.70-22.55ppm.Overall, the experimental and computed results had a lot of similarities, proving the robustness of the theoretical methods for simulating the experimental structure (Figures 2 and 3).

Anti-bacterial activity
All newly synthesized compounds were evaluated by determining their minimum inhibitory concentrations (MICs) using the Broth Dilution Method, and their results are shown in Table 2. Ampicillin, Chloramphenicol, Ciprofloxacin, Norfloxacin, and Gentamicin are reference antibiotics.The in vitro antibacterial activity of novel piperazine-chrysin conjugates was tested against gram-positive bacteria, including Streptococcus pyogenes (MTCC 442), Staphylococcus aureus (MTCC 96), and Gram-negative bacteria, including Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 1688).Chrysin-piperazine hybrids showed moderate inhibition against E. coli.Compound 7 (50 mg/ml) was equipotent to standard chloramphenicol, 5 and 9 were found to be least active against E. coli, 250 mg/ml.Compounds 3, and 10 were as active as chloramphenicol with 50 mg/ml, and compound 4 was as active as ciprofloxacin with 25 mg/ml MIC, showing better inhibition than chloramphenicol against S. pyogenes.Compounds 4, 7, and 10 had equal potency as chloramphenicol and ciprofloxacin against S. aureus with MIC 50 mg/ml.It is also interesting to note that compound 4 showed excellent activity against S. pyogenes bacteria with a MIC value of 12.5 mg/ml, better than Ampicillin, Chloramphenicol, and Ciprofloxacin.Compound 12 demonstrated significant activity against both E. coli and S. aureus strains, with a MIC of 125 mg/ml and an excellent binding interaction within the active pocket of the residue.Compounds 4 and 10 have been identified as having the highest in vitro activity against S. pyogenes and E. coli, with 12.5 g/mL MIC, even though their docked scores against E. coli of À 5.351 and À 5.831 kcal/mol, respectively, are not at par with compound 12, revealing that compound 4 and 10 might be interacting with other active pockets of the residue.

Molecular docking
Polyphenolic chemicals known as flavonoids have been shown to possess antibacterial properties through various modes of action.Several studies have shown that flavonoids can inhibit energy metabolism, cytoplasmic membrane function, and nucleic acid production (Friesner et al., 2004;Xie et al., 2015).Also, it has been discovered that flavonoids inhibit the development of adhesion and biofilms, porin on cell membranes, membrane permeability, and pathogenicity, all of which are essential for bacterial growth (Cushnie & Lamb, 2005;Donadio et al., 2021;Friesner et al., 2004;Rauha , 2000;Wu et al., 2013;Xie et al., 2015;Zheng et al., 1996).Molecular docking of synthesized 7-O-substituted 5hydroxy flavone derivatives was performed to identify the binding sites on the structure of S. aureus tyrosyl-tRNA synthetase and E. coli DNA GyrB proteins.Tyrosyl-tRNA synthetase is a desirable target enzyme for discovering new antibacterial compounds (Khan et al., 2018).It is a member of the family of enzymes known as aminoacyl-tRNA synthetases (aaRSs) and is in charge of catalysing the covalent attachment of amino acids to their specific tRNAs to create charged tRNA.As a result of their crucial function in the protein production pathway, aaRS inhibition influences cell development.Another target enzyme is topoisomerase II DNA gyrase, flavone and coumarins derivatives have emerged as one of this enzyme's most researched families of inhibitors (Gibson et al., 2019).DNA topoisomerases are crucial for cell viability because they catalyse changes in the architecture of DNA.A type II topoisomerase called DNA gyrase may add negative supercoils to DNA by consuming ATP.It is a target for antibacterial medicines since it is necessary in all bacteria but lacking in higher eukaryotes (Matos et al., 2017).When compared to the reference Co-Crystal Ligand, all synthesized compounds had significant docking scores in the range of À 6.806 to À 4.685 and À 7.311 to À 5.092 Kcal/mol with the S. aureus TyrRS and E. coli DNA GyrB, respectively, Table 1.Compound 12, among the synthesized compounds, show a significant docking score in both targets with À 6.806 and À 7.129 Kcal/mol, respectively, as well as a significant MIC value of 125 lM against the S. aureus and E. coli strains.The 2D and 3D graphical representations of the ligand-protein interactions presented in Figure 4 were prepared using Maestro's ligand-interaction tool.The binding interaction of the representative compound, 12, is completely consistent with the Co-crystal ligand SB-239629.
Compound 12 forms one hydrogen bond through the hydroxyl group of the 5-hydroxy flavone scaffold and one p-cation through the protonated nitrogen of piperazine with the key residue Asp149 (Figure 4A).In this target, SB-239629 interacts with different crucial residues, including Tyr36, Gly38, Asp40, Hid47, Hid50, Lys84, Asp177, and Asp195.The association of compound 12 with the critical residue Asp195 indicating its antibacterial potential via TyrRS protein inhibition.

Molecular dynamic (MD) simulation analysis
MD is a fundamental and well-established structure-based approach for investigating the dynamic character of proteinligand complexes at the atomic level.As a result, potential compound 12 MD simulations in complex with S. aureus TyrRS (1JIJ) and E. coli DNA GyrB (6YD9) were run for 100 ns to investigate stability.Each small molecule or ligand may cause significant changes in the protein's conformation when it binds to the active site.After the binding of representative compound 12, we assess the root-mean-square deviation (RMSD) to explore these conformational changes in the structures 1JIJ and 6YD9.We calculated the RMSD value for the protein Ca atoms to verify the stability of complexes.12-1JIJ Complex showed a stable RMSD throughout the simulation with an average RMSD value of 2.25 Å.The RMSD of the 12-6YD9 Complex reached 1.40 to 2.67 Å within 50 ns.However, it was consistent and then stable until the very end (average RMSD ¼ 2.37 Å).The RMSD figure demonstrates that the 12-1JIJ Complex fluctuates less than the 12-6YD9 Complex.The time evolution of the system RMSD values is shown in Figure 5.The plot's minimal variability suggests system stability.
Additionally, principal component analysis (PCA) was performed to provide insight into the binding cluster variance assessments of the complex system.PCA calculates the correlation between statistically significant conformations (large global motions) measured along the trajectory.We used PCA on the MD simulation-generated trajectories of the 12-1JIJ and 12-6YD9 complexes to investigate the protein's conformational distribution over time and large-scale collective motions in the protein-ligand complex (Halder et al., 2022).PCA simulations of the complex system's atomic backbone were done in normal mode molecular dynamics, utilizing two conformations, PC1 and PC2. Figure 5D shows the PCA of the three complexes.The projection of the protein motion along PC1 and PC2 in phase space revealed a consistent distribution of conformations across the simulations.Proteins bound to compound 12 global motion are periodic in 1JIJ and 6YD9 because the groups along the PC1 and PC1 planes do not entirely cluster independently.The observation that the trajectories converge inside a single cluster shows that MD trajectories shift on a periodic basis as a consequence of steady conformational global motion.The sampling of both systems reveals the simulation's complexes' stability (Figure 5D).
A MM-GBSA analysis was conducted on both protein-ligand complexes to determine compound 12's affinity for the 1JIJ and 6YD9 receptors.The MM-GBSA-based binding free energy calculations were done on the 100 ns long MDS trajectories.For the selection of protein-ligand complexes, the binding energies calculated by this approach are more efficient than the glide score values.The main energy elements like H-bond interaction energy (DG Bind_Hbond), electrostatic solvation free energy (DG Bind_Solv), Coulomb or electrostatics interaction energy (DG Bind_Coul), lipophilic interaction energy (DG Bind_Lipo), and van der Waals interaction energy (DG Bind_vdW) altogether contribute to the calculation of MM-GBSA based relative binding affinity.In the Table S1, the binding energies and contributing factors estimated for the MDS trajectories are mentioned.The compound 12 showed high binding free energies with DG Bind of À 85.17 Kcal/mol with significant contributions from the DG Bind_Coul (À 38.79 kcal/mol), DG Bind_Lip (À 24.25 kcal/ mol), and DG Bind_vdw (À 76.56 kcal/mol) in 1JIJ protein.While in the case of 6YD9 protein has a least average binding energy of À 67.93 kcal/mol.According to the MMGBSA calculation, the system was most favourable in terms of the sum of various binding energy computations, such as Vander Waal energy, lipophilic energy, and columbic (electrostatic) energy, while the binding free energy (DG Bind) was reduced by the polar solvation energy (DG Bind sol_GB).

Drug likeness and toxicity prediction study
Poor pharmacokinetics is the main cause of pharmaceutical candidates failing in clinical trials.Early inclusion of pertinent  ADME traits will result in the best candidates to prevent attrition and successfully complete clinical trials.With this goal in mind, the pharmacokinetic profiles of the synthesized compounds were examined using Schrodinger's Qikprop module to further predict how drug-like they would be.All synthesized compounds have only one violation of Lipinski's Rule of five, except compounds 5, 8, and 10, which follow all Jorgensen's rule of three.Compounds may be prospective drug development leads, according to a general drug-likeness measure and in silico anticipated ADME characteristics, Table 3.Only few compounds such as 6 and 10, and 11 were inactive against AMES toxicity, mutagenicity, cytotoxicity, carcinogenicity, hepatotoxicity, and immunotoxicity, according to the ProtoxII web server's assessment of compound toxicity.

Conclusion
The effective synthesis of novel chrysin derivatives from phloroglucinol was complemented by an assessment of their in vitro and in silico antibacterial efficacy against two Grampositive and two Gram-negative bacterial strains.In in vitro study, compound 4 revealed substantial action against S. pyogenes with a 12.5 g/ml MIC, while compound 10 showed exceptional activity against E. coli with a 12.5 g/ml MIC, which outperformed the standards.At À 7.129 and À 6.806 kcal/mol, respectively, compound 12 had the highest dock score interaction with S. aureus and E. coli, demonstrating the presence of structural factors for efficient binding with the active sites of S. aureus TyrRS and E. coli DNA GyrB.It's in vitro activity was also established with a MIC of 125 g/ml.Compound 12 exhibits the identical docking interaction as the co-crystallized ligand SB-239629 with various critical residues, including Tyr36, Gly38, Asp40, Hid47, Hid50, Lys84, Asp177, and Asp195.The association of compound 12 with the critical residue Asp195 indicates its antibacterial potential via TyrRS protein inhibition.
A typical atomistic 100-ns dynamic simulation analysis revealed the significant binding stability of compound 12 in the binding sites of S. aureus TyrRS and E. coli DNA GyrB through the analysis of RMSD and RMSF parameters.

General remarks
All chemicals and solvents bought from Sigma, Finar, and TCI were utilized directly without purification.Reactions were monitored by thin layer chromatography (TLC) on a precoated silica gel plate using an appropriate solvent system and spot visualization using the iodine chamber and UV chamber.NMR spectra were taken using a Bruker AVANCE 400 instrument ( and ATR-FTIR.The purity of the compounds was also checked by high-performance liquid chromatography (HPLC).

Procedure for the synthesis of 5,7-dihydroxy-2-phenyl-4H-chromen-4-one (1)
A solution of phloroglucinol, ethyl benzoylacetate, and 4dimethylaminopyridine (DMAP) was stirred and then refluxed for 3 h at 200 � C in a nitrogen atmosphere.The reaction was monitored by TLC.After completion of the reaction, the reaction mixture was cooled to room temperature.The resulting precipitate of compound (1) was washed with ethyl acetate and recrystallized in THF (Torgal Martins & Marta Ramos Pinto Correia da Silva Carvalho Guerra, 2017).

General procedure for the synthesis of compound (2)
Compound (1) in acetone (50 ml), dibromopropane/butane (1.1 mmol), and potassium carbonate (1.1 mmol) were taken in an RBF, and the reaction mixture was refluxed for 8-10 h.The reaction was monitored by TLC.The reaction mixture was cooled to room temperature, and the ice was poured into it.The crude solid is obtained after evaporation of the organic layer under reduced pressure.The crude product was washed with water, filtered, dried, and recrystallized by hexane/EtOH to yield compound (2) (Liu et al., 2014).

Anti-bacterial activity
A stock solution of 2000 lg/ml was prepared of synthesized compounds and was further diluted to get various concentrations from the stock solution.Primary screening: In primary screening, 1000 lg/ml, 500 lg/ml, and 250 lg/ml concentrations of the synthesized compounds were used.The compounds that were found active in this primary screening were further tested in a second set of dilutions against microorganisms.Secondary screening: synthesized compounds found active in primary screening were similarly diluted to prepare 200 lg/ml 100 lg/ml, 50 lg/ml, 25 lg/ml, 12.5 lg/ml, and 6.25 lg/ml concentrations.Reading Result: The highest dilution showing at least a 99% inhibition zone is considered the MIC.

Molecular docking
Molecular docking studies were performed on synthesized 7-O-substituted 5-hydroxy flavone derivatives (3-12) against the crystal structure of S. aureus TyrRS (PDB ID: 1JIJ) and E. coli DNA Gyrase B (GyrB) (PDB ID: 6YD9) as a target enzyme.LigPrep was used to model the synthesized compounds by creating potential enantiomers, ionization, and tautomeric states at pH ¼ 7.0 ± 2, and then ligands were energy minimized using default OPLS3e force field parameters (Kumar et al., 2023;Zala et al., 2023).The Protein Preparation Wizard was used to prepare the protein; during this procedure, crystallographic water molecules were eliminated, bond ordering and partial charges were assigned, ionisation and tautomeric states of the residues were established, and H bonds were assigned (Desai et al., 2023).The Receptor Grid Generation tool was started by clicking on the co-crystalized ligand and the default grid box was created.The docking was done in the active site of a receptor protein, utilizing the SP (Standard precision) Glide simulation-based docking methodology (Sudevan et al., 2022).

MD simulation
MD simulations were performed using simulations on the OPLS3e force field with the Desmond package.The simulations protocol and other experimental details were used exactly as reported elsewhere (Agwupuye et al., 2022;Aljuhani et al., 2022).The simulation was carried out for a period of 100 ns, and trajectory snapshots were recorded at an interval of 100 ps.MD trajectories were analysed using Desmond's Simulation Interaction Diagram (SID) to predict the binding orientation and stability of the ligand.

Table 3 .
Drug likeness and ADMET study of synthesised compounds (