Quantum mechanical, virtual screening, molecular docking, molecular dynamics, ADME and antimicrobial activity studies of some new indole-hydrazone derivatives as potent agents against E. faecalis

Abstract In this study, a new series of indole-5-carbaldehyde hydrazone derivative compounds were designed, synthesized, and their antimicrobial activities were determined by the microdilution method, and the in vitro cytotoxic effects on Beas-2b cell lines were investigated by MTT assay. When the activity results were examined, 5i12 showed promising activity against E. faecalis with MIC: 2 µg/mL compared to ampicillin, gentamicin, and vancomycin, although the antimicrobial activities of the indole derivatives were generally weaker than those of the standard drugs. Compounds showed no cytotoxic activity on the A549, MCF-7, and Beas-2b cell lines. Molecular docking studies were performed on 15 different proteins to understand the mechanism of 5i12's good antimicrobial action against E. faecalis, and it was concluded that the compounds interacted with FabH, not enough other protein structures. Molecular dynamics simulations were performed to investigate the protein–ligand stability of the most active compound against E. faecalis. The RMSD value of 5i12 varied between 0.02 and 0.16 nm during the MD simulation. The apoprotein peaked at 0.55 nm at the beginning of the simulation and stabilized below 0.5 nm. The theoretical ADME profiles of all compounds were calculated and found to comply with Lipinski and other limiting rules. In addition, some theoretical quantum parameters (HOMO-LUMO) of compounds, and both MEP analysis and geometric optimization analysis for 5i12 were calculated using the 6-311 G (d,p) base set and DFT/B3LYP theory, and the results were visualized. Communicated by Ramaswamy H. Sarma Graphical Abstract


Introduction
Infectious diseases are severe threats to public health today, and among the drugs used for therapeutic purposes, those with antimicrobial effects rank first. The most important problem faced in the treatment of antimicrobial drugs is the fact that microorganisms gain resistance in a short time, and the biochemistry of these microorganisms is not yet sufficient and some side effects of antimicrobial drugs (Ahmad et al., 2013(Ahmad et al., , 2017Peters et al., 2019;Spellberg et al., 2013). Antibiotic resistance is a significant health problem that concerns the whole world and not only today but also the future. As a result of the increase in the frequency of international travel with the help of today's technological and economic conditions, the problem of antibiotic resistance occurring in any region of the world reaches a global scale in a concise time. The molecular mechanism of bacteriainduced resistance to antibiotics is diverse and complex, and bacteria can develop resistance to various antibiotics (Blair et al., 2015;Siddiqui et al., 2012;Ventola, 2015).
Although resistance mechanisms developing against different antibiotic groups are seen as an inevitable result of antibiotic use, researchers have stated that many different bacteria develop resistance against antibiotics with different mechanisms and this situation should be taken under control. According to statistical data, it is estimated that antimicrobial resistance is currently responsible for more than 700,000 deaths annually worldwide, and estimated economic cost of over $100 million per year and over 10 million deaths by 2050 (Jansen et al., 2018). This has led researchers to design and synthesize new antimicrobial drugs with more effective and fewer side effects.
The indole ring is one of the important structure investigated in drug discovery because it exhibits a wide range of biological activities including antifungal (Pagniez et al., 2020), antibacterial (Qin et al., 2020), antitubercular (Cihan-€ Ust€ unda g et al., 2019), anticancer (El-Sharief et al., 2019), antiviral (Wang et al., 2019), antioxidant (Demurtas et al., 2019), antidepressant (Wr obel et al., 2019), anti-inflammatory (Huang et al., 2019), and antidiabetic activities (Sravanthi et al., 2017). There are many indole-containing drugs on the market as well as derivatives in clinical evaluations (Figure 1). In addition, hydrazone derivatives show important and diverse pharmacological activities in compounds bearing (-NHN ¼ CH-). In new drug development studies, new compounds with more potent effects can be found with a combination of different pharmacophore groups in the same molecule.
Based on previous studies (Gurkok et al., 2009;Sayed et al., 2018;Shirinzadeh et al., 2011), new compounds with stronger antimicrobial activity can be found in combination with indole and hydrazone groups. In this study, a series of compounds with the general structure of indole-5-carbaldehyde hydrazone were designed, synthesized, and their structures were illuminated by HRMS, 1 H NMR, and 13 C NMR spectroscopy ( Figure 2). The antimicrobial activities of all compounds against a variety of Gram-positive, Gram-negative bacteria and fungi as well as their isolates, and in vitro cytotoxic activities on the A549, MCF-7 and Beas-2b cell line were determined. Estimated ADME profiles were calculated, and molecular docking studies were performed on 15 different proteins to understand the mechanism of action of the compounds against E. faecalis. Molecular dynamics simulations were performed to investigate the protein-ligand stability of the most active compound against E. faecalis. Some theoretical quantum parameters (HOMO-LUMO) of all compounds as well as both MEP analysis and geometric optimization analysis for 5i12, were calculated using the 6-311 G (d,p) base set and DFT/B3LYP theory, and the results were visualized.

Chemistry
Chemicals and solvents were purchased from Sigma-Aldrich, Acros Organics, Merck, Riedel de Haen, and Fluka and used without further purification. During the synthesis, Thin Layer Chromatography (TLC) was used to monitor the progression of the reactions and to determine the purity of the products obtained. For this purpose, Kieselgel-60 GF254 coated aluminum plates (Merck) were used. UV light (Camag UV Lamp) with a wavelength of 254 nm was used to determine the spots. Melting point determinations were made by the capillary method using an Electrothermal 9100 device and given uncorrected. The [M þ 1] peaks of the synthesized compounds were determined using an Agilent 6224 Line-Mass TOF LC/MS system (Agilent Technologies, CA, USA). 1 H-NMR and 13 C-NMR spectra were obtained using a Varian Mercury-400 FT-NMR spectrometer, and tetramethylsilane (TMS) was used as the internal standard and DMSO-d 6 as the solvent.

General procedure for synthesis of 5i1-5i19
Indole-5-carbaldehyde (0.1 mmol) was dissolved in EtOH and reacted with phenylhydrazine hydrochloride or its derivatives in the presence of an aqueous sodium acetate solution for approximately 3 h. The precipitate formed at the end of the reaction was filtered, and the crystals obtained were purified by recrystallization from ethanol.

Antimicrobial study
In this assay, the sensitivity of some microorganisms to 5i series compounds synthesized in the first stage of our experiment was examined according to the Clinical Laboratory Standards Institute (CLSI) M100-S28 protocol for bacteria (Clinical Institute LS, 2017) and the CLSI M27-A3 protocol for fungi (Wayne, 2008). Results were given as the minimum inhibitory concentration (MIC) value. MIC is expressed as the lowest concentration of an antimicrobial agent, usually expressed as mg/mL, which completely inhibits the apparent growth of a test strain of an organism under in vitro conditions (Ahmad et al., 2017; Kowalska-Krochmal & Dudek-Wicher, 2021). The potential antibacterial activity of these compounds was tested against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and isolates of these bacteria in Mueller Hinton Broth (MHB) medium. And also, the potential antifungal activity of these compounds was tested against Candida albicans ATCC 10231 and its isolate in RPMI-1640 medium. First, stock solutions of the compounds were prepared in DMSO at a concentration of 12.8 mg/mL The serial dilutions of each compound (128,64,32,16,8,4, 2, 1 mg/mL) were prepared in 96-well microplates, after placing medium in each microplate well. The suspension of microorganisms was prepared using 0.5 McFarland standard and inoculated into each well at a final density of 10 5 CFU/mL. Bacteria were incubated for 24 h at 37 C and fungus for 48 h at 35 C. The activity of the reference antibiotics prepared in serial dilutions was tested on the same microorganisms. In addition, growth control of microorganisms and sterilization control of the medium were also tested in each microplate. The MIC values, were determined by visual evaluation, using the MTT dye (Shi et al., 2007). All assays were performed in triplicates.

Molecular docking
Molecular docking studies were performed using the Maestro module of Schr€ odinger, Inc (Maestro, 2018). , penicillin-binding protein 4 (PDB: 6MKI) and topoisomerase IV (PDB: 3FV5) crystal structures were imported from protein data bank (http://www.rcsb.org/pdb/) into the 'Protein Preparation Wizard' module. Hydrogens were added, non-bonding command with metals, the formation of disulfide bonds, deletion of water at 5 Å distance from het groups, and preprocess by creating pH: 7.00 ± 2.00 het states using Epik. Subsequently, an appropriate chain was selected. Water molecules and metals contained in protein crystal structures were retained, and molecules other than the protein structure were deleted. pH: 7.00 ± 2.00 regenerate state was created with S2. Finally, H-bond determination was optimized using PROKA pH: 7.00 with water sample orientation and protein was prepared by minimizing OPLS3 field forces. Second, the active site of the E. faecalis' target protein structure was determined by the coordinates of the ligands found in protein-ligand complexes. Grid determination was performed by clicking any atom of the B82, and the default box was 20 Â 20 Â 20 Å prepared by the 'Receptor Grid Generation' module. Finally, the indole structures were drawn using Chem3D and saved the in SDF file format, and data were entered into the 'Virtual Screening Workflow' module. Ligand structures were prepared by preferring the OPLS3 force field, and ionization was carried out using Epik in the range of possible pH: 7.00 ± 2.00, desalinated, and tautomer-formed ligand structures were prepared. Grid files of protein structures prepared in the first stage were added multiple. Finally, for the calculation of theoretical ligand-protein interactions, molecular docking was performed with extra precision (XP) and flexible ligand options. Docking score, Glide score, and Glide emodel were calculated and evaluated. The 2 D and 3 D interactions of the ligand and protein were determined and exhibited.

Molecular dynamics simulations
Molecular dynamics simulations were performed using the Gromacs 2019.2 version to investigate the stability of the protein-ligand complex formed with 5i12 and FabH via WebGRO for macromolecular simulations (https://simlab.uams.edu/) (Bekker et al., 1993). The topology of 5i12 ligand structure was created using the GlycoBioChem PRODRG2 server, and the topology file of the FabH structure was created using the SCP water model with a Gromos54a7 force field (Abraham et al., 2015;Oostenbrink et al., 2004). The MD simulations were performed under the periodic boundary condition. The system was balanced with 0.3 ns NVT (amount of substance, N, volume, V and equilibrium steps temperature, T) and 0.3 ns NPT (amount of substance, N, pressure, P and temperature, T) stages at 1 am pressure and 300 K temperature according to V-rescale thermostat (Bussi et al., 2007) and Parrinello-Rahman barostat (Parrinello & Rahman, 1981). The standard MD simulation of 100 ns duration was performed with leap-frog MD integrator. Trajectory analysis was performed with gmx scripts, the root-mean-square deviation (RMSD) and the root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA) measurements were performed. The binding free energy calculation by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) was performed between 20 and 100 ns according to RashmiKumari's g_mmpbsa package (Homeyer & Gohlke, 2012;Kumari et al., 2014). MD trajectory analysis results were monitored with VMD -Visual Molecular Dynamics, Discovery Studio Visualizer, and graphs were generated with GraphPad Prism.

Theoretical ADME predictions
ADME parameters (absorption, distribution, metabolism, and excretion) of the synthesized compounds were calculated using the QikProp module in Maestro 10.5 (Release, 2017). Rotatable bonds, molecular weight, stars, QPlogS, QPPCaco, estimated octanol/water distribution coefficient, the number of violations of Lipinski's five rules, and the number of violations of Jorgensen's three properties and descriptive rule were determined.

Chemistry
In this study, indole 5-carboxaldehyde hydrazone derivatives were obtained, as shown in Scheme 1 using the method given in the literature. All compounds are original. 5i1-5i19 were obtained by reacting indole-5-carboxaldehyde with substituted phenylhydrazines in the presence of EtOH and aqueous sodium acetate. 5i20 was obtained by reaction of indole-5-carboxaldehyde with hydrazine hydrate and EtOH. The chemical structures were illuminated by HRMS, 1 H-NMR, and 13 C-NMR spectroscopy, respectively, and the results confirmed the structures. Also, melting point determinations were made, and results were given without correction. Physical and spectral data of the synthesized compounds were presented in Section 4.1.1. (see also Supplementary Information).
According to 1 H-NMR spectroscopy data results, aliphatic protons were observed in the range of 2.21-3.68 ppm, aromatic protons in the range of 6.37-8.40 ppm, hydrazone's N-H proton in the range of 9.25-10.50 ppm, and indole's N-H protons as single peaks in the range of 11.19-11.26 ppm. The 13 C-NMR spectra of the compounds are also compatible with the structures. HRMS analysis results showed [M þ H] þ peak consistent with the formulas of the compounds, and even peaks of [M þ H þ 1] þ and [M þ H þ 3] þ were observed in compounds containing isotopes such as chlorine and bromine. The chemical structures of the studied compounds were provided in Table 1.

Antimicrobial evaluation
The observed MIC values of indole derivatives (5i1-5i20) and reference antimicrobials were presented in Table 2. The antimicrobial activities of compounds were compared to reference antimicrobials (ampicillin, gentamicin, vancomycin for antibacterial and fluconazole for antifungal activity, respectively).
According to the observations, reference antibacterial drugs had a much better effect (1-2 mg/mL) against S. aureus ATCC 29213 than the indole compounds (16-128 mg/mL). 5i10 and 5i12 had the best antibacterial activity against S. aureus ATCC 29213 (16 mg/mL) among all compounds. No indole compound was able to act on S. aureus isolate, while reference agents had good antibacterial activity (4-32 mg/mL) against S. aureus isolate. 5i-12 affected (2 mg/mL) on E. faecalis ATCC 29212, equivalent to that of ampicillin, gentamycin, and vancomycin. Except that, 5i10 and 5i11 affected E. faecalis ATCC 29212 at concentrations of 16 mg/mL and 128 mg/mL respectively, although not as good as that of reference antibiotics. Only two compounds, 5i3 and 5i6, exhibited activity on E. faecalis isolate at a concentration of 128 mg/mL, but reference antibiotics had better activities (8-32 mg/mL) on this isolate. The antibacterial activity (128 mg/mL) of 5i6 on E. coli ATCC 25922 and E. coli isolate was not as good as those of reference antibiotics (1-16 mg/ Scheme 1. General procedure for the preparation of 5i1-5i20 4-chloro 5i20 mL). And, the antibacterial activity (128 mg/mL) of 5i7 on E. coli ATCC 25922 was not good as those of reference antibiotics (1-16 mg/mL). When evaluating the possible antibacterial activities of the compounds on P. aeruginosa ATCC 27853 and its isolate, it was observed that none of the compounds was effective on either P. aeruginosa ATCC 27853 or P. aeruginosa isolate. Ten of the indole derivatives showed antifungal activity (16-128 mg/mL) on C. albicans ATCC 10231, albeit with high MIC values compared to fluconazole (1 mg/mL). 5i6 had the best MIC value (16 mg/mL) among these ten compounds. Antifungal activity levels of indole derivatives on C. albicans isolate were found at 32-128 mg/mL Although the antifungal activities of some indole derivatives in this study have been determined, MIC values appear to be higher than fluconazole as a reference drug. When the microbiology findings were evaluated in general, it was found that the tested derivatives showed more good antimicrobial activity against Gram-positives than Gram-negatives, and the most remarkable result was for the 5i12 which gave the MIC value at the level of reference antibiotics (2 mg/mL) against E. faecalis ATCC 29212.

Molecular docking study
Molecular docking analysis is widely used to estimate protein-ligand interaction energies (Alam et al., 2016;Celı K et al., 2021;Guedes et al., 2014;Lyne et al., 2006). In this context, to investigate the antimicrobial activity pathway of indole 5-carboxaldehyde hydrazone derivatives on E. faecalis, molecular docking analysis was performed on beta-ketoacylacyl carrier protein synthase III (FabH), thymidylate synthase, prolyl-tRNA synthetase, FMN-Dependent azoreductase, 3 0 ,5"aminoglycoside phosphotransferase type IIIa, NADH peroxidase mutant, VanG D-Ala:D-Ser ligase, class II lanthipeptide synthetase, NAD þ dependent DNA ligase, thiamin pyrophosphokinase family protein, phosphopantetheine adenylyltransferase, alanine racemase, DNA ligase, MDDEF, FIC, penicillinbinding protein 4 and topoisomerase IV proteins in the E. faecalis's X-ray crystal structure in PDB. When all molecular docking results were analyzed, it was concluded that the indole 5-carboxaldehyde hydrazone derivatives high interacted with FabH and did not enough interaction with other protein structures (Supplementary information). Also, Wang et al. reported that hydrazone derivatives showed antibacterial activity by inhibiting FabH (Wang et al., 2012). FabH is 128  vital for initiating fatty acid biosynthesis, inhibiting the process by long-chain fatty acids for feedback control, and adjusting the fatty acid profile of the organism due to its substrate specificity (Gajiwala et al., 2009).
PDB:3IL5 has a good resolution of 2.60 Å. Of these amino acids, 94.01% are in the favored region according to the Ramachandran plot. As shown in Table 3, the docking energy of most of the synthesized compounds were less than about À8.00 on FabH active site by XP docking analysis. These values may indicate that there is sufficient interaction between protein and ligand in terms of Glide docking studies. Besides, the XP docking scores formed by B82 with the re-docking process have a value close to the indole derivatives, which may indicate that it creates antimicrobial activity via FabH. As with other protein crystal structures, higher scores may suggest that the interaction is not sufficient to provide activity. According to the XP molecular docking analysis, the most active compound 5i12 has À65.86 MM-GBSA value. In comparison, the B82 compound has À73.57 MM-GBSA value in the E. faecalis FabH active site, and the proximity between the values may indicate that FabH and indole derivatives sufficiently interact. For molecular docking results of other proteins, see Supplementary Information.

Molecular dynamics simulations
Molecular Dynamics (MD) provides a lot of information about the change of conformational structures of many biological macromolecules such as protein and DNA over time. In addition, important information about the kinetic and thermodynamic properties of these biological macromolecules can be obtained at a time scale that cannot be obtained in any other way using MD methods. With the knowledge of the initial conditions of the system, it is possible to examine the time-dependent behavior of the system for the next step. Especially with this aspect, MD simulations are a tool that will facilitate our understanding of the evolution of a system over time (Alonso et al., 2006). Molecular dynamics simulations of 100 ns were performed to examine the stability of the 5i12-FabH protein-ligand complex obtained from Schr€ odinger Glide ligand docking. Also, MD simulation was performed with the protein-ligand complex as the control group in the apo form of the protein without ligand under the same conditions. Thus, the changes resulting from the interaction of 5i12 with the FahH protein were also analyzed. The results were interpreted by the analysis of the trajectory coordinate file recorded as a frame in MD simulations. Therefore, RMSD, RMSF, Rg, and SASA analyzes of apo and holoprotein were performed.
RMSD measurements provide basic information about the time-dependent shifts and deviations of protein and ligand. The RMSD measurement was calculated based on the backbone atoms of the protein. As seen in Figure 4a, the FabH-5i12 complex was below 0.3 nm in the first 20 ns, remained stable around 0.4 nm from 20 ns to 60 nm, and stabilized below 0.5 nm after 70 ns. The RMSD value of 5i12 varied between 0.02 and 0.16 nm during the MD simulation. The apoprotein peaked at 0.55 nm at the beginning of the simulation and stabilized below 0.5 nm.
The other analysis parameter was measured relative to the RMSF c-alpha carbon atom, which gives information about the fluctuation and conformational change of the protein. As shown in Figure 4b, apoprotein (0.56 nm) and FabH-5i12 complex (0.95 nm) showed the highest fluctuation between residues 195 and 210. While 5i12 increased the fluctuation of FabH protein around these residues, it decreased it at some other active site residues. There were no significant changes in  the active site residues of the holoprotein such as Ser157, Ala252, Ile223, Arg255, Ile256, Leu161, Phe224, Met218, and Ala252 compared to the apoprotein. Rg measurements are another important analysis parameter expressing protein compactness and stability. The compactness of the apoprotein and FabH-5i12 complex was compared. As given in Figure 4c, after 25 ns the apoprotein stabilized at around 1.95 nm, and the holo form around 1.92 nm. The interaction of 5i12 with FabH stabilized the protein compared to apoprotein.
Finally, the SASA value of apo and holoprotein was measured to examine the change in solvent access area on FabH by protein-ligand interaction. As given in Figure 4d, the Apoprotein and FabH-5i12 complex exhibited a similar SASA profile. The mean SASA value of the apoprotein was measured as 147.31 nm 2 , while the protein-ligand complex was measured as 149.63 nm 2 .
To detect the variation of 5i12 at the FabH active site at the atomic level, conformational changes at the beginning, middle, and end of its simulation were analyzed. As shown in Figure 5, the hydrophobic interactions and H-bonds between 5i12 and the active site amino acids Leu194, Cys117, Ile256, Met218, Phe224, Ile223, Cys117, Ala252, Ser157, Ile223, Arg255, Ile256, Leu161, and Ala252 continued alternatingly, thus 5i12 remains stable in active site with an average RMSD:0.9 Å value ( Figure 5).
Measuring the free binding energy of protein-ligand complexes (MM-PBSA) is another important protein-ligand stability research method. The free binding energy of the 5i12-FabH complex measured taking the sum of Van der Waal energy, electrostatic energy, polar solvation energy and SASA energy, and was given in Table 4. Accordingly, 5i12-FabH complex formed À142.551þ/-12.672 kJ/mol. The free binding energy standard deviation value was 12,672, less than 10% of the À142,551 value, indicating the stability of the protein-ligand complex.

Geometric optimization, molecular electrostatic potentials analysis and frontier molecular orbital
Geometry optimization constitutes the first stage of quantum chemical calculations for a molecular system. The small displacements of the atoms in the molecule in optimization calculations cause changes in the coordinates of the initial geometry, and each of these changes corresponds to different energies. The purpose of geometry optimization is to determine the situations where the molecule is most stable, i.e. the energy is minimum. In light of previous studies, we concluded that the compounds are found as E isomer. Accordingly, the optimized geometric structures of 5i10 and 5i12 were determined and shown in Figure 6. Molecular electrostatic potential (MEP) can be defined as the energy of interaction between the charge distribution of the molecular system and the positive unit charge. It is an optical method that enables us to understand the electronegativity, charge, dipole moment, and molecular polarity of a compound, as well as providing information about the net electrostatic effect created by the total charge distribution in the molecule. It also provides extensive information in determining intramolecular hydrogen bonds MEP surface diagrams of molecules are important in terms of showing their appearance as positive, negative, and neutral electrostatic potential zones depending on the colour grading. Potential increases were listed as red < orange < yellow < green < blue (Mary et al., 2020). The region where the MEP of a molecule is most negative is the region most prone to electrophilic attack. In contrast, the region where it is most positive is the region most susceptible to nucleophilic attack. In these maps, while the regions indicated in red represent the negative region of the electrostatic potential, they also represent the region where the electron density is higher than the nucleus over the entire molecule and is prone to chemical reactions. Blue regions are regions with partial positive charges and unstable in terms of reaction. Regions with fewer electrons are shown in yellow, while almost neutral regions are shown in green . The MEP map of 5i10 and 5i12 were computed to estimate the reactive sites. Looking at the MEP map (Figure 6), the blue zones are mainly concentrated on carbon and hydrogen atoms, the yellow zones around nitrogen, and chlorine atoms.
All molecules have HOMO (Highest Occupied Molecular Orbital), which is the highest filled molecular orbital, and LUMO (Lowest Unoccupied Molecular Orbital), which is the lowest empty molecular orbital. Since HOMO and LUMO orbitals play an essential role in chemical reactions, these orbitals can also be called precursor orbitals. If the molecule receives an electron, it gives it from HOMO; if it gives an electron, it gives it from LUMO, and this energy difference between HOMO-LUMO provides information about the stability of the molecule. 5i7 has a high tendency to accept electrons, and 5i17 has a higher electron donation. The smaller the energy difference, the more polarized the molecule can be. On the other hand, if the energy difference is large, it can be said that the ability to react is low; that is, the molecule is stable (Erol et al., 2021). The most stable compounds are listed as 5i6 > 5i7 > 5i5 > 5i2, respectively. Also, HOMO-LUMO boundary orbitals have an important place in studies examining antimicrobial activities, and it is thought that the antimicrobial property of the compounds is a function of LUMO energy. When a molecule behaves like a Lewis acid, the incoming electrons are taken to its LUMO, and molecules with low energy LUMO accept more electrons than those with high energy LUMO, so they show higher activity (Khan et al., 2019). The most effective antimicrobial compounds 5i12 have the lowest LUMO value. HOMO-LUMO energy differences and other electronic parameters derived from these energy differences (ionization potential (IP), electron affinity (EA), electronegativity (X), chemical hardness (g), chemical softness (S), chemical potential (l) and electrophilic index (x)) were calculated for all compounds and presented in Table 5. 5i12, which is the most active antimicrobial  that fall outside the 95% range of similar values for known drugs (0-5); Rotor: Number of non-trivial (not CX3), nonhindered (not alkene, amide, small ring) rotatable bonds (0-15); mol MW: Molecular weight of the molecule (130-725); Donor HB: Estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution (recommended value: 0-6); Acceptor HB: Estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution (recommended value:2-20); QPlogPo/w: Predicted octanol/water partition coefficient (recommended value:-2-6.5); QPlogS: Predicted aqueous solubility, log S. S in mol dm-3 is the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid (-6.5-0.5); QPPCaco: Predicted apparent Caco-2 cell permeability in nm/s. Caco-2 cells are a model for the gut blood barrier. QikProp predictions are for non-active transport (<25 poor, >500 great); Rule of Five: Number of violations ofLipinski's rule of five (Lyne et al., 2006). The rules are: mol_MW < 500, QPlogPo/w < 5, donorHB 5, accptHB 10. Compounds that satisfy these rules are considered druglike. (The "five" refers to the limits, rule of three number of violations of Jorgensen's rule of three. The three rules are QPlogS>-5.7, QP PCaco > 22 nm/s, # primary metabolites < 7). compounds, is one of the compounds with the highest quantum parameters with HOMO¼ À5.3339, LUMO¼ À1.2949 and DE ¼ 4,0389. When the HOMO and LUMO plots were examined (Figure 6), it is determined that the electron density is distributed over the whole compound.
3.6. In silico ADME estimation Drug discovery and development is a time-consuming long process. In this process, many molecular structures that have the chance to be an effective drug for patients are examined according to various parameters to determine which compounds to synthesize and test. While molecules are required to exhibit high biological activity with low toxicity, access, and concentration to the therapeutic target in the organism are equally important. To be effective as a drug, a potent molecule must reach its target in the body in sufficient concentration and remain in a sufficiently long bioactive form for the expected biological events to occur. In the drug development and discovery process, the screening of countless compounds for ADME is increasingly accepted. Therefore, in silico studies allow us to learn about the possibility of a compound being a potentially good drug. Early prediction of ADME parameters has been shown to reduce significantly the rate of pharmacokinetic failure in clinical phases during the discovery phase Puskullu et al., 2020). For this purpose, some important physicochemical properties, and descriptors of the 5i1-5i20 were theoretically calculated using the QikProp module of Schr€ odinger Maestro and presented in Table 6. In these calculations, there should be no more than one violation of ADME profiles of drug candidates according to Lipinski's five rules and Jorgensen's three rules. It can be seen from the table that all compounds comply with these rules. These results increase the likelihood that the compounds are potential drug molecules, given their promising antimicrobial activity results.

Conclusion
In this study, a series of indole-5-carboxaldehyde hydrazone compounds were synthesized, and their antimicrobial and cytotoxic activities were evaluated. Compounds showed moderate to weak antimicrobial activity against the structures and isolate studied, while 5i12 against E. faecalis showed comparable activity to reference drugs at MIC: 2 mg/ mL. Almost all compounds didn't show a significant antiproliferative activity on MCF-7 and A549 cell lines. At the same time, they also did not show a cytotoxic effect on healthy cell Beas-2b (except for 5i20, which reduced cell viability to 68.9% in 100 mM). Molecular docking studies of compounds were performed on 15 different proteins of E. faecalis, and it was concluded that the compounds interacted with FabH. XP docking scores and MM-GBSA energies were calculated in the FabH active site, and 2 D/3D interactions of 5i12 were also shown. Throughout the 100 ns MD simulation, 5i12 remained constant at the FabH active site with an average RMSD value of 0.9 Å. Also, DFT calculations were performed to estimate the geometric structure and electronic properties of the compounds. 5i12, which is the most effective compound against E. faecalis, is one of the lowest values with LUMO¼ À1,2949. In addition, the ADME profiles of the compounds also comply with Lipinski and other restrictive rules. According to all these results, the compounds are promising anti-E without harming human cells. It can be said that they may be E. faecalis agents.