Eco-Friendly Synthesis, Biological Evaluation, and In Silico Molecular Docking Approach of Some New Quinoline Derivatives as Potential Antioxidant and Antibacterial Agents

A new series of quinoline derivatives 5–12 were efficiently synthesized via one-pot multicomponent reaction (MCR) of resorcinol, aromatic aldehydes, β-ketoesters, and aliphatic/aromatic amines under solvent-free conditions. All products were obtained in excellent yields, pure at low-cost processing, and short time. The structures of all compounds were characterized by means of spectral and elemental analyses. In addition, all the synthesized compounds 5–12 were in vitro screened for their antioxidant and antibacterial activity. Moreover, in silico molecular docking studies of the new quinoline derivatives with the target enzymes, human NAD (P)H dehydrogenase (quinone 1) and DNA gyrase, were achieved to endorse their binding affinities and to understand ligand–enzyme possible intermolecular interactions. Compound 9 displayed promising antioxidant and antibacterial activity, as well as it was found to have the highest negative binding energy of -9.1 and -9.3 kcal/mol for human NAD (P)H dehydrogenase (quinone 1) and DNA gyrase, respectively. Further, it complied with the Lipinski’s rule of five, Veber, and Ghose. Therefore, the quinoline analogue 9 could be promising chemical scaffold for the development of future drug candidates as antioxidant and antibacterial agents.

Recently, there is a growing demand for the development of organic reactions in eco-friendly media. Synthetic manipulations have to be made to minimize the use of hazardous chemicals by replacing the traditional organic solvents in reactions and their subsequent workup with other nontoxic and environmentally benign solvents such as water.
For complementing this way and in continuation of our search work Haredi Abdelmonsef et al., 2020;Noser et al., 2020;Rashdan et al., 2020;Shehadi et al., 2020; toxicity (ADME/T) properties of the newly synthesized compounds were also calculated.

Chemistry
All melting points were measured by a Stuart SMP10 Melting point apparatus. IR spectra (KBr) were recorded by using a Bruker spectrometer (ν, cm −1 ). 1 H-NMR, 13 C-NMR, and DEPT 135 spectra were recorded on 400 and 100 MHz, DMSO-d 6 at AVANCE-III 400 MHz High performance FT-NMR Spectrum BRUKER. Bio Spin International AG-Switzerland at Sohag University. Elemental analysis was carried out at the Microanalytical Research Center, Faculty of Science, Cairo University. All the chemicals were commercially available from Sigma-Aldrich and El-Gomhouria Company, Egypt.

Biological Study
In-Vitro Antioxidant Assay The total antioxidant capacity of the compounds was evaluated according to the method described by Prieto et al. (1999). An aliquot of 0.5 ml of sample solution was combined with 4.5 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium The pharmacokinetic and physicochemical properties of the molecules (5-12). The agreeable ranges are as follows: Mol wt.: (<500); %Human oral absorption: >80% high, <25% low. logp, logarithm of partition coefficient between n-octanol and water <5; TPSA, topological polar surface area ≤140; nON, number of hydrogen bond acceptors 2.0-20.0; nOHNH, number of hydrogen bond donors 0.0-6.0; RBs, number of rotatable bonds ≤10.
Frontiers in Chemistry | www.frontiersin.org June 2021 | Volume 9 | Article 679967 phosphate, and 4 mM ammonium molybdate). In case of blank, 0.5 ml of 45% DMSO (dimethyl sulphoxide) has been used. The tubes incubated in a boiling water bath at 95°C for 90 min. After the samples cooled at RT, the absorbance of the aqueous solution of each sample was measured at 695 nm against blank by using a UV-2450 spectrophotometer (Shimadzu, Japan). The total antioxidant activity was expressed as the absorbance of the sample at 695 nm. The higher absorbance value indicated higher antioxidant activity (Wan et al., 2011).

Antimicrobial Assay
The antimicrobial activity of compounds was tested in vitro against various bacterial strains, Gram-negative (Escherichia coli), and Gram-positive (Staphylococcus haemolyticus, Kocuria kristinae, Enterococcus casseliflavus, and Bacillus subtilis) identified in Al-Azhar University, Regional Center for Mycology and Biotechnology. Amikacin, levofloxacin, and gentamicin were used as standard antibacterial agents obtained from Bioanalyse ® Ltd. (Turkey) for the comparison of biological activity of newly synthesized molecules. The method applied is "modified agar diffusion" (Bauer et al., 1966) using 2.0 mg per disc used to determine the antimicrobial activity. Nutrient agar (ready for use from EDM company,Egypt) inoculated with microbial cell suspension into sterile Petri dishes (200 µl in 20 ml medium). Sterile paper discs of 6 mm diameter saturated with a tested compound placed on the surface of the inoculated agar plates and negative control done using paper discs loaded with 20 µl of DMSO. Incubate overnight (24 h) at 37°C. Inhibition zone was measured at the end of the incubation period.

In Silico Docking Protocol
The molecular docking study of all compounds was carried out to identify their plausible mode of action against the active site residues of the target enzymes.
The 2D structures of the newly synthetic compounds were accurately drawn using ChemDraw Ultra 7.0 software and then converted to SDF format using Open Babel GUI tool (O'Boyle et al., 2011). An in-house library of ten synthesized compounds was generated for further study. The enzymes of NQO1: human NAD (P)H dehydrogenase (quinone 1) (PDB code 1DXO) (Faig et al., 2000) and DNA gyrase (PDB code 1JA6) (Holdgate et al., 1997) were selected as targets for docking simulation. The crystal structures of the targets were retrieved from the RCSB Protein Data Bank web server. The protein files were optimized by removing the ligands and water molecules. The grid box was generated around the active site pocket. Subsequently, the docking process was achieved using PyRx, virtual screening tool of AutoDock 4 software (Dallakyan and Olson, 2015). Among the nine confirmations of these ligand molecules obtained from the docking simulation, the pose with the lowest binding energy was selected for further study (HA and SP, 2016;Hussein et al., 2018). In addition, 7,8-Dihydroxyflavone (tropoflavin) and novobiocin were selected as standard drugs to compare the docking score with that of the synthesized compounds. Discovery Studio 3.5 was then used to visualize the intermolecular interactions between the ligand molecules and enzymes.

Chemistry
The present study entails the synthesis of a novel series of quinoline analogues 5-12 via one-pot multicomponent reaction (MCR) under solvent-free conditions.
The chemical structures of the products 5a-d were established on the basis of IR, 1 H-NMR, 13 C-NMR spectral data, and elemental analysis.
For example, IR spectrum of compound 5d showed new absorption bands at 3,351, 3,329, 3,294, 3,260 cm -1 for OH, NH, and NH 2 , respectively. The 1 H-NMR spectrum of compound 5d revealed triplet signal at 1.20 ppm for -CH 2 -CH 3 , singlet signal at 2.32 ppm for -CH 3 attached to phenyl ring, quartet signal at 4.08 ppm for -CH 2 , singlet signal at 5.71 ppm for CH-pyridine, singlet signal at 7.58 ppm for NH group, and singlet signal at 8.77 ppm for OH group, beside the appearance of the amino protons in interference with the aromatic protons at 6.27-7.28 ppm as a singlet. In addition, 13 C-NMR spectrum of compound 5d showed signals at 21. 02, 21.49, 59.96, and 168.18 ppm assigned to 2CH 3 and CH 2 and acetyl carbonyl group, respectively. Moreover, a negative signal of the CH 2 group was obtained at 61.59 ppm in DEPT 135 spectrum.
IR spectrum of compound 6 showed band at 1,668 cm -1 referred to carbonyl group; in addition, its 1 H-NMR spectrum exhibited beside the aromatic signals; new singlet signals at 11. 12, 9.98, 7.27, 5.95, and 2.38 ppm were consistent with the OH, 2NH, NH 2 , and sp 3 CH groups, respectively.
By adding ethyl acetoacetate 3b instead of ethyl cyanoacetate 3a, afforded the quinoline analogue 7 (Scheme 3). The chemical structure of compound 7 was established by elemental analysis and spectroscopic data, where IR spectrum showed amide carbonyl at 1,662 cm -1 . 1 H-NMR spectrum revealed a singlet signal at 1.87 ppm referred to CH 3 , 4.87 ppm for sp 3 CHpyrane, and OH at 8.64 ppm beside signals at 6.22-6.94 ppm for aromatic protons. Moreover, 13 C-NMR spectrum represented a signal at 33.68 ppm referred to CH 3 , in addition to other signals which confirmed the chemical structure of 7.
By the same way, a multicomponent reaction of resorcinol 1 with aromatic aldehyde 2e, ethyl acetoacetate 3b, and p-aminophenol 4b in the presence of sodium carbonate under solvent-free conditions afforded the quinoline derivative 9. This result was also achieved by the reaction of 3-oxobutanamide 8 with resorcinol 1 and salicylaldehyde 2e in the presence of sodium carbonate which makes the medium alkaline, as declared in Scheme 4.
IR spectrum confirmed the chemical structure of compound 9 by disappearance of the characteristic bands for acetyl carbonyl and the presence of new bands referred to imide carbonyl at 1,635 cm -1 . 1 H-NMR spectrum showed a singlet signal for CH 3 at 1.94 ppm, at 5.27 ppm a singlet signal referred to CH-pyridine, NH appeared at 8.90 ppm, and OH found at 11.53 ppm 13 C-NMR spectrum revealed signals at 19.32 and 160.64 ppm referred to CH 3 and imide carbonyl groups, respectively. Mass spectrum confirmed the molecular formula C 23 H 17 NO 4 by the molecular ion peak at m/z 371.
By the same way, the treatment of resorcinol 1 with 4chlorobenzaldehyde 2b and 3-oxobutanamide 8 afforded the quinoline derivative 10, as shown in Scheme 5.
IR spectrum of compound 10 showed new bands for acetyl and two OH groups at 1,710, 3,335, and 3,273 cm -1 , respectively. Its 1 H-NMR showed a singlet signal at 1.95 cm -1 for CH 3 , a singlet signal at 5.02 ppm for CH-pyridine, and two singlet signals at 8.42 and 11.56 ppm referred to two hydroxyl groups, beside multiplet signals of aromatic protons. Further, 13 C-NMR spectrum confirmed the presence of methyl group at 19.75 ppm, imide carbonyl at 157 ppm, and acetyl carbonyl at 172.53 ppm.
The reaction of resorcinol 1 with aldehyde, ethyl cyanoacetate, and aromatic amine was also studied to afford the quinoline analogues 11 and 12. For compound 11 stopped at the step of cyclization but compound 12 formed by additional reaction with hydroxyl group on aldehyde with ester and H 2 O get out. Ester form disappeared from compound 12 while it revealed at compound 11, as represented in Schemes 6, 7.
The spectral data of the newly synthesized compounds are represented as Supplementary Figures S1-S27 in supplementary information file.

Biological Evaluation
In-Vitro Antioxidant Assay The total antioxidant activity was determined using the phosphor molybdenum blue complex with a maximum absorption at 695 nm. The data presented in Table 1 showed that the tested compound 9 is the most active as represented in the following order: vit C > 9 > 12 > 5d > 11 > 5b.

Antimicrobial Evaluation
All the tested compounds showed good activity against bacterial strains tested with inhibition zones in range 9.0-20.0 mm. Amikacin showed inhibition zone 15.0 mm, levofloxacin 14.0-16.0 mm, and gentamicin 20.0 mm, as shown in Table 2. (Supplementary Figure S28 in supplementary information file). The compound 9 with electron donating groups like (-OH) and (-CH 3 ), piperidine, and pyran moieties showed the strongest activity against Gram-negative (Escherichia coli) and Gram-positive (Enterococcus casseliflavus) strains.

Molecular Docking Protocol
To understand as well as to support the in vitro antioxidant and antibacterial activity of the newly synthesized compounds for the rational design of novel and potential inhibitor molecules, molecular docking studies were performed (Abdelmonsef et al., 2016;Dasari et al., 2017;Rondla et al., 2017;Abdelmonsef, 2019).
Here, in silico molecular docking simulation of standard drugs and the new ten molecules with the active site of the target enzymes 1DXO and 1AJ6 was carried out to evaluate their binding affinities and to understand ligand-enzyme possible intermolecular interactions. The docking energies (ΔG bind ) and amino acid interactions for the screened compounds were summarized in Table 3. The 2D and 3D representation of the best docked complexes were represented in Figures 1, 2.

Antioxidant Activity
Human NAD (P)H dehydrogenase (quinone 1) is an enzyme that combats the oxidative stress conditions (Dinkova-Kostova and Talalay, 2010; Atia and Abdullah, 2020) as a gene highly expressed in human adipocytes and performing its antioxidant activity (Palming et al., 2007). In the present study, the docking studies were performed against the crystal structure of human NAD (P)H dehydrogenase (quinone 1) with PDB code 1DXO. All the docked compounds were fit on the enzyme active site with the docking scores (ΔG bind ) of the range -9.1--6.9 kcal/mol; in addition, the standard drug exhibited binding energy (ΔG bind ) -7.5 kcal/mol, ( Table 3). Compound 9 with the highest binding energy (-9.1 kcal/mol) docked to the target enzyme 1DXO through hydrogen bond and π-π stacking interactions with the amino acid residues Gly235, Tyr132, and Phe228 at the distances 2. 33, 5.83, 4.97, 3.76, 3.63, 5.25, 4.46, and 5.97 Å, respectively. In addition, compound 12 with − 8.7 kcal/mol showed four π-cation interactions with the residue Arg272 at distances 5. 36, 5.98, 5.76, and 5.94 Å, respectively (Figure 1). On the other hand, the standard drug (tropoflavin) with the binding energy − 7.5 kcal/mol binds with the target enzyme through similar amino acid residues Asn267, Arg272, and Pro264at 2. 97, 2.95, 4.95, 5.95, 5.98, and 3.75 Å, respectively. The rest of compounds are shown in supplementary file section as Supplementary Figure S30.

Antibacterial Activity
The DNA gyrase is a topoisomerase enzyme that controls the DNA's topological transition (Samadpour and Merrikh, 2018). In addition, the enzyme DNA gyrase has been considered as an essential for bacterial survival that catalyzes ATP-dependent negative super-coiling of bacterial chromosome (Reece et al., 1991;Tanitame et al., 2004). In this regard, in the present work, DNA gyrase has been selected as antibacterial drug target. The molecular docking simulation of the compounds 5-12 was carried out to identify their binding pattern with bacterial DNA gyrase. The compounds were observed to have the binding energies (ΔG bind ) ranging from -9.3 to -7.7 kcal/mol; in addition the standard drug exhibited binding energy (ΔG bind ) -8.3 kcal/mol, as shown in Table 3. The screened compounds 5-12 docked to the target enzyme through various intermolecular interactions as hydrogen bond and πstacking. Compound 9 has the best docking score (-9.3 kcal/mol) and exhibited four hydrogen bond interactions with the active site residues Asn46, Gly77, and Thr165 at the distances 2.76, 2.82, 3.08, and 2.29 Å, respectively. Moreover, the analogue 12 with -9.2 kcal/mol, showed intermolecular interactions through two hydrogen bond, two π-cation, and π-sigma at the distances of 3.07, 2.68, 4.39, 4.05, and 3.43 Å, respectively (Figure 2). On the other hand, the standard drug (novobiocin) with the binding energy -8.3 kcal/mol docked to the target through similar residues Arg76, His99, Ser121, Ile94, and Val97 at distances 2. 94, 2.97, 2.95, 2.19, 2.06, 5.15, and 4.13 Å, respectively (Figure 2). The other docked compounds with the target enzyme are shown in Supplementary Figure S2 (Supplementary file section).

Structure Activity Relationship Analysis
From the obtained results, we can conclude that the compound 9 with electron donating groups like (-OH) and (-CH 3 ), piperidine, and pyran moieties showed the best docking score (ΔG bind ) toward both target enzymes NQO1 and DNA gyrase (Haredi Abdelmonsef et al., 2020;Gomha et al., 2021). Comparing the standard drugs (tropoflavin and novobiocin), it has been found that they possess the same functional groups (-OH), (-CH 3 ), and pyran moieties. The docking scores of the synthesized quinoline molecules were in agreement with the experimental results which showed that the compound 9 could be used as potent inhibitor of NQO1 and DNA gyrase enzymes. Overall, the newly synthesized quinoline scaffolds have potential antioxidant and antibacterial activity and could be optimized to use as potent lead compounds as antioxidant and antibacterial agents.

ADMET/Pharmacokinetic Prediction Studies
In silico ADME/T and druglikeness prediction of the molecules 5-12, in addition to the standard drugs (tropoflavin and novobiocin), was computationally calculated in terms of absorption, distribution, metabolic, excretion, and toxicity via admetSAR (Cheng et al., 2012) and Molinspiration Web-based servers. The ADME/T analysis for different synthesized molecules was found to be in acceptable ranges ( Table 4). All compounds have molecular weight in the range of 279.30-405.84 g/mol (<500). The % oral intestinal drug absorption of all compounds was in the acceptable range (>80), indicating their possibilities in oral drug formulation for the treatment of bacterial infections. In addition, the new compounds exhibited little chance to cross the blood-brain barrier. The topological surface areas (TPSA) were found to be in the acceptable range (<140). In addition, H-bond acceptors (HBA) and donors (HBD) were found to be in the range of 3-6 and 2-4, respectively. Moreover, the newly synthesized compounds had high numbers of rotatable bonds (0-5), which indicates that they are flexible. Finally, the evaluation of toxicity and carcinogenic profiles for the compounds 5-12 declared that they are nontoxic and noncarcinogenic. Overall, the druglikeness study revealed that the new compounds fulfill the requirements of Lipinski's rule of five (Ro5) (Lipinski et al., 1997), Veber (Veber et al., 2002), and Ghose (Ghose et al., 2012) without any violations, suggesting that these compounds theoretically have ideal oral bioavailability. From all these results, we can conclude that all molecules exhibited good solubility and oral bioavailability.

CONCLUSION
In conclusion, we described a rapid, efficient, and low-cost method for synthesis of some quinoline analogues by using four components under solvent-free conditions. In addition, all synthesized compounds were in vitro screened for their antioxidant and antibacterial activity. Further, in silico molecular docking studies were achieved to support the biological experiments. The compound 9 displayed promising antioxidant and antibacterial activity, which was well supported by the in silico binding score, which showed it to have the highest binding energy of -9.1 and -9.3 kcal/mol against the target enzymes 1DXO and 1AJ6, respectively. In addition, compound 9 obeyed the Lipinski's rule of five, Veber, and Ghose . The experimental and in silico findings indicated that compound 9 could be used as a promising inhibitor of enzymes NQO1 and DNA gyrase.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.