Biphenyl Backbone-Based (Bis)Urea and (Bis)Thiourea Derivatives as Antimicrobial and Antioxidant Agents and Evaluation of Docking Studies and ADME Properties

Abstract In the present study, a new series of (bis)ureas 11(a–f) and (bis)thioureas 13(a–f) of 3,3'-dimethoxy-[1,1'-biphenyl]-4,4'-diamine dihydrochloride (9) has been synthesized. This precursor intermediate 9 in high yields was preparedfrom the commercial starting material, 4,4'-dinitro-[1,1'-biphenyl]-3,3'-diol (7) by following the chemical transformations such as methylation of hydroxyl groups and reduction of nitro to an amine functionality. Antimicrobial and antioxidant activities of the synthesized compounds were evaluated to screen for their biological significance. Antimicrobial activity screening results against four bacteria and two fungi unveiled that a few compounds, 11 b/13b, 11e/13e, and 11d (MIC 3.125 − 6.25 µg/ml) against all the tested bacterial strains and compound 11d against Cladosporium species (MIC 3.125 µg/ml) having potential activity. Especially, (bis)urea compounds 11a, 11 b, and 11e, and (bis)thiourea compounds 13a, 13d, and 13f (MIC 3.125 µg/ml) are proved the comparable activity with standard, cefixime (MIC 3.125 µg/ml) against P. aeruginosa. On the other hand, a few compounds 11c/13c, 13d, and 11e/13e were found, by using DPPH and FRAP methods, to exhibit a good antioxidant activity (IC50 range 25.0–32.5 µg/ml). The synthesized compounds were docked well in the active site of an enzyme, DNA Gyrase A, and the predictions of in silico-ADME and pharmacokinetic parameters suggest that the compounds have good oral bioavailability. The results demonstrate that this class of compounds has an expedient scope to use as antibacterial and antioxidant agents upon further development.


Introduction
Antibiotics were recognized in the middle of the 20th century as a great discovery to treat numerous infectious diseases. Ultimately, their use becomes more prevalent as modern medicine in various crucial surgeries like transplantation, cancer, and orthopedics. Because the abundant usage of antibiotic drugs, antibiotic resistance in bacteria is intensifying. [1][2][3] Gram-positive bacteria such as methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci faecalis (VRE), and Gram-negative bacteria Escherichia coli (E. coli), Klebsiella pneumonia and Pseudomonas aeruginosa are found as multidrug-resistant bacterial strains, and it has resembled as one of the most critical solicitudes faced in public health problems throughout the world. 4,5 Owing to this increased multidrug bacterial resistance, the discovery of new classes of antimicrobial agents with a wide range of activity against multi-resistance pathogens is a crucial demand. The reported specifics suggested that structural modifications of well-known existing antimicrobial agents and the development of new classes of antimicrobial candidates are two powerful strategies to overcome the resistance problems. 6,7 For example, recently discovered antibiotic candidates like linezolid and daptomycin have been serving potential antibiotics with novel modes of action against resistant bacteria. [8][9][10] Therefore, continuing research is essential to discover new classes of antibacterial agents that can circumvent the current resistance problem.
Biphenyls, being neutral molecules, are very worthwhile to introduce active functional groups that lead to produce novel molecules with collectively new activity. 11 They possess substantial photophysical properties and the sense to alter the geometry through internal rotation of the C-C single bond. 12,13 Thus, biphenyl scaffolds are privileged building blocks in numerous applications such as chiral reagents, inflexible 'spacers' between two parts of a molecule, and fluorescent layers in OLEDs. [14][15][16][17] Also, biphenyl derivatives have been employed as synthetic intermediates in the production of emulsifiers, optical brighteners, crop protection products, and plastics. 18 In addition, biphenyl derivatives exhibited numerous biological activities such as antimicrobial, antihypertensive, anti-inflammatory, diuretic, antidiabetic, antipsychotic, and anxiolytic. 11 Scientific analysis of scaffolds for pharmacologically active molecules has approved that biphenyl is a dominant backbone in the vast number of drug molecules, accounting for 2.1%. 19,20 Biphenyl derivative's importance is proved by their existence in several natural products like the antibacterial compounds MC21-A (1) and MC21-B (2), which were isolated from the marine bacterium Pseudoalteromonas phenolica. 21,22 The biphenomycin-A and -B (3) (Figure 1) were isolated from the cultured broth of Streptomyces griseorubiginosus 43608, as well. 23,24 Moreover, the biphenyl unit, based on the statistical analysis of NMR-based screening, is distinguished as the privileged substructure in a wide range of proteins. 25 Therefore, the overview results indicate that a biphenyl-containing structure can be a suitable framework for the designing and discovery of potential antimicrobial candidates.

Materials and instrumentation
All the chemicals and reagents employed in the present study were purchased from Sigma Aldrich, and the palladium on activated carbon (10% w/w) was obtained from Alfa-Aesar. The solvents like ethyl acetate (EtOAc), acetone, methanol (MeOH), isopropyl alcohol (IPA), tetrahydrofuran (THF), dichloromethane (DCM), and hexane were procured from Taiwan Field-rich Corporation. Thin Layer Chromatography (TLC) on silica gel plates, by visualization with UV light or in iodine vapor, was used routinely to monitor the progress of the reaction and the purity level of synthesized compounds. The compounds were purified by subject to the column chromatography packed with Merck 120 mesh silica gel as stationary phase, and a mixture of different ratios of DCM and MeOH, based upon the elution of desired product, was used as mobile phase. Guna melting point apparatus with an increasing temperature rate of 3 C/min was used to determine the melting points of the synthesized compounds and are uncorrected. NMR spectra were recorded on a BRUKER-400 MHz spectrometer, and it was operated at 400 MHz frequency for 1 H NMR and 100 MHz for 13   mass spectra. Elemental analysis was performed on Thermo Finnigan Flash 1112 analyzer. Highresolution mass spectra (HRMS) were obtained for a few compounds using the Synapt V R G2 HDMS V R system with electrospray ionization-hybrid quadrupole ion-mobility time-of-flight mass spectrometry (ESI-QTOF-MS). Bruker FT-IR spectrophotometer using KBr disks was used to record IR spectra, and the values are given in cm À1 . International principles and regulations were taken into consideration during biological activity screening. Selectivity index (S.I.) was calculated by dividing the zones of inhibition of compounds against organism(s) with the zones of inhibition of the standard, cefixime against organism(s).

2.2.3.
General procedure for the preparation of title products 11(a-f) and 13(a-f) To a reaction mixture of 3,3 0 -dimethoxy-[1,1 0 -biphenyl]-4,4 0 -diamine dihydrochloride (9) (300 mg, 0.95 mmol) in THF (5 mL) was added a solution of 1,4-dimethylpiperazine (DMPiz) (437 mg, 3.8 mmol) in THF (3 mL) at ambient temperature for 30 min. The reaction mass was agitated for 30 min at ambient temperature. A solution of substituted aryl isocyanates 10(a-f)/isothiocyanates 12(a-f) (1.6 mmol) in THF (3 mL) was added slowly to the above reaction mass at ambient temperature. The reaction mixture was heated to 55-60 C, and then it was agitated for 3-6 h. After completion of the reaction that was monitored by TLC, the reaction mixture was filtered to remove salts (1,4-dimethylpiperazine dihydrochloride), and the filtrate was concentrated under a vacuum to obtain the crude residue. It was subjected to column chromatography using Merck 120 mesh silica gel as a stationary phase and a mixture of different ratios of MeOH (0-10% v/v) and DCM (90-100% v/v), based upon the elution of desired product, to obtain the pure products.

Antibacterial and antifungal assays
The bacterial strains such as Staphylococcus aureus (ATCC 6538), Bacillus megatarium (ATCC 14581) (Gram-positive organisms) and Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) (Gram-negative organisms), and fungal strains like Aspergillus species (ATCC 16888) and Cladosporium species (ATCC 16022) were received from the Department of Zoology, Sri Venkateswara University, Tirupati, India. The disk diffusion method was followed to screen the activities. 39,40 The antibiotics, cefixime, and ketoconazole (Positive controls) were used as the reference drugs in the evaluation of antibacterial and antifungal activities, respectively, for comparison of the activities of the title products. The solvent, dimethyl sulfoxide (DMSO) used in the preparation of the test samples, and the stock solution was used as a negative control. Sterile filter paper disks (6 mm diameter) saturated with the test samples of the specific concentrations, 100 and 200 mg/disk were used in the screening of antimicrobial activities. The culture of bacteria/ fungi having approximately 24/48 h old was mixed with sterile physiological saline, and their turbidity was adjusted to the standard inoculum of Mc-Farland scale 0.5, 10 6 colony forming unit (CFU) per mL. The relative culture was aseptically transferred to the sterile Muller Hinton Agar plates (Hi-media) in the screening of antibacterial activity and potato dextrose agar plates (Himedia) in antifungal activity (each plate containing 20-25 mL of the sterile medium), and a sterile glass spreader was used for even distribution of the inoculum. The test samples of sterile filter paper disks (6 mm diameter) (100 and 200 mg/disk), positive controls, and negative control were deliberately placed on the culture plates that have been previously inoculated separately with the microorganisms. The bacteria inoculated plates for 24 h at 37 C, and fungal inoculated plates for 72 h at 25 C were incubated. The zone of inhibition of bacteria/fungi around the disk was measured edge to edge zone of the confluent growth which corresponded to the sharpest edge of the zone and was measured in millimeters. All determinations were made in duplicate for each compound. The average of two independent readings for each microorganism was shown as a final reading.

Minimum inhibitory concentrations (MICs)
The modified micro broth dilution method established by the National Committee for Clinical Laboratory Standards was used for the determination of MICs. 39 42 Ascorbic acid was used as a standard drug. FRAP reagent was prepared freshly by addition of acetate buffer (pH 3.6), TPPZ (2,4,6-tripyridyl-s-triazine) (10 mmol) solution in HCl (40 mmol) and ferric chloride (20 mmol) in the ratio of 10:1:1 (v/v) respectively. The FRAP solution (3 mL) was added to an aliquot of the standard sample (BHT) and test samples of various concentrations (25,50,75, and 100 mg/mL), and the blank (DMSO) in a set of test tubes and then they were incubated at 37 C in an incubator for 40 min. The absorbance of the blue color developed by the change of the ferric complex to the ferrous complex was measured spectrophotometrically at 593 nm against blank.

Half maximal inhibitory concentration (IC 50 )
The half-maximal inhibitory concentration (IC 50 ) is a quantitative measurement that signifies the required concentration of each chemical compound to inhibit a given biological process by half (50%). The IC 50 value of the synthesized compounds was determined using the DPPH method. Different concentrations of the synthesized compounds (with a difference of 5 mg/mL of each) were prepared based on the test results obtained in the above DPPH method at the concentrations of 25 and 50 mg/mL. The DPPH solution (4 mL, 0.004% (w/v)) was added to an aliquot of standard solution (BHT) and the tested sample solutions (1 mL of each) of various concentrations, and the test solutions were shaken vigorously. The absorbance of the tested samples was measured against blank at 517 nm, after incubating the test solutions for 30 min in the dark. The concentration of the test samples was recorded which inhibited the given biological process by half.
The experiments performed in the investigation of antioxidant activity of the title products were replicated, and the average value was taken as a result.

Molecular modeling
Three-dimensional chemical structures of the synthesized compounds and the standard, cefixime were built in ChemBioDraw (ChemBioDraw Ultra 14.0 version), and all these ligands structures were converted into Pdbqt file format. The atomic coordinates of these structures were built using Pyrex 2010.12. The three-dimensional structure of DNA Gyrase A (PDB: 3LPX) ( Figure S1) was downloaded from the RCSB Protein Data Bank. The atomic coordinates of the protein were alienated, and Argus Lab 4.0.1 was used for geometry optimization. Grid dimensions of the enzyme, 3LPX were predicted as˚X: 28.27, Y: 27.13, and Z: 28.51. The polar hydrogens were added to the binding site residues, and then energy minimization has done with the MMFF94x force field to get the stable conformer of the protein. Flexible docking was employed, and the inhibitor binding site residues were softened and highlighted through the 'Site Finder' module implemented in the Pymol software. Molecular docking of the synthesized compounds was performed against 3LPX protein using Pyrx 2010.12 by following the default parameters, i.e. placement: triangle matcher, recording 1: London dG, and refinement: force field. The maximum of ten docking binding modes or conformations of each compound was assessed, and the highest-ranked binding pose was used for graphical representation in the Pymol viewer tool (www.pymol.org). 43,44 Binding energy, the binding affinity of the protein-ligand complexes, and the bonding interactions, after the docking process, were analyzed, and their results were summarized in Table 1.

ADME calculations
Molinspiration online property toolkit was used to determine the various ADME properties like topological polar surface area (TPSA), molecular weight (MW), molecular volume (MV), number of rotatable bonds (n-ROTB), number of hydrogen bond donors, number of hydrogen bond acceptors, the logarithm of the partition coefficient (mi Log P) and violations of Lipinski rule. The %ABS was calculated by using the formula: %ABS ¼ 109 -(0.345 9 TPSA). 45 ADME prediction properties like HIA%, caco2 permeability, PPB%, and blood-brain barrier (BBB) were predicted by using a pre-ADMET online server (http://preadmet.bmdrc.org/). 46

Chemistry
Integration of more than one pharmacophore, each with a different mode of action in a single molecule, could be advantageous to explore potential activity. Considering the overview importance of biphenyl derivatives and urea and thiourea compounds and our continuing research on the discovery of biologically active molecules, we assumed that the combining of biphenyl scaffold with urea and thiourea functionalities may afford new chemical entities, which would possess potential antimicrobial activity. Therefore, we planned to prepare biphenyl backbone (bis)urea and (bis)thiourea derivatives, 1, dihydrochloride (9). Finally, the desired urea and thiourea derivatives of 3,3 0 -dimethoxy-[1,1 0biphenyl]-4,4 0 -diamine were synthesized in good to high yields by reacting 9 with substituted aryl isocyanates 10(a-f) and aryl thioisocyanates 12(a-f), respectively in the presence of an excess amount of base, 1,4-dimethyl piperazine at 55-60 C. The crude residues of title products obtained after the removal of solvent were subjected to column chromatography using a mixture of different ratios of DCM (90-100% v/v) and MeOH (0-10% v/v), based upon the elution of desired product, to obtain the pure title products.

Spectroscopic data analysis
The structures of newly synthesized compounds were elucidated based on the spectroscopic data such as IR, NMR ( 1 H and 13 C), mass, and HRMS. The appearance of a new peak, in the 1 H NMR spectrum of compound 8, at the chemical shift value, 4.01 ppm resembling O-CH 3 protons that confirmed the methylation reaction in the transformation of compounds 7-8. The chemical shift values of aromatic protons in the 1 H NMR spectrum of compound 9 (6.84-7.32 ppm) are shielded when compared with compound 8 (7.42-8.47 ppm), implying the reduction of the nitro group (NO 2 ) to amine (NH 2 ).
In IR spectra of all the synthesized compounds 11(a-f) and 13(a-f), the characteristic broad bands at the range of 3207-3365 cm À1 are attributed due to the presence of N-H stretching of urea/thiourea functionalities, and the bands in between 1201-1250 cm À1 are corresponding to C-O stretching of Ar-O-CH 3 . The absorption bands resonated at around 1630-1734 cm À1 in the urea compounds 11(a-f), and 1024-1081 cm À1 in thiourea compounds 13(a-f) are corresponding to the carbonyl group C¼O and thiocarbonyl functionality C¼S, respectively, which signifies the formation of the title products. 1

Biology
All the synthesized urea 11(a-f) and thiourea 13(a-f) derivatives were evaluated for their in vitro antimicrobial activity at two different concentrations (100 and 200 mg/disk) using bacterial strains such as Gram-positive (Staphylococcus aureus (ATCC 6538) and Bacillus megatarium (ATCC 14581)) and Gram-negative (Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 9027)) and fungal strains like Aspergillus species (ATCC 16888) and Cladosporium species (ATCC 16022). The disk diffusion method was followed in the screening of antimicrobial activity. 39,40 The antibiotics, cefixime, and ketoconazole (Positive controls) were used as the reference drugs in the evaluation of antibacterial and antifungal activities, respectively. The diameter of the growth inhibition zones shown by the title compounds at two different concentrations, 100 and 200 mg/ disk was measured as an indicator activity of each compound, and the observed results were summarized in Figure 2 (antibacterial activity) and Figure 3 (antifungal activity). The antimicrobial activity results were classified, for easy understanding, as moderate to excellent activity (if showed the activity at 100 mg/disk) and poor activity (not showed the activity at 100 mg/disk). The minimum inhibitory concentration (MIC) is defined as the lowest concentration/highest dilution of each chemical compound required to exhibit no growth of inoculated bacteria/fungi. Few compounds displayed remarkable microbial growth of zone of inhibition (ZOI); hence, the MIC of the active compounds was further screened using the micro-broth dilution method, 39,40 , and the found experimental results have been presented in Table 2. The antioxidant nature of the synthesized compounds was assessed by the radical scavenging activity using the DPPH 41 method and ferric reducing antioxidant power (FRAP), 42 and the observed results were summarized in Table  S1. The half-maximal inhibitory concentration (IC 50 ) that indicates the required concentration of each chemical compound to inhibit a given biological process by half (50%) was also investigated for the title products using the DPPH method, and the results were shown in Figure 4.

Antibacterial and antifungal activities
The preliminary antibacterial activity results of the synthesized compounds 11(a-f) and 13(a-f) are presented in Figure 2  Most of the compounds 11a, 11c, 11d, 13a, 13d, and 13f showed considerably promising activity against P. aeruginosa (ATCC 9027). Particularly, (bis)urea compound 11b (ZOI at 200 mg/ disk, 41.66 mm) and (bis)thiourea derivative 13b (ZOI at 200 mg/disk, 41.82 mm) and 13d linked with 3,4-dichlorophenhylring (ZOI at 200 mg/disk, 44.06 mm) exhibited higher growth of inhibition against P. aeruginosa (ATCC 9027) when compared with the standard (ZOI at 200 mg/disk, 40.77 mm). These observations hinting the substitution of chlorine on the phenyl ring could be a reason to explore higher activity because of its inductive effect. 47 Likewise, compounds 13d against B. megatarium (ATCC 14581) displayed good activity. Compounds 11c/13c bound with the 4-bromophenyl ring, 11f consisting of a 3-methoxyphenyl ring, and 11a/13a holding a 4-fluorophenyl ring exhibited poor and moderate activity against all the tested bacterial strains, respectively.
Pseudomonas aeruginosa (ATCC 9027) has been characterized as severe problematic bacteria in patients, with mortality rates of 34-48%, who required a tracheotomy for continued mechanical ventilation. 48 Most of the title (bis)urea/thiourea derivatives synthesized in the present study displayed excellent P. aeruginosa (ATCC 9027) growth of inhibition. Thus, the selectivity index (S.I.) of the synthesized (bis)urea/thiourea derivatives 11(a-f)/13(a-f) in comparison with cefixime was investigated against P. aeruginosa (ATCC 9027) ( Figure 5) by considering the experimental results at the concentration of 200 mg/disk. The results disclosed that compounds 11b, 13b, and 13d had a better selectivity index (S.I. >1), compounds 11a and 11e had almost similar Table 2. Minimum inhibitory concentrations (MICs) of the compounds 11(a-f) and 13(a-f).
Compd.  selectivity index and the remaining compounds possessed a lesser selectivity index (S.I. <1) when compared with reference drug, cefixime in the growth inhibition on P. aeruginosa (ATCC 9027). These significant antibacterial activities of the synthesized bisurea/thiourea derivatives may give the scope to prepare lead antibacterial agents, particularly against, P. aeruginosa for further modifications. As seen in Figure 3, all the synthesized compounds except compound 11d showed very poor activity and did not show any activity against Aspergillus species (ATCC 16888) at the concentration of 100 mg/disk. Few (bis)urea compounds such as 11 b having a 3-chlorophenyl ring and 11d bound with the 2,4-diflurophenyl ring, and (bis)thiourea derivative 13e linked with 4-nitrophenyl ring exhibited potential growth of inhibition (ZOI range at 200 mg/disk, 29.21-32.66 mm) against Cladosporium species (ATCC 16022). These compounds' activities are almost closer to the standard, ketoconazole (ZOI value at 200 mg/disk, 34.51 mm). While the compounds 11e associated  with 4-nitrophenyl ring and 13a bound with 4-fluorophenyl ring explored the moderate activity and the rest of the compounds possessed very poor activity against Cladosporium species (ATCC 16022).

Minimum inhibitory concentrations in mg/ml
The MIC values of the active compounds as given in Table 2 demonstrated that most of the synthesized compounds 11a, 11 b, 11e, 13a, 13d, and 13f exhibited excellent inhibitory activity (MIC 3.125 mg/mL) against P. aeruginosa (ATCC 9027), and these are very related to the activity of standard, cefixime (MIC 3.125 mg/mL). Compounds 11 b/13b and 11e/13e showed good to excellent inhibitory activity in the MIC range of 3.125-12.50 mg/mL against all the tested bacterial strains. Furthermore, it was found that all the synthesized compounds having no activity against Aspergillus species (ATCC 16888) and compound 11d showed comparable activity with the reference drug, ketoconazole against Cladosporium species (ATCC 16022) (MIC 3.125 mg/mL).

Structural-activity relationship
Overall, the synthesized (bis)urea/(bis)thiourea derivatives 11(a-f)/13(a-f) exhibited moderate to excellent activity against bacterial strains, in particular, more potent against Gram-negative bacteria than that of Gram-positive bacteria, and poor activity showed toward fungal strains. Since all the synthesized compounds are structurally related at their biphenyl ring and the phenyl ring congeners end but it consists of variations in either functionality such as urea and thiourea or substituents on the phenyl ring. Therefore, the diversity in the exhibition of antibacterial activity could be subjected to the nature of functionalities and the substituents on the phenyl ring. As shown in Table 1, no remarkable difference in the antibacterial activity of (bis)urea derivatives 11(a-f) and (bis)thiourea compounds 13(a-f) even the compounds having related substituents on the phenyl ring, suggesting the functionalities of both urea and thiourea having a similar tendency in the exhibition of activity. The most active compounds among all the synthesized compounds were 11 b/13b (3-chlorophenyl ring), and 11e/13e (4-nitrophenyl ring) against all the tested bacterial strains indicating the substituents chlorine and nitro groups have played the prompt role to show the increased antibacterial activity. Among the series of synthesized compounds, the chloro substituted derivatives 11 b/13b (3-chlorophenyl ring), and 13d (3,4-dichlorophenhyl ring) displayed higher activity against P. aeruginosa (ATCC 9027) than that of the reference drug, demonstrating that the chlorine might be shown a significant effect on the growth inhibition of P. aeruginosa (ATCC 9027). Also, fluoro and nitro-substituted compounds had moderate to good activity, and bromo and methoxy substituted derivatives except for compound 13f against P. aeruginosa (ATCC 9027) explored poor activity. This study unveiled that the heedful selection of substituents on the phenyl ring might be provided potent antibacterial agents, particularly against P. aeruginosa (ATCC 9027). Generally, the alteration of substituents on the phenyl ring at different positions could be diversified the biological activity, and it is beyond the scope of this paper and a discussion of future research.

Antioxidant activity
Generally, the mechanistic pathway of antioxidants to exhibit antioxidant activity is complex, and it could be involved in either chain initiation prevention, complex formation with transition metal ion, decomposition of peroxides, prevention of continued hydrogen abstraction, or reductive capacity, or radical scavenging. 49 Because of its stable free radical nature and ability to accept free electrons or hydrogen radicals to become a stable diamagnetic molecule, the DDPH radical model is an imperative and extensively used method to investigate the antioxidant tendency of target molecules. 50,51 FRAP method, which is mainly performed on the principle of the reduction of Fe(III) to Fe(II) by donating the electron from the antioxidant, is also a widely used protocol to investigate reducing the capacity of antioxidants. Therefore, these two methods have been used to investigate the antioxidant capacity of the (bis)urea 11(a-f) and (bis)thiourea 13(a-f) derivatives. Butylated hydroxytoluene (BHT) was used as the standard. Few compounds showed promising antioxidant activity in both the methods; therefore, the interest has been continued to evaluate IC 50 values by the DPPH method, and the results were shown in Figure 4.
Results as shown in Table S1, all the compounds showed moderate to good antioxidant activities in both methods at two different concentrations, 25 and 50 mg/mL. It might be the presence of many heteroatoms like N, O, S, and halogens, and they can donate the electrons easily and/or have a tendency to accept hydrogen. Among the series of compounds, bromo-substituted derivatives, 11d/13d (IC 50 range 25-30 mg/mL), dichloro-substituted bis(thiourea) compound 13d (IC 50 25 mg/mL), and nitro-substituted derivatives 11e/13e (IC 50 range 30-32.5 mg/mL) showed promising radical scavenging activity in DPPH method when compared with other compounds in this series. The same compounds explored good absorbance at 593 nm in the FRAP method as well which suggests that these compounds have a good ability to reduce Fe 3þ to Fe 2þ . Because of the natural tendency to donate valence electrons and lower electronegativity, the functional groups, Br, Cl, and NO 2 could be a reason to explore potential activity. It was found, based on the comparison of overall antioxidant results, that (bis)thiourea derivatives having better activities than that of (bis)urea compounds even consist of similar substitutions, demonstrating thiourea functionality is also an object to provide the activity. The activity of the synthesized compounds is slightly lower than the standard, BHT; even though there is a scope to enrich the activity of compounds by alteration of substituents on the phenyl ring, induction of the new class of isocyanates/isothiocyanates, and association of new molecules on biphenyl ring. However, it is beyond this present study and a subject of future research.

Molecular docking studies
DNA gyrase and topoisomerase IV are the two types II topoisomerases in bacteria, and both enzymes are implicated in fundamental processes of DNA replication, and they are indispensable for cell viability. 52,53 Each enzyme is independently catalyzing DNA topology modifications by breaking and rejoining double standard DNA. In particular, DNA gyrase is a heterotetramer that contains two GyrA and GyrB subunits, and is primarily accountable for introducing negative supercoils in DNA in front of the replication fork, while topoisomerase IV is essential for decatenation during DNA replication. 54 Therefore, DNA gyrase is the intracellular target of several antibacterial agents and as a paradigm for other DNA topoisomerases as well as this topic has attractive wave interest in the challenging search for new bacterial agents. Recently, many studies on urea and thiourea derivatives are detailed as potential antimicrobial agents, and they presented significant binding energies in the active site of DNA Gyrase A enzyme. 55 Because the potent antimicrobial activity of the synthesized urea and thiourea derivatives 11(a-f)/13(a-f) in the present study, the interest has been extended to interpret the binding mode of these compounds and with reference antibiotic drug, cefixime, in the active site of DNA Gyrase A (PDB: 3LPX) by performing the molecular docking study.
The three-dimensional structure of DNA Gyrase A (PDB: 3LPX) ( Figure S1) was downloaded from the RCSB Protein Data Bank and then switched the water molecules and ligands from the protein file. Argus Lab 4.0.1 was used for the geometry optimization, and the energy minimization has done with the MMFF94x force field to attain the stable conformer of the protein. Site Finder module implemented in the Pymol software was employed to soften the inhibitor binding site residues. Pyrx 2010.12 docking tool was used to perform the docking studies against 3LPX protein by following the default parameters, i.e. placement: triangle matcher, recording 1: London dG, and refinement: force field. After the docking, the binding energy, bond length, bond angle, and the bonding interactions of the protein-ligand complexes were analyzed, and their results are compiled in Table 1. The highest ten docking binding modes or conformations of each compound were evaluated, and the highest-ranked binding pose was used for graphical representation in the Pymol viewer tool (www.pymol.org). 43,44 The docking conformers of most active compounds and the remaining compounds are outlined in Figure 6(a-e) and Figure S2(a-g), respectively.
Based on the ligand-protein interactions as represented in Table 1, Figure 6(a-e) and Figure  S2(a-g), the chain residues such as Thy218, Thr219, Pro218, Gly110, Gly107, Asn108, His261, Ser111, Val112, Phe109, and Lys270 of the enzyme are associating with target compounds. They were majorly interacting with -NH portion of urea and thiourea functionalities in most of the compounds along with NO of the nitro group in 11e/13e and CO of the methoxy group in 11a, 13a, 13 b, and 13f, and with chlorine (Cl) group in 11 b. The active compound 11 b was bound with DNA Gyrase A by forming one favorable hydrogen bond with the amino acid residue, Asn108 through an electronegative chlorine atom (Figure 6(a)). Compound 13 b interacted remarkably through NH of thiourea group and CO of methoxy group with the enzyme by the formation of four hydrogen bonds with Pro218, Gly110 (two interactions), and Val112 ( Figure  6(b)), and compound 11d bound with the enzyme by forming one hydrogen bond with residue, His262 through NH of urea group (Figure 6(c)). Likewise, compounds 11e ( Figure 6(d)) and 13e ( Figure 6(e)) formed hydrogen bonds with amino acids of Gly107 and Gly110, and Phe109, Pro218, and Thr218, respectively in the active site of the enzyme through NH of urea/thiourea group and NO of nitro functionality. The standard drug, cefixime drug conferred a different interacting pattern (Asp297, Lys270, Leu264, and Pro218) with DNA Gyrase A enzyme ( Figure  6(f)). However, the only amino acid residue, Pro218 in the protein commonly interacts with most of the title compounds as well as the reference drug.

ADME calculations
Drug-likeness appears as a promising paradigm of the desired target compound that optimizes their adsorption, distribution, metabolism, excretion, and toxicity (ADMET) in the human body. 44 All these ADME and pharmacokinetic properties such as partition coefficient of n-octanol and water (LogP), aqua solubility, topological polar surface area (TPSA), number of rotatable bonds (n-ROTB), number of hydrogen bond donors (Don-HB)/acceptors (Acp-HB), colon adenocarcinoma (caco-2) cell permeability, percentage of absorption (%ABS), percentage of human intestinal absorption (%HIA), percentage of plasma protein binding (%PPB) and blood/ brain partition coefficient (BBP)/blood-brain barrier (BBB) of the synthesized compounds were computed using Molinspiration online property toolkit and a pre-ADMET online server (http:// preadmet.bmdrc.org/). The observed results are tabulated in Table 3.
To understand the drug-likeness of the title compounds, we have determined the compliance of the synthesized molecules to Lipinski's 'rule of five'. According to this rule, the molecule possesses Don-HB, and Acp-HB is less than or equal to five and ten, respectively, the molecular weight (Mol. Wt.) is more than 500, and the calculated LogP is less than 5, to have more absorption/bioavailability. Molecules violating more than one of these parameters may have predicaments with bioavailability and a high probability of failure to display drug-likeness. 46,56 TPSA is also another key property in estimating drug bioavailability was also calculated. Generally, compounds with a TPSA ! 140 A are believed to have low bioavailability. 57 It can be seen in Table  3; all the synthesized compounds obey Lipinski's 'rule of five' except for the molecular weight (all the synthesized compounds having more than 500 m. wt.) and TPSA property (78.86-92.86 A o ).
Moreover, the compounds exhibited a more significant percentage of absorption (%ABS) ranging from 90.30 to 96.20%, suggesting all the synthesized (bis)urea/thiourea derivatives in this study have excellent passive oral absorption and drug-likeness. Besides, the blood/Brain partition Table 3. Molecular descriptors in ADME study of the synthesized compounds 11(a-f) and 13(a-f). Compd.

Mol.
Wt coefficient/BBB value, which measures the ability of a drug to cross the blood-brain barrier of the synthesized molecules (0.118-0.322) is in the acceptable range. The predicted HIA% values for the title compounds are excellent in the range of 90.28-99.26%, which indicates the possibility of oral bioavailability of the target compounds. The caco-2 cell permeability values of the synthesized compounds are reasonable, ranging from 20.16-26.66 nm/s. Whereas, the active compounds 11 b/13b, 11d, and 13e (for except 11e) displayed higher permeability when compared with the remaining title products. Furthermore, all the compounds are firmly bound to plasma proteins with a %PPB penetration of more than 86.22%. Therefore, the summary of ADME and pharmacokinetic properties of the synthesized compounds may provide new insights into the further development of new chemotherapeutic drug candidates with advantageous oral bioavailability.

Conclusions
In conclusion, we have synthesized a new class of biphenyl backbone-based (bis)urea and (bis)thiourea derivatives from the precursor intermediate, 3,3 0 -dimethoxy-[1,1 0 -biphenyl]-4,4 0 -diamine dihydrochloride by reacting with substituted phenyl isocyanates/isothiocyanates. All these compounds were evaluated for their in vitro antimicrobial and antioxidant activities. The compounds, 11 b/13b, 11e/13e, and 11d against all the tested bacterial strains and compound 11d against Cladosporium species (ATCC 16022) displayed potential activity. Most of the compounds showed very poor to no activity against Aspergillus species (ATCC 16888). Interestingly, most of the compounds 11a, 11 b, 11e, 13a, 13d, and 13f (MIC 3.125 mg/ml) are proved comparable activity with standard, cefixime (MIC 3.125 mg/ml) against P. aeruginosa (ATCC 9027). Whereas, the title compounds possessed a chloro substituent on the phenyl ring showed the maximum activity. The antioxidant activity results accomplished in DPPH and FRAP methods unveiled that a few compounds substituted with bromo, chloro, and nitro groups (11c/13c, 13d, and 11e/13e) showed good antioxidant activity, however, they are slightly lower than that of the reference drug, BHT. Furthermore, all the compounds docked well in the active site of DNA Gyrase A with excellent binding energy than that of reference drug, cefixime, and those provide a good agreement to in vitro antibacterial results as well. The results of ADME predictions with one violation of Lipinski's rule of five and excellent pharmacokinetic parameters demonstrated these compounds possessed a superior oral bioavailability. Collectively, the results observed in the present study suggest that the biphenyl backbone-based urea and thiourea compounds have versatile properties that make them suitable for further testing, upon further development, as potential drug candidates.