Synthesis, DFT Analysis, and Evaluation of Antibacterial and Antioxidant Activities of Sulfathiazole Derivatives Combined with In Silico Molecular Docking and ADMET Predictions

Synthetic modifications of sulfathiazole derivatives become an interesting approach to enhance their biological properties in line with their applications. As a result, sulfathiazole derivatives become a good candidate and potential class of organic compounds to play an important role towards medicinal chemistry. In present study, one thiazole derivative and two new sulfathiazole derivatives are synthesized with 94% and 72–81% yields, respectively. Furthermore, the synthesized compounds were evaluated for their in vitro antibacterial activity against two Gram-negative (E. coli and P. aeruginosa) and two Gram-positive bacterial strains (S. pyogenes and S. aureus) by disk diffusion method. Among synthesized compounds, compound 11a showed potent inhibitory activity against Gram-negative, E. coli with 11.6 ± 0.283 mm zone of inhibition compared to standard drug sulfamethoxazole (15.7 ± 0.707 mm) at 50 mg/mL. The radical scavenging activities of these compounds were evaluated using DPPH radical assay, and compound 11a showed the strongest activity with IC50 values of 1.655 μg/mL. The synthesized compounds were evaluated for their in silico molecular docking analysis using S. aureus gyrase (PDB ID: 2XCT) and human myeloperoxidase (PDB ID: 1DNU) and were found to have minimum binding energy ranging from −7.8 to −10.0 kcal/mol with 2XCT and −7.5 to −9.7 with 1DNU. Compound 11a showed very good binding score −9.7 kcal/mol with both of the proteins and had promising alignment with in vitro results. Compound 11b also showed high binding scores with both proteins. Drug likeness and ADMET of synthesized compounds were predicted. The DFT analysis of synthesized compounds was performed using Gaussian 09 and visualized through Gauss view 6.0. The structural coordinates of the lead compounds were optimized using B3LYP/6–31 G (d,p) level basis set without any symmetrical constraints. Studies revealed that all the synthesized compounds might be candidates for further antibacterial and antioxidant studies.


Introduction
Most of the organic compounds including natural products possess heterocyclic rings as a core part of it, and they provide the ability to alter their molecular conformation, solubility, physicochemical, pharmaceutical, and biological activities. ese molecules perform remarkable functions in nature, medication, and innovation [1]. Heterocyclic compounds play an important role towards development of organic synthesis and have wide applications in the field of pharmaceutical science. Organic compounds with ring system containing sulfur, nitrogen, and oxygen as heteroatom are proven to be potent bioactive agents [2]. One of the most important groups of organic compounds among fivemember heterocyclic compounds containing S and N atoms are called thiazoles and they belong to the group of azole heterocycles.
iazole is structurally similar to imidazole and oxazole with the thiazole sulfur replaced by nitrogen in imidazole and oxygen in oxazole, respectively [3].
iazole derivatives act as antifungal [4], anti-inflammatory [5], analgesic [6], and anticancer agents [7]. On the other hand, sulfathiazole is an organic compound derived from sulfonamide by replacing one amide hydrogen atom with thiazole group. Sulfathiazole is the primary powerful bioactive agent efficiently utilized for the prevention and cure of bacterial infections.
Sulfonamides, known as sulfa drugs, are the oldest drugs commonly employed and systematically used as bioactive agents. Few of these drugs containing sulfonamide moiety are sulfathiazole (1), sulfadiazine (2), sulfamoxole (3), and sulfafurazole (4) [8] (Figure 1). Sulfathiazole derivatives having heterocyclic scaffold possess wide applications for pharmaceutical purpose, such as antibacterial [9], antifungal [10], anti-inflammatory [8], and cytotoxic agents [11]. Nowadays, sulfathiazole bearing heterocyclic moieties have been synthesized and explored for their biological activities with specific target of diseases. Sulfathiazole bearing five member heterocyclic compounds have been widely studied due to their interesting applications as bioactive molecules [12]. After literature review of these traditional sulfonamides, we are reporting here synthesis of modified new derivatives to achieve sustainability in this area.
Drug development is a process in which we should strive to get novel drugs with optimum utilization of resources. We need a direction through pharmacokinetics and dynamics so that a lead can be decided, and we would not end up with clueless and baseless laboratory wastage. e pharmacological parameters such as drug likeness, ADME, and toxicity are providing promising insights in identifying lead compounds [13,14]. In this process, molecular docking analysis and online ADMET predictions (SwissADME, Pro-Tox II and OSIRIS property explorer) are giving positive directions to researchers worldwide [15][16][17]. DFT analysis is helpful to optimize the geometry and identify the role of charge distribution to develop potential drug candidates [18][19][20]. DFT helps to get best binding mode during molecular docking studies as it minimizes the energy of the ligand and prepares it to get best fit within binding pocket of the enzyme. Synthesis and characterization of new sulfathiazole derivatives with detailed DFT study, as well as in vitro and in silico antibacterial and antioxidant analysis with pharmacological properties predictions such as drug likeness, ADME, and toxicity, are presented here for readers interest and benefit.

Materials and Methods
All solvents and chemicals were obtained commercially from fine chemicals PLC (Addis Ababa) and were used as received without further purification. Melting points were determined in an open capillary using digital melting point apparatus, expressed in oC. Reaction progress was checked on precoated TLC plates and spots were visualized using UV light at 254 nm. Silica gel (60-120 mesh, Merck grade) has been used for column chromatography. e column was subjected to gradient elution by increasing ethyl acetate in nhexane, and spots were visualized under UV lamp (254 nm). e synthesized compounds were characterized on the basis of physical and spectral analysis. e UV-Vis spectra of synthesized compounds were recorded on double-beam UV-Vis spectrophotometer using methanol as blank solvents for studying antioxidant activity. e 1 H and 13 C NMR spectra of the synthesized compounds were recorded on Bruker Avance 400 MHz NMR spectrophotometer using DMSO-d 6 as the solvent, and the values are expressed in δ ppm.  iazol-2-Amine (7). 4-(4′-Nitrophenyl) thiazol-2-amine (7) was synthesized using reported procedure developed by Abedi-Jazin et al. [21] (Scheme 1). Commercially available thiourea (3.04 g, 40 mmol), p-nitro acetophenone (3.3 g, 20 mmol), iodine (5.08 g, 20 mmol), and pyridine (2 drops) were mixed together and refluxed in ethanol (10 mL) at 100°C for 10 hr. e progress of the reaction was monitored using TLC in ethyl acetate/n-hexane (2 : 3) solvent system. After completion of the reaction, the mixture was cooled, extracted with diethyl ether to remove excess of acetophenone, and then washed with aqueous sodium thiosulfate to remove excess iodine and later with cold water. e crude product was dissolved in hot water and filtered to remove sulfonate, and the filtrate was basified with aqueous Na 2 CO 3 to yield the corresponding 4-(4′-nitro phenyl) thiazol-2-amine (7). e crude product was purified by recrystallization from ethanol and provided high yield (94%). e spectroscopic and analytical data of compound are as follows:

Antibacterial Activity.
e synthesized compounds were evaluated for their in vitro antibacterial activity against two Gram-negative (E. coli and P. aeruginosa) and two Grampositive bacteria (S. pyogenes and S. aureus). e bacterial cultures were inoculated into the nutrient broth (inoculation medium) and incubated overnight at 37°C. Inoculated medium was added aseptically to the nutrient medium and mixed thoroughly to get a uniform distribution. A solution of approximately 20 mL of sterile MHA was poured in sterile culture plates and allowed to attain room temperature. Sterile agar-disc diffusion previously soaked in a known concentration (50 mg/100 μL, 25 mg/100 μL, and 12.5 mg/ 100 μL) of synthesized compounds and standard drug sulfamethoxazole were prepared in DMSO using nutrient agar tubes and carefully placed at the center of the labelled seeded plate. e zones of growth inhibition around the disks were measured after 24 hours of incubation at 37°C. e inhibition zones were measured with a ruler and compared with the positive control disk (disk containing sulfamethoxazole) and expressed in millimeter [23].

Antioxidant Activities of Sulfathiazole Derivatives.
e free radical scavenging activities of the synthesized compound were measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. With this method, it is possible to determine the radical scavenging power of an antioxidant by measuring the decrease in the absorbance of DPPH at 517 nm. As a result of the color changing from purple to yellow, the absorbance was decreased when the DPPH radical is scavenged by an antioxidant through donation of hydrogen to form a stable DPPH molecule. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity [1].

Preparation of Ligands.
e 2D structures (.mol) of synthesized compounds (7, 11a-b) were drawn, and each individual structure was analyzed by using ChemDraw 16.0. Biochemistry Research International e selected molecules were treated quantum mechanically by applying DFT method using the Gaussian 09 program suite at the Becke-3-Lee-YangPar (B3LYP) level combined with the standard 6-31G (d,p) basis set. During the optimization procedure, all the parameters were set in order to obtain a stable structure with minimum energy. e global minimum energy of the title compound was determined from the structure optimization procedure. e 3D coordinates (.PDB) of each molecule were obtained through optimized structure.

Preparation of Macromolecules.
e crystal structure of receptor molecules S. aureus gyrase (PDB ID: 2XCT) and human myeloperoxidase (PDB ID: 1DNU) were downloaded from protein data bank. As per standard protocol and practice worldwide, protein preparation was done. Water molecules and cofactors were selected to eliminate. Previously attached ligands were detached, and protein was prepared by adding polar hydrogens using auto preparation of target protein file AutoDock 4.2.6 (MGL tools 1.5.6).

AutoDock Vina Analysis.
e graphical user interface program AutoDock 4.2.6 was used to set the grid box for docking simulations. We tried several different docking pockets and poses, and finally the grid was generated as per best results achieved. e docking algorithm provided with AutoDock Vina was used to search for the best docked conformation between ligand and protein. A maximum of nine conformers were generated for each ligand. e conformations with the most favorable (least) free binding energy were selected for analyzing the interactions between the target protein and ligands by Discovery Studio Visualizer and PyMOL.
Auto Dock Vina with standard protocol was used to dock the protein S. aureus gyrase (PDB ID: 2XCT) and human myeloperoxidase (PDB ID: 1DNU) and synthesized ligands (7, 11a-b) into the active site of proteins. e molecular docking studies were carried out using AutoDock Tools (ADT) [17], which is a free graphic user interface (GUI) for the AutoDock Vina program. e grid box was constructed using 20 × 20 × 20, pointing in x-, y-, and zdirections, respectively, with a grid point spacing of 0.375Å. e center grid box was of 62 × 30 × 62Å for 2XCT and of 65 × 40 × 65Å for 1DNU. Nine different conformations were generated for each ligand scored using AutoDock Vina functions and were ranked according to their binding energies. Binding pockets, H-bonds, and other hydrophobic and electrostatic interactions are shown by using different colours, sticks, ribbons, and lines.

In Silico Drug Likeness and Toxicity Predictions.
is prediction directs users in the direction of drug efficiency and provides insights that studied ligand has properties consistent with being an orally active drug or not. is prediction is based on an already established concept by Lipinski et al., called Lipinski's rule of five [15]. e chemical structure of the compounds (7, 11a-b) was converted to their canonical simplified molecular input line entry system (SMILE) and submitted to SwissADME tool to estimate in silico pharmacokinetic parameters. SwissADME predictor provides information on the number of hydrogen donors, hydrogen acceptors and rotatable bonds, and total polar surface area of a compound. e ligands were also subjected to Lipinski et al., screened using SwissADME and Pre-ADMET predictors. e organ toxicities and toxicological endpoints of the ligands and their LD 50 were predicted using Pro Tox II and OSIRIS Property Explorer [15,16]. e analyses of the compounds were compared with that of sulfathiazole and ascorbic acid standard drugs.

Quantum Computational Studies.
e DFT (density functional theory) analysis of synthesized compounds was performed using Gaussian 09 and visualized through Gauss view 6.0. e structural coordinates of the lead compounds were optimized using B3LYP/6-31 G (d,p) level basis set without any symmetrical constraints. e molecular electrostatic potential map and energies of the compounds were obtained from the optimized geometry. Koopman's approximation was used to estimate the HOMO-LUMO energy gap and related reactive parameters (electronegativity, chemical potential, hardness, softness, and electrophilicity) [24,25].

Statistical Data Analysis.
e antimicrobial analysis data generated by triplicate measurements were reported as mean ± standard deviation. GraphPad Prism version 5.00 for Windows was used to perform the analysis (GraphPad Software, San Diego, California, USA, https://www. graphpad.com). Groups were analyzed for significant differences using a linear model of variance analysis (ANOVA) test, with significance accepted for p < 0.05.
e plausible reaction mechanism of the compounds 11 (a-b) starts with nucleophilic substitution reaction of amino thiazole with benzene sulfonyl chloride/toluene sulfonyl chloride as depicted in Scheme 3.

Antibacterial Activity of Synthesized Compounds.
e in vitro antibacterial activities of synthesized compounds were done against two Gram-negative (E. coli and P. aeruginosa) and two Gram-positive bacterial strains (S. pyogenes and S. aureus) by disk diffusion assay (Table 1).
e results showed that all the tested compounds displayed potent to moderate antibacterial activity with inhibition zone of 6.00 ± 0.011 to 11.6 ± 0.283 mm (Figure 2). Compound 11a displayed potent inhibitory activity with inhibition zone of 11.6 ± 0.283 mm. Comparing with compound 11b, compound 11a showed highest inhibitory activities; however, the difference between the structures of the two compounds differs only by methyl group attached to the benzene sulfonamide. From these results, it can be assumed that antibacterial activities of synthesized compounds increase with the number of carbons attached to the benzene sulfonamide increases.

Antioxidant Activity of Synthesized Compounds.
DPPH is a simple method and is used to determine the radical scavenging power of an antioxidant by measuring the decrease in the absorbance of DPPH at 517 nm. In the DPPH scavenging assay, synthesized compounds were investigated for their free radical scavenging activities via their reaction with the stable DPPH radicals. e reduction of the DPPH was followed via the decrease in absorbance at 517 nm. Synthesized compounds significantly reduced the DPPH. e DPPH radical scavenging activities of synthesized compounds 7, 11a, and 11b were found to be 82.37%, 94.05%, and 78.85%, respectively, at 10 μg/ml ( Table 2) and ascorbic acid was found to be 97.57%. It was observed that the DPPH scavenging activity increased with increasing concentration of the samples in the assay. For the various concentrations, compound 11a exhibited slightly highest percent inhibition of the DPPH with IC 50 of 1.655 as compared to the other synthesized compounds. e positive control, ascorbic acid showed maximum scavenging effect at very high concentration with IC 50 of 1.526.

Molecular Docking Studies.
To understand the binding mode of the ligands, all the synthesized compounds were subjected to molecular docking studied against selected proteins, namely, S. aureus gyrase (PDB ID: 2XCT) and human myeloperoxidase (PDB ID: 1DNU) using AutoDock Vina [17].

Binding Mode of Analysis of Synthesized Compounds (7, 11a-B) Docked against S. aureus Gyrase (PDB ID: 2XCT).
Bacterial gyrase is paramount for bacterial survival and therefore necessary to disrupt as an antibacterial drug target [27]. erefore, in this study, the molecular docking analysis of the synthesized compounds was carried out to investigate their binding pattern with bacterial gyrase and the results were compared with standard antibacterial agent sulfathiazole (see Supplementary Data). e synthesized compounds (7, 11a-b) were found to have minimum binding energy ranging from −7.8 to-10.0 kcal/mol (Table 3), with the best result achieved using compound 11a (-9.7 kcal/ mol) and 11b (-10.0 kcal/mol) (Figures 3 and 4). Comparing to sulfathiazole (-7.4 kcal/mol), the synthesized compounds (7, 11a-b) have shown better binding affinity and similar residual interaction profile with amino acid residues. Hydrogen bonding interactions with various amino acids and bacterial DNA are also shown (Table 3). Based on the molecular docking analysis results, all the synthesized compounds have shown comparable residual interactions and docking scores with sulfathiazole. erefore, these compounds might have potential to be promising antibacterial agents. e binding affinity, H-bond, and residual interaction of all the synthesized compounds are summarized in Table 3. e in silico results are in good agreement with in vitro results.

Binding Mode of Analysis of Synthesized Compounds (7, 11a-B) Docked against Human Myeloperoxidase (PDB ID: 1DNU).
e molecular docking of the synthesized compounds (7, 11a-b) within the binding sites of human myeloperoxidase was analyzed, and the results were compared with standard antioxidant agent ascorbic acid and sulfathiazole (see Supplementary Data). e synthesized compounds (7, 11a-b) were found to have minimum binding energy ranging from −7.5 to -9.7 kcal/mol (Table 4). Comparing with ascorbic acid (-8.1 kcal/mol) and sulfathiazole (-6.9 kcal/mol), the synthesized compounds (7, 11a-b) have shown comparable and even better binding affinity and similar residual and DNA interaction profile with various amino acid residues. e in silico interaction results showed that all the synthesized compounds (7, 11ab) have comparable binding affinity with ascorbic acid; Biochemistry Research International among them, compounds 11a (−9.7 kcal/mol) and 11b (−9.6 kcal/mol) revealed good binding affinity (Figures 5 and  6). Based on the molecular docking analysis results, all the synthesized compounds have shown comparable residual interactions and comparable docking scores with ascorbic acid. Hence, these compounds might prove to be good antioxidant agents. e binding affinity, H-bond, and residual interaction of all the synthesized compounds are summarized in Table 4. e in silico results are in promising agreement with in vitro results.

In Silico Pharmacokinetics (Drug Likeness) and Toxicity
Analysis.
e drug likeness of the synthesized compounds (7, 11a-b) was characterized according to "Lipinski's rule of five." As per Lipinski's rule, the potential molecules should have the following physicochemical properties [28], such as (i) less than 5 hydrogen bond donors (HBDs), (ii) less than 10 hydrogen bond acceptors (HBAs), (iii) a molecular mass less than 500 Da, and (iv) log P not greater than 5 and (v) total polar surface area (TPSA) should not be > 140Å. e SwissADME computed results showed that all the synthesized compounds (7, 11a-b) in the present study are satisfying Lipinski's rule of five with zero violations (Table 5) [29]. Hence, all the synthesized compounds might be candidates for antioxidant and antibacterial studies. e in silico computed results of absorption, distribution, metabolism, and excretion (ADME) for synthesized compounds 7 and 11a-b reference drugs sulfathiazole and ascorbic acid are given in Tables 5 and 6.
As per toxicity class classification [15,16], none of the ligands has shown acute toxicity, and they were found The mean inhibition zone (mm) Figure 2: Mean inhibition zone of synthesized compounds in mm (mean ± SD) at 50 mg/mL. similar to standard drugs. e synthesized compound 7 has shown toxicity class classification 3, while compounds 11a and 11b showed much better toxicity class 5. e toxicological prediction gives results of endpoints, such as hepatotoxicity, carcinogenicity, mutagenicity, immunogenicity, and cytotoxicity. All the synthesized compounds were predicted to be nonimmunotoxic, nonirritant, and noncytotoxic. However, compound 7 has shown mutagenicity. Pro-Tox II and OSIRIS property explorer prediction analyses have shown in Table 7. Based on ADMET prediction analysis, none of the compounds have shown acute toxicity and so might be proven as good drug candidates.

Molecular Geometry.
e optimized structures of the synthesized compounds (7, 11a-b) along with force on nucleus, that is, 0.000, are shown in Supplementary Data.  e global minimum energy obtained by the DFT structure optimization procedure for the investigated compounds is summarized in Table 8. e bond lengths, Mulliken charges, molecular electrostatic potential surface, and 2D contour, HOMO-LUMO structures for all the ligands are shown in Supplementary Data. However, parameters for the best performing ligand 11a are shown in Figures 7 and 8.

Frontier Molecular Orbital Analysis.
e energy difference between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) is a parameter which provides excitation energy of a molecule, and it is an excellent indicator of electronic transition absorption in the molecular systems. ese molecular orbitals provide insight into the reactivity nature and the physical and structural properties of molecules. e positive and negative phase is represented in red and green color in the figures. e HOMO-LUMO energies and the energy gap for the investigated compounds are calculated using B3LYP/ 6-31G (d,p) method. Owing to the HOMO-LUMO orbital interaction, LP-LP, and LP-bond pair type interactions were observed to be predominant in the investigated compounds according to the molecular orbital theory. e calculated HOMO-LUMO energies, the energy gap, and dipole moment are shown in Table 8. e molecular orbital analysis for the investigated compounds based on their optimized geometry indicates that the Frontier molecular orbitals are mainly composed of p type-atomic orbitals. An electronic system with larger HOMO-LUMO gap should be less reactive than one with a smaller gap. Moreover, the HOMO-LUMO energy gap clearly explains the eventual charge transfer taking place within the molecule. e power of an electronegative atom in a compound to attract an electron towards was introduced by Pauling. e parameters such as hardness (ɳ), ionization potential (I), electronegativity (χ), chemical potential (μ), electron affinity (A), global softness (σ), and global electrophilicity (ω) are calculated. e ionization energy (IE) can be expressed through HOMO orbital energies, and electron affinity (EA) can be expressed through LUMO orbital energies. e hardness (ɳ) corresponds to the gap between HOMO and LUMO orbital energies. e hardness has been associated with the stability of the chemical system. All the calculated values of quantum chemical parameters of the investigated molecules using the B3LYP method with 6-31G (d,p) basis set are summarized in Table 8. From the results in

Mulliken Population Analysis.
e Mulliken population analysis of the title compounds was performed at DFT-B3LYP/ 6-31G (d,p) level to obtain the values of the atomic charges, and the results are shown in Supplementary Data. All calculated values indicate the extensive charge delocalization in the investigated molecules. e positive charges are localized over the hydrogen atoms.

Electric Charge Distribution and Electron Density.
It is basic chemistry principle that electrons and nuclei attract each other, while electrons repel themselves and the same is the case with nuclei. In the equilibrium geometry of a molecule, these electrostatic forces just balance. e fundamentally important Hellman-Feynman theorem [30] states that the force on a nucleus in a molecule is the sum of the Coulombic forces exerted by the other nuclei and by the electron density distribution ρ. On the basis of these mathematical algorithms and calculations, ESP surface and 2D contours are generated through gaussian software. Molecular electrostatic potential surface and 2D contour diagrams representing electronic charge distribution are shown in Supplementary Data. It is clearly indicated that all these compounds (7, 11a-b) have shown balanced charge distribution, which make them adhesive towards various biological enzymes.

Conclusion
Sulfathiazole derivatives were successfully synthesized with 72-81% yield through nucleophilic substitution reaction. e synthesized compounds were fully characterized using melting point and spectroscopic techniques ( 1 H and 13 C NMR). e in vitro antibacterial activities of synthesized compounds were evaluated against four bacterial strains E. coli, P. aeruginosa, S. pyogenes, and S. aureus with the best activity displayed by compound 11a against E. coli with an inhibition zone of 11.6 ± 0.283 and 11.1 ± 0.141 at 50 and 25 mg/mL, respectively. Antioxidant activity of synthesized compounds was examined. Out of the synthesized compounds, 11a showed better % radical scavenging activity. e synthesized compounds were evaluated for their in silico molecular docking analysis using S. aureus gyrase and human myeloperoxidase. e in silico molecular docking analysis has shown minimum binding energy ranging from -7.8 to -10.0 kcal/mol and -7.5 to -9.7 kcal/mol using S. aureus gyrase and human myeloperoxidase, respectively. Compound 11a showed very good binding score -9.7 kcal/ mol with both of the proteins and had perfect alignment with in vitro results. Compound 11b also showed promising  binding scores with both proteins. e results of in silico molecular docking study of the synthesized compounds have shown comparable residual interactions and better docking scores than sulfathiazole, and all the docking results are in good agreement with in vitro analysis. e drug likeness of the synthesized compound satisfies Lipinski's rule of five with zero violations. Hence, all the synthesized compounds might be candidates for further in vivo antibacterial and antioxidant studies.

Data Availability
e data used to support the findings of this study are included within the manuscript and also submitted as supporting information and, if needed more, can be asked to submit more by corresponding author.

Conflicts of Interest
e authors assert that there are no conflicts of interest.