Sigma Aldrich (USA) supplied all of the chemicals and solvents. The R_f values were calculated using a combination of ethanol and acetic acid (7:3). This was based on the solubility of the compounds. The Gallenkamp apparatus, Shimadzu 440 spectrometer, Varian Gemini 500/125 MHz spectrometer, GCMS/QP 1000 Ex mass spectrometer (70 eV), and VARIO El Elementer apparatus were used to obtain the melting point (°C), FTIR spectra (KBr, ν_max in cm^-1), ¹H-NMR (DMSO-d₆, 500 MHz, δ in ppm), ¹³C-NMR (DMSO-d₆, 125 MHz, δ in ppm), mass spectra (MS, m/z), and the elemental analysis (EA, provided as Calcd. (Found)), respectively.

Preparation of intermediates (1-3) and compounds (5a-5l)

The intermediates (1-3) used for the preparation of 5a-5l were prepared according to Figure S1 (Supplementary data). The preparation of 1 and 2 from 1-bromo-4-nitrobenzene is disclosed in the literature [9]. The preparation of 2-bromo-N-(4-morpholinophenyl)acetamide (3) from 2 is also provided in prior publications [7]. The compounds 5a-5l were prepared utilizing compound 3 and different aryl thioamides 4 (Figure S2) (Supplementary data). The structures of 5a-5l were established based on their spectral data (Supplementary data).

In vitro antimicrobial activity

It was done by utilizing the well-known serial plate dilution process against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Klebsiella pneumoniae employing nutrient agar medium, which was prepared according to the reported method [10]. The serial dilutions (200 to 6.25 μg/mL) of 5a-5l and ciprofloxacin were formulated in sterile DMSO. The DMSO deprived of drugs was utilized as a control. The plates were prepared with the agar medium, microorganisms were spread on them, cups were made and formulations of test/control/standard compounds were supplemented to the cups. The MIC (minimum inhibitory concentration) was ascertained (Table 1).

DNA gyrase inhibitory activity

The assay kit of E. coli DNA gyrase supercoiling was obtained from TopoGEN, Inc. (Cat. No. TG1003, Port Orange, FL). The instructions provided by the supplier were followed to perform the assay [11]. In short, the dilutions (0.1-50 μM) of 5a-5l and ciprofloxacin were formulated in DMSO along with the specified quantities of other ingredients (buffer, DTT, KCl, MgCl₂, spermidine, acetylated BSA, glycerol, ATP, album, and pBR322 substrate). The DMSO dilution (3 µL) of the desired compound was mixed with 2 U of E. coli DNA gyrase and kept at 37^oC for 30 min. The reaction was ended by adding 3X gel-loading buffer (10 ml). The resultant mixture (20 ml) was packed on agarose-TAE gel (1%) and run (3h, 60 V). The gel was blotted with ethidium bromide (0.5 mg/l in TAE) for a half-hour and then destained with water for 20 min. The fluorescent images were obtained at 300 nm employing a UV transilluminator imaging system. The fluorescence intensity of the supercoiled plasmid reaction product was quantitated utilizing ImagQuant software (Molecular Dynamics, Sunnyvale, CA, USA). The experiments were run in triplicate for each sample, and the IC₅₀ values were calculated by nonlinear regression analysis (Table 1).

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay

The MTT test was employed to assess the toxicity profile of 5a-5l concerning HepG2 cell (HCL) and Vero cell (VCL) lines [12,13]. The VCL (10⁴ cells/well) and HCL (5 x 10³ cells/well) were positioned in well plates and incubated (37^oC) for 24 h. The working formulations of the test and standard (200, 150, 100, and 50 µg/ml) were formulated in Dulbecco′s Modified Eagle′s Medium (DMEM). The blank formulation deprived of standard/test compounds was formulated. The working/standard/blank formulation was supplemented to wells encompassing VCL and HCL. The VCL (72 h) and HCL (24 h) well plates were incubated. The MTT component (50 µl, 2 mg/ml) was supplemented to well plates and incubated (4h). The sterile DMSO (50 µl) was supplemented to each well to liquefy formazan crystals. The OD (optical density) of the wells was quantified at 540 nm employing an Elisa reader. The %cell viability (test’s OD x 100 / blank’s OD), and %cell inhibition (100 – %cell viability) were determined. The curve fitting program was utilized to calculate the CC₅₀ (smallest concentration required for 50% cell killing) values (Table 1).

Molecular docking

It was accomplished by MOE software utilizing the chain A of DNA gyrase protein (PDB ID: 6F86) [14,15]. The selected chain was prepared for docking exercising MOE’s Quickprep functionality. The ligands (5a-5l and ciprofloxacin) were made and reserved (mdb files). The docking was executed by MOE’s default docking programming (10 poses for each compound). The DS (docking score, kcal/mol), and the RMSD (root mean square deviation) of the compounds are supplied in Table 2.

In silico toxicity prediction

It was performed by utilizing the ProTox-II web server [16,2]. The molefiles of 5a-5l and ciprofloxacin were uploaded to the software. The toxicity prediction button was pressed, and the toxicity data of 5a-5l and ciprofloxacin were recorded (Table 3).

In silico drug-likeliness and ADME predictions

These parameters were determined by the Swiss web server [17]. The molefiles of 5a-5l and ciprofloxacin were uploaded in the software, the run button was pressed, and the data were collected (Table 4).

Statistical analysis

It was performed utilizing SPSS software (version 20, Chicago, IL, USA), wherein p-value < 0.05 represents a statistically significant result.

Results

The compounds 5a-5l were prepared utilizing compound 3 and different aryl thioamides 4 (Figure S2) (Supplementary data). The antibacterial activity of 5a-5l was assessed against S. aureus, B. subtilis, E. coli, and K. pneumoniae (Table 1).

The docking of 5a-5l was executed to understand the reason for the excellent antibacterial activity of 5a-5l in comparison to ciprofloxacin (Table 2). The RMSD value of all ligands was < 1.5, whereas the DS ranged from -6.78 to -7.42 kcal/mol. An RMSD value < 2 and a higher negative DS indicates a strong binding of the molecule with the selected protein, and a strong inhibitory effect of the molecule, respectively [18]. The toxicity profile of 5a-5l was assessed by the ProTox-II webserver (Table 3). This web server provides the possible toxicity profile of a molecule based on the reported toxicity profile of similar compounds [15,16]. The LD₅₀ of the prepared compounds ranged from 681-900 mg/kg (class 4, 300 < LD₅₀ ≤ 2000), except 5j which had an LD₅₀ value of 222 mg/kg (class 3, 50 < LD50 ≤ 300) [2]. The SwissADME software was utilized to predict drug-likeliness and pharmacokinetic parameters of 5a-5l (Table 4) [17].

Figures & Tables

Discussion

It was surprisingly observed that the MIC values of 5a-5l were equal to the MIC values of ciprofloxacin (12.5 µg/ml) against the tested bacteria (Table 1). This observation reflected an equipotency of 5a-5l with ciprofloxacin. The IC₅₀ values of 5a-5l against DNA gyrase increased as 5h < 5g < 5f < 5e < 5l < 5b < 5k < 5i < 5d < ciprofloxacin < 5c < 5a < 5j. This demonstrates 5h, 5g, 5f, 5e, 5l, 5b, 5k, 5i, and 5d as more potent DNA gyrase inhibitors than ciprofloxacin.

This reflection also implies that the presence of 2-alkypyridin-4-yl provides potent DNA gyrase inhibitors, wherein the DNA gyrase inhibitory activity of compounds improves with the size of the alkyl group increases (5f < 5g < 5h). The unsubstituted pyridin-2-yl (5b) and pyridin-4-yl (5d) rings also provide potent DNA gyrase inhibitors. The presence of thiophene ring (5l) provides better inhibitors of DNA gyrase than pyrrole (5k) and pyrazine (5i) based compounds. The replacement of the pyridin-2-yl ring (5b) with pyridin-3-yl (5c) or pyrimidin-2-yl ring (5j) causes a decrease in activity. The MTT assay implied the non-cytotoxic nature of 5a-5l against HCL and VCL up to a concentration of 200 µg/ml.

The docking results indicate that 5a-5l and ciprofloxacin bind to the same active pocket of DNA gyrase. However, 5a-5l had a better binding affinity with the amino acids of the active site of DNA gyrase. This is evident from the DS of 5a-5l. This may be the reason that 5a-5l provided equal or better IC₅₀ values than ciprofloxacin against DNA gyrase. The 2D interaction of 6F86 with ciprofloxacin and three compounds (5f, 5g, and 5h) with least IC₅₀ values against DNA gyrase has been depicted in Figure S3, Figure S4, Figure S5, and Figure S6, respectively (Supplementary file). Some of the compounds displayed additional interaction with the residual amino acids, for example, 5a (Val 120), 5c (Val 120), 5d (Val 120), 5f (Val 120), and 5g (Met 95, Leu 132, Val 120). However, the presence of these additional interactions does not have any influence on the binding affinity of 5a, 5c, 5d, 5f, and 5g in the active site of DNA gyrase.

The toxicity data indicated no hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxic effect of 5a-5l. These results supported our MTT assay results, wherein 5a-5l displayed a non-toxic effect on HCl and VCl up to a concentration of 200 µg/ml. However, the author recommends in vivo toxicity studies to ascertain the non-toxic nature of 5a-5l.

The drug-likeliness determination is based on the physicochemical parameters of a molecule that would make it orally active [19]. The data of Table 4 indicate that 5a-5l possess all drug-likeliness properties, and also have a high gastrointestinal absorption. This is also evident from the bioavailability radar of 5f, 5g, and 5h (Figure S7, Supplementary data) that were obtained from SwissADME software. This observation suggests an orally active nature of 5a-5l. Most of the prepared compounds have been predicted as inhibitors of some metabolizing enzymes, unlike ciprofloxacin. Accordingly, these compounds might interact with the metabolism of other drugs, especially those metabolized by CYP3A4, CYP1A2, CYP2C9, and CYP2D6. However, for the clarity of such interaction, further studies need to be carried out.

Ciprofloxacin (Figure 1) is a well-known DNA gyrase inhibitor. In its chemical structure, the carbonyl group and the carboxylic acid group are at ortho-position. A similar position of these two groups is also present in the chemical structures of enoxacin, fleroxacin, lomefloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, levofloxacin, sparfloxacin, tosufloxacin, gatifloxacin, and moxifloxacin. These two groups bind with the bivalent metal, for example, magnesium, that is needed for the activity of DNA gyrase [20]. These groups also bind with food/drugs containing the bivalent metal, for example, milk and calcium gluconate. This interaction causes a decrease in the gastrointestinal absorption of ciprofloxacin and its structurally related DNA gyrase inhibitors [20-22]. The chemical structures of 5a-5l (Figure S2, (Supplementary data) neither possess the carbonyl group nor the carboxylic acid group. Therefore, the author believes that 5a-5l must not pose an interaction with food/drugs containing bivalent metals like calcium, unlike ciprofloxacin.

In conclusion, this study provides morpholine-based thiazole derivatives (5a-5l) as DNA gyrase inhibitors possessing promising safety and efficacy profiles. The compounds demonstrated equal MIC values along with ciprofloxacin (12.5 µg/ml) against S. aureus, B. subtilis, E. coli, and K. pneumoniae. The 5h, 5g, 5f, 5e, 5l, 5b, 5k, 5i, and 5d displayed potent DNA gyrase inhibitory activity than ciprofloxacin. This observation was supported by the molecular docking data of 5a-5l. The MTT assay and ProTox-II webserver data indicated the non-toxic nature of 5a-5l. The SwissADME software further indicated the drug-likeliness properties and high gastrointestinal absorption of 5a-5l. The 5h, 5g, 5f, 5e, 5l, 5b, 5k, 5i, and 5d have been identified as potent DNA gyrase inhibitors with promising safety and efficacy profiles. However, detailed toxicity studies are also recommended to establish the non-toxic nature of 5a-5l.

Supplementary Data

Supplementary data is available on demand.

Author Contributions

Mohd Imran, Abida, and Basheeruddin Asdaq conceptualized, supervised, and edited the article. All other members performed in the literature search, and experiments (silico studies, preparation, and in vitro studies), and participated in first manuscript draft writing.

Conflict of Interest

The author declare that there is no conflict of interest regarding the publication of this paper.

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