Design, synthesis and docking studies of novel thiazole derivatives incorporating pyridine moiety and assessment as antimicrobial agents

A novel series of substituted 4,6-dimethyl-2-oxo-1-(thiazol-2-ylamino)-1,2-dihydropyridine-3-carbonitrile derivatives 6, 9, 13, 15, and 17 was synthesized in a good to excellent yield from the reaction of 1-(3-cyano-4,6-dimethyl-2-oxopyridin-1(2H)-yl)thiourea with 2-oxo-N'-arylpropanehydrazonoyl chloride, chloroacetone, α-bromoketones, ethyl chloroacetate, and 2,3-dichloroquinoxaline, respectively. The potential DNA gyrase inhibitory activity was examined using in silico molecular docking simulation. The novel thiazoles exhibit dock score values between − 6.4 and − 9.2 kcal/mol and they were screened for their antimicrobial activities. Compound 13a shown good antibacterial activities with MIC ranged from 93.7–46.9 μg/mL, in addition, it shown good antifungal activities with MIC ranged from 7.8 and 5.8 μg/mL.

The structure of the compounds 6a-c and 9 was confirmed. The IR spectrum of compound 6b, as a representative example, exhibited the lack of NH 2 and C=S peak at 3261, 3219, and 1269 cm −1 . The 1 H-NMR spectrum of 6b showed new singlet signals at δ 2.37, 2.49 ppm assigned to two methyl, additionally, two doublet signals at δ 7.20 and 7.55 ppm attributable to 4-methylbenzene. Its 13 C-NMR spectrum revealed the lack of C=S signal at 185.5 ppm and appearance 17 carbon signals. Moreover, the mass spectra of 6b revealed [M + -15] ion peak at m/z 383. This clearly indicates the thioamide moiety was involved in cyclization reaction with hydrazonyl chlorides 3a-c to give 1,3-thiazole derivatives 6a-c.
Molecular docking studies and antimicrobial activity. The innovative arylthioureas were docked to the active site of DNA gyrase enzyme using Autodock 4. We studied the hypothetical binding approach of 9 derivatives at the clorobiocin binding site via molecular docking. Molecular docking was accomplished for arylthiourea derivatives to comprehend their possible intermolecular interactions with the receptor. Clorobiocin is a based coumarin antibiotics, which prohibits the cell division of bacteria by inhibition of the DNA gyrase enzyme [51][52][53][54] . Table 1 summarizes the binding depiction of the arylthioureas with DNA gyrase. The poses obtained from the docking procedure was selected due to their binding energy (~ − 6 -− 9 kcal/mol). Figures 3 and 4 showed 3D schematic interactions of compounds 13a and 9 into the chlorobiocin binding site and showed that the   The docking results exhibited that some compounds (9, 13a and 13b) can produce a strong hydrophobic interaction and hydrogen bonds with Arg136 and Asn46 in the binding site. It is exciting that more complex stabilization could result from the hydrogen bonds between these compounds and Arg136 via cyano group in the pyridone ring (Figs. 3 and 4). Although these interactions were also observed for some other derivatives, but we think that the hydrophobic interaction is responsible for the activity variations.
Docked compounds also stabilize the DNA gyrase via hydrophobic interactions with Ala47, Glu50, Val71, Asp73, Arg76, Gly77, Ile78, Pro79, Met91, Val43, Thr165, and Val167. Compounds 9 and 13a were pointedly embedded into the hydrophobic part of the pocket. All compounds showed that the hydrophobic pocket of the inhibitor pocket was occupied by pyridine, phenyl or substituted phenyl.
The docking method approved in this study was validated by redocking of chlorobiocin to the DNA gyrase protein. The residues Asp73, Asn46, and Arg136 are vital in making hydrogen bonds and are very important for the biological activity 55 and in our study some compounds also displayed a strong hydrogen bond with Asn46. The highest dock score for our derivatives was − 9.2 and − 8.8 kcal/mol for compounds 13a and 9, respectively. The remainder molecules exhibited a docking scores ranging from − 8.5 to − 6.4 kcal/mol. Thus, the binding model stated here, proposes that arylthiourea derivatives act as DNA gyrase inhibitors and display some key structural points to be used in further optimization.
The biological assay (Tables 2 and 3), some compounds exhibited a strong activity against both the Grampositive and Gram-negative bacterial. Gained results confirmed that compounds 9 had high activities against E. coli and P. aeruginosa with MIC 93.7 μg/mL. Also, compound 13a showed the superlative activity against E. coli, P. aeruginosa, S. aureus, and B. subtilis with MIC 93.7, 62.5, 46.9, and 62.5 μg/mL, respectively. Also compound 13a has shown the highest activity with MIC 7.8 and 5.8 μg/mL against C. albicans and Aspergillus flavus, respectively.
The observed results displayed that compound 13a has better biological results than other arylthioureas. Existence of electron-withdrawing group (bromine) at p-position of the phenyl ring could be accountable for good activities due to its size and inductive effect.

Experiment
General. Melting points were recorded on digital Gallen-Kamp MFB-595 apparatus and are uncorrected. IR spectra were recorded on Schimadzu FTIR 440 spectrometer using KBr pellets. Mass spectra were performed at 70 eV on an MS-50 Kratos (A.E.I.) spectrometer provided with a data system. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were recorded on a Bruker model Ultra Shield NMR spectrometer using CDCl 3 or DMSO-d 6 with TMS as an internal standard. Chemical shifts are reported as δ ppm units. The monitoring of the progress of reactions and homogeneity of the products was carried out using thin layer chromatography (TLC).   Table 2. In vitro antimicrobial activity of the synthesized compounds a,b . a Antimicrobial activity expressed as inhibition diameter zones in millimeters (mm) of synthesized compounds against the pathological strains based on well diffusion assay. b The experiment was carried out in triplicate and the average zone of inhibition was calculated. c NA No activity.   CH 3 ), 100, 101.4, 109, 116, 154.9, 156, 159.4, 181.7

Method 2
Synthesis of compound 6b from compound 9 57 . To a stirred solution of compound 9 (0.5206 g, 2 mmol) in ethanol (30 mL) sodium acetate trihydrate (0.26 g, 2 mmol) was added. After stirring for 15 min, the mixture was chilled at 0 °C and treated with a cold solution of p-toluidine (0.2 g, 2 mmol) in 6 M hydrochloric acid (1.5 mL) with sodium nitrite solution (0.14 g, 2 mmol) in water (3 mL). The addition of the diazonium salt was stirred for an additional 2 h at 0-5 °C and then left for 8 h in a refrigerator (4 °C). The resulting solid was collected by filtration, washed thoroughly with water and dried. The crude product was crystallized from ethanol. Molecular docking studies. The structure of our target enzyme (PDB code 1KZN) was chosen as the protein model for this study 58 . The heteroatoms were taken away from the protein file and the resulting structure was introduced to AutoDock. The binding image of 9 new arylthioureas with DNA gyrase were assessed in the same way of binding of clorobiocin.
Docking was executed by the default parameters of molecular docking AutoDock 4.2 and employed empirical free energy function 59 . In the docking procedure, compounds were supposed to be flexible and the docking software was allowed to rotate all rotatable bonds of them to obtain the best conformer within the active site of the enzyme. Clorobiocin was redocked to the binding site to evaluate our method.
The grid box was positioned with the coordinates x = 19.172, y = 30.465, z = 34.697 for DNA gyrase (PDB code 1KZN). Grid box sizes were 60 × 60 × 60 with a 0.5 Å grid points space. Grid maps were calculated by Autogrid4. A lamarckian genetic algorithm within the Autodock was used to estimate the diverse ligand conformers. Conformations were clustered by the root mean square deviation tolerance of 2.0 Å and were ranked according to the binding free energy 59 . Discovery Studio 2020 Visualizer was used to explore the hydrophobic and hydrogen bonding interactions of the compound with DNA gyrase. www.nature.com/scientificreports/ Antimicrobial evaluation. The agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts. Similar to the procedure used in disk-diffusion method, the agar plate surface is inoculated by spreading a volume of the microbial inoculum over the entire agar surface then, a hole with a diameter of 6 to 8 mm is punched aseptically with a sterile corkborerora tip A volume (20-100 mL) of the antimicrobial agent or extract solution at desired concentration is introduced into the well and agar plates are then incubated under suitable conditions depending upon the microorganism. The antimicrobial agent diffusion the agar medium and inhibits the growth of the microbial strain 60 . www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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