Designing, synthesis and characterization of 2-aminothiazole-4-carboxylate Schiff bases; antimicrobial evaluation against multidrug resistant strains and molecular docking

Background 2-Aminothiazoles are significant class of organic medicinal compounds utilized as starting material for the synthesis of diverse range of heterocyclic analogues with promising therapeutic roles as antibacterial, antifungal, anti-HIV, antioxidant, antitumor, anthelmintic, anti-inflammatory & analgesic agents. Experimental Eight compounds 1a, 2a–2g were synthesized and characterized by FTIR and NMR (1H and 13C). Evaluation of antibacterial potential against multi-drug resistant clinical isolates was performed and minimum inhibitory concentration (MIC) values were determined. Antifungal activity was also performed. Protein–ligand interactions of compounds with target enzyme were evaluated through docking studies. Results Resistance profiling of bacterical clinical isolates (MDRs) depicted that some standard drugs used were not active against these MDRs while our synthesized compounds showed good MIC values. Among all the synthesized compounds, 2a and 2b showed significant antibacterial potential towards gram-positive Staphylococcus epidermidis and gram-negative Pseudomonas aeruginosa at MIC 250 µg/mL and 375 µg/mL respectively. Likewise, compound 2d and 2g exhibited inhibitory potential against gram-positive Staphylococcus aureus and gram-negative Escherichia coli at MIC values of 250 and 375 µg/mL respectively. Compound 2b showed maximum antifungal potential against Candida glabrata (ATCC 62934) with a zone of inhibition 21.0 mm as compared to the reference drug nystatin which showed lesser antifungal potential with a zone of inhibition of 19.1 mm. Candida albicans (ATCC 60387) showed maximum sensitivity to compound 2a with a zone of inhibition 20.0 mm. Its antifungal activity is more in comparison to reference drug nystatin with exhibited the zone of inhibition of 19.3 mm. Designed compounds were docked with the target enzyme UDP-N-acetylmuramate/l-alanine ligase. The compound 2b showed highest binding affinity (− 7.6 kcal/mol). Conclusions The synthesized compounds showed moderate to significant antibacterial and antifungal potential. It is clear from the binding affinities that compounds having hydroxyl group substituted on benzene ring possess strong binding affinity as compared to other analogues. These designed compounds could be considered to act as antagonists against target UDP-N-acetylmuramate/l-alanine ligase.

The registered drugs Mirabegron (Anticholinergic agent) and Cefdinir (Antibacterial agent) belong to aminothiazole analogues [14,15]. Since it is well known that 2-aminothiazoles are significant class of organic medicinal compounds utilized as starting material for the synthesis of diverse range of heterocyclic analogues. Therefore, the design and synthesis of ethyl-2-aminothiazole-4-carboxylate Schiff bases was targeted in this study.
Designing of evident drugs can be achieved through virtual screening and molecular docking strategies. Likewise, interpretation of probable drug specificity with target enzyme or protein can be assessed [16]. In the present study the selected uridine diphosphate-N-acetylmuramate/l-alanine ligase was selected as antimicrobial target for docking [17]. It is the bacterial enzyme that catalyzes the reaction of peptidoglycan synthesis by first amino acid addition to peptidoglycan sugar moiety. Peptidoglycan is the key element for bacterial cell wall [18]. By targeting this ligase enzyme, the bacterial cell integrity can be dismantled ultimately resulting in bacterial cell death.
Analogues of ethyl -2-aminothiazole-4-carboxylate were designed by altering the amino group as illustrated in Fig. 1. Four aldehydes and two ketones were used to design the compounds form 2a-2h. Molecular properties were determined using ChemSketch Tool (https ://www. acdla bs.com/resou rces/freew are/). Chemo-informatics, ADME properties and drug likeliness were determined by following Lipinski rule while associated parameters of designed ligands were predicted by Molinspiration (https ://www.molin spira tion.com/) and pkCSM online tool [19,20]. The toxicity profile was obtained using TOXTREE and pkCSM online tool [21].

General procedure for the synthesis of ethyl 2-aminothiazole-4-carboxylate derivatives
Ethyl 2-aminothiazole-4-carboxylate (0.05 mol) and aldehyde/ketone (0.05 mol) in absolute ethanol (30 mL) were dissolved and few drops of glacial acetic acid were added. Reaction mixture was stirred and refluxed for 12 h. Progress of reaction was monitored by TLC. After cooling the excess solvent was evaporated by using rotary evaporator. The residue was dissolved in ethyl acetate, crystal growth was observed after few days [27,28]. Physical data of the synthesized compounds is given in Table 1.

Resistance profiling
Analysis of resistance pattern of pathogenic microbe (resistant, intermediate, susceptible), antibiotic sensitivity assay was performed. Fresh cultures of bacterial strains were made on nutrient agar and incubated for 24 h at 37 °C. The following day isolated colonies from bacterial cultures were picked and dissolved in 1 mL of PBS (autoclaved normal saline) and inoculum turbidity was then confirmed with 0.5% McFarland standard [29]. Dried Muller Hinton (MH) agar plates were used for resistance profiling. With the help of sterilized syringe antibiotic discs of known concentration were placed on MH agar plates namely Cefepime, Ciprofloxacin, Imipenem, Cefoxitin, Ampicillin, Aztreonam, Tetracycline, Ceftazidime, Minocyclin, Gentamycin, Co-trimoxazole, Colistin, Clindamycin, Vancomycin, Doxycycline, Erythromycin and Chloramphenicol. Later these plates were incubated for 24 h at 37 °C. Afterwards, resistance pattern of particular strain was studied based upon the zone of inhibition measurement following the CLSI 2017 guideline [30,31].

Quantification by nanophotometer
For quantification purpose Multiskan ™ GO Microplate Spectrophotometer was used. The standard stock solution of 2 mg/mL was prepared for each active compound by adding 0.02 g in 10 mL distilled water. From this stock solution various working dilutions ranging from 40 to 2000 μg/mL were prepared in triplicate. Wave scan of the dilutions was performed between 200-900 nm and λ max value was obtained from absorbance spectra. Afterwards, particular active compound λ max was used to measure optical density of dilutions and standard curve was plotted.

Antibacterial activity and minimum inhibitory concentration
Antimicrobial activity was assessed by broth dilution method. Clinical isolates were obtained from Microbiology and Public Health Laboratory culture collection, COMSATS University, Islamabad. Staphylococcus epidermidis (MDR) and Staphylococcus aureus (MDR) were the gram-positive and Escherichia coli (MDR) and Pseudomonas aeruginosa (MDR) were the gram-negative strains selected for this study.
Broth dilution method was performed for antibacterial activity of synthesized compounds against selected clinical microbes. MIC was calculated by broth dilution method, stock solution of 2 mg/mL for each active compound with DMSO 20% as solvent was prepared which was further consumed for preparing working dilutions ranged from 40 to 1000 μg/mL. The selected model bacterial strains were subjected to these active compounds dilutions separately and OD values were obtained at 595 nm [32].

Antifungal assay
Agar well diffusion method was utilized for antifungal assay using nystatin as positive control while DMSO (20%) was employed as a negative control. Sabouraud dextrose agar (SDA) and nutrient broth were prepared and sterilized by autoclaving at 121 °C for 15 min. Sterile agar plates were prepared by pouring the sterile agar in disposable sterile plates and incubated for 24 h for sterility check at 28 °C after congealing. The fungal strains Candida albicans (ATCC 60387) and Candida glabrata (ATCC 62934) were refreshed by inoculation in nutrient broth followed by 24 h incubation at 28 °C. Lawn of both fungal strains were made on nutrient agar plates and wells were made by employing sterile borer (6 mm). 50 μL of the synthesized compounds, nystatin and DMSO (20%) were poured through micropipette into individual well. The concentration of synthesized compounds, positives and negative controls used were 10 mg/mL and 750 μg/ mL respectively. Plates were sealed by using paraffin film (Parafilm M) and incubated for 3-7 days at 28 °C to be examined for zone of inhibition that reflects the antifungal potential [33].
Purity of all the synthesized compounds was ensured by recrystallization in appropriate solvents and checked by thin layer chromatography plates using ethyl acetate: petroleum ether (3:1) solvent system. Single spot yielded by each synthesized compound was obtained.
Synthesis of compound 1a was confirmed through FTIR and 1 H NMR spectral data. Strong peak of C=O (ester) at 1690 cm −1 , NH 2 (amine) 3300-3150 cm −1 and C-H 3000 cm −1 stretch was observed in FTIR spectrum. Strong peak of C=O (ester) 1690 cm −1 , C=N (imine) 1617 cm −1 and C-H stretch 2985 cm −1 were observed in FTIR spectrum of compound 2f. Singlets of CH 3 -C=N imine at 3.38 ppm and thiazole proton at 7.64 ppm were observed. Moreover, quartet and triplet of CH 2 and CH 3 were observed at 4.25 ppm and 1.26 ppm respectively. While in compound 2g peak of C=O (ester) was observed at 1685 cm −1 . C=N (imine) 1615 cm −1 and C-H stretch 2990 cm −1 were also perceived in FTIR spectrum of compound 2g. 1 H NMR spectral data showed singlets of CH 3 -C=N imine at 3.34 ppm and thiazole proton at 7.45 ppm were observed. Moreover, quartet and triplet of CH 2 and CH 3 were observed at 4.21 ppm and 1.23 ppm respectively.

Antibacterial assay of synthesized compounds
All experiments were conducted in the boundaries of ethical principles and there was no involvement of human or animal samples in this project.
Selected clinical gram-negative and gram-positive bacteria were subjected to antibiotic disks diffusion by Kirby Bauer assay and their pattern of resistance were determined. The resistance pattern of synthesized derivatives is given in Table 2.

Quantification
Spectrophotometric analysis was utilized for the quantification of compounds 2a, 2b, 2d and 2g. After performing wave scan the values obtained for λ max were 2a at 320 nm, 2b at 420 nm, 2d at 310 nm and 2g at 325 nm. λ max values were used to measure dilutions optical density (OD). These OD values were used to plot the standard curve and coefficient of determination (R 2 ) value was calculated. The curve was plotted between OD values on y-axis while the concentration values on horizontal x-axis. From regression analysis, the R 2 values obtained for compound 2a, 2b, 2d and 2g were 0.976, 0.999, 0.951 and 0.997 respectively. Values closer to 1 indicated the fitness of the data against the regression line. The graphs plotted are given in Fig. 2.

Minimum inhibitory concentration (MIC)
All the synthesized compounds manifested mild to moderate antibacterial potential. The lowest concentration of the drug exhibiting bacteriostatic action is stated to be the Minimum inhibitory concentration (MIC) of the drug. The MIC of synthesized derivatives is given in Table 3. Compound 2a and 2b showed quite good MIC of 250 µg/mL against gram-positive Staphylococcus epidermidis (MDR). Likewise compound 2d and 2g exhibited MIC of 250 µg/mL against gram-positive bacteria S. aureus (MDR). Compounds 2d and 2g exhibited MIC of 375 µg/mL against E. coli (gram-negative) (MDR). While compounds 2a and 2b showed MIC of 375 µg/mL against P. aeruginosa (gram-negative) (MDR).

Insilico studies
Best compounds are selected computationally by analyzing them for their chemo-informatics and ADMET properties as given in Tables 5 and 6. According to literature, established qualifying range for log P value is (− 0.4 to 5.6). All compounds log p value lie within the limits. Molar refractivity should lie between (40 to 130) while molecular weight limits are (160 to 480). Moreover, the required number of atoms of compounds should lie between (20 to 70). Number of rotatable bonds should be less than 10. All compounds have 4 rotatable bonds except the compounds 2e and 2h that are having 5 rotatable bonds. All the compounds are fulfilling the set criteria for above parameters. Moreover, the drug likeliness following the Lipinski rule is also fulfilled. That's why these compounds can be used as drug candidates. Toxicity profile was determined by using TOXTREE and pkCSM online tools. All the compounds employed were having a therapeutically safe profile excluding compound 2h, which holds genotoxic potential. Therefore, compound 2h was not utilized in the docking step. Table 7 contains the toxicity profile of designed compounds.
Our designed compounds were docked with the target enzyme UDP-N-acetylmuramate/l-alanine ligase and binding affinities were determined. The binding pocket of target enzyme was identified through Dogsitescorer.
respectively. While the pocket possesses quite good drug score of 0.77. Figure 3 illustrates the druggable pocket of UDP-N-acetylmuramate/l-alanine ligase target enzyme.
Results of docking of compounds are displayed ( Table 8) in terms of free binding energy of ligand also stated as binding affinity (kcal/mol), root mean square deviation (RMSD) upper bound along with values of RMSD lower bound of all the docked ligands as well. Strength of binding interactions between ligand and receptor is termed as affinity. Calculation of RMSD values is based on the best binding pose and it employs movable heavy atoms only. Among the compounds 2a, 2b, 2d and 2g; 2b shown the strongest binding affinity of − 7.6 kcal/mol against UDP-N-acetylmuramate/lalanine ligase. While compounds 2a, 2d and 2g exhibited binding affinity of − 6.8, − 7.3, − 7.1 and − 6.9 kcal/ mol respectively. It is inferred from the binding affinities that the compounds having hydroxyl gyoup substituted on benzene ring possess strong binding affinity as compared to others. These designed compounds could be considered as antagonist lead molecules for target UDP-N-acetylmuramate/l-alanine ligase. The enzyme-ligand binding interactions of compounds 2b, 2c, 2d, and 2g with target UDP-N-acetylmuramate/l-alanine ligase are shown in Figs. 4, 5, 6, 7 respectively.

Conclusion
The aim of the study is to design and synthesize novel Schiff base derivatives of ethyl-2-aminothiazole-4-carboxylate. Characterization was done by FTIR, 1 H NMR and 13 C NMR spectral data and quantification is done through Multiskan ™ GO Microplate Spectrophotometer. Synthesized derivatives are screened for their antibacterial potential against Multi drug resistant (MDR) clinical isolates. Moreover, synthesized derivatives also exhibited good antifungal activity against ATCC fungal strains. It is anticipated that these synthesized compounds are promising potent antibacterial therapeutic agents. In future it is aimed to develop a pharmacophore model from these