New 1,3-diphenyl-1H-pyrazol-5-ols as anti-methicillin resistant Staphylococcus aureus agents: Synthesis, antimicrobial evaluation and in silico studies

In the present work, two hybrid series of pyrazole-clubbed pyrimidine and pyrazole-clubbed thiazole compounds 3–21 from 4-acetyl-1,3-diphenyl-1H-pyrazole-5(4H)-ole 1 were synthesized as novel antimicrobial agents. Their chemical structures were thoroughly elucidated in terms of spectral analyses such as IR, 1H NMR, 13C NMR and mass spectra. The compounds were in vitro evaluated for their antimicrobial efficiency against various standard pathogen strains, gram -ive bacteria (Pseudomonas aeruginosa, Klebsiella pneumonia), gram + ive bacteria (MRSA, Bacillus subtilis), and Unicellular fungi (Candida albicans) microorganisms. The ZOI results exhibited that most of the tested molecules exhibited inhibition potency from moderate to high. Where compounds 7, 8, 12, 13 and 19 represented the highest inhibition potency against most of the tested pathogenic microbes comparing with the standard drugs. In addition, the MIC results showed that the most potent molecules 7, 8, 12, 13 and 19 showed inhibition effect against most of the tested microbes at low concentration. Moreover, the docking approach of the newly synthesized compounds against DNA gyrase enzyme was performed to go deeper into their molecular mechanism of antimicrobial efficacy. Further, computational investigations to calculate the pharmacokinetics parameters of the compounds were performed. Among them 7, 8, 12, 13 and 19 are the most potent compounds revealed the highest inhibition efficacy against most of the tested pathogenic microbes comparing with the standard drugs.


Introduction
Fleming was the first who admonish about the risk of the global antibiotic resistance [1][2][3].The antibiotic resistance was first identified in Shigella, Salmonella and E. Coli [4][5][6].Owing to the antibiotic misuse, where these antibiotics are available and used without prescriptions, the bacteria acquired antibiotic resistance against most of the commercially available antibiotics [4,5,7,8].Consequently, resistance of the antibiotics is considered as a global public health concern by the world health organization (WHO) [9].Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a serious clinical and financial burden to people all over the world.MRSA is turning into a fatal sickness for humans because it is easily transmitted, causes severe skin infections, and resists most recognized antibiotics, including vancomycin.Consequently, identify potential antimicrobial drug candidates to overcome MRSA isolates is urgently required [10].
Heterocyclic compounds incorporating pyrazole moiety constitute an essential class of biologically active compounds that are gaining an attention in medicinal bioorganic chemistry [11][12][13][14].Many clinically used drugs contain pyrazole moiety, as shown in Fig. 1.The most important class of pyrazole-containing drugs is tyrosine kinase inhibitors like ruxolitinib (Jakafi®) which is used in myelofibrosis, and polycythemia vera, crizotinib (Xalkori®) which is indicated in treatment of non-small cell lung cancer, ibrutinib (Imbruvica®) which is indicated in chronic lymphocytic leukemia and finally axitinib (Inlyta®) and pazopanib (Votrient®) which are indicated in advanced renal cell carcinoma [15].Vicinal diaryl azoles were utilized as scaffolds for selective cyclooxygenase-2 (COX-2) inhibition [16].One of these azoles is a pyrazole which was found in celecoxib (Celebrex®) which is still clinically used till now.Metamizole (Novalgin®) is a sulfonic acid-containing pyrazole derivative which is still used as antipyretic till now despite its several adverse effects.Sildenafil (Viagra®) is a pyrazolopyrimidine phosphodiesterase 5 (PDE5) inhibitor which is used in the treatment of Scheme 1. Synthesis of compounds 3, 5, 7 and 8.
Pyridine was incorporated in plenty of reported antimicrobial derivatives [17].Several pyrazole-pyridine hybrids were reported to have markable antimicrobial activity especially against MRSA [18], as declared in Fig. 1.Thiazolidine was fused with a β-lactam ring to form a penam ring which is the main pharmacophoric element of penicillin antibiotics as well as β-lactamase inhibitors like sulbactam and tazobactam [19].Moreover, aminothiazole moiety was incorporated in 3rd and 4th generation cephalosporines like cefotaxime, ceftriaxone and cefepime to improve their activity against gram -ive bacteria.The 5th generation cephalosporine like ceftaroline with thiazole ring is characterized by its antibacterial activity against MRSA [19].Additionally, molecular hybridization of pyrazole ring with thiazole afforded several hybrids with remarkable antibacterial and antifungal activity [20], as represented in Fig. 1.Thiosemicarbazone and its isostere hydrazinecarboximidamide were reported to be involved in several antimicrobial derivatives especially with pyrazole ring that have a remarkable activity against MRSA [21], as represented in Fig. 1.
DNA gyrase is considered as a crucial target for design and development of effective antibacterial inhibitors due to its ability to induce negative supercoiling of DNA or relieve positive supercoiling [20].Gyrase is present in prokaryotes and specific eukaryotes, albeit with variations in structure and sequence, resulting in different affinities for diverse derivatives [22][23][24].Furthermore, it is an extensively researched objective that is essential for bacterial DNA replication [25].Consequently, the justification for utilizing DNA gyrase is mainly substantiated by reported pyrazole-containing compounds, which have strong affinity for this protein.
The molecular docking approach is a technique used to discover structures for the active sites of proteins, in addition to identify the potential mechanism of action [26][27][28].
Viewing the importance of heterocyclic compounds incorporating pyrazole ring in medicinal chemistry field and to highlight the scope of compounds containing pyrazole, pyridine and/or thiazole moiety, herein, synthesis of two hybrid series of pyrazole-clubbed pyrimidine and pyrazole-clubbed thiazole derivatives 3-21 from compound 1 is described.The antimicrobial potency of the newly prepared compounds was estimated against the various standard pathogen strains, gram -ive bacteria (P.aeruginosa, K. pneumonia), gram + ive bacteria (MRSA, B. subtilis), and fungi (C.albicans) microorganisms.Further, in silico docking approaches [25,[29][30][31] of the molecules were performed to investigate their binding interactions with DNA gyrase enzyme.
The thiazolidinone 16 was produced via reaction of 13 with ethyl-2-chloroacetate and/or chloroacetic. 1H NMR spectrum represented the existence of a singlet peak at ™ 1.25 ppm referred to -CH 3 group, multiple peaks at ™ 7.28-7.86ppm for Ar-H, along with a singlet peak at ™ 5.02 ppm referred to -CH 2 group, a peak of NH proton at ™ 6.06 ppm, and hump one at ™ 11.80 ppm corresponding to -OH group.However, the MS of 16 showed the parent peak at 393 (M + +2), assignable to C 20 H 17 N 5 O 2 S.

In vitro biological activity
The antimicrobial potency of the tested molecules was estimated against the various standard pathogen strains, gram -ive bacteria (P.aeruginosa, K. pneumonia), gram + ive bacteria (MRSA, B. subtilis), and fungi (C.albicans) microorganisms.The inhibition zone diameter was determined using agar-well diffusion and the results were stated below in (Figs. 2 and 3).The results showed that most of the tested molecules exhibited moderate to high inhibition efficacy.Where 7, 8, 12, 13 and 19 are the most potent compounds revealed the highest inhibition efficacy comparing to the standard drugs.
The MIC of the most potent molecules 7, 8, 12, 13 and 19 was determined and mentioned in Table 1.The obtained results represented that the most potent molecules exhibited inhibition effect against most of the tested microbes at low concentration.In which compounds 12 and 13 showed strong effect against C. albicans at concentration 5 μg/mL.Compound 8 showed strong efficacy against C. albicans at concentration 10 μg/mL.Compound 8, 12, 13 and 19 exhibited a strong antimicrobial potency against B. subtilis at low concentration 5 μg/mL.Otherwise, compound 12 showed strong efficacy against MRSA at concentration 10 μg/mL.

In silico approaches 2.3.1. Molecular docking techniques
The docking approach is utilized discover structures for the active sites of proteins, in addition to identify the potential mechanism of action [40,41].In the present work, to go deeper into the suitable binding pose and molecular mechanism of antimicrobial activity of compounds, molecular docking studies [42] were conducted using MOE software.Molecular docking studies revealed good interactions of compounds with DNA gyrase enzyme [43], as shown in Figs.Finally, N-phenylpyrazole moiety of compound 19 (score = − 12.01 kcal/mol) formed four π-H interactions (4.33, 3.82, 4.37 and 3.85 Å) with DT16 (Fig. 12).Phenyl ring at thiazole C4 of compound 19 formed π-H interactions (3.89 Å) with DT14.Additionally, phenyldiazenyl mioety at thaizole C5 formed two π-π interactions (3.84 and 3.83 Å) with DA15.Out of all synthesized derivatives, compound 19 showed the highest binding score which aligns with its good binding strength against MRSA (MIC = 0.068 μg/mL), B. subtilis (MIC = 0.008 μg/mL) and K. pneumonia (MIC = 0.034 μg/mL).

Pharmacokinetics prediction
Aside from efficacy and safety profiles, several drug candidates have failed to reach clinic due to their poor physicochemical characters and pharmacokinetics [26,27].SwissADME website was utilized to estimate several physicochemical and pharmacokinetic properties of molecules (Supplementary Material).None of the compounds was predicted to be P-gp substrate.Compounds were predicted to have a little inhibitory activity on different CYP450 isozymes like 1A2, 2C19, 2C9, 2D6 and 3A4.Consequently, compounds were predicted to have few drug-drug interactions.Compounds 1, 3, 5, 10, 13, 15, 16 and 19 showed high GI absorption while other compounds 7, 8, 9, 11, 12 and 21 showed low GI absorption.The BOILED-Egg is a sturdy model that precisely predicts both GI absorption and blood brain barrier (BBB) permeability by calculating both the WLOGP (lipophilicity) and TPSA (polarity) (Supplementary Material).Compounds 1, 3, 5, 10, 13 and 16 were predicted to have a high GI absorption while compounds 9, 11, 12, 15 and 21 were predicted to have a low GI absorption.Except compound 1, all compounds were predicted not to pass BBB, indicating their good CNS safety profile.
There are 6 physicochemical characters that were considered in "bioavailability radar" viz, solubility, polarity, lipophilicity, saturation, flexibility, and size, which construct together a hexagon shape (Supplementary Material).The inner pink colored hexagon indicates the optimal values for acceptable bioavailability.that enhance their flexibility.To conclude, we can say that compounds 1, 3, 4, 10, 13, 15, 16 and 21 are predicted to have acceptable bioavailability.

Chemistry
Kofler Block instrument was utilized to determine the melting points of the prepared compounds.The FTIR 5300 spectrometer (ν, cm − 1 ) was used to record IR spectra.In addition, 1 H and 13 C NMR spectra were performed by a Varian Gemini spectrometer (400 and 100 MHz, respectively) in DMSO-d 6 as solvents.Tetramethylsilane (TMS), was used as an internal reference, is used to express the chemical changes in parts per million (ppm).1000 EX mass spectrometer at 70 eV.Using n-hexane and EtOAc, thin layer chromatography (TLC) on aluminum sheets was used to determine the purity of the produced compounds.The elemental analyses were carried out at Microanalytical Research Center, Faculty of Science, Cairo University, Egypt.

2-Amino-4,6-bis(5-hydroxy-1,3-diphenyl-1H-pyrazol-4-yl)benzonitrile (12)
Method A: In the presence of EtONa (30 mL), malononitrile (0.01 mol) was added to 9 (0.01 mol).For 24 h, the solution was refluxed.Once it had stopped, it was treated with ice-cold water then acidified with HCl.The solid formed was collected and recrystallized from ethanol to furnish (12; 66 %).Method B: compounds 1 (0.01 mol) and 10 (0.01 mol) were mixed together in (30 mL) of EtONa.For 24 h, the solution was heated under reflux.After completion the reaction, a cold diluted HCl was added to the reaction mixture, and the solid precipitated was filtered off, and recrystallized from ethanol to produce (12; (13) Thiosemicarbazide (0.01 mol) and 1 (0.01 mol) were refluxed for 7 h in (20 mL) absolute ethanol with 2 drops of HCl.After completion the reaction, the solid precipitated was collected by filtration and recrystallized from ethanol to yield (13; 75 %) as yellowish-white crystals; m.p.170-172   (15) Method A: In dioxane (30 mL) containing 3 drops of TEA, a suspension of 13 (0.01 mol) and ethyl 2-chloro-2-(2-(4-methoxyphenyl) hydrazono)acetate 14 (0.01 mol) was refluxed for 24 h.After allowing the solution to stop, a cold diluted HCl was added to the mixture.The solid formed was filtered off, dried, then recrystallized from ethanol to afford (15; 75 %).Method B: Aryldiazonium chloride 17 was added dropwise to a cold solution of 16 (0.01 mol) in ethanol (20 mL) containing excess of sodium acetate (2 gm).After completion the addition, the reaction mixture was allowed to stirrer for further 1 h in ice bath, then left overnight.The solid formed was collected by filtration, and recrystallized from ethanol to yield (15; 82 %) as yellowish brown crystals, m.p.120-122

Antimicrobial efficacy of the tested compounds
The ability of the molecules to prevent the microbial growth was investigated against the standard pathogen strains, gram -ive bacteria (P.aeruginosa, K. pneumonia), gram + ive bacteria (S. aureus, B. subtilis), and Unicellular fungi (C.albicans).Pre-activation of pathogens were performed by inoculating in the Nutrient broth medium for 24 h at 37 • C for bacterial strains, while fungal pathogen was inoculating in Potato Dextrose Broth (PDB) medium for 48 h at 28 • C under shaking condition.Screening of the tested material with different concentrations was preliminary occur at a constant concentration of the tested compounds using turbidometry method for each microbial pathogen except for multicellular fungi, which was evaluated by the Colony Forming Unite (CFU).In addition, the evaluation of tested compounds to inhibition the microbial proliferation was assessed using agar well diffusion method in terms of the inhibition zone diameter (mm) [44,45].

Determination of minimum inhibition concentration (MIC)
The MIC of highly potent molecules were performed to estimate the lowest concentration that inhibit the visible microbial growth after 24 h applying microdilution assay technique according to CLSI [46,47].In the experiment, different concentrations of the tested molecules were investigated in comparison to the classical antimicrobial agents.Briefly, the tested pathogens were cultivated in Mueller Hinton Broth (MHB) at 37 • C for 24 h, then the growth was diluted with sterilized bi-distilled water corresponding to 2 × 10 7 Colony Forming Unit (CFU)/mL, and the prepared cultures were diluted 10-folds in MHB for used as inoculum.The selected molecules were added into 100 μL aliquots into MHB, which were sequentially dispensed into the microdilution (96-well microtiter plate) in order to determine a known concentrations ranging from 5 to 200 μg/ml.A known inoculum of the bacterial cells was then incorporated with 100 μL to each well in triplicate trails.The positive control using three antibacterial agents was adjusted with the same concentrations of the tested samples [24,48].The MHB mixed with DMSO without and with compounds were also served as the control samples.

Molecular docking assessment
Modeling simulations that included docking study of the synthesized compounds were achieved.At first, the native ligand, moxifloxacin, was redocked with the active region of target protein (PDB ID: 5BS8) to validate our docking methodology (RMSD = 0.9812) [49].After that a library of 2D structures of the prepared derivatives and moxifloxacin were sketched using ChemDraw Professional 16.0, then converted to mol format.The energies of compounds and moxifloxacin were minimized and organized.The 3D structure of the target protein was prepared by removing solvent molecules and bound substances (ligands and cofactors).Docking process was conducted by default setting of MOE software [40].SwissADME website [50] was utilized to estimate several physicochemical and pharmacokinetic properties of molecules.

Conclusion
Two hybrid series of pyrazole-clubbed pyrimidine and pyrazole-clubbed thiazole compounds were prepared and in vitro screened for their antimicrobial activities against various standard pathogen strains.Moreover, the docking approach was achieved for understanding the antimicrobial efficacy in terms of binding affinity and intermolecular interactions with DNA gyrase enzyme.The findings revealed that compounds 7, 8, 12, 13 and 19 are considered as hopeful antimicrobial agents and could be the lead compounds for potential drug candidates.

Fig. 2 .
Fig.2.Antimicrobial activities of the prepared molecules using agar-well diffusion.

Fig. 3 .
Fig. 3. Two-way ANOVA showed a high significance between different samples and type of microbe used in the antimicrobial test.(pvalue <0.0001).

Table 1
Minimum inhibitory concentration (MIC) of the potent prepared pyrazole derivatives.