Antibacterial Evaluation, In Silico Characters and Molecular Docking of Schiff Bases Derived from 5-aminopyrazoles

A series of Schiff bases 14–25 were designed and synthesized for evaluation of their antibacterial properties against multi-drug resistant bacteria (MDRB). The antibacterial activities of Schiff bases 14–25 showed that most of the synthesized compounds displayed a significant antibacterial activity. Assessment of in silico ADMET properties (absorption, distribution, metabolism, excretion and toxicity) of Schiff bases illustrates that all derivatives showed agreement to the Lipinski’s rule of five. Further enzymatic assay aided by molecular docking study demonstrated that compound 18 is a potent inhibitor of staphylococcus aureus DNA gyrase and dihydrofolate reductase kinases. This study could be valuable in the discovery of new potent antimicrobial agents.


Introduction
Antibiotics, anti-microbial drugs, and anti-infectious agents are used for treating infectious with micro-organism diseases and able to kill or inhibit the growth of microbes by inhibition of cell membranes synthesis, protein synthesis, nucleic acid synthesis, or cytoplasmic membranes. Recently, the resistance of microbes to antibiotics can be observed and classified into internal resistance and acquired resistance. Inactivation of drugs by bacterial enzymes or the drug cannot bind are the reasons which explained the biochemical mechanisms of internal and acquired resistances. Therefore, there is an urgent need for production of new antimicrobial drugs or develop the used drugs to oppose the mutation of the microbes to solve the resistance.

Chemistry
5-Amino-1H-pyrazoles 12a-c were prepared via the sequence reaction of N-substituted cyanoacetamides 8 with 4-methoxyisothicyanate (9), methyl iodide and then with hydrazine hydrate in ethanol refluxing. A series of Schiff bases 14-25 were synthesized by the condensation of 5-aminopyrazoles 12a-c with aromatic aldehydes 13a-d and the chemical structures wer confirmed via spectral data (Scheme 1 and Table 1).

Chemistry
5-Amino-1H-pyrazoles 12a-c were prepared via the sequence reaction of N-substituted cyanoacetamides 8 with 4-methoxyisothicyanate (9), methyl iodide and then with hydrazine hydrate in ethanol refluxing. A series of Schiff bases 14-25 were synthesized by the condensation of 5-aminopyrazoles 12a-c with aromatic aldehydes 13a-d and the chemical structures wer confirmed via spectral data (Scheme 1 and Table 1). Ph

Antibacterial Evaluation
In vitro antibacterial activities against multi-drug resistant bacteria (MDRB) of Schiff bases 14-25 were performed at botany and microbiology department, Faculty of Science, Al-Azhar University, Cairo, Egypt. The antibacterial potential of 14-25 were investigated towards the multi-drug resistant bacteria (MDRB). The results were summarized as the diameter of the inhibition zones in mm [40] and minimal inhibitory concentration (MIC, µg/mL) [41] values in Table 2.

Antibacterial Evaluation
In vitro antibacterial activities against multi-drug resistant bacteria (MDRB) of Schiff bases 14-25 were performed at botany and microbiology department, Faculty of Science, Al-Azhar University, Cairo, Egypt. The antibacterial potential of 14-25 were investigated towards the multi-drug resistant bacteria (MDRB). The results were summarized as the diameter of the inhibition zones in mm [40] and minimal inhibitory concentration (MIC, µg/mL) [41] values in Table 2.

In Silico ADMET Properties of Schiff Bases 14-25
The physical properties and the ADMET parameters (absorption, distribution, metabolism, excretion and toxicity) of Schiff bases 14-25 were computed using the freely accessible web server Swiss ADME (http://swissadme.ch/index.php#undefined). The results of in silico ADMET properties of Schiff bases 14-25 are listed in Table 3. The

Structure-Activity Relationship (SAR)
From the results of antibacterial activities of Schiff bases 14-25 against multi-drug resistant bacteria, it was found that, in case of Ar = Ph, 4-CH 3 -C 6 H 4 or 4-Cl-C 6 H 4 , the order of antibacterial activity Ar 2 = Ph > 4-CH 3 -C 6 H 4 and Ar 2 = 4-Cl-C 6 H 4 > 4-F-C 6 H 4 was observed upon screening of Schiff bases 14-25 against the screening organisms ( Figure 3).

In Silico ADMET Properties of Schiff Bases 14-25
The physical properties and the ADMET parameters (absorption, distribution, metabolism, excretion and toxicity) of Schiff bases 14-25 were computed using the freely accessible web server Swiss ADME (http://swissadme.ch/index.php#undefined). The results of in silico ADMET properties of Schiff bases 14-25 are listed in Table 3.
The molecular weight (MW), the number of hydrogen bond acceptors (nHBA), donors (nHBD), the number of rotatable bonds (nRB) and the topological polar surface area (TPSA) for all the Schiff bases were in accordance with the Lipinski's rule of five. The lipophilicity property (expressed as MLogP

In Silico ADMET Properties of Schiff Bases 14-25
The physical properties and the ADMET parameters (absorption, distribution, metabolism, excretion and toxicity) of Schiff bases 14-25 were computed using the freely accessible web server Swiss ADME (http://swissadme.ch/index.php#undefined). The results of in silico ADMET properties of Schiff bases 14-25 are listed in Table 3.
The molecular weight (MW), the number of hydrogen bond acceptors (nHBA), donors (nHBD), the number of rotatable bonds (nRB) and the topological polar surface area (TPSA) for all the Schiff bases were in accordance with the Lipinski's rule of five. The lipophilicity property (expressed as MLogP ≤ 4.15) was in the range for all the Schiff bases excluding 20, 21, 23, 24 and 25. The highly lipophilic character (MLogP > 4.15) of the compounds 20, 21, 23, 24 and 25 may be because of the presence of chloro or fluoro atoms in their structures which may make difficult their transport through the blood strain [42].

In Vitro Kinase Assessment
In an effort to study the preliminary mechanism of the compound 18 with potent antibacterial activity, an enzyme inhibitory assay was performed towards staphylococcus aureus DNA gyrase, topoisomerase IV and dihydrofolate reductase enzymes. The obtained results were presented as IC 50 and provided in Table 4 using suitable positive controls, Ciprofloxacin and Methotrexate. From Table 4, it was observed that compound 18 demonstrated a nearly equipotent inhibitory activity towards DNA gyrase and weak activity against topoisomerase IV in comparison with the reference Ciprofloxacin (IC 50 = 1.68 ± 0.10, 74.55 ± 1.20, 1.51 ± 0.18 and 24.14 ± 1.01 µM, respectively). Moreover, compound 18 revealed two folds increase in the suppression effect towards dihydrofolate reductase comparing with Methotrexate (IC 50 = 0.08 ± 1.15 and 0.14 ± 1.07 µM, respectively).

Molecular Docking Study
Molecular docking studies concerning the in vitro kinase assessment were performed to understand the interactions of compound 18 with Staphylococcus aureus DNA gyrase and Dihydrofolate reductase enzymes. The binding modes of compounds 18 were investigated through using Molecular Operating Environment (MOE ® ) 2008.10 [43]. The X-ray crystal structures of Staphylococcus aureus DNA gyrase (PDB code: 2XCT) [44] and dihydrofolate reductase (PDB code: 1DLS) [45] were downloaded from the Protein Data Bank. In the present study, the proposed docking algorithms were initially validated by self-docking of the co-crystallized ligands Ciprofloxacin and Methotrexate to each of the aforementioned targets and exhibited root mean square deviation (RMSD) values of 0.86 and 0.92 Å, respectively. Subsequently, docking procedures have been achieved for compound 18 and the corresponding 2D and 3D representations of the binding modes are illustrated in Figures 4 and 5.
As shown in Figure 4, compound 18 linked tightly with amino acid residues of Staphylococcus aureus DNA gyrase. There were arene-arene interactions established between the centroids of DG9 and 4-methoxyphenyl, and arene-cation interactions between the centroid of benzylidene at p-5 of pyrazole moiety and Arg458. Moreover, the oxygen of 4-methoxy group supported the binding with two hydrogen bonds with the sidechain of Ser1084 (distance: 2.36 and 3.27 Å). As shown in Figure 4, compound 18 linked tightly with amino acid residues of Staphylococcus aureus DNA gyrase. There were arene-arene interactions established between the centroids of DG9 and 4-methoxyphenyl, and arene-cation interactions between the centroid of benzylidene at p-5 of pyrazole moiety and Arg458. Moreover, the oxygen of 4-methoxy group supported the binding with two hydrogen bonds with the sidechain of Ser1084 (distance: 2.36 and 3.27 Å). Considering the binding interaction of compound 18 with dihydrofolate reductase illustrated in Figure 5, it was noticed that N1 and N2 of pyrazole scaffold participated by two hydrogen bonds with the sidechain and the backbone of Ser59 (distance: 2.62 and 2.34 Å, respectively). Additionally, the tolyl moiety formed arene-arene interaction with the centroid of Ph34 passing through Ph31. Considering the binding interaction of compound 18 with dihydrofolate reductase illustrated in Figure 5, it was noticed that N1 and N2 of pyrazole scaffold participated by two hydrogen bonds with the sidechain and the backbone of Ser59 (distance: 2.62 and 2.34 Å, respectively). Additionally, the tolyl moiety formed arene-arene interaction with the centroid of Ph34 passing through Ph31.
The chemical structures of Schiff bases 14-25 was confirmed via spectral data [47].
The chemical structures of Schiff bases 14-25 was confirmed via spectral data [47].

Test Microorganisms
The synthesized compounds, Schiff bases 14-25, were in vitro evaluation of their antibacterial properties against multi-drug resistant bacteria (MDRB). Examples of Gram-positive bacteria are Staphylococcus aureus (MRSA, Sa), Staphylococcus epidermis (Sp), and Enterococcus faecalis (Ef). Examples of Gram-negative bacteria are Acinetobacter baumannii (Ab), Enterobacter cloaca (Ecl), and Escherichia coli (Ec). All the tested strain was identified by Vitek ® 2 system. The multi-drug resistant to antibiotics such as Ampicillin, Cephalexin, Colisin, Ipemenem, and Meropenem was verified.

Antibacterial Activity
In vitro antibacterial activities were performed at botany and microbiology department, Faculty of Science, Al-Azhar University, Cairo, Egypt. The antibacterial potential of Schiff bases 14-25 were investigated towards multi-drug resistant bacteria (MDRB) and expressed as the diameter of the inhibition zones according to the agar plate diffusion method [40].

Minimum Inhibitory Concentration (MIC) of the Active Compounds
The minimal inhibitory concentration (MIC) of the most potent Schiff bases was determined by the conventional paper disk diffusion method [41].

In Vitro Kinase Assessment
The in vitro enzyme inhibition determination for compound 18 was carried out in the confirmatory diagnostic unit, Vacsera, Egypt. The evaluation performed profiling of compound 18 against Staphylococcus aureus DNA gyrase, topoisomerase IV, and dihydrofolate reductase enzymes using Ciprofloxacin and Methotrexate as reference drugs according to the previously reported method [45,48].

Conclusions
In this work, a series of Schiff bases 14-25 were synthesized by the condensation of 5-aminopyrazoles 12a-c with aromatic aldehydes 13a-d, with high yields for evaluation of their in vitro antibacterial activities against multi-drug resistant bacteria (MDRB). In general, most of Schiff bases 14-25 displayed better antibacterial activity. In addition, a positive result of kinase inhibition was implicated by molecular docking study against Staphylococcus aureus DNA gyrase and dihydrofolate reductase enzymes. Furthermore, drug-likeness data revealed that the studied compounds fulfill Lipinski's rule requirements and have good drug score values. These preliminary results of Schiff bases against multi-drug resistant bacteria (MDRB) could provide an exceptional model that may lead to the discovery of new antibiotics by derivatization or modification.