Synthesis, Molecular Docking, MEP and SAR Analysis, ADME-Tox Predictions, and Antimicrobial Evaluation of Novel Mono- and Tetra-Alkylated Pyrazole and Triazole Ligands

Newly synthesized compounds of N-alkylated heterocyclic compounds were prepared by condensation of amine with alcohol which undergoes a reaction of SN2. These newly synthesized derivatives were characterized by spectral analysis. The objective is to prepare new potent nontoxic antimicrobial agents which are easy to synthesize and could be scaled up in pharmaceutical industries. Thirteen new heterocyclic compounds containing a pyrazole moiety were synthesized with good yields (29.79 to 99.6%) and were characterized by FTIR, 1H NMR, 13C NMR, and CG-MS techniques. The compounds were divided into two series—monoalkylated compounds (1–11) and tetra-alkylated compounds (12 and 13)—and then evaluated for their in vitro antifungal and antibacterial activities against several fungal and bacterial strains. None of the monoalkylated compounds had antibacterial or antifungal activity. However, the two tetra-alkylated pyrazole ligands displayed strong antibacterial potential. Moreover, compound 12 was more potent against all tested bacterial strains than compound 13. Interestingly, compounds 12 and 13 acted as weak antifungal agents against Saccharomyces cerevisiae. ADME-Tox studies suggested that compounds 12 and 13 exhibit better toxicity profiles than the commercial antibiotic streptomycin. MEP studies suggested that compounds 12 and 13 have the same charge locations but differ in their values which are due to the condensed geometry of compound 13 that make it more polarizable than compound 12. Of particular interest, these different MEPs were evident in ligand protein docking, suggesting that compound 12 has better affinity with MGL enzyme than compound 13. All these findings suggested that these novel compounds represent promising antibacterial lead compounds.

In context of fighting against these diseases, nitrogenous compounds have big interest either as antibacterial and/or antifungal agents. Particularly, pyrazole, triazole, benzotriazole, thiazole, imidazole, pyridine, and pyrimidine are very important moieties for the preparation of multiple interesting ligands [17][18][19][20].
In objective to look for nontoxic small molecules as new antimicrobial agents, mono-and tetra-N-alkylated heterocyclic derivatives based on pyrazole cores were prepared, characterized, and then evaluated for their in vitro antibacterial and antifungal potential. Additionally, molecular electrostatic potential (MEP) as well as molecular docking investigations was performed on the most active compounds to rationalize the antibacterial results obtained.

Experimental
All the chemicals were of analytical grades (Sigma-Aldrich, purity >99%), and the melting points were measured on Koffler bank, and FTIR analysis was performed using the FTIR-8400S spectrometer using KBr pellets. 1 H and 13 C NMR spectra were recorded on Bruker Avance 300, 400, and 500 MHz using TMS as internal standard in deuterated solvents such as CDCl 3 , CD 3 OD, DMSO-d 6 , and CD 2 Cl 2 .
Compounds 1, 2, and 6-9 were recrystallized from diethyl ether, compounds 3-5, 12, and 13 were recrystallized from DMSO/water (1 : 10), while compounds 10 and 11 were purified using a DCM/water (3 : 1) mixture. e structures of all the target compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) were established on the basis of FTIR, 1 H NMR, 13    e antibacterial effect was evaluated using the broth macrodilution method with phenol red indicator [23] against two Gram-negative bacterial strains, Escherichia coli and Citrobacter freundii, and two Gram-positive bacterial strains, Staphylococcus aureus and Listeria monocytogenes. e bacterial isolate was cultivated overnight in liquid Luria-Bertani medium (LB) at 37°C under aeration. After that, a suspension containing 106 CFU/mL of bacterial cells was prepared. en, 250 μL of this bacterial suspension was used to inoculate test tubes containing phenol red medium and the compound to be tested. After 24 hours of incubation at 37°C, bacterial growth was determined by a change of the colour of phenol red indicator from red to yellow. In the presence of antibacterial activity, the phenol red indicator R 1 R 1 = R 2 = R 3 = R 4 = H; R 5 = CH 3 ; X = CH R 1 = R 3 = R 4 = H; R 2 = Br; R 5 = CH 3 ; X = CH R 1 = R 5 = CH 3 ; = R 2 = R 3 = R 4 = H; X = CH R 1 = R 3 = R 5 = R 4 = H; R 2 = Br; X = N R 1 = R 3 = R 5 = R 4 = H; R 2 = Br; X = CH R 2 R 3 Journal of Chemistry remains red. While in the absence of antibacterial activity, the colour of the culture becomes yellow following the acidification of the medium due to bacterial growth. e compounds have been tested at 500 µM, and all experiments were repeated three times for each drug.

Determination of the Minimum Inhibitory Concentration (MIC).
Cultures were performed in the presence of phenol red indicator using different concentrations of the active compound. Bacterial growth was determined by visual observation of the red colour indicator as described above.
We defined the MIC as the lowest drug concentration that inhibits bacterial growth after incubation at 37°C for 24 hours. All experiments were carried out in triplicate, and means were calculated.

Determination of the Minimum Bactericidal Concentration (MBC).
e MBC is the lowest drug concentration that kills 99% of bacteria after 24 h of incubation. MBCs were determined as described in [24,25].

Determination of the Antifungal Activity.
Liquid cell culture method was used for the evaluation of the antifungal activity of the studied compounds against two fungal strains: Candida glabrata and Saccharomyces cerevisiae.
e absorbance of the cells at 600 nm (OD 600 ) was measured to monitor the growth rate of fungal cells in liquid culture using a V-1200 spectrophotometer (Shanghai Mapada Instruments Co., Ltd.). With this spectrophotometer, the reading is proportional to the cell number for an OD 600 <2.5. erefore, when culture was very overgrown, the OD 600 measurement was carried out after dilution of the culture, and then the dilution factor was used to calculate the OD in the original culture. e methodology used for the determination of the antifungal activity of a compound is as follows: firstly, cells were grown overnight in yeast peptone dextrose medium (YPD) at 30°C in a shaking incubator. en, cells were diluted from the overnight culture to an OD 600 of ∼0.08 and allowed to grow until the OD 600 reached ∼0.14, to ensure that the cells were in the logarithmic phase. e compound was then added, and the growth rate of fungal cells was determined every two hours by measuring OD 600 . All compounds were diluted in DMSO, and all assays, including the "no drug" control, contained 1% DMSO. Optical density (OD 600 ) was measured every 2 h to follow cell growth. All experiments were carried out in triplicate, and the curves shown are an average of three experiments. Error bars are presented, and where they are not visible, they are smaller than the data symbols. All experiments were repeated three times, and means were calculated.

DFT Study: MEP Surfaces.
e chemical structures of the studied molecules were sketched using GaussView 6.0 and then optimized by the DFT/B3LYP method with 6-31G (d,p) basis sets using Gaussian 09W software [26].

Molecular Docking Study.
e biological target selected for this study is the crystal structure of L-methionine c-lyase from Citrobacter freundii in complex with norleucine (PDB: 3JWB). Actually, methionine c-lyase (MGL) is an essential enzyme involved in the catalytic reaction of c-elimination and c-substitution of L-methionine and its derivatives, which play important roles in several biological processes. e presence of this enzyme in many bacteria including pathogenic ones and its absence in human (generally in mammals) makes it a potential target to design novel antibacterial drugs. Figure 2 displays the 3D structure of this enzyme and its active pocket. e docking study was carried out utilizing Molecular Operating Environment (MOE) 2015.10) software.

Antimicrobial Activity.
e antibacterial potential of the synthesized mono-and tetra-alkylated pyrazole and triazole derivatives shown in Figure 1 was evaluated against four bacterial strains (two Gram-positive, Staphylococcus aureus and Listeria monocytogenes, and two Gram-negative Escherichia coli and Citrobacter freundii). All the compounds were tested at 500 µM as described in Section 2.2.1. As shown in Table 1, all the screened monodentate ligands displayed no antimicrobial effect against all used strains. is result could be explained by the fact that these compounds lack pharmacophore sites which can act by inhibiting bacterial growth. e compounds have been tested at 500 µM. All experiments were repeated three times, and the result obtained for each time is presented. − − − indicates no inhibition of bacterial growth, and +++ indicates inhibition of bacterial growth.
Together, these results suggest that tetra-alkylated ligands 12 and 13 act as weak antifungal agents, but strong antibacterial agents, and might represent promising lead compounds for the development of specific antibacterial drugs. erefore, further investigation is required to better understand their antibacterial activity and their mode of action. us, theoretical investigations such as SAR, ADME-Tox, and molecular docking were performed.

SAR Analysis of Compounds 12 and 13.
e structure activity relationship (SAR) analysis of the tetra-alkylated pyrazole and triazole derivatives 12 and 13 revealed that the antibacterial activity of these compounds depends essentially on the nature of the starting materials (the diamine) which is benzene-1,3-diamine for compound 12 and pyridine-2,3- + + + + + + + + + + + + 13 + + + + + + + + + + + + Journal of Chemistry diamine for compound 13. Investigation of (R) substituents, showed that the presence of methyl makes compounds 12 and 13 active against Listeria monocytogenes, and also, the presence of the pyridine ring instead of benzene one and the positions of the amines on the starting material increase the MBC/MIC ratio from 1 to 1.6 ( Table 2). In the case of E. coli, MBC/MIC is equal to 1 for the two compounds either they have different structure resulting in their inhibition mechanism of action on the bacteria. Otherwise, compound 13 shows a higher MBC/MIC ratio of 1.25 against Staphylococcus aureus which is probably due to the presence of the nitrogen of the pyridine rings in addition to the effect of the methyl substituents on the pyrazole rings and the difference of the diamine starting material.

DFT Study.
MEP surfaces: molecular electrostatic potential (MEP) gives information about the reactive regions of nucleophilic and electrophilic attacks on a molecular system. It is often generated by mapping the electrostatic potential on the isoelectron density surface of the molecule, which gives us the opportunity to show the distribution of the electronic charge over all the structure. Currently, this technique becomes a useful tool to understand the molecule environment and the hydrogen bond interactions, as well as the biological recognition processes. Figure 6 shows the MEP surfaces generated by the DFT optimized geometries for compounds 12 and 13 that displayed strong antibacterial activity.
In this study, the MEP surfaces of compounds 12 and 13 showed negative charges located on sp 2 -nitrogen regions of the pyrazole ring with values of − 1.1690 and − 1.4598 eV, respectively. Moreover, compound 12 showed another negative charge around the phenyl ring with a value of − 0.7042 eV, while compound 13 has another one around the sp 3 -nitrogen with value of − 1.1488 eV. In contrast, the positive charges were located on the methyl substituents on the pyrazole rings with a value of 2.1137 and 0.4522 eV, respectively. erefore, both compounds have the same charge locations but difference in their values which is due to the condensed geometry of compound 13 that makes it more polarizable than compound 12.

ADME-Tox Predictions.
e in silico predictions of the physicochemical properties such as ADME (absorption, distribution, metabolism, and excretion) and toxicity risks (Tox) are important in the drug discovery process [27]. It can predict the nature of a studied compound within the human body. erefore, in the present work, swissADME web tool is used to check the ADME-Tox properties of the most active compounds 12 and 13 as well as streptomycin. e results are collected in Tables 3 and 4.
Overall, excepting the molecular weight which is found to be higher than 500 daltons, the two compounds 12 and 13 displayed good ADME profiles and were found to follow the Lipinski rule of five for drug likeness in terms of their logP (<5), H-donor (<5), H-acceptor (<10), nrotb (<10), and TPSA (<140Å 2 ). ese compounds were found to display better ADME profiles compared to streptomycin which showed more violations for the Lipinski rules.
On the other hand, the toxicity prediction reveals the nonmutagenic, nontumorigenic, and nonirritant proprieties and none risk on the reproductivity of compounds 12 and 13, whereas it showed a high irritant effect of streptomycin.

Molecular Docking Study.
e two compounds 12 and 13 were docked with the active site of L-methionine c-lyase enzyme (MGL) to generate their binding mode. After docking, it was found that both of the selected ligands can bind to the receptor residues via two types of interactions: π-H (in violet) and Van der Waals (in orange) interactions (Figure 7). Compound 12 was found to involve via the pyrazole rings in two π-H interactions with r354 and Arg374 amino acids with distances of 2.79 and 3.67Å, respectively, and via carbon atoms in three Van der Waals interactions with Llp210, Gln348, and r354 residues. However, the study reveals that compound 13 has formed only one π-H interaction with Gly114 (2.65Å) residue and two Van der Waals interactions with Gln348 and Cys115 residues (2.25 and 2.33Å, respectively). Apart from this, previous works [28,29] demonstrated that some of the amino acids located in the binding pocket of MGL are very essential for the catalytic reaction of this enzyme such as cysteine (Cys) and arginine (Arg), a fact that allows us to suggest that our compounds can probably be potential leads to block the MGL action which can lead to the inhibition of the bacteria.     Journal of Chemistry e binding scores (ΔG binding ) were also analyzed to measure the affinity of the selected ligands for the receptor.
e most negative value of ΔG binding corresponds to the best binding pose of the protein-ligand complex, giving an insight about the most active compound. From the results displayed in Table 5, compound 12 was found to be associated with the highest binding energy value (− 6.86 kcal/ mol) compared to compound 13 which showed a ΔG binding value of − 6.71 kcal/mol. In the light of these outcomes, compound 12 seems more active than compound 13, which is in good agreement with the experimental antibacterial findings.
Overall, these docking results revealed clearly that the interaction between ligand 12 and MGL is higher and stronger (ΔG binding � − 6.86 kcal/mol) than that between compound 13 and MGL (ΔG binding � − 6.71 kcal/mol), suggesting it as a potential MGL inhibitor. However, more computational calculations and suitable experimental investigations are required to confirm these good outcomes.

Conclusions
From thirteen heterocyclic compounds, only the two tetra-N-alkylated heterocyclic compounds (12 and 13) have excellent antimicrobial activities especially against C. freundii, with no antifungal activity. Furthermore, based on MEP and SAR analysis, the presence of the pyridine ring as a core with close pyrazole rings (closed cavity between the pyrazole rings) where the negative charges are located makes compound 13 more polarizable and best bactericidal candidate against Citrobacter freundii with close activity against studied bacterial strains. Otherwise, our compounds were found to display better ADME profile than streptomycin, and they showed no toxicity risks. e ligand protein docking study against L-methionine c-lyase enzyme (MGL) in the case of C. freundii strain implies that compound 12 has a better affinity (more active) to the selected receptor than compound 13 which is in good accordance with the experimental antibacterial results. Further experimental studies are needed as perspectives to verify their MGL inhibition activity.