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

Authors: Y. Kaddouri; B. Bouchal; F. Abrigach; M. El Kodadi; M. Bellaoui; R. Touzani
Journal: Journal of Chemistry
 (2021)

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## Abstract

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.


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## Body

## 1. Introduction

According to the World Health Organization, microbial infections are the main threat in the world’s deadly diseases, especially in developing countries [1]. Heterocyclic moieties are important building blocks for various interesting compounds used in many fields such as medicine [2], agriculture, and industries [3]. Currently, resistance to first-line antibiotic agents is a severe problem [4]. Infections caused by resistant microbes fail to respond to treatment resulting in prolonged illness and greater risk of death [5]. Among those bacteria, Listeria monocytogenes is a Gram-positive bacterium responsible for invasive listeriosis [6, 7] which typically affects old people, pregnant women, neonates, and immunosuppressed individuals, causing gastroenteritis with diarrhea, vomiting, fever, meningitis, encephalitis, miscarriage, and stillbirth [8–10]. Staphylococcus aureus causes bacteremia, infective endocarditis (IE), osteoarticular-, skin-, soft tissue-, pleuropulmonary-, and device-related infections with 30% human population colonized [11]. Otherwise, Gram-negative Escherichia coli causes septicemia and meningitis in neonates, diarrhea and urinary tract infections, and also hemolytic uremic syndrome [12]. Citrobacter freundii causes infections in urinary tract, liver, biliary tract, peritoneum, intestines, bone, respiratory tract, endocardium, wounds, soft tissue, meninges, and bloodstream [13]. Fungal infections caused by Saccharomyces cerevisiae are fungemia, pneumonia, empyema, liver abscess, peritonitis, and vaginitis [14], whereas Candida species cause the commonly known candidiasis [15] and bloodstream infections [16].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 [17–20].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.


## 2. 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.1H and 13C NMR spectra were recorded on Bruker Avance 300, 400, and 500 MHz using TMS as internal standard in deuterated solvents such as CDCl3, CD3OD, DMSO-d6, and CD2Cl2.


### 2.1. Chemistry

#### 2.1.1. General Procedure for Synthesis of Mono- and Tetra-Alkylated Pyrazole and Triazole ligands.

The title compounds (1–13) (Figure 1) were synthesized in one-pot condensation reaction following the known methodology described in literature [17–22].Figure 1
Chemical structure of compounds1–13 studied in this paper.Compounds1, 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. The structures of all the target compounds (1–13) were established on the basis of FTIR, 1H NMR, 13C NMR, and CG-MS.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-2-amine (1) 89.85% yield, m.p. 136–138°C; FTIR (KBr,ν (cm−1)): 3288 (N-H); 2950 (C-H); 1614 (C=C); 1532 (C-C); 1386 (C-N); 1269 (C=N); 1107 (N-N); 772 (=C-H); 1H NMR (CDCl3, 500 MHz)δ ppm: 8.03 (d, J = 5 Hz, 1 H); 7.33 (dd, J = 5 Hz, 1 H); 6.56 (d, J = 5 Hz, 1 H); 6.47 (dd, J = 5 Hz, 1 H); 5.68 (s, 1 H); 5.47 (t, J = 5 Hz, 1 H); 5.52 (s, 2 H); 2.34 (s, 3 H); 2.13 (s,  3H); 13C NMR (CDCl3, 125 MHz)δ ppm: 156.53, 147.39, 137.59, 114.31, 109.06, 106.29, 54.42, 13.49, 11.12, MS found: m/z 281.2 for [M + DMSO]+ peak and calculated for C11H14N4 is 202.26.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-6-methylpyridin-2-amine (2): 30% yield, m.p. 136–138°C; FTIR (KBr,ν (cm−1)): 3283 (N-H); 2880 (C-H); 1607 (C=C); 1537 (C-C); 1336 (C-N); 1291 (C=N); 1107 (N-N); 782 (=C-H); 1H NMR (CD2Cl2, 500 MHz)δ ppm: 7.21 (dd, J = 10 Hz, 1 H); 6.40 (d, J = 5 Hz, 1 H); 6.25 (d, J = 10 Hz, 1 H); 5.64 (s, 1 H); 5.43 (s, 2 H); 2.36 (s, 3 H); 2.27 (s, 3 H); 2.06 (s, 3 H); 13C NMR (CD2Cl2, 125 MHz)δ ppm: 157.01, 155.95, 146.57, 139.51, 137.98, 112.49, 105.95, 104.97, 53.79, 24.39, 13.78, 11.17, MS found: m/z 281.2 for [M + NH4Cl]+ peak and calculated for C12H16N4 is 216.29.Synthesis of N-((1H-1,2,4-triazol-1-yl)methyl)-5-bromopyridin-2-amine(3): 62.14% yield, m.p. 64–66°C; 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.56 (s, 1 H); 8.06 (s, 1 H); 7.93 (s, 1 H); 7.67 (t, J = 6.9 Hz, 1 H); 7.63 (d, J = 8.9 Hz, 1 H); 6.62 (d, J = 7.5 Hz, 1 H); 5.67 (s, 2 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm:δ 155.68, 151.11, 147.63, 144.07, 111.06, 107.64, 54.55, MS found: m/z 370.9 for [M + 2ACN + NH4] + peak and calculated for C8H8BrN5 is 254.09.Synthesis of N-((1H-pyrazol-1-yl)methyl)-5-bromopyridin-2-amine (4): 62.36% yield, m.p. 114–116°C; FTIR (KBr,ν (cm−1)): 3290 (NH); 2945 (CH alkene); 1541 (C=C); 1491 (C-C); 1260 (C=N); 1217 (C-N); 960 (C-C); 756 (=C-H); 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.15 (s, 2 H); 7.98 (t, J = 6.9 Hz, 1 H); 7.62 (d, J = 8.6 Hz, 1 H); 7.45 (s, 1 H); 6.64 (d, J = 8.9 Hz, 1 H); 6.22 (s, 1 H); 5.59 (s, 2 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 156.11, 147.63, 139.62, 138.55, 129.47, 110.78, 107.16, 106.16, 56.44, MS found: m/z 372.8 for [M + DMSO + K+2H]+ peak and calculated for C9H9BrN4 is 253.10.Synthesis of 5-bromo-N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-2-amine (5): 93.6% yield, m.p. 150–152°C.; FTIR (KBr,ν (cm−1)): 3430 (NH); 3260–2922 (CH); 1573 (C=N); 1512 (C=C); 1355 (C–N benzene); 1289 (C–N alkyl); 619 (C–Br); 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.11 (s, 1 H); 7.84 (t, J = 8.6 Hz, 1 H); 7.62 (d, J = 8.5 Hz, 1 H); 6.63 (d, J = 7.5 Hz, 1 H); 5.76 (d, J = 8.9 Hz, 2 H); 2.56 (s, 3 H); 2.35 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 156.06, 147.44, 146.16, 144.07, 110.66, 104.75, 53.42, MS found: m/z 213 for [M + CH3OH]+ peak and calculated for C11H13BrN4 is 281.16.Synthesis of N-((1H-pyrazol-1-yl)methyl)thiazol-2-amine (6): 53.08% yield, m.p. 108–110°C; FTIR (KBr,ν (cm−1)): 3239 (N-H); 3020 (C-H); 1560 (C=C); 1540 (C-C); 1386 (C-N); 1159 (C=N); 1050 (N-N); 757 (=C-H); 700 (C-S). 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.69 (d, J = 5 Hz, 1 H); 7.80 (d, J = 5 Hz, 1 H); 7.46 (d, J = 5 Hz, 1 H); 7.09 (d, J = 5 Hz, 1 H); 6.74 (dd, J = 5 Hz, 1 H); 6.23 (t, J = 5 Hz, 1 H); 5.56 (s, 2 H); 13C NMR (DMSO-d6, 125 MHz)δ ppm: 58.93, 105.27, 108.02, 129.94, 138.47, 138.83, MS found: m/z 213 for [M + CH3OH]+ peak and calculated for C7H8N4S is 180.23.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)thiazol-2-amine(7): 37.12% yield, m.p. 152–154°C; FTIR (KBr,ν (cm−1)): 3209 (N-H); 2995 (C-H); 2368 (C=N); 1562 (C=C); 1535 (C-C); 1301 (C-N); 1177 (C=N); 1135 (N-N); 787 (=C-H); 700 (C-S). 1H NMR (CDCl3, 500 MHz)δ ppm: 7.05 (d, J = 5 Hz, 1 H); 6.43 (d, J = 5 Hz, 1 H); 5.70 (s, 1 H); 5.45 (s, 2 H); 2.21 (t, J = 5 Hz, 1 H); 2.13 (s, 3 H); 2.10 (s, 3 H). 13C NMR (CDCl3, 125 MHz)δ ppm: 167.52, 148.68, 140.07, 138.67, 108.07, 105.57, 56.74, 13.46, 11.11, MS found: m/z 293.1 for [M+2ACN + H]+ peak and calculated for C9H12N4S is 208.28.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-4-amine (8): 29.79% yield: m.p. 154–156°C; FTIR (KBr,ν (cm−1)): 3436 (N–H); 2932 (C-H); 2364 (C=N); 1653 (C=C); 1559 (C-C); 1273 (C-N); 1218 (C=N); 986 (N-N); 824 (=C-H). 1H NMR (CD3OD, 300 MHz)δ ppm: 8.06 (d, J = 8.6 Hz, 2 H); 6.81 (d, J = 7.5 Hz, 2 H); 6.79 (t, J = 8.9 Hz, 1 H); 6.55 (s, 1 H); 5.39 (s, 2 H); 2.32 (s, 3 H); 2.17 (s, 3 H). 13C NMR (CD3OD, 75 MHz)δ ppm: 155.50, 148.49, 147.45, 139.91, 108.89, 105.87, 65.58, 11.91, 9.64, MS found: m/z 281.4 for [M + DMSO + H]+ peak and calculated for C11H14N4 is 202.26.Synthesis of Ethyl 1-((1H-imidazol-1-yl)methyl)-5-methyl-1H-pyrazole-3-carboxylate (9): 99.6% yield: m.p. 74–76°C; FTIR (KBr, ν (cm−1)): 3237 (C=C-H); 1702 (C=O); 1431 (C-C); 1169 (C-O); 1393 (C-N); 1172 (C=N); 1066 (N-N); 754 (=C-H). 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7.67 (s, 1 H); 7.04 (s, 2 H); 7.04 (s, 1 H); 6.50 (s, 2 H); 4.25 (q, J = 7.5 Hz, 2 H); 2.59 (s, 3 H); 1.28 (t, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 161.62, 135.10, 121.63, 106.55, 59.88, 14.13, 10.68, MS found: m/z 310 for [M + ACN + CH3OH + H]+ peak and calculated for C11H14N4O2 is 234.26.Synthesis of 2-(((1H-pyrazol-1-yl)methyl)amino)-6-methylpyrimidin-4-ol (10): 93.82% yield: m.p. 238–240°C, FTIR (KBr,ν (cm−1)): 3398 (NH); 3112 (OH); 3004 (CH alkene); 1640 (C=C); 1500 (C-C); 1304 (C=N); 1194 (C-N), 1044 (C-C), 786 (=C-H), 1H NMR (DMSO-d6, 500 MHz):δ 11.3 (s, 1 H); 6.71 (s, 1 H); 6.11 (s, 2 H); 5.18 (s, 2 H); 1.89 (s, 3 H); 1.80 (s, 1 H); 1.76 (s, 3 H); 1.63 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 164.80 (C-OH); 155.47 (C-CH3); 153.96 (C-NH); 138.96 (CH (3, pyrazole)); 129.30, 105.64, 100.34, 73.24, 23.6, MS found: m/z 234.1 for [M + H] + peak and calculated for C11H15N5O is 233.28.Synthesis of 2-(((3,5-dimethyl-1H-pyrazol-1-yl)methyl)amino)-6-methylpyrimidin-4-ol (11): 90.65% yield: m.p. 100–102°C, FTIR (KBr,ν (cm−1)): 3386 (NH); 3123 (OH); 2986 (CH alkene); 1628 (C=C); 1450 (C-C); 1370 (C=N); 1188 (C-N), 1035 (C-C), 794 (=C-H), 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7.34 (s, 1 H); 6.71 (s, 1 H); 6.11 (s, 2 H); 5.18 (s, 2 H); 1.89 (s, 3 H); 1.80 (s, 1 H); 1.76 (s, 3 H); 1.63 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 175.56, 172.05, 171.78, 163.62, 161.41, 99.54, 25.16, 23.20, 22.41, MS found: m/z 206.1 for [M + H]+ peak and calculated for C9H11N5O is 205.22.Synthesis of N1,N1,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene-1,3-diamine (12): 24.2% yield, m.p. 87–89°C. FTIR (KBr, ν (cm−1)): 3276–2916 (CH); 1548 (C=N); 1505 (C=C); 1337 (C-N (phenyl)); 1128 (C-N (pyrazole)), 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7,52 (s, 1 H); 7.15 and 6.80 (s, 2 H); 6.29 (s, 4 H); 6.28 (s, 1 H); 5.88 (s, 8 H); 3.42 (s, 12 H); 2.55 (s, 12 H), 13C NMR (DMSO-d6, 125 MHz)δ ppm: 129.66, 118.93, 115.73, 105.57, 66.14.Synthesis of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13): 73.91% yield, m.p. 116–118°C, FTIR (KBr,ν (cm−1)): 3000 (=C-H); 2363 (C-H); 1555 (C=C); 1532 (C-C); 1396 (C-N); 1304 (C=N); 1132 (N-N); 782 (=C-H). 1H NMR (CD2Cl2, 500 MHz)δ ppm: 7.22 (d, J = 5 Hz, 1 H); 7.11 (s, 1 H); 6.71 (d, J = 5 Hz, 1 H); 5.89 (dd, J = 15 Hz, 1 H); 5.51 (s, 8 H); 2.35 (s, 12 H); 2.20 (s, 12 H). 13C NMR (CD2Cl2, 125 MHz)δ ppm: 149.11, 148.42, 140.09, 138.99, 109.53, 107.94, 105.13, 70.45, 56.48, 13.14, 10.82.


### 2.2. Antimicrobial Activity

#### 2.2.1. Determination of the Antibacterial Activity

The 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.The 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. Then, 250μL 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 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. The compounds have been tested at 500 µM, and all experiments were repeated three times for each drug.


#### 2.2.2. 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.


#### 2.2.3. Determination of the Minimum Bactericidal Concentration (MBC)

The MBC is the lowest drug concentration that kills 99% of bacteria after 24 h of incubation. MBCs were determined as described in [24, 25].


#### 2.2.4. 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. The absorbance of the cells at 600 nm (OD600) 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 OD600 <2.5. Therefore, when culture was very overgrown, the OD600 measurement was carried out after dilution of the culture, and then the dilution factor was used to calculate the OD in the original culture. The 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. Then, cells were diluted from the overnight culture to an OD600 of ∼0.08 and allowed to grow until the OD600 reached ∼0.14, to ensure that the cells were in the logarithmic phase. The compound was then added, and the growth rate of fungal cells was determined every two hours by measuring OD600. All compounds were diluted in DMSO, and all assays, including the “no drug” control, contained 1% DMSO. Optical density (OD600) 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.


### 2.3. DFT Study: MEP Surfaces

The 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].


### 2.4. ADME-Tox Predictions

In silico prediction of the ADME properties (absorption, distribution, metabolism, and excretion) and the toxicity risks (mutagenicity, tumorigenicity, irritation, and reproduction) was performed using SwissADME web tool (http://www.swissadme.ch).


### 2.5. Molecular Docking Study

The biological target selected for this study is the crystal structure of L-methionineγ-lyase from Citrobacter freundii in complex with norleucine (PDB: 3JWB). Actually, methionine γ-lyase (MGL) is an essential enzyme involved in the catalytic reaction of γ-elimination and γ-substitution of L-methionine and its derivatives, which play important roles in several biological processes. The 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.Figure2 displays the 3D structure of this enzyme and its active pocket. The docking study was carried out utilizing Molecular Operating Environment (MOE) 2015.10) software.Figure 2
The 3D structure of L-methionineγ-lyase enzyme and the chosen active site for C. freundii (PDB: 3JWB).


## 2.1. Chemistry

### 2.1.1. General Procedure for Synthesis of Mono- and Tetra-Alkylated Pyrazole and Triazole ligands.

The title compounds (1–13) (Figure 1) were synthesized in one-pot condensation reaction following the known methodology described in literature [17–22].Figure 1
Chemical structure of compounds1–13 studied in this paper.Compounds1, 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. The structures of all the target compounds (1–13) were established on the basis of FTIR, 1H NMR, 13C NMR, and CG-MS.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-2-amine (1) 89.85% yield, m.p. 136–138°C; FTIR (KBr,ν (cm−1)): 3288 (N-H); 2950 (C-H); 1614 (C=C); 1532 (C-C); 1386 (C-N); 1269 (C=N); 1107 (N-N); 772 (=C-H); 1H NMR (CDCl3, 500 MHz)δ ppm: 8.03 (d, J = 5 Hz, 1 H); 7.33 (dd, J = 5 Hz, 1 H); 6.56 (d, J = 5 Hz, 1 H); 6.47 (dd, J = 5 Hz, 1 H); 5.68 (s, 1 H); 5.47 (t, J = 5 Hz, 1 H); 5.52 (s, 2 H); 2.34 (s, 3 H); 2.13 (s,  3H); 13C NMR (CDCl3, 125 MHz)δ ppm: 156.53, 147.39, 137.59, 114.31, 109.06, 106.29, 54.42, 13.49, 11.12, MS found: m/z 281.2 for [M + DMSO]+ peak and calculated for C11H14N4 is 202.26.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-6-methylpyridin-2-amine (2): 30% yield, m.p. 136–138°C; FTIR (KBr,ν (cm−1)): 3283 (N-H); 2880 (C-H); 1607 (C=C); 1537 (C-C); 1336 (C-N); 1291 (C=N); 1107 (N-N); 782 (=C-H); 1H NMR (CD2Cl2, 500 MHz)δ ppm: 7.21 (dd, J = 10 Hz, 1 H); 6.40 (d, J = 5 Hz, 1 H); 6.25 (d, J = 10 Hz, 1 H); 5.64 (s, 1 H); 5.43 (s, 2 H); 2.36 (s, 3 H); 2.27 (s, 3 H); 2.06 (s, 3 H); 13C NMR (CD2Cl2, 125 MHz)δ ppm: 157.01, 155.95, 146.57, 139.51, 137.98, 112.49, 105.95, 104.97, 53.79, 24.39, 13.78, 11.17, MS found: m/z 281.2 for [M + NH4Cl]+ peak and calculated for C12H16N4 is 216.29.Synthesis of N-((1H-1,2,4-triazol-1-yl)methyl)-5-bromopyridin-2-amine(3): 62.14% yield, m.p. 64–66°C; 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.56 (s, 1 H); 8.06 (s, 1 H); 7.93 (s, 1 H); 7.67 (t, J = 6.9 Hz, 1 H); 7.63 (d, J = 8.9 Hz, 1 H); 6.62 (d, J = 7.5 Hz, 1 H); 5.67 (s, 2 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm:δ 155.68, 151.11, 147.63, 144.07, 111.06, 107.64, 54.55, MS found: m/z 370.9 for [M + 2ACN + NH4] + peak and calculated for C8H8BrN5 is 254.09.Synthesis of N-((1H-pyrazol-1-yl)methyl)-5-bromopyridin-2-amine (4): 62.36% yield, m.p. 114–116°C; FTIR (KBr,ν (cm−1)): 3290 (NH); 2945 (CH alkene); 1541 (C=C); 1491 (C-C); 1260 (C=N); 1217 (C-N); 960 (C-C); 756 (=C-H); 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.15 (s, 2 H); 7.98 (t, J = 6.9 Hz, 1 H); 7.62 (d, J = 8.6 Hz, 1 H); 7.45 (s, 1 H); 6.64 (d, J = 8.9 Hz, 1 H); 6.22 (s, 1 H); 5.59 (s, 2 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 156.11, 147.63, 139.62, 138.55, 129.47, 110.78, 107.16, 106.16, 56.44, MS found: m/z 372.8 for [M + DMSO + K+2H]+ peak and calculated for C9H9BrN4 is 253.10.Synthesis of 5-bromo-N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-2-amine (5): 93.6% yield, m.p. 150–152°C.; FTIR (KBr,ν (cm−1)): 3430 (NH); 3260–2922 (CH); 1573 (C=N); 1512 (C=C); 1355 (C–N benzene); 1289 (C–N alkyl); 619 (C–Br); 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.11 (s, 1 H); 7.84 (t, J = 8.6 Hz, 1 H); 7.62 (d, J = 8.5 Hz, 1 H); 6.63 (d, J = 7.5 Hz, 1 H); 5.76 (d, J = 8.9 Hz, 2 H); 2.56 (s, 3 H); 2.35 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 156.06, 147.44, 146.16, 144.07, 110.66, 104.75, 53.42, MS found: m/z 213 for [M + CH3OH]+ peak and calculated for C11H13BrN4 is 281.16.Synthesis of N-((1H-pyrazol-1-yl)methyl)thiazol-2-amine (6): 53.08% yield, m.p. 108–110°C; FTIR (KBr,ν (cm−1)): 3239 (N-H); 3020 (C-H); 1560 (C=C); 1540 (C-C); 1386 (C-N); 1159 (C=N); 1050 (N-N); 757 (=C-H); 700 (C-S). 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.69 (d, J = 5 Hz, 1 H); 7.80 (d, J = 5 Hz, 1 H); 7.46 (d, J = 5 Hz, 1 H); 7.09 (d, J = 5 Hz, 1 H); 6.74 (dd, J = 5 Hz, 1 H); 6.23 (t, J = 5 Hz, 1 H); 5.56 (s, 2 H); 13C NMR (DMSO-d6, 125 MHz)δ ppm: 58.93, 105.27, 108.02, 129.94, 138.47, 138.83, MS found: m/z 213 for [M + CH3OH]+ peak and calculated for C7H8N4S is 180.23.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)thiazol-2-amine(7): 37.12% yield, m.p. 152–154°C; FTIR (KBr,ν (cm−1)): 3209 (N-H); 2995 (C-H); 2368 (C=N); 1562 (C=C); 1535 (C-C); 1301 (C-N); 1177 (C=N); 1135 (N-N); 787 (=C-H); 700 (C-S). 1H NMR (CDCl3, 500 MHz)δ ppm: 7.05 (d, J = 5 Hz, 1 H); 6.43 (d, J = 5 Hz, 1 H); 5.70 (s, 1 H); 5.45 (s, 2 H); 2.21 (t, J = 5 Hz, 1 H); 2.13 (s, 3 H); 2.10 (s, 3 H). 13C NMR (CDCl3, 125 MHz)δ ppm: 167.52, 148.68, 140.07, 138.67, 108.07, 105.57, 56.74, 13.46, 11.11, MS found: m/z 293.1 for [M+2ACN + H]+ peak and calculated for C9H12N4S is 208.28.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-4-amine (8): 29.79% yield: m.p. 154–156°C; FTIR (KBr,ν (cm−1)): 3436 (N–H); 2932 (C-H); 2364 (C=N); 1653 (C=C); 1559 (C-C); 1273 (C-N); 1218 (C=N); 986 (N-N); 824 (=C-H). 1H NMR (CD3OD, 300 MHz)δ ppm: 8.06 (d, J = 8.6 Hz, 2 H); 6.81 (d, J = 7.5 Hz, 2 H); 6.79 (t, J = 8.9 Hz, 1 H); 6.55 (s, 1 H); 5.39 (s, 2 H); 2.32 (s, 3 H); 2.17 (s, 3 H). 13C NMR (CD3OD, 75 MHz)δ ppm: 155.50, 148.49, 147.45, 139.91, 108.89, 105.87, 65.58, 11.91, 9.64, MS found: m/z 281.4 for [M + DMSO + H]+ peak and calculated for C11H14N4 is 202.26.Synthesis of Ethyl 1-((1H-imidazol-1-yl)methyl)-5-methyl-1H-pyrazole-3-carboxylate (9): 99.6% yield: m.p. 74–76°C; FTIR (KBr, ν (cm−1)): 3237 (C=C-H); 1702 (C=O); 1431 (C-C); 1169 (C-O); 1393 (C-N); 1172 (C=N); 1066 (N-N); 754 (=C-H). 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7.67 (s, 1 H); 7.04 (s, 2 H); 7.04 (s, 1 H); 6.50 (s, 2 H); 4.25 (q, J = 7.5 Hz, 2 H); 2.59 (s, 3 H); 1.28 (t, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 161.62, 135.10, 121.63, 106.55, 59.88, 14.13, 10.68, MS found: m/z 310 for [M + ACN + CH3OH + H]+ peak and calculated for C11H14N4O2 is 234.26.Synthesis of 2-(((1H-pyrazol-1-yl)methyl)amino)-6-methylpyrimidin-4-ol (10): 93.82% yield: m.p. 238–240°C, FTIR (KBr,ν (cm−1)): 3398 (NH); 3112 (OH); 3004 (CH alkene); 1640 (C=C); 1500 (C-C); 1304 (C=N); 1194 (C-N), 1044 (C-C), 786 (=C-H), 1H NMR (DMSO-d6, 500 MHz):δ 11.3 (s, 1 H); 6.71 (s, 1 H); 6.11 (s, 2 H); 5.18 (s, 2 H); 1.89 (s, 3 H); 1.80 (s, 1 H); 1.76 (s, 3 H); 1.63 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 164.80 (C-OH); 155.47 (C-CH3); 153.96 (C-NH); 138.96 (CH (3, pyrazole)); 129.30, 105.64, 100.34, 73.24, 23.6, MS found: m/z 234.1 for [M + H] + peak and calculated for C11H15N5O is 233.28.Synthesis of 2-(((3,5-dimethyl-1H-pyrazol-1-yl)methyl)amino)-6-methylpyrimidin-4-ol (11): 90.65% yield: m.p. 100–102°C, FTIR (KBr,ν (cm−1)): 3386 (NH); 3123 (OH); 2986 (CH alkene); 1628 (C=C); 1450 (C-C); 1370 (C=N); 1188 (C-N), 1035 (C-C), 794 (=C-H), 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7.34 (s, 1 H); 6.71 (s, 1 H); 6.11 (s, 2 H); 5.18 (s, 2 H); 1.89 (s, 3 H); 1.80 (s, 1 H); 1.76 (s, 3 H); 1.63 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 175.56, 172.05, 171.78, 163.62, 161.41, 99.54, 25.16, 23.20, 22.41, MS found: m/z 206.1 for [M + H]+ peak and calculated for C9H11N5O is 205.22.Synthesis of N1,N1,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene-1,3-diamine (12): 24.2% yield, m.p. 87–89°C. FTIR (KBr, ν (cm−1)): 3276–2916 (CH); 1548 (C=N); 1505 (C=C); 1337 (C-N (phenyl)); 1128 (C-N (pyrazole)), 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7,52 (s, 1 H); 7.15 and 6.80 (s, 2 H); 6.29 (s, 4 H); 6.28 (s, 1 H); 5.88 (s, 8 H); 3.42 (s, 12 H); 2.55 (s, 12 H), 13C NMR (DMSO-d6, 125 MHz)δ ppm: 129.66, 118.93, 115.73, 105.57, 66.14.Synthesis of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13): 73.91% yield, m.p. 116–118°C, FTIR (KBr,ν (cm−1)): 3000 (=C-H); 2363 (C-H); 1555 (C=C); 1532 (C-C); 1396 (C-N); 1304 (C=N); 1132 (N-N); 782 (=C-H). 1H NMR (CD2Cl2, 500 MHz)δ ppm: 7.22 (d, J = 5 Hz, 1 H); 7.11 (s, 1 H); 6.71 (d, J = 5 Hz, 1 H); 5.89 (dd, J = 15 Hz, 1 H); 5.51 (s, 8 H); 2.35 (s, 12 H); 2.20 (s, 12 H). 13C NMR (CD2Cl2, 125 MHz)δ ppm: 149.11, 148.42, 140.09, 138.99, 109.53, 107.94, 105.13, 70.45, 56.48, 13.14, 10.82.


## 2.1.1. General Procedure for Synthesis of Mono- and Tetra-Alkylated Pyrazole and Triazole ligands.

The title compounds (1–13) (Figure 1) were synthesized in one-pot condensation reaction following the known methodology described in literature [17–22].Figure 1
Chemical structure of compounds1–13 studied in this paper.Compounds1, 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. The structures of all the target compounds (1–13) were established on the basis of FTIR, 1H NMR, 13C NMR, and CG-MS.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-2-amine (1) 89.85% yield, m.p. 136–138°C; FTIR (KBr,ν (cm−1)): 3288 (N-H); 2950 (C-H); 1614 (C=C); 1532 (C-C); 1386 (C-N); 1269 (C=N); 1107 (N-N); 772 (=C-H); 1H NMR (CDCl3, 500 MHz)δ ppm: 8.03 (d, J = 5 Hz, 1 H); 7.33 (dd, J = 5 Hz, 1 H); 6.56 (d, J = 5 Hz, 1 H); 6.47 (dd, J = 5 Hz, 1 H); 5.68 (s, 1 H); 5.47 (t, J = 5 Hz, 1 H); 5.52 (s, 2 H); 2.34 (s, 3 H); 2.13 (s,  3H); 13C NMR (CDCl3, 125 MHz)δ ppm: 156.53, 147.39, 137.59, 114.31, 109.06, 106.29, 54.42, 13.49, 11.12, MS found: m/z 281.2 for [M + DMSO]+ peak and calculated for C11H14N4 is 202.26.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-6-methylpyridin-2-amine (2): 30% yield, m.p. 136–138°C; FTIR (KBr,ν (cm−1)): 3283 (N-H); 2880 (C-H); 1607 (C=C); 1537 (C-C); 1336 (C-N); 1291 (C=N); 1107 (N-N); 782 (=C-H); 1H NMR (CD2Cl2, 500 MHz)δ ppm: 7.21 (dd, J = 10 Hz, 1 H); 6.40 (d, J = 5 Hz, 1 H); 6.25 (d, J = 10 Hz, 1 H); 5.64 (s, 1 H); 5.43 (s, 2 H); 2.36 (s, 3 H); 2.27 (s, 3 H); 2.06 (s, 3 H); 13C NMR (CD2Cl2, 125 MHz)δ ppm: 157.01, 155.95, 146.57, 139.51, 137.98, 112.49, 105.95, 104.97, 53.79, 24.39, 13.78, 11.17, MS found: m/z 281.2 for [M + NH4Cl]+ peak and calculated for C12H16N4 is 216.29.Synthesis of N-((1H-1,2,4-triazol-1-yl)methyl)-5-bromopyridin-2-amine(3): 62.14% yield, m.p. 64–66°C; 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.56 (s, 1 H); 8.06 (s, 1 H); 7.93 (s, 1 H); 7.67 (t, J = 6.9 Hz, 1 H); 7.63 (d, J = 8.9 Hz, 1 H); 6.62 (d, J = 7.5 Hz, 1 H); 5.67 (s, 2 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm:δ 155.68, 151.11, 147.63, 144.07, 111.06, 107.64, 54.55, MS found: m/z 370.9 for [M + 2ACN + NH4] + peak and calculated for C8H8BrN5 is 254.09.Synthesis of N-((1H-pyrazol-1-yl)methyl)-5-bromopyridin-2-amine (4): 62.36% yield, m.p. 114–116°C; FTIR (KBr,ν (cm−1)): 3290 (NH); 2945 (CH alkene); 1541 (C=C); 1491 (C-C); 1260 (C=N); 1217 (C-N); 960 (C-C); 756 (=C-H); 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.15 (s, 2 H); 7.98 (t, J = 6.9 Hz, 1 H); 7.62 (d, J = 8.6 Hz, 1 H); 7.45 (s, 1 H); 6.64 (d, J = 8.9 Hz, 1 H); 6.22 (s, 1 H); 5.59 (s, 2 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 156.11, 147.63, 139.62, 138.55, 129.47, 110.78, 107.16, 106.16, 56.44, MS found: m/z 372.8 for [M + DMSO + K+2H]+ peak and calculated for C9H9BrN4 is 253.10.Synthesis of 5-bromo-N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-2-amine (5): 93.6% yield, m.p. 150–152°C.; FTIR (KBr,ν (cm−1)): 3430 (NH); 3260–2922 (CH); 1573 (C=N); 1512 (C=C); 1355 (C–N benzene); 1289 (C–N alkyl); 619 (C–Br); 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.11 (s, 1 H); 7.84 (t, J = 8.6 Hz, 1 H); 7.62 (d, J = 8.5 Hz, 1 H); 6.63 (d, J = 7.5 Hz, 1 H); 5.76 (d, J = 8.9 Hz, 2 H); 2.56 (s, 3 H); 2.35 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 156.06, 147.44, 146.16, 144.07, 110.66, 104.75, 53.42, MS found: m/z 213 for [M + CH3OH]+ peak and calculated for C11H13BrN4 is 281.16.Synthesis of N-((1H-pyrazol-1-yl)methyl)thiazol-2-amine (6): 53.08% yield, m.p. 108–110°C; FTIR (KBr,ν (cm−1)): 3239 (N-H); 3020 (C-H); 1560 (C=C); 1540 (C-C); 1386 (C-N); 1159 (C=N); 1050 (N-N); 757 (=C-H); 700 (C-S). 1H NMR (DMSO-d6, 500 MHz)δ ppm: 8.69 (d, J = 5 Hz, 1 H); 7.80 (d, J = 5 Hz, 1 H); 7.46 (d, J = 5 Hz, 1 H); 7.09 (d, J = 5 Hz, 1 H); 6.74 (dd, J = 5 Hz, 1 H); 6.23 (t, J = 5 Hz, 1 H); 5.56 (s, 2 H); 13C NMR (DMSO-d6, 125 MHz)δ ppm: 58.93, 105.27, 108.02, 129.94, 138.47, 138.83, MS found: m/z 213 for [M + CH3OH]+ peak and calculated for C7H8N4S is 180.23.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)thiazol-2-amine(7): 37.12% yield, m.p. 152–154°C; FTIR (KBr,ν (cm−1)): 3209 (N-H); 2995 (C-H); 2368 (C=N); 1562 (C=C); 1535 (C-C); 1301 (C-N); 1177 (C=N); 1135 (N-N); 787 (=C-H); 700 (C-S). 1H NMR (CDCl3, 500 MHz)δ ppm: 7.05 (d, J = 5 Hz, 1 H); 6.43 (d, J = 5 Hz, 1 H); 5.70 (s, 1 H); 5.45 (s, 2 H); 2.21 (t, J = 5 Hz, 1 H); 2.13 (s, 3 H); 2.10 (s, 3 H). 13C NMR (CDCl3, 125 MHz)δ ppm: 167.52, 148.68, 140.07, 138.67, 108.07, 105.57, 56.74, 13.46, 11.11, MS found: m/z 293.1 for [M+2ACN + H]+ peak and calculated for C9H12N4S is 208.28.Synthesis of N-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridin-4-amine (8): 29.79% yield: m.p. 154–156°C; FTIR (KBr,ν (cm−1)): 3436 (N–H); 2932 (C-H); 2364 (C=N); 1653 (C=C); 1559 (C-C); 1273 (C-N); 1218 (C=N); 986 (N-N); 824 (=C-H). 1H NMR (CD3OD, 300 MHz)δ ppm: 8.06 (d, J = 8.6 Hz, 2 H); 6.81 (d, J = 7.5 Hz, 2 H); 6.79 (t, J = 8.9 Hz, 1 H); 6.55 (s, 1 H); 5.39 (s, 2 H); 2.32 (s, 3 H); 2.17 (s, 3 H). 13C NMR (CD3OD, 75 MHz)δ ppm: 155.50, 148.49, 147.45, 139.91, 108.89, 105.87, 65.58, 11.91, 9.64, MS found: m/z 281.4 for [M + DMSO + H]+ peak and calculated for C11H14N4 is 202.26.Synthesis of Ethyl 1-((1H-imidazol-1-yl)methyl)-5-methyl-1H-pyrazole-3-carboxylate (9): 99.6% yield: m.p. 74–76°C; FTIR (KBr, ν (cm−1)): 3237 (C=C-H); 1702 (C=O); 1431 (C-C); 1169 (C-O); 1393 (C-N); 1172 (C=N); 1066 (N-N); 754 (=C-H). 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7.67 (s, 1 H); 7.04 (s, 2 H); 7.04 (s, 1 H); 6.50 (s, 2 H); 4.25 (q, J = 7.5 Hz, 2 H); 2.59 (s, 3 H); 1.28 (t, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 161.62, 135.10, 121.63, 106.55, 59.88, 14.13, 10.68, MS found: m/z 310 for [M + ACN + CH3OH + H]+ peak and calculated for C11H14N4O2 is 234.26.Synthesis of 2-(((1H-pyrazol-1-yl)methyl)amino)-6-methylpyrimidin-4-ol (10): 93.82% yield: m.p. 238–240°C, FTIR (KBr,ν (cm−1)): 3398 (NH); 3112 (OH); 3004 (CH alkene); 1640 (C=C); 1500 (C-C); 1304 (C=N); 1194 (C-N), 1044 (C-C), 786 (=C-H), 1H NMR (DMSO-d6, 500 MHz):δ 11.3 (s, 1 H); 6.71 (s, 1 H); 6.11 (s, 2 H); 5.18 (s, 2 H); 1.89 (s, 3 H); 1.80 (s, 1 H); 1.76 (s, 3 H); 1.63 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 164.80 (C-OH); 155.47 (C-CH3); 153.96 (C-NH); 138.96 (CH (3, pyrazole)); 129.30, 105.64, 100.34, 73.24, 23.6, MS found: m/z 234.1 for [M + H] + peak and calculated for C11H15N5O is 233.28.Synthesis of 2-(((3,5-dimethyl-1H-pyrazol-1-yl)methyl)amino)-6-methylpyrimidin-4-ol (11): 90.65% yield: m.p. 100–102°C, FTIR (KBr,ν (cm−1)): 3386 (NH); 3123 (OH); 2986 (CH alkene); 1628 (C=C); 1450 (C-C); 1370 (C=N); 1188 (C-N), 1035 (C-C), 794 (=C-H), 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7.34 (s, 1 H); 6.71 (s, 1 H); 6.11 (s, 2 H); 5.18 (s, 2 H); 1.89 (s, 3 H); 1.80 (s, 1 H); 1.76 (s, 3 H); 1.63 (s, 3 H). 13C NMR (DMSO-d6, 125 MHz)δ ppm: 175.56, 172.05, 171.78, 163.62, 161.41, 99.54, 25.16, 23.20, 22.41, MS found: m/z 206.1 for [M + H]+ peak and calculated for C9H11N5O is 205.22.Synthesis of N1,N1,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzene-1,3-diamine (12): 24.2% yield, m.p. 87–89°C. FTIR (KBr, ν (cm−1)): 3276–2916 (CH); 1548 (C=N); 1505 (C=C); 1337 (C-N (phenyl)); 1128 (C-N (pyrazole)), 1H NMR (DMSO-d6, 500 MHz)δ ppm: 7,52 (s, 1 H); 7.15 and 6.80 (s, 2 H); 6.29 (s, 4 H); 6.28 (s, 1 H); 5.88 (s, 8 H); 3.42 (s, 12 H); 2.55 (s, 12 H), 13C NMR (DMSO-d6, 125 MHz)δ ppm: 129.66, 118.93, 115.73, 105.57, 66.14.Synthesis of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13): 73.91% yield, m.p. 116–118°C, FTIR (KBr,ν (cm−1)): 3000 (=C-H); 2363 (C-H); 1555 (C=C); 1532 (C-C); 1396 (C-N); 1304 (C=N); 1132 (N-N); 782 (=C-H). 1H NMR (CD2Cl2, 500 MHz)δ ppm: 7.22 (d, J = 5 Hz, 1 H); 7.11 (s, 1 H); 6.71 (d, J = 5 Hz, 1 H); 5.89 (dd, J = 15 Hz, 1 H); 5.51 (s, 8 H); 2.35 (s, 12 H); 2.20 (s, 12 H). 13C NMR (CD2Cl2, 125 MHz)δ ppm: 149.11, 148.42, 140.09, 138.99, 109.53, 107.94, 105.13, 70.45, 56.48, 13.14, 10.82.


## 2.2. Antimicrobial Activity

### 2.2.1. Determination of the Antibacterial Activity

The 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.The 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. Then, 250μL 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 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. The compounds have been tested at 500 µM, and all experiments were repeated three times for each drug.


### 2.2.2. 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.


### 2.2.3. Determination of the Minimum Bactericidal Concentration (MBC)

The MBC is the lowest drug concentration that kills 99% of bacteria after 24 h of incubation. MBCs were determined as described in [24, 25].


### 2.2.4. 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. The absorbance of the cells at 600 nm (OD600) 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 OD600 <2.5. Therefore, when culture was very overgrown, the OD600 measurement was carried out after dilution of the culture, and then the dilution factor was used to calculate the OD in the original culture. The 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. Then, cells were diluted from the overnight culture to an OD600 of ∼0.08 and allowed to grow until the OD600 reached ∼0.14, to ensure that the cells were in the logarithmic phase. The compound was then added, and the growth rate of fungal cells was determined every two hours by measuring OD600. All compounds were diluted in DMSO, and all assays, including the “no drug” control, contained 1% DMSO. Optical density (OD600) 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.


## 2.2.1. Determination of the Antibacterial Activity

The 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.The 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. Then, 250μL 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 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. The compounds have been tested at 500 µM, and all experiments were repeated three times for each drug.


## 2.2.2. 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.


## 2.2.3. Determination of the Minimum Bactericidal Concentration (MBC)

The MBC is the lowest drug concentration that kills 99% of bacteria after 24 h of incubation. MBCs were determined as described in [24, 25].


## 2.2.4. 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. The absorbance of the cells at 600 nm (OD600) 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 OD600 <2.5. Therefore, when culture was very overgrown, the OD600 measurement was carried out after dilution of the culture, and then the dilution factor was used to calculate the OD in the original culture. The 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. Then, cells were diluted from the overnight culture to an OD600 of ∼0.08 and allowed to grow until the OD600 reached ∼0.14, to ensure that the cells were in the logarithmic phase. The compound was then added, and the growth rate of fungal cells was determined every two hours by measuring OD600. All compounds were diluted in DMSO, and all assays, including the “no drug” control, contained 1% DMSO. Optical density (OD600) 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.


## 2.3. DFT Study: MEP Surfaces

The 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].


## 2.4. ADME-Tox Predictions

In silico prediction of the ADME properties (absorption, distribution, metabolism, and excretion) and the toxicity risks (mutagenicity, tumorigenicity, irritation, and reproduction) was performed using SwissADME web tool (http://www.swissadme.ch).


## 2.5. Molecular Docking Study

The biological target selected for this study is the crystal structure of L-methionineγ-lyase from Citrobacter freundii in complex with norleucine (PDB: 3JWB). Actually, methionine γ-lyase (MGL) is an essential enzyme involved in the catalytic reaction of γ-elimination and γ-substitution of L-methionine and its derivatives, which play important roles in several biological processes. The 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.Figure2 displays the 3D structure of this enzyme and its active pocket. The docking study was carried out utilizing Molecular Operating Environment (MOE) 2015.10) software.Figure 2
The 3D structure of L-methionineγ-lyase enzyme and the chosen active site for C. freundii (PDB: 3JWB).


## 3. Results and Discussion

### 3.1. Chemistry

As an example, for details of the characterization of compound13, where the 1H NMR (CD2Cl2, 500 MHz) spectrum (Figure 3) of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13) contains the following peaks:(i)
Doublet peak at 7.13 for proton ofCH (1)(ii)
Doublet peak at 6.57 for the protonCH (3)(iii)
Doublet bidoublet peak at 6.06 for the proton ofCH (2)(iv)
Singlet peak at 5.89 and 5.80 for the proton ofCH (4′) on the pyrazole ring(v)
Singlet peak at 5.51 and 5.42 for the protons ofCH2)(vi)
Singlet peak at 2.45 for the protons ofCH3(a, b)Figure 3
1H NMR spectrum of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl) methyl)pyridine-2,3-diamine (13).The13C NMR (CD2Cl2, 125 MHz) spectrum (Figure 4) of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13) contain the following peaks at 140.09 for carbon C1; 138.68 for carbon C4 and C5’; 148.42 and 138.99 for carbon C3’; 109.53 for carbon C2; 107.94 for carbon C3; 105.89, 105.13, and 105.05 for carbon C4’; 56.48 for carbon CH2; and 13.14, 10.82, and 10.37 for carbon CH3.Figure 4
13C NMR spectrum N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13).


### 3.2. Antimicrobial Activity

The antibacterial potential of the synthesized mono- and tetra-alkylated pyrazole and triazole derivatives shown in Figure1 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. This result could be explained by the fact that these compounds lack pharmacophore sites which can act by inhibiting bacterial growth.Table 1
Antibacterial activity of the newly synthesized mono- and tetra-alkylated pyrazole and triazole derivatives determined by the broth macrodilution assay and using the phenol red indicator.
EntryL. monocytogenesE. coliS. aureusC. freundii1− − −− − −− − −− − −2− − −− − −− − −− − −3− − −− − −− − −− − −4− − −− − −− − −− − −5− − −− − −− − −− − −6− − −− − −− − −− − −7− − −− − −− − −− − −8− − −− − −− − −− − −9− − −− − −− − −− − −10− − −− − −− − −− − −11− − −− − −− − −− − −12+ + ++ + ++ + ++ + +13+ + ++ + ++ + ++ + +The compounds have been tested at 500µM. 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.In contrast, the two tetra-alkylated derivatives exhibit potent antibacterial activity against all the studied strains (Table2). Therefore, MIC and MBC of these compounds were determined as described in Sections 2.2.2 and 2.2.3. The MIC and MBC of compound 12 against L. monocytogenes, E. coli, and S. aureus were 300 µM. Interestingly, this compound was much more active against C. freundii with an MIC of 100 µM and MBC of 200 µM (Table 2). Similarly, C. freundii was the most sensitive to compound 13 (MIC = 300 µM, MBC = 400 µM), followed by S. aureus (MIC = 400 µM, MBC = 500 µM), E. coli (MIC = 500 µM, MBC = 500 µM), and then L. monocytogenes (MIC = 500 µM, MBC = 800 µM).Table 2
MIC (inµM) and MBC (in µM) values of active compounds 12 and 13 against the bacterial strains L. monocytogenes, E. coli, S. aureus, and C. freundii.
EntryL. monocytogenesE. coliS. aureusC. freundii12MIC300300300100MBC300300300200MBC/MIC111213MIC500500400300MBC800500500400MBC/MIC1.611.251.3All the prepared compounds were screened for their antifungal activity against two fungal strains, namely,Candida glabrata and Saccharomyces cerevisiae as described in Section 2.2.4. All the mono- and tetradentate pyrazole ligands tested (1–13) showed no antifungal activity against Candida glabrata. Figure 5 shows the result obtained with monodentate pyrazole ligands 9, 10, and 11 and tetradentate ones 12 and 13. In contrast, while monodentate pyrazole ligands were not toxic to Saccharomyces cerevisiae, tetradentate pyrazole ligands 12 and 13 exhibited significant antifungal activity against this strain (Figure 5). Interestingly, compound 12 showed stronger antifungal activity against Saccharomyces cerevisiae cells than compound 13 (Figure 5), consistent with their antibacterial activities demonstrated above.Figure 5
Activity of mono- and tetradentate pyrazole ligands againstCandida glabrata and Saccharomyces cerevisiae. Cells were cultured in the absence (“no drug” control) or in the presence of 500 µM of compounds 9–13.
(a)(b)Together, these results suggest that tetra-alkylated ligands12 and 13 act as weak antifungal agents, but strong antibacterial agents, and might represent promising lead compounds for the development of specific antibacterial drugs. Therefore, further investigation is required to better understand their antibacterial activity and their mode of action. Thus, theoretical investigations such as SAR, ADME-Tox, and molecular docking were performed.


### 3.3. SAR Analysis of Compounds12 and 13

The structure activity relationship (SAR) analysis of the tetra-alkylated pyrazole and triazole derivatives12 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-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.


### 3.4. 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. Figure6 shows the MEP surfaces generated by the DFT optimized geometries for compounds 12 and 13 that displayed strong antibacterial activity.Figure 6
MEP surfaces presentation for the compounds12 (a) and 13 (b).
(a)(b)In this study, the MEP surfaces of compounds12 and 13 showed negative charges located on sp2-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 sp3-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. Therefore, 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.


### 3.5. ADME-Tox Predictions

The 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. Therefore, 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. The results are collected in Tables 3 and 4.Table 3
Physicochemical properties of the most potent antibacterial pyrazole derivatives and the antibiotic control streptomycin.
EntryPhysicochemical propertiesMWLog PH-donH-accnrotbTPSA (Å2)12540.714.87041077.7613541.694.25051090.65Streptomycin581.578−6.65141511331.43MW: molecular weight expressed in daltons. Log P: octanol/water partition coefficient characterizing lipophilicity. H-don: number of hydrogen bond donors. H-acc: number of hydrogen bond acceptors. nrotb: number of rotable bonds. TPSA: total polar surface area.Table 4
Toxicity risks of the most potent antibacterial pyrazole derivatives and streptomycin.
EntryToxicityMutagenicTumorigenicReproductivityIrritant12NoneNoneNoneNone13NoneNoneNoneNoneStreptomycinNoneNoneNoneHighOverall, excepting the molecular weight which is found to be higher than 500 daltons, the two compounds12 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 (<140 Å2). These 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 compounds12 and 13, whereas it showed a high irritant effect of streptomycin.


### 3.6. Molecular Docking Study

The two compounds12 and 13 were docked with the active site of L-methionine γ-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 Thr354 and Arg374 amino acids with distances of 2.79 and 3.67 Å, respectively, and via carbon atoms in three Van der Waals interactions with Llp210, Gln348, and Thr354 residues. However, the study reveals that compound 13 has formed only one π-H interaction with Gly114 (2.65 Å) residue and two Van der Waals interactions with Gln348 and Cys115 residues (2.25 and 2.33 Å, 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.Figure 7
Interaction network of compound12 (a) and compound 13 (b) with L-methionine γ-lyase enzyme residues.
(a)(b)The binding scores (ΔGbinding) were also analyzed to measure the affinity of the selected ligands for the receptor. The most negative value of ΔGbinding 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 ΔGbinding 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.Table 5
Score, interactions, and bond length obtained for compounds12 and 13 with MGL enzyme.
EntryΔGbinding in kcal/molInteractionsBond length (Å)12−0.86CH3—H(Llp210)C(N-C-N)—H(Cys115)CH3—H(Gln348)5-ring—H(Thr354)CH3—O(Thr354)5-ring—H(Arg374)2.292.322.442.792.803.6713−6.71CH3—H(Gln348))C(N-C-N)—H(Cys115)5-ring—H(Gly114)2.252.332.65Overall, these docking results revealed clearly that the interaction between ligand12 and MGL is higher and stronger (ΔGbinding = −6.86 kcal/mol) than that between compound 13 and MGL (ΔGbinding = −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.


## 3.1. Chemistry

As an example, for details of the characterization of compound13, where the 1H NMR (CD2Cl2, 500 MHz) spectrum (Figure 3) of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13) contains the following peaks:(i)
Doublet peak at 7.13 for proton ofCH (1)(ii)
Doublet peak at 6.57 for the protonCH (3)(iii)
Doublet bidoublet peak at 6.06 for the proton ofCH (2)(iv)
Singlet peak at 5.89 and 5.80 for the proton ofCH (4′) on the pyrazole ring(v)
Singlet peak at 5.51 and 5.42 for the protons ofCH2)(vi)
Singlet peak at 2.45 for the protons ofCH3(a, b)Figure 3
1H NMR spectrum of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl) methyl)pyridine-2,3-diamine (13).The13C NMR (CD2Cl2, 125 MHz) spectrum (Figure 4) of N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13) contain the following peaks at 140.09 for carbon C1; 138.68 for carbon C4 and C5’; 148.42 and 138.99 for carbon C3’; 109.53 for carbon C2; 107.94 for carbon C3; 105.89, 105.13, and 105.05 for carbon C4’; 56.48 for carbon CH2; and 13.14, 10.82, and 10.37 for carbon CH3.Figure 4
13C NMR spectrum N2,N2,N3,N3-tetrakis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2,3-diamine (13).


## 3.2. Antimicrobial Activity

The antibacterial potential of the synthesized mono- and tetra-alkylated pyrazole and triazole derivatives shown in Figure1 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. This result could be explained by the fact that these compounds lack pharmacophore sites which can act by inhibiting bacterial growth.Table 1
Antibacterial activity of the newly synthesized mono- and tetra-alkylated pyrazole and triazole derivatives determined by the broth macrodilution assay and using the phenol red indicator.
EntryL. monocytogenesE. coliS. aureusC. freundii1− − −− − −− − −− − −2− − −− − −− − −− − −3− − −− − −− − −− − −4− − −− − −− − −− − −5− − −− − −− − −− − −6− − −− − −− − −− − −7− − −− − −− − −− − −8− − −− − −− − −− − −9− − −− − −− − −− − −10− − −− − −− − −− − −11− − −− − −− − −− − −12+ + ++ + ++ + ++ + +13+ + ++ + ++ + ++ + +The compounds have been tested at 500µM. 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.In contrast, the two tetra-alkylated derivatives exhibit potent antibacterial activity against all the studied strains (Table2). Therefore, MIC and MBC of these compounds were determined as described in Sections 2.2.2 and 2.2.3. The MIC and MBC of compound 12 against L. monocytogenes, E. coli, and S. aureus were 300 µM. Interestingly, this compound was much more active against C. freundii with an MIC of 100 µM and MBC of 200 µM (Table 2). Similarly, C. freundii was the most sensitive to compound 13 (MIC = 300 µM, MBC = 400 µM), followed by S. aureus (MIC = 400 µM, MBC = 500 µM), E. coli (MIC = 500 µM, MBC = 500 µM), and then L. monocytogenes (MIC = 500 µM, MBC = 800 µM).Table 2
MIC (inµM) and MBC (in µM) values of active compounds 12 and 13 against the bacterial strains L. monocytogenes, E. coli, S. aureus, and C. freundii.
EntryL. monocytogenesE. coliS. aureusC. freundii12MIC300300300100MBC300300300200MBC/MIC111213MIC500500400300MBC800500500400MBC/MIC1.611.251.3All the prepared compounds were screened for their antifungal activity against two fungal strains, namely,Candida glabrata and Saccharomyces cerevisiae as described in Section 2.2.4. All the mono- and tetradentate pyrazole ligands tested (1–13) showed no antifungal activity against Candida glabrata. Figure 5 shows the result obtained with monodentate pyrazole ligands 9, 10, and 11 and tetradentate ones 12 and 13. In contrast, while monodentate pyrazole ligands were not toxic to Saccharomyces cerevisiae, tetradentate pyrazole ligands 12 and 13 exhibited significant antifungal activity against this strain (Figure 5). Interestingly, compound 12 showed stronger antifungal activity against Saccharomyces cerevisiae cells than compound 13 (Figure 5), consistent with their antibacterial activities demonstrated above.Figure 5
Activity of mono- and tetradentate pyrazole ligands againstCandida glabrata and Saccharomyces cerevisiae. Cells were cultured in the absence (“no drug” control) or in the presence of 500 µM of compounds 9–13.
(a)(b)Together, these results suggest that tetra-alkylated ligands12 and 13 act as weak antifungal agents, but strong antibacterial agents, and might represent promising lead compounds for the development of specific antibacterial drugs. Therefore, further investigation is required to better understand their antibacterial activity and their mode of action. Thus, theoretical investigations such as SAR, ADME-Tox, and molecular docking were performed.


## 3.3. SAR Analysis of Compounds12 and 13

The structure activity relationship (SAR) analysis of the tetra-alkylated pyrazole and triazole derivatives12 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-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.


## 3.4. 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. Figure6 shows the MEP surfaces generated by the DFT optimized geometries for compounds 12 and 13 that displayed strong antibacterial activity.Figure 6
MEP surfaces presentation for the compounds12 (a) and 13 (b).
(a)(b)In this study, the MEP surfaces of compounds12 and 13 showed negative charges located on sp2-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 sp3-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. Therefore, 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.


## 3.5. ADME-Tox Predictions

The 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. Therefore, 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. The results are collected in Tables 3 and 4.Table 3
Physicochemical properties of the most potent antibacterial pyrazole derivatives and the antibiotic control streptomycin.
EntryPhysicochemical propertiesMWLog PH-donH-accnrotbTPSA (Å2)12540.714.87041077.7613541.694.25051090.65Streptomycin581.578−6.65141511331.43MW: molecular weight expressed in daltons. Log P: octanol/water partition coefficient characterizing lipophilicity. H-don: number of hydrogen bond donors. H-acc: number of hydrogen bond acceptors. nrotb: number of rotable bonds. TPSA: total polar surface area.Table 4
Toxicity risks of the most potent antibacterial pyrazole derivatives and streptomycin.
EntryToxicityMutagenicTumorigenicReproductivityIrritant12NoneNoneNoneNone13NoneNoneNoneNoneStreptomycinNoneNoneNoneHighOverall, excepting the molecular weight which is found to be higher than 500 daltons, the two compounds12 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 (<140 Å2). These 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 compounds12 and 13, whereas it showed a high irritant effect of streptomycin.


## 3.6. Molecular Docking Study

The two compounds12 and 13 were docked with the active site of L-methionine γ-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 Thr354 and Arg374 amino acids with distances of 2.79 and 3.67 Å, respectively, and via carbon atoms in three Van der Waals interactions with Llp210, Gln348, and Thr354 residues. However, the study reveals that compound 13 has formed only one π-H interaction with Gly114 (2.65 Å) residue and two Van der Waals interactions with Gln348 and Cys115 residues (2.25 and 2.33 Å, 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.Figure 7
Interaction network of compound12 (a) and compound 13 (b) with L-methionine γ-lyase enzyme residues.
(a)(b)The binding scores (ΔGbinding) were also analyzed to measure the affinity of the selected ligands for the receptor. The most negative value of ΔGbinding 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 ΔGbinding 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.Table 5
Score, interactions, and bond length obtained for compounds12 and 13 with MGL enzyme.
EntryΔGbinding in kcal/molInteractionsBond length (Å)12−0.86CH3—H(Llp210)C(N-C-N)—H(Cys115)CH3—H(Gln348)5-ring—H(Thr354)CH3—O(Thr354)5-ring—H(Arg374)2.292.322.442.792.803.6713−6.71CH3—H(Gln348))C(N-C-N)—H(Cys115)5-ring—H(Gly114)2.252.332.65Overall, these docking results revealed clearly that the interaction between ligand12 and MGL is higher and stronger (ΔGbinding = −6.86 kcal/mol) than that between compound 13 and MGL (ΔGbinding = −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.


## 4. 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. The ligand protein docking study against L-methionine γ-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.



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*Source: 6663245-2021-02-24.xml*