Synthesis, Molecular Docking and Characterization of Pyrazole N-Mannich Base Derivatives as Antimicrobial Agents

 

Arathi K N¹*, Sindhu T J¹, Vishnu M V², Basith M A³, Anitha S V¹, Annlisa Roy¹,
Arundhathi T¹, Ashly George¹, Asish S¹

¹Department of Pharmaceutical Chemistry, Sanjo College of Pharmaceutical Studies,

Vellapara, Kuzhalmannam, Palakkad, Kerala - 678702, India.

²Department of Pharmaceutical Chemistry, Chemists College of Pharmaceutical Sciences and Research, Puthencruz, Ernakulam, Kerala - 682308, India.

³Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER) S.A.S. Nagar, Sector 67, S.A.S. Nagar, Mohali, Punjab -160062, India.

 

ABSTRACT:

In this work we aimed to design synthesis and evaluate the N-Mannich bases of pyrazole. A novel series of N-Mannich bases of pyrazole analogues were designed and synthesized with an effort to overcome the increasing antibiotic resistance. Tyrosyl-tRNA synthetase (TyrRS) comprises an N-terminal domain, which has the fold of the class I aminoacyl-tRNA synthetases. Computational Autodock 4.2 tools will be employed in this study for docking of pyrazole ligand molecules against Tyrosyl-tRNA synthetase (TyrRS) of Escherichia coli (PDB code: 1x8x) and Staphylococcus aureus (PDB code: 1jil.pdb). Molinspiration server was used for lead optimization. The ligand molecules were subjected to molecular docking studies with enzyme Tyrosyl-tRNA synthetase. The molecular docking studies are supported to compare in-vitro antibacterial activity by the use of binding energy of the docked ligand molecules. The newly synthesized compounds were characterized by UV, IR and various physico-chemical methods. Further, the antibacterial activity of N-Mannich bases of pyrazole compounds were assessed with zone of inhibition by agar well diffusion method using gram negative bacterial strain Escherichia coli and gram-positive strain staphylococcus aureus. These same compounds were subjected to find the antifungal activity against Aspergillus fumigates and Aspergillus Niger.

 

 

 

INTRODUCTION: 

Pyrazole^1-10 is the simple organic compound of the heterocyclic diazole series characterized by a five-membered ring structure composed of three carbon atoms and two nitrogen atoms in adjacent positions. Now a day’s vast numbers of compounds with Pyrazole^11-14 nucleus have been reported to show a broad spectrum of biological activity including antimicrobial, antiviral, anti-tumour, anti-histaminic, anti-depressant, insecticides and fungicides.

 

Due to its wide range of biological activity, pyrazole ring constitutes a relevant synthetic route in pharmaceutical industry.

 

The condensation of a compound having one or more active hydrogen atoms with formaldehyde and ammonia or a primary or secondary amine usually as hydrochloride, resulting in the formation of β-aminocarbonyl compound is known as Mannich reaction. The products of the Mannich reaction are known as Mannich bases and many are useful as intermediates in synthesis. Tyrosyl-tRNA synthetase (TyrRS)^15,16 comprises an N-terminal domain, which has the fold of the class I aminoacyl-tRNA synthetases, followed by idiosyncratic domains, which differ in eubacteria, archaebacteria and eukaryotes. The tRNA binding site spans the two subunits of TyrRS, which interacts with tRNATyr according to a class II mode. The conserved sequences of class I, HIGH and KMSKS, are involved in the catalysis of tyrosine activation.

 

MATERIALS AND METHODS:

Molecular modeling^17-20:

Softwares and databases used include Auto Dock 4.2 combines, Python 2.5, Accelrys discovery studio viewer, Molinspiration server, RCSB protein data bank, Online SMILES translator. The leads selected from literature were substituted with various substituents and they were optimized for the pharmacokinetic parameters by evaluating the oral absorption property using molinspiration server and preADMET software. After the lead has been optimized, the protein was subjected to docking studies using Auto Dock 4.2 for evaluating the binding interactions. The protein downloaded from PDB for tyrosyl tRNAsynthetase of E. coli (1x8x) and tyrosyl tRNA synthetase of S.Aureus (1jil.pdb) was used for the present docking studies. The final docked conformations were ranked according to their binding free energy. The pharmacophore modelling of the docked pose were generated.

 

Preparation of dimethyl pyrazole^21,22:

A mixture of 10mmol of acetyl acetone and 10mmol of hydrazine hydrate in 20ml ethanol was refluxed for 3 hours. Distil the ethanol and precipitate crystallizes.

 

Preparation of N-mannich base of pyrazole^23-26:

To a solution of (step 1) 0.01 mol DMF, formaldehyde (0.02 mol) was added under stirring. The reaction mixture was stirred at room temperature for 30 minutes to complete the reaction of formaldehyde and to yield methylol derivative. To this solution primary and secondary amine was added drop by drop and refluxed for 2 hours to 8 hours.The reaction mixture was poured into ice cold water and filtered washed with hot water. Finally, dried recrystallised from alcohol.

 

N-[(3,5-dimethyl-1H-pyrazol-1-yl) methyl] aniline:

Solid, % yield: 78, m.p: 100, Rf: 0.872, λmax: 298, IR(KBr, cm-1) vmax: 3043.59 (Aromatic CH str), 3287.87 (2°amine NH), 1451(methylene CH2).

 

4-{[(3,5-dimethyl-1H-pyrazol-1-yl) methyl] amino} benzoic acid:

Solid, % yield: 74, m.p: 110, Rf:0.75, λmax: 393,IR (KBr, cm-1) vmax: 1022.32 (C=N str), 3382.96 (2°amine NH), 1493.40 (methylene CH2), 1653.65 (C=O), 1413.48 (OH bend), 1595.42 (aromatic C-N), 1171.56 (N=N str)

 

 

4-{[(3,5-dimethyl-1H-pyrazol-1-yl) methyl] amino} benzenesulfonic acid:

Solid, % yield: 65, m.p: 120, Rf:0.75, λmax: 290, IR(KBr, cm-1) vmax:2880 (2°amine NH), 1933.38 (methylene CH2),1319.33 (aromatic C-N), 1319.33 (O=S=O), 1422.94 (OH bend).

 

N-[(3,5-dimethyl-1H-pyrazol-1-yl) methyl] benzene-1,2-diamine:

Solid, % yield: 70, m.p: 165, Rf:0.707, λmax: 285, IR(KBr, cm-1) vmax:3261.58 (2°amine NH), 1676.40 (Aromatic CH bend), 1423.59 (methylene CH2), 1172.70 (N=N).

 

Antibacterial screening^27-29:

The antibacterial activity of synthesized compounds (A1-A4) was assessed against Escherichia coli and Staphylococcus aureus agar disc diffusion method. The sterilized (autoclave at 120°C for 30 minutes) nutrient agar medium (40-50°C) was inoculated with the suspension of microorganism and mixture was transferred to sterile Petri dishes and allowed to solidify. In each plate 10mm disc was placed. Ciprofloxacin disc of conc. 1µg placed kept as standard and 20µl synthesized compounds (200µg/µl) solutions in DMF is poured. The plates were kept in refrigerator for 30 minutes to allow the diffusion of sample to the surrounding agar medium. The plates were incubated at 37±2°C for 24 hours and observed for antibacterial activity. The diameter of zone of inhibition were measured and compared with that of standard.

 

Antifungal screening^30-33:

The antifungal activity of synthesized compounds (A1-A4) was assessed against Aspergillus fumigates and Aspergillus niger agar disc diffusion method the sterilized (autoclaved at 120 ̊C for 30 minutes) Mueller Hinton agar was inoculated with the suspension of microorganism and mixture was transferred to sterile Petri dishes and allowed to solidify. In each plate cup of 6 mm diameter bores were made at equal distances using sterile steel borer. All the cups were filled with 20µl of standard drug i.e. cotrimoxazole, and 20µl of synthesized compounds (200µg/µl) solutions in DMF is poured. Then the plates were kept in refrigerator for 30 minutes to allow the diffusion of surrounding medium. The plates were incubated at 37±2°C for 48 hours and observed for anti-fungal activity. The diameter was measured compared with that of standard the values are tabulated.

 

RESULTS:

For the present study, we have selected tyrosyl tRNA synthetase of E. Coli and S. Aureus as target enzyme for antibacterial activity based on literature review. Safety and efficacy of the lead compounds were evaluated by observing the computation of drug like properties. Four compounds (A1-A4) were subjected to in-silico lead optimisation. Oral bioavailability was evaluated by using Molinspiration server and preADMET software. All the compounds possessed better bioavailability and permeability mentioned in table 1 and 2.

 

Table 1: Drug likeness scores of A_1-4using molinspiration server

 

Table 2: ADME data

 

For the antibacterial docking study, E. coli tyrosyl tRNA synthetase (1x8x.pdb) Aureus tyrosyl tRNA synthetase (1jil.pdb) proteins were used. Docking of the ligands was done by the use of AutoDock 4.2 software and the interactions with the active site of enzyme were studied. The derivatives were subjected to interactions with E.coli tyrosyl tRNA synthetase (inhibited by tyrosine) and S. Aureus tyrosyl tRNA synthetase (inhibited by SB284485) mentioned in table 3 and 4. Most of the derivatives were interacting with the key active site of the tyrosyl tRNA synthetase with superior binding energy. In N-Mannich base of dimethyl Pyrazole series, all the compound shown moderate binding interaction against E. coli tyrosyl tRNA synthetase. Among these, sulphonic acid derivative of dimethyl Pyrazole (A3) shown the excellent binding energy -7.68 kcal/mol compared to standard ciprofloxacin (-9.14 kcal/mol). Other derivatives such as A2 (para-amino benzoic acid derivative) shown good binding energy -6.84 kcal/mol. Remaining A4 (orthophenylene diamine derivative) and A1 (Aniline derivative) shown nearly similar and least binding energy -6.27 and 6.21 kcal/mol. In N-Mannich base of dimethyl Pyrazole series, all the compound shown excellent binding interaction against Aureus tyrosyl tRNAsynthetase. Among these, para amino benzoic acid derivative of dimethyl Pyrazole (A2) shown the excellent binding energy -7.88kcal/mol compared to standard ciprofloxacin (-7.67kcal/mol). Other derivatives such as A3 (sulphonic acid derivative) shown very good binding energy -7.58 kcal/mol. A4 (orthophenylene diamine derivative) shown moderate binding energy of -6.27 kcal/mol and A1 (Aniline derivative) shown least binding energy -6.14kcal/mol. Snapshots of docked poses were shown in figure 1 and 2.

 

 

Table 3: Binding energies of A_1-4with tyrosyl tRNAsynthetase of E. coli (1x8x.pdb)

 

 

Table 4: Binding energies of A_1-4with tyrosyl tRNA synthetase of S.aureus (1jil.pdb)

 

Figure 1: Docked poses of ligand and standard ciprofloxacin against tyrosyl tRNA synthetase of E.Coli

 

Figure 2: Docked poses of ligand and standard ciprofloxacin against tyrosyl tRNA synthetase of S.aureus (1jil.pdb)

 

In this scheme (figure 3), the N-Mannich base of primary amine is prepared by two step process. At the first step we have prepared dimethyl pyrazole from acetyl acetone and hydrazine hydrate. Then dimethyl pyrazole from the step 1 is treated with formaldehyde and primary amine under reflux for 2-8 hrs gives the product. Melting point of all the newly synthesised compounds was determined. Rf values were determined to confirm the completion of the reaction. The structures of newly synthesized compounds were characterised by physical, UV and IR spectral studies.

 

Figure No. 3: Synthetic route of N-mannich bases of pyrazole

 

Antibacterial screening of the synthesized compounds were done by Agar well plate method in nutrient agar medium and ciprofloxacin was used as the standard. All newly synthesized compounds A1-4 showed antibacterial activity towards the tested clinical strain of Escherichia coli and Staphylococcus aureus. Among these, N-Mannich bases compound against E. coli, A3 (sulphanilic acid derivative) shown very good zone of inhibition of 10mm as compared to standard (ciprofloxacin) zone of 13mm. A4 (orthophenylene diamine) also shown good activity of Zone of inhibition 8mm. Remaining A1(Aniline derivative derivative) and A2 (para amino benzoic acid derivative) shown least activity of 7mm.Among these, N-Mannich bases compound against. S. Aureus, A2 (Paraamino benzoic acid derivative) shown very excellent zone of inhibition of 12mm. A1 (aniline derivative) also showed good zone of inhibition of 9mm. Remaining A3(sulphanilic acid derivative) and A4(orthophenylene diamine) shown least activity of 7mm.Antifungal screening of the synthesized compounds was done by Agar well plate method in Mueller Hinton nutrient agar medium and cotrimoxazole was used as the standard. All newly synthesized compounds A1-4 showed anti-fungal activity towards the tested clinical strain of Aspergillus fumigatus and Aspergillus niger. Among these, N-Mannich bases compound against Aspergillus fumigatus, A2(Para amino benzoic acid derivative) shown same zone of inhibition that of standard zone of 10mm. A1(aniline derivative) shown moderate Zone of inhibition 6mm. Remaining A3 and A4has no zone of inhibition. Among these, N- Mannichbases compound against Aspergillus Niger, A2 (Para aminobenzoic acid derivative) shown good zone of inhibition of 13mm. A3 (sulphanilic acid derivative) also showed good zone of inhibition of 9mm. Remaining A1(aniline derivative) and A4 (orthophenylene diamine) shown least activity of 7mm and 6mm. Zone of inhibition (mm) showed in table 5.

 

 

Table No. 5: Antimicrobial activity of test compounds against Escherichia coli, Staphylococcus aureus, Aspergillus fumigates and Aspergillus niger

 

DISCUSSION:

CADD helps to minimise the tedious drug discovery process over the traditional method. In silico drug likeness score and preADMET data of the ligands observed helped to predict a better pharmacokinetic activity and oral bioavailability of the designed leads. The binding energies obtained from docking study of tyrosyl tRNAsynthetase of E. coli (1x8x.pdb) and tyrosyl tRNA synthetase of Aureus (1jil.pdb) confirms that the lead compound inhibits the enzymes present in Escherichia coli and Staphylococcus aureus. Designed schemes were utilised in the preparation of novel dimethyl pyrazole incorporated N-mannich bases of primaryamine.The screening of synthesized compounds for antibacterial study against Escherichia coli revealed that sulphanilic acid derivative of N-mannich base of dimethyl pyrazole showed excellent activity. Similarly, activity against Staphylococcus aureus reveals that Para amino benzoic acid derivative showed excellent activity. The results of activity study coincide the molecular docking study. From this work we can conclude that ligands have potential activity against bacterial strains.Thus, the present study depicts that the utilization of computeraided drug design is an efficient tool in predicting the effectiveness of a series of compounds under study and thus can result in the design of potent antibacterial agent.

 

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Accepted on 21.07.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1047-1052.