Synthesis, antimicrobial evaluation and docking studies of new pyrazolone derivatives

Purpose: To synthesize new antimicrobial azo-pyrazolone derivatives III & IV and evaluate their antimicrobial activities using a combination of in vitro and molecular docking studies. Methods: Azopyrazolone compounds were prepared from the reaction of substituted aniline diazonium with ethyl acetoacetate to give azoxobutyric acid derivatives (II) which were then reacted with phenyl hydrazine or hydrazine hydrate. The pyrazolone derivatives (IV) were acetylated with glacial acetic acid to yield new acetylated pyrazolones (V). An agar dilution method was used to demonstrate the antimicrobial activities of the pyrazolone derivatives and their minimum inhibitory concentration (MIC) values calculated. Molecular docking studies were employed to further evaluate the most active compounds (on the basis of the MICs obtained). Results: The new pyrazolone derivatives showed varying antimicrobial activities (from negligible to strong) against a number of microorganisms. Derivatives IIIb and Vb showed potent activities against Bacillus subtilis, Sarcina lutea, Staphylococcus aureus and Enterococcus faecalis. However, the new compounds did not show antifungal activity. Molecular docking results for compounds IIIb and Vb were consistent with their antimicrobial activities and proved that the compounds inhibited glucosamine-6phosphate synthase. Conclusion: The new dichloropyrazolone compounds IIIa and Vb possess potent antimicrobial activities. These compounds have promising potential for use as new antibacterial agents or as templates for the design of new antimicrobial drugs.


INTRODUCTION
Literature survey has revealed that pyrazolone compounds exhibit a variety of biological activities [1][2][3][4][5].For example, the azo-pyrazolone derivatives have potent cytotoxic [6,7], anti-inflammatory [8-10] and antibacterial activities [11].Interestingly, pyrazolones 1 and 2 have antimicrobial activities and are considered novel types of non-nucleoside reverse transcriptase inhibitors against HIV-1 [12].However, drazoxolon 3 is an azo compound containing isoxazolone ring (ring isostere of pyrazoles) attached to phenyl hydrazone, and is used as a commercial fungicidal agent [13].In the review of reported biological activities of these derivatives, it was seen that a number of substituted phenylhydrazones bearing a pyrazole ring (IIIa-c, IVa-c and Va-c) have been synthesized (Figure 1).The synthesis of new pyrazolone compounds may be a valuable way for producing novel antimicrobial drugs.

EXPERIMENTAL Instrumentation
Melting points, NMR, IR, mass spectroscopy and elemental composition were measured in accordance with standard procedures reported in the literature [6][7][8][9].All reagents used in this study were purchased from Aldrich Chemical Company (Milwaukee, WI).The authors synthesized compounds II b and IIc, and compounds IVb and IIc in line with reported methods [14][15][16].

Synthesis of 2-[(3, 4-dimethoxy-phenyl)hydrazono]-3-oxo-butyric acid ethyl ester (II a)
To a mixture 3,4-dimethoxyaniline (0.01 mol) in aqueous HCl (10 %, 10 mL), an equal volume of ice-cooled NaNO 2 solution was added in aliquots for over 20 min with vigorous stirring to afford the diazonium salt.To an ice-cooled solution of the ethyl acetoacetate (0.01 mol) and sodium acetate (0.02mol) in aqueous ethanol (50%, 20 mL), the diazonium salt was added for 3 h with stirring.Following filtration, the product was rinsed in water and purified by crystallization from m ethanol to yield compound IIa.

Method used for synthesizing IIIa-c
A mixture of IIa-c (0.015 mol) in absolute ethanol (20 mL), and phenyl hydrazine (0.03 mol) was continually refluxed under heat for 8 h.On cooling, the product was rinsed in water, and after drying, it was subjected to crystallization from acetic acid.

General procedure for synthesis of Va-c
Pyrazolone compounds IV a-c (0.01 mol) were heated under reflux in glacial acetic acid (30 mL) for 8 h.The reaction mixture was cooled to room temperature and added to ice-cooled water (100 mL).The resultant precipitate was filtered, dried and crystallized from ethanol.

Molecular modeling studies
Docking experiments were performed using MOE ver.

Antimicrobial activity of synthesized compounds (MIC)
The compounds showed good antibacterial activities against Pseudomonas aeruginosa especially samples IIIb and Vb, which showed high activity against gram positive (Enterococcus faecalis, Staphylococcus aureus and Sarcina lutea) and gram negative (Salmonella typhi, E. coli and Pseudomonas aeruginosa) organisms, as well as Gram positive rods (Bacillus subtilis).All compounds were inactive against fungi (Candida albicans).Their MICs are shown in Table 1.

DISCUSSION
Compound IIa had the characteristic NMR peaks of methyl and ethyl groups.The cyclization of compounds IIa-c with phenyl hydrazine afforded the poly-substituted pyrazolone III.The HNMR of pyrazolones III a-c showed increasing number of aromatic proton peaks and disappearance of the ethyl group peaks.The butyric diazo compound II was reacted with hydrazine hydrate to give the new pyrazolone compounds IV a-c.Disappearance of aliphatic ethyl group HNMR peaks and appearance of NH peak confirmed the structure of compound IV a.The pyrazolone compounds IV a-c were acetylated with glacial acetic acid to produce the new acetylated pyrazolones V a-c.The appearance of new methyl of acetyl group peak and disappearance of NH peak confirmed the structure of compounds Va-c.
The dichlorophenyl group in pyrazolones IIIb and Vb has important role in the antibacterial activities of the pyrazolone compounds.In addition, the substitution at the nitrogen atom of pyrazolone with acetyl or phenyl ring enhances their activities.
To identify the possible mechanism of action of the newly synthesized compounds, they were subjected to molecular docking studies with glucosamine-6-phosphate synthase using the program of MOE site finder.D-glucosamine-6phosphate (D-Glcm6P) is produced from Dfructose-6-phosphate using L-glutamine as nitrogen source in a reaction catalyzed by glucosamine-6-phosphate synthase.Thus, the inhibition of glucosamine-6-phosphate synthase results in inhibition of production of Nacetylglucosamine which is a major unit in bacterial cell wall building.Consequently, the enzyme is an essential target for antibacterial agents.The docking study was performed using reported methods [6][7][8][9].In this study, the most active compounds IIIb and Vb from antimicrobial studies were docked into MurA-F binding site to confirm the ability of the novel candidates to act as antibacterial agents.Glucosamine-6-phosphate synthetase in complex with Glcm6P as a ligand was sourced from a data bank (PDB: ID 2VF5).The energy (S) of re-docking of Glcm6P into glucosamine-6phosphate synthase was-15.39kcal/mol.Ala602, Val399, Thr302, Gln348, Ser303, Ser349, Ser347 and Thr352 contributed nine Hbonds involving -NH 2 , -OH and -PO 4 groups (Figure 2).

Finally, the
candidate Vb showed an energy score of -16.33 Kcal/mol and exhibited four hydrogen bonding interactions as follows: (i) Ala602 with NH, (ii) Ser303 with C=O, (iii) Gln348 with C=O and (iv) Thr352 with pyrazole C=O (Figure-4).