Green One-pot Synthesis of Novel Polysubstituted Pyrazole Derivatives as Potential Antimicrobial Agents

Various biological properties of natural and synthetic pyrazole derivatives such as anti-inflammatory, antimicrobial, neuroprotective, anticonvulsant, antidepressant and anticancer activities encouraged us to propose a new, fast, green and eco-friendly procedure for the preparation of some novel 5-amino-3-(aryl substituted)-1-(2,4-dinitrophenyl)-1H-pyrazole-4-carbonitriles. They were efficiently synthesized via one-pot two-step process reaction of malononitrile, 2,4-dinitrophenylhydrazine and different benzaldehydes in deep eutectic solvent (DES) glycerol/potassium carbonate. The products yield and reaction times were considerably improved in the presence of applied DES. Antibacterial effects of all newly synthesized pyrazoles in comparison with several common antibiotics were evaluated against a variety of Gram-positive and Gram-negative pathogenic bacteria. In addition to, their inhibitory activities on three fungi were compared to some current antifungal agents. The moderate to good antimicrobial potentials particularly against fungi were observed in the major heterocyclic compounds according to the IZD, MIC, MBC and MFC results.


Introduction
Pyrazoles are an important class of azoles containing two adjacent nitrogen atoms, which are found as major or minor scaffolds in various medicinal compounds and natural products. L-α-Amino-β-(pyrazolyl-N)-propanoic acid and withasomnine, which were isolated from Citrullus vulgaris (watermelon) juice and from the roots of Withania somnifera Dun (Solanaceae), in fact, are two of the few naturally occurring pyrazoles that have found potential use as anti-diabetic and depressant agents in medicinal chemistry. 1,2 Pyrazofurin and formycin are natural C-nucleoside antibiotics that are used to treat viral infections as well as inhibition of tumor cells growth. Stanozolol is a synthetic anabolic steroid that can be applied for treatment of anaemia and hereditary angioedema. In addition to, the pyrazole ring as a part of the chemical structure of drugs such as antipyrine, celecoxib and betazole, plays an essential role in the relief of ear pain and swelling, improvement of osteoarthritis signs, and treatment of bacterial and fungal infections ( Figure 1).
Compounds containing pyrazole moiety exhibit a wide variety of biological and pharmacological activities including analgesic, neuroprotective, anticonvulsant, angiotensin converting enzyme (ACE) inhibitory, anti-angiogenesis, antioxidant and antiviral activities. [3][4][5][6][7][8][9] Numerous studies have also focused on antibacterial and antifungal properties of pyrazole derivatives. [10][11][12] In a research project, inhibitory activities of some heterocyclic Schiff bases derived from thiocarbohydrazide were assessed against various pathogenic bacterial and fungal strains via measurement of their inhibition zone diameters. One of the synthesized 1,2,4-triazines could block the growth of all selected microorganism. 13 Various methods were proposed for the synthesis of pyrazole and their analogues. [14][15][16][17] In this regard, a solution of the appropriate triethylamine in 1,4-dioxane efficiently catalysed synthesis of pyridine, thiophene and 4H-pyrane Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted ... derivatives via one-pot or multicomponent protocols. 18 Similar procedures were designed to prepare pyrazole derivatives. [19][20][21][22][23][24] Most of these methods include simultaneous or multistep reaction of aldehyde, hydrazine and active methylene compounds under different conditions. [25][26][27][28][29] Recently, deep eutectic solvents (DESs) were widely applied as eco-friendly media or efficient catalysts in organic synthesis especially for the preparation of pyrazoles. [30][31][32] Glycerol/potassium carbonate is a new class of DES having its physical properties, such as surface tension, viscosity, density and refractive index, carefully measured. 33 In order to apply glycerol/K 2 CO 3 system in organic synthesis, some novel 5-amino-1-(2,4-dinitrophenyl)-1H-pyrazole-4-carbonitrile derivatives were prepared via the reaction of malononitrile, 2,4-dinitrophenylhydrazine and various benzaldehydes. The in vitro antimicrobial activities of synthesized derivatives were studied against a variety of pathogenic bacteria and fungi, as well as structure-activity relationships were expanded.

1. Chemicals
All reagents, solvents, antibiotics and antifungal agents were purchased from commercial sources (Merck, Sigma and Aldrich), and used without further purification. The bacterial and fungal culture media were obtained from HiMedia. Melting points were determined with Kruss type KSP1N melting point meter and are uncorrected. Reaction progress was monitored by aluminium TLC plates pre-coated by silica gel with fluorescent indicator F254 using CH 2 Cl 2 /CH 3 OH (9:1, v/v) as the mobile phase, being visualized under UV radiation (254 nm). FT-IR spectra of the products were collected using Bruker Tensor-27 FT-IR spectrometer. 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker FT-NMR Ultra Shield-400 spectrometer. Elemental analyses were performed for C, H and N on a Thermo Finnigan Flash EA microanalyzer. DESs were prepared in various ratios of glycerol/K 2 CO 3 according to the procedures reported by Naser et al. 33 as follows: the mixture of different molar ratios of potassium carbonate and glycerol were vigorously stirred at 80 o C for 2 h to gain homogenous transparent colorless liquids.
The reaction conditions were optimized in terms of solvent, presence or absence of the catalyst and temperature. 1 mmol each of malononitrile (1), 4-acetamidobenzaldehyde (2a) and 2,4-dinitrophenylhydrazine (3) were reacted under different conditions (Table 1). Glycerol as a green, cheap, non-toxic, inflammable and readily available solvent was the component present in all reactions. No target products were obtained when the reaction mixture was stirred at room temperature. The solubility of reagents was improved as the viscosity of glycerol largely decreased with increasing temperature to 80 °C. All efforts to perform three-component reaction in media containing glycerol alone were unsuccessful (Entries 1, 2). The formation of Schiff bases as major products in glycerol showed that the presence of K 2 CO 3 catalyst is required for the synthesis of pyrazoles (Entries 3-6). There are two possible mechanisms to form the products, but it seems that only route b will afford the final compounds 4a-f (Scheme 2). Schiff-base condensation reaction was observed in route a under some of the applied conditions. Colorless solutions of various molar ratios of potassium carbonate to glycerol (DES1, 1:4; DES2, 1:5; DES3, 1:6) were selected because their physical properties, including conductivity, surface tension, viscosity, refractive index, density and pH have been evaluated very well in the temperature range of 10-80 °C. 33 Three-component reaction in 0.5 g of each of these three DESs at room temperature have resulted in the Schiff bases and benzylidenemalononitriles as the major products (Entries 7, 9, 11), pyrazoles were obtained in 35-40% yields due to the increase of temperature to 80 °C (Entries 8, 10, 12). One-pot twostep process was screened according to route b. Two-step procedure was carried out in all DESs, the increase in molar ratios of glycerol reduced the product yield (Entries [13][14][15][16][17][18]. This can be caused by the higher pH of DES1. The first stage reaction did not proceed completely at room temperature or at higher temperature even after 8 h, due to the lack of appearance of the intermediary benzylidenemalononitriles in the reaction media. Therefore, the next stage reaction of unconsumed reagents especially aldehydes with hydrazine was inevitable under these conditions (Entries 13,15,17). Adding water to achieve the final DES1/H 2 O ratios of 1:2, 1:1 and 3:1 (w:w) has improved reaction time and product yield, the condensation reaction of malononitrile with aldehyde The molecular structures and purity of the newly synthesized compounds were identified by NMR ( 1 H and 13 C), FT-IR and elemental analysis (CHN). In FT-IR spectra, absorption bands attributed to symmetric and asymmetric stretching vibrations of amino groups appeared within ν = 3428-3456 and 3281-3326 cm -1 , as well as stretching vibrations of nitro groups were recorded within ν = 1514-1543 and 1318-1331 cm -1 . The presence of nitrile groups was deduced both from IR bonds and 13 C NMR signals appearing at ν = 2206-2228 cm -1 and δ 113.67-117.37 ppm. In addition to these, 1 H NMR spectra and microanalytical data are in agreement with the chemical structures.

2. Antimicrobial Evaluation
The in vitro inhibitory activities of the newly synthesized derivatives were evaluated against a variety of pathogenic bacteria and fungi. Amikacin, ceftriaxone and penicillin belonging to aminoglycoside, cephalosporin and penicillin antibiotics, respectively, were used as positive antibacterial controls, as well as antifungal agents including terbinafine, fluconazole and nystatin. The antimicrobial effects were presented as IZD, MIC, MBC and MFC values in Tables 3 and 4.
According to the data reported in Table 3, the derivatives were ordered based on the spread of inhibitory properties and the MIC values as follows: 4b > 4e > 4d > 4c > 4f > 4a. The 3-phenyl ring in pyrazole derivative 4b was substituted by a methoxy group at para position, it was the only compound synthesized effective against Streptococcus pyogenes and Proteus vulgaris. The pyarazole 4a containing p-acetamidophenyl substituent was effective only against Gram-negative Salmonella typhi. The inhibitory effects of derivative 4e including 2,4-dichlorophenyl substituent were more significant than those of the derivative 4f with 2,6-dichlorophenyl substituent. Among pyrazoles 4a-f, the antibacterial properties against Proteus mirabilis and  Three-component, rt  240  -2  Gly  Three-component, 80°C  120  Schiff base  3 Gly One-pot two-step process (route a), rt 240 -4 Gly One-pot two-step process (route a), 80 °C 180 Schiff base 5 Gly One-pot two-step process (route b), rt 240 -6 Gly One-pot two-step process ( One-pot two-step process (route b), 80 °C 40 68 a Gly as glycerol; b Ratios as w:w; The amount 0.5 g of solvents containing glycerol was used. Under the optimized conditions, mono and disubstituted benzaldehydes 2b-f were also reacted with malononitrile (1) and 2,4-dinitrophenylhydrazine (3) to afford pyrazoles 4b-f. The results are presented in Table 2.  Shigella dysenteriae were observed for the compounds 4d and 4f, respectively. Amikacin in comparison with two other antibiotics could block the growth of all bacteria. The in vitro antifungal activities of prepared pyrazoles were also evaluated and the results were promising. No inhibitory effect was observed with derivative 4d containing 2-hydroxy-3-methoxyphenyl substituent at the 3-position of the pyrazole ring. The dichloro compounds 4e and 4f had the same antifungal properties despite their different stereochemistry. Data gathered in Table 4 show that terbinafine has more remarkable effects than the others.

Conclusions
An efficient, one-pot two-step procedure was proposed and the synthesis of polysubstituted pyrazoles has been carried out. Some deep eutectic solvents including different molar ratios of potassium carbonate to glycerol were prepared and applied as reaction media and catalyst in this synthesis. The best results in terms of product yields and reaction times were achieved in molar ratios 1:4:14 of K 2 CO 3 / glycerol/H 2 O. Efficiency of DES K 2 CO 3 /glycerol in organic synthesis is currently under our investigation, and will be in focus of our future research. Furthermore, antimicrobial ac-tivities of all synthesized derivatives were evaluated against a broad range of pathogenic bacteria and fungi. Based on the broad-spectrum inhibitory effects of the pyrazole 4b, including 4-methoxy group on 3-aryl ring, it is suggested that benzaldehydes with small para electron donating substituents should be used to synthesize future active analogues.

Acknowledgements
This work was supported by the University of Zabol under Grant number UOZ-GR-9517-15.