Synthesis, Characterization, Antimicrobial Screening and Free-Radical Scavenging Activity of Some Novel Substituted Pyrazoles

The present work deals with the synthesis of acetoxysulfonamide pyrazole derivatives, substituted 4,5-dihydropyrazole-1-carbothioamide and 4,5-dihydropyrazole-1-isonicotinoyl derivatives starting from substituted vanillin chalcones. Acetoxysulfonamide pyrazole derivatives were prepared from the reaction of chalcones with p-sulfamylphenylhydrazine followed by treatment with acetic anhydride. At the same time 4,5-dihydropyrazole-1-carbothioamide and 4,5-dihydropyrazole-1-isonicotinoyl derivatives were prepared from the reaction of chalcones with either thiosemicarbazide or isonicotinic acid hydrazide, respectively. The synthesized compounds were structurally characterized on the basis of IR, 1H-NMR, 13C-NMR spectral data and microanalyses. All of the newly isolated compounds were tested for their antimicrobial activities. The antimicrobial screening using the agar well-diffusion method revealed that the chloro derivatives are the most active ones. Moreover, the antioxidant and anti-inflammatory activity of these chloro derivatives are also studied using the DPPH radical scavenging and NO radical scavenging methods, respectively.


Chemistry
The synthesis of chalcones 1a-e was accomplished by a one-pot Claisen-Schmidt condensation [44,45] in 60% ethanol between the appropriate acetophenone derivative and 4-hydroxy-3-methoxybenzaldehyde (vanillin). In all the synthesized chalcones, only the trans double bond was obtained (on the basis of the corresponding NMR coupling constant). All synthesized compounds were characterized by spectral data (IR, 1 H-NMR and 13 C-NMR) that was consistent with the proposed structures. The IR spectra of 1a-e revealed the characteristic bands for C=O at 1661-1684, vinyl CH=CH that appeared at 1593-1618 and OH at 3424-3441 cm −1 . The 1 H-NMR spectra showed the presence of a broad singlet at δ = 11.22-11.72 ppm for the phenolic OH proton, multiplets at δ = 7. 30 All of our synthesized compounds, chalcones 1a-e, hydrazones 2a-e, pyrazoles 3a-e, and dihydropyrazoles 4a-e and 5a-e were tested for their antimicrobial activity against four test organisms, namely Staphylococcus aureus ATCC6538P, Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC9027, and Candida albicans ATCC2091 using rifampicin (5 μg/disc) and ampicillin (10 μg/disc) as standard drugs. The agar well-diffusion method [46] was used for studying the potential activities of these compounds. Pyrazoles 3a-e showed no significant effect, whereas all other compounds showed potent activity only against Staphylococcus aureus and Candida albicans in the following order: 2a-e > 4a-e > 1a-e ≥ 5a-e. Minimum inhibitory concentration (MIC) values for the individual compounds that showed inhibition zones > 10 mm were determined by means of the agar well-diffusion method in DMSO. The trend of activity was observed as follows: X > H > OMe > NO2 where X = Cl, Br. It is obvious that the presence of pharmacophores such as chloro and bromo substituents with lipophilic properties increases the antimicrobial activity. The activity results of our synthesized compounds against S. aureus, C. albicans are shown in Table 1 as zone of inhibition (in mm) and minimum inhibitory concentration, MIC (mg/mL). Also, minimum bactericidal concentrations (MBC) were determined for all the chloro derivatives 1c, 2c, 4c and 5c which exhibited high activities. These results were listed in Table 2.

Evaluation of Antioxidant and Anti-inflammatory Activities
Two pharmacological activities, namely antioxidant and anti-inflammatory activities, were tested for the chloro derivatives 1c, 2c, 3c, 4c, 5c. These activities vary according to their structures and functional groups.
Resulting from a color change from purple to yellow, the absorbance decreased when the DPPH is scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH molecule, in the radical form this molecule had an absorbance at 517 nm, which disappeared after acceptance of an electron or hydrogen radical from an antioxidant compound to form the reduced DPPH-R (Scheme 2). Moreover antioxidants are known to interrupt the free-radical chain of oxidation and to donate hydrogen from phenolic hydroxy groups, thereby, forming stable free radicals, which do not initiate further oxidation [51]. Furthermore, substitution in the aromatic ring system with halogens like chlorine or bromine sharply enhanced the antioxidant potency [52], it is thought that the chlorine atom because of its lone pair electron as well as its electronegative power enhanced the formation and subsequent stabilization of the nitrogen-ring radical through intervening aromatic system property, it might have enhanced the power to absorb free radicals, especially reactive oxygen and reactive nitrogen species (ROS and RNS), this is explain why we select this series of our synthesized compounds. The present investigation emphasized mainly on the chloro derivatives which showed significant antioxidant activity, the screening of the selected synthesized compounds through structure-activity relationship (SAR) showed that compound 2c was found to be the most efficacious antioxidant among all the listed compounds. The antioxidant activity of 2c is directly proportional to the concentrations used. Antioxidant results of the synthesized compounds 1c, 2c, 3c, 4c and 5c are reported in Table 3. As reported in literature some substances can serve as either antioxidants or pro-oxidants, depending on conditions [53,54]. All the other tested compounds act as antioxidants at low concentrations (0.25 mg/mL) in the following order: 4c > 5c > 3c > 1c, while converted to pro-oxidant compounds at higher concentrations.

Anti-Inflammatory Activity (Scavenging of Nitric Oxide Radical)
Nitric oxide (NO) is a potent inhibitor of physiological processes such as smooth muscle relaxation, neuronal signaling, and inhibition of platelet aggregation and regulation of cell mediated toxicity [55]. In addition to reactive oxygen species, nitric oxide is also implicated in inflammation, cancer and other pathological conditions [56,57]. NO is known to be a ubiquitous free-radical moiety, which is distributed in tissues or organ systems and is supposed to have a vital role in neuromodulation or as a neurotransmitter in the CNS [58]. In our study all the chloro derivatives of the synthesized compounds 1c, 2c, 3c, 4c, 5c were tested for in vitro anti-inflammatory activity compared to the standard vitamin C, showing acceptable anti-inflammatory activity. All tested compounds act as anti-inflammatory in a concentration dependent matter. Among all the tested compounds, 4c was the most potent compound, followed by: 5c > 3c > 1c > 2c. The in vitro anti-inflammatory activity of tested compounds is summarized in Table 4.

General Information
Melting points were determined in open capillary tubes using Electrothermal apparatus 9100 (Fisher Scientific, Leicestershire, UK) and are uncorrected. Microanalyses were operated at Faculty of Science, Cairo University, Cairo, Egypt, using an Elementary Vario El III C, H, N, S Analyzer (Shimadzu, Berlin, Germany). IR spectra were recorded using the potassium bromide method on a Tensor 37 FT-IR spectrometer (Bruker, Ettlingen, Germany): and expressed in wave number (υmax) cm −1 . 1 H-NMR and 13 C-NMR spectra were measured in deuterated chloroform (CDCl3) or deuterated dimethyl sulphoxide (DMSO-d6) on an EAC 500 MHz FT-NMR spectrophotometer (Jeol, Eching, Germany). Chemical Shifts were recorded in δ as parts per million (ppm) downfield from tetramethylsilane (TMS) used as internal standard. Reaction progress and compound purity were monitored by Thin Layer Chromatography (TLC) using Alugram Sil G/UV254 silica gel plates (Macherey-Nagel, Easlon, PA, USA) and chloroform or chloroform-ethanol (9:1) or (19:1) as eluent systems. The spots were visualized using an ultraviolet lamp (Vilber Lourmet, Marine La Vallee, France) at λ = 254 and 266 nm. The antimicrobial activities were determined at the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University. Antioxidant and anti-inflammatory activity tests were performed at the Biochemistry Lab, Faculty of Science, Alexandria University.

General Procedure for the Preparation of 1a-e
The appropriate p-substituted acetophenone (10 mmol) was added to a cold stirred solution of sodium hydroxide (3 g in 50 mL of 60%aqueous ethanol) followed by addition of vanillin (10 mmol) dropwise with continuous stirring for five hours. The resulting crude solid was filtered, washed successively with water, dried and crystallized from ethanol (95%) [44,45,[59][60][61].

General Procedure for the Preparation of 3a-e
A mixture of the appropriate hydrazone 2a-e (10 mmol) and acetic anhydride (15 mL) was heated under reflux for three hours. After the reaction mixture attained room temperature, it was poured into crushed ice and the oily product deposited was decanted from water and extracted with ether. The ether layer was washed three times with water, dried over anhydrous sodium sulphate and evaporated. The precipitate obtained was crystallized from ethanol (95%) to afford the corresponding pyrazoles 3a-e as needles.

General Procedure for the Preparation of 4a-e
A mixture of the appropriate chalcone 1a-e (10 mmol) in ethanol (30 mL) was heated under reflux with thiosemicarbazide (12 mmol) in glacial acetic acid (2 mL) for 7 h, then the reaction mixture was poured into crushed ice and kept overnight at room temperature. The separated crude solid was filtered off, washed successively with water, dried and crystallized from ethanol/chloroform to give 4a-e as needles.

General Procedure for the Preparation of 5a-e
A mixture of the appropriate chalcone 1a-e (10 mmol) in ethanol (95%) (30 mL) was heated under reflux with isonicotinic acid hydrazide (10 mmol) in glacial acetic acid (2 mL) for five hours. The reaction mixture was treated as mentioned for the preparation of 4a-e to give the corresponding 4,5-dihydropyrazoles 5a-e.

Determination of Antimicrobial Activity
All compounds were tested against four different microorganisms Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans. The agar well-diffusion method was applied for the determination of inhibition zone and minimum inhibitory concentration (MIC). Briefly, 0.75 mL of broth culture containing ca. 10 6 colony-forming units (CFU) per mL of the test strain was added to 75 mL of nutrient agar medium at 45 °C, mixed well, and then poured into a 15 cm sterile metallic Petri plate. The medium was allowed to solidify, and 8 mm wells were dug with a sterile metallic borer. Then, a DMSO solution of the test sample (1 mL) at 1 mg/mL was added to the respective wells. DMSO served as negative control, and the standard antimicrobial drugs Rifampicin (5 μg/disc) and Ampicillin (10 μg/disc) were used as positive controls. Triplicate plates for each microorganism strain were prepared and were incubated aerobically at 37 °C for 24 h. The activity was determined by measuring the diameter of zone showing complete inhibition (mm), thereby, the zones were precisely measured with the aid of a Vernier caliper (precision 0.1 mm). The growth inhibition was calculated with reference to the positive control. For the individual compounds that showed inhibition zones >10 mm, MIC values were determined by means of the agar well-diffusion method for concentrations of 1.0, 0.50, 0.25, 0.125, 0.063 and 0.031 mg/mL in DMSO. The tests were performed in triplicate, and the results were averaged. Also minimum bactericidal concentrations (MBC) were determined for all chloro derivatives which exhibited high activities (compounds 1c, 2c, 4c and 5c) for concentrations of 1.0, 0.50, 0.25, 0.125, 0.063 and 0.031 mg/mL in DMSO. All our results are listed in Tables 1 and 2.

DPPH Based Free Radical Scavenging Activity
Since DPPH is a stable free radical containing an odd electron in its structure, it is usually utilized for detection of the radical scavenging activity. Aliquots of different concentrations (20-100 µg/mL) of the test sample is added to 100 µL solution DPPH (4 mg/100 mL methanol). Absorbance at 517 nm is determined after 30 min. Each experiment was done in triplicate and average is taken. Vitamin C was used as a positive control and percentage of free radical scavenging was expressed as inhibition from the given formula: Calculated antioxidant data of all the tested samples were summarized in Table 3.

Nitric Oxide Radical Scavenging Activity
Nitric oxide was generated from sodium nitroprusside and measured by Griess' reaction [62,63]. Reagents are sodium nitroprusside (10 mM), phosphate buffer saline and Griess reagent (1 g of sulphanilic acid + 0.1 g naphthylethylene diamine dihydrochloride). 20 µL sodium nitroprusside, 5 µL phosphate buffer and 5 µL of compound were incubated at 25 °C for 2.30 h. After incubation, 20 µL of griess reagent was added to the previous mixture and allowed to stand for 30 min. The absorbance of the colour developed during diazotization of nitrite with sulphanilamide and its subsequent coupling with napthylethylenediamine hydrochloride was observed at 550 nm on spectrophotometer. Each experiment was done in triplicate and average is taken. Vitamin C was used as positive control and percentage of free radical scavenging was expressed as inhibition from the formula: Abs. of control Abs. of sample % inhibition of NO radical = 100 Abs. of control Efficacy was calculated for 0.25 mg/mL of each compound by using the following equations: NO scavenging % of compound at 0.25 mL Efficacy NO scavenging % of vitamin C at 0.2 = 5 mL (4) Calculated anti-inflammatory data of all the tested samples were summarized in (Table 4).

Conclusions
This work demonstrates a rapid, efficient method for the synthesis of new pyrazole and dihydropyrazole derivatives. All synthesized compounds were characterized by spectral data (IR, 1 H-NMR and 13 C-NMR) and the structures were consistent with the data. All the synthesized compounds were tested for their antimicrobial activity against four test organisms. The results showed that the compounds that having pharmacophores with lipophilic properties such as chloro and bromo substituents exhibited the greatest antimicrobial activities. Also two pharmacological activities namely antioxidant and anti-inflammatory activity, were tested for the chloro derivatives 1c, 2c, 3c, 4c, 5c. These activities vary according to their structures and functional groups.