Synthesis and biological evaluation of newer analogues of 2,5-disubstituted 1,3,4-oxadiazole containing pyrazole moiety as antimicrobial agents

2.1 Materials and methods

All the laboratory grade reagents were obtained commercially. The reaction was monitored by thin layer chromatography, which was performed on Merck precoated plates (silica gel. 60 F₂₅₄, 0.25 mm) and was visualized by fluorescence quenching under UV light (254 nm). Melting points were determined by the open capillary method and were uncorrected. The IR spectra were recorded on a Thermo Nicolet avatar 330-FT-IR spectrophotometer. ¹H-NMR and ¹³C-NMR spectra were recorded (DMSO-d₆) on a Bruker (400 and 100 MHz). Chemical shift values are given in δ scales. The mass spectra were recorded on LC–MS-Agilent 1100 series. Elemental analyses were performed on a Flash EA 1112 series CHNS–O Analyzer.

2.2 Synthesis

2.3 Characterization of synthesized compounds

2.4 Antimicrobial activity

The following bacteria and fungi were used for the experiment. Bacteria: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853. All bacterial strains were maintained on nutrient agar medium at ±37 °C. Fungi: Aspergillus flavus, Chrysosporium keratinophilum and Candida albicans MTCC 227 are used in this study. These cultures are obtained from the Department of Microbiology, Kuvempu University, Shimoga, India. All fungus strains were maintained on potato dextrose agar (PDA) at ±25 °C. After measuring the zone of inhibition, the values for Dimethylsulfoxide (DMSO) were subtracted to get the actual values.

The antibacterial activity of newly synthesized compounds (4a–n) was determined by the well plate method in Mueller–Hinton Agar (Arthington-Skaggs et al., 2000; Rocha et al., 1995). The compounds were tested against a panel of pathogenic microorganisms, including E. coli, S. aureus and P. aeruginosa. Microorganism strains were maintained on nutrient agar medium at ±37 °C. The cultures were inoculated in fresh 10 ml Nutrient Broth to yield an initial suspension of approximately 10–100 cfu/ml. All broths were then incubated statically at the aforementioned temperatures for microorganisms, for 18–24 h so that all cells were in the stationary phase. Susceptibility of the test organism to the compounds was determined by employing in the well plate technique. The bacterial suspensions were diluted ten times with distilled water, and 0.1 ml diluted culture was spread plated on nutrient agar in order to give a population of approximately 10⁶ cfu/plate. Six millimeter diameter well were then punched carefully using a sterile cork borer and 30 μl of test solutions of different concentrations (1000 μg/ml and 500 μg/ml) was added into each labeled well. The same procedure was repeated for different micro-organisms. Each experiment was carried out in triplicate. After the inoculation of the organism and compound, the Petri plates were incubated for 24 h at ± 37 °C. After the incubation, the inhibition zone was measured and the values for DMSO were subtracted to get the actual values. Streptomycin was used as standard drug.

Antifungal studies of newly synthesized compounds 4a-n were determined by the well plate method (Mac. Lowry et al., 1970; Portillo et al., 2001) against A. flavus, C. keratinophilum and C. albicans. Sabourands agar media were prepared by dissolving peptone (10 g), d-glucose (40 g) and agar (20 g) in distilled water (1000 ml) and adjusting the pH to 5.7. Normal saline was used to make a suspension of spore of fungal strains for lawning. A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of corresponding species. Twenty milliliters of agar media was poured into each petri dish. Excess of suspension was decanted and plates were dried by placing in an incubator at ±37 °C for 1 h. Six millimeter diameter well was then punched carefully using a sterile cork borer and 30 μl of test solutions of different concentrations (1000 μg/ml and 500 μg/ml) was added into each labeled well. A control was also prepared for the plates in the same way using solvent DMSO. The Petri dishes were prepared in triplicate and maintained at ±25 °C for 72 h. Antifungal activity was determined by measuring the diameter of inhibition zone. The activity of each compound was compared with fluconazole as standard.

3.1 Chemistry

The synthetic route has been outlined in Scheme 1. In the current work, aromatic esters (1) were synthesized from the appropriate aromatic acids by treating with ethanol in the presence of catalytic amount of sulfuric acid. Reaction of compound (1) with hydrazine hydrate yielded corresponding acid hydrazides (2) (Husain and Ajmal, 2009). Similarly, 3-substituted-pyrazole-4-carboxylic acids were synthesized as per the reported procedure (Lebedev et al., 2005). Subsequently condensation of substituted acid hydrazides (2) with various 3-substituted-pyrazole-4-carboxylic acids (3) in the presence of phosphorous oxychloride afforded a series of 2,5-disubstituted-1,3,4-oxadiazoles (4a-n). Newly synthesized compounds (4a–n) were characterized by IR, NMR, mass spectral and C, H, N analyses. Analytical and spectral data of all synthesized compounds were in full agreement with the proposed structures. IR spectrum of compound 4a showed absorption bands at 3143, 3053, 1593, 1531, 1087 cm⁻¹ which was due to the N–H, C–H, C⚌N, C⚌C and C–O–C groups, respectively. In ¹H-NMR spectra, all protons were seen according to the expected chemical shift and integral values. The ¹H-NMR spectrum of 4a showed a singlet at δ 13.79 corresponds to pyrazole NH proton. A singlet at δ 8.7 was due to pyrazole 5H proton. Also, at δ 7.55–8.0 a multiplet was observed which was due to aromatic protons. The mass spectrum of 4a showed a molecular ion peak at m/z = 321 (M-1), which is in agreement with the molecular formula C₁₇H₁₁ClN₄O. When the para position of the phenyl ring attached to pyrazole nucleus was substituted by electron donating and electron withdrawing groups, a change in delta values of aromatic protons was observed. In the presence of the electron donating group, delta value shifted to lower wavelength side whereas in the presence of electron withdrawing groups, delta value shifted to higher end. In compound 4d, due to the presence of the –OCH₃ group an additional singlet peak was observed at δ 3.82. Due to the presence of two chlorine atoms in mass spectrum of compound 4b, M+, M+2 and M+4 chlorine patterns were clearly observed. Similarly, the presence of one chorine atom in compound 4d showed M+1 and M+2 chlorine pattern which further confirms the structure. Similarly the spectral values for all the compounds and C, H, N analyses are given in the experimental part.

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Scheme 1

Schematic diagram showing the synthesis of 1,3,4-oxadiazole derivatives (4a–n) where, R = 4-Cl and R¹ = H (4a); R = 4-Cl and R¹ = 4-Cl (4b); R = 4-Cl and R¹ = 4-F (4c); R = 4-Cl and R¹ = 4-OCH₃ (4d); R = 4-Cl and R¹ = 2,4-Cl₂ (4e); R = 4-OCH₃ and R¹ = H (4f); R = 4-OCH₃ and R¹ = 4-Cl (4g); R = 4-OCH₃ and R¹ = 4-F (4h); R = 2-Cl and R¹ = H (4i); R = 2-Cl and R¹ = 4-Cl (4j); R = 2-Cl and R¹ = 4-F (4k); R = 2-Cl and R¹ = 4-OCH₃ (4l); R = 4-NO₂ and R¹ = H (4m); R = 4-NO₂ and R¹ = Cl (4n).

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3.2 Antimicrobial activity

E. coli is a common micro-organism which causes secondary infection, food poisoning in human. S. aureus causes septic arthritis, staphylococcal endocarditis and pneumonia. On the other hand P. aeruginosa causes skin and soft tissue infection, gastrointestinal infection, urinary tract infection and septic shock pneumonia. These pathogens are commonly causing harmful effects on human life. The evaluation of antimicrobial activity of 1,3,4-oxadiazole moiety against E. coli, S. aureus and P. aeruginosa has been carried out by many researchers and reported good results with respect to the above mentioned microorganisms (Sahin et al., 2002; de Oliveira et al., 2012; Li et al., 2012). Hence it was thought worthwhile to evaluate antimicrobial activity using these microorganisms.

The newly synthesized compounds 4a–n were tested for their antibacterial activity (in vitro) against E. coli, S. aureus and P. aeruginosa and their activity was compared to a well-known commercial antibiotic, streptomycin. Antibacterial activity was carried out by the well plate method by measuring its zone of inhibition. The compounds 4a–n were screened for their antibacterial activity in triplicate against E. coli, S. aureus and P. aeruginosa at two different concentrations of 1000, 500 μg/ml as shown in Table 1. The investigation of antibacterial screening data revealed that most of the tested compounds showed moderate to good bacterial inhibition. Compound 4i exhibited equipotent activity as that of standard against E. coli and S. aureus at 500 μg/ml. Compound 4j exhibited equipotent activity as that of standard against E. coli whereas slightly less than that of standard against S. aureus and P. aeruginosa at 500 μg/ml. 4l also inhibited the growth of S. aureus similarly as that of standard whereas slightly less than that of standard against E. coli and P. aeruginosa at 500 μg/ml. Compounds 4e and 4f were found to be active against all the tested bacterial strains. Compound 4b showed moderate activity against E. coli and S. aureus. Remaining compounds showed fair or poor activity against tested bacterial strains.

All the synthesized compounds were also tested for their antifungal activity (in vitro) against A. flavus, C. keratinophilum and C. albicans by measuring their average zone of inhibition (Table 2). Fluconazole was used as standard for antifungal activity. Among the tested compounds, 4i and 4j showed a good antifungal profile against A. flavus, and C. keratinophilum at a concentration of 500 μg/ml when compared with the standard. 4e, 4f and 4l showed moderate activity against A. flavus but showed poor activity against rest of two microorganisms. Remaining compounds showed poor activity against the tested microorganisms.

The enhanced activity of 4i and 4j can be attributed to the presence of 2-chlorophenyl substituent attached to the 5th position of the oxadiazole ring and the presence of phenyl and p-chlorophenyl substituents on the pyrazole ring which is attached to the 2nd position of the oxadiazole ring. The presence of 2-chlorophenyl substituent (5th position of oxadiazole) along with 4-fluorophenyl, 4-methoxyphenyl substituent on the pyrazole ring in 4k and 4l respectively may be the reason for its enhanced activity. Compounds 4e and 4f contain 4-chlorophenyl and 4-methoxyphenyl substituent (5th position of oxadiazole) along with 2,4-dichlorophenyl and phenyl substituents on a pyrazole ring which is attached to the 2nd position of the oxadiazole ring which may be the reason for its activity.

It can be concluded that the compounds 4i, 4j, 4k and 4l which contain 2-chlorophenyl substituent on the 5th position of the oxadiazole ring may increase the antimicrobial profile of the compound.