Design, synthesis and biological evaluation of 1,3,4-oxadiazoles/thiadiazoles bearing pyrazole scaffold as antimicrobial and antioxidant candidates

Article history: Received October 21, 2015 Received in revised form December 20, 2015 Accepted 12 Februray 2016 Available online 12 February 2016 A series of semicarbazones, thiosemicarbazones, 1,3,4-oxadiazoles/thiadiazoles bearing pyrazole scaffold were designed and synthesized. All the synthesized new compounds were characterized by 1H NMR, 13C NMR, MS and elemental analysis. The synthesized compounds were screened to probe their in vitro antimicrobial activity against bacteria and fungi species. The structure-activity relationship of the synthesized compounds was studied. The compounds displayed good to excellent potency against tested microorganisms. The in vitro antioxidant activities of the 1,3,4-oxadiazoles/thiadiazoles were evaluated by DPPH, hydroxyl and nitric oxide radical scavenging assay. Among the tested compounds, compound with chloro substitution showed good antioxidant potential. © 2016 Growing Science Ltd. All rights reserved.


Introduction
The pyrazole motif makes up the core structure of numerous biologically active compounds.Compounds bearing pyrazole nucleus exhibit versatile range of biological activities such as antimicrobial 1 , anti-inflammatory 2 , analgesic 3 and GABA receptor antagonists and insecticides. 4In addition to the diverse biological activities of pyrazoles, other heterocycles in association with pyrazoles play a prime role in chemical and pharmacological fields.The prevalence of pyrazole cores in biologically active molecules has stimulated the need for elegant and efficient ways to make these heterocyclic lead.
Further, semicarbazone and thiosemicarbazone are excellent prototypes for the design and development of novel amino oxadiazole and thiadiazole respectively.In addition semicarbazone have received significant attention from pharmaceutical industry due to their wide spectrum of biological activity such as anticonvulsant, 5 antioxidant, 6 antimicrobial activities 7 etc.Compounds containing 1,3,4-oxadiazole core were known to possess pharmacological properties such as antimicrobial, 8 COX-2 inhibitors and anti-inflammatory, antitumor and analgesic. 9The widespread use of 1,3,4-oxadiazoles as a scaffold in medicinal chemistry established this moiety as a member of the privileged structures class, among them the synthesis of 2-amino-5-substituted-1,3,4-oxadiazole has received a lot of interest.
Sulpha drugs are well recognized for their various physiological activities.Thiosemicarbazone derivatives have been the focus of medicinal chemists because of their potential biological activities. 10hiosemicarbazones are very useful intermediate for the development of molecules of pharmaceutically important molecules, as well as they themselves possess antimicrobial, antiviral, 11 anti-HIV 12 and anticancer 13 properties.1,3,4-Thiadiazoles have gained importance as they constitute the structural features of many bioactive compounds.These compounds are of great interest in chemistry owing to their bioactivity of certain plant growth regulating effects as well as antimicrobial activity. 141,3,4-Thiadiazoles possess a wide range of therapeutic activities like antioxidant, anticancer, anti-inflammatory and hyperlipidaemia. 15 the pursuit and design of new drugs, the development of hybrid molecules through the combination of different pharmacophores in one frame may lead to compounds with interesting biological profiles.In view of these facts and as a part of our extensive research program, the synthesis of 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives incorporating with pyrazole nucleus as hybrid molecule possessing antimicrobial and antioxidant activity is aimed.
The synthesized new compounds were characterized by spectral analysis before being evaluated for their in vitro antimicrobial and antioxidant activities.The compounds 3a-l showed a singlet for two protons in the region δ 3.566-4.112ppm for NH2 protons in their 1 H NMR spectra.The signals are observed for CONH/CSNH and CH=N protons of 2a-l were found absent.In their 13 C NMR spectra, the signals due to two N=C-O and N=C-S carbons appeared in the region δ 164.15-176.14ppm.Further, all compounds showed signals due to aromatic, substituent protons and carbons in the expected region.Synthesized new molecules showed M+1 ion peak as a base peak in their mass spectra.Further, the analytical data obtained for the compounds 3a-l were in good agreement with theoretically calculated data.All these spectral and analytical results confirmed the formation of the products.

Antimicrobial activity
Microbial studies of synthesized compounds were assessed by minimum inhibitory concentration (MIC) by serial dilution method 17 .The compounds were screened for their antimicrobial activities against Gram-negative bacteria species Escherichia coli, Pseudomonas aeruginosa, Gram-positive bacteria Staphylococcus aureus, fungi species Aspergillus nigar, Aspergillus flavus and Candila albicans.The experiments were carried out in triplicate; the results were taken as a mean of three determinations.Known antibiotics ciprofloxacin and fluconazole were used as standards for antibacterial and antifungal studies respectively.The results of MIC's were summarized in Table 1 and Table 2 The synthesized semicarbazones and thiosemicarbazones exerted a wide range of modest to good in vitro antibacterial activity against the tested organisms.Compounds 2a, 2g having no substitutions, and 2d, 2j with methoxy substitution on the aromatic ring showed moderate activity against tested species.Compounds 2c, 2e and 2k having CH3 substituent showed moderate activity and compound 2i containing CH3 substitution exhibited equal activity compared with that of standard.It has been interesting from the results of the study that chloro substitution in the synthesized compounds enhanced the activity to the greater extent.2b demonstrated excellent activity against all and 2h against S.aureus organisms.Nitro substitution present in compounds 2f and 2l retarded the inhibitory effect against the organism tested.
Compounds 2a and 2g showed moderate antifungal activity against the tested species.Compounds 2c, 2e, 2i and 2k having methyl and 2d, 2j having methoxy substitution showed moderate activity.Compounds 2b and 2h having chloro substitution exhibited inhibition to a remarkable extent; while 2f and 2l with electron withdrawing nitro substitution showed lesser activity against the tested organisms.
The synthesized new 1,3,4-oxadiazoles and 1,3,4-thiadiazoles demonstrated moderate to excellent antibacterial and antifungal activity by inhibiting the tested organisms.Compounds, 3a, 3g showed moderate activity, chloro substituted compound 3b showed excellent antibacterial activity against all the tested organisms.Compound 3h showed the highest activity against Staphylococcus aureus compared with standard ciprofloxacin.Compounds 3c, 3e, 3i and 3k having methyl substitution exhibited moderate to good activity, 3d and 3j having methoxy substitution showed good activity, Compounds 3f and 3l having nitro substitution exhibit lesser activity against the organisms tested.
Compounds 3a, 3g having no substitution, and compounds 3d, 3j having methoxy substitution exhibited moderate activity against the fungal species tested.However, compounds 3c, 3e, 3i and 3k having methyl substitution showed moderate to good activity.Chloro substitution present in 3b and 3h demonstrated excellent activity and 3f and 3l having nitro substitution exhibited lesser activity against the fungal organisms tested.
In an attempt to interpret and correlate the molecular parameters of the small molecules with the potency of inhibition against the various microorganisms, detailed quantitative structure-activity relationship (QSAR) analysis was carried out.Physicochemical parameters for the small molecules were computed 18 and both pair-wise and multivariate analysis was carried out as specified in the literature 19,20 .Further, parameters like LogP, aromatic bond content, molecular weight, number of atoms and bonds were negatively correlated with inhibition potency.
Analysis of the results indicates that the small-molecule features that likely contribute to increased potency of inhibition vary across different microorganisms.This is an encouraging observation since specific variation of a particular molecular feature would lead to increased specificity towards a particular kind of microorganism.Further, this analysis also points out to the parameters that can be modulated to increase the potency of these compounds in general across the different microorganisms employed.However, care must be exercised in interpreting these results given the small sample size that was employed across compounds 2a-l and 3a-l and the fact that MIC was considered as the dependent variable.
The effect of substitution in the aromatic ring of synthesised compounds has been studied based on their in vitro antimicrobial activity results.Monochloro substitution in carbazone, 2b and thiosemicarbazine, 2h; 1, 3, 4-oxadiazole, 3b and 1, 3, 4-thiadiazole, 3h bearing pyrazole scaffold showed good antimicrobial activity.Among these scaffolds, ortho substitution 3b and 3h showed high efficiency then para substitution 2b and 2h, in antimicrobial.So this suggest that ortho monochloro substitution plays a very vital role in hamper the cellular architecture of E. coli, P. aeruginosa, S. aureus, fungi species A. niger, A. flavus and C. albicans.Results suggest that 3h could actively inhibit the growth of gram positive (S.aureus) and gram negative (E. coli and P. aeruginosa).
Comparative analysis illustrate that, among 2b/3b and 2h/3h shows that, sulfur moiety in 1, 3, 4thiadiazoles, 2h/3h act as potent inhibitor for both gram positive as well as gram negative bacteria.In case of moderate electronegative elements like sulfur and chlorine containing compounds, 3h showed better in vitro activities, comparatively then at of higher electronegative element oxygen, 3b.Therefore, the substitution and position of chloro plays a very important role in enhancing the bioactivities of the compound.Thus the in vitro data suggest that monochloro substitution at ortho position, 3h is most favorable for enhancing the antimicrobial activity.

Antioxidant activities 2.3.1. DPPH radical scavenging activity
Antioxidants are characterized by their ability to scavenge free radicals.Proton radical scavenging action is an important attribute of antioxidants, which are measured by DPPH scavenging assay.This assay was performed by a method reported by Renuka et al. 17 1 mL of DPPH solution (0.1 mM in 95% methanol) was mixed with different aliquots of test samples (25, 50, 75 and 100 μg/ml) in methanol.The mixture was shaking vigorously and allowed to stand for 20 min at room temperature.The absorbance was read against blank at 517 nm in an ELICO SL 159 UV visible spectrophotometer.The free radical scavenging potential was calculated as a percentage (I %) of DPPH decoloration using the equation: where A0 is the absorbance of the control reaction mixture excluding the test compounds, and A1 is the absorbance of the test compounds.Tests were carried out in triplicate and the results are expressed as I% ± Standard Deviations and were summarized in Table 3.

Nitric oxide radical scavenging assay
This assay was performed by a method reported by Padmaja et al. 21Nitric oxide (NO) was generated from sodium nitroprusside (SNP) and it was measured by the Griess reaction.Nitric oxide was generated by the sodium nitroprusside in phosphate buffer at physiological pH and then nitric oxide was reacted with oxygen, produced the nitrite ions, which can be estimated by the Griess Reagent. 1 mL of Sodium nitroprusside (10 mM), 1.5 ml of phosphate buffer (pH 7.4) was mixed with the test solution (25, 50, 75 and 100 µg/ml) and incubated 25 °C for 150 min, to this 1 mL of Griess reagent (1 % sulfanilamide in 2 % phosphoric acid and 0.1% N-(1-naphthyl) ethylenediaminedihydrochloride) was added and allowed to stand for 3 min, the absorbance of the chromatophore was read at 546 nm.Ascorbic acid was used as standard.The experiments were carried out at four different concentrations in triplicates and the results are expressed as I% ± Standard Deviations and were summarized in Table 4.

Hydroxyl radical scavenging assay
Hydroxy radical scavenging assay was carried out according to the reported procedure. 22Product formed by the degraded deoxyribose was on heating with thiobarbituric acid (TBA) form a pink colored chromogen.This confirms the formation of OH•.The addition of the tested compound with the reaction mixture, they distant the hydroxy radicals from the deoxyribose and prevent their degradation.This experiment was performed by mixing 0.1 mL of phosphate buffer; 0.2 mL of 2-deoxyribose, test solution (25, 50, 75 and 100 µg/ml), 0.1 mL of H2O2 (10 mM), 0.1 mL of ascorbic acid (1 mM), 0.1 mL of EDTA and 0.01 mL of FeCl3 (100 mM) was incubated at 37 °C for 60min.Thereafter, the reaction was terminated by adding 1 mL of cold 2.8% trichlroacetic acid and the reaction product was measured by adding 1 mL of 1% thiobarbituric acid (1g in 100mL of 0.05 N NaOH) in boiling water for 15 min.The absorbance was measured at 535 nm.BHA was used as a positive control.Decreased absorbance of the reaction mixture indicates increased hydroxyl radical scavenging activity.The experiment was carried out in triplicate and the results were expressed as I% ± standard deviations and were summarized in Table 5.A freshly prepared DPPH solution shows a deep purple color with an absorption maximum at 517 nm.Changes in the purple color to yellow indicate decreased in the absorbance.This is because of the antioxidant molecule reduce the DPPH free radical through donation of a hydrogen atom.Hence, instantaneously or concomitant decrease in absorbance was found, which indicates that the more potent antioxidant activity of the compound.Table 3 shows all the newly synthesized compounds were exhibited moderate to good activity because of their H-donating capacity.Compounds 3b and 3h having chloro substituent and compounds 3d and 3j having methoxy substituent showed the stronger DPPH scavenging activity than others.Nitro substituent compound 3f and 3l have shown less activity compared with the standard ascorbic acid, while the remaining compounds exhibited moderate activity.Nitric oxide plays a significant role in inflammatory processes.In the immunological system, it fights against tumor cells and infectious agents.During inflammatory reactions, nitric oxide is produced by the inducible enzyme nitric oxide synthase in cells like macrophages and renal cells after stimulation by lipopolysaccharide.NO react with oxygen or superoxide anion radical to form even stronger oxidant peroxynitrite. 20Compounds, 3b, 3h having chloro substitution in the phenyl ring showed greater ability to scavenge NO radical.Compounds, 3f, 3l having nitro substituent showed least activity compare to standard and the remaining compounds displayed moderate activity.The hydroxy radical is a highly reactive free radical formed in biological systems and it is able to damage the biomolecule found in living cells. 23The hydroxy radical has the ability to break DNA and cause strand breakage, which contributes to carcinogenesis, mutagenesis and cytotoxicity.In the present investigation, compounds 3a-l were found to be stronger to weak hydroxyl radical scavenging activity.Among the samples studied, compound 3b and 3h exhibited the remarkable capacity for scavenging hydroxyl radical which was significantly higher than that of the standard of BHA.Remaining compounds exhibited moderate activity.
The hydroxyl group present parental scaffolds (1, 3, 4-oxadiazoles/thiadiazoles) acts as good free radicals scavenger.Sulfur-antioxidant paradox is well established in many bioactives like glutathione, thioredoxin and glutaredoxin efficiently form a line of defense against reactive oxygen and nitrogen species 24 .Hence sulfur containing compounds are known to be a very important in maintaining redox potentials in the system.Thus the in vitro data suggest that monochloro substitution at ortho position, 3h is most favorable for enhancing the antioxidant activity.Whereas in case of mono methyl substitution or mono/di nitro substitutions no appreciable amount of activity, suggesting that hydrophobicity in case of 3c and 3i and increased electronegative atoms in case of 3f and 3l doesn't have any role in enhancing antioxidant activity of the presented novel 1, 3, 4-oxadiazoles/thiadiazoles bearing pyrazole scaffolds.

Conclusion
The simple easy accessible procedure for the synthesis of 1,3,4-oxadiazole and 1,3,4-thiadiazole incorporating pyrazole nucleus and their in vitro antimicrobial and antioxidant activity results revealed the significance of the study.All newly synthesized compounds exhibited moderate to good antimicrobial activity against the tested microorganisms, compounds having chloro substituent demonstrated potent antimicrobial activity.Compounds 3b and 3h showed significant antioxidant activity in all the assays.The compounds, particularly 4b exhibited greater activity in comparison to the standard drug.The SAR study of the synthesized compounds remains the topic of interest.

Materials and methods
Melting points were determined by an open capillary tube method and are uncorrected.Purity of the compounds was checked on thin layer chromatography (TLC) plates pre-coated with silica gel using the solvent system ethyl acetate: n-hexane (1:4 v/v).The spots were visualized under iodine vapors and UV light.The 1 H NMR and 13 C NMR spectra was recorded on a Spect 500 MHz and 125.6 MHz spectrophotometer respectively using DMSO as solvent and TMS as internal standard.The chemical shifts are expressed in δ ppm.Mass spectra were obtained on Shimadzu LCMS-2010A spectrophotometer (ESI).Elemental analysis was obtained on a Thermo Finnigan Flash EA 1112 CHN analyzer.Purification of compounds was done by column chromatography on silica gel (70-230 mesh, Merck).

General procedure
General procedure for the synthesis of semicarbazones, 2a-f and thiosemicarbazones, 2g-l.
To a solution of semicarbazide hydrochloride (1.115 g, 0.01 mol) and 3-(2-hydroxyphenyl)-1-aryl-1H-pyrazole-4-carbaldehyde, 1a-f (2.64 g, 0.01 mol) in ethyl alcohol, 3-4 drops of acetic acid was added.The mixture was refluxed on a water bath for 2-3 h, the progress of the reaction was checked by TLC.After completion of the reaction, the mixture was poured in to crushed ice and mixed well; the solid separated was filtered, washed with water and recrystallized from ethyl alcohol to obtain the products 2a-f in 80-88% yield.Under similar conditions 1a-f with thiosemicarbazide hydrochloride yielded 2g-l in 80-86%.
2-(4-(5-Amino-1,3,4-oxadiazol/thiadiazol-2-yl)-1-aryl-1H-pyrazol-3-yl)phenol 3a-l were prepared by the oxidative cyclization of substituted semicarbazones/thiocarbazones 2a-l (0.01 mol) and sodium acetate were dissolved in 25mL glacial acetic acid taken in a two necked round bottomed flask fitted with a dropping funnel which was supplied with (0.01 mol) of bromine dissolved in (8 mL) of glacial acetic acid.Bromine was added drop wise with stirring magnetically.The reaction mixture was stirred at room temperature for 2-3 h.The progress of the reaction was monitored by TLC, after completion of the reaction the solution was poured into crushed ice and swirled well.The resulting solid was filtered, washed with water and dried under vacuum to obtain a crude product, which was purified by column chromatography on silica gel (60-120 mesh) using ethyl acetate and hexane (1:4 v/v) as eluent.

Table 1 .
. MIC's of the test compounds 2a-l against bacterial and fungal species

Table 2 .
MIC's of the test compounds 3a-l against bacterial and fungal species

Table 3 .
Antioxidant activity of compounds 3a-l in DPPH method

Table 4 .
Antioxidant activity of compounds 3a-l in nitric oxide method