Synthesis of 2-aryl-5-(arylsulfonyl)-1,3,4-oxadiazoles as potent antibacterial and antioxidant agents

Ten novel 2-aryl-5-(arylsulfonyl)-1,3,4-oxadiazoles were produced and assessed for their in vitro antibacterial and antioxidant activities. Diverse spectroscopic methods like 1H NMR, 13C NMR, IR, and LCMS were used for the characterization of the prepared samples and all the data was in good agreement with the anticipated structures. The prepared compounds 6a–j were screened for their in vitro antibacterial activity against bacterial strains Pseudomonas aeruginosa, Enterobacter aerogenes, Escherichia coli (gram-positive), and Bacillus cerus, Staphylococcus aureus, Bacillus subtilis (gram-negative). The antimicrobial screening outcome revealed that the prepared 2-(3,4-dimethylphenyl)-5-tosyl-1,3,4-oxadiazole (6j), 2-(3-isopropylphenyl)-5-tosyl-1,3,4-oxadiazole (6c), and 2-(2-ethylphenyl)-5-tosyl-1,3,4-oxadiazole (6i) are most potent among all the examined compounds. Furthermore, the antioxidant activity of the prepared compounds was also investigated by DPPH radical scavenging method and the results showed that some of the compounds were moderately active.


Introduction
Heterocyclic compounds are a vital part of most of the bioactive molecules used as drugs and are the key motifs for the novel drug discovery.The heterocyclic compounds enhance their activity when fused with other ring systems [1][2].Oxadiazoles with plethora of biological applications are identified as important construction motifs for the advance of innovative drug design [3][4][5], thus grabbing the attention of medicinal chemists around the world.With its capability to bind with a ligand, the oxadiazole ring can be used as a significant part of the pharmacophore.In certain instances, it behaves like a flat aromatic linker that affords the proper orientation of the molecule [6].In the oxadiazole family, 1,3,4-oxadiazoles occupied a unique position in medicinal chemistry due to their multi-purpose utility in designing many bioactive compounds.In medicinal chemistry 1,3,4-oxadiazole and its derivatives are playing a vital role with broad range of biological applications.Oxadiazoles are the bioisostere of compounds with carbonyl function, like carboxylic acids, amides, and esters capable to form superior hydrogen bonding interactions with various receptors thereby augmenting the biological responses to a notable extent [7][8].

Materials and methods
All the chemicals and reagents were acquired from Aldrich, Merck and utilized with no extra purification.After purchase, the solvents are dried prior to use by standard procedure [45].The melting point of all the prepared compounds was recorded using the Cintex melting point apparatus in open capillaries.Precoated thin layer chromatography (TLC) plates (0.25 mm, Merck, silica gel 60 F 254 ) were utilized to monitor all the reactions.A Varian-400 spectrometer was used to record the NMR (400MHz) spectra.All our experimental procedures were carried out by using a Centrifuge machine (VKSI-Medico) for the construction of the titled compounds.

General procedure for the synthesis of 2-aryl-1,3,4-oxadiazoles
The preparation of 2-(4-methylphenyl)-1,3,4-oxadiazole (3a) is exemplary for the construction of titled 2-aryl-5-(arylsulfonyl)-1,3,4-oxadiazoles.In a dried 100 mL two-necked reaction flask 30 mL of dry DMF, 3.5 g of 4-methylbenzoic acid (25.0 mmol) and 4.2 g of K 2 CO 3 (30.0mmol) were added and stirred well.Next, to this stirred solution, methyl iodide (2.5 mL, 30.0 mmol) was added dropwise for 10 min.The total mixture was stirred for 10 h at ambient temperature.Then the crude mixture was poured into ice water and extracted with hexane/EtOAc (20:5, v/v).After that, the organic layer was evaporated by utilizing a rotator evaporator.Silica gel (60-120 mesh) column chromatography was employed to purify the product with hexane/ethyl acetate (10:2, v/v) as eluent giving 4-methylbenzoate (3.4 g) in quantitative yield.Further, the obtained 4-methylbenzoate (3.4 g, 25.0 mmol), hydrazine monohydrate (7.5 g, 150.0 mmol), and EtOH (25 mL) were placed in a round bottom flask equipped with a condenser.The total mixture was stirred at reflux for 8 h and the reaction mixture was cooled to room temperature.Next, the mixture was concentrated using a rotary evaporator.The resulting residue was filtered with hexane and dried, affording 2.8 g of benzhydrazide 2a as the product in 90% yield [46].

Procedure for antibacterial activity [48]:
DMSO solution of all the prepared compounds at a concentration of 1mg/mL was prepared individually.In sterile Mueller Hinton medium each bacterium was inoculated and kept at 37 o C for 24 h to develop inoculums.The bacterial suspension was diluted by utilizing sterile saline to regulate the turbidity to the 0.5 McFarland standards.Next, on sterile Mueller Hinton agar plates, diluted suspension (200 µL) of every pathogen was inoculated.Wells were punched in the agar medium.Next, 100 µL of every compound solution was placed in a separate well with a micropipette.In addition, to check the activity of DMSO against the pathogenic culture, 100 µL of pure DMSO solution was also placed in a well and the entire petri dishes were incubated at 37 o C for 24 h.A clear zone around the well was regarded as positive results.The antimicrobial potency of the examined compounds was calculated after inclusive incubation.Finally, the zone of inhibition was calculated and recorded in millimetres (mm).

Procedure for antioxidant activity:
Ai Lan Chew et al. method [49] was used to determine the 2,2-Diphenyl-1-picryl hydrazyl (DPPH) free radical scavenging activity of the various extracts.The crude extracts in diverse concentrations viz., 25 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL were prepared in dimethyl sulphoxide (DMSO). 1 mL of every concentration was mixed with 4 mL of 0.004% (w/v) solution of DPPH prepared in CH 3 OH.The reaction mixture was set aside in dark for incubation for 30 min.CH 3 OH was used as control and ascorbic acid was employed as positive control.The absorbance was calculated at 517 nm.The following formula was used to determine the DPPH scavenging activity (%).DPPH scavenging activity (%) = [(AO-AS)/ AO] × 100, where, AO = absorbance of the control, AS = absorbance of the plant sample.
The structure of all the prepared compounds was attributed with IR, NMR ( 1 H and 13 C), and LC-MS spectral analyses and the spectroscopic and analytical data was in complete agreement with the anticipated structures.For example, in the IR spectrum of compound 6h, the appearance of a peak at 2224 (s) cm -1 indicates the presence of -C ≡ N group, formation of distinguishing peaks at 3064 (w) cm -1 , 1111 (s) cm -1 owing to Ar C-H, and C-O-C groups of oxadiazole frame.The appearance of IR peaks at 1400(s) cm -1 and 1262 (w) cm -1 are due to -C = N and N-N stretching's.Next, IR peaks at 1607(m), 1569 (s), and 1531 (w) cm -1 are due to aromatic C = C stretching.Further, a peak of 2922(s) cm -1 characterize the -C-H stretching of CH 3 group, asserted the formation of title compound 6h.
Next, the emergence of a signal in the 1 H-NMR spectrum of 6h at chemical shift value d 3.80 ppm as a singlet, integrating for three protons were assigned to -O-CH 3 group, doublets at d 7.78, 7.41 ppm integrating for two protons, multiplet at d 7.56-7.48ppm integrating for three protons and another doublet at d 6.85 ppm integrating for two protons were assigned for aromatic protons.Moreover, the 13

Biological evaluation 3.2.1. Antimicrobial activity
The well diffusion method [48] was used to study the in vitro bacterial growth inhibition activity of the test compounds 6a-j on gram-positive bacterial strains Pseudomonas aeruginosa, Enterobacter aerogenes, Escherichia coli and gram-negative bacterial strains Bacillus cerus, Staphylococcus aureus, Bacillus subtilis.The antibacterial activity screening outcome reveals that the compounds 6b, 6c, 6e, 6i, and 6j are active against all the six bacterial strains examined with a good zone of inhibition values (Table 1).The compounds 6j, 6i, and 6e were found to inhibit the growth of Pseudomonas aeruginosa with Zone of inhibition values 22mm, 19mm, and 16mm respectively.The compounds 6j, 6c, 6e, and 6f showed good activity against Enterobacter aerogenes with a consecutive zone of inhibition of 23mm, 19mm, 18mm, and 18mm.Test compounds 6j and 6i with a zone of inhibition of 20 mm and 15 mm, effectively inhibit the growth of microorganism Escherichia coli.Compounds 6c and 6j exhibited good activity against the gram-negative organisms Bacillus cerus, Staphylococcus aureus, and Bacillus subtilis with a zone of inhibition range of 19-23 mm and 18-22 mm consecutively.In addition, the antibacterial screening (Table 1) discloses that the titled compounds are more potent against the gram-negative bacteria compared to gram-positive bacteria.
Based on the zone of inhibition values, next the minimum inhibitory concentration (MIC) value (mg/mL) was determined for the compounds that showed significant growth inhibition zones with the use of serial dilution method and the MIC values recorded in Table 2.The MIC results indicate that most of the tested compounds displayed variable inhibitory effects on the growth of tested bacterial strains.The MIC was deduced by following the method and guidelines of the Clinical and Laboratory Standard Institute (CLSI) (Table 2).In this study, the MIC was determined for the most potent selected antimicrobial compounds 6b, 6c, 6e, 6i, and 6j.The investigation reveals that the MIC value of test compounds Table1.Antibacterial activity in Zone of inhibition (mm) of the final compounds (6a-6j). is in the range of 132-98 µg/mL against Enterobacter aerogenes and 72-150 µg/mL against Bacillus subtilis.Among the test compounds, 6j exhibited potent antibacterial activity with a minimum inhibitory concentration value of 98 µg/mL against Enterobacter aerogenes, and 72 µg/mL against Bacillus subtilis.Compound 6i disclosed the minimum inhibitory concentration values of 103 µg/mL and 75 µg/mL against Enterobacter aerogenes, and against Bacillus subtilis.However, all the test compounds are less potent than the reference drug streptomycin.

Antioxidant activity
The in vitro antioxidant activity of the prepared compounds 6a-6j was evaluated by a standard literature protocol [49].For this, different extracts were tested for their 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity according to the literature protocol.Dissimilar concentrations of the crude extracts with 25μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL concentrations were examined, by using ascorbic acid as a standard positive control.This investigation outcome is shown in Table 3.
The antioxidant activity screening outcomes (Table 3) reveal that all the prepared oxadiazole motifs showed good antioxidant activity.The title compounds 6a-6j exhibited concentration reliant increase in antioxidant activity, i.e. their antioxidant activity was increased as the concentration increased.Compounds 6j and 6b displayed the highest and lowest antioxidant activities at the concentrations of 100 µg, 50 µg, and 25µg, respectively.But, compounds 6f and 6a exhibited utmost and least antioxidant activity respectively at the concentration of 200 µg.The compound 6c displayed almost similar antioxidant activity at the concentration of 200 µg.All the remaining eight compounds except 6a and 6b, exhibited more than 40% level of antioxidant properties at 200 µg concentration.However, in comparison with standard ascorbic acid, all the prepared compounds displayed significantly lower antioxidant activity at all the tested concentrations.
From the results of antibacterial and antioxidant studies, it was assumed that (i) presence of electron donating functionality at 2 nd and 5 th position of benzene ring in 6j is responsible for significant activity against the tested bacterial strains because the presence of +I effect groups in benzene ring system amplifies the lipophilicity and thus enhance cell penetration rate, that is accountable for antibacterial drug efficiency; (ii) the physicochemical characters such as position and kind of substituent on the aromatic ring of sulphoxide influence the antimicrobial activity of the examined compounds; (ii) presence of the electron donating groups are also responsible for their better antioxidant activities also; (iii) the electron withdrawing nitro group is responsible for the moderate antioxidant activity of compound 6d.

Conclusion
A sequence of 2-aryl-5-(arylsulfonyl)-1,3,4-oxadiazole scaffolds were synthesized and evaluated for their antibacterial and antioxidant activities.The data obtained from spectroscopic techniques like IR, NMR, and LC-MS affirmed the structure of all the obtained compounds.The antimicrobial screening outcome of all these titled compounds revealed that the examined compounds 6j, 6c, and 6i were the most potent among all prepared compounds.Moreover, the obtained compounds exhibited good antioxidant activity also.The target compounds 6j and 6i showed the highest antioxidant activity.
C-NMR spectrum of compound 6h revealed the presence of 11 different carbons in the compound.The peak at d 55.0 ppm has been allocated to the methoxy carbon.The signals at d 155.1 and 153.1 ppm were due to the carbon of C-O core nuclei respectively.The signals at d 133.6-113.5 ppm have been consigned to the aromatic carbons of the compound.All the above spectral data indicate that compound 6h is 2-((4-methoxyphenyl) sulfonyl)-5-phenyl-1,3,4-oxadiazole.Moreover, the evolution of molecular ion peak at 317.19 (M+H) + in the mass spectrum (EI) supported the formation of compound 6h.
Mass spectrum of compound 6b IR spectrum of compound 6b Mass spectrum of compound 6c IR spectrum of compound 6c Mass spectrum of compound 6e IR spectrum of compound 6e Mass spectrum of compound 6f IR spectrum of compound 6f Mass spectrum of compound 6i IR spectrum of compound 6i

Diameter of Zone of Inhibition in mm
e -Staphylococcus aureus; BS f -Bacillus subtilis; -: No inhibition.