Synthesis, characterization and pharmacological evaluation of (Z)-2-(5-(biphenyl-4-yl)-3-(1-(imino)ethyl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)phenol derivatives as potent antimicrobial and antioxidant agents

1 Introduction

One of the main objectives of organic and medicinal chemists is to design and synthesize molecules having therapeutic values (Verma and Saraf, 2008). The dramatically rising prevalence of multi-drug resistant microbial infection in the past few decades has become a serious health care problem. In, the past 25 years, the incidence of microbial infection has increased in alarming levels all over the world as a result of antimicrobial resistance. A growing number of immuno-compromised patients (immune response attenuated by administration of immunosuppressive drugs) as a result of cancer chemotherapy, organ transplantation and HIV infection are the major factors contributing to this increase (Koca et al., 2005; Bonde and Gaikwad, 2004; Yu and Huiyuan, 2002; Ram, 1988). Hence there will always be a vital need to discover new chemotherapeutic agents to avert the emergence of resistance and ideally shorten the duration of therapy (Dolman et al., 2006; Murphy et al., 2007).

A wide variety of heterocyclic systems have been explored for developing pharmaceutically important molecules. Nitrogen containing heterocyclic molecules constitutes the largest portion of chemical entities, which are part of many natural products, fine chemicals, and biologically active pharmaceuticals (Khalil et al., 1980). Among them the derivatives of oxadiazoles played a very important role in the medicinal chemistry. Oxadiazoles have been described as bio-isosteres for amides and esters (Jonathan and Robert, 1998). Due to increased hydrolytic (Clapp, 1976) and metabolic stabilities of the oxadiazole ring, improved pharmacokinetic and in vivo performance is often observed, which make these heterocycles an important structural motif for the pharmaceutical industry. 1,3,4-oxadiazoles are an important class of heterocyclic compounds with a variety of biological activities. Substituted 1,3,4-oxadiazoles have shown antibacterial and antifungal (Gaonkar et al., 2006), antioxidant (Padmavathi et al., 2011), anticancer (Bhatt et al., 1994), antimycobacterial (Tangallapally et al., 2007), antimalarial (Kagthara et al., 1999), antihypoglycemic (Hussian et al., 1986), anti-inflammatory (Palaska et al., 2002), anticonvulsant (Zarghi et al., 2005) and muscle relaxant (Yale and Losee, 1966), genotoxic (Maslat et al., 2002), and insecticidal activities (Shi et al., 2001). They are also used extensively in the symptomatic treatment of rheumatic fever, arthritis (rheumatoid, osteo and jaundice arthritis), and management of primary dysmennorrhoea (Cao et al., 2003). Oxadiazole pharmacophore has the key property that influences the ability of a drug to reach the target by transmembrane diffusion and show potent antimicrobial activity (Testa et al., 2000).

The wide range of therapeutic values of 1,3,4-substituted oxadiazole ring systems promoted us to synthesize the title compounds and screen them for their antimicrobial and antioxidant activities. Therefore, it was envisaged that chemical entities with Schiff base and oxadiazole moieties would result in compounds of interesting biological activities. In view of these findings, we have attempted to incorporate all two biologically active components together to give the title compounds for evaluating their antimicrobial and antioxidant activities inspired by the above facts and in continuation of our ongoing research programme in the field of synthesis and antimicrobial activity of medicinally important compounds (Deep et al., 2010, 2012; Madhukar et al., 2009). In this communication we are reporting the synthesis of 1,3,4-substituted oxadiazole derivatives and evaluated them for antimicrobial and antioxidant activities.

2 Materials and methods

Melting points of the synthesized compounds were determined in open-glass capillaries on a Stuart SMP10 melting point apparatus and were recorded as uncorrected. The purity of the compounds was checked by thin layer chromatography (TLC). Silica gel plates kieselgel 0.25 mm, 60 GF₂₅₄, precoated sheets obtained from Merck, Darmstadt (Germany) were used for TLC and the spots were visualized by iodine vapours/ultraviolet light as visualizing agent. The IR spectra (υ, cm⁻¹) were obtained with a Perkin-Elmer 1600 FTIR spectrometer in KBr pellets. ¹H-NMR spectra (δ, ppm) were recorded in DMSO-d6 solutions on a Varian–Mercury 300 MHz spectrometer using tetramethylsilane as the internal reference. ¹³C-NMR spectra were recorded in dimethylsulphoxide (DMSO)-d6 solutions on a Bruker Avance II 400 spectrometer at 400 MHz using tetramethylsilane as the internal reference. Elemental analyses were performed on an ECS 4010 Elemental Combustion System. The necessary chemicals were purchased from Loba Chemie, Fluka and Sigma Aldrich.

3 Chemistry

The syntheses of targeted compounds were carried out as outlined in synthetic scheme. Compounds (6a–6r) were readily prepared in good yields and purity. Initially biphenyl-4-carboxylic acid (1) and excess of methanol with a catalytic amount of sulphuric acid were refluxed for 5 h to form biphenyl-4-carboxylic acid methyl ester (2) which on treatment with excess of hydrazine hydrate forms biphenyl-4-carboxylic acid hydrazide (3). Then biphenyl-4-carboxylic acid hydrazide and 2-hydroxybenzaldehyde were refluxed to form N′-(2-hydroxybenzylidene)biphenyl-4-carbohydrazide (4) which on reaction with acetic anhydride results in the formation of 1-(5-(biphenyl-4-yl)-2-(2-hydroxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone (5). Finally an equimolar amount of 1-(5-(biphenyl-4-yl)-2-(2-hydroxyphenyl)-1,3,4-oxadiazol-3(2H)-yl)ethanone and appropriate aromatic amines were refluxed with a catalytic amount of glacial acetic acid to form substituted oxadiazole derivatives (6a–6r). The purity of the compounds was checked by thin layer chromatography (TLC) and elemental analyses. The physical constants of synthesized compounds are given in Table 1.

Table 1 Physical constants of the synthesized compounds.

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Compound no.	R	Molecular formula	Molecular weight	Yield (%)	Mp (°C)
6a	2F	C28H22FN3O	451.49	72	192–194
6b	3F	C28H22FN3O	451.49	69	183–185
6c	4F	C28H22FN3O	451.49	64	214–216
6d	2Cl	C28H22ClN3O2	467.95	65	177–179
6e	3Cl	C28H22ClN3O2	467.95	58	182–184
6f	4Cl	C28H22ClN3O2	467.95	68	219–221
6 g	2Br	C28H22BrN3O2	512.40	75	227–229
6 h	3Br	C28H22BrN3O2	512.40	63	211–213
6i	4Br	C28H22BrN3O2	512.40	61	217–219
6j	2NO2	C28H22N4O4	478.50	77	225–227
6 k	3NO2	C28H22N4O4	478.50	63	208–210
6 l	4NO2	C28H22N4O4	478.50	73	224–226
6 m	2CH3	C29H25N3O2	447.23	58	197–199
6n	3CH3	C29H25N3O2	447.23	63	209–211
6o	4CH3	C29H25N3O2	447.23	65	206–208
6p	2OCH3	C29H25N3O3	463.53	55	214–216
6q	3OCH3	C29H25N3O3	463.53	63	207–209
6r	4OCH3	C29H25N3O3	463.53	67	231–233

4 Antimicrobial Evaluation

The newly synthesized compounds were screened for their antibacterial activity against Bacillus subtilis (MTCC 96), Staphylococcus aureus (MTCC 121), Pseudomonas aeruginosa (MTCC 2453) and Escherichia coli (MTCC 40) bacterial strains by the disc-diffusion method (Cruickshank et al., 1975; Collins, 1976). A standard inoculum (1–2 × 10⁷ c.f.u./ml 0.5 McFarland standards) was introduced on the surface of sterile agar plates, and a sterile glass spreader was used for even distribution of the inoculum. A disc measuring 6.25 mm in diameter was prepared from Whatman No. 1 filter paper and sterilized by dry heat at 140 °C for 1 h. The sterile disc previously soaked with the test compound solution in DMSO of specific concentrations 100 μg and 200 μg/disc was carefully placed on the agar culture plates. The plates were inverted and incubated for 24 h at 37 °C. Ciprofloxacin was used as a standard drug. Inhibition zones were measured and compared with the controls. The bacterial zones of inhibition were given in Table 2. Minimum inhibitory concentrations (MICs) were determined by the broth dilution technique. The nutrient broth, which contained logarithmic serially two fold diluted amount of the test compound and control was inoculated with approximately 5 × 10⁵ c.f.u (colony forming unit) of actively dividing bacteria cells. The cultures were incubated for 24 h at 37 °C and the growth was monitored visually and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as minimum inhibitory concentration (MIC).

To obtain the minimum bactericidal concentration (MBC), 0.1 ml volume was taken from each tube and spread on agar plates. The number of c.f.u was counted after 18–24 h of incubation at 35 °C. MBC was defined as the lowest drug concentration at which 99.9% of the inoculum was killed. The minimum inhibitory concentration and minimum bactericidal concentrations are given in Table 3.

The newly synthesized compounds were screened for their antifungal activity against Candida albicans (MTCC 8184) and Aspergillus niger (MTCC 8189) in DMSO by the agar diffusion method (Khan, 1997; Varma, 1998) Sabouraud’s agar medium was prepared by dissolving peptone (1 g), d-glucose (4 g) and agar (2 g) in distilled water (100 ml) and adjusting pH to 5.7. Normal saline was used to make a suspension of spore of fungal strain for lawing. A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of the corresponding species. Twenty millilitres of agar media was poured into each Petri dish. Excess of suspension was decanted and the plates were dried by placing in an incubator at 37 °C for 1 h. Using an agar punch, wells were made and each well was labelled. A control was also prepared in triplicate and maintained at 37 °C for 3–4 days. The fungal activity of each compound was compared with voriconazole as a standard drug. Inhibition zones were measured and compared with the controls. The fungal zones of inhibition values are given in Table 4.

The nutrient broth, which contained logarithmic serially two fold diluted amount of test compound and controls was inoculated with approximately 1.6 × 10⁴–6 × 10⁴ c.f.u./ml. The cultures were incubated for 48 h at 35 °C and the growth was monitored. The lowest concentration (highest dilution) required to arrest the growth of fungus was regarded as minimum inhibitory concentration (MIC). To obtain the minimum fungicidal concentration (MFC), 0.1 ml volume was taken from each tube and spread on agar plates. The number of c.f.u was counted after 48 h of incubation at 35 °C. MFC was defined as the lowest drug concentration at which 99.9% of the inoculum was killed. The minimum inhibitory, minimum bactericidal and minimum fungicidal concentrations are given in Table 5.

5 Antioxidant activity

Antioxidant activity is determined in terms of hydrogen peroxide scavenging activity. The solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4). Different concentrations (100, 300, and 500 μg/mL) of all the synthesized compounds were added to a hydrogen peroxide solution (0.6 mL, 40 mM). The absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage scavenging of hydrogen peroxide of the synthesized compounds and the standard compounds was calculated using the following formula: Percentage scavenging [H₂O₂] = [(A₀ − A₁)/A₀] × 100, where A₀ was the absorbance of the blank, and A₁ was the absorbance in the presence of the sample and standards (Gulcin et al., 2005). The percentage scavenging of hydrogen peroxide by the synthesized compounds at 100, 300 and 500 μg/mL concentrations was calculated and results are summarized in Table 6.

6 Result and discussion

Nevertheless, the structures of all new compounds synthesized were confirmed by (IR, ¹H NMR and ¹³C NMR) spectra. The IR spectra of all compound (6a–6r) showed absorption band at around 3459-3425, 2995-2958, 2865-2835, 1688-1675, 1578-1561, 1188-1171, and 1173-1118 cm⁻¹ regions, conforming the presence of OH, CH, CH₂, C⚌N, C⚌C, C–O, and C–N respectively. In the ¹H NMR spectra, the signals of the respective prepared derivatives were verified on the basis of their chemical shifts, multiplicities, and coupling constants. The spectra of most compounds showed the characteristic 1H proton of OH at around δ 9.95-9.71, 9H proton of biphenyl at around δ 8.69-7.55, characteristic protons of phenyl at δ 7.82-7.28 ppm, 3H proton of phenol appearing at 6.95-6.37, 1H proton of oxadiazole at 5.87-5.43, and 3H proton of N⚌C–CH₃ around δ 2.49-2.09 ppm. ¹³C-NMR spectra of compounds showed characteristic signals appearing for N⚌C–CH₃ at δ 163.94-163.18, oxadiazole ring at δ 155.84-68.95, phenyl ring at δ 162.83-108.91, biphenyl moiety at δ 131.94-127.03, phenol at δ 160.35-114.22 and N⚌C–CH₃ at δ 22.89-19.35 ppm.

The investigation of antibacterial screening data revealed that all the tested compounds showed moderate to good antibacterial and antifungal activities against B. subtilis, S. aureus, P. aeruginosa, E. coli, C. albicans and A. niger respectively. The compounds 6f, 6l and 6r displayed excellent antibacterial activity while 6e, 6j and 6k showed moderate antibacterial activity and the compounds 6b, 6m and 6p are less active as compared to standard drug ciprofloxacin. In case of antifungal activity compounds 6c, 6l and 6q exhibit significant activity while the compounds 6h, 6j, 6m, 6o and 6k showed moderate activity. Among all the synthesized derivatives the compound 6n was found to be the least active compound against fungal strain. All the synthesized compounds exhibited potent hydrogen peroxide scavenging activities. From all the synthesized compound analogues 6l with nitro moiety was the most active with scavenging of hydrogen peroxide of 58.18 at 500 μg/mL concentration, followed by compound 6q with methoxy group with hydrogen peroxide scavenging of 57.36 at 500 μg/mL.

The main aim of the present investigation is to synthesize and investigate the antimicrobial and antioxidant activity of newly synthesized oxadiazole containing compounds that are structurally related to the famous antimicrobial oxadiazole pharmacophore, with the hope of discovering new structure leads serving as potential broad spectrum antimicrobial and antioxidant activities. The obtained results revealed that 18 compounds were able to display growth inhibitory effects on the tested Gram-positive B. subtilis, S. aureus and Gram-negative bacteria P. aeruginosa, E. coli. Meanwhile, three compounds 6f, 6l and 6r displayed excellent activity against Gram-positive bacteria and Gram-negative bacteria meanwhile, three compounds 6c, 6l and 6q exhibit promising antifungal activity against C. albicans and A. niger. Structurally, the antimicrobial and antioxidant potential of the active compound depends on the nature of the substituents: remarkabe antibacterial and antifungal activities were encountered with the p-nitro phenyl moiety of the oxadiazole counterpart 6l, while the obtained antifungal activity conformed to those comprising compounds having p-fluoro, p-nitro and m-methoxy substituted oxadiazole derivatives 6c, 6l and 6q. While the compound 6l proved to be the most active antimicrobial member within this study with a considerable broad spectrum activity against all bacterial and fungal strains. From the results it is concluded that the introduction of p-nitro phenyl moiety along with oxadiazole pharmacophore 6l resulted in potent hydrogen peroxide scavenging activity. It may be due to nitro substitution at the para position of phenyl nucleus along with oxadiazole moiety which supports its therapeutic activity. So, from the results it has revealed that the nature and position of the substituent has shown a marked effect on antimicrobial and antioxidant activities. (See Scheme 1).

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Scheme 1 Reagents and conditions: (i) Methanol, reflux, 5 h, 84.43%; (ii) NH₂NH₂.H₂O, reflux, 3 h, 86.79%; (iii) 2-OHC₆H₄, ethanol, CH₃COOH, reflux, 5 h, 81% (iv) (CH₃CO)₂O, reflux, 7 h, 77% (v) Ethanol, CH₃COOH, aromatic amines, reflux, 9–11 h, 55–77%.

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7 Conclusion

This study reports the synthesis of (Z)-2-(5-(biphenyl-4-yl)-3-(1-(imino)ethyl)-2,3-dihydro-1,3,4 oxadiazol-2-yl)phenol derivatives as potent antimicrobial and antioxidant agents (6a–6r) and were characterized by spectral analysis. The obtained results revealed that most of the synthesized analogues have shown prominent antimicrobial and antioxidant activities. It was observed that the analogues (6f) (Z)-2-(5-(biphenyl-4-yl)-3-(1-(4-chlorophenylimino)ethyl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)phenol and (6l) (Z)-2-(5-(biphenyl-4-yl)-3-(1-(4-nitrophenylimino)ethyl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)phenol have shown excellent antibacterial activity rather in case of antifungal activity the analogues (6c) (Z)-2-(5-(biphenyl-4-yl)-3-(1-(4-fluorophenylimino)ethyl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)phenol, (6l) and (6q) (Z)-2-(5-(biphenyl-4-yl)-3-(1-(3-methoxyphenylimino)ethyl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)phenol exhibit prominent activity. Among all the synthesized compounds the analogue 6l exhibits potent antioxidant activity. So, these new therapeutic agents could be considered as lead molecule for the future development of drugs which could be used as antimicrobial and antioxidant agents.