Synthesis and antimicrobial evaluation of some 2,5 disubstituted 1,3,4-oxadiazole derivatives.

1,3,4-oxadiazoles are interesting compounds because of their valuable biological effects such as cytotoxic, antibacterial, antifungal, and anti-tubercular activities. Ethyl mandelate was treated with hydrazine hydrate to yield the corresponding acylhydrazide. Some of the 2,5 disubstituted 1,3,4-oxadiazole derivatives were prepared from acylhydrazide using three different procedures. In the first procedure, acylhydrazide was reacted with nitro or chloro aroyle chloride to afford a diacylhydrazide which was cyclized to 2,5-disubstituted 1,3,4-oxadiazole in the presence of phosphoryl chloride as dehydrating agent. In the second procedure, furan-oxadiazole derivative was directly prepared from carboxylic acid and acylhydrazide in one step. In the third procedure, acyl hydrazide was condensed with 5-nitrofuraldehyde to yield 5-nitrofuran-2-yl) methylene)-2-phenyl acetohydrazide intermediate which was cyclized to form the nitrofuran-oxadiazole derivative by acetic anhydride as dehydrating agent. The structures of these compounds have been elucidated by spectral IR and 1H-NMR analysis. All the newly synthesized compounds were screened for their antibacterial and antifungal activities. Compounds F3 and F4 showed remarkable antibacterial activities against Staphylococcus aureus and Escherichia coli bacteria.

INTRODUCTION

Infectious diseases are responsible for nearly one-half of all deaths in tropical countries. Although deaths from bacterial and fungal infections have declined in the developed countries, these infections are still major problem in undeveloped territories. One of the attractive backbones for scientists in production of new therapeutic agents is oxadiazole structure (1234). There are four known isomers of this five-membered heterocycle including: 1,2,4-, 1,2,3-, 1,2,5-, and 1,3,4-oxadiazole (56). However, 1,3,4-oxadiazole is more important because of its remarkable biological activities. Compounds containing 1,3,4-oxadiazole structure possess various pharmacological effects including antibacterial, antifungal (145678910), anti-tubercular (11), anticonvulsant, anti-allergic, anti-inflammatory, cytotoxic, and insecticidal activities (1612). This structure can be used as a bioisoester for carboxylic acids, esters, and carboxamides (6). Tiodazosin, nosapidil, and furamizole are derivatives of oxadiazole which have been introduced as antihypertensive and antibacterial agents, respectively (12). Various 2,5 substituted 1,3,4-oxadiazoles have been shown to be active against a wide range of gram-positive and gram-negative bacteria (61314).

It is generally accepted that lipophilic substitutions may facilitate transport of drug molecules through biological membrane of microorganisms thereby improving their antimicrobial activities (1015). The presence of electronegative groups such as Cl or NO2 on phenyl ring enhances the antimicrobial activities of drug moieties (101415).

Substitution of oxadiazoles with nitro furan ring has shown significant antibacterial activities against staphylococcal strains at 4 to 32 μg/mL (6).

Many methods have been reported for the synthesis of both symmetrical and asymmetrical 2,5-diaryl(alkyl)-1,3,4-oxadiazoles. One of the methods involves hydrazinolysis of the ethyl ester with hydrazine hydrate to yield the corresponding acylhydrazide. The resultants can be reacted with different aroyle chloride to afford intermediates which can be subsequently cyclized to 2,5-disubstituted 1,3,4-oxadiazole in the presence of a suitable dehydrating agent like phosphoryl chloride. Other dehydrating agents commonly used are CF3COOH, H2SO4, P2O5, H3PO4, PCl5 (1013). Literature survey revealed that some oxadiazoles could be directly prepared from carboxylic acids and acylhydrazides in one step which involves longer reaction time and production of by-products (131416). Another method is reaction of tetrazoles with acyl chlorides in the presence of pyridine (12). Therefore, the procedure started with N,N-diacyl hydrazines intermediate which is easier and more convenient to get target compounds.

Mandelic acid (MA) is an alpha-hydroxy acid used as an antibacterial in the treatment of urinary tract and skin infections such as acne. Some of the MA derivatives have shown antibacterial activities against Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Candida albicans (C. albicans) (1718).

Due to the remarkable antimicrobial effects of many 1,3,4-oxadiazole and MA derivatives, some novel structural hybrids of 1,3,4-oxadiazole derivatives and benzyl group derived from ethylmandelate were synthesized and evaluated for their antibacterial and antifungal activities.

MATERIALS AND METHODS

Chemistry

Melting points were determined in open capillaries using electrothermal 9200 melting point apparatus (England) and are uncorrected. IR (KBr discs) was recorded with a WQF-510 FT-IR spectrophotometer (China). 1H-NMR spectra were recorded on Bruker 400 MHz spectrometers (Germany) using tetramethyl-silane (TMS) as internal standard and either DMSO-d6 or CDCl3 as solvents. Mass spectra were recorded on Finnigan TSQ-70 Mass spectrometer (United States). All chemicals were purchased from Merck Company (Germany).

Synthesis of compounds

In this study, we have synthesized some new 1,3,4-oxadiazole derivatives substituted at 2,5 positions. Ethyl mandelate was reacted with hydrazine hydrate to obtain acylhydrazide as crystalline product. Acylhydrazide was reacted with 4-chlorobenzoyl chloride or 4-nitro benzoyl chloride to afford intermediate which subsequently was cyclized to 2,5-disubstituted 1,3,4-oxadiazole in the presence of a suitable dehydrating agent like phosphoryl chloride. These compounds were re-crystallized by ethanol to obtain target compounds (1213). In another procedure, acylhydrazide and furan-2-carboxylic acid refluxed in POCl3 for 1 h on water bath. The reaction mixture was poured into crushed ice with stirring. The resultant solid was collected, and purified by column chromatography (19202122) (Fig. 1).

Fig. 1

General reaction scheme for preparation of the final compounds; GAA stands for glacial acetic acid.

Reaction between acylhydrazide and 5-nitro-2-furaldehyde in ethanol (95%) with glacial acetic acid as the catalyst produced N-acylhydrazone in high yield and purity. Treatment of N-acylhydrazone with acetic anhydride afforded oxadiazole derivative substituted with nitrofuran (2324) (Fig. 1). The structures of synthesized compounds were confirmed by IR and 1H-NMR.

Details of preparation procedures of synthesized compounds

2-hydroxy-2-phenyl-acetohydrazide (I)

Ethyl mandelate (100 mmol, 13.8 mL) was added dropwise to a solution of hydrazine hydrate (100 mmol, 3.2 mL) in ethanol (8 mL) and was refluxed for 4 h. The ethanol was partially removed by vacuum distillation. On cooling the resultant mixture, white needle crystals of 2-hydroxy-2-phenyl-cetohydrazide began to separate. It was collected, filtered, and recrystallized from ethanol.

Diacyl hydrazide intermediates

A solution of the 4-chloro benzoyl chloride (2 mmol, 350 mg) or 4-nitro-benzoyl chloride (2 mmol, 370 mg) in tetrahydrofuran (THF) (3 mL) was added dropwise to the mixture of 2-hydroxy-2-phenyl-acetohydrazide (2 mmol, 330 mg) and THF (5 mL) over 1 h, while stirring at 0 °C. The reaction mixture was stirred for 3 h at room temperature. The solvent was removed in vacuum to furnish a semisolid material which was washed with saturated aqueous NaHCO3 solution then extracted with ethyl acetate. The organic layer was evaporated to afford the intermediate.

2,5-disubstituted 1,3,4-oxadiazoles (F)

A mixture of obtained diacyl hydrazide of 4-chloro benzoyl chloride (0.66 mmol, 200 mg) or 4-nitro-benzoyl chloride (0.67 mmol, 200 mg) and POCl3 (4 mL) was refluxed for 2-3 h. After cooling to room temperature, the reaction mixture was gradually poured into crushed ice with stirring. The aqueous layer was extracted with ethyl acetate. The extract was washed with water, dried over anhydrous sodium sulfate, and finally distilled under reduced pressure to remove the solvent. The resulting solid was recrystallized using ethanol to give the target compounds.

2-(Chloro(phenyl)methyl)-5-(furan-2-yl)-1,3,4-oxadiazole (F)

A mixture of 2-hydroxy-2-phenyl-acetohydrazide (6 mmol, 990 mg) and the furan-2-carboxylic acid (5 mmol, 560 mg) in POCl3 (4 mL) was heated to 70 °C for about 3 h on water bath. The progress of the reaction was monitored on TLC. The reaction mixture was poured into crushed ice with stirring. The resultant solid was collected, washed with water, and purified by column chromatography chloroform:methanol 19:1 to afford an orange powder (40% yield).

2-Hydroxy-N’-5-nitrofuran-2-yl) methylene)-2-phenylacetohydrazide (I)

To a stirred solution of 2-hydroxy-2-phenyl-acetohydrazide (1 mmol, 160 mg) and 5-nitrofuraldehyde (1 mmol, 140 mg) in ethanol, glacial acetic acid (0.2 mL) was added dropwise.

The resulting mixture was refluxed for 5 h, after which the solution was poured into ice water. The precipitate was collected by filtration and dried under reduced pressure to give hydrazone intermediate as brown solid (89% yield).

1-(5-(Hydroxy (phenyl) methyl)-2-(5-nitrofuran-2-yl)-1,3,4-oxadiazole-3(2H)-yl)ethanone (F)

5-Nitrofuran derivative of oxadiazole was synthesized by addition of acetic anhydride (2 mL) to hydrazone (0.34 mmol, 100 mg), and the resulting solution was refluxed for 1 h. The reaction mixture was poured into ice water, and the resulting solid was filtered, washed with water, and purified by PTLC to obtain a yellow powder, (54% yield).

Antimicrobial activity

The employed microorganisms for antimicrobial studies were S. aureus, PTCC (Persian Type Culture Collection) 1337 as gram-positive; Escherichia coli (E. coli), PTCC 1338 and P. aeruginosa, PTCC 1074 as gram-negative bacteria; and C. albicans, PTCC 5027 as fungus. Mueller Hinton agar, Mueller Hinton broth and Sabouraud dextrose agar were purchased from Merck (Germany). RPMI1640 culture medium was purchased from Gibco (USA).

Microplate alamar blue assay for antimicrobial evaluation

The inocula of bacterial and fungal strains (1.5 × 108 CFU/mL) were prepared from Mueller Hinton agar and Sabouraud dextrose agar cultures, respectively. Prepared suspensions of bacteria were adjusted to 0.5 McFarland standard turbidity and the fungal suspensions turbidity was measured spectrometrically at 580 nm.

Finally, prepared inoculum densities for bacterial and fungal strains were equal to 1.0 × 105 CFU/mL and 1.0 × 106 CFU/mL, respectively.

The Synthesized compounds were dissolved in DMSO (0.5 mL) and diluted with water up to 1 mL to obtain concentration of 5120 μg/mL as stock solutions. The stock solution was serially diluted to give concentrations of 2560 to 40 μg/mL.

Bacterial suspension (20 μL) was distributed in all 96 wells of microplate. Then 20 μL of each concentration of the compounds were added to wells with the exception of those wells acting as positive control (containing standard antibiotic) and growth control (containing culture media without testing materials). After adding alamar blue (20 μL) to all 96 wells the total volume in each well reached to 200 μL using culture medium. The final concentrations of the compounds in the wells were 512, 256, 32, 16, 8, and 4 μg/mL. After incubation, the lowest concentration which required inhibiting the growth of microorganism was regarded as minimum inhibitory concentration (MIC). The test was carried out in triplicates.

Following a broth micro dilution MIC test, from each well that showed no growth, contents were removed and spread onto Mueller Hinton agar plates for bacteria and Saboraud dextrose agar for fungi to determine minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC).

RESULTS

Chemistry of synthesized compounds

2-Hydroxy-2-phenyl-acetohydrazide (I)

White crystal, (89% yield), m.p.: 125-128 °C (lit.132-133 °C), (C8H10N2O2); IR (KBr, cm-1), 3338 (NH2), 3208 (N-H), 3062 (C-H Ar), 1695 (C=O), 1HNMR, δH (400 MHz; DMSO), 9.24 (1H, s, NH), 7.50 (2H, d, J = 7.2 Hz, Ha, Ar), 7.40 (2H, t, J = 7.2 Hz, Hb, Ar), 7.34 (1H, t, J = 7.2 Hz, Hc, Ar), 6.05 (1H, d, J = 5.2 Hz, CH), 5.01 (1H, d, J = 5.2 Hz, OH), 4.3 (2H, s, NH2).

2-(Chloro (phenyl) methyl)-5-(4-chlorophenyl)-1, 3, 4-oxadiazole (F)

Light yellow, (50% yield), m.p.: > 265 °C, MS (m/z,): found: 305 C15H10Cl2N2O; requires: 305, IR (KBr, cm-1), 3010 (CH, Ar), 1012 (C-O-C, ether), 1599 (C=C), 850 (C-Cl). 1HNMR, δH (400 MHz; CDCl3), 8.1 (2H, d, J = 8 Hz, Ha, Ar), 7.74 (2H, dd, J = 2 Hz, J = 6.4 Hz, Hb, Ar), 7.6 (2H, d, J = 8Hz, Hc, Ar), 7.54 (3H, m, Hd, Ar), 6.3 (1H, s, oxadiazole ring -CH-Cl).

2-(Chloro (phenyl) methyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole (F)

Pale yellow (45% yield), m.p.:154-155 °C, MS (m/z): found 315 C15H10ClN3O3; requires: 315, IR (KBr, cm-1), 3103 (CH, Ar), 1338, 1516 (NO2), 1012 (COC, ether), 700 (C-Cl), 1H-NMR, δH (400 MHz; CDCl3),8.38 (2H, d, J = 7.2Hz, Ha, Ar), 8.26 (2H, d, J = 7.2 Hz, Hb, Ar), 7.64 (2H, dd, J = 2Hz, J = 8 Hz, Hc, Ar), 7.45 (3H, m, Hd, Ar), 6.30 (1H, s, oxadiazole ring -CH-Cl).

2-(Chloro(phenyl)methyl)-5-(furan-2-yl)-1,3,4-oxadiazole (F)

Orange powder (40% yield), m.p.: 227.5-228.3 °C, C13H9ClN2O2, IR (KBr, cm-1), 1103, 1024 (C-O-C, ether), 806 (C-Cl). 1HNMR, δH (400 MHz; CDCl3),7.58 (1H, dd, J = 4 Hz, J = 0.4 Hz, Ha, furan), 7.55 (2H, dd, J = 8 Hz, J = 4 Hz, Hb, Ar), 7.35 (3H, m, Hc, Ar), 7.1 4 (1H, dd, J = 4 Hz, J = 0.4 Hz, Hd, furan), 6.53 (1H, m, He, furan), 6.19 (1H, s, oxadiazole ring-CH-Cl).

2-Hydroxy-N’-5-nitrofuran-2-yl) methylene)-2-phenylacetohydrazide (I)

Brown solid (89% yield), m.p.: 178-180.5 °C, (C13H11N3O5); IR (KBr, cm-1), (3400, OH), 3216 (NH), 1674 (C=N), 1300, 1550 (NO2), 1HNMR, δH (400 MHz; DMSO), 11.89 (1H, s, NH,),8.44 (1H, s, Ha),7.77 (1H, d, J = 4Hz, Hb, nitro furan ring), 7.48 (2H, d, J = 7.2 Hz, Hc, Ar), 7.36 (2H, t, J = 7.2 Hz, Hd, Ar), 7.3 (1H, t, J = 7.2 Hz, He, Ar), 7.19(1H, d, J = 4 Hz, Hf, nitrofuran ring), 6.52 (1H, d, J = 4.4 Hz, CH), 5.13 (1H, d, J = 4.4 Hz, O H).

1-(5-(Hydroxy (phenyl) methyl)-2-(5-nitrofuran-2-yl)-1,3,4-oxadiazole-3(2H)-yl)ethanone (F)

Yellow powder, (54% yield), m.p.:182-183 °C, (C15H13N3O6); IR (KBr, cm-1), 3427 (OH), 1685 (C=N), 1236 (COC, ether), 1352, 1560, (NO2), 1HNMR, δH (400 MHz; CDCl3), 9.07 (1H, s, OH),7.63 (2H, d, J = 8Hz, Ha, Ar),7.54 (1H, s, Hb),7.36-7.43 (4H, m, Hc and Hd),6.85 (1H, d, J = 4Hz, He, nitrofuran ring), 6.69 (1H, s, Hf), 2.22 (3H, s, CH3).

Antimicrobial results

The MIC of all the tested compounds was evaluated at concentrations of 4 to 512 μg/mL. Compounds F, F, and I showed significant inhibition at 8, 4, and 4 μg/mL concentrations, respectively against S. aureus as gram-positive bacteria. F, F, and I exhibited maximum activity at 16, 16, and 8 μg/mL, respectively, against E. coli as gram-negative bacteria. Compounds F and F displayed high fungi static and fungicidal activities against C. albicans at 32 μg/mL concentration. Results of MIC, MBC, and MFC are depicted in Tables 1 and 2.

Table 1

MIC results of synthesized compounds against bacteria and fungi.

Table 2

MFC and MBC results of synthesized compounds against bacteria and fungi.

DISCUSSION

Oxadiazoles constitute an important class for new drug development with various biological properties (1456789101112). Substituted 1,3,4-oxadiazoles at 2,5 positions are particularly important because of their antimicrobial activities against a wide range of gram-positive and gram-negative bacteria (613142526).

Structure-activity relationships have shown that presence of electronegative groups such as Cl or NO2 on phenyl ring can enhance their antimicrobial effects (101415). Substituted oxadiazole with nitro furan or furan ring showed significant antimicrobial activities (6). Due to valuable antimicrobial effects of oxadiazole scaffold, in this study, a new series of 2,5 disubstituted 1,3,4-oxadiazoles derivatives were synthesized and their antimicrobial activity assessed using microplate alamar blue assay.

Substitution of chlorine or nitro group on the phenyl ring (F and F) rendered reasonable antifungal activity compared to other tested compounds. While the furan-derivatives (F and F) showed remarkable antibacterial activities.

The results of antibacterial screening showed that compounds F, F, and I (furan and nitro furan derivatives) had the highest activities against S. aureus at 8, 4, and 4 μg/mL concentrations. It seems that presence of hydroxyl group of mandelate residue in compound F and I might improve activities in gram-positive bacteria. These compounds F, F, and I showed the best activities against E. coli at 16, 16, and 8 (μg/mL) concentrations, respectively. Presence of a para-substituted-phenyl group at position 5 of oxadiazole in compounds F and F may be responsible for fungistatic and fungicidal activities against C. albicans in comparison with furan derivative of oxadiazole.

CONCLUSION

Oxadiazole derivatives preparation from N, N-diacylhydrazine intermediate in two steps is easier and more convenient than direct preparation from carboxylic acids and acylhydrazides in one step. Direct one-step method with mild condition was used for the synthesis of furan-oxadiazole derivative. Condensation reaction between the 2-hydroxy-2-phenyl-acetohydrazide and 5-nitro-2-furaldehyde in ethanol (95%) with glacial acetic acid as the catalyst produced N-acylhydrazone in high yields. Nitro-furan-oxadiazole compound easily obtained by reacting N-acylhydrazone with acetic anhydride in moderate yield. According to the antimicrobial results, furan-derivatives of oxadiazoles showed potential antibacterial activities and a para-substituted phenyl derivatives of oxadiazole exhibited significant antifungal activities.