Synthesis and antimicrobial evaluation of isoxazole-substituted 1,3,4-oxadiazoles

Chemistry

Synthesis of N-(5-methylisoxazol-3-yl)-2-(5-phenyl-1,3,4-oxadiazol-2-yl)acetamides 5a–k is shown in Scheme 1. Condensation of 3-amino-5-methylisoxazole (1) with diethyl malonate in ethanol under reflux afforded ethyl 2-(5-methyl-3-isoxazolylcarbamoyl)acetate [28] (2). Treatment of ester 2 with excess of hydrazine hydrate in ethanol furnished 3-hydrazinyl-N-(5-methyl-3-isoxazolyl)-3-oxopropanamide (3). The hydrazide 3 was condensed with aromatic aldehydes in methanol to furnish (E/Z)-3-(2-benzylidenehydrazinyl)-N-(5-methylisoxazol-3-yl)-3-oxopropanamides 4a–k. Compounds 4a–k on treatment with chloramine-T underwent oxidative cyclization to give N-(5-methylisoxazol-3-yl)-2-(5-aryl-1,3,4-oxadiazol-2-yl)acetamides 5a–k.

Scheme 1

Reagents and conditions: (i) diethyl malonate, ethanol, reflux 12 h, 80%; (ii) N₂H₄H₂O, ethanol, reflux 6 h, 92%; (iii) Ar-CHO, methanol, glacial AcOH, reflux 4–6 h, 90–95%; (iv) chloramine-T, ethanol, reflux 4–6 h, 68–76%.

The structures of compounds 3–5 were established based on IR, ¹H NMR, ¹³C NMR, ESI-MS and analytical data. In particular, ¹H NMR spectra of N-acylhydrazones 4a–k show characteristic double signals for each of the proton around the C=N bond, suggesting that these compounds exist in two isomeric forms. Two geometric isomers may be present in the ratio of 3:1; based on the integration values of proton signals. The results of the NMR experiments are discussed as follows.

Compound 4a was used for the analysis of nuclear Overhauser effect (nOe) and two-dimensional (2D)-¹H NMR-¹³C heteronuclear multiple bond correlation (HMBC) spectra. In the nOe experiment, irradiation of the methylene protons (10-CH₂) at δ_H 3.72 gives rise to an enhancement for both NH protons, which demonstrates that the methylene group is flanked by two amidic NH groups (Figure 1). The difference in nOe enhancement observed for the two amidic NH protons is due to their spatial arrangement. On irradiation of the NHCO proton at δ_H 11.07, only a signal for methylene protons is enhanced and irradiation of the olefinic proton NN=CH at δ_H 7.93 gives rise to nOe enhancement for phenyl ring protons and a hydrazine NH proton. Furthermore, irradiation of the other NHN=C proton at δ_H 11.49 gives nOe enhancement for both the methylene protons of the major and minor isomers. There is also a strong enhancement for the olefinic proton of the major isomer while the proton of the minor isomer is unaffected. These results demonstrate that the compounds exist as a mixture of E- and Z-isomeric forms.

Figure 1

Z (minor) and E (major) isomers of compound 4a.

This conclusion was further confirmed by 2D-¹H-¹³C HMBC experiment. HMBC data unambiguously show that the connectivity, as expected, between protons and carbons of the major and minor isomers is intact, and the assignments of chemical shift values for compound 4a are as follows: correlation of H-6 (δ_H 2.3, 2.4) with C₂ (δ_c 170.0, 170.1) and C₁ (δ_c 96.7); H₇ (δ_H 11.0, 11.1) with C₁ (δ_c 96.6) and C₈ (δ_c 166.3, 165.9); H₁₃ (δ_H 11.5, 11.6) with C₁₅ (δ_c 143.5, 147.3) and C₁₀ (δ_c 43.0, 43.7) and C₁₁ (δ_c 163.0 and 169.1) and H₁₅ (δ_H 7.9, 8.2) with C₁₆ (δ_c 134.5) and C₁₇ and C₂₁ (δ_c 127.3, 127.6). The key points from HMBC data are the correlations between H₇-C₁, H₁₃-C₁₅ and H₁₀-C₈ and H₁₀-C₁₁, which indicate the absence of keto-enol tautomerism. On the basis of both 1D and 2D NMR data, it can be concluded that these compounds exist in the mixture of Z and E geometrical isomers.

The IR spectrum of N-(5-methylisoxazol-3-yl)-2-(5-phenyl-1,3,4-oxadiazol-2-yl)acetamide (5a) shows a characteristic band at 1237 cm⁻¹ due to C-O-C stretching vibration confirming the formation of 1,3,4-oxadiazole ring. ¹H NMR spectrum of 5a does not exhibit signals due to the CH=N proton, and the NH protons of the hydrazone, which are present in its precursor 4a at δ 7.93, 8.16, 11.49 and 11.55, confirming the oxadiazole ring formation. The absence of azomethine carbon signals at δ 143.5 and 147.3 in ¹³C NMR spectrum of 5a also supports the formation of the oxadiazole ring. The mass spectrum of 5a also agrees with its structure by exhibiting the protonated molecular ion [M+H]⁺ peak at m/z 285.

Antibacterial activity

Compounds 5a–k exhibit good antibacterial activity in comparison to the activity of the standard drug ciprofloxacin (Table 1). Some of the compounds exhibit excellent minimum inhibitory concentration values (MIC). Compounds 5b and 5f are highly active. The exceptional activity of compound 5d may be due to the presence of a nitro group on the phenyl ring. The remaining compounds 5a, 5c, 5e, 5g, 5h, 5i, 5j and 5k show moderate activity. However, the degree of inhibition varies both with the test compound and with the bacteria used in the present investigation.

Antifungal activity

Compounds 5a–k are significantly toxic toward all five pathogenic fungi and are lethal even at 100 μg/mL concentration when compared to the standard drug clotrimazole (Table 2). The activity data are indicated as a zone of inhibition at 100 μg/mL concentration. Compounds 5b and 5f exhibit high activity and they inhibit the growth of fungi to a remarkable extent, which may be due to the presence of chloro, nitro and bromo substituents on the benzene ring, besides the presence of isoxazole and oxadiazole skeletons. Compound 5d bearing a nitro group on the benzene ring shows good toxicity against the fungi used. Compounds 5a, 5c, 5e, 5g, 5h, 5i, 5j and 5k are moderate in their toxicity and they are less active compared to other compounds in the present study, but better than the standard drug clotrimazole. The degree of spore germination inhibition varies with test compounds and with the type of fungi.

Synthesis of ethyl 2-(5-methyl-3-isoxazolylcarbamoyl)acetate (2)

A mixture of 3-amino-5-methylisoxazole (1, 0.1 mmol) and diethyl malonate (0.1 mmol) in ethanol (20 mL) was heated under reflux for 12 h. The reaction was monitored by TLC analysis. After the mixture was concentrated and cooled, the separated solid product was filtered under suction, dried and crystallized from ethanol; yield 80%; mp 112–113°C; IR: υ_max 3266, 1743, 1700 cm⁻¹ ; ¹H NMR (CDCl₃): δ 1.30 (t, J=8.0 Hz, 3H, OCH₂-CH₃), 2.41 (s, 3H, isoxazole-CH₃), 3.52 (s, 2H, CH₂), 4.25 (q, J=8.0 Hz, 2H, OCH₂-CH₃), 6.70 (s, 1H, isoxazole-H), 10.19 (s, 1H, NH); ¹³C NMR (CDCl₃): δ 12.6, 14.0, 42.0, 62.0, 96.6, 157.7, 163.4, 168.2, 170.1; MS: m/z 213, (M+H)⁺. Anal. Calcd for C₉H₁₂N₂O₄: C, 50.94; H, 5.70; N, 13.20. Found: C, 50.92; H, 5.68; N, 13.21.

Synthesis of 3-hydrazino-N-(5-methylisoxazol-3-yl)-3-oxopropanamide (3)

A mixture of compound 2 (0.1 mmol) in ethanol (15 mL) and hydrazine hydrate (0.5 mmol) (98%) was heated under reflux for 6 h, and the progress of the reaction was monitored by TLC. The excess ethanol was removed under reduced pressure and the residue of 3 was washed with cold water and cold methanol and crystallized from methanol; yield 92%; mp 152–153°C; IR: υ_max 3211–3337, 3141, 1652 cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.35 (s, 3H, isoxazole-CH₃), 3.19 (s, 2H, CH₂), 4.26 (s, 2H, NH₂), 6.57 (s, 1H, isoxazole-H), 9.13 (s, 1H, NH), 10.94 (s, 1H, NH); ¹³C NMR (DMSO-d₆): δ 12.5, 42.5, 96.6, 158.3, 165.9, 166.1, 170.0; MS: m/z 199.15 (M+H)⁺. Anal. Calcd for C₇H₁₀N₄O₃: C, 42.42; H, 5.09; N, 28.27. Found: C, 42.40; H, 5.08; N, 28.25.

General procedure for the synthesis of 3-(2-arylidenehydrazino)-N-5-(methyl-3-isoxazolyl)-3-oxopropanamides 4a–k

A mixture of hydrazide 3 (0.1 mmol) and an aromatic aldehyde (0.1 mmol) was heated under reflux in methanol (20 mL) in the presence of a catalytic amount of glacial acetic acid for 4–6 h. The progress of the reaction was monitored by TLC. The reaction mixture was cooled, and the separated solid was filtered and crystallized from methanol.

General procedure for the synthesis of N-(5-methylisoxazol-3-yl)-2-(5-aryl-1,3,4-oxadiazol-2-yl)acetamides 5a–k

A mixture of hydrazone 4 (0.1 mmol), chloramine-T (0.5 mmol) and ethanol (15 mL) was heated under reflux for 4–6 h. The progress of the reaction was monitored with TLC. Afterward, the mixture was poured into water and extracted with ethyl acetate. The extract was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. After removal of the solvent, the crude product was passed over silica gel column. The product was eluted with 40% ethyl acetate in n-hexane.

Antibacterial activity

The antibacterial activity was assayed by the broth dilution method [29] and expressed as minimum inhibitory concentration. The nutrient broth medium (HiMedia, 24 g) was suspended in distilled water (100 mL) and heated to boiling until it dissolved completely. The medium and test tubes were autoclaved at a pressure of 15 lb/inch² for 20 min. A set of sterilized test tubes with nutrient broth medium was capped with cotton plugs. The test compound 5a–k was dissolved in acetone and the concentration of 100 μg/mL of the test compound was added to the first test tube, which was serially diluted. A fixed volume of 0.5 mL was added to all test tubes, and the tubes were incubated at 37°C for 24 h. Then, the tubes were measured for turbidity. Bacterial strains used were Pseudomonas aeruginosa (MTCC 741), Klebsiella aerogenes (MTCC 39) Chromobacterium violaceum (MTCC 2656) (Gram-negative) and Bacillus subtilis (MTCC 441), Bacillus sphaericus (MTCC 511) and Staphylococus aureus (MTCC 96) (Gram-positive). Ciprofloxacin was used as the standard drug for comparison.

Antifungal activity

The antifungal activity was assayed using agar cup bioassay method [30]. The potato dextrose agar (PDA) medium (HiMedia, 39 g) was suspended in distilled water (1 L) and the mixture was heated to boiling until a solution was formed. The medium and Petri dishes were autoclaved at a pressure of 15 lb/inch² for 20 min. The medium was poured into sterile Petri dishes under aseptic conditions in a laminar flow chamber. When the medium in the plates solidified, 0.5 mL of (week old) culture of the test organism was inoculated and uniformly spread over the agar surface with a sterile L-shaped rod. A solution was prepared by dissolving the compound 5a–k in acetone (100 μg/mL). Agar inoculated cups were scooped out with a 6-mm sterile cork borer, and the lids of the dishes were scooped out with a 6-mm sterile cork borer and the lids of the dishes were replaced. To each cup, 100 μg/mL concentration of the test solution 5a–k was added. Controls were maintained with acetone and clotrimazole (100 μg/mL). The treated mixtures and the controls were kept at room temperature for 72–96 h. The inhibition zone was measured as a diameter (mm). Three to four replicates were maintained for each treatment. The fungal strains Aspergillus niger (MTCC 282), Chrysosporium tropicum (MTCC 2821), Rhizopus oryzae (MTCC 262), Fusarium moniliforme (MTCC 1848) and Curvularia lunata (MTCC 2030) were used.