Functionalized isoxazoles are actively used in medicine and agriculture. Substituted isoxazoles are components of veterinary medicines, herbicides, pesticides, and fungicides such as hymexazol, pyrisoxazole, oxathiapiprolin, and drazoxolon [1, 2]. Compounds of the isoxazole class proved to be effective in fighting fungal diseases of plants [2–5]. Different approaches to preparing isoxazoles with alkyl(aryl)sulfanyl or alkyl(aryl)sulfonyl fragments have been developed recently [6–10]; these compounds show promise as antimicrobial [2] and fungicidal [9, 10] agents. Isoxazoles with diarylsulfonyl substituents [9] exhibit higher activity than related compounds with diaryl fragments, and sulfur-containing isoxazoles [10] surpass sulfur-containing pyrazoles in antimicrobial properties.

This study was aimed at preparing new (alkylsulfanyl)-containing isoxazoles by the microwave-assisted reaction of hydroxylamine with 3-[(alkylsulfanyl)methyl]pentane-2,4-diones, which, in turn, can be prepared by ternary condensation of acetylacetone with formaldehyde and thiols, and at evaluating the fungicidal activity of the compounds obtained toward Bipolaris sorokiniana, Rhizoctonia solani, and Fusarium oxysporum phytopathogenic fungi.

EXPERIMENTAL

Chemicals, methods, and devices. The reactions were performed in a Discover system 908010 microwave synthesis reactor (CEM Matthews NC) with the maximal radiation power of 300 W and frequency of 2455 MHz. The IR spectra were recorded with a Shimadzu JR Prestige-21 spectrometer in thin film. The 1Н and 13С NMR spectra were recorded with a Bruker Avance III 500 MHz spectrometer operating at 500 and 125 MHz, respectively, using СDСl3 as a solvent and the solvent signal (7.27 ppm for residual protons, 77.1 ppm for 13С) as an internal reference. The reaction completeness and the product purity were checked by gas–liquid chromatography with a Khromos 1000 chromatograph (Khromos, Russia) using a 1 m × 3 mm column; stationary phase 5% SE-30 on Chromaton N-AW-DMCS (0.16–0.20 mm), working temperature 50–300°С, flame ionization detector, carrier gas helium. The mass spectra were recorded with a Shimadzu LCMS-2010 EV liquid chromatograph–mass spectrometer with a single quadrupole in the mode of recording positive ions at a capillary potential of 4.5 kV with electrospray ionization; eluent MeCN–H2O (95 : 5). Elemental analysis was performed with a Euro ЕА 3000 CHNS analyzer (HEKAtech GmbH). Chromatographic separation was performed on columns packed with MN Kieselgel 60 silica gel (0.063–0.2 μm). Ethanol (chemically pure grade, Bashspirt, Russia), hexane, ethyl acetate, and chloroform (chemically pure grade, EKOS-1) were used as solvents. The solvents were purified by standard procedures [11]. Hydroxylamine hydrochloride (analytically pure grade, Reakhim, Russia) was used without additional purification. Pentane-2,4-diones 1a–1g were prepared by the procedure described in [12], and compounds 3–5, by those described in [13]. The purity of reactants 3–5 was confirmed by elemental analysis and IR and NMR spectroscopy; the spectroscopic characteristics agreed with the published data [13].

Synthesis of 4-[(alkylsulfanyl)methyl]-3,5-dimethylisoxazoles 2a–2g (general procedure). (a) To a solution of 1.5 mmol of 1a–1g in 10 mL of ethanol, a solution of 1.8 mmol of hydroxylamine hydrochloride in 0.5 mL of water was added with stirring. The mixture was refluxed for 5 h, after which it was diluted with water (~1 : 8), and the reaction product was extracted with chloroform (3 × 20 mL). The combined organic phase was washed with water (2 × 10 mL) and dried over MgSO4. The solvent was distilled off under reduced pressure, and the residue was chromatographed on a silica gel column (eluent ethyl acetate–hexane, 1 : 5).

(b) The microwave-assisted synthesis was performed in a 10-mL reaction vessel. To a solution of 0.5 mmol of 1a–1g in 5 mL of ethanol, 0.6 mmol of hydroxylamine hydrochloride was added, and the mixture was stirred at 78°С for 5 min. The microwave radiation power was varied from 50 W at the start of the reaction to 4–5 W on reaching the temperature of 78°С. This temperature was reached in 30 s. After the reaction completion, the mixture was worked up similarly to method a.

4-[(Ethylsulfanyl)methyl]-3,5-dimethylisoxazole (2а). Yield 0.23 g (88%, a), 0.082 g (95%, b). IR spectrum (thin film), ν, cm–1: 2968, 2927, 2870, 1637 (CN), 1452, 1423, 1375, 1267, 1240, 1195, 1041, 977, 889, 738. 1Н NMR spectrum (CDCl3), δ, ppm: 1.24 t (3H, CH3CH2, 3J = 7.4 Hz), 2.28 s (3H, CH3C3), 2.35 s (3H, CH3C5), 2.45 q (2H, CH3CH2, 3J = 7.4 Hz), 3.44 s (2H, CH2S). 13С NMR spectrum, δ, ppm: 10.09, 10.98 (СH3C3, CH3C5), 14.37 (CH3CH2), 23.20, 25.50 (CH2SCH2), 110.62 (С4), 159.59 (C5), 165.71 (C3). Mass spectrum, m/z (Irel, %): 172 [M + H]+ (47), 213 [M + H + MeCN]+ (100). Found, %: С 56.09, Н 7.62, N 8.10, S 18.78. С8Н13NОS. Calculated, %: С 56.10, Н 7.65, N 8.18, S 18.72.

4-[(2-Propylsulfanyl)methyl]-3,5-dimethylisoxazole (2b). Yield 0.23 g (82%, a), 0.084 g (91%, b). IR spectrum (thin film), ν, cm–1: 2960, 2927, 2866, 1637 (CN), 1454, 1425, 1382, 1365, 1271, 1253, 1238, 1195, 1155, 1053, 889, 738. 1Н NMR spectrum (CDCl3), δ, ppm: 1.26 d [6H, (CH3)2CH, 3J = 6.7 Hz], 2.27 s (3H, CH3C3), 2.34 s (3H, CH3C5), 2.79 septet [2H, (CH3)2CH, 3J = 6.7 Hz], 3.45 s (2H, CH2S). 13С NMR spectrum, δ, ppm: 10.19, 11.07 (СH3C3, CH3C5), 22.33 (CH2S), 23.17 [(CH3)2CH], 34.82 [(CH3)2CH], 112.00 (С4), 160.98 (C5), 166.94 (C3). Mass spectrum, m/z (Irel, %): 186 [M + H]+ (68), 227 [M + H + MeCN]+ (100). Found, %: С 58.29, Н 8.13, N 7.51, S 17.36. С9Н15NОS. Calculated, %: С 58.34, Н 8.16, N 7.56, S 17.31.

4-[(Butylsulfanyl)methyl]-3,5-dimethylisoxazole (2c). Yield 0.25 g (83%, a), 0.094 g (94%, b). The IR and 1H and 13C NMR spectra agree with the published data [13].

4-{[(1,1-Dimethylpropyl)sulfanyl]methyl}-3,5-dimethylisoxazole (2d). Yield 0.25 g (77%, a), 0.095 g (89%, b). IR spectrum (thin film), ν, cm–1: 2966, 2929, 2877, 1639 (CN), 1454, 1423, 1379, 1363, 1271, 1238, 1195, 1157, 1134, 1008, 887, 740. 1Н NMR spectrum (CDCl3), δ, ppm: 0.97 t (3H, CH3CH2, 3J = 7.4 Hz), 1.30 s [6H, (CH3)2C], 1.59 q (2H, CH3CH2, 3J = 7.4 Hz), 2.28 s (3H, CH3C3), 2.35 s (3H, CH3C5), 3.38 s (2H, CH2S). 13С NMR spectrum, δ, ppm: 9.13 (CH3CH2), 10.09, 11.06 (СH3C3, CH3C5), 19.55 (CH2S), 28.02 [(СH3)2C], 34.55 (CH3CH2), 46.33 [(СH3)2C], 110.14 (С4), 159.67 (C5), 165.78 (C3). Mass spectrum, m/z (Irel, %): 214 [M + H]+ (100), 255 [M + H + MeCN]+ (91). Found, %: С 61.85, Н 8.95, N 6.49, S 15.11. С11Н19NОS. Calculated, %: С 61.93, Н 8.98, N 6.57, S 15.03.

4-[(Pentylsulfanyl)methyl]-3,5-dimethylisoxazole (2e). Yield 0.25 g (77%, a), 0.101 g (94%, b). IR spectrum (thin film), ν, cm–1: 2956, 2927, 2870, 2858, 1637 (CN), 1454, 1423, 1379, 1271, 1242, 1193, 1037, 1028, 979, 889, 742. 1Н NMR spectrum (CDCl3), δ, ppm: 0.86 t [3H, CH3(CH2)4, 3J = 7.0 Hz], 1.24–1.35 m [4H, CH3(CH2)2], 1.54 quintet [2H, CH3(CH2)2СH2, 3J = 7.4 Hz], 2.25 s (3H, CH3C3), 2.32 s (3H, CH3C5), 2.38 t [2H, CH3(CH2)3СH2S, 3J = 7.4 Hz], 3.40 s (2H, CH2S). 13С NMR spectrum, δ, ppm: 10.05, 10.93 (СH3C3, CH3C5), 13.86 [CH3(CH2)4], 22.19, 23.49 (CH2S, CH3CH2), 28.92, 31.03, 31.58 (CH3CH2СH2CH2CH2S), 110.65 (С4), 159.53 (C5), 165.62 (C3). Mass spectrum, m/z (Irel, %): 214 [M + H]+ (39), 255 [M + H + MeCN]+ (100). Found, %: С 61.87, Н 8.96, N 6.51, S 15.08. С11Н19NОS. Calculated, %: С 61.93, Н 8.98, N 6.57, S 15.03.

4-[(Cyclohexylsulfanyl)methyl]-3,5-dimethylisoxazole (2f). Yield 0.28 g (84%, a), 0.102 g (90%, b). IR spectrum (thin film), ν, cm–1: 2929, 2852, 1637 (CN), 1448, 1423, 1381, 1340, 1269, 1242, 1193, 1028, 999, 887, 740. 1Н NMR spectrum (CDCl3), δ, ppm: 1.20–1.38 m (5H, CH2), 1.57–1.63 m (1H, СH), 1.73–1.79 m (2H, CH2), 1.90–1.97 m (2H, CH2), 2.27 s (3H, CH3C3), 2.34 s (3H, CH3C5), 2.51 tt (1H, CH, 3J = 10.4 Hz, 3J = 3.5 Hz), 3.45 s (2H, CH2S). 13С NMR spectrum, δ, ppm: 10.15, 11.01 (СH3C3, CH3C5), 21.80 (CH2S), 25.78, 26.04, 33.42 (C2ʹ,6ʹH2, C3ʹ,5ʹH2, С4ʹH2), 43.29 (C1ʹH), 110.90 (С4), 159.68 (C5), 165.59 (C3). Mass spectrum, m/z (Irel, %): 226 [M + H]+ (39), 267 [M + H + MeCN]+ (100). Found, %: С 63.90, Н 8.46, N 6.27, S 14.27. С12Н19NОS. Calculated, %: С 63.96, Н 8.50, N 6.22, S 14.23.

4-[(Hexylsulfanyl)methyl]-3,5-dimethylisoxazole (2g). Yield 0.24 g (70%, a), 0.100 g (88%, b). The IR and 1H and 13C NMR spectra agree with the published data [13].

Evaluation of the antifungal activity. As test objects we used phytopathogenic fungi from the collection of the Ufa Institute of Biology, Ufa Federal Research Center, Russian Academy of Sciences: Bipolaris sorokiniana (IB G-12), Fusarium oxysporum (VKM F-137 IB G-20), and Rhizoctonia solani (VKM F-895 IB G-62). The antifungal activity toward pathogens was evaluated by the method of diffusion into potato glucose agar [14]. 100-μL portions of a test culture suspension were applied onto the surface of potato glucose agar (20-mL portions poured into standard Petri dishes 90 mm in diameter). The suspension portions were thoroughly distributed over the surface with a spatula to ensure uniform continuous growth of the fungus. Four holes were made in the medium with a rubber stopper hole puncher, and 100-μL portions of 2c and 3–5 were added into these holes. Compounds 2c and 3–5 were tested as 0.5% solutions in dimethylformamide. Dimethylformamide did not negatively affect the test culture growth. Sterile tap water was used as a control; its 100-μL portions were added into the holes instead of the test substance. Tap water was sterilized in a VK-75-01 steam sterilizer (Mediko, Russia) under a pressure of 1 atm for 20 min. As a positive control we used fluconazole [Diflucan®, infusion solution, Pfizer; composition per milliliter: 2.0 mg of 2-(2,4-difluorophenyl)-1,3-bis(1Н-1,2,4-triazol-1-yl)-2-propanol, 9.0 mg of NaCl, water for injections to 1 mL]. 100-μL portions of the fluconazole solution were added into the holes similarly to the test substances. The results were evaluated by the diameter of the growth suppression zone of the phytopathogenic fungi after 7-day incubation at 22°C. Measurements were performed in two mutually perpendicular directions, and the suppression zone diameter was calculated as the arithmetic mean of the values obtained. Statistical processing of the results was performed using the Student’s t-test at the critical significance level p = 0.05.

RESULTS AND DISCUSSION

The reaction of 3-[(alkylsulfanyl)methyl]pentane-2,4-diones 1a–1g with 1.2 equiv of hydroxylamine hydrochloride on refluxing in ethanol for 5 h gives the corresponding 4-[(alkylsulfanyl)methyl]-3,5-dimethylisoxazoles 2a–2g in 70–88% yields. In the microwave-assisted synthesis, the heterocyclization is complete in 15 min, and the yields of target products 2a–2g reach 89–95%.

where (1, 2) R = Et (a), i-Pr (b), Bu (c), t-C5H11 (d), n-C5H11 (e), cyclo-C6H11 (f), n-C6H13 (g).

Isoxazoles 2c and 2g and new compounds 2а, 2b, and 2d–2f were prepared by the procedure described in [13] with decreased amount of hydroxylamine without a base and without purification of the starting compounds. As demonstrated by the example of pentane-2,4-diones 1e and 1g, with a decrease in the hydroxylamine amount the target product yields decrease or change insignificantly; microwave activation allows the reaction time to be reduced by a factor of 20 and the isoxazole yields to be increased almost to quantitative (Table 1). For example, under the conditions of refluxing in ethanol, 5 h is required to complete the reaction, whereas the microwave-assisted reaction is complete in 5–15 min. The reaction does not occur without ethanol, but hydroxylamine hydrochloride can be used without preliminary dissolution in water.

Table 1. Characteristics of the condensation of 3-[(pentylsulfanyl)methyl]- and 3-[(hexylsulfanyl)methyl]pentane-2,4-diones with hydroxylamine hydrochloride in refluxing ethanol
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The structure of isoxazoles 2a–2g was confirmed by the IR and 1H, 13C NMR data. In the IR spectra of all isoxazoles 2a–2g, there is a strong absorption band of C=N stretching vibrations at 1637–1639 cm–1. A characteristic feature of the 1H NMR spectra of 2a–2g is the presence of three singlets from СH3С3 and СH3С5 methyl protons (δ 2.25–2.28, 2.32–2.35 ppm) and SC1ʹH2 methylene protons (δ 3.38–3.45 ppm) along with the signals from protons of the alkylsulfanyl substituent. In the 13С NMR spectra, the С3 and С5 atoms of the isoxazole ring give characteristic signals at 165.62–166.94 and 159.53–160.98 ppm, and the С4 atoms, at 110.14–112.00 ppm. In the mass spectra of positive ions of compounds 2a–2g, recorded in the chemical ionization mode, there are peaks of protonated molecular ions [M + H]+ and ions [(M + H) + МеСN]+.

For the experiments on evaluating the biological activity, we chose 4-[(hexylsulfanyl)methyl]-3,5-dimethylisoxazole 2g, and also 4-[(butylsulfanyl)methyl]-3,5-dimethylisoxazole 2c, the corresponding sulfone 3, and pyrazoles 4 and 5 with the same 4-butylsulfanylmethyl substituent (Table 2). As we found, 1H-pyrazoles 4 and 5 exert no inhibiting effect on the development of Bipolaris sorokiniana, Rhizoctonia solani, and Fusarium oxysporum phytopathogenic fungi. In contrast to pyrazoles, isoxazole 2c exhibits fungistatic activity toward Fusarium oxysporum fungi and fungicidal activity with the sterile zone formation toward Rhizoctonia solani fungi. Isoxazole 2c inhibits growth of Rhizoctonia solani fingi to a lesser extent than fluconazole (a triazole derivative) does. However, compound 2c, in contrast of fluconazole, exhibits fungistatic activity toward Fusarium oxysporum fungi, being, however, inferior to hymexazol (5-methylisoxazol-3-ol) in this respect [15]. With an increase in the size of the hydrocarbon radical in the alkylsulfanyl substituent in isoxazoles 2, the fungicidal activity decreases. Oxidation of the sulfur atom in 4-[(butylsulfanyl)methyl]-3,5-dimethylisoxazole to sulfone leads to the disappearance of the fungicidal properties.

Table 2. Antifungal activity of some 4-[(alkylsulfanyl)methyl]-3,5-dimethylisoxazoles and -1H-pyrazoles
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CONCLUSIONS

A procedure was developed for preparing 4-[(alkylsulfanyl)methyl]-3,5-dimethylisoxazoles by the reaction of 3-[(alkylsulfanyl)methyl]pentane-2,4-diones with hydroxylamine under the conditions of microwave irradiation, which considerably reduces the reaction time (to 15 min) and ensures high product yield (89–95%). The synthesized 4-[(butylsulfanyl)methyl]-3,5-dimethylisoxazole exerts a fungistatic effect on Fusarium oxysporum and a fungicidal effect on Rhizoctonia solani phytopathogenic fungi. Replacement of the thioether sulfur atom by the sulfonyl group in the isoxazole studied leads to the loss of antifungal properties.