An efficient synthesis and antimicrobial evaluation of 5-alkenyl-and 5-styryl-1,2,4-oxadiazoles

The cyclodehydration of O -acylamidoximes at room temperature in the superbase system KOH/DMSO represents a simple and efficient way to 5-alkenyl-and 5-styryl-1,2,4-oxadiazoles. This method is suitable for the preparation of 5-(4-vinylphenyl)-1,2,4-oxadiazoles as well. Results of the antimicrobial tests demonstrated that the synthesized compounds exhibit a moderate antimicrobial effect against E.coli , S.aureus and C.albicans strains

At the onset of our study, we investigated the cyclodehydration of the acrylic acid derivative 3a (Table 1, entries 1 and 2).Initially, an equimolar ratio of the O-acylamidoxime and KOH was used 38 , and the reaction was carried out for 10 minutes.7][28][29][30] The isolated white powder contained no carbon-carbon double bond according to the IR spectrum.Moreover, the molecular weight of this solid is ~450 kDa (by GPC), suggesting that the material formed is a product of the base-initiated anionic polymerization (anionic polymerization of Michael acceptors in DMSO is known) 39,40 Reducing the amount of KOH to 0.1 equiv.decreases the reaction rate and allows isolation of the desired 4a after column chromatography, albeit in low (11%) yield (Table 1, entry 2).Similar results were obtained for the methacrylic derivative 3b (Table 1, entries 3 and 4).On the contrary, crotonic and methylcrotonic derivatives 4c and 4d were obtained in good yields (79 and 80%, respectively) with 0.1 equiv. of KOH (Table 1, entries 6 and 8).The cyclodehydration of O-acylamidoximes 3e-n was successful with 1 equiv. of KOH (with the single exception of 3k), furnishing good to excellent yields of desired 1,2,4-oxadiazoles (Table 1, entries 9-21).Our results demonstrate that the presence of alkyl, aryl or heteroaryl substituents at the terminal carbon atom of the double bond reduces the polymerization tendency of 1,2,4-oxadiazoles.
Furthermore, we applied our protocol for the preparation of 5-(4-vinylphenyl)-1,2,4-oxadiazoles.These compounds are only poorly studied, presumably because their synthesis is rather tedious and given that the vinyl group is unstable under thermal cyclodehydration conditions. 41For this reason, alternative approaches such as the Wittig and Stille reactions or the reduction of the appropriate alkyne 41,42 were utilized for the synthesis of 1,2,4-oxadiazoles with vinylphenyl moiety.Noteworthy, compounds 4o and 4p were prepared in good yields according to our method (Table 1, entries 22 and 23).
Table 1.Results of cyclodehydration of O-acylamidoximes (3a-p) to the corresponding oxadiazoles (4a-p) A broad spectrum of compounds based on the 1,2,4-oxadiazole scaffold are well known as potent antimicrobial agents. 43No antibacterial studies of 1,2,4-oxadiazoles containing a C=C double bond, however, have, hitherto, been conducted.Motivated by this, we assessed the antibacterial and antifungal activities of synthesized heterocycles against Staphylococcus aureus, Escherichia coli, and Candida albicans by the broth double microdilution method. 44,45try
The experimental data revealed that all of the tested 1,2,4-oxadiazoles exhibited low to medium activity against both Gram-positive and Gram-negative bacterial strains (Table 2).The screening set included twelve compounds (4d-g and 4i-p).Three synthesized 1,2,4-oxadiazoles (4a-4c) were too unstable at assay conditions, while 4h was insoluble in the assay medium.Therefore these compounds were excluded from the study.Pefloxacin and Fluconazole were used as reference standards for the comparison of antimicrobial activity.The minimum inhibitory concentration (MIC) for all compounds was determined.The experimental data (Table 2) revealed that all tested 1,2,4-oxadiazoles exhibit low to medium activity against both Gram-positive and Gram-negative bacterial strains.
Overnight cultures were grown at 37 °C in Lysogeny broth (LB) and diluted to obtain an opacity equivalent to 0.5 on the McFarland scale.The compounds are weighed and dissolved in DMSO to give concentrations equal to 20.7 µmol/mL.The pefloxacin and miconazole were used to antimicrobial standard.The concentration of 2.07 µmol/mL was used as the starting one, providing a final concentration equal to 0.03 µmol/mL.The tubes were incubated for 12 h at 37 °C.Growth inhibition detection was considered as positive results.It was verified that DMSO as completely inactive against the tested microorganisms in the less than 10% concentrations.A series of tubes is prepared by mixing one part of each dilution of the compound with some parts of the LB, previously inoculated and incubated for 24 h at 37 °C.All experiments were carried out in triplicate.The MIC was considered the lowest drug concentration for which there is no microbial growth.

Conclusions
We have developed a simple, speedy and efficient method for the synthesis of 5-alkenyl-and 5-styryl-1,2,4oxadiazoles. A series of 16 compounds was obtained, 10 of which have not been previously reported in the literature.The novel synthetic protocol offers several advantages, including short reaction times, high yields of products, and simple work-up procedure.In addition, the twelve 1,2,4-oxadiazoles synthesized were evaluated agaist both Gram-positive and Gram-negative bacteria strains as well as one fungi strain, and displayed moderate antimicrobial activity.

Experimental Section
General.Starting O-acylamidoximes (3a-p) were synthesized from commercial nitriles in two steps (for more details see Supplemental Materials).All other reagents and solvents, including DMSO, were purchased and used without further purification.All reactions were conducted in an open flask.. Reactions were monitored by analytical thin layer chromatography (TLC) Macherey-Nagel, TLC plates Silufol UV-254 using UV light for detection.Column chromatography was carried out with silica gel grade 60 (0.040-0.063 mm) 230-400 mesh with a hexane/ethyl acetate mixture as eluent (4/1).NMR spectra were recorded on Bruker Avance DPX 400 (400.13MHz and 100.61MHz for 1 H and 13 C, respectively) or on Bruker Avance III 500 MHz (500.03MHz for 1 H and 125.73 MHz for 13 C) in DMSO-d 6 or in CDCl 3 .Chemical shifts are reported as parts per million (δ, ppm).The solvent peaks were used as internal standards: 2.50 ppm for residual 1 H, 39.50 ppm for 13 C in DMSO-d 6 ; 7.26 ppm for residual 1 H, 77.16 ppm for 13 C in CDCl 3 .Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad and coupling constants, J, are reported in Hertz (Hz).The signals of second-order proton systems AA'XX' of the para-substituted and AA'XX'Y of the monosubstituted phenyl rings are not seen clearly in the 1 H NMR spectra, therefore, "apparent" coupling constants for observed "doublets" and "triplets" of appropriate aromatic protons are provided.Melting points were determined in open capillary tubes on Electrothermal IA 9300 series Digital Melting Point Apparatus.High-resolution mass spectra (HRMS) were measured on Bruker Maxis HRMS-ESI-qTOF (ESI Ionization).IR spectra were measured with an IRF Perkin Elmer Spectrum RX1 spectrometer using suspension in Nujol or as in thin films (microlayer) between KBr discs.Gel permeation chromatography was performed on DIONEX UltiMate-3000 (column: l = 15 сm, d = 2 mm).A mixture of acetonitrile and water (1:4) was used as eluent."Th" is the abbreviation for the thiophen ring.

Antimicrobial assay
Overnight cultures were grown at 37 °C in Lysogeny broth (LB) and diluted to obtain an opacity equivalent to 0.5 on the McFarland scale.The compounds are weighed and dissolved in DMSO to give concentrations equal to 21.4 µmol/mL.The pefloxacin and fluconazole were used as to antimicrobial drug standards.The concentration of 2.14 µmol/mL was used as the starting one, providing a final concentration equal to 0.03 µmol/mL.The tubes were incubated for 12 h at 37 °C.Growth inhibition detection was considered as positive results.It was verified that DMSO as completely inactive against the tested microorganisms in the less than 0.5 % concentrations.A series of tubes is prepared by mixing one part of each dilution of the compound with some parts of the LB, previously inoculated and incubated for 24 h at 37 °C.All experiments were carried out in triplicate.Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent which will inhibit the visible growth of a micro-organism after overnight incubation.

Table 2 .
MIC values in µg/mL µmol/mL for compounds