Green multicomponent synthesis, antimicrobial and antioxidant evaluation of novel 5-amino-isoxazole-4-carbonitriles

Background Design and synthesis of new inhibitor agents to deal with pathogenic microorganisms is expanding. In this project, an efficient, environmentally friendly, economical, rapid and mild procedure was developed for the synthesis of novel functionalized isoxazole derivatives as antimicrobial potentials. Methods Multicomponent reaction between malononitrile (1), hydroxylamine hydrochloride (2) and different aryl or heteroaryl aldehydes 3a–i afforded novel 5-amino-isoxazole-4-carbonitriles 4a–i in good product yields and short reaction times. Deep eutectic solvent K2CO3/glycerol was used as catalytic reaction media. Structure of all molecules were characterized by different analytical tools. In vitro inhibitory activity of all derivatives was evaluated against a variety of pathogenic bacteria including both Gram-negative and Gram-positive strains as well as some fungi. In addition, their free radical scavenging activities were assessed against DPPH. Results Broad-spectrum antimicrobial activities were observed with isoxazoles 4a, b, d. In addition, antioxidant activity of isoxazole 4i was proven on DPPH. Conclusions In this project, compounds 4a, b, d could efficiently inhibit the growth of various bacterial and fungal pathogens. Antioxidant properties of derivative 4i were also significant. These biologically active compounds are suitable candidates to synthesize new prodrugs and drugs due to the presence of different functional groups on their rings. Electronic supplementary material The online version of this article (10.1186/s13065-018-0488-0) contains supplementary material, which is available to authorized users.


Background
Isoxazoles are five-membered aromatic heterocycles containing adjacent oxygen and nitrogen atoms. The isoxazole ring system is found in a variety of naturally occurring compounds and biologically active molecules [1]. They are especially useful in medicine, since many antifungal drugs belong to the isoxazole class [2]. Sulfisoxazole and sulfamethoxazole are two bacteriostatic sulfonamide antibiotics that applied alone or combined with others in the treatment of infections caused Gram-positive and Gram-negative bacteria [3,4]. Acivicin is a γ-glutamyl transferase inhibitor with anticancer, anti-parasitic and antileishmania activities [5]. Isoxazole derivatives possess a broad variety of biological activities viz. antifungal, anti-inflammatory, antiplatelet, anti-HIV, anti-Alzheimer and analgesic [6][7][8][9][10][11].
Deep eutectic solvents (DES) play an essential key in green chemistry. They can be used as safe, low-cost, nontoxic, reusable, catalytic and environmentally friendly media in the most reactions [24]. Their applications are expanding in the field of materials, energy and environmental science [25]. Glycerol is a valuable green, nontoxic, low flammable and available solvent that applied as anti-freezer, sweetener, humectant, lubricant and thickener in industry [26]. This natural polyol as hydrogen bond donor is present in DESs with hydrogen bond acceptors such as choline chloride, methyl triphenyl phosphonium bromide, benzyl triphenyl phosphonium chloride, allyl triphenyl phosphonium bromide, N,Ndiethylethanolammonium chloride, and tetra-n-butylammonium bromide [27]. Glycerol/potassium carbonate is a low cost and environmentally friendly DES that recently its efficiently was proven in the preparation of pyrazole derivatives [28].
In order to develop applications of Gly/K 2 CO 3 to other heterocycles, it was successfully used as catalytic media in the synthesis of novel 5-amino-isoxazole-4-carbonitrile derivatives via multicomponent reaction of malononitrile, hydroxylamine and various aryl aldehydes (Fig. 1). In vitro inhibitory activity of all derivatives was evaluated against some pathogenic bacteria including both Gram-negative and Gram-positive strains as well as some fungi. In addition, their antioxidant potentials were assessed against DPPH.

Evaluation of the bioactivity of isoxazoles 4a-i
All synthesized compounds were assessed for their antimicrobial efficiency as well as antioxidant activity. Inhibitory effects of isoxazoles 4a-i were presented as MIC, MBC and MFC values in Tables 1 and 2.

Chemistry
The effects of variations in solvent, temperature and order mixing reactants were studied on product yield and reaction time. Aldoximes were produced as major products in glycerol at different conditions. They were also formed in Gly/K 2 CO 3 deep eutectic solvents under one-pot two-step procedures involving initial mixing hydroxylamine and aldehydes, followed by malononitrile. In addition, oximes were present as by-products in one-pot two-step processes involving initial mixing malononitrile and aldehydes. There are two possible mechanisms to form the products (Scheme 2). A reaction pathway, that does not lead to the target products, includes the reaction of aldoximes produced from aldehydes and hydroxylamine with malononitrile. On another path, the Knoevenagel condensation of aldehydes with malononitrile gives arylidene malononitriles, which react with hydroxylamine to form isoxazoles. The best results were obtained via simultaneous reaction of reagents in Gly/K 2 CO 3 (4:1 molar) as green catalytic media at room temperature, which considered as optimal conditions. Increase in Gly/K 2 CO 3 ratio and temperature led to a decrease in yields.
Multicomponent reaction of hydroxylamine derivatives, aldehydes and active methylene compounds is an efficient procedure to synthesize isoxazoles. Some recently proposed protocols were presented in Table 3. According to the data in the Table 3, reaction times decreased in the presence of catalysts at room temperature or under heating or UV radiation. It seems that basic catalysts are more effective than acidic equivalents. Our newly modified process provides an efficient, simple, economical, safe and eco-friendly reaction under mild conditions at acceptable products yields.
The chemical structure of isoxazoles 4a-i was characterized by spectral data. Nitrile groups were detected by FT-IR (~ 2220 cm −1 ) and 13 C NMR (~ 115 ppm). Amino groups were also identified based on their absorption bands in region of ~ 3430-3330 cm −1 and proton chemical shifts appeared approximately 8.50 ppm.

Conclusion
In summary, some novel 5-amino-isoxazole-4-carbonitriles were prepared via a green and efficient multicomponent procedure in acceptable product yields and short reaction times. Antimicrobial activity of isoxazoles was studied against a variety of bacterial and fungal pathogens. Significant inhibitory potentials were observed with compounds 4a, b, d. Isoxazole 4i also showed considerable antioxidant activities. These functionalized biologically active compounds could applied as prodrugs in future researches.

Materials
All reagents, solvents, antibiotics, DPPH and antifungal agents were purchased from commercial sources  (Merck, Sigma and Aldrich), and used without further purification. The bacterial and fungal culture media were obtained from (HiMedia). Melting points were determined with Kruss type KSP1N melting point meter and are uncorrected. Reaction progress was monitored by aluminium TLC plates pre-coated by SiO 2 with fluorescent indicator F254 using CHCl 3 /CH 3 OH (9:1, v/v) as mobile phase, which were visualized under UV radiation (254 nm). The absorption spectra were determined using a UV-2100 RAY Leigh UV-Vis spectrophotometer. FT-IR spectra of the products were collected using a Bruker Tensor-27 FT-IR spectrometer. 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker FT-NMR Ultra Shield-400 spectrometer. Elemental analyses (CHNS/O) were performed on a Thermo Finnigan Flash EA microanalyzer. DESs were prepared in various ratios of glycerol/K 2 CO 3 according to the published procedure [30] (Additional file 1).

MIC determination
Broth microdilution methods according to CLSI guidelines M07-A9 and M27-A2 were used for the determination of MIC values [32,33]. Bacterial and fungal suspensions at 0.5 McFarland standard were prepared in MHB and SDB, respectively. They were diluted to 150 and 250 times with MHB and SDB, respectively. 20 μl of each isoxazoles 4a-i with concentration of 20,480 μg ml −1 in DMSO was added to first and second wells in a row of a 96-well microtiter plate. 20 μl DMSO was added to wells 2-12, and two-fold serial dilutions were carried out in them. 170 μl of MHA or SDB with 10 μl of diluted microbial suspensions were added to all wells. Finally, a concentration range of 2048-1 μg ml −1 of the derivatives was prepared in each row; in addition, the concentration of DMSO did not exceed 10% (v/v). Microtiter plates were incubated with shaking at 100 rpm at 37 °C for 24 h. Fungi must be incubated in the relative humidity (45-55%). The lowest concentration of derivatives that resulted in no visible turbidity was considered as the MIC value.

MBC and MFC determination
Time-kill test according to CLSI guideline M26-A was applied to determine MBC and MFC values [32,33]. Samples of all wells that showed no growth in the MIC test, were cultured in MHA or SDA media plates. Dishes were incubated at 37 °C for another 24 h under similar conditions. The MBC or MFC was identified as the lowest concentration of derivatives at which no microorganisms survived.

IC 50 identification
Free radical scavenging activity of all synthesized heterocycles were evaluated against DPPH [34]. 1 ml of various concentrations of all compounds (25,50,75, and 100 µg ml −1 ) in methanol was added to 4 ml of 0.004% (w/v) methanolic solution of freshly prepared DPPH. Solutions were shaken and left to stand for 30 min at room temperature in darkness. A solution including 1 ml of methanol and 4 ml of 0.004% (w/v) methanolic