AQUEOUS PHASE SYNTHESIS, CRYSTAL STRUCTURE AND ANTIMICROBIAL ACTIVITY OF 4-(SUBSTITUTED PHENYLAZO)-3-METHYL-4H-ISOXAZOL-5-ONE AZO DYES

3-Methyl-4H-isoxazol-5-one was synthesized at room temperature by simple stirring method from ethyl acetoacetate and hydroxylamine hydrochloride in aqueous medium and coupled with diazotized substituted amine to form series of 4-(substituted phenylazo)-3-methyl-4H-isoxazol-5-ones through green chemistry. All the compounds formed were characterized by IR, H and C NMR spectroscopy, MS and elemental analysis. Crystal structure of novel 4-(4-fluorophenylazo)-3-methyl-4H-isoxazol-5-one was determined by the X-ray diffraction. Antibacterial and antifungal activity was studied against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus pyogenus and Candida albicans, Aspergillus niger, Aspergillus clavatus, respectively. All synthesized compounds were found to be active against a gram-positive bacterium Staphylococcus aureus. Two compounds showed antifungal activity against Candida albicans close to standard greseofulvin.


INTRODUCTION
Azo dyes are the most widely used class of coloring materials because of their fruitful applications in various fields of science and technology [1][2][3] and also for their interesting structural and physicochemical properties [4][5][6]. Azo dyes are synthesized by diazotization of aromatic amines and coupling reagent, which include one or more azo groups (-N=N-) attached to one or more aromatic moieties [7]. Currently, heteroarylazoisoxazol compounds have received the attention of many research groups [8] because of their wide spread potential applications in fields of catalysis [9], cancer treatment and as antibacterial and antiviral agents, agricultural fungicides as well as other biological uses [10][11][12]. Intramolecular N-H•••O hydrogen bonding study assisted by resonance for 1-ketone-2-arylhydrazones derivatives suggest that heterocyclic five member ring introduces geometrical constraint which hinders the strengthening of hydrogen bond [13]. Tautomerism and spectroscopic properties of some heteroarylazoisoxazolone dyes were studied earlier [14]. With these objects in view we focus on the synthesis and antimicrobial activity of 4-(substituted phenylazo)-3-methyl-4H-isoxazol-5one azo dyes.

EXPERIMENTAL
All the starting materials and solvents were purchased from commercial sources and were used without further purification. Melting points were determined in open capillaries using electro thermal melting point apparatus and are uncorrected. Progress of reactions was monitored by TLC. Infrared (IR) spectra (4000-600 cm -1 ) of the samples were recorded using a Perkin-Elmer Spectrum 100, equipped with a Specac Golden Gate Diamond ATR as a solid sample support. 1 H NMR spectra were recorded with a Bruker Avance III 500 nuclear magnetic resonance spectrometer with TMS as internal reference. 13 C NMR spectra were recorded with a Bruker Avance III 500 spectrometer at 125 MHz and were referenced against the central line of the solvent signal (CDCl 3 triplet at 77.0 ppm or DMSO-d 6 septet at 39.5 ppm). MS spectra were recorded with an Agilent 6624 Accurate Mass TOF LC/MS instrument (ESI ionization).
Single-crystal X-ray diffraction data for 6c•0.3H 2 O were collected on an Agilent Technologies SuperNova Dual diffractometer with the Atlas detector using monochromated Mo-Kα radiation (λ = 0.71073 Å) at room temperature. The data were processed using CrysAlis Pro. [15]. The structure was solved by direct methods using the program Superflip [16] and refined on F 2 using full-matrix least-squares procedures using SHELXL2014 [17]. All nonhydrogen atoms were refined anisotropically. Hydrogen atoms were readily located in a difference Fourier maps and were subsequently treated as riding atoms in geometrically idealized positions, with C-H = 0.93 (aromatic) or 0.96 Å (CH 3 ), N-H = 0.86 Å and with U iso (H) = kU eq (C or N), where k = 1.5 for methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms. The water H-atoms were refined with a distance restraint and with U iso (H) = 1.5U eq (O). Initially, the occupation factor for a water solvate molecule was refined, but was set as fixed at 0.30 in the final stage of the refinement. Structure was refined as a twin. Crystallographic data are listed in Table 1.

Determination of minimal inhibition concentrations by micro broth dilution for antibacterial and antifungal activity
All the synthesized drugs were used for antibacterial test procedures. All necessary controls like drug control, vehicle control, agar control, organism control, known antibacterial drugs control, all MTCC cultures were tested against above mentioned known and unknown drugs. Mueller Hinton broth was used as nutrient medium to grow and dilute the drug suspension for the test bacteria. Inoculum size for test strain was adjusted to 10 8 Cfu [Colony Forming Unit] per milliliter by comparing the turbidity. Common standard strains were used for screening of antibacterial and antifungal activities: The strains were procured from Institute of Microbial Technology, Chandigarh: E. coli (MTCC443), P. aeruginosa (MTCC1688), S. aureus (MTCC96), S. pyogenus (MTCC442), C. albicans (MTCC227), A. niger (MTCC282), A. clavatus (MTCC1323). DMSO was used as diluents / vehicle to get desired concentration of drugs to test upon standard bacterial strains.
The main advantage of the 'Broth Dilution Method' for minimal inhibition concentration (MIC) determination lies in the fact that it can readily be converted to determine the MIC as well [18][19][20][21][22]. Serial dilutions were prepared in primary and secondary screening. The control tube containing no antibiotic is immediately sub cultured (before inoculation) by spreading a loopful evenly over a quarter of plate of medium suitable for the growth of the test organism and put for incubation at 37 o C overnight. The tubes are then incubated overnight. The MIC of the control organism is read to check the accuracy of the drug concentrations. The lowest concentration inhibiting growth of the organism is recorded as the MIC. The amount of growth from the control tube before incubation is compared [23].
Each synthesized drug was diluted obtaining 2000 μg/mL concentration, as a stock solution. In primary screening 1000, 500, and 250 μg/mL concentrations of the synthesized drugs were taken. The active synthesized drugs found in this primary screening were further tested in a second set of dilution against all microorganisms. The drugs found active in primary screening were similarly diluted to obtain 200, 100, 50, 25, 12.5, 6.250 μg/mL end concentrations. The highest dilution showing at least 99% inhibition zone is taken as MIC.

General procedure for synthesis of 3-methyl-4-H-isoxazol-5-one
Hydroxylamine hydrochloride (1.041 g, 15 mmol) was dissolved in 20 mL of water and cooled to about 10 o C, added ethyl acetoacetate (1.95 g, 15 mmol). The mixture was shaken thoroughly in absence of any catalyst till the appearance of pale yellow color solution of 3-methyl-4Hisoxazol-5-one [24].

RESULTS AND DISCUSSION
A series of 4-(substituted phenylazo)-3-methyl-4-H-isoxazol-5-one azo dyes were obtained from easily available, economically feasible chemicals like ethyl acetoacetate, hydroxylamine hydrochloride and some substituted amines by environmentally benign, green route in aqueous medium without using any catalyst. In the first step the reaction of ethyl acetoacetate (1) and hydroxylamine hydrochloride (2) resulted in the formation of oxime, which on further ring closure formed 3-methyl-4H-isoxazol-5-one (3) (Scheme 1). Electron withdrawing effect of N atom in 3-methyl-4H-isoxazol-5-one along with electron donating effect of O atom and presence of electron releasing methyl and keto groups makes it more reactive [15]. In the second step diazotization of substituted amine 4a-e was carried out in the presence of sodium nitrite and hydrochloric acidwhich then coupled with active hydrogen atom of isoxazol nucleus under cold condition to give desired product (Scheme 1). All compounds 6a-e show singlet for NH proton in the range of 12.50-12.60 ppm. Multiplets for aromatic ring were observed between 7-8 ppm. A singlet for three protons in the range of 2.26-2.29 is attributed to CH 3 group of isoxazol. Compound 6d shows a singlet for two protons of NH 2 group related to sulphanilamide moiety at 4.71 ppm and compound 6e show a singlet for three protons related to COCH 3 group at 2.51 ppm. 13    Single-crystal X-ray structures of 4-(4-fluorophenylazo)-3-methyl-4H-isoxazol-5-one (6c•0.3H 2 O) was determined ( Figure 2, Table 1). Table 3 and 4 describes significant bond distances and bond angles. All the bond lengths and bond angles of compounds are within normal ranges for this type of structure as reported earlier [13]. Molecule 6c is almost planar, maximum deviation from the mean plane described by all atoms of 6c is -0.132(3) Å for atom C10. The dihedral angle between the isoxazolone and phenyl rings is 7.81(18) o . In the crystal structure bond distances indicates that the isoxazolone ring contains one double bond, i.e. C1-N1. Also, atoms C2 and N2 are connected through double bond and intramolecular N3-H3•••O2 hydrogen bonding is present between NH group and the carbonyl group of the isoxazole ring. A water solvate molecule present in the crystal lattice with 30% occupancy forms O3-H3A•••O2 hydrogen bond with two adjacent molecules of 6c. Furthermore, molecules are linked into chains via C4-H4A···O2 interactions between methyl group of isoxazole ring and carbonyl oxygen atom of isoxazole moiety of the adjacent molecule and C10-H10···O1 interactions between the phenyl moiety and the oxygen atom of isoxazole moiety of the adjacent molecule ( Figure 3).    All synthesized compounds 6a-e were screened for antibacterial and antifungal study against E. coli, P. aeruginosa, S. aureus and S. pyogenus as bacterial strain and C. albicans, A. niger, A. clavatus as fungal strain. All synthesized compounds 6a-e showed antibacterial activity against S. aureus. Compound 6c and 6e showed antibacterial activity against E. coli whereas compound 6b showed antibacterial activity against S. pyogenus close to activity of standard ampicillin (Table 5). It was found that compounds 6b and 6e show antifungal activity against C. albicans close to standard greseofulvin (Table 6). Norfloxain 10 10 10 10 Table 6. Antifungal activity study of 6a-e with standard drugs.

CONCLUSION
The method of synthesis of 6a-e is aqueous phased, environmentally friendly and gives product easily in good yield without using any hazardous chemicals. Spectroscopic study of 6a-e and Xray crystallographic study of 6c shows azo dye formation with tautemerism to retain aromaticity of isoxazole ring. All synthesized compounds showed antibacterial activity against S. aureus close to standard ampicillin. Compounds 6c and 6e showed antibacterial activity against E. coli close to ampicillin and 6b and 6e showed antifungal activity against C. albicans close to gresiofulvin. The crystal structure of 6c shows hydrogen bonding interaction with water molecule. Oxygen atom attached to isoxazole ring shows intermolecular as well as intramolecular hydrogen bonding. Nystatin 100 100 100 10 Griseofulvin 500 100 100