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ICAST 2018 Nano BF3

May 1, 2019 0 Comment

ICAST 2018
Nano BF3.SiO2 catalysed, microwave assisted Michaelis-Arbuzov reaction to synthesize biologically active phophonates under solvent-free condition
D. Ravi Kumar, Ch. Subramanyam, xxxxxxxxxx, K. Prasada Rao*
Department of Chemistry, Bapatla Engineering College, Bapatla, AP, INDIA-522101
Abstract
Nano-BF3.SiO2 is an efficient, reusable and eco-friendly catalyst for various organic reactions. So, this catalyst was applied for the synthesis of biologically active phosphonates by Michaelis-Arbuzov reaction in good to excellent yields. Key advantageous of this procedure is high yielding, easy work-up procedure, short reaction time and solvent free condition. All the title compounds were characterized by spectral and elemental analysis. They were further screened for their ability towards in vitro antibacterial and antifungal activity. Majority of the title compounds showed good inhibition towards bacteria and fungi.
Keywords: Nano-BF3.SiO2, phosphonates, Michaelis-Arbuzov reaction, antibacterial and antifungal activity

*Corresponding author Tel: +918008514677, E-mail address: [email protected]
1. Introduction
Phosphonates represent a class of stable organophosphorus compounds contain a single carbon-phosphorus (C–P) bond, are one of several pentavalent phosphorus compounds of considerable synthetic interest due to their utility as reagents in the Wadsworth-Emmons reaction1 and their applications in bioorganic chemistry.2 They are resistant to chemical and enzymatic hydrolysis, thermal decomposition 3 and photolysis.4 They are widely used as herbicides,5 pesticides,6 detergents,7 reagents for Wittig-Horner reactions,8 hybrid organic-inorganic supports and catalysts,9 antiviral agents,10 agents with antitumor activity11 or as chemical weapons of mass destruction.12 Various biomolecules which contains phosphonate motif are proved to be inhibitors of certain biosynthetic pathways. The high chemical stability of phosphonates together with their resistance to biodegradation, makes this class of compounds of particular interest for the drug design.

The Michaelis–Arbuzov rearrangement is one of the most extensively investigated reactions in organophosphorus chemistry. It is widely used in many fields of chemistry from organic synthesis to catalyst design to prepare phosphonates13 by simple SN2 reaction of nucleophilic trialkylphosphite with alkyl/aryl/arylmethyl halides to give a phosphonium intermediate which further led to phosphonate ester along with another alkyl halide. The reaction has some drawbacks such as the need for elevated temperature, removal of the trialkylphosphites used in excess, and weaker electrophiles aryl/heteroaryl halides or vinyl halides that give lower yields.

Recently, various methods were developed using different Lewis acid catalysts,14 bases such as Cs2CO3,15 and Pd-mediated cross-coupling reaction 16a-c that stimulate the M-A reaction effectively and minimize the troubles. But, they have one or more shortcomings such as long reaction time, elevated temperature, removal of the trialkylphosphites used in excess, toxic catalyst and lower yields. Recently, solid-supported heterogeneous acid catalysts are unique and have become more trendy over the past two decades. A heterogeneous catalyst, Nano BF3.SiO2 17 is a bench-top catalyst that has many advantages such as simple preparation, reusability, large surface area, strong Lewis acid character, easy handling and being environmentally benign.

On the other hand, microwave-assisted (MW-assisted) organic synthesis has been shown to provide a number of advantages over the standard heating techniques such as clean reactions, improved reaction yields and shortened reaction times, easy work-ups and/or solvent free reaction conditions.18
Keeping in mind the above points and in continuation of our research for the for the synthesis of biologically active phosphonate derivatives; we developed a new ecofriendly method by the reaction of various urea/thiourea derivatives with triethyl phosphite using 37% nano-BF3-SiO2 as ecofriendly, recyclable catalyst under microwave irradiation.

2. Experimental
2.1. Materials and methods
The reactions were carried out in a round bottom 50 mL flask fitted with reflux condenser, a dropping funnel and in Nitrogen atmosphere. A magnetic stirrer cum hot plate was used for stirring and heating the reaction mixtures. Rota evaporator was used for removing the solvent from the reaction mixture. All the chemicals used in the present work were obtained from Sd. Fine Chem. Ltd., India; Qualigens, Mumbai and were used after purifying them by following the established procedures. All the solvents and reagents were dried and purified before use by adopting the standard procedures and techniques. Rota evaporator was used for removing the solvent from the reaction mixture. Progress of the reaction and purity of the compounds were monitored by thin layer chromatography (TLC) on aluminium sheet of silica gel 60F254, E-Merck, Germany using iodine as visualizing agent. The 1H, 13C and 31P NMR Spectra were recorded on Bruker AMX spectrometer operating at 400 MHz for 1H, 100 MHz for 13C and 161.9 MHz for 31P NMR. All compounds were dissolved in DMSO-d6 and chemical shifts were referenced to TMS (1H and 13C NMR) and 85% H3PO4 (31P NMR) and Mass spectra were recorded on API 2000 Perkin-Elmer PE-SCIEX Mass spectrometer. IR spectra were recorded on an FTIR spectrometer Bruker IFS 55 (Equinox) in KBr. Micro-analytical data were obtained from University of Hyderabad, Hyderabad, India. The 1H chemical shifts were expressed in parts per million (ppm) with reference to tetramethylsilane (TMS). The following abbreviations were used while presenting the NMR data: s = singlet, d = doublet, t = triplet, q = quartet and m = multiplet. All microwave irradiation experiments were carried out in the commercially available single-mode microwave synthesis apparatus equipped with a high sensitivity infrared sensor for temperature control and measurement.
2.2. Preparation of the catalyst 24a, 29
0.37 g of BF3 (0.7 mL of BF3.Et2O) was added drop-wise to a mixture of 0.63 g of silica gel or nano-silica gel and 5 ml of chloroform. The mixture was stirred for 1 h at room temperature. The resulted suspension was filtered. The obtained solid was washed with chloroform and dried at room temperature.

2.3. Synthesis of urea/thiourea derivatives of 4-bromo-3-methylisoxazol-5-amine (3a-j)
To a stirred solution of 4-bromo-3-methylisoxazol-5-amine (1) (0.002 mol) in dry THF (20 mL), 3-isocyanatopyridine (2a) (0.002 mol) in dry THF (30 mL) was added drop wise in presence of triethylamine with stirring at 10 °C for a period of 15 minutes. The reaction mixture was stirred further at room temperature for 3 hours. The progress of the reaction was monitored by TLC and the solvent was removed in a rota-evaporator to obtain crude product. The product was purified by column chromatography on silica gel using petroleum ether-ethyl acetate (7:3) as eluent to afford 1-(4-bromo-3-methylisoxazol-5-yl)-3-(pyridin-3-yl)urea (3a). Same experimental procedure was adopted for the preparation of remaining compounds (3b-j).

2.4. Conventional procedure for the synthesis of phosphonates (5a-j)
The mixture of urea/thiourea derivatives (3a-j) (0.010 mol), triethyl phosphite (4) (3.4 mL, 0.020 mol) and 37% nano-BF3.SiO2 (0.25 g) were placed in a round bottomed flask. This mixture was heated to 70 °C and agitated for 2h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature. Dichloromethane (DCM) (15 mL) was added to the reaction content and stirred for 10 min. The catalyst, nano BF3.SiO2 was separated by filtration as residue, washed with DCM (2 ×10mL) and the residue was dried under vacuum at 100 °C to utilize in further studies. The organic layer was washed with water (20 mL), dried over anhydrous Na2SO4 and concentrated under vacuum at 50 °C to obtain crude product. The pure phosphonates (5a-j) were obtained by column chromatography using ethyl acetate: n-hexane (7:3) as eluent.
2.5. Microwave assisted synthesis of phosphonates (5a-j):
The mixture of urea/thiourea derivatives (3a-j) (0.010 mol), triethyl phosphite (4) (3.4 mL, 0.020 mol) and 37% nano BF3.SiO2 (0.25 g) were placed in a round bottomed flask. The mixture was microwave radiated at 420 W under room temperature for about 9-17 minutes. The progress of the reaction was monitored by TLC (ethylacetate: n-hexane, 7:3). After completion of the reaction as checked by TLC, the reaction mixture was cooled to room temperature. After completion of the reaction, the reaction mixture was cooled to room temperature. Dichloromethane (DCM) (15 mL) was added to the reaction content and stirred for 10 min. The catalyst, nano BF3.SiO2 was separated by filtration as residue, washed with DCM (2 ×10mL) and the residue was dried under vacuum at 100 °C to utilize in further studies. The organic layer was washed with water (20 mL), dried over anhydrous Na2SO4 and concentrated under vacuum at 50 °C to obtain crude product. The pure compound (5a-j) was obtained by column chromatography using ethyl acetate: n-hexane (7:3) as eluent.
2.6. Spectral data of title compounds (5a-j)

Diethyl 3-methyl-5-(3-pyridin-3-ylureido)isoxazol-4-ylphosphonate (5a). Yield: 90%; semi solid. 31P NMR spectrum (DMSO-d6): ? 22.5 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 9.25 (s, 1H, Ar-NH), 8.15 (d, 1H, Ar-H), 8.03 (d, 1H, Ar-H), 7.35 (s, 1H, NH), 7.17 (t, 1H, Ar-H), 4.09 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.26 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 166.5 (C-4), 154.2 (C-8), 153.1 (C-2), 138.3 (C-22), 137.8 (C-20), 134.6 (C-18), 133.4 (C-17), 125.9 (C-21), 101.8 (C-3), 63.5 (C-12, C-15), 16.6 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3295, 3221 (NH), 1702 (C=O), 1222 (P=O), 1019 (P-O-Calip); LCMS (m/z, %): 360 (M+H+, 100); Anal. Calcd for C13H18N3O5PS: C, 43.45; H, 5.05; N, 11.69%; found: C, 43.52; H, 5.12; N, 11.62%.

Diethyl 3-methyl-5-(3-thiophen-2-ylureido)isoxazol-4-ylphosphonate (5b): Yield: 93%; semi solid. 31P NMR spectrum (DMSO-d6): ? 24.2 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 10.75 (s, 1H, Ar-NH), 7.37 (s, 1H, NH), 6.95 (d, 1H, Ar-H), 6.89 (d, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 4.08 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.24 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 166.7 (C-4), 154.4 (C-8), 153.1 (C-2), 139.3 (C-17), 126.4 (C-18), 119.9 (C-19), 114.8 (C-20), 101.8 (C-3), 63.3 (C-12, C-15), 16.5 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3298, 3224 (NH), 1716 (C=O), 1224 (P=O), 1013 (P-O-Calip); LCMS (m/z, %): 360 (M+H+, 100); Anal. Calcd for C13H18N3O5PS: C, 43.45; H, 5.05; N, 11.69%; found: C, 43.52; H, 5.15; N, 11.60%.

Diethyl 5-(3-(3,5-dimethylisoxazol-4-yl)ureido)-3-methylisoxazol-4-ylphosphonate (5c): Yield: 91%; semi solid. 31P NMR spectrum (DMSO-d6): ? 22.9 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 10.75 (s, 1H, Ar-NH), 7.31 (s, 1H, NH), 4.09 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 2.42 (s, 3H, -CH3), 2.38 (s, 3H, -CH3), 1.25 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 166.4 (C-4), 154.5 (C-21), 154.5 (C-8), 153.1 (C-2), 148.4 (C-18), 118.2 (C-17), 101.8 (C-3), 63.2 (C-12, C-15), 16.4 (C-13, C-16), 14.3 (C-6), 10.2 (C-22), 9.4 (C-23); IR (KBr) (?max cm-1): 3312, 3225 (NH), 1718 (C=O), 1226 (P=O), 1014 (P-O-Calip); LCMS (m/z, %): 373 (M+H+, 100); Anal. Calcd for C14H21N4O6P: C, 45.16; H, 5.69; N, 15.05%; found: C, 45.20; H, 5.65; N, 15.09%.

Diethyl 5-(3-(furan-2-ylmethyl)ureido)-3-methylisoxazol-4-ylphosphonate (5d):
Yield: 94%; semi solid. 31P NMR spectrum (DMSO-d6): ? 25.3 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 7.94 (d, 1H, Ar-H), 7.35 (s, 1H, NH), 6.84 (t, 1H, Ar-H), 6.74 (s, 1H, CH2-NH), 6.35 (d, 1H, Ar-H), 5.31 (d, 2H, -CH2), 4.05 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.23 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 166.6 (C-4), 154.1 (C-8), 153.1 (C-2), 144.5 (C-18), 142.2 (C-21), 112.4 (C-20), 111.6 (C-19), 101.8 (C-3), 63.5 (C-12, C-15), 36.2 (C-17), 16.5 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3325, 3228 (NH), 1726 (C=O), 1217 (P=O), 1014 (P-O-Calip); LCMS (m/z, %): 358 (M+H+, 100); Anal. Calcd for C14H20N3O6P: C, 47.06; H, 5.64; N, 11.76%; found: C, 47.14; H, 5.69; N, 11.79%.

diethyl 3-methyl-5-(3-tosylureido)isoxazol-4-ylphosphonate (5e):
Yield: 90%; semi solid. 31P NMR spectrum (DMSO-d6): ? 24.5 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 10.84 (s, 1H, SO2-NH), 7.83 (d, 2H, Ar-H), 7.42 (d, 2H, Ar-H), 7.35 (s, 1H, NH), 2.65(s, 3H, -CH3), 4.09 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.26 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 166.4 (C-4), 154.2 (C-8), 153.1 (C-2), 139.5 (C-18), 139.1 (C-21), 129.4 (C-20, C-22), 128.6 (C-19, C-23), 101.8 (C-3), 63.6 (C-12, C-15), 23.2 (C-24), 16.4 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3299, 3265 (NH), 1743 (C=O), 1229 (P=O), 1016 (P-O-Calip); LCMS (m/z, %): 432 (M+H+, 100); Anal. Calcd for C16H22N3O7PS: C, 44.55; H, 5.14; N, 9.74%; found: C, 44.61; H, 5.19; N, 9.69%.

Diethyl 5-(3-(furan-2-ylmethyl)thioureido)-3-methylisoxazol-4-ylphosphonate (5f):
Yield: 92%; semi solid. 31P NMR spectrum (DMSO-d6): ? 26.1 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 7.92 (d, 1H, Ar-H), 6.35 (s, 1H, NH), 6.83 (t, 1H, Ar-H), 6.60 (s, 1H, CH2-NH), 6.32 (d, 1H, Ar-H), 5.33 (d, 2H, -CH2), 4.08 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.24 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 175.5 (C-8), 166.5 (C-4), 153.1 (C-2), 147.5 (C-18), 144.3 (C-21), 112.4 (C-19), 111.8 (C-20), 101.8 (C-3), 63.4 (C-12, C-15), 46.2 (C-17), 16.6 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3335, 3235 (NH), 1222 (P=O), 1094 (C=S), 1019 (P-O-Calip); LCMS (m/z, %): 374 (M+H+, 100); Anal. Calcd for C14H20N3O5PS: C, 45.04; H, 5.40; N, 11.25%; found: C, 45.11; H, 5.49; N, 11.19%.

Diethyl 3-methyl-5-(3-(4-morpholinophenyl)thioureido)isoxazol-4-ylphosphonate (5g):
Yield: 95%; semi solid. 31P NMR spectrum (DMSO-d6): ? 18.7 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 11.43 (s, 1H, Ar-NH), 6.85 (d, 2H, Ar-H), 6.32 (s, 1H, NH), 6.46 (d, 2H, Ar-H), 3.73 (t, 4H, -CH2), 3.45 (t, 4H, -CH2), 4.09 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.26 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 175.3 (C-8), 166.5 (C-4), 153.1 (C-2), 148.6 (C-20), 130.3 (C-17), 129.4 (C-18, C-22), 112.8 (C-19, C-21), 101.8 (C-3), 68.3 (C-25, C-27), 63.1 (C-12, C-15), 52.6 (C-24, C-28), 16.2 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3319, 3232 (NH), 1226 (P=O), 1097 (C=S), 1015 (P-O-Calip); LCMS (m/z, %): 455 (M+H+, 100); Anal. Calcd for C19H27N4O5PS: C, 50.21; H, 5.99; N, 12.33%; found: C, 50.28; H, 5.94; N, 12.39%.

Diethyl 3-methyl-5-(3-(4-(trifluoromethyl)phenyl)thioureido)isoxazol-4-ylphosphonate (5h):
Yield: 91%; semi solid. 31P NMR spectrum (DMSO-d6): ? 19.4 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 11.47 (s, 1H, Ar-NH), 7.45 (d, 2H, Ar-H), 6.32 (s, 1H, NH), 7.23 (d, 2H, Ar-H), 4.12 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.27 (t, J = 7.2 Hz, 6H, O-CH2CH3); 13C NMR spectrum (100 MHz, DMSO-d6): ? 175.8 (C-8), 166.5 (C-4), 153.1 (C-2), 142.4 (C-17), 133.5 (C-20), 127.4 (C-18, C-22), 126.8 (C-19, C-21), 123.3 (C-23), 101.8 (C-3), 63.5 (C-12, C-15), 16.6 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3345, 3274 (NH), 1228 (P=O), 1098 (C=S), 1021 (P-O-Calip); LCMS (m/z, %): 438 (M+H+, 100); Anal. Calcd for C16H19F3N3O4PS: C, 43.94; H, 4.38; N, 9.61%; found: C, 43.99; H, 4.44; N, 9.53%.

Diethyl 3-methyl-5-(3-(4-nitrophenyl)thioureido)isoxazol-4-ylphosphonate (5i):
Yield: 93%; semi solid. 31P NMR spectrum (DMSO-d6): ? 22.6 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 11.49 (s, 1H, Ar-NH), 8.13 (d, 2H, Ar-H), 6.35 (s, 1H, NH), 6.29 (d, 2H, Ar-H), 4.11 (m, 4H, O-CH2CH3), 2.54 (s, 3H, -CH3), 1.26 (t, J = 7.2 Hz, 6H, O-CH2CH3);13C NMR spectrum (100 MHz, DMSO-d6): ? 175.6 (C-8), 166.5 (C-4), 153.1 (C-2), 143.6 (C-17), 142.8 (C-20), 126.4 (C-18, C-22), 125.8 (C-19, C-21), 101.8 (C-3), 63.8 (C-12, C-15), 16.7 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3340, 3265 (NH), 1226 (P=O), 1097 (C=S), 1019 (P-O-Calip); LCMS (m/z, %): 415 (M+H+, 100); Anal. Calcd for C15H19N4O6PS: C, 43.48; H, 4.62; N, 13.52%; found: C, 43.49; H, 4.69; N, 13.45%.

Diethyl 5-(3-(4-methoxyphenyl)thioureido)-3-methylisoxazol-4-ylphosphonate (5j):
Yield: 93%; semi solid. 31P NMR spectrum (DMSO-d6): ? 21.6 ppm; 1H NMR spectrum (400 MHz, DMSO-d6): ? 11.49 (s, 1H, Ar-NH), 6.87 (d, 2H, Ar-H), 6.45 (d, 2H, Ar-H), 6.33 (s, 1H, NH), 4.07 (m, 4H, O-CH2CH3), 3.74 (s, 3H, -OCH3), 2.54 (s, 3H, -CH3), 1.24 (t, J = 7.2 Hz, 6H, O-CH2CH3);13C NMR spectrum (100 MHz, DMSO-d6): ? 175.2 (C-8), 166.5 (C-4), 155.8 (C-20), 153.1 (C-2), 132.6 (C-17), 128.7 (C-18, C-22), 115.3 (C-19, C-21), 101.8 (C-3), 63.3 (C-12, C-15), 56.4 (C-23), 16.5 (C-13, C-16), 14.3 (C-6); IR (KBr) (?max cm-1): 3294, 3243 (NH), 1220 (P=O), 1092 (C=S), 1013 (P-O-Calip); LCMS (m/z, %): 400 (M+H+, 100); Anal. Calcd for C16H22N3O5PS: C, 48.11; H, 5.55; N, 10.52%; found: C, 48.19; H, 5.62; N, 10.45%.

2.7. Biology
2.7.1.Antibacterial activity
Antibacterial activity screening of all the synthesized compounds was carried out against two Gram positive bacteria such as Staphylococcus aureus (ATCC-19433), Bacillus subtilis (ATCC-23857) and two Gram negative bacteria such as Escherichia coli (ATCC-10148), Pseudomonas marginalis (MTCC-2758) by using agar well diffusion method 19 at two different concentrations 100 ?g/mL and 200 ?g/mL. Norfloxacin was used as a standard drug for the comparison of the antibacterial activity. All the newly synthesized compounds exhibited moderate to good activity. Different concentrations (100 and 200 ?g/mL) of test compounds and the standard, Norfloxacin were prepared in dimethylsulfoxide (DMF) and DMF was used as control. The selected 24 h old bacteria cultured with 0.5 mL containing 1 × 107 CFU/mL in nutrient broth were spread on nutrient broth-agar in Petri dishes. The paper discs (6 mm diameter, Whatman No. 2) were dried and dipped in known concentration of the prepared test samples. Then, these discs were placed on the culture and incubating for 24 h at 37 ºC. The clear zones of inhibition around the disc were measured (in mm). The experiments were performed in triplicate and the average value was taken as final result and the results were presented in Table 1.
2.7.2.Antifungal activity
The antifungal activity of synthesized compounds was tested against two pathogenic fungi’s such as Fusarium oxysporum and Aspergillus niger using poison plate technique 20 at two different concentrations 100 and 200 µg/mL. Griseofulvin was used as a standard for the comparison of the antifungal activity.
The fungal strains Helminthosporium oryzae (ATCC 11000) and Aspergillus niger (ATCC 16404) were selected to screen the antifungal activity of the newly synthesized compounds by agar disc-diffusion method. The fungal strains were maintained on potato dextrose agar (PDA) medium (Hi-Media). A loop full of culture from the slant was inoculated into the potato dextrose broth and incubated at 37 ºC for 48-72 h. 0.1 mL of this culture was spread on the potato dextrose agar plate using a sterile glass spreader for even spreading of the inoculum. Sterile discs of Whatmann No.1 filter paper of about 6 mm diameter were impregnated on the surface of the media. A blank test was carried out which showed that DMF solvent used in the preparations of the test solutions does not affect the test organism. 100 and 200 ?g/mL concentrations of various test compounds were prepared, applied on the discs and incubated for 48-72 h at 37 ºC. The zone of inhibition around the disc was calculated edge to edge zone of the confluent growth which corresponds to the sharpest edge of the zone and was measured in millimeters. All tests were assayed in triplicate and average data were taken as final result. Griseofulvin was used as a standard drug and the inhibition zones of the test compounds were compared with control and the results were presented in Table 2.
3. Results and discussion
3.1. Chemistry
In continuation of our research in the development of new biologically active phosphonate derivatives,xxx we report an efficient and environmentally benign protocol for the synthesis of phosphonates (5a-j) by the reaction of urea/thiourea derivatives (3a-j) and triethylphosphite (4) using 37% nano-BF3.SiO2 (0.25 g) under solvent-free conditions using conventional and microwave irradiation methods (Scheme 1). Urea/thiourea derivatives (3a-j) were prepared from 4-bromo-3-methylisoxazol-5-amine (1) by the reaction with various isocyanates/isothiocyanates (2a-j) using triethylamine as base.

To find the optimal reaction conditions for carrying out the Michaelis-Arbuzov reaction, we have selected 1-(4-bromo-3-methylisoxazol-5-yl)-3-(pyridin-3-yl)urea (3a) and diethyl phosphite (4) as model substrates. The progress of the reaction was investigated in the presence of different Lewis acid catalysts (10mol%) such as AlCl3, FeCl3, LaCl3.7H2O, CeCl3.7H2O, ZnCl2, SiO2.ZnCl2, BF3.Et2O and 37% nano-BF3.SiO2 under solvent-free conditions using both conventional as well as microwave irradiation methods (Table 1, entry 1-8). The results revealed that, nano-BF3.SiO2 is a more efficient catalyst for the synthesis of phosphonates under microwave assisted neat reaction conditions with respect to reaction times and yields as compared to the conventional method (Table 1, entry 8).

Table 1. Synthesis of compound 5a under various conditions

Entry Catalyst
Conventional methodaMicrowave irradiation methodbTime (h) Yieldc (%) Time (min) Yieldc (%)
1 AlCl3 (10mol%) 6 58 30 63
2 FeCl3 (10mol%) 5 61 28 67
3 LaCl3.7H2O 4 63 26 69
4 CeCl3.7H2O (10mol%) 4 65 24 70
5 ZnCl2 (10mol%) 5 62 27 66
6 SiO2.ZnCl2 (10mol%) 3 68 20 74
7 BF3.Et2O (10mol%) 2 71 19 78
8 37% nano-BF3.SiO2 (0.30g) 1 80 9 95
aReaction of 1-(4-bromo-3-methylisoxazol-5-yl)-3-(pyridin-3-yl)urea and triethyl phosphite in presence of 37% nano-BF3.SiO2 in solvent-free condition at 70 ºC
bReaction of 1-(4-bromo-3-methylisoxazol-5-yl)-3-(pyridin-3-yl)urea and triethylphosphite in presence of 37% nano-BF3.SiO2 in solvent-free condition under microwave irradiation at room temperature
c Isolated yield.

On further modification of the reaction conditions, we examined the effect of the amount of the catalyst on the model reaction by altering the loading of catalyst (Table 2, entries 1-8) under solvent-free conditions using microwave irradiation. It was observed that good yield of the product was obtained when used 0.25 g of 37% nano-BF3.SiO2 catalyst. High loading of the catalyst did not lead to the significant variation in the yield of product, but lower yields of the product.

Table 2. The effect of the amount of the catalyst, 37% nano-BF3-SiO2 to promote the Michaelis-Arbuzov reactionaEntry Amount of Catalyst (gm) Time (min) Yieldb (%)
1 0.05 9 56
2 0.1 9 66
3 0.15 9 75
4 0.2 9 83
5 0.25 9 96
6 0.3 9 95
7 0.35 9 92
8 0.40 9 87
aReaction of 1-(4-bromo-3-methylisoxazol-5-yl)-3-(pyridin-3-yl)urea and triethylphosphite in presence of 37% nano-BF3.SiO2 in solvent-free condition under microwave irradiation at room temperature
bIsolated yield.

The reusability of the nano-BF3·SiO2 catalyst was also examined. After each run, the product was filtered and the residue of catalyst was washed with CHCl3 to remove stains from the catalyst surface and reused up to three cycles for the synthesis of compound 5a and re-examined these reactions (Table 3, entry 1-5). It is remarkable that the yield was diminished when the catalyst was reused in the forth cycle; hence, we did not reuse the catalyst more than three times for the reaction.
Table 3. Reusability of the catalyst, 37% nano-BF3.SiO2 for the synthesis of compound 5aa
Entry 37% nano BF3.SiO2 (0.30g) Time (min.) Yieldb (%)
1 1st run 8 96
2 2nd run 8 94
3 3rd run 8 94
4 4th run 8 89
5 5th run 8 85
a Reaction of 1-(4-bromo-3-methylisoxazol-5-yl)-3-(pyridin-3-yl)urea and triethylphosphite in presence of 37% nano-BF3.SiO2 (0.25g) in solvent-free condition under microwave irradiation at room temperature
b Isolated yield.

The model reaction was optimized by screening at 180, 220, 350, 420, 455 and 560 Watts to find out the effect of microwave power on the reaction. A microwave power 420 W was found to be optimal (Table 4).

Table 4. Effect of microwave oven power (Watt) on the yield of the compound 5a.a
Entry Microwave Power (Watts) Yieldb1 180 70
2 220 85
3 350 86
4 420 96
5 455 87
6 490 84
7 570 76
a Reaction of 1-(4-bromo-3-methylisoxazol-5-yl)-3-(pyridin-3-yl)urea and triethylphosphite in presence of 37% nano-BF3.SiO2 (0.25g) in solvent-free condition under microwave irradiation at room temperature
b Isolated yield.

After optimization of the reaction conditions by various examinations, the scope of the reaction was investigated by changing various urea/thiourea derivatives and the results are summarized in Table 5.

Table 5. Effect of microwave irradiation on the synthesis of phosphonates (5a-j)
Entry Product Time (min) Yieldb (%)
5a 9 96
5b 11 94
5c 16 90
5d 12 91
5e 13 95
5f 12 89
5g 10 92
5h 15 91
5i 17 94
5j 13 93
a Reaction of various urea/thiourea derivatives of 4-bromo-3-methylisoxazol-5-amine (3a-j) and triethylphosphite(4) in presence of 37% nano-BF3.SiO2 (0.25g) in solvent-free condition under microwave irradiation at room temperature
b Isolated yield.

The structures of the synthesized compounds were characterized by NMR (31P, 1H, 13C), IR, mass and elemental analysis. 31P NMR signals appeared in the region 26.1-18.7 ppm for all the compounds (5a-j). The 1H NMR spectra of the compounds (5a-j) gave signals due to Ar-H in the range of 8.15-6.32 ppm. The proton signal at 11.49-9.25 and 7.36-7.32 ppm are assigned to NH group attached to heterocyclic moiety and isoxazole moiety respectively for the compounds. The methylene protons of P-O-CH2CH3 gave a multiplet and methyl protons of P-O-CH2CH3 resonated as a triplet in the region ? 4.09-4.05 and ? 1.27-1.23 respectively for the compounds (5a-j). In the 13C NMR spectra of compounds (5a-j), the P-O-CH2-CH3 and P-OCH2-CH3 were resonated at ? 63.8-63.1 and 16.7-16.2 respectively. 13C NMR chemical shift for C=O of compounds (5a-e) and C=S of compounds (5f-j) were observed in the region ? 154.5-154.1 and 175.8-175.2 ppm. The compounds (5a-j) showed characteristic infrared absorption bands in the region 3345-3221, 1229-1220 and 1021-1013 cm-1 for NH, P=O and P-O-Calip stretching frequencies. The compounds (5a-e) exhibited characteristic absorption bands for C=O functional group in the region 1743-1702 cm-1. The compounds (5f-j) showed characteristic infrared absorption bands in the region 1098-1092 cm-1 for C=S stretching frequencies. In their mass spectra, M+. ions were observed in the expected m/z values.

3.2. Antibacterial activity
Antibacterial activity screening of all the synthesized compounds was carried out against two Gram positive bacteria such as Staphylococcus aureus (ATCC-19433), Bacillus subtilis (ATCC-23857) and two Gram negative bacteria such as Escherichia coli (ATCC-10148), Pseudomonas marginalis (MTCC-2758) by using agar well diffusion method at two different concentrations 100 ?g/mL and 200 ?g/mL. Norfloxacin was used as a standard drug for the comparison of the antibacterial activity. All the newly synthesized compounds exhibited moderate to good activity.
Table 6. Antibacterial activity of pyrazole derivatives 5a-j
CompdZone of inhibition (mm)
S. aureus(ATCC-19433) B. subtilis(ATCC-23857) E. coli
(ATCC-10148) P. marginalis(MTCC-2758)
100
?g/mL200
?g/mL100
?g/mL200
?g/mL100
?g/mL200
?g/mL100
?g/mL200
?g/mL5a 13.1 18.5 12.2 18.3 11.4 17.2 12.6 18.5
5b 12.5 18.9 11.5 16.4 16.9 20.8 16.2 20.1
5c 11.5 17.3 12.1 18.4 13.1 19.1 12.8 18.6
5d 15.2 21.8 15.6 22.2 15.0 20.8 15.6 21.9
5e 14.3 20.5 16.4 21.6 17.2 22.8 16.5 22.0
5f 12.4 18.3 13.4 19.2 13.1 18.8 13.0 20.2
5g 13.4 19.3 15.1 20.0 12.4 18.4 12.8 18.9
5h 17.2 23.1 19.5 24.3 18.0 25.8 16.9 22.7
5i 14.5 21.0 16.3 21.5 15.3 19.5 14.3 21.4
5j 15.8 22.1 17.6 23.4 16.4 20.3 17.4 23.1
Std. 18.2 24.5 20.2 25.6 22.1 26.0 19.3 24.3
DMF – – – – – – – –
*Std. – Norfloxacin; S. aureus – Staphylococcus aureus; B. subtili – Bacillus subtilis; E. coli – Escherichia coli; P. marginali – Pseudomonas marginalis.
3.3. Antifungal activity
The antifungal activity of synthesized compounds was tested against two pathogenic fungi’s such as Fusarium oxysporum and Aspergillus niger using poison plate technique at two different concentrations 100 and 200 µg/mL. Griseofulvin was used as a standard for the comparison of the antifungal activity. All the compounds exhibited moderate to good antifungal activity.

Table 6. Antibacterial activity of pyrazole derivatives 5a-j
 
CompdZone of inhibition (mm)
A. nigerF. oxysporum100
?g/mL200
?g/mL100
?g/mL200
?g/mL5a 10.1 18.3 7.5 15.5
5b 9.4 17.4 7.9 16.3
5c 10.3 18.5 9.0 17.4
5d 9.7 15.4 8.5 16.3
5e 6.4 12.6 6.2 13.3
5f 6.7 14.1 8.8 16.6
5g 5.8 12.5 6.2 11.3
5h 8.0 15.1 7.8 14.1
5i 7.1 13.2 7.2 15.0
5j 5.5 11.2 5.3 10.1
Std. 12.4 20.2 10.5 19.4
Control (DMF) – – – –
Std.- Standard (Gresiofulvin); A. niger – Aspergillus niger; F. oxysporum – Fusarium oxysporum4. Conclusion
In summary, we have demonstrated that the reactions of a urea/thiourea derivatives with triethyl phosphite under solvent free condition in the presence of nano BF3.SiO2 using microwave irradiation proceeded rapidly and cleanly to give the corresponding substituted phosphonates. Mild, non-hazardous and environment friendly reaction conditions, recyclability of the catalyst, good selectivity giving excellent yield in short reaction time are significant advantages of this method. The compounds 5d and 5h and 5j showed promising antibacterial activity when compared with the remaining title compounds, and were closer to standard drug. The compounds 5a and 5c showed exhibited potent antifungal activity against the two tested fungal strains when compared with the remaining compounds.
Acknowledgements
Authors express their thanks to Department of Biochemistry, S. V. University, Tirupati for providing biological assay results and Osmania University, Hyderabad and Hyderabad Central University, Hyderabad for providing spectral data.

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