Synthesis and Antimicrobial Studies of Some New 3-Isoxazoline Substituted Phthalazine Methylsulfonyl Oxadiazoles

A series of new 3-isoxazoline substituted phthalazine methylsulfonyloxadiazoles were prepared from methyl 2-(4-oxo-3,4-dihydrophthalazin-1-yl) acetate. The structure of synthesized compounds were characterized by spectral data and screened for their antimicrobial activities against various bacteria and fungi strains. Several of these compounds showed antimicrobial activity.


Introduction
Phthalazin-1(2H)-ones, methylsulfanyl oxadiazoles and isoxazolines serves as some important building blocks for biological active molecules.Figure 1 has been revealed by these three important biological active pharmacophore component systems.Phthalazine isoxazolines scaffold Phthalazin-1(2H)-one derivatives are of considerable interest due to their antidiabatic 1 antiallergic 2 , antiasthmatic 3 , antihypertensive 4 , vasorelaxant 5 , aldose reductase inhibitor 6 , and antimicrobial 7 activities.Moreover a number of established drug molecules like hydralazine 8 , budralazine 9 , azelastine 10 , ponalrestat 11 and zopolrestat 12 are accessible starting from the corresponding phthalazinones.The second pharmacophore component is isoxazoline, which represents an important class of heterocyclic compounds with broad spectrum of biological activities.Substituted isoxazolines have revealed anti-influenza virus 13 , antifungal 14 , antitubercular 15 , spermicidal and anti-HIV 16 , β-adrenergic receptor antagonist properties 17 .Similarly the third pharmacophore methylsulfanyl oxadiazole unit is also one of important biologically active building block.The substituted methylsulfanyloxadiazoles have been found to exhibit diverse biological activities such as inhibitors of glycogen synthase kinase-3β 18 , antifungal 19 , antibacterial and anti-HIV 20 , insecticidal 21 , antiinflammatory and analgesic 22 , anticonvulsants and muscle relaxants 23 .
The diverse biological activities of these pharmacophores; phthalazin-1(2H)-one, methylsulfanyl oxadiazole and isoxazoline pharmacophores, encouraged us to discover a new lead compounds, which contains all these pharmocophores in a single molecules, that may exhibit higher pharmacological activities.By combining these pharmacophore components in a single molecule, to give a compact system, we designed and synthesized a series of phthalazin-1(2H)-one derivatives containing methylsulfanyl oxadiazole and isoxazoline moieties.The synthesized new phthalazin-1(2H)-one derivatives were characterized by mass, IR and NMR spectral data's.

Experimental
NMR spectra were recorded on 400 MHz Varian-AS NMR spectrometer using TMS as an internal standard.IR spectra were recorded by using Perkin Elmer Spectrum 100 Series FT-IR spectrometer.Mass spectra were recorded on Agilent 1200 Series LC/MSD VL system.Melting points were determined by using Buchi melting point B-545 instrument and are uncorrected.All reactions were monitored by thin layer chromatography (TLC) using Merck pre-coated TLC silica gel plates.The crude compounds were purified by using CombiFlash® Companion® flash chromatography system, Teledyne Isco, Inc USA.BPL Sanyo domestic microwave oven was used for reactions.potassium carbonate (69.0 g, 0.5 mol) at room temperature and heated to 60-65 o C for 6 h.After completion of reaction, filtered the inorganics, the filtrate obtained was distilled completely under reduced pressure at 60-65 o C. The residue obtained was diluted with ice water (500 mL) and stirred for 30 minutes.The precipitated product was filtered, dried and recrystallized using isopropyl alcohol to yield compound 2 as white solid.M.p.: 90.

General method for the preparation of 3-substituted phthalazin-1(2H)-one derivatives 6(a-i)
An equimolar mixture of aldoxime (10.0 mmol) and N-chlorosuccinimide (10.0 mmol) were made paste with dimethylformamide (2 mL) and alumina (2 g) in a 10 mL beaker.The mixture was irradiated at 600 W for 2 min in domestic microwave oven.After adding compound (5) (9 mmol), the reaction mixture was irradiated further for 6 min.After completion of reaction, diluted with water (25 mL) and extracted with ethyl acetate (3x50 mL).The organic layer was washed with water (10 mL) and then with saturated sodium chloride solution.After drying with anhydrous sodium sulphate and filtration, the solvent was evaporated to get crude product, which was purified by using flash chromatography eluting with ethyl acetate / hexane (1: 5) to get the corresponding phthalazin-1(2H)-ones 6(a-i).

Biological screening
The in vitro antimicrobial activity was carried out against 24 h old cultures of two bacteria and two fungi by cup-plate method in duplicate.Compounds (6a-i) has been tested for their antimicrobial activity against E. coli and S. aureus and antifungal activity against M. gypsum and A. flavus at a concentration of 100 µg/mL in DMSO using cup plate diffusion method.Nutrient agar and potato dextrose agars were used to culture the bacteria and fungus respectively.The solution of Amoxicillin 100 µg/mL and metronidazole at 100 µg/mL were prepared in DMSO and used as standard for comparison of antibacterial and antifungal activities respectively the results were discussed in Table 1.

Table 1. Antimicrobial activity of compounds 6(a-i)
The compounds 6b and 6f-g exhibiting good activity against E. coli and compounds 6b, 6c, 6f and 6g show good activity of against S. Aureus.Similarly the compounds 7g showed good activity against M.gypsum and the compounds 6b and 6g showed activity against A. and all remaining compounds exhibiting moderate activity against all the four organisms used for screening

Conclusion
In this article we report the synthesis of (6a-i), new phthalazine isoxazolines substituted at C 4 position with methylsulfonyl oxadiazole starting from commercially available phthalic anhydride.Investigation of their antimicrobial activity revealed that isoxazolines with substitution with thiophene (6h) shows the most active compound although it was significantly less than that of positive control.The fact that the compounds prepared in this study are chemically unrelated to the current medication suggests that the further work is clearly warranted.

Figure 1 .
Figure 1.Phthalazine isoxazolines scaffoldPhthalazin-1(2H)-one derivatives are of considerable interest due to their antidiabatic 1 antiallergic 2 , antiasthmatic3 , antihypertensive4  , vasorelaxant5  , aldose reductase inhibitor6  , and antimicrobial 7 activities.Moreover a number of established drug molecules like hydralazine 8 , budralazine9 , azelastine 10 , ponalrestat11 and zopolrestat12 are accessible starting from the corresponding phthalazinones.The second pharmacophore component is isoxazoline, which represents an important class of heterocyclic compounds with broad spectrum of biological activities.Substituted isoxazolines have revealed anti-influenza virus13  , antifungal14 , antitubercular15 , spermicidal and anti-HIV16 , β-adrenergic receptor antagonist properties17 .Similarly the third pharmacophore methylsulfanyl oxadiazole unit is also one of important biologically active building block.The substituted methylsulfanyloxadiazoles have been found to exhibit diverse biological activities such as inhibitors of glycogen synthase kinase-3β18 , antifungal19 , antibacterial and anti-HIV20 , insecticidal21 , antiinflammatory and analgesic22 , anticonvulsants and muscle relaxants23 .The diverse biological activities of these pharmacophores; phthalazin-1(2H)-one, methylsulfanyl oxadiazole and isoxazoline pharmacophores, encouraged us to discover a new lead compounds, which contains all these pharmocophores in a single molecules, that may exhibit higher pharmacological activities.By combining these pharmacophore components in a single molecule, to give a compact system, we designed and synthesized a series of phthalazin-1(2H)-one derivatives containing methylsulfanyl oxadiazole and isoxazoline moieties.The synthesized new phthalazin-1(2H)-one derivatives were characterized by mass, IR and NMR spectral data's.