Synthesis and Bioevaluation of 4-[ ( 2 / 4-chloro-/ 2 , 3-dichloro-/ 2 / 4-bromo-/ 2 , 4-dinitro-/ 4-nitrophenyl ) Anilinomethyl ]-6-t-butyl-2 H-1-benzopyran-2-ones

Synthesis of 4-[(2/4-chloro-/2,3-dichloro-/2/4-bromo-/2,4-dinitro-/4-nitrophenyl) anilinemethyl]6-t-butyl-2H-1-benzopyran-2-ones have been carried out. Synthesized compounds were characterized by 1HNMR, IR and other physical and analytical data. All of the synthesized compounds were evaluated for their antifungal activity against Aspergillus awamori and Sclerotium rolfsii and antibacterial activity against a bacterium of Bacillus species in vitro at 10, 50, 100 and 200 μg/ml concentrations. Compounds 4-[(2-chlorophenyl)anilinomethyl]-6-t-butyl-2H-1-benzopyran-2-one(11) and 4-[(4chlorophenyl) anilinomethyl]-6-t-butyl-2H-1-benzopyran-2-one(12) exhibited good antifungal activity among tested compounds. Compound 4-[(2-bromophenyl) anilinomethyl]-6-t-butyl-2H-1-benzopyran2-one(14) showed significant antibacterial activity against the test bacterium.


INTRODUCTION
Since a long time, men have been struggling to reduce the adverse effects of pests on crop production and storage by using pesticides.On the other hand, pesticides and their residues are creating environmental problems, which include toxicity to non-target organisms.So there is a need to evolve and assess alternative compounds which may be effective, easily degradable, cheap and ecofriendly to get residues free food.Coumarin derivatives belong to one of the most important t-butyl-2H-1-benzopyran-2-ones and evaluation of synthesized compounds for their antimicrobial activity to establish structure activity relationship (SAR).

Materials and Methods
The melting points were determined in open capillaries on a Genson electrical melting point apparatus.Homogeneity of the compounds was routinely checked on silica gel-G TLC plates using ethyl acetate: hexane (3:7) as irrigant.IR spectra were recorded on Perkin Elmer FTIR spectrophotometer in KBr and frequencies are expressed in cm -1 .The NMR spectra were recorded on "Bruker Avance II 400-F" (400MHz) NMR spectrophotometer in DMSO-d 6 using tetramethylsilane (TMS) as internal reference.
The chemical shift values are expressed in ´ (ppm) units while J values in Hz and are compatible with the assigned structures.Only those spectral data have been mentioned which have a direct bearing on the assignment of the structure and are discussed here.

Bioevaluation Test for fungi toxicity
Amongst the several methods available, poisoned food technique 6 was used to test the antifungal activity of synthesized compounds against pathogenic fungi Aspergillus awamori and Sclerotium rolfsii.The test fungi was grown on Czapek's agar medium (pH 6.0) containing logarithmic concentrations series of each compound (10-200 µg/ml medium).The required amount of chemical dissolved in 1 ml of acetone was incorporated aseptically into 49 ml aliquotes of sterilized Czapek's agar cooled at 45 o C after brief shaking.Each lot of medium was poured into Petri dishes and allowed to solidify.Each dish was inoculated centrally with a 10 mm mycelial disc cut from the periphery of 2-3 days old fungal colonies.Inoculated Petri plates were incubated in the dark at 30±1 o C and colony diameters were measured periodically till the central dishes were nearly completely covered with fungus growth.Three replicates were used for each concentration of a chemical together with three dishes containing only the solvent and no toxicant.The degree of inhibition of growth was calculated from the mean differences between treatments and the control as percentage of latter by using the formula Where C = mycelial growth in control dish T = mycelial growth in treated dish

Test of Antibacterial activity
The antibacterial activity of synthesized compounds was tested by zone inhibition method 7 on bacterial culture of bacillus species on agar medium.30 ml of the medium was taken in 100 ml round bottom flask and autoclaved at 15 lbs pressure for 20 minutes.The bacterial growth (48h old) from five slants was taken and mixed in 100 ml sterilized distilled water aseptically.This gave the concentration of approximately 10 4 cells/ml.The medium was melted and cooled to 43 °C, needed medium was poured aseptically in sterilized Petri dishes and was allowed to solidify.10, 50, 100 and 200 ppm concentrations of the test compounds were prepared from the stock solution by diluting with acetone.
The sterile filter paper discs (Whatman's No. 1) of 10 mm diameter were dipped in different concentrations of the test compounds and a set of two paper discs was dipped in acetone.Such soaked discs were placed aseptically on inoculated Petri plates.Two paper discs were used for each concentration of a chemical.Each concentration and chemical was replicated 4 times.Such Petri plates were inverted and kept at 5°C for two h for better diffusion of the chemicals in agar medium.Later on, the Petri plates were incubated at 30°C ±1 for 48 h and zone of inhibition was recorded in mm, after 48h of incubation.
The compounds containing halogens such as chlorine and bromine as substitutents showed good antibacterial activity.Among them, Compound 14 containing bromo group at position-2 of phenyl ring showed the comparative best antibacterial activity with inhibition zone of 7mm at lowest concentration, followed by compound 13 with 6.50 inhibition zone containing chloro group at position-2 and 3. On the other hand, addition of bromo group at position 4 of phenyl ring in compound 15 makes it inactive.Again, introduction of NO 2 group in compounds drastically reduces the antibacterial activity with no growth inhibition even up to 200 µg/ml.