1. Introduction
Isocoumarins are the secondary metabolites of fungi, bacteria, plants and insect venoms and pheromones. A huge number of them have been isolated from fungi, lichens and bacteria. Some higher plants, insect and marine organisms are also the rich source of these secondary metabolites [1] [2] . They exhibit a broad range of pharmacological activities including antimalarial, antimicrobial, immunomodulatory, antifungal, anti-inflammatory, cytotoxic, antiangiogenic and antiallergic [3] -[9] . Isocoumarins are also used as a lead compound for the identification of insecticides which selectively bind at the insect GABA receptor [10] .
3-Substituted isocoumarins also show anti HIV activity in vitro, diuretic, antihypertensive, antiarrythmics, β-sympatholytics, anticorrosive, laxatives, asthmolytic, phytotoxic, and are useful in the treatment of emphysema [11] . Isocoumarin derivatives are potently inhibits endothelial cell proliferation, migration, sprouting, tube formation in vitro, and tumor growth in vivo [12] .
1(2H)-isoquinolones are the nitrogen analogues of isocoumarins. 1(2H)-isoquinolone derivatives are found in several bioactive natural product such as thalifolin, doryphorine [13] , ruprechstyril [14] , narciclasine [15] , lycoricidine [16] and the alkaloids coryaldine and thalflavine. Substitutd isoquinolones exhibit antidepressant, antiinflamatory, analgesic, hypolipidimic, and analeptic activities have also been reported.
Though a number of new antibiotics have been produced in the last three decades, yet resistance to these drugs by microorganisms has developed. Some antibiotics have become almost obsolete because of drug resistance [17] . In general, bacteria have the genetic ability to transmit and acquire resistance to drugs utilized as therapeutic agents [18] . Consequently new drugs must be synthesized and assayed against these pathogenic resistant microorganisms for the sake of life.
The present study has been designed to determine the in vitro antibacterial and antifungal activity of 3-(substituted phenyl)isocoumarins, 1-thioisocoumarins and 1(2H)-isoquinolones against some pathogenic and nonpathogenic strains. A comparison of the efficacy of these classes of compounds and then establishes a structure activity relationship between differently substituted analogues of isocoumarins, 1-thioisocoumarins and isoquinolones is also discussed.
2. Materials and Method
In this in vitro antimicrobial assay, we used three series of the compounds i.e. 3-(substituted phenyl) isocoumarins (1H-2-benzopyran-1-ones) (1 - 10), (isocarbostyrils 1(2H)-isoquinolones, the nitrogen analogues of isocoumarins (1H-2-enzopyran-1-ones) (11 - 20) and 1-thioisocoumarins (1H-isochromenes-1-thiones) (21 - 30). Each series contains ten compounds having different functionality at position 3. All of the compounds used in this study have been synthesized, purified and characterized by the author [19] [20] . All chemical used are of analytical grade. The purified samples were dissolved in DMSO 5 mg/ml which is the negative control in this bioassay. The antibiotic chloramphenicol and fluconazole were used as standard drugs for antibacterial and antifungal activity respectively.
2.1. Antibacterial Activity
The antibacterial assay was performed by agar well diffusion method against ten different Gram positive and Gram negative bacterial strains [21] . The bacterial strains Escherichia coli, Klebsiella pneumonae (ATCC 6633), Staphylococcus aureus (ATCC 29213), Micrococcus luteus (ATCC 9341), Pseudomonas aeruginosa (ATCC 33347), Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 19430), Lactobacillus bulgaricus, (ATCC 25929), Pasteurella multocida A (ATCC 9150), Staphylococcus epidermidis (ATCC 29232) and Proteus vulgaris (ATCC 49565) were selected in this study. Micrococcus luteus, Staphylococcus aureus and Staphylococcus epidermidis are the Gram positive whilst the remaining seven are Gram negative bacteria. All of the tested microorganisms were maintained on nutrient agar at 4˚C and sub-cultured before use.
The bacteria studied are clinically important ones causing several infections and it is essential to overcome them through some active therapeutic agents. Each tested bacterium was sub-cultured in nutrient broth at 37˚C for 24 h. One hundred microliters of each bacterium was spread with the help of sterile spreader on to a sterile Muller-Hinton agar plate so as to achieve a confluent growth. The plates were allowed to dry and wells (6mm diameter) were punched in the agar with the help of cork borer. 0.1 mL of the each compound solution (5 mg/ mL) in DMSO was introduced in to the well and the plates were incubated overnight at 37˚C.
The antimicrobial spectrum of the compounds was determined for the bacterial species in terms of size of the zones around each well and results are presented in tables 1-3. The diameters of the zone of inhibition produced by the compounds were compared with those produced by the commercial antibiotic chloramphenicol (5 mg/mL). This is the common antibiotic used for the treatment of infections caused by gram positive and gram negative bacteria. The control activity was deducted from the test and the results obtained were plotted. The experiment was performed three times to minimize the error and the mean values are presented.
2.2. Antifungal Activity
Antifungal activity of these three series of compounds was determined by using three fungal strain; Aspergillus flavus, Aspergillus nigar and Aspergillus pterus using poison plate method [22] . Potato dextrose agar (PDA) plates were equipped by using pour plate technique for each compound. A 2% concentration of the synthesized compounds in DMSO as a solvent was used. A 2% solution of fluconazole was used as standard. A drug free control was included and plates were observed for growth after 48 h of static incubation at 30˚C and results are presented in tables 4-6. All of the synthesized compounds showed poor antifungal activity against the selected fungal strains. The 1-thioisocoumarins showed good growth inhibition than the parent isocoumarins and isoquinolones. The Aspergillus niger is the most resistant strain against these isocoumarins derivatives.
3. Results and Discussion
The antibacterial activity of the 3-phenylsubstituted isocoumarins, 3-phenylsubstituted isoquinolin-1(2H)-ones and 3-phenylsubstituted 1H-isochromenes-1-thiones was determined against ten bacterial strains and reported in tables 1-3 respectively. The results of the antibacterial assay of these three series of compound reflect that the 3-phenyl substituted isocoumarins are more active as compared to their nitrogen analogues but less active as compared to their thio analogues. The structures of the compounds of these three series are shown in figure 1.
Table 1. In vitro Antibcterial activity of 3-substituted isocoumarins (1 - 10).
Table 2. In vitro Antibcterial activity of 3-substituted isoquinolones (11 - 20).
Table 3. In vitro Antibcterial activity of 3-substituted-1-thioisocoumarins (21 - 30).
*Activity of each sample is measured by subtracting the activity of DMSO, (-) No activity. Escherichia coli (E. c.), Klebsiella pneumonae (K. p.), Lactobacillus bulgaricus (L. b.), Micrococcus luteus (M. l.), Pasteurella multocida (P. m.), Proteus vulgaris (P. v.), Pseudomonas aeruginosa (P. a.), Salmonella typhi (S. t.), Staphylococcus aureus (S. a.) and Staphylococcus epidermidis (S. e.).
Table 4. In vitro Antifungal activity of 3-substituted isocoumarins (1 - 10).
Table 5. In vitro Antifungal activity of 3-substituted isoquinolones (11 - 20).
Table 6. In vitro Antifungal activity of 3-substituted-1-thioisocoumarins (21 - 30).
(-) No activity.
Among all the ten differently 3-phenylsubstituted isoquinolin-1(2H)-ones only the 3-(2,4-dichlorophenyl)- 1(2H) isoquinolone (18) shows moderate to potent activity against these tested microorganisms. It shows potent activity against K. pneumonae and have moderate efficacy against L. bulgaricus and P. multocida. These results indicate that in case of 1(2H) isoquinolones presence of two electronegative halogen (chlorine) functionality is important in showing antibacterial activity. It is inactive against gram negative bacteria and all of the remaining 1(2H) isoquinolones are inactive against both selected gram positive and gram negative bacterial strains.
Most of the 3-phenylsubstituted isocoumarins are inactive against these tested gram positive and gram negative bacterial strains. The 3-(4-nitrophenyl) isocoumarin (7) exhibits moderate activity against all the selected gram positive and gram negative bacterial strains. The compound 3-(3-flourophenyl)isocoumarin (9) shows moderate activity against L. bulgaricus and P. auriginosa which are gram positive bacterial strains but is inactive against all the remaining gram positive and gram negative bacterial strains.
It was found that 3-phenylsubstituted 1H-isochromenes-1-thiones show potent activity against gram positive bacteria and three derivatives also exhibit activity against gram negative bacteria. 3-(3-Iodophenyl)-1H-isochromenes-1-thiones is most active against E. coli but inactive towards all other tested microorganisms. Similarly