EVALUATION OF ANTIFUNGAL AND ANTIBACTERIAL ACTIVITIES OF MONOESTERS OF SUCCINIC ANHYDRIDE

Monoester of succinic acid (1-29), synthesised and characterised at our laboratory, were investigated with reference to their antifungal and antibacterial activities. The results concluded that though almost all the compounds were bioactive but the degree of activity was dependent over the substituent attached to benzyl group and order of their bioactivity was iodo > chloro > methoxy > nitro substituted monoesters against the considered microbes.


INTRODUCTION
Application of synthetic compounds as a substitute for natural medicines for the treatment of ailment has brought a revolution in synthetic chemistry. At present evaluation of synthetic compounds for their antifungal and antibacterial activity is the subject of active chemistry research [1][2][3]. In spite of the fact diseases caused by microbes are increasing with the passage of time, the microbes have become resistant to the available drugs and hence posing a great threat to human being but no considerable antifungal and antibacterial drugs have been synthesised/discovered from the last two decades. Therefore, it is need of the day to synthesise new antifungal/antibacterial compounds which may ultimately be used as drugs. Literature revealed that natural and synthetic esters of succinic acid had a wide range of applications like industrial, agrochemical and pharmaceutical [4][5][6][7]. Some of these are the treatment of HIV, tumours, as antiseptic agents, antioxidants, as enzyme inhibition, resolving the racemic mixtures and in the synthesis of bioactive compounds [8][9][10][11][12][13][14][15][16].
Therefore, quite a good number of scientists are involved in the synthesis of biologically active compounds and characterise them with reference to their antimicrobial properties [18][19][20]. In the last report the monoesters of succinic acid (1-29) were synthesised and characterised [19] whereas, in the present study the biological activities of these compounds (1-29) against various microbes are reported. All the reported compounds were bioactive, however, only the halogenated esters 11-20 displayed activity equivalent to standard drugs chloramphenicol and ketoconazole.

EXPERIMENTAL
General procedure for the preparation of 1-29. The aryl hydrogen succinates (1-29) were synthesized and characterised by following the standard protocol [19]. Briefly, 15 mmol of corresponding alcohol was added to succinic anhydride (15 mmol), anhydrous p-toluenesulfonic acid (0.06 mmol) and toluene (15 mL) under the atmosphere of nitrogen in a single-necked round-bottom flask (100 mL). The flask was equipped with magnetic stirrer, Dean-Stark trap and a reflux condenser. The solution was refluxed for 14 h and allowed to cool up to 25 o C. The product was then poured into saturated aqueous solution of NaHCO 3 (12.5 mL) and the organic layer was extracted with hexane (3 × 25 mL). The organic phase was then washed with brine (10 mL), dried over anhydrous Na 2 SO 4 and the excess of the solvent was removed under vacuum to give a resinous product. It was then subjected to column chromatography to get pure monoesters . The target substrates were characterized by UV, IR, 1 H-NMR and 13 C-NMR and mass measurement. The structure of prepared monoesters (1-29) so obtained is presented in Scheme 1.

Scheme 1. Structures of monoeasters 1-29.
Antifungal and antibacterial activities. Antifungal and antibacterial activities of synthesised compounds   Antifungal activity of monoesters. The antifungal activities of the monoesters (1-29) were determined by employing hanging drop method considering ketoconazole as standard [21]. Briefly, 500 µg/mL solution of the compounds was employed on the germinating fungal spores. The plates were incubated at 37 o C for 20 h and the antifungal activity was determined by measuring the diameter of the inhibition zone in mm ( Figure 1). The percentage inhibition of spore germination was calculated by observing the germination of the spores under microscope after 8 hours of incubation at 30 o C using Equation 1. Antibacterial activity of monoesters. The antibacterial activity of the monoesters (1-29) was determined by following the agar well diffusion method [22] using chloramphenicol as standard. Briefly, wells were dug in the media using a sterile borer. Using a sterile cotton swab, the surface of the agar nutrient was covered with eight-hour bacterial inoculum containing 10 4 -10 6 colony forming units (CFU/mL). Monoesters (6-16 mg in DMSO 1 mL) were placed in the wells. Pure DMSO (1 mL) and chloramphenicol (6 mg/mL DMSO) were introduced into two other wells for negative and positive controls, respectively. The plates were incubated immediately at 37 o C for 20 h. The activity was determined by measuring the diameter of the inhibition zone (in mm). Growth inhibition zone was calculated with reference to the positive control.
Minimum inhibitory concentration (MIC) of monoesters. The minimum inhibitory concentration (MIC) was determined by agar dilution method [22]. Twenty-five mL of the sterilized Mueller-Hinton agar (Oxoid) was added to sterilized test tube containing 1 mL of 6-16 µg/mL of monoesters at 25 o C. The mixture was then thoroughly mixed and poured into sterilized petri plates. The microbial suspension with density adjusted to 0.5 McFarland turbidity standard was inoculated (0.05 µL) on to the series of agar plates using micropipette. The plates were then incubated at 37 o C for 24 h and MIC values were calculated. - - The prepared compounds showed interesting structure activity relationships while exploring their antifungal and antibacterial activity. Some interesting trends that were noticed included low activities of the compounds having substituents linked through oxygen and having substituent at three position of benzene ring (Tables 1 and 2). Relatively high activity was observed for compounds with substituent at 2 and 4 position of benzene ring. Highest activity was revealed by halogenated monoesters (12)(13)(14)(15)(16)(17)(18)(19)(20) in general and iodinated monoesters in particular (18)(19)(20). Monoesters having substituents linked through oxygen to benzene ring (1)(2)(3)(4)(5)(6)(7)(8) and (24-29) displayed relatively less activity as compared to halogenated monoesters. This could be explained on the basis that the presence of halogens could be responsible for enhanced activity. Also substituents like methyl, methoxy and hydroxyl, having +M effect that increased electronic density on the benzene ring, decreased the activity of the compounds (Tables 1 and  2). As is evident from Tables 1 and 2, iodosubstituted monoesters exhibited values close to ketoconazole and chloramphenicol standards. Therefore, the compounds are potential sources as antibacterial and antifungal agents and can find use in biomedical area in near future. Moreover, meta-substituted isomeric monoesters showed lower activities than their ortho-and paraanalogues (Tables 1 and 2).

CONCLUSION
The prepared compounds except 9-11were found to be noticeably bioactive. The highest activity was observed for iodinated monoesters. It can be concluded that the compounds may be candidates for antifungal and antibacterial drugs. It is recommended that in vivo studies of these compounds may be carried out and their mode of action against these microbes be explored.