THE SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME 4-HYDROXYCOUMARIN DERIVATIVES

Due to exceptional reactivity of -hydroxycoumarin, the synthesis of new coumarin derivatives of dimer and tetramer type has been carried out. Th e synthesis was carried out from -hydroxycoumarin and various aromatic aldehydes. In this way, compounds of the dimer ,’-(benzilidene)bis (-hydroxycoumarin) type, as well as of the tetramer ,’ ,’’ ,’’’-(,-dimethylenphenyl)tetra (-hydroxycoumarin) type were prepared. Th e newly synthesized derivatives contain diff erent functional groups, and as such they could exhibit microbiological activity. Th erefore, we tested the microbiological activity of these derivatives on various species of bacteria and fungi. Th e tested compounds have shown diff erent activity in terms of growth inhibition of microorganisms. Newly synthesized derivatives exhibit antibacterial activities, manifested as growth inhibition on Grampositive bacteria types (Bacillus, Staphylococcus), while the activity against Candida was much weaker. Th e same compound did not show any antimicrobial activity against two Gram-negative bacteria types (Escherichia coli, Pseudomonas aeruginosa). Th e compound  showed the best microbiological activity. Th e obtained results confi rmed its good antibacterial and antimycotic activities against diff erent microorganisms.


Introduction
Studies of natural and synthetic coumarins and its derivatives have been present for a number of years. Coumarins and their derivatives are characterized by excellent chemical reactivity and different bioactivity (-). Their remarkable biological potential is the reason for synthesis of many new products, suitable for application in modern therapy. A great number of synthesized derivatives have shown pharmacological activity, and many of them are applied in therapy as anticoagulant (), antibacterial () and antifungal agents (). The interest in coumarins has recently increased significantly because it was found that they reduce the HIV activity (, ). Further, coumarin derivatives have shown cytostatic activity and therefore can be considered as potential candidates for anti-cancer therapy (). Recently, coumarin derivatives of dimer and tetramer type were reported to possess HIV- integrase inhibitory activity. These facts urged the researches to undertake the synthesis of coumarin derivatives and study their biological activity.

Syntheses of dimers of -hydroxycoumarin
Th e synthesis was carried out from -hydroxycoumarin and various aromatic aldehydes (). The first step of the synthesis is aldol condensation, followed by dehydration, resulting in creation of a stabile chromon. In the last step, the chromon conjugates in the presence of -hydroxycoumarin, the result of which is the creation of dimers of ,'-(benzylidene)bis (-hydroxycoumarin) type, as well as of tetramers of ,','','''- (,dimethylenphenyl)tetra (-hydroxycoumarin) type.
Th e general procedure for the preparation of dimers and tetramers of -hydroxycoumarin  mmol for dimers synthesis or  mmol for tetramers synthesis of -hydroxycoumarin were dissolved in  ml of hot ethanol and  mmol of the corresponding aldehyde was added. Th e reaction mixture was heated under refl ux for  hours. After the completion of the reaction, the reaction mixture was cooled at room temperature, which resulted in crystallization of the target compound. Pure compounds with constant melting points were obtained by recrystallization from chloroform.

Physical-chemical characteristics
Microanalyses for C, H and N were preformed on Perkin Elmer  elementary analyzer (Germany), IR spectra were recorded on Perkin Elmer FT-IR  (Germany) in KBr discs. The  H NMR spectra were recorded at , MHz, in CDCl and DMSO, on NMR Spectrometer, Varian Unity Plus  MHz and Bruker Advance DPX  MHz (Varian, UK).

Antimicrobial activity
Th e antimicrobial activity was tested by the diff usion method () against various bacteria such as Bacillus subtilis ATCC , Staphylococcus aureus ATCC P, Escherichia coli ATCC , Pseudomonas aeruginosa ATCC , while antifungal activity was tested against Candida albicans ATCC . Th e results of our tests were presented as the inhibition zones, given in millimeters (mm). Th e compound that showed best antimicrobial activity was further tested by the dilution method (). For determination of antimicrobial activity (diff usion method) Müller-Hinton and Sabouraud nutritious bases were used. Casein soya bean digest broth (Triptic soya bujon) was used in the dilution method. When using the diff usion method, the test samples were dissolved in dimethyl sulfoxide (,  DMSO) to obtain a  mg/ml stock solution. Th e inhibition zones for bacteria were measured in millimeters at the end of an incubation period of  h at °C, and for fungi after  h at °C. As reference substances gentamycin sulphate and clotrimazole were used. Solution of gentamycin sulphate was prepared in phosphate buff er (pH ,) at standard concentration , I.U. ( μg/ml). Solution of clotrimasole standard was prepared by solving  mg clotrimasole in  ml dimethylsulfoxyde ( μg/ml). For the analysis by the dilution method solution of the compound  was prepared, followed by formation of a series of  dilutions with liquid nutritious base. Th e , ml casein soya bean digest broth was added to the , ml starting solution of test material and thus the fi rst dilution was formed. Subsequently, , ml of this solution was diluted with , ml casein soya bean digest broth to give the second dilution and so on until  dilutions were obtained. Th e fi nal concentration of DMSO in liquid nutritious base was < , . After the incubation for  h, the last tube with no growth of microorganisms was taken to represent MIC expressed in mg/ml. Th e concentration of the prepared solutions were: , mg/ml, , mg/ml, , mg/ml, , mg/ml, , mg/ml, , mg/ml, , mg/ml, , mg/ml, , mg/ml, , mg/ml, , mg/ml and , mg/ml. Th e aim of this method was to determine the exact concentration of the investigated compound which will have an inhibitory effect on the growth of selected microorganisms. This concentration was considered as minimal inhibition concentration (MIC).

Results
Derivatives of dimer and tetramer type were prepared by condensation of -hydroxycoumarin with corresponding aromatic aldehyde. The synthesized compounds have the same basic structure, while they differ in substitutents at the phenyl ring. The structures of prepared dimers and tetramers of -hydroxycoumarin are shown in Table .
The results of yield, elemental analysis and spectral data for synthesized compounds shown in Table .
Antimicrobial activity data are presented in Table . and Table .

Discussion
The obtained results have shown that the newly synthesized derivatives possess antibacterial and antimycotic activities, tested on various bacteria species (B. subtilis, S. aureus) and Candida. Th e same compounds did not show any antimicrobial activity against two Gram-negative bacteria types (E. coli, P. aeruginosa). ,'-(-brombenzylidene--hydroxy)bis(-hydroxycoumarin) compound  has shown the best activity against S. aureus, with the inhibition zone of , mm. The same compound has also shown good activity against B. subtilis, with the inhibition zone of  mm. ,'-(-brombenzylidene)bis(-hydroxycoumarin) compound , the dimer with only bromine in structure at C  , did not have as good activity against Gram-positive bacteria, (inhibition zone of , mm against S. aureus and inhibition zone of , mm against B. subtilis) as compound . Possible explanation lies in the fact that oxygenation of the benzilidene ring contributes to an increase of antibacterial activity (). Already published studies have shown that the introduction of chlorine as a substituent contributes to the antimicrobial activity. Th us, the compound with chlorine at the position C  compound , has shown a good activity against S. aureus with the inhibition zone of , mm, while the activity against B. subtilis, with the inhibition zone of , mm, was signifi cantly weaker. Th e compound with the methoxy group at the position C  compound , shows good activity against S. aureus with the inhibition zone of , mm, while the compound with the same group at the position of C  compound , shows a considerably weaker eff ect (inhibition zone of , mm). Again, this could be explained in terms of oxygenation of the coumarin ring. Another compound that has shown a very good activity against the Grampositive bacteria (S. aureus, inhibition zone of , mm) and against B. subtilis, (inhibition zone of , mm) has dimethylamino group at the position C  compound . Th e tetramer structures have given relatively good inhibition zone against S. aureus, (compound  and compound  with inhibition zone of , mm and , mm). Both tetramers have shown much weaker eff ect against B. subtilis (inhibition zone of , and , mm). Th e obtained results do not suggest that the increase of the size of molecules entails the increase in antimicrobial activity. Th e inhibition zones of most of the analyzed compounds are greater than the inhibition zone of the gentamycin sulphate (inhibition zone of , mm). It is  known that the Gram-negative bacteria are much more resistant than the Gram-positive bacteria. In view of the fact that the Gram-negative bacteria membrane is also more lipophylic than the Gram-positive bacteria membrane, it was to be expected that the compounds, which are lipophylic in their nature, should penetrate the cell membrane of these bacteria. However, the Gram-negative bacteria remained resistant to the analyzed compounds. Th e results of antimycotic activity show that almost all synthesized derivatives have similar activity. However, the dimers with bromine, hydroxy group on benzilidene ring (compound ) and dimethylamino group on benzilidene ring (compound ) (inhibition zones of , and , mm, respectively) have shown the best activity. For other tested compounds (compounds , , ) zones of inhibition of growth of fungus Candida albicans are much smaller (inhibition zone of , mm -, mm, respectively). All tested compounds showed weaker activity than clotrimazole standard (inhibition zone of , mm). Th e compound, which showed the best microbiological activity according to the diff usion method (compound ) was analyzed also by the dilution method. Using the dilution method the same compound showed very good activity in a very small concentration against previously tested microorganisms: ◊ MIC for Staphylococcus aureus of , mg ml - , MBC , mg ml - , ◊ MIC for Candida albicans of , mg ml - , MBC , mg ml - ◊ MIC for Bacillus subtilis of , mg ml - , MBC , mg ml -

Conclusion
Th e tested compounds have shown diff erent activity in terms of growth inhibition of microorganisms. Newly synthesized derivatives have shown antibacterial activities, manifested as growth inhibition of various Gram-positive bacteria (B. subtilis, S. aureus), while the activity against Candida was much weaker. Th e same compound did not show any antimicrobial activity against two Gram-negative bacteria types (E. coli, P. aeruginosa). Th e compound  showed the best microbiological activity when tested by the diff usion method, and was tested also by the dilution method. Obtained results confi rmed its antibacterial and antimycotic activities against various microorganisms.