SYNTHESIS OF O-ALKYL DERIVATIVES OF DEHYDROZINGERONE ANALOGUES

Vanillin and isobuthyl methyl ketone (4-methylpent a -2-one) reacts under Claisen–Schmidt conditions yielding corresponding d ehydrozingerone analogue, ( E)-1(4-hydroxy-3-methoxyphenyl)-5-methylhex-1-en-3-one. A small series of its O-alkyl derivatives was prepared by alkylation of free phen olic group with corresponding alkyl halides. Products had been tested for their biologi cal activity and demonstrated relatively strong in vitro antimicrobial activity towards different strains o f bacteria and fungi. All new compounds were well characterized by IR, H and C NMR spectroscopy and physical data.


INTRODUCTION
Chalcones, 1,3-diaryl-2-propen-1-ones, are one of the important classes of organic compounds, which have a unique chemical structure with conjugated double bonds and a completely delocalized π-electron system on both aromatic rings.
Starting from this fact we supposed that vanillin is suitable substrate for further transformation, due its easy modification, by O-alkylation [29][30], by coupling reactions and forming of divanillin [31], formylations in position 5- [32].
In continuation of our interest in synthesis of vanillin derivatives we synthesized, starting from vanillin, dehydrozingerone analogue (E)-1-(4hydroxy-3-methoxyphenyl)-5-methylhex-1-en-3-one 2a. Starting from this enone compound several O-alkyl derivatives were synthesized, and all new products were characterized by their spectral data (IR, 1 H NMR and 13 C NMR). Their biological activity toward some strains of microorganisms wave been tested.

General remarks
All starting chemicals were commercially available and used as received, except that the solvents were purified by distillation. Chromatographic separations were carried out using silica gel 60 (Merck, 230-400 mesh ASTM) whereas silica gel on Al plates, layer thickness 0.2 mm (Merck), was used for TLC. IR spectra were recorded on a Perkin-Elmer One FT-IR spectrometer with a KBr disc, ν in cm -1 ; NMR spectra were recorded on a Varian Gemini 200 MHz spectrometer (200 MHz for 1 H and 50 MHz for 13 C), using CDCl 3 as solvent and TMS as the internal standard. 1 H and 13 C NMR chemical shifts were reported in parts per million (ppm) and were referenced to the solvent peak; CDCl 3 (7.26 ppm for 1 H and 76.90 ppm for 13 C). Multiplicities are represented by s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Coupling constants (J) are in Hertz (Hz).
The antimicrobial activity was estimated by determination of the minimal inhibitory concentration (MIC) using the broth microdilution method against five species of bacteria and five species of fungi.

Microorganisms and media
The following bacteria were used as test organisms in this study: Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), B. cereus (ATCC 10987), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). All of the bacteria used were obtained from the American Type Culture Collection (ATCC). The bacterial cultures were maintained on Müller-Hinton agar substrates (Torlak, Belgrade). The fungi used as test organisms were: Aspergillus flavus (ATCC 9170), A. fumigatus (ATCC 1022), Candida albicans (ATCC 10259), Penicillium purpurescens (ATCC 48987), P. verucosum (ATCC 48959). All of the fungi were from the American Type Culture Collection (ATCC). The fungal cultures were maintained on potato dextrose (PD) agar, except for C. albicans that was maintained on Sabourad dextrose (SD) agar (Torlak, Belgrade). All of the cultures were stored at 4°C and subcultured every 15 days.
Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37°C on Müller-Hinton agar substrates and brought up by dilution according to the 0.5 McFarland standard to approximately 10 8 CFU/mL. Suspensions of fungal spores were prepared from freshly mature (3-to 7-day-old) cultures that grew at 30°C on a PD agar substrate. The spores were rinsed with sterile distilled water, used to determine turbidity spectrophotometrically at 530 nm, and were then further diluted to approximately 10 6 CFU/mL according to the procedure recommended by NCCLS (1998).

Minimal inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) was determined by the broth microdilution method using 96-well micro-titer plates [43]. A series of dilutions with concentrations ranging from 20 to 0.004 mg/mL of the tested compounds was used in the experiment against every microorganism tested. The starting solutions of tested compounds was obtained by measuring off a certain quantity of the compounds and dissolving it in 5% dimethyl sulphoxide (DMSO). Two-fold dilutions of the compounds were prepared in a Müller-Hinton broth for bacterial cultures and a SD broth for fungal cultures. The MIC was determined with resazurin. Resazurin is an redox indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. The boundary dilution without any changing color of resazurin was defined as the MIC for the tested microorganism at a given concentration. As a positive control of growth inhibition, streptomycin and ketoconazole was used. A 5% DMSO solution was used as a negative control for the influence of the solvents.
Synthesized compounds 2a-g was well characterized by spectral data and microbiological activity. The tested compounds 2a-c demonstrated relatively strong antimicrobial activity inhibiting all tested microorganisms. The MIC for these compounds relative to the tested microorganisms ranged from 0.009 to 5 mg/mL. The strongest antibacterial activity was found in 2a component, which inhibited all the species of bacteria, especially B. subtilis where measured MIC value was extremely low (0.009 mg/mL). This compound also inhibited the growth of the tested fungi but in slightly higher concentrations (MIC values were from 0.312 to 0.625 mg/mL). Compound 2d inhibited only B. subtilis and B. cereus. Other tested components (2e-g) did not inhibite any of the test microorganisms. Among the bacteria, the highest resistance was shown in E. coli, while the most sensitive was B. subtilis. Among the fungi, the most sensitive appeared to be C. albicans.
The antimicrobial activity was compared with the standard antibiotics, streptomycin (for bacteria) and ketoconazole (for fungi). The results showed that standard antibiotics had stronger activity than tested samples as shown in Table 1. In these experiments, the compounds examined at the same concentrations showed a slightly stronger antibacterial than antifungal activity. These results could be expected due to the fact that numerous tests proved that bacteria are more sensitive to the antibiotic compared to fungi [44]. The reason for different sensitivities between fungi and bacteria can be found in different permeabilities of the cell wall. The cell wall of the gram-positive bacteria consists of peptidoglycans (murein) and teichoic acids, while the cell wall of gram-negative bacteria consists of lipopolysaccharides and lipopoliproteins [45], whereas, the cell wall of fungi consists of polysaccharides such as chitin and glucan [46].
Compounds 2b-c have substituent with short carbon chain on oxygen (Me and Et), whereas compounds 2d-g have longer carbon chain on oxygen (i-Pr, n-Pr, n-Bu and Bz). We suppose that structure of alkyl group is responsible for the lack of their activity.
From this point, the results of this study suggest that dehydrozingerone analogue derivatives 2a-g are promising candidates, after some modification, for testing of some other activities.