J. Chil. Chem. Soc, 52, N° 4 (2007), págs: 1330-1331

REGIOSELECTIVE SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF O-ALKYLATED PHYSCION'S DERIVATIVES

¹N. MANOJLOVIC* ²Z. MARKOVIC AND ²M.DURIC

¹Department of Pharmacy, Medical Faculty, University of Kragujevac, S.Markovica 69, 34000 Kragujevac, Serbia
²Faculty of Agronomy, University of Kragujevac, 32000 Cacak, Serbia

ABSTRACT

Regioselectivity in the reaction of microwave promoted O-alkylation of physcion in the presence of montmorillonite clay K-10 was discussed. The mechanism of formation of corresponding anions of physcion was investigated using semiempirical molecular-orbital methods. The results of antibacterial and antifungal activity of the obtained derivatives showed that the introduction of different substituents (OCF£₃, OCH₂C(CH₃)=CH₂, OCH₂CH₂CH₃ and OCH₂C₆H₅), at the C-1 and C-8 atoms led to a remarkable change in their antimicrobial activity.

Key words: physcion, O-alkylation, regio selective synthesis, antimicrobial activity

1,8-Dihydroxy-3-methoxy-6-methyl-9,10-anthraquinone (physcion) has expressed certain moderate biological activities including antibacterial, antifungal and purgative activities¹'⁶. Physcion could be isolated from a variety of sources like Xanthoria and Parmelia (lichens), Aspergillus (fungus) and Cassia, Chrusarobinium, Rhamnus, Rheum, and Rumex (higher plants)¹.

Our recent studies⁷ have shown the O-alkylation of physcion with different alkyl halides (methyl iodide, propyl bromide, 3-chloro-2-methylpropene and benzyl bromide), by using microwave promoted reaction in the presence of montmorillonite clay K-10 as a catalyst⁸. The first part of this paper deals with regioselectivity in above-mentioned reactions. The second part describes the antifungal and antibacterial activity of physcion's derivatives obtained.

The mechanism of formation corresponding anions of physcion (I) was investigated using semiempirical molecular-orbital methods PM3 including the program package MOP AC 7.0⁹. Although, it is well known, that AMI method is giving good results for geometries and energies for similar class of compounds^10-11. This method is not suitable for evaluation of transition states. For this purpose, we used PM3 method^10-12 for optimizing all the structure in neutral form and all possible ionic forma as well as transition states. Our reason for this decision lies in the fact that PM3 method is more suitable for determination geometry and energy of transition state¹⁰. Stable geometries were fully optimized from approximate starting geometries, and the absolute energy minimum was calculated, for each compound, exploring the angles defined by orientation of the hydro xyl and methoxy groups. The transition states for those reactions were found using appropriate MOP AC facilities (TS, SADDLE). Eventually, the geometries of the transition states were refined by means of Bartel's method (non-linear least squares gradient minimization routine) and then checked by means of vibrational analyses for the existence of a single 'negative' vibration.

All molecular structures were optimized according to the PM3 method in vacuum. For the geometry optimization in a polar medium, we have modeled the solvent as a dielectric continuum (COSMO model)^13-14 with dielectric constant for acetone 35.9. The solvent is treated as a perturbation on the gas phase system. Significant difference between optimized structure in gas phase and in the solution has been conformed. In the molecule of physcion OH anion can attack (Fig. 1) any of the two OH bond. These two possible attacks of the OH anion (a, b) are considered as the first step of the substitution reaction. Our calculations show that OH anion can react with OH groups in molecule of physcion via transition states TS 1 and TS2, their geometries are presented in Fig. 2. In this reaction the intermediate anions^15,16 are formed (1a^- and 1b^-), their optimized geometries are presented in Fig. 2.

In the next step of the reaction, the electrophilic alkyl group, attacks oxygen anions giving mono-products la and lb (Fig 1). Formation of di-O-alkylated product as well as the corresponding di-anion is not considered. For conversions 1 in the corresponding anions Ia^-and Ib^-, the activation energies of 7.62 and 12.01 kJ/mol, respectively, are found in gas phase, while values 25.40 and 27.36 kJ/mol respectively are found in acetone. According to the Curtin-Hammond principle, the product distribution is determined by the difference of free energies of the two transition states. The 1.05 kcal/mol difference in free energy of the first and second anion products results in about 5.9:1 ratio of the product concentrations in gas phase, on the other side 1.97 kJ/mol there is difference in free energy in acetone, that results in about 2.2:1 ratio of the corresponding product concentrations. The result obtained for reactions performed in acetone is in good agreement with experimental ratio of formation corresponding mono O-alkylated products of physcion.

All of the physcion's derivatives (I_a1-I_a4) were screened for their antimicrobial activities against Aspergillus niger, Doratomyces stemonitis, Trichoderma viride, Penicillium verrucosum (fungi) and Bacillus subtilis, Bacillus mycoides (Gram positive bacteria), Pseudomonas fluorescens and Pseudomonas phaseohcola (Gram negative bacteria). The disc diffusion method⁵ was used for the screening of antifungal (300 µg/disk) and antibacterial activity (200 µg/disk) of the anthraquinone derivatives (I_a1, I_a2 and I _a3). The inoculum used for all the assays reached the microbial density of 10⁶-10⁷ CFU/ml for the microorganisms tested. One hundred micro liter of test organisms grown in nutrient broth media (10⁶ cfu/ml) were spread overl thesurface of sterile Mueller-Hinton agar (for the bacteria) and Sabouraud Dextrose agar (for the fungi) in 9 cm diameter petri dishes. After incubation at 37⁺ 1°C for 18-24 h for bacteria and 10 days at room temperatures for fungi, the diameters of inhibition zone were measured in mm. Nystatin (100 µg/disc for antifungal testing) and penicilline G (50 µg/disc for antibacterial testing) were used as positive controls.

The results and the statistical analysis are summarized in Tables 1 and 2.

These physcion's derivatives exhibited a moderate antifungal activity against both fungi and bacteria tested, at least compared to the positive control. The results of antibacterial and antifungal activity of the obtained derivatives showed that the introduction of different substituents (OCH₃, OCH₂C(CH₃)=CH₂, OCH₂CH₂CH₃ and OCH₂C₆CH₅ at the C-1 and C-8 atoms led to a remarkable change in their antimicrobial activity. Among them, compounds I and I containing 2-methyl-propenyloxy moiety and benzyloxy moiety, respectively, displayed strong activity against the four fungi tested. As shown in Table 2, antibacterial activity of physcion's derivatives was mainly decreased compared to the physcion.

In conclusion, the antifungal activity was better with the derivatives than with physcion. The derivatives with large substituents improved the activity. On the other hand, antibacterial activity was not improved and physcion exhibited the highest activity. On the basis of theoretical results and experimental studies, it can be concluded that microwave promoted O-alkylation of physcion in the presence of montmorillonite clay K-10, is better than the conventional method⁷ in terms of regioselectivity and produces the anthraquinone derivatives whose antimicrobial activities were tested for the first time.

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e-mail: nmt@kg.ac.yu