Farmakohemijski aspekti delovanja odabranih 4-fenil hidroksikumarina-integrisana in vitro i komjuterska studija

Abstract

Introduction. Coumarins are widely present in the plant’s kingdom and represent a class of bioactive molecules with different functional groups attached to the basic coumarin structure which can be defined as a lactone where benzene ring is connected to α-piron. It was confirmed that molecules with hydroxyl groups attached to the basic coumarin structure exhibit antioxidant activity since they have the ability to react with free radicals. Coumarins are also considered as potential antibacterial agents which inhibit the ATPase activity of bacterial DNA gyrase. Results from a large number of studies have shown that the spectrum of biological activity of coumarins includes anticancer, anti-inflammatory, antibacterial, anticoagulant and immunomodulatory activity. One of the major goals of medicinal chemistry is to find new molecules with the antioxidant activities, which are capable of reacting with free radicals and neutralize their harmful effects. On the other hand, the morbidity and mortality caused by infectious diseases are still a major health issue. The phenomenon of bacterial resistance to existing antibiotics is one of the biggest problems of today's medicine so that rational use of antibiotics in clinical practice and the development of new antibacterial agents with a broad spectrum of activity present contemporary scientific challenges. The isolation of natural compounds and their application as a leading structure for the chemical modification and further pharmacochemical activity improvement, pharmacophore identification and the establishment of quantitative structure activity relationship (QSAR) parallel with in vitro and computer studies are the subject of modern research. Neoflavones present a group of natural compounds with the basic structure of 4- phenylcoumarin. 4-phenyl hydroxycoumarins have a phenyl group at position 4 and hydroxyl groups attached to the basic coumarin structure and coumarins selected for the study in this doctoral dissertation are: 7-Hydroxy-4-phenyl coumarin (7C), 5,7-dihydroxy-4-phenyl coumarin (5,7C), 7,8-dihydroxy-4-phenyl coumarin (7,8C). Aims of this research were to determine the antioxidant activity of selected 4-phenyl hydroxycoumarins, to establish a relationship between the structure and antioxidant activity with the application of DFT descriptors and to determine the mechanism of antioxidant action on the basis of physico-chemical parameters obtained with DFT calculations, to investigate the antibacterial activity and to establish a correlation between the potential antibacterial activity and the binding affinity of the studied molecules to the active site of bacterial enzymes with the application of computational molecular docking methods, to establish a QSAR models for the studied coumarins’ selected pharmacochemical activity and to assess the physico-chemical and molecular properties in order to predict the bioavailability of the studied 4-phenyl hydroxycoumarins. Methods. For the determination of antioxidant and antibacterial activity of selected 4-phenyl hydroxycoumarins in vitro methods have been used. DPPH•, ABTS•+, ferric thiocyanate method, FRAP and CUPRAC were applied for the determination of antioxidant activity. In addition to the studied compounds, for comparison, the antioxidant activities of commercial synthetic antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trolox, and α-tocopherol) were determined and used further as a control group. The significant differences between the antioxidant activity of studied coumarins and standard antioxidants was determined with the application of Dunett’s test (p<0.05 was considered as significant and p<0.01 as a very significant difference). Microdilution method was applied for the determination of antibacterial activity. Modern computer (in silico) methods (DFT, molecular docking, QSAR) are applied for establishing the correlation between the structure and the activity of the studied compounds and to determine the mechanisms by which this correlation is realized. In order to gain insight into the possible mechanisms of antibacterial and anti-HIV activities studied 4-phenyl hydroxycoumarins were subjected to docking simulations. S. aureus tyrosyl-tRNA synthetase, E. coli topoisomerase II DNA gyrase β and HIV-1 integrase have been chosen as target enzymes for the study of inhibitory activity. Results. 7,8C showed significantly higher antioxidant activity (p <0.01) in comparison to standard antioxidants determined with DPPH• and ABTS•+ method, while 7C and 5,7C didn’t show that activity. All tested molecules showed inhibitory effect on lipid peroxidation. After 60 h reaction time the percentage of the inhibition of lipid peroxidation (with 95% confidence interval) was: 56.61 (52.67 to 60.55)% for 7C, 82.48 (75.24 to 89.72)% for 5,7C and 90.939 (88.542 to 93.324)% for 7,8C. Of all the studied coumarin molecules only 7C did not show reducing power determined by FRAP and CUPRAC methods. The slope values (with a confidence interval of 95% and a correlation coefficient r2), which are directly proportional to reducing power were 0.0068 (0.00578-0.00777), 0.929 for 5,7C and 0.0269 (0.0235-0.0303), 0.985 for 7,8C determined with FRAP method and 0.0314 (0.0275-0.0354), 0.994 for 5,7C and 0.059 (0.0548-0.06316), 0.99924 for 7,8C determined with CUPRAC method. The calculated values for the DFT descriptors (HOMO, LUMO, the energy difference, IP, ΔEiso and dipole moment) for the studied coumarins in vacuum, n-octanol, ethanol and water show that of all studied 4-phenyl hydroxycoumarins 7,8C has the highest antioxidant activity, which is in agreement with obtained experimental data. The analysis of the parameters obtained with computational calculations indicate that the HAT is thermodynamically dominant mechanism in the gas phase, while in other studied systems, the most likely mechanism is SPLET. The obtained results for 7,8C show that the thermodynamically favored position of the hydroxyl group in the reactions involving BDE, PDE and ETE parameters is 8 and in the reactions which include the PA parameter is position 7. For 5,7C in the gas-phase favored position of the hydroxyl group in all mechanisms is position 5. In other studied systems, in the reaction mechanisms involving thermodynamic parameters BDE, PDE and ETE position 5 is favored, while in the mechanism that include parameter PA the position 7 is favored. It was determined that studied coumarins show better inhibitory effect against Gram (+) bacteria (MIC values range was from 0.01 to 2.50 mg/mL) in comparison to the Gram (-) (MIC values range was 0.16-10 0 mg/mL). 5,7 C and 7,8C show the weakest inhibitory effect against P. aeruginosa and P. mirabilis, while 7C even at the highest tested concentration of 10 mg/mL didn’t show inhibitory effect. All studied coumarins had the strongest inhibitory effect on the growth of strains of S. aureus, L. monocytogenes, L. innocua and M. luteus. The lowest activity among studied coumarin had 7C. 5,7C showed better inhibitory effect on the majority of the tested strains in comparison to 7,8C. For the interpretation of these results, data obtained by molecular docking studies of the studied compounds for the active site of characteristic enzymes were used. MolDock score values for the binding of the studied 4-phenyl hydroxycoumarins to the active site of tyrosyl tRNA synthetase demonstrated that 5,7C has the highest binding affinity, while 7,8C showed the highest binding affinity to the active site of topoisomerase II DNA gyrase β. Hbond values for the tyrosyl tRNA synthetase showed that 7,8C has the strongest interaction, whereas for the topoisomerase II DNA gyrase β 5,7C has strongest interaction. According to all Score values 7C shows the weakest interactions with the enzymes. Best QSAR models for the inhibition of HIV-1 integrase by a coumarin molecules have the following statistical parameters - for the 3' Processing activity: R2 = 0.9980 and Q2 = 0.9977 for the training set, and R2 = 0.9788 for the test set and for the Integration activity: R2 = 0.9999, and Q2 = 0.9998 for training set and R2 = 0.9213 for the test set. QSAR models are applied to the studied 4- phenyl hydroxycoumarins for calculation and evaluation of their inhibition activities on HIV- 1 integrase. All the obtained score values for the studied coumarins binding to the active site of HIV-1 integrase have shown that 7,8C has the highest binding affinity. Calculated physico-chemical and molecular characteristics showed that the investigated 4-phenyl hydroxycoumarins obey Lipinski rules. Conclusion. 7,8C showed a much more pronounced antioxidant activity in comparison to 7C and 5,7C which indicates that the ortho position of the hydroxyl groups in 4-phenyl hydroxycoumarins significantly contributes to their antioxidant activities. HOMO and LUMO energies and their energy difference of 4-phenyl hydroxycoumarins examined in this study also indicate that 7,8C has the highest antioxidant activity. Between 5,7C and 7C better antioxidant activity has 5,7C. ΔEiso values indicate that 7,8C has the greatest ability to capture free radicals. Total spin density indicates that for all examined hydroxyl groups least favored position in the basic structure of the 4-phenylcoumarins for free-radical reactions is position 7. DFT descriptors as well as obtained experimental data confirm that 7,8C has the highest antioxidative activity in comparison to other studied coumarins. HAT is thermodynamically dominant mechanism in the gas phase, while in other studied systems, the most likely antioxidative mechanism of action of studied coumarins is SPLET. The antibacterial activity of the compounds can be summarized in the following sequence: 7C<7,8C<5.7C. The molecular docking was applied for the evaluation of antibacterial activity on the basis of binding affinity of the studied coumarins in the active sites of tyrosyl tRNA synthetase (S. aureus) and topoisomerase II DNA gyrase β (E.coli). Based on the score values obtained with molecular docking, the binding affinity of the investigated molecules was determined and these results are in agreement with the results of their antibacterial activity. QSAR method was successfully applied for building of a mathematical model for calculating the anti-HIV activity of coumarin molecules on the basis of inhibition of HIV-1 integrase. All studied 4-phenyl hydroxycoumarins obey the Lipinski rules indicating their satisfactory pharmacokinetic profile.