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 mo...rtality 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.
