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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.58 no.4 Concepción Dec. 2013 





1 Departamento de Ciencias del Ambiente, Facultad de Química y Biología, Universidad de Santiago de Chile.
2 Departamento de Química, Facultad de Ciencias, Universidad de Chile
* e-mail:


Several natural compounds containing a coumarin moiety have been reported. They have multiple biological activities, e.g., as scavengers of reactive oxygen species (ROS) and to reduce the inflammatory processes by inhibition of enzymes like COX or LOX.

A study was made of the inhibitory capacity of seven natural coumarins and one derivative of esculetin, against soybean 15-lipoxygenase (15-sLOX). The studied coumarins showed interesting degrees of inhibition compared to quercetin, a known LOX inhibitor. Docking studies of some compounds into the binding site of 15-sLOX and 5-hLOX were also made.

Keywords: Soybean lipoxygenase, human lipoxygenase, coumarin inhibitors of LOX, percent inhibition values, docking.



Lipoxygenases (LOX) are a family of non-heme iron-containing enzymes that catalyse the dioxygenation of polyunsaturated fatty acids in lipids containing a cis,cis-1,4-pentadiene structure, which are classified with respect to their positional specificity of arachidonic acid oxydation and are referred to as 5-, 8-, 12- and 15-LOX.1-3

The biological properties of LOX's have been widely studied because they are involved in the biosynthesis of leukotrienes (LTs) and lipoxins (LPs) which participate in different human pathologies. For example, reticulocyte 15-lipoxygenase (15-LOX-1) has been implicated in colorectal4 and prostate cancer5, whereas platelet 12-lipoxygenase (12-LOX), in pancreatic6, breast7,8 and prostate cancers9,10. The most prominent isoform in LT production is 5-LOX, which also participate in inflammation11.

Due to the great importance of simple, prenyl and oxypentenyl coumarins as anti-inflammatories, considerable efforts have been made to isolate natural compounds and to prepare synthetic derivatives. A few examples of simple natural coumarins are esculetin, daphnetin and fraxetin, recognized as inhibitors of the isoform pro-inflammatory 5-LOX and COX. Simple prenyl coumarins like osthole are moderate and selective 5-LOX inhibitors11. Finally, prenylated coumarins such as auraptene12 and umbelliprenin exhibit remarkable and potent inhibition against 15-sLOX13.

Searching for new LOX inhibitors, we evaluated in-vitro seven coumarins previously isolated in our Laboratories from Haplopappus multifolius14,15 and one diacetyl derivative of esculetin (5), against soybean lipoxygenase (15-sLOX). Some of those compounds presented better inhibitory activity than quercetin, taked as comparison compound. It is important to mention that 15-sLOX assay may be used as preliminary study of 15-hLOX, due to the similarities between both enzymes16.Besides, some structures were docked in the active site of the crystal structure of 15-sLOX. In relation to the internal binding mode inside the 5-hLOX enzyme, the most interesting compound turned out to be 2. The values obtained were related to the structural features present in those molecules.


Melting Points were recorded on a capillary Microthermal instrument and are not corrected. Kinetics were run on a Lambda 25 UV/Vis spectrophotometer. The enzyme 15-sLOX (15-lipoxygenase soybean-P1) was purchased from Cayman Chemical.

Coumarins 1-7 were isolated from leaves of Haplopappus multifolius and their structures had been reported previously14, 15. Compound 8 was prepared from 5, by an acetylation in the common way. 1NMR spectra were obtained on a Bruker Avance DR 400 spectrometer with TMS as internal standard.

Percent inhibition assays

Percent I values were obtained by a modification of a previously described method17 using 25 mM Hepes buffer (pH 7.5) with 0.01% Triton X-100 for 15-s LOX and arachidonic acid as substrate (10μM). Enzyme rates were determined by monitoring the formation of hydroperoxyeicosatetraenoic acid (HPETE) at 234 nm on a Lambda 25 UV/Vis spectrophotometer at 25±0.1 °C. The inhibitors (1 mg/mL in DMSO) were added to the buffered substrate and the reaction was initiated by addition of the 15-s LOX enzyme (10 μL). Control rates were measured using the same volumes of DMSO and of inhibitor. Percent I was determined by measuring absorbance vs time.

Docking studies

All structures were built with the GaussianView software18. Restrained electrostatic potential (RESP) charges were obtained at the B3LYP/6-31G** level of theory employing the Gaussian 03 package19. Docking of inhibitors into the active site of the crystal structure of 15-lipoxygenase soybean-P1 (PDB code 1N8Q) at 2.1 A of resolution was performed with the AutoDock 4 package20 using a Lamarckian algorithm and assuming total flexibility of the inhibitors and partial flexibility for the His residues coordinated with Fe3+ inside the binding site. The grid maps were made up of 60 x 60 x 60 points, with a grid-point spacing of 0.375 A. The AutoTors option was used to define the ligand torsions, and the docking results were then obtained by a ranked cluster analysis, resulting in conformations with the highest overall binding energy (most negative -∆Gbinding value.). Finally, a docking study using human 5-lipoxygenase (PDB code: 3O8Y) at 2.39 A resolution on derivative 2 was obtained.


The structures of all the natural coumarins isolated from Haplopappus multifolius14,15, and the derivative 8, were characterized by spectroscopic methods and comparison with known compounds. The structures of the compounds are shown in Figure 1:

Figure 1: Structures of coumarins and derivative 1-8 and quercetin 9.

1 7-O-prenyl-coumarin; 2 6-hydroxy-7-[(E,E)-3',7'-dimethyl-7'-hydroxy-2',5'-octodienyloxy] coumarin; 3 6-hydroxy-7-[(E,E)-3',7'-dimethyl-5'-hy-droxy-2',6'-
octodienyloxy] coumarin; 4 6-hydroxy-7-prenyl coumarin (pre-nyletin); 5 6,7-dihydroxy (esculetin); 6 7-hydroxy coumarin (umbelliferone; 7 6[p-D-glucopyrasyl]
-7-hydroxy coumarin (esculin); 8 6,7 diacetyl-esculetin. The percent inhibition of all of them was compared with that of quercetin 9, a known LOX inhibitor21.




Table 1 shows % I values. This values show the importance of concentration in relation to % .The best compound was 2 with 34.75 % of inhibition at a concentration of 12.7 μM. 8 and 1 gave the worst values (16.06 and 23,47% of inhibition and 19,1 and 17.4 μM of concentration, respectively). For the sake of comparison, the % I of 2 was better than that of quercetin.

Table 1: Percent inhibition values of 15-sLOX inhibitors 1-9

The results besides show the importance of the presence of the catechol group in the aromatic systems or a location of hydroxyl groups in the geranyl chain .Their type and location accounts clearly for the value of % I of compound 2 compared to quercetin. Compound. 2 has well interaction of alcoholic geranyl group with non-heme iron (Fe3+) Besides a two hydrogen bridges interaction with amino acid residues, due to a flat structure of the aromatic system, causing inhibition of the enzyme. In contrast, in quercetin this rigidity is changed into a B ring that is flexible and whose conformation does not allow good interaction in the active site, and this takes place before the quercetin can degrade to 3,4-dihydroxybenzoic acid, which according to the literature is a potent inhibitor of 15-sLOX21-23.

Going over the whole table, the importance of the catechol group becomes evident, and therefore the loss of that group decreases the action against LOX. This happens when the hydroxyl groups are reduced, as seen with 7-hydroxy-coumarin or in 6,7 diacetyl-esculetin 8. The simple coumarins with a terpenoid chain bonded to the C of benzene or to the O of carbon 7' forming ether group, have not been studied as LOX inhibitors, and neither there are many studies on docking. In our results, O-prenylation of the phenolic hydroxyl causes a drop of the activity. Nevertheless, the inhibition increased in our new compounds 2 and 314,15, with an ether group of ten atoms chain and an aliphatic hydroxyl groups in different locations. Both coumarins are better inhibitors than quercetin. In these compounds is clear that the action of the phenolic hydroxyl group at C-6 is favored by the alcoholic hydroxyl of the geranyl chain ..

Most LOX inhibitors are antioxidants or free radical scavengers, since lipoxygenation occurs via a carbon-centered radical. In other hand, some studies suggest a relationship between LOX inhibition and the ability of the inhibitors to reduce Fe3+ in the active site21. In previous studies, the coumarins ofH.multifolius showed very good antioxidant activity15. Therefore, compounds with antioxidant properties could be estimated to offer protection in rheumatic arthritis and other inflammatory diseases21 and this would be better if the coumarins have phenolic hydroxyl or cathecol groups. D o ckin g results showed that in the potent coumarin 2 the aliphatic hydroxyl is near of Fe3and create hydrogen bridges

When carrying out docking with compounds 5 and 7 it was seen that both structures were located in the bonding site of arachidonic acid. Compound 5 was located at a distance of 3.5 Å from Fe3+ (active form of the enzyme) and the bond energy was -3.51 kcal/mol. In the case of compound 7, it was located in a very similar manner as in compound 5, but since the catechol group no longer exists, instead there is a glucose residue on C-7 (the distance to Fe3+ is 3.3 Å). Some decrease of the activity is because the hydroxyls are unable to join efficiently in the bonding site, and the docking energy drops to positive values of 3.3 kcal/mol. Therefore, suppression ofthe catechol group in compound 7 is a determining factor in the low inhibition decrease

To get an idea of how the best candidate of the coumarin derivatives would behave with the key enzyme in the inflammatory processes, we performed the docking of compound 2 with 15-sLOX and 5-hLOX. The bond energy of 2 at the bonding site with the substrate in 15-sLOX was -0.19 kcal/mol. shown in Figure 2.

Figure 2: Docking of compound 2 with 15- sLOX

At the same time, docking of compound 2 with 5-hLOX gave a bond energy of 3.11 kcal/mol, and the location of the alcoholic hydroxyl of the chain generated two hydrogen bridges with Gln363 and Asn425. Also, the phenolic hydroxyl group at position 6 was located at a distance of 3.82 Å from Fe+3 (see Figure 3), and this may also be due to the fact that the size of the cavity of 5-LOX is greater than that of 15-sLOX, and this would account for the variation of the interaction energy values within the active sites24. Finally, these preliminary % I and docking studies may suggest that compound 2 can have greater affinity for 5-hLOX and may be a good candidate as inhibitor of that enzyme. Finally, this work is in development and doing biological studies against 5-human lipoxygenase of geranyl coumarins and yours derivatives as potents anti-inflammatories.

Figure 3: Docking of compound 2 with 5-hLOX


Financial support from DICYT-UdeSantiago, proyect N° 0211141TG and Fondecyt N° 1120379 are gratefully acknowledged.



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(Received: July 18, 2013 - Accepted: September 4, 2013)

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