To evaluate the ability of both

To evaluate the ability of both sets of compounds to inhibit the activity of 15-LOX an initial screen was performed, whose results are shown in . The comparison of inhibition values would seem to indicate that, in the HYD series, an electron donor group at the position increases and an electron attractor decreases activity, as is particulary noticeable with methyl and methoxyl substitution. When the central nitrogen-nitrogen bond is oxidized to diazene, this trend seems to be reversed, although the electron-withdrawing cyano group has no apparent effect on the inhibitory activity. This behavior is more evident when the values of inhibition for each HYD and its DIA counterpart are compared. If the inhibitory activity is mediated by hydrogen bond formation in the enzyme’s active site, it may be supposed that there is a correlation between the effect of the electron donor group and the basicity of the secondary amine in the inhibitor’s dinitrogen bridge. This tendency indicates that arylhydrazide derivatives with electron donating aryl groups are better candidates for the design of 15-LOX inhibitors. In contrast, although DIA-4H and DIA-4CN are practically as potent as HYD-4Me and HYD-4OMe, the screening data do not suggest a design strategy for additional diazene analogs, although the smaller size of the cyano group (E=−0.51) as compared to methyl and bromine (E=−1.24 and −1 to 34 respectively) might explain the strong inhibition by DIA-4CN. The most potent 15-LOX inhibitors in our series were chosen to obtain their IC values and kinetic parameters (). The most potent inhibitor of the series (HYD-4Me), shows an IC value five and three times higher than the well-known inhibitors boswellic Fumagillin mg (IC=1μM) and baicalein (IC=1.6μM), respectively, but it is 3.4 times more potent than the commercial 15-LOX inhibitor 4-methyl-2-(4-methylpiperazinyl)-pyrimido[4,5]benzothiazine. The diazene derivatives DIA-4H and DIA-4CN inhibit 15-LOX to a somewhat lesser extent but in the same range as HYD-4Me. Even though the IC values of these compounds lie far from the desirable nanomolar range, they incorporate good molecular scaffolds that could be used to introduce a broad variety of structural modifications taking advantage of their simple synthesis and the ready availability of many differently substituted benzoic acids and arylhydrazines. To study the type of inhibition induced by both series of compounds, HYD-4Me and DIA-4CN were selected (). The Lineweaver-Burk plots indicate that the inhibition was competitive for both derivatives, suggesting that these inhibitors compete with arachidonic acid to reach the active site without affecting the theoretical maximum rate of conversion to product. The active site of this oxidoreductase enzyme has a non-heme iron atom which plays a key role in the introduction of a hydroxyl group at the C-15 position of the fatty acid by generating the allyl radical in the fatty acid chain and subsequently stabilizing the hydroperoxide radical formed by addition of molecular oxygen. With these results, it is possible to hypothesize that the inhibitor blocks the access of arachidonic acid to the active site by generating specific interactions with the iron and/or an amino acid residue present in the active site of 15-LOX, lowering the K. As an approach to understand the likely molecular interactions in the environment of the metal we built molecular models of possible complexes with the inhibitors (). Our results indicate that both inhibitors are able to reach the active site of 15-LOX, which is in agreement with the competitive inhibition found by kinetics experiments. A detailed analysis of the models shows HYD-4Me establishing basically two different interactions. One of these arises between the aromatic amine NH group and the iron atom (3.6Å). The second interaction found is a hydrogen bond (2.3Å) between the hydrogen of the amide NH group and the carboxyl of the C-terminal amino acid residue Ile-839. It is interesting to note that if hydrogen bonding contributes to the strength of binding in the active site, and/or if subsequently this capability is enhanced by electron donor substituents at the position of the phenyl ring, it would be possible to determine some grade of correlation between the presence of the substituents and the effective acidity parameter of Abraham, which indicates precisely the ability to donate a hydrogen atom toward a hydrogen acceptor. In order to evaluate which interaction would be mainly responsible for the inhibition, the series of 4-substituted anilines proposed by Abraham was taken as a model due to their structural analogy with the phenylhydrazide derivatives of this work. In the aniline series, higher values of acidity are observed for 4-chloroaniline (0.30) and lower values for 4-methoxy- and 4-methylaniline (0.23), which means that electron donor groups weaken the hydrogen donating ability. This tendency is opposite to the behavior observed in the HYD series, which seems to suggest that the higher affinity for the active site of the phenylhydrazides with electron donor groups obeys mainly to complex formation between the NH group and iron rather than hydrogen bond formation between –N′H- and Ile-839.