The phylogenetic relationship demonstrates that both bovine

The phylogenetic relationship demonstrates that both bovine and porcine 12/15-LO is more related to the human enzyme than the rabbit enzyme, despite the fact that the rabbit reticulocyte enzyme is a 15-lipoxygenase. The rabbit is one single species that expresses a 12/15 LO with 15-LO activity while in its most related species the corresponding enzyme has 12-LO activity. In fact, not even all primates express an enzyme with 15-LO activity. This seems to be restricted to the hominids which are the members of the biological family Hominidae including humans, chimpanzees, gorillas and orang-utans. If the restricted expression of a 15-LO-1 enzyme in hominids can be coupled to formation of eoxins or certain airway inflammatory diseases, such as asthma, in these species remains to be investigated. The expression of the second form of 15-LO, i.e. 15-LO-2, in rhesus and orang-utan has in contrast to human cells [35], [36], so far not been described. The relevance for the difference between man and macaca in using anandamide as a substrate is presently unclear but it might indicate differences in metabolism of endocannabinoids in these two species. In conclusion, we identify in this report the molecular nature of the 12- and 15-lipoxygenases in M. mulatta and P. pygmaeus and also that the expression of the rhesus enzyme in lung epithelial cells is regulated by interleukin-4. The switch from a mainly 12-LO enzyme to a mainly 15-LO enzyme during evolution indicates that the 15-lipoxygenating activity of the enzyme in higher primates is of importance for the function of this enzyme. Also, our results show that 12/15-LO from primates can have distinct differences in using endocannabinoids as substrate.
Acknowledgement
Lipoxygenases are a well-studied class of non-heme, iron containing oxidative enzymes that insert molecular oxygen into long-chain unsaturated fatty acids and derivatives thereof having a –-1,4-pentadiene motif. The human 15-lipoxygenase enzyme has its name due to the insertion of oxygen at position 15 of arachidonic X-Gal ((5,8,11,13)- 5,8,11,14-eicosatetraenoic acid) to give 15()-hydroperoxy-5,8,11,13)-5,8,11,14-eicosatetraenoic acid (15-HPETE) and exists in two variants, 15-LOX-1 and 15-LOX-2.Several lines of evidence indicate that inhibition of 15-LOX-1 can be effective in the treatment of respiratory inflammatory diseases, in particular asthma., In the preceding Letter in this series we described the discovery and synthesis of 1-substituted pyrazole-3-carboxanilides as potent and selective inhibitors of 15-LOX-1 (). Unfortunately, but not unexpectedly, the propensity of the pyrazole acting as a leaving group made compounds with an alkyl or an aryl group linked to the 1-nitrogen of the pyrazole, via a carbonyl or a sulfonyl group, were chemically unstable. Initial studies demonstrated that the intermediates in the syntheses, that is, pyrazoles lacking 1-substituents (, ) where at least as active as the desired products. Due to a complex patentability landscape, they were at that point not further investigated, but when we realized that the chemical stability issue could not be solved, all efforts in the development program were redirected to 1--pyrazoles. This Letter will describe how the synthetic program evolved into the development of clinical candidates based on pyrazol-3-carboxanilides and triazole-4-carboxanilides. The synthesis of the carboxanilides from pyrazole-3-carboxylic acids is described in the preceding Letter and in the patent literature, which also contains experimental details., , , , , When pyrazole-3-carboxylic acids () are readily available, the preferred route for the synthesis of is the one depicted in , steps a and b. Simply heating in SOCl with a catalytic amount of DMAP (4-dimethylaminopyridine) gives the diketopiperazines . After evaporation of the SOCl, crude is obtained which is used as such in the following step, where it is treated with the appropriate aniline. This reaction is preferably performed by heating with the aniline in the presence of DMAP in CHCl, or neat with an excess of the aniline. Compounds can also be obtained directly from and an aniline using a coupling reagent, but although not optimized, in a lower total yield. In some cases pyrazole carboxylic acids esters () are more readily obtainable than the acids themselves. Although the esters were readily hydrolyzed, we later developed a more direct route to () involving a trimethylaluminium-mediated amidation (, step d).