Structure–activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase
Graphical abstract
Introduction
Arachidonic acid, a ω-6 polyunsaturated fatty acid, plays important roles in cellular signaling as a lipid second messenger and is also a precursor in the production of oxidative metabolites known as eicosanoids by enzymes such as cyclooxygenase, lipoxygenase, and cytochrome P450. Prostanoids1, 2 and leukotrienes3 are major metabolic products of arachidonic acid by cyclooxygenase and lipoxygenase, respectively. These two pathways are largely inflammatory and induce inflammation, pain, and asthma,1, 2, 3 making the both enzymes current pharmaceutical targets for relief from the symptoms. The third branch of arachidonic acid cascade involves oxidation by cytochrome P450 to produce several inflammatory hydroxylated metabolites and the corresponding lipid epoxides formed at the olefinic centers and known as epoxyeicosatrienoic acids (EETs). EETs have been reported as a new class of lipid mediators with important biological functions.4 The endogenous epoxy lipids, EETs, influence blood pressure by modulating cardiac output, vascular resistance, and urinary composition.5, 6, 7, 8, 9, 10, 11 In addition, vascular inflammation and pain are modulated by the action of EETs.9, 10 However, the metabolism of the epoxy functionality of the EETs to the vicinal diols by soluble epoxide hydrolase (sEH) dramatically diminishes the biological activities.6 Many reports have shown that the treatment of potent human sEH inhibitors increases EET levels and reduces blood pressure and inflammatory responses in in vitro and in vivo experimental models,5, 6, 7, 8, 9, 10, 11 suggesting that human sEH is a promising pharmacological target for the treatment of cardiovascular and other diseases.
A number of urea compounds with a variety of substituents are highly potent inhibitors of the human sEH.12, 13, 14, 15, 16, 17, 18, 19, 20, 21 The best optimization of urea derivatives affords specific inhibition potency for the target enzyme in a range of less than 1 nM. Structure–activity relationship studies indicate that a carbonyl group and a single proton donating NH group of urea function are essential for making it an effective primary pharmacophore to inhibit the enzyme activity. Functionalities such as amides and carbamates with both a carbonyl group and an NH group are, therefore, known to produce potent inhibition as a primary pharmacophore, while ester or carbonate functions without a proton donating NH group yield no inhibition for the target enzyme.12, 22, 23, 24 Many of these compounds are difficult to formulate because they are high melting liphophilic solids. These formulation problems can be solved by reducing the melting point and crystal stability, increasing water solubility, and increasing potency. On the other hand, when a variety of functionalities including amides, esters, ketones, and ethers are incorporated as a secondary pharmacophore remote from the catalytic site in potent urea inhibitors, dramatic changes in inhibition potency are not observed, rather significant improvement in physical properties is often obtained,12 implying that primary inhibition of the human sEH depends on the structure of primary pharmacophores and secondary pharmacophores are useful for improving physical properties and potency. In the present study, we first investigated replacement of the primary pharmacophore with a series of substituted oxyoxalamides and then used oxyoxalamides as a second series to replace the secondary pharmacophore using the classical amide and urea primary pharmacophores. In both series, potent compounds were found with improved water solubility.
Section snippets
Chemistry
Substituted oxyoxalamide derivatives (3–15) and N-(benzyloxy)-2-(adamant-2-ylamino)acetamide (16) in Table 1, Table 2 were synthesized as outlined in Scheme 1. Ethyl (chlorocarbonyl)formate was reacted with an alkyl- or a cycloalkyl-amine (Scheme 1A) or adamant-2-ylamine (Scheme 1B) in dichloromethane, followed by hydrolysis with 1 N NaOH in ethanol to provide the corresponding (carbamoyl)formic acid in approximately 80–95% yield. The formic acid was then coupled with benzyloxyamine (Scheme 1A)
Conclusions
This study investigated whether the oxyoxalamide function works as an effective primary and/or secondary pharmacophore to inhibit the human sEH. In order to first see its potential to be a primary pharmacophore, a series of oxyoxalamides substituted with alkyl, cycloalkyl, aryl, or substituted aryl groups were synthesized (Table 1 and Fig. 2A). The inhibition results indicated that a 2-adamantyl group (6) is the most effective left side substituent of the oxyoxalamide function for producing
Chemistry
Unless otherwise noted, all materials were purchased from commercial suppliers and used without further purification. Purity and characterization of compounds were established by a combination of TLC, LC–MS, melting point, and NMR analysis described below. All melting points were determined with a Stuart SMP3 apparatus (A.H. Thomas Co.) and are uncorrected. 1H NMR spectra were recorded on a Digital Avance 400 MHz spectrometer (Bruker Analytik GmbH), using tetramethylsilane (TMS) as an internal
Enzyme preparation
Recombinant human sEH was prepared by using baculovirus expression system as previously reported.25 Briefly, Sf9 insect cells were infected by recombinant baculovirus harboring human sEH gene fused with a 6xHis tag. At 72 h post-infection, the infected cells were homogenized and the recombinant protein was purified by immobilized metal affinity chromatography. After removing the 6xHis tag using the tobacco etch virus protease, human sEH was further purified by anion-exchange chromatography.
IC50. assay conditions
Acknowledgments
This work was supported by Hyundai Pharm Research Grant (HOB-024). Partial support was from NIEHS Grant R01 ES002710 and a Grant-in-aid for Young Scientists (B) 23710042 from Japan Society for the Promotion of Science.
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