Elsevier

Bioorganic & Medicinal Chemistry

Volume 22, Issue 3, 1 February 2014, Pages 1163-1175
Bioorganic & Medicinal Chemistry

Structure–activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase

https://doi.org/10.1016/j.bmc.2013.12.027Get rights and content

Abstract

We explored both structure–activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.

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 (315) 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.

References and notes (26)

  • D.R. Robinson

    Am. J. Med.

    (1983)
  • S.B. Miller

    Semin. Arthritis Rheum.

    (2006)
  • S.L. Pfister et al.

    Adv. Pharmacol.

    (2010)
  • I.-H. Kim et al.

    Bioorg. Med. Chem.

    (2007)
  • T. Kasagami et al.

    Bioorg. Med. Chem. Lett.

    (2009)
  • P.D. Jones et al.

    Bioorg. Med. Chem. Lett.

    (2006)
  • S.-K. Anandan et al.

    Bioorg. Med. Chem. Lett.

    (2010)
  • H.C. Shen et al.

    Bioorg. Med. Chem. Lett.

    (2009)
  • H.Y. Lo et al.

    Bioorg. Med. Chem. Lett.

    (2010)
  • I.-H. Kim et al.

    Bioorg. Med. Chem. Lett.

    (2012)
  • K. Nishi et al.

    Protein Expr. Purif.

    (2010)
  • M. Peters-Golden et al.

    Clin. Exp. Allergy

    (2006)
  • J.D. Imig et al.

    Hypertension

    (2002)
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