Design of bioavailable derivatives of 12-(3-adamantan-1-yl-ureido)dodecanoic acid, a potent inhibitor of the soluble epoxide hydrolase

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Abstract

The soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in blood pressure regulation and vascular inflammation. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA, 1) is a very active inhibitor of sEH both in vitro and in vivo. However, its relatively high melting point and limited solubility in either water or oil-based solvents leads to difficulties in formulating the compound and often results in poor in vivo availability. We investigated the effect of derivatization of the acid functional group of inhibitor 1 on the inhibition potencies, physical properties, and pharmacokinetic properties. For human sEH, similar inhibition potency was obtained when the acid of compound 1 was modified to esters (215). The resulting compounds exhibited improved physical properties (23–66 °C lower melting point and 5-fold better solubility in oil). Pharmacokinetic studies showed that the esters possess improved oral bioavailability in mice. On the other hand, amide derivatives of AUDA 1 did not show significant improvement in inhibition potencies or physical properties (higher melting points and lower solubility). The esterification of 1 results in compounds that are easier to formulate in animal food and in triglycerides for gavage and other routes of administration, making it easier to study the biological effects of sEH inhibition in vivo.

Introduction

In mammals, the soluble epoxide hydrolase (sEH) is involved in the metabolism of endogenous mediators such as epoxides of arachidonic acid,1, 2, 3 linoleic acid,4 and other lipid epoxides.5, 6 Epoxides of arachidonic acid (epoxyeicosatrienoic acids or EETs) are effective regulators of blood pressure7 and have anti-inflammatory properties in vivo.8, 9 However, the dihydroxyeicosatrienoic acids (DHETs), the sEH hydrolyzed metabolites of the EETs, often have reduced biological activity, and are more water soluble and more easily conjugated.7 The blood pressure of spontaneous hypertensive and angiotensin II induced hypertensive rats treated with effective sEH inhibitors is dramatically reduced.10, 11, 12, 13 In addition, the EETs have further vascular protective effects such as anti-inflammatory properties in endothelial cells,14, 15, 16, 17 suppression of reactive oxygen species following hypoxia-reoxygenation,18 attenuation of vascular smooth muscle migration,19 and enhancement of a fibrinolytic pathway.20 In cellular and animal models, the EET-mediated regulation of hypertension and cardioprotective effects are dependent in part on the extent of epoxide hydrolysis by sEH,4, 21 suggesting that the inhibitors of the sEH are worth exploring as pharmaceuticals for the treatment of hypertension, inflammation, and other disorders that can be induced by changing the in vivo concentration of EETs.

1,3-Disubstituted ureas and related compounds are very potent and stable inhibitors of sEH. These compounds efficiently reduce epoxide hydrolysis in several in vitro and in vivo models.11, 12, 22 However, limited solubility in either water or organic solvents and high melting points of some of these inhibitors likely affect their in vivo efficacy and make formulation difficult.10, 23 Toward solving these problems, we previously showed that a polar functional group on one of the alkyl chains of the urea inhibitors improved solubility in water 2-4-fold while retaining inhibition potency.24 A polar group such as an ester, alcohol, ether, or ketone located on the fifth/sixth atom from the carbonyl group of the urea function, or an acid function present on the thirteenth atom from the urea pharmacophore were both effective for making soluble inhibitors in either water or oil-based solvents often without a drop in inhibition potencies.23, 24 These functional groups incorporated into lipophilic compounds are also useful in improving the binding selectivity of the inhibitors to the enzyme possibly by establishing additional hydrogen bonding.25, 26 Such compounds, for example, 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), were used to inhibit sEH in both cultured cells and animals.27, 28, 29 However, AUDA is difficult to formulate, and was administered with a co-solvent (up to 10% of DMSO),28, 29 or in the drinking water with a sizable amount of 2-hydroxypropyl β-cyclodextrin.27 Unless AUDA and similar compounds are in true solution when administered to animals, the availability is very low. Although a valuable improvement in the solubility was obtained from the inhibitors functionalized with an acid group or another polar group, their relatively high melting points (>100 °C) still limited solubility and speed of dissolution in both water and oil-based solvents, leading to relatively poor observed in vivo oral availability.23, 24, 27, 28 Therefore, in the present study, we investigated modifications of the acid function of AUDA on the inhibition potency and physical properties (e.g., melting point and solubility) with the goal of improving the availability of AUDA and its pharmacokinetic parameters in mice. We explored two kinds of modification: making alkyl esters to obtain compounds more soluble in oil, and various polar amides to obtain compounds more soluble in water.

Section snippets

Synthesis

While the synthesis of compound 1 (12-(3-adamantan-1-yl-ureido)dodecanoic acid; AUDA) was previously described,24 we report herein a simplified (see Scheme 1), more efficient method of producing this compound. Reaction of 1-adamantyl isocyanate with 12-aminododecanoic acid in 1,2-dichloroethane provided compound 1 in 95–100% yield. However, when this reaction was performed in DMF or THF, approximately 20% of the yield was 1,3-diadamantylurea, a crystalline by-product that has also been reported

Conclusion

This investigation focused on producing potent and bioavailable inhibitors of human sEH by the chemical modification of the acid function of compound 1. We found that esterification of the acid function of compound 1 did not change the inhibition potency for either murine or human sEHs. Further, the esterification reduced the melting point up to 66 °C and improved solubility in oil (Table 2). This increased solubility makes the resulting compound easier to formulate in oil for oral gavage,

Syntheses

All melting points were determined with a Thomas-Hoover apparatus (A.H. Thomas Co.) and are uncorrected. Mass spectra were measured by LC-MS/MS (Waters 2790) using positive mode electrospray ionization. Elemental analyses (C, H/N) were performed by Midwest Microlab (Indianapolis, IN); analytical results were within ±0.4% of the theoretical values for the formula given unless otherwise indicated (see Supplementary data for detailed results). 1H NMR spectra were recorded on QE-300 using

Acknowledgments

This work was supported, in part, by NIEHS Grant R37 ES02710, NIEHS Center for Environmental Health Sciences P30 ES05707, NIH/NIEHS Superfund Basic Research Program P42 ES04699, NIH/NHLBI R01 HL59699-06A1, and UCDMC Translational Technology Research Grant.

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    Present address: Food and Drug Safety Center, Hatano Research Institute, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan.

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