Incorporation of piperazino functionality into 1,3-disubstituted urea as the tertiary pharmacophore affording potent inhibitors of soluble epoxide hydrolase with improved pharmacokinetic properties

Abstract

The inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertension, inflammation and other disorders. However, the problems of limited water solubility, high melting point and low metabolic stability complicated the development of 1,3-disubstituted urea-based sEH inhibitors. The current study explored the introduction of the substituted piperazino group as the tertiary pharmacophore, which resulted in substantial improvements in pharmacokinetic parameters over previously reported 1-adamantyl-urea based inhibitors while retaining high potency. The SAR studies revealed that the meta- or para-substituted phenyl spacer, and N⁴-acetyl or sulfonyl substituted piperazine were optimal structures for achieving high potency and good physical properties. The 1-(4-(4-(4-acetylpiperazin-1-yl)butoxy)phenyl)-3-adamantan-1-yl urea (29c) demonstrated excellent in vivo pharmacokinetic properties in mice: T_1/2 =14 h, Cmax = 84 nM and AUC = 40200 nM • min with an IC₅₀ value of 7.0 nM against human sEH enzyme.

Introduction

Epoxide hydrolases (EHs, E.C.3.3.2.3) are a group of ubiquitous enzymes present in most living organisms. They catalyze the addition of water to an epoxide, resulting in the formation of a vicinal diol.¹ In mammals, several types of EHs have been identified including leukotriene A₄ hydrolase, cholesterol epoxide hydrolase,² hepoxilin hydrolase,³ microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH),⁴ which differ in their substrate specificity. The first three enzymes are not α/β fold family, while the latter two are. Among the α/β fold family, the sEH is of particular therapeutic interest because of its involvement in the metabolism of endogenously derived fatty acid epoxides and other lipid epoxides.⁵

The sEH promotes the hydrolysis of the biologically active epoxyeicosatrienoic acids (EETs) to the pharmacologically less active and more rapidly cleared dihydroxy epoxyeicosatrienoic acids (DHETs).^4,6 As the primary metabolites of cytochrome P450 epoxygenases of arachidonic acid,⁷ EETs are known to regulate blood pressure and inflammation.^8,9 In addition, the EETs have vascular protective effects such as suppression of reactive oxygen species following hypoxia-reoxygenation,¹⁰ attenuation of vascular smooth muscle migration,¹¹ and enhancement of a fibrinolytic pathway.¹² However, the metabolism of EETs to DHETs by sEH often leads to reductions in these biological activities.¹³ Thus, stabilizing the in vivo concentration of EETs through pharmacological intervention by sEH inhibitors is a novel and potentially therapeutic avenue to treat hypertension, inflammation, and other cardiovascular disorders.¹⁴ It has been reported that sEH inhibitors significantly reduce blood pressure of most varieties of the spontaneous hypertensive rats and angiotensin II induced hypertensive rats.^5,15–18 As such, an sEH inhibitor, AR9281 currently began clinical phase IIa trial for the treatment of type 2 diabetes mellitus and hypertention,¹⁹ which has in turn fueled the recent surge of interest in the development of sEH inhibitors.^20–27

To date, the most successful sEH inhibitors are 1,3-disubstituted ureas, which display anti-hypertension and anti-inflammatory effects through inhibition of EET hydrolysis in several cellular and animal models.^5,17 Common structural features of these inhibitors are the large hydrophobic domains flanking their central urea pharmacophore, which is believed to engage in the hydrogen bond formation with the active site residues Tyr381, Tyr465 and Asp333 of sEH enzyme.⁴ However, the urea-based inhibitors often suffer from poor solubility and bioavailability, which hinders their pharmacological use in vivo. During efforts to improve the potency and physiochemical properties, a new concept of a secondary pharmacophore was proposed, referring to a polar functional group located on the fifth/sixth atom (7.5 Å) from the carbonyl group of the urea primary pharmacophore to help improve the binding and the water solubility (Fig. 1).^28,29 In addition, there also existed a tertiary pharmacophore which located on the fourteenth to sixteenth atom (ca. 17Å) from the carbonyl group of the urea primary pharmacophore (Fig. 1). An acidic or ester group as the tertiary pharmacophore or a polar group such as ester, sulfonamide, ketone, alcohol or ether as the secondary pharmacophore was effective for producing soluble inhibitors in either water or oil while retaining inhibitory potency (Fig. 2). However, relatively short half-lives and in some cases poor bioavailability were still observed with these inhibitors.^30,31

Figure 1.

The proposed pharmacophore model for urea-based sEH inhibitors

Figure 2.

The representative urea-based sEH inhibitors and our designed piperazino-containing series

In order to improve the physical and pharmacokinetic properties of these urea-based sEH inhibitors while retaining potency, we were intrigued to incorporate a constrained heterocycle as a polar functionality on the 1,3-dialkyl urea platform. Piperazine is an interesting heterocycle structure present in many biologically active molecules, conferring metabolic stability and potentiating interactions with macromolecules.^32,33 We have tried a substituted piperazino group as the secondary pharmacophore on the 1-adamantanyl-urea scaffold.³⁴ As expected, these piperazino-containing sEH inhibitors displayed excellent water solubility and low melting point, but their inhibitory activity was remarkably decreased. Since the piperazino functionality is beneficial for the physical properties, we decided to try the piperazino group as the tertiary pharmacophore in the urea-based sEH inhibitors, to gain an improvement in the solubility and bioavailability without any loss of inhibitory activity. Combining the favorable structures obtained from the previous SAR studies, we designed a new class of 1,3-disubstituted ureas functionalized with piperazino groups on the tertiary pharmacophore while retaining ether as the secondary pharmacophore and choosing the phenyl ring and the alkyl chain as the linkers between the primary and secondary and tertiary pharmacophores, respectively (Figure 2). In this study, the substituted piperazino pharmacophore, the orientation of the phenyl spacer and the length of the alkyl chain were investigated with respect to the effect on the activity, physical and pharmacokinetic properties of the 1,3-disubstitued urea-based sEH inhibitiors.

Chemistry

Scheme 1 outlines the syntheses of the 1-adamantan-1-yl-3-substituted ureas having a phenyl ring between the urea group and the piperazino group in a para-, meta- or ortho-substitution pattern, separately. The piperazino group was located on the tertiary pharmacophore site and connected with the phenyl ring via an alkoxy linker with various chain lengths. These 1-adamantan-1-yl-3-(piperazin-1-yl)alkoxyphenyl ureas were synthesized by coupling 1-adamantyl isocyanate with various amines in DCM. In this article, when the piperazino ring was N-substituted unsymmetrically, N¹ represents the left-hand side nitrogen atom closer to the urea pharmacophore, while N⁴ represents the right-hand side nitrogen atom.

Scheme 1. Synthesis of 1-adamantan-1-yl urea derivatives with piperazine group present in the tertiary pharmacophore region^a.

^aReagents and conditions: (a) K₂CO₃, DMF, rt; (b) 1-methylpiperazine (for compound 7b, 8a-c, 9a-c and 10b) or tert-butyl piperazine-1-carboxylate (11b, 12a-c, 13a-b and 14b), THF, reflux; (c) (i) H₂ (1 atm), 10% Pd/C, CH₃OH, rt; (ii) 1-adamantyl isocyanate, DCM, 0 °C to rt; (d) TFA, DCM, 0°C to rt; (e) Ac₂O, DCM, rt; (f) Et₃N, DMF, reflux; (g) di-tert-butyl dicarbonate, CH₃OH, rt.

The preparation of various amines was commenced with commercially available o-, m- or p-nitrophenol and α,ω-dibromoalkane (i.e., 1,2-dibromoethane for compound 3b, 1,3-dibromopropane for compounds 4a-c, 1,4-dibromobutane for compounds 5a-c, 1,5-dibromopentane for compound 6b). The nucleophilic substitution followed by the alkylation with 1-methylpiperazine (for compounds 7b, 8a-c, 9a-c and 10b) or tert-butyl piperazine-1-carboxylate (for compounds 11b, 12a-b, 13a-c, and 14b) gave compounds bearing a piperazino group. Sequential reduction of the nitro group and condensation with 1-adamantyl isocyanate furnished the N, N′-disubstituted ureas (15-22)a-c. Further deprotection and acetylation on the N⁴ atom of the piperazino ring yielded the corresponding N⁴-unsubstituted piperazine-containing compounds (23-26)a-c and the N⁴-acetyl protected derivatives (27-30)a-c, respectively.

In order to optimize the synthetic route for an extensive SAR study, we established alternative approaches to synthesize the substituted piperazine-containing ureas, as depicted in Scheme 1B and 1C. First, the piperazine unit was introduced from the reaction of 1-(ω-bromoalkoxy)-2(3, or 4)-nitrobenzene 3-6 with free piperazine. Then the N⁴-unsubstituted piperazino moiety was transformed into variously N⁴-substituted analogs in a similar manner as Scheme 1A indicated. Alternatively, the N⁴-Boc protected intermediate 7-14 could be used to perform the N⁴-protecting group exchange prior to the coupling with the isocyanate.

On the other hand, we were interested in introducing functionality on the alkyl linker between the ether secondary pharmacophore and the piperazine tertiary pharmacophore. Scheme 2 describes the synthesis of such an analogue bearing a carbonyl group on the N¹-piperazine. Reaction of m-nitrophenol with tert-butyl 4-iodobutanoate generated the precursor tert-butyl 4-(3-nitrophenoxy)butanoate 31. The subsequent deprotection of the carboxyl group followed by the amidation with tert-butyl piperazine-1-carboxylate in the presence of HOAt and EDCI gave the tert-butyl 4-(4-(3-nitrophenoxy)butanoyl)piperazine-1-carboxylate (33). Then similar strategy as Scheme 1 indicated was employed, affording the target compound 1-(3-(4-(4-acetylpiperazin-1-yl)-4-oxobutoxy)phenyl)-3-adamantan-1-ylurea (37).

Scheme 2. Synthesis of 1-adamantan-1-yl-3-(3-(4-oxo-4-(piperazin-1-yl)butoxy)phenyl)urea derivatives^a.

^aReagents and conditions: (a) K₂CO₃, DMF, rt; (b) CF₃COOH, CH₂Cl₂, rt; (c) tert-butyl piperazine-1-carboxylate, HOAt, EDCI, DIPEA, CH₂Cl₂, rt; (d) H₂ (1 atm), 10% Pd/C, CH₃OH, rt; (e) 1-adamantyl isocyanate, CH₂Cl₂, 0°C to rt; (f) CF₃COOH, CH₂Cl₂, 0°C to rt; (g) (CH₃CO)₂O, CH₂Cl₂, rt.

The substitution effect on N⁴-piperazine of the urea inhibitors was investigated. The preparation of various N⁴-substituted piperazine-containing 1-adamantan-1-yl-3-subsituted ureas is depicted in Scheme 3. Starting from 1-adamantan-1-yl-3-(3-(4-(piperazin-1-yl)butoxy)phenyl)urea (25b), reaction with various acylating agents furnished the target products 38-43 in 50–88% yield.

Scheme 3. Synthesis of 1-adamantan-1-yl-3-(3-(4-(piperazin-1-yl)butoxy)phenyl)urea derivatives with different N⁴-substituents on piperazine ring (A) and 1-adamantan-1-yl-3-(3(or 2)-(3-(piperazin-1-yl)propoxy)phenyl)urea derivatives with N⁴-methoxycarbonyl (B) ^a.

^aReagents and conditions: (a) trifluoroacetic anhydride, Et₃N, DCM, rt for compound 38; 2-chloroacetyl chloride, Et₃N, DCM, rt for compound 39; methanesulfonyl chloride, Et₃N, DCM, rt for compound 40; 3,4,5-trimethoxybenzoic acid, HOAt, EDCI, DIPEA, DCM, rt for compound 41; 4-(trifluoromethyl)benzoic acid, HOAt, EDCI, DIPEA, DCM, rt for compound 42; 4-methylbenzene-1-sulfonyl chloride, Et₃N, DCM, rt for compound 43; (b) methyl carbonochloridate, Et₃N, DCM, rt

Results and Discussion

To obtain potent sEH inhibitors with improved physical and pharmacokinetic properties, a variety of 1,3-disubstituted ureas functionalized with piperazine as the tertiary pharmacophore were explored in this study. As a constrained hydrophilic heterocycle, the piperazino group has been reported to be beneficial for the water solubility and metabolic stability for many drugs.^32,33 Our previous effort to incorporate the piperazino group into the secondary pharmacophore of the urea inhibitors dramatically improved the physical properties of the compounds at the cost of lost potency.³⁴ Based on the pharmacophore model, herein we incorporated the piperazino group into the tertiary pharmacophore of 1-adamantan-1-yl-3-alkoxyphenyl urea platform and examined the optimal positioning and substitution of the piperazino functionality.

In the design of the scaffold, we combined the favorable structures obtained from the previous SAR studies, e.g. the ether as the secondary pharmacophore, the phenyl ring as the linker between the primary and secondary pharmacophores, the flexible carbon chain as the linker between the secondary and tertiary pharmacophores. The phenyl ring can provide ortho-, meta- and para-substitution patterns to determine the optimal orientation of the secondary ether bearing a piperazine tail relative to the primary urea. For the linker between the piperazine and the oxygen atom in the ether group, we initially selected propyl and butyl chains to meet the distance requirment between the secondary and tertiary pharmacophore. We investigated varying carbon chain length further to determine an appropriate location for the incorporated piperazine functionality. In addition, the substituent on the N⁴-atom of the piperazine was investigated to achieve an optimal balance between the hydrophilicity and the hydrophobicity for the tertiary pharmacophore, in which N⁴- and N¹-substitution varied in a range of acyl group and alkyl group.

The meta-substituted phenyl linker is favored for the sEH inhibition while the ortho-substitution is detrimental

The effect of the piperazine pharmacophore on enzyme inhibition and physical properties was investigated with regard to the phenyl substitution pattern and the N⁴-piperazino substitution. The activity data of these ureas is summarized in Table 1. In general, the inhibitory activity of this series of ureas bearing piperazine as the tertiary pharmacophore was far superior to that of the analogs carrying piperazine as secondary pharmacophore reported previously.³⁴ Furthermore, the melting point was desirably low to moderate for this class. Although melting point is usually a minor consideration, high melting point and lipophilic compounds are not soluble and hard to formulate. Usually, ~70°C is high enough to crystallize if needed & not melt in tablet press. The lower the melting point, the less chance of polymorphs and for lipophilic compounds the more easily soluble.

Table 1.

Inhibition of human sEH and melting point of 1-adamantan-1-yl-3-(piperazin-1-ylalkoxyphenyl)urea derivatives with variation on the phenyl substitution position and piperazin-4-yl substitution.

n	Substitution mode	Compd.	R	Mpa (°C)	IC50b (nM)
3	ortho-	24a	H	162~164	97460
		16a	CH3	154~156	2880
		28a	CH3CO	86~88	994
	meta-	24b	H	84~86	27.2
		16b	CH3	144~146	23.1
		28b	CH3CO	96~98	3.6
	para-	24c	H	104~106	156
		16c	CH3	98~100	17.9
		28c	CH3CO	170~172	3.3
4	ortho-	25a	H	147~149	72770
		17a	CH3	120~122	5290
		29a	CH3CO	98~100	3150
	meta-	25b	H	94~96	14.8
		17b	CH3	115~117	13.3
		29b	CH3CO	76~78	2.7
	para-	25c	H	87~90	126
		17c	CH3	118~121	44.8
		29c	CH3CO	150~152	7.0

^aMp, melting point;

^bThe IC₅₀ values were determined by a sensitive fluorescent assay on human sEH.³⁵

Obviously, the spacial arrangement of the primary urea and the secondary ether bearing a piperazine tail played an important role in the inhibitory potency against human sEH enzyme. As shown in Table 1, the meta-substituted ureas (e.g. 24b, 16b, 28b) were remarkably more potent than the ortho-substituted counterparts (24a, 16a, 28a), whereas the para-substituted analogs (24c, 16c, 28c) were nearly as potent as the meta-substituted ones when the N⁴ on the piperazine ring was substituted. As to the meta derivatives, the acetamide was more potent than the tertiary amine.

To understand the observation that the ortho-substitution caused a significant loss of the inhibitory potency on the enzyme compared to the meta- and para-substitution, we calculated the possible conformations of the three isomers (24a-c) by running MM2/minimize energy through Chemoffice Chem3D Ultra 9.0. As demonstrated in Figure 3, the steric hindrance conferred by the ortho-substituent on the phenyl ring might prevent the primary urea pharmacophore from interacting with the key residues in the binding pocket of sEH properly, resulting in the low potency.

Figure 3.

The possible conformations of the o, m, p-substituted phenyl containing ureas with minimized energy

Besides the substitution pattern of the phenyl spacer, the N⁴-substituent on the piperazine pharmacophore also affected the inhibitory activity dramatically. As indicated in Table 1,N⁴-acetyl substituted piperazine containing ureas (28a-c, 29a-c) were much more potent than the corresponding N⁴-unsubstituted analogs (24a-c, 25a-c), while the N⁴-methyl substituted piperazine containing ureas (16a-c, 17a-c) were slightly less potent than the N⁴-acetyl substituted counterparts. Taking the meta-substituted phenyl bridged ureas as example, the N⁴-acetylpiperazine containing urea (28c, IC₅₀ = 3.3 nM) exhibited a 47.2- and 5.4-fold increase in the potency relative to the N⁴-unsubstituted (24c, IC₅₀ = 156 nM) and N⁴-methyl substituted (16c, IC₅₀ = 17.9 nM) analogs, respectively. The differentiating effect might be attributed to the presence of the hydrogen bond acceptor, i.e. carbonyl group on the piperazine and the reduction of the polarity at this site. Previous studies have demonstrated that increasing lipophilicity trended toward more potent sEH inhibitors,^36,37 as expected from the hydrophobic catalytic site shown by crystal structures.³⁸ Therefore, reducing the polarity of the N⁴-piperazine by introducing N⁴-substituent such as methyl and acetyl group resulted in the increase of the potency in the context of piperazine as the tertiary pharmacophore.

Inspired by the potentiating effect of N⁴-acetyl substitution, we started to investigate the impact of the acylating N¹ atom in the piperazine pharmacophore on the enzyme inhibition. Taking the m-phenoxybutyl bridged urea as a convenient platform, we employed the synthetic route as presented in Scheme 2 to obtain N¹-acylated piperazine containing compounds and tested the enzyme IC₅₀ values. However, all the N¹-acylated compounds turned out 2.0–6.2-fold less potent than the corresponding N¹-alkylated analogs with respect to the sEH inhibition regardless of the N⁴-substitution (Table 2, 36, IC₅₀ = 91.4 nM vs 25b, IC₅₀ = 14.8 nM; 35, IC₅₀ = 21.5 nM vs 21b, IC₅₀ = 7.1 nM and 37, IC₅₀ = 5.4 nM vs 29b, IC₅₀ = 2.7 nM). Possibly, for the N¹-acylated ureas 35-37, the presence of N¹-carbonyl group may not form a new hydrogen bond within the enzyme catalytic site, but increase the hydrophilicity instead, which is disadvantageous for the interaction with the lipophilic region in the enzyme near the tertiary pharmacophore binding site.

Table 2.

Inhibition of human sEH and melting point of 1-adamantan-1-yl-3-(3-(4-(piperazin-1-yl)butoxy)phenyl)urea and 1-adamantan-1-yl-3-(3-(4-oxo-4-(piperazin-1-yl)butoxy)phenyl)urea derivatives with N⁴-unsubstituted or N⁴-acetylated piperazine.

Compd.	X	R	Mpa (°C)	IC50b (nM)
25b	H2	H	94~96	14.8
36	O	H	78~80	91.4
21b	H2		135~137	7.1
35	O		100~104	21.5
29b	H2		76~78	2.7
37	O		90~92	5.4

^aMp, melting point;

^bThe IC₅₀ values were determined by the sensitive fluorescent assay on human sEH.³⁵

Moreover, the alkyl chain length between the secondary and tertiary pharmacophore was observed to play a subtle role in the sEH inhibitory activity. In general, the butyl linker was advantageous for the meta-substituted urea activity, whereas for the ortho- and para-substituted analogs, the inhibitory potency of propyl linked ureas was higher than that of butyl ones. For example, the para-phenoxypropyl bridged compounds 16c (IC₅₀ = 17.9 nM) and 28c (IC₅₀ = 3.3 nM) displayed comparable potency to the m-phenoxybutyl bridged counterparts 17b (IC₅₀ = 13.3 nM) and 29b (IC₅₀ = 2.7 nM), respectively. However, for the potent N⁴-acetyl analogs, the activity difference between the butyl and propyl-linked ureas was slight (28b, IC₅₀ = 3.6 nM vs. 29b, IC₅₀ = 2.7 nM).

The effect of the chain length to attach the piperazine pharmacophore was dependent on the N⁴-substitution, while the methylsulfonyl group as N⁴-substituent is favored for producing potent sEH inhibitors

Since the preliminary structure activity relationship study indicated that the linker between the secondary ether and the tertiary piperazine pharmacophores as well as the N⁴-substitution on the piperazine were critical for the enzyme inhibition, the favored meta-substitution scaffold was selected as an effective platform to further compare the effect of the chain length and the N⁴-substitution on the potency. The enzyme IC₅₀ values of the ureas with variation on the alkyl chain length and the N⁴-piperazino substitution, and the calculated octanol-water partition coefficient (Log P) of every molecule, are summarized in Table 3.

Table 3.

Inhibitory activity against human sEH and melting point of 1-adamantan-1-yl-3-(3-(alkoxy)phenyl)urea derivatives with variation on the carbon chain length between the ether and piperazine pharmacophores and the N⁴-substitution on the pieperazine ring.

Compd	R	n	Mpa (°C)	logPb	IC50c (nM)	Compd	R	n	Mpa (°C)	logPb	IC50c (nM)
23b	H	2	80~82	1.93	67.1	19b		2	78~80	3.04	2.8
24b		3	84~86	2.03	27.2	20b		3	150~153	3.14	2.9
25b		4	94~96	2.49	14.8	21b		4	135~137	3.60	7.1
26b		5	82~84	2.91	13.8	22b		5	72~74	4.01	11.7
15b	CH3	2	94~96	2.31	7.8	27b		2	73~75	1.58	6.0
16b		3	144~146	2.41	23.1	28b		3	96~98	1.68	3.6
17b		4	115~117	2.87	13.3	29b		4	76~78	2.14	2.7
18b		5	134~137	3.28	11.5	30b		5	136~138	2.55	2.8

^aMp, melting point;

^blog P (octanol/water partition coefficient) calculated by Crippen’s method by using CS ChemDraw Ultra 9.0 version.

^cThe IC₅₀ values were determined by the fluorescent substrate-based assay on human sEH.³⁵

The linker length was examined from two to five methylene carbons with respect to the effect on the enzyme inhibition. As shown in Table 3, the effect was dependent on the N⁴-substitution of the piperazine pharmacophore. Basically, the polar piperazino functionality at this site was good for improving the physical properties as a “solubilizing” group, but the introduction of the carbonyl and the lipophilic group on N⁴-atom were beneficial for retaining the potency due to reducing the polarity and providing a hydrogen bond acceptor. So an optimal balance between the polarity and the lipophilicity is favored to achieve excellent potency.

By analyzing the structure and the activity data in Table 3, we proposed the overall hydrophobicity (indicated by the octanol-water partition coefficient, i.e. Log P) of the molecule and the presence of hydrogen bond acceptor structure as two major factors to determine the potency. For the N⁴-unsubstituted piperazine-containing ureas (compounds 23b-26b), the enzyme inhibitory activity was highly correlated with the net hydrophobicity, while for the N⁴-carbonyl substituted piperazine-contianing analogs (compounds 19b-22b, 27b-30b), the hydrophobicity and the hydrogen bond acceptor both contributed to the final enzyme IC₅₀ values. Especially for the N⁴-acetyl substituted piperazine series (compounds 27b-30b), the resulting optimal LogP value and the presence of hydrogen bond acceptor conferred an attractive scaffold with low nanomolar IC₅₀ values against human sEH.

Apparently, the N⁴-substituent played an important role in the inhibitory potency of the ureas with piperazine as tertiary pharmacophore. So, we selected the overall best inhibitor 29b as the template to extend the structural diversity of N⁴-substituent on piperazine. Because the acylation of N⁴ atom was beneficial for achieving high potency, we focused on the N⁴-acylation with different acyl groups and sulfonyl groups. As shown in Table 4, the sulfonyl substituted ureas (compounds 40 and 43) were generally more potent than the acyl substituted analogs. The bulkier structure adjacent to the carbonyl or sulfonyl center was disfavored (21b, 41 vs 29b, 43 vs 40). The introduction of halogen into the acetyl group enhanced the interaction with the enzyme (38, IC₅₀ = 1.8 nM; 39, IC₅₀ = 1.4 nM), possibly due to a new halogen bonding formation between the fluoro or chloro and the key residues in sEH.³⁹ The structural optimization on the N⁴-substitution afforded the most potent sEH inhibitor in the present work with improved physical properties (40, IC₅₀ = 1.0 nM, Mp= 63~65°C and S = 2.97 mg/mL), with the moderate melting points being low enough to make formulation easy but high enough that crystalization could be used for industrial production.

Table 4.

Inhibition of human sEH and melting point of 1-adamantan-1-yl-3-(3-(4-(piperazin-1-yl)butoxy)phenyl)urea derivatives with variation on the N⁴-substituent in piperazine.

Compd	R	Mpa (°C)	IC50b (nM)
29b		76–78	2.7
21b		135–137	7.1
38		156–160	1.8
39		196–200	1.4
40		63–65	1.0
41		140–142	9.6
42		174–176	13.6
43		86–89	3.3

^aMp, melting point;

^bThe IC₅₀ values were determined by a recently developed sensitive fluorescence-based assay on human sEH.³⁵

The bulky hydrophobic N⁴-substituent on piperazine tertiary pharmacophore helped improve the potency of the ortho-substituted ureas

We noticed that there were differences between the tert-butoxycarbonyl (Boc) and acetyl substituted ureas bearing 2–5 methylene carbon chain linker with regard to the influence of the N⁴-substituent structure on the potency (Table 3). We were intrigued to incorporate the Boc and Ac group into N⁴-piperazine on the ortho-, meta- and para-substituted phenyl linked ureas. Interestingly, a distinct enhancing effect was observed on the ortho-isomer from the para- and meta-isomers. As shown in Table 5, the replacement of Ac group with Boc group on N⁴-piperazine of ortho-substituted series resulted in a significant enhancement in the potency by a fator of 25–191 fold, affording nanomolar sEH inhibitors (20a, IC₅₀ = 39.4 nM; 21a, IC₅₀ = 16.5 nM). However, for meta- or para-substituted analogs, the displacement of Ac by Boc on N⁴-atom suffered an activity decrease by a factor of 2–4 fold (29b, IC₅₀ = 2.7 nM vs 21b, IC₅₀ = 7.1 nM; 28c, IC₅₀ = 3.3 nM vs 20c, IC₅₀ = 14.6 nM; 29c, IC₅₀ = 7.0 nM vs 21c, IC₅₀ = 20.1 nM).

Table 5.

Inhibition of human sEH, and their chang fold of IC₅₀ values of 1-adamantan-1-yl urea derivatives possessing piperazine group in the tertiary pharmacophore region, with N⁴-acetyl and N⁴-boc substitution.

Substitution mode	Compd.	n	R	IC50a (nM)	change foldb
ortho-	28a	3	CH3CO	990	25.2
	20a		tBuOCO	39.4
	29a	4	CH3CO	3150	191
	21a		tBuOCO	16.5
meta-	28b	3	CH3CO	3.6	1.2
	20b		tBuOCO	2.9
	29b	4	CH3CO	2.7	−2.6
	21b		tBuOCO	7.1
para-	28c	3	CH3CO	3.3	−4.4
	20c		tBuOCO	14.6
	29c	4	CH3CO	7.0	−2.9
	21c		tBuOCO	20.1

^aThe IC₅₀ values were determined by the sensitive fluorescence-based assay on human sEH.³⁵

^bChange fold was the ratio of IC₅₀ (R = CH₃CO)/IC₅₀ (R = ^tBuOCO), the negative fold represents the ratio of IC₅₀ (R = ^tBuOCO)/IC₅₀ (R = CH₃CO).

Although it was not clear why the N⁴-Boc substituent significantly improved the potency of the ortho-substituted ureas, we speculated that the increasing hydrophobicity supplied by the tert-butyl carbamate group might be a major contribution factor. As a comparison, we synthesized an analog containing an N⁴-methoxycarbonyl group (Figure 4). It was found that when the N⁴-piperazine was substituted by the small and relatively hydrophilic methyl carbamate, the enzyme inhibitory activity was sharply decreased by a factor of 231-fold (44, IC₅₀ = 9100 nM) relative to the N⁴-tertbutoxycarbonyl analog (20a, IC₅₀ = 39.4 nM). As introduced above, the congestion of the neighboring ether and urea pharmacophores might lead to the poor potency of the ortho-substituted ureas. Correspondingly, the large hydrophobic group at N⁴-piperazine might compensate for the impaired interaction of the ortho-ureas with the enzyme. However, for the potent meta-substituted analogs (Figure 4B), the three acyl groups made slight difference, ending up with almost equal potency (20b, IC₅₀ = 2.9 nM; 28b, IC₅₀ = 3.6 nM; 45, IC₅₀ = 1.1 nM).

Figure 4.

The structure and activity of the ortho- and meta-substituted ureas bearing different N⁴-substituents

The ureas containing an N⁴-substituted piperazine as the tertiary pharmacophore were potent sEH inhibitors with improved physical and pharmacokinetic properties

The water solubility of some selected ureas was experimentally measured, and in vivo pharmacokinetic properties of six potent inhibitors (with IC₅₀ < 10 nM) were tested following oral administration in mice.³⁷ Encouragingly, the incorporation of N⁴-substituted piperazine into the urea-based sEH inhibitor as the tertiary pharmacophore conferred excellent water solubility and in vivo pharmacokinetic parameters with retention of potent enzyme inhibitory activity (Table 6).

Table 6.

The water solubility of some selected ureas with a piperazine present on the tertiary pharmacophore region and the in vivo pharmacokinetic parameters in mice.⁴⁰

Compd	IC50a (nM)	Sb (mg/mL)	Mp (°C)	Tmaxc (h)	Cmaxd (nM)	T1/2e (h)	AUCf (nM·min)
16b	23.1	2.21	144–146
20b	2.9	0.54	150–153	0.5	6	23	6000
21b	7.1	2.78	135–137	9	6	59	5100
24b	27.2	0.81	84–86
27b	6.0	0.52	73–75
28a	990	1.78	86–88
28b	3.6	0.34	96–98	0.5	32	8	11100
28c	3.3	0.32	170–172	0.5	112	6	34800
29b	2.7	1.82	76–78
29c	7.0	0.70	150–152	0.5	84	14	40200
40	1.0	2.97	63–65	0.5	160	3.5	20000
AUDAg	3	<0.125	142–143	1.3	14	2.1	3000

^aThe IC₅₀ values were determined by the sensitive fluorescence-based assay on human sEH;³⁵

^bExperimentally obtained solubility in sodium phosphate buffer (0.1 M, pH 7.4) at 25 ± 1.0 °C;³⁴

^cT_max, time of maximum concentration;

^dC_max, maximum concentration;

^eT_1/2, half-life;

^fAUC, area under the curve estimated from a plot of the inhibitor concentration in plasma (nM) versus time (minutes) following an oral dose of 5 mg/kg of the indicated compound given to mice in triglyceride (N=4).⁴⁰

^gThis compound was previously reported by Hammock’s laboratory⁴¹ and used here as a reference.

As far as the structural effect on the solubility is concerned, the comparison of compounds 28a, 28b and 28c which just differed in the substitution pattern of the phenyl ring indicated that the ortho-substituted urea exhibited the best water solubility (28a, S = 1.78 mg/mL). Meanwhile, among the meta-substituted ureas of 27b, 28b and 29b bearing different carbon chain length linker, the four-methylene-carbon-linked urea displayed the highest water solubility (29b, S = 1.82 mg/mL) which was also the most potent sEH inhibitor in this series. With respect to the influence of the N⁴-substituent on the solubility, meta-substituted ureas 16b, 20b, 24b and 28b with variation on the N⁴-piperazine substitution pattern were tested and the N⁴-methyl-substituted one (16b, S = 2.21 mg/mL) showed the highest water solubility. Finally, among all the tested compounds, the N⁴-sulfonyl substituted 4-methylene-carbon linked urea (40, S = 2.97 mg/mL) exhibited the highest water solubility.

We further investigated the pharmacokinetics of selected potent compounds (with an IC₅₀ < 10 nM) in mice. As shown in Table 6, the in vivo pharmacokinetic parameters [time of maximum concentration (T_max), maximum concentration (C_max), half-life (T_1/2) and area under the curve (AUC)] of six compounds dissolved in a triglyceride of oleic oil (containing 3% EtOH) were determined following oral administration to mice at 5 mg/kg body weight.

The AUC is an expression of how much and how long a drug stays in the body and it is related to the amount of drug absorbed systemically as well as the amount of drug metabolized, sequestered, and eliminated; while the T_1/2 is more indicative of the rates of degradation, distribution, and elimination.⁴¹ Overall, the piperazino substituted ureas significantly improved the pharmacokinetics in comparison to the earlier inhibitor AUDA,⁴² in terms of the AUC and T_1/2. In the N⁴-Boc-piperazine ureas (20b and 21b), relatively low AUC were obtained, while the half-lives were long, suggesting that the presence of an N⁴-Boc group might decrease the absorption. Interestingly, a significant increase in the C_max and AUC were observed with the compounds that carried a para-substituted phenyl ring (28c and 29c) compared to the corresponding meta-substituted analog (28b), indicating that para-substituted pattern might be effective for producing excellent pharmacokinetic properties. Consistently, the most potent inhibitor 40 with a meta-substitution pattern exhibited high AUC value (AUC = 20,000 nM • min ) but still less than the para-substituted analogs (28c, AUC = 34,800 nM • min and 29c, AUC = 40,200 nM • min). However, the best compound 40 produced the highest C_max value (Cmax = 160 nM) among the tested urea compounds. Furthermore, it is worthwhile noting that adamantane is often rapidly metabolized;^43,44 however, in these more polar compounds containing the piperazino group as the tertiary pharmacophore, it does not appear to be as much a metabolic liability as in more lipophilic compounds.

Conclusion

This work focused on producing inhibitors of human sEH with improved physical and pharmacokinetic properties by incorporating a piperazine functionality into the 1,3-disubstituted ureas as the tertiary pharmacphore. By investigating the structural effect on the inhibition potency, physical properties (e.g., water solubility and melting point) and pharmacokinetic properties, a series of potent sEH inhibitors were identified with the meta- or para-substituted phenyl spacer, and N⁴-acetyl or sulfonyl substituted piperazine being the optimal structures. The corresponding N⁴-methanesulfonyl derivative (compound 40) showed the lowest IC₅₀ value and the best water solubility. There was an apparent trend that the bioavailability was closely related with the N⁴-substituent in piperazine, the substitution mode of phenyl spacer and the carbon chain length between the secondary ether pharmacophore and piperazine. We demonstrated that 1-(4-(4-(4-acetylpiperazin-1-yl)butoxy)phenyl)-3-adamantan-1-ylurea (29c) and 1-(4-(3-(4-acetylpiperazin-1-yl)propoxy)phenyl)-3-adamantan-1-ylurea (28c) showed the best AUC, long half-life (T_2/1) and good maximum concentration (C_max) with a nanomolar IC₅₀ value against human sEH enzyme. So, the N⁴-substituted piperazine as the tertiary pharmacophore was significantly effective for improving the solubility and bioavailability of urea-based sEH inhibitors without any loss in potency. These findings are an important basis for the design and development of improved, orally available sEH inhibitors as therapeutic agents for the treatment of hypertension and inflammation.

Experimental Section

The ¹H NMR spectra were recorded on a Varian Mercury-300 MHz spectrometer. The data are reported in parts per million relative to TMS and referenced to the solvent in which they were run. Signal multiplicities are represented as singlet (s), doublet (d), double doublet (dd), triplet (t), quartet (q), quintet (quint), multiplet (m), broad (br), and broad singlet (brs). Melting points (uncorrected) were determined on a Buchi-510 capillary apparatus. EI-MS spectra were obtained on a Finnigan MAT 95 mass spectrometer. ESI-MS spectra were obtained on a Finnigan LCQ Deca mass spectrometer. The purity of products was characterized by analytical HPLC and was more than 95% as determined by HPLC analyses with Vydac C18 column (4.6 mm × 250 mm) in two diverse systems (system 1, solvent A, 0.05% TFA in water; solvent B, 0.05% TFA in 95% acetonitrile; system 2, solvent A, 0.05% TFA in water; solvent B 0.05% TFA in methol) and methods (Method A, detection wavelength: 254 nm, flow: 2.0 mL/min, gradient: 10–90% of solvent B in 25 min; Method B, detection wavelength: 254 nm, flow: 2.0 mL/min, gradient: 10–40% of solvent B in 2 min, then 40%–90% of solvent B in 16 min; Method C, detection wavelength: 225 nm, flow: 2.5 mL/min, gradient: 10–20% of solvent B in 2 min, then 20%–60% of solvent B in 20 min, then 60%–90% of solvent B in 2 min; Method D, detection wavelength: 225 nm, flow: 2.5 mL/min, gradient: 10–20% of solvent B in 2 min, then 20%–60% of solvent B in 16 min, then 60%–90% of solvent B in 2 min).

The general procedures are exemplified by the preparation of compounds 16a, 24a, 28a, 28b and 35 herein. The modified synthesis and the physical data for other target compounds are provided as the supporting information.

1-Adamantan-1-yl-3-(2-(3-(4-methylpiperazin-1-yl)propoxy)phenyl)urea (16a)

The solution of 1-methyl-4-(3-(2-nitrophenoxy)propyl)piperazine 8a (200 mg, 0.72 mmol) in methanol was stirred under hydrogen overnight with palladium-charcoal (10%; 20 mg) as a catalyst. After filtration and removal of the solvent, 2-(3-(4-methylpiperazin-1-yl)propoxy)aniline was obtained as an unstable brown solid (174 mg, 97% yield). The aniline was used in the next step without further purification. The solution of 1-isocyanato-adamantane (85 mg, 0.48 mmol) in dry CH₂Cl₂ (10 mL) was added to 2-(3-(4-methylpiperazin-1-yl)propoxy)aniline (100 mg, 0.40 mmol) in dry CH₂Cl₂ (10 mL). The mixture was stirred at room temperature overnight. The solvent was removed at reduced pressure and the residue was purified by silica gel chromatography (CH₂Cl₂ : CH₃OH = 10 : 1) to afford 16a as a white powder (134 mg, 79% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.69 (s, 6H), 2.07–2.09 (m, 11H), 2.31 (s, 3H), 2.53–2.61 (m, 10H), 4.05 (t, 2H, J = 6.3 Hz), 4.73 (brs, 1H), 6.78–6.85 (m, 2H), 6.88–6.93 (m, 2H), 8.01–8.05 (m, 1H); ESI-MS m/z calcd for C₂₅H₃₉N₄O₂ [M+H]⁺, 427.6; found [M+H]⁺, 427.3; mp 154–156°C; Purity: system 1, 97.8% (method C, t_R = 19.92 min); system 2, 100% (method D, t_R = 23.48 min).

1-Adamantan-1-yl-3-(2-(3-(piperazin-1-yl)propoxy)phenyl)urea (24a)

To a stirred solution of tert-butyl 4-(3-(2-(3-(adamantan-1-yl)ureido)phenoxy)propyl)piperazine-1-carboxylate 20a (101 mg, 0.2 mmol) in CH₂Cl₂ (6 mL) was added 0.3 mL of CF₃COOH at 0°C. The solution was stirred at 0°C overnight, and then was evaporated to dryness in vacuum. The residue was dissolved in water (10mL) and then basified with 5% NaOH, and extracted with CH₂Cl₂. The organic layer was washed with brine and dried over Na₂SO₄. Evaporation in vacuum gave 1-adamantan-1-yl-3-(2-(3-(piperazin-1-yl)propoxy)phenyl)urea 24a (70 mg, 86% yield) as a white powder. ¹H NMR (300 MHz, CDCl₃) δ 1.69 (s, 6H), 2.04 (s, 6H), 2.08–2.17 (m, 3H), 2.57–2.65 (m, 6H), 2.98 (t, 4H), 4.05 (t, 2H), 5.29 (br s, 1H), 6.80–6.83 (m, 1H), 6.89–6.92 (m, 3H), 8.04–8.07 (m, 1H); EI-MS m/z calcd for C₂₄H₃₆N₄O₂ [M]⁺, 412; found [M]⁺, 412; mp 162–164°C; Purity: system 1, 98.6% (method A, t_R = 17.89 min); system 2, 96.9% (method B, t_R = 25.43 min).

1-Adamantan-1-yl-3-(2-(3-(4-acetylpiperazin-1-yl)propoxy)phenyl)urea (28a)

A solution of acetic anhydride (13 mg, 0.13 mmol) and 1-adamantan-1-yl-3-(2-(3-(piperazin-1-yl)propoxy)phenyl)urea 24a (43 mg, 0.10 mmol) in DMF (8 mL) was stirred for 10 h at room temperature. Then the solvent was evaporated. The residue was partitioned between ether (30 mL) and water (30 mL). The ether layer was dried over Na₂SO₄ and evaporated. The residue was purified by column chromatography on silica gel eluting with (CH₂Cl₂ : CH₃OH = 10 : 1) to yield 21 mg (44%) of 28a as a white powder. ¹H NMR (300 MHz, CDCl₃) δ 1.69 (s, 6H), 2.04–2.11 (m, 14H), 2.75 (m, 4H), 2.87 (t, 2H, J = 7.8 Hz), 3.63 (t, 2H, J = 5.1 Hz), 3.99 (t, 2H, J = 5.4 Hz), 6.22 (s, 1H), 6.72–6.75 (m, 1H), 6.82–6.93 (m, 2H), 7.37 (s, 1H), 8.23–8.26 (m, 1H); EI-MS m/z calcd for C₂₆H₃₈N₄O₃ [M]⁺, 454; found [M]⁺, 454; mp 86–88°C; Purity: system 1, 100% (method A, t_R = 20.13 min); system 2, 100% (method B, t_R = 24.95 min).

1-Adamantan-1-yl-3-(3-(3-(4-acetylpiperazin-1-yl)propoxy)phenyl)urea (28b)

1-(4-(3-(3-nitrophenoxy)propyl)piperazin-1-yl)ethanone 12e (54 mg, 0.18 mmol) dissolved in methanol was stirred in an atmosphere of hydrogen overnight with palladium-charcoal (10%; 6 mg) as a catalyst. After filtration and removal of the solvent, 1-(4-(3-(3-aminophenoxy)propyl)piperazin-1-yl)ethanone was obtained as a yellow oil (42 mg, 86% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.95–1.98 (m, 2H), 2.09 (s, 3H), 2.43–2.50 (m, 4H), 2.53–2.58 (m, 2H), 3.49 (t, 2H, J = 4.2 Hz), 3.65 (t, 2H, J = 4.8 Hz), 3.99 (t, 2H, J = 6.3 Hz), 6.23–6.24 (m, 1H), 6.27–6.33 (m, 2H), 7.04 (t, 1H, J = 7.8 Hz). The solution of 1-isocyanato-adamantane (29 mg, 0.17 mmol) in dry CH₂Cl₂ (5 mL) was added to 1-(4-(3-(3-aminophenoxy)propyl)piperazin-1-yl) ethanone (42 mg, 0.15 mmol) in dry CH₂Cl₂ (10 mL) at 0°C. The mixture was stirred at room temperature overnight. The solvent was removed at reduced pressure and the residue was purified by silica gel chromatography (CH₂Cl₂ : CH₃OH = 10 : 1) to afford 28b as a white powder (39 mg, 57% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.68 (s, 6H), 1.95–2.01 (m, 8H), 2.09 (m, 6H), 2.45–2.59 (m, 6H), 3.50 (t, 2H, J = 4.5 Hz), 3.64–3.67 (m, 2H), 4.01 (t, 2H, J = 6.0 Hz), 4.51 (s, 1H), 6.12 (s, 1H), 6.57 (dd, 1H, J =2.7 Hz, J = 5.1 Hz), 6.72 (d, 1H, J = 8.1 Hz), 7.15 (t, 1H, J = 8.4 Hz); EI-MS m/z calcd for C₂₆H₃₈N₄O₃ [M]⁺, 454; found [M]⁺, 454; mp 96–98°C; Purity: system 1, 98.7% (method A, t_R = 19.93 min); system 2, 100% (method B, t_R = 25.18 min).

tert-Butyl 4-(4-(3-nitrophenoxy)butanoyl)piperazine-1-carboxylate (33)

A solution of compound 32 (148 mg, 0.66 mmol), EDCI (127 mg, 0.66 mmol), and HOAt (90 mg, 0.66 mmol) in dry CH₂Cl₂ was stirred at 0°C for 30 min. To this solution was added tert-butyl piperazine-1-carboxylate (135 mg, 0.72 mmol). The reaction mixture was allowed to warm up to room temperature and added DIPEA (0.13 mL). The solution was stirred for 5h, and then extracted with dichloromethane. The combined organic layers were washed with saturated NaHCO₃ aq. solution and brine, and dried over Na₂SO₄. The concentration provided the residue which was purified by chromatography using CH₂Cl₂/CH₃OH (10:1) as eluent to give compound 33 (237 mg, 91%) as a white solid. ¹H NMR (CDCl₃): δ 1.47 (s, 9H), 2.19 (t, 2H, J = 6.6 Hz), 2.55 (t, 2H, J = 7.2 Hz), 3.40–3.45 (m, 6H), 3.61 (t, 2H, J = 4.5Hz), 4.12 (t, 2H, J = 6.0 Hz), 7.20–7.24 (m, 1H), 7.42 (t, 1H, J = 8.1 Hz), 7.23 (t, 1H, J = 2.1 Hz), 7.79–7.83 (m, 1H).

tert-Butyl 4-(4-(3-aminophenoxy)butanoyl)piperazine-1-carboxylate (34)

The tert-butyl 4-(4-(3-nitrophenoxy)butanoyl)piperazine-1-carboxylate 33 (98 mg, 0.25 mmol) dissolved in methanol was stirred in an atmosphere of hydrogen overnight with palladium-charcoal (10%; 10 mg) as a catalyst. After filtration and removal of the solvent, tert-butyl 4-(4-(3-aminophenoxy)butanoyl)piperazine-1-carboxylate 33 was obtained as a white solid (174 mg, 97% yield) which was used in next step without further purification.

tert-Butyl 4-(4-(3-(3- adamantan-1-yl ureido)phenoxy)butanoyl)piperazine-1-carboxylate (35)

The solution of 1-isocyanato-adamantane (49 mg, 0.275 mmol) in dry CH₂Cl₂ was added dropwise to the solution of 34 (0.25 mmol) in dry CH₂Cl₂ at 0°C. The mixture was stirred at room temperature overnight. The solvent was removed at reduced pressure and the residue was purified by silica gel chromatography (CH₂Cl₂ : CH₃OH = 30 : 1) to afford 35 as a pale brown powder (118 mg, 87% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.47 (s, 9H), 1.68 (s, 6H), 2.08 (s, 6H), 2.10–2.13 (m, 5H), 2.52 (t, 2H, J = 7.5 Hz), 3.40–3.44 (m, 6H), 3.58–3.61 (m, 2H), 3.99 (t, 2H, J = 5.7 Hz), 4.63 (s, 1H), 6.32 (s, 1H), 6.55 (dd, 1H, J = 8.1 Hz, J = 2.1 Hz), 6.75 (dd, 1H, J = 8.1 Hz, J = 1.2 Hz), 6.99 (s, 1H), 7.14 (t, 1H, J = 7.8 Hz); EI-MS m/z calcd for C₃₀H₄₄N₄O₅ [M]⁺, 540; found [M]⁺, 540; mp 100–104°C; Purity: system 1, 99.7% (method A, t_R = 32.20 min); system 2, 100% (method B, t_R = 33.14 min).

Solubility

Water solubility was determined experimentally in sodium phosphate buffer (0.1 M, pH 7.4) as previously described at 25±1.5°C.³⁴

Enzyme Preparation

Recombinant human sEH was produced in a polyhedron positive baculovirus expression system following cloning and sequencing in this laboratory and was purified by affinity chromatography as previously reported.^45–46

IC₅₀ Assay Conditions

IC₅₀ values were determined as described using a sensitive fluorescent-based assay,³⁵ and a brief description of the procedure is as follows: cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-phenyl-oxyran-2-yl) methyl carbonate (CMNPC) was used as a fluorescent substrate. Human sEH (1 nM) was incubated with inhibitors for 5 min in pH 7.0 Bis-Tris/HCl buffer (25 mM) containing 0.1 mg/mL of BSA at 30°C prior to substrate introduction ([S] = 5 μM). Activity was measured by determining the appearance of the 6-methoxy-2-naphthaldehyde with an excitation wavelength of 330 nm and an emission wavelength of 465 nm for 10 min. IC₅₀ results are averages of three replicates. The fluorescent assay as performed here has a standard error between 10 and 20%, suggesting that differences of 2-fold or greater are significant.

In Vivo Pharmacokinetic Studies

In vivo experiments were performed following protocols approved by the U.C.D. Animal Use and Care Committee. Mice were treated with test compounds orally at 5 mg/kg. Compounds were given by oral gavage in 0.1 mL of oleic oil solution containing 3% EtOH. 10 μL of blood was collected from mice tail vein before drug administration and 30, 60, 90, 120, 240, 450, 600, and 1440 Mins after drug administration (N=3). The samples were centrifuged at 3000 rpm at 4°C for 10 min and the plasma samples were collected for instrumental analysis. Blood sample preparation and LC/MS/MS analysis were performed as previously reported.⁴⁰ Pharmacokinetic parameters (AUC and t_1/2) were calculated by fitting the blood concentration-time data to a noncompartmental model with WinNonlin 5.0 (Pharsight, CA). Data are average results obtained from at least three different animals.

Abbreviations

sEH

soluble epoxide hydrolase

EET

epoxyeicosatrienoic acid

DHET

dihydroxy epoxyeicosatrienoic acid

AEPU

1-adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea

t-AUCB

trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid

AUDA

12-(3-adamantan-1-ylureido)dodecanoic acid

APAU

N-(1-acetylpiperidin-4-yl)-N′-(adamant-1-yl)urea

AMCU

1-Adamantan-1-yl-3-(4-(3-morpholinopropoxy)cyclohexyl)urea

T_1/2

half-life

C_max

maximum concentration

IC₅₀

half maximal inhibitory concentration

AUC_t

area under the concentration-time curve to terminal time