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

Article abstract of DOI:10.1021/jm101087u

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 stabil

Full text of DOI:10.1021/jm101087u

8376 J. Med. Chem. 2010, 53, 8376–8386
DOI: 10.1021/jm101087u
Incorporation of Piperazino Functionality into 1,3-Disubstituted Urea as the Tertiary Pharmacophore
Affording Potent Inhibitors of Soluble Epoxide Hydrolase with Improved Pharmacokinetic Properties
Shao-Xu Huang,† Hui-Yuan Li,† Jun-Yan Liu,‡ Christophe Morisseau,‡ Bruce D. Hammock,‡ and Ya-Qiu Long*,†
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Shanghai 201203, China, and ‡Department of Entomology and University of California Davis Cancer Center, University of California,
One Shields Avenue, Davis, California 95616, United States
Received August 23, 2010
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-adamantylurea 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: T1/2 =14 h, Cmax=84 nM, AUC=40200 nM min,
and IC50 =7.0 nM against human sEH enzyme.
3
Introduction
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, sta-
bilizing the in vivo concentration of EETs through pharma-
cological intervention by sEH inhibitors is a novel and
potentially therapeutic avenue to treat hypertension, inflam-
mation, and other cardiovascular disorders.14 It has been
reported that sEH inhibitors significantly reduce blood pres-
sure 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 hyper-
tention,19 which has in turn fueled the recent surge of interest
in the development of sEH inhibitors.20-27
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, cho-
lesterol epoxide hydrolase,2 hepoxilin hydrolase,3 microsomal
epoxide hydrolase (mEH), and soluble epoxide hydrolase
(sEHa),4 which differ in their substrate specificity. The first
three enzymes are not R/β fold family, while the last two are.
Among the R/β fold family, the sEH is of particular therapeutic
interest because of its involvement in the metabolism of endo-
genously 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 dihydroxyepoxyeicosa-
trienoic 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
To date, the most successful sEH inhibitors are 1,3-disub-
stituted ureas, which display antihypertension 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 How-
ever, the urea-based inhibitors often suffer from poor solubil-
ity and bioavailability, which hinders their pharmacological
use in vivo. During efforts to improve the potency and physio-
chemical properties, a new concept of a secondary pharma-
cophore was proposed, referring to a polar functional group
*To whom correspondence should be addressed. Phone: 86-21-
50806876. Fax: þ86-21-50806876. E-mail: yqlong@mail.shcnc.ac.cn.
a Abbreviations: sEH, soluble epoxide hydrolase; EET, epoxyeicosa-
trienoic acid; DHET, dihydroxyepoxyeicosatrienoic acid; AEPU, 1-ada-
mantan-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-acetylpiperi-
din-4-yl)-N0-(adamant-1-yl)urea; AMCU, 1-adamantan-1-yl-3-(4-(3-mor-
pholinopropoxy)cyclohexyl)urea; T1/2, half-life; Cmax, maximum concen-
tration; IC50, half maximal inhibitory concentration; AUCt, area under the
concentration-time curve to terminal time.
˚
located on the fifth/sixth atom (7.5 A) from the carbonyl
group of the urea primary pharmacophore to help improve
the binding and the water solubility (Figure 1).28,29 In addi-
tion, there also existed a tertiary pharmacophore that is loca-
˚
ted on the 14th to 16th atom (∼17 A) from the carbonyl group
of the urea primary pharmacophore (Figure 1). An acidic or
r
pubs.acs.org/jmc
Published on Web 11/11/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8377
ester group as the tertiary pharmacophore or a polar group
such as ester, sulfonamide, ketone, alcohol, or ether as the sec-
ondary pharmacophore was effective for producing soluble
inhibitors in either water or oil while retaining inhibitory
potency (Figure 2). However, relatively short half-lives and
in some cases poor bioavailability were still observed with
these inhibitors.30,31
effect on the activity and physical and pharmacokinetic pro-
perties 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)alkoxy-
phenylureas were synthesized by coupling 1-adamantyl iso-
cyanate with various amines in DCM. In this article, when the
piperazino ring was N-substituted unsymmetrically, N1 repre-
sents the left-hand side nitrogen atom closer to the urea phar-
macophore while N4 represents the right-hand side nitrogen
atom.
The preparation of various amines was commenced with
commercially available o-, m-, or p-nitrophenol and R,ω-
dibromoalkane (i.e., 1,2-dibromoethane for compound 3b,
1,3-dibromopropane for compounds 4a-c, 1,4-dibromobu-
tane for compounds 5a-c, 1,5-dibromopentane for com-
pound 6b). Thenucleophilicsubstitutionfollowed by the alky-
lation 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 furn-
ished the N,N0-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 pro-
tected derivatives (27-30)a-c, respectively.
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 het-
erocycle as a polar functionality on the 1,3-dialkylurea plat-
form. Piperazine is an interesting heterocyclestructure present
in many biologically active molecules, conferring metabolic
stability and potentiating interactions with macromole-
cules.32,33 We have tried a substituted piperazino group as
the secondary pharmacophore on the 1-adamantanylurea
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 favor-
able structures obtained from the previous SAR studies, we
designed a new class of 1,3-disubstituted ureas functionalized
with piperazino group on the tertiary pharmacophore while
retainingether as the secondary pharmacophoreand choosing
the phenyl ring and the alkyl chain as the linkers between the
primary and secondary and tertiary pharmacophores, respec-
tively (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
In order to optimize the synthetic route for an extensive
SAR study, we established alternative approaches to synthe-
size the substituted piperazine-containing ureas, as depicted in
Scheme 1B and Scheme 1C. First, the piperazine unit was
introduced from the reaction of 1-(ω-bromoalkoxy)-(2, 3, or 4)-
nitrobenzenes (3-6)a-c with free piperazine. Then the
N4-unsubstituted piperazino moiety was transformed into
variously N4-substituted analogues in a similar manner as
Scheme 1A indicated. Alternatively, the N4-Boc protected inter-
mediates (11-14)a-c could be used to perform the N4-protect-
ing 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
Figure 1. Proposed pharmacophore model for urea-based sEH
inhibitors.
Figure 2. Representative urea-based sEH inhibitors and our designed piperazino-containing series.

8378 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Huang et al.
Scheme 1. Synthesis of 1-Adamantan-1-ylurea Derivatives with Piperazine Group Present in the Tertiary Pharmacophore Regiona
a Reagents and conditions: (a) K2CO3, DMF, room temp; (b) 1-methylpiperazine (for compounds 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, room temp; (ii) 1-adamantyl isocyanate, DCM, 0 °C to
room temp; (d) TFA, DCM, 0 °C to room temp; (e) Ac2O, DCM, room temp; (f) Et3N, DMF, reflux; (g) di-tert-butyl dicarbonate, CH3OH, room temp.
Scheme 2. Synthesis of 1-Adamantan-1-yl-3-(3-(4-oxo-4-(piperazin-1-yl)butoxy)phenyl)urea Derivativesa
a Reagents and conditions: (a) K2CO3, DMF, room temp; (b) TFA, DCM, room temp; (c) tert-butyl piperazine-1-carboxylate, HOAt, EDCI,
DIPEA, DCM, room temp; (d) H2 (1 atm), 10% Pd/C, CH3OH, room temp; (e) 1-adamantyl isocyanate, DCM, 0 °C to room temp; (f) Ac2O, DCM,
room temp.
pharmacophore and the piperazine tertiary pharmacophore.
Scheme 2 describes the synthesis of such ananalogue bearing a
carbonyl group on the N1-piperazine. Reaction of m-nitro-
phenol 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 a similar strat-
egy as Scheme 1 indicated was employed, affording the target
compound 1-(3-(4-(4-acetylpiperazin-1-yl)-4-oxobutoxy)phe-
nyl)-3-adamantan-1-ylurea (37).
The substitution effect on N4-piperazine of the urea inhibitors
was investigated. The preparation of various N4-substituted

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8379
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-Methoxycar-
bonyl (B)a
a Reagents and conditions: (a) trifluoroacetic anhydride, Et3N, DCM, room temp for compound 38; 2-chloroacetyl chloride, Et3N, DCM, room temp
for compound 39; methanesulfonyl chloride, Et3N, DCM, room temp for compound 40; 3,4,5-trimethoxybenzoic acid, HOAt, EDCI, DIPEA, DCM,
room temp for compound 41; 4-(trifluoromethyl)benzoic acid, HOAt, EDCI, DIPEA, DCM, room temp for compound 42; 4-methylbenzene-1-sulfonyl
chloride, Et3N, DCM, room temp for compound 43; (b) methyl carbonochloridate, Et3N, DCM, room temp.
Table 1. Inhibition of Human sEH and Melting Points of 1-Adaman-
tan-1-yl-3-(piperazin-1-ylalkoxyphenyl)urea Derivatives with Variation
on the Phenyl Substitution Position and Piperazin-4-yl Substitution
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 var-
ious acylating agents furnished the target products 38-43 in
50-88% yield.
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 pharma-
cophore were explored in this study. As a constrained hydro-
philic 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 On the basis of
the pharmacophore model, herein we incorporated the piper-
azino group into the tertiary pharmacophore of 1-adaman-
tan-1-yl-3-alkoxyphenylurea platform and examined the
optimal positioning and substitution of the piperazino func-
tionality.
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 piper-
azine 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 pharmaco-
phore. We investigated varying carbon chain length further to
determine an appropriate location for the incorporated piper-
azine 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 groups and alkyl groups.
n
substitution mode compd
R
mp a (°C) IC50 b (nM)
3
ortho
meta
para
24a
16a
28a
24b
16b
28b
24c
16c
28c
25a
17a
29a
25b
17b
29b
25c
17c
29c
H
CH3
162-164
154-156
97460
2880
994
CH3CO 86-88
H
CH3
84-86
144-146
27.2
23.1
3.6
CH3CO 96-98
H
CH3
104-106
98-100
156
17.9
3.3
CH3CO 170-172
H
CH3
4
ortho
meta
para
147-149
120-122
72770
5290
3150
14.8
13.3
2.7
CH3CO 98-100
H
CH3
94-96
115-117
CH3CO 76-78
H
CH3
87-90
126
118-121
44.8
7.0
CH3CO 150-152
a mp, melting point. b The IC50 values were determined by a sensitive
fluorescent assay on human sEH.35
The meta- or para-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-piper-
azino substitution. The activity data of these ureas are
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 analogues
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

8380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Huang et al.
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
Figure 3. Possible conformations of the ortho-, meta-, and para-
substituted phenyl containing ureas with minimized energy.
compounds are not soluble and hard to formulate. Usually,
∼70 °C is high enough to crystallize if needed and not melt in
tablet press. The lower the melting point, the less chance
there is of polymorphs and the more easily soluble are the
lipophilic compounds.
a Mp, melting point. b The IC50 values were determined by the sen-
sitive fluorescent assay on human sEH.35
N4-piperazine by introducing an N4-substituent such as a
methyl or 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 substitu-
tion, we started to investigate the impact of the acylating the
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 pre-
sented in Scheme 2 to obtain N1-acylated piperazine contain-
ing compounds and tested the enzyme IC50 values. However,
all the N1-acylated compounds turned out 2.0- to 6.2-fold
less potent than the corresponding N1-alkylated analogues
with respect to the sEH inhibition regardless of the
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 analogues (24c, 16c, 28c) were nearly as
potent as the meta-substituted ones when the N4 on the
piperazine ring was substituted. As for 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 Che-
moffice 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 properly with the key residues in the binding
pocket of sEH, resulting in a low potency.
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 analogues (24a-c, 25a-c), while the N4-meth-
yl substituted piperazine containing ureas (16a-c, 17a-c)
were slightly less potent than the N4-acetyl substituted coun-
terparts. Taking the meta-substituted phenyl bridged ureas
as examples, 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) analogues,
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-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; 37 IC50=
5.4 nM vs 29b IC50=2.7 nM). Possibly, for the N1-acylated
ureas 35-37, the presence of the N1-carbonyl group may not
form a new hydrogen bond within the enzyme catalytic site
but increase the hydrophilicity instead, which is disadvanta-
geous for the interaction with the lipophilic region in the
enzyme near the tertiary pharmacophore binding site.
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 analogues, the
inhibitory potency of propyl linked ureas was higher than
that of the butyl ones. For example, the p-phenoxypropyl
bridged compounds 16c (IC50 =17.9 nM) and 28c (IC50
=
3.3 nM) displayed comparable potency to the m-phenoxy-
butyl bridged counterparts 17b (IC50 = 13.3 nM) and 29b
(IC50 = 2.7 nM), respectively. However, for the potent
N4-acetyl analogues, 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

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8381
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
a Mp, melting point. b log P (octanol/water partition coefficient) calculated by Crippen’s method by using CS ChemDraw Ultra, version 9.0.
c The IC50 values were determined by the fluorescent substrate-based assay on human sEH.35
secondary ether and the tertiary piperazine pharmaco-
phores as well as the N4-substitution on the piperazine was
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.
tive 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 diver-
sity of the N4-substituent on piperazine. Because the acyla-
tion of the N4 atom was beneficial for achieving high poten-
cy, we focused on the N4-acylation with different acyl groups
and sulfonyl groups. As shown in Table 4, the sulfonyl sub-
stituted ureas (compounds 40 and 43) were generally more
potent than the acyl substituted analogues. 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
because of 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
crystallization could be used for industrial production.
The bulky hydrophobic N4-substituent on piperazine ter-
tiary pharmacophore helped improve the potency of the ortho-
substituted ureas. We noticed that there were differences
between the tert-butoxycarbonyl (Boc) and acetyl substi-
tuted ureas bearing a two to five 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 linker length was examined from two to five methy-
lene carbons with respect to the effect on the enzyme inhibi-
tion. 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 was beneficial for retaining the potency because
of reduction of the polarity and the provision of 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 mole-
cule 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 corre-
lated with the net hydrophobicity, while for the N4-carbonyl
substituted piperazine-contianing analogues (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 log P and
the presence of hydrogen bond acceptor conferred an attrac-

8382 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Huang et al.
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 Varia-
tion on the N4-Substituent in Piperazine
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 sup-
plied by the tert-butyl carbamate group might be a major
contribution factor. As a comparison, we synthesized an
analogue 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-tert-
butoxycarbonyl analogue (20a IC50 = 39.4 nM). As intro-
duced above, the congestion of the neighboring ether and
urea pharmacophores might lead to a poor potency of the
ortho-substituted ureas. Correspondingly, the large hydro-
phobic group at N4-piperazine might compensate for the
impaired interaction of the ortho-ureas with the enzyme.
However, for the potent meta-substituted analogues (Figure 4B),
the three acyl groups made a slight difference, ending up with
almost equal potency (20b IC50=2.9 nM; 28b IC50=3.6 nM;
45 IC50=1.1 nM).
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 mea-
sured, 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 inhibi-
tor as the tertiary pharmacophore conferred excellent water
solubility and in vivo pharmacokinetic parameters with
retention of potent enzyme inhibitory activity (Table 6).
As far as the structural effect on the solubility is con-
cerned, 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 linkers, 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 sub-
stitution pattern were tested and the N4-methyl-substituted
one (16b S=2.21 mg/mL) showed the highest water solubi-
lity. Finally, among all the tested compounds, the N4-sulfo-
nyl substituted four-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 IC50 < 10 nM) in mice. As shown in
Table 6, the in vivo pharmacokinetic parameters [time of
maximum concentration (Tmax), maximum concentration
(Cmax), half-life (T1/2), and area under the curve (AUC)] of
six compounds dissolved in a triglyceride of oleic oil (con-
taining 3% EtOH) were determined following oral adminis-
tration 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 and the amount of drug metabolized, sequestered,
and eliminated, while the T1/2 is more indicative of the rates of
degradation, distribution, and elimination.41 Overall, the piper-
azino substituted ureas significantly improved the pharmacoki-
netics in comparison to the earlier inhibitor AUDA,42 in terms of
the AUC and T1/2. IntheN4-Boc-piperazine ureas (20b and 21b),
a Mp, melting point. b The IC50 values were determined by a recently
developed sensitive fluorescence-based assay on human sEH.35
Table 5. Inhibition of Human sEH and Their Change Fold of IC50 Values
of 1-Adamantan-1-ylurea Derivatives Possessing Piperazine Group in the
Tertiary Pharmacophore Region, with N4-Acetyl and N4-Boc Substitution
substitution mode compd
n
R
IC50 a (nM) change foldb
ortho
meta
para
28a
20a
29a
21a
28b
20b
29b
21b
28c
20c
29c
21c
3
CH3CO
tBuOCO
CH3CO
tBuOCO
CH3CO
tBuOCO
CH3CO
tBuOCO
CH3CO
tBuOCO
CH3CO
tBuOCO
990
39.4
3150
16.5
3.6
25.2
4
3
4
3
4
191
1.2
2.9
2.7
-2.6
-4.4
-2.9
7.1
3.3
14.6
7.0
20.1
a The IC50 values were determined by the sensitive fluorescence-based
assay on human sEH.35 b Change fold is the ratio of IC50(R=CH3CO)/
IC50(R=tBuOCO). The negative fold represents the ratio of IC50(R=
tBuOCO)/IC50(R=CH3CO).
the ortho-isomer from the para- and meta-isomers. As
shown in Table 5, the replacement of a Ac group with Boc
group on N4-piperazine of the ortho-substituted series
resulted in a significant enhancement in the potency by a
fator of 25- to 191-fold, affording nanomolar sEH inhibitors
(20a IC50 = 39.4 nM; 21a IC50 = 16.5 nM). However, for
meta- or para-substituted analogues, the displacement of Ac
by Boc on N4-atom suffered an activity decrease by a factor
of 2- to 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).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8383
Figure 4. Structure and activity of the ortho- and meta-substituted ureas bearing different N4-substituents.
Table 6. Water Solubility of Some Selected Ureas with a Piperazine Present on the Tertiary Pharmacophore Region and the in Vivo Pharmacokinetic
Parameters in Mice40
compd
IC50 a (nM)
S b (mg/mL)
mp (°C)
Tmax c (h)
Cmax d (nM)
T1/2 e (h)
AUC f (nM min)
3
16b
20b
21b
24b
27b
28a
28b
28c
23.1
2.9
7.1
27.2
6.0
990
3.6
3.3
2.7
7.0
1.0
3
2.21
0.54
2.78
0.81
0.52
1.78
0.34
0.32
1.82
0.70
2.97
<0.125
144-146
150-153
135-137
84-86
73-75
86-88
96-98
170-172
76-78
150-152
63-65
142-143
0.5
9
6
6
23
59
6000
5100
0.5
0.5
32
112
8
6
11100
34800
29b
29c
0.5
0.5
1.3
84
14
40200
20000
3000
40
AUDAg
160
14
3.5
2.1
a The IC50 values were determined by the sensitive fluorescence-based assay on human sEH.35 b Experimentally obtained solubility in sodium
34 c
phosphate buffer (0.1 M, pH 7.4) at 25 ( 1.0 °C.
T
max
, time of maximum concentration. d Cmax, maximum concentration. e T1/2, 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 g This compound was previously reported by Hammock’s laboratory41 and used here as a
reference.
relatively low AUC values 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 Cmax and AUC was observed with the com-
pounds that carried a para-substituted phenyl ring (28c and
29c) compared to the corresponding meta-substituted ana-
logue (28b), indicating that the 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=
incorporating a piperazine functionality into the 1,3-disubsti-
tuted ureas as the tertiary pharmacphore. By investigation of
the structural effect on the inhibition potency, physical prop-
erties (e.g., water solubility and melting point), and pharma-
cokinetic properties, a series of potent sEH inhibitors were
identified with the meta- or para-substituted phenyl spacer,
with 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 to the N4-substituent in
piperazine, the substitution mode of phenyl spacer, and the
carbon chain length between the secondary ether pharmaco-
phore and piperazine. We demonstrated that 1-(4-(4-(4-acet-
ylpiperazin-1-yl)butoxy)phenyl)-3-adamantan-1-ylurea (29c)
and 1-(4-(3-(4-acetylpiperazin-1-yl)propoxy)phenyl)-3-ada-
mantan-1-ylurea (28c) showed the best AUC, long half-life
(T1/2), and good maximum concentration (Cmax) 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 bio-
availability 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
20 000 nM min) but still less than the para-substituted
3
analogues (28c AUC = 34 800 nM min and 29c AUC =
3
40 200 nM min). However, the best compound, 40, pro-
3
duced the highest Cmax value (Cmax = 160 nM) among the
tested urea compounds. Furthermore, it is worthwhile not-
ing that adamantane is often rapidly metabolized;43,44 how-
ever, in these more polar compounds containing the pipera-
zino 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

8384 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Huang et al.
as therapeutic agents for the treatment of hypertension and
inflammation.
(t, 4H), 4.05 (t, 2H), 5.29 (brs, 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 C24-
H36N4O2 [M]þ, 412; found [M]þ, 412; mp 162-164 °C. Purity:
system 1, 98.6% (method A, tR =17.89 min); system 2, 96.9%
(method B, tR=25.43 min).
Experimental Section
1H NMR spectra were recorded on a Varian Mercury 300 MHz
spectrometer. Thedataare reportedinpartspermillionrelative 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 ana-
lytical HPLC and was more than 95% as determined by HPLC
analyses with Vydac C18 column (4.6 mm ꢀ 250 mm) in two
diverse systems and methods. System 1 consisted of the following:
solvent A, 0.05% TFA in water; solvent B, 0.05% TFA in 95%
acetonitrile. System 2 consisted of the following: solvent A,
0.05% TFA in water; solvent B, 0.05% TFA in methanol.
Method A parameters were as follows: detection wavelength,
254 nm; flow rate, 2.0 mL/min; gradient, 10-90% of solvent B in
25 min. Method B parameters were as follows: detection wave-
length, 254 nm; flow rate, 2.0 mL/min; gradient, 10-40% of
solvent B in 2 min, then 40-90% of solvent B in 16 min. Method
C parameters were as follows: detection wavelength, 225 nm; flow
rate, 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 parameters were as follows: detection wave-
length, 225 nm; flow rate, 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.
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 puri-
fied 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]þ, 454; found [M]þ, 454; mp 86-88 °C. Purity: system 1, 100%
(method A, tR=20.13 min); system 2, 100% (method B, tR=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)-
1
ethanone was obtained as a yellow oil (42 mg, 86% yield). H
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). A solution of
1-isocyanatoadamantane (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=
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 in Supporting Information.
1-Adamantan-1-yl-3-(2-(3-(4-methylpiperazin-1-yl)propoxy)-
phenyl)urea (16a). A solution of 1-methyl-4-(3-(2-nitrophenoxy)-
propyl)piperazine8a (200 mg, 0.72 mmol) inmethanolwasstirred
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. A solution of
1-isocyanatoadamantane (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
1
10:1) to afford 28b as a white powder (39 mg, 57% yield). H
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]þ, 454;
found [M]þ, 454; mp 96-98 °C. Purity: system 1, 98.7% (method
A, tR=19.93 min); system 2, 100% (method B, tR=25.18 min).
tert-Butyl 4-(4-(3-Nitrophenoxy)butanoyl)piperazine-1-carbo-
xylate (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 to room tempera-
ture, and DIPEA (0.13 mL) was added. The solution was stirred
for 5 h and then extracted with dichloromethane. The combined
organic layers were washed with saturated NaHCO3 aqueous
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
1
as a white powder (134 mg, 79% yield). H 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 þ H]þ, 427.6; found [M þ H]þ,
427.3; mp 154-156 °C. Purity: system 1, 97.8% (method C, tR=
19.92 min); system 2, 100% (method D, tR=23.48 min).
1
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 (10 mL) 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%
(237 mg, 91%) as a white solid. H NMR (300 MHz, 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 = 4.5 Hz), 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-carbo-
xylate (34). tert-Butyl 4-(4-(3-nitrophenoxy)butanoyl)piper-
azine-1-carboxylate 33 (98 mg, 0.25 mmol) dissolved in metha-
nol 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
1
yield) as a white powder. H 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

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8385
References
white solid (174 mg, 97% yield) which was used in next step
without further purification.
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tert-Butyl 4-(4-(3-(3-Adamantan-1-ylureido)phenoxy)butanoyl)-
piperazine-1-carboxylate (35). A solution of 1-isocyanatoada-
mantane (49 mg, 0.275 mmol) in dry CH2Cl2 was added drop-
wise to a 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
(2) Levin, W.; Michaud, D. P.; Thomas, P. E.; Jerina, D. M. Distinct
rat hepatic microsomal epoxide hydrolases catalyze the hydration
of cholesterol 5,6-alpha-oxide and certain xenobiotic alkene and
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(5) Imig, J. D.; Hammock, B. D. Soluble epoxide hydrolase as a
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(6) (a) Capdevila, J. H.; Falck, J. R.; Harris, R. C. Cytochrome P450
and arachidonic acid bioactivation: molecular and functional
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1
as a pale brown powder (118 mg, 87% yield). H 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]þ, 540; found [M]þ, 540; mp 100-104 °C.
Purity: system 1, 99.7% (method A, tR=32.20 min); system 2,
100% (method B, tR=33.14 min).
Solubility. Water solubility was determined experimentally in
sodium phosphate buffer (0.1 M, pH 7.4) as previously descri-
bed at 25 ( 1.5 °C.34
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Enzyme Preparation. Recombinant human sEH was pro-
duced 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-methoxyn-
aphthalen-6-yl)methyl trans-(3-phenyloxyran-2-yl) methyl car-
bonate (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 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 com-
pounds orally at 5 mg/kg. Compounds were given by oral gavage in
0.1 mL of oleic oil solution containing 3% EtOH. Then 10 μL of
blood was collected from mice tail vein before drug administration
and 30, 60, 90, 120, 240, 450, 600, and 1440 min 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 analyses were performed as previously reported.40
Pharmacokinetic parameters (AUC and T1/2) were calculated
by fitting the blood concentration-time data to a noncompart-
mental model with WinNonlin 5.0 (Pharsight, CA). Data are
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Acknowledgment. The work in Y.-Q.L.’s lab was sup-
ported by National Science and Technology Major Project
“Key New Drug Creation and Manufacturing Program”,
China (Grants 2009ZX09301-001 and 2009ZX09103-067).
Partial support was obtained from NIEHS Grant R01
ES002710 and NIH/NHLBI Grant Ro1 HL059699. B.D.H.
is a George and Judy Marcus Senior Fellow of the American
Asthma Foundation.
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Supporting Information Available: Details of the synthetic
procedures; physical data for compounds 15b, 16b-c, 17a-c,
18b, 19b, 20a-c, 21a-c, 22b, 23b, 24b-c, 25a-c, 26b, 27b, 28c,
29a-c, 30b, 36-45. This material is available free of charge via
the Internet at http://pubs.acs.org.

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