Design, Synthesis, and Biological Activity of 1,3-Disubstituted Ureas as Potent Inhibitors of the Soluble Epoxide Hydrolase of Increased Water Solubility

Article abstract of DOI:10.1021/jm030514j

The soluble epoxide hydrolase (sEH) is involved in the metabolism of endogenous chemical mediators that play an important role in blood pressure regulation and inflammation. 1,3-Disubstituted ureas are potent inhibitors of sEH that are active both in vitro and in vivo. However, their poor solubility in either water or lipid reduces their in vivo efficacy and makes them difficult to formulate. To improve these physical properties, the effect of incorporating polar functional groups into one of the alkyl chains was evaluated on their inhibitor potencies, water solubility, octanol/water partition coefficients (log P), and melting points. No loss of inhibition potency was observed when a polar functional group was incorporated at least five atoms (～7.5 ?) from the central urea carbonyl. In addition, the presence of a polar group at least 11 atoms away from the urea carbonyl group for the mouse and human sEHs, respectively, did not alter the inhibitor potency. The resulting compounds have better water solubility and generally lower log P values and melting points than nonfunctionalized liphophilic ureas. These properties will make the compounds more bioavailable and more soluble in either water- or oil-based formulations.

Full text of DOI:10.1021/jm030514j

2110
J . Med. Chem. 2004, 47, 2110-2122
Design , Syn th esis, a n d Biologica l Activity of 1,3-Disu bstitu ted Ur ea s a s P oten t
In h ibitor s of th e Solu ble Ep oxid e Hyd r ola se of In cr ea sed Wa ter Solu bility
In-Hae Kim, Christophe Morisseau, Takaho Watanabe, and Bruce D. Hammock*
Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue,
Davis, California 95616
Received October 10, 2003
The soluble epoxide hydrolase (sEH) is involved in the metabolism of endogenous chemical
mediators that play an important role in blood pressure regulation and inflammation. 1,3-
Disubstituted ureas are potent inhibitors of sEH that are active both in vitro and in vivo.
However, their poor solubility in either water or lipid reduces their in vivo efficacy and makes
them difficult to formulate. To improve these physical properties, the effect of incorporating
polar functional groups into one of the alkyl chains was evaluated on their inhibitor potencies,
water solubility, octanol/water partition coefficients (log P), and melting points. No loss of
inhibition potency was observed when a polar functional group was incorporated at least five
atoms (∼7.5 Å) from the central urea carbonyl. In addition, the presence of a polar group at
least 11 atoms away from the urea carbonyl group for the mouse and human sEHs, respectively,
did not alter the inhibitor potency. The resulting compounds have better water solubility and
generally lower log P values and melting points than nonfunctionalized liphophilic ureas. These
properties will make the compounds more bioavailable and more soluble in either water- or
oil-based formulations.
In tr od u ction
results in inflammation that is modulated by nitric oxide
synthase and endothelin-1.15,16 Micromolar concentra-
tions of leukotoxin reported in association with inflam-
mation and hypoxia17 depress mitochondrial respiration
in vitro18 and cause mammalian cardiopulmonary toxic-
ity in vivo.15,19,20 Leukotoxin toxicity presents symptoms
suggestive of multiple organ failure and acute respira-
tory distress syndrome (ARDS).17 In both cellular and
organismal models, leukotoxin-mediated toxicity is de-
pendent upon epoxide hydrolysis,5,21 suggesting a role
for sEH in the regulation of inflammation. The bio-
activity of these epoxy-fatty acids suggests that inhi-
bition of vic-dihydroxy-lipid biosynthesis may have
therapeutic value, making sEH a promising pharma-
cological target.
Epoxide hydrolases (EHs, EC 3.3.2.3) catalyze the
hydrolysis of epoxides or arene oxides to their corre-
sponding diols by the addition of water.1 There are two
well-studied EHs in mammals, the microsomal epoxide
hydrolase (mEH) and the soluble epoxide hydrolase
(sEH). These enzymes are very distantly related, have
different subcellular localization, and have different but
partially overlapping substrate selectivities.2,3 The mi-
crosomal EH is known to detoxify a wide array of
mutagenic, toxic, and carcinogenic xenobiotic epoxides
and it complements the sEH in substrate selectivity.2,3
The sEH is also involved in the metabolism of arachi-
donic acid,4 linoleic acid,5 and other lipid epoxides, some
of which are endogenous chemical mediators.6 Epoxides
of arachidonic acid (epoxyeicosatrienoic acids or EETs)
are known effectors of blood pressure7 and modulators
of vascular permeability.8,9 Hydrolysis of the epoxides
by sEH diminishes this activity.7 We have reported that
treatment with selective sEH inhibitors significantly
reduces the blood pressure of spontaneous hypertensive
rats (SHRs) or angiotensin II induced hypertension in
rats.10,11 In addition, male knockout sEH mice have
significantly lower blood pressure than wild-type mice,12
further supporting the role of sEH in blood pressure
regulation. sEH hydrolysis of EETs also regulates their
incorporation into coronary endothelial phospholipids,
suggesting a regulation of endothelial function by sEH.10
The EETs have also demonstrated antiinflammatory
properties in endothelial cells.13,14 In contrast, diols
derived from epoxylinoleate (leukotoxin) perturb mem-
brane permeability and calcium homeostasis,5 which
We reported 1,3-disubstituted ureas, carbamates, and
amides as new, potent, and stable inhibitors of sEH.
These compounds are competitive, tight-binding inhibi-
tors with nanomolar KI values that interact stoichio-
metrically with purified recombinant sEH.21 On the
basis of the X-ray crystal structure, the urea inhibitors
were shown to establish hydrogen bonds and to form
salt bridges between the urea function of the inhibitor
and residues of the sEH active site, mimicking features
encountered in the reaction coordinate of epoxide ring
opening by this enzyme.22,23 These inhibitors efficiently
reduced epoxide hydrolysis in several in vitro and in vivo
models.11,21,24 However, these dialkyl ureas have limited
solubility in water and high melting points,10 which
likely affect their in vivo efficacy and certainly make
formulation difficult. Therefore, we investigated the
effect of changes in the structure of 1,3-disubstituted
ureas, especially incorporating polar functional groups
into one of the alkyl chains, to design potent sEH
inhibitors with improved physical properties.
* Corresponding author. Tel: 530-752-7519 (office). Fax: 530-752-
1537. E-mail: bdhammock@ucdavis.edu.
10.1021/jm030514j CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/17/2004

Ureas as Inhibitors of Epoxide Hydrolase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2111
Sch em e 1. Synthesis of 1-(3-Chlorophenyl)-3-(4-oxo- or -hydroxydecyl)urea (8 and 17)a
a
Reagents and conditions: (a) Benzophenone imine, CH2Cl2, rt; (b) DIBAL, THF, -78 °C; (c) Mg/I2, bromohexane, THF, rt; (d) acetic
anhydride, DMSO, rt; (e) 1 M HCl, rt; (f) 3-chlorophenyl isocyanate, TEA, DMF, rt.
Sch em e 2. Syntheses of 4-[3-(Aryl or -Cycloalkyl)ureido]butyryl Compoundsa
a
Reagents and conditions: (a) aryl or cycloalkyl isocyanate, DMF, rt; (b) bromopentane, K2CO3, NaI, acetonitrile, reflux; (c) di-tert-
butyl dicarbonate, dioxane, 50 °C; (d) pentylamine, isobutyl chloroformate, NMM, DMF, rt; (e) 4 M HCl, dioxane; (f) 3-chlorophenyl
isocyanate, TEA, DMF, rt.
Ch em istr y
of 4-aminobutyric acid with 3-chlorophenyl, cyclohexyl,
or 1-adamantyl isocyanate gave the corresponding urea
acid (IV), which was alkylated with bromopentane in
the presence of K2CO3 and NaI (catalytic amount) in
acetonitrile to afford 4-[3-(3-chlorophenyl-, -cyclohexyl-,
or -adamantan-1-yl)ureido]butyric acid pentyl esters (9,
18, and 19) in 60-70% yield. For the preparation of 10,
N-protection of 4-aminobutyric acid was first carried out
by using di-tert-butyl dicarbonate and TEA in DMF at
50 °C to provide tert-butoxycarbonylated amino acid (V,
54%).30 The acid was then coupled with pentylamine by
using NMM and isobutyl chloroformate in DMF to give
the amide (VI, 33%),31 followed by N-deprotection of the
amino group with 4 M hydrochloric acid in dioxane.
Reaction of 3-chlorophenyl isocyanate with the amine
in the presence of TEA provided 4-[3-(3-chlorophenyl)-
ureido]-N-pentylbutyramide (10) in 100% yield.
Syn t h eses. Compounds 1-7, 20, and 21 were
prepared according to the procedures reported previ-
ously.25
Compounds 8 and 17 were synthesized by the proce-
dure outlined in Scheme 1. The benzophenone Schiff
base derivative (I) was prepared from ethyl 4-amino-
butylrate hydrochloride using benzophenone imine in
methylene chloride in 61% yield.26 Treatment of the
Schiff base with DIBAL solution in pentane at -78 °C
under nitrogen,27 followed by hexylmagnesium bromide
in THF and warming to room temperature, provided the
alkylated alcohol (II, 60%).28 Oxidation of the alcohol
with acetic anhydride in DMSO gave the corresponding
ketone (III, 100%).29 Hydrolyses of III and II with 1 N
HCl aqueous solution yielded the N-deprotected amine
salts, which reacted with 3-chlorophenyl isocyanate in
the presence of TEA in DMF to afford 8 and 17 (30-
50%), respectively.
The syntheses of 2-[3-(3-chlorophenyl)ureido]ethyl
esters (11-13) are described in part A of Scheme 3 and
2-[3-(3-chlorophenyl)ureido]ethyl compounds with an
amide, carbamate, or urea moiety (14, 15, and 16) were
Scheme 2 shows syntheses of 4-[3-(aryl or cycloalkyl)-
ureido]butyryl compounds (9, 10, 18, and 19). Reaction

2112 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Kim et al.
Sch em e 3. Syntheses of 2-[3-(3-Chlorophenyl)ureido]ethyl Compoundsa
a
Reagents and conditions: (a) 3-chlorophenyl isocyanate, DMF, rt; (b) heptanoic anhydride (11), chloroformic acid pentyl ester (12), or
pentyl Isocyanate (13), TEA, DMF, rt; (c) di-tert-butyl dicarbonate, dioxane, rt; (d) heptanoic anhydride (14), chloroformic acid pentyl
ester (15), or pentyl isocyanate (16), DMF, rt; (e) 4 M HCl, dioxane; (f) 3-chlorophenyl isocyanate, TEA, DMF, rt.
Ta ble 1. Inhibition of Mouse and Human sEH by
1-cycloalkyl-3-alkylurea
synthesized by the procedure outlined in part B of
Scheme 3. Reaction of 2-aminoethanol with 3-chloro-
phenyl isocyanate in DMF gave urea alcohol VII (40%).
Alkylation of the alcohol (VII) with heptanoic anhydride,
chloroformic acid pentyl ester, or pentyl isocyanate in
the presence of TEA afforded 11 (Y ) CH2), 12 (Y ) O),
and 13 (Y ) NH) in 70-85% yield, respectively. For the
syntheses of aminoethyl ureas (B in Scheme 3), first
mono-N-protected diamine (VIII) was prepared by using
di-tert-butyl dicarbonate in dioxane in 95% yield,32
which was followed by alkylation with heptanoic an-
hydride, chloroformic acid pentyl ester, or pentyl iso-
cyanate to provide IX (67%). N-Deprotection with 4 M
HCl in dioxane and reaction with 3-chlorophenyl iso-
cyanate afforded 14 (Y ) CH2), 15 (Y ) O), and 16 (Y )
NH) in 70-85% yield.
From 4-(3-adamantan-1-yl-ureido)butyric acid (22)
prepared by the reaction of 4-aminobutanoic acid and
1-adamantyl isocyanate in DMF, various [4-(3-adaman-
tan-1-yl-ureido)butyryloxy derivatives in Tables 5 and
6 were synthesized (Scheme 4). Alkylation of compound
22 with iodomethane in the presence of K2CO3 in DMF
provided the methyl ester of compound 22 (23) in 95%
yield. Five ethyl bromoalkanoates reacted with com-
pound 22 to afford 29 (n ) 1), 30 (n ) 2), 31 (n ) 3), 32
(n ) 4), and 24 (n ) 6) in a range of 30-95% yield.
Coupling of compound 22 with 3,7-dimethyl-oct-6-en-
1-ol in the presence of DMAP as a base by using ECDI
gave compound 25 in 65% yield.33 Ethyl 8-bromo-
octanoate, which was prepared by the coupling reaction
of 8-bromooctanoic acid and ethanol using EDCI and
a
Enzymes (0.12 µM mouse sEH and 0.24 µM human sEH) were
DMAP, reacted with compound 22 in the presence of
incubated with inhibitors for 5 min in sodium phosphate buffer
K2CO3 as a base in DMF to give compound 33 (75%).
(pH 7.4) at 30 °C before substrate introduction ([S] ) 40 µM).
For the syntheses of compounds 34, 35, and 36, 10-
hydroxydecanoic acid, 11-hydroxyundecanoic acid, and
12-hydroxydodecanoic acid were used as starting ma-
terials, respectively. First, the ω-hydroxyalkanoic acids
were esterified with bromoethane in the presence of
lithium carbonate as a base in DMF, which were coupled
with compound 22 by using EDCI and DMAP in DMF
to provide 34 (n ) 9), 35 (n ) 10), and 36 (n ) 11) in
50-60% yield.
Results are means ( SD of three separate experiments.
Scheme 5 (A and B) describes the syntheses of 4-[4-
(3-adamantan-1-yl-ureido)butyryloxy]aryl derivatives.
Esterification of 4-formylbenzoic acid with bromoethane
and K2CO3 in acetonitrile yielded ethyl 4-formylben-
zoate, followed by reduction of the aldehyde by using
sodium borohydride in ethanol. The reduced alcohol was
coupled with compound 22 by using EDCI and DMAP

Ureas as Inhibitors of Epoxide Hydrolase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2113
Ta ble 2. Inhibition of Mouse and Human sEH by
Ta ble 3. Inhibition of Mouse and Human sEH by
1-(3-Chlorophenyl)-3-(2-alkylated ethyl)ureas
1-(3-Chlorophenyl)-3-(4-hydroxydecyl)urea (17)
a
Enzymes (0.12 µM mouse sEH and 0.24 µM human sEH) were
incubated with inhibitors for 5 min in sodium phosphate buffer
(pH 7.4) at 30 °C before substrate introduction ([S] ) 40 µM).
Results are means ( SD of three separate experiments.
Micromass Quattro Ultima triple quadrupole tandem
mass spectrometer (Micromass, Manchester, UK)]. The
absolute amount of each compound obtained by LC-
MS/MS analysis was converted to a concentration (M),
and the experimental log P value (eLogP) was obtained
with the following equation: eLogP ) log [octanol]/
[water]. On the basis of the log P value, log S value
(solubility in water) was also obtained with the following
equation, which has been suggested by Banerjee et al.34:
log P ) 6.5 - 0.89(log S) - 0.01(melting point). The
cLogP value estimated by Crippen’s method was gener-
ated by using CS ChemDraw Ultra version 6.0. The
results reported in Table 4 are averages of triplicate
analyses.
Wa ter Solu bility (S, m g/m L). Water solubility was
determined using the following procedure at 23 ( 1.5
°C. An excess of the test compound (18-23) was added
to a vial containing sodium phosphate buffer, 0.1 M, pH
7.4 (1 mL), and a suspension of the mixture was
equilibrated during 24 h of shaking, followed by cen-
trifugation (5 min, 200g). The water supernatant (2.5µL)
was dissolved in 0.5 mL of methanol, and to the
methanol solution was added 0.5 mL of internal stand-
ard solution (CTU; 1000 ng/mL in methanol). Then, the
absolute amount of each compound was obtained in the
same procedure as described above.
En zym e P r ep a r a tion . Recombinant mouse sEH and
human sEH were produced in a baculovirus expression
system and purified by affinity chromatography.35-37
The preparations were at least 97% pure as judged by
SDS-PAGE and scanning densitometry. No detectable
esterase or glutathione transferase activities, which can
interfere with this sEH assay, were observed.38 Protein
concentration was quantified by using the Pierce BCA
assay using Fraction V bovine serum albumin as the
calibrating standard.
a
Enzymes (0.12 µM mouse sEH and 0.24 µM human sEH) were
incubated with inhibitors for 5 min in sodium phosphate buffer
(pH 7.4) at 30 °C before substrate introduction ([S] ) 40 µM).
Results are means ( SD of three separate experiments.
to afford compound 27 (Scheme 5A) in 46% yield.
4-Hydroxybenzoic acid, 4-hydroxyphenylacetic acid, and
4-hydroxy acrylic acid were coupled with compound 22
by using EDCI and DMAP, respectively, which were
alkylated with bromoethane and K2CO3 in DMF to give
compounds 28 and 26 in 20-50% yield (Scheme 5B).
Octa n ol/Wa ter P a r tition Coefficien ts (P ). Parti-
tion coefficients were determined using the following
procedure at 23 ( 1.5 °C. The test compound (18-23)
was dissolved in buffer-saturated octanol (5 mL).
Octanol-saturated sodium phosphate buffer, 0.1 M, pH
7.4 (5 mL), was then added to the solution of octanol,
and the mixture was equilibrated during 24 h of
shaking, followed by centrifugation (5 min, 200g). The
octanol fraction (2.5µL) was dissolved in 0.5 mL of
methanol, and to the methanol solution was added 0.5
mL of internal standard solution (1-cyclohexyl-3-tetra-
decylurea (CTU); 1000 ng/mL in methanol). A regression
curve for each compound was obtained from five stand-
ard stock solutions (r ) 0.99) by using LC-MS/MS [a
Waters 2790 liquid chromatograph equipped with a 30
× 2.1 mm 3 µm C18 Xterra column (Waters) and a
IC50 Assa y Con d ition s. IC50 values were determined
as described by using racemic 4-nitrophenyl-trans-2,3-
epoxy-3-phenylpropyl carbonate as substrate.38 En-
zymes (0.10 µM mouse sEH or 0.20 µM human sEH)
were incubated with inhibitors for 5 min in sodium
phosphate buffer, 0.1 M, pH 7.4, containing 0.1 mg/mL

2114 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Kim et al.
Ta ble 4. Inhibition of Mouse and Human sEH by 1-Cylclohexyl or adamantyl-3-alkylureas
a
Enzymes (0.12 µM mouse sEH and 0.24 µM human sEH) were incubated with inhibitors for 5 min in sodium phosphate buffer (pH
b
7.4) at 30 °C before substrate introduction ([S] ) 40 µM). Results are means ( SD of three separate experiments. Solubility in sodium
phosphate buffer (0.1 M, pH 7.4) at 25 °C. Results are means ( SD of three separate experiments. c eLogP: experimental log P (log
[octanol]/[water]). Sodium phosphate buffer (0.1 M, pH 7.4) was used as an aqueous phase. Results are means ( SD of three separate
experiments. cLogP: log P calculated by Crippen’s method by using CS ChemDraw version 6.0. e Solubility in water. It was calculated
d
according to the following equation suggested by Banerjee et al.: log P ) 6.5 - 0.89(log S) - 0.015mp, where mp is melting point. 1H
f
NMR δ (CDCl3) 0.88 (3H, t, J ) 6.9 Hz), 1.11-1.16 (2H, m) 1.26-1.37 (16H, m), 1.46-1.48 (2H, m), 1.58-1.60 (2H, m), 1.67-1.72 (2H,
m), 1.91-1.95 (2H, m), 3.13 (2H, q, J ) 6.9 Hz), 3.45-3.55 (1H, m), 4.19 (1H, s), 4.27 (1H, s); LC-MS (ESI) m/z calcd for C17H34N2O [M
g
+ H]+ 283.27, found [M + H]+ 283.27. 1H NMR δ (CDCl3) 0.88 (3H, t, J ) 6.9 Hz), 1.16-1.36 (12H, m), 1.44-1.49 (2H, m), 1.58-1.59
(2H, m), 1.66-1.68 (6H, m), 1.90-1.96 (6H, m), 2.05-2.07 (3H, m), 3.09 (2H, q, J ) 6.9 Hz), 3.98 (1H, s), 4.03 (1H, s); LC-MS (ESI) m/z
calcd for C21H38N2O [M + H]+ 335.30, found [M + H]+ 335.29.
of BSA, at 30°C before substrate introduction ([S] ) 40
µM). Activity was assessed by measuring the appear-
ance of the 4-nitrophenolate anion at 405 nm at 30 °C
during 1 min (Spectramax 340 PC, Molecular Devices).
Assays were performed in triplicate. IC50 is a concentra-
tion of inhibitor that reduces enzyme activity by 50%
and was determined by regression of at least five datum
points with a minimum of two points in the linear region
of the curve on either side of the IC50. The curve was
generated from at least three separate runs, each in
triplicate, to obtain the standard deviation (SD) given
in Tables 1-7.
phobic regions on many biological molecules. In addi-
tion, the stability of the crystal structure of our first-
generation compounds, indicated by their high melting
point, led to a general lack of solubility, even in organic
solvents. These properties make it difficult to formulate
the compounds in either an aqueous or oil base.
The addition of a polar functional group at the end of
a long aliphatic chain of urea inhibitors made com-
pounds less liphophilic while their inhibitory potencies
were retained.25 To further improve the physical prop-
erty of urea inhibitors, we first investigated the effect
of the addition of polar acid and corresponding methyl
ester moieties onto short aliphatic chains (less than
seven carbons) (Table 1). The free carboxylic acids (1,
3, 5, and 7) failed to show valuable inhibition activity
for either murine or human sEHs. However, the conver-
sion of the carboxylic acid function to the methyl ester
(2, 4, and 6) increased inhibition potency for both mouse
and human sEHs, indicating that the free acid func-
tionality at the end of short aliphatic chains negatively
impacts inhibitor potency. In particular, the methyl
ester of butanoic acid (6) showed 8-100-fold higher
activity for both enzymes than the esters of acetic and
propanoic acids (2 and 4), suggesting that a polar
Resu lts a n d Discu ssion
Recently, we described that 1,3-disubstituted ureas
(and related carbamates and amides) with various alkyl,
cycloalkyl, and aryl groups are potent sEH inhibi-
tors.21,25,39 These urea inhibitors efficiently reduced
epoxide hydrolysis in several in vitro and in vivo models.
However, the lipophilicity of these ureas caused limited
solubility in water, which probably affects their in vivo
efficacy. Often high lipophilicity results in undesirable
pharmacokinetic properties and a lack of specificity,
since lipophilic molecules have high affinity for hydro-

Ureas as Inhibitors of Epoxide Hydrolase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2115
Ta ble 5. Inhibition of Mouse and Human sEH by 4-(3-Adamantan-1-ylureido)butyryloxy Compounds
a
Enzymes (0.12 µM mouse sEH and 0.24 µM human sEH) were incubated with inhibitors for 5 min in sodium phosphate buffer (pH
b
7.4) at 30 °C before substrate introduction ([S] ) 40 µM). Results are means ( SD of three separate experiments. cLogP: log P calculated
by Crippen’s method by using CS ChemDraw version 6.0.
Ta ble 6. Inhibition of Mouse and Human sEH by 4-(3-Adamantan-1-ylureido)butyryloxy Compounds
a
b
The total number of atoms extending from the carbonyl group of the primary urea pharmacophore, TA ) n + 7. Enzymes (0.12 µM
mouse sEH and 0.24 µM human sEH) were incubated with inhibitors for 5 min in sodium phosphate buffer (pH 7.4) at 30 °C before
substrate introduction ([S] ) 40 µM). Results are means ( SD of three separate experiments. c cLogP: log P calculated by Crippen’s
method by using CS ChemDraw version 6.0.

2116 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Kim et al.
Sch em e 4. Syntheses of 4-(3-Adamantan-1-yl-ureido)butyryloxy Compoundsa
a
Reagents and conditions: (a) 1-adamantyl isocyanate, DMF, rt; (b) iodomethane (23) or bromoalkanoic acid ethyl esters, K2CO3,
DMF, rt; (c) 3,7-dimethyl-oct-6-en-1-ol, DMAP, EDCI, DMF, rt; (d) 8-bromooctanoic acid, ethanol, DMAP, EDCI, DMF, rt; (e) bromoethane,
K2CO3, DMF, rt; (f) 10-hydroxydecanoic acid (34), 11-hydroxyundecanoic Acid (35), or 12-hydroxydodecanoic acid (36), bromoethane, Li2CO3,
DMF, 70 °C. The corresponding ethyl esters were coupled with 22 by Using DMAP and EDCI.
Sch em e 5. Syntheses of 4-[4-(3-Adamantan-1-yl-ureido)butyryloxy]aryl Compoundsa
a
Reagents and conditions: (a) bromoethane, K2CO3, acetonitrile, reflux; (b) NaBH4, ethanol, rt; (c) 22, DMAP, EDCI; (d) bromoethane,
K2CO3, DMF, rt.
functional group located on the fifth atom (approxi-
mately 7.5 Å) from the carbonyl group of the primary
urea pharmacophore may be effective for preparing
potent sEH inhibitors of improved water solubility
(Figure 1).
As one adds polar groups to lipophilic inhibitors, the
biological activity is often dramatically reduced, because
the water shell around the polar functionality reduces
binding at the active site. However, if the new polar
groups can hydrogen bond to the enzyme within the
active site, without disturbing the binding of the pri-
mary pharmacophore, this offsets the energy required
to strip the water shell. Thus, the addition of an
additional polar group, which binds within the protein
target, can be expected to dramatically increase the
specificity of the resulting inhibitor. On the basis of the
above results, we designed urea compounds with a polar
carbonyl group on the fifth atom (approximately 7.5 Å)
from the primary urea pharmacophore to improve the
water solubility of lipophilic sEH inhibitors. Table 2
shows various functionalities, including ketone, ester,
amide, carbonate, carbamate, and urea, that contribute
a carbonyl group, and we term these groups as second-
ary pharmacophores (Figure 1). Because in this kind of
time-consuming combinatorial chemistry, a fast monitor
of chemical reactions is required; as shown in Table 2,
we selected a 3-chlorophenyl group as one of substitu-
ents of the urea pharmacophore, which is useful for
monitoring chemical reactions quickly on a TLC plate
and produces sufficient inhibition to compare relative
activity. For example, the 3-chlorophenyl urea is 2-fold
less potent than the cyclohexyl urea with the recombi-
nant murine and human enzymes. After optimizing the
secondary pharmacophore, the aryl substituent can be

Ureas as Inhibitors of Epoxide Hydrolase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2117
shown in Table 4, the substitution on the left side of
the urea pharmacophore with a cyclohexyl (18) or
adamantyl (19) group increased inhibitor potency 10-
fold over the 3-chlorophenyl analogue (9), indicating
that a bulky cycloalkyl group such as cyclohexyl or
adamantyl is a more adequate substitutent of the urea
pharmacophore than an aryl group for inducing potent
inhibition among functionalized ureas. Furthermore,
these compounds functionalized with a polar group were
as active as potent nonfunctionalized lipophilic inhibi-
tors (20 and 21) for both murine and human enzymes.
Comparing the inhibition potency of the two function-
alized compounds (18 and 19), the inhibitor with an
adamantyl substituent (19) had a 10-fold better potency
for human sEH than the cyclohexyl analogue (18). Also,
the two adamantyl analogues with a polar group (22
and 23 in Table 4) had 3-25-fold better potencies than
the corresponding cyclohexyl analogues (5 and 6 in
Table 1).
F igu r e 1. A representative sEH inhibitor with primary,
secondary, and tertiary pharmacophores indicated. The trivial
nomenclature used to describe the inhibitors in this study
consists of these three polar pharmacophores and three R
groups (R). The primary pharmacophore consists of a polar
carbonyl group shown in this case as a urea with R )
adamantane. The secondary pharmacophore consists of a polar
carbonyl group located on the fifth atom (7.5 ( 1 Å) from the
carbonyl group of the primary pharmacophore. A second R
group, termed R′, joins these two pharmacophores. The tertiary
pharmacophore used in this study is another carbonyl shown,
above as an ethyl ester group. It is illustrated linearly from
the carbonyl group of the primary pharmacophore. The tertiary
pharmacophore is located between the eighth and 16th atoms
from the primary phamacophore and isseparated from the
secondary pharmacophore by R′′. In the current study R′′
consists of an aliphatic alkyl carbon or substituted aryl carbon
chain.
When the water solubility of functionalized inhibitors
(S) was measured, a 3-4-fold better solubility was
shown in compounds 18, 19, and 23 with an ester group
than in nonfunctionalized inhibitors (20 and 21), and
in compound 22 with a carboxylic acid group, a 11-16-
fold increased water solubility was obtained. As ex-
pected, decreased experimental log P values (between
0 and 1) were obtained in these functionalized inhibi-
tors, indicating that a polar functional group incorpo-
rated into nonfunctionalized inhibitors improved solu-
bility in water and that the log P value might be a useful
factor to estimate water solubility. The log P values of
neutral immiscible liquids generally run parallel with
their solubility in water; however, for solids solubility
also depends on the energy required to break the crystal
lattice. Banerjee et al. have suggested an empirical
equation [log P ) 6.5 - 0.89(log S) - 0.015mp] to relate
solubility, melting point (mp), and log P.34 It is possible
to have compounds with high log P values that are still
relatively soluble because of their low melting point.
Similarly it is possible to have a low log P compound
with a high melting point that is very insoluble in both
aqueous and organic solutions. As shown in Table 4,
dramatically increased water solubility (log S), which
was calculated according to the above equation, was
obtained in functionalized compounds (18, 19, 22, and
23), and a 1.5-9.5-fold better solubility was also ob-
served in compound 18 than in the other functionalized
inhibitors (19, 22, and 23), while there was no change
in the experimental solubility of the compounds func-
tionalized with an ester group (18, 19, and 23), indi-
cating that the water solubility estimated through
Banerjee’s equation was not always consistent with the
experimental solubility of urea compounds in water.
On the other hand, when log P values calculated by
Crippen’s method (clogP) were generated, decreased
values (between one and three) were observed in the
functionalized compounds with an ester group, which
was consistent with the improved experimental water
solubility, indicating that the clogP value would be
useful to estimate the solubility in water quickly.
replaced by a cyclohexyl, adamantyl or other group
leading to more potent inhibitors.25 When the left of the
carbonyl (X) of the secondary pharmacophore is a
methylene carbon, the best inhibition was obtained if a
methylene carbon (ketone, 8) or oxygen (ester, 9) is
present in the right position (Y). The presence of a
nitrogen (amide, 10) dropped the activity by >10-fold.
In compounds with an oxygen to the left of the carbonyl
group (X), a >10-fold drop in activity was observed and
there was not any change in the activity even if the right
position, Y, was modified with a methylene carbon
(ester, 11), oxygen (carbonate, 12), or nitrogen (carbam-
ate, 13), respectively. All compounds (14, 15, and 16)
with a nitrogen in the left position had >200-fold lower
activities than 8 or 9. Comparing compounds 9 and 11,
the presence of a methylene carbon around the carbonyl
group showed a very different effect on the inhibition
activity. The compound with a methylene carbon to the
left of the carbonyl (9) showed a 20-fold better inhibition
than that to the right (11). While the rank-order potency
of this inhibitor series was equivalent with mouse and
human sEHs, a 3-5-fold higher inhibition potency was
observed for the murine enzyme. In addition, when the
carbonyl group of compound 8 located on the fifth atom
from the urea pharmacophore was modified to the
corresponding hydroxyl group (17 in Table 3), a 3-fold
better inhibition potency was obtained for the mouse
enzyme, while a 1.5-fold decreased inhibition was shown
for the human enzyme.
The data in Tables 2 and 3 indicate that the best
functionality for the secondary pharmacophore is a
ketone (8), ester (9), or alcohol (17). Because among
these three functionalities the ester functional group can
be prepared more easily and in a higher yield than the
ketone or alcohol, the ester functionality was main-
tained as the secondary pharmacophore in this study,
and the 3-chlorophenyl group on the left side of the urea
pharmacophore of the ester compound (9) was then
replaced by a potent cyclohexyl or adamantyl group. As
Adding polar groups to compounds generally increases
their water solubility, and this was the case when one
compares compounds (18 or 19 to 20 or 21 in Table 4).
In addition, stripping water of hydration out of the

2118 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Kim et al.
enzyme catalytic site requires about the same amount
of energy that is gained by forming a new hydrogen bond
between the inhibitor and the enzyme. Thus, addition
of polar groups that hydrogen bond to a target enzyme
may not dramatically increase potency if the inhibitor
is already potent. However, the presence of an ad-
ditional polar group can be expected to dramatically
increase specificity by decreasing the hydrophobic bind-
ing to biological molecules other than the primary target
(sEH). In this way, combining several active pharma-
cophores into a single molecule often has a massive
increase in biological specificity in complex biological
systems. Therefore, investigations were continued to
identify additional positions within the sEH inhibitor
structure that could allow the inclusion of another polar
group (Table 5).
13th-18th (24 and 33-36) atom showed 1.3-25-fold
decreased potencies. On the other hand, for the human
enzyme a carbonyl group on the 13th (24), 14th (33), or
16th (34) atom induced 2-15-fold better inhibition than
on the 8th-11th (29-32) and 17th or 18th (35 and 36)
atom. In addition, compounds 24, 33, and 34 have lower
melting points (58-67 °C) than compound 19 (114 °C)
and similar or a little higher clogP values than that of
compound 19, indicating that improved solubility may
result from these inhibitors as compound 19.
This investigation was implemented with the funda-
mental goal of producing sEH inhibitors with improved
physical properties (e.g. solubility and melting points).
We have found that urea sEH inhibitors possessing an
ester carbonyl group (secondary pharmacophore) on the
fifth atom (approximately 7.5 Å) from the urea phar-
macophore produced excellent inhibition (18 and 19 in
Table 4) and also had improved solubility. In addition,
a tertiary pharmacophore did not lower the inhibition
potency when it was located 12th atom from the urea
pharmacophore in an aryl derivative (27). In aliphatic
derivatives, selectivity between two enzymes was ob-
served: a carbonyl group on the eighth and 11th atoms
(29 and 32 in Table 6) from the carbonyl group of the
primary phamacophore provided potent inhibition for
the murine enzyme, while a carbonyl group on the 13th
(24), 14th (33), or 16th (34) atom induced potent
inhibition for the human enzyme. These results will be
the basis for the design of specific and selective sEH
inhibitors. Furthermore, all results obtained from the
present study will be useful for the design of intravenous
or orally available therapeutic agents for hypertension,
vascular and renal inflammation, and other disorders
that can be addressed by changing the in vivo concen-
tration of chemical mediators that contain an epoxide.
We have reported that ureas with an ester functional
group on the 12th or 13th atom from the urea pharma-
cophore are potent sEH inhibitors.25 In addition, as
shown in Table 4, the adamantyl urea compound (19)
with an ester carbonyl group on the fifth atom from the
urea pharmacophore is also a potent inhibitor and has
improved water solubility. Therefore, one ester carbonyl
group on the fifth atom was maintained, and the right
side of the carbonyl group was varied with aliphatic or
aryl groups with a polar carbonyl group corresponding
roughly to the 13th atom from the urea phamacophore
(Table 5, Figure 1). This corresponds roughly to a
secondary and tertiary pharmacophore in linear dis-
tance from the carbonyl of the primary pharmacophore.
When two carbonyl groups were present on the fifth and
13th atoms from the primary pharmacophore (24),
potencies were maintained for both the murine and
human enzymes, and in the methyl-branched olefin (25),
no loss of inhibition was also observed. A phenyl
derivative (26) with an ethyl ester carbonyl group on
the 13th atom showed a slightly decreased inhibition
for human sEH. However, in two aryl derivatives with
a carbonyl group on the 12th atom (27 and 28),
respectively, no drop of inhibition was observed, indicat-
ing that in aryl derivatives the 12th position from the
urea pharmacophore leads to better inhibition of the two
enzymes tested. In addition, similar clogP values to
those of compounds 18 and 19 in Table 4 were calcu-
lated, indicating that increased solubility in water may
be also obtained in these compounds.
Exp er im en ta l Section
All melting points were determined with a Thomas-Hoover
apparatus (A. H. Thomas Co.) and are uncorrected. Mass
spectra were measured by LC-MS/MS (Waters 2790) using
positive mode electrospray ionization. Elemental analyses (C,
H, N) were performed by Midwest Microlab; analytical results
were within (0.4% of the theoretical values for the formula
given, unless otherwise indicated. 1H NMR spectra were
recorded on a QE-300 spectrometer, using tetramethylsilane
as an internal standard. 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). Synthetic methods are described for representa-
tive compounds.
Syn th esis of 1-(3-Ch lor oph en yl)-3-(4-oxodecyl)u r ea (8).
Benzophenone imine (1.00 g, 5.52 mmol), ethyl 4-amino-
butyrate hydrochloride (0.94 g, 5.52 mmol), and methylene
chloride (20 mL) were stirred at room temperature for 24 h.
The reaction mixture was filtered to remove NH4Cl and
evaporated to dryness. The benzophenone Schiff base of ethyl
4-aminobutyrate (I) was extracted with ether (20 mL), and the
ether solution was washed with water (20 mL), dried over
sodium sulfate (Na2SO4), and concentrated. The residue was
purified by column chromatography on silica gel eluting with
hexane and ethyl acetate (5:1) to give I (1.00 g, 61%) as an oil.
To the solution of the benzophenone Schiff base (I) in tetra-
hydrofuran (THF, 20 mL) was added 1 M diisobutylaluminum
hydride (DIBAL, 3.7 mL) solution in pentane (3.73 mmol) at
-78 °C under nitrogen, and the reaction was stirred for 2 h
at this temperature. To magnesium turnings (0.10 g, 4.07
mmol) and I2 (catalytic amount) in THF (10 mL) was added
hexyl bromide (0.48 mL, 3.39 mmol) at room temperature
under nitrogen. After stirring for 1 h, this reaction solution
The presence of an ester carbonyl group on the 13th
atom in the aliphatic compound (24) did not decrease
the inhibition potency, while the potency was decreased
in an aryl analogue (26). So investigations were con-
tinued to identify other possible positions for the tertiary
pharmacophore in aliphatic inhibitors. All compounds
shown in Table 6 have the secondary phamacophore
(ester functionality) on the fifth atom from the urea
primary pharmacophore, and the atom units between
the secondary pharmacophore and tertiary pharma-
cophore were varied. As shown in Table 6, nine com-
pounds tested had 6-90-fold higher inhibition for mouse
sEH than for human sEH. For the mouse enzyme, a
carbonyl group on the eighth (29) or 11th (32) atom from
the primary pharmacophore provided the most potent
inhibitors, while 15-90-fold reduced potencies were
observed for the human enzyme. Compounds with a
carbonyl group on the ninth (30) or 10th (31) and the

Ureas as Inhibitors of Epoxide Hydrolase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2119
was added dropwise to the above reaction mixture at -78 °C,
and the solution was allowed to warm to room temperature
with stirring. After stirring for 5 h at room temperature, 5%
aqueous NaHCO3 aqueous solution (10 mL) was added to the
reaction, then the alkylated alcohol (II) was extracted with
ether (20 mL), and the ether solution was washed with water
(20 mL), dried over Na2SO4, and concentrated to give 0.26 g
(60%) of the alcohol product (II).
ether (2 × 30 mL). The organic extract was dried over Na2SO4
and evaporated to give 1.00 g (54%) of V as an oil.
A solution of V and 4-methylmorpholine (NMM, 0.54 mL,
4.92 mmol) in DMF (10 mL) was treated at room temperature
with isobutyl chloroformate (0.64 mL, 4.92 mmol). After 30
min, pentylamine (0.57 mL, 4.92 mmol) was added. The
reaction mixture was stirred for 12 h. The solvent was
evaporated, and the residue was partitioned between ethyl
acetate (25 mL) and water (25 mL). The ethyl acetate layer
was washed with 5% NaHCO3 (10 mL) and brine (20 mL),
dried over Na2SO4, and evaporated. The residue was chro-
matographed on silica gel eluting with hexane and ethyl
acetate (2:1) to give 0.33 g (33%) of tert-butoxycarbonylated
amino amide (VI). A solution of VI in dioxane (10 mL) was
treated with 4 M hydrochloric acid (2 mL) in dioxane, and the
mixture was stirred for 1 h at room temperature. Then the
solvent was evaporated to dryness, and the residual solid was
dissolved in DMF (10 mL) and treated with TEA (0.51 mL,
3.63 mmol) and 3-chlorophenyl isocyanate (0.15 mL, 1.21
mmol) at room temperature. After stirring for 5 h, the product
was extracted with ether (30 mL), and the ether was washed
with water (30 mL), dried over Na2SO4, and evaporated to
dryness. The residue was purified by column chromatography
on silica gel eluting with hexane and ethyl acetate (3:1) to
afford 0.39 g (100%) of 10 as a solid: 1H NMR δ (CDCl3) 0.89
(3H, t, J ) 6.9 Hz), 1.26-1.28 (4H, m), 1.46-1.50 (2H, m),
1.86 (2H, quint, J ) 6.9 Hz), 2.30 (2H, t, J ) 6.9 Hz), 3.23
(2H, q, J ) 6.9 Hz), 3.30 (2H, q, J ) 6.9 Hz), 5.87 (1H, s), 6.06
(1H, s), 6.93-6.97 (1H, m), 7.12-7.23 (2H, m), 7.49 (1H, m),
7.73 (1H, s); LC-MS (ESI) m/z calcd for C16H24ClN3O2 [M +
Acetic anhydride (2 mL) was added to a solution of II (0.77
mmol) in dimethyl sulfoxide (DMSO, 5 mL). The mixture was
allowed to stand at room temperature for 12 h and concen-
trated under reduced pressure. The residue was extracted with
ether (20 mL), and the ether was washed with water (20 mL),
dried over Na2SO4, and evaporated to provide 0.26 g (100%)
of the ketone compound (III). To a solution of III in diethyl
ether (5 mL) was added 1 N HCl (1 mL) aqueous solution at
room temperature. The reaction mixture was stirred for 2 h,
the water phase was concentrated to dryness, and then the
residue was dissolved in dimethylformamide (DMF, 5 mL) and
treated with triethylamine (TEA, 0.27 mL, 1.95 mmol) and a
solution of 3-chlorophenyl isocyanate (0.10 mL, 0.78 mmol) in
DMF (3 mL) at room temperature. After stirring for 5 h, the
product was extracted with ether (30 mL), and the ether was
washed with water (30 mL), dried over Na2SO4, and evapo-
rated to dryness. The residue was purified by column chro-
matography on silica gel eluting hexane and ethyl acetate (3:
1) to afford 75 mg (30%) of 8 as a solid. 1H NMR δ (CDCl3)
0.88 (3H, t, J ) 6.9 Hz), 1.21-1.29 (6H, m), 1.53-1.58 (2H,
m), 1.81 (2H, quint, J ) 6.9 Hz), 2.43 (2H, t, J ) 6.9 Hz), 2.49
(2H, t, J ) 6.9 Hz), 3.23 (2H, q, J ) 6.9 Hz), 5.10 (1H, s), 6.93
(1H, s), 6.98-7.02 (1H, m), 7.10-7.23 (2H, m), 7.49 (1H, s);
LC-MS (ESI) m/z calcd for C17H25ClN2O2 [M + H]+ 325.16,
found [M + H]+ 325.21; mp 62-64 °C. Anal. (C17H25ClN2O2)
C, H, N.
H]+ 326.16, found [M + H]+ 326.16; mp 109 °C. Anal. (C16H24
ClN3O2) C, H, N.
-
Syn th esis of Hep ta n oic Acid 2-[3-(3-Ch lor op h en yl)-
u r eid o]eth yl Ester (11). To a solution of 2-aminoethanol
(2.98 g, 48.8 mmol) in DMF (30 mL) was added 3-chlorophenyl
isocyanate (2.50 g, 16.3 mmol) at 0 °C. The reaction mixture
was stirred for 5 h at room temperature. The solvent was
evaporated, the residue was partitioned between ether (30 mL)
and 1 N hydrochloric acid (20 mL), and the ether layer was
washed with brine, dried over Na2SO4, and evaporated. The
residue was purified by column chromatography on silica gel
eluting with hexane and ethyl acetate (1:1) to provide 1.49 g
(40%) of urea alcohol (VII) as a white solid.
Compound 17 was synthesized with the same procedure as
that used for the preparation of compound 8 by using II and
3-chlorophenyl isocyanate instead of III.
Syn th esis of 4-[3-(3-Ch lor op h en yl)u r eid o]bu tyr ic Acid
P en tyl Ester (9). To a suspension of 4-aminobutyric acid (1.41
g, 13.7 mmol) in DMF (25 mL) was added 3-chlorophenyl
isocyanate (0.70 g, 4.56 mmol; cyclohexyl isocyanate for 18 and
1-adamantyl isocyanate for 19) at room temperature. The
reaction mixture was stirred for 24 h. Then, 1 N HCl aqueous
solution (30 mL) was added into the reaction, and the solution
was stirred for 30 min. The solid product was filtered and
washed with water (20 mL) and ethyl acetate (20 mL). The
resulting solid was dried in the vacuum oven to give 1.17 g
(100%) of urea acid (IV). A mixture of IV (0.50 g, 1.95 mmol),
potassium carbonate (K2CO3, 0.54 g, 3.90 mmol), bromo-
pentane (0.37 mL, 2.92 mmol), and sodium iodide (60 mg, 0.39
mmol) in DMF (20 mL) was stirred at room temperature for
20 h. Then the product was extracted with ether (20 mL), and
the ether was washed with 1 N NaOH aqueous solution (20
mL) and brine (20 mL), dried over Na2SO4, and evaporated to
afford 0.59 g (92%) of 9 as a solid: 1H NMR δ (CDCl3) 0.90
(3H, t, J ) 6.9 Hz), 1.26-1.34 (4H, m), 1.62-1.65 (2H, m),
1.88 (2H, quint, J ) 6.9 Hz), 2.41 (2H, t, J ) 6.9 Hz), 3.30
(2H, q, J ) 6.9 Hz), 4.08 (2H, t, J ) 6.9 Hz), 4.96 (1H, s), 6.62
(1H, s), 7.01-7.04 (1H, m), 7.18-7.22 (2H, m), 7.47 (1H, s);
LC-MS (ESI) m/z calcd for C16H23ClN2O3 [M + H]+ 327.14,
found [M + H]+ 327.12; mp 98 °C. Anal. (C16H23ClN2O3) C, H,
N.
Compounds 18 and 19 were synthesized in the same manner
as that described above using cyclohexyl isocyanate and
adamantyl isocyanate instead of 3-chlorophenyl isocyanate,
respectively.
Syn th esis of 4-[3-(3-Ch lor op h en yl)u r eid o]-N-p en tyl-
bu tyr a m id e (10). To a suspension of 4-aminobutyric acid
(2.84 g, 27.5 mmol) in DMF (30 mL) was added TEA (3.86 mL,
27.5 mmol). To this mixture, di-tert-butyl dicarbonate (2.00 g,
9.17 mmol) was added with stirring. The reaction mixture was
heated to 50 °C for 12 h, and then stirred with ice-cold dilute
hydrochloric acid (15 mL) for 10 min. The tert-butoxycar-
bonylated amino acid (V) was immediately extracted with
To a solution of VII (1.00 g, 4.60 mmol) and TEA (0.97 mL,
6.90 mmol) in DMF (15 mL) was added a solution of heptanoic
anhydride (2.23 g, 9.20 mmol) in DMF (5 mL) at room
temperature. The reaction was stirred for 12 h, and the solvent
was evaporated. The residue was partitioned between ether
(30 mL) and cold 1 N hydrochloric acid (20 mL). The ether
layer was washed with brine, dried over Na2SO4, and evapo-
rated. The residual solid was purified using silica gel column
chromatography (hexane:ethyl acetate ) 3:1) to afford 1.05 g
(70%) of 11 as an oil: 1H NMR δ (CDCl3) 0.87 (3H, t, J ) 6.9
Hz), 1.20-1.29 (6H, m), 1.60-1.62 (2H, m), 2.22-2.29 (2H,
m), 3.50-3.55 (2H, m), 4.09-4.20 (2H, m), 5.32 (1H, s), 7.01-
7.06 (2H, m), 7.16-7.22 (2H, m), 7.40 (1H, s); LC-MS (ESI)
m/z calcd for C16H23ClN2O3 [M + H]+ 327.14, found [M + H]+
327.15. Anal. Calcd for C16H23ClN2O3: C, 58.80; H, 7.09; N,
8.57. Found: C, 60.99; H, 8.50; N, 7.09.
Compounds 12 and 13 were prepared in the same manner
as that used for compound 11 from chloroformic acid pentyl
ester and pentyl isocyanate instead of heptanoic anhydride.
Syn th esis of Hep ta n oic Acid {2-[3-(3-Ch lor op h en yl)-
u r eid o]eth yl}a m id e (14). A solution of di-tert-butyl dicar-
bonate (0.50 g, 2.29 mmol) in dioxane (20 mL) was added over
a period of 1 h to a solution of 1,2-diaminoethane (1.10 g, 18.3
mmol) in dioxane (20 mL). The mixture was allowed to stir
for 22 h and the solvent was evaporated to dryness. Water
(30 mL) was added to the residue and the insoluble bis-
substituted product was removed by filtration. The filtrate was
extracted with methylene chloride (3 × 30 mL) and the
methylene chloride was evaporated to yield VIII as an oil (0.35
g, 95%).

2120 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Kim et al.
A solution of heptanoic anhydride (0.91 g, 3.75 mmol;
chloroformic acid pentyl ester for 15 and pentyl isocyanate for
16) and VIII (0.50 g, 3.13 mmol) in DMF (20 mL) was stirred
for 2 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 evapo-
rated. The residue was purified by column chromatography
on silica gel eluting with hexane and ethyl acetate (1:1) to yield
0.57 g (67%) of alkylated N-tert-butoxycarbonylamine (IX).
A solution of IX in dioxane (10 mL) was treated with 4 M
hydrochloric acid (2 mL) in dioxane, and the mixture was
stirred for 1 h at room temperature. Then the solvent was
evaporated to dryness, and the residual solid was dissolved
in DMF (10 mL) and treated with TEA (0.58 mL, 4.19 mmol)
and 3-chlorophenyl isocyanate (0.32 g, 2.10 mmol) at room
temperature. After stirring for 5 h, the product was extracted
with ether (30 mL), and the ether was washed with water (30
mL), dried over Na2SO4, and evaporated to dryness. The
residue was purified by column chromatography on silica gel
eluting with hexane and ethyl acetate (1:1) to afford 0.68 g
(100%) of 14 as a solid: 1H NMR δ (CDCl3) 0.84 (3H, t, J )
6.9 Hz), 1.16-1.25 (6H, m), 1.55-5.61 (2H, m), 2.21-2.24 (2H,
m), 3.31-3.40 (4H, m), 6.27 (1H, s), 6.90-6.95 (2H, m), 7.18-
7.20 (2H, m), 7.56 (1H, s), 8.07 (1H, s); LC-MS (ESI) m/z calcd
for C16H24ClN3O2 [M + H]+ 326.16, found [M + H]+ 326.25;
mp 133-135 °C. Anal. Calcd for C16H24ClN3O2: C, 58.98; H,
7.42; N, 12.90. Found: C, 58.18; H, 7.37; N, 12.64.
1.60 (9H, m), 1.67-1.69 (8H, m), 1.81 (2H, quint, J ) 6.9 Hz),
1.94-1.97 (6H, m), 2.05-2.07 (3H, m), 2.35 (2H, t, J ) 6.9
Hz), 3.16 (2H, q, J ) 6.9 Hz), 4.05 (1H, s), 4.11 (2H, t, J ) 6.9
Hz), 4.21 (1H, s), 5.09 (1H, t, J ) 6.9 Hz); LC-MS (ESI) m/z
calcd for C25H42N2O3 [M + H]+ 419.32, found [M + H]+ 419.22;
mp 49 °C. Anal. Calcd for C25H42N2O3: C, 71.73; H, 10.11; N,
6.69. Found: C, 70.27; H, 9.83; N, 6.39.
Syn t h esis of 8-[4-(3-Ad a m a n t a n -1-yl-u r eid o)b u t yr yl-
oxy]octa n oic Acid Eth yl Ester (33). To a solution of
8-bromooctanoic acid (0.20 g, 0.89 mmol), DMAP (0.12 g, 0.99
mmol), and ethanol (0.05 g, 0.99 mmol) in methylene chloride
(20 mL) was added EDCI (0.19 g, 0.99 mmol) at room
temperature. After stirring for 12 h, the reaction mixture was
washed with 1 N NaOH aqueous solution (15 mL) and water
(30 mL), and the organic layer was dried over Na2SO4 and
evaporated to give 8-bromooctanoic acid ethyl ester (0.17 g,
75%). This bromide reacted with 22 in the same manner as
that used for the preparation of 23 to provide 33 (0.19 g, 65%)
as a solid: 1H NMR δ (CDCl3) 1.26 (3H, t, J ) 6.9 Hz), 1.32-
1.35 (6H, m), 1.59-1.66 (10H, m), 1.82 (2H, quint, J ) 6.9 Hz),
1.94-1.97 (6H, m), 2.05-2.07 (3H, m), 2.28 (2H, t, J ) 6.9
Hz), 2.36 (2H, t, J ) 6.9 Hz), 3.16 (2H, q, J ) 6.9 Hz), 4.05-
4.14 (5H, m), 4.31 (1H, s); LC-MS (ESI) m/z calcd for
C
25H42N2O5 [M + H]+ 451.31, found [M + H]+ 451.20; mp 58-
59 °C. Anal. (C25H42N2O5) C, H, N.
Syn th esis of 10-[4-(3-Ad a m a n ta n -1-yl-u r eid o)bu tyr yl-
oxy]d eca n oic Acid Eth yl Ester (34). A mixture of 10-
hydroxydecanoic acid (0.25 g, 1.33 mmol; 11-hydroxyundec-
anoic acid for compound 35 and 12-hydroxydodecanoic acid for
compound 36), ethyl bromide (0.16 g, 1.46 mmol), and lithium
carbonate (0.11 g, 1.46 mmol) in DMF (25 mL) was stirred at
70 °C for 6 h. Then the product was extracted with ether (30
mL), and the ether solution was washed with 1 N NaOH
aqueous solution (20 mL) and water (30 mL), dried over
Na2SO4, and concentrated. The residue was purified by column
chromatography on silica gel eluting with hexane and ethyl
acetate (3:1) to give 10-hydroxydecanoic acid ethyl ester (80
mg, 28%). This alcohol was coupled with 22 by using EDCI/
DMAP coupling reagent to give 34 (0.11 g, 60%) as a solid:
1H NMR δ (CDCl3) 1.24-1.32 (13H, m), 1.62-1.68 (10H, m),
1.80 (2H, quint, J ) 6.9 Hz), 1.94-1.97 (6H, m), 2.05-2.07
(3H, m), 2.28 (2H, t, J ) 6.9 Hz), 2.36 (2H, t, J ) 6.9 Hz), 3.16
(2H, q, J ) 6.9 Hz), 4.05-4.14 (5H, m), 4.25 (1H, s); LC-MS
(ESI) m/z calcd for C27H46N2O5 [M + H]+ 479.34, found [M +
H]+ 479.29; mp 60-61 °C. Anal. Calcd for C27H46N2O5: C,
67.75; H, 9.69; N, 5.85. Found: C, 68.33; H, 9.92; N, 5.97.
Compounds 15 and 16 were prepared in the same manner
as that described above using chloroformic acid pentyl ester
and pentyl isocyanate instead of heptanoic anhydride, repec-
tively.
Syn th esis of 4-(3-Ad a m a n ta n -1-yl-u r eid o)bu tyr ic Acid
Meth yl Ester (23). To a suspension of 4-aminobutyric acid
(2.79 g, 27.1 mmol) in DMF (40 mL) was added 1-adamantyl
isocyanate (1.20 g, 6.77 mmol) at room temperature. The
reaction mixture was stirred for 24 h. Then 1 N HCl aqueous
solution (40 mL) was added into the reaction, and the mixture
was stirred for 30 min. The solid crystalline product was
filtered and washed with water (20 mL) and ethyl acetate (20
mL). The resulting solid was dried in a vacuum oven to give
1.90 g (100%) of 4-(3-adamantan-1-yl-ureido)butyric acid (22)
as a white solid: 1H NMR δ (CD3OD): 1.66-1.75 (8H, m),
1.94-1.97 (6H, m), 2.05-2.07 (3H, m), 2.30 (2H, t, J ) 6.9
Hz), 3.08 (2H, q, J ) 6.9 Hz), 3.32 (2H, s); LC-MS (ESI) m/z
calcd for C15H24N2O3 [M + H]+ 281.18, found [M + H]+ 281.25;
mp 165 °C. Anal. (C15H24N2O3) C, H, N.
Compound 22 was coupled with 11-hydroxyundecanoic acid
ethyl ester and 12-hydroxydodecanoic acid ethyl ester prepared
from corresponding acids to get compounds 35 and 36,
respectively.
A mixture of 22 (0.15 g, 0.54 mmol), K2CO3 (0.09 g, 0.64
mmol), and iodomethane (0.04 mL, 0.59 mmol) in DMF (20
mL) was stirred at room temperature for 20 h. Then the
product was extracted with ether (20 mL), and the ether was
washed with 1 N NaOH aqueous solution (20 mL) and brine
(20 mL), dried over Na2SO4, and evaporated to afford 0.15 g
(95%) of 23: 1H NMR δ (CDCl3) 1.66-1.68 (6H, m), 1.81 (2H,
quint, J ) 6.9 Hz), 1.94-1.97 (6H, m), 2.05-2.07 (3H, m), 2.37
(2H, t, J ) 6.9 Hz), 3.16 (2H, q, J ) 6.9 Hz), 3.68 (3H, s), 4.09
(1H, s), 4.25 (1H, s); LC-MS (ESI) m/z calcd for C16H26N2O3
[M + H]+ 295.19, found [M + H]+ 295.24; mp 114 °C. Anal.
(C16H26N2O3) C, H, N.
Syn t h esis of 4-[4-(3-Ad a m a n t a n -1-yl-u r eid o)b u t yr yl-
oxym et h yl]b en zoic Acid E t h yl E st er (27). A mixture of
4-formylbenzoic acid (1.00 g, 6.66 mmol), bromoethane (1.09
g, 9.99 mmol), and K2CO3 (1.10 g, 7.99 mmol) in acetonitrile
(30 mL) was refluxed for 6 h. After evaporation of the solvent,
4-formylbenzoic acid ethyl ester was extracted with ether (30
mL), and the organic solution was washed with 1 N NaOH
aqueous solution (20 mL) and water (30 mL), dried over
Na2SO4, and concentrated to give the ethyl ester product (0.65
g, 55%). Without further purification, to a solution of the ester
was added sodium borohydride (NaBH4; 0.05 g, 3.65 mmol) in
ethanol (20 mL) at 0 °C. After stirring for 5 h at room
temperature, the product was extracted with ether (30 mL),
and the ether solution was washed with water (30 mL), dried
over Na2SO4, and concentrated. The residue was purified by
using column chromatography on silica gel eluting with hexane
and ethyl acetate (3:1) to give 4-hydroxymethylbenzoic acid
ethyl ester (0.30 g, 46%) as an oil.
Compounds 29, 30, 31, 32, and 24 were prepared in the
same manner using the corresponding ethyl bromoalkanoates
instead of iodomethane to yield 30-95%.
Syn th esis of 4-(3-Ad a m a n ta n -1-yl-u r eid o)bu tyr ic Acid
3,7-Dim eth yl-oct-6-en yl Ester (25). To a solution of 22 (0.10
g, 0.36 mmol), 4-(dimethylamino)pyridine (DMAP; 44 mg, 0.36
mmol), and 3,7-dimethyl-oct-6-en-1-ol (61 mg, 0.39 mmol) in
methylene chloride (20 mL) was added 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (EDCI; 75 mg, 0.39
mmol) at room temperature. After stirring for 12 h, the
reaction mixture was washed with 1 N NaOH aqueous solution
(15 mL) and water (30 mL), and the organic layer was dried
over Na2SO4 and concentrated. The residue was purified by
column chromatography on silica gel eluting with hexane and
ethyl acetate (3:1) to give 25 (97 mg, 65%) as a solid: 1H NMR
δ (CDCl3) 0.91 (3H, d, J ) 6.9 Hz), 1.34-1.37 (2H, m), 1.56-
To a solution of 22 (1.23 g, 0.83 mmol), DMAP (0.05 g, 0.42
mmol), and the above alcohol (0.15 g, 0.83 mmol) in methylene
chloride (30 mL) was added EDCI (0.16 g, 0.83 mmol) at room
temperature. After stirring for 12 h, the reaction mixture was
washed with 1 N NaOH aqueous solution (15 mL) and water
(30 mL), and the organic layer was dried over Na2SO4 and

Ureas as Inhibitors of Epoxide Hydrolase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 2121
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concentrated. Then the residue was purified by column chro-
matography on silica gel eluting hexane and ethyl acetate
(5:1) to provide 27 (0.28 g, 75%) as a white solid: 1H NMR δ
(CDCl3) 1.40 (3H, t, J ) 6.9 Hz), 1.66-1.68 (6H, m), 1.84 (2H,
quint, J ) 6.9 Hz), 1.94-1.96 (6H, m), 2.05-2.07 (3H, m), 2.44
(2H, t, J ) 6.9 Hz), 3.17 (2H, q, J ) 6.9 Hz), 4.02 (1H, s), 4.17
(1H, s), 4.38 (2H, q, J ) 6.9 Hz), 5.17 (2H, s), 7.40 (2H, d, J )
7.8 Hz), 8.00 (2H, d, J ) 7.8 Hz); LC-MS (ESI) m/z calcd for
C
25H34N2O5 [M + H]+ 443.25, found [M + H]+ 443.25; mp 96-
99 °C. Anal. (C25H34N2O5) C, H, N.
Syn th esis of 4-(3-Ad a m a n ta n -1-yl-u r eid o)bu tyr ic Acid
4-Eth oxyca r bon ylm eth ylp h en yl Ester (28). To a solution
of 22 (0.15 g, 0.54 mmol), DMAP (0.07 g, 0.54 mmol), and
4-hydroxyphenylacetic acid (0.09 g, 0.59 mmol) in methylene
chloride (20 mL) was added EDCI (0.11 g, 0.59 mmol) at room
temperature. After stirring for 12 h, the reaction mixture was
washed with water (20 mL), and the methylene chloride
solution dissolving the product was dried over Na2SO4 and
concentrated to give conjugated product. This crude mixture
in DMF (30 mL) was treated with bromoethane (0.15 g, 1.34
mmol) and K2CO3 (0.18 g, 1.34 mmol) at room temperature
and stirred for 12 h at room temperature. The ethyl ester
product was extracted with ether (30 mL), and the ether
solution was washed with 1 N NaOH aqueous solution (20 mL)
and water (30 mL), dried over Na2SO4, and concentrated. The
residue was purified by column chromatography on silica gel
eluting hexane and ethyl acetate (5:1) to give 28 (47 mg, 20%)
as a white solid: 1H NMR δ (CDCl3) 1.40 (3H, t, J ) 6.9 Hz),
1.66-1.68 (6H, m), 1.89-1.95 (8H, m), 2.05-2.07 (3H, m), 2.62
(2H, t, J ) 6.9 Hz), 3.25 (2H, q, J ) 6.9 Hz), 3.60 (2H, s), 4.07
(1H, s), 4.16 (2H, q, J ) 6.9 Hz), 4.29 (1H, s), 7.08-7.10 (2H,
m), 7.28-7.30 (2H, m); LC-MS (ESI) m/z calcd for C25H34N2O5
[M + H]+ 443.25, found [M + H]+ 443.25; mp 95-97 °C. Anal.
(C25H34N2O5) C, H, N.
Compound 26 was prepared in the same manner by using
4-hydroxyphenylacrylic acid instead of 4-hydroxyphenylacetic
acid.
Ack n ow led gm en t. This work was supported, in
part, by NIEHS Grant R37 ES02710, NIEHS Center
for Environmental Health Sciences P30 ES05707,
NIH/NIEHS Superfund Basic Research Program P42
ES04699, UC Systemwide Biotechnology Research and
Education Training Grant #2001-07, NIEHS Center for
Children’s Environmental Health & Disease Prevention
P01 ES11269, and NIH/NIDDK UC Davis Clinical
Nutrition Research Unit P30 DK35747, Pilot Project.
Su p p or tin g In for m a tion Ava ila ble: Detailed analytical
data (NMR, LC-MS, elemental analysis, and melting point)
for 12, 13, 15-19, 24, 26, 29-32, 35, 36. This material is
available free of charge via the Internet at http://pubs.acs.org.
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