Optimization of amide-based inhibitors of soluble epoxide hydrolase with improved water solubility

Article abstract of DOI:10.1021/jm0500929

Soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in the regulation of blood pressure and inflammation. 1,3-Disubstituted ureas with a polar group located on the fifth atom from the carbonyl group of urea function are active inhibitors of sEH both in vitro and in vivo. However, their limited solubility in water and relatively high melting point lead to difficulties in formulating the compounds and poor in vivo efficacy. To improve these physical properties, the effect of structural modification of the urea pharmacophore on the inhibition potencies, water solubilities, octanol/water partition coefficients (log P), and melting points of a series of compounds was evaluated. For murine sEH, no loss of inhibition potency was observed when the urea pharmacophore was modified to an amide function, while for human sEH 2.5-fold decreased inhibition was obtained in the amide compounds. In addition, a NH group on the right side of carbonyl group of the amide pharmacophore substituted with an adamantyl group (such as compound 14) and a methylene carbon present between the adamantyl and amide groups were essential to produce potent inhibition of sEH. The resulting amide inhibitors have 10-30-fold better solubility and lower melting point than the corresponding urea compounds. These findings will facilitate synthesis of sEH inhibitors that are easier to formulate and more bioavailable.

Full text of DOI:10.1021/jm0500929

J. Med. Chem. 2005, 48, 3621-3629
3621
Optimization of Amide-Based Inhibitors of Soluble Epoxide Hydrolase with
Improved Water Solubility
In-Hae Kim, Fenton R. Heirtzler, Christophe Morisseau, Kosuke Nishi, Hsing-Ju Tsai, and Bruce D. Hammock*
Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue,
Davis, California 95616
Received January 31, 2005
Soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous
chemical mediators involved in the regulation of blood pressure and inflammation. 1,3-
Disubstituted ureas with a polar group located on the fifth atom from the carbonyl group of
urea function are active inhibitors of sEH both in vitro and in vivo. However, their limited
solubility in water and relatively high melting point lead to difficulties in formulating the
compounds and poor in vivo efficacy. To improve these physical properties, the effect of
structural modification of the urea pharmacophore on the inhibition potencies, water solubilities,
octanol/water partition coefficients (log P), and melting points of a series of compounds was
evaluated. For murine sEH, no loss of inhibition potency was observed when the urea
pharmacophore was modified to an amide function, while for human sEH 2.5-fold decreased
inhibition was obtained in the amide compounds. In addition, a NH group on the right side of
carbonyl group of the amide pharmacophore substituted with an adamantyl group (such as
compound 14) and a methylene carbon present between the adamantyl and amide groups were
essential to produce potent inhibition of sEH. The resulting amide inhibitors have 10-30-fold
better solubility and lower melting point than the corresponding urea compounds. These findings
will facilitate synthesis of sEH inhibitors that are easier to formulate and more bioavailable.
Introduction
stable inhibitors of sEH. These compounds are competi-
tive, tight-binding inhibitors with nanomolar KI values
that interact stoichiometrically with purified recombi-
nant sEH.16 These inhibitors efficiently reduce epoxide
hydrolysis in several in vitro and in vivo models.6,16,17
However, these dialkyl inhibitors have limited solubility
in water and high melting point, which likely affect their
in vivo efficacy and make formulation difficult.5,18
Recently, we reported that compounds with a polar
functional group located on the fifth/sixth atom from the
carbonyl group of the urea pharmacophore are potent
inhibitors, and their solubility in water is also improved
without drop of the inhibition potency.19 Analysis of the
X-ray crystal structure of the murine sEH with 1-cy-
clohexyl-3-dodecylurea (CDU), a sEH dialkylurea in-
hibitor, that the urea is bound by hydrogen bonding
between the phenol groups of Tyr381 and Tyr465, and the
carbonyl of the urea, while a salt bridge is formed
between one of the urea N-H and Asp333 (Figure 1).20,21
As illustrated in Figure 1, Gln382 and Trp334, both
conserved residues, form a possible hydrogen-bonding
site for a polar group present at around the fifth or sixth
atom (approximately 7.5 Å) from the urea carbonyl
group. The presence of a functionalized group at this
position helps improve the binding of the inhibitor to
the enzyme due to additional hydrogen binding. How-
ever, the positive effect on the solubility in water of the
inhibitor is still quite limited (<2 µg/mL) and the
resulting compounds still have relatively high melting
points (>100°C).19 Therefore, in the present study, we
report the effect of changes in the structure of inhibitors
functionalized with a polar group on the inhibition
potency and physical properties (e.g. water solubility,
log P, and melting point), specifically modifying the urea
In humans, soluble epoxide hydrolase (sEH; EC
3.3.2.3) is involved in the metabolism of endogenous
chemical mediators such as arachidonic acid,1 linoleic
acid,2 and other lipid epoxides.3 Epoxides of arachidonic
acid (epoxyeicosatrienoic acids or EETs) are known as
effective modulators of blood pressure.4 However, hy-
drolyzed metabolites of EETs, dihydroxyeicosatrienoic
acids (DHETs), generated by sEH diminish this activ-
ity.4 We have reported that treatment with effective
sEH inhibitors significantly reduces the blood pressure
of spontaneous hypertensive rats and angiotensin II
induced hypertension in rats.5-7 Furthermore, male
knockout sEH mice have significantly lower blood
pressure than wild-type mice,8 supporting the role of
sEH in blood pressure regulation. In addition, the EETs
have demonstrated further vascular protective effects
such as antiinflammatory properties in endothelial
cells,9-12 suppression of reactive oxygen species follow-
ing hypoxia-reoxygenation,13 attenuation of vascular
smooth muscle migration,14 and enhancement of a
fibrinolytic pathway.15 In both cellular and animal
models, continued efficacy of EET-mediated hyperten-
sion and cardioprotective effects including inflammation
control is dependent upon epoxide hydrolysis by sEH,2,16
suggesting that the inhibitors of the sEH will be
effective therapeutic agents for hypertension, inflam-
mation, and other disorders that can be addressed by
changing the in vivo concentration of EET.
We previously reported 1,3-disubsituted ureas (and
related carbamates and amides) as very potent and
* To whom correspondence should be addressed at the Department
of Entomology. Phone: 530-752-7519. Fax: 530-752-1537. E-mail:
bdhammock@ucdavis.edu.
10.1021/jm0500929 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/22/2005

3622 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Kim et al.
Scheme 1. Syntheses of 3-Chlorophenyl or
Adamantan-1-ylcarbamoylpentanoic Acid Pentyl Ester (7
and 13) and 3-Chlorophenyl- or
Adamantan-1-ylacetylaminobutyric Acid Pentyl Ester (8
and 14)a
Figure 1. Diagram of the murine sEH active site and a sEH
inhibitor: Distance of potential binding interaction from
primary urea pharmacophore. The primary pharmacophore
consistents of a polar carbonyl group, shown in this case as a
urea with R ) cyclohexyl, adamantyl, or 3-chlorophenyl. The
secondary pharmacophore consistents of a polar group (ester,
ketone, alcohol, or ether) located five/six atoms away from the
carbonyl group of the primary pharmacophore.
a Reagents: (a) 3-chloroaniline (for compound 7) or 1-adaman-
taneamine (for compound 13), EDCI, DMAP, dichloromethane, rt;
(b) 1-bromopentane, potassium carbonate, DMF, rt; (c) (1) 3-chlo-
rophenylacetic Acid (for compound 8), 1-adamantaneacetic acid
(for compound 14), or 1-adamantanecarboxylic acid (for com-
pound 15), EDCI, DMAP, dichloromethane, rt, (2) 1 N aq NaOH,
ethanol, rt.
central pharmacophore to design potent sEH inhibitors
with improved solubility in water.
Chemistry
Syntheses. Compounds in Tables 1 and 2, and
compounds 5 and 6 in Table 3 were prepared according
to the procedures reported previously.19 The syntheses
of 3-chlorophenyl or 1-adamantylcarbarmoylpentanoic
acid pentyl esters (7 and 13) are described in part A of
Scheme 1, and 3-chlorophenylacetyl-, 1-adamantyl-
acetyl-, or 1-adamantylcarbonylaminobutyric acid pen-
tyl ester (8, 14, and 15) and amides without a polar
group (16 and 17) were synthesized by the procedures
outlined in part B of Scheme 1. Coupling of adipic acid
with 3-chloroaniline (for compound 6) or 1-adamantyl-
amine (for compound 13) using 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (EDCI) and
4-(dimethylamino)pyridine (DMAP) in dichloromethane,
followed by alkylation of the acid with bromopentane
in the presence of potassium carbonate (K2CO3) as a
base in dimethylformamide (DMF), yielded compounds
7 and 14, respectively, in 65% yield.19 As described in
part B of Scheme 1, 3-chlorophenylacetic acid (for
compound 8), 1-adamantylacetic acid (for compound 14),
or adamantane-1-carboxylic acid (for compound 15) were
coupled with ethyl 4-aminobutyrate hydrochloride in the
presence of EDCI and DMAP in dichloromethane to give
3-chlorophenyl- or 1-adamantylacetylaminobutyric acid
ethyl ester intermediate (45%), which was hydrolyzed
with an aqueous solution of 1 N NaOH in ethanol and
then alkylated with bromopentane in the presence of
K2CO3 as a base in DMF to afford compounds 8, 14, and
15 in 75-87% yield. For preparation of 16 and 17, the
same coupling reaction as described above (using EDCI
and DMAP) was carried out from the corresponding
starting materials (1-adamantanamine and dodecanoic
acid for compound 16 (93%), and adamantan-1-ylacetic
acid and decylamine for compound 17 (88%)).
Compounds 9, 11, and 12 were synthesized by the
procedures outlined in Scheme 2. Alkylation of succinic
anhydride with pentanol and DMAP as a base in DMF,23
followed by reduction of carboxylic acid with BH3-THF
complex in THF, provided 4-hydroxybutyric acid pentyl
ester (I) in 85% yield.24 Reaction of I with 3-chlorophenyl
isocyanate in DMF yielded 9, in 50% yield. I was also
coupled with 3-chlorophenylacetic acid by using isobutyl
chloroformate and triethylamine as a base to provide
11 (55%).25 Alkylation of I with 4-nitrophenyl chloro-
formate in the presence of triethylamine as a base in
DMF gave 4-nitrophenyl carbonate intermediate (II) in
32% yield, and reaction of II with 3-chlorophenol and
K2CO3 as a base in acetonitrile provided 12 in 85% yield.
Scheme 3 shows synthesis of 4-(3-chlorophenoxycar-
bonylamino)butyric acid ethyl ester (10). Reaction of
ethyl 4-aminobutyrate hydrochloride with triphosgene
in NaHCO3 aqueous solution and dichloromethane gave
4-isocyanato intermediate III (95%),26 which was alky-
lated with 3-chlorophenol in the presence of triethyl-
amine in DMF to afford 10 (45%).
Water Solubility (S, µg/mL). Water solubility was
determined experimentally using the following proce-
dure at 25 ( 1.5 °C. An excess of the test compound (1,

Amide-Based Inhibitors of Epoxide Hydrolase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3623
Scheme 2. Syntheses of 4-Substituted Butyric Acid Pentyl Esters (9, 11, and 12)a
a Reagents: (a) pentanol, DMAP, DMF, rt; (b) BH3-THF complex, THF, -10 °C, N2; (c) 3-chlorophenyl isocyanate, triethylamine,
DMF; (d) 3-chlorophenylacetic acid, isobutyl chloroformate, triethylamine, DMF, rt; (e) 4-nitrophenyl chloroformate, triethylamine, DMF,
rt; (f) 3-chlorophenol, potassium carbonate, acetonitrile, rt.
Scheme 3. Synthesis of
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.34 Protein
concentration was quantified by using the Pierce BCA
assay using fraction V bovine serum albumin (BSA) as
the calibrating standard.
4-(3-Chlorophenoxycarbonylamino)butyric Acid Ethyl
Ester (10)a
IC50 Assay Conditions. IC50 values were determined
as described by using racemic 4-nitrophenyl-trans-2,3-
epoxy-3-phenylpropyl carbonate as substrate.34 En-
zymes (0.12 µM mouse sEH or 0.24 µM human sEH)
were incubated with inhibitors for 5 min in sodium
phosphate buffer (200 µL), 0.1 M, pH 7.4, containing
0.1 mg/mL of BSA, at 30 °C before substrate introduc-
tion ([S] ) 40 µM). Activity was assessed by measuring
the appearance 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
the concentration 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 1-4.
a Reagents: (a) triphosgene, aq NaHCO3/dichloromethane, rt;
(b) 3-chlorophenol, triethylamine, DMF, rt.
4, 13, 14, 16, and 17 in Table 4) 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 1 h of sonication and 24 h of shaking, followed
by centrifugation (5 min, 200g). The water supernatant
(2.5 µL) 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-tetradecylurea (CTU);
1000 ng/mL in methanol]. A regression curve for each
compound was obtained from five standard stock solu-
tions (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 Micromass
Quattro Ultima triple quadrupole tandem mass spec-
trometer (Micromass, Manchester, UK)]. Then, the
absolute amount of each compound was calculated.19,30
Calculated Octanol/Water Partition Coefficients
(P) and Water Solubility (log S). The log P value
estimated by Crippen’s method was generated by using
CS ChemDraw Ultra version 6.0. On the basis of this
log P value, log S value (solubility in water) was also
calculated with the following equation, which has been
suggested by Banerjee et al.:29 log P ) 6.5-0.89(log S)
- 0.015(melting point).
Results and Discussion
1,3-Disubstituted ureas with various alkyl, cycloalkyl,
and aryl groups were reported as potent sEH inhibi-
tors.16,18.28 These urea inhibitors stabilize endogenous
regulatory epoxides in several in vitro and in vivo
models, resulting in the expected biological changes.6,16,17
However, the liphophilicity of 1,3-dialkylureas causes
limited solubility in water, which probably affects their
in vivo efficacy.18,19 In addition, the stability of the
crystals of the urea compounds, indicated by their high
melting point, led to a general lack of solubility, even
in organic solvents. These poor physical properties of
the 1,3-dialkylureas result in undesirable pharmacoki-
netic properties and difficulty in compound formulation
in either an aqueous or oil base.19
Enzyme Preparation. Recombinant murine sEH
and human sEH were produced in a baculovirus expres-
sion system and purified by affinity chromatography.31-33
Morisseau et al.18 showed that the addition of a polar
functional group at the end of a long liphophilic aliphatic

3624 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Kim et al.
Table 1. Inhibition of Mouse and Human sEH by 1-Adamantyl-3-alkylureas
a Enzymes (0.12 µM mouse sEH or 0.24 µM human sEH) were incubated with inhibitors for 5 min in sodium phosphate buffer (200 µL;
pH 7.4) at 30 °C before substrate introduction ([S] ) 40 µM). Results are means ( SD of three separate experiments.
Table 2. Inhibition of Mouse and Human sEH by Ureas and the Corresponding Amides Substituted with Liphophilic Groups
a Enzymes (0.12 µM mouse sEH or 0.24 µM human sEH) were incubated with inhibitors for 5 min in sodium phosphate buffer (200 µL;
pH 7.4) at 30 °C before substrate introduction ([S] ) 40 µM). Results are means ( SD of three separate experiments. b mp: melting point.
chain (12-13 atoms away from the urea function) of
1-(adamantan-1-yl)-3-alkylureas makes compounds more
hydrophilic while retaining their inhibitory potencies.
We assume that such polar groups mimic the carboxylic
acids of suspected endogenous substrates. Furthermore,
we reported recently that urea compounds functional-
ized with a polar group (e.g. ester (1) or alcohol (2) in
Table 1) located on the fifth atom from the urea carbonyl
are as active as nonfunctionalized liphophilic inhibitor
(4), and the resulting functionalized compounds show
a 3-fold better water solubility than the liphophilic
inhibitor. As a following structure of compound 1, an
ether derivative (3) with a polar group on the sixth
atom has also the same inhibition as compounds 1 and
4, indicating that a polar group located on the fifth
and/or sixth atom (Figure 1; termed herein as the
secondary pharmacophore, with the urea moiety being
the primary pharmacophore) from the urea carbonyl
group is effective for preparing potent sEH inhibitors
with improved water solubility.19 However, relatively
high melting point and low water solubility still result
in inhibitors that are difficult to formulate and show
low bioavailability.
We previously reported that compounds with an
amide functionality as the primary pharmacophore
show potent inhibitory activity on murine and human
sEHs, as well.3,18 As described in Table 2, the inhibitory
potency (IC50) of amide compounds disubstituted with
liphophilic groups was observed in a range of 0.1-38
µM with both enzymes, while the corresponding ureas
showed inhibition (IC50) in a range of 0.06-1.4 µM,
implying that an amide functionality would be also
useful in the synthesis of potent inhibitors. As shown
in Table 2, a cyclohexylurea with a substitutent of
2-phenylethyl on the 3-position of the urea function
(a-1) showed very potent inhibition with both murine
and human enzymes. As the substitutent on the 3-posi-
tion of the urea function of compound a-1 was replaced

Amide-Based Inhibitors of Epoxide Hydrolase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3625
Table 3. Inhibition of Mouse and Human sEH by 1-(3-Chlorophenyl)-3-substituted alkylureas
a Enzymes (0.12 µM mouse sEH or 0.24 µM human sEH) were incubated with inhibitors for 5 min in sodium phosphate buffer (200 µL;
pH 7.4) at 30 °C before substrate introduction ([S] ) 40 µM). Results are means ( SD of three separate experiments.
by 3-chlorophenyl (b-1), 4-methoxyphenyl (c-1), and
phenyl (d-1), the potencies were decreased by 2-20-fold
on both enzymes. In the urea substituted with 2-hy-
droxymethylphenyl (e-1), no inhibition was obtained.
Interestingly, when the urea functionality of compounds
a-1, b-1, c-1, and d-1 was modified to the corresponding
amides (a-d-2 and -3), 2-300-fold reduction in inhibi-
tion potency was also obtained as observed in urea
inhibitors. In addition, the amides (e-2 and e-3) corre-
sponding to the urea (e-1) with no inhibition had no
inhibitory potency on either enzyme, suggesting that the
SAR trend of amide inhibitors substituted with lipho-
philic groups was similar to that of ureas. Often, a high
melting point, like the ones of ureas disubstutited with
liphophilic groups, induces bad physical properties such
as low solubility in water and organic solvents.19 The
amide derivatives shown in Table 2 have lower melting
points (30-100°C) than the corresponding urea com-
pounds, suggesting that functionalities such as amides
will be worth examining as the primary pharmacophore
to develop potent inhibitors with improved physical
properties.
tionality with amide, carbamate, ester, and carbonate
derivatives, as shown in Table 3. Compounds with a
3-chlorophenyl group on one side (5-12 in Table 3) were
first synthesized, because the ultraviolet-dense aromatic
group simplifies monitoring their formation and puri-
fication, and the corresponding ureas are relatively good
sEH inhibitors (5 and 6). After optimizing the primary
pharmacophore, the aryl substitutent can be replaced
by an adamantyl group, leading to more potent but
generally UV-transparent inhibitors. In general, 3-chlo-
rophenyl derivatives are 2-5-fold less potent than
adamantyl derivatives with the recombinant murine
and human enzymes.16 In addition, it was shown in the
previous study that an ester (5), alcohol (6), or ketone
located on the fifth atom from the primary urea carbonyl
group is a good functionality for the secondary phar-
macophore and that among these functionalities the
ester functional group can be prepared easily and in a
higher yield than the alcohol or ketone. Therefore, the
ester functionality was maintained as the secondary
pharmacophore in the present study (Table 3). The
replacement of the urea function (5) by an amide
function (7 and 8) results in 5-fold loss of inhibition
potency for the murine sEH and a 2-fold loss for the
human enzyme. Interestingly, for compounds without
Therefore, to further improve the physical properties
of previously reported urea inhibitors, we investigated
the effect of structural modification of the urea func-

3626 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Kim et al.
Table 4. Inhibition of Mouse and Human sEH by 1-Adamantyl-3-alkylureas
a Enzymes (0.12 µM mouse sEH or 0.24 µM human sEH) were incubated with inhibitors for 5 min in sodium phosphate buffer (200 µL;
pH 7.4) at 30 °C before substrate introduction ([S] ) 40 µM). Results are means ( SD of three separate experiments. b Experimentally
obtained solubility in sodium phosphate buffer (0.1 M, pH 7.4) at 25 ( 1 °C. Results are means ( SD of three separate experiments.c log
P calculated by Crippen’s method by using CS ChemDraw 6.0 version. d mp: melting point. e Solubility in water. It was calculated 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. f ND: not determined.
a secondary pharmacophore, at least a 20-fold lower
inhibition was observed for amides compared to the
corresponding urea inhibitor,18 underlying the positive
effect on inhibitor binding of the secondary pharma-
cophore. The presence of the nitrogen atom at the left
side of the carbonyl group of the amide (7) induced 1.5-
fold better inhibition than at the right position (8) for
the murine sEH, while for the human sEH no change
in the potency was observed between these two amides.
The presence of oxygen to the right side of carbonyl
group, yielding carbamate (9), dropped the activity by
10-fold, and interestingly, in the carbamate (10) with
the oxygen in the left of the carbonyl, no inhibition was
observed, confirming that a nitrogen atom (N-H) on the
left side of the carbonyl group is important for potent
inhibition in this series. Replacement of the carbamate
nitrogen on the right side of the carbonyl of compound
9 by a methylene carbon (ester, 11) resulted in total loss
of inhibition for both murine and human sEHs. More-
over, the structure with two oxygen atoms on both sides
of the carbonyl group (carbonate, 12) showed no inhibi-
tion, confirming that the presence of at least one
nitrogen atom (N-H) in the primary pharmacophore is
essential for inhibiting sEHs. These results overall
agreed with previous observation by Morisseau et al.
that at least one free amine (N-H) of the urea function
is necessary for the inhibition potency.16
The data in Table 3 indicate that the best functional-
ity for direct inhibition of pure sEH for the primary
pharmacophore is a urea (5 and 6); however, good
inhibition was obtained with amides (7 and 8). So the
amide functionality was maintained as primary phar-
macophore, and the 3-chlorophenyl group on the left side
of the amide function was then replaced by a potent
adamantyl group. As shown in Table 4, the adamantyl
substitution on the left side of the amide pharmacophore
increased inhibitor potency 2-50-fold (13 and 14) over
that of the 3-chlorophenyl analogue (7 and 8) for both
enzymes, indicating that a bulky cycloalkyl group is a
more efficient substituent of the amide pharmacophore
than an aryl group for inducing potent inhibition, which
is consistent with previous results obtained for urea
derivatives.19 A nitrogen atom present on the right side
of the carbonyl of the amide function (14) produced
10- and 5-fold better inhibition on murine and human
sEHs, respectively, than that on the left side of the
carbonyl group (13). Similarly, the right side nitrogen
atom in the liphophilic amide analogue (17) induced
50-fold better inhibition on human sEH than the nitro-
gen atom on the left side of carbonyl group (16), while

Amide-Based Inhibitors of Epoxide Hydrolase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3627
no change in inhibition potency on the murine sEH was
observed with either compound (16 and 17). On the
other hand, in compounds with a cyclohexyl group on
one side of the amide function in Table 2 and compounds
6 and 7 substituted with a 3-chlorophenyl group, no
reasonable changes in the inhibition potency between
the amide functions as that shown in adamantyl deriva-
tives (13, 14, 16, and 17) were obtained, suggesting that
a bulky adamantyl group makes a nitrogen atom
present on the right side of the amide function more
optimal for producing potent inhibition than the left side
nitrogen atom. Interestingly, the same inhibition po-
tency as observed for urea 1 was obtained for amide 14
for the murine sEH, while for the human sEH a 2.5-
fold lower inhibition was observed for the amide inhibi-
tor (14) over the urea analogue (1). Furthermore, the
activities obtained for compounds 14 and 1 are as potent
as the enzyme inhibition observed for the liphophilic
urea 4, suggesting that structural modifications of the
urea primary pharmacophore can be effective in produc-
ing potent inhibitors for murine and human enzymes
in the presence of a secondary pharmacophore. In
addition, the amide function of compound 15, without
a methylene carbon between the adamantane and amide
groups, dropped the inhibition 50- and 20-fold for
murine and human sEHs, respectively, implying that
a methylene carbon present between those groups is
important to make the amide inhibitors potent.
The water solubility of these inhibitors was experi-
mentally measured (Table 4). Around 10-fold better
solubility was obtained for compounds 13 and 14 with
the amide primary pharmacophore than with the urea
functionality (1). However, no improvement in water
solubility was observed in nonfunctionalized compounds
with the amide primary pharmacophore (16 and 17)
compared to the corresponding urea (4). When inte-
grated, these results indicate that a big improvement
in water solubility can be gained when the liphophilic
tail is functionalized with the secondary pharmacophore
and when the primary pharmacophore is an amide. As
expected, decreased log P values (∼3) and much lower
melting points (oils) were obtained in the functionalized
amide inhibitors (13 and 14), indicating that the amide
function of substituted inhibitors improved solubility in
water and that the calculated log P values and melting
points are useful factors to estimate water solubility,
which is a result consistent with that previously re-
ported in our lab.19 The log P values of neutral im-
miscible liquids generally run parallel with their water
solubility; however, estimating solubility of solids is
complex, because it 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: 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.29 As
shown in Table 4, a 10-fold increase in calculated water
solubility (log S), which was determined according to
the above equation, was obtained in functionalized
amide inhibitors (13 and 14) compared to the corre-
sponding urea (1), which was consistent with the
solubility obtained experimentally. However, consis-
tency between calculated and experimentally deter-
mined solubility was not observed for the higher melting
compounds lacking the secondary pharmacophore. These
data suggest that Banerjee’s equation might be limited
to estimating the water solubility of more polar com-
pounds with lower melting points.
This investigation was directed toward producing
potent sEH inhibitors with improved physical properties
(e.g. solubilities and melting points). We found that no
loss of inhibition potency for sEHs was observed when
the urea primary pharmacophore was replaced with an
amide functionality, as long as an ester secondary
pharmacophore is present on the fifth atom from the
carbonyl of the primary pharmacophore. Under these
conditions for the primary pharmacophore, a 10- and
5-fold better inhibition of murine and human sEHs,
respectively, is obtained if the nitrogen of the amide
function is placed on the right of the carbonyl. Further-
more, the presence of a methylene carbon between the
amide function and a bulky cycloalkyl group (compound
14) is essential to produce potent amide inhibitors. In
addition, the resulting amide inhibitors had 10-30-fold
better solubility in water and lower melting point than
the corresponding urea compounds. Also in general
there is a predictive correlation between the structure-
activity relationships (SARs) among compounds with a
urea and amide central pharmacophore. The ester
derivatives at the secondary pharmacophore provide a
facile synthetic route to exploring the steric and elec-
tronic effects of the right distal structural components
on enzyme binding. However, it is likely that an
unhindered ester would be too unstable in vivo to give
effective blood levels. However, such compounds may
prove valuable as soft drugs that can be administered
directly to the target tissue such as skin, air way, or
intestinal mucosa. These findings are important for the
design of potent inhibitors with improved physical
properties that will lead to obtaining intravenous or
orally available therapeutic agents for hypertension,
inflammation, and other disorders that can be addressed
by changing the in vivo concentration of chemical
mediators that contain an epoxide.
Experimental Section
Syntheses. All melting points were determined with a
Thomas-Hoover apparatus (A. H. Thomas Co.) and are uncor-
rected. 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, IN;
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 multiplici-
ties 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 representative compounds.
5-(3-Chlorophenylcarbamoyl)pentanoic Acid Pentyl
Ester (7). To a solution of adipic acid (0.5 g, 3.42 mmol) and
DMAP (0.42 g, 3.42 mmol) in dichloromethane (30 mL) and
DMF (3 mL) was added 3-chloroaniline (0.44 g, 3.42 mmol) at
room temperature. After stirring 10 min, 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide hydrochloride (0.65 g, 3.42 mmol;
EDCI) was added portionwise to the mixture at room temper-
ature. The reaction was stirred for 12 h. A 1 N aqueous HCl
solution (20 mL) was poured into the reaction mixture, and

3628 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10
Kim et al.
5-(3-chlorophenylcarbomoyl)pentanoic acid was extracted with
dichloromethane (30 mL). The organic solution was washed
with water (50 mL), dried over Na2SO4, and concentrated. This
residue was used for the next reaction without further
purification.
[M + H]+ 336.25, found [M + H]+ 336.34. Anal. (C20H33NO3)
C, H, N.
4-(3-Chlorophenylcarbamoyloxy)butyric Acid Pentyl
Ester (9). To a solution of succinic anhydride (3.58 g, 35.7
mmol) and DMAP (4.16 g, 34.0 mmol) in DMF (40 mL) was
added pentanol (3.0 g, 34.0 mmol) at room temperature under
nitrogen. After stirring for 12 h, succinic acid pentyl ester was
extracted with ether (40 mL), and the ether solution was
washed with 1 N aqueous HCl solution (20 mL) and water (40
mL), dried over sodium sulfate (Na2SO4), and concentrated.
The residue was purified by column chromatography on silica
gel eluting with hexane and ethyl acetate (3:1) to give the
succinic acid pentyl ester (6.07 g, 95%) as an oil. To the solution
of this acid in tetrahydrofuran (THF, 60 mL) was added 1 M
BH3-THF complex (64.53 mL, 64.5 mmol) at -10 °C under
nitrogen, and the reaction mixture was allowed to warm to
room temperature with stirring. After stirring for 12 h at room
temperature, 5% NaHCO3 aqueous solution (50 mL) was added
to the reaction and then the reduced alcohol (I) was extracted
with ethyl acetate (50 mL). The ethyl acetate solution was
dried over Na2SO4 and concentrated to give I (5.06 g, 90%).
4-Hydroxybutyric acid pentyl ester (I; 100 mg, 0.57 mmol)
was added to a solution of 3-chlorophenyl isocyanate (88 mg,
0.57 mmol) and triethylamine (0.12 mL, 0.86 mmol; TEA) in
DMF (15 mL) at room temperature. The mixture was allowed
to stand at room temperature for 12 h, the product was
extracted with ether (20 mL), and the ether solution was
washed with 1 N aqueous HCl 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 (5:1) to afford 9 (94 mg, 50%) 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), 1.71 (2H, quint, J ) 6.9 Hz), 2.66
(2H, t, J ) 6.9 Hz), 2.74 (2H, q, J ) 6.9 Hz), 4.10 (2H, t,
J ) 6.9 Hz), 7.05-7.08 (1H, m), 7.18-7.22 (3H, m), 7.35
(1H, s); LC-MS (ESI) m/z calcd for C16H22ClNO4 [M + H]+
328.12, found [M + H]+ 328.13; mp 82 °C. Anal. (C16H22ClNO4)
C, H, N.
To the solution of the above carbamoylpentanoic acid (0.72
g, 2.80 mmol) in DMF (15 mL) was added K2CO3 (0.58 g, 4.21
mmol) and 1-bromopentane (0.64 g, 4.21 mmol) at room
temperature. After stirring for 12 h, the product was extracted
with ether (30 mL), and the ether solution was washed with
an aqueous solution of 1 N NaOH (15 mL) and water (40 mL),
dried over Na2SO4, and concentrated. The residue was purified
using silica gel column chromatography (hexane:ethyl acetate
) 5:1) to afford 7 (0.59 g, 65%): 1H NMR δ (CDCl3) 0.91 (3H,
t, J ) 6.9 Hz), 1.29-1.37 (4H, m), 1.60-1.66 (2H, m), 1.70-
1.78 (4H, m), 2.35-2.43 (4H, m), 4.08 (H, t, J ) 6.9 Hz), 7.05-
7.09 (1H, m), 7.21-7.23 (1H, m), 7.37-7.40 (1H, m), 7.52-
7.55 (1H, m), 7.68 (1H, s); LC-MS (ESI) m/z calcd for
C17H24ClNO3 [M + H]+ 326.14, found [M + H]+ 326.16, mp 82
°C. Anal. (C17H24ClNO3) C, H, N.
Compound 13 was prepared with the same method used for
the preparation of compound 7 using adamantylamine instead
of 3-chloroaniline: 1H NMR δ (CDCl3) 0.91 (3H, t, J ) 6.9 Hz),
1.29-1.43 (4H, m), 1.64-1.69 (12H, m), 1.94-1.98 (6H, m),
2.06-2.13 (5H, m), 2.32 (2H, t, J ) 6.9 Hz), 4.06 (H, t, J )
6.9 Hz), 5.16 (1H, s); LC-MS (ESI) m/z calcd for C21H35NO3
[M + H]+ 350.26, found [M + H]+ 350.30. Anal. (C21H35NO3)
C, H, N.
4-[2-(3-Chlorophenyl)acetylamino]butyric Acid Pentyl
Ester (8). To a solution of 3-chlorophenylacetic acid (0.5 g,
2.93 mmol) and DMAP (0.36 g, 2.93 mmol) in dichloromethane
(30 mL) was added ethyl 4-aminobutyrate hydrochloride (0.49
g, 2.93 mmol) at room temperature. After stirring for 10 min,
EDCI (0.56 g, 2.93 mmol) was added portionwise to the
mixture at room temperature. The reaction was stirred for 12
h. A 1 N aqueous HCl solution (20 mL) was poured into the
reaction mixture, and 4-[2-(3-chlorophenyl)acetylamino]butyric
acid ethyl ester was extracted with ether (30 mL). The ether
solution was washed with water (50 mL), dried over Na2SO4,
and concentrated. To the residue dissolved in ethanol (10 mL)
was added 1 N aqueous NaOH solution (6 mL), and after 12 h
of stirring at room temperature, the product was extracted
with dichloromethane (30 mL). The organic solution was
washed with water (30 mL), dried over Na2SO4, and concen-
trated to give 4-[2-(3-chlorophenyl)acetylamino]butyric acid
(0.6 g, 80%). A mixture of this acid (0.6 g, 2.35 mmol), K2CO3
(0.49 g, 3.52 mmol), and 1-bromopentane (0.53 g, 3.52 mmol)
in DMF (20 mL) was stirred overnight at room temperature.
The product was extracted with ether (40 mL), and the ether
solution was washed with water (50 mL), dried over Na2SO4,
and concentrated. The residue was purified using silica gel
column chromatography (hexane:ethyl acetate ) 3:1) to afford
8 as an oil (0.74 g, 97%): 1H NMR δ (CDCl3) 0.91 (3H, t, J )
6.9 Hz), 1.26-1.33 (4H, m), 1.59-1.63 (2H, m), 1.80 (2H, quint,
J ) 6.9 Hz), 2.31 (2H, t, J ) 6.9 Hz), 3.27 (2H, q, J ) 6.9
Hz), 3.52 (2H, s), 4.04 (2H, t, J ) 6.9 Hz), 5.72 (1H, s), 7.13-
7.17 (2H, m), 7.27-7.30 (2H, m); LC-MS (ESI) m/z calcd for
C17H24ClNO3 [M + H]+ 326.14, found [M + H]+ 326.15. Anal.
(C17H24ClNO3) C, H, N.
Acknowledgment. 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 ES-
04699, NIH/NHLBI R01 HL59699-06A1, UC System-
wide Biotechnology Research and Education Training
Grant #2001-07, NIEHS Center for Children’s Environ-
mental Health & Disease Prevention P01 ES11269, and
NIH/NIDDK UC Davis Clinical Nutrition Research Unit
P30 DK35747, Pilot Project.
Supporting Information Available: Syntheses and de-
tailed analytical data for compounds 3, a-2, a-3, b-2, b-3, c-2,
c-3, d-2, d-3, 10-12, 16, and 17. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Zeldin, D. C.; Kobayashi, J.; Falck, J. R.; Winder, B. S.;
Hammock, B. D.; Snapper J. R.; Capdevila, J. H. Regio- and
enantiofacial selectivity of epoxyeicosatrienoic acid hydration by
cytosolic epoxide hydrolase. J. Biol. Chem. 1993, 268, 6402-
6407.
(2) Moghaddam, M. F.; Grant D. F.; Cheek, J. M.; Greene, J. F.;
Williamson, K. C.; Hammock, B. D. Bioactivation of leukotoxins
to their toxic diols by epoxide hydrolase. Nat. Med. 1997, 3, 562-
567.
(3) Carroll, M. A.; McGiff, J. C. A new class of lipid mediators:
Cytochrome P450 arachidonate metabolites. Thorax 2000, 55,
S13-16.
(4) Capdevila, J. H.; Falck, J. R.; Harris, R. C. Cytochrome P450
and arachidonic acid bioactivation: Molecular and functional
properties on the arachidonate monooxygenase. J. Lipid Res.
2000, 41, 163-181.
Compounds 14 and 15 were prepared with the same method
used for the preparation of compound 8 using 1-adamanty-
lacetic acid or adamantane-1-carboxylic acid instead of 3-chlo-
rophenylacetic acid. Compound 14: 1H NMR δ (CDCl3) 0.91
(3H, t, J ) 6.9 Hz), 1.29-1.35 (4H, m), 1.35-1.72 (14H, m),
1.84 (2H, quint, J ) 6.9 Hz), 1.91 (2H, s), 1.97 (3H, m), 2.37
(2H, t, J ) 6.9 Hz), 3.29 (2H, q, J ) 6.9 Hz), 4.07 (2H, t, J )
6.9 Hz), 5.66 (1H, s); LC-MS (ESI) m/z calcd for C21H35NO3
[M + H]+ 350.26, found [M + H]+ 350.29. Anal. (C21H35NO3)
C, H, N.
(5) Yu, Z.; Xu, F.; Huse, L. M.; Morisseau, C.; Draper, A. J.;
Newman, J. W.; Parker, C.; Graham, L.; Engler, M. M.; Ham-
mock, B. D.; Zeldin, D. C.; Kroetz, D. L. Soluble epoxide
hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic
acids. Circ. Res. 2000, 87, 992-998.
Compound 15: 1H NMR δ (CDCl3) 0.91 (3H, t, J ) 6.9 Hz),
1.29-1.35 (4H, m), 1.60-1.86 (16H, m), 2.03 (3H, m), 2.35
(2H, t, J ) 6.9 Hz), 3.29 (2H, q, J ) 6.9 Hz), 4.07 (2H, t, J )
6.9 Hz), 5.85 (1H, s); LC-MS (ESI) m/z calcd for C20H33NO3

Amide-Based Inhibitors of Epoxide Hydrolase
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 10 3629
(6) Imig, J. D.; Zhao, X.; Capdevila, J. H.; Morisseau, C.; Hammock,
B. D. Soluble epoxide hydrolase inhibition lowers arterial blood
pressure in angiotensin II hypertension. Hypertension 2002, 39,
690-694.
(7) Zhao, X.; Yamamoto, T.; Newman, J. W.; Kim, I. H.; Watanabe,
T.; Hammock, B. D.; Stewart, J.; Pollock, J. S.; Pollock, D. M.;
Imig, J. D. Soluble epoxide hydrolase inhibition protects the
kidney from hypertension-induced damage. J. Am. Soc. Nephrol.
2004, 15, 1244-1253.
(8) Sinal, C. J.; Miyata, M.; Tohkin, M.; Nagata, K.; Bend, J. R.;
Gonzalez, F. J. Targeted disruption of soluble epoxide hydrolase
reveals a role in blood pressure regulation. J. Biol. Chem. 2000,
275, 40504-40510.
(9) Oltman, C. L.; Weintraub, N. L.; VanRollins, M.; Dellsperger,
K. C. Epoxyeicosatrienoic acids and dihydroxyeicosatrienoic
acids are potent vasodilators in the canine coronary microcir-
culation. Circ. Res. 1998, 83, 932-939.
(10) Fisslthaler, B.; Popp, R.; Kiss, L.; Potente, M.; Harder, D. R.;
Fleming, I.; Busse, R. Cytochrome P450 2C is an EDHF synthase
in coronary arteries. Nature 1999, 401, 493-497.
(11) Node, K.; Huo, Y.; Ruan, X.; Yang, B.; Spiecker, M.; Ley, K.;
Zeldin, D. C.; Liao, J. K. Antiinflammatory properties of cyto-
chrome P450 epoxygenase-derived eicosanoids. Science 1999,
285, 1276-1279.
(12) Campbell, W. B. New role for epoxyeicosatrienoic acids as
antiinflammatory mediators. Trends Pharmacol. Sci. 2000, 21,
125-127.
(13) Yang, B.; Graham, L.; Dikalov, S.; Mason, R. P.; Falck, J. R.;
Liao, J. K.; Zeldin, D. C. Overexpression of cytochrome P450
CYP2J2 protects against hypoxia-reoxygenation injury in cul-
tured bovine aortic endothelial cells. Mol. Pharmacol. 2001, 60,
310-320.
(14) Sun, J.; Sui, X.; Bradbury, J. A.; Zeldin, D. C.; Conte, M. S.; Liao,
J. K. Inhibition of vascular smooth muscle cell migration by
cytochrome P450 epoxygenase-derived eicosanoids. Circ. Res.
2002, 90, 1020-1027.
(15) Node, K.; Ruan, X.; Dai, J.; Yang, S.; Graham, L.; Zeldin, D. C.;
Liao, J. K. Activation of Gs mediates induction of tissue-type
plasminogen activator gene transcription by epoxyeicosatrienoic
acids. J. Biol. Chem 2001, 276, 15983-15989.
(16) Morisseau, C.; Goodrow, M. H.; Dowdy, D.; Zheng, J.; Greene,
J. F.; Sanborn, J. R.; Hammock, B. D. Potent urea and carbamate
inhibitors of soluble epoxide hydrolases. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 8849-8854.
(17) Newman, J. W.; Denton, D. L.; Morisseau, C.; Koger, C. S.;
Wheelock C. E.; Hinton, D. E.; Hammock, B. D. Evaluation of
fish models of soluble epoxide hydrolase inhibition. Environ.
Health Perspect. 2001, 109, 61-66.
(18) Morisseau, C.; Goodrow, M. H.; Newman, J. W.; Wheelock, C.
E.; Dowdy, D. L.; Hammock, B. D. Structural refinement of
inhibitors of urea-based soluble epoxide hydrolases. Biochem.
Pharmacol. 2002, 63, 1599-1608.
(19) Kim, I. H.; Morisseau, C.; Watanabe, T.; Hammock, B. D. Design,
synthesis, and biological activity of 1,3-disubstituted ureas as
potent inhibitors of the soluble epoxide hydrolase of increased
water solubility. J. Med. Chem. 2004, 47, 2110-2122.
(20) Argiriadi, M. A.; Morisseau, C.; Hammock, B. D.; Christianson,
D. W. Detoxification of environmental mutagens and carcino-
gens: Structure, mechanism, and evolution of liver epoxide
hydrolase. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10637-10642.
(21) Argiriadi, M. A.; Morisseau, C.; Goodrow, M. H.; Dowdy, D. L.;
Hammock, B. D.; Christianson, D. W. Binding of alkylurea
inhibitiors to epoxide hydrolase implicates active site tyrosines
in substrate activation. J. Biol. Chem. 2000, 275, 15265-15270.
(22) Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. Esterification of
N-protected alpha-amino acids with alcohol/carbodiimide/4-
(dimethylamino)pyridine. Racemization of aspartic and glutamic
acid derivatives. J. Org. Chem. 1982, 47, 1962-1965.
(23) Larsen, S. D.; Barf, T.; Liljebris, C.; May, P. D.; Ogg, D.;
O’Sullivan, T. J.; Palazuk, B. J.; Schostarez, H. J.; Stevens, F.
C.; Bleasdale, J. E. Synthesis and biological Activity of a novel
class of small molecular weight peptidomimetic competitive
inhibitors of protein tyrosine phosphatase 1B. J. Med. Chem.
2002, 45, 598-622.
(24) Yoon, N. M.; Cho, B. T. Rapid reduction of carboxylic acid salts
with borane in tetrahydrofuran. Tetrahedron Lett. 1982, 23,
2475-2478.
(25) Balboni, G.; Guerrini, R.; Salvadori, S.; Bianchi, C.; Rizzi, D.;
Bryant, S. D.; Lazarus, L. H. Evaluation of the Dmt-Tic
pharmacophore: Conversion of a potent δ-opioid receptor an-
tagonist into a potent δ agonist and ligands with mixed proper-
ties. J. Med. Chem. 2002, 45, 713-720.
(26) Weisenfeld, R. B. Synthesis of disiloxanediyl diamines via a facile
homocondensation of amino silanols. J. Org. Chem. 1986, 51,
2434-2436.
(27) Ellison, R. A.; Lukenbach, E. R.; Chiu, C. Cyclopentenone
synthesis via aldol condensation. Synthesis of a key prostag-
landin intermediate. Tetrahedron Lett. 1975, 16, 499-502.
(28) McElroy, N. R.; Jurs, P. C.; Morisseau, C.; Hammock, B. D.
QSAR and classification of murine and human soluble epoxide
hydrolase inhibition by urea-like compounds. J. Med. Chem.
2003, 46, 1066-1080.
(29) Banerjee, S.; Yalkowsky, S. H.; Valvani, S. C. Water solubility
and octanol/water partition coefficients of organics. Limitations
of the solubility-partition coefficient correlation. Environ. Sci.
Technol. 1980, 10, 1227-1229.
(30) Watanabe, T.; Hammock, B. D. Rapid determination of soluble
epoxide hydrolase inhibitors in rat hepatic microsomes by high-
performance liquid chromatography with electrospray tandem
mass spectrometry. Anal. Biochem. 2001, 299, 227-234.
(31) Grant, D. E.; Storms, D. H.; Hammock, B. D. Molecular cloning
and expression of murine liver soluble epoxide hydrolase. J. Biol.
Chem. 1993, 268, 17628-17633.
(32) Beetham, J. K.; Tian, T.; Hammock, B. D. cDNA cloning and
expression of a soluble epoxide hydrolase from human liver.
Arch. Biochem. Biophys. 1993, 305, 197-201.
(33) Wixtrom, R. N.; Silva, M. H.; Hammock, B. D. Affinity purifica-
tion of cytosolic epoxide hydrolase using derivatized epoxy-
activated Sepharose gels. Anal. Biochem. 1998, 169, 71-80.
(34) Dietze, E. C.; Kuwano, E.; Hammock, B. D. Spectrophotometric
substrates for cytosolic epoxide hydrolase. Anal. Biochem. 1994,
216, 176-187.
JM0500929