Substituted adamantyl-urea inhibitors of the soluble epoxide hydrolase dilate mesenteric resistance vessels

Article abstract of DOI:10.1124/jpet.106.103556

The epoxyeicosatrienoic acids (EETs) have been identified as endothelium-derived hyperpolarizing factors. Metabolism of the EETs to the dihydroxyeicosatrienoic acids is catalyzed by soluble epoxide hydrolase (sEH). Administration of urea-based sEH inhibitors provides protection from hypertension-induced renal injury at least in part by lowering blood pressure. Here, we investigated the hypothesis that a mechanism by which sEH inhibitors elicit their cardiovascular protective effects is via their action on the vasculature. Mesenteric resistance arteries were isolated from Sprague-Dawley rats, pressurized, and constricted with the thromboxane A2 agonist U46619 (9,11-dideoxy-11,9-epoxymethano-prostaglandin F2α). Mesenteric arteries were then incubated with increasing concentrations of the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA). AUDA resulted in a concentration-dependent relaxation of mesenteric arteries, with 10 μM resulting in a 48 ± 7% relaxation. Chain-shortened analogs of AUDA had an attenuated vasodilatory response. Interestingly, at 10 μM, the sEH inhibitors 1-cyclohexyl-3-dodecylurea, 12-(3-cyclohexylureido) dodecanoic acid, and 950 [adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea] were significantly less active, resulting in a 25 ± 8%, 10 ± 9%, and -8 ± 3% relaxation, respectively. Treatment of mesenteric arteries with tetraethylammonium, iberiotoxin, ouabain, or glibenclamide did not alter AUDA-induced relaxation. The AUDA-induced relaxation was completely inhibited when constricted with KCl. In separate experiments, denuding mesenteric resistance vessels did not alter AUDA-induced relaxation. Taken together, these data demonstrate that adamantyl-urea inhibitors have unique dilator actions on vascular smooth muscle compared with other sEH inhibitors and that these dilator actions depend on the adamantyl group and carbon chain length. Copyright

Full text of DOI:10.1124/jpet.106.103556

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022-3565/06/3183-1307–1314$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
JPET 318:1307–1314, 2006
Vol. 318, No. 3
103556/3134742
Printed in U.S.A.
Substituted Adamantyl-Urea Inhibitors of the Soluble Epoxide
Hydrolase Dilate Mesenteric Resistance Vessels
Jeffrey J. Olearczyk, Mary B. Field, In-Hae Kim, Christophe Morisseau,
Bruce D. Hammock, and John D. Imig
Vascular Biology Center (J.J.O, M.B.F, J.D.I.) and Department of Physiology (J.D.I.), Medical College of Georgia, Augusta,
Georgia; and Department of Entomology and Cancer Research Center (I.-H.K., C.M., B.D.H.), University of California, Davis,
California
Received February 24, 2006; accepted June 12, 2006
ABSTRACT
The epoxyeicosatrienoic acids (EETs) have been identified as 7% relaxation. Chain-shortened analogs of AUDA had an at-
endothelium-derived hyperpolarizing factors. Metabolism of tenuated vasodilatory response. Interestingly, at 10 ␮M, the
the EETs to the dihydroxyeicosatrienoic acids is catalyzed by sEH inhibitors 1-cyclohexyl-3-dodecylurea, 12-(3-cyclohexyl-
soluble epoxide hydrolase (sEH). Administration of urea-based ureido)dodecanoic acid, and 950 [adamantan-1-yl-3-{5-[2-(2-
sEH inhibitors provides protection from hypertension-induced ethoxyethoxy)ethoxy]pentyl}urea] were significantly less active,
renal injury at least in part by lowering blood pressure. Here, we resulting in a 25 Ϯ 8%, 10 Ϯ 9%, and Ϫ8 Ϯ 3% relaxation,
investigated the hypothesis that a mechanism by which sEH respectively. Treatment of mesenteric arteries with tetraethyl-
inhibitors elicit their cardiovascular protective effects is via their ammonium, iberiotoxin, ouabain, or glibenclamide did not alter
action on the vasculature. Mesenteric resistance arteries were AUDA-induced relaxation. The AUDA-induced relaxation was
isolated from Sprague-Dawley rats, pressurized, and con- completely inhibited when constricted with KCl. In separate
stricted with the thromboxane A2 agonist U46619 (9,11- experiments, denuding mesenteric resistance vessels did not
dideoxy-11,9-epoxymethano-prostaglandin F2␣). Mesenteric alter AUDA-induced relaxation. Taken together, these data
arteries were then incubated with increasing concentrations of demonstrate that adamantyl-urea inhibitors have unique dilator
the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic actions on vascular smooth muscle compared with other sEH
acid (AUDA). AUDA resulted in a concentration-dependent re- inhibitors and that these dilator actions depend on the adaman-
laxation of mesenteric arteries, with 10 ␮M resulting in a 48 Ϯ tyl group and carbon chain length.
The soluble epoxide hydrolase (sEH) enzyme is found in a sponding diols and/or glycols by catalyzing the addition of
variety of mammalian tissues, with the highest activity mea- water to the epoxide moiety (Morisseau and Hammock, 2005;
sured in the liver, kidney, intestine, and vascular tissue Newman et al., 2005). An endogenously produced, biologi-
(
Wang et al., 1982; Yu et al., 2004; Newman et al., 2005). cally active group of epoxides that serve as a substrate for
Within these tissues, sEH metabolizes endogenous and/or sEH are the epoxyeicosatrienoic acids (EETs). EETs are cy-
exogenous epoxide-containing compounds to their corre- tochrome P450 epoxygenase metabolites of arachidonic acid
that have been identified as important regulatory molecules
in the cardiovascular and renal circulations. EETs have been
Institutional National Research Service Award Grants 5T32HL066993, identified as potential endothelial-derived hyperpolarizing
This work was supported in part by the National Institutes of Health
HL059699, HL074167, and DK38226; by National Institute of Environmental
Health Sciences (NIEHS) Grant ES02710; by NIEHS Superfund Grant
factors and have been reported to have specific anti-inflam-
matory properties (Campbell et al., 1996; Fisslthaler et al.,
1999; Node et al., 1999; Falck et al., 2003). The EETs are
metabolized by the sEH into their corresponding dihy-
droxyeicosatrienoic acids, thereby resulting in partial or com-
ES04699; and by NIEHS Center Grants ES05707 and ESO013933. J.I. is an
Established Investigator of the American Heart Association.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.103556.
ABBREVIATIONS: sEH, soluble epoxide hydrolase; EET, epoxyeicosatrienoic acid; AUDA, 12-(3-adamantan-1-yl-ureido)dodecanoic acid;
CUDA, 12-(3-cyclohexyl-ureido)dodecanoic acid; AUOA, 8-(3-adamantyl-ureido)-octanoic acid; AUHA, 6-(3-adamantyl-ureido)-hexanoic
acid; AUBA, 4-(3-adamantan-1-yl-ureido)butanoic acid; AADU, 1-adamantyl-3-(12-aminododecyl)urea; 950, adamantan-1-yl-3-{5-[2-(2-
ethoxyethoxy)ethoxy]pentyl}urea; DMSO, dimethyl sulfoxide; CDU, 1-cyclohexyl-3-dodecylurea; IbTX, iberiotoxin; MS-PPOH, N-methylsulfonyl-
6-(2-propargyloxyphenyl)hexanamide; PBS-T, phosphate-buffered saline containing 0.1% Tween 20; TEA, tetraethylammonium; DMF, dimethyl
formamide; E-64, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; LC, liquid chromatography; MS, mass spectrometry; TOF, time-of-
flight; U46619, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2␣.
1307

1
308
Olearczyk et al.
plete loss of activity (Moghaddam et al., 1997; Zeldin, 2001; 950 were dissolved in dimethyl sulfoxide (DMSO) and sonicated to
Ϫ1
Spector et al., 2004).
make 10
1
M stock solutions to be added to the vessel chamber.
-Cyclohexyl-3-dodecylurea (CDU) was dissolved in 75% ethanol to
As a result of the renal and cardiovascular protective ef-
fects afforded by the EETs, inhibiting their metabolism has
identified the sEH enzyme as a potential therapeutic target
in diseases like hypertension and inflammation. A number of
sEH inhibitors have been developed, and their administra-
tion in animal models of disease has proved to have beneficial
effects. In the spontaneously hypertensive rat, administra-
Ϫ2
make a 10 M stock. Doses of each sEH inhibitor (0.1, 1.0, 10, and
00 ␮M) were added to the vessel chamber (on the exterior of the
1
vessel) every 5 min, and responses were calculated as the mean
lumen diameter during the last 2 min of each dosage. Acetylcholine
10 ␮M, Sigma-Aldrich) was given at the end of the experimental
protocol to ensure endothelial integrity of the vessel, and the final
lumen diameter was calculated as the mean diameter over the last 2
(
tion of the sEH inhibitor N,NЈ-dicyclohexylurea lowered min of the acetylcholine response. To ensure that the vehicle into
blood pressure (Yu et al., 2000). Another sEH inhibitor, 1-cy- which the drugs were dissolved did not alter vessel reactivity, exper-
clohexyl-3-dodecylurea, also lowered blood pressure in an iments were conducted using either DMSO or ethanol in the absence
of drug. DMSO did not change vascular reactivity at any of the
animal model of angiotensin-dependent hypertension (Imig
concentrations used in these experiments. The addition of ethanol to
et al., 2002; Zhao et al., 2004). The sEH inhibitor 12-(3-
the vessel chamber also did not result in a significant change in
adamantan-1-yl-ureido)dodecanoic acid (AUDA) lowered
vascular reactivity at any of the concentrations used in these exper-
blood pressure and decreased renal damage associated with
iments.
angiotensin-dependent, salt-sensitive hypertension (Imig et
al., 2005). In addition to lowering blood pressure, it was
demonstrated that sEH inhibition also inhibits the inflam-
In inhibitor experiments, tetraethylammonium (1 mM; Sigma-
Aldrich), ouabain (100 ␮M; Sigma-Aldrich), and glibenclamide (1
M; Sigma-Aldrich) were used postconstriction and for 10 to 20 min
␮
matory response. Administration of 12-(3-adamantane-1-yl- prior to dosing with AUDA. Iberiotoxin (IbTX; Tocris Bioscience,
ureido)-dodecanoic acid n-butyl ester to spontaneously hyper- Ellisville, MO) was reconstituted with deionized water, and a 100 nM
tensive rats exposed to tobacco smoke significantly dose was given 15 min before constriction with U46619. N-methyl-
attenuated the tobacco smoke-induced infiltration of proin- sulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) was dis-
solved in 50% ethanol, and a 1 ␮M dose was given 30 min before
flammatory cells into the lung (Smith et al., 2005). The same
constriction with U46619. Potassium chloride (40 mM; Sigma-
sEH inhibitor was also reported to decrease lipopolysaccha-
Aldrich) was used to constrict vessels 20 min prior to addition of
ride-induced tissue injury and mortality (Schmelzer et al.,
AUDA. Endothelium-denuded vessels were prepared by inserting a
2
005).
human hair about which the vessel was rotated using a rolling
motion atop a raised, crowned surface in a dish of Krebs physiolog-
ical salt solution. To ensure that the vessel was no longer capable of
an endothelium-related dilation, acetylcholine (100 ␮M for 5 min)
Many of the renal and cardiovascular protective effects
elicited with sEH inhibition are attributed to the measured
decrease in blood pressure. To better understand the mech-
anism by which sEH inhibition decreases blood pressure, we was given. If an acetylcholine-induced relaxation was not observed,
turned our attention to the vasculature. In the present study, the vessel was rinsed and allowed to equilibrate for 10 min before
we investigated the hypothesis that a mechanism by which constriction with U46619 and treatment with AUDA. At the end of
the experimental protocol, sodium nitroprusside (100 ␮M) was added
sEH inhibitors elicit their cardiovascular protective effects is
to the chamber to demonstrate that the endothelium-denuded vessel
via their direct action on the vasculature.
was capable of relaxation.
Western Blot Analysis. Mesenteric artery segments were ob-
tained from five male Sprague-Dawley rats (250–310g) of the same
size that would be used in the isolated mesenteric resistance vessel
Materials and Methods
Isolated Mesenteric Resistance Vessel Preparation. All an- preparation. The vessels were homogenized in cell lysis buffer [0.05
imal studies were conducted in accordance with the National Insti- M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 0.1%
tutes of Health Guide for the Care and Use of Laboratory Animals. In NP-40, 1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluo-
addition, all experimental procedures were approved by the Medical ride, 0.8 ␮M aprotinin, 20 ␮M leupeptin, 40 ␮M bestatin, 15 ␮M
College of Georgia Animal Care and Use Committee. Mesenteric pepstatin, and 14 ␮M E-64], and the homogenate centrifuged at
artery segments were obtained from male Sprague-Dawley rats 10,000g for 10 min at 4°C to sediment any unhomogenized material.
(
250–310 g) and mounted between two cannulae in a pressure myo- The supernatant was removed, and the protein concentration was
graph system (Danish Myo Technology model 111P). The interior determined using a bicinchoninic acid protein assay (Pierce Biotech-
and exterior of the vessel were oxygenated in (95% O /5% CO ) Krebs nology, Inc., Rockford, IL). Protein was solubilized in Laemmli sam-
2
2
physiological salt solution (119.0 mM NaCl, 25.0 mM NaHCO , 4.6 ple buffer and resolved by electrophoresis using a 10% stacking
3
mM KCl, 1.2 mM KH PO , 1.2 mM MgSO , 1.8 mM CaCl , 11.0 mM Tris-glycine acrylamide gel. Protein was electrophoretically trans-
2
4
4
2
glucose; Sigma-Aldrich) at pH 7.4 and 37°C. Under no flow condi- ferred to polyvinylidene difluoride membrane and blocked with 3%
tions, over a span of 18 min, the pressure within the vessel was
increased at 10 mm Hg increments from 20 to 65 mm Hg. The vessel
bovine serum albumin and 10% dry milk in phosphate-buffered
saline containing 0.1% Tween 20 (PBS-T). After blocking, the mem-
was then equilibrated at 65 mm Hg for 30 min and remained at that branes were washed several times with PBS-T and incubated with
pressure for the duration of the experiment. Lumen diameter mea- primary antibody for the sEH enzyme (1:2000; a gift from Dr. Bruce
surements were acquired and logged using the MyoView 1.2P user
D. Hammock, University of California, Davis, CA). Membranes were
interface (DMT, Aarhus, Denmark). The control lumen diameter was washed with PBS-T and incubated with the appropriate secondary
calculated as the mean diameter during the last 15 min of the 30-min antibody conjugated to horseradish peroxidase (1:25,000; Pierce Bio-
equilibration. Vessels were constricted with U46619 (1–3 ␮M), and technology, Inc.). Protein was visualized using ECL Western Blot-
the diameter of the constricted vessel was calculated as the mean ting substrate (Pierce Biotechnology, Inc.).
during the last 2 of 15 min after the addition of U46619. sEH
inhibitors AUDA, 12-(3-cyclohexyl-ureido)dodecanoic acid (CUDA), CUDA, CDU, and AUBA are described elsewhere (Morisseau et al.,
-(3-adamantyl-ureido)-octanoic acid (AUOA), 6-(3-adamantyl-ure- 2002; Kim et al., 2004).
ido)-hexanoic acid (AUHA), 4-(3-adamantan-1-yl-ureido)butanoic 950. To a solution of adamantyl isocyanate (0.20 g, 1.13 mmol) in
acid (AUBA), 1-adamantyl-3-(12-aminododecyl)urea (AADU), and DMF (15 ml) was added a solution of 5-amino-1-pentanol (0.17 g,
sEH Inhibitors. The synthesis and characterization of AUDA,
8

sEH Inhibitors Dilate Mesenteric Resistance Vessels
1309
1
.69 mmol) in DMF (15 ml) at 0°C. After stirring for 12 h, an aqueous (19H, m), 1.42–1.49 (4H, m), 1.55–1.62 (12H, m), 2.06 (2H, s), 2.68
solution of 1 N HCl (40 ml) was added into the reaction at 0°C, and (2H, t, J ϭ 7.2 Hz), 3.13 (2H, q, J ϭ 6.6 Hz), 4.02 (1H, s), 4.10 (1H,
ϩ
the mixture was stirred for 30 min. The solid product crystalized was
filtered and washed with water (40 ml) and ethyl acetate (20 ml). The 337.1440, found [M ϩ H] 378.1221 Da.
resulting solid was dried in the vacuum oven to give 1-adamantan-
Statistical Analysis. All data are presented as mean Ϯ S.E.M. In
-yl-3-(5-hydroxypentyl)urea as a white solid (I; 0.75 g, 100%). To a experiments using AUDA, statistical significance between concen-
s) ppm; LC-MS (TOF) m/z calculated for C23H44N O [M ϩ H]
3
ϩ
1
solution of diethylene glycol monoethyl ether (1 Eq) in tetrahydro- trations was determined using an analysis of variance followed by a
furan (30 ml) was added portion-wise triphenylphosphine (1.1 Eq) least significance difference test to identify individual differences.
and carbon tetrabromide (1.1 Eq) at 0°C. After stirring for 12 h at The statistical significance between experimental groups was deter-
room temperature, hexane (60 ml) was added to the reaction mix- mined using a two-way analysis of variance and Bonferroni post
ture. This crude mixture was filtered to get rid of triphenylphospine tests to compare replicate means. P Յ 0.05 was considered statisti-
oxide, and the organic solvent dissolving the product was washed
cally significant.
with water (60 ml), dried over MgSO , and concentrated. The residue
4
was purified using silica gel column chromatography (hexane only
and hexane:ethyl acetate ϭ 3:1) to give the corresponding bromide II
Results
(
87%) as an oil. This bromide (1.5 Eq) was added portion-wise to a
The sEH has been identified as a pharmacological target
for diseases such as hypertension and inflammation. Car-
bamate- and urea-substituted sEH inhibitors have been dem-
onstrated to decrease blood pressure in animal models of
hypertension. To determine whether or not sEH inhibitors
suspension of 95% sodium hydride (3 Eq) and I (1 Eq) in DMF at
room temperature. After stirring for 12 h, water was poured into the
reaction mixture, and the product was extracted with ether. The
organic solution was dried over MgSO4 and concentrated. The resi-
due was purified using silica gel column chromatography (hexane:
1
ethyl acetate ϭ 3:1) to afford 950 (52%) as a solid. H NMR ␦ (CDCl ) have effects on vascular reactivity, isolated, pressurized, and
3
1
1
(
.22 (3H, t, J ϭ 6.9 Hz), 1.37–1.43 (2H, m), 1.46–1.53 (2H, m), preconstricted mesenteric vessels from Sprague-Dawley rats
.56–1.61 (2H, m), 1.64–1.69 (6H, m), 1.95–1.99 (6H, m), 2.05–2.08 were treated with increasing concentrations of AUDA, and
3H, m), 3.11 (2H, q, J ϭ 6.9 Hz), 3.46 (2H, t, J ϭ 6.9 Hz), 3.48–3.67
10H, m), 4.21 (1H, s), 4.26 (1H, s), LC-MS (ESI) m/z calculated for
the vascular diameter was measured. In experiments using
sEH inhibitors or analogs of AUDA, the vessel starting di-
ameter measured 233.6 Ϯ 4.9 ␮m (n ϭ 51) and measured
(
ϩ
ϩ
C22H40N O [M ϩ H] 397.30, found [M ϩ H] 397.31, mp 75°C,
2
4
Anal. (C22H40N O ) C, H, N.
2
4
79.0 Ϯ 5.3 ␮m (n ϭ 51) after treatment with U46619. Start-
AUOA. To a suspension of 8-aminooctanoic acid (0.48 g, 3.0 mmol)
in DMF (20 ml) was added 1-adamantyl-isocyanate (0.48 g, 2.7
mmol) at room temperature. The reaction mixture was stirred and
heated at 100°C for 3 h. After cooling down to room temperature, 1 N
ing vessel diameters and vessel diameters after treatment
with U46619 were not different between the experimental
groups. Treatment with AUDA resulted in a concentration-
HCl aqueous solution (25 ml) was added to the reaction and the dependent relaxation in mesenteric resistance vessels (Fig.
mixture was stirred for 30 min. The solid crystalline product was 1). These data obtained using AUDA are repeated on subse-
filtered and washed with water (20 ml) and ethyl acetate (20 ml). The quent figures for ease of comparison. To determine whether
resulting solid was recrystallized from methanol. The obtained crys-
these vasoactive effects were unique to AUDA, identical ex-
tal was dried in a vacuum oven to give 0.90 g (98%) of 8-(3-adaman-
periments were conducted using two additional sEH inhibi-
tors, (CDU), Table 1) and 950 (Table 1). Increasing concen-
trations of CDU also resulted in dilation of mesenteric
resistance arteries (Fig. 1). Interestingly, at a dose of 10 ␮M,
1
tyl-ureido)-octanoic acid as a white solid: mp 140°C; H NMR (CDCl /
3
TMS) 1.37–1.40 (6H, m), 1.55–1.58 (2H, m), 1.70–1.77 (9H, m),
1.98–2.05 (5H, m), 2.10–2.16 (3H, m), 2.40 (2H, t, J ϭ 7.4 Hz), 3.13
(
2H, t, J ϭ 6.5 Hz), 4.28 (1H, s), 4.47 (1H, s) ppm; LC-MS (TOF) m/z
ϩ
ϩ
CDU had a statistically lower percent dilation compared with
calculated for C19H33N O [M ϩ H] 337.2491, found [M ϩ H]
2
3
3
37.2508 Da.
AUHA. To a suspension of 6-aminohexanoic acid (0.98 g, 7.5
mmol) in DMF (15 ml) was added 1-adamantyl-isocyanate (0.89 g,
.0 mmol) at room temperature. The reaction mixture was stirred
and heated at 100°C for 3 h. After cooling down to room temperature,
N HCl aqueous solution (25 ml) was added to the reaction, and the
5
1
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 1.05 g (68%) of
1
6
-(3-adamantyl-ureido)-hexanoic acid as a white solid: mp 159°C; H
NMR (CDCl /TMS): 1.60–1.67 (13H, m), 1.90–1.96 (5H, m), 2.02–
3
2.07 (3H, m), 2.36 (2H, t, J ϭ 7.2 Hz), 3.09 (2H, t, J ϭ 6.9 Hz), 4.15
(
1H, s), 4.33 (1H, s) ppm; LC-MS (TOF) m/z calculated for
ϩ
ϩ
C17H29N O [M ϩ H] 309.2178, found [M ϩ H] 309.2164 Da.
2
3
AADU. To a solution of 1,12-diaminododecane (1.0 g, 5.0 mmol) in
DMF (20 ml), adamantyl-isocyanate (0.21 g, 1.1 mmol) at room
temperature was added. The reaction mixture was stirred and
heated at 100°C for 3 h. After cooling down to room temperature, 150
ml of water was added to the reaction, and the mixture was stirred
for 30 min. The yellowish solid was filtered and washed with water
(
20 ml). The targeted product was purified by chromatography on a
Fig. 1. Vascular reactivity of sEH inhibitors. Mesenteric resistance ves-
sels were isolated from Sprague-Dawley rats. The vessels were pressur-
ized, preconstricted with U46619, and treated with increasing concentra-
tions of the sEH inhibitors AUDA (n ϭ 10), CDU (n ϭ 7), and 950 (n ϭ 6).
ء, P Ͻ 0.05, different from previous dose of the same drug; †, P Ͻ 0.05,
different from other drugs at the same dose.
silica column equilibrated with ethyl acetate. Product was eluted
with a 50:50 mixture of ethyl acetate:ammonia-saturated methanol.
The solvent was evaporated, and the obtained crystal was dried in a
vacuum oven to give 0.20 g (49%) of 1-adamantyl-3-(12-aminodode-
cyl) urea as a white solid: mp 80°C; H NMR (CDCl /TMS) 1.22–1.33
1
3

1
310
Olearczyk et al.
TABLE 1
Structures and IC50 of inhibitors of the soluble epoxide hydrolase enzyme
R
Human
sEH IC50
n
Z
Identifier
nM
1
1
7
5
3
2
5
1
COOH
COOH
AUDA
AUOA
AUHA
AUBA
AADU
950
3.2
12.9
170.7
684.2
2.6
14.1
9.5
COOH
COOH
NH2
1
(O(CH ) ) OC H
2
2 2
2
5
1
1
COOH
CUDA
1
CH3
CDU
7.0
AUDA; 25 Ϯ 8% versus 48 Ϯ 7% for CDU and AUDA, respec- tural analogs of AUDA were prepared. These analogs, the
tively. At the highest dose, CDU resulted in a dilation that structures of which are provided in Table 1, were designed to
was not statistically different from that measured with identify the contributions of the adamantyl group, the car-
AUDA; 91 Ϯ 7% versus 77 Ϯ 7% dilation for CDU and AUDA, boxylic acid functional group, and the length of the aliphatic
respectively. Treatment of mesenteric resistance vessels with carbon chain on vascular reactivity. Changing the adamantyl
the sEH inhibitor 950 did not induce a measurable change in group of AUDA to a cyclohexyl group, which is the structure
vessel diameter at any of the concentrations tested.
of the sEH inhibitor CUDA, significantly attenuated the va-
The sEH enzyme is expressed in a variety of tissues includ- sodilatory response (Fig. 3). CUDA resulted in a 10 Ϯ 5%
ing the kidney, liver, and blood vessels (Enayetallah et al., relaxation of mesenteric resistance vessels at the 10 ␮M dose
2
006). To determine whether or not the sEH enzyme is ex- and a 52 Ϯ 13% relaxation at the 100 ␮M dose. Replacing the
pressed in mesenteric resistance vessels from male Sprague- carboxylic acid at the end of the aliphatic chain to a primary
Dawley rats, vessels were obtained, and the expression of the amine, AADU, resulted in concentration-dependent dilations
sEH enzyme was measured by Western blot. The sEH en- of mesenteric resistance vessels that were not different from
zyme was identified in the mesenteric resistance vessels from that of AUDA (Fig. 3). At 10 ␮M, AADU induced a 55 Ϯ 14%
Sprague-Dawley rats (Fig. 2). However, compared with the increase in vessel diameter, whereas AUDA induced a 48 Ϯ
kidney cortex from Sprague-Dawley rats, sEH protein ex- 7% increase in vessel diameter. Finally, shortening the ali-
pression in mesenteric resistance vessels was less per micro- phatic side chain from 12 carbons to eight carbons, AUOA, or
gram of total protein.
The unique structure of adamantyl-urea inhibitors of the
sEH enzyme imparts specific vasoactive properties that are
not observed with other sEH inhibitors that could contribute
to the reported blood pressure-lowering effects and renal and
cardiovascular protective properties. Adamantyl-urea inhib-
itors are 1,3-disubstituted ureas with an adamantyl group on
one side of the urea pharmacophore and a saturated 11-
carbon aliphatic chain on the other (Table 1). To investigate
the structural elements of adamantyl-urea inhibitors that
are responsible for the observed vasoactive actions, struc-
Fig. 2. Expression of the sEH enzyme in rat mesenteric resistance ves-
sels. Mesenteric resistance vessels of the size used in the isolated mes-
enteric resistance vessel preparation or kidney cortex were obtained from
male Sprague-Dawley rats. The vessels were homogenized and increasing
concentrations of total protein loaded onto a gel and resolved by electro-
phoresis. Protein was transferred to polyvinylidene difluoride membrane
and the sEH enzyme identified using an anti-sEH primary antibody
followed by a secondary antibody conjugated to horseradish peroxidase.
sEH was visualized using ECL Western blotting substrate.
Fig. 3. The effects of the adamantyl group and the carboxylic acid moiety
on the vascular reactivity of adamantyl-urea inhibitors of sEH. Mesen-
teric resistance vessels were isolated from Sprague-Dawley rats. The
vessels were pressurized, preconstricted with U46619 and treated with
increasing concentrations of CUDA (n ϭ 5) or AADU (n ϭ 6). ء, P Ͻ 0.05,
different from previous dose of the same drug; †, P Ͻ 0.05, different from
other drugs at the same dose.

sEH Inhibitors Dilate Mesenteric Resistance Vessels
1311
to four carbons, AUBA, significantly attenuated the vasodi-
latory response (Fig. 4). AUOA resulted in a Ϫ2 Ϯ 4% relax-
ation, and AUBA resulted in a 3 Ϯ 11% relaxation of mesen-
teric resistance vessels at the 10 ␮M dose. Interestingly,
shortening the aliphatic side chain from 12 carbons to six
carbons, AUHA, altered the tendency for the chain-shortened
analogs to decrease activity (Fig. 4). At 10 ␮M, AUHA in-
duced a 22 Ϯ 9% relaxation of mesenteric resistance vessels.
After treatment with the highest dose of AUDA or its ana-
logs, acetylcholine (10 ␮M) was added to the isolated mesen-
teric resistance vessel preparation to ensure endothelial in-
tegrity. Acetylcholine increased vessel diameter to 242.5 Ϯ
5
.0 ␮m, a 106 Ϯ 3% dilation.
Inhibition of the sEH enzyme results in the accumulation
of the EETs that are then able to elicit their vasoactive
effects. If the mechanism by which adamantyl-urea inhibi-
tors induce vasoreactivity is via increasing EETs levels, then
inhibiting EET synthesis would be expected to attenuate the
effects of AUDA. To investigate this hypothesis, mesenteric AUDA. Mesenteric resistance vessels were isolated from Sprague-Dawley
resistance vessels were pretreated with the epoxygenase in- rats. The vessels were pressurized and pretreated with MS-PPOH (1 ␮M,
Fig. 5. The effect of epoxygenase inhibition on the vascular reactivity of
3
0 min). The vessels were constricted with U46619 and treated with
hibitor MS-PPOH. Pretreatment of mesenteric resistance
vessels with MS-PPOH did not alter the average starting
vessel diameter, 224.9 Ϯ 9.1 ␮m before MS-PPOH treatment
and 225.2 Ϯ 9.5 ␮m after treatment (n ϭ 4). After the addi-
tion of U46619, the MS-PPOH-pretreated vessels had an
average diameter of 150 Ϯ 1.1 ␮m. MS-PPOH did not statis-
tically alter AUDA-induced vascular reactivity (Fig. 5). In the
presence of MS-PPOH, AUDA induced a 43.7 Ϯ 12.0% relax-
ation at the 10 ␮M dose. These data suggest that the mech-
anism by which AUDA induces its vascular reactivity could
be independent of its actions on the sEH enzyme.
Epoxyeicosatrienoic acids are reported to activate calcium-
activated potassium channels located in the endothelium and
smooth muscle cells, resulting in a decrease in the membrane
potential. To determine whether or not the adamantyl-urea
inhibitor-induced vasodilation was the result of these inhib-
itors to activate potassium channels, mesenteric resistance
vessels were pretreated with either tetraethylammonium
increasing concentrations of AUDA (n ϭ 4). ء, P Ͻ 0.05, different from
previous dose of the same drug.
(
TEA), a nonspecific potassium channel blocker, or IbTX, a
specific large-conductance calcium-activated potassium
channel blocker. In experiments using TEA, the average
vessel diameter measured 242.7 Ϯ 17.4 ␮m and measured
74.7 Ϯ 16.3 ␮m after treatment with U46619 (n ϭ 5). Addi-
tion of TEA resulted in a stable 22 Ϯ 8% increase in vessel
diameter. Treatment with IbTX did not alter the average
starting vessel diameter, 205.2 Ϯ 5.9 ␮m before IbTX treat-
ment and 200.1 Ϯ 5.2 ␮m after treatment (n ϭ 6). The
average diameter of IbTX-pretreated vessels after the addi-
tion of U46619 was 75.7 Ϯ 11.8 ␮m. Neither TEA nor IbTX
pretreatment inhibited the observed AUDA-induced relax-
ation (Fig. 6). In the presence of TEA or IbTX, AUDA induced
a 29 Ϯ 23% or a 34 Ϯ 9% dilation of mesenteric resistance
vessels at the 10 ␮M dose. The AUDA-induced dilation, how-
Fig. 4. The effects of chain-shortened adamantyl-urea inhibitors on vas-
cular reactivity. Mesenteric resistance vessels were isolated from
Sprague-Dawley rats. The vessels were pressurized, preconstricted with
U46619, and treated with increasing concentrations of AUOA (n ϭ 5),
AUHA (n ϭ 6), and AUBA (n ϭ 5) from Table 1. ء, P Ͻ 0.05, different from
previous dose of the same drug; †, different from AUOA and AUBA.
Fig. 6. The effects of potassium channel inhibitors on the vascular reac-
tivity of AUDA. Mesenteric resistance vessels were isolated from
Sprague-Dawley rats. The vessels were pressurized, preconstricted with
U46619, and pretreated with TEA (n ϭ 5), IbTX (n ϭ 6), or KCl (n ϭ 6).
ء, P Ͻ 0.05, different from previous dose of the same drug; †, different
from other drugs at the same dose.

1
312
Olearczyk et al.
ever, was completely blocked when mesenteric vessels were statistically alter the vessel diameter, which averaged 93.9 Ϯ
pretreated with potassium chloride (KCl). In these experi- 14.3 ␮m. Disrupting the endothelium did not affect the
ments, the starting vessel diameter measured 220.8 Ϯ 14.7 AUDA-induced relaxation (Fig. 8). At the 10 ␮M dose, AUDA
␮
m and decreased to 131.4 Ϯ 14.2 ␮m after KCl treatment elicited 48.1 Ϯ 7.0% dilation in nondenuded vessels and
n ϭ 6). At a dose of 10 ␮M, AUDA did not significantly 39.5 Ϯ 15.4% dilation in denuded vessels. The addition of
change the vessel diameter, which measured 117.5 Ϯ 17.4 sodium nitroprusside (10 ␮M) to the denuded mesenteric
m (Fig. 6). resistance vessel at the end of the experiment increased the
Inhibition of AUDA-induced increases in vessel diameter vessel diameter to 163.6 Ϯ 10.4 ␮m, a 158 Ϯ 29% dilation.
(
␮
by KCl suggests that adamantyl-urea inhibitors could be
influencing potassium flux or altering the activity of some
other membrane channel. The epoxyeicosatrienoic acids have
Discussion
ϩ
ϩ
been reported to activate the Na /K -ATPase as well as
The sEH enzyme metabolizes epoxide-containing com-
ϩ
ATP-sensitive K (KATP) channels (Pratt et al., 2001; Yang et pounds by catalyzing the addition of water to the epoxide
al., 2005; Ye et al., 2005, 2006). To investigate the possible moiety (Morisseau and Hammock, 2005; Newman et al.,
roles of these channels on AUDA-induced vascular reactivity, 2005). EETs, cytochrome P450 epoxygenase metabolites of
mesenteric resistance vessels were treated with ouabain, an arachidonic acid, are endogenously produced epoxides that
ϩ
ϩ
inhibitor of the Na /K -ATPase, or glibenclamide, an inhib- serve as substrates for the sEH enzyme. The metabolism of
itor of the KATP channel. In experiments using ouabain, the EETs by sEH into the corresponding dihydroxyeicosatrienoic
starting vessel diameter measured 216.4 Ϯ 9.7 ␮m and mea- acids results in partial or complete loss of activity
sured 51.8 Ϯ 2.7 ␮m after treatment with U46619 (n ϭ 5). (Moghaddam et al., 1997; Zeldin, 2001; Spector et al., 2004).
Treatment with ouabain did not significantly alter the vessel The EETs have been identified as important regulatory mol-
diameter, 79.2 Ϯ 20.8 ␮m. AUDA-induced relaxation was not ecules in the cardiovascular and renal circulations. As a
affected by ouabain treatment (Fig. 7). At the 10 ␮M dose, result, the sEH enzyme has been identified as a therapeutic
AUDA induced a 42 Ϯ 12% increase in vessel diameter (Fig. target for diseases like hypertension and inflammation. In-
7
). In experiments using glibenclamide, the starting vessel hibitors of the sEH enzyme have been successfully employed
diameter measured 194.2 Ϯ 15.8 ␮m and measured 102.3 Ϯ in animal models of hypertension where sEH inhibition was
4.0 ␮m after treatment with U46619 (n ϭ 4). Treatment reported to decrease blood pressure and decrease hyperten-
1
with glibenclamide resulted in a 28 Ϯ 4% increase in vessel sion-induced renal damage. These renal and cardiovascular
diameter. Glibenclamide treatment did tend to inhibit in- protective effects are attributed, at least in part, to the blood
creases in vessel diameter induced by AUDA; however, these pressure-lowering effects of the sEH inhibitor. Within the
decreases were not statistically significant (Fig. 7).
present study, we addressed the hypothesis that a mecha-
To determine whether adamantyl-urea inhibitors induce nism by which sEH inhibitors elicit their cardiovascular pro-
vasodilation indirectly via their actions on the endothelium tective effects is via their direct actions on the vasculature.
or directly via their actions on the vascular smooth muscle,
We demonstrated, using isolated mesenteric resistance
experiments were conducted using mesenteric resistance vessels, that treatment with adamantyl-urea inhibitors of
vessels where the endothelium was disrupted. The average the sEH enzyme resulted in a concentration-dependent re-
vessel diameter measured 168.4 Ϯ 12.9 ␮m and measured laxation of the preconstricted vessel. The dilator responses
8
5.0 Ϯ 8.6 ␮m after treatment with U46619. Treatment of observed in mesenteric resistance vessels occur at concentra-
the preconstricted, denuded vessel with acetylcholine did not
ϩ
ϩ
Fig. 7. The effects of inhibition of the Na /K -ATPase or the K
Fig. 8. The effects of endothelial cell disruption on the vascular activity
of AUDA. Mesenteric resistance vessels were isolated from Sprague-
Dawley rats. The endothelium was disrupted using a human hair and
then pressurized, constricted with U46619, and treated with increasing
concentrations of AUDA (n ϭ 5). ء, P Ͻ 0.05, different from previous dose
of the same drug; †, different from other drugs at the same dose.
ATP
channel on the vascular reactivity of AUDA. Mesenteric resistance ves-
sels were isolated from Sprague-Dawley rats. The vessels were pressur-
ized, preconstricted with U46619, and treated with either ouabain (n ϭ 4)
or glibenclamide (n ϭ 4). ء, P Ͻ 0.05, different from previous dose of the
same drug.

sEH Inhibitors Dilate Mesenteric Resistance Vessels
1313
tions that are above the reported IC50 for the sEH enzyme. also had the least amount of vasoactivity. AUHA had an
This response was also demonstrated with the sEH inhibitor intermediate IC50 for the human sEH enzyme, and although
CDU but was not observed with the more sterically hindered the responses look attenuated compared with those mea-
sEH inhibitor 950. These data suggest that the vasoactive sured with AUDA, they are not statistically different. Each ␴
properties measured with adamantyl-urea inhibitors are bond that comprises the aliphatic carbon side chain can
unique to their structure and are independent of their ability freely rotate giving the side chain significant flexibility.
to inhibit the sEH enzyme itself. Adamantyl-urea inhibitors Therefore, it is possible that the compounds with the 12-
are 1,3-disubstituted ureas with an adamantyl group on one carbon (AUDA) and six-carbon (AUHA) side chains are able
side of the urea pharmacophore and an 11-carbon aliphatic to assume a configuration that allows them to induce the
chain on the other (Table 1). To investigate what structural observed relaxation. Conversely, these configurations might
elements of adamantyl-urea inhibitors are responsible for not be possible in the eight-carbon (AUOA) or four-carbon
these unique vasoreactive responses, analogs of AUDA were (AUBA) side chains. In addition, 950, which has a carbon side
prepared (Table 1). Each of the analogs of AUDA had IC50
for the human sEH enzyme in the nanomolar range.
s
chain that is significantly more sterically hindered by the
addition of oxygen atoms to the carbon side chain and there-
The sEH inhibitor CUDA exhibited significantly decreased fore cannot assume conformations that are available to
vasoreactivity in mesenteric vessels compared with that AUDA, does not induce changes in vessel diameter. Taken
measured with AUDA. The structural difference between together, these data suggest that the length of the aliphatic
CUDA and AUDA is that CUDA has a cyclohexyl group on carbon chain and its flexibility are important structural fea-
one side of the urea pharmacophore, whereas AUDA has an tures of adamantyl-urea inhibitors that are responsible for
adamantyl group. These data would suggest that the ada- the observed vasoreactive properties.
mantyl group is an important structural feature that is
Inhibitors of the sEH enzyme increase endogenous EET
unique to adamantyl-urea inhibitors and is responsible for levels by decreasing the metabolism of the EETs to less
the observed vasoactive properties. Interestingly, the ada- active diols. If the mechanism by which adamantyl-urea in-
mantyl functional group was determined to be an important hibitors induce vasorelaxation is by increasing endogenous
structural feature for the inhibition of the sEH enzyme itself EET levels, then it would be expected that inhibiting EET
(
Morisseau et al., 2002). CUDA shares some structural sim- synthesis would attenuate the AUDA-induced response.
ilarity to CDU in that both sEH inhibitors have a cyclohexyl However, pretreatment of mesenteric resistance vessels with
group occupying one side of the urea pharmacophore. This the epoxygenase inhibitor MS-PPOH did not alter AUDA-
could explain why CDU exhibits a similar reactivity as induced increases in vessel diameter. One interpretation of
CUDA in this preparation. However, in addition to having a these data is that the mechanism by which AUDA induces
cyclohexyl group on one side of the urea pharmacophore, the vascular reactivity is not only due to increasing EET levels
structure of CDU also differs from AUDA in that it has an but may have effects independent of sEH inhibition.
aliphatic chain terminating with a methyl group instead of a
carboxylic acid. This could explain why at the highest con- cardiovascular effects is via activation of calcium-activated
centration, CDU demonstrated similar reactivity to AUDA. potassium channels in the endothelium and smooth muscle
One mechanism by which the EETs exert their renal and
When the negatively charged carboxylic acid at the end of cells, resulting in a decrease in the membrane potential
the aliphatic carbon chain of AUDA was changed to a posi- (Chataigneau et al., 1998; Wu et al., 2000). Neither TEA, a
tively charged amine group (Fig. 3, AADU), the vascular nonspecific potassium channel blocker, nor IbTX, a specific
reactivity of AUDA was not altered. These data suggest that calcium-activated potassium channel blocker, attenuated the
the terminal functional group at the end of the aliphatic observed AUDA-induced dilation (Fig. 4). These data suggest
carbon chain is not an important structural feature for the that a possible mechanism by which adamantyl-urea inhibi-
vascular reactivity observed with adamantyl-urea inhibitors. tors exert their vasoactive effects is not through their action
Interestingly, altering this functional group also had little on these specific potassium channels. In addition, these data
effect on the IC50 for the human sEH enzyme. This finding suggest that the dilation observed with AUDA may not due to
would also suggest that the inability of CDU to induce sim- the inhibition of sEH and the resulting increase in vascular
ilar vasodilations was mainly the result of the cyclohexyl EET levels.
functional group, not the methyl group at the end of the
The EETs have also been reported to alter the activity of
aliphatic carbon chain. Changing the length of the aliphatic other channels that might participate in the observed vaso-
ϩ
ϩ
carbon chain, however, did alter the observed vasoreactivity reactivity, the Na /K -ATPase and the K
channel (Pratt
ATP
of adamantyl-urea inhibitors. AUOA, which shortened the et al., 2001; Yang et al., 2005; Ye et al., 2005, 2006). The
length of the aliphatic carbon chain from 12 to eight carbons, observed AUDA-induced vasorelaxation was not altered with
significantly attenuated the vasoactive properties of AUDA. ouabain treatment; however, glibenclamide did tend to at-
The same result was observed with AUBA, which shortened tenuate the AUDA-induced response, although this decrease
the length of the aliphatic carbon chain from 12 to four did not reach statistical significance. Therefore, the mecha-
carbons. AUHA, which has an aliphatic carbon chain length nism by which adamantyl-urea inhibitors induce increases
ϩ
ϩ
of six carbons, demonstrated a slightly attenuated vasoreac- in vessel diameter is not by their actions on the Na /K -
tivity compared with AUDA. The human sEH enzyme IC50
s
ATPase or the KATP channel.
for AUOA, AUHA, and AUBA did not directly correspond to
Denuded mesenteric resistance vessels were used to deter-
the observed vasoreactivity. Of the chain-shortened analogs, mine whether adamantyl-urea inhibitors are acting on the
AUOA has the lowest IC50 for the human sEH but demon- endothelium or directly on the smooth muscle to induce the
strated very little vasoreactivity compared with AUDA. observed vasodilation. Disrupting the endothelium did not
AUBA had the highest IC50 for the human sEH enzyme and affect AUDA-induced relaxation at the lower concentrations

1
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more robust relaxation in response to 100 ␮M AUDA com-
pared with vessels where the endothelium remained intact.
These data provide evidence that AUDA acts directly on the
vascular smooth muscle in an endothelium-independent
manner to induce its vascular reactivity. In addition, AUDA-
induced relaxation was completely abolished when mesen-
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Pratt PF, Li P, Hillard CJ, Kurian J, and Campbell WB (2001) Endothelium-
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have unique vasodilator actions compared with other sEH
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(
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2
ϩ
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length. AUDA does not act on potassium channels, the Na /
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quires further investigation.
5
769–5776.
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(
2ϩ)
(ϩ)
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Address correspondence to: Dr. Jeffrey J. Olearczyk, The Medical College
of Georgia, Vascular Biology Center, 1120 15th Street, Augusta, GA 30912.
E-mail: jolearczyk@mail.mcg.edu