Design of bioavailable derivatives of 12-(3-adamantan-1-yl-ureido)dodecanoic acid, a potent inhibitor of the soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmc.2006.09.057

The soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators involved in blood pressure regulation and vascular inflammation. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA, 1) is a very active inhibit

Full text of DOI:10.1016/j.bmc.2006.09.057

Bioorganic & Medicinal Chemistry 15 (2007) 312–323
Design of bioavailable derivatives of
2-(3-adamantan-1-yl-ureido)dodecanoic acid, a potent
inhibitor of the soluble epoxide hydrolase
1
a
a
a
b
b
In-Hae Kim, Kosuke Nishi, Hsing-Ju Tsai, Tanya Bradford, Yasuko Koda,
a,ꢀ
a,
b
*
Takaho Watanabe, Christophe Morisseau, Joanne Blanchfield,
b
a
Istvan Toth and Bruce D. Hammock
a
Department of Entomology and University of California Davis Cancer Center, University of California,
One Shields Avenue, Davis, CA 95616, USA
b
School of Molecular and Microbial Sciences, University of Queensland, St. Lucia 4072, Australia
Received 14 July 2006; revised 21 September 2006; accepted 26 September 2006
Available online 29 September 2006
Abstract—The soluble epoxide hydrolase (sEH) plays an important role in the metabolism of endogenous chemical mediators
involved in blood pressure regulation and vascular inflammation. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid (AUDA, 1) is a
very active inhibitor of sEH both in vitro and in vivo. However, its relatively high melting point and limited solubility in either water
or oil-based solvents leads to difficulties in formulating the compound and often results in poor in vivo availability. We investigated
the effect of derivatization of the acid functional group of inhibitor 1 on the inhibition potencies, physical properties, and pharma-
cokinetic properties. For human sEH, similar inhibition potency was obtained when the acid of compound 1 was modified to esters
(
2–15). The resulting compounds exhibited improved physical properties (23–66 ꢀC lower melting point and 5-fold better solubility
in oil). Pharmacokinetic studies showed that the esters possess improved oral bioavailability in mice. On the other hand, amide
derivatives of AUDA 1 did not show significant improvement in inhibition potencies or physical properties (higher melting points
and lower solubility). The esterification of 1 results in compounds that are easier to formulate in animal food and in triglycerides for
gavage and other routes of administration, making it easier to study the biological effects of sEH inhibition in vivo.
ꢁ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
blood pressure of spontaneous hypertensive and angio-
tensin II induced hypertensive rats treated with effective
1
0–13
In mammals, the soluble epoxide hydrolase (sEH) is
involved in the metabolism of endogenous mediators
sEH inhibitors is dramatically reduced.
the EETs have further vascular protective effects such as
In addition,
1
–3
4
14–17
such as epoxides of arachidonic acid,
linoleic acid,
and other lipid epoxides. Epoxides of arachidonic acid
epoxyeicosatrienoic acids or EETs) are effective regula-
anti-inflammatory properties in endothelial cells,
suppression of reactive oxygen species following hypox-
5
,6
1
8
(
ia-reoxygenation,
attenuation of vascular smooth
muscle migration, and enhancement of a fibrinolytic
7
19
tors of blood pressure and have anti-inflammatory
8
,9
20
properties in vivo. However, the dihydroxyeicosatrie-
noic acids (DHETs), the sEH hydrolyzed metabolites of
the EETs, often have reduced biological activity, and are
pathway. In cellular and animal models, the EET-
mediated regulation of hypertension and cardioprotec-
tive effects are dependent in part on the extent of
4,21
7
more water soluble and more easily conjugated. The
epoxide hydrolysis by sEH,
suggesting that the inhib-
itors of the sEH are worth exploring as pharmaceuticals
for the treatment of hypertension, inflammation, and
other disorders that can be induced by changing the
in vivo concentration of EETs.
Keywords: Soluble epoxide hydrolase (sEH); sEH inhibitors; 1,3-dis-
ubstituted ureas; Pharmacokinetic properties.
*
Corresponding author. Tel.: +1 530 752 6571; fax: +1 530 752
537; e-mail: chmorisseau@ucdavis.edu
1
1,3-Disubstituted ureas and related compounds are very
potent and stable inhibitors of sEH. These compounds
ꢀ
Present address: Food and Drug Safety Center, Hatano Research
Institute, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan.
0
968-0896/$ - see front matter ꢁ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.057

I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
313
efficiently reduce epoxide hydrolysis in several in vitro and
1
the 1,3-diadamantyl urea could be removed from the
AUDA derivatives by trituration in hexane. Careful
recrystallization of the products removes the last traces
of the diadamantyl urea. All products were examined
for the presence of diadamantyl urea by LC-MS/MS
and no products contained more than 0.01% w/w of this
impurity.
1,12,22
in vivo models.
However, limited solubility in either
water or organic solvents and high melting points of some
of these inhibitors likely affect their in vivo efficacy and
1
0,23
make formulation difficult.
Toward solving these
problems, we previously showed that a polar functional
group on one of the alkyl chains of the urea inhibitors im-
proved solubility in water 2-4-fold while retaining inhibi-
2
4
tion potency. A polar group such as an ester, alcohol,
ether, or ketone located on the fifth/sixth atom from the
carbonyl group of the urea function, or an acid function
present on the thirteenth atom from the urea pharmaco-
phore were both effective for making soluble inhibitors
in either water or oil-based solvents often without a drop
Scheme 1 shows the syntheses of 1 and also the corre-
sponding ester (2–15) and amide (16–19) derivatives.
Alkylation of compound 1 with an alkyl or aryl bromide
(2: methyl, 3: ethyl, 4: propyl, 5: allyl, 6: propagyl, 7: iso-
propyl, 8: butyl, 9: 1-methylpropyl, 10: 2-methylpropyl,
11: benzyl, and 12: 2-chlorobenzyl) in the presence of
potassium carbonate as a base in DMF afforded the cor-
responding ester derivatives in 35–95% yield. Coupling
of compound 1 with an alcohol or amine (13: tert-buta-
nol, 14: 1-adamantylmethanol, 15: 1-naphthylmethanol,
16: ethylamine, and 17: isopropylamine) using 1-[3-(dim-
ethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDCI) and 4-dimethylaminopyridine (DMAP) in
dichloromethane or isobutyl chloroformate and triethyl-
amine in THF yielded the corresponding ester and
2
3,24
in inhibition potencies.
These functional groups
incorporated into lipophilic compounds are also useful
in improving the binding selectivity of the inhibitors to
the enzyme possibly by establishing additional hydrogen
2
5,26
bonding.
mantan-1-yl-ureido)dodecanoic acid (AUDA), were used
Such compounds, for example, 12-(3-ada-
2
7–29
to inhibit sEH in both cultured cells and animals.
However, AUDA is difficult to formulate, and was
administered with a co-solvent (up to 10% of DMSO),
or in the drinking water with a sizable amount of 2-
2
8,29
2
4,30
amide derivatives in 20–95% yield.
2
7
hydroxypropyl b-cyclodextrin. Unless AUDA and sim-
ilar compounds are in true solution when administered to
animals, the availability is very low. Although a valuable
improvement in the solubility was obtained from the
inhibitors functionalized with an acid group or another
polar group, their relatively high melting points
As outlined in Scheme 1, two sulfonamide derivatives
(18 and 19) were prepared through the dioxopyrrolidinyl
ester intermediate (I). Coupling of compound 1 with
N-hydroxysuccinimide using 1,3-dicyclohexylcarbodii-
mide in THF provided I in 72% yield. This activated
intermediate (I) was reacted with methanesulfonamide
(for compound 18) or benzenesulfonamide (for com-
pound 19) in the presence of DMAP to yield compounds
18 and 19 in 65–92% yield.
(
in both water and oil-based solvents, leading to relatively
>100 ꢀC) still limited solubility and speed of dissolution
2
3,24,27,28
poor observed in vivo oral availability.
There-
fore, in the present study, we investigated modifications
of the acid function of AUDA on the inhibition potency
and physical properties (e.g., melting point and solubility)
with the goal of improving the availability of AUDA and
its pharmacokinetic parameters in mice. We explored two
kinds of modification: making alkyl esters to obtain com-
pounds more soluble in oil, and various polar amides to
obtain compounds more soluble in water.
Scheme 2 describes the synthesis of 2-[12-3-(adamantan-
1-yl-ureido)dodecanoylamino]-decanoic acid (22). Meth-
yl 2-aminodecanoate (II) prepared by alkylation of
diethyl acetamidomalonate was coupled with compound
0
0
1 using O-benzotrizol-1-yl-N,N,N ,N -tetramethyluroni-
um hexafluorophosphate (HBTU) and diisopropylethyl-
amine (DIEA) in DMF to give the corresponding amide
ester (III) in 66% yield. Hydrolysis of III in the presence
of LiOH provided compound 22 in 45% yield.
2
. Results and discussion
2
.1. Synthesis
Compound 22 synthesized as shown in Scheme 2 was
used for the preparation of compound 20 (Scheme 3).
Azidation of protected glucopyranose with trimethylsi-
lyl azide in the presence of tin (IV) chloride as a catalyst
produced the corresponding azide (IV) in 82% yield.
Reduction of IV under an atmosphere of hydrogen in
the presence of palladium on carbon, followed by cou-
pling with compound 22 using HBTU and DIEA in
DMF, yielded V (59%). The acetyl protecting group of
V was removed in the presence of sodium in methanol
to afford compound 20 (95%). Synthesis of compound
21 was conducted in the same method as that described
above from compound 1 instead of compound 22 (64%).
While the synthesis of compound 1 (12-(3-adamantan-1-
yl-ureido)dodecanoic acid; AUDA) was previously
described, we report herein a simplified (see Scheme 1),
more efficient method of producing this compound.
Reaction of 1-adamantyl isocyanate with 12-aminodo-
decanoic acid in 1,2-dichloroethane provided compound
2
4
1
in 95–100% yield. However, when this reaction was
performed in DMF or THF, approximately 20% of the
yield was 1,3-diadamantylurea, a crystalline by-product
that has also been reported as a potent inhibitor of
sEH. Chlorinated solvents such as 1,2-dichloroethane
used for the preparation of 1 effectively produced a pure
compound in a high yield without contamination by the
bis urea. It must be cautioned that adamantyl isocyanate
contains up to 5% of the 1,3-diadamantylurea even when
freshly opened under nitrogen. Low concentrations of
2
3
2.2. Biological activity
At first, we examined the inhibitory potency of the
AUDA ester derivatives using our standard spectral

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I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
OH
O
H N
2
(a)
2-10, 13-14
O
(b)
OH
N
H
N
H
1
O
(c)
1
1-12, 15-17
(d)
O
O
O
O
N
H
N
N
H
(I)
O
(e)
O
H O
N
R
N
H
N
H
S
O
O
1
8: R = methyl
9: R = phenyl
1
Scheme 1. Syntheses of alkyl ester (2–17) and sulfonamide (18 and 19) derivatives of 12-(3-adamantan-1-yl-ureido)dodecanoic acid. Reagents and
conditions. (a) 1-adamantyl isocyanate, 1,2-dichloroethane, reflux; (b) alkyl bromide, K CO , DMF, rt; (c) EDCI/DMAP or isobutyl chloroformate/
2
3
triethylamine, corresponding alcohol or amine; (d) N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, THF, rt; (e) methanesulfonamide (for
compound 18) or benzenesulfonamide (for compound 19), DMAP, HMPA, 90 ꢀC.
O
OH
N
H
N
H
1
O
NH HCl
2
O
(a)
(II)
O
O
O
H
N
N
H
N
H
O
O
( )7
(
III)
(
b)
O
O
H
N
N
N
OH
H
H
O
( )7
2
2
Scheme 2. Synthesis of 2-[12-3-(adamantan-1-yl-ureido)dodecanoylamino]decanoic acid (22). Reagents and conditions: (a) 1—methyl 2-amino
decanoate (II) was prepared from diethyl acetamido malonate and 1-bromooctane; 2—II, HBTU, DIEA, DMF, rt; (b) LiOHÆH O, DME, H O, rt.
2
2
2
4
assay. However, as seen in Table 1, IC50 values for
most of the derivatives on the recombinant murine
and human enzymes were identical. Because such tight
binding inhibitors approach stoichiometric interaction
with the target enzyme, the observed IC s, which are
50
in part dependent on enzyme concentration, approach
[E]/2. Clearly the spectral assay was not able to distin-
guish relative potency among these inhibitors. Thus,

I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
315
O
O
H
N
N
N
OH
H
H
O
( )7
2
2
OAc
(a)
AcO
OAc (IV)
OAc
O
N
3
O
O
H
OAc
N
AcO
N
OAc
OAc
O
N
N
H
H
H
(
)7
(V)
O
(b)
O
O
OH
H
N
HO
N
OH
OH
O
N
N
H
H
H
(
)7
O
2
0
Scheme 3. Synthesis of 12-(3-adamantan-1-yl-ureido)dodecanoic acid [1-(2,3,4-trihydroxy-5-hydroxymethyl-cyclohexylcarbamoyl)nonyl]amide (20).
Reagents and conditions. (a) 1—2,3,4,6-tetra-O-acetyl-D-glucopyranosyl azide (IV) was prepared from 1,2,3,4,6-penta-O-acetyl-D-glucopyranose in
the presence of trimethylsilyl azide (4.24 mL, 32.1 mmol) and tin (IV) chloride, 2—IV, H
2
, Pd/C, THF, rt, 3—compound 20, HBTU, DIEA, DMF,
rt. For the preparation of compound 21, the same methods were used but from compound 1 instead of 22 in the first step.
we reran the inhibitors using a recently reported fluores-
cent-based assay which uses a much lower enzyme con-
centration and can distinguish among IC s below
have a lower melting point than 1. Their solubility in
water and oil was first examined. The more hydrophobic
ester inhibitors had 4- to 20-fold lower water solubility
than the acid form compound 1 (Table 2), while 2- to
5-fold improvement in oil solubility was observed with
the esters. The improved oil solubility would be useful
for the formulation of the compounds for delivery for
slow release by subcutaneous injection, by gavage, in
the food, or following topical treatment. Furthermore,
one could envision that the ester derivatives that have
better solubilities could be used as prodrugs for AUDA.
In general, esters are common functional groups used
for the prodrug approach due to its rapid hydrolysis
5
0
3
1
5
0 nM. Like the spectral assay, the fluorescent assay
as performed here has a standard error between 10%
and 20%, suggesting that differences of twofold or great-
er are significant. The data in Table 1 illustrated the
power of this new assay, and they also demonstrate that
synthesis of esters with various alkyl groups (2–15) pro-
duces inhibitors that are as potent as the free acid
(
AUDA, 1) on the human sEH. Even the presence of
a bulky ester (12–15) did not influence the inhibitory
potency in vitro. Furthermore, all of the ester derivatives
in Table 1, except for compound 11 with a tert-butyl
group, had 23–66 ꢀC lower melting points than the cor-
responding acid. These results agreed with previous re-
ports that a significant improvement in the inhibition
potency and reduction in melting point are observed
for esters of an acid functionality of urea inhibitors
3
2
to active agents by esterases in vivo, and prodrugs of
3
3
numerous drugs, such as meperidine analgesics and
3
4,35
an anticancer agent CPT-11,
are used as esters.
Thus, we examined the stability of the ester functionality
of compounds 7–10 with two recombinant human carb-
oxylesterases (hCE-1 and hCE-2). As shown in Table 2,
both enzymes were able to hydrolyze the four esters test-
ed to yield AUDA. Furthermore, the ester hydrolysis
was time- and enzyme concentration-dependent as one
could expect for a biocatalytic reaction. Overall these
experiments suggest that the ester compounds could
act as a prodrug-like inhibitor with improved physical
properties that may be metabolized in vivo to afford
the free acid (1) with increased bioavailability.
2
3,24
located around 5–6 atoms away from the urea.
Based on the inhibition potency results, in a preliminary
experiment we tested the small esters (2–11) for bioavail-
ability in a fast in vivo assay (1 mouse per compounds).
We found that 3–4 carbon ester alcohols (4 and 8) give a
longer lasting AUDA concentration in the bloodstream
than smaller one (2 and 3) (results are given in Supple-
mentary data). Also, we observed that branch chain es-
ters (7, 9, and 10) have beneficial effect compared to
straight chain esters (4 and 8), while the presence of
unsaturation (5 and 6) does not lead to major changes.
Furthermore, the propargyl alcohol 6 could be toxic.
From these results we selected four AUDA esters (com-
pounds 7–10) that are both more potent inhibitors and
Finally, we investigated the pharmacokinetics of these
four esters in vivo. Each compound dissolved in olive
oil was administered to mice via oral gavage at 5 mg/
kg body weight. Compared to 1, this dose corresponds
to a molar dose 10% and 13% lower for 7 and 8–10,
respectively. The parent esters are undetectable or only

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I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
Table 1. Inhibition of murine and human sEH by 12-(3-adamantan-1-yl-ureido)dodecanoate derivatives.
O
O
N
N
H
R
H
O
c
Compound
R
H
IC50 (lM)
IC50 (nM)
Human sEH
3.2
Mp (ꢀC)
a
a
b
Murine sEH
Human sEH
1
0.05 ± 0.01
0.1 ± 0.01
114
75
2
3
0.05 ± 0.01
0.05 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
2.3
1.6
82
4
5
0.05 ± 0.01
0.05 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
1.0
1.0
86
81
6
7
8
0.05 ± 0.01
0.05 ± 0.01
0.05 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
1.8
1.1
0.8
79
90
65
9
0.05 ± 0.01
0.05 ± 0.01
0.05 ± 0.01
0.05 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
0.23 ± 0.01
0.7
1.2
1.3
1.5
65
91
1
1
1
0
1
2
150
68
1
1
1
3
4
5
0.07 ± 0.01
0.05 ± 0.01
0.09 ± 0.01
0.13 ± 0.01
0.29 ± 0.01
0.21 ± 0.01
2.0
1.7
2.4
49
48
52
Cl
a
Spectrometric-based assay: enzymes (0.12 lM murine sEH or 0.24 lM human sEH) were incubated with inhibitors for 5 min in 0.1 M sodium
phosphate buffer (200 lL; pH 7.4) at 30 ꢀC before spectrometric substrate (NEPC) introduction ([S] = 40 lM), results are means ± SD of three
separate experiments.
b
c
Fluorescent-based assay: human sEH (0.96 nM) was incubated with inhibitors for 5 min in 25 mM Bis-Tris/HCl buffer (200 lL; pH 7.0) at 30 ꢀC
before fluorescent substrate (CMNPC) introduction ([S] = 5 lM), results are averages of three separate measurements.
Melting point.

I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
317
Table 2. Physical properties, hydrolysis, and pharmacokinetic parameters of 12-(3-adamantan-1-yl-ureido)dodecanoic acid alkyl ester derivatives
Compound
1
7
8
9
10
Solubility in water (lg/mL)
Solubility in oil (mg/mL)
35
5
7
12
5
25
5
27
1.7
17
a
hCE-1 hydrolysis (%)
100
100
72
39
41
39
50
26
55
33
b
hCE-2 hydrolysis (%)
c,f
C
max (ng/mL)
26.9
87
2.3
1.0
29.6
84
39.5
96
13.1
47
42.9
d,f
AUC (ng Æ h/mL)
110
e,f
1/2 (h)
T
1.9
1.1
1.5
1.3
3.8
0.6
2.9
1.4
g
Relative bioavailability
a,b
Percentage (%) of compound 1 produced after 1 h incubation of compounds 1 and 7–10 with human carboxylesterase-1 (hCE-1) and human
carboxylesterase-2 (hCE-2), respectively. Enzymes (333 nM) were incubated with inhibitors ([S] = 1.0 lM) for 0, 30, and 60 min with data shown for
6
0 min in sodium phosphate buffer (0.1 M; pH 7.4) at 37 ꢀC. Results are averages of three separate measurements.
Maximum concentration.
Area under the concentration–time curve to terminal time. In each case AUDA 1 was the predominate metabolite detected with the esters only
c
d
minor products.
Elimination half-life.
e
f
Because in each case AUDA 1 was the predominant metabolite with the parent esters only minor products, the parameters were calculated based on
the blood concentration of AUDA released from the esters.
Ratio of molar corrected AUC calculated after administration of each ester (7–10) to the AUC obtained after administration of AUDA 1.
g
50
45
40
35
30
25
20
15
10
5
0
1
7
8
9
1
0
0
5
10
15
20
25
Time after administration (hr)
Figure 1. Blood concentration of AUDA 1 in mice as a function of time following oral gavage of sEH inhibitors 1 and 7–10. All compounds were
dissolved in olive oil and were given at 5 mg/kg body weight. Data are averages of results obtained from three different mice. Standard deviations
were between 5% and 10%.
at trace levels in the blood of orally treated animals, but
AUDA released from the esters was observed (Fig. 1),
confirming that the esters could act as prodrugs for
AUDA. In order to determine if the ester derivatives
have improved bioavailability, pharmacokinetic param-
eters such as maximum concentration (Cmax), area under
the curve (AUC), and half-life (T1/2) were determined
using the blood concentrations of AUDA (Table 2). A
purpose. Half-lives of AUDA released from the four es-
ters (1.5–3.8 h) were similar to the one (2.3 h) obtained
by directly giving AUDA to the animals, as one could
expect. The short half-life of AUDA in the blood is
likely due to b-oxidation and also movement of the lipo-
philic compound into refractory pools as both the free
acid and biological esters. This may account for the long
beta phase of blood clearance. The observed changes in
Cmax and AUC for the various esters tested probably re-
flect differential absorption by the gut. The esters may
encourage penetration of the gut epithelium and thus
more rapid and complete movement into the blood-
stream. Since no or very low levels of esters were found
in the blood, ester hydrolysis could be almost immediate
or one could have the more lipophilic esters move quick-
ly from the blood into more refractory pools.
1
.5-fold increase in Cmax was observed with a substitu-
ent of an n-butyl (8) or isobutyl group (10). Improved
AUC results were also determined for these two com-
pounds (8 and 10), while a similar or a 2-fold lower Cmax
or AUC was observed for compounds 7 and 9, respec-
tively, compared to compound 1. It resulted in a 30–
4
0% increase of the relative bioavailability of AUDA
for 8 and 10. This implies that esterification of the acid
function of compound 1 is useful in improving bioavail-
ability, and that n-butyl (8) or isobutyl (10) esters are
more effective than other alkyl groups tested for this
In a second part of this work, we investigated the effect
of changing the acid function to an amide on inhibition

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I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
potency and physical properties, toward obtaining a
more stable and bioavailable sEH inhibitor. For this
purpose, seven amide derivatives, of compound 1 with
various functionalities were synthesized. As shown in
Table 3, alkyl, sulfonyl, lipoamino acid, and glucosyl
amide derivatives were prepared, and their inhibition
potency on sEHs, melting point, and solubility in water
or oil were examined. For murine sEH, there was no
change in the inhibition activity when the acid function
of compound 1 was substituted by an alkyl (16 and 17),
sulfonyl (18 and 19), glucosyl (20 and 21), and lipoami-
no acid (22) groups. Similarly, for human sEH, no big
differences in inhibition potencies, which were measured
by the spectrophotometric and fluorescent-based assays,
were observed when replacing the acid of compound 1
by the substituents except for compound 22 where inhi-
bition of the sEH decreased by 18-fold. When physical
properties were examined, no decreased melting point
was observed in the alkyl amides (16 and 17), while
the esters 3 and 7 had 25–32 ꢀC lower melting point than
that of compound 1, suggesting that alkyl amide func-
tion is not useful for improving physical properties of
the free acid 1. Because it was expected that a polar sul-
fonyl group can mimic the carboxylic acid function of
compound 1 in binding to the target enzyme, resist b-ox-
idation, and is also useful for making water soluble com-
pounds, two sulfonamide compounds with a methyl (18)
or phenyl (19) group were prepared. However, in both
compounds high melting points were measured, which
led to a 3.5-fold decrease in water solubility of com-
pound 18, and no improvement in oil solubility in either
case (18 and 19). This implies that the sulfonamides are
Table 3. Inhibition and physical properties of murine and human sEH by 12-(3-adamantan-1-yl-ureido)dodecanoic acid substituted amide
derivatives
O
H
N
N
N
R
H
H
O
c
Compound
R
IC50 (lM)
IC50 (nM)
Mp (ꢀC) Solubility in
Solubility in
water (lg/mL) oil (mg/mL)
a
a
b
Murine sEH
Human sEH
Human sEH
d
1
6
7
0.05 ± 0.01
0.1 ± 0.01
4.3
119
115
ND
ND
ND
ND
1
0.05 ± 0.01
0.05 ± 0.01
0.05 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
0.1 ± 0.01
2.9
2.1
1.9
O
S
1
1
8
9
103
100
10
4
5
O
O
ND
S
O
O
OH
OH
OH
HO
2
2
0
1
O
0.05 ± 0.01
0.05 ± 0.01
0.1 ± 0.01
7.5
7.6
130–140
100–102
38
2
N
H
(
)7
OH
OH
0.22 ± 0.01
ND
ND
HO
O
OH
O
22
0.05 ± 0.01
0.21 ± 0.01
55
oil
63
ND
OH
(
)7
a
Spectrometric-based assay: enzymes (0.12 lM murine sEH or 0.24 lM human sEH) were incubated with inhibitors for 5 min in 0.1 M sodium
phosphate buffer (200 lL; pH 7.4) at 30 ꢀC before substrate (NEPC) introduction ([S] = 40 lM), results are means ± SD of three separate
experiments.
b
Fluorescent-based assay: human sEH (0.96 nM) was incubated with inhibitors for 5 min in 25 mM Bis-Tris/HCl buffer (200 lL; pH 7.0) at 30 ꢀC
before substrate (CMNPC) introduction ([S] = 5 lM), results are averages of three separate measurements.
Melting point.
Non-determined.
c
d

I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
319
not effective for improving physical properties of com-
pound 1. The sulfonamides were insoluble in the triglyc-
eride formulation used for absorption, distribution,
metabolism, and elimination experiments described here
and in a separate protocol where the compounds were
dosed in triglyceride at 0.1 mg/kg body weight. In both
cases, the resulting blood levels were very low. We fur-
ther investigated the use of glycosylation as a method
of improving water solubility of compound 1. The con-
jugation of sugar moieties such as glucose has the
advantage of introducing a highly hydrophobic group
in a compact, biologically compatible unit. In addition,
the introduction of the glucose moiety at the opposite
end of the molecule to the hydrophobic adamantyl
group has the potential to provide the resulting molecule
with surfactant properties because it now possesses a po-
lar head group and a lipophilic tail. Surfactants are well
known to exhibit improved membrane permeability, and
thus this property may greatly enhance the delivery of
the drug in vivo. The sugar moiety was conjugated to
the AUDA (1) molecule via an amide linkage that is rel-
atively stable in vivo. As shown in compounds 20 and
ing amide pharmacophore. The base structure of
AUDA (1) with carboxylic acid as a tertiary pharmaco-
phore raises the possibility of dramatically altering
physical properties with salts, esters of varying hydro-
phobicity, prodrugs, and isosters.
3. Conclusion
This investigation focused on producing potent and
bioavailable inhibitors of human sEH by the chemical
modification of the acid function of compound 1. We
found that esterification of the acid function of com-
pound 1 did not change the inhibition potency for
either murine or human sEHs. Further, the esterifica-
tion reduced the melting point up to 66 ꢀC and im-
proved solubility in oil (Table 2). This increased
solubility makes the resulting compound easier to for-
mulate in oil for oral gavage, subcutaneous or intra-
peritoneal injection, or in food. Furthermore, in vitro
and in vivo measurements clearly showed that com-
pound 1 is readily released from the esters, suggesting
that the esters reported herein could be good prodrugs
for AUDA (1). The esters also slightly improved the
pharmacokinetic properties of AUDA (higher Cmax
and AUC) in mice following oral gavage. On the other
hand, modification of the acid of compound 1 to an
amide function did not provide significant improve-
ment in inhibition potency, and it did not yield valu-
able improvements in physical properties. Overall, the
results indicate that modification of the chemical struc-
ture of 1 to esters, such as the n-butyl (8) or isobutyl
(10) derivatives is a very useful method to obtain po-
tent, more bioavailable inhibitors for the human
sEH. However, the half-life of AUDA was not im-
proved for these two esters, and it may limit their ther-
apeutic utilities for this particular inhibitor.
Nevertheless, such derivatives should be of interest
for inhibitors that have longer in vivo half-life but
poor physical properties. Furthermore, understanding
the metabolism of AUDA will highlight the source of
its relative in vivo instability and allow the design of
more stable derivatives.
2
1, higher melting points than that of 1 were observed.
Interestingly, no improved water solubility was obtained
from the glucosylated compound 20, which might result
from relatively high melting point of the compound
although a very polar glucose moiety is present in the
molecule. As with sulfonamides, a 2.5-fold decreased
solubility in oil was observed in the polar glucose deriv-
ative (20), suggesting that highly polar functional groups
like glucose are not effective for improving physical
properties in this series of structures. On the other hand,
a huge decrease in melting point was gained when the
glucose moiety was removed from the high melting com-
pound 20 (22). However, as mentioned above, a huge
decrease in the inhibition potency was exhibited by the
lipoamino acid compound (22), which is 18-fold less ac-
tive than compound 1, implying that a free acid function
present around sixteen atoms away from the urea func-
tion is not alone effective for retaining the inhibition on
the human enzyme. When compound 22 was incubated
with human carboxylesterases (hCE-1 and -2), no
hydrolysis by hCE-1 and -2 was exhibited, indicating
that the sterically hindered amide linkage of compound
2
2 is stable to hydrolysis by esterases. Unless there is
The free acid AUDA in aqueous 2-hydroxypropyl b-cy-
clodextrin or an oil solution of n-butyl or other esters of
AUDA are simple ways to administer these compounds
orally, intraperitoneally, or subcutaneously (see Supple-
mentary data for detailed instructions on formulation).
These formulations were successfully used for the inhibi-
hydrolysis by an amidase, there is a very low chance
for compound 22 to behave like a prodrug in vivo that
is hydrolyzed to produce the active component com-
pound 1. Although no significant improvements in the
physical properties of the amide derivatives listed in Ta-
ble 3 were observed, their relative stability and inhibi-
tion potency are sufficient to encourage the further
exploration of other amide compounds to develop bio-
available, potent inhibitors with improved physical
properties. We have reported that modification of the
urea pharmacophore of potent sEH inhibitors to an
amide functionality does not dramatically alter the inhi-
bition potency and that at least a 10-fold improvement
in water solubility and a decrease in the melting points
9
,27,37,38
tion of sEH in vivo in recent work.
The n-butyl
ester of AUDA 8 was used in a number of animal mod-
els for a combination of reasons. It has high potency on
the human enzyme as both the ester and the acid metab-
olite. It is inexpensive and can be made in high yield.
The melting point (65 ꢀC) is high enough to facilitate
purification by recrystallization, but low enough to facil-
itate rapid dissolution in oil-based formulations.
Acceptable pharmacokinetic profiles for 8 were found
following oral administration, intraperitoneal and sub-
cutaneous injections in triglyceride. These findings are
important for the ease of use of sEH inhibitors with in vi-
vo models of hypertension and inflammatory diseases,
3
6
of these amide inhibitors are observed. This suggests
that the inhibition potency and physical properties of
the amide derivatives in Table 3 might be improved with
the modification of the urea function to the correspond-

320
I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
and further for designing inhibitors of improved physi-
cal properties and bioavailability for the treatment of
the diseases.
pound 8 by using the corresponding alkyl bromide in-
stead of 1-bromobutane.
4
.1.3.
N-[12-(3-Adamantan-1-yl-ureido)dodecano-
yl]methanesulfonamide (18). To a solution of compound
1 (0.2 g, 0.51 mmol) and N-hydroxysuccinimide (60 mg,
4. Experimental
0
.56 mmol) in THF (10 mL) were added 1,3-dic-
4
.1. Syntheses
yclohexylcarbodiimide (0.12 g, 0.56 mmol) at room
temperature. The reaction mixture was stirred for
12 h and filtered. And then, the filtrate was purified
by column chromatography (hexane/ethyl ace-
tate = 1:1) to give 2,5-dioxopyrrolidinyl ester (I)
(0.18 g, 0.37 mmol) in 72% yield. To this intermediate
(I) dissolved in HMPA (10 mL) was added portionwise
4-dimethylaminopyridine (54 mg, 0.44 mmol; DMAP)
and methanesulfonamide (0.35 g, 3.7 mmol). After stir-
ring for 2 h at 90 ꢀC, the product was extracted with
ether (30 mL) and washed with water (30 mL). The
organic solution was dried over magnesium sulfate
and evaporated, and then the residue was purified
using column chromatography eluting with hexane
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 ioniza-
tion. Elemental analyses (C, H/N) were performed by
Midwest Microlab (Indianapolis, IN); analytical results
were within ±0.4% of the theoretical values for the for-
mula given unless otherwise indicated (see Supplementa-
1
ry data for detailed results). H NMR spectra were
recorded on QE-300 using tetramethylsilane as an inter-
nal standard. Signal multiplicities are represented as sin-
glet (s), doublet (d), double doublet (dd), triplet (t),
quartet (q), quintet (quint), multiplet (m), broad (br),
and broad singlet (br s). Synthetic methods are de-
scribed for representative compounds.
and ethyl acetate (1:1) to afford compound 18 (0.16 g,
1
0.34 mmol) in 92% yield. H NMR d (CDCl ) 1.23–
3
1.35 (12H, m), 1.44–1.52 (4H, m), 1.57–1.61 (2H, m),
1
.65–1.69 (6H, m), 1.92–1.98 (6H, m), 2.07–2.09 (3H,
4
.1.1. 12-(3-Adamantan-1-yl-ureido)dodecanoic acid (1).
m), 2.38 (2H, t, J = 6.9 Hz), 3.11 (2H, q, J = 6.9 Hz),
3.20 (3H, s), 4.40 (1H, s), 4.48 (1H, s), 10.52 (1H, s)+,
LC-MS (ESI) m/z calcd for C H N O S [M+H]
A mixture of 12-aminododecanoic acid (6.1 g, 28 mmol)
and 1-adamantyl isocyanate (5.0 g, 28 mmol) in 1,2-di-
chloroethane (100 mL) was refluxed for 6 h under N2.
After cooling the reaction mixture to room temperature,
the solid product produced was filtered, triturated and
then washed with hexane (50 mL), and dried in the vac-
2
4
43
3
4
+
469.30,
(C H N O S) C, H, N.
found
[M+H] ,
mp
103 ꢀC,
Anal.
2
4
43
3
4
Compound 19 was synthesized with the same method
used for the preparation of compound 18 using benzene-
sulfonamide instead of methanesulfonamide. H NMR
uum oven at 40 ꢀC to afford pure compound 1 as a white
solid in 95–100% yield. H NMR d (CDCl ) 1.20–1.36
1
1
3
(
1
16H, m), 1.42–1.48 (2H, m), 1.57–1.65 (6H, m), 1.82–
.90 (6H, m), 1.94–1.98 (3H, m), 2.18 (2H, t,
J = 6.9 Hz), 2.90 (2H, q, J = 6.9 Hz), 3.45 (1H, br s),
.43 (1H, s), 5.58 (1H, s), LC-MS (ESI) m/z calcd for
d (CDCl ) 1.23–1.35 (12H, m), 1.44–1.52 (4H, m),
3
1.57–1.61 (2H, m), 1.65–1.69 (6H, m), 1.94–1.98 (6H,
m), 2.06–2.09 (3H, m), 2.28 (2H, t, J = 6.9 Hz), 3.10
(2H, q, J = 6.9 Hz), 4.39 (1H, s), 4.93 (1H, s), 5.45
(1H, s), 7.50–7.55 (2H, m), 7.60–7.62 (1H, m), 7.80–
7.83 (1H, m), 8.05–8.08 (1H, m), LC-MS (ESI) m/z calcd
5
+
+
C H N O [M+H] 393.30, found [M+H] 393.28,
3
mp 114 ꢀC, Anal. (C H N O ) C, H, N.
2
3
40
2
2
3
40
2
3
+
+
for C H N O S [M+H] 532.31, found [M+H]
2
9
45
3
4
4
.1.2. 12-(3-Adamantan-1-yl-ureido)dodecanoic acid butyl
ester (8). To a solution of 1 (0.2 g, 0.51 mmol) in DMF
20 mL) were added potassium carbonate (84 mg,
.61 mmol) and 1-bromobutane (86 mg, 0.61 mmol),
532.34, mp 100 ꢀC, Anal. (C H N O S) C, H, N.
2
9
45
3
4
(
0
4.1.4.
2-[12-3-(Adamantan-1-yl-ureido)dodecanoylami-
no]decanoic acid (22). Sodium metal (3.9 g, 0.17 mol)
was dissolved in ethanol (100 mL) under an inert atmo-
sphere in a round-bottomed flask fitted with a water
condenser. Diethyl acetamido malonate (30.4 g,
0.14 mol) was then added followed by 1-bromooctane
(36.7 g, 0.19 mol). The solution was refluxed overnight
under an inert atmosphere. The reaction mixture was
poured onto crushed ice (600 mL) and stirred. The
aminodiester product precipitated and was collected
by filtration. The crude product was then refluxed
overnight in a solution of HCl/DMF (9:1, 200 mL).
The precipitated product was collected by filtration,
washed with ice water, and air-dried in a vacuum des-
iccator to afford the a-amino acid hydrochloride in
>90% crude yield. The crude amino acid (3.0 g,
24.8 mmol) was then dissolved in methanol (100 mL)
and cooled to 0 ꢀC. Thionyl chloride (5.0 mL,
25.8 mmol) was added dropwise, and the reaction mix-
ture was stirred at 0 ꢀC for 10 min and then refluxed
and the reaction mixture was stirred for 24 h at room
temperature. The product was extracted with diethyl
ether (30 mL) and washed with water (30 mL). Then
the organic solution was dried over magnesium sulfate
and evaporated. The residue was purified using silica
gel column chromatography eluting with hexane and
ethyl acetate (5:1) to provide compound 8 as a white sol-
1
id in 94% yield. H NMR d (CDCl ) 0.95 (3H, t,
3
J = 6.9 Hz), 1.23–1.35 (12H, m), 1.44–1.52 (4H, m),
1.57–1.61 (4H, m), 1.66–1.69 (6H, m), 1.96–2.00 (8H,
m), 2.07–2.09 (3H, m), 2.30 (2H, t, J = 6.9 Hz), 3.11
(
(
2H, q, J = 6.9 Hz), 4.02–4.10 (4H, m), LC-MS
+
ESI) m/z calcd for C H N O [M+H] 449.37,
2
7
48
2
3
+
found [M+H] 449.36, mp 65 ꢀC, Anal. (C H N O )
C, H, N.
2
7
48
2
3
Compounds 2–7, 9, 10, 13, and 14 were prepared in the
same method as that used for the preparation of com-

I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
321
overnight. The reaction mixture was cooled to room
temperature and the volatiles removed under reduced
pressure, and the crude product was triturated in meth-
anol to afford racemic methyl 2-amino decanoate (II),
the reaction flask, and the reaction mixture was stirred
under N overnight. The resulting suspension was fil-
2
tered through Celite, which was washed well with ethyl
acetate (ꢀ100 mL), and the filtrate was washed with
4
.44 g, 89% yield.
5 M HCl (50 mL), saturated NaHCO (2· 50 mL),
3
and brine (1· 50 mL). The organic solution was dried
over magnesium sulfate, filtered, and concentrated.
The residue was purified using a silica gel column chro-
Compound 1 (1.04 g, 2.65 mmol) and HBTU (1.0 g,
.64 mmol) were dissolved in THF (60 mL). DIEA
2
(0.5 mL, 2.87 mmol), DMF (ꢀ2 mL), and methyl 2-ami-
no decanoate II (1.26 g, 5.30 mmol) were added, and the
matography with 10% methanol/dichloromethane to
1
yield V (0.16 g, 59.33). H NMR d (CDCl ) 0.78 (3H,
3
solution was stirred under N at room temperature over-
2
night. The yellow oil produced was diluted with 5% cit-
ric acid (100 mL) and extracted with ethyl acetate
t, J = 7 Hz), 1.16–1.23 (32H, br m), 1.35 (2H, m) 1.52
(2H, m) 1.57 (6H, br s), 1.86 (6H, br s,), 1.91–2.00
(15H, m), 2.14 (2H, m), 2.99 (2H, t, J= 7 Hz), 3.75
(2H, m), 3.95–4.07 (3H, m), 4.15–4.29 (4H, m), 4.43–
4.38 (1H, m) 4.80 (1H, dd, J = 5, 11 Hz), 4.84–4.89
(2H, m), 4.95–5.02 (3H, m), 5.12–5.24 (3H, m), 5.35
(1H, d, J = 3 Hz), 5.47 (1H, t, J = 9.8 Hz), 6.28 (1H,
dd, J = 7.6, 19 Hz), 6.60 (1H, d, J = 9.3 Hz), 7.18
(1H, d, J = 9.1 Hz), 7.34 (1H, d, J = 9.1 Hz). LC-MS
(
3· 50 mL). Organic layers were combined and washed
with 5% citric acid (2· 50 mL), saturated sodium bicar-
bonate (NaHCO ) (2· 50 mL), and brine (1· 50 mL).
3
The organic layer was dried over magnesium sulfate
and evaporated to yield an oil. The crude product was
purified by column chromatography with 1–2% metha-
nol/dichloromethane to yield a yellow oil III (0.77 g,
+
(ESI) m/z calcd for C H N O [M+H] 891.56,
4
7
78
4
12
+
found [M+H] 891.69.
66% yield). The methyl ester III (0.77 g, 1.34 mmol)
was dissolved in DME (25 mL) and water (10 mL). Solid
LiOHÆH O (0.33 g, 7.86 mmol) was added, and the solu-
tion was stirred at room temperature overnight. Reac-
tion mixture was acidified (pH 4) with 5% citric acid
To a solution of V (0.16 g, 0.18 mmol) in methanol
(15 mL) was added sodium metal (0.05 g), and the mix-
ture was stirred at room temperature overnight. The
reaction mixture was filtered through amberlite and
washed with methanol (ꢀ150 mL). The solvent was
evaporated under vacuum to produce compound 20 as
a fine white powder (0.12 g, 95%). LC-MS (ESI) m/z
2
(
ꢀ20 mL), and the product was extracted with ethyl ace-
tate (3· 30 mL). Combined organic layers were washed
with brine (30 mL), dried over MgSO , filtered, and
evaporated to yield compound 22 as a yellow oil
4
1
+
+
(
0.34 g, 45% yield). H NMR d (CDCl ) 0.82 (3H, t,
calcd for C H N O [M+H] 724.50, found [M+H]
39 70 4 8
3
J = 7 Hz), 1.16–1.23 (28H, br m), 1.41 (2H, m), 1.57
2H, br m), 1.60 (6H, br s), 1.89 (6H, br s), 2.01 (3H,
724.6, mp 130–140 ꢀC.
(
br s), 2.18 (2H, t, J = 7 Hz), 2.98 (2H, m), 4.52 (1H,
dd, J = 7.3, 13.3 Hz), 6.46 (2H, d, J = 7.6 Hz) 8.16
Compound 21 was prepared with a method similar to
the one used for the preparation of compound 20, but
starting from compound 1 instead of compound 22.
1
3
(
1H, br s). C NMR d (CDCl ) 14.0, 22.5, 25.0, 25.6,
3
2
2
5
6.7, 28.85, 28.00, 29.06, 29.10, 29.13, 29.24, 29.31,
9.44, 29.56, 31.73, 32.22, 36.27, 36.41, 40.68, 42.29,
0.96, 52.31, 158.70, 173.65, 175.39. LC-MS (ESI) m/z
4.2. Enzyme preparation
+
+
calcd for C H N O [M+H] 562.45, found [M+H]
4
Recombinant murine and human sEHs, as well as hu-
man carboxylesterases (hCE-1 and hCE-2), were pro-
duced in a baculovirus expression system and were
3
3
59
3
5
62.51.
3
9–42
4
(
.1.5. 12-(3-Adamantan-1-yl-ureido)dodecanoic acid [1-
2,3,4-trihydroxy-5-hydroxymethyl-cyclohexylcarbamoyl)-
purified as previously reported.
nonyl]amide (20). 1,2,3,4,6-Penta-O-acetyl-a-D-glucopyr-
anose (5.00 g, 12.8 mmol) was dissolved in 10–15 mL
dry CH Cl under an inert atmosphere. Trimethylsilyl
4.3. IC50 assay conditions
Spectrometric-based assay: enzymes (0.12 lM murine
sEH or 0.24 lM human sEH) were incubated with
inhibitors for 5 min in 0.1 M sodium phosphate buffer
(200 lL; pH 7.4) at 30 ꢀC before spectrometric substrate
(4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbon-
2
2
azide (4.24 mL, 32.1 mmol) and tin (IV) chloride
0.75 mL, 6.41 mmol) were added, and the reaction
(
mixture was stirred for 18 h at room temperature.
The reaction mixture was diluted with CH Cl
2
2
(
(
30 mL) and washed twice with saturated NaHCO3
20 mL) and with brine (20 mL). The organic phase
ate; NEPC) introduction ([S] = 40 lM). The IC s were
50
calculated from at least three separate runs, each in trip-
licate, to obtain the standard deviation given in the re-
was dried, filtered, and concentrated to leave 2,3,4,6-
tetra-O-acetyl-b-D-glucopyranosyl azide (IV) as a white
solid (3.92 g, 82%). IV (0.30 g, 80 mmol) was then dis-
solved in dry THF (15 mL), and Pd/C was added
(
H overnight. TLC revealed the reduction was com-
4
3
sults section.
Fluorescent-based assay: human sEH (0.96 nM) was
incubated with inhibitors for 5 min in 25 mM Bis-Tris/
HCl buffer (200 lL; pH 7.0) at 30 ꢀC before fluorescent
ꢀ5%) to this solution. The mixture was stirred under
2
pleted (Rfazide = 0.80, Rfamine = 0.10). In a separate
flask, compound 22 (0.17 g, 0.30 mmol) and 0.5 M
HBTU in DMF (1.20 mL, 0.60 mmol) were combined,
followed by the addition of DMF (ꢀ2 mL) and DIEA
substrate
trans-(3-phenyl-oxyran-2-yl)methyl
CMNPC) introduction ([S] = 5 lM) as that described
(cyano(2-methoxynaphthalen-6-yl)methyl
carbonate;
3
1
in a previous report. IC s results are averages of three
5
0
(
104 lL, 0.60 mmol). This solution was then added to
replicates.

3
22
I.-H. Kim et al. / Bioorg. Med. Chem. 15 (2007) 312–323
6. Newman, J. W.; Morisseau, C.; Hammock, B. D. Prog.
Lipid Res. 2005, 44, 1–51.
4
.4. Solubility
7
. Capdevila, J. H.; Falck, J. R.; Harris, R. C. J. Lipid Res.
2
Water solubility was determined experimentally in 1.0
mL of sodium phosphate buffer (0.1 M, pH 7.4) at
000, 41, 163–181.
2
4,36
8. Smith, K. R.; Pinkerton, K. E.; Watanabe, T.; Pedersen,
T. L.; Ma, S. J.; Hammock, B. D. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 2186–2191.
2
5 ± 1.5 ꢀC as previously reported.
Solubility in oil
was also determined experimentally (see Supplementary
data for details).
9
. Schmelzer, K. R.; Kubala, L.; Newman, J. W.; Kim, I.-H.;
Eiserich, J. P.; Hammock, B. D. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 9772–9777.
4
.5. Hydrolysis by esterases
1
0. Yu, Z.; Xu, F.; Huse, L. M.; Morisseau, C.; Draper, A. J.;
Newman, J. W.; Parker, C.; Graham, L.; Engler, M. M.;
Hammock, B. D.; Zeldin, D. C.; Kroetz, D. L. Circ. Res.
Hydrolysis of ester compound (7–10; 100 lM) to its acid
metabolite (1) was examined using purified hCE-1 (5 lg)
or hCE-2 (5 lg). A percentage of free acid metabolite (1)
was calculated using LC-MS/MS. Detailed experimental
procedures are available in the Supplementary data.
2
000, 87, 992–998.
1
1
1. Imig, J. D.; Zhao, X.; Capdevila, J. H.; Morisseau, C.;
Hammock, B. D. Hypertension 2002, 39, 690–694.
2. 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. J. Am. Soc. Nephrol. 2004,
4
.6. In vivo pharmacokinetic study
1
5, 1244–1253.
Male Swiss Webster mice, 7 weeks old, were treated with
test compounds orally at 5 mg/kg body weight. Blood
sample preparation and LC-MS/MS analysis were per-
13. Imig, J. D.; Zhao, X.; Zaharis, C. Z.; Olearczyk, J. J.;
Pollock, D. M.; Newman, J. W.; Kim, I.-H.; Watanabe,
T.; Hammock, B. D. Hypertension 2006, 46,
4
4
9
75–981.
4. Oltman, C. L.; Weintraub, N. L.; VanRollins, M.;
formed as previously reported.
Pharmacokinetic
1
1
1
parameters (Cmax, AUC, and T1/2) were calculated using
AUDA blood concentrations. Data are average results
obtained from at least three different mice.
Dellsperger, K. C. Circ. Res. 1998, 83, 932–939.
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This work was supported, in part, by NIEHS Grant R37
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ences P30 ES05707, NIH/NIEHS Superfund Basic Re-
search Program P42 ES04699, NIH/NHLBI R01
HL59699-06A1, and UCDMC Translational Technolo-
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