Salicylate-urea-based soluble epoxide hydrolase inhibitors with high metabolic and chemical stabilities

Article abstract of DOI:10.1016/j.bmcl.2009.01.069

We investigated N-adamantyl-N′-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester derivatives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabilities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo.

Full text of DOI:10.1016/j.bmcl.2009.01.069

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1784–1789
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Salicylate–urea-based soluble epoxide hydrolase inhibitors with high
metabolic and chemical stabilities
,à
à
*
Takeo Kasagami , In-Hae Kim , Hsing-Ju Tsai, Kosuke Nishi , Bruce D. Hammock, Christophe Morisseau
Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
We investigated N-adamantyl-N0-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester deriv-
atives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The
methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabil-
ities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 16 December 2008
Revised 20 January 2009
Accepted 21 January 2009
Available online 27 January 2009
Keywords:
Soluble epoxide hydrolase
Urea
Salicylate
Intramolecular hydrogen bond
Metabolic and chemical stability
Hypertension
Anti-inflammation
Epoxide hydrolases (EH, EC 3.3.2.3) catalyze the hydrolysis of
epoxides and arene oxides to their corresponding diols.1 The mam-
malian soluble EH (sEH) is involved in maintenance of homeostasis
through hydration of endogenous lipid epoxides such as epoxyei-
cosatrienoic acids (EETs).1,2 EETs, derived from arachidonic acid
by cytochrome P450 epoxygenation, have various biological activ-
ities. EETs have effect on vascular tone. In addition, it is known that
the 11,12- and 14,15-regioisomers of EETs have anti-inflammatory
activity.2 The EETs inhibit activation of nuclear factor kappa B
Figure 1. General schematic structure of urea-based human sEH inhibitors.
cyclohexyl, alkyl or aryl groups (R1, Fig. 1). The secondary pharma-
cophore (P2) is a polar group, such as ketone, ester, alcohol, sulfox-
ide, sulfonamide or ether located five or six atoms away from the
carbonyl group of the urea. A hydrophobic linker (L) joins the urea
or amide central pharmacophore to the secondary pharmacophore.
Unlike straight alkyl chains, the presence of cyclic linker (L), such
as a cyclohexane or benzene ring, between the primary (urea)
and secondary (P2) pharmacophores, seemed to increase the bio-
availability in a canine model.5 Such cyclic linkers also reduce flex-
ibility and are thought to make compounds more ‘‘drug-like”.6 In
addition, the introduction of polar group(s) on such linkers likely
will improve solubility, ease of formulation, and often their bio-
availability. The nature of group R2 situated between P2 and P3 is
more open to variation in term of size and polarity as long as P3
that is a polar group is at least 12 Å away from the urea carbonyl.5
Therefore, in this study, we designed simple adamantyl ureas
with a phenyl linker. These sEH inhibitors focus on the secondary
pharmacophore. Usage of a phenyl moiety has several advantages
for drug design, (1) the benzene ring, which is UV active, enables
one to easily trace the target molecule during the synthesis or later
(NF-jB) and its nuclear translocation. This means that, under cer-
tain conditions, EETs will reduce activation of a variety of pro-
inflammatory peptides and proteins such as cyclooxygenase 2.3
Since sEH converts EETs to the less biological active corresponding
diols (dihydroxyeicosatrienoic acids), sEH inhibition can result in
anti-inflammatory and anti-hypertensive effects.
1,3-Disubstituted ureas, and the corresponding amides and car-
bamates are strong sEH inhibitors.4,5 Our previous findings sug-
gested that ureas with two additional pharmacophores (P2 and
P3, Fig. 1) have strong inhibition in vitro as well as good bioactivity
in vivo.5 The primary pharmacophore is the urea group bearing a
bulky and/or hydrophobic substituent such as the adamantyl,
* Corresponding author. Tel.: +1 530 752 6571; fax: +1 530 752 1537.
E-mail address: chmorisseau@ucdavis.edu (C. Morisseau).
These authors contributed equally to this work.
à
Present address: Institute of Molecular Science, Chonnam National University,
300 Yongbong-dong, Buk-gu, Gwangju 500-757, South Korea.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.069

T. Kasagami et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1784–1789
1785
scale-up and upon HPLC analysis; (2) as mentioned above, a ben-
zene ring will play a role as a linker to keep a rigid distance be-
were prepared from the corresponding phenol (3 or 4) and appro-
priate acyl chloride in a conventional manner.
tween the primary and secondary pharmacophores; (3)
a
The inhibitory activity of the ureas with a benzene ring bearing
a hydroxyl, ether, ester, carboxylic acid or amide functional group
are given in Table 1. When a hydroxyl group was introduced in the
meta (3) or para (4) position on the benzene ring, the inhibitory
activity against human sEH was almost the same level as the
non-functionalized compound 1. However, the introduction of hy-
droxy group as an ortho isomer (2) reduced the inhibitory potency
by roughly 10-fold. It was found that the introduction of methoxy
group in the ortho position (5) reduced the inhibitory activity dra-
matically although meta (6) and para (7) isomers possessed strong
inhibition against sEH. These data indicated that the meta or para
position on the benzene ring is a preferable position for the intro-
duction of a functional group without adversely affecting sEH inhi-
bition. We previously observed that only compounds bearing
hydrogen or fluoride atoms on the ortho positions have sEH inhibi-
tion activity.4d The compounds bearing ester (8–13) as well as
ether groups (6 and 7) were around 2- to 6-fold better sEH inhib-
itors than 3 and 4, and even better than 1, suggesting that an ether
or ester group with hydrophobic properties helps bind to the active
site of the human sEH enzyme. In previous studies,4,5 it was found
that butyrate and caproate derivatives substituted in the 3 position
of the urea were inactive as inhibitors of the sEH. However, their
esters as secondary pharmacophores were highly active and could
be used as soft drugs. In contrast, substitution of the 3 position of
the urea with long chain acids such as dodecyl gave a tertiary phar-
macophore as active as the ester, the acid or an acid mimic.5 In
these cases, the ester is a pro-drug increasing ease of formulation
and absorption. In the series of compounds described herein, most
esters were very active (8–13). The dramatic decrease in the activ-
ity of the free acids is illustrated by compounds 14 and 15. Espe-
cially, compound 15, which had the acid group in the para
position, is 83-fold less active towards sEH than the un-substituted
phenyl 1 or the corresponding ester 13. Cyclic amides with a mor-
pholine (16 and 17) or piperidine (18) were also synthesized.
Unfortunately, up to 9-fold reduction in inhibitory potencies re-
benzene ring, unlike a cycloalkane ring, has no stereoisomers, thus
simplifying synthesis; and finally (4) numerous substituted ani-
lines are commercially available simplifying the synthesis of ureas
with functionalized phenyl rings, such as carboxylic acids that will
increase the water solubility of the molecule. Many of these inter-
mediates are inexpensive facilitating design of drugs for use in
developing countries. Further, a carboxylic acid group can be con-
nected with other functional group like alcohol or amine to design
the tertiary pharmacophore. Herein, we report the sEH inhibitory
activity as well as metabolic and chemical stabilities of simple N-
adamantyl-N0-phenyl urea derivatives.
The urea compounds (1–4) were synthesized through the di-
rect reaction with adamantyl isocyanate and the appropriate
amine (Scheme 1). Although the isocyanate functional group
can react with many nucleophiles such as alcohols, carboxylic
acids and amines, the hydroxy group of aminophenol did not re-
act significantly under our reaction conditions. However, for the
synthesis of the carboxylic acid group-containing compound
(14, 15, 19, 21, 23, 25 and 27), it was necessary to protect the
acid function with a methyl ester that was removed following
purification by alkaline hydrolysis (Scheme 1). The starting
methyl esters (I–VII) were synthesized from the corresponding
acids (i–vii) in a conventional manner. After the reaction with
the isocyanate and appropriate amine followed by evaporation,
the unreacted starting materials were removed by washing the
crude product with a hexane:ethyl acetate mixture. This opera-
tion was simple but effective to remove not only the residual
starting materials but also the N,N0-bisadamantyl urea which is
not only present in the commercially available adamantyl isocya-
nate but can be formed as a side product during urea synthesis.
This relatively insoluble high melting solid also shows strong
inhibition towards sEH.4 After washing, the desired compound
was purified through silica gel column chromatography and
recrystallization as final purification stage. The compounds 8–11
R1
H2N
O
O
O
R2
b
R1
a
NCO
N
H
N
H
N
H
N
H
O
1-7
8-11
(R1=H, OH or OCH3)
(R2=CH3 or phenyl)
a
n
n
COOH
n
COOCH3
O
O
d
COOCH3
COOH
N
H
N
H
N
H
N
H
R3
H2N
R3
R3
I-VII
16, 18, 20, 22, 24, 26, 28
15, 17, 19, 21, 23, 25, 27
c
e
Y
n
O
X
N
H2N
N
H
N
H
R3
O
i-vii
12-14
n=0~3
R3=H or OH
X=O, C or N
Y=CH3 or 4-morpholinyl
Scheme 1. Syntheses of the urea compounds used in this study. Reagents and conditions: (a) 1,2-dichloroethane, 45–50 °C, overnight; (b) THF, NaH, acetyl or benzoyl
chloride, 45–50 °C, overnight; (c) methanol, concd H2SO4, overnight; (d) THF, 5% NaOH aq, reflux, 2–4 h; (e) dichloromethane, 4-dimethylaminophenol, 1-ethyl-3-[3-
(dimethylamino)propyl] carbodiimide, appropriate amine, room temperature.

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T. Kasagami et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1784–1789
Table 1
Inhibitory activity of the ureas with a benzene ring containing a hydroxy, an ester, a carboxylic acid, amide or no functional group, against human sEH
a
a
Compound
Human sEH IC50 (nM)
Compound
Human sEH IC50 (nM)
O
O
N
H
N
H
N
H
N
H
O
1
2
17
10
11
3.9
11
O
O
139
O
O
OH
3
4
28
24
12
13
14
OH
OH
O
O
4.1
O
OH
5
6
7
8
8544
14
15
16
17
365
1411
25
O
O
O
4.7
OH
N
O
O
O
14
O
O
N
O
5.8
29
N
N
O
O
O
O
O
N
9
5.1
18
35
a
As determined via a kinetic fluorescent assay.13
sulted from these amides compared with ester substitutions, sug-
gesting that such amides are not suitable for yielding potent inhib-
itors. Interestingly, such heterocyclic groups when attached at the
end of an alkyl chain yield ureas that have good pharmacokinetic
properties in dogs while maintaining inhibitory potency.5f
Compared to 15, inhibition potency on sEH is regained by mov-
ing the carboxylic acid away from the benzene ring (Table 2). A
similar effect was observed with a series of alkyl-acids in place
of the phenyl-acids.4d On the other hand, the corresponding methyl
esters (13, 20, 22 and 24) showed strong inhibitory activities
against sEH independently of the alkyl chain length between the
ring and the methyl carboxylate. Recent X-ray analysis of the com-
plex between the human sEH and urea inhibitors7 indicated that a
basic amino acid residue(s) such as histidine and/or tryptophan, is
present in the catalytic tunnel away from the active center of sEH.
Such residues could make a salt bridge with an ionized carboxylate
as the tertiary pharmacophore, and the salt formation would affect
the orientation and affinity of such an ionizable acid molecule un-
der physiological pH. Taken together, these data suggested that a
free carboxylic acid or other ionizable polar group can be consid-

T. Kasagami et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1784–1789
1787
Table 2
The presence of a polar functional group in either the meta or
para position on the phenyl group linker appeared beneficial for
inhibition (Table 1). Thus, we designed and synthesized molecules
containing both acid and hydroxyl functions (Table 3). The methyl
salicylates 26 and 28 inhibited sEH strongly, with IC50s similar to
those of 12 and 13, suggesting that for the methyl ester, the adja-
cent phenolic group did not negatively influence inhibitor binding
to the enzyme. Surprisingly, the salicylic acids 25 and 27 showed
3- and 20-fold better inhibitory activity against sEH than 14 and
15, respectively. Infrared analysis of course shows strong internal
hydrogen bonds of the salicylates (25 and 27) in contrast to the
free carboxylic acids (14 and 15). Furthermore, the hydroxyl group
by itself placed on meta 3 or para 4 position did not improved the
inhibitor potency of 1, suggesting that the hydroxyl group plays an
important role in binding with the active site of sEH only if a car-
boxylic function is placed on the adjacent carbon. In such cases, a
hydrogen bond between the carboxylate and the hydroxyl is prob-
ably formed that certainly reduces the negative effect of the acid
function on the inhibition potency.
In general, the metabolic stability of a candidate compound will
influence drug efficacy in vivo. Although all esters synthesized in
this study showed strong inhibitory activities against the recombi-
nant human sEH, it was anticipated that they would be hydrolyzed
easily in the body. Thus, six compounds (10–13, 26 and 28) were
tested for in vitro stability with human hepatic S9 fraction with
or without NADPH, a cofactor necessary for cytochrome P450
activity.8 For all the compounds tested the results obtained were
similar with or without NADPH, suggesting that the principal route
of metabolism of these chemicals did not involve P450s. As ex-
pected, for most compounds only a small amount of the parent
compound was left after 60 min of incubation (Table 4), and large
amounts of the corresponding acids were detected. Unexpectedly,
90% or more of the methyl esters 13 and 28 remained after the
reaction, and the levels of the corresponding acid metabolites (15
and 27, respectively) were below the limit of detection, indicating
that, for this series of compounds, esters present on the para posi-
tion are more metabolically stable than on a meta position. The
quasi-absence of ester hydrolysis for the para compounds (13
and 28) is probably due to steric interactions that do not permit
an optimal binding into the liver esterases for hydrolysis. Com-
pared to 12, the presence of a hydroxyl group in para 26 increased
the metabolic stability of the resulting compound 10-fold. Other-
wise, 10 and 11, which give the phenol 4, were also decomposed
easily under the same reaction conditions. The acetate 10 was
hydrolyzed completely in an hour to give the corresponding phenol
Inhibitory activity of the urea compounds with alkyl chains (C0–C3) between
benzene ring and a carboxylate functional group, against human sEH
a
OR
n
O
O
N
N
H
H
R = H
R = CH3
a
a
Numbers of carbon (n)
Compound
IC50 (nM)
Compound
IC50 (nM)
0
1
2
3
15
19
21
23
1411
392
13
20
22
24
4.1
4.0
2.2
3.1
126
19.9
a
As determined via a kinetic fluorescent assay.13
ered distal from the urea carbonyl group as the tertiary but not the
secondary pharmacophore. When plotting the distance between
the urea carbonyl and the acid carbonyl as a function of the inhibi-
tion potency for both a series of alkyl-acids and phenyl-acids
(Fig. 2), we observed that both series of compounds followed a
similar hyperbolic curve. These plots suggested that such acid
functions as a tertiary pharmacophores should be roughly 12 Å
form the urea carbonyl.
Figure 2. Effect of the distance between the urea carbonyl and the acid carbonyl on
the inhibition potency. The distances were measured after minimizing the free
energy of the molecule in gas phase, using the Chem3DÒ 8.0 software (Cambridge-
Soft, Cambridge, MA).
Table 4
In vitro stability of the urea compounds containing an ester group on a phenyl ring
with human liver S9 incubation
R1
R2
Compound
NADPH
Residual parent
compound (%)
Table 3
O
Inhibitory activity of the benzoate- or the salicylate-based urea compounds against
human sEH
N
N
a
H
H
Compound
Human sEH IC50 (nM)
R1
R2
R1 (para)
R2 (meta)
ꢀ
3.5
46
97
93
+
O
12
26
13
28
10
11
H
OH
COOCH3
COOCH3
OC(O)CH3
OC(O)Ph
COOCH3
COOCH3
H
OH
H
6.9
51
95
89
<0.1
31
N
N
H
H
<0.1
32
R1 (para)
R2 (meta)
H
29a
30a
H
O(CH2CH2O)2C2H5
H
N.D.b
N.D.b
56
88
25
26
27
28
OH
OH
COOH
COOCH3
COOH
COOCH3
OH
116
12
71
O(CH2CH2O)2C2H5
a
IC50s on the human sEH are 1.8 and 1.1 nM for 29 and 30, respectively.
Not determined. Since the polyethoxylates are ethers not esters, incubation in
OH
2.8
b
a
As determined via a kinetic fluorescent assay.13
the absence of NADPH were not performed.

1788
T. Kasagami et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1784–1789
4. Compound 11 was hydrolyzed slower than 10 to produce around
70% of the phenol 4, while 30% of the starting compound 11 was
found. Imai et al.9 also reported that a phenyl acetate derivative
was hydrolyzed more rapidly by liver microsomal carboxylesteras-
es than methyl salicylate and benzoate derivatives. An earlier ser-
ies of ether containing compounds with a 5 or 6 carbon alkyl
spacer yielded potent inhibitors with excellent physical properties
but poor stability.5a Compounds 29 and 30 indicate that the methyl
ethers of 6 and 7 can be replaced successfully with polyethylene
glycol chains.
To investigate the surprising enzymatic stability of the benzoate
esters (13 and 28), and because most esterases have a nucleophilic
mechanism of action, we compared the chemical stability of these
two compounds in alkaline solution (40 mM NaOH; 22 °C) with the
para-phenolic esters (10 and 11) (Fig. 3). As observed with liver S9
(Table 4), 10 and 11 were hydrolyzed very fast with complete con-
version to the corresponding phenol 4 in less than 15 min (data not
shown). While 13 and 28 have similar metabolic stability with liver
S9 (Table 4), they were hydrolyzed at a different rate in alkaline
solution (Fig. 3). The methyl benzoate 13 was hydrolyzed gradually
to the corresponding acid 17 with a 2-h half-life. By contrast, the
methyl salicylate 28 was still more stable under the same reaction
conditions, with ꢁ80% remaining after 7 h. These results suggest a
different mechanism of hydrolysis for both compounds. In effect,
the neighboring phenolate was shown to influence salicylate ester
hydrolysis in alkaline conditions.10 This result suggested that intro-
ducing a hydroxyl group on the carbon adjacent to the benzoate es-
ter improved not only metabolic but also chemical stability of a
molecule by influencing its chemical reactivity. Furthermore, addi-
tional metabolic stability is obtained due to steric restrictions if the
ester is placed on the para position.
In addition to target potency and stability, a compound usually
needs good solubility to give good exposure to the biochemical tar-
get in vivo. As shown in Table 5, the compounds with a free acid
(15 and 27) have higher water solubility than the corresponding
methyl esters (13 and 28). Furthermore, as expected the addition
of a hydroxyl group on 27 led to a compound 20-fold more soluble
in water than 17. Interestingly, no such effect was observed for the
methyl esters 13 and 28. On the other hand, the hydroxyl group in-
creased solubility in octanol, which is a model of solubility in bio-
logical membranes,11 but only for the methyl ester 28 not its
corresponding acid 27. This result suggests that for 28 there is
probably an intramolecular bond between the hydroxyl group
and the ester carbonyl leading to increase miscibility with oil,
but not in water. These properties could aid absorption in the
gut. Finally, for the methyl esters (13 and 28), the measured logP
are very similar to the one calculated ones (clogP); however, for
the acids (17 and 27), the measured logP are between the clogP
of the acids and corresponding base forms. It suggests that both
forms of 17 and 27 are present at pH 7.4.
Finally, to test their bioavailability, the methyl salicylate 28 that
had good in vitro metabolic and chemical stability in addition to
potent sEH inhibitory activity, and its corresponding acid 27 were
administered orally to a dog. Blood levels of 28 and 27 were deter-
mined by LC/MS–MS (Fig. 4). We used AUDA, which has been uti-
lized in various biological studies,3,12 for comparison. As shown in
Figure 4, the blood concentration of 28 was higher than that of the
classical sEH inhibitor AUDA, indicating a good bioavailability.
While 28 and AUDA have similar IC50s (ꢁ3 nM) for the human
sEH, 28 is more water soluble and metabolically stable than AU-
DA.5d Thus, put together, 28 should be more efficient than AUDA
to inhibit sEH in vivo. Surprisingly, no 27 was detected in the blood
at all, indicating poor absorption.
In conclusion, benzoate–urea-based sEH inhibitors used in this
study had simple chemical structures and were easily synthesized.
The aromatic group made them easy to follow on thin layer chro-
matography. Furthermore, among them, the methyl salicylate 28
which is quite a potent sEH inhibitor has high metabolic and chem-
ical stabilities, as well as a good bioavailability. These data suggest
that such inhibitor is a potential lead molecule for a bioactive com-
pound in vivo. Finally, Schmelzer et al. reported recently that co-
Figure 3. Time-dependent hydrolysis of the methyl benzoate- and salicylate-based
urea compounds (13 and 28) under alkaline conditions (40 mM NaOH) at 22 °C.
Table 5
Physicochemical properties of the benzoate- or the salicylate-based urea compounds
Compound
Solubility in watera (mg/mL)
Mp (°C)
Experimental parameters and logP
R1
R2
O
N
N
H
H
R1 (para)
R2 (meta)
Concentrationb
Octanol
(
l
M)
logPc
clogPd
Buffer
13
17
27
28
COOCH3
COOH
COOH
H
H
OH
OH
0.4
10
199
0.2
219–220
>300
205–206
212–213
2445
2720
1641
3886
<0.3
17
39
>3.9
2.2
1.6
4.3
3.8/ꢀ0.5e
3.9/ꢀ1.1e
4.1
COOCH3
<0.3
>4.1
a
Solubility in 0.1 M sodium phosphate buffer (pH 7.4) at 23 1.5 °C.
b
c
Buffer saturated 1-octanol and 0.1 M sodium phosphate buffer (pH 7.4) were used. See Supplementary information in detail.
logP value was calculated following the equation; logP = log10 ([conc. of compound]octanol/[conc. of compound]buffer).
clogP was calculated using ChemDrawÒ software (CambridgeSoft, Cambridge, MA).
d
e
The clogP of both the acid and base forms of the carboxylic acid are given.

T. Kasagami et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1784–1789
1789
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Figure 4. Blood concentration–time profiles of sEH inhibitors (27, 28 and AUDA) in
dogs following oral gavage at a dose of 0.3 mg/kg in 6 mL of tri-olein rich oil.
Pharmacokinetic parameters are given in Supplementary materials.
administration of non-steroidal anti-inflammatory drugs and sEH
inhibitors enhanced anti-inflammatory and anti-nociceptional ef-
fects through dramatic suppression of prostaglandin E2 produc-
tion.3 Since the salicylate–urea compound in this study is also a
prospective cyclooxygenase inhibitor, this or related molecules
may have additional in vivo benefits with biological activities as
dual sEH-COX inhibitors. However, using a fluorescent inhibitor
screening assay kit (#700100, Cayman Chemicals, Ann Arbor, MI),
we were not able to show any inhibition of COX-1 and -2 with
11. Dennis, A. S.; van de Waterbeemd, H.; Walker, D. K.. In Pharmacokinetics and
Metabolism in Drug Design; Mannhold, R., Kubinyi, H., Timmerman, H., Eds.;
Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2006; Vol. 31, pp 1–15.
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Busse, R.; Fleming, I. Hypertension 2005, 45, 759; (b) Dorrance, A. M.; Rupp, N.;
Pollock, D. M.; Newman, J. W.; Hammock, B. D.; Imig, J. D. J. Cardiovasc.
Pharmacol. 2005, 46, 842; (c) 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.
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compound 28 at 10 lM.
Acknowledgments
The authors thank Dr. Jun-Yan Liu for his assistance on MS anal-
yses. This study was supported in part by NIEHS Grant R37
ES02710, NIEHS Superfund Grant P42 ES004699, and NIH/NHLBI
R01 HL059699. H.J.T. is a recipient of a Howard Hughes fellowship.
13. (a) Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock, B.; Hammock,
B. D. Anal. Biochem. 2005, 343, 66; (b) Morisseau, C.; Hammock, B. D. In
Techniques for Analysis of Chemical Biotransformation, Current Protocols in
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Eds.; John Wiley & Sons: New Jersey, 2007; pp 4.23.1–4.23.18.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.069.