Orally bioavailable potent soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1021/jm070270t

A series of N,N′-disubstituted ureas having a conformationally restricted cis- or trans-1,4-cyclohexane α to the urea were prepared and tested as soluble epoxide hydrolase (sEH) inhibitors. This series of compounds showed low nanomolar to picomolar activi

Full text of DOI:10.1021/jm070270t

J. Med. Chem. 2007, 50, 3825-3840
3825
Orally Bioavailable Potent Soluble Epoxide Hydrolase Inhibitors
Sung Hee Hwang, Hsing-Ju Tsai, Jun-Yan Liu, Christophe Morisseau, and Bruce D. Hammock*
Department of Entomology and UCD Cancer Center, UniVersity of California, One Shields AVenue, DaVis, California 95616-8584
ReceiVed March 9, 2007
A series of N,N′-disubstituted ureas having a conformationally restricted cis- or trans-1,4-cyclohexane R to
the urea were prepared and tested as soluble epoxide hydrolase (sEH) inhibitors. This series of compounds
showed low nanomolar to picomolar activities against recombinant human sEH. Both isomers showed similar
potencies, but the trans isomers were more metabolically stable in human hepatic microsomes. Furthermore,
these new potent inhibitors show a greater metabolic stability in vivo than previously described sEH inhibitors.
We demonstrated that trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid 13g (t-AUCB, IC50
) 1.3 ( 0.05 nM) had excellent oral bioavailability (98%, n ) 2) and blood area under the curve in dogs
and was effective in vivo to treat hypotension in lipopolysaccharide challenged murine models.
Introduction
appear to be polar binding sites in the catalytic tunnel. However,
the usefulness of these compounds was limited by the difficulties
encountered during formulation as well as their rapid metabolism
in vivo.12 Because the flexible alkyl chain of these later
compounds is susceptible to metabolism by â oxidation and
cytochrome P450 oxidation,13 we decided to investigate if more
conformationally restricted compounds could be made that were
more metabolically stable. To achieve this goal, we recently
reported piperidine-based conformationally restricted sEH in-
hibitors such as TPAU, APAU, or AMAU that showed
improved bioavailability in a canine model.14 These piperidine-
based inhibitors, however, still suffer from a short in vivo half-
life. In addition, those compounds in piperidine-based series
that showed optimal area under the curve (AUC) in a canine
model did not have optimal potency on the human enzyme. The
most potent compounds in this series, such as TPAU, did not
show a good AUC.14 Furthermore, we recently also reported
conformationally restricted N,N′-disubstituted ureas harboring
polar groups as potent sEH inhibitors.15 Thus, in this study, we
further explore conformationally restricted sEH inhibitors based
on ACU as a simple scaffold in which a cyclohexane ring serves
not only as a linker between a urea group and a polar group
but also as a template to restrict the structure (Figure 2).
To be effective in vivo, in addition to potency, compounds
need to have good metabolic stability and pharmacokinetic and
distribution properties. Thus, the metabolic stability of synthe-
sized potent sEH inhibitors was determined in human hepatic
microsomes.16 To determine the oral bioavailability of potent
compounds, we screened the compounds in a canine model for
the selection of compounds with good pharmacokinetic proper-
ties. Finally, the efficacy and the oral bioavailability of the best
inhibitor 13g in this series of compounds have been determined
in mice and canines, respectively.
The soluble epoxide hydrolase (sEH, EC 3.3.2.3) belongs to
the R/â hydrolase fold family of enzymes1 and is involved in
the metabolism of endogenously derived fatty acid epoxides and
other lipid epoxides.2 Epoxyeicosatrienoic acids (EETs),3 the
primary metabolites of cytochrome P450 epoxygenases of
arachidonic acid, are known to act at vascular, renal, and cardiac
levels of blood pressure regulation.4 EETs have also been shown
to possess anti-inflammatory properties.5 The sEH enzyme
catalytically hydrolyzes EETs into dihydroxyeicosatrienoic acids
(DHETs), which show reduced biological activity.6 We have
demonstrated that sEH inhibition significantly reduces the blood
pressure of the spontaneously hypertensive rats (SHRs)7 as well
as angiotensin II induced hypertensive rats.8 Recently, we also
demonstrated that sEH inhibitors not only dramatically synergize
nonsteroidal anti-inflammatory drugs (NSAIDs) but also shift
oxylipin metabolomic profiles away from propagation of
inflammation.9
We initially reported conformationally restricted N,N′-disub-
stituted ureas, e.g., DCU or ACU (Figure 1) as simple sEH
inhibitors.10 Even though these compounds were very potent
(Ki in the low nanomolar range), they were very difficult to
use for in vivo studies because of their poor physical properties
such as water solubility. Thus, a second homologous series of
flexible sEH inhibitors such as AUDA,a AUDA-BE, and AEPU
were investigated.10b,11 These flexible compounds improved
water solubility over DCU and ACU, thus facilitating efficacy
studies in several animal models.7-9 AEPU illustrates that a
polar group can be added to the molecule roughly five carbons
away from the central urea carbonyl group, increasing solubility
without reducing potency.11a Similarly a series of polar residues
such as esters, sulfones, amides, and carbamates can be placed
roughly 5-7 Å from the central pharmacophore where there
Chemistry
* To whom correspondence should be addressed. Phone: 530-752-7519
(office). Fax: 530-752-1537. E-mail: bdhammock@ucdavis.edu.
a Abbreviations: t-AUCB, trans-4-[4-(3-adamantan-1-ylureido)cyclo-
hexyloxy]benzoic acid; AUDA, 12-(3-adamantan-1-ylureido)dodecanoic
acid; AUDA-BE, 12-(3-adamantan-1-ylureido)dodecanoic acid butyl ester;
DCU, N,N′-dicyclohexylurea; ACU, N-adamantyl-N′-cyclohexylurea; AEPU,
1-adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea; APAU, N-(1-
acetylpiperidin-4-yl)-N′-(adamant-1-yl)urea; TPAU, N-(1-(2,2,2-trifluoro-
ethanoyl)piperidin-4-yl)-N′-(adamant-1-yl)urea; AMAU, N-((1-acetylpiperidin-
4-yl)methyl)-N′-(adamant-1-yl)urea; c-FCTU, 1-[4-(4-fluorophenoxy)cyclo-
hexyl]-3-(4-trifluoromethoxyphenyl)urea; c-FCUB, 4-{3-[4-(4-fluorophenoxy)-
cyclohexyl]ureido}benzoic acid.
Scheme 1 outlines the general synthesis of N,N′-disubstituted
ureas having a cis- or trans-1,4-cyclohexane ring between the
carbonyl group on the urea and an oxygen atom in a benzyloxy
or a phenoxy group. All compounds 3a-g, 9a-g, 13a-g,
16a-k were synthesized as a single isomer starting from
commercially available trans-4-aminocyclohexanol hydrochlo-
ride 1 (Scheme 1).
The Mitsunobu coupling reaction17 was used for the inversion
of secondary alcohol configuration on the cyclohexane ring and
10.1021/jm070270t CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/06/2007

3826 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hwang et al.
Figure 1. Common inhibitors of sEH. IC50 is for in vitro inhibition of the recombinant human sEH.
alcohol on a cyclohexane ring. A common intermediate 5 for
the synthesis of cis isomers 9a-g or trans isomers 13a-g was
obtained by the reaction of trans isomer 4 with p-nitrobenzoic
acid in the presence of diisopropyl azodicarboxylate (DIAD)
and triphenylphosphine (PPh3) in THF in excellent yield.
Hydrazinolysis of the phthalic group in the intermediate 5,
followed by the reaction of amine 6 with 1-adamantyl isocyanate
in DMF, provided compound 7 having the required cis config-
uration, which after hydrolysis under basic conditions afforded
the desired cis alcohol 8. The reaction of the cis alcohol 8 with
various substituted benzyl bromides gave compounds 9a-g. 9h
and 9i were also synthesized from 2 reacting with iodomethane
and cyclohexanemethyl methanesulfonate, respectively. By
modification of a previously published method,23 the cis alcohol
10 was obtained from 5 after saponification followed by a
microwave-assisted phthalimide ring-closing reaction in the
presence of excess triethylamine in DMF.24 Mitsunobu coupling
of the cis isomer 10 with various substituted phenols gave
compounds 11a-f having the desired trans configuration in
moderate to excellent yields. This was followed by the hydrazi-
nolysis of the phthalic group and urea formation resulting in
trans isomers 13a-f. Saponification of the ester 13f led to the
target acid 13g. Compounds 16a-f, 16h, and 16j were
synthesized by reaction of 1-adamantyl isocyanate with amines
15a-f, 15h, and 15j that were obtained by the Mitsunobu
reaction of trans alcohol 4 with various substituted phenols
followed by the removal of the phthalic group. Saponification
of the esters 16f, 16h, and 16j also led to the target acids 16g,
16i, and16k, respectively.
Figure 2. General structures of the new series of compounds.
allowed us to avoid tedious column chromatographic separations
of a cis/trans mixture that are time-consuming and not practical
for large-scale preparation of these compounds because of
solubility limitations. The configurations of the isomers (3a-g
vs 9a-g; 13a-g vs 16a-k) and purity could be easily
confirmed by comparisons of their NMR spectra. The relative
configurations of trans and cis isomers were established by the
comparison of peak assignments such as chemical shifts in 1H
and 13C NMR with previous reported cis- and trans-1,4-
cyclohexane isomers.18 We also observed that 1H NMR spectra
of these compounds showed that trans isomers have the expected
stable conformation in which both non-hydrogen substituents
are equatorial, but cis isomers exist in rapid equilibrium between
the two chair forms, which is consistent with the conformational
mobility reported by Johnston et al.19 and Hill et al.20 Trans
isomers 3a-g were prepared by the reaction with various
substituted benzyl bromides from the alcohol 2 that was obtained
by the reaction of 1 with 1-adamantyl isocyanate in the presence
of Et3N in DMF.
Compounds 3h and 3i also were synthesized from 2 by
reacting with iodomethane and cyclohexanemethyl methane-
sulfonate, respectively. Initial attempts to synthesize cis isomers
9a-g and 16a-k through the direct inversion of the configu-
ration of the hydroxyl group from the trans-cyclohexanol 2 was
problematic. In the presence of the urea group, the Mitsunobu
coupling reaction gave a dehydrated product exclusively.21 The
protection of the amino group in 1 by a phthalic (Phth) group
that is void of a hydrogen, however, minimized the dehydration
problem, suggesting that a hydrogen on the urea group is likely
responsible for the â-elimination of the hydroxyl group on the
cyclohexane ring in the compound 2.22 Thus, compounds 9a-
g, 13a-g, and 16a-k were synthesized from the Phth-protected
aminocyclohexanol 4 by alternating the configuration of the
Compounds 20a-c were synthesized by urea formation
starting from the isocyanates 18a or 18b by reacting with amines
12f or 15f followed by soponification of corresponding esters
19a-c (Scheme 2). Corresponding amides 22 and 23 of the
urea-based inhibitors 16d and 13d, respectively, were synthe-
sized as shown in Scheme 3. The EDC coupling of 1-adaman-
tylacetic acid 21 with amines 2415 and 15d gave amides 22 and
23, respectively. Compounds having different linkers such as a
n-butyl, acetylenyl, or phenyl group between a urea group and
an oxygen atom were synthesized by the procedure outlined in
Scheme 4. Compounds 27, 29, and 31 were synthesized starting
from commercially available 4-fluorophenol 25. Compounds 27
and 29 were synthesized by the reaction of 1-adamantyl
isocyanate with the amines derived from compounds 26 and
28 that were obtained by Mitsunobu coupling of 25 with
alcohols 3225 and 33,26 respectively. The reaction of 1-adamantyl

Epoxide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3827
Scheme 1. Synthesis of cis- and trans-1,4-Cyclohexane Based Urea Derivativesa
a Reagents and conditions: (a) 1-adamantyl isocyanate, Et3N, DMF, room temp, 6 h; (b) R1-PhCH2Br, NaH, DMF, 0 to room temp, 12 h; (c) Nefken’s
reagent, K2CO3, H2O, room temp, 30 min; (d) PPh3, p-nitrobenzoic acid, DIAD, THF, room temp, 12 h; (e) 35% hydrazine, CH2Cl2, MeOH, room temp,
1 day; (f) 1-adamantyl isocyanate, DMF, room temp, 12 h; (g) 1 N NaOH, CH3CN, room temp; (h) NaH, R2-PhCH2Br, DMF; (i) R4-PhOH, PPh3, DIAD;
THF, room temp, 12 h; (j) (i) 1 N NaOH; (ii) Et3N, MW, 110 °C, DMF, 30 min; (k) R3-PhOH, PPh3, DIAD, THF, room temp, 12 h; (l) aqueous 1 N NaOH,
CH3CN, 90 °C, 6 h.
Scheme 2a
Scheme 3. Synthesis of Amide Derivatives 22 and 23a
a Reagents and conditions: (a) 12f (for 19a) or 15f (for 19b,c), DMF,
room temp, 12 h; (b) 1 N NaOH, acetonitrile, water, 90 °C, 6 h.
isocyanate with aniline 30, which was obtained starting from
25 by following the procedure of Fotsch et al.,27 gave the desired
urea 31. Scheme 5 shows the syntheses of cis/trans mixed ureas
36 having a methylene unit instead of an oxygen atom on the
cyclohexane ring of either 13d or 16d. The ketone 34 was
obtained by the PDC oxidation of the alcohol 2. Wittig
olefination of ketone 34 with 4-fluorobenzyltriphenylphospho-
nium bromide28 afforded the olefin 35, which was subjected to
a Reagents and conditions: (a) 24, EDC, CH2Cl2, room temp, 2 h; (b)
15d, EDC, CH2Cl2, room temp, 2 h.
hydrogenation on 10% Pd/C generating desired compound 36
as an inseparable 1.25:1 (trans/cis) mixture.

3828 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hwang et al.
Scheme 4. Synthesis of Urea Compounds Having Different Linker in Place of the Cyclohexanea
a Reagents and conditions: (a) PhthNCH2(CH2)2CH2OH (32), DIAD, PPh3, THF; (b) PhthNCH2CtCCH2OH (33), DIAD, PPh3, THF; (c) (i) 1-fluoro-
4-nitrobenzene, K2CO3, DMF, 150 °C; (ii) 10% Pd/C, H2 (1 atm), EtOAc, room temp; (d) (i) 35% hydrazine, CH2Cl2, MeOH, room temp, 1 day; (ii)
1-adamantyl isocyanate, DMF; (e) 1-adamantyl isocyanate, DMF.
Table 1. SAR of Various Substituents on cis- or trans-Cyclohexane
Ring
Scheme 5. Synthesis of Compounds Having a Carbon Isostere
in Place of an Oxygen Atoma
a Reagents and conditions: (a) PDC, DMF, room temp, 12 h; (b) n-BuLi,
(4-F-Ph)CH2PPh3Br, -78 °C to reflux, 12 h; (c) 10% Pd/C, H2 (1 atm),
MeOH, room temp, 2 h.
Results and Discussion
Incorporation of Ether Groups on ACU. We have previ-
ously shown that ureas with a linear alkyl chain having a variety
of polar groups such as an ester, amide, or ether about five to
seven atoms away from the urea moiety increase water solubility
without changing the potency on the human sEH.11 Because of
the instability of most esters in vivo, they are most appropriate
as “soft drugs”. Thus, we turned our attention to ethereal
derivatives. Incorporation of a hydroxyl group at position 4 on
the cyclohexane ring in ACU such as trans-2 resulted in higher
IC50 value than the parent ACU (Table 1). However, compound
38, which replaces the cyclohexane ring with a linear n-butyl
chain, resulted in even greater reduction of inhibition activity,
suggesting that restriction of the linear chain by a cyclohexane
is beneficial. Because of the existence of conformational isomers
of 1,4-disubstituted cyclohexane, the corresponding cis isomer
8 was also synthesized. Interestingly, this cis isomer 8 was 5-fold
less potent compared to trans isomer 2. Both isomers, however,
became almost equally potent upon etherification of alcohol (3h
vs 9h, 3i vs 9i, and 3a vs 9a), and the cis isomers became more
potent when etherified with large groups such as 3i, 9i, 3a, and
9a. These results suggested that the absence of a hydrogen donor
at position 4 on the cyclohexane is crucial for obtaining highly
potent inhibitors. In support of this hypothesis we observed that
an ester analogue (7, 4-nitrobenzoic acid 4-(3-adamantan-1-
ylureido)cyclohexyl ester) also showed excellent potency (IC50
) 1.3 nM).
a Values are the mean ( SD of three independent experiments. At least
three concentrations above and below the repeated IC50 were used to
generate the data.
resulted in increased solubility while maintaining or enhancing
the inhibition potency.11 Thus, we tested whether adding a third
polar function will have a similar effect on these conforma-
tionally restricted urea inhibitors (Table 2). In general, as
previously observed, the addition of a third polar group relatively
far away from the urea carbonyl did not significantly influence
the inhibition potency of the human sEH except for a carboxylic
acid at the ortho position of the phenyl ring in 16k, for which
the IC50 is decreased ∼100-fold. Furthermore, the cis isomers
9a-g and 16a-g were nearly as potent as the trans isomers
3a-g and 13a-g.
Comparison of Cis and Trans Isomers. We previously
showed that the addition of a third polar group about 11 atoms
away from the urea carbonyl on a linear chain of a urea inhibitor

Epoxide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3829
Table 2
would be anticipated to be valuable physiological tools leading
to constant exposure following subcutaneous injection in oil or
administration as a wax plug. Surprisingly, the incorporation
of a free carboxylic acid group in 13g and 16g not only
dramatically increased the water solubility but also increased
the metabolic stability of the compounds. The improved
metabolic stability of the compounds 13g and 16g is most likely
caused by the decreased lipophilicity of these compounds, which
reduces their affinity for or accessibility to metabolic enzymes,
particularly the cytochrome P450 family.31 The relatively high
inhibitor potency of the free carboxylic acid containing com-
pounds at para and meta positions (13g, 16g, 16i) is partly due
to having a sufficient distance between the urea group and the
carboxylic acid.32 Moving the carboxylic acid group to the ortho
position (16k), however, dramatically decreased inhibitory
activity. In general, cis isomers showed poorer metabolic
stability against human hepatic microsomes than the corre-
sponding trans isomers. Presumably, cis isomers were more
susceptible to metabolism by CYP 450s than the trans isomers.
stabilityb
solubilityc
a
IC50
compd
n
R2
(nM)
(% remaining)
(µM)
trans
3a
3b
3c
3d
3e
3f
3g
13a
13b
13c
13d
13e
13g
cis
1
1
1
1
1
1
1
0
0
0
0
0
0
H
1.7 ( 0.1
1.7 ( 0.2
1.6 ( 0.1
2.7 ( 0.2
1.7 ( 0.2
1.7 ( 0.1
3.5 ( 0.1
2.0 ( 0.1
0.87 ( 0.03
0.64 ( 0.03
0.80 ( 0.05
1.0 ( 0.1
1.3 ( 0.05
67
41
67
54
51
43
34
23
82
nd
69
nd
>99
31 < x < 63
31 < x < 63
16 < x < 31
16 < x < 31
31 < x < 63
16 < x < 31
63 < x < 125
31 < x < 63
16 < x < 31
31 < x < 63
16 < x < 31
31 < x < 63
>500
4-Br
2-Me
2-Cl
2,6-diCl
2,6-diF
2,6-diF, 4-OiPr
4-Br
4-OMe
4-NO2
4-F
3,5-diF
4-CO2H
Docking Compounds 13g and 16g with sEH Enzyme. To
understand the observation that the cis isomers were, in general,
more potent than the trans isomers, we manually docked
inhibitors 13g and 16g into the active site of sEH. For this, we
used the published X-ray crystal structure of human sEH
complexed with a urea-based ligand (4-(3-cyclohexylureido)-
butyric acid, CU4, PDB accession number 1ZD3).32
9a
9b
9c
9d
9e
9f
9g
16a
16b
16c
16d
16e
16g
16i
16k
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
H
4-Br
2-Me
2-Cl
0.9 ( 0.1
2.1 ( 0.1
3.4 ( 0.1
2.0 ( 0.1
1.5 ( 0.1
1.1 ( 0.1
3.5 ( 0.2
1.3 ( 0.1
0.55 ( 0.06
0.72 ( 0.05
1.0 ( 0.1
0.82 ( 0.01
0.89 ( 0.04
1.9 ( 0.03
330 ( 30
nd
20
14
25
26
19
29
35
14
15
21
20
98
nd
nd
31 < x < 63
31 < x < 63
31 < x < 63
31 < x < 63
16 < x < 31
31 < x < 63
125 < x < 250
31 < x < 63
63 < x < 125
31 < x < 63
16 < x < 31
31 < x < 63
>500
2,6-diCl
2,6-diF
2,6-diF, 4-OiPr
4-Br
4-OMe
4-NO2
4-F
3,5-diF
4-CO2H
3-CO2H
2-CO2H
Between two plausible binding modes for 13g and 16g, the
orientation given in Figure 3 is more favorable. The other
binding mode that is used to explain the increased activity of
previous inhibitors, making an H-bond with Gln382, resulted in
steric clashes between the phenyl group of the inhibitors and
>500
>500
the residues of the binding site, such as Met337
.
a Values are the mean ( SD of three experiments. b Metabolic stability
in human hepatic microsomes. The remaining percentage of the parent
compounds was measured after incubation for 60 min. nd, not determined.
c Solubilities were measured in sodium phosphate buffer (pH 7.4, 0.1 M)
containing 1% of DMSO. The data present a range where the solubility is
greater than the lower value. Results are the means of three separate
experiments.
Our previous report also showed that this methionine residue
plays an important role in binding of the inhibitor into the active
site.33 To test this hypothesis, we made the trans,trans-1,3-bis-
(4-hydroxycyclohexyl)urea 39. We found that the potency (IC50
) 1400 ( 200 nM) of 39 is dramatically decreased compared
to DCU (IC50 ) 52 nM) in which both hydroxyl groups of 39
are replaced by hydrogens. These results clearly demonstrated
that a hydroxyl group on a cyclohexane in 39 interacts
unfavorably with one of the active site residues, presumably
with Met337. The observation that 2 and 8, which possess only
one 4-hydroxycyclohexyl group, showed almost equal or greater
inhibitory potency compared to DCU suggested that these
compounds, and presumably other compounds in the same series
(Table 2), orient themselves to avoid an unfavorable interaction
with Met337 (Figure 3).
As seen in Figure 3, compounds 13g and 16g were bound
primarily through interactions with Tyr381, Tyr465, and Asp333
with the urea pharmacophore. We also found that the carboxy-
late of compounds 13g and 16g could form hydrogen bonds
with Met418 and with Arg408 and Trp524, separately. Presumably,
the presence of extra H-bonding in 16g might explain the slightly
increased activity of cis isomers compared to trans isomers.
Structural Contribution to Inhibition. Before moving onto
the determination of the pharmacokinetic properties of these
inhibitors, we evaluated the effect of structural components of
each inhibitor by dividing them into four parts (P1, P2, P3, and
P4, Table 3).
First, as we demonstrated previously,15 replacement of the
adamantyl group with a p-trifluoromethoxyphenyl group at P1
in c-FCTU, 20a, and 20b showed no changes in potency
compared to 16d, 13g, and 16g. In contrast to 20a and 20b,
introduction of a free carboxylic acid close to the urea group
The metabolic stability of these compounds was determined
against human hepatic microsomes fortified with NADPH, and
their water solubility was determined in sodium phosphate buffer
(Table 2). We previously showed that metabolism and water
solubility are important factors contributing sEH inhibitor
potency in vivo.29 To produce more metabolically stable
inhibitors, we first hypothesized that metabolism on the benzylic
position is the most susceptible in these inhibitor skeletons.30
We thus decided to take two approaches to improve the
metabolic stability of the inhibitors: introduction of steric shields
and removal of the metabolically susceptible position. Therefore,
compounds were synthesized by introducing a methyl group or
halogen atom(s) as steric shields on ortho position(s) of the
phenyl ring, such as 3c-g and 9c-g, and by eliminating a
methylene unit, such as 13a-g and 16a-g. Both approaches
failed to stabilize the compounds except for 13g and 16g,
suggesting that the benzylic position is not a susceptible
metabolic group in this series of compounds. Moreover, phenoxy
derivatives having electron-withdrawing group(s) such as 16c-f
showed metabolic instability, also supporting the hypothesis that
the phenyl group is not susceptible to metabolism. In addition,
despite the favorable in vitro activity and metabolic stability of
compounds such as 13b (0.87 ( 0.03 nM, stability ) 82%),
they were found to be poor candidates for in vivo studies
because of minimal water solubility (in general less than 31
µM). Compounds such as the p-fluoro derivative 13d, however,

3830 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hwang et al.
Figure 3. (a) Superposition of the compounds 13g (green) and 16g (magenta) docked into the active site of human sEH. The residues Tyr465 and
Leu498 are omitted for clarity. (b) H-bonding of 13g with residue Met418. (c) H-bondings of 16g with residues Arg408 and Trp524. Black lines indicate
possible hydrogen bonds.
Table 3. Contribution of Each Portion (P1, P2, P3, and P4) in the Inhihitor
compd
R
X
L
Y
Z
IC50 a (nM)
c-FCTUb
20a
20b
c-FCUBb
20c
22
23
27
29
p-CF3O-Ph
p-CF3O-Ph
p-CF3O-Ph
p-CO2H-Ph
p-CO2H-Ph
adamantyl
adamantyl
adamantyl
adamantyl
adamantyl
adamantyl
NH
NH
NH
NH
NH
CH2
CH2
NH
NH
NH
NH
cis-cHex
trans-cHex
cis-cHex
cis-cHex
cis-cHex
trans-cHex
cis-cHex
n-Bu
O
O
O
O
O
OCH2
O
O
F
0.9 ( 0.1
0.9 ( 0.1
0.6 ( 0.1
220 ( 5
8300 ( 200
3.5 ( 0.3
9.1 ( 0.8
3.9 ( 0.3
42 ( 2
2.7 ( 0.2
2.5 ( 0.3
p-CO2H-Ph
p-CO2H-Ph
p-F-Ph
p-CO2H-Ph
2,6-diF
4-F
4-F
4-F
4-F
4-F
CH2CtCCH2
1,4-Ph
cHex
O
O
CH2
31
36
a Values are the mean ( SD of three experiments. b Reference 15.
as in c-FCUB and 20c dramatically decreased potency. These
results also support the poor potency of 16k compared to 16g
and 16i. These data suggested that the potency of these
compounds could be optimized by focused libraries at P1.15
Second, to test the importance of the urea group as a
pharmacophore, amide derivatives 22 and 23 were synthesized.
They showed 3.5- and 9-fold less potency compared to the
corresponding urea compounds 3f and 16d, respectively, which
was consistent with previous comparisons between amide-based
and urea-based compounds,11b suggesting that the urea phar-
macophore is generally more active than the amide group
(Scheme 2). However, the amides generally show greater
solubility and reduced melting point.
Third, to validate the choice of a cyclohexane group as a
conformationally restricted spacer at the P3 region, compounds
having different linkers such as a n-butyl, acetylenyl, or phenyl
group between the urea group and the oxygen atom on a
cyclohexane linker, were synthesized to evaluate the importance
of the cyclohexane ring as a linker as illustrated by analogues
27, 29, and 31 (Scheme 3). When the cyclohexane linker is

Epoxide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3831
Table 4. Comparison of Potencies of Selected Inhibitors for sEH from Various Animal Speciesa
IC50 (nM)
solubility
(µM)
AUCd
compd
mouse sEHb
rat sEHb
hamster sEHc
cat sEHc
dog sEHc
human sEHb
mp (°C)
(×104 nM‚min)
AUDA
AEPU
APAU
13g
10
3
9
11
5
6
5
2
2
2
3
27
450
6
3
86
500
1
3
14
15
2
63 < x < 125
250 < x < 500
>500
142-143
74-75
205-206
250-255
0.4
1.9
3.7
8
8
>500
14.2
a Full data on the activity of TPAU on the sEH from other species are not included because of the instability of the compound on storage (IC50 ) 1.1 nM,
AUC ) 0.33 × 104 nM·min). b Measured with fluorescent assay. c Measured with radioactive assay. d Area under the curve (AUC) estimated from a plot of
the inhibitor concentration in plasma (nM) versus time (minutes) following an oral dose of 0.3 mg/kg of the indicated compounds given to dogs in triglycerides.
chosen to assess oral bioavailability. The oral bioavailability
of 13g was determined after a single oral (po) and intravenous
(iv) administration in the dogs. It was dosed at 0.3 mg/kg iv (n
) 2) in 1% morpholine saline and 0.3 mg/kg po (n ) 2) in 1%
morpholine saline in two dogs via syringe. After a single
administration of compound 13g to female dogs, plasma samples
were collected over 24 h and plasma concentrations were
determined by HPLC-MS/MS. Its oral bioavailability in dogs
under these conditions was 98% (n ) 2) with a Tmax of 8 h and
a T1/2 of 19 h.
Comparison of Inhibitory Activity for sEH from Different
Animal Species. At this point, we decided to investigate the
inhibitory activity of selected inhibitors to sEH enzyme from
various animal species. AEPU, TPAU, and APAU, especially
APAU, showed poor inhibitory activities against sEH enzymes
from cat and dog. AUDA and 13g were potent against sEH
enzymes regardless of the species of origin, but 13g showed
better water solubility and metabolic stability than AUDA (Table
4).
Efficacy of 13g in Mice. With a compound having good
pharmacokinetic property and good oral bioavailability in hand,
our attention moved to its in vivo activity. The ability of 13g
to regulate murine blood pressure was evaluated. Administration
of lipopolysaccharide (LPS) to mice causes profound and often
fatal hypotension in addition to other symptoms. In this study,
the administration of only 1 mg/kg of 13g to mice treated with
LPS returned the blood pressure to normal, while more than 10
mg/kg of AUDA-BE were required to have a similar effect.
Even 0.5 mg/kg of 13g returned blood pressure to 60% of
normal values. These efficacy data strongly supported the
increased efficacy of 13g compared to AUDA-BE,37 suggesting
that our approach to optimize this lead was successful. To
support the hypothesis that reversal of hypotension is directly
derived from the inhibition of sEH, we determined the plasma
EETs/DHETs ratio by LC-MS/MS. As seen in Figure 5, the
increase in blood EETs/DHETs ratio caused by a low dose of
13g supports the argument that 13g is reducing hypotension by
inhibiting the sEH.
Figure 4. Pharmacokinetic profile data for selected compounds as
obtained via oral administration in a canine model. Area under the curve
(AUC) was estimated from a plot of inhibitor plasma concentration
(nM) versus time (min) following an oral dose of 0.3 mg/kg of the
indicated compounds in triglycerides.34
switched to a flexible linear n-butyl group, the inhibitory activity
dropped by about 4-fold. Interestingly, enhancing the rigidity
by incorporating a phenyl group or an acetylene group also
decreased inhibitory activity by at least 3-fold and 40-fold,
respectively.
The importance of the P4 region was tested by making
analogue 36 as a mixture by changing an oxygen atom to its
isostere, a methylene unit. Removal of an oxygen atom in either
13d or 16d, however, decreased potency, suggesting that the
oxygen atom helps not only to increase water solubility but also
to maintain potency (Scheme 4).
Pharmacokinetic Screening. At this point, we investigated
in vivo properties of these inhibitors. This pharmacokinetic
screening was performed following oral administration in dogs.34
As can be seen in Figure 4, 13g, 16g, and 20b, which have
improved metabolic stability and water solubility, are more
bioavailable than 16d or 3f. Meanwhile, ester 13f was metabo-
lized quickly to the corresponding acid 13g as a its major
metabolite, presumably by esterases.35 Compound 13g showed
an almost 40-fold increase in AUC compared to AUDA (or
TPAU) and a 4-fold increase compared to APAU. Since it has
been demonstrated that the gastrointestinal absorption in humans
and dogs is very similar, it is hoped that these results will be
transferable to humans.36 Compounds that are both poorly
soluble in water and have a stable crystal structure as indicated
by a high melting point are difficult to formulate. This situation
is made even more difficult with urea structures such as AUDA
that are also poorly soluble in common formulating reagents.
The increased potency of 13g over AUDA partially addresses
the above issue because less material needs to be delivered.
Although 13g possessed a relatively high melting point, this
problem is offset by the dramatically increased water solubility.
In addition, the benzoic acid of 13g was not susceptible to the
â oxidation that causes rapid metabolism of AUDA.
Conclusion
We have described the synthesis and structure-activity
relationships of a series of conformationally restricted sEH
inhibitors using a cis/trans-cyclohexane spacer. Our efforts to
optimize the cyclohexane portion of the lead ACU successfully
improved pharmacokinetic properties and potency as well. We
demonstrated that compound 13g, which has excellent bioavail-
ability, showed improved physical properties such as increased
water solubility and metabolic stability compared to previously
reported sEH inhibitors and enabled us to formulate the
compound easily for animal studies. 13g also showed good
inhibitory activity against sEH enzymes from several different
animal species, which will allow for the investigation of a variety
of animal models using the same compound. In addition, 13g
was shown to be more than 10 times effective in vivo in
Bioavailability of 13g in Dogs. Compound 13g had the best
blood levels from the pharmacokinetic screening, so it was

3832 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hwang et al.
3.58-3.41 (m, 1H), 3.37-3.24 (m, 1H), 2.11-1.81 (m, 13H),
1.50-1.33 (m, 6H), 1.50-1.33 (m, 2H), 1.17-0.99 (m, 2H). 13C
NMR (CDCl3): δ 156.65, 139.01, 128.49, 127.63, 127.57, 76.56,
70.14, 51.04, 48.64, 42.66, 36.57, 31.68, 30.82, 29.68. MS (ESI)
m/z: 383.3 (M + H+). Anal. (C24H34N2O2) C, H, N.
trans-1-Adamantan-1-yl-3-[4-(4-bromobenzyloxy)cyclohexyl]-
urea (3b). The general method above was used with p-bromobenzyl
bromide to afford a white solid (0.42 g, 90% yield). Mp 250-251
1
°C. H NMR (CDCl3): δ 7.45 (d, J ) 8 Hz, 2H), 7.20 (d, J ) 8
Hz, 2H), 4.48 (s, 2H), 3.95 (s, 1H), 3.90 (d, J ) 8 Hz, 1H), 3,-
58-3.43 (m, 1H), 3.35-3.22 (m, 1H), 2.12-1.89 (m, 13H), 1.70-
1.60 (m, 6H), 1.49-1.33 (m, 2H), 1.17-1.01 (m, 2H). 13C NMR
(DMSO-d6): δ 156.43, 138.77, 131.09, 129.37, 120.14, 76.03,
68.16, 49.32, 47.04, 42.04, 36.14, 30.70, 30.26, 28.93. MS (ESI)
m/z: 461.2 (M + H+).
trans-1-Adamantan-1-yl-3-[4-(2-methylbenzyloxy)cyclohexyl]-
urea (3c). The general method above was used with 2-methylbenzyl
bromide to afford a white solid (0.34 g, 86% yield). Mp 247-248
°C. 1H NMR (CDCl3): δ 7.34-7.29 (m, 1H), 7.21-7.13 (m, 3H),
4.51 (s, 2H), 3.96 (s, 1H), 3.91 (d, J ) 8 Hz, 1H), 3.57-3.43 (m,
1H), 3,38-3.25 (m, 1H), 2.32 (s, 3H), 2.13-1.87 (m, 13H), 1.69-
1.62 (m, 6H), 1.51-1.35 (m, 2H), 1.19-1.03 (m, 2H). 13C NMR
(CDCl3): δ 156.59, 136.76, 130.36, 128.58, 127.83, 125.95, 76.80,
68.76, 51.12, 48.76, 42.70, 36.60, 31.75, 30.83, 29.72, 18.96. MS
(ESI) m/z: 397.5 (M + H+).
Figure 5. LPS exposure produced temporal changes in plasma
oxylipins derived from epoxygenase and soluble epoxide hydrolase
pathways. (/) Results (average ( SD; n ) 4) in LPS-exposed mice
treated with vehicle, AUDA-BE, or 13g are depicted as the ratio of
(∑EETs/∑DHETs)observed/(∑EETs/∑DHETs)vehicle only. Doses are as fol-
lows: AUDA-BE, 10 mg/kg; 13g, 1 mg/kg. Only the 8,9-, 11,12-, and
14,15-epoxides and corresponding diols were summed because the 5,6-
regioisomer is a poor sEH substrate and spontaneously lactonized.
ameliorating hypotension than AUDA-BE in an LPS-sepsis
model. Further studies on the pharmacology of these and related
inhibitors of the sEH are underway.
trans-1-Adamantan-1-yl-3-[4-(2-chlorobenzyloxy)cyclohexyl]-
urea (3d). The general method above was used with 2-chlorobenzyl
bromide to afford a white solid (0.36 g, 86% yield). Mp 243-254
Experimental Section
1
°C. H NMR (CDCl3): δ 7.50 (dd, J ) 7 and 2 Hz, 1H), 7.35-
General. All reagents and solvents were obtained from com-
mercial suppliers and were used without further purification. All
reactions, unless otherwise described, were performed under an inert
atmosphere of dry nitrogen. Melting points were determined on an
7.17 (m, 3H), 4.62 (s, 2H), 3.95 (s, 1H), 3.90 (d, J ) 8 Hz, 1H),
3.60-3.45 (m, 1H), 3.41-3.31 (m, 1H), 2.17-1.85 (m, 13H),
1.71-1.61 (m, 6H), 1.54-1.36 (m, 2H), 1.21-1.04 (m, 2H). 13C
NMR (CDCl3): δ 156.58, 136.77, 132.79, 129.30, 128.98, 128.60,
126.93, 77.40, 67.38, 51.11, 48.67, 42.67, 36.58, 31.68, 30.83,
29.70. MS (ESI) m/z: 417.9 (M + H+).
1
OptiMelt melting point apparatus and are uncorrected. H NMR
and 13C NMR spectra were recorded at 300 and 75 MHz,
respectively. Elemental analyses were determined at Midwest
Microlab, Indianapolis, IN. Microwave reactions were performed
in an ETHOS SEL labstation (Milestone Inc., Shelton, CT). Mass
spectra were measured by LC-MS equipped with a Waters 2790
and a Waters PDA 996 using electrospray (+) ionization. Flash
chromatography was performed on silica gel. The following
reagents were prepared by literature methods: benzyl bromide,
methanesulfonic acid cyclohexylmethyl ester,38 4-fluorobenzyl-
triphenylphosphonium bromide,29 4-phthalylamino-1-butanol,26 4-
phthalimidobut-2-yn-1-ol.27
trans-1-Adamantan-1-yl-3-[4-(2,6-dichlorobenzyloxy)cyclohexyl]-
urea (3e). The general method above was used with 2,6-dichlo-
robenzyl bromide to afford a white solid (0.39 g, 86% yield). Mp
1
259-260 °C. H NMR (CDCl3): δ 7.33-7.24 (m, 2H), 7.21-
7.12 (m, 1H), 4.75 (s, 2H), 3.95 (s, 1H), 3.91 (d, J ) 8 Hz, 1H),
3.58-3.31 (m, 4H), 2.17-1.82 (m, 13H), 1.76-1.56 (m, 6H),
1.52-1.34 (m, 2H), 1.22-1.04 (m, 2H). 13C NMR (CDCl3): δ
156.57, 136.96, 133.98, 129.91, 128.56, 77.37, 65.14, 51.14, 48.73,
42.69, 36.60, 31.76, 30.79, 29.73. MS (ESI) m/z: 451.2 (M + H+).
trans-1-Adamantan-1-yl-3-[4-(2,6-difluorobenzyloxy)cyclohexyl]-
urea (3f). The general method above was used with 2,6-difluo-
robenzyl bromide to afford a white solid (0.36 g, 86% yield). Mp
trans-1-Adamantan-1-yl-3-(4-hydroxycyclohexyl)urea (2). To
a solution of 1-adamantyl isocyanate (5 g, 28 mmol) in DMF (35
mL) were added trans-4-aminocyclohexanol hydrochloride (6.4 g,
42 mmol) and Et3N (5.9 mL, 42 mmol) at 0 °C. The reaction
mixture was warmed to room temperature and stirred overnight.
After addition of 1 N HCl (40 mL) and water, the resulting white
precipitates were collected by suction filtration. The collected solid
was thoroughly washed with water. Recrystallization from methanol
afforded 7.5 g (92%) of the title compound as a white solid. Mp
1
244-246 °C. H NMR (CDCl3): δ 7.33-7.20 (m, 1H), 6.96-
6.83 (m, 2H), 4.59 (s, 2H), 3.94 (s, 1H), 3.88 (d, J ) 9 Hz, 1H),
3.55-3.42 (m, 1H), 3.40-3.27 (m, 1H), 2.11-1.89 (m, 13H),
1.69-1.60 (m, 6H), 1.48-1.31 (m, 2H), 1.20-1.03 (m, 2H). 13C
NMR (CDCl3): δ 162.12 (d, J ) 250 and 8 Hz), 156.54, 130.11
(t, J ) 10 Hz), 114.46 (t, J ) 19 Hz), 111.43 (dd, J ) 25 and 10
Hz), 77.37, 57.57, 51.13, 48.71, 42.68, 36.58, 31.71, 30.73, 29.71.
MS (ESI) m/z: 419.2 (M + H+). Anal. (C24H32F2N2O2) C, H, N.
trans-1-Adamantan-1-yl-3-[4-(2,6-difluoro-4-isopropoxyben-
zyloxy)cyclohexyl]urea (3g). The general method above was used
with 2-bromomethyl-1,3-difluoro-5-isopropoxybenzene 17 to afford
a white solid (0.37 g, 78% yield). Mp 182-186 °C. 1H NMR
(CDCl3): δ 6.39 (d, J ) 10 Hz, 2H), 4.49 (s, 2H), 4.49-4.41 (m,
1H), 3.95 (s, 1H), 3.91 (d, J ) 8 Hz, 1H), 3.54-3.39 (m, 1H),
3.35-3.23 (m, 1H), 2.08-1.89 (m, 13H), 1.67-1.61 (m, 6H),
1.45-1.24 (m, 2H), 1.31 (d, J ) 6 Hz, 6H), 1.18-1.01 (m, 2H).
13C NMR (DMSO-d6): δ 161.93 (dd, J ) 246 and 12 Hz), 158.98
(t, J ) 15 Hz), 156.43, 105.92 (t, J ) 21 Hz), 99.16 (dd, J ) 29
and 10 Hz), 76.03, 70.37, 56.41, 49.33, 47.00, 42.04, 36.15, 30.65,
30.11, 28.94, 21.53. MS (ESI) m/z: 477.4 (M + H+).
1
254-257 °C. H NMR (DMSO-d6): δ 5.48 (d. J ) 9 Hz, 1H),
5.38 (s, 1H), 4.48 (d, J ) 5 Hz, 1H), 3.42-3.28 (m, 1H), 3.28-
3.13 (m, 1H), 2.02-1.93 (m, 3H), 1.87-1.68 (m, 10H), 1.63-
1.54 (m, 6H), 1.24-0.93 (m, 4H). 13C NMR (DMSO-d6): δ 156.48,
68.16, 49.32, 47.24, 42.05, 36.16, 33.97, 31.17, 28.96. MS (ESI)
m/z: 293.2 (M + H+). Anal. (C17H28N2O2) C, H, N.
General Procedure for the Synthesis of Trans Isomers 3a-h
and 9h. To a solution of compound 2 (1 mmol) in DMF (10 mL)
was added 60% sodium hydride in oil (1.5 equiv) at 0 °C. After 10
min, benzyl bromide (1.2 equiv) was added. The reaction mixture
was allowed to slowly warm to room temperature overnight. The
reaction was quenched by adding water, and the resulting white
precipitates were collected and washed with water. The solids were
chromatographed by a dry loading method.
trans-1-Adamantan-1-yl-3-(4-benzyloxycyclohexyl)urea (3a).
The general method above was used with benzyl bromide to afford
a white solid (0.35 g, 91% yield). Mp 244-245 °C. 1H NMR
(CDCl3): δ 7.40-7.23 (m, 5H), 4.52 (s, 2H), 4.10-3.92 (m, 2H),
trans-1-Adamantan-1-yl-3-(4-methoxycyclohexyl)urea (3h).
The general method above was used with iodomethane to afford a
white solid (0.23 g, 75% yield). Mp 212-214 °C. 1H NMR
(CDCl3): δ 4.12-3.98 (m, 2H), 3.58-3.42 (m, 1H), 3.34 (s, 3H),

Epoxide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3833
3.18-3.06 (m, 1H), 2.13-1.88 (m, 13H), 1.74-1.58 (m, 6H),
1.41-1.24 (m, 2H), 1.20-1.03 (m, 2H). 13C NMR (DMSO-d6): δ
156.43, 77.59, 55.06, 49.33, 47.08, 42.04, 36.14, 30.66, 29.83,
28.93. MS (ESI) m/z: 307.4 (M + H+). Anal. (C18H30N2O2) C, H,
N.
(d, J ) 8 Hz, 1H), 4.02 (s, 1H), 3.66-3.51 (m, 2H), 2.23-1.07
(m, 23H). 13C NMR (CDCl3): δ 156.70, 139.19, 128.45, 127.54,
127.50, 73.02, 69.81, 50.96, 47.63, 42.69, 36.58, 29.69, 28.64,
28.51. MS (ESI) m/z: 383.3 (M + H+). Anal. (C24H34N2O2) C, H,
N.
trans-1-Adamantan-1-yl-3-(4-cyclohexylmethoxycyclohexyl)-
urea (9h). The general method above was used with methane-
sulfonic acid cyclohexylmethyl ester to afford a white solid (0.21
cis-1-Adamantan-1-yl-3-[4-(4-bromobenzyloxy)cyclohexyl]-
urea (9b). 9b was synthesized from compound 8 by the general
procedure as that described for the synthesis of trans isomers 3a-h
and 9h with p-bromobenzyl bromide. Yield: 85% (0.39 g). Mp
1
g, 54% yield). Mp 249-250 °C. H NMR (CDCl3): δ 3.95 (s,
1
1H), 3.91 (d, J ) 8 Hz, 1H), 3.56-3.41 (m, 1H), 3.22 (d, J ) 6
Hz, 2H), 3.19-3.08 (m, 1H), 2.13-0.99 (m, 32H), 0.96-0.79 (m,
2H). 13C NMR (CDCl3): δ 156.78, 77.30, 74.44, 50.98, 48.64,
42.67, 38.42, 36.58, 31.73, 30.83, 30.34, 29.68, 26.80, 26.00. MS
(ESI) m/z: 389.5 (M + H+). Anal. (C24H40N2O2) C, H, N.
cis-4-Nitrobenzoic Acid 4-(1,3-Dioxo-1,3-dihydroisoindol-2-
yl)cyclohexyl Ester (5).23 To a solution of trans-2-(4-hydroxycy-
clohexyl)isoindole-1,3-dione 4 (38 g, 155 mmol), triphenylphos-
phine (65 g, 248 mmol), and 4-nitrobenzoic acid (41 g, 248 mmol)
in 1500 mL of THF was added dropwise diisopropyl azodicar-
boxylate (50 g, 248 mmol) at room temperature. The reaction
mixture was stirred overnight. The solvent was evaporated, and
the resulting solid was recrystallized from methanol to afford 53 g
207-208 °C. H NMR (CDCl3): δ 7.46 (d, J ) 8 Hz, 2H), 7.22
(d, J ) 8 Hz, 2H), 4.44 (s, 2H), 4.09 (d, J ) 6 Hz, 1H), 4.01 (s,
1H), 3.67-3.50 (m, 2H), 2.19-1.35 (m, 23H). 13C NMR (CDCl3):
δ 156.59, 138.26, 131.55, 129.16, 121.29, 73.20, 69.12, 51.03,
47.70, 42.70, 36.58, 29.70, 28.65, 28.47. MS (ESI) m/z: 461.1 (M
+ H+).
cis-1-Adamantan-1-yl-3-[4-(2-methylbenzyloxy)cyclohexyl]-
urea (9c). 9c was synthesized from compound 8 by the general
procedure as that described for the synthesis of trans isomers 3a-
gh and 9h with 2-methylbenzyl bromide. Yield: 66% (0.26 g).
1
Mp 156-158 °C. H NMR (CDCl3): δ 7.41-7.07 (m, 4H), 4.46
(s, 2H), 4.11 (d, J ) 8 Hz, 1H), 4.03 (s, 1H), 3.66-3.51 (m, 2H),
2.33 (s, 3H), 2.18-1.43 (m, 23H). 13C NMR (CDCl3): δ 156.68,
137.03, 136.50, 130.24, 128.30, 127.67, 125.89, 73.35, 68.44, 50.94,
47.63, 42.68, 36.57, 29.68, 28.69, 28.57, 18.98. MS (ESI) m/z:
397.2 (M + H+).
1
(87%) of the title compound as a white solid. H NMR (CDCl3):
δ 8.40-8.36 (m, 4H), 7.79 (ddd, J ) 0.12, 0.02, and 0.02 Hz, 4H),
5.39 (s, 1H), 4.37-4.22 (m, 1H), 2.82-2.65 (m, 2H), 2.27-2.16
(m, 2H), 1.84-1.65 (m, 4H).
cis-1-Adamantan-1-yl-3-[4-(2-chlorobenzyloxy)cyclohexyl]-
urea (9d). 9d was synthesized from compound 8 by the general
procedure as that described for the synthesis of trans isomers 3a-h
and 9h with 2-chlorobenzyl bromide. Yield: 82% (0.34 g). Mp
cis-4-Nitrobenzoic Acid 4-Aminocyclohexyl Ester (6). An
amount of 35 wt % hydrazine hydrate (0.93 g, 10.1 mmol) was
added to a solution of compound 5 (2.0 g, 5.1 mmol) in CH2Cl2
(50 mL) followed by MeOH (50 mL) at room temperature. The
reaction mixture was allowed to stir overnight. The resulting white
precipitates were filtered off, and the solvent was removed in vacuo.
The resulting white solids were dissolved in aqueous 1 N HCl
solution and washed with CH2Cl2. The aqueous layer was basified
with excess 1 N NaOH solution and then extracted with CH2Cl2.
After the aqueous layer was dried with MgSO4, the solvent was
evaporated affording crude trans-4-nitrobenzoic acid 4-aminocy-
clohexyl ester 6 as a white solid (1.1 g, 89% yield), which was
used in the next step without further purification. 1H NMR (DMSO-
d6): δ 8.26 (dd, J ) 44 and 9 Hz, 4H), 6.72 (d, J ) 7 Hz, 2H),
5.08 (s, 1H), 2.00-1.36 (m, 9H).
cis-4-Nitrobenzoic Acid 4-(3-Adamantan-1-ylureido)cyclo-
hexyl Ester (7). To a solution compound 6 (0.66 g, 2.5 mmol) in
DMF was added 1-adamantyl isocyanate (0.4 g, 2.3 mmol) followed
by triethylamine (0.35 mL, 2.5 mmol) at 0 °C. The reaction mixture
was stirred overnight. The reaction mixture was poured into water,
and the resulting precipitates were collected and washed with water.
The crude product was recrystallized from CH2Cl2/hexanes to afford
0.9 g (89%) of the title compound as a white solid. Mp 194-217
°C. 1H NMR (CDCl3): δ 8.24 (dd, J ) 29 and 9 Hz, 4H), 5.23 (s,
1H), 4.13 (d, J ) 7 Hz, 1H), 4.05 (s, 1H), 3.75-3.61 (m, 1H),
2.17-1.41 (m, 23H). MS (ESI) m/z: 442.2 (M + H+).
1
168-172 °C. H NMR (CDCl3): δ 7.31-7.20 (m, 1H), 6.93-
6.83 (m, 3H), 4.54 (s, 2H), 4.04 (d, J ) 7 Hz, 1H), 3.96 (s, 1H),
3.65-3.51 (m, 2H), 2.12-1.39 (m, 23H). 13C NMR (CDCl3): δ
156.72, 136.93, 132.77, 129.28, 128.88, 128.52, 126.85, 73.72,
67.05, 50.98, 47.69, 42.69, 36.58, 29.69, 28.73, 28.54. MS (ESI)
m/z: 417.1 (M + H+).
cis-1-Adamantan-1-yl-3-[4-(2,6-dichlorobenzyloxy)cyclohexyl]-
urea (9e). 9e was synthesized from compound 8 by the general
procedure as that described for the synthesis of trans isomers 3a-h
and 9h with 2,6-dichlorobenzyl bromide. Yield: 58% (0.26 g). Mp
1
160-163 °C. H NMR (CDCl3): δ 7.40 (m, 3H), 4.71 (s, 2H),
4.08 (d, J ) 8 Hz, 1H), 4.01 (s, 1H), 3.67-3.54 (m, 2H), 2.13-
1.44 (m, 23H). 13C NMR (CDCl3): δ 156.68, 136.92, 134.12,
129.82, 128.49, 74.22, 64.97, 50.94, 47.55, 42.66, 36.58, 29.68,
28.73, 28.49. MS (ESI) m/z: 452.1 (M + H+).
cis-1-Adamantan-1-yl-3-[4-(2,6-difluorobenzyloxy)cyclohexyl]-
urea (9f). 9f was synthesized from compound 8 by the general
procedure as that described for the synthesis of trans isomers 3a-h
and 9h with 2,6-difluorobenzyl bromide. Yield: 63% (0.26 g). Mp
1
121-131 °C. H NMR (CDCl3): δ 7.31-7.19 (m, 1H), 6.93-
6.84 (m, 2H), 4.54 (s, 2H), 4.04 (d, J ) 7 Hz, 1H), 3.96 (s, 1H),
3.66-3.52 (m, 2H), 2.11-1.42 (m, 23H). 13C NMR (CDCl3): δ
162.03 (dd, J ) 250 and 8 Hz), 156.61, 130.03 (t, J ) 10 Hz),
114.44 (t, J ) 20 Hz), 111.37 (dd, J ) 26 and 10 Hz), 73.56, 57.21,
50.98, 47.57, 42.68, 36.59, 29.70, 28.58, 28.36. MS (ESI) m/z:
419.2 (M + H+).
cis-1-Adamantan-1-yl-3-(4-hydroxycyclohexyl)urea (8). To a
solution of ester 7 (1 g, 2.3 mmol) in THF (100 mL) was added 1
N NaOH solution (4.6 mL, 4.6 mmol) at room temperature. The
reaction mixture was stirred overnight, at which time the reaction
was quenched by addition of 1 N HCl solution (5.5 mL). The
resulting white precipitate was collected by filtration and recrystal-
lized from methanol/water to afford 0.63 g (95% yield) of the title
cis-1-Adamantan-1-yl-3-[4-(2,6-difluoro-4-isopropoxybenzyl-
oxy)cyclohexyl]urea (9g). 9a was synthesized from compound 8
by the general procedure as that described for the synthesis of trans
isomers 3a-h and 9h with 2-bromomethyl-1,3-difluoro-5-isopro-
1
1
compound as a white solid. Mp 205-207 °C. H NMR (DMSO-
poxybenzene 17. Yield: 53% (0.25 g). Mp 78-80 °C. H NMR
d6): δ 5.67 (d, J ) 8 Hz, 1H), 5.45 (s, 1H), 4.41 (s, 1H), 3.63-
3.51 (m, 1H), 3.46-3.36 (m, 1H), 2.00-1.92 (m, 3H), 1.87-1.78
(m, 6H), 1.62-1.53 (m, 6H), 1.51-1.36 (m, 8H). 13C NMR
(DMSO-d6): δ 156.39, 65.51, 49.29, 45.04, 42.08, 36.16, 30.95,
28.95, 28.24. MS (ESI) m/z: 293.2 (M + H+). Anal. (C17H28N2O2)
C, H, N.
(CDCl3): δ 6.41 (d, J ) 9 Hz, 2H), 4.54-4.42 (m, 1H), 4.46 (s,
2H), 4.20-3.97 (m, 2H), 3.65-3.50 (m, 2H), 2.11-1.45 (m, 23H),
1.32 (d, J ) 7 Hz, 6H). 13C NMR (CDCl3): δ 162.68 (dd, J ) 247
and 12 Hz), 159.41 (t, J ) 14 Hz), 156.70, 106.28 (t, J ) 21 Hz),
99.23 (dd, J ) 29 and 10 Hz), 73.41, 70.83, 56.97 (t, J ) 3 Hz),
50.88, 47.35, 42.63, 36.58, 29.68, 28.51, 28.39, 21.90. MS (ESI)
m/z: 477.2 (M + H+).
cis-1-Adamantan-1-yl-3-(4-benzyloxycyclohexyl)urea (9a). Com-
pound 9a was synthesized from compound 8 by the general
procedure as that described for the synthesis of trans isomers 3a-h
and 9h with benzyl bromide. Yield: 86% (0.33 g). Mp 181-182
cis-1-Adamantan-1-yl-3-(4-methoxycyclohexyl)urea (3i). 3i
was synthesized from compound 8 by the general procedure as that
described for the synthesis of trans isomers 3a-h and 9h with
1
1
°C. H NMR (CDCl3): δ 7.43-7.24 (m, 5H), 4.49 (s, 2H), 4.11
iodomethane. Yield: 85% (0.26 g). Mp 218-220 °C. H NMR

3834 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hwang et al.
(CDCl3): δ 4.11 (d, J ) 7 Hz, 1H), 4.06 (s, 1H), 3.65-3.52 (m,
1H), 3.38-3.24 (m, 1H), 3.30 (s, 3H), 2.16-1.38 (m, 23H). 13C
NMR (CDCl3): δ 156.67, 75.07, 55.69, 50.97, 47.57, 42.70, 36.59,
29.70, 28.36, 28.22. MS (ESI) m/z: 307.2 (M + H+). Anal.
(C18H30N2O2) C, H, N.
cis-1-Adamantan-1-yl-3-(4-cyclohexylmethoxycyclohexyl)-
urea (9i). 9i was synthesized from compound 8 by the general
procedure as that described for the synthesis of trans isomers 3a-h
and 9h with methanesulfonic acid cyclohexylmethyl ester. Yield:
70% (0.27 g). Mp 210-217 °C. 1H NMR (CDCl3): δ 4.15 (d, J )
7 Hz, 1H), 4.07 (s, 1H), 3.67-3.50 (m, 1H), 3.43-3.34 (m, 1H),
3.18 (d, J ) 7 Hz, 2H), 2.17-1.06 (m, 32H), 1.00-0.82 (m, 2H).
13C NMR (CDCl3): δ 156.68, 73.86, 73.32, 50.97, 47.71, 42.69,
38.46, 36.59, 30.38, 29.70, 28.68, 28.51, 26.83, 26.05. MS (ESI)
m/z: 389.2 (M + H+). Anal. (C24H40N2O2) C, H, N.
4.29 (m, 1H), 4.33 (q, J ) 7 Hz, 2H), 4.28-4.18 (m, 1H), 2.61-
2.19 (m, 4H), 1.88-1.79 (m, 2H), 1.69-1.56 (m, 2H), 1.38 (t, J )
7 Hz, 3H).
General Procedure for the Synthesis of Trans Isomers 13a-
f. Hydrazine hydrate (35 wt %, 2 equiv) was added to a solution
of compound 11 in CH2Cl2 followed by MeOH at room temperature.
The reaction mixture was allowed to stir for 1 day. The resulting
white precipitates were filtered off, and the solvent was removed
in vacuo. The resulting white solids were dissolved in aqueous 1
N HCl solution and washed with CH2Cl2. The aqueous layer was
basified with excess 1 N NaOH solution and then extracted with
CH2Cl2. After the aqueous layer was dried with MgSO4, the solvent
was evaporated affording crude amine 12, which was used in the
next step without further purification. To a solution compound 12
in DMF was added 1-adamantyl isocyanate (0.9 equiv) followed
by triethylamine (1 equiv) at 0 °C. The reaction mixture was stirred
overnight. The reaction mixture was poured into water, and the
resulting precipitates were collected and washed with water. The
crude product was purified by column chromatography by a dry
loading method.
cis-2-(4-Hydroxycyclohexyl)isoindole-1,3-dione (10).23 A 1 N
NaOH solution (19 mL, 19 mmol) was added at room temperature
to a solution of ester 5 (5 g, 12.7 mmol) in THF (100 mL). The
mixture was stirred overnight at room temperature, at which time
the reaction was quenched by addition of 1 N HCl solution (40
mL). The solvent was removed under reduced pressure, and the
resulting white precipitate that formed was collected by filtration
and dissolved in DMF. After adding triethylamine (6.5 g, 64 mmol)
at room temperature, the reaction mixture was heated at 150 °C
for 30 min in the microwave. After cooling to room temperature,
the reaction mixture was poured into water and then extracted with
ether. The organic layer was washed with water thoroughly. After
the organic layer was dried with MgSO4, the solvent was removed
in vacuo. The resulting white solids were recrystallized from CH2-
Cl2/hexanes. Yield: 60% (1.9 g). 1H NMR (CDCl3): δ 7.76 (ddd,
J ) 38, 5, and 3 Hz, 4H), 4.21-4.07 (m, 2H), 2.72-2.55 (m, 2H),
1.96 (d, J ) 14 Hz, 2H), 1.73-1.50 (m, 4H).
General Procedure for the Synthesis of Trans Isomers
(Phenoxy Intermediates) 11a-f. trans-2-[4-(4-Bromophenoxy)-
cyclohexyl]isoindole-1,3-dione (11a). Compound 11a was prepared
in 35% yield from compound 10 using the procedure detailed for
compound 5. 1H NMR (CDCl3): δ 7.83 (dd, J ) 5 and 3 Hz, 2H),
7.71 (dd, J ) 5 and 3 Hz, 2H), 7.39-7.33 (m, 2H), 6.83-6.77 (m,
2H), 4.32-4.13 (m, 2H), 2.49-2.20 (m, 4H), 1.88-1.77 (m, 2H),
1.65-1.49 (m, 2H).
trans-1-Adamantan-1-yl-3-[4-(4-bromophenoxy)cyclohexyl]-
1
urea (13a). 84% yield. Mp 205-210 °C. H NMR (CDCl3): δ
7.33 (d, J ) 9 Hz, 2H), 6.75 (d, J ) 9 Hz, 2H), 4.18-4.04 (m,
1H), 4.02-3.90 (m, 2H), 3.65-3.50 (m, 1H), 2.20-1.80 (m, 13H),
1.77-1.42 (m, 8H), 1.29-1.11 (m, 2H). 13C NMR (DMSO-d6): δ
156.65, 156.45, 132.16, 117.93, 111.67, 74.48, 49.36, 46.79, 42.05,
36.15, 30.35, 29.71, 28.95. MS (ESI) m/z: 447.1 (M + H+).
trans-1-Adamantan-1-yl-3-[4-(4-methoxyphenoxy)cyclohexyl]-
1
urea (13b). 82% yield. Mp 226-237 °C. H NMR (CDCl3): δ
6.89-6.80 (m, 4H), 5.59 (d, J ) 7 Hz, 1H), 5.40 (s, 1H), 4.19-
4.08 (m, 1H), 3.69 (s, 3H), 3.39-3.28 (m, 1H), 2.02-1.48 (m,
19H), 1.44-1.29 (m, 2H), 1.23-1.08 (m, 2H). 13C NMR (CDCl3):
δ 156.55, 154.18, 151.73, 117.89, 114.74, 76.62, 55.83, 51.13,
48.47, 42.68, 36.57, 31.47, 30.63, 29.70. MS (ESI) m/z: 399.26
(M + H+).
trans-1-Adamantan-1-yl-3-[4-(4-nitrophenoxy)cyclohexyl]-
1
urea (13c). 95% yield. Mp 205-227 °C. H NMR (CDCl3): δ
8.16 (d, J ) 9 Hz, 2H), 6.90 (d, J ) 9 Hz, 2H), 4.35-4.23 (m,
1H), 4.17-3.90 (m, 2H), 3.68-3.54 (m, 1H), 2.20-1.87 (m, 13H),
1.71-1.51 (m, 8H), 1.34-1.16 (m, 2H). 13C NMR (DMSO-d6): δ
162.95, 156.43, 140.44, 125.91, 115.70, 75.29, 49.36, 46.65, 42.04,
36.14, 30.22, 29.55, 28.95. MS (ESI) m/z: 414.24 (M + H+).
trans-1-Adamantan-1-yl-3-[4-(4-fluorophenoxy)cyclohexyl]-
trans-2-[4-(4-Methoxyphenoxy)cyclohexyl]isoindole-1,3-di-
one (11b). Compound 11b was prepared in 38% yield from
1
compound 10 using the procedure detailed for compound 5. H
1
NMR (CDCl3): δ 7.85-7.77 (m, 2H), 7.74-7.65 (m, 2H), 6.92-
6.76 (m, 4H), 4.28-4.11 (m, 2H), 3.77 (s, 3H), 2.47-2.19 (m,
4H), 1.88-1.75 (m, 2H), 1.64-1.45 (m, 2H).
trans-2-[4-(4-Nitrophenoxy)cyclohexyl]isoindole-1,3-dione (11c).
Compound 11c was prepared in 28% yield from compound 10 using
the procedure detailed for compound 5. 1H NMR (CDCl3): δ 8.21
(d, J ) 9 Hz, 2H), 7.88-7.69 (m, 4H), 6.98 (d, J ) 9 Hz, 2H),
4.53-4.40 (m, 1H), 4.32-4.18 (m, 1H), 2.56-2.20 (m, 4H), 1.95-
1.82 (m, 2H), 1.73-1.52 (m, 2H).
trans-2-[4-(4-Fluorophenoxy)cyclohexyl]isoindole-1,3-dione
(11d). Compound 11d was prepared in 40% yield from compound
10 using the procedure detailed for compound 5. 1H NMR
(CDCl3): δ 7.77 (ddd, J ) 38, 5, and 3 Hz, 4H), 7.00-6.84 (m,
4H), 4.30-4.15 (m, 2H), 2.48-2.31 (m, 2H), 2.26 (d, J ) 11 Hz,
2H), 1.89-1.77 (m, 2H), 1.65-1.49 (m, 4H).
urea (13d). 84% yield. Mp 242-245 °C. H NMR (CDCl3): δ
6.98-6.91 (m, 2H), 6.85-6.79 (m, 2H), 4.12-3.94 (m, 3H), 3.66-
3.51 (m, 1H), 2.17-1.88 (m, 12H), 1.73-1.45 (m, 9H), 1.28-
1.11 (m, 2H). 13C NMR (DMSO-d6): δ 156.43, 155.81 (d, J )
236 Hz), 153.42 (d, J ) 2 Hz), 117.17 (d, J ) 8 Hz), 115.81 (d,
J ) 23 Hz), 74.91, 49.35, 46.81, 42.04, 36.14, 30.37, 29.82, 28.93.
MS (ESI) m/z: 387.24 (M + H+).
trans-1-Adamantan-1-yl-3-[4-(3,5-difluorophenoxy)cyclohexyl]-
urea (13e). 87% yield. Mp 255-258 °C. 1H NMR (DMSO-d6): δ
6.76-6.64 (m, 3H), 5.56 (d, J ) 8 Hz, 1H), 5.32 (s, 1H), 4.43-
4.30 (m, 1H), 3.45-3.31 (m, 1H), 2.06-1.73 (m, 13H), 1.69-
1.35 (m, 8H), 1.32-1.15 (m, 2H). 13C NMR (CDCl3): δ 163.91
(dd, J ) 246 and 16 Hz), 159.85, 156.50, 99.67, 99.54, 99.42, 99.29,
96.42 (t, J ) 26 Hz), 76.00, 51.29, 48.32, 42.75, 36.64, 31.38, 30.30,
29.77. MS (ESI) m/z: 405.24 (M + H+).
trans-2-[4-(3,5-Difluorophenoxy)cyclohexyl]isoindole-1,3-di-
one (11e). Compound 11e was prepared in 31% yield from
compound 10 using the procedure detailed for compound 5. H
trans-4-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
Ethyl Ester (13f). 68% yield. Mp 187-189 °C. 1H NMR (DMSO-
d6): δ 7.88 (d, J ) 9 Hz, 2H), 7.04 (d, J ) 9 Hz, 2H), 5.61 (d, J
) 7 Hz, 1H), 5.40 (s, 1H), 4.49-4.38 (m, 1H), 4.27 (q, J ) 8 Hz,
2H), 3.43-3.29 (m, 1H), 2.08-1.78 (m, 13H), 1.65-1.53 (m, 6H),
1.52-1.36 (m, 2H), 1.29 (t, J ) 8 Hz, 3H), 1.33-1.15 (m, 2H).
13C NMR (CDCl3): δ 166.59, 161.64, 156.73, 131.68, 122.75,
115.12, 75.21, 60.79, 51.03, 48.11, 42.65, 36.56, 31.26, 30.28,
29.66, 14.51. MS (ESI) m/z: 441.4 (M + H+). Anal. (C26H36N2O4)
C, H, N.
1
NMR (CDCl3): δ 7.93-7.64 (m, 4H), 6.51-6.31 (m, 4H), 4.38-
4.06 (m, 2H), 2.56-2.11 (m, 4H), 1.96-1.36 (m, 4H). 13C NMR
(CDCl3): δ 168.42, 165.59, 165.38, 162.33, 162.12, 159.72, 134.10,
132.04, 123.32, 99.64, 99.51, 99.39, 99.26, 96.76, 96.42, 96.08,
75.64, 49.51, 31.14, 27.44.
trans-4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)cyclohexyloxy]-
benzoic Acid Ethyl Ester (11f). Compound 11a was prepared in
34% yield from compound 10 using the procedure detailed for
compound 5. 1H NMR (CDCl3): δ 7.99 (d, J ) 9 Hz, 2H), 7.85-
7.81 (m, 2H), 7.75-7.69 (m, 2H), 6.93 (d, J ) 9 Hz, 2H), 4.46-
trans-4-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
(13g). To a solution of urea in CH3CN was added lithium hydroxide
(3 equiv) followed by water at room temperature. The reaction

Epoxide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3835
mixture was stirred overnight. The solvent was evaporated in vacuo
and washed with EtOAc. The aqueous layer was acidified with 1
N HCl to give white precipitates. The resulting white solids were
collected by suction filtration and washed with water. The crude
product was recrystallized from MeOH to give 13g (88% yield) as
a white solid. Mp 250-255 °C. 1H NMR (DMSO-d6): δ 12.58 (s,
1H), 7.86 (d, J ) 9 Hz, 2H), 7.02 (d, J ) 9 Hz, 2H), 5.62 (d, J )
8 Hz, 1H), 5.41 (s, 1H), 4.48-4.36 (m, 1H), 3.63-3.54 (m, 1H),
2.11-1.34 (m, 21H), 1.32-1.11 (m, 2H). 13C NMR (DMSO-d6):
δ 166.99, 161.11, 156.43, 131.37, 122.61, 115.08, 74.39, 49.36,
46.75, 42.04, 36.14, 30.34, 29.73, 28.93. MS (ESI) m/z: 413.3 (M
+ H+). Anal. (C24H32N2O4‚CH4O) C, H, N.
Synthesis of Cis Isomers (Phenoxy Intermediates) 14a-f,h,j.
cis-2-[4-(4-Bromophenoxy)cyclohexyl]isoindole-1,3-dione (14a).
Compound 14a was prepared in 68% yield from compound 4 using
the procedure detailed for compound 5. 1H NMR (CDCl3): δ 7.83
(dd, J ) 5 and 3 Hz, 2H), 7.71 (dd, J ) 5 and 3 Hz, 2H), 7.43-
7.36 (m, 2H), 6.95-6.87 (m, 2H), 4.56 (s, 1H), 4.30-4.13 (m,
1H), 2.80-2.61 (m, 2H), 2.25-2.14 (m, 2H), 1.72-1.52 (m, 4H).
cis-2-[4-(4-Methoxyphenoxy)cyclohexyl]isoindole-1,3-dione
(14b). Compound 14b was prepared in 52% yield from compound
4 using the procedure detailed for compound 5. 1H NMR (CDCl3):
δ 7.83 (dd, J ) 6 and 3 Hz, 2H), 7.71 (dd, J ) 6 and 3 Hz, 2H),
7.00-6.73 (m, 4H), 4.51-4.46 (m, 1H), 4.26-4.14 (m, 1H), 3.77
(s, 3H), 2.84-2.61 (m, 2H), 2.29-2.10 (m, 2H), 1.72-1.46 (m,
4H).
by the general procedure for the synthesis of trans isomers 13a-f.
82% yield. Mp 203-204 °C. 1H NMR (CDCl3): δ 7.35 (d, J ) 9
Hz, 2H), 6.76 (d, J ) 9 Hz, 2H), 4.43-4.36 (m, 1H), 4.17 (d, J )
7 Hz, 1H), 4.09 (s, 1H), 3.71-3.57 (m, 1H), 2.21-1.43 (m, 23H).
13C NMR (CDCl3): δ 156.85, 156.62, 132.40, 117.86, 112.78,
71.86, 51.90, 47.46, 42.67, 36.56, 29.65, 28.40, 28.26. MS (ESI)
m/z: 447.3 (M + H+).
cis-1-Adamantan-1-yl-3-[4-(4-methoxyphenoxy)cyclohexyl]-
urea (16b). Compound 16b was synthesized from compound 14
through 15 by the general procedure for the synthesis of trans
1
isomers 13a-f. 86% yield. Mp 188-189 °C. H NMR (DMSO-
d6): δ 6.98-6.74 (m, 4H), 5.76 (d, J ) 8 Hz, 1H), 5.42 (s, 1H),
4.34-4.24 (m, 1H), 3.69 (s, 3H), 3.55-3.41 (m, 1H), 2.05-1.33
(m, 23H). 13C NMR (CDCl3): δ 156.57, 154.06, 151.51, 117.76,
114.78, 72.55, 55.83, 51.08, 47.82, 42.71, 36.59, 29.70, 28.63,
28.35. MS (ESI) m/z: 399.3 (M + H+). Anal. (C24H34N2O3) C, H,
N.
cis-1-Adamantan-1-yl-3-[4-(4-nitrophenoxy)cyclohexyl]urea
(16c). Compound 16c was synthesized from compound 14 through
15 by the general procedure for the synthesis of trans isomers 13a-
f. 79% yield. Mp 213-216 °C. 1H NMR (CDCl3): δ 8.28 (d, J )
8 Hz, 2H), 8.19 (d, J ) 8 Hz, 2H), 5.27-5.18 (m, 1H), 4.24 (d, J
) 6 Hz, 1H), 4.1 (s, 1H), 3.75-3.60 (m, 1H), 2.18-1.44 (m, 23H).
13C NMR (CDCl3): δ 162.90, 156.52, 141.29, 126.16, 115.44,
72.40, 51.13, 47.58, 42.69, 36.56, 29.69, 28.47, 28.17. MS (ESI)
m/z: 414.2 (M + H+). Anal. (C23H31N3O4) C, H, N.
cis-2-[4-(4-Nitrophenoxy)cyclohexyl]isoindole-1,3-dione (14c).
Compound 14c was prepared in 44% yield from compound 4 using
the procedure detailed for compound 5. 1H NMR (CDCl3): δ 8.21
(d, J ) 9 Hz, 2H), 7.86-7.67 (m, 4H), 7.05 (d, J ) 9 Hz, 2H),
4.78-4.69 (m, 1H), 4.30-4.17 (m, 1H), 2.78-2.60 (m, 2H), 2.29-
2.17 (m, 2H), 1.80-1.53 (m, 4H).
cis-2-[4-(4-Fluorophenoxy)cyclohexyl]isoindole-1,3-dione (14d).
Compound 14d was prepared in 80% yield from compound 4 using
the procedure detailed for compound 5. 1H NMR (CDCl3): δ 7.84-
7.80 (m, 2H), 7.71-7.67 (m, 2H), 6.98-6.94 (m, 4H), 4.51 (s,
1H), 4.26-4.12 (m, 1H), 2.76-2.60 (m, 2H), 2.18 (d, J ) 13 Hz,
2H), 1.79-1.49 (m, 4H).
cis-2-[4-(3,5-Difluorophenoxy)cyclohexyl]isoindole-1,3-di-
one (14e). Compound 14e was prepared in 48% yield from
compound 4 using the procedure detailed for compound 5. 1H NMR
(CDCl3): δ 7.90-7.64 (m, 4H), 6.61-6.47 (m, 2H), 6.46-6.34
(m, 1H), 4.59-4.50 (m, 1H), 4.29-4.13 (m, 1H), 2.77-2.58 (m,
2H), 2.29-2.12 (m, 2H), 1.76-1.52 (m, 4H).
cis-4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)cyclohexyloxy]-
benzoic Acid Ethyl Ester (14f). Compound 14f was prepared in
78% yield from compound 4 using the procedure detailed for
compound 5. 1H NMR (CDCl3): δ 8.00 (d, J ) 9 Hz, 2H), 7.86-
7.65 (m, 4H), 7.01 (d, J ) 9 Hz, 2H), 4.73-4.65 (m, 1H), 4.34 (q,
J ) 7 Hz, 2H), 4.28-4.15 (m, 1H), 2.80-2.62 (m, 2H), 2.28-
2.17 (m, 2H), 1.75-1.52 (m, 2H), 1.38 (t, J ) 7 Hz, 3H).
cis-3-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)cyclohexyloxy]-
benzoic Acid Ethyl Ester (14h). Compound 14h was prepared in
70% yield from compound 4 using the procedure detailed for
compound 5. 1H NMR (CDCl3): δ 7.83 (dd, J ) 6 and 3 Hz, 2H),
7.70 (dd, J ) 6 and 3 Hz, 2H), 7.65-7.61 (m, 2H), 7.36 (t, J ) 8
Hz, 1H), 7.23 (ddd, J ) 8, 3, and 1 Hz, 1H), 4.70-4.65 (m, 1H),
4.22 (tt, J ) 12 and 4 Hz, 1H), 3.92 (s, 3H), 2.86-2.58 (m, 2H),
2.28-2.16 (m, 2H), 1.75-1.53 (m, 4H).
cis-1-Adamantan-1-yl-3-[4-(4-fluorophenoxy)cyclohexyl]-
urea (16d). Compound 16d was synthesized from compound 14
through 15 by the general procedure for the synthesis of trans
1
isomers 13a-f. 92% yield. Mp 206-209 °C. H NMR (CDCl3):
δ 6.98-6.91 (m, 2H), 6.84-6.78 (m, 2H), 4.34 (s, 1H), 4.30 (d, J
) 10 Hz, 1H), 4.20 (s, 1H), 3.71-3.56 (m, 1H), 2.13-1.44 (m,
23H). 13C NMR (CDCl3): δ 158.89, 156.70, 155.74, 153.60, 153.57,
117.50, 117.39, 116.12, 115.82, 72.44, 51.00, 47.66, 42.69, 36.58,
31.72, 29.68, 28.52, 28.30. MS (ESI) m/z: 387.2 (M + H+). Anal.
(C23H31FN2O2) C, H, N.
cis-1-Adamantan-1-yl-3-[4-(3,5-difluorophenoxy)cyclohexyl]-
urea (16e). Compound 16e was synthesized from compound 14
through 15 by the general procedure for the synthesis of trans
1
isomers 13a-f. 83% yield. Mp 193-198 °C. H NMR (CDCl3):
δ 6.45-6.34 (m, 3H), 4.43-4.35 (m, 1H), 4.18-3.93 (m, 2H),
3.71-3.57 (m, 1H), 2.13-1.43 (m, 23H). 13C NMR (CDCl3): δ
164.52, 164.36, 162.10, 161.94, 159.45, 156.36, 99.68, 99.61, 99.48,
99.40, 96.14, 95.88, 95.62, 73.01, 49.33, 44.87, 42.06, 36.14, 28.94,
28.03, 27.19. MS (ESI) m/z: 405.5 (M + H+).
cis-4-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
Ethyl Ester (16f). Compound 16f was synthesized from compound
14 through 15 by the general procedure for the synthesis of trans
1
isomers 13a-f. 69% yield. Mp 101-136 °C. H NMR (CDCl3):
δ 7.89 (d, J ) 9 Hz, 2H), 7.04 (d, J ) 9 Hz, 2H), 5.78 (d, J ) 8
Hz, 1H), 5.40 (s, 1H), 4.61-4.53 (m, 1H), 4.27 (q, J ) 7 Hz, 2H),
3.56-3.46 (m, 1H), 2.02-1.93 (m, 3H), 1.88-1.39 (m, 20H), 1.30
(t, J ) 7 Hz, 3H). 13C NMR (CDCl3): δ 166.62, 161.46, 156.86,
131.70, 122.63, 115.22, 71.57, 60.80, 50.87, 47.35, 42.68, 36.58,
29.67, 28.47, 28.28, 14. 52. MS (ESI) m/z: 441.28 (M + H+).
cis-4-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
(16g). Compound 16g was prepared from 16f according to the same
procedure described for compound 13g. 88% yield. Mp 178-187
°C.
1H NMR (DMSO-d6): δ 12.60 (s, 1H), 7.87 (d, J ) 9 Hz,
2H), 7.01 (d, J ) 9 Hz, 2H), 5.80 (d, J ) 7 Hz, 1H), 5.42 (s, 1H),
4.62-4.52 (m, 1H), 3.59-3.45 (m, 1H), 2.09-1.38 (m, 23H). 13
cis-2-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)cyclohexyloxy]-
benzoic Acid Ethyl Ester (14j). Compound 14j was prepared in
68% yield from compound 4 using the procedure detailed for
compound 5. 1H NMR (CDCl3): δ 7.81 (dd, J ) 5 and 3 Hz, 2H),
7.80-7.77 (m, 1H), 7.69 (dd, J ) 5 and 3 Hz, 2H), 7.43-7.38 (m,
1H), 7.00-6.94 (m, 2H), 4.68-4.64 (m, 1H), 4.54 (q, J ) 7 Hz,
2H), 4.19 (tt, J ) 13 and 4 Hz, 1H), 2.74 (dq, J ) 13 and 4 Hz,
2H), 2.30-2.22 (m, 2H), 1.72-1.55 (m, 4H), 1.43 (t, J ) 7 Hz,
3H).
C
NMR (DMSO-d6): δ 166.97, 160.91, 156.36, 131.44, 122.63,
115.22, 72.24, 49.35, 44.90, 42.07, 36.15, 28.94, 28.09, 27.32. MS
(ESI) m/z: 413.2 (M + H+). Anal. (C24H32N2O4‚0.5CH4O) C, H,
N.
cis-3-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
Methyl Ester (16h). Compound 16d was synthesized from
compound 14 through 15 by the general procedure for the synthesis
1
Synthesis of Cis Isomers (Phenoxyureas) 16a-f,h,j. cis-1-
Adamantan-1-yl-3-[4-(4-bromophenoxy)cyclohexyl]urea (16a).
Compound 16a was synthesized from compound 14 through 15
of trans isomers 13a-f. 67% yield. Mp 153-156 °C. H NMR
(DMSO-d6): δ 7.54-7.48 (m, 1H), 7.45-7.39 (m, 2H), 7.25-
7.20 (m, 1H), 5.78 (d, J ) 8 Hz, 1H), 5.41 (s, 1H), 4.56-4.47 (m,

3836 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hwang et al.
1H), 3.84 (s, 3H), 3.57-3.44 (m, 1H), 2.01-1.94 (m, 3H), 1.86-
1.82 (m, 6H), 1.76-1.54 (m, 12H), 1.52-1.39 (m, 2H). 13C NMR
(DMSO-d6): δ 165.94, 157.07, 156.27, 130.96, 129.95, 121.20,
120.77, 116.00, 72.11, 52.11, 49.24, 45.04, 41.97, 36.05, 28.86,
27.93, 27.29. MS (ESI) m/z: 427.3 (M + H+).
cis-4-{4-[3-(4-Methoxycarbonylphenyl)ureido]cyclohexyloxy}-
benzoic Acid Ethyl Ester (19c). H NMR (CDCl3): δ 7.96 (t, J
1
) 9 Hz, 1H), 7.42 (d, J ) 9 Hz, 1H), 7.16 (s, 1H), 6.87 (d, J ) 9
Hz, 1H), 5.21 (d, J ) 8 Hz, 1H), 4.57-4.51 (m, 1H), 4.36 (q, J )
7 Hz, 1H), 3.92-3.77 (m, 1H), 3.88 (s, 1H), 2.07-1.95 (m, 1H),
1.88-1.56 (m, 1H), 1.39 (t, J ) 7 Hz, 1H). 13C NMR (CDCl3): δ
167.09, 167.01, 161.49, 154.39, 143.88, 131.78, 131.12, 123.95,
122.63, 118.00, 115.31, 71.31, 61.07, 52.12, 47.81, 28.41, 28.01,
14.52. MS (ESI) m/z: 441.2 (M + H+).
cis-3-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
(16i). Compound 16i was prepared from 16h according to the same
procedure described for compound 13g. 91% yield. Mp 219-222
1
°C. H NMR (DMSO-d6): δ 12.98 (s, 1H), 7.54-7.35 (m, 3H),
General Procedure for the Hydrolysis of 19a-d To Synthesize
20a-c. To a solution of urea in CH3CN was added lithium
hydroxide (3 equiv for 20a,b, 6 equiv for 20c) followed by water
at room temperature. The reaction mixture was stirred overnight
or warmed to 90 °C for 6 h. The solvent was evaporated in vacuo
and washed with EtOAc. The aqueous layer was acidified with 1
N HCl to give white precipitates. The resulting white solids were
collected by suction filtration and washed with water. The crude
product was recrystallized from MeOH.
trans-4-{4-[3-(4-Trifluoromethoxyphenyl)ureido]cyclohexyl-
oxy}benzoic Acid (20a). Mp 244-273 °C. 1H NMR (DMSO-d6):
δ 12.59 (s, 1H), 8.51 (s, 1H), 7.86 (d, J ) 9 Hz, 1H), 7.47 (d, J )
9 Hz, 1H), 7.22 (d, J ) 9 Hz, 1H), 7.03 (d, J ) 9 Hz, 1H), 6.19
(d, J ) 9 Hz, 1H), 4.52-4.38 (m, 1H), 3.61-3.45 (m, 1H), 2.12-
1.87 (m, 1H), 1.58-1.28 (m, 1H). 13C NMR (DMSO-d6): δ 167.07,
161.10, 154.47, 141.98, 139.87, 131.43, 122.74, 121.69, 118.55,
115.10, 74.31, 47.17, 30.01, 29.66. MS (ESI) m/z: 439.1 (M +
H+). Anal. (C21H21F3N2O5) C, H, N.
7.22-7.16 (m, 1H), 5.78 (d, J ) 8 Hz, 1H), 5.41 (s, 1H), 4.56-
4.45 (m, 1H), 3.57-3.43 (m, 1H), 2.02-1.94 (m, 3H), 1.90-1.37
(m, 20H). 13C NMR (DMSO-d6): δ 167.14, 157.09, 156.36, 132.25,
129.85, 121.49, 120.51, 116.14, 72.17, 49.34, 45.08, 42.06, 36.15,
28.95, 28.07, 27.38. MS (ESI) m/z: 413.2 (M + H+). Anal.
(C24H32N2O4) C, H, N.
cis-2-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
Ethyl Ester (16j). Compound 16d was synthesized from compound
14 through 15 by the general procedure for the synthesis of trans
1
isomers 13a-f. 88% yield. Mp 157-160 °C. H NMR (CDCl3):
δ 7.74 (dd, J ) 8.00 and 2 Hz, 1H), 7.42-7.35 (m, 1H), 6.95-
6.89 (m, 2H), 4.57-4.50 (m, 2H), 4.46 (s, 1H), 4.31 (q, J ) 7 Hz,
2H), 3.73-3.58 (m, 1H), 2.10-1.90 (m, 11H), 1.76-1.60 (m, 12H),
1.35 (t, J ) 7 Hz, 3H). 13C NMR (CDCl3): δ 166.69, 157.00,
156.92, 133.18, 131.70, 121.62, 120.06, 114.76, 72.16, 60.84, 50.89,
47.64, 42.61, 36.58, 29.66, 28.66, 28.17, 14.50. MS (ESI) m/z:
441.3 (M + H+).
cis-2-[4-(3-Adamantan-1-ylureido)cyclohexyloxy]benzoic Acid
(16k). Compound 16k was prepared from 16j according to the same
procedure described for compound 13g. 85% yield. Mp 215-221
cis-4-{4-[3-(4-Trifluoromethoxyphenyl)ureido]cyclohexyloxy}-
1
benzoic Acid (20b). Mp 210-212 °C. H NMR (DMSO-d6): δ
12.60 (s, 1H), 8.48 (s, 1H), 7.87 (d, J ) 8 Hz, 1H), 7.47 (d, J )
9 Hz, 1H), 7.21 (d, J ) 9 Hz, 1H), 7.03 (d, J ) 8 Hz, 1H), 6.35
(d, J ) 8 Hz, 1H), 4.66-4.57 (m, 1H), 3.73-3.60 (m, 1H), 1.87-
1.50 (m, 1H). 13C NMR (DMSO-d6): δ 167.04, 160.90, 154.36,
141.99, 139.84, 131.48, 122.77, 121.67, 118.57, 115.28, 71.95,
45.66, 27.71, 27.36. MS (ESI) m/z: 439.1 (M + H+). Anal.
(C21H21F3N2O5) C, H, N.
1
°C. H NMR (DMSO-d6) δ 12.49 (s, 1H), 7.62-7.58 (m, 1H),
7.47-7.40 (m, 1H), 7.11 (d, J ) 8 Hz, 1H), 6.95 (t, J ) 8 Hz,
1H), 5.67 (d, J ) 7.62 Hz, 1H), 5.51 (s, 1H), 4.58-4.49 (m, 1H),
3.52-3.37 (m, 1H), 2.02-1.74 (m, 11H), 1.71-1.44 (m, 12H). 13
C
NMR (DMSO-d6): δ 167.14, 157.09, 156.36, 132.25, 129.85,
121.49, 120.51, 116.14, 72.17, 49.34, 45.08, 42.06, 36.15, 28.95,
28.07, 27.38. MS (ESI) m/z: 413.2 (M + H+). Anal. (C24H32N2O4)
C, H, N.
cis-4-{4-[3-(4-Carboxyphenyl)ureido]cyclohexyloxy}ben-
zoic Acid (20c). Mp 250-257 °C. 1H NMR (DMSO-d6): δ 13.04
(br s, 1H), 8.69 (s, 1H), 8.07-8.05 (m, 1H), 7.80 (d, J ) 9 Hz,
1H), 7.66 (d, J ) 1 Hz, 1H), 7.48 (d, J ) 9 Hz, 1H), 6.46 (d, J )
8 Hz, 1H), 4.70-4.62 (m, 1H), 3.73-3.60 (m, 1H), 1.93-1.50 (m,
1H). 13C NMR (DMSO-d6): δ 167.14, 167.02, 160.89, 154.05,
144.79, 131.48, 130.57, 122.79, 122.74, 116.52, 115.29, 71.95,
45.64, 27.66, 27.33. Anal. (C21H22N2O6‚1/3CH4O) C, H, N.
General Procedure for the Synthesis of Amide Derivatives
22 and 23. To a solution of 1-adamantineacetic acid (1 mmol) in
CH2Cl2 (10 mL) were added an appropriate amine (1 mmol), Et3N
(1.5 mmol), and EDCI (1.1 mmol) at 0 °C. The reaction mixture
warmed up to room temperature and was stirred overnight. The
solvent was evaporated, and the remaining residue was dissolved
in EtOAc and washed with water. The organic layer was dried with
MgSO4 and filtered. After the solvent was evaporated, the residue
was purified with column chromatography with 20-30% EtOAc
in hexanes.
trans-2-Adamantan-1-yl-N-[4-(2,6-difluorobenzyloxy)cyclohexyl]-
acetamide (22). The general method was used with 24 to afford a
white solid (0.26 g, 62% yield). Mp 169-171 °C. 1H NMR
(DMSO-d6): δ 7.54 (d, J ) 8 Hz, 1H), 7.49-7.38 (m, 1H), 7.15-
7.05 (m, 2H), 4.51 (s, 2H), 3.58-3.43 (m, 1H), 3.35 (s, 2H), 3.35-
3.25 (m, 1H), 2.04-1.47 (m, 19H), 1.29-1.09 (m, 4H). 13C NMR
(DMSO-d6): δ 169.01, 161.20 (dd, J ) 248 and 8 Hz), 130.81 (t,
J ) 10 Hz), 114.09 (t, J ) 20 Hz), 111.56 (d, J ) 25 Hz), 111.55
(d, J ) 13 Hz), 76.52, 56.66, 49.98, 46.67, 42.13, 36.50, 32.21,
30.22, 30.05, 28.07. MS (ESI) m/z: 418.3 (M + H+). Anal.
(C25H33F2NO2) C, H, N.
2-Bromomethyl-1,3-difluoro-5-isopropoxybenzene (17). To a
solution of 2,6-difluoro-4-hydroxylbenzyl alcohol39 (0.55 g, 2.72
mmol) in THF (27 mL) were added PPh3 (1.07 g, 4.08 mmol) and
CBr4 (1.35 g, 4.08 mmol) at 0 °C. The reaction mixture was warmed
to room temperature and the stirred for 5 h. The solvent was
evaporated in vacuo and the residue was purified by column
chromatography to give 0.63 g (88%) of the titled compound as
1
colorless oil. H NMR (CDCl3): δ 6.42 (d, J ) 10 Hz, 2H), 4.51
(s, 2H), 4.54-4.44 (m, 1H), 1.34 (d, J ) 6 Hz, 6H).
General Procedure for the Synthesis of 19a-c. To a solution
of the appropriate amine (1.1 equiv) in DMF was added the
indicated isocyanate (1 mmol) followed by triethylamine (1.1 equiv)
at 0 °C. The reaction mixture was stirred overnight. The reaction
mixture was poured into water, and the resulting precipitates were
collected and washed with a 1 N HCl solution followed by water.
The crude product was recrystallized from CH2Cl2/hexanes or was
purified by silica gel chromatography using 30% EtOAc in hexanes
as an eluent.
trans-4-{4-[3-(4-Trifluoromethoxyphenyl)ureido]cyclohexyl-
1
oxy}benzoic Acid Ethyl Ester (19a). H NMR (CDCl3): δ 7.97
(d, J ) 9 Hz, 2H), 7.34 (d, J ) 9 Hz, 2H), 7.15 (d, J ) 9 Hz, 2H),
6.88 (d, J ) 9 Hz, 2H), 6.52 (s, 1H), 4.67 (d, J ) 8 Hz, 1H), 4.34
(q, J ) 7 Hz, 2H), 4.30-4.20 (m, 1H), 3.84-3.69 (m, 1H), 2.21-
2.08 (m, 4H), 1.69-1.54 (m, 2H), 1.38 (t, J ) 7 Hz, 3H), 1.33-
1.23 (m, 2H). 13C NMR (CDCl3): δ 166.69, 161.61, 154.67, 137.51,
131.74, 122.89, 122.16, 121.35, 115.17, 74.99, 60.90, 48.48, 31.03,
30.16, 14.53. MS (ESI) m/z: 467.2 (M + H+).
cis-4-{4-[3-(4-Trifluoromethoxyphenyl)ureido]cyclohexyloxy}-
benzoic Acid Ethyl Ester (19b). H NMR (CDCl3): δ 7.98 (d, J
) 9 Hz, 2H), 7.35 (d, J ) 9 Hz, 2H), 7.13 (d, J ) 9 Hz, 2H), 6.87
(d, J ) 9 Hz, 2H), 6.85 (s, 1H), 5.01 (d, J ) 8 Hz, 1H), 4.57-4.51
(m, 1H), 4.36 (q, J ) 7 Hz, 2H), 3.89-3.72 (m, 1H), 2.07-1.52
(m, 8H), 1.39 (t, J ) 7 Hz, 3H). MS (ESI) m/z: 467.2 (M + H+).
cis-2-Adamantan-1-yl-N-[4-(4-fluorophenoxy)cyclohexyl]acet-
amide (23). The general method was used with 15d to afford a
white solid (0.27 g, 70% yield). Mp 151-152 °C. 1H NMR
(DMSO-d6): δ 7.64 (d, J ) 8 Hz, 1H), 7.09 (t, J ) 9 Hz, 2H),
6.97-6.90 (m, 2H), 4.46-4.38 (m, 1H), 3.74-3.62 (m, 1H), 1.95-
1.78 (m, 9H), 1.70-1.48 (m, 16H). 13C NMR (DMSO-d6): δ
1

Epoxide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3837
169.02, 156.37 (d, J ) 236 Hz), 153.38 (d, J ) 2 Hz), 117.29 (d,
J ) 8 Hz), 115.88 (d, J ) 23 Hz), 71.91, 49.88, 45.60, 42.14,
36.51, 32.24, 28.08, 27.57, 27.12. MS (ESI) m/z: 386.3 (M + H+).
Anal. (C24H32FNO2) C, H, N.
in THF (5 mL) was added dropwise over 10 min to the reaction
mixture at room temperature and heated to reflux overnight. After
the mixture was cooled to room temperature, the reaction was
quenched by adding water. THF was removed in vacuo. The
remained aqueous layer was extracted with EtOAc (2×). The
combined organic layers were dried over MgSO4 and concentrated
in vacuo, and the residue was purified by column chromatography
(3:7 EtOAc/hexanes) to give 0.56 g (70%) of the titled compound
2-[4-(4-Fluorophenoxy)butyl]isoindole-1,3-dione (26). Com-
pound 26 was prepared in 85% yield from compound 32 using the
procedure detailed for compound 5. Mp 84-87 °C. 1H NMR
(CDCl3): δ 7.83 (dd, J ) 5 and 3 Hz, 2H), 7.71 (dd, J ) 5 and 3
Hz, 2H), 6.97-6.89 (m, 2H), 6.83-6.76 (m, 2H), 3.94 (t, J ) 6
Hz, 2H), 3.76 (t, J ) 7 Hz, 2H), 1.94-1.75 (m, 4H). 13C NMR
(CDCl3): δ 168.49, 157.19 (d, J ) 238 Hz), 155.05 (d, J ) 2 Hz),
134.01, 132.13, 123.26, 115.78 (d, J ) 23 Hz), 115.46 (d, J ) 8
Hz), 67.80, 37.687, 26.67, 25.36. MS (ESI) m/z: 314.1 (M + H+).
Anal. (C18H16FNO3) C, H, N.
1
as a white solid. Mp 193-199 °C. H NMR (CDCl3): δ 7.15-
7.08 (m, 2H), 7.02-6.94 (m, 2H), 6.20 (s, 1H), 4.12-4.02 (m,
2H), 3.82-3.66 (m, 1H), 2.79-2.66 (m, 2H), 2.42-2.22 (m, 2H),
2.15-1.88 (m, 9H), 1.74-1.59 (m, 6H), 1.37-1.07 (m, 4H). 13C
NMR (CDCl3): δ 162.96, 159.71, 156.72, 141.02, 134.08, 130.49,
130.39, 122.17, 115.18, 114.90, 51.01, 48.59, 42.70, 36.57, 35.27,
34.97, 34.23, 29.68, 27.05.
1-Adamantan-1-yl-3-[4-(4-fluorophenoxy)butyl]urea (27). Com-
pound 27 was synthesized from compound 26 by the general
procedure for the synthesis of trans isomers 13a-f. Mp 138-141
1-Adamantan-1-yl-3-[4-(4-fluorobenzyl)cyclohexyl]urea (36).
To a solution of 35 (0.3 g, 0.78 mmol) in EtOAc was added 10%
palladium on carbon. The solution was filled with H2, and the
reaction mixture was stirred for 2 h. After the solution was filtered
through Celite, the filtrate was concentrated in vacuo. Purification
by column chromatography (3:7 EtOAc/hexanes) gave the title
1
°C. H NMR (DMSO-d6): δ 7.09 (dt, J ) 9 and 2 Hz, 2H), 6.92
(ddd, J ) 9, 4, and 2 Hz, 2H), 5.66 (t, J ) 5 Hz, 1H), 5.44 (s, 1H),
3.92 (t, J ) 6 Hz, 2H), 3.01-2.92 (m, 2H), 2.01-1.94 (m, 3H),
1.86-1.82 (m, 6H), 1.70-1.56 (m, 8H), 1.52-1.40 (m, 2H). 13C
NMR (100 MHz, DMSO-d6): δ 156.37 (d, J ) 235 Hz), 157.07,
154.96 (d, J ) 2 Hz), 115.71 (d, J ) 31 Hz), 115.65, 67.79, 49.35,
42.06, 38.45, 36.15, 28.96, 26.74, 26.22. MS (ESI) m/z: 361.23
(M + H+).
1
compound, 0.28 g (95%), as a white solid. Mp 185-187 °C. H
NMR (CDCl3): δ 7.10-6.88 (m, 4H), 5.20 (d, J ) 8 Hz, 0.56H),
4.85 (s, 0.56H), 4.72 (d, J ) 8 Hz, 0.44H), 4.66 (s, 0.44H), 3.83-
3.74 (m, 0.56H), 3.50-3.34 (m, 0.44H), 2.46 (d, J ) 7 Hz, 1.12H),
2.42 (d, J ) 7 Hz, 0.88H), 2.07-1.90 (m, 9H), 1.72-1.44 (m,
11H), 1.29-1.13 (m, 2H), 1.10-0.95 (m, 2H). 13C NMR (CDCl3):
δ 161.33 (d, J ) 243 Hz), 156.92, 156.84, 136.74 (d, J ) 3 Hz),
136.59 (d, J ) 3 Hz), 130.42 (d, J ) 8 Hz), 130.40 (d, J ) 8 Hz),
115.01 (d, J ) 21 Hz), 114.95 (d, J ) 21 Hz), 50.90, 50.86, 49.36,
45.84, 42.72, 42.68, 41.60, 39.31, 38.18, 36.58, 33.90, 31.80, 30.20,
29.68, 27.64. MS (ESI) m/z: 385.3 (M + H+). Anal. (C24H33FN2O)
C, H, N.
2-[4-(4-Fluorophenoxy)but-2-ynyl]isoindole-1,3-dione (28). Com-
pound 28 was prepared in 90% yield from compound 33 using the
1
procedure detailed for compound 5. Mp 119-121 °C. H NMR
(300 MHz, CDCl3): δ 7.90-7.86 (m, 2H), 7.76-7.73 (m, 2H),
6.97-6.84 (m, 4H), 4.62 (t, J ) 2 Hz, 2H), 4.49 (t, J ) 2 Hz, 2H).
13C NMR (CDCl3) δ 167.05, 157.74 (d, J ) 239 Hz), 153.71 (d, J
) 2 Hz), 134.33, 132.00, 123.64, 116.30 (d, J ) 8 Hz), 115.88 (d,
J ) 23 Hz), 81.17, 78.05, 56.82, 27.33. MS (ESI) m/z: 310.1 (M
+ H+). Anal. (C18H12FNO3) C, H, N.
1-Adamantan-1-yl-3-(4-hydroxybutyl)urea (38). Compound 38
was prepared in 90% yield from 1-adamantyl isocycnate and
4-amino-1-butanol using the procedure detailed above for 7 except
that the titled compound (93%) was recrystallized from MeOH.
Mp 149-152 °C. 1H NMR (DMSO-d6): δ 5.60 (t, J ) 5 Hz, 1H),
5.44 (s, 1H), 4.39 (t, J ) 5, 1H), 3.41-3.33 (m, 2H), 2.94-2.86
(m, 2H), 2.01-1.94 (m, 3H), 1.88-1.80 (m, 6H), 1.62-1.56 (m,
6H), 1.42-1.27 (m, 4H). 13C NMR (DMSO-d6): δ 157.09, 60.56,
49.34, 42.08, 38.73, 36.18, 30.01, 28.97, 26.75. MS (ESI) m/z:
267.2 (M + H+). Anal. (C15H26N2O2) C, H, N.
1-Adamantan-1-yl-3-[4-(4-fluorophenoxy)but-2-ynyl]urea (29).
Compound 29 was synthesized from compound 28 by the general
procedure for the synthesis of trans isomers 13a-f. Mp 135-136
°C. 1H NMR (CDCl3): δ 7.02-6.85 (m, 4H), 4.64 (dd, J ) 2 and
2 Hz, 2H), 4.29-4.06 (m, 2H), 4.01-3.94 (m, 2H), 2.13-2.02 (m,
3H), 2.01-1.90 (m, 6H), 1.71-1.62 (m, 6H). 13C NMR (DMSO-
d6): δ 156.76 (d, J ) 239 Hz), 156.24, 153.67 (d, J ) 2 Hz),
116.11 (d, J ) 8 Hz), 115.82 (d, J ) 23 Hz), 85.82, 76.58, 56.27,
49.60, 41.94, 36.10, 28.95, 28.48. MS (ESI) m/z: 357.2 (M + H+).
Anal. (C21H25FN2O2) C, H, N.
trans,trans-1,3-Bis(4-hydroxycyclohexyl)urea (39). To a solu-
tion of 1,1′-carbonyldiimidazole (0.2 g, 1 mmol) in CH3CN (10
mL) were added trans-4-aminocyclohexanol (0.76 g, 5 mmol) and
triethylamine (0.7 mL, 5 mmol) followed by water (5 mL) at room
temperature. The reaction mixture was stirred overnight, and the
organic solvent was evaporated in vacuo. The resulting white
precipitate was filtered and washed with water thoroughly. The
resulting sample was purified by recrystallization from methanol
to give the titled compound (30%) as a white solid. Mp 285-290
°C. 1H NMR (DMSO-d6): δ 5.51 (d, J ) 8 Hz, 2H), 4.49 (d, J )
4 Hz, 2H), 3.41-3.17 (m, 4H), 1.81-1.66 (m, 8H), 1.25-0.95 (m,
8H). 13C NMR (DMSO-d6): δ 156.86, 68.19, 47.54, 33.99, 31.19.
MS (ESI) m/z: 257.2 (M + H+). Anal. (C13H24N2O3) C, H, N.
Molecular Modeling. Molecular modeling was performed using
“BioMedCAChe 5.0” software (Fujitsu Computer Systems Corpo-
ration). The atomic coordinates of the crystal structure of human
sEH complexed with CU4 were retrieved from Protein Data Bank
(PDB) (entry 1ZD3).33 Compounds 13g and 16g were docked into
the ligand-binding pocket manually by superposition with the parent
molecule (CU4). The ligand and the amino acid residues within
8.0 Å from the ligand were minimized on MM geometry (MM3).
Metabolic Stability Assay. Human hepatic microsomes were
purchased from BD diagnostics (Sparks, MD). The incubation
mixture consisted of microsomal protein in potassium phosphate
buffer (100 mM, pH 7.4) and 1 µM test compound in a final volume
of 1 mL. The concentration of hepatic microsomal protein was 0.05
mg/mL. An NADPH-generating system containing 50 mM MgCl2,
57 mM glucose 6-phosphate, 2 mM â-NADP+, and 5 unit/mL
1-Adamantan-1-yl-3-[4-(4-fluorophenoxy)phenyl]urea (31).
Compound 31 was prepared in 85% yield from compound 30 using
the procedure detailed for compound 2 except the product was
1
purified by column chromatography. Mp 169-207 °C. H NMR
(DMSO-d6): δ 8.24 (s, 1H), 7.34 (d, J ) 9 Hz, 2H), 7.18 (t, J )
9 Hz, 2H), 6.99-6.86 (m, 4H), 5.82 (s, 1H), 2.09-1.85 (m, 9H),
1.66-1.59 (m, 6H). 13C NMR (CDCl3): δ 158.70 (d, J ) 241 Hz),
155.11, 153.50 (d, J ) 2 Hz), 153.33, 134.59, 122.61, 119.92 (d,
J ) 8 Hz), 119.43, 116.34 (d, J ) 23 Hz), 51.37, 42.39, 36.49,
29.63. MS (ESI) m/z: 381.2 (M + H+). Anal. (C23H25FN2O2) C,
H, N.
1-Adamantan-1-yl-3-(4-oxocyclohexyl)urea (34). To a solution
of 34 (1.5 g, 5.1 mmol) in DMF (50 mL) was added PDC (4.8 g,
12.8 mmol) at room temperature. After being stirred overnight, the
reaction mixture was poured into water. The resulting white
precipitates were filtered and washed with water thoroughly. The
resulting solid was purified by recrystallization from DCM/hexanes
to give 1.26 g (85%) of the titled compound. Mp 240-460 °C. 1H
NMR (DMSO-d6): δ 5.95-5.71 (br s, 1H), 5.55-5.33 (br s, 1H),
3.88-3.68 (m, 1H), 2.47-2.30 (m, 2H), 2.28-2.14 (m, 2H), 2.11-
1.77 (m, 11H), 1.70-1.45 (m, 8H). 13C NMR (DMSO-d6): δ
210.03, 156.48, 49.42, 45.22, 42.02, 38.50, 36.14, 31.92, 28.94.
1-Adamantan-1-yl-3-[4-(4-fluorobenzylidene)cyclohexyl]urea
(35). To a solution of 4-fluorobenzyltriphenylphosphonium bromide
(2.33 g, 5.2 mmol) in THF (15 mL) was added 1.6 M n-butyllithium
in THF (3.25 mL, 5.2 mmol) at -78 °C, and the mixture was
warmed to room temperature. A solution of 34 (0.5 g, 1.72 mmol)

3838 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Hwang et al.
glucose 6-phosphate dehydrogenase (Sigma, St. Louis) was prepared
and added to the incubation mixture with a 10% volume of the
reaction mixture. After the addition of the NADPH-generating
system, the mixture was incubated at 37 °C for 0 and 60 min. The
reaction was terminated by the addition of one volume of ethanol.
All incubations were made in triplicate. The concentrations of the
test compound in the reaction mixture were measured by LC/MS-
MS, using a Micromass Quattro Premier triple quadrupole tandem
mass spectrometer (Micromass, Manchester, U.K.) equipped with
an atomospheric pressure ionization source [atomospheric z-spray
pressure chemical ionization (APcI) or electrospray ionization (ESI)
interface]. The separation was performed by Ultra Performance LC
(UPLC; Waters Corporation, Milford, MA) with an Aquity column
(BEH 2.1 mm × 50 mm, 1.7 µm; Waters Corporation) at a flow
rate of 0.3 mL/min at ambient temperature. Solvent A was 10%
acetonitrile and 90% water containing 0.1% formic acid, and solvent
B was acetonitrile containing 0.1% formic acid. Mobile phases were
mixed with a linear gradient from 30% B to 100% B for 5 min and
then isocratic for an additional 8 min with 100% B. Five microliters
of standard and the extracted microsome samples were injected onto
the column. Data were analyzed with MassLynx software (version
4.0).
Bioavailability Protocol. All in vivo experiments were done
following protocols approved by the Animal Use and Care
Committee of University of CaliforniasDavis. Two dogs weighing
between 19 and 22 kg were used in the exposure studies (n ) 2
per time point). For pharmacokinetic screening, oral doses of 0.3
mg/kg were administered via syringe in 1 mL of tristerate at 40
°C. For the determination of the bioavailability, 13g was separately
administered in saline containing 1% morpholine at 0.3 mg/kg orally
and intravenously. Dogs were bled via the retro-orbital route at 0,
15, 30, 60, 120, 180, 240, 300, 360, 480, and 1440 min after oral
dosing and at 0, 5, 10, 15, 30, 45, 60, 120, 150, 180, 240, 300,
360, and 1440 min after intravenous dosing. All blood samples
were collected into EDTA tubes. Plasma was separated by
centrifugation and stored at -78 °C until used. Plasma was assayed
by electrospray LC-MS/MS as described above. Least-squares
regression of the standard concentrations was used for sample
quantitation.
For the cytosolic preparation (hamster, cat, and dog), we used a
radioactive based assay to determine IC50 values.45 Enzyme extracts
were incubated with inhibitors for 5 min in pH 7.4 sodium
phosphate buffer at 30 °C prior to substrate (racemic [3H]-trans-
1,3-diphenylpropene oxide) introduction ([S]final ) 50 µM; ∼12000
dpm/assay). The enzymes were incubated at 30°C for 5-10 min.
The reaction was quenched by addition of 60 µL of methanol and
200 µL of isooctane, which extracts the remaining epoxide from
the aqueous phase. The activity was followed by measuring the
quantity of radioactive diol formed in the aqueous phase using a
liquid scintillation counter (Wallac model 1409; Gaithersburg, MD).
Conditions used gave rates that were linear with both time and
enzyme concentration and that resulted in at least 5% but not more
than 30% hydrolysis of the substrate. Assays were performed in
triplicate. IC50 results are the averages of three replicates.
Blood Pressure Determination and Oxylipin Profile. Male
mice (C57BL/6, 8 weeks old, Charles River Laboratories) between
22 and 25 g were used in all treatments. The LPS was from
Escherichia coli serotype 0111:B4 purchased from Sigma-Aldrich.
13g was dissolved in trioleine containing 1% ethanol and AUDA-
BE was dissolved directly in trioleine. 13g and AUDA-BE were
administered by oral gavage immediately after ip injection of LPS
(10 mg/kg) in saline. Blood pressure was determined with a Visitech
BP-2000 (Visitech Systems, Apex, NC) described by Schmelzer
et al.35
Blood was collected by cardiac puncture with an EDTA-rinsed
syringe. An amount of 10 µL of a mixture of triphenyphosphine,
butylated hydroxytoluene, and indomethacin (0.2% for each, w/w)
was added to each collection tube. Each sample was spun
immediately and the plasma separated. All samples were stored at
-80 °C until analysis. The ratio of EETs to DHETs was determined
using a previously reported method.46
Acknowledgment. The authors thank Dr. James Sanborn
for many helpful discussions. We also thank Dr. Jozsef Lango
for assistance with mass spectral determinations. This work was
supported in part by NIEHS Grant ES02710, NIEHS Superfund
Grant P42 ES04699, and NIEHS Center Grant P30 ES05707.
Enzyme Preparation. Recombinant murine, rat, and human
sEHs were produced in a baculovirus expression system40,41 and
were purified by affinity chromatography as previously reported.42
Supporting Information Available: Elemental analysis data
for the selected compounds and HPLC and H NMR data for the
evaluation of isomeric purity of compounds 13f and 16f. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1
Liver samples from hamster, dog, and cat are generous gifts from
Dr. W. Yokoyama (USDA), Dr. G. Gross (University of Wiscon-
sin), and Dr. A. Hill (University of California, Davis), respectively.
Cytosolic enzyme extracts were prepared in sodium phosphate
buffer (100 mM, pH 7.4) as described.43 The preparations were
frozen at -80 °C until used. Protein concentration was quantified
using the Pierce BCA assay (Pierce, Rockford, IL) using bovine
serum albumin (BSA) as the calibrating standard.
IC50 Assay Conditions. For the spectrometric-based assay,
enzymes (0.12 µM murine sEH or 0.24 µM human sEH) were
incubated with inhibitors for 5 min in 0.1 M sodium phosphate
buffer (200 µL, pH 7.4) at 30 °C before spectrometric substrate
(4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC)
introduction ([S] ) 40 µM).40
For the recombinant affinity purified sEHs (human, mouse, and
rat), we used a fluorescent-based assay to determine IC50 values.5
Enzymes (∼2nM mouse sEH, ∼1nM rat sEH, or ∼1 nM human
sEH) were incubated with inhibitors for 5 min in 25 mM bis-Tris-
HCl buffer (200 µL, pH 7.0) at 30 °C before substrate (cyano(2-
methoxynaphthalen-6-yl)methyl trans-(3-phenyloxyran-2-yl)meth-
ylcarbonate, CMNPC) was added ([S]final ) 5 µM). Activity was
assessed by measuring the appearance of the fluorescent 6-meth-
oxynaphthaldehyde product (λem ) 330 nm, λex ) 465 nm) at 30
°C during a 10 min incubation (Spectramax M2; Molecular Device,
Inc., Sunnyvale, CA).44 The IC50 values that are the concentrations
of inhibitors that reduce activity by 50% were calculated from at
least three separate runs, each in triplicate, to obtain the standard
deviation given in the results section.
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