1-Aryl-3-(1-acylpiperidin-4-yl)urea inhibitors of human and murine soluble epoxide hydrolase: Structure-activity relationships, pharmacokinetics, and reduction of inflammatory pain

Article abstract of DOI:10.1021/jm100691c

1,3-Disubstituted ureas possessing a piperidyl moiety have been synthesized to investigate their structure-activity relationships as inhibitors of the human and murine soluble epoxide hydrolase (sEH). Oral administration of 13 1-aryl-3-(1-acylpiperidin-4-yl)urea inhibitors in mice revealed substantial improvements in pharmacokinetic parameters over previously reported 1-adamantylurea based inhibitors. For example, 1-(1-(cyclopropanecarbonyl) piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (52) showed a 7-fold increase in potency, a 65-fold increase in C_max, and a 3300-fold increase in AUC over its adamantane analogue 1-(1-adamantyl)-3-(1-propionylpiperidin-4-yl) urea (2). This novel sEH inhibitor showed a 1000-fold increase in potency when compared to morphine by reducing hyperalgesia as measured by mechanical withdrawal threshold using the in vivo carrageenan induced inflammatory pain model.

Full text of DOI:10.1021/jm100691c

J. Med. Chem. 2010, 53, 7067–7075 7067
DOI: 10.1021/jm100691c
1-Aryl-3-(1-acylpiperidin-4-yl)urea Inhibitors of Human and Murine Soluble Epoxide Hydrolase:
Structure-Activity Relationships, Pharmacokinetics, and Reduction of Inflammatory Pain
Tristan E. Rose, Christophe Morisseau, Jun-Yan Liu, Bora Inceoglu, Paul D. Jones, James R. Sanborn, and
Bruce D. Hammock*
Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue,
Davis, California 95616
Received June 8, 2010
1,3-Disubstituted ureas possessing a piperidyl moiety have been synthesized to investigate their structure-
activity relationships as inhibitors of the human and murine soluble epoxide hydrolase (sEH). Oral
administration of 13 1-aryl-3-(1-acylpiperidin-4-yl)urea inhibitors in mice revealed substantial improve-
ments in pharmacokinetic parameters over previously reported 1-adamantylurea based inhibitors. For
example, 1-(1-(cyclopropanecarbonyl)piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (52) showed a
7-fold increase in potency, a 65-fold increase in Cmax, and a 3300-fold increase in AUC over its adamantane
analogue 1-(1-adamantyl)-3-(1-propionylpiperidin-4-yl)urea (2). This novel sEH inhibitor showed a 1000-
fold increase in potency when compared to morphine by reducing hyperalgesia as measured by mechanical
withdrawal threshold using the in vivo carrageenan induced inflammatory pain model.
Introduction
pharmacokinetics.24,25 Although earlier studies found that
trisubstituted ureas had reduced potency,9,26 with proper
substituents piperidine based trisubstituted ureas have been
found to be potent inhibitors of the enzyme.27-30 In the past
year several other promising pharmacophores have been
reported.17,18,21,28,31 We previously reported inhibitors incor-
porating a polar moiety, such as an N-acylpiperidine or cyclo-
hexyloxybenzoic acid, to one side of the urea, which yielded a
substantial improvement in water solubility and oral bioavail-
ability while retaining excellent potency.24,25,32,33 The ada-
mantyl moiety retained in many of these potent second-
generation sEH inhibitors provided sensitive characteristic
mass spectral fragmentation. However, this moiety is prone to
rapid metabolism, often leading to low drug concentrations in
the blood and short in vivo half-life.
Replacement of the adamantyl group with a phenyl ring
has been explored in our earlier work and has yielded se-
veral highly potent inhibitors, warranting further investiga-
tion.25,33,34 Thus, in this study, we further investigated urea
based sEH inhibitors by optimizing the 1-aryl-3-(1-acylpiper-
idin-4-yl)urea core structure. On the basis of the reported
2-fold increase in potency of the N-propionylpiperidine over
the N-acetylpiperidine in a group of adamantylureas,25,32
a series of inhibitors conserving the N-propionylpiperidine
moiety was synthesized to probe the SAR of the aryl group.
Additional inhibitors conserving 4-trifluoromethoxyphenyl
as the aryl group and varying in N-acylpiperidine or N-sulfo-
nylpiperidine substitution were synthesized to examine the
effects of polar and basic side chains on potency.
The soluble epoxide hydrolase (sEH,a EC 3.3.2.3) con-
verts epoxides to the corresponding diols by the catalytic addi-
tion of a water molecule. The enzyme is implicated in several
disease states for its ability to metabolize fatty acid epoxides
such as epoxyeicosatrienoic acids (EETs) and leukotoxin,
important endogenous signaling lipids, to less active dihydro-
xyeicosatrienoic acids (DHETs)1 and toxic, proinflammatory
leukotoxin diols,2 respectively. sEH inhibitors are of growing
interest for therapeutic use because they have been shown to
increasetheinvivoconcentrationofEETsandotherfatty acid
epoxides, resulting in anti-inflammatory,3 antihypertensive,4
neuroprotective,5 and cardioprotective effects.6-8 Several re-
views have been published concerning the mechanism of
action and diverse biological roles of EETs and the sEH inhi-
bitors that stabilize them.9-16 Of particular note, Marino17
recently reviewed the chemistry of sEH inhibitors and Shen18
summarized the patent literature in the sEH field.
The prototypical inhibitors dicyclohexylurea and 12-(3-
adamantane-1-ylureido)dodecanoic acid (AUDA), while po-
tent in vitro, suffer from poor physical properties and poor
in vivo stability. Amides, carbamates, and other pharmaco-
phores17,18 havebeenexploredas alternative pharmacophores
in an attempt to improve physical properties and show
structure-activity relationships similar to those of ureas,
but the disubstituted ureas remain the most studied class of
inhibitors because of their high potency19-23 and promising
*To whom correspondence should be addressed. Phone: 530-752-
7519. Fax: 530-752-1537. E-mail: bdhammock@ucdavis.edu.
a Abbreviations: sEH, soluble epoxide hydrolase; EETs, epoxyeico-
satrienoic acids; DHETs, dihydroxyeicosatrienoic acids; AUDA, 12-(3-
adamantane-1-ylureido)dodecanoic acid; SAR, structure-activity rela-
tionship; t1/2, half-life; Cmax, maximum concentration; IC50, half
maximal inhibitory concentration; AUC, area under the curve; LOQ,
limit of quantitation.
Inhibitors were first screened for potency in vitro against
homogeneous recombinant murine sEH and human sEH,
and their octanol-water partition coefficients were deter-
mined to help direct our search for more “druglike” mole-
cules. Pharmacokinetic screening by oral cassette dosing
was then undertaken in mice for 13 piperidines exhibiting
r
2010 American Chemical Society
Published on Web 09/02/2010
pubs.acs.org/jmc

7068 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Scheme 1. Synthesis of N-Propionylpiperidine Analoguesa
Rose et al.
a Reaction conditions: (a) triphosgene, DCM, sat. NaHCO3 or 1 N NaOH, sat. NaCl, 0 °C, 10 min; (b) 4-nitrophenyl chloroformate, Et3N, THF,
0-50 °C, 1-3 h; (c) 1, THF, 0 °C to room temp, 1-24 h; (d) 1, DMF, 70 °C, 4 h.
Scheme 2. Synthesis of N-Acyl and N-Sulfonylpiperidine Analoguesa
a Reagents and conditions: (a) 1-BOC-4-aminopiperidine, THF, 0 °C to room temp, 12 h; (b) 1 N HCl in MeOH, reflux, 3 h; (c) R1SO2Cl, Et3N, THF,
0 °C to room temp, 12 h; (d) EDCI, R2COOH, DMAP, DCM, room temp, 12-24 h.
good in vitro potency and desirable structural characteris-
tics. In addition several bridging compounds were studied to
relate this study to previous publications. The in vivo anti-
inflammatory and analgesic bioactivity of one sEHi were
evaluated using carrageenan induced inflammatory pain
model.
18-21, and 24-40. Saponification of methyl ester 21 with
methanolic NaOH afforded benzoic acid 22. Phenol 23 was
prepared via 4-benzyloxy isocyanate to avoid formation of a
carbamate side product.
Compounds 17 and 31 were prepared by conversion of the
corresponding aniline to the intermediate 4-nitrophenyl car-
bamate, which was then reacted with amine 1 to give the
desired urea. Intermediate 41 (Scheme 2) was prepared by the
reaction of 4-trifluoromethoxyphenyl isocyanate with 1-BOC-
4-aminopiperidine. BOC deprotection gave piperidine 42,
which was converted to N-acyl compounds 47-50 and 52 by
an EDCI mediated coupling reaction with the respective car-
boxylic acid.37 Acetylpiperazine 51 was prepared by debenzy-
lation of 50 and subsequent N-acetylation. Trifluoroacetyl
compound 53 was prepared by the reaction of intermediate
42 with ethyl trifluoroacetate. Trihydroxybenzoyl compound
54 was prepared by coupling of 42 with tris-O-benzyl protected
gallic acid (44) followed by hydrogenolysis.38,39 Intermediate
42 was also converted to N-sulfonyl compounds 55-59 by
reaction with the respective sulfonyl chlorides.
Chemistry
Scheme 1 outlines the two general synthetic routes used to
form the unsymmetrical 1,3-disubstituted urea pharmacophore.
Aryl isocyanates were purchased or formed from their corre-
sponding anilines by reaction with triphosgene in the presence of
aqueous base.35 The heptafluoroisopropylanilines required for
compounds 38 and 39 were prepared as described.36
Amine 1 was prepared from 4-aminopiperidine by protec-
tion of the primary amine as its benzylimine,37 reaction with
propionyl chloride in the presence of triethylamine, and sub-
sequent deprotection. All isocyanates were reacted with amine
1 to give the desired (1-propionylpiperidin-4-yl)ureas 2-16,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7069
Table 2. Substituted Phenyl Analogues
Table 1. Alkyl, Carbocycle, and Unsubstituted Aryl Analogues
a IC50 values were determined with a fluorescent assay using homo-
geneous recombinant murine and human enzymes(see methods section).
b Confidence refers to log P value. See Supporting Information for full
explaination. c The HPLC method used is limited to log P values greater
than zero.
Results and Discussion
Effects of Phenyl Substitution on Potency. In Tables 1-4 a
partial structure is shown at the top and the R groups are
detailed. Full structures are shown in the Supporting Informa-
tion along with detailed synthetic methods. Previous studies
have shown that the steric bulk of groups adjacent to the urea
is positively correlated with inhibitor potency,26 as was again
observed for compounds 2-8 (Table 1). This effect is more
directly attributed to the hydrophobicity of these groups,
as indicated by the positive correlation between potency
(-log IC50) and log P values (r = 0.67/0.87, human/murine)
for simple side chains in 2-8. Although replacing the cyclo-
hexyl ring (4) with a more compact phenyl ring (7) caused an
11-fold drop in potency against the human enzyme, sub-
stitution of the phenyl ring allowed access to electronically
and sterically diverse structures (Tables 2 and 3). Com-
pound 6 provides a bridging structure to recent literature
compounds.27 Inclusion of a pyridine on the left side of the
molecule as in compound 9 resulted in a dramatic reduction
in potency in the piperidine series, although good potency of
such pyridines are reported in the patent literature,17,18 and
other reports.22,27-30 Inclusion of such polar groups are
attractive to improve physical properties, pharmacokinetic
properties, and ease of formulation.
Compared to the unsubstituted phenyl compound 7, the
inhibition potencies increased dramatically when small non-
polar meta or para (11, 12) substituents were added. Their
presence at the ortho position (10) has a clear negative effect
on potency. Increasing the size of the hydrophobic para sub-
stituent in compounds 12-16 yielded a 3- to 46-fold increase
in potency over 7. However, the presence of polar para

7070 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 3. Halophenylurea Analogues
Rose et al.
Table 4. N-Acyl and N-Sulfonylpiperidine Analogues
a nd = Not determined.
substituents (17-23) resulted in less potent inhibitors. The
phenol 23, a likely metabolite of 15, was a poor inhibitor
presumably because of unfavorable electronic character and
polarity. Compound 20 was far less potent than anticipated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7071
because of the high polarity of the nitro functionality, des-
pite having a favorable electron deficient urea. Methyl ester
and corresponding carboxylic acid compounds 21 and 22
showed similarly diminished potency. The poor performance
of highly polar substituents led us to investigate halophenyl
analogues (Table 3). Halogens can increase polarity as a
result of their inherent electronegativity and can also serve to
block metabolism at particularly reactive sites and reduce
metabolism of the aromatic group by decreasing its electron
density.
Table 5. Pharmacokinetic Screening Resultsa
AUCt
compd
AUDAb
Cmax (nM)
Tmax (min)
t1/2 (min)
(ꢀ104 nM min)
0.3
1.9
3
14
138
80
45
126
78
2
3
2770
4600
5570
4810
5860
13000
8410
18300
3790
5940
17400
8900
60
50
50
56
35
58
4
12
13
14
15
24
25
27
35
40
52
30
30
72
56
92
74
50
70
58
82
95
283
467
1360
375
516
1400
985
Thus, compounds 24-27 were synthesized to slow meta-
bolic oxidation of the aromatic ring by cytochrome P450
enzymes (CYPs). These compounds also revealed a slight
electronic effect on potency, which was less clear in previous
studies.34,40 The observed increase in potency (-log IC50)
was correlated with electron withdrawing characteristics
according to classical Hammett substituent constants (r =
83
378
381
881
814
980
1180
188
440
230
220
190
a Values are from single oral cassette dosing at 5 mg/kg in 120-150 μL
of 20% PEG400 v/v in oleic ester rich triglyceride. Full pharmacokinetic
profiles are shown in Figures S1-S3, and plots of murine AUC as a
function of the IC50 on the human enzyme and the murine enzyme are
shown in Figures S4 and S5 in the Supporting Information. b AUDA
gave biphasic kinetics.
1
0.82) and the H NMR chemical shifts of the urea N-H
adjacent to the phenyl ring (r = 0.77).41 This effect, in the ab-
sence of confounding steric effects, was well revealed in com-
paring para versus meta fluorination. m-Fluorophenyl (28,
σ = 0.337, 1H NMR δ 8.57) showed a 2-fold and 5-fold lower
IC50 against the human and murine enzymes, respectively,
than p-fluorophenyl (24, σ = 0.062, 1H NMR δ 8.36). Along
these lines the 3,5-dichloro substituents yielded a particularly
potent sEH inhibitor (33, σ = 0.746, 1H NMR δ = 8.74). An
electron withdrawing group presumably strengthens hydro-
gen bonding interaction of the urea hydrogen with Asp334
at the catalytic site of the human enzyme (or Asp333 of the
murine enzyme) by inductive withdrawal of the nitrogen lone
electron pair, further polarizing the urea N-H bond. Fine
optimization of 3,4- and 3,5-disubstituted electron with-
drawing groups should yield additional potent compounds.
The fluorinated p-isopropyl derivative 38 showed an increase
in activity over the corresponding isopropylphenyl deriva-
tive 14. As observed previously40 and for the o-tolyl com-
pound 10, ortho mono- or dihalogenation in compounds 29,
31, and 34 drastically decreased potency. However, this
effect may be mitigable by the addition of a large hydro-
phobic para substituent, such as perfluoroisopropyl in com-
pound 39. It is difficult to discern between hydrophobic and
electronic contributions to inhibitor potency in vitro. Ex-
perimental log P values and calculated molar volumes (data
not shown) are highly predictive of the relative potencies of
the carbocyclic, alkylphenyl, and phenyl ether analogues.
However, these criteria do not fully account for the high
potency observed for halophenyl compounds, highlighting
an electronic contribution to inhibitor potency.
51, 54, 57-59) did not substantially improve potency over
such compounds as 40 or 56. Although bulky N-substitution
leads to some increases in potency, previous studies in
canines showed that large N-substituents decreased blood
levels dramatically following oral administration.25
Small N-acyl or N-sulfonyl substituents may improve
hydrophobic interaction with the enzyme while minimizing
the need for conformational change upon entry into the cata-
lytic tunnel. The presence of a small hydrophobic group (52,
53) improved potency approximately 9-fold over compound
40 and reached the LOQ of our in vitro assay. Although
highly potent, the hydrolytic instability of the trifluoroace-
tamide 53 makes it unsuited for in vivo use. Cyclopropane-
carboxamide 52, however, is a reasonable modification that
improves metabolic stability over propionamide 40 without
significantly increasing the molecular weight or log P value.
Proper substitution on the phenyl ring is crucial for attain-
ing good potency. A varying degree of polarity, bulk, and
basicity is extremely well tolerated by the target enzyme if
attached to the piperidine nitrogen, away from the urea
pharmacophore.
Pharmacokinetic Screening in Mice. Carbocycle and phen-
yl substituted ureas significantly improved pharmacokinetics
in comparison to the earlier inhibitors AUDA,34 AEPU (1-[1-
acetypiperidin-4-yl]-3-adamantanylurea),24,32 and 1-(1-
adamantyl)-3-(1-propionylpiperidin-4-yl)urea compound 232
(Table 5 and Supporting Information). Our results suggest
that the adamantane ring is generally less favorable, in terms of
ADME, than other groups. The results also demonstrate that
molecules can be fine-tuned to optimize ADME and physical
properties while retaining high inhibitory potency on the
enzyme.
Comparison of Piperidine N-Substituents. The 4-trifluoro-
methoxyphenyl moiety was used as a metabolically stable re-
placement for the adamantyl ring of our earlier generation of
inhibitors25,33,40 and was thus conserved in order to investigate
N-substitution of the piperidine moiety. N-Acyl and N-sulfonyl
substitution of the 1-(piperidin-4-yl)-3-(4-(trifluoromethoxy)-
phenyl)urea core structure (intermediate 42) yielded multiple
highly potent inhibitors (Table 4).
Compounds 47-51 demonstrated the feasibility of intro-
ducing a basic nitrogen to allow formulation of the inhibitor
as a salt. Analogues 55 and 56 indicate that the sulfonamide
group is a good isosteric replacement for the amide, yielding
comparably potent inhibitors. These two functional groups
may provide valuable pharmacokinetic and pharmacodynamic
differences. While bulky substituents on the phenyl moiety
improve potency, bulky N-substitution on the piperidine (50,
Replacing the adamantane with cycloalkanes in com-
pounds 2-4 resulted in substantially higher blood levels.
For example, substitution of the adamantyl ring (2) with a
cyclohexyl ring (4) resulted in a 33-fold and 81-fold increase
in Cmax and AUC, respectively, while the cycloheptyl ring (3)
increased exposure while retaining potency on the recombi-
nant enzymes (Figures S4 and S5). Likewise, the 4-alkylphe-
nyl compounds (12-14) showed improved PK profiles
similar to that of compound 4. Interestingly, homologation
of the para-alkyl group altered potency in vitro, with 14

7072 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Rose et al.
threshold of animals. This state was sustained over the course
of the experiment. The sEHi (10 ng/paw) and morphine
(10000 ng/paw) significantly reversed carrageenan induced
pain in a timedependent manner, althoughthe sEHi was more
stable than morphine (sEHi vs morphine, ANOVA followed
by Dunnett’s two-sided t test, p > 0.3) 270 min after treatment
even thoughthe doseof the sEH inhibitor was 1000lowerthan
the morphine dose.
Conclusion
The sEH inhibitors are promising pharmacological leads
based on their successful use in multiple models of human
disease including prevention and reversal of cardiac hyper-
trophy16,44 and a dramatic reduction of inflammation and pain
when used either alone or with nonsterodial anti-inflammatory
drugs.3,45 As discussed in the Introduction, there are several
excellent lead structures under investigation as sEH inhi-
bitors.17,18 Disubstituted urea sEH inhibitors with substituted
piperidines as the secondary pharmacophore were selected as
the focus of this study because they are small, druglike,
conformationally restricted structures that are synthetically
straightforward.25,32 Earlier compounds in this series showed
attractive pharmacokinetic properties.24,25 Although many
criteria are used for the selection of an investigational new
drug candidate or a probe for investigating the arachidonic
acid cascade in experimental animals, an estimate of expo-
sure indicated by blood levels (Table 5, Figures S1-S3) and
enzyme inhibition as an indication of potency (Tables 1-4
and S2) are important considerations as are physical proper-
ties. When the potential efficacy of compounds is expressed
as a function of exposure in murine blood and potency on
the recombinant human enzyme, compound 52 represents
an improvement of over 104 from the bridging compound
AUDA reported 10 years ago (Figure S4).9,19 It is important
to look at a compound’s potency in the model species being
used, and a similar trend with a 105 improvement is seen when
murine potency data are used (Figure S5). Evaluation of
potency in the model species used is critical. For example,
there is a good correlation between data on the recombinant
mouse and rat enzymes, while there is not a perfect correla-
tion between IC50 on human and murine enzymes (r2 = 0.77;
F =0.80; Figure S6), and a poor correlation among canine,
rodent, and human enzymes particularly for the piperidine
containing sEH inhibitors.25
Figure S3 compares the blood pharmacokinetic profile of
compound 52 with that of the closely related bridging com-
pound APAU previously published from this laboratory.32
The activity of these acylpyperidine compounds is similar on
the human and murine recombinant enzymes, but compound
52 is about 30 times more potent on the human than the
murine enzyme.25,32 The data in Figure S3 indicate a very
short half-life and small AUC for APAU. When the AUC/
IC50 is used as a metric, there is a 5500-fold improvement with
compound 52 over the earlier APAU.
Thus, multiple compounds in this series and particularly
compounds such as 40 and 52 should be reasonable com-
pounds to use in murine models of disease. Both inhibitors
have similar potencies on the rodent and human enzyme and
reasonable pharmacokinetics. In particular the data in Fig-
ure 1 demonstrate the efficacy of compound 52 in a pain
model. More broadly, these data demonstrate rational ways
to fine-tune inhibitors in this series as potential therapeutics. It
is possible that combinations of these and related structural
Figure 1. The soluble epoxide hydrolase inhibitor 52 (sEHi) re-
duces local inflammatory pain at a far lower dose than morphine.
The inflammatory agent carrageenan was administered at time 0,
resulting in a stable hyperalgesic response for the duration of the
experiment. Zero time points represent normal response to pain.
Treatment at 180 min after carrageenan with either morphine or
compound 52 reversed symptoms. Standard deviation represents
the average of six animals with regard to mechanical withdrawal
threshold.
exhibiting the lowest IC50 against both enzymes, but did not
significantly alter PK. The 4-methoxyphenyl (15) surpris-
ingly showed a further increase in Cmax and AUC by approx-
imately 2-fold and 3-fold, respectively, over compounds 3 and
12-14, suggesting that an ether substituent helps to improve
absorption and favorably alters metabolism. Compounds
2-4 and 12-15 were largely cleared in approximately 8 h.
The 4-halophenyl compounds 24, 25, and 27 are more per-
sistent than the former materials and have drastically in-
creased Cmax and AUC, supporting our hypothesis that
metabolism of the 1-aryl-3-(1-propionylpiperidin-4-yl)urea
core structure occurs predominantly at the phenyl moiety.
To bridge these data to the anticancer drug sorafenib,42
which is also a potent sEH inhibitor,43 compound 35 was
tested.
The sorafenib-like compound 35 exemplifies excellent
stability with two electron withdrawing groups on the phenyl
ring.43 Interestingly, the chlorophenyl compound 25 showed
significantly better PK than the other monohalogenated
analogues tested. While the lower Cmax and AUC of 4-iodo-
phenyl compound 27 is likely a consequence of its poor
solubility, the large difference between 4-fluorophenyl (24)
and 4-chlorophenyl (25) suggests that the improved ADME
of 25 is a result of favorable polarity and electronics.
Pain Reduction in Mice. Compound 52 was selected for
biological studies as showing both high potency and good
pharmacokinetics in mice (Figures S1-S5). Several studies
have demonstrated a dramatic reduction of inflammation
by sEH inhibitors both alone and when combined with aspirin
and nonsteroidal anti-inflammatory drugs.3 Thus, compound
52 was tested in an inflammation driven pain model (Figure 1).
For this study local inflammatory pain was induced by a
single, intraplantar injection of carrageenan in rats. The
animals’ mechanical withdrawal thresholds, expressed as a
percent of control, were measured before (baseline) and 3 h
after carrageenanadministration. At this time vehicle or com-
pounds were administered into the plantar surface of the
inflamed paw, indicated by an arrow, in a volume of 10 μL.
Mechanical withdrawal thresholds were then monitored (n = 6
per group). Intraplantar carrageenan led to significant pain
observed as a dramatic decrease in mechanical withdrawal

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7073
units will yield highly potent inhibitors and with pharmaco-
kinetics fine-tuned for use in various species.
dry THF was added Et3N (1.3 equiv), and the appropriate
aniline (1 equiv) was dissolved in dry THF. The mixture was
allowed to warm to room temperature, stirred for 30 min, and
then filtered. The filtrate was evaporated and dissolved in DMF.
Amine 1 was added and the mixture warmed to 50 °C for 1-3 h.
The mixture was cooled to room temperature, diluted with ethyl
acetate, and the organic phase washed with 1 N NaOH until the
wash was free of yellow p-nitrophenol. The organic phase was
dried, evaporated, and purified.
Method D: Synthesis of N-Acylpiperidine Analogues. To a
solution of 41 (1 equiv) in DCM were added the correspond-
ing carboxylic acid (1.1 equiv), DMAP (1 equiv), and EDCI (1.1
equiv). The mixture was stirred for 12-24 h at room tempera-
ture. Neutral products were worked up by partition with EtOAc
and 1 N HCl (basic products by partition with saturated sodium
bicarbonate and EtOAc), and the organic phase was dried,
evaporated, and purified.
Method E: Synthesis of N-Sulfonylpiperidine Analogues. To
a solution of 41 (152 mg, 0.5 mmol) in dry THF (5 mL) were
added Et3N (1.3 equiv) and the corresponding sulfonyl chloride
(1 equiv) in dry THF (1 mL). The mixture was stirred for 12 h,
quenched with 1 N HCl, and filtered to collect the resulting
precipitate, which was further purified.
Enzyme Purification. Recombinant murine and human sEH
were produced in a polyhedron positive baculovirus expression
system, and they were purified by affinity chromatography as
previously reported.46-48
IC50 Assay Conditions. IC50 values were determined using a
sensitive fluorescent based assay.49 Cyano(2-methoxynaphthalen-6-
yl)methyl trans-(3-phenyloxyran-2-yl)methylcarbonate (CMNPC)
was used as the fluorescent substrate. Human sEH (1 nM) or
murine sEH (1 nM) was incubated with the inhibitor for 5 min in
pH 7.0 Bis-Tris/HCl buffer (25 mM) containing 0.1 mg/mL BSA
at 30 °C prior to substrate introduction ([S]=5 μM). Activity
was determined by monitoring the appearance of 6-methoxy-2-
naphthaldehyde over 10 min by fluorescence detection with an
excitation wavelength of 330 nm and an emission wavelength
of 465 nm. Reported IC50 values are the average of three repli-
cates with at least two data points above and at least two below
the IC50. The fluorescent assay as performed here has a standard
error between 10% and 20%, suggesting that differences of 2-fold
or greater are significant.49
Pharmacokinetic (PK) Studies. Male CFW mice (7 week old,
24-30 g) were purchased from Charles River Laboratories. All
the experiments were performed according to protocols ap-
proved by the Animal Use and Care Committee of University
of California, Davis. Inhibitors (1 mg each) were dissolved in
1 mL of oleic ester-rich triglyceride containing 20% polyethylene
glycol (average molecular weight: 400) to give a clear solution for
oral administration. Since many of these compounds are high
melting and relatively water insoluble, it is important that they
are in true solution to study their pharmacokinetics. The 20%
PEG 400 in oleic ester rich triglyceride gave true solutions for all
compounds reported. To avoid ill defined levels of linoleate esters
(18:2) in the vehicle, we used the synthetic triglyceride of oleic
esters (18:1) or triolein for vehicle. Cassette 1 contained com-
pounds 2, 24, 25, and 27, cassette 2 compounds 12, 13, and 14,
cassette 3 compounds 3, 4, and AUDA, cassette 4 compounds 2,
15, and 35, and cassette 5 compounds 40 and 52. Each cassette
was orally administered to three or four mice at a dose of 5 mg/kg
in 120-150 μL of vehicle depending on animal weight. Blood
(10 μL) was collected from the tail vein using a pipet tip rinsed
with 7.5% EDTA (K3) at 0, 0.5, 1, 1.5, 2, 4, 6, 8, 24 h after oral
dosing with the inhibitor. The blood samples were prepared
according to the methods detailed in our previous study.24 Blood
samples were analyzed using an Agilent 1200 series HPLC instru-
ment equipped with a 4.6 mm ꢀ 150 mm Inertsil ODS-4 3 μm
column (GL Science Inc., Japan) held at 40 °C and coupled with
an Applied Biosystems 4000 QTRAP hybrid, triple-quadrupole
mass spectrometer. The instrument was equipped with a linear
Experimental Section
General. All reagents and solvents were purchased from
commercial suppliers and were used without further purifica-
tion. All reactions were performed under an inert atmosphere
of dry nitrogen. Flash chromatography was performed on silica
gel using a dry loading technique, where necessary for poorly
soluble products, and elution with the appropriate solvent sys-
tem. Melting points were determined using an OptiMelt melting
1
point apparatus and are uncorrected. H NMR spectra were
collected using a Bruker Avance 500 MHz spectrometer or
Varian Mercury 300 MHz spectrometer. Accurate masses were
measured using a Micromass LCT ESI-TOF-MS equipped with
a Waters 2795 HPLC. The log P and purity analyses were
performed using a Hewlett-Packard 1100 HPLC instrument
equipped with a diode array detector. A Phenomenex Luna
150 mm ꢀ 4.6 mm, 5 μm, C-18 column was used for all HPLC
analyses. For purity analysis, final products were dissolved in
MeOH/H2O (3:1, v/v) at 10 μg/mL, and 100 μL injections were
analyzed in triplicate by HPLC-UV with detection at 210, 230,
254, and 290 nm. HPLC conditions were the same as those
for log P determination (see below). Purity was judged as the
percent of total peak area for each wavelength. The lowest ob-
served purity is reported. Compounds were also judged to be
pure based on thin layer chromatography visualized with short
wave UV and stained with basic potassium permanganate.
Compounds were g95% pure by HPLC-UV except where
specifically noted (see Supporting Information). All compounds
were evaluated by LC--MS and NMR specifically to ensure
that there was no symmetrical urea impurity present, since these
compounds can be very active.
log P Determination. Octanol-water partition coefficients
were determined by an HPLC method following OECD Guide-
line 117. The accepted error for this method is (0.5 log unit
of shake flask values (OEDC Guideline 107). Isocratic MeOH/
H2O (3:1, v/v), 50 mM ammonium acetate in MeOH/H2O
(3:1, v/v) adjusted to pH 9.0, and MeOH/H2O (3:1, v/v) adjusted
to pH 3.0 with H3PO4 were used for neutral, basic, and acidic
analytes, respectively, with a flow rate of 0.75 mL/min. The
HPLC method was validated using compounds 24 and 54, which
were found to have log P values of 1.9 and 2.3, respectively,
using the shake flask method (OECD Guideline 107).25 Many
alogrithms for calculation of log P values experience difficulty
with urea compounds. For example the r2 for the correlation
for the ClogP values and shake flask values (OECD Guidelines
107 and 122) was <0.3,25 while the correlation with the HPLC
method used herein was ∼0.7 (Figure S7).
Method A: Synthesis of Aryl and Alkyl Isocyanates. The
aniline or amine (1 mmol) was added to an ice cold, stirred
biphasic mixture of DCM (10 mL) and saturated sodium bicar-
bonate (10mL) or1 N NaOH (3 mL) inbrine(7 mL) where noted.
Stirring was stopped momentarily, triphosgene (0.37 equiv) in
DCM (1 mL) added via syringe to the lower DCM layer, and
stirring continued for 10 min. The DCM layer was removed and
filtered through a bed of magnesium sulfate. The filtrate was
evaporated to afford the corresponding isocyanate, which was
used without further purification.
Method B: Synthesis of Ureas via Isocyanate. The isocyanate
(1 mmol) was dissolved or suspended in dry THF (3-5 mL) and
cooled in an ice bath. The amine (1 mmol) was dissolved in dry
THF (1 mL) and slowly added to the reaction. Stirring was
continued for 1-24 h at room temperature. The reaction was
quenched with dilute HCl (or water where the BOC group was
present) and extracted into ethyl acetate. The combined organic
phase was dried, evaporated, and purified.
Method C: Synthesis of Ureas via 4-Nitrophenylcarbamate. To
an ice cold solution of 4-nitrophenyl chloroformate (1 equiv) in

7074 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Rose et al.
ion trap and a Turbo V ion source and was operated in positive
ion MRM mode (see Table S1). The solvent system consisted
of water/acetic acid (999/1 v/v, solvent A) and acetonitrile/
acetic acid (999/1 v/v, solvent B). The gradient was begun at
30% solvent B and was linearly increased to 100% solvent B in
5 min. This was maintained for 3 min, then returned to 30%
solvent B in 2 min. The flow rate was 0.4 mL/min. The injection
volume was 10 μL, and the samples were kept at 4 °C in the
autosampler. Optimized conditions for mass spectrometry are
in Table S1.
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Acknowledgment. We thank Dr. Joszef Lango for assis-
tance with accurate mass determination. This work was
supported in part by NIEHS Grant R01 ES002710, NIEHS/
Superfund Basic Research Program Grant P42 ES004699,
and NIH/NHLBI Grant R01 HL059699. B.D.H. is a George
and Judy Senior Fellowofthe American Asthma Foundation.
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Supporting Information Available: Blood PK profiles, mass
spectrometer parameters, figures of exposure/potency ratios for
selected compounds, correlation between IC50 values for human
and murine sEH, correlation between experimental and ClogP
values, conditions and fragmentation patterns for mass spectro-
metric analysis, cumulative table of structures, results, and pro-
perties for all inhibitors presented, and synthetic details and
analytical data. This material is available free of charge via the
Internet at http://pubs.acs.org.

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