Optimized inhibitors of soluble epoxide hydrolase improve in vitro target residence time and in vivo efficacy

Article abstract of DOI:10.1021/jm500694p

Diabetes is affecting the life of millions of people. A large proportion of diabetic patients suffer from severe complications such as neuropathic pain, and current treatments for these complications have deleterious side effects. Thus, alternate therapeu

Full text of DOI:10.1021/jm500694p

Article
pubs.acs.org/jmc
Open Access on 07/31/2015
Optimized Inhibitors of Soluble Epoxide Hydrolase Improve in Vitro
Target Residence Time and in Vivo Efficacy
Kin Sing Stephen Lee,† Jun-Yan Liu,† Karen M. Wagner,† Svetlana Pakhomova,∥ Hua Dong,†
Christophe Morisseau,† Samuel H. Fu,† Jun Yang,† Peng Wang,†,§ Arzu Ulu,† Christina A. Mate,†
Long V. Nguyen,† Sung Hee Hwang,† Matthew L. Edin,⊥ Alexandria A. Mara,⊥ Heike Wulff,‡
Marcia E. Newcomer,∥ Darryl C. Zeldin,⊥ and Bruce D. Hammock*,†
†Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, One Shields
Avenue, Davis, California 95616, United States
‡Department of Pharmacology, University of California Davis, Davis, California 95616, United States
§Department of Applied Chemistry, China Agricultural University, Beijing 100193, People’s Republic of China
∥Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, United States
⊥Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research
Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: Diabetes is affecting the life of millions of
people. A large proportion of diabetic patients suffer from
severe complications such as neuropathic pain, and current
treatments for these complications have deleterious side
effects. Thus, alternate therapeutic strategies are needed.
Recently, the elevation of epoxy-fatty acids through inhibition
of soluble epoxide hydrolase (sEH) was shown to reduce
diabetic neuropathic pain in rodents. In this report, we
describe a series of newly synthesized sEH inhibitors with at
least 5-fold higher potency and doubled residence time inside both the human and rodent sEH enzyme than previously reported
inhibitors. These inhibitors also have better physical properties and optimized pharmacokinetic profiles. The optimized inhibitor
selected from this new series displayed improved efficacy of almost 10-fold in relieving pain perception in diabetic neuropathic
rats as compared to the approved drug, gabapentin, and previously published sEH inhibitors. Therefore, these new sEH inhibitors
could be an attractive alternative to treat diabetic neuropathy in humans.
reduced side effects is still imperative for these patients often
suffering multiple comorbid conditions.
INTRODUCTION
■
A recent survey from the Centers for Disease Control and
Prevention indicates that diabetes affects 25.8 million people in
the United States which is 8.3% of the U.S. population.1 Most
diabetic patients will ultimately develop kidney failure,
hypertension, and/or suffer stroke. In addition, about two-
thirds of diabetic patients will develop peripheral neuropathy.2,3
People suffering from diabetic neuropathic pain experience
spontaneous pain (pain sensation in the absence of
stimulation), hyperalgesia (increased pain sensation to painful
stimuli), and allodynia (pain sensation to innocuous stimuli),
which greatly affect their quality of life. Hyperglycemia has been
suggested to be the initiating cause of peripheral nerve fiber
degeneration, which results in pain.4 However, aggressive
glycemic control can only control the progression of neuronal
degeneration but not reverse the neuropathy.4 Current
treatments of diabetic neuropathy include tricyclic antidepres-
sants, anticonvulsants, selective serotonin reuptake inhibitors,
and opioids, however they often have side effects that limit their
use.5 Therefore, an alternative therapy with no or greatly
Epoxy fatty acids (EpFAs), formed by cytochrome P450
(CYP450) from polyunsaturated fatty acids, are important lipid
mediators.6 Epoxy-eicosatrienoic acids (EETs), epoxy-eicosate-
traenoic acids (EpETEs), and epoxy-docosapentaenoic acids
(EpDPEs), from arachidonic acid, eicosapentaenoic acid, and
docosahexaenoic acid, respectively, have analgesic properties in
inflammatory pain models.7,8 Although these EpFAs are very
potent lipid mediators, they are rapidly metabolized by soluble
epoxide hydrolase (sEH EC 3.3.2.10) to their corresponding
1,2-diols and to a lesser extent by other enzymes in vivo.9 The
in vivo biological activities of these natural chemical mediators
appear limited by their rapid degradation. Stabilization of
EpFAs through inhibition of sEH has shown anti-inflammatory,
antihypertensive, and analgesic effects. Recent studies also
indicate that sEH inhibition is analgesic in chronic diabetic
Received: May 5, 2014
© XXXX American Chemical Society
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Figure 1. (A) The general scaffold of sEH inhibitors used in this report. (B) The structure of inhibitor 18 (TPPU/UC1770). (C) The name
structures of previously published inhibitors.
neuropathic pain in animal models. In fact, it is more efficacious
than gabapentin, a clinically approved drug for this con-
dition.10,11 In nonmodel species, sEH inhibitors have reduced
the inflammatory and devastating neuropathic pain in laminitis
horses,12 reduced blood pressure in forearm blood flow studies
in man,13 and reduced neuropathic pain in human diabetics
(www.sphaerapharma.com). Thus, sEH is a potentially
important pharmaceutical target.6,8,9,12,14−20
concentrations of the ligand and the target are constant.
However, an in vivo system is an open system, thus the target is
exposed to a varying concentration of ligand after dosing
because of circulation, metabolism, and excretion. Therefore,
drug efficacy is no longer correlated with the in vitro potency
(IC50 or Ki) that is determined in a closed system but rather
depends on the duration the target is occupied by the ligand.
This residence time can be calculated from the reciprocal of the
dissociation rate constant (koff) of the target−ligand complex.
In this report, inhibitors with improved potency have been
designed and synthesized based on the holo-structure of the
recombinant human sEH with published sEH inhibitor: TPPU
(UC1770)23 (Figure 1B). Their residence times have been
determined and their in vivo efficacies have been tested in a
diabetic neuropathic pain model.
Over the years, several groups have reported the synthesis
and evaluation of sEH inhibitors with different central
pharmacophores with potency varying from micromolar to
nanomolar ranges.21−27 The 1,3-disubstituted urea is one of the
more potent central pharmacophores being used to inhibit sEH
because the urea forms tight hydrogen bonds with the active
residue Asp335 and the chemistry is easily accessible.21,23,28−30
The physical properties of many of the most potent
compounds are generally poor. Efforts to improve physical
properties including water solubility, hydrophilicity, decreased
clogP, and lowered melting point of sEH inhibitors have
generally resulted in a decrease in potency and less desirable
pharmacokinetics. These physical properties can also result in
poor absorption and inferior pharmacokinetic properties and
can demand heroic formulation.26,30−32 Therefore, it is
necessary to further optimize the structures of the inhibitors
and improve the oral bioavailability of the sEH inhibitors
carrying a 1,3-disubstituted urea as a central pharmacophores.
Recent reports in drug discovery suggest that the residence
time of a drug in its target is an important parameter to predict
in vivo drug efficacy.33 Residence time is defined as the
duration of time which the target, either enzyme or receptor, is
occupied by the ligand.33 The traditional IC50 and Ki or Kd is
determined in a closed in vitro system in which the
RESULTS AND DISCUSSION
■
Inhibitor Design and Synthesis. Piperidyl-urea inhibitors
of sEH were first described in 2006 and have the general
scaffold shown in Figure 1A.23,32 It has been demonstrated that
large amide substituents at R2 improve the potency of the sEH
inhibitors (Figure 1A).23 However, sEH inhibitors with large
amide substituents at R2 (Figure 1A) exhibit poor pharmaco-
kinetic profiles in dogs.34 To further improve both the potency
and pharmacokinetic profiles of the sEH inhibitors, we started
with a published inhibitor having a small substituent at R2:
TPPU (inhibitor 18) (Figure 1B), which resulted in good
potency and a good pharmacokinetic profile. Because of the
favorable properties of inhibitor 18, an X-ray structure of
human sEH with inhibitor 18 was obtained and used to predict
structural modifications of inhibitors leading to improved
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Figure 2. (A) Holo-crystal structure of human sEH (green) with inhibitor 18 (TPPU) (cyan) (PDB code: 4OD0). (B) The left side of the tunnel of
human sEH with inhibitor 18 (cyan). The arrow indicated the valley of the left side of the tunnel for potential additional binding for new inhibitors.
(C,D) The right binding pocket of human sEH with UC1770 from the view of the front and back (cyan). The graphics were prepared by the
PyMOL Molecular Graphics System, version 1.3 edu, Schrodinger, LCC.
potency and properties. The X-ray structure indicates that there
is a small secondary binding site (valley) next to the α-carbon
of the amide of inhibitor 18 (Figure 2A,B), which can provide
additional binding possibilities. We therefore hypothesized that
the potency of the inhibitors could be significantly improved by
incorporating a small hydrophobic substituent at the α-carbon
of the amide (Figure 2B). In addition, the 4-trifluoromethox-
yphenyl− group on the urea of inhibitor 18 fits closely to the
“right side” of the binding pocket with only a little room for
additional binding (Figure 2C,D). Thus, only substituents
which are similar to the size of the 4-trifluoromethoxyphenyl−
group were used for inhibitor optimization. On the basis of
these findings, a series of 30 piperidyl-ureas was made using
four different methods described previously.23 These methods
with slight modifications are summarized in Scheme 1 (detailed
synthetic procedures are provided in the Supporting
Information).
Optimization of the Potency (Ki) of sEH Inhibitors. A
new series of sEH inhibitors was synthesized with various
substituents at R2 on the amide (Table 1) while maintaining R1
as a 4-trifluoromethylphenyl− group. In general, as the size of
the substituent increases, the potency of the inhibitors against
human sEH increases. As we hypothesized, addition of a
methyl− group on the α-carbon of the amide (Figure 1A)
greatly improves the potency by almost 5-fold (inhibitors 2 vs 4
and inhibitors 3 vs 6). Interestingly, replacing the isopropyl−
(4) with cyclopropyl− group (5) also results in a slight increase
in potency, an increase in water solubility, and a decrease in
melting point. The elongation of the aliphatic chain by one
carbon (inhibitors 2 vs 3) slightly enhances (30%) the potency
(Table 1). To investigate whether the enhanced potency of
inhibitor 6 is stereospecific, we tested the corresponding S-
isomer (inhibitor 7), which is 2-fold more potent than its
racemic mixture (Table 1), suggesting that the R-isomer is less
active. The related inhibitor 8 is less potent than inhibitors 6
and 7, which indicates that the addition of methyl− group not
only is stereospecific but is also regiospecific. An X-ray structure
of human sEH with inhibitor 4 shows that the methyl− group
on the α-carbon of the amide of inhibitor 4 effectively fits into
the additional binding site on the right side of the binding
pocket (Figure 3). This additional binding site was predicted
from the crystal structure of human sEH with inhibitor 18
(Figure 2B).
Although the addition of an alkyl group enhances the
potency of this series of sEH inhibitors, their metabolic stability
will likely decrease because the added alkyl group at the α-
position is expected to be metabolized faster. This also
increases the lipophilicity of the inhibitors, which may increase
their affinity to CYP450 enzymes that are responsible for drug
oxidation.35 Because fluoride replacement is known to decrease
metabolism,36 we synthesized inhibitor 9,36 and it shows
potency similar to inhibitor 8. The size of the CF3− group is
similar to the size of the isopropyl−, group and the potency of
inhibitor 9 is maintained. This could provide an approach to
increase the stability of the inhibitors without hampering the
potency of the inhibitors.
We then further optimized the potency of sEH inhibitors by
modifying the substituent at R1 while maintaining the 2-methyl
butanoyl group at R2 (Table 2). The binding pocket is very
hydrophobic (Supporting Information, Figure S2) with limited
space (Figure 2C,D). Therefore, the size of R1 was modified
slightly in order to test if increasing the hydrophobic surface of
the inhibitors could enhance their potency. Consequently,
inhibitors 11 and 12 were synthesized (Table 2). The result
indicates that there is steric hindrance within the right binding
pocket (Figure 2C,D). Thus, the potency of the inhibitor
dropped when the size of the substituent at R1 (Figure 1A) was
increased from a 4-iso-propylphenyl group to a 4-tert-
butylphenyl- group (inhibitors 11 and 12) (Table 2). It is
interesting that the inhibitor 6, which has CF3− group in place
of an iso-propyl− and tert-butyl− group,37 is more potent as an
inhibitor when compared to inhibitors 11 and 12. To
C
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Scheme 1. Synthetic Schemes for sEH Inhibitors Synthesis
investigate whether the enhanced potency of inhibitor 6 is due
to fluorine induced interactions or better occupancy of the
binding pocket, the 4-t-butylphenyl− group of inhibitor 12 was
replaced by its isostere: a 4-heptafluoro-iso-propyl-phenyl group
at R1 of inhibitor 13.37 This compound is 20-fold more potent
than inhibitor 6, which indicates that the fluorine induced
interactions in the left binding pocket are very strong (Table 2).
These data suggest that the left side of the binding pocket is
likely fluorophilic. Interestingly, replacement of the 4-
trifluoromethylphenyl− at R1 of inhibitor 6 by a 4-
trifluoromethoxyphenyl− group (inhibitor 14) did not alter
potency, but the replacement improved solubility by 10-fold
(Table 1). Furthermore, inhibitors with a cycloalkyl− group at
R1 were synthesized, and the addition of carbon atoms
(inhibitor 15 to 17) enhances the binding toward human
sEH by more than 4 times (Table 2) and placement of
cycloalkyl− group at R1 greatly enhances their solubility.
It has been reported that inhibitors with 4-trifluoromethox-
yphenyl− at R1 are more potent than inhibitors with 4-
trifluoromethylphenyl− at R1 and have better physical proper-
ties.23 To investigate whether such substitution could enhance
the potency of the inhibitors and improve physical properties in
general,38 a series of inhibitors with 4-trifluoromethylphenyl
group at R1 was replaced with a 4-trifluoromethoxyphenyl−
group (Figure 1A). The results suggest that substitution of 4-
trifluoromethylphenyl− with 4-trifluoromethoxyphenyl− en-
hances the potency of certain inhibitors (2 vs 18 and 4 vs 19 in
Tables 1 and 3), but there is no substantial improvement with
inhibitors with the 2-methyl butanoyl group at R2 (6 vs 14 and
7 vs 21 in Table 1−3) or the cyclopropyl group at R2 (5 Vs
20). This may be due to the possibility of different binding
orientations than the one shown in Figure 2A and in previous
crystallography.39 Therefore, two inhibitors which are structur-
ally similar to 21 but with urea substituted by the amide (22
and 23) were synthesized. In general, structure−activity
relationships developed with the urea central pharmacophore
are predictive of enzyme inhibition with amide and carbamate.
Both inhibitors 22 and 23 are less potent than their
corresponding urea (21) (Table 3). Compound 22, which
has amide nitrogen (N″H) on the piperidine side (Figure 1A),
is far less potent than the amide 23, which has an amide
nitrogen (N′H) on the R1 side (Figure 1A) (Table 2). It
indicates that the urea N′H−Asp(O) interaction is stronger
than the urea N″H−Asp(O) interaction by 5.7 kJ mol−1,
calculated based on the Ki of both inhibitors. This result is
consistent with the crystal structure where the distance between
urea N′H and Asp(O) is shorter than the distance between
urea N″H and Asp(O) hydrogen bonding (Figure 2A). In some
cases, the placement of 4-trifluoromethoxyphenyl− at R1
(Figure 1A) enhances the potency of sEH inhibitors, but in
D
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e
Table 1. Physical Properties and Potency of sEH Inhibitors against Human sEH (Modification of R2)
a
Solubility was measured with sodium phosphate buffer (0.1 M, pH 7.4) according to the method described by Tsai et al. and described in detail in
Supporting Information.34 N.D. means “not determined”. elogP was determined by HPLC method calibrated with elogP of six selected inhibitors
b
c
d
determined by the shake-flask method (Supporting Information, Figure S3). Ki was determined by FRET-based displacement assay described by
Lee et al.38 The results are the average of duplicates with SEM. Abbreviation: elogP stands for experimental Log P.
e
It was reported that replacement of amide with sulfonamide
at R2 was shown to enhance the potency of the inhibitors.
However, such observations were based on very few
comparisons.23,34 Therefore, a new series of inhibitors with a
different sulfonamide at R2 was synthesized for a more detailed
study (Table 4). In general, the potency of the inhibitors
increases with the size of R2 (7-fold better potency from a
methyl 25 to a butyl 31). However, unlike the inhibitor with an
amide at R2 (Table 1), the placement of an iso-propylsulfona-
mide at R2 (29 vs 26) does not significantly enhance the
potency against human sEH. This is probably because the
sulfonamide exists as a tetrahedron while amide is trigonal
planar and the S(O)2−C bond is at least 0.28 Å longer than the
C(O)−C bond.40 These data further indicate that the enhanced
potency of the methyl substitution at the α-position of the
amide is structurally specific. Overall, unlike previously
Figure 3. Overlay structures of human sEH with inhibitor 18 (cyan)
reported, the inhibitors with a sulfonamide at R2 are less
potent than the inhibitors with amides at R2. In addition, the
physical properties (solubility and melting point) of inhibitors
with a sulfonamide at R2 are generally poor as compared to
inhibitors with amides at R2.
and inhibitor 4 (orange). This figure suggests that the design principle
is valid and the methyl− group at α-position of the amide provides
extra binding toward the valley of the left binding pocket. The graphics
were prepared by the PyMOL Molecular Graphics System, version 1.3
edu, Schrodinger, LCC.
Here, we have identified several structural changes that can
significant enhance the potency and improve the physical
properties of the inhibitors. We have also demonstrated that the
sulfonamide at R2 is less attractive than the corresponding
amide.
all cases, this substitution decreases the melting point and
improves the solubility of the inhibitors (Tables 1−3). These
improvements of physical properties ease the drug formulation
process.
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Table 2. Physical Properties and Potency of sEH Inhibitors against Human sEH (Modification of R1)
a
Solubility was measured with sodium phosphate buffer (0.1 M, pH 7.4) according to the method described by Tsai et al. and described in detail in
Supporting Information.34 N.D. means “not determined”. elogP was determined by HPLC method calibrated with elogP of six selected inhibitors
b
c
d
determined by the shake-flask method (Supporting Information, Figure S3). Ki was determined by FRET-based displacement assay described by
Lee et al.38 The results are the average of duplicates with SEM. Abbreviation: elogP stands for experimental Log P.
e
New sEH Inhibitors Have Improved Dissociation Rate
Constants (koff) against Human sEH. Recent studies have
suggested that koff, a kinetic parameter on enzyme inhibition,
from the enzyme is a better indicator for in vivo potency than
Ki.33 This is because inhibitors are only effective in blocking
catalysis when the target proteins are occupied by the
inhibitors. The koff can provide more detailed information,
about the duration of time the inhibitors are bound to the
target enzyme (target occupancy) than the Ki, an equilibrium
parameter on enzyme inhibition, and ultimately this translates
into in vivo efficacy.40 Therefore, a small set of new potent
inhibitors together with several potent published inhibitors
(Figure 1C) were selected to determine their koff against sEH
using a recently developed FRET-based assay.38
The result indicates that the koff of inhibitors decreases with
the size of R2 increased (Table 5 and Figure 1C, inhibitor 4 < 7;
TPAU < 18 (TPPU) < 19 < 21; and TUPS < 32). However,
the koff does not substantially change when the R1 was varied
among a 4-trifluoromethylphenyl− group (inhibitor 6), a 4-
trifluoromethoxyphenyl− group (inhibitor 14), and a 4-
isopropylphenyl− group (inhibitor 11) (Table 5). The 4-
heptafluoroisopropylphenyl− group at R1 (inhibitor 13 and
inhibitor 24) significantly decreased the koff of the inhibitors.
This further supports our hypothesis that the pocket is
fluorophilic and the added fluorines can induce several
interactions with the nearby residues within the binding
pocket. The koffs of new inhibitors are slower overall, indicating
a longer residence time in the target and have at least a 2-fold
slower off rate than any of the previously published inhibitors
(Table 5 and Figure 1C, APAU, TPAU, 18 (TPPU), TUPS, t-
TUCB).
To investigate relationship of koff with Ki and other physical
properties of the inhibitors, correlation graphs of koff with these
parameters were plotted (Supporting Information, Figure S1A).
The results show no correlation (R2 = 0.21) between the
number of non-hydrogen atoms and koff. However, there is a
trend showing (R2 = 0.52) that an increase in elogP results in a
decrease of koff (Supporting Information, Figure S1B). This
may due to the fact that the binding pocket of the human sEH
is hydrophobic (Supporting Information, Figure S2). An
increase of hydrophobicity of the ligand increases the lipophilic
interactions with the binding pocket. Therefore, it requires
higher activation energy to break up the interactions between
protein and the bound inhibitors. In addition, a correlation
between Ki and koff was plotted (Supporting Information,
Figures S1C, S1D). The plot indicates that there is a good
correlation between Ki and koff (R2 = 0.88) over a wide range of
potencies (from 0 to 20 nM). However, when we focused on a
narrower range of potencies (from 0 to 1.4 nM), the correlation
is moderate (R2 = 0.44). Because Ki is a ratio of koff over kon, Ki
should be inversely proportional to koff. Therefore, the poor
correlation over a moderate range Ki values suggests that the
differences are due to the kon. These results indicate that it is
possible to specifically modulate koff without greatly affecting Ki.
An Improved Pharmacokinetic Profile of New Series
of sEH Inhibitors. To investigate the oral bioavailability of
sEH inhibitors, we determined the pharmacokinetic profiles
post oral administration. Cassette dosing was used as a
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Table 3. Physical Properties and Potency of sEH Inhibitors against Human sEH (Modification of R1)
a
Solubility was measured with sodium phosphate buffer (0.1 M, pH 7.4) according to the method described by Tsai et al. and described in detail in
Supporting Information.34 N.D. means “not determined”. elogP was determined by HPLC method calibrated with elogP of six selected inhibitors
b
c
d
determined by the shake-flask method (Supporting Information, Figure S3). Ki was determined by FRET-based displacement assay described by
Lee et al.38 The results are the average of duplicates with SEM. Abbreviation: elogP stands for experimental Log P.
e
screening tool to select the compounds for more detailed study
to allocate limited resources to the most promising compounds.
There are cautions with cassette dosing including changes in
pharmacokinetic behavior due to competition for xenobiotic
metabolism. The high potency and thus low doses used in this
study make this artifact less likely. A comparison of
pharmacokinetic profiles of sEH inhibitors between the cassette
dose and single dose carried out in nonhuman primate. The
results show that there was no statistically significant difference
in the pharmacokinetic behaviors between cassette and
individual dosing of sEH inhibitors; therefore, cassette dosing
was predictive for the compounds reported here.41
The pharmacokinetic profiles of this series of selected potent
compounds also determined the effect of the position or
addition of aliphatic carbons on the inhibitors. In general, the
results indicate that the sEH inhibitors with a 4-trifluorome-
thylphenyl− group or a 4-trifluoromethoxyphenyl− group at R1
(Figure 1A) have good drug exposure levels based on the area
under the curve of the pharmacokinetic kinetic profile (PK-
AUC) after oral administration (Table 6). As we replaced the
4-trifluoromethylphenyl− group or 4-trifluoromethoxyphenyl−
group at R1 with a 4-isopropylphenyl− group (inhibitors 6 or
14 vs 11), the potency of inhibitor 14 decreases (Table 2). As
we anticipated, the pharmacokinetic T1/2 (PK-T1/2) and oral
drug exposure level estimated by PK-AUC also decreases
(Table 6). Further replacement of the phenyl group at R1 with
a cycloalkyl− group (inhibitors 16 and 17) greatly decreases
the oral drug exposure level (Table 6, Supporting Information,
Figures S6, S8, and S9). It is likely that each addition of an
aliphatic carbon renders the molecule more susceptible to
metabolism by CYP450 enzymes.42 Such a phenomenon was
also observed as the alkyl chain length varied at R2. As we
hypothesized, the PK-T1/2 of the inhibitors also decrease when
the alkyl chain length at R2 of the amide and sulfonamide series
increases (Table 6 for amide inhibitors, 18, 19, and 21; for
sulfonamide inhibitors, 25 and TUPS (Figure 1C) vs 31 and
32). The result also show that the PK-AUC estimated drug
exposure levels of the sulfonamide inhibitors 31 and 32 are, in
general, worse than the amide inhibitors (Table 6).
We then synthesized inhibitors 9 and 24 in order to study
whether the addition of fluorine could enhance the stability of
our inhibitors as has been previously suggested.36 The
replacement of a terminal methyl group with a CF3− group
at the R2 of the amide inhibitors (inhibitors 9 vs TPPU and 24
vs 13) not only improves the PK-T1/2 of the inhibitors but also
increases their oral drug exposure levels (Table 6). A similar
result was obtained with the sulfonamide inhibitor. Inhibitor 30
shows at least a 20-fold better bioavailability based on oral PK-
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Table 4. Physical Properties and Potency of sEH Inhibitors against Human sEH (Modification of R1 and R2 with sulfonamide at
e
R2)
a
Solubility was measured with sodium phosphate buffer (0.1 M, pH 7.4) according to the method described by Tsai et al. and described in detail in
Supporting Information.34 N.D. means “not determined”. elogP was determined by HPLC method calibrated with elogP of six selected inhibitors
b
c
d
determined by the shake-flask method (Supporting Information, Figure S3). Ki was determined by FRET-based displacement assay described by
Lee et al.38 The results are the average of duplicates with SEM. Abbreviation: Sol. stands for Solubility; elogP stands for experimental Log P.
e
Table 5. Study in Vitro Target Occupancy (koff) of Selected sEH Inhibitors against Human sEH
a
b
a
b
entry
inhibitor
APAU
koff (× 10−4 s−1
)
t1/2 (min)
entry
inhibitor
17
koff (× 10−4 s−1
)
t1/2 (min)
1
2
3
4
5
6
7
8
9
10
19.2 0.70
10.5 0.20
6.57 0.30
7.91 0.31
5.76 0.26
5.19 0.09
4.75 0.11
5.79 0.43
3.13 0.06
5.39 0.39
6.03 0.2
11.0 0.2
17.6 0.8
14.6 0.6
20.1 0.9
22.3 0.4
24.3 0.6
20.0 1.5
37.0 0.7
21.5 1.6
11
12
13
14
15
16
17
18
19
20
3.51 0.20
6.14 0.18
5.05 0.02
4.39 0.43
23.1 1.1
10.3 0.1
8.90 0.35
26.4 2.30
20.0 0.64
7.19 0.36
33.0 1.9
18.8 0.6
22.9 0.1
26.5 2.6
5.02 0.3
11.0 0.1
13.0 0.5
4.40 0.4
5.79 0.2
16.1 0.8
18 (TPPU)
19
4
21
5
24
6
25
7
31
9
32
11
13
14
33 (TPAU)
TUPS
t-TUCB
a
koff was determined by FRET-based displacement assay described by Lee et al.38 and described in detail in Supporting Information. Brieftly, a
preincubated human sEH−inhibitor complex (8 μM) was diluted by ×40 times by fluorescent reporter−APCU (2 μM, 0.1 M sodium phosphate, pH
7.4). The fluorescent enhancement (λexcit = 280 nm, λemit = 450) was measured over time (5100 s). The results are the average of triplicates with
b
SD t1/2 = ln(2)/koff, which describes the half-life of enzyme−inhibitor complex.
AUC with a substantially longer PK-T1/2 as compared to
inhibitors 31 and 32 (Table 6). These data support the
hypothesis that the CF3− substitution effectively blocks
metabolism at R2. However, addition of fluorine to the
fluorinated substituents could not further enhance the stability
of the inhibitors. When the inhibitors with 4-trifluoromethyl−
H
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because it is associated with poor pharmacokinetic behavior and
nontargeted related side effects. Although not conclusive, the
pharmacokinetic studies indicated that the 4-(heptafluoro-iso-
propyl-)phenyl group likely enhances the affinity of inhibitors
toward xenobiotic metabolizing enzymes and cell membranes,
potentially leading to the poor oral drug exposure of inhibitor
13 (Table 6).
It was reported that very high plasma protein binding can not
only hamper the efficacy of drugs or signaling molecules but
also can alter their pharmacokinetic profile.44,45 However,
modulating plasma protein binding can help drug solubilization
and drug distribution.46 Therefore, the plasma protein binding
of the selected inhibitors were measured with an assay carried
out with a rapid equilibrium dialysis device based on
manufacturer’s protocol (Table 7). These new inhibitors
Table 6. Pharmacokinetic Parameters of Selected sEH
Inhibitors after Oral Dosing on Mice
a
a
b
c
b
c
inhibitor
PK-AUC (nM·h)
Cmax (nM)
T1/2 (h) Tmax(h)
APAU10
183
5300
10650
19650
24630
3295
4940
8845
200
30
370
495
1160
960
320
435
330
90
3
15
12.1
6
1
TUPS18
3.3
8
18 (TPPU)
4
4
5
17
6
6
6
0.5
1
7
5
9
15.4
2
8
11
13
14
16
17
19
21
24
25
30
31
32
0.5
2.5
2
1600
3300
75
135
235
3
4.5
6
8.4
12
9
0.5
0.5
5
37
5
Table 7. Testing for Nonspecific Protein Binding with
Selected sEH Inhibitors
19500
2530
9600
4675
4270
245
900
195
365
130
100
30
7.7
18.9
22
25
7.4
2
4
human
plasma
protein
human
CYP2C
8
hERG
human CYP
2J remaining
8
channel
remaining
8
inhibition at binding at 1 activity at 10 activity at 10
a
a
b,
d
b,d
inhibitor
APAU
50 μM (%)
μM (%)
μM (%)
μM (%)
1
220
65
1.2
4.5 0.5
50.0 2.0
79.0 1.0
85.0 0.5
95.1 0.5
96.3 0.2
89.0 2.5
92.4 0.1
140.0 5.0
91.9 2.2
81.6 3.0
104.0 1.0
118.0 2.4
110.0 2.0
a
18 (TPPU)
26.0 1.0
16.5 1.5
41.5 1.5
The mice (n = 4) were treated by oral dosing with a cassette of 3 to 5
4
compounds (0.3 mg/kg per each compounds dissolved in 20%
b
c
c
PEG400 in oleic acid rich triglycerides). The pharmacokinetic profiles
5
N.D.
N.D.
c
of the inhibitors were calculated by Winonlin based on the model of
7
N.D.
82.6 3.1
73.4 0.3
60.6 8.2
107.0 3.0
103.0 1.0
108.0 1.0
c
one compartmental analysis. The pharmacokinetic profiles of the
19
21
32.5 1.5
c
inhibitors were calculated by Winonlin based on the model of
noncompartmental analysis.
N.D.
a
The result is the average of duplicates with standard error shown.
The result is the average of triplicates with standard derivation
b
groups at R1 were substituted by a heptafluoro-isopropyl−
group (inhibitor 6 vs 13), both the PK-T1/2 and bioavailability
decreased (Table 6). This is probably due to the fact that the
increase of elogP enhances the affinity toward xenobiotic
metabolizing enzymes and cell membranes. These results
suggest that in order to enhance the stability of the inhibitors,
addition of fluorine should be carefully positioned.35,43
In general, inhibitors with 4-trifluoromethylphenyl−, 4-
trifluoromethoxyphenyl−, and 4-(heptafluoro-iso-propyl)-
phenyl− groups at R1 with amide substituents at R2 showed
good pharmacokinetic profiles. Inhibitors 16, 17, 31, and 32,
although very potent, are not optimal candidates for testing in
animal chronic disease models because of poor oral drug
exposure levels. Our data also suggest that these inhibitors (16,
17, 31, and 32) may not be top candidates to be optimized for
human drugs. The inhibitors with a piperidyl moiety attached at
N2 of the urea show greatly improved oral pharmacokinetic
profiles compared to inhibitors with a cyclohexyl moiety
attached at N2 of the urea also (Supporting Information, Figure
S6).30 Overall, our data suggested that several newly
synthesized sEH inhibitors have an improved and optimized
pharmacokinetic profile for diseases requiring chronic treat-
ment.
c
d
shown. N.D. means “not determined”. The experiments were
conducted according to the Graves et al. procedure.50
demonstrated a moderate level of plasma protein binding
ranging from 85 to 96%. This level of plasma protein binding is
unlikely to alter their bioavailability. Given the inhibitors had
Kis approaching the subnanomolar range, the plasma proteins
could be considered as carrier proteins that facilitate the their
distribution.
Previously, sEH inhibitors with similar structures failed to
show significant inhibition of the CYP450s which are highly
involved in xenobiotic metabolism.47 In this study, we
examined the inhibition of CYP2C and CYP2J2 because
CYP2C is an important drug metabolizing enzyme but
principally because both enzymes are implicated in the
synthesis of epoxy fatty acids.48−50 The results indicated that
only minor inhibition of both CYP450 enzymes is observed at
10 μM of the sEH inhibitors (Table 7). These data suggested
that the sEH inhibitors do not affect the biosynthesis of EpFAs
by inhibition of CYP2C and CYP2J2. This in turn suggests that
the in vivo efficacy of the inhibitors is not due to inhibition
upstream in the CYP450 pathway.
New Series of sEH Inhibitors Show No Significant
Nonspecific Binding toward Other Pharmacologically
Important Proteins. Several of the newly synthesized sEH
inhibitors selected for potency and good oral drug exposure
levels were tested against a set of proteins to evaluate potential
off-target side effects and nonspecific binding. Inhibitor 24 was
excluded because of its relatively high elogP (Table 3), which
was suspected to lead to high nonspecific binding. High
lipophilicity is avoided when possible in medicinal chemistry
Inhibition of the hERG channel is a very important
toxicology screen due to demonstrated association with
cardiotoxicity. Most of the tested inhibitors show very minor
inhibition on hERG at 50 μM except for inhibitors 5 and 19
(Table 7). However, even inhibitors 5 and 19, which have 0.49
and 0.31 nM Ki against recombinant human sEH, have more
than an 10000-fold selectivity over the hERG channel.
Therefore, these selected inhibitors are considered not to
present a risk with hERG inhibition.
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In summary, we have demonstrated that our sEH inhibitors
having Ki value in the lower subnanomolar are unlikely to
induce unanticipated side effects due to nonspecific binding to
other pharmacologically important proteins. However, this does
not completely rule out idiopathic off-target effects.
sEH Inhibitors Are Effective against an Animal Model
of Type 1 Diabetic Neuropathic Pain. Several new potent
sEH inhibitors with good pharmacokinetic profiles and no
significant nonspecific binding on human proteins and enzymes
were selected for further in vitro testing prior to in vivo studies.
Because the in vivo studies were to be conducted in rat, we
determined the potency (Ki and koff) against the recombinant
rat sEH (Table 8). The data suggest that the inhibitors have a
Table 9. Pharmacokinetic Parameters of Selected sEH
Inhibitors Followed by Oral Dosing on Rat
a
a
b
b
b
b
structure
PK-AUC (nM·h)
Cmax (nM)
T1/2 (h)
Tmax(h)
4
7304
5716
510
790
890
297
5.4
4.9
3.5
4.2
4
1
1
4
7
19
21
12000
2590
a
The rat (n = 3 or 4) were treated by oral dosing with a cassette of
four compounds (0.3 mg/kg per each compound dissolved in 20%
b
PEG400 in oleic acid rich triglycerides). The pharmacokinetic profiles
of the inhibitors were calculated by Winonlin based on the best fit
model of one compartmental analysis.
Table 8. In Vitro Protency of Selected sEH Inhibitors against
Rat sEH
pain behavior increases dose dependently from 0.1 to 0.3 mg/
kg (Figure 4A,B, p < 0.05). In addition, there is a good
correlation between the blood concentration of inhibitor 7 and
increased withdrawal thresholds (Figure 4C,D).
a
,
b
a,
c
a,d
inhibitor
IC50 (nM)
koff (× 10−4 s−1
)
t1/2 (min)
13.6 0.8
18 (TPPU)
29.1 4.5
79 2.5
1.8 0.1
<1.25
8.52 0.47
9.70 0.14
3.26 0.72
5.37 0.45
6.87 0.11
4.89 0.28
TPAU
11.9 0.18
36.6 8.2
21.6 1.8
16.8 0.3
23.7 1.4
Our data indicate that inhibitor 7 shows better in vitro
efficacy in terms of both IC50 and koff than the previously
published inhibitor TPAU (Figure 1C) as well as having good
pharmacokinetic profiles in both mouse and rat (Table 6 and
9). Therefore, we compared the in vivo efficacy of inhibitor 7 as
previously reported results for TPAU in the same model. Our
results indicate that using a 10-fold lower dose of inhibitor 7 is
as efficacious as TPAU in the nociceptive bioassay (Figure 5).11
It is also reassuring that the relative in vivo potency of these
two sEH inhibitors in the nociceptive assays correlates with
their relative in vitro potency on the target enzyme. These pain
assays in the rat appear valuable for broad ranking of the
analgesic activity of sEH inhibitors. However, the differences in
pharmacokinetic and target site affinities caution against
extrapolating these data to predict the fine ranking of the
potency of different sEH inhibitors in man.
4
7
19
21
13.3 1.5
20 0.1
a
The result is the average of triplicates with standard derivation (SD)
b
shown. IC50 is determined by radiometric assay using [3H]-t-DPPO
(50 μM) as a substrate and rat sEH (2.5 nM) incubated at 30 °C for
c
10 min koff was determined by FRET-based displacement assay
described by Lee et al.38 Briefly, a preincubated rat sEH−inhibitor
complex (8 μM) was diluted by ×40 times by fluorescent reporter−
APCU (2 μM, 0.1 M sodium phosphate, pH 7.4). The fluorescent
enhancement (λexcit = 280 nm, λemit = 450) was measured over time
d
(5100 s). The results are the average of triplicates with SD. t1/2
=
ln(2)/koff, which describes the half-life of enzyme−inhibitor complex.
slightly different structure−activity relationship (SAR) on the
recombinant rat sEH compared to human sEH. The inhibitors
with a 4-trifluoromethoxyphenyl− group at R1 are less potent
than the inhibitors with a 4-trifluoromethylphenyl− group at R1
by at least 10-fold (Table 8). These results are opposite to the
SAR obtained from the human recombinant sEH (Table 1 and
3). In addition, there is not a clear SAR based on the koff of the
inhibitors (Table 8). However, the results indicate that the new
inhibitors are more potent, both in IC50 and koff, than the
previously published inhibitors 18 (TPPU) and TPAU (Figure
1C and Table 8). Additionally, the pharmacokinetic profiles in
rat were obtained for this new set of inhibitors (Supporting
Information, Figure S11). All of the selected inhibitors show
good oral bioavailability (PK-AUC) except inhibitor 21, which
demonstrated at least 2× lower blood concentration than the
other inhibitors (Table 9). Elimination is an important
parameter of overall pharmacokinetics, and it has been
previously reported that inhibitors with short elimination PK-
T1/2 are not well suited to use for chronic treatment.51,52
Several of the new inhibitors demonstrate good pharmacoki-
netic profiles with moderate elimination PK-T1/2 (≥5 h) in
both mice and rats. Therefore, these inhibitors were selected for
further in vivo efficacy test on rat.
Overall, our data show that the new inhibitor 7 exhibits a
strong correlation between in vivo efficacy with dose and drug
level in blood, is more efficacious than the previously reported
TPAU, and has promise as a therapy for use in treating chronic
pain conditions.
CONCLUSION
■
Here, a new series of 1,3-disubstituentd urea sEH inhibitors was
synthesized with the design based on the recently obtained
holo-structure of human sEH with inhibitor 18 (TPPU). The
SAR of this new series indicates that the right side binding
pocket of the sEH enzyme has limited space for optimization
and is fluorophilic (Figures 1A, 2A,B), with an additional
binding site identified in the left side binding pocket (Figures
1A, 2D). The newly synthesized inhibitors are at least 10 times
more potent against human sEH than a previously published
inhibitor (TPAU, Figure 1C, Ki(human sEH) = 9.4 0.3 nM) with
a minimum 2-fold longer residence time on the human sEH.
These new inhibitors have been demonstrated selective for sEH
with no significant specific binding toward several other human
proteins (plasma protein, CYP450 enzymes, and hERG channel
protein). The poor physical properties limit the oral
bioavailability of inhibitors with a 1,3-disubstituted
urea.18,30,31,34 This new series of inhibitors has improved
physical properties translating into good pharmacokinetic
profiles in the rat and mouse (Supporting Information, Figure
S5−S11) increases their druglikeness compared to previous
sEH inhibitors.
Because inhibitor 7 has the best in vitro potency (IC50 and
koff) against rat sEH with a good pharmacokinetic profile
(Table 8 and 9), it was tested in a model of type 1 diabetic
neuropathic pain. At as low as 0.1 mg/kg, inhibitor 7 effectively
increases mechanical withdrawal thresholds (reduces pain) in
neuropathic rats (Figure 4A). Importantly, this reduction in
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Figure 5. Newly optimized inhibitor 7 shows better in vivo efficacy in
disease model. AUC describes an area under the curve of the
withdrawal threshold post-oral dosing of the diabetic rat with sEH
inhibitors vs time. A comparison of the newly synthesized sEH
inhibitor 7 (IC50 rat sEH = <1.25 nM, t1/2 = 21.6 min) to previously
published analogue TPAU (IC50 rat sEH = 79 nM, t1/2 = 11.9 min)
showed a significantly higher response of inhibitor 7 at 0.3 mg/kg
compared to the same 0.3 mg/kg dose of TPAU in a model of diabetic
neuropathy (t test, t = 2.31 with 9 degrees of freedom, p = 0.046, *
shows significant difference to 0.3 mpk of inhibitor 7). However, there
was no significant difference between the 10-fold lower dose of
inhibitor 7 at 0.3 mg/kg and TPAU at 3 mg/kg (Mann−Whitney rank
sum test, U = 14.00, n1 = 5 n2 = 6, ns p = 0.931). The graphic and
statistics were prepared by SigmaPlot (SysTat Software, San Jose, CA).
Figure 4. (A) Inhibitor 7 (IC50 rat sEH = <1.25 nM) improves
mechanical withdrawal thresholds in a model of diabetic neuropathy.
Oral dosing of 0.1 and 0.3 mg/kg dose dependently increased MWT,
indicating pain relief (n = 5, mean SEM). The neuropathic baseline
is normalized to 100% to show the response to a single dose of sEH
inhibitor over the time course. The response to treatments depended
on the time, but when the treatments were compared, there was a
statistically significant increase from the 0.1 to the 0.3 mg/kg dose
(Mann−Whitney rank sum test, U = 454.5, n1 = n2 = 45, p = <0.001).
(B) Inhibitor 7 shows dose dependent pain relief in diabetic rats. AUC
describes an area under the curve of the MWT post-oral dosing of the
inhibitor vs time. The bar chart depicts the AUC of MWTs after oral
dosing of inhibitor 7. An increased in AUC of MWT is interpreted as
an increase in pain relief. When the AUC for these doses were
compared, this relationship maintained statistical significance (Mann−
Whitney rank sum test, U = 2.00, n1 = n2 = 5, p = 0.032). (C) Efficacy
in nociceptive assays relates to blood concentration. A plot of the
nociceptive responses (n = 5, mean SEM) vs blood concentration (n
= 4, mean SEM) reveals increasing efficacy with increasing blood
concentration. (D) The efficacy of inhibitor 7 is dependent on blood
concentration. When compared, the nociceptive responses (n = 5,
mean SEM) and the pharmacokinetic profile after oral dosing (n =
4, mean SEM) of 7 follow the same trend while revealing a slight
delay to in the behavioral assay. The graphics and statistics were
prepared by KaleidaGraph version 4.1 (Synergy Software, Reading,
PA).
the selected inhibitor 7 demonstrated strong correlation
between drug level in blood and dose with in vivo efficacy.
This new series of inhibitors has demonstrated enhanced
potency with slow koff, improved pharmacokinetic profiles
(moderate to long elimination T1/2 and high AUC), and more
importantly, improved efficacy against diabetic neuropathic
pain in a rat model. With increased potency and bioavailability,
there are decreases in the required effective dose and greatly
simplified formulation. The new sEH inhibitors are good
candidates for chronic treatment of diabetic neuropathic pain.
EXPERIMENTAL SECTION
■
General. All reagents and solvent were purchased from commercial
suppliers and were used directly without further purifications. All
syntheses were carried out in a dry nitrogen atmosphere unless
otherwise specified. Reactions were monitored by thin-layer
chromatography (TLC) on Merck F254 silica gel 60 aluminum sheets,
and spots were either visible under light or UV light (254 mm) or
stained with an oxidizing solution (KMnO4 stain). The same TLC
system was used to test purity, and all final products showed a single
spot on TLC. Column chromatography was performed with silica gel.
1H NMR spectra were recorded on a Varian QE-300 spectrometer
with deuterated chloroform (CDCl3; δ = 7.24 ppm) or deuterated
dimethyl sulfoxide (DMSO-d6) containing TMS an internal standard.
13C NMR spectra were recorded on a Varian QE-300 spectrometer at
75 MHz.
Inhibiting sEH has been demonstrated to be more efficacious
than gabapentin and celecoxib in alleviating modeled diabetic
neuropathic pain in rat with no apparent side effect such as
impaired mobility, cognition, or motor skill.10,11 However, the
sEH inhibitors reported in those studies had either poor
physical properties (poor water solubility and high melting
point) or poor in vivo stability. In this report, the new more
potent sEH inhibitors are close to 10 times more efficacious
than TPAU in a diabetic neuropathic pain model. In addition,
The purity of the inhibitors reported in this manuscript was
determined either by (1) HPLC-UV using Agilent 1200 series HPLC
series equipped with Phenomenex Luna2 C18 reverse phase column
(C18, 4.6 mm × 150 mm, 5 μm) coupled with Agilent G1314 UV−vis
detector (detection at 230 nm) with isocratic flow at methanol:water
(2:1 by volume) for 90 min, or by (2) H NMR. The lowest obtained
purity was reported. The inhibitor was dissolved in EtOH at 100 μM
and 10 μL was injected on HPLC. Purity was based on the percent of
total peak area at 230 nm using HPLC-UV. This purity estimate was
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Journal of Medicinal Chemistry
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compared with that from the H NMR. The presence of anilines in the
final product was estimated from H NMR. The lowest obtained purity
was reported. The purity was also further supported as described in the
Supporting Information by LC/MS with monitoring of total ion
current, TLC in several systems, a sharp melting point, and occasional
other technique. The elemental analysis was conducted by
MIDWESTMICRO lab, LCC.
The synthesis of tert-butyl 4-(3-(4-(trifluoromethyl)phenyl)ureido)-
piperidine-1-carboxylate, 1-(piperidin-4-yl)-3-(4-(trifluoromethyl)-
phenyl)urea, 1-(piperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea
and tert-butyl 4-(3-(4-(trifluoromethoxy)phenyl)ureido)piperidine-1-
carboxylate have been reported elsewhere.23,30,53 The synthesis of
inhibitors 1,38 2,23 3,23 6,38 APAU,32 TPAU,23 18 (TPPU),23 20
(TPCU),23 and t-TUCB34 were reported elsewhere. Experimental of
each individual inhibitor is described in Supporting Information in
detail.
The experimental procedures of enzyme preparation, IC50
determination for sEH inhibitors, and Ki determination for sEH
inhibitors followed the published procedures and are described in
detail in Supporting Information.38,54−58
Synthetic Method 1. Step 1. Corresponding isocyanate (1 equiv)
and 4-amino-1-Boc-piperidine (1.1 equiv) were dissolved in CH2Cl2
(50 mM, corresponding to isocyanate) and stirred at rt for 12 h. The
reaction was quenched by addition of water. The organic layer was
isolated, and the aqueous layer was extracted with EtOAc
(EtOAc:aqueous layer/1:1) four times. The combined organic layer
was dried over anhydrous magnesium sulfate and was concentrated
under vacuo and was further purified by flash chromatography,
yielding corresponding Boc-protected urea.
Step 2. The BOC protected urea from the step 1 was dissolved in
HCl solution (2M, MeOH) to make reaction mixture (186 mM, BOC
protected urea). The resulting solution was refluxed for 2 h. The
solvent was removed in vacuo, and the crude reaction product was
adjusted to pH 12 with NaOH solution (6N). The precipitates were
filtered and dried under high vacuum. The final product unprotected
urea was served as a scaffold for the next step of synthesis.
Step 3. Unless specified, the unprotected urea (1 equiv) from step
2, EDCI (1.5 equiv), DMAP (1.5 equiv), and corresponding carboxylic
acid (1.5 equiv) were dissolved in CH2Cl2 (8.3 mM, unprotected urea)
and stirred overnight (12 h) at rt. The reaction was quenched by
addition of HCl solution (1M). The organic layer was collected, and
the aqueous layer was extracted with EtOAc (EtOAc:aqueous layer/
1:1) four times. The combined organic layer was dried over anhydrous
magnesium sulfate and was concentrated in vacuo and further purified
by flash chromatography.
Synthetic Method 2. The corresponding isocyanate (1 equiv) was
added to a suspension of targeted piperidine (1.1 equiv) in CH2Cl2
(20 mM, corresponding isocyanate). The reaction was stirred
overnight (12 h) at rt. The reaction was quenched with the addition
of HCl solution (2M). The organic layer was collected, and the
aqueous layer was further extracted with EtOAc (EtOAc:aqueous
layer/1:1) three times. The combined organic layer was washed with
satd NaCl solution. The organic layer was dried over anhydrous
magnesium sulfate and was concentrated in vacuo. The product was
purified by flash chromatography.
Synthetic Method 3. Corresponding amine (1 equiv) and
triethylamine (1.2 equiv) was dissolved in CH2Cl2 (54 mM
corresponding to amine) and stirred at −78 °C. Triphosgene (0.37
equiv) dissolved in CH2Cl2 (20 mM, corresponding triphosgene) was
added dropwise at −78 °C. The reaction was then warm to rt and was
stirred for 30 min. The reaction was cooled to 0 °C. Corresponding
piperidine (1.1 equiv) dissolved in CH2Cl2 (54 mM, corresponding
piperidine) was added slowly, and the reaction was further stirred at rt
for 12 h. The reaction was quenched with the addition of HCl solution
(2M). The organic layer was collected, and the aqueous layer was
further extracted with EtOAc (EtOAc:aqueous layer/1:1) three times.
The combined organic layer was washed with satd NaCl solution. The
organic layer was dried over anhydrous magnesium sulfate and was
concentrated in vacuo. The product was purified by flash
chromatography.
Synthetic Method 4. The first two steps are the same as synthetic
method 1, steps 1 and 2, unless specified.
Step 3. The unprotected urea (1 equiv) and triethylamine (1.2
equiv) was dissolved in CH2Cl2 (8.3 mM, corresponding unprotected
urea), and corresponding sulfonyl chloride was added dropwise at 0 °C
and the reaction was stirred overnight (12 h) at rt. The reaction was
quenched by addition of HCl solution (1M). The organic layer was
collected, and the aqueous layer was extracted with EtOAc
(EtOAc:aqueous layer/1:1) four times. The combined organic layer
was dried over anhydrous magnesium sulfate and was concentrated in
vacuo and further purified by flash chromatography.
Protein Crystallization. Crystals of the enzyme were obtained
using the hanging drop vapor-diffusion method by mixing equal
volumes of protein (8−12 mg/mL concentration in 100 mM sodium
phosphate pH 7.4, 3 mM DTT) and the reservoir solution (30% PEG
3350, 0−10% sucrose) at 4 °C. The crystals grew in approximately 1
week and belonged to the hexagonal space group P6522.
Complexes of sEH with inhibitors 8/UC1770 or 4 have been
obtained by soaking sEH crystals grown as described above in
modified mother liquor (35% PEG 3350, 50 mM sodium phosphate
pH 7.4) supplemented with 1 mM solution of inhibitor for 1−7 days
Data Collection. Prior to the data collection, a suitable crystal was
dipped for 30 s in a modified mother liquor solution (35% PEG 3350)
with the addition of 10% glycerol as a cryoprotectant. Diffraction data
were collected at 100 K at the XP station at the Center for Advance
Microstructures and Devices at Louisiana State University with a MAR
charge-coupled device camera (structure TPPU/UC1770) or the NE-
CAT beamline 24-ID-C at the Advanced Photon Source equipped
with the Pilatus 6 M detector (structure 4). The images were
processed and scaled using the HKL2000 (structure TPPU/
UC1770)59 or Xia2 program suit60 (structure 4). Data collection
and data processing statistics are given in Table 1.
Crystal Structure Determination. The molecular replacement
procedure was applied to locate a solution using the program
MOLREP.61 A monomer of human sEH (PDB accession code 1S8O)
was used as a search model. The positioned MR model was refined
using the maximum likelihood refinement in REFMAC61 with the TLS
parameters generated by the TLSMD server.62 Program Coot was
used for model building throughout the refinement.63
HERG Patch Clamp Measurement. Cell Culture for Channel
Analysis. HEK-293 cells stably expressing hKv11.1 (hERG) under
G418 selection were a generous gift from Craig January (University of
Wisconsin, Madison). Cells were cultured in DMEM containing 10%
fetal bovine serum, 2 mM glutamine, 1 mM Na+ pyruvate, 100 U/mL
penicillin and 100 μg/mL streptomycin, and 500 mg/mL G418.
Before electrophysiological experiments, cells were grown to 60%
confluency and 10 mM astemizole was added to the culture for 24 h to
increase Kv11.1 surface expression.
Electrophysiology. All experiments were conducted with an EPC-
10 amplifier (HEKA, Lambrecht/Pfalz, Germany) in the whole-cell
configuration of the patch-clamp technique with a holding potential of
−80 mV. Pipette resistances averaged 2.0 MΩ. Compound solutions in
Na+-Ringer were freshly prepared during the experiments from 10
mM stock solutions in DMSO. The final DMSO concentration never
exceeded 1%. The external solution contained in mM: 160 NaCl, 4.5
KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4, osmolality 300 mmol/
kg. The internal pipet solution contained in mM: 120 KCl, 10 HEPES,
4 Na2ATP, 10 EGTA, 5.374 CaCl2, 1,75 MgCl2, pH 7.2, osmolality
295 mmol/kg (free Ca2+ concentration 150 nM calculated with
MaxChelator assuming a temperature of 25 °C, a pH of 7.2, and an
ionic strength of 160 mM). HERG (Kv11.1) currents were elicited
with a two-step pulse (applied every 10 s) from −80 mV first to 20 mV
for 1 s and then to −50 mV for 2 s, and the percent reduction of both
peak and tail current by the drug were determined.
Pharmacokinetic Study of Inhibitors Using Oral Dosing in
Mice. All the animal experiments were performed according to the
protocols approved by the Animal Use and Care Committee of
University of CaliforniaDavis. Male Swiss Webster mice (8 week
old, 24−30 g) purchased from Charles River Laboratories were used
for PK studies. Inhibitors for oral administration were dissolved in
L
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oleic acid-rich triglyceride containing 20% PEG400 (v/v) to give a
clear solution. 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, and
48 h after administration of the inhibitor in a cassette of 3−5
compounds (Supporting Information, Table S1) (0.3 mg/kg per
compounds, 100−110 uL). Each group contained 3−4 animals. Each
blood sample was immediately transferred to a tube containing 50 μL
of water containing 0.1% EDTA. After being mixed strongly on a
Vortex for 1 min, all samples were stored at −80 °C until analysis. The
blood samples were prepared for the measurement of sEH inhibitors
according to the previously reported method by Liu et al.18 The details
LC/MS−MS methods are described in detail in the Supporting
Information.
Pharmacokinetic Study of Inhibitors Using Oral Dosing in
Rat. All the animal experiments were performed according to the
protocols approved by the Animal Use and Care Committee of
University of CaliforniaDavis. Male Sprague−Dawley rats (n = 4, 8
week old, 250−300 g) were used for pharmacokinetic study for sEH
inhibitors. A cassette of four inhibitors (inhibitors 4, 7, 19, and 21, 0.3
mg/kg per inhibitors, 0.9−1.2 mL) was given by oral administration.
Inhibitor was dissolved in oleic oil containing 5% polyethylene glycol
400 to form a clear solution. Blood (10 μL) was collected from the tail
vein by using a pipet tip rinsed with 7.5% EDTA(K3) at 0, 0.5, 1, 1.5, 2,
4, 6, 8, and 24 h after oral dosing with the inhibitor. Each blood sample
was immediately transferred to a tube containing 50 μL of water and
mixed by vortex for 1 min, and all samples were stored at −80 °C until
analysis. The blood samples were prepared for the measurement of
sEH inhibitors according to the previously reported method by Liu et
al.18 The details LC/MS−MS methods are described in detail in the
Supporting Information.
Diabetic Neuropathic Pain Model. A diabetic neuropathic pain
modeled was generated using streptozocin which targets and kills the
pancreatic beta islet cells, rendering the animals with type I diabetes.
The rats were acclimated for 1 h and tested for baseline thresholds
before inducing diabetes. The baseline mechanical withdrawal
thresholds were established using the von Frey mechanical nociceptive
test with an electronic anesthesiometer (IITC, Woodland Hills, CA).
Subsequently, streptozocin (55 mg/kg) in saline was injected via tail
vein per previously reported methods.64 After 5 days, the allodynia of
diabetic rats was confirmed with the von Frey nociceptive assay. Rats
were placed in clear acrylic chambers on a steel mesh floor. The hind
paw of the rat was probed through the mesh with a rigid tip probe
connected to the electronic readout pressure meter set to the
maximum hold setting. The withdrawal thresholds per rat were
measured 3−5 times at 1 min intervals for each time point.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 530-752-7519. Fax: 530-752-1537. E-mail:
bdhammock@ucdavis.edu.
Notes
The authors declare the following competing financial
interest(s): The University of California holds patents on the
sEH inhibitors used in this study as well as their use to treat
inflammation, inflammatory pain, and neuropathic pain. B.D.
Hammock is a co-founder of Eicosis L.L.C., a start up company
advancing sEH inhibitors into the clinic. All the others co-
authors declare no competing financial interest..
ACKNOWLEDGMENTS
■
This work was partially funded by NIEHS grant R01 ES002710,
NIAMS grant R21 AR062866, NIH CounterAct U54
NS079202, and NIEHS Superfund Research Program grant
P42 ES004699. B.D.H. is a George and Judy Marcus Senior
Fellow of the American Asthma Foundation. This work was
supported in part by NIH R01 HL107887 (M.E.N.) and by the
Louisiana Governors’ Biotechnology Initiative. Data for the
structure TPPU (UC1770) were collected at the Gulf Coast
Protein Crystallography Beamline at the Center for Advanced
Microstructures and Devices. This beamline is supported by the
National Science Foundation grant DBI-9871464 with
cofunding from the National Institute of General Medical
Sciences. Data for the structure of inhibitor 4 (UC2389) were
collected at the Advanced Photon Source on the Northeastern
Collaborative Access Team beamlines, which are supported by
grants from the National Center for Research Resources
(P41RR015301-10) and the National Institute of General
Medical Sciences (P41 GM103403-10) from the National
Institutes of Health. Use of the Advanced Photon Source, an
Office of Science User Facility operated for the U.S.
Department of Energy (DOE) Office of Science by Argonne
National Laboratory, was supported by the U.S. DOE under
contract no. DE-AC02-06CH11357. We thank Dr. Henry
Bellami and Dr. David Neau for assistance with data collection.
The baseline diabetic allodynia was quantified again at the
beginning of all test days. The rats were then administered vehicle
or sEH inhibitor via oral gavage and tested at 30 min, 1, 2, 3, 4, 5, 6,
and 8 h for mechanical withdrawal thresholds. The reported scores are
the grams of force required to elicit a hind paw withdrawal averaged
with standard error of the mean (SEM) per a group of rats (n = 5).
The baseline diabetic neuropathic scores were normalized to 100% to
reflect the response to treatments which are reported as % of post
diabetic neuropathic baseline.
ABBREVIATIONS USED
■
sEH, soluble epoxide hydrolase; EpFAs, epoxy fatty acids;
CYP450, cytochrome P450; EETs, epoxy-eicosatrienoic acids;
EpETEs, epoxy-eicosatetraenoic acids; EpDPEs, epoxy-docosa-
pentaenoic acids; Ki, inhibition constant; Kd, dissociation
constant; TPPU, 1-trifluoromethoxyphenyl-3-(1-propionylpi-
peridin-4-yl) urea; koff, dissociation rate constant; elogP,
experimental Log P; PK-AUC, area under the curve of the
pharmacokinetic profiles; TUPS, 1-(1-methylsulfonyl-piperidin-
4-yl)-3-(4-trifluoromethoxyphenyl)-urea; TPAU, 1-trifluorome-
thoxyphenyl-3-(1-acetylpiperidin-4-yl)-urea; APAU, 1-(1-acety-
piperidin-4-yl)-3-adamantanylurea; KMnO4, potassium per-
manganate; t-TUCB, trans-4-{4-[3-(4-trifluoromethoxyphen-
yl)-uredio]-cyclohexyloxy}benzoic acid; EDCI, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide; EtOAc, ethyl acetate;
MeOH, methanol; HCl, hydrochloric acid; NaOH, sodium
hydroxide; DMAP, 4-dimethylaminopiperidine; MRM, multiple
reaction monitoring; MWT, mechanical withdrawal thresholds
ASSOCIATED CONTENT
■
S
* Supporting Information
Correlation plot between koff against number of non-hydrogen
atom, experimental log P and Ki; plots of pharmacokinetic
profiles of selected inhibitors in mice and rat; detailed
experimental on chemistry, biochemistry, and animal studies;
tables of optimized conditions for monitoring sEH inhibitors by
MRM. This material is available free of charge via the Internet
at http://pubs.acs.org.
Accession Codes
REFERENCES
PDB accession codes: 4OD0 (human sEH with inhibitor 18
(UC1770/TPPU)) and 4OCZ (human sEH with inhibitor 4
(UC2389)).
■
(1) National Diabetes Fact Sheet: National Estimates and General
Information on Diabetes and Prediabetes in the United States; U.S.
M
dx.doi.org/10.1021/jm500694p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Department of Health and Human Services, Centers for Disease
Control and Prevention: Atlanta, GA, 2011.
(2) Calcutt, N. A. Tolerating diabetes: an alternative therapeutic
approach for diabetic neuropathy. ASN Neuro 2010, 2, 216−217.
(3) Said, G. Diabetic neuropathya review. Nature Clin. Pract.
Neurol. 2007, 3, 331−340.
(4) Veves, A.; Backonja, M.; Malik, R. A. Painful diabetic neuropathy:
epidemiology, natural history, early diagnosis, and treatment options.
Pain Med. 2008, 9, 660−674.
(5) Wong, M.-c.; Chung, J. W. Y.; Wong, T. K. S. Effects of
treatments for symptoms of painful diabetic neuropathy: systematic
review. BMJ n(Br. Med. J.) 2007, 335, 87.
Chiamvimonvat, N.; Imig, J. D.; Hammock, B. D. Anti-inflammatory
Effects of omega-3 Polyunsaturated Fatty Acids and Soluble Epoxide
Hydrolase Inhibitors in Angiotensin-II-Dependent Hypertension. J.
Cardiovasc. Pharmacol. 2013, 62, 285−297.
(21) Shen, H. C.; Hammock, B. D. Discovery of Inhibitors of Soluble
Epoxide Hydrolase: A Target with Multiple Potential Therapeutic
Indications. J. Med. Chem. 2012, 55, 1789−1808.
(22) Shen, H. C.; Ding, F.-X.; Wang, S.; Deng, Q.; Zhang, X.; Chen,
Y.; Zhou, G.; Xu, S.; Chen, H.-S.; Tong, X.; Tong, V.; Mitra, K.;
Kumar, S.; Tsai, C.; Stevenson, A. S.; Pai, L.-Y.; Alonso-Galicia, M.;
Chen, X.; Soisson, S. M.; Roy, S.; Zhang, B.; Tata, J. R.; Berger, J. P.;
Colletti, S. L. Discovery of a Highly Potent, Selective, and Bioavailable
Soluble Epoxide Hydrolase Inhibitor with Excellent ex Vivo Target
Engagement. J. Med. Chem. 2009, 52, 5009−5012.
(6) Spector, A. A. Arachidonic acid cytochrome P450 epoxygenase
pathway. J. Lipid Res. 2009, 50, S52−S56.
(7) Morisseau, C.; Inceoglu, B.; Schmelzer, K.; Tsai, H.-J.; Jinks, S. L.;
Hegedus, C. M.; Hammock, B. D. Naturally occurring monoepoxides
of eicosapentaenoic acid and docosahexaenoic acid are bioactive
antihyperalgesic lipids. J. Lipid Res. 2010, 51, 3481−3490.
(8) Wagner, K.; Inceoglu, B.; Gill, S. S.; Hammock, B. D.
Epoxygenated Fatty Acids and Soluble Epoxide Hydrolase Inhibition:
Novel Mediators of Pain Reduction. J. Agric. Food Chem. 2011, 59,
2816−2824.
(23) Rose, T. E.; Morisseau, C.; Liu, J.-Y.; Inceoglu, B.; Jones, P. D.;
Sanborn, J. R.; Hammock, B. D. 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. J. Med. Chem. 2010, 53, 7067−7075.
(24) Reema, K. T.; McAtee, J. J.; Belyanskaya, S.; Brandt, M.; Brown,
G. D.; Costell, M. H.; Ding, Y.; Dodson, J. W.; Eisennagel, S. H.; Fries,
R. E.; Gross, J. W.; Harpel, M. R.; Holt, D. A.; Israel, D. I.; Jolivette, L.
J.; Krosky, D.; Li, H.; Lu, Q.; Mandichak, T.; Roethke, T.;
Schnackenberg, C. G.; Schwartz, B.; Shewchuk, L. M.; Xie, W.;
Behm, D. J.; Douglas, S. A.; Shaw, A. L.; Marino, J. P., Jr. Discovery of
1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble
epoxide hydrolase. Bioorg. Med. Chem. Lett. 2013, 23, 3584−3588.
(25) Podolin, P. L.; Bolognese, B. J.; Foley, J. F.; Long, E., III; Peck,
B.; Umbrecht, S.; Zhang, X.; Zhu, P.; Schwartz, B.; Xie, W.; Quinn, C.;
Qi, H.; Sweitzer, S.; Chen, S.; Galop, M.; Ding, Y.; Belyanskaya, S. L.;
Israel, D. I.; Morgan, B. A.; Behm, D. J.; Marino, J. P., Jr.; Kurali, E.;
Barnette, M. S.; Mayer, R. J.; Booth-Genthe, C. L.; Callahan, J. F. In
vitro and in vivo characterization of a novel soluble epoxide hydrolase
inhibitor. Prostaglandins Other Lipid Mediators 2013, 104, 25−31.
(26) Kim, I.-H.; Nishi, K.; Tsai, H.-J.; Bradford, T.; Koda, Y.;
Watanabe, T.; Morisseau, C.; Blanchfield, J.; Toth, I.; Hammock, B. D.
Design of bioavailable derivatives of 12-(3-adamantan-1-yl-ureido)-
dodecanoic acid, a potent inhibitor of the soluble epoxide hydrolase.
Bioorg. Med. Chem. Lett. 2007, 15, 312−323.
(27) Huang, S.-X.; Cao, B.; Morisseau, C.; Tin, Y.; Hammock, B. D.;
Long, Y.-Q. Structure-based optimization of the piperazino-containing
1,3-disubstituted ureas affording sub-nanomolar inhibitors of soluble
epoxide hydrolase. MedChemComm 2012, 3, 379−384.
(28) Morisseau, C.; Goodrow, M. H.; Dowdy, D.; Zheng, J.; Greene,
J. F.; Sanborn, J. R.; Hammock, B. D. Potent urea and carbamate
inhibitors of soluble epoxide hydrolases. Proc. Natl. Acad. Sci. U. S. A.
1999, 96, 8849−8854.
(29) Kim, I.-H.; Tsai, H.-J.; Nishi, K.; Kasagami, T.; Morisseau, C.;
Hammock, B. D. 1,3-Disubstituted ureas functionalized with ether
groups are potent inhibitors of the soluble epoxide hydrolase with
improved pharmacokinetic properties. J. Med. Chem. 2007, 50, 5217−
5226.
(30) Hwang, S. H.; Tsai, H.-J.; Liu, J.-Y.; Morisseau, C.; Hammock,
B. D. Orally bioavailable potent soluble epoxide hydrolase inhibitors. J.
Med. Chem. 2007, 50, 3825−3840.
(9) Morisseau, C.; Hammock, B. D. Impact of Soluble Epoxide
Hydrolase and Epoxyeicosanoids on Human Health. Annu. Rev.
Pharmacol. Toxicol. 2013, 53, 37−58.
(10) Wagner, K.; Inceoglu, B.; Dong, H.; Yang, J.; Hwang, S. H.;
Jones, P.; Morisseau, C.; Hammock, B. D. Comparative efficacy of 3
soluble epoxide hydrolase inhibitors in rat neuropathic and
inflammatory pain models. Eur. J. Pharmacol. 2013, 700, 93−101.
(11) Inceoglu, B.; Wagner, K. M.; Yang, J.; Bettaieb, A.; Schebb, N.
H.; Hwang, S. H.; Morisseau, C.; Haj, F. G.; Hammock, B. D. Acute
augmentation of epoxygenated fatty acid levels rapidly reduces pain-
related behavior in a rat model of type I diabetes. Proc. Natl. Acad. Sci.
U. S. A. 2012, 109, 11390−11395.
(12) Guedes, A. G. P.; Morisseau, C.; Sole, A.; Soares, J. H. N.; Ulu,
A.; Dong, H.; Hammock, B. D. Use of a soluble epoxide hydrolase
inhibitor as an adjunctive analgesic in a horse with laminitis. Vet.
Anaesth. Analg. 2013, 40, 440−448.
(13) Tran, L.; Kompa, A. R.; Wang, B. H.; Krum, H. Evaluation of
the Effects of Urotensin II and Soluble Epoxide Hydrolase Inhibitor on
Skin Microvessel Tone in Healthy Controls and Heart Failure Patients.
Cardiovasc. Ther. 2012, 30, 295−300.
(14) Ai, D.; Pang, W.; Li, N.; Xu, M.; Jones, P. D.; Yang, J.; Zhang,
Y.; Chiamvimonvat, N.; Shyy, J. Y. J.; Hammock, B. D.; Zhu, Y.
Soluble epoxide hydrolase plays an essential role in angiotensin II-
induced cardiac hypertrophy. Proc. Natl. Acad. Sci. U. S. A. 2009, 106,
564−569.
(15) Brenneis, C.; Sisignano, M.; Coste, O.; Altenrath, K.; Fischer, M.
J.; Angioni, C.; Fleming, I.; Brandes, R. P.; Reeh, P. W.; Woolf, C. J.;
Geisslinger, G.; Scholich, K. Soluble epoxide hydrolase limits
mechanical hyperalgesia during inflammation. Mol. Pain 2011, 7, 78.
(16) Imig, J. D.; Hammock, B. D. Soluble epoxide hydrolase as a
therapeutic target for cardiovascular diseases. Nature Rev. Drug
Discovery 2009, 8, 794−805.
(17) Inceoglu, B.; Jinks, S. L.; Schmelzer, K. R.; Waite, T.; Kim, I. H.;
Hammock, B. D. Inhibition of soluble epoxide hydrolase reduces LPS-
induced thermal hyperalgesia and mechanical allodynia in a rat model
of inflammatory pain. Life Sci. 2006, 79, 2311−2319.
(18) Liu, J.-Y.; Lin, Y.-P.; Qiu, H.; Morisseau, C.; Rose, T. E.; Hwang,
S. H.; Chiamvimonvat, N.; Hammock, B. D. Substituted phenyl groups
improve the pharmacokinetic profile and anti-inflammatory effect of
urea-based soluble epoxide hydrolase inhibitors in murine models. Eur.
J. Pharm. Sci. 2013, 48, 619−627.
(31) Liu, J. Y.; Tsai, H. J.; Hwang, S. H.; Jones, P. D.; Morisseau, C.;
Hammock, B. D. Pharmacokinetic optimization of four soluble
epoxide hydrolase inhibitors for use in a murine model of
inflammation. Br. J. Pharmacol. 2009, 156, 284−296.
(32) Jones, P. D.; Tsai, H. J.; Do, Z. N.; Morisseau, C.; Hammock, B.
D. Synthesis and SAR of conformationally restricted inhibitors of
soluble epoxide hydrolase. Bioorg. Med. Chem. Lett. 2006, 16, 5212−
5216.
(33) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Opinion
Drug−target residence time and its implications for lead optimization.
Nature Rev. Drug Discovery 2006, 5, 730−739.
(34) Tsai, H.-J.; Hwang, S. H.; Morisseau, C.; Yang, J.; Jones, P. D.;
Kasagami, T.; Kim, I.-H.; Hammock, B. D. Pharmacokinetic screening
(19) Schmelzer, K. R.; Kubala, L.; Newman, J. W.; Kim, I. H.;
Eiserich, J. P.; Hammock, B. D. Soluble epoxide hydrolase is a
therapeutic target for acute inflammation. Proc. Natl. Acad. Sci. U. S. A.
2005, 102, 9772−9777.
(20) Ulu, A.; Harris, T. R.; Morisseau, C.; Miyabe, C.; Inoue, H.;
Schuster, G.; Dong, H.; Iosif, A.-M.; Liu, J.-Y.; Weiss, R. H.;
N
dx.doi.org/10.1021/jm500694p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
of soluble epoxide hydrolase inhibitors in dogs. Eur. J. Pharm. Sci.
2010, 40, 222−238.
(52) Chiang, P.-C.; La, H.; Zhang, H.; Wong, H. Systemic
Concentrations Can Limit the Oral Absorption of Poorly Soluble
Drugs: An Investigation of Non-Sink Permeation Using Physiologi-
cally Based Pharmacokinetic Modeling. Mol. Pharmacol. 2013, 10,
3980−3988.
(53) Jones, P. D.; Tsai, H.-J.; Do, Z. N.; Morisseau, C.; Hammock, B.
D. Synthesis and SAR of conformationally restricted inhibitors of
soluble epoxide hydrolase. Bioorg. Med. Chem. Lett. 2006, 16, 5212−
5216.
(54) Borhan, B.; Mebrahtu, T.; Nazarian, S.; Kurth, M. J.; Hammock,
B. D. Improved radiolabeled substrates for soluble epoxide hydrolase.
Anal. Biochem. 1995, 231, 188−200.
(55) Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock,
B.; Hammock, B. D. Fluorescent substrates for soluble epoxide
hydrolase and application to inhibition studies. Anal. Biochem. 2005,
343, 66−75.
(56) Matveeva, E. G.; Morisseau, C.; Goodrow, M. H.; Mullin, C.;
Hammock, B. D. Tryptophan Fluorescence Quenching by Enzyme
Inhibitors As a Tool for Enzyme Active Site Structure Investigation:
Epoxide Hydrolase. Curr. Pharm. Biotechnol. 2009, 10, 589−599.
(57) Schebb, N. H.; Huby, M.; Morisseau, C.; Hwang, S. H.;
Hammock, B. D. Development of an online SPE-LC-MS-based assay
using endogenous substrate for investigation of soluble epoxide
hydrolase (sEH) inhibitors. Anal. Bioanal. Chem. 2011, 400, 1359−
1366.
(58) Wolf, N. M.; Morisseau, C.; Jones, P. D.; Hock, B.; Hammock,
B. D. Development of a high-throughput screen for soluble epoxide
hydrolase inhibition. Anal. Biochem. 2006, 355, 71−80.
(59) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Macromol. Crystallogr., Part A 1997, 276,
307−326.
(35) Lewis, D. F. V.; Jacobs, M. N.; Dickins, M. Compound
lipophilicity for substrate binding to human P450s in drug metabolism.
Drug Discovery Today 2004, 9, 530−537.
(36) Hagmann, W. K. The many roles for fluorine in medicinal
chemistry. J. Med. Chem. 2008, 51, 4359−4369.
(37) Jeschke, P. The Unique Role of Halogen Substituents in the Design
of Modern Crop Protection Compounds;Wiley-VCH Verlag GmbH &
Co. KGaA: Weinheim, Germany, 2012.
(38) Lee, K. S. S.; Morisseau, C.; Yang, J.; Wang, P.; Hwang, S. H.;
Hammock, B. D. Forster resonance energy transfer competitive
displacement assay for human soluble epoxide hydrolase. Anal.
Biochem. 2013, 434, 259−268.
(39) Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D.
W. Human soluble epoxide hydrolase: structural basis of inhibition by
4-(3-cyclohexylureido)-carboxylic acids. Protein Sci. 2006, 15, 58−64.
(40) Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.; Orpen,
A. G.; Taylor, R. Tables of bond lengths determined by x-ray and
neutron diffraction. Part 1. Bond lengths in organic compounds. J.
Chem. Soc., Perkin Trans. 1987, 2, S1−S19.
(41) Ulu, A.; Appt, S. E.; Morisseau, C.; Hwang, S. H.; Jones, P. D.;
Rose, T. E.; Dong, H.; Lango, J.; Yang, J.; Tsai, H. J.; Miyabe, C.;
Fortenbach, C.; Adams, M. R.; Hammock, B. D. Pharmacokinetics and
in vivo potency of soluble epoxide hydrolase inhibitors in cynomolgus
monkeys. Br. J. Pharmacol. 2012, 165, 1401−1412.
(42) Vickers, A. E. M.; Sinclair, J. R.; Zollinger, M.; Heitz, F.; Glanzel,
U.; Johanson, L.; Fischer, V. Multiple cytochrome P-450s involved in
the metabolism of terbinafine suggest a limited potential for drug−
drug interactions. Drug Metab. Dispos. 1999, 27, 1029−1038.
(43) Austin, R. P.; Barton, P.; Cockroft, S. L.; Wenlock, M. C.; Riley,
R. J. The influence of nonspecific microsomal binding on apparent
intrinsic clearance, and its prediction from physicochemical properties.
Drug Metab. Dispos. 2002, 30, 1497−1503.
(44) O’Brien, A. J.; Fullerton, J. N.; Massey, K. A.; Auld, G.; Sewell,
G.; James, S.; Newson, J.; Karra, E.; Winstanley, A.; Alazawi, W.;
Garcia-Martinez, R.; Cordoba, J.; Nicolaou, A.; Gilroy, D. W.
Immunosuppression in acutely decompensated cirrhosis is mediated
by prostaglandin E-2. Nature Med. 2014, 20, 522−527.
(45) Wu, J.; LoRusso, P. M.; Matherly, L. H.; Li, J. Implications of
Plasma Protein Binding for Pharmacokinetics and Pharmacodynamics
of the gamma-Secretase Inhibitor RO4929097. Clin. Cancer. Res. 2012,
18, 2066−2079.
(46) Smith, D. A.; Di, L.; Kerns, E. H. The effect of plasma protein
binding on in vivo efficacy: misconceptions in drug discovery. Nature
Rev. Drug Discovery 2010, 9, 929−939.
(60) Winter, G. xia2: an expert system for macromolecular
crystallography data reduction. J. Appl. Crystallogr. 2010, 43, 186−190.
(61) Bailey, S. The CCP4 Suiteprograms for Protein Crystallog-
raphy. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760−763.
(62) Painter, J.; Merritt, E. A. TLSMD web server for the generation
of multi-group TLS models. J. Appl. Crystallogr. 2006, 39, 109−111.
(63) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and
development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,
66, 486−501.
(64) Aley, K. O.; Levine, J. D. Rapid onset pain induced by
intravenous streptozotocin in the rat. J. Pain 2001, 2, 146−150.
(47) Morisseau, C.; Merzlikin, O.; Lin, A.; He, G.; Feng, W.; Padilla,
I.; Denison, M. S.; Pessah, I. N.; Hammock, B. D. Toxicology in the
fast lane: application of high-throughput bioassays to detect
modulation of key enzymes and receptors. Environ. Health Perspect.
2009, 117, 1867−1872.
(48) Zeldin, D. C.; Moomaw, C. R.; Jesse, N.; Tomer, K. B.;
Beetham, J.; Hammock, B. D.; Wu, S. Biochemical characterization of
the human liver cytochrome P450 arachidonic acid epoxygenase
pathway. Arch. Biochem. Biophys. 1996, 330, 87−96.
(49) Wu, S.; Moomaw, C. R.; Tomer, K. B.; Falck, J. R.; Zeldin, D. C.
Molecular cloning and expression of CYP2J2, a human cytochrome
P450 arachidonic acid epoxygenase highly expressed in heart. J. Biol.
Chem. 1996, 271, 3460−3468.
(50) Graves, J. P.; Edin, M. L.; Bradbury, J. A.; Gruzdev, A.; Cheng,
J.; Lih, F. B.; Masinde, T. A.; Qu, W.; Clayton, N. P.; Morrison, J. P.;
Tomer, K. B.; Zeldin, D. C. Characterization of four new mouse
cytochrome P450 enzymes of the CYP2J subfamily. Drug Metab.
Dispos. 2013, 41, 763−773.
(51) Chiang, P.-C.; Ran, Y.; Chou, K.-J.; Cui, Y.; Wong, H.
Investigation of utilization of nanosuspension formulation to enhance
exposure of 1,3-dicyclohexylurea in rats: preparation for PK/PD study
via subcutaneous route of nanosuspension drug delivery. Nanoscale
Res. Lett. 2011, 6, 413.
O
dx.doi.org/10.1021/jm500694p | J. Med. Chem. XXXX, XXX, XXX−XXX