Discovery of a highly potent, selective, and bioavailable soluble epoxide hydrolase inhibitor with excellent ex vivo target engagement

Article abstract of DOI:10.1021/jm900725r

4-Substituted piperidine-derived trisubstituted ureas are reported as highly potent and selective inhibitors for sEH. The SAR outlines approaches to improve activity against sEH and reduce ion channel and CYP liability. With minimal off-target activity an

Full text of DOI:10.1021/jm900725r

J. Med. Chem. 2009, 52, 5009–5012 5009
DOI: 10.1021/jm900725r
antiinflammatory activities, apparently by decreasing the
expression of cytokine induced endothelial cell adhesion
molecules, such as VCAM-1.6 In addition, EETs prevented
leukocyte adhesion to the vascular wall, presumably by
inhibiting NFκB and IKB kinases.7 Hence, it is conceivable
that EET elevation resulting from sEH inhibition could
inhibit the NFκB pathway and thus ameliorate vascular
inflammation. These properties suggest a potential applica-
tion of sEH inhibitors to the mitigation of endothelial dys-
function.
Recent studies in preclinincal species indicated that sEH
inhibition could be useful for the treatment of hypertension
and disease-modifying end organ protection. For example, it
has been reported that sEH inhibitors could significantly
reduce blood pressure in angiotensin II treated rats8 and
SHRs.9 Additionally, treatment with an sEH inhibitor in
angiotensin II infused hypertensive rats attenuated the affer-
ent kidney arteriolar diameter and reduced urinary albumin
secretion, a marker of compromised renal function. As such,
an sEH inhibitor will soon be evaluated in a phase II clinical
trial for metabolic syndrome indications.10
Discovery of a Highly Potent, Selective, and
Bioavailable Soluble Epoxide Hydrolase
Inhibitor with Excellent Ex Vivo Target
Engagement
Hong C. Shen,* Fa-Xiang Ding, Siyi Wang, Qiaolin Deng,
Xiaoping Zhang, Yuli Chen, Gaochao Zhou, Suoyu Xu,
Hsuan-shen Chen, Xinchun Tong, Vincent Tong,
Kaushik Mitra, Sanjeev Kumar, Christine Tsai,
Andra S. Stevenson, Lee-Yuh Pai,
Magdalena Alonso-Galicia, Xiaoli Chen,
Stephen M. Soisson, Sophie Roy, Bei Zhang,
James R. Tata, Joel P. Berger, and Steven L. Colletti
Merck Research Laboratories, Merck & Co., Inc., Rahway,
New Jersey 07065-0900
Received March 5, 2009
Abstract: 4-Substituted piperidine-derived trisubstituted ureas are
reported as highly potent and selective inhibitors for sEH. The SAR
outlines approaches to improve activity against sEH and reduce ion
channel and CYP liability. With minimal off-target activity and a
good PK profile, the benchmark 2d exhibited remarkable in vitro
and ex vivo target engagement. The eutomer entA-2d also elicited
vasodilation effect in rat mesenteric artery.
Despite some known sEH inhibitors,11 there remained a
significant challenge to develop sEH inhibitors with enhanced
potency, improved solubility and PK, and high selectivity
against other biological targets, particularly ion channels,
CYP enzymes, and targets that could lead to undesirable
pharmacological consequences. Herein, we report the identi-
fication of 4-substituted piperidine-derived trisubstituted ur-
eas as potent and selective sEH inhibitors (Figure 1), which
served as exploratory tools to examine sEH pharmacology. It
is noted that disubsituted ureas are typically associated with
poor PK and physical properties. The trisubstituted ureas,
however, clearly demonstrated improved PK and superior
physical properties.
All analogues were tested in human and rat sEH inhibition
assays. Potent compounds were also subject to cell-based
DHET production assay to examine their cellular sEH in-
hibitory activity. Because of the significant roles of mEH in
xenobiotic detoxication and steroid metabolism, the inhibi-
tion of this enzyme posed a safety concern. Therefore, mEH
became a major target for counterscreen, although few com-
pounds were known to be inhibitors of both enzymes.12
Additionally, CYP2C9, 2D6, and 3A4 were selected as routine
counterscreening targets. Of particular importance within this
cohort of enzymes is CYP2C9, as it is involved in producing
EETs via the monooxygenase-mediated epoxidation of ara-
chidonic acid.3a Therefore, elevation of EETs by sEH inhibi-
tion couldbe diminished by simultaneousCYP2C9inhibition.
Furthermore, cardiac potassium (IKr),13 calcium (DLZ bind-
ing or Cav1.2 functional assays),14 and sodium (Nav1.5)15
channels were scrutinized on the basis of their potential blood
pressure and cardiac effects.
Epoxide hydrolases are a group of ubiquitous enzymes
detected in species ranging from plants to mammals.1 These
enzymes catalyzethe additionofwater toanepoxide, resulting
in the formation of a vicinal diol. Several types of epoxide
hydrolases have been identified including sEH,a mEH,
cholesterol epoxide hydrolase, LTA4 hydrolase, and hepoxilin
hydrolase.2 These epoxide hydrolases differ in their substrate
specificity. For example, sEH is selective for aliphatic epox-
ides such as fatty acid epoxides while mEH is more selective
for cyclic and arene epoxides. The major known physiological
substrates of sEH are the four regioisomeric epoxides of
arachidonic acid, namely, 5,6-, 8,9-, 11,12-, and 14,15-epox-
yeicosatrienoic acids (also known as EETs) and epoxides of
linoleic acid.1 The corresponding diol products of sEH-
mediated hydrolysis of EETs are referred to as DHETs.
EETs elicit a wide range of biological effects.3 They are
known to vasodilate coronary arterioles and renal microves-
sels via the activation of BKCa ion channels.4 Notably, 11,12-
EET has been identified as EDHF, a long-sought endogenous
metabolite with robust vascular smooth muscle relaxing
activity.5 EETs, and especially 11,12-EET, have also exhibited
*To whom correspondence should be addressed. Phone: 732-594-
1755. Fax: 732-594-9473. E-mail: hong_shen@merck.com.
a Abbreviations: sEH, soluble epoxide hydrolase; mEH, microsomal
epoxide hydrolase; LTA4, leukotriene A4; EDHF, endothelial derived
hyperpolarization factor; EET, epoxyeicosatrienoic acid; DHET, dihy-
droepoxyeicosatrienoic acid; SHR, spontaneously hypertensive rats;
VCAM-1, vascular cell adhesion molecule 1; NFκB, nuclear factor κB;
COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; PGD2, prostaglan-
din D2; SHR, spontaneously hypertensive rats; PK, pharmacokinetics;
CYP, cytochrome P450; DLZ, diltiazem; SAR, structure-activity re-
lationship; SFC, supercritical fluid chromatography; 20-HETE, 20-
hydroxyeicosatetraenoic acid.
To the best of our knowledge, the previous reports on sEH
inhibitors did not disclose whether they had ion channel or
CYP inhibitory activities. This is a critical omission, since in
order to evaluate the antihypertensive effects of sEH inhi-
bitors, it is a prerequisite that they demonstrate strong
sEH engagement, without off-target activity that could by
itself impact blood pressure change. In addition, a telemetry
r
2009 American Chemical Society
Published on Web 07/31/2009
pubs.acs.org/jmc

5010 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Figure 1. Scaffold of trisubstituted urea sEH inhibitors.
Shen et al.
Table 2. In Vitro SAR on sEH and mEH Inhibition, DHET Produc-
tion, CYP and Ion Channelsa
Table 1. In Vitro SAR on sEH and mEH Inhibition, DHET
Production, CYP and Ion Channelsa
a Activity was determined in triplicate with 20% deviation. b The
conversion of 14,15-EET to 14,15-DHET was measured in HEK293
cells. c Human mEH was used.
a Activity was determined in triplicate with 20% deviation. b The
conversion of 14,15-EET to 14,15-DHET was measured in HEK293
cells. c Human mEH was used.
method was employed for the SHR model, thus providing a
reliable tool to monitor blood pressure.
issues but introduced a 40-fold right shift of the IC50 in the
DHET assay vs its human sEH IC50. Among three pyridine
regioisomers (2d, 2e, and 2f), 2d displayed the cleanest off-
target profiles and the best enzyme activity. Of the two
enantiomers of 2d, the eutomer (entA-2d) was 4-fold more
active than the distomer (entB-2d) against the human sEH.
Not only was entA-2d inactive in ion channel and CYP
enzyme assays, it was also selective against sEH vs a panel
of 166 counterscreening targets (IC50 >10 μM) including a
subset involved in blood pressure regulation. The X-ray
crystallographic structure of entA-2d established its absolute
configuration shown in the first entry of Table 3. The pyrazine
analogue 2g had a similar overall profile as 2d, but pyrimi-
dine analogue 2h was less active in inhibiting sEH and more
active in inhibiting CYP2C9. Finally, bicyclic heterocycles
such as isoquinoline or quinoline resulted in increased ion
channel and CYP activity (2i and 2j).
First, the 4-position of piperidine was systematically stu-
died with benzene and various six-membered heterocycles.
These analogues generally gave poor enzyme inhibitory activ-
ity (data not shown). In contrast, use of five-membered polar
heterocycles led to improved activity (Table 1). In particular,
oxadiazole regioisomer 1i offered the best enzymatic and
cellular activity, indicating its good cell permeability. It is
noted that most compounds with a 4-heteroaryl group in
Table 1 provided very clean off-target profiles with regard to
mEH (IC50 > 100 μM), ion channels (IC50 > 10 μM), and
CYP enzyme (IC50 > 10 μM) inhibition.
The choice of heterocycle proved to be critical, as small
changes in structure resulted in massive changes in sEH acti-
vity. Adopting the requisite oxadiazole shownin1i as the opti-
mized fragment, additional aryl substitution of oxadiazole
further diminished the sEH IC50 to the low nanomolar range
(Table 2). Small changes in structure, such as a substituted
phenyl group, led to more ion channel activity (2a and 2b).
The employment of an acid moiety (2c) removed ion channel
Table 3 summarizes the optimization of the right hand of
the molecule toward the identification of the phenylcyclopro-
pyl group as the optimized moiety, likely due to its aromatic
nature and conformational rigidity. Although aniline

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5011
Table 3. In Vitro SAR on sEH and mEH Inhibition, DHET Produc-
tion, CYP and Ion Channelsa
a Activity was determined in triplicate with 20% deviation. b The
conversion of 14,15-EET to 14,15-DHET was measured in HEK293
cells. c Human mEH was used.
Figure 3. Ex vivo target engagement of 2d in SHR blood (top,
50 mpk, po; bottom, 300 mpk, po). DHET production rate = (14,
15-DHET concentration (2d))/(14,15-DHET concentration (vehicle)).
after mixing each of the compounds (1 μM) with 14,15-EET
(10 μM) in blood. Given the good PK profile of 2d
(bioavailability (77%), clearance (29 (mL/min)/kg), normal-
ized oral exposure (1.2 μM h kg/mg)) and in vitro whole
3
3
blood activity, the ex vivo target engagement of 2d was
evaluated after a single dose of inhibitor. At 0.5, 2, 6, 24 h
postdosing, 14,15-EET was added to the blood freshly drawn,
and the 14,15-DHET production rate was measured. At
50 mpk, 2d reduced the DHET production rate to less than
10% of the control group during a time course of 0.5-6 h. In
addition, an inverse correlation of the compound level and the
DHET production was established (Figure 3, top). At
300 mpk, the DHET production rate in blood was diminished
to less than 5% from 2 to 24 h postdose, with over 10 μM
compound coverage throughout this period (Figure 3,
bottom). Furthermore, 2d demonstrated marked target en-
gagement in SHR kidney, as evidenced by an 85% and 90%
reduction in 14,15-DHET production at 50 and 300 mpk,
respectively. The high dose of 2d to achieve good target
engagement reflected its serum shift, which was corroborated
by rat plasma protein binding (11% free fraction in 100% rat
plasma at 2.5 μM 2d).
Figure 2. Minimized docking model of entA-2d (blue) in human
sEH. The dashed lines illustrate atom pairs within hydrogen bond
distance. The pictures were prepared by PyMOL (Delano Scientific
LLC, South San Francisco, CA). The model is based on the X-ray
crystallographic structure of 1ZD3 (PDB code): human sEH 4-(3-
cyclohexyluriedo)butyric acid complex.16
analogues such as 3a provided better activity in sEH and
DHET assays, they were associated with more ion channel
issues and potentially carcinogenic metabolites. Other ben-
zene-fused cycloalkyl groups gave worse sEH inhibition or
more off-target activity.
To rationalize its good inhibitory activity, a docking model
of entA-2d in human sEH was established on the basis of the
X-ray crystallographic structure of the previously reported
protein-inhibitor complex (Figure 2).16 Besides the antici-
pated hydrogen bond interactions between the urea N-H and
Asp333 and between the urea carbonyl and Tyr381/Tyr465,
an interesting hydrogen bond was identified between the
oxidazole oxygen atom and Gln382. Furthermore, a π-π
stacking interaction of the oxidazole and the indole ring of
Trp334 may further contribute to the inhibitor’s good binding
affinity.
With excellent target engagement and specificity, the acute
action of entA-2d on vascular tone was evaluated by SHR
mesenteric artery assay. Both entA-2d and entB-2d were able
to reverse vascular contraction induced by methoxamine
(IC50 = 6.5 and 15 μM for entA-2d and entB-2d, respectively),
an R1-adrenergic agonist.17 No vasoconstriction was obser-
ved on basal tension with either compound up to 100 μM.
These results were in line with the vasodilatory effect of EETs
previously reported in literature.18 However, in telemetrized
SHRs, entA-2d failed to demonstrate antihypertensive activ-
ity acutely over 24 h or chronically (300 mpk, po, q.d., 7 d).
A parallel PK study in SHRs confirmed excellent compound
exposure on the eighth day of dosing: 35 and 20 μM at 4 and
24 h postdose, respectively. Meanwhile the endogenous ep-
oxide/diol ratios in kidney were increased 2- to 9-fold (Figure 4,
An in vitro DHET production assay was employed to
determine sEH inhibitory activity in SHR whole blood.
Analogues 1i, 2d, and 2g exhibited excellent inhibition of
DHET production (91%, 92%, and 94%, respectively) 1 h

5012 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Shen et al.
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top). Moreover, 20-hydroxyeicosatetraenoic acid(20-HETE),
a potent vasoconstrictor,19 was measured in SHR kidney
(Figure 4, bottom). The lack of systolic blood pressure reduc-
tion by entA-2d may be explained by invoking the 2.5-fold
increase of 20-HETE, which could negate the vasodilatory
effect of elevated EETs. It remains unclear whether the 20-
HETE elevation resulted from the increased EETs. It is also
likely that sEH inhibition by itself does not elicit a robust
blood pressure effect in telemetrized SHRs.
The synthesis of racemate 2d commenced with cyanopyr-
idine 4 (Scheme 1). The hydroxyamine addition to cyanide 4
provided hydroxyamidine 5, which then underwent a cycliza-
tion with acid 6 to afford oxadiazole 7. The Boc-deprotection
followed by the urea formation and a chiral SFC separation
yielded two enantiomers entA-2d and entB-2d.
In summary, several 4-substituted piperidine-based trisubsti-
tuted ureas were discovered as highly selective and potent sEH
inhibitors. In particular, 2d inhibited sEH activity effectively in
vitro, ex vivo, and in vivo. This compound also demonstrated
significant vasodilatory activity in rat mesenteric artery.
Supporting Information Available: Experimental procedures
for compound preparation, characterization data, and biological
assay protocols. This material is available free of charge via the
Internet at http://pubs.acs.org.
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