Discovery of spirocyclic secondary amine-derived tertiary ureas as highly potent, selective and bioavailable soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.05.036

Spirocyclic secondary amine-derived trisubstituted ureas were identified as highly potent, bioavailable and selective soluble epoxide hydrolase (sEH) inhibitors. Despite good oral exposure and excellent ex vivo target engagement in blood, one such compoun

Full text of DOI:10.1016/j.bmcl.2009.05.036

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3398–3404
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of spirocyclic secondary amine-derived tertiary ureas as highly
potent, selective and bioavailable soluble epoxide hydrolase inhibitors
a
a
b
b
b
Hong C. Shen a, , Fa-Xiang Ding , Siyi Wang , Suoyu Xu , Hsuan-shen Chen , Xinchun Tong ,
Vincent Tong b, Kaushik Mitra b, Sanjeev Kumar b, Xiaoping Zhang c, Yuli Chen c, Gaochao Zhou c,
Lee-Yuh Pai d, Magdalena Alonso-Galicia d, Xiaoli Chen e, Bei Zhang c, James R. Tata a, Joel P. Berger c,
Steven L. Colletti a
*
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Drug metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
c Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
d Department of Cardiovascular Diseases, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
e Department of Pharmacology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Spirocyclic secondary amine-derived trisubstituted ureas were identified as highly potent, bioavailable
and selective soluble epoxide hydrolase (sEH) inhibitors. Despite good oral exposure and excellent
ex vivo target engagement in blood, one such compound, rac-1a, failed to lower blood pressure acutely
in spontaneously hypertensive rats (SHRs). This study posed the question as to whether sEH inhibition
provides a robust mechanism leading to a significant antihypertensive effect.
Received 17 April 2009
Revised 7 May 2009
Accepted 11 May 2009
Available online 18 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Soluble epoxide hydrolase
sEH
Spirocyclic secondary amines
Spontaneously hypertensive rats
Tertiary ureas
Human soluble epoxide hydrolase (sEH, EPHX2) is a bifunctional
and homodimer enzyme located in the cytosol and peroxisomes.1
The highest specific activity of sEH is observed in liver, followed
by kidney.2 sEH mediates the metabolism of arachidonic acid and
linoleic acid epoxides. Specifically, the C-terminus of sEH converts
epoxyeicosatrienoic acids (EETs), which are derived from metabo-
lism of arachidonic acid, to the corresponding dihydroxyeicosatri-
enoic acids (DHETs).3
atherosclerosis.8 In ischemia-reperfusion injured rats, EETs have
been shown to effectively reduce the myocardial infarct size.9
Therefore, the inhibition of sEH, as a strategy to elevate the levels
of EETs, could have a significantly beneficial impact on diseases such
as hypertension and atherosclerosis.1 Recently, several sEH inhibi-
tors have demonstrated a blood pressure lowering effect in an angio-
tensin II-induced hypertensive rat model.10 They also protected the
rats’kidneys from hypertension-induced damage.11 Arete has devel-
oped an sEH inhibitor for metabolic syndromes that will soon enter
Phase II clinical trials.12 These advancements prompted us to dis-
cover highly selective, orally bioavailable, and structurally novel
sEH inhibitors for an antihypertensive indication.
Our design of sEH inhibitors was informed by the X-ray crystal
structure of sEH (1ZD3 (PDB code): human sEH 4-(3-cyclohexylu-
riedo)-butyric acid complex).13 It has been revealed that the hydro-
lase catalytic site of sEH contains two tyrosine residues (Tyr381
and Tyr465) which act as hydrogen bond donors interacting with
the epoxide oxygen of EETs, thereby facilitating the epoxide ring
opening by Asp333. The resulting ester is then rapidly hydrolyzed
into DHETs. Amides and ureas are excellent mimics of epoxides in
that the amide or urea carbonyl oxygen acts as a hydrogen bond
EETs elicit a wide range of physiological effects.4 In particular,
EETs display vasodilatory effects in conduit vessels, renal afferent
arterioles, and coronary resistance vessels indicating a potential
role of EETs in blood pressure regulation and myocardial perfu-
sion.5 In rats, 14,15-EET transiently reduced arterial blood pres-
sure.6 In addition to dilating coronary arteries and arterioles,7
EETs also modulate adhesion molecule expression, platelet aggre-
gation, vascular smooth muscle migration, and thrombolytic prop-
erties thus representing
a
protective mechanism against
* Corresponding author. Tel.: +1 732 594 1755; fax: +1 732 594 9473.
E-mail address: hong_shen@merck.com (H.C. Shen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.036

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3398–3404
3399
Table 1
In vitro SAR on sEH and mEH inhibition, DHET production, CYP and ion channel inhibitions
Compds
sEH IC50 (h, r), nMa
DHET, IC50, nMb
mEH, IC50
,
l
Mc
CYP2C9, 2D6, 3A4 IC50
,
lM
DLZ, IKr Nav1.5, IC50, lM
O
Ph
N
H
3, 9
3
>100
ꢀ10,
>10,
>10
>30,
14,
>10
O
O
N
entA-1a
11, 29
3
>100
>10,
>10,
>10
>30,
17,
>10
O
O
N
O
entB-1a
1, 7
—
>100
<10,
<10,
<10
4,
0.2,
>10
N
O
N
rac-1b
4, 21
<1
15
>10,
>10,
>10
28,d
4,
>10
O
N
N
Me
O
N
rac-1c
3, 13
<1
>100
ꢀ10,
>10,
>10
26,d
2,
>10
O
H
N
Me
N
N
O
N
rac-1d
4, 12
—
—
>10,
>10,
>10
>26,
4,
>10
O
S
O
O
O
N
rac-1e
4, 11
3, 18
—
>100
>100
<10,
>10,
>10
6,
15,
>10
O
N
rac-1f
<1
<10,
>10,
>10
2,
7,
>10
F
O
N
rac-1g
3, 23
<1
24
>10,
>10,
>10
28,
15,
>10
O
S
O
O
N
ent-1h
(continued on next page)

3400
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3398–3404
Table 1 (continued)
Compds
sEH IC50 (h, r), nMa
DHET, IC50, nMb
mEH, IC50
,
l
Mc
CYP2C9, 2D6, 3A4 IC50
,
lM
DLZ, IKr Nav1.5, IC50, lM
O
Ph
N
H
11, 49
3
55
>10,
>10,
>10
>30,d
2,
>10
O
S
O
O
N
ent-1i
8, 20
4
>100
—,
>10,
>10
>30,d
>30,
>10
O
N
O
N
ent-1j
8, 31
8
>100
>100
>10,
>10,
>10
>30,
>30,
>10
OH
N
O
N
ent-1k
5, 21
<1
>10,
>10,
>10
20,d
>30,
>10
N
O
N
ent-1l
a
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
Studies were performed with HEK293 cells.
Human mEH was used.
b
c
d
Cav1.2 functional assay was performed.
acceptor for Tyr381 and Tyr465, whereas the hydrogen atom of
be performed with compounds lacking any obvious off-target
activity that could impact vascular tone.
amide or urea N–H group serves as a hydrogen bond donor to
Asp333. Therefore, various ureas and amides have been developed
as sEH inhibitors.14
We designed and prepared novel spirocyclic piperidine-based
trisubstituted ureas as sEH inhibitors. Herein, we report the struc-
ture–activity relationship (SAR), off-target profiles, and target
engagement of this class of compounds. Furthermore, we report
data from a blood pressure study conducted in spontaneously
hypertensive rats (SHRs), a common preclinical hypertension mod-
el that responds to various antihypertensive drugs.
To evaluate sEH inhibitors, human and rat sEH inhibition, and
human whole cell DHET production were utilized to determine
their intrinsic enzymatic and cellular activities. Counterscreening
were routinely performed on microsomal epoxide hydrolase
(mEH), several CYP enzymes and selected ion channels. Due to
its important roles in xenobiotic detoxication and steroid metabo-
lism, mEH inhibition was a key safety concern. Additionally, many
ureas have been reported to inhibit this enzyme.15 Amongst the
CYP2D6, 3A4, and 2C9 counterscreen targets, the latter is of partic-
ular importance due to its role in producing EETs via the monoox-
ygenase-mediated epoxidation of arachidonic acid;3 CYP2C9
inhibition could result in a diminution in EET elevation. Lastly, ef-
fects of compounds on ion channels, including potassium (IKr), cal-
cium (DLZ binding or Cav1.2 functional assays) and sodium
(Nav1.5) channels, were scrutinized due to their potential con-
founding cardiovascular effects.16 To the best of our knowledge,
previous literature reports did not disclose their status of ion chan-
nel or CYP inhibitory effects. We strongly believe that a proper
evaluation of the antihypertensive actions of sEH inhibitors should
The SAR study was conducted in two directions including the
left (Table 1) and right portion of the scaffold (Table 2) shown in
brown and blue in Figure 1 respectively. Racemic compounds
contain the prefix ‘rac’ while enantiomers are designated with
‘ent’.
In our first set of studies, the trans-2-phenyl-1-cyclopropylami-
no group was adopted as the common right-hand motif (Table 1).
All compounds in Table 1 demonstrated excellent nanomolar IC50
s
against the human sEH. In the rat sEH inhibition assay, compounds
were typically 2–10-fold less active. The whole cell DHET assay re-
sults were in agreement with enzyme activity suggesting good cell
permeability of compounds. A large window between sEH and
mEH potency (>10,000-fold) was achieved for most analogs. In
addition, ureas derived from one enantiomer of trans-2-phenyl-1-
cyclopropylamine were consistently 3–8-fold more active than
their enantiomeric counterparts, and both enantiomers gave very
similar off-target profiles (data not shown).
The two enantiomers of 1a were designated as entA-1a and
entB-1a. Despite a threefold difference of the IC50 in both human
and rat sEH inhibition assays, they displayed similar off-target pro-
files and whole cell activity in DHET production inhibition. The
chroman or chromanone moiety could be substituted with various
groups ranging from heterocycles (1b, 1c) and aminoheterocycles
(1d) to halides (1g) and methylsulfone (1e) without compromising
sEH potency. Unfortunately, these substitutions were found to
introduce moderate potassium channel (IKr) activity. The replace-
ment of chromanones with chromans did not significantly alter
sEH IC50s, but, in certain cases, ion channel activity was notably

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3398–3404
3401
Table 2
In vitro SAR on sEH and mEH inhibition, DHET production, CYP and ion channel inhibitions
Compds
sEH IC50 (h, r), nMa
DHET, nMb
mEH,
l
Mc
CYP2C9, 2D6, 3A4 IC50
,
lM
DLZ, IKr Nav1.5,IC50, lM
A
R
O
N
O
4, 10
2, 19
1, 4
<1
<1
<1
22
>10,
>10,
>10
2,
1,
>10
CF3
2a R = H, A = H, H
>100
>100
7.6,
>10,
>10
>30,
25,
>10
CF3
2b R = SO2Me, A = H, H
ꢀ10,
ꢀ10,
>10
>30,
0.8,
>10
Cl
Cl
2c R=MeSO2, A = H, H
7, 10
3
>100
<10,
<10,
>10
3,
6,
<10
2d R=F, A = H, H
3, 9
3
>100
ꢀ10,
>10,
>10
>30,
14,
>10
N
H
entA-1a R = H, A = O
5, 39
—
26
>10,
>10,
>10
9,
12,
ꢀ1
H
N
rac-2e R = H, A = O
20, 35
30
28
<10,
>10,
>10
6,
6,
<10
H
N
rac-2f R = H, A = O
8, 28
7, 21
—
—
>100
<10,
>10,
<10
5,
5,
<10
HN
rac-2g R = H, A = O
10
<10,
>10,
>10
18,
3,
>10
H
N
rac-1,3-trans-2h
R = SO2Me, A = O
a
Values are based on one or two experiments, each in triplicate, and within 20% deviation upon repeat.
Studies were performed with HEK293 cells.
Human mEH was used.
b
c

3402
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3398–3404
G
O
2
1.8
1.6
1.4
1.2
1
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
A
E
B
D
DHET
1a
p
H
N
N
R
m
O
Figure 1. Pharmacophore of urea analogs.
0.8
0.6
0.4
0.2
0
changed. For example, the DLZ binding affinity of chroman 1f was
higher than either chromanone entA-1a or entB-1a. On the other
hand, the IKr activity of chroman 1h was less than that of chroma-
none 1e. The piperidine moiety could be replaced by a homopiperi-
dine group, accompanied by a significant increase of IKr activity
(1i). Lastly, the replacement of chroman benzene moiety with pyr-
idine, shown in analogs 1j–l, provided excellent enzyme and whole
cell sEH inhibition, yet devoid of significant off-target activity
against CYPs and ion channels. The pyridine regioisomers shown
in 1j–l provided at least a threefold greater potency of sEH inhibi-
tion than the other three regioisomers (data not shown).
In subsequent studies, the right fragment (in blue) of the urea
structure shown in Figure 1 was probed. After screening a variety
of primary amines or anilines, it was found that phenyl cyclopropyl
amine (ent-1a) gave the best overall profile, on the basis of sEH,
CYP, DLZ and IKr activities.
Predose 0.50
2.00
6.00
24.00
Time (hr)
Figure 2. Ex vivo whole blood target engagement of rac-1a measured by 14,15-
DHET production (50 mpk, po).
rac-1a at a 50 mpk dose (po). At 0 h, 0.5 h, 2 h, 6 h and 24 h, blood
was drawn and combined with 14,15-EET; 14,15-DHET and the
compound concentration was then assayed. An excellent inverse
correlation was established between the plasma level of rac-1a
and DHET production (Fig. 2). The drug level remained above
0.8
post dose.
At a 300 mpk oral dose, which should presumably provide even
lM and DHET production was suppressed >95% from 0.5–6 h
To prioritize compounds for in vivo PD studies, an in vitro DHET
production assay was conducted in which each of the compounds
was incubated in rat blood for 1 h before the addition of substrate
(14,15-EET) and the measurement of DHET production rate.
Despite similar enzymatic and cellular activity, inhibitors were dif-
ferentiated in this assay, presumably reflecting their differential
affinity for blood proteins. Compounds rac-1a, ent-1j, ent-1k and
ent-1l clearly exhibited more potent inhibition of DHET production
than analogs rac-1f, rac-1h, 2a and 2b (Table 3).
The pharmacokinetic (PK) profiles of five analogs in rats are
presented in Table 4. Excellent bioavailability, moderate to good
half life, and good to excellent oral exposure were observed for
all compounds. In addition, most compounds displayed low clear-
ance except rac-1a. Despite the inferior PK of compound rac-1a re-
flected by its relatively lower oral exposure and higher clearance,
the excellent whole blood activity of rac-1a seemed to indicate that
this compound had a good free fraction in plasma. The high free
fraction of rac-1a could account for its higher apparent clearance.
Therefore, compound rac-1a was selected for an ex vivo target
engagement evaluation. In this study, SHRs were treated with
more substantial sEH engagement than demonstrated above at
50 mpk, rac-1a failed to induce statistically significant change of
systolic or diastolic blood pressure in SHRs (Fig. 3). A parallel
one-day PK study at 300 mpk dose showed that the drug level of
rac-1a remained above 1.7
dosing (3.6 M at 2 h, 2.7
l
M during the period of 2–24 h post
l
lM at 5 h, and 1.7 M at 24 h. Based
l
on the whole blood target engagement results depicted in Figure
2, the DHET production in blood should be reduced more than
95% compared with the control group throughout the PD study
timeframe.
Heart rate and pulse pressure differences between the treated
and vehicle (labrasol) groups were also negligible (Fig. 3).
The representative synthesis of
a spirocyclic secondary
amine-derived urea is shown in Scheme 1.17 An efficient one-step
conversion of carboxylic acid 3 into methyl ketone 4, followed by a
pyrrolidine-mediated spirocyclization afforded intermediate 6. The
ketone group in 6 was then reduced to CH2 via a reduction, dehy-
dration, and hydrogenation sequence. The resulting amine 9
reacted with the p-nitrophenyl carbamate generated in situ from
the phenyl cyclopropyl amine enantiomer 10 (i.e., the enantiomer
Table 3
In vitro DHET production in whole rat blooda
Compd
rac-1a
rac-1f
rac-1h
ent-1j
ent-1k
ent-1l
2a
2b
DHET production(% of vehicle)
8.6
20
32
12
7.3
9.2
48
66
a
DHET production was measured at 1 h after the addition of 14,15-EET and compound at 1
l
M concentration.
Table 4
Rat PKa
Compd
F (%)
Cl (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
AUCNpo
(lM h.kg/mg)
rac-1a
rac-1e
Rac-1h
2b
61
49
86
61
67
47
1
0.7
0.9
0.7
3.1
0.2
0.23
1.6
0.15
1.5
2.6
3.9
18
2.6
0.96
20
44
26
31
2c
a
Formulation: 1 mg/mL ethanol/PEG/water (20:40:40). iv dose: 1 mg/kg (n = 2). po dose: 2 mg/kg (n = 3). Blood concentration was determined by LC/MS/MS following
protein precipitation with acetonitrile.

H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3398–3404
3403
NBoc
Systolic Blood Pressure
OH
O
OH
OH
O
20
10
a
b
N
N
N
O
Boc
N
O
O
4
3
6
5
0
NBoc
O
NH
-10
-20
-30
O
c
e
d
Labrasol
N
N
Rac-1a 300 mpk
OH
8
7
0
0
0
2
4
6
8
8
8
O
NH
Time (h)
f
O
N
N
H
O
N
Diastolic Blood Pressure
N
9
20
10
ent-1l
H2N
ent-10
Scheme 1. Reagents and conditions: (a) MeMgBr, HCO2Me, THF, reflux, 2 h, 87%; (b)
pyrrolidine, 5, MeOH, reflux, 0.5 h, 75%; (c) NaBH4, EtOH, 0 °C to rt, 2 h, 99%; (d) p-
toluenesulfonic acid, toluene, Dean-Stark, 24 h; (e) H2, Pd/C, MeOH,16 h; (f) i-
Pr2NEt, CH2Cl2, ent-10, p-nitrophenyl chloroformate, 12 h, rt, then 9, 2 h, rt, 4.5% in
three steps.
0
-10
Labrasol
-20
-30
Rac-1a 300 mpk
2
4
6
References and notes
Time (h)
1. For a leading review: Newman, J. W.; Morisseau, C.; Hammock, B. D. Prog. Lipid
Res. 2005, 44, 1.
2. Pacifici, G. M.; Temellini, A.; Giuliani, L.; Rane, A.; Thomas, H.; Oesch, F. Arch.
Toxicol. 1988, 62, 254.
3. Yu, Z.; Xu, F.; Huse, L. M.; Morisseau, C.; Draper, A. J.; Newman, J. W.; Parker, C.;
Graham, L.; Engler, M. M.; Hammock, B. D.; Zeldin, D. C.; Kroetz, D. L. Circ. Res.
2000, 87, 992.
4. For a leading review: Spector, A. A.; Fang, X.; Snyder, G. D.; Weintraub, N. L.
Prog. Lipid Res. 2004, 43, 55.
5. For a leading review: Larsen, B. T.; Gutterman, D. D.; Hatoum, O. A. Eur. J. Clin.
Invest. 2006, 36, 293.
6. Lin, W. K.; Falck, J. R.; Wong, P. Y. Biochem. Biophys Res. Commun. 1990, 167, 977.
7. Fisslthaler, B.; Popp, R.; Kiss, L.; Potente, M.; Harder, D. R.; Fleming, I.; Busse, R.
Nature 1999, 401, 493.
8. Spiecker, M.; Liao, J. K. Arch. Biochem. Biophys. 2005, 433, 413.
9. Gross, G. J.; Hsu, A.; Flack, J. R.; Nithipatikom, K. J. Mol. Cell Card. 2007, 42,
687.
Change in Pulse Pressure
10
8
6
4
2
Labrasol
Rac-1a 300 mpk
0
-2
-4
-6
-8
-10
2
4
6
Time (h)
10. (a) Imig, J. D.; Zhao, X.; Zaharis, C. Z.; Olearczyk, J. J.; Pollock, D. M.; Newman, J.
W.; Kim, I.-H.; Watanabe, T.; Hammock, B. D. Hypertension 2005, 46, 975; (b)
Jung, O.; Brandes, R. P.; Kim, I.-H.; Schmidt, R.; Hammock, B. D.; Busse, R.;
Fleming, I. Hypertension 2005, 45, 759; (c) Imig, J. D.; Zhao, X.; Capdevila, J. H.;
Morisseau, C.; Hammock, B. D. Hypertension 2002, 39, 690.
11. Zhao, X.; Yamamoto, T.; Newman, J. W.; Kim, I.-H.; Watanabe, T.; Hammock, B.
D.; Stewart, J.; Pollock, J. S.; Pollock, D. M.; Imig, J. D. J. Am. Soc. Nephrol. 2004,
15, 1244.
% Change in Heart Rate
40
30
Labrasol
20
Rac-1a 300 mpk
10
12. http://www.aretetherapeutics.com.
13. Gomez, G. A.; Morisseau, C.; Hammock, B. D.; Christianson, D. W. Protein Sci.
2006, 15, 58.
0
-10
-20
-30
-40
14. (a) Delombaert, S.; Eldrup, A. B.; Kowalski, J. A.; Mugge, I. A.; Soleymanzadeh,
F.; Swinamer, A. D.; Taylor, S. J. WO2007106705 A1, 2007; (b) Ingraham, R. H.
WO2007106706 A1, 2007; (c) Hammock, B. D.; Jones, P. D.; Morisseau, C.;
Huang, H.; Tsai, H.-J.; Gless, R. WO2007106525 A1, 2007; (d) Hammock, B. D.;
Kim, I.-H.; Morisseau, C.; Watanabe, T.; Newman, J. W. WO2006045119 A2,
2006; (e) Eldrup, A. B.; Farrow, N. A.; Kowalski. J. A.; Delombaert, S.; Mugge, I.
A.; Soleymanzadeh, F.; Swinamer, A. D.; Taylor, S. J. WO2007098352 A2, 2007;
(f) Takahashi, H.; Ota, T.; Kakinuma, H. WO2007043652 A1, 2007; (g) Ota, T.;
Kakahashi, H.; Kakinuma, H.; Busujima, T. WO2007043653 A1, 2007; (h)
Ingraham, R. H.; Proudfoot, J. R. WO2003002555 A1, 2003; (i) Cardozo, M. G.;
Ingraham, R. H. WO2006121684 A2, 2006; (j) Cywin, C. L.; De Lombaert, S.;
Eldrup, A. B.; Ingraham, R. H.; Taylor, S.; Soleymanzadeh, F. WO2006121719
A2, 2006; (k) Kroetz, D.; Zeldin, D. C.; Hammock, B. D.; Morisseau, C.
US20030119900 A1, 2003; (l) Kroetz, D.; Zeldin, D. C.; Hammock, B. D.;
Morisseau, C. US20040092487 A1, 2004; (m) Hammock, B. D.; Kim, I.-H.;
Morisseau, C.; Watanabe, T.; Newman, J. W. US20050026844 A1, 2005; (n)
Erickson, D.; Hoffman, A. F.; Warren, T. C. WO200023060, 2000; (o) Hwang, S.
H.; Tsai, H.-J.; Liu, J.-Y.; Morisseau, C.; Hammock, B. D. J. Med. Chem. 2007, 50,
3825; (p) Kim, I.-H.; Morrisseau, C.; Watanabe, T.; Hammock, B. D. J. Med. Chem.
2004, 47, 2110; (q) Kim, I.-H.; Heirtzler, F. R.; Morisseau, C.; Hishi, K.; Tsai, H.-J.;
Hammock, B. D. J. Med. Chem. 2005, 48, 3621; (r) Morisseau, C.; Newman, J. W.;
Tsai, H.-J.; Baecker, P. A.; Hammock, B. D. Bioorg. Med. Chem. Lett. 2006, 16,
5439; (s) Li, H.-Y.; Jin, Y.; Morisseau, C.; Hammock, B. D.; Long, Y.-Q. Bioorg.
Med. Chem. Lett. 2006, 14, 6586.
0
2
4
6
8
Time (h)
Figure 3. Blood pressure, pulse and heart rate effects of rac-1a in SHRs (300 mpk,
po) (green curve: labrasol; blue curve: rac-1a).
leading to entA-1a) to give the desired urea ent-1l. Analogous
synthetic routes led to other analogs shown in Tables 1 and 2.
In conclusion, we have identified a novel spirocyclic amine-de-
rived urea class as potent and selective sEH inhibitors with good PK
and ex vivo target engagement. However, rac-1a, a representative
compound of this series, failed to elicit a significant blood pressure
lowering effect in SHRs. More structurally diverse sEH inhibitors
will be reported in due course.

3404
H. C. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3398–3404
15. Morisseau, C.; Newman, J. W.; Dowdy, D. L.; Goodrow, M. H.; Hammock, B. D.
Chem. Res. Toxicol. 2001, 14, 409.
16. (a) Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. J. Med. Chem. 2006, 49,
5029. and references therein; (b) Fisslthaler, B.; Hinsch, N.; Chataigneau, T.;
Popp, R.; Kiss, L.; Busse, R.; Fleming, I. Hypertension 2000, 36, 270; (c)
Viswanathan, P. C.; Balser, J. R. Trends Cardiovasc. Med. 2004, 14, 28.
6.2 mmol) and the resulting mixture was stirred for 2 h at rt. The reaction was
quenched by the addition of saturated NaHCO3 solution, and extracted with
EtOAc twice, the combined organic phase was washed with saturated NaHCO3
solution, dried over Na2SO4 to give 7 (0.98 g, 31 mmol, 99%) as an oil. To the
solution of
7 (260 mg, 0.73 mmol) in 40 mL toluene was added p-
toluenesulfonic acid (1.3 g, 7.2 mmol) and the solution was heated at reflux
in a Dean-Stark trap overnight. After removing the solvent, the residue was
treated with methanol (50 mL) and 10% palladium on carbon (50 mg), and the
resulting suspension was subjected to hydrogenation overnight. After
filtration, the solvent was removed and the residue was partitioned between
30% of isopropanol in 10% Na2CO3 solution. The organic phase was dried over
Na2SO4 and concentrated to give 9 as a crude product. To the solution of p-
nitrophenyl chlorofomate (163 mg, 0.8 mmol) in THF (5 mL) at 0 °C was added
17. To the solution of methyl magnesium bromide in THF (86 mL, 3 M, 260 mmol)
was added THF (100 mL). To the resulting solution at 64 °C was added a
solution of 3-hydroxypicolinic acid (7.2 g, 52 mmol) and triethylamine (26 g)
in 100 mL THF dropwise. The resulting mixture was heated under reflux for 2 h
before it was cooled down to 5 °C. At this temperature, to the mixture was
added 6.4 mL of methyl formate followed by quenching with 3 N HCl at 5 °C to
pH 3–4. The mixture was extracted with THF twice. The combined THF phase
was concentrated to 10 mL before the addition of 10 mL of water. The mixture
continued to concentrate until the removal of THF. The aqueous solution was
standing in a À10 °C refrigerator for 2 h. The resulting solid was collected by
filtration and then dissolved in Et2O. The Et2O solution was dried over Na2SO4,
concentrated to get 4 as a grey solid. (6.2 g, 45 mmol, 87%). The solution of 4
(6.2 g, 45 mmol), 5 (9.0 g, 45 mmol) and pyrrolidine (4.8 mL, 57 mmol) in
methanol (200 mL) was heated at reflux for 20 min. After removing the
solvents, the residue was purified on silica gel using 40–60% EtOAc/hexanes to
a solution of ent-10 (106 mg, 0.8 mmol) and diisopropylethyl amine (180 lL,
1.0 mmol) in DCM (5 mL). After stirring overnight, the solution was transferred
to a flask containing 9. The resulting solution was stirred at rt for 2 h. After
concentration, the residue was purified on silica gel chromatography using 40–
100% EtOAc/hexanes to give 12 mg of ent-1l (12 mg, 0.033 mmol, 4.5% overall
yield in 3 steps form 7 to ent-1l). 1H NMR (acetone-d6, 500 MHz) d = 8.09–7.08
(m, 1H), 7.26–7.10 (m, 7H), 6.38 (s, 1H), 3.82 (dd, J = 13.0, 1.5 Hz, 2H), 3.21 (t,
J = 10.8 Hz, 2H), 2.90 (t, J = 3.0 Hz, 2H), 2.83 (t, J = 3.2 Hz, 1H), 1.99–1.93 (m,
3H), 1.74 (d, J = 13.6 Hz, 2H), 1.65–1.59 (m, 2H), 1.19–1.09 (m, 2H); m/z: calcd.
for C22H25N3O2: 363.46; found [M+H+]: 364.63.
afford
6 (10.7 g, 34 mmol, 75%) as a solid. To the solution of 6 (0.98 g,
3.1 mmol) in EtOH (50 mL) at 0 °C was added sodium borohydride (0.23 g,