Design, synthesis and evaluation of non-urea inhibitors of soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2011.10.074

Inhibition of soluble epoxide hydrolase (sEH) has been proposed as a new pharmaceutical approach for treating hypertension and vascular inflammation. The most potent sEH inhibitors reported in literature to date are urea derivatives. However, these compounds have limited pharmacokinetic profiles. We investigated non-urea amide derivatives as sEH inhibitors and identified a potent human sEH inhibitor 14-34 having potency comparable to urea-based inhibitors.

Full text of DOI:10.1016/j.bmcl.2011.10.074

Bioorganic & Medicinal Chemistry Letters 22 (2012) 601–605
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of non-urea inhibitors of soluble
epoxide hydrolase
Stevan Pecic a, , Shi-Xian Deng a, Christophe Morisseau b, Bruce D. Hammock b, Donald W. Landry a
⇑
a Department of Medicine, Columbia University, 650 W 168th Street, BB 1029, New York, NY 10032, USA
b Department of Entomology and UCD Cancer Center, University of California, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of soluble epoxide hydrolase (sEH) has been proposed as a new pharmaceutical approach for
treating hypertension and vascular inflammation. The most potent sEH inhibitors reported in literature to
date are urea derivatives. However, these compounds have limited pharmacokinetic profiles. We inves-
tigated non-urea amide derivatives as sEH inhibitors and identified a potent human sEH inhibitor 14–34
having potency comparable to urea-based inhibitors.
Received 22 September 2011
Revised 20 October 2011
Accepted 21 October 2011
Available online 28 October 2011
Published by Elsevier Ltd.
Keywords:
Soluble epoxide hydrolase
sEH
Non-urea inhibitors
Hypertension
Anti-inflamatory
Soluble epoxide hydrolase (sEH) is an enzyme that is involved
in the metabolism of lipid epoxides.1 This enzyme is found in var-
ious mammalian tissues and is mostly located in liver, kidneys and
vascular tissues.2 It converts endogenous substrate—epoxyeicosar-
ienoic acids (EETs)—to dihydroxy eicosatrienoic acids (DHETs),
which show abolished or diminished or changed biological activ-
ity.3 EETs act as vasodilators in various arteries (renal, cerebral,
and coronary),4 protect from ischemic injury5 and manifest anti-
inflammatory properties.6 sEH inhibition increases cellular EETs
levels and promote this activity. Several preclinical studies suggest
that inhibition of sEH may represent a novel approach to treat
hypertension, organ protection and inflammation.7–10 Further-
more, it has been shown that sEH inhibition reduces pain in several
animal models.11
Initial sEH inhibitors with IC50 in the lower nanomolar range in-
cluded N,N0-disubstituted ureas, N,N0-dicyclohexylurea (DCU) and
N-adamantyl-N0-cyclohexylurea (ACU) (Fig. 1).12 However, poor
water solubility and limited in vivo studies13 led to a design of the
second generation of sEH inhibitors, 12-(3-adamantan-1-ylurei-
do)dodecanoic acid (AUDA) and 1-adamantan-1-yl-3-{5-[2
-(ethoxyethoxy) ethoxy]pentyl}urea (AEPU) with improved water
solubility and maintained inhibition.14,15 However, these inhibitors
suffered from rapid metabolism in vivo.16 Recent studies have
focused on piperidine-based di- and trisubstituted ureas, such as
N-(1-(2,2,2-trifluoroethanoyl)piperidin-4-yl)-N0-(adamant-1-yl)ur-
ea (TPAU) and N-(1-acetylpiperidin-4-yl)-N0-(adamant-1-yl)urea
(APAU).17,18 Some of these piperidine-based compounds possess
low nano to picomolar activity and good pharmacokinetic proper-
ties in different animal models.17–21
These extensive structure–activity relationship (SAR) studies
suggested that the pharmacophore for sEH inhibitors should in-
clude a central urea moiety for hydrogen bonding to two tyrosine
O
O
O
N
R
R
N
H
N
H
N
H
N
H
DCU (R=cHexyl)
IC50=52 nM
APAU(R=CH3)
TPAU (R=CF3)
IC50=7.0 nM
IC50=1.1 nM
ACU (R=Adamantyl) IC50=1.6 nM
O
OH
N
H
N
H
O
AUDA
AEPU
IC50=3.0 nM
O
O
O
N
H
N
H
O
IC50=14 nM
⇑
Corresponding author. Tel.: +1 212 3055839; fax: +1 212 3053475.
E-mail addresses: ar2230@columbia.edu, dwl1@columbia.edu (S. Pecic).
Figure 1. Chemical structures of known sEH inhibitors.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.10.074

602
S. Pecic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 601–605
compounds were prepared by standard synthetic methods from
Cl
O
O
commercially available starting materials (Scheme 1). Thus, per
Scheme 1, cycloheptylamine 3 was condensed with Boc-isonipe-
cotic acid 4 under standard peptide coupling conditions. The Boc
group of the amide 5 was removed with TFA to afford amine 6,
which was reacted with 2,4,6-trimethylbenzoyl chloride to yield
amide analog 7. In order to synthesize the methylene analog of
compound 2, amine 6 was treated with mesitaldehyde under
reductive amination conditions, which gave the final product 8.
The reversed amide analog was synthesized in similar fashion.
Commercially available cycloheptanecarboxylic acid 9 and 4-ami-
no-1-Boc-piperidine 10 were condensed under EDC coupling con-
ditions to afford amide 11, which was subsequently deprotected
to furnish amine 12. The amine 12 was sulfonylated with 2-mes-
itylenesulfonyl chloride to afford analog 13 (Scheme 2). This first
set of compounds demonstrated that the sulfonamide group is
important for recognition by sEH, since replacement with an amide
group, as in compound 7, resulted in a sixfold decrease in binding
affinity (IC50 = 46.1 nM). Compound 8, which contains a methylene
group, suffered a 15-fold loss of inhibitory potency (IC50 = 120 nM).
The effect of reversing amide group in compound 13 resulted in a
small, 2.5-fold decrease in the binding affinity for sEH
(IC50 = 22 nM), supporting previous studies that the proton in NH
(both in urea and amide sEH inhibitors) is important in sEH inhibi-
tion, ostensibly forming a salt bridge with the catalytic nucleophile
Asp333.26
These results returned our attention to the SAR of the right-hand
side of the sulfonamide group of the original lead compound 2. Thus,
amine 6 was reacted with a variety of sulfonyl chlorides to yield
products 14–1 to 14–51. This group of analogs will allow us to eval-
uate the role of different substituent on this part of the molecule. We
introduced as well several different polar groups on the aromatic
ring in order to obtain compounds that will be easier to formulate
and administer. The results are summarized in Table 1. As illustrated
in Table 1, the diverse right-hand side modifications led to improved
or similar potency (14–7, 14–27, 14–31 and 14–34) over the original
sulfonamide analog 2 and several structure–activity relationships
can be discerned in this series of analogs. First, the nonpolar group
N
H
N
H
N
O
N
O
Cl
S
S
O
O
1
IC50=20 nM
2
IC50=7.9 nM
Figure 2. Chemical structures of non-urea sEH inhibitors.
residues (Tyr381 and Tyr465) and one Asp333 residue—all three
located in the hydrolase catalytic pocket of sEH.22
Several common urea modifications such as thiourea, sulfonyl
urea, amide and aminomethylene amide have been incorporated
into central pharmacophore in order to maintain similar binding
profile to that of urea but improve potential therapeutic
applications.23
We sought to identify potent, selective non-urea sEH inhibitors
effective in vivo with good metabolic stability and pharmacoki-
netic and distribution properties, to test as an anti-hypertensive
and anti-inflammatory drug candidate. A sensitive fluorescent
based assay29 was employed to determine IC50 values. Using high
throughput screening we have previously reported a series of
non-urea sEH inhibitors with low micromolar to nanomolar po-
tency.24 From the compound collection provided by the NIH Road-
map project we identified sulfonyl isonipecotamide 1, a potent sEH
inhibitor (IC50 = 20 nM).25 In addition, we synthesized a secondary
library of compounds based on 1 and SAR revealed the most potent
non-urea sEH inhibitor in that study, compound
2 with
IC50 = 7.9 nM (Fig. 2).24 Herein we report design, synthesis and bio-
logical evaluation of non-urea sEH inhibitors based on compound
2.
Our evaluation of the SAR for 2 started with replacement of the
sulfonamide moiety with both an amide and methylene group, in
order to better understand what structural feature of the ‘second-
ary’ pharmacophore is important for sEH inhibition. Furthermore,
amides have in general better properties for formulation. Addition-
ally, we designed a compound in which the sulfonamide group was
retained, but the central amide moiety was reversed. These three
COOH
N
Boc
b
X
NH2
NH
N
H
N
N
H
N
a
c or d
f
H
N
O
O
O
Boc
3
4
5
6
7: X= -CO- IC50=46.1 nM
8: X= -CH2- IC50=120 nM
e
O
O
N
O
R
S
N
R'
H
N
H
N
14-1 to 14-51
15-1 to 15-12
O
O
Scheme 1. Reagents and conditions: (a) EDC, CH2Cl2, rt, 24 h, 68%; (b) TFA, CH2Cl2, rt, 24 h, 89%; (c) 2,4,6-trimethylbenzoyl chloride, Et3N, CH2Cl2, rt, 24 h, 85%; (d)
mesitaldehyde, NaBH(OAc)3, CH2Cl2, rt, 12 h, 82%; (e) R-SO2Cl, Et3N, CH2Cl2, rt, 24 h, 70–91%; (f) R0OCl, Et3N, CH2Cl2, rt, 24 h, 85–92%.
O
N
O
NH2
N
Boc
b
COOH
S
O
NH
O
N
O
a
c
N
H
N
H
N
H
Boc
9
10
11
12
13 IC50=22 nM
Scheme 2. Reagents and conditions: (a) EDC, CH2Cl2, rt, 24 h, 65%; (b) TFA, CH2Cl2, rt, 24 h, 90%; (c) 2,4,6-trimethylbenzoylsulfonyl chloride, Et3N, CH2Cl2, rt, 24 h, 85%.

S. Pecic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 601–605
603
Table 1
The biological results for sulfonamide analogs 14–1 to 14–52
R
O
N
S
O
NH
O
a,b
Compound
R
IC50 (nM)
Compound
R
IC50 (nM)
Br
Cl
F
14–1
340
14–27
5.4
F3C
Cl
F
14–2
14–3
960
490
14–28
14–29
15
18
F
F
F
14–4
14–5
450
950
14–30
14–31
27
7.7
O2N
Br
Br
Br
14–6
14–7
510
6.7
14–32
14–33
12
56
Br
CF3
F
14–8
14–9
14–10
78
24
12
14–34
14–35
14–36
1.6
1700
120
O2N
NO2
OCH3
O
14–11
14–12
450
340
14–37
14–38
150
32
COOH
NO2
Br
14–13
62
14–39
17
CF3
CN
OCH3
14–14
14–15
2100
240
14–40
14–41
360
OCH3
COOH
2000
CF3
Br
iPr
F
iPr
14–16
14–17
2100
560
14–42
14–43
16
iPr
Cl
6000
COOH
(continued on next page)

604
S. Pecic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 601–605
Table 1 (continued)
a,b
Compound
R
IC50 (nM)
Compound
R
IC50 (nM)
9.6
NCO
14–18
14–19
150
14–44
NO2
O
50000
14–45
224
O
OCF3
14–20
14–21
1100
140
14–46
14–47
35
OH
870
S
SO2CH3
Cl
14–22
14–23
14–24
13000
570
14–48
14–49
14–50
4200
710
Cl
Br
S
S
O
O
COOH
1300
6000
OCH3
S
F
14–25
1900
470
14–51
21
Cl
S
Cl
14-26
a
Reported IC50 values are the average of three replicates. The fluorescent assay as performed here has a standard error between 10 and 20% suggesting that differences of
two fold or greater are significant.27
b
t-AUCB that has an IC50 between 1 and 2 nM was used as positive control.13
Table 2
The biological results for amide analogs 15–1 to 15–12
R'
O
O
N
NH
a,b
Compound
15–1
R0
IC50 (nM)
Compound
15–7
R0
IC50 (nM)
780
OCH3
F
1200
1400
1200
630
15–2
15–3
15–4
15–5
15–6
15–8
1200
200
F
NO2
Cl
15–9
Cl
15–10
15–11
15–12
1100
2000
590
CN
Br
950
F
92
a
Reported IC50 values are the average of three replicates. The fluorescent assay as performed here has a standard error between 10 and 20% suggesting that differences of
twofold or greater are significant.27
b
t-AUCB that has an IC50 between 1 and 2 nM was used as positive control.13

S. Pecic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 601–605
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activities was observed if ortho-group is deleted (14–14, 14–16, 14–
19, 14–20, 14–22, 14–24, 14–25, etc.). Para-substitution is generally
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14–49 and 14–50).
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12, as it is outlined in Scheme 1. However, the various modifications
of the aromatic ring did not improve potency further (Table 2).
In conclusion, we have successfully improved the potency of non-
urea sulfonamide analogs through SAR-guided modification. Com-
pound 14–34,30 with an IC50 of 1.6 nM, represents the most potent
non-ureasEHinhibitorreportedtodate.Pharmacokineticevaluation
and pre-clinical studies of selected potent inhibitors are planned.
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Acknowledgments
28. Kim, I. H.; Heirtzler, F. R.; Morisseau, C.; Nishi, K.; Tsai, H. J.; Hammock, B. D. J.
Med. Chem. 2005, 48, 3621.
29. IC50 Assay Conditions: Cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-
phenyloxyran-2-yl) methyl carbonate (CMNPC) was used as the fluorescent
substrate. Human sEH (1 nM) or murine sEH (1 nM) was incubated with the
inhibitor for 5 min in pH 7.0 Bis–Tris/HCl buffer (25 mM) containing 0.1 mg/mL
This work was supported in part by NIEHS R01 ES002710. B.D.H.
is a George and Judy Senior fellow of the American Asthma
Foundation.
References and notes
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least two below the IC50
.
30. Analytical data for the compound 14–34: 1H NMR (300 MHz, CDCl3): d 7.81–7.78
(d, J = 8.1 Hz, 1H) 7.12 (s, 2H), 5.35 (br, 1H), 3.92–3.89 (m, 1H), 3.72–3.68 (d,
J = 12 Hz, 2H), 2.74–2.67 (t, J = 21 Hz, 2H), 2.57 (s, 3H), 2.37 (s, 3H), 2.13–2.05
(m, 1H), 1.88–1.84 (m, 2H), 1.78–1.69 (m, 6H), 1.62–1.39 (m, 8H), 13C NMR
(75 MHz, CDCl3) d 172.6, 143.8, 138.1, 133.7, 133.0, 130.7, 126.8, 50.6, 44.8,
43.0, 35.5, 28.8, 28.3, 24.4, 21.6, 20.8; ESI-MS (M++H): 393.
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