Discovery of potent non-urea inhibitors of soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmcl.2008.09.066

Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflammation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS) and chemical modification. IC₅₀s of the most potent compounds range from micromolar to low nanomolar.

Full text of DOI:10.1016/j.bmcl.2008.09.066

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2354–2359
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent non-urea inhibitors of soluble epoxide hydrolase
Yuli Xie a, Yidong Liu a, Gangli Gong a, Deborah H. Smith b, Fang Yan b, Alison Rinderspacher a, Yan Feng a,
a
a
a
b
c
c
´
Zhengxiang Zhu , Xiangpo Li , Shi-Xian Deng , Lars Branden , Dušica Vidovic , Caty Chung ,
Stephan Schürer c, Christophe Morisseau d, Bruce D. Hammock d, Donald W. Landry a,
*
a Department of Medicine, Columbia University, 650 W 168th Street, BB 1029, New York, NY 10032, USA
b Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA
c Scientific Computing, The Scripps Research Institute, Jupiter, FL 33458, USA
d 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:
Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflam-
mation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS)
and chemical modification. IC50s of the most potent compounds range from micromolar to low
nanomolar.
Received 29 August 2008
Revised 15 September 2008
Accepted 16 September 2008
Available online 20 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
sEH
Non-urea
HTS
Nanomolar
Soluble epoxide hydrolase (sEH) plays an important role in the
metabolism of lipid epoxides. Endogenous substrates of sEH in-
clude epoxides of arachidonic acid and the epoxyeicosatrienoic
acids or EETs, the latter are effective regulators of blood pressure
and inflammation. Hydrolysis by sEH converts EETs to the corre-
sponding diols with diminished vasodilatory and anti-inflamma-
tion effects.1,2 Inhibition of sEH leads to accumulation of active
EETs and thus provides a novel approach to the treatment of hyper-
tension and vascular inflammation.3 To date, the most successful
sEH inhibitors are 1,3-disubstituted ureas, which display anti-
hypertension and anti-inflammatory effects through inhibition of
EET hydrolysis. However, urea-based inhibitors often suffer from
poor solubility and bioavailability4 and new scaffolds are needed
for therapeutic applications. Here we describe the HTS, design
and synthesis of a series of potent non-urea sEH inhibitors.
From the compound collection provided by the NIH Roadmap
project, a variety of hits were identified with low micromolar to
nanomolar potency.6 A large proportion of hits were ureas, but sev-
eral non-urea compounds showed substantial activities. The most
potent compound among these non-ureas was the sulfonyl isonip-
ecotamide 1, a nanomolar inhibitor (IC50 = 20.0 nM) with some
structural similarity to the previously reported piperidine-contain-
ing urea AMAU (Fig. 2).7
A secondary library based on 1 was prepared by modifying
either the amide head group or the sulfonamide tail group. The syn-
thesis of the sulfonamide-modified analogs is outlined (Scheme 1).
Methyl isonipecotate 2 was first protected with benzyl chlorofor-
mate, and then converted to the acid chloride 4 by hydrolyzing
the methyl ester and then treating with oxalyl chloride. Coupling
of 4 with 2,4-dichlorobenzylamine followed by palladium cata-
lyzed hydrogenation afforded amine 5, which was reacted with a
variety of sulfonyl chlorides, carbonyl chlorides and chlorofor-
mates to yield products 6-1 to 6-37.
A fluorescent assay5 was employed for HTS using recombinant
human sEH and
a water soluble a-cyanocarbonate epoxide
(PHOME) as the substrate. As shown in Figure 1, sEH-catalyzed
hydrolysis of the non-fluorescent substrate is followed by sponta-
neous cyclization to a cyanohydrin that under basic conditions,
rapidly decomposes to a highly fluorescent naphthaldehyde. Fluo-
rescence with excitation at 320 nm and emission at 460 nm was
recorded at the endpoint of the reaction cascade.
Modification of the amide head is shown in Scheme 2. Thus, 2
was treated with mesitylenesulphonyl chloride and similarly con-
verted into the acid chloride 7. In parallel, reaction of 7 with vari-
ous amines led to the products 8-1 to 8-51.
The secondary library8 was screened at concentration 200 nm
using the fluorescence assay as above. The IC50 values were deter-
mined for those compounds displaying greater than 50% inhibition
at 200 nm. The results for the tail and head modification are sum-
marized in Tables 1 and 2, respectively.
* Corresponding author. Tel.: +1 212 3055839; fax: +1 212 3053475.
E-mail address: dwl1@columbia.edu (D.W. Landry).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.066

Y. Xie et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2354–2359
2355
CN
O
OH
CN
O
O
sEH
O
O
OH
O
O
PHOME
non-fluorescent
H
CN
O
O
O
O
6-methoxy-2-naphthaldehyde
cyanohydrin
CN-
fluorescent
Figure 1. Reaction mechanism of the fluorescent assay used for the HTS.
O
Cl
O
N
H
N
H
N
H
N
O
N
S
O
Cl
1
AMAU
O
IC50=20nm
IC50=5nm
Figure 2. The structures of compounds AMAU and 1.
O
O
1.2.4-dichlorobenzylamine
CH2Cl2,Et3N,rt,24 h
O
1.LiOH,MeOH,
CBZ-Cl,Et3N
rt,24 h
O
O
Cl
NH
N
2. H2/Pd-C,MeOH,
CH2Cl2,rt,24 h
2. (COCl)2,DMF,
CH2Cl2,rt,24 h
N
CBZ
CBZ
rt,24 h
3
4
2
Cl
O
Cl
O
RCl
CH2Cl2,Et3N,rt,24 h
N
N
H
6-1 to 6-37
H
N
NH
Cl
R
Cl
5
Scheme 1. The synthesis of compounds 6-1 to 6-37.
1.Mesitylenesulphonyl
O
chloride
O
O
CH2Cl2,Et3N,rt,24 h
R'-NH2,Et3N
R'
Cl
N
H
O
2.LiOH,MeOH,
rt,24 h
N
O
CH2Cl2,rt,24 h
S
N
O
NH
S
O
O
3. (COCl)2,DMF,
CH2Cl2,rt,24 h
7
2
8-1 to 8-51
Scheme 2. The synthesis of compounds 8-1 to 8-51.
As illustrated in Table 1, the diverse tail modifications did not
lead to improved potency over the original mesitylenesulfonamide.
However, several structure activity relationships can be observed
in this series. First, the sulfonamide moiety is important for potent
inhibition. Great loss of activities was observed for amides (6-27
and 6-30) compared with the corresponding sulfonamides. Aro-
matic sulfonamides appeared to be more favorable as all tested
alkylsulfonamides were poor inhibitors. Substituents on the aro-
matic ring modulated activities synergistically. Hydrophobic alkyl
groups and halides were generally preferred with a positive effect
more pronounced at the ortho-position. For example, bromo or flu-
oro substitution at the ortho -position (6-7 and 6-10) conferred
nanomolar potency in contrast to para-substitution (6-20). Deletion
of one or both of the ortho-methyl groups in 1 led to less active
compounds 6-1 and 6-25.
In the optimization of the head group, we first screened a set of
structurally diversified amides. All disubstituted amides tested
were inactive (results not shown), suggesting the proton in NH
could be important to sEH inhibition. Previous studies with urea-
based inhibitors have shown that one NH of the urea, forming a salt

2356
Y. Xie et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2354–2359
Table 1
The biological results for the tail modification
Cl
O
N
H
N
Cl
R
a
a
Compound
R
Inhibition (%) at 200 nm
97
IC50 (nM)
Compound
R
Inhibition (%) at 200 nm
37
IC50 (nM)
O2S
CF3
Br
1
20.0b
6-19
ND
O2S
O2N
6-1
6-2
6-3
6-4
15
47
45
21
NDc
ND
ND
ND
6-20
6-21
6-22
6-23
32
28
85
97
ND
O2S
O2S
O2S
O2S
ND
O2N
O2N
O2S
O2S
SO2
164.0
52.1
O2S
O2S
tBu
Cl
NO2
SO2
6-5
34
ND
6-24
98
23.9
O2S
OCH3
N
SO2
6-6
43
63
31
63
37
66
ND
6-25
6-26
6-27
6-28
6-29
6-30
97
88
34
18
45
0
46.9
44.5
ND
S
O2S
Br
tBu
Br
O2S
6-7
91.1
ND
O2S
tBu
tBu
6-8
O2S
O2S
Cl
O
NO2
F
6-9
150.0
ND
ND
O2N
O
SO2
Br
O2S
6-10
6-11
ND
O
N
F
87.6
ND
Br
O
O2S

Y. Xie et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2354–2359
2357
Table 1 (continued)
a
a
Compound
R
Inhibition (%) at 200 nm IC50 (nM) Compound
R
Inhibition (%) at 200 nm IC50 (nM)
O
O
N S
O
6-12
45
ND
6-31
33
ND
O2S
O
SO2
OCH3
OCH3
6-13
52
200.0
6-32
71
75.3
O
S
O2N
Cl
O
O
O2S
6-14
6-15
27
13
ND
ND
6-33
6-34
41
16
ND
ND
NO2
O
O2S
S
F3C
6-16
6-17
39
ND
6-35
55
200.0
O
O
O2S
N
SO2
O2N
30
53
ND
ND
6-36
6-37
44
3
ND
ND
O
Cl
Ph
6-18
P
O2S
NHAc
Ph
a
IC50 values are determined from a single experiment performed in triplicate.
IC50 values are determined from two independent experiments performed in triplicate.
ND, not determined.
b
c
Table 2
The biological results for the head modification
O
R'
N
H
N
O
S
O
a
Compound
R0
Inhibition (%) at 200 nm
87
IC50 (nM)
25.1
Compound
R0
Inhibition (%) at 200 nm
44
IC50 (nM)
8-1
8-26
ND
O
8-2
8-3
8-4
55
78
ꢀ4
39.4
35
8-27
8-28
8-29
62
90
65
49.1
Cl
Ph
27.4
Ph
Cl
NDc
64.0
(continued on next page)

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Y. Xie et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2354–2359
Table 2 (continued)
a
Compound
R0
Inhibition (%) at 200 nm
IC50 (nM)
ND
Compound
R0
Inhibition (%) at 200 nm
IC50 (nM)
Cl
8-5
ꢀ1
8-30
98
59.1
32.2
Cl
F
F
8-6
8-7
ꢀ16
ND
ND
8-31
8-32
97
96
F3C
3
173.0
Cl
F3C
8-8
8-9
48
65
ND
8-33
8-34
94
94
28.7
29.1
Cl
42.0
Cl
Cl
8-10
8-11
50
44
200.0
8-35
8-36
7
ND
Ph
Cl
ND
98
54.1
F
Cl
Cl
F
8-12
8-13
8-14
8-15
8-16
ꢀ7
ꢀ29
87
ND
ND
16.4
ND
ND
8-37
8-38
8-39
8-40
8-41
97
95
97
97
77
12.7b
128.0
50.0
Cl
O
N
Cl
Cl
Cl
N
S
ꢀ17
ꢀ18
32.0
Cl
O
N
S
37.2b
N
S
8-17
8-18
ꢀ6
ND
ND
8-42
8-43
90
55
7.9b
F
23
200.0
N
N
(continued on next page)

Y. Xie et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2354–2359
2359
Table 2 (continued)
a
Compound
R0
Inhibition (%) at 200 nm
49
IC50 (nM)
ND
Compound
R0
Inhibition (%) at 200 nm
88
IC50 (nM)
8-19
8-44
11.0b
Boc N
N
Cl
8-20
8-21
8-22
8-23
8-24
62
67
33.6
43.3
ND
8-45
8-46
8-47
8-48
8-49
8-50
79
86
92
75
14
15
20.1
39.6b
12.6b
37.3b
ND
NH
NH
Ph
16
NO2
ꢀ25
9
ND
Cl
F
ND
O
N
NH
8-25
ꢀ6
ND
ND
HN
O
a
IC50 values are determined from a single experiment performed in triplicate.
IC50 values are determined from two independent experiments performed in triplicate.
ND, not determined.
b
c
bridge with the catalytic nucleophile Asp333 in the active site of
sEH, is absolutely required for the activity.9 Hypothesizing that
the amide in our hit corresponded to the urea functionality serving
as the primary pharmacophore, we selected a variety of primary
amines to replace 2,4-dichloride benzylamine moiety, including
those frequently found in urea-based inhibitors. As shown in Table
2, the primary amide was quite tolerant to modification and nano-
molar potency was retained with various substitutions. N-Benzyla-
mide 8-9 (IC50 = 42.0 nM) and N-cyclohexylamide 8-14
(IC50 = 16.4 nM) were selected for further optimization. Extensive
modification of the benzyl group led to 8-37 with a significant
improvement of potency (IC50 = 12.7 nM). The most noticeable
SAR illustrated by this modification is that halides such as chloride
and fluoride seem favored for the substitution on the benzyl group.
The modification of cyclohexylamide 8-14 yielded the most potent
compounds 8-42 (IC50 = 7.9 nM) and 8-47 (IC50 = 12.6 nM). As
demonstrated by this result, adding an extra methylene to the
cyclohexyl ring significantly enhanced the activities. In contrast,
analogs with polar atoms (N and O) in the ring decreased the inhib-
itory potency to micromolar range (8-49 and 8-50). These SARs are
consistent with previous results obtained for urea derivatives.10
In summary, we have successfully identified a series of potent
non-urea sEH inhibitors via high throughput screens. Improved po-
tency was sought through SAR-guided modification. Compound 8-
42 with an IC50 of 7.9 nm represents the most potent non-urea sEH
inhibitor identified in this study. Physical and pharmacokinetic
properties of these promising compounds are under investigation.
Acknowledgment
This work was supported by the NIH MLSCN Grant.
References and notes
1. Smith, K. R.; Pinkerton, K. E.; Watanabe, T.; Pedersen, T. L.; Ma, S. J.; Hammock,
B. D. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2186.
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B. D. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9772.
3. Chiamvimonvat, N.; Ho, C. M.; Tsai, H. J.; Hammock, B. D. J. Cardiovasc.
Pharmacol. 2007, 50, 225.
4. Kim, I. H.; Tsai, H. J.; Nishi, K.; Kasagami, T.; Morisseau, C.; Hammock, B. D.
J. Med. Chem. 2007, 502, 5217.
5. Wolf, N. M.; Morisseau, C.; Jones, P. D.; Hock, B.; Hammock, B. D. Anal. Biochem.
2006, 335, 71.
6. See the screen results published in Pubchem (AID:1026).
7. Jones, P. D.; Tsai, H. J.; Do, Z. N.; Morisseau, C.; Hammock, B. D. Bioorg. Med.
Chem. Lett. 2006, 16, 5212.
8. Analytical data for the representative compounds: 8-42: 1H NMR (300 MHz,
CDCl3) d 6.94 (s, 2H), 5.38 (br, 1H), 3.92–3.89 (m, 1H), 3.62–3.57(m, 2H), 2.83–
2.63 (t, 2H), 2.61 (s, 6H), 2.30 (s, 3H), 2.17–2.10 (m, 1H), 1.87–1.83 (m, 4H),
1.74–1.35 (m, 12H); 13C NMR (75 MHz, CD3 OD) d 172.9, 142.9, 140.6, 132.1,
131.6, 50.6, 44.0,43.1, 35.4, 28.5, 28.3, 24.5, 23.1, 21.3; ESI-MS(M++H): 407.2. 8-
47: 1H NMR (300 MHz, CDCl3) d 6.94 (s, 2H), 5.48 (br, 1H), 3.60 (d, 2H), 3.07 (t,
2H), 2.81 (t, 2H), 2.61 (s, 6H), 2.30 (s, 3H), 2.20–2.05 (m, 1H), 1.88–1.84 (m, 2H),
1.77–1.64 (m, 6H); 1.50–1.38 (m, 1H); 1.35–1.05 (m, 4H); 0.95–0.82 (m, 2H);
13C NMR (75 MHz, CD3OD) d 174.2, 142.9, 140.7, 132.1, 131.5, 45.9, 44.0, 43.2,
38.2, 31.1, 28.6, 26.7, 26.1, 23.2, 21.3; ESI-MS(M++H): 407.3.
9. Argiriadi, M. A.; Morisseau, C.; Goodrow, M. H.; Dowdy, D. L.; Hammock, B. D.;
Christianson, D. W. J. Biol. Chem. 2000, 275, 15265.
10. Kim, I. H.; Heirtzler, F. R.; Morisseau, C.; Nishi, K.; Tsai, H. J.; Hammock, B. D.
J. Med. Chem. 2005, 48, 3621.