Solid-phase combinatorial approach for the optimization of soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.08.078

A 192-member library of N,N′-disubstituted urea inhibitors was synthesized by a solid-phase method. The ureas were tested for their inhibitory activities against recombinant human soluble epoxide hydrolase. Simple carbocyclic or para/meta-substituted phen

Full text of DOI:10.1016/j.bmcl.2006.08.078

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5773–5777
Solid-phase combinatorial approach for the optimization of soluble
epoxide hydrolase inhibitors
Sung Hee Hwang, Christophe Morisseau, Zung Do and Bruce D. Hammock*
Department of Entomology and UCD Cancer Center, University of California, One Shields Avenue, Davis, CA 95616-8584, USA
Received 24 July 2006; revised 16 August 2006; accepted 17 August 2006
Available online 1 September 2006
Abstract—A 192-member library of N,N0-disubstituted urea inhibitors was synthesized by a solid-phase method. The ureas were
tested for their inhibitory activities against recombinant human soluble epoxide hydrolase. Simple carbocyclic or para/meta-substi-
tuted phenyl groups showed inhibition potencies that were equal to or greater than adamantane-based sEH inhibitors, while the
presence of bulky or ionizable groups close to the urea group dramatically decreased their activities.
Ó 2006 Elsevier Ltd. All rights reserved.
Soluble epoxide hydrolase (sEH, EC 3.3.2.3) is involved
in the metabolism of endogenously derived fatty acid
epoxides, such as arachidonic acid, linoleic acid, and
other lipid epoxides.1 Epoxyeicosatrienoic acids (EETs),
the cytochrome P450 epoxygenase products of arachi-
donic acid, act at vascular, renal, and cardiac levels of
blood pressure regulation. Recent studies have showed
that EETs are involved in antihypertensive effects as
an endothelium-derived hyperpolarizing factor (EDHF)
that mediates vasodilation by activating Ca2+-activated
K+ channels in smooth muscle cells.1,2 TRPV4, a Ca2+
entry channel belonging to the vanilloid subfamily of
the transient receptor potential (TRP) channels, acts as
an extracellular receptor for EETs.3 The sEH enzyme
catalytically hydrolyzes EETs into dihydroxyeicosatrie-
noic acids (DHETs) which show reduced biological
activity.1 We have demonstrated that sEH inhibition sig-
nificantly reduces the blood pressure of the spontaneous
hypertensive rats (SHRs) and angiotensin II-induced
hypertensive rats.4,5 EETs also possess anti-inflammato-
ry properties in endothelial cells by inhibiting the expres-
sion of tumor necrosis factor-a (TNF-a)-induced
vascular cell adhesion molecule-1 (VCAM-1) which is
a pro-atherogenic mediator or by activating PPARc
which suppresses the NF-jB-mediated expression of
molecules, that is, VCAM-1, intercellular adhesion mol-
ecule-1 (ICAM-1), and endothelin-1.6 In addition, diols
derived from epoxy-linoleate (leukotoxin) via sEH
hydrolysis perturb membrane permeability and calcium
homeostasis, which results in inflammation.7 These re-
sults suggest that sEH inhibition may represent a novel
approach for the treatment of hypertension and inflam-
matory diseases.
Earlier inhibitors developed for sEH were substrate-like
compounds, such as chalcone oxides.8 More recently, we
have explored urea, amide, and carbamate-based
transition state analog inhibitors of sEH.9 Several recent
studies have emphasized adamantane-based urea com-
pounds due to their ease of synthesis, high potency,
and ease of analysis. The recent development of fluores-
cent substrates10,11 for sEH which both discriminate
among low nanomolar and picomolar inhibitors and
facilitate high-throughput analysis makes the develop-
ment of inhibitors through a combinatorial approach
attractive. In this report we illustrate this combinatorial
approach in the further exploration of the role of R1 (see
compound D, Scheme 1) as it influences the potency of
sEH inhibitors.
A focused library of 192-ureas was designed in order to
optimize R1 group as well as to avoid the tedious purifica-
tion step caused by the main contaminant during urea
formation, a symmetrical urea (R1NHC(@O)NHR1,
by-product from the step b in Scheme 1), which also has
significant inhibitory activity against the human sEH
enzyme.
The solid-phase procedure was set up using a previously
reported urea formation method starting from the com-
mercially available acid-labile formylindole resin A.12
Keywords: Soluble epoxide hydrolase; Urea; Hypertension; Anti-
inflammation.
*
Corresponding author. E-mail: bdhammock@ucdavis.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.078

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S. H. Hwang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5773–5777
O
O
R1
R2
a
c
b
H
R1
N
R2
N
R2
N
H
CHO
N
H
N
H
A
B
C
D
R1-NCO:
48 isocyanates (see Supplementary data)
O
F
O
S
Me
H
N
O
H2N
H2N
O
R2-NH2 : H2N
F
N
F
H2N
1
2
3
4
Scheme 1. Reagents and conditions: (a) i—R2-NH2, TEOF, THF, rt, 8 h; ii—1 M NaBH3CN in THF, AcOH, THF, rt, 4 h; (b) R1-NCO, Et3N,
CH2Cl2, rt, 1 d; (c) 1% TFA in CH2Cl2, rt, 4 h.
The reactions were performed on an IRORI AccuTagTM
Combinatorial Chemistry System13 according to the
general reaction pathway outlined in Scheme 1. Four
amines 1–4 and commercially available 48 isocyanates
were used to construct the 192-member urea library.
member library, 181 compounds having over 90% purity
were tested for their inhibitory activity at a 100 nM con-
centration using recombinant human sEH by an end-
point assay.11,17
Overall, compounds derived from amines 1 and 2
showed good inhibitory activity compared to those
made from amines 3 and 4 (see Table 1 in Supplementa-
ry data). These results strongly suggest that the tested
compounds orient in the same direction as the adaman-
tane-based inhibitors do when they bind at the active
site, which can be predicted from the recent X-ray crys-
tal structure of human sEH with urea-based inhibitors.14
The four amines 1–4 were selected based on the follow-
ing criteria. First, selection was based on inhibitory
activities of the corresponding adamantane-based ureas
(see IC50 values in Table 1). Second, three amines are
relatively large (amine 1, 2, and 3), thus driving their ori-
entation in the catalytic tunnel, while one amine is rela-
tively small (amine 4).14 Finally, amines containing a
benzene ring were chosen in order to evaluate the purity
of the products by UV detection on HPLC.
In order to determine the influence on the inhibitory
activity by R1 group itself, compounds having similar
activity compared to the corresponding adamantane-
based ureas (1{1}, 2{1}, 3{1}, and 4{1}) are highlighted
in black squares in Figure 1.
Based on the previous SAR analysis of the chalcone
oxide derivatives8 and other urea inhibitors,15 R1 groups
in the isocyanates were selected as follows: (1) an ada-
mantane as a control, (2) simple carbocyclic rings, (3)
para-, meta-, and ortho-mono-substituted phenyl rings,
(4) sterically hindered groups, such as tert-butyl or
ortho-di-substituted phenyl rings, (5) 1- and 2-naphthyl
group, and (6) phenylalkyl groups having various
lengths of alkyl spacers.16 The purity was assessed by
HPLC and all mass spectra were consistent with the
anticipated product structure. Out of the total 192-
From the illustration in Figure 1, it is apparent that car-
bocyclic groups ({2–6}) and para-substituted phenyl
groups ({8–17}) showed the best results, regardless of
R2 group. There was no strong correlation between
the inhibition and the r values. It is likely that the bind-
ing site in which the R1 group resides is a narrow, hydro-
phobic tunnel. This is further supported by the fact that
overall para-substituted phenyl groups are better than
meta-substituted phenyl groups ({18–26}), the 2-naph-
thyl group ({45}) is better than the 1-naphthyl group
({44}), and the phenyl groups having longer alkyl spac-
ers ({47–48}) gave the better activities. Meanwhile, ste-
rically hindered derivatives, such as inhibitors from the
isocyanates ({27–44 and 46}), showed poor inhibition.
Based on these results, poor activities most likely came
from steric effects of groups on R1. In other words,
the adamantyl group, which is generally considered as
a bulky group, might be the marginal biggest group as
the R1.
Table 1. Inhibitory activities of the adamantane-based ureas 1{1},
2{1}, 3{1}, and 4{1} derived from the amines 1 to 4
O
R2
N
N
H
H
Compounda
R2-NH2
% inhibitionc
86
75 12
b
IC50 (nM)
1{1}
2{1}
3{1}
4{1}
1
2
3
4
0.5
0.5
9
30
100
19
10
7
3
In addition, as seen in Figure 2, ureas derived from
amines 1 and 2 generally show similar inhibition; one
can expect such results because of the similar IC50 values
obtained for their corresponding adamantane-based
ureas (Table 1). However, for ureas having sterically
hindered groups on R1 (R1 = {27–44}), ureas derived
a The notation for the compound number: amine number {isocyanate
number}, for example, 2{1} indicates a compound made by the
combination of amine 2 with isocyanate 1.
b As determined via a kinetic fluorescent assay.10
c Determined via an end-point fluorescent assay, results are
means SD of three separate experiments.11

S. H. Hwang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5773–5777
5775
in the active site tunnel. Such altered positioning seems
to result in the further destabilization effect for ureas de-
rived from amine 2 than for those from amine 1.
O
R1
R2
N
N
H
H
R2
R2
To validate our high-throughput screening results, a
subset of compounds having high percent inhibition,
that is, 1{4}, 1{12}, 1{13}, 1{14}, 1{15}, 1{16} and hav-
ing low percent inhibition, that is, 1{34}, 1{38}, 1{39},
1{40}, were resynthesized. Their IC50 values were then
determined using a continuous fluorescent assay.10 In
general, data from both methods showed good correla-
tion as shown in Table 2. These results confirm that ste-
rically less hindered para- or meta-substituted phenyl
R1
1 2 3 4 R1
1 2 3 4
adamantyl
1
2
3
4
5
6
7
8
9
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
carbocyclic
phenyl
ortho-
mono-
substituted
phenyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Table 2. IC50 results for a selected subset of urea compounds from
Schemes 1 and 2
para-
substituted
phenyl
O
O
R1
sterically
hindered
phenyl or
alkyl
N
N
F
H
H
a
Compound IC50
(nM)
Inhibitionb R1
(%)
Mp (°C)
1{1}
1{4}
1{12}
1{13}
1{14}
1{15}
1{16}
1{34}
1{38}
1{39}
1{40}
7
0.5 0.1 86
0.5 0.1 87
1.0 0.1 76
0.7 0.1 81
0.6 0.1 80
0.9 0.1 84
1.2 0.2 82
9
8
7
8
6
7
5
3
3
1
3
Adamantyl
c-Hep
4-I–Ph–
242–245
158–167
198–201
177–182
192–194
157–158
171–174
158–160
meta-
mono-
naphthyl
substituted
phenyl
4-Cl–Ph
4-Br–Ph
phenyl-
alkyl
4-OCF3–Ph
4-CF3–Ph
2-OCF3–Ph
100
5
2
4
1
5
Figure 1. Relative inhibition of the 192-member urea library at
100 nM concentration compared to the corresponding adamantane-
based ureas 1{1}, 2{1}, 3{1}, and 4{1}, respectively. Black square:
equal to or greater than 80% of inhibition obtained when R1 is an
adamantyl group; grey square: less than 80% of the inhibition obtained
when R1 is an adamantyl group; white square: compounds were not
tested due to the poor purity (less than 90%).
1200 100
940 60
50500 500
2,6-Di-Me–Ph 196–199
2,6-Di-Cl–Ph 181–187
2,6-Di-i-Pr–Ph 218–222
2.9 0.2 62 13
220 nd
2.0 0.2 72
590 60 nd
4-CO2Me–Ph
4-CO2H–Ph
3-CO2Me–Ph
3-CO2H–Ph
162–163
269–285
116–123
240–254
8
5
9
10
6
nd denotes not determined.
a Determined via a kinetic fluorescent assay, results are means SD of
three separate experiments.
b Determined via an end-point fluorescent assay, results are
means SD of three separate experiments.
from amine 1 showed overall much better inhibition. It
is likely that a repulsion caused by sterically hindered
groups on R1 influences the positioning of the R2 group
O
R2-NH2:
R1
R2
1
2
100
80
60
40
20
0
N
H
N
H
0
5
10
15
20
25
30
35
40
45
1
R
Figure 2. Percent inhibition of ureas derived from amines 1 and 2 at 100 nM concentration. Note, ureas 2{2}, 2{4}, 2{5}, 2{6}, 2{11}, and 2{43}
were not tested for inhibition because of their low purity.

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S. H. Hwang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5773–5777
O
O
O
(a)
R
N
H
N
H
F
H2N
F
1
7, R = 4-CO2Me
9, R = 3-CO2Me
(b)
8, R = 4-CO2H
10, R = 3-CO2H
Scheme 2. Reagents and conditions: (a) Ar-NCO, DMF, rt, 12 h; (b) LiOH, acetonitrile, water, 90 °C, 6 h.
groups, simple non-rigid carbocyclic groups, 2-naph-
thyl, and phenyl alkyl groups having at least an ethyl
spacer are good alternatives to the adamantyl group.
These results are also consistent with our previous
SAR of chalcone oxide derivatives and other urea-based
inhibitors.8,15
potency. In addition, having ionizable free carboxylic
acid on R1 dramatically decreased the inhibitory activi-
ty. These observations strongly suggest that the side of
the active site tunnel where R1 of the N,N0-disubstituted
urea binds favors sterically unhindered lipophilic
groups.9c
The coincidence between the current SAR with that of
the chalcone oxide derivatives encouraged us to explore
the carboxylated analogs to investigate the effect in the
presence of the ionizable group on the R1. The required
acid compounds 8 and 10 were synthesized using amine
1 as outlined in Scheme 2.
Acknowledgments
The authors thank Dr. Mark J. Kurth for many helpful
discussions. We also thank Dr. Jozsef Lango and Dr.
Paul Whetstone for assistance with mass spectral deter-
minations. This work was supported in part by NIEHS
Grant ES02710, NIEHS Superfund Grant P42 ES04699,
NIEHS Center Grant P30 ES05707, and NHLBI STTR
Grant R41 HL078016.
In a similar manner as observed for chalcone oxide
derivatives,8 the introduction of the free carboxylic acid
at the para-position of the phenyl ring dramatically
decreased its activity about 440-fold compared to
compound 1{1}. Having a free carboxylic acid at the
meta-position decreased inhibition potency even more
dramatically. Corresponding ester compounds 7 and 9
are, however, only 2- to 6-fold less active compared to
compound 1{1}. The reason for the poor inhibitory
activities of inhibitors having the free carboxylic acid
on R1 is at present unclear. Possible explanations in-
clude: (1) water solvation of the carboxylate might either
prevent access of the inhibitor into the active site or
cause the repulsion with the residues at the active site,
(2) ionic interactions between the carboxylate anion
and protonated imidazole on His523 preventing the opti-
mal binding of the inhibitor at the active site. Recent
X-ray crystal structure data of human sEH complexed
with different dialkylurea inhibitors bearing pendant
carboxylate of varying length supported the latter
explanation.14
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.08.078.
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these diversity reagents.
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