Non-urea functionality as the primary pharmacophore in soluble epoxide hydrolase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.01.013

Inhibition of soluble epoxide hydrolase has been proposed as a promising new pharmaceutical target for diseases involving hypertension and vascular inflammation. The most potent sEH inhibitors reported to date contain a urea or amide moiety as the central or 'primary' pharmacophore. We evaluated replacing the urea pharmacophore with other functional groups such as thiourea, sulfonamide, sulfonylurea, aminomethylene amide, hydroxyamide, and ketoamide to identify novel and potent inhibitors. The hydroxyamide moiety was identified as a novel pharmacophore affording potency comparable to urea.

Full text of DOI:10.1016/j.bmcl.2009.01.013

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1066–1070
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Non-urea functionality as the primary pharmacophore in soluble epoxide
hydrolase inhibitors
*
Sampath-Kumar Anandan , Zung N. Do, Heather K. Webb, Dinesh V. Patel, Richard D. Gless
Arête Therapeutics, Inc., 3912 Trust Way, Hayward, CA 94545, 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 has been proposed as a promising new pharmaceutical target for
diseases involving hypertension and vascular inflammation. The most potent sEH inhibitors reported to
date contain a urea or amide moiety as the central or ‘primary’ pharmacophore. We evaluated replacing
the urea pharmacophore with other functional groups such as thiourea, sulfonamide, sulfonylurea, ami-
nomethylene amide, hydroxyamide, and ketoamide to identify novel and potent inhibitors. The hydrox-
yamide moiety was identified as a novel pharmacophore affording potency comparable to urea.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 26 November 2008
Revised 6 January 2009
Accepted 7 January 2009
Available online 10 January 2009
Keywords:
sEH inhibitors
Non-urea
Primary pharmacophore
Soluble epoxide hydrolase (sEH) has been proposed as a poten-
tial pharmaceutical target in a number of disease indications
including hypertension,1 stroke,2 inflammatory disease,3 and met-
abolic syndrome.4 The sEH enzyme is found in a variety of mam-
malian tissues, with the highest activity in liver, kidney,
intestinal and vascular tissue.5 sEH metabolizes endogenously pro-
duced epoxides of arachidonic acid, epoxyeicosatrienoic acids
(EETs), to the corresponding dihydroxyeicosatrienoic acids
(DHETs), by catalyzing the addition of water to the epoxide moi-
ety.6 EETs produce vasodilation in various vascular beds such as
renal, mesenteric, cerebral, pulmonary and coronary arteries.7
Hydrolysis of EETs by sEH to the corresponding DHETs significantly
diminishes this activity,8 suggesting that inhibition of sEH may be
a promising new therapy in the treatment of diseases involving
hypertension and vascular inflammation.
sEH inhibition. Subsequent attempts to improve solubility proper-
ties of such prototype molecules as dicyclohexyl urea (DCU), cyclo-
hexyl dodecyl urea (CDU), and adamantyl dodecyl urea (ADU) led
to incorporation of a solubilizing group at the end of the extended
alkyl chain to afford molecules like 12-[3-adamantane-1-yl-ure-
ido]-dodecanoic acid (AUDA). Additional studies targeting
improved solubility and pharmacokinetic properties showed that
polar functionality such as ether, ester, and amide moieties, desig-
nated as the ‘secondary’ pharmacophore,13 could be incorporated
into the alkyl chain ca. 7.5 Å distant from the ‘primary’ urea phar-
macophore without loss in potency.14 In some cases appropriate
selection of the secondary pharmacophore and other structural
features in the molecule could afford amide based scaffolds of
comparable potency to urea based scaffolds.11 Recent reports
describe potent sEH inhibitors, which have improved pharmacoki-
netic profiles, containing conformationally restricted ligands such
as a cyclohexane or piperidine ring linking the urea and the sec-
ondary pharmacophore.15
sEH inhibitors based on epoxide,9 urea,10 carbamate,10 amide,11
and acyl hydrazone,12 scaffolds have been reported. The most
potent sEH inhibitors reported to date for which enzyme IC50 data
have been reported contain a urea or amide as the central or ‘pri-
mary’ pharmacophore (Fig. 1).13 Early epoxide based inhibitors
that were designed based on the endogenous substrate proved to
be of limited use for in vivo studies prompting research that led
to the discovery of dicyclohexyl urea (DCU), a potent inhibitor of
the sEH enzyme.10 Evaluation of structural analogs of DCU includ-
ing amide, thiourea, guanidine, carbamate, thiocarbamate, ketone,
ester, and carbonate indicated that urea, amide, and carbamate
showed the greatest potential as the required functionality for
The ether scaffold 1 was chosen as a test scaffold for varying the
primary pharmacophore P1 based on relative ease of synthesis,
Linker
L2
Linker
L1
R
P3
P1
P2
Primary
Pharmacophore
Secondary
Pharmacophore
Tertiary
Pharmacophore
* Corresponding author. Tel.: +1 510 300 1870; fax: +1 510 785 7061.
E-mail address: skumar@aretetherapeutics.com (S.-K. Anandan).
Figure 1. Pharmacophore model.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.013

S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1066–1070
1067
excellent potency of such compounds in the urea series
(P1 = NHCONH),14 improved solubility, and potential for drugability.
Dess–Martin periodinane to the corresponding aldehyde 13 which
on treatment with trimethylsilylcyanide gave cyanohydrin 14.
Intermediate 14 on treatment with hydrochloric acid in the pres-
ence of methanol resulted in methyl ester which upon treatment
with aqueous lithium hydroxide yielded the hydroxy acid 15. Cou-
pling amine 4 with hydroxy acid 15 gave hydroxyamide 16. Oxida-
tion of hydroxyamide 16 with Dess–Martin periodinane afforded
the desired ketoamide 17.
R
P1
O
N
1
O
A further series of hydroxyamides and ketoamides (19 and 20)
with a more lipophilic benzyl ether on the right hand side and
incorporating different spacer lengths on either side of the P1
hydroxyamide or ketoamide were prepared (Scheme 5). Coupling
of hydroxy acid 15 with 4-benzyloxyphenyl amine 18 afforded
hydroxyamide 19, which on further oxidation with Dess–Martin
reagent yielded ketoamide 20. Intermediate 15 was prepared from
the corresponding adamantanol by sequential oxidation to alde-
hyde and cyanide addition followed by hydrolysis of nitrile to
the corresponding acid. The amine intermediate 18 was prepared
from the corresponding 4-benzyloxybenzyl alcohol by sequential
mesylation, displacement with sodium azide and reduction to
the corresponding amine.
Comparison of sEH enzyme inhibitor potency for selected R
groups in the amide series with the corresponding urea series is
shown in Table 1. Amide analogs 6 in general were less potent than
the corresponding urea 5 in a fluorescence-based enzyme assay.18
While adamantyl urea analog 5a was found to be very potent, the
corresponding amide analog 6a was found to be ca. two orders of
magnitude less active. However, the potency could be improved
in the amide series by inserting a methylene between the adaman-
tyl group and the amide, essentially replacing the urea NH with a
CH2 (e.g., 6b), maintaining a similar distance between the carbonyl
and the left hand substituent as in the urea. A similar improvement
was noted with the cyclohexylmethyl analogs 6d and 6c as well as
with the 4-trifluoromethylphenylmethyl analogs 6f and 6e. Inser-
tion of a methylene group between the urea NH and the urea car-
bonyl to afford the corresponding aminomethylene amide analog
11 resulted in diminished potency.
On the basis of the X-ray crystal structure, the urea based inhibitors
are postulated to establish hydrogen bonds between the urea moi-
ety and residues of the sEH enzyme mimicking features of the tran-
sition state of epoxide ring opening.16 Based on this rationale we
decided to explore other functionality that could have a similar
binding motif to that of urea. We first explored common urea mod-
ifications such as thiourea and sulfonyl urea followed by sulfon-
amide and amide as well as aminomethylene amide. We also
prepared hydroxyamide and ketoamide modifications that have
additional hydrogen binding possibilities.
The general synthetic route for the preparation of compounds
with urea, amide and sulfonamide as the primary pharmacophore
is shown in Scheme 1. Alkylation of morpholine with 1-bromo-3-
chloropropane afforded N-(3-chloropropyl)-morpholine 2.17
Reaction of 2 with m-nitrophenol gave nitroether 3 which upon
reduction yielded aniline intermediate 4. Treatment of intermedi-
ate aniline 4 with the corresponding isocyanates resulted in urea
analogs 5. Treatment of intermediate 4 with various acids or sulfo-
nyl chlorides afforded the desired amides 6 or sulfonamides 7.
Thiourea analog 8 was prepared by reacting adamantyl isothio-
cyanate with aniline intermediate 4 under reflux. Sulfonyl urea 9
(P1 = NHSO2NH) was prepared by sequential reaction of the requi-
site amine with sulfuryl chloride followed by treatment with inter-
mediate 4 (Scheme 2).
The aminomethylene amide analogs 11 were prepared by treat-
ing aniline intermediate 4 with chloroacetyl chloride followed by
alkylation with the desired amine (Scheme 3).
The synthesis of hydroxyamides 16 and ketoamides 17 is
presented in Scheme 4. Adamantanol 12 was oxidized using
Cl
N
HN
+
a)
Cl
Br
O
O
2
b)
Cl
N
+
O
N
O2N
OH
O2N
O
O
2
3
5
O
O
c)
RNCO
R
d)
H2N
O
N
N
H
N
H
N
4
O
O
RCO2H
RSO2Cl
f)
e)
O
O
O
S
R
N
O
N
N
H
O
N
R
H
7
6
O
O
Scheme 1. General synthetic scheme for the preparation of ureas, amides, and sulfonamides. Reagents and conditions: (a) K2CO3, DMF, rt, 12 h, 83%; (b) NaH, THF, 4 h, 75%;
(c) Fe, HCO2NH4, toluene, 12 h, 91%; (d) THF, reflux, 12 h, 58–73%; (e) Et3N, DCM, 12 h, 60–83%; (f) EDCI, HOBT, DIEA, DMF, 4 h, 50–65%.

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S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1066–1070
NCS
S
R
a)
N
H
N
H2N
O
N
N
H
O
8
4
O
O
O
O
O
O
4
RNH2
+
R
S
SO2Cl2
R
S
b)
N
Cl
c)
N
N
N
O
H
H
H
9
O
Scheme 2. Preparation of thioureas and sulfonylureas. Reagents and conditions: (a) THF, reflux, 12 h, 68%; (b) Et3N, DCM, rt, 12 h, 65%; (c) Et3N, DCM, rt, 16 h, 72%.
O
Cl
Cl
O
Cl
a)
N
H
O
N
H2N
O
N
O
O
10
4
RNH2
b)
O
H
N
N
N
H
O
R
O
11
Scheme 3. Preparation of aminomethylene amides. Reagents and conditions: (a) Et3N, DCM, rt, 12 h, 84% ; (b) Et3N, DCM, rt, 16 h, 65–79%.
OH
b)
a)
OH
CN
O
n
n
n
14
12
13
c, d)
OH
OH
O
O
H
N
4
OH
O
O
N
n
n
e)
O
O
16
15
f)
O
H
N
N
n
O
17
Scheme 4. Preparation of hydroxyamides and ketoamides. Reagents and conditions: (a) Dess–Martin periodinane, DCM, rt, 4 h, 52–84%; (b) TMSCN, THF, rt, 12 h, 58–83%; (c)
HCl, MeOH, reflux, 12 h, 63–72%; (d) LiOH, MeOH, rt, 12 h, 56–79%; (e) EDCI, HOBT, DIEA, DCM, rt, 12 h, 60–75%; (f) Dess–Martin periodinane, DCM, rt, 6 h, 65%.

S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1066–1070
1069
O
OH
a)
OH
+
H2N
m
n
O
18
15
O
O
b)
OH
H
N
m
n
n
O
O
19
O
H
N
m
20
Scheme 5. Preparation of hydroxyamides and ketoamides. Reagents and conditions: (a) EDCI, HOBT, DIEA, DCM, rt, 12 h, 58–73%; (b) Dess–Martin periodinane, DCM, rt, 6 h,
54–68%.
Table 2
Table 1
Enzyme IC50 values for hydroxyamides and ketoamides.
Enzyme IC50 values for urea, amide, and sulfonamides.
P1
IC50 (nM)a
Compound
n
R Group
P1
IC50 (nM)a
Compound R Group
16a
16b
16c
17a
17b
17c
0
1
2
0
1
2
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Hydroxyamide
Hydroxyamide
Hydroxyamide
Ketoamide
Ketoamide
Ketoamide
>2000
23
530
>2000
49
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
7
Adamantyl
Cyclohexyl
Urea
Urea
Urea
Urea
Amide
Amide
Amide
Amide
Amide
0.8
3.9
1.2
8.2
88
2.5
390
28
93
55
15,000
22
3600
4-Trifluoromethylphenyl
4-Trifluoromethoxyphenyl
Adamantyl
Adamantylmethyl
Cyclohexyl
Cyclohexylmethyl
4-Trifluoromethylphenyl
4-Trifluoromethylphenylmethyl Amide
48
a
IC50 values for all sEH inhibitors were determined using a fluorescence assay.18
4-Trifluoromethylphenyl
Adamantyl
Adamantyl
Sulfonamide
Thio urea
Sulfonyl urea
Aminomethylene amide 110
exhibit improved potency in the enzyme assay. This analog proved
to be the most potent within the hydroxyamide series. Ketoamide
analogs with one or two methylene spacers (17b or 17c) afforded
substantially improved and equally potent sEH enzyme inhibition.
While hydroxyamides 16 and ketoamides 17 are novel sEH
inhibitors, their in vitro potencies were somewhat inferior to the
corresponding urea or amide analogs. In an attempt to improve
the in vitro potency for the hydroxy- and ketoamide series, we pre-
pared a further set of compounds containing a benzyl moiety on
the right hand side. Incorporation of such a lipophilic group has
been reported to afford improved potency in a series of benzamide
sEH inhibitors.19 sEH enzyme IC50 values for hydroxyamides 19
and ketoamides 20 are presented in Table 3.
8
9
11
Adamantyl
a
IC50 values for all s-EH inhibitors were determined using a fluorescence assay.18
Substitution of common replacements for urea and amide such
as thiourea and sulfonamide led to less potent compounds. Thio-
urea analog 8 was found to be about 10-fold less potent than the
corresponding urea analog 5a. Sulfonamide analog 7 was found
to have substantially less potency in comparison to the corre-
sponding amide analog 6e or urea analog 5c. Replacing the sulfon-
amide with sulfonyl urea (e.g., 9) as the primary pharmacophore
afforded improved potency relative to the sulfonamide, but the
sulfonyl derivatives, in general, exhibited greatly diminished sEH
enzyme activity compared to the urea and amide analogs.
Hydroxyamides 16 and ketoamides 17 were found to be less
potent sEH inhibitors compared to urea analogs (Table 2). Based
on the observation that improved potency was obtained with the
insertion of a methylene group between the amide primary phar-
macophore and the R group in compounds 6a and 6b, hydroxya-
mide 16b with one methylene spacer between the adamantyl
group and the hydroxyamide moiety was prepared and found to
Table 3
Enzyme IC50 values for hydroxyamides and ketoamides.
Compound
m
n
P1
IC50 (nM)a
19a
19b
19c
19d
20a
20b
20c
20d
0
1
1
2
0
1
1
2
0
0
2
2
0
0
2
2
Hydroxyamide
Hydroxyamide
Hydroxyamide
Hydroxyamide
Ketoamide
Ketoamide
Ketoamide
Ketoamide
310
270
185
11
210
1110
1540
685
a
IC50 values for all sEH inhibitors were determined using a fluorescence assay.18

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S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1066–1070
In this series the hydroxyamide analogs 19 were found to be
References and notes
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Increasing the distance between the hydroxyamide pharmaco-
phore and the adamantyl and benzyloxyphenyl group afforded
improved sEH potency in the hydroxyamide series (19a–d), but
no such trend was observed in the ketoamide series (20a–d).
Hydroxyamide 19d with two methylene spacer on both sides of
the hydroxyamide primary pharmacophore was found to be the
most potent sEH inhibitor in the series, exhibiting potency compa-
rable to urea analog 21 (IC50 = 15 nM). The corresponding ketoa-
mide 20d was found to be ca. 60-fold less potent.
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Acknowledgment
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We wish to acknowledge Dawn Chen for verifying some of the
enzyme data.