Structure-activity relationships of cycloalkylamide derivatives as inhibitors of the soluble epoxide hydrolase

Article abstract of DOI:10.1021/jm101431v

Structure-activity relationships of cycloalkylamide compounds as inhibitors of human sEH were investigated. When the left side of amide function was modified by a variety of cycloalkanes, at least a C6 like cyclohexane was necessary to yield reasonable inhibition potency on the target enzyme. In compounds with a smaller cycloalkane or with a polar group on the left side of amide function, no inhibition was observed. On the other hand, increased hydrophobicity dramatically improved inhibition potency. Especially, a tetrahydronaphthalene (20) effectively increased the potency. When a series of alkyl or aryl derivatives of cycloalkylamide were investigated to continuously optimize the right side of the amide pharmacophore, a benzyl moiety functionalized with a polar group produced highly potent inhibition. A nonsubstituted benzyl, alkyl, aryl, or biaryl structure present on the right side of the cycloalkylamide function induced a big decrease in inhibition potency. Also, the resulting potent cycloalkylamide (32) showed reasonable physical properties.

Full text of DOI:10.1021/jm101431v

ARTICLE
pubs.acs.org/jmc
Structure-Activity Relationships of Cycloalkylamide Derivatives
as Inhibitors of the Soluble Epoxide Hydrolase
In-Hae Kim,† Yong-Kyu Park,‡ Bruce D. Hammock,§ and Kosuke Nishi*,||
†Industry and Academic Cooperation Laboratory for Drug Development, Mokpo National University, Muan, Jeonnam 534-729,
Republic of Korea
‡Department of Medicinal Chemistry, Hyundai Pharma, Suwon, Gyeonggi 443-270, Republic of Korea
§Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue,
Davis, California 95616, United States
Department of Applied Bioscience, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan
S Supporting Information
b
ABSTRACT: Structure-activity relationships of cycloalkyla-
mide compounds as inhibitors of human sEH were investigated.
When the left side of amide function was modified by a variety
of cycloalkanes, at least a C6 like cyclohexane was necessary to
yield reasonable inhibition potency on the target enzyme. In
compounds with a smaller cycloalkane or with a polar group on
the left side of amide function, no inhibition was observed. On the other hand, increased hydrophobicity dramatically improved
inhibition potency. Especially, a tetrahydronaphthalene (20) effectively increased the potency. When a series of alkyl or aryl
derivatives of cycloalkylamide were investigated to continuously optimize the right side of the amide pharmacophore, a benzyl
moiety functionalized with a polar group produced highly potent inhibition. A nonsubstituted benzyl, alkyl, aryl, or biaryl structure
present on the right side of the cycloalkylamide function induced a big decrease in inhibition potency. Also, the resulting potent
cycloalkylamide (32) showed reasonable physical properties.
’ INTRODUCTION
and many of the resulting compounds still have relatively high
melting points.19 Interestingly, dramatic improvement in melting
points and/or solubility in water is obtained when the corre-
sponding urea central pharmacophore is modified by a series of
functional groups such as amides, carbamates, carbonates, and
esters.18,19 Among them, alkylamide function with a polar group
is effective for producing potent inhibitors with improved phy-
sical properties,19 suggesting that the amide structure is a very
useful functionality as one of central pharmacophores for devel-
oping bioavailable potent inhibitors of human sEH. There is a
strong correlation between the potency of sEH inhibitor with
urea and amide central pharmacophores. However, the range of
substituents for generating optimum amide sEH inhibitors
appears more restricted and slightly different from that with a
urea central pharmacophore.18-27 Thus, investigation of the
relationships of the structure and inhibition potency of amide
compounds is important to further develop highly potent
inhibitors with improved physical properties and bioavailability.
In the present study, we report structure-activity relationships
of amide derivatives, specifically investigating the effect of various
structural modifications of cycloalkylamide compounds on in-
hibition potency for human sEH to design potent inhibitors with
cycloalkylamide function as a central pharmacophore.
Epoxyeicosatrienoic acids (EETs), which are produced from
arachidonic acid by cytochrome P450 epoxygenases, have im-
portant roles in the regulation of hypertension,1-6 inflam-
mation,7-11 and other cardiovascular related diseases.12-14
However, metabolism of EETs to their corresponding hydrated
products by soluble epoxide hydrolase (sEH) generally reduces
these biological activities.1 Both in vitro and In Vivo studies have
indicated that the antihypertensive and cardioprotective effects
mediated by the EETs are reversibly dependent on the extent of
sEH hydrolysis of the EETs.2-4,6-8,14,15 Thus, maintaining the
In Vivo concentration of EETs through sEH inhibition is a
promising therapeutic pathway to treat cardiovascular inflamma-
tory and other diseases.
Urea compounds substituted with hydrophobic groups are
very potent and stable inhibitors of sEH with significant biolo-
gical activities in both in vitro and In Vivo models.3,4,16 However,
poor physical properties of the early compounds, such as low
solubility and high melting points, likely resulted in limited
In Vivo availability.17 The addition of a polar functional group
on specific positions of one of the urea substituents is effective in
increasing solubility in either water or organic solvents and also in
improving In Vivo availability while maintaining the inhibition
potency on the target enzyme.18-21 However, the positive effect
on the solubility in water of the inhibitor is generally quite limited
Received: November 8, 2010
Published: February 21, 2011
r
2011 American Chemical Society
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Table 1. Inhibition of Human sEH by Cycloalkylamide Derivatives
a No steric isomers or mixtures of cis/trans or R/S unless otherwise indicated. b Human sEH (1 nM) was incubated with inhibitors for 10 min in 25 mM
Bis-Tris/HCl buffer (200 μL, pH 7.0) at 30 °C before fluorescent substrate (CMNPC) introduction ([S] = 5 μM). Results are triplicate averages.
’ CHEMISTRY
carbonate as a base in N,N-dimethylformamide (DMF) gave
compounds 30 and 31 in 90-95% yield (Scheme 1B). For the
syntheses of compounds 11 and 14 (Scheme 1C), methyl
4-oxocyclohexanecarboxylate for compound 11 and methyl
tetrahydro-2H-pyran-4-carboxylate for compound 14 were hy-
drolyzed with 1 N NaOH in methanol to give the corresponding
acids in 100% yield, followed by the above EDCI/DMAP
coupling reaction with 3-phenylpropylamine (90%).
Cycloalkylamide and substituted cycloalkylamide compounds
in Tables 1 and 2 were synthesized as outlined in Scheme 1. The
corresponding cycloalkanecarboxylic acid was coupled with
3-phenylpropylamine (Scheme 1A) or with a substituted
alkyl- or aryl-amine (Scheme 1B) using 1-[3-(dimethylamino)-
propyl]-3-ethylcarbodiimide (EDCI) in the presence of 4-di-
methylaminopyridine (DMAP) in dichloromethane to provide
the corresponding nonsubstituted and substituted cycloalkyla-
mide derivatives in approximately 40-85% yield.19 Alkylation of
carboxylic acid with iodomethane in the presence of potassium
Compounds 15, 16, and 17 were prepared by the procedures
depicted in Scheme 2. Alkylation of cyclohexane-1,4-dicarboxylic
acid with iodomethane in the presence of potassium carbonate
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Table 2. Inhibition of Human sEH by Adamantane- and Naphthaleneamide Derivatives
a Human sEH (1 nM) was incubated with inhibitors for 10 min in 25 mM Bis-Tris/HCl buffer (200 μL, pH 7.0) at 30 °C before fluorescent substrate
(CMNPC) introduction ([S] = 5 μM). Results are triplicate averages.
as a base in DMF yielded dialkylated ester in 100% yield.
Monohydrolysis of the diester with barium hydroxide octahy-
drate in 80% methanol in H2O produced 4-(methoxycar-
bonyl)cyclohexanecarboxylic acid in approximately 80% yield.28
Coupling of this acid with 3-phenylpropylamine using EDCI and
DMAP in dichloromethane provided compound 15 in 90% yield.
Hydrolysis of the ester group of compound 15 with 1 N NaOH in
methanol afforded the corresponding acid product (16) in 100%
yield. For the synthesis of compound 17, cyclohexane-1,2-dicar-
boxylic acid was used instead of cyclohexane-1,4-dicarboxylic
acid in the first step, which was followed by the monohydrolysis
and coupling reaction to yield compound 17 in approximately
45% yield.
Naphthalene derivatives (32-35) were prepared as depicted
in Scheme 3A. 1,2,3,4-Tetrahydronaphthalene-2-carboxylic acid
was coupled with methyl 4-aminomethylbenzoate in the pre-
sence of EDCI and DMAP in dichloromethane to give com-
pound 32 in 95% yield. In addition, 2-naphthoic acid or
6-hydroxy-2-naphthoic acid was reacted with the same reagents
used for the synthesis of compound 32 to afford compounds 33
and 34, respectively, in about 90% yield. Methylation of the
hydroxyl group of compound 34 with iodomethane in the pre-
sence of potassium carbonate as a base in DMF provided compo-
und 35 in 95% yield. As seen in Scheme 3B, the N-methylated
analogue of compound 19 (36) was synthesized in the reaction of
iodomethane with compound 19 in the presence of sodium
hydride as a base in DMF in 95% yield.
’ RESULTS AND DISCUSSION
1,3-Dialkylureas have been found as highly potent inhibitors of
human sEH.17-23 As seen in compound 1 in Figure 1, relatively
large alkyl groups attached to the urea pharmacophore often lead
to effective inhibitors (0.5-5 nM IC50 on human sEH). Gen-
erally, the corresponding alkylamide analogues (2) of urea
inhibitors exhibit approximately a 10- to 20-fold decreased
inhibition potency for human sEH.19 However, interestingly, a
>200-fold drop in inhibition was observed when substituents
similar to that attached to compound 1 were incorporated into
the amide function as seen in compound 3. This suggests that a
cycloalkylamide structure like compound 3 is much less potent
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Scheme 1. Syntheses of Cycloalkylamide Derivativesa
a (a) 3-Phenylpropylamine (in parts A and C) or substituted benzyl- or phenylamine (in part B), DMAP, EDCI, CH2Cl2, room temp; (b) 4-(2-
aminoethyl)benzoic acid (for 30) or 6-aminonaphthalene-2-carboxylic acid (for 31), DMAP, EDCI, CH2Cl2, room temp; (c) CH3I, K2CO3, DMF,
room temp; (d) 1 N NaOH, MeOH, room temp.
than alkylamide inhibitors in having the substituents effective for
making potent inhibitors in ureas or alkylamides and that
syntheses of a series of cycloalkylamides with various substitu-
tions are necessary to improve inhibition potency of cycloalk-
ylamide compounds against the target enzyme. In order to first
investigate the effect of modifications in cycloalkyl substituents
on the left side of the amide function on inhibition potency for
the human enzyme, various cycloalkylamide structures were syn-
thesized as listed in Table 1. The right side of the amide was fixed
with a 3-phenylpropyl group, which is one of useful right side
substituents of urea inhibitors for producing potent inhibitions
and is also useful for monitoring chemical reactions quickly on a
TLC plate. As seen in compounds 4 and 5, relatively small cyclo-
alkyl groups such as cyclopropyl (4) or cyclopentyl (5) present
on the left side of the amide function led to poor inhibitions,
which is different from the result that urea derivatives show
significant inhibition potency for human sEH (<500 nM) when a
C5-carbon as a pentyl or a cyclopentyl is present on the left side
of the urea function.17,18 However, at least a >5-fold improve-
ment in inhibition was exhibited when the cycloalkane of 4 or 5
was replaced by a cyclohexyl (6) or a cyclohexenyl (7) group.
This indicates that at least a six-membered cycloalkane structure
on the left side of the amide is necessary for cycloalkylamide
derivatives to produce potent inhibitors of the target enzyme.
Further, the addition of a hydrophobic group such as trifluoromethyl
(8) or tert-butyl (9) on the cyclohexane moiety of compound 6
resulted in improved inhibitors (a 3.5- to 15-fold improvement)
over the nonsubstituted derivatives (4-7), indicating that a
hydrophobic substitution on cycloalkane is effective to further
improve inhibition potency in cycloalkylamide compounds. On
the other hand, incorporation of a hydroxyl group on the cyclo-
hexyl (10) led to a poor inhibitor and conversion of the hydroxyl
group of compound 10 to a ketone (11) also negatively impacted
inhibition potency. However, when a methylene carbon was ad-
ded between the hydroxyl group and the cyclohexane of com-
pound 10, no drop in inhibition potency was observed (12). This
indicates that direct incorporation of a polar group on the cyclo-
hexane dramatically reduces inhibition potency, but a polar
hydroxymethyl group on the cyclohexane does not interrupt
the binding of the amide compound to the sEH. As shown in
compounds 13 and 14, no significant inhibition was exhibited
when an oxygen atom is incorporated into the cycloalkanes
making cyclic ethers, implying that hydrophobicity yielded by a
cycloalkane like cyclohexane on the left side of the amide func-
tion is highly important to produce inhibition for the target
human enzyme, and the presence of a polar substitution incor-
porated into the cycloalkane dramatically decreases inhibition
potency unless it is in the correct position as shown later. When
an ester group was substituted on the 4-position of the cyclohexa-
ne (compound 15), inhibition similar to that of 8 or 9 resulted.
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Scheme 2. Syntheses of Methyl 3-Phenylpropylcyclohexane-1,4-dicarboxylate (15) and Its Acid Analogue (16)a
a (a) CH3I, K2CO3, DMF, room temp; (b) Ba(OH)2 8H2O, 80% MeOH in H2O, room temp; (c) 3-phenylpropylamine, EDCI, DMAP, CH2Cl2, room
3
temp; (d) 1 N NaOH, MeOH, room temp. For the synthesis of compound 17, cyclohexane-1,2-dicarboxylic acid was used instead of cyclohexane-1,4-
dicarboxylic acid in (a).
However, no inhibition was observed with the corresponding
acid analogue (16). Also, the presence of an ester function on the
2-position of cycloalkanes as shown in compounds 17 and 18
provided no significant inhibition for the human sEH. This result
indicates that an ester group on the 4-position of hydrophobic
cyclohexane is effective for producing potent inhibition, while an
ester on the 2-position close to the amide pharmacophore dis-
turbs the binding of the amide compound to the target enzyme.
Compound 19 with an adamantane in the left side of the amide
function exhibited a 1.5- to 2-fold increase in inhibition potency
when compared to compounds with substituted cyclohexanes
(8, 9, and 15), which is similar to the previous results with urea
central pharmacophores.17-19,21 Interestingly, when the ada-
mantane group of compound 19 was replaced by a tetrahydro-
naphthalene (20), a 3-fold improvement in inhibition potency
was exhibited, indicating that cycloalkanes fused with an aryl
group would be useful to produce highly potent cycloalkylamide
inhibitors. On the other hand, a >30-fold loss in inhibition
potency was obtained in phenyl substituted cycloalkyl derivatives
(21-23) compared to compound 20, implying that the fused
structure of tetrahydronaphthalene as shown in compound 20 is
necessary to retain highly potent inhibition for the target enzyme.
Overall these results indicate that relatively bulky hydrophobic
cycloalkyl substitution on the left side of the amide pharmaco-
phore is important to yield significant inhibition potency in
cycloalkylamide compounds. An adamantane (19) was an effec-
tive substituent for providing potent inhibition for the human
sEH with the tetrahydronaphthalene in compound 20 still more
effective.
Scheme 3. Syntheses of Naphthaleneamide Derivatives
(32-35) and N-Methyl-N-(3-phenylpropyl)-1-adamantane-
carboxamide (36)a
a (a) RCOOH, methyl 4-(aminomethyl)benzoate hydrochloride, EDCI,
DMAP, CH2Cl2, room temp; (b) compound 34, CH3I, K2CO3, DMF,
room temp; (c) NaH, CH3I, DMF, room temp.
implying that the presence of an aryl on the right side of the
amide function is more effective for producing better inhibition
for human sEH compared to the normal alkane. We found a
similar result previously that aryls present in an optimal position
of the right side of urea pharmacophore often lead to better
inhibition potency than alkyls on the right side of the urea
function.21 So cycloalkylamides with an aryl group in the right
side of the amide function were synthesized to further increase
inhibition potency for the target enzyme (Table 2). When the
methylene carbon chain length between the amide function and
the benzene ring of compound 19 was then modified, at least a
5-fold decrease in inhibition was observed in compounds 24-26.
No significant activity was measured in compound 24 with one
methylene carbon between the two moieties. Moreover, the
In order to further optimize the right side of the amide
pharmacophore, the 3-phenylpropyl group in compound 19 or
20 was modified with alkyl, substituted alkyl, aryl, or substituted
aryl groups (Figure 1 and Table 2). Replacement of the 3-phenyl-
propyl of compound 19 by a dodecyl group (compound 3 in
Figure 1) decreased inhibition potency approximately 2.0-fold,
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Figure 1. Structures of 1,3-disubstituted ureas (1) and the corresponding alkyl- (2) and cycloalkylamides (3): n = 3-8; R1 = H, alkyl, or substituted
alkyl. A variety of substituents, such as carboxylic acid, ester, or amide, can be placed on the benzene ring or cyclohexane in compound 1. The IC50 of
compound 3 on human sEH is 97 nM.
the right position of the amide function is more effective than
functionalized phenethyl to improve inhibition potency. Non-
substituted phenethyl (25) provided better inhibition than
Table 3. Melting Point and Water Solubility of Adamantane-
and Naphthaleneamide Derivatives
compd
melting point (°C)
solubility in water (μM)a
nonsubstituted benzyl (24). Compound 29 with a benzyl linker
between the amide pharmacophore and the ester on the benzene
ring had approximately a 10-fold better inhibition than com-
pound 27 with a one carbon chain shorter linker. This further
supports that the presence of a benzyl linker functionalized with
an ester group on the right side of the amide pharmacophore
provides highly potent inhibitors for human sEH. On analogy
with previous urea derivatives a variety of polar groups could be
placed in this secondary pharmacophore position.18-21 Modify-
ing the benzyl linker of compound 29 with a naphthalene (31)
resulted in a 7-fold loss in inhibition potency. This suggests that
the functionalized benzyl moiety is more effective than a biaryl
structure for producing potent inhibitors for human sEH.
Tetrahydronaphthalene (20), which was found to be a promising
substitution for the left side of the amide function in Table 1, was
then combined with the benzyl group, an effective right side
substituent of the amide pharmacophore, to see if an improved
inhibitor is produced. As expected, replacement of the adaman-
tane group on the left side of the amide function of compound 29
by a tetrahydronaphthalene resulted in a highly potent inhibitor
32 (a 5-fold improvement), which is consistent with the results in
Table 1. This suggests that the naphthalene structure is very
useful for producing highly potent inhibitions for human sEH. As
shown in compound 33 with a 2-naphthalene on the left side of
the amide function, relatively potent inhibition was also obtained
for the target enzyme. Compound 33, however, was 3-fold less
potent when compared to that of the tetrahydronaphthalene
derivative (32). Interestingly, substituted 2-naphthalene deriva-
tives with a hydroxy (34) or a methoxy (35) group exhibited an
increase in inhibition potency that is similar to that of the tetra-
hydronaphthalene analogue (32). In general, polar groups on
this side of the central pharmacophore dramatically reduce the
potency of the resulting inhibitors. However, compound 34 in
particular shows that the polar functionality present correctly on
the left side of the primary pharmacophore can result in potent
compounds. On the other hand, a dramatic decrease in inhibition
potency was observed when the amide function was substituted
with a methyl group, as seen in compound 36. This indicates that
a free NH of the amide function of cycloalkylamide compounds
investigated in the present study plays an important role in
inhibiting the target enzyme in the binding pocket. Analysis of
the X-ray crystal structure shows that the urea central pharma-
cophore is bound by hydrogen bonding between the phenol
groups of Tyr381 and Tyr465 and the carbonyl of the urea while a
29
152
115
125
125
31
32
33
151
34
>200
172
31
35
AUDAb
15
114
63
a Water solubility was determined by adding varying concentrations of a
test compound in DMSO to 0.1 M sodium phosphate buffer (pH 7.4)
in a final ratio of 5:95 (v/v). The turbidity of the water solution
was measured at 650 nm to determine solubility in water. Results
are triplicate averages. b 12-(3-Adamantan-1-ylureido)dodecanoic acid,
which was synthesized in the reaction of 1-adamantane isocyanate
with 12-aminododecanoic acid in 1,2-dichloroethanol as previously
described.20 1H NMR δ (CDC13) 1.20-1.36 (16H, m), 1.42-1.48
(2H, m), 1.61 (6H, s), 1.86 (6H, s), 1.96 (3H, s), 2.18 (2H, t, J = 6.9 Hz),
2.90 (2H, t, J = 6.9 Hz), 3.45 (1H, s), 5.43 (1H, s), 5.58 (1H, s).
presence of two (25) or four (26) methylene carbons between
the amide and the benzene ring reduced inhibition potency (4- to
6-fold) for the target enzyme, implying that the phenylpropyl on
the right side of the amide function in compound 19 is more
effective than phenethyl (25) or phenylbutyl (26) for inhibition
potency. In previous reports, we indicated that introducing
functional groups in specific positions in hydrophobic inhibitors
is generally useful for improving physical properties such as
solubility or melting point while maintaining inhibition potency
on human sEH.18,20,21 This suggests that optimal incorporation
of polar functional groups is important for increasing both
potency and physical properties. Thus, a series of functionalized
compounds were investigated to see if inhibition potency is
affected by functionalization as observed previously. When an
ester (27) or methoxy group (28) was incorporated on a benzene
ring, a >10-fold better inhibition was obtained in the ethyl ester
compound (27) in comparison with that of compound 28 with a
methoxy group, indicating that an ester group is one of useful
functionalities to be incorporated to improve inhibition potency
for the target enzyme. Interestingly, a large improvement in
inhibition was gained in adding an ester function on the benzene
ring of compound 24 with poor inhibition potency (29). On the
other hand, similar potency was observed in both compounds 25
and 30 with phenethyl on the right side of the amide function,
implying that functionalization of the benzene ring is highly
effective for increasing inhibition potency for the target enzyme.
These results also show that the functionalized benzyl group on
salt bridge is formed between one of the urea NH and Asp333 19,29
.
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The free NH and the carbonyl of the amide pharmacophore may
also be bound by the hydrogen bonding and the salt bridge in the
pocket in inhibiting the enzyme.
26), a >5-fold decreased potency was yielded, suggesting that the
presence of a functional group, such as an ester, on the right side
of the amide pharmacophore is important to produce highly
potent inhibition for human sEH in cycloalkylamide derivatives.
Moreover, modification of the benzyl linker of compound 29 by
ethylphenyl (30) or biaryl (31) induced a decrease in inhibition
potency at least >7-fold, demonstrating that a benzyl group as a
linker is important for making potent inhibitors in cycloalkyla-
mide compounds. In a combined structure (32) with the
optimized left and right units of the amide function in Tables 1
and 2 further improved inhibition was obtained while having
improved physical properties. In general, either low water solu-
bility or high melting point can be addressed with careful
formulation. However, low water solubility and high melting
point are hard to address by formulation unless a compound is
highly potent. Amides are generally more soluble in a variety of
reagents used for formulation and are generally lower melting
than similar ureas. The results obtained from the present study
will be the basis for the design of specific and selective sEH
inhibitors containing cycloalkylamide pharmacophores. Further-
more, the results will be useful for the design of intravenous or
orally available therapeutic agents for hypertension, vascular and
renal inflammation, and other disorders that can be addressed by
changing the In Vivo concentration of chemical mediators that
contain an epoxide.
The results in Table 2 indicate that a benzyl linker substituted
with a functional group (e.g., ester) on the right side of the amide
function of cycloalkylamide compounds produces potent inhibi-
tors of the target enzyme (29), and the presence of naphthalene
structures (32-35) on the left side of the amide pharmacophore
improves inhibition potency. In general, liphophilicity of com-
pounds causes limited solubility in water, which probably affects
their In Vivo efficacy.17,21,23 In addition, the stability of the
crystals of compounds, indicated by their high melting points, led
to a general lack of solubility even in organic solvents. These poor
physical properties result in undesirable pharmacokinetic proper-
ties and difficulty in compound formulation in either an aqueous
or oil base.21,23 So we continuously examined the physical
properties of the above potent derivatives in Table 2. As seen
in Table 3, relatively high melting points (>150 °C) were
measured in the aryl derivatives (33-35), while those of
cycloalkylamide compounds (29 and 32) were observed in a
lower range (115-125 °C). In addition, the inhibitors with an
adamantane (29) or tetrahydronaphthalene (32) group on the
left side of amide pharmacophore showed higher solubility (4- to
8-fold) than the corresponding aryl derivatives (33, 34), suggest-
ing that a cycloalkyl substituent is better than an aryl group in
producing improved physical properties. When compared to that
of 12-(3-adamantan-1-ylureido)dodecanoic acid (AUDA), a
representative urea inhibitor used for sEH related biological
experiments in vitro and In Vivo, a 2-fold better solubility was
observed in compounds 29 and 32, indicating that the cycloalk-
ylamide structure is highly promising for developing potent
inhibitors with improved physical properties and bioavailability.
’ EXPERIMENTAL SECTION
Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Purity and characteriza-
tion of compounds were established by a combination of elemental
analysis, TLC, LC-MS, melting point, and NMR analytical techniques
described below. All melting points were determined with a Stuart SMP3
apparatus (A. H. Thomas Co.) and are uncorrected. 1H NMR spectra
were recorded on a Digital Avance 400 MHz spectrometer (Bruker
Analytik GmbH), using tetramethylsilane as an internal standard.
Mass spectra were measured by LC-MS/MS (EsquireHCT, Bruker
Daltonics) using positive mode electrospray ionization. Elemental
analyses (C, H, N) were performed for seven novel key compounds
by Central Lab of Mokpo National University, Korea. Thin-layer
chromatography (TLC) was performed on EMD precoated silica gel
60 F254 plates, and spots were visualized with UV light and stained with
basic KMnO4. The purity of all final compounds was determined to be
greaer than 95% unless otherwise indicated. Synthetic methods are
described for representative compounds.
’ CONCLUSIONS
This work focused on investigating structure-activity rela-
tionships of cycloalkylamide derivatives as human sEH inhibi-
tors, which provide an important base for developing valuable
compounds that are not only highly potent inhibitors but show
improved physical properties. First, these data indicate that sEH
inhibitors with an cycloalkylamide central pharmacophore can be
of the same order of potency as the best inhibitors with a urea
central pharmacophore. The inhibition studies indicated that at
least six-membered cycloalkyl groups on the left side of the amide
function are necessary to produce reasonable inhibition potency
for the target human enzyme (6 and 7). In compounds sub-
stituted with a smaller cycloalkyl group such as cyclopropane (4)
or cyclopentane (5) on the left side of the amide pharmacophore
no inhibition of the target enzyme was observed. In addition,
direct incorporation of a polar group such as hydroxyl (10) or
carbonyl (11) on the cycloalkane induced a dramatic decrease in
inhibition potency. On the other hand, a hydrophobic (8 and 9)
or an ester group (15) added to the cycloalkane made inhibitors
with improved inhibition potency. Furthermore, a sterically
hindered cycloalkane (e.g., adamatane, 19) present on the left
side of the amide function was useful for enhancing inhibition
potency. We found that a tetrahydronaphthalene (20) is the
most promising left side substituent for cycloalkylamide deriva-
tives. When a substituted benzyl linker with an ester group was
present on the right side of the amide pharmacophore, highly
enhanced inhibition potency for the target enzyme was obtained
(29 and 37). In nonfunctionalized derivatives (19, 24, 25, and
Synthesis of 4-Oxo-N-(3-phenylpropyl)cyclohexanecar-
boxamide (11). To a solution of ethyl 4-oxocyclohexanecarboxylate
(2.0 g, 11.7 mmol) in ethanol (5 mL) was added an aqueous solution of
1 N NaOH (15 mL) at room temperature. After being stirred overnight,
the reaction mixture was acidified with an aqueous solution of 1 N HCl
to pH 2. The water solution was extracted with ethyl acetate (60 mL ꢀ 3)
and dichloromethane (60 mL), respectively. The combined organic
solution was washed with water (60 mL ꢀ 2), dried over MgSO4, and
evaporated to dryness. To the residue (0.89 g, 6.25 mmol) in dichlor-
omethane (50 mL) was added 4-dimethylaminopyridine (DMAP; 0.76
g, 6.25 mmol) and 3-phenylpropylamine (0.93 g, 6.88 mmol) at room
temperature. After the mixture was stirred for 5 min, 1-[3-(dimethy-
lamino)propyl]-3-ethylcarbodiimide (EDCI; 1.31 g, 6.88 mmol) was
added portionwise to the reaction mixture. Extraction and purification of
the product were performed with the same method used for the
1
preparation of compound 32 to afford 11 as a solid (85%). H NMR
δ (CDCl3): 1.80-1.98 (4H, m), 2.05-2.17 (2H, m), 2.28-2.57 (5H,
m), 2.63 (2H, t, J = 6.8 Hz), 3.32 (2H, t, J = 6.8 Hz), 5.56 (1H, s),
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7.15-7.20 (3H, m), 7.26-7.30 (2H, m). LC-MS (ESI) m/z calcd for
C16H21NO2 [M þ H]þ 260.16, found [M þ H]þ 260.52. Mp 70 °C.
Compound 14 was synthesized with the same method used for the
preparation of compound 11 using methyl tetrahydro-2H-pyran-4-car-
boxylate instead of ethyl 4-oxocyclohexanecarboxylate.
NMR δ (CDCl3): 1.77 (6H, s), 2.01 (6H, s), 2.13 (3H, s), 3.97 (3H, s),
7.48-7.50 (2H, m), 7.80 (1H, d, J = 6.8 Hz), 7.89 (1H, d, J = 6.8 Hz),
8.01 (1H, d, J = 6.8 Hz), 8.36 (1H, s), 8.53 (1H, s). LC-MS (ESI) m/z
calcd for C23H25NO3 [M þ H]þ 364.18, found [M þ H]þ 364.77. Mp
>200 °C.
Compound 30 was synthesized with the same procedure used for the
preparation of 31 using the corresponding cycloalkanecarboxylic acid,
alkylamine, and iodomethane.
Synthesis of Methyl 4-(3-Phenylpropylcarbamoyl)cyclo-
hexanecarboxylate (15). A mixture of 1,4-cyclohexanedicarboxylic
acid (2.0 g, 11.6 mmol), potassium carbonate (4.82 g, 34.8 mmol), and
iodomethane (4.95 g, 34.8 mmol) in DMF (10 mL) was stirred
overnight at room temperature. The product was extracted with diethyl
ether (60 mL). The organic solution was washed with water (60 mL ꢀ
2), dried over MgSO4, and evaporated. The residue was washed with
cold hexane (20 mL) and concentrated to yield the corresponding
diethyl ester (100%).
A mixture of the above diester (1.77 g, 8.84 mmol) and barium
hydroxide octahydrate (1.39 g, 4.42 mmol) in 80% aqueous methanol
(30 mL) was stirred overnight at room temperature. Then the reaction
mixture was diluted with water (50 mL) and washed with hexane (50 mL
ꢀ 2) to remove remaining diester. The aqueous layer was acidified with
concentrated HCl to pH 2, and the product was isolated by extracting
with dichloromethane (50 mL). This organic layer was washed with
water (50 mL), dried over MgSO4, and evaporated to give 4-(meth-
oxycarbonyl)cyclohexanecarboxylic acid (65%). Without further purifi-
cation the residue was used for the next reaction.
Synthesis of Methyl 4-((1,2,3,4-Tetrahydronaphthalene-
2-carboxamido)methyl)benzoate (32). To a solution of 1,2,3,4-
tetrahydronaphthalenecarboxylic acid (0.4 g, 2.26 mmol) in dichloro-
methane (30 mL) was added DMAP (0.28 g, 2.26 mmol) and methyl
4-(aminomethyl)benzoate hydrochloride (0.46 g, 2.26 mmol) at room
temperature. To this mixture was then added portionwise EDCI (0.44 g,
2.26 mmol) at room temperature. After the mixture was stirred for 4 h,
the product was extracted with diethyl ether (50 mL) twice. The ether
solution was washed with an aqueous solution of 0.5 N HCl (50 mL) and
water (80 mL), dried over MgSO4, and evaporated. The residue was
recrystallized in hexane to afford compound 32 as a solid in 95% yield.
1H NMR δ (CDCl3): 1.93-1.94 (1H, m), 2.09-2.10 (1H, m), 2.55-
2.57 (1H, m), 2.82-3.04 (4H, m), 3.90 (3H, s), 4.50 (2H, s), 6.12 (1H,
s), 7.06-7.12 (4H, m), 7.30 (2H, d, J = 6.8 Hz), 7.97 (2H, d, J = 6.8 Hz).
LC-MS (ESI) m/z calcd for C20H21NO3 [M þ H]þ 324.15, found [M
þ H]þ 324.54. Mp 115 °C.
To a solution of the above monoacid (0.89 g, 4.78 mmol), DMAP
(0.58 g, 4.78 mmol), and 3-phenylpropylamine (0.64 g, 5.25 mmol) in
dichloromethane (30 mL) was added EDCI (1.0 g, 5.25 mmol) at room
temperature. The reaction mixture was then stirred for 4 h at room
temperature. The product was extracted and purified with the same
method used for the synthesis of compound 32 to yield 15 as a solid
(90%). 1H NMR δ (CDCl3): 1.52-1.68 (5H, m), 1.81 (2H, quint, J =
6.8 Hz), 2.03-2.16 (4H, m), 2.52-2.57 (1H, m), 2.62 (2H, t, J = 6.8
Hz), 3.23 (2H, t, J = 6.8 Hz), 3.64 (3H, s), 5.92 (1H, s), 7.15-7.20 (3H,
m), 7.26-7.30 (2H, m). LC-MS (ESI) m/z calcd for C18H25NO3 [M
þ H]þ 304.18, found [M þ H]þ 304.53. Mp 63 °C.
Compounds 3-14, 18-29, and 33-35 were synthesized with the
same procedure used for the preparation of 32 using the corresponding
cycloalkanecarboxylic acid and alkyl- or arylamine instead of 1,2,3,4-
tetrahydronaphthalenecarboxylic acid and methyl 4-(aminomethyl)-
benzoate hydrochloride.
Synthesis of N-Methyl-N-(3-phenylpropyl)-1-adamanta-
necarboxamide (36). To a suspension of sodium hydride 60%
(60 mg, 1.61 mmol) in DMF (10 mL) was added compound 19 (0.4 g,
1.34 mmol) at room temperature. After the mixture was stirred for 5 min,
iodomethane (0.23 g, 1.61 mmol) was added to the reaction mixture at the
same temperature. The product was extracted with diethyl ether (50 mL)
after stirring overnight. The ether solution was washed with water (50 mL)
twice, dried over MgSO4, and evaporated. The residue was purified by
using column chromatography (hexane/ethyl acetate = 5:1) to give
compound 36 as an oil. 1H NMR δ (CDCl3): 1.69 (6H, s), 1.88 (2H, t,
J = 6.8 Hz), 1.93 (6H, s), 1.97 (3H, s), 2.61(2H, t, J= 6.8 Hz), 3.02 (3H, s),
3.41 (2H, t, J = 6.8 Hz), 7.17-7.19 (3H, m), 7.26-7.29 (2H, m). LC-MS
(ESI) m/z calcd for C21H29NO [M þ H]þ 312.21, found [M þ H]þ
312.34.
Enzyme Preparation. Recombinant human sEH was produced in
baculovirus expression system as previously reported.30 Briefly, Sf9
insect cells were infected by recombinant baculovirus harboring human
sEH gene fused with a 6xHis tag. At 72 h postinfection, the infected cells
were homogenized and the recombinant protein was purified by
immobilized metal affinity chromatography. After treatment with to-
bacco etch virus protease to remove the 6xHis tag, human sEH was
further purified by anion-exchange chromatography.
Compound 17 was synthesized in the same manner used for the
preparation of compound 15 using cyclohexane-1,2-dicarboxylic acid
instead of cyclohexane-1,4-dicarboxylic acid.
Synthesis of 4-(3-Phenylpropylcarbamoyl)cyclohexane-
carboxylic Acid (16). To a solution of compound 15 (0.20 g, 6.59
mmol) in methanol (5 mL) was added an aqueous solution of 1 N
NaOH (3 mL). After being stirred overnight at room temperature, the
reaction mixture was acidified by adding an aqueous solution of 1 N HCl
to pH 2. The product was extracted with ethyl acetate (50 mL ꢀ 2). The
organic layer was washed with water (50 mL), dried over MgSO4, and
evaporated. The residue was recrystallized in ethyl acetate to afford 16 as
a solid in 95% yield. 1H NMR δ (CDCl3): 1.44-1.59 (2H, m), 1.67-
1.73 (3H, m), 1.78-1.86 (3H, m), 2.06-2.19 (3H, m), 2.59-2.68 (3H,
m), 3.28 (2H, t, J = 6.8 Hz), 5.44 (1H, s), 7.16-7.21 (3H, m), 7.26-7.31
(2H, m). LC-MS (ESI) m/z calcd for C17H23NO3 [M þ H]þ 290.17,
found [M þ H]þ 290.57. Mp 127 °C.
Synthesis of Methyl 6-(1-Adamantanecarboxamido)-
naphthalene-2-carboxylate (31). 6-(1-Adamantanecarboxami-
do)naphthalene-2-carboxylic acid intermediate was prepared with the
same method used for the synthesis of compound 32 using the
corresponding 1-adamantanecarboxylic acid (0.50 g, 2.77 mmol) and
6-aminonaphthalene-2-carboxylic acid (0.52 g, 2.77 mmol) in 60% yield.
To a mixture of the acid intermediate and potassium carbonate (1.0 g,
6.93 mmol) was added iodomethane (1.0 g, 6.93 mmol) in DMF (25
mL) at room temperature. After the mixture was stirred overnight, the
product was extracted with diethyl ether (60 mL). The organic solution
was washed with water (60 mL) twice, dried over MgSO4, and
evaporated. The residue was purified using silica gel column chroma-
tography (hexane/ethyl acetate = 3:1) to give 31 as a solid (95%). 1H
IC50 Assay Conditions. Standard solutions of compounds in
Figure 1 and Tables 1 and 2 were prepared in DMSO. Fluorescent
assays were performed by using a substrate (cyano(2-methoxynaphtha-
len-6-yl)methyl trans-(3-phenyloxyran-2-yl)methylcarbonate, CMNPC)
to determine IC50 values of the human sEH inhibitors according to
a published protocol31 except for the final enzyme concentration
(1 nM).30,32 Activity was measured by determining the appearance of
the 6-methoxy-2-naphthaldehyde with an excitation wavelength of
330 nm and an emission wavelength of 465 nm for 10 min on a
fluorometer (Victor3, PerkinElmer).30,32 The IC50 values were deter-
mined by regression of at least six data points with a minimum of three
points in a linear region of the curve. IC50 results are averages of three
separate measurements.
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Solubility. Water solubility of compounds in Table 3 was deter-
mined experimentally by light scattering method in sodium phosphate
buffer at 25 ( 1.5 °C. In brief, aqueous solubility was determined by
adding varying concentrations of inhibitor in DMSO to 0.1 M sodium
phosphate buffer (pH 7.4) in a final ratio of 5:95 (v/v). Insolubility of
the inhibitor was indicated by the increase in turbidity of the water
solution. The turbidity was measured as optical density at 650 nm on a
SH-8000 microplate reader (Corona Electric, Ibaraki, Japan) at 25 (
1.5 °C. Results are averages of three separate measurements.
inflammation by treatment with a soluble epoxide hydrolase inhibitor.
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’ ASSOCIATED CONTENT
S
Supporting Information. Analytical data for compounds
b
3-10, 12-14, 17-30, and 33-35; table of elemental analyses
for compounds 19, 20, 29, 32, and 33-35. This material is
available free of charge via the Internet at http://pubs.acs.org.
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Hammock, B. D.; Chiamvimonvat, N. Prevention and reversal of cardiac
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Sci. U.S.A. 2006, 103, 18733–18738.
(15) Moghaddam, M. F.; Grant, D. F.; Cheek, J. M.; Greene, J. F.;
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(16) Newman, J. W.; Denton, D. L.; Morisseau, C.; Koger, C. S.;
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’ AUTHOR INFORMATION
Corresponding Author
*Phone/fax: þ81-89-946-9818. E-mail: knishi@agr.ehime-u.ac.jp.
’ ACKNOWLEDGMENT
This work was supported by Hyundai Pharm Research
Grant HOB-024. Partial support was from NIEHS Grant R01
ES002710.
’ ABBREVIATIONS USED
EET, epoxyeicosatrienoic acid; sEH, soluble epoxide hydrolase;
EDCI, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide; DMAP,
4-dimethylaminopyridine; DMF, N,N-dimethylformamide; CM-
NPC, cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-pheny-
loxyran-2-yl)methylcarbonate; AUDA, 12-(3-adamantan-1-ylur-
eido)dodecanoic acid
(18) Kim, I.-H.; Morisseau, C.; Watanabe, T.; Hammock, B. D.
Design, synthesis, and biological activity of 1,3-disubstituted ureas as
potent inhibitors of the soluble epoxide hydrolase of increased water
solubility. J. Med. Chem. 2004, 47, 2110–2122.
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