Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmc.2013.12.027

We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors

Full text of DOI:10.1016/j.bmc.2013.12.027

Accepted Manuscript
Structure-Activity Relationships of Substituted Oxyoxalamides as Inhibitors of
the Human Soluble Epoxide Hydrolase
In-Hae Kim, In-Hee Lee, Hisashi Nishiwaki, Bruce D. Hammock, Kosuke Nishi
PII:
S0968-0896(13)01021-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.12.027
BMC 11289
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 October 2013
6 December 2013
7 December 2013
Please cite this article as: Kim, I-H., Lee, I-H., Nishiwaki, H., Hammock, B.D., Nishi, K., Structure-Activity
Relationships of Substituted Oxyoxalamides as Inhibitors of the Human Soluble Epoxide Hydrolase, Bioorganic &
Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.12.027
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December 6, 2013
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Structure-Activity Relationships of Substituted Oxyoxalamides
as Inhibitors of the Human Soluble Epoxide Hydrolase
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In-Hae Kim,a In-Hee Lee,b Hisashi Nishiwaki,a Bruce D. Hammock,c and Kosuke Nishia*
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aDepartment of Applied Bioscience, Faculty of Agriculture, Ehime University, Matsuyama, Ehime
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790-8566, Japan
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bDepartment of Medicinal Chemistry, Hyundai Pharm Co., Ltd., Suwon, Gyonggi 443-270, Republic
of Korea
cDepartment of Entomology & UCD Comprehensive Cancer Center, University of California Davis
One Shields Ave, Davis, CA 95616, USA
*Correspondence Address:
Department of Applied Bioscience, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi,
Matsuyama, Ehime 790-8566, Japan
Phone/fax: +81-89-946-9818. E-mail: knishi@agr.ehime-u.ac.jp (K. Nishi)
Abbreviations: EETs, epoxyeicosatrienoic acids; sEH, soluble epoxide hydrolase; EDCI, 1-[3-
(dimethylamino)propyl]-3-ethyl-carbodiimide; DMAP, 4-dimethylaminopyridine; DMF, N,N-
dimethylformamide; CMNPC, cyano-(2-methoxynaphthalen-6-yl)-methyl trans-(3-phenyl-oxyran-2-
yl)-methyl carbonate.
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Abstract
We explored both structure-activity relationships among substituted oxyoxalamides used as
the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to
improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety
of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found
to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising
primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the
development of further improved oxyoxalamide or other related derivatives. In addition, introduction
of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced
significant improvements in inhibition potency and water solubility. In particular, the N,N,O-
trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among
those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide
function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the
resulting structures will be an important basis for the development of bioavailable sEH inhibitors.
Key words: Substituted oxyoxalamides, human soluble epoxide hydrolase, inhibitors
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1. Introduction
Arachidonic acid, a ω-6 polyunsaturated fatty acid, plays important roles in cellular
signaling as a lipid second messenger and is also a precursor in the production of oxidative
metabolites known as eicosanoids by enzymes such as cyclooxygenase, lipoxygenase, and
cytochrome P450. Prostanoids1,2 and leukotrienes3 are major metabolic products of arachidonic acid
by cyclooxygenase and lipoxygenase, respectively. These two pathways are largely inflammatory
and induce inflammation, pain, and asthma,1-3 making the both enzymes current pharmaceutical
targets for relief from the symptoms. The third branch of arachidonic acid cascade involves oxidation
by cytochrome P450 to produce several inflammatory hydroxylated metabolites and the
corresponding lipid epoxides formed at the olefinic centers and known as epoxyeicosatrienoic acids
(EETs). EETs have been reported as a new class of lipid mediators with important biological
functions.4 The endogenous epoxy lipids, EETs, influence blood pressure by modulating cardiac
output, vascular resistance, and urinary composition.5-11 In addition, vascular inflammation and pain
are modulated by the action of EETs.9,10 However, the metabolism of the epoxy functionality of the
EETs to the vicinal diols by soluble epoxide hydrolase (sEH) dramatically diminishes the biological
activities.6 Many reports have shown that the treatment of potent human sEH inhibitors increases
EET levels and reduces blood pressure and inflammatory responses in in vitro and in vivo
experimental models,5-11 suggesting that human sEH is a promising pharmacological target for the
treatment of cardiovascular and other diseases.
A number of urea compounds with a variety of substituents are highly potent inhibitors of
the human sEH.12-21 The best optimization of urea derivatives affords specific inhibition potency for
the target enzyme in a range of less than 1 nM. Structure-activity relationship studies indicate that a
carbonyl group and a single proton donating NH group of urea function are essential for making it an
effective primary pharmacophore to inhibit the enzyme activity. Functionalities such as amides and
carbamates with both a carbonyl group and an NH group are, therefore, known to produce potent
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inhibition as a primary pharmacophore, while ester or carbonate functions without a proton donating
NH group yield no inhibition for the target enzyme.12,22-24 Many of these compounds are difficult to
formulate because they are high melting liphophilic solids. These formulation problems can be
solved by reducing the melting point and crystal stability, increasing water solubility, and increasing
potency. On the other hand, when a variety of functionalities including amides, esters, ketones, and
ethers are incorporated as a secondary pharmacophore remote from the catalytic site in potent urea
inhibitors, dramatic changes in inhibition potency are not observed, rather significant improvement
in physical properties is often obtained,12 implying that primary inhibition of the human sEH
depends on the structure of primary pharmacophores and secondary pharmacophores are useful for
improving physical properties and potency. In the present study, we first investigated replacement of
the primary pharmacophore with a series of substituted oxyoxalamides and then used oxyoxalamides
as a second series to replace the secondary pharmacophore using the classical amide and urea
primary pharmacophores. In both series, potent compounds were found with improved water
solubility.
2. Results and Discussion
2.1. Chemistry
Substituted oxyoxalamide derivatives (3-15) and N-(benzyloxy)-2-(adamant-2-
ylamino)acetamide (16) in Tables 1 and 2 were synthesized as outlined in Scheme 1. Ethyl
(chlorocarbonyl)formate was reacted with an alkyl- or a cycloalkyl-amine (Scheme 1A) or adamant-
2-ylamine (Scheme 1B) in dichloromethane, followed by hydrolysis with 1 N NaOH in ethanol to
provide the corresponding (carbamoyl)formic acid in approximately 80-95% yield. The formic acid
was then coupled with benzyloxyamine (Scheme 1A) or with a substituted oxyamine (Scheme 1B)
using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDCI) and 4-dimethylaminopyridine
(DMAP) in dichloromethane to yield compounds 3-12, 14, and 15 in 50-85% yield.23 As seen in
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Scheme 1B, substituted oxyamines for compounds 10-12 were prepared by the reaction of a
corresponding bromide with N-hydroxyphthalimide in N,N-dimethylformamide (DMF), which was
followed by the addition of hydrazine in trichloromethane (40-50%). N-Methylation of compound 6
with iodomethane (CH3I) in the presence of potassium carbonate (K2CO3) in DMF afforded
compound 13 in 55% yield (Scheme 1C). Substitution of methyl 2-bromoacetate with adamant-2-
ylamine in dichloromethane and hydrolysis of the ester with 1 N NaOH in methanol gave the
corresponding adamantylaminoacetic acid in approximately 85% yield. Then, coupling of this acid
with benzyloxyamine using EDCI/DMAP in dichloromethane produced compound 16 in 70% yield
(Scheme 1D).
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Amide-oxyoxalamide derivatives 18-26 in Table 3 were synthesized by the procedure
outlined in Scheme 2. Coupling reaction of 4-aminobenzylamine with adamant-1-ylcarboxylic acid
(for compounds 18-20 and 22) or with adamant-1-ylmethylcarboxylic acid (for compound 23) using
EDCI/DMAP in dichloromethane afforded an amide-amine intermediate, which was used for the
reaction with ethyl (chlorocarbonyl)formate in the presence of triethylamine (Et3N) in
dichloromethane to make substituted carbonylformate in about 60% yield. Hydrolysis of the ester
function of this intermediate with 1 N NaOH in ethanol gave the corresponding (carbamoyl)formic
acid, which was further reacted with a substituted oxyamine in the presence of EDCI/DMAP
coupling reagents in dichloromethane to produce compounds 18-20, 22, and 23 in a range of 50-90%
yield (Scheme 2A). As depicted in Scheme 2B, coupling of adamant-1-ylmethylcarboxylic acid with
mono-N-protected 4-aminoaniline using EDCI/DMAP and following N-de-protection using 4 N HCl
provided the corresponding amide-amine intermediate in about 80% yield. This amide-amine was
reacted with ethyl (chlorocarbonyl)formate in dichloromethane to afford the corresponding formate,
which was hydrolyzed with 1 N NaOH in ethanol to give a formic acid intermediate in 85% yield.
Coupling of the acid function with methoxymethylamine using EDCI/DMAP yielded compound 25
in 70% yield. In addition, compounds 21, 24, and 26 were synthesized by the alkylation of
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compounds 20, 23, 25, respectively, with CH3I in the presence of K2CO3 as a base in DMF in
approximately 60% yield (Scheme 2C).
Urea compounds with substituted oxyoxalamide were synthesized as outlined in Scheme 3.
The reaction of 4-aminobenzylamine with adamant-1-yl isocyanate in DMF gave 1-adamantyl-3-(4-
aminobenzyl)urea in 100% yield.13 Compounds 28, 29, 30, and 32 were produced by the above
reactions used for the syntheses of compounds 18, 19, 20, and 22 from this urea-amine intermediate
in a range of 40-70% yield (Scheme 3A). N-Methylation of compound 30 using CH3I and K2CO3 as
a base in DMF yielded compound 31 in 50% yield (Scheme 3B).
2.2. Structure-activity relationships
In order to first investigate whether the oxyoxalamide function can act as an effective
primary pharmacophore to inhibit the human sEH, a series of substituted oxyoxalamides were
synthesized. Our previous studies show that potent inhibition of the target enzyme is obtained with
the substitution of relatively hydrophobic alkyl or cycloalkyl groups and substituted alkyl or
aromatic groups on the left side and the right side of urea or amide pharmacophores, respectively (1
and 2 in Figure 1).12,14,15,21,23 Thus, hydrophobic alkyls and substituted alkyl or aryl groups were
incorporated in the both sides of the diketo of oxyoxalamide moiety, and inhibition potency of the
oxyoxalamide derivatives for the human sEH was evaluated (Table 1). As seen in compounds 3 and 4,
3-phenylpropyl (3) and 1-adamantyl (4) groups in the left side of the diketo, which are useful for
making potent inhibitors with urea or amide functions, were not effective for producing inhibition
activity for the target enzyme with oxyoxalamide function. However, in replacing the substituent in
the left side of the diketo of compounds 3 and 4 by an adamantylmethyl group (5), moderate
inhibition was gained. Moreover, interestingly, introduction of a 2-adamantyl group (6) in the left
side of the diketo moiety afforded a high improvement (16-fold) in inhibition potency compared to
that of compound 5. Comparing compound 6 with 2-adamantane to compound 4 with 1-adamantane,
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improvement in inhibition activity was dramatic, indicating that the 2-adamantyl group is
particularly suitable for being the left side substituent of oxyoxalamide function to yield significant
inhibition potency. It is also implied that slight changes in orientation or size of the alkyl groups in
the left side of oxyoxalamide function as shown in compounds 3-6 result in a large variation in
inhibition for the target enzyme, which is different from the results that similarly potent inhibition is
observed in urea or amide derivatives substituted with the alkyl groups.14,15,22,23
Based on the above results, the 2-adamantyl was fixed on the left side of the diketo moiety
of the oxyoxalamide, and then a benzyl group in the right side of the diketo of compound 6 was
further modified with phenyl and several arylalkyl groups. Because aryl containing groups in the
right side of urea or amide pharmacophores (e.g. 2 in Figure 1) provide much higher binding activity
than aliphatic alkyl groups,15,21,23 compounds with aryl substituent (7-13) were synthesized. As seen
in compound 7, replacement of the benzyl group of compound 6 by a phenyl group resulted in no
inhibition. This implies that the methylene benzyl carbon in the right side of the oxyoxalamide is
necessary for producing inhibition of the target enzyme. When a functional group such as a methyl
ester (8), nitro (9), or chloro (10) was incorporated on the 4-position of the benzyl group of
compound 6, > 300-fold drop in inhibition was also observed, indicating that these substituents on
the benzyl group of compound 6 are not effective for increasing inhibition potency. These results are
not consistent with previous observations with urea or amide primary pharmacophores.21,23 Because
we previously showed that a longer alkyl chain like an ethyl or a propyl between primary amide
pharmacophore and benzene ring in the right side of the amide function provides improved inhibition
for the target enzyme,23 2-phenylethyl and 3-phenylpropyl were introduced instead of the benzyl
group of compound 6. As seen in compounds 11 and 12, a propyl chain (12) resulted in
approximately 8-fold better inhibition than an ethyl chain (11). However, this still gave
approximately 10-fold drop in inhibition when compared to the potency of compound 6, supporting
that one methylene carbon of the benzyl group of compound 6 is important for yielding significant
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inhibition potency. N-Substitution of compound 6 with a methyl group (13) dropped inhibition
potency approximately 60-fold. Inhibition was further lost in the presence of a dimethyl or a polar
substituent as observed in compounds 14 and 15, respectively, indicating that a benzyl group next to
an unsubstituted NH function is effective as a right side substituent for yielding promising inhibitors
with an oxyoxalamide function as a primary pharmacophore. Furthermore, when one carbonyl group
of compound 6 was deleted as seen in compound 16 in Table 2, no inhibition was observed,
suggesting that the carbonyl group of oxyoxalamide function plays an important role in inhibiting the
target human enzyme like that of urea or amide functions.12 The inhibition potency of compound 6
was approximately 15-fold lower when compared to that of a very potent urea-based inhibitor
(AUDA),13 indicating that the oxyoxalamide 6 can be used as a new lead structure for structure-
activity relationship studies to further produce as potent oxyoxalamide inhibitors as urea-based
compounds. Overall, the results in Tables 1 and 2 show that the substituted oxyoxalamides are
promising novel functionalities as primary pharmacophores to yield inhibitors of significant potency
for the human sEH.
A functional group incorporated on around the 5th to 7th atom from the carbonyl of primary
pharmacophores such as ureas or amides can play a role as a secondary pharmacophore to modify
inhibition potency and/or physical properties of urea or amide inhibitors.12,14,22,23 In general,
functions such as amides, esters, ketones, ethers, sulfonamides, sulfoxides, and sulfones are useful
secondary pharmacophores for improving physical properties without decreasing inhibition potency
of urea inhibitors.12,14,23 In order to investigate whether the oxyoxalamide function can act as an
effective secondary pharmacophore for improving inhibition potency and/or physical properties, a
series of amide and urea derivatives with substituted oxyoxalamide groups were synthesized as
shown in Tables 3 and 4. We previously showed that incorporation of a functional group like an ester
as a secondary pharmacophore on a benzene ring in the right side of amide inhibitors like compound
17 in Table 3 is highly useful for enhancing inhibition potency for the target enzyme (2 in Table 2).23
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Thus, the oxyoxalamide function was introduced to compound 17 to see if it works as an effective
secondary pharmacophore for improving inhibition potency or the physical properties of amide
inhibitor. When an oxyoxalamide group was substituted on the 4-position of the benzene ring of
compound 17 as seen in compound 18, at least a 3-fold enhancement in inhibition was gained,
indicating that the oxyoxalamide function is also a useful secondary pharmacophores for improving
inhibitory potency of amide compounds. Because a polar functional group like an oxyoxalamide
inserted as a secondary pharmacophore plays an important role in improving physical properties (e.g.
water solubility) of hydrophobic inhibitors as well, oxyoxalamides with relatively less hydrophobic
groups were incorporated on the amide inhibitors. As seen in compound 19, substitution of a t-butyl
group on the oxygen atom of the oxyoxalamide instead of a methyl group of compound 18 lowered
inhibition a 1.5-fold, showing that a smaller alkyl group on the oxygen atom is better for inhibition.
In addition, oxyoxalamides with N,O-dimethyl (20) or N,N,O-trimethyl (21) yielded a further
improved potency (1.5-fold) compared to that of O-methyl derivative (18), implying that the N-
substitution of the oxyoxalamide is useful for enhancing inhibition for the target enzyme. On the
other hand, an oxyoxalamide with a polar group on the oxygen atom (22) led to a complete loss in
inhibition, suggesting that non-polar small alkyl groups such as a methyl group are optimal
substituents for the oxyoxalamide function to produce significant inhibition potency. Interestingly,
when the adamantyl group in the left side of the amide primary pharmacophore of compounds 20 and
21 was modified by an adamantylmethyl group (23 and 24), inhibition potency was enhanced up to
25-fold. In addition, replacement of the benzyl group in the right side of the amide function of
compounds 23 and 24 with a phenyl group (25 and 26) improved inhibition potency approximately
2-fold. This is approximately 50-fold enhanced inhibition compared to that of compound 21,
indicating that optimizing around the amide pharmacophore is a useful approach for yielding potent
amide-oxyoxalamide inhibitors. Production of highly potent amide-oxyoxalamide inhibitors depends
on the structure of the primary pharmacophore more than that of secondary pharmacophore. It also
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was found that N-methylation of the both nitrogen atoms of the oxyoxalamide is especially important
for affording potent amide-oxyoxalamide derivatives as shown by comparing compound 24 to 23.
Compound 26 with N,N,O-trimethylated oxyoxalamide had an 8-fold better inhibition than
compound 25 with N,O-dimethylated oxyoxalamide, again showing the effectiveness of N-
substitution of the both nitrogen atoms of the oxyoxalamide for the production of further improved
amide-oxyoxalamide inhibitors. When water solubility of the potent compounds (23-26) was
measured, a 2-15-fold enhancement was obtained in comparison with that of the amide inhibitor with
no oxyoxalamide function (17). Furthermore, the solubility was comparable to those of previously
reported soluble amide- (2)23 and urea-based (IK950)14 inhibitors. It was also found that inhibitions
obtained from compounds 24 and 26 were similar to those of potent inhibitors 2 and IK950. This
implies that the incorporation of the substituted oxyoxalamide function as a secondary
pharmacophore is effective for not only improving inhibition potency, but also significantly
increasing water solubility of amide compounds. Comparing compound 24 to 23, it was found that
N-methylation of the both nitrogen atoms of the oxyoxalamide function provides better water
solubility. A similar result was also observed between compounds 25 and 26, suggesting that the
trimethylated oxyoxalamide is especially effective for improving solubility in water.
The results in Table 3 show that attachment of a substituted oxyoxalamide as a secondary
pharmacophore to amide inhibitors results in significant enhancements in inhibition potency and
water solubility. Next, in order to see if the incorporation of the oxyoxalamide function to urea
inhibitors affects their inhibition and water solubility, several urea-oxyoxalamide derivatives were
synthesized as shown in Table 4. As seen in compound 28, an O-methyl-oxyoxalamide substitution
resulted in a 2.5-fold better inhibitor when incorporated to urea compound 27, indicating that the
oxyoxalamide function is useful for further improving inhibition potency of potent urea compounds.
On the other hand, O-t-butyl-oxyoxalamide (29) did not provide an increase in inhibition, implying
that a smaller alkyl group like a methyl on the oxygen atom is better than a relatively bulky t-butyl
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group for yielding improved urea inhibitors, which is similar to that observed in the corresponding
amide derivatives 18 and 19 in Table 3. N,O-Dimethyl (30) and N,N,O-trimethyl (31) oxyoxalamides
made a 3-fold and a 13-fold improvements in inhibition, respectively, in comparison with that of
compound 27. Interestingly, N-methylation of the both nitrogen atoms of the oxyoxalamide function
was also found to be important for yielding highly potent inhibition in urea-oxyoxalamide
derivatives as seen in compound 31, which is the same result as that observed in the corresponding
amide derivative (26). On the other hand, a polar substitution in the oxyoxalamide (32) was not
effective for making an improved urea inhibitor, which is also similar to that gained with amide
derivative 22 in Table 3. Furthermore, approximately 2-30-fold enhancement in water solubility was
gained from the substituted urea compounds (28-32) when compared to that of compound 27,
making the substituted oxyoxalamide function an effective secondary pharmacophore for improving
both inhibition and solubility. Among the derivatives tested, the most potent compound 31 with
N,N,O-trimethyl-oxyoxalamide was most soluble in water, which is a 10-fold higher solubility than
that of a urea inhibitor, AUDA. Moreover, the solubility of compound 31 was same as that of IK950
known as a soluble urea inhibitor useful for various in vivo studies. In addition, a 3-12-fold
enhancement in inhibition was observed in compound 31 when compared to those of AUDA and
IK950. These results suggest that N-methylation of the both nitrogen atoms of the oxyoxalamide
function is highly effective for both of inhibition and solubility of urea compounds as well, as that
observed in the corresponding amide derivatives 24 and 26.
3. Conclusions
This study investigated whether the oxyoxalamide function works as an effective primary
and/or secondary pharmacophore to inhibit the human sEH. In order to first see its potential to be a
primary pharmacophore, a series of oxyoxalamides substituted with alkyl, cycloalkyl, aryl, or
substituted aryl groups were synthesized (Table 1). The inhibition results indicated that a 2-
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adamantyl group (6) is the most effective left side substituent of the oxyoxalamide function for
producing significant inhibition potency for the target human enzyme. Interestingly, the 1-adamantyl
group (4) or other hydrophobic groups (3 and 5), which are highly useful for yielding potent
inhibition in amide or urea inhibitors, led to dramatically reduced or total lack of inhibition potency
with the oxyoxalamide function. When the right side of the oxyoxalamide function was optimized
with aryl containing substituents effective for affording potent amide or urea inhibitors, significant
inhibition potency was gained from a non-substituent benzyl group (6). In the presence of a
functional group on the benzene ring (8-10) or a longer alkyl group (11 and 12) between the benzene
ring and the oxyoxalamide function, which are reported as necessary elements for the production of
potent amide or urea inhibitors, a dramatic loss in inhibition was induced with the oxyoxalamide
pharmacophore. In addition, N-substitution of the oxyoxalamide function of compound 6 was not
effective for improving inhibition for the target enzyme (13 and 14). Overall structure-activity
relationship (SAR) results demonstrate that the substituted oxyoxalamide function is a promising
primary pharmacophore for human sEH inhibitors and the structural requirements for producing
significant inhibition from oxyoxalamide pharmacophore are different from those for other primary
pharmacophores such as amides and ureas. Furthermore, compound 6 found in the present study can
be a novel lead structure for the development of further improved oxyoxalamide and other related
derivatives. Next, a series of substituted oxyoxalamides were incorporated into inhibitors with an
amide or urea primary pharmacophore to investigate whether the oxyoxalamide function acts as an
effective secondary pharmacophore for improving inhibition potency and/or water solubility of them.
The SAR results in Tables 3 and 4 indicated that substituted oxyoxalamide function is a useful
secondary pharmacophore for enhancing inhibition potency of amide and urea inhibitors.
Substitution of the oxyoxalamide with smaller alkyl groups such as a methyl group was highly useful
for improving inhibition potency (18, 20-21, 23-26, 28, 30, and 31). However, oxyoxalamides with a
larger alkyl group (19 and 29) or a polar group (22 and 32) led to a significant loss in inhibition.
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These data also indicate that a N,N,O-trimethylated oxyoxalamide is especially effective for making
potent amide- or urea-oxyoxalamide derivatives (24, 26, and 31). In compounds with N,O-
dimethylated (23, 25, and 30) or O-mono-methylated (28) oxyoxalamide, reduced inhibition of up to
a 10-fold was observed, suggesting that the incorporation of three methyl groups on the
oxyoxalamide is important for yielding highly potent amide- or urea-oxyoxalamide compounds. In
addition, the potent compounds with oxyoxalamide function in Tables 3 and 4 had a 2-20-fold
enhanced water solubility. Especially, the highest increase in water solubility was gained from
compounds with the trimethylated oxyoxalamide function, suggesting that substituted oxyoxalamides
incorporated into amide or urea inhibitors as a secondary pharmacophore are useful for improving
not only inhibition but also solubility. The resulting compounds found in the present study will be the
basis for the design of selective amide- or urea-oxyoxalamide inhibitors with improved physical
properties, which will be useful for the development of intravenous or orally available compounds
for hypertension, vascular inflammation, and other cardiovascular disorders related by endogenous
mediators, including EETs and other epoxyfatty acids.
4. Experimental Section
4.1. Chemistry
Unless otherwise noted, all materials were purchased from commercial suppliers and used
without further purification. Purity and characterization of compounds were established by a
combination of TLC, LC-MS, melting point, and NMR analysis 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 (TMS) as an internal standard. 13C-NMR spectra were recorded on a JEOL JNM-
EX400 spectrometer (JEOL Ltd, Japan), using TMS as an internal standard. High resolution mass
spectra were measured by LC-MS (Xevo Q-TOFMS; Waters, UK) using positive mode electrospray
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ionization. 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 greater than 95% unless otherwise indicated. Synthetic methods
are described for representative compounds.
4.1.1. N1-(adamant-2-yl)-N2-(benzyloxy)oxalamide (6)
To a solution of adamant-2-ylamine hydrochloride (1.00 g, 5.53 mmol) and triethylamine
(1.47 g, 10.6 mmol) in dichloromethane (20 mL) was added ethyl (chlorocarbonyl)formate (0.73 g,
5.32 mmol) in dichloromethane (2 mL) at 0°C. After stirring overnight at room temperature, the
product was extracted with diethyl ether (50 mL), washed with an aqueous solution of 1 N HCl (30
mL) and water (50 mL X 2), and dried over MgSO4. The ether solution was evaporated to dryness
and the residue was used for the next reaction without purification. An aqueous solution of 1 N
NaOH (2 mL) was added dropwise to a solution of the above residue in ethanol (10 mL) and the
reaction mixture was stirred for 30 min at room temperature. After the reaction was acidified to pH 2
by adding an aqueous solution of 1 N HCl, the acid product was extracted with dichloromethane (50
mL X 2). The combined organic solution was washed with water (50 mL X 2), dried over MgSO4,
and evaporated to dryness. To the residue (0.19 g, 0.85 mmol) in dichloromethane (20 mL) was
added 4-dimethylaminopyridine (DMAP; 0.10 g, 0.85 mmol) and benzyloxyamine hydrochloride
(0.14 g, 0.85 mmol) at room temperature. After the reaction was stirred for 5 min, 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide (EDCI; 0.16 g, 0.85 mmol) was added to the reaction
mixture at room temperature. After stirring overnight, the product was extracted with diethyl ether
(50 mL). The organic layer was washed with an aqueous solution of 1 N HCl (30 mL X 2) and water
(50 mL X 2), dried over MgSO4, and evaporated. The residue was purified by using silica gel column
chromatography (hexane/ethyl acetate = 3:1) to afford compound 6 as a solid in 85% yield. 1H NMR
δ (CDCl3): 1.65-1.68 (3H, m), 1.84-1.88 (9H, m), 1.94 (2H, s), 3.99 (1H, s), 4.97 (2H, s), 7.37-7.43
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(5H, m), 7.70 (1H, s), 9.64 (1H, s). 13C NMR δ (CDCl3): 26.9, 31.6, 36.9, 37.3, 53.8, 78.7, 128.7,
129.1, 129.2, 134.3, 156.9, 157.7. Purity: > 90%. HRMS (ESI) m/z calcd for C19H24N2O3 [M + H]+
329.1865, found [M + H]+ 329.1862, Mp137ºC.
Compounds 3-5 and 7-15 were synthesized in the same procedure used for the preparation of
compound 6 by using a corresponding alkyl- or cycloalkyl-amine and a substituted oxyamine instead
of adamant-2-ylamine and benzyloxyamine hydrochloride.
N1-(Benzyloxy)-N2-(3-phenylpropyl)oxalamide (3)
1H NMR δ (CDCl3): 1.89 (2H, t, J = 7.3 Hz), 2.66 (2H, t, J = 7.3 Hz), 3.32 (2H, q, J = 7.3 Hz), 4.96
(2H, s), 7.16-7.26 (4H, m), 7.27-7.31 (3H, m), 7.37-7.40 (5H, m), 9.56 (1H, s). HRMS (ESI) m/z
calcd for C18H20N2O3 [M + H]+ 313.1552, found [M + H]+ 313.1543, Mp 123ºC.
N1-(Adamant-1-yl)-N2-(benzyloxy)oxalamide (4)
1H NMR δ (CDCl3): 1.68 (6H, s), 2.00 (6H, s), 2.10 (3H, s), 4.86 (2H, s), 7.10 (1H, s), 7.37-7.42 (5H,
m), 9.58 (1H, s). HRMS (ESI) m/z calcd for C19H24N2O3 [M + H]+ 329.1865, found [M + H]+
329.1855, Mp 178ºC.
N1-(Adamant-1-ylmethyl)-N2-(benzyloxy)oxalamide (5)
1H NMR δ (CDCl3): 1.49 (6H, brs), 1.73 (6H, brs), 1.99 (3H, brs), 2.97 (2H, d, J = 6.8 Hz), 4.97 (2H,
s), 7.38-7.42 (5H, m), 9.60 (1H, s). HRMS (ESI) m/z calcd for C20H26N2O3 [M + H]+ 343.2022,
found [M + H]+ 343.2023, Mp 195ºC.
N1-(Adamant-2-yl)-N2-(phenyloxy)oxalamide (7)
1H NMR δ (CDCl3): 1.66-1.69 (3H, m), 1.87-1.90 (9H, m), 1.98 (2H, s), 4.04 (1H, s), 7.07-7.11 (3H,
m), 7.31-7.35 (2H, m), 7.70 (1H, s), 10.14 (1H, s). HRMS (ESI) m/z calcd for C18H22N2O3 [M + H]+
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315.1709, found [M + H]+ 315.1711, Mp 187ºC.
N1-(Adamant-2-yl)-N2-(4-methoxycarbonylbenzyloxy)oxalamide (8)
1H NMR δ (CDCl3): 1.63-1.68 (3H, m), 1.86-1.89 (9H, m), 1.94 (2H, s), 3 .93 (3H, s), 4.01 (1H, s),
5.02 (2H, s), 7.50 (2H, d, J = 8.3 Hz), 7.71 (1H, s), 8.06 (2H, d, J = 8.3 Hz), 9.70 (1H, s). HRMS
(ESI) m/z calcd for C21H26N2O5 [M + H]+ 387.1920, found [M + H]+ 387.1916, Mp 175ºC.
N1-(Adamant-2-yl)-N2-(4-nitrobenzyloxy)oxalamide (9)
1H NMR δ (CDCl3): 1.64-1.67 (3H, m), 1.86-1.88 (9H, m), 1.94 (2H, s), 4.00 (1H, s), 5.09 (2H, s),
7.52 (2H, d, J = 8.3 Hz), 7.76 (1H, s), 8.23 (2H, d, J = 8.3 Hz), 11.74 (1H, s). HRMS (ESI) m/z calcd
for C19H23N3O5 [M + H]+ 374.1716, found [M + H]+ 374.1714, Mp >210ºC.
N1-(Adamant-2-yl)-N2-(4-chlorobenzyloxy)oxalamide (10)
To a solution of 4-chlorobenzyl bromide (4.00 g, 0.01947 mol) and N-hydroxyphthalimide
(3.18 g, 0.01947 mol) in DMF (40 mL) was added triethylamine (3.94 g, 0.03894 mol) at 0°C. After
stirring overnight at room temperature, the product was extracted with diethyl ether (80 mL X 2).
The organic layer was washed with water (80 mL X 2), dried over MgSO4, and evaporated to dryness.
To the residue in 10% methanol in trichloromethane (30 mL) was added hydrazine hydrate (0.93 g,
0.02912 mol) at room temperature. The reaction was stirred overnight and the product was extracted
with diethyl ether (80 mL X 2). The combined organic layer was washed with water (80 mL X 2),
dried over MgSO4, and evaporated. The residue was purified by using silica gel column
chromatography (hexane/ethyl acetate = 5:1) to give (4-chlorobenzyl)oxyamine in 65% yield. Then,
compound 10 was prepared in the same manner used for the preparation of compound 6 by using this
oxyamine. 1H NMR δ (CDCl3): 1.65-1.68 (3H, m), 1.83-1.89 (9H, m), 1.94 (2H, s), 4.00 (1H, s),
4.93 (2H, s), 7.26 (2H, s), 7.36 (2H, s), 7.68 (1H, s), 9.60 (1H, s). HRMS (ESI) m/z calcd for
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C19H23ClN2O3 [M + H]+ 363.1475, found [M + H]+ 363.1472, Mp 173ºC.
N1-(Adamant-2-yl)-N2-(2-phenylethyloxy)oxalamide (11)
(2-Phenylethyl)oxyamine was prepared in the same procedure used for the preparation of
compound 10 by using 2-phenylethyl bromide instead of 4-chlorobenzyl bromide. Compound 11 was
then synthesized in the same manner described in the preparation of compound 6 by using (2-
phenylethyl)oxyamine. 1H NMR δ (CDCl3): 1.65-1.68 (3H, m), 1.83-1.89 (9H, m), 1.95 (2H, s), 3.02
(2H, t, J = 7.1 Hz), 4.01 (1H, s), 4.20 (2H, t, J = 7.1 Hz), 7.22-7.24 (3H, m), 7.26-7.33 (2H, m), 7.70
(1H, s), 9.71 (1H, s). Purity: > 90%. HRMS (ESI) m/z calcd for C20H26N2O3 [M + H]+ 343.2022,
found [M + H]+ 343.2015, Mp >210ºC.
N1-(Adamant-2-yl)-N2-(3-phenylpropyloxy)oxalamide (12)
(3-Phenylpropyl)oxyamine was prepared in the same procedure used for the preparation of
compound 10 by using 3-phenylpropyl bromide instead of 4-chlorobenzyl bromide. Compound 12
was then synthesized in the same manner described in the preparation of compound 6 by using (3-
phenylpropyl)oxyamine. 1H NMR δ (CDCl3): 1.66-1.69 (3H, m), 1.83-1.89 (9H, m), 1.98 (2H, s),
2.08 (2H, quint, J = 7.3 Hz), 2.74 (2H, t, J = 7.3 Hz), 4.06 (1H, s), 4.29 (2H, t, J = 7.3 Hz), 7.18-7.26
(3H, m), 7.28-7.30 (2H, m), 7.42 (1H, s), 9.70 (1H, s). HRMS (ESI) m/z calcd for C21H28N2O3 [M +
H]+ 357.2178, found [M + H]+ 357.2178, Mp 53ºC.
4.1.2. N1-(adamant-2-yl)-N2-(benzyloxy)-N2-methyloxalamide (13)
A mixture of compound 6 (0.51 g, 1.55 mmol), potassium carbonate (0.43 g, 3.10 mmol),
and iodomethane (0.33 g, 2.32 mmol) in DMF (20 mL) was stirred overnight at room temperature.
The product was extracted with diethyl ether (80 mL). The organic solution was washed with water
(80 mL X 2), dried over MgSO4, and evaporated. The residue was purified by using silica gel column
chromatography (hexane/ethyl acetate = 4:1) to afford compound 13 as a solid in 55% yield. 1H
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NMR δ (CDCl3): 1.61-1.66 (3H, m), 1.84-1.88 (9H, m), 1.92 (2H, s), 4.01 (1H, s), 4.10 (3H, s), 5.06
(2H, s), 6.93 (1H, s), 7.35-7.38 (5H, m). HRMS (ESI) m/z calcd for C20H26N2O3 [M + H]+ 343.2022,
found [M + H]+ 343.2016, Mp 110ºC.
N1-(Adamant-2-yl)-N2-methyl-N2-(methyloxy)oxalamide (14)
1H NMR δ (CDCl3): 1.64-1.67 (3H, m), 1.86-1.88 (9H, m), 1.96 (2H, s), 3.76 (3H, s), 3.79 (3H, s),
4.00 (1H, s), 6.81 (1H, s). Purity: > 90%. HRMS (ESI) m/z calcd for C14H22N2O3 [M + H]+ 267.1709,
found [M + H]+ 267.1707, Mp 148ºC.
N1-(Adamant-2-yl)-N2-(tetrahydro-2H-pyran-2-yloxy)oxalamide (15)
1H NMR δ (CDCl3): 1.62-1.70 (8H, m), 1.81-1.88 (10H, m), 1.95 (2H, s), 3.67 (1H, s), 3.99-4.04 (2H,
m), 5.04 (1H, s), 7.72 (1H, s), 9.96 (1H, s). HRMS (ESI) m/z calcd for C17H26N2O4 [M + H]+
323.1971, found [M + H]+ 323.1973, Mp 137ºC.
4.1.3. N-(benzyloxy)-2-(adamant-2-ylamino)acetamide (16)
A mixture of methyl 2-bromoacetate (1.22 g, 7.99 mmol), adamant-2-ylamine hydrochloride
(1.50 g, 7.99 mmol), and potassium carbonate (2.21 g, 15.9 mmol) in DMF (30 mL) was stirred
overnight at room temperature. The product was extracted with diethyl ether (80 mL X 2). The
organic solution was washed with water (100 mL X 2), dried over MgSO4, and evaporated to dryness.
To the residue in methanol (15 mL) was added an aqueous solution of 1 N NaOH (4 mL) at room
temperature, and the reaction was stirred for 1 hr. After evaporating the reaction mixture to dryness,
the residue was used for the next reaction without further purification. A mixture of the acid residue
(0.79 g, 3.75 mmol), DMAP (0.46 g, 3.75 mmol), and benzyloxyamine hydrochloride (0.60 g, 3.75
mmol) in dichloromethane (30 mL) was stirred for 5 min at room temperature. To this reaction
mixture was added EDCI (0.72 g, 3.75 mmol) at room temperature. After stirring overnight, the
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product was extracted with diethyl ether (80 mL X 2). The combined organic solution was washed
with an aqueous solution of 1 N HCl (40 mL) and water (80 mL X 2), dried over MgSO4, and
evaporated. The residue was purified by using silica gel column chromatography (hexane/ethyl
acetate = 1:1) to give compound 16 as a yellowish oil in 70% yield. 1H NMR δ (CDCl3): 1.44-1.47
(2H, m), 1.61-1.72 (10H, m), 1.83-1.89 (4H, m), 2.53 (1H, s), 3.33 (2H, s), 4.95 (2H, s), 7.35-7.42
(5H, m). Purity: > 90%. HRMS (ESI) m/z calcd for C19H26N2O2 [M + H]+ 315.2072, found [M + H]+
315.2071.
N-(4-(N2-methyloxyoxalamido)benzyl)adamantanecarboxamide (18)
1H NMR δ (CDCl3): 1.68-1.71 (6H, m), 1.88 (6H, s), 2.05 (3H, s), 3.88 (3H, s), 4.42 (2H, s), 5.88
(1H, s), 7.27 (2H, d, J = 10 Hz), 7.57 (2H, d, J = 10 Hz), 9.09 (1H, s), 9.82 (1H, s). HRMS (ESI) m/z
calcd for C21H27N3O4 [M + H]+ 386.2080, found [M + H]+ 386.2076, Mp >210ºC.
N-(4-(N2-t-Butyloxyoxalamido)benzyl)adamantanecarboxamide (19)
1H NMR δ (CDCl3): 1.35 (9H, s), 1.68-1.72 (6H, m), 1.88 (6H, s), 2.04 (3H, s), 4.43 (2H, s), 5.88
(1H, s), 7.29 (2H, d, J = 10 Hz), 7.57 (2H, d, J = 10 Hz), 9.16 (1H, s), 9.42 (1H, s). HRMS (ESI) m/z
calcd for C24H33N3O4 [M + H]+ 428.2549, found [M + H]+ 428.2546, Mp >210ºC.
N-(4-(N2-methyl- N2-(methyloxy)oxalamido)benzyl)adamantanecarboxamide (20)
1H NMR δ (CDCl3): 1.66-1.76 (6H, m), 1.88 (6H, s), 2.04 (6H, s), 3.29 (3H, s), 3.83 (3H, s), 4.41
(2H, s), 7.25 (2H, d, J = 8.3 Hz), 7.55 (2H, d, J = 8.3 Hz), 9.34 (1H, s). HRMS (ESI) m/z calcd for
C22H29N3O4 [M + H]+ 400.2236, found [M + H]+ 400.2239, Mp 181ºC.
N-(4-(N1-methyl-N2-methyl- N2-(methyloxy)oxalamido)benzyl)adamantanecarboxamide (21)
1H NMR δ (CDCl3): 1.68-1.72 (6H, m), 1.88 (6H, s), 2.04 (6H, s), 2.96 (3H, s), 3.31 (3H, s), 3.79
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(3H, s), 4.43 (2H, s), 5.92 (1H, s), 7.24-7.26 (4H, m). HRMS (ESI) m/z calcd for C23H31N3O4 [M +
H]+ 414.2393, found [M + H]+ 414.2386, Mp 170ºC.
N-(4-(N2-(Tetrahydro-2H-pyran-2-yloxy)oxalamido)benzyl)adamantanecarboxamide (22)
1H NMR δ (CDCl3): 1.61-1.65 (6H, m), 1.68-1.72 (6H, m), 1.88 (6H, s), 2.04 (6H, s), 3.66-3.70 (2H,
m), 4.02 (1H, t, J = 6.8 Hz), 4.42 (2H, s), 5.90 (1H, s), 7.25 (2H, d, J = 8.3 Hz), 7.57 (2H, d, J = 8.3
Hz), 9.15 (1H, s), 9.98 (1H, s). Purity: > 90%. HRMS (ESI) m/z calcd for C25H33N3O5 [M + H]+
456.2498, found [M + H]+ 456.2495, Mp 195ºC.
N1-(4-((2-Adamantylacetamido)methyl)phenyl)-N2-methyl-N2-methyloxyoxalamide (23)
1H NMR δ (CDCl3): 1.62-1.71 (12H, m), 1.96-1.97 (5H, s), 3.29 (3H, s), 3.83 (3H, s), 4.42 (2H, s),
5.58 (1H, s), 7.27 (2H, d, J = 8.3 Hz), 7.56 (2H, d, J = 8.3 Hz), 9.33 (1H, s). HRMS (ESI) m/z calcd
for C23H31N3O4 [M + H]+ 414.2393, found [M + H]+ 414.2390, Mp 203ºC.
N1-(4-((2-Adamantylacetamido)methyl)phenyl)-N1-methyl-N2-methyl-N2-methyloxyoxalamide (24)
1H NMR δ (CDCl3): 1.63-1.72 (12H, m), 1.96-1.97 (5H, s), 2.95 (3H, s), 3.33 (3H, s), 3.80 (3H, s),
4.43 (2H, s), 5.65 (1H, s), 7.23-7.30 (4H, m). 13C NMR δ (CDCl3): 28.6, 31.2, 32.8, 35.7, 36.7, 42.6,
42.9, 51.7, 62.2, 126.3, 128.8, 138.6, 140.7, 164.3, 165.6, 170.9. HRMS (ESI) m/z calcd for
C24H33N3O4 [M + H]+ 428.2549, found [M + H]+ 428.2545, Mp 97ºC.
4.1.4. N1-(4-(2-adamantylacetamido)phenyl)-N2-methyl-N2-(methyloxy)oxalamide (25)
After a mixture of adamant-1-ylacetic acid (1.87 g, 9.60 mmol), DMAP (1.17 g, 9.60 mmol),
and 4-(tert-butyloxycarbonyl)aminoaniline (2.00 g, 9.60 mmol) in dichloromethane (30 mL) was
stirred for 10 min at room temperature, EDCI (1.84 g, 9.60 mmol) was added portionwise to the
reaction mixture. The reaction was stirred overnight and the product was then extracted with ethyl
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acetate (80 mL X 2). The organic solution was washed with an aqueous solution of 1 N HCl (50 mL)
and water (80 mL X 2), dried over MgSO4, and evaporated. To the residue washed with diethyl ether
(20 mL) in dichloromethane (30 mL) was added an aqueous solution of 4 N HCl (10 mL) at room
temperature. After stirring overnight, the product was extracted with ethyl acetate (80 mL X 2). The
organic layer was washed with water (80 mL X 2), dried over MgSO4, and evaporated to dryness.
The residue was used for the next reaction without further purification. To a solution of the residue
(1.42 g, 5.27 mmol) and triethylamine (0.80 g, 7.90 mmol) in dichloromethane (30 mL) was added
ethyl (chlorocarbonyl)formate (0.72 g, 5.27 mmol) at 0°C. After stirring overnight at room
temperature, the product was extracted with ethyl acetate (80 mL X 2). The organic solution was
washed with an aqueous solution of 1 N HCl (40 mL) and water (80 mL X 2), dried over MgSO4,
and evaporated to dryness. An aqueous solution of 1 N NaOH (5 mL) was added to a solution of the
residue in ethanol (10 mL) at room temperature. After the reaction was stirred overnight at room
temperature and acidified to pH 2 by adding an aqueous solution of 1 N HCl, the product was
extracted with ethyl acetate (80 mL X 2). The organic solution was washed with water (80 mL X 2),
dried over MgSO4, and evaporated to dryness. Continuously, to a solution of the residue (1.22 g, 3.42
mmol), DMAP (0.42 g, 5.13 mmol), methoxymethylamine hydrochloride (0.50 g, 5.13 mmol), and
triethylamine (0.52 g, 5.13 mmol), which was stirred for 10 min, was added EDCI (0.98 g, 5.13
mmol) at room temperature. After stirring overnight, the product was extracted with ethyl acetate (80
mL X 2). The organic solution was washed with water (80 mL X 2), dried over MgSO4, and
evaporated. The residue was purified by using silica gel column chromatography (hexane/ethyl
acetate = 1:1) to afford compound 25 in 45% yield. 1H NMR δ (CDCl3): 1.63-1.69 (12H, m), 2.04
(3H, s), 2.09 (2H, s), 3.28 (3H, s), 3.82 (3H, s), 7.06 (1H, s), 7.51-7.57 (4H, m), 9.33 (1H, s). HRMS
(ESI) m/z calcd for C22H29N3O4 [M + H]+ 400.2236, found [M + H]+ 400.2239, Mp 208ºC.
Compounds 18-23, 28-30 and 32 were prepared in the same manner used for the synthesis of
compound 25 by using 4-aminobenzylamine, adamant-1-ylcarboxylic acid or adamant-1-yl
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isocyanate, and a corresponding substituted oxyamine instead of 4-(tert-butyloxycarbonyl)-
aminoaniline and methoxymethylamine hydrochloride.
4.1.5. N1-(4-(2-adamantylacetamido)phenyl)-N1-methyl-N2-methyl-N2-(methyloxy)oxalamide
(26)
A mixture of compound 25 (0.30 g, 0.75 mmol), potassium carbonate (0.16 g, 1.12 mmol),
and iodomethane (0.11 g, 1.12 mmol) in DMF (15 mL) was stirred overnight at room temperature.
The product was extracted with diethyl ether (80 mL). The organic solution was washed with water
(80 mL X 2), dried over MgSO4, and evaporated. The residue was purified by using silica gel column
chromatography (hexane/ethyl acetate = 1:1) to give compound 26 in 60% yield. 1H NMR δ (CDCl3):
1.64-1.69 (12H, m), 1.99 (3H, s), 2.09 (2H, s), 2.98 (3H, s), 3.30 (3H, s), 3.80 (3H, s), 7.18 (2H, d, J
= 8.3 Hz), 7.48 (2H, d, J = 8.3 Hz). 13C NMR δ (CDCl3): 28.6, 31.3, 33.3, 35.7, 36.6, 42.5, 52.7, 62.2,
120.2, 126.8, 136.9, 137.9, 164.2, 165.7, 169.6. Purity: > 90%. HRMS (ESI) m/z calcd for
C23H31N3O4 [M + H]+ 414.2393, found [M + H]+ 414.2391, Mp 179ºC.
Compounds 21, 24, and 31 were synthesized in the same manner used for the preparation of
compound 26 by using compound 20, 23, or 30, respectively, instead of compound 25.
1-(4-(N2-Methyloxyoxalamido)benzyl)-3-adamantylurea (28)
Compound 28 was prepared in the same methods described in the syntheses of compounds 25
and 30. 1H NMR δ (CDCl3): 1.65 (6H, s), 1.95 (6H, s), 2.06 (3H, s), 3.83 (3H, s), 4.09 (1H, s), 4.30
(2H, s), 4.44 (1H, s), 7.25 (2H, d, J = 8.3 Hz), 7.53 (2H, d, J = 8.3 Hz), 9.12 (1H, s), 9.35 (1H, s).
HRMS (ESI) m/z calcd for C21H28N4O4 [M + H]+ 401.2189, found [M + H]+ 401.2187, Mp 177ºC.
1-(4-(N2-t-Butyloxyoxalamido)benzyl)-3-adamantylurea (29)
Compound 29 was prepared in the same methods described in the syntheses of compounds 25
22

542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
and 30. 1H NMR δ (CDCl3): 1.56 (9H, s), 1.66 (6H, s), 1.95 (6H, s), 2.05 (3H, s), 4.08 (1H, s), 4.30
(2H, s), 4.43 (1H, s), 7.30 (2H, d, J = 8.3 Hz), 7.57 (2H, d, J = 8.3 Hz), 9.16 (1H, s), 9.45 (1H, s).
HRMS (ESI) m/z calcd for C24H34N4O4 [M + H]+ 443.2658, found [M + H]+ 443.2654, Mp >210ºC.
1-(4-(N2-Methyl-N2-(methyloxy)oxalamido)benzyl)-3-adamantylurea (30)
To a solution of adamant-1-yl isocyanate (0.73 g, 4.09 mmol) in DMF (15 mL) was added
dropwise 4-aminobenzylamine (0.50 g, 4.09 mmol) in DMF (2 mL) at 0°C. After stirring overnight
at room temperature, the product was extracted with diethyl ether (80 mL X 2). The organic layer
was washed with water (80 mL X 2), dried over MgSO4, and evaporated to give 1-(adamant-1-yl)-3-
(4-aminobenzyl)urea in 100% yield. Compound 30 was synthesized in the same method described in
the preparation of compound 25 by using 1-(adamant-1-yl)-3-(4-aminobenzyl)urea instead of the
amide-amine intermediate. 1H NMR δ (CDCl3): 1.66 (6H, s), 1.95 (6H, s), 2.04 (3H, s), 3.28 (3H, s),
3.82 (3H, s), 4.09 (1H, s), 4.30 (2H, s), 4.40 (1H, s), 7.30 (2H, d, J = 8.3 Hz), 7.55 (2H, d, J = 8.3
Hz), 9.33 (1H, s). HRMS (ESI) m/z calcd for C22H30N4O4 [M + H]+ 415.2345, found [M + H]+
415.2347, Mp 202ºC.
1-(4-(N1-Methyl-N2-Methyl-N2-(methyloxy)oxalamido)benzyl)-3-adamantylurea (31)
Compound 31 was prepared in the same methods described in the syntheses of compounds
25 and 30. 1H NMR δ (CDCl3): 1.66 (6H, s), 1.95 (6H, s), 2.04 (3H, s), 2.95 (3H, s), 3.30 (3H, s),
3.80 (3H, s), 4.08 (1H, s), 4.31 (2H, s), 4.44 (1H, s), 7.22-7.29 (4H, m). 13C NMR δ (CDCl3): 29.5,
31.2, 35.7, 36.4, 42.4, 43.5, 51.0, 62.2, 126.1, 128.3, 139.9, 140.4, 157.0, 164.3, 165.7. HRMS (ESI)
m/z calcd for C23H32N4O4 [M + H]+ 429.2502, found [M + H]+ 429.2505, Mp >210ºC.
1-(4-(N2-(Tetrahydro-2H-pyran-2-yloxy)oxalamido)benzyl)-3-adamantylurea (32)
Compound 32 was prepared in the same methods described in the syntheses of compounds 25
23

567
568
569
570
571
572
573
574
575
576
577
578
579
and 30. 1H NMR δ (CDCl3): 1.61-1.64 (12H, m), 1.93 (6H, s), 2.04 (3H, s), 3.69 (1H, s), 4.09-4.14
(2H, m), 4.25 (2H, s), 4.70 (1H, s),5.19 (1H, s), 7.17 (2H, d, J = 8.3 Hz), 7.49 (2H, d, J = 8.3 Hz),
9.36 (1H, s), 10.56 (1H, s). HRMS (ESI) m/z calcd for C25H34N4O5 [M + H]+ 471.2607, found [M +
H]+ 471.2608, Mp 202ºC.
4.2. Biology
4.2.1. Enzyme preparation
Recombinant human sEH was prepared by using baculovirus expression system as
previously reported.25 Briefly, Sf9 insect cells were infected by recombinant baculovirus harboring
human sEH gene fused with a 6xHis tag. At 72 hr post-infection, the infected cells were
homogenized and the recombinant protein was purified by immobilized metal affinity
chromatography. After removing the 6xHis tag using the tobacco etch virus protease, human sEH
was further purified by anion-exchange chromatography.
580
581
582
583
584
585
586
587
588
589
590
591
4.2.2. IC50 assay conditions
Standard solutions of compounds in Tables 1-4 were prepared in DMSO. Fluorescent assays
were performed by using a substrate (cyano-(2-methoxynaphthalen-6-yl)-methyl trans-(3-phenyl-
oxyran-2-yl)-methyl carbonate; CMNPC; [S] = 5 µM) to determine IC50 values of the derivatives.26
Inhibition activity against human sEH (1 nM) was determined by measuring the appearance of the 6-
methoxy-2-naphthaldehyde with an excitation wavelength of 330 nm and an emission wavelength of
465 nm for 10 minutes on a fluorometer (Victor3; PerkinElmer).25,26 The IC50 values were gained by
regression of at least six datum points with a minimum of three points in a linear region of the curve.
IC50 results are averages of three separate measurements. 12-(3-Adamantan-1-yl-ureido)dodecanoic
acid (AUDA)13 in Table 2 was used as a positive control for the inhibition assay in the present study.
24

592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
4.3. Solubility
Water solubility of amide and urea derivatives in Tables 3 and 4 was determined
experimentally by light scattering method in sodium phosphate buffer at 25±1.5ºC. In brief, aqueous
solubility was determined by adding varying concentrations of a test compound prepared in DMSO
to 0.1 M sodium phosphate buffer (pH 7.4) in a final ratio of 5:95 (v/v). Insolubility of the compound
was shown 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±1.5ºC.
Results are averages of three separate measurements.
Acknowledgments
This work was supported by Hyundai Pharm Research Grant (HOB-024). Partial support was
from NIEHS Grant R01 ES002710 and a Grant-in-aid for Young Scientists (B) 23710042 from Japan
Society for the Promotion of Science.
Supplementary data
1H-NMR of compounds 6, 26, and 31, LC-MS analyses, preparation of human sEH, and
inhibition assays.
References
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(2)
(3)
(4)
Robinson, D. R. Am. J. Med. 1983, 75, 26-31.
Miller, S. B. Semin. Arthritis Rheum. 2006, 36, 37-49.
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(5)
Imig, J. D.; Zhao, X.; Capdevila, J. H.; Morisseau, C.; Hammock, B. D. Hypertension 2002,
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617
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619
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621
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623
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626
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628
629
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631
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636
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641
(6)
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(8)
(9)
Zhao, X.; Yamamoto, T.; Newman, J. W.; Kim, I.-H.; Watanabe, T.; Hammock, B. D.; Stewart,
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I.-H.; Watanabe, T.; Hammock, B. D. Hypertension 2006, 46, 975-981.
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P.; Singapuri, A.; Davis, B. B.; Low, R.; Hammock, B. D.; Chiamvimonvat, N. Proc. Natl.
Acad. Sci. USA 2006, 103, 18733-18738.
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(13) Kim, I. -H.; Nishi, K.; Tsai, H.-J.; Bradford, T.; Koda, Y.; Watanabe, T.; Morisseau, C.;
Blanchfield, J.; Toth, I.; Hammock, B. D. Bioorg. Med. Chem. 2007, 15, 312-323.
(14) Kim, I. -H.; Tsai, H.-J.; Nishi, K.; Kasagami, T.; Morisseau, C.; Hammock, B. D. J. Med.
Chem. 2007, 50, 5217-5226.
(15) Kasagami, T.; Kim, I.-H.; Tsai, H.-J.; Nishi, K.; Hammock, B. D.; Morisseau, C. Bioorg.
Med. Chem. Lett. 2009, 19, 1784-1789.
(16) Jones, P. D.; Tsai, H.-J.; Do, Z; Moresseau, C.; Hammock, B. C. Bioorg. Med. Chem. Lett.
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26

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651
652
653
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655
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656
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658
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Jersey) 2007, 4.23.1-4.23.18.
27

A.
O
H
N
O
O
(a), (b)
(c)
R
OH
R
O
O
N
Cl
N
H
H
O
O
O
O
4:
5:
R= 3:
B.
O
(a)
(b)
O
OH
Cl
N
H
6: R1 =
O
O
R2 = H
( )n
R1 =
R2 = H
Br
O
(d)
( )n
( )n
NH2
11: n = 1; 12: n = 2
R =
7:
(c)
1
R2 = H
R3
R3
(for 10-12)
CH
CH3
14: R1 =
3
R1 =
R =
2
R2 = H
O
R2
N
R3
8:
9: R =
10:
R3 = COOCH3
R1
15: R1 =
O
NO2
R = Cl
O
N
3
H
R2 = H
3
O
C.
O
CH3
N
O
H
N
(e)
O
O
N
N
H
H
O
O 6
13
D.
H
OCH3
N
(a)
(b)
(c)
OH
Br
N
O
N
H
H
O
O 16
O
659
660
Scheme 1. Syntheses of substituted oxyoxalamides (3-15) and N-(benzyloxy)-2-(adamant-2-
661
662
663
664
ylamino)acetamide (16). (a) an alkyl- or a cycloalkyl-amine, Et3N, CH2Cl2, room temp; (b) 1 N
NaOH, EtOH or MeOH, room temp; (c) benzyloxyamine (in parts A and D) or a substituted
oxyamine (in part B), EDCI, DMAP, CH2Cl2, room temp; (d) i) N-hydroxyphthalimide, Et3N, DMF,
room temp, ii) H2NNH2, 10% MeOH in CHCl3, room temp; (e) CH3I, K2CO3, DMF, room temp.
28

O
A.
R
N
O
R2
N
H2N
H
R3
N
O
NH2
H
O
20: R =
19: R =
18: R =
(a)
(b), (c), (d)
CH
R2 =
R =
3
R =
H
H
2
2
CH
R3 =
R3 =
R =
CH3
3
O
3
23:
R =
22:
R =
R
N
H
NH2
CH
R2 =
R3 =
3
R =
H
2
CH
3
R3 =
O
B.
H2N
H
H
N
N
(b), (c), (d)
(a), (e)
O
CH3
N
O
O
Boc
N
OCH3
NH2
N
H
H
25
O
O
C.
20
23
25
N ( )n
(f)
R
O
CH3
N
24:
21: R =
26: R =
R =
H
CH3
N
O
CH3
O
n = 0
n = 1
n = 1
665
666
667
668
669
670
671
672
Scheme 2. Syntheses of amide derivatives with substituted oxyoxalamide. (a) adamant-1-
ylcarboxylic acid (for compounds 18-20 and 22) or adamant-1-ylmethylcarboxylic acid (for
compounds 23 and 25), EDCI, DMAP, CH2Cl2, room temp; (b) ethyl (chlorocarbonyl)formate, Et3N,
CH2Cl2, room temp; (c) 1 N NaOH, EtOH, room temp; (d) a corresponding oxyamine (in parts A and
B), EDCI, DMAP CH2Cl2, room temp; (e) 4 N HCl, CH2Cl2, room temp;(f) CH3I, K2CO3,DMF,
room temp.
29