Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmc.2015.10.016

Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore.

Full text of DOI:10.1016/j.bmc.2015.10.016

Bioorganic & Medicinal Chemistry 23 (2015) 7199–7210
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of amide–phosphonate derivatives
as inhibitors of the human soluble epoxide hydrolase
In-Hae Kim a, Yong-Kyu Park b, Hisashi Nishiwaki a, Bruce D. Hammock c, Kosuke Nishi a,
⇑
a Department of Applied Bioscience, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan
b Department of Medicinal Chemistry, Hyundai Pharm Co., Ltd, Suwon, Gyonggi 443-270, Republic of Korea
c Department of Entomology and Nematology, and UCD Comprehensive Cancer Center, University of California, One Shields Ave, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure–activity relationships of amide–phosphonate derivatives as inhibitors of the human soluble
epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on
the carbon alpha to the phosphonate function in amide compounds to see whether substituted phospho-
nates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to
yield most potent inhibition on the target enzyme. A 4–50-fold drop in inhibition was induced by other
substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the
O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable
for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized
diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene
(31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent
amide–phosphonate compounds had reasonable water solubility, suggesting that substituted phospho-
nates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility
as a secondary pharmacophore.
Received 6 August 2015
Revised 1 October 2015
Accepted 12 October 2015
Available online 22 October 2015
Keywords:
Soluble epoxide hydrolase
Amide–phosphonate derivatives
Inhibitors
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
epoxy functionality of EETs to the vicinal diols by soluble epoxide
hydrolase (sEH) results in a huge reduction in the biological
It is well known that cyclooxygenase and lipoxygenase utilize
arachidonic acid as an endogenous substrate to form oxidized
and largely inflammatory metabolites such as prostanoids and leu-
kotrienes, which increase symptoms such as inflammation, pain,
and asthma.1–3 Inhibitors of these pathways and the enzymes
involved are thus current pharmacological targets for the treat-
ment of multiple medical conditions. On the other hand, other
metabolites of arachidonic acid derived by the action of cyto-
chrome P450 oxidases, known as epoxyeicosatrienoic acids (EETs),
are largely analgesic and anti-inflammatory. Stabilization of EETs
has been reported to have various valuable effects on human
health such as blood pressure regulation.4–8 In addition, several
reports indicate that vascular inflammation and pain are modu-
lated by the action of EETs.9,10 However, the hydrolysis of the
effects.6 Inhibition of human sEH has been demonstrated to be
effective to restore the biological actions of EETs in in vitro and
in vivo experimental models.5–10 This suggests that inhibitors of
the human enzyme are a useful tool for the treatment of EET
related disorders.
A number of substituted urea or amide compounds have been
reported as highly potent inhibitors of the human sEH.11–23 Most
promising inhibition among these compounds is observed in a
range of less than 5 nM.13,14 Many of urea or amide inhibitors con-
tain relatively hydrophobic substituents to retain their inhibition
potency on the target enzyme. This often causes poor physical
properties of the inhibitors such as high melting point and low
water solubility. Compounds with poor physical properties tend
to be poorly bioavailable and are difficult to formulate. On the
other hand, when a variety of functionalities including amides,
esters (e.g., 1 in Fig. 1), ketones, and ethers are incorporated onto
optimal positions of the hydrophobic substituents of urea or
amide inhibitors as a secondary pharmacophore, a significant
improvement in physical properties is observed without dramatic
changes in inhibition potency.11,22,23 This implies that secondary
Abbreviations: EETs, epoxyeicosatrienoic acids; sEH, soluble epoxide hydrolase;
EDCI, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; DMAP,
4-dimethylaminopyridine; DMF, N,N-dimethylformamide; CMNPC, cyano-(2-
methoxynaphthalen-6-yl)-methyl trans-(3-phenyl-oxyran-2-yl)-methyl carbonate.
⇑
Corresponding author. Tel./fax: +81 89 946 9818.
E-mail address: knishi@agr.ehime-u.ac.jp (K. Nishi).
http://dx.doi.org/10.1016/j.bmc.2015.10.016
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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Table 1
Primary pharmacophore
Inhibition of human sEH by adamantaneamide–phosphonate derivatives
O
O
O
Linker
Linker
O
P
OEt
OEt
R
N
H
O
N
H
N
H
N
O
N
H
O
N
H
2
1
O
O
a
No.
R
IC50 (nM)
Secondary pharmacophore
3
45
Figure 1. Amide compounds with an adamantyl group and a substituted benzyl
group. The amide function is primary pharmacophore. An ester (1) or an
a
oxyoxalamide (2) function attached to a benzyl group (a linker) is a secondary
pharmacophore. The IC50 on the human sEH of compounds 1 and 2 is 9.1 nM and
190 nM, respectively.22,23
4
5
130
192
199
pharmacophores are useful for improving physical properties
without reducing inhibition potency and that primary inhibition
of the human sEH is more directly related to the structure of pri-
mary pharmacophores (e.g., amides; 1 and 2 in Fig. 1). Our recent
report indicates that oxyoxalamide (2 in Fig. 1) is one of several
functions to be incorporated as a secondary pharmacophore to pro-
duce potent inhibitors with improved physical properties.23 In
addition, unexpectedly, the structure–activity relationship results
showed that the substituted oxyoxalamides are a promising func-
tion as a primary pharmacophore,23 suggesting that investigating a
wide range of structures are worthwhile to find out new pharma-
cophors for sEH inhibitors. In the present study, we reported struc-
6
7
8
O
O
230
120
O
F
9
108
185
ture–activity relationship results of
a series of substituted
CN
phosphonates as a secondary pharmacophore in amide inhibitors.
Highly potent amide–phosphonate compounds were found, and
the resulting compounds possessed reasonable water solubility.
10
N
2. Results and discussion
2.1. Chemistry
11
69
O
12
13
14
186
21
A series of adamantaneamide–dialkyl phosphonates 3–22 in
Table 1 were synthesized as depicted in Scheme 1. 1-Adaman-
tanecarboxylic acid was coupled with 4-aminobenzylamine by
using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (EDCI) in the presence of 4-dimethylaminopyridine
(DMAP) in dichloromethane to give an amide–aniline in 95% yield.
Kabachnik-fields reaction was then performed to introduce a phos-
phonate containing group to the amine function. First, an imine
intermediate was synthesized by adding a corresponding aldehyde
(3–15 and 17) or pivalaldehyde (16 and 18–22) to a suspension of
the amide–aniline and magnesium sulfate in dichloromethane.
Heating of the imine with diethyl phosphite (3–17) or a correspond-
ing substituted phosphite (18–22) provided amide–phosphonate
derivatives in approximately 80% yield. In order to synthesize urea
compounds 23–28 in Table 2, 4-aminobenzylamine was first
reacted with 1-adamantyl isocyanate in N,N-dimethylformamide
(DMF) to give a urea–aniline in 98% (Scheme 2). Then, the same
reactions as those described by Scheme 1 were conducted by using
the urea–aniline intermediate, aldehydes, and substituted phos-
phites to afford urea derivatives 23–28 in a range of 55–80% overall
yield. A series of modified amide–phosphonate derivatives 29–34,
36, and 37 in Table 3 were also synthesized in the same reactions
as those outlined in Scheme 1 by using the corresponding reactants
for each step (Scheme 3). For example, cyclohexanecarboxylic acid,
4-aminomethylaniline, pivalaldehyde, and diethyl phosphite
were used for the synthesis of compound 29. Syntheses of diethyl
1-(4-(2-(1-adamantylaminocarbonyl)ethyl)phenylamino)-2,2-
dimethylpropylphosphonate (35) and diethyl 1-(4-((1-adaman-
46
15
16
58
5.1
17
41
a
Test compounds dissolved in DMSO were incubated with human sEH for 10 min
in 25 mM Bis–Tris/HCl buffer (202 L; pH 7.0) at 30 °C. The fluorescent substrate
(CMNPC; [S] = 5 M) was then introduced to the incubation mixture. Inhibition
potency against the human sEH was determined by measuring the concentration of
the fluorophore, 6-methoxy-2-naphthaldehyde, with an excitation wavelength of
330 nm and an emission wavelength of 465 nm for 10 min on a microplate reader.
Results are averages of three separate measurements. See the Supplementary data
for the detailed procedures.
l
l
tanecarbonyloxy)methyl)phenylamino)-2,2-dimethylpropylphos-
phonate (38) were done as outlined in Scheme 4. The reaction of
3-(4-aminophenyl)propanoic acid with pivalaldehyde in the pres-
ence of magnesium sulfate gave an acid–imine intermediate, which
was alkylated by using iodomethane in the presence of potassium
carbonate as a base to afford an ester–imine. Heating of the ester–
imine with diethyl phosphite gave an ester–phosphonate interme-
diate in 42% yield. The ester function of the ester–phosphonate
was hydrolyzed by adding an aqueous solution of 1 N NaOH in

I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
7201
5:
4:
6:
7:
3:
8:
R =
O
O
OH
O
O
9:
10:
11:
16:
12:
N
F
CN
O
a,
b
13:
14:
15:
17:
O
O
O
P
OEt
c
N
H
N
H
OEt
R
N
H
N
R
d
O
O
P
R1
=
F
19:
20:
OR1
OR1
18:
21:
F
F
N
H
N
H
22:
Scheme 1. Syntheses of 1-adamantaneamide-substituted phosphonate derivatives. Reagents and conditions: (a) 4-aminobenzylamine, EDCI, DMAP, dichloromethane, rt; (b)
pivalaldehyde or a corresponding aldehyde, magnesium sulfate, dichloromethane, rt; (c) diethyl phosphite, 170 °C; (d) di-(2,2,2-trifluoroethyl) phosphite (18), diisopropyl
phosphite (19), dibutyl phosphite (20), diisobutyl phosphite (21), or dibenzyl phosphite (22), 170 °C.
Table 2
Inhibition of human sEH by amide- or urea-substituted phosphonate derivatives
O
O
O
P
O
P
OR
OR
OR
OR
N
H
N
H
N
H
R
N
H
N
H
a
a
No.
IC50 (nM)
No.
IC50 (nM)
16
5.1
23
0.96
F
F
F
18
30
24
3.8
19
20
21
6.7
26
16
25
26
27
1.7
3.0
1.8
22
42
28
2.6
a
Test compounds dissolved in DMSO were incubated with human sEH for 10 min in 25 mM Bis–Tris/HCl buffer (202
lL; pH 7.0) at 30 °C. The fluorescent substrate
(CMNPC; [S] = 5 M) was then introduced to the incubation mixture. Inhibition potency against the human sEH was determined by measuring the concentration of the
l
fluorophore, 6-methoxy-2-naphthaldehyde, with an excitation wavelength of 330 nm and an emission wavelength of 465 nm for 10 min on a microplate reader. Results are
averages of three separate measurements. See the Supplementary data for the detailed procedures.
F
25:
NCO
26:
23:
27:
24:
R =
F
F
a,
b
28:
O
O
O
P
c
OR
OR
N
H
N
H
N
H
N
H
N
N
H
Scheme 2. Syntheses of 1-adamantaneurea-substituted phosphonate derivatives. Reagents and conditions: (a) 4-aminobenzylamine, DMF, rt; (b) pivalaldehyde, magnesium
sulfate, dichloromethane, rt; (c) diethyl phosphite (23), di-(2,2,2-trifluoroethyl) phosphite (24), diisopropyl phosphite (25), dibutyl phosphite (26), diisobutyl phosphite (27),
or dibenzyl phosphite (28), 170 °C.

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I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
Table 3
imine. Coupling of the imine with 1-adamantanecarboxylic acid
by using EDCI/DMAP made an ester–imine. In order to incorporate
diethyl phosphorous onto the imine carbon, the imine intermediate
was heated with diethyl phosphite to produce compound 38 in
approximately 63%.
Inhibition of human sEH by amide–diethylphosphonate derivatives
O
OEt
P
OEt
R
N
H
2.2. Structure–activity relationships
a
No.
R
IC50 (nM)
In order to first investigate whether the substituted phospho-
nate functions can be a secondary pharmacophore, a series of phos-
phonate derivatives were synthesized. Our previous reports
suggest that substituted amides are highly effective as a primary
pharmacophore among other functions such as ureas and carba-
mates to provide not only potent inhibition on the human sEH,
but also favorable physical properties such as lower melting points
and better water solubility, which may lead to bioavailable and
potent inhibitors.21,22 The diethylphosphonate function was first
incorporated in an amide inhibitor substituted with an adamantyl
group and a benzyl group as a linker (Table 1). The carbon alpha to
the diethylphosphonate was then substituted with aryls,
substituted aryls, cycloalkyls, and alkyls, and the effects of these
substituents on the inhibition of the human sEH were determined.
When substituents containing an aryl or a substituted aryl (3–12)
were introduced on the alpha carbon, inhibitory activity ranged
from 45 nM to 230 nM. A phenyl group (3) provided moderate
inhibition potency for the target enzyme. However, the incorpora-
tion of an alkyl group such as a methyl (4) or a tert-butyl (5) on the
benzene ring of compound 3 lowered inhibition potency approxi-
mately 4-fold. Substituting the phenyl with a methoxy (6) or a
methoxycarbonyl (7) group also dropped potency up to 5-fold.
Other functional groups like a fluoro (8) or a cyano (9) did not
improve inhibition potency, either. In addition, a decrease in inhi-
bitory activity was exhibited in compounds with pyridine (10),
phenylketone (11), or naphthalene (12) on the alpha carbon. This
result indicates that a non-substituted phenyl group is most opti-
mal among the aryl groups tested for inhibiting the target enzyme
and that modifying the phenyl group of compound 3 is not effec-
tive in improving inhibitory potency. On the other hand, in the
compound with a cyclopropyl group (13) on the carbon alpha to
the diethylphosphonate moiety, approximately 2-fold improve-
ment in inhibition was gained comparing to that of compound 3.
However, reduced inhibition was observed when the cyclopropyl
of compound 13 was switched to a cyclohexyl (14) or a propyl
(15) group, indicating that a smaller cycloalkyl group like a cyclo-
propyl is suitable over a bigger cycloalkyl group, a straight alkyl, or
an aryl group for inhibiting the enzyme. Interestingly, a further
increase (4-fold) in inhibition was obtained when the cyclopropyl
of compound 13 was replaced by a tert-butyl group (16), which
gave a 10-fold enhancement in inhibition compared to that of com-
pound 3. However, the replacement of the tert-butyl of compound
16 by a 2,2-dimethylpropyl group (17) reduced potency by about
10-fold again, suggesting that a tert-butyl group on the carbon
alpha to the diethylphosphate moiety of compound 16 is specifi-
cally effective to make potent phosphonate derivatives for the tar-
get human sEH. This was confirmed again in comparing
compounds 15 and 16. When compound 16 was compared to pre-
viously reported amide inhibitors with an ester (1 in Fig. 1) or an
oxyoxalamide (2 in Fig. 1) function as a secondary pharmacophore,
better inhibition potency (up to 38-fold) was observed in the phos-
phonate derivative (16), implying that substituted phosphonates
are one of several promising functions as a secondary pharma-
cophore.22,23 The results in Table 1 imply that alkyl substituents
are more useful than aryls to be incorporated on the carbon alpha
to the diethylphosphonate function for producing improved
inhibition of the target enzyme. Especially, a tert-butyl group on
the alpha position was highly effective for yielding a potent
O
N
H
16
5.1
23
24
O
29
30
N
H
O
N
H
F3CO
O
31
32
3.0
17
N
H
O
N
H
HO
H
N
33
34
35
37
8.2
43
O
H
N
O
O
N
H
O
36
37
38
1.9
N
H
O
3.4
N
H
O
O
>1000
a
Test compounds dissolved in DMSO were incubated with human sEH for 10 min
in 25 mM Bis–Tris/HCl buffer (202 L; pH 7.0) at 30 °C. The fluorescent substrate
(CMNPC; [S] = 5 M) was then introduced to the incubation mixture. Inhibition
potency against the human sEH was determined by measuring the concentration of
the fluorophore, 6-methoxy-2-naphthaldehyde, with an excitation wavelength of
330 nm and an emission wavelength of 465 nm for 10 min on a microplate reader.
Results are averages of three separate measurements. See the Supplementary data
for the detailed procedures.
l
l
methanol to afford a corresponding acid–phosphonate, followed by
coupling with 1-adamantaneamine by using EDCI/DMAP to produce
compound 35 in 52% yield. For the preparation of compound 38,
(4-aminophenyl)methanol was first reacted with pivalaldehyde in
the presence of magnesium sulfate in dichloromethane to give an

I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
7203
O
O
O
O
P
a, b
c
OEt
OEt
( )n
( )n
R
N
H
R
N
H
R
OH
N
H
N
32: R =
31: R =
30:
R =
29:
R =
HO
n = 1
F CnO= 1
3
n = 1
36: R =
n = 1
37: R =
33: R =
34: R =
n = 1
n = 2
n = 1
n = 0
Scheme 3. Syntheses of a series of amide–diethyl phosphonate derivatives. Reagents and conditions: (a) 4-aminomethylaniline (29, 30, 31, 32, 36, and 37), 1,4-
diaminobenzene (33), or 4-aminoethylaniline (34), EDCI, DMAP, dichloromethane, rt; (b) pivalaldehyde, magnesium sulfate, dichloromethane, rt; (c) diethyl phosphite,
170 °C.
(A)
O
O
a, b
c, d
O
HO
N
NH2
O
O
O
P
O
P
OEt
OEt
OEt
OEt
e
HO
N
H
N
H
N
H
35
(B)
O
O
O
P
a, f
e
HO
OEt
OEt
O
O
NH2
N
N
38
H
Scheme 4. Syntheses of diethyl 1-(4-(2-(1-adamantylaminocarbonyl)ethyl)phenylamino)-2,2-dimethylpropylphosphonate (35) and diethyl 1-(4-((1-adamantanecarbony-
loxy)methyl)-phenylamino)-2,2-dimethylpropylphosphonate (38). Reagents and conditions: (a) pivalaldehyde, magnesium sulfate, dichloromethane, rt; (b) iodomethane,
potassium carbonate, DMF, rt; (c) diethyl phosphite, 170 °C; (d) 1 N NaOH, methanol, rt; (e) 1-adamantaneamine, EDCI, DMAP, dichloromethane, rt;
(f) 1-adamantanecarboxylic acid, EDCI, DMAP, dichloromethane, rt.
amide–phosphonate inhibitor, suggesting that substituted phos-
phonates with a tert-butyl group on the alpha carbon are a useful
secondary pharmacophore for the human sEH inhibitors.
on the phosphonate function gives relatively large variations in
inhibition potency in amide compounds. As shown in Table 2, we
further prepared the corresponding urea derivatives (23–28) to
compare inhibition between the two series of compounds. As seen
in compound 23, ethyl groups on the phosphonate function yielded
the most potent inhibitors in the urea series, as was observed in
the corresponding amide analog (16). Approximately 2-fold
decrease in inhibition was gained in isopropyl (25) and isobutyl
(27) derivatives. In addition, 2,2,2-trifluoroethyl (24), butyl (26),
and benzyl (28) groups decreased inhibition by 4-fold, indicating
that the preference of the substituents on the phosphonate moiety
is similar in both series of amide and urea compounds. However,
variations in inhibition noticed in urea derivatives were relatively
smaller than that in amide compounds (16 and 18–22) when the
substituents on the phosphonate function were changed.
The results in Table 2 indicate that ethyl groups on the phos-
phonate moiety are most effective for inhibition potency in both
amide (16) and urea (23) derivatives. Interestingly, highly potent
inhibition on the human sEH was yielded in the urea compound
(23) with the ethyl substitution on the phosphonate function. On
the other hand, approximately 5-fold decreased potency was
observed in the corresponding amide compound 16. Relatively
decreased inhibition was observed in other amide derivatives, as
well, implying that further optimization is necessary for amide–
phosphonate compounds to improve their inhibition potency
Based on the above results, a tert-butyl group was fixed on the
alpha position, and the two O-ethyl groups on the phosphonate
function were then optimized by using several alkyl groups. As
shown in Table 2, five amide derivatives (18–22) with alkyls or
substituted alkyls on the phosphonate moiety were synthesized.
When the ethyl groups in compound 16 were replaced by
2,2,2-trifluoroethyl groups (18), inhibition potency was decreased
by 6-fold, indicating that the introduction of functionalized alkyl
groups on the phosphonate function is not effective for improving
inhibition on the human sEH. On the other hand, similar inhibition
to that of compound 16 was observed in compound with isopropyl
groups on the phosphonate (19). However, butyl (20) and isobutyl
(21) groups lowered inhibition potency by 5-fold, implying that
larger alkyl chains are not useful for affording improved inhibition.
Moreover, the dibenzyl ester (22) had approximately 8-fold lower
inhibition than that of compound 16 with ethyl groups, indicating
that aryl containing substituents seem to be not suitable for pro-
ducing potent inhibitors. The above results suggest that relatively
short alkyl chains like an ethyl (16) or an isopropyl (19) need to
be placed on the phosphonate function in amide derivatives to
yield potent inhibition for the human sEH. It was also discovered
that a subtle change in the alkyl chain length of the substituents

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I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
towards the target enzyme. This result is consistent with previous
reports that amide inhibitors generally yield approximately 10-
fold lower inhibition than urea derivatives.13,21 Thus, compound
16 with the highest inhibition potency among the amide deriva-
tives in Tables 1 and 2 was further optimized as a lead structure.
As seen in Table 3, the diethylphosphonate moiety with a tert-butyl
group of compound 16 was fixed based on the results in Tables 1
and 2. Then, an adamantyl group attached to the amide function
of compound 16 was modified to cycloalkyl or aryl groups (29–
32). When the adamantyl group of compound 16 was replaced
by a cyclohexyl group (29), approximately 5-fold loss in activity
was observed. The introduction of a 4-trifluoromethylphenyl group
(30) resulted in the same reduction, which is similar to previous
results that sterically hindered cycloalkyl substituents, especially
an adamantyl group linked to amide or urea primary pharma-
cophores is more effective than other cycloalkyl or aryl sub-
stituents for inhibition potency.11,13 Interestingly, compound 31
with tetrahydronaphthalene had approximately 2-fold improved
inhibition over compound 16. However, hydroxynaphthalene
(32) induced a 5-fold drop in activity. On the other hand, it was
previously found that both tetrahydronaphthalene and substituted
naphthalenes are equally effective in amide inhibitors with an
ester function as a secondary pharmacophore.22 This suggests that
a secondary pharmacophore seems to affect the preference of the
substituents attached to the amide primary pharmacophore. More-
over, inhibition potency dropped approximately 7-fold when the
benzyl linker of compound 16 was modified with a phenyl group
as seen in compound 33. On the other hand, a little reduction in
inhibition (1.5-fold) was observed when a phenethyl group (34)
was placed instead of the benzyl linker of compound 16. This indi-
cates that the length of a linker between the amide primary phar-
macophore and the phosphonate secondary pharmacophore is
important for yielding potent inhibitors and that the space pro-
vided by a benzyl group seems to be near optimal for increasing
inhibition potency on the target enzyme in amide inhibitors. As
seen in compound 35, switch of the nitrogen atom in the right side
of the amide function of compound 34 to the left side dropped
potency 5-fold, suggesting that the presence of the nitrogen atom
of the amide function in the right side is important for affording
potent inhibition. Surprisingly, approximately 3-fold enhanced
potency was gained when a methylene carbon was incorporated
between the amide function and the adamantyl group of com-
pound 16 (36). A naphthalene derivative extended with a methy-
lene carbon (37) also had very potent inhibition. This result
implies that the extended length by one methylene carbon is
highly useful to produce very potent amide–phosphonate inhibi-
tors. Similar dramatic enhancement in inhibition was previously
observed by same incorporation of a methylene carbon in the opti-
mization of amide compounds substituted with an oxyoxalamide
secondary pharmacophore, supporting that the adaman-
tanemethyl group is one of essential substitutions to yield highly
potent amide inhibitors with a secondary pharmacophore.23 Mod-
ifying the amide function of compound 16 by a corresponding ester
group (38), as expected, led to a dramatic drop in inhibition, sug-
gesting that the amide function plays a critical role in the inhibi-
tion of the target enzyme as a primary pharmacophore and that
the substituted phosphonate moiety has a secondary role in con-
trolling the enzyme activity.
other hand, the combined structures with the above optimizations
may have poor physical properties such as low water solubility due
to the hydrophobicity of the substituents (e.g., adamantyl and ben-
zyl groups) although a relatively polar substituted phosphonate
was incorporated in the structures. In general, poor water solubil-
ity leads to undesirable pharmacokinetic properties in vivo like
decreased absorption, which can affect bioavailability. So we
examined water solubility of the potent inhibitors in Tables 1–3.
As seen in Table 4, water solubility of the compounds (16, 23, 36,
and 37) ranged from 62 lM to 125 lM. Compound 16 had higher
solubility in comparison with that of the corresponding urea
derivative (23) or two amides with adamantanemethyl (36) or
naphthalenemethyl (37). When the solubility of compound 16
was compared to that of a potent amide inhibitor with an ester sec-
ondary pharmacophore (1),22 the same solubility was observed,
indicating that the phosphonate function is effective for yielding
not only potent inhibition, but also reasonable solubility as a sec-
ondary pharmacophore. Moreover, it was 2-fold better than that
of AUDA,22 a first generation soluble urea inhibitor. However, it
was found to be a 5-fold lower solubility than that of amide–oxy-
oxalamide (HD98)23 and urea–ethyleneglycol (IK950)13 deriva-
tives that are reported as potent inhibitors with high water
solubility. Of course with improved potency, target occupancy,
and target site exposure, problems with crystal stability, lipophilic-
ity, and poor water solubility are minimizing. The results imply
that structures of amide–phosphonates need to be further opti-
mized to make enhanced inhibitors with improved physical prop-
erties based on the structure–activity relationship results found in
this study.
3. Conclusions
This report focused on exploring structure–activity relation-
ships among amide–phosphonate derivatives as human sEH inhi-
bitors to see whether the substituted phosphonate function
works as an effective secondary pharmacophore in amide and urea
sEH inhibitors. First, a variety of aryl and alkyl substituents were
introduced on the carbon alpha to the phosphonate function in
amide compounds (Table 1). The inhibition data indicated that
alkyl substitutions (13–17) on the alpha carbon make better
inhibitors than aryls or substituted aryls (3–12). Approximately
10–45-fold enhanced inhibitors were gained with the alkyl sub-
stituents on the alpha position. Especially, a tert-butyl group (16)
on the alpha carbon was found to be highly effective for the pro-
duction of potent inhibition of the target enzyme. Based on this
result, the tert-butyl substituent was fixed on the carbon alpha to
the phosphonate moiety, and then alkyl groups on the phospho-
nate function were optimized by testing a series of alkyl groups
(Table 2). The results in Table 2 suggested that ethyl groups (16)
on the phosphonate function are most effective for inhibition and
that longer alkyl (18–21) or aryl containing alkyl (22) groups drop
inhibition up to 8-fold. In addition, the preference of the sub-
stituents on the phosphonate function was similar between amide
(16 and 18–22) and urea (23–28) derivatives. In order to further
improve inhibition potency of amide compounds with the opti-
mized diethylphosphonate function, we modified the substituent
of the amide primary pharmacophore and the linker between the
primary pharmacophore and the phosphonate function (Table 3).
The results in Table 3 indicated that amide–phosphonate com-
pound 36 with an adamantanemethyl group and a benzyl linker
exhibited most promising inhibition potency among other amide
derivatives, which is the same order of potency as that of the best
inhibitors with a urea primary pharmacophore (23–28). Approxi-
mately 2–20-fold drop in inhibition was observed in modifying
the adamantanemethyl and the benzyl linker of compound 36 with
The above results indicate that cycloalkanes such as adaman-
tane and tetrahydronaphthalene are suitable substituents for the
amide primary pharmacophore. Especially, substituents extended
by the incorporation of a methylene carbon like an adaman-
tanemethyl group were promising to afford highly potent amide–
phosphonate inhibitors. In addition, it was found that a benzyl
group as a linker between the amide function and the phosphonate
moiety provides optimal length for improved inhibition. On the

I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
7205
M)
Table 4
Inhibition potency on the human sEH and water solubility of amide and urea inhibitors
a
No.
IC50 (nM)
Solubilityb
125
(l
O
O
OEt
N
H
P
16
23
36
5.1
OEt
N
H
O
O
O
OEt
OEt
N
N
H
P
H
0.96
1.9
78
62
78
N
H
O
P
OEt
OEt
N
H
N
H
O
O
P
OEt
OEt
N
H
37
3.4
N
H
O
N
1c
9.1
3.2
125
63
H
O
O
O
O
AUDAc
OH
N
H
N
H
O
N
O
HD98c
IK950c
H
7.9
14
625
625
N
N
O
O
H
O
O
O
(
)
3
O
N
H
N
H
a
Test compounds dissolved in DMSO were incubated with human sEH for 10 min in 25 mM Bis–Tris/HCl buffer (202
lL; pH 7.0) at 30 °C. The fluorescent substrate
(CMNPC; [S] = 5 M) was then introduced to the incubation mixture. Inhibition potency against the human sEH was determined by measuring the concentration of the
l
fluorophore, 6-methoxy-2-naphthaldehyde, with an excitation wavelength of 330 nm and an emission wavelength of 465 nm for 10 min on a microplate reader. Results are
averages of three separate measurements. See the Supplementary data for the detailed procedures.
b
Water solubility was determined by adding a variety of 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). The turbidity of the water solution was measured at 650 nm to determine solubility in water. Results are the average of triplicate determinations.
c
Previously reported amide and urea inhibitors. AUDA, 12-(3-adamantan-1-yl-ureido)dodecanoic acid, was used as a quality control for the inhibition assay. IK950 was
used as a quality control for the measurement of water solubility in the present study.
other alkyls, cycloalkyls, or substituted aryls, implying that an
adamantanemethyl group and a benzyl linker are highly useful to
design very potent amide inhibitors with a phosphonate function.
In addition, the potent inhibitors listed in Table 4 had reasonable
water solubility, suggesting that substituted phosphonates incor-
porated into amide inhibitors are effective for improving not only
inhibition but also solubility as a secondary pharmacophore. The
compounds resulting from this study provide insight into mapping
the catalytic site of the target enzyme and positions to combine
functionalities inhibiting other enzymes such as cyclooxygenase,
phosphodiesterase, lipoxygenase, and other pharmacophores
shown to synergize therapeutically with the sEH inhibitors and
thus open door to polypharmacology approaches to drug design.
In the shorter term, these phosphonate derivatives are the basis
for the syntheses of further improved amide–phosphonate
inhibitors with better physical properties, which will be useful
to develop bioavailable compounds for the treatment of
hypertension, vascular inflammation, and other disorders related
to the lipid mediators, EETs.
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 determined by using TLC,
LC–MS, melting point, and NMR analysis described below. All melt-
ing 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 spectrome-
ter (JEOL Ltd, Japan), using TMS as an internal standard. High

7206
I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
resolution mass spectra were measured by LC–MS (Xevo Q-TOFMS;
Waters, UK) using positive mode electrospray ionization. Thin-
layer chromatography (TLC) was performed on EMD precoated sil-
ica 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.
All of synthesized compounds are racemic mixtures.
m), 3.90–3.94 (1H, m), 4.08–4.13 (2H, m), 4.23 (2H, d, J = 5.7 Hz),
4.66–4.79 (2H, m), 5.67 (1H, s), 6.54 (2H, d, J = 8.4 Hz), 7.11 (2H,
d, J = 8.4 Hz), 7.21 (2H, d, J = 8.4 Hz), 7.33 (2H, d, J = 8.4 Hz). HRMS
(ESI) m/z calcd for C30H41N2O4P [M+H]+ 525.2882, found [M+H]+
525.2879, mp 173 °C.
4.1.4. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(4-tert-butylphenyl)methyl-phosphonate (5)
1H NMR d (CDCl3): 1.06 (3H, t, J = 7.3 Hz), 1.24–1.30 (12H, m),
1.66 (6H, s), 1.83 (6H, s), 2.03 (3H, s), 3.64–3.67 (1H, m), 3.90–
3.93 (1H, m), 4.07–4.14 (2H, m), 4.25 (2H, d, J = 5.7 Hz), 4.92 (1H,
dd, J = 18 Hz, 5.7 Hz), 4.92–4.94 (1H, m), 5.72 (1H, s), 6.56 (2H, d,
J = 8.4 Hz), 7.00 (2H, d, J = 8.4 Hz), 7.32–7.36 (4H, m). HRMS (ESI)
4.1.1. Diethyl 1-(4-((1-adamantanecarboxamido)methyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (16)
To a solution of 4-aminobenzylamine (0.50 g, 4.09 mmol),
4-dimethylaminopyridine (DMAP; 0.50 g, 4.09 mmol), and
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDCI; 0.78 g, 4.09 mmol) in dichloromethane (20 mL) was added
1-adamantanecarboxylic acid (0.73 g, 4.09 mmol) in dichloro-
methane (10 mL) 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 Â 2)
and water (50 mL Â 2), dried over magnesium sulfate, and
evaporated. The residue was purified by using silica gel column
chromatography (hexane/ethyl acetate = 1:2) to afford an amide–
amine intermediate in 95% yield. To a mixture of this amine
(0.70 g, 2.46 mmol) and magnesium sulfate (4.0 g) in dichloro-
methane (20 mL) pivalaldehyde (0.32 g, 3.69 mmol) was added at
room temperature. The reaction mixture was stirred overnight.
The imine product was extracted with diethyl ether (50 mL Â 2).
The combined ether solutions were washed with water
(100 mL Â 2), dried over magnesium sulfate, and evaporated. The
residue was washed with cold diethyl ether (10 mL) and used for
the next reaction without further purification. To the imine inter-
mediate (1.94 g, 22.5 mmol) was added diethyl phosphite excess.
The mixture was heated at 170 °C for 1 h and then cooled down
to room temperature. The product was extracted with diethyl ether
(80 mL Â 2). The ether solutions were washed with an aqueous
solution of saturated sodium bicarbonate (80 mL Â 4) and water
(80 mL Â 2), dried over magnesium sulfate, and evaporated. The
residue was purified by using column chromatography (hexane/
ethyl acetate = 1:2) to give compound 16 in 78%. 1H NMR d (CDCl3):
1.12 (9H, s), 1.13 (3H, t, J = 7.3 Hz), 1.27 (3H, t, J = 7.3 Hz), 1.71 (6H,
s), 1.86 (6H, s), 2.03 (3H, s), 3.47 (1H, dd, J = 18 Hz, 8 Hz), 3.90–4.00
(2H, m), 4.07–4.14 (3H, m), 4.29 (2H, d, J = 5.7 Hz), 5.72 (1H, s),
6.59 (2H, d, J = 8.4 Hz), 7.05 (2H, d, J = 8.4 Hz). 13C NMR d (CDCl3):
16.3, 16.5, 27.5, 27.6, 28.1, 35.6, 36.5, 39.3, 40.5, 42.9, 59.3, 60.8,
61.6, 62.6, 113.1, 127.5, 128.9, 147.2, 177.6. HRMS (ESI) m/z calcd
for C27H43N2O4P [M+H]+ 491.3038, found [M+H]+ 491.3034, mp
143 °C.
m/z calcd for
C
33H47N2O4P [M+H]+ 567.3351, found [M+H]+
567.3355, mp 147 °C.
4.1.5. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(4-methoxyphenyl)methyl-phosphonate (6)
1H NMR d (CDCl3): 1.12 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.68 (6H, s), 1.82 (6H, s), 2.01 (3H, s), 3.60–3.66 (1H, m), 3.78 (3H,
s), 3.90–3.98 (1H, m), 4.09–4.14 (2H, m), 4.24 (2H, d, J = 5.7 Hz),
4.74 (1H, dd, J = 18 Hz, 5.7 Hz), 4.79–4.81 (1H, m), 5.65 (1H, s),
6.54 (2H, d, J = 8.4 Hz), 6.86 (2H, d, J = 8.4 Hz), 7.00 (2H, d,
J = 8.4 Hz), 7.37 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd for
C
30H41N2O5P [M+H]+ 541.2831, found [M+H]+ 541.2826, mp
123 °C.
4.1.6. Diethyl (4-(methoxycarbonyl)phenyl)(4-((1-adamantane-
carboxamido)methyl)phenyl-amino)methylphosphonate (7)
1H NMR d (CDCl3): 1.12 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.68 (6H, s), 1.82 (6H, s), 2.01 (3H, s), 3.60–3.66 (1H, m), 3.90 (3H,
s), 3.93–3.98 (1H, m), 4.09–4.14 (2H, m), 4.24 (2H, d, J = 5.7 Hz),
4.80 (1H, dd, J = 18 Hz, 5.7 Hz), 4.84 (1H, s), 5.65 (1H, s), 6.50
(2H, d, J = 8.4 Hz), 6.99 (2H, d, J = 8.4 Hz), 7.54 (2H, d, J = 8.4 Hz),
8.00 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd for C31H41N2O6P [M
+H]+ 569.2780, found [M+H]+ 569.2773, mp 122 °C.
4.1.7. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(4-fluorophenyl)methyl-phosphonate (8)
1H NMR d (CDCl3): 1.14 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.69 (6H, s), 1.83 (6H, s), 2.03 (3H, s), 3.72–3.75 (1H, m), 3.93–3.97
(1H, m), 4.08–4.14 (2H, m), 4.25 (2H, d, J = 5.7 Hz), 4.69–4.75 (2H,
m), 5.74 (1H, s), 6.52 (2H, d, J = 8.4 Hz), 6.99–7.05 (4H, m), 7.41–
7.44 (2H, m). HRMS (ESI) m/z calcd for C29H38FN2O4P [M+H]+
529.2631, found [M+H]+ 529.2635, mp 133 °C.
4.1.8. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(4-cyanophenyl)methyl-phosphonate (9)
Compounds 3–15 and 17–22 were synthesized in the same pro-
cedure used for the synthesis of compound 16 by using a corre-
1H NMR d (CDCl3): 1.17 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.68 (6H, s), 1.83 (6H, s), 2.04 (3H, s), 3.83–3.88 (1H, m), 4.03–4.07
(1H, m), 4.08–4.15 (2H, m), 4.25 (2H, d, J = 5.7 Hz), 4.77 (1H, dd,
J = 18 Hz, 5.7 Hz), 4.89 (1H, s), 5.84 (1H, s), 6.49 (2H, d,
J = 8.4 Hz), 7.00 (2H, d, J = 8.4 Hz), 7.62 (4H, m). HRMS (ESI) m/z
calcd for C30H38N3O4P [M+H]+ 536.2678, found [M+H]+ 536.2676,
mp 168 °C.
sponding aldehyde or
a substituted phosphite instead of
pivalaldehyde and diethyl phosphite.
4.1.2. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(phenyl)methyl-phosphonate (3)
1H NMR d (CDCl3): 1.10 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.67 (6H, s), 1.82 (6H, s), 2.01 (3H, s), 3.60–3.66 (1H, m), 3.88–3.96
(1H, m), 4.09–4.14 (2H, m), 4.24 (2H, d, J = 5.7 Hz), 4.74 (1H, dd,
J = 18 Hz, 5.7 Hz), 4.81–4.83 (1H, m), 5.65 (1H, s), 6.54 (2H, d,
J = 8.4 Hz), 7.01 (2H, d, J = 8.4 Hz), 7.25–7.34 (3H, m), 7.45–7.47
(2H, m). HRMS (ESI) m/z calcd for C29H39N2O4P [M+H]+ 511.2725,
found [M+H]+ 511.2726, mp 98 °C.
4.1.9. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(pyridin-4-yl)methyl-phosphonate (10)
1H NMR d (CDCl3): 1.18 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.68 (6H, s), 1.83 (6H, s), 2.01 (3H, s), 3.80–3.90 (1H, m), 4.01–4.06
(1H, m), 4.10–4.15 (2H, m), 4.25 (2H, d, J = 5.7 Hz), 4.78 (1H, dd,
J = 18 Hz, 5.7 Hz), 4.80 (1H, m), 5.65 (1H, s), 6.49 (2H, d,
J = 8.4 Hz), 7.00 (2H, d, J = 8.4 Hz), 7.39 (2H, d, J = 5.8 Hz), 7.85
(2H, d, J = 5.8 Hz). HRMS (ESI) m/z calcd for C28H38N3O4P [M+H]+
512.2678, found [M+H]+ 512.2679.
4.1.3. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(p-tolyl)methyl-phosphonate (4)
1H NMR d (CDCl3): 1.12 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.66 (6H, s), 1.83 (6H, s), 2.01 (3H, s), 2.31 (3H, s), 3.64–3.68 (1H,

I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
7207
4.1.10. Diethyl 1-(4-((1-adamantanecarboxamido)methyl)phenyl-
amino)-2-oxo-2-phenylethylphosphonate (11)
4.1.17. Diisopropyl (4-((1-adamantanecarboxamido)methyl)
phenylamino)-2,2-dimethylpropyl-phosphonate (19)
1H NMR d (CDCl3): 1.12–1.19 (6H, m), 1.68 (6H, s), 1.86 (6H, s),
2.03 (3H, s), 4.06–4.12 (4H, m), 4.28 (2H, d, J = 5.7 Hz), 5.02 (1H, s),
5.52 (1H, dd, J = 18 Hz, 8.0 Hz), 5.75 (1H, s), 6.72 (2H, d, J = 8.4 Hz),
7.06 (2H, d, J = 8.4 Hz), 7.49 (2H, d, J = 8.4 Hz), 7.61 (1H, d,
J = 8.4 Hz), 8.06 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd
for C30H39N2O5P [M+H]+ 539.2675, found [M+H]+ 539.2685.
1H NMR d (CDCl3): 1.02 (3H, d, J = 7.3 Hz), 1.11 (9H, s), 1.21 (3H,
d, J = 7.3 Hz), 1.28 (6H, t, J = 7.3 Hz), 1.72 (6H, s), 1.86 (6H, s), 2.04
(3H, s), 3.41 (1H, dd, J = 18 Hz, 8 Hz), 3.99–4.01 (1H, m), 4.28 (2H, d,
J = 5.7 Hz), 4.63–4.68 (2H, m), 5.70 (1H, s), 6.58 (2H, d, J = 8.4 Hz),
7.03 (2H, d, J = 8.4 Hz). 13C NMR d (CDCl3): 23.9, 24.1, 24.5, 27.8,
28.1, 35.9, 36.5, 39.3, 40.5, 42.9, 59.7, 61.2, 70.5, 71.4, 113.1,
127.3, 128.9, 147.6, 177.6. HRMS (ESI) m/z calcd for C29H47N2O4P
[M+H]+ 519.3351, found [M+H]+ 519.3354, mp 175 °C.
4.1.11. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(naphthalene-2-yl)methylphosphonate (12)
1H NMR d (CDCl3): 1.07 (3H, t, J = 7.3 Hz), 1.28 (3H, t, J = 7.3 Hz),
1.66 (6H, s), 1.80 (6H, s), 1.99 (3H, s), 3.67–3.71 (1H, m), 3.90–3.94
(1H, m), 4.10–4.15 (2H, m), 4.24 (2H, d, J = 5.7 Hz), 4.92 (1H, dd,
J = 18 Hz, 5.7 Hz), 4.93 (1H, m), 5.68 (1H, s), 6.58 (2H, d,
J = 8.4 Hz), 6.97 (2H, d, J = 8.4 Hz), 7.45–7.48 (2H, m), 7.59 (1H, d,
J = 8.4 Hz), 7.80–7.84 (3H, m), 7.92 (1H, s). HRMS (ESI) m/z calcd
for C33H41N2O4P [M+H]+ 561.2882, found [M+H]+ 561.2883, mp
110 °C.
4.1.18. Dibutyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (20)
1H NMR d (CDCl3): 0.82 (3H, t, J = 7.3 Hz), 0.88 (3H, t, J = 7.3 Hz),
1.12 (9H, s), 1.23–1.30 (4H, m), 1.41–1.48 (2H, m), 1.52–1.59 (2H,
m), 1.71 (6H, s), 1.86 (6H, s), 2.03 (3H, s), 3.48 (1H, dd, J = 18 Hz,
8 Hz), 3.85–3.89 (1H, m), 4.00–4.14 (4H, m), 4.29 (2H, d,
J = 5.7 Hz), 5.71 (1H, s), 6.58 (2H, d, J = 8.4 Hz), 7.05 (2H, d,
J = 8.4 Hz). HRMS (ESI) m/z calcd for
C
31H51N2O4P [M+H]+
547.3664, found [M+H]+ 547.3667, mp 109 °C.
4.1.12. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(cyclopropyl)methyl-phosphonate (13)
4.1.19. Diisobutyl (4-((1-adamantanecarboxamido)methyl)
phenylamino)-2,2-dimethylpropyl-phosphonate (21)
1H NMR d (CDCl3): 0.78–0.80 (6H, m), 0.85–0.87 (6H, m), 1.13
(9H, s), 1.69–1.85 (8H, m), 1.86 (6H, s), 2.04 (3H, s), 3.48 (1H, dd,
J = 18 Hz, 8 Hz), 3.60–3.64 (1H, m), 3.74–3.82 (3H, m), 4.01–4.03
(1H, m), 4.28 (2H, d, J = 5.7 Hz), 5.70 (1H, s), 6.58 (2H, d,
J = 8.4 Hz), 7.05 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd for
1H NMR d (CDCl3): 0.38–0.42 (2H, m), 0.59–0.63 (2H, m), 1.21
(3H, t, J = 7.3 Hz), 1.32 (3H, t, J = 7.3 Hz), 1.72 (6H, s), 1.86 (6H, s),
2.05 (3H, s), 3.29–3.31 (1H, m), 3.80–3.82 (1H, m), 4.03–4.05 (1H,
m), 4.11–4.18 (3H, m), 4.30 (3H, m), 5.74 (1H, s), 6.60 (2H, d,
J = 8.4 Hz), 7.05 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd for
C
26H39N2O4P [M+H]+ 475.2725, found [M+H]+ 475.2724, mp 125 °C.
C
31H51N2O4P [M+H]+ 547.3664, found [M+H]+ 547.3668, mp 147 °C.
4.1.13. Diethyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)(cyclohexyl)methyl-phosphonate (14)
4.1.20. Dibenzyl (4-((1-adamantanecarboxamido)methyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (22)
1H NMR d (CDCl3): 1.18 (3H, t, J = 7.3 Hz), 1.23–1.30 (7H, m),
1.71 (10H, s), 1.86 (7H, s), 2.04 (4H, s), 3.61–3.64 (1H, m), 3.89–
3.99 (2H, m), 4.03–4.05 (1H, m), 4.06–4.13 (3H, m), 4.29 (2H, d,
J = 5.7 Hz), 5.74 (1H, s), 6.60 (2H, d, J = 8.4 Hz), 7.04 (2H, d,
1H NMR d (CDCl3): 1.12 (9H, s), 1.72 (6H, s), 1.86 (6H, s), 2.04
(3H, s), 3.56 (1H, dd, J = 18 Hz, 8 Hz), 3.99–4.01 (1H, m), 4.28
(2H, s), 4.80–4.82 (1H, m), 4.95–5.03 (3H, m), 5.70 (1H, s), 6.55
(2H, d, J = 8.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 7.11–7.13 (2H, m),
7.22–7.24 (2H, m), 7.26–7.29 (3H, m), 7.35–7.37 (3H, m). HRMS
(ESI) m/z calcd for C37H47N2O4P [M+H]+ 615.3351, found [M+H]+
615.3343.
J = 8.4 Hz). HRMS (ESI) m/z calcd for
C
29H45N2O4P [M+H]+
517.3295, found [M+H]+ 517.3295, mp 156 °C.
4.1.14. Diethyl 1-(4-((1-adamantanecarboxamido)methyl)phenyl-
amino)butylphosphonate (15)
1H NMR d (CDCl3): 0.91 (3H, t, J = 7.3 Hz), 1.18–1.30 (8H, m),
1.67 (6H, s), 1.85 (6H, s), 2.05 (3H, s), 3.68 (1H, m), 4.00–4.01
(1H, m), 4.07–4.14 (6H, m), 4.30 (2H, d, J = 5.7 Hz), 5.73 (1H, s),
6.60 (2H, d, J = 8.4 Hz), 7.06 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd
for C26H41N2O4P [M+H]+ 477.2882, found [M+H]+ 477.2883, mp
119 °C.
4.1.21. Diethyl 1-(4-((3-adamantan-1-ylureido)methyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (23)
To a solution of 4-aminobenzylamine (0.50 g, 4.09 mmol) in dry
N,N-dimethylformamide (15 mL) was added adamantyl isocyanate
(0.73 g, 4.09 mmol) in dry N,N-dimethylformamide (3 mL) at room
temperature. The reaction was stirred overnight. The product was
extracted with ethyl acetate (50 mL). The organic solution was
washed with water (50 mL Â 2), dried over magnesium sulfate,
and evaporated. The residue was recrystallized in hexane to afford
a urea–amine intermediate in 98% yield. The same reactions as
those conducted for the preparation of compound 16 were used
for the synthesis of compound 23 by using pivalaldehyde and
diethyl phosphite. 1H NMR d (CDCl3): 1.10 (9H, s), 1.13 (3H, t,
J = 7.3 Hz), 1.27 (3H, t, J = 7.3 Hz), 1.64 (6H, s), 1.93 (6H, s), 2.04
(3H, s), 3.47 (1H, dd, J = 18 Hz, 8 Hz), 3.90–4.00 (2H, m), 4.12–
4.18 (6H, m), 4.38 (1H, s), 6.58 (2H, d, J = 8.4 Hz), 7.08 (2H, d,
J = 8.4 Hz). 13C NMR d (CDCl3): 16.3, 16.4, 27.6, 29.5, 35.7, 36.3,
42.1, 42.5, 44.1, 50.9, 59.3, 60.8, 61.7, 62.7, 113.1, 128.1, 128.8,
147.2, 157.1. HRMS (ESI) m/z calcd for C27H44N3O4P [M+H]+
506.3147, found [M+H]+ 506.3147.
4.1.15. Diethyl 1-(4-((adamantan-1-ylcarboxamido)methyl)phe-
nylamino)-3,3-dimethylbutylphosphonate (17)
1H NMR d (CDCl3): 0.93 (9H, s), 1.14 (3H, t, J = 7.3 Hz), 1.26 (3H,
t, J = 7.3 Hz), 1.75 (8H, s), 1.87 (6H, s), 2.05 (3H, s), 3.50 (1H, d,
J = 8.0 Hz), 3.81–3.83 (2H, m), 4.06–4.11 (3H, m), 4.30 (2H, d,
J = 5.7 Hz), 5.72 (1H, s), 6.59 (2H, d, J = 8.4 Hz), 7.05 (2H, d,
J = 8.4 Hz). 13C NMR d (CDCl3): 16.5, 28.1, 29.9, 30.8, 30.9, 36.5,
39.3, 40.6, 43.0, 44.1, 47.8, 49.4, 62.0, 62.8, 113.0, 127.4, 128.9,
146.0, 177.6. HRMS (ESI) m/z calcd for C28H45N2O4P [M+H]+
505.3195, found [M+H]+ 505.3190, mp 153 °C.
4.1.16. Di-(2,2,2-trifluoroethyl) (4-((1-adamantanecarboxamido)
methyl)phenylamino)-2,2-dimethylpropyl-phosphonate (18)
1H NMR d (CDCl3): 1.12 (9H, s), 1.72 (6H, s), 1.86 (6H, s), 2.03
(3H, s), 3.67 (1H, dd, J = 18 Hz, 8 Hz), 3.84–3.90 (2H, m), 4.30–
4.45 (5H, m), 5.76 (1H, s), 6.63 (2H, d, J = 8.4 Hz), 7.10 (2H, d,
Compounds 24–34 and 36–37 were synthesized in the
same procedure used for the preparation of compound 23 by
using a corresponding substituted phosphite and an alkylcar-
boxylic acid instead of diethyl phosphite and adamantanecar-
boxylic acid.
J = 8.4 Hz). HRMS (ESI) m/z calcd for
C
27H37F6N2O4P [M+H]+
599.2473, found [M+H]+ 599.2472, mp 90 °C.

7208
I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
4.1.22. Di-(2,2,2-trifluoroethyl) 1-(4-((3-adamantan-1-ylureido)
methyl)phenylamino)-2,2-dimethylpropyl-phosphonate (24)
1H NMR d (CDCl3): 1.16 (9H, s), 1.65 (6H, s), 1.93 (6H, s), 2.04
(3H, s), 3.67 (1H, dd, J = 18 Hz, 8 Hz), 3.88–3.92 (2H, m), 4.18
(2H, s), 4.30–4.42 (4H, m), 4.65 (1H, s), 6.63 (2H, d, J = 8.4 Hz),
7.20 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd for C27H38F6N3O4P
[M+H]+ 614.2582, found [M+H]+ 614.2586, mp 154 °C.
4.1.29. Diethyl 1-(4-((1,2,3,4-tetrahydronaphthalen-2-carbox-
amido)methyl)phenylamino)-2,2-dimethyl-propylphosphonate
(31)
1H NMR d (CDCl3): 1.12 (9H, s), 1.13 (3H, t, J = 7.3 Hz), 1.27 (3H,
t, J = 7.3 Hz), 1.92–1.94 (1H, m), 2.10–2.17 (1H, m), 2.48–2.50 (1H,
m), 2.86–3.05 (4H, m), 3.47 (1H, dd, J = 18 Hz, 8 Hz), 3.94–4.08 (5H,
m), 4.34 (2H, s), 5.71 (1H, s), 6.60 (2H, d, J = 8.4 Hz), 7.06–7.12 (6H,
m). HRMS (ESI) m/z calcd for C27H39N2O4P [M+H]+ 487.2725, found
[M+H]+ 487.2729, mp 135 °C.
4.1.23. Diisopropyl 1-(4-((3-adamantan-1-ylureido)methyl)phen-
ylamino)-2,2-dimethylpropyl-phosphonate (25)
1H NMR d (CDCl3): 1.02 (3H, d, J = 7.3 Hz), 1.10 (9H, s), 1.24 (3H,
d, J = 7.3 Hz), 1.26–1.28 (6H, m), 1.65 (6H, s), 1.93 (6H, s), 2.05 (3H,
s), 3.41 (1H, dd, J = 18 Hz, 8 Hz), 3.98–4.00 (1H, m), 4.06 (1H, s),
4.16 (2H, s), 4.30 (1H, s), 4.63–4.67 (2H, m), 6.60 (2H, d,
J = 8.4 Hz), 7.09 (2H, d, J = 8.4 Hz). 13C NMR d (CDCl3): 24.1, 24.5,
27.8, 29.5, 36.0, 36.4, 42.4, 44.2, 50.9, 59.6, 61.1, 70.5, 71.5, 113.0,
127.8, 128.8, 147.5, 157.0. HRMS (ESI) m/z calcd for C29H48N3O4P
[M+H]+ 534.3460, found [M+H]+ 534.3455, mp 100 °C.
4.1.30. Diethyl 1-(4-((6-hydroxynaphthalene-2-carboxamide)
methyl)phenylamino)-2,2-dimethylpropylphosphonate (32)
1H NMR d (CDCl3): 1.12 (9H, s), 1.16 (3H, t, J = 7.3 Hz), 1.26 (3H,
t, J = 7.3 Hz), 3.50 (1H, dd, J = 18 Hz, 8 Hz), 3.96–4.11 (5H, m), 4.55
(2H, s), 6.37 (1H, s), 6.62 (2H, d, J = 8.4 Hz), 7.15–7.22 (5H, m), 7.67
(1H, s), 7.75–7.79 (2H, m), 8.19 (1H, s). HRMS (ESI) m/z calcd for
C
27H35N2O5P [M+H]+ 499.2362, found [M+H]+ 499.2360, mp
110 °C.
4.1.24. Dibutyl 1-(4-((3-adamantan-1-ylureido)methyl)pheny-
lamino)-2,2-dimethylpropyl-phosphonate (26)
4.1.31. Diethyl 1-(4-(1-adamantanecarboxamide)phenylamino)-
2,2-dimethylpropylphosphonate (33)
1H NMR d (CDCl3): 0.82 (3H, t, J = 7.3 Hz), 0.88 (3H, t, J = 7.3 Hz),
1.10 (9H, s), 1.21–1.27 (4H, m), 1.30–1.32 (2H, m), 1.52–1.59 (2H,
m), 1.65 (6H, s), 1.93 (6H, s), 2.04 (3H, s), 3.48 (1H, dd, J = 18 Hz,
8 Hz), 3.91–3.93 (1H, m), 3.94–4.01 (4H, m), 4.15 (2H, s), 4.41
(1H, s), 4.69 (1H, s), 6.57 (2H, d, J = 8.4 Hz), 7.07 (2H, d,
1H NMR d (CDCl3): 1.10 (9H, s), 1.15 (3H, t, J = 7.3 Hz), 1.25 (3H,
t, J = 7.3 Hz), 1.75 (6H, s), 1.95 (6H, s), 2.08 (3H, s), 3.45 (1H, dd,
J = 18 Hz, 8 Hz), 3.92–3.93 (2H, m), 4.04–4.10 (3H, m), 6.60 (2H,
d, J = 8.4 Hz), 7.11 (1H, s), 7.31 (2H, d, J = 8.4 Hz). HRMS (ESI) m/z
calcd for C26H41N2O4P [M+H]+ 477.2882, found [M+H]+ 477.2885,
mp 167 °C.
J = 8.4 Hz). HRMS (ESI) m/z calcd for
C
31H52N3O4P [M+H]+
562.3773, found [M+H]+ 562.3779, mp 95 °C.
4.1.32. Diethyl 1-(4-(2-(1-adamantanecarboxamido)ethyl)phen-
ylamino)-2,2-dimethylpropyl-phosphonate (34)
4.1.25. Diisobutyl 1-(4-((3-adamantan-1-ylureido)methyl)pheny-
lamino)-2,2-dimethylpropyl-phosphonate (27)
1H NMR d (CDCl3): 1.12 (9H, s), 1.13 (3H, t, J = 7.3 Hz), 1.23 (3H,
t, J = 7.3 Hz), 1.67 (6H, s), 1.77 (6H, s), 2.00 (3H, s), 2.67 (2H, t,
J = 7.3 Hz), 3.43–3.50 (3H, m), 3.94–3.96 (2H, m), 4.04–4.09 (3H,
m), 5.55 (1H, s), 6.60 (2H, d, J = 8.4 Hz), 6.97 (2H, d, J = 8.4 Hz).
HRMS (ESI) m/z calcd for C28H45N2O4P [M+H]+ 505.3195, found
[M+H]+ 505.3197, mp 138 °C.
1H NMR d (CDCl3): 0.79–0.82 (6H, m), 0.85–0.88 (6H, m), 1.12
(9H, s), 1.65–1.81 (8H, m), 1.95 (6H, s), 2.05 (3H, s), 3.58 (1H, dd,
J = 18 Hz, 8 Hz), 3.67–3.69 (1H, m), 3.82–3.86 (4H, m), 4.00–4.02
(1H, m), 4.16 (2H, s), 4.38 (1H, s), 6.61 (2H, d, J = 8.4 Hz), 7.09
(2H, d, J = 8.4 Hz). HRMS (ESI) m/z calcd for C31H52N3O4P [M+H]+
562.3773, found [M+H]+ 562.3774, mp 140 °C.
4.1.33. Diethyl 1-(4-(2-(1-adamantylaminocarbonyl)ethyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (35)
4.1.26. Dibenzyl 1-(4-((3-adamantan-1-ylureido)methyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (28)
To a solution of 3-(4-aminophenyl)propanoic acid (0.80 g,
6.05 mmol) in dichloromethane (40 mL) was added pivalaldehyde
(0.84 g, 12.1 mmol) and magnesium sulfate (4 g) at room temper-
ature. The reaction mixture was stirred overnight. The product
was extracted with dichloromethane (80 mL Â 2). The organic
solutions were washed with water (80 mL Â 2), dried over magne-
sium sulfate, and evaporated to dryness. To a solution of the resi-
due in DMF (5 mL) was added iodomethane (3.44 g, 24.2 mmol)
and potassium carbonate (3.35 g, 24.2 mmol) at room temperature.
The mixture was stirred overnight, and the product was extracted
with diethyl ether (50 mL Â 2). The organic solutions were washed
with water (50 mL Â 2), dried over magnesium sulfate, and evapo-
rated to dryness. The residue was stirred with diethyl phosphite
excess at 170 °C for 1 h. After cooling down the reaction to room
temperature, the product was extracted with diethyl ether
(50 mL Â 2). The combined ether solutions were washed with an
aqueous solution of saturated sodium bicarbonate (80 mL Â 4)
and water (80 mL Â 2), dried over magnesium sulfate, and evapo-
rated. The residue was purified by using column chromatography
(hexane/ethyl acetate = 1:2) to give an ester–phosphonate inter-
mediate in 42% yield. This intermediate in methanol (10 mL) was
added with an aqueous solution of 1 N NaOH (5 mL) at room tem-
perature. The reaction was stirred for 3 h. After adjusting the pH of
the reaction mixture to pH 2 by adding an aqueous solution of 1 N
HCl, the product was extracted with diethyl ether (50 mL Â 2). The
combined ether solutions were washed with water (50 mL Â 2),
dried over magnesium sulfate, and evaporated. The residue was
1H NMR d (CDCl3): 1.11 (9H, s), 1.64 (6H, s), 1.95 (6H, s), 2.05
(3H, s), 3.54 (1H, dd, J = 18 Hz, 8 Hz), 4.04–4.06 (1H, m), 4.15 (2H,
s), 4.36 (1H, s), 4.62 (1H, s), 4.77–4.79 (1H, m), 4.89–4.96 (3H,
m), 6.53 (2H, d, J = 8.4 Hz), 7.06 (2H, d, J = 8.4 Hz), 7.19–7.21 (2H,
m), 7.26–7.28 (2H, m), 7.29–7.31 (6H, m). HRMS (ESI) m/z calcd
for C37H48N3O4P [M+H]+ 630.3460, found [M+H]+ 630.3467, mp
73 °C.
4.1.27. Diethyl 1-(4-((cyclohexanecarboxamido)methyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (29)
1H NMR d (CDCl3): 1.11 (9H, s), 1.13 (3H, t, J = 7.3 Hz), 1.24 (5H,
t, J = 7.3 Hz), 1.40–1.48 (2H, m), 1.64–1.66 (2H, m), 1.77–1.78 (2H,
m), 1.85–1.88 (2H, m), 2.05–2.07 (1H, m), 3.47 (1H, dd, J = 18 Hz,
8 Hz), 3.88–3.94 (2H, m), 3.99–4.04 (3H, m), 4.29 (2H, s), 5.57
(1H, s), 6.59 (2H, d, J = 8.4 Hz), 7.06 (2H, d, J = 8.4 Hz). HRMS (ESI)
m/z calcd for
C
23H39N2O4P [M+H]+ 439.2725, found [M+H]+
439.2718, mp 127 °C.
4.1.28. Diethyl 1-(4-((4-(trifluoromethoxy)benzamide)methyl)
phenylamino)-2,2-dimethyl-propylphosphonate (30)
1H NMR d (CDCl3): 1.12 (9H, s), 1.15 (3H, t, J = 7.3 Hz), 1.37 (3H,
t, J = 7.3 Hz), 3.48 (1H, dd, J = 18 Hz, 8 Hz), 3.92–9.94 (1H, m), 3.99–
4.04 (4H, m), 4.50 (2H, s), 6.23 (1H, s), 6.62 (2H, d, J = 8.4 Hz), 7.15
(2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.4 Hz), 7.82 (2H, d, J = 8.4 Hz).
HRMS (ESI) m/z calcd for C24H32F3N2O5P [M+H]+ 517.2079, found
[M+H]+ 517.2076.

I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
7209
used for the next reaction without further purification. To a solu-
tion of the acid residue (90 mg, 0.24 mmol), 1-adamantanamine
(37 mg, 0.24 mmol), and DMAP (30 mg, 0.24 mmol) in dichloro-
methane (15 mL) was added EDCI (46 mg, 0.24 mmol) at room
temperature. The reaction was stirred overnight. The product
was extracted with diethyl ether (60 mL Â 2). The organic layer
was washed with water (60 mL Â 2), dried over magnesium sul-
fate, and evaporated. The residue was purified by using silica gel
column chromatography (hexane/ethyl acetate = 1:2) to afford
compound 35 in 52% yield. 1H NMR d (CDCl3): 1.11 (9H, s), 1.26
(3H, t, J = 7.3 Hz), 1.31 (3H, t, J = 7.3 Hz), 1.64 (6H, s), 1.90 (6H, s),
2.04 (3H, s), 2.30 (2H, t, J = 7.3 Hz), 2.79 (2H, t, J = 7.3 Hz), 3.47
(1H, dd, J = 18 Hz, 8 Hz), 3.85–3.93 (2H, m), 4.04–4.17 (3H, m),
4.93 (1H, s), 6.56 (1H, d, J = 8.4 Hz), 6.72 (1H, d, J = 8.4 Hz), 6.93–
6.99 (2H, m). HRMS (ESI) m/z calcd for C28H45N2O4P [M+H]+
505.3195, found [M+H]+ 505.3193.
1.05–1.14 (12H, m), 1.23 (3H, t, J = 6.9 Hz), 1.68 (6H, s), 1.88 (6H, s),
1.99 (3H, s), 3.49 (1H, dd, J = 18 Hz, 8 Hz), 3.92–3.95 (1H, m), 4.04–
4.09 (2H, m), 4.23 (2H, s), 4.96 (2H, s), 6.62 (2H, d, J = 7.5 Hz), 7.15
(2H, d, J = 7.5 Hz). HRMS (ESI) m/z calcd for C27H42NO5P [M+H]+
492.2879, found [M+H]+ 492.2878.
5. Biology
5.1. Enzyme preparation
Recombinant human sEH was produced by using baculovirus
expression system as previously reported.24 Briefly, Sf9 insect
cells were infected by recombinant baculovirus harboring human
sEH gene fused with a 6xHis tag. At 72 h post-infection, the
infected cells were homogenized and the recombinant protein
was purified by using immobilized metal affinity chromatogra-
phy. The 6xHis tag was removed by using the tobacco etch virus
protease and then human sEH was further purified by using
anion-exchange chromatography.
4.1.34. Diethyl 1-(4-((2-adamantan-1-ylacetamido)methyl)pheny-
lamino)-2,2-dimethylpropyl-phosphonate (36)
1H NMR d (CDCl3): 1.11 (9H, s), 1.13 (3H, t, J = 7.3 Hz), 1.25 (3H,
t, J = 7.3 Hz), 1.61 (6H, s), 1.71 (6H, s), 1.93 (2H, s), 1.96 (3H, s), 3.45
(1H, dd, J = 18 Hz, 8 Hz), 3.89–3.96 (2H, m), 4.01–4.05 (3H, m), 4.29
(2H, s), 5.47 (1H, s), 6.59 (2H, d, J = 8.4 Hz), 7.08 (2H, d, J = 8.4 Hz).
13C NMR d (CDCl3): 16.3, 16.5, 27.8, 28.6, 32.8, 35.7, 36.7, 42.6, 43.1,
51.9, 59.3, 60.8, 61.8, 62.6, 113.0, 127.4, 129.2, 147.3, 170.6. HRMS
(ESI) m/z calcd for C28H45N2O4P [M+H]+ 505.3195, found [M+H]+
505.3190, mp 158 °C.
5.2. IC50 assay conditions
Standard solutions of compounds in Tables 1–3 were prepared
in DMSO. Fluorescent assays were performed by using a substrate
(cyano-(2-methoxynaphthalen-6-yl)-methyl trans-(3-phenyl-oxy-
ran-2-yl)-methyl carbonate; CMNPC; [S] = 5 lM) to determine
IC50 values of the synthesized compounds.25 Inhibition activity
against human sEH (1 nM) was determined by measuring the con-
centration of the fluorophore, 6-methoxy-2-naphthaldehyde, with
an excitation wavelength of 330 nm and an emission wavelength
of 465 nm for 10 min on a microplate reader (Corona Electric, Ibar-
aki, Japan). The IC50 values were determined by regression of at
least five datum points with a minimum of three points in a linear
region of the curve. IC50 results are averages of three separate mea-
surements. 12-(3-Adamantan-1-yl-ureido)dodecanoic acid (AUDA)
in Table 4 was used as a quality control for the inhibition assay in
the present study.
4.1.35. Diethyl 1-(4-((2-(naphthalene-2-yl)acetamido)methyl)
phenylamino)-2,2-dimethylpropyl-phosphonate (37)
1H NMR d (CDCl3): 1.06–1.10 (12H, m), 1.20 (3H, t, J = 6.9 Hz),
3.45 (1H, dd, J = 18 Hz, 8 Hz), 3.77 (2H, s), 3.87–9.94 (2H, m),
4.01–4.05 (3H, m), 4.28 (2H, s), 5.57 (1H, s), 6.53 (2H, d,
J = 7.5 Hz), 6.96 (2H, d, J = 7.5 Hz), 7.38 (1H, d, J = 7.5 Hz), 7.47–
7.49 (2H, m), 7.71 (1H, s), 7.81–7.83 (3H, m). 13C NMR d (CDCl3):
16.3, 27.6, 35.6, 43.2, 44.0, 59.3, 60.7, 61.6, 62.6, 113.0, 126.0,
126.4, 126.9, 127.3, 127.6, 127.7, 128.2, 128.7, 128.8, 132.3,
132.5, 133.5, 147.3, 170.5. HRMS (ESI) m/z calcd for C28H37N2O4P
[M+H]+ 497.2569, found [M+H]+ 497.2575, mp 133 °C.
6. Solubility
4.1.36. Diethyl 1-(4-((1-adamantanecarbonyloxy)methyl)phenyl-
amino)-2,2-dimethylpropyl-phosphonate (38)
Water solubility of amide derivatives in Table 4 was determined
experimentally by a light scattering method in sodium phosphate
buffer at 25 1.5 °C.22,26 In brief, aqueous solubility was measured
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. A signal-to-noise ratio
equals to or >2 was considered insoluble. The turbidity was mea-
sured as optical density at 650 nm on a SH-8000 microplate reader
(Corona Electric) at 25 1.5 °C. Results are averages of three sepa-
rate measurements. IK950 in Table 4 was used as a quality control
for water solubility.
To a mixture of (4-aminophenyl)methanol (1.00 g, 8.11 mmol)
and magnesium sulfate (4.0 g) in dichloromethane (40 mL)
pivalaldehyde (1.05 g, 12.1 mmol) was added at room temperature.
The reaction mixture was stirred overnight. The imine product was
extracted with diethyl ether (80 mL Â 2). The combined ether solu-
tions were washed with water (100 mL Â 2), dried over magnesium
sulfate, and evaporated. The residue was used for the next reaction
without further purification. A solution of the imine (1.00 g,
5.23 mmol), 1-adamantanecarboxylic acid (0.94 g, 5.23 mmol),
and DMAP (0.64 g, 5.23 mmol) in dichloromethane (40 mL) was
stirred for 5 minutes at room temperature. To this reaction mixture
was added EDCI (1.00 g, 5.23 mmol). The reaction was stirred over-
night at room temperature. The product was extracted with diethyl
ether (80 mL Â 2). The ether layer was washed with an aqueous
solution of 1 N HCl (50 mL) and water (80 mL Â 2), dried over mag-
nesium sulfate, and evaporated to dryness. To the residue in
dichloromethane (10 mL) was added diethyl phosphite excess.
The reaction was heated at 170 °C for 1 h and then cooled down
to room temperature. The product was extracted with diethyl ether
(80 mL Â 2). The ether solutions were washed with an aqueous
solution of saturated sodium bicarbonate (80 mL Â 4) and water
(80 mL Â 2), dried over magnesium sulfate, and evaporated. The
residue was purified by using column chromatography (hexane/
ethyl acetate = 1:2) to give compound 38 in 63%. 1H NMR d (CDCl3):
Acknowledgments
This work was supported by Hyundai Pharm Research Grant
(HOB-024). Partial support was from a JSPS KAKENHI Grant No.
23710042 from Japan Society for the Promotion of Science. The
authors thank Ms. Jung-Joo Kim for her support in the purification
and characterization of some of synthesized compounds.
Supplementary data
Supplementary data (1H NMR and 13C NMR spectra of com-
pounds 16, 19, 23, 36, and 37, LC–MS analyses, preparation of

7210
I.-H. Kim et al. / Bioorg. Med. Chem. 23 (2015) 7199–7210
12. 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.
13. Kim, I.-H.; Tsai, H.-J.; Nishi, K.; Kasagami, T.; Morisseau, C.; Hammock, B. D. J.
Med. Chem. 2007, 50, 5217.
human sEH, and inhibition assays) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.
2015.10.016.
14. Kasagami, T.; Kim, I.-H.; Tsai, H.-J.; Nishi, K.; Hammock, B. D.; Morisseau, C.
Bioorg. Med. Chem. Lett. 2009, 19, 1784.
15. Jones, P. D.; Tsai, H.-J.; Do, Z.; Moresseau, C.; Hammock, B. D. Bioorg. Med. Chem.
Lett. 2006, 16, 5212.
16. Hwang, S. H.; Tsai, H.-J.; Liu, J.-Y.; Morisseau, C.; Hammock, B. D. J. Med. Chem.
2007, 50, 3825.
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