Discovery of enantioselectivity of urea inhibitors of soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.ejmech.2016.04.015

Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) in the metabolic pathway of arachidonic acid and has been considered as an important therapeutic target for chronic diseases such as hypertension, diabetes and inflammation. Although many urea derivatives are known as sEH inhibitors, the enantioselectivity of the inhibitors is not highlighted in spite of the stereoselective hydrolysis of EETs by sEH. In an effort to explore the importance of enantioselectivity in the urea scaffold, a series of enantiomers with the stereocenter adjacent to the urea nitrogen atom were prepared. The selectivity of enantiomers of 1-(α-alkyl-α-phenylmethyl)-3-(3-phenylpropyl)ureas showed wide range differences up to 125 fold with the low IC₅₀ value up to 13 nM. The S-configuration with planar phenyl and small alkyl groups at α-position is crucial for the activity and selectivity. However, restriction of the free rotation of two α-groups with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity between the enantiomers, despite the increase in activity up to 13 nM. The hydrophilic group like sulfonamido group at para position of 3-phenylpropyl motif of 1-(α-alkyl-α-phenylmethyl-3-(3-phenylpropyl)urea improves the activity as well as enantiomeric selectivity. All these ureas are proved to be specific inhibitor of sEH without inhibition against mEH.

Full text of DOI:10.1016/j.ejmech.2016.04.015

Accepted Manuscript
Discovery of Enantioselectivity of Urea Inhibitors of Soluble Epoxide Hydrolase
Manoj Manickam, Thanigaimalai Pillaiyar, PullaReddy Boggu, Eeda
Venkateswararao, Hitesh B. Jalani, Nam-Doo Kim, Seul Ki Lee, Jang Su Jeon, Sang
Kyum Kim, Sang-Hun Jung
PII:
S0223-5234(16)30297-5
10.1016/j.ejmech.2016.04.015
EJMECH 8530
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 February 2016
Revised Date: 5 April 2016
Accepted Date: 6 April 2016
Please cite this article as: M. Manickam, T. Pillaiyar, P. Boggu, E. Venkateswararao, H.B. Jalani, N.-
D. Kim, S.K. Lee, J.S. Jeon, S.K. Kim, S.-H. Jung, Discovery of Enantioselectivity of Urea Inhibitors
of Soluble Epoxide Hydrolase, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.04.015.
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Graphical Abstract
Discovery of Enantioselectivity of Urea Inhibitors of Soluble Epoxide Hydrolase

ACCEPTED MANUSCRIPT
Discovery of Enantioselectivity of Urea Inhibitors of Soluble Epoxide Hydrolase
Manoj Manickama, Thanigaimalai Pillaiyara, PullaReddy Boggua, Eeda Venkateswararaoa,
Hitesh B. Jalania, Nam-Doo Kimb, Seul Ki Leea, Jang Su Jeona, Sang Kyum Kima, Sang-Hun
Junga*
aCollege of Pharmacy and Institute of Drug Research and Development, Chungnam National
University, Daejeon 34134, Korea
bDGMIF, New Drug Development Center, 80, Cheombok-ro, Dong-gu, Daegu, Korea, 41061
Abstract
Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) in the metabolic
pathway of arachidonic acid and has been considered as an important therapeutic target for
chronic diseases such as hypertension, diabetes and inflammation. Although many urea
derivatives are known as sEH inhibitors, the enantioselectivity of the inhibitors is not highlighted
in spite of the stereoselective hydrolysis of EETs by sEH. In an effort to explore the importance
of enantioselectivity in the urea scaffold, a series of enantiomers with the stereocenter adjacent to
the urea nitrogen atom were prepared. The selectivity of enantiomers of 1-(α-alkyl-α-
phenylmethyl)-3-(3-phenylpropyl)ureas showed wide range differences up to 125 fold with the
low IC50 value up to 13 nM. The S-configuration with planar phenyl and small alkyl groups at α-
position is crucial for the activity and selectivity. However, restriction of the free rotation of two
α-groups with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity
between the enantiomers, despite the increase in activity up to 13 nM. The hydrophilic group like
sulfonamido group at para position of 3-phenylpropyl motif of 1-(α-alkyl-α-phenylmethyl-3-(3-
phenylpropyl)urea improves the activity as well as enantiomeric selectivity. All these ureas are
proved to be specific inhibitor of sEH without inhibition against mEH.

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Key words: EETs, soluble epoxide hydrolase, inhibitors, urea derivatives, enantioselectivity .
*Corresponding author: jungshh@cnu.ac.kr. Tel.; +82 42 821 5939; Fax +82 42 823 6566;
Present address: College of Pharmacy and Institute of Drug Research and development,
Chungnam National University, Daejeon 34134, Korea
1. Introduction
In humans, soluble epoxide hydrolase (sEH) plays an important role in the metabolism of
endogenous chemical mediators such as epoxides of arachidonic acid [1], linoleic acid [2] and
other lipids [3]. sEH hydrolyzes epoxides of arachidonic acid (epoxyeicosatrienoic acids, EETs)
to the corresponding diols (dihydroxyeicosatrienoic acids, DHETs) [4]. EETs are known as
effective modulators of blood pressure and inflammation [4]. The cardiovascular protective
actions of sEH inhibitors were first described in animal models of hypertension in 2000 [5,6].
Since then the evidences have been accumulated to demonstrate that sEH inhibitors lower blood
pressure in several animal models of hypertension, such as spontaneously hypertensive rats as
well as angiotensin II-induced hypertensive models [7-9]. In these regards, the sEH enzyme has
gained considerable attention as a therapeutic target for cardiovascular diseases over the last few
years [10-13] and thus the inhibition of sEH provides the novel strategy for the treatment of
hypertension and vascular inflammation [5-7,14].
In addition, the sEH enzyme regulates many other metabolic pathways and thus the inhibitors
display anti-fibrotic [15], anti-platelet aggregation properties [16,17], and could be an alternative
to treat diabetic neuropathy [18] and inflammation-induced carcinogenesis [19,20]. Therefore,
these also prove sEH as a potentially important pharmaceutical target. Several groups have

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reported the synthesis and evaluation of sEH inhibitors with diverse pharmacophoric scaffolds
with potency varying from nanomolar to micromolar ranges (Fig. 1) [14, 21-26].
Figure 1. Representative examples of sEH inhibitors
Among them, the 1,3-disubstituted ureas are the most potent pharmacophore for the inhibition of
sEH [27-30]. The binding mode (Fig. 2) of urea pharmacophore with human sEH was
determined by X-ray crystallography and mimics the transition state of epoxide ring opening
catalyzed by the enzyme (Fig. 2A), which shows the formation of tight hydrogen bonds with the
active site residue Asp333 as shown in Fig. 2B. [31-33].

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Figure 2. A) Transition state of epoxide ring opening catalyzed by sEH, B) Binding mode of
urea which mimic the transition state31
Hydrolysis of epoxides or EETs by sEH showed diverse features depending on structures of
substrates. The sEH catalyzed hydrolysis of substituted racemic epoxides gives a mixture of
unreacted epoxides and the corresponding diols as shown in Fig. 3 [34]. For example, the methyl
and butyl substituent in the racemic epoxide gives 16 % ee of unreacted 2R,3S epoxide and 10 %
ee of the 2R,3R diol, whereas the hydroxyethyl and pentyl substituted epoxide gives >98 % ee of
unreacted 3R,4S epoxide and >98 % of 2R,3R diol, which demonstrate that the sEH allow
stereoselective ring opening of the epoxide and the stereochemistry of the products depends upon
the substitution pattern on the epoxide ring (Fig. 3A). The arachidonic acid metabolism by
CYP450 gives four regio isomers namely 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET, each
having a mixture of S/R- and R/S-enantiomers with different ratio. Among the regio isomers, the
14,15-EET is the most preferred substrate for the hydrolysis catalyzed by sEH, whereas the 5,6-
EET is the least preferred one. The 8,9-EET and 11,12-EET being the moderately preferred
substrates [35]. The sEH catalyzed enantioselective hydrolysis of 14(R),15(S)-EET on rabbit
liver or lung cytosol gives 82 % 14(R),15(R) diol whereas the same hydrolysis of 14(S),15(R)-
EET gives only 35 % 14(R),15(R) diol, which also illustrate that stereochemistry of substrate
influences the formation of stereoisomers of diol products (Fig. 3B). Moreover the hydration rate
of 14(R),15(S)-EET (7994 nmol/mg protein/min) is four times faster than that of 14(S),15(R)-
EET (1992 nmol/mg protein/min) [35].

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Figure 3. A) Stereoselective hydrolysis of epoxides by sEH, B) Stereoselective hydrolysis of
EET by sEH
All the above points confirm that stereochemistry and substituents of epoxide ring influence the
rate and the ratio of formation of the stereoisomers of the diol products by sEH. Therefore we
inferred that stereoisomers of sEH inhibitor might differentiate the inhibitory activity and

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accordingly investigated the possibility of different inhibitory activity of enantiomers of
di(phenylalkyl)urea scaffold containing a stereocenter at α-position as shown in Fig. 4.
Figure 4. Designed urea 8
2. Chemistry
The urea derivatives 8a-j were prepared by the reaction of optical isomers of amines 9
(commercially available) and 3-phenylpropyl isocyanate 10 (Scheme 1) in high yields (80 -
90 %). Ureas 8k(S) and 8k(R) with sulfonamide substitution were prepared as illustrated in
Scheme 2. Protection of 3-phenylpropylamine 11 was initially conducted with trifluoroacetic
anhydride to give 12. The following chlorosulfonation reaction of 12 generated 13, which was
converted by the treatment with ammonia gas to sulfonamide 14. Deprotection of 14 by the
reaction with K2CO3 in methanol/water gave sulfamoyl substituted amine 15 which was used for
the preparation of ureas 8k(S) and 8k(R). The R- or S- isomer of 1-phenylethylamine was treated
with triphosgene in the presence of N,N-diisopropylethylamine for 0.5 h followed by the addition
of the sulfamoyl substituted amine 15 to obtain the ureas 8k(S) and 8k(R).
Scheme 1. Synthesis of urea derivatives 8a-j
Reagents and conditions: a) acetonitrile, RT, 8 h

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Scheme 2. Synthesis of sulfonamide substituted ureas 8k(S) and 8k(R).
Reagents and conditions: a) (CF3CO)2O, TEA, MC, 3 h; b) ClSO3H, MC, 0 °C-RT, 3 h; c) NH3
gas, 0.5 h; d) K2CO3, MeOH/H2O, 10 h; e) (R)- or (S)-1-phenylethylamine, triphosgene, DIPEA,
THF, 0 °C-RT, 8 h
3. Results and Discussion
The in vitro sEH inhibition activity was determined using 14,15-EET as a substrate and the
results are summarized in Table 1. The assay results for the inhibition of mEH enzyme are also
included in Table 1. The assay protocols are described in the experimental section.
Table 1. Synthesized compounds 8 with sEH and mEH inhibitory activity
Comp. No.
(configuration)
sEH inhibition
IC50 (uM)
mEH
activity at 10 uM
(%)
R3
3.26
H
(2.53 to 4.20) †
105 + 0.8
8a

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0.044
H
(0.037 to 0.051) †
84.6 + 0.7
8b(S)
1.73
H
H
(1.44 to 2.07) †
102 + 0.0
107 + 0.8
8b(R)
8c(S)
19.99
(8.69 to 45.98) †
2.74
H
(2.05 to 3.66) †
102 + 3.7
8c(R)
0.182
H
H
H
H
H
(0.151 to 0.219) †
99.4 + 2.3
103 + 0.0
106 + 2.7
103 + 1.5
102 + 1.1
8d(S)
8d(R)
8e(S)
8e(R)
8f(S)
0.302
(0.196 to 0.465) †
0.036
(0.022 to 0.057) †
4.50
(3.26 to 6.21) †
0.043
(0.034 to 0.054) †
0.364
H
H
(0.327 to 0.404) †
98.7 + 2.2
112 + 6.9
8f(R)
0.065
(0.051 to 0.083) †
8g(S)
8g(R)
0.022
H
H
(0.017 to 0.028) †
110 + 3.5
106 + 0.9
0.013
(0.010 to 0.016) †
8h(S)
8h(R)
8i(S)
0.013
H
(0.010 to 0.017) †
106 + 0.3
99.8 + 1.2
0.332
(0.253 to 0.428) †

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1.51
(1.15 to 1.98) †
101 + 0.2
101 + 1.6
8i(R)
8j(S)
0.063
H
H
(0.055 to 0.072) †
0.650
(0.053 to 0.797) †
102 + 0.5
8j(RS)
0.022
SO2NH2
SO2NH2
(0.019 to 0.025) †
99.9 + 4.2
101 + 1.1
8k(S)
8k(R)
2.03
(1.57 to 2.63) †
0.059
(0.042 to 0.083)
N.D.
AUDA
N.D.
50.9 + 3.7
Valpromide
† indicates 95% confidence interval of the IC50. mEH activity is expressed as the percentage of activity, relative to a
control samples containing no inhibitor (100%).
Initially the inhibitory activity (IC50 = 3.26 ꢀM) of 1-benzyl-3-(3-phenylpropyl)urea (8a) against
sEH was discovered in our laboratory. Compound 8a is linear type of 1,3-dialkylsubstituted urea
and thus ideal for the introduction of stereocenter at benzylic position (α-position) for the
examination of selective inhibition of enantiomers of urea analogs. The introduction of methyl
substituent at α-position showed a good enantioselectivity. The (S)-1-(1-phenylethyl)-3-(3-
phenylpropyl)urea (8b(S), IC50 = 0.044 ꢀM) showed 80 fold more potent than the parent benzyl
analog 8a and 40 fold more active than its R-isomer 8b(R) (IC50 = 1.73 ꢀM). As shown in Fig. 5,
the molecular docking analysis suggests that, compared to the parent benzyl analog 8a, the α-
methyl substituent of the 8b(S) provide additional hydrophobic interaction to Try336, thus 80
fold more active. This additional binding increases the activity.

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B
A
Y383
Y383
F267
Q384
W336
Q384
W336
F267
Y466
Y466
L408
L408
W524
W524
M339
M339
D335
D335
T360
T360
W525
W525
Figure 5. Binding model prediction data of
A) compound 8a, B) compound 8b(S). The yellow
dashed line denoted hydrogen bond interactions.
In the docking model of the enantiomers 8b(S) and 8b(R) to sEH, the
α-methyl substituent of the
8b(R) is located to Glu384 (Fig. 6B) whereas the same of the -isomer 8b(S) is located to
S
Try336 (Fig. 6A) and therefore experiences the stronger hydrophobic interaction, which results
in 40 fold stronger activity.

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A
B
Y383
Y383
F267
Q384
W336
Q384
F267
Y466
Y466
W336
L408
L408
W524
M339
M339
W524
D335
D335
T360
T360
W525
W525
C
Y383
Q384
F267
Y466
W336
L408
W524
M339
D335
T360
W525
Figure 6. Binding model prediction data of
A) 8b(S), B) 8b(R), C) Overlay of the predicted
docking model of 8b(S) and 8b(R). The yellow dashed line denoted hydrogen bond interactions.
Encouraged by these results, our next effort was to introduce additional hydrogen bonding
property (HBA and HBD). Therefore, we introduced hydroxymethyl function at the
shown in 8c(S) and 8c(R). Unfortunately this additional hydrogen bonding function reduced the
inhibitory activity compared to 8b(S). However, the -isomer 8c(R) (IC50 = 2.74 M) showed 10
fold more active than its -isomer (8c(S), IC50 = 19.99 M). Although 8c(R) is designated as R-
α-position as
R
ꢀ
S
ꢀ

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isomer because of priority rule, the absolute configuration of 8c(R) is the same as that of 8b(S)
.
These results indicated that the absolute configuration at the α-position as shown in 8b(S) is
important for the selectivity between the enantiomers of these urea scaffolds in their inhibition of
sEH.
In order to find the importance of phenyl ring at
ring which decreased the selectivity as well as activity. 8d(S) (IC50 = 0.182
fold more activity compared to 8d(R) (IC50 = 0.302 M). From the binding model prediction
α-position of 8b, it was changed to cyclohexyl
ꢀ
M) showed only 1.5
ꢀ
data, it is evident that, despite the phenyl and cyclohexyl rings are oriented towards the Met339,
the planar phenyl group of 8b(S) can bind more stably when compared to the bulky cyclohexyl
group of 8d(S) (Fig. 7).
Figure 7. Binding model prediction data of
A) compound 8b(S), B) compound 8d(S). The
yellow dashed line denoted hydrogen bond interactions.
In the next set of experiment, we focused on identification of the optimum size of the two groups
at the -position. To determine the effect of increment of size of alkyl function at -position,
α
α

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methyl group of 8b was changed to ethyl as shown in 8e. The
showed the 125 fold more potent activity than its -isomer 8e(R) (IC50 = 4.50
enhanced activity compared to 8b(S). These results indicated that the increase in size of alkyl
S
-isomer 8e(S) (IC50 = 0.036
ꢀM)
R
ꢀM) and the
function at
activity of
α
-position form methyl group to ethyl group improved the selectivity as well as the
-isomer. However, the increase in the size of the phenyl group with naphthyl ring
M) and
S
displayed a poor selectivity, though retained the activity as shown in 8f(S) (IC50 = 0.043
8f(R) (IC50 = 0.364 M). Thus the phenyl group is the optimum size for this position.
Next, to restrict the free rotation between the two -groups, the -group and the phenyl ring were
rigidified with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety as shown in 8g and 8h
Surprisingly, the -isomer 8g(S) (IC50 = 0.065 M) of the indan-1-yl analog showed 3 times less
activity compared to its -isomer 8g(R) (IC50 = 0.022 M) whereas the -isomer of the
tetrahydronaphthalen-1-yl analog 8h(S) (IC50 = 0.013 M) showed nil activity difference
compared to its -isomer 8h(R) (IC50 = 0.013 M). These results demonstrated that the
rigidification of the -position and the phenyl ring abolished the selectivity of these enantiomers
for the inhibition of sEH. Unlike open chain analogs such as 8b(R) or 8e(R), the activity of
isomer 8h(R) was remarkably enhanced to the activity level of its -isomer 8h(S). The reason for
ꢀ
ꢀ
α
α
.
S
ꢀ
R
ꢀ
S
ꢀ
R
ꢀ
α
R-
S
the tetrahydronaphthalen-1-yl analog to show nil selectivity between the enantiomers can be
explained from the molecular docking analysis as the binding mode of the two enantiomers is
quite similar unlike the open chain isomers (Fig.8). On the other hand, the rigidification of the
position and the urea nitrogen as shown in 8i also demonstrated poor selectivity and weak
activity. The -isomer 8i(S) (IC50 = 0.332 M) showed only 5 fold increase in activity compared
to its -isomer 8i(R) (IC50 = 1.51 M). These above results suggest that the rigidification on
either side of the -position has no advantage for the selectivity.
α-
S
ꢀ
R
ꢀ
α

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A
B
Y383
Y383
F267
Q384
W336
Q384
F267
Y466
Y466
W336
L408
L408
W524
W524
M339
M339
D335
D335
T360
T360
W525
W525
C
Y383
Q384
F267
Y466
W336
L408
W524
M339
D335
T360
W525
Figure 8. Binding model prediction of the six membered bicyclic enantiomers
A) 8h(S), B)
8h(R), C) Overlay of the predicted docking model of 8h(S) and 8h(R)
.
Next objective of our work was to study the effect of two small alkyl α-groups. Accordingly, the
methyl and ethyl substituents at the
and 8j(RS). The -isomer 8j(S) (IC50 = 0.063
8b(S) and 20 fold more activity when compared to the
calculated from the IC50 of -isomer 8j(S) and IC50 (0.650
α
-position were introduced as shown in compounds 8j(S)
M) showed nearly the same level of activity as
-isomer 8j(R) (IC50 = 1.24 M:
M) of the racemic compound
S
ꢀ
R
ꢀ
S
ꢀ

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8j(RS)). Although enantiomeric selectivity of
S-isomer 8j(S) was reduced, these results opened
the high possibility of replacement of phenyl of 8b(S) with unbranched alkyl group for
improvement of the selectivity as well as the activity.
Finally, to improve the water solubility, the hydrophilic sulfonamido group was introduced on
the para position of phenyl group on propyl linkage of 8b as shown in 8k. The
(IC50 = 0.022 M) showed 100 fold more activity compared to the -isomer 8k(R) (IC50 = 2.03
M) and more potent activity than 8b(S). Thus hydrophilic group enhanced the selectivity as
well as the activity.
S-isomer 8k(S)
ꢀ
R
ꢀ
Since microsomal epoxide hydrolase (mEH) located in liver is essential for metabolic process,
the selective inhibitor for sEH is necessary for the development. Thus the inhibitory effect of all
compounds
8
against mEH were measured as shown in Table 1 and showed no activity at 10
were proved as the specific inhibitors for sEH.
µM.
Therefore urea analogs
8
Figure 9. SAR analysis of the novel urea derivatives

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4. Conclusion
In conclusion, the enantioselectivity in urea scaffold for the inhibition of sEH was first time
investigated. Structure activity relationship study of the enantiomers demonstrated that the
absolute configuration of the 1-(
crucial for the selectivity as well as activity. The structure activity relationship is depicted in
Figure 9. The selectivity of enantiomers of ureas showed a wide range difference up to 125
fold with the low IC50 value up to 13 nM. The -configuration with planar phenyl and small alkyl
groups at -position of 1-( -alkyl- -phenylmethyl)-3-(3-phenylpropyl)urea is crucial for the
activity and selectivity. However, restriction of the free rotation of two -groups with indan-1-yl
α-alkyl-α-phenylmethyl)-3-(3-phenylpropyl)urea scaffold is
8
S
α
α
α
α
or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity between the enantiomers,
despite the increase in activity of 8h(S) and 8h(R) up to 13 nM. The hydrophilic function like
sulfonamido group at para position of 3-phenylpropyl of 1-(
phenylpropyl)urea increases the activity as well as enantiomeric selectivity. All the synthesized
ureas were proved to be specific inhibitors of sEH without inhibition against mEH. Therefore
the selectivity of the sEH inhibition between the two enantiomers of 1-( -alkyl- -phenylmethyl-
α-alkyl-α-phenylmethyl-3-(3-
8
α
α
3-(3-phenylpropyl)urea provides new structural insights toward the binding site of soluble
epoxide hydrolase that will lead to the discovery of novel and potent inhibitors.
5. Experimental section
5.1 Chemistry
Melting points were determined on Electro thermal 1A 9100 MK2 apparatus and are
uncorrected. All commercial chemicals were used as obtained and all solvents were purified by
the standard procedures [36] prior to use. All the chiral amines purchased were optically pure
(>98 % ee). Thin layer chromatography was performed on E Merck silica gel GF-254 pre-coated

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plates and the identification was done with UV light and colorization with spray 10 %
phosphomolybdic acid followed by heating. Flash column chromatography was performed with
E Merck silica gel (230-400 mesh). Infrared spectrum was recorded by using sample as such on
FT-IR spectrum with Nicolet-380 models. NMR spectra were measured against the peak of
tetramethylsilane by JEOL, JNM-AL-400 (Alice) 400 FT-NMR spectrometer. High resolution
mass spectra (HRMS) were measured in ESI ionization using AB Sciex TripleTOF 5600 LCMS
instrument.
5.1.1. General synthetic procedure for the preparation of urea derivatives 8a-j: To the
corresponding amine
9 (0.710 mmol) in acetonitrile (5 mL), 3-phenylpropyl isocyanate 10 (0.645
mmol) was added and allowed to stir for 8 h at ambient temperature. The resulting mixture was
portioned between water and ethyl acetate. The organic layer was dried over anhydrous Na2SO4
and evaporated under reduced pressure. The crude mixture was subjected to column
chromatography to obtain the pure compounds.
5.1.1.1. 1-Benzyl-3-(3-phenylpropyl)urea (8a). Yield 89 %; White solid; mp 96-98 °C; IR (neat):
1
3308, 2868, 1620, 1559, 1494, 1453 cm-1; H-NMR (CDCl3)
δ 1.76-1.84 (m, 2H), 2.61 (t, J =
7.80 Hz, 2H), 3.17-3.21 (m, 2H), 4.33 (d,
J = 6.00 Hz, 2H), 4.39 (bs, 1H), 4.66 (bs, 1H), 7.13-
7.19 (m, 3H), 7.24-7.34 (m, 7H); 13C-NMR (CDCl3)
δ
31.70, 33.02, 39.97, 44.36, 125.94,
127.28, 127.38, 128.38, 128.44, 128.84, 139.32, 141.62, 158.44; HRMS Calcd for C17H20N2O
m/z [M+H] 269.1654, found 268.1682.
5.1.1.2. (S)-1-(1-Phenylethyl)-3-(3-phenylpropyl)urea (8b(S)).Yield 86 %; White solid; mp 78-
80 °C; IR (neat): 3314, 3025, 2867, 1622, 1560, 1493, 1452 cm-1; 1H-NMR (CDCl3)
δ
1.42 (d,
J
= 7.20 Hz, 3H), 1.67-1.74 (m, 2H), 2.51 (t,
J = 7.80 Hz, 2H), 3.06-3.18 (m, 2H), 4.44 (bs, 1H),
4.69-4.74 (m, 1H), 4.83 (d,
J
= 7.00 Hz, 1H), 7.07-7.34 (m, 10H); 13C-NMR (CDCl3)
δ
23.51,

ACCEPTED MANUSCRIPT
31.41, 32.90, 40.05, 50.81, 125.81, 125.95, 127.58, 128.32, 128.42, 128.90, 141.31, 143.57,
157.90; HRMS Calcd for C18H22N2O m/z [M+H] 283.1810, found 283.1840
5.1.1.3. (R)-1-(1-Phenylethyl)-3-(3-phenylpropyl)urea (8b(R)).Yield 88 %; White solid; mp 78-
80 °C; IR (neat): 3314, 3025, 2869, 1622, 1560, 1493, 1452 cm-1; 1H-NMR (CDCl3)
δ
1.45 (d,
J
= 7.20 Hz, 3H), 1.70-1.78 (m, 2H), 2.51 (t,
J = 7.80 Hz, 2H), 3.10-3.17 (m, 2H), 4.40-4.45 (bs,
1H), 4.64-4.71 (m, 1H), 4.83 (d,
J
= 7.00 Hz, 1H), 7.07-7.34 (m, 10H); 13C-NMR (CDCl3)
δ
23.50, 31.35, 32.88, 40.10, 50.90, 125.81, 125.97, 127.66, 128.31, 128.43, 128.93, 141.25,
143.38, 157.92; HRMS Calcd for C18H22N2O m/z [M+H] 283.1810, found 283.1837
5.1.1.4. (S)-1-(2-Hydroxy-1-phenylethyl)-3-(3-phenylpropyl)urea (8c(S)). Yield 84 %; Pale white
1
solid; mp 104-106 °C; IR (neat): 3415, 3310, 3021, 2924, 1625, 1563, 1489, 1450 cm-1; H-
NMR (DMSO-d6
)
δ
1.59-1.69 (m, 2H), 2.55 (t,
J
= 7.50 Hz, 2H), 2.94-3.01 (m, 2H), 3.52-3.57
= 7.60 Hz, 1H), 6.30 (d, = 7.80
31.82, 32.45, 38.69, 55.21, 65.22, 125.67,
(m, 2H), 4.62-4.69 (m, 1H), 4.85 (t,
J
= 7.20 Hz, 1H), 6.08 (t,
J
J
Hz, 1H), 7.14-7.32 (m, 10H); 13C-NMR (DMSO-d6
)
δ
126.43, 126.72, 127.91, 128.25, 128.26, 141.79, 142.64, 157.66. HRMS Calcd for C18H22N2O2
m/z [M+H] 299.1760, found 299.1791
5.1.1.5. (R)-1-(2-Hydroxy-1-phenylethyl)-3-(3-phenylpropyl)urea (8c(R)). Yield 82 %; Pale
white solid; mp 105-107 °C; IR (neat): 3415, 3312, 3021, 2924, 1625, 1561, 1489, 1450 cm-1;
1H-NMR (DMSO-d6
)
δ
1.58-1.69 (m, 2H), 2.55 (t,
J
= 7.50 Hz, 2H), 2.95-3.02 (m, 2H), 3.52-
= 7.20 Hz, 1H), 6.06-6.10 (m, 1H), 6.30 (d, = 7.80
31.82, 32.45, 38.69, 55.21, 65.22, 125.65,
3.57 (m, 2H), 4.62-4.68 (m, 1H), 4.85 (t,
J
J
Hz, 1H), 7.14-7.32 (m, 10H); 13C-NMR (DMSO-d6
)
δ
126.42, 126.71, 127.90, 128.24, 128.25, 141.78, 142.63, 157.65; HRMS Calcd for C18H22N2O2
m/z [M+H] 299.1760, found 299.1790

ACCEPTED MANUSCRIPT
5.1.1.6. (S)-1-(1-Cyclohexylethyl)-3-(3-phenylpropyl)urea (8d(S)). Yield 80 %; White solid; mp
1
106-108 °C; IR (neat): 3331, 3020, 2866, 1625, 1564, 1498, 1448 cm-1; H-NMR (300 MHz,
CDCl3)
δ 0.91-1.02 (m, 2H), 1.06 (d, J = 6.80 Hz, 3H), 1.12-1.30 (m, 4H), 1.63-1.76 (m, 5H),
1.79-1.89 (m, 2H), 2.66 (t,
J
= 7.60 Hz, 2H), 3.19 (t,
J
= 7.20 Hz, 2H), 3.50-3.58 (m, 1H), 7.17-
18.31, 26.09, 26.31, 28.87, 31.53, 33.08,
7.21 (m, 3H), 7.26-7.31 (m, 2H); 13C-NMR (CDCl3)
δ
40.22, 43.41, 50.82, 126.06, 128.43, 128.54, 141.49, 158.13. HRMS Calcd for C18H28N2O m/z
[M+H] 289.2280, found 289.2311
5.1.1.7. (R)-1-(1-Cyclohexylethyl)-3-(3-phenylpropyl)urea (8d(R)). Yield 82 %; White solid; mp
1
106-108 °C; IR (neat): 3330, 3020, 2866, 1625, 1563, 1498, 1448 cm-1; H-NMR (CDCl3)
δ
0.91-1.04 (m, 2H), 1.06 (d,
J = 6.80 Hz, 3H), 1.10-1.29 (m, 4H), 1.64-1.67 (m, 2H), 1.72-1.75
(m, 3H), 1.81-1.88 (m, 2H), 2.66 (t,
J
= 7.60 Hz, 2H), 3.19 (t,
J
= 7.20 Hz, 2H), 3.52-3.55 (m,
18.32, 26.11, 26.33, 28.88,
1H), 7.17-7.21 (m, 3H), 7.27-7.31 (m, 2H); 13C-NMR (CDCl3)
δ
31.55, 33.10, 40.24, 43.43, 50.84, 126.08, 128.45, 128.55, 141.50, 158.15; HRMS Calcd for
C18H28N2O m/z [M+H] 289.2280, found 289.2311
5.1.1.8. (S)-1-(1-Phenylpropyl)-3-(3-phenylpropyl)urea (8e(S)). Yield 85 %; White solid; mp
100-102 °C; IR (neat): 3318, 3019, 2878, 1621, 1565, 1492, 1451 cm-1; 1H-NMR (CDCl3)
(t, = 7.20 Hz, 3H), 1.66-1.74 (m, 2H), 1.75-1.80 (m, 2H), 2.48 (t, = 7.50 Hz, 2H), 3.04-3.21
δ 0.89
J
J
(m, 2H), 4.38 (t,
J = 7.20 Hz, 1H), 4.56-4.97 (bs, 2H), 7.05-7.07 (m, 2H), 7.14-7.23 (m, 3H),
7.27-7.37 (m, 5H); 13C-NMR (CDCl3)
δ
10.57, 30.48, 31.38, 32.87, 40.06, 57.07, 125.95,
126.36, 127.68, 128.31, 128.42, 128.86, 141.30, 142.18, 158.13; HRMS Calcd for C19H24N2O
m/z [M+H] 297.1967, found 297.1993
5.1.1.9. (R)-1-(1-Phenylpropyl)-3-(3-phenylpropyl)urea (8e(R)). Yield 80 %; White solid; mp
99-101 °C; IR (neat): 3320, 3019, 2880, 1621, 1566, 1492, 1451 cm-1; 1H-NMR (CDCl3)
δ 0.88

ACCEPTED MANUSCRIPT
(t,
J
= 7.20 Hz, 3H), 1.67-1.73 (m, 2H), 1.74-1.80 (m, 2H), 2.48 (t, J = 7.50 Hz, 2H), 3.04-3.19
(m, 2H), 4.41 (t,
J = 7.20 Hz, 1H), 4.60-5.20 (bs, 2H), 7.06-7.08 (m, 2H), 7.15-7.25 (m, 3H),
7.27-7.36 (m, 5H); 13C-NMR (CDCl3)
δ
10.52, 30.43, 31.39, 32.81, 39.96, 56.98, 125.99,
126.41, 127.69, 128.37, 128.42, 128.86, 141.40, 142.32, 158.24; HRMS Calcd for C19H24N2O
m/z [M+H] 297.1967, found 297.1994
5.1.1.10. (S)-1-(-Naphthalen-1-yl)ethyl)-3-(3-phenylpropyl)urea (8f(S)). Yield 88 %; White
1
powder; mp 137-139 °C; IR (neat): 3326, 3011, 2881, 1623, 1565, 1493, 1450 cm-1; H-NMR
(CDCl3)
δ 1.61 (d, J = 6.90 Hz, 3H), 1.64-1.73 (m, 2H), 2.41-2.49 (m, 2H), 3.05-3.14 (m, 2H),
5.52-5.58 (m, 1H), 6.98-7.00 (m, 2H), 7.11-7.22 (m, 3H), 7.43-7.57 (m, 4H), 7.78 (d,
J = 8.10
13
Hz, 1H), 7.88 (dd,
J
= 7.80 Hz, 1.80 Hz, 1H), 8.13 (d,
J
= 8.40 Hz, 1H); C-NMR (CDCl3)
δ
22.33, 31.24, 32.73, 40.02, 46.94, 122.58, 122.76, 125.51, 125.92, 126.65, 128.24, 128.39,
128.42, 129.04, 129.05, 130.54, 134.01, 138.21, 141.11, 157.80; HRMS Calcd for C22H24N2O
m/z [M+H] 333.1967, found 333.2000
5.1.1.11. (R)-1-(Naphthalen-1-yl)ethyl)-3-(3-phenylpropyl)urea (8f(R)). Yield 86 %; White
1
powder; mp 137-139 °C; IR (neat): 3325, 3010, 2886, 1623, 1561, 1495, 1450 cm-1; H-NMR
(CDCl3)
δ 1.61 (d, J = 6.90 Hz, 3H), 1.64-1.71 (m, 2H), 2.41-2.47 (m, 2H), 3.03-3.11 (m, 2H),
4.72-4.90 (bs, 2H), 5.49-5.53 (m, 1H), 6.96-6.98 (m, 2H), 7.12-7.21 (m, 3H), 7.44-7.58 (m, 4H),
13
7.79 (d,
J
= 8.10 Hz, 1H), 7.89 (dd,
J
= 7.80 Hz, 1.80 Hz, 1H), 8.09 (d,
J
= 8.40 Hz, 1H); C-
NMR (CDCl3)
δ
22.29, 31.23, 32.68, 39.91, 46.77, 122.58, 122.84, 125.56, 125.96, 126.65,
128.31, 128.40, 128.43, 129.06, 129.07, 130.54, 134.03, 138.57, 141.24, 157.88; HRMS Calcd
for C22H24N2O m/z [M+H] 333.1967, found 333.1997
5.1.1.12. (S)-1-(2,3-Dihydro-1H-inden-1-yl)-3-(3-phenylpropyl)urea (8g(S)). Yield 85 %; White
1
solid; mp 148-150 °C; IR (neat): 3310, 2936, 1620, 1571, 1523, 1454, 956 cm-1; H-NMR

ACCEPTED MANUSCRIPT
(CDCl3)
δ
1.72-1.79 (m, 1H), 1.81-1.89 (m, 2H), 2.51-2.58 (m, 1H), 2.65 (t, J = 7.80 Hz, 2H),
2.78-2.86 (m, 1H), 2.91-2.98 (m, 1H), 3.20 (t,
J
= 7.20 Hz, 2H), 4.72-4.91 (bs, 2H), 5.21 (t,
J =
13
7.60 Hz, 1H), 7.16-7.30 (m, 9H); C-NMR (CDCl3) 29.97, 31.36, 32.99, 34.37, 40.25, 55.91,
δ
124.00, 124.88, 126.08, 126.81, 128.04, 128.40, 128.54, 141.32, 143.23, 143.21, 158.29; HRMS
Calcd for C19H22N2O m/z [M+H] 295.1810, found 295.1841
5.1.1.13. (R)-1-(2,3-Dihydro-1H-inden-1-yl)-3-(3-phenylpropyl)urea (8g(R)). Yield 86 %; White
1
solid; mp 148-150 °C; IR (neat): 3312, 2936, 1620, 1571, 1523, 1455, 956 cm-1; H-NMR (300
MHz, CDCl3)
δ
1.72-1.79 (m, 1H), 1.81-1.88 (m, 2H), 2.50-2.58 (m, 1H), 2.65 (t,
J
= 7.80 Hz,
2H), 2.78-2.86 (m, 1H), 2.91-2.98 (m, 1H), 3.20 (t,
= 7.60 Hz, 1H), , 7.15-7.29 (m, 9H); 13C-NMR (CDCl3)
J = 7.20 Hz, 2H), 4.40-4.50 (bs, 2H), 5.21 (t,
J
δ 29.96, 31.32, 32.97, 34.33, 40.26,
55.93, 123.99, 124.88, 126.09, 126.82, 128.06, 128.40, 128.54, 141.29, 143.12, 143.21, 158.37;
HRMS Calcd for C19H22N2O m/z [M+H] 295.1810, found 295.1842
5.1.1.14. (S)-1-(3-Phenylpropyl)-3-(1,2,3,4-tetrahydronaphthalen-1-yl)urea (8h(S)). Yield 84 %;
1
White solid; mp 133-135 °C; IR (neat): 3315, 2998, 2940, 1621, 1571, 1498, 1450 cm-1; H-
NMR (CDCl3)
2H), 3.18 (t,
NMR (CDCl3)
δ
1.74-1.89 (m, 6H), 1.97-2.04 (m, 1H), 2.65 (t,
J = 7.80 Hz, 2H), 2.73-2.76 (m,
J
= 7.20 Hz, 2H), 4.50-4.60 (bs, 1H), 4.90-4.95 (m, 1H), 7.06-7.31 (m, 9H); 13C-
δ
19.92, 29.19, 30.68, 31.44, 33.05, 40.30, 48.89, 126.03, 126.25, 127.28, 128.34,
128.48, 128.61, 129.12, 136.94, 137.50, 141.23, 157.80; HRMS Calcd for C20H24N2O m/z
[M+H] 309.1967, found 309.2000
5.1.1.15. (R)-1-(3-Phenylpropyl)-3-(1,2,3,4-tetrahydronaphthalen-1-yl)urea (8h(R)). Yield 81
%; White solid; mp 133-135 °C; IR (neat): 3315, 2999, 2942, 1621, 1571, 1498, 1450 cm-1; 1H-
NMR (CDCl3)
2H), 3.19 (t,
δ
1.74-1.88 (m, 6H), 1.99-2.04 (m, 1H), 2.65 (t,
J = 7.80 Hz, 2H), 2.70-2.81 (m,
J
= 7.20 Hz, 2H), 4.60-4.75 (bs, 1H), 4.92-4.94 (m, 1H), 7.07-7.31 (m, 9H); 13C-

ACCEPTED MANUSCRIPT
NMR (CDCl3)
δ
19.92, 29.19, 30.68, 31.44, 33.05, 40.30, 48.89, 126.03, 126.25, 127.28, 128.34,
128.48, 128.61, 129.12, 136.94, 137.50, 141.23, 157.80; HRMS Calcd for C20H24N2O m/z
[M+H] 309.1967, found 309.1999
5.1.1.16. (S)-2-Phenyl-N-(3-phenylpropyl)pyrrolidine-1-carboxamide (8i(S)). Yield 82 %;
Yellow oil; IR (neat): 3335, 3025, 2864, 1628, 1527, 1494, 1346 cm-1; 1H-NMR (CDCl3)
δ 1.60-
1.67 (m, 2H), 1.82-194 (m, 3H), 2.35-2.42 (m, 3H), 3.04-3.11 (m, 1H), 3.15-3.22 (m, 1H), 3.63-
3.72 (m, 2H), 4.01 (bs, 1H), 4.64-4.67 (m, 1H), 7.01 (d,
13C-NMR (CDCl3)
J = 7.60 Hz, 2H), 7.13-7.37 (m, 8H);
δ
23.02, 31.58, 32.84, 36.78, 39.87, 47.27, 60.84, 125.74, 125.78, 127.57,
128.33, 128.34, 128.97, 141.76, 143.33, 157.12; HRMS Calcd for C20H24N2O m/z [M+H]
309.1967, found 309.1996
5.1.1.17. (R)-2-Phenyl-N-(3-phenylpropyl)pyrrolidine-1-carboxamide (8i(R)). Yield 86 %;
Yellow oil; IR (neat): 3335, 3025, 2863, 1628, 1528, 1494, 1346 cm-1; 1H-NMR (CDCl3)
δ 1.60-
1.68 (m, 2H), 1.83-1.94 (m, 3H), 2.34-2.43 (m, 3H), 3.03-3.12 (m, 1H), 3.15-3.24 (m, 1H), 3.65-
3.72 (m, 2H), 4.64-4.68 (m, 1H), 7.00-7.02 (m, 2H), 7.12-7.29 (m, 6H), 7.33-7.39 (m, 2H). 13C-
NMR (CDCl3) δ 23.11, 31.58, 32.90, 36.74, 40.10, 47.58, 61.18, 125.76, 127.65, 128.30, 128.31,
128.97, 141.63, 142.85, 156.96; HRMS Calcd for C20H24N2O m/z [M+H] 309.1967, found
309.1995
5.1.1.18. (S)-1-sec-Butyl-3-(3-phenylpropyl)urea (8j(S)). Yield 83 %; White solid; mp: 86-88 °C;
IR (neat): 3331, 3020, 2866, 1625, 1564, 1498, 1448, 752, 687 cm-1; 1H NMR (CDCl3)
δ
0.90 (t,
= 6.60 Hz, 3H), 1.39-1.49 (m, 2H), 1.79-1.89 (m, 2H), 2.66 (t,
J = 7.20 Hz, 2H), 3.57-3.64 (m, 1H), 4.40-4.65 (bs, 2H), 7.16-7.23 (m,
J
= 7.20 Hz, 3H), 1.10 (d,
J
J =
7.80 Hz, 2H), 3.19 (t,
13
3H), 7.26-7.32 (m, 2H); C NMR (75 MHz, CDCl3)
δ 10.23, 20.72, 29.95, 31.33, 33.09, 40.45,

ACCEPTED MANUSCRIPT
48.48, 126.06, 128.36, 128.51, 141.11, 158.29; HRMS Calcd for C14H22N2O m/z [M+H]
235.1810, found 235.1835
5.1.1.19. 1-sec-Butyl-3-(3-phenylpropyl)urea (8j(RS)). Yield 80 %; White solid; mp: 86-88 °C;
IR (neat): 3330, 3020, 2866, 1625, 1564, 1498, 1449, 752, 687 cm-1; 1H NMR (CDCl3)
δ 0.90 (t,
J
= 7.20 Hz, 3H), 1.10 (d, = 6.60 Hz, 3H), 1.38-1.46 (m, 2H), 1.79-1.89 (m, 2H), 2.66 (t, J =
J
7.80 Hz, 2H), 3.19 (t,
J = 7.20 Hz, 2H), 3.56-3.64 (m, 1H), 7.16-7.21 (m, 3H), 7.27-7.31 (m,
2H); 13C NMR (75 MHz, CDCl3)
δ
10.36, 20.93, 30.10, 31.67, 33.15, 40.19, 47.86, 125.96,
128.37, 128.45, 141.48, 158.36. HRMS Calcd for C14H22N2O m/z [M+H] 235.1810, found
235.1835
5.1.2. Preparation of (S)-4-(3-(3-(1-Phenylethyl)ureido)propyl)benzenesulfonamide (8k(S)) and
(R)-4-(3-(3-(1-Phenylethyl)ureido)propyl)benzenesulfonamide (8k(R))
5.1.2.1. Preparation of 2,2,2-trifluoro-N-(3-phenylpropyl)acetamide (12): To a cooled solution
of 3-phenylpropylamine 11 (6.20 mmol) in methylene chloride (10 mL) at 0 °C, triethylamine
(9.30 mmol) was added. After 2 minutes, trifluoroacetic anhydride (9.30 mmol) was added at the
same temperature and allowed to stir for 3 h at ambient temperature. The reaction was monitored
by TLC. After the completion of the reaction, the reaction mixture was quenched with water and
extracted using methylene chloride. The organic layer was washed with water, dried over
anhydrous Na2SO4 and concentrated under vacuum to get the crude product 12. Without
1
purification, 12 was taken for the next step. Yield 82 %; White solid; H NMR (CDCl3)
δ 1.89-
1.99 (m, 2H), 2.69 (t, J = 7.40 Hz, 2H), 3.36-3.43 (m, 2H), 6.19 (bs, 1H), 7.17-7.33 (m, 5H).
5.1.2.2. Preparation of 4-(3-(2,2,2-trifluoroacetamido)propyl)benzene-1-sulfonylchloride (13):
To a cooled solution of chlorosulfuric acid (2 mL) at 0 °C, compound 12 (3.33 mmol) dissolved
in methylene chloride (2 mL) was added slowly and allowed to stir for 3 h to attain ambient

ACCEPTED MANUSCRIPT
temperature. The reaction was monitored by TLC. After the completion of the reaction, the
reaction mixture was slowly added to crushed ice with vigorous stirring. After all ice has melt
down, the reaction mass was extracted using methylene chloride and concentrated. The obtained
crude residue 13 was taken for the next step. Yield 65 %; Pale yellow solid; 1H NMR (CDCl3)
1.94-2.04 (m, 2H), 2.81 (t, = 7.40 Hz, 2H), 3.41-3.48 (m, 2H), 6.36 (bs, 1H), 7.44 (d, = 8.40
Hz, 2H), 7.98 (d, = 8.40 Hz, 2H).
δ
J
J
J
5.1.2.3. Preparation of 2,2,2-trifluoro-N-(3-(4-sulfamoylphenyl)propyl)acetamide (14): To a
solution of 13 (2.66 mmol) in methylene chloride (10 mL), ammonia gas was passed for a
period of 30 minutes. The resulting precipitate was filtered, washed with water and dried to
1
obtain 14. Yield 85 %; White solid; H NMR (DMSO-d6
)
δ
1.76-1.86 (m, 2H), 2.66 (t,
J = 7.40
Hz, 2H), 3.17-3.23 (m, 2H), 7.28 (s, 2H), 7.40 (d,
9.46 (bs, 1H).
J
= 8.40 Hz, 2H), 7.74 (d, = 8.40 Hz, 2H),
J
5.1.2.4. Preparation 4-(3-aminopropyl)benzenesulfonamide (15): To a solution of 14 (1.20
mmol) dissolved in methanol : water mixture (1:1, 10 mL), potassium carbonate (2.40 mmol)
was added and stirred at ambient temperature for 10 h. The completion of the reaction was
monitored by TLC. The reaction mixture was evaporated under vacuum to obtain white solid.
The resulting solid was dissolved in methanol (20 mL) and the residue was filtered. The filterate
was evaporated to get the amine 15 which was used for the next step. Yield 80 %; White solid;
1H NMR (D2O)
8.40 Hz, 2H), 7.76 (d,
5.1.2.5. Preparation of ureas 8k(S) and 8k(R): To a cooled solution of triphosgene (0.29 mmol)
in THF at 0 °C, a mixture of amine (0.88 mmol) and N,N-diisopropylethylamine (1.76 mmol)
was added. The reaction mixture was stirred at the same temperature for 30 minutes and then the
δ
1.75-1.82 (m, 2H), 2.63 (t,
= 8.40 Hz, 2H).
J = 7.40 Hz, 2H), 2.70-2.75 (m, 2H), 7.39 (d, J =
J
9

ACCEPTED MANUSCRIPT
amine 15 (0.88 mmol) was added. The reaction mixture was slowly allowed to attain ambient
temperature and further stirred for 8 h. After the completion of the reaction, water was added and
the reaction mixture was extracted using ethyl acetate. The organic layer was dried over
anhydrous Na2SO4 and evaporated under reduced pressure. The crude mixture was subjected to
column chromatography to obtain the 8k(S) and 8k(R)
.
5.1.2.6. (S)-4-(3-(3-(1-Phenylethyl)ureido)propyl)benzenesulfonamide (8k(S)). Yield 62 %;
White solid; mp: 164-166 °C; IR (neat): 3340, 3016, 2969, 1623, 1570, 1452, 1338, 1323, 1160
cm-1; 1H NMR (DMSO-d6
) δ 1.30 (d, J = 6.90 Hz, 3H), 1.61-1.71 (m, 2H), 2.61 (d, J = 7.80 Hz,
2H), 2.94-3.01 (m, 2H), 4.68-4.77 (m, 1H), 5.84-5.88 (m, 1H), 6.27 (d,
J = 8.10 Hz, 1H), 7.17-
7.31 (m, 7H), 7.36 (d,
J
= 8.40 Hz, 2H), 7.72 (d,
J
= 8.40 Hz, 2H); 13C NMR (DMSO-d6
) δ
23.11, 31.33, 32.06, 38.53, 48.46, 125.55, 125.60, 126.22, 127.99, 128.51, 141.59, 145.69,
145.93, 157.18; HRMS Calcd for C18H23N3O3S m/z [M+H] 362.1538, found 362.1575
5.1.2.7. (R)-4-(3-(3-(1-Phenylethyl)ureido)propyl)benzenesulfonamide (8k(R)). Yield 65 %;
White solid; mp: 164-166 °C; IR (neat): 3340, 3015, 2969, 1623, 1570, 1452, 1338, 1322, 1160
cm-1; 1H NMR (DMSO-d6
) δ 1.31 (d, J = 6.90 Hz, 3H), 1.62-1.72 (m, 2H), 2.61 (d, J = 7.80 Hz,
2H), 2.95-3.02 (m, 2H), 4.68-4.78 (m, 1H), 5.82-5.84 (m, 1H), 6.23 (d,
J = 8.10 Hz, 1H), 7.17-
7.31 (m, 7H), 7.35 (d,
J
= 8.40 Hz, 2H), 7.72 (d,
J
= 8.40 Hz, 2H). 13C NMR (DMSO-d6
) δ
23.18, 31.39, 32.09, 38.55, 48.49, 125.59, 125.64, 126.27, 128.04, 128.56, 141.61, 145.74,
145.97, 157.21; HRMS Calcd for C18H23N3O3S m/z [M+H] 362.1538, found 362.1573
6. Biology
6.1. Evaluation of sEH inhibition with LC-MS/MS method
sEH inhibition was determined using an LC-MS/MS method of Lee et al [37]. The physiological
substrate 14,15-EET (25 µM) was incubated at 30°C with various concentrations of test

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compound and 0.1 µg/mL sEH in 25 mM bis-Tris-HCl buffer (pH = 7.0) containing 0.1 mg/mL
of BSA in a total volume of 200 µL. The reactions were terminated after 20 min by the addition
of 200 µL of methanol containing 200 nM 9,10-DiHOME as an internal standard. Following
centrifugation (13,000 rpm for 10 min), the supernatant was used for LC-MS/MS analysis. The
enzyme inhibition parameter IC50 was calculated by fitting the Hill equation to the data using
nonlinear regression (least squares best fit modeling) of the plot of percentage control activity
versus concentration of the test inhibitors using GraphPad Prism 5.0 (GraphPad Software Inc.,
San Diego, CA).
The LC-MS/MS experiments were performed using a ProminenceTM UFLC system (Shimadzu,
Japan) coupled with an API3200QTRAPTM LC-MS/MS system (Applied Biosystems, Foster
City, CA) equipped with a Turbo V IonSpraysourceTM operated in negative-ion mode. The
sample injection volume was 10
C18 column (2.1 × 150 mm, 5
µ
L, and the separation was performed on a XTerraTM MS C18
m; Waters, Milford, MA, USA) with a SecuityGuardTM C18
ꢀ
guard column (2.0
× 4.0 mm i.d.; Phenomenex) maintained at 30°C. The mobile phase consisted
of deionized water (A) and acetonitrile (B). A linear gradient of the two solvents was used: start
at 70 % A and hold for 0.1 min, ramp to 40 % A to 0.5 min and hold until 3 min, and ramp to 10
% A to 6 min and hold for 1 min. The flow rate was set at 0.4 mL/min, and the overall
chromatographic run time was 7 min. The retention times for 14,15-DHET and the internal
standard (9,10-DiHOME) were 4.4 and 4.3 min, respectively. The auto-sampler compartment
was maintained at 10°C throughout the analysis. Ion source conditions for 14,15-DHET and
9,10-DiHOME were set as follows: CUR = 20, CAD = Medium, IS = −4500, TEM = 600,
GS1 = 50, and GS2 = 50. Quadrupoles Q1 and Q3 were set on unit resolution. The samples were
analyzed with multiple reaction monitoring. The monitoring ions were m/z 337
→207 for 14,15-

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DHET and m/z 313
→
201 for 9,10-DiHOME. In this study, the limit of detection and limit of
quantification values were 1.4 and 4.2 pg/mL with the signal-to-noise ratios over 2 and 10,
respectively.
6.2. HPLC method for evaluating mEH inhibition
mEH inhibition was determined using an HPLC method of Lee et al [37]. The substrate styrene
oxide (25 µM) was incubated with 10 µM test compound and pooled human liver microsomes
(0.2 mg/mL) in 0.1 M Tris-HCl (pH 9.0) in a total volume of 200 µL. Valpromide was used as a
positive mEH inhibitor. The reactions were incubated at 37°C for 10 min and terminated by the
addition of 200 µL of n-hexane. Following centrifugation (13,000 rpm, 10 min), the supernatant
was used for UV-HPLC analysis. The mEH activity of each sample was calculated according to
the amount of styrene glycol produced and is expressed as the percentage of the control activity.
The determination of styrene glycol was performed using a Shimadzu LC-10AD (Kyoto, Japan)
equipped with a UV detector (Shimadzu SPD-20A), controller (Shimadzu SIL-20AC), and
column oven (Shimadzu CTO-10AS). A Phenomenex Luna C18 column (150 × 4.5 mm, 5
ꢀm)
was used at 30 C with 40% methanol as the mobile phase, at a flow rate of 1.5 mL/min. Standard
°
and extracted samples of 10
monitored at 208 nm.
ꢀL were injected onto the column. The UV absorbance was
7. Binding Model Prediction of sEH Inhibitors
The crystal structure of human soluble epoxide hydrolase from Protein Data Bank (PDB code;
4OCZ [18], a complex with the 1-(1-isobutylpiperidin-4-yl)-3-(4-(trifluoromethyl)-phenyl)urea)
was used for docking simulation. The sEH inhibitors were built using a Maestro build panel in
the Schrodinger Package. The compounds were minimized using the Impact module of Maestro
in the Schrödinger Suite Program. The starting coordinate of the human peroxidase was further

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modified for binding model prediction. The protein structure was minimized using the Protein
Preparation Wizard by applying an OPLS-5 force field [38]. After complete preparation of the
ligands and protein for docking, receptor-grid files were generated. Ligand docking into the
active site of human sEH was carried out using the Schrödinger docking program, Glide [39].
The energy minimized sEH inhibitors were docked into the prepared receptor grid. The best-
docking models of sEH inhibitors with the lowest Glide docking score were adopted as the
binding conformation.
ACKNOWLEDGMENT
This work was supported by Priority Research Centers Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
(2009-0093815).
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