The 5-substituted piperazine as a novel secondary pharmacophore greatly improving the physical properties of urea-based inhibitors of soluble epoxide hydrolase

Article abstract of DOI:10.1016/j.bmc.2006.06.005

The inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertention and inflammation. The problems of limited water solubility and high melting points commonly displayed by the active 1,3-disubstituted ureas prevent the further development of potent urea-based sEH inhibitors. Therefore, a new class of potent inhibitors of sEH were designed and synthesized by the introduction of a polar constrained piperazino group in the right side of adasmantyl urea to increase the water solubility. A facile and general synthesis was established to prepare a series of 1-adamantan-1-yl-3-(2-piperazin-2-yl-ethyl)-ureas (1a-d) with various 5-substitutions on the 2-piperazino ring, which will advance the SAR study by the efficient making of structurally diverse analogs. The effect of the 5-substitution on the activity and the water solubility was examined. The best potency was exhibited by the 5-benzyl-substituted-piperazine-containing urea with an IC₅₀ value of 1.37 μM against human sEH and good water solubility (S = 7.46 mg/mL) and low melting point, in which the 5-substituted piperazine serves as a favorable secondary pharmacophore and a water-solubility enhancing group. Our present work provides a promising new template for the design of orally available therapeutic agents for the disorders that can be addressed by changing the in vivo concentration of the chemical mediators that contain an epoxide.

Full text of DOI:10.1016/j.bmc.2006.06.005

Bioorganic & Medicinal Chemistry 14 (2006) 6586–6592
The 5-substituted piperazine as a novel secondary pharmacophore
greatly improving the physical properties of urea-based
inhibitors of soluble epoxide hydrolase
Hui-Yuan Li,a Yi Jin,a Christophe Morisseau,b Bruce D. Hammockb and Ya-Qiu Longa,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
bDepartment of Entomology and UC Davis Cancer Center, University of California, One Shields Avenue, Davis, CA 95616, USA
Received 19 April 2006; revised 2 June 2006; accepted 3 June 2006
Available online 19 June 2006
Abstract—The inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hyper-
tention and inflammation. The problems of limited water solubility and high melting points commonly displayed by the active 1,3-
disubstituted ureas prevent the further development of potent urea-based sEH inhibitors. Therefore, a new class of potent inhibitors
of sEH were designed and synthesized by the introduction of a polar constrained piperazino group in the right side of adasmantyl
urea to increase the water solubility. A facile and general synthesis was established to prepare a series of 1-adamantan-1-yl-3-(2-pip-
erazin-2-yl-ethyl)-ureas (1a–d) with various 5-substitutions on the 2-piperazino ring, which will advance the SAR study by the effi-
cient making of structurally diverse analogs. The effect of the 5-substitution on the activity and the water solubility was examined.
The best potency was exhibited by the 5-benzyl-substituted-piperazine-containing urea with an IC50 value of 1.37 lM against human
sEH and good water solubility (S = 7.46 mg/mL) and low melting point, in which the 5-substituted piperazine serves as a favorable
secondary pharmacophore and a water-solubility enhancing group. Our present work provides a promising new template for the
design of orally available therapeutic agents for the disorders that can be addressed by changing the in vivo concentration of the
chemical mediators that contain an epoxide.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
acid, and other lipid epoxides. Epoxides of arachidonic
acid (epoxyeicosatrienoic acids or EETs) are known
effectors of blood pressure3 and modulators of vascular
permeability.4,5 Hydrolysis of the epoxides by sEH
diminishes this activity.3 In both cellular and animal
models, continued efficacy of EET-mediated hyperten-
sion and cardioprotective effects including inflammation
control is dependent upon epoxide hydrolysis by sEH,6,7
suggesting that inhibition of the mammalian sEH is a
promising new therapy in the treatment of disorders
resulting from hypertension and vascular inflamma-
tion.8–10
The epoxide hydrolases (EHs) are enzymes present in
most living organisms, which transform epoxide-con-
taining compounds to their corresponding diols by the
addition of water. The soluble epoxide hydrolase ap-
pears selective for epoxides of lipids. In plants and ani-
mals, many of these lipid substrates have potent
biological activities, such as host defenses, control of
development, and regulation of inflammation and blood
pressure.1 There are two well-studied EHs in mammals,
the microsomal epoxide hydrolase (mEH) and the solu-
ble epoxide hydrolase (sEH).2 In humans, the sEH plays
an important role in the metabolism of endogenous
chemical mediators such as arachidonic acid, linoleic
Based on the transition state of the epoxide hydrolysis
reaction catalyzed by sEH,11,12 1,3-disubstituted ureas
were designed and evaluated as potent sEH inhibitors,
which efficiently reduced epoxide hydrolysis in several
in vitro and in vivo models.7,13,14 The ureas mimicked
the structural features of the epoxide ring opening by
sEH, with similar charge distribution to the epoxide.11
However, the dialkyl ureas have limited solubility in
Keywords: Soluble epoxide hydrolase; 1-adamantanyl-urea-based
inhibitors; Piperazine; Chiral pool synthesis; Secondary pharmaco-
phore; Water solubility; Hypertension; Vascular inflammation.
*
Corresponding author. Tel./fax: +86 21 50806876; e-mail:
yqlong@mail.shcnc.ac.cn
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.005

H.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 6586–6592
6587
water and high melting points, leading to poor in vivo
efficacy and difficulty in formulation.15,16 During the
efficient efforts to improve the physical properties, a
new concept of a secondary pharmacophore was pro-
posed which consists of a polar functional group located
activity and the water solubility was examined. The best
potency was exhibited by the 5-benzyl-substituted-piper-
azine-containing urea with an IC50 value of 1.37 lM
against human sEH, and an exciting water solubility
(S = 7.46 mg/mL) and low melting point (77–78 °C),
providing a promising new scaffold for the further devel-
opment of orally available sEH inhibitors.
˚
on the fifth/sixth atom (7.5 A) from the carbonyl group
of the urea primary pharmacophore to help improve the
binding and the water solubility (Fig. 1).17,18 Previous
efforts were focused on ester, ketone, alcohol, and ether
functionality to determine a favorable secondary phar-
macophore. However, the positive effect on the solubil-
ity is still quite limited (<2 mg/mL) and the resulting
compounds still have relatively high melting points
(>100 °C) when the secondary pharmacophore is a polar
carbonyl group. How to tackle the problem of the poor
physical properties which prevents the potent urea-
based sEH inhibitors from entering clinical treatment?
We are interested in incorporating a constrained hetero-
cycle as the secondary pharmacophore into the 1,3-dial-
kyl urea platform. Piperazine is an interesting
heterocyclic structure present in many biologically
active molecules.19,20 Constraining the polar nitrogen
atoms into the piperazino ring can confer more drug-like
properties to molecules and enhance favorable interac-
tions with macromolecules. In this article, we designed
a new class of sEH inhibitors by introducing a substitut-
ed piperazino group into the right side of 1-adamanta-
nyl-urea to improve the activity and water solubility.
A chiral pool approach was established to synthesize
the target compounds with various substitutions on
the 5-position of the 2-piperazino ring (Fig. 1, com-
pounds 1a–d). The effect of the 5-substitution on the
2. Chemistry
An efficient and convenient synthetic approach was
developed to prepare the 1-adamantan-1-yl-3-(2-(5-
substituted piperazin-2-yl)-ethyl)-ureas. As depicted in
Scheme 1, the substituted piperazino moiety was con-
structed via a chiral pool approach employing the amino
acid as the starting material. The free asparagine was
easily converted into the Na-Boc-protected form, then
the coupling with another amino acid methyl ester affor-
ded the dipeptide 3a–d allowing the introduction of var-
ious substituents of R as the side chain of the amino
acid. The Boc removal of 3a–d followed by treatment
with the methanolic NH3 provided the 2,5-diketopiper-
azine intermediate 4a–d in good yield via an intramolec-
ular cyclization. Complete reduction of all amide bonds
with BH3ÆTHF21 produced the piperazine-containing
amine 5a–d, in which the amide side chain of the aspar-
agine of the starting material was judiciously converted
into the primary amine while the piperazino ring was
constructed. The free amine of the side chain became
the joining point which was reacted with 1-adamantyl
isocyanate to furnish the final product 1a–d. In this
Figure 1. A representative sEH inhibitor with primary and secondary pharmacophores indicated, and the structures of the designed new sEH
inhibitors with substituted piperazine as the secondary pharmacophore.
Scheme 1. Reagents and conditions: (a) (BOC)2O, dioxane/H2O; (b) HOBt, EDCI, DIPEA, L-amino acid methyl ester, CH2Cl2; (c) 1—CF3COOH
50% in CH2Cl2; 2—NH3/CH3OH; (d) BH3ÆTHF; (e) 1-adamantyl isocyanate, CH2Cl2.

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H.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 6586–6592
study, we just synthesized four representative structures,
but the methodology is efficient and applicable to the
general synthesis of various 5-substituted piperazine-
containing analogs for future SAR study. Furthermore,
the choice of the starting amino acid can adjust the dis-
tance of the urea functionality and the secondary phar-
macophore of piperazine. So, from the view point of
chemistry, we have established an efficient methodology
to synthesize a focused library of piperazine-containing
1,3-dialkyl ureas. Now, the next work is to investigate
whether the 5-substituted piperazine is an effective sec-
ondary pharmacophore which can enhance the physical
properties of this class of sEH inhibitors.
introducing a polar piperazine group as a secondary
pharmacophore, the water solubility (S, mg/mL) was
determined experimentally at 25 1.0 °C in PBS buffer
(0.1 M, pH 7.4).23 The octanol/water partition coeffi-
cient (P) is an important indication of the lipophilicity
of an organic molecule. The logP values were estimated
by Crippen’s method using CS ChemDraw Ultra version
6.0. On the basis of this logP value, logS value (solubil-
ity in water) was also calculated with the equation sug-
gested by Banerjee et al.24 as reference. All the activity
and physical property data are reported in Table 1.
In general, the high melting point was the indication of
high stability of crystal structure, which led to lack of
solubility in oil and in water. These properties make it
difficult to formulate the compounds in either an aque-
ous or oil base.17 As shown in Table 1, the piperazine-
containing 1-adamantyl-3-alkyl ureas (compounds
1a–d) pleasantly display improved physical properties:
lower melting point (mp < 80 °C) and lipophilicity
(logP = 2.09–0.54), and better water solubility
(S = 4.44–8.52 mg/mL) compared to the previously
reported best urea inhibitor of sEH (compound 1e:
mp = 114 °C, logP = 2.77, S = 1.69 mg/mL), which pos-
sess an ester functional group as the secondary pharma-
cophore. It is evident that the conformationally
constrained polar piperazine is really an excellent phar-
macophore on this position to enhance the drug-like
properties in the context of 1,3-dialkyl ureas as sEH
inhibitors.
3. Results and discussion
Four novel piperazine-containing dialkyl ureas were de-
signed and synthesized, in which the effect of the substi-
tuent on the 5-position of the 2-piperazino moiety on the
activity and the water solubility was examined. Four
representative structures were chosen as the 5-substitu-
ent: the aromatic ring (compound 1a), the conforma-
tional constrained heterocycle (compound 1b), the
alkyl chain (compound 1c), and the polar hydroxyl
group (compound 1d). We want to determine which type
of structure is favored for the binding with the sEH pro-
tein when attached on the piperazino ring. A recently
developed sensitive fluorescence-based assay22 was used
to evaluate the inhibitory activity of these new com-
pounds against human sEH enzyme.
With respect to the inhibitory potency, the 5-substitu-
tion on the 2-piperazino ring was found to play an
important role. When the substituent was a hydropho-
Since the goal of this work is to increase the water solu-
bility of the sEH inhibitors without loss of activity by
Table 1. Inhibition of human sEH by piperazine-containing 1-adamantyl ureas and the related physical properties
Compound
Structure
Human sEHa IC50 (lM)
logPb
Mpc (°C)
logSd
Se (mg/mL)
O
O
HN
HN
1a
NH
N
1.37
2.09
77–78
3.65
7.46 0.16
N
N
H
H
1b
1c
93.8
12.6
0.77
1.30
56–58
69–70
5.47
4.67
4.44 0.15
8.11 0.64
N
H
N
H
O
HN
HN
NH
N
H
N
H
O
O
OH
1d
100
0.54
2.77
66–68
114
5.56
2.27
8.52 0.04
1.69 0.44
NH
O
N
H
N
H
1ef
0.17 0.01
N
H
N
H
C5H11
O
a The IC50 values were determined by a recently developed sensitive fluorescence-based assay on human sEH.22
b logP (octanol/water partition coefficients) calculated by Crippen’s method by using CS ChemDraw 6.0 version.
c Mp, melting point.
d Solubility in water. It was calculated according to the following equation suggested by Banerjee et al.24: logP = 6.5–0.89 (logS)-0.015 mp, where mp
is melting point.
e Experimentally obtained solubility in sodium phosphate buffer (0.1 M, pH 7.4) at 25 1.0 °C.23 Results are means SD of two independent
experiments.
f The compound was reported previously17 and used here as a reference. Its activity was measured by an NEPC (4-nitrophenyl-trans-2,3-epoxy-3-
phenylpropyl carbonate) yellow water assay,25 which provides a relatively higher IC50 values compared to the fluorescence-based assay.

H.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 6586–6592
6589
bic group, for example, benzyl group, or isopropyl
group, the inhibition against human sEH by the result-
ing piperazine-containing adamantyl ureas was retained
potent, with an IC50 value of 1.37 lM for 1a and
12.6 lM for 1c, respectively. However, an additional po-
lar substituent such as propanol group on this position
remarkably reduced the inhibitory activity (1d,
IC50 = 100 lM), while the high hydrophilicity confers
the best water solubility to compound 1d (S = 8.52 mg/
mL). The fused pyrrolo ring with the piperazine endures
more rigid constraint and resulted in the lowest melting
point in this series (mp = 56–58 °C) at the loss of the
potency (1c, IC50 = 93.8 lM). So, we anticipate that
the optimal combination of the hydrophilic piperazine
ring and the hydrophobic substitution on the piperazino
ring would confer good physical properties with high
potency.
new template for further development of sEH inhibitors
as anti-hypertension and anti-inflammation drugs.
5. Experimental
5.1. Synthesis
The 1H NMR spectra were recorded on a Varian Mercu-
ry-400 MHz spectrometer. The data are reported in
parts per million relative to TMS and referenced to
the solvent in which they were run. Elemental analyses
were obtained using Vario EL spectrometer. Melting
points (uncorrected) were determined on a Buchi-510
capillary apparatus. EI-MS spectra were obtained on a
Finnigan MAT 95 mass spectrometer. ESI-MS spectra
were obtained on a Finnigan LCQ Deca mass spectro-
meter. Specific rotations (uncorrected) were determined
in a Perkin-Elmer 341 polarimeter.
Even though the best compound in our series (com-
pound 1a) is much less potent than the best one (com-
pound 1e) of previously reported classes which bear an
ester as the secondary pharmacophore, the physical
properties are significantly improved by the introduction
of the novel piperazine group. Since the poor water
solubility and high melting point are the remaining
drawbacks of the urea-based inhibitors of sEH, the
incorporation of the piperazino group provides a prom-
ising new strategy to circumvent the major problem.
Furthermore, the initial SAR study has disclosed that
the hydrophobic substitution on the 5-position of the
piperazino ring is favored for the binding with the
sEH, further structural optimization would definitely
improve the activity of this new series which inherit
good physical properties, and our well-established syn-
thesis in this work will benefit the making of various
analogs. In summary, our present work paved a new
way for the design of specific and potent sEH inhibitors
with enhanced drug-like properties, and will be useful
for the design of orally available therapeutic agents for
hypertension, vascular and renal inflammation, and
other disorders that can be addressed by changing the
in vivo concentration of chemical mediators that contain
an epoxide.
5.2. S-2-tert-Butoxycarbonylamino-succinamic acid (2)26
To a suspension of L-AsnÆH2O (1.5 g, 10 mmol) in the
solution of dioxane and H2O (1:1), (BOC)2O (6.5 g,
30 mmol) was added, with DMF to help increase solu-
bility. Then NaHCO3 (2.5 g, 30 mmol) was added. The
mixture was stirred at room temperature for 24 h, then
the solvent was removed at reduced pressure. The resi-
due was acidified with 6 N HCl to pH 2 and filtered.
The solid was recrystallized from CH3OH to afford
product 2 as white powder (1.5 g, 65%).
5.3. S,S-2-(2-tert-Butoxycarbonylamino-3-carbamoyl-
propionylamino)-3-phenyl-propionic acid methyl ester
(3a)27
Under ice-salt bath, to the solution of the amino acid
methyl ester L-Phe-OMeÆHCl (1.3 g, 6 mmol), com-
pound 2 (1.4 g, 6 mmol), and HOBt (1.35 g, 10 mmol)
in 20 mL of dry CH2Cl2 was added DIPEA (3 mL).
The reaction mixture was stirred at 0 °C, added the solu-
tion of EDCI (1.91 g, 10 mmol) in 20 mL of dry CH2Cl2
dropwise. Then the solution was stirred at room temper-
ature for 24 h. A lot of solid appeared, filtered, and the
solid was washed with saturated NH4Cl aq, 1 N HCl aq,
and saturated NaHCO3 aq, respectively. The crude
4. Conclusions
product was recrystallized from CH3OH to provide
22
Based on the mode of action of sEH and the structure of
the urea inhibitor complexed with the enzyme,11,12 we
designed and synthesized a new class of sEH inhibitors
with remarkably improved physical properties by the
introduction of substituted piperazino ring in the urea
platform. A practical and efficient synthesis is established
to prepare a range of piperazine-containing dialkyl ureas
with various 5-substitutions on the 2-piperazino ring,
which will benefit further structural elaboration and
extensive SAR study. The best potency was exhibited
by the 5-benzyl-substituted-piperazine-containing urea
with an IC50 value of 1.37 lM against human sEH and
an improved water solubility (S = 7.46 mg/mL) and low
melting point, in which the 5-substituted piperazine
serves as a favorable secondary pharmacophore and a
water-solubility enhancing group, providing a promising
white powder 3a (1.6 g, 68%). ½aꢀ ꢁ9 (c = 0.19,
D
MeOH). 1H NMR (CDCl3, 400 MHz): d 7.32–7.14
(5H, m), 6.02–5.99 (1H, m), 5.79–5.78 (1H, m), 5.35–
5.34 (1H, m), 4.80–4.78 (1H, m), 4.47–4.45 (1H, m),
3.69 (3H, s), 3.15–3.04 (2H, m), 2.93–2.89 (1H, d,
J = 16 Hz),
2.57–2.52
(1H,
dd,
J1 = 16 Hz,
J2 = 6.4 Hz), 1.38 (9H, s). Mp: 194–195 °C.
5.4. S,S-1-(2-tert-Butoxycarbonylamino-3-carbamoyl-
propionyl)-pyrrolidine-2-carboxylic acid methyl ester (3b)
Prepared from compound 2 (3.4 g, 15 mmol) and L-Pro-
OMeÆHCl (2.5 g, 15 mmol) according to the similar pro-
cedure to 3a. The residue was purified by silica gel chro-
matography (CH2Cl2/CH3OH 10:1) to afford 3b as
white foam (2.2 g, 44%). ½aꢀ ꢁ69.7 (c = 1.3, CH2Cl2).
22
D

6590
H.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 6586–6592
22
D
1
1H NMR (CDCl3, 400 MHz): d 6.59 (1H, blunt), 5.85–
5.83, 5.49–5.47 (2H, blunt), 4.80–4.79 (1H, m), 4.53–
4.50 (1H, m), 3.78–3.73 (2H, m), 3.73 (3H, s), 2.71–
2.55 (2H, m), 2.25–2.18, 2.05–1.96 (4H, m), 1.45 (9H,
s). Mp: 56–58 °C. EI-MS: m/z 343 (M+).
obtained (1.6 g, 63.8%). ½aꢀ ꢁ119.9 (c 1.0, H2O). H
NMR (DMSO-d6, 400 MHz): d 7.96 (1H, s), 7.412,
6.91 (2H, s, s), 4.38–4.36 (1H, t, J1 = J2 = 6 Hz), 4.23–
4.19 (1H, t, J1 = 6.4 Hz, J2 = 8 Hz), 3.40–3.31 (2H, m),
2.76–2.70, 2.34–2.28 (2H, dq, J1 = 6.0 Hz, J2 = 6.4 Hz,
J3 = 16 Hz), 2.12–2.10, 1.89–1.79 (4H, m). Mp:
>200 °C. EI-MS: m/z 211 (M+).
5.5. S,S-2-(2-tert-Butoxycarbonylamino-3-carbamoyl-
propionylamino)-3-methyl-butyric acid methyl ester (3c)
5.9. S,S-2-(5-Isopropyl-3,6-dioxo-piperazin-2-yl)-acetam-
ide (4c)
Prepared from compound 2 (2.9 g, 12.5 mmol) and L-
Val-OMeÆHCl (2.1 g, 12.5 mmol) according to the simi-
lar procedure to 3a. The crude product was purified by
Prepared from compound 3c (960 mg, 2.8 mmol)
according to the similar procedure to 4a. The crude solid
was recrystallized from H2O, and white crystal was ob-
silica gel chromatography (CH2Cl2/CH3OH 20:1) to af-
22
ford 3c as white foam (3.0 g, 70%). ½aꢀ +0.2 (c 0.775,
D
22
CH2Cl2). 1H NMR (CDCl3, 400 MHz): d 7.50 (1H,
blunt), 6.20–6.16 (2H, blunt), 5.64 (1H, blunt), 4.52–
4.51, 4.47–4.44 (2H, m), 3.73 (3H, s), 2.93–2.89 (1H,
dd, J1 = 4 Hz, J2 = 15.6 Hz), 2.65–2.59 (1H, dd,
J1 = 6.4 Hz, J2 = 16 Hz), 2.22–2.15 (1H, m), 1.50–1.43
(9H, s), 0.96–0.92 (6H, dd). Mp: 139–141 °C. (Mp28:
141–150 °C). EI-MS: m/z 345 (M+).
tained (520 mg, 88%). ½aꢀ ꢁ79 (c 0.23, H2O). 1H NMR
D
(D2O, 400 MHz): d 4.58–4.55 (1H, t, J1 = 5.2 Hz,
J2 = 8 Hz), 4.09–4.08 (1H, d, J = 6.4 Hz), 3.00–2.94,
2.85–2.79 (2H, dq, J1 = 4.8 Hz, J2 = 8 Hz), 2.36–2.32
(1H, m), 1.12–0.98 (6H, dd, J = 6.8 Hz). Mp: >200 °C.
EI-MS: m/z 213 (M+). Anal. Calcd for C9H15N3O3: C,
50.69; H, 7.09; N, 19.71. Found: C, 50.86; H, 7.02; N,
19.68.
5.6. S,S-2-(2-tert-Butoxycarbonylamino-3-carbamoyl-
propionylamino)-pentanedioic acid dimethyl ester (3d)
5.10. 3-(5-Carbamoylmethyl-3,6-dioxo-piperazin-2-yl)-
propionic acid methyl ester (4d)
Prepared from compound 2 (5.6 g, 24 mmol) and L-
Glu(OMe)-OMeÆHCl (5.1 g, 24 mmol) according to the
similar procedure to 3a. The crude product was purified
Prepared from compound 3d (4 g, 10.3 mmol) according
to the similar procedure to 4a. The crude solid was
recrystallized from H2O, and white crystal was obtained
(1.4 mg, 52.1%). ½aꢀ ꢁ34.2 (c 0.49, H2O). H NMR
(D2O, 400 MHz): dD4.49–4.44 (1H, m), 4.27–4.23 (1H,
m), 3.72 (3H, s), 2.89–2.87 (2H, d, J = 7.6 Hz), 2.58–
2.52 (2H, m), 2.23–2.16 (2H, m). Mp: >200 °C. EI-
MS: m/z 257 (M+). Anal. Calcd for C10H15N3O5: C,
46.69; H, 5.88; N, 16.33. Found: C, 46.81; H, 5.82; N,
16.28.
by silica gel chromatography (CH2Cl2/CH3OH 10:1) to
22
22
1
afford 3d as white foam (8.5 g, 91%). ½aꢀ +17.2 (c 1.1,
D
CH2Cl2). 1H NMR (CDCl3 400 MHz): d 7.48–7.47
(1H, blunt), 6.07 (2H, blunt), 5.70 (1H, blunt), 4.58–
4.56, 4.49–4.48 (2H, m), 3.73 (3H, s), 3.67 (3H, s),
2.95–2.91 (1H, dd, J1 = 3.2 Hz, J2 = 15.6 Hz), 2.61–
2.56 (1H, dd, J1 = 6.4 Hz, J2 = 15.6 Hz), 2.42–2.37
(2H, m), 2.23–2.19 (1H, m), 2.00–1.95 (1H, m), 1.44
(9H, s). Mp: 122–123 °C. EI-MS: m/z 389 (M+). Anal.
Calcd for C16H27N3O8: C, 49.35; H, 6.99; N, 10.79.
Found: C, 49.17; H, 7.04; N, 10.5.
5.11. S,S-2-(5-Benzyl-piperazin-2-yl)-ethylamine (5a)
To the suspension of compound 4a (260 mg, 1 mmol)
in 20 mL THF was added 10 mL of BH3–THF com-
plex (10 mmol) dropwise with stirring under N2 over
1 h. Gas emitted and the mixture became nearly solu-
tion at the end of the addition. The mixture was re-
fluxed for 40 h. It was cooled to room temperature
and 20 mL of methanol was added over 1 h. The reac-
tion mixture was concentrated to dryness. The residue
was dissolved in methanol (10 mL) and excess metha-
nolic HCl solution was added and the solution was re-
fluxed for 2 h. It was cooled and stirred at room
temperature overnight. The solvent was removed at re-
duced pressure, and then a solution of 10% NaOH in
water was added into the residue to pH 11. Then H2O
was removed at reduced pressure and dissolved in
methanol, and dried with anhydrous Na2SO4. Filtered
and removed the solvent, purified by silica gel chro-
matography (CH2Cl2/CH3OH 8:1) to afford 5a as light
yellow oil (177 mg, 80.9%). The methanol saturated
5.7. S,S-2-(5-Benzyl-3,6-dioxo-piperazin-2-yl)-acetamide
(4a)
Compound 3a (1.6 g, 4.2 mmol) was added into the
solution of CF3COOH in CH2Cl2 at 0 °C and then stir-
red for 4 h. The solvent was removed at reduced pres-
sure. The residue was dissolved in NH3-saturated
methanol at 0 °C and stirred for 4 h at room tempera-
ture. White solid appeared. The solvent was removed
at reduced pressure. The crude solid was recrystallized
from H2O. White powder was obtained (1 g, 92%).
22
1
½aꢀ ꢁ14 (c 0.2, H2O). H NMR (CD3OD, 400 MHz):
D
d 7.34–7.21 (5H, m), 4.31–4.29 (1H, m), 4.13–4.11 (1H,
m), 3.32–3.22 (1H, m), 3.03–2.99 (1H, m), 2.62–2.57
(1H, dq, J1 = 4 Hz, J2 = 6.4 Hz), 2.36–2.31 (1H, m).
Mp: >200 °C. (Mp29: 268–270 °C) EI-MS: m/z 261 (M+).
5.8. S,S-2-(1,4-Dioxo-octahydro-pyrrolo[1,2-a]pyrazin-3-
yl)-acetamide (4b)
with HCl was added to the oil, white solid was ob-
22
D
tained. ½aꢀ +2.6 (c 0.57, MeOH). 1H NMR (D2O,
Prepared from compound 3b (4.9 g, 14 mmol) according
to the similar procedure to 4a. The crude solid was
recrystallized from methanol, and white crystal was
400 MHz): d 7.46–7.33 (5H, m), 3.13–2.73 (10H, m),
1.85–1.73 (2H, m). Mp: >200 °C. ESI-MS: m/z 220.2
[M+H]+.

H.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 6586–6592
6591
5.12. S,S-2-(Octahydro-pyrrolo[1,2-a]pyrazin-3-yl)-ethyl-
amine (5b)
5.16. S,S-1-Adamantan-1-yl-3-[2-(octahydro-pyrrolo[1,2-
a]piperazin-3-yl)-ethyl]-urea (1b)
Prepared from compound 4b (422 mg, 2 mmol)
according to the similar procedure to 5a. The crude
product was purified by silica gel chromatography
(CH2Cl2/CH3OH 2:1) to afford 5b as light yellow
oil (145 mg, 45%). The methanol solution saturated
Prepared from compound 5b (78 mg, 0.46 mmol)
according to the similar procedure to 1a. The procedure
was similar as above. The crude product was purified by
silica gel chromatography (CH2Cl2/CH3OH 10:1) to af-
22
D
ford 1b as light yellow solid (60 mg, 44%). ½aꢀ +3.1 (c
1
with HCl was added to the oil, white solid was ob-
0.4, CH2Cl2). H NMR (CD3OD, 400 MHz): d 3.34–
3.00 (10H, m), 2.7–2.6 (2H, m), 2.04–1.70 (21H, m).
Mp: 56–58 °C. HRESI-MS m/z: 347.2806 [M+H]+, calcd
347.2811.
22
D
tained. ½aꢀ
ꢁ5.5 (c 0.83, MeOH). 1H NMR
(DMSO-d6, 400 MHz): d 3.04–2.47 (10H, m), 2.11–
1.94 (2H, m), 1.78–1.25 (6H, m). ESI-MS: m/z 170
[M+H]+.
5.17. S,S-1-Adamantan-1-yl-3-[2-(5-isopropyl-piperazin-
2-yl)-ethyl]-urea (1c)
5.13. S,S-2-(5-Isopropyl-piperazin-2-yl)-ethylamine
(5c)
Prepared from compound 5c (120 mg, 0.7 mmol)
according to the similar procedure to 1a. The crude
product was purified by silica gel chromatography
Prepared from compound 4c (426 mg, 2 mmol)
according to the similar procedure to 5a. The crude
product was purified by silica gel chromatography
(CH2Cl2/CH3OH 2:1) to afford 5c as light yellow
oil (150 mg, 43.9%). The methanol solution with sat-
urated hydrochloric acid was added to the oil, white
(CH2Cl2/CH3OH 10:1) to afford 1c as light yellow solid
22
D
(30 mg, 14%). ½aꢀ ꢁ95 (c 0.43, CH2Cl2). 1H NMR
(CD3OD, 400 MHz): d 3.34–3.00 (8H, m), 2.7–2.6 (1H,
m), 2.04–1.70 (18H, m), 1.206–0.969 (6H, dd). Mp:
69–70 °C. HRESI-MS m/z: 349.2951 [M+H]+, calcd
349.2967.
22
1
solid was obtained. ½aꢀ ꢁ13 (c 0.1, H2O). H NMR
D
(D2O, 400 MHz): d 4.05 3.76–3.49 (6H, m), 3.30–3.26
(2H, t), 2.37–2.31 (2H, m), 2.21–2.19 (1H, m), 1.18–
1.12 (6H, dd). Mp: >200 °C. ESI-MS: m/z 172
[M+H]+.
5.18. S,S-1-Adamantan-1-yl-3-{2-[5-(3-hydroxy-propyl)-
piperazin-2-yl]-ethyl}-urea (1d)
5.14. S,S-3-[5-(2-Amino-ethyl)-piperazin-2-yl]-propan-1-
ol (5d)
Prepared from compound 5d (320 mg, 1.73 mmol)
according to the similar procedure to 1a. The crude
product was purified by silica gel chromatography
(CH2Cl2/CH3OH 2:1) to afford 1d as light yellow solid
Prepared from compound 4d (1.3 g, 5.1 mmol)
according to the similar procedure to 5a. The crude
product was purified by silica gel chromatography
(CH2Cl2/CH3OH 2:1) to afford 5d as light yellow
oil (640 mg, 68%). The methanol solution saturated
22
1
(150 mg, 27%). ½aꢀ +7.8 (c 0.43, CH2Cl2). H NMR
D
(CDCl3, 400 MHz): d 4.77 (1H, blunt), 4.45 (1H, blunt),
3.65–3.55 (2H, m), 3.33 (1H, m), 3.14–3.10 (1H, m),
2.86–2.69 (6H, m), 2.32–2.10 (2H, blunt), 2.10–1.52
(21H, m). Mp: 66–68 °C. HRESI-MS: m/z 365.2921
[M+H]+, calcd 365.2917.
with HCl was added to the oil, white solid was ob-
22
D
tained. ½aꢀ ꢁ3.5 (c 0.42, H2O). 1H NMR (D2O,
400 MHz): d 3.962–3.955 (1H, m), 3.871–3.865 (1H,
m), 3.73–3.70 (2H, m), 3.67–3.59 (4H, m), 3.29–3.25
(2H, m,), 2.35–2.29 (2H, m), 2.00–1.96 (2H, m),
1.77–1.73 (2H, m). Mp: >200 °C. ESI-MS: m/z
188.2 [M+H]+.
Acknowledgment
Financial supports from Ministry of Science and Tech-
nology of China (No. 2004CB518903) are greatly appre-
ciated. Partial support was provided by NIEHS Grant
R37 ES02710.
5.15. S,S-1-Adamantan-1-yl-3-[2-(5-benzyl-piperazin-2-
yl)-ethyl]-urea (1a)
The solution of 1-isocyanato-adamantane (81 mg,
0.45 mmol) in dry CH2Cl2 was added dropwise 5a
(109 mg, 0.5 mmol) in 10 mL of dry CH2Cl2 at 0 °C.
Then 1.74 lL DIPEA was added. The mixture was
stirred at 0 °C for 3 h and then stirred at room tem-
perature overnight. The solvent was removed at re-
duced pressure and then purified by silica gel
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2006.
06.005.
chromatography (CH2Cl2/CH3OH 8:1) to afford 1a
References and notes
22
as light yellow solid (44 mg, 24%). ½aꢀ ꢁ11 (c 0.56,
D
CH2Cl2). 1H NMR (CDCl3, 400 MHz): d 7.32–7.18
(5H, m), 5.1 (1H, blunt), 4.67 (1H, blunt), 3.35 (1H,
m), 3.15–3.11 (1H, m), 2.97–2.75 (8H, m), 2.95–2.71
(7H, m), 2.32–2.28 (2H, m), 2.04–1.58 (17H, m).
Mp: 77–78 °C. HRESI-MS m/z: 397.2982 [M+H]+,
calcd 397.2967.
1. Newman, J. W.; Morisseau, C.; Hammock, B. D. Prog.
Lipid Res. 2005, 44, 1–51.
2. Fretland, A. J.; Omiecinski, C. J. Chem. Biol. Interact.
2000, 129, 41–59.
3. Capdevila, J. H.; Falck, J. R.; Harris, R. C. J. Lipid Res.
2000, 41, 163–181.

6592
H.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 6586–6592
4. Oltman, C. L.; Weintraub, N. L.; VanRollins, M.;
18. Kim, I. H.; Heirtzler, F. R.; Morisseau, C.; Nishi, K.;
Tsai, H.-J.; Hammock, B. D. J. Med. Chem. 2005, 48,
3621–3629.
19. Todorovic, A.; Haskell-Luevano, C. Peptides 2005, 26,
2026–2036.
Dellsperger, K. C. Circ. Res. 1998, 83, 932–939.
5. Fisslthaler, B.; Popp, R.; Kiss, L.; Potente, M.; Harder, D.
R.; Fleming, I.; Busse, R. Nature 1999, 401, 493–497.
6. Moghaddam, M. F.; Grant, D. F.; Cheek, J. M.; Greene,
J. F.; Williamson, K. C.; Hammock, B. D. Nat. Med.
1997, 3, 562–567.
20. Donnelly, L. E.; Rogers, D. F. Curr. Opin. Drug Discov.
Dev. 2003, 6, 815–826.
7. Morisseau, C.; Goodrow, M. H.; Dowdy, D.; Zheng, J.;
Greene, J. F.; Sanborn, J. R.; Hammock, B. D. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 8849–8854.
8. Morisseau, C.; Hammock, B. D. Annu. Rev. Pharmacol.
Toxicol. 2005, 45, 311.
9. Schmelzer, K. R.; Kubala, L.; Newman, J. W.; Kim, I. H.;
Eiserich, J. P.; Hammock, B. D. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 9772–9777.
10. Liu, Y.; Zhang, Y. J.; Schmelzer, K.; Lee, T. S.; Fang, X.;
Zhu, Y.; Spector, A. A.; Gill, S.; Morisseau, C.; Ham-
mock, B. D.; Shyy, J. Y. J. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 16705–16747.
11. Argiriadi, M. A.; Morisseau, C.; Hammock, B. D.;
Christianson, D. W. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 10637–10642.
12. Argiriadi, M. A.; Morisseau, C.; Goodrow, M. H.;
Dowdy, D. L.; Hammock, B. D.; Christianson, D. W.
J. Biol. Chem. 2000, 275, 15265–15270.
13. Imig, J. D.; Zhao, X.; Capdevila, J. H.; Morisseau, C.;
Hammock, B. D. Hypertension 2002, 39, 690–694.
14. Newman, J. W.; Denton, D. L.; Morisseau, C.; Koger, C.
S.; Wheelock, C. E.; Hinton, D. E.; Hammock, B. D.
Environ. Health Perspect. 2001, 109, 61–66.
15. Yu, Z.; Xu, F.; Huse, L. M.; Morisseau, C.; Draper, A. J.;
Newman, J. W.; Parker, C.; Graham, L.; Engler, M. M.;
Hammock, B. D.; Zeldin, D. C.; Kroetz, D. L. Circ. Res.
2000, 87, 992–998.
21. Brown, H.; Heim, P. J. Org. Chem. 1973, 38, 912–916.
22. Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.;
Hock, B.; Hammock, B. D. Anal. Biochem. 2005, 343, 66–75.
23. The water solubility (S, mg/mL) was determined experi-
mentally at 25 1.0 °C. For compound 1a with a UV
chromophore, an excess of the test compound was added to
a vial containing sodium phosphate buffer, 0.1 M, pH 7.4
(200 lL), and a suspension of the mixture was equilibrated
during 1 h of sonication and 1 h of shaking, followed by
centrifugation (5 min, 8000 rpm). The water supernatant
was scanned on UV spectrophotometer (HITACHI U-
2010) to get its UV absorbance at the wavelength of 254 nm.
A regression curve for compound 1a was obtained from six
standard stock solutions (r = 0.99) by using UV spectrom-
etry at the same wavelength. Then, the absolute amount of
compound 1a was calculated. For compounds 1b–c without
a UV chromophore, to an exactly weighed amount of test
compound was added PBS by an increment of 5 lL with
pipet until the solid was completely dissolved accompanied
with frequent shaking at 25 1.0 °C.
24. Banerjee, S.; Yalkowsky, S. H.; Valvani, S. C. Environ.
Sci. Technol. 1980, 14, 1227–1229.
25. Dietze, E. C.; Kuwano, E.; Hammock, B. D. Anal.
Biochem. 1994, 216, 176–187.
26. Broadbent, W.; Morley, J. S.; Stone, B. E. J. Chem. Soc. C
1979, 15, 2632–2636.
27. Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron
Lett. 1975, 14, 1219–1221.
28. Abiko, T.; Sekino, H. Chem. Pharm. Bull. 1989, 37, 391–396.
29. Pickenhagen, W.; Dietrich, P.; Keil, B.; Polinsky, J.;
Nouaille, F.; Lederer, E. Helv. Chim. Acta 1975, 58, 1078–
1084.
16. Morisseau, C.; Goodrow, M. H.; Newman, J. W.;
Wheelock, C. E.; Dowdy, D. L.; Hammock, B. D.
Biochem. Pharmacol. 2002, 63, 1599–1608.
17. Kim, I. H.; Morisseau, C.; Watanabe, T.; Hammock, B.
D. J. Med. Chem. 2004, 47, 2110–2122.