Pyrazole derivatives as new potent and selective 20-hydroxy-5,8,11,14- eicosatetraenoic acid synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.08.047

Introduction of a piperazino group to the selective and potent 20-HETE synthase inhibitor 1 to obtain potential therapeutic agents with suitable water-solubility for injectable formulation is described. Improvement of the physical properties of pyrazole derivative 1, which we reported previously as a potent and selective 20-HETE synthase inhibitor (IC₅₀ 5.7 nM), is described. Introduction of a sufficient substituted-amino group on the side chain enhanced the water-solubility of 1 (0.014 mg/mL at pH 6.8). Among the products, 2-piperazinoethoxy derivatives 3e and 6b showed solubility suitable for injection and potent inhibitory activity toward 20-HETE synthase (IC ₅₀ 21.2 and 14.0 nM, respectively).

Full text of DOI:10.1016/j.bmc.2004.08.047

Bioorganic & Medicinal Chemistry 12 (2004) 6209–6219
Pyrazole derivatives as new potent and selective
20-hydroxy-5,8,11,14-eicosatetraenoic acid synthase inhibitors
Toshio Nakamura,* Hiroyuki Kakinuma, Hideaki Amada, Noriyuki Miyata,
Kazuo Taniguchi, Ayumi Koda and Masakazu Sato*
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd, 403, Yoshino-Cho 1-Chome, Kita-ku, Saitama-Shi,
Saitama 331-9530, Japan
Received 9 July 2004; revised 31 August 2004; accepted 31 August 2004
Available online 18 September 2004
Abstract—Improvement of the physical properties of pyrazole derivative 1, which we reported previously as a potent and selective
20-HETE synthase inhibitor (IC50 5.7nM), is described. Introduction of a sufficient substituted-amino group on the side chain
enhanced the water-solubility of 1 (0.014mg/mL at pH6.8). Among the products, 2-piperazinoethoxy derivatives 3e and 6b showed
solubility suitable for injection and potent inhibitory activity toward 20-HETE synthase (IC50 21.2 and 14.0nM, respectively).
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
H
N
NH
N
20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE)
is a major metabolite of arachidonic acid (AA) produced
in the kidney.1 Its biological properties have recently
been extensively studied. 20-HETE is synthesized from
AA by oxidation with cytochrome P450 (CYP) 4A iso-
zymes (CYP4A1, 4A2, 4A3, and 4A8) in rat kidney,2
and with CYP4A11 and 4F2in human liver and kid-
ney.3 20-HETE plays an important role in the regulation
of renal vascular and tubular functions.4–6 20-HETE
contributes to the control of arterial blood pressure.7
More recent studies have indicated that 20-HETE is also
produced in the brain, where it regulates vascular tone
and contributes to the autoregulation of cerebral blood
flow.8 Therefore, the inhibition of 20-HETE is now con-
sidered to be a promising new target for the treatment of
renal and cerebral diseases. Some compounds are
known to inhibit the production of 20-HETE (Fig. 1),
however, these compounds lack sufficient potency and
specificity for further investigation as therapeutics. 1-
Aminobenzotriazole (1-ABT)9 has been reported to be
a 20-HETE synthase inhibitor with an IC50 value of
5lM, however, it inhibits drug-metabolizing enzymes
at almost the same concentration. 17-Octadecynoic acid
COOH
17-ODYA
IC50>5000nM
NH2
1-ABT
IC50>5,000nM
Br
Br
Br
CONHSO2Me
Br
COOH
DDMS
IC50=2,000nM
DBDD
IC50=2,000nM
H
N
N
OH
NaO
O
O
S
Me
HET0016
IC50=35.2nM
O
10-SUYS
IC50=7,500~12,700nM
Figure 1. Structures of 1-ABT, 17-ODYA, DDMS, DBDD, 10-SUYS
and HET0016. IC50 values are for 20-HETE production from AA by
rat renal microsome.
(17-ODYA),10,11 N-methylsulfonyl-12,12-dibromodo-
dec-11-enamide (DDMS),12 12,12-dibromododec-11-
enoic acid (DBDD)12 and sodium 10-undecynyl sulfate
(10-SUYS)13 deactivate 20-HETE synthase at micro-
molar concentrations by forming a covalent bond at
the active site via their chemically-reactive oxidized
product. In previous papers, we reported HET0016
(N-hydroxy-N0-(4-n-butyl-2-methylphenyl)formamidine)
as the first potent and selective 20-HETE synthase inhib-
itor.14 HET0016 inhibited the formation of 20-HETE by
Keywords: 20-HETE; Pyrazole.
*
Corresponding authors. Tel.: +81 48 669 3029; fax: +81 48 652
7254; e-mail: toshio.nakamura@po.rd.taisho.co.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.047

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T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
rat renal microsomes (IC50 = 35.2 4.4nM) and by
human renal microsomes (IC50 = 8.9 2.7nM),15 with
remarkable selectivity against CYP2C9, CYP2D6,
CYP3A4, and cyclooxygenases (COX-1,COX-2).15
HET0016 prevented the acute fall in cerebral blood flow
following subarachnoid hemorrhage (SAH) in the rat
when administrated by intravenous injection.16 How-
ever, despite its promising pharmacological properties,
the therapeutic potency of HET0016 as an injectable
formulation is limited due to its low water-solubility
under neutral conditions (0.0037mg/mL at pH6.8) and
instability under acidic conditions. Replacement of the
N-hydroxyformamidine moiety of HET0016 by a pyraz-
ole ring gave compound 1, which retains potent 20-
HETE synthase inhibitory activity (IC50 = 26.2nM)
while showing improved solubility and stability under
acidic conditions. However, its water-solubility under
neutral conditions (0.026mg/mL at pH6.8) is still inad-
equate for injectable formulations.17 In this study, we
investigated the introduction of a polar substituent the
hydrophilic side chain of compound 1 to improve its
water-solubility.
2. Chemistry
Compounds 3a–m were prepared in yields of 4–85% by
the alkylation of 4-(3-pyrazolyl)phenol 2 with corre-
sponding alcohols by the Mitsunobu reaction, as re-
ported for 1 (Scheme 1). Compounds 4a,b were
prepared by the alkylation of N-methylpiperazine and
N-isopropylpiperazine by 3m with Et3N in DMF at
120°C (Scheme 2). Deprotection of 1-[2-(4-tert-butoxy-
carbonylpiperazino)ethoxy]-4-(3-pyrazolyl)benzene 3g
with HCl and successive treatment of the resulting
mono-alkyl piperazine derivative 5 with acyl chlorides
or condensation with carboxylic acid gave amide deriv-
atives 6a–c, respectively. Reductive alkylation of 5 by
cyclohexanecarboxaldehyde with NaBH(OAc)3 gave 6d
(Scheme 3). Introduction of a 4- or 5- amino-substituted
1) R1OH
PPh3 / DEAD
THF
N
N
HN
HN
nHCl*
R1O
2) HCl
HO
2
3a-3m
4~85%
*3d was p-toluenesulfonate
Scheme 1.
R2
N
N
N
HN
HN
NH
R2
N
Cl
N
DMF
3HCl
O
O
then HCl
3m
4a: R2=Methyl
57~97%
4b: R2=i-Propyl
Scheme 2.
N
O
HN
R3
N
6a: R3=methyl
6b: R3=n-propyl
2HCl
N
1) R3COCl
DMF
O
35~60%
2) HCl
N
N
HN
HN
N
1) t-BuCOOH
EDC / HOBt
DMF
O
HN
HCl
BocN
HN
N
N
N
THF
83%
N
2HCl
O
O
2) HCl
O
80%
3g
5
6c
N
HN
1)
CHO
N
THF / AcOH
NaBH(OAc)3
N
O
3HCl
2) HCl
84%
6d
Scheme 3.

T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
6211
N
HN
O
O
R4OH
HCOOEt H2NNH2
NaH
THF
NaH
DMF
36~60%
F
R4O
R4O
8a-8c
7
9a-c
26~71%
Scheme 4.
O
EtO
O
O
O
R5
N
N
O
HN
R5
R5
N
HCOOEt H2NNH2
OH
EtO
N
EtO
N
HO
N
N
then HCl
32~91% 2 steps
O
NaH
THF
O
PPh3
DEAD
THF
2HCl
10a-10j
11a-j
12a-j
Scheme 5.
O
EtO
N
HN
O
O
N
NH
HCOOEt H2NNH2
EtOOCN
66%
EtOOCN
Br
N
N
then HCl
NaH
2HCl
Cs2CO3
DMF
THF
82%
15
13
14
Scheme 6.
alkyl group to phenol 2 failed, since intramolecular
nucleophilic attack of the nitrogen atom took the place
of the formation of intermolecular ether. Therefore,
compounds 9a–c were prepared as shown in Scheme 4.
Nucleophilic substitution of 4-fluoroacetophenone 7
with 3equiv of the corresponding alkoxide in N,N-
dimethylformamide at room temperature afforded 8a–c
in moderate yields, which were then treated with ethyl
formate and sodium hydride, and then hydrazine to give
corresponding pyrazole derivatives 9a–c in yields of 26–
58%.17 Compounds 12a–j were prepared as shown in
Scheme 5. The reaction of hydroxyacetophenone deriv-
atives 10a–j with 2-(4-ethoxycarbonylpiperazino)ethanol
under Mitsunobu conditions afforded 11a–j, which were
converted to the corresponding pyrazole derivatives
12a–j by the same method as used in the synthesis of
9a–c. Compound 15 was prepared as shown in Scheme
6. The reaction of 1-bromo-3-(4-acetylphenyl)propane
1318 with N-ethoxycarbonylpiperazine gave amino
derivative 14, which was converted to the corresponding
pyrazole derivative 15 in the same way.
drew upon them for drug design. Calculated ACD-log D
values were also shown in Tables 1–3.
The introduction of a polar functional group such as an
amino group or ether group to the hydrophobic side
chain of 1 improved its water-solubility (3a, 3c, 4a, 4b,
5, 9a, 9b, and 9c, Table 1) as expected. The solubility
of compounds with an acyclic aliphatic amino substitu-
ent (9a, 9b, and 9c) and a cyclic aliphatic amino substit-
uent (3c, 4a, 4b, 5) was about 100-fold better than that
of simple alkoxy derivative 1, however, this was accom-
panied by a great loss of activity. In contrast, methoxy
derivative 3a and some 4-substituted piperazine deri-
vatives (3d, 3e, and 6d) maintained potent activity with
improved solubility. Among these compounds, the sub-
stituent at the 4-position of the piperazine ring obvi-
ously affects their activities. While nonsubstituted (5),
methyl (4a), and isopropyl (4b) derivatives did not show
activity at 100lM, cyclohexylmethyl (6d), 4-fluoro-
benzyl (3d), and ethoxycarbonyl (3e) derivatives showed
potent activities. These results suggest that a rather
bulky substituent for the R group is necessary for potent
activity. However, this hypothesis does not explain the
difference between 3b and 4b. There are slight differences
between the bulkiness of the 4-dimethylaminophenyl
group of 3b and the 4-isopropylpiperazino group of
4b. Another possible explanation is the difference in
the lipophilicity of these compounds. All of the ACD-
log D values of the active compounds shown in Table
1 were greater than 2, and those of the inactive com-
pounds were less than 1. With regard to their ability
to reduce the lipophilicity of xenobiotic chemicals
by oxidation, drug-metabolizing CYPs generally have
3. Results and discussion
All of the compounds synthesized were evaluated with
regard to their inhibitory activity against human renal
microsomal synthesis of 20-HETE17 and water-solubil-
ity at pH6.8. The obtained IC50 values are shown in
Tables 1–3 along with the water-solubility values. We
took particular note between the inhibitory activity
toward 20-HETE synthase and lipophilicity of the com-
pounds, therefore, we calculated ACD-log D values and

6212
T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
Table 1. Inhibition of AA metabolism human 20-HETE synthesizing enzyme by new heterocyclic compounds and their calculated ACD-log D values
(1)
N
HN
(HX)
R1
O
Compound
R1
HX
IC50 (nM)a
Solubility (mg/mL)
at pH6.8
ACD-log Db at pH6.8
1
n-Bu
26.2
67.9
0.014
0.573
3.63
2.42
CH3
3a
HCl
H3C
O
Me 2N
9a
9b
111.0
>300
0.945
>1.05
ꢀ0.03
ꢀ0.90
O
Me2N
Me
N
9c
>300
0.935
0.41
Me2N
Me2N
3b
3c
5.7
0.009
4.22
HCl
>300
>1.021.06
N
HN
5
>300
>300
>1.12
ꢀ0.87
N
N
MeN
4a
3HCl
3HCl
>0.978
0.18
CH3
H3C
4b
>300
>1.020.95
N
N
N
6d
3d
3e
3HCl
32.8
38.2
21.2
>1.12
0.240
0.783
2.88
2.37
2.51
N
N
p-TsOH
N
F
O
H3C
O
2HCl
N
N
a IC50 value for 20-HETE production from AA by human renal microsome (n = 2 ).
b ACD-log D was calculated by ACD/log P DB version 5.0, Advanced Chemistry Development Inc.
higher affinity for more lipophilic compounds.19
Accordingly, 20-HETE synthase, which is a member of
the CYP family, should require appropriate lipophilicity
for its substrate and inhibitors. On the other hand, it is
well known that increased lipophilicity is generally asso-
ciated with a decrease in water-solubility.20 This infor-
mation suggests that a suitable range of lipophilicity is
needed to improve of the water-solubility of 1 while
retaining 20-HETE synthase inhibitory activity. The
preferable lipophilicity of the inhibitor is estimated to
have an ACD-log D value of between 2and 3.
can not explain these results, and accordingly the intro-
duction of a bulky group at this position should be
responsible for the loss of activity. In the case of amide
derivatives 6a–c, the introduction of a bulky substituent
resulted in a loss of activity as in the case of carbamate
derivatives. The introduction of a pivaloyl group (6c) re-
sulted in an almost complete loss of activity, while acetyl
(6a) and butyryl (6b) derivatives maintained potent
activity. These results indicate that the introduction of
a branched alkyl group around the 4-position of the pip-
erazine ring should result in a decrease in activity, and
suggest that there should be steric restriction around
the corresponding 20-HETE synthase active site. Substi-
tution of the carbamate group of 3e with a group other
than an amide group, such as a urea (3h,i) or sulfona-
mide (3j) group, was also shown to be associated with
potent activity, even though the potency of the urea
derivatives was less than that of the corresponding car-
bamate derivatives due to the decrease in lipophilicity.
As the next step in the optimization of the compounds,
the substituent at the 4-position of the piperazine ring of
3e was examined. The ACD-log D values of the com-
pounds were calculated before synthesis, and com-
pounds with an ACD-log D value between 2and 3
were examined.
The solubilities of the isopropyl carbamate (3f) and tert-
butyl carbamate (3g) derivatives were slightly less than
that of the ethyl carbamate derivative (3e) in accordance
with the slight increase in lipophilicity, however, these
compounds showed drastically decreased activity. The
slight difference in the lipophilicity of these compounds
Further modification of 3e was performed, and the re-
sults are shown in Table 3. While extension of the 1,2-
ethylene group linkage between the benzene ring and
the piperazine ring of 3e to a 1,3-propylene group (3k)
did not strongly affect activity, further extension to a

T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
6213
Table 2. Inhibition of AA metabolism human 20-HETE synthesizing
enzyme by new heterocyclic compounds and their calculated ACD-log
D values (2)
group on the benzene ring of 3e was also found to be
critically important for the activity, as in the case of re-
lated isoxazole derivatives we have reported previ-
ously,17 based on a complete loss of activity upon
moving the substituent from the 4-position to the 3-posi-
tion (12a) or 2-position (12b).
N
HN
R2
N
N
(HX)
O
Com- R2
pound
HX
IC50
Solubility ACD-
Finally, the introduction of a substituent on the benzene
ring of 3e was examined. The introduction of a methyl
group to the 3-position (position A) of 3e increased
the activity (12e; IC50 = 3.2nM)), however, the introduc-
tion of a chlorine atom at the same position decreased
the activity (12d; IC50 = 5.6nM), and a fluorine atom
and a methoxy group resulted in a complete loss of
activity (12c, 12f). The effects of the introduction of
these substituents were rather complicated. These results
cannot be simply explained by electrostatic ability, the
size of the substituent, or the lipophilicity of the com-
pounds. Introduction of the same substituents at the 2-
position (position B) of 3e resulted in a great decrease
in activity in all of the compounds (12g–j). These results
indicate that the spatial tolerance around the benzene
ring of 3e has a narrow range in coordination to the
enzyme, especially around the B-position. In addition
to the above examinations, the ether linkage of the 4-
position of the benzene ring was substituted with a
methylene group (15). In contrast to our previous results
with N-hydroxyformamidine derivatives,14 the activity
of 15 (IC50 = 502nM) was decreased to 1/30 of that of
the corresponding ether derivative 3e. This result also
suggests the activity is sensitive to the electrostatic abil-
ity of the substituent on the benzene ring of these
compounds.
(nM)a (mg/mL) log Db
at pH6.8 at pH6.8
O
CH3
3f
2HCl
231.0
0.684
0.119
2.86
3.21
H3C
O
O
3g
>300
t-Bu
O
O
6a
2HCl
2HCl
22.4
14.0
1.15
0.890
>1.15
>1.21
1.31
2.40
2.53
0.94
H3C
O
6b
H3C
O
6c
2HCl >300
2HCl >300
t-Bu
O
3h
H3C
N
H
O
3i
2HCl
49.9
0.980
2.01
H3C
N
H
O
O
3j
2HCl
2HCl
72.5
21.2
0.101
0.783
1.97
2.51
S
H3C
O
3e
H3C
O
a IC50 value for 20-HETE production from AA by human renal
microsome (n = 2 ).
b ACD-log D was calculated by ACD/log P DB version 5.0, Advanced
Chemistry Development Inc.
1,4-butylene group (3l) resulted in a complete loss of
activity. These results indicate that the distance between
the benzene ring and the piperazine ring of 3e strongly
affects the activity and a methylene chain number of 2
or 3 is acceptable. The position of the piperazinoethoxy
Among the compounds synthesized, compounds 3e and
6b were selected for further investigations based to their
potent activity and good solubility (Table 4). Both of
Table 3. Inhibition of AA metabolism human 20-HETE synthesizing enzyme by new heterocyclic compounds and their calculated ACD-log D values
(3)
O
N
EtO
HN
B
N
A
Y
N
(CH2)n
2HCl
Compound
Position
n
Y
A
B
IC50 (nM)a
ACD-log Db
3k
3l
4-
4-
3-
2-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
3
O
O
H
H
H
H
16.3
2.91
3.26
4
>300
.46
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
15
2O
2
H
H
>300
2
O
H
H
>300
.49
2.36
2O
2O
2O
2O
2O
2O
2O
2O
2CH
2
F
Cl
H
H
>1000
2
3.03
85.6
.97
CH
CH
H
H
H
3.22
3
3O
>300
2.19
F
Cl
>1000
>1000
.97
3.15
3.2
H
2
H
CH
CH
3722
>1000
5023.35
21.2
3
H
3O
2.06
2.51
H
H
H
H
2
3e
O
a IC50 value for 20-HETE production from AA by human renal microsome (n = 2 ).
b ACD-log D was calculated at pH6.8 by ACD/log P DB version 5.0, Advanced Chemistry Development Inc.

6214
T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
Table 4. Effects of 3e and 6b on cytochrome P450 (CYP) enzymes
Compound
IC50 (nM) for CYP enzymes
2C19
1A2
2C9
2D6
38,990
>100,000
3A4
86,060
>100,000
3e
6b
>100,000
>100,000
>100,000
49,770
98,860
>100,000
them showed more than 1000-fold selectivity against
major drug-metabolizing CYPs. They did not inhibit
the activity of COX-1 and COX-2at 100 lM. The log
D value of 3e was 2.60, and the log D value of 6b was
2.22. These values corresponded with the ACD-log D
values of them (3e: 2.51; 6b: 2.40). Along with these re-
sults, pharmacological examinations were conducted in
animal models of cerebrovascular disease and these indi-
cated that 3e and 6b possess attractive characteristics as
potential therapeutic agents. The detailed findings
regarding the pharmacological properties of these com-
pounds will be reported in a separate paper.
136.0°C; 1H NMR (300MHz, DMSO-d6) d 1.14 (d,
J = 6.1Hz, 3H), 1.79–1.93 (m, 2H), 3.23 (s, 3H), 3.46–
3.55 (m, 1H), 4.07 (t, J = 6.8Hz, 2H), 6.75 (d,
J = 2.3Hz, 1H), 7.01 (d, J = 8.9Hz, 2H) 7.77 (d,
J = 8.9Hz, 1H), 7.85 (d, J = 2.3Hz, 1H), 9.81 (br s,
1H); MS (ESI) m/z 247 (M+H); Anal. (C14H18-
N2O2ÆHCl) C, H, N. Compounds 3b–m were synthesized
with the corresponding alcohols in the same way.
5.2. 3-(4-Dimethylaminophenethyloxyphenyl)pyrazole
(3b)
1
Yield: 76%, a colorless powder: mp 112.5–113.5°C; H
NMR (200MHz, CDCl3) d 2.94 (s, 6H), 3.02 (t, J =
7.3Hz, 2H), 4.16 (t, J = 7.3Hz, 2H), 6.54 (d,
J = 2.2Hz, 1H), 6.74 (d, J = 8.8Hz, 2H), 6.96 (d, J =
8.8Hz, 2H), 7.18 (d, J = 8.8Hz, 2H), 7.60 (d,
J = 2.2Hz, 1H), 7.65 (d, J = 8.8Hz, 2H); MS (ESI) m/z
330 (M+Na); Anal. (C19H21N3O) C, H, N.
4. Conclusion
The introduction of a polar functional group to a proto-
type pyrazole derivative, the 20-HETE synthase inhibi-
tor 1, led to some promising compounds with potent
activity and good solubility. Among them, compounds
3e and 6b were selected for further investigation for
the treatment of cerebrovascular diseases.
5.3. 3-{4-[2-(1-Piperidino)ethoxy]phenyl}pyrazole hydro-
chloride (3c)
1
Yield: 34%, a colorless powder: H NMR (300MHz,
5. Experimental
DMSO-d6) d 1.38–1.68 (m, 6H), 2.38–2.59 (m, 4H),
2.80 (t, J = 6.0Hz, 2H), 4.15 (t, J = 6.0Hz, 2H), 6.53
(d, J = 2.3Hz, 1H), 6.95 (d, J = 8.9Hz, 2H), 7.59 (d,
J = 2.3Hz, 1H), 7.65 (d, J = 8.9Hz, 2H); MS (ESI) m/z
272 (M+H); Anal. (C16H21N3OÆHClÆ3/2H2O) C, H, N.
Melting points were determined on a Mettler FP-61 or a
Yanaco MP-500D melting point apparatus. NMR spec-
tra were recorded at 200MHz or 300MHz using a Var-
ian Instruments Gemini 2000 or a Varian Instruments
INOVA 300 with tetramethylsilane as an internal stand-
ard. Electron impact (EI) mass spectra were taken on a
Perkin Elmer Sciex API-300 mass spectrometer. Electro-
spray ionization (ESI) mass spectra were taken on a
Micromass Platform LC mass spectrometer. Elemental
analyses were performed on EA2400 elemental analyz-
ers, and the results were within 0.4% of calculated val-
ues. Reactions were monitored by TLC analysis using
Merck silica gel 60F-254 thin-layer plates. Column chro-
matography was carried out on silica gel Wako Pure
Chemical C-200 and NH silica gel Fuji Silicia chromat-
orex DM1020.
5.4. 1-(4-Fluorobenzyl)-4-{2-[4-(pyrazol-3-yl)phenyl]eth-
oxy}piperazine p-toluenesulfonate (3d)
Yield: 47%, a colorless powder: 1H NMR (200MHz,
DMSO-d6) d 2.38 (s, 3H), 6.55 (d, J = 2.2Hz, 1H), 6.87
(d, J = 8.8Hz, 2H), 7.05 (d, J = 8.8Hz, 2H), 7.20–7.40
(m, 4H), 7.60 (d, J = 2.2Hz, 1H), 7.66 (d, J = 8.8Hz,
2H), 7.81 (d, J = 8.4Hz, 2H); MS (ESI) m/z 381
(M+H); Anal. (C22H25FN4OÆC7H8O3SÆ3/2H2O) C, H, N.
5.5. 1-Ethoxycarbonyl-4-{2-[4-(pyrazol-3-yl)phenyl]eth-
oxy}piperazine dihydrochloride (3e)
5.1. 3-[4-(3-Methoxybutoxy)phenyl]pyrazole hydrochlo-
ride (3a)
Yield: 73%, a colorless powder: mp 217.0–218.0°C
(dec); 1H NMR (300MHz, DMSO-d6) d 1.27 (t,
J = 7.1Hz, 3H), 2.58 (m, 4H), 2.86 (m, 2H), 3.54 (m,
4H), 4.11–4.20 (m, 2H), 4.15 (q, J = 7.1Hz, 2H), 6.55
(d, J = 2.4Hz, 1H), 6.97 (d, J = 8.6Hz, 2H), 7.61 (d,
J = 2.4Hz, 1H), 7.68 (d, J = 8.6Hz, 2H); MS (ESI) m/z
345 (M+H); Anal. (C18H24N4O3Æ2HClÆH2O) C, H, N.
To a mixture of 2 (0.25g, 1.57mmol) and 3-methoxybut-
anol (0.294g, 2.83mmol; 1.8equiv) in THF (7.5mL) was
added
a mixture of triphenylphosphine (0.823g,
3.14mmol; 2equiv) and diethylazodicarboxylate
(0.494mL, 3.14mmol; 2equiv), and the mixture was stir-
red at room temperature for 16h. The reaction mixture
was evaporated in vacuo and then purified by silica gel
column chromatography to give free base of 3a
(0.220g, yield: 47%), which was treated with 4M HCl
in EtOAc to give 3a as a colorless powder: mp 133.5–
5.6. 1-Isopropoxycarbonyl-4-{2-[4-(pyrazol-3-yl)phen-
yl]ethoxy}piperazine dihydrochloride (3f)
1
Yield: 43%, a colorless powder: mp 206.0–208.0°C; H
NMR (300MHz, DMSO-d6) d 1.21 (d, J = 6.2Hz,

T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
6215
6H), 3.04–3.22 (m, 2H), 3.26–3.44 (m, 2H), 3.49–3.65
(m, 4H), 4.05 (d, J = 13.4Hz, 2H), 4.48 (t, J = 4.8Hz,
2H), 4.77–4.85 (m, 1H), 6.72 (d, J = 2.2Hz, 1H), 7.07
(d, J = 8.9Hz, 2H), 7.77–7.83 (m, 3H), 11.47 (br s,
1H); MS (ESI) m/z 359 (M+H); Anal. (C19H26-
N4O3Æ2HCl) C, H, N.
MS (ESI) m/z 381 (M+Na); Anal. (C19H26N4O3Æ2H-
ClÆ1/2H2O) C, H, N.
5.12. 1-Ethoxycarbonyl-4-{4-[4-(pyrazol-3-yl)phenyl]but-
oxy}piperazine dihydrochloride (3l)
1
Yield: 4%, colorless amorphous: H NMR (300MHz,
5.7. 1-tert-Butoxycarbonyl-4-{2-[4-(pyrazol-3-yl)phen-
yl]ethoxy}piperazine (3g)
DMSO-d6) d 1.21 (t, J = 7.2Hz, 3H), 3.17 (m, 2H),
3.35 (m, 2H), 3.56 (m, 4H), 4.05–4.10 (m, 2H), 4.08 (q,
J = 7.2Hz, 2H), 4.48 (t, J = 5.0Hz, 2H), 6.74 (d, J =
2.2Hz, 1H), 6.94 (dd, J = 1.6, 8.0Hz, 1H), 7.36
(t, J = 8.0Hz, 1H), 7.44–7.46 (m, 2H), 7.74 (d,
J = 2.2Hz, 1H); MS (ESI) m/z 373 (M+H). HPLC:
Purity 95.08%.
1
Yield: 26%, a colorless powder: H NMR (300MHz,
CDCl3) d 1.46 (s, 9H), 2.53–2.56 (m, 4H), 2.84 (t,
J = 5.8Hz, 2H), 3.45–3.48 (m, 4H), 4.15 (t, J = 5.8Hz,
2H), 6.53 (d, J = 2.3Hz, 1H), 6.96 (m, 2H), 7.60 (d,
J = 2.3Hz, 1H), 7.67 (m, 2H); MS (ESI) m/z 373
(M+H); Anal. (C20H28N4O3Æ1/2H2O) C, H, N.
5.13. 3-[4-(2-Chloroethoxy)phenyl]pyrazole (3m)
5.8. 1-Ethylaminocarbonyl-4-{2-[4-(pyrazol-3-yl)phen-
yl]ethoxy}piperazine dihydrochloride (3h)
72% yield, colorless powder: 1H NMR (200MHz,
CDCl3) d 3.85 (t, J = 5.5Hz, 2H), 4.28 (t, J =5.5Hz,
2H), 6.58 (d, J = 2.0Hz, 1H), 6.98 (m, JAB = 9.5Hz,
2H), 7.61 (d, J = 2.0Hz, 1H), 7.69 (m, JAB = 9.5Hz, 2H).
1
Yield: 50%, a colorless powder: mp 138.0–144.0°C; H
NMR (300MHz, DMSO-d6) d 1.02(t, J = 7.2Hz, 3H),
2.98–3.08 (m, 2H), 3.06 (q, J = 7.2Hz, 2H), 3.25 (t,
J = 12.4Hz, 2H), 3.46–3.61 (m, 4H), 4.07 (d,
J = 13.8Hz, 2H), 4.49 (m, 2H), 6.80 (d, J = 2.3Hz,
1H), 7.09 (d, J = 8.9Hz, 2H), 7.84 (d, J = 8.9Hz, 2H),
7.88 (d, J = 2.3Hz, 1H), 11.45 (br s, 1H); MS (ESI)
m/z 344 (M+H); Anal. (C18H25N5O2Æ2HClÆ2H2O) C,
H, N.
5.14. 3-{4-[2-(4-Methylpiperazino)ethoxy]phenyl}pyraz-
ole trihydrochloride (4a)
To a solution of 3m (0.3g, 0.35mmol) in DMF (3mL)
was added N-methylpiperazine (0.406g, 4.06mmol,
3equiv) and triethylamine (0.411g, 4.06mmol, 3equiv)
and the mixture was stirred for 9h at 120°C. The reac-
tion mixture was concentrated in vacuo and the residue
was purified by silica gel column chromatography (elu-
ent: hexane/ethyl acetate = 1:5) to give 0.374g (yield:
97%) of free base of 4a as a colorless oil, which was trea-
ted with 4M HCl in EtOAc to give 4a as a colorless
powder: mp 159.0–163.0°C; 1H NMR (300MHz,
DMSO-d6) d 2.84 (s, 3H), 3.43–3.92 (m, 10H), 4.42–
4.51 (m, 2H), 6.69 (d, J = 2.2Hz, 1H), 7.08 (d,
J = 8.9Hz, 2H), 7.74 (d, J = 2.2Hz, 1H), 7.78 (d,
J = 8.9Hz, 2H), 11.95 (br s, 1H); MS (ESI) m/z 287
(M+H); Anal. (C16H22N4OÆ3HClÆ3H2O) C, H, N.
5.9. 1-Butylaminocarbonyl-4-{2-[4-(pyrazol-3-yl)phen-
yl]ethoxy}piperazine dihydrochloride (3i)
1
Yield: 85%, a colorless powder: mp 202.0–204.5°C; H
NMR (300MHz, DMSO-d6) d 0.87 (t, J = 7.23Hz,
3H), 1.09–1.53 (m, J = 133.05Hz, 4H), 2.99–3.23 (m,
6H), 3.51–3.63 (m, 4H), 3.97–4.14 (m, 2H), 4.40–4.46
(m, 2H), 6.64 (d, J = 2.18Hz, 1H), 7.05 (d,
J = 8.86Hz, 2H), 7.69 (d, J = 2.33Hz, 1H), 7.76 (d,
J = 8.70Hz, 2H); MS (ESI) m/z 372(M+H); Anal.
(C20H29N5O2Æ2HClÆH2O) C, H, N.
5.15. 1-Isopropyl-4-{2-[4-(pyrazol-3-yl)phenyl]eth-
oxy}piperazine trihydrochloride (4b)
5.10. 1-Ethylsulfonyl-4-{2-[4-(pyrazol-3-yl)phenyl]eth-
oxy}piperazine dihydrochloride (3j)
Yield: 57%, a colorless powder: mp 215.0–220.0°C
(dec); 1H NMR (300MHz, DMSO-d6) d 1.31 (d,
J = 6.5Hz, 6H), 3.48–3.93 (m, 11H), 4.41–4.50 (m,
2H), 6.70 (d, J = 2.2Hz, 1H), 7.09 (d, J = 9.0Hz, 2H),
7.76 (d, J = 2.3Hz, 1H), 7.79 (d, J = 8.9Hz, 2H), 11.96
(br s, 1H); MS (ESI) m/z 315 (M+H); Anal. (C18H26-
N4OÆ3HClÆ2H2O) C, H, N.
Yield: 69%, a colorless powder: mp 196.0–198.0°C
(dec); 1H NMR (300MHz, DMSO-d6) d 1.23 (t,
J = 7.4Hz, 3H), 3.13–3.84 (m, 10H), 3.19 (q,
J = 7.4Hz, 2H), 4.44–4.51 (m, 2H), 6.69 (d, J = 2.3Hz,
1H), 7.07 (d, J = 8.9Hz, 2H), 7.74 (d, J = 2.2Hz, 1H),
7.78 (d, J = 8.9Hz, 2H), 11.45 (br s, 1H); MS (ESI)
m/z 387 (M+Na); Anal. (C17H24N4O3SÆ2HClÆH2O) C,
H, N.
5.16. 1-{2-[4-(Pyrazol-3-yl)phenyl]ethoxy}piperazine (5)
5.11. 1-Ethoxycarbonyl-4-{3-[4-(pyrazol-3-yl)phen-
yl]propoxy}piperazine dihydrochloride (3k)
To a solution of 3g (5g, 13.42mmol, 1equiv) in THF
(25mL) was added 4M HCl in EtOAc (33.6mL,
10equiv). The resulting mixture was stirred at room
temperature for 4h, and 5N NaOH (26mL) was then
added to the reaction mixture, and extracted with chlo-
roform. The organic layer was dried over magnesium
sulfate and evaporated in vacuo. The residue was recrys-
tallized from diethyl ether to give 3.04g (yield: 83%) of 5
1
Yield: 22%, a colorless powder: mp 202.0–203.0°C; H
NMR (300MHz, DMSO-d6) d 1.20 (t, J = 7.2Hz, 3H),
3.64 (br t, J = 4.8Hz, 2H), 4.08 (q, J = 7.2Hz, 2H),
4.56 (br t, J = 4.8Hz, 2H), 6.81 (d, J = 2.2Hz, 1H),
7.10 (m, 1H), 7.20 (d, J = 7.7Hz, 1H), 7.40 (m, 1H),
7.79 (dd, J = 1.8, 7.7Hz, 1H), 7.84 (d, J = 2.2Hz, 1H);
1
as a slightly red powder. Mp 148.0–151.0°C; H NMR

6216
T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
(200MHz, CDCl3) d 2.55–2.59 (m, 4H), 2.82 (t,
J = 5.9Hz, 2H), 2.91–2.96 (m, 4H), 4.15 (t, J = 5.9Hz,
2H), 6.54 (d, J = 2.2Hz, 1H), 6.96 (m, 2H), 7.60 (d,
J = 2.2Hz, 1H), 7.66 (m, 2H); MS (ESI) m/z 273
(M+H); Anal. (C15H20N4OÆ1/4H2O) C, H, N.
5.20. 1-Cyclohexylmethyl-4-{2-[4-(pyrazol-3-yl)phen-
yl]ethoxy}piperazine trihydrochloride (6d)
To a mixture of 5 (0.25g, 0.925mmol), acetic acid
0.167g (2.77mmol, 3equiv) and THF (1.5mL) were
added in succession cyclohexanecarbaldehyde (0.156g,
1.39mmol, 1.5equiv) and sodium triacetoxyborohydride
(0.588g, 2.77mmol, 3equiv), and the mixture was then
stirred at room temperature for 2.5h. To the reaction
mixture was added 5N NaOH (2mL), and the mixture
was extracted with ethyl acetate. The organic layer was
dried over magnesium sulfate, evaporated in vacuo,
and purified by silica gel column chromatography (elu-
ent: chloroform/methanol = 10:1) to give free base of
6d (0.285g, yield: 84%) as a colorless powder, which
was treated with 4M HCl in EtOAc to give 6d as a col-
orless powder: mp 272.0–273.0°C (dec); 1H
NMR (300MHz, DMSO-d6) d 0.86–1.04 (m, 2H),
1.07–1.31 (m, 3H), 1.55–1.96 (m, 6H), 3.00 (br s, 2H),
5.17. 1-Acetyl-4-{2-[4-(pyrazol-3-yl)phenyl]ethoxy}piper-
azine (6a)
To a solution of 5 (0.25g, 0.925mmol) in DMF (7.5mL)
was added acetyl chloride (0.087g, 1.02mmol,
1.1equiv), and the mixture was stirred at 0°C for 2h.
To the reaction mixture was added 5N NaOH (2mL),
and the mixture was extracted with toluene. The organic
layer was dried over magnesium sulfate, evaporated in
vacuo, and then purified by NH silica gel column chro-
matography (eluent: chloroform/methanol = 10:1) to
give free base of 6a (0.10g, yield: 35%) as a colorless
amorphous, which was treated with 4M HCl in EtOAc
to give 6a as a colorless powder. Mp 221.5–225.0°C
3.36–3.92(m, 10H), 4.45–4.51 (m, H2 ), 6.72(d,
J =
1
2.2Hz, 1H), 7.08 (d, J = 8.9Hz, 2H), 7.78 (d, J =
2.2Hz, 1H), 7.80 (d, J = 9.0Hz, 2H), 11.38 (br s, 1H);
MS (ESI) m/z 369 (M+H); Anal. (C22H32N4OÆ3HClÆ
2H2O) C, H, N.
(dec); H NMR (300MHz, DMSO-d6) d 2.05 (s, 3H),
2.96–3.28 (m, 3H), 3.47–3.67 (m, 5H), 3.93–4.07 (m,
1H), 4.35–4.47 (m, 1H), 4.48 (t, J = 4.9Hz, 2H), 6.72
(d, J = 2.3Hz, 1H), 7.06 (d, J = 9.0Hz, 2H), 7.76–7.83
(m, 3H), 11.52(br s, 1H); MS (ESI) m/z 315 (M+H);
Anal. (C17H22N4O2Æ2HClÆH2O) C, H, N.
5.21. 40-(4-Dimethylaminobutoxy)acetophenone (8a)
To a stirred suspension of sodium hydride (60% in oil,
1.74g, 43.5mmol, 3equiv) in DMF (29mL) was added
5.18. 1-Propionyl-4-{2-[4-(pyrazol-3-yl)phenyl]eth-
oxy}piperazine dihydrochloride (6b)
a
solution of 4-dimethylamino-1-butanol (5.10g,
43.5mmol, 3equiv) in DMF (7.2mL) at room tempera-
ture. The reaction mixture was stirred at room tempera-
ture for 30min, and 40-fluoroacetophenone (2g,
14.5mmol, 1equiv) was then added to the mixture at
0°C. After stirring for 3h, the reaction mixture was di-
luted with water, and extracted with ethyl acetate. The
organic layer was washed with brine, dried over magne-
sium sulfate, evaporated in vacuo, and purified by NH
silica gel column chromatography (eluent: hexane/ethyl
acetate = 2:1) to give 2.03g (yield: 60%) of 8a as a yellow
Yield: 60%, a colorless powder: mp 204.0–207.0°C
(dec); 1H NMR (200MHz, DMSO-d6) d 0.97 (t,
J = 7.5Hz, 3H), 1.66 (sext, J = 7.5Hz, 2H), 2.31 (t,
J = 7.5Hz, 2H), 2.54–2.64 (m, 4H), 2.85 (t, J = 5.6Hz,
2H), 3.51 (t, J = 5.0Hz, 2H), 3.67 (t, J = 5.0Hz, 2H),
4.15 (t, J = 5.6Hz, 2H), 6.54 (d, J = 2.3Hz, 1H), 6.95
(m, 2H), 7.60 (d, J = 2.3Hz, 1H), 7.67 (m, 2H); MS
(ESI) m/z 343 (M+H); Anal. (C19H26N4O2Æ2HClÆ
3/2H2O) C, H, N.
1
oil: H NMR (200MHz, CDCl3) d 1.56–1.94 (m, 4H),
5.19. 1-Pivaloyl-4-{2-[4-(pyrazol-3-yl)phenyl]ethoxy}pip-
erazine (6c)
2.24 (s, 6H), 2.33 (t, J = 7.0Hz, 2H), 2.56 (s, 3H), 4.05
(t, J = 6.0Hz, 2H), 6.93 (m, JAB = 9.5Hz, 2H), 7.93
(m, JAB = 9.5Hz, 2H). Compounds 8b and 8c were syn-
thesized in the same way.
To a mixture of 5 (0.248g, 0.918mmol), pivaric acid
(0.094g, 0.918mmol, 1equiv) and HOBt 0.149g
(1.10mmol, 1.2equiv) in DMF (1.5mL) was added
WSCÆHCl (0.211g, 10mmol, 1.2equiv), and the mixture
was stirred at room temperature for 19h. To the reac-
tion mixture was added 5N NaOH solution (2mL),
and the mixture was extracted with ethyl acetate. The
organic layer was dried over magnesium sulfate, evapo-
rated in vacuo, and then purified by silica gel column
chromatography (eluent: chloroform/methanol = 10:1)
to give free base of 6c (0.26g, yield: 80%) as a colorless
powder, which was treated with 4M HCl in EtOAc
to give 6c as a colorless powder: mp 272.0–273.0°C
5.22. 40-[2-(2-Dimethylaminoethoxy)ethoxy]acetophenone
(8b)
1
Yield: 36%, a yellow oil: H NMR (200MHz, CDCl3) d
2.28 (s, 6H), 2.55 (t, J = 5.5Hz, 2H), 2.58 (s, 3H), 3.66 (t,
J = 6.0Hz, 2H), 3.85 (t, J = 5.0Hz, 2H), 4.21 (t,
J = 5.0Hz, 2H), 6.95 (m, JAB = 8.5Hz, 2H), 7.93 (m,
JAB = 8.5Hz, 2H).
5.23. 40-{2-[N,N-(2-Dimethylaminoethyl)methyl-
amino]ethoxy}acetophenone (8c)
1
(dec); H NMR (200MHz, DMSO-d6) d 1.210 (s, 9H),
3.01–3.65 (m, 8H), 4.34–4.52(m, 4H), 6.69 (d,
J = 2.2Hz, 1H), 7.07 (m, JAB = 8.8Hz, 2H), 7.75 (d,
J = 2.2Hz, 1H), 7.78 (m, JAB = 8.8Hz, 2H), 11.43 (br
s, 1H); MS (ESI) m/z 379 (M+Na); Anal. (C20H28N4O2Æ
2HClÆ1/2H2O) C, H, N.
Yield: 43%, a light yellow oil: 1H NMR (200MHz,
CDCl3) d 2.25 (s, 6H), 2.38–2.49 (m, 2H), 2.39 (s, 3H),
2.54–2.68 (m, 2H), 2.56 (s, 3H), 2.83–2.93 (m, 2H),
4.14 (t, J = 6.0Hz, 2H), 6.94 (m, JAB = 9.5Hz, 2H),
7.94 (m, JAB = 9.5Hz, 2H).

T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
6217
5.24. 3-[4-(4-Dimethylaminobutoxy)phenyl]pyrazole (9a)
5.28. 1-Ethoxycarbonyl-4-{2-[3-(pyrazol-3-yl)phenyl]eth-
oxy}piperazine dihydrochloride (12a)
Ethyl formate (0.378g, 5.10mmol; 4equiv) was added to
a suspension of sodium hydride (60% in oil, 0.101g,
2.54mmol, 2equiv) in THF (1mL) at room temperature.
After stirring for 10min, a solution of 8a (0.3g,
1.27mmol, 1equiv) in THF (2mL) was added, and the
mixture was stirred for an additional 30min. To the
reaction mixture was then added 1M HCl (2.7mL),
and the mixture was separated. The aqueous layer was
washed with diethyl ether, hydrazine monohydrate
(0.636g, 12.7mmol, 10equiv) was added, and the mix-
ture was stirred for 30min. The reaction mixture was
made alkaline by adding of 6N NaOH (2mL) and ex-
tracted with ethyl acetate. The organic layer was washed
with brine, dried over magnesium sulfate, evaporated in
vacuo, and purified by NH silica gel column chromato-
graphy (eluent: ethyl acetate) to give a colorless solid,
which was recrystallized from hexane/ethyl acetate to
give 0.19g (yield: 58%) of 9a as a colorless powder.
A
mixture of 11a (0.50g,1.56mmol), which was
prepared from 10a by Mitsunobu alkylation, and
dimethylaminomethyl-tert-butylether (1.36g, 7.8mmol;
5equiv) was stirred at 90°C for 30min. The reaction
mixture was then cooled to room temperature and
immediately purified by silica gel column chromatogra-
phy. The resulting yellow oil was diluted with THF
(10mL) and hydrazine monohydrate (0.32g, 6.39mmol;
4.1equiv) was added. The mixture was then stirred at
room temperature for 16h. The reaction mixture was di-
luted with brine and extracted with ethyl acetate. The
organic layer was dried over magnesium sulfate, evapo-
rated in vacuo, and purified by NH silica gel column
chromatography to give free base of 12a (0.325g, yield:
61%), which was treated with 4M HCl in EtOAc to give
12a as a colorless powder: mp 133.5–136.0°C; 1H NMR
(300MHz, DMSO-d6) d 1.21 (t, J = 7.2Hz, 3H), 3.05–
3.63 (m, 8H), 4.01–4.10 (m, 2H), 4.08 (q, J = 7.2Hz,
2H), 4.44–4.52 (m, 2H), 6.74 (d, J = 2.2Hz, 1H), 6.87–
6.98 (m, 1H), 7.36 (t, J = 8.0Hz, 2H), 7.42–7.48
(m, 2H), 7.71–7.76 (m, 1H), 11.10 (br s, 1H); MS
(ESI) m/z 345 (M+H); Anal. (C18H24N4O3Æ2HClÆ
3/2H2O) C, H, N. Compounds 12b–j were synthesized
in the same way.
1
Mp 76.0–78.0°C; H NMR (200MHz, CDCl3) d 1.60–
1.91 (m, 4H), 2.25 (s, 6H), 2.34 (t, J = 7.3Hz, 2H),
4.02(t, J = 6.2Hz, 2H), 6.54 (d, J = 2.2Hz, 1H), 6.95
(m, JAB = 8.8Hz, 2H), 7.60 (d, J = 2.2Hz, 1H), 7.65
(m, JAB = 8.6Hz, 2H); MS (ESI) m/z 260 (M+H); Anal.
(C15H21N3OÆ1/4H2O) C, H, N. Compounds 9b and 9c
were prepared in the same way.
5.29. 1-Ethoxycarbonyl-4-{2-[2-(pyrazol-3-yl)phenyl]eth-
oxy}piperazine dihydrochloride (12b)
5.25. 3-{4-[2-(2-Dimethylaminoethoxy)ethoxy]phen-
yl}pyrazole (9b)
1
Yield: 42%, a colorless powder: H NMR (200MHz,
1
Yield: 71%, a brown powder: mp 130.0–138.5°C; H
DMSO-d6) d 1.20 (t, J = 7.2Hz, 3H), 3.01–3.98 (m,
8H), 3.64 (br t, J = 4.8Hz, 2H), 4.08 (q, J = 7.2Hz,
2H), 4.56 (br t, J = 4.8Hz, 2H), 6.81 (d, J = 2.2Hz,
1H), 7.10 (m, 1H), 7.20 (d, J = 7.7Hz, 1H), 7.35–
7.44 (m, 1H), 7.79 (dd, J = 1.8, 7.7Hz, 1H), 7.84
(d, J = 2.2Hz, 1H), 11.75 (br s, 1H); MS (ESI)
m/z 367 (M+H); Anal. (C18H24N4O3Æ2HClÆ1/2H2O) C,
H, N.
NMR (200MHz, DMSO-d6) d 2.76 (s, 3H), 2.78 (s,
3H), 3.28 (q, J = 5.2 Hz, 2 H), 3.78–3.89 (m, 4H),
4.15–4.24 (m, 2H), 6.75 (d, J = 2.2Hz, 1H), 7.02 (m,
JAB = 8.8Hz, 2H), 7.79 (m, JAB = 8.6Hz, 2H), 7.84
(d, J = 2 .2 Hz, 1H), 10.52 (br s, 1H); MS (ESmI)/z
276 (M+H); Anal. (C15H21N3O2Æ2HClÆ1/2H2O) C,
H, N.
5.26. 3-{4-[2-(N-Methyl-2-dimethylaminoethylamino)eth-
oxy]phenyl}pyrazole (9c)
5.30. 1-Ethoxycarbonyl-4-{2-[2-fluoro-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12c)
Yield: 26%, a colorless powder: mp 38.5–41.0°C; 1H
NMR (200MHz, CDCl3) d 2.30 (s, 6H), 2.39 (s, 3H),
2.49 (m, 2H), 2.65 (m, 2H), 2.87 (t, J = 5.9Hz, 2H),
4.13 (t, J = 5.9Hz, 2H), 6.55 (d, J = 2.2Hz, 1H), 6.96
(d, J = 8.8Hz, 2H), 7.60 (d, J = 2.2Hz, 1H), 7.66 (d,
J = 8.8Hz, 2H); MS (ESI) m/z 311 (M+Na); Anal.
(C16H24N4OÆ1/2H2O) C, H, N.
1
Yield: 83%, a colorless powder: mp 207.0–208.5°C; H
NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.07Hz,
3H), 3.07–3.66 (m, 8H), 3.99–4.14 (m, 2H), 4.09 (q,
J = 7.15Hz, 2H), 4.51–4.59 (m, 2H), 6.74 (d,
J = 2.33Hz, 1H), 7.29 (t, J = 8.70Hz, 1H), 7.61–7.77
(m, 3H), 11.49 (br s, 1H); MS (ESI) m/z 363 (M+H);
Anal. (C18H23FN4O3Æ2HCl) C, H, N.
5.27. 1-Ethoxycarbonyl-4-{3-[4-(pyrazol-3-yl)phenyl]prop-
yl}piperazine dihydrochloride (15)
5.31. 1-Ethoxycarbonyl-4-{2-[2-chloro-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12d)
1
Yield: 82%, a light yellow powder: mp 175.0–177.5°C;
1H NMR (200MHz, DMSO-d6) d 1.20 (t, J = 7.03Hz,
3H), 1.91–2.19 (m, 2H), 2.67 (t, J = 7.47Hz, 2H),
2.85–3.15 (m, 4H), 3.23–3.55 (m, 4H), 3.93–4.07 (m,
2H), 4.08 (q, J = 7.18Hz, 2H), 6.72 (d, J = 2.20Hz,
1H), 7.30 (d, J = 8.35Hz, 2H), 7.73–7.81 (m, 3H),
11.16 (br s, 1H); MS (ESI) m/z 343 (M+H); Anal.
(C19H26N4O2Æ2HClÆH2O) C, H, N.
Yield: 59%, a colorless powder: mp 202.0–203.0°C; H
NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.2Hz, 3H),
3.13–3.48 (m, 4H), 3.53–3.66 (m, 4H), 4.00–4.14 (m,
4H), 4.57 (br t, J = 4.8Hz, 2H), 6.76 (d, J = 2.3Hz,
1H), 7.26 (d, J = 8.7Hz, 1H), 7.75 (d, J = 2.2Hz, 1H),
7.78 (dd, J = 2.2, 8.5Hz, 1H), 7.91 (d, J = 2.0Hz, 1H);
MS (ESI) m/z 379 (M+H); Anal. (C18H23ClN4O3Æ2HClÆ
H2O) C, H, N.

6218
T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
5.32. 1-Ethoxycarbonyl-4-{2-[2-methyl-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12e)
5.37. 1-Ethoxycarbonyl-4-{2-[3-methoxy-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12j)
1
Yield: 32%, a colorless powder: mp 203.5–204.5°C;
1H NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.2Hz,
3H), 2.24 (s, 3H), 3.10–3.45 (m, 4H), 3.50–3.65
(m, 4H), 4.03–4.16 (m, 4H), 4.44 (t, J = 4.5Hz, 2H),
6.64 (d, J = 2.2Hz, 1H), 7.01 (d, J = 8.2Hz, 2H), 7.58–
7.65 (m, 2H), 7.70 (d, J = 2.2Hz, 1H); MS (ESI)
m/z 359 (M+H); Anal. (C19H26N4O3Æ2HClÆH2O) C,
H, N.
Yield: 69%, a colorless powder: mp 185.0–186.0°C; H
NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.07Hz,
3H), 3.04–3.64 (m, 8H), 3.89 (s, 3H), 4.05–4.09 (m,
2H), 4.09 (q, J = 7.10Hz, 2H), 4.49 (m, 2H), 6.66–6.80
(m, 3H), 7.71–7.79 (m, 2H), 11.32 (br s, 1H); MS
(ESI) m/z 375 (M+H); Anal. (C19H26N4O4Æ2HClÆH2O)
C, H, N.
5.38. 1-Ethoxycarbonyl-4-[3-(4-acetylphenyl)propyl]pip-
erazine (14)
5.33. 1-Ethoxycarbonyl-4-{2-[2-methoxy-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12f)
N-Ethoxycarbonylpiperazine (1.513g, 9.56mmol, 1equiv)
and cesium carbonate (6.40g, 19.6mmol, 2.05equiv)
were added to a solution of 13 (2.81g, 11.66mmol,
1.22equiv) in DMF (15mL), and the mixture was stirred
at room temperature for 16h.The reaction mixture was
diluted with ethyl acetate (200mL) after insoluble mate-
rial was removed by filtration, washed with brine, con-
centrated in vacuo, and then purified by silica gel
column chromatography (eluent: hexane/ethyl ace-
tate = 4:1 to ethyl acetate) to give 2.00g (yield: 66%)
Yield: 71%, a colorless powder: mp 189.5–191.0°C;
1H NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.2Hz,
3H), 3.08–3.25 (m, 2H), 3.28–3.45 (m, 2H), 3.51–
3.68 (m, 4H), 3.86 (s, 3H), 4.00–4.15 (m, 4H), 4.44
(br t, J = 4.8Hz, 2H), 6.75 (d, J = 2.2Hz, 1H), 7.10
(d, J = 8.2Hz, 1H), 7.38 (dd, J = 1.9, 8.2Hz, 1H), 7.49
(d, J = 1.9Hz, 1H), 7.77 (d, J = 2.2Hz, 1H); MS
(ESI) m/z 375 (M+H); Anal. (C19H26N4O4Æ2HClÆH2O)
C, H, N.
1
of 14 as a yellow oil: H NMR (300MHz, CDCl3) d
1.26 (t, J = 6.5Hz, 3H), 1.84 (m, 2H), 2.31–2.44 (m,
6H), 2.59 (s, 3H), 2.70 (t, J = 7.0Hz, 2H), 3.41–3.56
(m, 4H), 4.14 (q, J = 6.5Hz, 2H), 7.28 (d, J = 7.5Hz,
2H), 7.88 (d, J = 8.0Hz, 2H).
5.34. 1-Ethoxycarbonyl-4-{2-[3-fluoro-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12g)
1
Yield: 91%, a colorless powder: mp 199.5–201.0°C; H
5.39. 4-[3-(N0-Ethoxycarbonylpiperazino)propyl]-1-(3-
pyrazolyl)benzene (15)
NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.10Hz,
3H), 3.04–3.43 (m, 4H), 3.49–3.61 (m, 4H), 3.99–4.05
(m, 2H), 4.09 (q, J = 7.10Hz, 2H), 4.42–4.51 (m, 2H),
6.55–6.59 (m, 1H), 6.91–7.06 (m, 2H), 7.75 (d,
J = 2.18Hz, 1H), 7.87 (t, J = 8.86Hz, 1H), 11.02(br s,
1H); MS (ESI) m/z 363 (M+H); Anal. (C18H23-
FN4O3Æ2HClÆ1/2H2O) C, H, N.
This compound was prepared from 14 (0.732g,
2.30mmol) as described in the procedure for synthesiz-
ing 9a to give 0.646g (1.89mmol, 82%) of 15 as a light
yellow amorphous (2HCl salt, light yellow powder):
1
mp 175.0–177.5°C; H NMR (200MHz, DMSO-d6) d
1.20 (t, J = 7.03Hz, 3H), 1.91–2.19 (m, 2H), 2.67 (t,
J = 7.47Hz, 2H), 2.85–3.15 (m, 4H), 3.23–3.55 (m,
4H), 3.93–4.07 (m, 2H), 4.08 (q, J = 7.18Hz, 2H), 6.72
(d, J = 2.20Hz, 1H), 7.30 (d, J = 8.35Hz, 2H), 7.73–
7.81 (m, 3H), 11.16 (br s, 1H); MS (ESI) m/z 343
(M+H); Anal. (C19H26N4O2Æ2HClÆH2O) C, H, N.
5.35. 1-Ethoxycarbonyl-4-{2-[3-chloro-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12h)
1
Yield: 85%, a colorless powder: mp 193.0–195.0°C; H
NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.07Hz,
3H), 3.07–3.40 (m, 4H), 3.50–3.63 (m, 4H), 4.03–4.10
(m, 2H), 4.09 (q, J = 7.05Hz, 2H), 4.44–4.51 (m, 2H),
6.65 (d, J = 2.18Hz, 1H), 7.07 (dd, J = 8.78, 2.56Hz,
1H), 7.21 (d, J = 2.64Hz, 1H), 7.71 (d, J = 8.70Hz,
1H), 7.75 (d, J = 2.18Hz, 1H), 10.88 (br s, 1H); MS
(ESI) m/z 379 (M+H); Anal. (C18H23ClN4O3Æ2HClÆ1/
2H2O) C, H, N.
6. Measurement of the inhibition of 20-HETE formation
from AA
Test compounds were dissolved in DMSO, diluted with
50mM MOPS/5mM MgCl2/1mM EDTA (pH7.4) buf-
fer (final concentration of DMSO: 1%), and incubated
with human renal microsome (HCCC, Laurel, MD,
USA) (100lg/mL) at 37°C for 5min. [3H]-Arachidonic
acid (2lC/mL) and NADPH (1mM) were added to
the mixture, which was then incubated at 37°C for
10min. The reaction was terminated by adding formic
acid (pH3.5). Metabolites of AA were separated on col-
umn filled with ODS and the radioactivity of the eluate
containing [3H]-20-HETE was measured by a liquid
scintillation counter. IC50 values were determined by
curve-fitting and parameter estimation using Origin
6.0J (OriginLab Corp., MA, USA).
5.36. 1-Ethoxycarbonyl-4-{2-[3-methyl-4-(pyrazol-3-
yl)phenyl]ethoxy}piperazine dihydrochloride (12i)
1
Yield: 83%, a colorless powder: mp 196.5–199.0°C; H
NMR (300MHz, DMSO-d6) d 1.21 (t, J = 7.15Hz,
3H), 2.41 (s, 3H), 3.03–3.63 (m, 8H), 4.01–4.10 (m,
2H), 4.09 (q, J = 7.15Hz, 2H), 4.42 (m, 2H), 6.44 (d,
J = 2.18Hz, 1H), 6.87–6.95 (m, 2H), 7.46 (d,
J = 8.39Hz, 1H), 7.71 (d, J = 2.18Hz, 1H), 10.89 (br s,
1H); MS (ESI) m/z 359 (M+H); Anal. (C19H26N4O3Æ
2HClÆH2O) C, H, N.

T. Nakamura et al. / Bioorg. Med. Chem. 12 (2004) 6209–6219
6219
8. Gebremedhin, D.; Lange, R. D.; Lowry, T. F.; Taheri, M.
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7. Measurement of solubility
About 2mg of each compound was added to 2mL of
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ken at room temperature for 24h. The suspension was
then centrifuged for 10min at 11,000rpm. The superna-
tant was diluted with 50% aqueous acetonitrile for
HPLC analysis. The concentration of the compounds
was determined by a Shimadzu HPLC system composed
of an LC-10AD, SPD-10AV, and SIL-10A. The condi-
tions for HPLC were as follows: mobile phase,
10mmol/L aqueous ammonium acetate solution/aceto-
nitrile = 45:55; flow rate, 1.0mL/min; column, reverse-
phase (Capcell Pak UG120, 4.6mm I.D. + 150mm;
Shiseido) at 40°C; and detection wavelength, 255nm.
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Acknowledgements
The authors thank Dr. Eiichi Kunioka for evaluating
inhibitory activities toward COX-1 and COX-2, and
Kagumi Bando for evaluating inhibitory activities to-
ward drug-metabolizing CYP enzymes.
Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/
j.bmc.2004.08.047.
14. Sato, M.; Ishii, T.; Kobayashi-Matsunaga, Y.; Amada,
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N0-hydroxyphenylformamidine derivative HET0016 as a
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