Design and synthesis of Rho kinase inhibitors (II)

Article abstract of DOI:10.1016/j.bmc.2006.09.052

In a previous study, we identified several structurally unrelated scaffolds of the Rho kinase inhibitor using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. 1H-Indazole is one of the candidate scaffolds on which a new series of potent Rho kinase inhibitors could be developed. In this study, the detailed structure-activity relationship of 1H-indazole analogues was studied. During this study, we found that the cell-free enzyme inhibitory potential of Rho kinase inhibitors having the 1H-indazole scaffold did not necessarily correlate with their inhibitory potential toward the chemotaxis of cultured cells. The choice of the linker substructure was shown to be an important factor for the 1H-indazole analogues to inhibit the chemotaxis of cells. Optimization of the 1H-indazole inhibitors with respect to the in vitro inhibition of monocyte chemotaxis induced by MCP-1 was carried out. The inhibitory potential was improved both in the cell-free enzyme assay and in the chemotaxis assay.

Full text of DOI:10.1016/j.bmc.2006.09.052

Bioorganic & Medicinal Chemistry 15 (2007) 350–364
Design and synthesis of Rho kinase inhibitors (II)
,à
Masayuki Iwakubo, Atsuya Takami,à Yuji Okada, Takehisa Kawata,
Yoshimichi Tagami, Hiroshi Ohashi, Motoko Sato, Terumi Sugiyama,
Kayoko Fukushima and Hiroshi Iijima*
Pharmaceutical Research Laboratories, Kirin Brewery Co. Ltd, 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan
Received 15 August 2006; revised 22 September 2006; accepted 23 September 2006
Available online 12 October 2006
Abstract—In a previous study, we identified several structurally unrelated scaffolds of the Rho kinase inhibitor using pharmaco-
phore information obtained from the results of a high-throughput screening and structural information from a homology model
of Rho kinase. 1H-Indazole is one of the candidate scaffolds on which a new series of potent Rho kinase inhibitors could be devel-
oped. In this study, the detailed structure–activity relationship of 1H-indazole analogues was studied. During this study, we found
that the cell-free enzyme inhibitory potential of Rho kinase inhibitors having the 1H-indazole scaffold did not necessarily correlate
with their inhibitory potential toward the chemotaxis of cultured cells. The choice of the linker substructure was shown to be an
important factor for the 1H-indazole analogues to inhibit the chemotaxis of cells. Optimization of the 1H-indazole inhibitors with
respect to the in vitro inhibition of monocyte chemotaxis induced by MCP-1 was carried out. The inhibitory potential was improved
both in the cell-free enzyme assay and in the chemotaxis assay.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
H
NH
O
S
NH2
N
H
O
The small GTPase, Rho, is involved in a variety of cel-
lular functions, including cell migration and cell adhe-
sion. Rho kinase, which is also designated ROCK II,
is the best characterized among the Rho effectors.1
Rho kinase is considered to be a potential target kinase
where an inhibitor could be of therapeutic value. For
example, it is known that Rho kinase inhibitors can
inhibit the migration of monocyte cells induced by
MCP-1 (monocyte chemoattractant protein 1). Such
Rho kinase inhibitors could be useful compounds for
the treatment of atherosclerotic plaque formation and
inflammation.2,3 Several Rho kinase inhibitors have
been reported as illustrated in Figure 1. Fasudil dihy-
drochloride (HA-1077)4 has been used clinically as a
cerebrovascular contraction inhibitor.5 It is also under
clinical studies for the treatment of cardiac remodeling
N
2HCl
2HCl
N
O
N
HA-1077 (Fasudil)
Figure 1. Known Rho kinase inhibitors.
Y-27632
because the inhibition of Rho kinase is expected to sup-
press the migration of vascular smooth muscle cells.3
Uehata and colleagues have reported pyridine com-
pounds having a cyclohexanecarboxyamide or benzam-
ide moiety. Among them, Y-27632 (Fig. 1) was
representative as a specific Rho kinase inhibitor, by
which it was demonstrated that the Rho kinase inhibitor
might be useful in the prevention of invasion and metas-
tasis of cancer cells.6
We have previously reported several novel chemical
scaffolds as potential platforms for developing specific
Rho kinase inhibitors based on a homology model
Keywords: Rho kinase; Inhibitor; CCR2; MCP-1; Structure–activity
relationship.
*
Corresponding author. Tel.: +81 27 346 9776; fax: +81 27 346
8418; e-mail: iijimah@kirin.co.jp
of the kinase.7 N-(1H-5-indazolyl)-N0-benzylurea
1
(Fig. 2) is one of the new inhibitors designed in the pre-
vious study. The ligand-binding pocket of Rho kinase is
composed of three regions, namely the A, F and D
regions (Fig. 2). The A region is the bottom of the
Present address: Liquid Crystal Materials Technical Department,
Dainippon Ink and Chemicals, Inc., 4472-1 Komuro, Ina-machi,
Kitaadachi-gun, Saitama 362-8577, Japan.
à
These authors equally contributed to this work.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.052

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
351
prepared as follows: first,
2
was acylated with
2-chloroacetylchloride, then the resulting a-chloroamide
was coupled with N-methylbenzylamine using K2CO3 as
a base in CH3CN at room temperature. Finally, the
amide group was reduced by treatment with the BH3–
THF complex in THF at room temperature. Com-
pounds 5a–c were prepared as follows: 2 was treated
with 1-benzyl-4-piperidone, 1-benzyl-3-pyrrolidone, or
1-benzyl-3-piperidone. The resulting Schiff bases were
reduced by the BH3–pyridine complex in the presence
of a catalytic amount of acetic acid. Compound 6 was
prepared by reducing the Schiff base formed by 2 with
1-benzyl-4-piperidinecarbaldehyde. The aldehyde was
prepared by the oxidation of 1-benzyl-4-piperidinemeth-
anol with the sulfur trioxide–trimethylamine complex
(SO3–Me3N) in DMSO at room temperature. Com-
pounds 7a and 7b were synthesized by the condensation
of 2 with N-benzylisonipecotic acid or N-benzylnipecot-
ic acid using HOBT and WSC-HCl. Compound 8 was
prepared by the reaction of 2 and 1-benzyl-4-aminopi-
peridine using triphosgene with Et3N in CHCl3 at room
temperature. Compound 9 was prepared as follows: 1,4-
cyclohexanedione monoethylene ketal was reacted with
2, and the deprotection of the ketal group was then car-
ried out under acidic conditions. The resulting ketone
was reacted with benzylamine and reduced by NaB-
H(OAc)3 to give 9. Compound 9 was obtained as a mix-
ture of two diastereomers in the ratio of ca. 1:1. Each
diastereomer (9a and 9b) was separated using reverse-
phase chromatography. Compound 10 was prepared
from 5a. Compound 5a was acetylated with Ac2O using
a catalytic amount of DMAP in pyridine. The resulting
diacetylated intermediate of 5a was hydrolyzed under
acidic conditions. The N1 acetyl group was successfully
removed to produce 10.
Figure 2. Orthographic view of the ligand-binding pocket of Rho
kinase. A docking model of the 1H-indazole inhibitor 1 is shown. The
pocket is composed of three regions, A, F, and D. The possible
hydrogen-bonding sites are colored on the surface representation of
the pocket (blue, donor sites; red, acceptor sites). The backbone NH
group of Met167 is the hydrogen bond donor site of Rho kinase that
interacts with the N2 of the 1H-indazole ring. The backbone C@O
group of Glu165 acts as the hydrogen bond acceptor site. Hydrogen
bonds between the Rho kinase and 1 suggested by the docking
simulation are indicated by purple dashed lines.
pocket and offers an essential hydrogen bond donor (the
backbone NH of Met167) which forms a hydrogen bond
with the N2 of the 1H-indazole ring. The 1H-indazole
scaffold fits the flat shape of this region. The urea moiety
in the F region is a linker substructure that connects the
1H-indazole ring and the hydrophobic substructure in
the D region.
The inhibitory potency of the 1 analogues, such as 3a–c
(Table 1), determined by the cell-free Rho kinase en-
zyme assay (ICE50NZ) was in the 100–300 nM range. How-
ever, these compounds exhibited no inhibitory potential
for the chemotaxis of CCR2 over-expressing lymphoma
The syntheses of analogues having a 1H-indazole moiety
other than 2 are shown in Scheme 2. The cyclization of
2-methylanilines (11 and 14) to 1H-indazole was per-
formed using the method of Ruchart and Hassmann.9
4-Amino-3-cresol 11 was treated with acetic anhydride
and then reacted with isoamylnitrite to give N,O-diace-
tyl-5-hydroxy-1H-indazole. Deacetylation afforded
5-hydroxy-1H-indazole 12, which was then converted
to compounds 13a or 13b under Mitsunobu condi-
tions.10 The intermediate, 1H-indazole-5-carboxylic acid
15, was synthesized by the Ruchart and Hassmann cycli-
zation.9 Condensation of intermediate 15 with 1-benzyl-
4-aminopiperidine was carried out for the preparation of
16 using WSC-HCl and HOBT in DMF at room
temperature.
MCP
50
cells induced by MCP-1 (IC
HA-1077 showed a weak but significant inhibitory activ-
> 100 lMÞ. In contrast,
MCP
50
ity in the chemotaxis assay with an IC
value of
of HA-1077 determined for the cell-
ENZ
50
35 lM. The IC
free enzyme was 180 nM and was similar to that of the
ENZ
50
. Biophysical
analogues of 1. Species having comparative IC
exhibited a distinct difference in IC
MCP
50
properties other than the enzyme inhibitory potential
appeared necessary for the 1H-indazole analogues to
possess a high biological efficacy. The aim of this study
was the structural optimization of the 1H-indazole
derivatives based on the chemotaxis assay.
2. Chemistry
The intermediate, 3-methyl-1H-indazole 18, was synthe-
sized by diazotization of 2-aminoacetophenone 17. A ni-
tro group was introduced at the 5 position of 18 to yield
the intermediate 19, which was reduced to an aminoin-
dazole 20. Compound 21a was obtained by reacting
the intermediate 20 with 1-benzyl-3-piperidone. Com-
pound 21b was synthesized according to the literature.7
Analogues with various linker substructures between the
1H-indazole ring and the hydrophobic moiety were syn-
thesized from 5-amino-1H-indazole 2 as shown in
Scheme 1. A urea analogue 3a was prepared by reacting
2 with benzylisocyanate at 110 ꢁC. Compounds 3b and
3c were also synthesized by reacting 2 with 2,6-difluoro-
or 2,4-dichlorobenzylisocyanate which were prepared
from 2,6-difluoro- or 2,4-dichlorophenylacetic acid
using diphenylphosphoryl azide under the Curtius rear-
rangement conditions, respectively.8 Compound 4 was
Scheme 3 shows the synthesis of N-(3-piperidyl)-N-(1H-
5-indazolyl)amine analogues with substitutions at N-1
of the piperidine moiety. Analogues 24a–o were

352
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
Table 1. Modification of linker moiety
{ Linker}
R
N
N
H
ENZ
50
MCP
50
Compound
Rho kinase IC
(nM)
CCR2/MCP1 IC
35
(lM)
{Linker}
R
HA-1077 (Fasudil)
180
1
20
260
85
>100
>100
>100
>100
2,6-Cl–Ph
Ph
H
N
H
N
3a
3b
3c
2,6-F–Ph
2,4-Cl–Ph
O
220
H
N
5a
240
30
Ph
N
5b
5c
32
20
10
2
H
Ph
Ph
n = 1
n = 2
N
N
( )n
H
N
4
6
7900
nt
nt
Ph
N
N
H
N
>10,000
4-F–Ph
N
H
N
7a
16
7b
8
136
540
nt
nt
1
Ph
Ph
Ph
Ph
O
O
N
N
N
H
H
N
18
O
O
H
N
H
N
>10,000
nt
N
H
N
syn
9a
9b
320
520
nt
nt
Ph
Ph
anti
N
H
O
O
13a
13b
100
10
6
1
Ph
Ph
Ph
N
N
Ac
N
10
>10,000
>100
N
nt, not tested.

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
353
R
H
R
H
H
N
N
N
5a
10
R = H
R = Ac
n
Ph
Ar
N
Ar
N
Ar
Ph
N
O
4
H
N
n = 1
n = 2
5b
5c
3b
3c
Ar
R = 2,6-F
R =2,4-Cl
N
Ph
( )n
f
c, d, e
b
H
H
Ph
N
N
N
Ph
H
Ar
NH2
g, h
a
N
O
Ar
N
Ar NH2
=
6
N
3a
2
H
i
k, l ,m
N
Ph
j
H
N
Ar
Ar
7a
H
H
N
H
N
O
N
Ar
Ar
H
N
Ph
N
Ph
O
N
Ph
N
H
9
8
O
7b
Scheme 1. Synthetic routes of 1H-indazole analogues. The 1H-5-indazolyl group is abbreviated to Ar. Reagents and conditions: (a) benzylisocyanate,
DMF; (b) 2,6-difluorophenylacetic acid or 2,4-dichlorophenylacetic acid, (PhO)2PON3, Et3N; (c) 2-chloroacetylchloride, K2CO3, EtOAc, H2O, rt; (d)
N-methylbenzylamine, K2CO3, CH3CN, rt; (e) (1) BH3–THF, THF, 60 ꢁC, 1 h; (2) 1 N HCl aq, 60 ꢁC; (f) 1-benzyl-4-piperidone, 1-benzyl-2-
pyrrolidinone, or 1-benzyl-3-piperidone, BH3-pyridine, MeOH, AcOH, rt; (g) (1-benzyl-4-piperidiyl)methanol, SO3–Me3N, DMSO, Et3N, rt; (h) 2,
NaBH(OAc)3, MeOH, rt; (i) N-benzylnipecotic acid or N-benzylisonipecotic acid, WSC-HCl, HOBT, DMF, rt; (j) triphosgene, Et3N, CHCl3, rt, then
1-benzylpiperidin-4-amine; (k) 1,4-cyclohexane dione monoethylene ketal, BH3–pyridine, MeOH, AcOH, rt; (l) 50% AcOH, 80 ꢁC; (m) benzylamine,
NaBH(OAc)3, rt; (n) (1) Ac2O, pyridine, DMAP, rt; (2) 6 N HCl aq.
OH
OH
O
O
Ph
N
Ar
Ar
a
b
N
N
Ph
N
H
H2N
12
13b
11
13a
O
O
OH
OMe
Ph
N
O
a,c
d
N
N
H2N
Ar
N
H
H
14
15
16
NH2
g
NO2
e
f
O
H2N
N
N
N
N
N
H
N
H
H
17
18
19
20
H
N
h
N
N
N
H
21a
Scheme 2. Synthetic routes of analogues having a 1H-indazole moiety other than 5-amino-1H-indazole. The 1H-5-indazolyl group is abbreviated to
Ar. Reagents and conditions: (a) (1) Ac2O, AcOK, CHCl3, 0 ꢁC, then isoamylnitrite, 80 ꢁC; (2) 6 N HCl aq, MeOH, 40 ꢁC; (b) 1-benzyl-4-
hydroxypiperidine or 1-benzyl-3-hydroxypiperidine, DEAD, PPh3, THF, rt; (c) 3N NaOH aq; (d) (1-benzyl-4-piperidyl)-amine, WSC-HCl, HOBT,
DMF, rt; (e) 50% H2SO4, NaNO2, then SnCl2, 0 ꢁC; (f) 50% H2SO4, NaNO3, 0 ꢁC; (g) 50% HCl, SnCl2, rt; (h) 1-benzyl-3-piperidone, BH3–pyridine,
MeOH, AcOH, rt.
prepared as follows: alkylchlorides 23a–b and substitut-
ed benzylchlorides 23c–o were reacted with 3-hydroxypi-
peridine 22. The secondary alcohols were then oxidized
to ketones. The Schiff bases formed by the ketones with
5-amino-1H-indazole 2 were reduced using BH3-pyri-
dine to give 24a–o. Analogues 24p–r and 24s–x were
prepared as follows: the t-Boc-protected N-(3-pyper-
idyl)-N-(1H-5-indazolyl)amine 25 was first synthesized,
then t-Boc was removed by treating 25 with TFA. The
resulting N-(3-piperidyl)-N-(1H-5-indazolyl)amine 26
was converted to analogues 24p–r by a substitution reac-
tion with alkylchlorides 23p–r, or to analogues 24s–x by

354
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
entry
24k 2-NH2-PhCH2
3-NH2-PhCH2
24m 4-NH2-PhCH2
3-NO2-PhCH2
24o 4-NO2-PhCH2
R
entry
24c
24d
24e
24f
R
entry
24a
24b
R
n-propyl
isopentyl
2-Cl-PhCH2
3-Cl-PhCH2
4-Cl-PhCH2
4-F-PhCH2
3,4-F-PhCH2
4-Me-PhCH2
24l
H
O
N
R
R
HO
c
a,b
Ar
N
N
NH
24n
24g
24h
R Cl
23a-o
Ar-NH2
2
24i 4-MeO-PhCH2
22
3,5-MeO-PhCH2
24j
f
H
N
N
entry
24p
24q
24r
R
2-pyridyl
3-pyridyl
4-pyridyl
Ar
N
H
N
N
R
N
Cl
Ar
HO
c
d, b
NH
23p-r
Ar-NH2
2
entry
24s
24t
24u
24v
24w
24x
R
g
H
22
2-pyrrolyl
3-pyrrolyl
2-thienyl
3-thienyl
2-furyl
R = t-Boc 25
R = H 26
N
e
Ar
N
O
X
HO
X
23s-x
3-furyl
Scheme 3. Synthetic routes of N-(3-piperidyl)-N-(1H-5-indazolyl)amine analogues. The 1H-5-indazolyl group is abbreviated to Ar. Reagents and
conditions: (a) 23a–o, K2CO3, CH3CN, rt; (b) SO3–Me3N, DMSO, Et3N, rt; (c) 2, BH3–pyridine, MeOH, AcOH, rt; (d) (t-BuOCO)2O, 3 N NaOH
aq rt; (e) TFA, CHCl3, rt; (f) 23p–r, K2CO3, CH3CN, rt; (g) (1) 23s–x, WSC-HCl, HOBT, DMF, rt; (2) BH3–THF, THF, 60 ꢁC, 1 h, then 1 N HCl
aq, 60 ꢁC.
amide coupling with aryl carboxylates 23s–x followed
by reduction with the BH3–THF complex.
be due to the differing biophysical properties of 3a–c
and 5a–c.
We assumed that water-solubility might be a good sur-
rogate index for the inhibitors’ ability to inhibit the che-
motaxis of CCR2/U937 cells. In fact, the solubility of 3b
was poor; 5 lg/mL in WFI (water for injection, Otsuka
Pharmaceutical, Japan), 5 lg/mL in JP1st (0.086 N HCl
containing 34 mM NaCl), and less than 1 lg/mL in
JP2nd (50 mM KH2PO4, 24 mM NaOH). The solubility
of 5a was 794 lg/mL in WFI, 881 lg/mL in JP1st, and
495 lg/mL in JP2nd. Inhibitors potent in cell-free en-
zyme inhibition but having extremely poor water-solu-
bility would be less potent in inhibiting Rho kinase
inside the cells.
3. Structure optimization
The cell-free Rho kinase assay was performed according
to the methods described in a previous report7 using the
ribosomal S6 kinase substrate, S6 231–239, as the sub-
strate. The chemotaxis assay was performed using
CCR2-overexpressing human-derived histiocyte lym-
phoma (U937) cells and MCP-1 by the Boyden Cham-
ber method.11 HA-1077 was prepared by the published
method and used as the positive control.12
3.1. Inhibitory potency of 1H-indazole analogues for
chemotaxis
A docking study of the 1H-indazole inhibitors 5a–c indi-
cated that aminopiperidine or aminopyrrolidine would
show a goof fit with the F region of the ligand-binding
pocket of the Rho kinase, the shape of which is spherical
and spacious (Fig. 2). These cyclic aliphatic amines were
considered appropriate linker substructures both for the
interaction with the enzyme by full occupation of the F
region and for the physical properties of the inhibitor.
Enzyme inhibition and a suitable degree of solubility
would be necessary for the inhibitors to reach and inhib-
it Rho kinase in cells. Compound 4 possessed a good de-
gree of solubility but lost its affinity for the ligand-
binding pocket of the enzyme due to the loss of shape
complementarities with the F region (Table 1).
In a previous study, we carried out a structure-based de-
sign of Rho kinase inhibitors.7 One important bit of
information obtained in the initial study was that several
different substructures could be introduced as the linker
moiety at the 5 position of 1H-indazole scaffold to pro-
duce potent Rho kinase inhibitors.
In this study, the 1H-indozole inhibitors were initially
tested for their inhibition of MCP-1-induced cell migra-
tion of CCR2 over-expressing U937 cells. HA-1077 is
used as a positive control because HA-1077 is a com-
pound in clinical usage that has the required biophysical
properties. The inhibitory potential of HA-1077 in a
cell-free enzyme assessment, ICE50NZ, was 180 nM. HA-
1077 inhibited the chemotaxis of CCR2/U937 cells with
an ICM50CP of 35 lM. However, compounds 3a–c that car-
ry the urea substructure exhibited poor inhibition of
3.2. Optimization of the linker substructure
To develop potent 1H-indazole inhibitors effective with-
in cells, we first focused on modifying the linker moiety
of 1 (Fig. 2) by considering its effects on the solubility
and the shape complementarities with the spherical F re-
gion. The results are shown in Table 1. Compound 6, in
which the linker length was extended by the insertion of
chemotaxis in spite of the fact that they had comparable
ENZ
50
IC values with HA-1077 (Table 1). On the other
hand, compounds 5a–c were found to be potent inhibi-
tors of the chemotaxis (Table 1). This difference might

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
355
Table 3. Modification of IH-indazole ring
a methylene unit, lost its enzyme inhibition, but an
extension by an amide bond (7a, and 16) retained the
inhibitory potency, although the potency was not very
good. By adjusting the topology of the benzyl group
(7b), a good enzyme inhibitory potential was recovered
along with an inhibitory potential for chemotaxis. Fur-
ther extension with a urea substructure was not success-
ful (8). In compounds 9a and 9b, the amine nitrogen
atom was placed outside the ring. These compounds
were active but with a low potency. Compounds 13a
and 13b, in which the nitrogen atom between the1H-in-
dazole ring and the cyclic amine of 5a or 5c was replaced
H
N
N
{Indazole}
Rho kinase IC
20
ENZ
50
Compound
(nM)
{Indazole}
N
5c
N
H
Me
21a
21b
150
ENZ
50
MCP
and IC
50
N
with an oxygen atom, exhibited good IC
N
H
values. Compound 10 was the N-acetylated analogue of
5a. As shown in the previous study,7 introduction of a
substitution on the nitrogen atom between the 1H-inda-
zole ring and cyclic amine abolished the cell-free enzyme
inhibitory activity, because the substitution would not
allow 10 to dock to the ligand-binding pocket. This
compound was tested by the chemotaxis assay to deter-
mine if cells might incorporate this compound and if
N
N
>10,000
Me
of the substitution at the 3 position of the 1H-indazole
ring (21a). Introduction of a methyl group at the 3 posi-
subsequent hydrolysis might produce an active 5a inside
the cells. This intent was not successful. The IC
MCP
50
ENZ
50
of 10
tion decreased the activity from an IC
of 20 nM (5c)
of 150 nM (21a), while the decrease in the
MCP
50
ENZ
50
was quite inferior to the IC
of 5a.
to an IC
potency of the corresponding 4-amino-1-benzyl-piperi-
Pyrrolidine and piperidine seemed to be desirable linker
substructures. Table 2 compares the IC
dine derivative, N-(1-benzyl-4-piperidyl)-N-(3-methyl-
1H-5-indazolyl)amine, was more than 40-fold; the
ENZ
50
MCP
50
and IC
of
ENZ
analogues of 5a–c in order to investigate the effect of
ring size and substitutions of the cyclic amines. The ana-
logues having the 1,3-disubstituted piperidine (5c and
24c–e) ring as the linker indicated higher potency than
the 5a and 5b analogues.
ICE50NZ of 5a was 240 nM, while the IC
of the 3-methyl-
50
ated 5a was higher than 10 lM.7 It seemed that modifica-
tion ofthe 1H-indazole ring cannot effectively enhance the
inhibitory potency for the analogues employing the piper-
idine ring as the linker substructure.
We next examined the effect of modification on the 1H-
indazole ring of 5c. Table 3 shows the effects of the substi-
tution group at the 1 and 3 positions of the 1H-indazole
ring. Substitution with a methyl group at the 1 position
(21b) diminished the activity. We have reported the struc-
ture–activity relationship of 3-amino-1-benzyl-piperidine
derivatives in a previous study.7 A difference observed be-
tween the 3-amino-1-benzyl-piperidine derivatives and
the 4-amino-1-benzyl-piperidine derivatives was the effect
3.3. Optimization of the hydrophobic substructure for the
D region
Finally, the hydrophobic group for the D region of 5c
was optimized. The results are illustrated in Table 4.
The replacement of the benzyl group with an n-propyl
group (24a) or isopentyl group (24b) decreased the
Rho kinase inhibitory activity (IC 24a; 780 nM,
24b; 340 nM), suggesting that aromatic hydrophobic
ENZ
50
Table 2. Cyclic amine linkers
{ Linker}
R
N
N
H
Compound
Rho kinase
ENZ
50
CCR2/MCP1
MCP
50
{Linker}
R
IC
(nM)
IC
(lM)
5a
240
440
290
290
30
nt
nt
nt
H
N
Ph
5a2
5a3
5a4
2-Cl–Ph
3-Cl–Ph
4-Cl–Ph
N
N
5b
32
45
20
24
10
7
Ph
H
N
5b2
5b3
5b4
2-Cl–Ph
3-Cl–Ph
4-Cl–Ph
5
1
5c
20
13
19
11
2
8
8
1
Ph
H
N
24c
24d
24e
2-Cl–Ph
3-Cl–Ph
4-Cl–Ph
N
nt, not tested.

356
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
Table 4. Effect of the distal hydrophobic group
10 lM). Good inhibitory potential in the cell-free Rho
kinase inhibition and inhibition of the migration of
CCR2/U937 cells was observed in analogues that carried
H
N
R
N
N
the 2- or 3-thiophene ring (24u and 24v). The 2-furyl
analogue, 24w, exhibited a higher IC
N
MCP
50
H
value than
ENZ
50
expected from the ICE50NZ, while the IC
of the 3-furyl
Compound
Rho kinase
ENZ
50
CCR2/MCP1
MCP
50
R
analogue, 24x, was low. A similar relationship was ob-
served in the pyrrole analogues, 24s and 24t.
IC
(nM)
IC
(lM)
5c
20
2
PhCH2
n-Propyl
Isopentyl
24a
24b
24c
24d
24e
24f
24g
24h
24i
780
340
13
19
11
25
10
9
nt
nt
8
4. Conclusion
2-Cl–PhCH2
3-Cl–PhCH2
4-Cl–PhCH2
4-F–PhCH2
8
The inhibitory potential of the 1H-indazole analogues
against chemotaxis did not correlate directly with their
enzyme inhibitory potential, suggesting that biophysical
properties are relevant for in vivo efficacy.
1
4
1
3,4-F–PhCH2
4-Me–PhCH2
4-MeO–PhCH2
3,5-MeO–PhCH2
2-NH2–PhCH2
3-NH2–PhCH2
4-NH2–PhCH2
3-NO2–PhCH2
4-NO2–PhCH2
2-PyridylCH2
3-PyridylCH2
4-PyridylCH2
2-PyrrolylCH2
3-PyrrolylCH2
2-ThienylCH2
3-ThienylCH2
2-FurylCH2
1
16
80
85
35
10
3
1
24j
>30
1
We optimized the 1H-indazole derivatives based on the
inhibition of chemotaxis of CCR2 over-expressing cells.
First, we found that inhibitors employing the piperidine
and pyrrolidine linkers displayed an inhibitory activity
in the MCP-1/CCR2 chemotaxis assay as well as in the
cell-free Rho kinase enzyme assay, while those employ-
ing the urea linker failed to exhibit inhibition in the che-
motaxis assay. Second, we optimized the substitution
group on the 1H-indazole ring and substructures for
24k
241
24m
24n
24o
24p
24q
24r
24s
24t
24u
24v
24w
24x
3
2
19
19
nt
20
>30
10
nt
6
87
240
72
190
11
>1000
the D region of the ligand-binding pocket of the Rho
values in the
ENZ
50
5
3
kinase. 1H-indazole inhibitors with IC
low nanomolar range were attained.
1
65
180
5
nt
3-FurylCH2
Rho kinase has been reported to be involved in the che-
motaxis of eosinophiles.13 Compound 24g was submit-
ted to the eotaxin/CCR3 chemotaxis assay. The IC50
value of 24g for the inhibition of the chemotaxis of
CCR3 over-expressing cells induced by eotaxin
nt, not tested.
groups were more suitable for maintaining the activity
in the low nanomolar range than aliphatic groups. This
might be because the D region has a cleft-like shape. The
introduction of a chlorine atom at the 2, 3, or 4 position
(ICE50otaxin) was 0.7 lM, while the ICEotaxin of HA-1077
50
was 9.8 lM. This result indicated that Rho kinase inhib-
itors could suppress a wide range of leucocyte migra-
tions. A series of these 1H-indazole Rho kinase
inhibitors might be useful for the treatment of inflam-
mation associated with leucocyte migrations.
of the phenyl ring did not affect the cell-free Rho kinase
ENZ
50
inhibitory activity very significantly (IC 24c; 13 nM,
24d; 19 nM, and 24e; 11 nM). With regard to the inhibi-
tion of the chemotaxis, an analogue substituted at the 4
MCP
50
position (24e) was the best (IC
logues that were substituted at the 2 or 3 position had a
1 lM), whereas ana-
We observed that a variety of substructures could be uti-
lized for the 5-substituted 1H-indazole scaffold.7 Indeed,
other Rho kinase inhibitors having 1H-indazole scaffolds
have been reported.14 The chemical structures of these
inhibitors differ largely in the linker substructure. Accord-
ingly, their biophysical properties would also differ. 1H-
Indazole analogues might be a good source to seek Rho
kinase inhibitors with a high biological efficacy.
slightly decreased inhibitory potential for the chemotax-
is (IC
MCP
50
8 lM). The 4-fluoro- (24f) and 3,4-difluoro-
MCP
50
(24g) analogues showed an IC
substituted with methyl (24h) or methoxyl groups (24i)
of 1 lM. Analogues
at the 4 position exhibited a similar potency. A topolog-
ENZ
50
ical effect was observed in the IC of the analogues
substituted with a less hydrophobic amino group
ENZ
50
(IC
in contrast to the chlorine-substituted analogues, there
24k; 85 nM, 24l; 35 nM, 24m; 10 nM). However,
5. Experimental
MCP
50
was little difference in the IC
amine-substituted analogues. Analogues with a nitro
values of these
Commonly used abbreviations; AcOH (acetic acid),
DEAD (diethyl azodicarboxylate), DMAP (N, N-dimeth-
yl-4-aminopyridine), DMF (N,N-dimethylformamide),
DMSO (dimethylsulfoxide), EtOAc (ethyl acetate),
EtOH (ethanol), HOBT (5-hydroxybenzotriazole),
MeOH (methanol), t-Boc (tert-butoxycarbonyl group),
TFA (trifluoroacetic acid), THF (tetrahydrofuran),
ODS (octadecylated silica gel), WSC-HCl (1-ethyl-3-(3-
group substitution, 24n and 24o, exhibited lower
MCP
50
ENZ
.
50
IC
values than expected from their IC
Replacement of the phenyl ring in the D region with
3-pyridine and 2-pyrrole led to a moderate change in
ENZ
50
the Rho kinase inhibitory activities (IC 24q; 72 nM,
24s; 11 nM), but their inhibitory activities for the che-
MCP
50
motaxis were not improved (IC
24q; 20 lM, 24s;
dimethylaminopropyl)carbodiimide
hydrochloride),

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
357
BSA (bovine serum albumin), CCR2 (C–C chemokine
receptor type 2 or MCP-1 receptor), MCP (monocyte che-
moattractant protein).
mixture was stirred at 110 ꢁC for 1 h followed by the addi-
tion of 5-amino-1H-indazole 2 (100 mg, 0.75 mmol) and
DMF (0.2 mL). The resulting mixture was stirred at
110 ꢁC for 3 h and diluted with EtOAc and water. The
resulting precipitate was filtered and dried to give the
title compound as a colorless solid (14 mg, 0.05 mmol,
5.1. Chemotaxis assays
1
Mouse-derived CCR2 overexpressing human-derived
histiocyte lymphoma (U937) cells were suspended in a
0.1% BSA-containing RPMI 1640 medium (107 cells/
mL). A test compound was added, and the solution
was incubated for 20 min. Five hundred microliters each
of MCP-1 (1 nM) diluted in RPMI 1640 medium supple-
mented with 0.1% BSA was placed in the wells of 24-well
plates. A 24-well microchemotaxis chamber (Chemo-
taxicellTM, Kurabo Co., Osaka) was placed thereon,
and 200 lL of the cell suspension was added to the
top of the layer, followed by migration under a 5%
CO2 atmosphere at 37 ꢁC for 1 h. The number of cells
which had migrated to the lower chamber was counted
with a particle count analyzer (CDA-500, SYSMEX
Co.), and the percent inhibition of migration was calcu-
lated using the following equation; Migration inhibition
(%) = {1 ꢀ (the number of migrated cells in the presence
of the test compound/the number of migrated cells in the
absence of the test compound)} · 100. Chemokines were
purchased from Peprotech (London, UK).
6% yield in this step). H NMR (CDCl3, 400 MHz) d
4.39 (d, J = 5.8 Hz, 2H), 6.51 (t, J = 5.7 Hz, 1H), 7.10 (t,
J = 8.2 Hz, 2H), 7.21 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.35–
7.44 (m, 2H), 7.83 (d, J = 1.2 Hz, 1H), 7.92 (d,
J = 1.0 Hz, 1H), 8.36 (s, 1H), 12.8 (s, 1H). MS (ESI) m/z
303 (M+1)+.
5.2.3.
N-(2,4-Dichlorobenzyl)-N0-(1H-5-indazolyl)urea
(3c). Compound 3c was prepared from 2,4-dichlorophe-
nylacetic acid in a manner similar to that described for
3b with a yield of 8% as a colorless solid. H NMR
1
(CDCl3, 400 MHz) d 4.35 (d, J = 5.9 Hz, 2H), 6.40 (t,
J = 5.9 Hz, 1H), 7.26 (dd, J = 1.8 Hz, 8.9 Hz, 1H), 7.39–
7.47 (m, 3H), 7.56 (d, J = 1.7 Hz, 1H), 7.85 (s, 1H), 7.93
(s, 1H), 8.63 (s, 1H), 12.8 (s, 1H). MS (ESI) m/z 334
(Mꢀ1)+, 336 (M+1)+.
5.2.4.
N1-Benzyl-N1-methyl-N2-(1H-5-indazolyl)-1,2-
ethanediamine (4). To a mixture of 5-amino-1H-indazole
2 (3.99 g, 30 mmol) and K2CO3 (8.28 g, 60 mmol) in
THF/water (50:50 mL), 2-chloroacetylchloride (10.60 g,
50 mmol) was added dropwise at rt. The reaction mixture
was stirred at rt for 18 h, evaporated to remove the THF,
and diluted with EtOAc. The resulting precipitate, crude
N-(1H-5-indazolyl)-2-chloroacetamide, was filtered and
dried. To a mixture of the precipitate (105 mg, 0.5 mmol)
and K2CO3 (276 mg, 2 mmol) in CH3CN (1 mL), a solu-
tion of N-methylbenzylamine (121 mg, 1.0 mmol) in
CH3CN (1 mL) was added at rt. The reaction mixture
was stirred at rt for 18 h, diluted with EtOAc, and filtered
through Celite. The filtrate was concentrated to give N-
(1H-5-indazolyl)-2-(methylbenzylamino)acetamide. The
residue was suspended in THF (1 mL), and the BH3–
THF complex (2.5 mL, 2.5 mmol) was added dropwise
at 0 ꢁC. The reaction mixture was stirred at 60 ꢁC for
3 h and evaporated to remove the THF. HCl (1 N) aq
(1 mL) was added to the residue. The resulting mixture
was stirred at 60 ꢁC for 1 h and basified by the addition
of saturated NaHCO3 aq at 0 ꢁC. The reaction mixture
was extracted with EtOAc. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was then concentrated. The residual oil was subject-
ed to flash chromatography on silica gel, eluting with
CHCl3/MeOH (95:5) to give the title compound as a dense
yellow oil (57 mg, 0.20 mmol, 40% yield in this step). 1H
NMR (CDCl3, 400 MHz) d 2.40 (s, 3 H), 2.89 (t,
J = 5.6 Hz, 2H), 3.19 (t, J = 5.2 Hz, 2H), 3.76 (s, 2H),
6.74 (s, 1H), 6.78 (d, J = 8.8 Hz, 1H), 7.18–7.24 (m, 6H),
7.81 (s, 1H). MS (ESI) m/z 281 (M+1)+.
5.2. Chemistry
Analytical data were recorded for the compounds de-
scribed below using the following general procedures.
Proton NMR spectra were recorded using a Lambda
400 (Nippon Densi Datum, JEOL); chemical shifts were
recorded in ppm (d) based on the internal tetramethylsi-
lane standard as 0 ppm or on internal chloroform as
7.24 ppm in deuterochloroform. Coupling constants
(J) were recorded in Hertz. Mass spectra (MS) were
recorded using a PLATFORM-LC (Micromass) spec-
trometer. Melting points were taken using an electro-
thermal melting point apparatus and are uncorrected.
5.2.1. N-Benzyl-N0-(1H-5-indazolyl)urea (3a). To
a
solution of 5-amino-1H-indazole 2 (66 mg, 0.50 mmol)
in toluene (1 mL) and DMF (1 drop) was added benzylis-
ocyanate (0.061 mL, 0.50 mmol). The reaction mixture
was stirred at 110 ꢁC for 3 h and quenched with water.
The reaction mixture was extracted with EtOAc. The
EtOAc layer was washed with water and saturated NaCl
aq, dried over anhydrous Na2SO4, and filtered. The fil-
trate was then concentrated. The crude was purified by sil-
ica gel column chromatography eluting with CHCl3/
MeOH (95:5) to give the title compound (21 mg, 16%
1
yield) as a colorless solid. H NMR (CDCl3, 400 MHz)
d 4.31 (d, J = 5.9 Hz, 2H), 6.53 (t, J = 5.9 Hz, 1H), 7.21–
7.28 (m, 2H), 7.29–7.36 (m, 4H), 7.41 (d, J = 8.8 Hz,
1H), 7.85 (s, 1H), 7.93 (s, 1H,), 8.46 (s, 1H), 12.84 (s,
1H). MS (ESI) m/z 267 (M+1)+.
5.2.5. N-(1-Benzyl-4-piperidyl)-N-(1H-5-indazolyl)amine
(5a). To a mixture of 1-benzyl-4-piperidone (635 mg,
3.4 mmol), 5-amino-1H-indazole 2 (532 mg, 4.0 mmol),
and AcOH (0.20 mL) in MeOH (10 mL), the BH3–pyri-
dine complex (0.51 mL, 5.1 mmol) was added dropwise
at 0 ꢁC. The reaction mixture was stirred at rt for 18 h
and quenched by the addition of saturated NaHCO3
5.2.2.
N-(2,6-Difluorobenzyl)-N0-(1H-5-indazolyl)urea
(3b). To a mixture of 2,6-difluorophenylacetic acid
(129 mg, 0.75 mmol) and triethylamine (0.4 mL) in tolu-
ene (2 mL), diphenylphosphoryl azide (248 mg,
0.90 mmol) was added dropwise at rt. The reaction

358
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
1
with a yield of 35% as a dense yellow oil. H NMR
aq. The reaction mixture was extracted with CHCl3. The
combined organic layer was dried over anhydrous
Na2SO4 and filtered. The filtrate was then concentrated.
The residual oil was subjected to flash chromatography
on silica gel, eluting with CHCl3/MeOH (95:5) to give
the title compound as a yellow solid (1.00 g, 3.2 mmol,
(CDCl3, 400 MHz) d 1.66–1.78 (m, 1H), 2.30–2.41 (m,
1H), 2.44–2.53 (m, 1H), 2.61–2.38 (m, 1H), 2.77–2.87
(m, 2H), 3.66 (s, 2H), 4.00–4.08 (m, 1H), 6.73–6.76 (m,
1H), 6.77–6.83 (m, 1H), 7.24–7.36 (m, 6H), 7.88 (s,
1H). MS (ESI) m/z 293 (M+1)+.
1
82% yield). Mp 166 ꢁC. H NMR (CDCl3, 400 MHz) d
1.46–1.59 (m, 2H), 2.05–2.12 (m, 2H), 2.15–2.25 (m,
2H), 2.85–2.93 (m, 2H), 3.27–3.37 (m, 1H), 3.56 (s,
2H), 6.77–6.82 (m, 2H), 7.24–7.35 (m, 6H), 7.88 (s,
1H). MS (ESI) m/z 307 (M+1)+.
5.2.10.
N-[1-(2-Chlorobenzyl)-3-pyrrolidyl]-N-(1H-5-
indazolyl)amine (5b2). The compound 5b2 was prepared
from 3-hydroxypyrrolidine and 2-chlorobenzylchloride
in a manner similar to that described for the compound
24g with a yield of 41% as yellow dense oil. 1H NMR
(CDCl3, 400 MHz) d ppm: 1.66–1.75 (m, 1H), 2.23–
2.34 (m, 1H), 2.44–2.53 (m, 1H), 2.63–2.70 (m, 1H),
2.77–2.88 (m, 2H), 3.73 (s, 2H), 3.95–4.03 (m, 1H),
6.68–6.71 (m, 1H), 6.72–6.77 (m, 1H), 7.11–7.18 (m,
2H), 7.20–7.24 (m, 1H), 7.26–7.30 (m, 1H), 7.40 (d,
J = 6.8 Hz, 1H), 7.82 (s, 1H). MS (ESI) m/z 327 (M+1)+.
5.2.6. N-[1-(2-Chlorobenzyl)-4-piperidyl]-N-(1H-5-indaz-
olyl)amine (5a2). To a mixture of 4-piperidone hydro-
chloride monohydrate (77 mg, 0.50 mmol) and K2CO3
(138 mg, 1.0 mmol) in CH3CN (1.0 mL), a solution of
2-chlorobenzylchloride (81 mg, 0.50 mmol) in CH3CN
(0.5 mL) was added dropwise at 0 ꢁC. The reaction mix-
ture was stirred at rt for 18 h and diluted with EtOAc.
The resulting mixture was filtered through Celite. The
filtrate was concentrated to give 1-(2-chlorobenzyl)-4-
piperidone. To a mixture of the residue, 5-amino-1H-in-
dazole 2 (53 mg, 0.40 mmol), and AcOH (0.02 mL) in
MeOH (1.0 mL), a BH3–pyridine complex (0.05mL,
0.50 mmol) was added dropwise at 0 ꢁC. The reaction
mixture was stirred at rt for 18 h and quenched by the
addition of saturated NaHCO3 aq. The aqueous layer
was extracted with CHCl3. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated. The residual oil was subjected
to flash chromatography on silica gel, eluting CHCl3/
MeOH (95:5), to give the title compound as a yellow sol-
id (109 mg, 0.32 mmol, 80% yield in this step). 1H NMR
(CDCl3, 400 MHz) d ppm: 1.47–1.60 (m, 2H), 2.06–2.16
(m, 2H), 2.25–2.36 (m, 2H), 2.87–2.97 (m, 2H), 3.30–
3.40 (m, 1H), 3.67 (s, 2H), 6.78–6.84 (m, 2H), 7.16–
7.34 (m, 3H), 7.35 (d, J = 7.8 Hz, 1H), 7.49 (d,
J = 6.8 Hz, 1H), 7.88 (s, 1H). MS (ESI) m/z 341 (M++1).
5.2.11.
N-[1-(3-Chlorobenzyl)-3-pyrrolidyl]-N-(1H-5-
indazolyl)amine (5b3). The compound 5b3 was prepared
from 3-hydroxypyrrolidine and 3-chlorobenzylchloride
in a manner similar to that described for the compound
1
24g with a yield of 72% as yellow dense oil. H NMR
(CDCl3, 400 MHz) d ppm: 1.68–1.79 (m, 1H), 2.30–
2.41 (m, 1H), 2.42–2.51 (m, 1H), 2.61–2.67 (m, 1H),
2.76–2.86 (m, 2H), 3.62 (s, 2H), 4.01–4.08 (m, 1H),
6.74–6.77 (m, 1H), 6.79–6.83 (m, 1H), 7.18–7.26 (m,
3H), 7.29 (d, J = 8.8 Hz, 1H), 7.35 (s, 1H), 7.89 (s,
1H). MS (ESI) m/z =327 (M++1).
5.2.12.
N-[1-(4-Chlorobenzyl)-3-pyrrolidyl]-N-(1H-5-
indazolyl)amine (5b4). The compound 5b4 was prepared
from 3-hydroxypyrrolidine and 4-chlorobenzylchloride
in a manner similar to that described for the compound
1
24g with a yield of 67% as yellow dense oil. H NMR
(CDCl3, 400 MHz) d ppm: 1.67–1.77 (m, 1H), 2.30–
2.41 (m, 1H), 2.41–2.50 (m, 1H), 2.59–2.65 (m, 1H),
2.75–2.85 (m, 2H), 3.61 (s, 2H), 4.00–4.07 (m, 1H),
6.73–6.76 (m, 1H), 6.77–6.82 (m, 1H), 7.24–7.30 (m,
5H), 7.88 (s, 1H). MS (ESI) m/z =327 (M++1).
5.2.7. N-[1-(3-Chlorobenzyl)-4-piperidyl]-N-(1H-5-indaz-
olyl)amine (5a3). The compound 5a3 was prepared from
3-chlorobenzylchloride in a manner similar to that de-
scribed for the compound 5a2 with a yield of 79% as yel-
low dense oil.1H NMR (CDCl3, 400 MHz) d ppm: 1.45–
1.58 (m, 2H), 2.05–2.14 (m, 2H), 2.15–2.25 (m, 2H),
2.82–2.90 (m, 2H), 3.28–3.37 (m, 1H), 3.52 (s, 2H),
6.78–6.83 (m, 2H), 7.18–7.26 (m, 3H), 7.29 (d,
J = 8.5 Hz, 1H), 7.35 (s, 1H), 7.88 (s, 1H). MS (ESI)
m/z 341 (M++1).
5.2.13. N-(1-Benzyl-3-piperidyl)-N-(1H-5-indazolyl)amine
(5c). Compound 5c was prepared from 1-benzyl-3-pip-
eridone in a manner similar to that described for 5a with
1
a yield of 78% as a dense yellow oil. H NMR (CDCl3,
400 MHz) d 1.40–1.60 (m, 2H), 1.70–1.80 (m, 2H), 2.10–
2.50 (m, 4H), 3.16–3.22 (m, 2H), 3.45 (s, 1H), 6.75 (m,
2H), 7.15–7.28 (m, 6H), 7.78 (s, 1H). MS (ESI) m/z
307 (M+1)+.
5.2.8. N-[1-(4-Chlorobenzyl)-4-piperidyl]-N-(1H-5-indaz-
olyl)amine (5a4). The compound 5a4 was prepared from
4-chlorobenzylchloride in a manner similar to that de-
scribed for the compound 5a2 with a yield of 79% as yel-
low dense oil.1H NMR (CDCl3, 400 MHz) d ppm: 1.45–
1.59 (m, 2H), 2.05–2.13 (m, 2H), 2.13–2.25 (m, 2H),
2.81–2.91 (m, 2H), 3.25–3.37 (m, 1H), 3.51 (s, 2H),
6.78–6.82 (m, 2H), 7.27–7.32 (m, 5H), 7.87 (s, 1H).
MS (ESI) m/z 341 (M++1).
5.2.14. N-[1-(4-Fluorobenzyl)-4-piperidyl]methyl-N-(1H-
5-indazolyl)amine (6). To a mixture of 4-piperidylmetha-
nol (345 mg, 3.0 mmol) and K2CO3 (414 mg, 3.2 mmol)
in CH3CN (2 mL), a solution of 4-fluorobenzylchloride
(378 mg, 3.0 mmol) in CH3CN (2 mL) was added drop-
wise at 0 ꢁC. The reaction mixture was stirred at rt for
5 h and diluted with EtOAc. The resulting mixture was
filtered through Celite. The filtrate was then concentrat-
ed to give 1-(4-fluorobenzyl)-4-piperidylmethanol. This
material was dissolved in anhydrous DMSO (2 mL),
and triethylamine (0.5 mL) was added. Sulfur trioxide–
trimethylamine complex (700 mg, 5 mmol) was then
5.2.9. N-(1-Benzyl-3-pyrrolidyl)-N-(1H-5-indazolyl)amine
(5b). Compound 5b was prepared from 1-benzyl-3-pyr-
rolidone in a manner similar to that described for 5a

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
359
added to the mixture portionwise at 0 ꢁC. The reaction
mixture was stirred at rt for 18 h and quenched by the
addition of saturated NaHCO3 aq. The reaction mixture
was extracted with EtOAc. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was then concentrated to give 1-(4-fluorobenzyl)-
4-piperidylcarbaldehyde. This intermediate was dis-
solved in MeOH (3 mL), and 5-amino-1H-indazole 2
(276 mg, 2.0 mmol) was added. NaBH(OAc)3 (666 mg,
3.0 mmol) was then added at 0 ꢁC. The reaction mixture
was stirred at rt for 18 h and quenched by the addition
of saturated NaHCO3 aq. The reaction mixture was
extracted with CHCl3. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated. The residual oil was purified
by silica gel chromatography eluting with CHCl3/MeOH
(95:5) to give the title compound as a dense yellow oil
in CHCl3 (1 mL) was added dropwise at rt. The mixture
was stirred at rt for 30 min and followed by the portion-
wise addition of 5-amino-1H-indazole
2 (67 mg,
0.50 mmol) at rt. The reaction mixture was stirred at
rt for 18 h and quenched by the addition of saturated
NaHCO3 aq. The reaction mixture was extracted with
CHCl3. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was then
concentrated. The residual oil was purified by silica gel
chromatography, eluting with CHCl3/MeOH (95:5) to
give the title compound as a yellow solid (128 mg,
1
0.13 mmol, 73% yield). Mp 90 ꢁC. H NMR (CDCl3,
400 MHz) d 1.40–154 (m, 1H), 1.58–1.70 (m, 1H),
1.90–1.98 (m, 1H), 1.98–2.06 (m, 1H), 2.10–2.23 (m,
2H), 2.77–2.88 (m, 2H), 3.50 (s, 2H), 4.90–5.00 (m,
1H), 6.98 (d, J = 8.3 Hz, 1H), 7.27–7.33 (m, 6H), 7.91
(s, 1H), 8.23 (d, J = 8.8 Hz, 1H). MS (ESI) m/z 350
(M+1)+.
1
(328 mg, 1.0 mmol, 50% yield in this step). H NMR
(CDCl3, 400 MHz) d 1.03–1.15 (m, 1H), 1.62–1.77 (m,
2H), 1.80–1.87 (m, 1H), 1.93–2.20 (m, 3H), 2.85–2.93
(m, 1H), 3.00 (d, J = 6.6 Hz, 2H), 3.03–3.12 (m, 1H),
3.60 (d, J = 16.6 Hz, 1H), 6.69 (s, 1H), 6.71 (dd,
J = 8.8 Hz, 1H), 6.90–6.98 (m, 2H), 7.21–7.28 (m, 3H),
7.83 (s, 1H) MS (ESI) m/z 339 (M+1)+.
5.2.18. N1-Benzyl-N4-(1H-5-indazolyl)-1,4-cyclohexan-
ediamine (9a and 9b). To a mixture of 1,4-cyclohexane
monoethyleneketal (3.90 g, 25 mmol), 5-amino-1H-in-
dazole 2 (2.66 g, 20 mmol), and AcOH (0.5 mL), a
BH3–pyridine complex (2.50 mL, 25 mmol) was added
dropwise at 0 ꢁC. The mixture was stirred at rt for
18 h and concentrated for the removal of MeOH. The
residue was dissolved in 50% AcOH (50 mL). The reac-
tion mixture was stirred at 80 ꢁC for 3 h and quenched
by the addition of saturated NaHCO3 aq. The reaction
mixture was extracted with CHCl3. The combined
organic layer was dried over anhydrous Na2SO4 and fil-
tered. The filtrate was then concentrated. The residual
oil was purified by silica gel chromatography, eluting
with CHCl3 to give 4-(1H-5-indazolylamino)-1-cyclo-
hexanone (3.21 g, 14 mmol). To a mixture of 4-(1H-5-
indazolylamino)-1-cyclohexanone (57 mg, 0.25 mmol),
benzylamine (52 mg, 0.5 mmol) in MeOH (1 mL), NaB-
H(OAc)3 (111 mg, 0.5 mmol) was added at 0 ꢁC. The
reaction mixture was stirred at rt for 18 h and quenched
by the addition of saturated NaHCO3 aq. The reaction
mixture was extracted with CHCl3. The combined
organic layer was dried over anhydrous Na2SO4 and fil-
tered. The filtrate was then concentrated. The residual
oil was subjected to reverse-phase ODS HPLC (YMC-
Pack ODS-AQ, 20 mm X 150 mm), eluting with 5%
TFA aq/acetonitrile (80:20) to give the title compound
as a dense yellow oil (14 mg, syn isomer 9a, 24 mg,
anti-isomer 9b). Syn isomer: 1H NMR (CDCl3,
400 MHz) d 1.50–1.82 (m, 8H), 2.68 (tt, J = 3.9 Hz,
7.6 Hz, 1H), 3.44–3.50 (m, 1H), 3.90 (s, 2H), 6.70–6.77
(m, 2H), 7.20–7.30 (m, 6H), 7.80 (s, 1H). MS (ESI)
m/z 321 (M+1)+. Anti-isomer: 1H NMR (CDCl3,
400 MHz) d 1.05–1.32 (m, 4H), 1.95–2.03 (m, 2H),
2.05–2.20 (m, 2), 2.52 (tt, J =3.9, 11.0 Hz, 1H), 3.20
(tt, J = 307, 11.0 Hz, 1H), 3.70 (s, 2H), 6.68–6.77 (m,
2H), 7.22 (d, J = 8.8 Hz, 1H), 7.25–7.30 (m, 5H), 7.81
(s, 1H). MS (ESI) m/z 321 (M+1)+.
5.2.15. 1-Benzyl-N-(1H-5-indazolyl)piperidine-4-carbox-
amide (7a). To a solution of isonipecotic acid (129 mg,
1.0 mmol) in 3 N NaOH aq (1 mL), a solution of benzyl-
chloride (127 mg, 1.0 mmol) in DMF (1 mL) was added
dropwise at rt. The reaction mixture was stirred at rt for
2 h followed by a portionwise addition of 5-amino-1H-
indazole
2
(133 mg, 1.0 mmol), HOBT (230 mg,
1.5 mmol), WSC-HCl (288 mg, 1.5 mmol), and dimeth-
ylaminopyridine (5 mg) at 0 ꢁC. The reaction mixture
was stirred at rt for 18 h and quenched by the addition
of saturated NaHCO3 aq. The reaction mixture was
extracted with CHCl3. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was then concentrated. The residual oil was puri-
fied by silica gel column chromatography, eluting with
CHCl3/MeOH (95:5) to give the title compound as a yel-
low solid (86 mg, 0.26 mmol, 26% yield). Mp 218 ꢁC. 1H
NMR (CD3OD, 400 MHz) d 1.76–85 (m, 4H), 2.02–2.12
(m, 2H), 2.32 (dt, J = 7.7 Hz, 15.4 Hz, 1H), 2.93 (d,
J = 11.7 Hz, 2H), 3.50 (s, 2H), 7.19 (dd, J = 2.8 Hz,
5.1 Hz, 1H), 7.22–7.28 (m, 4H), 7.36 (dd, J = 9.0 Hz,
17.6 Hz, 2H), 7.88 (s, 1H), 7.93 (s, 1H). MS (ESI) m/z
335 (M+1)+.
5.2.16. 1-Benzyl-N-(1H-5-indazolyl)piperidine-3-carbox-
amide (7b). Compound 7b was prepared from nipecotic
acid in a manner similar to that described for 7a with
1
a yield of 35% as a yellow solid. Mp 175 ꢁC. H NMR
(CDCl3, 400 MHz) d 1.53–1.65 (m, 2H), 1.69–1.82 (m,
1H), 2.00–2.16 (m, 2H), 2.26–2.34 (m, 1H), 2.59–2.65
(m, 1H), 2.91–3.01 (m, 1H), 3.06–3.15 (m, 1H), 3.50
(d, J = 10.7 Hz, 2H), 7.21–7.38 (m, 7H), 7.96 (s, 1H),
7.98 (s, 1H). MS (ESI) m/z 335 (M+1)+.
5.2.19.
N1-(1-Benzyl-4-piperidyl)-N1-(1H-5-indazol-
5.2.17. N-(1-Benzyl-4-piperidyl)-N0-(1H-5-indazolyl)urea
(8). To a mixture of 4-amino-1-benzylpiperidine (95 mg,
0.50 mmol) and triethylamine (0.25 mL) in CHCl3
(1 mL), a solution of triphosgene (148 mg, 0.50 mmol)
yl)acetamide (10). To a mixture of N-(1-benzyl-4-piper-
idyl)-N-(1H-5-indazolyl)amine 5a (307 mg, 1.0 mmol)
and pyridine (190 mg, 2.4 mmol) and DMAP (15 mg,
0.12 mmol) in CHCl3 (1 mL), acetic anhydride (245 mg,

360
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
2.4 mmol) was added dropwise at rt. The reaction mixture
was stirred at rt for 18 h and quenched by the addition of
saturated NaHCO3 aq. The reaction mixture was extract-
ed with CHCl3. The combined organic layer was dried
over anhydrous Na2SO4 and filtered. The filtrate was then
concentrated. The crude material was dissolved in MeOH
(1 mL). To the solution was added HCl–MeOH (1 mL).
The reaction mixture was stirred at rt for 5 h and basified
by the addition of saturated NaHCO3 aq. The reaction
mixture was extracted with EtOAc. The combined organ-
ic layer was dried over anhydrous Na2SO4 and filtered.
The filtrate was then concentrated. The residual oil was
purified by silica gel chromatography, eluting with CHCl3
to give the title compound as a dense yellow oil (321 mg,
2H), 1.92–2.00 (m, 2H), 2.20–2.30 (m, 2H), 2.65–2.75
(m, 2H), 3.48 (s, 2H), 4.16–4.28 (m, 1H), 6.96–7.03 (m,
1H), 7.07–7.09 (m, 1H), 7.20–7.28 (m, 5H), 7.30 (d,
J = 9.0 Hz, 1H), 7.89 (s, 1H). MS (ESI) m/z 308 (M+1)+.
5.2.22. 1-Benzyl-3-piperidyl-(1H-5-indazolyl)ether (13b).
Compound 13b was prepared from 1-benzyl-3-hydrox-
ypiperidine in a manner similar to that described for
1
13a with a yield of 33% as dense yellow oil. H NMR
(CDCl3, 400 MHz) d 1.46–1.57 (m, 1H), 1.57–1.70 (m,
1H), 1.73–1.88 (m, 1H), 2.05–2.15 (m, 1H), 2.15–2.34
(m, 2H), 2.63–2.68 (m, 1H), 3.04 (m, 2H), 3.63 (s, 1H),
4.26–4.38 (m, 1H), 7.02 (d, J = 9.0 Hz, 1H), 7.13 (s,
1H), 7.20–7.35 (m, 6H), 7.88 (s, 1H). MS (ESI) m/z
308 (M+1)+.
1
0.95 mmol, 95% yield in this step). H NMR (CDCl3,
400 MHz) d 1.23–1.40 (m, 1H), 1.50–1.70 (m, 1H), 1.70–
1.80 (m, 1H), 1.82–1.92 (m, 1H), 2.02 (s, 3H), 2.11–2.28
(m, 2H), 2.91–3.08 (m, 2H), 3.48 (d, J = 12.7 Hz, 1H),
3.55 (d, J = 13.0 Hz, 1H), 4.66–4.76 (m,1H), 6.92–6.98
(m, 1H), 7.20–7.27 (m, 6H), 7.52 (s, 1H), 8.05 (s, 1H).
MS (ESI) m/z 349 (M+1)+.
5.2.23. 1H-Indazol-5-carboxylic acid (15). To a mixture
of methyl 4-amino-3-methylbenzoate 14 (0.85 g,
5.2 mmol) and potassium acetate (1.47 g, 15 mmol) in
CHCl3 (20 mL), acetic anhydride (1.42 mL, 15 mmol)
was added dropwise at 0 ꢁC. The resulting mixture was
stirred at rt for 30 min, heated to 80 ꢁC, followed by
the dropwise addition of isoamyl nitrite (1.17 g,
10 mmol) at 80 ꢁC. The reaction mixture was stirred at
80 ꢁC for 18 h and quenched by the addition of saturat-
ed NaHCO3 aq. The reaction mixture was extracted
with CHCl3. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was then
concentrated. The residual oil was purified by silica gel
column chromatography, eluting with CHCl3–MeOH
to give the intermediate, N1-acetyl-indazol-5-carboxylic
acid. The intermediate was hydrolyzed in 3 M NaOH aq
(5 mL)–MeOH (5 mL) at rt for 18 h and evaporated to
remove MeOH and water. The residual solid was sub-
jected to reverse-phase chromatography on ODS, elut-
ing with 0.5% TFA aq/CH3CN (95:5) to give the title
compound as a yellow solid (0.32 g, 2.0 mmol, 38%
yield).
5.2.20. 1H-5-Indazolol (12). To a mixture of 4-amino-3-
cresol 11 (12.3 g, 100 mmol) and potassium acetate
(24.4 g, 244 mmol) in CHCl3 (200 mL), acetic anhydride
(47.1 mL, 450 mmol) was added dropwise at 0 ꢁC. The
resulting mixture was stirred at rt for 30 min, heated
to 80 ꢁC, followed by the dropwise addition of iso-
amylnitrite (12.7 g, 110 mmol) at 80 ꢁC. The reaction
mixture was stirred at 80 ꢁC for 18 h and quenched by
the addition of saturated NaHCO3 aq. The reaction
mixture was extracted with CHCl3. The combined
organic layer was dried over anhydrous Na2SO4 and fil-
tered. The filtrate was then concentrated. The residual
oil was subjected to flash chromatography on silica gel
eluting with CHCl3–MeOH to give N,O-diacetyl-5-
indazolol, which was then treated with HCl–MeOH
(200 mL) at 80 ꢁC for 5 h and evaporated to remove
HCl–MeOH. The resulting mixture was basified with
saturated NaHCO3 aq. The mixture was extracted with
EtOAc. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was then
concentrated. The precipitate was washed with CHCl3
to give the title compound as a brown solid (7.99 g,
5.2.24.
N5-(1-Benzyl-4-piperidyl)-1H-5-indazolecarb-
oxyamide (16). To a mixture of 4-amino-1-benzylpiperi-
dine (280 mg, 1.47 mmol), 1H-5-indazolecarboxylic acid
15 (243 mg, 1.81 mmol), HOBT (306 mg, 2.0 mmol),
and dimethylaminopyridine (5 mg) in DMF (3 mL)
was added WSC-HCl (383 mg, 2.0 mmol) at 0 ꢁC. The
reaction mixture was stirred at rt for 18 h and quenched
by the addition of saturated NaHCO3 aq. The reaction
mixture was extracted with CHCl3. The combined
organic layer was dried over anhydrous Na2SO4 and fil-
tered. The filtrate was then concentrated. The residual
oil was purified by silica gel column chromatography,
eluting with CHCl3/MeOH (95:5) to give the title com-
pound as a yellow solid (42 mg, 0.13 mmol, 9% yield).
1
60 mmol, 60% yield). H NMR (CDCl3, 400 MHz) d
6.95 (d, J = 8.8 Hz, 1H), 7.03 (s, 1H), 7.31 (d,
J = 8.8 Hz, 1H), 7.89 (s, 1H).
5.2.21. 1-Benzyl-4-piperidyl-(1H-5-indazolyl)ether (13a).
To a mixture of 1H-5-indazolol 12 (52 mg, 0.40 mmol),
1-benzyl-4-hydroxypiperidine (96 mg, 0.50 mmol), and
triphenylphosphine (131 mg, 0.50 mmol) in THF
(1 mL), a solution of DEAD (87 mg, 0.50 mmol) in tol-
uene (1 mL) was added dropwise at 0 ꢁC. The reaction
mixture was stirred at rt for 18 h and quenched by the
addition of saturated NaHCO3 aq. The reaction mixture
was extracted with CHCl3. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was then concentrated. The residual oil was sub-
jected to silica gel column chromatography eluting
with CHCl3/MeOH (95:5) to give the title compound
as a yellow solid (35 mg, 0.11 mmol, 28% yield). Mp
90 ꢁC. 1H NMR (CDCl3, 400 MHz) d 1.71–184 (m,
1
Mp 126 ꢁC. H NMR (CDCl3, 400 MHz) d 1.60–1.80
(m, 2H), 1.95–2.08 (m, 2H), 2.20–2.32 (m, 2H), 2.90–
2.98 (m, 2H), 3.07 (s, 2H), 3.80–3.86 (m, 1H), 7.16–
7.40 (m, 5H), 7.46 (d, J = 8.8 Hz, 1H), 7.75 (d,
J = 8.8 Hz, 1H), 8.07 (s, 1H), 8.14 (s, 1H). MS (ESI)
m/z 335 (M+1)+.
5.2.25. N-(1-Benzyl-3-piperidyl)-N-(3-methyl-1H-5-ind-
azolyl)amine (21a). (1) 5-Amino-3-methyl-1H-indazole
(20). To a suspension of 20-aminoacetophenone 17

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
361
(1.35 g, 10 mmol) in 50% H2SO4 aq (20 mL), sodium ni-
trite (0.82 g, 12 mmol) was slowly added at 0 ꢁC. The
resulting mixture was stirred at rt for 1 h, followed by
the addition of tin(II)chloride dihydrate (6.76 g,
30 mmol). The reaction mixture was stirred at 0 ꢁC for
1 h and diluted with water. The white solid was collected
by filtration and dried to give 3-methyl-1H-indazole 18
(1.20 g, 9 mmol).
5.2.29.
N-[1-(2-Chlorobenzyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24c). Compound 24c was prepared
from 2-chlorobenzylchloride in a manner similar to that
described for 24g with a yield of 63% as a yellow solid.
Mp 72 ꢁC. 1H NMR (CDCl3, 400 MHz) d 1.42–1.74 (m,
4H), 2.31–2.53 (m, 3H), 2.65–2.75 (m, 1H), 3.51–3.60
(m, 3H), 6.71–6.79 (m, 2H), 7.07–7.18 (m, 2H), 7.21
(d, J = 8.8 Hz, 1H), 7.28 (dd, J = 1.7 Hz, 7.6 Hz, 1H),
7.34–7.42 (m, 1H), 7.78 (s, 1H). MS (ESI) m/z 341
(M+1)+.
Compound 18 was suspended in 50% H2SO4 aq
(5 mL) and sodium nitrate (0.85 g, 10 mmol) at 0 ꢁC.
The mixture was stirred at rt for 1 h and quenched
by the addition of saturated NaHCO3 aq. The reac-
tion mixture was extracted with EtOAc. The organic
layer was dried over anhydrous Na2SO4 and filtered.
The filtrate was then concentrated. The residual solid
was washed with CHCl3 to give 3-methyl-5-nitro-1H-
indazole 19. The nitro group of 19 was reduce to an
amino group: 19 was suspended in 50% HCl aq
(10 mL) and tin(II) chloride dihydrate (4.1 g, 18 mmol)
was added slowly at 0 ꢁC. The resulting mixture was
stirred at rt for 5 h and quenched by the addition of
saturated NaHCO3 aq. The reaction mixture was
extracted with EtOAc. The organic layer was dried
over anhydrous Na2SO4 and filtered. The filtrate was
then concentrated. The residual solid was washed with
CHCl3 to give 20 with a yield of 88% as a brown
solid.
5.2.30. N-[1-(3-Chlorobenzyl)-3-piperidyl]-N-(1H-5-ind-
azolyl)amine (24d). Compound 24d was prepared from
3-chlorobenzylchloride in a manner similar to that de-
scribed for 24g with a yield of 46% as a yellow solid.
Mp 85 ꢁC. 1H NMR (CDCl3, 400 MHz) d 1.49–1.65
(m, 2H), 1.68–1.78 (m, 2H), 2.33–2.54 (m, 3H), 2.60–
2.73 (m, 1H), 3.42–3.54 (m, 2H), 3.54–3.64 (m, 1H),
6.78–6.86 (m, 2H), 7.16–7.22 (m, 3H), 7.27 (d,
J = 8.8 Hz, 1H), 7.34 (s, 1H), 7.85 (s, 1H). MS (ESI)
m/z 341 (M+1)+.
5.2.31. N-[1-(4-Chlorobenzyl)-3-piperidyl]-N-(1H-5-ind-
azolyl)amine (24e). Compound 24e was prepared from
4-chlorobenzylchloride in a manner similar to that de-
scribed for 24g with a yield of 29% as a yellow solid.
1
Mp 139 ꢁC. H NMR (CDCl3, 400 MHz) d 1.43–1.57
(m, 2H), 1.62–1.74 (m, 2H), 2.20–2.40 (m, 3H), 2.63–
2.70 (m, 1H), 3.33–3.48 (m, 2H), 3.48–3.58 (m, 1H),
6.72–6.78 (m, 2H), 7.18–7.24 (m, 5H), 7.79 (s, 1H).
MS (ESI) m/z 341 (M+1)+.
(2) Compound 21a was prepared from 5-amino-3-meth-
yl-1H-indazole 20 and 1-benzyl-3-piperidone in a man-
ner similar to that described for 5a with a yield of 55%
1
as a dense yellow oil. H NMR (CDCl3, 400 MHz) d
5.2.32. N-[1-(4-Fluorobenzyl)-3-piperidyl]-N-(1H-5-ind-
azolyl)amine (24f). Compound 24f was prepared from
4-fluorobenzylchloride in a manner similar to that
described for 24g with a yield of 56% as a yellow solid.
1.44–1.61 (m, 3H), 1.65–1.80 (m, 2H), 2.20–2.30 (m,
1H), 2.43 (s, 3H), 2.72–2.81 (m, 2H), 3.48 (d,
J = 4.1 Hz, 2H), 3.53–3.60 (m, 1H), 6.65 (d,
J = 2.0 Hz, 1H), 6.73 (dd, J = 2.2 Hz, 8.8 Hz, 1H), 7.14
(d, J = 8.8 Hz, 1H), 7.22–7.30 (m, 5H). MS (ESI) m/z
321 (M+1)+.
1
Mp 166 ꢁC. H NMR (CDCl3, 400 MHz) d 1.43–1.58
(m, 2H), 1.60–1.75 (m, 2H), 2.20–2.40(m, 3H), 2.60–
2.75 (m, 1H), 3.34–3.47 (m, 2H), 3.47–3.58 (m, 1H),
6.72–6.78 (m, 2H), 6.89–6.96 (m, 2H), 7.19–7.25 (m,
3H), 7.79 (s, 1H). MS (ESI) m/z 325 (M+1)+.
5.2.26. N-(1-Benzyl-3-piperidyl)-N-(1-methyl-5-indazol-
yl)amine (21b). Compound 21b was synthesized in the
same way as that in the Ref. 7.
5.2.33. N-[1-(3,4-Difluorobenzyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24g). To a mixture of 3-hydroxypiperi-
dine (71 mg, 0.7 mmol) and K2CO3 (268 mg, 2.0 mmol)
in DMF (1 mL), a solution of 3,4-difluorobenzylchlo-
ride (119 mg, 0.7 mmol) in CH3CN (0.5 mL) was added
dropwise at 0 ꢁC. The reaction mixture was stirred at rt
for 18 h and diluted with EtOAc. The resulting mixture
was filtered through Celite. The filtrate was then concen-
trated to give 1-(3,4-difluorobenzyl)-3-hydroxypiperi-
dine. To a mixture of the residue, triethylamine
(0.25 mL) in anhydrous DMSO (1 mL) was slowly add-
ed a sulfur trioxide–trimethylamine complex (209 mg,
2.0 mmol) at 0 ꢁC. The reaction mixture was stirred at
rt for 18 h and quenched by the addition of saturated
NaHCO3 aq. The reaction mixture was extracted with
EtOAc. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was then
concentrated to give 1-(3,4-difluorobenzyl)-piperidine-
3-one. To a mixture of this intermediate, 5-amino-1H-
indazole 2 (53 mg, 0.4 mmol), and AcOH (0.02 mL) in
MeOH (1 mL), the BH3–pyridine complex (0.05 mL,
5.2.27. N-(1-Propyl-3-piperidyl)-N-(1H-5-indazolyl)amine
(24a). Compound 24a was prepared from 3-bromopro-
pane in a manner similar to that described for 24g with
1
a yield of 35% as a dense yellow oil. H NMR (CDCl3,
400 MHz) d 0.90 (t, J = 7.6 Hz, 3H), 1.51–2.07 (m, 4H),
2.07–2.21 (m, 4H), 2.36–2.40 (m, 2H), 2.96–2.99 (m,
2H), 3.29–3.36 (m, 1H), 6.75–6.81 (m, 2H), 7.25 (d,
J = 12.0 Hz, 1H), 7.88 (s, 1H). MS (ESI) m/z 259
(M+1)+.
5.2.28. N-(1-Isopentyl-3-piperidyl)-N-(1H-5-indazolyl)-
amine (24b). Compound 24b was prepared from
1-chloro-3-methylbutane in a manner similar to that de-
scribed for 24g with a yield of 32% as a dense yellow oil.
1H NMR (CDCl3, 400 MHz) d 0.89–0.91 (m, 6H), 1.35–
1.41 (m, 3H), 1.54–1.76 (m, 6H), 2.32–2.37 (m, 3H),
2.45–2.50 (m, 1H), 3.55–3.63 (m, 1H), 6.82–6.97 (m,
2H), 7.28–7.32 (m, 1H), 7.88 (s, 1H). MS (ESI) m/z
287 (M+1)+.

362
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
0.5 mmol) was added dropwise at 0 ꢁC. The reaction
mixture was stirred at rt for 18 h and quenched by the
addition of saturated NaHCO3 aq. The mixture was
extracted with CHCl3. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was then concentrated. The residual oil was sub-
jected to flash chromatography on silica gel, eluting
with CHCl3/MeOH (95:5) to give the title compound
as a yellow solid (85 mg, 0.25 mmol, 63% yield in this
step). Mp 166 ꢁC. 1H NMR (CDCl3, 400 MHz) d
1.30–1.40 (m, 1H), 1.59–1.69 (m, 1H), 1.74–1.80 (m,
1H), 1.91–2.00 (m, 2H), 2.18 (t, J = 7.0 Hz, 1H), 2.58–
2.68 (m, 1H), 2.93 (t, J = 10.0 Hz, 1H), 3.30–3.32 (m,
1H), 3.48 (q, J = 8.0 Hz, 2H), 6.85 (s, 1H), 6.92 (dd,
J = 2.2, 8.8 Hz, 1H), 7.07–7.10 (m, 1H), 7.14 (dd,
J = 8.1 Hz, 10.2 Hz, 1H), 7.26 (ddd, J = 2.0, 8.1,
10.5 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.77 (s, 1H).
MS (ESI) m/z 342 (M+1)+.
5.2.38.
N-[1-(3-Aminobenzyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24l). To a solution of N-[1-(3-nitroben-
zyl)-3-piperidyl]-N-(1H-5-indazolyl)amine 24n (88 mg,
0.25 mmol) in concd HCl (0.25 mL) and MeOH
(0.50 mL),
tin(II)chloride
dihydrate
(113 mg,
0.50 mmol) was slowly added at rt. The resulting mix-
ture was stirred at rt for 3 h and basified by the addition
of saturated NaHCO3 aq. The mixture was extracted
with EtOAc. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was then
concentrated. The residual oil was subjected to flash
chromatography on aluminum oxide, eluting with
CHCl3/MeOH (95:5) to give the title compound as a
dense yellow oil (64 mg, 0.23 mmol, 92% yield in this
steps). 1H NMR (CDCl3, 400 MHz) d 1.42–1.68 (m,
3H), 1.70–1.88 (m, 1H), 2.35–2.47 (m, 3H), 2.60–2.80
(m, 1H), 3.38–3.50 (m, 2H), 3.55–3.70 (m, 1H), 6.58
(d, J = 8.5 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.71 (d,
J = 7.3 Hz, 1H), 6.82 (br s, 2H), 7.09 (t, J = 7.7 Hz,
1H), 7.29 (d, J = 8.8 Hz, 1H), 7.86 (s, 1H). MS (ESI)
m/z 322 (M+1)+.
5.2.34.
N-[1-(4-Methylbenzyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24h). Compound 24h was prepared
from 4-methylbenzylchloride in a manner similar to that
described for 24g with a yield of 22% as a yellow solid.
5.2.39.
N-[1-(4-Aminobenzyl)-3-piperidyl]-N-(1H-5-
1
Mp 158 ꢁC. H NMR (CDCl3, 400 MHz) d 1.42–1.57
indazolyl)amine (24m). Compound 24m was prepared
from N-(1H-5-Indazolyl)-N-[1-(3-nitrobenzyl)-3-piper-
idyl]amine 24o in a manner similar to that described
(m, 2H), 1.60–1.72 (m, 2H), 2.26 (s, 3H), 2.24–2.40 (m,
3H), 2.62–2.75 (m, 1H), 3.35–3.48 (m, 2H), 3.48–3.58
(m, 1H), 6.71–6.78 (m, 2H), 7.05 (d, J = 7.8 Hz, 2H),
7.14 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.8 Hz, 1H), 7.79
(s, 1H). MS (ESI) m/z 321 (M+1)+.
1
for 24l with a yield of 99% as a dense yellow oil. H
NMR (CDCl3, 400 MHz) d 1.53–1.65 (m, 2H), 1.65–
1.80 (m, 2H), 2.26–2.49 (m, 3H), 2.70–2.82 (m, 1H),
3.45 (d, J = 9.3 Hz, 2H), 3.61 (br s, 1H), 6.58 (d,
J = 8.0 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.82 (s, 1H),
6.83 (d, J = 8.8 Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 7.25
(s, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.86 (s, 1H). MS
(ESI) m/z 322 (M+1)+.
5.2.35.
N-[1-(4-Methoxybenzyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24i). Compound 24i was prepared from
4-methoxybenzylchloride in a manner similar to that de-
scribed for 24g with a yield of 19% as a yellow solid, Mp
1
184 ꢁC. H NMR (CDCl3, 400 MHz) d 1.43–1.56 (m,
2H), 1.60–1.73 (m, 2H), 2.23–2.40 (m, 3H), 2.61–2.80
(m, 1H), 3.33–3.47 (m, 2H), 3.48–3.58 (m, 1H), 3.72 (s,
3H), 6.74–6.78 (m, 2H), 6.78 (d, J = 8.8 Hz, 2H), 7.16
(d, J = 8.5 Hz, 2H), 7.21 (d, J = 8.5 Hz, 1H), 7.79 (s,
1H). MS (ESI) m/z 337 (M+1)+.
5.2.40. N-[1-(3-Nitrobenzyl)-3-piperidyl]-N-(1H-5-indaz-
olyl)amine (24n). Compound 24n was prepared from
3-nitrobenzylchloride in a manner similar to that de-
scribed for 24g with a yield of 43% as a yellow solid.
Mp 80 ꢁC. 1H NMR (CDCl3, 400 MHz) d 1.52–1.66
(m, 2H), 1.71–1.84 (m, 2H), 2.26–2.50 (m, 3H), 2.70–
2.80 (m, 1H), 3.52–3.65 (m, 3H), 6.77–6.85 (m, 2H),
7.27 (d, J = 8.8 Hz, 1H), 7.44 (dd, J = 7.8, 8.1 Hz, 1H),
7.63 (d, J = 6.8 Hz, 1H), 7.83 (s, 1H), 8.07 (d,
J = 8.3 Hz, 1H), 8.23 (s, 1H). MS (ESI) m/z 352 (M+1)+.
5.2.36. N-[1-(3,5-Dimethoxybenzyl)-3-piperidyl]-N-(1H-
5-indazolyl)amine (24j). Compound 24j was prepared
from 3,5-dimethoxybenzylchloride in a manner similar
to that described for 24g with a yield of 38% as a yellow
1
solid. Mp 205 ꢁC. H NMR (CDCl3, 400 MHz) d 1.40–
1.57 (m, 2H), 1.62–1.75 (m, 2H), 2.20–2.43 (m, 3H),
2.70–2.80 (m, 1H), 3.35–3.47 (m, 2H), 3.50–3.60 (m,
1H), 3.73 (s, 6H), 6.29 (s, 1H), 6.45 (s, 2H), 6.72–6.79
(m, 2H), 7.22 (d, J = 8.5 Hz, 1H), 7.79 (s, 1H). MS
(ESI) m/z 367 (M+1)+.
5.2.41. N-[1-(4-Nitrobenzyl)-3-piperidyl]-N-(1H-5-indaz-
olyl)amine (24o). Compound 24o was prepared from
4-nitrobenzylchloride in a manner similar to that de-
scribed for 24g with a yield of 25% as a yellow solid.
Mp 85 ꢁC. 1H NMR (CDCl3, 400 MHz) d 1.51–1.68 (m,
2H), 1.69–1.86 (m, 2H), 2.26–2.47 (m, 3H), 2.71–2.83
(m, 1H), 3.52–3.65 (m, 3H), 6.79–6.83 (m, 2H), 7.28 (d,
J = 9.5 Hz, 2H), 7.49 (d, J = 8.5 Hz, 1H), 7.84 (s, 1H),
8.15 (d, J = 8.8 Hz, 2H). MS (ESI) m/z 352 (M+1)+.
5.2.37. N-[1-(2-Aminobenzyl)-3-piperidyl]-N-(1H-5-ind-
azolyl)amine (24k). Compound 24k was prepared from
N-[1-(2-nitrobenzyl)-3-piperidyl]-N-(1H-5-indazolyl)-
amine in a manner similar to that described for 24l with
1
a yield of 90% as a dense yellow oil. H NMR (CDCl3,
5.2.42.
indazolyl)amine (24p). To a solution of tert-butyl 3-
(1H-5-indazolylamino)-1-piperidinecarboxylate 25
N-[1-(2-Pyridylmethyl)-3-piperidyl]-N-(1H-5-
400 MHz) d 1.35–1.68 (m, 2H), 1.73–1.95 (m, 2H), 2.00–
2.35 (m, 3H), 2.35–2.62 (m, 1H), 2.80–2.96 (m, 1H),
3.45–3.37 (m, 2H), 6.65 (d, J = 6.8 Hz, 1H), 6.67 (d,
J = 7.6 Hz, 1H), 6.79 (s, 1H), 6.98 (d, J = 6.8 Hz, 1H),
7.09 (t, J = 7.7 Hz, 1H), 7.29 (d, J = 9.3 Hz, 1H), 7.86
(s, 1H). MS (ESI) m/z 322 (M+1)+.
(93 mg, 0.30 mmol) in CHCl3 (1 mL), TFA (1 mL) was
added dropwise at rt. The resulting mixture was stirred
at rt for 3 h and concentrated. To the residue, K2CO3
(138 mg, 1.0 mmol) and CH3CN (1 mL) were added.

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
363
To the mixture, a solution of 2-chloromethyl-pyridine
(50 mg, 0.30 mmol) in CH3CN (0.5 mL) was added
dropwise at 0 ꢁC. The reaction mixture was stirred at
rt for 18 h and quenched by the addition of water. The
reaction mixture was extracted with EtOAc. The com-
bined organic layer was dried over anhydrous Na2SO4
and filtered. The filtrate was then concentrated. The
residual oil was subjected to flash chromatography on
silica gel, eluting with CHCl3/MeOH (95:5) to give the
title compound as a dense yellow oil (80 mg, 0.26 mmol,
was then concentrated. The residual oil was subjected to
flash chromatography on silica gel, eluting with CHCl3/
MeOH (95:5) to give the title compound as a yellow solid
(12 mg, 0.04 mmol, 16% yield in three steps). Mp 93 ꢁC.
1H NMR (CDCl3, 400 MHz) d 1.25–1.58 (m, 2H), 1.64–
1.82 (m, 2H), 2.15–2.34 (m, 2H), 2.38–2.48 (m, 1H),
2.70–2.84 (m, 1H), 3.49 (d, J = 15.8 Hz, 2H), 3.63 (s,
1H), 5.96 (s, 1H), 6.03 (s, 1H), 6.67 (s, 1H), 6.74 (s, 1H),
6.75 (d, J = 3.4 Hz, 1H), 7.21 (d, J = 9.5 Hz, 1H), 7.79
(s, 1H). MS (ESI) m/z 296 (M+1)+.
1
87% yield in this step). H NMR (CDCl3, 400 MHz) d
1.50–1.72 (m, 2H), 1.73–1.88 (m, 2H), 2.32–2.62 (m,
3H), 2.80–2.93 (m, 1H), 3.60–380 (m, 3H), 6.82 (s,
1H), 6.84 (d, J = 8.5 Hz, 1H), 7.16 (t, J = 5.7 Hz, 1H),
7.28 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.65
(t, J = 7.6 Hz, 1H), 7.86 (s, 1H), 8.55 (d, J = 4.9 Hz,
1H). MS (ESI) m/z 308 (M+1)+.
5.2.46. N-[1-(1H-3-Pyrrolylmethyl)-3-piperidyl]-N-(1H-
5-indazolyl)amine (24t). Compound 24t was prepared
from pyrrole-3-carboxylic acid in a manner similar to
that described for 24s with a yield of 22% as a yellow
solid. Mp 187 ꢁC. 1H NMR (CD3OD, 400 MHz)
d 1.46–1.65 (m, 4H), 1.73–1.81 (m, 2H), 2.01–2.11 (m,
2H), 2.75–3.00 (m, 1H), 3.25–3.40 (m, 1H), 4.16 (d,
J = 13.2 Hz, 2H), 6.22 (s, 2H), 6.64 (s, 2H), 6.84 (d,
J = 8.3 Hz, 1H), 7.03 (s, 1H), 7.23 (d, J = 9.0 Hz, 1H),
7.68 (s, 1H). MS (ESI) m/z 296 (M+1)+.
5.2.43.
N-[1-(3-Pyridylmethyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24q). Compound 24q was prepared
from 3-chloromethyl-pyridine in a manner similar to
that described for 24p with a yield of 67% as a dense yel-
1
low oil. H NMR (CDCl3, 400 MHz) d 1.52–1.65 (m,
5.2.47.
N-[1-(2-Thienylmethyl)-3-piperidyl]-N-(1H-5-
2H), 1.70–1.84 (m, 2H), 2.30–2.50 (m, 3H), 2.73–2.85
(1H), 3.50–3.68 (m, 3H), 6.82 (s, 1H), 6.83 (d,
J = 8.8 Hz, 1H), 7.23–7.27 (m, 1H), 7.29 (d,
J = 8.8 Hz, 1H), 7.70 (br s, 1H), 7.86 (s, 1H), 8.51 (d,
J = 4.6 Hz, 1H), 8.56 (s, 1H). MS (ESI) m/z 308 (M+1)+.
indazolyl)amine (24u). Compound 24u was prepared
from thiophene-2-carboxylic acid in a manner similar
to that described for compound 24s with a yield of
19% as a yellow solid. Mp 136 ꢁC. H NMR (CDCl3,
400 MHz) d 1.52–1.63 (m, 2H), 1.64–1.80 (m, 2H),
1.88–1.97 (m, 1H), 2.35–2.55 (m, 1H), 2.72–2.78 (m,
2H), 3.14–3.24 (m, 2H), 3.56–3.64 (m, 1H), 6.80 (s,
1H), 6.82 (d, J = 8.8 Hz, 1H), 6.91 (dd, J = 3.4, 5.1 Hz,
1H), 7.21 (d, J = 5.1 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H),
7.84 (s, 1H). MS (ESI) m/z 313 (M+1)+.
1
5.2.44.
N-[1-(4-Pyridylmethyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24r). Compound 24r was prepared
from 4-chloromethyl-pyridine in a manner similar to
that described for 24p with a yield of 68% as a dense yel-
1
low oil. H NMR (CDCl3, 400 MHz) d 1.54–1.67 (m,
2H), 1.74–1.85 (m, 2H), 2.32–2.50 (m, 3H), 2.76–2.87
(m, 1H), 3.54 (dd, J = 14.3, 25.3 Hz, 2H), 3.65 (br s,
1H), 6.83 (s, 1H), 6.84 (d, J = 7.8 Hz, 1H), 7.27 (d,
J = 8.8 Hz, 2H), 7.31 (d, J = 8.1 Hz, 1H), 7.87 (s, 1H),
8.54 (d, J = 5.9 Hz, 2H). MS (ESI) m/z 308 (M+1)+.
5.2.48.
N-[1-(3-Thienylmethyl)-3-piperidyl]-N-(1H-5-
indazolyl)amine (24v). Compound 24v was prepared
from thiophene-3-carboxylic acid in a manner similar
to that described for 24s with a yield of 23% as a yellow
solid. Mp 149 ꢁC. H NMR (CDCl3, 400 MHz) d 1.48–
1
1.65 (m, 2H), 1.67–1.80 (m, 2H), 2.24–2.50 (m, 3H),
2.68–2.84 (m, 1H), 3.49–3.64 (m, 3H), 6.80 (s, 1H),
6.79–6.83 (m, 1H), 7.05 (d, J = 4.4 Hz, 1H), 7.10 (s,
1H), 7.25 (s, 1H), 7.27 (d, J = 6.8 Hz, 1H), 7.85 (s,
1H). MS (ESI) m/z 313 (M+1)+.
5.2.45. N-[1-(1H-2-Pyrrolylmethyl)-3-piperidyl]-N-(1H-
5-indazolyl)amine (24s). To a solution of tert-butyl
3-(1H-5-indazolylamino)-1-piperidinecarboxylate 25
(82 mg, 0.25 mmol) in CHCl3 (1 mL), TFA (1 mL) was
added dropwise at rt. The mixture was stirred at rt for
3 h and concentrated. To the residue, pyrrole-2-carboxyl-
ic acid (56 mg, 0.50 mmol), HOBT (77 mg, 0.5 mmol), tri-
ethylamine (0.2 mL) and dimethylaminopyridine (5 mg)
in CH3CN (1 mL) were added. WSC-HCl (86 mg,
0.5 mmol) was then added at 0 ꢁC. The reaction mixture
was stirred at rt for 18 h and quenched by the addition
of saturated NaHCO3 aq. The reaction mixture was
extracted with EtOAc. The combined organic layer was
dried over anhydrous Na2SO4 and filtered. The filtrate
was then concentrated. To a solution of the residue in
THF (1 mL) was added the BH3–THF (2.0 mL,
2.0 mmol) at 0 ꢁC. The resulting mixture was stirred at
80 ꢁC for 3 h. After cooling to rt, the reaction mixture
was acidified by the addition of 1 N HCl aq and stirred
at 80 ꢁC for 1 h. The reaction mixture was basified by
the addition of saturated NaHCO3 aq. The mixture was
extracted with EtOAc. The combined organic layer was
dried over anhydrous Na2SO4 and filtered. The filtrate
5.2.49. N-[1-(2-Furylmethyl)-3-piperidyl]-N-(1H-5-indaz-
olyl)amine (24w). Compound 24w was prepared from fur-
an-2-carboxylic acid in a manner similar to that described
for 24s with a yield of 4% as a yellow solid. Mp 167 ꢁC. 1H
NMR (CDCl3, 400 MHz) d 1.40–1.52 (m, 1H), 1.54–1.66
(m, 1H), 1.68–1.84 (m, 2H), 1.98–2.30 (m, 1H), 2.30–2.40
(m, 1H), 2.46–2.60 (m, 1H), 2.80–2.94 (m, 1H), 3.57 (d, J
=12.9 Hz, 2H), 3.54–3.62 (m, 1H), 6.17 (d, J = 2.9 Hz,
1H), 6.29 (dd, J = 1.2, 3.2 Hz, 1H), 6.78–6.83 (m, 2H),
7.27 (d, J = 9.5 Hz, 1H), 7.36 (s, 1H), 7.84 (s, 1H). MS
(ESI) m/z 297 (M+1)+.
5.2.50. N-[1-(3-Furylmethyl)-3-piperidyl]-N-(1H-5-indaz-
olyl)amine (24x). Compound 24x was prepared from
furan-3-carboxylic acid in a manner similar to that de-
scribed for 24s with a yield of 24% as a yellow solid.
1
Mp 149 ꢁC. H NMR (CDCl3, 400 MHz) d 1.45–1.65
(m, 2H), 1.67–1.80 (m, 2H), 2.22–2.52 (m, 3H), 2.70–

364
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 350–364
Nakano, T.; Okawa, K.; Iwamatsu, A.; Kaibuchi, K.
EMBO J. 1996, 15, 2208; (c) Ishizaki, T.; Maekawa, M.;
Fujisawa, K.; Okawa, K.; Iwamatsu, A.; Fujita, A.;
Watanabe, N.; Saito, Y.; Kakizuka, A.; Morii, N.;
Narumiya, S. EMBO J. 1996, 15, 1885.
2.84 (m, 1H), 3.41 (d, J = 10.3 Hz, 2H), 3.55–3.64 (m,
1H), 6.38 (s, 1H), 6.80 (s, 1H), 6.81 (d, J = 3.9 Hz,
1H), 7.27 (d, J = 8.8 Hz, 1H), 7.31 (s, 1H), 7.36 (s,
1H), 7.85 (s, 1H). MS (ESI) m/z 297 (M+1)+.
2. (a) Saito, H.; Minamiya, Y.; Saito, S.; Ogawa, J. J.
Leuk. Biol. 2002, 72, 829; (b) Liu, L.; Schwartz, B. R.;
Lin, N.; Winn, R. K.; Harlan, J. M. J. Immunol. 2002,
169, 2330; (c) Hauert, A. B.; Martinelli, S.; Marone, C.;
Niggli, V. Int. J. Biochem. Cell Biol. 2002, 34, 838; (d)
Ashida, N.; Arai, H.; Yamasaki, M.; Kita, T. J. Biol.
Chem. 2001, 276, 16555; (e) Niggli, V. FEBS Lett. 1999,
445, 69.
3. (a) Masumoto, A.; Mohri, M.; Shimokawa, H.; Urakami,
L.; Usui, M.; Takeshita, A. Circulation 2002, 105, 1545;
(b) Shimokawa, H. J. Cardiovasc. Pharmacol. 2002, 39,
319.
4. (a) Satoh, S.; Suzuki, Y.; Harada, T.; Ikegaki, I.; Asano,
T.; Shibuya, M.; Sugita, K.; Hidaka, H. Eur. J. Pharma-
col. 1992, 220, 243; (b) Shibuya, M.; Suzuki, Y.; Sugita,
K.; Saito, I.; Sasaki, T.; Takakura, K.; Nagata, I.;
Kikuchi, H.; Takemae, T.; Hidaka, H.; Nakashima, M.
J. Neurosurg. 1992, 76, 571.
5. Tsutiya, M.; Asano, T.; Sako, K.; Yonemasu, Y. Jpn.
Pharmacol. Ther. 1992, Suppl. 20, s1525. and references
cited therein.
6. (a) Uehata, M.; Ishizaki, T.; Satoh, H.; Ono, T.; Kawa-
hara, T.; Morishita, T.; Tamakawa, H.; Yamagami, K.;
Inui, J.; Maekawa, M.; Narumiya, S. Nature 1997, 389,
990; (b) Itoh, K.; Yoshioka, K.; Akedo, H.; Uehata, M.;
Ishizaki, T.; Narumiya, S. Nat. Med. 1999, 5, 221; (c)
Ishizaki, T.; Uehata, M.; Tamechika, I.; Keel, J.; Nonom-
ura, K.; Maekawa, M.; Narumiya, S. Am. Soc. Pharmacol.
Exp. Ther. 2000, 57, 976; (d) Narumiya, S.; Ishizaki, T.;
Uehata, M. Methods Enzymol. 2000, 325, 273.
7. Takami, A.; Iwakubo, M.; Okada, Y.; Kawata, T.; Odai,
H.; Takahasi, N.; Shindo, K.; Kimura, K.; Tagami, Y.;
Miyake, M.; Fukushima, K.; Inagaki, M.; Amano, M.;
Kaibuchi, K.; Iijima, H. Bioorg. Med. Chem. 2004, 12,
2115.
5.2.51. tert-Butyl 3-(1H-5-indazolylamino)-1-piperidine-
carboxylate (25). To a mixture of 3-hydroxypiperidine
(1.01 g, 10 mmol) in 3 N NaOH aq (10 mL), a solution
of di-tert-butyl dicarbonate (2.40 g, 11 mmol) in THF
(10 mL) was added dropwise at 0 ꢁC. The reaction mix-
ture was stirred at rt for 1 h and evaporated to remove
the THF. The aqueous layer was extracted with EtOAc.
The combined organic layer was dried over anhydrous
Na2SO4 and filtered. The filtrate was then concentrated.
To a mixture of the residue and triethylamine (2 mL) in
anhydrous DMSO (10 mL), the sulfur trioxide–trimeth-
ylamine complex (4.44 g, 20 mmol) was slowly added at
0 ꢁC. The reaction mixture was stirred at rt for 18 h and
quenched by the addition of saturated NaHCO3 aq. The
reaction mixture was extracted with EtOAc. The com-
bined organic layer was dried over anhydrous Na2SO4
and filtered. The filtrate was then concentrated. To a
mixture of the residue, 5-amino-1H-indazole 2 (0.98 g,
7.4 mmol), and AcOH (0. 2 mL) in MeOH (10 mL),
the BH3–pyridine complex (1.0 mL, 10 mmol) was slow-
ly added at 0 ꢁC. The reaction mixture was stirred at rt
for 18 h. The reaction was quenched by the addition of
saturated NaHCO3 aq. The mixture was extracted with
CHCl3. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was then
concentrated. The residual oil was purified by silica gel
column chromatography, eluting with CHCl3/MeOH
(95:5) to give the title compound as a dense yellow oil
1
(2.30 g, 7.20 mmol, 72% yield in three steps). H NMR
(CDCl3, 400 MHz) d 1.44 (s, 9H), 1.83–2.02 (m, 2H),
2.15–2.25 (m, 1H), 3.30–3.56 (m, 4H), 3.98–4.10 (m,
1H), 4.40–4.46 (m, 1H), 6.77–6.81 (m, 2H), 7.30 (d,
J = 8.3 Hz, 1H), 7.88 (s, 1H).
8. Crutius Thornton, T. J. Synthesis 1990, 295.
9. Ruchardt, C.; Hassmann, V. Liebigs Ann. Chem. 1980,
908.
10. Mitsunobu, O. Synthesis 1981, 1.
11. Ohashi, H.; Takei, M.; Ide, Y.; Ishii, H.; Kita, H.; Gleich,
G. J.; Ishikawa, M.; Fukamachi, H. Int. Arch. Allergy
Immunol. 1999, 118, 44.
12. Morikawa, A.; Sone, T.; Asano, T. Chem. Pharm. Bull.
1992, 40, 770.
13. Adachi, T.; Vita, R.; Sannohe, S.; Stafford, S.; Alam,
R.; Kayaba, H.; Chihara, J. J. Immunol. 2000, 167,
4609.
Acknowledgments
The authors thank Ms. Takemi Tokieda for her techni-
cal assistance and Dr. Kazuo Kubo for reading the man-
uscript. We are grateful to Dr. Takeshi Ohtani and Dr.
Kazuta Takemura who contributed to the initiation of
this project.
14. (a) Nagarathnam, D.; Asgari, D.; Shao, J.; Liu, X.-G.;
Khire, U.; Wang, C.; Hart, B.; Boyer, S.; Weber, O.;
Lynch, M.; Bankston, D. WO 02/076976 A2; (b) Imazaki,
N.; Kitano, M.; Ohashi, N.; Matsui, K. WO 02/100833
A1; (c) Hu, E.; Lee, D. Expert Opin. Ther. Targets 2005, 9,
715; (d) Mueller, B. K.; Mack, H.; Teush, N. Nat. Rev.
Drug Discovery 2005, 4, 387.
References and notes
1. (a) Fukata, Y.; Amano, M.; Kaibuchi, K. Trends Phar-
macol. Sci. 2001, 22; (b) Matsui, T.; Amano, M.;
Yamamoto, T.; Chihara, K.; Nakafuku, M.; Ito, M.;