Design and synthesis of rho kinase inhibitors (III)

Article abstract of DOI:10.1016/j.bmc.2006.10.028

The structure-activity relationship of Rho kinase inhibitors bearing an isoquinoline scaffold was studied. N-(1-Benzyl-3-pyrrolidyl)-N-(5-isoquinolyl)amine analogues were optimized with respect to their inhibitory potencies for the enzyme and for chemotaxis. The potent analogues were further evaluated by an ex vivo test in which the selected compounds were orally administered to rats, and the Rho kinase inhibitory potency observed in the rat serum was evaluated 3 h after the administration. Compound 23g showed a high level of Rho kinase inhibitory activity in the rat serum and was stable in an in vitro metabolic test using a microsomal cytochrome preparation. The (R)-isomer of 23g displayed a higher level of inhibitory potency than the (S)-isomer in a cell-free kinase assay and in the cell migration assay (IC₅₀^ENZ = 25 nM and IC₅₀^MCP = 1 μ M). The (R)-isomer successfully inhibited the phosphorylation of MBS (myosin-binding subunit) in cells.

Full text of DOI:10.1016/j.bmc.2006.10.028

Bioorganic & Medicinal Chemistry 15 (2007) 1022–1033
Design and synthesis of rho kinase inhibitors (III)
Masayuki Iwakubo,a, ,à Atsuya Takami,a, Yuji Okada,a Takehisa Kawata,a
Yoshimichi Tagami,a Motoko Sato,a Terumi Sugiyama,a Kayoko Fukushima,a
Shinichiro Taya,b Mutsuki Amano,b Kozo Kaibuchib and Hiroshi Iijimaa,*
aPharmaceutical Research Laboratories, Kirin Brewery Co. Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan
bDepartment of Cell Pharmacology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku,
Nagoya, Aichi 466-8550, Japan
Received 15 August 2006; revised 12 October 2006; accepted 14 October 2006
Available online 18 October 2006
Abstract—The structure–activity relationship of Rho kinase inhibitors bearing an isoquinoline scaffold was studied. N-(1-Benzyl-3-
pyrrolidyl)-N-(5-isoquinolyl)amine analogues were optimized with respect to their inhibitory potencies for the enzyme and for che-
motaxis. The potent analogues were further evaluated by an ex vivo test in which the selected compounds were orally administered
to rats, and the Rho kinase inhibitory potency observed in the rat serum was evaluated 3 h after the administration. Compound 23g
showed a high level of Rho kinase inhibitory activity in the rat serum and was stable in an in vitro metabolic test using a microsomal
cytochrome preparation. The (R)-isomer of 23g displayed a higher level of inhibitory potency than the (S)-isomer in a cell-free
ENZ
50
phosphorylation of MBS (myosin-binding subunit) in cells.
MCP
50
kinase assay and in the cell migration assay (IC
¼ 25 nM and IC
¼ 1 lM). The (R)-isomer successfully inhibited the
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
inactivating the phosphatase. These direct and indirect
effects act in coordination to regulate the phosphoryla-
tion state of MLC that results in the contraction of
smooth muscle cells and stress fiber formation in non-
muscle cells.1
Rho kinase is a serine/threonine protein kinase that is
composed of the kinase domain at the N-terminal fol-
lowed by the coiled-coil domain, the Rho-binding do-
main, and the PH-like domain at the C-terminal. Rho
kinase is one of the best-studied Rho effectors.1 Regula-
tion of the phosphorylation of myosin light chain
(MLC) by Rho is mediated by the activation of Rho ki-
nase. Rho kinase in the free state is auto-inhibited.
Association of the GTP-binding form of Rho with the
Rho-binding domain of Rho kinase induces a structural
change and the enzyme is activated.
Rho kinase inhibitors would hold great potential as nov-
el therapies for a number of diseases because the kinase
plays an important role in stress fiber formation, focal
adhesion, and smooth muscle contraction as described
above. It was shown that Fasudil dihydrochloride
(HA-1077), which has been used clinically as a cerebro-
vascular contraction inhibitor,2 is a potent Rho kinase
inhibitor and that the action of HA-1077 could be ex-
plained by the inhibition of Rho kinase. Evidence is
accumulating that Rho kinase inhibitors would be useful
for the treatment of myocardial ischemia,3 hyperten-
sion,4 penile corpus cavernosum,5 glaucoma,6 cancer
migration,7 and kidney diseases.8,9 Recently, it has been
suggested that the inhibition of Rho kinase might accel-
erate the regeneration of nerve fibers after spinal-cord
injury and might be effective in neurological disorders
such as spinal-cord injury, Alzheimer’s disease, and mul-
tiple sclerosis.10 Recent progress in the development of
Rho kinase inhibitors and their potential applications
have been reviewed.11
The activated Rho kinase directly phosphorylates MLC,
and simultaneously Rho kinase phosphorylates the
myosin-binding subunit of myosin phosphatase, thereby
Keywords: Rho kinase; Inhibitor; Structure–activity relationship;
Myosin-binding subunit phosphorylation.
*
Corresponding author. Tel.: +81 27 346 9776; fax: +81 27 346
8418; e-mail: iijimah@kirin.co.jp
These authors equally contributed to this work.
à
Present address: Liquid Crystal Materials Technical Department,
Dainippon Ink and Chemicals, Inc., 4472-1 Komuro, Ina-machi,
Kitaadachi-gun, Saitama 362-8577, Japan.
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.10.028

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
1023
In order to develop potent and selective Rho kinase
inhibitors, we carried out a structure-based drug design
of inhibitors using a homology model of the kinase and
have succeeded in identifying the seed compounds of
several different scaffolds.12 We have reported the prop-
erties and the structural optimization of 1H-indazole
analogues.13 Here we report on our investigation of
the structure–activity relationship (SAR) of isoquinoline
analogues.
were prepared by reductive alkylation of 5-aminoiso-
quinoline with 3a–c using Ti (OiPr)4 as a Lewis acid cat-
alyst and NaBH4 as a reducing agent.
Reductive alkylation of 5-aminoisoquinoline with 1,4-
cyclohexanedione monoethylene ketal 5 using the
BH3–pyridine complex in the presence of a catalytic
amount of acetic acid gave the corresponding com-
pound. This step was then followed by hydrolysis of
the ketal group with 50% acetic acid to give the interme-
diate 6. 1,4-Diaminocyclohexane analogues 8a–c were
prepared by reductive amination of 6 with several alkyl
amines using NaBH(OAc)3.
2. Chemistry
The intermediates, 1-alkyl-4-piperidone 3a–c, were ob-
tained by alkylation of 4-piperidone hydrochloride 1
with several alkyl bromides 2a–c (Scheme 1). N-(1-Al-
kyl-4-piperidyl)-N-(5-isoquinolyl)amine analogues 4a–c
The intermediate10 was prepared by reaction with 3-
hydroxypyrrolidine 9 and 1-chloro-3-methylbutane.
The corresponding ketone 11 was synthesized by oxida-
Scheme 1. Synthetic routes of isoquinoline analogues. Reagents and conditions: (a) K2CO3, CH3CN, rt, then reflux; (b) 5-aminoisoquinoline,
Ti(OiPr)4, rt then MeOH, NaBH4, rt; (c) (1) 5-aminoisoquinoline, BH3/pyridine, AcOH, MeOH, rt; (2) 50% AcOH, reflux; (d) NaBH(OAc)3, MeOH,
rt; (e) 1-chloro-3-methylbutane, K2CO3, DMF, 0 ꢁC; (f) SO3–Me3N, DMSO, Et3N, rt; (g) di-tert-butyl dicarbonate, THF, 3 N NaOH aq, rt; (h)
(1) 5-aminoisoquinoline, Na2SO4, AcOH, rt; (2) NaBH(OAc)3, rt; (i) (1) 95% TFA, CHCl3, rt; (2) K2CO3, CH3CN, rt; (j) SnCl2/H2O, HCl,
reflux.

1024
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
tion of 10 using the sulfur trioxide-trimethylamine com-
plex (SO3–Me3N) in DMSO at room temperature. 12
was prepared by reductive alkylation of 5-aminoisoquin-
oline with 11 using Ti (OiPr)4and NaBH4.
yield (< 5%). The t-Boc group was of 18 and 19 were re-
moved using TFA. The N-alkylated compounds 21a–c
and 22a–h were then prepared by alkylation with substi-
tuted benzylchlorides. The amines 23f–h were synthe-
sized by reduction of the corresponding nitro
compounds 22f–h with tin(II) chloride monohydrate.
The secondary amines, 3-hydroxypiperidine 13 and 3-
hydroxypyrrolidine 9, were protected by the tert-butox-
ycarbonyl (t-Boc) group (14 and 15). The ketones 16 and
17 were synthesized by oxidation of 14 and 15 using the
same method for the synthesis of 11. Compounds 18 and
19 were, respectively, obtained from 16 and 17 by reduc-
tive alkylation with 5-aminoisoquinoline using NaB-
H(OAc)3 at room temperature in glacial acetic acid.
Under other conditions such as Ti(OiPr)4/NaBH4 or
BH3/Pyridine/AcOH, this alkylation resulted in a low
The enantiomers of 23g were synthesized as shown in
Scheme 2. Trifluoromethanesulfonate 25 was prepared
from 5-hydroxyisoquinoline 24 by treatment with triflu-
oromethanesulfonic anhydride. (3R)-(+)-3-(t-Boc-ami-
no)-pyrrolidine 26 was allowed to react with
3-nitrobenzylchloride to afford 27. The intermediate 28
was prepared by cleavage of the t-Boc group of 27.
Compound 29 was synthesized from 28 and 25 by the
N
N
a
OH
OTf
24
25
H
N
H2N
N
H
N
N
b
t-BOC
c
NO2
NO2
NH
t-BOC
26
27
28
N
d
N
R = NO2
NH2
29
30-R
25
e
N
H
R
Scheme 2. Synthesis of 30-R. Reagents and conditions: (a) Tf2O, CHCl3, pyridine, rt; (b) 3-nitrobenzylchloride, K2CO3, CH3CN, rt; (c) TFA,
CHCl3, rt; (d) cat. Pd(OAc)2, BINAP, Cs2CO3, toluene, 80 ꢁC; (e) SnCl2/H2O, HCl, reflux.
H
6
χ1
χ2
N
1'
3'
5
N
5'
NH2
N
2
model
a
b
c
color of carbon atoms
cyan
magenta
yellow
packering position of
pyrrolidine ring
5'
S
1'
S
3'
S
pseudochirality of N1'
χ1 (6-5-N-3')
-72
87
-17
94
-20
154
χ2 (5-N-3'-2')
Figure 1. Docking models of 30-R. The molecular surface of the ligand-binding pocket was visualized. Three representative conformer models of 30-
R, a–c, are shown in a superimposed view. In the A region, the isoquinoline ring took a constant position, and the essential hydrogen bond between
the backbone NH of Met167 and the N2 of isoquinoline was well conserved (yellow dashed line). In the F region, different conformations were
possible for the pyrrolidine ring. The torsion angles that determine the orientation of the pyrrolidine ring relative to the isoquinoline ring (v1 and v2)
were not conserved. However, the center of the mass of the ring was conserved in the center of the F region. The sidechain methyl group of Ala226 is
shown.

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
Table 1. Inhibitory potencies of isoquinoline derivatives
1025
ENZ
50
Entry
Rho kinase IC
(nM)
Isoquinoline analogues
N
O
O
N
HA-1077
180
S
NH
R
4a
4b
4c
345
210
140
CH2CH2CH3
CH2CH(CH3)2
CH2-Ph
N
H
N
N
R
R
anti
syn
8a
8b
8c
180
180
CH2CH2CH3
N
H
N
anti
syn
70
195
CycloPr
NHR
anti
syn
440
580
CH2CH2-Ph
N
N
H
N
12
270
N
N
X
21a
21b
21c
210
265
185
2-Cl
3-Cl
4-Cl
H
N
X
X
22a
22b
22c
22d
22e
23f
23g
23h
100
65
2-Cl
3-Cl
N
65
95
4-Cl
4-F
H
N
N
75
2,6-F
2-NH2
3-NH2
4-NH2
X
205
275
235
Buchwald-Hartwig reaction with a catalytic amount of
Pd(OAc)2 and ( )-BINAP in the presence of Cs2CO3
at 80 ꢁC in toluene in a moderate yield.14 The corre-
sponding amine 30-R was obtained from 29 by reduc-
tion with tin(II) chloride monohydrate. The other
isomer, 30-S, was synthesized from (3S)-(ꢀ)-3-(t-Boc-
amino)-pyrrolidine in the same manner.
3.1. Enzyme inhibitory potency
The ligand-binding pocket of Rho kinase is composed of
three regions, namely the A, F, and D regions. The A
region is the bottom of the pocket and offers the essen-
tial hydrogen bond donor (the backbone NH of Met167,
Fig. 1). Planar aromatic scaffolds that have a hydrogen
bond acceptor atom inside or outside of the aromatic
ring, such as pyridine, indazole, isoquinoline, phthali-
mide, and benzamide, were found compatible with the
flat shape of the A region.12 The F region is spherical
and spacious. In the case of inhibitors employing 1H-in-
dazole as the substructure for the A region, docking sim-
ulation indicated that several chemical fragments could
possibly occupy the F region. Actually, 1H-indazole
derivatives that have 3-aminopiperidine, 4-aminopiperi-
dine, 3-aminopyrrolidine, and urea as the substructures
occupying the F region were all potent inhibitors.12,13 In
3. Structure optimization
The cell-free Rho kinase assay was performed according
to the methods described in a previous report.12 A che-
motaxis assay was performed by the Boyden Chamber
method using CCR2-overexpressing human-derived his-
tiocyte lymphoma (U937) cells and MCP-1.13 HA-1077
was prepared by the published method15 and was used
as the positive control.

1026
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
contrast, as the isoquinoline ring occupies more space
than a 1H-indazole ring in the A region, the choice of
the substructure in the F region was expected to be lim-
ited. In fact, it was demonstrated that the use of urea or
hydrophobic amino group on the phenyl ring indicated
MCP
50
IC
e. The inhibitory potency (IC
values comparative with those of 21c and 22a–
Þ of compounds 23f–h
MCP
50
is several times the value expected from their in vitro en-
ENZ
50
amide substructures leads to the loss of the inhibitory
activity of isoquinoline analogues (IC
zyme inhibitory potency (IC Þ based on Eq. 1. Hydro-
ENZ
50
> 10 lMÞ,
philic substitution on the phenyl ring would be
advantageous for cell permeability, but not for enzyme
inhibition potency. We had previously observed a simi-
lar SAR during the course of the structure optimization
of 1H-indazole inhibitors.13
and that a linker substructure consisting of an amine
nitrogen atom and a saturated ring was suitable.12 Con-
sidering the results from the previous study12, we exam-
ined the enzyme inhibitory potency of isoquinoline
analogues carrying a cyclic aliphatic ring of different
chemical structures.
3.3. Ex vivo test
In Table 1, the kinase inhibitory potency of isoquinoline
derivatives having 4-aminopiperidine (4a–c), 1,4-diami-
nocyclohexane (8a–c), 3-aminopiperidine (12 and 21a–
c), and 3-aminopyrrolidine (22a–h) substructures is
shown. 4-Aminopiperidine and 3-aminopiperidine deriv-
atives showed potency comparative to that of HA-1077
(4a–c, 12, and 21a–c). In the case of 1,4-diaminocyclo-
hexane derivatives, only 8b (anti) showed improved
potency, and other derivatives had comparative poten-
To estimate their efficacy in vivo, we measured the inhib-
itory potency of the serum 3 h after oral administration
of the isoquinoline inhibitors to SD rats at doses of
30 mg/kg (ex vivo test). The inhibition rate (%) was cal-
culated relative to that of the control rats (water was
administered). The result is shown in Table 2. The inhi-
bition rate of HA-1077 was 20%. Compounds 22a–e
ENZ
50
MCP
values than HA-1077,
50
have better IC
and IC
but showed poorer inhibition (<20%). In contrast, com-
pound 23g with an amino group at the 3-position on the
phenyl ring showed superior inhibition (57%). The ana-
logues having an amino group at the 2- or 4-position on
the phenyl ring showed weak inhibition in the ex vivo
test (23f: 9%, 23h: 15%), though they indicated a suffi-
cient inhibitory potency for the enzyme reaction and
for chemotaxis as did 23g. It was considered that the sol-
ubility or permeability of the compounds was not the
major reason for this difference.
cy. The 3-aminopyrrolidyl substructure (22a–c;
ENZ
50
IC
idyl substructure (12, 21a–c; IC
< 100 nMÞ was more favorable than the 3-piper-
ENZ
50
> 100 nMÞ. This
preference of an aminopyrrolidine substructure over
an aminopiperidine substructure was the opposite of
the SAR found in the 1H-indazole analogues.13
We examined the effect of the substitutions on the phen-
yl ring of 3-aminopyrrolidine analogues. Analogues
substituted with halogen atoms (22a–e) indicated a high-
er potency than 23f–h, which had an amino group on the
phenyl ring (Table 1). Hydrophobic substitution on the
phenyl ring would be advantageous for interaction with
the enzyme.
Thus, the metabolic stability of the inhibitors was eval-
uated using a rat liver microsome preparation. Com-
pounds were incubated with an excess of microsome
and NADPH for 30 min. The residual concentration
of 23g was 67%, while that for HA-1077 it was 45%.
Analogues that exhibited poorer efficacy in the ex vivo
test all indicated a higher susceptibility to microsome
oxidation (<15%). Stability against metabolism by liver
cytochromes might be a possible explanation why the
serum of the rats administered with 23g retained good
inhibitory potency. However, because the results of the
ex vivo test would reflect many complex pharmacokinet-
ic processes (for example, absorption, distribution, and
the possible existence of active metabolites), we should
refrain from attributing the good ex vivo property of
23g only to its metabolic stability.
3.2. Chemotaxis inhibitory potency
Potent isoquinoline-pyrrolidine derivatives also showed
good CCR2/MCP-1 chemotaxis inhibitory potency (Ta-
ble 2). Among the compounds 21c and 22a–e, the Rho
kinase inhibition potency roughly correlated with the
inhibition potency of chemotaxis;
MCP
50
ENZ
50
logðIC
Þ ¼ 0:69 logðIC Þ ꢀ 0:17ðr2 ¼ 0:61Þ ð1Þ
This fact would suggest that these halogenated ana-
logues have nearly equal permeability into the cells.
On the other hand, compounds 23f–h, which have a less
3.4. Profile of the (R)-isomer of 23g
Table 2. Properties of isoquinoline analogues
Compound 23g has a chiral center on the C3 of the pyr-
rolidine ring. As shown in Table 3, the (R)-isomer, 30-R,
has 12-fold Rho kinase inhibitory activity and 30-fold
chemotaxis inhibitory activity relative to the (S)-isomer,
Compound
Rho kinase
EHZ
50
CCR2/MCP-1
MCP
50
Ex vivo (%)
IC
(nM)
IC
(lM)
HA-1077
21c
22a
180
185
100
65
35
23
17
14
11
22
10
14
20
15
20
18
0
22b
22c
11
0
Table 3. Properties of 30-R and 30-S
65
95
Compound Rho kinase
EHZ
CCR2/MCP-1
MCP
50
Ex vivo (%)
22d
22e
4
IC
(nM) IC
(lM)
50
75
2
1h 4h 8h
52 42 26
23f
23g
205
275
235
9
30-R
30-S
25
310
1
30
57
15
30 17
0
23h

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
1027
30-S. Compound 30-R fortunately indicated higher ex vi-
vo efficacy. Although a time-dependent decrease in inhi-
bition was shown in the ex vivo test, sufficient inhibition
(26%) was observed even after 8 h (Table 3).
lial cells with tetradecanoylphorbol-13-acetate (TPA) in-
duced the phosphorylation of MBS under conditions
where membrane ruffling and cell migration were in-
duced.19 As shown in Figure 2, phosphorylation of
MBS induced by TPA was inhibited by pretreatment
with 30-R in a dose dependent manner. It should be not-
ed that the inhibition occurred at a concentration be-
The docking study of 30-R (Fig. 1) indicated that the
isoquinoline ring fully occupies the A region of the li-
gand-binding pocket and that the N2 atom of the iso-
quinoline ring interacts with NH of Met167. The
stereochemistry of C3 of the pyrrolidine ring, which
links the nitrogen atom of 5-aminoisoquinoline to its
ring system, is important. It is noted that all stable con-
formers of 30-R could certainly occupy the volume of
the ligand-binding pocket. Such a conformational
advantage was not possible with the (S)-isomer. Only
a proportion of the stable conformers of the (S)-isomer
could be docked into the pocket. The conformational
freedom in the ligand-binding pocket might explain
the higher potency of the (R)-isomer than the (S)-
isomer.
tween 1 and 10 lM. These concentrations correspond
MCP
50
to the IC
of 30-R.
4. Conclusion
The Rho kinase inhibitory potency of the serum of rats
administered with isoquinoline analogues was measured
and 23g was identified as an optimized lead Rho kinase
inhibitor having in vivo efficacy. The (R)-isomer, 30-R,
had 12- to 30-fold Rho kinase inhibitory potency rela-
ENZ
50
tive
IC
to
the
other
isomer
(IC
¼ 25 nM,
MCP
50
¼ 1 lM). 30-R exhibited enhanced metabolic sta-
bility. It inhibited MBS-phosphorylation in cells. Thus,
30-R would be a useful molecular probe to study the
function of Rho kinase in vivo. Application of 30-R to
several disease animal models is underway and will be
reported elsewhere.
Crystal structures of ROCK-I,16 an isoform of Rho ki-
nase, and Rho kinase17 have been elucidated recently.
It seemed that good use of the F region is important
for inhibitors to have a high affinity for and selectivity
against Rho kinase. The F region of Rho kinase is wider
than c-AMP dependent protein kinase A (PKA) because
the residue corresponding to Ala226 of Rho kinase is
Thr183 in PKA (Fig. 1). A mutation study of PKA18
strongly suggested that Ala226 is the key residue that af-
fects the inhibitor’s affinity and selectivity between PKA
and Rho kinase.
5. Experimental
Commonly used abbreviations: Ab (antibody), CCR-2
(C–C chemokine receptor type 2), MBS (myosin-binding
domain), MCP-1 (monocyte chemoattractant protein 1),
TPA (tetradecanoylphorbol-13-acetate), PBS (Na2H-
PO4 81 mM, KH2PO4 15 mM, 137 mM NaCl, 2.7 mM
KCl, pH 7.4), DMEM (Dulbecco’s modified Eagle’s
medium), DTT (dithiothreitol), PVDF (polyvinylidene
fluoride), SDS (sodium dodecylsulfonate), TBS (Tris–
HCl buffer 25 mM, pH 7.4, containing 137 mM NaCl
and 23 mM KCl), Ac (acetyl group), AcOH (acetic
acid), cycloPr (cyclopropyl group), DMF (N,N-dimeth-
ylformamide), DMSO (dimethylsulfoxide), EtOAc (eth-
yl acetate), EtOH (ethanol), MeOH (methanol), ODS
(octadecylated silica gel), TFA (trifluoroacetic acid), t-
Boc (tert-butoxycarbonyl group), Tf (trifluoromethane
sulfonyl group), THF (tetrahydrofuran).
3.5. Inhibition of MBS phosphorylation
Rho kinase phosphorylates the myosin-binding subunit
(MBS) of myosin phosphatase and thereby inactivates
the phosphatase activity. The sites of phosphorylation
of MBS by Rho kinase are Thr697, Ser854, Thr855,
and several other residues. By using an antibody that
specifically recognizes MBS phosphorylated at Ser854,
it has been found that the stimulation of MDCK epithe-
(-)
30-R (μM)
(-)
0.1
1
10 100
(+)
(+) (+)
(-) (+)
MW (KD)
208
(+)
TPA
5.1. Serum Rho kinase inhibitory activity (ex vivo test)
Rho kinase inhibitors (30 mg/kg) were orally adminis-
tered to SD rats (N = 4). The blood samples were ob-
tained from the tail artery 3 h after the administration.
The serum was collected by centrifugation and frozen
at ꢀ20 ꢁC until analysis. The serum samples were dilut-
ed 8-fold in water, and the Rho kinase inhibitory activ-
ity of the samples was determined. The inhibition rate
(%) was determined by comparison of the inhibitory
activity of the serum of the control rats (water was
administered).
119
Phosphorylated
MBS
94
208
119
Total MBS
94
Figure 2. Inhibition of Rho kinase by 30-R. Serum-deprived MDCK
II cells pretreated with 30-R were stimulated with 10 nM TPA for
10 min. The cell lysates were resolved by SDS–PAGE followed by
immunoblot analysis with anti-phosphorylated-MBS antibody (top) or
anti-MBS antibody (bottom). Arrowheads indicate the position of
phosphorylated MBS and total MBS.
5.2. Rat liver microsomal assays
The reaction mixture consisted of 100 lL of microsomal
protein (prepared from rat liver, protein concentration

1028
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
5 mg/mL), 100 lL of NADPH generation cofactors
(80 mM glucose-6-phosphate, 3.3 mM NADP, and
10 U/mL of glucose-6-phosphate dehydrogenase in
60 mM MgCl2), 350 lL of 250 mM potassium phos-
phate buffer (pH 7.4), 350 lL of water, and 100 lL of
the inhibitor (100 lM). The final concentration of the
test compound was 10 lM in a total reaction volume
of 1000 lL. The mixture was pre-incubated for 5 min
at 37 ꢁC. The reaction was initiated by the addition of
NADPH generation cofactor solution and incubated
for 30 min. The reaction was terminated by the addition
of EtOAc (6 mL). An internal standard was added to the
mixture. The organic phase (4 mL) was extracted, dried,
and diluted with the appropriate solvent. Drug concen-
trations were determined by LC analysis (Inatsil-ODS-3,
GL-Science, / 4.6 · 250 mm; flow rate, 1.0 mL/min;
Temp, 40 ꢁC; UV 240 nm; isocratic mobile phase con-
sisting of appropriate ratios of CH3CN and 100 mM
ammonium acetate) and were corrected by comparison
with the internal standard.
conformational models for the 1-benzyl-3-aminopyrroli-
dine substructure. Forty corresponding models of 1-ben-
zyl-3-aminopyrrolidine were built, and stable structures
were searched using the GA Conformational Search of
the SYBYL package for each of the 40 models. Because
the GA Search employs the same conformation genera-
tion algorithm as Flexi Dock, the ring conformation and
stereochemistry of the models are not altered. After min-
imization by Maxmin2 (Tripos force field), the models
were further optimized using a semi empirical molecular
orbital method (MOPAC) with a structure optimization
option. After this calculation, some structures con-
verged to identical structures. Ten stable conformational
models were finally obtained for the 1-benzyl-3-amino-
pyrrolidine substructure. Based on the 10 substructure
models, the corresponding models of 30-R were built.
These models were subjected to Flexi Dock simulation.
5.5. Chemistry
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 an internal chloroform as
7.24 ppm in deuterochloroform. Coupling constants
(J) were recorded in Hz. Mass spectra (MS) were record-
ed using a PLATFORM-LC (Micromass) spectrometer.
HA-1077 was prepared by the published method.15
5.3. Myosin-binding subunit phosphorylation assay
Serum-deprived MDCK II cells (5 · 105/ 60 mm dish) in
DMEM (4 mL) were treated with various doses of Rho ki-
nase inhibitors in PBS (300 lL). The cells were incubated
for 10 min, followed by the addition of TPA (tetradeca-
noylphorbol-13-acetate, 200 nM, 250 lL) at 37 ꢁC for
10 min. The cells were washed with PBS while cooled on
ice and treated with 10% trichloroacetic acid containing
2 mM DTT. Cells were scraped off while cooled on the
ice and centrifuged. The pellet was washed with acetone
containing 2 mM DTT, suspended in SDS-buffer, soni-
cated, and centrifuged. The supernatant was boiled at
100 ꢁC for 5 min and stored at ꢀ20 ꢁC. The specimen
(40 lL) was subjected to 8% SDS–polyacrylamide elec-
trophoresis. Proteins were transferred electrophoretically
to a PVDF membrane. Membranes were treated with
skimmed milk and successively treated with anti-pS854
Ab (a polyclonal rabbit antibody to MBS phosphorylated
at Ser854) or anti-MBS Ab (a polyclonal rabbit anti-MBS
antibody). They were then treated with TBS buffer con-
taining 0.1% Tween 20, skimmed milk, and anti-rabbit
IgG-HRP (horseradish peroxidase) for 1 h. After washing
with TBS buffer, detection was accomplished with an en-
hanced chemiluminescence (ECL+) detection system. De-
tails of the assay are described elsewhere.15
5.5.1. N-(1-Propyl-4-piperidyl)-N-(5-isoquinolyl)amine (4a).
To a mixture of 4-piperidone monohydrate 1 (300 mg,
2.6 mmol) and K2CO3 (540 mg, 3.9 mmol) in CH3CN
(5 mL), a solution of n-propylbromide 2a (359 mg,
2.7 mmol) in CH3CN (2 mL) was added dropwise at 0 ꢁC.
The reaction mixture was stirred at rt for 18 h and diluted
with EtOAc. The mixture was filtered through Celite to give
1-propyl-4-piperidone 3a. The filtrate was concentrated. A
mixture of the crude 3a and 5-aminoisoquinoline (208 mg,
1.4 mmol) was dissolved in titanium tetraisopropoxide
(5 mL), and the reaction mixture was stirred at rt for
30 min. The mixture was diluted with MeOH (5 mL), and
sodium borohydride (101 mg, 2.6 mmol) was added at
0 ꢁC. The mixture was stirred at rt for 3 h, filtered through
Celite, and quenched by the addition of saturated NaHCO3
aq. The mixture was extracted with EtOAc. The organic
layer was dried over anhydrous Na2SO4 and filtered. The
filtrate was 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
5.4. Docking simulation of 30-R with Rho kinase homol-
ogy model
1
oil (101 mg, 0.37 mmol, 26% yield). H NMR (CDCl3,
400 MHz) d 0.85 (t, J = 7.3 Hz, 3H), 1.47 (q, J = 7.6 Hz,
2H), 1.57 (dq, J = 4.2 Hz, 10.7 Hz, 2H), 2.05–2.18 (m,
4H), 2.28 (t, J = 7.8 Hz, 2H), 2.87 (t, J = 12.2 Hz, 2H),
3.38–3.50 (m, 1H), 6.71 (d, J = 7.6 Hz, 1H), 7.22 (d,
J = 8.1 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.46 (d,
J = 6.1 Hz, 1H), 8.39 (d, J = 5.9 Hz, 1H), 9.07 (s, 1H).
MS (ESI) m/z 270 (M+1)+.
The docking simulation was carried out using Flexi
Dock available in the SYBYL software package in a
manner similar to that described in a previous report.12
The Flexi Dock algorithm allows ligand molecule single
bond rotation but ring conformation is fixed. Thus,
when a ligand contains a flexible ring system, multiple
models of the ligand should be considered one by one.
30-R was such a case. Many conformational models
could be expected for the five-member ring (pyrrolidine
ring) of 30-R. Ring puckering (up and down) at the five
positions of the pyrrolidine ring and the pseudo-chirality
at the two nitrogen atoms produced 40 (2 · 5 · 2 · 2)
5.5.2. N-(1-Isobutyl-4-piperidyl)-N-(5-isoquinolyl)amine
(4b). The compound 4b was prepared from 1-bromo-2-
methylpropane 2b in a manner similar to that described
for the compound 4a with a yield of 35% as a dense
yellow oil. 1H NMR (CDCl3, 400 MHz) d 0.84

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
1029
(d, J = 6.6 Hz, 6H), 1.56 (dq, J = 3.2 Hz, 10.5 Hz, 2H),
1.68-1.77 (m, 1H), 2.06 (d, J = 7.3 Hz, 4H), 2.09 (d,
J = 10.7 Hz, 2H), 2.79 (d, J = 11.9 Hz, 2H), 3.38–3.45
(m, 1H), 6.70 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 8.1 Hz,
1H), 7.37 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 5.9 Hz, 1H),
8.38 (d, J = 5.9 Hz, 1H), 9.07 (s, 1H). MS (ESI)
m/z = 284 (M++1).
5.5.5. N1-Cyclopropyl-N4-(5-isoquinolyl)-1,4-cyclohex-
anediamine (8b). The compound 8b was prepared from
cyclopropylamine 7b in a manner similar to that de-
scribed for the compound 8a as a dense yellow oil. Anti
isomer: H NMR (CDCl3, 400 MHz) d ꢀ0.16 to ꢀ0.09
1
(m, 2H), ꢀ0.04 to 0.20 (m, 2H), 0.75–0.93 (m, 4H),
1.58–1.72 (m, 3H), 1.75–1.85 (m, 2H), 2.16–2.27 (m,
1H), 2.87–3.03 (m, 1H), 3.60–3.85 (m, 1H), 6.29 (d,
J = 7.6 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 7.06 (t,
J = 8.0 Hz, 1H), 7.16 (t, J = 6.1 Hz, 1H), 8.08 (t,
J = 5.8 Hz, 1H), 8.76 (s, 1H); MS (ESI) m/z 282
(M+1)+. Syn isomer: 1H NMR (CDCl3, 400 MHz) d
ꢀ0.04 to 0.02 (m, 2H), 0.07–0.13 (m, 2H), 0.90–1.58
(m, 8H), 1.68–1.78 (m, 1H), 2.48 (tt, J = 3.9 Hz,
7.8 Hz, 1H), 3.28–3.40 (m, 1H), 3.95–4.13 (m, 1H),
6.39 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 7.06
(t, J = 8.0 Hz, 1H), 7.16 (t, J = 6.1 Hz, 1H), 8.08 (d,
J = 5.8 Hz, 1H), 8.76 (s, 1H). MS (ESI) m/z 282 (M+1)+.
5.5.3.
N-(1-Benzyl-4-piperidyl)-N-(5-isoquinolyl)amine
(4c). The compound 4c was prepared from benzylbro-
mide 2c in a manner similar to that described for the
compound 4a with a yield of 25% as a dense yellow
oil. 1H NMR (CDCl3, 400 MHz) d 1.5–1.7 (m, 4H),
2.1–2.2 (m, 2H), 2.2–2.3 (m, 2H), 3.59 (s, m, 3H), 6.77
(d, J = 7.8 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.2–7.4
(m, 5H), 7.43 (dd, J = 7.8 Hz, 1H), 7.52 (d, J = 6.0 Hz,
1H), 8.46 (d, J = 6.0 Hz, 1H), 9.14 (s, 1H). MS (ESI)
m/z 318 (M+1)+.
5.5.4. N1-Propyl-N4-(5-isoquinolyl)-1,4-cyclohexanedi-
amine (8a). To a mixture of 1,4-cyclohexanedione
monoethyleneketal 5 (6.20 g, 40 mmol), 5-aminoiso-
quinoline (4.33 g, 30 mmol), and AcOH (0.5 mL) in
MeOH (50 mL), BH3–pyridine complex (4.0 mL,
40 mmol) was added dropwise at 0 ꢁC. The mixture
was stirred at rt for 18 h and concentrated for remov-
al of MeOH. The residue was dissolved in 50% AcOH
(50 mL). The solution was stirred at 80 ꢁC for 3 h and
concentrated, then quenched by the addition of satu-
rated NaHCO3 aq. The aqueous layer was extracted
with CHCl3. The organic layer was dried over anhy-
drous Na2SO4 and filtered. The filtrate was concen-
trated. The residual oil was purified by silica gel
column chromatography, eluting with CHCl3, to give
5.5.6. N1-Phenylethyl-N4-(5-isoquinolyl)-1,4-cyclohexan-
ediamine (8c). The compound 8c was prepared from 2-
phenylethylamine 7c in a manner similar to that
described for the compound 8a as a dense yellow oil.
1
Anti isomer: H NMR (CDCl3, 400 MHz) d 1.15–1.30
(m, 2H), 1.92–2.03 (m, 2H), 2.12–2.25 (m, 2H), 2.45–
2.55 (m, 1H), 2.76 (t, J = 7.1 Hz, 2H), 2.88 (t, J
=7.1 Hz, 2H), 3.29–3.42 (m, 1H), 4.05–4.18 (m, 1H),
6.69 (d, J = 7.6 Hz, 1H), 7.12–7.26 (m, 6H), 7.37 (d,
J = 7.9 Hz, 1H), 7.43 (t, J = 6.1 Hz, 1H), 8.37 (d,
J = 6.1 Hz, 1H), 9.06 (s, 1H); MS (ESI) m/z 346
(M+1)+. Syn isomer: 1H NMR (CDCl3, 400 MHz) d
1.46–1.58 (m, 2H), 1.64–1.74 (m, 4H), 1.76–1.86 (m,
2H), 2.65 (tt, J = 3.7 Hz, 8.1 Hz, 1H), 2.76 (tt,
J = 6.8 Hz, 7.1 Hz, 2H), 3.60–3.70 (m, 1H), 4.28–4.42
(m, 1H), 6.69 (d, J = 7.6 Hz, 1H), 7.10–7.26 (m, 6H),
7.37 (t, J = 7.9 Hz, 1H), 7.46 (d, J = 6.1 Hz, 1H), 8.38
(d, J = 6.1 Hz, 1H), 9.07 (s, 1H). MS (ESI) m/z 346
(M+1)+.
4-(isoquinolin-5-ylamino)-cyclohexanone
24 mmol).
6
(5.80 g,
To a mixture of 6 (60 mg, 0.25 mmol) and propylamine
7a (52 mg, 0.5 mmol) in MeOH (1 mL), NaBH(OAc)3
(111 mg, 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 aqueous
layer was extracted with CHCl3. The organic layer was
dried over anhydrous Na2SO4 and filtered. The filtrate
was concentrated. The residual oil was subjected to a
reverse-phase HPLC (ODS), eluting with 5%TFA aq–
CH3CN (4:1), to give the title compound as a dense yel-
low oil (18 mg, syn isomer; 22 mg, anti isomer). Anti iso-
5.5.7. N-(1-Isopentyl-3-pyrrolidyl)-N-(5-isoquinolyl)amine
(12). To a mixture of 3-hydroxypyrrolidine 9 (1.0 g,
10 mmol) and K2CO3 (2.68 g, 20 mmol) in DMF
(10 mL), a solution of 1-chloro-3-methylbutane (1.07 g,
10 mmol) in CH3CN (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 fil-
tered through Celite. The filtrate was then concentrated
to give 1-isopentyl-3-hydroxypyrrolidine 10. To a solu-
tion of 10 and triethylamine (1.78 mL) in anhydrous
DMSO (8 mL), a sulfur trioxide-trimethylamine com-
plex (2.45 g, 23 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 combined
organic layer was dried over anhydrous Na2SO4 and fil-
tered. The filtrate was then concentrated to give crude 1-
isopentyl-3-pyrrolidone 11 (1.55 g). 775 mg of the crude
11 and 5-aminoisoquinoline (304 mg, 1.9 mmol) were
dissolved in titanium tetraisopropoxide (5 mL), and
the reaction mixture was stirred at rt for 30 min. The
mixture was diluted with MeOH (5 mL), and sodium
borohydride (101 mg, 2.6 mmol) was added at 0 ꢁC.
The mixture was stirred at rt for 3 h, filtered through
1
mer: H NMR (CDCl3, 400 MHz) d 0.87 (t, J = 7.4 Hz,
3H), 1.18–1.32 (m, 4H), 1.41–1.52 (m, 2H), 1.94–2.06
(m, 2H), 2.14–2.16 (m, 2H), 2.44–2.58 (m, 2H), 2.57 (t,
J =7.5 Hz, 2H), 3.31–3.44 (m, 1H), 4.06–4.20 (m, 1H),
6.70 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.38
(t, J = 7.7 Hz, 1H), 7.44 (t, J = 6.1 Hz, 1H), 8.38 (t,
J = 5.9 Hz, 1H), 9.07 (s, 1H); MS (ESI) m/z 284
(M+1)+. Syn isomer: 1H NMR (CDCl3, 400 MHz) d
0.86 (t, J = 7.3 Hz, 3H), 1.40–1.50 (m, 2H), 1.50–1.60
(m, 2H), 1.68–1.76 (m, 4H), 1.80–1.90 (m, 2H), 2.57 (t,
J =7.3 Hz, 2H), 2.58–2.68 (m, 1H), 3.60–3.70 (m, 1H),
4.33–4.45 (m, 1H), 6.68 (d, J = 7.8 Hz, 1H), 7.20 (d,
J = 7.6 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.47 (t,
J = 5.8 Hz, 1H), 8.37 (t, J = 6.1 Hz, 1H), 9.07 (s, 1H).
MS (ESI) m/z 284 (M+1)+.

1030
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
Celite, and quenched by the addition of saturated NaH-
CO3 aq. The mixture was extracted with EtOAc. The
organic layer was dried over anhydrous Na2SO4 and fil-
tered. The filtrate was 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 (172 mg, 0.56 mmol, 29% yield).
1H NMR (CDCl3, 400 MHz) d 1.52–1.80 (m, 6H),
2.61–2.71 (m, 2H), 3.68 (s, 2H), 3.76–3.84 (m, 1H),
5.06–5.19 (m, 1H), 6.63–6.72 (m, 3H), 7.24 (s, 1H),
7.42 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 6.0 Hz, 1H), 8.47
(d, J = 6.0 Hz, 1H), 9.07 (s, 1H). MS (ESI) m/z 284
(M+1)+.
1H), 7.44 (d, J = 8.3 Hz, 1H), 7.58 (t, J = 7.9 Hz, 1H),
7.80–7.90 (m, 1H), 8.42 (d, J = 6.4 Hz, 1H), 9.20 (s,
1H). MS (ESI) m/z = 314 (M++1).
5.5.10. N-[1-(2-Chlorobenzyl)-3-piperidyl]-N-(5-isoquin-
olyl)amine (21a). To a solution of 3-(5-isoquinolylami-
no)-1-piperidinecarboxylic acid tert-butyl ester 18
(66 mg, 0.20 mmol) in CHCl3 (1 mL), TFA (1 mL) was
added dropwise at rt. The reaction mixture was stirred
at rt for 3 h. The mixture was concentrated under a re-
duced pressure. CH3CN (1 mL) and K2CO3 (69 mg,
0.50 mmol) were added to the residue, and 2-chloroben-
zylchloride 20a (40 mg, 0.25 mmol) in CH3CN (0.5 mL)
was added dropwise at rt. The reaction mixture was stir-
red at rt for 18 h. The reaction was quenched by the
addition of water (2 mL). The mixture was concentrated
under a reduced pressure to remove CH3CN, and the
residue was extracted with EtOAc. The organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated. The residue was dissolved in a
minimum amount of CHCl3–MeOH (10:1) and briefly
purified using a small silica gel column. The crude mate-
rial was concentrated and subjected to chromatography
on silica gel, eluting first with CHCl3, then with CHCl3–
MeOH (10:1) to give the titled compound as a dense yel-
low oil (19 mg, 0.054 mmol, 27% yield). 1H NMR
(CDCl3, 400 MHz) d 1.50–1.90 (m, 5H), 2.30–2.40 (m,
1H), 2.65–2.80 (m, 1H), 2.75 (m, 1H), 3.62 (d, J =
13.4 Hz, 1H), 3.67 (d, J = 13.4 Hz, 1H), 3.82 (br s,
1H), 6.70 (d, J = 7.8 Hz, 1H), 7.21–7.27 (m, 3H), 7.39–
7.43 (m, 3H), 7.60 (d, J = 5.9 Hz, 1H), 8.47 (d, J =
5.9 Hz, 1H), 9.13 (s, 1H). MS (ESI) m/z = 353 (M++1).
5.5.8. 3-(Isoquinolin-5-ylamino)-piperidine-1-carboxylic
acid tert-butyl ester (18). To a solution of 3-hydroxypi-
peridine 13 (10.0 g, 100 mmol) in 3 M NaOH aq
(100 mL),
a solution of di-tert-butyl dicarbonate
(25.0 g, 120 mmol) in THF (100 mL) was added drop-
wise at 0 ꢁC. The reaction mixture was stirred at rt for
1 h and evaporated to remove THF. The aqueous layer
was extracted with EtOAc. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated. To a mixture of the residue
and triethylamine (20 mL) in anhydrous DMSO
(100 mL), a sulfur trioxide-trimethylamine complex
(44.4 g, 200 mmol) was added portionwise 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 EtOAc. The combined organic
layer was dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated. The crude mixture was
purified by silica gel column chromatography, eluting
with CHCl3 to give 3-oxo-piperidine-1-carboxylic acid
tert-butyl ester 16 (15.6 g). To a mixture of the crude
16 (3.7 g, 20 mmol), 5-aminoisoquinoline (2.5 g,
17 mmol), and anhydrous Na2SO4 (14.2 g, 100 mmol)
in AcOH (100 mL), NaBH(OAc)3 (4.5 g, 20 mmol) was
added dropwise at 0 ꢁC. The reaction mixture was stir-
red at rt for 18 h and evaporated to remove AcOH.
The resulting mixture was basified with saturated NaH-
CO3 aq. The aqueous layer was extracted with EtOAc.
The combined organic layer was dried over anhydrous
Na2SO4 and filtered. The filtrate was concentrated.
The residual oil was purified by flash chromatography
on silica gel, eluting with CHCl3–MeOH (95:5), to give
the title compound as a dense yellow oil (3.7 g, 12 mmol,
5.5.11. N-[1-(3-Chlorobenzyl)-3-piperidyl]-N-(5-isoquin-
olyl)amine (21b). Compound 21b was prepared from 3-
chlorobenzylchloride 20b in a manner similar to that de-
scribed for the compound 21a with a yield of 33% as a
1
dense yellow oil. H NMR (CDCl3, 400 MHz) d 1.45–
1.81 (m, 5H), 2.35–2.50 (m, 1H), 2.60–2.68 (m, 2H),
3.48 (d, J = 13.4 Hz, 1H), 3.60 (d, J = 13.4 Hz, 1H),
3.80 (br s, 1H), 6.73 (d, J = 7.8 Hz, 1H), 7.26–7.30 (m,
4H), 7.42 (t, J = 7.8 Hz, 1H), 7.44 (s, 1H), 7.57 (d,
J = 5.9 Hz, 1H), 8.50 (d, J = 5.9 Hz, 1H), 9.14 (s, 1H).
MS (ESI) m/z = 353 (M++1).
5.5.12. N-[1-(4-Chlorobenzyl)-3-piperidyl]-N-(5-isoquin-
olyl)amine (21c). Compound 21c was prepared from 4-
chlorobenzylchloride 20c in a manner similar to that de-
scribed for the compound 21a with a yield of 36% as a
1
60% yield). H NMR (CDCl3, 400 MHz) d 1.44 (s, 9H),
1.48–1.68 (m, 1H), 1.78–1.83 (m, 2H), 1.90–2.10 (m,
1H), 3.10–3.32 (m, 2H), 3.52–3.65 (m, 2H), 3.92–3.98
(m, 1H), 6.86 (d, J = 7.6 Hz, 1H), 7.30 (d, J = 8.3 Hz,
1H), 7.45 (t, J = 7.9 Hz, 1H), 7.54 (d, J = 5.8 Hz, 1H),
8.42 (d, J = 5.8 Hz, 1H), 9.13 (s, 1H). MS (ESI) m/
z = 328 (M++1).
1
dense yellow oil. H NMR (CDCl3, 400 MHz) d 1.45–
1.76 (m, 5H), 2.30–2.45 (m, 1H), 2.46–2.60 (m, 1H),
2.61–2.75 (m, 1H), 3.45–3.60 (m, 2H), 3.80 (br s, 1H),
6.73 (d, J = 8.2 Hz, 1H), 7.25–7.35 (m, 4H), 7.42 (t,
J = 8.2 Hz, 1H), 7.50–7.58 (m, 2H), 8.49 (d,
J = 6.0 Hz, 1H), 9.15 (s, 1H). MS (ESI) m/z = 353
(M++1).
5.5.9. 3-(Isoquinolin-5-ylamino)-pyrrolidine-1-carboxylic
acid tert-butyl ester (19). The compound 19 was pre-
pared from 3-hydroxypyrrolidine 9 in a manner similar
to that described for the compound 18 with a yield of
5.5.13. N-[1-(2-Chlorobenzyl)-3-pyrrolidyl]-N-(5-isoquin-
olyl)amine (22a). To a solution of 19 (62 mg, 0.20 mmol)
in CHCl3 (1 mL), TFA (1 mL) was added dropwise at rt.
The resulting mixture was stirred at rt for 3 h and con-
centrated. To the residue, K2CO3 (69 mg, 0.50 mmol)
and CH3CN (1 mL) were added. A solution of 2-chlo-
65% as
a
dense yellow oil. 1H NMR (CDCl3,
400 MHz) d 1.46 (s, 9H), 1.75–1.94 (m, 1H), 2.02–2.10
(m, 1H), 3.35–3.55 (m, 3H), 3.75–3.86 (m, 1H), 4.17–
4.24 (m, 1H), 4.75–4.90 (m, 1H), 6.91 (d, J = 7.6 Hz,

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
1031
robenzylchloride 20a (40 mg, 0.25 mmol) in CH3CN
(0.5 mL) was then added dropwise at 0 ꢁC. The reaction
mixture was stirred at rt for 18 h and quenched by the
addition of water. The aqueous layer was extracted with
EtOAc. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was concen-
trated. The residual oil was subjected to flash chroma-
tography on silica gel, eluting CHCl3–MeOH (95:5), to
give the title compound as a dense yellow oil (28 mg,
5.5.18. N-[1-(2-nitrobenzyl)-3-pyrrolidyl]-N-(5-isoquinol-
yl)amine (22f). This compound was prepared from
2-nitrobenzylchloride 20f in a manner similar to that
described for the compound 22a with a yield of 38%
1
as a dense yellow oil. H NMR (CDCl3, 400 MHz) d
1.72–1.88 (m, 1H), 2.10–2.50 (m, 2H), 2.56–2.85 (m,
2H), 2.85–3.02 (m, 1H), 3.99 (s, 2H), 4.05–4.17 (m,
1H), 6.65 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H),
7.40 (t, J = 7.8 Hz, 1H), 7.45–7.60 (m, 2H), 7.67 (s,
1H), 7.75–7.90 (m, 2H), 8.49 (d, J = 6.0 Hz, 1H), 9.12
(s, 1H). MS (ESI) m/z = 349 (M++1).
1
0.08 mmol, 42% yield in this step). H NMR (CDCl3,
400 MHz) d 1.98–2.10 (m, 1H), 2.40–2.52 (m, 1H),
2.70–2.90 (m, 1H), 3.00–3.86 (m, 3H), 4.00–4.15 (m,
2H), 4.25–4.35 (m, 1H), 6.59 (d, J = 7.6 Hz, 1H), 7.20–
7.28 (m, 3H), 7.32–7.38 (m, 2H), 7.63–7.83 (m, 2H),
8.44 (d, J = 5.8 Hz, 1H), 9.07 (s, 1H). MS (ESI) m/
z = 339 (M++1).
5.5.19. N-[1-(3-nitrobenzyl)-3-pyrrolidyl]-N-(5-isoquinol-
yl)amine (22g). This compound was prepared from
3-nitrobenzylchloride 20g in a manner similar to that de-
scribed for the compound 22a with a yield of 38% as a
1
dense yellow oil. H NMR (CDCl3, 400 MHz) d 1.78–
5.5.14. N-[1-(3-Chlorobenzyl)-3-pyrrolidyl]-N-(5-isoquin-
olyl)amine (22b). Compound 22b was prepared from
3-chlorobenzylchloride 20b in a manner similar to that
described for the compound 22a with a yield of 21% as
a dense yellow oil. 1H NMR (CDCl3, 400 MHz) d 2.00–
2.10 (m, 1H), 2.45–2.55 (m, 1H), 2.70–2.80 (m, 1H),
3.00–3.45 (m, 3H), 3.80–3.90 (m, 2H), 4.25–4.36 (m,
1H), 6.64 (d, J = 7.6 Hz, 1H), 7.28–7.34 (m, 3H), 7.37–
7.43 (m, 3H), 7.75–7.85 (m, 1H), 8.48 (d, J = 6.1 Hz,
1H), 9.12 (s, 1H). MS (ESI) m/z = 339 (M++1).
1.90 (m, 1H), 2.40–2.57 (m, 2H), 2.72–2.79 (m, 1H),
2.85–2.96 (m, 2H), 3.77 (s, 2H), 4.14–4.24 (m, 1H),
4.55–4.67 (m, 1H), 6.69 (d, J = 7.6 Hz, 1H), 7.31 (d,
J = 8.3 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.49 (d,
J = 7.8 Hz, 1H), 7.58 (t, J = 6.1 Hz, 1H), 7.69 (d,
J = 6.7 Hz, 1H), 8.11 (d, J = 7.3 Hz, 1H), 8.23 (s, 1H),
8.47 (d, J = 6.1 Hz, 1H), 9.14 (s, 1H). MS (ESI) m/
z = 349 (M++1).
5.5.20. N-[1-(4-nitrobenzyl)-3-pyrrolidyl]-N-(5-isoquinol-
yl)amine (22h). This compound was prepared from 4-
nitrobenzylchloride 20h in a manner similar to that
described for the compound 22a with a yield of 29% as
a dense yellow oil. 1H NMR (CDCl3, 400 MHz) d
1.86–1.94 (m, 1H), 2.43–2.54 (m, 1H), 2.84–3.06 (m,
3H), 3.85 (s, 2H), 4.20–4.28 (m, 1H), 4.76–4.90 (m,
1H), 6.66 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H),
7.41 (t, J = 7.9 Hz, 1H), 7.57 (d, J = 8.7 Hz, 2H), 7.63
(t, J=5.8 Hz, 1H), 8.18 (d, J = 8.7 Hz, 2H), 8.46 (d, J =
5.8 Hz, 1H), 9.13 (s, 1H). MS (ESI) m/z = 349 (M++1).
5.5.15. N-[1-(4-Chlorobenzyl)-3-pyrrolidyl]-N-(5-isoquin-
olyl)amine (22c). Compound 22c was prepared from
4-chlorobenzylchloride 20c in a manner similar to that
described for the compound 22a with a yield of 15%
1
as a dense yellow oil. H NMR (CDCl3, 400 MHz) d
2.00–2.10 (m, 1H), 2.45–2.55 (m, 1H), 2.60–2.90 (m,
1H), 3.00–3.40 (m, 3H), 3.85–4.00 (m, 2H), 4.28–4.50
(m, 1H), 6.62 (d, J = 7.3 Hz, 1H), 7.28–7.44 (m, 6H),
7.80–8.00 (m, 1H), 8.48 (d, J = 6.1 Hz, 1H), 9.12 (s,
1H). MS (ESI) m/z = 339 (M++1).
5.5.21. N-[1-(2-aminobenzyl)-3-pyrrolidyl]-N-(5-isoquin-
olyl)amine (23f). To a solution of N-[1-(2-nitrobenzyl)-
5.5.16. N-[1-(4-Fluorobenzyl)-3-pyrrolidyl]-N-(5-isoquin-
olyl)amine (22d). Compound 22d was prepared from 4-
fluorobenzylchloride 20d in a manner similar to that de-
scribed for the compound 22a with a yield of 27% as a
3-pyrrolidyl]-N-(5-isoquinolyl)amine
22f
(27 mg,
0.07 mmol) in 3 M HCl aq (1 mL), SnCl2/H2O
(113 mg, 0.50 mmol) was added dropwise at 80 ꢁC.
The reaction mixture was stirred at the same tempera-
ture for 3 h and basified by the addition of 30% wt
NH3 aq. The resulting mixture was diluted with EtOAc
and filtered through Celite. The aqueous layer was
extracted with EtOAc. The combined organic layer
was dried over anhydrous Na2SO4 and filtered. The fil-
trate was concentrated. The residual oil was purified
by flash chromatography on aluminum oxide, eluting
with CHCl3–MeOH (95:5), to give the title compound
as a dense yellow oil (20 mg, 0.06 mmol, 84% yield in
this step). 1H NMR (CDCl3, 400 MHz) d 1.75–1.84
(m, 1H), 2.38–2.54 (m, 2H), 2.65 (dd, J = 3.5 Hz,
10.0 Hz, 1H), 2.82 (dt, J = 5.0 Hz, 8.6 Hz, 1H), 2.87
(dd, J = 6.3 Hz, 9.8 Hz, 1H), 3.65 (d, J = 12.4 Hz, 1H),
3.69 (d, J = 12.2 Hz, 1H), 4.10–4.18 (m, 1H), 4.45–4.55
(m, 1H), 6.64–6.70 (m, 3H), 7.03 (d, J = 7.3 Hz, 1H),
7.09 (t, J = 7.6 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.43
(t, J = 8.3 Hz, 1H), 7.52 (d, J = 5.9 Hz, 1H), 8.46 (d,
J = 6.1 Hz, 1H), 9.15 (s, 1H). MS (ESI) m/z = 319
(M++1).
1
dense yellow oil. H NMR (CDCl3, 400 MHz) d 1.80–
2.10 (m, 1H), 2.40–2.53 (m, 1H), 2.57–2.75 (m, 1H),
2.90–3.25 (m, 3H), 3.75–3.88 (m, 2H), 4.21–4.31 (m,
1H), 6.64 (d, J = 7.6 Hz, 1H), 7.03 (t, J = 8.5 Hz, 2H),
7.30 (d, J = 8.0 Hz, 1H), 7.35–7.43 (m, 3H), 7.65–7.78
(m, 1H), 8.47 (d, J = 6.1 Hz, 1H), 9.12 (s, 1H). MS
(ESI) m/z = 322 (M++1).
5.5.17. N-[1-(2,6-Difluorobenzyl)-3-pyrrolidyl]-N-(5-iso-
quinolyl)amine (22e). Compound 22e was prepared from
2,6-difluorobenzylchloride 20e in a manner similar to
that described for the compound 22a with a yield of
50% as a dense yellow oil. 1H NMR (CDCl3,
400 MHz) d 1.78–1.90 (m, 1H), 2.35–2.45 (m, 1H),
2.52–2.65 (m, 1H), 2.88–3.00 (m, 3H), 3.90 (s, 2H),
4.11–4.20 (m, 1H), 4.75–4.85 (m, 1H), 6.66 (d,
J = 7.6 Hz, 1H), 6.85–6.95 (m, 3H), 7.29 (d,
J = 8.3 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.58 (d,
J = 6.1 Hz, 1H), 8.44 (d, J = 6.1 Hz, 1H), 9.13 (s, 1H).
MS (ESI) m/z = 340 (M++1).

1032
M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
5.5.22. N-[1-(3-aminobenzyl)-3-pyrrolidyl]-N-(5-isoquin-
olyl)amine (23g). Compound 23g was prepared from
N-[1-(3-nitrobenzyl)-3-pyrrolidyl]-N-(5-isoquinolyl)amine
22g in a manner similar to that described for the com-
pound 23f with a yield of 71% as a dense yellow oil.
1H NMR (CDCl3, 400 MHz) d 1.80–1.94 (m, 1H),
2.38–2.48 (m, 1H), 2.52–2.62 (m, 1H), 2.75–2.86 (m,
1H), 2.87–3.02 (m, 2H), 3.64 (s, 2H), 4.14–4.24 (m,
1H), 4.75–4.90 (m, 1H), 658 (d, J = 7.6 Hz, 1H), 6.66
(d, J = 7.6 Hz, 1H), 6.70–6.95 (m, 2H), 7.09 (t,
J = 8.1 Hz, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.41 (t, J =
7.8 Hz, 1H), 7.59–7.66 (m, 1H), 8.46 (d, J = 6.1 Hz,
1H), 9.13 (s, 1H). MS (ESI) m/z = 319 (M++1).
palladium acetate (113 mg, 0.50 mmol), racemic-BINAP
(311 mg, 0.50 mmol), and Cs2CO3 (1.63 g, 5.0 mmol) in
toluene (5 mL) was stirred at 80 ꢁC for 18 h. The result-
ing mixture was diluted with EtOAc, filtered through
Celite, and the filtrate was concentrated. The residual
oil was purified by flash chromatography on silica gel,
eluting with CHCl3–MeOH (95:5), to give (3R)-N-[1-
(3-nirobenzyl)-3-pyrrolidyl]-N-(5-isoquinolyl)amine 29
as a dense yellow oil (629 mg, 1.68 mmol, 55% yield in
this step). The intermediate 29 was dissolved in 3N
HCl aq (3 mL). SnCl2/H2O (1.13 g, 5.0 mmol) was add-
ed to the solution portionwise at 0 ꢁC. The reaction mix-
ture was stirred at 80 ꢁC for 5 h and quenched by the
addition of 30% NH3 aq. The resulting mixture was
diluted with EtOAc and filtered through Celite. The
aqueous layer was extracted with EtOAc. The combined
organic layer was dried over anhydrous Na2SO4 and fil-
tered. The filtrate was concentrated. The residual oil was
purified by flash chromatography on aluminum oxide,
eluting with CHCl3, to give the title compound as a
dense yellow oil (462 mg, 1.45 mmol). 1H NMR
(CDCl3, 400 MHz) d 1.80–1.94 (m, 1H), 2.38–2.48 (m,
1H), 2.52–2.62 (m, 1H), 2.75–2.86 (m, 1H), 2.87–3.02
(m, 1H), 3.64 (s, 2H), 4.14–4.24 (m, 1H), 4.75–4.90 (m,
1H), 6.58 (d, J = 7.6 Hz, 1H), 6.66 (d, J = 7.6 Hz, 1H),
6.70–6.95 (m, 2H), 7.09 (t, J = 8.1 Hz, 1H), 7.29 (d,
J = 8.1 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.59–7.66 (m,
5.5.23. N-[1-(4-aminobenzyl)-3-pyrrolidyl]-N-(5-isoquin-
olyl)amine (23h). Compound 23h was prepared from
N-[1-(4-nitrobenzyl)-3-pyrrolidyl]-N-(5-isoquinolyl)amine
22h in a manner similar to that described for the
compound 23f with a yield of 88% as a dense yellow
1
oil. H NMR (CDCl3, 400 MHz) d 1.80–1.90 (m, 1H),
2.38–2.47 (m, 3H), 2.72–2.90 (m, 2H), 3.62(br s, 2H),
4.18–4.21 (m, 1H), 4.25–4.32 (m, 1H), 6.56–6.71 (m,
1H), 6.65 (d, J = 8.4 Hz, 2H), 6.95–6.98 (m, 1H), 7.15
(t, J = 8.4 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.41 (t,
J = 7.6 Hz, 1H), 7.65–7.72 (m, 1H), 8.47 (d,
J = 6.0 Hz, 1H), 9.14 (s, 1H). MS (ESI) m/z = 319
(M++1).
1H), 8.46 (d, J = 6.1 Hz, 1H), 9.13 (s, 1H). MS (ESI)
25
m/z = 319 (M++1). ½aꢁ = ꢀ103 (c = 0.25/CHCl3).
5.5.24.
(3R)-N-[1-(3-Aminobenzyl)-3-pyrrolidyl]-N-(5-
D
isoquinolyl)amine (30-R). To a mixture of 5-hydroxyiso-
quinoline 24 (4.35 g, 30 mmol) and pyridine (3.16 g) in
CHCl3 (50 mL), trifluoromethanesulfonic anhydride
(10.0 g, 35 mmol) was added dropwise at 0 ꢁC. The reac-
tion mixture was stirred at rt for 3 h and quenched by
the addition of saturated NaHCO3 aq. The aqueous
layer was extracted with EtOAc. The combined organic
layer was dried over anhydrous Na2SO4 and filtered.
The filtrate was concentrated. The residual oil was sub-
jected to flash chromatography on silica gel, eluting with
CHCl3, to give 5-isoquinolyl trifluoromethanesulfonic
acid (25) (7.55 g, 20 mmol).
5.5.25. (3S)-N-[1-(3-Aminobenzyl)-3-pyrrolidyl]-N-(5-iso-
quinolyl)amine (30-S). The compound 30-S was pre-
pared from (3S)-(ꢀ)-3-pyrrolidylcarbamic acid tert-
butyl ester in a manner similar to that described for
25
the compound 30-R as a dense yellow oil. ½aꢁ =+111
D
(c = 0.25/CHCl3).
Acknowledgments
The authors wish to thank Ms. Takemi Tokieda for
technical assistance, Mr. Kinya Kubo, Mr. Mitsunori
Kanazawa, and Mr. Shinichi Ohyama for their advice
and technical help in the microsome experiments. We
also thank Dr. Kazuo Kubo and Dr. Elizabeth A Kamei
for reading the manuscript. We are grateful to Dr. Take-
shi Ohtani and Dr. Kazuta Takemura who contributed
to the initiation of this project. This work was in part
supported by the grant from the National Institute of
Biomedical Innovation.
To a mixture of (3R)-(+)-3-pyrrolidylcarbamic acid tert-
butyl ester 26 (1.86 g, 10 mmol) and K2CO3 (2.07 g,
15 mmol) in CH3CN (10 mL), a solution of 3-nitroben-
zylchloride (1.88 g, 11 mmol) in CH3CN (10 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. The residual oil was sub-
jected to flash chromatography on silica gel, eluting with
CHCl3–MeOH (95:5), to give (3R)-(+)-[1-(3-nitroben-
zyl)-3-pyrrolidyl]carbamic acid tert-butyl ester 27 as a
dense yellow oil (2.70 g, 8.5 mmol). Compound 27
(1.00 g, 3.15 mmol) was dissolved in TFA/CHCl3
(2 mL/2 mL) and stirred at rt for 3 h. After evaporation
of TFA, the resulting mixture was basified with 3 N
NaOH aq. The aqueous layer was extracted with
EtOAc. The combined organic layer was dried over
anhydrous Na2SO4 and filtered. The filtrate was concen-
trated. The residual oil was purified by flash chromatog-
raphy on aluminum oxide, eluting with CHCl3, to give
1-(3-nitrobenzyl)-3-pyrrolidylamine 28 as a dense yellow
oil. A mixture of the triflate 25 (858 mg, 3.1 mmol), 28,
References and notes
1. (a) Fukata, Y.; Amano, M.; Kaibuchi, K. Trends Phar-
macol. Sci. 2001, 22, 1; (b) Narumiya, S. Nippon Yak-
urigaku Zasshi 1999, 114(Suppl. 1), 1–5.
2. Tsutiya, M.; Asano, T.; Sako, K.; Yonemasu, Y. Jpn
Pharmacol. Ther. 1992, 20, 1525, and references cited
therein.
3. (a) Mohri, M.; Shimokawa, H.; Hirakawa, Y.; Masumoto,
A.; Takeshita, A. J. Am. Coll. Cardiol. 2003, 41, 15; (b)
Matsumoto, A.; Mohri, M.; Shimokawa, H.; Urakami, I.;

M. Iwakubo et al. / Bioorg. Med. Chem. 15 (2007) 1022–1033
1033
Usui, M.; Takeshita, A. Circulation 2002, 105, 1545; (c)
Shimokawa, H. J. Cardiovasc. Pharmacol. 2002, 39, 319.
4. Uehata, M.; Ishizaki, T.; Satoh, H.; Ono, T.; Kawahara,
T.; Morishita, T.; Tamakawa, H.; Yamagami, K.; Inui, J.;
Maekawa, M.; Narumiya, S. Nature 1997, 389, 990.
5. (a) Wingard, C. J.; Johnson, J. A.; Holmes, A.; Prikosh,
A. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003, 284,
R1572; (b) Wang, H.; Eto, M.; Steers, W. D.; Somlyo, A.
P.; Somlyo, A. V. J. Biol. Chem. 2002, 277, 30614; (c)
Rees, R. W.; Ralpf, D. J.; Royle, M.; Moncade, S.; Cellek,
S. Br. J. Pharmacol. 2001, 133, 455; (d) Chitaley, K.;
Wingard, C. J.; Clinton, W. R.; Branam, H.; Stopper, V.
S.; Lewis, R. W.; Mills, T. M. Nat. Med. 2001, 1, 119.
6. (a) Waki, M.; Yoshida, Y.; Oka, T.; Azuma, M. Curr. Eye.
Res. 2001, 22, 470; (b) Rao, P. V.; Deng, P. F.; Kumar, J.;
Epstein, D. L. Invest. Ophtalmol. Vis. Sci. 2001, 42, 1029;
(c) Honjo, Y.; Tanihara, H.; Inatani, M.; Kido, N.;
Sawamura, T.; Yue, B. Y.; Narumiya, S.; Honda, Y.
Invest. Ophtjalmol. Vis. Sci 2001, 42, 137; (d) Honjo, Y.;
Inatani, M.; Kido, N.; Sawamura, T.; Yue, B. Y.; Honda,
Y.; Tanihara, H. Arch. Ophthalmol. 2001, 119, 1171.
7. (a) Takamura, M.; Sakamoto, M.; Genda, T.; Ichida, T.;
Asakura, H.; Hirohashi, S. Hepatology 2001, 33, 577; (b)
Imamura, F.; Mukai, M.; Ayaki, M.; Akedo, H. Jpn J.
Cancer Res. 2000, 91, 811.
10. Mueller, B. K.; Mack, H.; Teusch, N. Nat. Rev. 2005, 4,
387.
11. Hu, E.; Lee, D. Expert Opin. Ther. Targets 2005, 9, 715,
and the references therein.
12. 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.
13. Iwakubo, M.; Takami, A.; Okada, Y.; Kawada, T.;
Tagami, Y.; Ohashi, H.; Sato, M.; Sugiyama, T.; Fuku-
shima, K.; Iijima, H. Bioorg. Med. Chem. 2007, 15, 350–
367.
14. (a) Muci, A. R.; Buchwald, S. L. Top. Curr. Chem 2002,
219, 131; (b) Hartwig, J. F. In Modern Amination
Methods; Ricci, A., Ed.; Wiley-VCH: Weinheim:
Germany, 2000.
15. (a) Morikawa, A.; Sone, T.; Asano, T. Chem. Pharm. Bull.
1992, 40, 770; (b) Hidaka, H.; Sone, T.; Sasaki, Y.;
Sugihara, T. Japan Kokai Tokkyo Koho 57, 156463.
16. Jacobs, M.; Hayakawa, K.; Swenson, L.; Bellon, S.;
Fleming, M.; Taslimi, P.; Doran, J. J. Biol. Chem. 2006,
281, 260.
17. Yamaguchi, H.; Kasa, M.; Amano, M.; Kaibuchi, K.;
Hakoshima, T. Structure 2006, 14, 589.
8. (a) Satoh, S.; Yamaguchi, T.; Hitomi, A.; Sato, N.;
Shiraiwa, K.; Ikegaki, I.; Asano, T.; Shimokawa, H. Eur.
J. Pharmacol. 2002, 455, 169; (b) Nagatoya, K.; Moriyama,
T.; Kawada, N.; Takeji, M.; Oseto, S.; Murozono, T.;
Ando, A.; Imai, E.; Hori, M. Kidney Int. 2002, 61, 1684.
9. (a) Ishizaki, T.; Uehata, M.; Tamechika, I.; Keel, J.;
Nonomura, K.; Maekawa, M.; Narumiya, S. Am. Soc.
Pharmacol. Exp. Ther. 2000, 57, 976; (b) Narumiya, S.;
Ishizaki, T.; Uehata, M. Methods Enzymol. 2000, 325, 273.
18. (a) Breitenlechner, C.; Gassel, M.; Hidaka, H.; Kinzel,
V.; Huber, R.; Engh, R. A.; Bossemeyer, D. Structure
2003, 11, 1595; (b) Bonn, S.; Herrero, S.; Breitenlech-
ner, C. B.; Erlbruch, A.; Lehmann, W.; Engh, R. A.;
Gassel, M.; Bossemeyer, D. J. Biol. Chem. 2006, 281,
24818.
19. Kawano, Y.; Fukata, Y.; Oshiro, N.; Amano, M.;
Nakamura, T.; Ito, M.; Matsumura, F.; Inagaki, M.;
Kaibuchi, K. J. Cell Biol. 1999, 147, 1023.