Design and synthesis of Rho kinase inhibitors (I)

Bioorganic & Medicinal Chemistry 12 (2004) 2115–2137

Atsuya Takami,^a,Masayuki Iwakubo,^{a, ,à}Yuji Okada,^aTakehisa Kawata,^a

Hideharu Odai,^bNobuaki Takahashi,^bKazutoshi Shindo,^a,§Kaname Kimura,^a

Yoshimichi Tagami,^aMika Miyake,^bKayoko Fukushima,^aMasaki Inagaki,^c

Mutsuki Amano,^dKozo Kaibuchi^dand Hiroshi Iijima^a,*

^aPharmaceutical Research Laboratories, Kirin Brewery Co. Ltd, 3 Miyhara-cho, Takasaki-shi, Gunma 370-1295, Japan

^bCentral Laboratory for Key Technology, Kirin Brewery Co. Ltd, 1-13-5 Fukuura, Kanazawa-ku, Yokohama,

Kanagawa 236-0004, Japan

^cLaboratory of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681, Japan

^dDepartment of Cell Pharmacology, Nagoya University Graduate School of Medicine, 65 Maizuru-machi, Showa-ku,

Nagoya, Aichi 466-8500, Japan

Received 9 January 2004; revised 23 February 2004; accepted 23 February 2004

Abstract—Several structurally unrelated scaﬀolds of the Rho kinase inhibitor were designed using pharmacophore information

obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. A

docking simulation using the ligand-binding pocket of the Rho kinase model helped to comprehensively understand and to predict

the structure–activity relationship of the inhibitors. This understanding was useful for developing new Rho kinase inhibitors of

higher potency and selectivity. We identiﬁed several potent platforms for developing the Rho kinase inhibitors, namely, pyridine,

1H-indazole, isoquinoline, and phthalimide.

1. Introduction

for the cell adhesion process. Expression of the adhesion

device such as integrin on the cell surface requires

reorganization of the membrane proteins where actin

plays an important role in the process.

Cells sometime change their cytoskeletal structure in

response to external stimulation. For example, an

increase in the lysophosphatidic acid (LPA) concentra-

tion in plasma causes the contraction of the smooth

muscle of blood vessels. This contraction was explained

by the rearrangement of the cytoskeleton, which is a

result of an intracellar signal response initiated by the

LPA receptor at the cell surface that ﬁnally results in the

phosphorylation of the myosin light chain. It is also

known that such a cytoskeletal response is imperative

Rho kinase,¹which is also known as Rho-associated

coiled-coil forming protein serine/threonine kinase

(ROCK),²is one of the central regulatory molecules for

cytoskeleton control and the cell adhesion process.³Rho

kinase is activated by binding with the activated form of

a low molecular weight G protein, Rho.⁴It will then

phosphorylate myosin light chain and introduces the

formation of stress ﬁbers, adhesion cluster, and activa-

tion of adhesion devices on the cell surface. Rho kinase

also phosphorylates myosin phosphatase, which cata-

lyzes the dephosphorylation of the myosin light chain.

The phosphorylated phosphatase is inactive. As a result,

Rho kinase directly and indirectly enhances the phos-

phorylation of the myosin light chain.⁵It is interesting

that contraction of the smooth muscle by the Rho

pathway is independent from the Ca^2þpathway.⁶

Keywords: Rho kinase; Inhibitor; Structure–activity relationship;

Structure-based drug design.

* 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.

Present address: Department of Food and Nutrition, Japan Women’s

§

Rho kinase is thought to play an important role in a

variety of cellar functions such as stress ﬁber formation,

University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan.

doi:10.1016/j.bmc.2004.02.025

2116

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

focal adhesion formation, cell aggregation, cell mor-

phology, cytokinesis, cell migration, and Ca^2þ-sensiti-

zation in the smooth muscle. Recent experiments have

shown new functions of Rho kinase in cells, including

centrosome positioning and cell-size regulation.^7aMore

recently, it was shown that Rho kinase is involved in

regulation of the amyloid precursor protein process-

ing.^7bInhibitors of Rho kinase will be useful pharma-

ceutical candidates for a wide range of diseases such as

hypertension, inﬂammation, cancer, and injury caused

by ischemia and reperfuson.⁸Therefore, we have per-

formed a screening of the Rho kinase inhibitors.

Although any hit compounds did not indicate a high

inhibition potential, the commonality of pharmaco-

phores was observed among the hit compounds. Using

this information and a Rho kinase homology model, a

structure-based drug design was performed resulting in

the successful generation of potent Rho kinase inhibi-

tors of diverse scaﬀolds.

NH₂

H

N

R

NH

O

N

( )

N

O

n

S

Y27632

N

O

H

N

H

HN

H7 (n=1, R=Me)

HA-1077 (n=2, R=H)

O

N

Y30141

Figure 2. Known Rho kinase inhibitors.

(2 and 3).⁹Most of the ATP-competitive hits included

one or more hydrogen bond accepter atoms. To our

knowledge, the majority of potent low molecular weight

kinase inhibitors reported so far are ATP-competitive

and possess a hydrogen bond acceptor atom. It was also

found that ATP-competitive hits are less hydrophilic

than substrate-competitive ones, suggesting the possi-

bility of good membrane permeability and absorption in

vivo when administered. Therefore, we expected to

identify suitable compounds to develop from the ATP-

competitive hits.

2. Screening of inhibitors

We identiﬁed 417 hit compounds as Rho kinase inhibi-

tors by screening the in-house compound library (69,000

compounds). The ﬁrst screening was based on an

immobilized enzyme linked immunoassay. The hit

compounds were then examined by enzyme kinetical

assay employing [c-³²P]-ATP and a synthetic peptide (S6

peptide). The inhibitors were classiﬁed into ATP-com-

petitive, phosphate-acceptor-competitive, or other cate-

gories according to the inhibition mode. Figure 1 shows

a few examples of the hit compounds. Most of the

substrate-competitive inhibitors have high molecular

weights and high hydrophillicity (1). These structures

are contrary to the general understanding of drug-like

compounds. On the other hand, the structures of

inhibitors identiﬁed as ATP-competitive are drug-like

However, none of the screening hits indicated a strong

inhibitory activity (IC₅₀> 3 lM), while a standard

inhibitor, HA-1077 (Fasdil) (Fig. 2),¹⁰indicated an IC₅₀

of 0.1–0.3 lM. Furthermore, several cell based evalua-

tions, in which the Rho kinase plays an important role,

such as the formation of stress ﬁbers in mouse 3T3 cells

induced by LPA, aggregation of human B-cells by

phorbol ester, and aggregation of human platelets were

applied to the hit compounds. Here again HA-1077, the

reference compound, exhibited a signiﬁcant inhibition in

these evaluations, while none of the screening hits

exhibited clear and reproducible inhibitions for these

assays.

3. Homology modeling and docking simulation

Substrate-competitive

The crystal structure of a cAMP dependent protein ki-

nase (cAPK) complexed with an inhibitor H7 (Fig. 2)

was chosen as the template structure for homology

modeling (Protein Data Bank¹¹entry:1YDR¹²) because

cAPK was very homologous to Rho kinase among the

protein kinases whose three-dimensional structure are

known (37% sequence identity). The amino acid

sequence alignment of the Rho kinase and cAPK was

made using the Needleman and Wunsch algorithm¹³

(Fig. 3). The sequences of cyclin dependent protein

kinase 2 (CDK2, PDB entry:1HCL¹⁴) and insulin

receptor kinase (IRK, PDB entry:1IRK¹⁵) were also

added to the alignment and their side-chain conforma-

tion were referred to build the homology model as

described in the Experimental section.

O

-

Na⁺

O

Na⁺

S

O

Na⁺

-

S

-

O

S

O

HO

OH

N

-

N

O

S

O

N

O

Na⁺

N

HN

N

HN

1

ATP-competitive

H

N

H

N

O

N

The docking program used in this study, FlexiDock,¹⁶

can contain deﬁned rotatable bonds giving some ﬂexi-

bility to both the protein and the ligand, but the back-

bone conformation of the protein is ﬁxed. Docking

calculations of H7 and of ATP to the Rho kinase model

were run as tests. Both reproduced the binding struc-

tures of H7 and ATP that were found in the complex

N

O

NH

N

Cl

N

2

3

Figure 1. Example of hit compounds identiﬁed by screening Rho

kinase inhibitors in the chemical library.

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2117

Figure 3. Sequence alignment of human Rho kinase with bovine cAMP dependent protein kinase (PDB:1YDR), human insulin receptor kinase

(PDB:1IRK), and human cyclin dependent protein kinase 2 (PDB:1HCL). Sequences of the reference kinases were ones described in Protein Data

Bank. Secondary structures deﬁned in the crystal coordinates data are marked by wavy underlines for b-sheets and by double underlines for a-helices.

The activation loop is marked with asterisks. Conserved catalytic residues were indicated by ‘#’. Residues that deﬁne the ligand-binding site of the

Rho kinase are underlined.

with cAPK and CDK2, respectively. However, another

test run employing staurosporin as a ligand was a failure

because FlexiDock could not handle a conformational

change in the backbone structure. It was shown that the

shape of the ligand-binding pocket was wide-open in the

crystal structure of the staurosporin–cAPK complex

(PDB entry:1STC)¹⁷in order to accommodate the large

ligand molecule.

utilized to form a hydrogen bond with the N1 nitrogen

atom of the adenine ring of ATP. This hydrogen bond is

found in a number of protein kinase–inhibitor complex

structures reported to date and is considered to be

fundamental for the intermolecular interaction between

protein kinases and inhibitors. The second region of the

pocket is above the adenine region (F region). This

region is spherical in shape and also hydrophobic. This

region accepts the furanose ring of ATP. The third

region (D region) is formed by residues, Ile93:Arg95,

Ala97:Gly99, Gly170:Leu172, Asp209:Asn214, and

Asp227:Phe228. This distal region is open to the

molecular surface of the enzyme. A part of the nucleo-

tide-binding loop also contributes to determining the

shape of the D pocket.

It should be noted that the template cAPK structure is

the activated form and phosphorylated in the activation

loop. The activation loop positions outward from the

ligand-binding pocket. Recently, it was reported that

designing an inhibitor that binds to both inactive and

activated conformations of the kinase is advantageous

for enhancing the kinase speciﬁcity.¹⁸In this study,

however, the homology model of Rho kinase based on

the activated cAPK was employed because reliable

modeling of the inactive conformation of the activation

loop is not possible. It should be noted that the

recombinant Rho kinase used in this study lacked the

C-terminal regulatory subunits and was constitutively

active.¹⁹The design and experiments in this study are

under these limitations.

Comparison of the shapes of the ligand-binding pockets

of the Rho kinase and the cAPK indicated that the F

region of the Rho kinase is wider than that of the cAPK.

This diﬀerence could be explained by the amino acids

Ile95 and Ala226, which correspond to Leu47 and

Thr183 of cAPK, respectively. The D region of the Rho

kinase is wider than that of the cAPK. This is also

explained by the amino acid replacements to smaller

ones. Glu127 and Tyr330 of cAPK were replaced by

Asp171 and Ile382 in the Rho kinase model. The shape

of the A region is very similar.

4. Ligand-binding pocket

The ligand-binding pocket could be divided into three

regions (Fig. 4A). The bottom of the pocket is ﬂat in

shape and hydrophobic (A region). Characteristic is the

existence of an important hydrogen bond donor site

present in the backbone NH of Met167. This site is

5. Design of inhibitors

Pyridine derivatives with substitution at the 4-position

such as compounds 2 and 3 (Fig. 1) were representative

2118

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

Figure 4. (A) Ligand-binding pocket of Rho kinase homology model. The pocket was visualized by the channel surface representation calculated by

the Connolly module available in the ^SYBYLsoftware package.²⁶The surface was colored by lipophilicity; brown > green > blue. A docking model of

ATP is shown. Hydrogen bonds between N1of ATP and HN of Met167, and the amino group at the 6-position and C@O of Glu165 are indicated as

dashed lines. The pocket was partitioned into three parts; adenine region: A, franose region: F, and distal region: D. (B) Chemical substructures

designated for each division of the ligand-binding pocket. Arrows indicate attachment of fragments. Hatched circles indicate possible hydrogen bond

acceptor atom.

screening hits, though their inhibitory potencies were

low. A docking model of 2 (Fig. 5A) suggested that the

aromatic pyridine ring would ﬁt into the A region and

would be stabilized by a hydrogen bond between the

pyridine nitrogen atom and the backbone NH of

Met167. Based on the facts that 2 is an ATP-competitive

inhibitor and that the existence of a hydrogen bond

between an inhibitor and kinase at the bottom of the A

region is observed in many inhibitor–kinase complex

structures, we assumed that an aromatic ring and a

nitrogen atom of the aromatic ring are essential phar-

macophores of Rho kinase inhibitors.

The docking model proposed several hypotheses for

inhibitor design. First, molecular fragments that have a

hydrogen acceptor and that are planar would ﬁt the

shape of the A region. For example, one six-member

aromatic ring or fused 6–6 or 6–5 ring were candidate

substructures for the A region. Second, as the furanose

region (the F region) is relatively spacious, many

Figure 5. Docking models of Rho kinase inhibitors (stereo view). (A) Pyridine derivatives: 2 (carbon atoms colored in black), 4b (magenta), and 5a

(orange). (B) Indazole derivatives: 12a (carbon atoms colored in magenta), 14b (black), and 20b (orange). (C) Isoquinoline derivatives: 22c in

equatorial conformation (carbon atoms colored in orange), 22c in axial conformation (magenta), and H7 (black). (D) phthalimide derivatives and

benzamide: 29c (carbon atoms colored in magenta), 30c (black), and 31 (orange).

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2119

chemical substructures could be applied to this region of

the pocket. Third, the top part of the pocket (the D

region) is cleft-like in shape and a wide range of chem-

ical fragments would ﬁt this cleft. Fourth, a substructure

for the furanose region could be considered as a linker

to join an aromatic substructure for the A region and a

substructure for the D region. Figure 4B shows exam-

ples of the designed substructures for each region. We

built virtual inhibitors by combination of the substruc-

tures and then those virtual compounds were submitted

to the docking simulations. The resulted docking models

were interactively examined with the aid of computer

graphics.

the ligand-binding pocket are diﬀerent (Fig. 5A). The

dichlorophenyl group of 4b occupied the F region from

wall to wall in the docking model, while the dichloro-

phenyl group of 5a occupied the cleft of the D region,

allowing many types of substitutions (5b–f). Removal of

one chlorine atom from 4b drastically reduced the

inhibitory activity (4d) while a derivative lacking a

chlorine atom from 5a retained its inhibitory potency

(5c). The structure–activity relationship of the 4b

derivatives clearly indicated that 2,6-dichloro substitu-

tions is crucial (4a–c). Compound 4e exhibited no inhi-

bition while 5d retained the potency, indicating that full

use of the F region is important for 4b to interact with

the enzyme. This hypothesis was further supported by

the recovery of inhibitory activity found in 4f and 4g.

6. Pyridine scaﬀold

In the docking models of 4b and 5a, the urea bonds were

cis and in plane with the pyridine ring. Indeed, the

screening hit 2 could be considered as a conformation-

ally restrained cis urea derivative. The fact that

N-methylation abolished the inhibitory potency (8)

would support the importance of the conformation of

urea.

Pyridine derivatives that provide a linker moiety at the

4-position gave good docking models. Especially, linkers

that are planar in shape were good (Fig. 5A). Table 1

shows such examples (4a, 4b, 5a, 6a, and 6b) that

exhibited IC₅₀values of 0.2–0.9 lM. In contrast, pyri-

dine derivatives with linker substitution at the 3-position

failed to give docking models that retained the essential

hydrogen bond. 3-Substitued pyridine derivatives ex-

isted in the screening library and none indicated inhib-

itory activity (data not shown).

Because the A region is wide enough to contain both the

pyridine ring and a part of the linker moiety, the aro-

matic ring and the linker must be in the same plane.

Derivatives that include methylene (CH₂) in the

attachment at the 4-position (9a and 9b) were not

inhibitory and in good accordance with the fact that no

good docking model was obtained for these molecules.

On the other hand, derivatives that had a planar linker

The 2,6-dichlorophenyl group was a good substructure

fragment for phenylurea and benzylurea derivatives (4b

and 5a) than a free phenyl group (4e and 5d), although

the roles of the 2,6-dichlorophenyl group of 4b and 5a in

Table 1. Inhibitory potencies of pyridine derivatives

R

Cl

R3

R2

H

N

O

N

O

Cl

N

O

R1

N

R1

R3

R2

entry

R

entry

R1

R3

IC50 (µM)

IC50 ( M)

entry

R1

R2

R3

µ

IC50 ( M)

µ

0.5

0.9

F

H

6a

6b

0.2

0.9

>10

2.0

Cl

H

Cl

H

F

Cl

H

Cl

H

4a

4b

4c

4d

4e

4f

0.8

4.1

7.2

10.2

7.0

2.2

Cl

H

F

CF₃

Cl

H

F

H

Cl

H

5a

5b

5c

5d

5e

5f

F

CF₃

13.6

F

H

4g

O

Cl

O

Cl

R3

H

R2

N

O

N

O

N

H

N

H

O

N

Cl

R4 Cl

R1

entry

IC50 (µM)

>10

entry

IC50 (µM)

>10

entry

R1

R2

R3

R4

IC50 ( M)

µ

9a

9b

NH₂

Cl

H

Cl

NH₂

H

Cl

H

Cl

H

7a

7b

7c

7d

7e

>10

Cl

H

N

NH₂

H

N

O

H

N

>10

Cl

N

O

Cl

O

Cl

N

Cl

N

O

Cl

entry

IC50 (µM)

entry

IC50 (µM)

entry

IC50 (µM)

>10

11

>10

10

8

2120

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

(6a and 6b) were good ligands in the docking simulation

and were experimentally proven inhibitory. A known

Rho kinase inhibitor, Y27632 (Fig. 2), could be con-

sidered to belong to this family. Co-planarity between

the linker and the pyridine ring was decisive for the

pyridine derivatives.

7. 1H-Indazole scaﬀold

The 1H-indazole scaﬀold has a ﬁve-membered ring

system with a hydrogen bond acceptor atom at the 2-

position. Docking models suggested that the A region

could be better occupied by the indazole ring since the

indazole moiety is larger in size than the pyridine ring

system (Fig. 5B). There are two possible positions to

attach a linker substructure. Docking of the 5-subsituted

and 6-substituited analogues to the Rho kinase model

are both possible. The diﬀerence between the two ana-

logues was that an interaction between NH at the

1-position of indazole and C@O of Glu165 is possible

for the 5-substituted analogue in addition to the essen-

tial hydrogen bond between N-2 of indazole and NH of

Met-167. The docking model of the 6-substituted ana-

logues with two hydrogen bonds was not possible,

because there was no space available to the linker and

the peripheral moiety given that the indazole ring was

placed in the A region in the same manner allowed for

the 5-substituted analogues. An enzyme inhibition study

revealed that the 5-substituted analogues exhibited a

distinctively higher potency than those with the linker

substitution at the 6-position (12a vs 13a, 12b vs 13b, 12c

vs 13c, 14a vs 15, 16b vs 17, Table 2).

As the A region is spacious enough for a pyridine ring,

introduction of substitution might be possible. In fact, a

higher aﬃnity of Y30141 (K_i0.3 lM) than Y27632 (K_i

0.05lM) to Rho kinase was reported.²⁰This could be

explained by the full occupancy of the A region and/or

by formation of the additional hydrogen bond to the

C@O of Glu165 by the docking model. Thus the eﬀect of

substitutions at the 2- or 3-position of the pyridine rings

were investigated. Introducing the substitutions to the

3-position of the pyridine ring of 4b and 5a reduced

the inhibitory activity. Conformational analyses of the

3-substituted pyridine indicated that the substitutions

would destabilize the active conformation in which the

pyridine ring and urea linker is co-planar (7a, 7b, and

10) due to a steric interference with the linker urea

group. Substitutions at the 2-position were unfavorable

because they would orient toward the bottom wall of the

A region and cause unfavorable contact between the

inhibitor and the enzyme (7c, 7d, and 11). Introduction

of an amino group (hydrogen bond donor) at the 2-

position was intended to form an additional hydrogen

bond with C@O of Glu165, but this was not eﬀective

(7e). The docking model of 4b suggested that the use of

the urea linker would strictly determine the positioning

of the pyridine ring in the A region, which would not

allow placement of the amino group of 7e at the proper

distance from the C@O of Glu165.

The absence of N-2 lead to the loss of inhibitory

potency, conﬁrming the importance of the essential

hydrogen bond (18). If the additional hydrogen bond

between the NH-1 and C@O of Glu165 proposed by the

docking study existed, a substitution at the 3-position

should cause an unfavorable steric interaction with

Tyr166. Such an analogue (19e and 21a) indeed lost the

potency. Alkylation of N1, which disables the formation

Table 2. Inhibitory potencies of 1H-indazole derivatives

H

N

H

5

6

R

6

N

R

N

₁N

2

N

O

H

R2

N

R3

N

R4

entry IC50 (µM)

R

entry IC50 ( M)

R

µ

5

6

13a

13b

13c

>10

cycloHex

CH₂-cycloHex

CH₂-O-(2,6Cl,4F-Ph)

12a

12b

12c

0.5

0.9

0.5

cycloHex

CH₂-cycloHex

CH₂-O-(2,6Cl,4F-Ph)

N

R1

H

N

H

N

R

H

N

H

N

R

H

N

O

N

entry IC50 (µM) R1

R2

H

R3

H

R4

H

16c

19a

19b

19c

19d

19e

19f

0.2

0.4

>10

H

entry IC50 ( M)

R

µ

entry IC50 (µM)

15

>10

R

F

OMe

H

14a

14b

1

2,6Cl-Ph

CH₂-Ph

2,6Cl-Ph

0.3

(CO)Me

H

N

H

N

H

Et

H

Me

H

Me

N

R

N

Et

H

entry IC50 ( M)

µ

R

H

entry IC50 (µM)

17 >10

R1

H

16a

16b

16c

0.3

0.7

0.2

N

H

Et

CH₂-Ph

N

R

N

( )

H

N

n

R2

N

H

entry IC50 ( M) n

R

µ

20a

20b

20c

20d

0.8

0.02

0.1

2

1

nPr

CH₂-Ph

H

entry IC50 ( M) R1

R2

H

µ

N

H

20b

21a

21b

0.02

>10

H

Me

H

entry IC50 (µM)

18

>10

CH₂-Ph

0.03

Me

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

Table 3. Inhibitory potencies of isoquinoline derivatives

2121

R

X

5

N

1

N

R

N

H

N

₂N

entry

23a

23b

IC50( M)

R

entry

26

IC50( M)

>10

µ

entry

IC50( M)

X

N

CH

R

µ

CH₂Ph

nPr

NH-nPr

NHMe

CH₂Ph

(CH₂)₃CH(Me)₂

22a

22b

22c

22d

0.1

0.3

0.1

0.2

0.1

N

H

N

R

O

entry

27

IC50(µM)

>10

R

N

H

N

H

N

H

N

H

N

entry

24a

24b

24c

IC50(µM)

>10

R

N

H

entry

25a

25b

IC50(µM)

>10

R

2,6-F

2,6-Cl

4-(CO)Me

2,6-Cl

2,4,6-Cl

N

entry

28

IC50(µM)

>10

of the additional hydrogen bond, also resulted in the

loss of inhibitory potency (19f and 21b). Taken together,

the additional hydrogen bond would exist and it is an

important pharmacophore of the 5-substituted indazole

derivatives.

oline ring is planar, a planar linker is no longer neces-

sary. Here an amine nitrogen atom and a saturated ring

play a key role as the linker fragment (22a–d, 23a, and

23b, Table 3). Compound 22c, whose docking model is

shown in Figure 5C, is an example of a simple iso-

quinoline inhibitor. Note that both the axial and equa-

torial conformations were possible for the bond that

links the saturated ring and the nitrogen atom attached

to C5. The syn- and anti-isomers of 22c were equally

potent (IC₅₀¼0.1 lM), suggesting that the orientation of

the propyl group in the D region is not crucial. The X-

ray structure of H7¹¹and the docking model of 22c are

shown in Figure 5C.

As shown in Table 2, a variety of linker choices were

allowed for the 5-substituted indazole scaﬀold (e.g.,

12a–c, 14a–b, 16a–c, and 20a–d). In contrast to the

pyridine scaﬀold, a saturated ring system could be used

as a linker substructure in addition to the amide and

urea substructures. A modeling study indicated that the

planar linkers such as the amide or urea moieties posi-

tion its plane orthogonal to the indazole ring to ﬁll the

spherical F region and that the piperidine ring could

eﬀectively ﬁll the F region. See the docking model of

12a, 14b, and 20b in Figure 5B. Depending on the choice

of linker, the indazole ring orients diﬀerently in the A

region of the ligand-binding pocket. This observation

suggests that the indazole derivatives would diﬀer in

kinase speciﬁcity spectra based on the choice of the

linker moiety.

Employment of the isoquinoline scaﬀold would restrict

the choice of the linker substructures. A docking study

of compounds that include the urea linker (24a–c, 25a,

and 25b), an analogue with an insertion of a CH₂group

between N5 and a saturated ring (26 and 27) or

replacing the saturated ring with a planar aromatic ring

(28) indicated that these inactive derivatives did not

allow proper use of the F region.

N-alkylation of the linker nitrogen atom of 16c

decreased the inhibitory potency since no more space is

available for the alkyl group in the F region (19c and

19d). A large substitution at the 6-position of 16c

decreased the inhibitory potency (19b) while a smaller

substitution (19a) did not. This fact also suggests the full

occupation of the F region by indazole derivatives that

have an aminopiperidine as a linker substructure. In

fact, derivatives that have a cyclic aliphatic ring as the

linker substructure exhibited a higher potencies (20a–d)

than derivatives with the amide or urea linkers.

9. Phthalimide scaﬀold

Phthalimide is a ﬂat aromatic 5–6 ring system and has

hydrogen acceptors that are not included in the ring

system. As shown in Figure 4A, the essential hydrogen

bond donor (HN of Met167) is located oﬀ-center on the

bottom of the A region. Thus phthalimide was consid-

ered to utilize the A region of the ligand-binding pocket

in a diﬀerent way. The structure–activity relationship of

the phthalimide analogues is summarized in Table 4.

Compounds with a urea linker showed an inhibitory

potential (29a–d, and 30a–c). Figure 5D shows the

docking model of 29c and 30c. In addition to the

essential hydrogen bond between C@O at the 3-position

of the ligand and NH of Met167, NH of phthalimide

formed a hydrogen bond with C@O of Glu165. Another

carbonyl group at the 1-position was not utilized to

form a hydrogen bond.

8. Isoquinoline scaﬀold

The isoquinoline scaﬀold with substitution at the 5-po-

sition could be considered compatible with the pyridine

scaﬀold with a co-planar linker at the 4-position. HA-

1077 (Fig. 2) belongs to this category. As the isoquin-

2122

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

Table 4. Inhibitory potencies of phthalimide derivatives

tory potency was low for this scaﬀold. From 21 syn-

thesized benzamide derivatives, only the compound 31

exhibited an IC₅₀value less than 10 lM, while the

structurally corresponding phthalimide 30c exhibited an

IC₅₀value of 1.9 lM. This fact suggests that the

hydrophobic occupancy of the A region is very impor-

tant. It should be noted in the known crystal structures

of benzamides, the formylamide moiety is not co-planar

with the phenyl ring (dihedral plane angle approx. 20–

30°),²¹suggesting that the planarity of the hydrophobic

group is also important.

O

1

6

5

R2

O

HN

O³

R1

Y

N

H

X

entry

IC50( M)

X

Y

R1

R2

µ

CH₂

O

29a

29b

29c

29d

29e

29f

0.4

0.3

0.1

>10

NH

O

H

Cl

H

2-Cl

2,6-Cl

CH₂

O

10. The characterization of inhibitors

O

HN

R

N

H

N

Several Rho kinase inhibitors were tested for their

selectivity for other kinases such as MAP kinase, cAMP

dependent protein kinase, protein kinase C, and some

receptor tyrosine kinases. Table 5 shows the results,

indicating that the inhibitors designed in this study

indicated speciﬁcity to the Rho kinase and ATP-com-

petitive (Fig. 6).

H

O

entry

IC50( M)

R

nPr

Ph

µ

8.3

0.9

1.9

30a

30b

30c

2,6-Cl-Ph

N

*

>10

30d

30e

*

>10

Ph

NH₂

11. Conclusion

Cl

O

A high-throughput screening was carried out to discover

Rho kinase inhibitors. Based on a docking model of

screening hit compounds with a homolog model of Rho

kinase, a synthetically feasible virtual library shown in

Figure 4B was designed. With the aide of docking sim-

ulation, we selected possible active structures as well as

possible inactive structures from the virtual library. A

few of each of the possible active and the possible

inactive compounds were synthesized and submitted for

biological assessments. If the result was in accordance

with the docking simulation, we inferred that we

obtained an experimentally proven docking model.

Designing derivatives of the active compounds was then

a rather easy and safe task. Although accuracy or reli-

ability of a homology model is not easy to be evaluated,

Bossemeyer and co-workers experimentally demon-

strated that the cAPK crystal structure would be a

reliable template for homology modeling of the AGC

protein kinase family.²⁸Rho kinase belongs to the AGC

protein kinase family. We believe that our homology

model was a reliable one. This study demonstrated that

docking simulation with a structure of the target protein

is a robust method to explore a wider chemical space

with a smaller number of compounds.

N

H

Cl

entry

31

IC50(µM)

9

Analogues with an amide moiety as the linker were not

potent (29e and 29f). As far as using the urea linker, the

hydrophobic group for the D region should be aromatic

(30d and 30e). Introduction of a chlorine atom at the

6-position (29d) did not abolish the inhibitory potential

of 29c being in agreement with a molecular mechanical

calculation that the urea conformation (torsion angle of

C6–C5–N^linker–C^linker¼ꢀ175°) found in the docking

model of 29c would not be destabilized by the substi-

tution at the 6-position by a chlorine atom.

It was shown that the hydrogen bond acceptor of the

inhibitor should not necessarily be included in an aro-

matic ring system. We then investigated the benzamide

scaﬀold as an open form of the phthalimide scaﬀold.

The docking simulation of 31 gave a reasonable docking

model that is similar to the binding model of the

phthalimide inhibitors (Fig. 5D). However, the inhibi-

Table 5. Speciﬁcity of inhibitors (IC₅₀in lM)

Entry

Rho kinase

MAP kinase

cAMP dependent

protein kinase

PKC

PDFG–R kinase

SCF–R kinase

4a

5a

0.20

0.77

0.48

0.46

0.11

0.15

>30

>10

6a

>10

12c

29c

H-1077

>10

76% @ 10 lM

10% @ 10 lM

17% @ 10 lM

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2123

one aﬃnity chromatography.²²The GST fusion protein

of the head domain of glial ﬁbrillary acidic protein

(GFAP) was expressed in E. coli, puriﬁed by glutathione

aﬃnity chromatography and used as a substrate (phos-

phate acceptor).²³

40

30

20

µ

Fixing GFAP on microplates: A 100 lL aliquot of GFAP

solution (0.1 mg/mL) was added to each well of 96-well

ELISA plates and the plates were kept at 4 °C overnight.

The plates were washed three times with PBS (100 mM

sodium phosphate, pH 7.4), then 250 lL of blocking

buﬀer solution (5% bovine serum albumin, 5% sucrose in

10 mM sodium phosphate buﬀer of pH 8.0) was added

and stored at 4 °C overnight. The blocking buﬀer was

removed by suction and the wells were washed with PBS.

Ki^HA-1077= 0.15µM

Ki^4a= 0.32µM

10

0

Kinase reaction: Each 100 lL of the reaction buﬀer

(50 mM Tris–HCl buﬀer pH 7.5, 5 mM MgCl₂, 1 mM

EDTA, 10 lM ATP) was added to the wells. Test

compounds (1 mg/mL DMSO) were added to a ﬁnal

concentration of 10 lg/mL. The enzyme reaction was

started by adding recombinant Rho kinase (ﬁnal 5 lg/

mL). The reaction was carried out for 2 h at rt.

Km^ATP= 9.8µM

0.6 0.8

0

0.2

0.4

1

1/ATP (µM)

Figure 6. Lineweaver–Burk plot for 4a. K_ivalue for HA-1077 was

comparative with those previously reported.^6;27

The amount of phophorylated GFAP was determined

by the ELISA method. Microplates were washed three

times with PBS. The monoclonal antibody KT13 (mouse

monoclonal antibody speciﬁc to phosphorylated

GFAP)²³was added and stored at rt for 1 h. The wells

were washed ﬁve times with PBS followed by adding the

secondary antibody (goat anti-mouse IgG antibody

conjugated with horse radish peroxidase, Biorad 172-

1011). After 1 h, the wells were washed seven times with

PBS. The peroxidase activity was then measured by

adding 100 lL of peroxidase reaction solution (prepared

freshly with o-phenylenediamine 13 mg, 1.63 mL MeOH,

30.88 mL H₂O, and concd H₂O₂11 lL). The reaction

was stopped by adding 100 lL of 2 N H₂SO₄. Absor-

bance at 490 nm was measured.

The existence of a hydrogen bond acceptor and the

presence of an aromatic ring of a suitable size were

important for an inhibitor to interact with the A region

of the ligand-binding pocket. The combination of the

substructures for the A region and the F region would

determine the potency of the inhibitor and the enzyme

selectivity.

It was also shown that diﬀerent scaﬀolds were possible

as lead structures for the Rho kinase inhibitors. We

succeeded to design and synthesize novel lead com-

pounds. The optimization process and structure–activity

relationship of these lead scaﬀolds found in this study

will be reported elsewhere.

12.2. Rho kinase inhibitory activity

12. Experimental

The test compounds (10 lL, DMSO solution) were ad-

ded to the reaction buﬀer (Tris–HCl buﬀer 100 mM,

pH 7.5, containing MgCl₂10 mM, EGTA 2 mM, and

EDTA 2 mM, total volume 100 lL). Ribosomal S6

kinase substrate (S6 231–239 peptide, CarbioChem, ﬁnal

concentration 1 lM) was used as the substrate. The

enzymatic reaction was initiated by adding the

recombinant Rho kinase together with ATP ([c-³²P]-

ATP, ﬁnal concentration 10 lM). The reaction was

carried out at 30 °C and terminated by adding 50 lL of

150 mM phosphoric acid. Reaction solution (40 lL) was

adsorbed on a strip of ﬁlter paper (Whatman P81 ion-

exchange chromatography paper). The strips were

brieﬂy dried and transferred to a conical ﬂask ﬁlled with

an excess amount of phosphoric acid solution (75 mM)

and radioactive ATP was washed away from the strips.

This operation was repeated three times followed by a

rinse with water. The amount of the phosphorylated

substrate was measured by a liquid scintillation counter

(Wallac 1205 Beta Plate).

Commonly used abbreviations; DMF (dimethylforma-

mide), EtOAc (ethyl acetate), EtOH (ethanol), MeOH

(methanol), ATP (adenosine triphosphate), DMSO

(dimethylsulfoxide), Ph (phenyl group), Me (methyl

group), Et (ethyl group), nPr (normal propyl group),

cycloHex (cyclohexyl group), WSC–HCl (1-ethyl-3-(3-

dimethylaminopyropyl)carbodiimide

hydrochloride),

HOBt–H₂O (5-hydroxybenzotriazole mono hydrate),

PDGF (platelet derived growth factor), SCF (stem cell

factor), ELISA (enzyme linked immunoadsorbent

assay).

12.1. High-throughput screening

Enzyme and substrate: The catalytic domain of the

bovine Rho kinase was expressed as the GST (glutha-

thione-S-transferase) fusion protein in insect cells (Sf9)

using bacullovirus and puriﬁed by employing glutathi-

2124

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

12.3. P42-mitogen activated protein kinase

(MAP kinase-2) assay

100 ng/mL SCF for 15 min at 37 °C, respectively. The

cells were lysed with HNTG [20 mM HEPES (pH 7.5),

150 mM NaCl, 0.2% Triton X-100, 10% glycerol, 5 mM

Na₃VO₄, 2 mM Na₄P₂O₇, and 5 mM EDTA] and

transferred to 96-well ELISA plate pre-coated with anti-

phosphotyrosine antibody, PY20 (BD. Bioscience), and

incubated at rt for 1 h. After washing, the anti-PDGFR-

b antibody (Santa Cruz) or the anti-c-Kit polyclonal

antibody (Santa Curz) was added to the well and incu-

bated at rt for 1 h. The receptor–antibody complex was

probed with horseradish peroxidase conjugated anti-

rabbit-IgG antibody (Amersham) and the color devel-

oped with TMBZ (Sumitomo Bakelite).

The eﬀect of compounds on the MAP kinase activity

was examined using the in vitro kinase assay. Myelin

basic protein (MBP) was used as a kinase substrate and

6 lg of MBP was incubated in 30 lL of buﬀer containing

18 mM HEPES (pH 7.4), 10 mM MgCl₂, 1 mM vana-

date, 5 mM NaF, 1 mM b-glycerophosphate, 1 mM

EDTA, 20 lM [c-³²P]-ATP (5 lCi), and 0.2 lg of puri-

ﬁed activated GST-p42 MAP kinase (UBI) at 30 °C for

30 min in the presence or absence of compounds. The

proteins were then transferred onto P81 ion-exchange

chromatography papers (Whatman) and washed three

times with 75 mM phosphoric acid followed by a rinse

with water. The radioactivity of the proteins was mea-

sured using a liquid scintillation counter.

12.7. Homology modeling of Rho kinase catalytic domain

A crystal structure of cAMP dependent protein kinase

(a protein kinase A, cAPK) solved by Bossemeyer and

co-workers was chosen as the template structure (Pro-

tein Data Bank entry:1YDR).¹²The sequence of cyclin

dependent protein kinase 2 (CDK2: Protein Data Bank

entry:1HCL)¹⁴and the insulin receptor kinase

(IRK: Protein Data Bank entry:1IRK)¹⁵were also

aligned to help the side-chain modeling as described

below (Fig. 3).

12.4. cAMP dependent protein kinase assay

The catalytic subunit of bovine heart cAMP dependent

protein kinase (PKA) was purchased from Pierce. The

phosphorylation reaction was determined using the

ﬂuorescence intensity quenching method with a ﬂuoro-

phore-labeled kemptide as the substrate (IQ^ekinase

reagent from Pierce). The assay buﬀer contained 20 mM

Tris–HCl (pH 7.2), 10 mM MgCl₂, 2 mM ATP, 0.012%

Triton X-100, 60 lM labeled kemptide peptide and

inhibitor in a total volume of 30 lL. Fifteen units of the

enzyme was added and incubated at rt for 20 min. The

inhibitor was added as a DMSO solution. The ﬁnal

concentration of the DMSO was less than 10%.

First, the insertion/deletion was adjusted. There were

four loops where such an adjustment was required.

Four-residue insertion occurs in the ﬂexible activation

loop (Phe228:Pro249 in Rho kinase sequence corre-

sponding to Phe185:Pro202 of cAPK). Considering that

the conformation of the activation loop is not conserved

among the protein kinases whose three-dimensional

structures were known, we dare not model this loop and

left it as of cAPK. Thus our homology model is a chi-

merical model. Except for the activation loop, the other

insertion/deletion patches (one-residue deletion between

Gly136 and Ser139 of cAPK, four-residue insertion

between Gly214 and Tyr215 of cAPK and one-residue

insertion between Leu284 and Lys285 of cAPK) were

modeled by the loop search program available in the

SYBYL BIOPLYMER module. Since there were no further

insertion/deletions except the loops, the backbone atom

coordinates of the rest of the template protein were not

modiﬁed.

12.5. Protein kinase C inhibitory assay

Rat brain PKC was purchased from Sigma. Biotylnyl-

ated myelin basic protein peptide (Calbiochem) was

used as the substrate. The assay buﬀer contained 20 mM

Tris–HCl (pH 7.5), 10 mM MgCl₂, 0.5 mM CaCl₂,

15 lM [c-³²P]-ATP (25 lCi), 0.03% Triton X-100,

300 lg/mL of phosphatidyl- -serine, and 30 lg/mL of

L

1,3-diolein (diacylglycerol), 25 lM peptide, and inhibi-

tor in a total volume of 20 lL. The reaction was started

by the addition of PKC (25 mU, 5 lL). The reaction was

stopped by the addition of 10 lL of 8 M guanidine

hydrochloride. The phosphorylated peptide was recov-

ered as Avidin complex by using a PKC assay kit sup-

plied by Calbiochem. The inhibitor was added as a

DMSO solution. The ﬁnal concentration of the DMSO

was 10%.

Second, the amino acid residues of cAPK were altered

according to the sequence of the Rho kinase. The side-

chain conformation was determined by the following

rules.

(1) If an amino acid residue was identical between Rho

kinase and the temperate cAPK, the side-chain confor-

mation found in the cAPK structure was preserved for

the residue. Replacement of any amino acid to Ala or

Gly was accomplished by simply shortening the side

chain of a cAPK residue.

12.6. Receptor tyrosine kinase assay

The eﬀects of compounds on PDGFR-b and c-Kit

kinase activities were measured using sandwich ELISAs.

NIH3T3 and KU812F cells were used for the PDGFR-b

and the c-Kit assay, respectively. Serum starved cells

were treated with or without compounds at 30 °C for

30 min. NIH3T3 cells and KU812F cells were then

stimulated with 50 ng/mL PDGF-bb for 5 min and

(2) If amino acid replacement was ‘similar’ or ‘smaller’,

for example, replacement Ile with Val, Thr with Val, Lys

with Arg, replacing an aromatic amino acid residue with

aromatic one, replacing an amidic residue with the

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2125

corresponding acidic one (replacing Asn to Asp, Gln to

12.8. Docking simulation

Glu), etc., the v torsion angles of the cAPK side chain

were copied to the replaced residue. Replacing Asn115

of cAPK to Tyr159 belongs to this category by treating

the aromatic ring as an equivalent of the plane deﬁned

by the amide group of the asparaginyl residue. Mutating

an acidic amino acid to its corresponding neutral residue

was carried out by considering the formation of

hydrogen bonds or favorable electrostatic interactions

to locate the carbonyl and amide groups. Similarly, an

aromatic residue was converted to a diﬀerent aromatic

residue. An example is a mutation of Phe102 of cAMP

to Trp146 of Rho kinase. In this case, two possibilities

existed for matching the asymmetric indole ring to the

symmetric phenyl ring. Molecular mechanical energy

calculations were carried out for each possibility to

select the conformation.

A ligand molecule was placed in the active site in such

way that the hydrogen bond acceptor atom took the

position of N2 of H7 and the aromatic plane was in the

plane deﬁned by the isoquinoline ring of H7. The initial

conformation of the ligand was ﬁrst given interactively

so that the ligand would reside in the enzyme pocket.

The atomic charge of the ligand molecule was the

MOPAC charge. All single bonds of a ligand molecule

were selected as rotatable bonds except those in the ring

system. When a test ligand included a chiral center(s),

each conﬁgurational isomer model was built and con-

sidered independently. Similarly, when a cyclic system

such as the pyperidine ring was included in a test ligand,

the conformational models of the ring system, that is

boat and chair forms, were prepared and docked inde-

pendently. The cis/trans isomers were modeled for

ligands that include the urea moiety. Atoms in the

(3) Mutation to proline was conducted by keeping the /

and w torsional angles. Fortunately in this study, all

such mutations occurred for the amino acid residues

whose backbone conformation were compatible with

those allowed for proline;²⁴Ala124 to Pro168 (/ ¼ ꢀ60,

w ¼ ꢀ26); His260 to Pro311 (ꢀ101, ꢀ5); Lys295 to

Pro347 (ꢀ46, ꢀ44); Glu341 to Pro393 (ꢀ59, 138), and

Cys343 to Pro395 (ꢀ133, 57).

ꢀ

ligand-binding site and atoms within 4 A of the ligand-

binding site were used to calculate the docking interac-

tion energy (total of 988 atoms including hydrogen

atoms). Side-chain bonds of the amino acid residues that

orient toward the inside of the ligand-binding pocket,

Val101, Lys116, Glu135, Val148, Asp213, Asn214,

Leu216, Asp227, and Phe379 were selected as the

rotatable bonds. The atomic charge for the protein is

from the Kollman all-atom force ﬁled parameter avail-

able in the ^SYBYLpackage.

(4) If mutation from Gly, Ala, or Pro to other amino

acid residue or ‘diﬀerent/larger’ type mutation occurred,

the sequence alignment with CDK2 or IRK was

referred. If one found an identical amino acid type, the

side-chain structure was taken from either CDK2 or

IRK. For example, Val123 of cAPK corresponds to

Met167 of Rho kinase. In IRK, the corresponding

amino acid residue is Met1079. The side chain of the

methionyl residue was transplanted to the Rho kinase

model. Similarly Leu49 of cAPK was mutated using the

side-chain structure of the corresponding isoleucyl resi-

due of CDK2 (Ile10) to model Ile93 of Rho kinase.

12.9. Chemistry

Analytical data were recorded for the compounds

described 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 (r) from an internal tetramethylsilane

standard as 0 ppm or internal chloroform as 7.24 ppm in

deuterochloroform. Coupling constants (J) were

recorded in Hz. Mass spectra (MS) were recorded using

the PLATFORM-LC (Micromass) spectrometer. Melt-

ing points were taken using an electrothermal melting

point apparatus and are uncorrected. The reagents were

purchased from commercial sources.

(5) Residues whose side-chain conformation could not

be determined by the rule described above were modeled

by a conformational search (tweak search algorithm

available in ^SYBYLpackage).

After the modeling, energy minimization using ^SYBYL-

M

aximin2 software employing the force ﬁeld parameter

and atomic charge of Kollman was carried out.²⁵The

backbone atoms except for the modeled loops were ﬁxed

during this minimization.

12.9.1. N-(4-Pyridyl)-N⁰-(2,4,6-trichlorophenyl)urea (4a).

To a solution of 4-aminopyridine (500 mg, 3.56 mmol) in

toluene (5 mL) was added 2,4,6-trichlorophenylisocya-

nate (504 mg, 3.56 mmol) at rt. The reaction mixture was

stirred at 80 °C for 3 h and diluted with ether. The

resulting precipitate was collected by ﬁltration, washed

with ether, and dried up in vacuo to give the title com-

pound as a colorless solid (819 mg, 2.60 mmol, 87% yield

in this step). Mp 205 °C. ¹H NMR (CDCl₃, 400 MHz) d

7.43 (dd, J ¼ 1:7 Hz, 4.9 Hz, 2H), 7.62 (s, 2H), 8.35 (dd,

J ¼ 1:7 Hz, 4.9 Hz, 2H), 8.48 (s, 1H), 9.45 (s, 1H). MS

(ESI) m=z 315 (Mꢀ1)^ꢀ. Anal. Calcd for C₁₂H₈ON₃Cl₃:

C, 44.27; H, 2.79; N, 2.91. Found: C, 44.71; H, 2.63; N,

12.13.

The ligand-binding site was deﬁned by those residues

ꢀ

that locate within 4 A from a super-ligand molecule. The

super-ligand molecule was created by superimposing

ligand molecules, ATP, H7, and staurosporin taken

from the complex crystal structures of CDK2 (1HCL),

cAPK (1YDR), and cAPK (1STC), respectively. The

ligand-binding site involved 27 amino acid residues.

They were Ile93, Gly94, Arg95, Gly96, Ala97, Val101,

Ala114, Lys116, Glu135, Met139, Val148, Met164,

Glu165, Tyr166, Met167, Pro168, Gly170, Asp171,

Asp209, Lys211, Asp213, Asn214, Leu216, Ala226,

Asp227, Phe228, and Phe379.

2126

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

12.9.2. N-(4-Pyridyl)-N⁰-(2,6-dichlorophenyl)urea (4b).

Compound 4b was prepared from 2,6-dichlorophen-

ylisocyanate in a manner similar to that described for

4a, except washing the precipitate with n-hexane, with a

12.9.8. N-(2,6-Dichlorobenzyl)-N⁰-(4-pyridyl)urea (5a).

To a solution of 2,6-dichlorophnylacetic acid (150 mg,

0.78 mmol) in toluene (0.5 mL) was added triethylamine

(0.13 mL,

0.93 mmol),

diphenylphosphorylazide

1

yield of 88% as a colorless solid. Mp 168–170 °C. H

(0.2 mL, 0.93 mmol), stirred at 110 °C for 1 h, and

allowed to cool to rt. 4-Aminopyridine (128 mg,

0.78 mmol in DMF) was added and temperature was

raised to 110 °C for 2 h. Water and EtOAc were added

to the reaction mixture. The precipitate was ﬁltered and

washed with EtOAc to give the title compound as a

NMR (CDCl₃, 400 MHz) d 7.20 (t, J ¼ 7:8 Hz, 1H),

7.39 (d, J ¼ 8:3 Hz, 2H), 7.49 (dd, J ¼ 1:7 Hz, 4.9 Hz,

2H), 8.31 (dd, J ¼ 1:5 Hz, 5.1 Hz, 2H). MS (ESI) m=z

281 (Mꢀ1)^ꢀ.

1

white solid (76 mg, 32% yield). Mp 216 °C. H NMR

12.9.3. N-(4-Pyridyl)-N⁰-(2,4-dichlorophenyl)urea (4c).

Compound 4c was prepared from 2,4-dichlorophen-

ylisocyanate in a manner similar to that described

for 4a, except washing the precipitate with

n-hexane, with a yield of 93% as a colorless solid. Mp

244 °C. ¹H NMR (CDCl₃, 400 MHz) d 7.31 (dd,

J ¼ 2:4 Hz, 9.0 Hz, 1H), 7.48 (d, J ¼ 2:4 Hz, 1H), 7.53

(dd, J ¼ 1:7 Hz, 4.9 Hz, 2H), 8.17 (d, J ¼ 9:0 Hz, 1H),

8.33 (dd, J ¼ 1:7 Hz, 4.9 Hz, 2H). MS (ESI) m=z 281

(Mꢀ1)^ꢀ.

(CDCl₃, 400 MHz) d 6.08 (d, J ¼ 5:3 Hz, 2H), 6.73 (t,

J ¼ 5:4 Hz, 1H), 7.34 (dd, J ¼ 1:6 Hz, 4.9 Hz, 2H), 7.38

(dd, J ¼ 1:2 Hz, 7.3 Hz, 1H), 7.51 (d, J ¼ 7:8 Hz, 2H),

8.28 (dd, J ¼ 1:7 Hz, 4.9 Hz, 2H), 8.84 (s, 1H). MS (ESI)

m=z 295 (Mꢀ1)^ꢀ. Anal. Calcd for C₁₃H₁₁Cl₂N₃O: C,

52.72; H, 3.745; N, 14.19. Found: C, 52.65; H, 3.64; N,

13.98.

12.9.9. N-(2,4-Dichlorobenzyl)-N⁰-(4-pyridyl)urea (5b).

Compound 5b was prepared from 2,4-dichlorophen-

ylacetic acid in a manner similar to that described for 5a,

except puriﬁcation by silica gel column chromatography

(CHCl₃–MeOH), with a yield of 30% as a colorless solid.

12.9.4.

N-(4-Pyridyl)-N⁰-(2-chlorophenyl)urea

(4d).

Compound 4d was prepared from 2-chlorophenyliso-

cyanate in a manner similar to that described for 4a with

1

Mp 167–169 °C. H NMR (CDCl₃, 400 MHz) d 4.35 (d,

1

a yield of 76% as a colorless solid. H NMR (CDCl₃,

J ¼ 5:9 Hz, 2H), 6.94 (t, J ¼ 6:0 Hz, 1H), 7.37 (dd,

J ¼ 1:6 Hz, 4.8 Hz, 2H), 7.39 (d, J ¼ 8:4 Hz, 1H), 7.44

(dd, J ¼ 2:1 Hz, 8.4 Hz, 1H), 7.61 (d, J ¼ 2:2 Hz, 1H),

8.29 (dd, J ¼ 1:5 Hz, 4.9 Hz, 2H), 9.17 (s, 1H). MS (ESI)

m=z 295 (Mꢀ1)^ꢀ.

400 MHz) d 6.98 (m, 2H), 7.29 (dd, J ¼ 8:2 Hz, 1.5 Hz,

1H), 7.38 (d, J ¼ 6:2 Hz, 2H), 8.09 (dd, J ¼ 8:2 Hz,

1.5 Hz, 1H), 8.38 (d, J ¼ 6:2 Hz, 2H). MS (ESI) m=z 246

(Mꢀ1)^ꢀ.

12.9.5. N-(4-Pyridyl)-N⁰-phenylurea (4e). Compound 4e

was prepared from phenylisocyanate in a manner similar

to that described for 4a, except washing the precipitate

with n-hexane, with a yield of 88% as a colorless solid.

¹H NMR (CDCl₃, 400 MHz) d 7.2–7.3 (m, 5H), 7.33 (d,

J ¼ 5:7 Hz, 2H), 8.30 (d, J ¼ 5:7 Hz, 2H). MS (ESI) m=z

212 (Mꢀ1)^ꢀ.

12.9.10. N-(2-Chlorobenzyl)-N⁰-(4-pyridyl)urea (5c).

Compound 5c was prepared from 2-chlorophenylacetic

acid in a manner similar to that described for 5a, except

puriﬁcation by silica gel column chromatography

(CHCl₃–MeOH), with a yield of 38% as a colorless solid.

1

Mp 130–131 °C. H NMR (CDCl₃, 400 MHz) d 4.38 (d,

J ¼ 6:1 Hz, 2H), 6.90 (t, J ¼ 5:9 Hz, 1H), 7.28–7.41 (m,

4H), 7.37 (dd, J ¼ 1:5 Hz, 4.9 Hz, 2H), 7.45 (dd,

J ¼ 1:5 Hz, 7.7 Hz, 1H), 8.29 (dd, J ¼ 1:5 Hz, 4.9 Hz,

2H), 9.13 (s, 1H). MS (ESI) m=z 260 (Mꢀ1)^ꢀ.

12.9.6. N-(4-Pyridyl)-N⁰-(2,6-ﬂuorophenyl)urea (4f).

Compound 4f was prepared from 2,6-diﬂuorophenylis-

ocyanate in a manner similar to that described for 4a,

except washing the precipitate with n-hexane,

with a yield of 93% as a colorless solid. ¹H NMR

(CDCl₃, 400 MHz) d 6.95 (d, J ¼ 8:3 Hz, 1H), 6.97 (d,

J ¼ 8:1 Hz, 1H), 7.19 (t, J ¼ 8:3 Hz, 1H), 7.48 (dd,

J ¼ 1:5 Hz, 5.1 Hz, 2H), 8.31 (dd, J ¼ 1:5 Hz, 4.9 Hz,

2H). MS (ESI) m=z 248 (Mꢀ1)^ꢀ.

12.9.11. N-Benzyl-N⁰-(4-pyridyl)urea (5d). To a solution

of 4-aminopyridine (50 mg, 0.53 mmol) in toluene (1 mL)

was added benzylisocyanate (71 mg, 0.53 mmol) and

stirred at 110 °C for 2 h. Water and EtOAc were added

to the reaction mixture. EtOAc layer was washed with

water and saturated NaCl aq, dried over anhydrous

Na₂SO₄, and solvent was removed. The residue was

puriﬁed by silica gel column chromatography (CHCl₃–

MeOH). The title compound was obtained as a colorless

solid (18 mg, 15% yield). Mp 156–157 °C. ¹H NMR

(CDCl₃, 400 MHz) d 4.31 (d, J ¼ 5:9 Hz, 2H), 6.85 (t,

J ¼ 5:9 Hz, 1H), 7.24 (tt, J ¼ 1:7 Hz, 6.8 Hz, 1H), 7.28–

7.36 (m, 4H), 7.37 (dd, J ¼ 1:6 Hz, 4.8 Hz, 2H), 8.28 (d,

J ¼ 6:1 Hz, 2H), 9.01 (s, 1H). MS (ESI) m=z 226

(Mꢀ1)^ꢀ.

12.9.7.

N-(4-Pyridyl)-N⁰-[2-ﬂuoro-6-(triﬂuoromethyl)-

phenyl]urea (4g). Compound 4g was prepared from 2-

ﬂuoro-6-(triﬂuoromethyl)phenylisocyanate in a manner

similar to that described for 4a, except washing the

precipitate with EtOAc, with a yield of 44% as a col-

orless solid. Mp 231–232 °C. ¹H NMR (CDCl₃,

400 MHz) d 7.45 (dd, J ¼ 1:6 Hz, 4.8 Hz, 2H), 7.48–7.64

(m, 3H), 8.34 (s, 1H), 8.39 (dd, J ¼ 1:6 Hz, 4.8 Hz, 2H),

9.28 (s, 1H). MS (ESI) m=z 298 (Mꢀ1)^ꢀ.

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2127

12.9.12. N-(2,6-Diﬂuorobenzyl)-N⁰-(4-pyridyl)urea (5e).

Compound 5e was prepared from 2,6-diﬂuorophen-

ylacetic acid in a manner similar to that described for 5a,

except puriﬁcation by silica gel column chromatography

(CHCl₃–MeOH), with a yield of 57% as a colorless solid.

12.9.15. N1-(4-Pyridyl)-2-(2,6-dichlorophenoxy)acetam-

ide (6b). (1) 2,6-Dichloro-phenoxyacetic acid was pre-

pared from 2,6-dichlorophenol in a manner similar to

that described for 2,6-dichloro-4-ﬂuorophenoxyacetic

acid with a yield of 79% as a colorless solid.

1

Mp 153–155 °C. H NMR (CDCl₃, 400 MHz) d 4.39 (d,

J ¼ 5:4 Hz, 2H), 6.84 (t, J ¼ 5:7 Hz, 1H), 7.10 (t,

J ¼ 8:2 Hz, 2H), 7.33 (dd, J ¼ 1:6 Hz, 4.8 Hz, 2H), 7.40

(dt, J ¼ 1:6 Hz, 8.3 Hz, 1H), 8.27 (dd, J ¼ 1:6 Hz,

4.8 Hz, 2H), 8.87 (s, 1H). MS (ESI) m=z 262 (Mꢀ1)^ꢀ.

(2) The compound 6b was prepared from 4-aminopyr-

idine in a manner similar to that described for the

compound 6a with a yield of 10% as a colorless solid. ¹H

NMR (CDCl₃, 400 MHz) d 3.74 (d, J ¼ 2:7 Hz, 2H),

7.20–7.40 (m, 7H). MS m=z 297 (Mþ1)^ꢀ.

12.9.13.

N-[2-Fluoro-6-(triﬂuoromethyl)benzyl]-N⁰-(4-

pyridyl)urea (5f). Compound 5f was prepared from 2-

ﬂuoro-6-triﬂuoromethylphenylacetic acid in a manner

similar to that described for 5a, except puriﬁcation by

silica gel column chromatography (CHCl₃–MeOH),

with a yield of 55% as a colorless solid. Mp 138–139 °C.

¹H NMR (CDCl₃, 400 MHz) d 4.51 (d, J ¼ 4:9 Hz, 2H),

6.69 (t, J ¼ 5:0 Hz, 1H), 7.34 (dd, J ¼ 1:5 Hz, 4.9 Hz,

2H), 7.59–7.64 (m, 3H), 8.28 (s, 2H), 8.28 (d, J ¼ 6:3 Hz,

2H), 8.82 (s, 1H). MS (ESI) m=z 310 (Mꢀ1)^ꢀ.

12.9.16. N-(2-Amino-4-pyridyl)-N⁰-(2,6-dichlorophenyl)-

urea (7a). To a solution of 2,6-dichlorobenzoic acid

(2.0 g, 10.5 mmol) in toluene (10 mL) was added triethyl-

amine (1.89 mL, 12.6 mmol), diphenylphosphorylazide

(2.7 mL, 12.6 mmol) and stirred at 110 °C for 1 h and

allowed to cool to rt. 3,4-Diaminopyridine (1.14 g,

10.5 mmol in DMF) was added and temperature was

raised to 110 °C for 2 h. The solid precipitated in the

reaction mixture was ﬁltered, washed with EtOAc, and

dried up in vacuo to give the title compound as a col-

orless solid (440 mg, 14% yield). Mp 224–228 °C. ¹H

NMR (DMSO, 400 MHz) d 6.55 (br s, 2H), 6.78 (d,

J ¼ 5:9 Hz, 1H), 6.99 (t, J ¼ 7:6 Hz, 1H), 7.30–7.35 (m,

1H), 7.53 (d, J ¼ 8:1 Hz, 2H), 7.91 (d, J ¼ 6:1 Hz, 1H),

8.30 (s, 1H), 8.62 (br s, 1H). MS (ESI) m=z 296 (Mꢀ1)^ꢀ.

12.9.14. N1-(4-Pyridyl)-2-(2,6-dichloro-4-ﬂuorophenoxy)-

acetamide (6a). To a solution of 2,6-dichloro-4-ﬂuor-

ophenol (2.0 g, 11.05 mmol) in acetonitrile (10 mL),

K₂CO₃(1.83 g, 13.26 mmol), and methylbromoacetate

(1.69 g, 11.05 mmol) was added and stirred at 80 °C for

1 h. Water was added to the reaction mixture and the

mixture was extracted with EtOAc. The EtOAc layer

was washed with water and saturated NaCl aq, dried

over anhydrous Na₂SO₄, and the solvent was removed.

The resulting 2,6-dichloro-4-ﬂuorophenoxyacetic acid

ethyl ester was obtained as a white powder (2.66 g,

95.2%).

12.9.17. N-(3,5-Dichloro-4-pyridyl)-N⁰-(2,6-dichlorophen-

yl)urea (7b). To a solution of 2,6-dichlorobenzoic acid

(150 mg, 0.78 mmol) in toluene (0.5 mL) was added tri-

ethylamine (0.14 mL, 0.93 mmol), diphenylphosphoryl-

azide (0.2 mL, 0.93 mmol) and stirred at 110 °C for 1 h

and allowed to cool to rt. 4-Amino-3,5-dichloropyridine

(128 mg, 0.78 mmol in DMF) was added and tempera-

ture was raised to 110 °C for 2 h. Water was added to the

reaction mixture and the mixture was extracted with

EtOAc. The EtOAc layer was washed with water and

saturated NaCl aq, dried over anhydrous Na₂SO₄, and

dried up in vacuo. The residue was washed with CHCl₃

and collected by ﬁltration to give the title compound as

To the solution of the ester (2.66 g, 10.51 mmol) in

EtOH, 5% aqueous NaOH (20 mL) was added and

stirred at 80 °C for 1 h. The reaction mixture was con-

centrated, acidiﬁed to pH 4 with 5% aqueous HCl, and

extracted with EtOAc. The EtOAc layer was washed

with water and saturated NaCl aq, dried over anhydrous

Na₂SO₄, and dried in vacuo, to give 2,6-dichloro-4-ﬂu-

orophenoxyacetic acid (2.31 g, 4.18 mmol, 92%) as a

colorless solid.

1

a colorless solid (40 mg, 15% yield). Mp 250–255 °C. H

NMR (DMSO, 400 MHz) d 7.28–7.36 (m, 1H), 7.52 (d,

J ¼ 8:3 Hz, 2H), 7.54 (d, J ¼ 7:6 Hz, 2H), 8.44 (br s,

1H), 8.63 (s, 1H). MS (ESI) m=z 350 (Mꢀ1)^ꢀ.

To a solution of the acetic acid (2.31 g, 4.18 mmol) in

DMF (4 mL) was added 4-aminopyridine (393 mg,

4.18 mmol), WSC–HCl (946 mg, 5.0 mmol) and HOBt–

H₂O (678 mg, 5.0 mmol). The reaction mixture was

stirred for 3.5 h at rt. To the reaction mixture, water

was added and extracted with EtOAc. The organic layer

was washed with water and saturated NaCl aq, dried

over anhydrous Na₂SO₄, and concentrated in vacuo.

The crude material was puriﬁed by silica gel column

chromatography to aﬀord the title compound (155 mg,

11.8%) as colorless solid. Mp 126 °C. ¹H NMR (CDCl₃,

400 MHz) d 4.63 (s, 2H), 7.15 (d, J ¼ 7:8 Hz, 2H), 7.59

(dd, J ¼ 1:6 Hz, 4.8 Hz, 2H), 8.57 (dd, J ¼ 1:5 Hz,

4.9 Hz, 2H); MS (ESI) m=z 314 (Mꢀ1)^ꢀ. Anal. Calcd for

C₁₃H₉Cl₂FN₂O₂: C, 49.55; H, 2.88; N, 8.89. Found: C,

49.47; H, 2.98; N, 8.89.

12.9.18. N-(2,6-Dichloro-4-pyridyl)-N⁰-(2,6-dichlorophen-

yl)urea (7c). Compound 7c was prepared from

4-amino-2,6-diaminopyridine in a manner similar to

that described for 7a, with a yield of 4% as a colorless

solid. ¹H NMR (DMSO, 400 MHz)

d 7.38 (t,

J ¼ 8:3 Hz, 1H), 7.52 (d, J ¼ 8:3 Hz, 2H), 7.55–7.58 (m,

3H), 8.43 (br s, 1H). MS (ESI) m=z 350 (Mꢀ1)^ꢀ.

12.9.19. N-(2-Chloro-4-pyridyl)-N⁰-(2,4,6-trichlorophen-

yl)urea (7d). Compound 7d was prepared from 4-

amino-2-chloropyridine and 2,4,6-trichlorophenyl-

isocyanate, in a manner similar to that described for 4a

as a colorless solid. Mp 238 °C (dp). NMR (CD₃OD,

2128

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

400 MHz) d 7.29 (dd, J ¼ 5:8 Hz, 1.9Hz, 1H), 7.50 (s,

2H), 7.58 (d, J ¼ 1:9 Hz, 1H), 8.06 (d, J ¼ 5:8 Hz, 1H);

MS (ESI) m=z 350 (Mꢀ1)^ꢀ.

12.9.24. N-(2,6-Dichlorobenzyl)-N⁰-(3-amino-4-pyridyl)-

urea (10). To a solution of 2,6-dichlorophenyl acetic

acid (100 mg, 0.49 mmol) in toluene (0.5 mL) was added

triethylamine (0.08 mL, 0.54 mmol) and diphenyl-

phosphorylazide (162 mg, 0.63 mmol), stirred at 110 °C

for 1 h, and allowed to cool to rt. 3,4-Diaminopyridine

(53 mg, 0.49 mmol in DMF) was added and temperature

was raised to 110 °C for 2 h. The crystal precipitated in

the reaction mixture was ﬁltered. The titled compound

was obtained as colorless solid (35 mg, 23%). Mp 249–

250 °C. ¹H NMR (DMSO, 400 MHz) d 4.53 (d,

J ¼ 5:6 Hz, 2H), 5.85 (br s, 2H), 6.50 (t, J ¼ 5:4 Hz,

1H), 6.60 (d, J ¼ 5:6 Hz, 1H), 7.30–7.35 (m, 1H), 7.46

(d, J ¼ 8:3 Hz, 2H), 7.64 (br s, 1H), 7.78 (d, J ¼ 5:6 Hz,

1H), 8.11 (s, 1H). MS (ESI) m=z 310 (Mꢀ1)^ꢀ.

12.9.20. N-(2-Amino-4-pyridyl)-N⁰-(2,6-dichlorophenyl)-

urea (7e). Compound 7e was prepared from 2,4-dia-

mino-pyridine and 2,6-dichlorophenylisocyanate, in a

manner similar to that described for 4a as a colorless

solid. Mp 271–273 °C. NMR (CD₃OD, 400 MHz) d 4.50

(s, 2H), 7.03 (t, J ¼ 8:0 Hz, 1H), 7.29 (d, J ¼ 8:0 Hz,

2H), 7.40 (m, 2H), 7.77 (m, 1H); MS (ESI) m=z 296

(Mꢀ1)^ꢀ.

12.9.21.

N-(2,6-Dichlorophenyl)-N,N⁰-dimethyl-N⁰-(4-

pyridyl)urea (8). To a solution of N-(4-pyridyl)-N⁰-(2,6-

dichlorophenyl)urea (4b) (50 mg, 0.18 mmol) in DMF

(2 mL) was added sodium hydride (8.5 mg, 0.35 mmol)

portionwise at rt. The reaction mixture was stirred at rt

for 30 min and followed by addition of methyliodide

(28 mg, 0.20 mmol). The resulting mixture was stirred at

110 °C for 1 h and quenched by addition of water. The

aqueous layer was extracted with EtOAc. The combined

organic layer was washed with saturated NaCl aq, dried

over anhydrous Na₂SO₄, and concentrated in vacuo.

The crude material was puriﬁed by silica gel column

chromatography, eluting with CHCl₃–MeOH to aﬀord

12.9.25.

N1-(2-Chloro-4-pyridyl)-2-(2,6-dichlorophen-

oxy)acetamide (11). (1) 2,6-Dichloro-phenoxyacetic

acid was prepared from 2,6-dichlorophenol in a manner

similar to that described for 2,6-dichloro-4-ﬂuorophen-

oxyacetic acid with a yield of 79% as a colorless solid.

(2) The compound 11 was prepared from 4-amino-2-

chloropyridine in a manner similar to that described for

1

the compound 6a with a yield of 10% as a dense oil. H

NMR (CD₃OD, 400 MHz) d 7.03 (t, J ¼ 8:0 Hz, 1H),

7.29 (d, J ¼ 8:0, 2H), 7.40 (m, 2H), 7.77 (m, 1H).

1

the title compound (79 mg, 76.1%) as a colorless oil. H

NMR (CD₃OD, 400 MHz) d 3.06 (s, 3H), 3.09 (s, 3H),

6.93 (d, J ¼ 5:1 Hz, 2H), 7.01 (t, J ¼ 8:3 Hz, 1H), 7.16

(d, J ¼ 8:3 Hz, 2H), 8.11 (d, J ¼ 5:1 Hz, 2H). MS (ESI)

m=z 311 (Mþ1)^þ.

12.9.26.

N1-(1H-5-Indazolyl)-1-cyclohexanecarboxy-

amide (12a). To a solution of cyclohexanecarboxylic

acid (96 mg, 0.75 mmol) in DMF (1 mL) was added 5-

amino-1H-indazole (100 mg, 0.75 mmol), WSC–HCl

(173 mg, 0.9 mmol) and HOBt–H₂O (122 mg,

0.75 mmol). The reaction mixture was stirred for 16 h at

rt. Water was added to the reaction mixture and

extracted with EtOAc. The EtOAc layer was washed

with water and saturated NaCl aq, dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated. The title compound

was obtained (80 mg, 44% yield) by silica gel column

chromatography (CHCl₃–MeOH 95:5) Mp 230 °C (dp).

¹H NMR (CDCl₃, 400 MHz) d 1.14–1.34 (m, 3H), 1.44

(q, J ¼ 11:4 Hz, 2H), 1.62–1.69 (m, 1H), 1.73–1.84 (m,

4H), 2.33 (tt, J ¼ 3:4 Hz, 11.6 Hz, 1H), 7.42 (dd,

J ¼ 1:5 Hz, 9.0 Hz, 1H), 7.45 (d, J ¼ 9:0 Hz, 1H), 7.98

(s, 1H), 8.12 (s, 1H), 9.76 (s, 1H), 12.92 (s, 1H). MS

(ESI) m=z 242 (Mꢀ1)^ꢀ.

12.9.22. O-(4-Pyridylmethyl)-N-(2-chlorobenzyl)carba-

mate (9a). Compound 9a was prepared from 2-chlor-

ophenylacetic acid in a manner similar to that described

for 9b with a yield of 44% as a colorless solid. Mp 75 °C.

¹H NMR (CDCl₃–CD₃OD¼25:1, 400 MHz) d 4.49 (d,

J ¼ 6:4 Hz, 2H), 5.13 (s, 2H), 5.42 (br, 1H), 7.22–7.28

(m, 4H), 7.36–7.42 (m, 2H), 8.55–8.60 (m, 2H). MS

(ESI) m=z 275 (Mꢀ1)^ꢀ.

12.9.23. O-(4-Pyridylmethyl)-N-(2,6-dichlorophenyl)car-

bamate (9b). To a solution of 2,6-dichlorobenzoic acid

(150 mg, 0.78 mmol) in toluene (1 mL) was added tri-

ethylamine (0.13 mL, 0.86 mmol), diphenylphosphoryl-

azide (0.2 mL, 0.93 mmol) and stirred at 110 °C for 1 h

and allowed to cool to rt. 4-Pyridylcarbinol (86 mg,

0.78 mmol) was added and temperature was raised to

110 °C for 2 h. Water was added to the reaction mixture

and the mixture was extracted with EtOAc. The EtOAc

layer was washed with water and saturated NaCl aq,

dried over anhydrous Na₂SO₄, and dried up in vacuo.

The residue was puriﬁed by a silica gel column chro-

matography (CHCl₃–MeOH) to give the title compound

as a colorless solid (136 mg, 58% yield). Mp 114–116 °C.

¹H NMR (CDCl₃–CD₃OD¼25:1, 400 MHz) d 5.24 (s,

2H), 6.69 (br s, 1H), 7.20 (t, J ¼ 8:2 Hz, 1H), 7.23–7.28

(m, 2H), 7.39 (d, J ¼ 8:1 Hz, 2H), 8.60 (d, J ¼ 5:9 Hz,

2H). MS (ESI) m=z 296 (Mꢀ1)^ꢀ.

12.9.27.

N1-(1H-5-Indazolyl)-1-cyclohexylacetamide

(12b). Compound 12b was prepared from cyclohexane

acetic acid in a manner similar to that described for 12a

with a yield of 36% as a yellow solid. Mp 227–228 °C. ¹H

NMR (CDCl₃, 400 MHz) d 0.92–1.04 (m, 2H), 1.10–1.30

(m, 3H), 1.58–1.82 (m, 6H), 2.19 (d, J ¼ 7:1 Hz, 2H),

7.39 (dd, J ¼ 1:7 Hz, 9.0 Hz, 1H), 7.45 (d, J ¼ 8:8 Hz,

1H), 7.98 (s, 1H), 8.11 (s, 1H), 9.81 (s, 1H), 12.92 (s,

1H,). MS (ESI) m=z 256 (Mꢀ1)^ꢀ.

12.9.28. N1-(1H-5-Indazolyl)-2-(2,6-dichloro-4-ﬂuoro-

phenoxy)acetamide (12c). To a mixture of 2,6-dichloro-

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2129

4-ﬂuorophenol (1.39 g, 7.69 mmol) and K₂CO₃(1.40 g,

9.22 mmol) in acetonitrile (10 mL) was added methyl

2-bromoacetate (1.18 g, 7.69 mmol) dropwise at rt. The

reaction mixture was stirred at 80 °C for 1 h, and evap-

orated for removal of acetonitrile. Water was added to

the reaction mixture, and the aqueous layer was

extracted with EtOAc. The EtOAc layer was washed

with water, dried over anhydrous Na₂SO₄, and con-

centrated in vacuo. The residue was dissolved in EtOH

(10 mL) followed by addition of 10% NaOH aq (5 mL).

The mixture was stirred at 80 °C for 1 h and evaporated.

The resulting mixture was acidiﬁed by 5% HCl aq. The

aqueous layer was extracted with EtOAc. The combined

organic layer was dried over anhydrous Na₂SO₄

and ﬁltered. The ﬁltrate was concentrated in vacuo to

give 2,6-dichloro-4-ﬂuorophenoxyacetic acid (1.79 g,

7.48 mmol). To a mixture of 2,6-dichloro-4-ﬂuorophen-

oxyacetic acid (197 mg, 0.83 mmol), 5-amino-1H-indaz-

ole (100 mg, 0.75 mmol), HOBt–H₂O (122 mg,

0.90 mmol), and dimethylaminopyridine (5 mg) in DMF

(3 mL) was added WSC–HCl (178 mg, 0.90 mmol)

dropwise at 0 °C. The reaction mixture was stirred at rt

for 18 h and quenched by addition of saturated

NaHCO₃aq. The aqueous layer was extracted with

EtOAc three times. The combined organic layer was

dried over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate

was concentrated in vacuo. The residual oil was sub-

jected to ﬂash chromatography on silica gel, eluting with

CHCl₃–MeOH (95:5), to give the title compound

7.59 (s, 1H), 7.63 (s, 1H), 8.12 (s, 1H); (ESI) m=z 353

(Mꢀ1)^ꢀ.

12.9.32. N-(1H-5-Indazolyl)-N⁰-(2,6-dichlorophenyl)urea

(14a). 5-Amino-1H-indazole (100 mg, 0.75 mmol) and

2,6-dichlorophenylisocyanate (155 mg, 0,83 mmol) was

dissolved in toluene and stirred at 110 °C for 4 h. Solvent

was removed and the resulting residue was washed with

n-hexane to give the title compound (232 mg, 97% yield)

1

as a colorless solid. Mp 275 °C (dp). H NMR (CDCl₃,

400 MHz) d 7.29 (d, J ¼ 8:8 Hz, 1H), 7.33 (dd,

J ¼ 1:7 Hz, 8.8 Hz, 1H), 7.45 (d, J ¼ 8:8 Hz, 1H), 7.52

(d, J ¼ 8:8 Hz, 1H), 7.89 (dd, J ¼ 1:7 Hz, 8.8 Hz, 1H),

7.96 (s, 1H), 8.14 (s, 1H), 8.86 (s, 1H). MS (ESI) m=z 320

(Mꢀ1)^ꢀ.

12.9.33. N-(1H-5-Indazolyl)-N⁰-(benzyl)urea (14b). To a

solution of 5-amino-1H-indazole (66 mg, 0.50 mmol)

dissolved in toluene (1 mL) and trace amount of DMF,

benzylisocyanate (0.061 mL, 0.5 mmol) was added and

stirred at 110 °C for 3 h. Water was added to the reac-

tion mixture and extracted with EtOAc. The EtOAc

layer was washed with water and saturated NaCl aq,

dried over anhydrous Na₂SO₄, ﬁltered, and concen-

trated. The crude material was puriﬁed by silica gel

column chromatography (CHCl₃–MeOH 95:5) to give

the title compound (21 mg, 16% yield) as a colorless

1

(210 mg, 0.59 mmol, 79% yield) as a colorless solid. H

solid. ¹H NMR (CDCl₃, 400 MHz)

d 4.31 (d,

NMR (CDCl₃, 400 MHz) d 4.62 (s, 2H), 7.49 (t,

J ¼ 8:8 Hz, 1H), 7.53 (dd, J ¼ 1:7 Hz, 8.8 Hz, 1H), 7.59

(s, 1H), 7.61 (s, 1H), 8.03 (s, 1H), 8.16 (s, 1H), 10.06 (s,

1H), 12.99 (s, 1H). MS (ESI) m=z 353 (Mꢀ1)^ꢀ.

J ¼ 5:9 Hz, 2H), 6.53 (t, J ¼ 5:9 Hz, 1H), 7.21–7.28 (m,

2H), 7.29–7.36 (m, 4H), 7.41 (d, J ¼ 8:8 Hz, 1H), 7.85 (s,

1H), 7.93 (s, 1H,), 8.46 (s, 1H), 12.84 (s, 1H). MS (ESI)

m=z 265 (Mꢀ1)^ꢀ.

12.9.29.

N1-(1H-6-Indazolyl)-1-cyclohexanecarboxy-

12.9.34. N-(1H-6-Indazolyl)-N⁰-(2,6-dichlorophenyl)urea

(15). Compound 15 was prepared from 6-amino-1H-

indazole in a manner similar to that described for 14a as

a yellow solid. Mp 264–267 °C. NMR (CD₃OD,

400 MHz) d 6.91 (dd, J ¼ 7:9 Hz, 1.8Hz, 1H), 7.19 (t,

J ¼ 8:0 Hz, 1H), 7.39 (d, J ¼ 8:0 Hz, 2H), 7.58 (dd,

J ¼ 7:9 Hz, 0.8 Hz, 1H), 7.84 (m, 1H), 7.85 (s, 1H); MS

(ESI) m=z 320 (Mꢀ1)^ꢀ.

amide (13a). Compound 13a was prepared from 6-ami-

no-1H-indazole in a manner similar to that described for

12a with a yield of 10% as a colorless solid. Mp 230 °C

(dp). ¹H NMR (CDCl₃, 400 MHz) d 1.10–1.82 (m, 11H),

7.07 (d, J ¼ 8:8 Hz, 1H), 7.62 (d, J ¼ 8:5 Hz, 1H), 7.93

(s, 1H), 8.11 (s, 1H), 9.94 (s, 1H), 12.87 (br, 1H). MS

(ESI) m=z 242 (Mꢀ1)^ꢀ.

12.9.30.

N1-(1H-6-Indazolyl)-1-cyclohexylacetamide

12.9.35. N-(1H-5-Indazolyl)-N-(4-piperizyl)amine (16a).

To

(13b) . Compound 13b was prepared from 6-amino-1H-

indazole and cyclohexaneacetic acid in a manner similar

to that described for 12a with a yield of 30% as a yellow

solid. Mp 192–193 °C. H NMR (CDCl₃, 400 MHz) d

0.94–1.85 (m, 11H), 2.23 (d, J ¼ 7:1 Hz, 2H), 7.09 (dd,

J ¼ 1:7 Hz, 8.5 Hz, 1H), 7.66 (d, J ¼ 8:5 Hz, 1H), 7.97

(s, 1H), 8.16 (s, 1H), 10.03 (br, 1H), 12.91 (br, 1H). MS

(ESI) m=z 256 (Mꢀ1)^ꢀ.

a

suspension of 4-hydroxypiperizine (1.01 g,

10 mmol) in 3 M NaOH aq (10 mL) was added a solu-

tion of di-tert-butyl dicarbonate (2.40 g, 11 mmol) in

THF (10 mL) dropwise at 0 °C. The reaction mixture

was stirred at rt for 1 h and evaporated for removal of

THF. The aqueous layer was extracted with EtOAc. The

organic layer was dried over anhydrous Na₂SO₄and

ﬁltered. The ﬁltrate was concentrated in vacuo to give

4-(tert-butyloxycarbonyl)piperizinone. To the piperizi-

none, triethylamine (2 mL) in anhydrous DMSO

(10 mL) was added followed by addition of sulfur tri-

oxide–trimethylamine complex (4.44 g, 20 mmol) por-

tionwise at 0 °C. The reaction mixture was stirred at rt

for 18 h and quenched by addition of saturated

NaHCO₃aq. The aqueous layer was extracted with

EtOAc. The EtOAc layer was dried over anhydrous

1

12.9.31.

N1-(1H-6-Indazolyl)-2-(2,6-dichloro-4-ﬂuoro-

phenoxy)acetamide (13c). Compound 13c was prepared

from 6-amino-1H-indazole in a manner similar to that

described for 12c as a yellow solid. Mp 219–221 °C. H

NMR (DMSO-d₆, 400 MHz) d 5.43 (s, 2H), 6.69 (d,

J ¼ 8:5 Hz, 1H), 7.46 (s, 1H), 7.47 (d, J ¼ 8:5 Hz, 1H),

1

2130

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

Na₂SO₄and ﬁltered. The ﬁltrate was concentrated in

vacuo. To the residue, 5-amino-1H-indazole (0.98 g,

7.4 mmol) dissolved in MeOH (10 mL) and AcOH (0.

2 mL) were added. To the solution, BH₃–pyridine

complex (1.0 mL, 10 mmol) was added dropwise at 0 °C.

The reaction mixture was stirred at rt for 18 h and

quenched by addition of saturated NaHCO₃aq. The

aqueous layer was extracted with CHCl₃. The CHCl₃

layer was dried over anhydrous Na₂SO₄and ﬁltered.

The ﬁltrate was concentrated in vacuo. The residual oil

was subjected to ﬂash chromatography on silica gel,

eluting with CHCl₃–MeOH (95:5), to give tert-butyl 4-

(1H-5-indazol-ylamino)-1-piperizinecarboxylate as yel-

low dense oil (2.30 g, 7.20 mmol, 72% yield in three

steps).

12.9.39. N-(1-Benzyl-4-piperidyl)-N-(1H-5-indolyl)amine

(18). Compound 18 was prepared from 5-amino-1H-

indole in a manner similar to that described for 16c with

a yield of 73% as a yellow solid. ¹H NMR (CDCl₃,

400 MHz) d 1.5–1.7 (m, 4H), 1.9–2.1 (m, 2H), 2.80 (m,

2H), 3.25 (m, 1H), 3.49 (br s, 2H), 6.31 (m, 1H), 6.54

(dd, J ¼ 8:6 Hz, 2.1 Hz, 1H), 6.78 (d, J ¼ 2:1 Hz, 1H),

7.05 (dd, J ¼ 2:6 Hz, 1H), 7.13 (d, J ¼ 8:6 Hz, 1H), 7.2–

7.3 (m, 5H), 7.84 (br s, 1H). MS (ESI) m=z 306 (Mþ1)^þ.

12.9.40.

N-(1-Benzyl-3-piperidyl)-N-(6-ﬂuoro-1H-5-

indazolyl)amine (19a). (1) 5-Amino-6-ﬂuoro-1H-indaz-

ole. To a mixture of 5-ﬂuoro-o-toluidine (6.25 g,

50 mmol) and potassium acetate (6.00 g, 60 mmol) in

CHCl₃(50 mL) was added acetic acid anhydride (15.0 g,

150 mmol) dropwise at 0 °C. The mixture was stirred at

rt for 30 min, heated to 40 °C, and followed by addition

of isoamyl nitrite (11.7 g, 100 mmol) dropwise at 40 °C.

The reaction mixture was stirred at 60 °C for 18 h and

quenched by addition of saturated NaHCO₃aq. The

aqueous layer was extracted with CHCl₃. The organic

layer was dried over anhydrous Na₂SO₄and ﬁltered.

The ﬁltrate was concentrated in vacuo. The residual oil

was subjected to chromatography on silica gel, eluting

with CHCl₃–MeOH (9:1) to give 6-ﬂuoro-1-acetyl-

indazole.

To a solution of tert-butyl-4-(1H-5-indazolylamino)-1-

piperizinecarboxylate (450 mg, 1.42 mmol) in CHCl₃

(3 mL) was added 95% triﬂuoroacetic acid (3 mL)

dropwise at rt. The reaction mixture was stirred at rt for

3 h and concentrated in vacuo to give the title compound

as a colorless solid having mp of 172–176 °C (420 mg,

1

1.32 mmol, 93% yield in this step). H NMR (CDCl₃,

400 MHz) d 1.29 (m, 2H), 1.95 (m, 2H), 2.59 (m, 2H),

2.98 (m, 2H), 3.22 (m, 1H), 6.82 (s, 1H), 6.86 (d,

J ¼ 8:8 Hz, 1H), 7.24 (d, J ¼ 8:8 Hz, 2H), 7.73 (s, 1H);

MS (ESI) m=z 217 (Mþ1)^þ.

A mixture of the residue in HCl–MeOH (200 mL) was

stirred at 80 °C for 5 h and evaporated for removal of

HCl–MeOH. The resulting residue was basiﬁed by sat-

urated NaHCO₃aq. The aqueous layer was extracted

with EtOAc. The organic layer was dried over anhy-

drous Na₂SO₄and ﬁltered. The ﬁltrate was concentrated

in vacuo. The precipitate was washed with CHCl₃to

give 6-ﬂuoro-1H-indazole as a brown solid (4.80 g,

35 mmol, 70% yield).

12.9.36.

N-(1-Ethyl-4-piperidyl)-N-(1H-5-indazolyl)-

amine (16b). Compound 16b was prepared from

1-ethyl-4-piperidone in a manner similar to that

described for 16c with a yield of 41% as a yellow dense

1

oil. H NMR (CDCl₃, 400 MHz) d 1.06 (t, J ¼ 7:3 Hz,

3H), 1.42–1.53 (m, 2H), 2.02–2.17 (m, 4H), 2.42 (q,

J ¼ 7:1 Hz, 2H), 2.86–2.95 (m, 2H), 3.21–3.33 (m, 1H),

6.70–6.76 (m, 2H), 7.23 (d, J ¼ 8:5 Hz, 1H), 7.81 (s, 1H).

MS (ESI) m=z 245 (Mþ1)^þ.

To a mixture of 6-ﬂuoro-1H-indazole (4.80 g, 35 mmol)

in 50% H₂SO₄aq (20 mL) was added sodium nitrate

(3.40 g, 40 mmol) portionwise at 0 °C. The resulting

mixture was stirred at rt for 1 h, and quenched by

addition of saturated NaHCO₃aq. The aqueous layer

was extracted with EtOAc. The organic layer was dried

over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate was

concentrated in vacuo. The residual solid was washed

with CHCl₃to give 5-nitro-6-ﬂuoro-1H-indazole (3.00 g,

16 mmol).

12.9.37.

N-(1-Benzyl-4-piperidyl)-N-(1H-5-indazolyl)-

amine (16c). To a solution of 1-benzylpiperidone

(635 mg, 3.4 mmol), 5-amino-1H-indazole (532 mg,

4 mmol), and AcOH (0.20 mL) in MeOH (10 mL) was

added BH₃–pyridine complex (0.51 mL, 5 mmol) drop-

wise at 0 °C. The reaction mixture was stirred at rt for

18 h and quenched by addition of saturated NaHCO₃

aq. The aqueous layer was extracted with CHCl₃. The

CHCl₃layer was dried over anhydrous Na₂SO₄and

ﬁltered. The ﬁltrate was concentrated in vacuo. 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, 3.26 mmol, 82%

To a mixture of 5-ﬂuoro-5-nitro-1H-indazole (2.00 g,

11 mmol) in 50% HCl aq (10 mL) was added

tin(II)chloride dihydrate (4.50 g, 20 mmol) portionwise

at 0 °C. The resulting mixture was stirred at rt for 5 h,

and quenched by addition of saturated NaHCO₃aq.

The aqueous layer was extracted with EtOAc. The

organic layer was dried over anhydrous Na₂SO₄and

ﬁltered. The ﬁltrate was concentrated in vacuo. The

residual solid was washed with CHCl₃to give 5-amino-

6-ﬂuoro-1H-indazole (1.67 g, 11 mmol).

1

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 (Mþ1)^þ.

(2) Compound 19a was prepared from 5-amino-6-ﬂu-

oro-1H-indazole in a manner similar to that described

for 16c with a yield of 52% as a yellow solid. Mp 137–

12.9.38. N-(1-Ethyl-4-piperidyl)-N-(1H-6-indazolyl)-

amine (17). Purchased from ChemBridge.

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2131

1

140 °C. H NMR (CDCl₃, 400 MHz) d 1.52–1.64 (m,

was dried over anhydrous Na₂SO₄and ﬁltered. The ﬁl-

trate was concentrated in vacuo. The residual oil was

subjected to chromatography on silica gel, eluting with

CHCl₃, to give the title compound as yellow dense oil

(50 mg, 0.15 mmol, 75% yield). ¹H NMR (CDCl₃,

400 MHz) d 0.99 (br s, 3H), 1.3–2.2 (m, 8H), 3.05 (m,

2H), 3.20 (br s, 2H), 3.30 (m, 1H), 7.03 (s, 1H), 7.05 (s,

1H), 7.2–7.3 (m, 6H), 7.87 (s, 1H). MS (ESI) m=z 335

(Mþ1)^þ.

2H), 2.09 (br d, J ¼ 10:0 Hz, 2H), 2.23 (br t,

J ¼ 10:8 Hz, 2H), 2.91 (br d, J ¼ 11:6 Hz, 2H), 3.32 (br

s, 1H), 3.58 (s, 2H), 6.83 (d, J ¼ 8:0 Hz, 1H), 7.08 (d,

J ¼ 13:6 Hz, 1H), 7.23–7.27 (m, 1H), 7.28–7.35 (m, 4H),

7.88 (s, 1H). MS (ESI) m=z 325 (Mþ1)^þ.

12.9.41. N-(1-Benzyl-4-piperidyl)-N-(6-methoxy-1H-5-

indazolyl)amine (19b). (1) 5-Amino-6-methoxy-1H-in-

dazole. The compound was prepared from 5-methoxy-2-

methylaniline in a manner similar to that described for

the compound, 5-amino-6-ﬂuoro-1H-indazole, with a

yield of 75% as a brown solid.

12.9.44.

N-(1-Benzyl-4-piperidyl)-N-(3-methyl-1H-5-

indazolyl)amine (19e). (1) 5-Amino-3-methyl-1H-indaz-

ole. To a mixture of 2-aminoacetophenone (1.35 g,

10 mmol) in 50% H₂SO₄aq (20 mL) was added sodium

nitrite (0.82 g, 12 mmol) portionwise at 0 °C. The

resulting mixture was stirred at rt for 1 h, and followed

by addition of tin(II)chloride dehydrate (6.76 g,

30 mmol). The reaction mixture was stirred at 0 °C for

1 h and diluted by water. The white solid was collected

by ﬁltration, and dried up in vacuo to give 3-methyl-1H-

indazole (1.20 g, 9 mmol). The introduction of the amino

group at the 5-position was performed in a manner

similar to that described for the compound, 5-amino-6-

ﬂuoro-1H-indazole, with a yield of 88% as brown solid.

(2) Compound 19b was prepared from 5-amino-6-

methoxy-1H-indazole in a manner similar to that

described for 16c with a yield of 40% as a yellow solid.

Mp 161–164 °C. H NMR (CDCl₃, 400 MHz) d 1.51–

1.64 (m, 2H), 2.11 (br d, J ¼ 11:2 Hz, 2H), 2.23 (br t,

J ¼ 10:8 Hz, 2H), 2.90 (br d, J ¼ 11:6 Hz, 2H), 3.32 (br

s, 1H), 3.57 (s, 2H), 3.86 (s, 3H), 6.71 (s, 1H), 6.75 (s,

1H), 7.23–7.29 (m, 1H), 7.30–7.40 (m, 4H), 7.81 (s, 1H).

MS (ESI) m=z 337 (Mþ1)^þ.

1

(2) Compound 19e was prepared from 5-amino-3-me-

thyl-1H-indazole in a manner similar to that described

for 16c with a yield of 55% as yellow dense oil. ¹H NMR

(CDCl₃, 400 MHz) d 1.50–1.75 (m, 2H), 2.00–2.11 (m,

2H), 2.15–2.40 (m, 2H), 2.56 (s, 3H), 2.89–3.00 (m, 2H),

3.25–3.37 (m, 1H), 3.57–3.67 (m, 2H), 6.51 (t,

J ¼ 1:9 Hz, 1H), 6.81 (dd, J ¼ 1:9 Hz, 9.2 Hz, 1H), 7.23–

7.32 (m, 5H), 7.47 (d, J ¼ 9:0 Hz, 1H). MS (ESI) m=z

321 (Mþ1)^þ.

12.9.42. N1-(1-Benzyl-4-piperidyl)-N1-(1H-5-indazolyl)-

acetamide (19c). To a mixture of N-(1-benzyl-4-piper-

idyl)-N-(1H-5-indazolyl)amine (307 mg, 1.0 mmol) and

pyridine (220 mg, 2.4 mmol) in CHCl₃(1 mL) was added

acetylchloride (170 mg, 2.4 mmol) dropwise at rt. The

reaction mixture was stirred at rt for 18 h, and quenched

by addition of saturated NaHCO₃aq. The aqueous

layer was extracted with CHCl₃. The CHCl₃layer was

dried over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate

was concentrated in vacuo. The residual was dissolved

in MeOH (1 mL) and HCl–MeOH (1 mL) was added.

The reaction mixture was stirred at rt for 5 h and basi-

ﬁed by addition of saturated NaHCO₃aq. The aqueous

layer was extracted with EtOAc. The organic layer was

dried over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate

was concentrated in vacuo. The residual oil was sub-

jected to ﬂash chromatography on silica gel, eluting with

CHCl₃, to give the title compound as yellow dense oil

(321 mg, 0.95 mmol, 95% yield). ¹H 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 ¼ 12:7 Hz, 1H),

3.55 (d, J ¼ 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 (Mþ1)^þ.

12.9.45. N-(1-Benzyl-4-piperidyl)-N-(1-methyl-5-indaz-

olyl)amine (19f). Compound 19f was prepared from N-

(1-benzyl-4-piperidyl)-N-(1-H-5-indazolyl)amine (16c)

in a manner similar to that described for 21b as yellow

1

dense oil. H NMR (CDCl₃, 400 MHz) d 1.5–1.8 (m,

4H), 2.0–2.2 (m, 2H), 2.8–2.9 (m, 2H), 3.2–3.3 (m, 1H),

3.5–3.6 (br s, 2H), 3.98 (s, 3H), 6.76 (m, 1H), 6.78 (dd,

J ¼ 8:6 Hz, 2.2 Hz, 1H), 7.18 (d, J ¼ 8:6 Hz, 1H), 7.2–

7.3 (m, 5H), 7.75 (s, 1H). MS (ESI) m=z 321 (Mþ1)^þ.

12.9.46.

N-(1-Propyl-3-piperidyl)-N-(1H-5-indazolyl)-

amine (20a). 3-Hydroxypiperidine (1.0 g, 9.8 mmol),

K₂CO₃(2.76 g, 41 mmol), and 1-bromopropane (1.23 g,

10 mmol) were stirred in acetonitrile (10 mL) for 12 h at

rt, and quenched by water. The aqueous layer was

extracted with EtOAc. The EtOAc layer was washed

with saturated NaCl aq, dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo to give N-propyl-3-hydorxypy-

peridine (1.10 g, 41 mmol). To a mixture of the crude N-

propyl-3-hydorxypyperidine and triethyl amine (1.45 g,

15 mmol) in anhydrous DMSO (15 mL) was added sul-

fur trioxide trimethylamine complex (2.13 g, 15 mmol)

and the reaction mixture was stirred for 12 h. Water was

added to the reaction mixture and extracted with

EtOAc. The EtOAc layer was washed with saturated

12.9.43. N1-(1-Benzyl-4-piperidyl)-N1-(1H-5-indazolyl)-

ethylamine (19d). To a mixture of N1-(1-benzyl-4-pip-

eridyl)-N1-(1H-5-indazolyl)acetamide (19c) (70 mg,

0.2 mmol) in THF (1 mL) was added 1M BH₃/THF

(0.06 mL, 0.6 mmol) dropwise at rt. The reaction mix-

ture was stirred at 60 °C for 3 h, and quenched by

addition of 1 N HCl aq at 0 °C. The resulting mixture

was stirred at 60 °C for 1 h and cooled to rt. The aque-

ous layer was extracted with CHCl₃. The CHCl₃layer

2132

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

NaCl aq, dried over anhydrous Na₂SO₄, and concen-

trated in vacuo. The crude mixture was puriﬁed by silica

gel column chromatography (hexane–EtOAc 1:1) to give

N-propyl-3-piperidone (213 mg, 1.5 mmol). A mixture of

the N-propyl-3-piperidone and 5-aminoindazole

(200 mg, 1.5 mmol) in Ti(O-i-Pr)₄(3 mL) was stirred for

1 h and followed by addition of MeOH (3 mL) and

sodium borohydride (100 mg, 2.8 mmol). The reaction

mixture was quenched by addition of water at 0 °C. The

reaction mixture was extracted with CHCl₃. The CHCl₃

layer was washed with saturated NaCl aq, dried over

anhydrous Na₂SO₄, and concentrated in vacuo. The

residue was puriﬁed by silica gel column chromatogra-

phy (CHCl₃–MeOH 9:1) to give the title compound

J ¼ 2:0 Hz, 1H), 673 (dd, J ¼ 2:2 Hz, 8.8 Hz, 1H), 7.14

(d, J ¼ 8:8 Hz, 1H), 7.22–7.30 (m, 5H). MS (ESI) m=z

321 (Mþ1)^þ.

12.9.51. N-(1-Benzyl-3-piperidyl)-N-(1-methyl-5-indaz-

olyl)amine (21b). A mixture of N-(1-benzyl-3-piper-

idyl)-N-(1H-5-indazolyl)amine (20b) (100 mg, 0.33

mmol), K₂CO₃(50 mg, 0.36 mmol), and methyliodide

(0.02 mL, 0.33 mmol) in DMF (2 mL) was stirred at

110 °C for 1 h. The resulting mixture was diluted with

water. The aqueous layer was extracted with EtOAc.

The organic layer was dried over anhydrous Na₂SO₄

and ﬁltered. The ﬁltrate was concentrated in vacuo. The

residual oil was subjected to chromatography on silica

gel, eluting with CHCl₃, to give the title compound as

yellow dense oil (79 mg, 0.25 mmol, 76% yield). ¹H

NMR (CDCl₃, 400 MHz) d 1.4–1.6 (m, 4H), 1.6–1.8 (m,

2H), 2.3–2.4 (m, 2H), 3.4–3.6 (m, 3H), 3.93 (s, 3H), 6.71

(m, 1H), 6.77 (m, 1H), 7.13 (d, J ¼ 8:6 Hz, 1H), 7.2–7.3

(m, 5H), 7.68 (s, 1H). MS (ESI) m=z 321 (Mþ1)^þ.

1

(100 mg, 3.8%, total yield) as pale-yellow dense oil. H

NMR (CDCl₃, 400 MHz) d 0.90 (t, J ¼ 7:4 Hz, 3H),

1.59 (m, 4H), 2.11 (t, J ¼ 12:0 Hz, 2H), 2.18 (t,

J ¼ 10:0 Hz, 2H), 2.38 (dd, J ¼ 9:8 Hz, 2H), 2.97 (br d,

J ¼ 11:4 Hz, 2H), 3.33 (tt, J ¼ 9:8 Hz, 11.4 Hz, 1H),

6.76 (d, J ¼ 9:0 Hz, 1H), 6.81 (d, J ¼ 1:3 Hz, 1H), 7.25

(d, J ¼ 9:0 Hz, 1H), 7.88 (d, J ¼ 1:3 Hz, 1H). MS (ESI)

m=z 259 (Mþ1)^þ.

12.9.52. N-(1-Benzyl-4-piperidyl)-N-(5-isoquinolyl)amine

(22a). To a solution of 1-benzylpiperidone (150 mg,

0.79 mmol), 5-aminoisoquinoline (114 mg, 0.79 mmol),

and AcOH (1 drop) in MeOH (5 mL) was added BH₃–

pyridine complex (0.10 mL, 1 mmol) dropwise at 0 °C.

The reaction mixture was stirred at rt for 18 h and

quenched by addition of saturated NaHCO₃aq. The

aqueous layer was extracted with CHCl₃. The CHCl₃

layer was dried over anhydrous Na₂SO₄and ﬁltered.

The ﬁltrate was concentrated in vacuo. The residual oil

was subjected to ﬂash chromatography on silica gel,

eluting with CHCl₃–MeOH (95:5), to give the title

compound (47 mg, 0.15 mmol, 20% yield) as yellow

12.9.47.

N-(1-Benzyl-3-piperidyl)-N-(1H-5-indazolyl)-

amine (20b). Compound 20b was prepared from 1-ben-

zyl-3-piperidone in a manner similar to that described

for 16c with a yield of 78% as yellow dense oil. ¹H NMR

(CDCl₃, 400 MHz) d 1.40–1.60 (m, 3H), 1.70–1.80 (m,

2H), 2.10–2.50 (m, 3H), 3.45 (s, 2H), 3.16–3.22 (m, 1H),

6.75 (dd, J ¼ 2:4 Hz, 11.2 Hz, 2H), 7.15–7.28 (m, 6H),

7.78 (d, J ¼ 0:7 Hz, 1H). MS (ESI) m=z 305 (Mꢀ1)^ꢀ.

12.9.48. N-(1H-5-Indazolyl)-N-tetrahydro-1H-3-pyrro-

rylamine (20c). Compound 20c was prepared from 3-

pyrrolidinol in a manner similar to that described for

1

dense oil. H NMR (CDCl₃, 400 MHz) d 1.5–1.7 (m,

1

16a with a yield of 45% as yellow dense oil. H NMR

4H), 2.1–2.2 (m, 2H), 2.2–2.3 (m, 2H), 3.59 (s, m, 3H),

6.77 (d, J ¼ 7:8 Hz, 1H), 7.30 (d, J ¼ 7:8 Hz, 1H), 7.2–

7.4 (m, 5H), 7.43 (dd, J ¼ 7:8 Hz, 1H), 7.52 (d,

J ¼ 6:0 Hz, 1H), 8.46 (d, J ¼ 6:0 Hz, 1H), 9.14 (s, 1H).

MS (ESI) m=z 318 (Mþ1)^þ.

(CDCl₃, 400 MHz) d 1.6–1.9 (m, 2H), 2.53 (m, 1H), 2.64

(m, 1H), 2.82 (m, 1H), 3.26 (m, 1H), 3.39 (m, 1H), 6.75

(d, J ¼ 6:8 Hz, 1H), 6.78 (s, 1H), 7.26 (d, J ¼ 6:8 Hz,

1H), 7.80 (s, 1H); (ESI) m=z 203 (Mþ1)^þ.

12.9.49. N-(1-Benzyltetrahydro-1H-pyrrolyl)-N-(1H-5-

indazolyl)amine (20d). Compound 20d was prepared

from 1-benzyl-3-pyrrolidone in a manner similar to that

described for 16c with a yield of 35% as yellow dense oil.

¹H NMR (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), 7.14–7.28 (m, 6H), 7.88

(s, 1H). MS (ESI) m=z 293 (Mþ1)^þ.

12.9.53. N-(5-Isoquinolyl)-N-(1-propyl-4-piperidyl)amine

(22b). To a mixture of 4-piperidone monohydrate

(300 mg, 2.6 mmol) and K₂CO₃(540 mg, 3.9 mmol) in

acetonitrile (5 mL) was added a solution of n-propylbr-

omide (359 mg, 2.7 mmol) in acetonitrile (2 mL) drop-

wise at 0 °C. The reaction mixture was stirred at rt for

18 h and diluted with EtOAc. The mixture was ﬁltered

through Celite. The ﬁltrate was concentrated in vacuo.

A mixture of the residue and 5-aminoisoquinoline

(208 mg, 1.4 mmol) was dissolved in titanium tetraiso-

propoxide (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, ﬁl-

tered through Celite, and quenched by addition of sat-

urated NaHCO₃aq. The mixture was extracted with

EtOAc. The organic layer was dried over anhydrous

Na₂SO₄and ﬁltered. The ﬁltrate was concentrated in

vacuo. The residual oil was subjected to chromatogra-

12.9.50.

N-(1-Benzyl-3-piperidyl)-N-(3-methyl-1H-5-

indazolyl)amine (21a). Compound 21a was prepared

from 5-amino-3-methyl-1H-indazole and 1-benzyl-3-

piperidone in a manner similar to that described for 16c

with a yield of 55% as yellow dense oil. ¹H NMR

(CDCl₃, 400 MHz) d 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 ¼ 4:1 Hz, 2H), 3.53–3.60 (m, 1H), 6.65 (d,

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2133

phy on silica gel, eluting with CHCl₃–MeOH (95:5), to

give the title compound as yellow dense oil (101 mg,

0.37 mmol, 26% yield). H NMR (CDCl₃, 400 MHz) d

that described for 22c with a yield of 69%, as yellow

dense oil. A mixture of two diastereomers in a ratio of

1

ca. 1:1. MS (ESI) m=z 256 (Mþ1)^þ. H NMR (CDCl₃,

0.85 (t, J ¼ 7:3 Hz, 3H), 1.47 (q, J ¼ 7:6 Hz, 2H), 1.57

(dq, J ¼ 4:2 Hz, 10.7 Hz, 2H), 2.05–2.18 (m, 4H), 2.28 (t,

J ¼ 7:8 Hz, 2H), 2.87 (t, J ¼ 12:2 Hz, 2H), 3.38–3.50 (m,

1H), 6.71 (d, J ¼ 7:6 Hz, 1H), 7.22 (d, J ¼ 8:1 Hz, 1H),

7.37 (t, J ¼ 7:8 Hz, 1H), 7.46 (d, J ¼ 6:1 Hz, 1H), 8.39

(d, J ¼ 5:9 Hz, 1H), 9.07 (s, 1H). MS (ESI) m=z 268

(Mꢀ1)^ꢀ.

400 MHz) d 1.4–1.8 (m, 8H), 2.0–2.4 (m, 2H), 2.44 (s,

3H), 6.69 (d, J ¼ 7:5 Hz, 1H), 7.3–7.6 (m, 2H), 8.35 (d,

J ¼ 6:1 Hz, 1H), 9.07 (br s, 1H).

12.9.56. N-(1-Benzyl-3-piperizyl)-N-(5-isoquinolyl)amine

(23a). To a solution of 3-hydroxypiperizine (10.0 g,

100 mmol) in 3 M NaOH aq (100 mL) was added a

solution of di-tert-butyl dicarbonate (25.0 g, 120 mmol)

in THF (100 mL) dropwise at 0 °C. The reaction mixture

was stirred at rt for 1 h and evaporated for removal of

THF. The remaining aqueous layer was extracted with

EtOAc. The EtOAc layer was dried over anhydrous

Na₂SO₄and ﬁltered. The ﬁltrate was concentrated in

vacuo. To a mixture of the residue and triethylamine

(20 mL) in anhydrous DMSO (100 mL) was added sulfur

trioxide–trimethylamine complex (44.4 g, 200 mmol)

portionwise at 0 °C. The reaction mixture was stirred at

rt for 18 h and quenched by addition of saturated

NaHCO₃aq. The aqueous layer was extracted with

EtOAc. The EtOAc layer was dried over anhydrous

Na₂SO₄and ﬁltered. The ﬁltrate was concentrated in

vacuo. The crude mixture was puriﬁed by silica gel

column chromatography (CHCl₃) to give the interme-

diate, 3-(tert-butyloxycarbonyl)piperizinone (15.6 g). To

a mixture of the intermediate (3.7 g, 20 mmol), 5-am-

inoisoquinoline (2.5 g, 17 mmol), and anhydrous

Na₂SO₄(14.2 g, 100 mmol) in AcOH (100 mL), sodium

triacetoxyborohydride (4.5 g, 20 mmol) was added

dropwise at 0 °C. The reaction mixture was stirred at rt

for 18 h and evaporated for removal of AcOH. The

resulting mixture was basiﬁed by saturated NaHCO₃aq.

The aqueous layer was extracted with EtOAc. The

EtOAc layer was dried over anhydrous Na₂SO₄and

ﬁltered. The ﬁltrate was concentrated in vacuo. The

residual oil was subjected to chromatography on silica

gel, eluted with CHCl₃–MeOH (95:5), to give tert-butyl

12.9.54. N1-(5-Isoquinolyl)-N4-propyl-1,4-cyclohexane-

diamine (22c). To a mixture of 1,4-cyclohexane mono-

ethyleneketal (6.20 g, 40 mmol), 5-aminoisoquinoline

(4.33 g, 30 mmol), and acetic acid (0.5 mL) in MeOH

(50 mL) was added BH₃–pyridine complex (4.0 mL,

40 mmol) dropwise at 0 °C. The mixture was stirred at rt

for 18 h and concentrated for removal of MeOH. The

residue was dissolved in 50% acetic acid (50 mL). The

solution was stirred at 80 °C for 3 h and concentrated for

removal of AcOH. The mixture was basiﬁed by the

addition of saturated NaHCO₃aq. The aqueous layer

was extracted with CHCl₃. The CHCl₃layer was dried

over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate was

concentrated in vacuo. The residual oil was subjected to

chromatography on silica gel, eluting with CHCl₃, to

give 4-(5-isoquinolylamino)-1-cyclohexanone (5.80 g,

24 mmol).

To a mixture of 4-(5-isoquinolylamino)-1-cyclohexa-

none (60 mg, 0.25 mmol), propylamine (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

addition of saturated NaHCO₃aq. The aqueous layer

was extracted with CHCl₃. The CHCl₃layer was dried

over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate was

concentrated in vacuo. The residual oil was subjected to

a reverse phase HPLC (ODS), eluting with 5% TFA aq–

acetonitrile (4:1), to give the title compound as yellow

dense oil (18 mg, syn-isomer; 22 mg, anti-isomer). anti-

Isomer: ¹H NMR (CDCl₃, 400 MHz) d 0.87 (t,

J ¼ 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,

1H), 2.57 (t, J ¼ 7:5 Hz, 2H), 3.31–3.44 (m, 1H), 4.06–

4.20 (m, 1H), 6.70 (d, J ¼ 7:6 Hz, 1H), 7.21 (d,

J ¼ 8:0 Hz, 1H), 7.38 (t, J ¼ 7:7 Hz, 1H), 7.44 (t,

J ¼ 6:1 Hz, 1H), 8.38 (t, J ¼ 5:9 Hz, 1H), 9.07 (s, 1H).

MS (ESI) m=z 284 (Mþ1)^þ. syn-Isomer: ¹H NMR

(CDCl₃, 400 MHz) d 0.86 (t, J ¼ 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 ¼ 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 ¼ 7:8 Hz, 1H), 7.20 (d, J ¼ 7:6 Hz, 1H), 7.36 (t,

J ¼ 7:7 Hz, 1H), 7.47 (t, J ¼ 5:8 Hz, 1H), 8.37 (t,

J ¼ 6:1 Hz, 1H), 9.07 (s, 1H). MS (ESI) m=z 284

(Mþ1)^þ.

3-(5-isoquinolylamino)-1-piperizinecarboxylate as

a

yellow dense oil (3.7 g, 12 mmol, 60% yield in three

steps).

To a solution of tert-butyl 3-(5-isoquinolylamino)-1-

piperizinecarboxylate (66 mg, 0.20 mmol) in CHCl₃

(1 mL) was added triﬂuoroacetic acid (1 mL) dropwise at

rt. The resulting mixture was stirred at rt for 3 h and

concentrated in vacuo. To a mixture of residue and

K₂CO₃(69 mg, 0.50 mmol) in CH₃CN (1 mL) was added

a solution of 4-chlorobenzylchloride (40 mg, 0.25 mmol)

in CH₃CN (0.5 mL) dropwise at 0 °C. The reaction

mixture was stirred at rt for 18 h and quenched by

addition of water. The aqueous layer was extracted with

EtOAc. The EtOAc layer was dried over anhydrous

Na₂SO₄and ﬁltered. The ﬁltrate was concentrated in

vacuo. The residual oil was subjected to chromatogra-

phy on silica gel, eluted with CHCl₃–MeOH (95:5), to

give the title compound as yellow dense oil (18 mg,

1

12.9.55. N1-(5-Isoquinolyl)-N4-methyl-1,4-cyclohexan-

ediamine (22d). Compound 22d was prepared from

methylamine/MeOH solution in a manner similar to

0.057 mmol, 29% yield in this step). H NMR (CDCl₃,

400 MHz) d 1.40–2.05 (m, 5H), 2.25–2.98 (m, 3H), 3.37–

4.10 (m, 3H), 6.65–6.80 (m, 1H), 7.24–7.58 (m, 8H), 8.47

2134

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

(d, J ¼ 5:9 Hz, 1H), 9.12 (s, 1H). MS (ESI) m=z 318

(Mþ1)^þ.

12.9.60.

N-(2,6-Diﬂuorobenzyl)-N⁰-(5-isoquinolyl)urea

(25a). To a mixture of 2,6-diﬂuorophenylacetic acid

(86 mg, 0.50 mmol) and triethylamine (0.4 mL) in tolu-

ene (2 mL) was added diphenylphosphoryl azide

(165 mg, 0.60 mmol) dropwise at rt. The reaction mix-

ture was stirred at 110 °C for 1 h followed by addition of

5-aminoisoquinoline (65 mg, 0.45 mmol) and succes-

sively 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 up in vacuo

to give the title compound (85 mg, 0.27 mmol, 54%

12.9.57. N-[1-(4-Methylpentyl)-3-piperidyl]-N-(5-isoquin-

olyl)amine (23b). A mixture of 3-hydroxypiperidine

(1.0 g, 9.9 mmol), 1-bromo-4-methylpentane (1.65 g,

10 mmol), and K₂CO₃(2.76 g) in DMF (10 mL) was

stirred for 12 h and quenched by addition of water. The

aqueous layer was extracted with EtOAc. The organic

layer was washed with saturated aqueous NaCl, dried

over anhydrous Na₂SO₄, ﬁltered, and concentrated in

vacuo to give N-(4-methyl)pentane-3-hyroxypiperidine

(2.04 g, 11 mmol). To a mixture of the compound, N-(4-

methyl)pentane-3-hyroxypiperidine (760 mg, 4 mmol)

and 5-aminoisoquinoline (473 mg, 3.3 mmol) in Ti(O-i-

Pr)₄was added a solution of sodium borohydride

(77.6 mg, 2.3 mmol) suspended in MeOH (3.8 mL). The

reaction mixture was stirred for 18 h and quenched by

addition of water. The reaction mixture was extracted

with EtOAc. The EtOAc layer was washed with satu-

rated NaCl aq, dried over anhydrous Na₂SO₄, ﬁltered,

and concentrated in vacuo. The crude mixture was

puriﬁed by silica gel column chromatography, eluting by

CHCl₃–MeOH to give the title compound (17.4 mg, 2%

yield) as yellow dense oil. ¹H NMR (CDCl₃, 400 MHz) d

0.88–0.91 (m, 1H), 0.90 (dd, J ¼ 6:6 Hz, 6H), 1.25 (m,

2H), 1.55 (m, 4H), 1.76 (br s, 2H), 2.36 (m, 4H), 2.53 (br

s, 1H), 2.69 (br s, 1H), 3.79 (s, 1H), 6.77 (d, J ¼ 7:9 Hz,

1H), 7.26 (d, J ¼ 7:9 Hz, 1H), 7.44 (d, J ¼ 7:9 Hz, 1H),

7.57 (d, J ¼ 6:0 Hz, 1H), 8.45 (d, J ¼ 6:0 Hz, 1H), 9.14

(s, 1H). MS (ESI) m=z 312 (Mþ1)^þ.

1

yield) as a colorless solid. Mp 196–197 °C. H NMR

(CDCl₃, 400 MHz) d 4.69 (s, 2H), 7.21 (dd, J ¼ 7:4 Hz,

8.8 Hz, 1H), 7.35 (d, J ¼ 8:0 Hz, 1H), 7.55 (t,

J ¼ 7:9 Hz, 1H), 7.75 (dd, J ¼ 6:4 Hz, 8.3 Hz, 1H), 8.04

(d, J ¼ 7:6 Hz, 1H), 8.31 (d, J ¼ 6:1 Hz, 1H), 9.10 (s,

1H). MS (ESI) m=z 314 (Mþ1)^þ.

12.9.61.

N-(2,6-Dichlorobenzyl)-N⁰-(5-isoquinolyl)urea

(25b). Compound 25b was prepared from 2,6-dichlor-

ophenylacetic acid in a manner similar to that described

for the compound 25a with a yield of 52%, as a colorless

solid. Mp 215–220 °C. H NMR (CDCl₃, 400 MHz) d

4.75 (s, 2H), 6.90 (t, J ¼ 8:0 Hz, 2H), 7.26 (tt,

J ¼ 6:5 Hz, 8.5 Hz, 1H), 7.54 (t, J ¼ 7:9 Hz, 1H), 7.75 (t,

J ¼ 8:3 Hz, 2H), 7.99 (d, J ¼ 7:8 Hz, 1H), 8.31 (d,

J ¼ 6:1 Hz, 1H), 9.10 (s, 1H). MS (ESI) m=z 347

(Mþ1)^þ.

1

12.9.62. N-[1-(4-Fluoro-1-benzyl)-4-piperidylmethyl]-N-

(5-isoquinolyl)amine (26). To a mixture of 4-piperidi-

nemethnol (345 mg, 3.0 mmol) and K₂CO₃(414 mg,

3.2 mmol) in acetonitrile (2 mL) was added a solution of

4-ﬂuorobenzylchloride (432 mg, 3.0 mmol) in acetoni-

trile (2 mL) dropwise at 0 °C. The reaction mixture was

stirred at rt for 5 h and diluted with EtOAc. The mixture

was ﬁltered through Celite. The ﬁltrate was concentrated

in vacuo. To the residue, triethylamine (0.5 mL) in

anhydrous DMSO (2 mL) and sulfur trioxide–trimeth-

ylamine complex (700 mg, 5 mmol) was added portion-

wise at 0 °C. The reaction mixture was stirred at rt for

18 h and quenched by addition of saturated NaHCO₃

aq. The aqueous layer was extracted with EtOAc. The

EtOAc layer was dried over anhydrous Na₂SO₄and

ﬁltered. The ﬁltrate was concentrated in vacuo. To the

residue, 5-aminoisoquinoline (288 mg, 2.0 mmol) in

MeOH (3 mL) was added followed by addition of

NaBH(OAc)₃(666 mg, 3.0 mmol) at 0 °C. The reaction

mixture was stirred at rt for 18 h and quenched by

addition of saturated NaHCO₃aq. The aqueous layer

was extracted with CHCl₃. The CHCl₃layer was dried

over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate was

concentrated in vacuo. The residual oil was subjected to

chromatography on silica gel, eluting with CHCl₃–

MeOH (95:5), to give the title compound as yellow

dense oil (439 mg, 0.087 mmol, 44%). ¹H NMR (CDCl₃,

400 MHz) d 1.82 (br t, J ¼ 11:2 Hz, 2H), 1.95–2.05 (m,

2H), 2.14 (br t, J ¼ 11:2 Hz, 2H), 2.45–2.52 (m, 1H),

2.95 (br d, J ¼ 11:6 Hz, 2H), 3.43 (s, 2H), 3.51 (s, 2H),

6.85–7.02 (m, 2H), 7.01 (d, J ¼ 4:6 Hz, 1H), 7.25–7.37

(m, 2H), 7.78 (d, J ¼ 4:2 Hz, 1H), 7.95 (dd, J ¼ 1:5 Hz,

12.9.58. N-(4-Acetylphenyl)-N⁰-(5-isoquinolyl)urea (24a).

To

a

solution of 4-amino-isoquinoline (100 mg,

0.69 mmol) in toluene (2 mL) was added 4-acetylphenyl-

isocyanate (123 mg, 0.76 mmol) at rt. The reaction

mixture was stirred at 110 °C for 3 h and diluted with

ether. The resulting precipitate was collected by ﬁltra-

tion, washed with ether, and dried up in vacuo to give

the title compound as a colorless solid. ¹H NMR

(CD₃OD, 400 MHz) d 2.48 (s, 3H), 7.5–7.6 (m, 3H), 7.8–

7.9 (m, 4H), 8.12 (d, J ¼ 5:0 Hz, 1H), 8.39 (d,

J ¼ 5:0 Hz, 1H), 9.16 (s, 1H). MS (ESI) m=z 306

(Mþ1)^þ.

12.9.59. N-(2,6-Dichlorophenyl)-N⁰-(5-isoquinolyl)urea

(24b), and N-(2,4,6-trichlorophenyl)-N⁰-(5-isoquinolyl)-

urea (24c). Compound 24b and 24c were prepared

from 2,6-dichloroisocyanate or 2,4,6-trichloroisocya-

nate in a manner similar to that described for the

compound 24a with a yield of about 75%. 24b: ¹H NMR

(CDCl₃, 400 MHz) d 7.3–7.4 (m, 3H), 7.57 (dd,

J ¼ 7:7 Hz, 1H), 7.75 (d, J ¼ 5:9 Hz, 1H), 7.81 (d,

J ¼ 7:7 Hz, 1H), 7.98 (d, J ¼ 7:7 Hz, 1H), 8.51 (d,

J ¼ 5:9 Hz, 1H), 9.20 (s, 1H). MS (ESI) m=z 331

1

(Mꢀ1)^ꢀ. 24c: H NMR (CDCl₃, 400 MHz) d 7.43 (m,

1H), 7.51 (m, 1H), 7.60 (d, J ¼ 6:0 Hz, 1H), 7.6–7.8 (m,

2H), 8.47 (d, J ¼ 6:0 Hz, 1H), 9.18 (s, 1H). MS (ESI)

m=z 365 (Mꢀ1)^ꢀ.

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2135

4.0 Hz, 1H), 8.07 (d, J ¼ 2:6 Hz, 1H), 8.24 (d,

J ¼ 2:6 Hz, 1H), 8.40 (dd, J ¼ 1:5 Hz, 4.0 Hz, 1H). MS

(ESI) m=z 350 (Mþ1)^þ.

DMF (2 drops). The mixture was stirred at 110 °C for

2 h and diluted with water and EtOAc. The resulting

precipitate was collected by ﬁltration and washed with

EtOAc to give the title compound (37 mg, 23% yield) as

a colorless solid. Mp > 280 °C. ¹H NMR (CDCl₃,

400 MHz) d 4.40 (d, J ¼ 5:6 Hz, 2H), 6.94 (t, J ¼ 6:0 Hz,

1H), 7.28–7.38 (m, 2H), 7.41 (dd, J ¼ 1:7, 7.6 Hz, 1H),

7.46 (dd, J ¼ 1:7 Hz, 7.6 Hz, 1H), 7.61 (dd, J ¼ 1:8 Hz,

8.2 Hz, 1H), 7.68 (d, J ¼ 8:5 Hz, 1H), 8.01 (d,

J ¼ 1:5 Hz, 1H), 9.42 (s, 1H), 11.08 (s, 1H). MS (ESI)

m=z 328 (Mꢀ1)^ꢀ.

12.9.63. N-[1-(4-Fluoro-1-benzyl)-3-piperidylmethyl]-N-

(5-isoquinolyl)amine (27). Compound 27 was prepared

from 3-piperidinemethnol in a manner similar to that

described for 26 with a yield of 45%, as yellow dense oil

¹H NMR (CDCl₃, 400 MHz) d 1.40–1.80 (m, 3H),1.91–

1.98 (m, 1H), 1.14 (br t, J ¼ 4:6 Hz, 1H), 2.25 (br t,

J ¼ 10:4 Hz, 1H), 2.44 (br s, 1H), 2.72 (br s, 1H), 2.99

(br d, J ¼ 10:0 Hz, 1H), 3.45 (d, J ¼ 3:2 Hz, 2H), 3.49 (s,

2H), 6.90–7.01 (m, 2H), 7.03 (d, J ¼ 4:8 Hz, 1H), 7.22–

7.28 (m, 2H), 7.79 (d, J ¼ 4:0 Hz, 1H), 7.99 (dd,

J ¼ 1:4 Hz, 3.6 Hz, 1H), 8.07 (d, J ¼ 2:8 Hz, 1H), 8.24

(d, J ¼ 2:4 Hz, 1H), 8.40 (dd, J ¼ 1:4 Hz, 3.6 Hz, 1H).

MS (ESI) m=z 350 (Mþ1)^þ.

12.9.67. N-(2,6-Dichlorobenzyl)-N⁰-(1,3-dioxo-2,3-dihy-

dro-1H-5-isoindolyl)urea (29c). Compound 29c was

prepared from 2,6-dichlorophenylacetic acid in a man-

ner similar to that described for 29b with a yield of 67%

1

as a colorless solid. Mp 281 °C (dp). H NMR (CDCl₃,

400 MHz) d 4.60 (d, J ¼ 5:4 Hz, 2H), 6.78 (t, J ¼ 5:4 Hz,

1H), 7.39 (d, J ¼ 7:3 Hz, 1H), 7.51 (d, J ¼ 7:8 Hz, 2H),

7.55 (dd, J ¼ 2:0 Hz, 8.3 Hz, 1H), 7.67 (d, J ¼ 8:3 Hz,

1H), 8.01 (d, J ¼ 2:0 Hz, 1H), 9.14 (s, 1H), 11.1 (s, 1H).

MS (ESI) m=z 363 (Mꢀ1)^ꢀ. Anal. Calcd for

C₁₆H₁₁Cl₂N₃O₃: C, 52.77; H, 3.04; N, 11.54. Found: C,

52.62; H, 3.49; N, 11.25.

12.9.64. N-(4-tert-Butyl-phenyl)-N-(5-isoquinolyl)amine

(28). 5-Aminoisoquinoline (54 mg, 0.4 mmol) was dis-

solved in a mixture of triethylamine (0.5 mL) and DMF

(2 mL). To the solution, 4-tert-butylphenylbronic acid

(180 mg, 1.0 mmol) and cupric acetate (250 mg,

1.4 mmol) was added and stirred at rt for 3 days. The

reaction mixture was ﬁltered through Celite and diluted

with water. The aqueous layer was extracted with

EtOAc. The AcOEt layer was washed with water and

saturated NaCl aq, dried over anhydrous Na₂SO₄, ﬁl-

tered, and concentrated. Puriﬁcation by silica gel col-

umn chromatography eluted with hexane–EtOAc (9:1)

yielded the title compound (6.5 mg, 6% yield). ¹H NMR

(CDCl₃, 400 MHz) d 1.33 (s, 9H), 5.96 (s, 1H), 7.03 (d,

J ¼ 8:5 Hz, 2H), 7.33 (d, J ¼ 8:5 Hz, 2H), 7.47–7.49 (m,

2H), 7.57 (m, 1H), 7.76 (d, J ¼ 6:1 Hz, 1H), 8.51 (d,

J ¼ 5:9 Hz, 1H), 9.23 (s, 1H). MS (ESI) m=z 277

(Mþ1)^þ.

12.9.68. N-(2,6-Dichlorobenzyl)-N⁰-(6-chloro-1,3-dioxo-

2,3-dihydro-1H-5-isoindolyl)urea (29d). Compound 29d

was prepared from 4-amino-5-chlorophthalimide and

2,6-dichlorophenylacetic acid in a manner similar to that

described for 29b with a yield of 4% as a colorless solid.

1

Mp > 280 °C. H NMR (CDCl₃, 400 MHz) d 4.60–4.64

(m, 2H), 7.36–7.46 (m, 3H), 7.54 (d, J ¼ 7:8 Hz, 2H),

7.87 (s, 1H), 8.71 (s, 1H), 11.28 (s, 1H). MS (ESI) m=z

397 (Mꢀ1)^ꢀ.

12.9.69. O-(2,6-Dichlorobenzyl)-N-(1,3-dioxo-2,3-dihy-

dro-1H-5-isoindolyl)carbamate (29e). To a mixture of

4-aminophthalimide (81 mg, 0.50 mmol) and triethyl-

amine (0.25 mL) in CHCl₃(1 mL) was added a solution

of triphosgen (148 mg, 0.50 mmol) in CHCl₃(1 mL)

dropwise at rt. The mixture was stirred at rt for 30 min

and followed by addition of 2,6-dichlorobenzylalcohol

(89 mg, 0.50 mmol) portionwise at rt. The reaction

mixture was stirred at rt for 18 h and quenched by

addition of saturated NaHCO₃aq. The aqueous layer

was extracted with CHCl₃. The CHCl₃layer was dried

over anhydrous Na₂SO₄and ﬁltered. The ﬁltrate was

concentrated in vacuo. The residual oil was subjected to

chromatography on silica gel, eluting with CHCl₃–

MeOH (95:5), to give the title compound as a colorless

solid (42 mg, 0.12 mmol, 24% yield). Mp 257–260 °C. ¹H

NMR (DMSO, 400 MHz) d 3.30 (s, 2H), 6.38 (br s, 1H),

6.78 (d, J ¼ 12:2 Hz, 1H), 6.85 (s, 1H), 6.94–7.02 (m,

1H), 7.37 (d, J ¼ 13:0 Hz, 1H), 7.44 (d, J ¼ 12:2 Hz,

1H). MS (ESI) m=z 365 (Mþ1)^þ.

12.9.65. N-Benzyl-N⁰-(1,3-dioxo-2,3-dihydro-1H-5-iso-

indolyl)urea

(29a).

4-Aminophthalimide

(80 mg,

0.5 mmol) was dissolved in toluene (1 mL) and trace

amount of DMF. Benzylisocyanate (66 mg, 0.5 mmol)

was added and stirred at 110 °C for 2.5 h. Water and

EtOAc were added to the reaction mixture. The result-

ing precipitate was collected by ﬁltration, and washed

with EtOAc to give the title compound (96 mg, 66%

yield) as a colorless solid. Mp > 280 °C. ¹H NMR

(CDCl₃, 400 MHz) d 4.33 (d, J ¼ 4:4 Hz, 2H), 6.90 (t,

J ¼ 6:0 Hz, 1H), 7.22–7.35 (m, 5H), 7.61 (dd,

J ¼ 1:8 Hz, 8.2 Hz, 1H), 7.67 (d, J ¼ 8:3 Hz, 1H), 8.02

(dd, J ¼ 0:5 Hz, 2.0 Hz, 1H), 9.30 (s, 1H), 11.08 (s, 1H).

MS (ESI) m=z 294 (Mꢀ1)^ꢀ.

12.9.66. N-(2-Chlorobenzyl)-N⁰-(1,3-dioxo-2,3-dihydro-

1H-5-isoindolyl)urea (29b). To a mixture of 2-chlor-

ophenylacetic acid (85 mg, 0.5 mmol) and triethylamine

(61 mg, 0.6 mmol) in toluene (2 mL) was added diph-

enylphosphorylazide (165 mg, 0.6 mmol). The reaction

mixture was stirred at 110 °C for 1 h and followed by

addition of 4-aminophthalimide (81 mg, 0.5 mmol) and

12.9.70. 2-(2,6-Dichlorophenoxy)-N-(1,3-dioxo-2,3-dihy-

dro-1H-5-isoindolyl)acetamide (29f). To a mixture of

2,6-dichlorophenol (1.0 g, 6.13 mmol) and K₂CO₃

2136

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

(1.02 g, 7.36 mmol) in acetonitrile (8 mL) was added

methylbromoacetate (1.12 g, 6.75 mmol). The reaction

mixture was stirred at 80 °C for 3 h and quenched by

addition of water. The aqueous layer was extracted with

EtOAc. The EtOAc layer was washed with saturated

aqueous NaCl, dried over Na₂SO₄, ﬁltered, and solvent

was removed in vacuo. The residue was puriﬁed by silica

gel column chromatography (CHCl₃) to give 2,6-di-

chlorophenoxyacetic acid ethyl ester. A mixture of the

compound in 5% NaOH aq (10 mL) and EtOH (10 mL)

was stirred at 80 °C for 1 h. The reaction mixture was

concentrated, acidiﬁed to pH 4 with 5% HCl aq, and

extracted with EtOAc. The EtOAc layer was washed

with saturated NaCl aq, dried over anhydrous Na₂SO₄,

and concentrated in vacuo to give 2, 6-dichlorophen-

oxyacetic acid (1.08 g, 79%) as a colorless solid.

(CDCl₃, 400 MHz) d 7.01 (t, J ¼ 7:3 Hz, 1H), 7.31 (t,

J ¼ 7:9 Hz, 2H), 7.48 (dd, J ¼ 1:0 Hz, 8.5 Hz, 2H), 7.67

(dd, J ¼ 1:8 Hz, 8.2 Hz, 1H), 7.73 (d, J ¼ 8:3 Hz, 1H),

8.05 (d, J ¼ 1:5 Hz, 1H), 8.89 (s, 1H), 9.36 (s, 1H), 11.14

(s, 1H). MS (ESI) m=z 280 (Mꢀ1)^ꢀ.

12.9.73. N-(1,3-Dioxo-2,3-dihydro-1H-5-isoindolyl)-N⁰-

(2,6-dichlorophenyl)urea (30c). Compound 30c was pre-

pared from 4-amino-5-chlorophthalimide and 2,6-di-

chlorophenylacetic acid in a manner similar to that

described for 30b with a yield of 86% as a colorless solid.

1

Mp > 280 °C. H NMR (CDCl₃, 400 MHz) d 7.35 (dd,

J ¼ 7:8 Hz, 8.0 Hz, 1H), 7.60 (d, J ¼ 8:1 Hz, 2H), 7.72

(d, J ¼ 1:2 Hz, 2H), 8.04 (d, J ¼ 1:2 Hz, 1H), 8.50 (s,

1H), 9.69 (s, 1H), 11.13 (s, 1H). MS (ESI) m=z 349

(Mꢀ1)^ꢀ.

To a solution of the acetic acid (150 mg, 0.68 mmol) and

4-aminophthalimide (100 mg, 0.62 mmol) in DMF

(2 mL) was added WSC–HCl (147 mg, 0.74 mmol) and

HOBt–H₂O (100 mg, 0.74 mmol). The reaction mixture

was stirred at rt for 4 h and quenched by addition of

water. The aqueous layer was extracted with EtOAc.

The EtOAc layer was washed with saturated NaCl aq,

dried over anhydrous Na₂SO₄, ﬁltered, and concen-

trated. Puriﬁcation was carried out by silica gel column

chromatography (CHCl₃–MeOH 95:5). The title com-

pound (64 mg, 28% yield) was obtained as colorless

12.9.74. N-(1,3-Dioxo-2,3-dihydro-1H-5-isoindolyl)-N⁰-

(4-methylpiperizyl)urea (30d). To a mixture of 4-amino-

1H-phthlimide (200 mg, 1.23 mmol) and triethylamine

(1.5 mL) in CHCl₃(5 mL) was added a solution of tri-

phosgen (366 mg, 1.23 mmol) in CHCl₃(1 mL) dropwise

at rt. The resulting mixture was stirred at rt for 30 min

and followed by addition of 1-amino-4-methylpiper-

azine (142 mg, 1.23 mmol) portionwise at rt. The reac-

tion mixture was stirred at rt for 18 h and diluted by

water. The resulting precipitate was collected by ﬁltra-

tion, washed with ether, and dried up in vacuo to give

the title compound as a colorless solid. ¹H NMR

(CDCl₃, 400 MHz) d 1.8–2.0 (m, 2H), 2.2–2.3 (m, 2H),

2.8–2.9 (m, 2H), 3.0–3.1 (m, 2H), 3.42 (br s, 3H), 7.50

(m, 1H), 7.65 (m, 1H), 7.72 (br s, 1H). MS (ESI) m=z 302

(Mꢀ1)^ꢀ.

1

solid. Mp > 280 °C. H NMR (CDCl₃, 400 MHz) d 4.71

(s, 2H), 7.24 (t, J ¼ 8:5 Hz, 1H), 7.54 (d, J ¼ 8:1 Hz,

1H), 7.54 (d, J ¼ 8:1 Hz, 1H), 7.80 (d, J ¼ 8:3 Hz, 1H),

8.02 (dd, J ¼ 2:0 Hz, 8.1 Hz, 1H), 8.24 (d, J ¼ 2:0 Hz,

1H), 10.70 (s, 1H), 11.25 (s, 1H). MS (ESI) m=z 365

(Mþ1)^þ.

12.9.71. N-(1,3-Dioxo-2,3-dihydro-1H-5-isoindolyl)-N⁰-

propylurea (30a). To a solution of 4-aminophthalimide

(80 mg, 0.5 mmol) in toluene (1 mL) and a trace amount

of DMF, n-propylisocyanate (42 mg, 0.5 mmol) was

added and stirred at 110 °C for 2.5 h. Water was added

to the reaction mixture and extracted with EtOAc. The

EtOAc layer was washed with saturated NaCl aq, dried

over anhydrous Na₂SO₄, concentrated, and puriﬁed by

preparative TLC (CHCl₃–acetone 95:5) to give the title

compound (35 mg, 29% yield) as a colorless solid.

Mp > 280 °C. ¹H NMR (CDCl₃, 400 MHz) d 0.88 (t,

J ¼ 7:4 Hz, 3H), 1.41–1.51 (m, 2H), 3.07 (q, J ¼ 6:8 Hz,

2H), 6.40 (t, J ¼ 5:6 Hz, 1H), 7.58 (dd, J ¼ 2:1 Hz,

8.2 Hz, 1H), 7.66 (d, J ¼ 8:3 Hz, 1H), 8.00 (d,

J ¼ 1:7 Hz, 1H), 9.14 (s, 1H), 11.06 (s, 1H). MS (ESI)

m=z 246 (Mꢀ1)^ꢀ.

12.9.75. N-(1-Benzylpiperidin-4-yl)-N⁰-(1,3-dioxo-2,3-di-

hydro-1H-5-isoindolyl)urea (30e). Compound 30e was

prepared from 1-benzyl-4-aminopiperizine in a manner

1

similar to that described for 30d. H NMR (CD₃OD,

400 MHz) d 1.4–1.5 (m, 2H), 1.9–2.0 (m, 2H), 2.0–2.1

(m, 2H), 2.7–2.8 (m, 2H), 3.5–3.6 (m, 3H), 5.05 (br s,

2H), 7.2–7.3 (m, 5H), 7.53 (dd, J ¼ 8:1 Hz, 2.0 Hz, 1H),

7.59 (d, J ¼ 8:1 Hz, 1H), 7.88 (d, J ¼ 2:0 Hz, 1H). MS

(ESI) m=z 377 (Mꢀ1)^ꢀ.

12.9.76. 4-{[(2,6-Dichloroanilino)carbonyl]amino}benz-

amide (31). To a solution of 4-aminobenzdiamide

(100 mg, 0.73 mmol) in toluene (1 mL) was added 2,6-

dichloro-phenylisocyanate (138 mg, 0.73 mmol). The

reaction mixture was stirred at 110 °C for 2 h and diluted

with water and EtOAc. The precipitate was collected by

ﬁltration and washed with EtOAc to give the title

compound (210 mg, 88% yield) as a colorless solid.

12.9.72. N-(1,3-Dioxo-2,3-dihydro-1H-5-isoindolyl)-N⁰-

phenylurea (30b). To a solution of 4-aminophthalimide

(80 mg, 0.5 mmol) in toluene (1 mL) was added pheny-

lisocyanate (59 mg, 0.5 mmol) and DMF (1 drop). The

reaction mixture was stirred at 110 °C for 2.5 h. Water

and EtOAc was added to the reaction mixture. The

precipitated crystal was collected by ﬁltration and wa-

shed with EtOAc to give the title compound (95 mg, 68%

yield) as a colorless solid. Mp > 280 °C. ¹H NMR

1

Mp > 280 °C. H NMR (CDCl₃, 400 MHz) d 7.15 (br s,

1H), 7.31 (t, J ¼ 8:1 Hz, 1H), 7.49 (d, J ¼ 8:8 Hz, 2H),

7.52 (d, J ¼ 8:1 Hz, 2H), 7.79 (d, J ¼ 8:8 Hz, 2H), 7.87

(br s 1H), 8.31 (br s, 1H), 9.22 (br s, 1H). MS (ESI) m=z

323 (Mꢀ1)^ꢀ.

A. Takami et al. / Bioorg. Med. Chem. 12 (2004) 2115–2137

2137

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Acknowledgements

We thank Prof. Shyu Narumiya of Kyoto University

and Prof. Masaaki Ito of the Mie University School of

Medicine for their precious scientiﬁc advice. We deeply

thank Dr. Takeshi Ohtani and Dr. Kazuta Takemura of

the Kirin Brewery Research and Development Division

who ﬁrst took the initiative of this project and Dr.

Toshio Izawa for encouragement. We also thank Dr.

Teruyuki Sakai, Mr. Toshiyuki Shimizu, and Mr.

Yasunari Fujiwara for the synthesis of several important

chemicals. The inhibition activity against the PDGF

receptor kinase, the SCF receptor kinase (c-Kit) and the

MAP kinase were measured by Dr. Kazuhide Nakam-

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Article Doi

Design and synthesis of Rho kinase inhibitors (I)

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