AU5777801A - Pharmaceutical agent containing Rho kinase inhibitor - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): WELFIDE CORPORATION Invention Title: PHARMACEUTICAL AGENT CONTAINING Rho KINASE INHIBITOR The following statement is a full description of this invention, including the best method of performing it known to me/us:

SPECIFICATION

PHARMACEUTICAL AGENT CONTAINING Rho KINASE INHIBITOR Technical Field The present invention relates to treatment of various diseases by the use of a Rho kinase inhibitor as a pharmaceutical agent. Moreover, the present invention relates to use of a Rho kinase inhibitor as a reagent or a diagnostic.

Background Art Ever since the discovery of Ras in 1981, a number of small GTP binding proteins (small G proteins) similar to Ras have been found, and many physiological functions they possess have been studied. These small G proteins have a molecular weight of 20,000-30,000 and do not have a subunit structure. They all specifically bind GDP and GTP, and hydrolyze the thus-bound GTP (GTPase activity) (Hall, Science, 249, 635-640, 1990; Bourne, H. R et al., Nature, 349, 117-127, 1991).

To date, more than 50 kinds of genes encoding these small G proteins have e"e been found from yeast to mammals, forming a superfamily. These small G .oee proteins are largely divided into 5 groups of Ras, Rho, Rab, Arf and others, according to the similarity of amino acid sequences.

Of these, Rho was named so because its gene isolated in the form of cDNA from sea hare neuromuscle encodes a polypeptide having about 35% homology with Ras (Ras homologue) (Madaule, Cell, 41, 31-40, 1985).

Rho is specifically ADP ribosylated by C3 enzyme, which is one of the botulinum toxins, and Staphylococcal toxin EDIN, and inactivated (Narumiya, S.

and Morii, Cell Signal, 5, 9-19, 1993; Sekine, A. et al., J. Biol. Chem., 264, 8602-8605, 1989). Hence, the C3 enzyme and EDIN were used to study the involvement of Rho in cell functions from various aspects.

For example, phosphorylation by myosin light chain (MLC) kinase is considered to enable actin myosin interaction and initiate contraction of smooth muscle, and the structure of smooth muscle myosin phosphatase which dephosphorylates MLC has been clarified (Shimizu, H. et al., J. Biol. Chem., 269, 30407-30411, 1994). It has been clarified that the activity of myosin phosphatase is, like MLC kinase, under the control of the intracellular signal transduction system and Rho is involved in this mechanism. Moreover, an active 1A Rho bound with GTP has been found to enhance Ca-dependent contraction in a smooth muscle skinned fiber specimen (Hirata, J. Biol. Chem., 267, 8719-8722, 1992), thereby suggesting that the increase in Ca sensitivity in smooth muscle contraction is caused by the inhibition of myosin phosphatase activity via Rho.

In Swiss 3T3 cell and 3Y1 cell, moreover, Rho-dependent promotion of tyrosine phosphorylation (Kumagai, N. et al., J. Biol. Chem., 270, 8466-8473, 1993) and activation of many kinds of serine/threonine kinases (Kumagai, N. et al., FEBS Lett., 366, 11-16, 1995) have been acknowledged. From this, the presence of plural protein kinases in the downstream of Rho in the signal transduction pathway via Rho has been suggested and, actually, ROCa (Leung, T. et al., J. Biol.

Chem., 270, 29051-29054, 1995) [another name Rho-kinase, ROCK-II] and (Ishizaki, T. et al., The EMBO 15(8), 1885-1893, 1996) [another name ROC, ROCK-I] have been reported as serine/threonine kinase (Rho kinase) activated along with the activation of Rho. It has been also reported that i: biological distribution of the both enzymes is different (Nakagawa, O. et al., FEBS Lett. 392 189-193, 1996). In addition, it has been reported that this Rho kinase directly phosphorylates myosin phosphatase and inhibits its activity (Kimura, K. et al., Science, 273,245-248, 1996).

Rho has been documented to be responsible for the activation of not only protein kinase but also lipid kinase (Zang, J. et al., J. Biol. Chem., 268, 22251- 22254, 1993), and the presence ofphospholipase (PLD) activated by Rho has been also suggested (Siddiqi, A. R. et al, J. BioL Chem., 268, 24535-24538, 1995).

Control by Rho of the motility of Swiss 3T3 fibroblasts in the presence of serum, motility of keratinocyte 303R by HGF and TPA (12-O-tetradecanoylphorbol 13-acetate), spontaneously occurred and chemoatractant mediated motility of neutrophils have been reported (Takai, Y. et al., Trends Biochem. Sci., 20, 227-231, 1995), and control of the permeation of liver cancer cell (MM1 cell), which is one of the metastatic cancer models, through cultured mesothelial layer by the activation of Rho has been reported (Yoshioka, K. et al., FEBS Lett., 372, 25-28, 1995), thereby suggesting the involvement of Rho in cell motility.

Meanwhile, in the cells derived from nerves, such as neuroblastoma, PC- 12 cells and the like, retraction of neurite and rounding of the cell by lysophosphatidic acid, which is an activation stimulant of Rho, have been

L

acknowledged. Inasmuch as this retraction can be inhibited by C3 enzyme treatment (Jalink, K. et al., J. Cell Biol., 126, 801-810, 1994) and the formation of ringed structure of podosome, which separates the site where dissolution and absorption of bone take place in the clear zone of osteoclast from the surrounding, is inhibited by C3 enzyme treatment (Zhang, D. et al., J. Cell Sci., 108, 2285-2292, 1995), a deep involvement of Rho in the morphological changes in cells has been suggested.

In addition, C3 enzyme treatment reportedly inhibits activation of an adhesion molecule such as LFA (leukocyte function-associated antigen) and the like, and C3 enzyme treatment reportedly inhibits proliferation of Swiss 3T3 fibroblasts (Yamamoto, M. et al., Oncogene, 8, 1449-1455, 1993). Thus, Rho reportedly controls cell adhesion and cell division via actin cytoskeleton, and is also concerned with the transcription control of c-fos gene (Hill, C. S. et al., Cell, 81, 1159-1170, 1995) and transformation of cell (Khosravi-Far, R. et al., Mol. Cell Biol., 15(11), 6443-6453, 1995).

In view of the inhibition of invasion of dysentery bacillus into epithelial cells by C3 enzyme, a recent report has documented the deep involvement of Rho in bacterial infection (Adam, T. et al., The EMBO 15(13), 3315, 1996).

In pregnant rats, moreover, the levels of Rho and Rho kinase are reported to be higher as compared to nonpregnant rats (Niiro, N. et al, Biochem. Biophys.

Res. Commun., 230, 356-359, 1997), and deep involvement of Rho and Rho kinase in muscle contraction of uterus for childbirth has been known. Further, integrin (Sueoka, K. et al., Fertility Sterility, 67(5) 799-811, 1997) considered to *.be involved in the cell-cell and cell-extracellular matrix adhesion during the stages of fertilization, embryogenesis and embryonidation is known to be activated by Rho (Morii, N. et al., J. Biol. Chem., 267, 20921-20926, 1992).

Hence, it has been made clear that Rho is activated upon receipt of signals from various cell membrane receptors and the activated Rho functions as a molecule switch of a broad range of cell phenomena, such as smooth muscle contraction, cell motility, cell adhesion, morphological changes of cell, cell growth and the like, via actomyosin system.

Smooth muscle contraction is significantly involved in the disease states of hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, imminent immature birth and the like; cell motility plays an important role in invasion and metastasis of cancer, arteriosclerosis, retinopathy, immune response and the like; cell adhesion is deeply involved in metastasis of cancer, inflammation, autoimmune disease, AIDS, fertilization and nidation of fertilized egg and the like; morphological change of cell is deeply involved in brain function disorder, osteoporosis, bacterial infection of digestive tract and the like; and cell growth is deeply involved in cancer, arteriosclerosis and the like.

Therefore, a drug that blocks the functions of Rho is considered to make a therapeutic agent for these diseases in which Rho plays some role.

At present, however, only C3 enzyme and EDIN can inhibit the actions of Rho. These are proteins which cannot permeate cytoplasm, which prevents their development as a pharmaceutical agent.

On the other hand, inhibition of Rho kinase, which is considered to be present downstream of the signal transduction pathway via Rho, is considered to lead to the inhibition of responses of various cell phenomena due to Rho.

'However, a specific inhibitor of Rho kinase has not been known to date.

9o*.

SIt is expected, therefore, that by searching a compound that inhibits Rho kinase, such Rho kinase inhibitor will be an effective agent for the prophylaxis and/or treatment of the above-mentioned diseases and phenomena relating to ho, such as hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, immature birth, arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, fertilization and nidation of fertilized egg, osteoporosis, retinopathy, brain function disorder, bacterial infection of digestive tract and the like.

The compound of the formula is already known to be useful as an agent for the prophylaxis and treatment of circulatory disorder in coronary, cerebral, renal and peripheral arteries and the like a potent and long lasting therapeutic agent of hypertension, angina pectoris, renal and peripheral circulation disorder, and suppressive agent of cerebrovascular contraction and the like), as well as a therapeutic agent of asthma (Japanese Patent Unexamined Publication No. 62-89679, Japanese Patent Unexamined Publication No. 3- 218356, Japanese Patent Unexamined Publication No. 4-273821, Japanese Patent Unexamined Publication No. 5-194401, Japanese Patent Unexamined Publication No. 6-41080 and W095/28387 and the like).

The compound of the formula (II) is already known to be useful as a vasodilator, a therapeutic agent of hypertension, a brain function improving agent, an anti-asthma agent, a heart protection agent, a platelet aggregation inhibitor, a psychosyndrome treating agent, an anti-inflammatory agent and an agent for the prophylaxis and treatment of hyperviscosity syndrome (Japanese Patent Unexamined Publication No. 57-200366, Japanese Patent Unexamined Publication No. 61-227581, Japanese Patent Unexamined Publication No. 2- 256617, Japanese Patent Unexamined Publication No. 4-264030, Japanese Patent Unexamined Publication No. 6-56668, Japanese Patent Unexamined Publication No. 6-80569, Japanese Patent Unexamined Publication No. 6-293643, Japanese Patent Unexamined Publication No. 7-41424 and Japanese Patent Unexamined Publication No. 7-277979).

However, these compounds of the formula or (II) are not known to block the functions of Rho or to have Rho kinase inhibitory action.

.Disclosure of the Invention The present invention aims at providing a Rho kinase inhibitor as a novel pharmaceutical agent. As a result of intensive studies, the present inventors have found that a compound inhibiting Rho kinase has an antihypertensive action, an anti-angina pectoris action, a cerebrovascular contraction suppressive action, an anti-asthma action, a peripheral circulation improving action, an immature birth preventive action, an anti-arteriosclerosis action, an anti-cancer action, an antiinflammatory action, an immunosuppressive action, an autoimmune disease improving action, an anti-AIDS action, a preventive action on fertilization and nidation of fertilized egg, an osteoporosis treating action, a retinopathy treating action, a brain function improving action, a preventive action on bacterial infection of digestive tract and that the Rho kinase inhibitor is useful as a pharmaceutical agent, particularly as a therapeutic agent of hypertension, a therapeutic agent of angina pectoris, a suppressive agent of cerebrovascular contraction, a therapeutic agent of asthma, a therapeutic agent of peripheral circulation disorder, a prophylactic agent of immature birth, a therapeutic agent of arteriosclerosis, an anti-cancer drug, an anti-inflammatory agent, an immunosuppressant, a therapeutic agent of autoimmune disease, an anti-AIDS drug, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy, a brain function improving drug, a contraceptive and a prophylactic agent of digestive tract infection, which resulted in the completion of the present invention.

It has been also found that a compound which inhibits Rho kinase is useful as a reagent for the study of Rho and Rho kinase and as a diagnostic of the diseases relating to those, which resulted in the completion of the present invention.

Accordingly, the present invention provides the following.

A pharmaceutical agent containing a Rho kinase inhibitor.

A pharmaceutical agent containing a Rho kinase inhibitor, which is at least one member selected from the group consisting of a therapeutic agent of hypertension, a therapeutic agent of angina pectoris, a suppressive agent of cerebrovascular contraction, a therapeutic agent of asthma, a therapeutic agent of peripheral circulation disorder, a therapeutic agent of arteriosclerosis, an anti-cancer drug, an anti-inflammatory agent, an immunosuppressant, a therapeutic agent of autoimmune disease, an anti-AIDS drug, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy, a brain function improving drug, a prophylactic agent of immature birth, a contraceptive and a prophylactic agent of digestive tract infection.

A pharmaceutical composition containing a therapeutically effective amount of a Rho kinase inhibitor and a pharmaceutically acceptable additive.

A reagent containing a Rho kinase inhibitor.

A diagnostic containing a Rho kinase inhibitor.

A Rho kinase inhibitor containing an amide compound of the formula (I) o Rb II I Ra- C- N -Rc wherein Ra is a group of the formula

R

R N- (a)

R

1

R

3 N--A

R

4

R

L

N

or (c) in the formulas and R is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or a group of the formula a. a a a.

a.

a,

NR

7 (d) wherein R 6 is hydrogen, alkyl or formula: -NR'NR 9 wherein R 8 and R 9 are the same or different and each is hydrogen, alkyl, aralkyl or phenyl, R 7 is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R 6 and R 7 in combination show a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom, is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or in combination form, together with the adjacent nitrogen atom, a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom, is hydrogen or alkyl, are the same or different and each is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl or azide, and is a group of the formula Rand R' R2

R

3 and R 4

A

-(CH

2 )1(C)m(CH 2 )n

R

1 1 wherein R" 0 and R 1 are the same or different and each is hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R' 0 and R' 1 show a group which forms cycloalkyl in combination and 1, m. and n are each 0 or an integer of 1-3, in the formula L is hydrogen, alkyl, aminoalkyl, mono or dialkyLaminoalcyl, tetrahydrofurfuryl, carbamoylalkyl, phthalimidoalkyl, axnidino or a group of the formula 0

B-C-

:2!2 3 wherein B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl, hydroxyalkyl, alkanoyloxyalkyl, alkoxycarbonyllkyl, c-aminobenzyl, furyl, pyridyl, phenyl, phenylamino, styryl or imidazopyridyl, Q' is hydrogen, halogen, hydroxy, aralkyloxy or thienylmnethyl, W is alkylene,

Q

2 is hydrogen, halogen, hydroxy or aralkyloxy, X is alkylene,

Q

3 is hydrogen, halogen, hydroxy, alkoxy, nitro, amino, 2,3dihydrofuryl or 5-methyl-3-oxo-2,3,4,5-tetrahydropyridazin-6-yl; and Y is a single bond, alkylene or alkenylene, and in the formula a broken line is a single bond or a double bond, and

R

s is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkanoyloxy or aralkyloxycarbonyloxy; Rb is a hydrogen, an alkyl, an aralkyl, an aminoalkyl or a mono- or dialkylaminoalkyl; and Rc is an optionally substituted heterocycle containing nitrogen, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A pharmaceutical agent containing a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof, which is a therapeutic agent of at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma and peripheral circulation disorder, which are caused by Rho kinase.

A pharmaceutical agent containing a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof, which is at least one therapeutic agent selected from the group consisting of a therapeutic agent of arteriosclerosis, an anti-cancer drug, an anti-inflammatory agent, an immunosuppressant, a therapeutic agent of autoimmune disease, an anti-AIDS drug, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy, a brain function improving drug, a prophylactic agent of immature birth, a contraceptive and a prophylactic agent of digestive tract infection.

A reagent having a Rho kinase inhibitory activity, which contains a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A diagnostic of a disease caused by Rho kinase, which contains a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(11) A Rho kinase inhibitor containing a substituted isoquinolinesulfonamide derivative of the formula (II)

SO

2 N Alk N R" wherein

R

1 2 is a hydrogen, a chlorine or a hydroxy, and when R 1 2 is a hydrogen, Alk is an alkylene having 2 to 6 carbon atoms, which optionally has alkyl having 1 to 10 carbon atoms, aryl or aralkyl as a substituent; R is a hydrogen;

R

1 4 is a hydrogen, or a linear or branched alkyl having 1 to 6 carbon atoms, an aryl or an aralkyl;

R'

s is a hydrogen, a linear or branched alkyl having 1 to 6 carbon atoms, an aryl or an aralkyl, or a benzoyl, a cinnamyl, a cinnamoyl, a furoyl or a group of the following formula a a. a a

OR

16 wherein R 16 is linear or branched alkyl having 1 to 6 carbon atoms or a group of the following formula

NR

1 7

NHRM

N 18 wherein R 17 and R x 8 are hydrogen or directly bonded to form alkylene having 2 to 4 carbon atoms; or are directly bonded to form alkylene having 4 or less carbon atoms, which is optionally substituted by alkyl having 1 to 10 carbon atoms, phenyl or benzyl, or

R

13 and R 14

R

14 and R 1 5 directly or in combination via oxygen atom form a heterocycle together with the adjacent nitrogen atom, and when R 1 2 is a chlorine or a hydroxy, Alk is an alkylene having 2 to 6 carbon atoms, which is optionally substituted at the hydrogen bonded to carbon by alkyl having 1 to 6 carbon atoms,

R

13 and R 1 4 are each a hydrogen, a linear or branched alkyl having 1 to 6 carbon atoms or directly bonded to each other to form ethylene or trimethylene, wherein hydrogen bonded to carbon is optionally substituted by alkyl having 1 to 6 carbon atoms; or

R

s is a hydrogen, a linear or branched alkyl having 1 to 6 carbon atoms or an amidino, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(12) A pharmaceutical agent containing a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof, which is a therapeutic agent of at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, inflammation and, brain function disorder, which are caused by Rho kinase.

(13) A pharmaceutical agent containing a compound of the formula an isomer %006 thereof and/or a pharmaceutically acceptable acid addition salt thereof, which is at least one therapeutic agent selected from the group consisting of a therapeutic agent of peripheral circulation disorder, a therapeutic agent of arteriosclerosis, an anti-cancer drug, an immunosuppressant, a therapeutic agent of autoimmune disease, an anti-AIDS drug, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy, a prophylactic agent of immature birth, a contraceptive and a prophylactic agent of digestive tract infection.

(14) A reagent having a Rho kinase inhibitory activity, which contains a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A diagnostic for a disease caused by Rho kinase, which contains a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(16) A compound of the formula (III) Ndr Rb L- NN c- N Re'c (Im)

II

O

wherein Rc' is an optionally substituted heterocycle having nitrogen, which is other than pyridine of Rc, and other symbols are as defined above, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(17) The pharmaceutical agent of the above containing a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof as a Rho kinase inhibitor.

(18) A pharmaceutical agent containing a compound of the formula (III), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof, which is at least one therapeutic agent selected from the group consisting of a therapeutic agent of hypertension, a therapeutic agent of angina pectoris, a suppressive agent of cerebrovascular contraction, a therapeutic agent of asthma, a therapeutic agent of peripheral circulation disorder, a therapeutic agent of arteriosclerosis, an anti-cancer drug, an anti-inflammatory agent, an immunosuppressant, a therapeutic agent of autoimmune disease, an anti-AIDS drug, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy, a brain function improving drug, a prophylactic agent of immature birth, a contraceptive and a prophylactic agent of digestive tract infection.

(19) A pharmaceutical composition of the above containing a compound of the formula (III), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof as a Rho kinase inhibitor.

A reagent having a Rho kinase inhibitory activity, which contains a compound of the formula (III), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof as a Rho kinase inhibitor.

(21) A diagnostic for a disease caused by Rho kinase, which contains a compound of the formula (III), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(22) A method for treating a disease based on inhibition of Rho kinase, comprising administering a pharmaceutically effective amount of a Rho kinase inhibitor to a patient.

(23) The treating method of the above wherein the disease treatable by the inhibition of the Rho kinase is at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, a peripheral circulation disorder, arteriosclerosis, cancer, an inflammation, an immune disease, an autoimmune disease, AIDS, osteoporosis, retinopathy, a brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract.

(24) A method for treating at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma and a peripheral circulation disorder, which are caused by Rho kinase, and arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract, which comprises administering a pharmaceutically effective amount of a compound of S" the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(25) A method for treating at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, inflammation and brain function disorder, which are caused by Rho kinase, and a peripheral S circulation disorder, arteriosclerosis, cancer, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract, which comprises administering a pharmaceutically effective amount of a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(26) A method for treating at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract, which comprises administering a pharmaceutically effective amount of a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

(27) Use of a Rho kinase inhibitor for the production of a therapeutic agent of a 13 disease treatable by inhibiting Rho kinase.

(28) The use of a Rho kinase inhibitor of the above wherein the disease treatable by the inhibition of Rho kinase is at least one member selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract.

(29) The use of a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof for the production of a therapeutic agent of at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma and peripheral circulation disorder caused by Rho kinase, and arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract.

(30) Use of a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof for the production of a therapeutic agent of at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, inflammation and brain function disorder caused by Rho kinase, and peripheral circulation Sdisorder, arteriosclerosis, cancer, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract.

Use of a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof for the production of a therapeutic agent of at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract.

(32) A commercial package comprising a Rho kinase inhibitor and a written matter associated therewith, the written matter stating that the Rho kinase inhibitor can or should be used for treating at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract.

(33) A commercial package comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof and a written matter associated therewith, the written matter stating that the compound can or should be used for treating at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma and peripheral circulation disorder, which are caused by Rho kinase, and arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, *0ft fertilization and nidation of fertilized egg and infection of digestive tract.

0000 (34) A commercial package comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof and a written matter associated therewith, the written matter stating that the compound can or should be used for treating at least one disease selected from the group S. consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, inflammation and brain function disorder, which are caused by Rho kinase, and peripheral circulation disorder, arteriosclerosis, cancer, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, immature birth, S fertilization and nidation of fertilized egg and infection of digestive tract.

A commercial package comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof and a written matter associated therewith, the written matter stating that the compound can or should be used for treating at least one disease selected from the group consisting of hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, osteoporosis, retinopathy, brain function disorder, immature birth, fertilization and nidation of fertilized egg and infection of digestive tract Detailed Description of the Invention The Rho kinase inhibitory action, antihypertensive action, anti-angina pectoris action, cerebrovascular contraction suppressive action, anti-asthma action, peripheral circulation improving action, immature birth preventive action, anti-arteriosclerosis action, anti-cancer action, antiinflammatory action, immunosuppressive action, autoimmune disease improving action, anti-AIDS action, preventive action of fertilization and nidation of fertilized egg, preventive action on bacterial infection of digestive tract, osteoporosis treating action, retinopathy treating action and brain function improving action of the present invention can be confirmed by Rho kinase inhibitory activity, vasohypotonic action, trachea relaxing action, peripheral blood flow increasing action, cell adhesion induction inhibitory action, malignant tumor metastasis inhibitory action, bone resorption inhibitory action, mouse allogenic MLR inhibitory activity, tumor cell proliferation inhibitory action, angiogenesis inhibitory action, vascular smooth muscle cell proliferation inhibitory action and the like.

SOS.

SThe disease relating to Rho, on which the inventive Rho kinase inhibitor is effective include, for example, disease symptoms of hypertension, angina pectoris, cerebrovascular contraction, asthma, peripheral circulation disorder, immature birth, arteriosclerosis, cancer, inflammation, immune disease, autoimmune disease, AIDS, bacterial infection of digestive tract, osteoporosis, retinopathy, brain function disorder and the like, as well as biological phenomena such as fertilization and nidation of fertilized egg.

As used herein, by the Rho kinase of the present invention is meant serine/threonine kinase activated along with the activation of Rho, which is exemplified by the aforementioned ROCa(ROCKII), pl60ROCK(ROCp, ROCK-I) and other proteins having serine/threonine kinase activity.

Cancer includes bone marrow leukemia, lymphocytic leukemia, gastric cancer, colon cancer, lung cancer, pancreatic cancer, liver cancer, cancer of esophangus, ovarian cancer, breast cancer, skin cancer, cervical cancer, orchioncus, neuroblastoma, urinary epithelial cancer, multiple myeloma, uterine cancer, melanoma, cerebral tumor and the like, and anti-cancer means inhibition of formation, infiltration, metastasis, growth and the like of these tumors.

The immune disease includes allergic diseases, rejection in organ transplantation and the like.

The autoimmune disease includes articular rheumatism, systemic lupus erythematodes, Sj6gren's disease, multiple sclerosis, myasthenia gravis, type I diabetes, endocrine ophthalmopathy, primary biliary cirrhosis, Crohn's disease, glomerulonephritis, sarcoidosis, psoriasis, pemphigus, hyoplastic anemia, essential thrombocytopenic purpura and the like.

Bacterial infection of digestive tract means various diseases caused by the invasion of Salmonella, dysentery bacillus, intestinal pathogenic Escherichia coli and the like into intestinal mucosa epithelial cells.

Retinopathy means angiopathic retinopathy, arteriosclerosis retinopathy, central angiospastic retinopathy, central serous retinopathy, circinate retinopathy, diabetic retinopathy, dysproteinemic retinopathy, hypertensive retinopathy, leukemic retinopathy, lipemic retinopathy, proliferative retinopathy, renal retinopathy, sickle retinopathy, toxemic retinopathy of pregnancy and the like.

Brain function disorder includes psychotic condition due to cerebral hemorrhage, cerebral thrombus, cerebral embolus, subarachnoid hemorrhage, transient cerebral ischemic stroke, hypertensive encephalopathy, cerebral arteriosclerosis, subdural hematoma, extradural hematoma, cerebral hypoxia, cerebral edema, cerebritis, cerebral tumor, external injury in head, mental disease, metabolite poisoning, drug poisoning, temporal respiratory arrest, deep anesthesia during operation, physical disorder and the like, and sequelae, decreased attention, hyperactivity, logopathy, delayed mental development, lethe, dementia (inclusive of S"wandering, nocturnal delirium, aggressive behavior and the like associated with dementia) caused by the above-mentioned diseases.

Therefore, the Rho kinase inhibitor of the present invention is effective as a pharmaceutical agent, particularly as an agent for the prophylaxis and treatment of these diseases caused by Rho, such as a therapeutic agent of hypertension, a therapeutic agent of angina pectoris, a suppressive agent of cerebrovascular contraction, a therapeutic agent of asthma, a therapeutic agent of peripheral circulation disorder, a prophylactic agent of immature birth, a therapeutic agent of arteriosclerosis, an anti-cancer drug, an anti-inflammatory agent, an immunosuppressant, a therapeutic agent of autoimmune disease, an anti-AIDS drug, a contraceptive, a prophylactic agent of digestive tract infection, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy and a brain function improving drug.

The compounds of the formula and the formula (II) have high affinity for Rho kinase. Thus, the radioactive substance (radio ligand) thereof are industrially useful as a selective radio ligand of Rho kinase. The compounds of the formula and the formula (II) and modified compounds thereof radio ligand of these compounds and the like), which are Rho kinase inhibitors, are useful as reagents for the study of Rho and Rho kinase and as diagnostics of the diseases relating to them.

The compound to be used as the Rho kinase inhibitor of the present invention may be any as long as it has a Rho kinase inhibitory action. For example, the compounds of the formula and the formula (II) are used.

In the present specification, each symbol of the formula is defined as follows.

Alkyl at R and R' is linear or branched alkyl having 1 to 10 carbon atoms, which is exemplified by methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, *tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like, with preference given to alkyl having 1 to 4 carbon atoms.

Cycloalkyl at R and R 1 has 3 to 7 carbon atoms and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cydoheptyl and the like.

Cycloalkylalkyl at R and R 1 is that wherein the cycloalkyl moiety is the above-mentioned cycloalkyl having 3 to 7 carbon atoms and the alkyl moiety is linear or branched alkyl having 1 to 6 carbon atoms methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl and the like), which is exemplified by cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, *cycloheptylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, cycloheptylethyl, cyclopropylpropyl, cyclopentylpropyl, cyclohexylpropyl, cycloheptylpropyl, cyclopropylbutyl, cyclopentylbutyl, cyclohexylbutyl, cycloheptylbutyl, cyclopropylhexyl, cyclopentylhexyl, cyclohexylhexyl, cycloheptylhexyl and the like.

Aralkyl at R and R' is that wherein alkyl moiety is alkyl having 1 to 4 carbon atoms and is exemplified by phenylalkyl such as benzyl, 1-phenylethyl, 2- 18 phenylethyl, 3-phenylpropyl, 4-phenylbutyl and the like.

The substituent of optionally substituted cycloalkyl, cycloalkylalkyl, phenyl and aralkyl on the ring at R and R' is halogen chlorine, bromine, fluorine and iodine), alkyl (same as alkyl at R and alkoxy (linear or branched alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like), aralkyl (same as aramlkyl at R and or haloalkyl (alkyl at R and R' which is substituted by 1-5 halogen, and exemplified by fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl and the like), nitro, amino, cyano, azide and the like.

The group formed by R and R' in combination together with the adjacent nitrogen atom, which forms a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom is preferably a 5 or 6membered ring and bonded ring thereof. Examples thereof include 1-pyrrolidinyl, piperidino, 1-piperazinyl, morpholino, thiomorpholino, 1-imidazolyl, 2,3dihydrothiazol-3-yl and the like. The substituent of the optionally substituted nitrogen atom is exemplified by alkyl, aralkyl, haloalkyl and the like. As used herein, alkyl, aralkyl and haloalkyl are as defined for R and R 1 Alkyl at R 2 is as defined for R and R'.

Halogen, alkyl, alkoxy and aralkyl at R 3 and R are as defined for R and R'.

Acyl at R 3 and lR4 is alkanoyl having 2 to 6 carbon atoms acetyl, propionyl, butyryl, valeryl, pivaloyl and the like), benzoyl or phenylalkanoyl wherein the alkanoyl moiety has 2 to 4 carbon atoms phenylacetyl, phenylpropionyl, phenylbutyzyl and the like).

Alkylamino at R 3 and R 4 is that wherein the alkyl moiety is alkylamino having linear or branched alkyl having 1 to 6 carbon atoms. Examples thereof Sinclude methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, hexylamino and the like.

Acylamino at R' and R 4 is that wherein acyl moiety is alkanoyl having 2 to 6 carbon atoms, benzyl or the alkanoyl moiety is phenylalkanoyl having 2 to 4 carbon atoms and the like, which is exemplified by acetylamino, propionylamino, butyrylamino, valerylamino, pivaloylamino, benzoylamino, phenylacetylamino, phenylpropionylan-iino, phenylbutyrylamino and the like.

Alkylthio at W 3 and R 4 is that wherein the alkyl moiety is linear or branched alkyl having 1 to 6 carbon atoms, which is exemplified by methyiffijo, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio, hexylthio and the like.

Aralkyloxy at W 3 and W 4 is that wherein the alkyl moiety is alkyl having 1 to 4 carbon atoms, which is exemplified by benzyloxy, 1-phenylethyloxy, 2phenylethyloxy, 3-phenypropyloxy, 4-phenylbutyloxy and the like.

Aralkylthio, at RW and W 4 is that wherein the alkyl moiety is alkyl having 1 to 4 carbon atoms, which is exemplified by benzylthio, 1 -phenylethylthio,2phenylethylthio,3-phenylpropylthio,4-phenylbutylthio and the like.

Alkoxycarbonyl at RW and le is that wherein the alkoxy moiety is linear or branched alkoxy having 1 to 6 carbon atoms, which is exemplified by methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl and the like.

Alkylcarbamnoyl. at RW and le is carbamoiyl mono- or di-substituted by alkyl having 1 to 4 carbon atoms, which is exemplified by methylcarbanioyl, dimethylcarbamoyl, ethylcarbarmoyl, diethylcarbamoyl, propylcarbamnoyl, dipropylcarbamoyl, butylcarbamoqyL dibutylcarbamnoyl and the like.

Alkoxy at RW is as defined for R and R'.

Alkoxycarbonyloxy at RW is that wherein thle alkoxy moiety is linear or branched alkoxy having 1 to 6 carbon atoms, which is exemplified by methoxycarbonyoxy, ethoxycarbonyloxy, propoxycarbonyloxy, isopropoxycarbonyloxy, butoxycarbonyloxy, isobutoxycarbonyloxy, sec-butoxycarbonyloxy, tert- *.:butoxycarbonyloxy, pentyloxycarbonyloxy, hexyloxycarbonyloxy and the like.

~*Alkanoyloxy at R' is that wher-ein the alkanoyl, moiety is alkanoyl. having 2 to 6 carbon atoms, which is exemplified by acetyloxy, propionyloxy, butxyloxy, valeryloxy, pivaloyloxy and the like.

Aralkyloxycarbonyloxy at W 5 is that wherein the aralkyl moiety is aralkyl having C 1

-C

4 alkyl, which is exemplified by benzyloxycarbonyloxy, 1phenylethyloxycarbonyloxy, 2-phenylethyloxycarbonyloxy, 3phenypropyloxycarbonyloxy, 4-phenylbutyloxycarbonyloxy and the like.

Alkyl at W 6 is as defined for R and alkyl at W 8 and R 9 is as defined for R and and aralkyl at W 8 and R? is as defined for R and R'.

Alkyl at R 7 is as defined for R and R 1 and aralkyl at RW is as defined for R and R 1 The group formed by W 6 and R 7 in combination, which forms a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom, is imidazol-2-yl, thiazol-2-yl, oxazol-2-yl, imidazolin-2-yl, 3,4,5,6tetrahydropyridin-2-yl, 3,4,5,6-tetrahydropyrimidin-2-yl, 1,3-oxazolin-2-yl, 1,3thiazolin-2-yl or optionally substituted benzoimnidazol-2-yl, benzothiazol-2-yl, benzoxazol-2-yl and the like having a substituent such as halogen, alkyl, alkoxy, haloalkyl, nitro, amino, phenyl, aralkyl and the like. As used herein, halogen, alkyl, alkoxy, haloalkyl and aralkyl are as defined for R and R'.

The sub stituent of the above-mentioned optionally substituted nitrogen atom is exemplified by alkyl, aralkyl, haloalkyl and the like. As used herein, alkyl, aralkyl and haloalkyl are as defined for R and R'.

Hydroxyalkyl at R 10 and R" 1 is-linear or branched alkyl having 1 to 6 carbon atoms which is substituted byl1 to 3 hydroxy, which is exemplified by hydroxymethyl, 2-hydroxyethyl, 1-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl and the like. Alkyl at W 10 and W 11 is as defined for R and R 1 haloalky and alkoxycarbonyl at R" 0 and RW 1 are as defined for R and R 1 aralkyl at R 10 and R 11 is as defined for R and and cycloalkyl formed by R 10 and W 11 in combination is the same as cycloalkyl at R and R 1 Alkyl at L is as defined for R and R 1 Aminoalky at L is a linear or branched alkyl having 1 to 6 carbon atoms, which is substituted by amino, which is exemplified by aminomethyl, 2aminoethyl, 1-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6aninohexyl and the like.

Mono- or dialkylaminoalkyl at L is mono- or di-substituted aminoalkyl with alkyl having 1 to 4 carbon atoms, which is exemplified by methylaninomethyl, dimethylaminomnethyl, ethylamninomethyl, diethylaniinomnethyl, propylam-inomethyl, dipropylan-inomethyl, butylaminomethyl, dibutylaminomethyl, 2-dimethylaminoethyl, 2-diethylarninoethyl and the like.

Carbamnoylalkyl at L is linear or branched alkyl having 1 to 6 carbon atoms substituted by carbamoyl, which is exemplified by carbamoylmethyl, 2carbamoylethyl, 1 -carbamoylethyl, 3-carbamoylpropyl, 4-carbamoylbutyl, carbamoylpentyl, 6-carbamoylhexyl and the like.

Phthalimidoalkyl at L is linear or branched alkyl having 1 to 6 carbon atoms, which is substituted by phthalimide. Examples thereof include phthalimi~domethyl, 2-phthalimidoethyl, 1 -phthainidoethyl, 3-phthalimidopropyl, 4-phthalimidobutyl, 5-phthalimridopentyl, 6-phthaimidohexyl and the like.

Alkyl at Bis asdefinedfor Rand R 1 Alkoxy at B is as defined for R and R 1 Aralkyl at B is as defined for R and R'.

Aralkyloxy at B is as defined for RW andR.

Aminoalkyl at B is as defined for L.

Hydroxyalkyl at B is as defined for R' 0 and R" 1 Alkanoyloxyalkyl at B is that wherein linear or branched alkyl having 1 to 6 carbon atoms is substituted by alkanoyloxy having aikanoyl moiety having 2 to 6 carbon atoms, which is exemplified by acetyloxymethyl, propionyloxymethyl, butyzuyloxymethyl, valeryloxymethyl, pivaloyloxymnethyl, acetyloxyethyl, propionyloxyethyl, butyiyloxyethyl, valeryloxyethyl, pivaloyloxyethyl and the like.

Alkoxycarbonylalkyl at B is that wherein linear or branched alkyl having 1 to 6 carbon atoms is substituted by ailkoxycarbonyl having alkoxy moiety having 1 to 6 carbon atoms, which is exemplified by metho xycarbonylmethyl, ethoxycarbonylmethyl, propoxycarbonylmethyl. isopropoxycarbonylmethyl, butoxycarbonylmethyl, isobutoxycarbonylmethyl, sec-butoxycarbonylmethyl, tert-butoxycarbonylmnethyl, pentyloxycarbonylmethyl, hexyloxycarbonylmethyl, methoxycarbonylethyl, ethoxycarbonylethyl, propoxycarbonylethyl, **.:isopropoxycarbonylethyl, butoxycarbonylethyl, isobutoxycarbonylethyl, secbutoxycarbonylethyl, tert-butoxycarbonylethyl, pentyloxycarbonylethyl, hexyloxycarbonylethyl and the like.

Halogen at Q 2 and Q 3 is as defined for R and R 1 AralkyloXy at Q 1 and Q 2 is as defined for W? andR.

Alkoxy at Q 3 is as defined for R and R'.

Alkylene at W, X and Y is linear or br-anched ailkylene having 1 to 6 carbon atoms, which is exemplified by methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene and the like.

Alkenylene at Y is linear or branched alkenylene having 2 to 6 carbon atoms, which is exemplified by vinylene, propenylene, butenylene, pentenylene and the like.

Alkyl at Rb is as defined for R and R 1 Aralkyl at. Rb is as defined for R and R'.

Aminoalkyl at Rb is as defined for L.

Mono- or dialkyLarninoalkyl at Rb is as defined for L.

The heterocycle when single ring containing nitrogen at Rc is pyridine, pyrimidine, pyridazine, triazine, pyrazole, triazole and the like, and when it is a condensed ring, it is exemplified by pyrrolopyridine 1H-pyrrolo[2,3-bjpyridine, 1 H-pyrrolop3,2-blpyridine, 1 H-pyrrolo[3,4-bjpyridine and the like), pyrazolopyridine lH-pyrazoloP3,4-b~pyridine, 1H-pyrazolo[4,3-blpyridine and the like), iinidazopyridine 1H-imidazo[4,5-blpyridine and the like), pyrrolopyrimidine 1 H-pyrrolo[2,3-dlpyrimidine, 1 -pyrrolo[3,2-dipyrimidine, 1H-pyrrolo[3,4-d~pyriniidine and the like), pyrazolopyrimidine 1H-pyrazoloP3,4-djpyrimidine, pyrazolo[l ,5-alpyriniidine, 1H-pyr-azolo[4,3d~pyriinidine and the like), imidazopyrimidine. imidazoll,2-ajpyrimidine, 1Hand the like), pyrrolotriazine pyr-roio[i,2-al-i,3,5triazine, pyrrolo[2, 1,2,4-triazine), pyrazolotriazine pyrazolo[ 1,5-al- 1,3,5triazine and the like), triazolopyridine 1 H-1l,2,3-tiazolo[4,5-bjpyrIdine and the like), triazolopyrimidine 1,2,4-tiazolo[1,5-ajpyriniidine, 1,2,4tiazolo[4,3-alpyrimidine, 1H-1,2,3-tiazlo[4,5-dlpyrinijidine and the like), cinnoline, quinazoline, quinoline, pyridopyridazine pyrido[2,3-cipyridazine and the like), pyridopyrazine pyrido[2,3-blpyrazine and the like), pyridopyrimidine pyrido[2,3-djpyrimidine, pyrido[3,2-djpyrimidine and the like), pyrimidopyrimidine pyrimido[4,5-dipyrimidine, pyrimido[5,4dipyrimidine and the like), pyrazinopyrimidine pyrazino[2,3-dlpyriniidine and the like), naphthyridine 1,8-naphthyridine and the like), tetrazolopyrimidine tetrazolo[1,5-alpyrimidine and the like), thienopyridine thieno[2,3b~pyridine and the like), thienopyrimidine thieno[2,3-djpyrimidine and the like), thiazolopyridine thiazolo[4,5-blpyridine, thiazolo[5,4-blpyridine and the like), thiazolopyriinidine thiazolo[4,5-dlpyrimidine, thiazolo[5,4-djpyrimidine 23 and the like), oxazolopyridine oxazolo[4,5-blpyridine, oxazolo[5,4-blpyridine and the like), oxazolopyrimidine oxazolo[4,5-djpyrimidine, oxazolo[5,4d~pyrimidine and the like), fur-opyridine furo[2,3-blpyridine, furo[3,2..

bjpyridine and the like), fuiropyrimidine firo[2,3-dlpyrimidine, furo[3,2dipyrimidine and the like), 2,3-dihydropyrrolopyridine 2,3-dihydro- 1Rpyrrolo[2,3-bjpyridine, 2,3-dihydr-o-1lH-pyrrolo[3,2-bjpyridine and the like), 2,3dihydr-opyrr-olopyrimidine 2,3-dihydro- 1H-pyrrolo[2,3-dlpyrimidine, 2,3dihydrp-1H-pyrrolo[3,2-dlpyrimidine and the like), 5,6,7,8-tetrahydropyrido[2,3djpyrimidine, 5,6,7,8-tetr-ahydr-o- 1,8-naphthyridine, 5,6,7,8-tetrahydroquinMoline and the like. When these rings form a hydrogenated aromatic ring, the carbon atom in the ring may be carbonyl and includes, for example, 2,3-dihydro-2oxopyrrolopyridine, 2,3-dihydro-2 ,3-dioxopyrrolopyridine, 7,8-dihydro-7-oxo- 1,8naphthyridine, 5,6,7,8-tetrahydro-7-oxo- 1,8-naphthyridine and the like.

These rings may be substituted by a substituent such as halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, amino, alkylami[no, cyano, formyl, acyl, aminoalkyl, mono- or diakylaminoalkyl, azide, carboxy, alkoxycarbonyl,' carbamnoyl, alkylcarbamnoyl, alkoxyalkyl methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl and the like), optionally substituted hydrazino and the like.

As used herein, the substituent of the optionally substituted hydrazino includes alkyl, aralkyl, nitro, cyano and the like, wherein alkyl and aralkyl are as defined for R and R' and exemplified by methyl hydrazino, ethyl hydrazino, benzyl hydrazino and the like.

In the present specification, each symbol of the formula (II) is defined as follows.

The linear or branched alkyl having 1 to 6 carbon atoms at W 3

R'

4

,R'

~and R 6 ismtyehlropyl, isopropyl, butyl, isobutyl, sec-btyl, etbtl pentyl, hexyl and the like.

Aryl at R 14 and R 5 is phenyl, naphthyl and the like.

Aralkyl at R' 4 and R 15 is as defined for R and R'.

Alkylene having 4 or less carbon atoms, which is formed by R3 and R 14 directly bonded to each other, is methylene, ethylene, trimethylene, propylene, tetramethylene and the like.

Alkyl having 1 to 10 carbon atoms, which substitutes alkylene having 4 or less carbon atoms formed by R 13 and R' 4 directly bonded to each other, is linear or branched alkyl having 1 to 10 carbon atoms. Examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like.

Alkyl having 1 to 6 carbon atoms which substitutes ethylene and trimethylene formed by RU and R" directly bonded to each other is linear or branched alkyl having 1 to 6 carbon atoms, which is the same as those for R' 3 The heterocycle formed by R 1 4 and R 1 5 directly or via oxygen atom bonded together with the adjacent nitrogen atom is pyrrolidino, piperidino, morpholino, homopiperidino, homomorpholino and the like.

Alkylene having 2 to 4 carbon atoms formed by R 1 7 and R 18 directly bonded to each other is ethylene, trimethylene, propylene, tetramethylene and the like.

Alkylene having 2 to 6 carbon atoms at Alk is ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene and the like.

Alkyl having 1 to 6 carbon atoms and alkyl having 1 to 10 carbon atoms, which are the substituents of alkylene having 2 to 6 carbon atoms at Alk, are as i defined for R 13 SAryl and aralkyl, which are the substituents of alkylene having 2 to 6 carbon atoms at Alk, are as defined for R 4 SThe compound to be used as the Rho kinase inhibitor of the present invention is, for example, a compound of the formula which is exemplified by the following compounds.

4-(2-pyridylcarbamoyl)piperidine 1-benzyloxycarbonyl-4-(4-pyridylcarbamoyl)piperidine 1-benzoyl-4-(4-pyridylcarbamoyl)piperidine 1-propyl-4-(4-pyridylcarbamoyl)piperidine [3-(2-(2-thienylmethyl)phenoxy)-2-hydroxypropyl]-4-(4-pyridylcarbamoyl)piperidine 4-(4-pyridylcarbamoyl)piperidine 1-benzyl-4-(4-pyridylcarbamoyl)-1,2,5,6-tetrahydropyridine 3-(4-pyridylcarbamoyl)piperidine 1-benzyl-3-(4-pyridylcarbamoyl)piperidine 1 -(2-(4-benzyloxyphenoxy)ethyl)-4-(N-(2-pyridyl)-N-benzylcarbamoyl)pyridime (11) 1-formyl-4-(4-pyridylcarbamoyl)piperidine (12) 4-(3-pyridylcarbamoyl)piperidine (13) 1-isopropyl-4-(4-pyridylcarbamoyl)piperidine (14) 1-methyl-4-(4-pyridylcarbamoyl)piperidine 1-hexyl-4-(4-pyidylcarbamnoyl)piperidine (16) 1-benzyl-4-(4-pyridylcarbamoyl)piperidine (17) 1-(2-phenylethyl)-4-(4-pyridylcarbamoyl)piperidine (18) 1-(2-(4-methoxyphenyl)ethyl)-4-(4-pyridylcarbamoyl)piperidine (19) 1 -(2-(4-methoxyphenyl)ethyl)-4-(2-pyridylcarbamoyl)piperidine 1-(2-(4-chlorophenyl)ethyl)-4-(4-pyridylcarbamoyl)piperidine (21) 1-diphenylmethyl-4-(2-pyridylcarbamoyl)piperidine (22) 1-12-(4-(5-methyl-3-oxo-2,3 ,4,5-tetrahydropyridazin-6-yl)phenyl)ethyl-4-(2pyridylcarbamnoyl)piperidine (23) 1-(4-(4,5-dihydro-2-furyl)phenyl)-4-(4-pyridylcarbainoyl)piperidine 9* (24) 1-(2-nitrophenyl)-4-(4-pyridylcarbamoyl)piperidine 1-(2-aminophenyl)-4-(4-pyridylcarbamoyl)piperidine (26) 1-nicotinoyl-4-(4-pyridylcarbamoyl)piperidine (27) 1-isonicotinoyl-4-(4-pridylcarbamoyl)piperidine set 0 (28) 1-(3,4,5-trimethoxybenizoyl)-4-(4-pyridylcarbamoyl)piperidine (29) 1-acetyrl-4-(4-pyridylcarbamoyl)piperidine 193(-looezy~rpl--(-yiycraolpprd (30) 1-(3-(4-fluorobenzoyl)propyl)-4-(4-pyridylcarbamoyl)piperidine (31) 1-(3-(4-fluoroxbenzoyl)ropyl)-4-(2-pyridylcarbamoyl)piperidine (33 19l(-ezlxbnolehl-4(-yiycraolppzdn (32) 1-(4-hydroxybenoy)ethyl)-4-(2-pyridylcarbamoyl)piperidine (35) 1 -(4-(4-fluorophenyl)-4-hydroxybutyl)-4-(4-pyridylcarbamoyl)piperidine (36) 1-methyl-2-(4-hydroxyphenyl)-2-hydroxyethyl)-4-(2-pyridylcarbamoyl)piperidine (37) 1-cinnamylA4-(2-pyridylcarbamoyl)piperidine (38) 1-(2-hydroxy-3-phenoxypropyl)-4-(4-pyridylcarbamoyl)piperidine (39) 1 -(2-hydroxy-3-phenoxypropyl)-4-(3-pyridylcarbamoyl)piperidine 1 -(2-hydroxy-3-phenoxypropyl)-4-(2-pyridylcarbamoyl)piperidine 26 (41) 1-(2-phenylethyl)-4-[ N-(2-pyridyl)-N-(2-( N, N-dimethyLamino)ethyl)carbamoyljpiperidine (42) 1-benzyloxycarbonyl-4-(2-pyridylcarbamoyl)piperidine (43) 1-(3-chlorophenyl)carbamoYl-4-(4-pyridylcarbamoyl)piperidine (44) N-(2-pyridyl)-N-(2-( N, N-dimethylamino)ethyl)carbamoyljpiperidine 1-methyl-4-(4-pyridylcarbamoyl)- 1,2,5,6-tetrahydropyridine (46) 1-nicotinoyl-3-(4-pyridylcarbamoyl)piperidine (47) 1-[2-(4-fluorobenzoyl)ethylJ-4-(4-pyridylcarbamoyl)piperidine (48) 1-(6-chloro-2-methylliidazo[ 1,2-a~pyridine-3-carbonyl)-4-(4pyridylcarbamoyl)piperidine (49) 1-(4-nitr-obenzyl)-4-(4-pyridylcarbamoyl)piperidine 1-hexyl-4-(4-pyridylcarbamoyl)piperidine (51) 1-benzyloxycarbonyl-4-(2-chloro-4-pyridylcarbamoyl)piperidine (52) 4-(2-chloro-4-pyridylcarbamnoyl)piperidine (53) 1-(2-chloronicotinoyl)-4-(4-pyridylcarbamoyl)piperidine '0004 (54) 3-(2-chloro-4-pyridylcarbamoyl)piperidine 1-(4-phthalimidobutyl)-4-(4-pyridylcarbamoyl)piperdine 1-(3,5-di-tert-butyl-4-hydroxycinnamoyl)-4-(4-pyridylcarbamoyl)piperidine too. (57) 1-carbamoylmethyl-4-(4-pyridylcarbamoyl)piperidine (58) 1-benzyloxycarbonyl-4-(5-nitro-2-pyridylcarbamoyl)piperidine (59) 4-(5-nitro-2-pyridylcarbamoyl)piperidine trans-4-benzyloxycarboxamidomethyl-l1-(4-pyridylcarbamoyl)cyclohexane (61) tr-ans-4-aniinomethyl-l1-(4-pyridylcarbamoyl)cyclohexane (62) trans-4-fonnamidomethyl- 1-(4-pyridylcarbamoyl)cyclohexanc (63) trans-4-dimethylaniinomethyl- 1-(4--pyridylcarbamoyl)cyclohexane 8 (64) N-benzylidene-trans-(4-pyridylcarbamoyl)cyclohexylmethyLamine trans-4-benzylaminomethyl-1-(4-pyridylcarbamoyl)cyclohexane (66) tr-ans-4-isopropylaminomethyl- 1-(4-pyridylcarbamoyl)cyclohexane (67) tr-ans-4-nicotinoylaxninomethyl- 1-(4-pyridylcarbamoyl)cyclohexane (68) trans-4-cyclohexylaminomethyl- 1-(4-pyridylcarbamoyl)cyclohexane (69) trans-4-benzyloxycarboxamide- 1-(4-pyridylcarbanioyl)cyclohexane trans-4-amino- 1-(4-pyridylcarbamoyl)cyclohexane (71) tr-ans-4-( 1-aininoethyl)-l1-(4-pyridylcarbamoyl)cyclohexane (72) trans-4-aniinomethyl-cis-2-methyl- 1-(4-pyridylcarbamoyl)cyclohexane (73) 1-benzyloxycarboxamidopropyl)- 1-cyclohexanecarboxylic acid (74) (+)-trans-4-(l1-benzyloxycarboxamridopropyl)-l1-(4-pyridylcarbamoyl)cyclohexane 1-benzyloxycarboxaniidpropyl)- 1-(4-pyridylcarbamoyl)cyclohexane (76) (+)-trans-4-(l1-aminopr-opyl) -1-(4-pyridylcarbamoyl)cyclohexane (77) (-)-trans-4-(l1-aninopropyl)- 1-(4-pyridylcarbamoyl)cyclohexane (78) 1-benzyloxycarboxamridoethyl)- 1-(4-pyridylcarbamoyl)cyclohexane (79) 1-benzyloxycarboxamidoethyl)- 1-(4-pyridylcarbamoyl)cyclohexane 1-aninoethyl)- 1-(4-pyridylcarbamoyl)cyclohexane (81) 1-aninoethyl)-l1-(4-pyridylcarbamoyl)cyclohexane (82) trans-4-(4-chlorobenzoyl)aminomethyl- 1-(4-pyridylcarbamoyl)cyclohexarie (83) trans-4-aminomethyl- 1-(2-pyridylcarbamoyl)cyclohexane (84) trans-4-benzyloxycarboxamidomethyl- 1-(2-pyridylcarbarnoyl)cyclohexane o* (85) trans-4-methyaninomethyl- 1-(4-pyridylcarbamoyl)cyclohexane (86) trans-4-( N-benzyl-N-methylaxnino)methyl- 1-(4-pyridylcarbamoyl)cyclohexane o o: (87) trans-4-aninomethyl- 1-3-pyridylcarbamoyl)cyclohexane *000 (88) trans-1-aminomethyl-1-[(3-hydroxy-2-pyridyl)carbamoylicyclohexane 0 .0(89) trans-4-benzyloxycarboxaniidomethyl-1-(3-pyridylcarbamoyl)cyclohexane trans-4-benzyloxycarboxamidomethyl- 1-[(3-benzyloxy-2-pyridyl)carbamoyllcyclohexane (91) trans-4-phthaimidomethyl-l1-(4 -pyridylcarbamoyl)cyclohexane 0 (92) trans-4-benzyloxycarboxaniidomethyl-l1-(3-methyl-4-pyridylcarbamoyl)cyclohexane (93) trans-4-aminomethyl- 1-(3-methyl-4-pyridylcarbamoyl)cyclohexane (94) 4-(trans-4-benzyloxycarboxamiidomethylcyclohexylcarbonyl)amino-2,6dimethylpyrldine-N-oxide 4-(trans-4-aminomefliylcyclohexylcarbonyl)amiino-2,6-dimethylpyridine-Noxide (96) trans-4-aminomethyl-l1-(2 -methyl-4-pyridylcarbamoyl)cyclohexane 28 (97) trans-4-(l1-benzyloxycarboxamridoethyl)- 1-(4-pyridylcarbamoyl)cyclohexane (98) trans-4-( 1-amiino-i -methylethyl)-l1-(4 -pyridylcarbamoyl)cyclohexane (99) trans-4-(2-aniinoethyl)-l1-(4-pyridylcarbamoyl)cyclohexane (100) trans-4-(2-amino- 1-methylethyl)- 1-(4-pyridylcarbamnoyl)cyclohexane (101) trans-4-(l1-aniinopr-opyl)-l1-(4-pyridylcarbamoyl)cydlohexane (102) trans-4-aminomethyl-trans- 1-methyl-i -(4-pyridylcarbamoyl)cyclohexane (103) tr-ans-4-benzylamiinomethyl-cis-2-methyl-l1-(4-pyridylcarbamoyl)cyclohexane (104) trans-4-(l1-benzyloxycarboxamide-l1-methylethyl)- i-(4-pyridylcarbamoyl) cyclohexane (105) tr-ans-4-benzyloxycarboxamidomethyl- N-methyl-4-pyridylcarbamoyl)cyclohexane (106) trans-4-( 1-acetamide- 1-methylethyl)- 1-(4-pyridylcarbamoyl)cyclohexane (107) trans-N-(6-amlino-4-pyriniidyl)-4-aminomethylcyclohexanecarboxamide (108) tr-ans-N-( 1H-pyrrolo[2,3-bjpridin-4-yl)A4-aminomethylcyclohexanecarboxamide (109) (+)-trans-N-(l1H-pyrrolo[2,3-blpyridin-4-y)-4-( 1-aminoethlyl)cyclohexanecarboxamide (110) trans-N-( 1H-pyrrolo[2,3-b~pyridin-4-yl)-4-( 1-amino- 1-methylethlyl)cyclohexanecarboxamide (111) trans-N-( iH-pyrazolo[3,4-blpyridin-4-yl)-4-aminomethylcyclohexanecarboxamide (112)- (+)-tr-ans-N-(l1H-pyrazolo[3 ,4-bjpyridin-4-yl)-4-(l1-aminoethyl)cyclohexanecarboxamide tans-N-( 1H-pyrazolo[3,4-blpyridin-4-y1)-4-( 1-amino- 1-methylethlyl)cyclohexanecarboxamnide (114) (+)-trans-N-(2-amino-4-pyridyl)-4-( 1-amiinoethyl)cyclohexanecarboxamide (115) trans-N-( 1H-pyrazolo[3,4-dipyrimidin-4-yl)-4-aminomethylcyclohexanecarboxamide (116) (+)-trans-N-(l1H-pyrazolo[3 ,4-djpyrimidin-4-yl)-4-( 1-amlinoethyl)cyclohexanecarboxarrnide (117) trans-N-( 1H-pyrazolo[3,4-djpyrimidin-4-yl)-4-(l1-amino- 1-methylethyl)cyclohexanecarboxamnide (118) tr-ars-N-(4-pyrin-iidinyl)-4-aminomethylcyclohexanecarboxamride (119) trans-N-(3-amino-4-py-ridyl)-4-aminomethylcyclohexanecarboxamide (120) trans-N-(7H-imidazo[4,5-djpyrimidin-6-yl)-4-aminomethylcyclohexanecarboxamide (12 1) trans-N-(3H- 1,2 ,3-triazolol4,5-dlpyrimiidin-7-yl) -4-anilnomethylcyclohexanecarboxamide (122) trans-N-(l1-benzyl- 1H-pyrazolo[3,4-blpyridin-4-yl)-4-aminomethylcyclohexaecarboxan-ide (123) tr-ans-N-(l1H-5-pyrazolyl)-4-aminomethylcyclohexanecarboxamide (124) tr-ans-N-( lH-pyrazolo[,4-bjpyridin-4-yl)-4-aminomethylcyclohexanecarboxamide (125) traris-N-(4-pyridazinyl)-4-amidnomethylcyclohexanecarboxamide (126) trans-N-(7H-pyrrolo[2,3-d~pyrimidin-4-y1)-4-aminomethylcyclohexanecarboxamide (127) tr-ans-N-(2-amino-4-pyridyl)-4-aminomethylcyclohexanecarboxamide (128) tr-ans-N-(thieno[2 ,3-djpyriniidin-4-yl)-4-aminomethylcyclohexanecarboxamide (129) trans-N-(5-methyl- 1,2,4-tiazolo[ 1,5-ajpyrimidin-7-yl)-4-aminomethylcyclohexanecarboxaniide (130) trans-N-(3-cyano-5-methylpyrazolo[ 1,5-alpyrimidin-7-yl)-4-aminomethylcyclohexanecarboxamidde (131) trans-N-( 1H-pyrazolo[3,4-bjpyridin-4-yl)-4-( 1-amino- 1-methylethyl)cydlohexanecarboxamide (132) trans-N-(2-( 1-pyrrolidinyl)-4-pyridyl)-4-aminomethylcyclohexanecarboxamide (133) trans-N-(2,6-diamino-4-pyriniidyl)-4-aniinomethylcyclohexanecarboxamide (134) (+)-trans-N-(7-methyl- 1,8-naphthyridin-4-yl)-4-( 1-aniinoethyl)cyclohexanecarboxamide (135) trans-N-( 1-benzyloxymethylpyrrolo.[2 ,3-bjpyridin-4-yl)-4-amiinomethylcyclohexanecarboxanide (136) (-i)-trans-N-(l1-methylpyrrolo[2,3-bjpyridin-4-yl) 1-aminoethyl)cyclohexanecarboxanide (137) trans-N-benzyl-N-(2-benzylaniino-4-pyridyl)-4-( 1-amino- 1-methylethyl)cyclohexanecarboxaniide (138) trans-N-(2-azide-4-pyridyl)-4-aminomethylcyclohexanecarboxamide (139) trans-N-(2,3-dihydro- 1H-pyrrolo[2,3-bjpyridin-4-yl)-4-aminomethylcydlohexanecarboxamide (140) trans-N-(2 '3-dihydr-o- LH-pyrrolo[2,3 -bjpyridin-4-yl)-4-( 1-amino-i1methylethyl)cyclohexanecarboxarmde (141-1) trans-N-(2-carboxy-4-pyridyl)-4-aininomethylcyclohexanecarboxaniide (141-2) (R)-(+)-trans-N-(3-bromo- 1H-pyrrolo[2,3-blpyridin-4-yl)-4-( 1-aminoethyl)cyclohexanecarboxamide (142) trans-N-( lH-pyrrolo[2,3-b]pyridin-4-yl)-4-guanidinomethylcyclohexanecarboxanide (143) trans-N-( 1H-pyrazolo[3,4-bjpyridin-4-yl)-4-guanidinomethylcyclohexanecarboxamide (144) trans-N-(4-pyridyl)-4-guanidinomethylcyclohexanecarboxamnide (145) trans-N-( 1-methylpyrrolop2,3-bipyridin-4-yl)-4-(guanidinomethyl)cydlohexanecarboxamide (146) tr-ans-N-( lH-pyrrolo[2,3-bjpyridin-4-yl)-4-(2-imidazolin-2-yl)amiinomethlylcyclohexanecarboxam-ide (147) trans-N-( 1-benzyloxymethylpyrrolo[2,3-blpyridin-4-yl)-4-guanidinomethylcyclohexanecarboxaniide (148) trans-N-(2-amino-4-pyridyl)-4-guanidinomethylcyclohexanecarboxamfide (149) trans-N-(l1-benzyloxyrnethyl- 1H-pyrrolo[2,3-bjpyridi n4-yl)-4-(2-imidazolifl- 2-yl)aminomethylcyclohexanecarboxaxnide (150) trans-N-( lH-pyrrolo[2,3-bjpyridin-4-yl)-4-(3-benzylguaniidinomethyl)- 2. cyclohexanecarboxamide (151) trans-N-( 1H-pyrrolo[2,3-bjpyridin-4-yl)-4-(3-phenylguanidinomethyl)cydlohexanecarboxamide (152) trans-N-( 1H-pyrrolo[2,3-bjpyridin-4-yl)-4-(3-propylguanidinomethyl)cyclohexanecarboxaniide (153) trans-N-(l1H-pyrrolo[2,3-bjpyridin-4-yl)-4-(3-octylguanidinomethyl)cyclohexanecarboxamide (154) trans-N-(1-benzyloxymethylpyrrolo[2,3-blpyridin-4-yl)-4-(2-benzyl-3ethylguarnidinomethyl)cyclohexanecarboxamide (155) tr-ans-N-( 1H-pyrrolo[2,3-bjpyridin-4-yl)-4-(imidazol-2-yl)aminomethylcyclohexaecarboxamide (156) trans-N-(l1H-pyrrolo[2,3-bjpyridin-4-yl)-4-(thiazol-2-yl)aminomethylcyclohexanecarboxamnide (157) (R)-(+)-N-(4-pyridyl)-4-(l1-aminoethyl)benzamide (158) N-(4-pyridyl)-4-( 1-amino-i1 -methylethyl)benzaniide (159) N-(4-pyridyl)-4-amidnomethyl-2-benzyloxybenzamide (160) N-(4-pyridyl)-4-am-inomethyl-2-ethoxybenzamide (161) (R)-(-)-N-(4-pyridyl)-4-(l1-aminoethyl)-3-nitrobenizariide (162) (R)-(-)-N-(4-pyridyl)-3-amino-4-( 1-aminoethyl)benzaniide (163) 1-aminoethyl)-3-chlorobenzamide (164) N-(4-pyridyl)-3-aninomethylbenzamide (165) lH-pyrrolol2,3-blpyridin-4-yl) 1-aniinoethyl)benzamide (166) 1H-pyrazolo[3,4-blpyrridin-4-yl) 1-aminoethyl)benzamide a (167) N-(l1H-pyrazolo[,4-blpyridin-4-y)-4-guanidinomethylberizamride (168) N-(4-pyridyl)-4-guanidinomethylbenzamide (169) (R)-(+)-N-(4-pyridyl)-4-(l1-aminoethyl)-3-fluorobenza-ide (170) N-(4-pyridyl)-4-aminomethylbenzamide (171) N-(4-pyridyl)-4-aminomethyl-2-hydrloxybenzamide (172) N-(4-pyridyl)-4-(2-aniinoethyl)benzaniide.

(173) N-(4-pyridyl)-4-aninomethyl-3-nitrobenmide (174) N-(4-pyridyl)-3-amino-4-aminomethylbenzaniide (175) (S)-(-)-N-(4-pyridyl)-4-(l1-aminoethyl)benzam-ide (176) (S)-(-)-N-(4-pyridyl)-2-(l1-aminoethyl)benzaniide (177) (R)-(+)-N-(4-pyridyl)-4-(l1-aminoethyl)-2-chlorobenzamiide (178) lH-pyrrolo[2 ,3-blpyridin-4-yl)-4-( 1-(3-propylguanidino)ethyl)benzamide (179) (R)-(-)-N-(l1H-pyr-rolo[2 ,3-bjpyridin-4-Yl)-4-( 1-aniinoethyl)-3-azideberizamnide (180) (R)-(-')-N-(4-pyridyl)-4-(l1-aminoethyl)-2-nitrobenzamide (181) (R)-(-)-N-(4-pyridyl)-4-(l1-aniinoethyl)-3-ethoxybenzaniide (182) 1H-pyrrolo[2,3-bjpyridin-4-yl)-4-( 1-amiinoethyl)benzamude (183) 1H-pyrrolo[2,3-bjpyridin-4y)-4-( 1-aminoethyl)-3azidebenzamide (184) (R)-(-)-N-(4-pyridyl)-4-(l1-amrinoethyl)-3-hydroxybenzamide (185) N-(l1H-pyrazolo[3,4-bjpyridin-4-yl)-4-guanidinomethyl-3-nitrobenzaniide (186) (R)-N-(l1H-pyrazolo[3,4-blpyriclin-4-y).-4-(l1-guanidinoethyl)-3nitrobenzamide (187) (R)-N-(l1H-pyrazolo[3,4-blpyridin-4-yl)-4-( 1-aniinoethyl)-2-nitrobenzamide (188) N-(l1H-pyr-azolo[3,4-bjpyridin-4-yl)-4-guanidinobenzamide (189) (R)-N-(l1H-pyrazolo[3,4-blpyridin-4-yl)-4-( 1-amiinoethyl)-3-nitrobenzarnide (190) (R)-N-(l1H-pyrazolo[3,4-b]pyridin-4-yl)-4-( 1-guanidinoethyl)benzam-ide (191) 1H-pyrazolo[3,4-blpyridinA-yl)-4-(l1-amino-2-hydr-oxyethyl)benzamide (192) 1H-pyrazolo[3,4-blpyridin-4-yl)-4-aminomethyl-3-nitr-obenzamide (193) N-(l1H-pyrrolo[2,3-blpyridin-4-yl)-4-piperidinecarboxamide (194) N-(l1H-pyrazolo[3,4-bipyridin-4-yl)-4-piperidinecarboxam-ide (195) N-(l1H-pyrazolo[3,4-bjpyridin-4-yl)-l1-aminoacetyl-4-piperidinecarboxamide (196) N-(l1-methoxymethyl- 1H-pyrazolop3,4-blpyridin-4-yl)-4-piperidinecarboxanide (197) N-(2,3-dihydro- 1H-pyrrolo[2,3-bjpyridin-4-yl)-4-piperidinecarboxamide (198) N-(l1H-pyrrolo[2,3-blpyridin-4-y)-l1-(2-phenylethyl)-4-piperidinecarboxaxnide (199) N-(1H-pyrrolo[2,3-bpyridin-4-yl)--amidino-4-pipen ecarboxamide (200) N-(l1H-pyrrolo[2,3-blpyridin-4-yl)- 1-(3-phenylpropyl)-4-piperidinecarboxamide (201) N-(1 H-pyrrolo[2,3-bjpyridin-4-yl)-l1-benzyl-4-piperidinecarboxamide 9.9. (202) 1 H-pyrazolo[3,4-bjpyridin-4-yl)- 1-(2-phenylethyl)-4-piperidinecarboxamide (203) N-(l1H-pyrazolo [3,4-bjpyridin-4-yl)- 1-(3-phenylpropyl)-4-piperidinecarboxamide Preferred are compounds (109), (110), (112), (115), (142), (143), (144), (145), (153), (157), (163), (165), (166) and (179).

The compound to be used as the Rho inase ihibitor of the present invention is,. for example, a compound of the formnula. (MI, which is exemplified by the following compounds.

(204) 1 (205) 1 -(5-isoquinolinesulfonyl)-2-methylhomopiperazine (206) 1 -(5-isoquinolinesulfonyl)-3-methylhomopiperazine (207) 1 -(5-isoquinolinesulfonyl)-6-methylhomopiperazine (208) 1 -(5-isoquinolinesulfonyl) -2,3-dimethyihomopiperazine (209) 1 -(5-isoquinolinesulfonyl)-3,3-dimethylhomopiperazine (210) 1-(5-isoquinolinesulfonyl)-3-ethylhomopiperazine (211) 1 -(5-isoquinolinesulfonyl)-3-propylhomopiperazine (212) 1 -(5-isoquinolinesulfonyl)-3-isobutylhomopiperazine (213) 1 -isoquinolinesulfonyl) -3-phenylhomopiperazine (214) 1-(5-isoquinolinesuifonyl)-3-benzylhomopiperazine (215) 1 -(5-isoquinolinesulfonyl)-6-ethylhomopiperazine (216) 1 -(5-isoquinolinesulfonyl)-6-propylhomopiperazine (217) 1-(5-isoquinolinesulfonyl)-6-butylhomopiperazine (218) 1 -(5-isoquinolinesulfonyl)-6-pentylhomopiperazine (219) 1 -(5-isoquinolinesulfonyl) -6-hexyihomopiperazine (220) 1-(5-isoquinolinesulfonyl)-6-phenylhomopiperazine (221) 1 -(5-isoquinolinesulfonyl)-6-benzylhomopiperazine (22 *.ouioieuloy)4mehloopprzn (222) 1-(5-isoquinolinesulfonyl)-4-ethylhomopiperazine (223) 1-(5-isoquiriolinesulfonyl)-4-ethylhomopiperazine (224) 1-(5-isoquinolinesulfonyl)-4-proplhomopiperazine (225) 1 -(5-isoquinolinesulfonyl)-4-butylhomopiperazine (226) 1-(5-isoquinolinesulfonyl)-4-hexylhomopiperoan-e (227) N-(2-aminoethyl)- (228) N-(4-amino-1myethyl)-1-chlor)-5-isoquinolinesulfonamide (229) N-(2-aniino- 1-methyletyl)-1chloro-5-isoquinolineufnmd (230) N-(2-amino- 1-methylpentyl)- (231) N-(3-gainomethylyl)- (232) N-(3-gdinbutmnpoyl)-1-chloro-5-isoquinolinesulfona midee (234) N-(2-guanidino-lmtyethyl)- -chloro-5-isoquino nesulfona rride (235) N-(2-guanidinobuhyetyl)- (236) N-(2-guanidino- 1-methyltyl)- (237) N-(2-guanidino-2methylptyl)- (240) N-(4-guanidino-3-metylbuty) (241) 2-methyl-4-( 1-chloro-5-isoquinolinesulfony1~piperazine (242) 2-ethyl-4-( (243) 2-isobutyl-4-( (244) 2,5-dimethyl-4-(l1-chloro-5-isoquinolinesulfonyl)piperazine (245) 1-methylA4-( (246) 1-amidino-4-(l1-chloro-5-isoquinolinesulfonyl)piperazine (247) 1 -amidino-4-(l1-chloro-5-isoquinolinesulfonyl)homopiperazine (248) 1 -aniidino-3-methyl-4-(l1-chloro-5-isoquinolinesulfonyl)piperazine (249) 1 -amidino-2 ,5-dimethyl-4-( (250) N-(2-aminoethyl)- (251) N-(4-aniinobutyl)- (252) N-(2-amrino- 1-methylethyl)- (253) N-(2-aniino-1-methyiheptyl)- (254) N-(3-amino-2-methylbutyl)- (255) N, (256) N-cyclohexyl-N-methylaniino)ethyl]- :sulfonamide (257) N-(2-guanidinoethyl)- (258) N-(4-guanidinobutyl)- (29 N-2gaiiolmtyehl--yrx--sqioieufnmd (259) N-(2-guanidino- 1-ethyletyl)- (260) N-(21-guanidino-3methylntyl)- (261) N-(2-guanidino-3-methylbutyl)- (262) N-(3-guanidino-2-methylpropyl)- (263) N-4gadn--methyl buty)- -hydroxy-5-isoquinolnsloy~ieainesufnmd (264) 2-ethyl-4-( (265) 2-esthyl-4-( (266) 2,-ibtyl-4-(-hydroxy-5-isoquinolinesulfonyl)piperazine (267)2 (268) 1-methylno4-( (269) 1 -amidino-4-(l1-hydroxy-5-isoquinolinesulfonyl)hpiperazine (270) 1 -amidino-3mty-4-(l-hydr-oxy-5-isoquinolinesulfonyl)piperazine (272) 1 -amnidino-2 ,5-dimethyl-4-( (273) N-(2-methylaminoethyl)- (274) N-(2-ethylaminoethyl)- (275) N-(2-propylaminoethyl)-l1-chloro-5-isoquinolinesulfonamide (276) N-(2-butylaxninoethyl)- 1 (277) N-(2-hexylaminoethyl)-l1-chloro-5-isoquinolinesulfonamide (278) (279) 1 (280) N-(2-methylaniinoethyl)-1-hydroxy-5-isoquinolinesulfonamiide (281) N-(2-ethylamnoethyl)- (282) N-(2-propylaminoethyl)- (283) N-(2-butylaminoethyl)-l1-hydroxy-5-isoquinolinesulfonaxnide (284) N-(2-hexylaminoethyl)-l1-hydroxy-5-isoquinolinesufonamide (285) 1 (286) (27 **ouioieuloy)4-ehliprzn (287) 1-(5-isoquinolinesulfonyl)-4-m-ehylpiperazine (288)l1-(5-isoquinolinesulfonyl)-4-cnhexylpiperazine (290) 1-(5-isoquinolinesulfonyl)pi4-cinaypprzn (290) 1-(2aity)5-isoquinolinesuilfonlapiprazin (291) (293) (294) 1-(5-isoquinolinesulfonyl)-3-methylpiperazine (295) 1 -(5-isoquinolinesulfonyl)-3-isobutylpiperazine (296) 1-(5-isoquinolinesulfonyl)-2,5-dimethylpiperazine (297) N-(3-guanidino-2-phenylpropyl)-5-isoquinollnesulfonamide (298) N-(6-guanidino- (299) 2-[2-(5-isoquinolinesulfonamride)ethylamino]-2-im-idazoline (300) 2-amidino-1-(5-isoquinolinesulfonyl)piperazine (301) 4-amidino-2 ,5-dimethyl- (302) 4-amidino- (303) 4-(N 1, N2-dimethylamidino)- (304) 4-aniidino-3-butyl-l1-(5-isoquinolinesulfonyl)piperazine (305) 4-hexyl- (306) (307) (308) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine Preferred are compounds (204) and (308).

The compound to be used as the Rho kinase inhibitor of the present invention may be a pharmaceutically acceptable acid addition salt. The acid is exemplififed by inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid and the like and organic acid such as methanesulfonic acid, fumaric acid, maleic acid, mandelic acid, citric acid, tartaric acid, salicylic acid and the like.

The compound having a carboxyl group can be converted to a salt with a metal such as sodium, potassium, calcium, magnesium, aluminum and the like or a salt with amino acid such as lysine and the like. In addition, their monohydrate, dihydrates, 1/2 hydrates, 1/3 hydrates, 1/4 hydrates, 2/3 hydrates, 3/2 hydrates and the like are also encompassed in the present invention.

e*e.

The compound of the formula can be synthesized according to the method disclosed in Japanese Patent Unexamined Publication No. 62-89679, Japanese Patent Unexamined Publication No. 3-218356, Japanese Patent Unexamined Publication No. 5-194401, Japanese Patent Unexamined Publication No. 6-41080, W095/28387 and the like.

The compound of the formula can be synthesized according to the method disclosed in Japanese Patent Unexamined Publication No. 57-156463, Japanese Patent Unexamined Publication No. 57-200366, Japanese Patent Unexamined Publication No. 58-121278, Japanese Patent Unexamined *i Publication No. 58-121279, Japanese Patent Unexamined Publication No. 59- 93054, Japanese Patent Unexamined Publication No. 60-81168, Japanese Patent Unexamined Publication No. 61-152658, Japanese Patent Unexamined Publication No. 61-227581, Japanese Patent Unexamined Publication No. 62- 103066, USP-4678783 and the like.

Of the compounds of the formula a compound wherein Ra is a group of the formula and Rc is Rc', namely, an amide compound of the formula (Il)

R

C- N -R[n]

II

O

wherein Rc' is an optionally substituted heterocycle containing nitrogen of the above-mentioned Rc except pyridine, and other symbols are as defined above, is a novel compound which can be synthesized by the following methods.

Method 1 A compound of the formula (IV) Rc' -NH-Rb (IV) wherein each symbol is as defined above, and a compound of the formula (V)

RS

L- N

II

O H *0 wherein each symbol is as defined above, or a reactive derivative thereof are reacted to give the compound. The reactive derivative of carboxylic acid compound is exemplified by acid halide, ester, acid anhydride, mixed acid anhydride and the.like.

This reaction beneficially proceeds by stirring in the presence of a solvent inert to the reaction, such as tetrahydrofuran, dioxane, chloroform, dichloromethane, dimethylformamide, benzene, toluene, ethanol and the like.

Water, alcohol or acid liberated during the reaction is removed from the reaction mixture by a method known in the pertinent field, such as azeotropic distillation, forming a complex, converting to salt and the like.

Method 2 Of the compounds of the formula a compound wherein L has a substituent other than hydrogen can be produced by reacting a compound wherein L is hydrogen, with a compound of the formula (VI) L -M

(VI)

wherein L 1 is, of the aforementioned L, a substituent other than hydrogen and M is a reactive atom, according to N-alkylation or N-acylation known in this field.

Method 3 Of the compounds of the formula (III), a compound wherein L is alkyl or has a substituent having the formula can be produced by reductive amination reaction of a compound wherein L is hydrogen and a compound of the formula (VII)

L

2 =C=O

(VII)

wherein L 2 is a group that can be converted to alkyl or a group of the formula by reductive amination reaction.

Method 4 Of the compounds of the formula (III), a compound wherein L is a group of the formula (1) l/ o (1) wherein Q 1 is as defined above and W 1 is hydroxytrimethylene from among the substituents at W, can be produced by reacting a compound of the formula (III) wherein L is hydrogen and a compound of the formula (VIII)

CH

2 cH- CH 2

(VIII)

S

wherein Q- is as defined above.

The reaction advantageously proceeds in a suitable solvent which does not influence the reaction, such as alcohol methanol, ethanol, 2-propanol and the like), aliphatic or alicyclic ketone 2-propanone, 2-butanone, cyclohexane and the like) and the like. Addition of a suitable base such as alkali metal carbonate, hydrogencarbonate and the like enables acceleration of the reaction rate. The reaction temperature is rather elevating, which is preferably refluxing temperature of the reaction mixture.

Method Of the compounds of the formula a compound wherein L is hydrogen can be produced from a compound of the formula (III-a)

R

Bl I C Nr Rb 0 IB CjA (HI-a) C N R'

II

0 wherein B 1 is alkoxy or aralkyloxy, from among the aforementioned substituents B, and other symbols are as defined above.

Of the compounds (III-a), a compound wherein B 1 is alkoxy is stirred in a suitable organic solvent which does not influence the reaction, such as alcohol methanol, ethanol, 2-propanol and the like) and ether tetrahydrofuran and the like) in the presence of a suitable base, suchas hydroxide of alkali metal or alkaline earth metal, carbonate or hydrogencarbonate sodium hydroxide, potassium carbonate, sodium hydrogencarbonate and the like) and heated as necessary to give a compound of the formula (III) wherein L is hydrogen.

Of the compounds (III-a), a compound wherein B' is aralkyloxy is subjected to reductive decomposition reaction in a suitable organic solvent which does not influence the reaction in the presence of a suitable catalyst such as palladium carbon and the like using a hydrogen source of hydrogen, hydrazine, formic acid, ammonium formate and the like at normal temperature or under pressurization where necessary.

Moreover, a compound (III-a) is stirred in 5-35/o, preferably 15-30%, acetic acid in the presence of hydrogen bromide, whereby the compound can be converted. A compound of the formula (II-b)

R

SR(Ill-b)

A\

Rc'

II

o wherein Y' is methylene, from among the aforementioned substituents Y, and other symbols are as defined above, is subjected to catalytic hydrogenation decomposition reaction wherein the compound is stirred in a suitable organic solvent which does not influence the reaction in the presence of a suitable catalyst such as palladium carbon and the like under hydrogen to give a compound of the formula (III) wherein L is hydrogen.

The compound of the formula (III) thus obtained can be separated from the reaction mixture and purified by a method known in the field of art, such as recrystallization, chromatography and the like.

In addition, the compound of the formula (III) can form a pharmaceutically acceptable salt by a conventional method. The acid to be used for forming a salt can be appropriately selected from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and the like, organic acids such as methanesulfonic acid, fumaric acid, maleic acid, mandelic acid, citric acid, tartaric acid, salicylic acid and the like, amino acids such as lysine and the like, and metal such as sodium, potassium, calcium, magnesium, aluminum and the like. These acid addition salts can be converted to a corresponding free base by the reaction with alkali such as sodium hydroxide, potassium hydroxide and the like according to a known method. The salts can be also converted to quaternary ammonium.

The compound of the formula (III) may exist as optical isomer, racemate thereof or cis-trans isomer, all of which are encompassed in the present invention.

These isomers can be isolated by a conventional method or produced by using various starting compounds.

When the Rho kinase inhibitor of the present invention is used as a pharmaceutical agent, particularly as a therapeutic agent of hypertension, a therapeutic agent of angina pectoris, a suppressive agent of cerebrovascular contraction, a therapeutic agent of asthma, a therapeutic agent of peripheral circulation disorder, a prophylactic agent of immature birth, a therapeutic agent of arteriosclerosis, an anti-cancer drug, an anti-inflammatory agent, an immunosuppressant, a therapeutic agent of autoimmune disease, a contraceptive, a prophylactic agent of digestive tract infection, an anti-AIDS drug, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy or a brain function improving drug, it can be prepared as a general pharmaceutical agent. For example, the Rho kinase inhibitor of the present invention is mixed with a pharmaceutically acceptable carrier excipient, binder, disintegrator, corrective, corrigent, emulsifier, diluent, solubilizer and the like) to give a pharmaceutical composition or a pharmaceutical preparation in the form of tablet, pill, powder, granule, capsule, troche, syrup, liquid, emulsion, suspension, injection liquid, suspension and the like), suppository, inhalant, percutaneous absorber, eye drop, eye ointment and the like in the form suitable for oral or parenteral preparation.

When preparing a solid preparation, an additive such as sucrose, lactose, cellulose sugar, D-mannitol, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectines, tragacanth, gum arabic, gelatins, collagens, casein, albumin, calcium phosphate, sorbitol, glycine, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, glycerol, polyethyleneglycol, sodium hydrogencarbonate, magnesium stearate, talc and the like are used. Tablets can be applied with a typical coating, where necessary, to give sugar coated tablets, enteric tablets, film-coated tablets, twolayer tablets and multi-layer tablets.

When preparing a semi-solid preparation, animal and plant fats and oils olive oil, corn oil, castor oil and the like), mineral fats and oils petrolatum, white petrolatum, solid paraffin and the like), wax jojoba oil, carauba wax, bee wax and the like), partly or entirely synthesized glycerol fatty acid esters lauric acid, myristic acd, palmitic acid and the like), and the like are used.

Examples of commercially available products of these include Witepsol (manufactured by Dynamitnovel Ltd.), Farmazol (NOF Corporation) and the like.

When preparing a liquid preparation, an additive, such as sodium chloride, glucose, sorbitol, glycerol, olive oil, propylene glycol, ethyl alcohol and the like, is used. In particular, when preparing an injection, a sterile aqueous solution such as physiological saline, isotonizing liquid, oily liquid sesame oil and soybean oil) and the like is used. Where necessary, a suitable suspending agent such as sodium carboxymethylcellulose, nonionic surfactant, solubilizer benzyl benzoate and benzyl alcohol), and the like can be concurrently used. Moreover, when an eye drop is prepared, an aqueous liquid or solution is used, which is particularly a sterile injectable aqueous solution. The liquid for an eye drop can appropriately contain various additives such as buffer (preferred are borate buffer, acetate buffer, carbonate buffer and the like for less irritation), isotonizing agent, solubilizer, preservative, thickener, chelating agent, pH adjuster (preferably, pH is generally adjusted to about 6-8.5) and aromatic.

The content of the active ingredient in these preparation is 0.1-100 wt/o, suitably 1-50 of the preparation. While subject to variation depending on the condition, body weight, age and the like of patient, in general, about 1-500 mg of the active ingredient is orally administered day for an adult in a single dose or several doses.

Examples The present invention is described in more detail in the following by way of Examples, Formulation Examples and pharmacological action, to which the present invention is not limited.

In the following, the synthetic method of the novel compound of the formula (III) of the present inventi on is described by referring to examples.

Example 1 N-Benzyloxycarbonylisonipecotyl chloride (5 g) was added to a solution of 4amino-l1-tert-butoxycarbonyl- 1H-pyrolo[2,3.bJpyridine (3 g) and dilsopropylethyamine (2.16 g) in acetonitrile (40 ml) and the mixture was stirred at room temper-ature for 2 hours. The reaction mixture was poured into ice-water and extracted with chloroform. The residue obtained by water washing, drying and :~:then concentration under reduced pressure was purified by silica gel column chromatography to give 6.3 g of 1-tert-butoxycarbonyl- 1H-pyrrolo- [2,3-bjpyridin-4-yl) -benzyloxycarbonyl-4-piperidinecarboxamide.

.*.*PMR(CDC1 3 :1.67(9H, 1.79(2H, in), 1.95(2H, in), 2.53(1H, mn), 2.89(2H, in), 00.4.29(2H, in), 5,15(2H, 6.48(1H, dJ=4.4Hz), 7.36(5H, in), 7.59(1H, br), 7.6 1(1H, dJ=4.4H-z), 7.99(1H, dJ=5.4Hz), 8.43(1H, dJ=5.4Hz) o N-(l1-tert-Butoxycarbonyl- 1H-pyrrolo[2,3-bjpyridin-4-yl)- 1-benzyloxycarbonyl-4-piperidinecarboxamide (2 g) was dissolved in methanol (30 ml) and 0 10/6 palladium carbon hydroxide (0.5 g) was added for hydrogenation (normal pressure). After the completion of the reaction, the catalyst was filtered off and the filtrate was concentrated under reduced pressure to give 1.2 g of N-(1-tertbutoxycarbonyl-l1H-pyrrolo[2,3-blpyridin-4-yl)-4-piperidinecarboxarnjde.

PMR(DMSOAd) 1.59(9H, 1.83(2H, in), 2.01(2H, in), 2.89(2H, in), 3.0 1(1H, in), 3.32 (2H, in), 7.19(1H, dJ4.4Hz), 7.68(1H, dJ4.4Hz), 7.97(1H, dJ5.4Hz), 8.24(lH, dJ=5.4Hz), 8.8 1(1H, br), 10.45(1H, s) Formic acid (10 ml) was added to N-(1-tert-butoxycarbonyl-1H-pynrolo[2,3bjpyridin-4-yl)-4-piperidinecarboxanmide (1 g) and the mixture was sftired at roomn 43 temperature for 2 hours. The mixture was neutralized with aqueous 1N sodium hydroxide solution and extracted with chloroform. The crystals obtained by water washing, drying and then concentration under reduced pressure were dissolved in hydrochloric acid-methanol solution (5 ml). The crystals obtained by concentration of the resulting solution were recrystallized from ethanol-ethyl acetate to give 650 mg of N-(1H-pyrrolo[2,3-blpyridin-4-yl)-4-piperidinecarboxamide mono hydrochloride monohydrate, melting point 273*C (decomposition).

PMR(DMSO-d) 1.52(2H, 1.69(2H, 2.51(2H, 2.70(1H, 2.97(2H, m), 3.32(1H, br), 6.79(1H, d,J=3.4Hz), 7.31(1H, dJ=3.4Hz), 7.79(1H, d,J=5.4Hz), 8.04(1H, dJ=5.4Hz), 9.82(1H, 11.54(1H, br) Example 2 A solution of 1-tert-butoxycarbonyl- 1H-pyrrolo[2,3-b]pyridin-4-yl)-4piperidinecarboxamide (0.6 phenetyl bromide (390 mg) and potassium carbonate (290 mg) in dimethylformamide (10 ml) was stirred at 80*C for 2 hours.

The reaction mixture was poured into ice water and extracted with chloroform.

The residue obtained by water washing, drying and then concentration under reduced pressure was purified by silica gel column chromatography to give 550 mg of N-(1-tert-butoxycarbonyl- 1 H-pyrrolo[2,3-b]pyridin-4-yl)- 1-(2-phenylethyl)-4piperidinecarboxamide.

PMR(DMSO-d) 1.59(9H, 1.66(2H, 1.80(2H, 1.98(2H, 2.50(2H, m), 2.56(1H, 2.74(2H, 3.01(2H, 7.05(1H, dJ=4.4Hz), 7.23(5H, 7.68(1H, dJ=4.4Hz), 7.97(1HJ=5.4Hz), 8.23(1H, d,J=5.4Hz), 10.03(1H, s) Formnnic acid (5 ml) was added to N-(1-tert-butoxycarbonyl-1H-pyrolo[2,3b]pyridin-4-yl)-1-(2-phenylethyl)-4-piperidinecarboxamide (550 mg) and the mixture was stirred at room temperature for 2 hours. The mixture was neutralized with aqueous 1N sodium hydroxide solution and extracted with chloroform. The crystals obtained by water washing, drying and then concentration under reduced pressure were dissolved in 15% hydrochloric acidmethanol solution (1 ml). The crystals obtained by concentration of the resulting solution were recrystallized from ethanol-ethyl acetate to give 250 mg of N-(1Hpyrrolo[2,3-blpyridin-4-yl)-1-(2-phenylethyl)-4-piperidinecarboxamide dihydrochloride 1/4 hydrate, melting point 272*C (decomposition).

PMR(DMSO-d/TMS) :2.00-2.19(4H, in), 2.93-3.41(7H, mn), 3.63-3.68(2H, in), .7.22-7.37(5H, mn), 7.50(l1H, d,J=2.OHz), 7.56(lH, t,J=2.OHz), 8.25(lH, d,J- 6.8Hz), 8.33(1H, dJ6.8Hz), 10.86(1H, br), 1l.36(1H, 12.77(lH, br) Example 3 A solution of 1-tert-butoxycarbonyl- 1H-pyrrolo[2,3-blpyr idin-4-yl)-4piperidinecarboxamide (500 mg), benzyl bromide (370 mng) and potassium carbonate (300 mg) in dimethylformamide (10 ml) was stirred at 80*C for 4 hours.

The reaction mixture was poured into ice-water and extracted with chloroform.

The residue obtained by water washing, drying and then concentration under reduced pressure was purified by silica gel column, chromatography to give 300 mg of N-(l1-tert-butoxycarbonyl-l1H-pyrrolo pyridin-4-yl)- 1-benzyl-4piperidinecarboxamide.

PMR(CDC1 3 1.65(9H, 1.9 1(4H, in), 2.04(2H, in), 2.35(lH, mn), 2.97(2H, in), 3.51(2H, 6.44(1H, d,J=3.9Hz), 7.30(5H, mn), 7.49(1H, br), 7.57(1H, d,J=3.9Hz), 7.99( 1H, dJ=5.4Hz), 8.4 1( 1H, dJ=5.4Hz) Formic acid (4 ml) was added to N-(1-tert-butoxycarbonyl-lH-pyrrolo[2,3bjpyridin-4-yl)-1-benzyl-4-piperidinecarboxanmide (300 mg) and the mixture was stirred at room temperature for 1 hour. The mixture was neutralized with aqueous 1N sodium hydroxide solution and extracted with chloroformr. The crystals obtained by water washing, drying and then concentration under reduced pressure, were dissolved in 15% hydrochloric acid-methanol solution (1 ml). The crystals obtained by concentration of the resulting solution were, recrystallized from ethanol-ethyl acetate to give 120 mg of 1H-pyrrolo[2,3-bjpyridin-4-yl)- 1benzyl-4-piperidinecarboxarnide dihydrochloride monohydrate, melting point (decomposition).

PMR(DMSO-d6/TMS) :2.00-2.15(4H, in), 2.92-2.98(2H, mn), 3.13-3.19(1H, in), :e 3.36-3.43(2H, in), 4.32(2H, 7.55(1H, br), 7.63(2H, in), 8.20(lH, dJ=6.4Hz), 8.3 1(1H, dJ=6.4Hz), 10.76(1H, br), 11.25(1H, br), 12.69(lH, br) The following compounds can be obtained in the same manner as in the above Examples.

Exmple 4 1H-pyrazolop,4-bipyridin-4-yl)-4-piperidinecarboxaniide dihydrochloride 3/2 hydr-ate, melting point 277 0 C (decomposition) Example 1H-pyrazolo[3,4-blpyridin-4-yl)- 1-aininoacetyl-4-piperidinecarboxamide dihydrochioride 1/2 hydrate, melting point 26400 (decomposition) Example 6 1-methoxyrnethyl- 1 H-pyrazolo[3 ,4-blpyridin-4-yl)-4-piperidinecarboxamide monohydrate, melting point 240-24 100 Example 7 N-(2,3-dihydr-o- 1H-pyrrolo[2,3-bjpyridin-4-yl)-4-piperidinecarboxamide dihydrochioride 3/2 hydrate, melting point 23500 (decomposition) Example 8 1H-pyrrolo[2,3-bjpyridinA4-yl)- 1-amidino-4-piperidinecarboxamide dihydrochioride 5/4 hydrate, melting point 24600 (decomposition) Example 9 1H-pyrrolo[2,3-b]pyridin-4-yl)-l1-(3-phenylpr-opyl)-4-piperidinecarboxainide dihydrochioride, melting point 27600 (decomposition) Example N-(l1H-pyrazlo[3,4-blpyridin-4-yl)- 1-(2-phenylethyl)-4-piperidinecarboxamride dlhiydrochloride hydrate, melting point 259-26 100C (decomposition) Example 11 N-(l1H-pyrazolo[3,4-bjpyridin-4-yl)- 1-(3-phenylpropyl) -4piperidinecarboxamide dihydrochioride 1/2 hydrate, melting point 24O-244*C (decomposition) A method for preparing the pharmaceutical preparation of the pr'esent invention is explained in the following.

Formulation Example 1 tablets Inventive compound 10.0 mg Lactose 50.0 mg Corn starch 20.0 mg Crystalline cellulose 29.7 mg Polyvinylpyrrolidone K(30 5.0 Mg Talc Magnesium stearate 0.3mg 120.0 mg a 00.004 04 .0 a a.

a.

a The inventive compound, lactose, corn starch and crystalline cellulose were mixed, kneaded with polyvinylpyrrolidone K30 paste solution and passed through a 20-mesh sieve for granulation. After drying at 50°C for 2 hours, the granules were passed through a 24-mesh sieve, and talc and magnesium stearate were added. Using a <7 mm punch, tablets weighing 120 mg per tablet were prepared.

Formulation Example 2 Capsules Inventive compound 10.0 mg Lactose 70.0 mg Corn starch 35.0 mg Polyvinylpyrrolidone K30 2.0 mg Talc 2.7mg Magnesium stearate 0.3mg 120.0 mg The inventive compound, lactose, corn starch and crystalline cellulose were mixed, kneaded with polyvinylpyrrolidone K30 paste solution and passed through a 20-mesh sieve for granulation. After drying at 50°C for 2 hours, the granules were passed through a 24-mesh sieve and talc and magnesium stearate were added. The mixture was filled in hard capsules (No. 4) to give capsules weighing 120 mg.

The pharmacological action of the pharmaceutical preparation of the present invention is explained in the following by way of experimental examples.

Experimental Example 1: Rho kinase inhibitory action (inhibition of bovine aorta thoracia Rho kinase) The Rho kinase was prepared from bovine aorta of thorax by partial purification as in the following. The artery was minced and homogenized with a 9-fold amount of 50 mM Tris-hydroxymethylaminomethane (Tris) 1 mM dithiothreitol, 1 mM EGTA, 1 mM EDTA, 100 rM p-amidinophenylmethylsulfonyl fluoride, 5 gM E-64, 5 tM leupeptine and 5 pM pepstatin A. The homogenate was centrifuged (10,000 x g, 30 minutes) to give supernatant. The supernatant was adsorbed onto a hydroxyapatite column. The column was washed with 0.2M phosphate buffer (pH The standard product of Rho kinase was eluted with 0.4M phosphate buffer The Rho kinase was assayed as follows.

A reaction mixture (total amount 50 pl) containing 50 mM Tris, 1 mM EDTA, 5 mM MgCla, 50 pg/ml histone, 10 AM GTPy S, 100 pg/ml Rho, 2 tM 32 P]ATP, the Rho kinase (3 pi) prepared in the above and the test compound was reacted at 30°C for 5 minutes. The reaction was terminated by the addition of trichloroacetic acid (TCA) solution (1 ml) and the mixture was stood at 4°C for minutes. Then, the mixture was filtered through a membrane filter (HAWP type, Millipore), and the radioactivity of the filter was counted on a liquid scintillation counter. The inhibitory action of the test compound was calculated from the following formula based on the comparison of the radioactivity with the sample without the test compound (control). The results are shown in Table 1.

cpm in the presence cpm under control of test compound Inhibition pmudrcnolx 100 cpm under control Table 1 Test compound Inhibition Compound 109.2HC1 (1 pM) 81 (10 tLM) 100 Compound 165.2HC1.3/2H 2 0 (10 IpM) 100 Compound 80.2HCLH 2 O (10 LM) 100 Compound 204.2HC1 (10 JM) 93 Experimental Example 2: Rho kinase inhibitory action (inhibition of human platelet Rho kinase Human platelet pl60ROCK was isolated by the method of Ishizaki et al.

(Ishizaki T et al., The EMBO 15(8), 1885-1893, 1996).

Kinase assay included the following steps. That is, a reaction mixture (total amount 30 Il) containing 50 mM Hepes-NaOH 10 mM MgCl 2 mM MnC12, 2 mM dithiothreitol, 0.02% Brij35, 1 iM [y 3 2 PATP, 330 pg/ml histone, p 160ROCK (2 pl) isolated by the method of Ishizaki et al. and the test compound was incubated at 30*C for 20 minutes. The solution was mixed with a 1/3 amount of4X Laemmli sample buffer, boiled for 5 minutes and applied to SDS-PAGE. The gel was stained with Coomassie Brilliant Blue and dried. The 0@ .999 9 *9*9 *9 S 9 .9 9 band of histone was cut out and assayed for radioactivity. The test compound was evaluated in the same manner as in Experimental Example 1, and the concentration of each test compound necessary for 50% inhibition was calculated as IC50 The results are shown in Table 2.

Table 2 Test compound ICso (tM) Compound 80.2HC.H20 Compound 109.2HCI 0.11 Compound 143.2HC1.H 2 0 1.6 Compound 204.2HCI 3.8 Compound 308.2HCI Experimental Example 3: Rho kinase inhibitory action (inhibition of and ROCKII) The standard enzyme products of p160ROCK (Ishizaki T et al, The EMBO 15(8), 1885-1893, 1996) and ROCKI (Nakagawa O et al., FEBS Lett. 392 189- 193, 1996) were obtained in the following manner. COS cells were seeded in a cm dish and incubated overnight. Using lipofectamine, the expression vectors of pl60ROCK and ROCKII (pCAG-myc-pl60ROCK and pCAG-myc-ROCKH: see Ishizaki T et al., The EMBO 15(8), 1885-1893, 1996 and Nakagawa O et al., FEBS Lett. 392 189-193, 1996) were transfected. After incubation for 20 hours, the cells were washed once with ice-cooled PBS, and the cells were lysed on ice for minutes using a lysis buffer (20 mM Tris-HCl 1 mM EDTA, 1 mM S• EGTA, 5 mM MgCl 2 25 mM NaF, 10 mM p glycerophosphate, 5 mM sodium so* pyrophorphate, 0.2 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 0.2 mM sodium vanadate, 0.05% Triton X-100, 0.1 iM calyculin The lysate was *oss. centrifuged at 10,000 xg for 10 minutes and the supernatant was recovered. To the supernatant was added 9E10 anti-myc epitope antibody (see Ishizaki T et al., The EMBO 15(8), 1885-1893, 1996) and the mixture was shaken for 2 hours.

Then, protein G-Sepharose was added and the mixture was shaken for 2 more hours. The suspension was centrifuged at 1,000 x g for 5 minutes and the resulting pellets were washed 3 times with lysis buffer and once with kinase buffer mM Hepes-NaOH 10 mM MgC2, 5 mM MnCl 2 2 mM dithiothreitol, 0.02% Brij35). The pellets were suspended in kinase buffer to give a standard enzyme product. The kinase assay followed the method shown in Experimental- Example 2, wherein the standard enzyme product obtained in this Experimental Example was used instead of human platelet Rho kinase (pl60ROCK). The concentration of each test compound necessary for 50% inhibition was calculated as IC50 The results are shown in Table 3.

Table 3 Test compound ICso (iM)

ROCK-II

Compound 80.2HC1.H 2 0 0.63 0.56 Compound 109.2HC1 0.095 0.048 Compound 143.2HCl.H 2 0 0.88 0.47 Compound 204.2HC1 2.3 1.1 Experimental Example 4 vasodilating action Male rabbits (body weight 1.9-3.0 kg) were anesthetized with pentobarbital sodium and exsanguinated, whereafter thoractic aorta was removed. An about 2 mm width aortic ring samples were prepared and hung in a Magnus bath (40 ml) filled with Krebs-Henseleit solution (37°C, NaCl 117 mM KC1 4.7 mM CaCI 2 mM MgSO 4 1.2 mM NaHCO324.8 mM KH 2

PO

4 1.2 mM glucose 11.0 mM) at a load of 2 g. The Magnus bath was constantly bubbled with a mixed gas COz gas). The tension of the preparation was measured with an isomeric transducer (TB-61 T, Nippon Koden). The preparation was contracted with phenylephrine (10 6 M) and, after the contraction was stabilized, the test compound was added accumulatively and relaxing action was observed. The relaxing action of the test compound was calculated by expressing the concentration of the test compound necessary for 50% relaxation as IC50 (PM) against the contraction with phenylephrine as 100%. The results are shown in S. Table 4.

Experimental Example 5 Effect on contraction by acetylcholine of trachea specimen removed from guinea pig Male Hartley guinea pigs (body weight 260-390 g) were anesthetized by the peritoneal administration of pentobarbital sodium (100 mg/kg) and exsanguinated, whereafter trachea was removed. The anterior cartilage of the trachea was opened and the band was cut in a 3 mm width strip to give a specimen.

The specimen was hung in a Magnus bath (40 ml) filled with Krebs-Henseleit solution (NaCI 117 mM; KC14.7 mM; CaC1 2 2.5 mM; MgSO 4 1.2 mM; NaHCO 3 24.8 mM KH 2

PO

4 1.2 mM glucose 11.0 mM) at a load of 1 g. The Magnus bath was constantly bubbled with a mixed gas (95% 02+5% CO 2 gas). The tension of the strip was measured with an isomeric transducer (TB-611T, Nippon Koden) and depicted on a recorder (Ti-102, Tokai Irika). The strip was contracted with acetylcholine (10' M) and, after the contraction was stabilized, the test compound was added accumulatively and relaxing reaction was observed. The relaxing action of the test compound was calculated and expressed by the concentration of the test compound necessary for 50% relaxation as IC50 against the maximum response with papaverine (10 4 M) as 100%. The results are shown in Table 4.

Table 4 Test compound Vasorelaxing Trachea relaxing action (pM) action (gM) Compound 80.2HCLHzO 0.70 0.56 Compound 109.2HC1 0.1 0.043 Compound 165.2HC1.3/2H 2 0 0.051 0.066 Compound 179.2HBr.1/2H 2 0 0.03 0.029 Experimental Example 6 Peripheral blood flow increasing action Streptozotocin (STZ, 65 mg/kg) was intravenously injected to male SD rats (body weight 200-300 g) to prepare diabetic rats. One month later, STZ-induced diabetic rats were anesthetized with pentobarbital sodium and the blood flow in the hind limb skin was measured with laser blood flowmeter (ALF21R, Advance).

The test compound was intravenously administered via catheter dwelled in the carotid arteries, and hind limb skin blood flow increasing action was observed.

The blood flow increasing action of the test compound was expressed by increase percentage from the blood flow before administration. The results are shown in Table Table Test compound Increase in skin blood flow+ standard error Compound 80.2HCLH 2 0 (1 Pg) 135.0±13.4 Compound 157.HCI.H 2 0 (1 pg) 211.6±13.6 Compound 165.2HC1.3/2H 2 0 (0.03 jtg) 135.8± 0.0 (0.1 Rg) 144.7± 0.0 Compound 166.2HC1.H 2 0 (0.3 Ag) 143.2±25.4 (1 Fg) 165.9±42.5 Experimental Example 7 Inhibition of VLA (very late antigen) integrin activation As the index of the activation by VLA integrin, phorbol ester-induced adhesion of CEM cells (human T cell type established cell) to fibronectin, which is a ligand of VLA integrin, was measured. The inhibitory action on the induced adhesion by the test compound was determined by the following method.

CEM cells were washed with RPMI1640 medium containing 0.5% bovine serum albumin (BSA), 10 mM HEPES, 2 mM L-glutamin, 1 mM sodium pyruvate, pg/ml kanamycin sulfate and 1.5 mg/ml sodium hydrogencarbonate (hereinafter this medium is referred to as culture solution) and suspended in this i" medium for use in the following experiment. To each well of a 96 well plate coated with human fibronectin were added CEM cells (5 X 104) and the test compound Sdissolved in the culture solution (final concentration 1-100 tM) to the amount of 100 pl, and the plate was stood at 37°C for 1 hour. Then, PMA (phorbol 12myristate 13-acetate, TPA final concentration 10 ng/ml) and the test compound were added to the amount of 200 pi, and the plate was stood at 37°C for minutes. Each well was washed twice with the culture solution (200 Il) at 37°C, and the LDH (lactate dehydrogenase) activity of the cells adhered to the plate was determined, whereby the amount of the adhered cell was measured. Based on the results obtained by the above-mentioned method, the inhibitory action of the test compound on the induced adhesion was calculated by the following formula.

The results are shown in Table 6.

Inhibition of adhesion induction= X 100 a= number of cells adhered with the addition of PMA b= number of cells adhered with the addition of test compound and PMA c= number of cells adhered without stimulation Table 6 Test compound Concentration (riM) Adhesion induction inhibition Compound 80.2HC1.H 2 0 100 Compound 109.2HCI 100 .67 Compound 143.2HC1.H 2 0 100 77 Compound 165.2HC.3/2H 2 0 10 Compound 204.2HC1 100 82 Anti-pl antibody 20 Igg/ml 118 IgG1 20 gg/ml Experimental Example 8 Inhibition of bone resorption (in vitro) The determination of the in vitro inhibition of bone resorption using mouse femoral bone followed the method below.

The femoral bone of 3-6 week old male ICR mice was aseptically removed, and bone marrow cavity was washed with F12 medium, containing 10% heat inactivated fetal bovine serum, penicillin G calcium (100 units/ml), kanamycin sulfate (60 ig) and 0.15% sodium hydrogencarbonate (hereinafter the medium is to be referred to as culture solution). After washing the bone marrow cavity and S*then removing the soft tissue adhered to the bone, the bone was subjected to incubation. The test compound was once dissolved in dimethyl sulfoxide (DMSO) to give a 10 mg/ml solution, which was diluted 1000-fold with the culture solution to give a 10 Ilg/ml solution. The test compounds were respectively added to the concentration shown in Table 7 and, using this culture solution (1.2 ml), the ICR mouse femoral bone was incubated in a 24 well plate for 6 days under the conditions of 5% CO 2 gas, 95% air. After the completion of the incubation, the culture supernatant was recovered, and the amount of calcium suspending in the culture supernatant was quantitatively determined by the chelate method using o-cresolphthalein. The bone resorption inhibitory action of the test compound was calculated by the following formula using the incubation of the femoral bone without addition of the test compound as a control.

Amount of free Ca Amount of free Ca without addition of with addition of Inhibition of test compound test compound bone resorp- x 100 tion Amount of free Ca Amount of Ca without addition of in culture test compound This experiment was done with 4 cases in each group. As the control, the same amount of DMSO alone as in the case with the addition of the test compound was used. The results are shown in Table 7.

Experimental Example 9 Inhibition of mouse allogenic mixed lymphocyte reaction A mouse allogenic mixed lymphocyte reaction (hereinafter to be referred to as mouse allogenic MLR) was performed by mixed culture (equal ratio) of the spleen cell of BALB/c mice as the reaction cell and the spleen cell of C57BL/6 mice treated with mitomycin C as stimulated cell.

The reaction cells were prepared by the following method. Spleen was removed from 5-6 week old BALB/c mice and treated with RPMI1640 medium (containing kanamycin sulfate (60 pg/ml), penicillin G potassium (100 units/ml), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (10 mM), 0.1% sodium hydrogencarbonate and L-glutamin (2 mM)) supplemented with 5% heat S. inactivated fetal bovine serum (FBS) to give a single cell suspension of the spleen cell. After hemolysis treatment, the suspension was adjusted to107 cells/ml with RPMI1640 medium containing 104 M 2-mercaptoethanol and 10% FBS and used .as a reaction cell suspension.

The reaction cell suspension (50 id) prepared by the above method, stimulated cell suspension (50 id) and the test compound (100 Il) prepared using RPMI1640 medium containing 10/o FBS were added to a 96 well plate and S.i* incubated at 37°C under 5% CO 2 gas, 95% air for 4 days.

A pigment assay using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was applied for the determination of lymphocyte transformation reaction.

After the completion of culture, the supernatant (100 IL) in each well was removed, and 5 mg/ml MTT solution (20 dp) was added to each well, which was followed by incubation at 37C for 4 hours. Then, a 0.01 N hydrochloric acid

S

solution (100 pl) containing 10%/ sodium dodecyl sulfate was added and the mixture was incubated at 37°C overnight. The resulting purple crystals of formazan was dissolved and absorbance at 550 nm was measured using a microplate absorption meter, which was used as the index of lymphocyte transformation reaction of the mouse allogenic MLR. The inhibition of mouse allogenic MLR was evaluated by calculating the inhibition percentage by the following formula. The results are shown in Table 7.

SAbsorbance of MLR with absorbance of Iaddition of test compound reacted cells alone Absorbance of MLR without absorbance of addition of test compound reacted cells alone Table 7 Test compound Bone resorption Mouse allogenic MLR inhibition (pM) inhibitory activity ICso (IM) Compound 80.2HC1.H 2 0 40.9(100) 9.6 Compound 109.2HCI 42.6(100) 1.6 Compound 112.2HCI 75.7(100) 4.4 Compound 110.2HC1.H 2 0 74.0(100) 1.1 Compound 142.2HC1.H 2 0 44.2(100) Compound 143.2HC1.H 2 0 39.4(100) Compound 308.2HCI 13.9 Experimental Example 10 Inhibition of cell growth of SK-Mel-28 melanoma Human SK-Mel-28 melanomna (104 cells) and the test compound were suspended in RPMI1640 medium containing 100 tl of 10% FBS and incubated in a 96 well plate at 37*C under 5% CO 2 gas for 72 hours. After the incubation, td of MTT (5 mg/ml) was added to each well and the cells were incubated at 37°C under 5% CO 2 gas for 4 hours. Then, 10% sodium dodecyl sulfate and 0.01 N hydrochloric acid solution were each added by 10 ld to respective wells. After the plate was stood overnight, absorbance at 570 nm was measured using a microplate reader and the inhibition percentage cytotoxicity) was calculated by the following formula. The results are shown in Table 8.

The cytotoxicity against human cultured tumor cells was confirmed by pigment method (Carmichael et al., Cancer Res., 47, 936-942, 1987 Mosman, J.

Immunol. Methods, 65, 55-63, 1983) using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT).

The test compound was dissolved in dimethyl sulfoxide and diluted with RPMI1640 medium before use. The final dimethyl sulfoxide concentration was adjusted to not more than 0.25%.

Absorbance when test compound was added' Inhibition 1 x 100 Absorbance when test compound was not added Table 8 Test compound Cell growth inhibition ICso (PM) Compound 115.2HBr.1/4H 2 0 9 Compound 109.2HCI 58 Compound 142.2HC1.H 2 0 59 Compound 145.2HC1.H 2 0 62 Experimental Example 11 Inhibition of angiogenesis The inhibition of angiogenesis was evaluated by using the inhibition of lumen formation in vascular endothelial cell as an index. To be specific, normal human umberical vascular endothelial cells (KURABO INDUSTRIES LTD.) were suspended in E-GMUV medium at 5.5 x 104 cells/ml and 400 ld therefrom was added on matrigel plate (EHS sarcoma-derived reconstructed basement membrane, Collaborative Biomedical Products). Then, the test compound (1 mM solution, 4 pl) was added and the cells were incubated at 37°C under 5% CO 2 gas for 18 hours. After the completion of the incubation, the number of lumen per S. predetermined area was counted under a microscope. Inasmuch as the number of lumen increases by the inhibition of lumen formation, the test compound was evaluated by comparison of the number of lumen with the control. The results are shown inTable9.

,o are shown in Table 9.

Table 9 Test compound Number of lumen Compound 109.2HCI 153% Compound 80.2HC1.H 2 0 174% Compound 110.2HCl.H 2 0 203% Compound 165.2HC1.3/2H 2 0 222% Compound 204.2HCI 133% Experimental Example 12 Inhibition of growth of vascular smooth muscle cell The separation from the artery of rat and culture of smooth muscle cell (SMC) followed the explant method of Ross (Ross, R and Glomset, J. N. Engl. J.

Med., 295, 369-420, 1976). Male wistar rats (10 week old) was slaughtered by cutting the carotid arteries and aorta of thorax was removed. After removal of fat tissues around the tunica externa and peeling of tunica intima, the artery was minced and incubated in 10% fetal bovine serum (FBS)-containing DMEM medium at 37C under 5% CO2 gas. Seven days later, the out-grown cells were separated by trypsin treatment, washed with phosphate-buffered saline (PBS) and incubated in 10%/ FBS-containing DMEM medium in a 80 cm 2 culture flask. The cells of subculture 2 were suspended in 10% FBS-containing DMEM medium at x 10 4 cells/ml and 100 R1 thereof per well was added to 96 well collagen-coated plate, which was incubated at 37C under 5% CO 2 gas for one day. The test compound was appropriately diluted with dimethyl sulfoxide (DMSO) and added to the 96 well plate. The concentration of DMSO in the medium was adjusted to 1%.

After 48 hours, 10 gl of MTT solution (5 mg/ml) was added and, 4 hours later, sodium dodecyl sulfate-0.01 N hydrochloric acid (50 Pl) was added. The absorbance at 570 nm was measured the following day by an immunoreader.

The SMC growth inhibitory action of the test compound was shown by inhibition percentage calculated by the following formula. The results are shown in Table Absorbance when test compound was added' Inhibition x 100 Absorbance when test compound was not added Table Test compound ICso (IpM) compound 153.2HC1 27 compound 157.2HC1.H 2 0 compound 165.2HC1.3/2H 2 0 38 compound 163.2HBr 63 Experimental Example 13 :Acute toxicity The compound 109.2HC1 and compound143.2HCl.H 2 0 were respectively administered intraperitoneally to ddY mice and the mice were monitored for 5 days.

As a result, the intraperitoneal administration at 30 mg/kg did not cause death.

The foregoing Formulation Examples and pharmacological experiments reveal that the compounds of the formula and the formula (II) have strong Rho kinase inhibitory action. These Rho kinase inhibitors have vasodilating action, trachea relaxing action, peripheral blood flow increasing action, cell adhesion induction inhibitory action, tumor cell metastasis inhibitory action, bone resorption inhibitory action, mouse allogenic MLR inhibitory activity, tumor cell growth inhibitory action, angiogenesis inhibitory action, vascular smooth muscle cell growth inhibitory action and other various actions. Therefore, they are useful as pharmaceutical agents, particularly, a therapeutic agent of hypertension, a therapeutic agent of angina pectoris, a suppressive agent of cerebrovascular contraction, a therapeutic agent of asthma, a therapeutic agent of peripheral 9 circulation disorder, a prophylactic agent of immature birth, a therapeutic agent of arteriosclerosis, an anti-cancer drug, an anti-inflammatory agent, an immunosuppressant, a therapeutic agent of autoimmune disease, an anti-AIDS drug, a contraceptive, a prophylactic agent of digestive tract infection, a therapeutic agent of osteoporosis, a therapeutic agent of retinopathy and a brain function improving drug.

In addition, since Rho kinase inhibitors of the present invention have strong Rho kinase inhibitory activity, they are also useful as reagents for the study relating to Rho and Rho kinase and as diagnostics of the diseases related to them.

This application is based on application No. 212409/1996 filed in Japan, the contents of which are incorporated hereinto by reference.

It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising", or grammatical variations thereof, is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.

oooo 58a

Claims (127)

1. A pharmaceutical agent comprising a Rho kinase inhibitor.

2. A therapeutic agent of hypertension, comprising a Rho kinase inhibitor.

3. A therapeutic agent of angina pectoris, comprising a Rho kinase inhibitor.

4. A suppressive agent of cerebrovascular contraction, comprising a Rho kinase inhibitor.

A therapeutic agent of asthma, comprising a Rho kinase inhibitor.

6. A therapeutic agent of a peripheral circulation disorder, comprising a Rho kinase inhibitor.

7. A therapeutic agent of arteriosclerosis, comprising a Rho kinase inhibitor.

8. An anti-cancer drug comprising a Rho kinase inhibitor.

9. An anti-inflammatory agent comprising a Rho kinase inhibitor.

An immunosuppressant comprising a Rho kinase inhibitor.

11. A therapeutic agent of an autoimmune disease, comprising a Rho kinase inhibitor.

12. An anti-AIDS drug comprising a Rho kinase inhibitor.

13. A therapeutic agent of osteoporosis, comprising a Rho kinase inhibitor.

14. A therapeutic agent of retinopathy, comprising a Rho kinase inhibitor.

A brain function improving drug comprising a Rho kinase inhibitor.

16. A prophylactic agent of immature birth, comprising a Rho kinase inhibitor.

17. A contraceptive comprising a Rho kinase inhibitor.

18. A prophylactic agent of digestive tract infection, comprising a Rho kinase inhibitor.

19. A pharmaceutical composition comprising a therapeutically effective amount 59 of a Rho kinase inhibitor and a pharmaceutically acceptable additive.

A reagent comprising a Rho kinase inhibitor.

21. A diagnostic comprising a Rho kinase inhibitor.

22. A Rho kinase inhibitor comprising an amide compound of the formula (I) O Rb Ra- C- N -R (I) wherein Ra is a group of the formula R 2 R N-A (a) R R R R N-A R R 4 I (c) L- N N in the formulas and R is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or a group of the formula NR 7 (d) wherein R 6 is hydrogen, alkyl or formula: -NRNR 9 wherein R 8 and R 9 are the same or different and each is hydrogen, alkyl, aralkyl or phenyl, R 7 is hydrogen, alkyl, aralkyl, phenyl, nitro or R and R 1 R 2 R 3 and R 4 A cyano, or R 6 and R 7 in combination show a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom, is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralky, which optionally have a substituent on the ring, or in combination form, together with the adjacent nitrogen atom, a group forming a heterocycle optionally having, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom, is hydrogen or alkyl, are the same or different and each is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl or azide, and is a group of the formula .c I S(cH2)1(C)m(cH)n wherein R' 1 and R 1 are the same or different and each is hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R 1 0 and R" show a group which forms cycloalkyl in combination and 1, m and n are each 0 or an integer of 1-3, in the formula L is hydrogen, alkyl, aminoalkyl, mono or dialkylaminoalkyl, tetrahydrofurfuryl, carbamoylalkyl, phthalimidoalkyl, amidino or a group of the formula O B-C- Q o-w- 0 11 C x or Q 2 3 wherein B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl,. hydroxyalkl, alkanoyloxylkl, alkoxycarbonylalkyl, a-axninobenzyl, furyl, pyridyl, phenyl, phenylarnino, styryl or imidazopyridyl, is hydrogen, halogen, hydroxy, aralkyloxy or thienylmethyl, W is alkylene, :Q 2 is hydrogen, halogen, hydroxy or aralkyloxy, X is alkylene, Q 3 is hydrogen, haogen, hydroxy, alkoxy, nitro, amino, 2,3- dihydrofturyl or 5-methyl-3-oxo-2,3,4,5-tetrahydropyridazin-6-yl; and Y is a single bond, alkylene or alkenylene, and in the formula a broken line is a single bond or a double bond, and 5 is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkanoyloxy or aralkyloxycarbonyloxyr Rb is a hydrogen, an alkyl, an aralkyl, an aininoalkyl or a mono or dialkylamidnoalkyl; and Rc is an optionally substituted heterocycle containing nitrogen, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

23. A therapeutic agent of hypertension caused by Rho Inase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

24. A therapeutic agent of angina pectoris caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A suppressive agent of cerebrovascular contraction caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

26. A therapeutic agent of asthma caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

27. A therapeutic agent of peripheral circulation disorder caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

28. A therapeutic agent of arteriosclerosis, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

29. An anti-cancer drug comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

An anti-inflammatory agent comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

31. An immunosuppressant comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

32. A therapeutic agent of an autoimmune disease, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

33. An anti-AIDS drug comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

34. A therapeutic agent of osteoporosis, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A therapeutic agent of retinopathy, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

36. A brain function improving drug comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

37. A prophylactic agent of immature birth, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

38. A contraceptive comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

39. A prophylactic agent of digestive tract infection, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

40. A reagent having a Rho kinase inhibitory activity, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

41. A diagnostic of a disease caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

42. A Rho kinase inhibitor containing a substituted isoquinolinesulfonamide derivative of the formula (II) R13 R 14 SI s) SO 2 N Alk N R 1 R12 wherein R 12 is a hydrogen, a chlorine or a hydroxy, and when R 12 is a hydrogen, Alk is an alkylene having 2 to 6 carbon atoms, which optionally Has alkyl having 1 to 10 carbon atoms, aryl or aralkyl as a substituent; R 13 is a hydrogen; R 14 is a hydrogen, or a linear or branched alkyl having 1 to 6 carbon atoms, an aryl or an aralkyl; R' 5 is a hydrogen, a linear or branched alkyl having 1 to 6 carbon atoms, an aryl or an aralkyl, or a benzoyl, a cinnamyl, a cinnamoyl, a furoyl or a group of the following formula OR 16 CHCH 2 :....wherein R 16 is linear or branched alkyl having 1 to 6 carbon atoms or a group of the following formula NR 17 NHR 18 [wherein R 1 7 and R' 8 are hydrogen or directly bonded to form alkylene having 2 to 4 carbon atoms; or R 13 and R 1 4 are directly bonded to form alkylene having 4 or less carbon atoms, which is optionally substituted by alkyl having 1 to carbon atoms, phenyl or benzyl, or S" R 1 4 and R s 15 directly or in combination via oxygen atom form a heterocycle together with the adjacent nitrogen atom, and wherLR 2 is a chlorine or a hydroxy, Alk is an alkylene having 2 to 6 carbon atoms, which is optionally substituted at the hydrogen bonded to carbon by alkyl having 1 to 6 carbon atoms, R 1 3 and R 1 4 are each a hydrogen, a linear or branched alkyl having 1 to 6 carbon atoms or directly bonded to each other to form ethylene or trimethylene, wherein hydrogen bonded to carbon is optionally substituted by alkyl having 1 to 6 carbon atoms; or R i s is a hydrogen, a linear or branched alkyl having 1 to 6 carbon atoms or an amidino, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

43. A therapeutic agent of hypertension caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

44. A therapeutic agent of angina pectoris caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A suppressive agent of cerebrovascular contraction caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

46. A therapeutic agent of asthma caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

47. A therapeutic agent of a peripheral circulation disorder, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

48. A therapeutic agent of arteriosclerosis, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

49. An anti-cancer drug comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A therapeutic agent of inflammation caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

51. An immunosuppressant comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

52. A therapeutic agent of an autoimmune disease, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

53. An anti-AIDS drug comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

54. A therapeutic agent of osteoporosis, comprising a compound of the formula (II), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A therapeutic agent of retinopathy, comprising a compound of the formula (II), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

56. A medicament for improving a brain function disorder caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

57. A prophylactic agent of immature birth, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

58. A contraceptive comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

59. A prophylactic agent of digestive tract infection, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

A reagent having a Rho kinase inhibitory activity, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

61. A diagnostic of a disease caused by Rho kinase, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

62. A compound of the formula L N(III) C- N -Rc' II O wherein Rc' is an optionally substituted heterocycle having nitrogen, which is other than pyridine of Rc, and other symbols are as defined above, an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.

63. The pharmaceutical agent of any of claims 1.to 18, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof as a Rho kinase inhibitor.

64. The pharmaceutical composition of claim 19, comprising a compound of the formula (IlI), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof as a Rho kinase inhibitor. S*

65. The reagent of claim 20, comprising a compound of the formula an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof as a Rho kinase inhibitor.

66. The diagnostic of claim 21, comprising a compound of the formula (III), an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof as a Rho kinase inhibitor. 0. 00 *000

67. A method for inhibiting Rho kinase, comprising administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

68. A method for treating a disease caused by Rho kinase, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

69. A method for treating hypertension caused by Rho kinase, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

70. A method for treating angina pectoris caused by Rho kinase, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

71. A method for suppressing cerebrovascular contraction caused by Rho kinase, which comprises administering to a patient in need thereof a 15 therapeutically effective amount of a Rho kinase inhibitor.

72. A method for treating asthma caused by Rho kinase, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

73. A method for treating peripheral circulation disorder caused by Rho kinase, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

:74. A method for treating arteriosclerosis, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor. e 25

75. A method for treating cancer, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

76. A method for treating inflammation, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

77. A method for inducing immunosuppression, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

78. A method for treating an autoimmune disease, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

79. A method for treating AIDS, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

80. A method for treating osteoporosis, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

81. A method for treating retinopathy, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor. o

82. A method for improving brain function disorder, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

83. A method for preventing immature birth, which comprises .ooo@i administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

A method for contraception, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor. a

85. A method for preventing digestive tract infection, which comprises administering to a patient in need thereof a therapeutically effective amount of a Rho kinase inhibitor.

86. A method for inhibiting Rho kinase activity in a sample, which comprises contacting the sample with an effective amount of a Rho kinase inhibitor.

87. A method for diagnosing a disease caused by Rho kinase, which comprises contacting a sample with a Rho kinase inhibitor.

88. The use of a Rho kinase inhibitor for inhibiting Rho kinase.

89. The use of a Rho kinase inhibitor for the preparation of a medicament for inhibiting Rho kinase.

The use of a Rho kinase inhibitor for treating a disease caused by Rho kinase.

91. The use of a Rho kinase inhibitor for the preparation of a medicament for treating a disease caused by Rho kinase.

92. The use of a Rho kinase inhibitor for treating hypertension caused by Rho kinase.

93. The use of a Rho kinase inhibitor for the preparation of a medicament for treating hypertension caused by Rho kinase.

94. The use of a Rho kinase inhibitor for treating angina pectoris caused 15 by Rho kinase.

95. The use of a Rho kinase inhibitor for the preparation of a medicament for treating angina pectoris caused by Rho kinase.

96. The use of a Rho kinase inhibitor for suppressing cerebrovascular contraction caused by Rho kinase. 20

97. The use of a Rho kinase inhibitor for the preparation of a medicament for suppressing cerebrovascular contraction caused by Rho kinase.

98. The use of a Rho kinase inhibitor for treating asthma caused by Rho kinase.

99. The use of a Rho kinase inhibitor for the preparation of a medicament for treating asthma caused by Rho kinase.

100. The use of a Rho kinase inhibitor for treating peripheral circulation disorder caused by Rho kinase.

101. The use of a Rho kinase inhibitor for the preparation of a medicament for treating peripheral circulation disorder caused by Rho kinase.

102. The use of a Rho kinase inhibitor for treating arteriosclerosis.

103. The use of a Rho kinase inhibitor for the preparation of a medicament for treating arteriosclerosis.

104. The use of a Rho kinase inhibitor for

105. The use of a Rho kinase inhibitor for for treating cancer.

106. The use of a Rho kinase inhibitor for

107. The use of a Rho kinase inhibitor for for treating inflammation.

108. The use of a Rho kinase inhibitor for

109. The use of a Rho kinase inhibitor for for inducing immunosuppression. 15

110. The use of a Rho kinase inhibitor for disease.

111. The use of a Rho kinase inhibitor for treating cancer. the preparation of a medicament treating inflammation. the preparation of a medicament inducing immunosuppression. the preparation of a medicament treating an autoimmune the preparation of a medicament .4 9 4. 9 4. *4 4 9*9 4 4r treating an autoimmune disease.

112. The use of a Rho kinase inhibitor for treating AIDS. 4*

113. The use of a Rho kinase inhibitor for the preparation of a medicament for treating AIDS. 9**

114. The use of a Rho kinase inhibitor for treating osteoporosis.

115. The use of a Rho kinase inhibitor for the preparation of a medicament for treating osteoporosis.

116. The use of a Rho kinase inhibitor for treating retinopathy.

117. The use of a Rho kinase inhibitor for treating retinopathy.

118. The use of a Rho kinase inhibitor disorder.

119. The use of a Rho kinase inhibitor for improving brain function disorder.

120. The use of a Rho kinase inhibitor

121. The use of a Rho kinase inhibitor for preventing immature birth.

122. The use of a Rho kinase inhibitor

123. The use of a Rho kinase inhibitor for contraception.

124. The use of a Rho kinase inhibitor for the preparation of a medicament for improving brain function for the preparation of a medicament for preventing immature birth. for the preparation of a medicament for contraception. for the preparation of a medicament for preventing digestive tract .9 9999* 15 oooo 99 20 9.. infection.

125. The use of a Rho kinase inhibitor for the preparation of a medicament for preventing digestive tract infection.

126. The use of a Rho kinase inhibitor for inhibiting Rho kinase activity in a sample.

127. The use of a Rho kinase inhibitor for diagnosing a disease caused by Rho kinase. Dated this 2nd day of August 2001 WELFIDE CORPORATION By its Patent Attorneys GRIFFITH HACK