WO2001017562A1 - Osteogenesis promoting agents - Google Patents

Abstract

Osteogenesis promoting agents characterized by containing a compound having an Rho kinase inhibitory activity and a bone inducing factor; and osteogenesis induction potentiating agents characterized by containing a compound having an Rho kinase inhibitory activity as the active ingredient and being capable of potentiating the bone inducing effect of a foreign bone inducing factor. As an example of the compound having an Rho kinase inhibitory activity, (+)-trans-4-(1-aminoethyl)-1-(4-puridylcarbamoyl)cyclohexane may be cited. By using the compound having an Rho kinase inhibitory activity together with the bone inducing factor, the differentiation of mesenchymal cells into osteoblasts is more remarkably induced. The above-described osteogenesis promoting agents and osteogenesis induction potentiating agents are useful in preventing and treating various bone diseases, in particular, bone or cartilage injury, defect or hypogenesis.

Description

 Specification

 Bone formation promoter

 Technical field

 The present invention provides an osteogenesis promoting agent characterized by comprising a compound having Rho kinase inhibitory activity and an osteoinductive protein, and an exogenous osteoinductive factor comprising a compound having Rho kinase inhibitory activity as an active ingredient. The present invention relates to an osteogenesis-inducing enhancer characterized by enhancing the osteoinductive action of the compound.

 Background art

 In bone tissue, bone resorption and bone formation are constantly repeated in a balanced manner. Osteoblasts play a central role in bone formation, and osteoclasts play a central role in bone resorption.

 This balance of bone remodeling works by linking osteoclast and osteoblast cell lines. In other words, it is thought that systemic and local regulators form a complex network at each stage of bone reconstruction, act on osteoblasts and osteoclasts, and maintain bone turnover. I have.

 If the balance between bone resorption and bone formation breaks down, it is thought that bone loss will occur, and osteoporosis and other bone diseases will occur.

 At present, calcium preparations, active vitamin D3 preparations, estrogen preparations, calcitonin preparations, bisphosphonates, etc. are used as therapeutic agents for the prevention of bone diseases.Estrogen preparations, calcitonin preparations, bisphosphonate preparations, etc. Bone resorption inhibitors are mainly used.

 However, the administration of these bone resorption inhibitors may be limited or their effects may not be assured, and at present, sufficient therapeutic effects have not been obtained.

On the other hand, fluorine is a clinically usable substance that promotes bone formation. However, the safety margin of administration is narrow and there is a problem with long-term administration. In addition, growth factors such as parathyroid hormone (PTH), osteoinductive factor (BMP) ^ insulin-like growth factor (IGF), and fibroblast growth factor (FGF) are known as substances promoting bone formation. I have. Bone morphogenetic protein (BMP) acts on undifferentiated mesenchymal cells in the subcutaneous or muscular tissue and differentiates them into chondroblasts or osteoblasts to form cartilage or bone. Active protein. BMP was released as a substance exhibiting ectopic osteoinductive activity in the demineralized bone matrix, but was not purely isolated and its specific structure remained unclear.

 However, by genetic engineering, the gene encoding human BMP was cloned, and the amino acid sequence was revealed. Human BMPs have also been found to constitute a family of family proteins with similar amino acid sequences, and numerous types of recombinant human bone inducing factor (rhBMP) have been identified. Natl. Acad. Sci. USA Vol. 87, pp. 2220-2224 (1990); Progress in Growth Factor Research, Vol. 1, CScience Vol. 242, pp. 1528-1534 (1988); pp.267-280 (1989); Japanese Patent Publication No. 2-500241, Japanese Patent Publication No. 3-503649, Japanese Patent Publication No. 3-505098, WO 91/18098, WO 92/05199, Publications of WO 93Z092 229]. Production by transformants is also underway.

 Various methods have been studied for treating the bone, cartilage for damage, defect or dysplasia using the above-mentioned BMP.

 As for the mechanism of bone formation, undifferentiated cannabis cells first migrate locally. Next, transforming growth factor ^ (TGF- /?) Released from platelets and bone, osteoinduction factor (BMP), insulin-like growth M (IGF), fibroblast proliferation K Active site forces such as (FGF) induce the differentiation of these undifferentiated mesenchymal cells into chondrocytes and blasts. The differentiated osteoblast group consists of collagen, mainly type I, alkaline phosphatase, and osteonectin.

It produces extracellular matrix such as (osteonectin), osteopontin, bone sialoprotein, and osteocalcine to induce further differentiation and form bone tissue (Grainger, DJ-, et al _.: Nature Med. 1. p.932-937 (1995)). Therefore, first of all, for bone formation, it is important to induce differentiation of undifferentiated mesenchymal cells into osteoblasts that play a central role in bone formation.

 However, these undifferentiated mesenchymal cells are derived not only from osteoblasts, but also from other bone marrow stromal cells, including chondrocytes, muscle and fat cells, which make up the skeletal system. This suggests that it is necessary to consider the induction of the differentiation of the interstitial cells into osteoblasts along with the regulation of the differentiation into other cell lines.

 Rho, on the other hand, is activated by receiving signals from various cell membrane receptors, and activated Rho is activated by the actomyosin system, resulting in smooth muscle contraction, cell motility, cell adhesion, changes in cell morphology, cell proliferation, etc. It has been shown to function as a molecular switch for various cellular phenomena. It is now being clarified that Rho kinase present downstream of the Rho-mediated signal transduction pathway plays an important role in the response of Rho to the above-mentioned cellular phenomena.

 In addition, red differentiated mesenchymal cells migrate locally, and are then induced to differentiate into myotubes, leading to the formation of muscle and blood vessels, but to induce differentiation of these undifferentiated mesenchymal cells into myotubes. It has been shown that Rh is involved (Mol. Cell Biol., 18,

PP. 1580-1589 (1998), J. Biol. Chem., 46, pp. 30287-30294 (1998)). However, the relationship with bone differentiation is not yet clear.

 In recent years, a compound represented by the following general formula (I) has been reported as a compound having Rho kinase inhibitory activity (W98 / 06433). Certain isoquinoline sulfone amide derivatives and isoquinoline derivatives have also been reported to have Rho kinase inhibitory activity (WO 98Z0 6433 and Naunyn-Schmiedeberg's Archives of Pharmacology 385 (1 ) Supl. R219, (1998) 11).

The pharmaceutical uses of these compounds having Rh kinase inhibition are disclosed in WO 98/06433, and are widely used for treating hypertension, angina, cerebral vasospasm, asthma, peripheral circulatory disorders Therapeutics, premature birth prevention, arteriosclerosis treatment, anticancer drugs, anti-inflammatory drugs, immunosuppressants, autoimmune disease drugs, anti-AIDS drugs, osteoporosis drugs, net It is noted that it is useful as a drug for treating membrane colic, a brain function improving drug, contraceptive Ji, and gastrointestinal tract infection.

 Further, the compound represented by the general formula (I) is a potent and persistent drug for treating hypertension, angina pectoris, renal and peripheral circulatory disorders, cerebral vasospasm, etc. It is already known to be useful as a therapeutic agent for diseases of the circulatory system such as peripheral arteries, and as a therapeutic agent for asthma (Japanese Patent Application Laid-Open Nos. 62-89679 and 3-2-111). No. 8356, JP-A-4-127382, JP-A-5-194041, JP-A-6-41080 and WO 95/28387.

 In addition, the isokinoline sulfone amine derivative described in WO 98/06433 is a vasodilator, a therapeutic agent for hypertension, a brain function improving agent, an anti-asthmatic agent, a cardioprotective agent, a platelet aggregation HI harmful agent, Antipyretic, anti-inflammatory, hyperviscosity treatment or prevention, glaucoma, ocular hypoxia, cerebral thrombotic motor paralysis, viral infection prevention and transcriptional regulator inhibitor (Japanese Patent Application Laid-Open Nos. 57-200366, 61-227581, 62-25617, JP-A-4-264030 and JP-A-6-264030). No. 56668, JP-A-6-80569, JP-A-6-293643, JP-A-7-41424, JP-A-7-277 979, W097Z23222, JP-A-9-1223737, JP-A-Heisei 1 0-455 98 and entitled 1 0-8749 1).

 Furthermore, the isoquinoline derivatives described in the above three documents (Naunyn-Schmiedeberg's Archives or Pharmacology 385 (1) Suppl.R219, (1998)) may be useful as agents for preventing and treating brain tissue injury due to vasospasm. It is already known (WO 97/28130).

Further, as described above, WO 98/06433 describes that a compound having an Rho kinase inhibitory activity is useful as a therapeutic agent for osteoporosis. It is based on the inhibitory effects of integrin activation and cell adhesion induction, which are thought to be involved in bone resorption by Rho. There is no description of the effect of enhancing the osteoinductive effect of the inducer.

 Guidance of invention

 An object of the present invention is to provide a useful novel bone formation promoting agent and a bone formation induction enhancer capable of enhancing the osteoinductive action of an osteoinductive factor.

 The present inventors have conducted intensive studies and have found that the combined use of an osteoinductive K child and a compound having Rho kinase inhibitory activity dramatically improves bone formation ability, and completed the present invention. .

 That is, the present invention is as follows.

 (1) An osteogenesis promoting agent comprising a compound having Rh kinase inhibitory activity and an 'inducing factor'.

 (2) The compound having Rh kinase inhibition activity is represented by the general formula (I)

0 Rb

Ra——C-N- -Rc (I)

(Where Ra is the formula

Factory

〔式 （a) および （b) 中、 Rは水素、 アルキルまたは環上に置換基をおしてい てもよぃシクロアルキル、 シクロアルキルアルキル、 フエニルも しくはァラルキ ルを示すか、 あるいは式

Then N

[In the formulas (a) and (b), R may be hydrogen, alkyl, or a substituent on the ring. R represents cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, or

(Wherein R is hydrogen, alkyl or a formula: NR ⁸ R. (where R ^{8 is} the same or different and represents water, alkyl, aralkyl or phenyl).) R ⁷ represents hydrogen, alkyl, aralkyl, phenyl, nitro or cyano; or R ⁷ and R ⁷ may be bonded to each other to have an oxygen-, sulfur- or substituent in the ring. A group forming a heterocyclic group which may contain a good nitrogen atom.

R ¹ represents hydrogen, alkyl, or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on the ring.

Or R and R ¹ represent a group which, together with an adjacent nitrogen atom, forms a heterocyclic group which may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent. .

R ² represents hydrogen or alkyl.

RR ⁴ may be the same or different and may be hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, argavamoyl , Mono-dialkylcarbamoyl or azide.

 A is the formula

(Wherein, R ^{1 Q,} R ^{1 1} are the same or different and each is hydrogen, alkyl, haloalkyl, § aralkyl, hydroxyalkyl, showing a carboxy or alkoxycarbonyl _(or, R ^{1 (1} and R ^{1 1} is bonded to cyclo Represents an alkyl-forming group, 1, m, n represents 0 or an integer of 1 to 3 respectively. ).

 In the formula (c), L represents hydrogen, alkyl, aminoalkyl, mono'dialkylaminoalkyl, tetrahydrofurfurfuryl, fulvamoylalkyl, furymidylalkyl, amidino, or

B one (g)

(

 (: ^ Where B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl, hydroxyalkyl, alkanoyloxyalkyl, alkoxycarbonylalkyl, hyaminobenzyl, furyl, pyridyl, phenyl, phenylamino, styryl or Indicates midazopyridyl.

Q ¹ represents hydrogen, halogen, hydroxyl, aralkyloxy or cetylmethyl.

 W represents alkylene.

Q ² represents hydrogen, halogen, a hydroxyl group or aralkyloxy.

 X represents alkylene.

Q ³ represents hydrogen, halogen, hydroxyl, alkoxy, nitro, amino, 2,3-dihydrofuryl or 5-methyl-3-oxo-1,2,3,4,5-tetrahydropyridazine-6-yl.

 Y represents a bond, alkylene or alkenylene. ).

 Further, in the formula (c), a broken line indicates a-heavy bond or a single bond.

 R 5 represents a waterline, a hydroxyl group, an alkoxy, an alkoxycarbonyloxy, an alkanoyloxy or an aralkyloxycarbonyl. ] Is shown.

R b is hydrogen, alkyl, aralkyl, aminoalkyl or mono'dialkyl Represents luminoalkyl.

 R c represents a nitrogen-containing heterocyclic ring which may have a substituent. ]

The osteogenesis promoter according to the above (1), which is an amide compound represented by the formula: or an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof.

 (3) The compound having Rh kinase inhibitory activity is represented by the general formula (I ')

 O Rb

Ra'ichi C- -N- -Rc (Ι ')

[Wherein, a 'is a formula

[In the formula, R ′ represents hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on 璟.

R ¹ represents hydrogen, alkyl or cycloalkyl optionally having a substituent on a ring. Cycloalkylalkyl, phenyl or aralkyl.

Or R 'and R ¹ are bonded together with an adjacent nitrogen atom to form a heterocyclic ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent. Is shown.

R ² represents hydrogen or alkyl.

R ³ and R ⁴ are the same or different and are hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, ara This refers to ruquiloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, dirubamoyl, monodialkylcarbamoyl or azide.

 A is the formula

R 10 (CH ₂ h ( _m (CH ₂ ) _n ~-(e)

(Wherein, R ^{1 G,} R ^{1 1} are the same or different and each is hydrogen, alkyl, haloalkyl, § aralkyl, hydroxycarboxylic alkyl, or _t indicating the carboxy or alkoxycarbonyl, R ¹ ° and R ^{1 1} is bonded to 1, m and n each represent 0 or an integer of from 1 to 3.)]].

 Rb represents hydrogen, alkyl, aralkyl, aminoalkyl or mono'dialkylaminoalkyl.

 R c represents a nitrogen-containing heterocyclic group which may have a substituent. ]

The osteogenesis promoter according to the above (1) or (2), which is an amide compound represented by the formula: or an isomer thereof and Z or a pharmaceutically acceptable acid addition salt thereof.

(4) Compounds having Rh kinase inhibition activity include (+)-trans-4-1 (1-aminoethyl) 1-1- (4-pyridylcarbamoyl) cyclohexane, (+)-trans-N- ( 1 H-Pyro mouth [2,3-b] Pyridine-14-yl) 1-4- (1-Aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-Pyridyl) 1-41- (1-aminoethyl) benzamide and (R)-(+)-N- (1H-pyro mouth [2,3-b] pyridine-14-yl) 1-41- (1-aminoethyl) benzami And z or a pharmaceutically acceptable acid addition salt thereof, especially (+) — trans-4 -_ (1-aminoethyl) —1- (4-pyridylcarbamoyl) cyclo The bone formation promoting agent according to the above (1), which is xane and Z or a pharmaceutically acceptable acid addition salt thereof. (5) The bone formation promoting agent according to any one of (1) to (4) above, wherein the osteoinductive factor is recombinant human BMP-2.

 (6) The bone according to any one of the above (1) to (5), which is adapted for prevention-treatment of a bone disease, particularly a bone disease accompanied by damage, loss or dysplasia of bone or cartilage. Formation promoter.

 (7) An osteogenesis induction enhancer comprising a compound having Rh kinase inhibitory activity as an active ingredient, which enhances the osteoinduction effect of an exogenous osteoinductive factor.

 (8) The compound according to the above (7), wherein the compound having Rh kinase inhibitory activity is a amide compound represented by the general formula (I), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. Osteogenesis induction enhancer.

 (9) The compound according to the above (7) or (7), wherein the compound having a Rh kinase inhibition activity is an amide compound represented by the general formula (1 ′), an isomer thereof and Z or a pharmaceutically acceptable acid addition salt thereof. (8) The bone formation induction enhancer according to (8).

 (10) The compound having Rh kinase inhibitory activity is (+)-trans-4- (1-aminoethyl) _1- (4-viridylcarbamoyl) cyclohexane,

(+) 1-trans-N- (1H-pyrro [[2,3_b] pyridine-14-yl) -14- (1-aminoethyl) cyclohexanecarboxamide, (R) — (+) — N- (4-pyridyl) -14- (1-aminoethyl) benzamide and (R) ―

A compound selected from the group consisting of (+)-N- (1H-pyro mouth [2,3-b] pyridine-14-yl) -14- (1-aminoethyl) benzamide and Z or Pharmaceutically acceptable acid addition salts, especially (+) — trans-41- (1-aminoethyl) 1-1- (4-pyridylcarbamoyl) cyclohexane and / or its pharmaceutically acceptable acid addition salts The bone formation induction enhancer according to the above (7), which is

 (11) The osteogenesis-inducing enhancer according to any one of (7) to (10) above, wherein the osteoinductive W child is recombinant human BMP-2.

(12) The method according to any one of (7) to (11) above, which is adapted for prevention of bone disease, particularly bone disease accompanied by damage, loss or dysplasia of bone or cartilage. Osteogenesis induction enhancer.

 (13) A pharmaceutical composition for preventing or treating bone disease, comprising a compound having a Rh kinase inhibitory activity, an osteoinductive factor, and a pharmaceutically acceptable carrier.

 (14) The compound according to the above (13), wherein the compound having Rh kinase inhibitory activity is an amide compound represented by the general formula (I), a tertiary compound thereof, or a pharmaceutically acceptable acid addition salt thereof. Pharmaceutical composition for prevention and treatment of bone diseases.

 (15) The compound according to the above (13), wherein the compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (1), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. Or The pharmaceutical composition for prevention and treatment of bone disease according to (14). (16) The compound having Rho kinase inhibitory activity is (+)-trans-4- (1-aminoethyl) -1- (4-viridylcarbamoyl) cyclohexane, (+)-trans-N — (1 H—Pyro mouth [2,3-b] pyridine-14-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide, (R) — (+) —N-(4— Pyridyl) 1-4- (1-aminoethyl) benzamide and (R)-(+)-N- (1H-pyro mouth [2,3-b] pyridine-14-yl) 1-4-1 (1- Aminoethyl) A compound selected from the group consisting of benzamides and / or their pharmaceutically acceptable acid addition salts, especially (+) — trans-41- (11-aminoethyl) -11- (4-pyridyl) Carbamoyl) Bone disease according to (13), which is cyclohexane and Z or a pharmaceutically acceptable acid addition salt thereof. Pharmaceutical composition for prevention and treatment of

 (17) The pharmaceutical composition for preventing or treating bone diseases according to any one of (13) to (16) above, wherein the induced prisoner is a recombinant human BMP-2.

(18) The pharmaceutical composition for preventing or treating a bone disease according to any one of the above (13) to (17), wherein the bone disease is accompanied by damage, loss or dysplasia of bone or cartilage. (19) A bone disease comprising a compound having Rho kinase inhibitory activity as an active ingredient and a pharmaceutically acceptable bond, which enhances the osteoinductive action of an exogenous osteoinductive factor. Preventive 'therapeutic pharmaceutical composition. (20) The compound according to the above (19), wherein the compound having a Rh kinase inhibitory activity is an amide compound represented by the general formula (I), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. Bone disease prevention and treatment pharmaceutical composition.

 (21) The compound according to the above (19), wherein the compound having a Rh kinase inhibitory activity is an amide compound represented by the general formula (1 ′), an isomer thereof and Z or a pharmaceutically acceptable acid addition salt thereof. Or the pharmaceutical composition for preventing or treating a bone disease according to (20).

(22) Compounds having Rh kinase inhibitory activity include (+) — trans-41- (1-aminoethyl) -111- (4-viridylcarbamoyl) cyclohexane, and (x) -trans-N- ( 1 H-Pyro mouth [2,3-b] pyridine-4-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-pyridyl) 4- (1-aminoethyl) benzamide and (R)-(+)-N- (1H-pyro mouth [2,3-b] pyridine-14-yl) 14- (1-aminoethyl) A) a compound selected from the group consisting of benzamides and Z or a pharmaceutically acceptable acid addition salt thereof, especially (+) — trans-41- (11-aminoethyl) -11- (4-pyridylcarbamoyl) (19) The cyclohexane and / or a pharmaceutically acceptable acid addition salt thereof according to the above (19). Pharmaceutical composition for prevention and treatment of bone diseases.

 (23) The pharmaceutical composition for preventing or treating a bone disease according to any of (19) to (22) above, wherein the exogenous osteoinductive factor is recombinant human BMP-2.

 (24) The pharmaceutical composition for preventing or treating a bone disease according to any one of the above (19) to (23), wherein the bone disease is accompanied by damage, loss or dysplasia of bone or cartilage. (25) A method for preventing and treating a bone disease comprising administering to a patient a pharmaceutically effective amount of a compound having Rh kinase inhibition activity and a pharmaceutically effective amount of an osteoinductive factor.

 (26) The compound according to the above (25), wherein the compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (I), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. Child protection for bone disease · How to treat.

(27) The compound having Rho kinase inhibitory activity is represented by the general formula (1 ′): (25) or (26), wherein the amide compound is an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof.

 (28) The compound having Rho kinase inhibitory activity is (+)-trans- (1-aminoethyl) -111- (4-pyridylcarbamoyl) cyclohexane, (+)-trans-N- ( 1 H-Virolo [2,3-b] pyridine-4-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-pyridyl) -1 4- (1-aminoethyl) benzamide and (R)-(+)-1-N- (1H-pyrro [[2,3-b] pyridine-4-yl) 1-4- (1-aminoethyl) Compounds selected from the group consisting of benzamides and / or their pharmaceutically acceptable acid addition salts, especially (+) — trans-41- (1-aminoethyl) -1-1- (4-pyridylcarbamoyl) cyclo The above-mentioned xanth and oxane or a pharmaceutically acceptable acid addition salt thereof, 2 5) prevention of bone disease according 'treatment how.

 (29) The method for preventing or treating a bone disease according to any one of the above (25) to (28), wherein the osteoinductive factor is recombinant human BMP-2.

 (30) The method for preventing or treating a bone disease according to any one of the above (25) to (29), wherein the bone disease is accompanied by damage, loss or dysplasia of bone or cartilage.

 (31) Use of a compound having Rho kinase inhibitory activity and an osteoinductive factor for producing an osteogenesis promoter.

 (32) The compound according to (31), wherein the compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (I), an isomer thereof, and / or a pharmaceutically acceptable acid addition salt thereof. Use of the description.

 (33) The compound according to the above (31), wherein the compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (1 ′), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. Or Use as described in (32).

(34) The compound having Rho kinase inhibitory activity is (+)-trans-4-1- (1-aminoethyl) 1-1-1- (4-viridylcarbamoyl) cyclohexane, (+) — Trans-N- (1H-pyro-mouth [2,3-b] pyridine-1 41

4- (1-Aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-pyridyl) 1-41- (1-aminoethyl) benzamide and (R)-

(+)-N- (1H-Virolo [2,3-b] pyridine-14-yl) 1-4- (1-1aminoethyl) benzamide A compound selected from the group consisting of benzamides and / or pharmaceuticals thereof Pharmaceutically acceptable acid addition salts, especially (+) — trans-4- (1-aminoethyl) -1- (4-pyridylcarbamoyl) cyclohexane and / or its pharmaceutically acceptable acid addition salts. The use described in (31) above.

 (35) The use according to any one of (31) to (34), wherein the osteoinductive factor is recombinant human BMP-2.

 (36) The method according to any of (31) to (35) above, wherein the osteogenesis promoting agent is for preventing or treating a bone disease, particularly a bone disease associated with damage, loss or dysplasia of bone or cartilage. use.

 (37) Use of a compound having Rho kinase inhibitory activity for the production of a formation induction enhancer, which enhances the osteoinductive action of an exogenous osteoinductive factor.

(38) The use according to the above (37), wherein the compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (I), an isomer thereof and Z or a pharmaceutically acceptable acid addition salt thereof. .

 (39) The compound described in (37) or (38) above, wherein the compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (1 ′), an isomer thereof, or a pharmaceutically acceptable acid addition salt thereof. ) Use of the description.

(40) The compound having Rho quinase inhibitory activity is (+)-trans-41- (1-aminoethyl) 1-1- (4-pyridylcarbamoyl) cyclohexane, (+)-trans-N- (1 H-Virolo [2,3-b] pyridine-14-yl) 1-41- (1-aminoethyl) cyclohexancarboxamide, (R) — (+) — N- (4-pyridyl 1) 4- (1-aminoethyl) benzamide and (R)-(+)-N- (1H-birolo [2,3-b] pyridine-14-yl) 14- (1- A compound selected from the group consisting of aminoethyl) benzamide and / or a pharmaceutically acceptable acid addition salt thereof, （(（) -trans-4-1 (1-aminoethyl) 1-1-1 ((4-1) (Bidylcarbamoyl) The use according to the above (37), which is cyclohexane and / or a pharmaceutically acceptable acid addition salt thereof.

 (41) The use according to any of (37) to (40) above, wherein the exogenous osteoinductive factor is recombinant human BMP-2.

 (42) Any of (37) to (41), wherein the osteogenesis-inducing enhancer is used for the prevention or treatment of a bone disease, particularly a bone disease associated with damage, loss or dysplasia of bone or cartilage. Use of the description.

 (43) The pharmaceutical composition for preventing or treating a bone disease according to any one of (13) to (24) above, and the pharmaceutical composition can or should be used for the prevention and treatment of a bone disease. A commercial package containing a document stating that there is.

 BRIEF DESCRIPTION OF THE FIGURES

m 1 is the effect of BMP on ALP expression, and BMP in ALP expression and-is one of the compounds represented by the general formula (I) (+) — trans-1 41 (1 -aminoethyl) 1 1 — (4 one pyridylcarbamoyl) cyclohexane _{2 HC 1 - 1 H 2 0} ( hereinafter, Y- 2763 2 hereinafter) the combined effects of, illustrates the percentage of cells in a LP-positive. Figures la to e are micrographs showing the change in the number of ALP-positive cells when BMP alone or when BMP and Y-27632 were used in various concentrations. Fig. 1f is a graph of the results of Figs. 1a to 1e.

 FIG. 2 is a graph showing the combined effect of BMP and Y-27632 on ALP activity or osteocalcin production. Figure 2a shows ALP activity in 10T1 / 2 cells, Figure 2b shows osteocalcin production in 10T1 / 2 cells, and 1 ^ 12c shows ALP activity in ST2 cells.

Figure 3 shows the relationship between ALP activity and Rh activity. Figure 3a shows BMP-only expression in 10T1 / 2 cells with or without Rho expression. This figure shows changes in AL AL activity when administered in combination with Germany or BMP and Y-27632. 1 ^ 13b shows the expression of live cow ¾yRhο confirmed by the immunoblot method.

 Figure 4 shows the relationship between the ALP-positive or BMP-4 expression level and the ROCK activity. FIG. 4a shows the change in ALP activity in the presence and absence of Y-27632 in ST2 cells in which activated ROCK and dominant negative ROCK were forcibly expressed. FIG. 4b shows changes in BMP-4 mRNA levels in the presence and absence of Y-27632 in ST2 cells in which active R0CK and dominant negative ¾ROCK were forcibly expressed. FIG. 4c shows the expression of activated R〇CK and dominant negative ROCK which were forcibly expressed by the immunoblotting method.

 Fig. 5 is a photo showing an experiment for investigating the effect of Y-27632 on ectopic bone formation induced by BMP.

 Figure 6 shows the effect of Y-27632 on ectopic bone formation induced by BMP. Figure 6a is a photograph of the bone formed when BMP was used alone or in combination with BMP and Y-27632. Figure 6b is a lentogen photograph of the limb bone (femur) and formed bone when BMP alone or BMP and Y-27632 are used in combination.

 FIG. 7 shows the results of HE-staining of Y-27632 Sakugawa against ectopic bone formation induced by BMP. FIGS. 7a and 7c are photomicrographs of BMP alone examined by HE staining for the appearance of ectopic bone formation. Figures 7b and d are photomicrographs of the ectopic bone formation using BMP and Y-27632.

Fig. 8 shows one of the compounds represented by the general formula (I) for ectopic bone formation induced by BMP (+)-trans-N- (1H-pyromouth [2,3-b] It shows the effect of 4-pyridine (1-pyridine) 4- (1-aminoethyl) cyclohexanecarboxamide 2 HC1 (hereinafter referred to as Y-30141). Figure 8a is a photograph of the bone formed when BMP was used alone or in combination with BMP and Y-30141. Figure 8b is a radiograph of the bone formed when BMP alone or BMP and Y-30141 were used in combination.

 Figure 9 shows BMP-4 mRNA positive cells of cannabis cells accumulating around the BMP-2 pellet. Fig. 9A shows a case where only the BMP pellet is filled, and Fig. 9B shows an enlarged image. FIG. 9C shows a case where Y-27632 was administered in addition to the BMP pellet, and FIG. 9D shows an enlarged image thereof.

 Detailed description of the invention

 In the present invention, the osteogenesis promoter includes a compound having an Rho kinase inhibitory activity and an osteoinductive factor, and activates any part of osteogenesis as long as it has an activity of promoting bone formation. However, at least the method described in the experimental examples of the present invention may be used to perform the induction of osteoblast differentiation using mesenchymal cells, osteoblasts using Al-force phosphatase activity as an index, BMP-4, etc. It has an activity of inducing the production of osteoinductive factors and promoting bone formation.

 In the present invention, the osteogenesis-inducing enhancer is a osteoinductive effect possessed by the bone inducing factor f by using a compound having Rh kinase inhibitory activity as an active ingredient and using it in combination with an exogenous osteoinductive factor (described later). Is to enhance.

The “bone disease” in the present invention means a disease accompanied by symptoms such as damage, loss or dysplasia of bone or cartilage. “Treatment of bone disease” refers to the prevention or treatment of a disease that requires the formation of bone or cartilage tissue, or the improvement of a condition that requires the formation of bone or cartilage tissue-sleeve closure. Specifically, repair of bone or cartilage defect sites due to accidents, diseases, birth defects or surgery on each ear, promotion of healing of various fractures, artificial joints, bone around artificial implants such as artificial bones or artificial roots Bone or cartilage regeneration in the field of orthopedics, such as the formation of bones, promotion of fixation when using human implants, promotion of spinal fixation, leg extension, etc. Or restoration of the jawbone in the dental area, regeneration of the alveolar bone, restoration of cementum and bone enlargement for implant use. In addition, bone resorption and bone formation It also includes the prevention and treatment of metabolic bone diseases including osteoporosis, fibrous ostitis, osteomalacia, pageet's disease, etc., and osteoarthritis which occur when the lance is broken.

The osteoinductive factor (BMP) which can be used in the present invention is a protein having an activity of acting on undifferentiated mesenchymal cells to differentiate them into chondrocytes and osteoblasts and forming cartilage or bone. It is not particularly limited as long as the preparation method is not _limited.c However, it is manufactured by genetic recombination technology in that it is possible to obtain a large amount of materials with stable clinical safety such as immunity and quality. Human BMP is preferred. That is, a transformant (cell or microorganism) containing a recombinant DNA containing a nucleotide sequence encoding a human bone inducing factor is cultured, and the transformed human bone produced by the transformant is cultured. It is a recombinant human bone inducing factor (rhBMP) prepared by isolating and purifying the inducing factor. These human osteoinductive factors (rhBMP) include, for example, rhBMP-2, rhBMP-3, rhBMP-4 (also referred to as rhBMP-2B), rhBMP-5, rhBMP-6, rhBMP- 7, rhBMP-8, rhBMP-9, rhBMP-10, rhBMP-15, and rhBMP heterodimers, or modified or partially deleted forms thereof. These proteins can be used alone or as a mixture of two or more. Preferably, it is rhBM P-2.

 These rhBMPs can be expressed in mammalian cells (eg, CHO cells), microorganisms (eg, E. coli), yeast cells, and the like. Although rhBMP has already been established for mass production and purification, there is rhBMP—2 progress in Growth Factor Research, Vol. 1, pp. 267-280 (1989)], but other rhBMPs Similarly, it can be produced, purified and used.

 The already known purified rh BMP-2 is a bi :: body protein with a molecular weight of about 30,000. Each monomer has a high-mannose type sugar chain at Asn 56 residue (Abstract Sixth Interaction Symposium of the Protein Society, San Diego, CA (1992)).

In the present invention, “exogenous osteoinductive factor” refers to a bone endogenously expressed in a living body. Apart from the inducer, it is isolated or purified, or is genetically or chemically prepared, isolated or purified and administered to a subject for the purpose of inducing osteogenesis. Means something. The biochemical characteristics of the “exogenous osteoinductive factor” are as described above.

 The compound having Rho kinase inhibitory activity used in the present invention may be any compound having a Rh kinase inhibitory activity. [In the present description, Rho kinase means a serine / threonine kinase activated upon activation of Rho. For example, ROK II (RO CK II: Leung, T. et al., J. Biol. Chem., 270, pp. 29051-29054 (1995)), pi 60RO CK (ROK ?, ROCK—I: Ishizaki'T. Et al. , The EMB0 J., 15 (8), pp. 1885-1893 (1996)) and other proteins having serine / threonine kinase activity. ]

 Specific examples of the compound having Rh kinase inhibition activity used in the present invention include those described in WO98 / 06433 and WO097Z28130, and Naunyn-Schmiedeberg, Archives of Pharmacology 385 (1 ),

 Suppl., R219 (1998), amide compounds, isoquinoline sulfonamide derivatives and isoquinoline derivatives.

For example, as the amide compound, a compound represented by the above general formula (I), especially a compound represented by the general formula (1 ′), and as the isoquinoline sulfone amide derivative, fasudil hydrochloride [hexahydro] And 1- (5-isoquinolinylsulfonyl) -1H-1, 4-diazepine], and the isoquinoline derivative is hexahydryl 1-1 [(4-methyl-5-isoquinolinyl) sulfonyl. 1) 1 H—1,4-Dazebine dihydrochloride, (S) — (10) —Hexahydro-2-methyl-1 11 [(4-methyl-5-isoquinolinyl) sulfonyl] 1 1H—1: 4 —Dazepine hydrochloride, hexahydro 7-Methyl-11-((4-methyl-5-isoquinolinyl) sulfonyl) -1 1H-1,4-dazepine dihydrochloride, hexahydr-5-methyl- 1 [(4-methyl-5-isoquinolinyl) sulfonyl] l H— l, 4-Dazepine dihydrochloride, HexahiDraw 2-methyl-1 — [(4-Methyl-5-isoquinolinyl) sulfonyl] — 1 H—1,4-Dazepine hydrochloride, (R) − (1) 1-Hexahydro-2-methyl-1-[(4-methyl-5-isoquinolinyl) sulfonyl] -1 1H-1, 4-diazepine hydrochloride, (R)-(+)-hexahydro-1 5-methyl-1-[(4-methyl-5-isoquinolinyl) sulfonyl] —1H—1,4-dazepine hydrochloride and the like.

 Preferred are amide compounds represented by the general formula (I), and particularly preferred are amide compounds represented by the general formula (1 ′).

 In the present specification, the definition of each symbol of the general formula (I) and the general formula (1 ′) is as follows.

R, R ', the alkyl group in R ¹ an alkyl Number 1-1 0 straight-chain or branched carbon, methyl, Echiru, Purobiru, isoproterenol building, butyl, I Sobuchiru, secondary Examples thereof include butyl, tertiary butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Alkyl having 1 to 4 carbon atoms is preferable.

R, R ', denotes cyclopropyl Cycloalkyl in R ^1, cyclobutyl, cyclopentyl, cyclohexyl, carbon number 3-7 or cycloalkyl of cycloheptyl etc. cyclohexylene.

The cycloalkylalkyl in R, R ', and R' is the cycloalkyl moiety having 3 to 7 carbon atoms, and the alkyl moiety is a straight-chain or branched alkyl having 1 to 6 carbon atoms. (Methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, etc.) which is cycloalkylalkyl, cyclopropylmethyl, cyclobutylmethyl, cyclobentylmethyl, cyclohexylmethyl, cycloheptinolemethyl, cyclopropinoleethyl Rile, cyclopentylethyl, cyclohexylethyl, cycloheptylethyl, cyclopropyl, cyclopentylpropyl, cyclohexylpropyl, cycloheptylpropyl, cyclopropylbutyl, cyclopentylbutyl, cyclohexylbutyl, cycloheptyl · The Bed chill, cyclohexane professional buildings cyclohexyl, hexyl Shikurobenchiru, the cyclohexyl Syl, heptylhexyl and the like.

Aralkyl in R, R, and R ¹ has 1 to 4 carbon atoms as the alkyl moiety, and is benzyl, 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl And phenylalkyl.

R, R ', cycloalkyl which may have a substituent on the ring at R ^1, a cycloalkyl alkyl, phenyl, a substituted grave Ararukiru is halogen (chlorine, bromine, fluorine, iodine), alkyl (R, alkyl and synonym of \ R ^1), alkoxy (a carbon number of 1 to 6 straight-chain or branched alkoxy, main butoxy, ethoxy, Buropokishi, Isopurobokishi, butoxy, isobutanol butoxy, secondary butoxy, tertiary butoxy, Penchiruokishi shows Kishiruokishi like to.), Ararukiru (R, R ', Ararukiru synonymous) in R ^1, Haroaruki Le (R, R', the alkyl as indicated in R ¹ Substituted with 1 to 5 halogens, such as fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl , 2, 2, 3, 3, indicates to the 3-pen evening Furuoropuropiru like.), Nitro, Amino, Shiano, azides, and the like.

R and R ¹ or R 'and: ¹ are bonded together with an adjacent nitrogen atom, and the complex may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent. The ring-forming group is preferably a 5- to 6-membered ring or a bonded ring thereof. Specifically, 1-pyrrolidinyl, piperidino, 1-piperazinyl, morpholino, thiomorpholino, 1-imidazolyl, 2,3-dihidyl Drothiazol-3-yl and the like are exemplified. Examples of the substituent on the nitrogen atom which may have a substituent include alkyl, aralkyl, haloalkyl and the like. Wherein alkyl, § La alkyl, haloalkyl R, R ', the same meaning as in the R ^1.

The alkyl in R ² has the same meaning as the alkyl in R, R ′ and R ¹ .

Halogen, alkyl, alkoxy and aralkyl in RR ^{4 have} the same meanings as those described for R, R ′ and R ¹ . The acyl in R ³ and R ⁴ is an alkanol having 2 to 6 carbon atoms (such as acetyl, pulpionyl, butyryl, valeryl, and vivaloyl), benzoyl, or alkenyl having a carbon atom of 2 to 4 carbon atoms. (Such as phenylacetyl, phenylpropionyl, and phenylbutyryl).

The Arukiruamino in RR ^4, an alkyl amino having an alkyl number 1 of the 6 I-chain or branched carbon alkyl portion, methylamino, Echiruamino, professional buildings amino, isoproterenol building amino, Buchiruamino, Represents isobutylamino, secondary butylamino, tertiary butylamino, ventilamino, hexylamino and the like.

The Ashiruamino in R \ R ^4, a Ashiruamino having carbon number 2 to 6 pieces of Al force Noiru as Ashiru, Benzoiru or Arukanoiru portion charcoal ^ number 2-4 amino phenylene Luarca Noiru like, Asechiruamino, And propionylamino, butyrylamino, no, 'relylamino, bivaloylamino, penzylamino, phenylacetylamino, phenylpropionylamino, phenylbutyrylamino and the like.

And alkylthio in RR ^4, a alkylthio having an alkyl number 1 of 6 linear or branched carbon atoms in the alkyl part, methylthio, Echiru Chio, Purobiruchio, isoproterenol building, butylthio, isobutylthio, Secondary butylthio, tertiary butylthio, pentylthio, hexylthio, etc.

R ³ , R aralkyloxy is an aralkyl having an alkyl having 1 to 4 carbon atoms in the alkyl portion thereof, and is benzyloxy, 1-phenylethyloxy, 2-phenylethyloxy. Xy, 3-phenylpropyloxy, 4-phenylbutyloxy and the like.

The aralkylthio in R is one having an aralkyl having an alkyl group having 1 to 4 carbon atoms in the alkyl part thereof, such as benzylthio, 1-phenylethylthio, 2-phenylethylthio, 3-phenylpropylthio, Represents phenylbutylthio and the like. Alkoxycarbonyl at RR ^4, it is one having a carbon number of 1 to 6 straight-chain or branched alkoxy alkoxy part, main Tokishikaru Boniru, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl carbonylation Le Butoxycarbonyl, isobutoxycarbonyl, secondary butoxycarbonyl, tertiary butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl and the like.

The mono-dialkyl carbamoyl in R-\ R ⁴ is a carbamoyl mono- or di-substituted by an alkyl having 1 to 4 carbon atoms, and is methylcarbamoyl, dimethylcarbamoyl, ethylcarbamoyl, getylcarbamoyl, Probyl rubamoyl, dipropyl carbamoyl, butylcarbamoyl, dibutylcarbamoyl, etc.

The alkoxy in R ⁵ R, R ', the same meaning as alkoxy at R ^1. The alkoxycarbonyloxy represented by R5 has a straight-chain or branched alkoxy having 1 to 6 carbon atoms in the alkoxy moiety, and includes methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy. , Isopropoxycarbonyloxy, butoxycarbonyloxy, isobutoxycarbonyloxy, secondary butoxycarbonyloxy, tertiary butoxycarbonyloxy, benzyloxycarbonyloxy, hexyloxycarbonyloxy, etc. Is shown.

The Arca Noi Ruo Kin in R ^5, be one having from 2 to 6 § Rukanoiru carbon number Arukanoiru section shows Asechiruokishi, propionyl Ruo alkoxy, Petit Riruokishi, Bruno S Reriru old xylene, the Bibaroiruokishi like.

The aralkyloxycarbonyloxy represented by R ⁵ has an aralkyl having an alkyl group having 1 to 4 carbon atoms in the alkyl portion thereof, and is a benzyloxycarbonyloxy, 1-phenylethyloxy group. Examples include carbonyloxy, 2-phenylethoxycarbonylcarbonyl, 3-phenylpropyloxycarbonyloxy, 4-phenylbutyloxycarbonyloxy, and the like. The alkyl in has the same meaning as the alkyl in R, R ',: ¹ . The alkyl at R \ R ^y is R, R ', has the same meaning as alkyl at R ^1, Ararukiru in R ^8, R ⁹ is R, R', the same meaning as Ararukiru in R ^1.

Alkyl in R ⁷ is R, R ', it has the same meaning as alkyl at R ^1, Ararukiru the definitive in R ⁷ is R, R', the same meaning as Ararukiru in R ^1.

R "and R ⁷ combine to further oxygen atom in the ring, as a group forming a sulfur atom or a heterocyclic ring which may contain a nitrogen atom which may have a substituent, Lee Mida zone one 2-yl, thiazol-2-yl, oxazole-2-yl, imidazolin-2-yl, 3, 4, 5, 6-tetrahydroviridine 1-2-yl, 3, 4, 5, 6-tetrahydropyrimidine-1-yl, 1,3-oxazoline-12-yl-1,3-thiazoline-2-yl or halogen, alkyl, alkoxy, noroalkyl, nitro, amino, phenyl, aralkyl Benzoimidazole-1-yl, benzothiazol-2-yl, benzoxazol-1-yl, etc. which may have a substituent such as halogen, alkyl, alkoxy, haloalkyl, and the like. Aralchi The R, R ', the same meaning as in the R ^1, also, as the S substituent in a nitrogen atom which may have the above substituent, § alkyl, Ararukiru, haloalkyl and the like . here, the alkyl, Araru kill, and haloalkyl R, R ', the same meaning as in the R ^1.

The hydroxyalkyl in R ^lu and R ¹¹ is a straight-chain or branched-chain alkyl having 1 to 6 carbon atoms substituted with 1 to 3 hydroxy, such as hydroxymethyl, 2 —Hydroxyl, 1-hydroxyl, 3-hydroxyl mouth building, 4-hydroxylbutyl, etc.

Alkyl in R ¹ R ^{1 1} is R, R ', has the same meaning as alkyl at R ^1, R ^{l fl,} haloalkyl in R ^{1 1,} alkoxycarbonyl R, R', same meaning as that shown in had us to R ¹ , and the Ararukiru in R ^in, R ^{1 1} is R, R ', is synonymous with Ararukiru in R ^1.

Cycloalkyl R ¹ R ^{1 1} is formed by combining also R, consequent in R \ R ¹ Synonymous with mouth alkyl.

 The alkyl in L has the same meaning as the alkyl in R, R 'and R.

 The aminoalkyl in L is a substituted or branched chain alkyl having 1 to 6 carbon atoms, which is substituted with amino, such as aminomethyl, 2-aminoethyl, 1-aminoethyl, and 3-aminoalkyl. Aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl and the like.

 The mono-dialkylaminoalkyl in L is an aminoalkyl which is mono- or di-substituted by alkyl having 1 to 4 carbon atoms, such as methylaminomethyl, dimethylaminomethyl, ethylaminomethyl, acetylaminomethyl, and propylaminomethyl. Nomethyl, dibutylamino, butylaminomethyl, dibutylaminomethyl, 2-dimethylaminoethyl, 2-getylaminoethyl and the like.

 L-rubamoylalkyl in L is a straight- or branched-chain alkyl having 1 to 6 carbon atoms, which is substituted with l-rubamoyl, for example, l-rubamoylmethyl, 2--lumbamoylethyl, 1-lumbamoylethyl , 3-potassium rubamoyl propyl, 4-potassium rubamoyl butyl, 5-carpamoyl pentyl, 6-potassium rubamoyl hexyl and the like.

 The fluorimidalkyl in L is a straight- or branched-chain alkyl having 1 to 6 carbon atoms substituted by phthalimid. For example, phthalimidomethyl, 2-fluoroimidethyl Phthalimidethyl, 3-phthalimidopropyl, 4-phthalimidbutyl, 5-phthalimidopentyl, 6-phthalimidhexyl and the like.

Alkyl in B is R, R ', the same meaning as alkyl at R ^1.

Alkoxy at B is R, R ', alkoxy and MukaiYoshi in R ^1. Ararukiru in B is R, R ', the same meaning as Ararukiru in R ^1. The aralkyloxy in B is the same as the aralkyloxy in R ³ and R ⁴ .

Aminoalkyl in B is the same as aminoamino in L. The hydroxyalkyl in B has the same meaning as the hydroxyalkyl in R ⁱⁿ and R ¹¹ .

 The alkanoyloxyalkyl in B is a straight-chain or branched alkyl having 1 to 6 carbons substituted by an alkanoyloxy having an alkanoyl moiety having 2 to 6 carbons. Acetyloxymethyl, propionyloxymethyl, butyryloxymethyl, valeryloxymethyl, bivaloyloxymethyl, acetyloxechil, propionyloxyxetil, butyryloxethyl, valeryloxychetyl, vivaloyloxich Chill and the like.

 The alkoxycarbonylalkyl in B is a straight-chain or branched-chain alkyl having 1 to 6 carbons substituted by an alkoxycarbonyl having an alkoxy moiety having 1 to 6 carbons. Methoxycarbonylmethyl, ethoxycarbonylmethyl, propoxycarbonylmethyl, isopropoxycarbonylmethyl, butoxycarbonylmethyl, isobutoxycarbonylmethyl, secondary butoxycarbonylmethyl, tertiary butoxycarbonylmethyl, pentyloxycarbonylmethyl, to Xyloxycarbonylmethyl, methoxycarbonylethyl, ethoxycarbonylethyl, propoxycarbonylethyl, isopropoxycarbonylyl, butoxycarbonylethyl, isobutoxycarbonylethyl, secondary butoxy Aryloxycarbonyl E chill, tertiary butoxycarbonyl E chill, Benchiruo alkoxycarbonyl E chill, hexyl O butoxycarbonyl E chill like to.

The halogen in the QQ ^2, Q ^3! ?., R ', Ru halogen synonymous der in R ^1. The aralkyl group in Q ¹ Q ² is synonymous with the aralkyl group in R ³ and R ⁴ .

The alkoxy in Q ³ has the same meaning as the alkoxy in R, R \ R ¹ . The alkylene in W, X and Y is a linear or branched alkylene having 1 to 6 carbon atoms, such as methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene, etc. .

Alkenylene in Y is a linear or branched alkylene having 2 to 6 carbon atoms. A two lens shows vinylene, propenylene, butenylene, a pent two Ren et al. ₍

The alkyl in Rb R, R ', the same meaning as alkyl at R ^1.

The Ararukiru in Rb R, R ', the same meaning as Ararukiru in R ¹ <a Aminoarukiru in Rb is as defined Aminoarukiru in L. The mono-dialkylaminoalkyl for Rb has the same meaning as the mono-dialkylaminoalkyl for L.

 The nitrogen-containing heterocyclic ring represented by R c represents a monocyclic ring, such as pyridine, pyrimidine, viridazine, triazine, virazol, or triazole. , 3—b] pyridine, 1H-pyro [3,2-b] pyridine, 1H-pyro [3,4-b] pyridine, etc., virazolopyridine (1H-virazolo [3,4 1b) pyridine, 1H-birazolo [4,3-b] pyridine, etc.), imidazoviridine (1H-imidazo [4,5-b] pyridine, etc.), piro-mouth bilimidine (1H-pyro-mouth) [2,3-d] pyrimidine, 1 H-pyro mouth [3,2- <1] bilimidine, 1 H-virolo [3,4-d] pyrimidine, etc., bisazolopyrimidine (1H-birazolo) [3,4-d] Pyrimidine, virazolo [1,5-a] Pyrimidine, 1H-birazolo [4,3-d] Limidin), imidazobilimidine (imidazo [1,2—a] bilimidine, 1H-imidazo [4,5—d] pyrimidine, etc.), virolotriazine (pyro mouth [1,2— a] 1-1,3,5-triazine, virolo [2,1–f] 1 l, 2,4-triazine, etc., virazolo triazine (virazolo [1,5-a] 1-1,3,5 — Triazine, etc.), triazolopyridine (1H—1,2,3—triazolo [4,5—b] pyridine, etc.), triazolobirimidine (1,

2,4 Triazolo [1, 5—a] pyrimidine, 1, 2 ₃ 4 Triazolo [4,

3—a] birimidine, 1H—1,2,3-triazolo [4,5_d] birimidine, etc.), cinnoline, quinazoline, quinoline, and bilidopyridazine (pyrid [2,3—c ] Pyridazine, pyridopyrazine (pyrid [2,3-b] pyrazine, etc.), pyridopyrimidine (pyrid [2,3-d] pyrimidine, bilide [3,2-d] pyrimidine, etc. ), Pyrimidopyrimidine (pyrimido [4, 5—d] pyrimidine, virimi De [5,4-d] birimidine, etc.), birazino pyrimidine (birazino [2,3-d] birimidine, etc.), naphthyridine (1,8-naphthyridine, etc.), tetrazolopyrimidine (tetrazolo [1, 5-a] birimidine, etc.), chenoviridine (thieno [2,3-b] viridine, etc.), chenobilimidine (thieno [2,3-d] birimidine, etc.), thiazolopyridine (thiazolo [4, 5-] b] pyridine, thiazo-mouth [5,4-b] pyridine, etc.), thiazolovirimidine (thiazolo [4, 5-d] pyrimidine, thiazolo [5, 4-d] pyrimidine, etc.), oxazolobiridine (Oxazolo [4,5-b] pyridine, oxazolo [5,4-b] pyridine, etc.), oxazolo bilimidine (oxazolo [4, 5-d] virimidine, oxazolo [5, 4-d] pyrimidine, etc.) Furopyridine (furo [2, 3 _b] Pyridine, furo [3, 2 - b] pyridine, etc.), Furopiri spermidine (furo [2, 3- d] Billiton Mi Jin, furo [3:

2—d] birimidine, etc.), 2,3-dihydropyrroloviridine (2,3-dihydro 1H-virolo [2,3-b] pyridine, 2,3-dihydro 1H-virolo

[3,2-b] pyridine, etc.), 2,3-dihydropyrropyrimidine (2,3-dihydro 1H-birolo [2,3-d] pyrimidine, 2,3-dihydro 1H —Virolo [3,2—d] pyrimidine etc.), 5, 6, 7, 8—Tetrahydropyrrolide [2 _:

3—d] birimidine, 5, 6, 7, 8—tetrahydrodraw 1, 8, naphthyridine, 5, 6, 7, 8, 8-tetrahydroquinoline, etc., and these rings are hydrogenated When forming an aromatic ring, the carbon atom in the ring may be a carbonyl, for example, 2,3-dihydro-12-oxobiropyridine, 2,3-dihydro2,3-dioxobirolopyridine , 7,8-Dihydro 7-oxo-1,8_naphthyridine, 5,6,7,8-tetra and hydro-7-oxo-1,8-naphthyridine ^ are also included.

In addition, these rings may be halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, amino, alkylamino, cyano, formyl, acyl, aminoalkyl, mono-dialkylaminoalkyl, azide, carboxy, alkoxyl Lubamoyl, mono ■ Dialkyl power Lubamoyl, alkoxyalkyl (Eg, methoxymethyl, methoxyshethyl, methoxypropyl, ethoxymethyl, ethoxyshethyl, ethoxypropyl) and optionally substituted hydrazino.

Examples of the substituent of the human Dorajino be substituted, alkyl, Ararukiru, two Toro, but Shiano the like, alkyl, Ararukiru is R, R ', alkyl in R ^1, and Ararukiru Synonymous, for example, methylhydrazino, ethylhydrazino, penzylhydrazino and the like are exemplified.

 -Specific examples of the compound represented by the general formula (I) include the following compounds.

 (1) 4- (2-pyridylcarbamoyl) biperidine

 (2) 1-benzyloxycarboxyl 4- (4-pyridylcarbamoyl) biveridine

 (3) 1-benzoyl-1- (4-pyridylcarbamoyl) biperidine

 (4) 1-Propyl 4- (4-pyridylcarbamoyl) biperidine

 (5) 1-3- (2- (2-Chenylmethyl) phenoxy) -12-Hydroxypropyl] 4-14-1 (4-Vyridylcarbamoyl) biveridine

 (6) 4-(4 -viridylcarbamoyl) piperidine

 (7) 1-benzyl-41- (4-pyridylcarbamoyl) 1-1,2,5,6-tetrahydropyridine

 (8) 3— (4-pyridylcarbamoyl) biberidine

 (9) 1-benzyl-3- (4-biridylcarbamoyl) biperidine

 (10) 1- (2- (4-benzyloxyphenoxy) ethyl) 4- (N- (2-pyridyl) -1-N-benzylcarbamoyl) biperidine

 (1 1) 1-formyl-1-4- (4-pyridylcarbamoyl) biveridine

 (1 2) 4-(3-Viridylcarbamoyl) biperidine

 (1 3) 1-Isoprovir 1-4-1 (4-Viridylcarbamoyl) biperidine

(14) 1-Methyl-14- (4-pyridylcarbamoyl) piperidine (1 5 1 1-Hexyl- 1 4- (4-pyridylcarbamoyl) piperidine

 (1 6 1—Benzyl-1-41 (4-pyridylcarbamoyl) piperidine

 (1 7 1 1 (2-phenylethyl) 4- (4-biridylcarbamoyl) bi-lysine

 (1 8 1— (2— (4-Methoxyphenyl) ethyl) 4-(4—Pyridylkarubamyl) bidiridine

 (1 9 1— (2— (4—methoxyphenyl) ethyl) 1-4 (2—pyridylcarbamoyl) biperidine

 (20 1— (2— (4-chlorophenyl) ethyl) 1-4— (4-pyridylcarbamoyl) biperidine

 (2 1 1 diphenylmethyl-4- (2-pyridylcarbamoyl) piperidine (22 1— [2— (4— (5-methyl-13-oxo-1,2,3,4,5—tetrahydro D 1-pyridazine-6-yl) phenyl) ethyl] 1-4- (2-biridylcarbamoylbiperidine

 (2 3 1-(4-1 (4,5-dihydro-2-furyl) phenyl) 1-4 (4-1 pyridylcarbamoyl) bipyridine

 (24 (2-nitrophenyl) 1-41 (4-pyridylcarbamoyl) piperidine

 (2 5 1-(2 -aminophenyl) 1-(4-pyridylcarbamoyl) pyridine

 (2 6 1 1 nicotinol 1 4 1 (4 1 pyripiperidine (2 7 1 1 isonicotinoyl 4 4 1 (4 pyripyridine (28 1— (3, 4, 5— — (4 _ Viridylka Lubamoyl) Biberidine

 (2 91 1-acetyl-4_ (4-pyridylcarbamoyl) piperidine

(30 1- (3- (4-fluorobenzoyl) propyl) 1-4- (4-pyridylcarbamoyl) piperidine (3 1) 1— (3-(4 —fluorobenzoyl) propyl) 1 4— (2-pyridylcarbamoyl) piperidine

 (32) 1— (1— (4—Hydroxybenzoyl) ethyl) 1-4— (2—Pyridylcarbamoyl) piperidine

 (33) 1— (11- (4-benzyloxybenzoyl) ethyl) — 4 -— (2-pyridylcarbamoyl) piperidine

 (34) 1- (2- (4-Hydroxyphenoxy) ethyl) 1-4_ (2-Pyridylcarbamoyl) piperidine

 (35) 1- (4- (4-Fluorophenyl) 1-4-hydroxybutyl) 4- (4-pyridylcarbamoyl) biperidine

 (36) 1- (1-Methyl-2- (4-hydroxyphenyl) -1-hydroxyethyl) 1-4- (2-Pyridylcarbamoyl) biperidine

 (37) 1-cinnamyl 4- (2-pyridylcarbamoyl) piperidine

(38) 1- (2-Hydroxy-1-3-phenoxypropyl) 1-4- (4-pyridylcarbamoyl) piperidine

 (39) 1- (2-hydroxy-3-phenoxypropyl) 1-4- (3-pyridylcarbamoyl) biperidine

 (40) 1- (2-hydroxy-13-phenoxypropyl) 1-4- (2-pyridylcarbamoyl) piperidine

 (41) 1- (2-Phenylethyl) 1-41-CN- (2-pyridyl) — N— (2 (N, N-dimethylamino) ethyl)

 (42) 1-Benzyloxycarbonyl-1 4- (2-pyridylcarbamoyl) piperidine

 (43) 1— (3-phenyl phenyl) dirubamoyl 4- (4-pyridylcarbamoyl) piperidine

 (44) 4—CN- (2-pyridyl) -1-N— (2- (N, N-dimethylamino) ethyl) lvamoyl] piperidine

31 Corrected form (Rule 91) (45) 1-Methyl-4- (4-pyridylcarbamoyl) 1-1,2,5,6-Tetrahydropyridine

 (46) 1-nicotinol-1- (4-pyridylcarbamoyl) piperidine (47) 1- [2- (4-fluorobenzoyl) ethyl] 1-4- (4-pyridyl carbamoyl) biperidine

 (48) 1- (6-chloro-1--2-methylimidazo [1,2-a] pyridine-13-carbonyl) 1-4- (4-pyridylcarbamoyl) piperidine

 (49) 1- (4-12-trobenzyl) 1-4-1 (4-pyridylcarbamoyl) piperidine

 (50) 1-Hexyl-1- (4-Pyridylcarbamoyl) piperidine

 (5 1) 1-Benzyloxycarbonyl 4- (2-cyclopentyl-4-pyridylcarbamoyl) piperidine

 (52) 4— (2-chloro-1-4-pyridylcarbamoyl) piperidine

 (53) 1- (2-cyclonicotinol) 1-4- (4-pyridylcarbamoyl) piperidine

 (54) 3— (2-chloro-1-4-pyridylcarbamoyl) piperidine

 (55) 1— (4-phthalimidobutyl) 1-4— (4-pyridylcarbamoyl) biperidine

 (56) 1- (3,5-di-tert-butyl-4-hydroxycinnamoyl) —4- (4-pyridylcarbamoyl) piperidine

 (57) 1-Lumbamoylmethyl-1- (4-pyridylcarbamoyl) piperidine

 (58) 1-Benzyloxycarbonyl 4- (5-nitro-2-pyridylcarbamoyl) piperidine

 (59) 4- (5-Nitro-1-pyridylcarbamoyl) piperidine

32 Corrected Form (Rule 91) (60) trans-41-benzyloxycarboxamide methyl 1- (4-pyridylcarbamoyl) cyclohexane

 (6 1) trans 4-aminomethyl-1- (4-pyridylcarbamoyl) cyclohexane

 (62) trans-4-formamide methyl-1- (4-pyridylcarbamoyl)

 (63) trans-1-dimethylaminomethyl-1-(4-viridylcarbamoyl) cyclohexane

 (64) N-benzylidene-trans-I (4-viridylcarbamoyl) cyclohexylmethylamine

 (65) trans-4-benzylaminomethyl _ 1-(4-pyridylcarbamoyl) cyclohexane

 (66) trans-41-isopropylaminomethyl-1- (4-viridylcarbamoyl) cyclohexane

 (67) trans-1-nicotinoylaminomethyl 1- (4-pyridylcarbamoyl)

 (68) Trans-1-cyclohexylaminomethyl-11- (4-pyridylcarbamoyl) cyclohexane

 (69) trans 1-benzyloxycarboxamide 1-11 (4-pyridylcarbamoyl) cyclohexane

 (70) trans-4-amino-1-11 (4-pyridylcarbamoyl) cyclohexane

 (71) Trans-4-1 (1-aminoethyl) 1-1-1 (4-Viridylcarbamoyl) cyclohexane

 (72) trans-4-Aminomethyl-cis-2-methyl-11- (4-pyridyl sorbamoyl) cyclohexane

(73) (+) — trans-41 (1-benzyloxycarboxamide doprobi) 1) 1-cyclohexanecarboxylic acid

 (74) (+) — trans-4- (1-benzyloxycarboxamidopropyl) -1- (4-pyridylcarbamoyl) cyclohexane

 (75) (-) 1-trans-1- (1-benzyloxycarboxamide propyl) 1-1- (4-viridylcarbamoyl) cyclohexane

 (76) (+) — trans-4- (1-aminopropyl) 1-11 (4-pyridyl-powerbamoyl) cyclohexane

 (77) (-) — trans-1- (1-aminoprovir) 1-1- (4-pyridylcarbamoyl) cyclohexane

 (78) (-) — trans-4_ (1-benzyloxycarboxamidoethyl)-1- (4-pyridylcarbamoyl) cyclohexane

 (79) (+) 1-trans 1- (1-benzyloxycarboxamidoethyl)-1- (4-pyridylcarbamoyl) cyclohexane

 (80) (+) — 1-transyl 4- (1-aminoethyl) 1 1- (4-pyridylcarbamoyl) cyclohexane

 (8 1) (-) 1-trans-1- (1-aminoethyl) 1 1- (4-pyridylcarbamoyl) cyclohexane

 (82) Trans-1- (4-chlorobenzoyl) aminomethyl-1- (4-Bidylcarbamoyl) cyclohexane

 (83) trans-41-aminomethyl-11- (2-pyridylcarbamoyl) cyclohexane

 (84) trans-4-1-benzyloxycarboxamide methyl-1- (2-pyridylcarbamoyl) cyclohexane

 (85) trans-41-methylaminomethyl-1- (4-pyridylcarbamoyl) cyclohexane

(86) trans-1- (N-benzyl N-methylamino) methyl-11- (4-biridylcarbamoyl) cyclohexane (87) trans-41-aminomethyl-11- (3-pyridylcarbamoyl) cyclohexane

 (88) trans-1-aminomethyl-11 [(3-hydroxy-1-pyridyl) dirubamoyl] cyclohexane

 (89) trans-4-benzyloxycarboxamide methyl 1- (3-pyridylcarbamoyl) cyclohexane

 (90) trans-4-benzyloxycarboxamidomethyl-1-[(3 ^ ^ benzyloxy-1-pyridyl) cyclohexane

 (91) Trans-1-fluoromethyl-1- (4-pyridylcarbamoyl) cyclohexane

 (92) trans-41-benzyloxycarboxamide methyl-1-(3-methyl-4-viridylcarbamoyl) cyclohexane

 (93) trans 4-aminomethyl-1- (3-methyl-4-pyridylcarbamoyl)

 (94) 4-(trans-1-benzyloxycarboxamide methylcyclohexylcarbonyl) amino-2, 6-dimethylpyridine-N-oxide

 (95)-(trans-4-aminomethylcyclohexylcarbonyl) amino-1,2,6-dimethylpyridine N-methyl oxide

 (96) trans- 4-aminomethyl- 1- (2-methyl- 1-4-pyridylcarbamoyl) cyclohexane

 (97) Transformer 4_ (1—

 (4-pyridylcarbamoyl)

(98) 1 trans

Rucarbamoyl) cyclohexane

 (99) trans-1- (2-aminoethyl) -111 (4-pyridylcarbamoyl) cyclohexane

(100) trans-1 4-1 (2-amino-1 1-methylethyl) 1 1- (4 Jilcarbamoyl) cyclohexane

 (10 1) Trans-1-4-(1-Aminobu pill) 1-1-(4-Viridylcarbamoyl)

 (102) trans-1-aminomethyl-trans-1-methyl-1-(4-bilysylcarbamoyl) cyclohexane

 (103) trans-4-1-benzylaminomethyl-cis-2-methyl-1- (4-pyridylcarbamoyl) cyclohexane

 (104) trans-1-(1-benzyloxycarboxamido 1-methylethyl) 1-(4-viridylcarpamoyl) cyclohexane

 (105) trans-1-benzyloxycarboxamide methyl-11-(N-methyl-14-pyridylcarbamoyl) cyclohexane

 (106) trans-1- (1-acetamido 1-methylethyl) -1- (4-pyridylcarbamoyl) cyclohexane

 (107) trans-N- (6-amino-4-pyrimidyl) -14-aminomethylcyclohexanecarboxamide

 (108) trans-N- (1H-pyro-mouth [2,3-b] pyridine-14-yl) -14-aminomethylcyclohexanecarboxamide

 (109) (+) 1-trans-N_ (1 H-pyrro [2,3-b] pyridin-4-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide

 (1 10) trans-N_ (1H-pyrro [2,3-b] pyridine-4-41)-4-(1-amino-1-methylethyl) cyclohexanecarboxamide

(111) trans-N- (1H-birazolo [3,4-b] pyridine-14-yl) -14-aminomethylcyclohexanecarboxamide

 (1 1 2) (+) — trans-N-(1 H-virazolo [3,4-b] pyridine-14-yl)-41-(1-amino-ethyl) cyclohexanecarboxamide

(1 13) trans-N- (1H-birazolo [3,4-b] pyridine-14-yl) 1-4-1 (1-amino-1-methylethyl) cyclohexanecarboxamide (1 1 4) (+) — trans-N— (2-amino-4-pyridyl) 1-4 — (1-aminoethyl) cyclohexanecarboxamide

 (1 15) trans-N- (1H-pyrazo-mouth [3,4-d] pyrimidine-1-yl) 14-aminomethylcyclohexanecarboxamide

 (1 16) (+) — trans-N- (1 H-birazolo [3,4-d] pyrimidine-14-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide

(1 17) trans-N- (1H-pyrazo-mouth [3,4-d] pyrimidine-14-yl) -4-1 (1-amino-1-methylethyl) cyclohexanecarboxamide

(111) trans-N- (4-pyrimidinyl) -14-aminomethylcyclohexancarboxamide

 (111) trans-N- (3-amino-4-pyridyl) -14-aminomethylcyclohexanecarboxamide

 (1 20) trans-N- (7H-imidazo [4,5-d] pyrimidine-1-6-yl) -14-aminomethylcyclohexanecarboxamide

 (1 2 1) trans-N- (3H-1,2,3-triazolo [4,5-d] pyrimidine_7-yl) -41-aminomethylcyclohexanecarboxamide

 (122) Trans-N- (1-benzyl-1H-birazolo [3,4-b] pyridin-141-yl) -14-aminomethylcyclohexanecarboxamide

 (1 2 3) trans-N- (1H-5-virazolyl) -14-aminomethylcyclohexanecarboxamide

 (1 24) trans-N- (1H-pyrazo-mouth [3,4-1b] pyridine-14-yl) -14-aminomethylcyclohexanecarboxamide

 (125) trans-N- (4-pyridazinyl) -14-aminomethylcyclohexancarboxamide

 (126) trans-N- (7H-pyrro [2,3-d] pyrimidine_4-yl) 1-aminomethylcyclohexanecarboxamide

(1 2 7) trans-N- (2-amino 4-pyridyl) 1-4-aminomethyl Clohexanecarboxamide

 (1 2 8) trans-N-(thieno [2,3-d] birimidine-14-yl)-1-4-aminomethylcyclohexanol lipoxamide

 (122) trans-N- (5-methyl-1,2,4-triazo [1,5—a] pyrimidine-17-yl) -14-aminomethylcyclohexanecarboxamide ( 130) Trans-N- (3-Cyan-5-methylbiazolo [1,5-a] pyrimidine_7-yl) -14-Aminomethylcyclohexanecarboxamide (131) Trans 1 N— (1 H—Villa V-mouth [3,4-1b] Pyridine-14-yl) 1-4-1 (1-amino-1—methylethyl) Cyclohexanol lipoxamide (1 3 2) Trans-N— (2- (1-pyrrolidinyl) 1-41-pyridyl) 1-41-aminomethylcyclohexanecarboxamide

 (133) Trans-N- (2,6-diamino-4-pyrimidyl) -14-aminomethylcyclohexanecarboxamide

 (1 3 4) (+) — trans-N- (7-methyl-1,8-naphthyridine-14-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide

 (135) trans-N- (1-benzyloxymethylpyrro [2,3-b] biridin-4-yl) -14-aminomethylcyclohexanecarboxamide

 (1 3 6) (+) — trans-N- (1 -methylpyro [2,3-b] pyridine-4-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide (1 3 7) trans-N-benzyl-N- (2-benzylamino 4-bipyridine) 1-4- (1-amino-1-methylethyl) cyclohexanecarboxamide

(138) Trans-N- (2-azido-4-pyridyl) -14-aminomethylcyclohexanecarboxamide

 (13 9) trans-N- (2,3-dihydro-1H-pyro [2,3-b] biridin-4-yl) -14-aminomethylcyclohexanol lipoxamide

(1440) trans-N- (2,3-dihydro 1H-birolo [2,3-b] pyridine-1-4-isole) 1-4- (1 amino 1-methylethyl) Lupoxamide

 (14 1-1) trans-N-(2-carboxy-4-pyridyl) 14-aminomethylcyclohexanecarboxamide

 (14 1-2) (R) 1-(+) 1-trans-N-(3-bromo-1 H-pyro-mouth [2, 3-b] pyridine-1-4-yl) 14-(1-aminoethyl ) Cyclohexanecarboxamide

 (142) trans-N- (1 H-birolo [2,3-b] pyridine-14-yl) -14-guanidinomethylcyclohexanecarboxamide

 (143) trans-N- (1H-birazolo [3,4-b] pyridine-14-yl) -14-guanidinomethylcyclohexanecarboxamide

 (14) trans-N- (4-pyridyl) 14-guanidinomethylcyclohexancarboxamide

 (145) trans-N- (1-methylvirolo [2,3-b] pyridine-14-yl) 1-41- (guanidinomethyl) cyclohexanecarboxamide

 (146) trans-N-(1 H-virolo [2,3-b] pyridine-14-yl)-41-(2-imidazoline-1-2-yl) aminomethylcyclohexanecarboxamide

 (147) trans-N-(1-benzyl-xylmethylpyro-mouth [2,3-b] pyridin-14-1-yl)-14-guanidinomethylcyclohexanecarboxamide (148) trans-N- (2-amino 4- 4-pyridyl) 4-guanidinomethylcyclohexanecarboxamide

 (149) trans-N-(1-benzyloxymethyl-1 H-virolo [2,3-b] pyridine-1-4-yl) 14-(2-imidazoline-1-2-yl) aminomethylcyclo Hexanecarboxamide

 (150) trans _N— (1 H—Virolo [2,3-b] pyridine-1-yl) —4— (3-Benzylguanidinomethyl) cyclohexanecarboxamide

(1 5 1) Trans-N- (1H-Virolo [2,3-b] pyridin-1-yl) —41- (3-phenylguanidinomethyl) cyclohexanecarboxamide (152) trans-N_ (1H-pyro mouth [2,3-b] pyridine-14-yl)

--(3-Provirguanidinomethyl) cyclohexanecarboxamide (153) trans _N— (1H-birolo [2,3-b] pyridine-1-yl)-4-(3 —Octylguanidinomethyl) cyclohexanecarboxamide

(1 54) trans-N- (l-benzyloxymethylbirollo [2,3-b] pyridin-1-4-yl) 1-4- (2-benzyl-1-3-ethylethylguanidinomethyl ) Mouth hexane power lipoxamide

 (1 5 5) trans-N-(1 H-virolo [2,3-b] pyridine-41-yl)-41-(imidazole-2-yl) aminomethylcyclyl hexanecarboxamide

(1 56) trans-N-(1 H-virolo [2,3-b] pyridine-4-yl)-4-(thiazo-1-2-yl) aminomethylcyclohexanecarboxamide

(1 57) (R) — (+) — N_ (4-pyridyl) 1-41 (1 -aminoethyl) benzamide

 (1 58) N-(4-pyridyl)-4- (1-amino-1-methylethyl) benzamide

 (1 5 9) N- (4-Pyridyl) 1-4-Aminomethyl-12-Benzyloxybenzamide

 (160) N— (4-pyridyl) -1-aminomethyl-2-ethoxybenzamide '

 (16 1) (R) — (—) —N— (4-pyridyl) — 4- (1-aminoethyl) — 3-nitrobenzamide

 (166) (R) I (I) -N- (4-pyridyl) I 3-Amino 4-(1-Aminoethyl) benzamide

 (163) (R) 1 (+) -N- (4-pyridyl) 1 4-(1 -aminoethyl)-3-

(1 64) N- (4-pyridyl) -1-3-aminomethylbenzamide (16 5) (R)-(+)-N-(1 H-Pyro-mouth [2, 3-b] Pyridine-14-1) 14-1 (1-aminoethyl) benzamide

 (1 66) (R) 1 (+) — N— (1 H—Birazolo [3, 4—b] pyridine 1 4 —yl) 1 4— (1—aminominethyl) benzamide

 (1 67) N— (1 H—pyrazo mouth [3, 4-b] pyridine-1-4-yl) 1-4-guanidinomethylbenzamide

 (1 68) N— (4-pyridyl) 1-4-guanidinomethylbenzamide

 (1 69) (R) 1 (+) -N- (4-Pyridyl) 1 4 1 (1-Aminoethyl) — 3—Fluorobenzamide

 (170) N— (4-pyridyl) 1-41-aminomethylbenzamide

 (17 1) N— (4-pyridyl) 1 4-aminomethyl-2-hydroxyhydroxy

^ Do

 (172) N— (4-pyridyl) — 4- (2-aminoethyl) benzamide (173) N- (4-pyridyl) — 4-aminomethyl-3--2-benzamide (174) N— (4 1-Pyridyl) 1-Amino-4-aminomethylbenzamide (175) (S)-(-)-N- (4-Pyridyl) 1-41- (1-Aminoethyl) benzamide

 (176) (S) one (-) one N— (4-pyridyl) one 2— (1-aminoethyl) benzamide

 (177) (R)-(+)-N-(4-pyridyl) 1-4-(1-aminoethyl)-2-benzobenzamide

 (178) (R) 1 (+)-N-(1 H-Pyro-mouth [2,3-b] pyridine 1-41-yl) 1-4 (1-(3-Provirguanidino) ethyl) Benzamide

 (1 79) (R)-(I) I-N- (1H-Virolo [2,3-b] Pyridine- 14-yl) 14- (1- Aminoethyl) 1-3-Azidobenzamide

(180) (R) ― (+) -N- (4 -pyridyl) 1-41 (1 -aminoethyl) — 2--2-trobenzamide (18 1) (R) ― (-) 1 N— (4 pyridyl) – 4 1 (1-aminoethyl) 1 3 -ethoxybenzamide

 (182) (R)-(+) -N- (3-1-H-pyrro [[2,3-b] pyridin-4-yl) 1-4-(1 -aminoethyl) benzamide

 (183) (R)-(+) — N— (3-iodo 1 H—pyro mouth [2,3-b] pyridin-1-4-yl) 1-41 (1-aminoethyl) 1-3 —Aji Dovenzamide

(184) (R)-(I) -N- (4-pyridyl) 1-4-1 (1-aminoethyl) — 3-Hydroxybenzamide

 (185) N— (1 H—birazolo [3,4-b] pyridine-14-yl) 1-4-guanidinomethyl-1-nitrobenzamide

 (1 86) (R) -N- (1H- lazolo [3,4-b] pyridine-41-yl)-4-- (1-guanidinoethyl) -l-nitole vent amide

 (1 87) (R) -N- (1H-pyrazo-mouth [3,4-1b] pyridine-14-yl) 1-4- (1-aminoethyl) -12-nitrotropenzamide

 (188) N- (1H-pyrazo-mouth [3,4-b] pyridine-14-yl) -14-guanidinobenzamide

 (189) (R) — N— (1 H—Bilazolo [3,4-b] Pyridine-1-41-yl)-4-(1-Aminoethyl) -1 3-Nitrobenzamide

 (1 90) (R) -N- (1H-birazolo [3,4-1b] pyridine-14-yl) 1-41- (1-guanidinoethyl) benzamide

 (1 9 1) N— (1H—birazolo [3, 4—b] viridine 1-41) 1 4-— (1-amino-1 2-hydroxicetil) benzamide

 (1992) N- (1H-Vilazolo C3,4-b) Pyridine-14-yl) 1-4-Aminomethyl-3-2-2 Trobenzamide

 (193) N— (1H—Virolo [2,3-b] pyridine-14-yl) -14-biberidinecarboxamide

(194) N- (1H-birazolo [3,4-1b] pyridine-14-yl) -14-bi Beridinecarboxamide

 (1 95) N- (1H-Bilazolo [3,4-b] pyridine-1 4-yl) 1-1-aminoacetyl 4-biperidinecarboxamide

 (196) N- (1-Methoxymethyl-1H-birazolo [3,4-b] pyridine-14-yl) -14-Piridinecarboxamide

 (197) N- (2,3-dihydro-1H-pyrro [2,3_b] pyridine-14-yl) 1-4-bipyridine carboxamide

 (1 98) N- (1H-pyro mouth [2,3-b] pyridine-14-yl) 1-1- (2-phenylethyl) -14-biveridinecarboxamide

 (1 9 9) N- (1H-pyro mouth [2,3-b] pyridine-1-41) 1-1-amidino-4-bipyridine carboxamide

 (200) N— (1H—Virolo [2,3—b] pyridine-14-yl) ー 11- (3-phenylpropyl) -14-bipyridinecarboxamide

 (20 1) N- (1 H-pyrro [2,3-b] pyridine-4-yl) 11-benzylyl 4-pyrididinecarboxamide

 (202) N— (1H—pyrazo mouth [3, 4-b] pyridine-14-yl) 1-11 (2-phenylethyl) 1-4-biperidinecarboxamide

 (203) N- (1H-Bilazolo [3,4-b] pyridin-1-41) 1-11 (3-phenylpropyl) 1-4-biperidinecarboxamide

 (204) N- (1 H-Virolo [2,3-b] pyridine-14-yl) 1-4- (1-amino-1-methylethyl) benzamide

 Preferably, compounds (80), (109), (110), (112), (115), (1

42), (143), (144), (145), (153), (157), (163), (16

5), (166) and (179).

The compound used as the compound having the Rho kinase inhibitory activity of the present invention may be an acid addition salt which is acceptable in production, and the acid may be an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, or methane. Sulfonic acid, fumaric acid, maleic acid, mandelic acid, citric acid, Organic acids such as tartaric acid and salicylic acid are exemplified. Further, the compound having a carboxyl group may be a salt with a metal such as sodium, potassium, calcium, magnesium, or aluminum, or a salt with an amino acid such as lysine. In addition, those

Monohydrate, dihydrate, 1/2 hydrate, 1/3 hydrate, 1/4 hydrate, 2/3 hydrate, 3Z dihydrate, etc. are also included in the present invention. .

 Compounds represented by the general formula (I) are described in JP-A-62-89679, JP-A-3-218356, JP-A-5-1944 ° 1, JP-A-6-411080, W095 / 28387 and WO98 / 06433, and the like.

 When the above-mentioned compound having Rho kinase inhibitory activity has an optical isomer, a racemic rest or a cis-trans isomer thereof, all of them can be used in the present invention, and these isomers can be used. Can be produced by using the raw materials of each isomer.

 In the present invention, one kind of compound having Rh 0 kinase inhibitory activity may be contained alone, or if necessary, several kinds may be used in combination. Further, in addition to the compound having Rho kinase inhibitory activity and the osteoinductive factor, which are essential for the osteogenesis promoter of the present invention, other bone resorption inhibitors and Z or an osteogenesis promoter can be used in combination.

For example, calcium preparations, vitamin D, calcitonins, bisphosphonates, sex hormones such as estrogen, fluorine such as sodium fluoride, sub ^[Jo gland hormones (Rotauita acids), insulin-like Naruyoshi factor (I GF acids ) And fibroblast growth factors (FGFs).

 The osteogenesis-inducing enhancer of the present invention also includes, in addition to a compound that inhibits Rho kinase inhibitory activity, which is an active ingredient, and an osteoinductive factor used in combination with the osteogenesis-inducing enhancer, other bone resorption inhibitors, and An osteogenesis promoter can be used in combination. Specific examples include the same as described above.

The bone formation promoting agent of the present invention comprises a compound having a Rh kinase inhibition activity and an osteoinductive agent. It can be prepared by mixing with a pharmaceutically acceptable carrier (stabilizer, preservative, solubilizer, pH adjuster, thickener, etc.) if necessary. It is preferably a preparation comprising a compound having Rho kinase inhibitory activity and an osteoinductive factor in a suitable solid, paste-like or liquid carrier.

 Here, examples of the carrier include natural polymer materials such as collagen, gelatin, and hyaluronic acid, and artificial polymer materials such as polylactic acid (PLA), polyglycolic acid (PLG), and copolymers thereof (PLGA); Solid, pasty, viscous, or liquid materials combining one or two or more of the rest-adaptable materials, such as natural or artificial inorganic materials such as hydroxyapatite, phosphoric acid cement, etc. Carriers are examples.

 A collagen sponge and a gelatin sponge coated with PGL A (WO96Z104024) are particularly preferred.

 These carriers are already known as carriers of osteoinductive factors, and the osteogenesis promoter of the present invention can be supported on these carriers in the same manner as the method of supporting osteoinductive factors. .

 Further, the osteogenesis promoter of the present invention can also contain additional components useful for bone and cartilage formation, for example, fibronectin, osteonectin and the like. These desired components can be added by a suitable method at a suitable stage of preparation of the present bright composition.

 The osteogenesis-promoting agent of the present invention containing a carrier is administered to a site where bone or cartilage formation is expected by any method. Liquids can be administered by injection, pastes or clays can be implanted at the desired site, and solids can be administered by shaping into the desired shape.

The bone formation promoter of the present invention may be used not only alone but also in combination with other known implants. For example, when implanting an artificial root or an artificial tooth root, the bone formation promoting agent of the present invention can be used by coating the surface with an adhesive substance such as collagen-based fibrin glue. Or the table of these artificial bones and artificial roots It is also possible to form the surface to be porous and to use an osteogenesis promoter in that portion. In order to locally fix the bone formation promoting agent of the present invention, a life-compatible film, for example, a collagen film, a Gore-Tex film or a polylactic acid film used for the GTR method may be combined.

 The osteogenesis-inducing enhancer comprising the compound having Rho kinase inhibitory activity of the present invention as an active ingredient may be administered in any form as long as it can be used in combination with osteoinductive protein. It may be administered as a formulation or as a separately administered formulation by different methods of administration.

 Here, examples of the osteoinductive factor-containing preparation include the same preparation as the above-mentioned osteogenesis promoter.

 On the other hand, preparations to be administered separately from osteoinductive factors include, if necessary, pharmaceutically acceptable carriers (excipients, binders, disintegrants, flavoring agents, flavoring agents, emulsifiers, solubilizing agents, etc.) Various solid preparations, semi-solid preparations or liquid preparations prepared by mixing. These preparations can be administered by any administration method such as twisting, injection, or topical administration. Formulations that can be administered continuously are preferred, and for example, sustained release formulations, depots, or formulations for continuous administration such as intravenous drip are preferred.

 In the case of solid dosage forms, excipients such as sucrose, lactose, cellulose sugar, D-mannitol, maltitol, dextran, starches, agar, alginate, chitin, chitosan, pectin, Tran gums, gum arabic, gelatin, collagen, casein, albumin, calcium phosphate, sorbitol, glycine, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl cellulose, hydroxypropylmethylcellulose, Glycerin, polyethylene glycol, sodium bicarbonate, magnesium stearate, and silver are used.

In the case of semi-isotopic preparations, animal and vegetable oils (olive oil, corn oil, castor oil, etc.), mineral oils (vaseline, white petrolatum, solid paraffin, etc.), waxes (jojoba oil, carnapa wax, beeswax, etc.), Partially or totally synthesized glycerin Examples of commercially available products using fatty acid esters (eg, lauric acid, myristic acid, palmitic acid) include Witebsol (manufactured by Dynamid Nobel), Pharmasol (manufactured by Nippon Oil & Fats Co., Ltd.) and the like.

 In the case of a liquid preparation, additives such as sodium chloride, glucose, sorbitol, glycerin, olive oil, propylene glycol, ethyl alcohol and the like can be mentioned. In particular, in the case of an injection, a sterile aqueous solution such as a salt solution, an isotonic solution, or an oily solution such as sesame oil or soybean oil is used. If necessary, a suitable suspending agent such as sodium carboxymethylcellulose, a nonionic surfactant, a solubilizing agent such as benzyl benzoate or benzyl alcohol may be used in combination.

 In addition, as the exogenous osteoinductive factor used in combination with the osteogenesis induction enhancer of the present invention, a conventionally known osteoinductive factor preparation can be used. Examples of the exogenous osteoinductive factor preparation include an implantable preparation using the above-described carrier. Can be Preferred is an osteogenic implant described in W096 / 10426.

 In the osteogenesis promoting agent of the present invention, the amount of the compound having Rho kinase inhibitory activity added to the preparation (support) is not particularly limited as long as it is a concentration at which the osteogenesis promoting effect is exhibited. 0.1 g per preparation (support): 11000 mg, preferably 0.1 lg ~ 500 mg. The osteoinductive factor may be added at any concentration as long as it can induce osteo- and cartilage-inducing action. However, when rhBMP-2 is used, it is usually 0.000 per 1 ml of the preparation (support). The amount is 1 g or more, preferably from 0.01 to 500 QS, more preferably from 0.01 to 1 000 °.

In the osteogenesis-inducing enhancer of the present invention, the dose of the compound having Rho kinase inhibitory activity varies depending on the administration method, but in general, per dose, 0.1 to 1,000 mg / kg in the case of microdose administration 0.1 to 500 mg / kg for injection, 0.03 to 30 Omg / kg for injection, preferably 0.1 to: L 00 mg / kg, 0.1 to L for topical administration. mgZkg, preferably 0.1 to 500 mg / kg. At this time, the amount of the osteoinductive factor of the exogenous osteoinductive factor preparation to be used in combination is as described above. The same is true.

 The osteogenesis-promoting agent of the present invention and the osteogenesis-inducing enhancer used in combination with an exogenous osteoinductive factor are useful for preventing or treating the above-mentioned bone disease. It can be applied to the affected area in a manner known in the art to repair various bone and cartilage defects caused.

 In particular, when the present invention is used as a transplant, the transplant can be applied to various fields of treatment of bone disorders, and particularly, to the treatment of bone or cartilage caused by accidents, diseases, birth defects, or various operations. Repair of defective parts, promotion of healing of various fractures, formation of bone around human implants such as artificial joints, artificial bones or dental implants, promotion of fixation when using human implants, promotion of spinal fixation, leg extension Reconstruction or replacement of bone or cartilage in orthopedic field, reconstruction of joints, replacement of bone or cartilage in plastic surgery field, or restoration of jaw bone in dental area, regeneration of alveolar bone, restoration of cementum or in-plan It is suitable for increasing bones for use in animals.

 The effectiveness of the present invention for bone formation can be confirmed as follows. The effects on osteogenesis are as follows: 1) C3Η10 21/2 cells, an undifferentiated mesenchymal cell line that has been reported to have the ability to differentiate into osteoblasts (Growth Factors, 9, pp .57-71 (1993)) to evaluate the degree of differentiation promoting effect on osteoblasts. 2) It can be evaluated by examining the expression of differentiation function of primary cultured osteoblasts isolated from calvaria of chicken embryos. . In each case, as a differentiation marker, a change in the activity of Alkaline phosphatase (ALPase) of the cell, such as a change in the amount of genius steocalcin or osteopontin, can be used as an index.

Example

 Hereinafter, the present invention will be described more specifically with reference to formulation examples and experimental examples, but the present invention is not limited thereto.

In the following formulation examples and experimental examples, the compound having Rho kinase inhibitory activity is one of the compounds represented by the general formula (I). —Aminoethyl) 1 1— (4-pyridylcarbamoinole) cyclohexyl San 2 HC 1-1 H ₂ 0 (Y-27632) and (+) — trans — (1 H-pyro mouth [2,3-b] viridine 4-yl) 1 4 One (1-aminoethyl) cyclohexanecarboxamide 2 HC1 (Y-30141) was used. As an exogenous osteoinductive factor, a recombinant human BMP-2 protein (Genentics Institute, hereinafter referred to as rh BMP-2) was used.

Formulation example 1: Tablet

 Compound of the present invention (Y-27632) 10.0 mg-lactose 50.0 mg-corn starch 20.0 mg crystalline cellulose 29.7 mg polyvinyl vinylidone K 30 5.0 mg talc 5.0 mg stearin 0.3 mg of magnesium acid

 120.0 mg The compound of the present invention (Y-27632), lactose, corn starch and crystalline cellulose were mixed, kneaded using polyvinylpyrrolidone-30 size liquid, and granulated through a 20 mesh sieve. After drying at 50 ° C for 2 hours, the mixture was passed through a 24-mesh sieve, talc and magnesium stearate were mixed, and a tablet of 12 Omg was produced using a 7 mm diameter punch.

Formulation example 2: Capsules

 Compound of the present invention (Y-27632) 10.0 mg Lactose 70.0 mg Corn starch 35.0 mg Polyvinyl bicarbonate Ridon K 30 2.0 mg Talc 2.7 mg Magnesium stearate-0.3 mg

1 20.0 mg- The compound to be investigated (Y-27632), lactose and corn starch were mixed, kneaded using polyvinylidene-lidone-30 size liquid, and granulated through a 20-mesh sieve. After drying at 50 ° C for 2 hours, the mixture was passed through a 24-mesh sieve, talc and magnesium stearate were mixed, and the mixture was filled into hard capsules (No. 4) to produce a capsule formulation of 120 mg.

Formulation example 3: Injection

 Compound of the present invention (Y-27632) 5-0 mg Sodium chloride 18.0 mg Distilled water for injection Total volume 2.0 ml Sodium chloride was dissolved in about 80 parts of distilled water for injection, and then the compound of the present invention (Y -Add 27632) and dissolve to make the total amount (100 parts). After filtration using a membrane filter (0.2 Aim), the mixture was filled into a 2 ml 1 ampoule and sterilized at 115 ° C for 30 minutes to prepare a 2 ml injection.

Reference formulation example: Exogenous osteoinductive factor formulation example

D, L—Copolymer of lactic acid and glycolic acid (molar ratio = 50:50, molecular weight = 40,000: Boehringer Tangerheim) 12.0 g of polysorbate 80 (Kanto Was added to 1,4 dioxane (reagent manufactured by Kanto Chemical Co., Ltd.) to a concentration of 0.5% by weight, and dissolved by heating to 150 ml. After cooling this polymer solution to room temperature, a 7 cm x 1 O cm x 1 cm gelatin sponge (svonzel) is immersed. Next, the spongel was immersed in this polymer solution—frozen at 45 ° C., and then lyophilized at 0.1 mbar. After the polymer adhering to the periphery was cut off with a razor, it was subjected to dry heat sterilization at 135 ° C for 36 minutes. A rhBMP-2 solution [0.12 mg Zml to 5.9 mg / m 1; 2.5% glycine, 0.5% sucrose, 5 mM sodium chloride, 5 mM glutamate, 0.01% polysorbate 80; ρH4.5] was dropped into about 60 ml and absorbed to obtain a transplant containing rhBMP-2 (0.1 mgZml to 5.0 mg / m1). . Experimental Example 1: BMP and Y— 276 affect differentiation of undifferentiated mesenchymal cells to osteoblasts 32 actions

 (experimental method)

Using undifferentiated mesenchymal cells (mouse 10T1 / 2), alkaline phosphatase (ALP) activity and osteocalcin production were measured as indicators of osteoblast differentiation. Mesenchymal cells (10 T 1 Z 2 cells; purchased from RIKEN CE LL BANK (RCB0247)) were given rhBMP-2 (0, 30 ng / m1) and Y-27632 (0, 3, 5, 1 0, 20, was added by changing the various concentrations 30〃Myu), 5% C0 ₂ presence in Basal Medium Eagle + 1 0% FCS in, and incubated 37 ° C, 72 hours. After completion of the culture, the number of ALP-positive cells (Fig. 1a-f) by ALP staining (NBT-BCIP method, Promega), and cells by p-ditrophenyl phosphate substrate method (alkaline phospha B-test) The ALP activity (Fig. 2a) contained within (mostly bound to the cell membrane) and the amount of osteocalcin secretion in the culture supernatant (Fig. 2b) were measured by the RIA method (Biotec) < ALP activity (Fig. 2c) was measured for another undifferentiated mesenchymal cell, mouse ST2 cell (available from RI KEN CE LL BAN K (RBC0224)) in the same manner as 10T12 cells. did. For ST2 cells, a culture vessel coated with type 1 collagen, MEM + 10% FCS was used, and the culture time was 5 days.

 (Experimental result)

 The administration of BMP alone in 10 T 1/2 cells increased the number of ALP-positive cells more than 5-fold. Furthermore, by simultaneously ffling Y-27632, the effect of increasing the number of ALP-positive cells by rhBM P-2 was enhanced in a concentration-dependent manner (FIGS. La to f). In addition, Y-27632 enhanced the ALP activity increasing effect of rhBMP-2 in a concentration-dependent manner (Fig. 2a).

On the other hand, in terms of the amount of secretion of osteocalcin, administration of rh BMP-2 alone increased the secretion by about 4-fold. Furthermore, Y-27632 also increased the osteocalcin secretion increasing effect of BMP in a concentration-dependent manner (Fig. 2b). Y-27632 also enhanced rhBMP-2's ALP activity increasing effect on ST2 cells in a concentration-dependent manner (Fig. 2c).

 In all these experiments, the greatest osteoblast differentiation-inducing effect was observed when 10% of Y-27632 was added.

Experimental Example 2: Effect of BMP and Y-27632 on differentiation of mesenchymal cells into osteoblasts in which active Rh ο is forcibly expressed

 (experimental method)

 Activated R ho (Va 1 14 Rh o) expression vector in 10 T 1/2 cells

(Yoshioka, Κ ·, et al., J. Biol. Chem., Vol. 273, No. 9, Feb: pp. 5146-5155 (1998)) was introduced by a method using lipofectamine. To each cell, add rhBMP-2 (0, 30 ng / m1) and Y-27632 (0, 1◦〃Μ) as in Experimental Example 1, and add 5% C0 in Basal Medium Eagle + 10% FCS. After culturing at 37 ° C for 48 hours under ₂ , the change in ALP activity due to the expression of active Rh was examined. The expression of active Rho was measured by Western blotting using an antibody of Rho, and ALP activity was measured in the same manner as in Experimental Example 1. The results are shown in Figure 3.

 (Experimental result)

 10T1Z2 cells expressing activated Rho have reduced ALP activity and increased Rho activity with or without BMP stimulation, compared to mock cells with control expressing only vector. It was confirmed that differentiation into osteoblasts was suppressed (Fig. 3a). In addition, addition of 10-〃M Y-27632 partially restored the reduced ALP activity in activated Rho current cells (Fig. 3a, lane 6). Therefore, it is presumed that suppression of Rho activity by a compound having Rho kinase inhibitory activity (Y-27632) promotes differentiation induction into osteoblasts and promotes bone formation.

Experimental Example 3: Effect of hepatic cells expressing activating ROCK and dominant negative ROCK on osteoblast differentiation and BMP-4 production (experimental method)

 RO CK catalytic domain (amino acid sequence Nos. 1 to 477: num 4, active type) and a sequence in which Lys at position 105 was replaced with A1a to eliminate enzymatic activity (Κϋ 44, dominant negative type) cDNA encoding pcDNA NA3 encoding Ishizaki et al., EMBO J., 15, 1885 (1996)

(Invitrogen) was created. The expression vector was introduced into ST2 cells by a method using lipofectamine. Was added to each cell Y- 2 7 6 3 2 (0 , 1 0 MM), shed MEM + 1 5% C_〇 ₂ below in 0% FCS, 3 7 ° C , 4 8 after more hours culture, AL Changes in P activity and changes in BMP-4 mRNA expression levels were examined. Expression of RO CK (including mutant RO CK) was confirmed by a Western blotting method using an antibody against RO CK (Fujita et al., Biochem. J., 328, 769 (1997)). ALP activity was measured in the same manner as in Experimental Example 1. BM P-4 mRNA was prepared by the RNA blot method (Yoshioka, K., et al., J. Biol.

Chem., 273 (9), 5146 (1998)). Fig. 4 shows the results.

 (Experimental result)

ST2 cells (Δ4 cells) expressing activated RO CK had lower ALP activity than control mock cells expressing only vector (FIG. 4a). In contrast to this, in ST 2 cells expressing Dominan preparative negative form ROCK (KDA 4 cells) as compared to mo ck cells, AL P activity was increased (Fig. 4 a) _t These results This indicates that an increase in ROCK activity suppresses differentiation into osteoblasts. The decrease in ALP activity in the △ 4 cells was restored by the addition of 10 M Y—27632 (FIG. 4 a).

 On the other hand, BMP-4 mRNA expression level (normalized by GAPDH mRNA) was increased in ΚϋΔ4 cells, and was also increased by the addition of 2-27632 (FIG. 4b).

From these results, ROCK negatively regulates BMP-4 mRNA expression, and the R0CKP inhibitor suppresses this control to increase BMP-4 production. It was suggested that it promoted differentiation into osteoblasts.

 The expression of ROCK in each cell was confirmed using ROCK antibody (Fig. 4c) o

Experimental Example 4: Effects of Y—27663 and Y—31041 on ectopic bone formation induced by BMP

 (experimental method)

 BMP pellets (RhBMP-2 impregnated in freeze-dried collagen gel with a trade name of 5 mm) were buried under the left latissimus dorsi fascia of C 3 H mice (body weight about 30 g) and boned. An experimental system for examining formation (Mori, S., Yoshika, H., et al., Bone, Vol. 22, No. 2, Feb .: pp. 99-105 (1998)) was used. A compound having a kinase inhibitory activity (Y—27632: 50 mg or 15 mg Z30 g mouse / two weeks) was continuously administered using an osmotic pump implanted subcutaneously on the right back ^ (Itoh, K , Et al., Nature Med. Feb; 5 (2), pp.221-225 (1999)), and Y-27 against rhBMP-2 induced ectopic skeletal bone formation. The action of 632 was investigated. Figure 5 shows the appearance of the sudden test. Similarly, Y-301, having Rh kinase inhibition activity, was examined at a dose of 5 mg / 30 g mouse for 2 weeks.

 (Experimental result)

 A photograph of the bone formed by Y—2763 2 (50 mg / 30 g mouse) after two weeks is shown in FIG. 6a together with the drug-free group. The left column shows the group receiving only the BMP pellet, and the right column shows the group receiving the BMP pellet and further administering Y-27632. Implantation of BMP pellets resulted in ectopic bone formation with calcification, with an average wet weight of 78.4 ± 14.4 mg (n = 4) of the bone formed. Was. In contrast, a significant increase was observed in the Y-27663 group administration group, with an average of 274.1 ± 44.7 mg (n = 4).

In addition, even when the dose of Y-27632 was reduced to 15 mgZ30 g mice, the average weight of ectopically formed bone was 1 ◦ when the BMP pellet alone was administered. 5.8 ± 10.80 (n = 6), but a significant increase to the mean of 182 · 5 ± 40.28 (η = 6) when administered in combination with Y-27632. Indicated. Furthermore, when the calcium content of the formed bone was examined, the calcium content was significantly increased by administration of 27-27632 in both cases.

 J: Fig. 6b shows a lentogen photograph of the bone formed by the experiment. The upper four rows in the figure show the lower limb bones, and the lower four rows show the bones formed in the BMP embedding part (similar to Fig. 6a above). The results show that the effect of the compound with Rho kinase inhibitory activity (Y-27632) has no effect on the existing bone, the lower limb bone, and has a remarkable potentiating effect on ectopic bone formation induced by BMP. It was confirmed to have.

 The hematoxylin-eosin (HE) stained images of the bones formed in the experiment described above are shown in Fig. 7a (control; administration of BMP pellet only) and Fig. 7b (a compound having Rho kinase inhibitory activity (Y_ 27632) Combination group), and HE staining image showing bone tissue formed in the 埋 め 込 み -let embedded area ^] 7c (control; administration of BMP pellet only) and Fig. 7d (Rho kinase Compounds having inhibitory activity (Y-27632) group are shown. In the group combined with the compound having Rho kinase inhibitory activity (Y-27632), formation of well-differentiated bone marrow was observed.

 Furthermore, when Y-30141 (Smg / SOg mouse) was administered, the same results as in the Y-27632 administration group were obtained. Pictures of bones and lentogens formed two weeks later are shown in FIGS. 8a and 8b, respectively, together with the group filled with BMP pellet only. The upper row shows the group in which only the BMP pellet was filled, and the lower row shows the group in which the BMP pellet was filled and Y-30141 was administered.

The wet weight of the formed bone averaged 71.0 ± 4.7 mg (n = 5) in the group filled with BMP pellet only, whereas the average weight in the Y-30141 group was 1 The dose was 61.6 ± 13.8 mg (n = 5), and a significant (p = 0.0033) increasing effect was observed. The average calcium content of the formed bone was 3.61 ± 0.19 mg (n = 5) in the group filled with BMP pellet only, whereas the calcium content of the formed bone was The mean was 8.62 ± 0.34 mg (n = 5), and a significant (p = 0.◦01) increase effect was observed.

Experimental Example 5: Effect of Y-27632 on the number of BMP-4 mRNA positive cells in mesenchymal cells accumulating around BMP-2 cells

 (experimental method)

 In the same manner as in Experimental Example 4, Y-27632 was administered in the same manner as in Experimental Example 3 at 50 mgZ30 gZ for 2 weeks at ffl 鲎 using an experimental system for examining bone formation by burying a BMP pellet. Four days after filling the BMP pellet, the in situ hybridization method (Nakase, T., et al., J. Bone Miner. Res 9: pp.651-659 (1994)) was used to generate the BMP pellet. BMP-4mRNA-positive cells of mesenchymal cells accumulating in and surrounding tissues were observed.

 (Experimental result)

 Fig. 9 shows the results. BMP-4 mRNA was obtained when Y-27632 was administered (Figs. 9C and D; D is a magnified image) compared to the case where only the BMP beret was filled (Figs. 9A and B; B is a magnified image). Increased the number of cells expressing. The combined ffl of BMP and Y-27632 promotes the proliferation of accumulated interrogative cells and differentiation into osteoblasts, and this effect ultimately contributes to increased wet weight of bone. It was thought to be something.

 Industrial applications

According to the above experimental examples and other pharmacological experiments, the combination of a compound having Rho kinase inhibitory activity and an osteoinductive factor can be used in undifferentiated cannabis cells in alkaline phosphatase (ALP ) The activity and the amount of osteocalcin-producing cows are significantly increased, indicating that they have the effect of inducing differentiation into osteoblasts. In addition, in an ectopic bone formation test, a combination of a compound having Rho kinase inhibitory activity and an osteoinductive factor showed significantly better osteogenic ability than administration of osteoinductive factor alone. In addition, it was found that existing bones were not affected. Therefore, the osteogenesis-promoting agent of the present invention and the osteogenesis-inducing enhancer used in combination with the exogenous osteoinductive factor are useful because they have excellent osteogenesis-promoting action and osteogenesis-inducing strong action.

 The bone formation promoting agent of the present invention and the bone formation induction enhancer used in combination with the exogenous bone induction factor are used to repair various bone and cartilage defects caused by trauma, disease, or congenital abnormalities. Used for It induces bone and cartilage formation promptly in the body of mammals such as humans, dogs, dogs, dogs, mice, rats, etc., and forms (heals) good bones and cartilage. Can be.

 Therefore, it can be applied to various fields, for example, accidents, diseases, congenital abnormalities can be used to repair bone or cartilage defects due to various operations, promote healing of various fractures, artificial joints, artificial bones or artificial bones Bone and cartilage regeneration in orthopedic fields such as bone formation around human implants such as tooth roots, promotion of fixation when using human implants, promotion of spinal fixation, and leg extension, joint reconstruction, plastic surgery Bone syrup in the field is suitable for cartilage replacement, jaw bone restoration in the dental area, alveolar bone regeneration, cementum restoration and bone enlargement for implant use.

 It is also used for the prevention and treatment of metabolic bone disease and osteoarthritis, including osteoporosis, fibrous ostitis, osteomalacia, pageet's disease, etc., which occur when the loss of bone resorption and bone formation in bone tissue is disrupted. Can be used. The present application is based on Japanese Patent Application No. 2493983 filed in Japan, the contents of which are incorporated in full herein.

Claims

The scope of the claims  1. An osteogenesis promoter comprising a compound having Rho kinase inhibitory activity and an osteoinductive factor.  2. The compound having Rho kinase inhibitory activity has the general formula (I) O Rb Ra—— C —— Rc (I) (Where Ra is the formula Shi-N

[In the formulas (a) and (b), R represents hydrogen, alkyl, or a substituent which may have a substituent on the ring. R represents cycloalkyl, cycloalkylalkyl, phenyl or aralkyl;

(Wherein, R ^B is hydrogen, alkyl or formula:. In one NR ⁸ R ⁹ (wherein, R ^a, R Li is equal - to or different water尜, alkyl, to) the Ararukiru or phenyl, R ⁷ Represents hydrogen, alkyl, aralkyl, phenyl, nitro or cyano You. ^{Alternatively} , R ^fi and R ⁷ are combined to form a group which forms a heterocyclic group which may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent in the ring. ). R ⁵ represents hydrogen, alkyl, or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on the ring.  Or a group which forms a heterocyclic ring which may further contain an oxygen atom, a sulfur atom, or a nitrogen atom which may have an S substituent in the bridge together with an adjacent nitrogen atom by bonding to R and R. Show. R ² represents hydrogen or alkyl. R ³ and R ⁴ may be the same or different, and may be hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxy Represents carbonyl, carbamoyl, mono-dialkylcarbamoyl or azide.  A is the formula

(Wherein, R ^{1 Q} and R ¹¹ are the same or different and represent hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl <or R ¹ and R ¹¹ are 1, m, and n each represent 0 or an integer of 1 to 3). In the formula (c), L represents hydrogen, alkyl, aminoalkyl, mono-dialkylaminoalkyl, tetrahydrofurfuryl, carbamoylalkyl, furimidylalkyl, amidino, or

(i)

(Wherein B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl, hydroxyalkyl, alkanoyloxyalkyl, alkoxycarbonylalkyl, hyaminobenzyl, furyl, pyridyl, phenyl, phenylamino, styryl or imidazobilidyl Γ, す. Q ¹ represents hydrogen, halogen, hydroxyl, aralkyloxy or cetylmethyl.  W represents alkylene.  Represents hydrogen, halogen, hydroxyl group or aralkyloxy.  X represents alkylene. Q ³ represents hydrogen, halogen, hydroxyl group, alkoxy, nitro, amino, 2,3-dihydrofuryl or 5-methyl-13-oxo-1,2,3,4,5-tetrahydropyridazin-1-yl .  Y represents a single bond, alkylene or alkenylene. ).  Further, in the formula (c), a broken line indicates a single bond or a double bond. R ⁵ represents hydrogen, hydroxyl, alkoxy, alkoxycarbonyloxy, alkanoyloxy or aralkyloxycarbonyloxy. ] Is shown.  R b represents hydrogen, alkyl, aralkyl, aminoalkyl or mono'dialkylaminoalkyl.  R c represents a nitrogen heterocycle which may have a substituent. ] An amide compound represented by the formula: 2. The bone formation promoting agent according to claim 1, which is an acid addition salt.  3. The compound having Rho kinase inhibitory activity is represented by the general formula (I  O Rb Ra'ichi C- -N--c (Γ) Where R a 'is

[In the formula, R, represents hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on 璟.  R ′ represents hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on the ring. Or a group in which R ′ and R ¹ are bonded to form a heterocyclic ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent together with an adjacent nitrogen atom in the ring. Is shown. R ² represents hydrogen or alkyl. R-\ R ⁴ is the same or different and is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acetyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, Shows carbamoyl, mono'dialkylcarbamoyl or azide. A is the formula

(In the formula, R ^{1 D} and R ¹¹ are the same or different and represent hydrogen, alkyl, haloalkyl, aralkyl, hydroxydialkyl, carboxy or alkoxycarbonyl ₍ or R ¹ ° and R ¹¹ are 1, m, and n each represent 0 or an integer of 1 to 3.]).]].  Rb is hydrogen, alkyl, aralkyl, aminoalkyl or monodialkylaminoalkyl.  Rc represents a nitrogen-containing heterocyclic group which may have a substituent. ] 3. The bone formation promoting agent according to claim 1 or 2, which is an amide compound represented by the formula: or an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. 4. Compounds with Rho kinase inhibitory activity are (+)-trans-4- (1-aminoethyl) -11- (4-pyridylcarbamoyl) cyclohexane, (+)-trans-N- (1H) —Pyro mouth [2,3-b] pyridine-1-4-yl) 1-4- (1-aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-pyridyl) 14 — (1-Aminoethyl) benzamide and (R) — (+)-N- (1H-pyro-mouth [2,3-b] pyridine-1 4-yl) 1-4- (1-aminoethyl) benzamido 2. The bone formation promoting agent according to claim 1, which is a compound selected from the group consisting of amides and / or a pharmaceutically acceptable acid addition salt thereof. 5. Compounds having Rho kinase inhibitory activity include (+) _ trans-41- (1-aminoethyl) -11- (4-viridylcarbamoyl) cyclohexane and Z or a pharmaceutically acceptable acid thereof. The bone formation promoter C according to claim 1, which is an addition salt. 6. The bone formation promoting agent according to any one of claims 1 to 5, wherein the osteoinductive factor is recombinant human BMP-2. 7. The bone formation promoting agent according to any one of claims 1 to 6, which is adapted for prevention and treatment of bone disease.  8. The bone formation promoting agent according to claim 7, wherein the bone disease is accompanied by bone, cartilage damage, defect or hypoplasia.  9. An osteogenesis-inducing enhancer comprising a compound having a Rh kinase inhibitory activity as an active ingredient, which enhances the osteoinductive action of an exogenous osteoinductive factor.  10. The compound having Rho kinase inhibitory activity is represented by the general formula (I): 0 Rb Ra—— C—— N Rc (I)  (Where Ra is the formula

 [In the formulas (a) and (b), represents a hydrogen, an alkyl or a cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on the ring.

(Wherein, R "is hydrogen, alkyl or a formula: NR ⁸ R ⁹ (where R ⁸ and R ^H are the same or different and represent hydrogen, alkyl, aralkyl or phenyl). R ⁷ represents hydrogen, alkyl, aralkyl, phenyl, nitro or cyano. Alternatively, R ⁶ and R ⁷ are combined to form a heterocyclic ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent in the ring. ) Indicates "3  R ′ represents hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on the ring.  Or R and R ′ represent a group which, together with an adjacent nitrogen atom, forms a heterocyclic group which may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent. . R ² represents hydrogen or alkyl. R ³ and R ⁴ are the same or different and are hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, arylalkoxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl , Carbamoyl, mono'dialkylcarbamoyl or azide.  A is the formula

(Wherein, R ^{1 C)} and R ¹¹ are the same or different and represent hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R ^{1 G} and R ¹¹ are bonded Represents a group which forms cycloalkyl. 1, m and n each represent 0 or an integer of 1 to 3. ). In the formula (c), L represents hydrogen, alkyl, aminoalkyl, mono-dialkylaminoalkyl, tetrahydrofurfuryl, phorbamoylalkyl, fumidylalkyl, amidino, or O B—— C—— (f] (g)

(Wherein B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl, hydroxyalkyl, alkanoyloxyalkyl, alkoxycarbonylcarbonylalkyl, thioaminobenzyl, furyl, pyridyl, phenyl, phenylamino, styryl or imidazo Indicates pyridyl. Q ¹ represents hydrogen, halogen, hydroxyl, aralkyloxy or cetylmethyl.  W represents alkylene. Q ² represents hydrogen, halogen, a hydroxyl group or aralkyloxy.  X represents alkylene. Q ³ represents hydrogen, nitrogen, hydroxyl, alkoxy, nitro, amino, 2,3-dihydrofuryl or 5-methyl-13-oxo-1,2,3,4,5-tetrahydropyridazine-6-yl Show.  Y represents a ^ bond, alkylene or alkenylene. ).  Further, in the formula (c), a broken line indicates a single bond or a double bond. R ⁵ represents hydrogen, hydroxyl, alkoxy, alkoxycarbonyloxy, alkanoyloxy or aralkyloxycarbonyloxy. ] Is shown.  R b represents hydrogen, alkyl, aralkyl, aminoalkyl or mono'dialkylaminoalkyl.  R c represents a nitrogen-containing heterocyclic ring which may have a substituent. ] An amide compound represented by the formula: The osteogenesis induction enhancer according to claim 9, which is an acid addition salt.  11. The compound having Rho kinase inhibitory activity is represented by the general formula (I 0 Rb Ra'ichi C—— N—— Rc () [Expression B, R a 'is an expression

[In the formula, R, represents hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on 璟. R ¹ represents hydrogen, alkyl, or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl which may have a substituent on 璟. Or R and R ¹ are bonded together with an adjacent nitrogen atom to form a heterocyclic ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom which may have a substituent. Is shown. R ² represents hydrogen or alkyl. RR ⁴ may be the same or different and may be hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkoleamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acetyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, Shows carbamoyl, mono'dialkylcarbamoyl or azide. A is the formula

(Wherein, R ^{1 D,; 1} 'are the same or different and each is hydrogen, alkyl, haloalkyl, § aralkyl, hydroxycarboxylic alkyl, or _r shows a carboxy or alkoxycarbonyl, R ¹ "and R ^{1 1} is bonded to 1, m, and n each represent 0 or an integer of 1 to 3.]).  Rb represents hydrogen, alkyl, aralkyl, aminoalkyl or mono'dialkylaminoalkyl.  R c represents a nitrogen-containing heterocyclic ring which may have a substituent. ] The osteogenesis-inducing enhancer according to claim M9 or 10, which is an amide compound represented by the formula: or an isomer thereof and Z or a pharmaceutically acceptable acid addition salt thereof.  12. Compounds with Rh kinase inhibition activity include (+)-trans-4- (1-aminoethyl) -111 (4-pyridylcarbamoyl) cyclohexane, (+)-trans-N- (1H —Virolo [2,3—b] pyridine-14-yl) 14- (1-aminoethyl) cyclohexanecarboxamide, (R) — (+) —N— (4-pyridyl) 14 (1-aminoethyl) benzamide and (R)-(+)-N- (1H-birolo [2,3-b] pyridine-14-yl) 14- (11-aminoethyl) benzamide A bone formation induction enhancer according to claim 9, which is a compound selected from the group and a compound or a pharmaceutically acceptable acid addition salt thereof. 13. The compound having Rho kinase inhibitory activity is (+)-trans-41- (1-aminoethyl) -111- (4-viridylcarbamoyl) cyclohexane and / or a pharmaceutically acceptable acid addition salt thereof. An osteogenesis induction enhancer according to claim 9. 14. Any of claims 9 to 13 wherein the osteoinductive factor is recombinant human BMP-2 The bone formation induction enhancer described in ^.  15. The bone formation induction enhancer according to any one of claims 9 to 14, which is adapted for prevention and treatment of bone disease.  16. The osteogenesis-inducing enhancer according to claim 15, wherein the bone disease is accompanied by bone, cartilage damage, defect or dysplasia.  17. A composition for preventing and treating bone diseases, comprising a compound having Rh kinase inhibitory activity, an osteoinductive factor, and a pharmaceutically acceptable carrier.  18. The compound according to claim 17, wherein the compound having a Rh kinase inhibitory activity is a amide compound represented by the general formula (I), an isomer thereof, and Z or a pharmaceutically acceptable acid addition salt thereof. Pharmaceutical composition for prevention and treatment of bone diseases.  1 9. The compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (1 ′), an isomer thereof, and / or a pharmaceutically acceptable acid addition salt thereof. 7. A pharmaceutical composition for preventing or treating a bone disease according to 7 or 18.  20. Compounds with rhokinase inhibitory activity are (+) — trans — 4 — (1 aminoethyl) 1 1 — (4 viridylcarbamoyl) cyclohexane, (+) 1 trans _N — ( 1 H-Pyro mouth [2,3-b] pyridine-14-yl) 141- (1-aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-pyridyl) — 4— (1-Aminoethyl) benzamide and (R) — (+) — N— (1 H—Pyro mouth [2,3—b] Pyridine-1-4-yl) 1-41 (1-Aminoethyl 18. The pharmaceutical composition for preventing / treating a bone disease according to claim 17, which is a compound selected from the group consisting of benzamides and a pharmaceutically acceptable acid addition salt thereof.  2 1. A compound with Rh kinase inhibition activity is (+)-trans-41- (1-aminoethyl) -111- (4-biridylcarbamoyl) cyclohexane and its or pharmaceutically acceptable The pharmaceutical composition for preventing or treating bone disease according to claim 17, which is an acid addition salt. 22. Any of claims 17 to 21 wherein the osteoinductive H child is recombinant human BMP-2 A pharmaceutical composition for preventing or treating a bone disease according to any of the above items.  23. The pharmaceutical composition for preventing or treating a bone disease according to any one of claims 17 to 22, wherein the bone disease is accompanied by damage, loss, or dysplasia of bone or cartilage. 24. A bone disease comprising a compound having a ho kinase inhibitory activity and a pharmaceutically acceptable carrier as an active ingredient, which enhances the osteoinductive action of an exogenous osteoinductive factor. Preventive and therapeutic pharmaceutical compositions.  25. The bone according to claim 24, wherein the compound having a Rh kinase inhibitory activity is a amide compound represented by the general formula (I), an isomer thereof, and / or a pharmaceutically acceptable acid addition salt thereof. Pharmaceutical composition for prevention and treatment of diseases. 26. The compound according to claim 24 or 25, wherein the compound having a Rh kinase inhibition activity is an amide compound represented by the general formula (1), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. A pharmaceutical composition for the prevention and treatment of bone diseases listed above. 27. The compound having Rho kinase inhibitory activity is (+)-trans-4- (1-aminoethyl) 1-11- (4-pyridylcarbamoyl) cyclohexane, (+)-trans-N- (1H- Pyro mouth [2,3-b) pyridine-4-yl) 1-41- (1-aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-pyridyl) -14- ( 1-aminoethyl) benzamide and (R)-(+)-N- (1H-birolo [2,3-b] pyridine-14-yl) 1-41- (11-aminoethyl) benzamide 25. The pharmaceutical composition for preventing or treating a bone disease according to claim 24, which is a compound selected from the group and / or a pharmaceutically acceptable acid addition salt thereof.  28. The compound having Rho kinase inhibitory activity is (+)-trans-4- (1-aminoethyl) 1-1- (4-pyridylcarbamoyl) cyclohexane and / or a pharmaceutically acceptable acid addition salt thereof. 25. The pharmaceutical composition for prevention and treatment of bone disease according to claim 24. 29. The pharmaceutical composition according to any one of claims 24 to 28, wherein the exogenous osteoinductive factor is recombinant human BMP-2. 30. The pharmaceutical composition for prevention of bone disease according to any one of claims 24 to 29, wherein the bone disease is accompanied by damage, defect, or hypoplasia of bone or cartilage. 3 1. Pharmaceutically effective amount of a compound having Rho kinase inhibitory activity and ¾ medicinal イ:。 A method of preventing and treating bone diseases, which comprises administering to a patient an effective amount of an osteoinductive factor. 32. The compound according to claim 31, wherein the compound having Rho kinase inhibitory activity is a amide compound represented by the general formula (I), an isomer thereof and Z or a pharmaceutically acceptable acid addition salt thereof. How to prevent and treat bone disease.  33. The compound according to claim 31, wherein the compound having a Rh kinase inhibitory activity is an amide compound represented by the general formula (1 ′), an isomer thereof and Z or a pharmaceutically acceptable acid addition salt thereof. 32. A method for preventing or treating a bone disease according to 32.  34. Compounds with Rh kinase inhibitor activity are (+)-trans-4_ (1-aminoethyl) 1-1- (4-viridylcarbamoyl) cyclohexane, (+)-trans-N- (1 H-Pyro mouth [2,3-b] Pyridine-14-yl) 1-4- (1-Aminoethyl) cyclohexanecarboxamide, (R)-(+)-N- (4-pyridyl 1) 4- (1-aminoethyl) benzamide and (R) ― A compound selected from the group consisting of (+)-N- (1H-pyro [2,3-b] pyridine-14-yl) -14- (1-1 aminoethyl) benzamide and a compound selected from the group consisting of: 31. The method for preventing or treating a bone disease according to claim 31, which is a pharmaceutically acceptable acid addition salt. 35. When the compound having Rho kinase inhibitory activity is (+)-trans-41- (1-aminoethyl) -111- (4-viridylcarbamoyl) cyclohexane and / or its pharmaceutically acceptable acid addition 31. The method for preventing or treating bone disease according to claim 31, which is a salt.  36. The method for preventing or treating a bone disease according to any one of claims 31 to 35, wherein the osteoinductive factor is recombinant human BMP-2. 37. The method for preventing or treating a bone disease according to any one of claims 31 to 36, wherein the bone disease is accompanied by damage, loss or dysplasia of bone or cartilage. 38. Use of a compound having Rh kinase inhibitory activity and an osteoinductive factor for the production of a bone formation promoter.  39. The use according to claim 38, wherein the compound having a Rh kinase inhibitory activity is a amide compound represented by the general formula (I), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. .  40. The compound according to claim 38 or 3, wherein the compound having Rh kinase inhibition activity is an amide compound represented by the general formula (1 ′), an isomer thereof, and / or a pharmaceutically acceptable acid addition salt thereof. Use as described in 9.  4 1. The compound having R h0 kinase inhibitory activity is (10) 1-trans-1- (1-aminoethyl) -11- (4-pyridylcarbamoyl) cyclohexane, (+) 1-trans-1N— ( 1 H-Pyro mouth [2,3-b] Pyridine-14-yl) 1-4- (1-Aminoethyl) cyclohexane lipoxamide, (R)-(+) _ N- (4-Pyridyl)- 4— (1-Aminoethyl) benzamide and (R)-(+)-N- (1H—pyro mouth [2,3-b] pyridine-14-yl) 14- (1-aminoethyl) benzami 39. The use according to claim 38, which is a compound selected from the group consisting of amides and / or a pharmaceutically acceptable acid addition salt thereof. 42. Compounds having Rh kinase inhibition activity include (+)-trans-4- (1-aminoethyl) 1-1- (4-pyridylcarbamoyl) cyclohexane and Z or a pharmaceutically acceptable acid addition salt thereof. The use according to claim 38. 43. Use according to any one of claims 38 to 42, wherein the osteoinduction factor is recombinant human BMP-2.  44. The use according to any one of claims 38 to 43, wherein the osteogenesis promoting agent is for preventing or treating a bone disease.  45. The use according to claim 44, wherein said bone disease is associated with bone, cartilage damage, defect or dysplasia. 46. Use of a compound having R h0 kinase inhibitory activity for the manufacture of an osteogenic induction enhancer characterized by enhancing the osteoinductive effect of an exogenous osteoinductive factor. 47. The use according to claim 46, wherein the compound having Rho kinase inhibitory activity is a amide compound represented by the general formula (I), an isomer thereof, or a pharmaceutically acceptable acid addition salt thereof.  48. The compound according to claim 46 or 47, wherein the compound having Rho kinase inhibitory activity is an amide compound represented by the general formula (I ′), an isomer thereof and / or a pharmaceutically acceptable acid addition salt thereof. Use of.  49. Compounds with Rh kinase inhibition activity are (+)-trans-41- (1-aminoethyl) 1-1- (4-viridylcarbamoyl) cyclohexane, (+)-trans-N- (1 H-Virolo [2,3-b] pyridine-1-41) 1-41- (1-aminoethyl) cyclohexanecarboxamide, (R) — (+)-N- (4-pyridyl) 1-4 From (1—aminoethyl) penzamide and (R) — (+) — N— (1H—pyro mouth [2,3_b] pyridine-14-yl) 1-4- (11-aminoethyl) benzamide 47. The use according to claim 46, which is a compound selected from the group consisting of: and / or a pharmaceutically acceptable acid addition salt thereof. 50. The compound having Rh kinase inhibition activity is (+)-trans-41- (1-aminoethyl) -1-1- (4-viridylcarbamoyl) cyclohexane and / or a pharmaceutically acceptable acid addition thereof. 47. The use according to claim 46, which is a salt. 5 1. Use according to any of claims ffl46-50, wherein the exogenous osteoinductive factor is recombinant human BMP-2. 52. The use according to any one of claims 46 to 51, wherein the osteogenesis-inducing enhancer is for preventing or treating a bone disease.  53. The use according to claim 52, wherein the bone disease is associated with bone, cartilage damage, defect or dysplasia.  54. A pharmaceutical composition for preventing or treating a bone disease according to any one of claims 17 to 30, and the pharmaceutical composition can be used or should be used for the prevention and treatment of a bone disease. Commercial package containing documents stating