HK1164170A - Prophylactic or therapeutic agent for diseases associated with abnormal bone metabolism - Google Patents

Description

Preventive or therapeutic agent for diseases caused by abnormal bone metabolism

Technical Field

The present invention relates to a therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism, particularly to a prophylactic or therapeutic agent for osteoporosis, and more particularly to a therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism, particularly osteoporosis, which comprises an N-hydroxybenzamidine derivative represented by formula (I) or a salt thereof and active vitamin D or a prodrug thereof as active ingredients.

Background

The N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof has a bone resorption inhibitory effect and a bone formation promoting effect, and is expected as a therapeutic or prophylactic agent for osteoporosis. N-hydroxybenzamidine derivatives represented by the formula (I) or salts thereof are disclosed in patent documents 1, 2, 3 or 4.

One of the N-hydroxybenzamidine derivatives represented by the formula (I), namely N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } -benzamidine represented by the formula (II) or a salt thereof has a bone resorption inhibitory effect and a bone formation promoting effect, and is expected as a therapeutic or prophylactic agent for osteoporosis.

N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } -benzamidine or its salt is disclosed in patent document 4 and non-patent document 1.

Active vitamin D or a prodrug thereof is used as a bone-activating drug for regulating bone metabolism and is used as a therapeutic agent for diseases based on abnormal calcium metabolism, such as osteoporosis (non-patent document 2).

Currently, there are a variety of osteoporosis therapeutics available. However, the combined use of these drugs does not produce the same therapeutic effect. For example, it is known that the therapeutic effects in the combination of PTH (Parathyroid Hormone; Parathyroid Hormone) and certain bisphosphonates counteract each other. Although both are representative therapeutic agents for osteoporosis, there are reports that: as a result of preclinical tests and clinical tests, the efficacy of the combination administration was reduced as compared with the individual administrations (non-patent documents 3 and 4). In addition, as for active vitamin D or a prodrug thereof, there are also reports: in the combination with vitamin K having an effect of fixing calcium in bone, as with PTH and bisphosphonic acid described above, the efficacy of the combination administration is reduced in clinical trial results as compared with the case where each is administered alone (non-patent document 5).

From the above examples, it is not easy to find a more effective therapeutic method or prophylactic method using an existing or novel therapeutic agent for osteoporosis.

Patent document 1: japanese Kokai publication 2008-509134

Patent document 2: japanese Kohyo publication No. 2009-525321

Patent document 3: WO2009/017346 booklet

Patent document 4: japanese Kokai publication Hei-2004-537549

Non-patent document 1: plum 12477a, 12531 ウン, synthetic and Biological Activity of Natural products and Designed New Hybrid for the Treatment of LTB4 Related diseases, Proc. of university of Chaishan university, Proc. academic proceedings, 1999.8

Non-patent document 2: real mous in China, Junfu in pine, and surveilled by Gantumaoming, "flowers fade away と active type ビタミン D-to と cable in the future of そして - (bone metabolism and active type vitamin D-past and present, and future-)" published by ライフサイエンス, issued on 25/9/2006

Non-patent document 3: r Samadfam et al, Endocrinology, Vol.148, No. 6, 2007

Non-patent document 4: DM Black et al, The New England Journal of Medicine, volume 349, No. 13, 2003

Non-patent document 5: xiaolinqiayi, baimuzhenxiao, gaokangfu, "a major bone song for disease treatment に お け る ビタミン K merging (the current situation of vitamin K combination therapy in osteoporosis treatment)", CLINICAL calcum, No. 17, No. 11, 2007

Disclosure of Invention

The invention aims to: a therapeutic agent or prophylactic agent having a better effect than conventional agents on diseases caused by abnormal bone metabolism, particularly osteoporosis. The invention also aims to: a method for treating or preventing a disease caused by abnormal bone metabolism, particularly osteoporosis, which is more effective than conventional methods for treating or preventing the disease.

The present inventors have conducted intensive studies on ingredients effective as therapeutic or prophylactic agents for diseases caused by abnormal bone metabolism, particularly osteoporosis, and have found that by using specific drugs which can be used alone in combination, a drug effect which is remarkably superior to that obtained by using the drugs alone is obtained.

Namely, the present invention is as follows.

(1) A therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism, which comprises the following (a) and (b) as active ingredients.

(a) An N-hydroxybenzamidine derivative represented by the following formula (I) or a salt thereof.

(b) Active form of vitamin D or a prodrug thereof.

[ in the above-mentioned formula (I),

R¹represents: hydrogen; C1-C6 alkyl; C3-C6 cycloalkyl; C6-C12 aryl; a 5-12 membered ring heteroaryl group containing 1-3 heteroatoms selected from N, O and S; or NR⁵R⁶；

R²Represents: hydrogen; C1-C6 alkyl; C2-C6 alkenyl; C3-C6 cycloalkyl; an amino group; a C1-3 dialkylamino group; halogen, C3-C6 cycloalkyl, C6-C12 aryl, containing groups selected from N, O and S5-12 membered ring heteroaryl of 1-3 heteroatoms, C3-6 heterocycloalkyl-substituted C1-3 alkyl containing 1-3 heteroatoms selected from N, O and S; by NR⁷R⁸A substituted C1-3 alkyl group; or N-methylcarbamoyl;

R³and R⁴Independently represent: hydrogen; halogen; a hydroxyl group; c1-6 alkyl unsubstituted or substituted by halogen; c3-6 cycloalkylamino; c1-6 alkoxy; c1-6 alkanoyloxy; c2-6 alkenyloxy; phenyl-C1-6 alkoxy; a phenoxy group; c2-6 alkenoyloxy; phenyl-C1-6 alkanoyloxy; c3-6 cycloalkyloxy substituted with carboxyl, esterified carboxyl or amidated carboxyl; or aminooxy;

R⁵represents hydrogen or a C1-C6 alkyl group;

R⁶represents: hydrogen; C1-C6 alkyl; c1-3 alkyl substituted with hydroxy, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S;

R⁷represents: hydrogen; C1-C6 alkyl; or C3-C6 cycloalkyl;

R⁸represents: hydrogen; C1-C4 alkyl; c1-3 alkyl substituted with C1-3 alkoxy; or 4-pyridylcarbonyl;

X¹represents-O-, -S-, -NH-or-N (C1-6 alkyl) -;

X²represents a C3-7 alkylene group, a C3-7 alkylenecarbonyl group, or a C1-3 alkylene-phenylene-C1-3 alkylene group;

X³represents-O-, -S-, -NH-or-N (C1-6 alkyl) -.]

(2) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to (1),

1) when R is¹When the alkyl is C1-C6,

R²represents: C1-C6 alkyl; c2-a C6 alkenyl group; C3-C6 cycloalkyl; an amino group; a C1-3 dialkylamino group; c1-3 alkyl substituted with halogen, C3-C6 cycloalkyl, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S; or by NR⁷R⁸A substituted C1-3 alkyl group;

2) when R is¹When the alkyl is C3-C6 cycloalkyl,

R²represents a C1-C6 alkyl group or is NR⁷R⁸A substituted C1-3 alkyl group;

3) when R is¹When the aryl group is a C6-C12 aryl group or a 5-12 membered ring heteroaryl group containing 1-3 heteroatoms selected from N, O and S,

R²represents a C1-C3 alkyl group substituted with a halogen, a C3-C6 cycloalkyl group, a C6-C12 aryl group, a 5-12 membered ring heteroaryl group containing 1-3 heteroatoms selected from N, O and S, or a C3-6 heterocycloalkyl group containing 1-3 heteroatoms selected from N, O and S;

4) when R is¹Is NR⁵R⁶When the temperature of the water is higher than the set temperature,

R²represents: C1-C6 alkyl; c3-6 cycloalkyl; C1-C3 alkyl substituted with halogen, C3-C6 cycloalkyl, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S; or N-methylcarbamoyl.

(3) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to (1) or (2),

1) when R is⁵In the case of hydrogen, the acid is,

R⁶represents hydrogen or a C1-C6 alkyl group;

2) when R is⁵When the alkyl is C1-C6,

R⁶represents: C1-C6 alkyl; selected from hydroxyl, C6-C12 aryl, containing a group selected from N, O and S5-12 membered ring heteroaryl having 1-3 hetero atoms and C1-3 alkyl substituted with a C3-6 heterocycloalkyl group having 1-3 hetero atoms selected from N, O and S.

(4) (1) the therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (3),

1) when R is⁷In the case of hydrogen, the acid is,

R⁸represents: C1-C4 alkyl; c1-3 alkyl substituted with C1-3 alkoxy or C3-6 heterocycloalkyl;

2) when R is⁷When the alkyl is C1-C6,

R⁸represents a C1-C4 alkyl group;

3) when R is⁷When the alkyl is C3-C6 cycloalkyl,

R⁸represents a 4-pyridineformyl group.

(5) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (4), wherein R¹Represents hydrogen, methyl, ethyl, propyl, isopropyl, cyclohexyl, phenyl, 3-pyridyl or NR⁵R⁶。

(6) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (5), wherein R²Represents: hydrogen; a methyl group; an ethyl group; propyl; isopropyl group; a butyl group; a vinyl group; a cyclopentyl group; an amino group; a dimethylamino group; by chlorine, pentyl, phenyl, 1-imidazolyl, 4-thiomorpholinyl, 4-morpholinyl, NR⁷R⁸Substituted methyl or ethyl; or methyl or ethyl substituted by N-methylcarbamoyl.

(7) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (6), wherein R³Is hydrogen.

(8) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (7), wherein R⁴Is hydrogen or fluorine.

(9) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (8), wherein R⁵Is hydrogen, methyl, ethyl, propyl or isopropyl.

(10) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (9), wherein R⁶Represents: hydrogen; a methyl group; an ethyl group; propyl; isopropyl group; or methyl or ethyl substituted by hydroxy, phenyl, 3-pyridyl or 4-morpholinyl.

(11) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (10), wherein R⁷Is hydrogen, methyl, ethyl or cyclopropyl.

(12) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (11), wherein R⁸Is hydrogen, methyl, ethyl, isopropyl, 3-isopropoxypropyl or 4-picolinoyl.

(13) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (12), wherein X¹is-O-;

X²is butylene or pentylene;

X³is-O-, -NH-or-N (CH)₃)-。

(14) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (13), wherein,

X¹-X²-X³comprises the following steps:

(15) the therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to (1), wherein the N-hydroxybenzamidine derivative represented by the formula (I) is any one of the following compounds.

1) N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

2) N-hydroxy-4- {5- [4- (5-benzyl-2- (pyridin-3-yl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

3) N-hydroxy-4- {5- [4- (5- (2-chloroethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

4) N-hydroxy-4- {5- [4- (5-cyclopentylmethyl-2-ethyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

5) N-hydroxy-4- {5- [4- (5-vinyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

6) N-hydroxy-4- {5- [4- (5-cyclopentylmethyl-2-methylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

7) N-hydroxy-4- {5- [4- (2- (2-hydroxyethyl) methylamino-5-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

8) N-hydroxy-4- {5- [4- (2-benzylmethylamino-5-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

9) N-hydroxy-4- {5- [4- (5-methyl-2- (methyl (pyridin-3-ylmethyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

10) N-hydroxy-4- {5- [4- (2-benzylmethylamino-5-ethyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

11) N-hydroxy-4- {5- [4- (2-dipropylamino-5-ethyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

12) N-hydroxy-4- {5- [4- (5-isopropyl-2- (methyl (pyridin-3-yl-methyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

13) N-hydroxy-4- {5- [4- (5-butyl-2- (methyl (2- (4-morpholino) ethyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

14) N-hydroxy-4- {5- [4- (5-cyclopentylmethyl-2-dipropylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

15) N-hydroxy-4- {5- [4- (5-cyclopentyl-2- (methyl (2- (4-morpholino) ethyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

16) N-hydroxy-4- {5- [4- (5-isopropyl-2-dipropylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

17) N-hydroxy-4- {5- [4- (5-isopropyl-2-dimethylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

18) N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentanoylamino } benzamidine

19) N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentylmethylamino } benzamidine

20) N-hydroxy-2-fluoro-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

21) N-hydroxy-4- {4- [4- (2-cyclohexyl-5-ethyl-1, 3-thiazol-4-yl) phenoxy ] butoxy } benzamidine

22) N-hydroxy-4- {3- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxymethyl ] benzyloxy } benzamidine

23) N-hydroxy-4- {5- [4- (5- (2- (imidazol-1-yl) ethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

24) N-hydroxy-4- {5- [4- (5- (2- (isopropylamino) ethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

25) N-hydroxy-4- {5- [4- (5- (2- (3- (isopropoxy) propylamino) ethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

26) N-hydroxy-4- {5- [4- (5- (2-diethylaminoethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

27) N-hydroxy-4- {5- [4- (2-isopropyl-5- (2- (thiomorpholin-4-yl) ethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

28) N-hydroxy-4- {5- [4- (2-cyclohexyl-5- (2- (dimethylamino) ethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

29) N-hydroxy-4- {5- [4- (2-dimethylamino-5- (2- (morpholin-4-yl) ethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

30) N-hydroxy-4- {5- [4- (5- (2- (cyclopropyl (pyridine-4-carbonyl) amino) ethyl) -2-isopropyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

31) N-hydroxy-4- {5- [4- (2-amino-5- (N-methylcarbamoyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

32) N-hydroxy-4- {5- [4- (5- (imidazol-1-ylmethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

33) N-hydroxy-4- {5- [4- (2-ethylmethylamino-5- (morpholin-4-ylmethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

34) N-hydroxy-4- {5- {4- (2- (methyl (2- (morpholin-4-yl) ethyl) amino) -5- (thiomorpholin-4-yl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

35) N-hydroxy-4- {5- [4- (5-dimethylamino-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

(16) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to (1), wherein the N-hydroxybenzamidine derivative represented by formula (I) is N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine.

(17) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (16), wherein the disease caused by abnormal bone metabolism is a disease caused by abnormal bone resorption.

(18) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism as described in any one of (1) to (16), wherein the disease caused by abnormal bone metabolism is a disease caused by abnormal bone formation.

(19) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (16), wherein the disease caused by abnormal bone metabolism is osteoporosis.

(20) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (19), wherein (b) is 1 α, 25-dihydroxyvitamin D₃1 alpha, 24R-dihydroxy vitamin D₃Or 1 alpha-hydroxy vitamin D₃。

(21) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (19), wherein (b) is 1 α, 25-dihydroxyvitamin D₃Or 1 alpha-hydroxy vitamin D₃。

(22) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (21), wherein (a) and (b) constitute a mixture.

(23) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (21), wherein (a) and (b) are each independently a single agent.

(24) The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of (1) to (21), which is a kit comprising (a) and (b).

The present invention also relates to a method for treating or preventing a disease caused by abnormal bone metabolism, particularly osteoporosis, characterized in that: administering a medicament containing the above (a) and (b) as active ingredients.

Effects of the invention

The present invention relates to a therapeutic or prophylactic agent for diseases caused by abnormal bone metabolism, particularly osteoporosis, which comprises both an N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof and active vitamin D or a prodrug thereof as active ingredients. The present invention also relates to a method for treating or preventing a disease caused by abnormal bone metabolism, particularly osteoporosis, characterized in that: the above drugs are administered. By administering the two active ingredients in combination, a remarkably strong therapeutic or prophylactic effect can be obtained, as compared with the case where the N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof and the active vitamin D or a prodrug thereof are used alone.

Drawings

FIG. 1 shows the use of N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy]Pentoxy } -benzamidine (hereinafter referred to as Compound 1) solution, 1. alpha., 25-dihydroxy vitamin D₃Solution, dimethyl sulfoxide (hereinafter referred to as DMSO) (preparation solvent of Compound 1 solution), and ethanol (1. alpha., 25-dihydroxyvitamin D)₃Solution preparation solvent) of each two of the rat osteosarcoma cell lines (ROS17/2.8 cells) were jointly stimulated.

FIG. 2 shows the use of N-hydroxy-4-5- [4- (2-dipropylamino-5-ethyl-1, 3-thiazol-4-yl) phenoxy]Pentoxy-benzamidine (hereinafter referred to as compound 11) solution, 1 alpha, 25-dihydroxy vitamin D₃Solution, dimethyl sulfoxide (hereinafter referred to as DMSO) (preparation solvent of Compound 11 solution), and ethanol (1. alpha., 25-dihydroxyvitamin D)₃Solution preparation solvent) of each two of the rat osteosarcoma cell lines (ROS17/2.8 cells) were jointly stimulated.

Detailed Description

The N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof used in the present invention has an excellent bone resorption inhibitory effect and bone formation promoting effect, and is expected as a therapeutic or prophylactic agent for diseases caused by abnormal bone metabolism, particularly osteoporosis.

N-hydroxybenzamidine derivatives represented by the formula (I) are described in patent documents 1, 2 or 3 and non-patent document 1, and can be prepared according to the descriptions of these patent documents and the like.

In the present invention, the N-hydroxybenzamidine derivative represented by the formula (I) is the following compound.

[ in the above-mentioned formula (I),

R¹represents: hydrogen; C1-C6 alkyl; C3-C6 cycloalkyl; C6-C12 aryl; a 5-12 membered ring heteroaryl group containing 1-3 heteroatoms selected from N, O and S; or NR⁵R⁶；

R²Represents: hydrogen; C1-C6 alkyl; C2-C6 alkenyl; C3-C6 cycloalkyl; an amino group; a C1-3 dialkylamino group; c1-3 alkyl substituted with halogen, C3-C6 cycloalkyl, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S; by NR⁷R⁸A substituted C1-3 alkyl group; or N-methylcarbamoyl;

R³and R⁴Independently represent: hydrogen; halogen; a hydroxyl group; c1-6 alkyl unsubstituted or substituted by halogen; c3-6 cycloalkylamino; c1-6 alkoxy; c1-6 alkanoyloxy; c2-6 alkenyloxy; phenyl-C1-6 alkoxy; a phenoxy group; c2-6 alkenoyloxy; phenyl-C1-6 alkanoyloxy; c3-6 cycloalkyloxy substituted with carboxyl, esterified carboxyl or amidated carboxyl; or aminooxy;

R⁵is hydrogen or C1 ℃A C6 alkyl group;

R⁶represents: hydrogen; C1-C6 alkyl; c1-3 alkyl substituted with hydroxy, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S;

R⁷is hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl;

R⁸hydrogen, C1-C4 alkyl, C1-3 alkyl substituted by C1-3 alkoxy, or 4-picolinoyl;

X¹is-O-, -S-, -NH-or-N (C1-6 alkyl) -;

X²is a C3-7 alkylene group, a C3-7 alkylenecarbonyl group or a C1-3 alkylene-phenylene-C1-3 alkylene group;

X³is-O-, -S-, -NH-or-N (C1-6 alkyl) -.]

Furthermore, R¹Preferably hydrogen, methyl, ethyl, propyl, isopropyl, cyclohexyl, phenyl, 3-pyridyl or NR⁵R⁶(ii) a Further preferred is methyl, ethyl, isopropyl, cyclohexyl, 3-pyridyl or NR⁵R⁶。

R²Preferably: hydrogen; a methyl group; an ethyl group; propyl; isopropyl group; a butyl group; a vinyl group; a cyclopentyl group; an amino group; a dimethylamino group; by chlorine, pentyl, phenyl, 1-imidazolyl, 4-thiomorpholinyl, 4-morpholinyl or NR⁷R⁸Substituted methyl or ethyl; or methyl or ethyl substituted by N-methylcarbamoyl; still more preferably: a methyl group; an ethyl group; isopropyl group; a butyl group; a vinyl group; a cyclopentyl group; a dimethylamino group; by chlorine, pentyl, phenyl, 1-imidazolyl, 4-thiomorpholinyl, 4-morpholinyl or NR⁷R⁸Substituted methyl or ethyl; or methyl or ethyl substituted by N-methylcarbamoyl.

R³Hydrogen is preferred.

R⁴Hydrogen or fluorine are preferred.

R⁵Hydrogen, methyl, ethyl, propyl or isopropyl are preferred, and hydrogen, methyl, ethyl or propyl are more preferred.

R⁶Preferably: hydrogen; a methyl group; an ethyl group; propyl; isopropyl group; or methyl or ethyl substituted by hydroxy, phenyl, 3-pyridyl or 4-morpholinyl; still more preferably: hydrogen; a methyl group; an ethyl group; propyl; or methyl or ethyl substituted by hydroxy, phenyl, 3-pyridyl or 4-morpholinyl.

R⁷Hydrogen, methyl, ethyl or cyclopropyl are preferred.

R⁸Preference is given to hydrogen, methyl, ethyl, isopropyl, 3-isopropoxypropyl or 4-pyridineformyl.

X¹preferably-O-;

X²preferably butylene, pentylene;

X³preferably-O-, -NH-or N (CH)₃)-。

And, X¹-X²-X³Preference is given to

In particular, the N-hydroxybenzamidine derivatives represented by the formula (I) are preferably the derivatives shown in table 1 or salts thereof. Among them, N-hydroxy-4-5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy-benzamidine of Compound No. 1 is particularly preferred.

The above compounds have excellent bone resorption inhibitory effects and bone formation promoting effects, and are expected to be useful as therapeutic or prophylactic agents for diseases caused by abnormal bone metabolism, particularly osteoporosis, in combination with active vitamin D or a prodrug thereof, and agents containing both as active ingredients are effective as therapeutic or prophylactic agents for diseases caused by abnormal bone metabolism, particularly osteoporosis.

[ Table 1]

The dehydroxylation matrix of the N-hydroxybenzamidine derivative represented by formula (I) or a salt thereof, i.e., the benzamidine derivative or a salt thereof, is also excellent in bone resorption inhibitory effect and bone formation promoting effect, and is expected to be used as a therapeutic or prophylactic agent for diseases caused by abnormal bone metabolism, particularly osteoporosis, in combination with active vitamin D or a prodrug thereof, and a drug containing both as active ingredients is effective as a therapeutic or prophylactic agent for diseases caused by abnormal bone metabolism, particularly osteoporosis.

As the salt of the N-hydroxybenzamidine derivative represented by the formula (I), there is a pharmaceutically acceptable salt using an inorganic acid (hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid) or an organic acid (citric acid, acetic acid, lactic acid, tartaric acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, methanesulfonic acid, ethanesulfonic acid, benzoic acid, maleic acid, gluconic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, pamoic acid, glutamic acid or aspartic acid). In the present invention, as the inorganic acid, hydrochloric acid is preferably used; as the organic acid, methanesulfonic acid or ethanesulfonic acid is preferably used.

The dosage of the N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof is an amount effective for the prevention or treatment of a disease caused by abnormal bone metabolism, particularly osteoporosis, and cannot be generally determined depending on the age, body weight, type of combination therapy, frequency of treatment, type of desired effect, administration method, and the like of a patient, but may be an amount generally administered when used as a therapeutic agent or a prophylactic agent.

The active form of vitamin D or a prodrug thereof used in the present invention is not particularly limited as long as it has affinity with a vitamin D receptor and exhibits a physiological action based on the affinity. Examples of active forms of vitamin D are: 1 alpha, 25-dihydroxyvitamin D₃(calcitriol), 1 alpha, 24R-dihydroxy vitamin D₃(tacalcitol), 2 beta- (3-hydroxypropoxy) -1 alpha, 25-dihydroxyvitamin D₃(Elcalcitol, ED-71), 1 alpha, 25-dihydroxy-2-methylene-19-nor-vitamin D₃(2MD), 1 alpha, 25-dihydroxy-22-oxavitamin D₃(Maxacalcidol, OCT), 1 alpha, 24S-dihydroxy-22, 23:26, 27-Tetradehydro-vitamin D₃(calcipotriol, MC903), 1 alpha, 25-dihydroxy-26, 26, 26, 27, 27, 27-hexafluorovitamin D₃(Fluorocalciferol), (23E) -1 α -fluoro-25-hydroxy-16, 17, 23, 24-tetrahydro-26, 27-bishomo (bishomo) -20-epi-vitamin (epivitamin) D₃(elacalcitol, BXL-628), 19-nor-1 alpha, 25-dihydroxyvitamin D₂(paricalcitol), and the like; prodrug of active vitamin DExamples are: 1 alpha-hydroxy vitamin D₃(alpha-calciferol) 1 alpha-hydroxyvitamin D₂(doxercalciferol, ドキサカルシフエロ - ル, doxercalciferol), and the like.

As the active form of vitamin D or a prodrug thereof in the present invention, α -calciferol is preferable.

In the present invention, when the N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof is used in combination with active vitamin D or a prodrug thereof, one of the compounds may be used, or two or more of them may be used in combination at an arbitrary ratio. It is preferable to use one compound selected from the above-mentioned compounds in combination.

The term "prodrug" as used herein refers to any compound that undergoes hydrolysis, metabolism, derivatization, etc. in vivo to produce an active compound having a desired activity. Furthermore, the above prodrugs may be compounds which are converted into the active compounds under physiological conditions. Also, among vitamin D, it is known, for example, as in 1 alpha-hydroxy vitamin D₃It is found in (alpha-calciferol) that in vivo, mainly in the liver, 25-position is subjected to hydrogen oxidation and converted into the active compound, 1 alpha, 25-dihydroxyvitamin D₃The effect on bone metabolism is shown in the form of active vitamin D. Prodrugs also include 1 alpha-hydroxy vitamin D as such prior to receiving a hydrogen oxidation₃Such compounds (Shu "medicine and medicine having an open skeleton" No. 7 "character string 35336", 185 nd and 186 th pages, Guanchuan bookshop, 1990).

Certain active forms of vitamin D or prodrugs thereof may form salts. As the salt of an active form of vitamin D or a prodrug thereof used in the present invention, there can be mentioned: pharmaceutically acceptable salts of inorganic acids (hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid), organic acids (citric acid, acetic acid, lactic acid, tartaric acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, methanesulfonic acid, benzoic acid, maleic acid, gluconic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galactaric acid, pamoic acid, glutamic acid or aspartic acid), inorganic bases (alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, metal salts such as aluminum and zinc) or organic bases (ammonia, triethylamine, diethylamine, ethylenediamine, propylenediamine, pyrrolidine, piperidine, piperazine, pyridine, lysine, choline, ethanolamine, diethanolamine, N-dimethylethanolamine, 4-hydroxypiperidine, glucosamine and N-methylglucamine) are used.

The therapeutic or prophylactic agent for osteoporosis of the present invention can provide a drug having an effect of improving bone density and/or bone strength of a disease caused by abnormal bone metabolism, particularly a therapeutic agent for osteoporosis, by using two active vitamin D or a prodrug thereof and an N-hydroxybenzamidine derivative represented by formula (I) or a salt thereof as active ingredients in combination. In addition, the drug of the present invention has low toxicity and excellent stability. By using the combination, the dosage of each effective component can be reduced as compared with the single dosage.

The dose of active vitamin D or a prodrug thereof is an amount effective for the treatment or prevention of a disease caused by abnormal bone metabolism, particularly osteoporosis, and cannot be generally determined depending on the age, body weight, type of combination therapy, frequency of treatment, type of desired effect, administration method, and the like of a patient.

The therapeutic agent or prophylactic agent, or the therapeutic method or prophylactic method for a disease caused by abnormal bone metabolism, particularly osteoporosis, of the present invention may contain, as active ingredients, an N-hydroxybenzamidine derivative represented by formula (I) or a salt thereof, and active vitamin D or a prodrug thereof. The two active ingredients may be mixed and administered simultaneously, or may be administered separately and simultaneously or continuously, or separately at regular intervals. When administration is not simultaneous, for example, two active ingredients may be administered alternately, or one active ingredient may be administered sequentially and then the other active ingredient may be administered.

The drug of the present invention may be in any form as long as it contains, as active ingredients, the N-hydroxybenzamidine derivative represented by formula (I) or a salt thereof and active vitamin D or a prodrug thereof. For example, a mixture containing two active ingredients may be constituted, or each may be constituted in the form of a single dose. Here, the mixture refers to a preparation in which two or more active ingredients are mixed in one preparation, and the single agent refers to a preparation in which one active ingredient is contained in one preparation. In the case of a single dose form, when a plurality of active vitamin D or a prodrug thereof are used, each of the active vitamin D and the prodrug thereof may be used as a single dose or a mixture, but the active vitamin D and the prodrug thereof are preferably used as single doses.

When the two active ingredients are used as a single agent in the present invention, the therapeutic agent or prophylactic agent, or the therapeutic method or prophylactic method refers to an agent or method in which single agents that can be used alone are combined. Thus, the medicament containing the two active ingredients may each be in a different dosage form. For example, each preparation form may be a solid and a solid, or a liquid and a liquid, and may be a solid and a liquid, and is not particularly limited. When the two active ingredients are single agents, the two formulations may be brought into a state of being integrated, i.e., a form of a kit. A representative form of the kit includes a blister pack (blister pack) in which two kinds of preparations corresponding to a specific period (for example, 1 week or more) are collectively packed in one sheet according to a series of administration schedules. The packaging may be performed by PTP or the like at the end of the preparation production, or the packaging may be performed in the same bag at the time of prescription in a hospital, a pharmacy, or the like, and is not particularly limited.

As the mixture, for example, two kinds of active ingredients are combined in appropriate amounts to exert their respective effects, and the mixture is prepared into dosage forms such as tablets, capsules, and liquid preparations. In this case, the time for combining the two active ingredients to prepare a mixture may be at the stage of manufacturing the mixture preparation, or may be just before administration. When mixing is performed in the production stage, for example, appropriate amounts of the respective active ingredients may be mixed, followed by molding or mixing. The molding method is not particularly limited, and for example, various formulations may be mixed, or layers may be laminated. When the active ingredients are prepared as a mixture just before administration, for example, the active ingredients are stored in a state independent of each other until just before administration, and liquid preparations are mixed at the time of administration, or solid preparations such as tablets, pills, granules, powders, or capsules are dissolved in liquid preparations, or solid preparations such as granules or powders are mixed with each other. The mixing may be carried out by hand as a method of mixing just before administration, or a package obtained by simply mixing the two preparations by cutting, pulling, tearing, pulling, or the like may be used. The form of the mixture is as follows: tablet, pill, granule, powder, solution, suspension, syrup or capsule.

The therapeutic agent or prophylactic agent for diseases caused by abnormal bone metabolism, particularly osteoporosis, of the present invention may be administered daily or intermittently, and the number of administration per day may be 1 or 2 to 3. When the two active ingredients are each a single dose, the administration frequency may be the same or different. When the two formulations are single doses, the number of times each is usually administered may be combined. Even when two preparations are administered at different times, convenience can be expected to be improved by making the preparations in the form of a kit such as a blister pack.

The N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof, and the active vitamin D or a prodrug thereof of the present invention can be prepared into a preparation by a known method using only the active ingredients thereof or using the active ingredients together with appropriate additives described below. Specific examples of such dosage forms are: oral preparations such as soft capsule, hard capsule, tablet, syrup, etc.; injection or topical preparation.

The preparation containing the active ingredient of the present invention is prepared using additives generally used for the preparation of preparations. Examples of such additives include excipients such as lactose, sucrose, glucose, corn starch, potato starch, crystalline cellulose, light silicic anhydride, synthetic aluminum silicate, magnesium aluminum silicate, and calcium hydrogen phosphate in the case of solid preparations; binders such as crystalline cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone; disintegrants such as starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, and sodium carboxymethyl starch; lubricants such as talc and stearic acids; coating agents such as hydroxymethylpropylcellulose, hydroxypropylmethylcellulose phthalate, and ethylcellulose; a colorant; in the case of semisolid preparation, there are white vaseline and other matrix; in the case of liquid preparation, there are solvent such as ethanol, cosolvent such as ethanol, preservative such as parabens (パラオキシ benzoic acid エステル like), and penetrant such as glucose; buffers such as citric acids, antioxidants such as L-ascorbic acid, chelating agents such as EDTA, suspending agents such as polysorbate 80, and emulsifiers.

In addition, the diseases caused by abnormal bone metabolism in the present invention include not only osteoporosis but also diseases caused by abnormal bone resorption or diseases caused by decreased bone formation in bone metabolism. Examples of diseases caused by reduced bone formation include bone formation insufficiency, rickets, osteomalacia, diabetic osteoporosis classified as secondary osteoporosis, and glucocorticoid-induced osteoporosis. In addition, as a disease in which a significant therapeutic effect or preventive effect is expected by further promoting bone formation, there are fractures including delayed fracture healing (refractory fractures and pseudoarthrosis) and the like. Diseases caused by abnormal bone resorption include: rheumatic arthritis, osteoarthritis deformans, hypercalcemia, alveolar bone loss, renal osteodystrophy, osteolytic tumors, metastatic tumors, and the like. Osteoporosis is particularly preferred as a disease caused by abnormal bone metabolism in the present invention.

Examples

Example 1

Study of N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy]Pentoxy- Benzamidine (Compound 1) and 1 α, 25-dihydroxy vitaminD ₃ Cell proliferation on ROS17/2.8 cells Effect of reproduction (FIG. 1, Table 2)

ROS17/2.8 cells at 2X 10⁴Density of individual/well was seeded in 96-well plates. As the medium, an α MEM medium supplemented with 10% fetal calf serum (hereinafter referred to as 10% FCS- α MEM) was used. After overnight culture, the medium was removed from each well, and then α MEM medium supplemented with 0.5% fetal calf serum (hereinafter referred to as 0.5% FCS — α MEM) was added thereto. After culturing for about 6 hours, the medium was removed from each well, and DMSO and ethanol (hereinafter referred to as solution 1), a compound 1 solution and ethanol (hereinafter referred to as solution 2), and 1 α, 25-dihydroxyvitamin D were added thereto₃Solution and DMSO (hereinafter referred to as solution 3), or a solution of Compound 1 and 1 α, 25-dihydroxyvitamin D₃10% FCS-. alpha.MEM of the solution (hereinafter referred to as solution 4). The 10% FCS-. alpha.MEM containing the solution 1 was prepared by diluting DMSO and ethanol 1000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 2 was prepared by mixing 10^-5The compound 1 solution at a concentration of M and ethanol were diluted 1000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 3 was prepared by mixing 10^-41 alpha, 25-dihydroxyvitamin D at a concentration of M₃The solution and DMSO were diluted 1000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 4 was prepared by mixing 10^-5Compound 1 solution and 10 at concentration of M^-41 alpha, 25-dihydroxyvitamin D at a concentration of M₃The solution was diluted 1000-fold with 10% FCS-. alpha.MEM. After culturing for about 24 hours, Compound 1 and 1 α, 25-dihydroxyvitamin D were evaluated by the Amersham Cell Proliferation Biotrak ELISA System (2 nd edition) (product of GE ヘルスケアバイオサイエンス, accession No. RPN250)₃Effect on cell proliferation of ROS17/2.8 cells. That is, 5-bromo-2' -deoxyuridine (BrdU) was taken into proliferating cells and detected by ELISA using peroxidase-labeled anti-BrdU antibody. BrdU uptake into cells was quantified by measuring absorbance (450nm) with a microplate reader.

The results are shown in FIG. 1. The BrdU intake amount in each solution addition group was determined as a percentage of the average value of 4 wells relative to the BrdU intake amount in the solution 1 addition group. The figure is expressed as mean ± standard error of 4 wells for each solution addition group. There was no significant difference in cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 2 compared to ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 1 (n.s., Dunnett's multiple comparison test). Furthermore, cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 3 was also not significantly different from cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 1 (n.s., Dunnett's multiple comparison test). On the other hand, cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 4 was significantly increased compared to cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 1 (multiple comparative test of x: p < 0.01, Dunnett). That is, it is clear that: with only compound 1 and 1 alpha, 25-dihydroxyvitamin D₃The combined stimulation of (2) can enhance the cell proliferation of ROS17/2.8 cells.

Study of Compound 1 and 1 α, 25-dihydroxyvitamin D Using the method of bury (Gentianjilang, Xiaoze gloss, eds. "Pharmacology", pp. 18-19, Nanshan Tang, 1987)₃Synergistic effect on ROS17/2.8 cell proliferation, results are shown in Table 2. In the method of bury, the value of each position group is divided by the value of the reference group to calculate a relative index. Comparing the product of the relative indexes of the individual treatment groups with the relative index of the group actually treated in combination, and judging that the synergistic effect is achieved when the value of the group treated in combination is excellent; when the two are equal, the two are judged to have an addition effect. On the other hand, when the values of the treatment groups were different, the treatment groups were judged to have antagonistic action.

[ Table 2]

A relative index of BrdU intake of each solution-added group was calculated with the BrdU intake of 1.000 when solution 1 was added. Combined addition of Compound 1 and 1 alpha, 25-dihydroxyvitamin D₃The relative index (1.596) of the group (a) was larger than the product (1.233) of the relative indices of the individual addition groups, and a synergistic effect was observed when the two groups were used in combination.

Example 2

Investigation of N-hydroxy-4-5- [4- (2-dipropylamino-5-ethyl-1, 3-thiazol-4-yl) phenoxy]Pentaoxo Yl-benzamidine (Compound 11) and 1 α, 25-dihydroxy vitamin D ₃ Cells specific for ROS17/2.8 Effect of cell proliferation (FIG. 2, Table 3)

ROS17/2.8 cells at 5X 10³The density of individual/well was seeded on 96-well plates. The culture medium used was 10% FCS-. alpha.MEM. After overnight incubation, the medium was removed from each well and 0.5% FCS-. alpha.MEM was added. After culturing for about 6 hours, the medium was removed from each well, and DMSO and ethanol (hereinafter referred to as solution 1), a compound 11 solution and ethanol (hereinafter referred to as solution 5), and 1 α, 25-dihydroxyvitamin D were added₃Solution and DMSO (hereinafter referred to as solution 3), or a solution of Compound 11 and 1 α, 25-dihydroxyvitamin D₃10% FCS-. alpha.MEM of the solution (hereinafter referred to as solution 6). The 10% FCS-. alpha.MEM containing solution 1 was prepared by diluting DMSO and ethanol 1000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 5 was prepared by mixing 10^-5The compound 11 solution at a concentration of M and ethanol were diluted 1000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 3 was prepared by mixing 10^-41 alpha, 25-dihydroxyvitamin D at a concentration of M₃The solution and DMSO were diluted 1000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 6 was prepared by mixing 10^-5Compound 11 solution and 10 at concentration of M^-41 alpha, 25-dihydroxyvitamin D at a concentration of M₃The solution was diluted 1000-fold with 10% FCS-. alpha.MEM. About culturingAfter 24 hours, Compound 11 and 1 α, 25-dihydroxyvitamin D were evaluated using Amersham Cell Proliferation BiotrakELISA System (2 nd edition) (product of GE ヘルスケアバイオサイエンス, accession No. RPN250)₃Effect on cell proliferation of ROS17/2.8 cells. That is, 5-bromo-2' -deoxyuridine (BrdU) was taken into proliferating cells and detected by ELISA using peroxidase-labeled anti-BrdU antibody. BrdU uptake into cells was quantified by measuring absorbance (450nm) with a microplate reader.

The results are shown in FIG. 2. The BrdU intake amount of each solution addition group was determined as a percentage of the average value of 4 wells relative to the BrdU intake amount of the solution 1 addition group. The figure is expressed as mean ± standard error of 4 wells for each solution addition group. There was no significant difference in cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 5 compared to ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 1 (n.s., Dunnett's multiple comparison test). Furthermore, cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 3 was also not significantly different from cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 1 (n.s., Dunnett's multiple comparison test). On the other hand, cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 6 was not significantly different from cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 1, but a significant tendency to cell proliferation was observed (p ═ 0.066, Dunnett's multiple comparison test).

Study of Compound 11 and 1 α, 25-dihydroxyvitamin D Using the method of bury₃Synergistic effect on ROS17/2.8 cell proliferation, results are shown in Table 3. A relative index of BrdU intake of each solution-added group was calculated with the BrdU intake of 1.000 when solution 1 was added. Combined addition of Compound 11 and 1 alpha, 25-dihydroxyvitamin D₃The relative index (1.444) of the group (a) was larger than the product (1.223) of the relative indices of the individual addition groups, and a synergistic effect was confirmed by the combined use.

[ Table 3]

Example 3

The N-hydroxybenzamidine derivatives (hereinafter referred to as test compounds) and 1. alpha.25-bis (N-hydroxybenzamidine) shown in Table 1 were investigated Hydroxy vitamin D ₃ Effect on cell proliferation of ROS17/2.8 cells (tables 4 and 5)

100. mu.L of a solution containing DMSO and ethanol (hereinafter referred to as solution 7), a test compound solution and ethanol (hereinafter referred to as solution 8), and 1. alpha., 25-dihydroxyvitamin D were injected into each of the solutions₃Solution and DMSO (hereinafter referred to as solution 9), or a test compound solution and 1 alpha, 25-dihydroxyvitamin D₃10% FCS-. alpha.MEM of the solution (hereinafter referred to as solution 10). At 5X 10³One/100. mu.L/well was seeded with ROS17/2.8 cells. Evaluation of test Compound and 1 alpha, 25-dihydroxyvitamin D after about 48 hours of culture Using the MTT method₃Effect on cell proliferation of ROS17/2.8 cells. That is, after culturing the cells for about 44 hours, MTT solution (3- [4, 5-dimethylthiazol-2-yl) was added at a volume of 25. mu.L/well]2, 5-diphenyltetrazoleBromide 2mg/mL), and incubated for an additional 4 hours. After removing the medium, 100. mu.L/well of DMSO was added to dissolve the formazan formed(formalzan). The formazan formed was quantified by measuring the absorbance (560nm) with a microplate reader。

1 comprising solution 70% FCS-. alpha.MEM was prepared by diluting DMSO 1000-fold with 10% FCS-. alpha.MEM, and diluting ethanol 10000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 8 was prepared by mixing 2X 10^-5A test compound solution having a concentration of M was diluted 1000-fold with 10% FCS-. alpha.MEM, and ethanol was diluted 10000-fold with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 9 was prepared by mixing 2X 10^-31 alpha, 25-dihydroxyvitamin D at a concentration of M₃The solution was diluted 10000 times with 10% FCS-. alpha.MEM, and DMSO was diluted 1000 times with 10% FCS-. alpha.MEM. 10% FCS-. alpha.MEM containing solution 10 was prepared by mixing 2X 10^-5A test compound solution having a concentration of M was diluted 1000-fold with 10% FCS-. alpha.MEM, and 2X 10^-31 alpha, 25-dihydroxyvitamin D at a concentration of M₃The solution was diluted 10000-fold with 10% FCS-. alpha.MEM.

The results are shown in Table 4. A for each solution addition groupAmount of formed nail formed in the group added to solution 7The quantity is calculated as a percentage of the mean value of 4 wells. All quantitation values are expressed as mean ± standard deviation of 4 wells of each solution addition group. Cell proliferation of all ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 10 was confirmed to be significantly different from cell proliferation of ROS17/2.8 cells cultured in the presence of 10% FCS- α MEM containing solution 8 and solution 9, respectively (p < 0.05,; p < 0.01vs. test compounds added alone, Student's t test, #; p < 0.05, # #; p < 0.01vs. 1 α, 25-dihydroxyvitamin D)₃Group added alone, Student's t-test).

[ Table 4]

Study of test Compounds and 1 alpha, 25-dihydroxyvitamin D Using the method of bulbge₃Synergistic effect on ROS17/2.8 cell proliferation, results are shown in Table 5. With the first of solution 7 addedThe amount of produced was 1.00, and the formazan in each solution addition group was calculatedRelative index of production. Combined addition of test compound and 1 alpha, 25-dihydroxyvitamin D₃The relative index of the group (a) is greater than the product of the relative indices of each of the individually added groups, in all of the test compounds and 1 alpha, 25-dihydroxy vitamin D₃The synergistic effect was confirmed in all of the combinations.

[ Table 5]

Bone is continuously replaced with new bone by osteoblasts responsible for bone formation and osteoclasts responsible for bone destruction, and is maintained normal in quality and quantity. It is known that an increase in bone mass occurs when the balance between the amount or functionality of osteoblasts and osteoclasts is relatively inclined toward bone formation (history of "neobone formation science", dental medicine press, 2007). It is considered that, in particular, an increase in the amount and functionality of osteoblasts is directly related to the promotion of bone formation, and the ROS17/2.8 cells used in the examples of the present specification are osteoblast-like cells derived from osteosarcoma, and it can be said that the proliferation of these cells promotes the promotion of bone formation. Thus, the above results suggest: the combined use of an N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof and an active vitamin D or a prodrug thereof exhibits a strong bone formation-promoting effect as compared with the case where each is used alone.

Industrial applicability

In the present invention, the combined use of an N-hydroxybenzamidine derivative represented by the formula (I) or a salt thereof and active vitamin D or a prodrug thereof is useful as a therapeutic agent or a prophylactic agent, or a therapeutic method or a prophylactic method for a disease caused by abnormal bone metabolism, particularly osteoporosis.

Claims (24)

1. A therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism, which comprises as active ingredients the following (a) and (b):

(a) an N-hydroxybenzamidine derivative represented by the following formula (I) or a salt thereof; and

(b) active form of vitamin D or a prodrug thereof;

in the above-mentioned formula (I),

R¹represents: hydrogen; C1-C6 alkyl; C3-C6 cycloalkyl; C6-C12 aryl; a 5-12 membered ring heteroaryl group containing 1-3 heteroatoms selected from N, O and S; or NR⁵R⁶；

R²Represents: hydrogen; C1-C6 alkyl; C2-C6 alkenyl; C3-C6 cycloalkyl; an amino group; a C1-3 dialkylamino group; c1-3 alkyl substituted with halogen, C3-C6 cycloalkyl, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S; by NR⁷R⁸A substituted C1-3 alkyl group; or N-methylcarbamoyl;

R³and R⁴Independently represent: hydrogen; halogen; a hydroxyl group; c1-6 alkyl unsubstituted or substituted by halogen; c3-6 cycloalkylamino; c1-6 alkoxy; c1-6 alkanoyloxy; c2-6 alkenyloxy; phenyl-C1-6 alkoxy; a phenoxy group; c2-6 alkenoyloxy; phenyl-C1-6 alkanoyloxy; c3-6 cycloalkyloxy substituted with carboxyl, esterified carboxyl or amidated carboxyl; or aminooxy;

R⁵represents hydrogen or a C1-C6 alkyl group;

R⁶represents: hydrogen; C1-C6 alkyl; or C1-3 alkyl substituted with hydroxy, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S;

R⁷represents: hydrogen; C1-C6 alkyl; or C3-C6 cycloalkyl;

R⁸represents: hydrogen; C1-C4 alkyl; c1-3 alkyl substituted with C1-3 alkoxy; or 4-pyridylcarbonyl;

X¹represents-O-, -S-, -NH-or-N (C1-6 alkyl) -;

X²represents a C3-7 alkylene group, a C3-7 alkylenecarbonyl group, or a C1-3 alkylene-phenylene-C1-3 alkylene group;

X³represents-O-, -S-, -NH-or-N (C1-6 alkyl) -.

2. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to claim 1, wherein,

1) when R is¹When the alkyl is C1-C6,

R²represents: C1-C6 alkyl; C2-C6 alkenyl; C3-C6 cycloalkyl; an amino group; a C1-3 dialkylamino group; c1-3 alkyl substituted with halogen, C3-C6 cycloalkyl, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S; or by NR⁷R⁸A substituted C1-3 alkyl group;

2) when R is¹When the alkyl is C3-C6 cycloalkyl,

R²represents a C1-C6 alkyl group or is NR⁷R⁸A substituted C1-3 alkyl group;

3) when R is¹When the aryl group is a C6-C12 aryl group or a 5-12 membered ring heteroaryl group containing 1-3 heteroatoms selected from N, O and S,

R²represents a C1-C3 alkyl group substituted with a halogen, a C3-C6 cycloalkyl group, a C6-C12 aryl group, a 5-12 membered ring heteroaryl group containing 1-3 heteroatoms selected from N, O and S, or a C3-6 heterocycloalkyl group containing 1-3 heteroatoms selected from N, O and S;

4) when R is¹Is NR⁵R⁶When the temperature of the water is higher than the set temperature,

R²represents: C1-C6 alkyl; c3-6 cycloalkyl; C1-C3 alkyl substituted with halogen, C3-C6 cycloalkyl, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, or C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S; or N-methylcarbamoyl.

3. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to claim 1 or 2, wherein,

1) when R is⁵In the case of hydrogen, the acid is,

R⁶represents hydrogen or a C1-C6 alkyl group;

2) when R is⁵When the alkyl is C1-C6,

R⁶represents:C1-C6 alkyl; c1-3 alkyl substituted with a group selected from hydroxy, C6-C12 aryl, 5-12 membered ring heteroaryl containing 1-3 heteroatoms selected from N, O and S, and C3-6 heterocycloalkyl containing 1-3 heteroatoms selected from N, O and S.

4. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 3, wherein,

1) when R is⁷In the case of hydrogen, the acid is,

R⁸represents: C1-C4 alkyl; or C1-3 alkyl substituted with C1-3 alkoxy or C3-6 heterocycloalkyl;

2) when R is⁷When the alkyl is C1-C6,

R⁸represents a C1-C4 alkyl group;

3) when R is⁷When the alkyl is C3-C6 cycloalkyl,

R⁸represents a 4-pyridineformyl group.

5. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 4, wherein R is¹Represents hydrogen, methyl, ethyl, propyl, isopropyl, cyclohexyl, phenyl, 3-pyridyl or NR⁵R⁶。

6. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 5, wherein R is²Represents: hydrogen; a methyl group; an ethyl group; propyl; isopropyl group; a butyl group; a vinyl group; a cyclopentyl group; an amino group; a dimethylamino group; by chlorine, pentyl, phenyl, 1-imidazolyl, 4-thiomorpholinyl, 4-morpholinyl, or NR⁷R⁸Substituted methyl or ethyl; or methyl or ethyl substituted by N-methylcarbamoyl.

7. The method for treating bone metabolism disorder according to any one of claims 1 to 6Therapeutic or prophylactic agent for diseases, wherein R³Is hydrogen.

8. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 7, wherein R is⁴Is hydrogen or fluorine.

9. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 8, wherein R is⁵Is hydrogen, methyl, ethyl, propyl or isopropyl.

10. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 9, wherein R is⁶Represents: hydrogen; a methyl group; an ethyl group; propyl; isopropyl group; or methyl or ethyl substituted by hydroxy, phenyl, 3-pyridyl or 4-morpholinyl.

11. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 10, wherein R is⁷Is hydrogen, methyl, ethyl or cyclopropyl.

12. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 11, wherein R is⁸Is hydrogen, methyl, ethyl, isopropyl, 3-isopropoxypropyl or 4-picolinoyl.

13. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 12, wherein,

X¹is-O-;

X²is butylene or pentylene;

X³is-O-, -NH-or-N (CH)₃)-。

14. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 13, wherein,

X¹-X²-X³comprises the following steps:

15. the therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to claim 1, wherein the N-hydroxybenzamidine derivative represented by formula (I) is any one of the following compounds:

1) n-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

2) N-hydroxy-4- {5- [4- (5-benzyl-2- (pyridin-3-yl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

3) N-hydroxy-4- {5- [4- (5- (2-chloroethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

4) N-hydroxy-4- {5- [4- (5-cyclopentylmethyl-2-ethyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

5) N-hydroxy-4- {5- [4- (5-vinyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

6) N-hydroxy-4- {5- [4- (5-cyclopentylmethyl-2-methylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

7) N-hydroxy-4- {5- [4- (2- (2-hydroxyethyl) methylamino-5-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

8) N-hydroxy-4- {5- [4- (2-benzylmethylamino-5-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

9) N-hydroxy-4- {5- [4- (5-methyl-2- (methyl (pyridin-3-ylmethyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

10) N-hydroxy-4- {5- [4- (2-benzylmethylamino-5-ethyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

11) N-hydroxy-4- {5- [4- (2-dipropylamino-5-ethyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

12) N-hydroxy-4- {5- [4- (5-isopropyl-2- (methyl (pyridin-3-yl-methyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

13) N-hydroxy-4- {5- [4- (5-butyl-2- (methyl (2- (4-morpholino) ethyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

14) N-hydroxy-4- {5- [4- (5-cyclopentylmethyl-2-dipropylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

15) N-hydroxy-4- {5- [4- (5-cyclopentyl-2- (methyl (2- (4-morpholino) ethyl) amino) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

16) N-hydroxy-4- {5- [4- (5-isopropyl-2-dipropylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

17) N-hydroxy-4- {5- [4- (5-isopropyl-2-dimethylamino-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

18) N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentanoylamino } benzamidine

19) N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentylmethylamino } benzamidine

20) N-hydroxy-2-fluoro-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

21) N-hydroxy-4- {4- [4- (2-cyclohexyl-5-ethyl-1, 3-thiazol-4-yl) phenoxy ] butoxy } benzamidine

22) N-hydroxy-4- {3- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxymethyl ] benzyloxy } benzamidine

23) N-hydroxy-4- {5- [4- (5- (2- (imidazol-1-yl) ethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

24) N-hydroxy-4- {5- [4- (5- (2- (isopropylamino) ethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

25) N-hydroxy-4- {5- [4- (5- (2- (3- (isopropoxy) propylamino) ethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

26) N-hydroxy-4- {5- [4- (5- (2-diethylaminoethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

27) N-hydroxy-4- {5- [4- (2-isopropyl-5- (2- (thiomorpholin-4-yl) ethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

28) N-hydroxy-4- {5- [4- (2-cyclohexyl-5- (2- (dimethylamino) ethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

29) N-hydroxy-4- {5- [4- (2-dimethylamino-5- (2- (morpholin-4-yl) ethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

30) N-hydroxy-4- {5- [4- (5- (2- (cyclopropyl (pyridine-4-carbonyl) amino) ethyl) -2-isopropyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

31) N-hydroxy-4- {5- [4- (2-amino-5- (N-methylcarbamoyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

32) N-hydroxy-4- {5- [4- (5- (imidazol-1-ylmethyl) -2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

33) N-hydroxy-4- {5- [4- (2-ethylmethylamino-5- (morpholin-4-ylmethyl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

34) N-hydroxy-4- {5- {4- (2- (methyl (2- (morpholin-4-yl) ethyl) amino) -5- (thiomorpholin-4-yl) -1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine

35) N-hydroxy-4- {5- [4- (5-dimethylamino-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine.

16. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to claim 1, wherein the N-hydroxybenzamidine derivative represented by formula (I) is N-hydroxy-4- {5- [4- (5-isopropyl-2-methyl-1, 3-thiazol-4-yl) phenoxy ] pentoxy } benzamidine.

17. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 16, wherein the disease caused by abnormal bone metabolism is a disease caused by abnormal bone resorption.

18. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 16, wherein the disease caused by abnormal bone metabolism is a disease caused by abnormal bone formation.

19. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 16, wherein the disease caused by abnormal bone metabolism is osteoporosis.

20. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 19, wherein (b) is 1 α, 25-dihydroxyvitamin D₃1 alpha, 24R-dihydroxy vitamin D₃Or 1 alpha-hydroxy vitamin D₃。

21. Any one of claims 1 to 19The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism, wherein (b) is 1 α, 25-dihydroxyvitamin D₃Or 1 alpha-hydroxy vitamin D₃。

22. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 21, wherein the components (a) and (b) constitute a mixture.

23. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 21, wherein (a) and (b) are each independently a single agent.

24. The therapeutic or prophylactic agent for a disease caused by abnormal bone metabolism according to any one of claims 1 to 21, which is a kit comprising (a) and (b).