KR20100066870A - Novel 6-indazolylamino-3-nitropyridine derivative compounds, preparation thereof and pharmaceutical composition comprising the same - Google Patents

Abstract

PURPOSE: A novel 6-indazoleamino-3-nitropyridien derivative compound is provided to suppress osteoclast formulation and to prevent and treat osteoporosis. CONSTITUTION: A 6-indazolylamino-3-nitropyridine derivative compound is denoted by chemical formula 1. The 6-indazolylamino-3-nitropyridine derivative compound or pharmaceutically acceptable salt thereof comprises: a step of reacting 2,6-dichloro-3-nitropyridine and amine of chemical formula 3(HR_1) under the presence of base to 6-chloro-3-nitropyridien derivative compound of chemical formula 4: and a step of reacting the compound of chemical formula 4 with 5-aminoindazole or 6-aminoindazole under the presence of base.

Description

Novel 6-Indazolylamino-3-nitropyridine derivative compounds, preparations and pharmaceutical composition comprising the same

The present invention relates to novel 6-indazolylamino-3-nitropyridine derivatives, methods for their preparation and pharmaceutical compositions comprising the same.

Bone (骨) plays a role in preserving the bone mass and structure required as a physical support of the body, and as a storage of calcium (Ca ²⁺ ) and plays an important role in maintaining blood concentrations of calcium and the like. As a necessary response to carry out this function, the bone always implements coordination of decomposition and remodeling. Therefore, the bone tissue is a dynamic state of destruction by osteoclasts and formation of osteoblasts by osteoblasts, which is metabolic in terms of equilibrium. If the equilibrium relationship between bone absorption and bone formation is destroyed, bone absorption may be relatively higher than bone formation, resulting in a decrease in bone density or bone mass, resulting in osteoporosis in which bone strength is not maintained.

Osteoporosis is a disease most commonly seen in middle-aged and older women, with reduced bone mineralization resulting in thinning of dense bone, resulting in a wider bone marrow cavity and weakening bones as symptoms progress. Because of this, it is easy to fracture even with a small impact.

In the past, the study was mainly focused on bone minerals, namely, calcium and phosphorus metabolic abnormalities, but no significant progress was made in determining the pathogenesis.

Materials currently used for the treatment of osteoporosis include bisphosphonate preparations (alendronate, ethidronate), hormone preparations (raloxifene), vitamin D preparations, calcitonin preparations, calcium preparations, and the like. However, bisphosphonate preparations are poorly absorbed, difficult to take, and cause esophagitis. Hormonal preparations should be taken for a lifetime. Unclear, calcitonin preparations are expensive and difficult to administer, and calcium preparations have fewer side effects but are limited to preventive effects rather than treatment.

Osteoporosis cannot be treated with short-term administration of the drug, but long-term administration of the drug is essential. Therefore, there is a need for a new substance that does not have such side effects and has good efficacy when the drug is administered for a long time.

Therefore, the inventors of the present invention, while studying to develop an effective therapeutic for osteoporosis, have synthesized novel 6-indazolylamino-3-nitropyridine derivatives, and these compounds inhibit bone formation by inhibiting the formation of osteoclasts. In addition to effectively inhibiting at the same time to promote the activity of osteoblasts to increase the bone production effectively confirmed that there is an excellent effect in the treatment and prevention of osteoporosis and completed the present invention.

It is an object of the present invention to provide a 6-indazolylamino-3-nitropyridine derivative.

It is another object of the present invention to provide a method for preparing a 6-indazolylamino-3-nitropyridine derivative.

Another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of osteoporosis, comprising a 6-indazolylamino-3-nitropyridine derivative.

The present invention provides a 6-indazolylamino-3-nitropyridine derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 1]

In Chemical Formula 1,

일 수 있으며, 여기서 R₅는 직쇄 또는 분쇄상의 C_1~C₆의 알킬기, C₃~C₆의 고리화 알킬기, C₁~C₆의 직쇄 또는 분쇄 상 히드록시알킬기 또는 N, O 및 S 중에서 선택된 1 내지 3개의 헤테로 원자를 포함하는 불포화의 5~6원자 헤테로 고리화 화합물이며, 여기서 헤테로 고리화합물은 비치환되거나 C₁~C₆ 알킬기, 할로겐 또는 SCH₃로 치환되어있고, R ₁ is NR ₃ (CH ₂ ) n R ₄ , wherein R ₃ is H, a substituted or unsubstituted straight or crushed C ₁ -C ₆ alkyl group, or a substituted or unsubstituted C ₃ -C ₆ cyclic alkyl Compound, wherein the substituent is a hydroxy group or an amine group; R ₄ is H, a hydroxy group, a straight or pulverized C ₁ to C ₆ alkylamine group or a saturated or unsaturated 5 to 6 membered heterocyclic compound containing 1 to 3 heteroatoms selected from N, O and S at the terminal; ego; In addition, R ₃ and R ₄ are connected to each other, R ₁ may be a saturated or unsaturated 5 to 6 membered heterocyclic amino compound further comprising 0 to 3 hetero atoms selected from N, O and S, In this case they are unsubstituted or substituted at various positions of the C ₁ to C ₃ alkyl group, the amine group, the hydroxy group and the C ₁ to C ₂ May be substituted with one or more selected from hydroxyalkyl groups; In addition, R ₃ and R ₄ are connected to each other so that R ₁

Wherein R ₅ is a linear or pulverized C _{1 to} C ₆ alkyl group, C ₃ to C ₆ cyclic alkyl group, C ₁ to C ₆ linear or pulverized hydroxyalkyl group or N, O and S Unsaturated 5-6 membered heterocyclic compound containing 1 to 3 heteroatoms selected, wherein the heterocyclic compound is unsubstituted or substituted with a C ₁ -C ₆ alkyl group, halogen or SCH ₃ ,

n is an integer from 0 to 3;

R ₂ is an indazol-5-ylamine group or an indazol-6-ylamine group.

Halogen in the present invention means fluoro, chloro, bromo or iodo.

이며, 여기서 R₅는 히드록시메틸, 히드록시이소부틸, 피리딘-3-일 또는 (2-메틸티오)피리딘-3-일이며; n은 0 내지 3인 정수이고; R ₂ 는 인다졸-5-일아민기 또는 인다졸-6-일아민기이다.In Chemical Formula 1, preferably R ₁ NR ₃ (CH ₂ ) n R ₄ where R ₃ is H, isopropyl, isobutyl, t-butyl, cyclopropyl or 4-trans-hydroxycyclohexyl; R ₄ is H, piperidin-1-yl, morphonyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl or imidazol-1-yl; Or R ₃ and R ₄ are connected to each other so that R ₁ is piperazine, methylpiperazine, hydroxypiperidine or

Wherein R ₅ is hydroxymethyl, hydroxyisobutyl, pyridin-3-yl or (2-methylthio) pyridin-3-yl; n is an integer from 0 to 3; R ₂ is an indazol-5-ylamine group or an indazol-6-ylamine group.

Preferred compounds of the 6-indazolylamino-3-nitropyridine derivatives of the general formula (1) of the present invention are as follows:

1) 6- (1H-indazol-5-yl) amino-2-cyclopropylamino-3-nitropyridine,

2) 6- (1H-indazol-5-yl) amino-2-isopropylamino-3-nitropyridine,

3) 6- (1H-indazol-6-yl) amino-2-isopropylamino-3-nitropyridine,

4) 6- (1H-indazol-5-yl) amino-2-isobutylamino-3-nitropyridine,

5) 6- (1H-indazol-5-yl) amino-2- t -butylamino-3-nitropyridine,

6) 6- (1H-indazol-5-yl) amino-2- (2-piperidino) ethylamino-3-nitropyridine,

7) 6- (1H-indazol-5-yl) amino-2- (2-morpholino) ethylamino-3-nitropyridine,

8) 6- (1H-indazol-5-yl) amino-2- ( trans -4-hydroxy) cyclohexylamino-3-nitropyridine,

9) 6- (1H-indazol-5-yl) amino-2- (3-pyridyl) methylamino-3-nitropyridine,

10) 6- (1H-indazol-5-yl) amino-2- [2- (2-pyridyl) ethylamino] -3-nitropyridine,

11) 6- (1H-indazol-5-yl) amino-2- [3- (morpholin-1-yl) propylamino] -3-nitropyridine,

12) 6- (1H-indazol-5-yl) amino-2- [3- (imidazol-1-yl) propylamino] -3-nitropyridine,

13) 6- (1H-indazol-5-yl) amino-2- (4-hydroxypiperidin-1-yl) -3-nitropyridine,

14) 6- (1H-indazol-5-yl) amino-2- (4-methylpiperazin-1-yl) -3-nitropyridine,

15) 6- (1H-indazol-5-yl) amino-2- (piperazin-1-yl) -3-nitropyridine,

16) 6- (1H-indazol-5-yl) amino-2-[(hydroxyacetyl) piperazin-1-yl] -3-nitropyridine,

17) 6- (1H-indazol-5-yl) amino-2-[(dimethylhydroxyacetyl) piperazin-1-yl] -3-nitropyridine,

18) 6- (1H-indazol-5-yl) amino-2- {4-[(3-pyridyl) carbonyl] piperazin-1-yl} -3-nitropyridine and

19) 6- (1H-indazol-5-yl) amino-2- {4-[(3- (2-methylthio) pyridyl) carbonyl] piperazin-1-yl} -3-nitropyridine.

In the present invention, the pharmaceutically acceptable salts generally mean inorganic acids and organic acid salts used by pharmaceutical manufacturers to prepare pharmaceuticals. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, or the like may be used. Citric acid, acetic acid, lactic acid, tartaric acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, methanesulfonic acid, benzenesulfonic acid, maleic acid, benzoic acid, gluconic acid, glycolic acid, succinic acid, 4-morpholineethanesulfonic acid, camphorsulfonic acid, 4 -Nitrobenzenesulfonic acid, hydroxy-O-sulfonic acid, 4-toluenesulfonic acid, caluturonic acid, emboic acid, glutamic acid, aspartic acid and the like can be used. Hydrochloric acid is preferable as an inorganic acid, and methanesulfonic acid is preferable as an organic acid.

The present invention also provides a process for preparing a 6-indazolylamino 3-nitropyridine derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof, comprising the following steps:

1) reacting 2, 6-dichloro-3-nitropyridine with an amine of formula 3 in the presence of a base to prepare a 6-chloro-3-nitropyridine derivative compound of formula 4 wherein the amine is substituted in the 2-position; And

2) preparing a 6-indazolylamino-3-nitropyridine derivative compound of formula 1 by reacting the compound of formula 4 prepared in step 1 with 5-aminoindazole or 6-aminoindazole under a base.

(3)

HR ₁

[Formula 4]

[Formula 1]

In the above formula, R ₁ and R ₂ are as defined in the compound of formula (1).

2, 6-dichloro-3-nitropyridine and the amine of the formula (3) used as starting materials and reaction materials in steps 1 and 2 can be easily purchased and used as a commercially available material.

In addition, 5-aminoindazole and 6-aminoindazole of step 2 may also be easily purchased and used as a commercially available material.

In the above steps 1 and 2, the reaction solvent is selected from alcohols such as methanol, ethanol, isopropanol and acetonitrile, chloroform, methylene chloride, tetrahydrofuran, N, N-dimethylformamide, N-methylpyrrolidinone and the like. Preference is given to using a single solvent or a mixed solvent.

In the steps 1 and 2, the base may be appropriately selected from various organic bases generally known to those skilled in the art. For example, the organic salts used in the reaction may be triethylamine, N, N-diisopropylethylamine , N-methylmorpholine, DBU (1,8-Diazabicyclo [5.4.0] undec-7-ene), N-methylpiperidine, 4-dimethylaminopyridine, N, N-dimethylaniline, 2, 6- Preference is given to using at least one general tertiary organic base selected from lutidine and pyridine.

The present invention also provides a method for preparing a 6-indazolylamino-3-nitropyridine derivative compound of Formula 1B or a pharmaceutically acceptable salt thereof comprising the following steps:

1) 2, 6-dichloro-3-nitropyridine and 1- t -BOC- piperazine was reacted by the presence of a base 2- (4- t -BOC piperazin-1 of Formula 5-yl) -6-chloro- Preparing a 3-nitropyridine compound;

2) 6-indazolylamino-2- (4- t- BOC-piperazine- of formula 6 by reacting the compound of formula 5 prepared in step 1 with 5-aminoindazole or 6-aminoindazole in the presence of a base Preparing a 1-yl) -3-nitropyridine compound;

3) Deprotection of the amine protecting group "B0C" (tert-butoxy carbonyl) from the compound of Chemical Formula 6 prepared in Step 2 6-indazolylamino-2- (piperazin-1-yl) -3 Preparing a nitropyridine compound; And

4) preparing a compound of Formula 1B by reacting the compound of Formula 7 prepared in Step 3 with an acid chloride compound of Formula 8 in the presence of a base.

[Chemical Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 1B]

In the above formula,

R ₂ is an indazol-5-ylamine group or an indazol-6-ylamine group;

R ₅ is a linear or pulverized C _{1 to} C ₆ alkyl group; C ₃ -C ₆ cyclic alkyl group; C ₁ -C ₆ linear or pulverized hydroxyalkyl group; Unsaturated 5 to 6 membered heterocyclic compounds containing 1 to 3 heteroatoms selected from N, O and S, wherein the heterocyclic compound is unsubstituted or substituted with a C ₁ to C ₆ alkyl group, halogen or SCH ₃ Can be.

2, 6-dichloro-3-nitropyridine and t- BOC-piperazine used as starting materials and reaction materials in step 1 can be easily purchased and used as a commercially available material. In addition, the 5-aminoindazole or 6-aminoindazole of Step 2 and the ecitchloride compound of Formula 8 of Step 4 may also be easily purchased and used as a commercially available material.

In the steps 1 and 2, the reaction solvent is a single solvent selected from alcohols such as methanol, ethanol, isopropanol, acetonitrile, chloroform, methylene chloride, tetrahydrofuran, N, N-dimethylformamide, N-methylpyrrolidinone, and the like. Or it is preferable to use a mixed solvent, It is preferable that reaction temperature is 0-80 degreeC, and it is more preferable that it is 25-80 degreeC.

In the step 3, the reaction solvent is preferably a single solvent or a mixed solvent selected from alcohols such as methanol, ethanol, isopropanol and acetonitrile, chloroform, methylene chloride, tetrahydrofuran and the like. Deprotection of the protecting group “BOC” (t-butoxy carbonyl) in step 3 refers to a “BOC” deprotection reaction already known to those skilled in the art, for example, an acid treatment, and then a base treatment. The acid used to remove the protecting group “BOC” (t-butoxy carbonyl) is used by those skilled in the art to remove “BOC” such as hydrochloric acid, trifluoroacetic acid, methanesulfonic acid and sulfuric acid. Acid, and its equivalent may be adjusted according to the properties of the compound, and base may be used such as sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, sodium hydrogen carbonate and the like.

The reaction temperature of step 3 is also preferably 0 ~ 80 ℃.

In step 4, the reaction solvent is preferably used a single solvent or a mixed solvent selected from chloroform, methylene chloride, tetrahydrofuran, etc., the reaction temperature is preferably 0 ~ 80 ℃.

The base in step 1, step 2 or step 4 is triethylamine, N, N-diisopropylethylamine, N-methylmorpholine, DBU (1,8-Diazabicyclo [5.4.0] undec-7-ene ), N-methylpiperidine, 4-dimethylaminopyridine, N, N-dimethylaniline, 2, 6-lutidine and pyridine.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis comprising 6-indazolylamino-3-nitropyridine derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Here, the pharmaceutically acceptable salts are as described above.

In another aspect, the present invention provides a method for preventing or treating osteoporosis by administering to the mammal, including a human in need of preventing or treating osteoporosis.

The present invention also provides the use of the above-mentioned compound of formula 1 in the preparation of a pharmaceutical preparation for the prevention or treatment of osteoporosis.

In the present invention, the term "osteoporosis" refers to a state in which there is no abnormality in the remaining bones, and the amount of minerals and substrates that form the bones is excessively reduced, resulting in a lot of small holes such as sponges, resulting in soft and easily broken, Also called osteoporosis. In a specific embodiment, the 2-nitropyridine derivative compounds substituted with the 2, 6-position of Formula 1 of the present invention promote osteoblast activity and effectively increase bone formation to promote bone formation, as well as osteoclast Suppresses formation and suppresses bone resorption. Therefore, the 2-nitropyridine derivative compound substituted at the 2, 6-position of the present invention and a pharmaceutically acceptable salt thereof can be usefully used for the prevention and treatment of osteoporosis.

In addition, the composition of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the 6-indazolylamino-3-nitropyridine derivative, or a pharmaceutically acceptable salt thereof.

The composition of the present invention can be prepared by containing one or more pharmaceutically acceptable carriers in addition to the above-mentioned ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage of the 6-indazolylamino-3-nitropyridine derivative of the present invention is about 10 to 1,000 mg / kg, preferably 50 to 500 mg / kg, more preferably administered once to several times a day. Do.

The composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention and treatment of osteoporosis.

The novel 6-indazolylamino-3-nitropyridine derivatives of the present invention promote osteoblast activity, effectively increase bone formation, promote bone formation, and inhibit osteoclast formation to inhibit bone resorption. It is also excellent in its ability to be useful for the prevention and treatment of osteoporosis.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

In addition, the reagents and solvents mentioned below were purchased from Aldrich, or Cambridge Isotope Labortories, unless otherwise specified, and the ¹ H-NMR data were measured by a JNM-LA400 (JEOL) machine, and the mass data. Is the value measured with 1100MSD (Hewlett Packard) machine.

I. Synthesis of 6-indazolylamino-3-nitropyridine (1a) of the present invention using Scheme 1 below

Scheme 1

Preparation Example 1 Preparation of 6-chloro-2-cyclopropylamino-3-nitropyridine compound (4a) according to Scheme 1

To 50 ml of methylene chloride , 500 mg (2.59 mmol) of 2, 6-dichloro-3-nitropyridine (2) and 0.71 ml (5.18 mmol) of triethylamine were added, and 0.18 ml (2.59 mmol) of cyclopropylamine was added at 0 to 5 ° C. After slowly adding, the mixture was reacted at room temperature (20-30 ° C.) for about 30 hours. After completion of the reaction, 50 ml of methylene chloride was added, extracted with 100 ml of water, and the organic layer was distilled under reduced pressure, and then purified by column chromatography using n-nucleic acid: ethyl acetate = 6: 1 (v / v) as a developing solvent. After vacuum drying at about 40 ℃ to give the target compound 306mg (yield: 55%).

Mass (M +): 214.1

¹ H-NMR (DMSO-d ₆ ) (ppm): 0.70 (m, 2H), 0.81 (m, 2H), 2.97 (m, 1H), 6.84 (d, 1H), 8.41 (d, 2H).

Example 1 Preparation of 6- (1H-indazol-5-yl) amino-2-cyclopropylamino-3-nitropyridine compound (1a)

6-chloro-2-cyclopropylamino-3-nitropyridine obtained in Preparation Example 1 in 10 ml of acetonitrile. Compound (4a) 300mg (1.39 mmol) , triethylamine 0.2ml (1.52 mmol), DBU 0.42ml (1,8-Diazabicyclo [5.4.0] undec-7-ene, 2.78mmol) and 5-amino-indazole ( 5) After 194 mg (1.45 mmol) was added, the mixture was reacted at 65-82 ° C for 24 hours. After completion of the reaction, the solvent was distilled under reduced pressure, 5 ml of methanol was added to the concentrate, and the mixture was stirred at room temperature for 1 hour. The resulting solid was filtered, washed with 5 ml of methanol and dried in vacuo at about 40 ° C. to obtain 397 mg (yield: 91%) of the title compound.

Mass (M +): 311.1

¹ H-NMR (DMSO-d ₆ ) (ppm): 0.74 (m, 2H), 0.94 (m, 2H), 3.00 (m, 1H), 6.28 (d, 2H), 7.53 (d, 1H), 7.63 (d, 1H), 8.03 (s, 1H), 8.11 (d, 1H), 8.71 (brm, 1H0, 8.82 (d, 1H), 10.14 (s, 1H).

<Examples 2 to 14>

Except for using the amine compound (3a) shown in Table 1 in place of cyclopropylamine in Preparation Example 1 using the same method as Preparation Example 1 6-chloro-3-nitro substituted with various amines in the 2-position Pyridine Compound (4a) was synthesized, and the synthesized compound (4a) and 5-aminoindazole or 6-aminoindazole were reacted in the same manner as in Example 1 to obtain the target compound (1a) shown in Table 1 below.

In Table 1 below , the name of compound (1a) prepared in Examples 2 to 14, and the amine (3a) used in Preparation Example to prepare compound ( 4a) in Scheme 1, to prepare Scheme (1a) The names, yields, Mass and NMR results of the amine (5) used were shown.

TABLE 1

In the above table, * means the yield (%) when the synthesized compound (4a) compound and 5-aminoindazole or 6-aminoindazole in the same manner as in Example 1, ** ** compound (1a Of) Mass data.

II. Synthesis of 6-indazolylamino-3-nitropyridine (1b) of the present invention using Scheme 2 below

Scheme 2

Preparation Example 2-1 2- (4-) according to Scheme 2 t -BOC-piperazin-1-yl) -6-chloro-3-nitropyridine compound (4b)

5 g (25.9 mmol) of 2, 6-dichloronitro-3-pyridine (2) and 3.97 ml (28.5 mmol) of triethylamine were added to 300 ml of methylene chloride, and the temperature of the solvent was cooled to 0-5 ° C., followed by 1- ( t 5.06 g (27.1 mmol) of butoxycarbonyl) piperazine (3b) was slowly added and stirred for 3 to 4 hours at the same temperature (0 to 5 ° C). After completion of the reaction, the mixture was extracted with methylene chloride and water, water was removed using magnesium sulfate, and distilled under reduced pressure to obtain 8.7 g (yield: 99%) of the title compound.

Mass (M +): 343.1

¹ H-NMR (DMSO-d ₆ ) (ppm): 1.41 (s, 9H), 3.37 (m, 4H), 3.46 (m, 4H), 6.95 (d, 1H), 8.31 (d, 1H).

Production Example 2-2 6- (indazol-5-yl) amino-2- (4- t -BOC-piperazin-1-yl) -3-nitropyridine compound (6b)

8.7 g (25.4 mmol) of 2- (4- t- BOC-piperazin-1-yl) -6-chloro-3-nitropyridine compound (4b) obtained in Preparation Example 2-1 was dissolved in 150 ml of acetonitrile, 4.2 ml (36.4 mmol) of triethylamine were added thereto, and 4.8 g (36.4 mmol) of 5-aminoindazole (5 ) was added thereto, followed by stirring under reflux for 3 days. After the reaction was completed, the temperature was lowered to room temperature, and water was added dropwise to about 100 ml, followed by stirring at room temperature for about 1 hour. The resulting solid was filtered, washed with 50 ml of water and dried in vacuo at 40 ° C. to obtain 8.9 g (yield: 80%) of the title compound.

Mass (M +): 440.2

¹ H-NMR (DMSO-d ₆ ): 1.41 (s, 9H), 3.39 (brm, 4H), 3.47 (brm, 4H), 6.30 (d, 2H), 7.52 (m, 2H), 8.06 (s, 1H), 8.11 (m, 1H), 8.17 (d, 1H), 9.94 (s, 1H).

Example 15 Preparation of 6- (indazol-5-yl) amino-2- (piperazin-1-yl) -3-nitropyridine compound (7b)

300 mg (0.68 mmol) of 6- (indazol-5-yl) amino-2- (4- t- BOC-piperazin-1-yl) -3-nitropyridine compound (6b) obtained in Preparation Example 2-2. Was dissolved in 20 ml of methylene chloride, 0.5 ml (6.8 mmol) of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 8 hours. After completion of the reaction, the solvent was removed by distillation under reduced pressure, and 5 ml of methanol was added to dissolve the reaction. The temperature was lowered to 0-5 ° C., and 3N aqueous sodium hydroxide solution was added dropwise so that the pH of the solution was about 8-9, followed by stirring at the same temperature for about 30 minutes. The resulting solid was filtered, washed with 10 ml of water and dried in vacuo at 40 ° C. to obtain 220 mg of the target compound (yield: 95%).

Mass (M +): 440.2

¹ H-NMR (DMSO-d ₆ ) (ppm): 2.87 (brm, 4H), 3.11 (brm, 2H), 3.35 (brm, 2H), 6.25 (d, 2H), 7.50 (m, 2H), 8.03 (s, 1 H), 8.14 (m, 2 H), 9.89 (s, 1 H).

Example 16 Preparation of 6- (1H-indazol-5-yl) amino-2-[(hydroxyacetyl) piperazin-1-yl] -3-nitropyridine (1b)

500 mg (1.5 mmol) of 6- (indazol-5-yl) -2- (piperazin-1-yl) -3-nitropyridine compound (7b) obtained in Example 15 was dissolved in 10 ml of methylene chloride, and then triethyl 0.5 ml (3.52 mmol) of amine was added thereto, and the temperature was decreased to 0-5 ° C., and 0.19 ml (1.8 mmol) of acetoxyacetyl chloride (8 ) was slowly added dropwise to the reaction solution. After 3 hours of reaction at the same temperature, the mixture was extracted with methylene chloride and water, followed by 2-[(acetoxyacetyl) piperazin-1-yl] -6- (1H-indazol-5-yl) amino-3- Nitropyridine compound 650 mg (yield: 98%) were obtained, and the reaction proceeded to the next reaction.

Mass (M +): 440.2

645 mg (1.47 mmol) of 6- (1H-indazol-5-yl) amino-2-[(acetoxyacetyl) piperazin-1-yl] -3-nitropyridine compound obtained in the reaction was dissolved in 30 ml of methanol. , 460 mg (4.41 mmol) of sodium carbonate was dissolved in 20 ml of water, and added dropwise to the reaction solution. The reaction was carried out at 30 to 40 ° C. for 3 hours. After the reaction was completed, the resulting solid was filtered, washed with 10 ml of water and dried in vacuo at 40 ℃ to obtain 420 mg of the target compound (yield: 81%).

Mass (M +): 398.2

NMR (DMSO-d6) (ppm): 3.37 (brm, 4H), 3.52 (m, 2H), 3.62 (m, 2H), 4.13 (d, 2H), 4.63 (t, 1H), 6.31 (d, 1H ), 7.53 (m, 2H), 8.07 (s, 1H), 8.12 (s, 1H), 8.18 (d, 1H), 9.94 (s, 1H).

<Examples 17 to 19>

In Example 16, the compound was synthesized in the same manner as in Example 16 except that the acid chloride (8) shown in Table 2 was used instead of acetoxyacetyl chloride to obtain the target compound (1b) shown in Table 2 below. .

Table 2 below shows the names of compounds (1b) prepared in Examples 17-19, names of acid chlorides (8) used, yields, and Mass and NMR results.

TABLE 2

Example Used acid chlorides ( 8) Name of compound (1b) yield(%)* Of compound (1b) ^One H-NMR value (DMSO-d ₆ ) M (+) **

17 2-acetoxyisobutyl chloride 6- (1H-indazol-5-yl) amino-2- (dimethylhydroxyacetyl) piperazin-1-yl-3-nitropyridine 72 1.34 (s, 6H), 3.54 (brm, 4H), 3.71 (brm, 2H), 4.18 (bem, 2H), 5.26 (brs, m, 2H), 5.51 (s, 1H), 6.88 (s, 1H) , 7.99 (d, 1H), 7.41 (d, 1H), 8.27 (s, 1H), 8.30 (d, 1H), 8.46 (d, 1H).
426.1

18 Nicotinoyl chloride 6- (1H-indazol-5-yl) amino-2- [4-[(3-pyridyl) carbonyl] piperazin-1-yl] -3-nitropyridine 91 3.44 (m, 2H), 3.53 (brm, 4H), 3.80 (m, 2H), 6.32 (d, 1H), 7.50 (m, 3H), 7.89 (d, 1H), 8.04 (s, 1H), 8.10 (s, 1 H), 8.19 (d, 1 H), 8.66 (m, 2 H), 9.97 (s, 1 H).
445.2

19 2-methylthionicotinoyl chloride 6- (1H-indazol-5-yl) amino-2- {4-[(3- (2-methylthio) pyridyl) carbonyl] piperazin-1-yl} -3-nitropyridine 94 3.32 (m, 2H), 3.35 (s, 3H), 3.41 (m, 2H), 3.54 (m, 2H), 3.80 (m, 2H), 6.32 (d, 1H), 7.19 (t, 1H), 7.51 (m, 2H), 7.64 (d, 1H), 8.02 (s, 1H), 8.08 (s, 1H), 8.19 (d, 1H), 8.52 (d, 1H), 9.96 (s, 1H).
491.2

In the above table, * means the yield when the synthesized compound (4a) and 5-aminoindazole or 6-aminoindazole were reacted in the same manner as in Example 1, ** indicates the yield of Compound (1a) Mass data.

Experimental Example 1. Inhibitory Effect of Osteoclast Formation by Coexistence Culture>

In order to confirm the inhibitory effect of osteoclast formation, the co-culture system (Reference: Endocrinology 137 (1996), 2187-2190, E. Jimi et al.) Was used. Is the same as

1) Preparation of bone marrow cells and osteoblasts

Bone marrow cells were harvested by the normal method using a syringe by extracting the femur and tibia from 6-8 week old male ddY mice. In brief, tissues were removed from the extracted bone, scissors cut off at both ends, and bone marrow was isolated by pushing against one of the cut bones using a 23G syringe with medium. The separated bone marrow was repeatedly piston-driven to become single cells using a syringe. (Reference: Endocrinology 123 (1988), 2600-2602, Takahashi et al.). After erythrocytes were removed from the bone marrow, the bone marrow cells recovered by centrifugation were placed in α-MEM medium to which 10% fetal bovine serum was added, and nucleated cells were counted and used immediately for co-culture.

In the case of Calvarial cells, skulls were extracted from 1-2 days old ICR mice and osteoblasts were isolated by continuous reaction with 0.2% collagenase enzyme solution. Osteoblasts collected by centrifugation of the cell-suspended supernatant were grown until completely filled in α-MEM medium to which 10% fetal bovine serum was added, and then diluted to a desired cell number.

2) inhibition of osteoclast formation by co-culture

All cultures used in the co-culture were added to the α-MEM medium with 10% fetal bovine serum added differentiation factors 1α, 25-dihydroxyvitamin D ₃ (10 ^-8 M) and dexamethasone (10 ^-8 M). Differentiation medium was used to induce osteoclast formation. First, the compounds dissolved at 1 mM concentration in dimethyl sulfoxide (DMSO) solvent were diluted to 2 uM using the above-described differentiation medium, and prepared as 0.2% (v / v) DMSO as a solvent control group. 100 uL each was added to the wells. In addition, the bone marrow cells and osteoblasts prepared above were diluted to 1x10 ⁵ cells / 50 uL and 3,000 cells / 50 uL per 96 wells and added to each well. The total volume at this time was 200 uL per well, the final compound concentration was 1 uM and the control group was 0.1% (v / v) DMSO. The cells were exchanged every two to three days with fresh medium containing differentiation factor and test substance.

After 7 days from the start of the culture, it was confirmed by microscopic observation that osteoclasts with a multinucleus were formed, and then the medium was removed and the cells were fixed with 10% phosphate buffered formalin solution. The degree of formation of mature osteoclasts was determined using the characteristics of osteoclasts with a positive response to tartaric acid-resistant phosphatase (TRAP) stain. TRAP staining buffer solution was prepared by dissolving the substrate naphthol AS-MS phosphate and pigment (Fast Red Violet LB salt) in N, N-dimethylformamide and adding 0.1 N NaHCO ₃ buffer containing 50 mM tartaric acid. Under the light microscope, 6-7 or more multinucleated cells among TRAP-positive cells were recognized as mature osteoclasts.

The degree of inhibition of osteoclast formation was calculated using Equation 1 below. (The experiment was performed with the population of four wells (n = 4) in each experimental group, and the result was calculated by expressing the mean ± standard deviation.)

[Equation 1]

(Number of osteoclasts in the control group using only the number of osteoclasts / solvent observed in the experimental group) × 100 (%) = degradation rate of osteoclast formation (%)

[Table 3]

As shown in Table 3, the compounds of the present invention was observed to inhibit the formation of most osteoclasts. Therefore, it was confirmed that the compound of the present invention can be used as an excellent prevention and treatment of osteoporosis.

Experimental Example 2. ALP Activity Verification Experiment

The level of osteoblast differentiation and activity was indirectly evaluated by measuring the activity of ALP (Alkaline Phosphatase), which is closely related to bone formation.

The osteoblasts prepared in Experimental Example 1 were collected in α-MEM medium containing 10% fetal bovine serum, and the number of cells was measured. The cells were grown by dispensing 20,000 cells per well in a 24-well culture ware. . After 24 hours of culture, the existing medium was discarded and replaced with fresh medium (1 mL / well) in which the compound to be tested was diluted to a concentration of 1 uM. At this time, the solvent control group containing 0.1% DMSO was also treated. The cells were incubated at 37 ° C. and 5% CO ₂ inhibitor conditions for 3 days with the compound treated. The supernatant was removed at the end of the experiment, and the cells were washed three times with 4 ° C. cold phosphorylation buffer. 0.2% Triton X-100 was added to the washed cells, followed by freezing at -70 ° C and thawing at room temperature three times to completely lyse the cells. The cell supernatant collected by centrifugation of cell extracts was used for ALP activity and protein measurement. Protein concentration was measured using a BCA assay kit (manufactured by Sigma-Aldrich). The ALP activity was measured by adding p-nitrophenylphosphate to the cell supernatant and reacting at 37 ° C. for 30 minutes, and stopping the reaction by adding 50ul of 0.2N sodium hydroxide. P-nitrophenol was used as a standard to draw a standard curve at absorbance of 405 nm, and then the absorbance of the reacted test substance was measured to determine the amount of p-nitrophenol produced.

ALP activity was calculated by dividing p-nitrophenol produced by each test substance by protein amount and reaction time, and expressed the unit of ALP activity as p-nitrophenol / ug protein / min. The results are shown in Table 4 in which the ALP activity units of each test substance were compared with the solvent control group and displayed to varying degrees.

[Table 4]

As shown in Table 4, it was confirmed that the compound of the present invention has excellent ALP activity in Calvarial cells.

Experimental Example 3. Cytotoxicity Test

In order to determine the cytotoxicity of the compound of the present invention, the following experiment was performed.

The drug was diluted to a concentration of 2 uM in α-MEM culture medium containing 10% fetal bovine serum, and 0.2% DMSO was included as a solvent control group. After diluting the diluted drug into 96-well plates, 100 μl, the osteoblasts prepared in Experiment 1 were added to each well at 1.0 × 10 ⁴ cells / 100 μl. At this time, the concentration of the final compound in the cell culture solution is 1uM, the solvent control group contains 0.1% DMSO. Cells incubated in 5% CO ₂ inhibitor at 37 ° C. for 72 hours were treated with 2 mg / ml of MTT [3- (4,5-dimetyl-2-thiazolyl) -2,5 dissolved in PBS 4 hours before the end of the culture. 25 µl of -diphenyl-2H-tetrazolium bromide] was added to each cell culture medium. After completion of the reaction, the plate was centrifuged and the medium was removed. Formazan was dissolved in 100 μl of DMSO (dimethyl sulfoxide). Finally, the absorbed plate was measured at 540 nm. The survival rate of the cells was expressed as a% concentration compared to the solvent control (control), the results are shown in Table 5.

TABLE 5

As shown in Table 5, it was confirmed that the compound of the present invention has almost no cytotoxicity, and the compounds 5 to 10, 14 to 19 are not only non-toxic, but also inhibit the formation of osteoclasts and simultaneously form osteoblasts. It was confirmed that there is an promoting effect.

Claims (10)

6-indazolylamino-3-nitropyridine derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof: [Formula 1]

R ₁ is NR ₃ (CH ₂ ) n R ₄ , wherein R ₃ is H, a substituted or unsubstituted straight or crushed C ₁ -C ₆ alkyl group, or a substituted or unsubstituted C ₃ -C ₆ cyclic alkyl Compound, wherein the substituent is a hydroxy group or an amine group; R ₄ is H, a hydroxy group, a straight or pulverized C ₁ to C ₆ alkylamine group or a saturated or unsaturated 5 to 6 membered heterocyclic compound containing 1 to 3 heteroatoms selected from N, O and S at the terminal; ego; In addition, R ₃ and R ₄ are connected to each other, R ₁ may be a saturated or unsaturated 5 to 6 membered heterocyclic amino compound further comprising 0 to 3 hetero atoms selected from N, O and S, In this case they are unsubstituted or substituted at various positions of the C ₁ to C ₃ alkyl group, the amine group, the hydroxy group and the C ₁ to C ₂ May be substituted with one or more selected from hydroxyalkyl groups; In addition, R ₃ and R ₄ are connected to each other so that R ₁

Wherein R ₅ is a linear or pulverized C _{1 to} C ₆ alkyl group, C ₃ to C ₆ cyclized alkyl group, C ₁ to C ₆ linear or pulverized hydroxyalkyl group or N, O and S Unsaturated 5-6 membered heterocyclic compound containing 1 to 3 heteroatoms selected, wherein the heterocyclic compound is unsubstituted or substituted with a C ₁ -C ₆ alkyl group, halogen or SCH ₃ , n is an integer from 0 to 3; R ₂ is an indazol-5-ylamine group or an indazol-6-ylamine group. The 6-indazolylamino-3-nitropyridine derivative compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the substituent of Formula 1 has the following formula: R ₁ is NR ₃ (CH ₂ ) n R ₄ where R ₃ is H, isopropyl, isobutyl, t-butyl, cyclopropyl or 4-trans-hydroxycyclohexyl; R ₄ is H, piperidin-1-yl, morphonyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl or imidazol-1-yl; Or R ₃ and R ₄ are connected to each other so that R ₁ is piperazine, methylpiperazine, hydroxypiperidine or

Wherein R ₅ is hydroxymethyl, hydroxyisobutyl, pyridin-3-yl or (2-methylthio) pyridin-3-yl; n is an integer from 0 to 3; R ₂ is an indazol-5-ylamine group or an indazol-6-ylamine group. 3. The method of claim 2, 1) 6- (1H-indazol-5-yl) amino-2-cyclopropylamino-3-nitropyridine, 2) 6- (1H-indazol-5-yl) amino-2-isopropylamino-3-nitropyridine, 3) 6- (1H-indazol-6-yl) amino-2-isopropylamino-3-nitropyridine, 4) 6- (1H-indazol-5-yl) amino-2-isobutylamino-3-nitropyridine, 5) 6- (1H-indazol-5-yl) amino-2- t -butylamino-3-nitropyridine, 6) 6- (1H-indazol-5-yl) amino-2- (2-piperidino) ethylamino-3-nitropyridine, 7) 6- (1H-indazol-5-yl) amino-2- (2-morpholino) ethylamino-3-nitropyridine, 8) 6- (1H-indazol-5-yl) amino-2- ( trans -4-hydroxy) cyclohexylamino-3-nitropyridine, 9) 6- (1H-indazol-5-yl) amino-2- (3-pyridyl) methylamino-3-nitropyridine, 10) 6- (1H-indazol-5-yl) amino-2- [2- (2-pyridyl) ethylamino] -3-nitropyridine, 11) 6- (1H-indazol-5-yl) amino-2- [3- (morpholin-1-yl) propylamino] -3-nitropyridine, 12) 6- (1H-indazol-5-yl) amino-2- [3- (imidazol-1-yl) propylamino] -3-nitropyridine, 13) 6- (1H-indazol-5-yl) amino-2- (4-hydroxypiperidin-1-yl) -3-nitropyridine, 14) 6- (1H-indazol-5-yl) amino-2- (4-methylpiperazin-1-yl) -3-nitropyridine, 15) 6- (1H-indazol-5-yl) amino-2- (piperazin-1-yl) -3-nitropyridine, 16) 6- (1H-indazol-5-yl) amino-2-[(hydroxyacetyl) piperazin-1-yl] -3-nitropyridine, 17) 6- (1H-indazol-5-yl) amino-2-[(dimethylhydroxyacetyl) piperazin-1-yl] -3-nitropyridine, 18) 6- (1H-indazol-5-yl) amino-2- {4-[(3-pyridyl) carbonyl] piperazin-1-yl} -3-nitropyridine and 19) 6- (1H-indazol-5-yl) amino-2- {4-[(3- (2-methylthio) pyridyl) carbonyl] piperazin-1-yl} -3-nitropyridine 6-indazolylamino-3-nitropyridine derivative compound selected from the group consisting of or a pharmaceutically acceptable salt thereof. 4. The hydrochloride or methanesulfonate of any one of claims 1 to 3 of the 6-indazolylamino-3-nitropyridine derivative compound. A process for preparing the 6-indazolylamino 3-nitropyridine derivative compound of Formula 1 or a pharmaceutically acceptable salt thereof, comprising the following steps: 1) reacting 2, 6-dichloro-3-nitropyridine with an amine of formula 3 in the presence of a base to prepare a 6-chloro-3-nitropyridine derivative compound of formula 4 wherein the amine is substituted in the 2-position; And 2) preparing a 6-indazolylamino-3-nitropyridine derivative compound of formula 1 by reacting the compound of formula 4 prepared in step 1 with 5-aminoindazole or 6-aminoindazole under a base. (3) HR ₁ [Formula 4]

[Formula 1]

In the above formula, R ₁ and R ₂ are as defined in claim 1. The method of claim 5, wherein the base of step 1 or step 2 is triethylamine, N, N-diisopropylethylamine, N-methylmorpholine, DBU (1,8-Diazabicyclo [5.4.0] undec-7 -ene), N-methylpiperidine, 4-dimethylaminopyridine, N, N-dimethylaniline, 2, 6-lutidine and pyridine. A process for preparing the 6-indazolylamino-3-nitropyridine derivative compound of Formula 1B or a pharmaceutically acceptable salt thereof comprising the following steps: 1) 2- (4- t- BOC-piperazin1-yl) -6-chloro- of formula 5 by reacting 2, 6-dichloro-3-nitropyridine and 1- t- BOC-piperazine in the presence of a base Preparing a 3-nitropyridine compound; 2) 6-indazolylamino-2- (4- t- BOC-piperazine- of formula 6 by reacting the compound of formula 5 prepared in step 1 with 5-aminoindazole or 6-aminoindazole in the presence of a base Preparing a 1-yl) -3-nitropyridine compound; 3) De-protection of the amine protecting group "B0C" (tert-butoxy carbonyl) in the compound of Formula 6 prepared in Step 2 6-indazolylamino-2- (piperazin-1-yl) -3 Preparing a nitropyridine compound; And 4) preparing a compound of Formula 1B by reacting the compound of Formula 7 prepared in Step 3 with an acid chloride compound of Formula 8 in the presence of a base. [Chemical Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 1B]

In the above formula, R ₂ is an indazol-5-ylamine group or an indazol-6-ylamine group; R ₅ is a linear or pulverized C _{1 to} C ₆ alkyl group; C ₃ -C ₆ cyclic alkyl group; C ₁ -C ₆ linear or pulverized hydroxyalkyl group; Unsaturated 5 to 6 membered heterocyclic compounds containing 1 to 3 heteroatoms selected from N, O and S, wherein the heterocyclic compound is unsubstituted or substituted with a C ₁ to C ₆ alkyl group, halogen or SCH ₃ Can be. 8. The process according to claim 7, wherein the BOC deprotection reaction of step 3 is carried out through base treatment after acid treatment. The method of claim 7, wherein the base of step 1, 2 or 4 is triethylamine, N, N-diisopropylethylamine, N-methylmorpholine, DBU (1,8-Diazabicyclo [5.4.0] undec -7-ene), N-methylpiperidine, 4-dimethylaminopyridine, N, N-dimethylaniline, 2, 6-lutidine and pyridine. A pharmaceutical composition for the prevention or treatment of osteoporosis, comprising the 6-indazolylamino-3-nitropyridine derivative compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.