WO2010027114A1

WO2010027114A1 - Use of pyrazole-pyridine derivatives and its salts for treating or reventin osteoporosis

Info

Publication number: WO2010027114A1
Application number: PCT/KR2008/005259
Authority: WO
Inventors: Pann-Ghill Suh; Jeom-Yong Kim; Mi-Ra Ma; Jung-Min Kim; Yun-Hee Kim; Seong-Hui Jin; Sung-Ho Ryu; Hyung-Ho Ha
Original assignee: Choongwae Pharma Corporation; Postech Academy-Industry Foundation
Priority date: 2008-09-05
Filing date: 2008-09-05
Publication date: 2010-03-11

Abstract

The present invention provides a pharmaceutical composition comprising a pyrazole-pyridine derivative or a pharmaceutically allowable salt thereof for preventing or treating osteoporosis, and particularly relates to a use of a pyrazole-pyridine derivative or a salt thereof that has a bone formation activity by promoting differentiation from a mesenchymal stem cells into osteoblast cells as a preventing or treating agent of bone metabolism diseases, in particular, osteoporosis. The present invention relates to a use of a pyrazole-pyridine derivative or a pharmaceutically allowable salt thereof as an agent for preventing or treating osteoporosis, and the present invention is capable of being used in various diseases that relates to a bone metabolism such as regeneration of the alveolar bone, osteogenesis imperfecta, osteomalacia and the like in addition to prevention or curing effect of osteoporosis.

Description

Use of pyrazole-pyridine derivatives and its salts for treating or preventing osteoporosis

Technical Field

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, comprising a pyrazole-pyridine derivative that is represented by the following Formula I or a salt thereof, and more particularly to a use of a pyrazole-pyridine derivative that is represented by the following Formula I or a salt thereof, which has a bone formation activity by promoting differentiation from mesenchymal stem cells into osteoblast cells, as a preventing or treating agent of bone metabolism diseases, in particular, osteoporosis. <Formula I>

Background Art

Osteoporosis is a disease of bone which is characterized by reduced bone mass and disruption of the micro-architecture of bone, giving decreased bone strength that lead to an increased risk of fracture even with a weak impact.

In normal bones, bone resorption by osteoclast cells and bone formation by osteoblast cells take place and a dynamic state in views of metabolism in a normal state is maintained. Imbalance between bone resorption and bone formation may result in a bone disease. Excessive bone resorption relatively to bone formation leads to a reduction of the bone density or the bone amount, which may cause osteoporosis.

The osteoporosis may be classified into postmenopausal osteoporosis, age-related osteoporosis, and medication-related osteoporosis. Postmenopausal osteoporosis results from lack of estrogen, thereby the activity of osteoclast cells is increased and that of osteoblast cells is suppressed. Age-related osteoporosis is caused by the reduction of performance of osteoblast cells according to the aging, and the medication-related osteoporosis is caused by the reduction of bone formation because of the reduction of performance and lifespan of the osteoblast cells by glucocorticoides.

As a curing agent of the osteoporosis, bisphosphonate drugs have been the main pharmacological measures that reduce the performance of osteoclast cells to suppress the excessive bone absorption. However, it is known that the bisphosphonate drugs are difficult to take and have the high risk of the occurrence of gullet / gastrointestinal ulcer. Currently, it is known that PTH (Parathyroid Hormone, Forteo ) that is on the market as the osteoporosis curing agent promotes the bone formation by osteoblast to increase bone mass and restore the micro-architecture of bone, thus showing the curing effect of osteoporosis.

Stem cells are cells that have the ability to renew themselves and differentiating into specialized cell types according to an appropriate signal. Mesenchymal stem cells may be subjected to differentiate into bones, cartilages, adipocytes, muscles and nerves, and play an important role in regeneration and recovery of them. If the differentiation of the mesenchymal progenitors into osteoblast cells is controlled wrong, diseases that relates to bones occurs (BR Olsen et al., 2000.). The current study of stem cell suggests it as a curing agent for the regeneration of organs or tissues, as well as for treating various diseases such as a cardiovascular system disease, a nervous system disease, a musculoskeletal disease, diabetes, and a spinal cord injury. If the from the stem cells to the desired cells is capable of being promoted or controlled by using the small molecular compound, this may be developed as a new medicine to a specific disease. The compound that promotes the differentiation from the mesenchymal stem cells into osteoblast cells may be used as a preventing and curing agent of bone metabolism diseases, particularly, osteoporosis.

It is reported that strontium (Strontium ranelate, PROTELOS^®), which was approved in Europe as an osteoporosis curing agent, is able to suppress bone absorption by the osteoclast cells by binding to bone surface and being deposited on calcium hydroxyapatite crystals_through the calcium sensing receptor, and to increase pre- osteoblast replication, osteoblast differentiation, and synthesis of extracellular matrix such as collagen, through a calcium-sensing receptor-dependent mechanism, thereby it promotes the bone formation (Biochem Pharmacol 74; 438-447, 2007).

Xu Wu, et al. found purmorphamine that is a small molecule with osteogenesis- inducing activity from the multipotent mesenchyma stem cell, and reported that this material increases the ALP activity that plays an important role in mineralization in C3 H 1OT 1/2 cells which are mouse embryonic mesoderm fibroblasts and the human bone marrow-derived mesenchymal progenitor cells. In addition, purmorphamine does not affect the proliferation of the general cell but stimulate pre-osteoblast cells to promote the differentiation into osteoblast cells, and shows the possibility of the differentiation of the stem cells by the small molecular compound and the possibility of the application as the osteoporosis curing agent by increasing the mineralization (J. AM. CHEM. SOC. 2002, 124, 14520).

Recently, it is reported that bortezomib that is the proteasome inhibitor-based myeloma curing agent promotes the differentiation from mesenchymal stem cells into osteoblast cells and improves the bone density in an osteoporosis-induced mouse, thereby suggesting the possibility of development of the small molecular compound as the osteoporosis curing agent that activates the differentiation from stem cells into osteoblast cells (J. Clin. Invest. 111 :1771-1782, 2008).

Accordingly, it is deemed that the compound for promoting the differentiation of the osteoblast affects the diseases that relate to the bone metabolism, currently, studies thereof have been made. Disclosure of Invention

Technical Problem

It is an object of the present invention to provide an agent for preventing or treating osteoporosis, which comprises a small molecular compound that activates differentiation into osteoblast cells. Technical Solution

The present inventors found that the pyrazole-pyridine derivative of Formula 1 and a salt thereof has the effect promoting differentiation from mesenchymal stem cells into osteoblast cells, thereby accomplishing the present invention.

The present invention provides a use of a pyrazole-pyridine derivative that is represented by the following Formula I and a pharmaceutically allowable salt thereof as an agent for preventing or treating bone metabolism diseases, in particular, osteoporosis. <Formula I>

wherein, R₁ is hydrogen or halogen,

R₂, R₃, and R₃' are each independently hydrogen or -(Xi)-R₅, wherein X₁ is Ci-C₈ straight or branched alkylene, O, CO, (CO)₂, SO, or SO₂, R₅ is: Ci~C₈ straight or branched alkyl; hydroxy; carboxy; C₁-C₈ straight or branched alkanol, C₁-C₈ alkoxy; C₁-C₈ acetoxy; C₂-C₈ alkenyl; C₂-C₈ alkynyl; C₃-C₈ cycloalkyl; C₆-C₂₀ aryl; Cs-C_2O heterocycloalkyl; C₆-C₂₀ aryl that is substituted with halogen; C₆-C_2O aryl that is substituted with nitrile; C₁-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl; C₁-C₈ straight or branched alkyl that is substituted with C₆-C_2O aryl substituted with halogen; C₂-C₈ alkenyl that is substituted with C₆-C_2O aryl; C₂-C₈ alkenyl that is substituted with C₆-C₂₀ aryl substituted with halogen; C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl; C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl substituted with halogen; NH₂; nitrile; or NA₁A₂, wherein Ai or A₂ are each independently the same as or different from each other, and Ci-C₈ straight or branched alkyl that is unsubstituted or substituted with phenyl; C₂-C₈ alkenyl; or C₆-C₂₀ aryl that is unsubstituted or substituted with halogen, Ci-C₄ alkyl, or C₁-C₄ alkoxy;

R₄ is hydrogen or -(X₂)-R₆;

X₂ is a single bond, C₁-C₈ straight or branched alkylene, C₂-C₈ alkenylene, C₆-C₂₀ arylene, CO, or SO₂;

R₆ is C₁-C₈ straight or branched alkyl; Ci-C₈ alkoxy; C₂-C₈ alkenyl; C₂-C₈ alkynyl; C₃-C₈ cycloalkyl; C₆-C_2O aryl; C₆-C₂₀ aryl that is substituted with Ci-C₈ alkoxy; C₆-C₂₀ aryl that is substituted with halogen; Cs-C₂₀ heterocycloalkyl; C₆-C₂₀ aryl that is substituted with halogen; C₁-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl; Ci-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl substituted with halogen; C₆-C₂₀ aryl that is substituted with C₆-C₂₀ alkyl; C₆-C₂₀ aryl that is substituted with Ci-C₈ alkyl substituted with halogen; C₂-C₈ alkenyl that is substituted with C₆-C₂₀ aryl substituted with Ci-C₈ alkoxy; C₂-C₈ alkenyl that is substituted with C₆-C₂₀ aryl substituted with halogen; C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl; or C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl substituted with halogen. In the heterocycloalkyl, as the hetero atom, N, S, O and the like may be used. In the compound, it is more preferable that

R₁ is fluorine; R₂, R₃, and R₃' are each independently hydrogen or -(Xi)-R₅, wherein Xi is Ci-Cg straight or branched alkylene, O, or CO; and R₅ is Ci-C₈ straight or branched alkyl; hydroxy; carboxy; C₆-C₂₀ aryl; C₁-C₈ acetoxy; C₆-C₂₀ aryl that is substituted with halogen; C₆-C₂₀ aryl that is substituted with nitrile; nitrile; or C₁-C₈ alkylamine; R₄ is hydrogen or -(X₂)-R₆;

X₂ is a single bond, C₆-C₂₀ arylene, CH₂, CO or SO₂; and R₆ is C₁-C₈ straight or branched alkyl; Ci-C₈ alkoxy; C₆-C₂₀ aryl that is substituted with Ci-C₈ alkoxy; C₆-C₂₀ aryl that is substituted with halogen; Ci-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl that is substituted with halogen; or C₆-C₂₀ aryl that is substituted with Ci-C₈ alkyl substituted with halogen.

Advantageous Effects

As described above, the present invention relates to a use of a pyrazole-pyridine derivative as a preventing or treating agent of osteoporosis, and the present invention is capable of being used for osteoporosis, as well as in various diseases that relates to a bone metabolism such as the regeneration of the alveolar bone, osteogenesis imperfecta, osteomalacia and the like.

In addition, since the present invention has prevention or curing effect for osteoporosis with low toxicity, it is suitable for medicine products.

Mode for Invention

The present invention relates to a use of a compound that is represented by Formula 1 or a salt thereof. It shows the effect as an agent for preventing or curing osteoporosis, and the present invention relates to a compound that is represented by the following Formula I or a pharmaceutical composition that comprises a pharmaceutically allowable salt thereof.

Hereinafter, the present invention will be described in detail.

1. Compound The compound of the present invention is a compound that is represented by the following Formula I or a pharmaceutically allowable salt thereof. <Formula I>

Substituting V groups R₁, R₂, R₃, R₃', and R₄ of Formula I are as described above. Pharmaceutically allowable salt may include an inorganic acid salt such as hydrochloride, bromate, sulfate, and phosphate of the compound, and an organic acid salt such as citrate, acetate, lactate, tartrate, fumarate, alkyl formate, propionic acid salt, oxalate, trifluoro acetate, methane sulfonate, maleic acid benzoate, gluconic acid salt, glyconic acid salt, succinate, 4-toluene sulfonate, galacturonic acid salt, embonic acid salt, glutamate and aspartate of the compound, but not limited thereto. It is preferable that the hydrochloride may be used as the inorganic acid salt and methane sulfonate may be used as the organic acid salt.

2. Production method of the compound

The compound that is represented by Formula I according to the present invention may be produced by using the following Reaction Scheme 1. <Reaction Scheme 1>

R₁, R₂, R₃, R₃', and R₄ as defined above have the same definition as R₁, R₂, R₃, R₃', and R₄ of Formula I, and in R₂L, R₃L and R₃'L in the above, L is a leaving group. Examples of the leaving group include halogen, an alcohol group and the like, but are not limited thereto.

The compound according to the present invention may be produced by using any conventional method. The synthesis method of the compound is described in the following Preparation Examples.

In addition, for example, the compound that is represented by Formula I may be produced through a synthetic path of the following Reaction Scheme 2. <Reaction Scheme 2>

R₁, R₂, R₃, and R₄ as defined above have the same definition as R₁, R₂, R₃, and R₄ of Formula I, and in R₂L, and R₃L in the above, L is a leaving group. Examples of the leaving group include halogen, an alcohol group and the like, but are not limited thereto. The following pyridinium derivative is a raw material that is used in a second reaction step of Reaction Scheme 2. Hereinafter, the synthetic method of the pyridinium derivative will be described. I. Synthesis of a pyridinium derivative

Preparation Example 1 : Production of 2-(2,4-dinitro-phenoxy)-isoindole-l,3(2HVdione

Triethyl amine (21.5 ml, 0.154 mol) was added to the N-hydroxyphthalimide (25.0 g, 0.153 mol) suspension in 500 mi acetone, and the mixture thereof was agitated at room temperature. The reaction mixture became dark red, and N-hydroxyphthalimide was slowly dissolved. This reactant was agitated until a homogeneous solution (about 10 min) was prepared. 2,4-dinitrochlorobenzene (31 g, 0.153 mol) was added thereto, and the reactant was agitated for 2 hours at room temperature. Next, the light yellow suspension was formed, and the reaction mixture was poured to cold water (500 mi). The precipitate was filtered, and washed three times by using cold MeOH. This solid was washed with 100 mi of hexane three times, and dried under vacuum to obtain the product as a white solid (48 g, yield 98%).

¹H-NMR (300 MHz, CDCl₃): δ 7.43 (d, J=9.3 Hz, IH), 7.88-7.91 (m, 2H), 7.96- 7.99 (m, 2H), 8.41 (dd, J=2.5 Hz, J=9.3 Hz, IH), 8.97 (d, J=2.7 Hz, IH)

Preparation Example 2: Production of O-(2,4-dinitrophenyl^')hvdroxyl amine

Hydrazine hydrate (8.86 ml, 0.18 mmol) in MeOH was added to 2-(2,4-dinitro- phenoxy)-isoindole-l ,3(2H)-dione (20 g, 60.7 mmol) in CH₂Cl₂ at O⁰C. The reaction mixture rapidly became light yellow, and the precipitate was formed. The suspension was left for 30 min at O⁰C, cold water-soluble HCl (1 N, 400 ml) was added thereto, and the reactant was shaken rapidly at 0⁰C. The reaction mixture was filtered through a Buchner funnel, and the precipitate was washed by using ACN (50 mi) three times. The filtrate was poured in a separatory funnel, the organic phase was separated, and the aqueous phase was extracted by using CH₂Cl₂. The organic phase was combined, dried, filtered under Na₂SO₄, and concentrated under reduced pressure to obtain the title compound as an organic solid (12 g, yield 83%).

¹H-NMR (300 MHz, CDCl₃): 6.35(brs, 2H), 7.99(d, J=9.4 Hz, IH), 8.37(dd, J=2.7 Hz, J=9.4 Hz, IH), 8.76(d, J=2.8 Hz, IH)

Preparation Example 3: Production of 2,4-dinitro-phenolate l-amino-4-methoxy- pyridinium

4-methoxy pyridine (3.8 ml, 0.037 mmol) and O-(2,4- dinitrophenyl)hydroxylamine (8.19 g, 0.041 mmol) were mixed with each other in ACN. The reaction vessel was sealed and the reactant was agitated for 24 hours at 4O⁰C. The reactant was concentrated, and the residual was pulverized by using Et₂O. The solid was filtered, and dried under vacuum to obtain the product of 2,4-dinitro-phenolatel-amino-4- methoxy-pyridinium as a light orange solid (11 g, yield 95%).

¹H-NMR (300 MHz, DMSOd₆): 4.04(s, 3H), 6.31(d, J-9.7 Hz, IH), 7.51(d, J=7.4 Hz, 2H), 7.75-7.8 l(m, 3H), 8.58(d, J=3.2 Hz, IH), 8.65(d, J=7.5 Hz, 2H)

Preparation Example 4: 2,4-dinitro-phenolate l-amino-2-methyl-pyridinium

The same process as Preparation Example 3 was carried out to obtain the title compound as a light orange solid, except that 2-methyl pyridine was used as the starting material.

¹H-NMR (300 MHz, DMSO-d₆) : 2.71(s, 3H), 6.29(d, J=9.9 Hz, IH), 7.75(dd, J=2.9 Hz, J=9.6 Hz, IH), 7.86(dd, J=7.4 Hz, IH), 7.95(d, J=7.6 Hz, IH), 8.03(brs, 2H), 8.20(dd, =7.8 Hz, IH), 8.58(d, J=2.9 Hz, IH), 8.78(d, J=6.3 Hz, IH)

II. Synthesis of the pyrazole-pyridine derivative Preparation Example 5: Production of 2-chloro-6-ethvnyl-5-fluoro-nicotinonitrile

Triethyl amine (29 ml, 0.209 mol), copper iodine (1.99 g, 0.01 mol) and palladium chloride bis-triphenyl phosphine (3.67 g, 0.005 mol) were added to a 2,6- dichloro-5-fluoro-3-pyridine carbonitrile (20 g, 0.105 mol) solution. TMS-acetylene (17.4 ml, 0.12 mol) was slowly added to the reaction mixture, and the reaction mixture was agitated for one day at room temperature. The reaction mixture was diluted by using hexane, and the solid was filtered. The natural intermediate that was silylated in MeOH was added to potassium fluoride (6.08 g, 0.104 mol), and the reactant was agitated for 10 min at room temperature. It was concentrated under vacuum with silica gel column chromatography using ethyl acetate/hexane, to obtain 7.6 g of solid (yield 40%). 1H-NMR (300 MHz, CDCl₃): 3.70(s, IH), 7.72(d, J=7 Hz, IH) Preparation Example 6: Production of the 2-chloro-5-fluoro-6-pyrazole [l,5-a]pyridine-3- yl-nicotinonitrile derivative

R₄ as defined above is the same as the definition of R₄ of Formula I.

The pyridinium derivative (0.013 mol) and K₂CO₃ (0.033 mol) were added to the 2-chloro-6-ethynyl-5-fluoro-nicotinonitrile (0.011 mol) agitation solution in THF. The reaction mixture was agitated for one day at room temperature, the solvent was removed under vacuum, MC (Methylene chloride) was added thereto, and the reaction mixture was washed by using water. The organic phase was dried by using Na₂SO₄, and concentrated under vacuum. The desired product was obtained by using the column chromatography using ethyl acetate/hexane.

Preparation Example 7:

2,4-dinitro-phenolate l-amino-4-methoxy-pyridinium was used as the starting material to obtain the title compound as a light orange solid by the same process as Preparation Example 6 (yield 50%).

¹H-NMR (300 MHz, CDCl₃): 7.03(dd, J=6.99 Hz, IH), 7.48(dd, J=7 Hz, IH), 7.66(d, J=IO Hz, IH), 8.58-8.63(m, 2H), 8.67(d, J=9 Hz, IH)

The compound that was obtained from Preparation Example 3 was used as the starting material to obtain the title compound as a light orange solid by the same process as Preparation Example 6 (yield 35%).

¹H-NMR (300 MHz, CDCl₃): 3.98(s, 3H), 6.69(dd, J=2.9 Hz, J=7.6 Hz, IH), 7.60(d, J=10.1 Hz, IH), 8.02(d, J=2.7 Hz, IH), 8.37(d, J=7.6 Hz, IH), 8.51(d, J=3.9 Hz, IH)

The compound that was obtained from Preparation Example 4 was used as the starting material to obtain the title compound as a light orange solid by the same process as Preparation Example 6 (yield 35%).

¹H-NMR (300 MHz, CDCl₃): 2.83(s, 3H), 6.92(d, J=7 Hz, IH), 7.42(dd, J=8.9 Hz, IH), 7.63(d, J=IO Hz, IH), 8.58(d, J=8.9 Hz, IH), 8.63(d, J=3.9 Hz, IH)

Preparation Example 10: Production of 5-fluoro-6-pyrazole [l,5-a]pyridine-3-yl-lH- pyr azolo [3, 4-b1pyridine-3-yl-amine derivative

R₄ as defined above is the same as the definition of R₄ of Formula I.

Hydrazine hydrate (0.05 mol) was added to an agitated solution of 2-chloro-5- fluoro-6-pyrazole [1,5-a] pyridine-3-yl-nicotinonitrile derivative (0.01 mol) in 2-methoxy ethanol, and the reaction mixture was refluxed for one day. The solvent was concentrated, and the resulting residual was pulverized by using Et₂O. The solid was filtered, and dried under vacuum to obtain the desired product as a yellow solid.

Preparation Example 11 : 2

The compound that was obtained from Preparation Example 7 was used as the starting material to obtain the title compound as a light orange solid by the same process as Preparation Example 10.

¹H-NMR (300 MHz, DMSO-d6) : 5.53(brs, 2H), 7.1 l(dd, J=6.9 Hz, J=8.9 Hz, IH), 7.52(dd, J=6.4 Hz, J=6.8 Hz, IH), 8.02(d, J=I 1.8 Hz, IH), 8.55(d, J=4.2 Hz, IH), 8.68(d, J=9 Hz, IH), 8.82(d, J=7 Hz, IH), 11.98(brs, IH)

Preparation Example 12:

The compound that was obtained from Preparation Example 8 was used as the starting material to obtain the title compound as a light orange solid by the same process as Preparation Example 10.

¹H-NMR (300 MHz, DMSO-d6) : 3.06(s, 3H), 5.48(s, 2H), 6.79(dd, J=2.8 Hz, J=7.5 Hz, IH), 7.95(d, J=I 1.8 Hz, IH), 8.17(d, J=2.8 Hz, IH), 8.46(d, J=4.2 Hz, IH), 8.69(d, J=7.5 Hz, IH), 11.93(s, IH)

Preparation Example 13:

The compound that was obtained from Preparation Example 9 was used as the starting material to obtain the title compound as a light orange solid by the same process as Preparation Example 10.

¹H-NMR (300 MHz, DMSO-d6) : 2.76(s, 3H), 5.52(s, 2H), 7.03(d, J=6.9 Hz, IH), 7.45(dd, J=6.9 Hz, IH), 8.02(d, J=I 1.8 Hz, IH), 8.59(d, J=4.3 Hz, IH), 8.65(d, J=8.9 Hz, IH), 11.96(s, IH) Preparation Example 14:

R₄ and R₅ as defined above are the same as the definition of R₄ and R₅ of Formula I.

Acyl chloride (1.67 mmol) was added to an agitated solution of 5-fluoro-6- pyrazolo [1, 5-a] pyridine-3-yl-lH-pyrazolo [3,4-b]pyridine-3-ylamine derivative (1.11 mmol) in pyridine. After the reaction mixture was refluxed for one day, the solvent was concentrated, and IN HCl was added thereto. The residual was extracted by using ethyl acetate, and the organic layer was dried under Na₂SO₄. After the organic phase was concentrated, it was purified by the recrystallization using an appropriate solvent.

In the following Table 1 , representative examples that is produced by Reaction Scheme 2 and is represented by Formula I is described. M of Table 1 represents a molecular weight, and M + H represents a mass spectrum value that was measured by using a mass spectroscopy (ESI-MS).

The preparation data of the compound listed in Table 1 are as follows:

Compound No. 1-1: cyclopropanecarboxylic acid(5-fluoro-6-pyrazolo[l,5-a1 pyridine-3- yl-lH-pyrazolo [3,4-b] pyridine-3-vD-amide

¹H-NMR (300 MHz, DMSO-d6) : 0.82-0.89(m, 4H), 1.92-2.01(m, IH), 7.13(dd, J=6.8 Hz, IH), 7.55(dd, J=8.9 Hz, IH), 8.20(d, J=12.5 Hz, IH), 8.61(d, J=4.2 Hz, IH), 8.72(d, J=8.9 Hz, IH), 8.89(d, J=7 Hz, IH), 11.02(s, IH), 13.18(s, IH) Compound No. 1-2: cyclopentanecarboxylic acid(5-fluoro-6-pyrazolo[l,5-al pyridine-3-yl- lH-pyrazolo[3, 4-b] pyridine-3-yl)-amide

¹H-NMR (300 MHz, OMSO-dό) : 1.55-1.58(m, 2H), 1.62-1.81(m, 4H), 1.83- 1.91(m, 2H), 2.93(q, J=7.8 Hz, IH), 7.15(dd, J=6.8 Hz, IH), 7.58(dd, J=6.9 Hz, IH), 8.20(d, J=8.4 Hz, IH), 8.60(d, J=4.2 Hz, IH), 8.72(d, J=9 Hz, IH), 8.88(d, J=6.9 Hz, IH), 10.66(s, IH), 13.17(s, IH)

Compound No. 1-3: cyclopropanecarboxylic acid[5-fluoro-6-(7-methyl-pyrazolo [1,5-a] pyridine-3 -ylV 1 H-pyrazolo (^"3 ,4-b]pyridine-3 -yl]-amide

¹H-NMR (300 MHz, DMSO- d6) : 0.84-0.89(m, 2H), 1.96-2.01 (m, IH), 2.77(s, 3H), 7.07(d, J=6.9 Hz, IH), 7.53(dd, J=I Hz, IH), 8.19(d, J=12.5 Hz, IH), 8.63(d, J=4.4 Hz, IH), 8.69(d, J=9.1 Hz, IH), 11.01(s, IH), 13.17(s, IH)

Compound No. 1-4: cyclopentanecarboxylic acid[5-fluoro-6-(7-methyl-pyrazolo [1,5-a] pyridine-3-yl)-l H-pyrazolo r3,4-blpyridine-3-yll-amide ¹H-NMR (300 MHz, DMSO-Jd) : 1.55-1.59(m, 2H), 1.61-1.79(m, 4H), 1.81- 1.91(m, 2H), 2.74(s, 3H), 2.93(q, J=7.6 Hz, IH), 7.02(d, J=6.9 Hz, IH), 7.46(dd, J=7.1 Hz, IH), 8.17(d, J=12.5 Hz, IH), 8.60(d, J=4.3 Hz, IH), 8.63(d, J=8.9 Hz, IH), 10.66(s, IH), 13.14(s, IH)

Compound No. 1-5: 3-cvano-N-[5-fluoro-6-(7-methyl-pyrazolo [1, 5-a]pyridme-3-yD-lH- pyrazolo[3,4-blpyridine-3-yl]-benzamide

¹H-NMR (300 MHz, DMSO-J6) : 2.78(s, 3H), 7.10 (d, J=6.8 Hz, IH), 7.54(dd, J=7 Hz, IH), 7.78(d, J=7.8 Hz, IH), 8.09(d, J=7.7 Hz, IH), 8.24(d, J=12.2 Hz, IH), 8.36(d, J=7.9 Hz, IH), 8.52(s, IH), 8.66-8.71(m, 2H), 11.34(s, IH), 13.42(s, IH)

Compound No. 1-6: N-[5-fluoro-6-(7-methyl-pyrazolo [1,5-a] pyridine-3-vD-lH-pyrazolo [3,4-b]pyridine-3-yll-2-(4-fluoro-phenyl)-acetamide

¹H-NMR (300 MHz, CDCl₃) : 2.77(s, 3H), 3.75(s, 2H), 7.07(d, J=6.9 Hz, IH), 7.17(dd, J=6.8 Hz, IH), 7.42(dd, J=5.7 Hz, IH), 7.52(d, J=7 Hz, IH), 8.16(d, J=12.4 Hz, IH), 8.65(dd, J=10.3 Hz, 2H), 10.99(s, IH), 13.29(s, IH)

Compound No. 1-7: 4-dimethylamino-N-[5-fluoro-6-(7-methyl-pyrazolo [1, 5-a] pyridine- 3-yl~)-lH-pyrazolo [3,4-b1pyridine-3-yl]-benzamide 1H-NMR (300 MHz, CDCl₃) : 2.77(s, 3H), 3.01(s, 6H), 6.75(d, J=9 Hz, 2H), 7.06(d, J=6.8 Hz, IH), 7.53(dd, J=7 Hz, IH), 7.99(d, J=8.8 Hz, 2H), 8.16(d, J=12.3 Hz, IH), 8.65(d, J=4.2 Hz, IH), 8.68(d, J=8.9 Hz, IH), 10.71(s, IH), 13.25(s, IH)

Compound No. 1-8: cyclopropanecarboxylic acid [5-fluoro-6-(5-methoxy-pyrazolo [1,5-a] pyridme-3-vD-lH-pyrazolo |^"3,4-b1pyridine-3-yl^"|-amide

¹H-NMR (300 MHz, CDCl₃) : 0.86-0.92(m, 4H), 1.91-1.99(m, IH), 3.99(s, 3H), 6.82(dd, J=2.7 Hz, J=7.6 Hz, IH), 8.16-8.22(m, 2H), 8.50(d, J=4.3 Hz, IH), 8.72(d, J=7.6 Hz, IH), 11.02(s, IH), 13.14(s, IH) Compound No. 1-9: N-[5-fluoro-6-(5-methoxy-pyrazolo [1, 5-a] pyridine-3-yr)-lH- pyrazolo [3,4-blpyridine-3-yl]-2-(4-fluoro-phenyl^')-acetamide

¹H-NMR (300 MHz, CDCl₃) : 3.71(s, 2H), 3.94(s, 3H), 6.78(dd, J=2.8 Hz, J=7.5 Hz, IH), 7.13(d, J=8.8 Hz, 2H), 7.38(dd, J=5.7 Hz, 2H), 8.09(d, J=12.6 Hz, IH), 8.16(d, J=2.8 Hz, IH), 8.45(d, J=4.3 Hz, IH), 8.67(d, J=7.5 Hz, IH), 10.95(s, IH), 13.16(s, IH)

Compound No. 1-10: 2-(4-fluoro-phenyl)-N-(^'5-fluoro-6-pyrazolo [L 5-a] pyridine-3-yl- lH-pyrazolo [3, 4-blpyridine-3-yl)-acetamide

¹H-NMR (300 MHz, CDCl₃) : 3.75(s, 2H), 7.1 l-7.17(m, 3H), 7.42(d, J=5.8 Hz, IH), 7.45(d, J=5.9 Hz, IH), 7.57(d, J=6.9 Hz, IH), 8.16(d, J=8.4 Hz, IH), 8.59(d, J=4.2 Hz, IH), 8.71(d, J=9 Hz, IH), 8.86(d, J=6.9 Hz, IH), 10.99(s, IH), 13.23(s, IH)

Compound No. 1-11 : 5-fluoro-6-pyrazolo[l,5-alpyridine-3-yl-lH-pyrazolo[3,4-b1pyridine- 3-ylamine

IH-NMR (300 MHz, DMSO-d6) : 5.53(brs, 2H), 7.1 l(dd, J=6.9 Hz, J=8.9 Hz, IH), 7.52(dd, J=6.4 Hz, J=6.8 Hz, IH), 8.02(d, J=I 1.8 Hz, IH), 8.55(d, J=4.2 Hz, IH), 8.68(d, J=9 Hz, IH), 8.82(d, J=7 Hz, IH), 11.98(brs, IH)

Compound No. 1-12: 5-fluoro-6-(5-methoxy-pyrazolori,5-alpyridine-3-yl)-lH- p yrazolo [3 ,4-b1pyridine-3 -ylamine

IH-NMR (300 MHz, DMSO-d6) : 3.06(s, 3H), 5.48(s, 2H), 6.79(dd, J=2.8 Hz, J=7.5 Hz, IH), 7.95(d, J=I 1.8 Hz, IH), 8.17(d, J=2.8 Hz, IH), 8.46(d, J=4.2 Hz, IH), 8.69(d, J=7.5 Hz, IH), 11.93(s, IH)

Compound No. 1-13: 4-fluoro-N-(^'5-fluoro-6-(5-methoxyH-pyrazolo[l,5-alpyridin-3-yl)- lH-pyrazolof3,4-b]pyridin-3-yl)benzamide

IH-NMR (300 MHz, DMSO-d6) : 4.00 (s, 3H), 6.86(dd, J=2.7 Hz, J=7.6 Hz, IH), 7.39 (t,

J=8.7 Hz, 2H), 8.14-8.20(m, 2H), 8.24 (d, J=2.7 Hz, IH), 8.54 (d, J = 4.2 Hz, IH), 8.76 (d,

J=7.5 Hz, IH), 11.19 (s, IH), 13.36 (s, IH)

Compound No. 1-14: S-cyano-N-fS-fluoro-ό-rH-pyrazolofLS-aipyridin-S-vD-lH- pyrazolo[3,4-b]pyridin-3-yl)benzamide

IH-NMR (300 MHz, CDC13) : 7.14(dd, J=6.8 Hz, IH), 7.57(dd, J=6.9 Hz, IH), 7.78 (dd,

J=7.9 Hz, IH), 8.09(d, J=7.7 Hz, IH), 8.21(d, J=12.1 Hz, IH), 8.36(d, J=8 Hz, lH),8.52(s,

IH), 8.63(d, J=4.2 Hz, IH), 8.75(d, J=8.9 Hz, IH), 8.87(d, J=6.8 Hz, IH), 11.35 (s, IH),

13.43(s, IH)

In addition, Preparation Example according to Reaction Scheme 2 is described below. Preparation Example 15:

R₄ as defined above is the same as the definition OfR₄ of Formula I. The derivative that was obtained from Preparation Example 10 was used as the starting material, and Cs₂CO₃ (0.035 mol) was added to the starting material solution in DMF. The mixture was agitated for 30 min at room temperature. Next, 2-bromoethyl acetate (0.014 mol) was added thereto, and the solution was heated for one day at 50⁰C. Next, the reactant was extracted by using ethyl acetate and washed with water. The organic phase was dried by using Na₂SO₄, and concentrated under vacuum. Through the column chromatography using ethyl acetate/hexane, the desired product was obtained, and the obtained representative compounds are shown in the following Table 2.

Preparation Example 16:

R₄ and R₅ as defined above are the same as the definition OfR₄ and R₅ of Formula I.

The derivative that was obtained from Preparation Example 15 was used as the starting material, and DIEA (5.249 mmol) and acyl chloride (2.62 mmol) were added to an agitated solution of the starting material in dioxane. The reaction mixture was agitated for 4 hours at room temperature, IN HCl was added thereto, the formed solid was filtered, and it was washed by using Et₂O. The solid was concentrated and dried under vacuum to obtain the desired product as a yellow solid, and the obtained representative compounds are shown in the following Table 2.

Preparation Example 17:

The derivative that was obtained from Preparation Example 16 was used as the starting material, and K₂CO₃ (1.96 mmol) was added to an agitated solution of the starting material in MeOH. The reaction mixture was agitated for 1 hour at room temperature, the solvent was removed under vacuum, and the formed solid was extracted by using MC. The organic phase was dried by using Na₂SO₄, and concentrated under vacuum to obtain the product as a solid, and the obtained representative compounds are shown in the following Table 2.

In the following Table 2, representative examples that were obtained by Preparation Examples 19 to 21 according to Reaction Scheme 2 and is represented by Formula I is described. M of Table 2 represents a molecular weight, and M + H represents a mass spectrum value that is measured by using a mass spectroscopy (ESI-MS).

The preparation data of the above compounds are as follows:

Compound No. 1-15: [2-(3-amino-5-fluoro-6-(7-methylpyrazolo[l,5-alpyridin-3-yl)-lH- pyrazolo[3,4-b]pyridin-l -vDethyl acetate]

¹H-NMR (300 MHz, CDCl₃) : 1 83(s, 3H), 2.84(s, 3H), 4.04(s, 2H), 4.51(t, J=5.4 Hz, 2H), 4.63(t, J=5.2 Hz, 2H), 6.83(d, J=6.8 Hz, IH), 7.37(dd, J=7 Hz, IH), 7.56(d, J=ILl Hz, IH), 8.70(d, J=4.2 Hz, IH), 8.79(d, J=8.9 Hz, IH) Compound No. 1-16: r2-(5-fluoro-3-(4-fluorobenzamido)-6-(7-methylpyrazolori,5- a]pyridin-3 - yl)- 1 H-pyrazolo[^~3 ,4-b]pyridin- 1 - vDethyl acetate]

¹H-NMR (300 MHz, CDCl₃) : 1.85(s, 3H), 2.85(s, 3H), 4.57(t, J=5.3 Hz, IH), 4.75(t, J=5.4 Hz, IH), 6.87(d, J=6.9 Hz, IH), 7.19(d, J=8.5 Hz, IH), 7.39(dd, J=7 Hz, IH), 7.99(dd, J=5.2 Hz, J=8.8 Hz, IH), 8.38(d, J=12.1 Hz, IH), 8.52(s, IH), 8.75(dd, J=4.2 Hz, IH)

Compound No. 1-17: [2-(3-amino-5-fluoro-6-(pyrazolo[^"L5-a]pyridin-3-yl)-lH- pyrazolo[3 ,4-blpyridin- 1 -yDethyl acetate]

¹H-NMR (300 MHz, CDCl₃) : 1 83(s, 3H), 4.06(s, 2H), 4.53(t, J=5.2 Hz, IH), 4.60(t, J=5.1 Hz, IH), 6.96(dd, J=6.8 Hz, IH), 7.43(dd, J=7.8 Hz, IH), 7.56(d, J=ILl Hz, IH), 8.55(d, J=7 Hz, IH), 8.65(d, J=4.2 Hz, IH), 8.79(dd, J=8.9 Hz, IH)

Compound No. 1-18: [2-(3-amino-5-fluoro-6-(5-methoxypyrazolo|^"L5-a]pyridin-3-yl^')-lH- pyrazolo[3,4-b]pyridin-l -vDethyl acetate]

¹H-NMR (300 MHz, CDCl₃) : 1.79(s, 3H), 4.02(s, 3H), 4.05(s, 2H), 4.52(t, J=5.1 Hz, IH), 4.59(t, J=5.0 Hz, IH), 6.62(dd, J=2.8 Hz, J=7.5 Hz, IH), 7.53(d, J=11.2 Hz, IH), 8.16(d, J=2.8 Hz, IH), 8.36(d, J=7.6 Hz, IH), 8.57(d, J=4.3 Hz, IH)

Compound No. 1-19: [2-(3-(^'cyclopropanecarboxamido)-5-fluoro-6-(^'pyrazolo[l,5- a]pyridin-3-yl)- 1 H-pyrazolo[3 ,4-b]pyridin- 1 -vDethyl acetatei 1H-NMR (300 MHz, DMS(Wd) : 0.85-0.93(m, 4H), 1.76(s, 3H), 1.96-1.98(m, IH), 4.49(t, J=4.9 Hz, IH), 4.70(t, J=4.9 Hz, IH), 7.17(dd, J=6.8 Hz, IH), 7.59(t, J=7.2 Hz, IH), 8.22(d, J=12.5 Hz, IH), 8.62(d, J=4.0 Hz, IH), 8.77(d, J=8.9 Hz, IH), 8.87(d, J=6.9 Hz, IH), Compound No. 1-20: [^"2-(3-(cvclopentanecarboxamido)-5-fluoro-6-(pyrazolo[l,5- a]pyridin-3 -yP- 1 H-pyrazolo [3 ,4-b]pyridin- 1 - vPethyl acetate]

¹H-NMR (300 MHz, DMSO-Jd) : 1.56-1.59(m, 2H), 1.64-1.81(m, 7H), 1.89-1.91(m, 2H), 2.91-2.96(m, IH), 4.49(t, J=5 Hz, 2H), 4.70(t, J=5 Hz, 2H), 7.17(dd, J=6.8 Hz, IH), 7.59(dd, J=7.6 Hz, IH), 8.24(d, J=12.4 Hz, IH), 8.63(d, J=2.1 Hz, IH), 8.78(dd, J=8.9 Hz, IH), 8.87(dd, J=6.9 Hz, IH), 10.8(s, IH)

Compound No. 1-21 : [N-(5-fluoro-l-(2-hydroxyethyl)-6-(pyrazolo(^"l,5-a]pyridin-3-yπ-lH- pyrazolo[3,4-b]pyridin-3-yl)-2-(4-fluorophenyl)acetamide]

¹H-NMR (300 MHz, DMSO-Jd) : 3.75(s, 2H), 3.91(s, 2H), 4.50(t, J=5.5 Hz, 2H), 7.14- 7.20(m, 3H), 7.41(d, J=5.6 Hz, IH), 7.42(d, J=5.8 Hz, IH), 7.60(dd, J=8.6 Hz, IH), 8.19(d, J=12.4 Hz, IH), 8.63(d, J=4.3 Hz, IH), 8.78(d, J=8.9 Hz, IH), 8.89(d, J=6.9 Hz, IH), 11.10(s, IH)

Compound No. 1-22: [2-(3-amino-5-fluoro-6-(pyrazolo[L5-a]pyridin-3-yl)-lH- ρyrazolo[3,4-b]pyridin-l-yl)ethanol]

¹H-NMR (300 MHz, DMSO-Jd) : 3.81(t, J=6.0 Hz, 2H), 4.28(t, J=6.1 Hz, 2H), 4.8 l(s, IH), 5.61(s, 2H), 7.12(dd, J=6.8 Hz, IH), 7.55(dd, J=6.8 Hz, IH), 8.02(d, J=11.7 Hz, IH), 8.56(d, J=4.3 Hz, IH), 8.74(d, J=8.9 Hz, IH), 8.83(d, J=6.9 Hz, IH) Compound No. 1-23: [2-(3-benzamido-5-fluoro-6-(pyrazolo[L5-a]pyridin-3-yl)-lH- pyrazolo[3,4-b]pyridin-l -vPethyl acetate]

¹H-NMR (300 MHz, CDCl₃) : 1.86(s, 3H), 4.59(t, J=5.3 Hz, 2H), 4.76(t, J=5.4 Hz, 2H), 6.99(dd, J=6.9 Hz, IH), 7.44-7.62(m, 4H), 7.99(d, J=6.9 Hz, 2H), 8.45(d, J=12 Hz, IH), 8.57-8.60(m, 2H), 8.72(d, J=AA Hz, IH), 8.81(d, J-8.9 Hz, IH)

Compound No. 1-24: [N-(5-fluoro-l-(^'2-hvdroxyethyl^')-6-(pyrazolo[l,5-alpyridin-3-yl)-lH- pyrazolo[3,4-b1pyridin-3-yl)benzamide] 1H-NMR (300 MHz, DMSO- d6) : 3.97(q, J=5.4 Hz, 2H), 4.56(t, J=5.7 Hz, 2H), 4.96(t, J=5.5 Hz, IH), 7.17(dd, J=6.8 Hz, IH), 7.53-7.65(m, 4H), 8.10(d, J=7.3 Hz, 2H), 8.21(d, J=12.2 Hz, IH), 8.64(d, J=4.3 Hz, IH), 8.81(d, J=8.8 Hz, IH), 8.88(d, J=6.9 Hz, IH), 11.21(s, IH) Compound No. 1-25: [2-(3-amino-5-fluoro-6-f5-methoxypyrazolo[l,5-a]pyridin-3-yl)-lH- pyrazolo[3,4-blpyridin-l-yl)ethanol]

¹H-NMR (300 MHz, DMSO-J6) : 3.86(q, J=5.7 Hz, 2H), 3.94(s, 3H), 4.30(t, J=6.0 Hz, 2H), 4.60(t, J-5.5 Hz, 2H), 5.14(s, 2H), 6.64(dd, J-2.9 Hz, J=7.5 Hz, IH), 7.81(d, J=W.6 Hz, IH), 8.21(d, J=2.8 Hz, IH), 8.38(d, J=4.1 Hz, IH), 8.41(d, J=7.6 Hz, IH)

Compound No. 1-26: [2-(3-benzamido-5-fluoro-6-(5-methoxypyrazolo[l,5-a]pyridin-3- yl)-l H-pyrazolo [3, 4-b]pyridin-l-yl)ethyl acetate]

¹H-NMR (300 MHz, DMSO-Jd): 1.77(s, 3H), 4.05(s, 3H), 4.54(t, J=5.0 Hz, 2H), 4.77(t, J=5.0 Hz, 2H), 6.86(dd, J=2.8 Hz, J=7.5 Hz, IH), 7.56-7.64(m, 3H), 8.12(d, J=7.1 Hz, 2H), 8.20-8.25(m, 2H), 8.59(d, J=4.5 Hz, IH), 8.75(d, J=7.5 Hz, IH), 11.23(s, IH)

Compound No. 1-27: [N-(5-fluoro-l-(2-hvdroxyemyl)-6-(5-memoxypyrazolo|^"l,5- a]pyridin-3-yl)-lH-pyrazolo[3,4-blpyridin-3-yl)benzamidel

¹H-NMR (300 MHz, Methaol-^) : 3.97(s, 3H), 4.05(t, J=5.5 Hz, 2H), 4.58(t, J=5.3 Hz, 2H), 6.65(dd, J=2.8 Hz, J=7.5 Hz, IH), 7.46-7.56(m, 3H), 7.96(d, J=6.9 Hz, 2H), 8.18(d, J=I 1.9 Hz, 2H), 8.33(d, J=7.5 Hz, IH), 8.53(d, J=4.1 Hz, IH)

Compound No. 1-28: [4-fluoro-N-(5-fluoro-l-(4-fluorobenzoyl)-6-(7-methylpyrazolo[l,5- aipyridin-3 -yl)- 1 H-pyrazolo [3 ,4-b1pyridin-3 -ypbenzamide] IH-NMR (300 MHz, DMSO-d6) : 2.75(s, 3H), 7.07(d, J=6.9 Hz, IH), 7.37-7.46(m, 5H), 8.09(dd, J=5.6 Hz, J=8.6 Hz, 2H), 8.17(dd, J-5.6 Hz, J=8.6 Hz, 2H), 8.30(d, j=12 Hz, IH), 8.57(d, J=8.8 Hz, IH), 8.69(d, J=4.3 Hz, IH), 11.58(s, IH)

Compound No. 1-29: [N-(l-(cvclopentanecarbonyl)-5-fluoro-6-(pyrazolori,5-alpyridin-3- vD-lH-pyrazolofS^-blpyridin-S-vDcvclopentanecarboxamide]

IH-NMR (300 MHz, DMSO-d6) : 1.56-1.61(m, 2H), 1.65-1.82(m, 8H), 1.87-1.94(m, 4H),

2.01-2.09(m, 4H), 2.95-3.06(m, 2H), 7.17(dd, J=6.8 Hz, IH), 7.67(dd, J=6.8 Hz, IH),

8.34(d, J=12.1 Hz, IH), 8.68(d, J=4.2 Hz, IH), 8.91(d, J=6.9 Hz, IH), 9.19(d, j=8.6 Hz,

IH), 11.24(s, IH)

Compound No. 1-30: [2-(5-fluoro-3-(2-(4-fluorophenyl)acetamido)-6-(pyrazolo|^"l,5- a]pyridin-3 -yl)- 1 H-pyrazolo [3 ,4-blpyridin- 1 -yPethyl acetate]

IH-NMR (300 MHz, DMSO-d6) : 1.74(s,3H), 3.75(s, 2H), 4.50(t, J=4.8 Hz, 2H), 4.70(t, j=4.7 Hz, 2H), 7.14-7.19(m, 3H), 7.39(dd, J=7 Hz, 2H), 7.58(dd, J=7.3 Hz, IH), 8.19(d, J=12.5 Hz, IH), 8.62(d, J=4.1 Hz, IH), 8.79(d, J=8.9 Hz, IH), 8.88(d, J=6.8 Hz, IH),

11.26(s, IH)

Compound No. 1-31 : 2-(5-fluoro-3-(4-fluorobenzamido)-6-(H-pyrazolo[l,5-a]pyridin-3- vD- 1 H-pyrazolo [3 ,4-b]pyridin- 1 -yPethyl acetate

IH-NMR (300 MHz, CDC13) : 1.83(s, 3H), 4.57(t, J=5.1 Hz, 2H), 4.74(t, J=5.0 Hz, 2H), 6.97(dd, J=6.9 Hz, IH), 7.18(t, J=8 Hz, 2H), 7.45(dd, J=8.2 Hz, IH), 7.99(dd, J=5.2 Hz, J=7.5 Hz, 2H), 8.39(t, J=12 Hz, IH), 8.57-8.60(m, 2H), 8.70(d, J=4.0 Hz, IH), 8.79(d, J=8.5 Hz, IH)

Compound No. 1-32: N-(5-fluoro-l-(2-hvdroxyethyl)-6-(H-pyrazolo[l,5-alpyridin-3-yl^')- 1 H-pyrazolo[3 Λ-blpyridin-S-vDcyclopentanecarboxamide

IH-NMR (300 MHz, DMSO-dό) : 1.55-1.57(m, 2H), 1.59-1.74(m, 4H), 1.80-1.90(m, 2H), 2.89-2.94(m, IH), 3.90(s, 2H), 4.48(t, J=5.7 Hz, 2H), 7.16(dd, J=6.9 Hz, IH), 7.59(dd, J=7.2 Hz, IH), 8.22(d, J=12.4 Hz, IH), 8.62(d, J=4.3 Hz, IH), 8.78(d, J=8.9 Hz, IH), 8.86(d, J=6.9 Hz, IH), 10.76(s, IH)

Compound No. 1-33: 4-fluoro-N-(^'5-fluoro-6-(5-methoxypyrazolo[^'l,5-a1pyridin-3-yl)-lH- pwazolo[3,4-b]pyridin-3-yr)benzamide hydrochloride

¹H-NMR (300 MHz, DMSO-d6): 11.17 (s, IH), 8.75(d, J=7.5 Hz, IH), 8.53(d, J=4.2 Hz, IH), 8.24(d, J=3.0 Hz, IH), 8.20-8.14 (m, 3H), 7.39(t, J=8.7 Hz, 2H), 6.85(dd, J=2.7, 7.5 Hz, IH), 4.00(s, 3H) Compound No. 1-34: 4-fluoro-N-(5-fluoro-6-(5-methoxypyrazolo[l,5-a]pyridin-3-yl)-lH- pyrazolo [3 ,4-b]pyridin-3 -yl)benzamide dimethanesulfonate

¹H-NMR (300 MHz, DMSO-d6): 11.15 (s, IH), 8.74(d, J=7.5 Hz, IH), 8.53(d, J=4.2 Hz, IH), 8.24(d, J=2.7 Hz, IH), 8.20-8.14 (m, 3H), 7.38(t, J=8.7 Hz, 2H), 6.86(dd, J=2.7, 7.5 Hz, IH), 4.00(s, 3H), 2.37 (s, 6H)

3. Composition

The osteoporosis curing agent composition of the present invention includes a pyrazole-pyridine derivative that is represented by Formula I and a pharmaceutically allowable carrier. The pharmaceutically allowable carrier may be any one of a standard pharmaceutical carrier that is used in a known preparation such as a sterile solution, a pellet, a coating tablet, and a capsule. In general, the carrier may include starch, milk, saccharide, a specific type of clay, gelatin, a stearic acid, talc, vegetable oil, oil, gum, an excipient such as glycohols, or other known excipients. The carrier may further include a flavor, a pigment additive, and other components. The composition that includes the carrier may be formulated by using a known method.

The osteoporosis curing agent of the present invention may be administered through various administration routes in an amount that is effective to cure the osteoporosis, and formulation, dosage amount, and frequency may be easily determined by those who are skilled in the art in consideration of an addition object, administration routes, a characteristic of the preparation, a state and a weight of the subject to be administered and the like.

In the present invention, the osteoporosis curing agent that includes the pyrazole- pyridine derivative of Formula I or a salt thereof may be administered by using a known route, for example, orally, intravenously, intramuscularly, percutaneously and the like, but is not limited thereto.

The range of the dosage amount of the pyrazole-pyridine derivative according to the present invention for expecting the prevention or the curing effect of osteoporosis is very wide, but it is preferable that the daily dosage amount which is effective as the osteoporosis curing agent is 1 — 1000 mg. The dosage and the frequency (number of administration) may be easily determined by those who skilled in the art according to the characteristic of the preparation, a state and a weight of addition subject, and the administration route.

Experimental Example 1 : Bone Formation Activity Evaluation

The alizarin red S (AR-s) is a compound that is used to confirm the mineral accumulation of calcium, phosphorus, and the like which are deposited in cytoplasm during bone formation by using the characteristics of combining with calcium of the cell. The present inventors purchased the human bone marrow-derived mesenchymal stem cell, cultured it, and used it for the test in order to evaluate the effect of a compound to the differentiation and the activity of osteoblast cells.

1. Purchasing and culturing of mesenchymal stem cells Human bone marrow-derived mesenchymal stem cells, which are commercially available (Cambrex, Catalog No: PT-2501), was subcultured two or three times, the differentiation potential into osteoblast cells was confirmed, and they were used in the present test. As the culture medium, the human bone marrow-derived mesenchymal stem cell exclusive medium (MSCGM; Cambrex, Catalog No: PT-3001; contains Mesenchymal Cell Growth Supplement, L-Glutamine, and Penicillin/ Streptomycin) was used, and 18 times of subculture were used.

2. The osteoblast differentiation test of mesenchymal stem cells

The prepared mesenchymal stem cells were cultured in the MSCGM medium, and 1 x 10⁴ /well cells were plated in 96 well plate. After 24 hours, the medium was exchanged, and it was replaced by a medium in which factors that induces osteoblast differentiation were added (MSCGM to which 10 mM β-glycerophosphate, 100 nM dexamethasone, 50 μg/ml of ascorbic acid were added) on the following day.

In order to evaluate the inducement of compounds into osteoblast cells and the bone formation activity of the compound, the medium was exchanged one time per three days along with treatment with a compound. On days 10 to 12 after the start of the culture, the osteoblast differentiation and the degree of mineralization by the compound were measured, by dyeing it with the alizarin red S (AR-s) to measure the mineral accumulation of calcium, phosphorus and the like which are deposited in cytoplasm during the differentiation of mesenchymal stem cells into osteoblast cells. For the alizarin red S solution, the alizarin red powder (Sigma) was dissolved in distilled water so that the concentration of it was 1%, the pH was titrated by using 0.5% ammonium hydroxide to the range of 4.1 - 4.5. 3. Measurement of bone formation

After the culture, the cells in the 96 well plate were washed twice with PBS (Phosphate buffered saline), fixed for 15 min at normal temperature by using 4% paraformaldehyde, and washed with distilled water three times.

After the water was removed from the plate, 100 μl of the alizarin red solution was put into each well, and dyeing was performed for 20 min at normal temperature. After dyeing, the alizarin red solution was removed, and washed with distilled water five times to remove the residual dyeing solution. 100 μl of 10% acetic acid solution was put into each well, and it was strongly shaken for 30 min. The plate was vortexed and heated in a dry oven for 30 min at 60°C. At this time, in order to prevent the vaporization of liquid, the plate was sealed by using a tape. The plate was cooled for 5 min in ice, and 10% ammonium hydroxide was added to titrate it so that the pH was in the range of 4.1 ~ 4.5. The colorimetric absorbance was measured by Envision at 405 nm.

The differentiation medium was used as the control to which the compound of the present invention was not added but the differentiation induction factor for inducing osteogenic differentiation was added. The light absorbance of the alizarin red S of the test group in which the compound of Formula I was treated in the differentiation medium to which the differentiation induction factor was added was measured, the light absorbance of the alizarin red S of the test group was represented by the multiple in comparison with the control group, and the results are described in Table 3.

(Table 3)

As shown in the experimental data, it can be seen that the compounds of the present invention are greatly increased (about 3 ~ 18 times) in comparison with the control (the compound of the present invention is not added) in views of the AR-s light absorbance. This result means that by significantly increasing the osteoblast differentiation and the bone formation activity by the compound that is represented by Formula I of the present invention or a salt thereof, the mineral accumulation of calcium, phosphorus and the like is increased. It is known that the mineral accumulation is the most important process in the mineralization process (JBC 2005, 280, 4785-4791). Accordingly, the present inventors found that the compounds of the present invention promote bone formation, and this promotion of bone formation proves the effect as an osteoporosis prevention or curing agent. The composition of the present invention is expected to be capable of being used in various diseases that relates to a bone metabolism such as the regeneration of the alveolar bone, osteogenesis imperfecta, osteomalacia and the like as well as the prevention or the curing effect of osteoporosis by the action that is caused by the inducing of bone formation.

In addition, since the composition of the present invention has the osteoporosis curing effect and has the low toxicity, it is suitable to be used as medicine product.

Claims

1. A pharmaceutical composition for preventing or treating osteoporosis, comprising an effective amount of compound represented by the following Formula I or its pharmaceutically allowable salt: <Formula I>

wherein, R₁ is hydrogen or halogen,

R₂, R₃, and R₃' are each independently hydrogen or -(Xj)-R₅, wherein X₁ is C₁-C₈ straight or branched alkylene, O, CO, (CO)₂, SO, or SO₂,

R₅ is: Ci-C₈ straight or branched alkyl; hydroxy; carboxy; Ci-C₈ straight or branched alkanol, C₁-C₈ alkoxy; C₁-C₈ acetoxy; C₂-C₈ alkenyl; C₂-C₈ alkynyl; C₃-C₈ cycloalkyl; C₆-C₂₀ aryl; C₅-C₂₀ heterocycloalkyl; C₆-C₂₀ aryl that is substituted with halogen; C₆-C₂₀ aryl that is substituted with nitrile; C₁-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl; C₁-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl substituted with halogen; C₂-C₈ alkenyl that is substituted with C₆-C_2O aryl; C₂-C₈ alkenyl that is substituted with C₆-C₂₀ aryl substituted with halogen; C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl; C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl substituted with halogen; NH₂; nitrile; or NA₁A₂, wherein A₁ or A₂ are each independently the same as or different from each other, and Ci-C₈ straight or branched alkyl that is unsubstituted or substituted with phenyl; C₂-C₈ alkenyl; or C₆-C₂₀ aryl that is unsubstituted or substituted with halogen, C₁-C₄ alkyl, or C₁-C₄ alkoxy;

R₄ is hydrogen or -(X₂)-R₆;

X₂ is a single bond, Cj-C₈ straight or branched alkylene, C₂-C₈ alkenylene, C₆-C₂₀ arylene, CO, or SO₂;

R₆ is C₁-C₈ straight or branched alkyl; Ci-C₈ alkoxy; C₂-C₈ alkenyl; C₂-C₈ alkynyl; C₃-C₈ cycloalkyl; C₆-C₂₀ aryl; C₆-C₂₀ aryl that is substituted with Cj-C₈ alkoxy; C₆-C₂₀ aryl that is substituted with halogen; C₅-C_2O heterocycloalkyl; C₆-C_2O aryl that is substituted with halogen; C₁-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl; C₁-C₈ straight or branched alkyl that is substituted with C₆-C₂₀ aryl substituted with halogen; C₆-C₂₀ aryl that is substituted with C₆-C₂₀ alkyl; C₆-C₂₀ aryl that is substituted with Cj-C₈ alkyl substituted with halogen; C₂-C₈ alkenyl that is substituted with C₆-C₂₀ aryl substituted with Cj-C₈ alkoxy; C₂-C₈ alkenyl that is substituted with C₆-C₂₀ aryl substituted with halogen; C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl; or C₂-C₈ alkynyl that is substituted with C₆-C₂₀ aryl substituted with halogen.

2. The pharmaceutical composition for preventing or treating osteoporosis, wherein: Rj is fluorine; R₂, R₃, and R₃' are each independently hydrogen or -(XO-R₅, wherein Xi is Ci~C₈ straight or branched alkylene, O, or CO; and R₅ is C₁-C₈ straight or branched alkyl; hydroxy; carboxy; C₆-C_2O aryl; C₁-C₈ acetoxy; C₆-C₂₀ aryl that is substituted with halogen; C₆-C₂₀ aryl that is substituted with nitrile; nitrile; or C₁-C₈ alkylamine; R₄ is hydrogen or -(X₂)-R₆;

X₂ is a single bond, C₆-C₂₀ arylene, CH₂, CO or SO₂; and R₆ is C₁-C₈ straight or branched alkyl; C₁-C₈ alkoxy; C₆-C_2O aryl that is substituted with C₁-C₈ alkoxy; C₆-C₂₀ aryl that is substituted with halogen; Ci-C₈ straight or branched alkyl that is substituted with C₆-C₂O aryl that is substituted with halogen; or C₆-C₂₀ aryl that is substituted with C₁-C₈ alkyl substituted with halogen.