KR20090005842A - Novel carbonitrile compounds, process for preparing thereof, and pharmaceutical composition for treating or preventing articular rheumatism, osteoarthritis, paget's disease, hypercalcemia of malignancy, metabolic bone disease and cancers comprising the same - Google Patents

Abstract

A novel carbonitrile compound or its pharmaceutically acceptable salt, a method for preparing the compound, and a pharmaceutical composition containing compound are provided to prevent and treat osteoarthritis and various cancer without side effect. A carbonitrile compound is represented by the formula 1, wherein n is 1 or 2; R1 is H, halogen-substituted or unsubstituted linear or branched saturated or unsaturated C1-C10 alkyl, C3-C7 cycloalkyl, C1-C10 alkoxy, C1-C10 alkylthio, hydroxy-C1-C10 alkyl, C1-C10 alkylcarbonyl, C1-C10 alkylthio-C1-C10 alkyl, C3-C7 cycloalkyl-C1-C10 alkyl, C3-C7 cycloalkyl-C1-C10 alkyl, C1-C10 alkoxy-C1-C10 alkyl, phenyl, ar-C1-C10 alkyl, or ar-C1-C10 alkoxy group; and R2 linear or branched saturated or unsaturated C1-C10 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkyl-C1-C10 alkyl, phenyl, ar-C1-C10 alkyl, C1-C10 alkoxycarbonyl, or ar-C1-C10 alkoxycarbonyl group.

Description

Novel Carbonitrile Compounds, Process For Preparing Novel Carbonitrile Compounds, Methods for Producing Them, and Rheumatoid Arthritis, Osteoarthritis, Paget's Disease, Malignant Hypercalcemia, Metabolic Bone Diseases and Various Cancers Thereof, And Pharmaceutical Composition For Treating Or Preventing articular rheumatism, Osteoarthritis, Paget's disease, hypercalcemia of malignancy, Metabolic bone disease And Cancers Comprising The Same}

The present invention is a novel carbonitrile compound represented by Formula 1, a method for preparing the same, and a pharmaceutical for the treatment and prevention of rheumatoid arthritis, osteoarthritis, Paget's disease, malignant hypercalcemia, metabolic bone disease and various cancers It relates to a composition.

The present invention also relates to an inhibitor composition of cysteine protease and an inhibitor composition of cathepsin B, L and S, which contain a novel carbonitrile compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

[In Formula 1, n is 1 or 2.]

Cathepsin is a cysteine protease that is involved in the proteolysis of cellular lysosomes and is involved in the development and metastasis of arthritis, osteoporosis, various cancers, malaria, various inflammatory diseases, blood coagulation and hemorrhage, and myopathy (WO 2006-102535). , PCT / GB00 / 00529, WO 2005/039496, WO 2005/021487, WO 2007/003056, WO 2000/49007.

Cysteine proteases play important roles in human biology and diseases including atherosclerosis, emphysema, osteoporosis, chronic inflammation and immune diseases (H. A. Chanman et al., 1997, Ann. Rev. Physiol., 59, 63).

The literature on diseases related to cysteine protease is as follows, but is not limited thereto.

Osteoporosis, Osteoarthritis: Inui, T., O. Ishibashi, J Biol Chem 1997, 272 (13), 8109-12; Saftig, P., E. Hunziker, et al., Adv Exp Med Biol 2000 + ADs 2000, 477, 293-303; Saftig, P., E. Hunziker, et al., Proc Natl Acad Sci USA 1998, 95 (23), 13453-8)

Periodontal disease, Paget's disease, Atherosclerosis: Jormsjo, S., D. M. Wuttge, et al., Am J Pathol 2002 161 (3), 939-45.

Multiple Sclerosis: Beck, H., G. Schwarz, et al., Eur J Immunol 2001, 31 (12), 3726-36.

Arthritis: Nakagawa, T.Y., W. H. Brissette, et al, Immunity 1999, 10 (2), 207-17; Hou, W. S., Z. Li, et al, Am J Pathol 2001, 159 (6), 2167-77.

Asthma, lupus: Cimerman, N., P. M. Brguljan, et al, Pflugers Arch 2001, 442 (6 Suppl 1), R204-6.

Alzheimer's: Lemere, C. A., J. S. Munger, et al., Am J Pathol 1995, 146 (4), 848-60.

Parkinson's disease: Liu, Y., L. Fallon, et al., Cell 2002, 111 (2), 209-18.

Cancer: Fernadez, P. L., X. Farre, et al., Int J Cancer 2001, 95 (1), 51-5.

Malaria: Malhotra, P., P. V. Dasaradhi, et al., Mol Microbiol 2002, 45 (5), 1245-54.

Cathepsin B-related diseases include osteoarthritis, bone diseases such as osteoarthritis, cancer and asthma, and cathepsin K-related diseases include osteoporosis, lung disease, gum disease (e.g., gingivitis and periodontitis) and arthritis. Diseases related to the tapsin L include skin cancer, and diseases related to cathepsin S are known to be Alzheimer's disease, autoimmune disease, arthritis, asthma (WO 2006/102535).

It is an object of the present invention to provide a novel carbonitrile compound of formula (1) and a pharmaceutically acceptable salt thereof. It provides a pharmaceutical composition for the treatment and prevention of rheumatoid arthritis, osteoarthritis, Paget's disease, malignant hypercalcemia, metabolic bone diseases and various cancers.

Still another object of the present invention is to provide an inhibitor composition of cysteine protease and an inhibitor composition of cathepsin B, L and S, which contain a novel carbonitrile compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention is a novel carbonitrile compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, a preparation method thereof and rheumatoid arthritis, osteoarthritis, Paget's disease, malignant hypercalcemia, metabolic bone disease containing the same as an active ingredient And pharmaceutical compositions for the treatment and prevention of various cancers.

The present invention also relates to an inhibitor composition of cysteine protease and an inhibitor composition of cathepsin B, L and S, which contain a novel carbonitrile compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

[In Formula 1, n is 1 or 2;

R ₁ represents a hydrogen atom, straight or unsubstituted saturated or unsaturated (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, (C ₁ -C ₁₀ ) alkoxy, (C ₁ -C ₁₀ ) alkylthio, hydroxy (C ₁ -C ₁₀ ) alkyl, (C ₁ -C ₁₀ ) alkylcarbonyl, (C ₁ -C ₁₀ ) alkylthio (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl (C ₁ -C ₁₀ ) alkoxy, (C ₁ -C ₁₀ ) alkoxy (C ₁ -C ₁₀ ) alkyl , Phenyl, ar (C ₁ -C ₁₀ ) alkyl or ar (C ₁ -C ₁₀ ) alkoxy;

R ₂ is straight or branched saturated or unsaturated (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, (C ₃ -C ₇ ) cycloalkyl (C ₁ -C ₁₀ ) alkyl, phenyl, (C ₁ -C ₁₀ ) alkyl, (C ₁ -C ₁₀ ) alkoxycarbonyl or ar (C ₁ -C ₁₀ ) alkoxycarbonyl;

Provided that the phenyl, ar (C ₁ -C ₁₀ ) alkyl or ar (C ₁ -C ₁₀ ) alkoxy of R ₁ and R ₂ is (C ₁ -C ₁₀ ) alkyl, fluorine-substituted (C ₁ -C ₁₀ Or alkyl, (C ₁ -C ₁₀ ) alkoxy, halogen, nitro, cyano or hydroxy, respectively.]

In the above, “ar (C ₁ -C ₁₀ ) alkyl” is an alkyl group of (C ₁ -C ₁₀ ) substituted with an aryl group of (C ₆ -C ₁₄ ), benzyl, phenethyl, triphenylmethyl, 1-phenyl Ethyl, 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl, 1-naphthylmethyl, 2-naphthylmethyl, naphthylethyl, anthracenylmethyl, diphenylmethyl, and the like, but are not limited thereto. no.

“Ar (C ₁ -C ₁₀ ) alkoxy” is an alkoxy group of (C ₁ -C ₁₀ ) substituted with an aryl group of (C ₆ -C ₁₄ ), benzyloxy, phenylethoxy, phenylpropoxy, phenylbutoxy, Naphthyl methoxy, naphthylethoxy, anthracenyl methoxy, diphenyl methoxy etc. are included, but this is not restrict | limited.

As a range of specific compounds among the novel carbonitrile compounds represented by the general formula (1) according to the present invention, R ₁ is a linear or branched saturated or unsaturated (C ₁ -C ₁₀ ) alkyl, (C ₁ -C ₁₀ ) alkylthio ( C ₁ -C ₁₀ ) alkyl, phenyl or benzyl; R ₂ is phenyl, benzyl, t-butoxycarbonyl or benzyloxycarbonyl; However, the R ₁ and the phenyl or benzyl are (C ₁ -C ₁₀₎ is an alkyl, fluorine-substituted (C ₁ -C ₁₀₎ alkyl, (C ₁ -C ₁₀₎ alkoxycarbonyl of R _2, fluoride (F), chloro (Cl), nitro, cyano or hydroxy, respectively.

Scheme 1 is illustrated as a method of preparing the carbonitrile compound of Chemical Formula 1 according to the present invention, and the following preparation method does not limit the method of preparing the Chemical Formula 1 compound according to the present invention. As will be apparent to those skilled in the art, unless otherwise stated, the definitions of substituents in the following schemes are the same as in Formula 1.

The method for preparing a carbonitrile compound of Chemical Formula 1 according to the present invention is condensed with an aminoacetic acid compound ( 2 ) and a cyclic compound ( 3 ) and then reacted with cyanogen bromide (BrCN) as shown in Scheme 1 below. It is possible to prepare a novel carbonitrile compound represented by the formula (1) according to the invention.

Scheme 1

[Wherein R ₁ , R ₂ and n are the same as defined in formula (1).]

The amino acetic acid compound ( 2 ) can be synthesized by the following scheme 2.

Scheme 2

[In the above scheme, R ₁ and R ₂ are the same as defined in the formula (1).]

In addition, the cyclic compound ( 3 ) is a reflux reaction of 1,2-di-t-butoxycarbonylhydrazine (1,2-di-tert-butoxycarbonylhydrazine) and dibromo compound (5), as shown in Scheme 3 below. After the synthesis of the t-butoxycarbonyl ring compound (6) to the ring compound ( 3 ) can be synthesized by adding 4M HCl, but is not limited thereto, it can also be synthesized by other methods.

Scheme 3

[Wherein n is the same as defined in the formula (1).]

The carbonitrile compound represented by Chemical Formula 1 according to the present invention has excellent inhibitory activity and high selectivity against the enzymes of cathepsin B, L and S, which are biochemical and pharmacological tests, and excellent arthritis in disease model animal experiments. It was confirmed to exhibit a therapeutic effect.

The carbonitrile compound compound represented by Formula 1 according to the present invention is suitable as a pharmaceutical composition for the treatment and prevention of rheumatoid arthritis, osteoarthritis, Paget's disease, malignant hypercalcemia, metabolic bone diseases and various cancers. Pharmaceutically usable salts of compounds of formula 1 include all salts formed with acids or bases. Pharmaceutically acceptable acid addition salts include hydrochloric acid, bromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, triphenylacetic acid, phenylacetic acid, substituted phenylacetic acid, for example methoxy Phenylacetic acid, sulfamic acid, sulfanilic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, malic acid, glutamic acid, aspartic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, arylsulfonic acid (e.g., p-toluenesulfonic acid , Benzenesulfonic acid, naphthalenesulfonic acid or naphthalenedisulfonic acid), salicylic acid, glutaric acid, gluconic acid, tricavalylic acid, mandelic acid, cinnamic acid, substituted cinnamic acid (e.g., 4-methyl and 4-methoxycin Methyl, methoxy, halo, or phenyl substituted cinnamic acid, including Namic acid and α-phenyl cinnamic acid), ascorbic acid, oleic acid, naphthoic acid, hydroxynaphthoic acid (eg, 1- or 3-hydroxy- 2-naphthoic acid), naph Taleneacrylic acid (eg, naphthalene-2-acrylic acid), benzoic acid, 4-methoxybenzoic acid, 2 or 4-hydroxybenzoic acid, 4-chlorobenzoic acid, 4-phenylbenzoic acid, benzeneacrylic acid (eg, 1,4 Benzenediacrylic acid) and isethionic acid. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts (eg sodium and potassium salts), alkaline earth metal salts (eg calcium and magnesium salts) and organic bases (eg mono-, di-, or tri) Alkylammonium, dicyclohexylamine and salts of N-methyl-D-glucamine).

The compounds according to the invention also have the potential of combining long lasting effects and rapid onset of action. Certain compounds also exhibit improved therapeutic indicators in animal models compared to existing long-lasting cathepsin inhibitors. In addition, the compounds of the present invention may be suitable for once to three times daily administration.

The amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof used to achieve a therapeutic effect depends on the compound, the method of administration, the subject of treatment and the severity of the disease, or disease.

It is possible to administer the carbonitrile compound of Formula 1 or a pharmaceutically acceptable salt thereof alone, but preferably in a pharmaceutical formulation. Accordingly, the present invention is also preferably provided as a pharmaceutical formulation comprising a carbonitrile compound of formula 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient, and any one or more therapeutic ingredients. .

Accordingly, the carbonitrile compounds of the present invention are novel compounds having new chemical structures, and have excellent inhibitory activity against the cysteine proteases cathepsin B, L and S and high selectivity to cathepsin B, L and S. . In addition, animal experiments using the compounds showed excellent osteoarthritis treatment effect, preventing osteoarthritis diseases, rheumatoid arthritis, Paget's disease, malignant hypercalcemia, metabolic bone disease and various cancers based on cathepsin B, L and S inhibition And it can be used as a therapeutic agent without side effects.

The present invention as described above will be described in more detail based on the following examples, but the present invention is not limited thereto.

Preparation Example 1 Preparation of 3-methyl-2- (phenylamino) pentanoic acid (Compound A )

Bromobenzene (1.74) in a solution of 2-amino-3-methylpentanoic acid (2 g, 15 mmol), K ₂ CO ₃ (3.1 g, 22.5 mmol) and CuI (290 mg, 1.5 mmol) with anhydrous dimethylacetamide (35 ml) ml, 16.5 mmol) is added and stirred at 90 ° C. for 48 h. After cooling to room temperature, add EtOAc (60 ml) and water (30 ml) and dilute. Cool with ice-bath and conc. Adjust pH to 3 by adding HCl. Extraction with EtOAc followed by distillation under reduced pressure to remove the solvent and purification by column chromatography (EtOAc / Hexane = 1/4 to 1/1) gave the title compound 3-methyl-2- (phenylamino) pentanoic acid (Compound A ) (2.24). g, 72%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.19 (2H, t, J = 7.8 Hz), 6.77 (1H, t, J = 7.4 Hz), 6.59 (2H, d, J = 8.1 Hz), 3.94 (1H , d, J = 5.4 Hz), 2.00-1.92 (1H, m), 1.64-1.56 (1H, m), 1.38-1.23 (1H, m), 1.03 (3H, d, J = 6.9 Hz), 0.97 ( 3H, t, J = 7.4 Hz)

Preparation Example 2 Preparation of Pyrazolidine 2HCl (Compound C )

1,2- Bis - tert - butoxycarbonylpyrazolidine (compound B Manufacturing

1,2-di-t-butoxycarbonylhydrazine (1,2-di-tert-butoxycarbonylhydrazine) (2g, 8.6 mmol) is dissolved in toluene (20ml), 50% -NaOH (10ml) is added, and tetraethyl ammonium Bromide (2.71 g, 12.92 mmol) and dibromopropane (2.6 g, 12.92 mmol) were added and the mixture was refluxed at 110 ° C. for 5 hours. The reaction solution was cooled to room temperature, extracted with EtOAc, washed with brine and water, dried and concentrated under reduced pressure. Purified by column chromatography (EtOAc: Hexane = 1/5) to 1,2-Bis-tert-butoxycarbonylpyrazolidine (Compound B ) (2 g, 87%) was obtained.

¹ H NMR (300 MHz, CDCl ₃ ) δ 3.88 (2H, m), 3.23 (2H, m), 2.00 (2H, q, J = 7.2 Hz), 1.47 (18H, S)

Pyrazolidine 2HCl (Compound C Manufacturing

16 ml of 4M HCl / dioxane solution was added to 1,2-Bis-tert-butoxycarbonylpyrazolidine (Compound B ) (1.9 g, 6.99 mmol), and the mixture was stirred at room temperature for 2 hours. The resulting solid was filtered and washed with anhydrous diethyl ether to give the title compound Pyrazolidine 2HCl (Compound C ) as a white solid (1.01 g, 100%).

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.30 (4H, br), 3.01 (4H, t J = 7.2), 1.92 (2H, q, J = 7.2 Hz)

Preparation Example 3 Preparation of 3-Methyl-2-phenylamino-1-pyrazolidin-1-yl-pentan-1-one (Compound D )

Pyrazolidine 2HCl (Compound C ) (450 mg, 3.1 mmol) was dissolved in CH ₂ Cl ₂ (6 ml) and added to Et ₃ N (0.86 ml, 6.2 mmol), 3-methyl-2- (phenylamino) pentanoic acid ( Compound A ) (640 mg, 3.1 mmol) and EDCI (590 mg, 3.1 mmol) were added sequentially, followed by stirring at room temperature for 10 hours. Water was added, extraction was performed using CH ₂ Cl ₂ , and the organic layer was distilled under reduced pressure to remove the solvent, and then purified by column chromatography (EtOAc: Hexane = 1/1) to obtain the title compound, 3-Methyl-2-phenylamino-1- Pyrazolidin-1-yl-pentan-1-one (Compound D ) (520 mg, 64%) was obtained.

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.12 (2H, t, J = 7.8 Hz), 6.69-6.64 (3H, m), 4.70 (1H, d, J = 7.5 Hz), 3.57-3.33 (2H, m), 3.07-2.83 (2H, m), 2.05- 1.96 (2H, m), 1.75-1.67 (2H, m), 1.28-1.14 (1H, m), 0.96 (3H, d, J = 6.6 Hz) , 0.91 (3H, t, J = 7.2 Hz)

EXAMPLES Preparation of Compound of Formula 1 by Reaction of Compound (4)

Compound 4 (0.35 mmol) was dissolved in Methanol: H ₂ O (15: 1) (3 mL: 0.2 mL), sodium acetate (0.7 mmol) and cyanogen bromide (BrCN) (0.7 mmol) were added thereto, and the mixture was stirred at room temperature. The reaction solution was diluted with water, extracted twice with ethyl acetate, and the organic layer was washed with water and brine. The extracted organic layer was dried over anhydrous sodium sulfate (Na ₂ SO ₄ ), filtered and distilled under reduced pressure, and the residue was purified by silica gel column chromatography to obtain the target compound of Chemical Formula 1.

The structures of the compounds of Formula 1 and ¹ H NMR are shown in Tables 1 and 2, respectively.

Example 1

Preparation of [1- (2-Cyano-pyrazolidine-1-carbonyl) -2-methylbutyl] carbamic acid tert-butyl ether (Compound 101 )

Pyrazolidine 2HCl (Compound C ) (450 mg, 3.1 mmol) is dissolved in CH ₂ Cl ₂ (10 ml) and added to Et ₃ N (0.86 ml, 6.2 mmol), 3-methyl-2- (tert-butoxycarbonylamino) pentanoic acid (717 mg, 3.1 mmol) and EDCI (590 mg, 3.1 mmol) were added sequentially, followed by stirring at room temperature for 10 hours. Water was added, extraction was performed with CH ₂ Cl ₂ , and the organic layer was distilled under reduced pressure to remove the solvent, and then purified by column chromatography (EtOAc: Hexane = 1/1) to obtain 630 mg of [2-methyl-1- (pyrazolidine- 1-carbonyl) butyl] -carbamic acid tert-butyl ether was obtained and dissolved in MeOH / H ₂ O (20 ml / 1.3 ml), followed by NaOAc (0.36, 4.4 mmol) and CNBr (0.46 g, 4.4 mmol) at 10 ° C. Was added and stirred for 5 hours. It was concentrated by distillation under reduced pressure and purified by column chromatography (EtOAc / Hexane = 2/1) to obtain the title compound [1- (2-Cyano-pyrazolidine-1-carbonyl) -2-methylbutyl] carbamic acid tert-butyl ether ( Compound 101 ) (582 mg, 85%) was obtained.

¹ H NMR (300 MHz, CDCl ₃ ) δ 4.95 (d, 1H, J = 8.5 Hz), 4.69 (m, 1H), 4.09 (m, 1H), 3.60 (m, 1H), 3.49 (m, 1H) , 2.30 (m, 2H), 1.76 (m, 1H), 1.52 (m, 1H), 1.42 (s, 9H), 1.20 (m, 1H), 1.02 (d, 3H, J = 6.75 Hz), 0.92 ( t, 3H, J = 7.5 Hz).

Example 2

Preparation of 2- (3-Methyl-2-phenylamino-pentanoyl) -pyrazolidine-1-carbonitrile (Compound 102 )

3-Methyl-2-phenylamino-1-pyrazolidin-1-yl-pentan-1-one (130 mg, 0.50 mmol) and NaOAc (62 mg, 0.75 mmol) in MeOH / H ₂ O (2 ml / 0.1 ml) After dissolving in CNBr (80 mg, 0.75 mmol) was added at 10 ℃ and stirred for 5 hours. It was concentrated by distillation under reduced pressure and purified by column chromatography (EtOAc / Hexane = 3/4) to obtain the title compound 2- (3-Methyl-2-phenylamino-pentanoyl) -pyrazolidine-1-carbonitrile (Compound 102 ) (123 mg, 86%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.15 (2H, t, J = 7.8 Hz), 6.75-6.66 (3H, m), 4.45-4.38 (1H, m), 4.09 (1H, br), 4.95- 4.83 (1H, m), 3.56-3.43 (2H m), 3.21-3.10 (1H, m), 2.30-2.05 (2H, m), 1.83-1.68 (2H, m), 1.27-1.14 (1H, m) , 1.05 (3H, d, J = 6.9 Hz), 0.95 (3H, t, J = 7.4 Hz)

Example 3

Preparation of 2- (3-methyl-2- (benzyloxycarbonylamino) pentanoyl) piperazine-1-carbonitrile (Compound 142 )

Pyrazolidine 2HCl (Compound C ) (493 mg, 3.1 mmol) is dissolved in CH ₂ Cl ₂ (10 ml) and added to Et3N (0.86 ml, 6.2 mmol), 3-methyl-2- (benzyloxycarbonylamino) pentanoic acid (822 mg, 3.1 mmol) and EDCI (590 mg, 3.1 mmol) were added sequentially, followed by stirring at room temperature for 10 hours. Water was added, extraction was performed using CH ₂ Cl ₂ , and the organic layer was distilled under reduced pressure to remove the solvent, and then purified by column chromatography (EtOAc: Hexane = 1/1) to obtain 770 mg of 3-methyl-2- (benzyloxycarbonylamino)- 1-piperazine-1-yl-pentan-1-one was obtained and dissolved in MeOH / H ₂ O (20 ml / 1.3 ml), followed by NaOAc (0.37 g, 4.6 mmol) and CNBr (0.48 g, 4.6 mmol) at 10 ° C. ) Was added and stirred for 5 hours. It was concentrated by distillation under reduced pressure and purified by column chromatography (EtOAc / Hexane = 2/1) to obtain the title compound 2- (3-methyl-2- (benzyloxycarbonylamino) pentanoyl) piperazine-1-carbonitrile (Compound 142 ) (490 mg , 59%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.34-7.26 (m, 5H), 5.29 (d, 1H, J = 9.0 Hz), 5.14-5.02 (m, 2H), 4.81 (m, 1H), 4.51 ( d, 1H, J = 13.2), 3.55 (m, 2H), 3.19-3.15 (m, 1H), 2.03 (m, 1H), 1.82-1.65 (m, 3H), 1.56-1.49 (m, 1H), 1.32 (m, 1H), 1.01 (d, 3H, J = 6.6 Hz), 0.91 (t, 3H, J = 7.4 Hz).

Example 4

Preparation of 2- [2- (3- (trifluoromethyl) phenylamino) -4- (methylthio) butanoyl] pyrazolidine-1-carbonitrile (Compound 199 )

Pyrazolidine 2HCl (Compound C ) (493 mg, 3.1 mmol) is dissolved in CH ₂ Cl ₂ (10 ml) and added to Et ₃ N (0.86 ml, 6.2 mmol), 2- (3- (trifluoromethyl) phenylamino) -4- ( Methylthio) butanoic acid (0.91 g, 3.1 mmol) and EDCI (590 mg, 3.1 mmol) were added sequentially, followed by stirring at room temperature for 10 hours. Water was added, extraction was performed using CH ₂ Cl ₂ , and the organic layer was distilled under reduced pressure to remove the solvent, and then purified by column chromatography (EtOAc: Hexane = 1/1) to give 550 mg of 2- (3- (trifluoromethyl) phenylamino). 4- (methylthio) -1- (pyrazolidin-1-yl) butan-1-one was obtained and dissolved in MeOH / H ₂ O (20 ml / 1.3 ml), followed by NaOAc (0.25 g, 3.04 mmol) at 10 ° C. And CNBr (0.32 g, 3.04 mmol) were added and stirred for 5 hours. It was concentrated by distillation under reduced pressure and purified by column chromatography (EtOAc / Hexane = 2/1) to obtain the title compound 2- [2- (3- (trifluoromethyl) phenylamino) -4- (methylthio) butanoyl] pyrazolidine-1-carbonitrile (Compound 199 ) (420 mg, 71%) was obtained.

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.29-7.23 (m, 1H), 6.99 (d, 1H, J = 7.8 Hz), 6.85-6.83 (m, 2H), 4.93-4.86 (m, 1H), 4.47 (d, 1H, J = 10.2 Hz) 3.98-3.89 (m, 1H), 3.67-3.57 (m, 2H), 3.37 (m, 1H), 2.72-2.68 (m, 2H), 2.43-2.18 (m , 2H), 2.19-2.09 (m, 4H), 2.04-1.92 (m, 1H).

TABLE 1

TABLE 2

[ Cathepsin  Method for analyzing activity of B, L and S]

Inhibition of Cathepsin B Activity

Prepared by dissolving the compound (Compound 101-Compound 199) obtained using dimethyl sulfoxide (DMSO) to a concentration of 12.5mM, the substrate (Z-RR-pNA; biomol) used in the reaction is prepared by dissolving to 400μM concentration It was. Enzyme reactions were carried out in a buffer containing 100 mM NaOAcetate, 2 mM EDTA, 3 mM DTT (buffer pH was adjusted to pH 5.5 using 1M acetic acid). 5 μl of a substrate (Z-RR-PNA biomol) dissolved in 400 μM in a 96 well plate (Costar) and a compound were added, and recombinant Cathepsin B (human; 1-339a.a) was added to the buffer solution at a concentration of 5.88 nM. The reaction was carried out at 30 ° C. for 2 hours. After completion of the reaction, the absorbance was measured at 405 nm using benchmark plus (Bio-RAD). The enzyme inhibitory ability was evaluated by adding a solution prepared by dissolving the sample compound in dimethyl sulfoxide (DMSO) to within 5% of the total reaction solution. In addition, the enzyme inhibitory ability was expressed as a percentage of the measured value in the presence of the sample compound to the measured value in the absence of the test compound, and the concentration of the compound inhibiting 50% enzymatic activity was determined as an IC ₅₀ (μM) value. . IC ₅₀ values of the compounds are shown in Table 3 below.

Inhibition of Cathepsin L Activity

Prepared by dissolving the compound (Compound 101-Compound 199) obtained using dimethyl sulfoxide (DMSO) to a concentration of 12.5mM, the substrate (Z-FR-pNA; biomol) used in the reaction was prepared by dissolving to 400μM concentration It was. Enzyme reactions were carried out in a buffer containing 100 mM NaOAcetate, 2 mM EDTA, 3 mM DTT (buffer pH was adjusted to pH 5.5 using 1M acetic acid). 5 μl of a substrate (Z-FR-pNA biomol) dissolved in 400 μM in a 96 well plate (Costar) and a compound were added thereto, and the recombinant Cathepsin L (calbiochem) was added to the buffer at a concentration of 300 mU, followed by 2 hours at 30 ° C. Reacted. After the reaction was completed, absorbance was measured at 405 nm using benchmark plus (Bio-RAD). The enzyme inhibitory ability was evaluated by adding a solution prepared by dissolving the sample compound in dimethyl sulfoxide (DMSO) to within 5% of the total reaction solution. In addition, the enzyme inhibitory ability was expressed as a percentage of the measured value in the presence of the sample compound with respect to the measured value in the absence of the test compound, and the concentration of the compound that inhibited 50% enzymatic activity was determined as an IC ₅₀ (μM) value. IC ₅₀ values of the compounds are shown in Table 3 below.

Inhibition of Cathepsin S Activity

Prepared by dissolving the compound (Compound 101-Compound 199) obtained using dimethyl sulfoxide (DMSO) to a concentration of 12.5mM, the substrate (Z-FR-pNA; biomol) used in the reaction was prepared by dissolving to 400μM concentration It was. Enzyme reactions were carried out in a buffer containing 100 mM NaOAcetate, 2 mM EDTA, 3 mM DTT (buffer pH was adjusted to pH 5.5 using 1M acetic acid). 5 μl of a substrate (Z-FR-pNA biomol) dissolved in 400 μM in a 96 well plate (Costar) and compound were added, and recombinant Cathepsin S (human; 1-331a.a) was added to the buffer solution at a concentration of 100 nM. The reaction was carried out at 30 ° C. for 2 hours. After the reaction was completed, absorbance was measured at 405 nm using benchmark plus (Bio-RAD). The enzyme inhibitory ability was evaluated by adding a solution prepared by dissolving the sample compound in dimethyl sulfoxide (DMSO) to within 5% of the total reaction solution. In addition, the enzyme inhibitory ability was expressed as a percentage of the measured value in the presence of the sample compound to the measured value in the absence of the test compound, and the concentration of the compound that inhibited 50% enzymatic activity was determined as an IC ₅₀ (μM) value. IC ₅₀ values of the compounds are shown in Table 3 below.

TABLE 3

[Evaluation of Antiarthritis Efficacy of Kadipsin B Inhibitors in Collagen-Induced Arthritis Model Mice]

Arthritis induction by collagen is according to Trentham et al. (Trentham DE, Townes As, Kang AH (1997) Autoimmunity to type II collagen an experimental model of arthritis, J. Exp. Med., 146, 857-868). In other words, collagen (100 ug) / Freund's complete immune adjuvant (CFA) mixture was first injected into the dermis of the tail of 6-week-old male DBA / 1J mice. Arthritis was induced. At 25 days after the first immune response, the drug was treated daily in the abdominal cavity in an amount of 100 mpk and the symptoms of arthritis were scored for 2 weeks. Depending on the condition of each foot, 1-3 points (0: normal joint, 1: slight inflammation and redness, 2: swelling and erythema, 3: deformed joint, joint firmness and loss of function) were given and the scores of each of the four feet were summed. . Compound 145 of the present invention was used as the drug, and treated with Rofecoxib 20mpk as a control drug. When Compound 145 was treated at a 100 mpk dose, it showed a similar therapeutic effect as using Rofecoxib 20mpk (FIG. 1).

Figure 1-Graph showing the degree of arthritis treatment of Compound 145 in animal experiments

Claims (10)

A carbonitrile compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. [Formula 1]

[In Formula 1, n is 1 or 2; R ₁ represents a hydrogen atom, straight or unsubstituted saturated or unsaturated (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, (C ₁ -C ₁₀ ) alkoxy, (C ₁ -C ₁₀ ) alkylthio, hydroxy (C ₁ -C ₁₀ ) alkyl, (C ₁ -C ₁₀ ) alkylcarbonyl, (C ₁ -C ₁₀ ) alkylthio (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl (C ₁ -C ₁₀ ) alkoxy, (C ₁ -C ₁₀ ) alkoxy (C ₁ -C ₁₀ ) alkyl , Phenyl, ar (C ₁ -C ₁₀ ) alkyl or ar (C ₁ -C ₁₀ ) alkoxy; R ₂ is straight or branched saturated or unsaturated (C ₁ -C ₁₀ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, (C ₃ -C ₇ ) cycloalkyl (C ₁ -C ₁₀ ) alkyl, phenyl, (C ₁ -C ₁₀ ) alkyl, (C ₁ -C ₁₀ ) alkoxycarbonyl or ar (C ₁ -C ₁₀ ) alkoxycarbonyl; Provided that the phenyl, ar (C ₁ -C ₁₀ ) alkyl or ar (C ₁ -C ₁₀ ) alkoxy of R ₁ and R ₂ is (C ₁ -C ₁₀ ) alkyl, fluorine-substituted (C ₁ -C ₁₀ Or alkyl, (C ₁ -C ₁₀ ) alkoxy, halogen, nitro, cyano or hydroxy, respectively.] The method of claim 1, R ₁ is straight or branched saturated or unsaturated (C ₁ -C ₁₀ ) alkyl, (C ₁ -C ₁₀ ) alkylthio (C ₁ -C ₁₀ ) alkyl, phenyl or benzyl; R ₂ is phenyl, benzyl, t-butoxycarbonyl or benzyloxycarbonyl; However, the R ₁ and the phenyl or benzyl are (C ₁ -C ₁₀₎ is an alkyl, fluorine-substituted (C ₁ -C ₁₀₎ alkyl, (C ₁ -C ₁₀₎ alkoxycarbonyl of R _2, fluoride (F), chloro A carbonitrile compound or a pharmaceutically acceptable salt thereof, which can be substituted with (Cl), nitro, cyano or hydroxy, respectively. The method of claim 1, Carbonitrile compound or a pharmaceutically acceptable salt thereof, characterized in that it is selected from the following compounds.

The method of claim 1, Carbonitrile compound or a pharmaceutically acceptable salt thereof, characterized in that it is selected from the following compounds.

A carbonitrile compound represented by Chemical Formula 1 characterized by preparing a carbonitrile compound of Chemical Formula 1 according to claim 1 by condensing a compound of Chemical Formula 2 with a compound of Chemical Formula 3 and reacting with cyanogen bromide (BrCN) Manufacturing method. Scheme 1

[Wherein R ₁ , R ₂ and n are the same as defined in claim 1. A pharmaceutical composition for the treatment and prevention of rheumatoid arthritis, comprising the carbonitrile compound represented by the formula (1) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for treating and preventing osteoarthritis, characterized in that it comprises a carbonitrile compound represented by Formula 1 according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Drug for the treatment and prevention of Paget's disease, malignant hypercalcemia and metabolic bone disease, comprising the carbonitrile compound represented by the formula (1) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient Pharmaceutical composition. An inhibitor composition of cysteine protease, comprising the carbonitrile compound represented by Formula 1 according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Inhibitor composition of cathepsin B, L and S characterized in that it contains a carbonitrile compound represented by the formula (1) according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

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