KR100753017B1 - Pharmaceutical composition treating or preventing arthritis, atopic dermatitis, tumor and degenerative brain diseases comprising 7-(3,4-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidine compounds - Google Patents

Abstract

A pharmaceutical composition for treating or preventing arthritis, atopic dermatitis, tumor and degenerative brain diseases comprising 7-(3,4-Dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidine compounds is provided to inhibit activity of PDE-4(phosphodiesterase-4) and reduce side effects such as vomiting by reducing HARBS(high-affinity rolipram binding sites) affinity. The pharmaceutical composition for treating or preventing arthritis, atopic dermatitis, tumor and degenerative brain diseases comprises 7-(3,4-Dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidine compounds represented by the formula(1), wherein R1 and R2 are independently hydrogen atom, linear or branched saturated or unsaturated (C1-C7)alkyl, linear or branched saturated or unsaturated (C1-C7)alkyl containing halogen atom, (C3-C7)cycloalkyl, 3-membered to 7-membered heterocycloalkyl containing oxygen, nitrogen or sulfur in a hetero aromatic ring, (C4-C10)cycloalkyl(C1-C7)alkyl, 3-membered to 7-membered heterocycloalkyl(C1-C7)alkyl containing oxygen, nitrogen or sulfur in a hetero ring, phenyl, benzyl or allyl group; R3 is hydrogen atom, halogen atom, formyl, linear or branched and saturated or unsaturated (C1-C7)alkyl, (C1-C7)alkylketone, (C1-C7)alkoxy, (C1-C7)alkoxy (C1-C7)alkyl, hydroxy(C1-C7)alkyl group, carboxylic acid, carboxyl(C1-C7)alkylester, carboxyl(C1-C7)alkylamide, amino, or mono or di(C1-C7)alkylaminocarbonyl; and R4 is hydrogen atom, formyl, linear or branched saturated or unsaturated (C1-C7)alkyl, (C3-C7)cycloalkyl, (C1-C7)alkoxy, (C1-C7)alkoxy (C1-C7)alkyl, hydroxy(C1-C7)alkyl ketone, (C1-C10)alkylcarboxylic acid, carboxyl(C1-C10)alkylester, carboxyl(C1-C10)alkylamide, amino, mono or di(C1-C7)alkylamino, mono or di(C1-C7)alkylaminocarbonyl, (C3-C7)cycloalkylamine, morpholine, morpholine oxide, piperidine, piperazine, piperazine oxide or aryl group.

Description

For the treatment of brain diseases such as arthritis, atopic dermatitis, cancers including leukemia and Alzheimer's, depression or memory loss, including 7- (3,4-diakoxyphenyl) -pyrazolo [1,5-a] pyrimidine compounds, and Pharmaceutical composition treating or preventing arthritis, atopic dermatitis, tumor and degenerative brain diseases comprising 7- (3,4-Dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compounds}

The present invention relates to arthritis, atopic dermatitis, cancers including leukemia and Alzheimer's, including 7- (3,4-diakoxyphenyl) -pyrazolo [1,5-a] pyrimidine compounds, brain diseases such as depression or memory loss It relates to a pharmaceutical composition for the treatment and prevention of.

PDE-4 inhibitors on which the present invention is based, various compounds have been studied for the development of treatment for asthma and chronic obstructive pulmonary disease [Peter Norman, Expert Opin . Ther. Patents. 2002 , 12 (1) , pp 93-111], among which the most representative compounds are Rolipram [EP 0660711, July 5, 1995], Cilomilast [USP 6,013,827, January 11, 2000] and Roflumilast [USP 5,712,298] January 27, 1998]. Rolipram, the first of these compounds to enter the clinic, was stopped due to side effects such as weak clinical effects and vomiting. In addition, Cilomilast is a compound that has ceased development as an asthma treatment due to the low therapeutic effect on asthma, and is undergoing a clinical trial for Chronic Obstructive Pulmonary Disease (COPD) [Peter Norman, Expert Opin . Ther . Patents 2002 , 12 (1) , 93-111; Compton C., Edelson JD., Cedar E ,. Am. J. Respir . Crit . Care Med. 2001 , 163 , A909. Roflumilast (Zheng Huang, Yves Ducharme, Dwight Macdonald and Annette Robichaud, Current Opinion in Chemical Biology 2001 , 5, 432-438) developed by Altana, Germany, has the highest PDE-4 inhibitory effect. (IC ₅₀ = 0.8 nM) Animal studies have also shown excellent effects on asthma and COPD, but are described in [Armin Hatzelman and Christian Schudt, The Journal of Pharmacology and Experimental Therapeutics 2001 , 297 (1) , 267-279; Daniela S. Bundschuh, Manfrid Eltze, Johannes Barsig, Lutz Wollin, Armin Hatzelmann, and Rolf Beume, The Journal of Pharmacology and Experimental Therapeutics 2001 , 297 (1 ), 280-290] have.

PDE-4 inhibitors treat arthritis in addition to the inflammatory-related lung diseases such as asthma and chronic obstructive pulmonary disease (USP 2003/0092706 A1), atopic dermatitis, leukemia, various cancers (Miles D. Houslay, Peter Schafer and Kam) YJ Zhang, Drug Discovery Today , 2005 , 10 (22), 1503-1519) and depression treatment (USP 4,178,449, December 11, 1979), Alzheimer's and other brain diseases (Sophie L. Rovner, C & EN , 38, February 21, 2005).

2,7-diphenyl-pyrazolo [1,5-a] pyrimidine derivatives similar to the compounds of formula 1 used in the present invention have been reported for their synthesis (Mohamed H. Elnagdi and Ayman W. Erian, Bull and. Chem. Soc. Jpn., 1990, 63, 1854-1856), arthritis, prevention and treatment area of neurodegenerative diseases such as Alzheimer's and various cancers, depression, memory loss, including atopic dermatitis, leukemia is not related .

It is an object of the present invention to treat arthritis, atopic dermatitis, cancers including leukemia and Alzheimer's, depression or memory, comprising the 7- (3,4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound of formula 1 It is to provide a pharmaceutical composition for the treatment and prevention of brain diseases such as reduction.

Arthritis, atopic dermatitis, leukemia, comprising 7- (3,4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound represented by the following Chemical Formula 1 as an active ingredient It relates to a pharmaceutical composition for the treatment and prevention of cancer and Alzheimer's, brain diseases such as depression or memory loss.

7- (3,4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound represented by the following Chemical Formula 1 and a method of preparing the same are described in Korean Patent Application No. 10-2004-0101458 In the present invention, cancer, including arthritis, atopic dermatitis, leukemia, containing 7- (3,4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound represented by Formula 1 as an active ingredient; It is characterized by a pharmaceutical composition for the treatment and prevention of brain diseases such as Alzheimer's, depression or memory loss.

[Formula 1]

[In Formula 1, R ₁ And R ₂ may be the same or different, independently represent a hydrogen atom, a linear or saturated or unsaturated crushing each other (C ₁ - C ₇₎ alkyl, a saturated straight or branched chain containing a halogen group such as fluorine or unsaturated (C ₁ C ₇ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, 3- to 7-membered heterocycloalkyl containing oxygen, nitrogen or sulfur in the heterocycle, (C ₄ -C ₁₀ ) cycloalkyl (C ₁ -C ₇ ) alkyl, including oxygen, nitrogen or sulfur in the heterocycle A 3-7 membered heterocycloalkyl (C ₁ -C ₇ ) alkyl, phenyl, benzyl or allyl group or with R ₁ R ₂ may be linked with (C ₁ -C ₃ ) alkylene containing a halogen group such as (C ₁ -C ₃ ) alkylene or fluorine; R ₃ represents a hydrogen atom, a halogen atom, formyl, straight or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkyl ketone, (C ₁ -C ₇ ) alkoxy, (C _1- C ₇ ) alkoxy (C ₁ -C ₇ ) alkyl, hydroxy (C ₁ -C ₇ ) alkyl group, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkyl Amide, amino, mono or di (C ₁ -C ₇ ) alkylamino, mono or di (C ₁ -C ₇ ) alkylaminocarbonyl; R ₄ is hydrogen, formyl, straight or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, (C ₁ -C ₇ ) alkoxy, (C ₁ -C ₇ ) Alkoxy (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkylketone, (C ₁ -C ₁₀ ) alkylcarboxylic acid, carboxyl (C ₁ -C ₁₀ ) alkylester, carboxyl (C _1- C ₁₀ ) alkylamide, amino, mono or di (C ₁ -C ₇ ) alkylamino, mono or di (C ₁ -C ₇ ) alkylaminocarbonyl, (C ₃ -C ₇ ) cycloalkylamine, morpholine, Aryl groups represented by morpholine oxide, piperidine, piperazine, piperazine oxide or Ar below;

Wherein A is N or NO, m and n represent an integer from 0 to 4 independently of each other, X may be the same or different from each other, and independently of each other a hydrogen atom, a pulverized or straight chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, hydroxy, (C ₁ -C ₇ ) alkoxy, (C ₁ -C ₇ ) alkoxy (C ₁ -C ₇ ) alkyl, Halogen, cyano, nitro, amino, mono- or di (C ₁ -C ₇ ) alkyl amino, mono- or di (C ₁ -C ₇ ) alkyl aminocarbonyl, (C ₃ -C ₇ ) cycloalkylamine, Morpholine, Morpholine Oxide, Piperazine, Piperazine Oxide, Guanidine, Urea, Phenyl, Benzyl, Benzyloxy, Thiobenzyl, Carboxylic Acid, Carboxyl (C ₁ -C ₇ ) Alkylester, Carboxyl (C ₁ -C ₇ ) alkylamide, (C ₁ -C ₇ ) alkylketone, benzoyl; Provided that the alkyl group, cycloalkyl group, heterocycloalkyl group, heterocycloalkylalkyl group, phenyl group or benzyl group of R _{1 ,} R _{2 ,} R _3, and R ₄ is (C ₁ -C ₇ ) alkoxy, halogen including fluorine, nitro, And may be substituted with a cyano, hydroxy, carboxyl or amino group.]

The compound of Formula 1 has excellent activity and selectivity against PDE-4 enzymes, and has excellent in vivo effects in animal experiments such as arthritis, atopic dermatitis, cancers including leukemia and Alzheimer's, depression or memory loss, etc. It is useful as a therapeutic and preventive agent for diseases.

Specific examples of the 7- (3,4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound of Formula 1 or a pharmaceutically acceptable salt thereof used as an active ingredient in the pharmaceutical composition according to the present invention Examples of R ₁ are linear or saturation of the ground or unsaturated (C ₁ - C ₇₎ alkyl or a fluorine atom is a saturated substituted straight or branched chain or unsaturated - and (C ₁ C ₇₎ alkyl, R ₂ is a hydrogen atom, a linear or Saturated or unsaturated (C ₁ -C ₇ ) alkyl or linear or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl substituted by fluorine atoms, cyclopropylmethyl, cyclopropyl, cyclopentyl, phenyl, benzyl or allyl R ₃ is substituted with a hydrogen atom, halogen, formyl, (C ₁ -C ₇ ) alkoxy (C ₁ -C ₇ ) alkyl or hydroxy (C ₁ -C ₇ ) alkyl.

Preferred compounds of the formula (1) used as an active ingredient in the pharmaceutical composition according to the present invention include a straight or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl or a fluorine atom substituted with R ₁ is saturated or Unsaturated (C ₁ -C ₇ ) alkyl, R ₂ is straight or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl, cyclopropylmethyl, cyclopropyl, cyclopentyl, phenyl, benzyl or allyl group, R ₃ Is a hydrogen atom or halogen, R ₄ is an aryl group represented by

A is N or NO, m and n represent an integer of 0 to 3 independently of each other, X may be the same or different from each other, and independently of each other hydrogen, benzyloxy, hydroxy, thiobenzyl, carboxylic acid , Carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkylamide, halogen, crushed or straight-chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) Alkoxy, amino, di (C ₁ -C ₇ ) alkylamino or mono- or di (C ₁ -C ₇ ) alkylaminocarbonyl.

Particularly preferred ranges of compounds include the following general formula (2) compounds.

[Formula 2]

[A is N or NO, X may be the same or different from each other, and independently from each other hydrogen atom, hydroxy, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkylamide, halogen, crushed or straight chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkoxy, amino, m and n are each independently an integer from 0 to 3.

The most preferred compound used as an active ingredient in the pharmaceutical composition according to the present invention, X is a hydrogen atom, hydroxy, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl ( C ₁ -C ₇ ) alkylamide, halogen, milled or straight chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkoxy, amino or A is N or NO If it is.

[Formula 3]

[A is N or NO, X may be the same or different from each other, and independently from each other hydrogen atom, hydroxy, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkylamide, halogen, crushed or straight chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkoxy, amino, m and n are each independently 0 or 1.

Representative compounds of the general formula (3) include the following compounds.

As a method for preparing the compound of Formula 1, which is used as an active ingredient in the pharmaceutical composition according to the present invention, Scheme 1, Scheme 2 and Scheme 3 are illustrated, and the following preparation method is an active ingredient in the pharmaceutical composition according to the present invention. It is not intended to limit the method of preparing the compound of Formula 1 to be used as, modifications of the following preparation method will be apparent to those skilled in the art, and unless otherwise stated, the definition of the substituents in the following scheme is the same as the definition in formula (1).

As shown in Scheme 1, 3-amino-5- (substituted phenyl) pyrazole compound (3) and aminopropenone compound (4) are reacted in acetic acid medium to 7- (3,4-Dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compounds can be prepared.

Scheme 1

[Wherein, R ₁ , R ₂ , R ₃ and R ₄ are the same as defined in the formula (1).]

From 3-amino-5- (substituted phenyl) pyrazole compound (3) and aminopropenone compound (4) to 7- (3,4-dialkoxyphenyl) -pyrazolo [ In preparing a 1,5-a] pyrimidine compound, a base and an alcohol such as piperidine may be used (KM Al-Zaydi, MAA Al-Shiekh, EAA Hafez, J. Chem . Res. Synop, 2000 , 1 EI Al-Afaleq, Synth Commun , 2000 , 30 (11), 1985-1999), more preferably using acetic acid as a solvent.

In addition, as another method for preparing 7- (3,4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound of the formula (1), 3-amino-5- ( Substituted phenyl) pyrazole compound (3) can be prepared by reacting 3- (3,4-dialkoxyphenyl) -3-oxopropionaldehyde compound (5) in the presence of acetic acid.

Scheme 2

[Wherein, R ₁ , R ₂ , R ₃ and R ₄ are the same as defined in the formula (1).]

In addition, as another method for preparing 7- (3,4-diakoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound represented by Chemical Formula 1, 3-amino-5- The (substituted phenyl) pyrazole compound (3) can be prepared by reacting the 3- (3,4-dialkoxyphenyl) cinnaaldehyde compound (6) with acetic acid or other solvents and bases. In this case, Y represents halogen or alkoxy.

Scheme 3

[Wherein, R ₁ , R ₂ , R ₃ and R ₄ are the same as defined in the formula (1).]

Among the compounds prepared by the routes of Scheme 1, Scheme 2 and Scheme 3, a compound of Formula 1 including a new substituent may be prepared by a reaction of converting some substituents to other substituents, such as converting a carboxylic acid of the X substituent to a carboxylic acid ester. Can be.

3-amino-5- (substituted phenyl) pyrazole compound (3), which is a substance used for preparing the compound of Formula 1, reacts α -cyanomethylphenylketone compound (7) with hydrazine, as shown in Scheme 4. The aminopropene compound (4) can be prepared by reacting the catechol ketone compound (8) with an acetal of dimethylformamide. In addition, 6a, which is a representative compound of Compound 6, may be prepared by using POCl ₃ / DMF in Compound 8, and Compound 6b may be obtained by substituting alcohol for Compound 6a.

Scheme 4

[Wherein, R ₁ , R ₂ , R ₃ and R ₄ are the same as defined in the formula (1).]

In the process of preparing 3-amino-5- (substituted phenyl) pyrazole compound (3) from α -cyanomethylphenyl ketone compound (7) and hydrazine, alcohol is mainly used as a solvent, and a catechol ketone compound ( 8) and the reaction for preparing the aminopropene compound (4) from acetals of dimethylformamide can be synthesized in a similar manner as known in the literature (F. Al-Omran, N. Al-Awadhi, MM Abdel Khalik, K. Kaul, AA El-Khair, and MH Elnadgi, J. Chem . Res , Synop , 1997 , 3 , 84-85 ), where DMF-acetal is a reactant and a solvent.

In addition, the α -cyanomethylphenyl ketone compound (7) may be prepared according to the method exemplified in British Patent Publication No. 2,127,410, but is not limited thereto, and may be synthesized by other methods.

On the other hand, 7- (3,4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound of the formula (1) used as an active ingredient in the pharmaceutical composition according to the present invention is excellent for PDE-4 enzyme The side effects such as vomiting are expected to be negligible with low activity and enzyme selectivity and low HARBS affinity.

The pharmaceutical composition according to the present invention contains a compound represented by the formula (1) as an active ingredient, a pharmaceutical agent for the treatment and prevention of arthritis, atopic dermatitis, cancer treatment including leukemia and brain diseases such as Alzheimer's, depression or memory loss It is suitable as a use as a composition.

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. The following preparation is described in Korean Patent Application No. 10-2004-0101458.

[ Production Example  One]

3-amino-5- (substituted Phenyl ) Pyrazole  Compound (3) and 1- (3,4- Dialkoxyphenyl ) -3- Dimethylaminopropenone  Preparation of Compound of Formula 1 by Reaction of Compound (4)

5-amino-pyrazole derivatives (3) 0.69 mmol and 1- (3,4-diakoxyphenyl) -3-dimethylaminopropenone (1- (3,4-dialkoxyphenyl) -3 in acetic acid 0.69 mmol of -dimethyl amino-propenone) compound (4) was added thereto, followed by stirring at room temperature. NaHCO ₃ saturated aqueous solution was added to the reaction solution, extracted three times with ethyl acetate, and the organic layer was washed twice with brine. After drying over MgSO ₄ , the solvent was removed under reduced pressure. The organic layer was dried over MgSO _4, and then the solvent was removed under reduced pressure. The mixture was separated by silica gel column chromatography to obtain the target compound of formula (1). The structure of each compound of Formula 1 and ¹ H NMR are shown in Table 1 below.

[ Production Example  2]

3-amino-5- (substituted Phenyl ) Pyrazole  Compound (3) and 3- (3,4- Dialkoxyphenyl ) -3- Oxopropy Preparation of Compound of Formula 1 by Reaction of Onaldehyde Compound (5)

3-aminopyrazole derivatives in acetic acid (3) 0.60 mmol and 3- (3,4-dialkoxyphenyl) -3-oxopropionaldehyde (3- (3,4-Dialkoxy-phenyl) -3 0.60 mmol of -oxopropionaldehyde) compound (5) was added, followed by stirring at room temperature. NaHCO ₃ to the reaction solution Add saturated aqueous solution, extract three times with ethyl acetate, and wash the organic layer twice with brine. After drying over MgSO ₄ , the solvent was removed under reduced pressure. The mixture was separated by column chromatography (silica gel, n-hexene / ethyl acetate, 3/1) to obtain the desired compound of formula (1). The structure of each compound of Formula 1 and ¹ H NMR are shown in Table 1 below.

[ Production Example  3]

3-amino-5- (substituted Phenyl ) Pyrazole  Compound (3) and 3- (3,4- Dialkoxyphenyl ) Cinnamic Aldehyde  Preparation of the compound of formula 1 by the reaction of (6)

5-amino-pyrazole derivatives in acetic acid (3) 0.69 mmol and 3- (3,4-dialkoxyphenyl) cinnamaldehyde (3- (3,4-dialkoxyphenyl) cinnamic aldehyde) Compound (6) 0.69 mmol was added and stirred at room temperature. NaHCO ₃ saturated aqueous solution was added to the reaction solution, extracted three times with ethyl acetate, and the organic layer was washed twice with brine. After drying over MgSO ₄ , the solvent was removed under reduced pressure. The organic layer was dried over MgSO _4, and then the solvent was removed under reduced pressure. The mixture was separated by silica gel column chromatography to obtain the target compound of formula (1). The structure of each compound of Formula 1 and ¹ H NMR are shown in Table 1 below.

TABLE 1

PDE -4 active Inhibitory ability  analysis

The activity inhibition activity of PDE-4d was performed by modifying the method of Kate et al. (Kate, et al., JBC , 271 : 796 (1996)). PDE-4d used human PDE-4d prepared by genetic recombination technology.

The PDE4d gene was cloned into the GST fusion expression vector pGEX4T3 (APBiotech), and then transformed into E. coli BL21, DE3. After incubating the strain at 37 ° C. in LB medium, the temperature was lowered to 18 ° C. when 0.3-0.5 OD was induced to induce PDE4d expression with 0.5 mM IPTG. After 15 hours of expression induction, PDE4d-expressing cells were obtained, and the cells were suspended in a buffer solution (50 mM Tris, pH 8.0, 0.4 M NaCl, 5 mM DTT), disrupted, and purified by centrifugation. The columns used for purification are Q-Sepharose (APBiotech), Glutanion-Sepharose (APBiotech), and Superdex 200 gel filtration chromatography (APBiotech) in this order.

In order to measure the ability to inhibit PDE4d activity of each compound, 1 nM of PDE4d and 250 nM of cAMP (Sigma) as a substrate were added to the reaction buffer (50 mM Tris-HCl, pH 7.5) and 4 mM MgCl ₂ , and obtained in Example 1 above. Each compound was added at a concentration of 1 to 50 uM and then reacted at 34 ° C. for 45 minutes. The reaction total was 60 µl. At this time, cAMP was used by mixing a ratio of [ ³ H] cAMP (Amersham, 1 μCi / μl) and cAMP (hot: cold, that is, isotope: non-isotope) at 1: 200. The reaction was then stopped by heating at 95 ° C. for 2 minutes, then cooled for 3 minutes and 30 μl of 1 mg / ml Snake Venome (sigma V0376) was added. Subsequently, after 30 minutes of reaction at 34 ° C, 30 μl of the reaction solution was poured into 250 μl of DOWEX 1 × 2-100 ion exchange resin (Aldrich, pre-mixed at a ratio of 1 to 1.6 of distilled water) and stirred vigorously for 2 minutes. Then, the resin was allowed to settle, and 130 μl of the supernatant was added to 2 ml of scintillation cocktail (Packard), mixed well, and measured by beta-counter. The concentration of each test compound that inhibited 50% of the enzyme activity compared to the enzyme activity of the control group without the addition of the compound of Formula 1 was determined as IC ₅₀ . The structure and IC ₅₀ values of the compounds are shown in Table 2 below.

TABLE 2

PDE -4 Enzyme Selectivity Assay

In order to measure the enzyme selectivity against PDE4d, the enzyme activity inhibitory effect of each test compound against PDE-3a, PDE-5a, and PDE-7a was measured. PDE3a and PDE7a, which used cAMP as a substrate, measured the degree of activity inhibition using the Amplified Luminescent Proximity Homogeneous Assay (Alpha) Screen method, and PDE5a, which used cGMP as a substrate, used the enzyme of each test compound by FP (fluorescence polarization) method. Activity inhibition efficacy was measured.

PDE3a, PDE5a and PDE7a all used human enzymes prepared by genetic recombination techniques.

In order to measure the inhibitory activity of PDE3a and PDE7a of each test compound, first put each compound (final DMSO 1%) at the concentration to be tested in a 96 well half area white plate and follow the same conditions as the conditions for measuring PDE4d enzyme activity described above. Enzyme reaction at When measuring enzyme activity using Alpha Screen, the concentrations of PDE3a and PDE7a should be 10nM and 80nM, respectively, and were carried out according to the method proposed in the Alphascreen cAMP assay kit (Perkin Elmer life science). Briefly, after the enzymatic reaction between PDE3a and PDE7a is terminated, final 10 nM biotinylated-cAMP and 20ul / mL donor beads are added to the reaction mixture. The donor bead is sensitive to light, so be careful not to expose it to light and let it react for 30 minutes at room temperature. Then add acceptor beads to a final 20ul / ml. After reacting for 45 minutes at room temperature, the reaction was further reacted for 20 minutes in the Fusion Alpha-FP device, and the emission at 520-620 nm was measured.

In the enzymatic reaction of PDE3a and PDE7a, the signal increase and decrease is caused by the mutual competition between the remaining cAMP and the newly added biotinylated-cAMP, which is not used as a substrate. When the enzyme activity of PDE3a and PDE7a is inhibited by each test compound, signal is reduced.

PDE5a, which uses cGMP as an enzyme substrate, measured the enzyme inhibitory ability of each test compound according to the experimental method presented by IMAP ^TM Phosphodiesterase Assay Kit (Molecular Device), which measures enzyme activity by fluorescence polarization (FP) method. First, add each test compound (final DMSO 5%) at the concentration to be tested in 96 well black plate, and add FL-cGMP (Fluorescence-labeled derivatives of cGMP) and final 200 nM PDE5a. After reacting for 45 minutes at 30 degrees, add the IMAP binding reagent provided by the kit. After reacting at room temperature for 60 minutes, FP obtained by 485 nm exitation and 535 nm emission was measured. The more enzyme activity of PDE5a, the higher the FP signal. The inhibition of enzyme activity of PDE5a by each test compound can be determined by measuring the decreased FP signal.

BBB (blood-brain barrier) test

Three 7-week-old SD rats were grouped into each experimental group, and the experimental drug was intraperitoneally injected at 10 mg / 2 ml / kg. After 30 minutes, 1, 3, and 5 hours after administration, the animals were opened under ether anesthesia, and blood was collected from the abdominal vein. The rat skull was removed, the brains were separated, weighed, frozen and stored. The blood was centrifuged to separate plasma and frozen until stored.

In the pretreatment process for sample analysis, 100ul was taken after melting the plasma, an internal standard was added, and an organic solvent, which was found to have excellent recovery through solvent extraction experiment, was added and mixed. After centrifugation, the protein was removed, the supernatant was collected, the organic solvent was evaporated using an evaporator, and the pellet was dissolved and analyzed as a mobile phase used in LC-MS / MS analysis.

Brain tissues were homogenized with homogenizer with saline of 3 times the weight and extracted and analyzed in the same way as plasma.

Drug concentrations in plasma and brain were measured using LC-MS / MS to compare drug concentrations in blood and brain tissues over time.

Drug test method on memory

In order to measure the effectiveness of the drug, environmental fear conditioning, which is dependent on hippocampus, was conducted. Environmental fear conditioning learning is a widely known method of measuring memory improvement (Fanselow, 2000; Gewirtz et al ., 2000; Maren et al ., 2000). Adult mice were used 8-9 weeks old because they were more stable than young mice. C57BL / 6 male mice , which have been reported to be superior in learning and memory compared to other mice (Crawley et al., 1997), were isolated from pathogens, isolated from pathogens, and kept at 12 hours for animal breeding at Seoul National University Genetic Engineering Research Institute. It was raised by adjusting day / night. In addition, a freeze monitor (6610-0002-C, SanDiego instrument) was used to measure the freezing that occurs after memorizing the electric shock and the context. It is 26.5cm-26.5cm-24cm, and it is made of a transparent acrylic (plaxiglas). In addition, the grid, which gives a shock of 0.5 mA to the feet of the mice, has a distance of 0.8 cm and a width of 26 cm by 26 cm. The actual learning method was used by Barad et al ., 1998. First, the training is adapted to the human hand. Mice originally have a rejection of the human hand, so that the response is minimal in learning. Next, a pre-exposure that adapts to the environment is likewise done in an environment where you learn for three minutes per horse to minimize the fear that the environment itself can give. The drug was then intraperitoneally injected and learned 30 minutes later because the drug reached the brain after 30 minutes (Pietrzak et al., 2003). The drug is dissolved in DMSO (sigma chemical co.) And each drug is dissolved in DMSO in successive dilutions in sigma chemical co., Phosphate-buffered saline (PBS). The experiment was performed not to exceed%. 30 minutes after the medicine was injected, it was able to explore the environment for 120 seconds and gave a tone (dB, dB) for 30 seconds. In the last two seconds of the sounding 30 seconds, an electric shock of 0.5 mA was applied to the mouse's foot through the grid, allowed to settle for 30 seconds again, and then isolated from the environment. This is because, as described above, mice aim to learn by linking the environment with electrical shock. Lastly, after 24 hours of exposure to the same environment, learning and remembering, and watching for a fear response for 3 minutes every five seconds, the watch measured the two people directly and used the average value. Data analysis was performed using the PRISM program (Graph Pad).

As a result of the PDE-4 inhibitory activity test, as shown in Table 2, in the compound of the series in which R ₁ and R ₂ are substituted with methyl and cyclopentyl groups in Formula 1, the compounds in which X is substituted by meta position are substituted by ortho or para It can be seen that PDE-4 inhibitory activity is generally superior to the prepared compounds. In addition, it can be seen that compounds substituted with a meta position of X have excellent PDE-4 inhibitory activity in a series of compounds in which R ₁ and R ₂ are substituted with difluoromethyl and cyclopropylmethyl groups, respectively.

Among the compounds in which A is substituted with N or NO in Formula 1, meta-pyridine compounds (Compound 58, IC ₅₀ = 20 nM) this para-pyridne compound 93 (IC ₅₀ = Compared to 180 nM), but showed a very good activity, the activity of the meta-pyridine-based compound is very variable depending on the substituents of R ₁ and R ₂ (Compounds 60, 62, 65, 66). In addition, R ₃ In the case of substitution, the PDE-4 inhibitory activity was significantly higher than that of Compound 67 substituted with Br.

Table 2 shows the results of the inhibitory activity test for PDE-3, -5, and -7 of the representative compounds having excellent activity and highly pharmacokinetic test results as oral administration agents. As a result, the PDE-4 enzyme selectivity of the representative compounds tested among the synthesized compounds is 100 times or more, indicating that the selectivity for the target point of action PDE-4 is very high.

Table 3 PDE enzyme inhibitory activity IC ₅₀ (uM)

As a result of passing the BBB of the compound 59 of the compound showed a BBB passage rate of 50% or more as shown in Table 4.

Table 4 BBB Pass Test Results for Compound 59

The results of environmental fear conditioning dependent on hippocampus were measured in Table 5 to determine the memory-improving effect of Compound 59 obtained through this study. It can be seen that the effect is excellent.

TABLE 5 Memory Enhancing Effects of Compound 59 in Environmental Fear Conditioning Learning

Therefore, the pharmaceutical composition of the present invention contains 7- (3,4-diakoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound exhibiting excellent selective activity against PDE-4 enzymes as an active ingredient. Characterized in that, the pharmaceutical composition exhibits excellent brain permeability in the brain permeation model animal experiments, and shows the effect of improving memory in vivo, cancer and Alzheimer's disease including arthritis, atopic dermatitis, leukemia based on PDE-4 inhibition, It can be used for preventing and treating brain diseases such as depression or memory loss.

Claims (8)

Arthritis, atopic dermatitis, characterized in that it contains a 7- (3, 4- dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound represented by the formula (1) or a pharmaceutically acceptable salt thereof Pharmaceutical compositions for the treatment and prevention of cancer and brain diseases. [Formula 1]

[In Formula 1, R ₁ And R ₂ may be the same or different, independently represent a hydrogen atom, a linear or saturated or unsaturated crushing each other (C ₁ - C ₇₎ alkyl, a saturated straight or branched chain containing a halogen group such as fluorine or unsaturated (C ₁ C ₇ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, 3- to 7-membered heterocycloalkyl containing oxygen, nitrogen or sulfur in the heterocycle, (C ₄ -C ₁₀ ) cycloalkyl (C ₁ -C ₇ ) alkyl, including oxygen, nitrogen or sulfur in the heterocycle A 3-7 membered heterocycloalkyl (C ₁ -C ₇ ) alkyl, phenyl, benzyl or allyl group or with R ₁ R ₂ may be linked with (C ₁ -C ₃ ) alkylene containing a halogen group such as (C ₁ -C ₃ ) alkylene or fluorine; R ₃ represents a hydrogen atom, a halogen atom, formyl, straight or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkylketone, (C ₁ -C ₇ ) alkoxy, (C _1- C ₇ ) alkoxy (C ₁ -C ₇ ) alkyl, hydroxy (C ₁ -C ₇ ) alkyl group, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkyl Amide, amino, mono or di (C ₁ -C ₇ ) alkylamino, mono or di (C ₁ -C ₇ ) alkylaminocarbonyl; R ₄ is hydrogen, formyl, straight or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₃ -C ₇ ) cycloalkyl, (C ₁ -C ₇ ) alkoxy, (C ₁ -C ₇ ) Alkoxy (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkylketone, (C ₁ -C ₁₀ ) alkylcarboxylic acid, carboxyl (C ₁ -C ₁₀ ) alkylester, carboxyl (C _1- C ₁₀ ) alkylamide, amino, mono or di (C ₁ -C ₇ ) alkylamino, mono or di (C ₁ -C ₇ ) alkylaminocarbonyl, (C ₃ -C ₇ ) cycloalkylamine, morpholine, Aryl groups represented by morpholine oxide, piperidine, piperazine, piperazine oxide or Ar below;

Wherein A is N or NO, m and n represent an integer from 0 to 4 independently of each other, X may be the same or different from each other, and independently of each other a hydrogen atom, a pulverized or straight chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, hydroxy, (C ₁ -C ₇ ) alkoxy, (C ₁ -C ₇ ) alkoxy (C ₁ -C ₇ ) alkyl, Halogen, cyano, nitro, amino, mono- or di (C ₁ -C ₇ ) alkyl amino, mono or di (C ₁ -C ₇ ) alkylaminocarbonyl, (C ₃ -C ₇ ) cycloalkylamine, parent Porin, morpholin oxide, piperazine, piperazine oxide, guanidine, urea, phenyl, benzyl, benzyloxy, thiobenzyl, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkylester, carboxyl (C ₁ C ₇ ) alkylamides, (C ₁ -C ₇ ) alkylketones, benzoyl; Provided that the alkyl group, cycloalkyl group, heterocycloalkyl group, heterocycloalkylalkyl group, phenyl group or benzyl group of R _{1 ,} R _{2 ,} R _3, and R ₄ is (C ₁ -C ₇ ) alkoxy, halogen including fluorine, nitro, And may be substituted with a cyano, hydroxy, carboxyl or amino group.] The method of claim 1, R ₁ saturation of a straight or branched chain or unsaturated (C ₁ - C ₇₎ alkyl or a fluorine atom is a saturated substituted straight or branched chain or unsaturated - and (C ₁ C ₇₎ alkyl, R ₂ is a hydrogen atom, a straight or branched chain Saturated or unsaturated (C ₁ -C ₇ ) alkyl, or straight or branched saturated or unsaturated (C ₁ -C ₇ ) alkyl, substituted with fluorine atoms, cyclopropylmethyl, cyclopropyl, cyclopentyl, phenyl, benzyl or allyl groups , R ₃ is a hydrogen atom, halogen, formyl, 7 (wherein a is substituted by - (C ₁ C ₇₎ alkyl, _{3, (C 1 - - C} 7) alkoxy (C ₁ C ₇₎ alkyl or hydroxy Pharmaceuticals for the treatment and prevention of arthritis, atopic dermatitis, cancer and brain diseases characterized by containing 4-dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compounds or pharmaceutically acceptable salts thereof Pharmaceutical composition. The method of claim 2, R ₁ is straight-chain or a saturated or unsaturated grinding (C ₁ - C ₇₎ alkyl or a fluorine atom is substituted by a straight chain or a saturated or unsaturated grinding - and (C ₁ C ₇₎ alkyl, R ₂ is a straight chain or a saturated or unsaturated grinding (C ₁ -C ₇ ) alkyl, cyclopropylmethyl, cyclopropyl, cyclopentyl, phenyl, benzyl or allyl group, R ₃ is a hydrogen atom or a halogen, R ₄ is an aryl group represented by the following Ar,

A is N or NO, m and n represent an integer of 0 to 3 independently of each other, X may be the same or different from each other, and independently of each other hydrogen, benzyloxy, hydroxy, thiobenzyl, carboxylic acid , Carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkylamide, halogen, crushed or straight-chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) 7- (3,4-dialkoxyphenyl) -pyrazolo, characterized in that it is alkoxy, amino, di (C ₁ -C ₇ ) alkylamino or mono- or di (C ₁ -C ₇ ) alkylaminocarbonyl [ A pharmaceutical composition for the treatment and prevention of arthritis, atopic dermatitis, cancer and brain diseases, comprising 1,5-a] pyrimidine compound or a pharmaceutically acceptable salt thereof. The method of claim 2, It is characterized by containing a 7- (3, 4- dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound or a pharmaceutically acceptable salt thereof, which is represented by the following formula (2): Pharmaceutical compositions for the treatment and prevention of arthritis, atopic dermatitis, cancer and brain diseases. [Formula 2]

[A is N or NO, X may be the same or different from each other, and independently of each other a hydrogen atom, hydroxy, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C _1- C ₇ ) alkylamide, halogen, crushed or straight chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkoxy or amino, m and n are each independently an integer from 0 to 3. ] The method of claim 4, wherein X is hydrogen atom, hydroxy, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkylamide, halogen, crushed or straight chain saturated or unsaturated (C ₁ -C ₇ ) A pharmaceutical composition for the treatment and prevention of arthritis, atopic dermatitis, cancer and brain diseases, wherein the alkyl, (C ₁ -C ₇ ) alkoxy or amino group, m and n, independently of each other, are an integer from 0 to 3. . The method of claim 4, wherein It is characterized by containing a 7- (3, 4- dialkoxyphenyl) -pyrazolo [1,5-a] pyrimidine compound or a pharmaceutically acceptable salt thereof, which is represented by the following formula (3): Pharmaceutical composition for the treatment and prevention of arthritis, atopic dermatitis, cancer and brain diseases. [Formula 3]

[A is N or NO, X may be the same or different from each other, and independently from each other hydrogen atom, hydroxy, carboxylic acid, carboxyl (C ₁ -C ₇ ) alkyl ester, carboxyl (C ₁ -C ₇ ) alkylamide, halogen, crushed or straight chain saturated or unsaturated (C ₁ -C ₇ ) alkyl, (C ₁ -C ₇ ) alkoxy or amino group, m and n are independently 0 or 1 of each other.] The method according to any one of claims 1 to 6, The brain disease is Alzheimer's, depression or memory loss, pharmaceutical composition for the treatment and prevention of arthritis, atopic dermatitis, cancer and brain diseases. delete